CN116531486A - 一种人血清白蛋白负载奥曲肽纳米颗粒制备方法 - Google Patents
一种人血清白蛋白负载奥曲肽纳米颗粒制备方法 Download PDFInfo
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Abstract
本发明提出一种人血清白蛋白负载奥曲肽纳米颗粒制备方法,通过引入含有N‑羟基琥珀酰亚胺酯和马来酰亚胺两种官能团的异型双功能交联剂,能够有效实现人血清白蛋白与含有氨基的奥曲肽等多肽药物的复合;采用本发明建立的人血清白蛋白负载奥曲肽纳米颗粒制备方法,能够获得粒径细小均匀、稳定性好的多肽纳米制剂,可应用于口服制剂、干粉吸入制剂、皮下注射制剂等;纳米制剂能够在人体作用下,在特定时间释放出多肽药物,在多肽药物控释领域具有广阔的应用前景。
Description
技术领域
本发明属于药物制剂技术领域,具体涉及一种应用于多肽制剂、纳米制剂和控释制剂的人血清白蛋白负载奥曲肽纳米颗粒制备方法。
背景技术
多肽药物药理活性好、毒副作用小、作用专一性高,在疾病治疗上具有良好的应用前景,近年来在药物研发领域备受瞩目,已上市和研发中的多肽药物包括生长激素、促肾上腺皮质激素、胰岛素、胰高血糖素、降钙素、加压素、缩宫(催产)素、胸腺肽、干扰素等。在多肽药物及其制剂的开发过程中,主要限制因素在于多肽药物的化学结构和生物效应难以稳定保持、易被消化酶降解或首过效应消除、作用半衰期短、生物利用度低。目前多肽药物的主要制剂形式与给药方式为皮下或静脉注射,长期和频繁注射用药存在诸多使用不便,患者依从性低,且容易对细胞组织造成伤害和引发感染,对于长效作用制剂、控制释放制剂、非侵入式给药方式及其制剂需求迫切。
以奥曲肽(octreotide,OCT)为例,OCT化学名称为D-苯丙胺酰-L-半胱氨酰-L-苯丙胺酰-D-色氨酰-L-赖氨酰-L-苏氨酰-L-半胱氨酰-L-苏氨醇(2→7)环二硫化物,化学式为C49H66N10O10S,是首个人工合成的生长抑素类似物,对生长激素、胰高血糖素、胰岛素等分泌具有抑制作用,临床上是治疗肢端肥大症的首选药物,同时也用于治疗急慢性胰腺炎、上消化道出血、肠梗阻、胃肠胰神经内分泌肿瘤等疾病。目前批准上市的奥曲肽制剂有两种,一种是OCT注射液,经皮下注射,一天3次;另一种是采用生物可降解材料聚乳酸-羟基乙酸(PLGA)包覆OCT形成的缓释微球,经肌内注射,每28天注射一次。2018年,Hou等采用喷雾干燥技术制备了OCT可吸入微粉,干粉吸入剂为非侵入式给药方式,研究表明,微粉在肺部的有效摄入量为39.82%,生物利用度与皮下注射相当,但半衰期仅为15min(AilinH,LuL,YingH,etal.Fragmentedparticlescontainingoctreotideacetatepreparedby spraydryingtechniquefordrypowderinhalation[J].Drugdeliveryand translationalresearch,2018,8(3):693-701.)。2020年,向童欣分别采用超临界抗溶剂、冷冻干燥和喷雾冷冻干燥技术制备了OCT可吸入微粉,且较为系统地揭示了各制备技术的工艺条件对微粉性能的影响规律,研究表明,OCT微粉具有良好的吸入性能,体内作用效果与皮下注射相当,但半衰期也仅提高至30min(向童欣.奥曲肽可吸入微粉制备及其性能研究[D].军事科学院,2021.)。
