Posts Tagged ‘Cancer – General’

Nanoparticle Delivery to Cancer Drug Targets

Posted in Cancer and Current Therapeutics, Cell Biology, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Disease Biology, Drug Delivery Platform Technology, Drug Toxicity, Nanotechnology for Drug Delivery, tagged Cancer - General, nano–bio interaction, nanoparticles, nanotechnology, targeting on February 29, 2016| Leave a Comment »

Nanoparticle Delivery to Cancer Drug Targets

Curator: Larry H. Bernstein, MD, FCAP

UPDATED 8/05/2022

 

 

 

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Lipid-based drug delivery (LBDD) systems have gained much importance in the recent years due to their ability to improve the solubility and bioavailability of drugs with poor water solubility⁹. The absorption of drug from lipid based formulation depends on numerous factors, including particle size, degree of emulsification, rate of dispersion and precipitation of drug upon dispersion^4 and 10.

Diagram of liposome showing a phospholipid bilayer surrounding an aqueous interior

This diagram shows several ways in which transport across the BBB works. For nanoparticle delivery across the BBB, the most common mechanisms are receptor-mediated transcytosis and adsorptive transcytosis

Wikipedia

Targeted Polymeric Nanotherapeutics

Author: Jeff Hrkach

New drug-delivery strategies will lead to safer, more effective treatments for previously intractable diseases.

This paper provides an overview of steps being taken by BIND Biosciences Inc. to translate innovative research conducted at the Massachusetts Institute of Technology (MIT) and Harvard Medical School into novel, targeted, polymeric nanotherapeutics.

https://www.nae.edu/File.aspx?id=12467

Schematic diagram of a BIND targeted polymeric nanoparticle.

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Drugs delivered by nanoparticles hold promise for targeted treatment of many diseases, including cancer. However, the particles have to be injected into patients, which has limited their usefulness so far.

Now, researchers from MIT and Brigham and Women’s Hospital (BWH) have developed a new type of nanoparticle that can be delivered orally and absorbed through the digestive tract, allowing patients to simply take a pill instead of receiving injections.

The BIND Targeted Nanoparticle
BIND Biosciences Inc. (BIND), a biopharmaceutical company that was founded upon the research of two pioneers in nanoparticle drug delivery, Professor Robert Langer of MIT and Professor Omid Farokhzad of Brigham and Women’s Hospital of the Harvard Medical School, has developed methods of engineering targeted nanoparticles composed of biodegradable and biocompatible polymers with precise biophysicochemical properties optimized to deliver drugs for specific therapeutic applications (Gu et al., 2008).

The foundational research by Langer and Farokhzad put BIND in a position to pursue the development of targeted polymeric nanotherapeutics for treating several diseases. BIND’s lead program is focused on translating their innovative academic findings into improved treatments for patients with cancer. The BIND technology offers a unique combination of long-circulating nanoparticles with the capability of targeting diseased cells specifically and releasing drugs from nanoparticles in a programmable, controlled way.

Figure 1 is a schematic diagram of a BIND targeted nanoparticle. The targeting ligand enables the nano-particle to recognize specific proteins or receptors on the surface of cells involved in disease, or in the surrounding extracellular matrix, and bind, with high specificity and avidity, to its intended cellular target site. Many types of cancer have been shown to have cell-surface receptors that are highly expressed on the cancer cells (e.g., prostate cancer [prostate-specific membrane antigen, PSMA], breast cancer [human epidermal growth factor receptor 2, HER-2], and lung cancer [epidermal growth factor receptor, EGFR]), and many drugs are being evaluated that might improve treatment outcomes.

Surface Functionalization
Surface functionalization imparted by a PEG component shields the targeted nanoparticles from MPS immune clearance, while providing an attachment site for the targeting ligand on the particle surface at precise, controlled levels through proprietary linkage strategies. A key to the successful development of BIND targeted nanoparticles is the optimization of the nanoparticle surface, which requires a precise balance between the targeting ligand and PEG coverage so the nanoparticle surface is masked enough to provide circulation times long enough to reach the disease site and enough targeting ligand on the surface to effectively bind to the target cell surface receptors. This delicate balance requires precise control over the nanoparticle production process. It also requires the discovery and selection of ligands that are potent and specific enough to bind selectively to the targeted disease cells while remaining bound to the nanoparticle surface.

The polymer matrix, the bulk of the nanoparticle composition, encapsulates the drug in a matrix of clinically safe, validated biodegradable and biocompatible polymers that can be designed to provide appropriate particle size, drug-loading level, drug-release profile, and other critical properties. A variety of drugs or therapeutic payloads can be incorporated into the targeted nanoparticles, including small molecules, peptides, proteins, and nucleic acids, such as siRNA.

Composite magnetic nanoparticle drug delivery system
US 20120265001 A1

ABSTRACT

A composite magnetic nanoparticle drug delivery system provides targeted controlled release chemotherapies for cancerous tumors and inflammatory diseases. The magnetic nanoparticle includes a biocompatible and biodegradable polymer, a magnetic nanoparticle, the biological targeting agent human serum albumin, and a therapeutic pharmaceutical composition. The composite nanoparticles are prepared by oil-in-oil emulsion/solvent evaporation and high shear mixing. An externally applied magnetic field draws the magnetic nanoparticles to affected areas. The biological targeting agent draws the nanoparticles into the affected tissues. Polymer degradation provides controlled time release delivery of the pharmaceutical agent.

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Nanoparticle delivery systems for cancer therapy: advances in clinical and preclinical research.

Egusquiaguirre SP¹, Igartua M, Hernández RM, Pedraz JL.

Clin Transl Oncol. 2012 Feb;14(2):83-93.                      http://dx.doi.org:/10.1007/s12094-012-0766-6.

Nanoparticle drug delivery systems exploit the abnormal characteristics of tumour tissues to selectively target their payloads to cancer cells, either by passive, active or triggered targeting.

Drug delivery and nanoparticles: Applications and hazards

Wim H De Jong¹ and Paul JA Borm
Int J Nanomedicine. 2008 Jun; 3(2): 133–149.

The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly. Currently many substances are under investigation for drug delivery and more specifically for cancer therapy. Interestingly pharmaceutical sciences are using nanoparticles to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient. The kind of hazards that are introduced by using nanoparticles for drug delivery are beyond that posed by conventional hazards imposed by chemicals in classical delivery matrices. For nanoparticles the knowledge on particle toxicity as obtained in inhalation toxicity shows the way how to investigate the potential hazards of nanoparticles. The toxicology of particulate matter differs from toxicology of substances as the composing chemical(s) may or may not be soluble in biological matrices, thus influencing greatly the potential exposure of various internal organs. This may vary from a rather high local exposure in the lungs and a low or neglectable exposure for other organ systems after inhalation. However, absorbed species may also influence the potential toxicity of the inhaled particles. For nanoparticles the situation is different as their size opens the potential for crossing the various biological barriers within the body. From a positive viewpoint, especially the potential to cross the blood brain barrier may open new ways for drug delivery into the brain. In addition, the nanosize also allows for access into the cell and various cellular compartments including the nucleus. A multitude of substances are currently under investigation for the preparation of nanoparticles for drug delivery, varying from biological substances like albumin, gelatine and phospholipids for liposomes, and more substances of a chemical nature like various polymers and solid metal containing nanoparticles. It is obvious that the potential interaction with tissues and cells, and the potential toxicity, greatly depends on the actual composition of the nanoparticle formulation. This paper provides an overview on some of the currently used systems for drug delivery. Besides the potential beneficial use also attention is drawn to the questions how we should proceed with the safety evaluation of the nanoparticle formulations for drug delivery. For such testing the lessons learned from particle toxicity as applied in inhalation toxicology may be of use. Although for pharmaceutical use the current requirements seem to be adequate to detect most of the adverse effects of nanoparticle formulations, it can not be expected that all aspects of nanoparticle toxicology will be detected. So, probably additional more specific testing would be needed.

Recent years have witnessed unprecedented growth of research and applications in the area of nanoscience and nanotechnology. There is increasing optimism that nanotechnology, as applied to medicine, will bring significant advances in the diagnosis and treatment of disease. Anticipated applications in medicine include drug delivery, both in vitro and in vivo diagnostics, nutraceuticals and production of improved biocompatible materials (Duncan 2003; De Jong et al 2005; ESF 2005; European Technology Platform on Nanomedicine 2005; Ferrari 2005). Engineered nanoparticles are an important tool to realize a number of these applications. It has to be recognized that not all particles used for medical purposes comply to the recently proposed and now generally accepted definition of a size ≤100 nm (The Royal Society and Royal Academy of Engineering 2004). However, this does not necessarily has an impact on their functionality in medical applications. The reason why these nanoparticles (NPs) are attractive for medical purposes is based on their important and unique features, such as their surface to mass ratio that is much larger than that of other particles, their quantum properties and their ability to adsorb and carry other compounds. NPs have a relatively large (functional) surface which is able to bind, adsorb and carry other compounds such as drugs, probes and proteins. However, many challenges must be overcome if the application of nanotechnology is to realize the anticipated improved understanding of the patho-physiological basis of disease, bring more sophisticated diagnostic opportunities, and yield improved therapies. Although the definition identifies nanoparticles as having dimensions below 0.1 μm or 100 nm, especially in the area of drug delivery relatively large (size >100 nm) nanoparticles may be needed for loading a sufficient amount of drug onto the particles. In addition, for drug delivery not only engineered particles may be used as carrier, but also the drug itself may be formulated at a nanoscale, and then function as its own “carrier” (Cascone et al 2002; Baran et al 2002; Duncan 2003; Kipp 2004). The composition of the engineered nanoparticles may vary. Source materials may be of biological origin like phospholipids, lipids, lactic acid, dextran, chitosan, or have more “chemical” characteristics like various polymers, carbon, silica, and metals. The interaction with cells for some of the biological components like phospholipids will be quite different compared to the non biological components such as metals like iron or cadmium. Especially in the area of engineered nanoparticles of polymer origin there is a vast area of possibilities for the chemical composition.

