CN117531019A - 一种聚合物多酚载药纳米体系制备方法及其应用 - Google Patents
一种聚合物多酚载药纳米体系制备方法及其应用 Download PDFInfo
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- CN117531019A CN117531019A CN202311383969.7A CN202311383969A CN117531019A CN 117531019 A CN117531019 A CN 117531019A CN 202311383969 A CN202311383969 A CN 202311383969A CN 117531019 A CN117531019 A CN 117531019A
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Abstract
本发明公开了一种聚合物多酚载药纳米体系的制备方法及其应用,包括如下步骤:A)利用原子转移自由基聚合技术引发OEGMA单体和tBMA单体进行共聚,合成了一种两亲性共聚物P(OEGMA‑st‑tBMA);B)得到多羧基聚合物P(OEGMA‑st‑AA);C)得到多酚聚合物P(OEGMA‑st‑AA‑DA);D)得到DOX@POAD‑Fe@GOD NPs。采用本发明的制备方法制备的聚合物多酚载药纳米体系可以同时负载DOX,GOD和Fe3+,且重现性好,具有较高的稳定性。纳米粒中负载的DOX和GOD可加速Fe3+诱导的芬顿反应,产生有毒的・OH,进而增强化学动力学治疗(CDT)。
Description
技术领域
本发明涉及医用生物材料领域,尤其涉及一种聚合物多酚载药纳米体系的制备方法。
背景技术
化学动力学治疗(CDT)是利用肿瘤微环境的某些独特的内在特性配以过渡金属功能材料,特异性地在肿瘤细胞内催化芬顿/类芬顿反应使细胞内过氧化氢(H2O2)转化为细胞毒性羟基自由基(∙OH)的一种新兴的癌症治疗策略。由于正常细胞和组织中的轻度碱性条件和较低H2O2水平,芬顿/类芬顿反应将不能正常发生,这使得CDT对正常细胞和组织的细胞毒性可以被忽略。
然而,由于肿瘤微环境中有限的内源性H2O2、金属催化剂浓度以及轻度酸性等的影响,肿瘤细胞内芬顿/类芬顿反应缓慢,将导致CDT的治疗效果一般不理想。因此,亟待改善肿瘤部位 H2O2、金属离子浓度和 pH 值来增强 CDT 的抗肿瘤效果。
DOX是一种广泛应用的蒽环类抗癌药物,其可以通过抑制拓扑异构酶 II 来治疗多种癌症,并且它可以激活烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶(NOXs)。NOXs 可以催化 O2和 NADPH 转化为NADP+和超氧自由基(∙O2 -),并在超氧化物歧化酶(SOD)的催化下进行歧化反应生成H2O2。此外,葡萄糖氧化酶(GOD)被认为是多种H2O2生成剂中较好的选择,它可通过催化葡萄糖生成H2O2和葡萄糖酸,不仅提高了H2O2水平,同时降低了肿瘤微环境的pH值,显著加速肿瘤内芬顿反应。
金属催化剂是决定CDT中芬顿/类芬顿反应速率的另一个关键因素。在 CDT 研究中,铁通过触发肿瘤微环境中的芬顿反应来产生细胞毒性活性氧(ROS),常作为化学动力剂的核心成分被广泛应用。芬顿反应中报道的典型铁催化剂包括Fe2+、Fe3+、Fe3O4和二茂铁。其中,利用金属与多酚化合物之间的强配位相互作用来递送Fe2+和Fe3+是最简单的一种方法。
发明内容
