Study On Sustained-release Drug Delivery System Of Protein Drugs Based On Multivesicular Liposomes Technology

Multivesicular liposomes (MVLs) prepared by multiple emulsion method are distinguished from traditional liposomes by the characteristic structure. While traditional liposomes are liposomes with a single bilayer surrounding an aqueous compartment or with concentric lipid bilayers, MVLs are composed of non-concentric multiple lipid layers and have more envelopment volume and larger granularity. But the same as traditional liposomes, it has been proved to be unstable in vitro. Furthermore, MVLs are not suitable for subcutaneous or intramuscular injection because of their large granularity.This study that was based on liposome technology and emulsification technology, investigated the effect of process parameters on encapsulation efficiency and particles size of MVLs. And the effect of using vitamin E and tween-80 was discussed separately. The investigation helped to set up a protein drug delivery system with high encapsulation efficiency, smaller size and more stability in vitro compared with MVLs. Quality evaluation methods of this drug delivery system were established.①Bovine serum albumin (BSA) was used as a model drug, MVLs was prepared by multiple emulsion methods. Then the understanding of the effect of process parameters on encapsulation efficiency, particles size would be in favor of optimizing process conditions. The results showed that, the weight of model drug had little effect on encapsulation efficiency. Using chloroform to dissolve lipid component could heighten encapsulation efficiency, compared with using aether. In a mixture with cholesterol (Chol) and egg phosphatidylcholine (EPC)(1:1 mole ratio), maximal encapsulation efficiency was obtained. The first emulsion could be treated with ultrasonic to obtain a stable w/o emulsion in order to heighten encapsulation efficiency. Triolein (TO), the second emulsification and phase volume fractions had large effect on encapsulation efficiency. TO and the second emulsification had large effect on particles size.②Based on these results, MVLs with high encapsulation efficiency and small size were produced by a double emulsification process as follows. The first step was making a"water-in-oil"emulsion by dissolving 0.5mmol EPC, 0.5mmol Chol and 0.02mmol TO, in 3mL chloroform for lipid component, adding to the first aqueous component of glucose (4.5%) containing BSA (1mg/mL), and then emulsified by surging for 2 min and treating with ultrasonic for 60s. This w/o emulsion was subsequently emulsified with 15mL second aqueous solution containing 4.0% glucose, to get the w/o/w double emulsion. The chloroform was removed by flushing nitrogen at 37℃over the surface of the emulsion. The small size and narrow MVLs with high encapsulation efficiency and good spheroid were successfully obtained.③Based on the optimized process conditions, the use of vitamin E and tween-80, whether had changed encapsulation efficiency, morphology, size, distribution, stability and release performance was investigated. It was shown that stability of MVLs-VitE and MVLs-Tween80 was heightened, compared with that of MVLs.④HV2-MVLs were prepared and quality evaluation methods of HV2-MVLs were established, including morphology, size and distribution, encapsulation efficiency, protein integrality, biological activity, leakage, oxidation degree, release performance. It was shown that HV2-MVLs were white suspension. And morphologically, HV2-MVLs were spheroids with granular internal structures under the light microscope (Fig.4.1). The average size of particles was 8.5μm and span was 0.9. Particles that bigger than 15μm were less than 2% of total particles, and particles distribution was normal distribution (Fig.4.2). Encapsulation efficiency was over 50% (Tab.4.5 and Tab.4.6). The integrality of HV2 was not destroyed (Fig.4.5 and Fig.4.6) and biological activity of HV2 was not changed (Tab.4.7). Moreover, leakage of HV2-MVLs was low (Tab.4.8) and oxidation index was less than 0.2 (Tab.4.9). In vitro release profile (Fig.4.9) showed that, HV2-MVLs released less than 1% of HV2 in 0.5h and sustained-release was lasting over several days.