CN115227673B - 一种具有尺寸调控的纳米载体及其制备方法和应用 - Google Patents
一种具有尺寸调控的纳米载体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种具有尺寸调控的纳米载体及其制备方法和应用，该纳米载体是使用聚己内酯‑聚乙二醇‑氨基(PCL‑PEG‑NH2)，水包油乳化法(O/W)制备小尺寸纳米粒子(SNPs)；再使用两端羧基(‑COOH)化的金属基质蛋白酶响应(MMP)的肽段将SNPs交联得到的大尺寸纳米粒子(LNPs)。本发明制备的纳米载体具备尺寸调控功能，实现在体内长循环时大尺寸、肿瘤内小尺寸，解决纳米药物在肿瘤中富集与渗透对纳米药物尺寸的矛盾要求，从而促进肿瘤的穿透，大幅提升癌症治疗的效率。
Description
技术领域
本发明涉及一种具有尺寸调控的纳米载体及其制备方法和应用，属于生物医用高分子聚合物材料技术领域。
背景技术
有研究表明，纳米载体进入血液循环后，为了使纳米载体能够高效地在肿瘤内富集，纳米粒子的尺寸被限制在100～200nm之间，尺寸太小会因血管渗漏或肾代谢而快速排出体外，尺寸过大则会被体内的生物屏障滞留。然而小尺寸纳米载体在肿瘤内具有良好的组织穿透性是当下的一种共识，小尺寸纳米载体能够有效地穿透到肿瘤组织深处，并取得理想治疗效果。这种纳米药物尺寸上的矛盾性严重限制了普通纳米载体的治疗效率，因此探索智能纳米载体在确保纳米药物能够实现在肿瘤内富集的前提下，提高其向肿瘤其向肿瘤深处的递释效率，是纳米药物提高化疗治疗效果所必具的属性。
因此，通过对纳米材料的设计，使其在注射到体内时维持较大尺寸(100～200nm)，进入肿瘤后变为较小尺寸(＜50nm)，可以有效解决纳米材料在肿瘤内富集与渗透的难题。这一尺寸策略在无机纳米材料中得到了验证，通过肿瘤响应的肽段将粒径在5nm左右的W18O49纳米粒子交联成粒径约50nm的大球，从而实现了体内长循环时为大尺寸球，到达肿瘤后解体为小尺寸球，提升了纳米材料在肿瘤内的富集与渗透。然而，如何实现有机纳米材料在肿瘤内大小尺寸转换，是目前尚未解决的一个难题。
发明内容
发明目的：本发明的目的在于提供一种具有尺寸调控的纳米载体，该有机纳米载体能在肿瘤内进行大小尺寸转换，解决纳米递释系统在血液循环和肿瘤穿透过程中的尺寸矛盾问题；本发明的第二目的在于提供构一种具有尺寸调控的纳米载体的制备方法，本发明的第三目的在于提供该具有尺寸调控的纳米载体在制备治疗肿瘤药物中的应用。
技术方案：本发明所述一种具有尺寸调控的纳米载体，所述纳米载体为LNPs，结构似“集束炸弹”，所述纳米载体LNPs是通过两端羧基化的肽段与SNPs纳米颗粒的-NH2反应交联得到，所述SNPs纳米颗粒是由聚己内酯-聚乙二醇-氨基(PCL-PEG-NH2)采用水包油的方法合成。
其中，所述纳米载体LNPs含有肿瘤过表达的金属基质蛋白酶响应的肽段，利用此设计来进行尺寸调控。
其中，所述两端羧基化的肽段的氨基酸残基序列为HOOC-Pro-Leu-Gly-Val-Arg-Gly-COOH，其中，Pro：脯氨酸；Leu：亮氨酸；Gly：甘氨酸；Val：缬氨酸；Arg：精氨酸。
其中，所述SNPs纳米颗粒的直径为5～15nm，纳米载体LNPs的直径为80～150nm。
本发明所述的具有尺寸调控的纳米载体的制备方法，包括以下步骤：
(1)将PCL-PEG-NH2溶于二氯甲烷(DCM)中得到DCM溶液，取吐温80加入去离子水中得到水溶液，将DCM溶液逐滴加入水溶液中，超声搅拌乳化反应，真空抽去DCM，过滤，离心，得到SNPs；
