CN107028888B - 一种主动靶向肿瘤emt细胞的抗耐药脂质体及其制备方法 - Google Patents
一种主动靶向肿瘤emt细胞的抗耐药脂质体及其制备方法 Download PDFInfo
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Abstract
本发明提供一种主动靶向肿瘤EMT细胞的新型抗耐药脂质体及其制备方法，属于药物制剂领域。将二硬脂酰磷脂酰乙酰胺‐N‐羟基丁二酰亚胺‐聚乙二醇与环五肽ADH‑1为原料，制备环五肽‐聚乙二醇‐磷脂；将卵磷脂、胆固醇、甲氧基‐聚乙二醇‐磷脂、环五肽‐聚乙二醇‐磷脂按一定摩尔比溶于氯仿后，减压旋蒸至形成均匀透明的脂膜；加入预热磷酸盐缓冲液，涡旋、震荡、水化至脂膜完全脱落，将脂膜破碎后用葡聚糖凝胶柱进行纯化，得到新型抗耐药脂质体。本发明制备方法简单、技术成熟、具有广阔的应用前景，得到的新型抗耐药脂质体由环五肽‐聚乙二醇‐磷脂修饰，对肿瘤EMT细胞具有主动靶向性。
Description
技术领域
本发明属于药物制剂领域，提供了一种主动靶向肿瘤EMT细胞的新型抗耐药脂质体。
技术背景
现阶段，针对肿瘤细胞耐药，根据其耐药的发生机制，抑制通路中关键调控靶点，是改善和提高现有抗肿瘤药物敏感性的有效方法。肿瘤耐药包括原发性耐药和获得性耐药两大类。近年来的研究发现，上皮细胞间质转化(epithelial-mesenchymal transition,EMT)与肿瘤细胞耐药有密切的关系，一般发生EMT的肿瘤细胞可获得凋亡抵抗，而凋亡抵抗通常导致肿瘤放、化疗耐受。研究资料证明，EMT在多种肿瘤的化疗耐药中发挥重要作用，涉及的化疗药物包括紫杉醇、长春新碱等。因此，可通过靶向EMT来逆转肿瘤耐药，提高肿瘤细胞对药物的敏感性。
肿瘤细胞发生EMT时，细胞表面分子标志物的表达会发生变化：E-钙黏蛋白(E-cadherin)表达下降、N-钙黏蛋白(N-cadherin)表达增加、β-连环素 (β-catenin)由细胞膜上向细胞质中分布等等。因此，阻断或反转肿瘤细胞发生 EMT时产生的变化，能抑制肿瘤细胞EMT的发生，例如可通过针对肿瘤细胞发生EMT时特异表达的分子标志物，阻断其功能或者信号通路来实现这一目标。 N-cadherin是肿瘤细胞发生EMT时细胞膜上显著高表达的分子标志物，是肿瘤细胞获得耐药性的关键分子，因此N-cadherin是一个很有潜力的治疗靶标来阻断EMT。环五肽ADH-1，能够选择性地结合并阻断N-cadherin，可作为N-cadherin 的靶向分子用于肿瘤EMT细胞的靶向递送和治疗。
发明内容
针对现有技术存在的问题，本发明提供了一种主动靶向肿瘤EMT细胞的新型抗耐药脂质体，采用环五肽ADH-1作为主动靶向的靶头，利用受体与配体之间的相互作用，靶向作用于肿瘤EMT细胞表面的N-cadherin，进而阻断EMT 过程，有效地逆转肿瘤的耐药性。采用脂质体作为载体，脂质体由中性磷脂制备，其理化性质与细胞接近，具有良好的生物相容性，同时载体的表面电荷为中性，降低了载体对细胞的毒性。
为了达到上述目的，本发明的技术方案为：
一种主动靶向肿瘤EMT细胞的新型抗耐药脂质体，采用环五肽-聚乙二醇- 磷脂ADH-1-PEG2000-DSPE对脂质体进行修饰，该新型抗耐药脂质体对肿瘤EMT 细胞具有主动靶向性，能够提高肿瘤细胞对化疗药物的敏感性，逆转肿瘤耐药。
上述主动靶向肿瘤EMT细胞的新型抗耐药脂质体的制备方法为：
第一步，合成靶向材料环五肽-聚乙二醇-磷脂ADH-1-PEG2000-DSPE
