CN111388453A - 一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法 - Google Patents
一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法 Download PDFInfo
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Abstract
本发明公开了一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,属于药物制备领域。由该制备方法制的的舒尼替尼纳米药物胶囊,舒尼替尼表面有一层聚合物载体,聚合物载体的存在使胶囊在肿瘤微酸环境下表面电荷呈现较高正电性;舒尼替尼纳米药物胶囊的平均粒径在200~300nm之间,分散性较好;溶出速率快,溶出度高,具有高血运、高细胞摄取效率,且具备肿瘤微环境响应释药性。
Description
技术领域
本发明属于药物制备领域,特别涉及一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法。
背景技术
癌症是目前危害人类身体健康的最主要疾病,具有难发现、难治疗、高复发的问题。药物化疗是治疗癌症的一大手段,如何选择合适载体并制备一种高效的化疗药物就成为亟待解决的问题。
舒尼替尼是一种小分子多靶点酪氨酸激酶抑制剂,由于其可抑制多种生长因子受体(包括血管内皮生长因子受体、血小板衍生生长因子受体、干细胞因子受体等),具有显著的抑制肿瘤血管生成和抗肿瘤细胞生长的作用,是常见的抗肿瘤原料药物。其分子式为C22H27FN4O2,其结构如下:
但当前的药物制备领域里,舒尼替尼具有一定的临床应用局限性,且制剂多为口服,暂无纳米级注射制剂,且存在药物水溶性差、组织释放率低、经消化系统吸收之后毒副作用较大等问题。
聚合物包覆的舒尼替尼纳米级可注射药物也成为当前的热点研究领域。聚合物载体在抗癌药物的应用领域被广为探索,其具有高血运、高肿瘤蓄积和可以被刺激响应缓释药物等优点。然而,聚合物纳米胶囊也存在一些细胞摄取效率低、聚合物载体降解慢、药物释放效率不高的问题,导致聚合物纳米胶囊的治疗应用受到了阻碍。此外,大多数聚合物载体所制备的纳米药物呈现负电性,导致所制备纳米药物的细胞摄取效率和细胞溶酶体逃逸效率很低,很难在细胞质基质内释放药物发挥药效。
原位表面聚合反应应运而生,并被广泛用于纳米材料的合成和表面改性。与常规包覆手法相比,通过原位表面聚合制备的聚合物纳米胶囊的结构设计更易操控,可以选择不同聚合条件和功能性单体与交联剂来制备不同功能的纳米胶囊。此外,原位表面聚合是一种“涂覆方式”,聚合物胶囊层是从原位表面生长的,使得制备的纳米药物胶囊提纯过程简便,产品纯度高。
根据以上背景技术,本研究旨在构建一类同时具备高血运、高细胞摄取效率及肿瘤微环境响应释药性的舒尼替尼纳米药物胶囊。
发明内容
本发明的目的是建立一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,该方法操作简单,不仅可以构建稳定的纳米胶囊,具有较高载药量和较好的稳定性,还可以在肿瘤组织的酸性环境下,实现药物胶囊表面聚合物的级联反应降解,进而提高纳米药物的血液循环周期、细胞摄取效率和药物释放效率,改善药物舒尼替尼的给药途径及药物疗效。
本发明采用的技术方案为一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,该方法包括以下步骤,
