Gene therapy based on interleukin-12 loaded chitosan nanoparticles in a mouse model of fibrosarcoma

Document Type : Original Article


1 Immonuology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

2 Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

3 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran


Objective(s): Interleukin-12 (IL-12) as a cytokine has been proved to have a critical role in stimulating the immune system and has been used as immunotherapeutic agents in cancer gene therapy. Chitosan as a polymer, with high ability of binding to nucleic acids is a good candidate for gene delivery since it is biodegradable, biocompatible and non-allergenic polysaccharide. The objective of the present study was to investigate the effects of cells transfected with IL-12 loaded chitosan nanoparticles on the regression of fibrosarcoma tumor cells (WEHI-164) in vivo.
Materials and Methods: WEHI-164 tumor cells were transfected with IL-12 loaded chitosan nanoparticles and then were injected subcutaneously to inoculate tumor in BALB/c mice. Tumor volumes were determined and subsequently extracted after mice sacrifice. The immunohistochemistry staining was performed for analysis of Ki-67 expression (a tumor proliferation marker) in tumor masses. The expression of IL-12 and IFN-γ were studied using real-time polymerase chain reaction and immunoblotting.
Results: The group treated with IL-12 loaded chitosan nanoparticles indicated decreasing of tumor mass volume (P<0.001). The results of western blotting and real-time PCR showed that the IL-12 expression was increased in the group. Immunohistochemistry staining indicated that the Ki-67expression was reduced in the group treated with IL-12 loaded chitosan nanoparticles.
Conclusion: IL-12 gene therapy using chitosan nanoparticles has therapeutic effects on the regression of tumor masses in fibrosarcoma mouse model.


