| Liposomes are composed of phospholipid bilayer structures that encapsulate an aqueous interior. Liposomes are promising and effective vector in tumor treatment. Liposomal antineoplastic agents are characterized by targeting tumor and controlled-release, and thus they can increase therapeutic index, improve clinical effect and reduce toxicity. Liposome properties and biological behaviors can be controlled by surface modification. Attachments of specific ligands, such as monoclonal antibody, peptides, folate and galactosyl, make liposomes own active targeting effeet to specific cells or tissues.It was reported that asialoglycoprotein receptors (ASGPR) were expressed plentifully on the surface of hepatoma cells, and targeting could be accomplished through introduction of galactose residues which can bind specifically to the ASGPR on hepatoma cells, into drug carriers for the treatment of liver cancers. There are large amount of ASGPR in the mammalian hepatocytes, deasialylated proteins can be bound and internalized in the cell interior. Terminal P-D-galactose or N-acetylgalactosamine residues can be recognized by ASGPR and they were considered as specially targeting ligands for drug carriers.Glycyrrhetinic acid is an active principal aglycone of glycyrrhizin which has been shown to be hydrolyzed by glucuronidase in intestinal bacteria after oral administration. Glycyrrhetinic acid is a lipophilic drug with a very low solubility in water (<0.01 mg/ml), which may result in its poor bioavailability. After absorption in gut, glycyrrhetinic acid is eliminated fast in plasma. Glycyrrhetinic acid has been shown to possess several beneficial pharmacological activities, such as anti-inflammatory activity, direct and indirect antiviral activity, and an antihepatitis effect. Glycyrrhetinic acid may cause sodium retention and potassium loss, which are associated with hypertension while the adverse effects of glycyrrhetinic acid seem to be dose-dependent.In this research, glycyrrhetinic acid was chosen as the model drug and galactose as the targeting head. Asialoglycoprotein receptor-mediated active targeting liposomes were prepared. Liposomes could be targeted to liver through the interaction between ligand and receptor on the carcinoma cells of liver. The drug delivery system actively targeting to malignant liver cells is expected to be achieved.Lipid materials for preparing liver targeting liposomes, such as N-octadecyl-4-[(D-galactopyranosyl)oxy]-2,3,5,6-tetrahydroxy hexanamide (NOH), were enzymic synthesized. Most of the present strategies for the amidation were focused on chemical methods, which were hampered by the low regioselectivity, the tedious reaction procedures and the environmental concerns of the process. The possibility of the enzymatic regioselective acylation was explored in this dissertation. Also, the effects of some variables on the enzymatic reaction in different media and lipases were characterized. Additionally, novel enzymatic reaction systems which could be used for highly efficient and regioselective preparation of monoesters of N-octadecyl -4-[(D-galactopyranosyl)oxy]-2,3,5,6-tetrahydroxy hexanamide have been well established. The synthetic products were determined via TLC, IR, HPLC-MS,'H-NMR,13C-NMR, DSC etc.The pre-prescription of glycyrrhetinic acid was carried out via three steps: establishment of glycyrrhetinic acid by HPLC analysis method, oil-water distribution coefficient and determination method of encapsulation efficiency. The study of physico-chemical property showed that glycyrrhetinic acid was a poorly water-soluble drug. Its solubility in water was only about 6.32μg/mL. It was easy to dissolve in n-octanol and had a good liposolubility. After choosing several common methods of preparation of liposomes, such as film dispersion method and ethanol injection method, encapsulation efficiency and trait characteristics as indicators, liposomes were prepared by ethanol injection technique. The lgP value of glycyrrhetinic acid was about 4.69. Gglycyrrhetinic acid was high lipophilic compounds, which adapt to preparation of liposomes. The unencapsulated glycyrrhetinic acid and liposomes were separated by sephadex gel G-50, the encapsulation efficiency was detected by HPLC. Through a single factor and orthogonal design optimization, we achieve the best formulation. Ethanol injection technique is a good process to avoid using of toxic organic solvents, and the products have shown characteristic and good stability, fit encapsulation efficiency, appropriate particle size and be in accordance with the normal distribution.To observe the glycyrrhetinic acid liver targeting liposomes physical and chemical properties systematically, including particle size, morphology, Zeta potential, in vitro release rate, determination of encapsulation efficiency, stability reseach, determination of alcohol residue, and so on. The appearance of glycyrrhetinic acid liver targeting liposomes was slightly translucent opalescence, color uniformity, good mobility. The morphological examination of glycyrrhetinic acid liver targeting liposomes was performed using transmission electron microscopy. The liposomes were spherical or ellipsoidal shape. The particle size and Zeta potential of the liposomes were measured. the mean partical size of 195 nm, and Zeta potential of 38.7 mv. The unencapsulated glycyrrhetinic acid and liposomes were separated by sephadex gel G-50, the encapsulation efficiency was detected by HPLC. Entrapment efficiency is over 80%. Resarch on chemical stability show that oxidation index is low. Reseach on lofting stability results show that glycyrrhetinic acid liver targeting liposomes situated at (0-4)℃place for three months to maintain stability. The stability of glycyrrhetinic acid liver targeting liposomes was better. The release kinetics in vitro obeyed Higuchi equation. the glycyrrhetinic acid liver targeting liposomes have obviously sustained effect. Alcohol residue is under 0.5%.Furthermore, the pharmacokinetic behavior and distribution of glycyrrhetinic acid liver targeting liposomes were studied. Both the two formulations and free glycyrrhetinic acid were administered via tail vein. Glycyrrhetinic acid was separated from the plasma component by solvent extract. The plasma concentration and tissue distribution of the drug was determined by HPLC. Pharmacokinetic parameters of rats were as follow:t1/2 of GA, GA-LP and NOH-GA-LP were 3.91,46.49 and 50.18 h, respectively; AUC(0-24) of them were 1.95,12.85 and 15.41μg-h/mL, respectively; MRT(0-24) of them were 1.75,8.18 and 8.11 h, respectively. HPLC was employed to exmaine the tissue distribution of mice at diffetent times after liposomes were injected into their tail veins. Compared with glycyrrhetinic acid control solution, the relative targeting efficiencies of liver of glycyrrhetinic acid, GA-LP and NOH-GA-LP were 2.4 and 4.83 respectively, which indicated that the efficiency of liver targeting was improved when galactose ligand was introduced to liposomes.HepG2 cells were cultured in vitro, and the tumor cell inhibition test was conducted with MTT assay. The result showed that 10 %NOH-GA-LP was significantly more suppressive than the 5%NOH-GA-LP. There was significantly different between the 5%NOH-GA-LP and 10%NOH-GA-LP with MTT assay. HepG2 cells were suppressed when galactose ligand was introduced to liposomes.In conclusion, glycyrrhetinic acid active targeting liposomes were successfully prepared by using ethanol injection method and the method is repeatable with satisfactory results. Physical and chemical stability, particle size, encapsulation efficiency and the leakage rate of the products meet the requirements and so on. Glycyrrhetinic acid in NOH-GA-LP and in biological material was detemrination by HPLC. A good theoretical and practical foundation was established for the exploration of the liposomes' dosage formula, characteristics and work mechanism in this research, which not only enlarged research field of liposomes but also brought new ideas and new methods to treat cancer. Further more, this research has potential value in clinic practise.
|
PDF Full Text Request