S1提供具备pH响应的单体聚乙二醇-马来酸酐-N-丙烯酰胺单体(PEG-CDM-NAM),简称PCN;
S2将包含舒尼替尼的二甲基亚砜溶液和丙烯酸酯基辛基苯酚聚乙二醇醚(OP-AC)的二甲基亚砜溶液混合,得到表面修饰有乙烯基的舒尼替尼,再加入到含有一定量中性单体丙烯酰胺(AM)、具备pH响应单体PCN、氧化还原响应降解交联剂N,N’-双(丙烯酰基)胱氨酸(BAC)的缓冲液中,同时加入聚合反应引发剂过硫酸铵(APS)、乳化剂TEMED引发原位自由基聚合;
S3收集S2中反应产物,通过透析得到舒尼替尼纳米药物胶囊溶液。
OP-AC的合成方法为现有技术,此方法不再赘述。
具有pH响应的单体PCN是通过如下步骤合成的:
S1.1将2-丙-3-甲基马来酸酐(以下简称CDM)和草酰氯以一定比例添加到装有无水二氯甲烷(CH2Cl2)的烧瓶中,滴加四氢呋喃(DMF)催化剂并隔绝空气,将烧瓶置于冰水中搅拌1h,移至25℃继续反应3h;
S1.2使用旋转蒸发仪除去CH2Cl2,得到固体产物;
S1.3将S1.2中的固体产物与无水聚乙二醇单甲醚(以下简称mPEG)以一定比例添加到装有无水CH2Cl2的烧瓶中,滴加吡啶催化剂并隔绝空气,将烧瓶放置于25℃环境下反应3h;
S1.4取反应产物与两倍体积的饱和NH4Cl混合,分液以除去多余反应物,使用冻乙醚进行沉降并离心干燥,得到中间产物mPEG(CDM);
S1.5将N-(3-氨基丙基)甲基丙烯酸盐盐酸盐(以下简称NAM)溶于无水乙醇中并加入三乙胺(TEA)除去盐酸,与一定比例的S1.4产物mPEG(CDM)共同溶解于无水乙醇中,25℃环境下反应6h,使用冻乙醚进行沉降并离心干燥,得到具备pH响应单体PEG-CDM-NAM,即PCN。
反应式如下:
进一步地,S1.1中CDM和草酰氯的质量比为1/1.6。
进一步地,S1.3中固体产物与无水聚乙二醇单甲醚的质量比为1/1.35。
进一步地,S1.5中mPEG(CDM)与N-(3-氨基丙基)甲基丙烯酸盐-盐酸盐的质量比为13/1。
进一步地,缓冲液指的是浓度150mM,pH=7.4的磷酸二氢钠/磷酸氢二钠缓冲溶液PBS。
进一步地,OP-AC的二甲基亚砜溶液中的OP-AC和舒尼替尼的质量比为1/5;
进一步地,二甲基亚砜和反应环境缓冲液的体积比为1/20。
进一步地,交联剂为氧化还原响应降解交联剂N,N'-双(丙烯酰基)胱氨酸(BAC)或非可降解交联剂N'N-甲叉双丙烯酰胺(BIS),优选BAC;
进一步地,舒尼替尼/(单体+交联剂)质量比为1/0.4;单体和交联剂的质量比为1/0.4;酸敏感电荷反转单体PCN和中性单体AM的质量比为10/1;
进一步地,所述的舒尼替尼和过硫酸铵的质量比为1/0.04,单体和TEMED的质量比为1/5;
由该制备方法制的舒尼替尼纳米药物胶囊,舒尼替尼表面有一层聚合物载体,聚合物载体在偏酸性环境下表面电荷由负电性反转为正电性,舒尼替尼纳米药物胶囊在偏酸性环境下表面正电荷增大;舒尼替尼纳米药物胶囊的平均粒径在200~300nm之间,分散性较好;溶出速率快,溶出度高,具有良好的pH及氧化还原响应释药性。
与现有技术相比,本发明具有以下有益效果。
采用本发明的方法,可通过交联剂结合具备pH响应单体PCN,对比使用非可降解交联剂BIS后所得实验结果等,得到较小粒径分散均匀,溶出速率快溶出度高等优性能的舒尼替尼纳米药物胶囊。
该药物胶囊的结构是舒尼替尼的表面有一层聚合物载体,表面带有电势较低的正电荷,在肿瘤组织偏酸性环境下电势迅速增高;舒尼替尼纳米药物胶囊的平均粒径在200~300nm之间,分散性较好;溶出速率快,溶出度高,具有良好的氧化还原响应释药性。
附图说明
图1是实施例1中制得的PCN核磁图。
