CN112107542A - 具有肿瘤pH和H2O2特异性激活抗肿瘤活性多功能聚合物胶束及制备方法 - Google Patents
具有肿瘤pH和H2O2特异性激活抗肿瘤活性多功能聚合物胶束及制备方法 Download PDFInfo
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Abstract
本发明提供了一种具有肿瘤pH和H2O2特异性激活的抗肿瘤活性多功能聚合物胶束及制备方法,利用硼酸酯键在肿瘤部位的pH和H2O2双敏感性合成了一种可以在水相中自组装的聚合物胶束(TBPBA‑PEG‑VI‑FA),通过水热法合成了AglnS2量子点,使用具有配位功能的乙烯基咪唑搭载具有荧光成像和光动力学治疗的AglnS2量子点,同时使用叶酸靶向肿瘤细胞表面受体(以靶向过度表达的肿瘤细胞表面叶酸受体(FR‑α(+))。本发明体外模拟释放结果显示,核心负载DOX·HCl的聚合物可以肿瘤组织的微酸和H2O2环境中很好的释放药物,具有良好的pH和H2O2响应性,是一种具有广泛应用前景的智能纳米药物递送系统。
Description
技术领域
本发明涉及抗肿瘤活性的智能纳米药物递送载体领域,具体指一种具有肿瘤pH和H2O2特异性激活的抗肿瘤活性多功能型聚合物胶束和制备方法。
背景技术
癌症是全球人口主要的死亡原因,预计在未来20年内大约40%的人可能被诊断出患有癌症。目前针对癌症的临床治疗中多采用单一的化疗方式,然而,由于癌细胞的复杂性和多样性原因,使单一化学治疗方法容易导致肿瘤部位产生耐药性,不能随时诊断以及监测药物在肿瘤部位的分布情况,无法达到理想的肿瘤治疗效果,使得完全根治肿瘤变得困难。在肿瘤治疗学领域中,用于癌症治疗的智能药物输送系统(SDDS)引起了人们的极大兴趣,新的治疗型纳米颗粒的设计思维策略必须以对人类安全的利益为中心,因此开发具有早期诊断能力,增强的药物递送,有效的生物降解和多种治疗方式联合治疗的SDDS仍然是科学挑战。
聚合物胶束为将探针和生物活性化合物传递到肿瘤部位进行分子成像和治疗提供了特殊的优势。在过去的十年中,人们对聚合物胶束在癌症治疗中的用途进行了深入研究。刺激响应型聚合物胶束能够大大提高药物分子在水中的溶解度,延长体内循环时间,增强渗透和保留(EPR)效应来提高递送系统在肿瘤组织的富集程度,并在肿瘤部位能够被特异性激活特性而被广泛用于抗癌药物输送系统,再加上肿瘤细胞靶向分子的进一步缀合减少了对正常组织的细胞毒性并增加了药物在肿瘤组织的生物利用度,使药物更容易被肿瘤细胞吸收。两亲性聚合物胶束在较低浓度下会聚集并出现更稳定的核-壳纳米结构,有利于抗肿瘤药物的细胞内递送。苯基硼酸衍生物已成为越来越多的科学关注的对象,当将溶液调节至低于其pKa的弱碱性时,疏水性苯基硼酸衍生物可与OH-结合生成可溶但不稳定的物质,以及苯基硼酸酯对弱酸和H2O2的不稳定性,可以将苯基硼酸衍生物引入聚合物胶束构造出具有肿瘤特异性激活和可降解的的多功能型纳米药物递送系统是一种具有独特优势的药物递送平台。
为了解决癌症单一化学治疗的弊端,提高癌症治疗的有效性,出现了化学疗法与其他治疗方式联合的新范例,与单一的化疗相比联合治疗获得了更高的治疗效率。光动力学治疗(PDT)是一种新兴的抗肿瘤方法,光敏剂(PS)在光照条件下产生具有细胞毒性的活性氧(ROS),从而导致癌细胞死亡,增强了纳米载药递送系统的抗肿瘤功效。
发明内容
本发明的目的是提供一种具有肿瘤pH和H2O2特异性激活抗肿瘤活性多功能型聚合物胶束,是集CT治疗、PDT治疗、诊断和监测一体的纳米递送系统。
本发明的另一个目的是提供一种具有肿瘤pH和H2O2特异性激活抗肿瘤活性多功能型聚合物胶束的制备方法,可用于在生物体内荧光成像、光动力学治疗、定位纳米药物递送载体在肿瘤组织的分布。
本发明所采用的技术方案为:、
一种具有肿瘤pH和H2O2特异性激活的抗肿瘤活性多功能聚合物胶束,其结构如下:
2、一种具有肿瘤pH和H2O2特异性激活抗肿瘤活性的多功能型聚合物胶束的制备方法,具体过程如下:
(1)RAFT试剂TTC的合成:
将一定量丙酮,十二硫醇和甲基三辛基氯化铵加入至三颈烧瓶中,再向混合溶液中滴加NaOH溶液,然后加入CS2的丙酮溶液。当溶液变成红色后,加入氯仿,再继续滴加NaOH溶液,然后用浓盐酸酸化。真空泵抽滤之后用异丙醇进行萃取后,正己烷重结晶,即得目标产物TTC,其结构式为:
(2)pH及H2O2响应硼酸酯键疏水链段,即MA–TME-TBPBA的合成:
将一定量的1,1,1-三羟甲基乙烷和4-叔丁基苯硼酸(TBPBA)溶于无水甲苯置于Dean-Stark除水装置中,将混合物在120℃加热回流,将反应物过滤,洗涤,沉淀,真空干燥得粗产物;将上述粗产物充分溶于干燥的DCM中,再加入三乙胺,然后在冰浴下用恒压漏斗缓慢滴加甲基丙烯酰氯,再用缓冲溶液洗涤,用无水硫酸钠干燥,真空干燥得到白色固体;其结构式为:
(3)AA-FA单体的合成
将一定量叶酸FA加入到DMSO的圆底烧瓶中,首先置于55℃油浴锅中加热直至全部溶解,再加入二环己基碳二亚胺DCC将叶酸在避光条件下活化,待叶酸全部转化为叶酸活化酯,再向该反应液中加入烯丙基胺盐酸盐,催化剂4-二甲氨基吡啶DMAP,在室温条件下反应24小时,过滤除去固体,将过滤液继续用乙酸乙酯和丙酮沉淀,抽滤,真空干燥,得到黄色粉即为AA-FA单体;其结构式为:
(4)聚合物TTC-TBPBA的合成:
