CN115192735A - 一种亲水性药物载体靶向脑肿瘤荧光示踪剂及制备方法 - Google Patents
一种亲水性药物载体靶向脑肿瘤荧光示踪剂及制备方法 Download PDFInfo
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Abstract
一种亲水性药物载体靶向脑肿瘤特异性荧光示踪剂及制备方法。本发明提供一种包裹聚合物胶束‑IR780药物缀合物的磷脂纳米粒的制备方法，包括脂质体和磷脂胶束，这是一种易于进入原发性脑肿瘤中多形性胶质母细胞瘤的药物载体，它们具有生物相容性，能够通过增强细胞通透性和保留（EPR）效应改善药物的药代动力学和在实体肿瘤中的积聚情况。近红外染料七甲川菁染料IR780具有优异的肿瘤靶向性和成像特性，使用聚合物胶束‑药物偶联物能够有效解决七甲川菁染料IR780的缺点，并且弥补一些因素影响药物发挥实质效果或不能够准确送达靶向肿瘤细胞。基于共价载药聚合物的药物释放系统，即聚合物胶束‑药物偶联物，药物和聚合物通过相对稳定的连接物（例如酰胺和酯键）缀合，提高药物靶向特性、荧光特性、连续释放性和传输稳定性。
Description
技术领域
本发明属于生物医药技术领域，涉及靶向脑肿瘤亲水性药物载体特异性荧光示踪剂及制备方法。
背景技术
多形性胶质母细胞瘤是最常见、最致命的原发性脑肿瘤，由于其侵袭性生长而缺乏有效的治疗手段。转移的肿瘤细胞渗透到正常的软组织中，阻止了其完全的手术切除，其对化疗和放疗的高抵抗力促使多形性胶质母细胞瘤作为一种更具侵袭性的复发表型。近红外荧光成像技术具有非侵入、高灵敏度、无损伤、实时成像、成像效果好等优点。在荧光示踪剂的标记下，荧光成像技术可以实现对微小病灶的检测与荧光引导切除，包括肿瘤边界的完全切除及转移的肿瘤细胞的追踪。
七甲川菁染料IR780具有良好的脑肿瘤靶向性和成像特性，特别是对于多形性胶质母细胞瘤的靶向成像，但其疏水性限制了其临床应用。另外，IR780的低细胞毒性使其具有潜在的临床应用价值。另一方面，IR780也具有疏水性且不溶于医药上可接受的溶剂得缺点，因此临床应用需要合适的药物载体。
由脂质体或磷脂胶束形成的人工膜包裹IR780染料是有效的药物载体方式，但对于这些药物释放系统，还存在许多因素影响药物发挥实质效果或不能够准确送达靶向肿瘤细胞，如高稀释度、pH值、温度、离子强度、血液循环中的大剪切力以及大量带电血液成分的存在，都会影响其稳定性，这可能导致药物的突然释放。基于共价载药聚合物的药物释放系统，即聚合物胶束-药物偶联物，药物和聚合物通过相对稳定的连接物（例如酰胺和酯键）缀合，能够有效解决上述问题。与脂质体或磷脂胶束直接物理包埋方法相比，聚合物胶束药物缀合物通过物理封装载药更稳定，并且通常具有连续释放而无突发释放效应的特点。两亲性嵌段共聚物可以在水溶液中形成核壳胶束，再在其表面包裹人工膜脂质体或磷脂胶束能够大大地提高药物靶向特性、荧光特性、连续释放性和传输稳定性。
发明内容
有鉴于此，本发明的主要目的是为了解决靶向脑肿瘤的近红外七甲川菁染料IR780具有疏水性、同类荧光靶向示踪剂疏水性问题以及因素影响药物发挥实质效果或不能够准确送达靶向肿瘤细胞，而提供了聚合物胶束药物缀合物通过物理封装载药制备方法。
为实现上述目的，本发明与脑肿瘤细胞靶向结合的药物-聚合物偶联物的制备方法，包括以下步骤：
步骤1：以聚乙二醇-羟基和二溴异丁酯为原料，通过酯化反应制备了制备PEG-Br大引发剂。
