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Quantum dots target infections

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Photoactivated QDs Kill Antibiotic-Resistant ‘Superbugs’

BOULDER, Colo., Jan. 20, 2016 — A technique for treating bacterial infections has successfully used light-activated quantum dots (QDs) to kill multiple multidrug-resistant strains.

http://www.photonics.com/Article.aspx?AID=58218

e coli

http://www.photonics.com/images/Web/Articles/2016/1/20/PIC_QD2.jpg

Modified atomic force micrograph of multidrug-resistant E. coli. Courtesy of the Nagpal Group/University of Colorado Boulder.

 

The approach is adaptive to constantly evolving drug-resistant bacteria and avoids damage to surrounding cells, an issue encountered in earlier attempts that deployed metal nanoparticles such as gold and silver to combat bacteria.

“By shrinking these [QD] semiconductors down to the nanoscale, we’re able to create highly specific interactions within the cellular environment that only target the infection,” said professor Prashant Nagpal of the University of Colorado Boulder.

The QDs — which are inactive in darkness — were tailored to target particular infections thanks to their light-activated properties. The researchers said that by modifying the wavelength of light applied, they could activate the QDs to alter and kill infected cells with specificity.

Napgal and his team tested the QD therapy on mammalian tissue containing bacterial cells in mono- and cocultures. The bacteria under investigation were ethicillin-resistant Staphylococcus aureus, carbapenem-resistant E. coli, and extended-spectrum ß-lactamase-producing Klebsiella pneumoniae and Salmonella typhimurium.

They reported 92 percent of bacterial cells were killed, while leaving mammalian cells intact. The QDs could also be tuned to increase bacterial proliferation.

qd

http://www.photonics.com/images/Web/Articles/2016/1/20/PIC_QD1.jpg

Plated antibiotic resistant ‘superbugs’ before and after treatment with nanoparticles. Courtesy of the Nagpal Group/University of Colorado Boulder.

The team said the killing effect was independent of the QD material used; rather, it was controlled by the redox potentials of the photogenerated charge carriers, which selectively altered cellular redox states. Photoexcited QDs could be used in the study of the effect of redox states on living systems, and lead to clinical phototherapy for the treatment of infections, the researchers said.

The specificity of the treatment could help reduce or eliminate the potential side effects of other treatment methods, as well as provide a path forward for future development and clinical trials.

“Antibiotics are not just a baseline treatment for bacterial infections, but HIV and cancer as well,” said professor Anushree Chatterjee. “Failure to develop effective treatments for drug-resistant strains is not an option, and that’s what this technology moves closer to solving.”

Nagpal and Chatterjee are the cofounders of Praan Biosciences Inc., a startup that can sequence genetic profiles using a single molecule, and have filed a patent on the QD therapy technology.

The research was published in Nature Materials (doi: 10.1038/nmat4542).

 

Photoexcited quantum dots for killing multidrug-resistant bacteria

Colleen M. CourtneySamuel M. GoodmanJessica A. McDanielNancy E. MadingerAnushree ChatterjeePrashant Nagpal

Nature Materials(2016)      http://dx.doi.org:/10.1038/nmat4542

Multidrug-resistant bacterial infections are an ever-growing threat because of the shrinking arsenal of efficacious antibiotics1, 2, 3, 4. Metal nanoparticles can induce cell death, yet the toxicity effect is typically nonspecific5, 6, 7, 8. Here, we show that photoexcited quantum dots (QDs) can kill a wide range of multidrug-resistant bacterial clinical isolates, including methicillin-resistant Staphylococcus aureus, carbapenem-resistant Escherichia coli, and extended-spectrum β-lactamase-producingKlebsiella pneumoniae and Salmonella typhimurium. The killing effect is independent of material and controlled by the redox potentials of the photogenerated charge carriers, which selectively alter the cellular redox state. We also show that the QDs can be tailored to kill 92% of bacterial cells in a monoculture, and in a co-culture of E. coli and HEK 293T cells, while leaving the mammalian cells intact, or to increase bacterial proliferation. Photoexcited QDs could be used in the study of the effect of redox states on living systems, and lead to clinical phototherapy for the treatment of infections.

Figure 2: The effect of CdTe-2.4 is specific to the reduction and oxidation potentials.close

The effect of CdTe-2.4 is specific to the reduction and oxidation potentials.

a, Absorbance spectra for CdTe and CdSe of several sizes. Insets show transmission electron microscopy (TEM) images with colour-coded scale bars (50nm except for CdTe-2.4, which is 25nm). b, Scanning tunnelling spectroscopy (STS) meas…

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Vaccines, Small Peptides, aptamers and Immunotherapy [9]

Writer and Curator: Larry H. Bernstein, MD, FCAP

This contribution has the following structure:

9.1.1 Viruses in carcinogenesis

9.1.2   Simultaneous Humoral and Cellular Immune Response against Cancer–Testis Antigen NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2–binding Peptide Epitopes

9.1.3 Monoclonal Antibodies in Cancer Therapy

9.1.4 Aptamers

9.1.5 Tumor Suppressors

9.1 Vaccines

9.1.1  Viruses in carcinogenesis

  • HPV-associated cervical cancer
  • HPV-associated head and neck cancer: a virus-related cancer epidemic

The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide

Risk of pancreatic cancer among individuals with hepatitis C or hepatitis B virus infection: a nationwide study in Sweden.

HIV Infection and Cancer Risk

HIV and cancer of the cervix

Anal cancer: an HIV-associated cancer

The therapeutic potential of CXCR4 antagonists in the treatment of HIV infection, cancer metastasis and rheumatoid arthritis

Types of Cancer: AIDS/HIV related malignancies

Cytokines in cancer pathogenesis and cancer therapy

Dendritic Cells as Therapeutic Vaccines against Cancer

9.1.2   Simultaneous Humoral and Cellular Immune Response against Cancer–Testis Antigen NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2–binding Peptide Epitopes

9.1.3 Monoclonal antibodies

Monoclonal antibodies in cancer therapy

Monoclonal Antibodies in Cancer Therapy: 25 Years of Progress

9.1.4 Aptamers

Nanocarriers as an emerging platform for cancer therapy

Quantum Dot−Aptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing of Drug Delivery Based on Bi-Fluorescence Resonance Energy Transfer

Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

9.1 Vaccines

9.1.1  Viruses in carcinogenesis

HPV-associated cervical cancer

http://www.cancer.net/navigating-cancer-care/prevention-and-healthy-living/hpv-and-cancer

Human papillomavirus (HPV) is a virus that is usually passed on during direct skin-to-skin contact, most commonly sex. In fact, HPV is the most common sexually transmitted disease in the United States. Most men and women are not aware they have an HPV infection because they do not develop any symptoms or health problems. Certain HPV types can cause precancerous lesions (areas of abnormal tissue) or cancer.

More than 40 of the viruses are called “genital type” HPVs. These viruses are spread from person to person when their genitals come into contact, usually during vaginal or anal sex. They can also be passed on through oral sex.

Genital HPV types can infect the genital area of women, including the vulva (outer portion of the vagina), the vagina, and the cervix (the lower, narrow part of a woman’s uterus), as well as the genital area of men, including the penis. In both men and women, genital HPV can infect the anus and some areas of the head and neck.

Nearly all cervical cancers are caused by HPV infection. Strong scientific evidence shows that a lasting HPV infection is required for cervical cancer to begin developing. Whether a woman who is infected with HPV will develop cervical cancer depends on a number of factors, including the type of HPV infection she has. Of the cervical cancers related to HPV, about 70% are caused by two strains, HPV-16 or HPV-18. In women who have HPV, smoking may increase the risk of cervical cancer.

Warts and precancerous lesions can be removed through cryotherapy (freezing); loop electrosurgical excision procedure (LEEP), which uses electric current to remove abnormal tissue; or surgery.

Receiving an HPV vaccine reduces your risk of infection. The U.S. Food and Drug Administration (FDA) approved two vaccines that help prevent HPV infection: Gardasil and Cervarix. It is important to note that the vaccines cannot cure an existing HPV infection.

Purpose of the vaccines. The goal of HPV vaccination is to prevent a lasting HPV infection after a person is exposed to the virus. Gardasil, introduced in 2006, helps prevent infection from the two HPVs known to cause most cervical cancers and precancerous lesions in the cervix. The vaccine also prevents against the two low-risk HPVs known to cause 90% of genital warts. Gardasil is approved for the prevention of cervical, vaginal, and vulvar cancers in girls and women ages nine to 26. It is also approved to prevent anal cancer in women and men and genital warts in men and boys in the same age range. Cervarix, introduced in 2009, is approved for the prevention of cervical cancer in girls and women ages 10 to 25.

Effectiveness and safety of the vaccines. Data show the HPV vaccinations are safe and highly effective in preventing a lasting infection of the HPV types they target. Because it takes many years before a precancerous lesion develops into an invasive cancer, it will likely take several more years before there is evidence that the number of cancer cases in vaccinated individuals has been reduced.

HPV-associated head and neck cancer: a virus-related cancer epidemic
Shanthi Marur, Gypsyamber D’Souza, William H Westra, Arlene A Forastiere
Lancet Oncol 2010; 11: 781–89
http://dx.doi.org:/10.1016/S1470-2045(10)70017-6

A rise in incidence of oropharyngeal squamous cell cancer—specifically of the lingual and palatine tonsils—in white men younger than age 50 years who have no history of alcohol or tobacco use has been recorded over the past decade. This malignant disease is associated with human papillomavirus (HPV) 16 infection. The biology of HPV-positive oropharyngeal cancer is distinct with P53 degradation, retinoblastoma RB pathway inactivation, and P16 upregulation. By contrast, tobacco-related oropharyngeal cancer is characterized by TP53 mutation and downregulation of CDKN2A (encoding P16). The best method to detect virus in tumor is controversial, and both in-situ hybridization and PCR are commonly used; P16 immunohistochemistry could serve as a potential surrogate marker. HPV-positive oropharyngeal cancer seems to be more responsive to chemotherapy and radiation than HPV-negative disease. HPV 16 is a prognostic marker for enhanced overall and disease-free survival, but its use as a predictive marker has not yet been proven. Many questions about the natural history of oral HPV infection remain under investigation. For example, why does the increase in HPV-related oropharyngeal cancer dominate in men? What is the potential of HPV vaccines for primary prevention? Could an accurate method to detect HPV in tumor be developed? Which treatment strategies reduce toxic effects without compromising survival? Our aim with this review is to highlight current understanding of the epidemiology, biology, detection, and management of HPV-related oropharyngeal head and neck squamous cell carcinoma, and to describe unresolved issues.

Cancers of the head and neck arise from mucosa lining the oral cavity, oropharynx, hypopharynx, larynx, sinonasal tract, and nasophaynx. By far the most common histological type is squamous cell carcinoma, and grade can vary from well-differentiated keratinizing to undifferentiated non-keratinizing. An increase in incidence of oropharyngeal squamous cell carcinoma—specifically in the tonsil and tongue base—has been seen in the USA, most notably in individuals aged 40–55 years. Patients with oropharyngeal cancer are mainly white men. Unlike most tobacco-related head and neck tumors, patients with oropharyngeal carcinoma usually do not have a history of tobacco or alcohol use. Instead, their tumors are positive for oncogenic forms of the human papillomavirus (HPV), particularly 16 type. About 60% of oropharyngeal squamous cell cancers in the USA are positive for HPV 16. HPV-associated head and neck squamous cell carcinoma seems to be a distinct clinical entity, and this malignant disease has a better prognosis than HPV-negative tumors, due in part to increased sensitivity of cancers to chemotherapy and radiation therapy. Although HPV is now recognized as a causative agent for a subset of oropharyngeal squamous cell carcinomas, the biology and natural history of oropharyngeal HPV infection and the best clinical management of patients with HPV-related head and neck squamous cell tumors is not well understood.

Head and neck cancer is the sixth most common cancer worldwide, with an estimated annual burden of 563 826 incident cases (including 274 850 oral cavity cancers, 159 363 laryngeal cancers, and 52 100 oropharyngeal cancers) and 301 408 deaths.1 Although HPV has been long known to be an important cause of anogenital cancer, only in recent times has it been recognized as a cause of a subset of head and neck squamous cell carcinomas.2 More than 100 different types of HPV exist,3 and at least 15 types are thought to have oncogenic potential.4 However, most (>90%) HPV-associated head and neck squamous cell cancers are caused by one virus type, HPV 16, the same type that leads to HPV-associated anogenital cancers. The proportion of head and neck squamous cell carcinomas caused by HPV varies widely (figure 1),5–16 largely because of the burden of tobacco-associated disease in this population of tumors. Tobacco, alcohol, poor oral hygiene, and genetics remain important risk factors for head and neck tumors overall, but HPV is now recognized as one of the primary causes of oropharyngeal squamous cell cancers. In the USA, about 40–80% of oropharyngeal cancers are caused by HPV, whereas in Europe the proportion varies from around 90% in Sweden to less than 20% in communities with the highest rates of tobacco use (figure 1).

The incidence of head and neck cancers overall in the USA has fallen in recent years, consistent with the decrease in tobacco use in this region. By contrast, incidence of HPV-associated oropharyngeal cancer seems to be rising, highlighting the increasing importance of this causal association.17–19 In a US study in which data of the Surveillance, Epidemiology, and End Results (SEER) program were used, incidence of oropharyngeal tumors (which are most likely to be HPV-associated) rose by 1·3% for base of tongue cancers and by 0·6% for tonsillar cancers every year between 1973 and 2004. By contrast, incidence of oral cavity cancers (not associated with HPV) declined by 1·9% every year during the same period.17 The age-adjusted incidence of tonsillar cancer increased 3·5-fold in women and 2·6-fold in men between 1970 and 2002.24 Augmented incidence of HPV-associated oropharyngeal cancers represents an emerging viral epidemic of cancer.

Why is increased incidence of HPV-associated oropharyngeal cancer most pronounced in young individuals? This effect could be attributable to changes in sexual norms (i.e., more oral sex partners or oral sex at an earlier age in recent than past generations) combined with fewer tobacco-associated cancers in young cohorts, making the outcomes of HPV-positive cancers more visible. Can the higher rates of HPV-associated oropharyngeal cancers in men compared with women be accounted for solely by differences in sexual behavior, or are biological differences in viral clearance present that could contribute to the higher burden of these cancers in men? HPV prevalence in cervical rather than penile tissue might boost the chances of HPV infection when performing oral sex on a woman, contributing to the higher rate of HPV-associated oropharyngeal cancer in men.

Tobacco use has fallen in past decades, and the corresponding rise in proportion of head and neck cancers that are oropharyngeal in origin has been striking, both in the USA and internationally. SEER data suggest that about 18% of all head and neck carcinomas in the USA were located in the oropharynx in 1973, compared with 31% of such squamous cell tumors in 2004.19 Similarly, in Sweden, the proportion of oropharyngeal cancers caused by HPV has steadily increased, from 23% in the 1970s to 57% in the 1990s, and as high as 93% in 2007.13,25 These data indicate that HPV is now the primary cause of tonsillar malignant disease in North America and Europe.

Findings of initial studies suggest that oral HPV frequency increases with age. Prevalent oral HPV infection is detected in 3–5% of adolescents26–28 and 5–10% of adults.14,29 We do not yet know whether the natural history of oral HPV or risk factors for persistent HPV infection in the oropharynx differ from those known for anogenital HPV infection (table 1). Data suggest oral HPV prevalence is amplified with number of sexual partners and is more typical in men, in HIV-infected individuals, and in current tobacco users.26–28,30,31

In view of the importance of tobacco use in head and neck squamous cell carcinoma, most cases of this malignant disease seen in non-smokers are unsurprisingly HPV-related. However, oral HPV infection is common in smokers and non-smokers and is an important cause of oropharyngeal cancer in both groups. For example, in case series, only 13–16% of individuals with HPV-positive head and neck squamous cell cancer did not smoke or drink alcohol.32,33 Although a higher proportion of individuals with HPV-positive compared with HPV-negative tumors are non-smokers or neither smoke nor drink alcohol, many with HPV-positive disease have a history of alcohol and tobacco use. In fact, 10–30% of HPV-positive head and neck squamous cell carcinomas were recorded in heavy tobacco and alcohol users.32,33 This finding underscores that HPV-associated malignant disease not only arises in people who do not smoke or drink alcohol but also occurs in people with the traditional risk factors of tobacco and alcohol use.

HPV detection may ultimately serve a more comprehensive role than mere prognostication. Detection of HPV is emerging as a valid biomarker for discerning the presence and progress of disease encompassing all aspects of patients’ care, from early cancer detection,41 to more accurate tumor staging (e.g., localization of tumor origin),42,43 to selection of patients most likely to benefit from specific treatments,44 to post-treatment tumor surveillance.45,46 Consequently, there is a pressing need for a method of HPV detection that is highly accurate, reproducible from one diagnostic laboratory to the next, and practical for universal application in the clinical arena. Despite growing calls for routine HPV testing of all oropharyngeal carcinomas, the best method for HPV detection is not established. Various techniques are currently in use, ranging from consensus and type-specific PCR methods, real-time PCR assays to quantify viral load, type-specific DNA in-situ hybridization, detection of serum antibodies directed against HPV epitopes, and immunohistochemical detection of surrogate biomarkers (e.g., P16 protein). Although PCR-based detection of HPV E6 oncogene expression in frozen tissue samples is generally regarded as the gold standard for establishing the presence of HPV, selection of assays for clinical use will ultimately be influenced by concerns relating to sensitivity, specificity, reproducibility, cost, and feasibility. Development of non-fluorescent chromogens has enabled visualization of DNA hybridization by conventional light microscope; furthermore, adaptation of in-situ hybridization to formalin-fixed and paraffin-embedded tissues has made this technique compatible with standard tissue-processing procedures and amenable to retrospective analysis of archival tissue blocks. Most PCR-based methods, on the other hand, need a high level of technical skill and are best used with fresh-frozen samples.

Limitations of any one detection assay can be offset by algorithms that combine the strengths of complementary assays.50 A highly feasible strategy incorporates P16 immunohistochemistry and HPV in-situ hybridization. In view of sensitivity that approaches 100%, P16 immunostaining is a good first-line assay for elimination of HPV-negative cases from any additional analysis. Since specificity is almost 100%, a finding positive for HPV 16 on in-situ hybridization reduces the number of false-positive cases by P16 staining alone. A P16-positive, HPV 16-negative result singles out a subset of tumors that qualifies for rigorous analysis for other (i.e., non-HPV 16) oncogenic virus types.

HPV in-situ hybridisation and P16 immunostaining as a practical diagnostic approach to discernment of HPV status can be applied readily to cytological preparations, including fi ne-needle aspirates from patients with cervical lymph-node metastases.41,52 Further expansion of HPV testing to blood and other body fl uids would advance the role of HPV as a clinically relevant biomarker, but these specimens would need other detection platforms. PCR-based detection of HPV DNA in blood (53) and saliva (54) of patients after treatment of their HPV-positive cancers suggests a future role in tumour surveillance. Detection of serum antibodies to HPV-related epitopes can predict the HPV status of head and neck cancers, and this method has been advocated as a way to project clinical outcomes and guide treatment without the constraints of tissue acquisition.53,55

The increasing prevalence of oropharyngeal cancer in young populations and substantially amplified survival rates with current treatment approaches stands in contrast to survival achieved in older individuals with comorbid disorders associated with tobacco and alcohol history. Several characteristics of patients with head and neck cancer have been linked with favorable prognosis, including non-smoker, minimum exposure to alcohol, good performance status, and no comorbid disorders, all of which are related to HPV-positive tumor status. Findings of retrospective analyses suggest that individuals with HPV-positive oropharyngeal cancer have higher response rates to chemotherapy and radiation and increased survival62–65 compared with those with HPV-negative tumors. Augmented sensitivity to chemotherapy and radiotherapy has been attributed to absence of exposure to tobacco and presence of functional unmutated TP53.63,64,66 Increased survival of patients with HPV-positive cancer is also possibly attributable in part to absence of field cancerization related to tobacco and alcohol exposure.67

Survival outcomes for individuals with HPV 16-positive and P16-positive oropharyngeal tumors were similar. Failure data indicated significantly diminished rates of locoregional failure and second primary tumour in patients with HPV-positive oropharyngeal cancer compared with those with HPV-negative tumors; distant metastases did not differ between the two groups. When survival was assessed after adjustment for tobacco exposure, in individuals who smoked, those with HPV-positive oropharyngeal tumors and fewer than 20 pack-years had 2-year overall survival of 95%, compared with 80% in those with HPV-positive cancers and 20 pack-years or more, and 63% in HPV-negative cancers and 20 pack-years or more. By comparison with people with HPV-positive oropharyngeal tumors who smoked and had fewer than 20 pack-years, the hazard of death was raised for those with HPV-negative tumors and 20 pack-years or more (hazard ratio 4·33) and those with HPV-positive cancers and 20 pack-years or more (1·79). These data indicate clearly that tobacco exposure alters the biology of HPV-positive oropharyngeal tumors and is an important prognostic factor.

An association between HPV-positive, P16-positive oropharyngeal tumors and survival outcomes was reported in another retrospective analysis of a large phase 3 trial of chemoradiation, which included more than 800 patients enrolled from international sites.72 This substudy analysis looked at 195 available tumor samples in patients with an oropharyngeal primary cancer, of which 28% were HPV-positive and 58% were P16-positive. Individuals with HPV-positive cancers had 2-year overall survival of 94% and 2-year failure-free survival of 86% compared with 77% (p=0·007) and 75% (p=0·035), respectively, in those with HPV-negative tumors. When co-expression of HPV and P16 was correlated with survival outcomes, individuals with HPV-positive, P16-positive tumors had 2-year overall survival of 95% compared with 88% in those with HPV-negative, P16-positive cancers and 71% (p=0·003) in those with HPV-negative, P16-negative tumors. Similar results were noted for 2-year failure-free survival (89%, 86%, and 69%, respectively; p=0·002) and time to locoregional failure (93%, 95%, and 84%, respectively; p=0·051). By multivariable analysis, HPV 16 and P16 were identified as independent prognostic factors.

ECOG proposes induction chemotherapy with a triple drug regimen to reduce tumor burden to subclinical disease (clinical complete response at primary site) followed by lower dose radiation (total dose 54 Gy) and concurrent cetuximab. Overall survival and progression-free survival outcomes will be assessed and compared with results of the 2008 ECOG study.70 The main aim of this planned study is to assess potential for a lower dose of radiation to control disease and to investigate toxic effects and quality-of-life variables.

In summary, tumor HPV status is a prognostic factor for overall survival and progression-free survival and might also be a predictive marker of response to treatment. The method of in-situ hybridization provides a feasible approach for implementation in most diagnostic pathology laboratories, and immunohistochemical staining for P16 could be useful as a surrogate marker for HPV status. Seemingly, locoregional recurrence—but not the rate of distant disease—is diminished in patients with HPV-positive tumors. Smoking and tobacco exposure might modify survival and recurrence of HPV-positive tumors and should be considered in future trials for risk stratification of patients with HPV-positive malignant disease.

HCV and cancer

The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide
Joseph F. Perz, Armstrong GL, Farrington LA,  Hutin YJF, Bell BP
J Hepatol 2006; 45:529-538
http://dx.doi.org:/10.1016/j.jhep.2006.05.013

End-stage liver disease accounts for one in forty deaths worldwide. Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) are well-recognized risk factors for cirrhosis and liver cancer, but estimates of their contributions to worldwide disease burden have been lacking. Methods: The prevalence of serologic markers of HBV and HCV infections among patients diagnosed with cirrhosis or hepatocellular carcinoma (HCC) was obtained from representative samples of published reports. Attributable fractions of cirrhosis and HCC due to these infections were estimated for 11 WHO-based regions. Results: Globally, 57% of cirrhosis was attributable to either HBV (30%) or HCV (27%) and 78% of HCC was attributable to HBV (53%) or HCV (25%). Regionally, these infections usually accounted for >50% of HCC and cirrhosis. Applied to 2002 worldwide mortality estimates, these fractions represent 929,000 deaths due to chronic HBV and HCV infections, including 446,000 cirrhosis deaths (HBV: n = 235,000; HCV: n = 211,000) and 483,000 liver cancer deaths (HBV: n = 328,000; HCV: n = 155,000). Conclusions: HBV and HCV infections account for the majority of cirrhosis and primary liver cancer throughout most of the world, highlighting the need for programs to prevent new infections and provide medical management and treatment for those already infected.

Among primary liver cancers occurring worldwide, hepatocellular carcinoma (HCC) represents the major histologic type and likely accounts for 70% to 85% of cases [2]. Cirrhosis precedes most cases of HCC, and may exert a promotional effect via hepatocyte regeneration [3,4]. Compared with other causes of cirrhosis, chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) is associated with a higher risk of developing HCC [3,5]. Alcohol abuse represents a leading cause of cirrhosis and is also a major contributor. dietary aflatoxin exposure in parts of Africa and Asia has been associated with primary liver cancer, especially in hosts with chronic HBV infection [8].

An understanding of the relative contribution of various etiologies to disease burden is important for setting public health priorities and guiding prevention programs [10,11]. The World Health Organization’s Global Burden of Disease (GBD) 2000 project aims to quantify the burden of premature morbidity and mortality from over 130 major causes [1,12]. Liver cancer and cirrhosis are included in the analysis, but with the exception of alcohol, the etiologies underlying these diseases have not been well accounted for [1,11,13]. In particular, HBV and HCV infections have been poorly characterized in previous WHO estimates since these were based primarily on the acute effects of infection and omitted the associated burdens of chronic liver disease [10,11].

The attributable fraction represents the proportion of disease occurrence that potentially would be prevented by eliminating a given risk factor. For cirrhosis, a systematic analysis of attributable fractions has been lacking altogether. For HCC, previous estimates of the attributable fractions due to HBV and HCV are available but are not comprehensive and do not correspond to the regional designations and related conventions of the current GBD project [14].

The prevalence of HBV and HCV infection among cirrhosis and HCC patients varied considerably within and between regions (Tables 2 and 3). These variations tended to reflect known patterns of HBV and HCV infection endemicity [99,100]. For example, in countries where HCV infection has long been endemic, such as Japan and Egypt, there were high prevalences of HCV infection among cirrhosis and HCC patients. The same held true for China and most of the African nations in our sample regarding HBV infection. Areas such as these, where HBV infection predominated, appeared to have a younger population of HCC cases, which is thought to reflect the preponderance of infections acquired early in life (e.g., perinatal HBV transmission) [8]. Patterns of HBV and HCV co-infection were also notable.

When we applied the HBV- and HCV-attributable fractions we derived to 2002 worldwide mortality estimates [1], we found that approximately 929,000 deaths from cirrhosis (n = 446,000) and primary liver cancer (n = 483,000) were likely due to chronic viral hepatitis infections. HBV infection accounted for 563,000 deaths (235,000 from cirrhosis and 328,000 from liver cancer) and HCV infection accounted for 366,000 deaths (211,000 from cirrhosis and 155,000 from liver cancer).

We showed that chronic viral hepatitis infections likely account for the majority of both cirrhosis and HCC globally and in nearly all regions of the world. One of the strengths of our analysis was that it employed simple and transparent methods. Our estimates of attributable fractions were derived from reviews of published studies reporting the prevalence of HBV and HCV infections in patients with cirrhosis or HCC in all regions of the world. Alternate approaches rely on estimates of the prevalence of risk factors and corresponding relative risks in the source populations. However, errors associated with extrapolating exposure or hazard from one population to another are a major source of uncertainty in efforts to characterize international health risks [12]. Given the lack of representative data regarding HBV and HCV infection prevalences worldwide along with uncertainties in deriving region specific risk estimates, we believe ours is the preferred approach.

Our findings help illustrate the great need for programs aimed at preventing HBV or HCV transmission. In 1992, WHO recommended that all countries include hepatitis B vaccine in their routine infant immunization programs. As of 2003, WHO/UNICEF estimated 42% hepatitis B vaccination coverage among the global birth cohort [106]. Therefore, implementation of this strategy, which represents the most effective way of preventing chronic HBV infection and related end stage liver disease, is far from complete [107,108]. Other key primary prevention strategies include screening blood donors and maintaining infection control practices to prevent the transmission of healthcare-related HBV and HCV infections [105,109,110]. In countries where these activities have not been fully implemented, they should be given a high priority. In most developed countries, injection drug use and high-risk sexual behaviors represent the major risk factors for HCV infection and HBV infection, respectively, indicating the importance of related prevention efforts (e.g., reducing the numbers of new initiates to injection drug use).

The role of programs to identify, counsel, and provide medical management for the many persons already infected with HBV or HCV requires careful consideration [105,110]. Counseling that includes advice regarding avoidance of alcohol and education regarding modes of transmission can help reduce the risks for developing chronic disease or spreading infection to susceptible persons. The widespread application of therapeutic interventions also has the potential to accelerate the declines in end-stage liver disease that will eventually follow from hepatitis B vaccination and other primary prevention efforts [104,107]. Recent advances have occurred in the therapeutic management of chronic hepatitis B and chronic hepatitis C, but treatments are long and involve substantial costs and side effects [111–113]. Countries will need to consider the potential benefits of treatment while insuring that scarce healthcare resources are allocated in a manner that does not undermine primary prevention efforts [114].

Risk of pancreatic cancer among individuals with hepatitis C or hepatitis B virus infection: a nationwide study in Sweden.

Huang J1Magnusson MTörner AYe WDuberg AS.
Br J Cancer. 2013 Nov 26; 109(11):2917-23.
http://dx.doi.org:/10.1038/bjc.2013.689

A few studies indicated that hepatitis C and hepatitis B virus (HCV/HBV) might be associated with pancreatic cancer risk. The aim of this nationwide cohort study was to examine this possible association. Methods: Hepatitis C virus-
and hepatitis B virus-infected individuals were identified from the national surveillance database from 1990 to 2006, and followed to the end of 2008. The pancreatic cancer risk in the study population was compared with the general population by calculation of Standardized Incidence Ratios (SIRs), and with a matched reference population using a Cox proportional hazards regression model to calculate hazard ratios (HRs). Results: In total 340 819 person-years in the HCV cohort and 102 295 in the HBV cohort were accumulated, with 34 and 5 pancreatic cancers identified, respectively. The SIRHCV was 2.1 (95% confidence interval, CI: 1.4, 2.9) and the SIRHBV was 1.4 (0.5, 3.3). In the Cox model analysis, the HR for HCV infection was 1.9 (95% CI: 1.3, 2.7), diminishing to 1.6 (1.04, 2.4) after adjustment for potential confounders.
Conclusion: Our results indicated that HCV infection might be associated with an increased risk of pancreatic cancer but further studies are needed to verify such association. The results in the HBV cohort indicated an excess risk, however, without statistical significance due to lack of power.

Pancreatic cancer is one of the most rapidly fatal malignancies with a 5-year survival rate below 5%. The long-term survival is poor also for early diagnosed patients treated with resection surgery (Jemal et al, 2010). In Europe, it was estimated in a prediction model that in the year 2012 there would be 75 000–80 000 deaths from pancreatic cancer, which is the fourth most common cause of cancer-associated death for both men and women (Malvezzi et al, 2012). The incidence of pancreatic cancer is higher in the Nordic countries and Central Europe than in other parts of the world (Bosetti et al, 2012).

Tobacco smoking is a well-established risk factor for pancreatic cancer (Iodice et al, 2008), and a similar magnitude of excess risk as smoking was found among the users of Scandinavian snus (moist snuff) (Boffettaet al, 2005Luo et al, 2007). Besides, accumulating evidence consistently shows that old age, male sex, diabetes mellitus, hereditary pancreatitis, chronic pancreatitis and family history are positively associated with this carcinoma (Pandol et al, 2012). Albeit the biological mechanism is unclear, recent epidemiological studies indicated that some infections, such as exposure to Helicobacter pylori (Trikudanathan et al, 2011), poor oral health (Michaud et al, 2007), hepatitis C virus (HCV) (Hassan et al, 2008El-Serag et al, 2009) or hepatitis B virus (HBV) (Hassan et al, 2008Iloeje et al, 2010Wang et al, 2012a2012b) might be associated with pancreatic cancer risk.

Globally, ∼170 million people are chronically infected with HCV (World Health Organization, 1997) and an estimated 350 million with HBV (Custer et al, 2004). The prevalence rates of HCV and HBV infection vary widely in the world, and Sweden is a low endemic country with an estimated 0.5% of the population infected with HCV (Duberg et al, 2008a) and even lower rate for HBV infection. Both chronic HCV and HBV infections are main causes of hepatocellular carcinoma (HCC). Previous findings demonstrated that HBV may replicate within the pancreas (Shimoda et al, 1981Yoshimura et al, 1981) and that HCV could be associated with pancreatitis (Alvares-Da-Silva et al, 2000Torbenson et al, 2007). Some studies support that HCV and HBV may have a role in the development of pancreatic cancer, but the evidence is far from conclusive (Hassan et al, 2008El-Serag et al, 2009Iloeje et al, 2010Wang et al, 2012a2012b), and more studies are needed. Towards this end, we utilised Swedish population-based nationwide registers, with documentation of all diagnosed HCV- and HBV-infected individuals in Sweden, to explore the association of HCV or HBV infection and the risk of pancreatic cancer.

Baseline characteristics of the HCV and HBV cohorts are presented in Table 1. In the HCV and chronic HBV cohorts the mean follow-up time were 9.1 and 9.4 years, with a total of 360 154 and 107 986 person-years at risk, respectively. There was a clear male dominance in the HCV cohort, and median age at entry into the HCV or HBV cohorts (notification date) was 38 and 31 years, respectively. A marked difference between cohorts was observed regarding the aspect of country of origin; HCV-infected individuals were more likely from Nordic countries, but persons with chronic HBV infection were often immigrants from non-Nordic countries.

Hepatitis C virus cohort

In the HCV cohort, there were 34 pancreatic cancer cases observed during 340 819 person-years of follow-up (first 6 months of follow-up excluded), whereas 16.5 were expected, yielding a statistically significant increased risk of pancreatic cancer (SIR: 2.1; 95% CI: 1.4, 2.9). The SIR did not alter substantially across sex or estimated duration of HCV infection (Table 2). The majority of cases were among the patients who were born before 1960.

From the Cox regression model, an ∼90% excessive risk for pancreatic cancer (HR 1.9; 95% CI: 1.3, 2.7) was observed after adjustment for age, sex and county of residence, which is similar to the result from the SIR analysis. This excess risk diminished somewhat but remained statistically significant after further adjustment for potential confounders (HR 1.6; 95% CI: 1.04, 2.4). The results did not vary markedly when stratified by sex (Table 3). In the additional analyses, excluding all individuals ever hospitalized with acute and/or chronic pancreatitis, the results did not alter notably (data not shown).

In the HCV cohort, the Standardized Incidence Ratio (SIR) for lung cancer was 2.3 (95% CI: 1.9, 2.7) and the Hazard Ratio (HR) for lung cancer was 2.2 (95% CI: 1.8, 2.7), decreasing to 1.6 (95% CI: 1.3, 2.1) after adjustment for the potential confounders used in the pancreatic cancer analyses.

Chronic HBV cohort

A total of five pancreatic cancer cases were found during 102 295 person-years of follow-up (first 6 months excluded), whereas 3.5 were expected. Compared with the age- and sex-matched Swedish general population, a 40% excess risk of pancreatic cancer was found in the chronic HBV cohort (SIR: 1.4; 95% CI: 0.5, 3.3), but without statistical significance. Because of the small number of pancreatic cancer cases, there was not enough power for additional stratified analyses (Table 4).

The Cox regression model revealed similar results as the SIR analysis. The point estimates were somewhat higher (HR=2.0 from the model adjusted for only matching factors and HR=1.8 from the fully adjusted model), but still statistically non-significant (Table 5). The SIR for lung cancer in the chronic HBV infection cohort was 1.7 (95% CI: 1.1, 2.5).

This population-based large cohort study revealed a doubled risk of pancreatic cancer among HCV-infected patients compared with the Swedish general population. The excess risk was persistent across strata by sex or duration of infection. Although further adjustment for potential confounders, i.e., chronic obstructive pulmonary disease (related to smoking), diabetes mellitus, chronic pancreatitis and alcohol-related disease, resulted in an attenuated relative risk, this finding still supports the hypothesis that HCV infection might be associated with an increased risk of pancreatic cancer. Besides, the result indicated a moderate excessive risk of pancreatic cancer among HBV-infected patients according to different statistical approaches, but the size of the study cohort and the observed number of cancers were too small to draw a sound conclusion. Pancreatic cancer is more common in older age groups, and the small number of pancreatic cancers among the HBV cohort was probably an effect of the relatively young cohort, concordant with the epidemiology of chronic hepatitis B in Sweden.

The strengths of this register-based study include population-based cohort design, relatively large sample size, independently collected data on documentation of HCV/HBV notifications and pancreatic cancer occurrence and high completeness of follow-up.

The parallel (laboratory and clinician) notification system of HCV/HBV infections in Sweden has a high coverage of those with a diagnosed infection; it is estimated that about 75–80% of HCV infections are diagnosed, but there still remain unknown infections, not yet diagnosed or documented. In addition, a small portion of the reported patients could have a resolved infection, spontaneously or by treatment, this could (probably insignificant) lower the risk in the HCV and HBV cohort.

The number of unidentified HCV/HBV-coinfected individuals is probably low in the studied cohorts. However, in the HCV cohort there could be some patients who were never diagnosed with hepatitis B but have serologic markers of a past HBV infection. In these patients we cannot exclude the possibility of occult hepatitis B.

The biological mechanism of the association between HCV and pancreatic cancer is unclear. However, virtually, the pancreas and liver share the common blood vessels and ducts, and prior evidence demonstrated that the pancreas is a remote location for hepatitis virus inhabitation and replication (Hassan et al, 2008). HCV infection is associated with type 2 diabetes, which is both a risk factor and might be a consequence of pancreatic cancer (Mehta et al, 2000Sangiorgio et al, 2000). Besides, previous studies reported that subclinical/acute pancreatitis (Katakura et al, 2005) and hyperlipasemia (Yoffe et al, 2003) may be extrahepatic manifestations of HCV infection. In addition, pancreatic involvement was observed among patients who suffered from chronic hepatitis infection, resulting in mild pancreatic damage accompanied with increased serum levels of pancreatic enzyme (Taranto et al, 1989Katakura et al, 2005). Immune response may lead to chronic inflammation in the targeted organs after long time persistent infection with HCV. Therefore, hepatitis C virus conceivably serves as a biological agent that may indirectly have a role in inflammation-associated pancreatic carcinogenesis. Although still unclear to what extent chronic inflammation contributes to pancreatic cancer development, it is postulated that HCV can induce inflammatory microenvironment with high concentration of growth factors and cytokines. This may exert effects by accumulating alterations in driver genes and promoting cancer cell growth and proliferation.

HIV AIDS and Cancer

http://www.cancer.gov/cancertopics/causes-prevention/risk/infectious-agents/hiv-fact-sheet

Key Points

  • People infected with human immunodeficiency virus (HIV) have a higher risk of some types of cancer than uninfected people.
  • A weakened immune system caused by infection with HIV, infection with other viruses, and traditional risk factors such as smoking all contribute to this higher cancer risk.
  • Highly active antiretroviral therapy and lifestyle changes may reduce the risk of some types of cancer in people infected with HIV.
  • The National Cancer Institute (NCI) conducts and supports a number of research programs aimed at understanding, preventing, and treating HIV infection, acquired immunodeficiency syndrome-related cancers, and cancer-associated viral diseases.
  1. Do people infected with human immunodeficiency virus (HIV) have an increased risk of cancer?