将一定量TTC、AIBN和TBPBA溶于DMF加入Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封Schlenk瓶,在65℃油浴锅中聚合反应24小时,终止反应,再用透析袋透析48小时除去杂质,然后减压蒸馏除去水分,真空干燥得到白色固体粉末即为聚合物TTC-TBPBA;其结构式为:
(5)聚合物TBPBA-PEG的合成:
将TTC-TBPBA、AIBN和PEGMA溶于5mL DMF加入到Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封Schlenk瓶,在65℃油浴锅中聚合反应24小时后,终止反应,再将溶液转移至透析袋中透析48小时除去杂质,然后减压蒸馏除去水分,真空干燥得到淡黄色固体即为聚合物TBPBA-PEG;其结构式为:
(6)聚合物TBPBA-PEG-VI-FA的合成:
将TBPBA-PEG、AIBN、FA、VI溶于5mL DMSO加入Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封Schlenk瓶,在65℃油浴锅中聚合反应24小时后,终止反应,再将溶液转移至透析袋中透析48小时除去杂质,然后减压蒸馏除去水分,真空干燥得到棕黄色固体即为聚合物TBPBA-PEG-VI-FA;其结构式为:
(7)聚合物TBPBA-PEG-VI-FA@AglnS2的制备:
将一定量的聚合物TBPBA-PEG-FA-VI溶于1mL DMF中,取少量AglnS2量子点溶于1mL蒸馏水中使其充分溶解,将TBPBA-PEG-VI-FA和AglnS2量子点溶液加入到圆底烧瓶中,搅拌24小时后,将溶液转移至透析袋中透析48小时,然后冷冻干燥得到聚合物TBPBA-PEG-VI-FA@AglnS2。
3、根据权利要求2所述的具有肿瘤pH和H2O2特异性激活抗肿瘤活性的多功能型聚合物胶束的制备,其特征在于步骤(7)中所述量子点AglnS2的制备:
将AgNO3水溶液,MAA水溶液和NH4OH水溶液溶于50mL蒸馏水中,再加NH4OH使水溶液由浑浊淡黄色悬浮液变为透明至无色,使溶液pH=11;再向上述溶液中加入HNO3的lncl3水溶液,室温下再在另一个烧瓶中加Na2S水溶液,在油浴中加热30分钟;在旋转蒸发之前,再次加入少量MAA水溶液以防止溶剂萃取过程中胶体QD的聚集,将溶液减压蒸馏至最终体积约10mL,然后将其尺寸选择性沉淀;
其中AgNH3,lnCl3和Na2S的摩尔比10:7:1,反应时间为90-120分钟,反应温度为90-95℃。
本发明的聚合物胶束具有肿瘤TME(pH和H2O2)特异性激活的硼酸酯键,含有聚乙二醇(PEG)的纳米颗粒具有相对较大的粒径(50–150nm)并具有隐身能力,能够提高药物分子在水中的溶解度,利用增强的渗透性和保留(EPR)效应延长血液循环时间并有效改善纳米药物递送系统在肿瘤部位的蓄积,FA基团通过靶向细胞表面过表达叶酸受体将药物准确递送至肿瘤细胞,VI基团搭载的AglnS2QD用于PDT治疗和荧光成像,形成了具有CT治疗、PDT治疗、诊断和监测一体的纳米递送系统。
本发明中,通过可逆加成-断裂链转移(RAFT)聚合方法设计了一种先进的SDDS旨在在单个纳米结构中整合了多种不同的功能(诊断、治疗、靶向和监测),具有肿瘤特异性激活的多功能聚合物胶束,以协同杀死肿瘤部位的癌细胞。该功能型聚合物胶束在改善和监测给药后药物的递送方面显示出巨大的潜力,组成这种功能型纳米胶束颗粒的结构成分,与其所有添加的功能分子(包括DOX·HCl,AglnS2,VI和FA)兼容,同时仍能精确发挥预期的药物(DOX·HCl)控制释放,VI配位QDs光动力学治疗和荧光成像以及FA靶向细胞表面受体(以靶向过度表达的肿瘤细胞表面叶酸受体(FR-α(+)))的性能,这提高了肿瘤治疗功效并最大程度地降低脱靶毒性,能够准确定位药物在肿瘤部位的分布情况。这种纳米药物递送系统为治疗肿瘤带来了巨大的临床优势,这些功能使治疗性纳米颗粒能够最大程度地发挥药物功效,并改善癌症治疗开发方法的合理性,是一种具有广泛应用前景的智能纳米药物递送系统。
本发明的水热法合成AglnS2QDs的制备方法,可用于在生物体内荧光成像、光动力学治疗、定位纳米药物递送载体在肿瘤组织的分布。
附图说明
图1-1,1-2分别为本发明合成的TTC的核磁共振氢谱和核磁共振碳谱;
图2-1,2-2分别为本发明合成的MA-TME-TBPBA单体的核磁共振氢谱和核磁共振碳谱;
图3-1,3-2分别为本发明合成的AA-FA单体的核磁共振氢谱和核磁共振碳谱;
图4为本发明合成聚合物TTC-TBPBA的核磁共振氢谱;
图5为本发明合成聚合物TBPBA-PEG的核磁共振氢谱;
图6为本发明合成聚合物TBPBA-PEG-VI-FA的核磁共振氢谱;
图7-1,7-2分别为本发明测试AglnS2QDs的紫外吸收光谱和荧光光谱图;
图8为本发明量子点AglnS2的稳定性;
图9为本发明量子点AglnS2的XRD表征;
图10为本发明量子点AglnS2的透射电镜(TEM);
图11-1,11-2为本发明聚合物TBPBA-PEG-VI-FA@AglnS2活性氧(·OH)检测;
图12为本发明聚合物DOX@TBPBA-PEG-VI-FA@AglnS2的紫外吸收光谱;
图13为本发明聚合物DOX@TBPBA-PEG-VI-FA@AglnS2的Ze-Ta电位;