步骤2：以PEG-Br为引发剂，CuBr/PMDETA为催化体系，采用ATRP法合成了PEG-b-P（OEGEMA-co-AzPMA）嵌段共聚物。
步骤3：通过气密注射器引入AzPMA（0.95 mL，6.0 mmol）、OEGEMA（2.0 mL，10.0mmol）、干甲苯（10 mL）和PMDETA（0.16 mL，0.8 mmol）。
步骤4：通过三次冻融循环降解混合物，然后在80℃下浸入油浴中。聚合持续6小时，并通过将烧瓶浸入液氮中终止。混合液用四氢呋喃稀释，并通过一个短的中性氧化铝柱除去残余的铜催化剂。
步骤5：溶液在真空下浓缩并沉淀成冷己烷。经四氢呋喃溶解、冷己烷沉淀反复纯化，真空干燥过夜，得到含蜡固体PEG-b-P（OEGEMA-co-AzPMA）。
本发明公开了聚合物胶束-IR780药物偶联物中间物的制备方法，包括以下步骤：
步骤1：将一定量的苯氧丙酸和IR780加入到一定量的DMSO中，通过亲核取代胆碱化物的单分子亲核取代反应机制将苯氧丙酸加入到IR780碘化物。
步骤2：在65℃环境下，氮气氛围下将此反应持续5h。
步骤3：5h后将所得的沉淀物过滤、清洗、烘干得到聚合物胶束-IR780药物偶联物中间物。
本发明公开了制备聚合物胶束-IR780药物偶联物的制备方法，通过铜催化PEG-b-P（OEGEMA-co-AzPMA）与聚合物胶束-IR780药物偶联物中间物的叠氮化物-炔烃点击反应，合成了药物-聚合物偶联物OEGEMA-co-AzPMA-IR780，包括以下步骤：
步骤1:向10 mL烧瓶中添加聚合物胶束-IR780药物偶联物中间物（0.09 g，0.10mmol）、PEG-b-P（OEGEMA-co-AzPMA）（0.12 g，0.10 mmol ）和PMDETA（0.16 mL，0.8 mmol）并用2 mL DMF溶解。
步骤2:通过三次冻融循环降解混合物，然后添加CuBr（57.2mg，0.40mmol）。反应混合物在氮气气氛下在室温下搅拌24小时。
步骤3:然后用
稀释混合物，并用10 wt%EDTA水溶液和去离子水洗涤。
步骤4：有机相用硫酸钠干燥，然后沉淀成过量的乙醚。经
溶解、乙醚沉淀反
复纯化，真空干燥过夜，得到药物-聚合物偶联物OEGEMA-co-AzPMA-IR780。
基于上述技术方案可知，本发明的方法和由此制备的近红外荧光染料具有如下有益效果：本发明涉及的原料易获得，反应条件温和，具有较为简单的可操作性和可控性；制备的荧光染料药物稳定好，对于靶向目标细胞具有强靶向性、高特异性和高荧光灵敏度。制备的纳米制剂不仅可以使用在七甲川菁染料IR780使其获得亲水性，还可以使用在同种类型的靶向肿瘤细胞疏水性药物载体方面，有利于推动近红外荧光靶向探针在临床医学上的应用。
附图说明
图1为聚合物-IR780药物偶联物OEGEMA-co-AzPMA-IR780结构式。
图2为聚合物-IR780药物偶联物中间物制备流程图。
图3为二嵌段共聚物OEGEMA-co-AzPMA的制备流程图。
图4为聚合物-IR780药物偶联物制备流程图。
图5为靶向肿瘤两亲性药物载体示意图。
具体实施方式
为了更清楚的说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应当以此限制本发明的保护范围。
