CN114848834A - 一种双药物共递的复合多层纳米载体及其制备方法和应用 - Google Patents
一种双药物共递的复合多层纳米载体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种双药物共递的复合多层纳米载体的制备方法：(1)通过两层生长法合成亲水/疏水多层复合纳米递药载体HCP；(2)通过扩散‑富集封装法将疏水药物和亲水药物分别封装在亲水/疏水多层复合纳米递药载体HCP中，得到双药物共递的复合多层纳米载体。本发明还公开了上述制备方法得到的双药物共递的复合多层纳米载体及在制备抗肿瘤药物中的应用。该药物载体可以同时搭载亲水药物和疏水药物，从而实现不同性状药物的组合治疗，发挥药物协同作用；该药物可以通过调节聚合物的聚合度、亲疏水嵌段比例来实现对粒径大小和载药率的控制；该药物载体提高了抗肿瘤药物的理化稳定性并赋予药物pH响应性，提升药物治疗效果，减少毒副作用。
Description
技术领域
本发明涉及药物载体，具体涉及一种双药物共递的复合多层纳米载体及其制备方法和应用。
背景技术
恶性肿瘤的分子靶向治疗是当前的研究热点。单药分子靶向治疗的效果往往不够理想，其中原因包括复杂的肿瘤微环境、药物的耐药性等。通过研发新型纳米药物载体、探索针对肿瘤微环境的药物组合策略，可以提升肿瘤分子靶向药物的临床疗效。
纳米药物载体可以通过改善药物溶解度、延长药物在血浆中的循环时间、提供可控的药物释放、赋予药物靶向性等方式来增强药物的治疗效果。超支化聚缩水甘油(HPG)是一类分子内部富含醚键而分子表面富含羟基的超支化聚合物，具有优良的水溶性与生物相容性。HPG与PEG的结构相似，具有避免单核吞噬细胞系统(MPS)吸收的能力，并且在热和氧化方面比PEG更稳定。疏水衍生化HPG最初是作为血清白蛋白替代品开发的，用作血浆扩增剂。这些疏水修饰的单分子HPG胶束具有优势，因为它们在各种环境影响(如稀释，剪切力和pH值)下具有显着的稳定性。在既往的研究中，疏水修饰的HPGs药物载体已被成功验证为可通过局部滴注到膀胱中治疗膀胱癌。然而，针对HPG的静脉给药药物递送系统的研究仍待进一步展开。
肿瘤相关成纤维细胞(Cancer-Associated Fibroblasts，CAFs)是肿瘤微环境的重要组成部分。CAFs的激活受到多种因素调节，激活后能发挥多种重要功能。CAFs可以反馈作用于肿瘤细胞，进一步形成可促进肿瘤细胞恶性进展的肿瘤微环境；CAFs还可以保护肿瘤细胞抵抗化疗药物的细胞毒作用，增加肿瘤化疗耐药性；CAFs还可以影响细胞外基质(extracellular matrix，ECM)的构成，造成基质硬化、压迫血管、微环境缺氧，进而减少药物递送、促进肿瘤浸润转移。因此，针对CAFs的治疗可以潜在增强化疗药物的治疗效果。
卡泊三醇是维生素D受体(VDR)的激动剂，具有抗增殖能力，并能增强包括吉西他滨在内的多种抗癌药物的细胞毒性。作为VDR激动剂，卡泊三醇可以诱导肿瘤间质重塑，增加肿瘤内吉西他滨的投递。卡泊三醇的性状为白色或近白色结晶粉末，极易溶于乙醇，溶于氯仿和丙二醇，微溶于二氯甲烷，在水中的溶解度低。常被用作油脂质体的包裹对象。吉西他滨是是核酸合成抑制剂，是一类常用的化疗药物。吉西他滨盐酸盐极性较强，易溶于水。
综上所述，HPG具有成为理想药物载体的前景；适当的药物组合方案可以显著提升药物的治疗效果。
发明内容
本发明的目的在于提供了一种双药物共递的复合多层纳米载体及其制备方法和应用；该载体可以实现两种不同水溶性药物的联载和得到高负载量的纳米药物，可以提升药物在生物体内的稳定性，增强治疗效果。
本发明提供如下技术方案：
一种双药物共递的复合多层纳米载体的制备方法，所述制备方法包括：
(1)通过两层生长法合成亲水/疏水多层复合纳米递药载体HCP；
(2)通过扩散-富集封装法将疏水药物和亲水药物分别封装在亲水/疏水多层复合纳米递药载体HCP中，得到双药物共递的复合多层纳米载体；
