CN116333190A - 一种多重响应的前药聚合物、制备方法和应用 - Google Patents
一种多重响应的前药聚合物、制备方法和应用 Download PDFInfo
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Abstract
Description
技术领域
本发明属于聚合物前体药物合成技术领域,具体涉及一种多重响应的前药聚合物、制备方法和应用。
背景技术
免疫原性细胞死亡(ICD)指正在死亡的肿瘤细胞通过分泌损伤相关分子模式(DAMP),如钙网蛋白(CRT)、高迁移率族蛋白1(HMGB1))和肿瘤相关抗原(TAA),由此激发宿主免疫反应,以促进树突状细胞(DC)的识别、成熟和抗原递呈。小分子化学药物如阿霉素等可引起肿瘤ICD,但肿瘤微环境(TME)中高水平的活性氧(ROS)可损伤HMGB1的功能,降低ICD引起的免疫疗效。双硒键作为ROS的敏感键,可消耗TME中ROS水平增强ICD疗效。此外,刺激响应型前药纳米平台能赋予载体生物活性,减少药物泄露,并在特定部位触发释放药物,在肿瘤治疗中被广泛应用。
免疫抑制TME包括肿瘤相关成纤维细胞(CAFs)、调节性T细胞(Treg)和转化生长因子-β(TGF-β)等,也不利于肿瘤免疫治疗。成纤维细胞激活蛋白(FAP)是CAFs的典型标志物,在纤维化、细胞外基质(ECM)重塑和肿瘤进展中起关键作用。FAP抑制剂如talabostatmesylate(Tab)可下调免疫抑制细胞因子,已被开发用于抑制CAFs。
免疫检查点信号通路如程序性细胞死亡蛋白1(PD-1)、程序性细胞死亡配体1(PD-L1)可促进肿瘤细胞逃脱宿主免疫监视。近年来,D型肽DPPA-1等被报道可作为PD-L1拮抗剂,用于阻断肿瘤免疫检查点。
因此,为应对肿瘤免疫抑制微环境(如CAFs,高表达的ROS)及免疫检查点信号通路介导的免疫监督逃脱,同时协同增强肿瘤免疫原性细胞死亡,构建囊泡胶束可转换前药纳米平台以实现药物分别在肿瘤微环境及细胞内的顺序释放,并阻断免疫检查点,具有十分重要的意义。
发明内容
针对现有技术中的上述问题,本发明的目的在于提供一种多重响应的前药聚合物,其对ROS、pH及GSH具有多重响应性,能够自组装得到聚合物纳米囊泡,实现不同亲疏水性药物的不同时空可控释放,增强肿瘤ICD效应,适于肿瘤免疫治疗应用。
本发明的目的还在于提供一种多重响应的前药聚合物的制备方法。
本发明目的还在于提供上述多重响应的前药聚合物的应用。
为了实现以上目的,本发明采用的技术方案是:
一种多重响应的前药聚合物,记为dSe-PEG-PC7A-P(BAEMA-(AEMA-SS-R)),结构式如式Ⅰ所示:
如式Ⅰ所示的A-B-C型嵌段聚合物中,嵌段A为聚乙二醇链段,即PEG链段,对应的重复单元m为68~182;所述嵌段A占聚合物分子主链总链长的20%-40%;
嵌段B为聚甲基丙烯酸-2-(六甲撑亚胺)乙酯链段,即PC7A链段,对应的重复单元n为6~17;
嵌段C为含有聚甲基丙烯酸叔丁基氨基乙酯的复合链段,即P(BAEMA-(AEMA-SS-R))链段,对应的重复单元x为12~20,y为1~3;嵌段C中,基团R选自羟基或活性药物基团。
本发明提供的多重响应的前药聚合物,具有新颖的化学结构,并且针对活性氧(ROS)、酸碱度(pH)及谷胱甘肽(GSH)具有多重响应性。
其中,本发明前药聚合物在pH 7.4条件下,疏水部分(嵌段B和嵌段C)的分子量是亲水部分(嵌段A)的1.5倍及以上,适用于聚合物纳米囊泡的制备(双亲分子自组装的一种超分子聚集体)。其中,本发明前药聚合物的疏水段PBAEMA可通过接枝药物活性基团以引起肿瘤免疫原性细胞死亡。将本发明聚合物通过自组装过程制备所得聚合物纳米囊泡,在囊泡亲水性内腔中可装载Fab抑制剂Tab,用于抑制CAFs,重塑肿瘤微环境。pH 6.8条件下,嵌段PC7A由疏水性转变为亲水性,亲疏水比例发生变化,纳米粒由囊泡转变为胶束并释放Tab,抑制CAFs。而PEG处键接的二硒键,可消耗肿瘤微环境中的ROS,增强肿瘤ICD。