将多肽药物与纳米载体复合制备为纳米制剂,能够发挥纳米颗粒特有的性质,提高药物的组织滞留性和屏障穿透性,延长药物作用时间,提高药物治疗效果。人血清白蛋白(Humanserumalbumin,HSA)是制备成纳米载体并用于多肽制剂的理想载体材料,具体表现为:HSA为内源性运输蛋白,是人体血液中最丰富的血浆蛋白(约45mg/mL),具有维持血浆pH和渗透压等生理功能,主要在肝脏中合成,无毒副作用,生物相容性好且来源丰富;HSA分子的结构中具有3个同源结构域,每个结构域都包含两个子域,这些结构域能够与多种内源性或外源性物质发生特异性结合,加之HSA分子在生理pH下带负电荷,可以与带正电荷的药物相互作用,具有良好的载药性能;HSA的半衰期约为20天,在血液中带负电荷,不易被巨噬细胞清除,可延长药物在体内的滞留和作用时间;HSA分子由585个氨基酸残基组成,相对分子质量约为66KDa,HSA分子内含有17个二硫键,1个游离的巯基和59个游离的氨基,这些基团让HSA具有良好修饰性,使药物与其有更多的结合方式,同时可使递送系统具有更多功能。
白蛋白纳米颗粒(HSA-NPs)的制备方法较多,常见的有反溶剂法、乳化法、纳米喷雾干燥、热凝胶法和自组装技术等,其中反溶剂法工艺简单,制备的颗粒粒径较小且重复性较好,相较于其他方法具有明显的优势,是目前应用最广的HSA-NPs的制备方法。HSA在水中和有机溶剂中的溶解度有明显的差异,在水中具有较高的溶解度而不溶于有机溶剂,反溶剂化法就是利用这一特性,通过向HSA水溶液中添加有机溶剂(一般为乙醇),降低HSA的溶解度,使HSA溶液过饱和,进而脱溶析出生成纳米颗粒。由于HSA具有较多的极性基团,从水相中脱溶析出的纳米颗粒状态不够稳定,因此需要通过化学交联等方式进一步稳定,文献报道的交联剂包括戊二醛、1,6-己二异氰酸酯、京尼平、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、葡萄糖等。
HSA与多肽药物复合的方式可分为非共价结合和化学偶联结合两大类,其中吸附和包埋是非共价结合的主要形式。吸附载药过程是在获得空白HSA-NPs后,利用HSA-NPs携带电荷的静电作用或表面/孔道等的空间结构,实现多肽药物的装载,该方式工艺简单,载药量相对较高,但由于多肽药物与载体吸附的结合力较弱,长效作用或控制释放的效果有限。包埋载药则是在HSA-NPs形成的过程中,同时将药物包覆于载体内部,或者实现药物与载体的相互契合,该方式获得的制剂能够有效延长作用时间,达到缓释的效果,但是与小分子化药相比,多肽类药物的分子量较大且具有一定的空间结构,难于同时获得较高的载药量与包封率,且具有相对较高的技术门槛。
化学偶联结合主要是利用HSA游离的巯基或氨基,将多肽药物以共价键的方式直接连接于HSA的分子上,再将复合药物制备成纳米颗粒。该复合方式下多肽药物与HSA载体结合稳定,药物释放需要依靠化学键的断裂或HSA的分解,因此能够有效延长作用时间,可以设计为特定生理环境或溶出条件下的响应释放,或者设计为特定时间的突释型控释制剂。但HSA和多肽药物都具有一定空间结构,化学键合可能存在明显的空间位阻,并且多肽药物能够匹配HSA共价结合反应的官能基团有限。
本发明旨在建立一种以化学偶联方式制备HSA负载多肽药物纳米颗粒的新方法,以生产抑素类药物奥曲肽作为代表性多肽模型药物,通过引入异型双功能交联剂作为连接桥梁,使HSA和多肽药物能够更为普遍的以共价键的方式复合,并发挥纳米制剂特定时间突释的控释效果。
发明内容
(一)要解决的技术问题
本发明提出一种人血清白蛋白负载奥曲肽纳米颗粒制备方法,以解决生产抑素类药物奥曲肽作为代表性多肽模型药物的技术问题。