Yes. People infected with HIV have a substantially higher risk of some types of cancer compared with uninfected people of the same age (1). Three of these cancers are known as “acquired immunodeficiency syndrome (AIDS)-defining cancers” or “AIDS-defining malignancies”: Kaposi sarcomanon-Hodgkin lymphoma, and cervical cancer. A diagnosis of any one of these cancers marks the point at which HIV infection has progressed to AIDS.

People infected with HIV are several thousand times more likely than uninfected people to be diagnosed with Kaposi sarcoma, at least 70 times more likely to be diagnosed with non-Hodgkin lymphoma, and, among women, at least 5 times more likely to be diagnosed with cervical cancer (1).

In addition, people infected with HIV are at higher risk of several other types of cancer (1). These other malignancies include analliver, and lung cancer, and Hodgkin lymphoma.

People infected with HIV are at least 25 times more likely to be diagnosed with anal cancer than uninfected people, 5 times as likely to be diagnosed with liver cancer, 3 times as likely to be diagnosed with lung cancer, and at least 10 times more likely to be diagnosed with Hodgkin lymphoma (1).

People infected with HIV do not have increased risks of breastcolorectalprostate, or many other common types of cancer (1). Screening for these cancers in HIV-infected people should follow current guidelines for the general population

HIV and cancer of the cervix

Z.M. Chirenje
bestpracticeobgyn April 2005; 19(2):269–276
http://dx.doi.org/10.1016/j.bpobgyn.2004.10.002

Cancer of the cervix is the second most common cause of cancer-related death in women worldwide, and in some low resource countries accounts for the highest cancer mortality in women. The highest burden of the HIV/AIDS epidemic is currently in sub-Saharan Africa, where more than half of the people infected are women who have no access to cervical cancer screening. The association between HIV and invasive cervical cancer is complex, with several studies now clearly demonstrating an increased risk of pre-invasive cervical lesions among HIV-infected women. However, there have not been significantly higher incidence rates of invasive cervical cancer associated with the HIV epidemic. The highest numbers of HIV-infected women are in poorly-resourced countries, where the natural progression of HIV disease in the absence of highly active antiretroviral treatment sometimes results in deaths from opportunistic infections before the onset of invasive cervical cancer. This chapter will discuss the association of HIV and cervical intraepithelial neoplasia, the treatment of pre-invasive lesions, and invasive cervical cancer in HIV-infected women. The role of screening and the impact of antiretroviral treatment on the progression of pre-invasive and invasive cancer will also be discussed.

Anal cancer: an HIV-associated cancer

Klencke BJPalefsky JM
Hematology/oncology Clinics of North America [2003, 17(3):859-872]
http://dx.doi.org:/1016/S0889-8588(03)00039-X

Although not yet included in the Centers for Disease Control definition of AIDS, anal cancer clearly occurs more commonly in HIV-infected patients. An effective screening program for those groups who are at highest risk might be expected to impact rates of anal cancer just as significantly as did cervical Pap screening programs for the incidence of cervical cancer. Despite a relatively low rate of progression from AIN to invasive cancer, the scope of the problem is enormous based on the prevalence of anal HPV infection and the size of the HIV-infected, at-risk population. Thus, the potential benefits of screening, detection, and the development of more effective therapy also are enormous. Currently, therapeutic HPV vaccines for AIN represent an exciting avenue of research in HPV-related anogenital disease. Invasive anal cancer and HSIL (which is believed to be the precursor lesion) are expected to become increasingly important health problems for both HIV-infected men and women as their life expectancy lengthens. Although HAART may have improved the ability of many to tolerate CMT, it appears that toxicity of this therapy continues to be a problem for a proportion of HIV-infected subjects. The acute side effects present specific challenges to the clinician and patient, have an immediate impact on the patient’s plan of care and dose intensity of the treatment, and ultimately may impact the outcome of the planned treatment. Late toxicity may influence the long-term quality of life. Small patient numbers, variable radiation therapy doses, limited information about viral load, and a potential confounding effect of higher CD4+ levels make it difficult to draw any conclusions about the effect of HAART on anal cancer outcome. Large, prospective studies will be required before solid conclusions about the impact of various factors on anal cancer prognosis and outcome can be drawn.

The therapeutic potential of CXCR4 antagonists in the treatment of HIV infection, cancer metastasis and rheumatoid arthritis

Hirokazu Tamamura, and Nobutaka Fujii
Exp Opin on Ther Targets Dec 2005; 9(6): 1267-1282 http://dx.doi.org:/10.1517/14728222.9.6.1267

CXCR4 is the receptor of the chemokine CXCL12, which is involved in progression and metastasis of several types of cancer cells, HIV infection and rheumatoid arthritis. The authors developed selective CXCR4 antagonists, T22 and T140, initially as anti-HIV agents, which inhibit T cell line-tropic (X4-) HIV-1 infection through their specific binding to CXCR4. Recently, T140 analogues have also been shown to inhibit CXCL12-induced migration of breast cancer cells, leukaemia T cells, pancreatic cancer cells, small cell lung cancer cells, chronic lymphocytic leukaemia B cells, pre-B acute lymphoblastic leukaemia cells and so on in vitro. Biostable T140 analogues significantly suppressed pulmonary metastasis of breast cancer cells and melanoma cells in mice. Furthermore, these compounds significantly suppressed the delayed-type hypersensitivity response induced by sheep red blood cells and collagen-induced arthritis, which represent in vivo mouse models of arthritis. Thus, T140 analogues proved to be attractive lead compounds for chemotherapy of these problematic diseases. This article reviews recent research on T140 analogues, referring to several other CXCR4 antagonists.

Types of Cancer: AIDS/HIV related malignancies

http://cancer.northwestern.edu/cancertypes/cancer_type.cfm?category=1

People with HIV/AIDS are at high risk for developing certain cancers, such as Kaposi’s sarcoma, non-Hodgkin lymphoma, and cervical cancer. For people with HIV, these three cancers are often called “AIDS-defining conditions,” meaning that if a person with HIV has one of these cancers it can signify the development of AIDS. The connection between HIV/AIDS and certain cancers is not completely understood, but the link likely depends on a weakened immune system. Most types of cancer begin when normal cells begin to change and grow uncontrollably, forming a mass called a tumor. A tumor can be benign (noncancerous) or malignant (cancerous, meaning it can spread to other parts of the body). The types of cancer most common for people with HIV/AIDS are described in more detail below.

Kaposi’s sarcoma

Kaposi’s sarcoma is a type of skin cancer, which has traditionally occurred in older men of Jewish or Mediterranean descent, young men in Africa, or people who have received organ transplantation. Today, Kaposi’s sarcoma is found most often in homosexual men with HIV/AIDS and related to an infection with the human herpesvirus 8 (HHV-8). Kaposi’s sarcoma in people with HIV is often called epidemic Kaposi’s sarcoma. HIV/AIDS-related Kaposi’s sarcoma causes lesions to arise in multiple sites in the body, including the skin, lymph nodes, and organs such as the liver, spleen, lungs, and digestive tract.

Non-Hodgkin lymphoma

HIV/AIDS-related NHL is the second most common cancer associated with HIV/AIDS, after Kaposi’s sarcoma. There are many different subtypes of NHL. The most common subtypes of NHL in people with HIV/AIDS are primary central nervous system lymphoma (affecting the brain and spinal fluid), found in 20% of all NHL cases in people with HIV/AIDS, primary effusion lymphoma (causing fluid to accumulate around the lungs or in the abdomen), or intermediate and high-grade lymphoma. More than 80% of lymphomas in people with HIV/AIDS are high-grade B-cell lymphoma, while 10% to 15% of lymphomas among people with cancer who do not have HIV/AIDS are of this type. It is estimated that between 4% and 10% of people with HIV/AIDS develop NHL.

Other types of cancer

Other, less common types of cancer that may develop in people with HIV/AIDS are Hodgkin’s lymphoma, angiosarcoma (a type of cancer that begins in the lining of the blood vessels), anal cancer, liver cancer, mouth cancer, throat cancer, lung cancer, testicular cancer, colorectal cancer, and multiple types of skin cancer including basal cell carcinoma, squamous cell carcinoma, and melanoma.

Treatment options for the most common treatments for HIV/AIDS-related cancers are listed by the specific type of cancer. Treatment options and recommendations depend on several factors, including the type and stage of cancer, possible side effects, and the patient’s preferences and overall health.

Palliative care can help a person at any stage of illness. People often receive treatment for the cancer and treatment to ease side effects at the same time. In fact, patients who receive both often have less severe symptoms, better quality of life, and report they are more satisfied with treatment.

Palliative treatments vary widely and often include medication, nutritional changes, relaxation techniques, and other therapies. You may also receive palliative treatments similar to those meant to eliminate the cancer, such as chemotherapy, surgery, and radiation therapy.

It is extremely important that all patients with HIV/AIDS and an associated cancer receive treatment with highly active antiretroviral treatment (HAART) both during the cancer treatments and afterwards. HAART can effectively control the virus in most patients. Better control of the HIV infection decreases the side effects of many of the treatments, may decrease the chance of a recurrence, and can improve a patient’s chance of recovery from the cancer.

The treatment of HIV/AIDS-related Kaposi sarcoma usually cannot cure the cancer, but it can help relieve pain or other symptoms. This can be followed by palliative care for Kaposi sarcoma. Antiviral treatment for HIV/AIDS helps reduce a person’s chance of getting Kaposi sarcoma and can reduce the severity of Kaposi sarcoma. HAART helps treat the tumor and reduce the symptoms associated with Kaposi sarcoma for people with HIV/AIDS. It is usually used before other treatments, such as chemotherapy.

Curettage and electrodesiccation. In this procedure, the cancer is removed with a curette, a sharp, spoon-shaped instrument. The area can then be treated with electrodesiccation, which uses an electric current to control bleeding and kill any remaining cancer cells. Many patients have a flat, pale scar from this procedure.

Cryosurgery. Cryosurgery, also called cryotherapy or cryoablation, uses liquid nitrogen to freeze and kill cells. The skin will later blister and shed off. This procedure will sometimes leave a pale scar. More than one freezing may be needed.

In photodynamic therapy, a light-sensitive substance is injected into the lesion that stays longer in cancer cells than in normal cells. A laser is directed at the lesion to destroy the cancer cells.

Radiation therapy is the use of high-energy x-rays or other particles to destroy cancer cells. A doctor who specializes in giving radiation therapy to treat cancer is called a radiation oncologist. The most common type of radiation treatment is called external-beam radiation therapy, which is radiation given from a machine outside the body. When radiation therapy is given using implants, it is called internal radiation therapy or brachytherapy. External-beam radiation therapy may be given as a palliative treatment. A radiation therapy regimen (schedule) usually consists of a specific number of treatments given over a set period of time.

Side effects from radiation therapy may include fatigue, mild skin reactions, upset stomach, and loose bowel movements. Most side effects go away soon after treatment is finished. Learn more about radiation therapy.

Chemotherapy may help control advanced disease, although curing HIV/AIDS-related Kaposi sarcoma with chemotherapy is extremely rare. Usually, for HIV/AIDS-related Kaposi sarcoma, chemotherapy is used to help relieve symptoms and to lengthen a patient’s life. Common drugs for Kaposi sarcoma include: liposomal doxorubicin (Doxil), paclitaxel (Taxol, LEP-ETU, Abraxane), and vinorelbine (Navelbine, Alocrest).

The side effects of chemotherapy depend on the individual and the dose used, but they can include fatigue, risk of infection, nausea and vomiting, hair loss, loss of appetite, and diarrhea. These side effects usually go away once treatment is finished.

HIV/AIDS-related Kaposi sarcoma may receive alpha-interferon (Roferon-A, Intron A, Alferon), which appears to work by changing the surface proteins of cancer cells and by slowing their growth. Immunotherapy is generally used for people who are in the good-risk category in the immune system (I) factor of the TIS staging system (see Stages). The most common side effects of alpha-interferon are low levels of white blood cells and flu-like symptoms.

The main treatments for HIV/AIDS-related non-Hodgkin lymphoma are chemotherapy, targeted therapy, and radiation therapy.

Treatments for women with the precancerous condition called CIN (see   Overview) are generally not as effective for women with HIV/AIDS because of a weakened immune system. Often, the standard treatment for HIV/AIDS can lower the symptoms of CIN.

Women with invasive cervical cancer and HIV/AIDS that is well-controlled with medication, generally receive the same treatments as women who do not have HIV/AIDS. Common treatment options include surgery, radiation therapy, and chemotherapy.

Cytokines in cancer pathogenesis and cancer therapy

Glenn Dranoff
Nature Reviews Cancer Jan 2004; 4(11-22) http://dx.doi.org:/10.1038/nrc1252

The mixture of cytokines that is produced in the tumor microenvironment has an important role in cancer pathogenesis. Cytokines that are released in response to infection, inflammation and immunity can function to inhibit tumor development and progression. Alternatively, cancer cells can respond to host-derived cytokines that promote growth, attenuate apoptosis and facilitate invasion and metastasis. A more detailed understanding of cytokine–tumor-cell interactions provides new opportunities for improving cancer immunotherapy.

Dendritic Cells as Therapeutic Vaccines against Cancer
Jacques Banchereau and A. Karolina Palucka
Nature Reviews Immunology APR 2005; 5:296-306
http://cnc.cj.uc.pt/BEB/private/pdfs/2007-2008/Immunology/E/Rev_paper_E4.pdf

Mouse studies have shown that the immune system can reject tumours, and the identification of tumor antigens that can be recognized by human T cells has facilitated the development of immunotherapy protocols. Vaccines against cancer aim to induce tumor-specific effector T cells that can reduce the tumor mass, as well as tumor-specific memory T cells that can control tumor relapse. Owing to their capacity to regulate T-cell immunity, dendritic cells are increasingly used as adjuvants for vaccination, and the immunogenicity of antigens delivered by dendritic cells has now been shown in patients with cancer. A better understanding of how dendritic cells regulate immune responses will allow us to better exploit these cells to induce effective anti-tumor immunity.

Vaccines against infectious agents are one success of immunology and have spared countless individuals from diseases such as polio, measles, hepatitis B and tetanus8 . However, progress in the development of vaccines against infectious agents has been largely empirical and not always successful, as many infectious diseases still evade the immune system, particularly chronic infections such as tuberculosis, malaria and HIV infection. Further progress will be made through rational design based on our increased understanding of how the immune system works and how the induction of protective immunity is regulated. The same principle applies to vaccines against cancer, particularly as cancer is a chronic disease, and when it becomes clinically visible, tumor cells and their products have already been interacting with and affecting host cells for a considerable time to ensure the survival of the tumor. Ex vivo-generated, antigen-loaded DCs have now been used as vaccines to improve immunity9 . Numerous studies in mice have shown that DCs loaded with tumor antigens can induce therapeutic and protective antitumor immunity10. The immunogenicity of antigens delivered by DCs has been shown in patients with cancer9 or chronic HIV infection11, thereby providing proof of principle that using DCs as vaccines can work. Despite this, the efficacy of therapeutic vaccination against cancer has recently been questioned12 because of the undeniably limited rate of objective tumor regressions that has been observed in clinical studies so far. However, the question is not whether DC vaccines work but how to orient further studies to refine the immunological and clinical parameters of vaccination with DCs to improve its efficacy.

Vaccines against cancer Early studies in mice showed that the immune system can recognize and reject tumours13 and that immunodeficient mice (lacking interferon-γ (IFN-γ) and recombination-activating gene 2) have an increased incidence of cancer14 (BOX 1). In humans, the incidence of some cancers is increased in immunodeficient patients15 and is increased with age, owing to Immunosenescence16. These observations support the scientific rationale for immunotherapy for cancer. The term immunotherapy refers to any approach that seeks to mobilize or manipulate the immune system of a patient for therapeutic benefit17. In this regard, there are numerous strategies for improving the resistance of a patient to cancer. These include non-specific activation of the immune system with microbial components or cytokines, antigen-specific adoptive immunotherapy with antibodies and/or T cells, and antigen-specific active immunotherapy (that is, vaccination). The main limitation of using antibodies is that target proteins need to be expressed at the cell surface. By contrast, targets for T cells are usually peptides derived from intracellular proteins, which are presented at the cell surface in complexes with MHC molecules18. The identification of defined tumor antigens in humans19,20 prompted the development of adoptive T-cell therapy. Yet, the most attractive strategy is vaccination, which is expected to induce both therapeutic T-cell immunity (in the form of tumor-specific effector T cells) and protective T-cell immunity (in the form of tumor-specific memory T cells that can control tumor relapse)21–23. Numerous approaches for the therapeutic vaccination of individuals who have cancer have been developed, including the use of the following: autologous and allogeneic tumor cells (which are often modified to express various cytokines), peptides, proteins and DNA vaccines9,23–26. The observed results are variable; however, in many cases, a tumour-specific immune response has been induced, and tumor regressions, albeit limited, have occurred. These approaches rely on random encounter of the vaccine with host DCs. A lack of encounter of the vaccine antigen with DCs might result in the absence of an immune response. Alternatively, an inappropriate encounter — for example, with unactivated DCs or with the ‘wrong’ subset of DCs — might lead to silencing of the immune response27. Both of these situations could explain some of the shortcomings of current cancer vaccines. Furthermore, we do not know how tumor antigens need to be delivered to DCs in vivo to elicit an appropriate immune response.

Immature and mature dendritic cells have different functions. A | Immature dendritic cells (DCs) induce tolerance. Tissue DCs constantly sample their environment, capture antigens and migrate in small numbers to draining lymph nodes. In the absence of inflammation, the DCs remain in an immature state, and antigens are presented to T cells in the lymph node without costimulation, leading to either the deletion of T cells or the generation of inducible regulatory T cells. B | Mature DCs induce immunity. Tissue inflammation induces the maturation of DCs and the migration of large numbers of mature DCs to draining lymph nodes. The mature DCs express peptide–MHC complexes at the cell surface, as well as appropriate co-stimulatory molecules. This allows the priming of CD4+ T helper cells and CD8+ cytotoxic T lymphocytes (CTLs), the activation of B cells and the initiation of an adaptive immune response. To control the immune response, CD4+CD25+ regulatory T (TReg)-cell populations are also expanded. [ADCC, antibody-dependent  cell-mediated cytotoxicity; NK, natural killer; TCR, T-cell receptor].

Box 1 |

Mice

  • The immune system can reject tumors
  • Immune-mediated rejection of chemically induced tumours13
  • Increased cancer incidence in immunodeficient mice14

Humans

  • Increased cancer incidence in immunodeficient patients15
  • Increased cancer incidence with age (immunosenescence)16
  • Cancer regression in patients with paraneoplastic neurological disorders that are mediated by onconeuronal antibodies and specific CD8+ T cells136

Dendritic cells DC subsets. There are thought to be two main pathways of differentiation into DCs2,31 (FIG. 2). The myeloid pathway generates two subsets: Langerhans cells, which are found in stratified epithelia such as the skin; and interstitial DCs, which are found in all other tissues32. The lymphoid pathway generates plasmacytoid DCs (pDCs), which secrete large amounts of type I IFNs (IFN-α and IFN-β) after viral infection33,34. DCs and their precursors show remarkable functional plasticity. For example, pDCs form one of the first barriers to the expansion of intruding viruses, thereby functioning, through the release of type I IFNs, as part of the innate immune response. Subsequently, these cells differentiate into DCs that can present antigens to T cells, thereby functioning as members of the adaptive immune system35,36. Monocytes can differentiate into either macrophages, which function as scavengers, or DCs that induce specific immune responses37,38. Different cytokines skew the in vitro differentiation of monocytes into DCs with different phenotypes and functions (FIG. 3). So, after activation (for example, by granulocyte/ macrophage colony-stimulating factor, GM-CSF), monocytes that encounter interleukin-4 (IL-4) become DCs known as IL-4-DCs29,30,39. By contrast, after encounter with IFN-α, tumour-necrosis factor (TNF) or IL-15, activated monocytes differentiate into IFN-α-DCs40–43, TNF-DCs44 or IL-15-DCs45, respectively. Whether, in vivo, all interstitial DCs are derived from monocytes remains to be established, but myeloid DCs that are isolated from human peripheral blood also give rise to different DC types after exposure to different cytokines. Each of these DC subsets has both common and unique biological functions, which are determined by a unique combination of cell-surface molecule expression and cytokine secretion. For example, whereas IL-4-DCs are a homologous population of immature cells that is devoid of Langerhans cells, TNFDCs are heterogeneous and include both CD1a+ Langerhans cells and CD14+ interstitial DCs44.In vitro experiments showed that Langerhans cells and interstitial DCs that were generated from cultures of CD34+ hematopoietic progenitors differ in their capacity to activate lymphocytes: interstitial DCs induce the differentiation of naive B cells into immunoglobulin-secreting plasma cells4,32, whereas Langerhans cells seem to be particularly efficient activators of cytotoxic CD8+ T cells. They also differ in their cytokine-secretion pattern (only interstitial DCs produce IL-10) and their enzymatic activity4,32, which might be fundamental for the selection of peptides that are presented to T cells. Indeed, different enzymes are likely to degrade a given antigen into different sets of peptides, as has recently been shown for the HIV protein Nef 46. This then leads to different sets of peptide–MHC complexes being presented and thereby to distinct repertoires of antigen-specific T cells. So, these unique DCs are likely to yield unique immune effectors, thereby allowing the broad immune response that is required to combat permanently evolving microorganisms and tumors.

Distinct DC subsets induce distinct types of immune response. DCs have a crucial role in determining the type of response that is induced. There is evidence that either polarized DCs or distinct DC subsets might provide T cells with different signals that determine the class of immune response31. So, in mice, splenic CD8α+ DCs prime naive CD4+ T cells to produce TH1 cytokines in a process that involves IL-12, whereas splenic CD8α– DCs prime naive CD4+ T cells to produce TH2 cytokines47,48. Furthermore, this polarization into different T-cell subsets also depends on the signal received by a DC, as shown by the induction of IL-12 production and polarization towards TH1 cells when DCs are activated with Escherichia coli lipopolysaccharide (LPS), but the absence of IL-12 production and polarization towards TH2 cells when the same type of DC is exposed to LPS from Porphyromonas gingivalis 49. In humans, CD40 ligand (CD40L)-activated monocyte-derived DCs prime TH1-cell responses through an IL-12-dependent mechanism, whereas pDCs activated with IL-3 and CD40L have been shown to secrete negligible amounts of IL-12 and to prime TH2-cell responses50. So, both the type of DC subset and the activation signals to which DCs are exposed are important for polarization of T cells.

Mouse proof-of-principle in vivo studies

  • Ex vivo-generated, antigen-loaded dendritic cells (DCs) induce antigen-specific T-cell immunity137
  • Ex vivo gene-loaded DCs can induce humoral immunity138
  • Ex vivo-generated, antigen-loaded DCs induce tumor-specific immunity139,140
  • Ex vivo-generated DCs are superior to other types of vaccine141
  • Ex vivo-generated immature DCs induce tolerance142
  • Combination therapy with ex vivo-generated DCs improves vaccine efficacy112,113

This is an important parameter in vaccination against cancer, as type 1 immunity (including IFN-γ secretion) is desirable, whereas type 2 immunity (including IL-4 or IL-10 secretion) is considered deleterious. DCs and immune tolerance. DCs can induce and maintain immune tolerance27, both central and peripheral.

Central Tolerance depends on mature thymic DCs, which are essential for the deletion of newly generated T cells that have a receptor that recognizes self-components51. However, central tolerance might not be effective for all antigens. Furthermore, many self-antigens might not have access to the thymus, and others are only expressed later in life. So, there is a requirement for Peripheral Tolerance, which occurs in lymphoid organs and is mediated by immature DCs (FIG. 1a). Immature DCs present tissue antigens to T cells in the absence of appropriate co-stimulation, leading to T-cell Anergy or deletion27 or to the development of IL-10-secreting Inducible Regulatory T Cells52,53. The research groups of Nussenzweig and Steinman54 have elegantly shown that fusion proteins targeted to immature DCs lead to the induction of antigen-specific tolerance. By contrast, concomitant activation of these DCs with CD40- specific antibody results in a potent immune response, because the DCs are induced to express a large number of co-stimulatory molecules55. However, mature DCs might also contribute to peripheral tolerance by promoting the clonal expansion of naturally occurring CD4+CD25+ REGULATORY T (TReg) CELLS56, as discussed later. Therefore, the biology of DCs offers several targets for the control of cellular immunity. The parameters that need to be considered include DC-related factors, host-related factors and combining DC vaccines with other therapies.

Subsets of human dendritic cells. (Fig not shown). The population of dendritic cells (DCs) in the peripheral blood, which can be mobilized by treatment with FLT3L (fms-related tyrosine kinase 3 ligand), contains both CD11c+ myeloid DCs and CD11c– plasmacytoid DCs. So far, most studies of DCs have been carried out with DCs generated by culturing monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4); this simple procedure yields a homogenous population of DCs that resemble interstitial DCs, and the population is devoid of Langerhans cells. These DCs are immature and require exogenous factors for maturation. DCs can also be generated by culturing CD34+ haematopoietic progenitor cells (HPCs) or peripheral-blood monocytes with GM-CSF and tumour-necrosis factor (TNF). In this way, two DC subsets can be obtained: Langerhans cells, which might have improved efficacy for eliciting cytotoxic T lymphocytes; and interstitial DCs, which resemble monocyte-derived DCs. Adding IL-4 to CD34+ HPC cultures in the presence of GM-CSF and TNF inhibits the differentiation of Langerhans cells. [Green boxes indicate cell types that can be induced by culture with GM-CSF and TNF. Yellow boxes indicate cell types that can be induced by culture or mobilization with FLT3L].

Plasticity of monocyte-derived dendritic cells. (Fig not shown). Activated monocytes can differentiate into different types of dendritic cell (DC) after encounter with different cytokines. These distinct DCs will influence the differentiation of lymphocytes into immune effectors with different functions, leading to varied immune responses. For example, interleukin-15-DCs (IL-15- DCs) are remarkably more efficient at priming and maturation of rare antigen-specific cytotoxic T lymphocytes (CTLs) than are IL-4-DCs. Thymic stromal lymphopoietin-DCs (TSLP-DCs) induce CD4+ T cells to differentiate into pro-inflammatory T helper 2 (TH2) cells, which secrete large amounts of IL-13 and tumor-necrosis factor (TNF)143, whereas interferon-α-DCs (IFN-α-DCs) induce CD4+ T cells to differentiate into TH1 cells, which secrete IFN-γ and IL-10. The properties and function of TNF-DCs remain to be determined. [FLT3L, fms-related tyrosine kinase 3 ligand; GM-CSF, granulocyte/macrophage colony-stimulating factor].

Antigen loading. Loading MHC class I and class II molecules at the cell surface of DCs with peptides derived from defined antigens is the most commonly used strategy for DC-based vaccination22,87. Although this technique is important for proof-of-principle studies, the use of peptides has limitations: the restriction of a peptide to a given HLA type; the limited number of well-characterized Tumor-Associated Antigens; the relatively rapid turnover of exogenous peptide– MHC complexes, resulting in comparatively low antigen presentation by the time that the DC arrives in the draining lymph node after injection; and the induction of a restricted repertoire of T-cell clones, thereby limiting the ability of the immune system to control tumor-antigen variation. Yet another level of complexity is brought about by the use of MODIFIED HETEROCLITIC PEPTIDES. Some synthetic peptides, even those derived from immune-dominant antigens, do not bind MHC class I molecules with high affinity, possibly explaining their limited immunogenicity in vivo88. Therefore, the generation of peptide analogues with increased affinity for MHC class I molecules (known as heteroclitic peptides) could be used to improve peptide immunogenicity89,90. However, recent elegant studies in patients with malignant melanoma show that T cells elicited in vivo by vaccination with heteroclitic MART1 (melanoma antigen recognized by autologous T cells) or glycoprotein 100 (gp100) peptide show poor recognition of the endogenous melanoma-derived peptide and less efficient tumor-cell lysis compared with T cells specific for the native peptide91.

Immunoregulatory mechanisms

Naturally occurring CD4+CD25+ regulatory T cells

Cell-mediated suppression independent of interleukin-10 (IL-10) and/or transforming growth factor-β (TGF-β);
clonal expansion is regulated by mature dendritic cells (DCs)

Inducible regulatory T cells

Cytokine-mediated suppression through IL-10 and/or TGF-β; induction and clonal expansion is regulated by immature DCs

Natural killer T cells

Cytokine-mediated suppression through IL-13

Vaccine-induced B cells?

Cytokine-mediated regulation through IL-4, IL-6 and IL-10; competition with DCs for antigen uptake

Tumor-specific interferon-γ-secreting T cells?

Immunoediting and selection of escape variants (not discussed in main text)

Immune correlates of efficacy of dendritic-cell-based vaccines

  • Induction of broad tumour-specific T-cell immunity: T cells specific for several tumour antigens
  • Induction of effector T cells: T cells with immediate capacity to recognize tumour antigens and secrete cytokines such as tumour-necrosis factor and interferon-γ
  • Induction of memory T cells: T cells that secrete interleukin-2 and proliferate on re-exposure to tumour antigen
  • Induction of T cells that kill tumour cells
  • Decreased number of T cells with regulatory function

DCs are an attractive target for therapeutic manipulation of the immune system to increase otherwise insufficient immune responses to tumour antigens. However, the complexity of the DC system requires rational manipulation of DCs to achieve protective or therapeutic immunity. So, further research is needed to analyse the immune responses induced in patients by distinct ex vivo-generated DC subsets that are activated through different pathways. The ultimate ex vivo-generated DC vaccine will be heterogeneous and composed of several subsets, each of which will target a specific immune effector. These ex vivo strategies should help to identify the parameters for in vivo targeting of DCs, which is the next step in the development of DC-based vaccination. Indeed, distinct DC subsets express unique cell-surface molecules, such as different lectins131: Langerhans cells express langerin, which is crucial for the formation of Birbeck granules132,133; interstitial DCs express DCSIGN (dendritic-cell-specific intercellular-adhesionmolecule-3-grabbing non-integrin), which is involved in interactions with T cells and in DC migration but is also used by pathogens (such as HIV) to hijack the immune system; and pDCs express yet another lectin, BDCA2 (blood DC antigen 2)134,135. Such differential expression of cell-surface molecules might allow specific in vivo targeting of DC subsets for induction of the desired type of immune response.

9.1.2   Simultaneous Humoral and Cellular Immune Response against Cancer–Testis Antigen NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2–binding Peptide Epitopes

Elke JägerYao-Tseng ChenJan W. Drijfhout, Julia Karbach, et al.
J Exp Med. 1998 Jan 19; 187(2): 265–270.
A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I–restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the “cancer–testis” antigen NY-ESO-1. Concurrently, a strong MHC class I–restricted CTL reactivity against the autologous NY-ESO-1–positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2, NY-ESO-1–positive, but not NY-ESO-1–negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2–binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.

There is growing evidence for humoral and cellular immune recognition of cancer by the autologous human host (16). Based on CTL-dependent lysis of cultured melanoma cell lines, several categories of autoimmunogenic tumor antigens have been characterized, including differentiation antigens of specific cell lineages (79), individual antigens caused by point mutations (1011), and tumor antigens, such as MAGE, which are expressed in a variable proportion of different tumor types, but are silent in most normal tissues except the testis (12). CTL responses against melanoma antigens induced by peptide vaccines in vivo have been associated with a favorable development of advanced melanoma in some patients (613). As immunoselection of antigen-negative tumor cell variants has been observed during peptide vaccination (14), the molecular characterization of additional CTL-defined tumor antigens is needed to develop polyvalent vaccines with broader immunotherapeutic effects.

Sahin et al. have recently introduced a powerful new methodology for identifying human tumor antigens eliciting humoral immune response (5). The method has been called SEREX, for serological expression cloning of recombinant cDNA libraries of human tumors. Novel and previously defined tumor antigens have been identified by the SEREX method, including MAGE-1 and tyrosinase, both originally identified by cloning the epitopes recognized by CTLs. Thus, antibody screening of cDNA libraries prepared from human tumors can be used to identify antigens eliciting a cellular immune response, including CTLs, circumventing the need for established cultured autologous cell lines and stable CTL lines.

We have recently identified a novel human tumor antigen by SEREX analysis of a human esophageal cancer (15). The antigen, NY-ESO-1, belongs to a growing number of human tumor antigens we have called “cancer–testis” antigens that include MAGE, GAGE, BAGE (1), and SSX2 (HOM-MEL-40) (516). These antigens have the following characteristics: (a) they are expressed in a variable portion of a wide range of cancers, (b) their normal tissue expression is generally restricted to the testis, and (c) they are generally coded for by genes on the X chromosome. In a recent survey of sera from normal individuals and cancer patients, antibodies against NY-ESO-1 were found in ∼10% of patients with melanoma, ovarian cancer, and other cancers, but not in normal individuals (Stockert, E., manuscript in preparation). One patient with a high NY-ESO-1 antibody response was found to have specific CTL reactivity against cultured autologous melanoma cells. In the present study, we report that NY-ESO-1 encodes the CTL target in this patient and identify the NY-ESO-1 peptides that are recognized.

High-titer Antibody Reactivity against NY-ESO-1.

Melanoma patient NW38 presented with extensive metastases to inguinal lymph nodes having large areas of necrosis. Reverse transcriptase PCR of tumor RNA showed that this tumor expressed NY-ESO-1. Based on the hypothesis that exposure of the immune system to large amounts of intracellular tumor proteins released from the necrotic tumor might elicit a strong humoral immune response, the serum of patient NW38 was tested for specific reactivity against recombinant NY-ESO-1 protein. Fig. ​Fig.11 shows the reactivity of NW38 serum with the recombinant NY-ESO-1 protein, with a lysate of NY-ESO-1–transfected COS-7 cells, and with a lysate of the autologous NY-ESO-1 messenger RNA–positive tumor cell line NW-MEL-38. A 22-kD protein species was identified in both cell lysates, and comigrated with the purified recombinant NY-ESO-1 protein. The identity of this protein species as NY-ESO-1 was further confirmed by using an anti–NY-ESO-1 mouse monoclonal antibody. Reactivity against recombinant NY-ESO-1 protein was still detectable at a serum dilution of 1:100,000. No reactivity was detected against a lysate of untransfected COS-7 cells.
The correlation between NY-ESO-1 expression and NW38-IVS-1 reactivity suggested NY-ESO-1 as the antigenic target. To prove this, COS-7 cells were transfected with NY-ESO-1 cDNA and different MHC class I molecules and used as targets for NW38-IVS-1. Reactivity was measured in a standard TNF-α release assay. TNF release was found after stimulation of NW38-IVS-1 with COS-7 cells cotransfected with HLA-A2 and NY-ESO-1 cDNA. No reactivity was detected after stimulation with cotransfectants of pcDNA3.1(−)-NY-ESO-1 and pcDNA1Amp-HLA-A1 cDNA, COS-7 cells transfected with pcDNA3.1(−), or untransfected COS-7 cells (Fig. ​(Fig.3).3).

Peptide-specific CTLs.

26 different peptides encoded by NY-ESO-1 with theoretical binding motifs to the HLA-A2.1 molecule were tested for specific recognition by NW38-IVS-1. The target cells were peptide-pulsed T2 cells. Of these 26 peptides, three were recognized by NW38-IVS-1 as determined by a standard51Cr–release assay (Table ​(Table1).1). The peptide sequences SLLMWITQCFL, SLLMWITQC, and QLSLLMWIT are located between positions 155 and 167 of the NY-ESO-1 protein (15), and show overlapping sequences. The 11-mer SLLMWITQCFL (2 in Table ​Table1)1) and the 9-mer SLLMWITQC (12 in Table ​Table1)1) consist of identical amino acids at positions 1–9.

To provide additional confirmation of the peptide specificity, the 26 synthetic peptides were individually incubated with HLA-A2–transfected COS-7 cells and tested in the TNF release assay. Consistent with the results of 51Cr–release assay, specific TNF-α release was detected in tests with peptides SLLMWITQCFL, SLLMWITQC, and QLSLLMWIT. NY-ESO-1/HLA-A2 transfectants were used as a positive control in these assays (Fig. ​(Fig.4).4).

The search for tumor antigens that induce specific immune responses in cancer patients is the ongoing challenge in tumor immunology. Evidence for a specific humoral response to human cancer came from serological analysis of cell surface reactivity of sera from cancer patients for autologous cancer cells, an approach called autologous typing (4). However, with only a few exceptions, this approach did not allow for the structural definition of the antigenic target. An autologous typing system also provided the first evidence for the development of CTLs with specificity for human melanoma cells (3172124). Using specific antitumor CTLs as probes, a number of CTL targets have been cloned on the basis of MHC class I–restricted recognition (16). However, this approach involves cultured cancer cell lines and stable CTL lines from the same patient, two requirements that cannot easily be met with many tumor types. With the demonstration that genes coding for CTL-recognized tumor antigens elicit humoral immunity and can be cloned by SEREX methodology, a technically less demanding approach defining immunogenic tumor antigens is now available, one that extends the range of analysis to tumor types that are not easily adaptable to in vitro growth and are not sensitive targets for CTLs. A number of novel tumor antigens have been defined by SEREX, including two new members of the cancer–testis antigenic family, SSX2 (HOM-MEL-40) (516), and NY-ESO-1 (15).

In this study, we identified a melanoma patient, NW38, with high-titered antibody against NY-ESO-1. This patient had a large and highly necrotic tumor, and the sustained release of intracellular antigens that are usually inaccessible to the immune system may account for the high NY-ESO-1 titer. The establishment of an autologous cell line that typed NY-ESO-1 positive provided target cells for assessing CTL reactivity in this patient. A CTL line was established from this patient that lysed the autologous melanoma cell line in an HLA-A2–restricted fashion. Using target cells transfected with NY-ESO-1 and HLA-A2, the specificity of CTL reactivity was found to be coded by NY-ESO-1. Computer analysis of the NY-ESO-1 sequence identified 26 peptides with HLA-A2–binding motifs. Screening of these peptides presented by T2 cells identified three sequences that were confirmed to be specifically recognized by NW38-IVS-1. This is the first conclusive demonstration of simultaneous antibody and CTL responses against a cancer–testis antigen in a single patient.

The strategy used in this study to generate and analyze CTL reactivity to a SEREX-defined antigen can be used as a model for investigating cellular immune responses to the growing list of other SEREX antigens. Identification of clones in SEREX requires high-titered IgG antibody, and the development of such antibodies requires the help of CD4+ T cells. In this sense, SEREX can be thought of as a method to define the CD4+ T cell repertoire to human tumor antigens. Also, the presence of both NY-ESO-1 antibody and CTLs in patient NW38 suggests that screening for an antibody response may be a simple and effective way to identify patients with concomitant CTL reactivity, and this possibility is now being tested in other patients with NY-ESO-1 antibody. In the absence of autologous tumor cell lines, CD8+ T cells can be stimulated with autologous antigen-presenting cells that have been transfected with the coding gene or fed purified protein antigens. A similar strategy can be used to identify peptide targets for CD4+ T cells.