图2是实施例2中制得的OPAC-PCN聚合物核磁图(a)及其透射电镜照片(b)和粒径分布图(c)。
图3是实施例3中制得的舒尼替尼药物胶囊(A)的透射电镜照片(a)和粒径分布图(b)
图4是实施例4中制得的舒尼替尼药物胶囊(B)的透射电镜照片(a)和粒径分布图(b)
图5是实施例5中制得的舒尼替尼药物胶囊(C)的透射电镜照片(a)和粒径分布图(b)
图6是实施例6中制得的舒尼替尼药物胶囊(D)的透射电镜照片(a)和粒径分布图(b)
图7是实施例7中制得的酸化处理后OPAC-PCN聚合物的核磁图(a)、透射电镜照片(b)和粒径分布图(c),以及其电势分布图(d)。
图8是实施例8中制得的酸化处理后舒尼替尼药物胶囊的透射电镜照片(a)和粒径分布图(b),以及其电势分布图(c)。
图9是实施例3~实施例6中制得的舒尼替尼药物胶囊(A~D)在pH=7.4的磷酸盐缓冲溶液中的溶出曲线图(a),以及实施例3以及实施例6中制得的舒尼替尼药物胶囊(A~D)在包含15mM GSH的pH=7.4的磷酸盐缓冲溶液中的溶出曲线图(b)。
具体实施方式
以下结合附图和实施例对本发明进行详细说明。
本发明首次设计、合成了一种具有pH响应的聚乙二醇-马来酸酐-N-丙烯酰胺(PEG-CDM-NAM,简称PCN)单体,并将其与还原响应降解交联剂N,N’-双(丙烯酰基)胱氨酸(BAC)结合,通过反溶剂纳米沉淀技术和表面原位聚合技术制备了一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊。舒尼替尼纳米药物胶囊经静脉注射后,在到达肿瘤微酸环境之前,由PCN和BAC制备的聚合物胶囊层可以有效防止药物释放或泄露,而PCN中的PEG组分可有效地提高药物胶囊的体内循环的稳定性。当纳米胶囊到达微酸的肿瘤组织后,PCN的酰胺键在肿瘤的微酸环境下水解,暴露出PCN内部的氨基,使纳米药物胶囊表面呈现出较高的正电性,进一步有效提高肿瘤细胞对药物胶囊的摄取率及内涵提逃逸效率。纳米药物胶囊在被肿瘤细胞摄取后,聚合物胶囊层中的还原响应交联剂则会被细胞质中高表达的谷胱甘肽(GSH)还原降解,从而将胶囊层内部的舒尼替尼药物分子快速释放,大幅改善药物的抗肿瘤疗效。
PCN的分子式如下:
BAC的分子式如下:
具体来说
实施例1
将0.14g CDM与10mL CH2Cl2添加到单口烧瓶中,将单口烧瓶置于冰水中,使用电磁搅拌器搅拌。溶解完全后加入150μL草酰氯及40μL DMF,冰水浴反应1小时,温度保持在0~5℃,撤去冰水浴使继续在25℃温度下继续反应3小时。反应产物通过旋转蒸发仪除去未反应的草酰氯、CH2Cl2等,设置温度为45℃。将0.19g无水mPEG与9mL CH2Cl2添加到单口烧瓶中,将旋蒸产物使用1mL的CH2Cl2溶解后加入烧瓶中,滴加30μL吡啶25℃反应3小时,取反应产物与20mL饱和NH4Cl混合并使用分液漏斗分液,取下层溶液使用100mL冻乙醚沉降,取沉淀干燥,得到PEG(CDM)。
将50mg的mPEG(CDM)溶于2mL无水乙醇添加到反应瓶中,将6.5mg的N-(3-氨基丙基)甲基丙烯酸盐-盐酸盐溶于1mL无水乙醇中,加10μL TEA除去盐酸,加入反应瓶中,常温搅拌反应6h,取反应产物,用30mL冻乙醚沉降,取沉淀干燥,得到PCN。
从图1所示的核磁图中可以看出,在5~6ppm之间有明显特征峰,说明实验合成的PCN中具备双键。
实施例2