(二)技术方案
为了解决上述技术问题,本发明提出一种人血清白蛋白(HSA)负载奥曲肽纳米颗粒制备方法,该制备方法基于化学偶联复合技术,引入含有N-羟基琥珀酰亚胺酯(NHS酯)和马来酰亚胺(Mal)两种官能团的异型双功能交联剂;首先利用异型双功能交联剂中的NHS酯官能团与奥曲肽(OCT)中的氨基反应形成酰胺键,实现奥曲肽与异型双功能交联剂的复合;其次利用已复合奥曲肽的异型双功能交联剂中的马来酰亚胺官能团与人血清白蛋白中的巯基反应形成硫醚键,实现奥曲肽与人血清白蛋白的复合;最后采用水-乙醇反溶剂法,配合人血清白蛋白的氨基交联,经离心、洗涤、干燥得到人血清白蛋白负载奥曲肽纳米颗粒。
进一步地,异型双功能交联剂为6-(马来酰亚胺基)己酸琥珀酰亚胺酯(EMCS)或N-羟基琥珀酰亚胺酯-聚乙二醇-马来酰亚胺(SM(PEG)n);白蛋白氨基的交联剂为戊二醛或1,6-己二异氰酸酯。
进一步地,选用EMCS为异型双功能交联剂时,制备方法具体包括如下步骤:
S1.OCT-EMCS合成:按照摩尔比1:1称取OCT和EMCS,分别溶解于无水乙醇和乙腈中,将EMCS乙腈溶液滴加至OCT无水乙醇溶液中,室温下搅拌24h,反应生成OCT-EMCS,形成OCT-EMCS溶液;
S2.OCT-EMCS-HSA合成:称取HSA,用PBS缓冲液溶解,按摩尔比(1:1)~(1:1.5)向HSA溶液中加入OCT-EMCS溶液,调节pH至6.5~7.5,室温下搅拌24h,反应生成OCT-EMCS-HSA;
S3.OCT-EMCS-HSA纯化:将OCT-EMCS-HSA反应液置于透析袋中透析,去除有机溶剂和未反应的OCT-EMCS,透析液为纯水,每8h更换一次透析液,连续透析48h,将透析袋内溶液冻干得到纯化的OCT-EMCS-HSA;
S4.OCT-EMCS-HSA纳米颗粒制备:将纯化的OCT-EMCS-HSA复溶于纯水中,在搅拌下滴加反溶剂无水乙醇,待溶液转变为淡蓝色混悬液,加入交联剂,交联剂用量为HSA中氨基40%~100%发生交联,搅拌反应3~24h后,旋转蒸发除去混悬液中的有机溶剂,离心获取沉淀物,沉淀物用纯水反复洗涤和离心后,冷冻干燥,得到OCT-EMCS-HSA纳米颗粒。
进一步地,选用SM(PEG)n为异型双功能交联剂时,制备方法具体包括如下步骤:
S1.OCT-SM(PEG)n合成:按照摩尔比1:1称取OCT和SM(PEG)n,分别溶解于PBS缓冲溶液中,将SM(PEG)n溶液滴加至OCT溶液中,调节pH值至7~9,室温下搅拌24h,反应生成OCT-SM(PEG)n,形成OCT-SM(PEG)n溶液;
S2.OCT-SM(PEG)n-HSA合成:称取HSA,用PBS缓冲液溶解,按摩尔比(1:1)~(1:1.5)向HSA溶液中加入OCT-SM(PEG)n溶液,调节pH至6.5~7.5,室温下搅拌24h,反应生成OCT-SM(PEG)n-HSA;
S3.OCT-SM(PEG)n-HSA纯化:将OCT-SM(PEG)n-HSA反应液置于透析袋中透析,去除未反应的OCT-SM(PEG)n,透析液为纯水,每8h更换一次透析液,连续透析48h,将透析袋内溶液冻干得到纯化的OCT-SM(PEG)n-HSA;
S4.OCT-SM(PEG)n-HSA纳米颗粒制备:将纯化的OCT-SM(PEG)n-HSA复溶于纯水中,在搅拌下滴加反溶剂无水乙醇,待溶液变为淡蓝色混悬液,加入交联剂,交联剂用量为HSA中氨基40%~100%发生交联,搅拌反应24h后,旋转蒸发除去混悬液中的有机溶剂,离心获取沉淀物,沉淀物用纯水反复洗涤和离心后,冷冻干燥得到OCT-SM(PEG)n-HSA纳米颗粒。