其中HCP的结构式为：
本发明提供的方法可以赋予超支化聚缩水甘油药物载体以同时负载疏水药物和亲水药物的功能，实现了两种不同水溶性药物的联载；本发明提供的制备方法简单有效，可以得到具有高负载量的纳米药物；本发明提供的制备方法具有良好的可控性，可以通过调节聚合物的聚合度、亲疏水嵌段比例来实现对粒径大小和载药率的控制；本发明提供的方法可以提升药物在生物体内的稳定性；本发明提供的方法可以使药物获得pH响应性，促进药物更多地在低pH的肿瘤环境中释放，提升靶向性，增强治疗效果，减少毒副作用。
在步骤(1)中：利用了阴离子开环聚合法，以三羟甲基丙烷为起始剂，经甲醇钾的离子化后形成了活性阴离子；升温后加入缩水甘油和聚乙二醇缩水甘油基十二烷基醚的混合物，反应得到形成了具有亲水内核-疏水层-响应性亲水空间的结构的亲水/疏水多层复合纳米递药载体HCP。
所述三羟甲基丙烷、甲醇钾、缩水甘油和聚乙二醇缩水甘油基十二烷基醚的投料比为0.68g:0.98g:(20～40)mL:(10～20)mL，其中聚乙二醇缩水甘油基十二烷基醚中的聚乙二醇嵌段的相对分子质量为400～4000。
在步骤(2)中，所述扩散-富集封装法为：将HCP、疏水药物和亲水药物混合于共同良溶剂中，搅拌后疏水药物和亲水药物富集于HCP中的不同嵌段中；去除溶剂以及游离或不溶的疏水药物和亲水药物，旋干获得双药物共递的复合多层纳米载体。
优选地，所述HCP、疏水药物、亲水药物的质量比分别为1g:20～60mg:20～80mg。
进一步优选地，所述亲水药物为吉西他滨，所述疏水药物为卡泊三醇。
本发明还提供了一种根据上述制备方法得到的双药物共递的复合多层纳米载体。
所述双药物共递的复合多层纳米载体包括亲水/疏水多层复合纳米递药载体HCP，以及包埋于亲水/疏水多层复合纳米递药载体HCP中的吉西他滨(Gemcitabine，标记为Gem)和卡泊三醇(Calcipotriol，标记为Cal)，两者在HCP中的负载量分别为5～60mg/g(HCP)和2～50mg/g(HCP)。
本发明提供的制备方法合成方法简单，1g制备的双重药物载体中，抗肿瘤药物的负载量最高可达50～60mg/g，提高了药物的利用率和药效。
本发明还提供了一种上述制备方法得到的双药物共递的复合多层纳米载体在制备抗肿瘤药物中的应用。
同现有技术相比，本发明的有益效果体现在：
(1)本发明中，超支化聚缩水甘油药物载体包含疏水内核，可以同时搭载亲水药物和疏水药物，从而实现不同性状药物的组合治疗，发挥药物协同作用，提升药物的临床治疗效果。
(2)本发明提供的制备方法具有良好的可控性，可以通过调节聚合物的聚合度、亲疏水嵌段比例来实现对粒径大小和载药率的控制。
(3)本发明提供的方法所制备的药物具有良好的pH响应性，可促进药物更多地在酸性的肿瘤环境中释放，增强抗肿瘤效果。
(4)本发明提供的方法可以提升药物在生物体内的稳定性。
附图说明
图1为实施例1制备的HCP的合成机理图；
图2为实施例1制备的HCP的核磁共振氢谱分析图；
图3为实施例1、2制备的HCP、Gem@HCP、Cal-Gem@HCP的核磁共振氢谱分析图；
图4为实施例1制备的HCP的核磁共振碳谱分析图；
图5为实施例1制备的HCP的红外光谱图；
图6为酸性环境下实施例2制备的Cal-Gem@HCP的药物释放曲线图；
图7为实施例1、2分别制备的载药体系在抑制肿瘤细胞活性中的作用分析图。
具体实施方式
下面结合具体的实施例对本发明作进一步说明。
实施例1：制备HCP
聚合反应的机理如图1所示。反应在装有机械搅拌器的三颈圆底烧瓶中进行。三颈瓶经烘箱烘干后取出，加入计量好的三羟甲基丙烷，加热至60℃溶解后，以抽气-通氮气方式驱赶瓶内的空气与水气，而后以注射器注入经称量并溶于少量无水甲醇(2mL)的甲醇钾，磁力搅拌30min后抽真空除去甲醇，升温至95℃，并在氮气保护下利用注射连续24h滴加精制好的缩水甘油和聚乙二醇缩水甘油基十二烷基醚的混合物，随后继续反应3小时。加入50mL无水甲醇后，搅拌为均一的溶液。随后经以氢离子交换树脂柱中和获得中性的大分子。经过旋蒸蒸去大部分甲醇后倾入250mL无水乙醚中，经磁力搅拌半个小时得到粘稠的聚合物，弃去乙醚，重复2遍。再以去离子水溶解收集在截留分子量为3500的透析袋中，在去离子水中透析3天，每天更换3次水。最后透析袋中的液体在真空冻干机中冻干，得到白色的粘稠液体。
