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. 2012 Dec 27;18(1):120–136. doi: 10.2478/s11658-012-0043-2

A nanoformulation of siRNA and its role in cancer therapy: In vitro and in vivo evaluation

Hitesh Jagani 1, Josyula Venkata Rao 1,✉, Vasanth Raj Palanimuthu 1,2, Raghu Chandrashekar Hariharapura 1, Sagar Gang 1
PMCID: PMC6275763  PMID: 23271435

Abstract

Overexpression of anti-apoptotic Bcl-2 is often observed in a wide variety of human cancers. It prevents the induction of apoptosis in neoplastic cells and contributes to resistance to chemotherapy. RNA interference has emerged as an efficient and selective technique for gene silencing. The potential to use small interfering RNA (siRNA) as a therapeutic agent for the treatment of cancer has elicited a great deal of interest. However, insufficient cellular uptake and poor stability have limited its therapeutic applications. The purpose of this study was to prepare chitosan nanoparticles via ionic gelation of chitosan by tripolyphosphate for effective delivery of siRNA to silence the anti-apoptotic Bcl-2 gene in neoplastic cells. Chitosan nanoparticles loaded with siRNA were in the size range 190 to 340 nm with a polydispersive index ranging from 0.04 to 0.2. They were able to completely bind with siRNA, provide protection against nuclease degradation, and enhance the transfection. Cell culture studies revealed that nanoparticles with entrapped siRNA could efficiently silence the antiapoptotic Bcl-2 gene. Studies on Swiss albino mice showed that siRNA could be effectively delivered through nanoparticles. There was significant decrease in the tumor volume. Blocking the expression of anti-apoptotic Bcl-2 can enhance the sensitivity of cancerous cells to anti-cancer drugs and the apoptosis rate. Therefore, nanoformulations with siRNA can be promoted as an adjuvant therapy in combination with anti-cancer drugs.

Key words: Chitosan, RNA interference, siRNA, Bcl-2, Apoptosis, Cancer, Nanoformulation