图14为本发明聚合物DOX@TBPBA-PEG-VI-FA@AglnS2的粒径分布;
图15为本发明聚合物DOX@TBPBA-PEG-VI-FA@AglnS2胶束的临界胶束浓度(CMC);
图16为本发明聚合物DOX@TBPBA-PEG-VI-FA@AglnS2的透射电镜(TEM)表征;
图17为本发明聚合物DOX@TBPBA-PEG-VI-FA@AglnS2的体外药物释放曲线;
图18为本发明具有肿瘤pH和H2O2特异性激活抗肿瘤活性多功能型聚合物胶束的结构示意图。
具体实施方式
下面通过具体实施例对本发明抗肿瘤聚合物DOX@TBPBA-PEG-VI-FA@AglnS2胶束的合成作进一步的说明及验证。
一、具有肿瘤pH和H2O2特异性激活抗肿瘤活性多功能型聚合物胶束
一种具有肿瘤pH和H2O2敏感的抗肿瘤活性聚合物纳米载药胶束,是由4-叔丁基苯硼酸(TBPBA)和三羟甲基乙烷合成具有pH和H2O2响应的硼酸酯键作为纳米胶束的疏水链段,将聚(乙二醇)作为亲水链段,在生理pH下在水相中自组装成纳米胶束,再将可以配位AglnS2QDs的乙烯基咪唑和被动靶向细胞表面叶酸受体的叶酸引入聚合物链段,形成一种具有可靶向、可光动力学治疗、可荧光成像、可包埋药物的多功能纳米药物递送系统。其结构如下:
二、具有肿瘤pH和H2O2特异性激活抗肿瘤活性的多功能型聚合物胶束的制备方法,如下:
(1)RAFT试剂TTC的合成
将一定量丙酮,十二硫醇和甲基三辛基氯化铵加入至三颈烧瓶中,再向混合溶液中滴加NaOH溶液,然后加入CS2的丙酮溶液。当溶液变成红色后,加入氯仿,再继续滴加NaOH溶液,然后用浓盐酸酸化。真空泵抽滤之后用异丙醇进行萃取后,正己烷重结晶,即得目标产物TTC,其结构式为:
图1-1为上述制备RAFT试剂的核磁共振氢谱,图1-2为核磁共振碳谱。通过核磁共振氢谱分析可以得出,化学位移在3.26ppm,1.70ppm,1.69–1.62ppm,1.40–1.32ppm,1.32–1.18ppm,0.86ppm为TTC上氢的出峰。核磁共振氢谱和磁共振碳谱表明成功合成了RAFT试剂TTC。
(2)pH及H2O2响应硼酸酯键疏水链段(MA–TME-TBPBA)的合成
将一定量的1,1,1-三羟甲基乙烷(TME)和4-叔丁基苯硼酸(TBPBA)溶于无水甲苯置于Dean-Stark除水装置中,将混合物在120℃加热回流,将反应物过滤,洗涤,沉淀,真空干燥得粗产物。将上述粗产物充分溶于干燥的DCM中,再加入三乙胺,然后在冰浴下用恒压漏斗缓慢滴加甲基丙烯酰氯(MA),再用缓冲溶液洗涤,用无水硫酸钠干燥,真空干燥得到白色固体。其结构式为:
图2-1,2-2分别为上述制备TBPBA单体的核磁共振氢谱和核磁共振碳谱。通过核磁共振氢谱分析可以得出,化学位移在7.70ppm和7.36ppm为苯环上氢的出峰,5.57ppm和6.10ppm为碳碳双键上氢的出峰,1.03ppm为羟甲基上氢的出峰。核磁共振氢谱和核磁共振碳谱表明成功合成了MA–TME-TBPBA单体。
(3)AA-FA单体的合成
将一定量叶酸(FA)加入到DMSO的圆底烧瓶中,首先置于55℃油浴锅中加热直至全部溶解,再加入二环己基碳二亚胺(DCC)将叶酸在避光条件下活化,待叶酸全部转化为叶酸活化酯,再向该反应液中加入烯丙基胺盐酸盐,催化剂4-二甲氨基吡啶(DMAP,),在室温条件下反应24小时,过滤除去固体,将过滤液继续用乙酸乙酯和丙酮沉淀,抽滤,真空干燥,得到黄色粉即为AA-FA单体。其结构式为:
图3-1,3-2分别为上述制备AA-FA单体的核磁共振氢谱和核磁共振碳谱。通过核磁共振氢谱分析可以得出,化学位移在7.61ppm和6.61ppm为苯环上氢的出峰,5.26ppm和5.36ppm为碳碳双键上氢的出峰。核磁共振氢谱和核磁共振碳谱表明成功合成了AA-FA单体。
(4)聚合物TTC-TBPBA的合成
将一定量TTC,AIBN和TBPBA溶于DMF加入Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封Schlenk瓶,在65℃油浴锅中聚合反应24小时,终止反应,再用透析袋(MWCO=3000)透析48小时除去杂质,然后减压蒸馏除去水分,真空干燥得到白色固体粉末即为聚合物TTC-TBPBA。其结构式为:
图4为上述制备聚合物TTC-TBPBA的核磁共振氢谱。通过核磁共振氢谱分析可以得出,化学位移在7.70ppm和7.36ppm为苯环上氢的特征峰,1.26ppm为TTC上氢的特征峰。核磁共振氢谱表明成功合成了聚合物TTC-TBPBA。
(5)聚合物TBPBA-PEG的合成
将(TTC-TBPBA),AIBN和PEGMA溶于5mL DMF加入到Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封Schlenk瓶,在65℃油浴锅中聚合反应24小时后,终止反应,再将溶液转移至透析袋(MWCO=3000)中透析48小时除去杂质,然后减压蒸馏除去水分,真空干燥得到淡黄色固体即为聚合物TBPBA-PEG。其结构式为:
图5为上述制备聚合物TBPBA-PEG的核磁共振氢谱。通过核磁共振氢谱分析可以得出,化学位移在7.70ppm和7.36ppm为TBPBA苯环上氢的特征峰,3.63ppm为PEG上氢的特征峰。核磁共振氢谱表明成功合成了聚合物TBPBA-PEG。
(6)聚合物TBPBA-PEG-VI-FA的合成
