CN112076158A - 一种治疗慢性肾炎的脂质体-纳米粒复合体 - Google Patents
一种治疗慢性肾炎的脂质体-纳米粒复合体 Download PDFInfo
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- CN112076158A CN112076158A CN202010882221.1A CN202010882221A CN112076158A CN 112076158 A CN112076158 A CN 112076158A CN 202010882221 A CN202010882221 A CN 202010882221A CN 112076158 A CN112076158 A CN 112076158A
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Abstract
本发明提供了一种经抗体修饰的具有脂质外壳和纳米粒核心的脂质体‑纳米粒复合体(Liposome‑nanoparticle hybrids,LNHy),它以荷电的纳米粒作为内核,表面包被相反电荷的脂质体磷脂双分子层形成具有核‑壳的类细胞结构。该复合体具有靶向能力,可包载对慢性肾炎具有预防和/或治疗作用的抗炎药及抗纤维化药物,从而实现对慢性肾炎双调控的有效治疗作用。
Description
技术领域
本发明属于医药技术领域,具体涉及一种经抗体修饰的载双药脂质体-纳米粒复合体及其用于治疗慢性肾炎的用途。
背景技术
慢性肾炎(Chronic glomerulonephritis,CGN)是一种多病因引起的慢性肾小球病变,具有患病率高、隐匿性强等特点。部分患者首次确诊时,肾脏损害已处于不可逆转阶段。免疫炎症损伤是CGN的主要发病机制,通过诱发免疫复合体在肾小球沉积,逐步发展至肾脏纤维化,最终引发慢性肾衰竭,危及患者生命。目前,CGN尚无特效药物根治,为了控制炎症反应,需要长期服用糖皮质激素类或其他免疫抑制类药物。然而,长期服用上述药物不仅会引发严重的肾外毒性,还可能诱发终末期肾病。而且,单一使用这类药物抗炎治疗并不能逆转部分已经开始纤维化的病变组织,使得CGN难以获得良好的预后。
美国肾病协会的报道指出,肾小球系膜细胞(Mesangial cells,Mcs)增生及成纤维化作为CGN的基本病理过程,是对损伤作出的早期成纤维化反应,是CGN进展到终末期肾病的基本步骤,是肾脏纤维化的早期病灶,所以Mcs是治疗CGN的理想靶点。为了建立针对Mcs病灶的肾小球肾脏靶向给药系统,Tuffin等人首次提出用免疫脂质体靶向治疗Mcs病变的可能并阐述了研究意义(Tuffin,G.,Waelti,E.,Huwyler,J.,Hammer,C.,Marti,H.P.,2005.Immunoliposome targeting to mesangial cells:a promising strategy forspecific drug delivery to the kidney.Journal of the American Society ofNephrology:JASN 16,3295-3305);Scindia等人进一步提出利用肾小球生理结构特点,以Mcs为靶点,通过控制普通聚乙二醇(Polyethylene glycol,PEG)化的免疫脂质体粒径大小,但结果显示这种脂质体靶向效率比较低,易蓄积在肝、脾等器官(Scindia,Y.,Deshmukh,U.,Thimmalapura,P.R.,Bagavant,H.,2008.Anti-alpha8 integrinimmunoliposomes in glomeruli of lupus-susceptible mice:a novel system fordelivery of therapeutic agents to the renal glomerulus in systemic lupuserythematosus.Arthritis and rheumatism 58,3884-3891);Choi等人亦尝试制备系列不同粒径大小的普通PEG化金纳米粒(75±25nm),考察其在正常小鼠体内被Mcs摄取的规律(Choi,C.H.,Zuckerman,J.E.,Webster,P.,Davis,M.E.,2011.Targeting kidneymesangium by nanoparticles of defined size.Proc Natl Acad Sci U S A 108,6656-6661)。然而,以上研究不仅忽视了在肾小球损伤情况下,有效滤过区域和系膜支撑区域孔径会变化的特点,使所得到的规律不适用病理条件下治疗药物的剂型设计,而且由于网状内皮系统(RES)对普通PEG化纳米粒的大量捕获(约占给药剂量的40-80%),使得纳米粒在Mcs的蓄积仅占给药剂量的1-4%,远未达到靶向给药的目的。国内,樊梅采用了TRX配基修饰的PEG化脂质体探索靶向Mcs的可能,结果发现不仅配基毒性较大,而且脂质体靶细胞的蓄积也很不理想(樊梅.脒修饰的载雷公藤甲素阳离子脂质体的肾小球靶向给药系统研究.硕士毕业论文.2015)。目前,如何进一步减少机体RES对载体的捕获,提高靶向效率增加其在Mcs的蓄积,仍是一个关键性的问题。
