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==Possible therapeutic applications and challenges==
==Possible therapeutic applications and challenges==


Since many disease states are caused by the overexpression of a gene responsible for a certain biochemical pathway, siRNA has been gathered support as a therapeutic agent.
Since many disease states are caused by the [[overexpression]] of a gene responsible for a certain [[biochemical pathway]], siRNA has been gathered support as a therapeutic agent.
In a therapeutic application, an siRNA antisense to the gene that is overexpressed is introduced to the cell, which binds to the mRNA transcript and causes the biochemical pathway that the gene was responsible for to stop functioning. For example, it has been shown that depleting polo-like kinase 1 can lead to apoptosis in cancer cells, but not normal cells.<ref name="ref1" /><ref name="ref2" /> Therefore, introduction of an siRNA that is antisense to the gene coding polo-like kinase 1 will lead to down regulation of the kinase, which causes the death of only tumor cells and therefore can be used to treat cancer.<ref name="ref3"/>
In a therapeutic application, an siRNA [[antisense]] to the gene that is overexpressed is introduced to the cell, which binds to the [[mRNA]] transcript and causes the biochemical pathway that the gene was responsible for to stop functioning. For example, it has been shown that depleting [[polo-like kinase 1]] can lead to [[apoptosis]] in cancer cells, but not normal cells.<ref name="ref1" /><ref name="ref2" /> Therefore, introduction of an siRNA that is antisense to the gene coding polo-like kinase 1 will lead to [[Receptor_(biochemistry)#Role_in_Genetic_Disorders|down regulation]] of the kinase, which causes the death of only tumor cells and therefore can be used to treat cancer.<ref name="ref3"/>
Introducing siRNA, however, is not easy. The siRNA molecules are too small to stay in circulation, but are too large to enter cells passively. Moreover, the immune response induced by exogenous siRNA has a large effect on the therapeutic value of the treatment.<ref name="ref4"/> Research on the use of siRNA as a therapuetic agent is largely focused on the safe and effective delivery of the siRNA molecules.
Introducing siRNA, however, is not easy. The siRNA molecules are too small to stay in circulation, but are too large to enter cells passively. Moreover, the [[immune response]] induced by [[exogenous]] siRNA has a large effect on the therapeutic value of the treatment.<ref name="ref4"/> Research on the use of siRNA as a therapuetic agent is largely focused on the safe and effective delivery of the siRNA molecules.
===Lipid Nanoparticle (LNP) Systems===
===Lipid Nanoparticle (LNP) Systems===
The most mature technology is the Lipid Nanoparticle (LNP) delivery system. In this system, a positively-charged lipid is formulated in a way that it is protonated at physiological pH, and can therefore bind to the negative charges on the siRNA backbone. This forms a micelle around the siRNA molecule which acts as a charge-shielding envelope.<ref name="ref5"/> Because of this, the micelle is significantly more stable in circulation, and can be taken into the cell through endocytosis.
The most mature technology is the Lipid [[Nanoparticle]] (LNP) delivery system. In this system, a positively-charged lipid is formulated in a way that it is [[protonated]] at physiological [[pH]], and can therefore bind to the negative charges on the siRNA backbone. This forms a [[micelle]] around the siRNA molecule which acts as a charge-shielding envelope.<ref name="ref5"/> Because of this, the micelle is significantly more stable in circulation, and can be taken into the cell through [[endocytosis]].
Once inside the cell, the change in pH across the cell membrane can deprotonate the lipids, making the lipids neutral. The loss of positive charge causes the lipids to detach from the siRNA and the micelle begins to degrade. This allows the siRNA payload to be delivered to intracellular environments exclusively.
Once inside the cell, the change in pH across the cell membrane can [[deprotonate]] the lipids, making the lipids neutral. The loss of positive charge causes the lipids to detach from the siRNA and the micelle begins to degrade. This allows the siRNA payload to be delivered to [[intracellular]] environments exclusively.
====Recent Advancements====
====Recent Advancements====
Further, the LNP can be “doped” with various polymers and other lipophilic components to allow the attachment of targeting molecules<ref name="ref6"/> that allow the LNP to deliver only to specific tissue types. This is important because, without some form of targeting, the LNPs affect any cell that takes them up.<ref name="ref7" /> Therefore, the introduction of targeting groups reduces off-target effects in pathways that exist in both healthy and diseased cells.
Further, the LNP can be “doped” with various [[polymers]] and other [[lipophilic]] components to allow the attachment of [[targeting molecules]]<ref name="ref6"/> that allow the LNP to deliver only to specific [[cell type|tissue types]]. This is important because, without some form of targeting, the LNPs affect any cell that takes them up.<ref name="ref7" /> Therefore, the introduction of targeting groups reduces off-target effects in pathways that exist in both healthy and diseased cells.
Other molecules such as fusogenic peptides have been attached to the surfaces of the nanoparticles, which improve cellular uptake of the nanoparticles.<ref name="ref8" /> These peptides help the LNP penetrate the cell membrane by interacting with the charged surface of the phospholipid bilayer and inserting themselves into the membrane.<ref name="ref9" /> This helps the LNP initiate endocytosis, increasing the efficiency of the drug delivery.
Other molecules such as [[cell penetrating peptide|fusogenic peptides]] have been attached to the surfaces of the nanoparticles, which improve cellular uptake of the nanoparticles.<ref name="ref8" /> These peptides help the LNP penetrate the cell membrane by interacting with the charged surface of the [[phospholipid bilayer]] and inserting themselves into the membrane.<ref name="ref9" /> This helps the LNP initiate endocytosis, increasing the efficiency of the drug delivery.
