CN116421550A - 用于骨关节治疗的新型可注射水凝胶及其制备方法 - Google Patents
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Abstract
本发明公开了一种用于骨关节治疗的新型可注射水凝胶及其制备方法，涉及生物医学材料技术领域，其技术方案要点是：水凝胶由氧化透明质酸钠(OHA)、4‑氨基苯硼酸(4‑ABA)和鞣花酸(EA)按照特定的投料比例在特定pH范围的无菌纯水中制备而成。水凝胶包含两种pH响应的可逆共价交联结构，即OHA与4‑ABA之间的席呋碱键以及EA与4‑ABA之间的苯硼酸酯键。水凝胶原位注入膝骨关节炎关节腔后，响应pH环境持续降解释放具有润滑功能的OHA以及具有抗炎功能的EA，起到较好的骨关节炎治疗效果。
Description
技术领域
本发明涉及生物医学材料技术领域，更具体地说，它涉及用于骨关节治疗的新型可注射水凝胶及其制备方法。
背景技术
骨关节炎是目前全世界最为常见的一种与年龄相关的退行性关节疾病。据统计，我国60岁以上男性和女性患有骨关节炎的比例分别高达10％和13％。随着我国人口老龄化加剧，骨关节炎的潜在患者数量正在逐年快速增长。骨关节炎以软骨退化和滑膜囊炎症为主要特征，同时伴随软骨下骨硬化、骨刺形成以及滑膜/韧带增厚等现象。因此，骨关节炎患者需长期承受慢性疼痛、关节僵硬以及关节软骨丧失带来的活动功能障碍等痛苦，生活质量受到极大影响。
骨关节炎的主要发病机制为关节部位机械磨损诱发分解代谢分子(主要为基质金属蛋白酶和含凝血酶敏感基序的去整合素金属蛋白酶)的分泌增加，进而造成胶原蛋白和软骨可聚蛋白多糖的渐进性不可逆降解，随后降解产物扩散至整个关节引发持续性炎症反应，最终炎症介质(例如白细胞介素-1β、肿瘤坏死因子-α、活性氧种、活性氮种)进一步造成软骨细胞凋亡以及细胞外基质损伤，触发涉及整个关节的退行性变级联。骨关节炎治疗的关键在于：(i)增强关节润滑功能，减轻机械磨损；(ii)消除关节部位炎症反应，阻断退行性变级联。由于关节软骨是不可再生的，因此临床上针对晚期骨关节炎治疗的主要手段是使用假体材料来替换受损的关节。但是，对于早中期骨关节炎而言，由于软骨尚未完全丧失，关节活动功能不受限制，因此非外科式的精准治疗手段更符合患者的需求。口服或静脉注射等系统性给药方式可以缓解疼痛和炎症反应，但由于软骨血管网少，药物吸收率低，因此治疗效果不理想。尽管加大药物摄入量可以增强治疗效果，但这会诱发系统性毒性反应。原位微针注射改善了药物的生物利用度同时降低了系统性毒性风险，但注入关节腔内的药物会被机体快速清除，因此要想达到理想的治疗效果就必须频繁给药，这样就增加了感染的风险。鉴于此，近年来，一些能够延长药物释放周期的可注射递送平台(例如脂质体、固体脂质纳米颗粒、胶束、聚合物微米/纳米颗粒和水凝胶珠)也被开发出来用于骨关节炎治疗。但是，这些递送平台载药量低，在骨关节腔内的结构依从性低，靶向释药能力差。此外，有些载体材料本身是不可降解和代谢的，其残留在骨关节腔内可能会引起类似无菌免疫应激反应等副作用。
可注射水凝胶是一种兼具生物因子递送功能与自润滑功能的高黏流性材料，因此被认为是一种理想的早中期骨关节炎综合治疗平台。此外，可注射水凝胶在骨关节炎治疗中还具有以下优点：(i)侵入性小，感染风险低；(ii)患者依从性高；(iii)对不规则缺损结构依从性高；(iv)生物相容且系统毒性小；(v)关节滞留时间长，药物递送效率高；(vi)苯硼酸酯键的形成有效保护了EA的活性。截至目前，已有多种可注射水凝胶被开发出来用于骨关节炎治疗，但其临床疗效报道不一。