本发明的前药聚合物,应用后能够通过形貌转换,分别在肿瘤微环境中释放亲水性药物,在肿瘤细胞内释放疏水性药物,实现不同亲疏水性药物的不同时空可控释放。并且,本发明的前药聚合物的亲水段PEG的末端,可通过化学键偶联肿瘤特异性靶向分子,如DPPA-1,其可在肿瘤微环境中高ROS条件下被释放,从而有效阻断免疫检查点。因此本发明的聚合物在肿瘤免疫治疗中体现出良好的应用前景。
为优化前药聚合物的结构和各项性能,优选地,m为114,n为11,x为18,y为3;嵌段A占聚合物分子主链总链长的40%;所述活性药物基团为阿霉素基团阿霉素基团在多重响应的前药聚合物中的总个数为1、2或3。
本发明的多重响应的前药聚合物的制备方法,采用的技术方案是:
如上所述的多重响应的前药聚合物的制备方法,包括以下步骤:
(1)将羧基-聚乙二醇-氨基与4-氰基-4-(苯羰基硫代)戊酸-琥珀酰亚胺酯进行酰胺化反应,得到羧基-聚乙二醇-4-氰基-4-(苯羰基硫代)戊酰胺,记为COOH-PEG-CPPA;
(2)将COOH-PEG-CPPA与甲基丙烯酸-2-(六甲撑亚胺)乙酯、甲基丙烯酸叔丁基氨基乙酯先后发生聚合反应,得到产物A,记为HOOC-PEG-PC7A-PBAEMA;
(3)将HOOC-PEG-PC7A-PBAEMA与三氟乙酸反应,得到产物B,记为COOH-PEG-PC7A-P(BAEMA-AEMA);
(4)将COOH-PEG-PC7A-P(BAEMA-AEMA)与二硒二乙醇进行酯化反应,得到产物C,记为dSe-PEG-PC7A-P(BAEMA-AEMA);
(5)将dSe-PEG-PC7A-P(BAEMA-AEMA)加入到含有3,3二硫代二丙酸的溶液中进行酰胺化反应,得到产物D,记为dSe-PEG-PC7A-P(BAEMA-(AEMA-DTPA));
(6)将dSe-PEG-PC7A-P(BAEMA-(AEMA-DTPA))与活性药物进行酰胺化反应,即得所述多重响应的前药聚合物,记为dSe-PEG-PC7A-P(BAEMA-(AEMA-SS-R))。
本发明的前药聚合物的制备方法,以聚乙二醇为引发剂,通过活性RAFT聚合顺序共聚甲基丙烯酸-2-(六甲撑亚胺)乙酯和甲基丙烯酸叔丁基氨基乙酯单体,得到分子量可控、分子量分布较窄的聚合物,水解后暴露氨基,然后与二硒二乙醇发生酯化反应,再与二硫代二丙酸、阿霉素发生酰胺化反应,得到多重响应的前药聚合物。本发明的上述制备方法,工艺简单,能够有效制备得到多重响应的前药聚合物,适于聚合物的制备应用。
通过在疏水段PBAEMA接枝药物活性基团,从而达到引起肿瘤免疫原性细胞死亡的目的。本发明对于药物活性基团的种类不作特别限定,其只要能够导致肿瘤免疫原性细胞死亡并实现在疏水段的有效接枝即可。优选地,所述活性药物为阿霉素、紫杉醇、米托蒽醌中的一种。
进一步地,所述二硒二乙醇采用如下步骤制备:氮气条件下,将3.8g硼氢化钠溶于30mL水中,加入6.4g硒粉,然后将混合物在105℃下搅拌30min,再加入6.4g硒粉,继续搅拌30min,冷却至室温后,在氮气条件下,加入含有12.5g 2-溴乙醇的四氢呋喃溶液,搅拌24h后,经硅胶柱层析纯化,即得。
进一步优选地,步骤(6)中,所述活性药物为阿霉素;dSe-PEG-PC7A-P(BAEMA-(AEMA-DTPA))与阿霉素的反应摩尔比为3.33:10。
本发明还提供上述多重响应的前药聚合物的应用,具体是在制备形貌可转换的抗肿瘤纳米药物中的应用。
进一步地,所述抗癌纳米药物的制备方法是:将所述多重响应的前药聚合物通过溶剂置换法制备得到聚合物纳米囊泡,然后在聚合物纳米囊泡表面键接PD-L1阻断剂DPPA-1,得到DPPA-1修饰的聚合物纳米囊泡,将DPPA-1修饰的聚合物纳米囊泡作为抗肿瘤纳米药物。
更进一步地,抗肿瘤纳米药物的制备方法中还包括在DPPA-1修饰的聚合物纳米囊泡上进行Tab装载的步骤。
对于抗肿瘤纳米药物所作用的肿瘤细胞的种类,本发明不作特殊限定,具体应用时可进行不同的肿瘤细胞进行适应性设计。优选地,所述肿瘤为乳腺癌。