将(TBPBA-PEG)、AIBN、FA、VI溶于5mL DMSO加入Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封Schlenk瓶,在65℃油浴锅中聚合反应24小时后,终止反应,再将溶液转移至透析袋(MWCO=3000)中透析48小时除去杂质,然后减压蒸馏除去水分,真空干燥得到棕黄色固体即为聚合物TBPBA-PEG-VI-FA。其结构式为:
图6为上述制备聚合物TBPBA-PEG-VI-FA的核磁共振氢谱。通过核磁共振氢谱分析可以得出,化学位移在7.69ppm和6.63ppm为FA苯环上氢的特征峰,6.96ppm为咪唑上氢的特征峰。核磁共振氢谱表明成功合成了聚合物TBPBA-PEG-VI-FA。
(7)量子点AglnS2的制备
将AgNO3水溶液,MAA水溶液和NH4OH水溶液溶于50mL蒸馏水中,再加NH4OH使水溶液由浑浊淡黄色悬浮液变为透明至无色,使溶液pH=11。再向上述溶液中加入HNO3的lncl3水溶液,室温下再在另一个烧瓶中加Na2S水溶液,在油浴中加热30分钟。在旋转蒸发之前,再次加入少量MAA水溶液以防止溶剂萃取过程中胶体QD的聚集,将溶液减压蒸馏至最终体积约10mL,然后将其尺寸选择性沉淀。
其中AgNH3,lnCl3和Na2S的摩尔比10:7:1,反应时间为90-120分钟,反应温度为90-95℃。
(8)聚合物TBPBA-PEG-VI-FA@AglnS2的制备
将一定量的聚合物TBPBA-PEG-FA-VI溶于1mL DMF中,取少量AglnS2量子点溶于1mL蒸馏水中使其充分溶解,将TBPBA-PEG-VI-FA和AglnS2量子点溶液加入到圆底烧瓶中,搅拌24小时后,将溶液转移至透析袋(MWCO=3000)中透析48小时,然后冷冻干燥得到聚合物TBPBA-PEG-VI-FA@AglnS2。
三、聚合物DOX@TBPBA-PEG-VI-FA@AglnS2的pH及H2O2响应性实验
1、量子点AglnS2性能测试及表征
(1)量子点AglnS2的紫外吸收光谱和荧光光谱测试
取一定等量上述制备的量子点AglnS2,分别溶解在不同试管的蒸馏水中,分别测试其不同反应时间的紫外吸收光谱和荧光光谱。
图7-1和7-2分别为AglnS2QDs的紫外吸收光谱和荧光光谱图。其结果显示该量子点的紫外吸收峰约为585nm,随着反应时间的增加AglnS2量子点紫外吸收峰发生红移,其荧光强度液随之增加。说明该量子点具有较大的吸收波长,可以用于肿瘤部位的荧光成像。
(2)量子点AglnS2的稳定性测试
取一定量的量子点AglnS2,溶解在10mL蒸馏水中,按照一定的时间间隔测试其储存稳定性。
图8为量子点AglnS2的稳定性。通过AglnS2QDs的荧光谱图可以看出随着时间的变化该量子点的荧光强度值减小的很小,说明该量子点具有良好的储存稳定性。
(3)量子点AglnS2的X-射线衍射(XRD)表征
将制备的AglnS2QDs水溶液加异丙醇沉淀后离心,真空干燥。然后取一定量粉末测试其AglnS2QDs的XRD。
如图9所示通过XRD分析可得AglnS2QDs量子点在角2θ值为26.0、44.7和52.2附近有三个明显的衍射峰分别对应于(112)、(220)和(312)晶面表示所制备的AglnS2QDs具有立方晶系结构。表明成功合成了AglnS2QDs。
(4)量子点AglnS2的透射电镜(TEM)表征
将制备的AglnS2QDs水溶液加异丙醇沉淀后离心,真空干燥。然后取一定量干燥的AglnS2QDs溶解在蒸馏水中测试其形貌。
图10为AglnS2QDs的TEM,该图显示AglnS2QDs直径约为4nm呈球形而且分布均匀。
2、聚合物DOX@TBPBA-PEG-VI-FA@AglnS2胶束的制备和测试
(1)空白胶束的制备
采用透析法制备聚合物TBPBA-PEG-VI-FA@AglnS2空白胶束,首先称取10mg的Poly(TBPBA-PEG-VI-FA@AglnS2)胶束溶于1ml的DMF中,在室温下快速搅拌,超声使其完全溶解,然后将聚合物溶液缓慢滴加到9mL的去蒸馏水中(平均速度可以控制在15s/滴),溶液呈现淡蓝色乳光,在室温下搅拌24小时形成聚合物胶束,然后将聚合物溶液转移至透析袋(MWCO=3000)中,于蒸馏水中透析24小时,得到1mg/mL的聚合物TBPBA-PEG-VI-FA@AglnS2空白胶束。
(2)载药聚合物胶束的制备
将10mg的TBPBA-PEG-VI-FA@AglnS2溶于0.5mL的DMF中,在室温下快速搅拌2小时,再超声分散30分钟使其完全溶解,再将3mgDOX·HCl溶于0.5mlL DMF中超声30分钟使其完全溶解,再将TBPBA-PEG-VI-FA@AglnS2溶液和DOX·HCl溶液充分混合,然后将混合液超声30分钟后缓慢滴加到9mL的去蒸馏水中(平均速度可以控制在15s/滴),在室温下搅拌后将溶液转移至透析袋(MWCO=3000)中,于蒸馏水中透析24小时,每间隔4-6小时更换一次蒸馏水,得到载药聚合物胶束。
(3)聚合物TBPBA-PEG-VI-FA@AglnS2光照产生羟基自由基(·OH)测定