在药剂学研究中,模型药物的包载与释放情况一直是研究的重点问题。本来关于肾小球靶向给药系统的相关研究报道就不多,选用了模型药物的研究报道就更少。通过仅有的研究报道以及我们的前期研究可知,无论是选用的泼尼松龙,还是雷公藤内酯醇模型药物,在采用脂质体对它们分别进行包载时,低包封率和低载药量是一直无法回避的问题。而且,一些毒性大的药物(如:雷公藤内酯醇)被包载于脂质体或者纳米粒后,药物在体内的突释也是一个问题。同时,已有研究报道中,均只考虑到单一药物抗炎治疗,而忽略了病程中的纤维化预防和逆转问题,这并不利于CGN的预后,所以采用高效靶向的“抗炎/抗纤维化”双调控可能会实现对CGN的有效治疗。
发明内容
为克服上述现有技术的缺陷和不足,本发明提供了一种经抗体修饰的具有脂质外壳和纳米粒核心的脂质体-纳米粒复合体(Liposome-nanoparticle hybrids,LNHy),它以荷电的纳米粒作为内核,表面包被相反电荷的脂质体磷脂双分子层形成具有核-壳的类细胞结构。该复合体具有靶向能力,可包载对慢性肾炎具有预防和/或治疗作用的抗炎药及抗纤维化药物,从而实现对慢性肾炎双调控的有效治疗作用。
本发明的目的是提供一种载双药的integrin α8修饰的脂质体-纳米粒复合体,所述复合体具有脂质外壳和纳米粒核心,可同时包载抗炎药物和抗纤维化药物,所述复合体的脂质外壳经抗体修饰,所述抗体为巯基化修饰后的抗体integrinα8。
本发明所述的载双药的integrin α8修饰的脂质体-纳米粒复合体,其中纳米粒核心选自以下一种或多种:聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)纳米粒核心、金纳米粒(Gold nanoparticles,AuNPs)核心、介孔二氧化硅(Mesoporous silica,MS)纳米粒核心。
本发明所述的载双药的integrinα8修饰的脂质体-纳米粒复合体,其中所述脂质外壳由膜材DOPE、DOTAP、胆固醇、mPEG2000-DSPE和Mal-PEG2000-DSPE制备而成。本领域技术人员熟知,DOTAP是指(2,3-二油酰基-丙基)-三甲胺、HSPC是指氢化大豆卵磷脂、Chol是指胆固醇、DOPE是指二油酰磷脂酰乙醇胺、mPEG2000-DSPE是指二硬脂酰磷脂酰乙醇胺-聚乙二醇、Mal-PEG2000-DSPE是指磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺。
本发明所述脂质外壳中,膜材DOPE、DOTAP、胆固醇、mPEG2000-DSPE和Mal-PEG2000-DSPE的摩尔比为1~2:0.2~0.8:0.8~1.2:0.05~0.1:0.01~0.03,优选为1.5:0.5:1:0.08:0.02。
本发明所述的载双药的integrinα8修饰脂质体-纳米粒复合体,其中抗炎药物和抗纤维化药物以相同或不同的形式进行包载,例如可分别包载于纳米粒核心、包载于脂质外壳内部、也可包载于脂质外壳中。
本发明所述的载双药的integrinα8修饰的脂质体-纳米粒复合体,其中抗炎药物优选为糖皮质激素(Glucocorticoid,GC)类抗炎药,例如地塞米松、泼尼松、泼尼松龙、倍氯米松、倍他米松、可的松、氢化可的松、曲安西龙等,更优选为地塞米松。所述抗纤维化药物选自以下一种或多种:卡托普利、罗非昔布、美洛昔康、吡非尼酮、甘草甜素、丹参酮II A、汉防己甲素、川芎嗪、商陆皂甙甲、大黄素、小干扰RNA(small interfering RNA,siRNA)。
本发明所述的载双药的integrinα8修饰的脂质体-纳米粒复合体,其粒径控制在70~130之间。
本发明还提供所述载双药的脂质体-纳米粒复合体的制备方法,包括以下步骤:
(1)制备纳米粒核心;
(2)制备脂质体-纳米粒核心复合体;
(3)制备抗体修饰的脂质体-纳米粒复合体。
其中,所述步骤(1)中的纳米粒核心选自以下一种或多种:聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)纳米粒核心、金纳米粒(Gold nanoparticles,AuNPs)核心、介孔二氧化硅(Mesoporous silica,MS)纳米粒核心。
其中,所述步骤(2)的脂质体-纳米粒核心复合体中的脂质体外壳由膜材DOPE、DOTAP、胆固醇、mPEG2000-DSPE和Mal-PEG2000-DSPE制备而成。
其中,所述步骤(3)的抗体为巯基化修饰后的抗体integrinα8。
其中,所述抗炎药及抗纤维化药物分别在步骤(1)或步骤(2)中完成包载。
优选的,本发明提供一种载双药的integrinα8修饰的脂质体-PLGA纳米粒复合体(PLGA-ILS/双药)的制备方法,包括:
(1)纳米粒核心的制备
精密量取PLGA和抗炎药溶于有机溶剂中混合作为有机相溶液,将有机相溶液滴加到混合有少量表面活性剂的水相溶液中,室温搅拌10-30min后,于水浴旋转蒸发除去有机相,去除粒径较大的PLGA纳米粒,将溶液离心后回收上清液,再将回收上清液离心后移除上清,除去游离的抗炎药,加入去离子水重悬,即得载药的纳米粒核心(PLGA/药物)。
(2)载双药脂质体-纳米粒(PLGA-LNHy/双药)的制备
将脂质体的膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE溶于有机溶剂,旋转蒸发除去有机溶剂得到均一薄膜,将抗纤维化药物溶于步骤(1)获得的纳米粒核心溶液中,再将溶液加入均一薄膜,加热一段时间后水化脱膜,探头超声乳化后,用聚碳酸酯膜挤压控制粒径后,得载双药脂质体-纳米粒(PLGA-LNHy/双药)。
(3)integrin α8抗体修饰的载双药脂质体-纳米粒(PLGA-ILs/双药)的制备
