CN114366710B - 可注射型牛蒡微载体在治疗晚期骨性关节炎中的用途 - Google Patents
可注射型牛蒡微载体在治疗晚期骨性关节炎中的用途 Download PDFInfo
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Abstract
本发明涉及医学技术领域,提出了一种可注射型牛蒡微载体在治疗晚期骨性关节炎中的用途。以结冷胶(GG)为主体结构,通过EDC和NHS活化反应基团,进而引入氨基修饰的环糊精(HCD),充分反应后得到环糊精接枝的GG‑HCD,通过加入适当浓度的牛蒡苷(AC),最终获得可注射型牛蒡微载体。该方法制备得到的牛蒡微载体具有良好的可注射性和长效释放功能;为当前中药单体治疗骨关节炎的研究提供了新思路,填补了现有中药靶向性不足的缺陷,拓展了可注射型微载体在骨性关节炎中的应用前景。
Description
技术领域
本发明涉及医学技术领域,具体涉及一种可注射型牛蒡微载体在治疗晚期骨性关节炎中的用途。
背景技术
随着我国人口老龄化问题的进行性加重,老年性骨与软骨退行性疾病的发病率久高不下。作为骨关节的代表性疾病,骨关节炎不仅给病人带来巨大的痛苦,同时给社会造成严重的负担。作为中国传统文化的瑰宝,数千年来中药复方的治疗方式在不断地传承和发展。近年来,随着药物治疗精准化和靶向化的不断提高,基于中药单体的治疗策略逐渐得到重视,为顽固性疾病的治疗带来了新的曙光。
牛蒡,又名大力子,是一种菊科二年生草本植物,含菊糖、纤维素、蛋白质、钙、磷、铁等人体所需的多种维生素及矿物质。《本草纲目》记载,牛蒡能“通十二经脉,除五脏恶气”、“久服轻身耐老”。作为从成熟果实牛蒡子中提取分离而来的木脂素类化合物,牛蒡苷具有拮抗肿瘤、减轻炎症、对抗过度氧化等诸多的生物学功能。因此,基于中药单体牛蒡苷强大的生物活性的,结合生物材料的载药优势,开发出高效缓释治疗策略是治疗晚期骨关节炎的潜在方式。
结冷胶接枝改性技术为构建高效药物递送载体提供了平台。室温条件下,结冷胶具备一定的流动性,便于体内关节腔注射治疗。同时,良好的生物相容性利于基于结冷胶的生物治疗策略的进一步临床转化。另一方面,环状结构的低聚糖环糊精具有特定的锥形圆环结构,能包载各类脂溶性小分子药物。在环糊精外壳进行氨基化修饰增强其生物反应性,利于其与各类大分子聚合物共价结合。基于此,在结冷胶结构上接枝氨基化的环糊精不仅保留了结冷胶的物理特性,同时具备强大的药物包载性能,为多种脂溶性药物的体内递送创造了条件。然而现有的治疗晚期骨关节炎微载体具有疗效差等问题。
发明内容
鉴于当前晚期骨关节炎治疗方案的不足,本发明创新性地提出了一种可注射型牛蒡微载体的制备方法并证实了该系统体外调控软骨细胞代谢稳态,体内延缓关节软骨退变的强大功能。本发明提示该可注射型牛蒡微载体是一种高效的药物递送模式,为晚期骨性关节炎的临床治疗提供了新方向。
具体的,本发明的技术方案如下:
本发明第一个方面公开了一种牛蒡微载体的制备方法,包括以下步骤:
以结冷胶为主体结构,通过EDC和NHS活化反应基团引入氨基修饰的环糊精,反应后得到环糊精接枝的GG-HCD;接着加入牛蒡苷,包载得到牛蒡微载体。
优选的,包括:
S1:将结冷胶粉末溶于MES缓冲液,设置反应温度为50-70℃,充分搅拌至完全溶解;
S2:维持反应温度为50-70℃,向反应体系中加入EDC和NHS粉末,充分搅拌20-40min,使活性基团充分活化;
S3:维持50-70℃反应温度,向反应体系中加入HCD,避光反应确保接枝反应充分完成;
S4:使用透析袋充分透析5-6天,每日更换透析液;
S5:将透析结束后的溶液冷冻干燥后加入牛蒡苷,充分包载10-14 h后得到牛蒡微载体。
更优选的,在S2中,向反应体系中加入2当量的EDC和1当量的NHS粉末。
更优选的,在S3中,向反应体系中加入1当量的HCD。
本发明第二个方面公开了上述方法得到的牛蒡微载体。