进一步地,选用SM(PEG)n为异型双功能交联剂时,聚乙二醇的聚合度n=2~24。
采用该方法,人血清白蛋白能够负载其他含有游离氨基的多肽药物或化学药物。制备获得的人血清白蛋白负载奥曲肽纳米颗粒,具有延时释放的控释效果。
此外,本发明还提出一种人血清白蛋白负载奥曲肽纳米颗粒,该人血清白蛋白负载奥曲肽纳米颗粒采用上述方法制备得到。
此外,本发明还提出一种人血清白蛋白负载奥曲肽纳米颗粒的应用,该人血清白蛋白负载奥曲肽纳米颗粒应用于口服制剂、干粉吸入制剂、皮下注射制剂。
(三)有益效果
本发明提出一种人血清白蛋白负载奥曲肽纳米颗粒制备方法,通过引入含有N-羟基琥珀酰亚胺酯和马来酰亚胺两种官能团的异型双功能交联剂,能够有效实现人血清白蛋白与含有氨基的奥曲肽等多肽药物的复合;采用本发明建立的人血清白蛋白负载奥曲肽纳米颗粒制备方法,能够获得粒径细小均匀、稳定性好的多肽纳米制剂,可应用于口服制剂、干粉吸入制剂、皮下注射制剂等;纳米制剂能够在人体作用下,在特定时间释放出多肽药物,在多肽药物控释领域具有广阔的应用前景。
本发明中的6-(马来酰亚胺基)己酸琥珀酰亚胺酯(EMCS)、N-羟基琥珀酰亚胺酯-聚乙二醇-马来酰亚胺(SM(PEG)n)可替换为其他含有N-羟基琥珀酰亚胺酯和马来酰亚胺两种官能团的异型双功能交联剂;HSA负载的药物不局限于奥曲肽,可推广应用至其他含有游离氨基的多肽药物或化学药物。
附图说明
图1为OCT-EMCS-HSA化学合成路线图;
图2为OCT-SM(PEG)n-HSA化学合成路线图;
图3为合成OCT-EMCS的液相色谱-质谱联用质谱图;图中:OCT-EMCS的[M+H]+、[M+Na]+、双电荷峰的m/z分别为1212.3、1234.2和606.7;
图4为合成OCT-EMCS-HSA的基质辅助激光解析飞行时间质谱图;图中:OCT-EMCS-HSA的[M+H]+、双电荷峰的m/z分别为67561.14和33725.64;HSA的[M+H]+、双电荷、碎片峰的m/z分别为66437.52、33157.40和22078.25与44294.71;
图5为不同白蛋白氨基交联剂制备OCT-EMCS-HSA纳米颗粒平均粒径和储存稳定性;图中:横坐标为氨基交联剂的种类;纵坐标为纳米颗粒的平均粒径,单位为纳米(nm);交联剂用量为HSA中氨基100%发生交联;
图6为不同白蛋白氨基交联剂制备OCT-EMCS-HSA纳米颗粒扫描电镜图片;图中:A为氨基交联剂戊二醛,B为氨基交联剂1,6-己二异氰酸酯,C为1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC),交联剂用量为HSA中氨基100%发生交联;
图7为不同用量氨基交联剂戊二醛制备OCT-EMCS-HSA纳米颗粒透射电镜图;图中:A为HSA中氨基40%发生交联,B为HSA中氨基60%发生交联,C为HSA中氨基100%发生交联;
图8为不同异型双功能交联剂制备OCT-SM(PEG)n-HSA纳米颗粒扫描电镜图;图中:A为异型双功能交联剂SM(PEG)2,B为异型双功能交联剂SM(PEG)12,C为异型双功能交联剂SM(PEG)24;
图9为OCT-EMCS-HSA纳米制剂血药浓度曲线;图中:横坐标为时间,单位为小时(h);纵坐标为血药浓度,单位为纳克/毫升(ng/mL)。
具体实施方式
为使本发明的目的、内容和优点更加清楚,下面结合附图和实施例,对本发明的具体实施方式作进一步详细描述。
实施例1
选用6-(马来酰亚胺基)己酸琥珀酰亚胺酯(EMCS)为异型双功能交联剂,制备方法的具体步骤如下:
S1.OCT-EMCS合成:按照摩尔比1:1精密称取100mgOCT和30.24mgEMCS,分别溶解于无水乙醇和乙腈中,将EMCS乙腈溶液滴加至OCT无水乙醇溶液中,室温下搅拌24h,反应生成OCT-EMCS,形成OCT-EMCS溶液;
S2.OCT-EMCS-HSA合成:精密称取400mgHSA,用PBS缓冲液溶解,按摩尔比1:1.5向HSA溶液中加入OCT-EMCS溶液,调节pH至6.5~7.5,室温下搅拌24h,反应生成OCT-EMCS-HSA;
S3.OCT-EMCS-HSA纯化:将反应液置于透析袋中透析去除有机溶剂和未反应的OCT-EMCS,透析袋截留分子量5000~8000,透析液为纯水,每8h更换一次透析液,连续透析48h,将透析袋内溶液冻干得到纯化的OCT-EMCS-HSA;
S4.OCT-EMCS-HSA纳米颗粒制备:将OCT-EMCS-HSA复溶于纯水中,在搅拌下滴加反溶剂无水乙醇,待溶液转变为淡蓝色混悬液,加入HSA氨基交联剂,HSA氨基交联剂分别选择戊二醛、1,6-己二异氰酸酯、京尼平、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)、葡萄糖,交联剂用量为HSA中氨基100%发生交联,搅拌反应3h后,旋转蒸发除去混悬液中的有机溶剂,离心获取沉淀物,沉淀物用纯水洗涤和离心3次后,冷冻干燥得到OCT-EMCS-HSA纳米颗粒。
OCT-EMCS-HSA化学合成路线图如图1所示。
合成OCT-EMCS的液相色谱-质谱联用质谱图如图3所示,OCT-EMCS的[M+H]+、[M+Na]+、双电荷峰的m/z分别为1212.3、1234.2和606.7,验证合成得到了OCT-EMCS,且证明OCT与EMCS的反应具有良好的专属性,无其它副反应。
合成OCT-EMCS-HSA的基质辅助激光解析飞行时间质谱图如图4所示,图中m/z为67561.14、33725.64处,对应为OCT-EMCS-HSA的[M+H]+、双电荷峰,验证合成得到了OCT-EMCS-HSA,实现了奥曲肽与白蛋白的化学偶联复合。采用巯基试剂盒测定HSA原料和纯化后OCT-EMCS-HSA的巯基含量,结果显示HSA原料中仅有约30%的HSA存在巯基,因此基质辅助激光解析飞行时间质谱图中有HSA的特征峰,HSA的[M+H]+、双电荷、碎片峰的m/z分别为66437.52、33157.40和22078.25与44294.71;合成产物中不存在游离巯基,证明OCT-EMCS已结合了HSA中全部的游离巯基。
不同白蛋白氨基交联剂制备OCT-EMCS-HSA纳米颗粒的平均粒径和储存稳定性如图5所示,结果表明不加交联剂时,获得的颗粒粒径较大,且在储存后粒径进一步增大;选用京尼平或葡萄糖交联剂时,制备初期的颗粒粒径细小(100~200nm),但储存稳定性较差,经过10天、20天储存后,颗粒粒径迅速增大,团聚现象明显;选择戊二醛、1,6-己二异氰酸酯或EDC交联剂时,纳米颗粒粒径细小且经过10天、20天储存后保持稳定,其中选择戊二醛或1,6-己二异氰酸酯交联剂时,纳米颗粒更为细小,且粒径均一性更优。不同白蛋白氨基交联剂制备OCT-EMCS-HSA纳米颗粒扫描电镜图片如图6所示,选用戊二醛或1,6-己二异氰酸酯作为白蛋白氨基交联剂时,制备颗粒粒径100~200nm,形貌为规则的球状,颗粒间分散良好;而选用EDC交联剂时,颗粒形貌不够规则,且颗粒粒径均一性差。综上,戊二醛或1,6-己二异氰酸酯为优选的白蛋白氨基交联剂。
实施例2