为解决上述问题,本发明公开了一种聚合物多酚载药纳米体系的制备方法及其应用。采用本发明的制备方法制备的聚合物多酚载药纳米颗粒具有重现性好,体外稳定性好等特点,能以便捷的方式同时负载DOX、GOD和Fe3+,显著加速了肿瘤细胞内芬顿反应,进而增强CDT。基于上述设计原则,我们在本发明中公开了聚合物多酚载药纳米颗粒DOX@POAD-Fe@GOD NPs的构建,它们通过多酚-金属配位的策略同时负载DOX、GOD和Fe3+,用于增强CDT。
为实现上述目的,本发明的技术方案为:
一种聚合物多酚载药纳米体系的制备方法,包括如下步骤:
步骤一、制备两亲性共聚物 P(OEGMA-st-tBMA) ;
步骤二、由P(OEGMA-st-tBMA)脱叔丁基得到多羧基聚合物P(OEGMA-st-AA);
步骤三、将多巴胺(DA)通过酰胺反应接枝到P(OEGMA-st-AA)上得到多酚聚合物 P(OEGMA-st-AA-DA);
步骤四、利用多酚聚合物 P(OEGMA-st-AA-DA)中邻苯二酚结构与Fe3+的金属配位作用形成金属-酚类网络,同时包裹阿霉素(DOX)得到了DOX@POAD-Fe NPs。接着DOX@POAD-Fe NPs进一步负载葡萄糖氧化酶(GOD)得到DOX@POAD-Fe@GOD NPs。
进一步的改进,所述步骤一中,两亲性共聚物 P(OEGMA-st-tBMA)的制备方法如下:
取小分子引发剂2-溴异丁酸丙炔酯、PMDETA、tBMA和OEGMA溶于苯甲醚中。经过三次冷冻-抽气-解冻循环后,在氮气气氛下加入溴化亚铜。重复上述冷冻-抽气-解冻循环操作三次,将混合物密封封闭,放入45℃的油浴中聚合2 h。然后通过除去盖子终止聚合,将混合物在冰正己烷中沉淀三次并离心收集粗产物。随后将沉淀用 DMF溶解,装入透析袋中(MWCO:3.5 kDa),对去离子水透析纯化48 h,冻干,得到P(OEGMA-st-tBMA)。
进一步的改进,所加入的2-溴异丁酸丙炔酯、PMDETA、tBMA、OEGMA、溴化亚铜的摩尔比为1:1:50:50:1。
进一步的改进,,所述步骤二中,多羧基聚合物P(OEGMA-st-AA)的制备方法如下:
将P(OEGMA-st-tBMA)溶于9 mL无水二氯甲烷中,缓慢滴加三氟乙酸和二氯甲烷的混合溶液,在室温下反应48 h。反应结束后旋转蒸发去除大部分有机试剂,在冷乙醚沉淀三次并收集粗产物。将粗产物溶解于DMF中,装入透析袋中(MWCO:3.5 kDa),用甲醇透析12h和用去离子水透析48 h,冻干,得到P(OEGMA-st-AA)。
进一步的改进,所加入的二氯甲烷与三氟乙酸的体积比为20:3。
进一步的改进,所述步骤三的具体步骤如下:
将聚合物P(OEGMA-st-AA)用无水DMF充分溶解,然后在冰浴搅拌条件下加入EDCI,HOBt和三乙胺,反应3h。在氮气保护下加入DA·HCl以及三乙胺,在氮气保护下室温搅拌反应48h。反应结束后,将溶液在过量的冰乙醚中沉淀离心。将得到的粗产品用DMF溶解,装于透析袋中(MWCO:3.5 kDa),用去离子水透析72 h,冻干得到多酚聚合物P(OEGMA-st-AA-DA)。
进一步的改进,所加入的 EDCI,HOBt和DA·HCl的摩尔比为1:1:1。
进一步的改进,所述步骤四的具体步骤如下:
将P(OEGMA-st-AA-DA)溶解于DMF中,加入到含DOX的DMF溶液中避光搅拌30 min。随后,将该混合溶液和FeCl3溶液通过注射泵注入到超纯水中,避光搅拌1h。将得到的溶液转移至透析袋(MWCO:3.5 kDa)用超纯水透析48 h得到DOX@PODA-Fe NPs。接着将得到的DOX@PODA-Fe NPs和GOD在Tris-HCl溶液中避光搅拌12 h,然后用超滤管(300 kDa)超滤离心,浓缩,得到DOX@POAD-Fe@GOD NPs,离心条件为5000 rpm,10min。
进一步的改进,所述DOX为脱盐酸的疏水性DOX;FeCl3溶液中Fe3+与P(OEGMA-st-AA-DA)中DA的摩尔比为1:1~7。