试验证实:本发明的多重响应性聚合物前药,可在pH 7.4条件下,分别在有无DPPA-1修饰条件下,得到形貌可转换囊泡和阻断免疫检查点的形貌转换囊泡。聚合物囊泡在中性条件下,可装载亲水性药物;到达肿瘤后,在肿瘤微环境(TME)高ROS条件下,双硒键断裂,释放PD-L1拮抗剂,阻断免疫检查点;在肿瘤弱酸微环境条件下,PC7A嵌段由疏水性变为亲水性,链段亲疏水比例发生变化,囊泡转变为胶束,释放亲水药物调节TME;转变后形成的胶束经内吞作用进入细胞,在细胞内高还原条件下,二硫键断裂并释放药物,引起肿瘤ICD。
因此,本发明的多重响应性聚合物前药,能够有效应用于肿瘤免疫治疗。基于本发明聚合物制备的纳米药物为囊泡-胶束可转换的一类新型的抗癌纳米药物。
与现有技术相比,本发明的有益效果在于:
(1)本发明的多重响应性聚合物前药,可直接制备得到形貌可转换聚合物纳米囊泡,其可装载亲水性小分子FAP抑制剂如Tab,在模拟肿瘤微环境弱酸条件下,形貌转换为胶束并释放药物;接着胶束通过内吞作用进入细胞内,在高浓度还原条件下,二硫键断裂释放疏水药物,引起肿瘤ICD。通过不同时空条件下顺序释放药物,解决了亲水性药物在TME中用于免疫抑制逆转和细胞内释放疏水化疗药引起ICD之间的难题。
(2)本发明公开的多重响应性聚合物前药,制备得到的形貌可转换囊泡纳米药物,可通过ROS敏感键在其表面修饰免疫检查点拮抗剂DPPA-1,在TME的高ROS条件下,敏感键断裂释放拮抗剂阻断免疫检查点,有效解决了现有技术中需额外与抗体联用阻断免疫检查点的难题。
(3)本发明公开的多重响应性聚合物前药,所得生物相容性聚合物囊泡可在制备过程中形成还原敏感的纳米粒,制备方法简便,从而克服了现有技术中制备还原敏感纳米药物时需要复杂的操作和提纯过程等缺陷。
(4)本发明公开的多重响应性聚合物前药,自组装制备的形貌可转换囊泡,可同时用于亲、疏水小分子药物的控制释放体系,从而克服了现有纳米胶束载体仅适用装载疏水小分子药物的不足;并且可阻断免疫检查点的形貌可转换纳米囊泡,在癌症(如乳腺癌)免疫治疗方面具有广泛的应用价值。
附图说明
图1为本发明实施例2中COOH-PEG-PC7A-P(BAEMA-AEMA)的合成路线图;
图2为本发明实施例2中涉及的聚合物的核磁共振氢图谱;其中,图2A为COOH-PEG-CPPA;图2B为COOH-PEG-PC7A;图2C为COOH-PEG-PC7A-PBAEMA;图2D为COOH-PEG-PC7A-P(BAEMA-AEMA);
图3为本发明实施例2中dSe-PEG-PC7A-P(BAEMA-(AEMA-SS-DOX)的合成路线图;
图4为本发明实施例2中dSe-PEG-PC7A-P(BAEMA-AEMA)的氢核磁图谱(4A)及其红外图谱(4B);
图5为本发明实施例3中TME、pH和还原三响应性多聚体前药纳米平台的粒径分布图;
图6为本发明实施例3中DPPA-1修饰的TRPP(DPPA-TRPP)纳米平台的粒径分布图;
图7为试验例1中前药聚合物对过氧化氢的消耗结果图;
图8为试验例2中不同条件下24h内DOX和Tab的体外释放情况图;
图9为试验例3中用四甲基偶氮唑盐比色法(MTT)检测TRPP产生的细胞毒性结果图;
图10为试验例4中分别用激光共聚焦显微镜和流式细胞仪检测细胞内在化情况图;
图11为试验例5中DPPA-TRPP介导4T1细胞发生免疫原性细胞死亡的结果图;
图12为试验例6中DPPA-TRPP/DIR和TRPP/DIR在小鼠体内不同时间点的肿瘤蓄积结果图;
图13为试验例7中DPPA-TRPP/Tab在4T1肿瘤模型中的体内抗肿瘤活性和长期记忆免疫应答图;其中图13A为小鼠治疗后的肿瘤体积变化图,图13B为小鼠体重变化图,图13C为小鼠生存曲线图,图13D为二次接种肿瘤体积生长曲线图。
具体实施方式
以下结合具体实施方式,对本发明的技术方案作进一步描述。但是本领域技术人员应当理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。以下实施例中,涉及的原料和方法如无特殊说明,均为本领域常规的市售材料和常规工艺方法。
实施例1
本实施例的多重响应的前药聚合物,为A-B-C型嵌段聚合物,记为dSe-PEG-PC7A-P(BAEMA-(AEMA-SS-R)),结构式如下所示:
其中,嵌段A为聚乙二醇链段(PEG链段);嵌段B为聚甲基丙烯酸-2-(六甲撑亚胺)乙酯链段(PC7A链段);嵌段C为含有聚甲基丙烯酸叔丁基氨基乙酯的复合链段(P(BAEMA-(AEMA-SS-R))链段);嵌段C中,基团R为羟基和/或活性药物基团。
在不同的实施例中,阿霉素基团在多重响应的前药聚合物中的总个数为1个、2个或3个。当取代基R中只有1个阿霉素基团时,羟基的个数为2个;当含有2个阿霉素基团时,羟基的个数为1个;当含有3个阿霉素基团时,不含羟基。
实施例2
本实施例为多重响应的前药聚合物的制备方法的实施例,其进行如实施例1结构的前药聚合物的制备,具体方法包括如下步骤:
一、COOH-PEG-PC7A-P(BAEMA-AEMA)的合成
COOH-PEG-PC7A-P(BAEMA-AEMA)的合成路线图如图1所示。具体步骤如下:
(1)大分子可逆加成断裂转移自由基聚合(RAFT)试剂的合成
RAFT试剂即羧基-聚乙二醇-4-氰基-4-(苯羰基硫代)戊酰胺(COOH-PEG-CPPA)是由羧基-聚乙二醇-氨基(COOH-PEG-NH2,购自Biochempeg,分子量为5000,聚乙烯醇重复单元数114)与4-氰基-4-(苯羰基硫代)戊酸-琥珀酰亚胺酯(CPPA,TCI(上海)科技有限公司)发生酰胺化反应得到。
酰胺化反应的具体过程为:氮气及冰水浴条件下,将CPPA(7.52mg,0.03mmol)的二氯甲烷溶液逐滴加入到COOH-PEG-NH2(100mg,0.02mmol)与三乙胺(0.002mmol,0.20mg)的溶液中,室温反应24h。产物经冰***沉淀、抽滤及真空干燥得RAFT试剂HOOC-PEG-CPPA。
图2A为该步骤制备所得HOOC-PEG-CPPA的核磁共振氢谱图。根据图2A的核磁共振氢谱(1H NMR)的表征结果可知,PEG:δ3.23,3.51,CPPA:δ7.3-7.5可知,CPPA的键接率为94.6%。
(2)COOH-PEG-PC7A-PBAEMA的合成
大分子三嵌段共聚物羧基-聚乙二醇-聚甲基丙烯酸-2-(六甲撑亚胺)乙酯-聚甲基丙烯酸叔丁基氨基乙酯(COOH-PEG-PC7A-PBAEMA)由RAFT试剂聚合得到。
具体过程为:氮气条件下,HOOC-PEG-CPPA(100mg,0.02mmo1),甲基丙烯酸-2-(六甲撑亚胺)乙酯(MA-C7A,60mg,0.28mmo1),偶氮二异丁腈(AIBN,0.49mg,0.003mmo1)在1,4-二氧六环(2mL)中溶解。65℃下搅拌24h后,产物经冰***沉淀、抽滤、真空干燥,得两嵌段PEG-PC7A。
图2B为COOH-PEG-PC7A的核磁共振氢谱图。根据图2B中1H NMR(400MHz,DMSO-d6):PEG:δ3.23,3.51;PC7A:δ0.5-1.2的表征结果可知,上述结构式中,m=114,n=11。PEG-PC7A分子量为:5.0-2.3kg/mol,即PEG(5.0k)-PC7A(2.3k)。
进一步地,在氮气条件下,将上步骤制备所得两嵌段PEG-PC7A(140mg,0.019mmol),甲基丙烯酸叔丁基氨基乙酯(BAEMA,112mg,0.49mmol)和AIBN(0.49mg,0.003mmol)溶解在1,4-二氧六环中,密闭条件下反应48h。反应后将混合物用冰***沉淀,然后抽滤、真空干燥得产物,记为HOOC-PEG-PC7A-PBAEMA。
图2C为HOOC-PEG-PC7A-PBAEMA的核磁共振氢谱图。根据图2C中1H NMR(400MHz,DMSO-d6):PEG:δ3.23,3.51;PC7A:δ0.5-1.2;PBAEMA:δ1.4,0.5-0.9的表征结果可知,上述结构式中,m=114,n=11,p=21。HOOC-PEG-PC7A-PBAEMA分子量为5.0-2.3-4.7kg/mol,即COOH-PEG(5.0k)-PC7A(2.3k)-PBAEMA(4.7k)。
(3)COOH-PEG-PC7A-P(BAEMA-AEMA)的合成
将上述所得HOOC-PEG-PC7A-PBAEMA(200mg,0.017mmol)和三氟乙酸(TFA,2mL)溶于二氯甲烷(2mL)中。室温搅拌0.5h,旋转蒸发仪蒸发除去溶剂,去离子水(截留分子量MWCO=3500Da)透析并冷冻干燥。最终产物为淡黄色粉末,记为COOH-PEG-PC7A-P(BAEMA-AEMA)。