称取2mg的PCN与2mg的OP-AC在室温下溶解于2mL二甲基亚砜中混合均匀,打入50mL浓度为150mM,pH=7.4的PBS中。将0.8mg的BAC使用0.5mL的DMSO溶解,一次性打入PBS中,随后同时加入500μL过硫酸铵(APS,0.1g/mL)和85μL N,N,N',N'-四甲基乙二胺(TEMED),于常温下避光反应3小时,得到OPAC-PCN聚合物,反应结束后用透析袋(Mw:3500Da)在15mM pH=7.4的PBS缓冲液中透析以除去DMSO及未反应的反应物,每3-4h更换一次透析液,共四次。透析后在4℃下避光储存。
从图2所示的核磁图中可以看出,在5~6ppm之间的双键消失,说明成功引发了原位聚合反应。
从图2所示的透射电镜照片和粒径分布图中可以看出,OPAC-PCN聚合物为平均粒径127.6nm、形貌规则、淡灰色的球形颗粒,分布均匀,分散性较好。
实施例3
称取10mg的舒尼替尼与2mg的OP-AC,在室温下共同溶解于2mL的二甲基亚砜中混合均匀,打入50mL浓度为150mM,pH=7.4的PBS中,分散30分钟,按照舒尼替尼/(单体+BAC)为1/0.4(质量比)、单体和BAC的质量比为1/0.4、酸敏感单体PCN/中性单体AM质量比为10/1的比例,向体系内加入PCN与AM,10min后加入BAC,并同时添加500μL过硫酸铵(APS,0.1g/mL)及84μLTEMED,超声10分钟,反应3小时,反应期间每隔一小时超声一次,每次超声5分钟。反应结束后用透析袋(Mw:3500Da)在15mM pH=7.4的PBS缓冲液中透析以除去DMSO及未反应的反应物,每3-4h更换一次透析液,共四次。透析后在4℃下避光储存。
从图3所示的透射电镜照片和粒径分布图中可以看出,舒尼替尼胶囊(A)为平均粒径253.1nm、形貌规则,为淡灰色聚合物内含深色药物颗粒的球形结构,分布均匀,分散性较好。
实施例4
称取10mg的舒尼替尼与2mg的OP-AC,在室温下共同溶解于2mL的二甲基亚砜中混合均匀,打入50mL浓度为150mM,pH=7.4的PBS中,分散30分钟,按照舒尼替尼/(AM+BAC)为1/0.4(质量比)、AM和BAC的质量比为1/0.4的比例,向体系内加入AM,10min后加入BAC,并同时添加500μL过硫酸铵(APS,0.1g/mL)及84μLTEMED,超声10分钟,反应3小时,反应期间每隔一小时超声一次,每次超声5分钟。反应结束后用透析袋(Mw:3500Da)在15mM pH=7.4的PBS缓冲液中透析以除去DMSO及未反应的反应物,每3-4h更换一次透析液,共四次。透析后在4℃下避光储存。
从图4所示的透射电镜照片和粒径分布图中可以看出,舒尼替尼胶囊(B)为平均粒径176.9nm、形貌规则,为淡灰色聚合物内含深色药物颗粒的球形结构,分布均匀,分散性较好。
实施例5
称取10mg的舒尼替尼与2mg的OP-AC,在室温下共同溶解于2mL的二甲基亚砜中混合均匀,打入50mL浓度为150mM,pH=7.4的PBS中,分散30分钟,按照舒尼替尼/(单体+BAC)为1/0.4(质量比)、单体和BIS的质量比为1/0.4、酸敏感单体PCN/中性单体AM质量比为10/1的比例,向体系内加入PCN与AM,10min后加入BIS,并同时添加500μL过硫酸铵(APS,0.1g/mL)及84μL TEMED,超声10分钟,反应3小时,反应期间每隔一小时超声一次,每次超声5分钟。反应结束后用透析袋(Mw:3500Da)在15mM pH=7.4的PBS缓冲液中透析以除去DMSO及未反应的反应物,每3-4h更换一次透析液,共四次。透析后在4℃下避光储存。
从图5所示的透射电镜照片和粒径分布图中可以看出,舒尼替尼胶囊(C)为平均粒径472.7nm、为淡灰色聚合物内含深色药物颗粒的球形结构,分散性一般。
实施例6