====Problems and Further Study====
====Problems and Further Study====
One issue that plagues LNPs is that they activate a part of the immune system known as complement.<ref name="ref10" /><ref name="ref11" /> In complement, the immune system identifies a foreign particle, here an LNP, and initiates pre-programmed cell death. However, since the LNPs are taken up into cells and deliver siRNA faster than complement is activated, the therapeutic index is significantly greater than that for unformulated siRNA.<ref name="ref12" /> Current research focuses on lessening the immunostimulatory nature of these particles; as the immune response to the particles decreases, the therapeutic index increases.
One issue that affects LNPs is that they activate a part of the immune system known as [[complement system]].<ref name="ref10" /><ref name="ref11" /> In complement, the immune system identifies a foreign particle, here an LNP, and initiates pre-programmed cell death. However, since the LNPs are taken up into cells and deliver siRNA faster than complement is activated, the [[therapeutic index]] is significantly greater than that for unformulated siRNA.<ref name="ref12" /> Current research focuses on lessening the [[immune system|immunostimulatory]] nature of these particles; as the immune response to the particles decreases, the therapeutic index increases.
===Bifunctional Gold Nanoparticles===
===Bifunctional Gold Nanoparticles===
Small clusters of neutral gold atoms known as gold nanoparticles have also been effective in siRNA delivery.<ref name="ref13" /> In these systems, polymers are attached to the surface of the nanoparticle by gold-sulfur bonds, followed by the annealing of the nucleotide to the polymer by disulfide bonds.<ref name="ref14" /> These particles are very stable to circulation until taken up into the cell, where enzymes within the cell cut the siRNA away from the nanoparticle.
Small clusters of neutral gold atoms known as gold nanoparticles have also been effective in siRNA delivery.<ref name="ref13" /> In these systems, polymers are attached to the surface of the nanoparticle by gold-sulfur bonds, followed by the [[annealing (biology)|annealing]] of the siRNA molecule to the polymer by [[disulfide]] bonds.<ref name="ref14" /> These particles are very stable to circulation until taken up into the cell, where [[enzymes]] within the cell cut the siRNA away from the nanoparticle.
====Recent Advancements====
====Recent Advancements====
The benefit of using gold nanoparticles is that in addition to modifying the surface of the particle, the core can also be modified.
The benefit of using gold nanoparticles is that in addition to modifying the surface of the particle, the core can also be modified.
Some recent research has focused on adding a magnetic component to the particles.<ref name="ref15" /> In this way, the particle reacts to magnetic fields, allowing the particle to be manipulated in living systems using a magnet to attract or repel the particles.
Some recent research has focused on adding a [[magnetic]] component to the particles.<ref name="ref15" /> In this way, the particle reacts to magnetic fields, allowing the particle to be manipulated in living systems using a magnet to attract or repel the particles.
Other efforts have added imaging components to the particles.<ref name="ref16" /> By incorporating MRI contrast agents, such as amphiphol, into the nanoparticle, the particles can be visualized as they move through the body.<ref name="ref16" /><ref name="ref17" /> This allows the researcher or physician to be sure the particle is delivering the siRNA to the appropriate parts of the body during treatment.
Other efforts have added [[radiocontrast|imaging components]] to the particles.<ref name="ref16" /> By incorporating MRI contrast agents, such as amphiphol, into the nanoparticle, the particles can be visualized as they move through the body.<ref name="ref16" /><ref name="ref17" /> This allows the researcher or physician to be sure the particle is delivering the siRNA to the appropriate parts of the body during treatment.
====Problems and Further Study====
====Problems and Further Study====
The particles, however, face problems with excretion, and can cause liver failure due to buildup of the particles.<ref name="ref17" /> Because there is no dedicated method for breaking down gold in the body, the nanoparticles tend to collect in cells until they are finally passed to the liver, where they still cannot be broken down.<ref name="ref14" /><ref name="ref17" /> While the particles are generally not immunostimulatory, they can cause a response if they remain in the liver long enough,<ref name="ref17" /><ref name="ref18" /> and as large quantities build up, toxicity leading to liver failure can be observed.<ref name="ref17" /> Current research is focused on finding ways to maximize efficacy to limit the amount of gold introduced to the body, and to find ways to increase excretion of the particles to limit accumulation,<ref name="ref19" /> which will significantly improve the therapeutic index of the gold nanoparticle drugs.
The particles, however, face problems with [[excretion]], and can cause [[liver failure]] due to buildup of the particles.<ref name="ref17" /> Because there is no dedicated method for breaking down gold in the body, the nanoparticles tend to collect in cells until they are finally passed to the liver, where they still cannot be broken down.<ref name="ref14" /><ref name="ref17" /> While the particles are generally not immunostimulatory, they can cause a response if they remain in the liver long enough,<ref name="ref17" /><ref name="ref18" /> and as large quantities build up, toxicity leading to liver failure can be observed.<ref name="ref17" /> Current research is focused on finding ways to maximize efficacy to limit the amount of gold introduced to the body, and to find ways to increase excretion of the particles to limit accumulation,<ref name="ref19" /> which will significantly improve the therapeutic index of the gold nanoparticle drugs.




