Studies On The Relationship Between The Structure And SiRNA Delivery Efficiency Of PH-sensitive Amphiphilic Cationic Polymers

siRNA is a kind of biomacromolecules that can specifically inhibit the expression of target genes and is an ideal biological drug for the treatment of diseases caused by abnormal gene expression.However,siRNA is transported into cells and exerts the inhibitory effect on target genes with the assistance of a low-toxic and efficient delivery vector.Non-viral vectors become the research and development focus of siRNA delivery systems because of their diversity and non-immunity.However,their delivery efficiency is far lower than that of viral vector.Low intracellular delivery efficiency is also an urgent problem to be solved.The previous studies of our research group have proved that polymethylamino-ethyl methacrylate(PDMAEMA)modified by polyethylene glycol(PEG)and hydrophobic segment has potential application prospects,and this paper further explores effective ways to reduce the toxicity of PDMAEMA and improve the intracellular delivery efficiency.In this paper,biodegradable polycarbonate was introduced into the carrier.For the first time,a cyclic carbonate monomer,MTC-Br,was synthesized and used to modify polycarbonate.Comb polycation,m PEG-b-PC-g-PDMAEMA(GMDM)with PC as main chain and PDMAEMA as branch chain,was synthesized by ring opening polymerization(ROP)and atomic transfer radical polymerization(ATRP).GMDM could load and deliver siRNA effectively.Unfortunately,GMDM has not been improved in gene inhibition efficiency and biocompatibility.Next,the effective ways to reduce the toxicity and intracellular delivery efficiency of PDMAEMA vector were discussed.Hydroxylation was used to modify cationic units to reduce toxicity.And polyacetal that can be hydrolyzed in acidic solution was used as hydrophobic segment to promote siRNA endosomal escape efficiency.With m PEG as hydrophilic segment,polyacetal(PTTMA)as hydrophobic segment and as methacrylic acid glycidyl ether modified by N,N-dimethylamino mercaptan(PGMA_N)as cationic segment,pH-sensitive hydrolysable polymers were synthesized by chain transfer radical polymerization and epoxy-mercapto reaction.The results showed that the associated hydroxyl group could reduce the damage of ETGN to cell membrane and enhance the compatibility of ETGN cells.When N/P=50,the biological activity of ETGN/siRNA complex can still reach 90%.At the same time,siRNA can be delivered by ETGN efficiently.When N/P=50(50 n M siRNA),the gene inhibition efficiency of ETGN/siRNA in vitro can reach 95%.Moreover,ETGN obtained 80%gene inhibition efficiency in subcutaneous tissue and 92%gene inhibition efficiency in liver.Further studies showed that the pH-sensitive degradation performance of ETGN could effectively enhance the escape ability of siRNA from endosome and thus enhance the gene inhibition efficiency.However,the problem is that high gene silencing efficiency is required at high dose vectors(N/P=50).In view of the effect that pH sensitivity of hydrophobic kernels promotes the endosomal escape of siRNA,it is necessary to further discuss the effect to obtain a carrier with the most optimized structure.The study of this problem needs to avoid the interference of endocytosis efficiency.For this purpose,a series of triblock copolymers,PCB-b-P(DMAEMA-co-DPA)-b-PG(GDDC),were designed and synthesized.Among them,the ratio of DMAEMA(p Ka=8.4)and DPA(p Ka=6.0)can adjust the pH sensitivity of the hydrophobic section.In this system,hydrogen bond pairing of carboxyl group on hydrophilic chain polycarboxybetaine(PCB)and cationic polyguanidine(PG)inhibited cell endocytosis of GDDC/siRNA complex.Endocytosis assays showed that the endocytosis rate of GDDC/siRNA series complex was approximately 1/6 of that of commercial Lipo 2000/siRNA complexes.Gene silencing efficiency in vitro and endosomal escape ability of GDDC/siRNA complexes were related with the disassembly of GDDC series nanoparticles.The results showed that GDDC-4nanoparticles dissociated at pH=?6.8 showed the highest endosome escape efficiency and gene silencing efficiency.When N/P=8(50 n M siRNA),gene silencing efficiency almost reached?100%,which was significantly higher than Lipo 2000(65%).After in vivo subcutaneous injection,siRNA can fixed in subcutaneous tissue by GDDC-4for a long time and achieve 80%gene inhibition efficiency.Based on the optimal pH-sensitive kernel structure that was obtained from the above studies,the mechanism of action was further discussed.Proton buffering ability is considered to be the mechanism of promoting endosomal escape,but it is still controversial.In order to avoid the interference of cationic segment and PCB proton buffering effect,quaternized PDMAEMA(PTDMAEMA)with as cationic segment and PEG as hydrophilic chain was used to prepare a series of triblock cationic polymers,m PEG-b-P(DMAEMA-co-DPA)-b-PTDMAEMA(TDDE).The difference of endocytosis of TDDE series vectors can be ignored.The relationship between proton buffering domain of TDDE and endosomal escape ability was studied,and it was found that the polymer with stronger proton buffering ability in early endosome can better promote endosomal escape of siRNA.Further,the higher the proportion of pH variation difference of the early endosome in the polymer proton buffer domain,namely the higher the K value,the higher the endosomal escape efficiency of the siRNA.Therefore,we screened the polymer TDDE-3,which showed the highest escape efficiency of siRNA and high gene inhibition efficiency on various cells,including cardiac myocyte H2C9.In addition,quaternized PDMAEMA can significantly reduce the toxicity of TDDE series vectors,which is another effective way to reduce the toxicity of PDMAEMA class vectors.Finally,based on GDDC-4 and TDDE-3 mentioned above,the approach to balance cellular compatibility and delivery efficiency was further explored.P(DMAEMA-co-DPA)-b-PG(GDD-4)and m PEG-b-P(DMAEMA-co-DPA)-b-PG(GDDE-4)were synthesized.The siRNA delivery efficiency and cellular biocompatibility of GDDC-4,TDDE-3,GDDE-4 and GDD-4 were compared with GDDC-4 and TDDE-3.Among the four vectors,GDDE-4 has the strongest siRNA delivery capacity in vitro.When N/P=8,5nm siRNA,the gene inhibition efficiency of GDDE-4 can still reach 80%.TDDE-3 has the lowest cytotoxicity and the lowest intracellular delivery efficiency of siRNA.In addition,PEG modification was found to be an effective way to improve the delivery efficiency of cationic polymer siRNA.