优选的,所述牛蒡微载体为可注射型牛蒡微载体。
本发明第三个方面公开了一种用于治疗关节炎的药物,所述药物包括牛蒡微载体。
优选的,所述关节炎为晚期骨性关节炎。
本发明第四个方面公开了上述的牛蒡微载体在制备治疗关节炎药物中的应用。
本发明第五个方面公开了上述的牛蒡微载体在制备抑制软骨细胞外基质降解的药物或延缓体内关节炎进展药物中的应用。
与现有技术相比,本发明至少具有以下有益效果:
本发明以结冷胶(GG)为主体结构,通过EDC和NHS活化反应基团,进而引入氨基修饰的环糊精(HCD),充分反应后得到环糊精接枝的GG-HCD,通过加入适当浓度的牛蒡苷(AC),最终获得可注射型牛蒡微载体。该方法制备得到的牛蒡微载体具有良好的可注射性和长效释放功能。本发明为当前中药单体治疗骨关节炎的研究提供了新思路,填补了现有中药靶向性不足的缺陷,拓展了可注射型微载体在骨性关节炎中的应用前景。
附图说明
图1显示了可注射型牛蒡微载体调控软骨代谢稳态。
图2显示了可注射型牛蒡微载体无明显生物毒性。
图3显示了可注射型牛蒡微载体减轻软骨下骨硬化。
图4显示了可注射型牛蒡微载体缓解关节软骨退变。
具体实施方式
下面结合附图和实施例对本发明的技术方案进行详细描述,但并不因此将本发明限制在所述的实施例范围之中。
下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。本发明所用试剂和原料均市售可得。
实施例1
本实施例公开了可注射型牛蒡微载体的制备方法,具体包括:
(1)将结冷胶粉末溶于MES缓冲液配置成1%的使用浓度,设置反应温度为60℃,充分搅拌15 min至完全溶解。
(2)维持反应温度为60℃,向反应体系中加入2当量的EDC(1-(3-二甲基氨丙基)-3-乙基碳二亚胺盐酸盐)和1当量的NHS (N-羟基琥珀酰亚胺)粉末,充分搅拌30 min,使活性基团充分活化。
(3)维持60℃反应温度,向反应体系中加入1当量的HCD(单-(6-己二胺基-6-去氧)-β-环糊精),避光反应24 h,确保接枝反应充分完成。
(4)使用14000 MW的透析袋充分透析5日,每日更换透析液。
(5)将透析结束后的溶液冷冻干燥后,加入适当浓度的牛蒡苷,充分包载12 h后得到成品可注射型牛蒡微载体,用于后续实验。
实施例2
本实施例研究可注射型牛蒡微载体生物相容性评估,具体包括以下步骤:
(1)分离、培养得到胎鼠软骨细胞,使用CCK-8检测法测定牛蒡微载体对软骨细胞增殖能力的影响。具体实施步骤为,将P1代软骨细胞按照1000个/孔接种于96孔细胞培养板,使用DMEM/F12培养基培养。分组为:对照组、微载体组和牛蒡微载体组。分别于第1,3,5,7天加入10% CCK-8检测试剂,置于37℃培养箱中孵育60 min。使用全波长酶标仪在450 nm波长位置检测吸光度(OD)值。结果证实,牛蒡微载体无明显细胞毒性。
(2)采用细胞活/死染色试剂盒检测牛蒡微载体对软骨细胞的毒性效应。具体实施步骤为。将P1代软骨细胞按照5000个/孔接种于24孔细胞培养板,使用DMEM/F12培养基培养。分组为:对照组、微载体组和牛蒡微载体组。培养3天后,加入多聚甲醛固定细胞15 min。随后分别加入Calcein-AM (钙黄绿素)和PI (碘化丙啶)进行染色,使用倒置荧光显微镜观察并拍照。结果证实,牛蒡微载体对软骨细胞无显著细胞毒性,结果如图2所示。
实施例3
本实施例研究可注射型牛蒡微载体调控软骨细胞稳态,具体包括:
(1)使用RT-PCR,WB和免疫荧光法检测牛蒡微载体对软骨细胞合成代谢的影响。将P1代软骨细胞按照20000个/孔接种于6孔细胞培养板,使用DMEM/F12培养基培养。分组为:对照组、白介素组、白介素+牛蒡微载体组。培养3天后,分别进行免疫荧光染色、RT-PCR检测和蛋白凝胶电泳检测。主要观察指标为:II型胶原和糖胺聚糖表达量。结果证实:牛蒡微载体在体外促进软骨细胞外基质的合成。