A major objective in defining immunogenic human tumor targets is to explore their use in the development of cancer vaccines, and a number of clinical trials with various vaccine constructs are currently underway. Although tumor regression is the desired goal of a therapeutic vaccine, this end point cannot be expected to be an effective way to develop maximally immunogenic tumor vaccines. For this purpose, reliable immunological assays are needed to monitor the specificity and strength of specific immune reactions generated by the vaccine. With the exception of vaccines aimed at inducing a humoral immune response such as GM2 ganglioside vaccines, most vaccine trials are designed to stimulate cellular immunity, particularly the development of CTLs and CD4+ T cells. These have been difficult to detect in vaccine trials with MAGE peptides (25), and difficult to interpret in trials with vaccines containing melanocyte differentiation antigens, since CTLs against these antigens can be generated in vitro from nonvaccinated melanoma patients as well as normal individuals (2627). However, de novo induction and increase of preexisting CTL reactivity have been detected after vaccination with melanocyte differentiation antigens and observed to be associated with cancer regressions in a limited number of patients (13). The demonstration of a simultaneous antibody and CTL response to NY-ESO-1 in the same patient suggests that serological methods may be useful in monitoring vaccine trials with NY-ESO-1 and other tumor antigens eliciting a humoral immune response.

9.1.3 Monoclonal Antibodies in Cancer Therapy

R K Oldham
JCO September 1983; 1(9): 582-590
http://jco.ascopubs.org/content/1/9/582.short


The need for improved specificity in cancer therapy is apparent. With the advent of monoclonal antibodies, the possibility of specifically targeted therapy is being considered. Early trials of monoclonal antibody in experimental animals and humans have indicated its ability to traffic to specific tumor sites and to localize on or around the tumor cells displaying antigens to which the antibody is directed. This evidence of specific targeting, along with preliminary evidence of therapeutic efficacy for monoclonal antibodies and immunoconjugates with drugs, toxins, and isotopes is encouraging. The current status of clinical trials with monoclonal antibodies is reviewed and an example of the experimental approach for the development of immunoconjugates in animal models is presented.

Monoclonal Antibodies in Cancer Therapy: 25 Years of Progress

Robert K. Oldham, Robert O. Dillman
JCO Apr 10, 200826(11): 1774-1777
http://dx.doi.org:/10.1200/JCO.2007.15.7438

In 1983, it was apparent that a major problem with current modalities of cancer treatment was the lack of specificity for the cancer cell.1 It was predicted that a major advancement in treatment of cancer would be the development of a class of agents that would have a greater degree of specificity for the tumor cell. Based on many animal studies and the treatment of fewer than 100 patients, it was evident in 1983 that monoclonal antibodies would be that major advance.

The first patient treated in the United States with monoclonal antibody therapy was a patient with non-Hodgkin’s lymphoma.2 Nadler et al2 described the treatment using a murine monoclonal antibody designated AB 89. Although treatment was not successful in inducing a significant clinical response, it did represent the first proof of principle in humans that a monoclonal antibody could induce transient decreases in the number of circulating tumor cells, induce circulating dead cells, and form complexes with circulating antigen, all with minimal toxicity to the patient. Antibody could be detected on the surface of circulating lymphoma cells, and free antigen in the serum decreased with each infusion of antibody. After two courses of milligram doses of AB 89, a final and third course with 1.5 g of antibody was administered during a 6-hour period. A marked reduction in circulating antigen was noted, but these studies suggested to the authors that the quantity of circulating antigen was too great to effectively deliver AB 89 to the patient’s tumor cells in a therapeutically effective manner.2

In the Journal of Clinical Oncology review article cited earlier,1 evidence was reviewed from animal tumor models that clearly demonstrated both specificity and therapeutic efficacy with little serious toxicity. Whereas passive serotherapy of human cancer had shown little success,3 it was apparent in the earlier review that monoclonal antibodies could be used in the treatment of leukemia and lymphoma.4,5 In 1983, a review of the literature revealed approximately 10 published studies and one in-press article of therapeutic trials of monoclonal antibody therapy in humans. All of these studies used murine monoclonal antibodies and were phase I/II studies. Most were in leukemia or lymphoma, but the earliest solid tumor studies were also underway in melanoma6 and GI cancer.1

By 1983, the pioneers in monoclonal antibody research believed that a new era of cancer therapy had begun, and for the first time, true specific and targeted therapy was underway using hybridoma technology to produce monoclonal antibodies with exquisite specificity. It was also apparent, based on animal model studies, that monoclonal antibodies could be a vehicle to bring immunoconjugate therapy to the clinic by conjugating monoclonal antibodies to drugs, toxins, and radioisotopes using the specificity of the monoclonal antibody to carry enhanced killing capacity directly to the tumor cells. Thus, the era of monoclonal antibody therapy, as well as immunoconjugate therapy, had begun.

Although there was much excitement (and skepticism) about this new treatment modality (the use of a form of biologic therapy with great specificity in patients with advanced cancer) there were also problems and limitations. As presented in Table 1, there were clinical toxicities with murine monoclonal antibodies, most of which were secondary to the interaction with the target antigen.7 However, the major limitation was their immunogenicity. Murine proteins are highly immunogenic, and it was soon found that only a few infusions of these foreign proteins could be given to patients with cancer because of the development of human antimouse antibody.8 Another problem quickly became apparent, in that some of the antigens on cancer cell surfaces modulated off the surface and into the circulation when antibody attached. Modulation could also cause internalization of the complex. It was recognized that this could represent a therapeutic advantage by using the antibody as carrier to internalize the toxic component of an immunoconjugate, potentially making it more therapeutically active.

In 1983, few specific antigens found only in cancer cells had been identified, and there was much debate about the specificity of these antigens. Many of the antigens to which monoclonal antibodies were made were embryonic antigens or shared antigens found on cancer cells and some normal cells. Therefore, although the specificity of the antibody was exquisite for the antigen, the specificity for the antibody or immunoconjugate for cancer was not absolute. One fairly clear exception occurred early in the 1980s when Levy et al9 developed monoclonal antibodies to the idiotype of B-lymphoma cells. The first patient given this anti-idiotypic antibody had a complete response to therapy, and his lymphoma went into a sustained remission that lasted for years. As a direct result of these early studies with anti-idiotypic antibodies, there is now a series of idiotype vaccines that are in phase III trials in patients with low-grade follicular lymphomas.10 These anti-idiotype vaccines will likely be the first truly custom-tailored, personalized anticancer vaccines to be approved for therapeutic use.

The major limitation of murine monoclonal antibody therapy was the immunogenicity of the mouse protein; a variety of investigators postulated that for monoclonal antibody therapy to be truly successful, human or humanized antibodies would be necessary. It was also known 25 years ago that the half-life of murine antibodies in the circulation was brief, and because of human antimouse antibody, became briefer with each infusion of murine monoclonal antibody. Previous studies of human immunoglobulin in clinical trials had demonstrated a much longer half-life for human immunoglobulin, which predicted that once human or humanized antibodies were available, the therapeutic efficacy of monoclonal antibodies and their immunoconjugates might be considerably enhanced.1
How has the field of monoclonal antibody and immunoconjugate therapy fared since the predictions of the early 1980s? Twenty-five years later, considerable progress has been made in this field.11,12 The US Food and Drug Administration has approved 21 monoclonal antibody products, with six of these biologic drugs approved specifically for cancer (Table 2). It was a landmark date in November 1997 when rituximab became the first monoclonal antibody approved specifically for cancer therapy.13 In addition to these six unconjugated monoclonal antibody therapies, one drug immunoconjugate, gemtuzumab ozogamicin (Mylotarg; Wyeth-Ayerst, Madison, NJ), has been approved. This humanized monoclonal antibody to CD33 is approved for use in acute myelogenous leukemia and uses the antibody conjugated to calicheamicin, a potent enediyene antibiotic originally isolated from aMicromonospora echoinospora.14 Two radioisotope-antibody conjugates, ytrrium-90 ibritumomab tiuxetan (Zevalin; Cell Therapeutics Inc, Seattle, WA) and iodine-131 tositumomab (Bexxar; GlaxoSmithKline, Middlesex, United Kingdom) have been approved.15 The murine form of these antibodies was retained in order to expedite clearance from the circulation. Both radiolabeled antibodies target the CD20 antigen on lymphoma cells.

Unlike the immunoconjugates, which are currently infrequently used, each of the six unconjugated antibodies approved for cancer therapy is currently frequently used in the treatment of humans with cancer. The use of techniques to humanize or chimarize monoclonal antibodies to decrease their murine components has been an important advance in the field. These molecules have a long half-life in the blood stream, and can interact with human complement or effector cells of the patient’s immune system. They behave in a manner similar to naturally occurring immunoglobulin and work along the lines of our normal antibody-based immune response as effective agents in treating patients with cancer.16

Rituximab has become the largest-selling biologic drug in clinical oncology, and is active in a variety of human lymphomas and chronic lymphocytic leukemia.17,18 This is a chimeric monoclonal antibody targeting the CD20 antigen found on both normal B cells and on most low-grade and some higher grade B-cell lymphomas. It is effective as a single agent in induction and maintenance therapy. It is primarily used, however, in combination with standard chemotherapies in the treatment of patients with non-Hodgkin’s B-cell lymphomas and chronic lymphocytic leukemia.19-22

A second monoclonal antibody that has proven highly effective in the clinic is trastuzumab, a humanized antibody that reacts with the second part of the human epidermal growth factor receptor 2.23 Like rituximab, it is effective as a single agent in induction and maintenance therapy, but is used primarily in conjunction with chemotherapy for patients with human epidermal growth factor receptor 2/neu–positive breast cancer.24,25

Alemtuzumab is a humanized monoclonal antibody targeting the CD52 antigen found on B lymphocytes and is used primarily for chronic lymphocytic leukemia.26 Like the two previously cited monoclonal antibody therapies, alemtuzumab is effective as induction and maintenance therapy. Alemtuzumab is also reactive with T lymphocytes, and unlike the other two antibodies, it is typically not combined with chemotherapy because of the increased risk of infection.(26)

Another humanized monoclonal antibody, bevacizumab, has been applied more broadly in human solid tumors because it targets vascular endothelial growth factor, which is the ligand for a receptor found on blood vessels.(27) Because this receptor is on endothelial cells, bevacizumab seems to be effective by reducing the blood supply to tumor nodules, thereby slowing or interrupting growth. Initially approved for advanced colorectal cancer,(28) it is now used in a variety of human solid tumors including cancers of the lung, kidney, and breast.(29-31)

The last two antibodies approved for clinical use were cetuximab (a chimeric antibody), and panitumumab (a completely human antibody). Both target the epidermal growth factor receptors found on a variety of human tumors.(32,33) Cetuximab was originally approved for use in combination with chemotherapy in metastatic colorectal cancer.(34) It also enhances chemotherapy and radiation therapy of squamous cell cancers of the head and neck.(35) Panitumumab was approved based on its single-agent activity in refractory colorectal cancer and is being combined with chemotherapy as well.

At the end of 2007, 25 years of clinical studies have resulted in the approval of six unconjugated, humanized, or chimeric monoclonal antibodies for cancer therapy along with one drug immunoconjugate and two radioisotope immunoconjugates. Although few in number, these monoclonal antibodies are changing the face of cancer therapy, bringing us closer to more specific and more effective biologic therapy of cancer as opposed to nonspecific cytotoxic chemicals.

Modern recombinant techniques have made it possible to rapidly produce both chimeric antibodies and humanized antibodies, and totally human antibodies are also being produced. Identification of surface receptors that are integral to proliferation and apoptosis has provided more targets for monoclonal antibodies beyond those originally identified by the murine immune system. In 2008, there are more than 100 monoclonal antibody–based biologic drugs in hundreds of clinical trials. Many of these are in phase II and phase III and will be coming before the US Food and Drug Administration for approval in the next few months and years. At long last, immunoconjugates are proving efficacious with acceptable toxicity and will extend our diagnostic (36) and therapeutic armamentarium (37) from mainly unconjugated monoclonal antibodies to a broad array of highly active and specific immunoconjugates.

On this silver anniversary for our 1983 review, “Monoclonal Antibodies in Cancer Therapy, ” we can confidently predict that progress toward more specific and less toxic therapy for human cancer is in our near future. The developments during the past 25 years in both biologic drugs and targeted small molecules place us on the verge of more cures with less toxicity for our patients with cancer.

9.1.4 Aptamers

Nanocarriers as an emerging platform for cancer therapy

Dan Peer1,7, Jeffrey M. Karp2,3,7, Seungpyo Hong, et al. 
Nature Nanotechnology
 2, 751 – 760 (2007)
http://dx.doi.org:/10.1038/nnano.2007.387

Nanotechnology has the potential to revolutionize cancer diagnosis and therapy. Advances in protein engineering and materials science have contributed to novel nanoscale targeting approaches that may bring new hope to cancer patients. Several therapeutic nanocarriers have been approved for clinical use. However, to date, there are only a few clinically approved nanocarriers that incorporate molecules to selectively bind and target cancer cells. This review examines some of the approved formulations and discusses the challenges in translating basic research to the clinic. We detail the arsenal of nanocarriers and molecules available for selective tumor targeting, and emphasize the challenges in cancer treatment.

Quantum Dot−Aptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing of Drug Delivery Based on Bi-Fluorescence Resonance Energy Transfer
Vaishali Bagalkot, L Zhang, E Levy-Nissenbaum, S Jon, PW Kantoff, et al.
Nano Letters 2007; 7(10):3065-3070
http://dx.doi.org:/10.1021/nl071546n

We report a novel quantum dot (QD)−aptamer(Apt)−doxorubicin (Dox) conjugate [QD−Apt(Dox)] as a targeted cancer imaging, therapy, and

sensing system. By functionalizing the surface of fluorescent QD with the A10 RNA aptamer, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA), we developed a targeted QD imaging system (QD−Apt) that is capable of differential uptake and imaging of prostate cancer cells that express the PSMA protein. The intercalation of Dox, a widely used antineoplastic anthracycline drug with fluorescent properties, in the double-stranded stem of the A10 aptamer results in a targeted QD−Apt(Dox) conjugate with reversible self-quenching properties based on a Bi-FRET mechanism. A donor−acceptor model fluorescence resonance energy transfer (FRET) between QD and Dox and a donor−quencher model FRET between Dox and aptamer result when Dox intercalated within the A10 aptamer. This simple multifunctional nanoparticle system can deliver Dox to the targeted prostate cancer cells and sense the delivery of Dox by activating the fluorescence of QD, which concurrently images the cancer cells. We demonstrate the specificity and sensitivity of this nanoparticle conjugate as a cancer imaging, therapy and sensing system in vitro.

Semiconductor nanocrystals known as quantum dots (QDs)

have been increasingly utilized as biological imaging and labeling probes because of their unique optical properties, including broad absorption with narrow photoluminescence spectra, high quantum yield, low photobleaching, and resistance to chemical degradation. In some cases, these unique properties have conferred advantages over traditional fluorophores such as organic dyes.1-4 The surface modification of QDs with antibodies, aptamers, peptides, or small

molecules that bind to antigens present on the target cells or tissues has resulted in the development of sensitive and specific targeted imaging and diagnostic modalities for in vitro and in vivo applications.5-7 More recently, QDs have been engineered to carry distinct classes of therapeutic agents for simultaneous imaging and therapeutic applications.8,9 While these combined imaging therapy nanoparticles represent an exciting advance in the field of nanomedicine, it would be ideal to engineer “smart” multifunctional nanoparticles that are capable of performing these tasks while sensing the delivery of drugs in a simple and easily detectable manner. One way to achieve this goal is to develop multifunctional nanoparticles capable of sensing the release of the therapeutic modality by a change in the fluorescence of the imaging modality.

Figure 1. (a) Schematic illustration of QD-Apt(Dox) Bi-FRET system. In the first step, the CdSe/ZnS core-shell QD are surface functionalized with the A10 PSMA aptamer. The intercalation of Dox within the A10 PSMA aptamer on the surface of QDs results in the formation of the QD-Apt(Dox) and quenching of both QD and Dox fluorescence through a Bi-FRET mechanism: the fluorescence of the QD is quenched by Dox while simultaneously the fluorescence of Dox is quenched by intercalation within the A10 PSMA aptamer resulting in the “OFF” state. (b)

Schematic illustration of specific uptake of QD-Apt(Dox) conjugates into target cancer cell through PSMA mediate endocytosis. The release of Dox from the QD-Apt(Dox) conjugates induces the recovery of fluorescence from both QD and Dox (“ON” state), thereby sensing the intracellular delivery of Dox and enabling the synchronous fluorescent localization and killing of cancer cells.

Figure 3. Fluorescence spectra. (a) QD-Apt conjugate (1 µM) with increasing molar ratio of Dox (from top to bottom: 0, 0.1, 0.3, 0.6, 1, 1.5, 2.1, 2.8, 3.5, 4.5, 5.5, 7, and 8) at an excitation of 350 nm. (b) Dox (10 µM) with increasing molar ratio of QD-Apt conjugate (from top to bottom: 0.02, 0.04, 0.07, 0.09, 0.12, 0.14, and 0.16) at an excitation of 480 nm.

In conclusion, herein we report to our knowledge the first example of a multifunctional nanoparticle that can detect cancer cells at a single cell level while intracellularly releasing a cytotoxic dose of a therapeutic agent in a reportable manner. We demonstrate the specificity and sensitivity of this cancer imaging, therapy and sensing nanoparticle conjugate system in vitro by using PCa cell lines. By functionalizing the surface of fluorescent QD with the A10 PSMA aptamer, and intercalating Dox into the double-stranded CG sequence of the A10 PSMA aptamer, we developed a targeted QD-Apt(Dox) conjugate with reversible Bi-FRET properties. The incorporation of multiple CG sequences within the stem of the aptamers may further increase the loading efficiency of Dox on these conjugates. The presence of additional Dox may enhance the selfquenching effect of QD-Apt(Dox) conjugates thereby improving their imaging sensitivity, while the higher dose of Dox may enhance the therapeutic efficacy of the conjugates. Furthermore, through the use of other disease-specific aptamers or other targeting molecules, similar multifunctional nanoparticles may potentially be developed for additional important medical applications

Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

Hongguang Sun1, Xun Zhu2, Patrick Y Lu3, Roberto R Rosato, et al.
Molecular Therapy Nucleic Acids(2014) 3, e182;
http://dx.doi.org:/10.1038/mtna.2014.32

Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed “chemical antibodies.” In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.

The terms “aptamer” and “SELEX” were introduced by two independent groups in 1990.1,2 The term “aptamer” refers to small nucleic acid ligands that exhibit specific therapeutic functions and an unambiguous binding affinity for their targets. Conversely, Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology is the method used for aptamer development. Although using small molecule nucleic acids as therapeutics has been explored for decades, development of SELEX and aptamer technology revolutionized this field.

The most important property of an aptamer, from the Latin aptus (to fit), is its high target selectivity. These short, chemically synthesized, single-stranded (ss) RNA or DNA oligonucleotides fold into specific three-dimensional (3D) structures with dissociation constants usually in the pico- to nano-molar range.3 Moreover, in contrast to other nucleic acid molecular probes, aptamers interact with and bind to their targets through structural recognition (Figure 1), a process similar to that of an antigen-antibody reaction. Thus, aptamers are also referred to as “chemical antibodies.”

Figure 1.

Schematic diagram of aptamer binding to its target.

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Due to their small size and oligonucleotide properties, aptamers offer several advantages over protein antibodies in both their extensive clinical applicability and a less challenging industrial synthesis process. Specifically, (i) aptamers can penetrate tissues faster and more efficiently due to their significantly lower molecular weight (8–25 kDa aptamers versus ~150 kDa of antibodies). Therefore, aptamers penetrate tissues barriers and reach their target sites in vivo more efficiently than the larger-sized protein antibodies. (ii) Aptamers are virtually nonimmunogenic in vivo. In principal, as aptamers are oligonucleotides they should not be recognized by the immune system. In practice, a recent clinical study showed that aptamers did not stimulate an immune response in vivo,4,5 as compared to protein antibodies that are highly immunogenic, especially following repeat injections. (iii) Aptamers are thermally stable. Based on the intrinsic property of oligonucleotides, even after a 95 °C denaturation, aptamers can refold into their correct 3D conformations once cooled to room temperature. In comparison, protein-based antibodies permanently lose their activity at high temperatures. More importantly, a well-established synthesis protocol and chemical modification technology lead to (iv) rapid, large-scale aptamer synthesis and modification capacity that includes a variety of functional moieties; (v) low structural variation during chemical synthesis; and (vi) have lower production costs. Moreover, aptamers specifically recognize a wide range of targets, such as ions, drugs, toxins, peptides, proteins, viruses, bacteria, cells, and even tissues.6,7,8,9,10,11,12 In the clinic, aptamer-based therapeutics are gaining momentum. For example, Macugen, a modified RNA aptamer, specifically targets vascular endothelial growth factor. It has been approved by the US Food and Drug Administration (FDA)13 for the treatment of wet age-related macular degeneration and is under evaluation for other conditions.14 In the cancer setting, AS1411 targets nucleolin, a protein over-expressed in a variety of tumors. It is currently being evaluated as a potential treatment option in solid tumors and acute myeloid leukemia.15 An updated list of therapeutic aptamers undergoing clinical trials is included in ref. 16 and Table 1. Taken together, these clinical studies highlight many possible uses that aptamers may have in a variety of biomedical fields, including therapeutics.17
Table 1 – A list of therapeutic aptamers undergoing clinical trials.

Since aptamer technology was first introduced, the RNA-based sequence library has been widely used for SELEX. Based on the existing evidence, it is believed that the presence of a 2′-OH group and non-Watson-Crick base pairing allows RNA aptamer oligonucleotides to fold into more diverse 3D structures than ssDNA molecules. Consequently, using the more flexible RNA sequences simplifies the development of high-affinity and -specificity aptamers. Despite their advantages, RNA sequences are very sensitive to nucleases present in biological environments and can be rapidly degraded.18 To increase nuclease resistance of RNA-based aptamers, several chemical modifications have been investigated. Evidence shows that 2′-OH group and phosphodiester linkages of RNA sequences are the sites of nuclease hydrolysis. Subsequently, substitutions of the 2′-OH functional group by 2′-fluoro, 2′-amino, or 2′-O-methoxy motifs, and/or changes to the phosphodiester backbone with boranophosphate or phosphorothioate are the most common modifications aimed at increasing nuclease resistance.19 More recently, Wu et al. developed a novel chemical modification method to increase siRNA stability, in which phosphorodithioate and 2′-O-Methyl were simultaneously substituted in the same nucleotide.20
This modification method significantly enhanced siRNA stability and represents a potential new direction for utilization of RNA-based therapies in complex biological systems. Other effective modifications recently reported utilize the locked nucleic acid technology16,21 or generate “mirror” RNA sequence structures, termed spiegelmers.22 These modifications result in structural changes to the RNA sequences, which cannot be digested by nucleases.

In addition to RNA aptamers, ssDNA-based aptamers have also been developed. Due to their lack of 2′-OH groups, DNA molecules are naturally resistant to 2′-endonucleases and are stable in biological environments. Recently, our group developed a biostable DNA-based aptamer specific for CD30, a protein biomarker that is over-expressed in Hodgkin and anaplastic large cell lymphomas. Functional analysis demonstrated that this ssDNA-based aptamer exhibited high CD30 binding affinity as low as 2 nmol/l and was stable in human serum for up to 8 hours. Conversely, an RNA-based CD30 aptamer was digested within 10 minutes under similar conditions.23
In summary, unique chemical features and biological functions have made aptamers a very attractive tool in biomedical research over the past two decades. Currently, there are over 4,000 published articles referenced in the PubMed database that include the term “aptamer.” Research areas that include aptamer technology cover bioassays, drug development, cell detection, tissue staining, in vitro and in vivo imaging, nanotechnology, and targeted therapy. As chemical antibodies, aptamers represent an excellent alternative to replace or supplement protein antibodies, which have been extensively used in the clinic.

Aptamers Specifically Targeting Cell Surface Biomarkers

Using SELEX technology to develop aptamers for cell surface biomarkers

SELEX, the methodology used to develop aptamers specific for a target of interest, is based on a repetitive amplification and enrichment process. The SELEX process follows several steps: first, a random ssDNA oligonucleotide library is chemically synthesized to contain between 1014–1015 unique random sequences flanked by conserved primer binding sites. This step utilizes the following universal scheme: 5′-sense primer sequence-(random sequence)-antisense primer sequence-3′, where the primer sequence ranges from 18 to 22 bases and the random sequence contains 20–40 nucleic acids. The general procedure consists of labeling the 5′-sense primer with a fluorochrome reporter for monitoring aptamer selection, while the 3′-antisense primer is labeled with an affinity molecule, such as biotin, that is used to separate single-stranded oligonucleotides generated in each amplification round. This random ssDNA library can be used directly to select an initial pool of DNA aptamers. Conversely, generation of RNA aptamers requires two extra steps. Specifically, a pool of random ssDNA oligonucleotides is generated, T7 RNA polymerase promoter sequence is added to the 5′-sense primer, and the DNA is then used as a template for T7 RNA polymerase-based transcription in the 5′ to 3′ direction. During the second SELEX step, the oligonucleotide library is heated and rapidly cooled to promote the formation of 3D structures. The library is then mixed with the target of interest for specific binding enrichment. In the third step, the unbound sequences are discarded through the use of membranes, columns, magnetic beads, and capillary electrophoresis.6,24,25 In the fourth step, the enriched sequences are amplified in vitro by either PCR (DNA aptamers) or RT-PCR (RNA aptamers) to generate a new sequence library for the next round of SELEX. The amplified sequence library may go through further negative-target selection, which eliminates the nonspecific sequences generated by binding of nontarget moieties. Lastly, aptamer selection goes through 4–20 rounds of amplification and enrichment. The exact number of required amplification and selection steps depends on the aptamer target being a purified protein or a living cell, and on the evolution of the aptamer sequence library, as that established by gel electrophoresis, flow cytometry (for target binding), classical cloning or sequencing methods, or by high throughput Next-Generation Sequencing (NGS). In recent years, the traditional SELEX method had also been modified to include the capillary electrophoresis (CE) SELEX, toggle selection, photo-SELEX, bead-based selection, X-Aptamers, and Slow Off-rate Modified Aptamers (SOMAmers) in order to maximize affinity and specificity, to improve the speed of selection and success rate, and to provide additional properties to the selected aptamers.26,27,28,29,30,31

Similar to protein antibody development, purified recombinant proteins or peptides expressed in prokaryotic or eukaryotic systems can be used as targets for aptamers selected by the SELEX method. However, because of the posttranslational modifications, especially in the case of highly glycosylated proteins, purified proteins or peptides often cannot fold into the correct 3D structure that is formed under physiologic conditions.32 Consequently, the newly synthesized aptamers may not be able to selectively recognize and interact with their corresponding targets, which would result in failure of the biomedical application. As this is a common problem, it is very important to choose biomarkers in their native conformation for aptamers selection. Taking this issue into an account, a modified SELEX technology that uses whole living cells, Cell-based SELEX (or Cell-SELEX), was recently established.33 To develop cell-specific aptamers, the Cell-SELEX method uses whole living cells that express surface biomarkers of interest. However, the presence of many different cell surface molecules in addition to the target biomarker(s) results in the synthesis of many unrelated/unwanted aptamers. Therefore, in addition to all the SELEX steps described above, Cell-SELEX technology also utilizes control cells that do not express the target biomarker(s) during the counter-selection step.33

Well-characterized biomarkers that are endogenously expressed at high levels, such as the ErbB superfamily, MUC1, EpCAM, and CD30, offer the best potential for cell-based aptamer development. Subsequently, cell lines that have high endogenous expression of cell-specific or cancer type-specific biomarker(s) are commonly used for Cell-SELEX. However, if such cell lines are unavailable, a biomarker of interest could be over-expressed in a particular cell line via gene transfection and the parental cells used for counter-selection. Using this approach, aptamers targeting the cancer stem cell (CSC) biomarker CD133 have been recently developed.34 In this study, CD133 cDNA was transfected into HEK293T cells that were then used for aptamer enrichment, with the parental HEK293T cells serving as a negative control. Similarly, an aptamer specific for the human receptor tyrosine kinase was recently developed.35

Figure 2.

Schematic diagram of our hybrid-SELEX method for selection of CD30-specific ssDNA aptamer. In our experiment, the hybrid-SELEX process is divided into (a) the cell-based SELEX selection and (b) CD30 protein-based SELEX enrichment. First, CD30-expressing lymphoma cells are used for positive selection and CD30-negative Jurkat cells are used in negative counter-selection. After 20 rounds of selection, the enriched aptamer pool is incubated with CD30 protein immobilized on magnetic beads for five additional rounds of enrichment. SELEX, Systematic Evolution of Ligands by EXponential enrichment.

Full figure and legend (183K)

Aptamers specific for cell surface biomarkers

Cell surface biomarkers are functionally important molecules involved in many biological processes, such as signal transduction, cell adhesion and migration, cell–cell interactions, and communication between the intra- and extra-cellular environments. An abnormal expression of cell surface biomarkers is often related to tumorigenesis.50 Clinically, it is estimated that about 60% of cancer-targeting drugs, including therapeutic antibodies and small molecule inhibitors, target cell surface biomarkers,51 making them attractive for disease treatment. In the last decade, many aptamers targeting cell surface biomarkers have been developed through the advancement of both the protein- and/or cell-based SELEX technologies (see Table 2 for detailed list). These aptamers have been extensively studied for diagnosis and/or treatment of hematological malignancies,7,23,49 lung,52,53,54 liver,55 breast,56,57 ovarian,58 brain,59,60colorectal,61 and pancreatic cancers,46 as well as for identification and characterization of CSCs.34,62

Aptamer-Mediated Targeted Therapies

Traditional cancer treatment approaches, such as chemotherapy, radiotherapy, photodynamic therapy, and photothermal therapy can cause serious side effects in patients due to their associated nonspecific toxicity. To minimize these side effects, a concept of personalized, targeted therapy has been gaining momentum. One of the main clinical approaches for targeted cancer therapy employs antibody-based drugs. Although antibody-mediated therapy is highly specific and results in fewer side effects, potential immunogenicity and high cost of production may limit its clinical applications. To overcome these obstacles, oligonucleotide aptamer-based targeted therapeutics and specific drug delivery systems have recently been explored. These studies revealed numerous advantages offered by the aptamer technology over protein-based antibody therapies, with some of these described in the section below.
Aptamer-drug conjugates

Aptamer-drug conjugation (ApDC) is a very simple yet effective model of noncovalently or covalently conjugating aptamer sequences directly with therapeutic agents (Figure 3). For example, aptamer-conjugated Doxorubicin (Dox), a chemotherapeutic agent extensively used in the treatment of various cancers, has recently been shown to have enhanced therapeutic efficacy over Dox alone. Mechanistically, Dox cytotoxicity is caused by its intercalation into the nucleic acid structure at the preferred paired CG or GC sites with subsequent inhibition of cancer cell proliferation. Taking advantage of its propensity for intercalation, Dox can be noncovalently conjugated to oligonucleotide aptamers containing CG/GC sequences through a simple incubation step. A recent report by Subramanian et al. describes the effectiveness of aptamer-Dox conjugates in the treatment of retinoblastoma.63 In their study, a 2′-fluoro modified RNA aptamer EpDT3 (specific for EpCAM, a CSC marker), was noncovalently conjugated with Dox. After binding to EpCAM molecules expressed at the cancer cell surface, the EpDT3-Dox conjugates were preferentially internalized by the cancer and not by the healthy cells, greatly enhancing therapeutic efficacy and reducing treatment-associated side effects. Several other studies also utilized aptamer-Dox conjugates for cancer therapy, such as HER2 aptamer-Dox conjugates targeting breast cancer,64 MUC1 aptamer-Dox conjugates targeting lung cancer,65 and PSMA aptamer-Dox conjugates targeting prostate cancer.66 Despite their obvious advantages, several concerns related to the use of aptamer-Dox conjugate have been raised. These include (i) instability of the aptamer-drug conjugate due to the reversible nature of noncovalent conjugation process; (ii) short circulating half-life of aptamer-drug conjugates in vivo due to their low molecular weight; and (iii) poor drug payload capacity due to a very simple structure of aptamers. These three disadvantages and technological approaches to improve them are described in greater detail below.

Figure 3.

Schematic diagram of noncovalent or covalent aptamer-drug conjugation.

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To enhance the stability of drug loading, Dox can be covalently conjugated to aptamer sequences via a functional linker moiety. For example, the DNA aptamer sgc8 possesses a strong affinity for PTK7 kinase that is abundantly expressed on the surface of CCRF-CEM T-cell acute lymphoblastic leukemia cells. To enhance its stability, this aptamer was covalently conjugated with Dox through an acid-labile linker.67 Once the sgc8 aptamer-Dox conjugate was preferentially bound and internalized by the target cells, the acid-labile linker was easily cleaved in the acidic lysosomal environment, releasing Dox and effectively killing target cells.67 On the other side of the spectrum, covalent conjugation is the most commonly used method of aptamer-drug conjugation, especially for agents that cannot intercalate into the nucleic acid structure or whose intercalation would disrupt aptamer structure.68 Evidence suggests that these covalently conjugated aptamer-drug compounds are significantly more stable than the corresponding noncovalently conjugated intercalations.69

Conjugation of aptamers with high molecular weight polymers, such as polyethylene glycol (PEG), has been examined in order to increase aptamer molecular weight. Specifically, PEG has been widely used in drug modifications, including synthesis of Macugen aptamers. This modification, resulting in PEGylated aptamers, not only increased the aptamer molecular weight and prolonged its circulating half-life, but also enhanced its stability and decreased its toxic accumulation in nontarget tissues.70,71

Finally, in order to increase aptamer-drug payload capacity, an innovative model named aptamer-tethered DNA nanotrains (aptNTrs) was recently introduced by Zhu et al. to deliver Dox to cancer cells.72 In this study, structure of the sgc8 aptamer that targets PTK7 was modified by adding a DNA trigger probe on the 5′-end. Consequently, the modified aptamer acted as a locomotive for targeting, while two hairpin monomers containing Dox intercalation sites acted as boxcars to deliver the drug. After self-assembly, the newly synthesized sgc8 aptamer-NTrs displayed high drug payload capacity, with the drug/sgc8 aptamer-NTr molar ratio of 50:1. Importantly, sgc8 aptamer-NTrs-Dox conjugates were preferentially internalized by the target cells, thereby inhibiting tumor cell growth in vitro and in vivo.72

Another strategy for increasing the aptamer payload capacity involves the construction of polyvalent aptamers. Polyvalent aptamers exhibit an increased target affinity and are more rapidly internalized by their target cells. To demonstrate this, Boyacioglu et al. developed a new DNA aptamer they termed SZTI01 against PSMA.69 First, a dimeric aptamer complex (DAC) was created for specific delivery of Dox to PSMA-expressing cancer cells. Then, the SZTI01aptamer was modified on the 3′-terminus with either a dA16 or dT16 single-stranded tail that contained CpG sites for loading Dox, and the two monomers were annealed in a 1:1 ratio to form the DAC structure. The results of the study showed that DACs have a high Dox payload capacity with the Dox/DAC molar ratio of about 4:1, and the DACs-Dox conjugates were stable under physiological conditions for up to 8 hours.69 In another study, a DNA aptamer targeting MUC1 was truncated and an aptamer containing three repeats of the active targeting region, termed L3, was synthesized. Although the Dox payload capacity was not specifically modified in the L3 aptamer, the L3-Dox conjugates showed a stronger affinity to target cells and lower cytotoxicity to off-target cells than the parental MUC1 aptamer.73 Finally, polyvalent aptamers can also be constructed through the rolling circle amplification (RCA) technology. Using the RCA method and the sgc8 aptamer sequence as a circular template, a polyvalent sgc8 aptamer, termed Poly-Aptamer-Drug, was synthesized.74 It was determined that the Dox payload capacity of the polyvalent sgc8 aptamer increased tenfold, as compared to the monovalent sgc8 aptamer. Moreover, because of their 40-fold greater binding affinity, the Poly-Aptamer-Drug conjugates were more effective than their monovalent counterparts in targeting and killing leukemia cells.74

Although Dox presents itself as a very attractive chemotherapeutic agent for use in aptamer conjugation, other drugs, such as Gemcitabine (Gem) and photosensitizers, can also be targeted to cancer cells through the aptamer technology. Gem is an FDA-approved deoxycytidine analog (dFdC) used for anticancer therapy. To deliver Gem specifically to pancreatic cancer cells, Ray et al. developed a novel aptamer-Gem polymer model. In this model, a single-stranded RNA polymer contained Gem that was enzymatically synthesized through a mutant T7 RNA polymerase-mediated transcription reaction and fused with a nuclease-resistant 2′-fluoro-modified RNA aptamer (E07) that selectively binds to EGFR on pancreatic cancer cells. The E07 aptamer structure was modified by introducing a 24-nucleotide sequence at the 3′ end and using it as an adaptor for Gem polymer binding. Following an annealing step, the Gem polymer complementary bound with the E07 aptamer and preferentially targeted the EGFR-expressing pancreatic cancer cells, inhibiting cell proliferation.75

Compared with the traditional chemotherapeutic agents, controlled conditional prodrug photosensitizers have also been extensively used for aptamer-mediated drug delivery. In this therapeutic approach, termed photodynamic therapy, or photodynamic therapy, photosensitizers are activated by light irradiation and induce production of intracellular reactive oxygen species, resulting in cytotoxicity. A study by Ferreira et al. describes the development of a DNA aptamer specific for MUC1 and covalently conjugated at the 5′ end with the photosensitizer chlorin e6.76 Upon light irradiation, MUC1-expressing epithelial cancer cells were preferentially killed with cytotoxicity about 500-fold higher than that of the control cells. Similar studies have reported using a necleolin aptamer (AS1411)-TMPyP4 for targeting breast cancer77 and the EGFR aptamer (R13)-TF70 for treatment of lung cancer.78

Finally, approaches to extend the scope of aptamer application have also been developed. Similar to bi-specific antibodies, bi-specific or even tri-specific aptamers can be constructed. A bi-specific aptamer for targeting different cells was recently described by Zhu et al. In their study, specific DNA aptamers sgc8 and sgd5a were conjugated through a dsDNA linker. Compared to each mono-aptamer, this bi-specific aptamer (named SD) could recognize its target cell simultaneously with equal specificity and affinity, while Dox intercalation into the dsDNA induced target cell cytotoxicity.79 In the same study, a Y-shape dsDNA linker was used to construct a tri-specific aptamer that also recognized its target cells with high specificity and affinity.79 Clinically, Min et al. proposed using a bi-specific aptamer for prostate cancer therapy. It is well established that prostate tumors may contain both PSMA-positive and -negative cell types. Thus, this study utilized two aptamers, a 2′-fluoro modified RNA aptamer targeting PSMA-expressing cells and a DUP-1 peptide aptamer specific to PSMA-negative cells, conjugated through streptavidin. Moreover, intercalating Dox into the PSMA aptamer of this bi-specific aptamer model could serve as a tool to target all prostate cancer cell types.80

Aptamer-nanoparticle therapeutics

Nanoparticles (NPs) are attractive vehicles to increase both the half-life and the drug payload capacity of aptamer-mediated drug delivery. In addition to their common features, such as biocompatibility for clinical applications, large surface for enhanced aptamer and drug loading, and uniform size and shape for excellent biodistribution, NPs have other individual physical and chemical properties defined by their materials. For example, copolymers and liposomes are biodegradable, while metal materials offer exceptional photothermal and magnetic performance.

