聚乙丙交酯微球的制备及对曲安奈德的包载与释放研究.pdf

1266第 53 卷第 11 期2023 年 11 月Vol.53 No.11Nov.2023日 用 化 学 工 业（中英文）China Surfactant Detergent&Cosmetics Preparation of poly（lactic-co-glycolic acid）microsphere and its behaviors of encapsulation and release of triamcinolone acetonideWenboYuan1,HuatongZhang2,CongdeQiao2,LingWang2,MingmingLuan2,XiaodengYang2,*（1.Engineering Technology Center,Shandong Guyuchun Biotechnology Co.,Ltd.,Jinan,Shandong 250098,China;2.Shandong Key Laboratory of Molecular Engineering,Qilu University of Technology,Shandong Academy of Sciences,Jinan,Shandong 250353,China）Abstract:Triamcinolone acetonide has the advantages of strong medical efficacy and long duration,however,its oil solubility limits its application.Poly（lactic-co-glycolic acid）（PLGA）has excellent biocompatibility and biodegradability,which can be made into microspheres loaded with triamcinolone acetonide.In this work,the PLGA was prepared using trifluoromethanesulfonic acid as catalyst and glucose as initiator.The molecular structure,molecular weight and molecular weight distribution of PLGA were characterized by NMR and gel chromatography.The results showed that the mass ratio of lactic acid to glycolic acid segments in PLGA was 5050,and the molecules were terminated with carboxyl groups,and the molecular weight was between 36 000 and 41 000,exhibiting narrow molecular weight distribution（molecular weight distribution index was ca.1.60）.The microspheres were made by electrostatic spraying method,whose particle size was less than 20 m（the content of microspheres of diameter being smaller than 20 m was more than 92%）.The drug loading efficiency for triamcinolone acetonide was more than 44%,and the period of release was up to 120 h.In the initial stage of drug release,the relationship between drug release rate and time was linear.This work could provide basic data for the preparation of PLGA microspheres for drug-loading and expand the application of triamcinolone acetonide in the treatment of allergic dermatitis,neurodermatitis and chronic eczema.Moreover,this work could provide reference for the development of functional skin care products.Key words:triamcinolone acetonide;poly（lactic-co-glycolic acid）;microsphere;drug loadingReceived:October 24,2022;Revised:October 26,2023.*Corresponding author.Tel.:[phone],E-mail:.