将二硬脂酰磷脂酰乙酰胺-N-羟基丁二酰亚胺-聚乙二醇NHS-PEG2000-DSPE 和环五肽ADH-1按1.1～2:1的摩尔比溶于无水无氨的二甲基甲酰胺DMF后，置于茄型瓶中，加入适量三乙胺调节pH至8左右；室温、避光搅拌48h后，将反应液转移至截留分子量为3500的透析袋中，两端夹紧，用去离子水透析24h，以除去 DMF及反应液中其他杂质；将透析袋中的溶液冷冻干燥后，得到产物 ADH-1-PEG2000-DSPE，产物可用质谱进行鉴定。其中NHS-PEG2000-DSPE中聚乙二醇嵌段的分子量为2000。
第二步，合成主动靶向肿瘤EMT细胞的新型抗耐药脂质体
将卵磷脂、胆固醇、甲氧基-聚乙二醇-磷脂、ADH-1-PEG2000-DSPE室温下溶于氯仿后，置于茄型瓶中，37℃条件下减压旋蒸，除去有机溶剂，形成均匀透明的脂膜；再加入预热的磷酸盐缓冲液，涡旋震荡水化至脂膜完全脱落后，采用细胞破碎仪将脂膜破碎，破碎至溶液中出现蓝色乳光为止；最后将上述溶液用葡聚糖凝胶柱进行纯化，即得到该主动靶向肿瘤EMT细胞的新型抗耐药脂质体。所述的卵磷脂、胆固醇、甲氧基-聚乙二醇-磷脂、ADH-1-PEG2000-DSPE的摩尔比为65:20:4.35:0.5～1。
本发明的有益效果为：本发明方法制备方法简单、技术成熟、具有广阔的应用前景，也为相应给药系统的设计和发展打下基础。制备得到的主动靶向肿瘤EMT细胞的新型抗耐药脂质体能够显著增加对肿瘤EMT细胞的靶向性，提高肿瘤EMT细胞对药物的敏感性，有效逆转肿瘤耐药。制备载体采用中性磷脂，使其生物毒性明显降低。
附图说明
图1为ADH-1-PEG2000-DSPE的质谱结果图；
图2(a)为透射电镜观察到的主动靶向肿瘤EMT细胞的新型抗耐药脂质体的形态结果图；
图2(b)为采用动态光散射仪分析得到的主动靶向肿瘤EMT细胞的新型抗耐药脂质体的粒径分布图；
图3(a)为未经紫杉醇诱导的肿瘤MCF7细胞形态图；
图3(b)为紫杉醇诱导发生EMT的肿瘤MCF7细胞形态图；
图4(a)为采用激光共聚焦显微镜观察到的紫杉醇诱导发生EMT的MCF7细胞表面N-cadherin的表达情况；
图4(b)为采用激光共聚焦显微镜观察到未经紫杉醇诱导的肿瘤MCF7细胞表面N-cadherin的表达情况；
图5为免疫印迹分析未经紫杉醇诱导的肿瘤MCF7细胞和紫杉醇诱导发生EMT的肿瘤MCF7细胞相关蛋白的表达情况图；
图6为紫杉醇诱导发生EMT的肿瘤MCF7细胞对香豆素标记的未经ADH-1修饰的脂质体的摄取图，其中细胞核用DAPI进行染色。
图7为紫杉醇诱导发生EMT的肿瘤MCF7细胞对香豆素标记的主动靶向肿瘤 EMT细胞的新型抗耐药脂质体的摄取图，其中细胞核用DAPI进行染色。
图8为提前用ADH-1孵育细胞后，紫杉醇诱导发生EMT的肿瘤MCF7细胞对香豆素标记的主动靶向肿瘤EMT细胞的新型抗耐药脂质体的摄取图，其中细胞核用DAPI进行染色。，
图9为用CCK-8法测定的包载紫杉醇的主动靶向肿瘤EMT细胞的新型抗耐药脂质体A-LP、包载紫杉醇的未经ADH-1修饰的脂质体LP和游离紫杉醇PTX对紫杉醇诱导发生EMT的肿瘤MCF7细胞的细胞毒作用。
具体实施方式
以下结合具体实施方式对本发明作进一步说明。
实施例1
第一步，合成靶向材料—环五肽-聚乙二醇-磷脂(ADH-1-PEG2000-DSPE)
精密称取5mg ADH-1肽和10mg NHS-PEG2000-DSPE，加入新鲜蒸馏的无水无氨二甲基甲酰胺(DMF)将其溶解。在室温，磁力搅拌下，将NHS-PEG2000-DSPE 溶液逐滴加入到ADH-1溶液的茄型瓶中，同时加入适量三乙胺调节pH至8左右，室温、避光搅拌48h。反应结束后，将反应液转移至截留分子量为3500的透析袋中，两端夹紧，用去离子水透析24h，以除去DMF及反应液中其他杂质。随后将透析袋中的溶液冷冻干燥，即得到所需产物ADH-1-PEG2000-DSPE。