Conclusion

Antibody-based targeted therapeutics provide high target specificity and affinity. However, their potential for immunogenicity is of a great concern, as is their high production cost, both of which have limited their clinical applicability. As discussed in this review, when compared to protein antibodies, oligonucleotide aptamers offer many advantages, including simple chemical synthesis, virtual nonimmunogenicity, smaller size, faster tissue penetration, ease of modification with different functional moieties, low cost of production, and high biological stability. Therefore, aptamers have become a promising new class of molecular ligands that could replace or supplement protein antibodies. In summary, aptamer technology has a strong market value and may be applied in various biomedical fields, including in vitro cancer cell detection, in vivo tumor imaging, and targeted cancer therapy (Figure 7).

Figure 7.

Summary of various aptamer applications.

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Although aptamer technology has a great potential in the biomedical field, several technical challenges remain and must be addressed. These include: (i) how can aptamers be rapidly adapted for specific targets by decreasing false-positive/-negative selection? Primarily dependent on the natural properties of targets of interest, such as proteins versus cells or tissues, the process of aptamer selection is usually time-consuming, and the success rate is sometimes low. To improve the speed and success rate, novel methods for aptamer selection have been recently described. They include bead-based selection, that can select aptamers as rapidly as a single round of selection,27,28 and the SOMAmer, which improves the aptamer production success rate from less than 30% to over 50%.29,30 More recently, a study by Cho et al. devised a Quantitative Parallel Aptamer Selection System (QPASS) method, which integrates microfluidic selection, NGS, and in situ-synthesized aptamer arrays. This approach allows for the simultaneous measurement of affinity and specificity for thousands of candidate aptamers in parallel.116 In addition to QPASS, evolving modifications to the Cell-SELEX approach are beginning to address difficulties with successful removal of the influence stemming from the presence of dead cells, slow enrichment aptamers recognizing targets of interest, and contamination with unwanted aptamer sequences. As described above, utilization of the above-mentioned FACS-mediated SELEX44,45 and hybrid-SELEX23 offers novel approaches that address these technical challenges.

(ii) How can we select cancer-relevant targets for aptamer development and clinical applications? Tumorigenesis is a dynamic process that includes multiple constantly changing factors. Therefore, a one-size-fits-all cancer-specific biomarker is unlikely to ever be identified. Yet, it has been established that certain biomarkers present in healthy tissues are highly expressed in cancer cells. Moreover, certain biomarkers are associated with particular cancer cell types making them to be considered as useful targets for development of targeted cancer therapy. However, while use of cancer cells to identify biomarkers and to develop therapeutic agents is a reasonable approach, cultured cells, especially immortalized cell lines, greatly differ from tumor tissues in vivo. To overcome these limitations and to select more reliable cancer-relevant biomarkers for aptamer development, several innovative SELEX methods have been recently described. Of particular interest are the tissue-based SELEX117 and the in vivo-SELEX,118 which offer target selection under more relevant pathologic conditions. This cell/tissue-specific biomarker selection can also be utilized for development of noncancer related therapies, as shown for aptamers targeting the adipose tissue in obesity119 and for aptamers designed to penetrate the blood-brain barrier in order to combat brain diseases.120 Hence, we believe that the careful selection of cancer-associated biomarkers and cell/tissue type-specific biomarkers will expand the scopes of aptamer applicability and improve the feasibility of clinical applications.

(iii) What methods could improve aptamer biostability in vivo? Unmodified RNA-based aptamers are very susceptible to the nuclease-mediated degradation in vivo. Although many chemical modifications aimed at increasing biostability of the RNA aptamers have been developed, including 2′-modifications, 3′-modifications, phosphodiester backbone modifications,19,20 and utilizations of novel nucleic acids (locked nucleic acid and Spiegelmers),16,21,22 their effectiveness is still limited. When it was first described, PEGylation was a very attractive strategy for prolonging aptamer circulation half-life and enhancing their biostability. However, a recent report showed that the in vivo use of PEGylated aptamers induced production of anti-PEG antibodies,121 emphasizing the need for the development of alternative approaches.

(iv) How can aptamer technology be modified to achieve a more effective drug delivery? Many drug delivery systems described in this review are tested in vitroor in animal models. Yet, as with any compound that is translated from the bench to the bedside, aptamer-drug conjugates may behave differently in a human patient than they do in laboratory animals. Therefore, aptamer-drug conjugation remains an important challenge that must be considered. Specifically, various coupling approaches lead to different pharmacokinetics, biodistribution, and tolerability in vivo, which in turn greatly affect treatment effectiveness. In the same vein, we must consider the effectiveness of aptamer-mediated target gene therapy. Gene therapy, including siRNA and miRNA aimed at silencing specific genes, is considered the next generation therapeutic approach. However, silencing a single pathogenic gene may not be a viable therapeutic option because tumorigenesis is a process regulated by multiple genes and signaling pathways. Therefore, combining targeted therapeutics with gene therapy may represent the most effective strategy. Such combinational therapy approaches can greatly improve the therapeutic efficacy while reducing the required dosages of both drugs and small molecule RNAs,122 and, more importantly, may offer new alternatives to combat chemotherapy-resistant cancers.110

(v) The last important point to consider is whether aptamer-mediated biotherapies can become effective, FDA-approved medications. Following Macugen approval by the FDA, many aptamer-mediated biotherapies have been evaluated in clinical trials. Of particular interest is AS1411, an antitumor aptamer that has completed several Phase I clinical trials.15 Trial results are promising and offer useful insights into further modifications that could be applied to therapeutic aptamer development.

Taken together, although some technical challenges remain to be addressed, oligonucleotide aptamers have become an attractive and promising tool for targeted cancer therapy. As more clinical data are accumulated, we and others will be better equipped to optimize aptamer formulations, leading to the expansion of aptamer use in the clinic.

9.1.5 Tumor Suppressors

Intrinsic Disorder in PTEN and its Interactome Confers Structural Plasticity and Functional Versatility
Prerna Malaney, Ravi R Pathak, Bin Xue, VN UverskyVrushank Davé
Scientific Reports 20 June 2013; 3(2035)
http://dx.doi.org:/10.1038/srep02035

IDPs, while structurally poor, are functionally rich by virtue of their flexibility and modularity. However, how mutations in IDPs elicit diseases, remain elusive. Herein, we have identified tumor suppressor PTEN as an intrinsically disordered protein (IDP) and elucidated the molecular principles by which its intrinsically disordered region (IDR) at the carboxyl-terminus (C-tail) executes its functions. Post-translational modifications, conserved eukaryotic linear motifs and molecular recognition features present in the C-tail IDR enhance PTEN’s protein-protein interactions that are required for its myriad cellular functions. PTEN primary and secondary interactomes are also enriched in IDPs, most being cancer related, revealing that PTEN functions emanate from and are nucleated by the C-tail IDR, which form pliable network-hubs. Together, PTEN higher order functional networks operate via multiple IDP-IDP interactions facilitated by its C-tail IDR. Targeting PTEN IDR and its interaction hubs emerges as a new paradigm for treatment of PTEN related pathologies.

The concept of “Intrinsic Disorder” in proteins has rapidly gained attention as the preponderance and functional roles of IDPs are increasingly being identified in eukaryotic proteomes12. Structured proteins adopt energetically stable three-dimensional conformations with minimum free energy. In contrast, IDPs, due to their unique amino acid sequence arrangements, cannot adopt energetically favorable conformations and, thus, lack stable tertiary structure in vitro3. This structural plasticity allows IDPs to operate within numerous functional pathways, conferring multiple regulatory functions456. Indeed, mutations in and dysregulation of IDPs are associated with many diseases including cancer167, signifying that IDPs play vital roles in functional pathways. Evidence suggests that ~80% of proteins participating in processes driving cancer contain IDRs6. For example, tumor suppressor p53 as an IDP, functions via its C-terminal IDR, which simultaneously exists in different conformations, each of which function differently1. Since PTEN is the second most frequently mutated tumor suppressor with versatile functions8, we hypothesized that PTEN may contain IDR(s) that can be exploited for therapeutic targeting in cancers and diseases associated with pathogenic PI3K/Akt/mTOR (Phosphoinositide 3-Kinase/Akt/ mammalian Target of Rapamycin) signaling91011.

PTEN (phosphatase and tensin homolog), a 403 amino acid dual protein/lipid phosphatase converts phosphatidylinositol(3,4,5)-triphosphate (PIP3) to phosphatidylinositol(4,5)-bisphosphate (PIP2), thereby regulating the PI3K/Akt/mTOR pathway involved in oncogenic signaling, cell proliferation, survival and apoptosis12. PTEN, as a protein phosphatase, autodephosphorylates itself13. Deficiency or dysregulation of PTEN drives endometrial, prostate, brain and lung cancers, and causes neurological defects1415. PTEN is activated after membrane association16, providing conformational accessibility to the catalytic phosphatase domain (PD) that converts PIP3 to PIP216(Figure 1a). Because PTEN reduces PIP3 levels and inhibits pathogenic PI3K signaling, therapeutically targeting PTEN to the membrane to enhance its activity is of significance in treating several pathologies including cancer.

Figure 1: PTEN: A newly identified IDP.

PTEN - A newly identified IDP. srep02035-f1

PTEN – A newly identified IDP. srep02035-f1

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(a) Diagrammatic representation of PTEN structure. PTEN, a 403 amino acid protein, comprises of PBM: PIP2 Binding Module (AA 1–13; in green), a phosphatase Domain (AA 14–185; in pink), C2 Domain (AA 190–350; in blue), C-terminal region or Tail (AA 351–400; in orange) and a PDZ binding domain (AA 401–403; in dark blue). The PDZ-binding motif is considered as a part of the C-terminal region. *Figure not to scale. (b) Crystal structure of PTEN. Only the phosphatase (in pink) and C2 domain (in blue) are amenable to crystallization. The first seven residues and the last 50 residues represent unstructured/loosely-folded regions that are yet to be crystallized. These regions represent the N- and C-termini of PTEN, respectively. (Source: RCSB Protein Data Bank). (c) Disorder analysis of PTEN. PONDR-VLXT and PONDR-FIT prediction tools were used to determine the disorder score of PTEN. Any value above 0.5 indicates intrinsic disorder. There are several disordered stretches within the PTEN protein, however, the most prominent of these disordered regions is a 50 amino-acid stretch located at the C-terminus of the PTEN protein. (d) IDPs are enriched in polar (R, Q, S, T, E, K, D, H) and structure breaking (G, P) amino acids and are depleted in hydrophobic (I, L, V, M, A), aromatic (Y, W, F) and cysteine (C) and asparagine (N) residues. The amino acid sequence of PTEN highlights these classes of residues with their relative distribution. (e) Composition profiling for full-length PTEN (in green), its ordered domain (in yellow) and its IDR (in red). The tool used is Composition Profiler (Vacic et al, 2007). As shown in the graph, the disordered region in PTEN is enriched in polar residues (specifically H, T, D, S and E), structure breaking residues (specifically P) and is depleted in all hydrophobic residues, cysteine and all aromatic residues. (f) Histogram representing the percentage of hydrophobic, polar, aromatic, structure breaking, cysteine and asparagines residues in ordered vs. disordered regions. The disordered region has an amino acid composition in line with the definition of IDPs.

PTEN crystal structure revealed that the PD and membrane-binding C2 domains are ordered (Figure 1b); however, the structures of the N-terminus, the CBR3 loop and the 50 amino-acid C-tail remain undetermined17. The C-tail is of particular significance due to its ability to regulate PTEN membrane association, activity, function, stability18192021. Herein, we identify PTEN as an IDP with its C-tail being intrinsically disordered. The PTEN C-tail IDR is heavily phosphorylated by a number of kinases and regulates the majority of PTEN functions, including a large number of PPIs that forms the PTEN primary and secondary interactomes, comprising critical functional protein hubs, most of which are related to cancer. Our analysis provides a mechanistic insight into the functioning of the PTEN C-tail IDR at the systems level, including inter- and intra-molecular interactions that will aid in designing drugs to enhance the lipid phosphatase activity of PTEN for the pharmacotherapy of cancers and pathological conditions driven by hyperactive PI3K-signaling.

PTEN is an IDP

Utilizing two disorder prediction software programs, PONDR-VLXT and PONDR-FIT2223, we have identified PTEN as a bona fide IDP. PTEN has a highly disordered, functionally versatile, C-tail encompassing amino acids 351–403 (Figure 1a and 1c). A PDZ-binding motif (amino acids 401–403) is part of the disordered region. Thus, the PTEN C-tail IDR facilitates interactions with a vast repertoire of PDZ domain-containing proteins (Figs. 1a and 2d). The unique amino acid composition of IDRs dictates their structural plasticity32324. IDRs are enriched in polar and structure-breaking amino acid residues, depleted in hydrophobic and aromatic residues and, rarely, contain Cys and Asn residues12324. The ordered region of PTEN (AA 1–350) has 25% hydrophobic, 43% polar, 9% structure breaking, 13% aromatic and 9% Cys and Asn residues. In contrast, the PTEN C-tail (AA 351–403) is enriched in polar (66%) and structure breaking (11%) residues and is depleted in hydrophobic (11%), aromatic (6%) and Cys and Asn residues (6%), indicating an ideal profile for the IDR (Figs. 1d and 1f ). Further, compositional analysis of PTEN using the Composition Profiler24 reveals that the disordered region in PTEN is enriched in polar residues (specifically H, T, D, S and E) and structure breaking residues (specifically P) but is depleted in all aromatic and hydrophobic residues in addition to cysteine. (Figure 1e), again exhibiting universal characteristics of IDPs. Taken together, we establish the PTEN C-tail as a functional IDR and classify PTEN as a new IDP.

Figure 2: The functional relevance of the PTEN IDR.

The functional relevance of the PTEN IDR. srep02035-f2

The functional relevance of the PTEN IDR. srep02035-f2

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(a) The number of mutations observed in PTEN over its 403 amino-acid stretch is plotted. Fewer mutations are observed in the tail region (in red) possibly indicating the deleterious nature of mutations in the functionally critical C-terminal region. [Source: Sanger Institute Catalogue of Somatic Mutations in Cancer (COSMIC), Human Gene Mutation Database (HGMD)]. (b) Number of mutations in every successive 50 amino-acid stretch of the PTEN protein. The last 50 amino-acid stretch, representing the tail region has at least one-eighth the number of mutations seen in any other 50 amino-acid stretch along PTEN, pointing to its critical function in cell homeostasis. (c) Correlation of mutations with the amino acid composition of PTEN. The ratio of mutations in specific residues in the disordered vs. ordered region are represented in this graph. The residues considered here are those used to define IDRs: hydrophobic, polar, aromatic, structure-breaking, cysteine and asparagine residues. Compared to the other classes of residues, mutations in aromatic residues are much higher in the disordered region when compared to the ordered region. (d) The PTEN primary interactome. Forty proteins interact with known regions of PTEN. There are approximately 340 more proteins that interact with PTEN at sites that are yet to be determined (see Supplementary Table S2). Proteins shown in pink interact with the phosphatase domain, those in blue interact with the C2 domain and those in orange interact with the disordered tail. (Visualization tool: Cytoscape). (e) The PTEN C-tail has a higher propensity for PPIs. Of the 40 mapped proteins, 60% interact with the disordered indicating a strong correlation between degree of disorder and the number of protein interactions. (f) Most proteins within the PTEN interactome are highly disordered. Approximately 80% of PTEN-interacting proteins within the primary interactome are disordered, as indicated in red. The proteins within the interactome that are ordered are indicated in blue.

Low mutability of PTEN IDR suggests critical biological functions

Mutations in PTEN are associated with several types of cancers14. To correlate PTEN mutations to its structure, we analyzed all human PTEN mutations deposited in the COSMIC Database (http://www.sanger.ac.uk/genetics/CGP/cosmic/). The disordered PTEN C-tail IDR shows unusually low mutability (~8-fold less) compared to any other 50 amino-acid stretch of PTEN (Figure 2a and 2b). To confirm our finding of the low mutability of the C-tail region, we also analyzed all human PTEN mutations deposited in the Human Gene Mutation Database (HGMD,http://www.hgmd.cf.ac.uk/ac/index.php)25 (Figure 2a), cBioPortal for Cancer Genomics2627(Supplementary Figure S1) and the Roche Cancer Genome Database28 (Supplementary Figure S1) which was consistent with the COSMIC database mutational data. It is likely that evolutionary pressure maintains a survival advantage and ipso facto abrogates progeny with mutations in highly functional protein sequences293031. Thus, the functionally versatile PTEN C-tail IDR cannot afford mutations, hence showing least number of mutations. It is equally likely that mutations in individual residues within the IDR are well tolerated, as the evolutionary pressure may have shifted to maintaining global biophysical properties and structural malleability of the IDR to safeguard the critical protein function29. In either case, on a global scale, the versatile structural pliability of the PTEN IDR dictates functional diversity and biological activities29. Thus, the slightest functional perturbation in the PTEN IDR due to mutations, either within the IDR or in domains interacting with it, could disrupt cellular homeostasis as seen in cancers and neurodegenerative disorders associated with PTEN mutations. This is supported by our data indicating that PTEN, as an IDP when mutated, causes several cancers14.

Moreover, the PTEN C-tail IDR exhibits preferential mutations in aromatic residues compared to the ordered region (Figure 2c). The ratio of mutations in aromatic residues in the disordered to ordered region is much higher than any other class of residues (structure breaking, hydrophobic, polar, Cys and Asn), likely attributed to the structure-imparting property of aromatic residue32. Specifically, aromatic residues within IDRs engage in stacking interactions, enhancing nucleation between distinct residues at functional protein-protein interaction interfaces32. Thus loss of this critical structural and functional property imparted by aromatic residues is associated with a disease phenotype. In summary, the disordered PTEN C-tail IDR has functionally evolved to contain a combination of peptides that cannot tolerate mutations.

Disorderliness in PTEN primary interactome drives functional networks

Protein-Protein Interactions (PPIs) typically occur between conserved, structurally rigid regions of two or more proteins, particularly ordered proteins that display energetically favorable, highly-folded conformations. Intriguingly, IDPs lack tertiary structure, yet engage in PPIs, albeit with lower affinities but high specificity1. The lack of structure within IDPs enhances their biophysical landscape, conferring them with the ability to attain structural complementarities required for PPIs. Since IDPs do not conform to a stable structure, they are less compact, providing a larger physical interface and energetic adaptability to interact with multiple proteins17. Thus, conditional folding within IDPs is effectively utilized for interaction with a multitude of binding partners, enabling them to shuttle between several signaling cascades as efficient “cogs”, mediating and regulating PPIs4,733343536. Indeed, we discovered that PTEN, being an IDP, interacted with more than 400 proteins (Supplementary Table S1) when a combination of online software, literature search and database mining tools were used. Proteins with known PTEN interaction domains were classified as “mapped” (Figure 2d and Supplementary Table S1), whereas those with uncharacterized/predicted interactions were designated as “unmapped” proteins (Supplementary Table S1). Derivation of PTEN primary interactome from the mapped proteins using Cytoscape (http://www.cytoscape.org/) indicated that PTEN disorderliness is efficiently used for interaction with 40 proteins, most existing in distinct functional pathways (Figure 2d, 2e and Supplementary Table S2).

Interestingly, within the PTEN primary interactome, 60% of interactions occurred within the disordered C-tail region. Furthermore, disorder analysis on the primary interactome revealed that 33 proteins (>82%) were IDPs, of which two-thirds interacted with the C-tail IDR (Figure 2e, 2f andSupplementary Table S3), indicating a high propensity for disorder-disorder (D-D)-type interactions.

In order to study evolutionary conservation of the PTEN C-tail and its interactions across species, several sequence alignments were performed (Figure 3a). Sequence alignment of the entire PTEN protein from different animal species shows a good conservation of the catalytic phosphatase domain between vertebrates and invertebrates with 100% sequence conservation for the dual specificity phosphatase catalytic motif HCKAGKGR8 (Supplementary Figure S2). The C-tail shows good conservation in the vertebrate species, likely indicating the recent emergence of the function of PTEN C-tail region in regulating PTEN activity and enriching its PPI potential, translating to its versatile functions. In order to examine the conservation across species for the PTEN C-tail interacting proteins, a literature search was conducted to identify experimentally verified domains/motifs involved in interaction with the C-tail. The domains involved in these interactions with the C-tail for 13 proteins with relevant literature sources for these interactions are part of Supplementary Figure S3. Subsequent sequence alignments for these thirteen proteins (Supplementary Figure S3) shows good sequence homology for the domains/motifs involved in interaction with the PTEN C-tail. These findings support the concept that the PTEN C-tail has evolved in vertebrates to incorporate features that allow it to interact with these proteins.

Figure 3: Sequence conservation in PTEN and its interacting partners reflects functionality.

Sequence conservation in PTEN and its interacting partners reflects functionality. srep02035-f3

Sequence conservation in PTEN and its interacting partners reflects functionality. srep02035-f3

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(a) Sequence alignment of the PTEN protein for vertebrate and invertebrate animals. Green color indicates sequence similarity while red indicates sequence dissimilar amino acid residues. All comparisons are made with respect to the human PTEN protein. (b) Network analysis for PTEN was performed to assess its potential as a network hub. The network shows multiple secondary interactions within the 40 mapped proteins, indicating their role in multiple signaling cascades mediated via PTEN. The proteins SMAD2/3, AR, PCAF, ANAPC7, B-arrestin 1 and p53 appear to be critical within these signaling cascades and also happen to be intrinsically disordered (Supplementary Table S3), reinforcing the concept of preferential interactions between disordered proteins. (Analysis Tool: Metacore by GeneGo).

Further, to assess whether PTEN acts as a functional hub protein and regulates pathways through its protein-binding partners, we performed functional network analysis using the Analyze Network option from MetaCore (GeneGo Inc, Thomson Reuters, 2011) (Figure 3b). The PTEN primary interactome was used as input with PTEN as the central node. We identified multiple interactions not only between PTEN (node) and SMAD2/3, AR, PCAF, ANAPC3, ANAPC4, Caveolin, β-arrestin 1 and p53 (edges), but also amongst the edge proteins themselves (Figure 3b). Interestingly, all the edge proteins are themselves highly disordered (Supplementary Table S3). Further supporting this finding, our functional enrichment revealed that 13 proteins (one-third) of the PTEN primary interactome were cancer-related and highly disordered (Figure 4a, Supplementary Table S3 and S4).

Figure 4: Derivation and disorder analysis of the PTEN cancer interactome.

Derivation and disorder analysis of the PTEN cancer interactome. srep02035-f4

Derivation and disorder analysis of the PTEN cancer interactome. srep02035-f4

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  • Derivation of the PTEN Cancer Interactome. Functional enrichment of the PTEN primary interactome identified 13 cancer-related proteins which are also intrinsically disordered. Subsequently, the PTEN secondary interactome was derived from the primary PTEN interacting proteins. A subset of the secondary interactome was designated as the PTEN Cancer Interactome and it represents the proteins that interact with the 13 cancer-related proteins of the primary interactome. (b) PTEN Cancer Interactome. PTEN is the primary node that interacts with the 13 cancer-related proteins representing the partial primary interactome. Proteins that interact with each of the 13 cancer-related proteins comprise the secondary interactome. Disordered proteins are represented in red while ordered proteins are shown in blue. Cancer-related proteins in the PTEN primary interactome were identified using IPA (Ingenuity® Systems, ingenuity.com). (c) We identified 40 proteins that are part of the PTEN primary interactome of which 13 are highly disordered (IDP) and identified as potential cancer network hubs based on functional network analysis. We further identify 299 IDPS from the secondary PTEN interactome. A filter for cancer-related proteins revealed that approximately two-thirds of the IDPs that form the secondary interactome (193 out of 299) are involved in oncogenesis, suggesting a high degree of functional enrichment. (Functional network analysis was performed using IPA (Ingenuity® Systems,www.ingenuity.com).Full size image (805 KB)

Pliant PTEN secondary interactome relays function of the primary network

The disorderliness of the PTEN primary interactome prompted us to investigate the possibility that PTEN radiates its function via a malleable network of IDPs that extends beyond the primary interactome. Therefore, we derived the PTEN secondary interactome (Supplementary Table S5) and ascertained the interaction of 13 cancer-related proteins identified in the primary interactome (Figure 4a). The entire PTEN secondary interactome consisted of 299 IDPs, of which 193 IDPs (two-thirds) were associated with the 13 cancer-related proteins, generating a “PTEN-Cancer Interactome” (Figure 4Supplementary Table S5 and S6). Thus, two-third of the IDPs within the PTEN secondary interactome associates with one-third of the cancer related IDPs within the PTEN primary interactome, indicating that cancer-related functions are driven by IDPs in the PTEN interactome and that the flexibility of IDP-IDP interactions modulates diverse functions; dysregulation of which causes cancers.

Functional network analysis of the 193 cancer-related IDPs identified 31 proteins that shared multiple nodes (Figure 5a and Supplementary Table S6). We overlaid this network with the cancer-related IDPs of the primary interactome to predict functionally critical protein hubs (indicated in yellow circles in Figure 5a and b). Our analysis revealed 16 proteins as highly populated hubs, most enriched in disordered regions, again demonstrating that a high degree of structural and functional association between the hubs required IDP-IDP interactions (Figure 5b). The involvement of these hubs in multiple, critical oncogenic signaling pathways make them attractive drug targets in the field of clinical oncology. Our bioinformatic analysis resonates well with observed biological phenomena as seen in the case of MDM2 protein, which is a major PPI hub regulating p53. Interaction of the human androgen receptor (AR) protein and MDM2 influences prostate cell growth and apoptosis37. Mdm2-Daxx interaction activates p53 following DNA damage38, and Daxx binds and inhibits AR function39. Conversely, the breast cancer susceptibility gene 1 (BRCA1) interacts directly with AR and enhances AR target genes, such as p21(WAF1/CIP1), that may result in the increase of androgen-induced cell death in prostate cancer cells40. Further, BRCA1 complexes with Smad3 and is inactivated, leading to early-onset familial breast and ovarian cancer41. Within the same network, MDM2 inhibits the transcriptional activity of SMAD proteins including SMAD342, thereby, emerging as a major player in prostrate, breast and ovarian cancer. Loss of PTEN, on the other hand, results in resistance to apoptosis by activating the MDM2-mediated antiapoptotic mechanism. We also identified proteins like NCL, DAXX and SUMO that play critical roles in mediating cancers as being a part of the PTEN centric cancer interactome (Figure 5b). Interestingly, all of the 16 predicted hubs can be traced back to PTEN (either directly or through other signaling adaptors) reinforcing our analysis (Figure 5c). These findings support the prevailing concept of preferential interaction between disordered regions of two distinct proteins; with PTEN being the common disordered interacting hub, giving functional centrality to PTEN in many critical cellular pathways.

Figure 5: Predicting functionally relevant network hubs in the PTEN cancer interactome.

Predicting functionally relevant network hubs in the PTEN cancer interactome. srep02035-f5

Predicting functionally relevant network hubs in the PTEN cancer interactome. srep02035-f5

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(a) Methodology to identify functional hubs within the PTEN Cancer Interactome. The PTEN Cancer Interactome contains 193 IDPs that are potential hubs. Over-represented IDPs (or IDPs with multiple occurrences) in the PTEN Cancer Interactome would have a greater propensity to function as hubs. Upon sorting for over-represented IDPs the list of 193 proteins is brought down to 31 proteins. In order to assess the possibility of these 31 proteins as functional hubs a network analysis is warranted. (b) We identified 31 potential hubs based on multiple associations from within the 193 cancer-associated IDPs of the PTEN secondary interactome. Regulatory networks derived from these 31 proteins were overlaid with a similar network from the 13 cancer-related proteins. Based on the number of associations within the network, we identify 16 potential functional hubs in the PTEN cancer interactome (indicated in yellow). Regulatory interactions were generated using the Transcriptome Browser tool (Lopez et al, 2008). (c) Functional network analysis of the 16 predicted hubs. In order to assess the functional association of the 16 predicted hubs with PTEN – a network analysis with PTEN as a central node was done. The analysis identifies MDM2 protein, a major regulator of p53, as one of the major PPI hubs in the PTEN cancer interactome. A number of other critical cancer-related proteins, such as AR, SMAD2/3 and PDGFRB that are part of the PTEN primary interactome, feature prominently in the PTEN cancer interactome. We also identified proteins like NCL, DAXX and SUMO that play critical roles in mediating cancers as being a part of the PTEN centric cancer interactome. Interestingly, all of the 16 predicted hubs can be traced back to PTEN (either directly or through other signaling adaptors) reinforcing our analysis. (Functional network analysis was performed using IPA (Ingenuity® Systems, www.ingenuity.com).

To further validate our methodology in using intrinsic disorder and cancer as filters to identify key signaling hubs, we compared our data sets with a previously published cancer signaling data set. We derived 7 common hubs (Supplementary Table S7), which were extended using the expansive human signaling network described previously43444546 to obtain the PTEN associated cancer interactome (Figure 6a). An extensive disease associated network analysis using IPA validated our predictions as all the seven predicted hubs had an extensive cross-talk across multiple cancer disease types (Figure 6b).

Figure 7: Biochemical features modulating PTEN PPIs.

Biochemical features modulating PTEN PPIs. srep02035-f7

Biochemical features modulating PTEN PPIs. srep02035-f7

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(a) A PTEN linked cancer network was derived using seven of the 16 predicted cancer hubs that were common with the human cancer associated gene set. The associated partners of the seven hubs were extracted from the human signaling network (Cui et al, 2007, Awan et al, 2007, Li et al, 2012 and Newman et al, 2013). Red color denotes the potential cancer hubs and blue color are their associated partners. Topological analysis identifies p53 as the most significant network hub in the PTEN linked cancer network (Supplementary Table S7). (b) Disease associated network of PTEN cancer hubs. A functional network was constructed with the seven topologically relevant hubs identified previously using the Core Analysis function from the IPA suite to derive the primary network (denoted as MP). A disease network was constructed using the Path Designer option and disease associated biological functions were overlaid on the primary network. Fx denotes the different functions associated with the members of the networks.

Modulation of PTEN PPIs by linear binding motifs

Recent evidence has shown that IDPs mediate PPIs via short linear amino acid sequences (~20 residues) called Molecular Recognition Elements (MoREs) or Molecular Recognition Features (MoRFs)3547. MoRFs undergo disorder-to-order transitions upon binding and adopt thermodynamically stable well-defined structures47, increasing the propensity of IDPs to interact with a vast repertoire of proteins. MoRFs also display molecular recognition elements that capture the binding partner proteins with high specificity. These partner-dependent conformational differences are critical to imparting versatile binding properties to IDRs35.

Since the PTEN IDR engages in multiple PPIs, we tested the possibility for the existence of MoRFs. The MORFP red algorithm48 revealed that PTEN contains major MoRF sites at amino acids 273–279 (part of the disordered CBR3 loop of the C2 domain), amino acids 339–347 (in close vicinity of the disordered C-tail) and amino acids 395–403 (part of the disordered C-tail) (Figure 7a and Supplementary Figure S4). The primary restriction of MoRFs to the PTEN C-tail IDR or adjacent regions indicates that these MoRFs directly participate in modulating PPI functions (Figure 7a). However, mutational analysis within MoRFs is required to establish their active role in functional PPIs.

Figure 7: Biochemical features modulating PTEN PPIs.

Biochemical features modulating PTEN PPIs. srep02035-f7

Biochemical features modulating PTEN PPIs. srep02035-f7

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(a) MoRFs in the PTEN C-tail IDR. MoRFpred (Disfani et al, 2012), a computational tool, was used to identify MoRF regions within the PTEN protein (Supplementary Figure S4). The MoRFs in the vicinity of the C-tail IDR are highlighted in red. Interestingly, all of the major MoRFs (with a length greater than 5 residues) are observed in the vicinity of disordered regions (either part of the disordered CBR3 loop of the C2 domain or the C-tail IDR) indicating a positive correlation between intrinsic disorder and PPIs. (b) ELMs in PTEN C-tail IDR. Eukaryotic Linear Motifs (or ELMs) are 3–11 amino acid long sequences that mediate PPIs. IDRs are particularly enriched in ELMs (Dinkel et al, 2012). The linear motifs occurring in the disordered segment of PTEN (tail + PDZ domain) have been highlighted. The motifs with a high conservation score (>0.75) are indicated in red. Interestingly, all of the motifs with a high conservation score are restricted to the C-tail IDR. (c) Phosphorylation sites in the C-tail IDR. Phosphorylation of PTEN, particularly on serine and threonine residues in the disordered region, regulates the function and stability of PTEN. Phosphorylation occurs at Ser 362, Thr 366, Ser 370, Ser 380, Thr 382, Thr 383, Ser 385 by various enzymes such as Casein Kinase II, Glycogen synthase kinase 3-B and Polo-like kinase 3. Each of these phosphorylation events helps regulate the availability and stability of the PTEN molecule within the cell.

Protein-protein interactions are also facilitated by very short motifs (3–10 amino acids) called Short Linear Motifs (SLiMs) or Eukaryotic Linear Motifs (ELMs)4950. Because of their short sequences, ELMs arise/disappear by simple point mutations, providing the evolutionary plasticity that the ordered protein domains lack. Thus, ELMs easily adapt to novel interactions in signaling pathways, where rapid assembly/disassembly of multi-protein complexes is a prerequisite. The frequent occurrence of ELMs in a typical proteome indicates their critical cellular functions. Consistent with this notion, a higher density of ELMs are observed in hub proteins and IDPs50. Since ELMs have short sequences, they interact with low-affinity, however, they engage in highly cooperative binding in protein complexes, triggering productive signaling50. Therefore, at increased intracellular local concentrations they competitively bind to mutually overlapping physiological targets of each other as seen with PDZ, SH2 and PTB interaction domains found in cancer-associated proteins and in IDRs4950. As PTEN contains a PDZ-binding motif within the IDR (Figure 1a and c), we probed for the existence and features of ELMs in PTEN using The Eukaryotic Linear Motif Resource (http://elm.eu.org). We identified 34 different classes of ELMs in PTEN that mediate PPIs (Supplementary Figure S5). Interestingly, the four ELMs that are most conserved (conservation score>0.75) occurred within the PTEN C-tail IDR, indicating its high level of functional/biological significance (Figure 7b). ELM functions are further modulated by post-translational modifications, mainly by phosphorylation50. Indeed, the PTEN IDR possesses nine phosphorylation sites5152(Figure 7c).

PTEN phosphorylation modulates intramolecular association and PPI function

Post-translational Modifications (PTMs) in IDPs facilitate PPIs5. Modifying enzymes readily dock on structurally flexible IDRs, making them a hot spot for PTMs475354. Consistent with this notion, regulatory cancer-associated proteins have twice as much disorder and undergo more frequent phosphorylation/dephosphorylation than other cellular proteins as predicted by DISPHOS (a DISorder-enhanced PHOSphorylation prediction software)54, implicating a tight interconnection between protein phosphorylation and disorder. Consistent with the function of PTM in IDRs, clustering of Ser and Thr phosphorylation sites (Figure 7c) in the C-tail IDR regulates PTEN stability, membrane association and activity1920. Phosphorylation in the PEST [proline (P), glutamic acid (E), serine (S) and threonine (T)] domain within the C-tail IDR (amino acids 352 to 399) inhibits degradation of PTEN51. Casein kinase II (CK II), Glycogen synthase kinase 3-beta (GSK3-β) and PLK3 (Polo-like kinase 3) phosphorylate Ser and Thr residues within the IDR, each providing a distinct function51 (Figure 7c). The microtubule-associated serine/threonine (MAST), serine/threonine kinase 11(STK11) or LKB1 and casein kinase I (CKI) kinases have also been implicated in PTEN phosphorylation. STK11/LKB1 modifies T383, while CKI modifies T366, S370 and S38552. Indeed, our DISPHOS prediction for C-tail IDRs supports these experimental observations (Supplementary Figure S6).

Substrate-kinase interactions are typically of the disordered-ordered (D-O) type and are stabilized by hydrogen bonding (Figure 7c), a hallmark of IDRs54. Indeed, computational analysis revealed that large ordered regions comprising the catalytic domains of CKII, GSK3B, PLK3, Rak, and Src kinases interact with the C-tail IDR (Supplementary Table S8), indicating that PTEN engages in D-O type intermolecular interactions with the modifying kinases.

At the intramolecular level, phosphorylation at C-tail residues triggers a conformational change in PTEN, inhibiting its membrane association and, therefore, its lipid phosphatase activity18192155. The phosphorylated C-tail IDR folds onto the PD and C2 domains giving rise to the “closed-closed” conformation of PTEN (Figure 8a) that is incapable of interaction with the membrane1820. The “closed- closed” form of PTEN is enzymatically inactive and cannot convert PIP3 to PIP2. The identification of the exact resides involved in this intramolecular interaction remains an active area of research182056.

Figure 8: Targeting PTEN C-tail IDR.
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Most PTEN functions emanate from the C-tail IDR, including aberrant PPIs that hyper-activate oncogenic pathways. (a) Phosphorylation mediates an intramolecular interaction in the PTEN molecule. Phosphorylation causes a conformational change in PTEN converting it to the enzymatically inactive “closed closed ” form wherein the flexible tail folds onto residues in the C2 and phosphatase domain, thereby making it incapable of interacting with the membrane. Dephosphorylation (by an unknown phosphatase or via auto-dephosphorylation) converts PTEN to the “open-closed” form. Electrostatic interactions, mediated by the PBM, further convert PTEN to the “open-open” form wherein it binds to the membrane and acts as a lipid phosphatase converting PIP3 to PIP2, thereby, abrogating signaling via the PI3K/Akt/mTOR pathways. Subsequent to membrane binding, several E3 ubiquitin ligases polyubiquitinate PTEN marking it for proteasomal degradation. Phosphorylation, by inducing the intramolecular interaction, masks the ubiquitination sites thereby increasing the half-life of the PTEN protein within the cell. Therefore, phosphorylation negatively regulates PTEN function but positively regulates its stability. (b) PTEN IDR engages in PPIs of the disorder:order type (D-O type). As revealed in the present study, this occurs via the use of a MoRF or SLiM region. Therefore, designing a peptidomimetic drug molecule that competes with the PTEN MoRF/SLiM binding to the ordered protein will abrogate PTEN binding, therefore PTEN function. PTEN IDR is highly accessible to multiple kinases that phosphorylate and modulate PTEN function, mainly its inhibition via intra-molecular interactions. PTEN inhibition hyper-activates the PI3K/AKT/mTOR pathway, which increase the oncogenic potential of the cell and drives cancer growth. Therefore, targeting the PTEN C-tail IDR with small molecules that bind and sterically hinder PTEN phosphorylation and/or intra-molecular interactions will be an ideally adjunctive therapy to multiple inhibitor therapy targeting of the PI3/AKT/mTOR pathway.

It was recently shown that the phosphorylation events of PTEN occur in two independent cascades of ordered events, with the S380–S385 cluster being modified prior to the S361–S70 cluster52. Even within the two clusters, the phosphorylation events follow a specific pattern with a distributive kinetic mechanism. Not surprisingly, distributive kinetics is energetically favorable on protein domains that are highly disordered with multiple ensembles of flexible structures52. Thus the dynamic nature of these phosphorylation events is contingent to the inherent flexibility in the PTEN structure driven by intrinsically disordered C-tail crucial for PTEN stability and localization within the cell (Figure 8a).