称取10mg的舒尼替尼与2mg的OP-AC,在室温下共同溶解于2mL的二甲基亚砜中混合均匀,打入50mL浓度为150mM,pH=7.4的PBS中,分散30分钟,按照舒尼替尼/(AM+BIS)为1/0.4(质量比)、AM和BIS的质量比为1/0.4的比例,向体系内加入AM,10min后加入BIS,并同时添加500μL过硫酸铵(APS,0.1g/mL)及84μL TEMED,超声10分钟,反应3小时,反应期间每隔一小时超声一次,每次超声5分钟。反应结束后用透析袋(Mw:3500Da)在15mM pH=7.4的PBS缓冲液中透析以除去DMSO及未反应的反应物,每3-4h更换一次透析液,共四次。透析后在4℃下避光储存。
从图6所示的透射电镜照片和粒径分布图中可以看出,舒尼替尼胶囊(D)为平均粒径320.2nm、形貌规则,为淡灰色聚合物内含深色药物颗粒的球形结构,分布均匀,分散性较好。
实施例7
取实施例2中的OPAC-PCN聚合物10mL,在室温下转入反应瓶中,滴加稀盐酸调节pH至3.5~4.5,常温搅拌反应48小时。反应结束后,用透析袋(Mw:3500Da)在15mM pH=7.4的PBS缓冲液中透析以除去小分子产物及盐酸,每3-4h更换一次透析液,共四次。透析后在4℃下避光储存。
从图7所示的核磁图中可以看出,在2~3ppm之间的PEG烷基链特征峰消失,说明酸性条件造成了OPAC-PCN聚合物的破坏。
从图7所示的透射电镜照片和粒径分布图中可以看出,OPAC-PCN聚合物在酸性环境下48小时后,形貌发生明显变化,颗粒间团聚明显,平均粒径增大至967.3nm,分散性差。
从图7所示的电势分布图可以看出,酸性环境下48小时后,OPAC-PCN聚合物的电势有所变化,证明其具有一定的pH响应功能。
实施例8
取实施例3中的舒尼替尼胶囊10mL,在室温下转入反应瓶中,滴加稀盐酸调节pH至3.5,常温搅拌反应48小时。反应结束后,用透析袋(Mw:3500Da)在15mM pH=7.4的PBS缓冲液中透析以除去小分子产物及盐酸,每3-4h更换一次透析液,共四次。透析后在4℃下避光储存。
从图8所示的透射电镜照片(a)和粒径分布图(b)中可以看出,舒尼替尼胶囊在酸性环境下48小时后,形貌发生明显变化,结构破坏明显,平均粒径增大至2176.1nm,出现团聚现象。
从图8所示的电势分布图(c)可以看出,酸性环境下48小时后,舒尼替尼胶囊的电势增大,证实胶囊具有pH响应功能。
图9所示,BIS作交联剂的舒尼替尼药物胶囊和BAC作交联剂的舒尼替尼药物胶囊在无GSH的磷酸盐缓冲溶液中的溶出没有明显差距。在15mM GSH环境下,与BIS作交联剂的舒尼替尼药物胶囊(a)相比,使用BAC交联剂交联的纳米药物胶囊,溶出速率快,溶出度高,说明其具有氧化还原响应。
Claims (10)
1.一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,其特征在于:该方法包括以下步骤,
S1提供具备pH响应的单体聚乙二醇-马来酸酐-N-丙烯酰胺单体PEG-CDM-NAM,简称PCN;
S2将包含舒尼替尼的二甲基亚砜溶液和丙烯酸酯基辛基苯酚聚乙二醇醚OP-AC的二甲基亚砜溶液混合,得到表面修饰有乙烯基的舒尼替尼,再加入到含有一定量中性单体丙烯酰胺AM、具备pH响应单体PCN、氧化还原响应降解交联剂N,N’-双(丙烯酰基)胱氨酸BAC的缓冲液中,同时加入聚合反应引发剂过硫酸铵APS、乳化剂TEMED引发原位自由基聚合;
S3收集S2中反应产物,通过透析得到舒尼替尼纳米药物胶囊溶液。
2.根据权利要求1所述的一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,其特征在于:具有pH响应的单体PCN是通过如下步骤合成的:
S1.1将2-丙-3-甲基马来酸酐简称CDM和草酰氯以一定比例添加到装有无水二氯甲烷CH2Cl2的烧瓶中,滴加四氢呋喃DMF催化剂并隔绝空气,将烧瓶置于冰水中搅拌1h,移至25℃继续反应3h;
S1.2使用旋转蒸发仪除去CH2Cl2,得到固体产物;
S1.3将S1.2中的固体产物与无水聚乙二醇单甲醚简称mPEG以一定比例添加到装有无水CH2Cl2的烧瓶中,滴加吡啶催化剂并隔绝空气,将烧瓶放置于25℃环境下反应3h;
S1.4取反应产物与两倍体积的饱和NH4Cl混合,分液以除去多余反应物,使用冻乙醚进行沉降并离心干燥,得到中间产物mPEG(CDM);
S1.5将N-(3-氨基丙基)甲基丙烯酸盐盐酸盐(以下简称NAM)溶于无水乙醇中并加入三乙胺(TEA)除去盐酸,与一定比例的S1.4产物mPEG(CDM)共同溶解于无水乙醇中,25℃环境下反应6h,使用冻乙醚进行沉降并离心干燥,得到具备pH响应单体PEG-CDM-NAM,即PCN。
3.根据权利要求2所述的一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,其特征在于:S1.1中CDM和草酰氯的质量比为1/1.6。
4.根据权利要求2所述的一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,其特征在于:S1.3中固体产物与无水聚乙二醇单甲醚的质量比为1/1.35。
5.根据权利要求2所述的一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,其特征在于:S1.5中mPEG(CDM)与N-(3-氨基丙基)甲基丙烯酸盐-盐酸盐的质量比为13/1。
6.根据权利要求1所述的一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,其特征在于:缓冲液指的是浓度150mM,pH=7.4的磷酸二氢钠/磷酸氢二钠缓冲溶液PBS。
7.根据权利要求1所述的一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,其特征在于:OP-AC的二甲基亚砜溶液中的OP-AC和舒尼替尼的质量比为1/5。
8.根据权利要求1所述的一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,其特征在于:二甲基亚砜和反应环境缓冲液的体积比为1/20。
9.根据权利要求1所述的一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,其特征在于:交联剂为氧化还原响应降解交联剂N,N'-双(丙烯酰基)胱氨酸(BAC)或非可降解交联剂N'N-甲叉双丙烯酰胺(BIS);
舒尼替尼/(单体+交联剂)质量比为1/0.4;单体和交联剂的质量比为1/0.4;酸敏感电荷反转单体PCN和中性单体AM的质量比为10/1;
所述的舒尼替尼和过硫酸铵的质量比为1/0.04,单体和TEMED的质量比为1/5。
10.根据权利要求1所述的一种具有pH和还原级联双响应的舒尼替尼纳米药物胶囊的制备方法,其特征在于:由该制备方法制的舒尼替尼纳米药物胶囊,舒尼替尼表面有一层聚合物载体,聚合物载体在偏酸性环境下表面电荷由负电性反转为正电性,舒尼替尼纳米药物胶囊在偏酸性环境下表面正电荷增大;舒尼替尼纳米药物胶囊的平均粒径在200~300nm之间。
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