CN111560244A - 一种pH响应的壳聚糖荧光复合胶束及其制备方法和应用 - Google Patents
一种pH响应的壳聚糖荧光复合胶束及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种pH响应的壳聚糖荧光复合胶束及其制备方法和应用，由水溶性聚多糖与四羧基改性的四苯乙烯化合物弱碱性水中静电络合形成。本发明的壳聚糖荧光纳米胶束合成简单、快捷且条件温和，具有四苯乙烯结构聚集诱导产生较高的荧光，尺寸和光学强度可以调控。利用pH的介导控制，能够使构建荧光纳米胶束的非共价作用介于静电与氢键之间转换，使得纳米胶束可以在聚电解质络合物和氢键配合物两种形式间可逆转变，进而可以实现对复合胶束荧光性质的调整。因此，结合纳米胶束的亲水性和壳聚糖的生物相容性，本发明所提供的壳聚糖荧光胶束有望应用于环境监测、生物标记及示踪等领域。
Description
技术领域
本发明属于纳米高分子材料技术领域，具体涉及一种pH响应的壳聚糖荧光复合胶束及其制备方法和应用。
背景技术
荧光试剂主要在环境监测、生物和医学等领域应用，尤其是亲水性荧光材料具有广阔的应用前景。目前使用较多的传统荧光试剂包括有机染料和无机纳米材料等，却一直存在着光漂白、聚集诱导光淬灭和生物毒性等三大问题。因此，2001以来唐本忠等人发现的一类因分子内阻转而体现聚集诱导发光(AIE)性的分子，巧妙地为解决上述问题提供了一种思路。
壳聚糖是一种提取自虾蟹壳的可生物降解聚多糖，无毒、生物相容性和正电性赋予了其在生物、保健和医药等领域的应用潜力。相比于其它天然高分子，富含阳离子的壳聚糖对细胞膜具有更强的亲和力进而极易递送各种功能材料入细胞。另外，膜黏着性不仅保证其附着于目标基质上，而且延长了它的停留时间。壳聚糖携带丰富的官能团提供了其进一步修饰的平台，若被用作复合基质构造功能器件能以足够的封装空间与功能材料作用而稳定，因此，该聚多糖拥有为疾病检测、组织修复和生物活性分子递送等领域组建器件平台的巨大潜能。
利用多重弱可逆相互协同作用的非共价超分子方法构造纳米组装体，因器件功能特性而在生物系统显得非常有用而引起研究者的广泛关注。非共价的超分子作用通常有：疏水缔合、配位作用、π～π堆积、主客体作用、静电作用和氢键相互作用等。静电相互作用制备聚电解质络合物被作为构建诊疗制剂的常用工具，在生物传感标记和载药方面拥有应用空间。此外，天然及合成高分子还可以利用分子内或分子间氢键作用进行组装形成氢键配合物，这对构筑动态的生物医学材料也具有潜在的应用前景。而且，构建具有仿生功能材料的这些非共价作用相互转换可以维持并调节生物系统的生理功能。到目前为止，利用这些非共价超分子相互作用，已经设计构筑了具有各种功能取向的大量精致仿生纳米结构。
发明内容
本发明的目的在于提供一种pH响应的壳聚糖荧光复合胶束，利用聚集诱导发光分子与壳聚糖非共价组装的pH响应性复合纳米胶束，该粒子可以在pH介导下，由静电相互作用与氢键作用的可逆转变形成具有倒置结构的两种不同纳米胶束。
本发明另一目的在于提供一种pH响应的壳聚糖荧光复合胶束的制备方法。
本发明最后一个目的在于提供一种pH响应的壳聚糖荧光复合胶束的应用，用于环境监测、生物标记及示踪。
本发明具体技术方案如下：
本发明提供的一种pH响应的壳聚糖荧光复合胶束的制备方法，包括以下步骤：
