CN116392642A - 用于心衰治疗的细胞外基质/糖肽水凝胶及其制备方法 - Google Patents
用于心衰治疗的细胞外基质/糖肽水凝胶及其制备方法 Download PDFInfo
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- CN116392642A CN116392642A CN202310477348.9A CN202310477348A CN116392642A CN 116392642 A CN116392642 A CN 116392642A CN 202310477348 A CN202310477348 A CN 202310477348A CN 116392642 A CN116392642 A CN 116392642A
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Abstract
本发明公开了一种用于心衰治疗的细胞外基质/糖肽水凝胶及其制备方法，水凝胶具体成分包括心肌细胞外基质，多糖以及功能性多肽。本发明所得水凝胶具有仿细胞外基质的组分及结构，可长期替代受损组织的细胞外基质，提供细胞生在、浸润和增殖的微环境；本发明的细胞外基质/糖肽水凝胶具有良好可注射性，可直接注射到心肌内，具有良好的力学支撑特性，并且具有调控免疫炎症反应、促进血管新生的生物活性，调节心脏重构，缓解心衰进程。
Description
技术领域
本发明涉及生物医用材料和医疗器械研发技术领域，更具体的说是涉及一种用于心衰治疗的细胞外基质/糖肽水凝胶及其制备方法。
背景技术
心力衰竭简称心衰，是包括冠状动脉疾病(如心肌梗死)、高血压、心肌炎及瓣膜性心脏病等多种心血管疾病发展的终末状态，也是世界范围内的主要健康问题，造成了严重的社会和经济负担。对于终末期心衰患者，主要通过左心室辅助装置和心脏移植延长生存时间，但均存在费用昂贵、手术复杂和心脏移植供体来源稀缺等问题。多数终末期心衰患者无法得到有效治疗，亟需探索新的治疗方式。
近年来，随着材料科学和组织工程学的发展，新型生物材料被广泛应用于心血管治疗领域，其中具有代表性的生物材料为细胞外基质和多糖类的水凝胶。细胞外基质水凝胶保留了心脏细胞外基质原有的胶原、弹性蛋白、糖蛋白、粘多糖等活性成分和多孔的纤维网状结构，可以替代心梗后异常降解的细胞外基质，为细胞的黏附、分化提供三维骨架。然而，细胞外基质水凝胶力学稳定性和强度较低，无法为心梗后变薄的室壁提供足够的力学支持强度。同时，细胞外基质水凝胶仍容易被心肌损伤后炎症细胞持续分泌的金属蛋白酶迅速降解。在前期的临床试验中，细胞外基质水凝胶未能展现有效的心室重塑功效。多糖类水凝胶如海藻酸钠、透明质酸水凝胶，具有良好的力学稳定性、可注射性和生物相容性，心肌内注射后可以降低心肌壁张力。然而多糖基水凝胶结构成分较为单一，且缺乏心脏组织中丰富特殊的活性成分，无法充分调控复杂的细胞分化过程，在临床试验中也未能提高患者心脏功能和生存率。
因此，目前所用水凝胶未能满足心衰的治疗需求，在临床试验中结果仍不理想。基于此，开发一种新型的多功能水凝胶，可以满足重塑心脏细胞外基质、提供力学支撑、调控炎症反应、促进血管新生，提高心衰患者的心脏功能和生存率，具有十分重要的临床意义。
发明内容
本发明针对现有技术的上述不足，提供一种用于心衰治疗的细胞外基质/糖肽水凝胶的制备方法，所得水凝胶不仅具有仿细胞外基质的成分与结构，以长期替代受损组织的细胞外基质，降低室壁应力并提供有利的微环境；还具有调控炎症反应、促进血管新生的生物活性，从而抑制心衰的进展。
为实现上述目的，本发明的目的可通过如下技术方案实现：
一种用于心衰治疗的细胞外基质/糖肽水凝胶，其主要成分为心脏脱细胞基质、多糖及功能性多肽，其中心心脏脱细胞基质占体系总固含量的质量分数为10～50％；多肽占体系总固含量的质量分数为0.5～20％；多糖占体系总固含量的质量分数为10～90％。
优选地，心脏脱细胞基质的来源包括猪、牛、羊、鼠、兔或移植器官。