本发明提供一种亲水性药物载体靶向脑肿瘤特异性荧光示踪剂制备方法，这是一种易于进入原发性脑肿瘤中多形性胶质母细胞瘤的药物载体，它们具有生物相容性，能够通过增强细胞通透性和保留（EPR）效应改善药物的药代动力学和在实体肿瘤中的积聚情况。近红外染料七甲川菁染料IR780具有优异的肿瘤靶向性和成像特性，本发明提出创新的聚合物胶束-IR780药物偶联物以及脂质体和磷脂胶束封装，能够大大地提高药物靶向特性、荧光特性、连续释放性和传输稳定性。
具体地，本发明公开了药物聚合物共轭物PEG-b-P的制备方法，包括以下步骤：
步骤1：以聚乙二醇-羟基和二溴异丁酯为原料，通过酯化反应制备了制备PEG-Br大引发剂。
步骤2：以PEG-Br为引发剂，CuBr/PMDETA为催化体系，采用ATRP法合成了PEG-b-P（OEGEMA-co-AzPMA）嵌段共聚物。
步骤3：通过气密注射器引入AzPMA（0.95 mL，6.0 mmol）、OEGEMA（2.0 mL，10.0mmol）、干甲苯（10 mL）和PMDETA（0.16 mL，0.8 mmol）。
步骤4：通过三次冻融循环降解混合物，然后在80℃下浸入油浴中。聚合持续6小时，并通过将烧瓶浸入液氮中终止。混合液用四氢呋喃稀释，并通过一个短的中性氧化铝柱除去残余的铜催化剂。
步骤5：溶液在真空下浓缩并沉淀成冷己烷。经四氢呋喃溶解、冷己烷沉淀反复纯化，真空干燥过夜，得到含蜡固体PEG-b-P（OEGEMA-co-AzPMA）。
更具体地，本发明公开了聚合物胶束-IR780药物偶联物中间物的制备方法，包括以下步骤：
步骤1：将一定量的苯氧丙酸和IR780加入到一定量的DMSO中，通过亲核取代胆碱化物的单分子亲核取代反应机制将苯氧丙酸加入到IR780碘化物。
步骤2：在65℃环境下，氮气氛围下将此反应持续5h。
步骤3：5h后将所得的沉淀物过滤、清洗、烘干得到聚合物胶束-IR780药物偶联物中间物。
更具体地，本发明公开了制备聚合物胶束-IR780药物偶联物的制备方法，通过铜催化PEG-b-P（OEGEMA-co-AzPMA）与聚合物胶束-IR780药物偶联物中间物的叠氮化物-炔烃点击反应，合成了药物-聚合物偶联物OEGEMA-co-AzPMA-IR780，包括以下步骤：
步骤1:向10 mL烧瓶中添加聚合物胶束-IR780药物偶联物中间物（0.09 g，0.10mmol）、PEG-b-P（OEGEMA-co-AzPMA）（0.12 g，0.10 mmol ）和PMDETA（0.16 mL，0.8 mmol）并用2 mL DMF溶解。
步骤2:通过三次冻融循环降解混合物，然后添加CuBr（57.2mg，0.40mmol）。反应混合物在氮气气氛下在室温下搅拌24小时。
步骤3:然后用
稀释混合物，并用10 wt%EDTA水溶液和去离子水洗涤。
步骤4：有机相用硫酸钠干燥，然后沉淀成过量的乙醚。经
溶解、乙醚沉淀反
复纯化，真空干燥过夜，得到药物-聚合物偶联物OEGEMA-co-AzPMA-IR780。
更具体地，本发明公开了制备药物-聚合物偶联物OEGEMA-co-AzPMA-IR780脂质体特异性荧光染料的制备方法，包括以下步骤：
步骤1：将DSPC、胆固醇和DSPE-PEG2000以54.8:40:5的摩尔比溶解于氯仿（总脂质约15mg/ml）中，然后加入1mg/ml乙醇中的药物-聚合物偶联物OEGEMA-co-AzPMA-IR780，使染料与总脂质的摩尔比达到0.2%。
步骤2：溶液在黑暗中旋转蒸发至真空干燥。
步骤3：干燥的脂质膜在氮气中水合，用0.9%的盐水冲洗至最终总脂质浓度30mm，在氮气气氛下旋转并超声处理，以最终悬浮脂质颗粒。