Although solid NPs may be used for drug targeting, when reaching the intended diseased site in the body the drug carried needs to be released. So, for drug delivery biodegradable nanoparticle formulations are needed as it is the intention to transport and release the drug in order to be effective. However, model studies to the behavior of nanoparticles have largely been conducted with non-degradable particles. Most data concerning the biological behavior and toxicity of particles comes from studies on inhaled nanoparticles as part of the unintended release of ultrafine or nanoparticles by combustion derived processes such as diesel exhaust particles (reviewed by Oberdörster 1996; Donaldson et al 2001, 2004; Borm 2002;Donaldson and Stone 2003; Dreher 2004; Kreyling et al 2004; Oberdörster, Oberdörster et al 2005). Research has demonstrated that exposure to these combustion derived ultrafine particles/nanoparticles is associated with a wide variety of effects (Donaldson et al 2005) including pulmonary inflammation, immune adjuvant effects (Granum and Lovik 2002) and systemic effects including blood coagulation and cardiovascular effects (Borm and Kreyling 2004;Oberdorster, Oberdörster et al 2005). Since the cut-off size for both ultrafine and nanoparticles (100 nm) is the same, now both terms are used as equivalent. Based on the adverse effects of ultrafine particles as part of environmental pollution, engineered nanoparticles may be suspected of having similar adverse effects. It is the purpose of this review to use this database on combustion derived nanpoarticles (CDNP) obtained by inhalation toxicology and epidemiology and bridge the gap to engineered nanoparticles.

Nanoparticles and drug delivery

Drug delivery and related pharmaceutical development in the context of nanomedicine should be viewed as science and technology of nanometer scale complex systems (10–1000 nm), consisting of at least two components, one of which is a pharmaceutically active ingredient (Duncan 2003; Ferrari 2005), although nanoparticle formulations of the drug itself are also possible (Baran et al 2002; Cascone et al 2002; Duncan 2003; Kipp 2004). The whole system leads to a special function related to treating, preventing or diagnosing diseases sometimes called smart-drugs or theragnostics (LaVan et al 2003). The primary goals for research of nano-bio-technologies in drug delivery include:

• More specific drug targeting and delivery,
• Reduction in toxicity while maintaining therapeutic effects,
• Greater safety and biocompatibility, and
• Faster development of new safe medicines.

The main issues in the search for appropriate carriers as drug delivery systems pertain to the following topics that are basic prerequisites for design of new materials. They comprise knowledge on (i) drug incorporation and release, (ii) formulation stability and shelf life (iii) biocompatibility, (iv) biodistribution and targeting and (v) functionality. In addition, when used solely as carrier the possible adverse effects of residual material after the drug delivery should be considered as well. In this respect biodegradable nanoparticles with a limited life span as long as therapeutically needed would be optimal.

Table 1  presents some of the types of chemical structures and possibilities for the preparation of nanoscale materials used as pharmaceutical carrier system (reviewed in Borm and Muller-Schulte 2006). Certainly none of the so far developed carriers fulfill all the parameters mentioned above to the full extent; the progress made in nanotechnology inter alia emerging from the progress in the polymer-chemistry, however, can provide an intriguing basis to tackle this issue in a promising way.

Table 1

Overview of nanoparticles and their applications in Life Sciences

Particle class	Materials	Application
Natural materials or derivatives	Chitosan Dextrane Gelatine Alginates Liposomes Starch	Drug/Gene delivery
Dendrimers	Branched polymers	Drug delivery
Fullerenes	Carbon based carriers	Photodynamics Drug delivery
Polymer carriers	Polylactic acid Poly(cyano)acrylates Polyethyleinemine Block copolymers Polycaprolactone	Drug/gene delivery
Ferrofluids	SPIONS USPIONS	Imaging (MRI)
Quantum dots	Cd/Zn-selenides	Imaging In vitro diagnostics
Various	Silica-nanoparticles Mixtures of above	Gene delivery

Nanoparticle delivery system to tackle cancer

By Tim Sandle     Feb 23, 2016 in Science            http://www.digitaljournal.com/science/nanoparticle-delivery-system-to-tackle-cancer/article/458373

Directing drug treatment to tumors is a hit-or-miss activity. Considerable research efforts are going into improving targeted drug delivery. A new approach centers on nanotechnology

Nanoparticle delivery system to tackle cancer 

Cancer drugs are injected into the bloodstream and move through the body seeking out fast-growing cancer cells. One consequence of chemotherapy is the unintended effect on different parts of the body, including messing up the digestive system. Such side effects can be minimized if the drug is better targeted.

Another consequence of the poor targeting of some chemo drugs is that they miss cancer cells entirely.

For these reasons, different research groups are focusing on drug delivery: finding smart ways to direct the anti-cancer drug to the required target. One such research team is led by Professor Warren Chan of the University of Texas.

Professor Chan thinks the answer to more effective targeting is the use of nanoparticles. In trials, the research group has used nanoparticles attached to strands of DNA that can, remarkably, change shape to gain improved access to cancerous tissue.

Interviewed by Pharmaceutical Processing, Professor Chan explains: “Your body is basically a series of compartments.” He added: “Think of it as a giant house with rooms inside. We’re trying to figure out how to get something that’s outside, into one specific room.”

The complication with the approach is based on different cancers. Because different types of cancer differ in morphology, and cancers at different stages equally vary, selecting the appropriate nanoparticle is important. Here the research group have been looking at nanoparticles of varying sizes and shapes, as well as different coatings.

The solution is to create nanoparticles that can change shape to meet different types of tumors. This structural alteration makes the technology more versatile and means treatments could be delivered more quickly, rather than waiting for test results to assess the size and shape of the tumor.

The shape-shifting has been achieved by constructing the nanoparticles from tiny fragments of metal and then attaching DNA to them. The DNA acts as a means for marking the cancer, and then allowing the chemotherapy drug to attack the tumor.

The research is published in the journal Proceedings of the National Academy of Sciences. The paper is titled “Tailoring nanoparticle designs to target cancer based on tumor pathophysiology.”

Tailoring nanoparticle designs to target cancer based on tumor pathophysiology

Edward A. Sykes^a, Qin Dai^a, Christopher D. Sarsons^b, Juan Chen^c, Jonathan V. Rocheleau^a,^d, David M. Hwang^e, Gang Zheng^c, David T. Cramb^f, Kristina D. Rinker^b, and Warren C. W. Chan^a,^g,^h,ⁱ,^j,¹   Author Affiliations

Proc Nat Acad Sci  Feb 16, 2016;                                 http://dx.doi.org:/10.1073/pnas.1521265113www.pnas.org/lookup/suppl/doi:10.1073/pnas.1521265113/-/DCSupplemental.

Significance

Nanotechnology is a promising approach for improving cancer diagnosis and treatment with reduced side effects. A key question that has emerged is: What is the ideal nanoparticle size, shape, or surface chemistry for targeting tumors? Here, we show that tumor pathophysiology and volume can significantly impact nanoparticle targeting. This finding presents a paradigm shift in nanomedicine away from identifying and using a universal nanoparticle design for cancer detection and treatment. Rather, our results suggest that future clinicians will be capable of tailoring nanoparticle designs according to the patient’s tumor characteristics. This concept of “personalized nanomedicine” was tested for detection of prostate tumors and was successfully demonstrated to improve nanoparticle targeting by over 50%.

 

Abstract

Nanoparticles can provide significant improvements in the diagnosis and treatment of cancer. How nanoparticle size, shape, and surface chemistry can affect their accumulation, retention, and penetration in tumors remains heavily investigated, because such findings provide guiding principles for engineering optimal nanosystems for tumor targeting. Currently, the experimental focus has been on particle design and not the biological system. Here, we varied tumor volume to determine whether cancer pathophysiology can influence tumor accumulation and penetration of different sized nanoparticles. Monte Carlo simulations were also used to model the process of nanoparticle accumulation. We discovered that changes in pathophysiology associated with tumor volume can selectively change tumor uptake of nanoparticles of varying size. We further determine that nanoparticle retention within tumors depends on the frequency of interaction of particles with the perivascular extracellular matrix for smaller nanoparticles, whereas transport of larger nanomaterials is dominated by Brownian motion. These results reveal that nanoparticles can potentially be personalized according to a patient’s disease state to achieve optimal diagnostic and therapeutic outcomes.

 

 

Curr Pharm Des. 2013;19(37):6560-74.

Mechanisms for targeted delivery of nanoparticles in cancer.

Beech JR¹, Shin SJ, Smith JA, Kelly KA.  Author information     

With the evolution of the “omics” era, our molecular understanding of cancer has exponentially increased, leading to the development of the concept of personalized medicine. Nanoparticle technology has emerged as a way to combine cancer specific targeting with multifunctionality, such as imaging and therapy, leading to advantages over conventional small molecule based approaches. In this review, we discuss the targeting mechanisms of nanoparticles, which can be passive or active. The latter utilizes small molecules, aptamers, peptides, and antibodies as targeting moieties incorporated into the nanoparticle surface to deliver personalized therapy to patients.

PMID: 23621529

 

 

Nanoparticle-based targeted drug delivery

Rajesh Singh1 and James W. Lillard Jr.1
Exp Mol Pathol. 2009 June ; 86(3): 215–223.     http://dx.doi.org:/10.1016/j.yexmp.2008.12.004

Nanotechnology could be defined as the technology that has allowed for the control, manipulation, study, and manufacture of structures and devices in the “nanometer” size range. These nano-sized objects, e.g., “nanoparticles”, take on novel properties and functions that differ markedly from those seen from items made of identical materials. The small size, customized surface, improved solubility, and multi-functionality of nanoparticles will continue to open many doors and create new biomedical applications. Indeed, the novel properties of nanoparticles offer the ability to interact with complex cellular functions in new ways. This rapidly growing field requires crossdisciplinary research and provides opportunities to design and develop multifunctional devices that can target, diagnose, and treat devastating diseases such as cancer. This article presents an overview of nanotechnology for the biologist and discusses the attributes of our novel XPclad© nanoparticle formulation that has shown efficacy in treating solid tumors, for single dose vaccination, and oral delivery of therapeutic proteins.