将所得样品，用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS) 进行表征，图1为ADH-1-PEG2000-DSPE的MALDI-TOF-MS结果图：可以看到图中有两个峰，其中前峰是未反应完全的DSPE-PEG2000-NHS，为了使ADH-1充分反应它在反应中的加入是过量的。ADH-1的分子量是528，DSPE-PEG-NHS的分子量是2986，因此，材料的理论分子量为3514，质谱图后峰的分子量主要分布在3500，证明靶向材料的成功合成。
第二步，合成主动靶向肿瘤EMT细胞的新型抗耐药脂质体
将15.8mg卵磷脂、2.4mg胆固醇、4.0mg甲氧基-聚乙二醇-磷脂、0.6mg ADH-1-PEG2000-DSPE溶解于氯仿后，37℃条件下减压旋蒸，除去有机溶剂，使其在茄型瓶底形成均匀透明的脂膜；然后加入预热的磷酸盐缓冲液涡旋、震荡、水化至脂膜完全脱落，用细胞破碎仪，破碎至出现蓝色乳光；最后将该溶液加到葡聚糖凝胶柱中，用pH为7.4的磷酸盐缓冲液洗脱，收集脂质体部分，即得到主动靶向肿瘤EMT细胞的新型抗耐药脂质体。
将主动靶向肿瘤EMT细胞的新型抗耐药脂质体滴加于透射电子显微镜 (TEM)载样铜网上，待自然晾干后在TEM下观察，图2(a)中可见主动靶向肿瘤EMT细胞的新型抗耐药脂质体为类圆形纳米粒。
将主动靶向肿瘤EMT细胞的新型抗耐药脂质体用动态光散射仪进行粒径分析，图2(b)为其粒径分布，可以看出主动靶向肿瘤EMT细胞的新型抗耐药脂质体的粒径约为90nm，分布较为均一。
肿瘤EMT细胞模型的建立及表征
细胞模型的建立方法：先用初浓度为2nM的紫杉醇诱导MCF7细胞一周，再逐级增大紫杉醇浓度，进行诱导，至肿瘤MCF7细胞发生EMT(紫杉醇的终浓度为24nM，共诱导六个月)。图3(a)、(b)是用显微镜观察到的肿瘤MCF7发生EMT前后的形态图。
将未经紫杉醇诱导的肿瘤MCF-7细胞和紫杉醇诱导发生EMT的肿瘤MCF7 细胞接种于放置盖破片的24孔板中，培养过夜，使细胞爬片。移除培养基，用预冷磷酸盐缓冲液(500μL/孔)洗两次，每次5min；室温条件下，用4％多聚甲醛(500μL/孔)固定15min，再用磷酸盐缓冲液洗三次；用5％牛血清白蛋白封闭 1h；然后用N-cadherin的抗体孵育2h，同时设置阴性对照组(不加抗体，其他处理条件相同)，孵育后用磷酸盐缓冲液冲洗三次；再用FITC标记的二抗孵育1h，用磷酸盐缓冲液冲洗三次；再用5μg/mL的DAPI(400μL/孔)，避光条件下染核15min，最后用磷酸盐缓冲液冲洗三次；将盖玻片取出，置于载玻片上，用激光共聚焦显微镜进行观察。如图4所示，可以看出紫杉醇诱导发生EMT的肿瘤MCF7 细胞表面的N-cadherin的表达量显著高于未经紫杉醇诱导的肿瘤MCF7的表达量，说明紫杉醇诱导发生EMT的肿瘤MCF7细胞确实发生了上皮间质转化，同时也表明本发明设计的主动靶向肿瘤EMT细胞的新型抗耐药脂质体通过 N-cadherin这一靶标来实现靶向抑制肿瘤EMT细胞，提高对化疗药物敏感性的可行性。
接着，进行了免疫印迹分析，如图5所示，与未经紫杉醇诱导的肿瘤MCF7细胞相比，紫杉醇诱导发生EMT的肿瘤MCF7细胞的E-cadherin的表达量下降， vimentin的表达量增加，符合发生EMT的肿瘤细胞的变化，进一步证明了肿瘤 EMT细胞模型的成功建立。
紫杉醇诱导发生EMT的肿瘤MCF7细胞对主动靶向肿瘤EMT细胞的新型抗耐药脂质体的摄取