在现有技术中，有采用葡糖胺负载至由泊洛沙姆407和188构成的温敏可注射水凝胶中用于骨关节炎的治疗，但其释药周期不足三个小时；还有采用将负载了与睡眠相关环状RNA3503(cirRNA3505)的PDLLA-PEG-PDLLA三嵌段聚合物水凝胶注入关节腔，展现了优异的骨关节炎治疗效果。但是cirRNA3505的提取与分离过程复杂，价格昂贵，临床可操作性低；还有采用将负载了脂肪衍生干细胞的羊膜水凝胶注入骨关节腔，成功延缓了炎症诱导的软骨退化，但是，由于负载的软骨细胞和干细胞活性半衰期短，因此上述水凝胶的体外保存难度大，临床操作成本高。
因此，本发明旨在提供一种用于骨关节治疗的新型可注射水凝胶及其制备方法，以解决上述提到的相关问题。
发明内容
本发明的目的是提供用于骨关节治疗的新型可注射水凝胶及其制备方法，以开发一种制备简单、性价比高、易于保存、生物相容性好、易于体内降解与代谢且同时兼具自润滑功能和长效抗炎功能的可注射水凝胶。
本发明的上述技术目的是通过以下技术方案得以实现的：用于骨关节治疗的新型可注射水凝胶，包括如下成分：
氧化透明质酸钠(OHA)、4-氨基苯硼酸(4-ABA)、鞣花酸(EA)。
本发明进一步设置为：所述EA与4-ABA的摩尔比为1-2:2-3。
本发明还提供一种用于骨关节治疗的新型可注射水凝胶的制备方法：S1：将一定质量的OHA溶于超纯水中，标记为A液；
S2：称取EA和4-ABA溶于超纯水中，将混合物pH值调至预设值，标记为B液；
S3：将B液加入到A液中，混合均匀即得。
本发明进一步设置为：S2中所述预设值为8.5-9.5。
综上所述，本发明具有以下有益效果：在本发明中，其包含可用于润滑的OHA以及可用于抗炎的EA。并且由于在反应中OHA与4-ABA的氨基形成席呋碱键、EA与4-ABA的硼酸形成硼酸酯键，此两种化学键能够随pH的变化而发生断裂，具有良好的pH响应性。因此在pH发生改变的膝骨关节炎关节腔进行原位注射，能够起到较好的缓释效果。
附图说明
图1为制备得到的OHA/EA水凝胶凝胶化展示图；
图2为OHA/EA水凝胶的可注射性展示图；
图3为A组OHA0.1/EA0.065/4-ABA0.074溶液的储能模量和损耗模量对比图；
图4为B组OHA0.1/EA0.043/4-ABA0.050溶液的储能模量和损耗模量对比图；
图5为C组OHA0.1/EA0.032/4-ABA0.038溶液的储能模量和损耗模量对比图；
图6为D组OHA0.1/EA0.022/4-ABA0.025溶液的储能模量和损耗模量对比图；
图7为E组EA0.032/4-ABA0.038溶液的储能模量和损耗模量对比图；
图8为F组OHA0.1溶液的储能模量和损耗模量对比图；
图9为OHA/EA水凝胶在不同pH环境下的响应机制；
图10为OHA/EA水凝胶治疗SD大鼠骨关节炎后膝关节大体实物图；
图11为OHA/EA水凝胶治疗SD大鼠骨关节炎后大体形态学量化评分结果；
图12为OHA/EA水凝胶治疗SD大鼠骨关节炎后关节组织番红固绿染色切片；图13为OHA/EA水凝胶治疗SD大鼠骨关节炎治疗后关节软骨组织切片曼昆软骨组织量化评分结果；
图14为OHA/EA水凝胶治疗SD大鼠骨关节炎治疗后关节组织苏木精-伊红染色切片；
图15为OHA/EA水凝胶治疗SD大鼠骨关节炎治疗后关节滑膜组织炎症量化评分结果。
具体实施方式
以下结合附图1-15对本发明作进一步详细说明。
实施例1：可注射OHA/EA水凝胶的制备、可注射性的验证以及应用制备方法以及可注射性的验证如下：
(1)首先将透明质酸钠溶于37℃超纯水中，其间不断搅拌直至溶解。溶解后，等待冷却至室温，加入高碘酸钠，搅拌反应12h。随后加入乙二醇对未反应完全的高碘酸钠进行猝灭，反应1h。将溶液转移到3500D的透析袋中透析3d。3d后冷冻干燥即得到氧化透明质酸钠。
