CN109517162A - 可注射水凝胶及制备方法 - Google Patents
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Abstract
本发明公开了可注射水凝胶及制备方法,所述可注射水凝胶由6‑氰基苯并噻唑CBT修饰的亲水性支化聚合物和两端半胱氨酸Cys修饰的小分子构成。本发明选用亲水的支状聚合物上面修饰的6‑氰基苯并噻唑和小分子两端的半胱氨酸作为化学交联位点形成的水凝胶具有一定的强度,并且凝胶化的速率可控,其制备在生理条件下即可进行,不需要加入交联剂,毒副作用小,且反应具有专一性。获取的水凝胶具有良好的生物安全性和生物降解性,具有应用于药物载体和组织工程等方面的潜能。
Description
技术领域
本发明属于生物医学材料领域,具体涉及可注射水凝胶及制备方法。
背景技术
可注射水凝胶在用于生物医学应用方面具有许多优良的性质:例如微创、副作用小;同时,水凝胶作为载体不仅使各种生物活性分子和药物在组织有效的累积,还可以通过引入对pH、温度、酶和光敏感的化学键,使有效成分达到缓控释的目的。
在生物医学应用中,可注射水凝胶作为一种理想的载体或支架,有以下基本要求:(i)适当的凝胶速率:快速凝胶化会在注射过程中引起针头堵塞,或导致严重的毒性和显著的热效应,在注射后引起局部细胞凋亡和组织坏死。缓慢凝胶化可导致包封在水凝胶中的药物或细胞快速扩散到周围区域并降低治疗效果;(ii)合适的机械强度以抵抗损伤区域中的高压以及避免嵌入的生物分子或细胞发生突释;(iii)优异的生物相容性以支撑细胞生长,同时调节细胞分化。
制备可注射的水凝胶通常有以下三种方法:(i)聚合物或多肽对pH,温度和光刺激发生响应性自组装;(ii)乙烯基功能化的PEG,PVA或多糖发生自由基交联;(iii)由大分子单体发生迈克尔加成,Diels-Alder反应以及Schiff碱反应。其中,聚合物的自组装属于物理交联,这是一种相对温和的交联策略,不仅细胞毒性较小,并且成胶过程可逆,制备的水凝胶也具有良好的自我修复性能。但是,通过物理作用形成的水凝胶强度却不如化学作用制备的水凝胶。
由于化学键交联制备的水凝胶具有显著增强的机械性能,并且结构稳定。因此,针对一些应用,采用化学交联来制备水凝胶是更好的选择。然而,化学交联在制备水凝胶时凝胶化相对快速,交联剂的使用也通常会带来一定的毒性。
发明内容
本发明的目的在于提供可注射水凝胶及制备方法。
本发明通过以下技术方案来实现上述技术目的:
一种可注射水凝胶,所述可注射水凝胶由6-氰基苯并噻唑CBT修饰的亲水性支化聚合物和两端半胱氨酸Cys修饰的小分子构成。
进一步的,所述小分子为对基质金属蛋白酶或对谷胱甘肽敏感的小分子;
进一步的,所述对基质金属蛋白酶敏感的小分子为小分子多肽,其序列优选GPLGLAGK;所述对谷胱甘肽敏感的小分子为包含二硫键的胱胺。
进一步的,所述亲水性支化聚合物为四臂聚乙二醇羧酸或八臂聚乙二醇羧酸;
进一步的,所述所述亲水性支化聚合物分子量为5~40kDa。
本发明还提供了可注射水凝胶的制备方法,包括以下步骤:
1)对亲水性支化聚合物进行6-氰基苯并噻唑CBT修饰,获取组分A;
2)在MMP酶/GSH敏感的小分子两端修饰巯基和氨基双保护的半胱氨酸;
3)对步骤2)获取的产物进行脱保护,获取两端修饰半胱氨酸的小分子,为组分B;