Full Text

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Abbreviations used

DEPC

diethylpyrocarbonate

DMEM

Dulbecco’s modified Eagle’s medium

FBS

fetal bovine serum

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PDI

polydispersive index

RNAi

RNA interference

RTPCR

reverse transcriptase polymerase chain reaction

SDS-PAGE

sodium dodecyl sulphate-polyacrylamide

SEM

scanning electron microscopy

siRNA

small interfering RNA

TPP

pentasodium tripolyphosphate

References

  • 1.Lowe S.W., Lin A.W. Apoptosis in cancer. Carcinogenesis. 2000;21:485–496. doi: 10.1093/carcin/21.3.485. [DOI] [PubMed] [Google Scholar]
  • 2.Chandra J., Kaufmann S.H. Apoptotic pathways in cancer progression and treatment. In: Finkel T., Gutkind J.S., editors. Signal Transduction and Human Disease. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 2003. pp. 143–170. [Google Scholar]
  • 3.Fulda S., Meyer E., Debatin K.M. Inhibition of TRAIL-induced apoptosis by Bcl-2 overexpression. Oncogene. 2002;21:2283–2291. doi: 10.1038/sj.onc.1205258. [DOI] [PubMed] [Google Scholar]
  • 4.Reed J.C. Apoptosis-targeted therapies for cancer. Cancer Cell. 2003;3:17–30. doi: 10.1016/S1535-6108(02)00241-6. [DOI] [PubMed] [Google Scholar]
  • 5.Brown J.M., Attardi L.D. The role of apoptosis in cancer development and treatment response. Nat. Rev. Cancer. 2005;5:231–237. doi: 10.1038/nrc1560. [DOI] [PubMed] [Google Scholar]
  • 6.Vousden K.H. Activation of the p53 tumor suppressor protein. Biochim. Biophys. Acta-Rev. Cancer. 2002;1602:47–59. doi: 10.1016/S0304-419X(02)00035-5. [DOI] [PubMed] [Google Scholar]
  • 7.Brown J.M., Wouters B.G. Apoptosis, p53, and tumor cell sensitivity to anticancer agents. Cancer Res. 1999;59:1391–1404. [PubMed] [Google Scholar]
  • 8.Banic B., Nipic D., Suput D., Milisav I. DMSO modulates the pathway of apoptosis triggering. Cell. Mol. Biol. Lett. 2011;16:328–341. doi: 10.2478/s11658-011-0007-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Adams J. M., Cory S. The Bcl-2 protein family: arbiters of cell survival. Science. 1998;281:1322–1345. doi: 10.1126/science.281.5381.1322. [DOI] [PubMed] [Google Scholar]
  • 10.Cory S., Huang D.C.S., Adams J.M. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene. 2003;22:8590–8607. doi: 10.1038/sj.onc.1207102. [DOI] [PubMed] [Google Scholar]
  • 11.Reed J.C. Dysregulation of apoptosis in cancer. J. Clin. Oncol. 1999;17:2941–2961. doi: 10.1200/JCO.1999.17.9.2941. [DOI] [PubMed] [Google Scholar]
  • 12.Oda E., Ohki R., Murasawa H., Nemoto J., Shibue T., Yamashita T., Tokino T., Taniguchi T. Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science. 2000;288:1053–1061. doi: 10.1126/science.288.5468.1053. [DOI] [PubMed] [Google Scholar]
  • 13.Bouillet P., Cory S., Zhang L.C., Strasser A., Adams J.M. Degenerative disorders caused by Bcl-2 deficiency prevented by loss of its BH3-only antagonist Bim. Dev. Cell. 2001;1:645–653. doi: 10.1016/S1534-5807(01)00083-1. [DOI] [PubMed] [Google Scholar]
  • 14.Sattler M., Liang H., Nettesheim D., Meadows R.P., Harlan J.E., Eberstadt M., Yoon H.S., Shuker S.B., Chang B.S., Minn A.J. Structure of Bcl-xLBak peptide complex: recognition between regulators of apoptosis. Science. 1997;275:983–994. doi: 10.1126/science.275.5302.983. [DOI] [PubMed] [Google Scholar]
  • 15.Kelekar A., Thompson C.B. Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol. 1998;8:324–330. doi: 10.1016/S0962-8924(98)01321-X. [DOI] [PubMed] [Google Scholar]
  • 16.Meier P., Finch A., Evan G. Apoptosis in development. Nature. 2000;6805:796–801. doi: 10.1038/35037734. [DOI] [PubMed] [Google Scholar]
  • 17.Oltersdorf T., Elmore S.W., Shoemaker A.R., Armstrong R.C., Augeri D.J., Belli B.A., Bruncko M., Deckwerth T.L., Dinges J., Hajduk P.J. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature. 2005;435:677–681. doi: 10.1038/nature03579. [DOI] [PubMed] [Google Scholar]
  • 18.Tan F.L., Yin J.Q. Application of RNAi to cancer research and therapy. Front Biosci. 2005;10:1946–1960. doi: 10.2741/1670. [DOI] [PubMed] [Google Scholar]
  • 19.Abdelrahim M., Safe S., Baker C., Abudayyeh A. RNAi and cancer: Implications and applications. Int. J. RNA Gene Target Res. 2006;2:136–152. [PMC free article] [PubMed] [Google Scholar]
  • 20.Akhtar S., Benter I.F. Nonviral delivery of synthetic siRNAs in vivo. J. Clin. Invest. 2007;117:3623–3632. doi: 10.1172/JCI33494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Bozkir A., Saka O.M. Chitosan nanoparticles for plasmid DNA delivery: effect of chitosan molecular structure on formulation and release characteristics. Drug Deliv. 2004;11:107–112. doi: 10.1080/10717540490280705. [DOI] [PubMed] [Google Scholar]
  • 22.Mao H.Q., Roy K., Troung-Le V.L., Janes K.A., Lin K.Y., Wang Y., August J.T., Leong K.W. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. J. Control. Release. 2001;70:399–421. doi: 10.1016/S0168-3659(00)00361-8. [DOI] [PubMed] [Google Scholar]
