CN114848591A - 一种精氨酸多囊脂质体的制备方法 - Google Patents
一种精氨酸多囊脂质体的制备方法 Download PDFInfo
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- CN114848591A CN114848591A CN202210494016.7A CN202210494016A CN114848591A CN 114848591 A CN114848591 A CN 114848591A CN 202210494016 A CN202210494016 A CN 202210494016A CN 114848591 A CN114848591 A CN 114848591A
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- arginine
- lipid
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Abstract
本发明提供一种精氨酸多囊脂质体的制备方法，将精氨酸水溶液作为第一水相，溶有脂质成分的有机相作为油相，含有乳化稳定剂的等渗溶液作为第二水相，通过将第一水相和油相混合形成的W/O乳剂加入到第二水相中混合形成W/O/W复乳，再将所得W/O/W复乳稀释后，去除有机溶剂，低速离心收集得到。本发明利用生物安全性高、免疫原性弱、容易获得且生产成本低的脂质作为主要的载体材料，实现了对高浓度精氨酸水溶液的高效包封。该载体具备优良的载药和长效缓释能力，其制备方便、成本低廉、性质稳定，利于储存，局部注射后可长时间在病灶部位补充精氨酸，具有良好的生产和应用前景。
Description
技术领域
本发明属于药物制剂技术领域，涉及一种精氨酸多囊脂质体的制备方法，是一种具备长效缓释特征的精氨酸多囊脂质体的制备方法。
背景技术
L-精氨酸(L-arginine,L-arg)是一种半必需氨基酸，参与许多生化过程。它是不同酶促反应的底物，在体内通过三种主要的已知途径代谢：(1)精氨酸酶将L-精氨酸代谢为L-鸟氨酸；(2)L-精氨酸脱羧酶将L-精氨酸代谢为胍基丁胺；(3)一氧化氮(NO)合酶(NOS)使用L-精氨酸形成NO和瓜氨酸。而通过下游进一步反应，可以合成多胺、多肽、氨基酸和嘧啶等生命活动的必需物质。
因为其在生命活动中的重要代谢合成作用，L-精氨酸的可用性可以影响到许多生理过程，例如免疫反应、血管生成、细胞周期、代谢调控以及伤口修复等。研究证明，当L-精氨酸不足时，T细胞的增殖会陷入细胞周期阻滞(Blood.2007Feb 15；109(4):1568-73)。缺乏L-精氨酸的环境还会引起T细胞受体TCRζ链的下调，影响活化信号传导(JPEN JParenter Enteral Nutr.Jan-Feb 2001；25(1):23-9)，并减少相关免疫因子的分泌，严重影响T细胞的正常免疫功能。而在补充L-精氨酸后，不仅上述抑制现象会逆转，同时会引起活化的CD8+T细胞适应性整体改善和中央记忆细胞生成增加，表现为更强的免疫能力和更持久的免疫记忆(Cell.2016Oct 20；167(3):829-842)。大量研究表明，补充L-精氨酸能够显著增强T细胞免疫反应，继而提升对癌症、细菌或病毒感染等疾病的治疗效果(ACS ApplMater Interfaces.2020Sep 16；12(37):41127-41137；J Clin Immunol.2021Apr；41(3):515-525)。