CN105832705A - 一种特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒 - Google Patents
一种特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒 Download PDFInfo
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Abstract
本发明涉及一种特异加速肿瘤细胞凋亡及疗效自监测智能纳米粒的制备方法。该智能纳米粒以聚(D，L‑丙交酯‑co‑乙交酯)‑聚乙二醇、聚(D，L‑丙交酯‑co‑乙交酯)‑聚醚酰亚胺、二硬脂酰基磷脂酰乙醇胺‑聚乙二醇‑叶酸为载体，通过一步自组装过程包裹疏水性抗肿瘤药物和半胱氨酸天冬氨酸蛋白酶(caspase‑3)底物荧光肽制备得到。该纳米粒能特异性识别叶酸受体高表达的肿瘤细胞，通过受体介导的内吞进入溶酶体。借助聚醚酰亚胺的“质子海绵效应”，使溶酶体膜通透化，加快药物从溶酶体逃逸并诱导肿瘤细胞凋亡，激活细胞凋亡的标志性蛋白酶caspase‑3，剪切底物荧光肽，产生荧光信号用于疗效实时监测，实现肿瘤细胞的精准治疗，极具肿瘤诊疗应用前景。
Description
一、技术领域
本发明属于医药技术领域，具体涉及一种特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒及其制备方法。
二、背景技术
肿瘤严重威胁着人类的健康和生命，且发病率和死亡率呈现逐年上升趋势。目前化疗在肿瘤临床治疗中起着重要的作用，但仍存在以下几个尚未解决的关键问题：大部分抗肿瘤药物无靶向性，导致毒副作用大；大部分化疗药物的靶点位于细胞内，而药物进入细胞过程中通常会经历溶酶体降解途径，导致药物药效降低，发挥药效时间延长；传统的化疗难以及时反映治疗效果，导致疗效不佳或过度治疗。随着分子识别、荧光成像、纳米等技术的快速发展与融合，为克服上述问题，发展新型纳米药物、实现肿瘤精准治疗带来了新契机。
让药物选择性地在病变部位释放并加快药物与靶点的作用过程，及时反馈药物与靶点作用后引起的生物学变化，可以实现精准用药，有效避免治疗不足或过度治疗。本发明首次利用聚(D，L-丙交酯-co-乙交酯)-聚乙二醇、聚(D，L-丙交酯-co-乙交酯)-聚醚酰亚胺、二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸为载体，通过一步自组装过程包裹疏水性抗肿瘤药物和半胱氨酸天冬氨酸蛋白酶(caspase-3)底物荧光肽，合成了一种加速肿瘤细胞凋亡及疗效自监测的智能纳米粒。该智能纳米粒利用表面的叶酸分子对叶酸受体高表达的肿瘤细胞进行特异性识别，通过受体介导的内吞过程进入肿瘤细胞的溶酶体。溶酶体内部酸性的pH环境使聚醚酰亚胺质子化，导致纳米粒崩解释放出药物。同时，由于聚醚酰亚胺的“质子海绵效应”，使溶酶体膜通透化，药物从溶酶体逃逸并快速与靶点相互作用，激活细胞凋亡的标志性蛋白酶caspase-3，剪切底物荧光肽，产生荧光信号用于疗效实时监测，实现了肿瘤细胞的精准治疗。
三、发明内容
本发明的目的是：为了克服目前大部分抗肿瘤药物存在的靶向性差、疗效低、发挥药效过程慢、缺乏疗效监控等问题，提出一种特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒，具有良好的临床应用价值及前景。
本发明的目的可以通过以下技术方案来实现：
一种特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒，包括以聚(D，L-丙交酯-co-乙交酯)-聚乙二醇、聚(D，L-丙交酯-co-乙交酯)-聚醚酰亚胺、二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸为载体，通过一步自组装过程包裹疏水性抗肿瘤药物和半胱氨酸天冬氨酸蛋白酶(caspase-3)底物荧光肽制备得到。
所述的特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒为核-壳结构，平均粒径为50～90nm。
本发明涉及特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的制备方法，其特征在于，包括如下制备步骤：
取抗肿瘤药物1.6mg、caspase-3底物荧光肽0.2mg、聚(D，L-丙交酯-co-乙交酯)-聚乙二醇5mg、聚(D，L-丙交酯-co-乙交酯)-聚醚酰亚胺4mg，溶于2ml二甲基亚砜(DMSO)，得到第一混合液；