4)将组分A和组分B的溶液混合,制备获取三维空间网状结构的水凝胶。
进一步的,所述步骤1)中,CBT修饰亲水性支化聚合物的制备方式为:取亲水性支化聚合物溶于干燥的有机溶剂,以N-甲基吗啉和氯甲烷甲酯为活化剂,然后在反应液中加入6-氰基苯并噻唑Cys反应,反应结束后产物溶液在乙醚中沉淀,离心,沉淀物用水溶解,透析,冻干得组分A。
进一步的,所述步骤1)中,CBT与亲水性支化聚合物的羧基的摩尔比n>1;优选为1.25。
进一步的,所述步骤2)中,巯基和氨基双保护的半胱氨酸为N-叔丁氧羰基-S-三苯甲基-L-半胱氨酸;将小分子与N-叔丁氧羰基-S-三苯甲基-L-半胱氨酸在缩合剂的条件下进行酰胺缩合反应,获取两端修饰N-叔丁氧羰基-S-三苯甲基-L-半胱氨酸的小分子。
进一步的,所述步骤3)中,脱保护的方法为:在TFA的酸性条件下脱去巯基和氨基的保护基团,之后加入三异丙基硅烷以保护脱去保护后得到的中间体;旋蒸,乙醚沉淀,离心得到粗产品;之后水中透析纯化,冻干得组分B。
进一步的,所述步骤4)中,将组分A与组分B分别在pH 7.4的水中或者PBS磷酸盐缓冲液中溶解,之后将两者的溶解液均匀混合,得到化学交联的水凝胶。
进一步的,所述步骤4)中,组分A与组分B溶液质量浓度w>1%;优选为10%。
本发明通过6-氰基苯并噻唑与半胱氨酸的“click反应”将亲水性支化聚合物进行交联,获取三维网状的水凝胶,可将生物活性分子或药物嵌入三维网状结构中,实现水凝胶在作为药物载体中的应用。两端被半胱氨酸修饰的小分子序列可以在MMP酶/GSH的作用下发生断裂,从而实现水凝胶的降解,有效的释放出药物。此外,本发明选用亲水的支状聚合物上面修饰的6-氰基苯并噻唑和小分子两端的半胱氨酸作为化学交联位点形成的水凝胶具有一定的强度,并且凝胶化的速率可控,其制备在生理条件下即可进行,不需要加入交联剂,毒副作用小,且反应具有专一性。
附图说明
图1为本发明实施例1中所述4ARM PEG-CBT和Cys-MMP-Cys的合成路线图以及两者形成水凝胶的路线图。
图2为本发明实施例3中所述CBT的质谱。
图3为本发明实施例3中4ARM PEG-CBT的核磁氢谱。
图4为本发明实施例3中Cys-MMP-Cys的质谱。
图5为本发明实施例3中Cys-MMP-Cys的HPLC。
图6为本发明实施例3中所述制备的水凝胶。
图7为本发明实施例5中所述制备的水凝胶的内部形貌。
图8为本发明实施例6中所述水凝胶的流变学性质。
图9为本发明实施例7中所述制备水凝胶组分稀溶液的生物安全性评价。
具体实施方式
以下通过实施例的具体实施方式再对本发明的上述内容作进一步的详细说明。但不应当将此理解为本发明上述主题的范围仅限于以下的实例。在不脱离本发明的精神和原则之内做的任何修改,以及根据本领域普通技术知识和惯用手段做出的等同替换或者改进,均应包括在本发明的保护范围内。
实施例中使用的试剂与材料如下:
聚乙二醇羧酸,购自北京键凯科技有限公司;
多肽序列(GPLGLAGK),购自浙江昂拓莱斯生物技术有限公司;
胱胺二盐酸盐,购自西格玛奥德里奇;
四氢呋喃(THF),购自百灵威科技有限公司;
氯甲酸异丁酯(IBCF),购自阿拉丁;
6-氰基苯并噻唑(CBT),购自湖北信康医药化工有限公司;
N-叔丁氧羰基-S-三苯甲基-L-半胱氨酸(Boc-Cys(Trt)-OH),购自吉尔生化;