M2型巨噬细胞则高表达精氨酸酶1，将精氨酸分解代谢为鸟氨酸等产物，用于促进伤口愈合(Am J Physiol.1988Apr；254(4Pt 1):E459-67)。此外，对于表达诱导型一氧化氮合酶(iNOS)的M1型巨噬细胞而言，能正常的合成NO是其发挥抗菌和细胞毒性功能的前提，而L-精氨酸是其合成NO的必需底物(J Immunol.2003Nov 1；171(9):4561-8)。另外两种常见一氧化氮合酶亚型，分别表达于内皮细胞和神经元细胞，充足的L-精氨酸是这两种细胞正常功能的营养基础。已经证明，补充L-精氨酸在新生血管生成、动脉粥样硬化治疗等血管疾病治疗方面具有良好的应用前景(J Clin Invest.1998Jun 1；101(11):2567-78；NatClin Pract Cardiovasc Med.2007 May；4(5):274-83)。
综上所述，补充L-精氨酸对多种疾病的治疗具有积极的促进作用。但是目前的L-精氨酸补充多依赖口服途径，该途径对剂量要求较大，易导致胃肠道反应。此外，需要L-精氨酸补充的部位，其往往是局部的病灶，依赖血液循环供给L-精氨酸效率低下，不能实现有效的病灶部位富L-精氨酸化，对治疗的促进作用极低(Nature.2021Oct；598(7882):662-666)。因此亟需一种可以局部长效补充L-精氨酸的方法。
发明内容
本发明的目的是提供一种精氨酸多囊脂质体的制备方法，通过以下方案实现：
(1)溶液的制备
以精氨酸(或其药学上可接受的盐)的水溶液作为第一水相，以两亲性脂质、胆固醇和中性脂质的疏水有机溶液作为油相，以含有乳化稳定剂的等渗水溶液作为第二水相；
(2)将第一水相与油相混合制备油包水型(W/O)乳剂；
(3)将W/O乳剂与一定体积的第二水相混合制备水包油包水型(W/O/W)复乳；
(4)将W/O/W复乳再次与一定体积的第二水相或等渗水溶液混合稀释，水浴去除有机溶剂后低速离心，得到精氨酸多囊脂质体悬液；
(5)将所得精氨酸多囊脂质体悬液经低速离心后收集，4℃储存。
步骤(1)中精氨酸优选L-精氨酸或其药学上可接受的盐，精氨酸浓度为10～100mg/mL。
步骤(1)中，疏水有机溶液为易挥发疏水溶剂，选用醚类、烃类和卤代烃类中的一种或几种的混合物，如***、二氯甲烷和三氯甲烷。两亲性脂质选用两性离子型脂质、阴离子脂质和阳离子脂质中的一种或几种的混合物，如大豆磷脂、蛋黄卵磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰甘油、磷脂酰肌醇或二酰基二甲胺丙烷。其中两亲性脂质与胆固醇的质量比为1～10:1。
步骤(1)中，中性脂质选用甘油三酯、甘油二酯、甾醇、固醇和生育酚中的一种或几种的混合物，如三油酸甘油酯、三辛酸甘油酯、三癸酸甘油酯，维生素E或α-生育酚等。中性脂质占总脂质质量的1％～50％。
步骤(1)中，乳化稳定剂为O/W型乳化剂或助乳化剂中的单独一种或混合物，如十二烷基硫酸钠、吐温80、聚乙烯醇、赖氨酸等。
步骤(1)中，等渗水溶液的渗透压由氯化钠、蔗糖、葡萄糖等盐或糖类物质中的至少一种调节。
步骤(2)中，混合方法包括探头超声、搅拌、涡旋和均质中的任何一种。且混合过程中第一水相与油相的体积比为1:1～6。
步骤(3)中，W/O乳剂与第二水相的体积比为1:1～20。