Targeting intrinsic disorder in PTEN and its interactome

Drug targeting to critical protein regions can mitigate aberrant cellular processes driving oncogenesis57. However, despite numerous clinical trials with molecularly targeted therapies, failure rates for cancer treatments remain high. Conventional therapies targeting pathway-specific kinases suffer from “off-target effects” and often fail due to the emergence of compensatory and alternative pathways58. As a novel approach, facile drug targeting to IDRs within critical signaling hub proteins is highly plausible596061. Moreover, as IDRs undergo extensive PTMs53 and engage in PPIs43436, the multitude of resulting protein interactions (normal and aberrant) can be targeted concomitantly with a cocktail of distinct inhibitors, which dampens oncogenic signaling60.

Indeed, targeting PPIs is a more selective treatment strategy over conventional enzyme inhibitors60. However, disruption of multiple ordered interfaces within PPIs by small molecule inhibitors remains challenging62. The advantage of targeting IDPs engaged in PPIs is that, unlike ordered proteins, they engage in PPIs via MoRFs or ELMs, which are small peptide regions that bind with low affinity and thus are susceptible to disruption by small molecule inhibitors59. Consistent with this notion, small molecules disrupted highly disordered complexes of p53-Mdm2 and c-Myc-Max interactions by inducing order upon binding6063. Likewise, targeting the PTEN C-tail IDR may reduce its intra- and inter-molecular interactions and limit accessibility to enzymes mediating PTMs (Figure 8b), providing a means to increase PTEN activity. Our analysis shows that since the C-tail IDR is rich in conserved MoRFs/SLiMS, targeting these regions will prove to be a rational therapeutic modality for a large number of cancers that show compromised PTEN activity or hyperactivation of the oncogenic PI3K/AKT/mTOR pathway91011. Since reductions in the levels and activity of PTEN are sufficient to drive oncogenesis111415, increasing PTEN activity is an ideal therapy for cancers associated with hyperactive PI3K-signaling.

Discussion

Recent studies on genome- and proteome-wide molecular alterations in diseases indicate that pathological conditions are caused by perturbations in complex, highly interconnected biological networks64. Thus, current reductionist approach of studying structure-function relationship in diseases has limited our abilities to discover effective targeted therapeutics. In an attempt to overcome these limitations, in the current study, we have undertaken a novel approach to drug discovery that exploits systems and network biology at the structural, topological and functional level. Using PTEN, a tumor suppressor, we have applied computational and systems biology approaches and integrated extensive data-mining and biochemical properties of IDP interactions to reach a finer understanding of PTEN function. These results have identified PTEN C-tail IDR and several hub proteins in PTEN-driven molecular network implicated in human diseases as therapeutic targets, enhancing the repertoire of clinically relevant biological targets for pharmacotherapy.

Our derivation and analysis of PTEN primary and secondary interactome indicates that altered levels or interactions of IDPs perturb myriad cellular signaling pathways, leading to pathological conditions including cancer. IDPs have the propensity to aggregate and cause cellular toxicity65. Therefore, PTEN as an IDP has evolved a mechanism, wherein, the level of active PTEN, its cellular localization and PTEN-PPIs are regulated via phosphorylation of the C-tail IDR. Furthermore, evolutionarily conserved ELMs and MoRFs that we have identified within the C-tail IDR may play a critical role in orchestrating the formation and function of the PTEN interactome.

Increase in complexity of PPIs is either directed by the number and type of proteins or by increasing the number of interactions required to execute cellular functions66. To delineate how PTEN executes myriad functions, we first derived the PTEN primary interactome. We found 40 proteins to directly interact on the PTEN molecule, out of which 25 were associated with the C-tail IDR, consistent with the concept that disorderliness within PTEN executes its myriad functions. To enhance our understanding of PTEN functions in the context of multiple distinct pathways at the systems-level, we delineated functional networks operating within the primary interactome. Our findings showed a high degree of cross-talk between edges, implying that shared regulatory modules, comprised of multiple signaling cascades, operate via PTEN-mediated interaction networks. When these networks are altered, diseases ensue with extreme functional penalties. We also found that the edge proteins were themselves highly disordered indicating that disorderliness within the PTEN primary interactome confers functional versatility. Supporting this notion, 13 proteins that were functionally classified as cancer-related were also highly disordered forming a pliable “PTEN-Cancer Interactome”. Thus, PTEN lesions influence the flexibility of IDP-IDP interactions modulating diverse functions, likely causing cancer.

Owing to the inherent ability of PPIs to be flexible while being complex, specific cellular functions are readily fine-tuned as per the biological demands. Emerging evidence suggests that certain features on the IDRs are recognized as a way of conferring plasticity to protein interaction networks. Consistent with this concept, our data suggest that PTEN, a hub protein containing an IDR, likely utilizes MoRFs and ELMs, gets differentially modified via PTMs, acquiring complementary structures to engage and modulate PPI activity by facilitating adaptive binding to multiple protein partners in many cellular pathways. Thus, our present work provide a novel entrée in targeting intrinsic disorder in PTEN and its interactome to dampen the aberrant PI3K-signaling that drives many cancers. First, imparting order to the PTEN structure may help dampen multiple oncogenic signaling pathways mediated via the 16 hub proteins identified in the present study, by limiting their affinity for PPIs. Second, targeting intrinsic disorder in PTEN and its interactome can become an adjunctive or alternative approach to the use of various kinase inhibitors, which are toxic and have many off-target effects when used to mitigate the aberrant hyperactivation of PI3K/AKT/mTOR oncogenic signaling pathway. Taken together, the present findings provide a novel entrée to design strategies for drug discovery and may become a logical intervention in the pharmacotherapy of cancer and other PTEN-associated disease treatment modalities.

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Quantum dots

Writer and Curator: Larry H. Bernstein, MD, FCAP

7.1  Quantum dots

7.1.1 Bioconjugated quantum dots for cancer research: present status, prospects and remaining issues.

7.1.2 Bioconjugated quantum dots for in vivo molecular and cellular imaging

7.1.3 In vivo molecular and cellular imaging with quantum dots

7.1.4 Luminescent quantum dots for multiplexed biological detection and imaging

7.1.5 Multifunctional quantum dots

7.1.6 Potentials and pitfalls of fluorescent quantum dots for biological imaging

7.1.1 Bioconjugated quantum dots for cancer research: present status, prospects and remaining issues.

Biju VMundayoor SOmkumar RVAnas AIshikawa M.
Biotechnol Adv. 2010 Mar-Apr;28(2):199-213
http://dx.doi.org:/10.1016/j.biotechadv.2009.11.007

Semiconductor quantum dots (QDs) are nanoparticles in which charge carriers are three dimensionally confined or quantum confined. The quantum confinement provides size-tunable absorption bands and emission color to QDs. Also, the photoluminescence (PL) of QDs is exceptionally bright and stable, making them potential candidates for biomedical imaging and therapeutic interventions. Although fluorescence imaging and photodynamic therapy (PDT) of cancer have many advantages over imaging using ionizing radiations and chemo and radiation therapies, advancement of PDT is limited due to the poor availability of photostable and NIR fluorophores and photosensitizing (PS) drugs. With the introduction of biocompatible and NIR QDs, fluorescence imaging and PDT of cancer have received new dimensions and drive. In this review, we summarize the prospects of QDs for imaging and PDT of cancer. Specifically, synthesis of visible and NIR QDs, targeting cancer cells with QDs, in vitro and in vivo cancer imaging, multimodality, preparation of QD-PS conjugates and their energy transfer, photosensitized production of reactive oxygen intermediates (ROI), and the prospects and remaining issues in the advancement of QD probes for imaging and PDT of cancer are summarized.

Fluorescence imaging and photodynamic therapy (PDT) are advancing clinical trials for efficient detection and curing of cancers. Fluorescence imaging of cancer is facilitated by targeting tumor milieus using fluorescent dyes conjugated with anticancer antibodies followed by exciting the dyes with visible or NIR light sources.In PDT, cancers are treated by applying a photosensitizing (PS) drug followed by light.The principle underlying PDT is that a photoactivated PS drug transfers energy or electron to oxygen or other molecules, and creates reactive oxygen intermediates (ROI), which immediately react with and damage vital biomolecules in cell organelles resulting in cell death. The main advantage of fluorescence imaging over other biomedical imaging techniques such as X-rays, CT and PET is that visible and NIR excitation in fluorescence imaging is non-ionizing and less hazardous. The main advantage of PDT over chemotherapy and radiation therapy is that site-specific photoactivation of targeted PS drugs using visible or NIR light offers selective therapy, leaving the immune system and normal cells intact. However, fluorescence imaging and PDT of cancer are challenging due to the limited availability of photostable and NIR dyes as PS drugs. The center of fluorescence imaging and PDT of cancer is the selective delivery of fluorescent dyes and PS drugs in tumor milieu.The basic principle underlying PDT is that photoactivation of a PS drug results in the formation of ROI such as singlet oxygen (1O2), hydroxyl radical (UOH), superoxide anion(−∙O2) and hydrogen peroxide (H2O2) through a series of energy and electron transfer reactions initiated between PS and dissolved oxygen (3O2) [(Ochsner, 1997) and (Oleinick and Evans, 1998)].

Fig. 1 shows various photophysical and photochemical processes involved in PDT. Briefly, photoactivation of a PS drug places it at the excited singlet (S1) and triplet (T1) states.The lifetime of the T1 states for most PS drugs ranges from several hundred nanoseconds to milliseconds, much longer than the S1 lifetime. A PS drug in the T1 state either relaxesto the groundstate (S0) by transferring excess energy to molecular oxygen or transfers an electron (also, at S1 state) to oxygen, water or a proximal molecule and enters into a series of photochemical reactions [(Ochsner, 1997) and (Oleinick and Evans, 1998)]. By the energy transfer from a PS to 3O2, an electron in the πx */πy * orbital in 3O2 changes its spin quantum number and forms 1O2, for which the energy required is only 94.3 kJ/ mol. 1O2 is an unstable species and it reacts with water, generating a sequence of ROI such as UOH, −∙O2 and H2O2. On the other hand, electron transfer from a PS drug directly produces ROI. However, electron transfer creates the cation radical of a PS, which irreversibly reactswithothermoleculeandresultsinthechemicaltransformation of PS (Lachheb et al., 2002). On the other hand, photosensitized production of ROI through energy transfer is a renewable process. Thus, energy transfer is preferred over electron transfer for the durability of PS drugs. In both the mechanisms, cell death is initiated by the photochemical reactions of ROI with biomolecules and cell organelles such as amino acids, endoplasmic reticulum, mitochondrion, lysosomes and Golgi apparatus. Examples of standard PS drugs for PDT are porphyrins, phthalocyanines, and chlorine derivatives. In the earlier days, a mixture of porphyrins, called the first generation PS drugs was used for PDT. For example, Dougherty etal. (1975) successfully cured breast cancer in a mouse model by applying hematoporphyrin derivatives as the PS drug. Later, with the introduction of purified PS drugs, also called the second generation PS drugs, such as porphyrins, phthalocyanines and chlorine derivatives, research on PDT has infiltrated into clinical trials. For example, superficial bladder cancer was treated by non-specific administration of photofrin as the PS drug followed by illuminating the bladder with red light (Nseyoetal.,1998). However,this approach suffered from severe side effects due to non-specific drug delivery and photoactivation. Recently,with the advancements such as synthesis of new generation PS drugs, targeted drug delivery, image-guided PDT, and introduction of tunable and fiber-optic laser light sources, imaging and PDT of cancer have become more popular methods for skin cancers, Barrett’s esophagus, bronchial cancers, head and neck cancer, lung cancer, prostate cancer, and bladder cancer. Recently, metal, semiconductor, polymer and ceramic nanoparticles have gained much attraction in the imaging and PDT of cancer (Brigger et al. 2002). Polymer and ceramic nanoparticles have been widely employed as drug carriers, whereas metal and semiconductor nanoparticles act as probes for imaging and therapy. Among various nanoparticles, semiconductor quantum dots (QDs) attracted much attention as probes for bioimaging [(Chan and Nie, 1998), (Bruchez et al.,1998),(Alivisatosetal.,2005),(Gaoetal.,2005),(Paraketal.,2005), (Medintz et al., 2005), (Michalet et al., 2005), (Klostranec and Chan, 2006), (Bijuetal.,2007a), (Hoshinoetal.,2007), (Jamiesonetal.,2007), (Hild et al., 2008), (Biju et al., 2008), (Smith et al., 2008), (Anas et al., 2009), (Delehanty et al., 2009), and (Walling et al., 2009)] and PDT [(Samiaetal.,2003), (Lovricetal.,2005), (Shietal.,2006), (Hsiehetal., 2006), (Tsayetal.,2007), (Bagalkotetal.,2007),(Anasetal.,2008), (Ma etal.,2008), (Juzenasetal.,2008a), (Wallingetal.,2009), and (Yaghini et al., 2009)]. QDs are nanoparticles in which electrons and holes are three dimensionally confined within the exciton Bohr radius of the material,providinguniqueopticalproperties,suchasbroadabsorption and sharp emission bands and size-tunable photoluminescence color [(Brus,1984),(Murrayetal.,1993),(Alivisatos,1996),(Dabbousietal., 1997) and (Biju et al., 2008)]. Also, bright emission, exceptional photostability, large-surface area, large two-photon absorption crosssection, availability in multicolor and with NIR photoluminescence are the most attractive properties of QDs for imaging and PDT of cancer.

Fig. 1. Photophysical and photochemical processes involved in PDT.

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Surface functionalization of quantum dots
High quality core-only and core/shell QDs with absorption and photoluminescence in the visible and NIR regions can be prepared by the methods described above. However, surface of such QDs is covered by hydrophobic molecules such as TOPO, TOP and TBP. On the other hand, QDs with hydrophilic surface-molecules and reactive functional groups are necessary for biological applications. Thus, conversion of hydrophobic-capped core and core/shell QDs from organic phase into an aqueous phase was extensively investigated. The conversion was carried out by coating or conjugating hydrophilic and amphiphilic molecules such as mercapto acids, hydrophilic dendrimers, silica-shells, amphiphilic polymers, proteins, and sugars on the surface of core and core/shell QDs. These methods are gracefully summarized by Medintz et al. (2005). For example, Chan and Nie (1998) successfully converted CdSe QDs from an organic to aqueous phase by exchanging hydrophobic molecules on the surface of QDs with mercaptoacetic acid. By a similar approach, Uyeda et al. (2005) tethered bidentate dihydrolipoic acid (DHLA) on the surface of CdSe/ZnS QDs and prepared water-soluble QDs. Now, surface modification of QDs using DHLA has become a popular method. The formation of disulfide bond with ZnS shell is the key in these preparations. Conjugation of biomolecules on the surface of QDs dispersed in water is another important requirement for biological applications. For this purpose, antibodies, nucleic acids, peptides, etc. can be attached either covalently or non-covalently on the surface of QDs. In particular, conjugation of anticancer antibodies, peptides and PS drugs on the surface of QDs is required for imaging and PDT of cancer. QDs bearing surface functional groups such as carboxylic acids, primary amine and thiol can be conjugated with antibodies and peptides by exploiting cross-linking chemistry of carbodiimide, maleimide and succinimide. Also, avidin–biotin cross-linking is one of the most popular methods for conjugating biomolecules on the surface of QDs. These methods are summarized in Fig. 2.

Fig. 2. Schematic presentation of steps involved in the bioconjugation of QDs.

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Absorption and photoluminescence properties of quantum dots

Broad absorption bands, sharp and symmetrical photoluminescence bands, large two-photon absorption cross-section, size-tunable absorption and photoluminescence spectra, and exceptional photostability are the optical properties of QDs attractive for biological applications. These properties, in particular, the size-tunable absorption and photoluminescence spectra of QDs originate from the large surface to volume ratios and the quantum confinement effect [(Brus, 1984)]. Due to the broad absorption band and the large two-photon absorption cross-section, QDs can be photoactivated using one- or multi-photon excitation. Also, the sharp and size-tunable photoluminescence of QDs is beneficial for multiplexed bioimaging. The absorption spectra of semiconductor QDs are broad due to a combined effect of a distribution of electronic transitions in the bulk semiconductoranddiscreteelectronictransitionssuchass–s,p–pand d–d transitionsdueto the quantumconfinement effect. However,the sharp photoluminescence bands of QDs are contributed by carrier recombination in the band-edge states. The band-edge states are quantum confined or size-dependent, and are 8-fold degenerate in CdSe QDs due to asymmetric and crystal-filed splitting, and mixing of carrier exchange perturbations with angular momentum of the charge carriers [(Norris and Bawendi, 1995), (Nirmal et al., 1995), (Efros et al.,1996) and (Nirmal and Brus,1999)]. Thus, for example, in the case of CdSe QDs the photoluminescence color shifts from near visible to NIR region with an increase in the size of QDs. In CdSeQDs, the highest occupied states are contributed by the 4p orbitals of selenium and the lowest unoccupied states are contributed by the 5s orbitals of cadmium. Similar to the size-dependent absorption and photoluminescence spectra for a given QD, the absorption and photoluminescence spectra can be tuned from UV to NIR regions by varying the core material.Forexample,2.5 nmdiameterCdS,CdSe,InP,CdTe,PbS,PbSe and PbTe QDs show near visible to NIR band-edge absorption and photoluminescence.Thus,QDs with suitable absorption spectrum and photoluminescence color for bioimaging and PDT can be easily selected based on either the core size or the core material. The merits of the broad absorption and sharp photoluminescence bands of QDs for cancer imaging and PDT are many. For example, QDs can be photoactivated at any wavelength below the band-edge absorption.
Fig. 3. (A) Schematic presentation of an immunoliposome internalized with doxorubicin and conjugated with QDs and anti-Her2 antibody. (B) Fluorescence images of human pancreatic cancer cells incubated with (a) InP QD-anti-Claudin-4 antibody conjugate and (b) InP QD without antibody. Reprinted with permission from (A) Weng et al. (2008) and (B) Yong et al. (2009). Copyright (2008, 2009) American Chemical Society.

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Targeted imaging of cancer cells using quantum dot-ligand conjugates

Anticancer antibodies are specific but expensive agents for targeting certain over-expressed receptors in cancer cells. Thus, alternative bioconjugates of QDs for targeted imaging of cancer cells were investigated by many researchers. For example, biomolecules such as arginine–glycine–aspartic acid (RGD peptide), folic acid, epidermal growth factor, transferrin and a few aptamers were investigated for targeting particular cancer cells. Like in the case of antibodies, these biomolecules target specific receptors over-expressed in cancer cells. For example, Cai et al. (2006) targeted MDA-MB-435 human breast cancer cells and U87MG human glioblastoma cells using QD conjugated with RGD peptide. The advantage of QD-RGD peptide conjugate is that the peptide selectively labels over-expressed αvβ3 integrin in the above cell lines. They also found that RGD peptide effectively distinguishes MCF-7 human breast cancer cells, in which αvβ3 integrin is not upregulated, from other cancer cells such as MDA-MB-435 and U87MG cells. Bharali et al. (2005) successfully labeled human nasopharyngeal epidermal carcinoma cells (KB cells) using InPQDs conjugated with folic acid. The advantages of InPQD-folic acid conjugate are twofold: InPQD is less toxic than QDs derived from heavy metals such as Cd, Pb, and Hg, and folic acid selectively recognizes over-expressed folate receptor in KB cells.Onthe other hand, human lung carcinoma cells (A549), in which folate receptor is not up-regulated, were not labeled by QD-folic acid conjugates.Bagalkotetal.(2007)foundthatQDslabeledwithaptamers were selectively delivered in prostate cancer cells. They labeled PSMA positive LNCaP prostate cells using QDs conjugated with an A10 RNA aptamer, but not PSMA-negative PC3 prostate adenocarcinoma cells. The QD-aptamer conjugate was found to be equally efficient as QDPSMA antibody conjugate for selectively labeling and imaging prostate cancer cells. Thus, the aptamer-based targeting is cost effective than antibody-based targeting. Like antibodies, ligands for membrane receptors are ideal candidates for targeting cancer cells. For example, Lidke et al. (2004) and Kawashima et al. (2010) found that CHO and A431 cells were efficiently labeled by QD-epidermal growth factor(EGF) conjugates due to the specific binding of EGF to EGFR. The advantage of QD-EGF conjugate is that it can be utilized for labeling various cancer cells because EGFR is over-expressed in many cancers. Although the QD-conjugates discussed above efficiently label over-expressed receptors in various cancer cells, the receptors are signaling proteins important for the regular growth and functioning of normal cells as well.

In vivo targeted imaging of cancer using quantum dots

In vivo targeted imaging of cancer cells using quantum dot-antibody conjugates

The basic principles underlying in vitro targeting of cancer cells can be applied in vivo. However, the main challenges for in vivo targeting and imaging of cancers using QDs are biodistribution of QD bioconjugates, penetration depths of excitation light and photoluminescence, tissue autofluorescence, toxicity and pharmacokinetics. Bioconjugated QDs were applied in vivo either systemically for deep cancers or subcutaneously for peripheral cancers. However,compared with local administration, systemic administration needs more attention owing to possible interactions of QD-conjugates with blood components and stimulation of immune response. Although it was found that QDs conjugated with various anticancer antibodies were selectively and uniformly distributed in tumor milieu, little evidence supports that QDs have the ability to extravasate to reach tumor cells in vivo. Indeed, biodistribution of QDs and non-specific uptake in the reticulo endothelial system that includes the liver, spleen and lymphatic system is an important issue remaining in the in vivo applications of QDs. InvivoapplicationofQDswas firsttestedbyAkermanetal.(2002). They injected CdSe/ZnS QDs coated with peptides into the tail vein in mouse, and found that the injected QDs preferentially distribute in endothelial cells in the lung blood vessels. Also, based on ex vivo fluorescence microscopic imaging of tissue sections, they found that the QD-peptide conjugates were preferentially bound to tumors. Subsequently, QDs conjugated with various cancer markers such as PSMA antibody (Gao et al., 2004), RGD peptide (Cai et al., 2006), alpha-fetoprotein (Yu et al., 2007) and anti-Her2 antibody (Weng et al.,2008) were tested in vivo in mouse models.Gao etal.(2004) were the first to apply QD-antibody conjugates in vivo and perform whole animal cancer imaging. They systemically administered QD-PSMA antibody conjugates in mouse bearing subcutaneous human prostate cancer. The QD-antibody conjugate was efficiently and uniformly distributed in prostate tumor due to the specific binding between PSMA antigen in prostate cancer cells and PSMA antibody on QDs (Fig. 4A). By using RGD peptide conjugated NIR QDs, Cai et al. (2006) investigated in vivo targeting and imaging of cancers. They targeted glioblastoma with NIR QD-RGD peptide conjugate and investigated the selective targeting by in vivo whole animal imaging and ex vivo tumor imaging. As described in the previous section, the key factor underlying in this targeting is the selective binding of RGD peptide to over-expressed αvβ3 integrin in U87MG glioblastoma cells and MDAMB-435 human breast cancer cells. Fig. 4B shows the signal to background ratio for NIR QD-RGD peptide conjugates in the cancer. More recently, Yu et al. (2007) found that QDs conjugated with an antibody to alpha-fetoprotein (anti-AFP) is an ideal candidate for in vivotargetedimagingofHCCLM6humanhepatacarcinomacells.They subcutaneously implanted HCCLM6 cancer cells in mice, and intravenously injected the QD-anti-AFP antibody conjugates. AFP, a main component in mammalian serum, is an important marker protein for liver cancer. Thus, the systemically administered QD-antiAFP conjugate was effectively accumulated in human hepatocarcinoma cells. Weng et al. (2008) developed multifunctional immunoliposomes for in vivo targeted imaging of cancers, drug delivery, and chemotherapy. As discussed in the previous section, they conjugated NIR QDs and anti-Her2 antibody on the surface of a liposome, and encompassed the liposome with doxorubicin, ananticancerdrug. The immunoliposome was applied to MCF-7/Her2 Xenografts implanted
in nude mouse. This multimodal approach of targeted imaging of cancersand drug deliveryhas great potentialsfor imaging and PDT of cancer.
4.2.2. Non-specific imaging of tumor vasculature and lymph nodes using quantum dots Withtheclassicalworkonmulti-photoninvivo fluorescenceimaging using QDs by Larson et al. (2003), targeted and two-photon imaging of tumor vasculature and lymph node using bioconjugated QDs attracted much attention in cancer research. Larson et al. (2003) systemically administeredwater-solubleCdSe/ZnSQDsinlivingmice,andvisualized capillaries in the adipose tissue and skin using NIR excited two-photon fluorescence.Thelargetwo-photonabsorptioncross-sectionofQDsisthe keyforNIRexcitationofvisibleQDs.Soonafterthisreport,non-specificin vivoimagingoftumorvasculature,lymphnodes,andlymphaticdrainage using bioconjugated QDs emerged into active research topics. For example, Stroh et al. (2005) targeted and imaged tumor vasculature associatedwithMCaIVisogenicmouseadenocarcinomatumorimplants in C3H mice using PEG-phosphatidylethanolamine-labeled core/shell CdS/ZnS and CdSe/ZnCdS QDs and two-photon excitation. Kim et al. (2004) applied QDs for in vivo lymph node mapping. They subcutaneously injected oligo-phosphine coated NIR CdTe/CdSe QDs in a mouse and a pig, and found that the QDs were drained within a few minutes after the injection into the sentinel lymph node (SLN) 1cm below the skin. The NIR photoluminescence of QDs enabled them not only to visualize the drainage of QDs towards SLN, but image-guided resection of samples as well. More recently, Ballou et al. (2007) successfully imaged lymph nodes in mice model using QDs without any specific surface functional group.
Fig. 4. (A) Fluorescence image of human prostate cancer implanted in a mouse. The tumor is targeted with anti-PSMA antigen conjugated CdSe/ZnS QDs. Reprinted by permission from Mcmillan publishers Ltd: [Nature Biotechnology], Ref. Gao et al. (2004).(B)Histogramof fluorescencesignalfromU87MGtumor-bearingmiceinjected with an NIR QD-RGD peptide conjugate. Reprinted with permission from Ref. Cai et al. (2006). Copyright (2006) American Chemical Society.

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Quantum dots for multimodal imaging

Magnetic resonance imaging (MRI), radiography, and fluorescence imaging are powerful biomedical imaging modalities. Each imaging modality has its merits and demerits and hence cannot achieve comprehensive imaging. Quality imaging requires high spatial and temporal resolutions, 3D tomography, excellent signal-to-noise ratio, and noninvasiveness. Individual modalities lack one or more of these qualities and therefore, multimodality has been sought as active imaging technology in basic research and biomedical applications. Independent implementation of imaging probes for different modalities cannot be an ideal solution to achieve multimodal imaging because different probes very often differ in their biodistribution and other pharmacodynamic properties. Thus, grouping the properties for different imaging modalities in the same chemical entity has been sought after. Multimodal imaging probes have components that function synergistically, complementing and enhancing the functionality of each other. Notably, QDs are promising multimodal probes as it is possible to combine multiple probe characteristics in QDs. For example, fluorescence imaging using QDs can be combined with MRI and radiography imaging if interfaced with molecules/materials having paramagnetism and radioactivity on the surface of QDs [(Cheon and Lee, 2008) and (Jennings and Long, 2009)]. Examples of bimodal imaging using QD probes are MRI-fluorescence imaging and scintigraphy-fluorescence imaging.The main advantage of QDs for multimodal imaging is the durability of the probe.On the other hand, fluorescence imaging using multimodal probes based on organic dyes such as FITC and rhodamine is less promising due to photobleaching. Typical example for MR-fluorescence bimodal imaging using QDs was investigated by Mulder et al. (2006) using multifunctional CdSe/ ZnSQDprobes.They coated QDs with pegylated phospholipid micelle, a Gd-diethylene triamine pentaacetic acid (DTPA) conjugate as MRI probe, and an RGD peptide for targeting cancer cells. By using this multifunctional probe, they successfully targeted endothelial cells and detected both by fluorescence and MRI imaging. This approach was extended to QD-based bimoda lprobes contained in a silica nanoparticle which is known to improve biocompatibility (Koole et al., 2008). AnotherexampleforQD-basedMR-fluorescencebimodalimagingisthe detection of apoptosis in a culture of Jurkat cells as well as in a murine carotid artery injury model by using QDs conjugated with annexin A5 andaGd-DTPAconjugate(Prinzenetal.,2007).Similarly,bycombining fluorescence and radioactivity in a single nanoprobe, Kobayashi et al. (2007)demonstrated dualmodalinvivolymphatic imaging in mice. In another report, Duconge et al. (2008) successfully demonstrated the utility of CdSe/ZnS QDs encapsulated in Fluorine-18 labeled phospholipids micelle as bimodal imaging probes for combined positron emission tomography (PET) and in vivo fibered confocal fluorescence imaging in mice. In short, as individual imaging technologies are now well-developed, biomedical imaging of cancer should receive a new dimension and momentum with the design and synthesis of suitable multimodal probes based on QDs. This appears achievable in the context of the rapid growth in the field of QDs and the wealth of information on the molecular mechanisms of cancer and other diseases.

Quantum dots for photodynamic therapy of cancer

The quality of a PS drug for PDT depends on its efficiency for energy and or electron transfer to molecular oxygen and the subsequent production of ROI. Compared with electron transfer, energy transfer is desirable for PDT because electron transfer products such as cation and anion radicals undergo irreversible chemical transformations, which prevent subsequent photoactivation of a PS drug and continuous generation of ROI. The concept “QDs for PDT” was proposed and investigated first by Samia et al. (2003). Exceptional photostability of QDs is the most promising property for PDT. Additionally, broad absorption band and large two-photon absorption cross-section of QDs are advantages for photoactivation using various visible and NIR light sources. Despite these advantages, photosensitized production of ROI at high efficiency is the primary requirement for a standard PS drug. Although targeted delivery of QDs in cancer cells and tumor milieu by using anticancer antibodies and other biomolecules have became possible recently, compared with conventional PS drugs such as porphyrins and phthalocyanines, the efficiency of QDs to produce ROI under direct photoactivation is low. Thus, preparation of conjugates between QDs and conventional PS drugs, investigation of energy transfer efficiencies from QDs to PS drugs and ROI production by the conjugates are being widely investigated.

Quantum dots vs conventional PS drugs for PDT

Samia et al. (2003) found that direct photoactivation of QDs produces 1O2 due to energy transfer from the dark exciton state of QDs to 3O2.

Fig. 5. Nude mouse bearing M21 melanoma, dorsal view 3 min after injection into the tumor using 655nmPEG5k-COOH quantum dots. Left, visible light; right, fluorescence at 655nm. Reprinted with permission from Ballouetal. (2007). Copyright (2007) AmericanChemical Society.

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Despite the low efficiency for 1O2, QDs offer prolonged photoactivation and persistent production of 1O2 and other ROI owing to the incredible photostability. Thus, in contrast to conventional PS drugs that are less photostable, QDs offer cumulative effects in PDT. For example,Anasetal.(2008)foundthatprolongedphotoactivationofa QD-plasmid DNA conjugate at 512 nm results in the breakage and damage of DNA. The breakage and damage of DNA were due to the photosensitized production of ROI, which was determined using nitroblue tetrazolium (NBT) chloride as the ROI scavenger. Also, the strand breakage of DNA was characterized by atomic force microscopy imaging and nucleobase damage was characterized by gel electrophoresis and base excision repair enzyme assays. ROI such as hydroxyl radical abstract hydrogen atoms from the bases or pyranose ring and create radical centers in DNA. Subsequent rearrangement of free radicals in DNA results in the strand breakage and nucleobase damage in DNA. Fig. 6 shows the photoactivation of a QD, various relaxation processes in a photoactivated QD, ROI production and subsequent breakage and damage of DNA. The photosensitized strand breakage and nucleobase damage of DNA suggest that QDs are promising PS drugs for nucleus targeted PDT if combined with intranuclear delivery of QDs in cancer cells. Also, Liang et al. (2007) reported that UV illumination of a mixture of calf thymus DNA and CdSe QDs results in DNA nicking, which was attributed to the reactions of DNA with ROI. Similarly, Clarke et al. (2006) reported that photoactivation of QD dopamine complex internalized in A9 cells results in DNA damage due to the production of 1O2. However, the production of 1O2 was due to electron transfer from QD to dopamine followed by the oxidation of dopamine. More recently, the potential of QDs as PS drugs for PDT was investigated by Juzenas et al. (2008b). They found that NIR photoactivation of QDs in cancer cells results in the production of ROI and reactive nitrogen species (RNS) such as superoxide and peroxynitrite. They employed dihydrorhodamine 123 as a sensor for the oxidation, and found that RONS generated by QDs results in the breakage of lysosomes. In contrast to the reports by Samia et al. (2003) and Anas et al. (2008), specific tests made by Juzenas et al. (2008a,b) using 9,10-dimethylanthracene, a 1O2 scavenger, and 1O2 sensorgreenindicatedthat 1O2 wasnotproducedbyQDsunderdirect photoactivation. The properties of QDs such as photostability, photosensitized production of ROI and RNS, and damage and breakage of DNA and lysosomes show the potentials of QDs for PDT. However, cytotoxicity of QDs due to photo-oxidation and chemical degradation should be resolved. For example, Derfus et al. (2004) found that CdSe QDs release toxic levels of cadmium ions inside cells and result in cell death. Similarly, Cho et al. (2007) found that human breast cancer cells MCF-7 treated with cysteine or mercaptoacetic acid capped CdTe QDs results in severe mitochondrial impairment and cell death due to both the release of cadmium ions through surface-etching and the production of superoxide through electron transfer.
Fig. 6. Schematic presentation of ROI production by a QD (center part) and reactions of a DNA molecule with hydroxyl radical and subsequent nucleobase damage and strand breakage (peripheral part). Reprinted with permission from Anas et al. (2008). Copyright (2008) American Chemical Society.

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Quantum dot-PS hybrids as drugs for PDT

There are several advantages and limitations for both conventional PS drugs and QDs when individually applied for PDT. For example, the properties of QDs such as NIR absorption, large two-photon absorption cross-section,broad absorption band and photostability are promising for PDT. In contrast to these unique optical properties of QDs, narrow absorption band, poor photostability, visible light absorption and small two-photon absorption cross-section of conventional PS drugs are less attractive for PDT. However, the efficiency (N75%) for ROI production by PS drugs is superior to that by QDs (∼5%). In other words, the advantages and limitations of QDs and PS drugs complement each other. Thus, in order to utilize the photostability of QDs and improve the production of 1O2, several conjugates/hybrids of QDs and conventional PS drugs were investigated as new generation drugs for PDT.In such hybrid QD-PS systems, the excited singlet (1PS*) and triplet (3PS*) states of PS drugs are indirectly generated by nonradiative energy transfer, also called Förster resonance energy transfer (FRET) from photoactivated QDs (QD*). Due to the indirect photoactivation, photobleaching of PS drugs was minimized. Also, due to the large surface area and biocompatibility of QDs multiple PS drug molecules, which are hydrophobic, can be conjugated to QDs. The indirectly excited PS drugs form collision-complexes (QD-3PS*-3O2) with oxygen, transfer energy to 3O2, and generate 1O2 and other ROI. Fig.7  shows steps involved in the photoactivation of a QD-PS conjugate and the production of ROI. The concept of FRET-based production of 1O2 by QD-PS hybrid systems was first envisaged and demonstrated by Samia et al. (2003) by preparing a non-covalent mixture composed of CdSe QDs and a silicon phthalocyanine (Pc4). They selected Pc4 due to its high 1O2 efficiency (43%) under direct photoactivation. In the QD-Pc4 hybrid system, QD acts as the energy donor to Pc4, and Pc4 acts as both an energy acceptor from QD and an energy donor to 3O2. Thus, high quantum efficiency for 1O2 and stability for the hybrid system were anticipated. However,according to the principle underlyingFRET,the energy transfer efficiencyinversely varies withthe sixth powerof the distance between a donor and an acceptor [(Lakowicz, 1986), (Medintz and Mattoussi, 2009), (Biju et al., 2006), and (Kanemoto et al., 2008)]. Thus, close conjugation, typically within 10 nm, of PS drugs to QDs is necessary for efficient energy transfer and ROI production. Simply, the construction of energy donor–acceptor QD-PS systems should follow the standards described by Medintz and Mattoussi (2009) and in the reference therein. Following the first investigation of QD-PS system by Samia et al. (2003), many researchers were attracted to the energy transfer properties of covalent and non-covalent QD-PS systems composed of CdSe, CdSe/ CdS/ZnS, CdSe/ZnS, and CdTe QDs as energy donors and various chromophores such as porphyrins, phthalocyanines, inorganic complexes and other organic dyes as energy acceptors. Depending on the energy acceptor, the QD-PS systems can be classified into QDphthalocyanines, QD-porphines, QD-organic dyes, and QD-inorganic dyes.

Quantum dot-phthalocyanine conjugates for FRET and single oxygen production

Phthalocynaine-conjugated QDs (QD-Pc) were widely investigated for energy transfer and 1O2 production due to the high triplet quantum efficiency and long-living triplet state for Pc. Burda and coworkers extended investigations of energy transfer and 1O2 production into a large number of QD-Pc conjugates as functions of donor–acceptor distance, relative numbers of QDs and Pcs, terminal functional group in Pc, bulkiness of spacers between donors and acceptors, the mode of binding between QD and Pc, and the size and surface states of QDs [(Dayal et al., 2006), (Samia et al., 2006), (Dayal and Burda, 2007), and (Dayal et al., 2008)]. For example, they employed fluorescence up-conversion and transient absorption measurements, which are valuable methods for characterizing the energy transfer kinetics from various exciton-states in photoactivated QDs, and investigated energy transfer from CdSe QDs to silicon Pc molecules bearing one or two axial functional groups such as thiol, hydroxyl, tertiary alkyl and tertiary amine [(Dayal et al., 2006), and (Dayal et al., 2008)]. Examples of Pc molecules that were used as energy acceptors in QD-Pc systems are shown in Fig. 8. For these molecules, the energy transfer efficiency decreased with increase in both the length and the bulkiness of spacers between QD and Pc [(Dayal et al. (2006), and (Dayal et al., 2008)]. Also, they found that functional groups such as amine and thiol in Pc played important roles on both QD to Pc bonding and quenching of the excited state of QDs.In particular, the energy transfer efficiency was found higher when Pc molecules were linked to QDs through two axial amine or thiol groups. Dayal et al. (2006) detected up to 70% efficiency for energy transferfrom QDsto aprimary amine-terminatedPc. Also,quenching of QD’s photoluminescence was effective for 1:1 and 1:2 conjugates between QD and Pc, but the energy transfer efficiency has decreased with increase in the number of Pc per QD due to self absorbance (Dayaletal.,2006),indicatingthatalargenumberofPSonthesurface of a QD will be less attractive for PDT. One of the reasons for different energy transfer efficiencies for QD-Pc systems linked through bulky or amine/thiol/alkyl functional groups was different electronic coupling between the donor and acceptor.
Fig. 7. Energy transfer processes in a photoactivated QD-PS system, and the production of ROI.

