CN112891553A - 一种载药纳米颗粒及其制备方法与应用 - Google Patents

一种载药纳米颗粒及其制备方法与应用 Download PDF

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CN112891553A
CN112891553A CN202110228290.5A CN202110228290A CN112891553A CN 112891553 A CN112891553 A CN 112891553A CN 202110228290 A CN202110228290 A CN 202110228290A CN 112891553 A CN112891553 A CN 112891553A
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梁鸣
马鹏跃
杨显珠
曹紫洋
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Guangzhou First Peoples Hospital
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Abstract

本发明公开了一种载药纳米颗粒及其制备方法与应用，涉及化学药品技术领域。本发明所述载药纳米颗粒的制备方法包括如下步骤：(1)将疏水性药物和硫缩酮键交联的超支化聚磷酸酯材料溶解在有机溶剂中，得到混合液A；(2)将混合液A逐渐加入超纯水中，搅拌，然后浓缩、除去有机溶剂，离心，得到所述载药纳米颗粒。本发明所述载药纳米颗粒具有良好的生物相容性和可降解性，对活性氧敏感，可精准释放药物。

Description

一种载药纳米颗粒及其制备方法与应用
技术领域
本发明涉及化学药品技术领域，尤其涉及一种载药纳米颗粒及其制备方法与应用。
背景技术
目前，据中国慢性肾脏病病学调查结果估算，我国现有慢性肾脏病患者约1.3亿，约有2％的患者会进入终末期肾病阶段，这部分患者需要肾脏替代治疗，其中血液透析是最常见的治疗方法。自体动静脉内瘘(arteriovenous fistula，AVF)是目前国内外血液透析患者首选的血管通路，是血液透析患者顺利进行透析的必备条件，是血液透析患者的“生命线”。然而，临床实践中发现自体动静脉内瘘的两年通畅率仅为65％，4年通畅率降为48％，而内瘘内膜增生是导致内瘘狭窄的主要病理改变，是导致血液透析人群透析不充分、增加死亡率和高住院率的重要原因。目前临床上仍无有效治疗方法来减轻动静脉内瘘血管壁的过度增厚。因此，如何延缓或消除新生内膜增生是提高内瘘血管远期通畅率的关键。
目前，有潜力的药物如紫杉醇、雷帕霉素、他汀类、血管紧张素受体阻滞剂等药物，可部分减缓新生内膜增生，但因不可控性、全身用药副作用较多在应用上受到限制。随着纳米技术的最新进展，可将治疗药物封装到纳米颗粒上，这种载药纳米颗粒可实现在特定部位富集，从而减少药物副作用，是临床上一个有吸引力的给药策略。除此之外，温敏性水凝胶内部存在大量的孔隙，可作为药物负载和存储的载体，将载药纳米颗粒包裹于温度敏感性水凝胶可实现原位释放药物作用于动静脉内瘘部位，通过外界光源调控颗粒的降解和药物的释放，具有良好的可控性及高安全性等优势。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种活性氧敏感的载药纳米颗粒及其制备方法与应用。
为实现上述目的，本发明所采取的技术方案为：一种载药纳米颗粒的制备方法，包括如下步骤：
(1)将疏水性药物和硫缩酮键交联的超支化聚磷酸酯材料(TK-hbPPE)溶解在有机溶剂中，得到混合液A；
(2)将混合液A逐渐加入超纯水中，搅拌，然后浓缩、除去有机溶剂，离心，得到所述载药纳米颗粒。
本发明选用的硫缩酮键交联的超支化聚磷酸酯材料可在水中自组装，包覆疏水性药物，形成纳米颗粒。含有硫缩酮键的聚磷酸酯除了具有两亲性，可自组装外，还可生物降解，并且其最终降解产物对生物体无不良影响。此外，在活性氧环境中硫缩酮键会发生断裂，使药物颗粒快速释放。