步骤(4)中，W/O/W复乳的稀释体积比为1:2～50。去除有机溶剂的方法包括搅拌蒸发、旋转蒸发或氮吹法中的任一种。所述水浴的温度为20℃～60℃。太低不利于去除有机溶剂，太高不利于多囊脂质体稳定。
本发明构建了一种精氨酸多囊脂质体，利用生物安全性高、免疫原性弱、容易获得且生产成本低的脂质作为主要的载体材料，实现了对高浓度精氨酸水溶液的高效包封。该载体具备优良的载药和长效缓释能力，其制备方便、成本低廉、性质稳定，局部注射后可长时间在病灶部位补充精氨酸，具有良好的生产和应用前景。
本发明的有益效果在于：
本发明提供了一种精氨酸多囊脂质体的制备方法，制备获得的精氨酸多囊脂质体悬液为外观呈乳白色、均匀的混悬液，粒径均一，没有膜黏连现象，对精氨酸包封率高，载药量大，具备良好的长效缓释特性、生物相容性和生物降解性，通过局部注射可实现在病灶区域持续的精氨酸补充。在4℃保存150天后渗漏率低于1％，稳定性高，利于储存。本发明方法设计合理，工艺简单，物料成本低，稳定可靠，具备良好的商业化生产应用前景。
附图说明
图1是精氨酸多囊脂质体粒径分布。
图2是精氨酸多囊脂质体光学显微镜下的形态(比例尺：20μm)。
图3是精氨酸多囊脂质体冷冻扫描电子显微镜下的形态(左：表面；右：切面)。
图4是精氨酸多囊脂质体体外释放曲线。
图5是精氨酸多囊脂质体体外释放过程形态变化(比例尺：50μm)。
图6是ICG标记的精氨酸多囊脂质体的体内释放荧光图(左)及定量曲线(右)。
图7是精氨酸多囊脂质体保存过程的形态变化(比例尺：50μm)。
图8是精氨酸多囊脂质体保存过程中的药物泄漏率曲线。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例和附图对本发明进行具体描述，有必要指出的是，以下实施例仅仅用于对本发明进行解释和说明，并不用于限定本发明。本领域技术人员根据上述发明内容所做出的一些非本质的改进和调整，仍属于本发明的保护范围。
实施例1一种精氨酸多囊脂质体的制备方法
精氨酸多囊脂质体的组成：
处方量的L-精氨酸溶于2mL纯水中，作为第一水相；处方量的大豆卵磷脂S100、胆固醇和α-生育酚溶于3mL三氯甲烷中，作为油相。将第一水相加入油相中，涡旋乳化10分钟，制得W/O乳剂。将含1％赖氨酸的等渗葡萄糖水溶液作第二水相，将制得的W/O乳剂加入到10mL第二水相中，低速搅拌制得W/O/W型复乳，向其中加入等渗葡萄糖水溶液30mL，25℃水浴搅拌5小时，挥发去除有机溶剂，制得乳白色均匀状精氨酸多囊脂质体悬液。所得悬液经低速离心富集精氨酸多囊脂质体，弃去上清并加入20mL的等渗葡萄糖水溶液洗涤，重复3次。洗涤完毕后，收集精氨酸多囊脂质体，于4℃储存。
实施例2一种精氨酸多囊脂质体的制备方法
精氨酸多囊脂质体的组成：
处方量的L-精氨酸盐酸盐溶于1mL纯水中，作为第一水相；处方量的蛋黄卵磷脂E80、胆固醇和维生素E溶于2mL二氯甲烷中，作为油相。将第一水相加入油相中，200W探头超声乳化4分钟，制得W/O乳剂。将含2％十二烷基硫酸钠的等渗氯化钠水溶液作第二水相，将制得的W/O乳剂加入到10mL第二水相中，低速搅拌制得W/O/W型复乳，向其中加入等渗氯化钠溶液120mL，35℃旋转蒸发1小时，去除有机溶剂，制得乳白色均匀状精氨酸多囊脂质体悬液。所得悬液经低速离心富集精氨酸多囊脂质体，弃去上清并加入30mL的等渗氯化钠溶液洗涤，重复3次。洗涤完毕后，收集精氨酸多囊脂质体，于4℃储存。
实施例3一种ICG标记的精氨酸多囊脂质体的制备方法