The development of a wide spectrum of nanoscale technologies is beginning to change the scientific landscape in terms of disease diagnosis, treatment, and prevention. These technological innovations, referred to as nanomedicines by the National Institutes of Health, have the potential to turn molecular discoveries arising from genomics and proteomics into widespread benefit for patients. Nanoparticles can mimic or alter biological processes (e.g., infection, tissue engineering, de novo synthesis, etc.). These devices include, but are not limited to, functionalized carbon nanotubes, nanomachines (e.g., constructed from interchangeable DNA parts and DNA scaffolds), nanofibers, self-assembling polymeric nanoconstructs, nanomembranes, and nano-sized silicon chips for drug, protein, nucleic acid, or peptide delivery and release, and biosensors and laboratory diagnostics.

Nanotechnology-based Drug Delivery in Cancer

Drug delivery in cancer is important for optimizing the effect of drugs and reducing toxic side effects. Several nanotechnologies, mostly based on nanoparticles, can facilitate drug delivery to tumors.

Hydrogels

Hydrogel-nanoparticles are based on proprietary technology that uses hydrophobic polysaccharides for encapsulation and delivery of drug, therapeutic protein, or vaccine antigen. A novel system using cholesterol pullulan shows great promise. In this regard, four cholesterol molecules gather to form a self-aggregating hydrophobic core with pullulan outside. The resulting cholesterol nanoparticles stabilize entrapped proteins by forming this hybrid complex. These particles stimulate the immune system and are readily taken up by dendritic cells. Alternatively, larger hydrogels can encapsulate and release monoclonal antibodies.

Curcumin, a substance found in the cooking spice turmeric, has long been known to have anti-cancer properties. Nevertheless, widespread clinical application of this relatively efficacious agent has been limited due to its poor solubility and minimal systemic bioavailability. This problem has been resolved by encapsulating curcumin in a polymeric nanoparticle, creating “nanocurcumin” (Bisht et al., 2007). Further, the mechanism of action of nanocurcumin on pancreatic cancer cells mirrors that of free curcumin, including induction of apoptosis, blockade of nuclear factor kappa B (NFκB) activation, and downregulation of pro-inflammatory cytokines (i.e., IL-6, IL-8 and TNF-α). Nanocurcumin provides an opportunity to expand the clinical repertoire of this efficacious agent by enabling soluble dispersion. Future studies utilizing nanocurcumin are warranted in preclinical in vivo models of cancer and other diseases that might benefit from the effects of curcumin.

Micelles and liposomes

Block-copolymer micelles are spherical super-molecular assemblies of amphiphilic copolymer. The core of micelles can accommodate hydrophobic drugs, and the shell is a hydrophilic brush-like corona that makes the micelle water soluble, thereby allowing delivery of the poorly soluble contents. Camptothecin (CPT) is a topoisomerase I inhibitor that is effective against cancer, but clinical application of CPT is limited by its poor solubility, instability, and toxicity. Biocompatible, targeted sterically stabilized micelles (SSM) have been used as nanocarriers for CPT (CPT-SSM). CPT solubilization in SSM is expensive yet reproducible and is attributed to avoidance of drug aggregate formation. Furthermore, SSM composed of PEGylated phospholipids are attractive nanocarriers for CPT delivery because of their size (14 nm) and ability to extravasate through the leaky microvasculature of tumors and inflamed tissues. This passive targeting results in high drug concentration in tumors and reduced drug toxicity to the normal tissues (Koo et al., 2006).

Stealth micelle formulations have stabilizing PEG coronas to minimize opsonization of the micelles and maximize serum half-life. Currently, SP1049C, NK911, and Genexol-PM have been approved for clinical use (Sutton et al., 2007). SP1049C is formulated as doxorubicin (DOX)-encapsulated pluronic micelles. NK911 is DOX-encapsulated micelles from a copolymer of PEG-DOX-conjugated poly(aspartic acid), and Genexol-PM is a paclitaxelencapsulated PEG-PLA micelle formulation. Polymer micelles have several advantages over other drug delivery systems, including increased drug solubility, prolonged circulation halflife, selective accumulation at tumor sites, and lower toxicity. However, at the present time this technology lacks tumor specificity and the ability to control the release of the entrapped agents. Indeed, the focus of nano-therapy has gradually shifted from passive targeting systems (e.g., micelles) to active targeting.

Super paramagnetic iron oxide particles can be used in conjunction with magnetic resonance imaging (MRI) to localize the tumor as well as for subsequent thermal ablation. This has been used, for example, to target glioblastoma multiforme (GBM), a primary malignant tumor of the brain with few effective therapeutic options. The primary difficulty in treating GBM lies in the difficulty of delivering drugs across the BBB. However, nanoscale liposomal iron oxide preparations were recently shown to improve passage across the BBB (Jain, 2007).

 

Nanomaterial formulation

Nanomaterials have been successfully manipulated to create a new drug-delivery system that can solve the problem of poor water solubility of most promising currently available anticancer drugs and, thereby, increase their effectiveness. The poorly soluble anticancer drugs require the addition of solvents in order for them to be easily absorbed into cancer cells. Unfortunately, these solvents not only dilute the potency of the drugs but create toxicity. Researchers from the University of California Los Angeles California Nanosystem Institute have devised a novel approach using silica-based nanoparticles to deliver the anticancer drug CPT and other water insoluble drugs to cancer cells (Lu et al., 2007). The method incorporates the hydrophobic anticancer drug CPT into the pores of fluorescent mesoporous silica nanoparticles and delivers the particles into a variety of human cancer cells to induce cell death. The results suggest that the mesoporous silica nanoparticles might be used as a vehicle to overcome the insolubility of many anticancer drugs.

Nanosystems

Novel nanosystems can be pre-programmed to alter their structure and properties during the drug delivery process, allowing for more effective extra- and intra-cellular delivery of encapsulated drug (Wagner, 2007). This is achieved by the incorporation of molecular sensors that respond to physical or biological stimuli, including changes in pH, redox potential, or enzymes. Tumor-targeting principles include systemic passive targeting and active receptor targeting. Physical forces (e.g., electric or magnetic fields, ultrasound, hyperthermia, or light) may contribute to focusing and triggering activation of nano systems. Biological drugs delivered with programmed nanosystems also include plasmid DNA, siRNA, and other therapeutic nucleic acids.

Using a degradable, polyamine ester polymer, polybutanediol diacrylate co amino pentanol (C32), a diptheria toxin suicide gene (DT-A) driven by a prostate-specific promoter was directly injected into normal prostate and prostate tumors in mice (Peng et al., 2007). This C32/DT-A system resulted in significant size reduction, apoptosis in 50% of normal prostate. However, a single injection of C32/DT-A triggered apoptosis in 80% of tumor cells present in the tissue. It is expected that multiple nanoparticle injection would trigger a great percentage of prostate tumor cells to undergo apoptosis. These results suggest that local delivery of polymer/DT-A nanoparticles may have application in the treatment of benign prostatic hypertrophy and prostate cancer.

Multidrug resistance (MDR) of tumor cells is known to develop through a variety of molecular mechanisms. Glucosylceramide synthase (GCS) is responsible for the activation of the pro-apoptotic mediator, ceramide, to a nonfunctional moiety, glucosylceramide. This molecule is over-expressed by many MDR tumor types and has been implicated in cell survival in the presence of chemotherapy. A study has investigated the therapeutic strategy of co-administering ceramide with paclitaxel in an attempt to restore apoptotic signaling and overcome MDR in a human ovarian cancer cell line using modified poly(epsiloncaprolactone) (PEO-PCL) nanoparticles to encapsulate and deliver the therapeutic agents for enhanced efficacy (van Vlerken and Amiji, 2006). Results show that MDR cancer cells can be completely eradicated by this approach. Using this approach, MDR cells can be resensitized to a dose of paclitaxel near the IC50 of non-MDR cells. Molecular analysis of activity verified the hypothesis that the efficacy of this therapeutic approach is due to a restoration in apoptotic signaling, showing the promising potential for clinical use of this therapeutic strategy to overcome MDR.

Nanocells

Indiscriminate drug distribution and severe toxicity of systemic administration of chemotherapeutic agents can be overcome through encapsulation and cancer cell targeting of chemotherapeutics in 400 nm nanocells, which can be packaged with significant concentrations of chemotherapeutics of different charge, hydrophobicity, and solubility (MacDiarmid et al., 2007). Targeting of nanocells via bispecific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release. Doses of drugs delivered via nanocells are ∼1,000 times less than the dose of the free drug required for equivalent tumor regression. It produces significant tumor growth inhibition and regression in mouse xenografts and lymphoma in dogs, despite administration of minute amounts of drug and antibody. Indeed, reduced dosage is a critical factor for limiting systemic toxicity. Clinical trials are planned for testing this method of drug delivery.

Dendrimers

In early studies, dendrimer-based drug delivery systems focused on encapsulating drugs. However, it was difficult to control the release of drugs associated with dendrimers. Recent developments in polymer and dendrimer chemistry have provided a new class of molecules called dendronized polymers, which are linear polymers that bear dendrons at each repeat unit. Their behavior differs from that of linear polymers and provides drug delivery advantages because of their enhanced circulation time. Another approach is to synthesize or conjugate the drug to the dendrimers so that incorporating a degradable link can be further used to control the release of the drug.

DOX was conjugated to a biodegradable dendrimer with optimized blood circulation time through the careful design of size and molecular architecture (Lee et al., 2006). Specifically, the DOX-dendrimer controlled drug-loading through multiple attachment sites, solubility through PEGylation, and drug release through the use of pH-sensitive hydrazone dendrimer linkages. In culture, DOX-dendrimers were >10 times less toxic than free DOX toward colon carcinoma cells. Upon intravenous administration to tumor bearing mice, tumor uptake of DOX-dendrimers were nine-fold higher than intravenous free DOX and caused complete tumor regression and 100% survival of the mice after 60 days.