优选地，所述步骤(1)中，有机溶剂包含三氯甲烷。
优选地，所述步骤(1)中，疏水性药物包含维替泊芬、紫杉醇、蒽醌类中的至少一种。
优选地，所述步骤(1)中，疏水性药物与硫缩酮键交联的超支化聚磷酸酯材料的质量比为1:8～12。
优选地，所述步骤(2)中，搅拌时间为2～3h；以油泵进行浓缩，浓缩时间为0.8～1.2h；离心速率为1500～2000rpm，离心时间为5～10min。
优选地，所述载药纳米颗粒的平均粒径为280～320nm。
进一步优选地，所述载药纳米颗粒的平均粒径为300nm。
同时，本发明公开了一种由上述方法制备所得的载药纳米颗粒。
此外，本发明还公开了一种载药纳米颗粒水凝胶递送体系，由所述载药纳米颗粒和温敏性水凝胶混合，在35～39℃培养箱中保温，得到所述载药纳米颗粒水凝胶递送体系。
优选地，所述温敏性水凝胶为PLGA-PEG-PLGA温敏性水凝胶。
优选地，所述载药纳米颗粒和温敏性水凝胶的体积比为1:2～10；保温时间为1～3min。
另外，本发明还公开了所述载药纳米颗粒水凝胶递送体系在抑制动静脉内瘘内膜增生中的应用。
优选地，治疗时辅以光照，抑制内瘘内膜增生的效果更好。
相比于现有技术，本发明的有益效果为：本发明合成的活性氧敏感的超支化聚磷酸酯材料与温度敏感性水凝胶具有良好的生物相容性和可降解性。纳米颗粒水凝胶递送体系，通过在病灶部位直接注射成胶后，在特定波长激发下可以实现疏水性抗增殖药物的可控释放，具有时空可控性及良好的安全性，具有巨大的临床应用潜能。
附图说明
图1为TK-hbPPE的合成路线图；
图2为TK-hbPPE的1H NMR图；
图3为PLGA-PEG-PLGA的结构式图；
图4为实施例1载药纳米颗粒的粒径统计图；
图5为实施例1载药纳米颗粒的活性氧敏感性测试图；
图6为实施例1载药纳米颗粒的体外药物释放量测试图；
图7为载药纳米颗粒递送体系抑制动静脉内瘘内膜增生的机理图；
图8为血管平滑肌细胞对载药纳米颗粒水凝胶递送体系中药物的摄取能力测试图；
图9为血管平滑肌细胞对载药纳米颗粒水凝胶递送体系中药物的内吞性能测试图；
图10为载药纳米颗粒水凝胶递送体系中药物浓度与血管平滑肌细胞活力的关系图；
图11为血管平滑肌细胞的FITC/PI凋亡检测图；
图12为血管平滑肌细胞的迁移能力测试图；
图13为血管平滑肌细胞中YAP蛋白表达图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
实施例1
本发明所述载药纳米颗粒的制备方法的一种实施例，本实施例所述制备方法如下：
(1)合成TK-hbPPE：如图1所示，首先，通过半胱胺盐酸盐和丙酮发生丙酮叉反应后脱盐得到2,2'-(丙烷-2,2-二甲基双(二硫))双(乙烷-1-胺)单体(PDSE)。单体(PDSE)和三氯氧磷在无水反应条件下通过A2B3缩聚反应，合成所述活性氧敏感的TK-hbPPE。图2为TK-hbPPE的1H NMR表征结果，如图2所示，化学位移在2.69ppm和2.89ppm的两个峰分别归属PDSE的两个亚甲基，而1.59ppm的峰归属于PDSE的甲基，3.51ppm和3.23ppm分别为PEG主峰和甲氧基峰，上述结果证明了TK-hbPPE的成功合成。
(2)称取10mg TK-hbPPE和1mg疏水性抗增殖药物维替泊芬溶于1mL三氯甲烷中，得到混合液A；
(3)在搅拌过程中将上述混合液A缓慢加入10mL超纯水中，继续搅拌2h，然后将溶液以油泵浓缩至5mL除去三氯甲烷；浓缩结束后，以1500rpm的转速离心5min、除去未被包载的药物，得到载药纳米颗粒，标记为TKhbPPE-VER。