为提高纳米微粒的靶向性,将巯基化修饰后integrin α8与PLGA-LNHy/双药在室温孵育过夜,用Sepharose CL-4B除去未偶联巯基化抗体分子,得到抗体分子修饰的载双药脂质体-纳米粒(PLGA-ILs/双药)。
优选的,所述步骤(1)中,所述有机溶剂可为丙酮、二氯甲烷、三氯甲烷等。
优选的,所述步骤(1)中,所述表面活性剂可为泊洛沙姆188、聚乙烯醇(PVA)、维生素E聚乙二醇琥珀酸酯(TPGS)等,所述水相中表面活性剂的含量为0.5%~2%。
优选的,所述步骤(1)中,所述水浴旋转蒸发温度为30℃~50℃,蒸发5-20min,更优选为蒸发温度为35℃~40℃,蒸发8-15min。
优选的,所述步骤(2)中,膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE的摩尔比为1~2:0.2~0.8:0.8~1.2:0.05~0.1:0.01~0.03,更优选为1.5:0.5:1:0.08:0.02。
优选的,所述步骤(2)中,所述有机溶剂可为丙酮、二氯甲烷、三氯甲烷等。
优选的,所述步骤(2)中,水化脱膜在40℃~60℃恒温水浴中进行30-60min。
优选的,本发明提供一种载双药的integrin α8修饰的脂质体-金纳米粒复合体(Au-ILs/双药)的制备方法,包括:
(1)纳米粒核心的制备
精密量取适量的HAuCl4·4H2O溶液,搅拌加热至沸腾,缓慢加入一定体积的柠檬酸钠溶液,继续搅拌加热5~20min,再冷却搅拌10~30min,即得红色的AuNPs溶液,低温密封避光保存,将适量的抗纤维化药与AuNPs溶液混合,20-60℃条件下孵育。将反应产物转入透析袋中除去未结合的抗纤维化药物,去离子水重悬,即得载药的纳米粒核心(AuNPs/抗纤维化药)。
(2)载双药脂质体-纳米粒子(Au-LNHy/双药)的制备
将一定量的抗炎药和脂质体的膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE共溶于有机溶剂中,旋转蒸发除去有机溶剂得到均一薄膜,将步骤(1)得到的载药的纳米粒核心溶液加入薄膜,加热一段时间后水化脱膜,探头超声乳化后,用聚碳酸酯膜挤压控制粒径后,去离子水重悬,得载双药脂质体-纳米粒子(Au-LNHy/双药)。
(3)抗体修饰的载双药脂质体-纳米粒子(Au-ILs/载双药)的制备
为提高纳米微粒的靶向性,将巯基化修饰后的抗体integrin α8与Au-LNHy/双药在室温孵育过夜,用Sepharose CL-4B除去未偶联巯基化抗体分子,得到抗体分子修饰的载双药脂质体-纳米粒子(Au-ILs/双药)。
优选的,所述步骤(1)中,加入柠檬酸钠溶液后继续搅拌加热的温度为8~15min,再冷却搅拌15~20min.
优选的,所述步骤(1)中,孵育温度为30-40℃。
优选的,所述步骤(2)中,膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE的摩尔比为1~2:0.2~0.8:0.8~1.2:0.05~0.1:0.01~0.03,更优选为1.5:0.5:1:0.08:0.02。
优选的,所述步骤(2)中,所述有机溶剂可为丙酮、二氯甲烷、三氯甲烷等。
优选的,所述步骤(2)中,水化脱膜在40℃~60℃恒温水浴中进行30-60min。
优选的,本发明提供一种载双药的integrin α8修饰的脂质体-MS纳米粒复合体(MS-ILs/双药)的制备方法,包括:
(1)纳米粒核心的制备
称取一定量的十六烷基三甲基溴化铵(CTAB)溶于去离子水中,加入乙酸钠、适量乙醇和1mol·L-1的氢氧化钠,搅拌升温到一定温度至溶液澄清透明,在搅拌下滴加适量的正硅酸四乙酯(TEOS),恒温高速搅拌,随后用适量水和乙醇溶液反复多次离心洗涤、室温下干燥。称取适量干燥后的粉末悬于浓盐酸-乙醇混合液中回流,继续用适量水和乙醇溶液反复多次离心洗涤去除表面活性剂,终产物真空干燥,即得球形介孔二氧化硅纳米粒。精密称取一定量的抗纤维化药物至西林瓶中,加入一定量的溶剂超声溶解。精密称取一定量的介孔二氧化硅分散其中,在常温下密封搅拌,离心,移除上清除去游离的抗纤维化药物,加入去离子水重悬,即得载药纳米粒核心(MS/药物)。
(2)载双药脂质体-纳米粒子(MS-LNHy/载双药)的制备
将一定量的抗炎药和脂质体的膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE共溶于有机溶剂中,旋转蒸发除去有机溶剂得到均一薄膜,将步骤(1)得到的载药的纳米粒核心溶液加入薄膜,加热一段时间后水化脱膜,探头超声乳化后,用聚碳酸酯膜挤压控制粒径后,得载双药脂质体-纳米粒子(MS-LNHy/双药)。
(3)抗体修饰的载双药脂质体-纳米粒子(MS-ILs/双药)的制备
为提高纳米微粒的靶向性,将巯基化修饰后的抗体integrin α8与MS-LNHy/载双药在室温孵育过夜,用Sepharose CL-4B除去未偶联巯基化抗体分子,得到抗体分子修饰的载双药脂质体-纳米粒子(MS-ILs/双药)。
优选的,所述步骤(1)中,滴加正硅酸四乙酯(TEOS)后恒温高速搅拌的时间为1~3h。
优选的,所述步骤(1)中,称取干燥后的粉末悬于浓盐酸-乙醇混合液中回流的温度为80-100℃,时间为12-30h。
优选的,所述步骤(1)中,终产物于100℃真空干燥6-10h。