选用6-(马来酰亚胺基)己酸琥珀酰亚胺酯(EMCS)为异型双功能交联剂,制备方法的具体步骤如下:
S1.OCT-EMCS合成:按照摩尔比1:1精密称取100mgOCT和30.24mgEMCS,分别溶解于无水乙醇和乙腈中,将EMCS乙腈溶液滴加至OCT无水乙醇溶液中,室温下搅拌24h,反应生成OCT-EMCS,形成OCT-EMCS溶液;
S2.OCT-EMCS-HSA合成:精密称取400mgHSA,用PBS缓冲液溶解,按摩尔比1:1向HSA溶液中加入OCT-EMCS溶液,调节pH至6.5~7.5,室温下搅拌24h,反应生成OCT-EMCS-HSA;
S3.OCT-EMCS-HSA纯化:将反应液置于透析袋中透析去除有机溶剂和未反应的OCT-EMCS,透析袋截留分子量5000~8000,透析液为纯水,每8h更换一次透析液,连续透析48h,将透析袋内溶液冻干得到纯化的OCT-EMCS-HSA;
S4.OCT-EMCS-HSA纳米颗粒制备:将OCT-EMCS-HSA复溶于纯水中,在搅拌下滴加反溶剂无水乙醇,待溶液转变为淡蓝色混悬液,加入戊二醛作为HSA氨基交联剂,交联剂用量分别为HSA中氨基20%、40%、60%、100%发生交联,搅拌反应24h后,旋转蒸发除去混悬液中的有机溶剂,离心获取沉淀物,沉淀物用纯水洗涤和离心3次后,冷冻干燥得到OCT-EMCS-HSA纳米颗粒。
不同用量氨基交联剂戊二醛制备OCT-EMCS-HSA纳米颗粒透射电镜图如图7所示,氨基20%发生交联时,未能形成形貌规则的纳米颗粒;氨基40%~100%发生交联时,能够制备获得粒径约100nm的颗粒,形貌为规则的球状,颗粒间分散良好;随着氨基发生交联比例的增加,颗粒整体越为完整与规则。
实施例3
选用N-羟基琥珀酰亚胺酯-聚乙二醇-马来酰亚胺(SM(PEG)n)为异型双功能交联剂,制备方法的具体步骤如下:
S1.OCT-SM(PEG)n合成:按照摩尔比1:1精密称取100mgOCT和相应的SM(PEG)n,具体为SM(PEG)241.14mg、SM(PEG)1284.95mg、SM(PEG)24136.82mg,分别溶解于PBS缓冲溶液中,将SM(PEG)n溶液滴加至OCT溶液中,调节pH值至7~9,室温下搅拌24h,反应生成OCT-SM(PEG)n,形成OCT-SM(PEG)n溶液;
S2.OCT-SM(PEG)n-HSA合成:精密称取400mgHSA,用PBS缓冲液溶解,按摩尔比1:1.3向HSA溶液中加入OCT-SM(PEG)n溶液,调节pH至6.5~7.5,室温下搅拌24h,反应生成OCT-SM(PEG)n-HSA;
S3.OCT-SM(PEG)n-HSA纯化:将反应液置于透析袋中透析去除未反应的OCT-SM(PEG)n,透析袋截留分子量8000~12000,透析液为纯水,每8h更换一次透析液,连续透析48h,将透析袋内溶液冻干得到纯化的OCT-SM(PEG)n-HSA;
S4.OCT-SM(PEG)n-HSA纳米颗粒制备:将OCT-SM(PEG)n-HSA复溶于纯水中,在搅拌下滴加反溶剂无水乙醇,待溶液变为淡蓝色混悬液,加入戊二醛作为HSA氨基交联剂,交联剂用量分别为HSA中氨基100%发生交联,搅拌反应16h后,旋转蒸发除去混悬液中的有机溶剂,离心获取沉淀物,沉淀物用纯水洗涤和离心3次后,冷冻干燥得到OCT-SM(PEG)n-HSA纳米颗粒。
OCT-SM(PEG)n-HSA化学合成路线图如图2所示。