Revision as of 06:20, 19 March 2010

Possible therapeutic applications and challenges

Since many disease states are caused by the overexpression of a gene responsible for a certain biochemical pathway, siRNA has been gathered support as a therapeutic agent. In a therapeutic application, an siRNA antisense to the gene that is overexpressed is introduced to the cell, which binds to the mRNA transcript and causes the biochemical pathway that the gene was responsible for to stop functioning. For example, it has been shown that depleting polo-like kinase 1 can lead to apoptosis in cancer cells, but not normal cells.[1][2] Therefore, introduction of an siRNA that is antisense to the gene coding polo-like kinase 1 will lead to down regulation of the kinase, which causes the death of only tumor cells and therefore can be used to treat cancer.[3] Introducing siRNA, however, is not easy. The siRNA molecules are too small to stay in circulation, but are too large to enter cells passively. Moreover, the immune response induced by exogenous siRNA has a large effect on the therapeutic value of the treatment.[4] Research on the use of siRNA as a therapuetic agent is largely focused on the safe and effective delivery of the siRNA molecules.

Lipid Nanoparticle (LNP) Systems

The most mature technology is the Lipid Nanoparticle (LNP) delivery system. In this system, a positively-charged lipid is formulated in a way that it is protonated at physiological pH, and can therefore bind to the negative charges on the siRNA backbone. This forms a micelle around the siRNA molecule which acts as a charge-shielding envelope.[5] Because of this, the micelle is significantly more stable in circulation, and can be taken into the cell through endocytosis. Once inside the cell, the change in pH across the cell membrane can deprotonate the lipids, making the lipids neutral. The loss of positive charge causes the lipids to detach from the siRNA and the micelle begins to degrade. This allows the siRNA payload to be delivered to intracellular environments exclusively.

Recent Advancements

Further, the LNP can be “doped” with various polymers and other lipophilic components to allow the attachment of targeting molecules[6] that allow the LNP to deliver only to specific tissue types. This is important because, without some form of targeting, the LNPs affect any cell that takes them up.[7] Therefore, the introduction of targeting groups reduces off-target effects in pathways that exist in both healthy and diseased cells. Other molecules such as fusogenic peptides have been attached to the surfaces of the nanoparticles, which improve cellular uptake of the nanoparticles.[8] These peptides help the LNP penetrate the cell membrane by interacting with the charged surface of the phospholipid bilayer and inserting themselves into the membrane.[9] This helps the LNP initiate endocytosis, increasing the efficiency of the drug delivery.