合成过程中，所使用的三羟甲基丙烷、甲醇钾、缩水甘油和聚乙二醇缩水甘油基十二烷基醚的投料比为0.68g:0.98g:(20～40)mL:(10～20)mL，其中聚乙二醇嵌段的相对分子质量为400～4000。获得的HCP的数均相对分子质量为3000～16,000。
在本实施例中，所使用的三羟甲基丙烷、甲醇钾、缩水甘油和聚乙二醇缩水甘油基十二烷基醚的投料比为0.68g:0.98g:40mL:20mL。
实施例2：制备Gem@HCP、Cal@HCP、Cal-Gem@HCP
找到亲水性药物与疏水性药物两种药物和HCP的共同良溶剂甲醇，三者混合于甲醇中，持续搅拌3h。两种药物将根据其自身的极性富集于HCP中的不同嵌段中。随后，将上述甲醇溶液在60℃中旋干。氮气流通过小瓶内部以去除残留溶剂。所得产物先倾倒入乙酸乙酯中洗去游离的卡泊三醇。最后再用乙醇溶解产物，滤去不溶的吉西他滨颗粒，旋干后即可获得双药物共递的复合多层纳米载体(Cal-Gem@HCP)。Gem@HCP、Cal@HCP的合成过程同上，区别在于投料过程分别只投入吉西他滨或者卡泊三醇。
其中，HCP、疏水药物、亲水药物的投料比分别为1g:20～60mg:20～80mg。上述合成过程中，卡泊三醇的负载量为2～50mg/g(HCP)。吉西他滨的负载量为5～60mg/g(HCP)。
本实施例中，制备得到的Gem@HCP负载量为：39.6mg/g、Cal@HCP负载量为30.5mg/g、Cal-Gem@HCP的负载量为42.5mg(Gem)/g和36.3mg(Cal)/g。
表征试验1：核磁共振氢谱分析
以重水为溶剂，在500MHz核磁共振波谱仪上测HCP的1H NMR谱图(见图2)。由谱图中可知，δ4.2处为聚缩水甘油末端与酯基相连的亚甲基的质子吸收峰(HPG-CH2-OOCR)、δ3.0～4.0ppm的宽峰为聚缩水甘油核中的亚甲基和次甲基质子吸收峰；b峰δ2.3处为十六烷基酰氯酯化后靠近羰基的亚甲基质子吸收峰(HPG-COO-CH2)；c峰δ1.6ppm处为HPG-COO-CH2-CH2质子峰；d峰δ1.3处为烷基链中亚甲基的质子吸收峰((CH2)12)；e峰δ0.8处为烷基链尾甲基质子吸收峰。Gem@HCP和Cal-Gem@HCP两个体系的1H NMR谱图见图3。对比单载吉西他滨与双载的体系，在δ6.0～8.0ppm之间均出现有两种分子的信号峰，同时，在δ1.0～3.0ppm之间出现了较多的卡泊三醇的信号峰，这些结果均表明了吉西他滨和卡泊三醇的成功搭载。
表征试验2：核磁共振碳谱分析
采用核磁共振碳谱来表征，以进一步证实HCP的结构。其结果如图4所示。在13C NMR中，40ppm之前的三个峰归属于正十二烷基上的三种类型的碳的峰。而60～85ppm之间的稍宽的峰则属于聚合物结构的峰。因PEO与HPG一样，都是聚醚的骨架结构，因此在13C NMR上存在较大的重叠而在谱图中看起来是连绵不断的峰。
表征试验3：FTIR谱图分析
图5中的HCP的红外谱图则进一步证实了该聚合物的结构。从图中可知，3580cm-1处为聚缩水甘油及PEO端基上的羟基峰，1200cm-1处的中等强度信号峰为C-O-C的醚键不对称伸缩震动峰，这可归属于超支化聚缩水甘油基PEO链段上含的聚醚型的结构。另外，在2956cm-1和2810cm-1处饱和长链烷烃端基CH3的不对称与对称振动吸收峰，加上1460cm-1与720cm-1两处的尖峰为长链烷基中四个以上CH2叠加而生成的，则表明了饱和碳氢的正十二烷基的存在。
表征试验4：粒径及分布分析
