An Immunotherapy For Prostate Cancer Based On Peptide Modified Multi-walled Carbon Nanotube Loaded With CpG

Prostate cancer is one of the most major cause for male tumor incidence and mortality in western countries.The incidence of prostate cancer is also increasing annually in china.Although Androgen deprivation therapy(ADT)has been the most usual treatment on prostate cancer,but almost all prostate cancer would develop into androgen independent prostate cancer(AIPC)with the treatment of ADT.The traditional therapy received eligible curative effect on prostate cancer.Tumor immunotherapy is a type of cancer treatment in recent yearswhich has achieved remarkable effect in clinical treatment.In this study,we e xplore the effect of Oligodeoxynucleotides containing CpG motifs(CpG ODN)immu ne-stimulants which contain cytosine-guanine dinucleotide on AIPC in vivo and in v itro via the mediation of multi-walled carbon nanotubes Anti-andr-ogen-independent prostate cancer in vivo and in vitro.The first section of the research introduced the construction and characterization of peptide-modified muti-walled carbon nanotube vector loaded with CpG.The peptide H3R6 containing 3 histidines and 6 arginines was synthesized by solid-phase peptide synthesis.Purification was carried out by preparative liquid chromatography and purified by high performance liquid chromatography and mass spectrometry.The purity of H3R6 determined could meet the experimental requirements.Multi-walled carbon nanotubes(MWCNTs)were carboxylated with concentrated sulfuric acid and concentrated nitric acid,and then re-reacted with the polypeptide to form the carrier MHR.The successful synthesis of the complex was confirmed by 1H NMR,IR,TEM and thermal gravimetric analysis.The MHR / CpG nanocomposites was prepared through the electrostatic interaction between arginine-rich positive charges in MHR vectors and negatively charged nucleic acids CpGs.At N / P of 6,the MHR vector was able to efficiently load the CpG immunosuppressant.The potential could keep at a steady state at N / P of 20 which is approximately 25 ± 2.62 mV.Through HEK293 transfection experiments,we found that the efficiency of transfection of MHR vector at N / P = 20 was highest.The second part of this project reported the in vitro cytological evaluation of MHR / CpG nano-emulsions,The CpG Nano Immunization could be efficiently internalized by RAW264.7 cells,CpG can combined with intracellular endometrial TLR9 receptor.In CCK8 cytotoxicity assays,MHR vectors have low toxicity to DU145,RM-1 and RAW264.7 cells.In vitro immune activity ELISA and PCR results showed that MHR / CpG could significantly promote the secretion of IL-6 and TNF-? proinflammatory cytokines on RAW264.7 cells which is higher than that in monomeric CpG group and was comparable with LPS(10 ng / ml).The third part of this paper illustrated the anti-prostate cancer effect and immunological activity of MHR / CpG nano-immune preparations.We successfully constructed DU145 prostate cancer BALB / c nude mice and RM-1 prostate cancer BALB / c mouse models.The results of in vivo image system showed that MHR / CpG could efficiently accumulate in tumor and tumor draining lymph nodes through Targeting effect.In vivo pharmacodynamics results show that MHR / CpG has a strong inhibitory effect on the proliferation of prostate cancer.Flow cytometric analysis of mouse splenic lymphocytes shows that MHR / CpG can effectively stimulate the proliferation and differentiation of CD4 and CD8 T cells in vivo.As showed in the mouse kidney and lung HE staining picture,the safety of MHR / CpG is quite high in vivo.This research successfully prepared a multi-walled carbon nanotube vector which modified by a peptide and loaded with immune activator CpG.the strategy illustrated in the research provides a new therapy for AIPC,and offered valued reference for the treatment of other tumors at the same time.