Problems and Further Study

One issue that affects LNPs is that they activate a part of the immune system known as complement system.[10][11] In complement, the immune system identifies a foreign particle, here an LNP, and initiates pre-programmed cell death. However, since the LNPs are taken up into cells and deliver siRNA faster than complement is activated, the therapeutic index is significantly greater than that for unformulated siRNA.[12] Current research focuses on lessening the immunostimulatory nature of these particles; as the immune response to the particles decreases, the therapeutic index increases.

Bifunctional Gold Nanoparticles

Small clusters of neutral gold atoms known as gold nanoparticles have also been effective in siRNA delivery.[13] In these systems, polymers are attached to the surface of the nanoparticle by gold-sulfur bonds, followed by the annealing of the siRNA molecule to the polymer by disulfide bonds.[14] These particles are very stable to circulation until taken up into the cell, where enzymes within the cell cut the siRNA away from the nanoparticle.

Recent Advancements

The benefit of using gold nanoparticles is that in addition to modifying the surface of the particle, the core can also be modified. Some recent research has focused on adding a magnetic component to the particles.[15] In this way, the particle reacts to magnetic fields, allowing the particle to be manipulated in living systems using a magnet to attract or repel the particles. Other efforts have added imaging components to the particles.[16] By incorporating MRI contrast agents, such as amphiphol, into the nanoparticle, the particles can be visualized as they move through the body.[16][17] This allows the researcher or physician to be sure the particle is delivering the siRNA to the appropriate parts of the body during treatment.

Problems and Further Study

The particles, however, face problems with excretion, and can cause liver failure due to buildup of the particles.[17] Because there is no dedicated method for breaking down gold in the body, the nanoparticles tend to collect in cells until they are finally passed to the liver, where they still cannot be broken down.[14][17] While the particles are generally not immunostimulatory, they can cause a response if they remain in the liver long enough,[17][18] and as large quantities build up, toxicity leading to liver failure can be observed.[17] Current research is focused on finding ways to maximize efficacy to limit the amount of gold introduced to the body, and to find ways to increase excretion of the particles to limit accumulation,[19] which will significantly improve the therapeutic index of the gold nanoparticle drugs.


