Construction Of Nano-delivery System By Self-assembly Of Dualtargeted Small Molecule Prodrug And Its Anti-tumor Immune Effect

Colon cancer is a common clinical malignant tumor with relatively high immunogenicity.Theoretically,immunotherapy should achieve good curative effect,but the clinical therapeutic efficiency is very low.This may be related to the degree of T cell infiltration and the killing function of T cells in the tumor tissues of colon cancer patients.Induction of immunogenic cell death(ICD)in cancer cells results in the release of endogenous danger signals and tumor antigens to effectively initiate anticancer immunity and activate the anti-tumor immune response of T cells.In recent years,autophagy has been considered as an accomplice to tumor cell immune escape,and inhibition of autophagy may be a promising strategy for enhancing T cell immunotherapy.In addition,autophagy is also involved in the polarization of macrophages.The use of autophagy inhibitors can reprogram tumor-associated macrophages(TAMs)from a pro-tumor M2 phenotype to an anti-tumor M1 phenotype.Studies have shown that M2-type macrophages can promote abnormal tumor angiogenesis,which may affect T cell recruitment.Cancer immunotherapy alone usually has limited efficacy.Therefore,a combination of ICD induction,autophagy inhibition and polarized TAMs using nano-drug delivery systems may produce powerful anti-tumor T-cell immune effects.In this study,Doxorubicin(DOX)and Hydroxychloroquine(HCQ)were connected by disulfide bonds to form small molecular prodrug(DSSH).The small molecular prodrug can be self-assembled into uniform nanoparticles by one-step nano-precipitation method.To achieve an effective dose,a portion of the free HCQ is loaded during self-assembly.Finally,phospholipid polyethylene glycol mannose(DSPE-PEG-Man)was modified to obtain nano-delivery system by self-assembly DSSH(HCQ)/DSPE-PEG-Man to achieve dual targeting and long circulation in mouse colon cancer cells(CT26)and M2-type macrophages.1.Construction and characterization of DSSH(HCQ)/DSPE-PEG-ManThe successful synthesis,delivery system loading of HCQ and surface modification of DSPE-PEG-Man of DSSH were characterized by UV-vis,~1H NMR,FT-IR,particle size and zeta potential,and transmission electron microscopy.By optimizing the self-assembly conditions,nanoparticles with a particle size of about 180nm and a potential of about-26 m V were obtained.On this basis,we continued to investigate the stability of the preparation,and the results showed that the particle size of the modified phospholipid polyethylene glycol(DSPE-PEG)nanoparticles did not change significantly within 24 h,while the particle size of the unmodified nanoparticles increased with the time extension within 24 h.Finally,DSSH(HCQ)/DSPE-PEG-Man release in vitro was investigated,and it was found that DOX and HCQ could be released in both DSSH and DSSH(HCQ)/DSPE-PEG-Man release media containing glutathione(GSH),while DOX and HCQ could be released in GSH-free release media.DOX could not be released within 72 h due to the presence of disulfide bonds in the structure of nanoparticles,which can respond to GSH drug release.2.DSSH(HCQ)/DSPE-PEG-Man cytology study in vitroUsing CT26 and TAM2 as models,the in vitro uptake of DSSH(HCQ)/DSPE-PEG-Man in CT26 and TAM2 was investigated by Laser Confocal Fluorescence Microscopy(CLSM)and flow cytometry.The results showed that the system was mediated by mannose receptors highly expressed on the surface of CT26 and TAM2.And significantly increase intake.Secondly,the phenotypic reversion of TAMs by DSSH(HCQ)/DSPE-PEG-Man in vitro was determined by flow cytometry.The results showed that after treatment with DSSH(HCQ)/DSPE-PEG-Man for 12 h,the proportion of M1-type macrophages increased from 2.5%to 29.5%,and the proportion of M2 macrophages decreased from 55.0%to 21.9%compared with the control group.These results indicated that DSSH(HCQ)/DSPE-PEG-Man could significantly reverse M2-type macrophages into M1 phenotype.Enzyme linked immunosorbent assay(ELISA)was used to detect cytokine secretion of macrophages after drug treatment,to further verify the effect of DSSH(HCQ)/DSPE-PEG-Man nanoparticles on TAMs phenotype.Subsequently,CLSM was used to investigate the expression of CRT in CT26 cells,and ELISA kit was used to investigate the release of HMGB1.The results showed that the expression of CRT in CT26 cells in DSSH(HCQ)/DSPE-PEG-Man treatment group was significantly increased,and the release of HMGB1 was also significantly increased,with significant differences compared with the control group.These results indicate that DSSH(HCQ)/DSPE-PEG-Man can induce ICD successfully.Finally,the expression of autophagy related proteins was detected by Western Blot,and the quantitative results showed that the LC3-II/LC3-I ratio of the preparation group was significantly different from that of the control group,suggesting that DSSH(HCQ)/DSPE-PEG-Man could significantly inhibit the autophagy progression of CT26 cells.In conclusion,DSSH(HCQ)/DSPE-PEG-Man nanodrug delivery system has strong targeting of CT26 and M2 macrophages,and can effectively reverse TAMs phenotype,induce ICD and inhibit autophagy.3.In vivo targeting and pharmacodynamics of DSSH(HCQ)/DSPE-PEG-Man The tumor targeting and antitumor effects of DSSH(HCQ)/DSPE-PEG-Man in vivo were investigated in CT26 tumor-bearing mice.In vivo imaging results showed that IR783 labeled DSSH/DSPE-PEG-Man showed good tumor targeting.Pharmacodynamic results showed that DSSH(HCQ)/DSPE-PEG-Man had a higher tumor inhibition rate(65.7%)and significantly prolonged the survival time of mice.Hematoxylin-eosin staining(H&E)and in situ terminal transferase labeling(TUNEL)showed large area necrosis and apoptosis of DSSH(HCQ)/DSPE-PEG-Man tumor cells(70.1%).In addition,the body weight of mice in each group did not change significantly during administration,and the levels of blood biochemical indexes were all within the normal range.In conclusion,DSSH(HCQ)/DSPE-PEG-Man nano drug delivery system can play a good tumor targeting in CT26 tumor-bearing mouse model,and has good anti-tumor activity and biosafety.4.Study on the immune effect of DSSH(HCQ)/DSPE-PEG-Man in vivoIn view of the strong anti-tumor effect of DSSH(HCQ)/DSPE-PEG-Man nanodelivery system.Flow cytometry was used to detect the infiltration of immune cells in tumor tissues of CT26 tumor-bearing mice after administration.Beckman flow cytometry showed that DSSH(HCQ)/DSPE-PEG-Man significantly increased the recruitment and invasion of cytotoxic T lymphocytes(CTLs)and helper T cells(CD4~+T cells)in tumor microenvironment(TME).Significantly increased the proportion of M1macrophages(4.4%to 22.1%)and decreased the proportion of M2 macrophages(40.4%to 16.1%).ELISA was used to detect the expression of related cytokines in tumor tissues of mice after administration,and the results showed that immunostimulating cytokines IFN-γ(37.8 pg/m L to 99.5 pg/m L),TNF-α(55.4 pg/m L to 98.7 pg/m L)and IL-6(43.1 pg/m L to 97.4 pg/m L)were compared in the DSSH(HCQ)/DSPE-PEG-Man group compared with the control group,and IL10 secretion(188.0 pg/m L to 129.5pg/m L)decreased.In conclusion,the nano-delivery system by self-assembly of dual-targeted small molecule has good antitumor effect and can enhance the recruitment and invasion of immunoeffector cells,which has potential application value in tumor immunity.