Study On Preparation And Anti-tumor Efficacy Of Cholesterol-Galactoside Ligand-modified Liposomes Containing Paclitaxel

Objective:Galactose receptor widely distributes on the surface of mammalian liver parenchyma cells, known as Asialoglycoprotein receptor (ASGPR), which can be applied to establishing the liver-targeted liposomes. Paclitaxel (PTX) is extracted from Taxus belonging to tetranuclear diterpenoids; since its toxicity is lower compared with other chemotherapeutic drugs, PTX is taken as a kind of widely-used anti-cancer drugs. Clinical studies have revealed that the tumor-killing capacity of PTX is equal to fluorouracil with less side effects. To explore paclitaxel preparation for the treatment of liver cancer is a new target at present in the research and development of PTX. This study first designed enzymatic synthesis of cholesterol-galactoside as a novel ligand applying to the preparation of ligand modified liposomes containing PTX. Because of liver targeting property of galactose ligand, it is possible to make the modified liposomes gathering in the liver, and effectively played the role against liver cancer. At the same time, the ligand liposomes prescription, preparation technology, basic physical and chemical properties and stability were investigated with paclitaxel injection and conventional paclitaxel liposomes as the reference preparations; the pharmacokinetics in vivo, tissue distribution, and its antitumor efficacy in vitro of ligand modified liposomes were discussed in detail, which could provide the theoretical foundation for the research and development of PTX in the form of a new anti-tumor targeting preparation.Methods:1. Enzymatic synthesis, purification, structural characterization and stability investigation of cholesterol-galactoside ligandCholesterol-galactoside ligand was synthesized by the enzyme Novozym 435 under these conditions:lactitol and cholesteryl hemi-vinyl sebacate as the substrates, pyridine:acetone (2:1, V/V) as the reagents and moderate temperature. To screen and optimize the lipase-catalyzed synthesis of cholesterol-galactoside, reaction reagents, temperatures, kinds of lipases, enzyme amount and ratio of the substrates were investigated respectively by single-factor test to confirm the parameters of Box-Behnken Design. Then, the obtained product was purified by flash column chromatography and detected by HPLC analytical method. TLC, MS, IR, and NMR technologies accounted for the structural characterization of cholesterol-galactoside ligand. Stability investigation was carried out by influencing factors and accelerated tests.2. The preparation and physico-chemical properties of ligand modified liposomes containing PTXAdd appropriate proportion (w%) of Cholesterol-galactoside into lipid material. After thin-film dispersion, hydration incubation, ultrasonic probe and microporous membrane filter, the preparation of ligand modified paclitaxel liposomes was finished; the single-factor and orthogonal design was carried out for optimizing the prescription and obtaining stable liposomes; we also investigated the appearance, particle size, zeta potential, encapsulation efficiency of ligand modified paclitaxel liposomes.3. Pharmacokinetics and tissue distribution of ligand modified paclitaxel liposomesThree preparations of PTX injection, PTX liposomes and ligand modified paclitaxel liposomes was ready. SD rats, KM mice as animal model, through the tail intravenous injection, we investigated three drug preparations of pharmacokinetic parameters in vivo respectively, as well as the tissue distribution (heart, liver, spleen, lung, kidney); the extraction of PTX in plasma was dealt with methyl tertiary butyl ether (MTBE), Docetaxel (DOC) as internal standard (IS) was set to calculate PTX blood concentration; DAS 2.1 software analyzed all pharmacokinetic data.4. In vitro anti-tumor efficacy of ligand-modified liposomes containing PaclitaxelIn vitro anti-tumor experiment, a large number of human HepG2 cell lines were cultivated, then was inoculated in 96-well culture plate; there were four groups:negative control group, PTX injection group, PTX-L group and Gal-PTX-L group; after the treatment in 24-48-72 h, the CCK (tetrazolium mono-sodium salts) and LDH (lactate dehydrogenase) methods determined cellular survival rate, drug IC50 and the activity of LDH, and then the comprehensive evaluation on anti-tumor efficacy of Gal-PTX-L preparation