Nanotubes Even though it was previously possible to attach drug molecules directly to antibodies, attaching more than a handful of drug molecules to an antibody significantly limits its targeting ability because the chemical bonds that are used tend to impede antibody activity. A number of nanoparticles have been investigated to overcome this limitation. Tumor targeting single-walled carbon nano-tube (SWCNT) have been synthesized by covalently attaching multiple copies of tumor-specific monoclonal antibodies (MAbs), radiation ion chelates and fluorescent probes (McDevitt et al., 2007). A new class of anticancer compound was created that contains both tumor-targeting antibodies and nanoparticles called fullerenes (C60). This delivery system can be loaded with several molecules of an anticancer drug, e.g., Taxol® (Ashcroft et al., 2006). It is possible to load as many as 40 fullerenes onto a single skin cancer antibody called ZME-108, which can be used to deliver drugs directly into melanomas. Certain binding sites on the antibody are hydrophobic (water repelling) and attract the hydrophobic fullerenes in large numbers so multiple drugs can be loaded into a single antibody in a spontaneous manner. No covalent bonds are required, so the increased payload does not significantly change the targeting ability of the antibody. The real advantage of fullerene-based therapies vs. other targeted therapeutic agents is likely to be fullerene’s potential to carry multiple drug payloads, such as taxol plus other chemotherapeutic drugs. Cancer cells can become drug resistant, and one can cut down on the possibility of their escaping treatment by attacking them with more than one kind of drug at a time. The first fullerene immuno-conjugates have been prepared and characterized as an initial step toward the development of fullerene immunotherapy.

Polymersomes

Polymersomes, hollow shell nanoparticles, have unique properties that allow delivery of distinct drugs. Loading, delivery and cytosolic uptake of drug mixtures from degradable polymersomes were shown to exploit the thick membrane of these block copolymer vesicles, their aqueous lumen, and pH-triggered release within endolysosomes. Polymersomes break down in the acidic environments for targeted release of these drugs within tumor cell endosomes. While cell membranes and liposomes are created from a double layer of phospholipids, a polymersome is comprised of two layers of synthetic polymers. The individual polymers are considerably larger than individual phospholipids but have many of the same chemical features.

Polymersomes have been used to encapsulate paclitaxel and DOX for passive delivery to tumor-bearing mice (Ahmed et al., 2006). The large polymers making up the polymersome allows paclitaxel, which is water insoluble, to embed within the shell. DOX is water-soluble and stays within the interior of the polymersome until it degrades. The polymersome and drug combination spontaneously self-assembles when mixed together. Recently, studies have shown that cocktails of paclitaxel and DOX lead to better tumor regression that either drug alone, but previously there was no carrier system that could carry both drugs as efficiently to a tumor. Hence, this approach shows great promise.

Quantum dots

Single-particle quantum dots conjugated to tumor-targeting anti-human epidermal growth factor receptor 2 (HER2) MAb have been used to locate tumors using high-speed confocal microscopy (Tada et al., 2007). Following injection of quantum dot-MAb conjugate, six distinct stop-and-go steps were identified in the process as the particles traveled from the injection site to the tumor where they bound HER2. These blood-borne conjugates extravasated into the tumor, bound HER2 on cell membranes, entered the tumor cells and migrated to the perinuclear region. The image analysis of the delivery processes of single particles in vivo provided valuable information on MAb-conjugated therapeutic particles, which will be useful in increasing their anticancer therapeutic efficacy. However, the therapeutic utility of quantum dots remains undetermined.

XPclad® nanoparticles

The poor aqueous solubility of many drug candidates presents a significant problem in drug delivery and related requirements such as bioavailability and absorption. Recently, our laboratory has developed XPclad® nanoparticles that represent a novel formulation method that uses planetary ball milling to generate particles of uniform size (Figure 1), 100% loading efficiency of hydrophobic or hydrophilic drugs, subsequent coating for targeted delivery, and control of LogP for systemic, cutaneous, or oral administration of cancer drugs, vaccines, or therapeutic proteins (Figure 2).

The method for making XPclad® nanoparticles uses a novel and relatively inexpensive preparation technique (i.e., planetary ball milling), which allows for controlling the size of the particles (100 nm to 50 μm; ± 10% of mean size) with >99% loading efficiency, polymer- or ligand-coating for controlled-, protected-, and targeted-release and delivery of their contents. The nanoparticles produced thereby contain the desired biologically active agent(s) in a biopolymer excipient such as alginate, cellulose, starch or collagen and biologically active agents. Generally, there are two types of mills that have been employed for making particles: vibratory or planetary ball mills. The vibratory ball milling grinds powders by high velocity impact while planetary ball milling employs a grinding motion. Typically, planetary ball milling has been used only to generate micron-sized particles, while vibratory milling can yield nano-particles. However, the high impact resulting from the vibratory milling technique makes incorporating biologicals difficult. Planetary ball mills pulverize and mix materials ranging from soft and medium to extremely hard, brittle and fibrous materials. Both wet and dry grinding can be carried out. Minerals, ores, alloys, chemicals, glass, ceramics, plant materials, soil samples, sewage sludge, household and industrial waste and many other substances can be reduced in size simply, quickly and without loss. Planetary ball mills have been successfully used in many industrial and research sectors, particularly wherever there is high demand for purity, speed, fineness and reproducibility. The planetary ball mills produce extremely high centrifugal forces with very high pulverization energies and short grinding times. Because of the extreme forces exerted, the use of vibratory and planetary ball mills to formulate therapeutics has not been practiced until now. In general, XPclad® particle size can be engineered to range from 5 to 30 nm up to 10 to 60 μm by controlling the size and number of planetary balls, grinding speed, milling cycles, and centrifugal force by varying the revolutions per second and planetary jar velocity.

 

Nano delivery systems hold great potential to overcome some of the obstacles to efficiently target a number of diverse cell types. This represents an exciting possibility to overcome problems of drug resistance in target cells and to facilitate the movement of drugs across barriers (e.g., BBB). The challenge, however, remains the precise characterization of molecular targets and ensuring that these molecules only affect targeted organs. Furthermore, it is important to understand the fate of the drugs once delivered to the nucleus and other sensitive cells organelles.

UPDATED 8/05/2022

 

One step closer to cancer nanomedicine

High-throughput tool uncovers links between cell signaling and nanomaterial uptake

JESSICA O. WINTER

SCIENCE

21 Jul 2022

Vol 377, Issue 6604

pp. 371-372

DOI: 10.1126/science.add3666

Source: https://www.science.org/doi/10.1126/science.add3666 

The promise of chemotherapeutic nanomedicine has tantalized clinicians and patients for decades. Nanoparticles (NPs) can directly target tumor cells, which would reduce the amount of chemotherapy administered and its systemic toxicity, increasing patient quality of life and extending utility of therapies with lifetime dosing limits. However, these hopes remain largely unrealized. Liposomal drug carriers, which make up nearly all clinically approved nanomedicines, have not extended overall patient survival compared with treatment with the drugs alone (1). These failures have been attributed to poor delivery to target cells (2) because NPs must first traverse a series of biological barriers (3). Although nanocarrier composition, surface chemistry, size, and shape have been optimized to promote cell entry, progress has been confounded by heterogeneity in cell uptake signaling (4). On page 384 of this issue, Boehnke et al. (5) uncover the reciprocal relationship between NP material properties and cell internalization using nanoPRISM, a high-throughput screening approach.

The nanoPRISM technology uses the profiling relative inhibition simultaneously in mixtures (PRISM) (6) method to generate a screening library of ∼500 cancer cell lines that are barcoded with distinct DNA sequences that permit identification of cells with high-throughput genomic sequencing. This cell library is combined with a panel of 35 different fluorescently labeled NPs with varying core compositions, surface chemistries, and diameters to identify synergistic interactions for cell uptake. PRISM-tagged cells are separated into four groups according to uptake level, and their DNA is sequenced to identify them and screen for key drivers of NP internalization that can be attributed to either NP characteristics or cell signaling.

Boehnke et al. compared the uptake efficiency of NPs conjugated to antibodies targeting epidermal growth factor receptor (EGFR) versus EGFR antibodies alone in cell lines that overexpress this receptor. NanoPRISM revealed differences in cellular uptake, most likely resulting from the steric hindrance of NP conjugation. These results suggest that nanoPRISM may be suitable for evaluating antibody-drug conjugates (ADCs), a growing therapeutic category.

Boehnke et al. also use nanoPRISM to interrogate NPs with compositions most commonly applied to nanomedicine: spherical liposomes made of lipid bilayers and solid lipid and polymer NPs consisting of disordered, spherical lipid or polymer aggregates. They also examine NPs with or without polyethylene glycol (PEG) modification, which is used to reduce systemic uptake and improve circulation time (7). They find that NP core composition is a primary determinant in cellular uptake. This unexpected finding upends years of work on modulating NP surface chemistries to alter protein adsorption patterns and subsequent cell adhesion (8). Although cells first detect NPs through their surface chemistry, the findings of Boehnke et al. support early studies that showed that NP stiffness and deformability, which are dictated by core composition, are stronger modulators of the uptake process (9).

The power of the nanoPRISM method is further illustrated by combining these findings with the Cancer Cell Line Encyclopedia, which quantifies mutational genomic signatures of common cancer cell lines. Boehnke et al. identify genomic signatures and signaling networks most correlated with NP internalization. Many of the results are not surprising, such as involvement of the solute carrier (SLC) transporter or adenosine triphosphate (ATP)–binding cassette (ABC) families, which have previously been implicated in NP cellular entry and transport. The nanoPRISM screens also highlight gene networks associated with the plasma membrane and extracellular matrix that contribute to NP cellular entry processes (see the figure).

