CN114668772A - 负载两性霉素b仿生纳米系统的组合物、制备方法及应用 - Google Patents
负载两性霉素b仿生纳米系统的组合物、制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种负载两性霉素B仿生纳米系统的组合物、制备方法及应用；该组合物包括AmB、PLGA纳米颗粒、阴道上皮细胞膜；其中AmB封包在PLGA纳米颗粒中，封包的整体由阴道上皮细胞膜包裹；其制备方法为分离和纯化阴道上皮细胞的细胞膜；将AmB包封至PLGA纳米颗粒中；将阴道上皮细胞的细胞膜包裹到含有AmB的PLGA纳米颗粒表面；其应用为治疗白色念珠菌感染。本发明的方案能够克服现有技术中的制剂对于药物的靶向性和滞留时间差，减少为了治疗效果注射大剂量药物造成毒副作用的问题。
Description
技术领域
本发明涉及生物医药，具体为一种基于阴道上皮细胞膜构建的负载两性霉素B仿生纳米系统的组合物、制备方法及其应用。
背景技术
外阴阴道念珠菌病(VVC)是育龄期妇女最常见的疾病之一，其中85％-90％由白色念珠菌感染引起，患者会伴随外阴红肿、强烈瘙痒和阴道分泌物增多等症状。据统计，75％-80％的女性一生中至少有过一次白色念珠菌感染，是一种条件致病菌。临床治疗中，VVC可通过局部外用或口服抗真菌药物治疗。两性霉素B(AmB)被证明能与白色念珠菌细胞膜上的麦角固醇结合，增加细胞膜通透性，导致细胞内如K+，Mg2+等离子外流，破坏真菌的正常代谢从而抑制其生长。
有研究显示，白色念珠菌对两性霉素B(AmB)较为敏感，其最小抑制浓度(MIC)≤1μg/mL。然而，由于麦角固醇与胆固醇结构相似，故AmB也能结合到人正常细胞膜表面的胆固醇，造成毒副作用，比如急性肾功能损伤等。此外，AmB来源于链霉菌，能被单核细胞表面的Toll样受体2(TLR2)和跨膜信号蛋白CD14识别，引发寒战、恶心、呕吐和头痛等一系列不良反应。
近年来，纳米技术为提高AmB的治疗指数提供新思路。大量文献报道，纳米材料能有效改善AmB的溶解度，延长半衰期及降低免疫原性，如负载AmB的纳米脂质体、聚乳酸-羟基乙酸共聚物(PLGA)纳米颗粒等。然而，大部分负载AmB的纳米制剂均未能有效提高药物在念珠菌感染部位的靶向性和滞留时间，只能通过大剂量注射含有AmB纳米材料的方式获得一定的治疗效果，从而不可避免的引起剂量依赖的毒副作用。
因此现有的治疗方案实际上还不足以实现非常好的治疗效果，依旧存在治疗效果差或副作用明显的问题。
发明内容
针对现有技术存在的不足，本发明的目的在于提供一种负载两性霉素B仿生纳米系统的组合物、制备方法及应用，能够克服现有技术中的制剂对于药物的靶向性和滞留时间差，减少为了治疗效果注射大剂量药物造成毒副作用的问题。
为实现上述目的，本发明提供了如下技术方案：一种负载两性霉素B仿生纳米系统的制备方法，包括如下步骤：
分离和纯化阴道上皮细胞的细胞膜；
将AmB包封至PLGA纳米颗粒中；
将阴道上皮细胞的细胞膜包裹到含有AmB的PLGA纳米颗粒表面。
作为本发明的进一步改进，细胞膜的分离和纯化采用低渗和机械破碎法。
作为本发明的进一步改进，AmB采用纳米共沉淀法包封至PLGA纳米颗粒中。
作为本发明的进一步改进，阴道上皮细胞在分离和纯化之前还经过培养。
作为本发明的进一步改进，阴道上皮细胞的细胞膜通过水浴超声的方式进行包裹。
作为本发明的进一步改进，水浴超声通过120W超声5min完成包裹。
一种负载两性霉素B仿生纳米系统的组合物，包括AmB、PLGA纳米颗粒、阴道上皮细胞膜；其中AmB封包在PLGA纳米颗粒中，封包的整体由阴道上皮细胞膜包裹。
一种负载两性霉素B仿生纳米系统的应用，该仿生纳米系统由上述的制备方法获得或采用上述的组合物，并用于治疗白色念珠菌感染。
本发明的有益效果，制备的阴道上皮细胞仿生纳米材料能够通过阴道上皮细胞表面特有的黏附分子与念珠菌结合，提高仿生纳米材料在念珠菌感染部位的靶向性和滞留时间，提高局部AmB的浓度，延长AmB的作用时长，减小AmB的毒副作用，最终增强AmB杀伤念珠菌的作用。
附图说明
图1为本发明的AmB@PLGA@VEM仿生纳米系统的表征(A为AmB@PLGA纳米颗粒与AmB紫外分光光谱仪测定图；B为纳米系统表面形貌图；C为纳米系统的水力学尺寸大小和表面电位图)；