将四羧基四苯乙烯化合物溶液调节pH至弱碱性，搅拌条件下滴入壳聚糖溶液，过滤，即得。
四羧基四苯乙烯化合物包括但不限于四[4-乙酸基氨基苯]乙烯或四[4-羧基甲氧基苯]乙烯；
所述四[4-乙酸基氨基苯]乙烯的制备方法为：将四[4-氨基苯]乙烯与氯乙酸溶解于甲苯，搅拌下加入干燥的三乙胺后，加热升温回流，反应后冷却至室温，产物分离纯化，即得。其中，四[4-氨基苯]乙烯与氯乙酸质量比为1:1.5-3。
优选的，所述四[4-乙酸基氨基苯]乙烯的制备方法为：将1.0克四[4-氨基苯]乙烯与1.5-3克氯乙酸溶解于10-20毫升甲苯，搅拌下加入2-3毫升干燥的三乙胺后，升温至85℃回流，反应6-10小时后，冷却至室温，产物分离纯化，即得。
所述四[4-羧基甲氧基苯]乙烯的制备方法为：四[4-羟基苯]乙烯与氯乙酸溶解于甲苯，搅拌下加入干燥的三乙胺后，加热升温回流，反应后冷却至室温，产物分离纯化，即得。其中，四[4-羟基苯]乙烯与氯乙酸质量比为1:1.5-3。
进一步的，所述产物分离纯化具体为：产物过滤所得滤渣，用饱和碳酸氢钠水溶液溶解并抽滤，滤液在乙醚中沉淀出产物，以二氯甲烷溶解后用乙醚沉淀洗涤产品两次，真空干燥过夜，得到产品。
进一步的，将四羧基四苯乙烯化合物溶液调节pH至弱碱性优选为调节pH至7-8；调节pH采用0.5-1mol/L的氢氧化钠溶液。
滴入壳聚糖溶液速度为0.6-1ml/min。
进一步的，所述四羧基四苯乙烯化合物溶液是以去离子水为溶剂配置，浓度为1g/L。
所述壳聚糖溶液浓度为0.2g/L-1.0g/L。
搅拌条件下滴入壳聚糖溶液后，继续搅拌5-10min。
进一步的，四羧基四苯乙烯化合物溶液和壳聚糖溶液体积比为1:1，即等体积混合。
本发明提供的一种pH响应的壳聚糖荧光复合胶束，采用上述方法制备得到。产品胶束粒径随滴加复合所用壳聚糖浓度的变化而变化，形貌为球形。
本发明中，壳聚糖溶液缓慢滴入弱碱性四羧基四苯乙烯化合物溶液溶液中，滴入前后都在搅拌，温度为室温。在弱碱近中性的条件下，羧基化的四苯乙烯化合物滴入壳聚糖，羧基去质子化后与壳聚糖的氨基发生静电络合作用，从而形成难溶于水的复合物，四苯乙烯即被壳聚糖包裹在内部，壳聚糖因除氨基外还有大量的羟基，所以作为亲水外层存在，反应所得的就是荧光复合胶束。四苯乙烯因难溶于水而在胶束内聚集，发生聚集诱导发射荧光。
本发明由水溶性聚多糖与四羧基改性的四苯乙烯化合物弱碱性水中静电络合形成。本发明的壳聚糖荧光纳米胶束合成简单、快捷且条件温和，具有四苯乙烯结构聚集诱导产生较高的荧光，尺寸和光学强度可以调控。利用pH的介导控制，能够使构建荧光纳米胶束的非共价作用介于静电与氢键之间转换，使得纳米胶束可以在聚电解质络合物和氢键配合物两种形式间可逆转变，如图9所示，发明所指的荧光纳米胶束，中性偏碱性溶液中，去质子化的TPE-4COO-以负电荷羧基与壳聚糖所带正电荷氨基发生静电络合，即为聚电解质络合物；随体系pH值下降至酸性，TPE-4COOH被质子化，羧基与壳聚糖的氨基静电作用逐渐被他们之间形成的氢键代替，从而转化为氢键结合的配合物。静电与氢键作用力不同，胶束的这两种作用形式使得他们的水合粒径与荧光发射强度均具有规律性变化趋势，如图6和图7所示，进而可以实现对复合胶束荧光性质的调整。因此，结合纳米胶束的亲水性和壳聚糖的生物相容性，本发明所提供的壳聚糖荧光胶束有望应用于环境监测、生物标记及示踪等领域。