图2D为COOH-PEG-PC7A-P(BAEMA-AEMA)的核磁共振氢谱图。根据图2D中1H NMR(PEG:δ3.23,3.51;PC7A:δ0.5-1.2;P(BAEMA-AEMA):δ1.4)的表征结果可知,上述结构式中,m=114,n=11,x=18,y=3。
二、dSe-PEG-PC7A-P(BAEMA-AEMA)的合成
dSe-PEG-PC7A-P(BAEMA-AEMA)的合成路线图如图3所示。具体步骤如下:
氮气条件下,将硼氢化钠(3.8g,100mmol)溶于30mL水中,加入6.4g(81mmol)硒粉。将混合物在105℃下搅拌30min,再加入当量硒粉(6.4g,81mmol),继续搅拌30min。冷却至室温后,在氮气条件下,加入溶解于四氢呋喃(50mL)的2-溴乙醇(12.5g,100mmol)溶液。搅拌24h后,经硅胶柱层析纯化,得最终产物,记为HO-Se-Se-OH,简写为dSe,黄色液体(6.2g,产率21.3%)。
将步骤一得到的HOOC-PEG-PC7A-P(BAEMA-AEMA)(100mg,8.6μmol)溶解于二甲基甲酰胺(DMF,2mL)中,在氮气条件下,加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC·HCl,2.5mg,13.1μmol)和4-二甲氨基吡啶(DMAP,1.6mg,13.1μmol)活化1h。随后,氮气保护条件下,加入dSe(1mg,12.6μmol)。室温反应24h后,去离子水(MWCO=3.5kDa)透析并冷冻干燥,即得产物,记为dSe-PEG-PC7A-P(BAEMA-AEMA),为淡黄色粉末(75mg,产率74%)。
该步骤制备所得产物dSe-PEG-PC7A-P(BAEMA-AEMA)的氢核磁图谱(A)及其红外图谱(B)如图4所示。
由图4A中1H NMR(PEG:δ3.23,3.51;PC7A:δ0.5-1.2;P(BAEMA-AEMA):δ1.4;dSe:δ0.5-0.9)的表征结果可知,上述结构式中m=114,n=11,x=18,y=3,双硒成功连接到大分子共聚物中。图4B中,C-Se及Se-Se结构的出现,也证明了双硒的成功键接。
三、dSe-PEG-PC7A-P(BAEMA-(AEMA-SS-DOX)的合成
dSe-PEG-PC7A-P(BAEMA-(AEMA-SS-DOX)的合成路线图如图3所示。具体步骤如下:
氮气条件下,向3,3二硫代二丙酸(3.1mg,14.7μmol)的DMF(2mL)溶液中加入EDC·HCl(4.2mg,21.9μmol)和N-羟基琥珀酰亚胺(NHS,2.5mg,21.9μmol),室温条件下活化1h。氮气条件下,将步骤二得到的dSe-PEG-PC7A-P(BAEMA-AEMA)(55mg,4.7μmol)滴加到上述混合物中。室温反应24h后,去离子水(MWCO=3.5kDa)透析并冷冻干燥,得到的产物记为dSe-PEG-PC7A-P(BAEMA-(AEMA-DTPA)),为淡黄色粉末(50mg)。
将产物dSe-PEG-PC7A-P(BAEMA-(AEMA-DTPA))(40mg,3.33μmol)溶解在DMF(2mL)中,氮气条件下,分别加入EDC·HCl(2.9mg,15μmol)和NHS(1.7mg,15μmol)活化1h。随后,氮气条件下加入DOX(5.8mg,10μmol),室温反应24h后,去离子水(MWCO=3.5kDa)透析并冷冻干燥,得到的产物记为dSe-PEG-PC7A-P(BAEMA-(AEMA-SS-DOX),为红色粉末。
用紫外-可见光谱(UV-Vis)法测定不同浓度DOX(5、10、15、20、25、30、35、40μg/mL)的紫外吸收,绘制标准曲线。将上述制备得到的前药物共聚物dSe-PEG-PC7A-P(BAEMA-(AEMA-SS-DOX))溶于DMF中(浓度1mg/mL),通过UV-Vis测定其吸光度。根据吸收强度和标准曲线计算分子链中DOX的含量。结果表明,DOX质量分数为5%,即由于位阻效应,制备所得的多重响应的前药聚合物的共聚物链中,阿霉素DOX的平均接枝数目为1.2。