(2)使用RT-PCR,WB和免疫荧光法检测牛蒡微载体对软骨细胞分解代谢的影响。将P1代软骨细胞按照20000个/孔接种于6孔细胞培养板,使用DMEM/F12培养基培养。分组为:对照组、白介素组、白介素+牛蒡微载体组。培养3天后,分别进行免疫荧光染色、RT-PCR检测和蛋白凝胶电泳检测。主要观察指标为:基质金属蛋白酶和蛋白多糖酶表达量。结果如图1所示证实:牛蒡微载体在体外抑制软骨细胞外基质的降解。
实施例4
本实施例研究可注射型牛蒡微载体延缓体内关节炎进展,具体包括:
(1)实验动物选取C57/BL6小鼠,分组为:对照组、关节炎组、关节炎+微载体组。小鼠关节炎模型采用经典的内侧半月板失稳术(DMM)实现,术后一周开始进行关节腔注射治疗,频率为每周1次,于术后第10周收取膝关节标本进行后续检测。
(2)影像学评估可注射型牛蒡微载体对软骨下骨硬化的影响。具体实施步骤为,将小鼠膝关节置于福尔马林中固定48 h。之后采用micro-CT设备进行扫描,感兴趣区域为胫骨内侧平台。利用分析软件和三维重建平台进行定量分析。结果如图3所示,证实牛蒡微载体在体内抑制软骨下骨的异常硬化。
(3)组织学评估可注射型牛蒡微载体对关节软骨退变的影响。具体实施步骤为,将小鼠膝关节置于EDTA溶液中进行脱钙处理。石蜡包埋并切片后,进行组织学(苏木素伊红、番红快绿、甲苯胺蓝)和免疫组织化学(II型胶原)染色。结果如图4所示,证实牛蒡微载体在体内缓解关节软骨退变。
综上所述,本次发明得出了以下重要结论:(1)本发明得到的可注射型牛蒡微载体具有良好的生物相容性;(2)本发明得到的可注射型牛蒡微载体调控软骨代谢稳态;(3)本发明得到的可注射型牛蒡微载体减轻软骨下骨硬化;(4)本发明得到的可注射型牛蒡微载体缓解关节软骨退变。
本次发明创新性地提出了一种可注射型牛蒡微载体的制备方法,在体内和体外取得了满意的治疗效果。提示该可注射型牛蒡微载体应用于软骨组织工程和再生医学的前景,为今后临床开发高效策略治疗晚期骨关节炎提供了参考。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (9)
1.一种牛蒡微载体的制备方法,其特征在于,
包括以下步骤:
以结冷胶为主体结构,通过EDC和NHS活化反应基团引入氨基修饰的环糊精HCD,反应后得到环糊精接枝的GG-HCD;接着加入牛蒡苷,包载得到牛蒡微载体;
具体包括:
S1:将结冷胶粉末溶于MES缓冲液,设置反应温度为50-70℃,充分搅拌至完全溶解;
S2:维持反应温度为50-70℃,向反应体系中加入EDC和NHS粉末,充分搅拌20-40 min,使活性基团充分活化;
S3:维持50-70℃反应温度,向反应体系中加入HCD,避光反应确保接枝反应充分完成;
S4:使用透析袋充分透析5-6天,每日更换透析液;
S5:将透析结束后的溶液冷冻干燥后加入牛蒡苷,充分包载10-14 h后得到牛蒡微载体。
2.根据权利要求1所述的方法,其特征在于,
在S2中,向反应体系中加入2当量的EDC和1当量的NHS粉末。
3.根据权利要求1所述的方法,其特征在于,
在S3中,向反应体系中加入1当量的HCD。
4.根据权利要求1-3任一项所述方法得到的牛蒡微载体。
5.根据权利要求4所述的牛蒡微载体,其特征在于,
所述牛蒡微载体为可注射型牛蒡微载体。
6.一种用于治疗关节炎的药物,其特征在于,
所述药物包括利用权利要求1-3任一项所述方法得到的牛蒡微载体。
7.根据权利要求6所述的药物,其特征在于,
所述关节炎为晚期骨性关节炎。
8.根据权利要求4所述的牛蒡微载体在制备治疗关节炎药物中的应用。
9.根据权利要求4所述的牛蒡微载体在制备抑制软骨细胞外基质降解的药物或延缓体内关节炎进展药物中的应用。
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