精氨酸多囊脂质体的组成：
处方量的L-精氨酸和ICG溶于1mL纯水中，作为第一水相；处方量的蛋黄卵磷脂E80、胆固醇和α-生育酚溶于2mL二氯甲烷中，作为油相。将第一水相加入油相中，200W探头超声乳化4分钟，制得W/O乳剂。将含2％十二烷基硫酸钠的等渗氯化钠水溶液作第二水相，将制得的W/O乳剂加入到10mL第二水相中，低速搅拌制得W/O/W型复乳，向其中加入等渗的氯化钠溶液120mL，35℃旋转蒸发1小时，去除有机溶剂，制得ICG标记的精氨酸多囊脂质体悬液。所得悬液经低速离心富集多囊脂质体，弃去上清并加入30mL的等渗氯化钠溶液洗涤，重复3次。洗涤完毕后，收集ICG标记的精氨酸多囊脂质体，于4℃储存。
实施例4精氨酸多囊脂质体的形态考察
本实施例在实施例2的基础上，取精氨酸多囊脂质体稀释于10mL的等渗氯化钠溶液中，测定平均粒径为21.74±4.37μm(图1)分别使用光学显微镜和冷冻扫描电子显微镜观察所制得的精氨酸多囊脂质体的形态。在光学显微镜下其形态一致，边缘圆整，脂质膜层没有黏连(图2)。通过冷冻扫描电子显微镜(Cryo-SEM)观察，精氨酸多囊脂质体表面光滑，其内部由排列紧密的微小囊泡构成(图3)。
实施例5精氨酸多囊脂质体的包封率测定
本实施例在实施例2的基础上，取精氨酸多囊脂质体，用甲醇溶解后，经高效液相色谱(HPLC)测定包封的精氨酸含量(Me)，以实施例2中的处方量作总精氨酸量(Mt)，通过公式En＝Me/Mt×100％计算精氨酸的包封率(En)。计算所制得的精氨酸多囊脂质体中精氨酸的包封率为79.73±6.44％。
所用HPLC条件为：
流动相：甲醇：0.01M戊烷磺酸钠＝(10：90)，1ml/min；检测波长：206nm；色谱柱：Agilent ZORBAX C18 column(4.6×250mm,5μm)。
实施例6精氨酸多囊脂质体的体外释放考察
本实施例在实施例2的基础上，取精氨酸多囊脂质体，稀释于pH为7.4或6.6的PBS溶液中，置于37℃恒温振荡箱中，于设定时间取样，在光学显微镜下观察其形态并拍照后，低速离心后弃去上清，将收集的多囊脂质体用甲醇溶解后，经实施例5中所述的HPLC法测定精氨酸含量，检测精氨酸多囊脂质体体外释放行为。释放曲线表明，37℃条件下，精氨酸多囊脂质体在pH为7.4或6.6的PBS溶液中均表现出了长达216h的缓释能力(图4)，在pH6.6条件下制剂的释放前期具有更强的突释效应，可能是因为磷脂膜两侧更高的质子浓度差造成。对其跟踪的形态学记录结果与释放曲线相符(图5)，可观察到在体外释放过程中，精氨酸多囊脂质体发生了表面溶蚀以及内部囊泡融合等结构变化，表现为大量游离小型囊泡和内部大囊泡的产生、多囊脂质体的融合和破碎以及脂质固体的析出，该过程在pH 7.4的环境下发生得更为缓慢。
实施例7精氨酸多囊脂质体的体内释放考察
本实施例在实施例2和实施例3的基础上，取ICG标记的精氨酸多囊脂质体，稀释于等渗氯化钠溶液中，制成多囊脂质体悬液；另配制相同ICG浓度的等渗氯化钠溶液。将6只成年雌性ICR小鼠随机分为2组(每组n＝3)，分别在背部皮下注射50μL的ICG标记的精氨酸多囊脂质体悬液(ICG&L-arg@MVL)或ICG的等渗氯化钠溶液(ICG@Saline)。通过小动物成像仪测定皮下ICG的荧光强度随时间的变化，考察精氨酸多囊脂质体在皮下的释放持续时间。ICG&L-arg@MVL组小鼠皮下荧光至第18天才基本消失，释放维持时间远远超过游离ICG@Saline组(图6)。说明所制备的精氨酸多囊脂质体具备良好的体内长效缓释作用。