However, the nanoPRISM method also reveals involvement of an understudied gene that has not been associated with NP internalization: SLC46A3. This encodes a lysosomal transmembrane protein linked to lipid catabolism (10) that influences lysosomal trafficking of ADCs (11). Expression of SLC46A3 negatively regulated liposomal and solid lipid NP cellular uptake, whereas polymer NPs that lack lipids were unaffected. SLC46A3 association with lipid-based NPs was evidenced even when NP surfaces were coated with nonlipid molecules. This further indicates the importance of NP core composition in cellular uptake processes and also suggests that cells can detect core composition through surface coatings, which better resemble a porous net than a wall. This could have important implications for predicting the efficacy of nucleic acid vaccines and therapies that use lipid-based carriers, such as COVID-19 mRNA vaccines. For example, SLC46A3 biomarker testing could be implemented to identify patients most likely to respond to lipid-based nanotherapeutics.

GRAPHIC: V. ALTOUNIAN/SCIENCE

OPEN IN VIEWER

The results of the nanoPRISM screens are also confirmed in animal models, indicating that this technique could be used to identify the most promising formulations for downstream analysis, reducing preclinical animal testing demands. Such high-throughput approaches are critical to the rapid advancement of cancer nanomedicine, because US and European regulatory agencies have not established criteria for nanomedicine approval based on similarity to an existing product (12). Given the long timeline for drug development, which can span a decade or more, technologies to safely accelerate this process are desirable.

The nanoPRISM method represents a substantial advance over the less rigorous and qualitative studies of NP internalization that characterized the early years of the field. Studies that examined a few NP properties in a single cell line could not capture the complexities of NP cell entry. Combined with machine learning and iterative simulation and materials synthesis approaches, nanoPRISM could enable screening for nanomaterials that target specific cell types, similar to current biopanning methods for peptides or the systematic evolution of ligands by exponential enrichment (SELEX) method of aptamer discovery (13). Although the study of Boehnke et al. examines only 35 different NPs, additional nanomaterials could be added to the library, such as inorganic NPs (such as gold, silica, and carbon) and materials with complex geometries (such as DNA origamis). A limitation of nanoPRISM is its focus on cellular entry, the last step of the biodistribution process. However, it is easy to envision expanding this approach beyond cell uptake to study the relationship between NP material properties and gene expression in cell adhesion and trafficking. Additionally, with the template provided by Boehnke et al., similar methods could be integrated with microfluidics, organ-on-a-chip, or tumor organoid cultures to model other delivery barriers, such as circulation, extravasation, and tissue diffusion. Thus, the nanoPRISM approach could catalyze rapid materials optimization, accelerating nanocarrier design and bringing the promise of cancer nanomedicine closer to reality.

References and Notes

1

G. H. Petersen, S. K. Alzghari, W. Chee, S. S. Sankari, N. M. La-Beck, J. Control. Release 232, 255 (2016).

2

S. Wilhelm et al., Nat. Rev. Mater. 1, 16014 (2016).

3

S. Barua, S. Mitragotri, Nano Today 9, 223 (2014).

4

B. D. Chithrani, A. A. Ghazani, W. C. W. Chan, Nano Lett. 6, 662 (2006).

5

N. Boehnke et al., Science 377, eabm5551 (2022).

6

C. Yu et al., Nat. Biotechnol. 34, 419 (2016).

7

M. Eugene, Cell. Mol. Biol. 50, 209 (2004).

8

A. Albanese et al., ACS Nano 8, 5515 (2014).

9

X. Sun et al., Biomacromolecules 6, 2541 (2005).

10

J.-H. Kim et al., Nat. Commun. 12, 290 (2021).

11

K. J. Hamblett et al., Cancer Res. 75, 5329 (2015).

12

S. Soares, J. Sousa, A. Pais, C. Vitorino, Front Chem. 6, 360 (2018).

13

C. Tuerk, L. Gold, Science 249, 505 (1990).

 

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Is the Warburg effect an effect of deregulated space occupancy of methylome?

Posted in Anaerobic Glycolysis, Cancer - General, CANCER BIOLOGY & Innovations in Cancer Therapy, Curation, Genomic Expression, Oxidative phosphorylation, Pyruvate Kinase, Warburg effect, tagged anaerobic and aerobic gycolysis, ascorbate, Cancer - General, dihydroascorbate, glutamine, lactate dehydrogenase reaction, Warburg Effect on February 15, 2016| Leave a Comment »

Is the Warburg effect an effect of deregulated space occupancy of methylome?

Larry H. Bernstein and Radoslov Rosov, co-curation

LPBI

UPDATED

8/20/2024

Editor Correction:  One of the co-curators names on this article was misspelled. The co-curators should be Larry H. Bernstein and  Radislov Rosov.

Therefore the correction version of the article title and authors should read

Is the Warburg effect an effect of deregulated space occupancy of methylome?

Larry H. Bernstein and Radoslov Rosov, co-curation

The editors of LPBI Group apologize for the confusion.

 

It would appear that pyruvate is directly used by cancer cell machinery for sustaining genome independence, and that CRISP-Cas9 system is essentially a modified CAD protein for making small bases.

¹³C-labeled biochemical probes for the study of cancer metabolism with dynamic nuclear polarization-enhanced magnetic resonance imaging

Lucia Salamanca-Cardona and Kayvan R. Keshari

Cancer & Metabolism 2015; 3:9          http://dx.doi.org:/10.1186/s40170-015-0136-2

In recent years, advances in metabolic imaging have become dependable tools for the diagnosis and treatment assessment in cancer. Dynamic nuclear polarization (DNP) has recently emerged as a promising technology in hyperpolarized (HP) magnetic resonance imaging (MRI) and has reached clinical relevance with the successful visualization of [1-¹³C] pyruvate as a molecular imaging probe in human prostate cancer. This review focuses on introducing representative compounds relevant to metabolism that are characteristic of cancer tissue: aerobic glycolysis and pyruvate metabolism, glutamine addiction and glutamine/glutamate metabolism, and the redox state and ascorbate/dehydroascorbate metabolism. In addition, a brief introduction of probes that can be used to trace necrosis, pH changes, and other pathways relevant to cancer is presented to demonstrate the potential that HP MRI has to revolutionize the use of molecular imaging for diagnosis and assessment of treatments in cancer.

 

Since the hallmark discovery of the Warburg effect in cancer cells in the 1920s, it has been widely accepted that the metabolic properties of cancer cells are vastly different from those of normal cells [1]. Starting from the observation that many cancerous (neoplastic) cells have higher rates of glucose utilization and lactate production, the development of tools and methods to correlate specific cellular metabolic processes to different types of cancer cells has received increased research focus [2, 3]. Several imaging techniques are currently in use for this purpose, including radiography, scintigraphy, positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance (MR) [4, 5].

For more than 30 years, MR has been a revolutionary diagnostic tool, used in a wide range of settings from the central nervous system to cardiomyopathies and cancers. MR imaging (MRI) can outline molecular and cellular processes with high spatial resolution. Typically, MRI of body tissues is achieved via contrast visualization of the protons (¹H) of water, which are present in high abundance in living systems. This can be extended to MR spectroscopy (MRS), which can further differentiate between less abundant, carbon-bearing, biological metabolites in vivo utilizing ¹Hs of these compounds [6, 7]. However, despite its usefulness in imaging whole body tissues, ¹H MRS has low spectral resolution and poor sensitivity for these less abundant metabolites. In addition,¹³C MRS is increasingly difficult, in comparison to ¹H MRS, in that both the gyromagnetic ratio (approximately 25 % of ¹H) and natural abundance (1.1 % of ¹H) are significantly lower, making the detection of carbon-bearing compounds difficult [8, 9]. The low spectral resolution of ¹H MRS for metabolites can be addressed by using ¹³C-enriched compounds, and with this direct ¹³C MRS, metabolic processes can be traced, utilizing enriched tags on specific carbons in a given metabolite [10]. While enrichment of molecules in ¹³C can also moderately address the sensitivity limitation of MRS, recent work in hyperpolarization (HP) provides a means of dramatically increasing sensitivity and enhancing signals, well beyond that of the equilibrium state obtained via MRS. [11, 12]. The focus of this review will be the introduction of this approach in the setting of cancer metabolism, delineating probes of interest, which have been applied to study metabolic processes in vivo.

Obtaining a hyperpolarized probe

In MR, a desired target is placed in a magnetic field where the nuclear spins of molecules are aligned with or against the direction of the magnetic field. The nuclear spins have thus different energies, and an MR signal is detected upon relaxation of nuclear spins of higher energy. At thermal equilibrium, the number of spins aligned with the magnetic field nearly equals the number of spins opposing the direction of the magnetic field. Thus, at thermal equilibrium, spin polarization is in the order of >0.0005 % resulting in a limited signal. Signal increases on the order of 100,000-fold can be achieved by hyperpolarizing the system via the redistribution of the spin population levels found at equilibrium [10, 13]. There are several techniques that have been used to achieve hyperpolarization of various nuclei: spin-exchange optical pumping of ³He and ¹²⁹Xe, parahydrogen-induced polarization (PHIP), and dissolution dynamic nuclear polarization (DNP) [11,14, 15]. Both PHIP and DNP techniques can polarize biologically relevant nuclei like ¹³C and ¹⁵N, although there is a wider range of molecules that can be targeted for hyperpolarization using dissolution DNP [14, 16–18].