优选地，多糖为海藻酸、壳聚糖、羟甲基纤维素、葡聚糖、透明质酸、硫酸软骨素和肝素中的一种或多种。
优选的，多肽为Q11多肽(QQKFQFQFEQQ)RADA多肽(RADARADARADARADA)KK多肽(KKSLSLSLSLSLSLKK)KEF9多肽(KKFKFEFEF)中的一种或多种。
本发明的另一个目的在于提供上述的用于心衰治疗的细胞外基质/糖肽水凝胶的制备方法，包括以下步骤：
(1)制备细胞外基质水凝胶：将新鲜的心脏组织剪碎，在浓度为0.1-3wt％的十二烷基硫酸钠(SDS)中以20-100r/min速度搅拌5-10天，直到心脏组织呈白色透明状，完成脱细胞过程，将脱细胞的组织在去离子水中继续搅拌2-3天彻底去除残留的SDS，冻干并研磨成粉末，将细胞外基质粉末溶于0.1-1M氯化氢溶液(氯化氢溶液pH为1-2)中并用胃蛋白酶进行消化12-72小时，使用1-10M氢氧化钠溶液滴定到细胞外基质溶液pH值为7-8终止消化，静置形成细胞外基质水凝胶；
(2)制备马莱酰亚胺-多糖：将浓度为1-10wt％多糖糖溶解在二甲基亚砜中，加入马莱酰亚胺丁酸或马莱酰亚胺丙酸，在1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)和4-二甲氨基吡啶(DMAP)催化下反应48-72h，通过透析、冻干得到马莱酰亚胺-葡甘聚糖粉末。
(3)制备细胞外基质/糖肽水凝胶：在无菌条件下，将多肽溶液，细胞外基质水凝胶溶液与马莱酰亚胺-多糖溶液混合，搅拌均匀后，通过马克尔加成反应交联，静置5-60min后形成糖肽水凝胶。
优选地，步骤(1)中粉末状的细胞外基质与胃蛋白酶的质量比为5-20：1，以获得分子量适宜的细胞外基质降解产物。
优选地，步骤(1)中细胞外基质粉末溶解的浓度为10-100mg/mL，以获得粘度适中的凝胶前体溶液。
优选地，步骤(2)中多糖与马莱酰亚胺丁酸或马莱酰亚胺丙酸的摩尔比为1：1-100，与EDC的摩尔比为1：1-10；与DMAP的摩尔比为1：1-10。
优选地，水凝胶最终固含量为5～50％，以满足水凝胶用于心衰治疗的可注射性与力学强度。
经由上述的技术方案可知，与现有技术相比，本发明具有如下有益效果：
本发明通过细胞外基质复合的糖肽水凝胶，可以保留心脏细胞外基质的三维网状结构和活性成分，同时通过化学交联引入糖肽成分，增强水凝胶的力学稳定性，赋予免疫调控、促血管新生的特殊生物功能，多方面调控心梗后心肌重塑过程。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为脱细胞的细胞外基质(dECM)制备流程
图2为葡甘聚糖(GM)和接有马莱酰亚胺基团的葡甘聚糖(MAL-GM)的1H-NMR图
图3为细胞外基质/糖肽水凝胶(dECM/GP)与细胞外基质水凝胶(dECM)和糖肽水凝胶(GP)的流变学性质
图4为脱细胞外基质与细胞外基质/糖肽水凝胶的电镜扫描微观结构
图5为细胞外基质/糖肽水凝胶(dECM/GP)、细胞外基质水凝胶(dECM)和糖肽水凝胶(GP)治疗急性心梗大鼠模型的Masson染色结果
图6为治疗28天后，梗死区域心肌组织的CD68/CD206免疫荧光染色结果
图7为治疗28天后，梗死区域心肌组织的α-SMA/cTNT免疫荧光染色结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
选取新鲜猪心左室游离壁组织切为2mm3的小块，放入1wt％SDS溶液中快速搅拌5天，期间每隔一天更换SDS溶液，直至组织变为透明状，脱细胞后的组织放入去离子水中搅拌2天，期间连续更换去离子水，去除残留SDS。将组织冻干48h，用冷冻研磨机研磨成粉末，将细胞外基质粉末按50mg/mL溶解于0.1M氯化氢溶液，按10：1质量比加入猪胃蛋白酶，消化24h后用1M氢氧化钠溶液滴定，使溶液pH值为7.5，静置形成细胞外基质水凝胶。
