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Posts Tagged ‘molecular self-assembly’


Tumor Shrinking Triple Helices

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Tumor-Shrinking Triple-Helices

A braided structure and some adhesive hydrogel make therapeutic microRNAs both stable and sticky.

By Ruth Williams | April 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/45576/title/Tumor-Shrinking-Triple-Helices

 

MicroRNAs (miRs) are small, noncoding ribonucleic acids that control the translation of target messenger RNAs (mRNAs). Given their roles in development, differentiation, and other cellular processes, misregulation of miRs can contribute to diseases such as cancer. Indeed, “they are recognized as important modulators of cancer progression,” says Natalie Artzi of Harvard Medical School.

In addition to occasionally promoting cancer pathology, miRs also hold the potential to treat it—either by restoring levels of suppressed miRs, or by repressing overactive ones using antisense miRs (antagomiRs). While miRs are promising therapeutic molecules, says Daniel Siegwart of the University of Texas Southwestern Medical Center in Dallas, their use “is currently hindered by at least two issues: nucleic acid instability in vivo, and the development of effective delivery systems to transport miRs into tumor cells.”

Artzi and her team have now addressed both of these issues in one fell swoop. They first assembled two therapeutic miRs—one antagomiR and one that replaced a deficient miR—together with a third miR, a complement of the replacement strand, into triple-helix structures, which increased molecular stability without affecting function. They then complexed these helices with dendrimers—large synthetic branching polymer particles—and mixed these complexes with dextran aldehyde to form an adhesive hydrogel. The gel could then be applied directly to the surface of tumors to deliver the therapeutic miRs into cells with high efficiency.

In mice with induced breast tumors, the triple-helix–hydrogel approach led to dramatic tumor shrinkage and extended life span: the animals survived approximately one month longer than those treated with standard-of-care chemotherapy drugs. Because the RNA-hydrogel mixture must be applied directly to the tumor, the approach will not be suitable for all cancers. But one potential application, says Siegwart, is that “the hydrogel could be applied by a surgeon after performing bulk tumor removal…[and] might kill remaining tumor cells that would otherwise cause tumor recurrence.” (Nature Materials, http://dx.doi.org:/10.1038/NMAT4497, 2015)

STICKING IT TO TUMORS: To deliver therapeutic microRNAs (miRs) to tumors, braids of three microRNAs (miRs)—an antisense strand that blocks a miR overactive in cancer, a strand that replaces a deficient miR, and a stabilizing strand (1)—are added to a dendrimer (2) and mixed with a hydrogel scaffold (3). When researchers introduced the sticky gel onto mouse mammary tumors (4), the malignancies shrank and the animals lived longer (5)© GEORGE RETSECK; J.CONDE ET AL., NATURE MATERIALS

 

miR DELIVERY SYSTEM VEHICLE DOSE TUMOR TARGETING APPLICABLE TUMOR TYPES
Nanoparticles Examples: gold particles, liposomes, peptide nucleic acids, or polymers Usually multiple injections Combining miRs with aptamers or antibodies can guide nanoparticles to target cells, but systemic delivery inevitably leads to some off-target dispersion. Multisite or blood cancers
RNA–triple-helix-hydrogel Dendrimer-dextran hydrogel One Adhesive hydrogel sticks miRs to tumor site with minimal dispersion to other tissues. Solid Tumors

 

Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment

João CondeNuria OlivaMariana AtilanoHyun Seok Song & Natalie Artzi
Nature Materials15,353–363(2016)
                         http://dx.doi.org:/10.1038/nmat4497

The therapeutic potential of miRNA (miR) in cancer is limited by the lack of efficient delivery vehicles. Here, we show that a self-assembled dual-colour RNA-triple-helix structure comprising two miRNAs—a miR mimic (tumour suppressor miRNA) and an antagomiR (oncomiR inhibitor)—provides outstanding capability to synergistically abrogate tumours. Conjugation of RNA triple helices to dendrimers allows the formation of stable triplex nanoparticles, which form an RNA-triple-helix adhesive scaffold upon interaction with dextran aldehyde, the latter able to chemically interact and adhere to natural tissue amines in the tumour. We also show that the self-assembled RNA-triple-helix conjugates remain functional in vitro and in vivo, and that they lead to nearly 90% levels of tumour shrinkage two weeks post-gel implantation in a triple-negative breast cancer mouse model. Our findings suggest that the RNA-triple-helix hydrogels can be used as an efficient anticancer platform to locally modulate the expression of endogenous miRs in cancer.

