Polyamidoamine Derivative-mediated The Delivery Of PD-L1-targeted Gene Editing Plasmid And Dnazyme

Background:Unlike conventional chemotherapy,which acts directly on cancer cells,immunotherapy,which uses the patient's own immune system to destroy tumors,has become a popular form of cancer treatment.Immune checkpoint blockade therapies targeting programmed cell death receptor 1(PD-1)and its ligand programmed cell death-ligand 1(PD-L1)have provided clinical benefits for a variety of cancers including melanoma,non-small cell lung cancer,advanced renal cell carcinoma and liver cancer.Currently,PD-L1 antibodies such as atelelizumab,avelumab and devalumab have shown promising clinical applications.By blocking the PD-1/PD-L1signaling pathway,T cells can be reactivated,providing a new strategy for antitumor therapy.Although antibody-based immunotherapies can provide an exceptional and relatively safe anti-cancer strategy,PD-L1 antibodies still have several unavoidable drawbacks,such as low response rates,susceptibility to immune-related adverse events,poor tissue penetration,and high production and administration costs.Therefore,new drug forms and delivery modalities need to be developed to reduce treatment costs,improve therapeutic efficacy and safety of immunotherapy.Gene editing using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)technology has become an effective tool for PD-L1 immune checkpoint therapy.The site-specific cleavage of PD-L1 gene by Cas9 protein can inhibit PD-L1 expression in a long-lasting and irreversible manner.Another novel gene therapy strategy is the use of DNAzymes,which are single-stranded DNA molecules with high catalytic activity and stability that specifically cleave purine-pyrimidine linked oligonucleotides in single-stranded RNA and catalyze the degradation of RNA.With the advantages of low synthetic cost,high stability,programmability,and high activity,DNAzymes can be used as a potential therapeutic molecule to inhibit the expression of tumor-associated genes.Since both plasmids and DNAzymes are DNA in nature,their inherent negative electrical properties determine their lack of transport capacity and stability in the organism,and limited cellular uptake and bioavailability.Also,systemic administration can lead to off-target effects and unwanted adverse reactions.To address these limitations,efficient and safe delivery systems need to be developed.Polyamidoamine(PAMAM)dendrimers are non-viral gene delivery carriers that are safer and easier to prepare than traditional viral vectors,and the abundant amino groups in the outer layer of PAMAM provide a high density of cationic charge that allows them to efficiently bind and assemble nucleic acid molecules,protect them from nuclease degradation,and provide a high level of release through the“proton sponge”effect.The“proton sponge”effect enables the endosome escape of the nanocomplex.In addition,the amino surface of PAMAM provides favorable conditions for multifunctional modifications,and several modification strategies have been developed to improve PAMAM transfection efficiency,reduce cytotoxicity and enhance tumor targeting.Objective:To construct two delivery carriers with high transfection efficiency and low cytotoxicity based on dendrimer PAMAM and modify its surface with different modification strategies to achieve tumor site delivery of CRISPR/Cas9 plasmids and DNAzymes targeting PD-L1,respectively.The physical and chemical properties,in vitro functions and in vivo efficacy of the two nanocomplexes were investigated to preliminarily elucidate their roles and intrinsic mechanisms in melanoma immunotherapy,with the aim of providing theoretical and experimental bases for the development of new immunotherapeutic strategies.Part ?.Study of nucleobase-modified PAMAM mediated delivery of PD-L1 knockdown CRISPR/Cas9 plasmidsMethods:1.Construction and screening of efficient PD-L1 knockout plasmid pX459-sgPD-L1B using PD-L1 gene as the target.2.The surface of PAMAM molecule was modified using nucleobase modification strategy to prepare the ucleobase-modified PAMAM(AP-PAMAM),and its physicochemical properties were characterized by transmission electron microscopy analysis,particle size potential determination,gel blocking assay and MTT assay.The ratio of AP-PAMAM and plasmid transfection was optimized by fluorescence microscopy and flow cytometry,and the ability of AP-PAMAM/pX459-sgPD-L1B nanoparticles to enter the cell and escape from the endosome was examined by laser confocal microscopy.3.The PD-L1 expression level of transfected cells was assessed by protein immunoblotting assay,and the PD-L1 gene editing efficiency of AP-PAMAM/pX459-sgPD-L1B nanoparticles was evaluated by T7EI assay,Sanger sequencing and TIDE analysis.4.A mouse model of melanoma lung metastasis was constructed and AP-PAMAM/pX459-sgPD-L1B nanoparticles were injected into the tail vein,and the tumor inhibition effect of the nanoparticles was evaluated by observing the lung tumor growth,HE staining,Ki-67 immunofluorescence staining and TUNEL apoptosis analysis.The expression levels of