CN113694067B - 一种共载异烟肼和利福平的微球缓释制剂及其制备方法 - Google Patents
一种共载异烟肼和利福平的微球缓释制剂及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种共载异烟肼和利福平的微球缓释制剂及其制备方法,运用静电流体喷涂技术将异烟肼和利福平两种抗结核药物共同包载于PLGA微球中,微球平均粒径满足肺部给药有效粒径,包封率高。15天的体外药物释放实验表明微球中药物的累积释药率在40%~60%之间,达到了缓释的技术要求。本发明可有效提高结核药物在肺部的局部药物浓度,减少给药次数、降低毒副反应和提高药物的经济效益比。
Description
技术领域
本发明提供了一种能够将水溶性抗结核药物和脂溶性抗结核药物共载于同一微球的PLGA微球制剂及其制备方法,具体为一种共载异烟肼和利福平的微球缓释制剂及其制备方法。
背景技术
肺结核多为飞沫传播,结核菌被肺泡巨噬细胞吞噬后,引起组织干酪样坏死并液化,引起肺实质病变,如果淋巴结干酪样坏死破入支气管,病灶部位会有炎性渗出、干酪样坏死、肉芽肿等组织病理学改变,导致患者出现管腔狭窄、管腔闭塞,导致雾化吸入的治疗效果不佳。而肺部局部血运差,也会导致通过静脉给药的方式抗结核药物的肺靶向浓度较低。
异烟肼和利福平均为临床治疗肺结核的一线药物,也是气管镜介入治疗主要给药品种。单独应用时,结核菌易产生耐药性,故临床上将利福平和异烟肼联合应用后能够显著降低耐药性,增强药效。目前支气管镜下局部给药技术较为成熟,操作更为安全,注入药物剂量准确可控,并且经气管镜可以直视患者病灶部位的引流支气管,抗结核药物经导管直接注入病灶局部,对于支气管结核、耐多药肺结核及空洞型肺结核的靶向性更强,可使病灶局部达到有效的药物浓度,同时对血药浓度影响较小,从而避免相关合并症的发生几率。现阶段临床上应用较多的是先将抗结核药物制成注射剂或混悬剂,再通过介入技术直接注入肺结核病灶部位,但这种制剂肺部滞留性不佳,需频繁给药,患者的顺应性差。
肺部给药时,药物粒子大小影响药物到达的部位。微粒的沉积受到重力沉降、惯性嵌入和布朗运动三种作用的影响,大于10 μm的粒子沉积在上呼吸道中并很快被咳嗽、吞咽及纤毛运动清除,2~10 μm的粒子可到达支气管和细支气管,3~5 μm的粒子则主要在下呼吸道沉积,2~3 μm的粒子可到达肺泡,太小的粒子容易通过呼气排出,不能停留在呼吸道。所以0.5~5 μm的微粒大小最为适宜。而无论是从结核菌分支杆菌引起支气管感染的病因机制还是耐药结核菌的产生原理,肺靶向输送的宿主是巨噬细胞,直径1~3 μm的粒子可通过吞噬作用被肺泡巨噬细胞有效吸收。由此可见将载有抗结核药物的缓释制剂其粒径控制在1~3 μm范围内是维持肺泡巨噬细胞高药物水平的理想方法。
微球(microsphere)是微米级的固体球形颗粒,通常由天然或人工合成的聚合物制备而成缓释制剂,其粒径范围在1~250 μm之间,属于微米级,可满足肺靶向介入给药对药物微粒的粒径要求。PLGA微球的制备方法有乳化溶剂挥发法、盐析法、相分离法、膜乳化法、喷雾干燥法、静电流体喷涂技术和超临界流体技术。对于同时包载两种药物的微球制剂,其最为常用的方法是乳化溶剂挥发法,但其往往存在产量低、水溶性药物包封率低、微粒易粘连聚集以及有机溶剂残留等问题。静电流体喷涂技术是一种简便易行的制备核壳结构聚合物微球的方法,相比于传统方法,其理论上具有以下优势:(1)超微米颗粒尺寸;(2)尺寸分布较窄;(3)单分散性;(4)操作简单,可重复性好;(5)利于包裹;(6)高产率,在医药应用领域有较广的应用前景。
