CN115177790A - 透明质酸和润滑素蛋白协同修饰胶原蛋白基质的制备方法 - Google Patents
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Abstract
本发明公开了一种基于透明质酸和润滑素蛋白协同修饰胶原蛋白基质的制备方法，提升胶原蛋白基质的抗蛋白吸附和促润滑性质，属于生物材料技术领域。本发明利用透明质酸和润滑素蛋白的特殊分子结构，将透明质酸和润滑素蛋白直接连接到胶原蛋白表面，从而获得一种基于透明质酸和润滑素蛋白修饰的胶原蛋白基质。该方法制备的修饰型胶原蛋白表面具有优异的润滑性能、抗蛋白吸附性能以及良好的生物相容性，并可便捷自主修饰在胶原蛋白表面，能很好地结合到受损软骨胶原蛋白表面，克服了未修饰胶原蛋白表面润滑性能不足、抗蛋白性能差的缺点，而且该制备方法简便，具有很好的可行性和实用性。
Description
技术领域
本发明涉及生物材料技术领域，具体涉及一种透明质酸和润滑素蛋白协同修饰胶原蛋白基质的制备方法。
背景技术
骨关节炎是一种可致残的慢性关节疾病，在全球60岁以上人口中，有多达10％的男性和18％的女性患有骨关节炎。骨关节炎会导致软骨基质组织的损伤和丢失，如胶原蛋白(COL)、蛋白多糖、聚集蛋白聚糖等分子从软骨表面的降解和脱落，进而导致软骨基质表面蛋白的异常沉积和摩擦增加。健康的关节软骨是一种高效的水基摩擦学***，摩擦系数极低，其独特的力学性能取决于覆盖在软骨基质表面成分之间的作用。软骨基质由高达70-80％的水和复杂的大分子组成，COL基质约占成人关节软骨干重的60-80％，被认为是软骨基质的主要结构成分。病变关节软骨胶原蛋白结构表面会出现早期磨损和较高的摩擦力，同时有较多的蛋白异常沉积和软骨细胞凋亡。
透明质酸(HA)是由β-1,4糖苷键和β-1,3-糖苷键交替链接的D-葡萄糖醛酸和D-N-乙酰葡萄糖胺构成，既是软骨细胞外基质的重要糖胺聚糖，也是滑液的主要组成成分。研究发现HA具有优异的润滑性能以及良好的抗炎症因子性能，在治疗骨关节炎中发挥着不可或缺的作用。虽然单一的透明质酸可以暂时缓解软骨损伤的进程，但并不能从根本上再生修复软骨组织。
发明内容
为改善胶原蛋白表面抗蛋白非特异性性能差、润滑性能不足的缺点，达到更好治疗软骨损伤的目的，本发明提供一种透明质酸和润滑素蛋白协同修饰胶原蛋白基质的制备方法。
为此，本发明采用以下技术方案：
一种透明质酸和润滑素蛋白协同修饰胶原蛋白基质的制备方法，包括以下步骤：
1)制备胶原蛋白基质：
配制pH为7.3-7.5的磷酸盐缓冲液，将胶原蛋白溶解在适量磷酸盐缓冲液中，配制成50μg/mL的胶原蛋白溶液，随后将100μL所述胶原蛋白溶液滴加到基底表面，孵化1-3h，并用所述磷酸盐缓冲液冲洗，以除去未吸附在基底的分子，得到修饰在基底表面的胶原蛋白基质；
2)制备透明质酸和润滑素蛋白协同修饰的胶原蛋白基质：
在步骤1)得到的胶原蛋白基质表面滴加100μL透明质酸和润滑素蛋白的混合液，孵化1-3h，随后用所述磷酸盐缓冲液彻底冲洗表面并用氮气吹干，制得透明质酸和润滑素蛋白协同修饰的胶原蛋白基质，所述混合液中透明质酸和润滑素蛋白的浓度分别为1mg/mL和50μg/mL-200μg/mL。
其中，所述磷酸盐缓冲液的制备方法为：3.63g十二水合磷酸氢二钠、0.27g磷酸二氢钾、8g氯化钠和0.2g氯化钾溶于1L去离子水中，调节至需要的pH。
优选的是，所述透明质酸的分子量范围为9000-3000000。
优选的是，步骤2)的混合液中，所述润滑素蛋白的浓度为100μg/mL。