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Another important factor involved in the energy transfer efficiency is the surface states of QDs, which was identified by Dayal et al.(2008) from non-linear relationship between spectral overlap integral and energy transfer efficiency for QD-Pc systems. In short, Burda and coworkers have concluded that 1:1 or 1:2 complexes between QDs and PS molecules bearing two axial amine or thiol functional groups and non-bulky and short spacers would be ideal QD-PS donor–acceptor systems for efficient energy transfer and 1O2 production. Investigations such as preparation of QD-Pc systems, energy transfer from QD to Pc and the generation of 1O2 were further extended into complexes between CdTe QDs and tetrasulfonated aluminum Pc (AlTSPc) systems [(Idowu et al., 2008), (Moeno and Nyokong, 2008), and (Moeno and Nyokong, 2009)]. Here, Nyokong and coworkers prepared CdTe–AlTSPc mixtures by adding solutions of AlTSPc having varying concentrations to solutions of CdTe QDs tethered with mercaptocarboxylic acids such as thioglycolic acid (TDA), 3-mercaptopropionic acid (MPA) and L-lysine (Idowu et al., 2008). In this mixture, the excited state of QDs was quenched and resulted in an increase in the triplet yield for AlTSPc along with fluorescence emission from AlTSPc. Among the CdTe QDs with three different capping ligands stated above, MPA capped CdTe QDs provided long-living triplet state of AlTSPc, which was attributed to the strong binding between AlTSPc and MPA. Later, they found that the CdTe–AlTSPc complex produces 1O2 at 9.5–15% yield that was determined using phosphorescence decay of 1O2 in the presence and absence of sodium azide, a 1O2 scavenger (Moeno and Nyokong, 2008). Recently, they extended energy transfer investigations to various metallophthalocyanines (TSPc) linked to CdTe QDs through sulfonic acid, carboxylic acid, and pyridinium group (Moeno and Nyokong, 2009). By varying the metal ion and the functional groups in Pc, they obtained QD-Pc systems with exceptionally high triplet yields and energy transfer efficiencies (up to 80%). The most important properties of the CdTe-TSPc systems are their water solubility and photosensitized production of 1O2. However, the mode of binding between CdTe QDs and sulfonated Pcs, correlation between the quenching of QD’s excited state and the formation of both the triplet and singlet states of TSPcs, toxicity due to cadmium, and potentials of QD-Pc systems for in vitro and in vivo PDT need further attention.
Quantum dot-porphine conjugates for FRET and singlet oxygen production

Porphines are classical photosensitizers clinically applied for PDT of various cancers due to their high triplet yields and high efficiencies for ROI production. However, as with most phthalocyanines, poor water solubility, inadequate mechanism for selective delivery in tumor milieu and lack of NIR absorption are major drawbacks of porphines for PDT. Recently, Tsay et al. (2007) lifted most of these drawbacks by coating Chlorin e6 on the surface of CdSe/CdS/ZnS QDs either non-covalently using an alkylamine linker or covalently using a lysine-terminated peptide linker (Fig. 9). They found that the photoluminescence lifetime of QDs was decreased as a result of energy transfer from QDs to Chlorin e6. Also, in contrast to the previousreportbyDayaletal.2006),Tsayetal.(2007)foundthatthe energy transfer efficiency from QD to Chlorin e6 has increased with increase in the number of Chlorin e6 molecules attached to a single QD. The QD-Chlorin e6 conjugate provided 1O2 at 31% efficiency. Another example for water-soluble QD-porphine system for 1O2 production is CdTe QDs electrostatically coated by a meso-tetra(4sulfonatophenyl)porphine (TSPP), investigated by Shi et al. (2006). The CdTe-TSPP composite produced 1O2 at 43% efficiency when photoactivated at 355 nm. At this wavelength, both the donor and acceptor were directly photoactivated. Thus, the quantum efficiency for FRET-based 1O2 production was probably overrated. However, based on an assumption that QDs quench the directly-excited triplet stateofanacceptor,Tsayet al.(2007)ruledoutthe productionof 1O2 throughdirectphotoactivationofanacceptorintheproximityofaQD. Also,incontrasttotheproductionof1O2 andotherROIbyCdSeQDsas reportedbySamiaetal.(2003)andAnasetal.(2008), 1O2 production was not detected for CdTe QDs alone, indicating that QD-PS systems are ideal candidates for PDT compared with QDs alone. Despite the above two reports on QD-porphine systems for energy transfer and 1O2 production, systematic investigations of the relations among energy transfer, donor–acceptor distance, size of QDs, dielectric constant of the medium and the efficiency for 1O2 production remain.
Fig. 8. Examples of Pc molecules having different bridging units and terminal functional groups. With kind permission from Springer Science+Business Media; Dayal et al. (2006).

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Quantum dot-organic/inorganic dye systems for FRET and singlet oxygen production

Organic and inorganic dyes having high triplet quantum efficiencies are potential energy acceptors for the construction of QD-PS systems for 1O2 and other ROI production and PDT. Typical example for QD-dye conjugates was investigated by Tsay et al. (2007) by conjugating Rose Bengal on the surface of CdSe/CdS/ZnSQDs through alysine-terminated peptide linker (Fig. 9). As a result of the conjugation of Rose Bengal the photoluminescence lifetime of QD was considerably decreased, indicating efficient FRET from QD to Rose Bengal. Furthermore, they investigated the production of 1O2 by recording the steady-state absorption spectrum of anthracene dipropionic acid, a well-known 1O2 scavenger, and the phosphorescence spectrum of 1O2 at 1270nm. The 1O2 quantum efficiency for QD-Rose Bengal conjugate excited at 355nm was 17%. Here,the production of 1O2 through direct excitation of the acceptor was ruled as stated in the previous section. The low quantum efficiency for 1O2 production was attributed to inefficient energy transfer because of poor donor–acceptor spectral overlap integral.Interestingly,by selecting Chlorine6 as the energy acceptor, they achieved 31% quantum efficiency for 1O2 owing to better overlap between the photoluminescence spectrum of QDs and the absorption spectrum of Chlorin e6. Other examples of organic dyes for the preparation of QD-PS systems are Merocyanine 540 (MC540) and Toluidine Blue O (TBO) [(Narayanan et al., 2008), and (Narband et al., 2008)]. From steady-state and timeresolved fluorescence measurements, Narayanan et al. (2008) detected efficient FRET from CdSe/ZnS QDs to MC540, a chemotherapeutic drug. Here,FRET efficiency was determined from the quenching of the steady state and time-resolved photoluminescence of QDs. Narband et al.(2008) utilized the advantages of QD-PS systems for photodynamic killing of bacteria by applying a mixture of NIR QD and TBO. Photoactivation of QDs resulted in FRET from QD to TBO and the production of 1O2.Here,the high molar extinction coefficient of QDs in the short wavelength region and efficient overlap between the photoluminescence spectrum of QDs and the absorption spectrum of TBO were advantageous for the generation of various cytotoxic species including 1O2. Energy transfer, 1O2 production and bactericidal action for TBO:QD mixtures were discussed in terms of ionic interactions between QD andTBO.
Covalent conjugates and physical mixtures between QDs and inorganic dyes are another class of donor–acceptor systems with potentials for PDT. For example, Hsieh et al. (2006) conjugated iridium complexes with CdSe/ZnS QDs and prepared covalent donor– acceptor systems. Photoactivation of a de-oxygenated solution of the conjugate resulted in a weak phosphorescence emission with a 2.1 μs decay component from the Ir complex, which disappeared when the solution was aerated. Here, the excited state of the Ir complex was generated through FRET from QDs. The disappearance of the phosphorescence during aeration was due to the quenching of the excited state of Ir complex by 3O2 and the formation of 1O2. Although high quantum efficiency (97%) for 1O2 production was estimated for the QD-Ir complex system, the roles of non-radiative relaxations of QDs and the Ir complex, spectral overlap integral, anddonor–acceptor distance are yet to be addressed. Another example for QD-inorganic dye systems was investigated by Neuman et al.(2008) by preparing a physical mixture between CdSe/ZnS QDs and trans-Cr(cyclam)Cl2. Here, the excited state of QD was quenched by the Cr complex, evidenced from a non-linear Stern–Volmer quenching kinetics and a decrease in the photoluminescence lifetime of QDs with increase in the concentration of Cr complex. The spectral overlap integral for the QD-Cr complex was ideal for efficient FRET. Preparation of QD-PS systems such as non-covalent and covalent assemblies between QDs and organic chromophores as well as investigation of energy transfer and 1O2 production are emerging research topics with great potentials for environment and health management. The significance of QD-PS systems compared to conventional PS is that the exceptional photostability of QDs offers durability. Despite the reports discussed above, systematic investigationsofthedonor–acceptordistance,donor–acceptorspectraloverlap integral, donor–acceptor orientation, efficiency of 1O2 production, toxicity of the donor–acceptor systems and in vitro andi n vivo PDT are important issues remaining. Inparticular, QD-PS systems composed of QDs with small size and without heavy metals would bring radical changes to PDT of cancer.

Fig. 9. QD-Chlorin e6 (top) and QD-Rose Bengal (bottom) FRET pairs conjugated using peptide linkers. Reproduced with permission from Tsay et al. (2007). Copyright (2007) American Chemical Society

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7.1.2 Bioconjugated quantum dots for in vivo molecular and cellular imaging

Smith AMDuan HMohs AMNie S.
Adv Drug Deliv Rev. 2008 Aug 17;60(11):1226-40
http://dx.doi.org:/10.1016/j.addr.2008.03.015

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Semiconductor quantum dots (QDs) are tiny light-emitting particles on the nanometer scale, and are emerging as a new class of fluorescent labels for biology and medicine. In comparison with organic dyes and fluorescent proteins, they have unique optical and electronic properties, with size-tunable light emission, superior signal brightness, resistance to photobleaching, and broad absorption spectra for simultaneous excitation of multiple fluorescence colors. QDs also provide a versatile nanoscale scaffold for designing multifunctional nanoparticles with both imaging and therapeutic functions. When linked with targeting ligands such as antibodies, peptides or small molecules, QDs can be used to target tumor biomarkers as well as tumor vasculatures with high affinity and specificity. Here we discuss the synthesis and development of state-of-the-art QD probes and their use for molecular and cellular imaging. We also examine key issues for in vivo imaging and therapy, such as nanoparticle biodistribution, pharmacokinetics, and toxicology.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649798/   FREE PMC Article

The development of biocompatible nanoparticles for molecular imaging and targeted therapy is an area of considerable current interest [19]. The basic rationale is that nanometer-sized particles have functional and structural properties that are not available from either discrete molecules or bulk materials [13]. When conjugated with biomolecular affinity ligands, such as antibodies, peptides or small molecules, these nanoparticles can be used to target malignant tumors with high specificity [1013]. Structurally, nanoparticles also have large surface areas for the attachment of multiple diagnostic (e.g., optical, radioisotopic, or magnetic) and therapeutic (e.g., anticancer) agents. Recent advances have led to the development of biodegradable nanostructures for drug delivery [1418], iron oxide nanocrystals for magnetic resonance imaging (MRI) [19, 20], luminescent quantum dots (QDs) for multiplexed molecular diagnosis and in vivoimaging [2125], as well as nanoscale carriers for siRNA delivery [26, 27].

Due to their novel optical and electronic properties, semiconductor QDs are being intensely studied as a new class of nanoparticle probe for molecular, cellular, and in vivo imaging [1024]. Over the past decade, researchers have generated highly monodispersed QDs encapsulated in stable polymers with versatile surface chemistries. These nanocrystals are brightly fluorescent, enabling their use as imaging probes both in vitroand in vivo. In this article, we discuss recent developments in the synthesis and modification of QD nanocrystals, and their use as imaging probes for living cells and animals. We also discuss the use of QDs as a nanoscale carrier to develop multifunctional nanoparticles for integrated imaging and therapy. In addition, we describe QD biodistribution, pharmacokinetics, toxicology, as well as the challenges and opportunities in developing nanoparticle agents for in vivo imaging and therapy.

QD Chemistry and Probe Development

QDs are nearly spherical semiconductor particles with diameters on the order of 2–10 nanometers, containing roughly 200–10,000 atoms. The semiconducting nature and the size-dependent fluorescence of these nanocrystals have made them very attractive for use in optoelectronic devices, biological detection, and also as fundamental prototypes for the study of colloids and the size-dependent properties of nanomaterials [28]. Bulk semiconductors are characterized by a composition-dependent bandgap energy, which is the minimum energy required to excite an electron to an energy level above its ground state, commonly through the absorption of a photon of energy greater than the bandgap energy. Relaxation of the excited electron back to its ground state may be accompanied by the fluorescent emission of a photon. Small nanocrystals of semiconductors are characterized by a bandgap energy that is dependent on the particle size, allowing the optical characteristics of a QD to be tuned by adjusting its size. Figure 1 shows the optical properties of CdSe QDs at four different sizes (2.2 nm, 2.9 nm, 4.1 nm, and 7.3 nm). In comparison with organic dyes and fluorescent proteins, QDs are about 10–100 times brighter, mainly due to their large absorption cross sections, 100–1000 times more stable against photobleaching, and show narrower and more symmetric emission spectra. In addition, a single light source can be used to excite QDs with different emission wavelengths, which can be tuned from the ultraviolet [29], throughout the visible and near-infrared spectra [3033], and even into the mid-infrared [34]. However QDs are macromolecules that are an order of magnitude larger than organic dyes, which may limit their use in applications in which the size of the fluorescent label must be minimized. Yet, this macromolecular structure allows the QD surface chemistry and biological functionality to be modified independently from its optical properties.

Figure 1

Size-dependent optical properties of cadmium selenide QDs dispersed in chloroform, illustrating quantum confinement and size tunable fluorescence emission. (a) Fluorescence image of four vials of monodisperse QDs with sizes ranging from 2.2 nm to 7.3

2.1. QD Synthesis

QD synthesis was first described in 1982 by Efros and Ekimov [35, 36], who grew nanocrystals and microcrystals of semiconductors in glass matrices. Since this work, a wide variety of synthetic methods have been devised for the preparation of QDs in different media, including aqueous solution, high-temperature organic solvents, and solid substrates [28, 37, 38]. Colloidal suspensions of QDs are commonly synthesized through the introduction of semiconductor precursors under conditions that thermodynamically favor crystal growth, in the presence of semiconductor-binding agents, which function to kinetically control crystal growth and maintain their size within the quantum-confinement size regime.

The size-dependent optical properties of QDs can only be harnessed if the nanoparticles are prepared with narrow size distributions. Major progress toward this goal was made in 1993 by Bawendi and coworkers [39], with the introduction of a synthetic method for monodisperse QDs made from cadmium sulfide (CdS), cadmium selenide (CdSe), or cadmium telluride (CdTe). Following this report, the synthetic chemistry of CdSe QDs quickly advanced, generating brightly fluorescent QDs that can span the visible spectrum. As a result, CdSe has become the most common chemical composition for QD synthesis, especially for biological applications. Many techniques have been implemented to post-synthetically modify QDs for various purposes, such as coating with a protective inorganic shell [40, 41], surface modification to render colloidal stability [42, 43], and direct linkage to biologically active molecules [44, 45]. QD production has now become an elaborate molecular engineering process, best exemplified in the synthesis of polymer-encapsulated (CdSe)ZnS (core)shell QDs. In this method, CdSe cores are prepared in a nonpolar solvent, and a shell of zinc sulfide (ZnS) is grown on their surfaces. The QDs are then transferred to aqueous solution through encapsulation with an amphiphilic polymer, which can then be cross-linked to biomolecules to yield targeted molecular imaging agents.

In the design of a QD imaging probe, the selection of a QD core composition is determined by the desired wavelength of emission. For example, CdSe QDs may be size-tuned to emit in the 450–650 nm range, whereas CdTe can emit in the 500–750 nm range. QDs of this composition are then grown to the appropriate wavelength-dependent size. In a typical synthesis of CdSe, a room-temperature selenium precursor (commonly trioctylphosphine-selenide or tributylphosphine-selenide) is swiftly injected into a hot (~300°C) solution containing both a cadmium precursor (dimethylcadmium or cadmium oleate) and a coordinating ligand (trioctylphosphine oxide or hexadecylamine) under inert conditions (nitrogen or argon atmosphere). The cadmium and selenium precursors react quickly at this high temperature, forming CdSe nanocrystal nuclei. The coordinating ligands bind to metal atoms on the surfaces of the growing nanocrystals, stabilizing them colloidally in solution, and controlling their rate of growth. This injection of a cool solution quickly reduces the temperature of the reaction mixture, causing nucleation to cease. The remaining cadmium and selenium precursors then can grow on the existing nuclei at a slower rate at lower temperature (240–270°C). Once the QDs have reached the desired size and emission wavelength, the reaction mixture may be cooled to room temperature to arrest growth. The resulting QDs are coated in aliphatic coordinating ligands and are highly hydrophobic, allowing them to be purified through liquid-liquid extractions or via precipitation from a polar solvent.

Because QDs have high surface area to volume ratios, a large fraction of the constituent atoms are exposed to the surface, and therefore have atomic or molecular orbitals that are not completely bonded. These “dangling” orbitals serve as defect sites that quench QD fluorescence. For this reason, it is advantageous to grow a shell of another semiconductor with a wider bandgap on the core surface after synthesis to provide electronic insulation. The growth of a shell of ZnS on the surface of CdSe cores has been found to dramatically enhance photoluminescence efficiency [40, 41]. ZnS is also less prone to oxidation than CdSe, increasing the chemical stability of the QDs, and greatly decreasing their rate of oxidative photobleaching [46]. As well, the Zn2+ atoms on the surface of the QD bind more strongly than Cd2+ to most basic ligands, such as alkyl phosphines and alkylamines, increasing the colloidal stability of the nanoparticles [47]. In a typical shell growth of ZnS on CdSe, the purified cores are again mixed with coordinating ligands, and heated to an elevated temperature (140–240°C). Molecular precursors of the shell, usually diethylzinc and hexamethyldisilathiane dissolved in TOP, are then slowly added [40]. The (CdSe)ZnS nanocrystals may then be purified just like the cores.

More recently, it has become possible to widely engineer the fluorescence of QDs by changing the material composition while maintaining the same size. The technological advances that made this possible were the development of alloyed QDs [29, 30] and type-II heterostructures [32]. For example, homogeneously alloying the semiconductors CdTe and CdSe in different ratios allows one to prepare QDs of 5 nm diameter with emission wavelengths of 620 nm for CdSe, 700 nm for CdTe, and 800 nm for the CdSe0.34Te0.67 alloy [30]. Alternatively, type-II QDs allow one to physically separate the charge carriers (the electron and its cationic counterpart, known as the hole) into different regions of a QD by growing an appropriately chosen material on the QD as a shell [32]. For example, both the valence and conduction band energy levels of CdSe are lower in energy than those of CdTe. This means that in a heterostructure composed of CdTe and CdSe domains, electrons will segregate to the CdSe region to the lowest energy of the conduction band, whereas the hole will segregate to the CdTe region, where the valence band is highest in energy. This will effectively decrease the bandgap due to the smaller energy separating the two charge carriers, and emission will occur at a longer wavelength. By using different sizes of the core and different shell thicknesses, one can engineer QDs with the same size but different wavelengths of emission.

Surface Modification

QDs produced in nonpolar solutions using aliphatic coordinating ligands are only soluble in nonpolar organic solvents, making phase transfer an essential and nontrivial step for the QDs to be useful as biological reporters. Alternatively, QD syntheses have been performed directly in aqueous solution, generating QDs ready to use in biological environments [48], but these protocols rarely achieve the level of monodispersity, crystallinity, stability, and fluorescent efficiency as the QDs produced in high-temperature coordinating solvents. Two general strategies have been developed to render hydrophobic QDs soluble in aqueous solution: ligand exchange, and encapsulation by an amphiphilic polymer. For ligand exchange, a suspension of TOPO-coated QDs are mixed with a solution containing an excess of a heterobifunctional ligand, which has one functional group that binds to the QD surface, and another functional group that is hydrophilic. Thereby, hydrophobic TOPO ligands are displaced from the QD through mass action, as the new bifunctional ligand adsorbs to render water solubility. Using this method, (CdSe)ZnS QDs have been coated with mercaptoacetic acid and (3-mercaptopropyl) trimethoxysilane, both of which contain basic thiol groups to bind to the QD surface atoms, yielding QDs displaying carboxylic acids or silane monomers, respectively [44, 45]. These methods generate QDs that are useful for biological assays, but ligand exchange is commonly associated with decreased fluorescence efficiency and a propensity to aggregate and precipitate in biological buffers. More recently it has been shown that these problems can be alleviated by retaining the native coordinating ligands on the surface, and covering the hydrophobic QDs with amphiphilic polymers [10, 23,49]. This encapsulation method yields QDs that can be dispersed in aqueous solution and remain stable for long periods of time due to a protective hydrophobic bilayer surrounding each QD through hydrophobic interactions. No matter what method is used to suspend the QDs in aqueous buffers, they should be purified from residual ligands and excess amphiphiles before use in biological assays, using ultracentrifugation, dialysis, or filtration. Also, when choosing a water solubilization method, it should be noted that many biological and physical properties of the QDs may be affected by the surface coating, and the overall physical dimensions of the QDs are dependent on the coating thickness. Typically the QDs are much larger when coated with amphiphiles, compared to those coated with a monolayer of ligand.

Bioconjugation

Water-soluble QDs may be cross-linked to biomolecules such antibodies, oligonucleotides, or small molecule ligands to render them specific to biological targets. This may be accomplished using standard bioconjugation protocols, such as the coupling of maleimide-activated QDs to the thiols of reduced antibodies [22]. The reactivities of many types of biomolecules have been found to remain after conjugation to nanoparticles surfaces, although possibly at a decreased binding strength. The optimization of surface immobilization of biomolecules is currently an active area of research [50, 51]. The surfaces of QDs may also be modified with bio-inert, hydrophilic molecules such as polyethylene glycol, to eliminate possible nonspecific binding, or to decrease the rate of clearance from the bloodstream following intravenous injection. QDs have also emerged as a new class of sensor, mediated by energy transfer to organic dyes (fluorescence resonance energy transfer, FRET) [5254]. It has also recently been reported that QDs can emit fluorescence without an external source of excitation when conjugated to enzymes that catalyze bioluminescent reactions, due to bioluminescence resonance energy transfer (BRET) [55].

Figure 2 depicts the most commonly used and technologically advanced QD probes. Biologically nonfunctional QDs may be prepared by using a variety of methods. As shown from left to right (top), QDs coated with a monolayer of hydrophilic thiols (e.g. mercaptoacetic acid) are generally stabilized ionically in solution [45]; QDs coated with a cross-linked silica shell can be readily modified with a variety of organic functionalities using well developed silane chemistry [44]; QDs encapsulated in amphiphilic polymers form highly stable, micelle-like structures [23, 49]; and any of these QDs may be modified to contain polyethylene glycol (PEG) to decrease surface charge and increase colloidal stability [56]. Also, water-soluble QDs may be covalently or electrostatically bound to a wide range of biologically active molecules to render specificity to a biological target. As shown in Figure 2 (middle), QDs conjugated to streptavidin may be readily bound to many biotinylated molecules of interest with high affinity [23]; QDs conjugated to antibodies can yield specificity for a variety of antigens, and are often prepared through the reaction between reduced antibody fragments with maleimide-PEG-activated QDs [22, 57]; QDs cross-linked to small molecule ligands, inhibitors, peptides, or aptamers can bind with high specificity to many different cellular receptors and targets [58, 59]; and QDs conjugated to cationic peptides, such as the HIV Tat peptide, can quickly associate with cells and become internalized via endocytosis [60]. Further, QDs have been used to detect the presence of biomolecules using intricate probe designs incorporating energy donors or acceptors. For example, QDs can be adapted to sense the presence of the sugar maltose by conjugating the maltose binding protein to the nanocrystal surface (Figure 2, bottom) [53]. By initially incubating the QDs with an energy-accepting dye that is conjugated to a sugar recognized by the receptor, excitation of the QD (blue) yields little fluorescence, as the energy is nonradiatively transferred (grey) to the dye. Upon addition of maltose, the quencher-sugar conjugate is displaced, restoring fluorescence (green) in a concentration-dependent manner. QDs can also be sensors for specific DNA sequences [52]. By mixing the ssDNA to be detected with (a) an acceptor fluorophores conjugated to a DNA fragment complementary to one end of the target DNA and (b) a biotinylated DNA fragment complementary to the opposite end of the target DNA, these nucleotides hybridize to yield a biotin-DNA-fluorophore conjugate. Upon mixing this conjugate with QDs, QD fluorescence (green) is quenched via nonradiative energy transfer (grey) to the fluorophore conjugate. This dye acceptor then becomes fluorescent (red), specifically and quantitatively indicating the presence of the target DNA. Finally, QDs conjugated to the luciferase enzyme can nonradiatively accept energy from the enzymatic bioluminescent oxidation of luciferins on the QD surface, exciting the QDs without the need for external illumination [55].

nonfunctionalized and bioconjugated QD probes  nihms62165f2

nonfunctionalized and bioconjugated QD probes nihms62165f2

Schematic diagrams of nonfunctionalized and bioconjugated QD probes for imaging and sensing applications. See text for detailed discussion.
Live-Cell Imaging

Researchers have achieved considerable success in using QDs for in vitro bioassays [61, 62], labeling fixed cells [23] and tissue specimens [63, 64], and for imaging membrane proteins on living cells [58, 65]. However, only limited progress has been made in developing QD probes for imaging inside living cells. A major problem is the lack of efficient methods for delivering monodispersed (that is, single) QDs into the cytoplasms of living cells. A common observation is that QDs tend to aggregate inside cells, and are often trapped in endocytotic vesicles such as endosomes and lysosomes.

Imaging and Tracking of Membrane Receptors

QD bioconjugates have been found to be powerful imaging agents for specific recognition and tracking of plasma membrane antigens on living cells. In 2002 Lidke et al. coupled red-light emitting (CdSe)ZnS QDs to epidermal growth factor, a small protein with a specific affinity for the erbB/HER membrane receptor [58]. After addition of these conjugates to cultured human cancer cells, receptor-bound QDs could be identified at the single-molecule level (single QDs may be distinguished from aggregates because the fluorescent intensity from discrete dots is intermittent, or “blinking”). The bright, stable fluorescence emitted from these QDs allowed the continuous observation of protein diffusion on the cellular membrane, and could even be visualized after the proteins were internalized. Dahan et al. similarly reported that QDs conjugated to an antibody fragment specific for glycine receptors on the membranes of living neurons allowed tracking of single receptors [65]. These conjugates showed superior photostability, lateral resolution, and sensitivity relative to organic dyes. These applications have inspired the use QDs for monitoring other plasma membrane proteins such as integrins [50, 66], tyrosine kinases [67, 68], G-protein coupled receptors [69], and membrane lipids associated with apoptosis [70, 71]. As well, detailed procedures for receptor labeling and visualization of receptor dynamics with QDs have recently been published [72, 73], and new techniques to label plasma membrane proteins using versatile molecular biology methods have been developed [74, 75].

Intracellular Delivery of QDs

A variety of techniques have been explored to label cells internally with QDs, using passive uptake, receptor-mediated internalization, chemical transfection, and mechanical delivery. QDs have been loaded passively into cells by exploiting the innate capacity of many cell types to uptake their extracellular space through endocytosis [7678]. It has been found that the efficiency of this process may be dramatically enhanced by coupling the QDs to membrane receptors. This is likely due to the avidity-induced increase in local concentration of QDs at the surface of the cell, as well as an active enhancement caused by receptor-induced internalization [58, 77, 79]. However, these methods lead to sequestration of aggregated QDs in vesicles, showing strong colocalization with membrane dyes. Although these QDs cannot diffuse to specific intracellular targets, this is a simple way to label cells with QDs, and an easy method to fluorescently image the process of endocytosis. Nonspecific endocytosis was also utilized by Parak et al. to fluorescently monitor the motility of cells on a QD-coated substrate [78]. The path traversed by each cell became dark, and the cells increased in fluorescence as they took up more QDs. Chemically-mediated delivery enhances plasma membrane translocation with the use of cationic lipids or peptides, and was originally developed for the intracellular delivery of a wide variety of drugs and biomolecules [60, 8083]. The efficacy of these carriers for the intracellular deliver of QDs is discussed below (Section 3.3 and Section 3.4). Mechanical delivery methods include microinjection of QDs into individual cells, and electroporation of cells in the presence of QDs. Microinjection has been reported to deliver QDs homogeneously into the cytoplasms of cells [49, 83], however this method is of low statistical value, as careful manipulation of single cells prevents the use of large sample sizes. Electroporation makes use of the increased permeability of cellular membranes under pulsed electric fields to deliver QDs, but this method was reported to result in aggregation of QDs in the cytoplasm [83], and generally results in widespread cell death.

Despite the current technical challenges, QDs are garnering interest as intracellular probes due to their intense, stable fluorescence, and recent reports have demonstrated that intracellular targeting is not far off. In 2004, Derfus et al. demonstrated that QDs conjugated to organelle-targeting peptides could specifically stain either cellular mitochondria or nuclei, following microinjection into fibroblast cytoplasms [83]. Similarly, Chen et al. targeted peptide-QD conjugates to cellular nuclei, using electroporation to overcome the plasma membrane barrier [60]. These schemes have resulted in organelle-level resolution of intracellular targets for living cells, yielding fluorescent contrast of vesicles, mitochondria, and nuclei, but not the ability to visualize single molecules. Recently Courty et al. demonstrated the capacity to image individual kinesin motors in HeLa cells using QDs delivered into the cytoplasm via osmotic lysis of pinocytotic vesicles [84]. By incubating the cells in a hypertonic solution containing QDs, water efflux resulted in membrane invagination and pinocytosis, trapping extracellular QDs in endosomal vesicles. Then a brief incubation in hypotonic medium induced intracellular water influx, rupturing the newly formed vesicles, and releasing single QDs into the cytosol. All of the QDs were observed to undergo random Brownian motion in the cytoplasm. However if these QDs were first conjugated to kinesin motor proteins, a significant population of the QDs exhibited directional motion. The velocity of the directed motion and its processivity (average time before cessation of directed motion) were remarkably close to those observed for the motion of these conjugates on purified microtubules in vitro. Although this work managed to overcome the plasma membrane diffusion barrier, it highlighted a different problem fundamental to intracellular imaging of living cells, which is the impossibility of removing probes that have not found their target. In this report, the behavior of the QDs was sufficient to distinguish bound QDs from those that were not bound, but this will not be the case for the majority of other protein targets. Without the ability to wash away unbound probes, which is a crucial step for intracellular labeling of fixed, permeabilized cells, the need for activateable probes that are ‘off’ until they reach their intended target is apparent. However QDs have already found a niche for quantitative monitoring of motor protein transport and for tracking the fate of internalized receptors, allowing the study of downstream signaling pathways in real time with high signal-to-noise and high temporal and spatial resolution [58, 67, 68, 85, 86].

Tat-QD Conjugates

Cell-penetrating peptides are a class of chemical transfectants that have garnered widespread interest due to the high transfection efficiency of their conjugated cargo, versatility of conjugation, and low toxicity. For this reason, cell-penetrating peptides such as polyarginine and Tat have been investigated for their capacity to deliver QDs into living cells [81, 85, 87], but the delivery mechanism and the behavior of intracellular QDs are still a matter of debate. Considerable effort has been devoted to understanding the delivery mechanism of these cationic carrier, especially the HIV-1-derived Tat peptide, which has emerged as a widely used cellular delivery vector [8893]. The delivery process was initially thought to be independent of endocytosis because of its apparent temperature-independence [8993]. However, later research showed that the earlier work failed to exclude the Tat peptide conjugated cargos bound to plasma membranes, and was largely an artifact caused by cellular fixation. More recent studies based on improved experimental methods indicate that Tat peptide-mediated delivery occurs via macropinocytosis [94], a fluid-phase endocytosis process that is initiated by the binding of Tat-QD to the cell surface [90]. These new results, however, did not shed any light on the downstream events or the intracellular behavior of the internalized cargo. This kind of detailed and mechanistic investigation would be possible with QDs, which are sufficiently bright and photostable for extended imaging and tracking of intracellular events. In addition, most previous studies on Tat peptide-mediated delivery are based on the use of small dye molecules and proteins as cargo [8993], so it is not clear whether larger nanoparticles would undergo the same processes of cellular uptake and transport. This understanding is needed for the design and development of imaging and therapeutic nanoparticles for biology and medicine.

Ruan et al. have recently used Tat peptide-conjugated QDs (Tat-QDs) as a model system to examine the cellular uptake and intracellular transport of nanoparticles in live cells [95]. The authors used a spinning-disk confocal microscope for dynamic fluorescence imaging of quantum dots in living cells at 10 frames per second. The results indicate that the peptide-conjugated QDs are internalized by macropinocytosis, in agreement with the recent work of Dowdy and coworkers [90]. It is interesting, however, that the internalized Tat-QDs are tethered to the inner surface of vesicles, and are trapped in intracellular organelles. An important finding is that the QD-loaded vesicles are actively transported by molecular machines (such as dyneins) along microtubule tracks to an asymmetric perinuclear region called the microtubule organizing center (MTOC) [96]. Furthermore, it was found that Tat-QDs strongly bind to cellular membrane structures such as filopodia, and that large QD-containing vesicles are able to pinch off from the tips of filopodia. These results not only provide new insight into the mechanisms of Tat peptide-mediated delivery, but also are important for the development of nanoparticle probes for intracellular targeting and imaging.

QDs with Endosome-Disrupting Coatings

Duan and Nie [97] developed a new class of cell-penetrating quantum dots (QDs) based on the use of multivalent and endosome-disrupting (endosomolytic) surface coatings (Figure 3). Hyperbranched copolymer ligands such as PEG-grafted polyethylenimine (PEI-g-PEG) were found to encapsulate and stabilize luminescent quantum dots in aqueous solution through direct ligand binding to the QD surface. Due to the cationic charges and a “proton sponge effect” [98100] associated with multivalent amine groups, these QDs could penetrate cell membranes and disrupt endosomal organelles in living cells. This mechanism arises from the presence of a large number of weak bases (with buffering capabilities at pH 5–6), which lead to proton absorption in acidic organelles, and an osmotic pressure buildup across the organelle membrane [100]. This osmotic pressure causes swelling and/or rupture of the acidic endosomes and a release of the trapped materials into the cytoplasm. PEI and other polycations are known to be cytotoxic, however the grafted PEG segment was found to significantly reduce the toxicity and improve the overall nanoparticle stability and biocompatibility. In comparison with previous QDs encapsulated with amphiphilic polymers, the cell-penetrating QDs were smaller in size and exceedingly stable in acidic environments [56]. Cellular uptake and imaging studies revealed that these dots were rapidly internalized by endocytosis, and the pathways of the QDs inside the cells showed dependence on the number of PEG grafts of the polymer ligands. While higher PEG content led to QD sequestration in vesicles, the QDs coated by PEI-g-PEG with fewer PEG grafts are able to escape from endosomes and release into the cytoplasm.

Encapsulation and solubilization of core-shell CdSe-CdS-ZnS quantum dots  nihms62165f3

Encapsulation and solubilization of core-shell CdSe-CdS-ZnS quantum dots nihms62165f3

Encapsulation and solubilization of core-shell CdSe/CdS/ZnS quantum dots by using multivalent and hyperbranched copolymer ligands. (a) and (b) Chemical structures of PEI and 19 PEI-g-PEG copolymers consisting of two or four PEG chains per PEI polymer

Lovric et al. [101] recently reported that very small QDs (2.2 nm) coated with small molecule ligands (cysteamine) spontaneously translocated to the nuclei of murine microglial cells following cellular uptake through passive endocytosis. In contrast, larger QDs (5.5 nm) and small QDs bound to albumin remained in the cytosol only. This is fascinating because these QDs could not only escape from endocytotic vesicles, but were also subjected to an unknown type of active machinery that attracted the QDs to the nucleus. Nabiev et al. [102] studied a similar trend of size-dependent QD segregation in human macrophages, and found that small QDs may target nuclear histones and nucleoli after active transport across the nuclear membrane. They found that the size cut-off for this effect was around 3.0 nm. Larger QDs eventually ended up in vesicles in the MTOC region, although some QDs were found to be free in the cytoplasm. This group proposed that the proton sponge effect was also responsible for endosomal escape, as small carboxyl-coated QDs could buffer in the pH 5–7 range. These insights are important for the design and development of nanoparticle agents for intracellular imaging and therapeutic applications.

In Vivo Animal Imaging

Compared to the study of living cells in culture, different challenges arise with the increase in complexity to a multicellular organism, and with the accompanying increase in size. Unlike monolayers of cultured cells and thin tissue sections, tissue thickness becomes a major concern because biological tissue attenuates most signals used for imaging. Optical imaging, especially fluorescence imaging, has been used in living animal models, but it is still limited by the poor transmission of visible light through biological tissue. It has been suggested that there is a near-infrared optical window in most biological tissue that is the key to deep-tissue optical imaging [103]. The rationale is that Rayleigh scattering decreases with increasing wavelength, and that the major chromophores in mammals, hemoglobin and water, have local minima in absorption in this window. Few organic dyes are available that emit brightly in this spectral region, and they suffer from the same photobleaching problems as their visible counterparts, although this has not prevented their successful use as contrast agents for living organisms [104]. One of the greatest advantages of QDs for imaging in living tissue is that their emission wavelengths can be tuned throughout the near-infrared spectrum by adjusting their composition and size, resulting in photostable fluorophores that are stable in biological buffers [24].

Biodistribution of QDs

For most in vivo imaging applications using QDs and other nanoparticle contrast agents, systemic intravenous delivery into the bloodstream will be the main mode of administration. For this reason, the interaction of the nanoparticles with the components of plasma, the specific and nonspecific adsorption to blood cells and the vascular endothelium, and the eventual biodistribution in various organs are of great interest. Immediately upon exposure to blood, QDs may be quickly adsorbed by opsonins, in turn flagging them for phagocytosis. In addition, platelet coagulation may occur, the complement system may be activated, or the immune system can be stimulated or repressed (Figure 4). Although it is important for each of these potential biological effects to be addressed in detail, so far there are no studies that directly examine blood or immune system biocompatibility of QDs in vivo or ex vivo. However, a recent review article by Dobrovolskaia and McNeil addresses the immunological properties of polymeric, liposomal, carbon-based, and magnetic nanoparticles [105]. Considering the many factors that may affect systemically administered QDs, such as size, shape, charge, targeting ligands, etc., the two most important parameters that affect biodistribution are likely size and the propensity for serum protein adsorption.