纳米颗粒粒径的表征，将HCP、Gem@HCP和Cal-Gem@HCP三个体系与去离子水混合，配制成均一的溶液。取2mL溶液利用动态光散射法测定其中的粒径及其分布，得到如表1所示的结果。可以看出，三元复合的载药体系的粒径更小，且多分散系数较低。
表1三种纳米载体的粒径分析
性能试验1：酸性环境下两种药物的释放速度分析
根据吉西他滨与卡泊三醇的溶解性，选用了以下的色谱条件。色谱柱：IntersilC18(250mm×4.6mm，5μm)；流动相：甲醇-乙腈-磷酸盐缓冲液(0.01mol·L-1磷酸氢二铵溶液，用磷酸调pH至6.0)＝58:22:20；流速：1.0mL·min-1；柱温：25℃；检测波长：302nm；进样量：100μL。将Cal-Gem@HCP放置上述pH环境的流动相中进行透析，隔一段时间取外面的透析液测定。在弱酸性环境中，HCP上的PEO链段首先发生离子化，进而导致链段间的相互排斥，而使其中搭载的吉西他滨优先释放出来。而卡泊三醇的极性更接近于HCP上的疏水部分，而更易被负载在疏水部分，在流动相的环境中，卡泊三醇的释放速度要慢于吉西他滨。两种药物释放的曲线如图6所示。
性能试验2：药物载体在抗肿瘤治疗中的作用分析
将药物载体在中性液体环境中(pH 7.4，MEM培养基)测试细胞毒性。以人源性胆管细胞癌细胞株CCLP-1为对象，采用CCK-8试验分析药物对肿瘤细胞的作用(作用48小时)。在中性环境中，Gem@HCP、Cal-Gem@HCP等具有良好的肿瘤细胞杀伤功能，而HCP本身无明显细胞毒性(图7)。
Claims (9)
1.一种双药物共递的复合多层纳米载体的制备方法，其特征在于，所述制备方法包括：
(1)通过两层生长法合成亲水/疏水多层复合纳米递药载体HCP；
(2)通过扩散-富集封装法将疏水药物和亲水药物分别封装在亲水/疏水多层复合纳米递药载体HCP中，得到双药物共递的复合多层纳米载体；
其中HCP的结构式为：
2.根据权利要求1所述的双药物共递的复合多层纳米载体的制备方法，其特征在于，在步骤(1)中：利用了阴离子开环聚合法，以三羟甲基丙烷为起始剂，经甲醇钾的离子化后形成了活性阴离子；升温后加入缩水甘油和聚乙二醇缩水甘油基十二烷基醚的混合物，反应得到形成了具有亲水内核-疏水层-响应性亲水空间结构的亲水/疏水多层复合纳米递药载体HCP。
3.根据权利要求2所述的双药物共递的复合多层纳米载体的制备方法，其特征在于，所述三羟甲基丙烷、甲醇钾、缩水甘油和聚乙二醇缩水甘油基十二烷基醚的投料比为0.68g:0.98g:(20～40)mL:(10～20)mL，其中聚乙二醇缩水甘油基十二烷基醚中的聚乙二醇嵌段的相对分子质量为400～4000。
4.根据权利要求1所述的双药物共递的复合多层纳米载体的制备方法，其特征在于，在步骤(2)中，所述扩散-富集封装法为：将HCP、疏水药物和亲水药物混合于共同良溶剂中，搅拌后疏水药物和亲水药物富集于HCP中的不同嵌段中；去除溶剂以及游离或不溶的疏水药物和亲水药物，旋干获得双药物共递的复合多层纳米载体。
5.根据权利要求4所述的双药物共递的复合多层纳米载体的制备方法，其特征在于，所述HCP、疏水药物、亲水药物的质量比分别为1g:20～60mg:20～80mg。
6.根据权利要求1所述的双药物共递的复合多层纳米载体的制备方法，其特征在于，所述亲水药物为吉西他滨，所述疏水药物为卡泊三醇。
7.一种权利要求1-6任一所述的制备方法得到的双药物共递的复合多层纳米载体。
8.根据权利要求7所述的双药物共递的复合多层纳米载体，其特征在于，所述双药物共递的复合多层纳米载体包括亲水/疏水多层复合纳米递药载体HCP，以及包埋于亲水/疏水多层复合纳米递药载体HCP中的吉西他滨和卡泊三醇，两者在HCP中的负载量分别为5～60mg/g(HCP)和2～50mg/g(HCP)。
9.一种权利要求1-6任一所述的制备方法得到的双药物共递的复合多层纳米载体在制备抗肿瘤药物中的应用。
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