中国科学院过程工程研究所机构知识库

局部麻醉药罗哌卡因广泛用于外科手术中区域阻滞、术后镇痛以及慢性疼痛控制等适应症。但由于其半衰期短，单次注射难以达到期望的镇痛时间，临床上常通过频繁多次给药以及导管植入等方式来延长局部镇痛效果，这会造成患者顺应性差以及感染等风险。针对此问题，科学工作者对罗哌卡因缓释微球做了大量的研究工作，仍然存在以下问题难以解决：微球粒径不均一；载药率偏低；释放行为不理想等。为了克服这些关键问题，本论文通过快速膜乳化技术结合单乳法，优化了制备过程的处方工艺参数，并研究了其内在的科学规律与原理，制备了粒径均一性好、载药率、包埋率高、药物释放速率可控的载罗哌卡因缓释微球。具体研究内容分为以下四个部分。（1）针对传统技术制备局麻药缓释微球粒径不均一的问题，采用快速膜乳化技术结合单乳法，考察了过膜压力、过膜次数、PLGA浓度、PLGA分子量、外水相pH值、初乳制备方式、固化方式等因素对微球性质的影响，最终制备得到了粒径均一、包埋率高、低突释的载罗哌卡因微球。（2）在上述工艺优化的基础上，系统地地探讨了不同固化方法（真空负压与常温常压）对微球性质的影响。研究表明，常温常压条件制备的微球内部呈“蜂窝状”结构，突释较高，包埋率偏低。这是由于固化时间较长，乳滴内部的罗哌卡因逐渐形成晶体并逃逸至外水相引起的。在真空负压条件下制备的微球，突释较低，包埋率高，体外释放可以保持匀速释放。这是由于固化时间短，乳滴快速形成微球。罗哌卡因来不及形成晶体逃逸，与PLGA共同形成微球骨架。（3）系统地考察了使用不同端基的PLGA（端羟基、端羧基、端酯基）对于制备罗哌卡因微球的包埋率、体外释放、药代动力学、药效学等的影响。结果表明端羧基PLGA制备的载罗哌卡因微球包埋率高于端羟基与端酯基制备的微球，突释要低于端羟基与端酯基制备的微球。进一步在微观水平上，通过石英晶体微天平、原子力显微镜、pH探针等进行分析，研究表明PLGA的端基与罗哌卡因之间产生的吸附作用力的差异是导致微球性质不同的原因。随后在分子水平上，通过密度泛函计算与前线分子轨道理论确定了分子间反应活化中心，佐证了以上结论。（4）针对临床潜在的应用适应症，建立了SD大鼠神经阻滞麻醉模型和豚鼠局部浸润麻醉模型。对上述最优处方工艺下制备得到的罗哌卡因载药微球进行了药效行为学的评价。研究表明罗哌卡因载药微球可以显著延长局部镇痛时间。随后在体外细胞水平以及体内组织、血液生化水平上进行了载罗哌卡因微球的安全性评价。结果表明载罗哌卡因微球不会引起神经损伤以及局部组织炎症，对主要脏器也不会产生毒性。本文通过快速膜乳化技术结合单乳法，使罗哌卡因参与微球骨架的形成，制备了高载药率、粒径均一、释放行为趋近于零级释放的载罗哌卡因缓释微球，延长了局部镇痛效果。通过对分析处方工艺参数、研究不同固化方法、PLGA端基的呈球机理，为小分子药物的包埋提供了理论基础;Local Anesthetics, Ropivacaine, are widely used in postoperative analgesia, local nerve block in surgical and the therapy of chronic pain. Due to its short half-time, a single injection of ropivacaine just provides a nerve block lasting only 3-4 hours, making it difficult to achieve desirable analgesia effect. To meet the long-time analgesia requirement, multiple methods such as frequent injection, catheter implantation in vivo or patient controlled analgesia (PCA) device have been applied in clinical. The wide-spread use of these approaches has been hampered by poor patient compliance, the lack of portability and the risks of infection. Nowadays, a lot of work have focused on the development of ropivacaine loaded microspheres. Nevertheless, there are still several issues to be solved, such as particle size with broad distribution, low drug loading efficiency, undesirable release behavior. In order to overcome these key problems, we proposed to combine O/W emulsion method with novel premix membrane emulsification technique. After the optimization of fabrication procedure, ropivacaine loaded microspheres with narrow size distribution, high drug loading efficiency and an ideal constant release behavior have been obtained. This thesis is divided into four parts:The first part focused on the difficulty of preparing ropivacaine loaded microspheres with uniform size. Premix membrane emulsification technique combine with O/W emulsion method were developed. After the optimization of tran-membrane pressure, tran-membrane cycles, PLGA concentration, PLGA molecule weight, pH value of external water phase, primary emulsion preparation and solidification method, we obtained ropivacaine loaded microsphere with narrow size distribution, high drug loading efficiency and an ideal constant release behavior. Based on the above optimization, the influences caused by solidification method (ambient pressure and negative pressure) were investigated. It was found that high burst release and low encapsulation efficiency was achieved in ambient pressure condition. Because ropivacaine gradually aggregate into a crystal which might easily leak out from droplets, that would result in lower drug loading efficiency. As for in vitro release profile, the leaked drug crystal on the surface of the ASE microspheres could not easily be washed away completely, which led to strong initial burst release. With respect to NSE-MS, ropivacaine existed in amorphous state was difficult to escape and inclined to uniformly disperse inside polymer matrix, which led to a higher drug loading efficiency and constant release rate. The effect of terminal group of PLGA (OH, COOH, COOR) were systematically investigated. It was found that the encapsulation efficiency of ropivacaine loaded microsphere prepared by COOH-PLGA was higher than that of OH-PLGA and COOR-PLGA. Meanwhile, the initial burst of ropivacaine loaded microsphere prepared by COOH-PLGA was lower than that of OH-PLGA and COOR-PLGA. The reasons for the distinction in adsorption force between different terminal group of PLGA and ropivacaine were analyzed from the microscopic level by using QCM-D and AFM. Furthermore, the combination ability of molecule reaction activation center is analyzed by density functional theory and frontier molecule orbital theory. The pharmacodynamic of the optimized ropivacaine loaded microspheres were evaluated through the nerve block model SD rats and the local infiltration model of guinea pigs. It was found that ropivacaine loaded microspheres exhibited the good sustained release effect in terms of both intensity and duration of anesthetic effect. Additional, in vitro cytotoxicity and histological evaluation proved that ropivacaine loaded microspheres did not cause nerve damage and local tissue inflammation. Biochemical analysis results demonstrated the cardiotoxicity was reduced by encapsulation ropivacaine into microspheres because a steady release behavior in vivo.In this research, the ropivacaine loaded microsphere with high drug loading efficiency, narrow size distribution and prolonged local analgesic effect were obtained by premix membrane emulsification technique combined with O/W emulsion method. By optimizing the preparation process parameters, investigating the effect and mechanism of different solidification methods as well as different terminal group of PLGA, it provides a theoretical direction for encapsulating soluble small molecular drug