将紫杉醇诱导发生EMT的肿瘤MCF7细胞接种于放置盖破片的24孔板中，培养过夜，使细胞爬片。移除培养基，分别用包载香豆素的未经ADH-1修饰的脂质体和主动靶向肿瘤EMT细胞的新型抗耐药脂质体处理细胞2h，同时设置受体提前封闭对照组：先用游离的ADH-1处理细胞1h，然后再用包载香豆素的主动靶向肿瘤EMT细胞的新型抗耐药脂质体处理2h。用激光共聚焦显微镜观察各处理组脂质体在紫杉醇诱导发生EMT的肿瘤MCF7细胞内的摄取情况。从图6和图7 显示的结果可以看出：本发明设计的主动靶向肿瘤EMT细胞的新型抗耐药脂质体在紫杉醇诱导发生EMT的肿瘤MCF7细胞内的摄取量明显高于未经ADH-1修饰的脂质体的摄取量，说明该主动靶向肿瘤EMT细胞的新型抗耐药脂质体能主动靶向紫杉醇诱导发生EMT的肿瘤MCF7细胞。同时，从图8也可以看出提前用游离ADH-1处理的对照组，紫杉醇诱导发生EMT的肿瘤MCF7细胞对主动靶向肿瘤EMT细胞的新型抗耐药脂质体的摄取量与实验组相比有所下降，说明该主动靶向肿瘤EMT细胞的新型抗耐药脂质体是通过与紫杉醇诱导发生EMT的肿瘤 MCF7细胞表面的N-cadherin的特异性结合，主动靶向进入细胞的。图6，图7，图8的比例尺为50.0μm。
主动靶向肿瘤EMT细胞的新型抗耐药脂质体的细胞毒性测定
首先，用CCK-8法测定了游离紫杉醇对未经紫杉醇诱导的肿瘤MCF7细胞和紫杉醇诱导发生EMT的肿瘤MCF7细胞的半抑制浓度(IC50)，结果分别为77.7nM 和320.7nM，进一步证明上皮间质转化与肿瘤耐药确实有密切的联系，而且可以初步得出这样的结论：肿瘤细胞发生上皮间质转化后，会产生耐药，降低对化疗药物的敏感性。也再次证明，我们发明的主动靶向肿瘤EMT细胞的新型抗耐药脂质体通过靶向肿瘤EMT细胞来抗耐药的可行性。
接着，用CCK-8法测定了主动靶向肿瘤EMT细胞的新型抗耐药脂质体的细胞毒性：将紫杉醇诱导发生EMT的肿瘤MCF7细胞(5×103/孔)接种于96 孔板中，培养过夜。分别加入100μL/孔的一系列紫杉醇浓度梯度的游离紫杉醇，包载紫杉醇的未经ADH-1修饰的脂质体和主动靶向肿瘤EMT细胞的新型抗耐药脂质体，同时以加入100μL/孔的完全培养基作为空白对照组，将细胞在细胞培养箱内培养48h，然后加入10μL/孔的CCK-8溶液继续培养1h，最后在酶标仪上，于450nm测定OD值。按以下公式计算细胞生存率：
细胞生存率(％)＝(OD样品/OD空白)×100％
从图9显示的结果表明：紫杉醇诱导发生EMT的肿瘤MCF7细胞确实对紫杉醇产生了耐药，即使当游离紫杉醇的浓度达到96nM时，对肿瘤EMT细胞的生存影响也很小(生存率为90％)；而用包载紫杉醇的主动靶向肿瘤EMT的新型抗耐药脂质体处理紫杉醇诱导产生EMT的肿瘤MCF7细胞，在紫杉醇的浓度为 16nM时就与游离紫杉醇的生存率产生了显著性的差异，而且当紫杉醇的浓度为 96nM时其生存率仅为50％，经计算，包载紫杉醇的主动靶向肿瘤EMT的新型抗耐药脂质体处理的细胞半抑制浓度为96nM，这与游离紫杉醇对紫杉醇诱导发生 EMT的肿瘤MCF7细胞的半抑制浓度320.7nM相比有大幅度的下降，这说明：本发明的主动靶向肿瘤EMT的新型抗耐药脂质体显著提高了肿瘤EMT细胞对紫杉醇的敏感性，起到了逆转肿瘤耐药的效果。
实施例2
将第一步的ADH-1肽和NHS-PEG2000-DSPE的质量5mg和10mg改为5mg和 5.5mg；将第二步的ADH-1-PEG2000-DSPE的质量0.6mg改为0.9mg；其它制备条件不变，制备主动靶向肿瘤EMT细胞的新型抗耐药脂质体。
实施例3
将第一步的ADH-1肽和NHS-PEG2000-DSPE的质量5mg和10mg改为5mg和 7.5mg，将第二步的ADH-1-PEG2000-DSPE的质量0.6mg改为1.1mg，其它制备条件不变，制备主动靶向肿瘤EMT细胞的新型抗耐药脂质体。
Claims (5)
1.一种主动靶向肿瘤EMT细胞的抗耐药脂质体，其特征在于，该抗耐药脂质体由环五肽-聚乙二醇-磷脂修饰，抗耐药脂质体对肿瘤EMT细胞具有主动靶向性，能够提高肿瘤细胞对药物的敏感性；
所述主动靶向肿瘤EMT细胞的抗耐药脂质体的制备方法，包括以下步骤：
第一步，合成环五肽-聚乙二醇-磷脂