优选的,所述步骤(2)中,膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE的摩尔比为1~2:0.2~0.8:0.8~1.2:0.05~0.1:0.01~0.03,更优选为1.5:0.5:1:0.08:0.02。
优选的,所述步骤(2)中,所述有机溶剂可为丙酮、二氯甲烷、三氯甲烷等。
优选的,所述步骤(2)中,水化脱膜在40℃~60℃恒温水浴中进行30-60min。
本发明的目的还包括提供上述载双药的脂质体-纳米粒复合体在制备治疗慢性肾炎的疾病的药物中的用途。
本发明的另一个目的还包括提供一种药物组合物,所述药物组合物包含上述载双药的脂质体-纳米粒复合体和药学上可接受的载体。
本发明的其它目的还包括提供一种治疗慢性肾炎的方法,所述方法包括给有需要的人或动物提供治疗有效量的所述载双药的脂质体-纳米粒复合体或上述包含载双药的脂质体-纳米粒复合体的药物组合物。
本发明的脂质体-纳米粒复合体提供了更多的药物包载形式以优化药物包载与释放,从而达到“抗炎/抗纤维化”的双调控。其中,聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)包载模型药物成为纳米粒核心,此方法适合脂溶性强的药物;金纳米粒(Gold nanoparticles,AuNPs)与含有巯基的药物或者药物的巯基衍生物通过共价结合的方式实现载药,并且脂质体-纳米粒复合体的空囊中可包裹水溶性药物,脂质体磷脂双分子层可包载脂溶性药物;介孔二氧化硅(Mesoporous silica,MS)纳米粒吸附药物于介孔内制成核心,此方法适合包载水溶性药物,特别适合包载大分子核酸、蛋白质类生物技术药物。以上三种不同纳米粒核心的PEG和integrinα8共同修饰脂质体-纳米粒复合体,形成不同模型药物被同时包载,实现“抗炎/抗纤维化”的双调控,避免药物提前释放,顺利蓄积于Mcs,增强了疗效。
本发明的具有脂质外壳和纳米粒核心的脂质体-纳米粒复合体采用纳米微粒靶向Mcs的机制,利用肾小球生理结构特点,并依靠肾小球内皮细胞有效滤过区域(10~70nm)和系膜支撑区域(70~130nm,病理条件下,孔径会进一步扩大,甚至超过200nm)不同孔径大小差异而产生的通透性不同,使血液循环中的纳米微粒被阻隔在有效滤过区域之外,顺利进入系膜支撑区域(类似于肿瘤组织的EPR效应),顺利抵达Mcs。因此,将载体设计为粒径在70-130nm之间能够通过肾小球脉管系统渗出,并且不能进一步穿过内皮细胞有效滤过区域进入尿腔,从而被动沉积并保留在系膜空间中,并被Mcs所吸收。载体在受体介导的内吞作用下,形成内吞体进入Mcs中,随着内吞体中pH值的下降,阳离子脂质双分子层逐步破裂,释放出抗炎药物和抗纤维化药物。由于磷脂的质子海绵效应,内吞体膜破裂,药物分子从内吞体逃逸进入细胞质,抗炎药物进入细胞核发挥抗炎作用;抗纤维化药物发挥抗纤维化作用。最终实现抑制炎症反应期与预防纤维化形成期的并举,有效治疗肾炎,减缓甚至逆转肾小球纤维化进程,改善预后。
本发明的具有脂质外壳和纳米粒核心的脂质体-纳米粒复合体(Liposome-nanoparticle hybrids,LNHy),它以荷电的纳米粒作为内核,表面包被相反电荷的脂质体磷脂双分子层形成具有核-壳的类细胞结构,结合了纳米粒和脂质体的优点,即纳米粒核心可作为支撑骨架,赋予脂质层机械稳定性、可控的形态学、狭窄的粒径分布。并且采用Mcs表面特异抗原integrin α8的抗体分子作为靶分子制备免疫复合体(Immunoliposomes,ILs),以此克服了现有Mcs靶向给药载体无法耐受高密度PEG修饰的问题,从而显著减少微粒被RES系统捕获,体外研究表明免疫复合体具有良好的Mcs靶向性,而且复合体能耐受高密度PEG2000化修饰(5%~10%),显著降低了其在肝脏中的分布。
附图说明
图1 各组相对荧光强度比较
图2 MS-ILs透射电镜图
图3 MS-ILs激光粒度及电位分析仪结果
图4 各组肾脏PAS染色比较
图5 各组肾小球有核细胞数
图6 各组肾脏PCNA染色比较
图7 肾小球内细胞PCNA阳性率
图8 各组两种血清生化指标
图9 各组肾皮质组织中两种细胞因子的含量
图10 RT-PCR测定CCL2 mRNA的相对表达量
图11 Western blot法检测CCL2蛋白表达结果
图12 灰度分析Western blot检测结果
具体实施方式
下面结合实施例,更具体地说明本发明的内容。应当理解,本发明的实施并不局限于下面的实施例,对本发明所做的任何形式上的变通与/或改变都将落入本发明保护范围。
实施例1包载双药的integrin α8修饰的脂质体-PLGA纳米粒复合体PLGA-ILs/双药
(1)纳米内核的制备
精密量取10mg PLGA和3mg地塞米松(DXMS)溶于3mL丙酮溶液涡旋混合作为有机相溶液,以针头滴加的方式(0.5mL/min)将有机相溶液滴加到15mL 1%的泊洛沙姆188水相溶液(涡旋搅拌1000r/min),室温搅拌15min后,于37℃水浴旋转蒸发10min,除去有机相。去除粒径较大的纳米粒,将溶液于8000r/min离心5min,回收上清液,再将回收上清液于12000r/min离心10min,移除上清除去游离的DXMS,加入10mL去离子水重悬,即得纳米内核(PLGA/DXMS)。
(2)PLGA-LNHy/双药的制备
将脂质体的膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE按摩尔比1.5:0.5:1:0.08:0.02溶于氯仿,旋转蒸发除去氯仿得到均一薄膜,将一定量的抗纤维化药物卡托普利(CAP)溶于PLGA/DXMS溶液中,再将溶液加入薄膜,50℃下恒温水浴30~60min水化脱膜,探头超声乳化后,用聚碳酸酯膜挤压控制粒径后,得PLGA-LNHy/DXMS/CAP。