将2g的透明质酸(20kDa)溶解在100mL去离子水中，加入2.2g马莱酰亚胺丁酸，7gEDC和0.5gDMAP，在室温下反应48h，所得产物透析5天，冻干48h后得到马莱酰亚胺修饰的透明质酸，并以8wt％的浓度溶解形成多糖溶液。
将促血管新生多肽KK以4wt％的浓度配置溶液。将细胞外基质水凝胶(5wt％)、多糖溶液(8wt％)、多肽溶液按2：1：1的体积比混合，充分斡旋后静置，通过迈克尔加成反应交联，形成细胞外基质/糖肽水凝胶。
实施例2
选取新鲜猪心左室游离壁组织切为22mm3的小块，放入1wt％SDS溶液中快速搅拌5天，每隔一天更换SDS溶液，直至组织变为透明状。脱细胞后的组织放入去离子水中搅拌2天，期间连续更换去离子水，去除残留SDS。将组织冻干48h，用冷冻研磨机研磨成粉末。将细胞外基质粉末按20mg/mL溶解于0.1M氯化氢溶液，按10：1质量比加入猪胃蛋白酶，消化24h后用1M氢氧化钠溶液滴定，使溶液pH值为7.5，静置形成细胞外基质水凝胶。图1所示为细胞外基质水凝胶制备过程。
将2g的葡苷聚糖(100kDa)溶解在100mL去离子水中，加入2.2g马莱酰亚胺丁酸，7gEDC和0.5gDMAP，在室温下反应48h，所得产物透析5天，冻干48h，以10％的浓度溶解形成多糖水凝胶。图2所示为马莱酰亚胺修饰葡苷聚糖的1H-NMR，证明成功合成接有马莱酰亚胺基团的葡苷聚糖。
将促血管新生多肽RADA以2wt％的浓度配置溶液。将细胞外基质水凝胶(2wt％)、多糖(10wt％)、多肽(2wt％)按1：1：1的体积比混合，充分斡旋后静置，通过迈克尔加成反应交联，形成细胞外基质/糖肽水凝胶。
实施例3
对实施例2中所制备的细胞外基质/糖肽水凝胶、细胞外基质水凝胶、糖肽水凝胶进行流变测试。所用糖肽水凝胶为实施例1中多糖溶液和多肽溶液按体积比1：1混合的产物。
图3测试结果表明细胞外基质/糖肽水凝胶比单独的细胞外基质水凝胶、糖肽水凝胶有更高的储能模量(G′)和损耗模量(G″)，证明其更优的力学特性。
图4显示猪脱细胞外基质及实施例1所得细胞外基质/糖肽水凝胶的三维微观结构图，从图中可以看出，所制备的细胞外基质/糖肽水凝胶具有与脱细胞外基质类似的三维多孔的纳米纤维网络结构，从而有望替代受损组织的细胞外基质，提供细胞生长、增殖和浸润的有利微环境。
实施例4
考察细胞外基质/糖肽水凝胶对心衰的治疗效果，将实施例3中所制备的细胞外基质/糖肽水凝胶、细胞外基质水凝胶、糖肽水凝胶进行动物实验。取6周龄体重180-200g之间的Sprague Dawley(SD)大鼠制备急性心肌梗死模型。麻醉后气管插管，调整呼吸机参数。将大鼠固定在手术板上，沿胸骨旁左侧2mm做一长约2cm的纵行切口，依次剪开皮肤、筋膜，使用血管钳顿性分离胸大肌和前锯肌，于第3、第4肋间平行于肋骨剪开约1cm的横行切口，用牵开器撑开肋间，暴露心脏。使用镊子撕开心包，充分显露心脏结构，于动脉圆锥与左心耳连线中点下方2-3mm处使用6-0缝合线结扎冠状动脉左前降支，结扎后可观察到左室前壁心肌颜色变为苍白，室壁活动减弱，即为结扎成功。结扎缺血30min后，在梗死边缘区取4个均匀间隔的注射点，每个点注射25μl细胞外基质/糖肽水凝胶、细胞外基质水凝胶、糖肽水凝胶或PBS溶液，共计100μl。注射后使用无菌纱布充分止血。使用3-0缝合线间断缝合逐层关闭胸腔，并行胸腔排气，避免造成气胸。对合前锯肌、胸大肌，缝合皮肤。待大鼠恢复自主呼吸后拔出气管插管。腹腔注射青霉素20万U/只，预防感染。
图5为术后7天和术后28天心脏切片Masson染色结果。结果显示，与其他组相比，细胞外基质/糖肽水凝胶可以显著减少心梗面积，损伤部位壁厚更厚。此外，28天的Masson染色结果也表明，其他治疗组均不同程度的观察到广泛和密集的胶原沉积，表明心力衰竭的病理性变化，而细胞外基质/糖肽水凝胶组有效抑制了这种不良的心室重塑过程。
实施例5