国家自然科学基金青年基金（21904074）；山东省重点研发（2019JZZY011118）；齐鲁工业大学（山东省科学院）重大创新专项（2022JBZ02-04）DOI：10.3969/j.issn.2097-2806.2023.11.004HOOOOHHOOH+OHTFMS,Glu40,4 hOHH3CCO(A)COCCCH3OOmnOmn020406080100PLGA41PLGA36PLGA38Drug loading capacity/%Samples of PLGA(B)020406080100Drug encapsulating capacity/%20406080100120020406080100Drug releasing behavior/%t/h(C)PLGA36 PLGA38 PLGA41HH2SyntheticrouteofPLGAbypolymerization（A）,drugloadingcapacityanddrugencapsulation（B）,anddrugreleasebehavior（C）ofPLGAwithdifferentmolecularweights1267第 11 期开发与应用袁文博，等：聚乙丙交酯微球的制备及对曲安奈德的包载与释放研究 曲安奈德（Triamcinolone acetonide）是一种能够发挥长效作用的肾上腺皮质激素药物，具有抗炎性、抗病毒性和抗过敏性等作用，因药效强、作用时间持久，经常用于抗过敏和多种皮炎的治疗。然而，作为一种亲脂性药物，曲安奈德以乳膏制剂涂抹于病体表面容易引起衣服污渍，使用不方便。聚乙丙交酯（Poly（lactide-co-glycolide），PLGA）是一类由乳酸（Lactic acid，LA）和乙醇酸（Glycolic acid，GA）聚合而成、具有优良生物相容性、可在人体内生物降解的生物大分子1,2。PLGA的降解产物是H2O和CO2，通过人体普通的生理循环即可排出体 外3，是重要的生物医用材料和药物载体。PLGA可以以胶束、凝胶、微球、纳米粒子、膜等不同形式载药2-5，研究2,6,7发现，PLGA的分子量、分子组成（乳酸和乙醇酸的链段比）、分子量分布、分子端基种类等因素影响载药量和对药物的释放周期。PLGA微球的粒径分布越窄，微球对罗哌卡因的载药量越大。Li等6发现当微球的粒径分散性指数为0.469时，对罗哌卡因的载药量高达49%，持续释放周期长达168 h。乳酸与乙醇酸单元比例越接近5050，PLGA的降解速率越快，被包裹药物的释放速率也越快8，但是，通过介孔二氧化硅包覆后，乳酸单元和乙醇酸单元质量比为5050的PLGA微球对罗丹明B的释放周期可以长达4个月9。羧基封端的PLGA通过氢键与药物分子相互作用，增大对药物的载药量，同时延长药物释放时间；与之形成对比，醚基封端或者分子量大的PLGA易于与药物分子的疏水基团相互作用，影响药物的释放时间，两种情况对应两种药物释放机 理10。实际应用中，通过调整上述参数可以满足不同领域的应用需求。PLGA微球是指直径介于11 000 m的PLGA球形自聚集体，可以提供良好的药物缓释/控释效果和药物靶向传递性11，可以通过微乳液法、微流控法、喷雾干燥法、溶剂蒸发法和高分子聚合法等方法制 得12,13。美国食品和药物管理局（FDA）已经批准19种基于PLGA微球载药体系，但是，我国的PLGA微球载药尚处于理论研究阶段。本文在前期研究的基础上5，合成了分子量一定的PLGA，并通过NMR和凝胶色谱法对其分子结构、分子量和分子量分布进行表征；通过静电喷雾法制备PLGA载药微球，表征了微球的形貌、粒径和粒径分布，并对曲安奈德药物的释放行为进行了研究，计算了PLGA微球曲安奈德的载药量和包封率。以期为PLGA载药微球的制备和拓展曲安奈德在过敏性皮炎、神经性皮炎和慢性湿疹等皮肤疾病治疗提供基础数据，为功能性护肤品的开发提供参考。1 实验部分1.1 主要原料、试剂与仪器乳酸（LA，纯度大于98%）、乙醇酸（GA，纯度大于98%）、葡萄糖（生物试剂级）、三氯甲烷（纯度大于99.5%）、三氟甲磺酸（纯度大于98%），上海阿拉丁生化科技股份有限公司。所有试剂用前均未经过进聚乙丙交酯微球的制备及对曲安奈德的包载与释放研究袁文博 1，张华彤 2，乔从德 2，王 领 2，栾明明 2，杨效登 2,*（1.山东谷雨春生物科技有限公司 工程技术中心，山东 济南 250098；2.齐鲁工业大学（山东省科学院）山东省轻工助剂重点实验室，山东 济南 250353）摘要：曲安奈德具有药性好、药效强、作用时间持久的优点，但是，油溶性使其应用不方便。聚乙丙交酯（Poly（lactic-co-glycolic acid），PLGA）具有优良的生物相容性和生物降解性，可做成微球负载曲安奈德。本文以三氟甲磺酸为催化剂、葡萄糖为引发剂制备了聚乙丙交酯，并以核磁共振波谱和凝胶色谱法表征了聚乙丙交酯的分子结构、分子量和分子量分布。结果显示，所得聚乙丙交酯中乳酸和乙醇酸链段的质量比为5050，羧基封端，分子量介于36 00041 000，分子量分布窄，分散性指数约为1.60。以高压静电喷雾法制得粒径小于20 m（d2092%）的微球，该微球对曲安奈德亲脂性药物的载药量大于44%，能实现长达120 h的释放周期，而且在药物释放前期释放速率和时间呈现线性关系。文章为PLGA载药微球的制备和拓展曲安奈德在过敏性皮炎、神经性皮炎和慢性湿疹等皮肤疾病治疗提供基础数据，为功能性护肤品的开发提供参考。关键词：曲安奈德；聚乙丙交酯；微球；载药中图分类号：TQ658 文献标识码：A 文章编号：2097-2806（2023）11-1266-071268第 53 卷开发与应用日 用 化 学 工 业（中英文）一步处理。Bruker Advance 400核磁共振波谱仪，瑞士布鲁克公司；Axio Scope.A1光学显微镜，德国蔡司集团；LC-20A高效液相色谱仪、GL inertsil ODS-3色谱柱（C18，5 m，200 mm4.6 mm）、SPD-M20A二极管阵列检测器、RID-10A凝胶渗透色谱仪，岛津仪器有限公司。1.2 PLGA的合成向容积为100 mL的三口烧瓶中加入50 mL三氯甲烷，称取乳酸15 g和乙醇酸10 g于该三口烧瓶中。向三口烧瓶中加入0.050.10 mL三氟甲磺酸和0.10.2 g葡萄糖分别作为聚合反应的催化剂和引发剂。将三口烧瓶置于40 的油浴中，在氮气氛围中回流4 h后降温，终止反应并用甲醇沉淀粗产品。将粗产品置于40 的真空干燥箱中干燥24 h，然后，在索氏提取器中用乙酸乙酯抽提24 h。