CN112979935B - 线粒体靶向类高分子载体材料tpp-pla、荧光素纳米粒及制备方法和应用 - Google Patents
线粒体靶向类高分子载体材料tpp-pla、荧光素纳米粒及制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种线粒体靶向类高分子载体材料TPP‑PLA、荧光素纳米粒及制备方法和应用。本发明利用乳化‑溶剂挥发法制备纳米粒，TPP‑PLA作为高分子载体材料包载荧光素，应用荧光成像实验证明TPP‑PLA具有线粒体靶向性。实验证实该纳米粒能够在神经胶质瘤细胞中染色线粒体，说明该载体能够递送进入线粒体基质，可实现细胞线粒体的追踪，可用于线粒体的靶向定位给药，在靶向给药领域具有广泛的应用前景。
Description
技术领域
本发明属于医药技术领域，具体涉及一种线粒体靶向类纳米粒高分子载体材料TPP-PLA、荧光素纳米粒及制备方法和应用。
背景技术
线粒体是一种双层膜的亚细胞器，是细胞中制造能量的结构，同时还是活性小分子像活性氧、活性氮产生的主要场所，这些活性小分子与众多重大疾病，如阿尔茨海默病、帕金森病、神经细胞死亡和癌症等密切相关。
在癌症的治疗中，传统的给药途径使药物分散到全身，浓度高，毒性大，副作用强。而纳米颗粒在肿瘤血管中具有很强的渗透性，纳米粒携带药物后靶向作用可定位肿瘤组织释放的药物可以清除肿瘤细胞，减少药物全身分布，减少药物的副作用，并降低毒性。
目前尚未见以三苯基膦作为靶向配体，与聚乳酸结合以制备具有线粒体靶向性的高分子载体材料的相关报道。
发明内容
为了解决上述技术问题，本发明的目的在于提供一种靶向线粒体的TPP-PLA荧光素纳米粒、其制备方法及应用。
本发明提供的靶向线粒体的TPP-PLA荧光素纳米粒，以聚乳酸为母体，以三苯基膦为定位基团，设计合成了一类靶向定位于线粒体的纳米粒高分子载体材料。该载体材料可用于包裹药物分子以制成纳米粒，进而用于定位给药和靶向治疗，具有增加药物浓集浓度，减少药物全身毒副作用等作用。
本发明提供的技术方案如下：
第一方面，本发明提供一种线粒体靶向类高分子载体材料TPP-PLA，其结构如下：
第二方面，本发明提供线粒体靶向类高分子载体材料TPP-PLA的制备方法，包括以下步骤：
(1)中间体三苯基膦接3-溴丙酸的合成：
将三苯基膦与3-溴丙酸溶于乙腈中，在真空环境氮气饱和下，回流搅拌，再使用无水乙醚沉淀产物，将沉淀物用乙腈重结晶，真空干燥得微黄色粉末，即为产物TPP-COOH；
(2)中间体BOC-氨基乙醇的合成：
将(BOC)2O溶解在三氯甲烷中，0℃下缓慢滴加到含有适量的氨基乙醇的三氯甲烷溶液中，缓慢升至室温，继续搅拌反应至反应完全(反应24h)，除去溶剂得到无色粘稠状液体，即为BOC-氨基乙醇；
(3)中间体BOC-氨基乙醇-聚乳酸(BOC-NH-PLA)的合成：
将BOC-氨基乙醇和辛酸亚锡溶于三氯甲烷中，加入L-丙交酯，抽真空除去三氯甲烷，在干燥氮气保护下，升温反应，反应后再次加入三氯甲烷溶解，无水乙醚4℃沉淀过夜，取沉淀真空干燥得白色沉淀，即为BOC-氨基乙醇-聚乳酸(BOC-NH-PLA)；
(4)中间体氨基封端的聚乳酸(PLA-NH2)的合成：
将BOC-氨基乙醇-聚乳酸溶于二氯甲烷中，加入适量盐酸饱和的四氢呋喃溶液，室温搅拌反应，真空去除溶剂，再加入二氯甲烷溶解，溶液用碳酸氢钠溶液洗涤，再用去离子水洗至水相为中性，有机层用无水硫酸钠干燥，无水乙醚沉淀，干燥得白色固体，即得氨基封端的聚乳酸(PLA-NH2)；
(5)高分子载体材料TPP-PLA的合成：
将三苯基膦溴丙酸、N-羟基丁二酰亚胺(NHS)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDCI)溶于乙腈中，加入三乙胺，室温搅拌反应4小时来活化羧基后得到混合溶液；然后将氨基封端的聚乳酸溶于乙腈中，缓慢滴入混合溶液中，室温搅拌反应过夜；经去离子水洗涤反应物，将有机层醇沉，干燥即得载体材料TPP-PLA。