将二硬脂酰磷脂酰乙酰胺-N-羟基丁二酰亚胺-聚乙二醇和环五肽按1.1～2:1的摩尔比溶于无水无氨的二甲基甲酰胺DMF后，置于茄型瓶中，加入三乙胺调节pH至8左右；室温、避光搅拌后，将反应液转移透析袋中，采用去离子水透析，除去DMF及反应液中其他杂质；将透析袋中的溶液冷冻干燥后，所得产物为环五肽-聚乙二醇-磷脂；
第二步，合成主动靶向肿瘤EMT细胞的抗耐药脂质体
室温下，将卵磷脂、胆固醇、甲氧基聚乙二醇-磷脂、环五肽-聚乙二醇-磷脂按65:20:4.35:0.5～1的摩尔比溶于氯仿后，置于茄型瓶中，37℃条件下减压旋蒸除去有机溶剂，形成均匀透明的脂膜；再加入预热的磷酸盐缓冲液，涡旋震荡水化至脂膜完全脱落后，采用细胞破碎仪将脂膜破碎，破碎至溶液中出现蓝色乳光为止；最后将上述溶液用葡聚糖凝胶柱进行纯化，得到该主动靶向肿瘤EMT细胞的抗耐药脂质体。
2.根据权利要求1所述的抗耐药脂质体的制备方法，其特征在于，第一步中所述的室温、避光搅拌时间为48小时。
3.根据权利要求1或2所述的抗耐药脂质体的制备方法，其特征在于，第一步中所述透析袋的截留分子量为3500，去离子水透析时间为24小时。
4.根据权利要求1或2所述的抗耐药脂质体的制备方法，其特征在于，第一步中所述的二硬脂酰磷脂酰乙酰胺-N-羟基丁二酰亚胺-聚乙二醇的聚乙二醇嵌段分子量为2000。
5.根据权利要求3所述的抗耐药脂质体的制备方法，其特征在于，第一步中所述的二硬脂酰磷脂酰乙酰胺-N-羟基丁二酰亚胺-聚乙二醇的聚乙二醇嵌段分子量为2000。
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| CN101327190A (zh) * | 2008-07-29 | 2008-12-24 | 北京大学 | 一种供注射用的抗肿瘤长循环靶向脂质体 |
| CN102764234A (zh) * | 2012-08-03 | 2012-11-07 | 上海现代药物制剂工程研究中心有限公司 | 一种盐酸拓扑替康靶向脂质体制剂及其制备方法 |
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|---|---|---|---|---|
| WO2008039525A2 (en) * | 2006-09-27 | 2008-04-03 | Adherex Technologies, Inc. | Cadherin antagonists in combination with anticancer agents for use in cancer treatment |
| CN101113159A (zh) * | 2006-11-03 | 2008-01-30 | 江其生 | 预防与治疗肺癌细胞侵袭转移的反义寡核苷酸的结构及用途 |
| CN101327190A (zh) * | 2008-07-29 | 2008-12-24 | 北京大学 | 一种供注射用的抗肿瘤长循环靶向脂质体 |
| CN102764234A (zh) * | 2012-08-03 | 2012-11-07 | 上海现代药物制剂工程研究中心有限公司 | 一种盐酸拓扑替康靶向脂质体制剂及其制备方法 |
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| ADH-1 suppresses N-cadherin-dependent pancreatic cancer progression;Yasushi Shintani etal;《International Journal of Cancer》;20080101;第122卷(第1期) * |
| K237修饰的紫杉醇长循环靶向脂质体的制备及体外评价;胡戴;《湖北中医药大学硕士学位论文》;20131007 * |
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