1. Asadi H, Orangi M, Shanehbandi D, Babaloo Z, Delazar A, Mohammadnejad L, et al. Methanolic fractions of Ornithogalum cuspidatum induce apoptosis in PC-3 prostate cancer cell line and WEHI-164 fibrosarcoma cancer cell line. Adv Pharm Bull 2014; 4:455-458.
2. Helmy KY, Patel SA, Nahas GR, Rameshwar P. Cancer immunotherapy: accomplishments to date and future promise. Ther Deliv 2013; 4:1307-1320.
3. Yoshimoto T, Morishima N, Okumura M, Chiba Y, Xu M, Mizuguchi J. Interleukins and cancer immunotherapy. Immunotherapy 2009; 1:825-844.
4. Gajewski TF. Cancer immunotherapy. Mol Oncol 2012; 6:242-250.
5. Kowalczyk DW, Wysocki PJ, Mackiewicz A. Cancer immunotherapy using cells modified with cytokine genes. Acta Biochim Pol 2003; 50:613-624.
6. Zarour HM, Ferrone S. Cancer immunotherapy: progress and challenges in the clinical setting. Eur J Immunol 2011; 41:1510.
7. Ohno M, Natsume A, Wakabayashi T. Cytokine therapy.  Glioma: Springer; 2012. p. 86-94.
8. Hallaj-Nezhadi S, Valizadeh H, Baradaran B, Dobakhti F, Lotfipour F. Preparation and characterization of gelatin nanoparticles containing pDNA encoding IL-12 and their expression in CT-26 carcinoma cells. Future Oncol 2013; 9:1195-1206.
9. Lotfipour F, Hallaj-Nezhadi S, Valizadeh H, Dastmalchi S, Baradaran B, Jalali MB, et al. Preparation of chitosan-plasmid DNA nanoparticles encoding interleukin-12 and their expression in CT-26 colon carcinoma cells. J Pharm Pharm Sci 2011; 14:181-195.
10. Razi Soofiyani S, Kazemi T, Lotfipour F, Mohammad hosseini A, Shanehbandi D, Hallaj-Nezhadi S, et al. Gene therapy with IL-12 induced enhanced anti-tumor activity in fibrosarcoma mouse model. Artif Cells Nanomed Biotechnol 2016; 44:1988-1993.
11. Xu D, Gu P, Pan PY, Li Q, Sato AI, Chen SH. NK and CD8+ T cell‐mediated eradication of poorly immunogenic B16‐F10 melanoma by the combined action of IL‐12 gene therapy and 4‐1BB costimulation. Int J Cancer 2004; 109:499-506.
12. Beadling C, Slifka MK. Regulation of innate and adaptive immune responses by the related cytokines IL-12, IL-23, and IL-27. Arch Immunol Ther Exp 2006; 54:15-24.
13. Xu M, Mizoguchi I, Morishima N, Chiba Y, Mizuguchi J, Yoshimoto T. Regulation of antitumor immune responses by the IL-12 family cytokines, IL-12, IL-23, and IL-27. Clinical and Developmental immunology 2010; 2010
14.  A Engel M, F Neurath M. Anticancer properties of the IL-12 family-focus on colorectal cancer. Current medicinal chemistry 2010; 17:3303-3308.
15. Choi KJ, Zhang SN, Choi IK, Kim JS, Yun CO. Strengthening of antitumor immune memory and prevention of thymic atrophy mediated by adenovirus expressing IL-12 and GM-CSF. Gene Ther 2012; 19:711-723.
16. Suzuki R, Namai E, Oda Y, Nishiie N, Otake S, Koshima R, et al. Cancer gene therapy by IL-12 gene delivery using liposomal bubbles and tumoral ultrasound exposure. J Controll Release 2010; 142:245-250.
17. Bielawska-Pohl A, Blesson S, Benlalam H, Trenado A, Opolon P, Bawa O, et al. The anti-angiogenic activity of IL-12 is increased in iNOS−/− mice and involves NK cells. J Mol Med 2010; 88:775-784
18. Janát-Amsbury MM, Yockman JW, Lee M, Kern S, Furgeson DY, Bikram M, et al. Combination of local, nonviral IL12 gene therapy and systemic paclitaxel treatment in a metastatic breast cancer model. Mol Ther 2004; 9:829-836.
19. Hallaj-Nezhadi S, Dass CR, Lotfipour F. Intraperitoneal delivery of nanoparticles for cancer gene therapy. Future Oncol 2013; 9:59-68.
20. Dass C, Hallaj-Nezhadi S, Lotfipour F. Nanoparticle-mediated interleukin-12 cancer gene therapy. J Pharm Pharm Sci 2010; 13:472-485.
21. Tan H, Wu J, Lao L, Gao C. Gelatin/chitosan/-hyaluronan scaffold integrated with PLGA microspheres for cartilage tissue engineering. Acta Biomater 2009; 5:328-337.
22. Conwell CC, Huang L. Recent advances in non‐viral gene delivery. Adv Genet 2005; 53:1-18.
23. Wang JH, Liu XY. Targeting strategies in cancer gene therapy. Acta Biochim Biophys Sin 2003; 35:311-316.
24. Malvicini M, Ingolotti M, Piccioni F, Garcia M, Bayo J, Atorrasagasti C, et al. Reversal of gastrointestinal carcinoma-induced immunosuppression and induction of antitumoural immunity by a combination of cyclo-phosphamide and gene transfer of IL-12. Mol Oncol 2011; 5:242-255.
25. Newman C, Bettinger T. Gene therapy progress and prospects: ultrasound for gene transfer. Gene Ther 2007; 14:465-475.
26. Taniyama Y, Tachibana K, Hiraoka K, Namba T, Yamasaki K, Hashiya N, et al. Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation 2002; 105:1233-1239.
27. Shen Z, Brayman A, Chen L, Miao C. Ultrasound with microbubbles enhances gene expression of plasmid DNA in the liver via intraportal delivery. Gene Ther 2008; 15:1147-1155.
28. Walser TC, Ma X, Kundu N, Dorsey R, Goloubeva O, Fulton AM. Immune-mediated modulation of breast cancer growth and metastasis by the chemokine Mig (CXCL9) in a murine model. J Immunother 2007; 30:490-498.
29. You CX, Shi M, Liu Y, Cao M, Luo R, Hermonat PL. AAV2/IL-12 gene delivery into dendritic cells (DC) enhances CTL stimulation above other IL-12 applications: Evidence for IL-12 intracrine activity in DC. Oncoimmunology 2012; 1:847-855
30. Reay J, Gambotto A, Robbins P. The antitumor effects of adenoviral-mediated, intratumoral delivery of interleukin 23 require endogenous IL-12. Cancer Gene Ther 2012; 19:135-143.
31. Fallarino F, Ashikari A, Boon T, Gajewski TF. Antigen-specific regression of established tumors induced by active immunization with irradiated IL-12-but not B7-1-transfected tumor cells. Int Immunol 1997; 9:1259-1269.
32. Qin Z, Schwartzkopff J, Pradera F, Kammertœns T, Seliger B, Pircher H, et al. A critical requirement of interferon γ-mediated angiostasis for tumor rejection by CD8+ T cells. Cancer Res 2003; 63:4095-4100.
33. Wasungu L, Hoekstra D. Cationic lipids, lipoplexes and intracellular delivery of genes. J Controll Release 2006; 116:255-264.
34. Swain S, Babu S, Beg S, Jena J. Nanoparticles for Cancer Targeting: Current and Future Directions. Current drug delivery 2016.
35. Khaja F, Jayawardena D, Kuzmis A, Önyüksel H. Targeted sterically stabilized phospholipid siRNA nanomedicine for hepatic and renal fibrosis. Nanomaterials 2016; 6:8.
36. Sandbichler AM, Aschberger T, Pelster B. A method to evaluate the efficiency of transfection reagents in an adherent zebrafish cell line. Biores Open Access 2013; 2:20-27.
37. Chada S, Ramesh R, Mhashilkar AM. Cytokine-and chemokine-based gene therapy for cancer. Curr Opin Mol Ther 2003; 5:463-474.
38. Yang Y, Chen J, Li H, Wang Y, Xie Z, Wu M, et al. Porcine interleukin-2 gene encapsulated in chitosan nanoparticles enhances immune response of mice to piglet paratyphoid vaccine. Comp Immunol Microbiol Infect Dis 2007; 30:19-32.
39. Zhang DY, Shen XZ, Wang JY, Dong L, Zheng YL, Wu LL. Preparation of chitosan-polyaspartic acid-5-fluorouracil nanoparticles and its anti-carcinoma effect on tumor growth in nude mice. World J Gastroenterol 2008; 14:3554-3562.