一种聚合物多酚载药纳米体系的用途,所述聚合物多酚载药纳米体系如上所示;所述聚合物多酚载药纳米体系用于增强化学动力学治疗发挥抗癌作用。
本发明的优点:
本发明技术方案带来的有益效果
采用本发明的制备方法制备的聚合物多酚载药纳米体系重现性好,具有较高的稳定性,可以便捷的方式同时负载DOX、GOD和Fe3+,显著加速了肿瘤细胞内芬顿反应,进而增强CDT。
附图说明
图1 为多酚聚合物 P(OEGMA-st-AA-DA)的合成路线图。
图2 为PBiB的1HNMR谱图(CDCl3)。
图3为P(OEGMA-st-tBMA)的1HNMR谱图(CDCl3)。
图4 为P(OEGMA-st-AA)的1HNMR谱图(DMSO)。
图5 为P(OEGMA-st-AA-DA)的1HNMR谱图(DMSO)。
图6 为P(OEGMA-st-tBMA)和P(OEGMA-st-AA)的SEC淋洗曲线图。
图7 为P(OEGMA-st-tBMA)、P(OEGMA-st-AA)和P(OEGMA-st-AA-DA)的FT-IR图。
图8 为DOX@POAD-Fe@NPs(A)和DOX@POAD-Fe@GOD NPs(B)的TEM图。
图9 为POAD NPs、POAD-Fe NPs、DOX@POAD-Fe NPs、DOX@POAD-Fe@GOD NPs的粒径(A)和紫外吸收曲线(B)。
图10 为DOX@POAD-Fe@GOD NPs在24天的粒径变化图。
图11为聚合物纳米粒子在PBS(pH 7.4、pH 5.5)中的药物释放曲线(n=3)。
图12 为DOX和DOX@POAD-Fe@GOD NPs 的溶血率(A),DOX 和 DOX@POAD-Fe@GODNPs 处理 HUVEC(B)、L02(C)和 HK-2(D)24 h后的存活率(n=3)。
图13为DOX和DOX@POAD-Fe@GOD NPs与4T1细胞共孵育2h,4h和8h的荧光图像。
图14 为POAD NPs、POAD-Fe NPs、DOX@POAD-Fe NPs、DOX@POAD-Fe@GOD NPs 分别与 4T1 细胞共孵育 6 h 后细胞中ROS荧光成像图。
具体实施方式
以下结合附图及实施例对本发明做进一步说明。
实施例
本发明提供了一种聚合物多酚载药纳米体系的制备方法,包括以下步骤:
A)制备两亲性共聚物 P(OEGMA-st-tBMA);
B)由P(OEGMA-st-tBMA)脱叔丁基得到多羧基聚合物P(OEGMA-st-AA);
C)将多巴胺(DA)通过酰胺反应接枝到P(OEGMA-st-AA)上得到多酚聚合物 P(OEGMA-st-AA-DA);
D)利用多酚聚合物 P(OEGMA-st-AA-DA)中的邻苯二酚结构与Fe3+的金属配位作用形成金属-酚类网络,同时包裹阿霉素(DOX)得到了DOX@POAD-Fe NPs。接着DOX@POAD-FeNPs进一步负载葡萄糖氧化酶(GOD)得到DOX@POAD-Fe@GOD NPs。
本发明首先利用ATRP聚合反应合成了两亲性共聚物 P(OEGMA-st-tBMA)。具体来说就是利用丙炔醇和溴代异丁酰溴合成小分子引发剂PBiB,然后利用ATRP聚合合成了两亲性共聚物 P(OEGMA-st-tBMA)。
在本发明的实施例中,ATRP聚合反应溶剂为苯甲醚,在某些实施例中,溶剂为丙酮。
在本发明的实施例中,ATRP聚合反应的反应温度为45 ℃,反应时间为2 h。
在本发明的实施例中,分子内点击化学反应温度为100℃,时间为48 h,溶剂为DMF。
后续在两亲性共聚物 P(OEGMA-st-tBMA)的基础上,通过脱叔丁基和酰胺反应得到多酚聚合物 P(OEGMA-st-AA-DA)。具体来说就是使两亲性共聚物利用三氟乙酸脱去叔丁基合成了多羧基聚合物P(OEGMA-st-AA),然后利用聚合物侧链上的羧基与盐酸多巴胺发生酰胺反应得到多酚聚合物 P(OEGMA-st-AA-DA)。