(2)将两端羧基化的肽段加入制得的SNPs溶液中，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基硫代琥珀酰亚胺(sulfo-NHS)催化搅拌反应，得到LNPs。
其中，步骤(1)中，所述PCL-PEG-NH2与DCM的摩尔比为1：0.8～1.2。
其中，步骤(1)中，所述吐温80的质量分数为2％～8％。
其中，步骤(1)中，所述DCM与吐温80的体积比为1:0.2～5
其中，步骤(1)中，所述超声搅拌乳化反应过程需在冰浴条件下完成，乳化时间为2～20min，。
其中，步骤(1)中，离心的过程中洗涤1～5次。
其中，步骤(2)中，PCL-PEG-NH2与两端羧基化的肽段的质量比为1：0.8～2。
其中，步骤(2)中，两端羧基化的肽段、EDC与sulfo-NHS的摩尔比为1:(0.5～4)：(0.5～4)。
其中，步骤(2)中，催化搅拌反应的温度为室温，催化搅拌的时间为6～48h。
本发明还包括所述具有尺寸调控的纳米载体在制备化疗癌症药物中的应用。
其中，所述纳米载体包覆阿霉素、紫杉醇或喜树碱中的任一种化疗药物。
本发明通过对有机纳米药物的设计，实现纳米药物尺寸与递释关系的统一。所述纳米载体LNPs由SNPs交联而成集束炸弹结构，纳米载体LNPs是通过两端-COOH化肽段交联SNPs变大的；所述的纳米载体LNPs在体内长循环时具有与之相匹配的较大尺寸，到达肿瘤部位后，由于肿瘤微环境具有过表达金属基质蛋白酶的特性，故采用金属基质蛋白酶响应的肽段交联SNPs，纳米载体到肿瘤部位时达成由大变小的尺寸调控。肿瘤过表达的金属基质蛋白酶激发纳米载体进行尺寸调控，释放小型尺寸的纳米粒子，更有利于肿瘤穿透。本发明所述纳米药物通过尺寸调控在体内实现体内长循环与渗透统一的作用示意图如图1所示：由图1可以看出，大尺寸纳米载体LNPs实现肿瘤富集后，在肿瘤内被高表达的金属基质蛋白酶解体为小尺寸纳米载体SNPs，进而增强其在实体瘤内的穿透效果。
有益效果：本发明和现有技术相比，具有以下显著优点：
(1)本发明的纳米载体LNPs一种可进行尺寸调控的有机纳米载体。
(2)在肿瘤富集的前提下，具有金属基质蛋白酶激发的尺寸调控，释放小尺寸纳米粒子，提高纳米药物利用率的同时改善肿瘤的治疗效果。
附图说明
图1为本发明所述纳米药物通过尺寸调控在体内实现体内长循环与渗透统一的作用示意图；
图2为SNPs透射电镜(TEM)示意图；
图3为LNPs TEM示意图；
图4为血液中cy5.5的相对荧光度值随时间变化曲线图。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
实施例1
1)将20mg PCL-PEG-NH2溶于1mL二氯甲烷(DCM)中得到DCM溶液，取1mL吐温80(质量分数为6％)加入20mL去离子水中得到水溶液。将DCM溶液逐滴加入水溶液中，使用超声机中冰水浴一边乳化、一边搅拌10min；然后通过真空泵抽去DCM，使用注射器过滤头过滤，10000r/min离心，洗涤两次后获得SNPs溶液；
2)将20mg两端羧基化的肽段加入制得的SNPs溶液中，肽段的氨基酸残基序列为HOOC-Pro-Leu-Gly-Val-Arg-Gly-COOH，其中，Pro：脯氨酸；Leu：亮氨酸；Gly：甘氨酸；Val：缬氨酸；Arg：精氨酸。加入11.4mg EDC和12.4mg sulfo-NHS催化搅拌反应24h，10000r/min离心、洗涤两次后得到LNPs。
实施例2
