Polystyrene-acrylamide Nano-copolymer Co-delivering TGF-? Inhibitor And CpG Adjuvant As Immunotherapy For Hepatic Carcinoma

Nanocarriers can transport a variety of drugs, which have being widely used in cancer immunotherapy. Through hydrophilic modification, nanocarriers can obtain long circulating in vivo. Nanomedicine can be targeted to the tumor site passively through enhanced permeability and retention effect(EPR). Intelligent nanocarriers release the drugs at a controlled rate. The objective of this study was to construct a nano material containing immunosuppressive agents TGF-? inhibitor and immunoadjuvant CpG for immunotherapy of Hepatic carcinoma(HCC). The main research works including:(1) Polystyrene-acrylamide nano-copolymer(Pst-Amm) was prepared and modified by polyethyleneimine(PEI) and PEG. Then TGF-? inhibitor LY2157299(LY) or/and Cp G were loaded. Dynamic light scattering(DLS) and transmission electron microscopy(TEM) were used to characterize nanoparticles. The size of PS, PS-LY and PS-LY-CpG from TEM images were about 220-280 nm. PS, PS-LY and PS-LY-CpG were well dispersed spherical particles, the hydrodynamic diameter was 237.2nm, 261.4nm and 331.2nm respectively. Zeta potential was 37.9 mV, 39.9 mV and-25.5 mV respectively.(2) HPLC and UV spectrophotometer was used to detect the drug- loading content of LY and CpG in nanopartical respectively. The LY release behaviors were detected by HPLC. The results showed that 3mg PS could load LY 0.658 mg and CpG 70?g. 10.56% LY released at 24 h, followed by continuous steady release in vitro.(3) The biological safety of PS-LY-CpG was evaluated by MTT assay. No noticeable cytotoxic effect was observed even with high concentration of PS-LY-CpG at 24 h, 48 h and 72 h in HEK293 cell(Human embryonic kidney cell line).(4) Anti-tumor effect of the nanoaprticle was evaluated in vivo. Mouse liver cancer H22 subcutaneous tumor model was established at first. Seven groups were intratumoral injected PBS, LY, CpG, LY+CpG, PS-LY, PS-CpG, PS-LY-CpG respectively on day 8 post tumor cells inoculated. Drugs were adminstered every three days. Body weight and tumor volume were measured every two days. At the end of drug administered, sera were collected and animals were sacrificed. Tumor and spleen were taken out. Serum cytokine levels were tested by Elisa. CD3, CD4, CD8 cell levels in spleen was detected by flow cytometry and immunohistochemistry analysis. Subcutaneous tumors was weighed and sliced up for HE staining. Results: Tumor volume growth was the slowest and body weight almost no changes in PS-LY-CpG group, in which the inhibition rate reached to 99.67%. Flow cytometry and immunohistochemical results showed that CD3, CD8 T cell levels were higher than PBS group, CD4 T cell levels were less than PBS group. The levels of cytokine IL-12p70 and TNF-? were higher than PBS group.In summary, PS nanoparticals co-delivering LY and CpG has good physical and chemical properties. The mechanism probably was by increasing the level of CD8 T cells and enhancing cytokines of IL-12p70 and TNF-? to anti-cancer. PS-LY-CpG may have great potential in treating for liver cancer and other tumors.