不同异型双功能交联剂制备OCT-SM(PEG)n-HSA纳米颗粒扫描电镜图如图8所示,结果表明选用SM(PEG)n为异型双功能交联剂时,聚乙二醇(PEG)聚合度在n=2~24的范围内,均能够制备获得粒径50~100nm的颗粒,形貌为规则的球状,颗粒间分散良好;选用不同PEG聚合度的SM(PEG)n异型双功能交联剂,纳米制剂无显著差异。
实施例4
选用6-(马来酰亚胺基)己酸琥珀酰亚胺酯(EMCS)为异型双功能交联剂,制备方法的具体步骤如下:
S1.OCT-EMCS合成:按照摩尔比1:1精密称取100mgOCT和30.24mgEMCS,分别溶解于无水乙醇和乙腈中,将EMCS乙腈溶液滴加至OCT无水乙醇溶液中,室温下搅拌24h,反应生成OCT-EMCS,形成OCT-EMCS溶液;
S2.OCT-EMCS-HSA合成:精密称取400mgHSA,用PBS缓冲液溶解,按摩尔比1:1向HSA溶液中加入OCT-EMCS溶液,调节pH至6.5~7.5,室温下搅拌24h,反应生成OCT-EMCS-HSA;
S3.OCT-EMCS-HSA纯化:将反应液置于透析袋中透析去除有机溶剂和未反应的OCT-EMCS,透析袋截留分子量5000~8000,透析液为纯水,每8h更换一次透析液,连续透析48h,将透析袋内溶液冻干得到纯化的OCT-EMCS-HSA;
S4.OCT-EMCS-HSA纳米颗粒制备:将OCT-EMCS-HSA复溶于纯水中,在搅拌下滴加反溶剂无水乙醇,待溶液转变为淡蓝色混悬液,加入戊二醛作为HSA氨基交联剂,交联剂用量分别为HSA中氨基60%发生交联,搅拌反应24h后,旋转蒸发除去混悬液中的有机溶剂,离心获取沉淀物,沉淀物用纯水洗涤和离心3次后,冷冻干燥得到OCT-EMCS-HSA纳米颗粒。
以大鼠为模型动物,分别将OCT-EMCS-HSA纳米制剂以口服制剂、皮下注射制剂、干粉吸入制剂形式给药,采用液相色谱-质谱联用方式分析不同给药时间后的血药浓度,绘制血药浓度曲线(如图9所示),对照样品为OCT溶液肺部滴注给药。分析结果表明,OCT溶液肺部给药后药物迅速释放,0.5h达到血药浓度峰值;人血清白蛋白负载奥曲肽纳米制剂经口服、皮下注射、肺部吸入等形式给药后,分别于6h、12h、16h达到血药浓度峰值,表现出明显的延迟释放效果,在多肽药物控释领域具有广阔的应用前景。
本发明同时开展了OCT-EMCS-HSA纳米制剂的体外溶出性能评价,溶出温度37±1℃,溶出介质PBS缓冲溶液,溶出介质pH值分别为7.4、3、10,溶出时间48h。结果显示纳米制剂在中性、酸性、碱性介质中均没有药物释放,表明本发明方法制备的人血清白蛋白负载奥曲肽纳米颗粒制剂体外储存稳定,需要依赖人体内部的生理作用释放药物。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变形,这些改进和变形也应视为本发明的保护范围。
Claims (7)
1.一种人血清白蛋白负载奥曲肽纳米颗粒制备方法,所述制备方法基于化学偶联复合技术,其特征在于,引入含有N-羟基琥珀酰亚胺酯和马来酰亚胺两种官能团的异型双功能交联剂;首先利用异型双功能交联剂中的N-羟基琥珀酰亚胺酯官能团与奥曲肽中的氨基反应形成酰胺键,实现奥曲肽OCT与异型双功能交联剂的复合;其次利用已复合奥曲肽的异型双功能交联剂中的马来酰亚胺官能团与人血清白蛋白中的巯基反应形成硫醚键,实现奥曲肽与人血清白蛋白HSA的复合;最后采用水-乙醇反溶剂法,配合人血清白蛋白的氨基交联,经离心、洗涤、干燥,得到人血清白蛋白负载奥曲肽纳米颗粒。
2.如权利要求1所述的人血清白蛋白负载奥曲肽纳米颗粒制备方法,其特征在于,所述异型双功能交联剂为6-(马来酰亚胺基)己酸琥珀酰亚胺酯EMCS或N-羟基琥珀酰亚胺酯-聚乙二醇-马来酰亚胺SM(PEG)n;白蛋白氨基的交联剂为戊二醛或1,6-己二异氰酸酯。