PD-L1,CD8,granzyme B and immune-related cytokines in tumor tissues were examined by immunohistochemical staining and ELISA assay to investigate the degree of immune activation.Results:1.The efficient PD-L1 knockout plasmid pX459-sgPD-L1B was successfully constructed and obtained.2.The nucleobase-modified PAMAM(AP-PAMAM)was prepared,which has good plasmid binding and compression ability and can assemble with pX459-sgPD-L1B plasmid to form stable nanoparticles with low cytotoxicity.3.In the transfection of tumor cells,AP-PAMAM exhibited high transfection efficiency and endosome escape ability due to the introduction of natural base derivatives,which regulated the hydrogen bonding interaction between the carrier and DNA molecules;analysis of the genome sequence of the transfected mouse melanoma cells showed that AP-PAMAM/pX459-sgPD-L1B nanoparticles successfully disrupted the PD-L1 gene and reduced the expression of PD-L1 protein.4.In animal experiments,AP-PAMAM/pX459-sgPD-L1B nanoparticles demonstrated good antitumor activity,and AP-PAMAM-mediated delivery of pX459-sgPD-L1B plasmid produced more pronounced tumor regression compared with unmodified PAMAM;AP-PAMAM/pX459-sgPD-L1B nanoparticle treatment decreased PD-L1 expression levels in tumor tissues,increased CD8~+T cell infiltration,and promoted the proliferation viability and killing ability of CD8~+T cells with good biocompatibility.Part ?.Tumor-targeted PAMAM derivatives mediating targeted PD-L1 DNAzyme deliveryMethods:1.The PD-L1 mRNA secondary structure was simulated to select susceptible targets for PD-L1-targeted DNAzyme design.Subsequently,fluorescence quantitative PCR and protein immunoblotting were used to screen for DNAzyme Dz4,which can efficiently cleave PD-L1 mRNA.2.The tumor-targeting dendrimer APFP was constructed using various strategies including fluorinated modification,polyethylene glycol(PEG)coupling and surface modification of nucleophosmin aptamer AS1411;its physicochemical properties were characterized by NMR hydrogen spectroscopy,transmission electron microscopy,particle size potential measurement,gel blocking assay,DNase protection assay and MTT assay.The APFP/Dz4 nanocomplex was fluorescently labeled to examine its cell entry ability,lysosomal escape ability and tumor cell targeting.3.To evaluate the inhibitory effect of APFP/Dz4 complex transfection on PD-L1expression in B16-F10 cells by fluorescent quantitative PCR and protein immunoblotting assay.4.In melanoma lung metastasis mice,the anti-tumor effect of APFP/Dz4 complex was evaluated by observing lung tumor growth,HE staining,Ki-67immunofluorescence staining,and TUNEL apoptosis analysis system;PD-L1expression at tumor sites,infiltration of CD8~+T cells and related experiments were performed by immunohistochemical staining analysis,flow cytometry,and ELISA The immunological mechanism of APFP/Dz4 complex activation in the immune system was further investigated by immunohistochemical staining,flow cytometry and ELISA,and the distribution of APFP/Dz4 complex in tumor-bearing mice was tracked by small animal live imaging system to evaluate its tumor targeting ability.Results:1.The PD-L1 targeted DNAzyme Dz4 has efficient PD-L1 mRNA cleavage ability and can reduce PD-L1 expression at the mRNA and protein levels.2.APFP,a tumor-targeted dendrimer derivative,was successfully prepared;APFP has good DNAzyme binding and protection ability,can form homogeneous and stable nanoparticles with Dz4,and protect Dz4 from DNase degradation;Thanks to fluorination modification and PEG molecular coupling,APFP has low cytotoxicity;modification of aptamer AS1411 confers high tumor cell targeting to APFP,enabling efficient delivery of Dz4 into tumor cells and lysosomal escape.3.The APFP/Dz4 nanocomplex achieved PD-L1 mRNA cleavage in tumor cells,and the APFP/Dz4 complex had stronger PD-L1 expression inhibition compared with the non-targeting carrier PFP.4.In melanoma lung metastasis mice,APFP/Dz4 complex showed good biosafety,mediated proliferation inhibition and increased apoptosis in tumor tissues;APFP/Dz4complex efficiently increased the recruitment and activation of CD8~+T cells and the secretion of immune-related cytokines at tumor sites;APFP mediated tumor immune activation better than AS1411,thanks to the tumor targeting mediated by AS1411.mediated tumor immune activation better than the non-targeting carrier PFP,and thus achieved more significant tumor suppression and regression.Conclusion:In summary,we designed and constructed two types of PD-L1 immune checkpoint inhibition systems using dendrimer PAMAM derivatives as carriers using different modification strategies to achieve efficient and stable delivery of PD-L1 knockout plasmids and DNAzyme molecules,which restored the recognition and killing of melanoma by the immune system and achieved good tumor suppression.The development of these two tumor immunotherapy platforms has laid a good foundation for the subsequent research and development of PD-L1 immune checkpoint therapies,and is expected to provide new strategies for the clinical application of immunotherapy.