H6R6 Short Peptide Modified Chitosan Nanoparticles Are Used For The Delivery Of SiRNA And Its Use In Tumor Therapy

In recent years,gene therapy has become an important means for treating malignant tumors,while the small interfering RNA(siRNA)drugs have aroused extensive attention from the medical field.However,due to the instability of naked siRNA in vivo,it is apt to be degraded by nuclease with a short half-life.These deficiencies restrict the clinical application of siRNA in tumor therapy.As a result,one of the difficulties that should be urgently solved in the application of siRNA therapy is to develop carriers that can protect and deliver siRNA into cells effectively.Hence,a series of non-viral carriers were synthesized and the most efficient one was screened to deliver functional siRNA for anti-tumor therapy in vivo.Firstly,poly(histidine-arginine)6 peptides(H6R6)were covalently conjugated to the terminal of free amino groups in CS molecules,so as to produce siRNA delivery carrier through a coupling reaction.The structural composition of H6R6-CS copolymers were identified by 1HNMR analysis method.Subsequently,MTT assays were carried out to evaluate the cytotoxicity of H6R6-CS copolymers by selecting different kinds of cell lines.The results of cytotoxicity assay indicated the H6R6-CS copolymer was safe and non-toxic.The buffer capacity of H6R6-CS copolymers was investigated through acid-base titration,which was proved to be quite distinct within the pH range of 3.5-6.5.The complex coacevation method was applied to prepare nanoparticles for loading siRNA,and their physiochemical properties,such as surface morphology,particle size,zeta potential and stability were explored.The assay results showed that the particle size of the nanoparticle produced was about 175nm with a narrow polydispersity index,and the zeta potential was around +14.8mV,moreover,the surface of the nanoparticles was smooth.The 4T1Luc cells which can stably express luciferase reporter gene were selected to evaluate the inhibition effects of H6R6-CS/siRNA nanoparticles on endogenous genes at different transfection conditions.Compared with undecorated chitosan nanoparticle,H6R6 modified nanoparticles can inhibit the expression of endogenous genes more effectively with an inhibition efficiency of 50%.Meanwhile,cellular uptake of nanoparticles were estimated through flow cytometry and confocal laser scanning microscopy(CLSM),while the lysosome escape capability of nanoparticleswas examined by CLSM.Based on the research results,the efficiency of H6R6 modified nanoparticles to be uptaken by tumor cells is improved significantly,which can swiftly run away from endosome after being transfected for 6h.Nanoparticles were prepared by selecting Survivin and BRAF targeted siRNA,so as to investigate the anti-proliferation effect of nanoparticles on two kinds of tumor cells 4T1 and A375.The anti-proliferation efficiency of siRNA loaded nanoparticles was measured through MTT assays.The results showed that the anti-proliferation efficiency of two nanoparticles could reach up to as high as 49%and 51%respectively after being transfected for 48 hours,and the apoptosis rate of H6R6 modified nanoparticles is two times higher than that of undecorated chitosan nanoparticle.Nanoparticles were prepared by deliverying Survivin and BRAF targeted siRNA,so as to investigate the anti-tumor effect of nanoparticles on two kinds of tumor including breast cancer and melanoma.The research results indicated that H6R6-CS/siRNASur nanoparticles can not only inhibit tumor growth and metastasis significantly,but also prolong the survival time of tumor-bearing mice effectively.In addition,by intratumorally injecting nanoparticles prepared with BRAF targeted siRNAs into melanoma-bearing mice,the significant inhibition effects of H6R6-CS/siRNABRAF nanoparticles can also be verified.