进一步，所述所述步骤(1)中三苯基膦与3-溴丙酸的摩尔比为1:1。
进一步，所述步骤(3)中升温反应的温度为120℃，反应时间为1-10小时。
进一步，所述步骤(3)中BOC-氨基乙醇、辛酸亚锡和L-丙交酯的摩尔比为1:100:100。
进一步，所述步骤(4)中室温搅拌反应的时间为30min。
进一步，所述步骤(4)中碳酸氢钠溶液的浓度为0.05wt％。
进一步，所述步骤(5)中三苯基膦溴丙酸、N-羟基丁二酰亚胺(NHS)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDCI)。
第三方面，本发明提供一种荧光素纳米粒的制备方法，包括以下步骤：
(1)将第二方面制备的载体材料TPP-PLA溶于二氯甲烷中，加入油相，形成相A；
(2)取荧光素溶于无水乙醇中，再向里加入纯水，形成相B；
(3)边搅拌相A，边缓慢滴加相B，继续搅拌形成初乳；
(4)将SDS溶液缓慢滴加进初乳中，搅拌形成复乳；
(5)经搅拌固化挥发有机溶剂后，再经超声搅拌即得。
进一步，所述原料的用量如下：TPP-PLA载体材料0.01g，二氯甲烷3mL，油相4滴，荧光素0.003g，无水乙醇0.5mL，纯水1mL，2.5wt％SDS溶液5mL。
进一步，所述油相选自司盘-80、司盘-20、司盘-40、司盘-60、司盘-65和司盘-85中的一种或几种的混合。油相经绘制三元相图后选用司盘-80。
进一步，所述步骤(5)中固化时间为4小时；超声由超声细胞破碎仪提供，频率为195W，时间为15min。
第四方面，本发明提供利用第三方面方法制备的荧光素纳米粒。
第五方面，本发明第四方面所提供的荧光素纳米粒在线粒体的靶向药物载体中的应用。
本发明的有益效果：
(1)本发明以聚乳酸为母体，以三苯基膦为定位基团，通过工艺设计和参数优化合成了可靶向定位于线粒体的纳米粒高分子载体材料；该载体材料能够包裹药物进行靶向治疗，增加靶向作用部位的药物浓集浓度，减少其他部位的副作用；
(2)本发明提供的靶向线粒体高分子载体材料的制备方法具有较大应用前景和参考价值。
附图说明
图1是三苯基膦接3-溴丙酸的核磁共振氢谱图；
图2是BOC-氨基乙醇的核磁共振氢谱图；
图3是BOC-氨基乙醇的红外结构图；
图4是BOC-NH-PLA的核磁共振氢谱图；
图5是PLA-NH2的核磁共振氢谱图；
图6是TPP-PLA的核磁共振氢谱图；
图7：A是TPP-PLA纳米粒溶液的表观图；B是TPP-PLA纳米粒粒径分布图；C是各中间体及纳米粒的Zeta电位图；
图8是神经胶质瘤细胞与TPP-PLA纳米粒溶液共孵之后的荧光显像图。
具体实施方式
下面通过借助实施例更加详细地说明本发明，但以下实施例仅是说明性的，本发明的保护范围并不受这些实施例的限制。
实施例1
一、制备线粒体靶向类高分子载体材料TPP-PLA
(1)中间体三苯基膦接3-溴丙酸的合成：
三苯基膦(16.25mmoL)与3-溴丙酸(16.25mmoL)以1:1摩尔比溶于13mL的的乙腈中，真空环境下氮气饱和，80℃回流搅拌24小时，无水乙醚沉淀产物。将沉淀物用乙腈重结晶，挥干溶剂，真空干燥得微黄色粉末，即为产物TPP-COOH。
(2)中间体BOC-氨基乙醇的合成：
取20mmoL(BOC)2O溶解在40mL的三氯甲烷中，0℃下缓慢滴加到含有20mmoL的氨基乙醇的三氯甲烷溶液中(10mL)，缓慢升至室温，继续搅拌反应24小时后，除去溶剂得到无色粘稠状液体即为BOC-氨基乙醇。
(3)中间体BOC-NH-PLA的合成：