附图说明
图1为四[4-乙酸基氨基苯]乙烯核磁共振氢谱；
图2为壳聚糖、四[4-乙酸基氨基苯]乙烯和荧光纳米胶束的红外光谱；
图3为壳聚糖荧光纳米胶束的透射电子显微镜图片；
图4为不同壳聚糖与四[4-乙酸基氨基苯]乙烯投料比所制备的纳米胶束水合粒径变化；
图5为不同壳聚糖与四[4-乙酸基氨基苯]乙烯投料比所制备的纳米胶束荧光发射谱图；
图6为投料比0.8时(壳聚糖对TPE-4COOH质量比)时所制壳聚糖荧光胶束的水合粒径随介质的pH变化的趋势；
图7为投料比0.8时所制壳聚糖荧光胶束的荧光发射强度随介质的pH变化的趋势；
图8为四[4-氨基苯]乙烯为原料制备四[4-乙酸基氨基苯]乙烯的反应方程式；
图9为本发明制备的pH响应的壳聚糖荧光复合胶束在不同pH条件下的变化示意图；
图10为壳聚糖荧光复合胶束被摄入MCF-7癌细胞中的荧光成像结果图。
具体实施方式
实施例1
一种pH响应的壳聚糖荧光复合胶束的制备方法，包括以下步骤：
1)四[4-乙酸基氨基苯]乙烯(TPE-4COOH)的制备
将1.0克四[4-氨基苯]乙烯与2.0克氯乙酸溶解于15毫升甲苯，搅拌下加入3毫升干燥的三乙胺后，升温至85℃回流，反应6小时后冷却至室温，过滤混合物所得滤渣，用少量饱和碳酸氢钠水溶液溶解并抽滤，滤液在60毫升乙醚中沉淀出产物，以二氯甲烷溶解后用乙醚沉淀洗涤产品两次，真空干燥过夜，得到浅黄色的四[4-乙酸基氨基苯]乙烯产品，产率约为83.6％。图1为四[4-乙酸基氨基苯]乙烯的核磁共振氢谱，1H NMR(CDCl3,δ):6.82(d,8H)，6.43(d,8H)，4.7(s,4H)，4.1(s,8H)，羧基峰位置在12，图中没有延伸显示，图中，a和b分别归属于苯环上两个不同位置的氢6.82和6.43，与氨基相邻或相间，四个苯环，相邻氢为8个、相间位氢8个，d为四个氨基剩余的氢4.7，每个氨基一个氢、一共四个氢，c为乙酸基所连的亚甲基双氢4.1，四个亚甲基就8个氢，1.33和3.1位置的两个峰属于残留三乙胺峰，5.27为残留的二氯甲烷溶剂峰,7.26位置是氘代氯仿残留峰。
2)壳聚糖(CS)荧光复合胶束的制备
取0.5克上述四[4-乙酸基氨基苯]乙烯溶于去离子水中，配成1g/L，以1mol/L的氢氧化钠溶液调节pH至8，将水溶性壳聚糖溶解于去离子水中，配制成浓度0.2g/L，于搅拌下将壳聚糖溶液慢滴入等体积的四[4-乙酸基氨基苯]乙烯溶液，滴入壳聚糖溶液速度为1ml/min，滴完后继续搅拌5min。得到略显蓝光的乳液即产生了壳聚糖荧光纳米胶束。过滤除去难溶物，动态光散射检测显示所得胶束的水合粒径为155纳米。
重复实施例1，不同在于，将壳聚糖溶液替换为0.4g/L、0.6g/L、0.8g/L和1.0g/L，分别得到140纳米、126纳米、94纳米和66纳米粒径的胶束。荧光光谱研究证实所有纳米胶束均有以440纳米为中心的宽荧光发射，用荧光光谱仪测试产物胶束溶液所得。
图2为壳聚糖、四[4-乙酸基氨基苯]乙烯和所制备的荧光纳米胶束的红外光谱；荧光纳米胶束在3443cm-1处吸收峰属于壳聚糖羟基伸缩振动，此外，1650cm-1和1450cm-1处的特征峰与四[4-乙酸基氨基苯]乙烯的谱图酰胺基的1631cm-1和1400cm-1伸缩振动峰形貌及峰位接近，但向高波数移动，表明壳聚糖氨基和TPE-4COOH的羧基发生了静电络合作用，即荧光纳米胶束确实形成了。