(3)PLGA-ILs/DXMS/CAP的制备
为提高纳米微粒的靶向性,将巯基化修饰后的抗integrinα8与PLGA-ILs/DXMS/CAP在室温孵育过夜,用Sepharose CL-4B除去未偶联巯基化抗体分子,得到抗体分子修饰的PLGA-LNHy/DXMS/CAP(PLGA-ILs/DXMS/CAP)。
实施例2包载siRNA/DXMS的integrinα8修饰的脂质体-金纳米粒复合体(Au-ILs/siRNA/DXMS)
(1)纳米内核的制备
siRNA的合成:设计合成并筛选目的基因有效片段TGF-β1siRNA,合成的靶基因片段须带有巯基,使其易与AuNPs进行结合。
精密量取适量的0.11mg/mL的HAuCl4·4H2O溶液,搅拌加热至沸腾,缓慢加入一定体积的34mM柠檬酸钠溶液,继续搅拌加热10min,再冷却搅拌15min,即得红色的AuNPs溶液,于4℃密封避光保存。
AuNPs-siRNA复合体的制备:将10pmol siRNA与AuNPs按(1:0或1:1或1:2或1:5或1:10或1:20)不同质量比稀释,混合,37℃条件下孵育30min制备AuNPs-siRNA复合体,将反应产物转入透析袋中除去未结合的siRNA,加入10mL去离子水重悬,即得AuNPs-siRNA。
(2)Au-LNHy/DXMS/siRNA的制备
将一定量的DXMS和脂质体的膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE按摩尔比1.5:0.5:1:0.08:0.02共溶于氯仿,旋转蒸发除去氯仿得到均一薄膜,将AuNPs-siRNA复合体溶液加入薄膜,50℃下恒温水浴30~60min水化脱膜,探头超声乳化后,用聚碳酸酯膜挤压控制粒径后,得Au-LNHy/DXMS/siRNA。
(3)Au-ILs/DXMS/siRNA的制备
为提高纳米微粒的靶向性,将巯基化修饰后的抗integrinα8与Au-LNHy/DXMS/siRNA在室温孵育过夜,用Sepharose CL-4B除去未偶联巯基化抗体分子,得到抗体分子修饰的Au-LNHy/DXMS/siRNA(Au-ILs/DXMS/siRNA)。
实施例3包载双药的integrinα8修饰的脂质体-MS纳米粒复合体(MS-ILs/两种不同模型药物)
(1)纳米内核的制备
称取一定量的十六烷基三甲基溴化铵(CTAB)溶于去离子水中,加入0.3g乙酸钠、适量乙醇和1mol·L-1的氢氧化钠,搅拌升温到一定温度至溶液澄清透明,在搅拌下滴加适量的正硅酸四乙酯(TEOS),恒温高速搅拌2h,随后用适量水和乙醇溶液反复多次离心洗涤、室温下干燥。称取适量干燥后的粉末悬于浓盐酸-乙醇混合液(5∶90)中85℃回流24h,继续用适量水和乙醇溶液反复多次离心洗涤去除表面活性剂,终产物于100℃真空干燥8h,即得球形介孔二氧化硅纳米粒。精密称取一定量的抗纤维化药物丹参酮IIA(T IIA)至西林瓶中,加入一定量的溶剂超声溶解。精密称取一定量的介孔二氧化硅分散其中,在常温下密封搅拌24h后,于12000r/min离心10min,移除上清除去游离的T IIA,加入10mL去离子水重悬,即得纳米内核(MS/T IIA)。
(2)MS-LNHy/DXMS/T IIA的制备
将一定量的地塞米松DXMS和脂质体的膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE按摩尔比1.5:0.5:1:0.08:0.02共溶于氯仿,旋转蒸发除去氯仿得到均一薄膜,将MS/T IIA复合体溶液加入薄膜,50℃下恒温水浴30~60min水化脱膜,探头超声乳化后,用聚碳酸酯膜挤压控制粒径后,得MS-LNHy/DXMS/T IIA。
(3)MS-ILs/DXMS/T IIA的制备
为提高纳米微粒的靶向性,将巯基化修饰后的抗体integrinα8与MS-LNHy/DXMS/TIIA在室温孵育过夜,用Sepharose CL-4B除去未偶联巯基化抗体分子,得到抗体分子修饰MS-LNHy/DXMS/T IIA(MS-ILs/DXMS/T IIA)。
试验例以下试验例说明本发明产生的技术效果,不构成对本发明技术内容的任何限制。
试验例1 Mcs对PLGA-ILs纳米粒摄取能力研究
试验方法:制备未载药带有荧光标记的载体
(1)PLGA纳米粒的制备
精密量取10mg PLGA溶于3mL丙酮溶液涡旋混合作为有机相溶液,以针头滴加的方式(0.5mL/min)将有机相溶液滴加到15mL 1%的泊洛沙姆188水相溶液(涡旋搅拌1000r/min),室温搅拌15min后,于37℃水浴旋转蒸发10min,除去有机相。去除粒径较大的纳米粒,将溶液于8000r/min离心5min,回收上清液,再将回收上清液于12000r/min离心10min,移除上清除去游离的DXMS,加入10mL去离子水重悬,即得PLGA纳米粒。
(2)PLGA-LNHy的制备
将脂质体的膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE按摩尔比1.5:0.5:1:0.08:0.02溶于氯仿,旋转蒸发除去氯仿得到均一薄膜,将PLGA纳米粒胶体溶液加入薄膜,50℃下恒温水浴30~60min水化脱膜,探头超声乳化后,用聚碳酸酯膜挤压控制粒径后,得PLGA-LNHy。
