CN116570719A - 一种左旋肉碱-tpgs纳米靶向药物载体的制备方法及用途 - Google Patents
一种左旋肉碱-tpgs纳米靶向药物载体的制备方法及用途 Download PDFInfo
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Abstract
本发明公开了一种左旋肉碱‑TPGS（Lc‑TPGS）纳米靶向药物载体，结构式如（I）所示：本发明的新型纳米药物载体具有良好的生物相容性和抗肿瘤活性。通过细胞成像技术，活体成像技术和细胞毒活性实验表明，在实现靶向抗癌方面，Lc‑TPGS具有较高的应用潜能。
Description
技术领域
本发明涉及药物化学领域，特别涉及靶向纳米药物载体的设计合成及用途。
背景技术
化疗是目前治疗恶性肿瘤的主要方法，但由于细胞毒性药物代谢快及具有非特异性生物分散的特点，使化疗伴有强烈的副作用，导致治疗效果不佳。此外，许多化疗药物水溶性差，限制了其临床应用。
纳米载体是增强疏水性药物传递的常用策略之一。由两亲性分子形成的纳米胶束通常被用作疏水药物纳米载体，将活性药物成分装载在疏水核中，随后载药体系在血液循环过程中将药物释放到预定的作用部位。聚乙二醇维生素E琥珀酸酯（TPGS）是一种新型的非离子型表面活性剂，其在溶液状态下形成的TPGS胶束作为药物递送系统中的纳米材料有着广泛的应用。基于TPGS纳米载药体系不仅具有良好的生物相容性、促进内皮细胞吸收、延长血液循环时间和改善药物水溶性等先进而优异的特性，而且还具有依赖于肿瘤血管壁内皮细胞的高渗透性（EPR效应）主动靶向肿瘤组织。此外，TPGS中的维生素E作为p-糖蛋白抑制剂，可以降低由p-糖蛋白作用下的药物外流，减少细胞耐药性，提高药物利用率。因此，基于TPGS的给药纳米系统在抗癌领域得到了广泛的关注。通常，肿瘤组织中富集的纳米药物通过内吞作用被引入癌细胞。如果在纳米药物表面修饰某些癌细胞靶向分子，药物或其载体将被赋予与癌细胞主动结合的能力。这将有效提高癌细胞对药物的摄取速率和转运量，并减少对邻近正常组织的毒副作用。例如，Yao等人合成了甘草次酸修饰的TPGS聚合胶束，可用于肝细胞癌靶向治疗。
左旋肉碱是一种细胞进行正常生理活动所必需的维生素类似物。新型有机阳离子转运蛋白2（OCTN2）是依赖于Na+的左旋肉碱小肠吸收的转运体。研究发现，OCTN2蛋白不但广泛分布于各种正常器官和组织中，如肾脏、肝脏、上皮细胞、结肠上皮细胞，并且在大多数癌细胞表面呈现高表达。因此，基于左旋肉碱-OCTN2特异性相互作用，可以将具有抗癌作用的肉碱类似物或肉碱修饰的纳米抗癌药物进行口服递送，并最终实现靶向抗癌。
因此，为了增强TPGS纳米载体对负载的疏水性化疗药物的肿瘤靶向性，减少其副作用的产生，设计了左旋肉碱-TPGS聚合物，并研究了其对疏水性化疗药物喜树碱（CPT）的负载效率，及纳米药物负载体系的体外抗癌活性。
发明内容
为解决上述问题，本发明提出的技术方案为：一种左旋肉碱-TPGS（Lc-TPGS）纳米靶向药物载体，结构式如下所示：
（I）
一种Lc-TPGS纳米靶向药物载体的制备方法，包括如下步骤：
步骤（1）：将摩尔比为1:1:1.2 的TPGS、 4-二甲氨基吡啶（DMAP）和丁二酸酐摩尔溶于适量 1,4二氧六环中；将混合物在80℃下搅拌6小时；之后，真空蒸发系统中的1,4二氧六环；适量二氯甲烷（DCM） 重新溶解残余物并过滤去除杂质；滤液依次以HCl溶液，饱和碳酸氢钠溶液和饱和食盐水洗涤，干燥后滴加过量冰乙醚重结晶得TPGS-COOH；
步骤（2）：左旋肉碱酯化采用活化TPGS-COOH方法，首先， 将1.2mol的TPGS-COOH溶解于二氯亚砜中，添加催化剂N,N-二甲基甲酰胺，在70℃下回流活化4h，然后将溶剂二氯亚砜在真空中挥发，剩余物质重新溶于N,N-二甲基甲酰胺（DMF）中，将1.2mol的左旋肉碱溶解于DMF中，再滴加到上述反应体系中并在25℃下搅拌12h，反应结束后，通过透析袋（MWCO=1000）将粗产物的水溶液透析3天，并通过冷冻干燥获得黄白色粉末的Lc-TPGS最终产物。