QD interactions with blood immune cells and plasma proteins  nihms62165f4

QD interactions with blood immune cells and plasma proteins nihms62165f4

Schematic diagram showing QD interactions with blood immune cells and plasma proteins. The probable modes of interactions include (a) QD opsonization and phagocytosis by leucocytes (e.g., monocytes), (b) non-specific QD-cell membrane interactions (electrostatic

The number of papers published on quantum dot pharmacokinetics and biodistribution is limited, but several common trends can be identified. It has been consistently reported that QDs are taken up nonspecifically by the reticuloendothelial system (RES), including the liver and spleen, and the lymphatic system [106108]. These findings are not necessarily intrinsic to QDs, but are strictly predicated upon the size of the QDs and their surface coatings. Ballou and coworkers reported that (CdSe)ZnS QDs were rapidly removed from the bloodstream into organs of the RES, and remained there for at least 4 months with detectable fluorescence [107]. TEM of these tissues revealed that these QDs retained their morphology, suggesting that given the proper coating, QDs are stable in vivo for very long periods of time without degradation into their potentially toxic elemental components. A complimentary work by Fischer, et al. showed that nearly 100% of albumin-coated QDs were removed from circulation and sequestered in the liver within hours after a tail vein injection, much faster than QDs that were not bound to albumin [108]. Within the liver, QDs conjugated to albumin were primarily associated with Kupffer cells (resident macrophages). From a clinical perspective, it may be possible to completely inhibit the accumulation of QDs and avoid potential toxic effects if they are within the size range of renal excretion. Recent publications have focused on this insight. Frangioni and coworkers demonstrated that the renal clearance of quantum dots is closely related to the hydrodynamic diameter of the nanoparticle and the renal filtration threshold (~5–6 nm) [109]. Of equal importance to the QD size, is that the surface does not promote protein adsorption, which could significantly increase QD size above that of the renal threshold, and promote phagocytosis. However, it is unlikely that even small QDs could be entirely eliminated from the kidneys, as it has also been found that small QDs (~9 nm) may directly extravasate out of blood vessels, into interstitial fluid [110].

For targeted imaging, specific modulation of the biodistribution of QD contrast agents is the main goal. One way to increase the probability of bioaffinity ligand-specific distribution is to increase the circulation time of the contrast agent in the bloodstream. QD structure and surface properties have been found to strongly impact the plasma half-life. It was demonstrated by Ballou et al. [107] that the lifetime of anionic, carboxylated QDs in the bloodstream of mice (4.6 minutes half-life) is significantly increased if the QDs are coated with PEG polymer chains (71 minutes half-life). This effect has also been documented for other types of nanoparticles and small molecules, in part due to decreased nonspecific adsorption of the nanoparticles, an increase in size, and decreased antigenicity [111]. In a more recent study using perfused porcine skin in vitro, Lee, et al.demonstrated that carboxylated QDs were extracted more rapidly from circulation, and had greater tissue deposition than PEG coated QDs [112]. It is important to note that a bioaffinity molecule may also be prone to RES uptake, despite a strong affinity for its intended target. For example, Jayagopal et al. reported that QD-antibody conjugates have a significantly longer circulation time if the Fc antibody regions (non-antigen binding domains) are immunologically shielded to reduce nonspecific interactions [113].

In Vivo Vascular Imaging

One of the most immediately successful applications of QDs in vivo has been their use as contrast agents for the two major circulatory systems of mammals, the cardiovascular system and the lymphatic system. In 2003, Larson et. al demonstrated that green-light emitting QDs remained fluorescent and detectable in capillaries of adipose tissue and skin of a living mouse following intravenous injection [114]. This work was aided by the use of near-infrared two-photon excitation for deeper penetration of excitation light, and by the extremely large two-photon cross-sections of QDs, 100–20,000 times that of organic dyes [115]. In other work, Lim et al. used near-infrared QDs to image the coronary vasculature of a rat heart [116], and Smith et al. imaged the blood vessels of chicken embryos with a variety of near-infrared and visible QDs [117]. The later report showed that QDs could be detected with higher sensitivity than traditionally used fluorescein-dextran conjugates, and resulted in a higher uniformity in image contrast across vessel lumena. Jayagopal et al. [113] recently demonstrated the potential for QDs to serve as molecular imaging agents for vascular imaging. Spectrally distinct QDs were conjugated to three different cell adhesion molecules (CAMs), and intravenously injected in a diabetic rat model. Fluorescence angiography of the retinal vasculature revealed CAM-specific increases in fluorescence, and allowed imaging of the inflammation-specific behavior of individual leukocytes, as they freely floated in the vessels, rolled along the endothelium, and underwent leukostasis. The unique spectral properties of QDs allowed the authors to simultaneously image up to four spectrally distinct QD tags.

For imaging of the lymphatic system, the overall size of the probe is an important parameter for determining biodistribution and clearance. For example, Kim et al. [24] intradermally injected ~16–19 nm near-infrared QDs in mice and pigs. QDs translocated to sentinel lymph nodes, likely due to a combination of passive flow in lymphatic vessels, and active migration of dendritic cells that engulfed the nanoparticles. Fluorescence contrast of these nodes could be observed up to 1 cm beneath the skin surface. It was found that if these QDs were formulated to have a smaller overall hydrodynamic size (~9 nm), they could migrate further into the lymphatic system, with up to 5 nodes showing fluorescence [110]. This technique could have great clinical impact due to the quick speed of lymphatic drainage and the ease of identification of lymph nodes, enabling surgeons to fluorescently identify and excise nodes draining from primary metastatic tumors for the staging of cancer. This technique has been used to identify lymph nodes downstream from the lungs [106, 118], esophagus [119], and from subcutaneous tumors [120]. Recently the multiplexing capabilities of QDs have been exploited for mapping lymphatic drainage networks. By injection of QDs of different color at different intradermal locations, these QDs could be fluorescently observed to drain to common nodes [121], or up to 5 different nodes in real time [122]. A current problem is that a major fraction of the QDs remain at the site of injection for an unknown length of time [123].

In Vivo Tracking of QD-Loaded Cells

Cells can also be loaded with QDs in vitro, and then administered to an organism, providing a means to identify the original cells and their progeny within the organism. This was first demonstrated on a small organism scale by microinjecting QDs into the cytoplasms of single frog embryos [49]. As the embryos grew, the cells divided, and each cell that descended from the original labeled cell retained a portion of the fluorescent cytoplasm, which could be fluorescently imaged in real time under continuous illumination. In reports by Hoshino et al. [124] and Voura et al. [82], cells loaded with QDs were injected intravenously into mice, and their distributions in the animals were later determined through tissue dissection, followed by fluorescence imaging. Also Gao et al. loaded human cancer cells with QDs, and injected these cells subcutaneously in an immune-compromised mouse [10]. The cancer cells divided to form a solid tumor, which could be visualized fluorescently through the skin of the mouse. Rosen et al. recently reported that human mesenchymal stem cells loaded with QDs could be implanted into an extracellular matrix patch for use as a regenerative implant for canine hearts with a surgically-induced defect [125]. Eight weeks following implantation, it was found that the QDs remained fluorescent within the cells, and could be used to track the locations and fates of these cells. This group also directly injected QD-labeled stem cells into the canine myocardium, and used the fluorescence signals in cardiac tissue sections to elaborately reconstruct the locations of these cells in the heart. With reports that cells may be labeled with QDs at a high degree of specificity [80, 81], it is foreseeable that multiple types of cells may be simultaneously monitored in living organisms, and also identified using their distinct optical codes.

In Vivo Tumor Imaging

Imaging of tumors presents a unique challenge not only because of the urgent need for sensitive and specific imaging agents of cancer, but also because of the unique biological attributes inherent to cancerous tissue. Blood vessels are abnormally formed during tumorinduced angiogenesis, having erratic architectures and wide endothelial pores. These pores are large enough to allow the extravasation of large macromolecules up to ~400 nm in size, which accumulate in the tumor microenvironment due to a lack of effective lymphatic drainage [126129]. This “enhanced permeability and retention” effect (EPR effect) has inspired the development of a variety of nanotherapeutics and nanoparticulates for the treatment and imaging of cancer (Figure 5). Because cancerous cells are effectively exposed to the constituents of the bloodstream, their surface receptors may also be used as active targets of bioaffinity molecules. In the case of imaging probes, active targeting of cancer antigens (molecular imaging) has become an area of tremendous interest to the field of medicine because of the potential to detect early stage cancers and their metastases. QDs hold great promise for these applications mainly due to their intense fluorescent signals and multiplexing capabilities, which could allow a high degree of sensitivity and selectivity in cancer imaging with multiple antigens.

QDs involved in both active and passive tumor targeting  nihms62165f5

Schematic diagram showing QDs involved in both active and passive tumor targeting. In the passive mode, nanometer-sized particles such as quantum dots accumulate at tumor sites through an enhanced permeability and retention (EPR) effect [126

The first steps toward this goal were undertaken in 2002 by Akerman et al., who conjugated QDs to peptides with affinity for various tumor cells and their vasculatures [130]. After intravenous injection of these probes into tumor-bearing mice, microscopic fluorescence imaging of tissue sections demonstrated that the QDs specifically homed to the tumor vasculature. In 2004 Gao et al. demonstrated that tumor targeting with QDs could generate tumor contrast on the scale of whole-animal imaging [10]. QDs were conjugated to an antibody against the prostate-specific membrane antigen (PSMA), and intravenously injected into mice bearing subcutaneous human prostate cancers. Tumor fluorescence was significantly greater for the actively targeted conjugates compared to nonconjugated QDs, which also accumulated passively though the EPR effect. Using similar methods, Yu et al. were able to actively target and image mouse models of human liver cancer with QDs conjugated to an antibody against alpha-fetoprotein [131], and Cai et al. showed that labeling QDs with RGD peptide significantly increased their uptake in human glioblastoma tumors [132].

The development of clinically relevant QD contrast agents for in vivo imaging is certain to encounter many roadblocks in the near future (see Section 5), however QDs can currently be used as powerful imaging agents for the study of the complex anatomy and pathophysiology of cancer in animal models. Stroh et al.[133] demonstrated that QDs greatly enhance current intravital microscopy techniques for the imaging of tumor microenvironment. The authors used QDs as fluorescent contrast agents for blood vessels using two-photon excitation, and simultaneously captured images of extracellular matrix from autofluorescent collagen, and perivascular cell contrast from fluorescent protein expression. The use of QDs allowed stark contrast between the tumor constituents due to their intense brightness, tunable wavelengths, and reduced propensity to extravasate into the tumor, compared to organic dye conjugates. In this work, the authors also used QD-tagged beads with variable sizes to model the size-dependent distribution of various nanotherapeutics in tumors. As well, the authors demonstrated that bone marrow lineage-negative cells, which are thought to be progenitors for neovascular endothelium, were labeled ex vivo with QDs and imaged in vivo as they flowed and adhered to tumor blood vessels following intravenous administration. More recently, Tada et al. used QDs to study the biological processes involved in active targeting of nanoparticles. The authors used QDs labeled with an antibody against human epidermal growth factor receptor 2 (HER2) to target human breast cancer in a mouse model [134]. Through intravital fluorescence microscopy of the tumor following systemic QD administration, the authors could distinctly observe individual QDs as they circulated in the bloodstream, extravasated into the tumor, diffused in extracellular matrix, bound to their receptors on tumor cells, and then translocated into the perinuclear region of the cells. The combination of sensitive QD probes with powerful techniques like intravital microscopy and in vivo animal imaging could soon lead to major breakthroughs in the current understanding of tumor biology, improve early detection schemes, and guide new therapeutic designs.

Nanoparticle Toxicity

Great concern has been raised over the use of quantum dots in living cells and animals due to their chemical composition of toxic heavy metal atoms (e.g. Cd, Hg, Pb, As, Pb). Presently the most commonly used QDs contain divalent cadmium, a nephrotoxin in its ionic form. Although this element is incorporated into a nanocrystalline core, surrounded by biologically inert zinc sulfide, and encapsulated within a stable polymer, it is still unclear if these toxic ions will impact the use of QDs as clinical contrast agents. It may be of greater concern that QDs, and many other types of nanoparticles, have been found to aggregate, bind nonspecifically to cellular membranes and intracellular proteins, and induce the formation of reactive oxygen species. As previously stated, QDs larger than the renal filtration threshold quickly accumulate in the reticuloendothelial system following intravenous administration. The eventual fate of these nanoparticles is of vital importance, but so far has yet to be elucidated.

Cadmium Toxicity

In the only long-term, quantitative study on QD biodistribution to date, Yang, et al. showed that after intravenous administration of cadmium-based QDs, the concentration of cadmium in the liver and kidneys gradually increased over the course of 28 days, as determined via ICP-MS [135]. The cadmium levels in the kidneys eventually reached nearly 10% of the injected dose, compared to 40% in the liver. Although it was not apparent if the cadmium was in the form of a free ion, or remained in the nanocrystalline form, fluorescence microscopy revealed the presence of intact QDs in both the liver and kidneys. However the redistribution of the cadmium over time may signify the degradation of QDs in vivo, since the natural accumulation sites of Cd2+ ions are the liver and kidneys [79, 136, 137]. In acute exposures, free cadmium also may be redistributed to the kidneys via hepatic production of metallothionein [138]. Whether or not this is the specific mechanism observed in this report should be the focus of detailed in vivo validation studies. Nevertheless, these findings stress that (a) QD size and nonspecific protein interaction should be minimized to allow renal filtration, or else QDs will inevitably accumulate in organs and tissues of the RES, lung, and kidney, and (b) the potential release of the elements of the QD and their distribution in specific organs, tissues, cell types, and subcellular locations must be well understood.

In general, most in vitro studies on the exposure of cells to QDs have attempted to relate cytotoxic events to the release of potentially toxic elements and/or to the size, shape, surface, and cellular uptake of QDs. Because the toxicity of Cd2+ ions is well documented, a significant body of work has focused on the intracellular release of free cadmium from the QDs. Cd2+ ions can be released through oxidative degradation of the QD, and may then bind to sulfhydryl groups on a variety of intracellular proteins, causing decreased functionality in many subcellular organelles [139]. Several groups have investigated methods to quantify the amount of free Cd2+ ions released from QDs, either intracellularly or into culture media, by ICP-MS or fluorometric assays, leading to the conclusion that Cd2+ release correlates with cytotoxic manifestations [79,140, 141]. Derfus, et al. facilitated oxidative release of cadmium ions from the surface of CdSe QDs by exposure to air or ultraviolet irradiation [79]. Under these conditions, CdSe QD cores coated with small thiolate ligands were toxic. Capping these QDs with ZnS shells or coating with BSA rendered the QD cores less susceptible to oxidative degradation and less toxic to primary rat hepatocytes, implicating the potential role of cadmium in QDs cytotoxicity. The decrease in QD cytotoxicity of CdSe QDs with the overgrowth of a ZnS shell has since been verified in several reports [139, 142]. If it is revealed in the future that Cd2+release is a major hindrance for the use of QDs in cells and in animals, several new types of QDs that have no heavy metals atoms may be useful for advancing this field [143, 144].

Toxicity Induced by Colloidal Instability

Presently it is nearly impossible to drawing firm conclusions about the toxicity of QDs in cultured cells due to (a) the immense variety of QDs and variations of surface coatings used by different labs and (b) a technical disparity in experimental conditions, such as the duration of the nanoparticle exposure, use of relevant cell lines, media choice (e.g. with or without serum), and even the units of concentration (e.g. mg/ml versus nM). Nonetheless, the cytotoxicity of QDs reported in the literature has strongly correlated with the stability and surface coatings of these nanoparticles, which can be separated into three categories. (1) Core CdTe QDs that are synthesized in aqueous solution and stabilized by small thiolate ligands (e.g. mercaptopropionic acid or mercaptoacetic acid). These QDs have been widely used due to their ease of synthesis, low cost, and immediate utility in biological buffers. However, because these QDs are protected only by a weakly bound ligand, they are highly prone to degradation and aggregation, and their cytotoxicity toward cells in culture has been widely reported [140, 145]. (2) Core/shell CdSe/ZnS QDs synthesized in nonpolar solvents and transferred to water using thiolate ligands. CdSe is less prone to oxidation than CdTe, and ZnS is even more inert, and therefore these QDs are much more chemically stable. With direct comparison to CdTe QDs, these nanocrystals are significantly less cytotoxic, although high concentrations have been found to illicit toxic responses from cells [140]. Because these QDs are coated with a ZnS shell, the origin of this cytotoxicity is still unclear, whether it is from degradation of the shell, leading to cadmium release, or if it is caused by other effects. When coated with small ligands, these QDs have similar surface chemistries compared to aqueous CdTe QDs, burdened by significant dissociation of ligands from the QDs, rendering the nanoparticles colloidally unstable [146]. This propensity for aggregation may contribute to their cytotoxicity, even if free cadmium is not released. Importantly for the comparison between CdSe/ZnS QDs and their cadmium-only counterparts (CdSe or CdTe core QDs), thiolate ligands bind more strongly to zinc than to cadmium, which may contribute colloidal stability. (3) Core/shell CdSe/ZnS QDs synthesized in nonpolar solvents and transferred to water via encapsulation in amphiphilic polymers or cross-linked silica. These QDs have been found to be significantly more stable colloidally, chemically, and optically when compared to their counterparts coated in small ligands [56]. For this reason, they have been found to be nearly biologically inert in both living cells and living animals [10, 24, 49, 60, 79, 107, 114, 147]. Only when exposed to extreme conditions or when directly injected into cells at immensely high concentrations have these QDs been found to elicit toxic or inflammatory responses [49, 142].

It is feasible that a significant amount of toxicological data obtained for QDs thus far has been considerably influenced by the colloidal nature of these nanoparticles. The tendency for nanoparticles to aggregate, precipitate on cells in culture, nonspecifically adsorb to biomolecules, and catalyze the formation of reactive oxygen species (ROS) may be just as important as heavy metal toxicity contributions to toxicity. For example, Kircher et al. found that CdSe/ZnS QDs coated with an amphiphilic polymer shell induced the detachment of human breast cancer cells from their cell culture substrate [139]. This effect was found to also occur for biologically inert gold nanoparticles coated with the same polymer, thus ruling out the possibility of heavy metal atom poisoning. Microscopic examination of the cells revealed that the nanoparticles precipitated on the cells, causing physical harm. Indeed, carbon nanotubes, which are entirely composed of harmless carbon, have been found to be capable of impaling cells and causing major problems in the lungs of mammals [148]. Nonspecific adsorption to intracellular proteins may also impair cellular function, especially for very small QDs (3 nm and below), which can invade the cellular nucleus [101], binding to histones and nucleosomes [102], and damage DNA in vitro [149, 150]. QDs are also known to catalyze the formation of ROS [145, 151], especially when exposed to ultraviolet radiation. In fact, Cho et al. exposed cells to CdTe QDs in cell culture and determined that their cytotoxicity could only be accounted for with the effects of ROS generation, as there was no dose-dependent relationship with intracellular Cd2+ release, as determined with a cadmium-reactive dye [140]. However, protection of the surface of QDs with a thick ZnS shell may greatly reduce ROS production [152, 153]. Despite a significant surge of interest in the cytotoxicity of nanoparticles, there is still much to learn about the cytological and physiological mediators of nanoparticle toxicology. If it is determined that heavy metal composition plays a negligible role in QD toxicity, QDs will have as good of a chance as any other nanoparticle at being used as clinical contrast agents.

Dual-Modality QDs for Imaging and Therapy

In comparison with small organic fluorophores, QDs have large surfaces that can be modified through versatile chemistry. This makes QDs convenient scaffolds to accommodate multiple imaging (e.g., radionuclide-based or paramagnetic probes) and therapeutic agents (e.g. anticancer drugs), through chemical linkage or by simple physical immobilization. This may enable the development of a nearly limitless library of multifunctional nanostructures for multimodality imaging, as well as for integrated imaging and therapy.

Dual-Modality Imaging

The applications of QDs described above for in vivo imaging are limited by tissue penetration depth, quantification problems, and a lack of anatomic resolution and spatial information. To address these limitations, several research groups have led efforts to couple QD-based optical imaging with other imaging modalities that are not limited by penetration depth, such as MRI, positron emission tomography (PET) and single photon emission computed tomography (SPECT) [154158]. For example, Mulder et al. [154] developed a dual-modality imaging probe for both optical imaging and MRI by chemically incorporating paramagnetic gadolinium complexes in the lipid coating layer of QDs [154, 155]. In vitro experiments showed that labeling of cultured cells with these QDs led to significant T1 contrast enhancement with a brightening effect in MRI, as well as an easily detectable fluorescence signal from QDs. However, the in vivoimaging potential of this specific dual-modality contrast agent is uncertain due to the unstable nature of the lipid coating that was used. More recently, Chen and coworkers used a similar approach to attach the PET-detectable radionuclide 64Cu to the polymeric coating of QDs through a covalently bound chelation compound [158]. The use of this probe for targeted in vivo imaging of a subcutaneous mouse tumor model was achieved by also attaching αvβ3 integrin-binding RGD peptides on the QD surface. The quantification ability and ultrahigh sensitivity of PET imaging enabled the quantitative analysis of the biodistribution and targeting efficacy of this dual-modality imaging probe. However, the full potential of in vivo dual-modality imaging was not realized in this study, as fluorescence was only used as an ex vivo imaging tool to validate the in vivo results of PET imaging, primarily due to the lower sensitivity of optical imaging in comparison with PET. This imbalance in sensitivity is fundamental to the differences in the physics of these imaging modalities, and points to an inherent difficulty in designing useful multimodal imaging probes. The majority of these probes are still at an early stage of development. The clinical relevance of these nanoplatforms still needs further improvement in sensitivity and better integration of different imaging modalities, as well as validation of their biocompatibility and safety.

It is also noteworthy that recent advances in the synthesis of QDs containing paramagnetic dopants, such as manganese, have led to a new class of QDs that are intrinsically fluorescent and magnetic [159, 160]. However the utility of these new probes for bioimaging application is unclear because they are currently limited to the ultraviolet and visible emission windows, and their stability (e.g., photochemical and colloidal) and biocompatibility have yet to be systematically investigated [144]. As well, inorganic heterodimers of QDs and magnetic nanoparticles have generated dual-functional nanoparticles [161, 162]. Although these new materials are of great interest, they are still in development and have only recently shown applicability in cell culture, but not yet in living animals [160, 163].

Integration of Imaging and Therapy

Drug-containing nanoparticles have shown great promise for treating tumors in animal models and even in clinical trials [157]. Both passive and active targeting of nanotherapeutics have been used to increase the local concentration of chemotherapeutics in the tumor. Due to the size and structural similarities between imaging and therapeutic nanoparticles, it is possible that their functions can be integrated to directly monitor therapeutic biodistribution, to improve treatment specificity, and to reduce side effects. This synergy has become the principle foundation for the development of multi-functional nanoparticles for integrated imaging and cancer treatment. Most studies are still at a proof-of-concept stage using cultured cancer cells, and are not immediately relevant to in vivo imaging and treatment of solid tumors. However, these studies will guide the future design and optimization of multifunctional nanoparticle agents for in vivo imaging and therapy [164167].

In one example, Farokhzad et al. reported a ternary system composed of a QD, an aptamer, and the small molecular anticancer drug doxorubicin (Dox) for in vitro targeted imaging, therapy and sensing of drug release [165]. As illustrated in Figure 6, aptamers were conjugated to QDs to serve as targeting units, and Dox was attached to the stem region of the aptamers, taking advantage of the nucleic acid binding ability of doxorubicin. Two donor-quencher pairs of fluorescence resonance energy transfer occurred in this construct, as the QD fluorescence were quenched by Dox, and Dox was quenched by the double-stranded RNA aptamers. As a result, gradual release of Dox from the conjugate was found to “turn on” the fluorescence of both QDs and Dox, providing a means to sense the release of the drug. However it is clear that the current design of this conjugate will not be sufficient for in vivo use unless the drug loading capacity can be greatly increased (currently 7–8 Dox molecules per QD).

QD-Aptamer-Dox FRET system and its targeted delivery  nihms62165f6

QD-Aptamer-Dox FRET system and its targeted delivery nihms62165f6

Schematic illustration of QD-Aptamer-Dox FRET system and its targeted delivery through receptor-mediated endocytosis. (a) QDs-aptamer conjugates (QD-Apt) are fluorescent until they are mixed with the fluorescent drug doxorubicin (Dox), which intercalates
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649798/bin/nihms62165f6.jpg
QDs for siRNA Delivery and Imaging

QDs also provide a versatile nanoscale scaffold to develop multifunctional nanoparticles for siRNA delivery and imaging. RNA interference (RNAi) is a powerful technology for sequence-specific suppression of genes, and has broad applications ranging from functional gene analysis to targeted therapy [168172]. However, these applications are limited by the same delivery problems that hinder intracellular imaging with QDs (Section 3.2), namely intracellular delivery and endosomal escape, in addition to dissociation from the delivery vehicle (i.e. unpacking), and coupling with cellular machines (such as the RNA-induced silencing complex or RISC). For cellular and in vivo siRNA delivery, a number of approaches have been developed (see ref. [168] for a review), but these methods have various shortcomings and do not allow a balanced optimization of gene silencing efficacy and toxicity. For example, previous work has used QDs and iron oxide nanoparticles for siRNA delivery and imaging [27, 166, 167, 173], but the QD probes were either mixed with conventional siRNA delivery agents [166] or an exogenous compound, such as the antimalaria drug chloroquine, was needed for endosomal rupture and gene silencing activity [173].

Gao et al. have recently fine-tuned the colloidal and chemical properties of QDs for use as delivery vehicles for siRNA, resulting in highly effective and safe RNA interference, as well as fluorescence contrast [174]. The authors balanced the proton-absorbing capacity of the QD surface in order to induce endosomal release of the siRNA through the proton sponge effect (see Section 3.4). A major finding is that this effect can be precisely controlled by partially converting the carboxylic acid groups on a QD into tertiary amines. When both are linked to the surface of nanometer-sized particles, these two functional groups provide steric and electrostatic interactions that are highly responsive to the acidic organelles, and are also well suited for siRNA binding and cellular entry. As a result, these conjugates can improve gene silencing activity by 10–20 fold, and reduce cellular toxicity by 5–6 fold, compared with current siRNA delivery agents (lipofectamine, JetPEI, and TransIT). In addition, QDs are inherently dual-modality optical and electron microscopy probes, allowing real-time tracking and ultrastructural localization of QDs during transfection.

Concluding Remarks

Quantum dots have been received as technological marvels with characteristics that could greatly improve biological imaging and detection. In the near future, there are a number of areas of research that are particularly promising but will require concerted effort for success:

(1) Design and development of nanoparticles with multiple functions

For cancer and other medical applications, important functions include imaging (single or dual-modality), therapy (single drug or combination of two or more drugs), and targeting (one or more ligands). With each added function, nanoparticles could be designed to have novel properties and applications. For example, binary nanoparticles with two functions could be developed for molecular imaging, targeted therapy, or for simultaneous imaging and therapy. Ternary nanoparticles with three functions could be designed for simultaneous imaging and therapy with targeting, targeted dual-modality imaging, or for targeted dual-drug therapy. Quaternary nanoparticles with four functions can be conceptualized in the future to have the capabilities of tumor targeting, dual-drug therapy and imaging.

(2) Use of multiplexed QD bioconjugates for analyzing a panel of biomarkers and for correlation with disease behavior, clinical outcome, and treatment response

This application should begin with retrospective studies of archived specimens in which the patient outcome is already known. A key hypothesis to be tested is that the analysis of a panel of tumor markers will allow more accurate correlations than single tumor markers. As well, the analysis of the relationship between gene expression from cancer cells and the host stroma may help to define important cancer subclasses, identify aggressive phenotypes of cancer, and determine the response of early stage disease to treatment (chemotherapy, radiation, or surgery).

(3) Design and development of biocompatible nanoparticles to overcome nonspecific organ uptake and RES scavenging

There is an urgent need to develop nanoparticles that are capable of escaping RES uptake, and able to target tumors by active binding mechanisms. This in vivo biodistribution barrier might be mitigated or overcome by systematically optimizing the size, shape, and surface chemistry of imaging and therapeutic nanoparticles.

(4) Penetration of imaging and therapeutic nanoparticles into solid tumors beyond the vascular endothelium

This task will likely require active pumping mechanisms such as caveolin transcytosis and receptor-mediated endocytosis, or cell-based strategies such as nanoparticle-loaded macrophages.

(5) Release of drug payloads inside targeted cells or organs

This task will likely require the development of biodegradable nanoparticle carriers that are responsive to pH, temperature, or enzymatic reactions.

(6) Nanotoxicology studies including nanoparticle distribution, excretion, metabolism, pharmacokinetics, and pharmacodynamics in animal models in vivo

These investigations will be vital for the development of nanoparticles beyond their current use as research tools, toward clinical applications in cancer imaging and therapy.

7.1.3 In vivo molecular and cellular imaging with quantum dots

Gao XYang LPetros JAMarshall FFSimons JWNie S.
Curr Opin Biotechnol. 2005 Feb;16(1):63-72.
http://dx.doi.org:/10.1016/j.copbio.2004.11.003

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The structure of a multifunctional QD probe. Schematic illustration showing the capping ligand TOPO, an encapsulating copolymer layer, tumor-targeting ligands (such as peptides, antibodies or small-molecule inhibitors), and polyethylene glycol (PEG).

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Methods for conjugating QDs to biomolecules. (a) Traditional covalent cross-linking chemistry using EDAC (ethyl-3-dimethyl amino propyl carbodiimide) as a catalyst. (b) Conjugation of antibody fragments to QDs via reduced sulfhydryl-amine coupling. SMCC, succinimidyl-4-Nmaleimidomethyl-cyclohexane carboxylate. (c) Conjugation of antibodies to QDs via an adaptor protein. (d) Conjugation of histidine-tagged peptides and proteins to Ni-NTA-modified QDs, with potential control of the attachment site and QD:ligand molar ratios.

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Quantum dots (QDs), tiny light-emitting particles on the nanometer scale, are emerging as a new class of fluorescent probe for in vivo biomolecular and cellular imaging. In comparison with organic dyes and fluorescent proteins, QDs have unique optical and electronic properties: size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to the development of multifunctional nanoparticle probes that are very bright and stable under complex in vivo conditions. A new structural design involves encapsulating luminescent QDs with amphiphilic block copolymers and linking the polymer coating to tumor-targeting ligands and drug delivery functionalities. Polymer-encapsulated QDs are essentially nontoxic to cells and animals, but their long-term in vivo toxicity and degradation need more careful study. Bioconjugated QDs have raised new possibilities for ultrasensitive and multiplexed imaging of molecular targets in living cells, animal models and possibly in humans.

7.1.4 Luminescent quantum dots for multiplexed biological detection and imaging

Chan WC1Maxwell DJGao XBailey REHan MNie S.
Curr Opin Biotechnol. 2002 Feb;13(1):40-6.


http://dx.doi.org/10.1016/S0958-1669(02)00282-3

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Recent advances in nanomaterials have produced a new class of fluorescent labels by conjugating semiconductor quantum dots with biorecognition molecules. These nanometer-sized conjugates are water-soluble and biocompatible, and provide important advantages over organic dyes and lanthanide probes. In particular, the emission wavelength of quantum-dot nanocrystals can be continuously tuned by changing the particle size, and a single light source can be used for simultaneous excitation of all different-sized dots. High-quality dots are also highly stable against photobleaching and have narrow, symmetric emission spectra. These novel optical properties render quantum dots ideal fluorophores for ultrasensitive, multicolor, and multiplexing applications in molecular biotechnology and bioengineering.

7.1.5 Multifunctional quantum dots for Personalized Medicine

Pavel Zrazhevskiy and Xiaohu Gao
Nano Today. 2009 Oct 5; 4(5): 414–428.
http://dx.doi.org:/10.1016/j.nantod.2009.07.004

Successes in biomedical research and state-of-the-art medicine have undoubtedly improved the quality of life. However, a number of diseases, such as cancer, immunodeficiencies, and neurological disorders, still evade conventional diagnostic and therapeutic approaches. A transformation towards personalized medicine may help to combat these diseases. For this, identification of disease molecular fingerprints and their association with prognosis and targeted therapy must become available. Quantum dots (QDs), semiconductor nanocrystals with unique photo-physical properties, represent a novel class of fluorescence probes to address many of the needs of personalized medicine. This review outlines the properties of QDs that make them a suitable platform for advancing personalized medicine, examines several proof-of-concept studies showing utility of QDs for clinically relevant applications, and discusses current challenges in introducing QDs into clinical practice.

State-of-the-art medicine is an indispensable part of the human society. Wealth of medical knowledge accumulated over centuries of observation and experimentation, advanced diagnostic techniques made possible by the technological revolution, and innovative biomedical research done on the cellular and molecular levels provide a formidable weapon against nearly any threat to human health. However, the most devastating diseases, such as cancer, immunodeficiencies and neurological disorders to name a few, are notorious for their ability to evade current diagnostic methods and resist therapy. It is not easy to pinpoint the main reasons for poor success in combating these diseases, as they might range from a lack of understanding of patho-physiology to the absence of appropriate diagnostic techniques capable of addressing the complexity of these diseases. One potential issue is that utilization of generalized diagnostic and treatment approaches based on identifying and targeting disease symptoms (often with limited information about the underlying cause) is inefficient in addressing the great genetic and phenotypic variability of cancer and immune system disorders. Significant heterogeneity on molecular level, complex interlinking of subcellular mechanisms along with integrated pathophysiological effects on organs and systems of the human body, and an often unclear origin and cause of the disease represent major challenges for current biomedical research and clinical practice.

Personalized medicine, a practice of addressing individual diseases in a pathology-specific and patient-specific manner spanning all levels from whole-body symptoms down to molecular signatures of the disease, is an emerging field of medicine promising to provide efficient tools against cancer and other challenging diseases. A personalized approach offers unique opportunities to accurate diagnosis (i.e. pinpoint exact changes that occurred within healthy cells and tissues), prognosis (i.e. predict progression of a disease based on these changes), and treatment (i.e. specifically reverse the changes or, if not possible, target and kill the diseased cells without affecting healthy ones). Such an approach relies on advances in basic research as well as integration of novel diagnostic and therapeutic techniques into clinical practice.

Currently, attempts of introducing personalized approach in medicine rely on screening for genetic alterations in diseased cells; yet diagnostic and predictive power of genetic screening alone is questionable due to insufficient knowledge of how certain alterations on the DNA level propagate along the DNA-RNA-protein chain [1, 2] and the requirement of performing analysis on a homogenized mixture of different cell types, including a variety of healthy cells [3]. Therefore, complementary analysis of phenotypic changes (i.e. changes in protein expression) as well as assessment of the effect of diseased cells on the healthy tissues (e.g. activation of angiogenesis in tumors) is necessary for comprehensive analysis of a pathological process. Compilation of a database of genetic and phenotypic signatures of individual diseases will provide an access to a more accurate prognosis and personalized treatment targeted directly against the biomarkers expressed. Realizing this, significant research effort is being focused at understanding the physiology of normal cellular processes as well as patho-physiology of diseases in order to determine specific disease-causing changes in individual cells, organs, and systems.

A key challenge is presented by the complexity of inter- and intracellular networks with multiple inputs, controllers, and feedback loops, which is hard to assess using conventional biomedical techniques (such as immunohistochemistry, Western blot, ELISA, etc) that suffer from a limitation in the number of biomarkers that can be analyzed simultaneously, lack real-time monitoring capacity for intracellular processes, provide limited single-cell information resulting from the need to analyze signals averaged over many cells, and utilize qualitative rather than quantitative analytical techniques [46]. Consequently, diagnosis and prognosis are limited by the lack of knowledge about the predictive biomarkers that would unambiguously discriminate between disease and normal function as well as distinguish different disease types and provide information about possible progression of the pathological process.

Advances in nanotechnology have enabled the design of nanoparticle-based tools for improved diagnosis and personalized treatment of many complex diseases. In particular, semiconductor QDs have emerged as a new platform for high-throughput quantitative characterization of multiple biomarkers in cells and clinical tissue specimens ex vivo, detection of diseased cells in vivo, and potentially targeted and traceable drug delivery [3,7, 8].

Properties of quantum dots for addressing the needs of personalized medicine

QDs are semiconductor nanoparticles with size ranging between 2 and 10 nm in diameter (hydrodynamic size often larger). Restricting the mobility of charge carriers (electrons and holes) within the nanoscale dimensions generates the quantum confinement effect responsible for unique size-dependent photo-physical properties of QDs [911]. Additionally, nanometer-scale size of QDs comparable with the size of large proteins enables integration of nanoparticles and biomolecules yielding biologically functional nanomaterials suitable for probing physiological processes on a molecular level [1214]. While a relatively large size (compared to small drug molecules or organic fluorescent dyes) might be associated with slower diffusion, limited permeability, complex bio-distribution, and possible interference with intracellular processes [15], QDs possess a wide range of features essential for addressing the most urgent needs of personalized medicine. Among such features are size-tunable and spectrally narrow light emission, simultaneous excitation of multiple colors, improved brightness, resistance to photobleaching, and an extremely large Stokes shift.

The cornerstone of personalized medicine is the ability to uniquely identify the disease by its “molecular fingerprint” (i.e. pattern of biomarker expression), associate the fingerprint with possible progression of the disease, and assign a treatment which targets diseased cells with the identified fingerprint. Achieving this goal is not a trivial task – many diseased cells look very much like the healthy ones (especially in case of cancer), and screening for a large panel of biomarkers is required. It is quite possible that certain diseases have one or few biomarkers specific enough for unique identification, yet finding these biomarkers de novo using low-throughput conventional approaches is like looking for a needle in a haystack. QDs open access to a multi-parameter biomarker screening on intact specimens via multiplexed detection [16]. This feature is based on two properties of QDs: spectrally narrow size-tunable light emission [1719] and effective light absorption throughout a wide spectrum [12] (Fig. 1). Excitation of multiple QD probes with a single light source (e.g. laser) significantly reduces the complexity and cost of imaging instrumentation and simplifies data analysis. Utilization of hyperspectral imaging, a technique that allows deconvolution of an image into spectral components, further improves the multiplexing capabilities of QD technology (Fig. 2) [20]. It is worth mentioning that highly multiplexed molecular analysis would be limited if hyperspectral imaging or QDs are used separately. Combination of these two complementary technologies enhances each other’s capability.

Figure 1

Quantum dots possess unique photo-physical properties suitable for addressing the needs of personalized medicine. The ability to utilize multicolor QD probes (A) and tune the emission color by the particle size allows multiplexed biomarker detection.

Figure 2

Hyperspectral imaging represents a powerful technique for analysis of multiple QD-labeled biomarkers within a single specimen. While standard RGB camera cannot distinguish spectrally overlapping probes and is limited to analysis of few biomarkers, hyperspectral

An indispensable part of disease molecular profiling is the ability to quantify biomarker expression in an accurate and consistent manner. So far, this requirement has been only partially fulfilled. The problem lies in the fact that colorimetric assays usually rely on amplification mechanisms, which are difficult to control, thus providing inconsistent and mostly qualitative information about the biomarker expression. Quantitative analysis with fluorescence imaging using organic fluorophores is often compromised by the quick photobleaching of the dyes and unstable signal intensity. Destructive techniques, while allowing protein quantification (e.g. Western blot, RT-PCR, protein chips), do not preserve tissue morphology and cannot properly address the heterogeneity of specimens. QD probes, on the other hand, are well-suited for addressing these issues. First of all, QDs are highly resistant to photobleaching and photodegradation: in one example QDs retained constant signal intensity for over 30 minutes of illumination, while organic dyes faded by more than 90% in less than one minute under identical experimental conditions [21]. Second, QDs do not rely on chemical amplification (in contrast to assays such as horse radish peroxidase mediated color development and Au catalyzed Ag-enhancement) and have a promise of providing imaging probes with a 1:1 stoichiometry. It is necessary to note, though, that the intensity of different color QDs under identical illumination conditions differ significantly, showing enhancement of red QD signal over green/blue QDs. Such discordance has been observed by Ghazani and coworkers in a three-color staining of lung carcinoma xenografts for epidermal growth factor receptor, E-cadherin, and cytokeratin using QDs emitting at 655, 605, and 565 nm [22]. While quantitative analysis of individual QD signals was readily achievable, comparison between different QD signals was not possible through this study. The discordance in fluorescence intensity of individual probes directly relates to light absorption properties and the quantum yield of QDs (i.e. red particles having larger cross-section absorb light more efficiently) and can be accounted for in signal analysis algorithm. For example, Yezhelyev et al used bulk fluorescence measurement of equal concentrations of QDs and determined that QD655 were 8 times as bright and QD605 4 times as bright as QD565 [23]. However, other effects associated with high QD concentration, such as steric hindrance between the probes, self-quenching, and fluorescence resonance energy transfer (FRET) from smaller to larger particles [22], might be possible in cases of high biomarker density and deserves further investigation for achieving accurate quantitative analysis.