(3)PLGA-ILs的制备
为提高纳米微粒的靶向性,将巯基化修饰后的抗integrin α8与PLGA-ILs在室温孵育过夜,用Sepharose CL-4B除去未偶联巯基化抗体分子,得到抗体分子修饰的PLGA-ILs。
分为3组:步骤(1)得到的PLGA纳米粒组;步骤(2)得到PLGA-LNHy组;步骤(3)得到的PLGA-ILs组。在相同的测定条件下,DAPI与细胞核中的DNA结合后显深蓝色荧光用于标记细胞核,香豆素6(C6)在步骤(1)中包载于PLGA纳米粒中,在一定激发波长下显绿色,RHOD在步骤(2)中载入脂质体双分子层中,在一定激发波长下显红色。
测定结果:PLGA-ILs组的绿色荧光要强于PLGA-LNHy和PLGA纳米粒组,红色荧光要强于PLGA-LNHy组;PLGA-LNHy组的绿色荧光要强于PLGA纳米粒组。合并后绿色、红色荧光信号和绿色、红色荧光信号重叠成的明黄荧光信号基本都处于细胞核(蓝色荧光)周围,PLGA-ILs组荧光信号最强。由于integrin α8增加了Mcs对PLGA-ILs的滞留能力,提高了Mcs摄取的能力,说明PLGA-ILs对Mcs具有良好的靶向性。通过ImageJ软件分析各载体荧光图片的相对荧光强度,结果如图1所示:在同等检测条件下,PLGA-ILs的相对荧光强度为1.48;PLGA-LNHy为1.09;PLGA纳米粒为0.61。所以由此也可以判定Mcs摄取能力:PLGA-ILs>PLGA-LNHy>PLGA纳米粒。
试验例2 Au-ILs肾内靶向性研究试验
试验方法:制备未载药载体
(1)纳米内核的制备
精密量取适量的0.11mg/mL的HAuCl4·4H2O溶液,搅拌加热至沸腾,缓慢加入一定体积的34mM柠檬酸钠溶液,继续搅拌加热10min,再冷却搅拌15min,即得红色的AuNPs胶体溶液,于4℃密封避光保存。
(2)Au-LNHy的制备
将一定量的DXMS和脂质体的膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE按摩尔比1.5:0.5:1:0.08:0.02共溶于氯仿,旋转蒸发除去氯仿得到均一薄膜,将AuNPs胶体溶液加入薄膜,50℃下恒温水浴30~60min水化脱膜,探头超声乳化后,用聚碳酸酯膜挤压控制粒径后,得Au-LNHy。
(3)Au-ILs的制备
为提高纳米微粒的靶向性,将巯基化修饰后的抗integrin α8与Au-LNHy/DXMS/siRNA在室温孵育过夜,用Sepharose CL-4B除去未偶联巯基化抗体分子,得到抗体分子修饰的Au-ILs。
分为3组:步骤(1)得到的Au纳米粒组;步骤(2)得到Au-LNHy组;步骤(3)得到的Au-ILs组。将肾样品均匀浆成0.2g/mL组织浓度进行了ICP-MS测量处理,测定肾组织中Au浓度。
测定结果:如表1,Au-ILs肾内靶向性研究表明,AuNPs组平均每克肾组织中含Au49.59±4.68ng,Au-LNHy组和Au-ILs组则分别为83.93±5.10ng和93.71±4.93ng。结果表明Au-ILs显著提高AuNPs的肾靶向性。
表1肾内Au浓度测定(n=3)
试验例3 MS-ILs性状表征试验
对实施例3制备获得的包载双药的integrinα8修饰的脂质体-MS纳米粒复合体采用透射电子显微镜和激光粒度及电位分析仪进行表征,透射电镜表征结果如图2所示,可见所制备的MS-ILs为近球状且呈现出明显的核壳结构,外壳表面相光对滑,粒径在100nm左右。激光粒度及电位分析仪结果如图3所示,表明Zeta电位为-30.63mV,平均粒径为117.2nm,表明实施例3制备获得的包载双药的integrinα8修饰的脂质体-MS纳米粒复合体稳定性较好,且粒径分布均匀。
试验例4实施例1PLGA-ILs/DXMS/CAP对系膜增生性肾小球肾炎(MsPGN)模型的药效学研究。
通过血清生化指标(BUN、Scr)的检测、切片PAS染色和免疫组化PCNA染色的观察、ELISA法测定细胞因子(TGF-β、TNF-α)、RT-PCR和Western blot测定CCL2的表达。
将MsPGN模型小鼠随机分为五组,每组3只:第一组为模型组(B组),第二组为PLGA-ILs/DXMS/CAP治疗组(C组),第三组为DXMS、CAP混合原料药DXMS/CAP治疗组(D组),第四组为DXMS治疗组(E组),第五组为CAP原料药治疗组(F组),尾静脉注射给药(DXMS给药剂量为1mg/kg;CAP给药剂量1.5mg/kg),正式造模成功后第3天开始给药治疗,每次给药间隔1天,共给药三次。另设正常组(A组):3只正常小鼠。正常组和模型组给予等体积生理盐水。
通过对糖原染色(Periodicacid-schiff stain,PAS)结果观察(×400,25μm)得到(如图4所示):正常组:肾小球的一个毛细血管袢系膜区只有1~2个Mcs,系膜区宽度未超过外周袢毛细血管,肾小球轮廓清晰可见;模型组:病变的肾小球开始出现显著的Mcs团簇样增殖,各种特殊蛋白在系膜区沉积,致使系膜基质增生,甚至有的出现肾小球硬化;PLGA-ILs/DXMS/CAP组系膜区域清晰可见,系膜组织对肾小球毛细血管无影响,无明显Mcs增生,形态结构与正常组基本一样;CAP/DXMS组系膜区域较清晰可见,出现轻度Mcs增生,系膜组织对毛细血管已无明显压迫,病变呈节段分布;DXMS组系膜组织对毛细血管有一定压迫,Mcs增生明显,炎症细胞浸润较少;CAP组大量炎症细胞浸润,系膜组织对毛细血管压迫较小,呈弥散性分布。结果说明在三次给药后,PLGA-ILs/DXMS/CAP组治疗效果最好。
