Development Of Ionizable Nanocarrier For Delivery Of RNA-based Therapeutics

RNA-based therapeutics could specifically regulate the expression of target genes with high specificity and safety,and have broad application in the treatment of cancer,inflammation,infectious diseases and genetic disorders.However,the development of RNA-based therapeutics is hampered by their instability against nuclease,immune response activation and insufficient intracellular delivery.Therefore,the development of safe and potent RNA delivery system is essential for improving the stability and bioavailability of RNA drugs.Viral vectors have high transfection efficiency,but also have some potential side effects,such as mutagenesis risk and immunogenicity.Nonviral vectors have higher safety and are easily to be synthesized,while the transfection efficiency is needed to further improved.Based on the understanding and analysis of the physiochemical properties of RNA molecules and main biological barriers in the delivery of RNA,we synthesized a set of ionizable nanocarriers and optimizing the structure of ionizable lipids and the synthesis methods of nanocarriers to improve RNA encapsulation and delivery efficiency both in vitro and in vivo and further evaluated their therapeutic application in disease models.The main contents of this thesis are composed of the following five parts:In chapter Ⅰ,several commonly used RNA drugs were introduced,as well as their working mechanism and the main biological application.Subsequently,the main biological barriers of RNA drugs in vitro and vivo,along with the types of common RNA delivery system were introduced in detail.Finally,we summarized the basis and research significance of this thesis.In chapter Ⅱ,four ionizable lipid nanoparticles(LNPs)were prepared by nanoprecipitation method,and the top-performing lipid nanoparticles(LNP-114)was identified via in vitro experiments for the treatment of liver fibrosis.The ζ-potential of LNPs was positive,which could bind negative siRNA via electrostatic interaction.Optimizing the weight ratio of LNPs and siRNA could improve the encapsulation efficiency of LNPs.In addition,in vitro results showed that LNP-114/siRNA had the effective cellular uptake,endosomal escape and gene silencing efficiency in HeLa cells,and similar results were obtained in hepatic stellate cells LX-2.In vivo results demonstrated that LNP-114/siRNA had good biocompatibility and could selectively accumulated in liver organs.Moreover,LNP-114 loaded with therapeutic siRNA against collagen α1(Ⅰ)could effectively ameliorate liver injury,and inhibit the progression of fibrosis.In summary,in this part,we synthesized and identified a potent ionizable lipid nanoparticle for hepatic delivery,which showed good biocompatibility,high efficiency,and great potential in the treatment of liver fibrosis and other liver diseases.In chapter Ⅲ,compared with other types of cells,the immune properties of macrophages make it extremely difficult to be transfected.Therefore,on the basis of the above researches,we further explored the transfection efficiency of LNPs formulated with anti-Interleukin-1β siRNA(siIL-1β)in macrophages,and identified the top-performing LNP for the treatment of acute liver failure.In vitro results showed that LNP-114/siRNA could efficiently facilitate siRNA internalization via multiple endocytosis pathways,and inhibit the expression of pro-inflammatory cytokine,interleukin-1β in polarized macrophages.In addition,in acute liver failure mice model,LNP-114/siIL-1β could significantly attenuated hepatic inflammation and liver damage by reducing the expression of IL-1β.In summary,this work provides an efficient ionizable lipid nanoparticle for macrophages transfection,which has great potential application value in the treatment of inflammatory diseases.In addition,this nanoparticle can further encapsulate other functional molecules to expand its biological application.In chapter Ⅳ,we aimed to develop potent ionizable carriers for mRNA delivery.due to the significantly different physical properties between siRNA and mRNA,the experience from siRNA delivery might not be able to translate directly to mRNA delivery.In this part,we synthesized six ionizable lipid nanoparticles and thoroughly investigated the impact of amine head group on mRNA delivery both in vitro and in vivo.In vitro experiment results showed that all LNPs had high intracellular transport capacity and strong membrane penetration during transmembrane transport.In addition,the top-performance ionizable lipid nanoparticles(114-iLNP)composed of lipid with spermine as amine head,had high endosomal escape ability,and the gene expression efficiency in HeLa cells achieved 95%.In vivo results further demonstrated 114iLNP/mRNA had the liver-selective delivery capability and high biocompatibility.In addition,114-iLNP could deliver therapeutic gene human erythropoietin(EPO)mRNA into liver organs,and induced hEPO expression in a dose-dependent manner.In summary,in this work,we identified an ionizable lipid nanoparticle with highly mRNA delivery efficiency via optimizing the amino head groups,which has great potential for mRNA therapeutics development as protein-replacement therapy,such as vaccine delivery or gene editing.In chapter Ⅴ,to improve the stability of nanocarriers,we fabricated ionizable nanogels with three-dimensional network structure via chemical crosslinking method.The synthetic nanogels are composed of poly(2-(diisopropylamino)ethyl methacrylate)(PDPA)as the core and poly(carboxybetaine methacrylate)(PCBMA)as the shell,and functionalized with cyclic peptides containing Arginine-Glycine-Aspartic acid-DPhenylalanine-Lysine(RGD).The resulted ionizable nanogels(PDPA@PCBMA-RGD)can maintain the ionizable properties of PDPA(pKa～6.5)and low-fouling property of PCBMA.Under physiological condition,PDPA@PCBMA-RGD could significantly resist nonspecific interactions with Raw 264.7 and HeLa cells.In acidic environment,the charge reversal property of nanogels caused by protonation could change the physicochemical property and improve cellular uptake efficiency.Meanwhile,PDPA@PCBMA-RGD NPs could specifically target to αvβ3 integrin-expressed human glioblastoma(U87)cells.In summary,in this work,a multifunctional ionizable nanogels PAPD-PCBMA-RGD was synthesized via chemical crosslinking method,which can be further loaded with RNA drugs for targeted therapy of tumors.