Study On Synthesis And Properties Of Rhamnose-based Cationic Lipsomes And Design And Synthesis The Functional DNA Solid-phase Reagents

This article is divided into two parts:Part ?.Study on synthesis and properties of rhamnose-based cationic liposomes.The key to gene therapy is to build a targeted,safe and efficient delivery system to transfer foreign genes into target cells.Cationic liposomes have become hotspots as gene carriers due to their high transfection efficiency and good biocompatibility.In this paper,rhamnose is used as the raw material,three kinds of rhamnose cationic lipids,such as Rha-DiC14MA,Rha-DiC16MA and Rha-DiC18MA,whose hydrophobic chains attached to the nitrogen atom at the head of the quaternary ammonium salt,were synthesized via peracetylation,selective1-O-deacetylation,trichloroacetimidation esterification,glycosylation,azidation,deacetylation,Staudinger reduction,tertiary amimation,and quarternization.The three cationic lipids were dissolved in double-distilled water,and liposomes were formed by ultrasonic dispersion,then combined with the pEGFP-C3 plasmid to form liposome/pDNA complexes.Zetasizer Nano ZS was used to determine the zeta potential,average particle size and PDI values of cationic liposomes and their different N/P ratio pDNA complexes.The results showed that the zeta potential of the cationic liposomes was between 35-65 mv,the average particle size was between 150-180 nm and distributed uniformly;the particle size distribution of the liposome/pDNA complex was between 70-275 nm.The distribution is also relatively uniform,the zeta potential is between-10-46 mv,and gradually increases with the increase of N/P ratio.The ability of the liposome to bind to plasmid DNA was explored by gel retardation experiments.The results showed that as the N/P ratio increased,the blocking effect of plasmid DNA was enhanced,with the exception of the liposome Rha-DiC18MA,the remaining liposomes When the N/P ratio reaches 1,it has completely bound to the plasmid DNA.Using Lipo3000 as a positive control,the expression of three cationic liposome-carrying plasmid DNA in four different cell lines,HEK293,HeLa,Beas-2B and A549,was examined.The results showed that the liposome Rha-DiC16MA had good transfection efficiency in all four types of cells.The transfection efficiency of liposome Rha-DiC14MA was lower,and Rha-DiC18MA had no transfection effect.The CCK8 method was used to evaluate the cytotoxicity of the three cationic liposomes.The experimental results showed that Rha-DiC16MA was less toxic in HeLa,Beas-2B,and A549 cells,but slightly higher than Lipo3000;Rha-DiC18MA in HEK293 HeLa,Beas-2B and A549 are basically non-toxic.These experimental results can provide an important reference for the study of the gene carrier with rhamnose as the parent structure.Part ?.Design and synthesis of functional DNA solid-phase reagents.Solid-phase synthesis provides us with a new idea of using functional groups to modify DNA.That is,the structure of vicinal diols and functional groups such as click chemistry is introduced into biomolecules by chemical synthesis,and the adjacent hydroxyl groups are used 4,4-dimethoxytriphenyl and 2-cyanoethyl N,N-diisopropyl groups(necessary protecting group for solid phase synthesis)as protection respectively to prepare functional DNA solid phase synthesis reagents.To synthesis nucleic acids labeled with functional functional groups automately and modular.Introducing small-molecule drugs,fluorescent dyes,or other biomolecules in nucleic acids efficiently and targeting for biological research.In this study,two types DNA solid-phase synthesis reagents have been synthesized:one is using p-hydroxy benzoic acid reacting with 3-Amino propane-1,2-diol under dicyclohexylcarbodiimide and NHS to produce N-(2,3-dihydroxypropyl)-4-hydroxybenzamide,then reacted with tert-Butyldimethylsilyl chloride to prepare intermediate N-(2,3-di-tert-butyldimethylsiloxypropyl)-4-tert-butyldimethylsilyl Oxybenzamide(?).N-(2,3-dihydroxypropyl)-4-tert-butyldimethylsilyloxybenzamide(?)is obtained through the selective deprotection of ? under the action of iodine,then ? reacts with DMTrC1 to generate compounds N-(3-(4,4'-dimethoxytriphenyl)-2-hydroxypropy1)-4-tert-butyldimethylsil yloxybenzamide(?).Finally reacting with 2-cyanoethylated N,N-diisopropyl chlorinated phosphoramide to generate the DNA solid-phase reagents N-(3-(4,4'-dimethylformamide)Oxytriphenyl)-2-(2-cyanoethyl-N,N-diisopropylphosphoramido))-4-tert-butyldimethylsilylox ybenzamide(?)containing second generation of click chemistry.The other is using the hydroxy phenylethylamine as the raw material,firstly reacting with 2-methyl acryloyl chloride to generate N-(4-hydroxy phenyl)methyl acrylamide under the effect of TMSC1,then synthesizing intermediates N-(4-(2,3-dihydroxypropoxy)phenethyl)methacrylamide(?)with glycidol.? reacts with DMTrC1 to generate compounds N-(4-(3-(4,4'-dimethoxytriphenyl),2-hydroxypropoxy)phenethyl)methacr ylamide(VIII),subsequently with 2-cyanoethylated N,N-diisopropyl chlorinated phosphoramide,synthesizing the final product N-(4-(3-(4,4'-dimethoxytriphenyl),2-(2-cyanoethy1N,N-diisopropylphos-phoramido)propoxy)benzene Methacrylamide(?).