实施例3
本实施例为实施例2制备得到的多重响应的前药聚合物在制备囊泡-胶束可转换的抗癌纳米药物中的应用。
具体应用方式如下:
一、TME、pH和还原三响应性多聚体前药纳米聚合物囊泡的制备
采用溶剂交换法制备形貌可转换的前药纳米聚合物囊泡TRPP。将前药聚合物dSe-PEG-PC7A-P(BAEMA-(AEMA-SS-DOX))溶解于四氢呋喃中(200μL,5mg/mL),缓慢加到磷酸盐缓冲液(PBS,800μL,pH 7.4)中,得均匀分布的纳米聚合物囊泡。纳米聚合物囊泡TRPP的粒径分布图及透射电镜图如图5所示。
结合图5的动态光散射(DLS)结果,TRPP的流体力学尺寸为174±4nm,分布为0.13±0.02。
二、DPPA-1修饰TRPP(DPPA-TRPP)的制备及Tab装载
DPPA-1能够通过ROS敏感键修饰在前药纳米聚合物囊泡TRPP表面形成DPPA-TRPP。
修饰过程如下:
向DPPA-1(55μg,0.026μmol)溶液中分别加入EDC·HCl(13.6μg,0.071μmol),DMAP(8.6μg,0.071μmol)和三乙胺(TEA,10μL,0.071μmol),30℃下反应30分钟。将反应物滴加到TRPP(1mg/mL,1mL)中搅拌过夜。透析(MWCO,3500)后,得DPPA-TRPP。DPPA-1修饰的TRPP(DPPA-TRPP)纳米聚合物囊泡的粒径分布图及透射电镜图如图6所示。
由图6的DLS结果表明,DPPA-TRPP的流体力学尺寸为134±5nm,分布为0.19±0.05,透射电子显微镜证实了其中空形态。
进一步装载Tab后,可得DPPA-TRPP/Tab。其制备方法与DPPA-TRPP相似,只不过前药聚合物药物需加到含Tab的PBS溶液中。
试验例1前药聚合物对过氧化氢的消耗
将实施例2制备所得多重响应的前药聚合物溶解在DMF中,得到不同浓度(1mg/mL,5mg/mL)的溶液,并加入过氧化氢(100μM)。随后,将双氧水荧光探针(Oxi Vision GreenTM)加入上述溶液和双氧水标准溶液中,室温下用荧光分光光度计检测各样品中过氧化氢浓度的变化。结果如图7所示。
由图7可知,经前药聚合物处理后,520nm处的吸收减弱,说明过氧化氢浓度显著降低。随着前药浓度的增加,过氧化氢的消耗量随之增加。因此,该前药聚合物可以消耗过氧化氢,而且其消耗程度受聚合物浓度影响。
试验例2DOX和Tab的体外释放
将实施例3中的DPPA-TRPP放入释放袋(MWCO=12000)中进行体外释放研究。为获得漏槽条件,将装载TRPP(0.5mL)的释放袋置于25mL释放介质中,而后置于摇床中(37℃,200rpm)。在预先设定的时间点,取出释放介质5mL,并补加相同体积的新鲜介质。释放介质分别为PBS(pH 6.8,10mM GSH)、PBS(pH 6.8,0mM GSH)、PBS(pH 7.4,10mM GSH)、PBS(pH7.4,0mM GSH)、醋酸/醋酸钠缓冲液(HAc/NaOAc,pH 5.0,10mM GSH)、HAc/NaOAc(pH 5.0,0mM GSH)。用荧光分光光度计测定不同时间点DOX的释放量。结果如图8所示。
图8结果表明,DOX在pH 5.0同时含10mM GSH溶液中快速释放,24h累积释放量高达80.6%。不含GSH或10mM GSH(pH 7.4)时,DOX释放量为11.8-14.1%。上述结果表明,DOX的释放既需要低pH,又需要高浓度GSH(图8A)。
小分子药物Tab的释放研究,与DOX的类似,只不过释放介质不同。Tab的释放介质分别为PBS(pH 6.8,0μM H2O2),PBS(pH 6.8,100μM H2O2),PBS(pH 7.4,0μM H2O2),PBS(pH7.4,100μM H2O2)。其在不同时间点的释放量通过液相质谱检测得到(图8B)。
试验例3用四甲基偶氮唑盐比色法(MTT)检测TRPP细胞毒性