发明内容
本发明提供了一种共载异烟肼和利福平的微球缓释制剂及其制备方法,运用静电流体喷涂技术将异烟肼和利福平两种抗结核药物共同包载于PLGA微球中,微球平均粒径满足肺部给药有效粒径,故可有效提高结核药物在肺部的局部药物浓度,减少给药次数、降低毒副反应和提高药物的经济效益比。
本发明解决其技术问题所采用的技术方案是:一种共载异烟肼和利福平的微球缓释制剂的制备方法,步骤如下:
(1)微球制备过程需在避光下进行,以磷脂和胆固醇为乳化剂,聚乙烯醇(PVA)为助乳剂,聚(乳酸-羟基乙酸)共聚物(PLGA)为载体材料,乳酸-羟基乙酸比例为50:50或者75:25,重均分子量为3.7-5.2万;
(2)水相是将精密称量的异烟肼10~30 mg加入到0.5 mLPVA水溶液中,40℃水浴10 min;
(3)油相是将精密称量的利福平15~40 mg、PLGA 250~750 mg、磷脂120~240 mg和胆固醇20~40 mg溶于5 mL二氯甲烷和无水乙醇的混合溶液中,水浴超声5 min促进其溶解;
(4)将水相加入到油相中,油水体积比在1:8~1:12之间,经手持式均质机混合、均质、乳化2 min后,抽入5 mL注射器内;
(5)将注射器放置于静电纺丝机推注泵上,针头选择21#,在针头与接收装置之间建立高压电场;设定好适宜的电压、推注速度和接收距离后,运用静电喷涂技术制备共载双药的PLGA微球制剂,接收装置为铝箔纸,室温真空干燥12 h。
其中,步骤(2)PVA浓度在0.5%~2%之间;优选浓度为1.4%。
步骤(3)PLGA的加入量在250~750 mg之间,优选的加入量为350mg,乳酸与羟基乙酸的优选比例为75:25;磷脂和胆固醇的比例在6:1~9:1之间,优选比例为6:1。
步骤(4)油水体积比在1:8~1:12之间,优选比例为1:10;均质机转速在8000~12000 rpm,优选转速为10000 rpm。
步骤(5)静电纺丝机上电压在8~12kV,优选电压为8kV;接收距离在10~20cm之间,优选距离为10~15 cm;推注速度在0.1~0.3 mm/min,优选速度为0.2 mm/min。
本发明的有益效果是:本发明提供了可以共载水溶性药物异烟肼和脂溶性药物利福平的PLGA微球制剂,该制剂解决了水溶性药物包封率低的问题;制备的微球制剂可以满足肺靶向的较窄粒径范围要求1~3 μm,其平均粒径在1.85~2.36 μm。
附图说明
图1为微球制备过程中用载玻片在光学显微镜下观察的微球表面形貌。
图2为透射电镜下观察微球表面形貌。
图3为本发明异烟肼体外药物释放曲线。
图4为本发明利福平体外药物释放曲线。
具体实施方式
下面通过实验例和实施例来进一步说明本发明。应该正确理解的是:本发明的实验例和实施例仅仅用于说明本发明而给出,但不构成对本发明保护范围的限制。任何在本发明的方法前提下进行的简单改进均属于本发明要求保护范围。
一、仪器与试剂
1.1仪器:LC1000 型高效液相色谱仪(山东鲁南瑞虹化工仪器有限公司),FA3004G型电子分析天平(常州万泰天平仪器有限公司),KS-2500 型超声波清洗机(宁波科生仪器厂),SHZ-D(Ⅲ)循环水式真空泵(巩义市予华仪器有限责任公司),DF101-S集热式恒温磁力搅拌器(巩义市予华仪器有限责任公司),PHS-3C数显台式酸度计(上海浦春计量仪器有限公司),Evolution 300紫外可见分光光度计(赛默飞世尔科技(中国)有限公司),TG16G高速离心机(河南北弘实业有限公司),SS-2535DC静点纺丝设备(北京永康乐业科技发展有限公司),MT-30K 手持式均质机(杭州米欧仪器有限公司),NANO-ZS90纳米粒度仪(马尔文公司),RCZ-8B溶出试验仪(上海鲁硕实业有限公司)