1)将20mg PCL-PEG-NH2溶于0.8mL二氯甲烷(DCM)中得到DCM溶液，取0.8mL吐温80(质量分数为2％)加入20mL去离子水中得到水溶液。将DCM溶液逐滴加入水溶液中，使用超声机中冰水浴一边乳化、一边搅拌2min；然后通过真空泵抽去DCM，使用注射器过滤头过滤，离心，洗涤两次后获得SNPs溶液；
2)将16mg两端羧基化的肽段加入制得的SNPs溶液中，肽段的氨基酸残基序列为HOOC-Pro-Leu-Gly-Val-Arg-Gly-COOH，其中，Pro：脯氨酸；Leu：亮氨酸；Gly：甘氨酸；Val：缬氨酸；Arg：精氨酸。加入7.8mg EDC和9.3mg sulfo-NHS催化搅拌反应6h，10000r/min离心、洗涤两次后得到LNPs。
实施例3
1)将20mg PCL-PEG-NH2溶于1.2mL二氯甲烷(DCM)中得到DCM溶液，取1.2mL吐温80(质量分数为8％)加入20mL去离子水中得到水溶液。将DCM溶液逐滴加入水溶液中，使用超声机中冰水浴一边乳化、一边搅拌20min；然后通过真空泵抽去DCM，使用注射器过滤头过滤，离心，洗涤两次后获得SNPs溶液；
2)将24mg两端羧基化的肽段加入制得的SNPs溶液中，肽段的氨基酸残基序列为HOOC-Pro-Leu-Gly-Val-Arg-Gly-COOH，其中，Pro：脯氨酸；Leu：亮氨酸；Gly：甘氨酸；Val：缬氨酸；Arg：精氨酸。加入15.7mg EDC和16.8mg sulfo-NHS催化搅拌48h，10000r/min离心、洗涤两次后得到LNPs。
对比例1
按文献(Sen Liu,Xing-Yu Luo,Shi-Yi Liu,Pei-Pei Xu*,Jian-Quan Wang*,Yong Hu*，Acetazolamide-Loaded pH-Responsive Nanoparticles Alleviating TumorAcidosis to Enhance Chemotherapy Effects.Macromol.Biosci.2019,19(2):1800366.)报道，将20mg PCL-PEG-NH2、2mg荧光染料Cy 5.5溶于2mL二氯甲烷(DCM)中，并逐滴加入到20mL去离子水中，使用超声机中冰水浴一边乳化、一边搅拌10min；然后通过真空泵抽去DCM，使用注射器过滤头过滤，离心，洗涤两次后获得不具备尺寸调控的大尺寸纳米粒子，根据文献报道该纳米粒子尺寸约为200nm。
实施例4
将实施例1中所制备的SNPs和LNPs，使用透射电镜观察其尺寸形貌，结果如图2-3所示，图2为SNPs的TEM图，图3为LNPs的TEM图。由图2-3可见，SNPs尺寸约7nm且分布均匀，即成功制备小型尺寸的纳米粒子；LNPs尺寸约100nm，图3中显示了较好的交联效果，证明成功构建了大型尺寸的纳米粒子。本发明制备纳米载体为智能纳米系统的级联递释的尺寸调控创造了良好条件。
实施例5
制备过程同实施例1中所制备的SNPs和LNPs，制备过程中向DCM加入2mg荧光染料Cy 5.5，制备荧光染料标记的SNPs和LNPs。将20mg PCL-PEG-NH2、2mg Cy5.5溶于1mL二氯甲烷(DCM)中得到DCM溶液，取1mL吐温80加入20mL去离子水中得到水溶液。将DCM溶液逐滴加入水溶液中，使用超声机中冰水浴一边乳化、一边搅拌10min，然后通过真空泵抽去DCM，使用注射器过滤头过滤，10000r/min离心，洗涤两次后获得荧光染料标记的SNPs；将20mg两端羧基化的肽段加入上述SNPs溶液中，肽段的氨基酸残基序列为HOOC-Pro-Leu-Gly-Val-Arg-Gly-COOH，其中，Pro：脯氨酸；Leu：亮氨酸；Gly：甘氨酸；Val：缬氨酸；Arg：精氨酸。加入11.4mg EDC和12.4mg sulfo-NHS催化搅拌24h，10000r/min离心、洗涤两次后，得到荧光染料标记的LNPs。