步骤4：将悬浮液在56℃下通过100 nm Whatman核孔径迹蚀刻聚碳酸酯膜反复挤压16次。
步骤5：用氮气冲洗获得的透明蓝色悬浮液，密封在玻璃瓶中，并在4℃或−20℃的黑暗中储存。
作为优选，该制备方法还包括：
上述反应中，可以通过步骤干燥的脂质膜用氮气冲洗0.9%生理盐水水合，改变总脂质浓度形成磷脂胶束。
为了制备IR780磷脂胶束，具体实施例：
步骤1：将DSPC、胆固醇和DSPE-PEG2000以54.8:40:5的摩尔比溶解于氯仿（总脂质约15mg/ml）中，然后加入1mg/ml乙醇中的药物-聚合物偶联物OEGEMA-co-AzPMA-IR780，使染料与总脂质的摩尔比达到0.2%。
步骤2：溶液在黑暗中旋转蒸发至真空干燥，得到脂质薄膜。
步骤3：干燥的脂质膜用氮气冲洗0.9%生理盐水水合，最终总脂质浓度为15mM，在氮气气氛下在56°C下涡旋和超声处理15min，并通过100nm Whatman核孔跟踪聚碳酸酯膜挤压。
步骤4：获得的透明青色胶束悬浮液，密封在玻璃瓶中，并在4℃或−20℃的黑暗中储存。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (9)
1.一种聚合物胶束-IR780药物偶联物结构式，
2.一种聚合物胶束-IR780药物偶联物中间物的制备方法，其特征是将一定量的苯氧丙酸和IR780加入到一定量的DMSO在65℃环境下，氮气氛围下将此反应持续5h。
3.一种制备聚合物胶束-IR780药物偶联物的制备方法，其特征是通过铜催化PEG-b-P（OEGEMA-co-AzPMA）与聚合物胶束-IR780药物偶联物中间物的叠氮化物-炔烃点击反应，合成了药物-聚合物偶联物OEGEMA-co-AzPMA-IR780。
4.一种聚合物胶束-IR780药物偶联物药物载体靶向示踪剂，其特征是，磷脂纳米颗粒，包括脂质体和胶束，脂质体囊泡由包围一个水隔室的磷脂双层膜组成，磷脂胶束囊泡有一层磷脂心，表面上有亲水性PEG链涂层。
5.一种聚合物胶束-IR780药物偶联物脂质体药物载体靶向示踪剂的制备方法，其特征是，将DSPC、胆固醇和DSPE-PEG2000混合溶解于氯仿（总脂质约15mg/ml）中，然后加入1mg/ml乙醇中的IR780碘化物，使染料与总脂质的摩尔比达到0.2%。
6.如权利要求5所述的聚合物胶束-IR780药物偶联物脂质体药物载体靶向示踪剂的制备方法，其特征是，DSPC、胆固醇和DSPE-PEG2000的摩尔比为54.8:40:5，溶液在黑暗中旋转蒸发至真空干燥。
7.如权利要求5所述的聚合物胶束-IR780药物偶联物脂质体药物载体靶向示踪剂的制备方法，其特征是，干燥的脂质膜在氮气中水合，用0.9%的盐水冲洗至最终总脂质浓度30mM，在氮气气氛下旋转并超声处理，以最终悬浮脂质颗粒。
8.如权利要求5所述的聚合物胶束-IR780药物偶联物脂质体药物载体靶向示踪剂的制备方法，其特征是，将悬浮液在56℃下通过Whatman孔径100nm径迹蚀刻膜聚碳酸酯膜反复挤压16次，用氮气冲洗获得。
9.如权利要求5、6所述的聚合物胶束-IR780药物偶联物磷脂胶束药物载体靶向示踪剂，其特征是，干燥的脂质膜用氮气冲洗0.9%生理盐水水合，最终总脂质浓度为15mM，在氮气气氛56°C下涡旋和超声处理15min，并通过Whatman孔径100nm径迹蚀刻膜聚碳酸酯膜挤压获得。
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