  • 23.Katas H., Alpar H.O. Development and characterisation of chitosan nanoparticles for siRNA delivery. J. Control. Release. 2006;115:216–225. doi: 10.1016/j.jconrel.2006.07.021. [DOI] [PubMed] [Google Scholar]
  • 24.Agnihotri S.A., Mallikarjuna N.N., Aminabhavi T.M. Recent advances on chitosan-based micro-and nanoparticles in drug delivery. J. Control. Release. 2004;100:5–28. doi: 10.1016/j.jconrel.2004.08.010. [DOI] [PubMed] [Google Scholar]
  • 25.Roy K., Mao H.Q., Huang S.K., Leong K.W. Oral gene delivery with chitosan-DNA nanoparticles generates immunologic protection in a murine model of peanut allergy. Nat. Med. 1999;5:387–397. doi: 10.1038/7385. [DOI] [PubMed] [Google Scholar]
  • 26.Jagani H.V., Josyula V.R., Hariharapura R.C., Palanimuthu V.R., Gang S.S. M. Nanoformulation of siRNA silencing Bcl-2 gene and its implication in cancer therapy. Arzneimittel-Forsch. 2011;61:577–591. doi: 10.1055/s-0031-1300556. [DOI] [PubMed] [Google Scholar]
  • 27.Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  • 28.Denizot F., Lang R. Rapid colorimetric assay for cell growth and survival: Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods. 1986;89:271–277. doi: 10.1016/0022-1759(86)90368-6. [DOI] [PubMed] [Google Scholar]
  • 29.Mullis, K.B. Process for amplifying nucleic acid sequences. Google Patents (1987).
  • 30.Mullis K.B., Faloona F.A., Scharf S.J., Saiki R.K., Horn G.T., Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 1986;51:263–273. doi: 10.1101/SQB.1986.051.01.032. [DOI] [PubMed] [Google Scholar]
  • 31.Lanciotti R.S., Calisher C.H., Gubler D.J., Chang G. J., Vorndam A.V. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J. Clin. Microbiol. 1992;30:545–560. doi: 10.1128/jcm.30.3.545-551.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Burnette W.N. Western blotting: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 1981;112:195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  • 33.De Martimprey H., Bertrand J.R., Fusco A., Santoro M., Couvreur P., Vauthier C., Malvy C. siRNA nanoformulation against the ret/PTC1 junction oncogene is efficient in an in vivo model of papillary thyroid carcinoma. Nucleic Acids Res. 2008;36:e2–e2. doi: 10.1093/nar/gkm1094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Goodsell D.S. The molecular perspective: Bcl-2 and apoptosis. The Oncologist. 2002;7:259–260. doi: 10.1634/theoncologist.7-3-259. [DOI] [PubMed] [Google Scholar]
  • 35.Ma W.W., Adjei A.A. Novel agents on the horizon for cancer therapy. CA Cancer J. Clin. 2009;59:111–137. doi: 10.3322/caac.20003. [DOI] [PubMed] [Google Scholar]
  • 36.Howard K.A., Rahbek U.L., Liu X., Damgaard C.K., Glud S.Z., Andersen M.A., Hovgaard M.B., Schmitz A., Nyengaard J.R., Besenbacher F. RNA interference in vitro and in vivo using a chitosan/siRNA nanoparticle system. Mol. Ther. 2006;14:476–484. doi: 10.1016/j.ymthe.2006.04.010. [DOI] [PubMed] [Google Scholar]
  • 37.Lee D., Zhang W., Shirley S.A., Kong X., Hellermann G.R., Lockey R.F., Mohapatra S.S. Thiolated chitosan/DNA nanocomplexes exhibit enhanced and sustained gene delivery. Pharm. Res. 2007;24:157–167. doi: 10.1007/s11095-006-9136-9. [DOI] [PubMed] [Google Scholar]
  • 38.Verma N.K., Davies A.M., Long A., Kelleher D., Volkov Y. STAT3 knockdown by siRNA induces apoptosis in human cutaneous T-cell lymphoma line Hut78 via downregulation of Bcl-xL. Cell. Mol. Biol. Lett. 2010;15:342–355. doi: 10.2478/s11658-010-0008-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Akhtar S., Benter I.F. Nonviral delivery of synthetic siRNAs in vivo. J. Clin. Invest. 2007;117:3623–3643. doi: 10.1172/JCI33494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Mao S., Sun W., Kissel T. Chitosan-based formulations for delivery of DNA and siRNA. Adv. Drug Delivery Rev. 2010;62:12–27. doi: 10.1016/j.addr.2009.08.004. [DOI] [PubMed] [Google Scholar]
  • 41.Dass C.R., Choong P.F.M. The use of chitosan formulations in cancer therapy. J. Microencapsul. 2008;25:275–279. doi: 10.1080/02652040801970461. [DOI] [PubMed] [Google Scholar]
  • 42.De La Fuente M., Ravi A.M., Paolicelli P., Sanchez A., Seijo B., Alonso M.J. Chitosan-based nanostructures: a delivery platform for ocular therapeutics. Adv. Drug Delivery Rev. 2010;62:100–117. doi: 10.1016/j.addr.2009.11.026. [DOI] [PubMed] [Google Scholar]
  • 43.Kim T.H., Jiang H.L., Jere D., Park I.K., Cho M.H., Nah J.W., Choi Y.J., Akaike T., Cho C.S. Chemical modification of chitosan as a gene carrier in vitro and in vivo. Prog. Polym. Sci. 2007;32:726–753. doi: 10.1016/j.progpolymsci.2007.05.001. [DOI] [Google Scholar]
  • 44.Spandana, K., Hitesh, J., Vasanth, R.P., Jesil, M.A., Mallikarjuna, R.C. and Venkata R.J. In vitro and in vivo evaluation of the efficacy of nanoformulation of siRNA as an adjuvant to improve the anticancer potential of cisplatin. Exp. Mol. Pathol. 2012; 10.1016/j.yexmp.2012.10.007. [DOI] [PubMed]

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