The goal of DNP is the transfer of polarization from highly polarized unpaired electron spins to the nuclear spins of a desired target compound. This is achieved by applying an external magnetic field to a free-radical agent in order to polarize electron spins, followed by saturating the electron spin resonance via microwave irradiation in order to obtain polarization transfer. The free-radical agent is generally a stable organic compound that is compatible with aqueous buffers, which are used as solvent in order to obtain a homogeneous distribution of the radical [13]. Nearly 100 % of the electrons on the free-radical agent are polarized when the free-radical/solvent mixture is subjected to high magnetic fields (≥3.3 T) followed by rapid freezing to 1 K using liquid helium in order to obtain a sample frozen to an amorphous state, which is necessary for retention and transfer of polarization [18]. For biological applications, after transfer of electron spin polarization to the nuclei of interest has occurred, the preparation must exist in solution, which can be achieved utilizing a dissolution process in which the solid sample is rapidly melted via injection of a hot solvent, typically a biologically compatible buffer, into the frozen sample [13]. The dissolution process results in a liquid sample at room temperature, while still preserving the enhanced polarization obtained by the microwave irradiation of the frozen sample [8]. Additionally, the use of chelating agents (e.g., EDTA) with the solvent to eliminate trace metals and more recently the use of gadolinium (Gd) chelates with the DNP sample have been used to further enhance and retain polarization in the liquid sample, albeit with caution over potential toxic effects when applied in vivo and the potential for loss of hyperpolarization due to T ₁ shortening [11, 19, 20]. More in-depth exploration of the technical aspects of probe development has been previously reviewed [8, 11].

Considerations in probe selection and current research

The usefulness of a molecule for hyperpolarized MRS is dependent on the polarization lifetime of the nucleus of interest, and this property is determined by the spin-lattice relaxation constant (T₁) [21]. Dipolar coupling, the magnetic field range, and molecular size can also affect the T₁ of a given nucleus. In general, high magnetic fields and large molecular weights decrease the T₁. Dipole-dipole coupling of ¹³C with ¹H is common in biologically relevant molecules, and it shortens relaxation times; therefore, carbon atoms directly bound to ¹H are generally not useful as probes for HP. For example, all carbons present in glucose (an important substrate in cancer cells) have relaxation times shorter than 2 s [22]. On the other hand, carbonyl carbons of biologically relevant molecules generally have T₁’s above 20 s even at high magnetic fields like [1-¹³C] pyruvic acid, which has relaxation times of 67, 48, and 44 s at 3, 11.7, and 14.1 T, respectively [23–25]. Even carbons that are less oxidized than carbonyls, like the hemi-ketal in [2-¹³C] fructose have T₁’s one order of magnitude higher than glucose carbons. Short spin-lattice relaxation times can sometimes be increased by deuterium enrichment of the sample. With this technique, protons that are directly bound to carbons are exchanged for deuterium atoms which results in the reduction of dipole-dipole relaxation, further preserving the hyperpolarized state [26]. This has resulted in increased T₁’s of ¹³C nuclei in molecules such as glucose (T₁ increased from 2 s to 10–14 s), providing the possibility of utilizing them in future metabolic studies [27–29]. Despite the effect of deuterium enrichment, research efforts have largely focused on developing carbonyl-bearing molecules as molecular imaging probes. The focus of this review is to introduce representative compounds relevant to metabolism that are characteristic of cancer tissue and have been applied in the work of multiple groups: aerobic glycolysis, glutamine addiction, and the redox state.

Pyruvate and aerobic glycolysis

Of particular interest to cancer metabolism is the increased conversion of glucose to lactate as a result of modulated aerobic glycolysis. This process, also known as the Warburg effect, is characteristic of many tumors with altered metabolism where pyruvate generated from glucose metabolism via glycolysis is preferentially converted to lactate by lactate dehydrogenase (LDH) as opposed to entering the tricarboxylic acid cycle [1]. With this phenotype, cancer cells show a preference for lactate fermentation even in the presence of oxygen, thus bypassing oxidative respiration for ATP generation. Because of this, pyruvate has been the preferred probe for HP MRS research since it is an intermediate metabolite in pathways characteristic of aberrant metabolism in cancer cells, including increased lactate production as a result of aerobic glycolysis where detection of HP pyruvate-derived lactate can be used as a marker for cancer and response to treatment [30, 31] as well as an intermediate in amino acid metabolism (e.g., interconversion to alanine via transamination with glutamate) (Fig. 1). In addition, as mentioned before, carbonyl carbons in pyruvate have long relaxation times where even the methyl carbon can have T₁’s above 50 s after deuterium enrichment [32]. The interconversion of pyruvate to lactate has been exploited for MRI by using [1-¹³C] pyruvate and detecting the accumulation of increased lactate in cancerous tissue as compared to surrounding benign tissue. Increased conversion of pyruvate to lactate and alanine has been demonstrated to precede MYC-driven tumorigenesis by using HP [1-¹³C] pyruvate in murine models [33]. Furthermore, in the same study, a decrease in the flux of alanine was observed at the tumor stage while a decrease in lactate conversion was indicative of tumor regression [33]. In transgenic adenocarcinoma of mouse prostate (TRAMP) models, in vivo studies using HP [1-¹³C] pyruvate demonstrated that hyperpolarized pyruvate and its metabolic products can be used non-invasively and with high specificity to obtain a profile of the histologic grade of prostate cancers [34]. In vivo imaging following hyperpolarized pyruvate has also been used to evaluate the role of glutaminase and LDH in human lymphoma models [35] as well as to elucidate metabolism of pyruvate in breast cancer [36] and renal cell carcinoma with treatment [30, 37].

http://static-content.springer.com/image/art%3A10.1186%2Fs40170-015-0136-2/MediaObjects/40170_2015_136_Fig1_HTML.gif

Flux of hyperpolarized [1-¹³C] pyruvate to [1-¹³C] lactate in prostate regions. a MR image from patient with prostate cancer showing regions of cancerous tissue and surrounding normal tissue. b–d Localized dynamic hyperpolarized [1-¹³C]pyruvate and [1-¹³C]lactate spectral from voxels overlapping the contralateral region of prostate (turquoise), a region of prostate cancer (yellow), and a vessel outside the prostate (green). Adapted with permission from ref. [43]

Early work that utilized HP pyruvate to assess the response of tumors to treatment was conducted in mice xenografted with EL-4 lymphoma cells and treated with etoposide, a topoisomerase inhibitor that causes rapid cell death [38, 39]. In this study, tumor necrosis was correlated to a decrease in the flux of hyperpolarized lactate which was suggested to be due to a decrease in NAD+ and NADH in the intracellular pool as well as loss of LDH function. More recently, HP [1-¹³C] pyruvate has been used as a biomarker to evaluate early response to radiation therapy in glioma tumors by observing a decrease in hyperpolarized lactate suggested to be a result of changes in tumor perfusion which can be detected between 24 and 96 h following treatment [40, 41]. HP [1-¹³C] pyruvate has also been used to detect early response to temozolomide (TMZ) treatment on human glioblastoma rat models [42]. The study successfully showed for the first time detection of response to TMZ therapy 1 day after TMZ administration. The continued reports on using HP pyruvate as an imaging probe for assessing treatment response indicate the potential of the compound to become a standard in the field. Moreover, these studies demonstrate the usefulness of HP [1-¹³C] pyruvate as a tool for early assessment of therapy response, which can improve treatment selection at the clinical level. Pyruvate has also been validated as a metabolic imaging marker for use in humans [43]. In a two-phase study, patients with biopsy-proven prostate cancer of various histological grades were injected with HP [1-¹³C] pyruvate. In the first phase, a maximum dose level was determined to establish pharmacological safety of the HP probe while still injecting enough pyruvate to allow visualization. This addressed one of the major challenges faced in translating HP MRI to clinical applications: the potential toxicity of compounds that must be injected into patients. In the second phase, metabolism of pyruvate was visualized in real time and differences in the ratio of [1-¹³C] lactate to [1-¹³C] pyruvate between identified cancerous regions and normal tissue regions were successfully observed (Fig. 1a–d). [1-¹³C] lactate in regions that did not contain tumor was not detected, confirming previous biopsy and preclinical studies that demonstrated low flux of [1-¹³C] pyruvate to lactate and low concentrations of lactate in benign prostate tissues [44, 45]. Preliminary results indicated the possibility of detecting previously unobserved cancerous regions by HP [1-¹³C] pyruvate, later confirmed to be Gleason 4+3 cancer by biopsy, though further investigation into the relationship between grade and metabolism in prostate cancer patients is needed. While there are challenges associated with translation to clinical use for HP [1-¹³C] pyruvate, the first in human study demonstrated the feasibility of hyperpolarization technology as a safe diagnostic tool and provides the potential for utilizing this approach in preclinical models with direct translation to the clinic.