本发明中使用的润滑素蛋白为重组润滑素蛋白。
所述的基底可以是关节、软骨组织或培养皿的表面。
本发明的方法制得的透明质酸和润滑素蛋白协同修饰的胶原蛋白基质可用于生物医药领域中，特别适用于制备软骨修复材料，治疗骨关节炎，改善受损软骨表面胶原蛋白的抗蛋白吸附以及润滑性能。
本发明通过使用天然的透明质酸和润滑素蛋白与胶原蛋白的特殊分子间相互作用，将透明质酸和润滑素蛋白协同修饰在胶原蛋白表面，通过此方法制得的胶原蛋白基质既保留了胶原蛋白在软骨细胞迁移、生长和在受损软骨组织粘附的能力，同时又赋予了其优异的抗非特异性吸附性能和促润滑性能。润滑素蛋白(LUB)是一种高度糖基化的大分子蛋白，由软骨细胞和滑膜细胞分泌，是滑液和软骨表面的主要成分，与HA协同降低软骨的磨损。
与现有技术相比，本发明具有以下优点及有益效果：
1)本发明的方法制备的透明质酸和润滑素蛋白协同修饰的胶原蛋白基质，其表面具有很好的抗蛋白非特异性吸附性能，对于溶菌酶(带正电荷)、牛血清蛋白(带负电荷)的非特性吸附量分别低至0ng/cm2、0ng/cm2，远低于未修饰胶原蛋白基质的非特异性吸附量，可大大降低了因蛋白、细菌吸附而引起的各种副作用或感染问题。
2)本发明的方法制备的透明质酸和润滑素蛋白协同修饰的胶原蛋白基质，其表面具有优异的润滑性能，在磷酸盐缓冲液(PBS，模拟生理环境)中的摩擦系数可低至0.07左右，克服了未修饰胶原蛋白润滑性能差的缺点。
3)本发明的方法制备的透明质酸和润滑素蛋白协同修饰的胶原蛋白基质，其表面耐磨损性能高，在磷酸盐缓冲液(PBS，模拟生理环境)中的耐磨损压力可达10.83MPa，远超过未修饰胶原蛋白的耐磨损压力。
4)本发明制备方法简便、生物相容性好，有很好的可行性和实用性。
附图说明
图1是本发明中透明质酸和润滑素蛋白协同修饰的胶原蛋白基质的结构示意图；
图2是实施例3中胶原蛋白表面与透明质酸和润滑素蛋白协同修饰的胶原蛋白表面对2mg/mL的溶菌酶(LYS)的非特异性吸附实时曲线变化图；
图3是实施例3中胶原蛋白表面与透明质酸和润滑素蛋白协同修饰的胶原蛋白表面对牛血清蛋白(BSA)的非特异性吸附实时曲线变化图；
图4是实施例1中胶原蛋白与透明质酸和润滑素蛋白协同修饰的胶原蛋白的摩擦性能图；
图5是实施例2中胶原蛋白与透明质酸和润滑素蛋白协同修饰的胶原蛋白的摩擦性能图；
图6是实施例3中胶原蛋白与透明质酸和润滑素蛋白协同修饰的胶原蛋白的摩擦性能图。
具体实施方式
下面结合附图和实施例对本发明的制备方法进行详细说明。
透明质酸(HA)和润滑素蛋白(LUB)分别具有以下结构：
以下实施例中，磷酸盐缓冲液的制备方法为：
3.63g十二水合磷酸氢二钠、0.27g磷酸二氢钾、8g氯化钠和0.2g氯化钾溶于1L去离子水中，配制成pH＝7.4的10mM磷酸盐缓冲液。
以下实施例中，所述润滑素蛋白为重组润滑素蛋白，购自Lubris Biopharma(Weston，MA)。
本发明中的透明质酸为食品级。
本发明中使用的胶原蛋白为无毒、无热源、纯度>99.9％的胶原蛋白。以下实施例中使用的胶原蛋白购自关节动力安达(天津)生物科技有限公司。
本发明中透明质酸和润滑素蛋白协同修饰的胶原蛋白基质的结构示意图如图1所示。
为便于制备和进行性能测试，以下实施例中均以云母或耗散型石英晶体微天平***使用的带金膜的芯片为基底，该基底仅起支撑作用，对制得的胶原蛋白基质的功能不产生影响。可以理解，在实际应用中，可以以关节、软骨组织或培养皿的表面等作为基底。
实施例1