3.如权利要求2所述的人血清白蛋白负载奥曲肽纳米颗粒制备方法,其特征在于,选用EMCS为异型双功能交联剂时,制备方法具体包括如下步骤:
S1.OCT-EMCS合成:按照摩尔比1:1称取OCT和EMCS,分别溶解于无水乙醇和乙腈中,将EMCS乙腈溶液滴加至OCT无水乙醇溶液中,室温下搅拌24h,反应生成OCT-EMCS,形成OCT-EMCS溶液;
S2.OCT-EMCS-HSA合成:称取HSA,用PBS缓冲液溶解,按摩尔比(1:1)~(1:1.5)向HSA溶液中加入OCT-EMCS溶液,调节pH至6.5~7.5,室温下搅拌24h,反应生成OCT-EMCS-HSA;
S3.OCT-EMCS-HSA纯化:将OCT-EMCS-HSA反应液置于透析袋中透析,去除有机溶剂和未反应的OCT-EMCS,透析液为纯水,每8h更换一次透析液,连续透析48h,将透析袋内溶液冻干得到纯化的OCT-EMCS-HSA;
S4.OCT-EMCS-HSA纳米颗粒制备:将纯化的OCT-EMCS-HSA复溶于纯水中,在搅拌下滴加反溶剂无水乙醇,待溶液转变为淡蓝色混悬液,加入交联剂,交联剂用量为HSA中氨基40%~100%发生交联,搅拌反应3~24h后,旋转蒸发除去混悬液中的有机溶剂,离心获取沉淀物,沉淀物用纯水反复洗涤和离心后,冷冻干燥,得到OCT-EMCS-HSA纳米颗粒。
4.如权利要求2所述的人血清白蛋白负载奥曲肽纳米颗粒制备方法,其特征在于,选用SM(PEG)n为异型双功能交联剂时,制备方法具体包括如下步骤:
S1.OCT-SM(PEG)n合成:按照摩尔比1:1称取OCT和SM(PEG)n,分别溶解于PBS缓冲溶液中,将SM(PEG)n溶液滴加至OCT溶液中,调节pH值至7~9,室温下搅拌24h,反应生成OCT-SM(PEG)n,形成OCT-SM(PEG)n溶液;
S2.OCT-SM(PEG)n-HSA合成:称取HSA,用PBS缓冲液溶解,按摩尔比(1:1)~(1:1.5)向HSA溶液中加入OCT-SM(PEG)n溶液,调节pH至6.5~7.5,室温下搅拌24h,反应生成OCT-SM(PEG)n-HSA;
S3.OCT-SM(PEG)n-HSA纯化:将OCT-SM(PEG)n-HSA反应液置于透析袋中透析,去除未反应的OCT-SM(PEG)n,透析液为纯水,每8h更换一次透析液,连续透析48h,将透析袋内溶液冻干得到纯化的OCT-SM(PEG)n-HSA;
S4.OCT-SM(PEG)n-HSA纳米颗粒制备:将纯化的OCT-SM(PEG)n-HSA复溶于纯水中,在搅拌下滴加反溶剂无水乙醇,待溶液变为淡蓝色混悬液,加入交联剂,交联剂用量为HSA中氨基40%~100%发生交联,搅拌反应24h后,旋转蒸发除去混悬液中的有机溶剂,离心获取沉淀物,沉淀物用纯水反复洗涤和离心后,冷冻干燥得到OCT-SM(PEG)n-HSA纳米颗粒。
5.如权利要求4所述的人血清白蛋白负载奥曲肽纳米颗粒制备方法,其特征在于,选用SM(PEG)n为异型双功能交联剂时,聚乙二醇的聚合度n=2~24。
6.一种人血清白蛋白负载奥曲肽纳米颗粒,其特征在于,所述人血清白蛋白负载奥曲肽纳米颗粒采用如权利要求1-5任一项所述方法制备得到。
7.一种如权利要求6所述的人血清白蛋白负载奥曲肽纳米颗粒的应用,其特征在于,所述人血清白蛋白负载奥曲肽纳米颗粒应用于口服制剂、干粉吸入制剂、皮下注射制剂。
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