 

Figure 1: Self-assembled RNA-triple-helix hydrogel nanoconjugates and scaffold for microRNA delivery.

Self-assembled RNA-triple-helix hydrogel nanoconjugates and scaffold for microRNA delivery.

a, Schematic showing the self-assembly process of three RNA strands to form a dual-colour RNA triple helix. The RNA triplex nanoparticles consist of stable two-pair FRET donor/quencher RNA oligonucleotides used for in vivo miRNA inhibit…

 

Figure 4: Proliferation, migration and survival of cancer cells as a function of RNA-triple-helix nanoparticles treatment.close

Proliferation, migration and survival of cancer cells as a function of RNA-triple-helix nanoparticles treatment.

a, miR-205 and miR-221 expression in breast cancer cells at 24, 48 and 72h of incubation (n = 3, statistical analysis performed with a two-tailed Students t-test, , P < 0.01). miRNA levels were normalized to the RNU6B reference gene

 

  1. Kasinski, A. L. & Slack, F. J. MicroRNAs en route to the clinic: Progress in validating and targeting microRNAs for cancer therapy. Nature Rev. Cancer 11, 849864 (2011).
  2. Li, Z. & Rana, T. M. Therapeutic targeting of microRNAs: Current status and future challenges. Nature Rev. Drug Discov. 13, 622638 (2014).
  3. Yin, H. et al. Non-viral vectors for gene-based therapy. Nature Rev. Genet. 15, 541555(2014).
  4. Conde, J., Edelman, E. R. & Artzi, N. Target-responsive DNA/RNA nanomaterials for microRNA sensing and inhibition: The jack-of-all-trades in cancer nanotheranostics? Adv. Drug Deliv. Rev. 81, 169183 (2015).
  5. Chen, Y. C., Gao, D. Y. & Huang, L. In vivo delivery of miRNAs for cancer therapy: Challenges and strategies. Adv. Drug Deliv. Rev. 81, 128141 (2015).
  6. Yin, P. T., Shah, B. P. & Lee, K. B. Combined magnetic nanoparticle-based microRNA and hyperthermia therapy to enhance apoptosis in brain cancer cells. Small 10, 41064112(2014).
  7. Hao, L. L., Patel, P. C., Alhasan, A. H., Giljohann, D. A. & Mirkin, C. A. Nucleic acid–gold nanoparticle conjugates as mimics of microRNA. Small 7, 31583162 (2011).
  8. Endo-Takahashi, Y. et al. Systemic delivery of miR-126 by miRNA-loaded bubble liposomes for the treatment of hindlimb ischemia. Sci. Rep. 4, 3883 (2014).
  9. Chen, Y. C., Zhu, X. D., Zhang, X. J., Liu, B. & Huang, L. Nanoparticles modified with tumor-targeting scFv deliver siRNA and miRNA for cancer therapy. Mol. Ther. 18, 16501656(2010).
  10. Anand, S. et al. MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis. Nature Med. 16, 909914 (2010).

 

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Author: Tilda Barliya PhD

Peripheral nerve lacerations are common injuries and often cause long lasting disability (1a) due to pain, paralyzed muscles and loss of adequate sensory feedback from the nerve receptors in the target organs such as skin, joints and muscles (1b).

Nerve injuries are common and typically affect young adults with the majority of injuries occur from trauma or complication of surgery. Traumatic injuries can occur due to stretch, crush, laceration (sharps or bone fragments), and ischemia, and are more frequent in wartime, i.e., blast exposure. Domestic or occupational accidents with glass, knifes of machinery may also occur.

Statistics show that peripheral nervous system (PNS) injuries were 87% from trauma and 12% due to surgery (one-third tumor related, two-thirds non– tumor related). Nerve injuries occurred 81% of the  time in the upper extremities and 11% in the lower extremities, with the balance in other locations (4).

Injury to the PNS can range from severe, leading to major loss of function or intractable neuropathic pain, to mild, with some sensory and/or motor deficits affecting quality of life.

Functional recovery after nerve injury involves a complex series of steps, each of which may delay or impair the regenerative process. In cases involving any degree of nerve injury, it is useful initially to categorize these regenerative steps anatomically on a gross level. The sequence of regeneration may be divided into anatomical zones (4):

  1. the neuronal cell body
  2. the segment between the cell body and the injury site
  3. the injury site itself
  4. the distal segment between the injury site and the end organ
  5. the end organ itself

A delay in regeneration or unsuccessful regeneration may be attributed to pathological changes that impede normal reparative processes at one or more of these zones.