将1mg二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸溶于10mL超纯水得到第二混合液；
将上述第一混合液滴加至所述第二混合液中，于室温下搅拌3～5小时，超滤(4000g，15min)除去DMSO，超纯水洗涤并重悬即得特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒，于4℃条件下保存。
本发明涉及特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的制备方法，其特征在于，所述caspase-3底物多肽序列及其标记染料为：(4-二甲胺偶氮苯-4-羧酸)-赖氨酸-苯丙氨酸-苯丙氨酸-苯丙氨酸-天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-赖氨酸(荧光素)[(Dabcyl)-KFFFDEVDK(FAM)]。
本发明涉及特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的制备方法，其特征在于，所述的抗肿瘤药物包括喜树碱、紫杉醇、阿霉素、姜黄素、吴茱萸碱及水飞蓟宾。
本发明涉及特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的制备方法，其特征在于，所述的二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸、聚(D，L-丙交酯-co-乙交酯)-聚乙二醇、聚(D，L-丙交酯-co-乙交酯)-聚醚酰亚胺的质量比为1∶4～6∶3～5。
本发明的原料的简称及获得方式分别是：
聚(D，L-丙交酯-co-乙交酯)-聚乙二醇：简称PLGA-PEG，购自山东岱罡生物科技有限公司
聚(D，L-丙交酯-co-乙交酯)-聚醚酰亚胺：简称PLGA-PEI，购自山东岱罡生物科技有限公司
二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸：简称DSPE-PEG-FA，购自西安瑞禧生物科技有限公司
caspase-3底物荧光肽：简称FRET-Pep，由强耀生物有限科技公司合成
喜树碱：简称CPT，购自梯希爱(上海)化成工业发展有限公司
紫杉醇：简称PTX，购自梯希爱(上海)化成工业发展有限公司
阿霉素：简称DOX，购自梯希爱(上海)化成工业发展有限公司
姜黄素：简称CUR，购自百灵威科技有限公司
吴茱萸碱：简称EVO，购自百灵威科技有限公司
水飞蓟宾：简称SIL，购自百灵威科技有限公司
超滤管：购自Sigma-Aldrich
与现有技术相比，本发明具有以下特点：
本发明制得的特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒能同时实现对肿瘤细胞的快速靶向治疗及疗效实时监测。相对于现有的诊疗一体化试剂，具有以下特点：
1.利用纳米粒表面的叶酸对肿瘤细胞表面高表达的叶酸受体进行特异性识别，实现了抗肿瘤药物的的靶向给药。
2.本发明涉及的聚醚酰亚胺基团能使药物快速从溶酶体中逃逸，避免了药物被溶酶体的酸性环境及酶降解，而且使药物快速释放到胞浆中，加速药物与靶点结合，加快了肿瘤细胞凋亡进程。
3.本发明涉及的caspase-3底物荧光肽是在底物肽KFFFDEVDK两端分别修饰猝灭基团(4-二甲胺偶氮苯-4-羧酸)和荧光基团荧光素得到，利用荧光共振能量转移效应(FRET)使荧光信号“关闭”，在细胞凋亡过程中产生的标志性蛋白酶caspase-3激活荧光信号，从而实现对肿瘤细胞治疗效果的及时监测。
4.本发明所述的特异性加速肿瘤细胞凋亡及疗效自监测的智能纳米粒具有良好的肿瘤靶向性，快速从溶酶体中逃逸，加快肿瘤细胞凋亡进程，实时无损的疗效自反馈等多种功能。相对于现有的诊疗一体化试剂具有明显优势。
四、附图说明
图1.特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的结构示意图；
图2.特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的工作原理图；
图3.特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的透射电镜图(a)及动态光散射图(b)；
图4.Caspase-3底物荧光肽的荧光响应图
本发明的工作原理：