将载药纳米颗粒用2mL无菌水重悬，采用紫外分光光度计测试颗粒溶液中维替泊芬的浓度，取重悬的颗粒溶液100μL置于冷冻干燥机中冻干，得到粉末，之后用2mL的DMSO溶解。配制10mL的维替泊芬二甲亚砜溶液，稀释至10μg/mL，依次对半稀释至0.3125μg/mL，通过紫外分光光度计测定690nm处的维替泊芬特征吸收峰，将浓度与吸收值关联，建立标准曲线，纳米颗粒包载维替泊芬的包封效率(encapsulation efficiency，EE)通过以下公式算得：
Figure BDA0002956431410000051
通过实施例1制备得到的载药纳米颗粒的包封效率约为30％。
实施例2
本发明所述载药纳米颗粒水凝胶递送体系的一种实施例，本实施例所述载药纳米颗粒水凝胶递送体系的制备方法为：将实施例1制备的载药纳米颗粒和PLGA-PEG-PLGA温敏性水凝胶物理混合，于37℃培养箱中静置2min，制备出维替泊芬浓度分别为6.125μg/mL、12.5μg/mL、25μg/mL、50μg/mL、100μg/mL的载药纳米颗粒水凝胶递送体系。
本实施例所用PLGA-PEG-PLGA温敏性水凝胶中，PLGA-PEG-PLGA的浓度为20wt.％；其中，PEG段的分子量为1000～1500，每一段PLGA段的分子量为1200～1550，低温下为无色透明溶液，32℃以上为乳白色凝胶。其结构式如图3所示。
性能测试
1、实施例1制备的载药纳米颗粒的性能测试
1)粒径测试：使用动态光散射仪(Dynamic light scattering,DLS)检测载药纳米颗粒粒径，如图4所示，载药纳米颗粒的平均粒径在300nm左右。
2)活性氧敏感性测试：取载药纳米颗粒加入超纯水配成浓度为10μg/mL的颗粒水溶液，2μL的SOSG(单线态氧荧光探针)试剂盒(浓度为5mmol/L)加入到1mL的颗粒水溶液中。利用660nm激光器以0.05W/cm2开始照射样品(0、1、2、5、10、15min)，利用分光光度计测定520-650nm发射光谱，根据光谱的强度及面积可检测活性氧(ROS)产量。如图5所示，载药纳米颗粒水溶液在660nm激光照射下可产生ROS，且随光照时间延长，ROS产量增多。
3)体外药物释放能力测试：释放能力测试是在含有0.02mol/L的磷酸盐缓冲液(phosphate buffered saline，PBS，pH＝7.4)中进行的，做三组平行实验，取1mL的实施例1的水溶液(C维替泊芬＝100.0μg/mL)重悬于pH＝7.4的15mL 0.02M PBS中，利用660nm激光器0.05W/cm2照射5min，照射后将载药纳米颗粒置于透析袋中(Spectra/Por，Float-A-Lyzer，MWCO＝14000)，再将透析袋置于三组装有15mL的PBS缓冲液(pH＝7.4)50mL离心管中，释放于37℃摇床(80rpm)下进行。在指定的时间将释放外液全取出，并补充等量的新鲜缓冲液。将取出的释放外液冻干，DMSO溶解，荧光分光光度计分析释放外液中维替泊芬的浓度。如图6所示，载药纳米颗粒在pH＝7.4的环境下培养24h后，经过660nm光照后的载药纳米颗粒较非光照组相比，药物释放量提高15％左右，药物释放量明显增加。这些结果表明了载药纳米颗粒具有活性氧敏感性释放的特点，出现这一结果的原因可能是载药纳米颗粒具有大量的硫缩酮键，在活性氧环境下能发生断裂，最终导致颗粒崩解，快速释放药物。
2、实施例2载药纳米颗粒水凝胶递送体系的性能测试
1)抑制动静脉内瘘内膜增生性能测试：将载药纳米颗粒水凝胶递送体系注射在小鼠动静脉周围，在小鼠体温下水凝胶体系由液体改变为凝胶状，使其固定于动静脉内瘘周围，通过660nm激光光照，药物从载药纳米颗粒中释放出来，作用于血管平滑肌细胞，起到抑制动静脉内瘘内膜增生的作用。作用机理如图7所示。抑制效果从下述实验中可知。
