Synthesis Of Galactosyl Ligand-functionalized Pegylated Curdlan And Study Of Its Targeted SiRNA Delivery

Gene therapy is a proven treatment for major diseases such as monogenic genetic diseases and cardiovascular diseases.RNA interference（RNAi）is a mechanism by which endogenous or exogenous double-stranded RNA（ds RNA）mediates the specific degradation of target messenger RNA（m RNA）in cells,leading to the silencing of target genes resulting in corresponding functional phenotypic deletion.RNAi is a promising gene therapy.However,the main challenge for RNAi is how to deliver exogenous genes into cells.Cationic polymer carriers are widely used in RNAi due to their high biocompatibility,biodegradability,low toxicity and low raw material cost.In this thesis,the following scientific research has been carried out on the above-mentioned problems:1.Synthesis and Characterization of Galactosyl Ligand-functionalized Pegylated CurdlanCurdlan derivative 6AC-100 has good cell transfection efficiency but its concurrent excessive cytotoxicity limits the application of 6AC-100 in vivo.Therefore,in order to reduce the cytotoxicity of 6AC-100 and enhance its cellular targeting,In this paper,based on the previous work of the research group,the reducing D-（+）lactose-hydrate（lactose for short）was used as the raw material,reductive amination of the NH₂ at C6 position of 6AC-100 and the hemiacetal hydroxyl group in lactose using pyridine borane in boronic acid buffer gave 6AC-100-Lac with a galactose residue attached to the C6 position of 6AC-100.Studies have shown that galactosyl can target ASGPR receptors that are widely present on the surface of liver cells,and the disulfide bond can be cut off by glutathione,which is widely present in blood and cells,so that the cationic polymer nanoparticle carrier is slowly released to achieve the purpose of improving the transfection efficiency.Therefore,respectively using disulfide-bonded PEGylated PEG（2S PEG）and the activate PEG as control group,according to the four feeding ratios of 5%,10%,20%and 40%,after being condensed by EDC,connected to the 6AC-100-Lac backbone through an amide bond,eight PEGylated 6AC-100-Lac were obtained,named 6AC-100-Lac-2S PEG and 6AC-100-Lac-PEG,respectively.The structure,molecular weight,particle size and stability of 8 cationic polymers were determined by nuclear magnetic resonance spectroscopy,laser particle size,electric potential,gel permeation chromatography and elemental analysis,it lays the foundation for the next step of in vitro si RNA delivery experiments.2.Biocompatibility of PEGylated Curdlan Functionalized with Galactosyl LigandsBased on the above characterizations,the small interfering RNA（si RNA）binding and in vitro delivery capacity of the synthesized nanoparticle carriers were determined.The results show that the two galactosylated curdlan nanospheres synthesized in this paper have uniform particle size,strong si RNA loading capacity,low cytotoxicity and high biocompatibility,and due to the existence of disulfide bonds,6AC-100-Lac-2S PEG has a higher transfection ability than the control group 6AC-100-Lac-PEG and is much higher than the 6AC-100-2S PEG synthesized in the early stage of the research group.