Study On Lipase-catalyzed Synthesis Of Targeted Carrier Material：Galactosylated Cholesterol And Its Liver Targeting

ObjectiveAsialoglycoprotein receptor, as known as galactose receptor, a high-capacity C-type lectin receptor expressed on mammalian hepatocytes, plays an important role in the lysosomal processing of N-acetylgalactosamine and galactose containing glycopeptide substrates. Asialoglycoprotein receptor is a high-efficiency endocytosis receptor and it is only express on hepatocytes surface, thus it is the best receptor for the target of liver, it offer a good reach way for the therapy of liver cancer, the test of liver function and the target gene express for liver. We can design a new type of carrier material, which can load drug to liver and increase drug concentration, prolong detention time, decrease the dose of drug, reduce the toxicity of drug. The high-affinity galactosides described in this study might prove valuable in optimizing the strategy for hepatotrophic drug targeting.Enzyme catalysis in non-aqueous is a green and environmentally friendly synthetic method. Since researcher found that lipase can keep catalytic activity on organic solvent, this funding tremendously extent application area for lipase. Medical material is been synthesized by the use of lipase enzyme, which can not only save energy and protect environment, but also improve the safety of product immensely, hence it is a prospective synthesis method.Liposomes are composed by phospholipid bilayer structure that encapsulates an aqueous interior, which has good biocompatibility.It can raise the solubility of the drug in water; change the distribution of drug in the vivo; increase therapeutic index; improve clinical effect and reduce toxicity.Liposomes are prospective and effective vector in cancer treatment. Attachments of specific ligands make liposomes own active targeting effect to specific cells or tissures.Our research purpose:design a new ASGPR ligand compound; synthesize by enzymatic action; then couple the surface of liposomes to prepare ligand targeting liposomes; explore the property of targeting liposomes by the method of pharmacokinetics and molecular biology study.Methods1Design and synthesize a galactosyl ligand compound:GAL-C18by enzymatic synthesis; product was separated and purified by silica gel chromatographic column; the chemical structure of product was identified by MS and NMR method; the optimal reaction condition was achieved by single factor and tested by three times verified experiment.2Design and synthesize a compound:(5-Cholesten-3b-yl) vinyl Decanedioic acid (CHS-SE) by enzymatic synthesis; product was separated and purified by recrystallization method; HPLC analysis method was built to detect the content of CHS-SE; the chemical strucure of product was identified by MS and NMR method; the influencing factor on enzymatic reaction was inspected by single factor; Response surface methodology (RSM) was used to optimize reaction parameters.3Design and syntheize a galactosyl ligand compound:(5-Cholesten-3b-yl)[(4-0-b-D-galactopyranosyl)D-Glucitol-6] Decanedioic acid (CHS-SE-LA) by enzymatic synthesis; product was separated and purified by silica gel chromatographic column;HPLC analysis method was built to detect the content of CHS-SE; the chemical strucure of product was identified by MS and NMR method; the influencing factor on enzymatic reaction was inspected by single factor and verified by three sample; the reaction condition was optimized by RSM.4We prepared the normal DOC liposomes and galactosylated DOC liposomes. HPLC analysis method was built to detect the content of DOC in liposomes; two kind of encapsulation efficiency assay method are investigated by the factor of recover and degree of separation; the preparing of liposomes are assessed by the factors of appearance、partical size、zeta potential and encapsulation efficiency; Orthogonal experiment was used to optimize the preparation technics of liposomes. 