1.2试剂:甲醇(色谱纯)、乙腈(色谱纯)、磷酸、二氯甲烷、无水乙醇(天津市科密欧化学试剂有限公司),磷酸氢二钠(天津市凯通化学试剂有限公司),磷酸盐缓冲液(0.01MpH7.2~7.4北京索莱宝科技有限公司),聚乙烯醇(PVA124)(国药集团化学试剂有限公司)
1.3材料:利福平对照品(批号F0201A,大连美仑生物科技有限公司),异烟肼对照品(批号D1208A,大连美仑生物科技有限公司),PLGA(75:25,MW=42000,批号20200623017,50:50,MW=23000,批号 20190428278 济南岱罡生物工程有限公司),磷脂(Lipoid S 100德国),胆固醇(BWJ4217-2016 北京世纪奥科生物技术有限公司),BIOSHARP透析袋(MW14000)(北京兰杰柯科技有限公司)。
二、载药量和包封率
精密称取3份5 mg微球,在避光条件下用10 mL流动相溶解4 h,8000 rpm高速离心4 min取上清液0.22 μm微孔滤膜过滤,用HPLC检测药物峰面积,并将其代入标准曲线公式中计算出微球中药物的含量。按照以下公式计算出微球的载药量(Drug Loading DL)和包封率(Encapsulation Efficiency EE)。
载药量(DL,%)=微球中所含药物重量/微球的总重量×100%
包封率(EE,%)=微球中实际药物量/微球中理论投药量×100%。
实验例1 异烟肼、利福平体外分析方法实验
利福平、异烟肼紫外分光光度法:经过全波长扫描,利福平在238 nm、254 nm和334nm 波长处有最大吸收,在298 nm波长处有最小吸收。异烟肼在262 nm波长处有最大吸收。综合分析将262 nm 作为利福平、异烟肼分析方法的检测波长。
利福平、异烟肼HPLC检测方法学:色谱柱:Besil-C18(5 μm,4.6×250 mm),紫外分光光度计检测波长:262 nm,进样量:20 µL,柱温:25℃,流速:1.0 mL/min,流动相A:磷酸氢二钠溶液(0.02 mol/L)用磷酸调节pH值至4.0,流动相B:甲醇:乙腈(体积比为1:2)。前10min流动相A和B体积比恒定在95:5;10~20 min 流动相A由95%降到35%,流动相B由5%上升到65%;20~30 min 流动相A和B体积比恒定在35:65;30~35 min 流动相A和B体积比恒定在95:5。
实验例2 PLGA微球制备工艺的处方优化
1. 单因素考察:根据制备方法,选择磷脂和胆固醇比例、磷脂胆固醇加入量、转速、水相PVA浓度、油相PLGA浓度、油水体积比、电压、接收距离、推注速度、投药量、投药比例及油相中二氯甲烷与无水乙醇的比例作为微球质量控制的影响因素,以平均粒径和包封率为评价指标,进行单因素实验。由实验结果可知,磷脂胆固醇的加入量在1.4%~5.6%之间、油水体积比在1:8~1:10之间、接收距离在10~15 cm之间及投药比例对药物的包封率和微球粒径影响不大。而二氯甲烷和无水乙醇体积比在4:1和3:2时,形成带有纺丝加微球这种串珠结构的制剂。
2. Box-Behnken设计-响应面法优化处方:根据单因素试验结果,分别以利福平和异烟肼的载药量和包封率为评价指标,磷脂/胆固醇比例、PVA浓度、PLGA浓度、推注速度、投药量为因素,每个因素设置低、中、高3个水平,进行五因素三水平响应面分析试验。最终得到的最优工艺:磷脂/胆固醇比例(6:1)、PVA浓度(1.40%)、PLGA浓度(7.00%)、推注速度(0.20 mm/min)、投药量(52.64 mg)、INH载药量(5.98%)、RFP载药量(6.11%)、INH包封率(71.78%)、RFP包封率(84.73%)。