将6×10-2M对苯二甲酸(TA)溶于2×10-2MNaOH溶液中。将(8×10-6M)TBPBA-PEG-VI-FA@AglnS2纳米颗粒(2.87×10-1μg/mL)分散到50mL水中。向(2mL,8×10-6M)聚合物TBPBA-PEG-VI-FA@AglnS2中加入(2mL,6×10-3M)TA碱性溶液,以功率密度为100mW/cm2的均匀照明节能白炽灯作为光辐照源,辐照20min后检测TAOH的荧光光谱。再通过使用二甲基亚砜(0.6M,DMSO)作为羟基自由基清除剂,在白炽灯下光照20min后监测TBPBA-PEG-VI-FA@AglnS2产生的羟基自由基(·OH)将TA氧化为TAOH的荧光光谱,可确认TA被羟基自由基氧化为TAOH。
图11-1,11-2为聚合物TBPBA-PEG-VI-FA@AglnS2活性氧(ROS)的测试图。利用对苯二甲酸测定在光照及无氧条件下聚合物TBPBA-PEG-VI-FA@AglnS2产生的羟基自由基(·OH),以DMSO荧光猝灭样品作为对比,其结果显示在光照条件下AglnS2活能够产生ROS(·OH),并随着光照时间和样品浓度的增加其荧光强度越大,说明该聚合物产生的羟基自由基(·OH)量增加。说明该量子点可以用于肿瘤治疗,杀死癌细胞。
3、聚合物DOX@TBPBA-PEG-VI-FA@AglnS2性能测试及表征
(1)聚合物DOX@TBPBA-PEG-VI-FA@AglnS2的紫外吸收光谱测试
取一定等量的AglnS2QDs、聚合物TBPBA-PEG-VI-FA@AglnS2和聚合物DOX@TBPBA-PEG-VI-FA@AglnS2分别溶解在不同试管的蒸馏水中,分别测试其紫外吸收光谱。
从图12可以看出游离的AglnS2QDs、聚合物TBPBA-PEG-VI-FA@AglnS2和聚合物DOX@TBPBA-PEG-VI-FA@AglnS2紫外吸收峰发生了不同的偏移,说明了AglnS2QDs成功的通过咪唑的配位功能搭载到了聚合物TBPBA-PEG-VI-FA上。
(2)聚合物DOX@TBPBA-PEG-VI-FA@AglnS2的Ze-Ta电位测试
取一定等量的AglnS2QDs、聚合物TBPBA-PEG-VI-FA@AglnS2和聚合物DOX@TBPBA-PEG-VI-FA@AglnS2分别溶解在不同试管的蒸馏水中,利用动态光散射(DLS)测试聚合物的Ze-Ta电位。
图13为AglnS2QDs、聚合物TBPBA-PEG-VI-FA@AglnS2和聚合物DOX@TBPBA-PEG-VI-FA@AglnS2Ze-Ta电位。
(3)聚合物DOX@TBPBA-PEG-VI-FA@AglnS2的粒径分布测试
取一定浓度的聚合物DOX@TBPBA-PEG-VI-FA@AglnS2溶解在试管的蒸馏水中,测试体外载药胶束的粒径分布情况。
图14为聚合物DOX@TBPBA-PEG-VI-FA@AglnS2在生理条件下的粒径分布,可以看出在生理条件下聚合物DOX@TBPBA-PEG-VI-FA@AglnS2粒径约为115nm,PDI为0.066的纳米颗粒,分散比较均匀,说明能够在血液循环过程中稳定存在。
(4)聚合物DOX@TBPBA-PEG-VI-FA@AglnS2胶束的临界胶束浓度(CMC)测定
将(1mL,6×10-6M)芘的丙酮溶液加入到10mL容量瓶中,在黑暗条件下放置24h使丙酮完全蒸发。将不同浓度的聚合物(DOX@TBPBA-PEG-VI-FA@AglnS2)溶液加入到容量瓶中使芘的浓度均为6×10-7M,超声30min,然后将混合溶液在黑暗条件下静置24h。利用上海棱光F97Pro型荧光分光光度计以373nm为发射波长,扫描波长在300~400nm之间样品溶液的激发光谱(狭缝宽10nm),得到芘的激发光谱,以I339/I336比值与聚合物胶束质量浓度对数作图,得到聚合物DOX@TBPBA-PEG-VI-FA@AglnS2胶束的CMC。
图15为上述聚合物DOX@TBPBA-PEG-VI-FA@AglnS2临界胶束浓度,该图显示由I339/I336与lgρ作图可得-3.44,表明芘被包裹在该胶束的疏水核心中并且其荧光值发生变化。可以计算得到该纳米胶束具有较小的CMC值(3.63×10-4mg/mL),这表明即使在体内高度稀释的条件下该聚合物胶束也不容易解离。
(5)聚合物DOX@TBPBA-PEG-VI-FA@AglnS2的透射电镜(TEM)表征
首先称取10mg的Poly(DOX@TBPBA-PEG-VI-FA@AglnS2)胶束溶于1ml的DMF中,在室温下快速搅拌使其完全溶解,然后将聚合物溶液缓慢滴加到9mL的去蒸馏水中,在室温下搅拌24小时后形成聚合物胶束,然后将聚合物溶液转移至透析袋(MWCO=3000)中,于蒸馏水中透析24小时,得到1mg/mL的样品,利用JEM-1400型透射电子显微镜测试聚合物的TEM。
图16为聚合物DOX@TBPBA-PEG-VI-FA@AglnS2的TEM,从图中可以看出该聚合物计算具有均匀的球形分布,粒径约为50nm,与图13溶液中的粒径分布不同,这是因为在制备透射电子显微镜测试样品的过程中样品需要被冻干和脱水。
(6)聚合物DOX@TBPBA-PEG-VI-FA@AglnS2胶束的体外药物释放测试
将上述制备好的10mL DOX@TBPBA-PEG-VI-FA@AglnS2胶束溶液平均分成两等份,将其置于两个透析袋(MWCO=3000)中,分别将其置于100mL的pH为7.4磷酸盐缓冲溶液和pH为5.0的乙酸盐缓冲溶液(含1mMH2O2,模拟肿瘤部位的酸度和H2O2浓度研究体外药物释放性能)中进行透析,按照一定时间间隔进行取样,温度尽可能控制在37℃左右,每次从100mL烧杯中取样3ml进行测试,测完放回。当DOX·HCl释放48小时之后,为了计算聚合物胶束的释放速率,向烧杯中加入3滴盐酸和100mMH2O2使包埋的DOX·HCl尽可能完全释放,使用UV/Vis光谱法监测485nm处的吸光度,得到百分百释放的吸光强度。将485nm处的各吸光强度与加HCl的吸光强度作比较得到累计释放率,再将累计释放率与时间间隔作图,得到DOX·HCl的累计模拟释放曲线。可由以下公式计算聚合物胶束的药物加载效率(DLC,wt%):