Nanotechnology for regenerative nerves: by Gunilla Elam

Repairing nerve defects with large gaps remains one of the most operative challenges for surgeons. Incomplete recovery from peripheral nerve injuries can produce a diversity of negative outcomes, including numbness, impairment of sensory or motor function, possibility of developing chronic pain, and devastating permanent disability.

In the past few years several techniques have been used to try and repair nerve defects and include:

  • Coaptation
  • Nerve autograph
  • Biological or polymeric nerve conduits (hollow nerve guidance conduits)

For example, When a direct repair of the two nerve ends is not possible, synthetic or biological nerve conduits are typically used for small nerve gaps of 1 cm or less. For extensive nerve damage over a few centimeters in length, the nerve autograft is the “gold standard” technique. The biggest challenges, however, are the limited number and length of available donor nerves, the additional surgery associated with donor site morbidity, and the few effective nerve graft alternatives.

Degeneration of the axonal segment in the distal nerve is an inevitable consequence of disconnection, yet the distal nerve support structure as well as the final target must maintain efficacy to guide and facilitate appropriate axonal regeneration. There is currently no clinical practice targeted at maintaining fidelity of the distal pathway/target, and only a small number of researchers are investigating ways to preserve the distal nerve segment, such as the use of electrical stimulation or localized drug delivery. Thus development of tissue-engineered nerve graft may be a better matched alternative (6,7,9).

The guidance conduit serves several important roles for nerve regeneration such as:
a) directing axonal sprouting from the regenerating nerve
b) protecting the regenerating nerve by restricting the infiltration of fibrous tissue
c) providing a pathway for diffusion of neurotropic and neurotophic factors

Early guidance conduits were primarily made of silicone due to its stability under physiological conditions, biocompatibility, flexibility as well as ease of processing into tubular structures. Although silicone  conduits have proven reasonably successful as conduits for small gap lengths in animal models (<5 mm). The non-biodegradability of silicone conduits has limited its application as a strategy for long-term repair and recovery. Tubes also eventually become encapsulated with fibrous tissue, which leads to nerve compression, requiring additional surgical intervention to remove the tube. Another limiting factor with inert guidance conduits is that they provide little or no nerve regeneration for gap lengths over 10 mm in the PNS unless exogenous growth factors are used (6,7).

In animal studies, biodegradable nerve guidance conduits have provided a feasible alternative, preventing neuroma formation and infiltration of fibrous tissue. Biodegradable conduits have been fabricated from natural or synthetic materials such as collagen, chitosan and poly-L-lactic acid.

Nanostructured Scaffolds for Neural Tissue Engineering: Fabrication and Design

At the micro- and nanoscale, cells of the CNS/PNS reside within functional microenvironments consisting of physical structures including pores, ridges, and fibers that make up the extracellular matrix (ECM) and plasma membrane cell surfaces of closely apposed neighboring cells. Cell-cell and cell-matrix interactions contribute to the formation and function of this architecture, dictating signaling and maintenance roles in the adult tissue, based on a complex synergy between biophysical (e.g. contact-mediated signaling, synapse control), and biochemical factors (e.g. nutrient support and inflammatory protection). Neural tissue engineering scaffolds are aimed toward recapitulating some of the 3D biological signaling that is known to be involved in the maintenance of the PNS and CNS and to facilitate proliferation, migration and potentially differentiation during tissue repair (9).

Nanotechnology and tissue engineering are based on two main approaches:

  • Electrospinning (top-down) – involves the production of a polymer filament using an electrostatic force. Electrospinning is a versatile technique that enables production of polymer fibers with diameters ranging from a few microns to tens of nanometers.
  • Molecular self-assembly of peptides (bottom-up) – Molecular self-assembly is mediated by weak, non-covalent bonds, such as van der Waals forces, hydrogen bonds, ionic bonds, and hydrophobic interactions. Although these bonds are relatively weak, collectively they play a major role in the conformation of biological molecules found in nature.


Pfister et al (6) very nicely summarized the various polymeric fibers been used to achieve the goal of nerve regeneration, even in humans. These material include a wide array of polymers from silica to PLGA/PEG and Diblock copolypeptides.