在高倍镜(×400)下对肾小球PAS染色后,每组随机选取60个肾小球对其有核细胞进行计数并计算如图5所示:正常组:小鼠的肾小球内有核细胞数约为35个左右;模型组:细胞有核数增加到46个左右,表明肾小球由于病变各种细胞因子的刺激,大量细胞浸润或增生。治疗组:随着治疗的推进,细胞有核数逐步恢复到与正常组一样,其中PLGA-ILs/DXMS/CAP组最为接近正常组,表明经过药物治疗,各种细胞浸润和增生得到有效控制。
通过增殖细胞核抗原(Proliferating Cell Nuclear Antigen,PCNA)染色观察(×400,25μm),免疫阳性反应产物为胞核内棕褐色颗粒,部分胞浆内也可见阳性表达。利用免疫组织化学染色观察各组肾小球内PCNA的表达情况,正常组小鼠肾小球部分中仅见少数棕褐色颗粒,说明PCNA阳性表达量很少;模型组肾小球内棕褐色颗粒增多,说明模型组PCNA阳性细胞增多;治疗组肾小球内棕褐色颗粒有了明显减少,PLGA-ILs/DXMS/CAP组效果最为明显。如图6所示,模型组明显可见棕褐色颗粒聚集,而PLGA-ILs/DXMS/CAP组、DXMS/CAP组棕褐色颗粒分布较少,与正常组基本一致,DXMS与CAP组还有少许分布。
通过Image-Pro-Plus软件进行PCNA阳性细胞计数以及有核细胞计数,并计算PCNA阳性率结果如图7所示:模型组与正常组相比显著性极强(P<0.01),治疗组与模型组相比存在显著性(P<0.05),与正常组相比无显著性差异(*表示与模型组相比,#表示与正常组相比,*表示p<0.05,##表示p<0.01)。
通过生化指标的测定,各组尿素氮(BUN)、肌酐(Scr)测定结果如图8所示:BUN:模型组与正常组相比存在显著性(P<0.05),PLGA-ILs/DXMS/CAP组与模型组相比存在显著性(P<0.05),Scr:模型组、DXMS组与正常相比存在显著性(P<0.05),而其它组与正常组相比无显著性差别,PLGA-ILs/DXMS/CAP组与正常组Scr值最为接近(*表示与模型组相比,#表示与正常组相比,*、#表示p<0.05)。
通过ELISA试剂盒分别测定肾皮质组织细胞因子肿瘤坏死因子α(TNF-α)和转化生长因子-β(TGF-β)结果如图9所示:TNF-α:模型组与正常组有显著性(P<0.05),其余组TNF-α逐渐下降,PLGA-ILs/DXMS/CAP组与正常组最为接近,说明虽然PLGA-ILs/DXMS/CAP组与模型组相比并无显著性,但是还是具有抑制TNF-α的作用。TGF-β:模型组、DXMS组和CAP组与正常组相比存在显著性(P<0.05),PLGA-ILs/DXMS/CAP组和DXMS/CAP组已逐渐接近正常水平(#表示与正常组相比,#表示p<0.05=。
RT-PCR检测肾皮质中趋化因子(CCL2)mRNA的相对表达量结果如图10所示:PLGA-ILs/DXMS/CAP组与模型组相比存在显著性(P<0.05=,与正常组无显著性差异,说明PLGA-ILs/DXMS/CAP能够显著性降低CCL2的表达,而其它治疗组与正常组仍存在显著性差异(*表示与模型组相比,#表示与正常组相比,*、#表示p<0.05)。
Western blot法检测CCL2蛋白表达量实验结果如图11(a/b/c/d/e/f分别代表B组/A组/C组/D组/E组/F组)和图12所示:PLGA-ILs/DXMS/CAP组与模型组对比具有极强的显著性(P<0.01),与正常组无显著性,而其它治疗组与正常组还存在显著性差异,说明PLGA-ILs/DXMS/CAP能够显著性降低CCL2的蛋白表达,表明PLGA-ILs/DXMS/CAP能够通过对药物的靶向和缓释,达到显著抑制肾皮质区CCL2的蛋白表达(*表示与模型组相比,#表示与正常组相比,*表示p<0.05,**、##表示p<0.01)。
综上,构建的脂质体-纳米粒复合体具有良好的Mcs靶向性,通过药效学研究证明PLGA-ILs/DXMS/CAP具有高效的抗炎作用和一定的抗纤维化能力。
Claims (10)
1.一种载双药的脂质体-纳米粒复合体,所述复合体具有脂质外壳和纳米粒核心,可同时包载抗炎药物和抗纤维化药物,所述复合体的脂质外壳经抗体修饰,所述抗体为巯基化修饰后的抗体Anti-α8integrin。
2.如权利要求1所述的复合体,其中纳米粒核心选自以下一种或多种:聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)纳米粒核心、金纳米粒(Goldnanoparticles,AuNPs)核心、介孔二氧化硅(Mesoporous silica,MS)纳米粒核心。
3.如权利要求1或2所述的复合体,其中所述脂质外壳由膜材DOPE、DOTAP、胆固醇、mPEG2000-DSPE和Mal-PEG2000-DSPE制备而成。
4.如权利要求1-3任一项所述的复合体,其中所述脂质外壳中,膜材DOPE、DOTAP、胆固醇、mPEG2000-DSPE和Mal-PEG2000-DSPE的摩尔比为1~2:0.2~0.8:0.8~1.2:0.05~0.1:0.01~0.03,优选为1.5:0.5:1:0.08:0.02。
5.如权利要求1-4任一项所述的复合体,其中所述抗炎药物和抗纤维化药物以相同或不同的形式进行包载,例如可分别包载于纳米粒核心、包载于脂质外壳内部、也可包载于脂质外壳中,其中抗炎药物优选为糖皮质激素(Glucocorticoid,GC)类抗炎药,例如地塞米松、泼尼松、泼尼松龙、倍氯米松、倍他米松、可的松、氢化可的松、曲安西龙等,更优选为地塞米松,所述抗纤维化药物选自以下一种或多种:卡托普利、罗非昔布、美洛昔康、吡非尼酮、甘草甜素、丹参酮II A、汉防己甲素、川芎嗪、商陆皂甙甲、大黄素、小干扰RNA(smallinterfering RNA,siRNA)。