六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBop),购自阿拉丁;
1-羟基苯并三唑(HOBt),购自阿拉丁;
N,N-二异丙基乙胺(DIEA),购自阿拉丁;
实施例1
本实施例具体说明CBT修饰的亲水性支化聚合物的制备方法。本实施例中的亲水性支化聚合物为四臂聚乙二醇羧酸和八臂聚乙二醇羧酸。
(1)合成CBT为四臂PEG端基的化合物(4ARM PEG-CBT)。
称取0.25g的四臂聚乙二醇羧酸(4ARM PEG-COOH,10kDa),加入到25mL的支口瓶中备用;对反应瓶除水除氧后,进行氮气保护,再将2mL二氯甲烷(干燥)加入到反应瓶,得到澄清溶液;然后依次加入12μL的MMP与1mL的THF的混合溶液,13μL的IBCF与1mL的THF的混合溶液;反应在冰水浴中搅拌30min后,加入用的22.8mg CBT的THF溶液。反应在冰水浴下继续搅拌2h,之后室温反应3d。将上述反应液滴加至过量乙醚中进行搅拌,过滤得到沉淀;将该沉淀用蒸馏水溶解,用3500kDa的透析袋在水中进行透析24h,之后冷冻干燥得到4ARM PEG-CBT。使用400MHz核磁共振仪对所得化合物的结构进行确定,其核磁氢谱如图3所示。
选用不同分子量的四臂PEG为原料,同样合成得到合成CBT为四臂PEG端基的化合物(4ARM PEG-CBT)。
(2)合成CBT为八臂PEG端基的化合物(8ARM PEG-CBT)。
称取0.25g的八臂聚乙二醇羧酸(8ARM PEG-COOH 10kDa),加入到25mL的支口瓶中备用;对反应瓶除水除氧后,进行氮气保护,再将2mL二氯甲烷(干燥)加入到反应瓶,得到澄清溶液;然后依次加入的MMP(24μL)与1mL的THF的混合溶液,26μL的IBCF与1mL的THF的混合溶液;反应在冰水浴中搅拌30min后,加入用的45.5mg CBT的THF溶液。反应在冰水浴下继续搅拌2h,之后室温反应72h。将上述反应液滴加至过量乙醚中进行搅拌,过滤得到沉淀;将该沉淀用蒸馏水溶解,用3500kDa的透析袋在水中进行透析24h,之后冷冻干燥得到8ARM PEG-CBT。选用不同分子量的八臂PEG为原料,同样合成得到合成CBT为八臂PEG端基的化合物(8ARM PEG-CBT)。
实施例2
本实施例具体说明两端半胱氨酸修饰的小分子的制备方式。
(1)两端半胱氨酸修饰的MMP酶敏感的小分子(Cys-MMP-Cys):
称取0.63g氨基酸序列为GPLGLAGK的MMP酶敏感的小分子多肽、1g的Boc-Cys(Trt)-OH、1.3g的PyBop和0.35g的HOBt于反应瓶;在真空条件下,向反应瓶中加入8mL的DMSO溶液,之后在冰水浴条件下缓慢滴加1.4mL的DIEA。室温搅拌48h后反应终止,向反应液中加入50mL的二氯甲烷稀释,分别用饱和的NaCl溶液、1M的HCl溶液和饱和的NaHCO3水洗三次;将有机相干燥、过滤最后旋蒸,通过柱层析的方法分离得到Boc-(Trt)-Cys-MMP-Cys-(Trt)-Boc。
称取0.24g的Boc-(Trt)-Cys-MMP-Cys-(Trt)-Boc于25mL的反应,在除水除氧氮气保护的条件下,滴加94uL的TIS和1.5mL的二氯甲烷混合液,之后滴加1.5mL的TFA;反应10min后旋蒸,加入过量乙醚进行搅拌过夜,离心得到沉淀,得到粗产品Cys-MMP-Cys,然后蒸馏水透析,离心,冻干得最终产物。