(2)称取0.1g氧化透明质酸钠溶于0.5ml超纯水中，标记为A液。同样，称取鞣花酸和4-氨基苯硼酸溶于0.5ml超纯水中，调整pH至8.5-9.5，标记为B液。
(3)取0.5mlB液加入到A液中，几秒钟后倒置，凝胶聚集于离心管底部不随重力影响而流动。简单证明材料成功制备，如图1。
将OHA/EA水凝胶转移到注射器中，推动注射器，样品逐渐从针头流出，如图2，证明该材料具有可注射性。
实施例2：OHA/EA水凝胶的机械性能与润滑性表征
主要采用流变仪所给出的储能模量和损耗模量的对比得出该材料的机械性能和润滑性能。
因此，我们设置5组不同交联程度的样品分别为：(A)OHA/EA0.065/4-ABA0.074、(B)OHA/EA0.043/4-ABA0.050、(C)OHA/EA0.032/4-ABA0.038、(D)OHA/EA0.022/4-ABA0.025、(E)EA0.032/4-ABA0.038和(F)OHA，其中OHA的含量始终保持一致。
理论上，水凝胶的交联密度越强，结构稳定性越高，则力学性能越强。
正如图3所示，A组的交联密度是最强的，随着频率增加，其储能模量(G')和损耗模量(G')分别增加到19700和2108Pa，为凝胶状样品。
C组样品由于交联程度较低，结构稳定性相对较差，所以力学性能也相对较差，当频率小于14.7时，其储能模量(G')低于损耗模量(G')，呈粘弹性样品，达到可注射的要求(如图5)，符合我们预期的理论设计；当频率大于14.7时，其储能模量(G')高于损耗模量(G')，呈凝胶样品。
与此同时，我们还对EA0.032/4-ABA0.038和OHA0.1两种溶液也进行机械性能的测定，可以发现储能模量(G')和损耗模量(G')波动起伏较大，呈水样(图7和图8)。通过6组样品之间的比较，可以证明OHA、EA和4-ABA之间能够形成交联结构，并且随着质量的变化其机械性能也会发生相应的变化。
实施例3：OHA/EA水凝胶在不同pH环境下的响应机制表征
将OHA/EA水凝胶转移到500D的透析袋中，内部加入3ml不同pH(7.3和10.5)的PBS溶液，外部同样加入30ml不同pH的PBS溶液。每5min、10min、15min、20min、30min、45min、60min、90min、120min、3h、4h、5h、6h、7h、8h、9h、10h、11h、12h、24h随后1d取一次溶液，测定药物在不同pH下的响应机制(图9)。可以发现两者释放速率呈现明显区别，当pH＝7.3时，药物释放到第13天时，已经基本没有药物释放，释放曲线趋于平缓。而当pH＝10.5时，材料在21天内都能有效释放药物，达到一个长周期的缓释、治疗效果。
实施例4：OHA/EA水凝胶对SD大鼠骨关节炎治疗效果
本发明的体内动物实验是将新型可注射水凝胶(OHA/EA)用于大鼠膝骨关节炎(KOA)的治疗。具体实验方案如下：
将18只SD大鼠随机分为实验组、对照组、假手术组，每组6只；对照组和实验组予以切断前交叉韧带(ACL)来制备大鼠KOA模型；假手术组切开关节囊不切断ACL。
(1)术后4周，实验组大鼠KOA关节内注射OHA/EA，而对照组和假手术组则注射医用生理盐水。各组大鼠每10天注射一次，一次注射100微升。治疗40天后处死大鼠，取大鼠膝关节。
(2)治疗40天后处死大鼠，由3名专业人员按国际骨关节炎研究学会(OsteoarthritisResearchSocietyInternational，OARSI)大鼠膝关节大体形态学评分指南，盲法进行形态学评分。观察膝关节软骨表面光滑程度、颜色、软骨磨损程度及软骨下骨露出程度,选三者的平均值进行统计分析。
