摘要
聚乳酸作为优良的生物相容性和可生物降解性的聚合物，在药物控释体系得到了广泛的研究；超临界二氧化碳流体技术在细微颗粒的制备方面也具有独特的优点，其应用领域中最受研究者关注的莫过于生物和医药制剂以及特殊聚合物。本文主要采用超临界流体强制分散溶液法(SEDS法)进行聚乳酸药物载体微球的制备，并以溶菌酶为模型研究其在多肽、蛋白类大分子药物给药体系中的应用，为日后超临界流体技术在制备大分子类药物缓释微球方面的应用奠定基础。
首先，以L-聚乳酸为模型体系，超临界CO_2为抗溶剂，采用SEDS法制备聚乳酸微球。考察了压力、温度、溶液浓度、溶液流速、聚合物分子量等参数对制备微球的形态、粒径及其分布的影响。结果表明：改变工艺参数，可在一定范围内调控微球粒径，所制微球平均粒径0.67μm-6.64μm，有机溶液浓度及其流速为主要影响因素，SEDS过程的最佳操作条件是压力为12 MPa，温度33℃，有机溶液浓度0.5％(w／v)，流速0.5 ml-min~(-1)。在实验范围内，聚乳酸微球的粒径随着压力的增大先减小后增大；随温度升高而增大；随浓度增大而变大，且微球的团聚程度增大；随着有机溶液流速变大，聚乳酸微球粒径长大；四种分子量聚乳酸同种条件下制备得微球，分子量为5万、10万的聚乳酸能够形成球形度较好、粒度较均匀的微球。在优化后的操作条件下，又研究了不同方法(超临界CO_2流体抗溶剂法及SEDS法)和混合溶剂对聚乳酸微球形貌及粒径的影响。实验条件一定时，采用二氯甲烷-丙酮混合溶剂及强制分散溶液法制备得较小粒径微球
其次，利用SEDS法制备溶菌酶粉末；并对制备前后溶菌酶二级结构及活性的变化进行表征；红外光谱及圆二色光谱测试结果表明，经SEDS过程后，溶菌酶的二级结构没有明显变化，活性测试结果表明经SEDS过程后溶菌酶的活性提高了51.2％。
再次，以溶菌酶作为多肽、蛋白类大分子药物模型，制备了溶菌酶—聚乳酸复合微球。将制备所得溶菌酶粉末用于溶菌酶—聚乳酸复合微球的制备，考察聚乳酸分子量、聚乙二醇(PEG)含量、PEG分子量以及载药量等对复合微球药物释放性能的影响，结果表明：在实验范围内，随着载药量的增大，溶菌酶—聚乳酸复合微球的粒径逐步减小，药物释放速度逐步增大，载药量为8％的微球的球形度及分散度较好；微球的大小随着聚乳酸分子量的增大先增大后减小，药物的释放速度由聚乳酸分子量及复合微球粒径大小共同决定；PEG的含量越高，分子量越大，微球的粒径越小，药物的释放速度越快。
最后，对聚乳酸药物载体微球进行了体外降解与初步生物学评价实验。采用pH 6.8 PBS作为降解介质对经超临界二氧化碳流体技术制备出的不同分子量的聚乳酸微球进行体外降解实验；同时以MTT法考察了聚乳酸微球。实验结果表明，在37℃的pH 6.8 PBS浸泡10周后，起初表面光滑的聚乳酸微球在表面均出现一定程度的孔洞，但质量及降解介质pH均未发现显著变化，聚乳酸的分子量出现一定程度的下降，且分子量较大的聚乳酸其分子量的下降幅度也较大。MTT实验结果显示，聚乳酸微球无体外细胞毒性，具有良好的生物相容性。
In this study, the drug carries based on PLLA was prepared using solution-enhanced dispersion by supercritical CO_2 (SEDS), and its application in drug delivery for protein drugs was investigated.
Firstly, microparticles of poly-L-lactide (PLLA) were prepared with SEDS process by using supercritical carbon dioxide as an anti-solvent. The effects of several key factors on surface morphology, and particle size and particle size distribution were investigated. These factors included temperature (33-39°C), pressure (8-16 MPa), PLLA concentration (0.5-1.5%, w/v), flow rate of organic solution (0.5-1.5 ml·min~(-1)), mixing acetone into PLLA dichloromethane solution (1:1, v/v) and different molecular weight of PLLA. The results indicated that concentration of the organic solution and the flow rate of the solution played important roles on the properties of products; the mean particles size was decreased with the decreasing of PLLA concentration or flow rate of organic solution; the temperature and pressure had minor effects on the properties of products, however, the mean particles size was decreased slightly with the increasing of CO_2 density, and the agglomeration of products was increased with the increasing of temperature; the PLLA microparticles with MW of 50 kDa and 100 kDa exhibited rather spherical shape, smooth surface, and narrow particle size distribution. Various microparticles with the mean particle size ranging from 0.64μm to 6.64μm, could be prepared by adjusting the operational parameters. Fine microparticles were obtained in the SEDS process with dichloromethane/acetone mixture as solution.
Secondly, lysozyme was used as macromolecular drug model, the micronization of lysozyme was performed in SEDS process and both the change in the secondary structure and activity of lysozyme were studied. The results of Fourier-transform infrared (FTIR) spectra and Circular Dichoism (CD) spectra both showed that there was no significant change in the secondary structure of lysozyme after SEDS process, while the activity test indicated that its activity was increased 51.2% after this process;
Thirdly, the micronized lysozyme were used to prepare lysozyme-PLLA microparticles, the effect of molecular weight (MW) of PLLA, Polyethylene Glycol (PEG) ratio, PEG MW and drug loading on the properties of lysozyme-PLLA microparticles were studied. The results indicated that with the increasing of drug loading, PEG ratio, or PEG MW, the particle size of lysozyme-PLLA microparticles decreased, and its rate of drug release increased, while with the increasing of PLLA MW, the particle size of lysozyme-PLLA microparticles increased first and then decreased, and its drug-release rate was controlled by both particle size and PLLA MW.
Finally, the in vitro degradation of PLLA microparticles was investigated by incubating PLLA microparticles in 37℃pH 6.8 PBS, and the toxicity of PLLA microparticles was measured by MTT assay. It was found that after being incubated for 10 weeks, there were some tiny holes on the surface of PLLA microparticles, the PLLA MW was decreased, the larger was the MW, the bigger was the decreasing scope of MW; the MTT assay demonstrated that the PLLA microparticles had no cytotoxicity, they would be biocompatible.
引文
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