称取0.001mol的BOC-氨基乙醇和0.001mol的辛酸亚锡溶于1mL三氯甲烷中，加入0.1mol的L-丙交酯，抽真空除去三氯甲烷，干燥氮气保护下，100℃反应12-32小时，再次加入三氯甲烷溶解，无水乙醚4℃沉淀过夜，取沉淀真空干燥得白色沉淀，即为BOC-氨基乙醇-聚乳酸(BOC-NH-PLA)。
(4)中间体BOC-NH-PLA脱BOC：
1mmol BOC-氨基乙醇-聚乳酸用二氯甲烷溶解后，加入盐酸饱和的四氢呋喃溶液，室温搅拌30min，真空去除溶剂，再加入二氯甲烷溶解，溶液用0.05wt％碳酸氢钠溶液洗涤，再用去离子水洗至水相为中性，有机层用无水硫酸钠干燥，无水乙醚沉淀，干燥得白色固体，即得氨基封端的聚乳酸(PLA-NH2)。
(5)TPP-PLA的合成：
取0.23g三苯基膦溴丙酸、N-羟基丁二酰亚胺(NHS)(0.1636g)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDCI)(03090g)溶于乙腈中，加入20μL三乙胺，室温搅拌4小时来活化羧基。然后将氨基封端的聚乳酸溶于20mL乙腈中，缓慢滴入三苯基膦溴丙酸溶液中，室温搅拌反应过夜。用去离子水水洗涤反应物，将有机层醇沉，干燥即得载体材料TPP-PLA。
二、制备荧光素纳米粒TPP-PLA-NPs：
利用乳化-溶剂挥发法制备TPP-PLA-NPs纳米粒，内水相包载荧光素。取TPP-PLA载体材料0.04g溶解于3ml二氯甲烷中，加入4滴司盘80作为油相。取荧光素0.007g溶于1.5mL无水乙醇中，再向里加入1mL纯水，作为内水相。搅拌油相，缓慢将内水相滴加入油相中，继续搅拌3min形成W/O型初乳。反相滴加，将8mL 1.5％SDS溶液缓慢滴加进初乳中，搅拌5min形成W/O/W型复乳。在磁力搅拌器上固化4小时挥发有机溶剂后，超声(220W)7min。常温阴凉处储存。从图7中可以看出，纳米粒有明显的蓝色乳光，平均粒径67.6±0.19nm，分散性较好，Zeta为-32.5±2.69mv。
三、线粒体靶向测试：
将神经胶质瘤(C6)细胞接种于共聚焦小皿，孵育24h后弃去培养液，加入适量纳米粒溶液(荧光素标记的TPP-PLA纳米粒)，37℃孵育10h后弃去培养液，1mL PBS震摇，清洗3次，每次5min，细胞核由DAPI(蓝色荧光)染色15min，线粒体用Mito Tracker Red(红色荧光)染色，15min后吸弃染料，用1mL PBS震摇清洗3次；通过激光共聚焦显微镜观察纳米粒Fluorescein(绿色荧光)在细胞内的分布，得到载体材料的线粒体靶向情况。图8示出了荧光图像，图中第一列为蓝色荧光，第二列为红色荧光，第三列为绿色荧光，第四列为第二列和第三列的重叠，第五列为第四列中选取对象的放大。从图中可以看出，纳米颗粒的绿色荧光与线粒体染色的红色荧光共定位为黄色。黄色荧光强度的对比表明，C6细胞线粒体对TPP-PLLA NPs的总体摄取明显高于PLLA NPs。
实施例2
TPP-PLA和TPP-PLA-NPs的制备方法同实施例1，区别在于：
荧光素纳米粒TPP-PLA-NPs的制备中的TPP-PLA载体材料用量为0.02g，荧光素用量为0.005g，无水乙醇用量为1mL，SDS浓度为2wt％、用量为7mL，超声搅拌时间为9min，功率为215w。
实施例3
TPP-PLA和TPP-PLA-NPs的制备方法同实施例1，区别在于：
荧光素纳米粒TPP-PLA-NPs的制备中的TPP-PLA载体材料用量为0.03g，荧光素用量为0.004g，无水乙醇用量为1mL，SDS浓度为2wt％、用量为7mL，超声搅拌时间为10min，功率为210w。
实施例4
TPP-PLA和TPP-PLA-NPs的制备方法同实施例1，区别在于：
荧光素纳米粒TPP-PLA-NPs的制备中的TPP-PLA载体材料用量为0.02g，荧光素用量为0.004g，无水乙醇用量为1mL，SDS浓度为2wt％、用量为6mL，超声搅拌时间为10min，功率为205w。