在本发明的实施例中,脱叔丁基反应的溶剂为无水二氯甲烷,所述反应的时间为48 h。
在本发明的实施例中,酰胺反应的溶剂为无水DMF,所述反应的时间为48 h。
最终利用多酚聚合物 P(OEGMA-st-AA-DA)中的邻苯二酚结构与Fe3+的金属配位作用,并同时负载DOX和GOD得到DOX@POAD-Fe@GOD NPs。具体来说,利用多酚聚合物 P(OEGMA-st-AA-DA)中的邻苯二酚结构与Fe3+的金属配位作用通过自组装形成纳米粒,同时负载DOX得到DOX@POAD-FeNPs。接着将DOX@POAD-FeNPs通过与GOD避光混合负载GOD得到DOX@POAD-Fe@GOD NPs。
在本发明的实施例中,Fe3+与邻苯二酚的投料比为1:5,在某些实施例中,投料比为1:3、1:7。
在本发明的实施例中,DOX@POAD-FeNPs的纯化方法为超纯水透析。
在本发明的实施例中,GOD负载的反应溶剂为Tris-HCl溶液(pH=8.5),反应时间为12 h,在某些实施例中,反应时间为6h。
在本发明的实施例中,DOX@POAD-Fe@GOD NPs的纯化方法为超滤。
本发明对上文采用的原料的来源并无特殊的限制,可以为一般市售。
为了进一步说明本发明,以下结合实施例对本发明提供的一种聚合物多酚载药纳米体系的制备方法及其应用进行详细描述,但不能将其理解为对本发明保护范围的限定。
以下实施例中所用的原料均为一般市售。
实施例1
小分子引发剂(PBiB)的合成:称取 1.0 g 的丙炔醇加入装有 25 mL 无水二氯甲烷的 50 mL 圆底烧瓶中,加入 3.4 g 的三乙胺,在冰浴条件下搅拌 30 min。然后称取4.5 g 的溴代异丁酰溴,将其用无水二氯甲烷溶解,加入恒压滴定漏斗,在氮气保护下以一滴每秒的速度滴加到上述搅拌的 50 mL 圆底烧瓶中。待反应液滴加结束后,溶液继续在冰浴条件下搅拌 1 h。然后将圆底烧瓶转移到室温继续搅拌反应 12 h。反应结束后,将溶液进行抽滤,收集滤液,并用蒸馏水萃取 4 次,溶液加入无水硫酸钠进行干燥过夜。最后将溶液抽滤,经旋转蒸发浓缩,用乙酸乙酯和正己烷溶液(v/v 1:5)作为洗脱剂柱层析分离纯化粗产品,旋蒸除去有机溶剂,真空干燥得无色油状产物。如图1和图2所示,用核磁验证产物的成功合成(产率,85%)。
实施例2
两亲性共聚物 P(OEGMA-st-tBMA)的合成:准确称取 41.01 mg PBiB,34.68 mgPMDETA,1.422 g tBMA和 3 mg OEGMA溶于 8 mL 苯甲醚中,充分混匀,转入 50 mL 的聚合管中,用 2 mL 苯甲醚润洗转入聚合管中。经过 3 次冷冻-抽气-解冻循环后,在氮气的保护下加入 28.69 mg CuBr。再继续重复上述冷冻-抽气-解冻循环操作三次,将聚合管拧紧密封后,室温搅拌 10 min,最后将聚合管放入 45 ℃的油浴中开始进行聚合。反应 2 h后,开盖,将反应混合物暴露在空气中搅拌,终止聚合。用溶液 10 倍量的冰正己烷沉淀(分3 次,每次 4 mL),通过冷冻超速离心机离心收集粗产物,重复三次沉淀操作除去大部分未反应完的单体。将 3次离心后得到的粗产品用 2.0 mL 的 DMF 溶解,并将其装于截留分子量为 3.5 kDa 的透析袋中,进一步对去离子水透析 48 h,每 6 h 换一次水,去除未反应完的单体和铜催化剂,冷冻干燥得到产品。如图1、3所示,用核磁验证产物的成功合成(产率,70%)。
实施例3
