| Asialoglycoprotein receptor (ASGPR) also known as galactose receptor, mainly expressed on the surface of the liver parenchyma cells of the mammalian's liver sinusoid. Asialoglycoprotein receptor has characteristics that can recognize, bind and endocytose glycoproteins whose terminal has galactose and acetyl glycoproteins in blood circulation. These characteristics commonly are used for mediating drugs or drug carriers of hepatic targeted drug delivery system. Drugs or drug carriers modified with galactose can be recognized and bound by ASGPR on the surface of the liver parenchyma cells, and thus the drugs will accumulate in the liver, significantly improve the targeted effect of the drugs to the liver, this is an important way to develop new drugs in the treatment of liver diseases.Liposome modified with galactose ligand is a studying focus in the field of pharmaceutical preparations, with a very widely application prospect. Using liposomes as drug carriers modified with galactose on its surface, once inside our bodies it can be quickly accumulate in the liver, thereby enhance the targeted effect of drugs and reduce the harmfulness to normal cells.Currently, the common method of connecting the galactose ligand to the liposomes is covalent coupling. Which is couple the aliphatic hydrophobic chain hydrocarbons (such as vinyl stearate, etc.) or phospholipids with the ligand, using the function of its long-chain fatty to embed the galactose ligand into the surface of lipid bilayers, thus forming liposomes modified with galactose ligand. However, the synthesis methods of galactose ligand are generally chemical methods, there are some shortcomings such as harsh reaction conditions, much more by-products, complicated operational steps, organic solvents pollution. Compared to chemical methods, enzyme catalysis has characteristics of mild reaction conditions, high efficiency, selectivity, and much less by-product. Thus, we use non-aqueous enzymatic catalysis to catalyze hydroxyl groups of galactose and ester on the vinyl stearate to take the ester exchange reaction, and produce a liver ligand-targeted modifier that can be inserted the surface of liposomes.This dissertation showed the enzyme-catalyzed etherification of galactose and vinyl stearate and a kind of galactose ligand-targeted modifier which was used to insert the surface of liposomes has been synthesized. The structure of product has been confirmed by TLC, ESI-MS and 1H NMR. The factors of types and quantity of enzyme, organic solvents, molar ratio of substrate, temperature and time of reaction have been studied. Results showed when using acetone as reaction medium, the quantity of Novozym 435 immobilized lipase was 30 mg-mL-1, molar ratio of galactose to vinyl stearate was 1:5, and reacted in 60℃for 12 h, the transformation of vinyl stearate reached more than 70%. This study provides a novel and efficient route to the synthesis of ligand-targeted modifier. This synthetic method is high efficiency, regioselectivity, mild reaction conditions and so on.After we got the liver ligand-targeted modifier galactose esters by enzymatic catalysis, we choose docetaxel as a model drug, and galactose ligand-targeted modifier galactose esters as targeted head group to prepared asialoglycoprotein receptor-mediated active targeted liposomes. In the preparation process of the docetaxel liposomes modified with galactose esters (Gal-DOC-L), we studied the method for determination of liposomes entrapment efficiency at first. The results showed that the rdialysis method is much more accurate and reliable. Then Gal-DOC-L's formulation was optimized by single factor and orthogonal design. Finally, we determined to use the method of ethanol injection to prepare the docetaxel liposomes modified with galactose esters. Preparation processes of liposomes is as follows: weighed phospholipids, cholesterol, VE and drugs (mass ratio 6:2:1:0.3) accurately, added 10% galactose ligand-targeted, soluble in ethanol with water bath, then slowly injected the solution to the phosphate buffered saline (PBS) (pH 7.4,33.23 mmol-L-1) which maintained its temperature at 55℃, at the same time, the N2 was passed through the solution to remove the ethanol. After injection, the liposomes were incubated at 45℃for 20 min, extracted 6 min in ultrasonic disintegrator, filtrated by 0.45,0.22μm microspore filter, then the Gal-DOC-L was prepared. The ordinary docetaxel liposomes (DOC-L) was achieved with the same method.Gal-DOC-L and DOC-L's physical and chemical properties were investigated. The liposomes were suspension with slightly opalescence, translucent, uniform color and good fluidity. The results of TEM observation showed that, DOC-L and Gal-DOC-L had uniform distribution of particle size, round shape and good distribution. The results of laser scattering particle size measurement showed that, DOC-L and Gal-DOC-L were in the narrow range size distribution, with an average particle size of 126.8 nm,155.2 nm, a single peak distribution. The results of Zeta potential analyzer showed that DOC-L's surface Zeta potential was (-20.8±0.3) mV, Gal-DOC-L's surface Zeta potential was (-33.1±0.6) mV. The results of release experiments in vitro showed that, DOC-L and Gal-DOC-L had obvious sustained-release effects. The release kinetics in vitro obeyed with Weibull distribution function. The entrapment efficiency of liposomes were all above 80%, and the oxidation index was low, the degree of oxidation was small, under the conditions of 0-4℃for 3 months, the leakage rate of liposomes were less than 6%, particle size changed little and the stability of liposomes was well, liposomes were diluted in accordance with the clinical usage, the size and entrapment efficiency had no significant change.The pharmacokinetics of Gal-DOC-L and DOC-L in rabbits was investigated. The results showed that compare to an ordinary injection of docetaxel (DOC), the AUC of liposomes increased significantly (P<0.05), and the total body clearance (CL) decreased (P <0.05). Thus, Gal-DOC-L, DOC-L can maintain relatively high blood concentration in vivo, and extend the time of drug effect. To a certain extent, it can enhance the therapeutic effect.Finally, tissue distribution in mice and liver targeted of DOC-L, Gal-DOC-L were studied. DOC, DOC-L and Gal-DOC-L were injected respectively into the tail vein of mice intravenously, the determination of drug tissue distribution in mice, with DAS 2.0 pharmacokinetic program to calculate DOC, DOC-L and Gal-DOC-L in various tissues AUCo-12 and Cmax, using relative uptake rate (re), peak concentration ratio (Ce) and targeting efficiency (te) as the evaluation parameters to evaluate the DOC-L, Gal-DOC-L's liver targeted effect. The results showed, the relative uptake rate of DOC-L and Gal-DOC-L were 2.80 and 4.34, peak concentration ratio of DOC-L and Gal-DOC-L were 1.67 and 2.38, targeting efficiency of DOC-L and Gal-DOC-L were 28.44% and 38.91%. These indicated that the drug encapsulated by liposomes has a certain liver targeted effect, but liposomes modified with galactose ligand-targeted would further improve the hepatic targeted effect.In conclusion, this dissertation used techniques of non-aqueous enzymatic to modify the structure of galactose, and synthesized a galactose derivative (galactose esters) for hepatic targeted which can be embedded into the lipid bilayers of the surface of liposomes. Then docetaxel was used as a model drug and prepared asialoglycoprotein receptor-mediated Gal-DOC-L by ethanol injection. Pharmacological experiments showed that, compared to DOC-L, Gal-DOC-L had better liver targeting effect. Liposome modified with galactose was an ideal liver targeted carrier. This research will provide a kind of drug carrier for hepatic targeted drug delivery system, maybe it has certain academic value and application prospect.
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