Functional Nanoplatform Delivery Of CRISPR-Cas9 Ribonucleoprotein For Combined Tumor Therapy

The CRISPR-Cas9 system is a gene editing tool derived from the natural immune defense system of prokaryotes.It is a complex of a small noncoding RNA and a Cas9 protein,which can recognize and cut the target sequence by complementary pairing of only 20 specific nucleotides with it,and its simplicity and efficiency make it widely used and rapidly becoming the focus of many research fields.However,the CRISPRCas9 system relies mostly on plasmids as a form of delivery,which has a higher risk of off-target mutations,and to achieve higher gene editing efficiency requires the use of viruses as delivery vectors,which further increases the potential risk of the plasmid system.Especially in disease treatment,this problem will greatly affect the realization of clinical applications of CRISPR-Cas9 system.Therefore,it is important to enhance the safety and therapeutic efficacy of CRISPR-Cas9 gene editing tools in the field of disease treatment for their development in the biomedical field.In this study,the potential of this gene editing tool was exploited by Cas9 ribonucleoprotein(Cas9 RNP)combined with functional nanoparticles.This form of Cas9 RNP eliminates the process of protein expression such as transcription and translation compared to plasmids,and will not integrate into the genome of cells,thus greatly reducing the risk of off-target mutations.The functional nanoparticles can not only be used as Cas9 delivery vehicles,but also enhance the efficacy of gene editing tools as disease therapies through their own properties(e.g.photothermal,photodynamic effects,etc.).CuS nanoparticles were prepared by reacting copper chloride(CuCl2·2H2O),trisodium citrate(C6H5NaO7·2H2O)and sodium sulfide(Na2S·9H2O),which have the characteristics of small particle size and strong photothermal conversion.Cas9 RNP was loaded onto CuS nanoparticles by complementary pairing of sulfhydryl-modified single-stranded DNA with sgRNA,and wrapped with polyethyleneimine(PEI)by electrostatic adsorption to form a positively charged nanocomplex(CuS-RNP@PEI),allowing it to adhere with negatively charged cell membranes and enter cells through cytokinesis to accomplish the editing mission of the PTPN2 gene,an immunosuppressive target.The intracellular CuS nanoparticles can effectively convert light energy into heat energy under 808 nm NIR light irradiation to denature some cellular proteins,leading to co-aggregation with natural aggregation-sensitive proteins.This phenomenon can seriously affect enzyme activities,DNA synthesis and repair processes,etc.,which eventually leads to the death of some tumor cells and the production of tumor-associated antigens(TAA)to activate the immune response of the organism through antigen-presenting cells(APC).The results of in vivo experiments showed that this combined immune and photothermal treatment strategy significantly inhibited the growth rate of tumors in mice and exhibited excellent anti-tumor efficacy.On this basis,we also developed a combination strategy of photodynamic therapy based on the photosensitizer Chlorin e6(Ce6)with Cas9 RNP dual-targeted immunotherapy based on hypoxia-inducible factor α subunit(HIF-1α)and cell programmed death ligand 1(PD-L1).Using mesoporous silica(MSN)with high biosafety as the inner layer of the nano system,and ce6 modification on hyaluronic acid as the outer layer,to develop a combined photodynamic and immunotherapy system.The experiment showed that compared with the control group,the photodynamic and immune combination therapeutic effect of the MSM-RNP@HC nano system on tumor-bearing mice can significantly reduce the growth rate of tumors,and this combination treatment strategy provides a new idea for the application of CRISPR Cas9 gene editing technology.