一种透明质酸和润滑素蛋白协同修饰胶原蛋白基质的制备方法，包括以下步骤：
1)胶原蛋白基质的制备：
将胶原蛋白(COL)溶于在磷酸盐缓冲液(pH＝7.4)(PBS)中，配制成50μg/mL的COL溶液，随后将100μL COL溶液滴加到基底(芯片或云母)表面，孵化1h，并用PBS(pH＝7.4)冲洗，以除去未吸附在基底的分子，得到修饰在基底表面的胶原蛋白基质。
2)透明质酸和润滑素蛋白在胶原蛋白基质表面的协同修饰：
在步骤1)得到的COL基质表面滴加100μL透明质酸(HA)和润滑素蛋白(LUB)的混合液，孵化约1h，随后用PBS彻底冲洗表面并用氮气吹干，最终获得透明质酸和润滑素蛋白协同修饰的胶原蛋白基质(COL/HA/LUB-1h)。其中，所述混合液中HA和LUB的浓度分别为1mg/mL HA和100μg/mL。
抗非特异性吸附量测定：
对制得的透明质酸和润滑素蛋白协同修饰的胶原蛋白表面的抗非特异性吸附性能进行测试，具体方法如下：
将获得的具有透明质酸和润滑素蛋白协同修饰胶原蛋白基质的耗散型石英晶体微天平芯片(COL/HA/LUB)贴合到耗散型石英晶体微天平***的流通池内。以pH为7.4的磷酸缓冲液为流动相，流速为50μL/min。待基线平稳后，通入2mg/mL的牛血清白蛋白或溶菌酶，经流动相推动蛋白溶液到达芯片表面，由耗散型石英晶体微天平***测定实时变化曲线，读取频率变化值，并计算非特异性吸附量，见表1。
由表1的结果可知，实施例1制得的基于透明质酸和润滑素蛋白修饰胶原蛋白基质表面对牛血清蛋白和溶菌酶的非特异性吸附量分别为3.87ng/cm2和5.87ng/cm2，远低于对照组胶原蛋白表面吸附量。
摩擦系数测定与润滑性能评价：
采用表面力仪对制得的透明质酸和润滑素蛋白协同修饰胶原蛋白表面的摩擦系数进行测定，评价其润滑性能，具体方法如下：
将具有透明质酸和润滑素蛋白协同修饰胶原蛋白基质的云母片固定于表面力仪测试***内测量其摩擦系数，重复测量3次，结果汇总见图4。
由图4可见，实施例1中制得的基于透明质酸和润滑素蛋白修饰胶原蛋白基质(COL/HA/LUB-1h)，其表面的摩擦系数为0.07，远低于单一胶原蛋白表面的摩擦系数(0.48)，因此具有良好的润滑性能。
实施例2
一种透明质酸和润滑素蛋白协同修饰胶原蛋白基质的制备方法，包括以下步骤：
1)胶原蛋白基质的制备：
将COL溶解在PBS(pH＝7.4)中，配制成50μg/mL的溶液，随后将100μL COL溶液滴加到基底(芯片或云母)表面，孵化2h，并用PBS冲洗以除去未吸附在基底的分子，得到修饰在基底表面的胶原蛋白基质。
2)透明质酸和润滑素蛋白在胶原蛋白基质表面的协同修饰：
在步骤1)得到的COL基质表面滴加100μL HA和LUB的混合液，孵化2h，随后用PBS彻底冲洗表面并用氮气吹干，最终获得透明质酸和润滑素蛋白协同修饰的胶原蛋白基质(COL/HA/LUB-2h)。所述混合液中HA和LUB的浓度分别为1mg/mL的溶液和100μg/mL。
抗非特异性吸附量测定：
测试方法同实施例1。测试结果见表1。
由表1的结果可知，实施例2中制得的基于透明质酸和润滑素蛋白修饰的胶原蛋白基质，其表面对牛血清蛋白和溶菌酶的非特异性吸附量分别为1.22ng/cm2和2.31ng/cm2，远低于对照组胶原蛋白表面吸附量。
摩擦性能评价：
采用表面力仪对制得的透明质酸和润滑素蛋白修饰胶原蛋白表面的磨损速度进行测定，评价其润滑性能，具体方法如下：
将具有透明质酸和润滑素蛋白协同修饰胶原蛋白基质的云母片固定于表面力仪测试***内测量其磨损速度，重复测量3次，结果汇总见图5。