通过质谱确定Cys-MMP-Cys的分子量916,通过HPLC确定其达到纯度98%,分别如图4和5所示。
(2)两端半胱氨酸修饰的GSH敏感的小分子(Cys-SS-Cys):
称取0.20g的胱胺二盐酸盐、1g的Boc-Cys(Trt)-OH、1.35g的PyBop和0.35g的HOBt于反应瓶;在真空条件下,向反应瓶中加入8mL的DMSO溶液,之后在冰水浴条件下缓慢滴加1.4mL的DIEA。室温搅拌48h后反应终止,向反应液中加入50mL的二氯甲烷稀释,分别用饱和的NaCl溶液、1M的HCl溶液和饱和的NaHCO3水洗三次;将有机相干燥、过滤最后旋蒸,通过柱层析的方法分离得到Boc-(Trt)-Cys-SS-Cys-(Trt)-Boc。
称取0.16g的Boc-(Trt)-Cys-SS-Cys-(Trt)-Boc于25mL的反应,在除水除氧氮气保护的条件下,滴加94uL的TIS和1.5mL的二氯甲烷混合液,之后滴加1.5mL的TFA;反应10min后旋蒸,加入过量乙醚进行搅拌过夜,离心得到沉淀,得到粗产品Cys-MMP-Cys,然后蒸馏水透析,离心,冻干得最终产物。
实施例3:水凝胶的制备PEG-CBT/Cys-MMP-Cys
实施例3水凝胶制备的化学反应式如图1所示。
室温下,将10mg的4ARM PEG-CBT(10kDa)和1.83mg的Cys-MMP-Cys分别溶于200uL的蒸馏水或者PBS缓冲液,之后将两溶液充分混合,观察一段时间溶液发生凝胶化,如图6所示,将玻璃瓶垂直倒立后没有液体流下。除此,采用不同分子量的4ARM PEG-CBT(5,20,40kDa)和8ARM PEG-CBT(10,20,40kDa)与Cys-MMP-Cys都可以采用同样的方式制备一系列可注射水凝胶。
实施例4:水凝胶的制备PEG-CBT/Cys-SS-Cys
室温下,将10mg的4ARM PEG-CBT(10kDa)和0.72mg的Cys-SS-Cys分别溶于200uL的蒸馏水或者PBS缓冲液,之后将两溶液充分混合,观察一段时间溶液发生凝胶化,将玻璃瓶垂直倒立后没有液体流下。除此,采用不同分子量的4ARM PEG-CBT(5,20,40kDa)和8ARMPEG-CBT(10,20,40kDa)与Cys-SS-Cys都可以采用同样的方式制备一系列可注射水凝胶。
实施例5:水凝胶的形貌
将实施例3中制备得到的水凝胶冻干,通过SEM观察到水凝胶的内部结构,如图7所示,该水凝胶为三维空间网状结构,内部具有较多的孔状结构,可用于药物及生物活性分子的包载。
实施例6:水凝胶的流变力学特性
在25℃下,采用Modular Compact Rheometer302流变仪(Anton Paar公司)对实施例3制备的水凝胶力学特性进行考察。采用angle 1°的锥板,滴加400uL的4ARM PEG-CBT和Cys-MMP-Cys的样品溶液,在角频率为10rad s-1,应变力为10%的条件下,考察形成水凝胶过程中储能模量(G’)和损耗模量(G”)随时间的变化。
如图8所示,在t约为260s时,储能模量等于损耗模量,说明此时开始形成水凝胶,溶液由液态向固态转变;当时间达到1500s时,储能模量趋于稳定最终达到600Pa。说明在生理条件下,通过CBT与Cys的化学交联得到的水凝胶具有合适的成胶时间以及适宜的力学性能,这些有利于该水凝胶在皮肤方面的应用。