图2为本发明的AmB@PLGA@VEM仿生纳米系统在体外对白色念珠菌的靶向和黏附作用图；
图3为本发明AmB@PLGA@VEM仿生纳米系统对体内白色念珠菌小鼠感染模型的靶向和黏附作用图；
图4为本发明AmB@PLGA@VEM仿生纳米系统对白色念珠菌感染小鼠模型的治疗作用图；
图5为本发明AmB@PLGA@VEM仿生纳米系统的生物相容性(A为AmB@PLGA@VEM仿生纳米系统对红细胞溶血的作用图；B&C为AmB@PLGA@VEM仿生纳米系统对阴道上皮细胞以及对巨噬细胞的细胞毒性试验图)。
具体实施方式
下面将结合附图所给出的实施例对本发明做进一步的详述。
参照图1-5所示，
实施例1
阴道上皮细胞膜的提取
培养阴道上皮细胞VK2/E6E7(ATCC CRL-2616)：将VK/E6E7在T175培养瓶中用含0.1ng/mL人重组EGF，0.05mg/mL牛垂体后叶素提取物以及44.1mg/L氯化钙的角质细胞-无血清培养基(GIBCO-BRL 17005-042)培养细胞，培养至细胞汇合率达到80％以上，用0.02％EDTA溶液进行消化，收集阴道上皮细胞。配制含有D-甘露醇、蔗糖以及Tris-HCl的缓冲液，将其加入细胞，以800×g离心5分钟清洗细胞去除杂质，将细胞清洗干净后向缓冲液中加入蛋白酶抑制剂以及磷酸酶抑制剂，用其将细胞重悬，匀浆器进行匀浆，以7600rpm离心30分钟去除细胞中的细胞核、细胞器等，最后以29600rpm离心50分钟，收集沉淀即阴道上皮细胞膜(Vaginal epithelial membrane，VEM)。通过BCA蛋白定量试剂盒检测蛋白浓度，-80度储存以备后续实验所需。
实施例2
AmB@PLGA@VEM仿生纳米颗粒的制备以及表征
采用纳米共沉淀法制备AmB@PLGA颗粒：利用丙酮溶解50：50粘度、羧基端修饰的PLGA(0.67dL/g)至7.5mg/mL待用。将0.65mL的4.6mg/mL两性霉素B(AmB)的二甲基亚砜(DMSO)溶液与2mL的PLGA溶液混合，然后向混合物中迅速加入5mL含有0.5％(w/v)的聚乙烯醇(Polyvinyl alcohol，PVA)水溶液，搅拌挥发有机溶剂后，13300rpm离心30min去除多余的AmB以及DMSO，收集AmB@PLGA纳米颗粒。经紫外分光光谱仪测定AmB@PLGA纳米颗粒与AmB具有相同的特征峰，说明AmB已包封至PLGA纳米颗粒中(图1-A)。随后，将AmB@PLGA纳米颗粒与阴道上皮细胞膜(VEM)按照PLGA和VEM膜蛋白的质量比2∶1混合，利用水浴超声(120W超声5min)将VEM包覆到AmB@PLGA纳米颗粒表面构建AmB@PLGA@VEM仿生纳米系统。将制备的AmB@PLGA@VEM进行基本物理表征，包括利用透射电子显微镜(TEM)观察纳米系统表面形貌(图1-B)以及利用粒度电位仪测定纳米系统的水力学尺寸大小和表面电位(图1-C)。结果显示AmB@PLGA@VEM呈现核壳结构，水力学尺寸大小约101.8nm，表面电位约-28.4mV(图1)。
实施例3
AmB@PLGA@VEM仿生纳米系统在体外对白色念珠菌的靶向和黏附作用
按照DiD与PLGA质量比2∶1000的比例制备DiD荧光标记的AmB@PLGA-DiD@VEM。同理，制备DiD荧光标记的AmB@PLGA-DiD@RBCM(RBCM为红细胞膜)和AmB@PLGA-DiD@F127(F127是脂质膜)仿生纳米系统。将白色念珠菌的菌量调整为3×107CFU/mL与相同脂质浓度的AmB@PLGA-DiD@VEM、AmB@PLGA-DiD@RBCM及AmB@PLGA-DiD@F127分别共孵育30min，随后用双蒸水清洗去除未与白色念珠菌结合的纳米系统。利用扫描电子显微镜(SEM)观察3种仿生纳米系统对白色念珠菌的靶向黏附效果。结果表明，AmB@PLGA-DiD@VEM纳米系统与白色念珠菌结合最多，AmB@PLGA-DiD@RBCM纳米系统较少，AmB@PLGA-DiD@F127组纳米系统几乎观察不到(图2)。
实施例4
AmB@PLGA@VEM仿生纳米系统对体内白色念珠菌小鼠感染模型的靶向和黏附作用