3. 工艺验证
根据响应面试验筛选出的最优条件,制备5批微球制剂,结果见下表,其制备微球的利福平、异烟肼载药量和包封率实测值与预测值RSD<5%,可以作为异烟肼-利福平-PLGA微球制剂的最优处方。
三、粒径分布及微球表面形貌
1. 粒径分布及平均粒径:将最优处方制备出的微球溶于正己烷中冰浴超声30 s后置于NANO-ZS90纳米粒度仪检测池中,检测粒径为2.06±0.86 μm,PDI:0.38±0.06,粒径分布较为均匀,Zeta电位:-17.80±5.05 mV,溶液稳定。
2. 微球表面形貌:微球制备过程中用载玻片在光学显微镜下观察,如图1。将最优处方制备的微球混悬液滴至铜网2.0%磷钨酸负染,自然风干后于Hitachi7700型透射电镜下观察微球呈现圆球形,球形良好,大小分布较均匀,如图2。
实施例1
共载异烟肼和利福平的肺靶向PLGA微球制剂,制备方法为:
(1)水相是精密称量异烟肼26mg加入到0.5 mL1.4%PVA水溶液中,40℃水浴10min;
(2)油相是将精密称量的利福平26mg、7%PLGA(乳酸-羟基乙酸75:25)、磷脂60 mg和胆固醇10 mg溶于5 mL二氯甲烷和无水乙醇的混合溶液中,水浴超声5 min促进其溶解;
(3)将水相加入到油相中,手持式均质机1000 rpm混合、均质、乳化2 min后,抽入5mL注射器内;
(4)将注射器放置于静电纺丝机推注泵上,针头选择21#,在针头与接收装置之间建立高压电场。设定好电压8 kV、推注速度0.20 mm/min和接收距离10 cm后,运用静电喷涂技术制备共载双药的PLGA微球制剂,接收装置为铝箔纸,室温真空干燥12 h。
实施例2
共载异烟肼和利福平的肺靶向PLGA微球制剂,制备方法为:
(1)水相是精密称量异烟肼30mg加入到0.5 mL1.4%PVA水溶液中,40℃水浴10min;
(2)油相是将精密称量的利福平22mg、7%PLGA(75:25)、磷脂60 mg和胆固醇10 mg溶于5 mL二氯甲烷和无水乙醇的混合溶液中,水浴超声5 min促进其溶解;
(3)将水相加入到油相中,手持式均质机1000 rpm混合、均质、乳化2 min后,抽入5mL注射器内;
(4)将注射器放置于静电纺丝机推注泵上,针头选择21#,在针头与接收装置之间建立高压电场。设定好电压8 kV、推注速度0.20 mm/min和接收距离10 cm后,运用静电喷涂技术制备共载双药的PLGA微球制剂,接收装置为铝箔纸,室温真空干燥12 h。
实施例3
共载异烟肼和利福平的肺靶向PLGA微球制剂,制备方法为:
(1)水相是精密称量异烟肼30mg加入到0.5 mL1.4%PVA水溶液中,40℃水浴10min;
(2)油相是将精密称量的利福平22mg、7%PLGA(50:50)、磷脂60 mg和胆固醇10 mg溶于5 mL二氯甲烷和无水乙醇的混合溶液中,水浴超声5 min促进其溶解;
(3)将水相加入到油相中,手持式均质机1000 rpm混合、均质、乳化2 min后,抽入5mL注射器内;
(4)将注射器放置于静电纺丝机推注泵上,针头选择21#,在针头与接收装置之间建立高压电场。设定好电压8 kV、推注速度0.20 mm/min和接收距离10 cm后,运用静电喷涂技术制备共载双药的PLGA微球制剂,接收装置为铝箔纸,室温真空干燥12 h。
实验例3 体外药物释放的测定