将4T1细胞(5.0×103个/孔)接种到96孔板中,培养24h后,分别向其中加入游离的DOX和TRPP(TRPP用pH 6.8和100μM H2O2预处理)。药物浓度由低到高依次为0.16、0.32、0.62、1.25、2.5、5、10μg/mL。孵育48h后,加入MTT溶液(10μL,5mg/mL)。继续孵育4h,吸走培养基并向每孔加入150μL二甲基亚砜。最后,用酶标仪测定在570nm处的吸光度。细胞毒性检测结果如图9所示。
由图9结果可知,偶联DOX的TRPP对4T1细胞有较强的毒性作用,其IC50为1.39μg/mL,与游离DOX(1.25μg/mL)类似。该结果表明,键接到TRPP上的DOX可以在细胞内快速释放,并发挥毒性作用。
试验例4用激光共聚焦显微镜检测细胞内在化
激光共聚焦显微镜(CLSM)表征过程为:将4T1细胞(2.0×104个/孔)接种于24孔板中,培养24h后,分别加入DPPA-TRPP、TRPP和游离DOX(药物浓度为5μg/mL),孵育2、4、8h。随后,细胞经PBS洗涤、4%多聚甲醛固定(15min)、DAPI(2μg/mL)染色(10min)。最后,盖玻片覆盖细胞,并用指甲油密封,激光共聚焦显微镜获取细胞图像。结果如图10所示。
图10结果显示,DPPA-TRPP可被4T1细胞迅速内在化,从CLSM图像中可在细胞内观察到DOX的红色信号。被细胞摄取是纳米粒发挥作用的前提,该结果为纳米粒诱导肿瘤免疫原性死亡提供了基础。
试验例5DPPA-TRPP介导4T1细胞发生免疫原性细胞死亡
DPPA-TRPP介导肿瘤细胞免疫原性死亡可分别通过检测CRT暴露与HMGB1释放表征。对于CRT暴露的检测,用CLSM进行表征。具体过程是,4T1细胞接种于24孔板中,培养24h后,分别加入DPPA-TRPP、TRPP、TP和游离DOX。孵育24h后,细胞经PBS洗涤、4%多聚甲醛固定、Alexa488抗CRT染色。PBS洗涤后,细胞核经DAPI染色,盖玻片覆盖及指甲油密封,用CLSM获取细胞图像。结果表明,细胞经DPPA-TRPP、TRPP和游离DOX处理后,可明显观察到绿色荧光,而TP和PBS组则无明显荧光,说明DOX的存在介导了肿瘤细胞ICD。
在HMGB1释放研究方面,采用CLSM进行检测。首先,将4T1细胞接种于24孔板(2.0×104个/孔)中培养24h,分别加入DPPA-TRPP、TRPP、TP和游离DOX。孵育48h后,细胞经PBS清洗、4%多聚甲醛固定、Alexa647抗HMGB1染色和细胞核染色。用激光共聚焦显微镜获取细胞图像。上述测试的结果如图11所示。
由图11可知,PBS和TP处理后的细胞中,可明显看到红色荧光;而经DPPA-TRPP、TRPP和游离DOX处理后,红色荧光显著降低,表明DOX的键接促进了HMGB1的释放。
试验例6体内肿瘤蓄积研究
在体内肿瘤蓄积研究中,用亲脂性DIR取代Tab作为荧光模型包裹在DPPA-TRPP中,形成DPPA-TRPP/DIR。BALB/c小鼠右侧接种4T1细胞,当肿瘤体积增大到150~200mm3时,分别静脉注射DPPA-TRPP/DIR和TRPP/DIR。注射后2、4、8、24h,用IVIS成像***检测肿瘤富集情况,结果如图12所示。
图12结果表明,在24小时内注射TRPP/DIR或DPPA-TRPP/DIR的小鼠,肿瘤组织中的荧光信号随时间增加而增强,表明纳米制剂的保留时间延长。
试验例7DPPA-TRPP/Tab在乳腺癌模型中的抗肿瘤活性和长期记忆免疫应答
BALB/c小鼠右侧接种4T1细胞,对于初始肿瘤体积较小的治疗,当肿瘤体积在100mm3左右时,在接种后第6天静脉给药。观察并记录45d内小鼠的存活情况。肿瘤完全消除的小鼠再次接种4T1细胞,以进行记忆免疫反应研究。当未经治疗的小鼠肿瘤体积约为1500mm3时(第72天),将小鼠安乐死,试验结果如图13所示。
图13结果表明(图13A~13D),DPPA-TRPP/Tab治疗后,小鼠肿瘤体积增长被显著抑制,完全消除率高达60%,小鼠生存中值明显延长。再次接种后,与PBS组对比,DPPA-TRPP/Tab组肿瘤增长缓慢,表明其具有明显的免疫记忆作用。