was formed.Results:1. Enzymatic synthesis, purification, structural characterization and stability investigation of cholesterol-galactoside ligandCholesterol-galactoside ligand was synthesized by the enzyme Novozym 435. Reaction reagents, temperature, kinds of lipases, enzyme amount and ratio of the substrates were investigated respectively by single-factor test to confirm the parameters of Box-Behnken Design; the yield of CHS-DD-LA was utilized as its estimating index for further optimizing the lipase-catalyzed synthesis. The optimal conditions:molar ratio of lactitol to cholesteryl hemi-vinyl sebacate 1:3.7, Novozym 43522.8 mg, pyridine:acetone (2:1, V/V) 2.1 L, reaction temperature 55℃,reaction time 31.2 h, and the yield of CHS-DD-LA was above 94%. The purity of product could exceed 90% by flash column chromatography. The results of TLC, MS, IR, and NMR methods had confirmed the structural characterization of ligand. According to the results of the influencing factors, the ligand should be sealed, placed in a dry, cool, dark place.2. The preparation and physico-chemical properties of ligand modified liposomes containing PTXConventional PTX liposomes and galactosyl PTX liposomes were prepared. Through the linearity, specificity, precision, recovery, repeatability and stability test, it is proved that the established HPLC determination method is applicable and reliable. Selecting the ratio of drug to phospholipids, phospholipids to cholesterol and dosage of cholesterol-galactoside (w%) as the factors, and the encapsulation rate (%) as the evaluation index, orthogonal test was utilizing in the optimization of liposomal preparation technology, thus the optimal conditions were as follows:ratio of drug to phospholipids (1:4), phospholipids to cholesterol (5:1) and dosage of cholesterol-galactoside (w%=8%). Under the optimal process, the encapsulation rate (%) was above 86%, particle size remained at about 160 run.3. Pharmacokinetics and tissue distribution of ligand modified paclitaxel liposomesConcentration of PTX in plasma was detected by the LC-MS/MS method, the methodology investigation indicated that the method is applicable and feasible; after screening the sample extraction method, it was confirmed that the methyl tertiary butyl ether (MTBE) extraction was better consistent with the requirements of analysis; after i.v. administration of three preparations at the dosage of 5 mg/kg in rat, the half-life T1/2z (h) were 4.26±0.72 (PTX-i),2.83±1.28 (PTX-L),3.32±2.05 (Gal-PTX-L); the apparent volume of distribution Vz (L/kg) were 9.44±2.28 (PTX-i),3.89±1.03 (PTX-L),16.11±6.23 (Gal-PTX-L);CLz (L/h/kg) were 1.46±0.17 (PTX-i),1.12±0.35 (PTX-L),4.06±1.55 (Gal-PTX-L), respectively. In addition, after i.v. administration of three preparations in mice, the liver targeting parameters of PTX-L and Gal-PTX-L were as follows:Re of 0.105 and 4.103, Te (%) of 3.083 and 56.112, RTe of 0.117 and 2.105, Ce of 0.109 and 3.298.4. In vitro anti-tumor efficacy of ligand-modified liposomes containing PaclitaxelAt the dosage of 0.1μg/mL,24 h cell viability (%) of PTX-i, PTX-L and Gal-PTX-L groups were 50.08%,26.25% and 11.38%; 48 h cell viability (%) were 45.84%,32.25% and 19.17%; 72 h cell viability (%) were 43.03%,28.45% and 24.85%. In addition, IC50 of three preparations were respectively 0.438μg/mL,0.121μg/mL and 0.037μg/mL. After the intervention of three drug groups,24 h LDH viability were 20.14 IU/L,27.67 IU/L and 39.37 IU/L; 48 h LDH viability were 34.60 IU/L,51.74 IU/L and 65.11 IU/L; 72 h LDH viability were 48.98 IU/L,65.20 IU/L and 80.91 IU/L.Conclusions:Given the whole results of this paper, study on synthesis of cholesterol-galactoside ligand and anti-tumor efficacy of ligand-modified liposomes containing paclitaxel provides a novel, efficient and stable way to transport galactose liposomes to the targeted HepG2 cells, exhibiting the anti-tumor efficacy. The outcomes of pharmacokinetics and tissue distribution demonstrated that ligand modified paclitaxel liposomes has better liver targeting property than conventional preparation, easier to accumulate in the liver; in vitro study, ligand modified paclitaxel liposomes could effectively inhibited the growth of hepatic tumor cells, the difference was statistically significant (P< 0.01).Therefore, we have reason to believe that ligand modified paclitaxel liposomes possess broad application prospects in the field of liver disease.