上述实施例分别加入3 mL pH7.2~7.4PBS释放介质溶液,再装入预先处理好的透析袋中,两端扎紧后放入全自动溶出仪的转篮中,以pH7.2~7.4PBS溶液100 mL为释放介质,设定温度为(37±0.5)℃,转速为100 r/min,取样时间分别设定为0.5 h,1 h,2 h,4 h,8 h,12 h,24 h,2 d,3 d,5 d,7 d,10 d,15 d,HPLC法测定释放介质中异烟肼和利福平的药物浓度,计算累积药物释放百分数,绘制体外释放曲线,结果如图3和图4。
结论:本发明首次通过静电流体喷涂技术将利福平和异烟肼同时包载于PLGA微球中,其平均粒径满足肺靶向给药的粒径范围1~3 μm,其水溶性药物异烟肼包封率在71%以上,脂溶性药物利福平包封率在83%以上。扫描电镜下观察其为圆球状,表面光滑,大小分布均匀。体外药物释放实验表明24h两种药物均不存在突释现象,15d异烟肼的累积释药率为61.47±0.42%、利福平的累积释药率为39.23±0.74%。
Claims (8)
1.一种共载异烟肼和利福平的微球缓释制剂的制备方法,其特征在于:其步骤如下:
(1)微球制备过程需在避光下进行,以磷脂和胆固醇为乳化剂,聚乙烯醇PVA为助乳剂,聚乳酸-羟基乙酸共聚物PLGA为载体材料,乳酸-羟基乙酸比例为50:50或者75:25,重均分子量为2.3-5.2万;
(2)水相是将精密称量的异烟肼10~30 mg加入到0.5 mLPVA水溶液中,40℃水浴10min;
(3)油相是将精密称量的利福平15~40 mg、PLGA 250~750 mg、磷脂120~240 mg和胆固醇20~40 mg,磷脂和胆固醇的比例为6:1,溶于5 mL二氯甲烷和无水乙醇的混合溶液中,水浴超声5 min促进其溶解;
(4)将水相加入到油相中,油水体积比在1:8~1:12之间,经手持式均质机混合、均质、乳化2 min后,抽入5 mL注射器内;
(5)将注射器放置于静电纺丝机推注泵上,针头选择21#,在针头与接收装置之间建立高压电场;电压在8~12kV之间,接收距离在10~20cm之间,推注速度在0.1~0.3 mm/min之间,运用静电喷涂技术制备共载双药的PLGA微球制剂,接收装置为铝箔纸,室温真空干燥12h;
制备的微球制剂可以满足肺靶向的较窄粒径范围要求1~3 μm,其平均粒径在1.85~2.36 μm。
2.根据权利要求1所述的一种共载异烟肼和利福平的微球缓释制剂的制备方法,其特征在于:其中步骤(2)PVA浓度在0.5%~2%之间。
3.根据权利要求1所述的一种共载异烟肼和利福平的微球缓释制剂的制备方法,其特征在于:其中步骤(3)PLGA的加入量为350mg,乳酸与羟基乙酸的比例为75:25。
4.根据权利要求1所述的一种共载异烟肼和利福平的微球缓释制剂的制备方法,其特征在于:其中步骤(4)油水体积比为1:10。
5.根据权利要求1所述的一种共载异烟肼和利福平的微球缓释制剂的制备方法,其特征在于:其中均质机转速在8000~12000 rpm。
6.根据权利要求1所述的一种共载异烟肼和利福平的微球缓释制剂的制备方法,其特征在于:其中步骤(5)静电纺丝机上电压为8kV;接收距离为10~15 cm;推注速度为0.2 mm/min。
7.根据权利要求1所述的一种共载异烟肼和利福平的微球缓释制剂的制备方法,其特征在于:其中步骤(2)PVA浓度为1.4%。
8.根据权利要求1所述的一种共载异烟肼和利福平的微球缓释制剂的制备方法,其特征在于:其中均质机转速为10000 rpm。
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