实施例5
TPP-PLA和TPP-PLA-NPs的制备方法同实施例1，区别在于：
荧光素纳米粒TPP-PLA-NPs的制备中的TPP-PLA载体材料用量为0.02g，荧光素用量为0.003g，无水乙醇用量为0.5mL，SDS浓度为2.5wt％、用量为5mL，超声搅拌时间为13min，功率为195w。
实施例6
TPP-PLA和TPP-PLA-NPs的制备方法同实施例1，区别在于：
荧光素纳米粒TPP-PLA-NPs的制备中的TPP-PLA载体材料用量为0.01g，荧光素用量为0.003g，无水乙醇用量为0.5mL，SDS浓度为2.5wt％、用量为5mL，超声搅拌时间为15min，功率为195w。
Claims (4)
1.一种线粒体靶向类高分子载体材料TPP-PLA，其特征在于，结构如下：
2.一种权利要求1所述的线粒体靶向类高分子载体材料TPP-PLA的制备方法，其特征在于，包括以下步骤：
(1)中间体三苯基膦接3-溴丙酸的合成：
将三苯基膦与3-溴丙酸溶于乙腈中，在真空环境氮气饱和下，回流搅拌，再使用无水乙醚沉淀产物，将沉淀物用乙腈重结晶，真空干燥得微黄色粉末，即为产物TPP-COOH；所述三苯基膦与3-溴丙酸的摩尔比为1:1；
(2)中间体BOC-氨基乙醇的合成：
将(BOC)2O溶解在三氯甲烷中，0℃下缓慢滴加到含有适量的氨基乙醇的三氯甲烷溶液中，缓慢升至室温，继续搅拌反应至反应完全，除去溶剂得到无色粘稠状液体，即为BOC-氨基乙醇；
(3)中间体BOC-氨基乙醇-聚乳酸（BOC-NH-PLA）的合成：
将BOC-氨基乙醇和辛酸亚锡溶于三氯甲烷中，加入L-丙交酯，抽真空除去三氯甲烷，在干燥氮气保护下，升温反应，反应后再次加入三氯甲烷溶解，无水乙醚4℃沉淀过夜，取沉淀真空干燥得白色沉淀，即为BOC-氨基乙醇-聚乳酸（BOC-NH-PLA）；所述升温反应的温度为120℃，反应时间为1-10小时；所述BOC-氨基乙醇、辛酸亚锡和L-丙交酯的摩尔比为1:100:100；
(4)中间体氨基封端的聚乳酸（PLA-NH2）的合成：
将BOC-氨基乙醇-聚乳酸溶于二氯甲烷中，加入适量盐酸饱和的四氢呋喃溶液，室温搅拌反应30min，真空去除溶剂，再加入二氯甲烷溶解，溶液用碳酸氢钠溶液洗涤，再用去离子水洗至水相为中性，有机层用无水硫酸钠干燥，无水乙醚沉淀，干燥得白色固体，即得氨基封端的聚乳酸（PLA-NH2）；
(5)高分子载体材料TPP-PLA的合成：
将三苯基膦溴丙酸、N-羟基丁二酰亚胺（NHS）、1-（3-二甲氨基丙基）-3-乙基碳二亚胺（EDCI）溶于乙腈中，加入三乙胺，室温搅拌后得到混合溶液；然后将氨基封端的聚乳酸溶于乙腈中，缓慢滴入混合溶液中，室温搅拌反应过夜；经去离子水洗涤反应物，将有机层醇沉，干燥即得载体材料TPP-PLA。
3.一种荧光素纳米粒的制备方法，其特征在于，包括以下步骤：
将权利要求1所述的载体材料TPP-PLA溶于二氯甲烷中，加入油相，形成相A；
取荧光素溶于无水乙醇中，再向里加入纯水，形成相B；
边搅拌相A，边缓慢滴加相B，继续搅拌形成初乳；
将 SDS溶液缓慢滴加进初乳中，搅拌形成复乳；
经搅拌固化挥发有机溶剂后，再经超声搅拌即得；
所述原料的用量如下：TPP-PLA载体材料0.01 g，二氯甲烷3 mL，油相4滴，荧光素0.003g，无水乙醇0.5 mL，纯水1 mL，2.5wt %SDS溶液5 mL；所述油相选自司盘-80、司盘-20、司盘-40、司盘-60、司盘-65和司盘-85中的一种或几种的混合。
4.一种荧光素纳米粒，其特征在于：采用权利要求3所述的方法制备。
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