反应式如下：
本发明的有益效果为：本发明的新型纳米药物载体具有良好的生物相容性和抗肿瘤活性。通过细胞成像技术，活体成像技术和细胞毒活性实验表明，在实现靶向抗癌方面，Lc-TPGS具有较高的应用潜能。
说明书附图
图1为TPGS核磁共振氢谱图；
图2为TPGS-COOH核磁共振氢谱图；
图3为Lc-TPGS核磁共振氢谱图；
图4为Lc-TPGS(上图)及TPGS（下图）质谱图；
图5为利用活体成像仪探究Lc-TPGS纳米载体生物分散性，A）：Lc-TPGS，TPGS纳米载体的肿瘤靶向性。B，C）：Lc-TPGS,TPGS纳米载体在生物体内的分布；
图6探究细胞对Lc-TPGS的摄取行为研究，A，B）：LM880探究香豆素6介导的MCF-7对纳米载体的摄取行为。C,D）：探究MCF-7摄取纳米粒子的定量分析；
图7为利用MTT法表征的MCF-7细胞与不同浓度的喜树碱（CPT）、TPGS负载CTP的纳米药物TPGS-CPT、或Lc-TPGS负载CTP的纳米药物Lc-TPGS-CPT作用48小时后的细胞存活率。
具体实施方式
下面结合实施例对本发明作进一步说明。需要说明的是，下述实施例仅是用于说明，而并非用于限制本发明。本领域技术人员根据本发明的教导所做出的各种变化均应在本申请权利要求所要求的保护范围之内。
实施例1
Lc-TPGS的制备方法具体如下：
（1）将800 mg TPGS和54 mg丁二酸酐溶解于20ml 1,4二氧六环中，并加入65mgDMAP作催化剂，80℃下回流搅拌6h。溶剂1,4二氧六环蒸发后重新溶于冷的二氯甲烷（DCM）并析出固体后过滤，滤液依次用稀HCl溶液，饱和碳酸氢钠溶液和饱和食盐水洗三次后加入无水硫酸钠固体干燥，干燥后滴加过量冰乙醚得白色固体。
1H NMR (500 MHz, CDCl3) ：δ 2.4 pmm为特征峰。
（2）将500毫克步骤（1）中的得到的固体溶解在10毫升二氯亚砜，滴加2滴DMF作为催化剂，使得TPGS-COOH的羧基被活化，回流活化4小时后蒸干溶剂二氯亚砜并重新溶于DMF中。把101 mg的左旋肉碱溶解于1ml DMF中，然后滴加到上述反应体系中并在25℃下搅拌12h。反应结束后溶液以1000 mW透析袋以水透析3天，冻干得黄色固体。
1H NMR (500 MHz, CDCl3)：δ 3.0 pmm，4.6 pmm；
HR-MS[774.5156 + 44.0262n + 1 = (M+H+)]，观察值：1699.9529。
实施例2
为了探究Lc-TPGS在生物体内靶向肿瘤性能，Balb/c小鼠通过将5×1064T1细胞注射到右前肢腋下来建立异种移植物肿瘤模型。采用与Lc-TPGS-CPT药物类似的方法制备了疏水性近红外染料DiR负载的TPGS-DiR和Lc-TPGS-DiR系统，并用于证明Lc-TPGS载体有效靶向肿瘤的能力。当肿瘤体积接近500mm3时，灌胃方式将Lc-TPGS-DiR（200µL，0.4mg DiR/kg）、TPGS-DdR（200μL，0.4mgDIR/kg）或PBS（空白）分别注射到荷瘤小鼠中。如图5A所示，在不同的时间点（0.5、2、4、6、8、10、12和24小时），通过吸入异氟烷气体麻醉小鼠，然后使用PerkinElmer IVIS光谱体内成像系统进行荧光成像。最后如图5B,C所示，人道处死三只小鼠，采集它们的心脏、肝脏、脾脏、肺、肾器官和肿瘤组织进行离体荧光成像。实验证明，随着时间变化，Lc-TPGS实验组在肿瘤区域的荧光强度逐渐增强，然而TPGS在肿瘤区域的荧光变化较少。说明Lc-TPGS通过血液循环更容易累积并渗入肿瘤。
实施例3