多羧基聚合物P(OEGMA-st-AA)的合成:称取 1 g P(OEGMA-st-tBMA),用 20 mL的无水二氯甲烷充分溶解,加入到 50 mL 圆底烧瓶中充分溶解,再搅拌条件下缓慢加入 3mL 的三氟乙酸,反应 2 d。反应结束后,将溶液旋蒸,用 2 mL 的四氢呋喃将其溶解,在过量的冰乙醚中沉淀,通过冷冻超速离心机离心收集粗产物,重复三次。将 3 次离心后得到的粗产品用 3.0 mL 的 DMF 溶解,并将其装于透析袋中(MWCO,3.5 kDa),进一步对甲醇透析 12 h,对去离子水透析 36 h,去除脱掉的叔丁醇,冷冻干燥得到产品。如图4、6所示,用核磁和GPC验证产物的成功合成(产率,84%)。
实施例4
多酚聚合物P(OEGMA-st-AA-DA) 的合成:称取 68 mg 的聚合物P(OEGMA-st-AA),用 4 mL 的无水 DMF 充分溶解,加入到 50 mL 两颈烧瓶中,在冰浴搅拌条件下加入 44.2mg EDCI 和 31.2 mg HOBt,加入 32 μL 三乙胺,冰浴下反应 3h。在氮气保护下加入 45mg DA·HCl 以及 33 μL 三乙胺,在氮气保护下室温搅拌反应 2 d。反应结束后,将溶液在过量的冰乙醚中沉淀,然后离心。将得到的粗产品用 4.0 mL 的 DMF 溶解,并将其装于透析袋中(MWCO,3.5 kDa),用去离子水透析 72 h,冷冻干燥得到产品。如图5、7所示,用核磁和红外验证产物的成功合成(产率,40%)。
实施例5
(1)DOX@POAD-Fe NPs的制备:准确称取 2 mg 的 DOX·HCl,用 2 mL 的 DMF 充分溶解,加入 10 mL 圆底烧瓶中,再用 2 mL 的 DMF 润洗残余的液体加入圆底烧瓶中,缓慢转速下滴入45 μL 的三乙胺,然后在中等转速下避光搅拌过夜。将多酚聚合物P(OEGMA-st-AA-DA) 20 mg 溶于 4mL 的 DMF 中,将它们加入到上述 10 mL 的圆底烧瓶中避光搅拌混合 30 min,。再将多酚聚合物及 DOX 的混合溶液及 FeCl3溶液在高速搅拌的条件下用注射泵注入盛有 8 mL 超纯水的带磁子的 25 mL 圆底烧瓶中,避光继续搅拌 1 h。将液体转移至透析袋中(MWCO:3.5 kDa),避光条件下透析 36 h,得到 DOX@POADFe NPs。
(2)DOX@POAD-Fe@GOD NPs的制备:将 DOX@POAD-Fe NPs与 GOD 在 Tris-HCl 溶液(10 mM, pH=8.5)中避光搅拌 12 h,然后用超滤管(300 kD)离心(5000 rpm,10 min)洗涤 3 次,得到 DOX@POAD-Fe@GOD NPs。如图8、9所示,用透射电镜(TEM)、动态光散射(DLS)和紫外验证纳米药物的成功制备。
实施例6
利用动态光散射(DLS)考察了纳米药物的稳定性
纳米药物能够在一定的时间内保持一个相对的稳定对于药物的储存和放置尤为重要,因此我们通过DLS测定DOX@POAD-Fe@GOD NP在24天内的粒径,结果如图10所示。
实施例7
为了研究 37 ℃下纳米药物在不同 pH 值条件下的体外药物释放,将 PBS 溶液(pH7.4、pH5.5)提前一晚置于 37 ℃摇床水浴预热。将适量的纳米粒子转移至透析袋(MWCO=3.5 kDa),将透析袋浸没在 2 组不同的PBS 中(pH 7.4、pH 5.5),两种介质各做 3 个平行样。以 120 rpm 的速度振荡,在不同时间点(0 h、2 h、4h、8h、12h、24h、48h、72h)取 2 mL的释放介质,同时补充 2 mL新鲜的缓冲液。用紫外-可见分光光度计监测 DOX 的浓度,结果如图11所示。
实施例8
生物相容性评价:
(1)溶血试验:用生理盐水配制浓度为 0.04、0.4、4、8、16 和 32 μg/mL 的游离DOX 溶液和 DOX@POAD-Fe@GOD(等效 DOX)溶液,将红细胞混悬液与不同浓度的各溶液等体积进行混合。设置双蒸水和 PBS 分别作为阳性和阴性对照组。37 ℃ 恒温孵育 2 h 后,12000 rpm 离心 15 min,取上清置于 96 孔细胞培养板中,测量在各样品在 540 nm 处的吸光度值,结果如图12所示。
(2)MTT 法检测 DOX@POAD-Fe@GOD NPs 对HUVEC、L02及 HK-2 细胞的细胞毒性:每孔以8×103的细胞密度接种到 96 孔细胞培养板中,最外围加 200 μL 的PBS 溶液,培养 12 h 后取出培养板,去除原培养液后,分别每孔加入浓度为0.04、0.4、4、8、16、32 μg/mL 的 DOX 溶液和 DOX@POAD-Fe@GOD NPs(等效DOX)溶液,另外设置空白组(只有培养液、无细胞、无药物)和对照组(有细胞、无药物),继续孵育 24 h。在避光条件下,往培养板中加入 MTT溶液孵育 4 h 后,加 SDS 孵育过夜,测定在 450 nm 的吸光度,结果如图12所示。
实施例9
细胞摄取:通过荧光成像仪观察细胞摄取情况。每孔以 1×105个的 4T1 细胞接种到 24 孔细胞培养板中,培养 24 h 后将细胞培养板取出,加入 PBS 进行清洗,重复 3次。往孔中加入等量 DOX 浓度 2 μg/mL 的游离 DOX 和 DOX@POAD-Fe@GOD NPs,孵育特定时间 2 h、4 h、8h。培养到特定时间后,吸除 24 孔板中含药培养基,每孔加入 PBS 清洗,重复3 次后,每孔加入 200 μL 的 4%(w/v)多聚甲醛溶液,固定 10 min,到点后弃去多聚甲醛溶液,PBS 清洗 3 遍。然后往每孔加 200 μL DAPI 染色液,染核5min,到点后弃去DAPI 染色液,PBS 清 3 遍,吸尽培养板中的液体,将细胞板置于荧光成像仪成像,结果如图13所示。
实施例10
细胞内ROS水平的测定:通过荧光成像仪对细胞内 ROS 的水平进行评估。1.5×105个/孔的 4T1 细胞接种到 24 孔细胞培养板中,培养 24h 后将细胞培养板取出,加入PBS 进行清洗,重复 3 次。往孔中加入各组分 PBS、POAD NPs、POAD-Fe NPs、DOX@POAD-FeNPs、DOX@POAD-Fe@GOD NPs,孵育 6 h。培养到特定时间后,吸除 24 孔板中含药培养基,每孔加入冷 PBS 清洗细胞,重复 3 次后,将用无血清培养基稀释的 DCFH-DA 溶液加入孔中,200 μL/孔,孵育30 min,用 PBS 洗涤细胞 3 次,去除未进入细胞内的 DCFH-DA。用PBS 清洗3遍,加入多聚甲醛固定10 min,到点后弃去多聚甲醛溶液,用 PBS 清洗 3 遍,加入DAPI 染色液,染核10 min,到点后弃去 DAPI 染色液,PBS 清 3 遍。最后用荧光成像仪观察并拍照,结果如图14所示。
尽管本发明的实施方案已公开如上,但并不仅仅限于说明书和实施方案中所列运用,它完全可以被适用于各种适合本发明的领域,对于熟悉本领域的人员而言,可容易地实现另外的修改,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节和这里所示出与描述的图例。
Claims (10)
1.一种聚合物多酚载药纳米体系的制备方法,其特征在于,包括如下步骤:
步骤一、制备两亲性共聚物 P(OEGMA-st-tBMA) ;
步骤二、由P(OEGMA-st-tBMA)脱叔丁基得到多羧基聚合物P(OEGMA-st-AA);
步骤三、将多巴胺(DA)通过酰胺反应接枝到P(OEGMA-st-AA)上得到多酚聚合物 P(OEGMA-st-AA-DA);