Glutamine metabolism

Glutamine is an amino acid that plays an important cellular role as nitrogen donor in the form of an amide group for purine and pyrimidine biosynthesis, leaving a glutamate molecule in the process although glutamine can also be converted to glutamate by glutaminase in a reaction independent of nucleotide biosynthesis. Glutamate is the primary nitrogen donor for the biosynthesis of non-essential amino acids. Transaminases catalyze the transfer of the amine group from glutamate to α-ketoacids to synthesize alanine, aspartate (precursor for asparagine), serine (precursor for glycine and cysteine), ornithine (precursor for arginine), and proline which is derived from the glutamate carbon backbone. Glutamine is considered a non-essential amino acid as it can be recycled from glutamate and ammonia in a reaction catalyzed by glutamine synthetase; however, some cancer cells show increase consumption of glutamine and are unable to grow in the absence of exogenous glutamine [46, 47]. This metabolic characteristic of cells to require exogenous glutamine for growth has been termed “glutamine addiction” and has generated extensive research interest as an indicator of development of cancerous tissues [48]. In particular to the field of HP MRI, the conversion rate of glutamine to glutamate (Fig. 2) was explored in hepatocellular carcinoma (HCC) using a [5-¹³C] glutamine probe (Fig. 2) [49]. Using the ratio between [5-¹³C] glutamine and [5-¹³C] glutamate, it was demonstrated that HCC cells convert glutamine at a higher rate than normal cells supporting the notion of glutamine addiction. One important aspect of this study was the choice of [5-¹³C] glutamine as a probe as opposed to [1-¹³C] glutamine, which has a longer T₁ (16.1 vs. 24.6 s at 9.4 T) [49, 50]. [5-¹³C] glutamine was selected because the chemical shift change obtained from [1-¹³C] in glutamine and glutamate is far too small, which could prevent proper identification and quantification of the peaks. This highlights the importance of understanding not only the target compound to be hyperpolarized but also the metabolic products to be detected and their resulting spectra in MR. This is further emphasized with studies that demonstrate the usefulness of [1-¹³C] glutamine as a source for [1-¹³C] glutamate in order to follow the metabolism of α-ketoglutarate to observe the metabolic state of the TCA cycle in transformed cells [51]. Furthermore, [1-¹³C] α-ketoglutarate has been hyperpolarized and used to visualize other metabolic events involving [1-¹³C] glutamate such as mutations in IDH1 expression in glioma tumors and pathways dependent on hypoxia-inducible factor (HIF) [51–53]. More recently, [5-¹³C] glutamine has been used to visualize the metabolism of liver cancer in vivo and in vitro, as well as the treatment response of prostate cancer cells in vitro [54]. Based on the promise of glutamine as a biomarker for cancer diagnosis and treatment response, extending the spin-lattice relaxation time of the [5-¹³C] glutamine has been researched and successfully accomplished. The facile synthesis of [5-¹³C-4-²H₂] glutamine has been reported, and its study showed that by relying on the effect of deuterium enrichment to lessen dipolar coupling effects, the T₁ of [5-¹³C] glutamine could be increased from approximately 15 to 30 s [55]. Visualization of real-time conversion of glutamine to glutamate in SF188 cells was achieved using this probe, demonstrating the promise of [5-¹³C-4-²H₂] glutamine as a probe for molecular imaging of metabolic events in real time. Further investigation of this probe applied to in vivo preclinical models will lay the foundation for its clinical translational potential in the future.

http://static-content.springer.com/image/art%3A10.1186%2Fs40170-015-0136-2/MediaObjects/40170_2015_136_Fig2_HTML.gif

Metabolism of [5-¹³C] glutamine to [5-¹³C] glutamate. a Time-dependent spectral data following conversion of [5-¹³C] glutamine to [5-¹³C] glutamate. The signals are from ¹³C-enriched [5-¹³C]glutamate at 181.5 ppm and [5-¹³C]glutamine at 178.5 ppm and from natural abundance ¹³C label in [1-¹³C]glutamate at 175.2 ppm and [1-¹³C]glutamine at 174.7 ppm. b Plot of the ratio of the signal intensities of [5-¹³C]glutamate/[5-¹³C]glutamine showing the ratio in hepatoma cells (shaded circle), cell lysate (square), and control (triangle). These results demonstrated that hepatoma cancer cells convert glutamine to glutamine at a higher rate than normal cells. Adapted with permission from ref. [49]

Dehydroascorbate as a redox sensor

Reactive oxygen species (ROS) like the hydroxyl radical, superoxide, and hydrogen peroxide have been shown to cause DNA damage and can lead to mutations that transform normal cells into cancerous cells [56]. The reduction/oxidation (redox) state, which is dependent on the balance between oxidizing equivalents like ROS and reducing cofactors, can provide insight into the physiological condition of the cell with respect to potential cancer transformations. Furthermore, the presence of ROS in tissue has been implicated to be a factor in developing resistance to radiation therapies [57]. During oxidative stress (i.e., when there is an increase in ROS), redox homeostasis is maintained by the action of antioxidant compounds, such as ascorbate (or vitamin C, VitC), which can scavenge for ROS and reduce the compounds to rid the cells of damaging agents [58]. In this process, ascorbate that is available to cells in high concentrations can be oxidized to dehydroascorbate (DHA) while reducing ROS. DHA can then be transported into the cell where DHA is reduced back to ascorbate resulting in a process of recycling ascorbate and DHA (Fig. 3) [59]. In this sense, the ratio of DHA to ascorbate can be used as a molecular marker to investigate the redox state and thus the physiological state of tissues. Additionally, conversion of DHA to ascorbate can be enzymatically catalyzed in an NADPH-dependent manner or via oxidation of glutathione (GSH) to glutathione sulfide (GSSG); thus, visualization of ascorbate/DHA metabolism offers a method for probing in vivo metabolism of NADPH as well as determination of GSSG to GSH ratio, both of which have been implicated to be indicators of oxidative stress in the cells, particularly for neurodegenerative, cardiovascular, and cancer diseases [60–62]. Hyperpolarized [1-¹³C] DHA was successfully used in murine models to detect increased reducing capacity in prostate cancer with the purpose of developing a non-invasive, early diagnostic tool for improving selection of treatment therapies [62, 63]. DHA demonstrates a relatively long T₁ at clinically relevant field strengths (>50 s at 3 T) and adequate chemical shift separation between it and its metabolic product ascorbate (δ = 3.8 ppm). Increased reduction of HP [1-¹³C] DHA to ascorbate was observed in tumor tissue compared to normal tissue as well as other metabolic organs (Fig. 3). This was additionally demonstrated in lymphoma cells, further supporting the potential for using DHA as a probe in living systems [64]. A following study validated these results, and the correlation between increased DHA reduction and glutathione was established in vivo, thus showing the utility of [1-¹³C] DHA as a molecular imaging probe to detect events that go beyond the direct metabolism of DHA [63]. Notwithstanding the potential of HP DHA as a diagnostic probe, the toxicity of DHA remains to be validated. Earlier studies on mammalian cells showed DHA toxicity starting at 10 mM, while a study carried on rats demonstrated neurological effects of DHA starting at injections of 50 mg/kg [65, 66]. However, as outlined above, successful use of DHA injections in rats and mice for hyperpolarization has been demonstrated without reported side effects on the animals. More research is needed to determine the parameters regarding the toxicity of DHA in larger animal models using pure formulations to assess its potential for clinical trials. Further work in DHA could demonstrate its applicability for the study of ROS and redox changes in model systems.

http://static-content.springer.com/image/art%3A10.1186%2Fs40170-015-0136-2/MediaObjects/40170_2015_136_Fig3_HTML.gif

Determination of redox state by imaging of HP [1-¹³C] ascorbate (VitC) and [1-¹³C] dehydroascorbate (DHA). Oxidative stress caused by ROS (1.) can be alleviated by oxidation of ascorbate to DHA (2.), and recycling of DHA to ascorbate can occur indirectly with oxidation of glutathione (3.) or directly with oxidation of NADH (4.). The ratio of [ascorbate] to [DHA] has been successfully used in mice models as a biomarker to determine pH in vivo. Adapted with permission from ref. [62]

Other metabolic imaging probes

While the three probes discussed earlier are the most well studied in metabolic events that are characteristic of cancer cells in general, other molecules have been evaluated in their potential to be used as biomarkers. Hyperpolarized bicarbonate (H¹³CO₃⁻) has been successfully used to determine the pH in extracellular matrix of lymphoma tumors in mice, and a correlation between acidic environments and cancer was established [67]. The relaxation times for bicarbonate compounds at 3 T are between 34 and 50 s, which is enough time to detect the rapid conversion of H¹³CO₃⁻ and ¹³CO₂ catalyzed by carbonic anhydrase [23]. The attractive feature of this probe is based on how ubiquitous acidic extracellular environments are to a wide variety of diseases; thus, HP bicarbonate has the potential for clinical translation beyond cancer research, though extensive work will be necessary to generate a preparation which will result in an adequate dose for the clinic [68, 69]. More recently, the potential of α-ketoisocaproate (KIC) as a molecular probe for in vivo detection of branched chain amino acid transaminase (BCAT) has been explored. BCAT catalyzes the conversion of KIC to leucine, and its expression has been suggested to correlate to genetic characterization of certain tumors. In a pilot study, HP α-keto-[1-¹³C]-isocaproate was shown to have a T₁ of 100 s so its metabolism can be sensitively traced for over a minute after injection [70]. In the same study, metabolism of HP [1-¹³C] KIC to [1-¹³C] leucine by BCAT was observed in murine lymphoma tumor tissue but was absent in rat mammary adenocarcinoma with a correlation between BCAT expression and [1-¹³C] leucine signal detection [70]. Additionally, in the same models, [1-¹³C] pyruvate conversion to [1-¹³C] lactate and [1-¹³C] alanine was detected in both types of tumors. These findings show the promise of using [1-¹³C] KIC as a discriminative probe in addition to pyruvate in order to diagnose different types of cancer [71, 72]. Furthermore, the correlation between BCAT expression and [1-¹³C] leucine detection was also shown in rat brain tissue, confirming the usefulness of HP [1-¹³C] KIC in assessing BCAT activity in vivo [73]. Choline is another compound that has been evaluated as a molecular imaging probe since elevated choline and choline-derived metabolites have been correlated by ¹H-MRS imaging to cancer in the brain, breast, colon, cervix, and prostate [74–76]. Despite its potential as a global marker for cancer because of the long T₁ relaxation times that can be achieved with deuterium and ¹⁵N enrichment [77, 78], HP applications of ¹³C enriched choline are limited due to the small change in chemical shifts of choline and choline-derived metabolites as well as its potential toxicity [16, 79, 80]. It has been shown that choline toxicity occurs at doses of 53 mg/kg in mice, although a recent study successfully detected HP ¹³C choline in vivo without adverse effects in rats at doses of 50 mg/kg by using atropine to prevent cholinergic intoxication [81, 82] though metabolic products have been difficult to visualize in vivo. As mentioned earlier, the usefulness of glucose as a probe is limited due to the short relaxation times of all the carbons present in the molecule and although the T₁’s can be increased through deuterium enrichment, the lifetime of the probe remains a hurdle for clinical applications [27, 28]. Thus, fructose (a pentose analog of glucose) has been successfully used as an alternative to probe glycolytic pathways [83] in TRAMP models where differences in HP [2-¹³C] fructose uptake and metabolism was visualized in tumor regions compared to surrounding normal tissues. Like choline, the limiting factor in the usefulness of [2-¹³C] fructose for in vivo studies is in small chemical shifts between the metabolite and its phosphorylated product. Finally, tumor necrosis can be used as a measure of treatment response, particularly early necrosis. HP [1,4-,¹³C] malate has been visualized in lymphoma mice models after injection of HP [1,4-¹³C] fumarate [84]. In normal cells, fumarate has a slow rate of transport into the mitochondria; however, in necrotic cells where the mitochondrial membrane is degraded, fumarase has access to the HP fumarate and its ubiquitous cofactor, water, thus facilitating rapid conversion to malate. Preliminary studies have shown the potential for its use in animal models though further work is required to determine the necessary density of necrotic cells for detection and the timings required for adequate visualization in patients.