图17为聚合物DOX@TBPBA-PEG-VI-FA@AglnS2胶束的体外模拟药物释放曲线,由图可以看出在生理环境pH=7.4的磷酸盐缓冲溶液中,聚合物胶束在48小时之内的药物释放率为23%,释放速率比较缓慢,而在pH=5.0的乙酸盐缓冲溶液(含1mMH2O2)中,释放速率显著加快,在24小时之后达到86%,在48小时之后的药物释放率为92%。这表明该药物载体可以在人体的正常pH值下循环,并在肿瘤微环境条件下释放药物。并由DOX的标准曲线和上述公式可以得到该聚合物胶束的载药率为80%。
实施例1,一种具有肿瘤pH和H2O2特异性激活抗肿瘤活性的多功能型聚合物胶束的制备方法,具体如下:
(1)RAFT试剂TTC的合成
将十二硫醇(20.3g,0.1mol),丙酮(46.7g,0.8mmol)和甲基三辛基氯化铵(1.6g,0.004mol)依次加入250mL通氮气的三颈瓶中并搅拌。向上述反应混合物中滴加NaOH溶液(80g,2mol),20min后逐滴加入CS2的丙酮溶液(15.21mL,0.2mol),当溶液变成红色后,搅拌15min。再加入氯仿(35.625mL,0.30mol),再继续滴加NaOH溶液(40g,1mol),滴加完毕后搅拌过夜。加入600mL水,用100mL的浓盐酸酸化并剧烈搅拌。静置后用布氏漏斗抽滤,固体溶解在1L异丙醇中,用正己烷重结晶,即得到黄色晶体化合物0.68g。1H NMR(600MHz,CDCl3,δ,ppm)3.26(t,3H,CH3CH2(CH2)8CH2CH2-S-),1.70(s,6H,-C(CH3)2(C=O)OH),1.69–1.62(m,2H,CH3CH2(CH2)8CH2CH2-S-),1.40–1.32(m,2H,CH3CH2(CH2)8CH2CH2-S-),1.32–1.18(m,16H,CH3CH2(CH2)8CH2CH2-S-),0.86(t,J=7.1Hz,3H,CH3CH2(CH2)8CH2CH2-S-)。13C NMR(150MHz,CDCl3,δppm)220.70,179.19,55.56,37.04,31.89,29.61,29.57–29.27,29.02,27.80,25.18,22.66,14.09。
(2)pH和H2O2响应硼酸酯键疏水链段(MA-TME-TBPBA)的制备
1)250mL圆底烧瓶中,称取20mmol,2.4029g,1,1,1-三羟甲基乙烷,20mmol,3.5608g,4-叔丁基苯硼酸溶于100mL无水甲苯置于Dean-Stark除水装置中,将混合物在120℃回流加热12小时,通过真空除水除去部分甲苯,过滤,洗涤,沉淀,真空干燥得粗产物5.13g,产率为86%。
2)在100mL圆底烧瓶中,称取上述产物15mmol,3.9328g,充分溶于30mL干燥的二氯甲烷(DCM)中,再加入三乙胺,然后在冰浴下用恒压漏斗缓慢滴加甲基丙烯酰氯(15mmol,),在冰浴下搅拌反应24小时,将反应液用pH=8.0的磷酸盐缓冲溶液洗涤2-3次,用无水硫酸钠干燥之后,再用硅胶层析(石油醚:乙酸乙酯v/v=3:1)进一步纯化,旋蒸除去溶剂,真空干燥得到白色固体4.06g,产率为82%。1H NMR(400MHz,CDCl3,δ,ppm):7.71(d,J=8.2Hz,2H,-B-C6H2H2C(CH3)3),7.37(d,J=8.2Hz,2H,-B-C6H2H2C(CH3)3),6.10(s,1H,CHH=C(CH3)COOCH2C(CH3)(CH2-O-)2),5.58(s,1H,CHH=C(CH3)COOCH2C(CH3)(CH2-O-)2),4.11(s,2H,CH2=C(CH3)COOCH2C(CH3)(CH2-O-)2),4.03,3.84(d,J=11.1Hz,4H,CH2=C(CH3)COOCH2C(CH3)(CH2-O-)2),1.94(s,3H,CH2=C(CH3)COOCH2C(CH3)(CH2-O-)2),1.31(s,9H,-B-C6H2H2C(CH3)3),1.03(s,3H,CH2=C(CH3)COOCH2C(CH3)(CH2-O-)2).13C NMR(150MHz,CDCl3,δppm):167.02,153.97,135.98,133.80,125.88,124.55,68.03,66.21,35.73,34.80,31.22,18.28,17.77。
(3)AA-FA单体的制备