(3)将大鼠KOA关节标本进行脱钙、包埋后制成石蜡切片然后通过番红-固绿染色、苏木精-伊红(HE)染色。分别通过曼昆软骨组织形态学评分(Mankin’sscore)系统和OARSI膝关节软组织炎症评分系统量化评价不同组之间关节软骨损伤程度和滑膜内炎症反应程度。
治疗40天后，经解剖各组大鼠膝关节所见(图10)，假手术组较少有关节承重区软骨侵蚀，无软骨下骨暴露。对照组的关节软骨出现大面积肿胀变性，部分软骨缺损，软骨下骨暴露。与对照组相比，实验组大鼠膝关节表面软骨损伤较轻，虽然关节表面微黄变色，但是无大面积的承重区软骨变性，更无大面积的软骨下骨暴露。随后依据国际骨关节炎研究学会(OARSI)大体评分标准，由三名专业人员对各组膝关节情况盲法进行评分，每只标本最终评分取平均值进行统计分析，统计方法选择MannWhitney检验。统计结果显示，实验组大体评分总体上低于对照组，且两组间存显著差异，说明给予OHA/EA水凝胶治疗后，实验组膝关节大体解剖结构情况优于对照组(图11)。
治疗40天后，各组大鼠膝关节番红-固绿染色切片显示(图12)，假手术组的关节软骨结构正常，软骨细胞密度均匀，形态正常，潮线单一且完整。而实验组则出现承重区胫骨关节表面软骨部分区域全层缺损，突破钙化层，代之以增生的紊乱纤维组织。软骨细胞变形、缩小、排列紊乱，潮线周围有血管增生。而实验组与对照组相比，软骨缺损区域直径较小，软骨缺损未突破钙化层，软骨细胞形态也较对照组更好，潮线尚且完整。随后依据曼昆关节软骨组织评分(Mankin’ssocre)标准，由三名专业人员盲法对切片标本进行评分，每只标本取平均值进行统计分析，统计方法选择MannWhitney检验。统计结果显示，实验组曼昆评分总体上低于对照组，且两组间存在显著差异，说明给予OHA/EA水凝胶治疗后，实验组膝关节软骨损伤情况好于对照组(图13)。
治疗40天后，各组大鼠膝关节滑膜组织苏木精-伊红染色切片显示(图14)，假手术组滑膜可见轻度增生或未见明显增生，滑膜下组织未见明显异常。对照组滑膜细胞增生严重，滑膜紊乱无法分辨确切的关节腔间隙，滑膜下组织亦增生严重，疏松结缔组织被纤维组织所取代，甚至有炎症细胞浸润。而实验组滑膜细胞则只增加了1-2层，滑膜下组织未见明显的增生，较少有炎症细胞浸润。随后依据国际骨关节炎研究学会(OARSI)膝关节滑膜组织评分标准，由三名专业人士对大鼠膝关节滑膜组织进行打分后，每只标本取平均值进行统计分析，统计方法选择MannWhitney检验。统计结果显示，实验组OARSI滑膜炎症评分总体上低于对照组，且两组间存在显著差异，说明给予OHA/EA水凝胶治疗后，实验组膝关节滑膜炎症情况好于对照组(图15)。提示OHA/EA水凝胶对大鼠膝骨关节炎(KOA)治疗有效。
本具体实施例仅仅是对本发明的解释，其并不是对本发明的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本发明的权利要求范围内都受到专利法的保护。
Claims (4)
1.一种用于骨关节治疗的新型可注射水凝胶，其特征是：包括如下成分：
氧化透明质酸钠(OHA)、4-氨基苯硼酸(4-ABA)、鞣花酸(EA)。
2.根据权利要求1所述的用于骨关节治疗的新型可注射水凝胶，其特征是：所述EA与4-ABA的摩尔比为1-2:2-3。
3.一种用于权利要求1所述的用于骨关节治疗的新型可注射水凝胶的制备方法，其特征是：
S1：将一定质量的OHA溶于超纯水中，标记为A液；
S2：称取EA和4-ABA溶于超纯水中，将混合物pH值调至预设值，标记为B液；
S3：将B液加入到A液中，混合均匀即得。
4.根据权利要求3所述的用于骨关节治疗的新型可注射水凝胶的制备方法，其特征是：S2中所述预设值为8.5-9.5。
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