实施例7:制备水凝胶组分稀溶液的生物安全性评价
CCK-8法测定实施例3水凝胶组分稀溶液的细胞活性实验。选取L929细胞接种于96孔板中,培养24小时后,加入不同浓度的4ARM PEG-CBT和Cys-MMP-Cys的混合溶液。每个浓度设置5个复孔平行样,并设置对照组。继续孵育24小时后,每孔内培养基移除,细胞用PBS洗涤并更换新的培养基,加入CCK-8,将96孔板放入培养箱内孵育1.5小时。最后用酶标仪测定每孔在490nm波长处的吸光值,计算细胞存活率。
图9为水凝胶组分稀溶液的细胞活性实验结果,结果表明细胞存活率在80%以上,水凝胶的组分具有良好的生物相容性。
以上所述仅为本发明的优选实施例,对本发明而言仅是说明性的,而非限制性的;本领域普通技术人员理解,在本发明权利要求所限定的精神和范围内可对其进行许多改变,修改,甚至等效变更,但都将落入本发明的保护范围。
Claims (10)
1.一种可注射水凝胶,其特征在于,所述可注射水凝胶由6-氰基苯并噻唑CBT修饰的亲水性支化聚合物和两端半胱氨酸Cys修饰的小分子构成。
2.根据权利要求1所述的可注射水凝胶,其特征在于,所述小分子为对基质金属蛋白酶或对谷胱甘肽敏感的小分子。
3.根据权利要求2所述的所述的可注射水凝胶,其特征在于,所述对基质金属蛋白酶敏感的小分子为多肽序列;所述对谷胱甘肽敏感的小分子为包含二硫键的胱胺。
4.根据权利要求1所述的所述的可注射水凝胶,其特征在于,所述亲水性支化聚合物为四臂聚乙二醇羧酸或八臂聚乙二醇羧酸。
5. 根据权利要求1所述的所述的可注射水凝胶,其特征在于,所述亲水性支化聚合物分子量为5~40 kDa。
6.一种可注射水凝胶的制备方法,其特征在于,包括以下步骤:
1)对亲水性支化聚合物进行6-氰基苯并噻唑CBT修饰,获取组分A;
2)在基质金属蛋白酶/谷胱甘肽敏感的小分子两端修饰巯基和氨基双保护的半胱氨酸;
3)对步骤2)获取的产物进行脱保护,获取两端修饰半胱氨酸的小分子,为组分B;
4)将组分A和组分B的溶液混合,制备获取三维空间网状结构的水凝胶。
7.根据权利要求6所述的制备方法,其特征在于,所述步骤1)中,CBT修饰亲水性支化聚合物的制备方式为:取亲水性支化聚合物溶于干燥的有机溶剂,以N-甲基吗啉和氯甲烷甲酯为活化剂,然后在反应液中加入6-氰基苯并噻唑反应,反应结束后产物溶液在乙醚中沉淀,离心,沉淀物用水溶解,透析,冻干得组分A。
8.根据权利要求6所述的制备方法,其特征在于,所述步骤2)中,巯基和氨基双保护的半胱氨酸为N-叔丁氧羰基-S-三苯甲基-L-半胱氨酸;将小分子与N-叔丁氧羰基-S-三苯甲基-L-半胱氨酸在缩合剂的条件下进行酰胺缩合反应,获取两端修饰N-叔丁氧羰基-S-三苯甲基-L-半胱氨酸的小分子。
9.根据权利要求6所述的制备方法,其特征在于,所述步骤3)中,脱保护的方法为:在三氟乙酸TFA的酸性条件下脱去巯基和氨基的保护基团,之后加入三异丙基硅烷以保护脱去保护后得到的中间体;旋蒸,乙醚沉淀,离心得到粗产品;之后水中透析纯化,冻干得组分B。
10.根据权利要求6所述的制备方法,其特征在于,所述步骤4)中,将组分A与组分B分别在pH 7.4的水中或者PBS磷酸盐缓冲液中溶解,之后将两者的溶解液均匀混合,得到化学交联的水凝胶。
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