由图5可见，实施例2中制得的基于透明质酸和润滑素蛋白修饰胶原蛋白基质，其表面(COL/HA/LUB-2h)的磨损速度为19μm/s，远高于单一胶原蛋白表面的磨损速度(3μm/s)，说明其耐磨性高，具有优异的润滑性能。
实施例3
一种透明质酸和润滑素蛋白协同修饰胶原蛋白基质的制备方法，包括以下步骤：
1)胶原蛋白基质的制备：
将COL溶解在PBS(pH＝7.4)中，配制成50μg/mL的溶液，随后将100μL COL溶液滴加到基底(芯片或云母)表面，孵化3h，并用PBS冲洗以除去未吸附在基底的分子，最终获得修饰在基底表面的胶原蛋白基质。
2)透明质酸和润滑素蛋白在胶原蛋白表面的协同修饰：
在步骤1)得到的COL基质表面滴加100μL HA和LUB的混合液(浓度分别为1mg/mL、100μg/m)，孵化约3h，随后用PBS彻底冲洗表面并用氮气吹干，最终获得透明质酸和润滑素蛋白协同修饰的胶原蛋白基质(COL/HA/LUB-3h)。
抗非特异性吸附量测定：
测试方法同实施例1。测试结果见表1、图2和图3。
表1不同表面的非特异性吸附量
由表1的结果可知，实施例3中制得的基于透明质酸和润滑素蛋白协同修饰胶原蛋白基质表面(COL/HA/LUB-3h)对牛血清蛋白和溶菌酶的非特异性吸附量分别为0ng/cm2和0ng/cm2。
摩擦性能评价：
采用表面力仪对制得的透明质酸和润滑素蛋白协同修饰胶原蛋白表面的摩擦系数进行测定，评价其润滑性能，具体方法如下：
将具有透明质酸和润滑素蛋白协同修饰胶原蛋白表面的云母片固定于表面力仪测试***内测量其磨损压力，重复测量3次，结果汇总见图6。
由图6可见，实施例3中制得的基于透明质酸和润滑素蛋白修饰胶原蛋白表面(COL/HA/LUB-3h)的磨损压力为10.83MPa，远高于单一胶原蛋白表面的磨损速度(1.87MPa)，说明其耐磨性高，具有优异的润滑性能。
Claims (6)
1.一种透明质酸和润滑素蛋白协同修饰胶原蛋白基质的制备方法，包括以下步骤：
1)制备胶原蛋白基质：
配制pH为7.3-7.5的磷酸盐缓冲液，将胶原蛋白溶解在适量磷酸盐缓冲液中，配制成50μg/mL的胶原蛋白溶液，随后将100μL所述胶原蛋白溶液滴加到基底表面，孵化1-3h，并用所述磷酸盐缓冲液冲洗，除去未吸附在基底的分子，得到修饰在基底表面的胶原蛋白基质；
2)制备透明质酸和润滑素蛋白协同修饰的胶原蛋白基质：
在步骤1)得到的胶原蛋白基质表面滴加100μL透明质酸和润滑素蛋白的混合液，孵化1-3h，随后用所述磷酸盐缓冲液彻底冲洗表面并用氮气吹干，制得透明质酸和润滑素蛋白协同修饰的胶原蛋白基质，所述混合液中透明质酸和润滑素蛋白的浓度分别为1mg/mL和50μg/mL-200μg/mL。
2.根据权利要求1所述的制备方法，其特征在于，所述磷酸盐缓冲液的制备方法为：3.63g十二水合磷酸氢二钠、0.27g磷酸二氢钾、8g氯化钠和0.2g氯化钾溶于1L去离子水中，调节至需要的pH。
3.根据权利要求1所述的制备方法，其特征在于：所述透明质酸的分子量范围为9000-3000000。
4.根据权利要求1所述的制备方法，其特征在于：步骤2)的混合液中，所述润滑素蛋白的浓度为100μg/mL。
5.根据权利要求4所述的制备方法，其特征在于：所述润滑素蛋白为重组润滑素蛋白。
6.根据权利要求1-5中任一项所述的制备方法，其特征在于：所述的基底为关节、软骨组织或培养皿的表面。
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