经40 真空干燥24 h即得PLGA精品。聚合反应过程如图1所示。将精品PLGA装于自封袋中，自封袋置于干燥器中备用。1.3 PLGA分子结构表征将约0.05 g精品PLGA溶于1.0 mL三氯甲烷，配成质量浓度约为5 g/L的溶液，通过核磁共振波谱仪测得其分子中1H的核磁振动波谱，由乳酸（-CH3，1.48）和乙醇酸（-CH2-，5.05）的特征波谱峰积分面积求得乳酸和乙醇酸单元的比值，如式（1）所示。（1）1.4 分子量和分子量分布35 下，以四氢呋喃（THF）为流动相，流速为1.0 mL/min，用凝胶渗透色谱法测定PLGA的分子量和分子量分布。所有样品经过直径为0.22 m的过滤器过滤后方可注入检测器。本实验以分子量为1 300，3 350，13 000，25 000和50 000的单分散聚苯乙烯为标准品，制作标准工作曲线。1.5 PLGA微球的制备与表征采用高压静电喷雾法制备PLGA微球，喷丝不锈钢针头内径为0.4 mm，针头与接收桶间距15 cm，控制电压为1020 kV，溶液推进速度为0.55.0 mL/h，温度为室温。样品于80 的真空干燥箱中干燥24 h。电喷溶液为不同分子量和分子量分布的PLGA/三氯甲烷溶液，质量浓度为510 g/L。以光学显微镜观察PLGA微球的形貌，用测微尺测量微球的直径，统计数量不少于200个，计算微球5.05/21.48/3mGAmLA=的平均粒径和粒径分布。1.6 PLGA载药微球的制备将1.0 g曲安奈德和2.0 g PLGA溶于20 g三氯甲烷，配制曲安奈德和PLGA质量浓度分别为5和10 g/L的溶液。用高压静电喷雾法制备PLGA载药微球，控制电压为12 kV，溶液推进速度为2.0 mL/h，其他操作条件同“1.5”部分。1.7 PLGA微球载药量的测定参考已有方法6，称取0.5 g干燥的载药微球（记为m1+m2），将100 mL乙醇分3份冲洗微球表面的曲安奈德，将3份乙醇溶液混合，摇匀后通过高效液相色谱仪测量被洗掉的曲安奈德的量（记为m3）。按式（2）和式（3）分别计算PLGA微球的载药量（DLC）和包封率（DEE）。（2）（3）式中，m1为曲安奈德的初始质量，m2为PLGA的质量，m3为被乙醇洗掉的曲安奈德的质量，单位均为g。高效液相色谱仪用GL inertsil ODS-3色谱柱（C18，5 m，200 mm4.6 mm），以磷酸缓冲液（pH=7.2）/乙腈（体积比为6535）为流动相，进样流速为0.5 mL/min，单次进样量为20 L，进样前用直径为m2m1-m3DLC100%=m1m1-m3DEE100%=图1 三氟甲磺酸催化、葡萄糖引发乳酸和乙醇酸聚合制备PLGA示意图Fig.1 Sy nthetic route of PLGA by polymerization of lactic acid and glycolic acid using trifluoromethanesulfonic acid as catalyst and glucose as initiatorHOOOOHHOOH+OHTFMS,Glu40,4 hOHH3CCOCOCCCH3OOmnOmnHH21269第 11 期开发与应用袁文博，等：聚乙丙交酯微球的制备及对曲安奈德的包载与释放研究 0.22 m的过滤器过滤溶液，采用二极管阵列检测器，在35、波长254 nm处检测。检测前，配制同浓度的曲安奈德/甲醇溶液，制作曲安奈德工作曲线。1.8 载药微球药物释放参考文献7中的方法，用透析法研究曲安奈德的体外释放行为，取0.5 g用100 mL乙醇分3次冲洗过、干燥的载药微球转移到截留分子量为8 000 14 000的透析袋内，将透析袋密封，并放入盛有50 mL pH=7.2的磷酸盐缓冲溶液的具塞三角烧瓶中。将三角烧瓶置于（370.5）的恒温摇床，摇床振摇速度为100 r/min。摇床启动时开始计时，每隔一定时间取样2.0 mL，同时补充等体积的缓冲溶液。用直径为0.22 m的过滤器过滤后通过高效液相色谱仪测定曲安奈德的含量。平行测试3次，取平均值绘制曲安奈德的释放曲线。用乙醇冲洗掉吸附在PLGA微球表面的曲安奈德，对包载在微球内部的曲安奈德没有 影响。1.9 统计分析为了保证实验数据的准确性，每组实验最少平行测定3次，确保数据的显著性差异P92%）的PLGA微图4 不同分子量的PLGA对曲安奈德的包封率、载药量（A）和释放行为（B）Fig.4 Drug loading capacity,drug encapsulation（A）,and drug release behavior（B）of PLGA with different molecular weights020406080100PLGA41PLGA36PLGA38?/%PLGA?A020406080100?/%20406080100120020406080100?/%?/hB PLGA36 PLGA38 PLGA41 1272第 53 卷开发与应用日 用 化 学 工 业（中英文）球，该微球对油溶性药物曲安奈德有良好的包载率和持久释放性，包载率大于44%，释放周期长达120 h。参考文献：1 Jin S,Xia X,Huang J,et al.Recent advances in PLGA-based biomaterials for bone tissue regeneration J.Acta Biomaterialia,2021,127:56-79.2 Su Y,Zhang B,Sun R,et al.PLGA-based biodegradable microspheres in drug delivery:recent advances in research and application J.Drug Delivery,2021,28（1）:1397-1418.3 Chen X,Chen J,Li B,et 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