Smart Targeting Nanodelivery System Based On The Combined Application Of Gene-drug For Synergistic HCC Treatment

Hepatocellular carcinoma(HCC)is a malignant disease that seriously endangers human health and safety all over the world.In particular,patients in China have increased greatly in the past decade and remain a high mortality rate.In addition to the traditional strategies of surgery,radiotherapy and chemotherapy agents,including a variety of new molecular targeted drugs and natural products,new treatment strategy of cell therapy and immune therapy have also been widely applied to clinical treatment of HCC.However,on account of the complex mechanism of HCC occurrence and development,the therapeutic effect of monotherapy is not ideal enough.In comparison,the combination of multi-drugs or multi-therapies have been proved to be one of the most promising strategies for clinical application at present.In recent years,gene therapy based on the CRISPR/Cas9 combined with drug therapy has attracted extensive attention in the HCC treatment.According to the anti-cancer mechanism and characteristics of drugs,corresponding oncogenes could be selected for synergistic effects with precise and efficient gene editing by CRISPR,so as to realize more effective gene-drug combination HCC treatment.Meanwhile,the development of various non-viral nanodelivery systems has also facilitated the combination of CRISPR and drugs.Furthermore,besides the traditional inert nanocarriers,the preparation strategies of a variety of new nanodrugs,such as biomimetic nanosystem and carrier-free self-delivery system have also been studied in depth with great application potential.Based on the research above,this paper proposed a various of novel smart nanodelivery system based on the combined application of gene-drug,in order to realize the multi-mode combination treatment on HCC.The main contents are summarized as follows:Part one.The construction and optimization of CRISPR/Cas9 system(1)The green fluorescent protein EGFP stable expression HEK293 T cell lines was constructed by lentivirus technology.EGFP-targeted recombinant CRISPR plasmid was obtained by digestion and ligation,and bioactive Cas9 protein was prepared by prokaryotic expression,which was used to construct EGFP-targeted Cas9 RNP complex.(2)The gene editing characteristic and efficiency of two different preparation methods of CRISPR system(recombinant plasmid and Cas9 RNP complex)were analyzed by EGFP-HEK293 T cell lines,the results found that both of the systems have good effect of gene knockout,among Cas9 RNP complex works faster,while the recombinant plasmid works longer and slightly higher than Cas9 RNP complex in knockout efficiency.Based on that,the recombinant plasmid or Cas9 RNP complex targeting EGFR,PD-L1 gene were constructed,and it was verified that two targeted sg RNAs we designed had good biological activity.Part two.The study of combination gene-drug nanosystem based on hollow mesoporous silica for HCC treatment(1)The "core-shell" nanocomposite system for co-delivery of Sorafenib and CRISPR was successfully constructed based on the hollow mesoporous silica NPs,which was modified the dendritic molecules PAMAM and Ep CAM-aptamer.The complex system had high encapsulation efficiency of 78.3%,76.8% for drug and plasmid.NPs presented a spherical shape with feasible particle size of 184 nm,as well as had the good p H responsive and stability.(2)The nanodrug of SEHPA NPs achieved the significant targeted aggregation effect on HCC cells in vitro,as well as realized the efficient recognition and enrichment for tumor tissues in vivo.Meanwhile,the co-delivery nanosystem exhibited the synergistic cytotoxicity for HCC cells by EGFR gene therapy and Sorafenib,and showed an fourfold increase of tumor growth inhibition on HCC tumor-bearing mice compared to the monotherapy,which greatly improved the therapeutic effect.(3)The excellent biosafety of SEHPA NPs was verified by normal cytotoxicity evaluation,body weight detection of treated mice,HE staining of major tissues and blood biochemical analysis et al.It was worth noting that we found that EGFR gene therapy and Sorafenib synergistic reduced > 90% EGFR protein expression and effectively inhibited the downstream PI3K-Akt pathway,so as to limit the effect of tumor angiogenesis.Part three.The study of combination gene-drug biomimetic nanosystem based on the dual-drugs self-assembly for HCC treatment(1)The natural product ursolic acid and Sorafenib were used to construct the dual-drug self-assembled nanosystem,which was used to prepare the carrier-free co-assembled nanodrug of USPR NPs by combining with CRISPR system.On the basis,the bionic nanosystem of USPR@M NPs was successfully obtained by the homologous tumor cell membrane camouflage.USPR@M NPs appeared as spherical particle with particle size about 196 nm and good stability.(2)USPR@M NPs had obvious homologous tumor identification characteristics in vitro and in vivo,which greatly improved the targeted enrichment of nanodrugs and effectively enhanced the long circulation of 72 h of drugs in body.Meanwhile,USPR@M NPs showed a significant effect on HCC treatment,the 90% tumor growth was significantly inhibited in tumor-bearing mice.In addition,USPR@M NPs had no obvious toxic and side effect during in vivo and in vitro treatment was proved with the same test.(3)Studies had found that ursolic acid activated the innate immune TLR-2-My D88 pathway and a variety of immune-related cytokines,thereby induced the activation and proliferation of NK cells,which exhibited the synergistic activating for the generation of immune cytotoxicity T cells in combination with PD-L1 gene therapy,and finally realizing the effective immune infiltration of HCC tumor tissues.Meanwhile,the immunotherapy had obvious distal effect and reached to the 86%growth inhibition for distal tumors.In summary,this paper constructed two gene-drug combination systems for HCC treatment based on mesoporous silica and biomimetic co-assembly,which achieved significant targeted identification and enrichment of gene-drug for HCC,and reached to more than 90% tumor treatment effect with good biosafety.In addition,the study revealed the anti-HCC molecular mechanism of synergistic inhibition of PI3K-Akt pathway and repression of angiogenesis by EGFR gene therapy combined with Sorafenib,as well as the anti-HCC therapeutic mechanism of Sorafenib combined with synergistic immune activation by PD-L1 gene therapy and ursolic acid.This research could provide a new direction for the further study of gene-drug combination therapy system in the future,and provided an important theoretical basis for the advanced HCC clinical treatment,it also opened up the novel idea for the development of more cancer treatment strategy.