Many of these approaches also enlist many trophic factors that have been investigated in nerve conduits

Currently there are three general biomaterial approaches for local factor delivery:

  1. Incorporation of factors into a conduit filler such as a hydrogel
  2. Designing a drug release system from the conduit biomaterial such as microspheres
  3. Immobilizing factors on the scaffold that are sensed in place or liberated upon matrix degradation.

Maeda et al had a  creative approach to bridge larger gaps by using the combination of nerve grafts and open conduits in an alternating “stepping stone” assembly, which may perform better than an empty conduit alone (8).

Summary

Peripheral nerve repair is a growing field with substantial progress being made in more effective repairs. Nanotechnology and biomedical engineering have made significant contributions; from surgical instrumentation to the development of tissue engineered grafting substitutes.  However, to date the field of neural tissue engineering has not progressed much past the conduit bridging of small gaps and has not come close to matching the autograf. Much more studies are needed to understand the cell behaviour that can promote cell survival, neurite outgrowth, appropriate re-innervation and consequently the functional recovery post PNS/CNS injuries. This is since understanding of the cellular response to the combination of these external cues within 3D architectures is limited at this stage.

Ref:

1a. Jaquet JB, Luijsterburg AJ, Kalmijn S, Kuypers PD, Hofman A, Hovius SE.  Median, ulnar, and combined median-ulnar nerve injuries:functional outcome and return to productivity. J Trauma 2001 51: 687-692. http://www.ncbi.nlm.nih.gov/pubmed/11586160

1b. Lundborg G, Rosen B. Hand function after nerve repair. Acta Physiol (Oxf) 2007 189: 207-217. http://www.ncbi.nlm.nih.gov/pubmed/17250571

1. Chang WC., Kliot M and Stretavan DW. Microtechnology and Nanotechnology in Nerve Repair. Neurological Research 2008; vol 30: 1053-1062. http://vision.ucsf.edu/sretavan/sretavanpdfs/2008b-Chang%20&%20Sretavan.pdf

2. Biazar E., Khorasani MT and Zaeifi D. Nanotechnology for peripheral nerve regeneration. Int. J. Nano. Dim. 2010 1(1): 1-23.  http://www.ijnd.ir/doc/2010-v1-i1/2010-V1-I1-1.pdf

3. Albert Aguayo. Nerve regeneration revisited. Nature Reviews Neuroscience 7, 601 (August 2006).

http://www.nature.com/nrn/journal/v7/n8/full/nrn1974.html

4. Burnett MG and  Zager EL. Pathophysiology of Peripheral Nerve Injury: A Brief Review. Neurosurg Focus. 2004;16(5) .

http://www.medscape.com/viewarticle/480071_5

5. Dag Welin. Neuroprotection and axonal regeneration after peripheral nerve injury. MEDICAL DISSERTATIONS

Welin, D., Novikova, L.N., Wiberg, M., Kellerth, J-O. and Novikov, L.N. Survival and regeneration of cutaneous and muscular afferent neurons after peripheral nerve injury in adult rats. Experimental Brain Research, 186, 315-323, 2008.

http://link.springer.com/article/10.1007%2Fs00221-007-1232-5

6. Pfister BJ., Gordon T., Loverde JR., Kochar AS., Mackinnon SE and Cullen Dk. Biomedical Engineering Strategies for Peripheral Nerve Repair: Surgical Applications, State of the Art, and Future Challenges. Critical Reviews™ in Biomedical Engineering 2011, 39(2):81–124. http://www.med.upenn.edu/cullenlab/user_documents/2011Pfisteretal-PNIReviewArticleCritRevBME.pdf

7. Zhou K, Nisbet D, Thouas G,  Bernard C and Forsythe J. Bio-nanotechnology Approaches to Neural Tissue Engineering. Intechopen. Com. http://cdn.intechopen.com/pdfs/9811/InTech-Bio_nanotechnology_approaches_to_neural_tissue_engineering.pdf

8. Maeda T, Mackinnon SE, Best TJ, Evans PJ, Hunter DA, Midha RT. Regeneration across ‘stepping-stone’ nerve grafts. Brain Res. 1993;618(2):196–202. http://www.ncbi.nlm.nih.gov/pubmed/?term=Maeda+T+and+regeneration+across+stepping+stone

9. Sedaghati T., Yang SY., Mosahebi A., Alavijeh MS and Seifalian AM. Nerve regeneration with aid of nanotechnology and cellular engineering. Biotechnol Appl Biochem. 2011 Sep-Oct;58(5):288-300. http://www.ncbi.nlm.nih.gov/pubmed/21995532

 

 

 

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