1g/L壳聚糖溶液和1g/L四[4-乙酸基氨基苯]乙烯溶液，体积比为1:1制备的壳聚糖荧光纳米胶束的透射电子显微镜图片如图3。
图4为不同壳聚糖与四[4-乙酸基氨基苯]乙烯投料比(质量比)所制备的纳米胶束水合粒径变化；壳聚糖浓度越大，壳聚糖与四[4-乙酸基氨基苯]乙烯投料比升高，纳米胶束水合粒径越低。
图5为不同壳聚糖与四[4-乙酸基氨基苯]乙烯投料比(质量比)所制备的纳米胶束荧光发射谱图；
取壳聚糖对TPE-4COOH投料比(质量比)为0.8时所制的纳米胶束溶液105毫升，平均分成7等份，每份15毫升。使用0.5-1mol/L氢氧化钠溶液将这些胶束液pH值分别调节为2、3、4、5、6、7和8，在静置5-8小时后，以动态光散射粒度分析仪检测这些不同pH值溶液的胶束水合粒径，结果如图6所示；用荧光光谱仪测定不同pH值胶束溶液的荧光强度，归一化后结果如图7所示。
图6为投料比0.8时(壳聚糖对TPE-4COOH质量比)所制壳聚糖荧光胶束的水合粒径随介质的pH变化的趋势；
图7为投料比0.8时所制壳聚糖荧光胶束的荧光发射强度随介质的pH变化的趋势。
壳聚糖荧光胶束的细胞摄入成像
将MCF-7癌细胞以100000/孔的密度种在培养皿内，置于5％的CO2培养箱孵育24小时，弃培养基并以PBS缓冲液洗涤后，补加新鲜培养基和200微升本发明制备的壳聚糖荧光胶束溶液，37℃培养4小时后，用PBS液洗涤，随后用激光共聚焦显微镜在375nm激发波长下检测成像效果，结果见图10。
Claims (10)
1.一种pH响应的壳聚糖荧光复合胶束的制备方法，其特征在于，所述制备方法包括以下步骤：
将四羧基四苯乙烯化合物溶液调节pH至弱碱性，搅拌条件下滴入壳聚糖溶液，过滤，即得。
2.根据权利要求1所述的制备方法，其特征在于，所述四羧基四苯乙烯化合物包括但不限于四[4-乙酸基氨基苯]乙烯或四[4-羧基甲氧基苯]乙烯；
所述四[4-乙酸基氨基苯]乙烯制备方法为：将四[4-氨基苯]乙烯与氯乙酸溶解于甲苯，搅拌下加入干燥的三乙胺后，加热升温回流，反应后冷却至室温，产物分离纯化，即得。
3.根据权利要求2所述的制备方法，其特征在于，所述四[4-羧基甲氧基苯]乙烯的制备方法为：四[4-羟基苯]乙烯与氯乙酸溶解于甲苯，搅拌下加入干燥的三乙胺后，加热升温回流，反应后冷却至室温，产物分离纯化，即得。
4.根据权利要求1所述的制备方法，其特征在于，将四羧基四苯乙烯化合物溶液调节pH至7-8。
5.根据权利要求1所述的制备方法，其特征在于，滴入壳聚糖溶液速度为0.6-1ml/min。
6.根据权利要求1所述的制备方法，其特征在于，所述四羧基四苯乙烯化合物溶液浓度为1g/L。
7.根据权利要求1所述的制备方法，其特征在于，所述壳聚糖溶液浓度为0.2g/L-1.0g/L。
8.根据权利要求1所述的制备方法，其特征在于，四羧基四苯乙烯化合物溶液和壳聚糖溶液体积比为1:1。
9.一种权利要求1-8任一项所述制备方法制备的pH响应的壳聚糖荧光复合胶束。
10.一种权利要求1-8任一项所述制备方法制备的pH响应的壳聚糖荧光复合胶束的应用，其特征在于，用于环境监测、生物标记及示踪。
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