步骤四、利用多酚聚合物 P(OEGMA-st-AA-DA)中邻苯二酚结构与Fe3+的金属配位作用形成金属-酚类网络,同时包裹阿霉素(DOX)得到了DOX@POAD-Fe NPs。接着DOX@POAD-FeNPs进一步负载葡萄糖氧化酶(GOD)得到聚合物多酚载药纳米体系DOX@POAD-Fe@GOD NPs。
2.如权利要求1所述的聚合物多酚载药纳米体系制备方法,其特征在于,所述步骤一中,两亲性共聚物 P(OEGMA-st-tBMA)的制备方法如下:
取小分子引发剂2-溴异丁酸丙炔酯、PMDETA、tBMA和OEGMA于反应容器内溶于苯甲醚中,经过三次冷冻-抽气-解冻循环后,在氮气气氛下加入溴化亚铜,重复冷冻-抽气-解冻循环操作三次,将混合物密封封闭,放入45℃的油浴中聚合2 h,然后通过除去反应容器的盖子终止聚合得到混合物,将混合物在冰正己烷中沉淀三次并离心收集粗产物,随后将粗产物用 DMF溶解,装入透析袋中(MWCO:3.5 kDa),用去离子水透析纯化48h,冻干,得到P(OEGMA-st-tBMA)。
3.如权利要求2所述的制备方法,其特征在于,所加入的2-溴异丁酸丙炔酯、PMDETA、tBMA、OEGMA、溴化亚铜的摩尔比为1:1:50:50:1。
4.如权利要求1所述的聚合物多酚载药纳米体系制备方法,其特征在于,所述步骤二中,多羧基聚合物P(OEGMA-st-AA)的制备方法如下:
将P(OEGMA-st-tBMA)溶于无水二氯甲烷中,缓慢滴加三氟乙酸和二氯甲烷的混合溶液,在室温下反应48 h,反应结束后旋转蒸发去除大部分有机试剂,在冷乙醚沉淀三次并收集粗产物,将粗产物溶解于DMF中,装入MWCO:3.5 kDa透析袋中,用甲醇透析12h和用去离子水透析48h,冻干,得到P(OEGMA-st-AA)。
5.如权利要求4所述的制备方法,其特征在于,所述三氟乙酸和二氯甲烷的混合溶液中二氯甲烷与三氟乙酸的体积比为20:3。
6.如权利要求1所述的聚合物多酚载药纳米体系制备方法,其特征在于,所述步骤三的具体步骤如下:
将聚合物P(OEGMA-st-AA)用无水DMF充分溶解,然后在冰浴搅拌条件下加入EDCI,HOBt和三乙胺,反应3h,在氮气保护下加入DA·HCl以及三乙胺,在氮气保护下室温搅拌反应48h,反应结束后得到反应溶液,将反应溶液在过量的冰乙醚中沉淀离心得到粗产品,将得到的粗产品用DMF溶解,装于MWCO:3.5 kDa的透析袋中,用去离子水透析72h,冻干得到多酚聚合物P(OEGMA-st-AA-DA)。
7.如权利要求6所述的制备方法,其特征在于,所加入的 EDCI,HOBt和DA·HCl的摩尔比为1:1:1。
8.如权利要求1所述的聚合物多酚载药纳米体系制备方法,其特征在于,所述步骤四的具体步骤如下:
将P(OEGMA-st-AA-DA)溶解于DMF中,加入到含DOX的DMF溶液中避光搅拌30 min得到混合溶液,随后将混合溶液和FeCl3溶液通过注射泵注入到超纯水中,避光搅拌1 h,将得到的溶液转移至透析袋(MWCO:3.5 kDa)用超纯水透析48 h得到DOX@PODA-Fe NPs;接着将得到的DOX@PODA-Fe NPs和GOD在Tris-HCl溶液中避光搅拌12 h,然后用超滤管(300 kDa)超滤离心,浓缩,得到DOX@POAD-Fe@GOD NPs,离心条件为5000 rpm,10min。
9.如权利要求8所述的制备方法,其特征在于,所述DOX为脱盐酸的疏水性DOX;FeCl3溶液中Fe3+与P(OEGMA-st-AA-DA)中DA的摩尔比为1:1~7。
10.一种权利要求1~9任一项制备的聚合物多酚载药纳米体系用于增强化学动力学治疗发挥抗癌作用,所述癌症包括乳腺癌、肝癌和肺癌。
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