在100mL圆底烧瓶中,将(1.77g,4mmol)叶酸(FA)加入到50mL DMSO当中,首先置于55℃油浴锅中加热直至全部溶解,再加入二环己基碳二亚胺(DCC,0.824g,4mmol)将叶酸在避光条件下活化6-8小时,待叶酸全部转化为叶酸活化酯,再向该反应液中加入烯丙基胺盐酸盐(0.56g,6mmol),催化剂4-二甲氨基吡啶(DMAP,0.73g,6mmol),在室温条件下搅拌反应24小时,过滤除去固体N,N-二环己基脲(DCU),将过滤后的溶液继续用乙酸乙酯和丙酮沉淀,抽滤,真空干燥,得到黄色粉末1.98g,产率为76%。1H NMR(600MHz,DMSO-d6,δ,ppm)8.66–8.59(m,1H,-NH-CH2-C4N2H-),7.61(t,J=9.9Hz,2H,-Ar-CH of FA),6.62(t,J=8.3Hz,2H,-Ar-CH of FA),5.85(ddt,J=16.6,10.6,6.0Hz,1H,CH2=CHCH2NH-),5.40–5.21(m,2H,CH2=CHCH2NH-),4.46(t,J=8.5Hz,1,-CH2CH2CH(COOH)NH-),4.27(dd,J=13.7,8.2Hz,2H,-CH2NH-Ar-),3.42(d,J=6.0Hz,2H,CH2=CHCH2NH-),2.48(dd,J=5.7,4.0Hz,2H,CH2=C HCH2NH(C=O)CH2CH2-),2.28(dd,J=15.9,8.4Hz,2H,CH2=CHCH2NH(C=O)CH2CH2-)。13C NMR(150MHz,DMSO-d6δppm)174.80,170.05–169.48,166.54,161.74,154.74,151.15,148.85,143.59,131.46,129.27,128.36,121.97,120.11,111.65,107.21,52.80,46.37,41.31,31.31,27.14。
(4)聚合物TTC-TBPBA的制备
将TTC(25mg,0.069mmol),AIBN(25mg,0.15mmol),和TBPBA(550mg,1.67mmol)溶于5mlDMF加入Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封Schlenk瓶,在65℃油浴锅中聚合反应24小时后,置于冰浴中使反应终止,再将溶液转移至透析袋(MWCO=3000)中,于蒸馏水中透析48小时,每间隔4-6小时更换一次蒸馏水,然后旋蒸除去水分,真空干燥得到白色固体粉末500mg。1H NMR(600MHz,CDCl3,δ,ppm)7.70(2H,-B-(CC2H2C2H2C)C(CH3)3),7.36(2H,-B-(CC2H2C2H2C)C(CH3)33),3.70(4H,-OCH2C(CH3)(CH2O)2-B-),1.26(18H,CH3(CH2)9-).
(5)聚合物TBPBA-PEG的制备
将500mg Poly(TTC-TBPBA),AIBN(25mg,0.15mmol),和PEGMA(550mg,1.1mmol)溶于5mL DMF加入Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封Schlenk瓶,在65℃油浴锅中聚合反应24小时后,置于冰浴中使反应终止,再将溶液转移至透析袋(MWCO=3000)中,于蒸馏水中透析48小时,每间隔4-6小时更换一次蒸馏水,然后旋蒸除去水分,真空干燥得到淡黄色固体1.1g。1H NMR(600MHz,CDCl3,δ,ppm)7.70(2H,-B-(CC2H2C2H2C)C(CH3)3),3.70(4H,-OCH2C(CH3)(CH2O)2-B-),3.61(4H,CH3OCH2CH2O-)。
(6)聚合物TBPBA-PEG-VI-FA的制备
将500mgPoly(TBPBA-PEG),AIBN(35mg,0.21mmol),FA(340mg,069mmol),VI(65mg,069mmol)溶于5mLDMSO加入Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封,在65℃油浴锅中聚合反应24小时后,置于冰浴中使反应终止,再将溶液转移至透析袋(MWCO=3000)中,于蒸馏水中透析48小时,每间隔2-4小时更换一次蒸馏水,然后旋蒸除去水分,真空干燥得到棕黄色固体900mg。1H NMR(600MHz,DMSO-d6,δ,ppm)7.36(2H,-B-(CC2H2C2H2C)C(CH3)33),3.61(4H,CH3OCH2CH2O-),8.6(1H,-NH-CH2-C4N2H-),6.97(1H,-Ar-CH-of VI),以δ7.36ppm处的积分为基准计算。Mn=2.35×104g/mol(TBPBA26-PEG17-VI10-FA11)。
(7)AglnS2量子点的制备
1)将1.0mL,0.1M,AgNO3水溶液,2mL,1.0M,MAA水溶液和0.2mL,5.0M,NH4OH水溶液溶于50mLH2O中,然后再加5.0M的NH4OH水溶液于上述溶液中,当溶液由浑浊淡黄色悬浮液变为透明至无色,使溶液pH=11。
2)向上述溶液中加入0.7mL含有0.2M,HNO3的1.0M,lncl3水溶液)。
3)室温下在另一个烧瓶中加1.0mL,1.0M,Na2S水溶液,在95℃油浴锅中加热90分钟。
4)冷却终止反应,再向上述溶液中加入0.5mL,1.0M,MAA水溶液,以防止溶剂萃取过程中胶体QD的聚集,将溶液在40℃蒸发至最终体积约10mL,然后将其用异丙醇沉淀,真空干燥得到AglnS2QDs。
(8)聚合物TBPBA-PEG-VI-FA@AglnS2的制备