2)血管平滑肌细胞(VSMC)对药物的摄取效果测试：选取血管平滑肌细胞系(MOVAS)进行测试。首先在Transwell的24孔板中每孔种1×105个血管平滑肌细胞，每孔加入500μL的DMEM培养基(含10％FBS+1％双抗)，在CO2培养箱中(37℃，CO2浓度为5％)培养24h。将载药纳米颗粒和PLGA-PEG-PLGA混合，制备维替泊芬浓度为50μg/mL的混合液，然后将混合液快速加到Transwell上室中，先放于空24孔板，37℃培养箱中2分钟，得到载药纳米颗粒水凝胶递送体系，利用660nm激光器0.05W/cm2照射实验光照组5min，照射结束后将每组小室移至含有细胞的孔板中培养4h。4h后洗去未被摄取的药物，用流式细胞仪检测细胞内维替泊芬的荧光强度，结果见图8。由图8可见，与非光照组相比，光照实验组的细胞内维替泊芬荧光强度明显增强，表明超支化聚磷酸酯纳米颗粒能有效的输送维替泊芬，并且能提高血管平滑肌细胞内的药物的摄取。
3)载药纳米颗粒水凝胶递送体系的细胞内吞实验：用激光共聚焦扫描显微镜观察载药纳米颗粒水凝胶递送体系递送维替泊芬在细胞内的分布。和流式细胞摄取实验一样，首先在Transwell的24孔板中每孔种1.0×105血管平滑肌细胞，每孔添加500μL的DMEM培养基(含10vol.％FBS+1vol.％双抗)，在CO2培养箱中(37℃，CO2浓度为5％)培养24h。提前配制载药纳米颗粒和水凝胶混合液(维替泊芬浓度为50μg/mL)，将混合液快速加到Transwell上室中，先放于空24孔板，37℃培养箱中2分钟待其成胶，得到载药纳米颗粒水凝胶递送体系，利用660nm激光器0.05W/cm2照射实验光照组5min，照射结束后将每组小室移至含有细胞的孔板中培养4h。分别用鬼笔环肽(Alexa Fluor 488phalloidin)和4',6-二脒基-2-苯基吲哚(DAPI)染色细胞骨架的F-肌动蛋白和细胞核。如图9所示，疏水性抗增殖药物维替泊芬主要在细胞核周围富集，且实验光照组中维替泊芬的荧光明显强于非光照组，证明了载药纳米颗粒水凝胶递送体系能够有效输送维替泊芬并增强血管平滑肌细胞对维替泊芬的摄取，使维替泊芬在细胞内富集。
4)载药纳米颗粒水凝胶递送体系对血管平滑肌细胞的增殖抑制效果测试：采用MTT比色法进行测试。具体方法如下：首先在24孔板中每孔种3×104个细胞/500μL DMEM培养基(含有10vol.％FBS)，在CO2培养箱中(37℃，CO2浓度为5％)培养24h。并提前配制不同药物浓度的载药纳米颗粒和水凝胶混合液，将混合液快速加到Transwell上室中，先放于空24孔板，37℃培养箱中2分钟待其成胶，利用660nm激光器0.05W/cm2照射实验组5min，光照结束后将每组小室转移至含有新鲜培养液的24孔板中。8h后去掉transwell小室，将培养液转移至含有细胞的孔板中培养48h。最后用MTT法检测各组细胞活性。由图10可见，在四个浓度梯度的维替泊芬浓度下，非光照组对血管平滑肌细胞的杀伤效果并不好，肿瘤细胞活性均在80％以上。而光照组随着药物的增加，在光照条件下对血管平滑肌细胞的杀伤效果逐渐增加。由此我们推断，超支化载药纳米颗粒通过温敏性PLGA-PEG-PLGA水凝胶递送，通过660nm激光器可控制药物释放并对血管平滑肌细胞起到增殖抑制效果。
5)载药纳米颗粒水凝胶递送体系对血管平滑肌细胞的细胞凋亡实验：利用FITC/PI凋亡检测试剂盒评估上述制备的载药纳米颗粒水凝胶递送体系对血管平滑肌细胞(VSMC)的细胞杀伤效果。具体方法如下：首先在24孔板中每孔种3×104个细胞/500μL DMEM培养基(含有10vol.％FBS)，在CO2培养箱中(37℃，CO2浓度为5％)培养24h。并提前配制不同药物浓度的载药纳米颗粒和水凝胶混合液，将混合液快速加到Transwell上室中，先放于空24孔板，37℃培养箱中2分钟待其成胶，利用660nm激光器0.05W/cm2照射实验组5min，光照结束后将每组小室转移至含有新鲜培养液空的24孔板中，培养24h。用膜联蛋白V-FITC和碘化丙啶(PI)染色血管平滑肌细胞VSMC以评估细胞凋亡。如图11所示，载药纳米颗粒水凝胶经过光照后细胞杀伤效果明显优于非光照组，由此我们推断，载药纳米颗粒通过温敏性PLGA-PEG-PLGA水凝胶递送，通过660nm激光器光照可以促进药物释放，对血管平滑肌细胞起到细胞杀伤作用。