Studying patho-physiology with QD probes

A variety of nanomaterials have already shown utility in addressing tough questions posed by unmet clinical needs. In particular, QDs have proven to be well suited for sensitive quantitative molecular profiling of cells and tissues, holding tremendous promise for unraveling the complex gene expression profiles of diseases, accurate clinical diagnosis and personalized treatment of patients [3, 24]. Possessing advantageous photo-physical properties and being compatible with conventional biomedical assays, QDs have found use in most techniques where fluorescence or colorimetric imaging of target biomarker is utilized (e.g. cell and tissue staining, Western blot, ELISA, etc.) and have launched many novel applications (e.g. targeted in vivoimaging, single-molecule tracking, traceable drug delivery, etc.). The number of biomedical applications of QDs continues growing, ranging from ultrasensitive detection in vitro to targeted drug delivery and imagingin vivo.

Identification of molecular fingerprints of diseases

Molecular fingerprinting of diseases implies characterization of biomarker expression schemes in diseased cells in comparison to healthy ones. QD-based probes are uniquely suited for this task when employed by both multi-parameter flow-cytometry analysis of cell populations and quantitative multiplexed analysis of biomarker expression in intact tissue specimens. For example, Chattopadhyay et al, by utilizing a 17-parameter flow-cytometry (based on 8 QD probes and 9 organic fluorophores), revealed significant phenotypic differences between T-cells specific to distinct epitopes of the same pathogen (Fig. 3) [25]. Access to molecular profiles of individual cell populations not only improves our understanding of immune response, but also enables analysis of changes occurring during immune system disorders, sensitive detection of metastasizing cancer cells in a bloodstream, and accurate phenotyping of heterogeneous cell populations.

Figure 3

Seventeen-parameter flow-cytometry analysis of antigen-specific T-cell populations was achieved using 8 QD probes and 9 organic fluorophores. Significant heterogeneity in biomarker expression within a CD8+ T-cell population (shown in gray) emphasizes

Moving towards introducing QD technology into clinical diagnostics, five-parameter characterization of breast cancer tissue specimens obtained from biopsies has been demonstrated [23]. Comparison of the three specimens revealed distinct molecular profiles, where one tumor over-expressed such biomarkers as ER and PR, another tumor primarily expressed EGFR, and third tumor showed abundance of ER and HER2 (Fig. 4). Besides diagnostic and prognostic value of such analysis, potential targets for anti-cancer treatment can also be identified, thus enabling a “personalized” approach in therapy.

Figure 4

Five-parameter quantitative analysis of the three tissue specimens obtained from tumor biopsies clearly identified the differences in biomarker expression profiles between different types of breast cancers. Molecular fingerprinting might not only provide

Accuracy of molecular fingerprinting based on protein expression can be further improved by analysis of gene expression via quantification of mRNA using fluorescent in situ hybridization (FISH). Relying on binding of oligonucleotide probes to complimentary mRNA molecules in 1:1 probe-to-target ratio, this technique offers high level of specificity, yields direct quantitative correlation between gene amplification (i.e. number of mRNA molecules present) and signal intensity, and provides accurate information about mRNA localization within the cell. Similar to protein-based staining, quantitative potential and sensitivity of FISH might be significantly improved by utilization of QD probes [14]. In early proof-of-concept studies Xiao and Barker have used highly stable QD-Streptavidin bioconjugates for monochromatic visualization of biotinylated oligonucleotide probes in FISH analysis of amplification of clinically important erbB2 gene [26]. Using a slightly modified procedure, Tholouli et al have achieved multiplexed staining of 3 mRNA targets within one specimen [27]. In order to reduce the size of imaging probe and improve binding stoichiometry, Chan et al have developed a monovalent FISH probe by blocking extra streptavidin sites with biocytin (water-soluble biotin derivative) [28]. High-resolution multiplexed FISH has been demonstrated in simultaneous detection of four mRNA targets using two different QD probes and two different organic fluorophore probes within a single mouse midbrain neuron (Fig. 5). Notably, reduced size of FISH probes enabled staining in milder, protein-compatible specimen permeabilization conditions, which is essential for combined QD-based FISH and QD-based immunohistochemistry (IHC), thus offering the possibility of correlating gene expression at the mRNA level with the number of corresponding protein copies in diseased cells or tissue specimens [14].

Figure 5

Multi-parameter FISH using QD probes and organic fluorophores enables high-resolution imaging of different mRNA molecular within single cells, thus providing information about relative gene expression levels, localization of mRNA within cellular compartments,
Probing intracellular pathways

While molecular fingerprinting of diseases holds tremendous diagnostic and therapeutic value, uncovering intracellular pathways leading to disorder is essential for understanding the patho-physiology of a disease, identification of an underlying cause of the pathologic changes, and design of therapies targeting dysfunctional pathways on a molecular level. Study of patho-physiology on sub-cellular level involves the characterization of intracellular distribution and relative expression of biomarkers (proteins, mRNA, etc.), analysis of phenotypic changes in cells upon certain stimulation, and real-time monitoring of changes in intracellular processes (e.g. phagocytosis, intracellular trafficking, and cell motility) in live cells.

One interesting study of intracellular morphology was demonstrated by Matsuno et al who combined QD-based FISH and IHC along with confocal laser scanning microscopy for three-dimensional imaging of the intracellular localization of growth hormone (GH), prolactin (PRL), and of their mRNAs within tissue specimens [29]. With further improvements in design of QD probes suitable for multiplexed FISH and IHC, this technology will allow three-dimensional mapping of the relative position of biomarkers and corresponding mRNAs inside cells and tissues with high resolution and sensitivity, thus providing access to studies of intricate signaling pathways and mechanisms of pathogenesis.

Further improvement in imaging resolution can be achieved by utilization of transmission electron microscopy (TEM). For example, relatively high electron density of QDs was successfully employed by Giepmans et al for high-resolution study of intracellular biomarker distribution [30]. In this study initial optimization of staining conditions was achieved using fluorescence imaging, while further examination with TEM revealed intracellular localization of QD probes (and corresponding biomarkers) with respect to sub-cellular structures. Due to direct correlation between fluorescence emission color and QD size, detection of three QD-labeled biomarkers distinguishable at both fluorescence (by color) and TEM (by size) levels was achieved [30]. Enhancement in multiplexing functionality of this technique can be obtained from discrimination of QDs based on their elemental composition. Nisman et al have proposed the use of electron spectroscopic imaging (ESI, a technique for generating elemental maps of materials with high resolution and detection sensitivity) for mapping the distribution of QDs in cells and tissues based on QD internal chemistry in addition to discriminating probes by size [31].

Monitoring of intracellular processes in live cells, although more difficult and less flexible in terms of multiplexing, provides information about dynamics of cellular functioning and real-time cellular response to applied stimuli. Design of biocompatible coatings and unprecedented photostability render QDs well-suited for this task, as long exposure to excitation source and constant signal intensity are often not achievable with conventional techniques. The relatively large size of QD probes creates a barrier for intracellular targeting, yet biomarkers expressed on the cell membrane are readily accessible. As a result the majority of reports on real-time tracking describe dynamics of membrane proteins rather than intracellular targets. For example, Lidke et al used QDs conjugated to epidermal growth factor (EGF) to study erbB/HER receptor-mediated cellular response to EGF in living human epidermoid carcinoma A431 cells, assigning the mechanism of EGF-induced signaling to heterodimerization of erbB1 and erbB2 monomers and uncovering retrograde transport of endocytosed QD probes (Fig. 6) [32]. Murcia et al utilized QD-lipid bioconjugates for high-speed tracking of single-probe movement on cell surface and accurate measurement of diffusion coefficient [33], while Roullier et al labeled two subunits of type I interferon receptor with QD probes and monitored diffusion and interaction of these subunits in real-time [34].

Figure 6

Outstanding photostability and high brightness of QD probes enable long-term real-time monitoring of erbB receptor activation by QD-EGF and study the retrograde transport of these probes along the filopodia towards the cell body. Scale bars 5 um [32].

One highly informative method of intracellular tracking involves endocytosis of QD probes with consequent monitoring of endosome dynamics. Cui et al utilized pseudo-TIRF (total internal reflection fluorescence) microscopy for long-term real-time tracking of intracellular transport of QD-labeled nerve growth factor (NGF) along axons of rat dorsal root ganglion neurons and described the dynamics of axonal internalization and neuronal retrograde transport of QD-NGF [35]. In another example, Zhang et al induced single QD uptake into synaptic vesicles and monitored fluorescence of each QD probe to discriminate between complete vesicle fusion (full-collapse fusion) and transient fusion (so-called kiss-and-run behavior), thus characterizing dynamics of neuronal transmission with respect to time and frequency of impulse firing [36].

The challenge that is yet to be overcome is labeling of intracellular components in live cells. Integrity of cellular membrane and crowded intracellular environment have proven to be an obstacle for QD entry into live cells. While endosomal uptake of bare QDs is readily achievable, escape from endosomal compartments and labeling of specific components is challenging. Further, elimination of unbound probes from intracellular compartments to avoid false positive detection is often hampered, because, unlike fixed cells, unbound nanoparticles cannot be washed away. Recently a few reports on delivery of nanoparticles within live cells have been published. In mechano-chemical approach, Yum et al utilized gold-coated boron nitride nanotubes (with a diameter of 50 nm) to deliver QDs within the cytoplasm or nucleus of live HeLa cells with consequent 30-minute monitoring of QD diffusion within those compartments (Fig. 7A) [37]. Park et alengineered arrays of vertically aligned carbon nanosyringes for intracellular delivery of QDs and therapeutic agents (Fig. 7B) [38]. While efficiently delivering nanoparticles within cells, both techniques are quite labor-intensive and low-throughput. Design of nanoparticles capable of escaping endosomes or entering cells without inducing endocytosis remains the most promising approach for intracellular delivery [3942]. For example, Kim et al encapsulated multiple QDs within the biodegradable polymer poly(D,L-lactide-co-glycolide) (PLGA) that induced cellular uptake, endosomal escape, and release of QD load within the cytoplasm (Fig. 7C), providing efficient high-throughput method for intracellular delivery of multicolor QDs and enabling multiplexed staining within live cells [40]. In a single-particle approach, Qi and Gao coated individual QDs with a pH-responsive amphyphilic polymer [42]. Besides achieving efficient cellular uptake and endosomal escape facilitated by a proton sponge effect, polymer-coated QDs allowed delivery of intact siRNA inside the cells and monitoring of siRNA release within the cytoplasm.

delivery-of-qd-probes-inside-cells-represents-a-challenge-for-labeling-intracellular-target-nihms137729f7

delivery-of-qd-probes-inside-cells-represents-a-challenge-for-labeling-intracellular-target-nihms137729f7

Delivery of QD probes inside cells represents a challenge for labeling intracellular targets. Different modes of delivery are being developed to overcome this issue. A) Mechano-chemical modes of QD delivery involve utilization of mechanically strong materials
In vivo molecular imaging and profiling using quantum dots

In vivo imaging of diseased cells and tissues provides many benefits for personalized medicine, including high-throughput screening and potential for diagnosis at early stages of disease, obtaining patient-specific information about the localization and size of the disease core, assessment of adverse effects on healthy tissues, and monitoring of disease progression and response to therapy. Therefore, non-invasive in vivoimaging represents one of the major goals of current biomedical research. Conventional medical imaging techniques, such as ultrasound imaging, magnetic resonance imaging (MRI), and positron emission tomography (PET), in most cases do not offer sensitivity and resolution simultaneously for early-stage diagnosis (e.g. MRI provides high resolution, yet poor sensitivity; while PET offers high sensitivity with low resolution) as well as specificity for conveying disease molecular information.

Fluorescence microscopy remains the most potent technique for molecular profiling of diseased cells. However, presence of tissue barriers between disease sites and imaging equipment complicates the utilization of fluorescence microscopy for in vivo imaging, as biological tissues efficiently absorb and scatter visible light along with producing intense autofluorescence over a broad spectrum. Unlike organic fluorophores, QDs possess high brightness and multiplexing capabilities along with large Stokes shifts, thus representing a promising tool for in vivo molecular imaging and profiling [16, 22, 2931, 4345]. In particular, the spectral gap between excitation and emission for QDs is significantly larger than that of organic fluorophores and can be as large as 300–400 nm, depending on the wavelength of the excitation light [46, 47], thus moving QD signal into a region with reduced tissue autofluorescence. For example, in early studies Akerman et aldemonstrated targeted imaging of tumor vasculature using QD-peptide bioconjugates [48]. However, utilization of green and red QDs limited deep-tissue imaging in live animals, and post-mortem histological examination of tissue specimens was used to evaluate QD biodistribution. Taking advantage of large stokes shift, Gao et al have demonstrated the utility of PEG-coated red QDs (emission peak around 640 nm) conjugated to antibodies against prostate-specific membrane antigen (PSMA) for in vivo tumor imaging in mice [49]. Further signal processing with spectral unmixing algorithm allowed clear separation of QD signal from the background fluorescence (Fig. 8).

Figure 8

Utilization of large Stokes shift produced by red and NIR QD probes enables targeted in vivo imaging of subcutaneous tumors. Further image processing with spectral demixing allows efficient removal of tissue autofluorescence [49].

Utilization of near-infrared imaging probes might further reduce interference from tissue autofluorescence and enable in vivo imaging with deeper penetration and better resolution. Modeling studies performed by Limet al have identified two spectral windows in far-red (700–900 nm) and infrared (1200–1600 nm) regions suitable for nearly background-free deep-tissue imaging [50]. Kim et al utilized model predictions on practice in mapping sentinel lymph nodes (SLN) with NIR QDs, providing accurate identification and image-guided resection of SLN – an indispensable tool in surgical treatment of metastatic cancers [51]. Targeted in vivoimaging of human glioblastoma vasculature in mouse model was demonstrated by Cai et al, who used NIR CdTe/ZnS QDs conjugated to targeting peptide against integrin αvβ3, which is significantly up-regulated in tumors [52]. Recently, Diagaradjane et al reported on in vivo imaging and quantitative analysis of EGFR with NIR QDs (emission peak at 800 nm), showing QD capability to distinguish EGFR over-expression in tumor site compared to normal expression levels in surrounding tissues [53]. Meanwhile Shi et al used NIR QDs to achieve a deep-tissue high-sensitivity detection of prostate cancer xenografts grown in mouse tibia [54].

An alternative NIR QD probe was developed by So et al, who conjugated luciferase to QD surface to yield self-illuminating fluorescent probes via bioluminescence resonance energy transfer process (Fig. 9) [55]. By making external excitation unnecessary, bioluminescent QDs completely eliminated the tissue autofluorescence and provided higher sensitivity of detection. Increased size of luciferase-QD bioconjugates and requirement for supplying the substrate coelenterazine put certain limitations on in vivo imaging applications. Therefore, development of compact self-illuminating QDs utilizing naturally occurring biomolecules as a substrate might further advance this technology and provide high-sensitivity in vivoimaging probes.

shallow-depth-of-in-vivo-imaging-with-qd-probes-imposes-significant-limits-nihms137729f9

shallow-depth-of-in-vivo-imaging-with-qd-probes-imposes-significant-limits-nihms137729f9

Shallow depth of in vivo imaging with QD probes imposes significant limits on utilization of this technology for deep-tissue imaging. One way to improve the depth and sensitivity of imaging is to use self-illuminating QDs. QD probes conjugated with luciferase

Two-photon microscopy represents another promising alternative to standard in vivo fluorescence imaging. Despite some technical limitations, two-photon microscopy represents a powerful tool for multiplexed and highly sensitive in vivo imaging. This technique uses low-energy photons (in red and infrared regions) for excitation of QDs emitting in visible range, achieving dramatically reduced attenuation of excitation light by tissues along with reducing the autofluorescense, while allowing utilization of QDs emitting over a full visible spectrum. Moreover, the high two-photon cross-section of QDs enables deeper-tissue imaging with higher sensitivity. The first study of QD-based multiphoton fluorescence in vivo imaging was reported by Larson et al, when green CdSe/ZnS QDs were used for imaging of capillaries under the dermis layer of skin [56]. Levene et al have combined needle-like gradient index lens imaging setup together with multiphoton microscopy to obtain high-resolution microangiographs of deep brain blood vessels labeled with QDs [57]. In a recent in vivo study of tumor morphology Stroh et al utilized two-photon microscopy for simultaneous imaging of tumor vessels (stained with blue QDs) and perivascular cells (expressing GFP) [58]. Further incorporation of second harmonic generation signal emanating from collagen provided information about the distribution and morphology of extracellular matrix (Fig. 10).

Figure 10

Multi-photon microscopy represents a powerful tool for multiplexed in vivo imaging. By utilizing low-energy photons (minimally absorbed by tissues) for excitation of multicolor QD probes, this technique provides deeper tissue penetration and higher sensitivity

Overall, NIR QDs have already proven to be a great tool for characterization of disease models in small animals and post-mortem evaluation of tissue specimens. Moving towards in vivo imaging in human subjects, mapping of lymph nodes and image-guided resection of tumors represent most promising clinical applications of QD probes. Additionally, recent reports on decorating QD probes with TAT peptide [59] and wheat germ agglutinin [60] for improving QD transport over a blood-brain barrier and targeting cells of the central nervous system might enable highly accurate and conservative image-guided surgeries on brain tissue.

Targeted and traceable drug delivery

Accurate identification of key molecular targets distinguishing diseased cells from healthy ones enables targeted drug delivery with minimal side-effects. Nanoparticle-based drug carriers show great potential for efficient targeted delivery applications, as they can provide sufficiently long blood circulation, protect the cargo from degradation, possess large drug loading capacity and controlled drug release profile, and integrate multiple targeting ligands on their surface. Additionally, QD probes provide unique functionality of traceable drug delivery, as biodistribution of carriers and intracellular uptake can be monitored via fluorescence.

Several drug delivery applications employing tracing functionality of QDs have been developed recently. For example, Chen et al co-transfected QDs and siRNA using Lipofectamine 2000 and monitored transfection efficiency via QD fluorescence [61]. Mixing QDs with transfection reagent in 1:1 mass ratio provided correlation between the QD signal intensity and the degree of gene silencing. Such an approach enabled the collection of uniformly silenced cell population by fluorescence-activated cell sorting, while introducing minimal modifications into standard siRNA transfection protocol and requiring no chemical modifications of siRNA. Interestingly, additional co-transfection of different siRNA molecules with different QD colors might allow multiplexed monitoring of gene silencing. Yet, indirect link between siRNA and QD transfection imposes certain limitations on this technology, as there is a possibility of interference between QD probes and siRNA transfection resulting in inaccurate correlation of fluorescence signal with the degree of gene silencing. More reliable quantitative information about the number of siRNA molecules delivered into cells can be achieved by using QD-doped chitosan nanobeads developed by Tan et al [62]. In such an approach siRNA molecules are deposited on the surface of nanobeads, and intracellular delivery can be directly monitored by the nanobead fluorescence. Further improvement can be gained from a direct labeling approach demonstrated by Jia et al, who used carbon nanotubes for intracellular delivery of antisense oligonucleotides tagged with QDs [63]. This technology might not only enable a reliable method of quantification of intracellular oligonucleotide concentration, but also provide spatial information about the localization of oligonucleotides within the cell. For example, direct labeling of plasmid DNA with QDs followed by Lipofectamine-mediated transfection enabled long-term study of intracellular and intranuclear localization and transport of plasmid DNA, while preserving the ability of expressing reporter protein encoded by the plasmid [64].

Initial success of highly efficient and traceable intracellular drug delivery utilizing supplementary transfection reagents inspired the design of compact single-QD based carriers with integrated functionalities. Utilization of single-QD drug delivery vehicles for in vivo applications is desirable, as intermediate size of such carriers (~10–20 nm in diameter) reduces the renal clearance as well as uptake by reticulo-endothelial system (RES), thus increasing the blood circulation time and improving the delivery efficiency. Further, QD core can serve as a structural scaffold for loading of various types of drug molecules. For example, small-molecule hydrophobic drugs can be embedded between the inorganic core and the amphiphilic polymer coating layer [65], while hydrophilic therapeutic agents (such as siRNA and antisense oligonucleotides) can be deposited onto the hydrophilic exterior of the polymeric shell (Fig. 11) [41]. Flexibility of the shell design enables engineering of drug carriers with different physical properties (e.g. size, charge, biodegradability, etc), thus providing a large platform for a variety of specific applications.

Figure 11

QD-based drug carriers integrate drug delivery tracing, loading of various types of drugs (e.g. hydrophobic small-molecule drugs between the QD core and polymer coating or hydrophilic drugs on the exterior surface of the polymeric shell), and targeting

Integration of functionality for enhanced cellular uptake and endosomal escape within single-QD probes was demonstrated by Qi and Gao [42]. Encapsulation of QDs with zwitterionic amphipols enabled non-covalent deposition of up to 10 siRNA molecules on the surface of each particle via electrostatic interaction. Efficient endosomal uptake of such particles followed by decrease in pH and shift in particle surface charge resulted in endosomal escape and release of intact siRNA within the cells. While outperforming transfection efficiency of common reagents (such as PEI and Lipofectamine), QD carriers showed substantially lower toxicity in cell cultures. Further, real-time monitoring of particle uptake (via QD fluorescence) and release of siRNA (via labeling of individual siRNA molecules with FITC) was achieved. Targeted siRNA transfection to tumor cells was demonstrated by Defrus et al, who used PEG-coated QDs as a platform for deposition of siRNA and tumor-homing peptide [66]. Attachment of siRNA molecules via cleavable chemical bonds ensured efficient intracellular release of active siRNAs. However, deposition of targeting ligands and cargo molecules in a “parallel” manner introduced competition between the loading capacity and targeting capabilities of the delivery vehicles. In light of successful RNAi experiments with aptamer-siRNA chimeras performed by McNamara et al [67] it is reasonable to expect higher efficiency from vehicles with “serial” attachment of therapeutic molecule and targeting ligand. For small-molecule drug delivery, Bagalkot et al functionalized the QDs with targeting RNA aptamers and loaded anti-cancer drug Doxorubicin via intercalation within the aptamer [68]. Notably, bi-FRET from QD core to Doxorubicin and then to aptamer enabled monitoring of the vehicle disintegration and drug release within the cells via restoration of QD fluorescence.

In vivo drug delivery with QD carriers was demonstrated by Manabe et al [69]. Conjugation of an antihypertensive drug captopril to the QD surface provided the therapeutic effect similar to that of the free drug, while also enabling the monitoring of QD-drug biodistribution over a 96-hour period. With advancements in design of biocompatible QD surface coatings and identification of suitable molecular targets for therapy, QD-based drug delivery vehicles promise to provide an indispensable tool for modeling of pharmacokinetics and pharmacodynamics of nanoparticle-drug carriers.

Challenges of integrating QD technology into clinical practice

Nanotechnology represents a highly dynamic field of research developing novel platforms for a variety of applications. Unique properties of nanomaterials inspire enthusiasm for overcoming limitations of current technology and hold promise of advancing the field of personalized medicine. An increasing number of proof-of-concept studies along with more applied and clinically relevant QD-based tools appearing in a variety of fields ranging from ex vivo molecular fingerprinting of individual cells to in vivo diagnostics and image-guided therapy will undoubtedly make their way into clinical practice. However, there are still a number of challenges on the way of integrating QD technology into biomedical applications.

Unique behavior of nanomaterials compared with small molecules and lack of clinical experience of utilizing nanoparticle-based assays often raise concerns of result reproducibility, reliability, and comparability between each other and conventional techniques. However, increasing numbers of proof-of-concept studies are actively exploring a wide range of possible areas of QD applications. A forthcoming leap towards technologies working in clinical settings along with wide-scale “test-drives” of QD tools and training of technical personnel should encourage interest in QD-based tools, increase familiarity and hands-on experience working with QD probes, and establish confidence in this technology within scientific and medical communities. Among first steps towards this goal, standardization of QD-based assays will be beneficial for making data from different research centers comparable and enabling large-scale clinical studies.

Increasing efforts are focused on the study of the effect of QDs on human health and environment. Partially due to the novelty of nanotechnology, there is not much information about these effects available yet. Short-term and long-term toxicity and immunogenicity of nanoparticles as well as disposal of nanoparticle-containing waste remain a highly debatable area of research and deserve thorough investigation to ensure safety of QD technology in clinical practice [7072]. While early studies of QD toxicity by Derfus et alindicated significant cytotoxicity of unprotected CdSe QDs due to nanoparticle photo-oxidation upon exposure to UV light and release of toxic Cd2+ ions [73, 74], capping of CdSe core with ZnS layer and deposition of a stable coating seemed to dramatically reduce QD toxicity in cell cultures. In a more adequate model based on 3D cell culture (liver tissue spheroids) Lee et al observed substantially decreased nanoparticle-induced toxicity compared to 2D cell cultures, emphasizing the impact of tissue morphology on toxicity [75]. Sometimes conflicting toxicity data might also result from significant over- or under-estimation of cell toxicity determined with standard cell viability assays [76]. In addition to in vitro assays, greater complexity of live organisms with plethora of mechanisms for QD accumulation, degradation, and excretion might require more thorough in vivo toxicity studies. For example, Mancini et al suggested that hypochlorous acid together with hydrogen peroxide produced by phagocytes can diffuse through an otherwise stable secondary coating, causing solubilization of the QD core and release of toxic ions [77]. Additionally, the QD surface coating and particle size play important role in the particle biodistribution and toxicity [71, 78, 79]. Pharmacokinetics studies performed on rat models by Fischer et al have shown that QDs coated with bovine serum albumin (BSA) are efficiently eliminated from the bloodstream by liver uptake, while QDs lacking BSA on their surface are cleared at slower rate [80]. As each QD probe appears to be unique, development of comprehensive assays for QD toxicity assessment should improve our understanding of potential risks of this technology, provide guidance for design of QD probes with minimized adverse effects, and increase the public confidence in QD-based diagnostics and therapeutics.

As promising benefits of QD technology might be hampered by potential adverse effects, design of biocompatible and non-toxic QD probes has become an active area of research. One way of resolving an issue of heavy metal toxicity involves utilization of QD probes made of benign materials. For example, Yonget al recently prepared Cd-free InP/ZnS QDs and utilized these probes for targeting of pancreatic cancer cell lines [81]; however, low quantum yield (~30%) and large size (~30 nm in diameter) might limit utility of such probes for in vivo imaging. Higher-quality probes with quantum yield of up to ~60% and hydrodynamic diameter of 17 nm were developed by Li et al on the basis of CuInS2/ZnS QDs [82]. Further, engineering of low-cost, non-toxic, and potentially biodegradable in vivo imaging probes might become available through utilization of recently developed technology for preparation of water-soluble QDs made of silicon [83, 84] – intert, biocompatible, and abundant material.

While being an attractive approach, Cd-free QDs still suffer from poor stability and inferior photo-physical properties compared to high-quality QDs made of toxic materials (such as CdSe). Therefore, improving biocompatibility of potentially toxic QD probes remains a sound and highly promising alternative, and elimination or reduction of cadmium interaction with live cells seem to be the cornerstone of such approach. There are several feasible strategies to achieving this goal. The toxicity associated with cadmium poisoning comes from a quick release of large amounts of this metal into a bloodstream, its preferential accumulation in kidneys, and consequent nephrotoxicity. However, up to 30 ug/day of dietary Cd (coming from fish, vegetables, and other sources) can be consumed by a healthy adult without adverse effects on kidney function [85]. Therefore, slow degradation of QD probes within a human body followed by urinary excretion might offer a way of safe and efficient elimination of QDs. Adapting technology developed for controlled drug release and coating nanoparticles with biodegradable polymers might provide one strategy for gaining control over QD degradation and Cd release in vivo.

Complete and quick elimination of intact QD probes from the body via renal excretion represents another approach to overcoming possible toxicity. This approach seems especially favorable in light of sparse information on in vivo QD degradation mechanisms and long-term effect of QD accumulation in organs. Systematic investigation of the renal clearance of QDs on rat and mice models done by Choi et al has defined the renal clearance threshold of 5.5 nm and emphasized the role of zwitterionic surface coatings in preventing protein absorption and retaining the original nanoparticles size [79]. Working along this direction, Law et alprepared ultrasmall (3–5 nm in diameter) cysteine-coated CdTe/ZnTe QDs and tested biodistribution of these probes in mice, finding no QDs in liver and spleen 2 weeks post-injection [86]. However, bio-functionalization of QDs, required for targeted imaging and therapy, increases the QD size, thus making renal clearance of functional QD probes difficult. Further, quick renal clearance is often undesirable, as prolonged QD circulation is required for specific targeting, high-sensitivity imaging, and therapeutic efficiency. Therefore, high molecular weight coatings are routinely applied to QD probes to increase their circulation time and improve bioavailability. Ballou et al emphasized the importance of coating with high molecular weight PEG to reduce accumulation of QDs in liver and bone marrow [87], and Prencipe et alachieved remarkably long blood circulation of nanomaterials encapsulated with branched PEG [88]. Utilization of biodegradable ligands that would detach from QD probes after prolonged circulation in blood or due to degradation in target cells, thus releasing single nanoparticles with original size below 5.5 nm, might render renal excretion of functional QD probes feasible.

In some cases complete elimination of QD probes from the body via renal excretion or other means might prove challenging or undesirable. Engineering of ultra-stable QDs encapsulated with inert biocompatible materials might provide an alternative strategy for addressing Cd toxicity issue. If QD integrity within a human body can be retained for many years, biological systems might never be exposed to heavy metal components of the QD core. For example, Ballou et al indicating that intact PEG-coated QDs remained in bone marrow and lymph nodes of mice for several months after injection [87]. While organic coatings, such as polymers and lipids, might still degrade due to exposure to biological environment, utilization of more stable inorganic materials should protect the cores of QD probes for extended periods of time.

Summary

Advancement of personalized medicine is essential for making progress towards combating such complex diseases as cancer and immune system disorders, and incorporation of novel QD-based tools will undoubtedly play a major role in this process. Design of compact, stable, and biocompatible coatings functionalized with targeting agents have already converted QDs into multifunctional nanodevices suitable for in vitro as well as in vivo applications. While certain challenges and concerns regarding QD incorporation into clinical practice remain, and cautiously enthusiastic attitude towards QD-based tools prevails in scientific community, the benefits of this technology will ensure the increasing interest in QDs as more practical and clinically relevant applications are demonstrated and comprehensive toxicity data is made available. With further advances in design and engineering of biocompatible QD probes such applications as image-guided surgery, molecular fingerprinting of diseases, and personalized diagnosis and therapy will become widely available.

7.1.6 Potentials and pitfalls of fluorescent quantum dots for biological imaging

Jaiswal JKSimon SM.
Trends Cell Biol. 2004 Sep; 14(9):497-504.
http://dx.doi.org/10.1016/j.tcb.2004.07.012

Fluorescent semiconductor nanocrystals, known as quantum dots (QDs), have several unique optical and chemical features. These features make them desirable fluorescent tags for cell and developmental biological applications that require long-term, multi-target and highly sensitive imaging. The improved synthesis of water-stable QDs, the development of approaches to label cells efficiently with QDs, and improvements in conjugating QDs to specific biomolecules have triggered the recent explosion in their use in biological imaging. Although there have been many successes in using QDs for biological applications, limitations remain that must be overcome before these powerful tools can be used routinely by biologists.

Glossary
Fluorescence blinking: a property of a single fluorophore to transit between a fluorescent (on) and non-fluorescent (off) phase, which is caused by its transition between a singlet (fluorescent) and a triplet (non fluorescent) state. Blinking occurs in quantum dots because a specific process causes them to switch between their ionized and neutralized states.

Multiphoton microscopy: a process in which more than one photon, each with a fraction of the energy needed to excite fluorescent molecules, is simultaneously absorbed by the fluorophore, resulting in fluoresce emission. This process facilitates the use of infrared light (which, owing to its longer wavelength, penetrates deeper into the tissue) for animal imaging.

Quantum yield: the ratio of photons absorbed to photons emitted by a fluorescent molecule. The quantum yield quantifies the probability that a molecule in an excited state will relax by emitting fluorescence rather than by decaying non-radiatively.

Semiconductor: a material that is an insulator at very low temperature but has considerable electrical conductivity at room temperature. Stoke’s shift: the separation in energy (and thus wavelength) between the excitation and emission spectra.

Box 1. History of biocompatible quantum dots

Ekimov and Onuschenko [46] carried out the first controlled synthesis of semiconductor crystals of nanometer size by heating glass containers with supersaturated solutions of copper and chlorine compounds at high temperatures to cause the controlled precipitation of copper chloride (CuCl). They used additional heating to create, systematically, collections of small crystalline CuCl particles ranging from tens to hundreds of A ˚ ngstroms, which were initially called quantum droplets and later given other names including nanoparticles, nanocrystals, nanocrystallites and Q-dots. This approach provided particles that remained trapped in the glass and thus could not be easily manipulated after synthesis. In 1993, Bawendi’s group [47] developed an approach for quantum dot (QD) synthesis that facilitated the production of high-quality (see Ref. [2]) monodisperse nanoparticle QDs. Their approach allowed the synthesis of QDs that could be dispersed in various solvents and whose surface could be derivatized. These QDs still had poor fluorescence quantum yields (w10%). A subsequent approach led to the large-scale synthesis of more uniform and monodisperse QDs with higher quantum yields (O20%) [48]. It was, however, the approach of coating the QDs with a few layers of zinc sulfide (ZnS) that provided the greatest enhancement of quantum yield (Figure 1a) [3,49]. Because ZnS-coated QDs are hydrophobic, several methods have been used to stabilize them in aqueous solution and to facilitate their conjugation to biomolecules to make them useful for biological imaging. These include (i) embedding them in a silica or siloxane shell with a thickness of 1–5 nm and with amine, thiol or carboxyl functional groups on its surface [17,50]; (ii) derivatizing their surface with mercaptoacetic acid [18]; (iii) encapsulating them in phospholipid micelles [16]; (iv) derivatizing their surface with dihydroxylipoic acid [2]; and (v) coating them with an amine-modified polyacrylic acid [13].

http://ars.els-cdn.com/content/image/1-s2.0-S0962892404001916-gr1.sml

Figure 1. Properties of bioconjugatable quantum dots (QDs). (a) QDs are inorganic fluorophores and consist of a cadmium selenide (CdSe) core with several layers of a thick zinc sulfide (ZnS) shell to improve quantum yield and photostability. (b) The excitation spectrum (broken lines) of a QD (green) is very broad, whereas that of an organic dye (rhodamine, orange) is narrow. The emission spectrum (unbroken lines) is narrower for a QD (green) than for organic dyes (rhodamine, orange). Values indicate the full spectral width at half-maximum intensity (FWHM value). (c) The emission of the QDs can be tuned by controlling the size of the CdSe core: an increase in the size of the core shifts the emission to the red end of the spectrum. The combined size of the core and the shell of QDs emitting in the visible region of spectra are in the size range of commonly used fluorescent proteins such as green fluorescent protein (GFP) and DsRed. (d,e) To provide specificity of binding, QDs are conjugated with antibody molecules (blue) by using avidin (purple) or protein A (green) as linkers. Between 10 and 15 linker molecules can be attached covalently or electrostatically to a single QD, which facilitates the binding of many or a few (note the presence offree linker molecules) antibody molecules on each QD. Note that, although the QDs and molecules are drawn to size, their binding sites and relative topologies are shown hypothetically

http://ars.els-cdn.com/content/image/1-s2.0-S0962892404001916-gr2.sml

Figure 2. Specific labeling of live cells with quantum dots (QDs). (a) Positively charged avidin and maltose-binding protein containing a positively charged tail (MBPzb) selfassemble on the negatively charged surface of QDs capped with dihydrolipoic acid (DHLA) and can bind to biotinylated molecules such as antibodies specific for Pgp. (b) Transient transfection of HeLa cells with Pgp–GFP (green fluorescent protein) results in its expression in a subset of cells (not marked with arrows). The subsequent incubation of all cells with biotin-conjugated antibodies specific for Pgp, followed by avidin-conjugated QDs, leads to labeling of the cell membrane with the QD bioconjugates: only cells that express detectable levels of Pgp–GFP, and not those that do not express Pgp–GFP (marked with arrows), are labeled [48]. Yellow coloring in the fluorescence image indicates an overlap of green (Pgp–GFP) and red (QD bioconjugate) fluorescence emission. (See Ref. [2] for further details).

Box 2. Specific labeling of biomolecules in vitro

Quantum dots (QDs) have been used to tag molecules of interest both selectively and stably. One approach involves capping the surface of QDs with dihydroxylipoic acid (DHLA), which makes the QD surface negatively charged [2]; this enables QDs to bind to linker molecules, such as protein G engineered to carry a positively charged tail (PGzb) or avidin, which is innately positively charged. These linker molecules provide the specificity to bind the molecule of interest through interactions between either PGzb and antibody or avidin andbiotin (Figure2a). Such QDbioconjugateshave beenused to detect simultaneously as little as 10K9 g of single or multiple toxins and small molecules in vitro [6,20]. Specific biomolecules can be detected despite an excess of other nonspecific biomolecules; the specificity is limited only by the specificity of the antibody used [6]. Collectively, theseresults haveprovedthatQDscanbe conjugatedto biomolecules without compromising their biological activity. Because QDs are brighter than most conventional fluorophores, their use should increase the sensitivity of all fluorescence-based assays. In addition, QDs have been shown to be inert when conjugated via other approaches and when used to detect other molecules such as protein ligands [11,51]. For example, QDs have found a major application in the area of nucleic acid detection [52–54], where QD-tagged probes are being used for the simultaneous detection of multiple nucleic acids [52,53]. The ability to identify simultaneously (not sequentially) and specifically different molecules in a single solution significantly expedites high-throughput chemical screening and holds the potential to revolutionize microarray-based approaches for large-scale studies of the gene expression profiles of organisms.

Box 3. How to get quantum dots into cells

Owing to their size and chemical nature, quantum dots (QDs) cannot diffuse through the cell membrane. To use QDs for labeling and imaging cytoplasmic proteins, the QDs must be delivered by invasive approaches such as microinjection [16], cationic lipidbased reagents [7] or conjugation to membrane-permeable peptides [30]. However, these approaches can cause the intracellular QDs to aggregate in punctae or to end up in endosomes [26,55], instead of being dispersed in the cytosol. Crucial challenges to using QDs for intracellular imaging are (i) the development of non-invasive approaches for the efficient intracellular deliveryanddispersalofQDs;(ii)thedevelopmentofmethodstolabel intracellularproteinsthatarelocatedinanenvironmentvastlydifferent from the extracellular space; and (iii) the development of QDs that eitherareinerttothecytoplasmicenvironmentorrespondinadefined manner to selective changes of the cytoplasmic environment.

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