将10mg Poly(TBPBA-PEG-VI-FA)溶于1mLDMF中,2mg AglnS2量子点溶于1mL蒸馏水中,置于超声机使其充分溶解,将Poly(TBPBA-PEG-VI-FA)和AglnS2量子点溶液加入到8mL蒸馏水的圆底烧瓶中,避光搅拌24小时后,将橙黄色溶液转移至透析袋(MWCO=3000)中,于蒸馏水中透析48小时,每间隔2-4小时更换一次蒸馏水,然后冷冻干燥得到橙黄色固体。
Claims (3)
2.一种如权利要求1所述的具有肿瘤pH和H2O2特异性激活抗肿瘤活性的多功能型聚合物胶束的制备方法,其特征在于,具体过程如下:
(1)RAFT试剂TTC的合成:
将一定量丙酮,十二硫醇和甲基三辛基氯化铵加入至三颈烧瓶中,再向混合溶液中滴加NaOH溶液,然后加入CS2的丙酮溶液。当溶液变成红色后,加入氯仿,再继续滴加NaOH溶液,然后用浓盐酸酸化。真空泵抽滤之后用异丙醇进行萃取后,正己烷重结晶,即得目标产物TTC,其结构式为:
(2)pH及H2O2响应硼酸酯键疏水链段,即MA–TME-TBPBA的合成:
将一定量的1,1,1-三羟甲基乙烷和4-叔丁基苯硼酸(TBPBA)溶于无水甲苯置于Dean-Stark除水装置中,将混合物在120℃加热回流,将反应物过滤,洗涤,沉淀,真空干燥得粗产物;将上述粗产物充分溶于干燥的DCM中,再加入三乙胺,然后在冰浴下用恒压漏斗缓慢滴加甲基丙烯酰氯,再用缓冲溶液洗涤,用无水硫酸钠干燥,真空干燥得到白色固体;其结构式为:
(3)AA-FA单体的合成
将一定量叶酸FA加入到DMSO的圆底烧瓶中,首先置于55℃油浴锅中加热直至全部溶解,再加入二环己基碳二亚胺DCC将叶酸在避光条件下活化,待叶酸全部转化为叶酸活化酯,再向该反应液中加入烯丙基胺盐酸盐,催化剂4-二甲氨基吡啶DMAP,在室温条件下反应24小时,过滤除去固体,将过滤液继续用乙酸乙酯和丙酮沉淀,抽滤,真空干燥,得到黄色粉即为AA-FA单体;其结构式为:
(4)聚合物TTC-TBPBA的合成:
将一定量TTC、AIBN和TBPBA溶于DMF加入Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封Schlenk瓶,在65℃油浴锅中聚合反应24小时,终止反应,再用透析袋透析48小时除去杂质,然后减压蒸馏除去水分,真空干燥得到白色固体粉末即为聚合物TTC-TBPBA;其结构式为:
(5)聚合物TBPBA-PEG的合成:
将TTC-TBPBA、AIBN和PEGMA溶于5mL DMF加入到Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封Schlenk瓶,在65℃油浴锅中聚合反应24小时后,终止反应,再将溶液转移至透析袋中透析48小时除去杂质,然后减压蒸馏除去水分,真空干燥得到淡黄色固体即为聚合物TBPBA-PEG;其结构式为:
(6)聚合物TBPBA-PEG-VI-FA的合成:
将TBPBA-PEG、AIBN、FA、VI溶于5mL DMSO加入Schlenk瓶中,利用真空装置抽真空通氮气循环3-5次后密封Schlenk瓶,在65℃油浴锅中聚合反应24小时后,终止反应,再将溶液转移至透析袋中透析48小时除去杂质,然后减压蒸馏除去水分,真空干燥得到棕黄色固体即为聚合物TBPBA-PEG-VI-FA;其结构式为:
(7)聚合物TBPBA-PEG-VI-FA@AglnS2的制备:
将一定量的聚合物TBPBA-PEG-FA-VI溶于1mLDMF中,取少量AglnS2量子点溶于1mL蒸馏水中使其充分溶解,将TBPBA-PEG-VI-FA和AglnS2量子点溶液加入到圆底烧瓶中,搅拌24小时后,将溶液转移至透析袋中透析48小时,然后冷冻干燥得到聚合物TBPBA-PEG-VI-FA@AglnS2。
3.根据权利要求2所述的具有肿瘤pH和H2O2特异性激活抗肿瘤活性的多功能型聚合物胶束的制备,其特征在于步骤(7)中所述量子点AglnS2的制备:
将AgNO3水溶液,MAA水溶液和NH4OH水溶液溶于50mL蒸馏水中,再加NH4OH使水溶液由浑浊淡黄色悬浮液变为透明至无色,使溶液pH=11;再向上述溶液中加入HNO3的lncl3水溶液,室温下再在另一个烧瓶中加Na2S水溶液,在油浴中加热30分钟;在旋转蒸发之前,再次加入少量MAA水溶液以防止溶剂萃取过程中胶体QD的聚集,将溶液减压蒸馏至最终体积约10mL,然后将其尺寸选择性沉淀;
其中AgNH3,lnCl3和Na2S的摩尔比10:7:1,反应时间为90-120分钟,反应温度为90-95℃。
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CN114748634A (zh) * | 2020-12-29 | 2022-07-15 | 兰州大学 | 一种苯硼酸/叶酸双靶向纳米递送载体的制备与应用 |
CN113509550A (zh) * | 2021-06-10 | 2021-10-19 | 广州市第一人民医院(广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院) | 一种分子刷纳米粒子及其制备方法和应用 |
CN113509550B (zh) * | 2021-06-10 | 2023-04-14 | 广州市第一人民医院(广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院) | 一种分子刷纳米粒子及其制备方法和应用 |
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