PH-Sensitive Hydrophobic Segments Of Cationic Polymers Promote SiRNA Delivery Efficiency And Antitumor Effects

Nucleic acid drugs and chemotherapeutics are commonly used in clinical anti-tumor drugs,which effectively improve the survival time of tumor patients.However,due to their shortcomings,such as the electronegativity,large molecular weight,and instability of gene drugs,and the toxic of chemotherapeutics drugs,their clinical translation and application have been greatly limited.Although people have successively developed a variety of nano-delivery systems and made great progresses in tumor-targeted drug delivery,there is still an urgent need to improve delivery efficiency in vivo and at the intracellular level.Based on the acidic microenvironment of tumors,in order to solve the problem of low delivery efficiency of small interfering RNA(siRNA),the effect of different pH-sensitive hydrophobic segments on the siRNA delivery efficiency of amphiphilic cationic polymer carriers was studied,and the optimization structrures were evaluated.The application performance of the optimized carriers in tumor immunotherapy and anti-tumor multimodal diagnosis and treatment were discussed by siRNA/drug and drug/photosensitizer combination.However,the in vivo delivery efficiency and toxicity of such delivery systems still need to be addressed.This article first used aminoethyl methacrylate(AMA)as a cationic monomer,diisopropylaminoethyl methacrylate(DPA)as a pH-sensitive hydrophobic monomer,and polyethylene glycol(PEG)as the hydrophilic segment,to prepare three block copolymers through living polymerization,PEG-PAMA-PDPA(E-A-D),PEG-PDPA-PAMA(E-D-A),and PEG-P(AMA/DPA)(E-(A/D)).The siRNA delivery effect of the distribution of hydrophobic units D in the block copolymers was studied in vitro and in vivo.The results found that all the self-assembled nanoparticles of these cationic polymers could successfully load siRNA,and showed higher siRNA delivery efficiency and lower cytotoxicity at the cellular level.However,E-(A/D)showed a high degree of hemolysis.In vivo experiments found that the delivery efficiency of E-A-D siRNA was significantly better than that of E-D-A,which indicated that the amphiphilic cationic polymer distributed by E-A-D as a structural unit was beneficial to promote the delivery efficiency of siRNA in vitro and in vivo.Based on the structural optimization of the E-A-D block copolymer described above,the contribution of the pH sensitivity(p Ka)of the hydrophobic segments to siRNA delivery was next studied.We used 2-(pentamethyleneimino)ethyl methacrylate(C6A-MA),2-(hexamethyleneimino)ethyl methacrylate(C7A-MA),and methacrylic acid isopropylaminoethyl(DPA-MA)and dibutylaminoethyl methacrylate(DBA-MA)were chose as hydrophobic monomers.By adjusting the copolymerization unit and composition,a series of triblock cationic polymers EAAS with p Ka of 7.0,6.8,6.5,6.2,6.0,5.8,5.6 and 5.2,were synthesized respectively.The experiments in vitro found that EAAS cationic polymer carriers with p Ka in range of5.8-6.2 showed high potential in enhancing siRNA endosome escape and gene silencing efficiency.And the efficiencies of gene silencing in vivo by the peritumoral administration and intravenous administration were also obtained.We speculated the effect of p Ka value of the hydrophobic segments on the cationic carrier may be related to the proton buffering effect in the endosome.In order to reveal the mechanism,random copolymers of different proportions of diethylaminoethyl methacrylate(DEA)and dipentylaminoethyl methacrylate(D5A)were used as hydrophobic segments,to synsize series cationic polymer carriers,PEG-PAMA-P(DEAx-co-D5Ay)EAE5_x/y,which presented samecomposition,but different p Ka values.Then,according the pH gradient in the process of cell endocytosis,we analyzed the proton buffering abilities of EAE5_x/y series cationic copolymers nanoparticles in three ranges of pH 6.5-7.4,5.5-6.5 and 4.5-5.5,and correlated with their intracellular siRNA delivery efficiency.The results in vitro showed that EAE5_48/29 and EAE5_39/37,which have strong buffering capabilities in the pH 5.5-7.4 range,showed strong escape ability and thus led to higher gene silencing efficiency.The experiments in vivo found that the gene delivery efficiency of EAE5_39/37 was significantly higher than that of EAE5_48/29,either administered intravenously or subcutaneously.This was attributed to the proton buffer effect of EAE5_39/37 mainly occured in the pH 5.5-6.5 range,which suitable p Ka value and low critical micelle concentration(CMC)guaranteed better stability in vivo,while the proton buffering effect of EAE5_48/29 mainly occured in the pH range of 6.5-7.4,which had poor stability during delivery in vivo.In order to further improve the delivery efficiency and reduce the toxicity,the cationic polymer PEG-PAMA-PDPA described above,was co-assembled with phosphatidylcholine(PC)and cholesterol to construct hybrid cationic liposomes.The chemotherapeutic drug Doxorubicin(DOX)and siRNA(si PD-L1)for silencing the immune checkpoint PD-L1 were co-loaded to achieve the combined delivery of chemotherapeutic drugs and gene drugs.The results showed that DOX could successfully induce tumor cell immunogenic death through the PARP1 pathway,and successfully activated the immune response in vivo,which could prevent tumor lung metastasis.The in vivo evaluation on prevention experimental model found that the immunogenic death of DOX and the silencing of PD-L1 played a good synergistic role and could successfully prevent tumor occurrence.The results of tumor suppression experiments in vivo also showed that the hybrid liposome co-delivery system successfully achieved combination of chemotherapeutic drug-induced immunogenic death and PD-L1 blocking by si PD-L1 and effectively inhibited tumor growth.The design method of this carrier provides a good design idea for improving the stability of pH-sensitive materials in the body and achieving the combined delivery of genes and chemotherapy drugs.Finally,the application of block copolymers with pH-sensitive hydrophobic segments in tumor imaging and multimodal therapy was studied.The pH-sensitive material was further hydrophobized,and PEG-PDPA-PBMA(EPB-x)triblock polymers were synthesized by living polymerization.Disulfide bonds were successfully introduced to bond indocyanine green(ICG)to the polymers.The prepared EPB-3-ICG₁ nanoparticles enhanced tumor enrichment,and achieved the tumor-specific targeting"OFF/ON"imaging function by responding to the reducibility of high-concentration glutathione in tumor cells.In addition,EPB-3 or EPB-3-ICG₁ were used to package hydrophobic chemotherapeutic drug PTX and photosensitizer(ICG or Au rods)for multimodal treatment of tumor imaging and photothermal therapy,chem-photothermal therapy.Only one intravenous dose was needed to extend mouse survival.Therefore,EPB-3 provides a nano-delivery platform for the realization of anti-tumor multimodal therapy.