实施例8精氨酸多囊脂质体的储存稳定性考察
本实施例在实施例2的基础上，取精氨酸多囊脂质体稀释于20mL的等渗氯化钠溶液中，置于4℃冰箱中，于设定时间取样，在光学显微镜下观察其形态并拍照后，低速离心后取上清，经实施例5中所述的HPLC法测定精氨酸含量，检测精氨酸多囊脂质体在保存过程中的药物泄露行为。形态学结果表明(图7)，在为期150天的4℃保存过程中，精氨酸多囊脂质体没有发生明显的破碎或者膜融合等现象，始终保持不黏连的规则球形。对各时间点所取上清液中精氨酸含量的定量结果表明(图8)，在保存过程中，精氨酸多囊脂质体中所包载的精氨酸泄漏率低于1％。以上结果均说明，所制备的精氨酸多囊脂质体在4℃保存条件下，稳定性好，药物泄漏率低，易于储存。
Claims (8)
1.一种精氨酸多囊脂质体的制备方法，其特征在于，通过以下步骤实现：
(1)溶液的制备：以精氨酸或其药学上可接受的盐的水溶液作为第一水相，以两亲性脂质、胆固醇和中性脂质的疏水有机溶液作为油相，以含有乳化稳定剂的等渗水溶液作为第二水相；
(2)将第一水相与油相混合制备油包水型(W/O)乳剂；
(3)将W/O乳剂与一定体积的第二水相混合制备水包油包水型(W/O/W)复乳；
(4)将W/O/W复乳再次与一定体积的第二水相或等渗水溶液混合稀释，水浴去除有机溶剂后低速离心，得到精氨酸多囊脂质体悬液；
(5)将所得精氨酸多囊脂质体悬液经低速离心后收集，4℃储存。
2.根据权利要求1所述的制备方法，其特征在于，步骤(1)所述精氨酸选用L-精氨酸或其药学上可接受的盐，精氨酸浓度为10～100mg/mL。
3.根据权利要求1所述的制备方法，其特征在于，步骤(1)中，疏水有机溶液为易挥发疏水溶剂，选用醚类、烃类或卤代烃类中的一种或几种的混合物；两亲性脂质选用两性离子型脂质、阴离子脂质和阳离子脂质中的一种或几种的混合物；中性脂质选用甘油三酯、甘油二酯、甾醇、固醇和生育酚中的一种或几种的混合物；乳化稳定剂为O/W型乳化剂或助乳化剂中的单独一种或混合物。
4.根据权利要求3所述的制备方法，其特征在于，易挥发疏水溶剂选用***、二氯甲烷和三氯甲烷；两亲性脂质选用大豆磷脂、蛋黄卵磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰甘油、磷脂酰肌醇或二酰基二甲胺丙烷；中性脂质选用三油酸甘油酯、三辛酸甘油酯、三癸酸甘油酯，维生素E或α-生育酚；乳化稳定剂选用十二烷基硫酸钠、吐温80、聚乙烯醇、赖氨酸；等渗水溶液的渗透压由氯化钠、蔗糖、葡萄糖等盐或糖类物质中的至少一种调节。
5.根据权利要求1所述的制备方法，其特征在于，步骤(1)中两亲性脂质与胆固醇的质量比为1～10:1，中性脂质占总脂质质量的1％～50％。
步骤(2)中，混合方法包括探头超声、搅拌、涡旋和均质中的任何一种。且混合过程中第一水相与油相的体积比为1:1～6。
6.根据权利要求1所述的制备方法，其特征在于，步骤(3)中，W/O乳剂与第二水相的体积比为1:1～20。
7.根据权利要求1所述的制备方法，其特征在于，步骤(4)中，W/O/W复乳的稀释体积比为1:2～50。
8.根据权利要求1所述的制备方法，其特征在于，步骤(4)去除有机溶剂的方法选用搅拌蒸发、旋转蒸发或氮吹法中的一种，所述水浴的温度为20℃～60℃。
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