将荧光染料标记的SNPs和LNPs分别注射到荷瘤小鼠内，每隔10min取10μL血液，通过小动物活体成像仪测试其相对荧光强度(激发光波长：646nm；发射光波长：664nm)，以10min时荧光强度为1。结果如图4所示，图4为血液中cy5.5的相对荧光度值随时间变化曲线图。由图4可以看出，SNPs因其尺寸较小很快被清除出血液，而LNPs具有更好的血液半衰期，约为440min。
实施例6
实施例5中所制备的荧光染料标记的SNPs和LNPs以及按对比例1制备荧光标记的不具备尺寸调控的大尺寸纳米粒子。以肿瘤4T1细胞培育3D细胞球，分别加入上述三种纳米粒子，以及金属基质蛋白酶，2h后切片，通过激光共聚焦显微镜观察边缘、1/2半径、球心三个点的荧光强度，以边缘处荧光强度为1。结果如表1所示。
表1 3D细胞球内不同深度处的cy5.5的相对应荧光度值
由表1可以看出，SNPs有较好的肿瘤穿透效果。LNPs因其具有尺寸调控功能可以解体为小尺寸纳米粒子，因此也具有近似SNPs的肿瘤穿透效果。而对比例1中不具备尺寸调控的大尺寸纳米粒子因其尺寸较大，肿瘤穿透效果差。
Claims (7)
1.一种具有尺寸调控的纳米载体，其特征在于，所述纳米载体为LNPs，所述纳米载体LNPs是通过两端羧基化的肽段与SNPs纳米颗粒的-NH2反应交联得到，所述SNPs纳米颗粒是由PCL-PEG-NH2采用水包油的方法合成，所述两端羧基化的肽段的氨基酸残基序列为HOOC-Pro-Leu-Gly-Val-Arg-Gly-COOH，其中，Pro：脯氨酸；Leu：亮氨酸；Gly：甘氨酸；Val：缬氨酸；Arg：精氨酸。
2.根据权利要求1所述的具有尺寸调控的纳米载体，其特征在于，所述SNPs纳米颗粒的直径5~15 nm，纳米载体LNPs的直径为80~150 nm。
3.一种权利要求1-2任一项所述的具有尺寸调控的纳米载体的制备方法，其特征在于，包括以下步骤：
（1）将PCL-PEG-NH2 溶于DCM中得到DCM溶液，取吐温80加入去离子水中得到水溶液，将DCM溶液逐滴加入水溶液中，超声搅拌乳化反应，真空抽去DCM，过滤，离心，得到SNPs；
（2）将两端羧基化的肽段加入制得的SNPs溶液中，加入EDC和sulfo-NHS催化搅拌反应，得到LNPs。
4.根据权利要求3所述的具有尺寸调控的纳米载体的制备方法，其特征在于，步骤（1）中，所述PCL-PEG-NH2与DCM的摩尔比为1：0.8~1.2，所述吐温80的质量分数为2%~8%，所述DCM与吐温80的体积比为1:0.2~5，所述超声搅拌乳化反应过程需在冰浴条件下完成，乳化时间为2~20 min，离心的过程中洗涤1~5次。
5.根据权利要求3所述的具有尺寸调控的纳米载体的制备方法，其特征在于，步骤（2）中，PCL-PEG-NH2与两端羧基化的肽段的质量比为1：0.8~2，两端羧基化的肽段、EDC与sulfo-NHS的摩尔比为1: 0.5~4：0.5~4，反应温度为室温，催化搅拌的时间为6~48h。
6.权利要求1-2任一项所述具有尺寸调控的纳米载体在制备治疗肿瘤药物中的应用。
7.根据权利要求6所述的应用，其特征在于，所述纳米载体包覆阿霉素、紫杉醇或喜树碱中的任一种化疗药物。
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