6)划痕实验验证载药纳米颗粒水凝胶递送体系通过激光控制维替泊芬释放对血管平滑肌细胞的迁移影响：具体方法如下：首先在6孔板中每孔种50×104个细胞/2mL DMEM培养基(含有10vol.％FBS)，在CO2培养箱中(37℃，CO2浓度为5％)培养24h。次日，待细胞增殖至密度达90％-100％，用10μL微量移液器头在孔中划线，用PBS洗3遍，添加无血清培养液，用荧光显微镜拍照一次。提前配制载药纳米颗粒和水凝胶混合液(维替泊芬浓度为50μg/mL)，快速加到Transwell上室中，先放于空6孔板，37℃培养箱中2分钟待其成胶。利用660nm激光器0.05W/cm2照射实验组5min。照射结束后将小室转移到有细胞的孔板上继续培养8h，8h后将小室取出，继续培养。在各个时间点用荧光显微镜拍照，统计结果，如图12所示，光照组较非光照组相比，细胞迁移距离变小，说明光照后维替泊芬释放量增加，抑制平滑肌细胞的迁移能力。
7)Western-Blot验证载药纳米颗粒水凝胶递送体系通过激光控制维替泊芬释放降低VSMC中YAP蛋白表达：具体方法如下：首先在6孔板中每孔种10×104个细胞/2mL DMEM培养基(含有10vol.％FBS)，在CO2培养箱中(37℃，CO2浓度为5％)培养24h。提前配制载药纳米颗粒和水凝胶混合液(维替泊芬浓度为50μg/mL)，将混合液快速加到Transwell上室中，先放于空6孔板，37℃培养箱中2分钟待其成胶，利用660nm激光器0.05W/cm2照射实验组5min，光照结束后将每组小室转移至含有新鲜培养液空的24孔板中，培养48h。48h后收集各组细胞，通过WB技术检测各组细胞中YAP表达。如图13所示，光照组较非光照组相比，血管平滑肌细胞中YAP表达降低，由此我们可推断出，通过660nm激光器光照载药纳米颗粒水凝胶递送体系，可以促进药物从载药纳米颗粒中释放出来，并降低细胞中yes相关蛋白(YAP蛋白)的表达。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，但并不脱离本发明技术方案的实质和范围。

Claims (10)

1.一种载药纳米颗粒的制备方法，其特征在于，包括如下步骤：
(1)将疏水性药物和硫缩酮键交联的超支化聚磷酸酯材料溶解在有机溶剂中，得到混合液A；
(2)将混合液A加入超纯水中，搅拌，然后浓缩、除去有机溶剂，离心，得到所述载药纳米颗粒。
2.如权利要求1所述的载药纳米颗粒的制备方法，其特征在于，所述步骤(1)中，有机溶剂包含三氯甲烷。
3.如权利要求1所述的载药纳米颗粒的制备方法，其特征在于，所述步骤(1)中，疏水性药物包含维替泊芬、紫杉醇、蒽醌类中的至少一种。
4.如权利要求1所述的载药纳米颗粒的制备方法，其特征在于，所述步骤(1)中，疏水性药物与硫缩酮键交联的超支化聚磷酸酯材料的质量比为1:8～12。
5.如权利要求1所述的载药纳米颗粒的制备方法，其特征在于，所述步骤(2)中，搅拌时间为2～3h；以油泵进行浓缩；离心速率为1500～2000rpm，离心时间为5～10min。
6.一种载药纳米颗粒，其特征在于，由如权利要求1～5任一项所述的制备方法制备所得。
7.一种载药纳米颗粒水凝胶递送体系，其特征在于，所述载药纳米颗粒水凝胶递送体系的制备方法为：将如权利要求6所述的载药纳米颗粒与温敏性水凝胶混合，在35～39℃培养箱中保温，得到所述载药纳米颗粒水凝胶递送体系。
8.如权利要求7所述的载药纳米颗粒水凝胶递送体系，其特征在于，所述温敏性水凝胶为PLGA-PEG-PLGA温敏性水凝胶。
9.如权利要求7所述的载药纳米颗粒水凝胶递送体系，其特征在于，所述载药纳米颗粒和温敏性水凝胶的体积比为1:2～10；保温时间为1～3min。
10.一种如权利要求7～9任一项所述的载药纳米颗粒水凝胶递送体系在抑制动静脉内瘘内膜增生中的应用，其特征在于，治疗时辅以光照。
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