本发明的工作原理如图2所示，所述的特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒在生理条件下无荧光，在叶酸的靶向识别作用下，特异性地与肿瘤细胞表面高表达的叶酸受体结合，并经过受体介导的内吞途径到达溶酶体，在聚醚酰亚胺(PEI)的“质子海绵效应”下，溶酶体破裂并释放药物及FRET-Pep到胞浆中。抗肿瘤药物与相应的作用靶点结合后激活肿瘤细胞凋亡途径，诱导肿瘤细胞死亡。在凋亡过程中激活caspase-3，剪切FRET-Pep产生荧光信号，实现对肿瘤治疗效果的实时监测。本发明利用两亲性嵌段聚合物有效整合叶酸、疏水性抗肿瘤药物和FRET-Pep，实现了对叶酸受体高表达肿瘤细胞的高效、快速靶向治疗及疗效自监测。
五、具体实施方式
实施例1：结合图1，合成特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒
1. 1.6mg疏水性抗肿瘤药物(CPT、PTX、DOX、CUR、EVO或SIL)，0.2mg FRET-Pep，5mgPLGA-PEG及4mg PLGA-PEI溶于2mL DMSO，搅拌使之完全溶解；
2.将1mg DSPE-PEG-FA溶于10mL超纯水；
3.将上述步骤1的混合液滴加到上述步骤2的混合液中，室温下避光搅拌3～5h；
搅拌结束后，超滤(4000g，15min)除去有DMSO，超纯水洗涤并重悬即得特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒，于4℃条件下保存。
实施例2：特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的表征
特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的形貌用JEOL JEM-200CX透射电子显微镜(TEM)表征，加速电压为200kV。将样品溶液滴到碳支持膜铜网上，并用2.0％(w/v)磷钨酸溶液负染色，电镜观察；尺寸及分布用Mastersizer 2000particle size analyzer动态光散射(DLS)表征。由图3可见，该纳米粒为球形的核-壳结构，纳米粒子分散性良好，粒径均匀，平均粒径为50～90nm。
实施例3：FRET-Pep对caspase-3的荧光响应
100μL FRET-Pep加到900μL的反应缓冲液中，缓冲液pH 7.4，组成为50mM 4-羟乙基哌嗪乙磺酸(HEPES)，10mM二硫苏糖醇(DTT)，100mM氯化钠(NaCl)，1mM乙二胺四乙酸(EDTA)，0.1％w/v3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐(CHAPS)，10％v/v甘油(glycerol)。加入不同浓度的caspase-3，37℃反应1h。由图4可见随着caspase-3浓度的增加，该多肽底物的荧光逐渐增强，表明该多肽底物能特异性的响应caspase-3。
实施例4：特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的细胞毒性分析
以喜树碱为例，单纯的喜树碱与HeLa细胞孵育24h后细胞存活率为58％左右，而用智能纳米粒AGIP@CPT(同等的喜树碱当量)仅需8h就能达到相同的效果，因此该智能纳米粒能有效加快肿瘤细胞凋亡。
实施例5：特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的细胞成像分析
HeLa细胞接种于35mm共聚焦培养皿中并与该智能纳米粒在37℃下孵育不同的时间，利用激光共聚焦荧光显微镜观测。激发波长488nm，收集波长505～535nm。8h后能观察到由caspase-3激活的FAM荧光信号，证明该智能纳米粒具有疗效自监测功能。
Claims (6)
1.一种特异加速肿瘤细胞凋亡及疗效自监测的智能纳米，其特征在于，纳米粒以聚(D，L-丙交酯-co-乙交酯)-聚乙二醇、聚(D，L-丙交酯-co-乙交酯)-聚醚酰亚胺、二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸为载体，通过一步自组装过程包裹疏水性抗肿瘤药物和半胱氨酸天冬氨酸蛋白酶(caspase-3)底物荧光肽制得。
2.根据权利要求1所述的特异加速细胞肿瘤凋亡及疗效自监测的智能纳米，其特征在于，所述的纳米粒为核-壳结构，平均粒径为50～90nm。