References

  1. ^ Chopra, P.; Sethi, G.; Dastidar, S. G.; Ray, A., Polo-like kinase inhibitors: an emerging opportunity for cancer therapeutics. Expert Opin. Investig. Drugs 19 (1), 27-43.
  2. ^ Liu, X. Q.; Lei, M.; Erikson, R. L., Normal cells, but not cancer cells, survive severe Plk1 depletion. Mol. Cell. Biol. 2006, 26 (6), 2093-2108.
  3. ^ Hu, K. J.; Lee, C.; Qiu, D. X.; Fotovati, A.; Davies, A.; Abu-Ali, S.; Wai, D.; Lawlor, E. R.; Triche, T. J.; Pallen, C. J.; Dunn, S. E., Small interfering RNA library screen of human kinases and phosphatases identifies polo-like kinase 1 as a promising new target for the treatment of pediatric rhabdomyosarcomas. Mol. Cancer Ther. 2009, 8 (11), 3024-3035.
  4. ^ Frank Y. Xie, M. C. W., Patrick Y. Lu, Harnessing in vivo siRNA delivery for drug discovery and therapeutic development. Drug Discovery Today 2006, 11 (1-2), 67-73.
  5. ^ Semple, S. C.; Akinc, A.; Chen, J. X.; Sandhu, A. P.; Mui, B. L.; Cho, C. K.; Sah, D. W. Y.; Stebbing, D.; Crosley, E. J.; Yaworski, E.; Hafez, I. M.; Dorkin, J. R.; Qin, J.; Lam, K.; Rajeev, K. G.; Wong, K. F.; Jeffs, L. B.; Nechev, L.; Eisenhardt, M. L.; Jayaraman, M.; Kazem, M.; Maier, M. A.; Srinivasulu, M.; Weinstein, M. J.; Chen, Q. M.; Alvarez, R.; Barros, S. A.; De, S.; Klimuk, S. K.; Borland, T.; Kosovrasti, V.; Cantley, W. L.; Tam, Y. K.; Manoharan, M.; Ciufolini, M. A.; Tracy, M. A.; de Fougerolles, A.; MacLachlan, I.; Cullis, P. R.; Madden, T. D.; Hope, M. J., Rational design of cationic lipids for siRNA delivery. Nature Biotechnology 2010, 28 (2), 172-U18.
  6. ^ Yoshizawa, T.; Hattori, Y.; Hakoshima, M.; Koga, K.; Maitani, Y., Folate-linked lipid-based nanoparticles for synthetic siRNA delivery in KB tumor xenografts. Eur. J. Pharm. Biopharm. 2008, 70 (3), 718-725.
  7. ^ Singh, S. K.; Hajeri, P. B., siRNAs: their potential as therapeutic agents - Part II. Methods of delivery. Drug Discovery Today 2009, 14 (17-18), 859-865.
  8. ^ Hatakeyama, H.; Ito, E.; Akita, H.; Oishi, M.; Nagasaki, Y.; Futaki, S.; Harashima, H., A pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo. J. Control. Release 2009, 139 (2), 127-132.
  9. ^ Martin, I.; Ruysschaert, J. M., Common properties of fusion peptides from diverse systems. Biosci. Rep. 2000, 20 (6), 483-500.
  10. ^ Nakanishi, T.; Kunisawa, J.; Hayashi, A.; Tsutsumi, Y.; Kubo, K.; Nakagawa, S.; Nakanishi, M.; Tanaka, K.; Mayumi, T., Positively charged liposome functions as an efficient immunoadjuvant in inducing cell-mediated immune response to soluble proteins. J. Control. Release 1999, 61 (1-2), 233-240.
  11. ^ Scholer, N.; Hahn, H.; Muller, R. H.; Liesenfeld, O., Effect of lipid matrix and size of solid lipid nanoparticles (SLN) on the viability and cytokine production of macrophages. Int. J. Pharm. 2002, 231 (2), 167-176.
  12. ^ Christian Wolfrum, S. S., K Narayanannair Jayaprakash, Muthusamy Jayaraman, Gang Wang, Rajendra K Pandey, Kallanthottathil G Rajeev, Tomoko Nakayama, Klaus Charrise, Esther M Ndungo, Tracy Zimmermann, Victor Koteliansky, Muthiah Manoharan,Markus Stoffel, Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nature Biotechnology 2007, 25, 1149-1157.
  13. ^ Elbakry, A.; Zaky, A.; Liebkl, R.; Rachel, R.; Goepferich, A.; Breunig, M., Layer-by-Layer Assembled Gold Nanoparticles for siRNA Delivery. Nano Lett. 2009, 9 (5), 2059-2064.
  14. ^ a b DeLong, R. K.; Akhtar, U.; Sallee, M.; Parker, B.; Barber, S.; Zhang, J.; Craig, M.; Garrad, R.; Hickey, A. J.; Engstrom, E., Characterization and performance of nucleic acid nanoparticles combined with protamine and gold. Biomaterials 2009, 30 (32), 6451-6459.
  15. ^ Boyer, C.; Priyanto, P.; Davis, T. P.; Pissuwan, D.; Bulmus, V.; Kavallaris, M.; Teoh, W. Y.; Amal, R.; Carroll, M.; Woodward, R.; St Pierre, T., Anti-fouling magnetic nanoparticles for siRNA delivery. J. Mater. Chem. 2010, 20 (2), 255-265.
  16. ^ a b Qi, L. F.; Gao, X. H., Quantum dot-amphipol nanocomplex for intracellular delivery and real-time imaging of siRNA. ACS Nano 2008, 2 (7), 1403-1410.
  17. ^ a b c d e Longmire, M.; Choyke, P. L.; Kobayashi, H., Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats. Nanomedicine 2008, 3 (5), 703-717.
  18. ^ Massich, M. D.; Giljohann, D. A.; Seferos, D. S.; Ludlow, L. E.; Horvath, C. M.; Mirkin, C. A., Regulating Immune Response Using Polyvalent Nucleic Acid-Gold Nanoparticle Conjugates. Mol. Pharm. 2009, 6 (6), 1934-1940.
  19. ^ Reijnders, L., Hazard reduction in nanotechnology. J. Ind. Ecol. 2008, 12 (3), 297-306.
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