6.如权利要求1-5任一项所述的复合体,其粒径控制在70~130之间。
7.如权利要求1-6任一项所述载双药的脂质体-纳米粒复合体的制备方法,包括以下步骤:
(1)制备纳米粒核心;
(2)制备脂质体-纳米粒核心复合体;
(3)制备抗体修饰的脂质体-纳米粒复合体。
8.一种包载双药物的PEG化免疫脂质体-PLGA纳米粒复合体(PEG-PLGA-LNHy/双药),其特征在于,通过下述制备方法制备而成:
(1)纳米粒核心的制备
精密量取PLGA和抗炎药溶于有机溶剂中混合作为有机相溶液,将有机相溶液滴加到混合有少量表面活性剂的水相溶液中,室温搅拌10-30min后,于水浴旋转蒸发除去有机相,去除粒径较大的纳米粒,将溶液离心后回收上清液,再将回收上清液离心后移除上清,除去游离的抗炎药,加入去离子水重悬,即得载药的纳米粒核心溶液;
(2)载双药脂质体-纳米粒(PEG-PLGA-LNHy/抗炎药/抗纤维化药物)的制备
将脂质体的膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE溶于有机溶剂,旋转蒸发除去有机溶剂得到均一薄膜,将抗纤维化药物溶于步骤(1)获得的纳米粒核心溶液中,再将溶液加入均一薄膜,加热一段时间后水化脱膜,探头超声乳化后,用聚碳酸酯膜挤压控制粒径后,得载双药脂质体-纳米粒(PEG-PLGA-LNHy/抗炎药/抗纤维化药物);
(3)抗体修饰的载双药脂质体-纳米粒(PEG-PLGA-LNHy/抗炎药/抗纤维化药物)的制备
将巯基化修饰后的抗体Anti–α8integrin与PEG-PLGA-LNHy/抗炎药/抗纤维化药物在室温孵育过夜,用Sepharose CL-4B除去未偶联巯基化抗体分子,得到抗体分子修饰的载双药脂质体-纳米粒(PEG-PLGA-LNHy/抗炎药/抗纤维化药物)。
9.一种包载双药物的PEG化免疫脂质体-金纳米粒复合体(PEG-AuNPs-LNHy/抗纤维化药/抗炎药),其特征在于,通过下述制备方法制备而成:
(1)纳米粒核心的制备
精密量取适量的HAuCl4·4H2O溶液,搅拌加热至沸腾,缓慢加入一定体积的柠檬酸钠溶液,继续搅拌加热5~20min,再冷却搅拌10~30min,即得红色的AuNPs溶液,低温密封避光保存,将适量的抗纤维化药与AuNPs溶液混合,20-60℃条件下孵育。将反应产物转入透析袋中除去未结合的抗纤维化药物,即得载药的纳米粒核心;
(2)载双药脂质体-纳米粒子(载双药PEG-Au-LNHy)的制备
将一定量的抗炎药和脂质体的膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE共溶于有机溶剂中,旋转蒸发除去有机溶剂得到均一薄膜,将步骤(1)得到的载药的纳米粒核心溶液加入薄膜,加热一段时间后水化脱膜,探头超声乳化后,用聚碳酸酯膜挤压控制粒径后,得载双药脂质体-纳米粒子(载双药PEG-Au-LNHy);
(3)抗体修饰的载双药脂质体-纳米粒子(抗体修饰载双药PEG-Au-LNHy)的制备
将巯基化修饰后的抗体Anti–α8integrin与载双药PEG-Au-LNHy在室温孵育过夜,用Sepharose CL-4B除去未偶联巯基化抗体分子,得到抗体分子修饰的载双药脂质体-纳米粒子(抗体修饰载双药PEG-Au-LNHy)。
10.一种包载双药物的PEG化免疫脂质体-MS纳米粒复合体(PEG-MS-LNHy/抗纤维化药/抗炎药),其特征在于,通过下述制备方法制备而成:
(1)纳米粒核心的制备
称取一定量的十六烷基三甲基溴化铵(CTAB)溶于去离子水中,加入乙酸钠、适量乙醇和1mol·L-1的氢氧化钠,搅拌升温到一定温度至溶液澄清透明,在搅拌下滴加适量的正硅酸四乙酯(TEOS),恒温高速搅拌,随后用适量水和乙醇溶液反复多次离心洗涤、室温下干燥,称取适量干燥后的粉末悬于浓盐酸-乙醇混合液中回流,继续用适量水和乙醇溶液反复多次离心洗涤去除表面活性剂,终产物真空干燥,即得球形介孔二氧化硅纳米粒,精密称取一定量的抗纤维化药物至西林瓶中,加入一定量的溶剂超声溶解,精密称取一定量的介孔二氧化硅分散其中,在常温下密封搅拌,离心,移除上清除去游离的抗纤维化药物,加入去离子水重悬,即得载药纳米粒核心(MS-抗纤维化药物);
(2)载双药脂质体-纳米粒子(载双药PEG-MS-LNHy)的制备
将一定量的抗炎药和脂质体的膜材DOPE、DOTAP、胆固醇(Chol)、mPEG2000-DSPE和Mal-PEG2000-DSPE共溶于有机溶剂中,旋转蒸发除去有机溶剂得到均一薄膜,将步骤(1)得到的载药的纳米粒核心溶液加入薄膜,加热一段时间后水化脱膜,探头超声乳化后,用聚碳酸酯膜挤压控制粒径后,得载双药脂质体-纳米粒子(载双药PEG-MS-LNHy);
(3)抗体修饰的载双药脂质体-纳米粒子(抗体修饰载双药PEG-MS-LNHy)的制备
将巯基化修饰后的抗体Anti–α8integrin与载双药PEG-Au-LNHy在室温孵育过夜,用Sepharose CL-4B除去未偶联巯基化抗体分子,得到抗体分子修饰的载双药脂质体-纳米粒子(抗体修饰载双药PEG-MS-LNHy)。
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