3.根据权利要求1所述的特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒，其特征在于，所述caspase-3底物多肽序列及其标记染料为：(4-二甲胺偶氮苯-4-羧酸)-赖氨酸-苯丙氨酸-苯丙氨酸-苯丙氨酸-天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-赖氨酸(荧光素)[(Dabcyl)-KFFFDEVDK(FAM)]。
4.根据权利要求1所述的特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒，其特征在于，所述的疏水性抗肿瘤药物包括喜树碱、紫杉醇、阿霉素、姜黄素、吴茱萸碱及水飞蓟宾。
5.根据权利要求1-4中任一项所述的特异加速肿瘤细胞凋亡及疗效自监测的智能纳米粒的制备方法，其特征在于，包括如下步骤：
按质量比为8∶1∶20～30∶15～25将抗肿瘤药物、caspase-3底物荧光肽、聚(D，L-丙交酯-co-乙交酯)-聚乙二醇、聚(D，L-丙交酯-co-乙交酯)-聚醚酰亚胺溶解到二甲基亚砜(DMSO)中混合得到第一混合液；
将二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸溶解在超纯水中得到第二混合液；
将上述第一混合液滴加至所述第二混合液中，于室温下搅拌3～5小时，超滤(4000g，15min)除去DMSO，再用超纯水重悬至浓度1～3mg/mL。
6.根据权利要求5所述的特异加速肿瘤细胞凋亡及疗效自监测的智能纳米的制备方法，其特征在于，所述的二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸、聚(D，L-丙交酯-co-乙交酯)-聚乙二醇、聚(D，L-丙交酯-co-乙交酯)-聚醚酰亚胺的质量比为1∶4～6∶3～5。
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| WO2008157493A2 (en) * | 2007-06-15 | 2008-12-24 | The Research Foundation Of State University Of New York Stor Intellectual Property Division | Use of zno nanocrystals for imaging and therapy |
| CN103842472A (zh) * | 2011-09-01 | 2014-06-04 | 香港科技大学 | 用作荧光生物探针的具有聚集诱导发光性质的生物相容纳米粒子及其在体外／体内成像中的应用方法 |
| CN103566379A (zh) * | 2013-09-30 | 2014-02-12 | 中国药科大学 | 一种“胞内触发”式还原敏感型药物联合基因靶向共传递体的制备及应用 |
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| CN104434806A (zh) * | 2014-11-06 | 2015-03-25 | 中国人民解放军第四军医大学 | 一种具有高载药量及主动靶向效应的脂质混合plga纳米粒 |
| CN104840957A (zh) * | 2015-04-28 | 2015-08-19 | 长春健欣生物医药科技开发有限公司 | 赫赛汀修饰的载多西他赛的三重靶向纳米粒载体系统 |
| CN105055322A (zh) * | 2015-09-02 | 2015-11-18 | 厦门市壳聚糖生物科技有限公司 | 甲氨喋呤磷脂复合物、其纳米粒子及其靶向缓释制剂三者的制备方法 |
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| CN107389628A (zh) * | 2017-06-09 | 2017-11-24 | 福建医科大学孟超肝胆医院 | 一种细胞凋亡双色检测和成像探针及其应用 |
| CN107389628B (zh) * | 2017-06-09 | 2019-09-24 | 福建医科大学孟超肝胆医院(福州市传染病医院) | 一种细胞凋亡双色检测和成像探针及其应用 |
| CN114560984A (zh) * | 2022-04-07 | 2022-05-31 | 安徽科润美新材料科技股份有限公司 | 一种自监测聚氨酯及其制备方法和应用 |
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