5Pharmacokinetic characteristics was studied in vivo of SD rat on normal and galactosylated DOC liposomes; HPLC analysis method was built to detect the content of DOC in plasma; DOC was extracted from the plasma with Methyl Tertiary Butyl Ether;internal standard method was utilized to calculate the content of DOC in plasma; pharmacokinetic parameter was analysised with DAS2.0software.6We prepare fluorescently-labeled normal liposmes and galactosylated liposomes decorated by deferente ligand compounds. According to compare normal liposomes with galactosylated liposomes on the differences of combining rate with HepG2cell, the liver targetability of galactosylated liposomes was evaluated.Results1We synthesize GAL-C18by enzymatic method, and its chemical structure is confirmed by NMR and MS method. The reaction site is the C-6of galactose. According to the test of single factor on the types of enzyme, the molar ratio of the substrate(Sr)、the amount of enzyme (En)、reaction medium、reaction temperature and reaction time(t), the best reaction conditions are obtained: Novozym435as catalyst, Sr (1:4), reaction medium:DMSO:THF (1:3),8h reaction time on55℃. Esterif ication rate can achieve more than75%under these conditions.2(5-Cholesten-3b-yl) vinyl Decanedioic acid (CHS-SE) is synthesized by used of cholesterol and divinyl sebacate through enzymatic method. Synthetic products was isolated and purified by recrystallization and its purity is up to95%. The purified product structure was confirmed by MS and NMR. The determination of product content is established by HPLC method. The best lipase and solvent are Candida rugosa Lipase RCL and isooctane; t、En and Sr have significant effect on the enzymatic reaction. The optimal reaction conditions are:t,11.47h; Sr,8.94; En,7.7mg/mL, as expected, yield (92.6±0.8M%, n=3) was achieved.3CHS-SE-LA was synthesized with CHS-SE and lactitol through enzymatic method. Product is isolated and purified by silica gel column. The purified product structure was confirmed by MS and NMR. Only6-OH was acylated on lactitol by Lipase. The determination of CHS-SE-LA content is established by HPLC method. The best solvent and lipase are:pyridine:acetone(3:1) and Novozyme435. t. En and Sr have significant effect on the enzymatic reaction. The optimal reaction conditions are:En,22.8mg; t,31.2h; Sr,3.7:1, as expected, yield (95.6± 1.35M%, n=3) was achieved.4Conventional DOC liposoMes and galactosylated DOC liposomes were prepared. The content detection of docetaxel liposomes with HPLC method is proved feasible by applicability, precision and specificity investigation. Liposomes envelopment is measured by Gel column chromatography. Thin film method is obtained to prepare the liposomes by the comparing of two types of preparation methods on the basis of appearance, encapsulation efficiency and particle size. Liposome preparing method is optimized by orthogonal design, and the optimal parameters are obtained: the mass ratio of drug to lipid is1to15, phospholipid to cholesterol is4to1, and the amount of DSPE-PEG2000is6%. Through the validation experiments under this condition, the maximum encapsulation is93%and the particle size is150nm.5The Pharinacokinetie behavior of galactosylated liposomes were studied By using SD rats as an animal model. We established the mothod of determination of DOC concentration in plasma and proved to be feasible by methodological study. The best way of DOC extraction from plasma is that:DOC is extracted by tert-butyl methyl ether for3times, recovery rate is up to77%. After experimental data is analyzed by DAS2.0software, we concluded that the half-life period of galactosylated liposomes decrease obviously and clearance rate increase comparing with the conventional liposomes. It indicates that the incorporation of CHS-SE-LA into liposomes enhanced the liver targetability.6Conventional liposomes and galactosylated liposomes marked with fluorescent were prepared which particle size are under70nm and fluorescent tags rate is up to99%. ASGPR receptor recognition ability is associated with the distance between the targeted head with the surface of liposome. ASGPR receptor recognition ability can be inhibited by add lactose into cell culture fluid in advance. All of experimental data gave a strong evidence that the galactosylated liposomes have a potential for hepatocyte-targeting drug delivery system.In conclusion we have developed a safe, stable and efficient system using galactosylated liposomes to deliver DOC in HepG2cells as target cells. Firstly, we synthesized a novel glycolipid CHS-SE-LA by lipase-catalyzed, which is an amphiphilic molecule. Then, galactosylated liposomes were developed consisting of EPC, cholesterol and CHS-SE-LA. The uptake mechanism was confirmed to be recognition by the asialoglycoprotein receptors on hepatocytes accordint to cell binding and cellular uptakes test, and the specifcity of targeting is very high. These observations provide valuable information to help in the design of galactosylated carrier systems in order to optimize their targeting efficiency to hepatocytes.