Conclusions

The application of hyperpolarized ¹³C imaging has been extensively investigated in preclinical models, and the successful demonstration of HP [1-¹³C] pyruvate in patients with prostate cancer has validated the potential of HP MRI as a safe diagnostic and treatment assessment tool. Application of other probes beyond pyruvate is still in its infancy, particularly because of the need to further study the currently developed models under conditions that are relevant to a clinical setting (i.e., lower magnetic fields) as well as to study the necessary parameters (probe toxicity dose limits, safety limits for rapid injection) to withstand the necessary hurdles to translation. Nevertheless, these vast research findings are promising and indicate an eventual translation to humans. Furthermore, there is a large variety of biologically relevant molecules that have the potential to be hyperpolarized (Fig. 4), and molecular imaging of metabolic events in real time using not only one single probe but a combination of relevant probes could become an invaluable tool in elucidating so far undiscovered metabolic and proteomic interactions that play a role in cancer development and treatment. This gives HP MRI the great potential to revolutionize current molecular imaging technologies.

http://static-content.springer.com/image/art%3A10.1186%2Fs40170-015-0136-2/MediaObjects/40170_2015_136_Fig4_HTML.gif

Metabolic pathways with compounds that can be used as molecular imaging probes for HP MRI. A wide variety of metabolic pathways have already been visualized or have the potential to be visualized using hyperpolarization technology that can be applied to different pathological states of the cell including cardiovascular disease and a large variety of cancers. 1. Metabolism of C1 (red dots) in pyruvate. Theasterisks on selected compounds represent enrichment of ¹³C in the second pass of pyruvate in TCA cycle. 2. Metabolism of C1 (brown dots) in DHA using a pool of NADPH derived from the pentose phosphate pathway. 3. Metabolism of C1 (blue dots) and C5 (green dots) of glutamine. 4. Metabolism of C1 and C4 (purple dots) of fumarate unrelated to TCA metabolites. 5. Metabolism of extracellular bicarbonate (gray dots). MTC1 monocarboxylate transporter 1, MTC4 monocarboxylate transporter 4,System ASC amino acid transporter, GLUTs glucose transporters, DCT dicarboxylate transporter, DHARdehydroascorbate reductase, GR glutathione reductase, GSH glutathione, GSSG glutathione disulfide,LDH lactate dehydrogenase, ALT alanine transaminase, CA carbonic anhydrase, PC pyruvate carboxylase,PDH pyruvate dehydrogenase, CS citrate synthase, GLS glutaminase, GLDH glutamate dehydrogenase,IDH isocitrate dehydrogenase, OGDC oxoglutarate dehydrogenase complex, SCS succinyl CoA synthetase, SQR succinate dehydrogenase, FH fumarate hydratase, MDH malate dehydrogenase, FUMfumarase. Cofactors have been omitted for brevity

Abbreviations

ALT:   alanine transaminase;   BCAT:  branched chain amino acid transaminase;   DHA:  dehydroascorbate;   DNP:  dynamic nuclear polarization;   EDTA:  ethylenediaminetetraacetic acid;   GSH:  glutathione;   GSSG:   glutathione sulfide;   HCC:  hepatocellular carcinoma;   HIF:  hypoxia-inducible factor;   HP:  hyperpolarized/hyperpolarization;   IDH:  isocitrate dehydrogenase;   KIC:  ketoisocaproate;   LDH:  lactate dehydrogenase;   MR: magnetic resonance;   MRI:  Magnetic resonance imaging;   MRS:  magnetic resonance spectroscopy;   NAD(H):  nicotinamide adenine dinucleotide;   NADP(H):  nicotinamide adenine dinucleotide phosphate;   PET:  positron emission tomography;   ROS:  reactive oxygen species;   SPECT:  single-photon emission computed tomography;   TRAMP:  transgenic adenocarcinoma of mouse prostate

References

1. Warburg O. On the origin of cancer cells. Science. 1956;123:309–14.View ArticlePubMed
2. Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer. 2004;4(11):891–9.View ArticlePubMed
3. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324:1029–33.View ArticlePubMed CentralPubMed
4. Shie P, Cardarelli R, Brandon D, Erdman W, AbdulRahim N. Meta-analysis: comparison of F-18 fluorodeoxyglucose-positron emission tomography and bone scintigraphy in the detection of bone metastases in patients with breast cancer. Clin Nucl Med. 2008;33:97–101.View ArticlePubMed
5. Frangioni JV. New technologies for human cancer imaging. J Clin Oncol. 2008;26:4012–21.View ArticlePubMed CentralPubMed
6. Castillo M, Kwock L, Mukherji SK. Clinical applications of proton MR spectroscopy. Am J Neuroradiol. 1996;17:1–16.PubMed
7. Barker PB, Bizzi A, De Stefano N, Gullapalli R, Lin DD. Clinical MR spectroscopy: techniques and applications. Cambridge University Press; 2009.
8. Comment A, Merritt ME. Hyperpolarized magnetic resonance as a sensitive detector of metabolic function. Biochemistry. 2014;53:7333–57.View ArticlePubMed
9. Rider OJ, Tyler DJ. Clinical implications of cardiac hyperpolarized magnetic resonance imaging. J Cardiov Magn Reson. 2013;15:93.View Article
10. Golman K, Olsson LE, Axelsson O, Månsson S, Karlsson M, Petersson JS. Molecular imaging using hyperpolarized 13C. Br J Radiol. 2003;76 Suppl 2:S118–S27.View ArticlePubMed

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sjwilliamspa commented on Is the Warburg effect an effect of deregulated space occupancy of methylome?

Is the Warburg effect an effect of deregulated space occupancy of methylome? Larry H. Bernstein and Radoslav Bozov, …

It would be an interesting figure, although I am not sure anyone has been able to measure it, is the spatial distribution of lactate and pyruvate over the tumor as a function of diffusion distance such as a heat map to see if pyruvate and lactate levels have a gradiant over a solid tumor. I am not sure it would but interesting to see where tumor cells, which undergo Warburg type metabolic phenotype actually exist, if it is a function of angiogenesis or a function of the proliferative capacity of cells in situ.

Response by LHB…

Radoslav Bozov has repeatedly referred to the real problem of space/time in the required experimental view that is intractable, as seen by Erwin Schroedinger.  It is confounded by
the restrictions imposed by research, and to an extent also the dilemma of location and velocity.

I think it is to an extent also inherent in the modern revelations of autophagy and apoptosis that were not part of the view in the mid 20th century.  However, the work of B. Chance led to a substantially better understanding of the hydride transfer in the NAD/NADH.  What is overlooked is the important role cited by NO Kaplan of NADPH/NADP vs NADH/NAD associated with synthetic and, alternatively, catabolic processes in the cell. What role the pyridine nucleotide transhydrogenase would play is anyones guess.   In any case the proliferation of malignant cells is dependent on NADPH.  This would limit the NAD/NADH related reactions. The effect in the cytoplasm is PYR –> LAC, with generation of NAD from NADH.  In addition, the type of isoenzyme favored should be consequential.  For instance, the M-type LDH does not form an abortive ternary complex LDH*NAD+*PYR. In addition, Bernstein, Everse and Grisham showed that in cancer there is an aberrant cytoplasmic MDH.

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3 Dimensional Ex vivo Technique for In situ Tumor Growth

Posted in Cancer - General, tagged angiogensis, brain cancer, Cancer - General, Glioblastoma on February 15, 2016| Leave a Comment »

3 Dimensional Ex vivo Technique for In situ Tumor Growth

Reporter: Stephen J. Williams, Ph.D.

Brain tumors are both difficult to treat and hard to study because of the organ they affect. The structure of the brain is extremely sensitive to alterations. Until recently the study of architectural alterations and their effects was mostly restricted to in vivo experiments. Typical culturing of brain tissue requires disaggregation and manipulation into a 2-dimensional format, losing any anatomically relevant structure. To study the in situbrain structure, a new technique has been described by researchers from the University of Erlangen-Nürnberg. By carefully sectioning the brains of 4 day-old mice and placing them on a 0.4 uM pore-size transwell membrane 6 well plate insert within required culture medium, they were able to study the endogenous structure under varying conditions. They injected astrocytes or glioma cells with a micropipette into the slices, and investigated the structural changes brain tumors effect in their environment. Termed the Vascular Organotypic Glioma Impact Model (VOGIM), it revealed all the characteristic pathological alterations normally associated with the disease in vivo such as tumor size and borders, vessel length, vessel junctions, and vessel branches, microglia, cell survival, and neuronal modifications. As this method allows for live cell fluorescent observation, they employed the technique to observe cultures treated with the chemotherapeutic Temozolamide (TMZ, Temodal/Temcad^®). Indeed they found reduced tumor growth in treatment groups vs controls, but also revealed surprising reduction in microglial cells in the peritumoral region. Additionally, they were able to observe the lack of response TMZ elicited from microglial in healthy regions of the tissue, despite its overall reduction in vascularization towards normal levels. The VOGIM technique allows for ex vivo study of brain tissue requiring three dimensional measurements, but may also be extended to other tissues with unique morphology such as kidney, liver, and intestine.

Ghoochani, et al. (December, 2015) A versatile ex vivo technique for assaying tumor angiogenesis and microglia in the brainONCOTARGET

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