白色念珠菌感染模型的构建：将白色念珠菌菌量调整为7×106CFU/mL，取100μL注射到BalB/C雌鼠阴道后第三天开始后续实验。分别注射相同脂质剂量AmB@PLGA-DiD@VEM、AmB@PLGA-DiD@RBCM仿生纳米系统以及PBS作为阴性对照组，48h后暴露雌鼠阴道组织，利用小动物活体成像仪检测各组DiD荧光信号强度。小动物活体成像结果显示，AmB@PLGA-DiD@VEM纳米系统组雌鼠阴道组织内的荧光信号较强，而AmB@PLGA-DiD@RBCM纳米系统组的荧光信号与阴性对照组接近，表明AmB@PLGA-DiD@VEM纳米系统较AmB@PLGA-DiD@RBCM纳米系统对白色念珠菌感染的阴道部位具有靶向和富集效果(图3)。
实施例5
AmB@PLGA@VEM仿生纳米系统对白色念珠菌感染小鼠模型的治疗作用
构建BalB/C雌鼠白色念珠菌阴道感染模型。分别将PBS、AmB、AmB@PLGA、AmB@PLGA@VEM仿生纳米系统(含等剂量的AmB)注射到模型小鼠阴道内，24h后测量阴道液以及阴道组织匀浆液中白色念珠菌的菌量。结果显示，AmB@PLGA@VEM纳米系统治疗白色念珠菌感染小鼠的效果要明显优于其他组，表明AmB@PLGA@VEM纳米系统具有更好的白色念珠菌感染治疗作用(图4)。
实施例6
AmB@PLGA@VEM仿生纳米系统的生物相容性
AmB@PLGA@VEM仿生纳米系统对红细胞溶血的作用。收集新鲜小鼠全血，分离红细胞，并利用PBS稀释为5％(v/v)进行后续实验。向5％红细胞悬液中分别加入含有不同AmB浓度的AmB@PLGA和AmB@PLGA@VEM以及相对应的游离AmB，37℃水浴孵育30min后13300rpm离心5min，再利用酶标仪检测540nm吸光值。结果表明，游离的AmB在1.88μg/mL时，即可产生溶血，而AmB@PLGA与AmB@PLGA@VEM组即使达到15μg/mL也不会产生溶血，表明AmB@PLGA@VEM的安全性(图5-A)。
AmB@PLGA@VEM仿生纳米系统对阴道上皮细胞以及对巨噬细胞的细胞毒性。将VK2/E6E7人阴道上皮细胞以及RAW264.7小鼠巨噬细胞铺板于96孔板内，直到汇合率达到80％以上时，加入不同浓度的AmB、AmB@PLGA以及AmB@PLGA@VEM纳米系统(以AmB的浓度为准)。新鲜培养基组作为阴性对照组。培养24h后，利用Cell Counting Kit-8(CCK-8)方法检测细胞毒性。结果显示，高于3.75μg/mL的AmB浓度时，游离AmB对两种细胞均产生明显的细胞毒素(图5-B&C)。同时，在AmB@PLGA中高于3.75μg/mL的AmB浓度对RAW264.7小鼠巨噬细胞也产生一定细胞毒性(图5-C).然而，即使到达15μg/mL，AmB@PLGA@VEM纳米系统对两种细胞均不产生任何细胞毒性，表明AmB@PLGA@VEM纳米系统具有较好的生物安全性。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种负载两性霉素B仿生纳米系统的制备方法，其特征在于，包括如下步骤：
分离和纯化阴道上皮细胞的细胞膜；
将AmB包封至PLGA纳米颗粒中；
将阴道上皮细胞的细胞膜包裹到含有AmB的PLGA纳米颗粒表面。
2.根据权利要求1所述的制备方法，其特征在于，细胞膜的分离和纯化采用低渗和机械破碎法。
3.根据权利要求1所述的制备方法，其特征在于，AmB采用纳米共沉淀法包封至PLGA纳米颗粒中。
4.根据权利要求1所述的制备方法，其特征在于，阴道上皮细胞在分离和纯化之前还经过培养。
5.根据权利要求1所述的制备方法，其特征在于，阴道上皮细胞的细胞膜通过水浴超声的方式进行包裹。
6.根据权利要求5所述的制备方法，其特征在于，水浴超声通过120W超声5min完成包裹。
7.一种负载两性霉素B仿生纳米系统的组合物，其特征在于，包括AmB、PLGA纳米颗粒、阴道上皮细胞膜；其中AmB封包在PLGA纳米颗粒中，封包的整体由阴道上皮细胞膜包裹。
8.一种负载两性霉素B仿生纳米系统的组合物，其特征在于，该组合物由权利要求1～6任意一项制备方法获得。
9.一种负载两性霉素B仿生纳米系统的应用，其特征在于，该仿生纳米系统采用如权利要求7的组合物，并用于治疗白色念珠菌感染。
10.一种负载两性霉素B仿生纳米系统的应用，其特征在于，该仿生纳米系统采用如权利要求8的组合物，并用于治疗白色念珠菌感染。
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