如图6A所示，为了探究Lc-TPGS对肿瘤细胞的亲和力，用香豆素6（C6）负载的Lc-TPGS或TPGS系统（Lc-TPGS-C6或TPGS-C6），以MCF-7细胞研究Lc-TPGS-C6或TPGS-C6系统的细胞摄取行为。简单地说，将MCF-7细胞转移到几个细胞密度为8×103的共聚焦培养皿中。在37℃孵育24小时后，将相同浓度（300 ng/mL C6）的Lc-TPGS-C6或TPGS-C6体系分别加入不同的细胞样品中。处理后的细胞在培养箱中进一步培养并在不同的时间点（30、60和120分钟），随后用PBS依次洗涤细胞样品三次，并在激发波长为485nm的共聚焦激光扫描显微镜下观察。
通过类似的处理，流式细胞术分析了细胞样品的荧光强度。如图6B,C所示在用Lc-TPPS-C6或TPGS-C6系统处理后，首先用PBS在不同时间点（30、60、120、240、360和480分钟）洗涤每个MCF-7细胞样品三次，然后用胰蛋白酶消化。以3000rpm离心细胞悬浮液1分钟，除去上清液，然后加入不含BSA的培养基DMEM以重新悬浮细胞。最后，用绿色荧光通道（507nm）通过流式细胞术分析处理的细胞悬浮液。实验表明，Lc-TPGS对肿瘤细胞MCF-7亲和力远高于TPGS纳米粒子，流式细胞仪定量分析证明，Lc-TPGS的摄取效率明显高于TPGS。
实施例4
采用MTT法检测不同样本处理后人乳腺癌症细胞MCF-7的存活率。实验系统被分为三组。它们包括游离CPT组、空白胶束组（Lc-TPGS或TPGS）和CPT负载的纳米药物组（Lc-TPGS-CPT或TPGS-CPT）。
CPT负载纳米药物组的实验过程描述如下。MCF-7细胞在含有10% BSA的DMEM高葡萄糖培养基中在细胞培养箱（37ºC和5%CO2）中培养24小时。当细胞密度达到5×103时，去除培养基，加入100µL含有不同浓度Lc-TPGS-CPT和TPGS-CPT胶束的新培养基。实验系统中CPT的最终浓度分别为30、25、20、15、10和5μM。所有实验系统中Lc-TPGS或TPGS载体的相应浓度均低于200µg/ml。在培养箱中培养细胞48小时后，吸出培养基并加入2.5mg/ml MTT溶液再培养4小时。然后，去除培养基，加入100µl二甲基亚砜，并用微孔板读数器测量各组在490nm波长下的吸光度。如图7例所示，MTT实验证明，TPGS-CPT或Lc-TPGS-CTP处理的MCF-7细胞的存活率呈下降趋势，IC50分别为CPT：7.8μM，Lc-TPGS-CPT：3.12μM，TPGS-CPT：1.9μM。证明Lc-TPGS具有较高的抗肿瘤潜力。
Claims (3)
1.一种左旋肉碱-TPGS纳米靶向药物载体：结构式如下：
（I）。
2.根据权利要求1所述的通式（I）的纳米靶向药物载体在制备抗癌药物中的用途。
3.根据权利要求1所述的左旋肉碱-TPGS纳米靶向药物载体的制备方法，其特征在于，包括如下步骤：
步骤（1）：将摩尔比为1:1:1.2 的TPGS、 4-二甲氨基吡啶（DMAP）和丁二酸酐摩尔溶于适量 1,4二氧六环中；将混合物在80℃下搅拌6小时；之后，真空蒸发系统中的1,4二氧六环；适量二氯甲烷（DCM） 重新溶解残余物并过滤去除杂质；滤液依次以HCl溶液，饱和碳酸氢钠溶液和饱和食盐水洗涤，干燥后滴加过量冰乙醚重结晶得TPGS-COOH；
步骤（2）：将TPGS-COOH溶解于二氯亚砜中，添加催化剂N,N-二甲基甲酰胺，在70℃下回流活化4h，然后将溶剂二氯亚砜在真空中挥发，剩余物质重新溶于N,N-二甲基甲酰胺（DMF）中；将左旋肉碱溶解于N,N-二甲基甲酰胺中，再滴加到上述反应体系中并在25℃下搅拌12h，反应结束后，通过透析袋（MWCO=1000）将粗产物的水溶液透析3天，并通过冷冻干燥获得黄白色粉末的Lc-TPGS最终产物。
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