综上可知,本发明的多重响应性聚合物前药,可制备得到形貌可转换聚合物纳米囊泡,其可装载亲水性小分子FAP抑制剂如Tab,在模拟肿瘤微环境弱酸条件下,形貌转换为胶束并释放药物;接着胶束通过内吞作用进入细胞内,在高浓度还原条件下,二硫键断裂释放疏水药物,引起肿瘤ICD。通过不同时空条件下顺序释放药物,解决了亲水性药物在TME中用于免疫抑制逆转和细胞内释放疏水化疗药引起ICD之间的难题。并且,本发明公开的多重响应性聚合物前药,制备得到的形貌可转换囊泡纳米药物,可通过ROS敏感键在其表面修饰免疫检查点拮抗剂DPPA-1,在TME的高ROS条件下,敏感键断裂释放拮抗剂阻断免疫检查点,有效解决了现有技术中需额外与抗体联用阻断免疫检查点的难题,在癌症(如乳腺癌)免疫治疗方面具有广泛的应用价值。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (10)
1.一种多重响应的前药聚合物,其特征在于,所述多重响应的前药聚合物为A-B-C型嵌段聚合物,记为dSe-PEG-PC7A-P(BAEMA-(AEMA-SS-R)),结构式如式Ⅰ所示:
如式Ⅰ所示的A-B-C型嵌段聚合物中,嵌段A为聚乙二醇链段,即PEG链段,对应的重复单元m为68~182;嵌段A占聚合物分子主链总链长的20%-40%;
嵌段B为聚甲基丙烯酸-2-(六甲撑亚胺)乙酯链段,即PC7A链段,对应的重复单元n为6~17;
嵌段C为含有聚甲基丙烯酸叔丁基氨基乙酯的复合链段,即P(BAEMA-(AEMA-SS-R))链段,对应的重复单元x为12~20,y为1~3;嵌段C中,基团R选自羟基或活性药物基团。
4.如权利要求1所述的多重响应的前药聚合物的制备方法,其特征在于,包括以下步骤:
(1)将羧基-聚乙二醇-氨基与4-氰基-4-(苯羰基硫代)戊酸-琥珀酰亚胺酯进行酰胺化反应,得到羧基-聚乙二醇-4-氰基-4-(苯羰基硫代)戊酰胺,记为COOH-PEG-CPPA;
(2)将COOH-PEG-CPPA与甲基丙烯酸-2-(六甲撑亚胺)乙酯、甲基丙烯酸叔丁基氨基乙酯先后发生聚合反应,得到产物A,记为HOOC-PEG-PC7A-PBAEMA;
(3)将HOOC-PEG-PC7A-PBAEMA与三氟乙酸反应,得到产物B,记为COOH-PEG-PC7A-P(BAEMA-AEMA);
(4)将COOH-PEG-PC7A-P(BAEMA-AEMA)与二硒二乙醇进行酯化反应,得到产物C,记为dSe-PEG-PC7A-P(BAEMA-AEMA);
(5)将dSe-PEG-PC7A-P(BAEMA-AEMA)加入到含有3,3二硫代二丙酸的溶液中进行酰胺化反应,得到产物D,记为dSe-PEG-PC7A-P(BAEMA-(AEMA-DTPA));
(6)将dSe-PEG-PC7A-P(BAEMA-(AEMA-DTPA))与活性药物进行酰胺化反应,即得所述多重响应的前药聚合物,记为dSe-PEG-PC7A-P(BAEMA-(AEMA-SS-R))。
5.根据权利要求4所述的多重响应的前药聚合物的制备方法,其特征在于,所述活性药物为阿霉素、紫杉醇、米托蒽醌中的一种。
6.根据权利要求4或5所述的多重响应的前药聚合物的制备方法,其特征在于,步骤(6)中,所述活性药物为阿霉素;dSe-PEG-PC7A-P(BAEMA-(AEMA-DTPA))与阿霉素的反应摩尔比为3.33:10。
7.如权利要求1~3任一项所述的多重响应的前药聚合物的应用,其特征在于,在制备形貌可转换的抗肿瘤纳米药物中的应用。
8.根据权利要求7所述的多重响应的前药聚合物的应用,其特征在于,所述抗肿瘤纳米药物的制备方法是:将所述多重响应的前药聚合物通过溶剂置换法制备得到聚合物纳米囊泡,然后在聚合物纳米囊泡表面键接PD-L1阻断剂DPPA-1,得到DPPA-1修饰的聚合物纳米囊泡,将DPPA-1修饰的聚合物纳米囊泡作为抗肿瘤纳米药物。
9.根据权利要求8所述的多重响应的前药聚合物的应用,其特征在于,抗肿瘤纳米药物的制备方法中还包括在DPPA-1修饰的聚合物纳米囊泡上进行Tab装载的步骤。
10.根据权利要求7所述的多重响应的前药聚合物的应用,其特征在于,所述肿瘤为乳腺癌。
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