通过免疫荧光染色，考察细胞外基质/糖肽水凝胶调节心脏重塑愈合的基质。对于巨噬细胞极化研究，将切片与兔抗CD68(1:200)、小鼠抗CD206(1:200)、小鼠抗α-SMA(1:200)和兔抗心脏肌钙蛋白T(1:200)分别孵育。孵育过夜后，将切片与对应于一抗的AlexaFlour488驴抗小鼠IgG(1:200)和AlexaFlour594驴抗兔IgG(1:200)孵育2h。PBS清洗后，用含DAPI封片剂封片，通过共聚焦显微镜观察并分析。
图6中结果显示，细胞外基质/糖肽水凝胶组的心肌组织炎症细胞浸润更少，具有较多的M2型巨噬细胞分布，说明细胞外基质/糖肽水凝胶可以有效促进M2型巨噬细胞的极化并减轻心肌损伤后的炎症反应。图7中结果显示，细胞外基质/糖肽水凝胶组的心肌组织具有较多的α-SMA+血管新生以及更多的心肌组织出现在新生血管周围，证明细胞外基质/糖肽水凝胶可以促进血管新生和心肌细胞存活，从而抑制心衰的进展。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。对于实施例公开的装置而言，由于其与实施例公开的方法相对应，所以描述的比较简单，相关之处参见方法部分说明即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (10)
1.一种用于心衰治疗的细胞外基质/糖肽水凝胶，其特征在于：所述水凝胶包括心脏脱细胞基质、多糖和功能性多肽；
其中，所述心脏脱细胞基质占体系总固含量的质量分数为10～50％；所述功能性多肽占体系总固含量的质量分数为0.5～20％；所述多糖占体系总固含量的质量分数为10～90％。
2.根据权力要求1所述的一种用于心衰治疗的细胞外基质/糖肽水凝胶，其特征在于：所述多糖为海藻酸、壳聚糖、羟甲基纤维素、葡聚糖、透明质酸、硫酸软骨素和肝素中的一种或多种。
3.根据权利要求1所述的一种用于心衰治疗的细胞外基质/糖肽水凝胶，其特征在于，所述多肽为Q11多肽、RADA多肽、KK多肽和KEF9多肽中的一种或多种。
4.根据权利要求1所述的一种用于心衰治疗的细胞外基质/糖肽水凝胶，其特征在于，所述水凝胶的固含量为为5～50％。
5.一种如权利要求1-4任一项所述的用于心衰治疗的细胞外基质/糖肽水凝胶的制备方法，其特征在于：包括以下步骤：
(1)制备细胞外基质水凝胶：将心脏组织剪碎，加入到十二烷基硫酸钠中搅拌，直至心脏组织呈白色透明状，完成脱细胞过程，将脱细胞的组织在水中继续搅拌去除残留的十二烷基硫酸钠，然后冻干并研磨成粉末，将细胞外基质粉末溶于pH为1-2的氯化氢溶液中并用胃蛋白酶进行消化12-72h，然后使用氢氧化钠溶液滴定到细胞外基质溶液pH值为7-8终止消化，静置形成细胞外基质水凝胶；
(2)制备马莱酰亚胺-多糖：将多糖糖溶解在二甲基亚砜中，加入马莱酰亚胺丁酸或马莱酰亚胺丙酸，在1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和4-二甲氨基吡啶催化下反应48-72h，通过透析、冻干得到马莱酰亚胺-葡甘聚糖粉末；
(3)制备细胞外基质/糖肽水凝胶：在无菌条件下，将多肽溶液，细胞外基质水凝胶溶液与马莱酰亚胺-多糖溶液混合，搅拌均匀后，通过马克尔加成反应交联，静置5-60min后形成糖肽水凝胶。
6.根据权利要求5所述的一种用于心衰治疗的细胞外基质/糖肽水凝胶的制备方法，其特征在于，步骤(1)中所述十二烷基硫酸钠的质量浓度为0.1-3wt％；所述氯化氢溶液的浓度为0.1-1M；所述氢氧化钠溶液的浓度为1-10M；所述搅拌速度为20-100r/min。
7.根据权利要求5所述的一种用于心衰治疗的细胞外基质/糖肽水凝胶的制备方法，其特征在于，步骤(1)中所述粉末状的细胞外基质与胃蛋白酶的质量比为5-20：1。
8.根据权利要求5所述的一种用于心衰治疗的细胞外基质/糖肽水凝胶的制备方法，其特征在于，步骤(1)中所述细胞外基质粉末溶解的浓度为10-100mg/mL。
9.根据权利要求5所述的一种用于心衰治疗的细胞外基质/糖肽水凝胶的制备方法，其特征在于，步骤(2)中所述多糖溶液的质量浓度为1-10wt％。
10.根据权利要求4所述的一种用于心衰治疗的细胞外基质/糖肽水凝胶的制备方法，其特征在于，步骤(2)中所述多糖与马莱酰亚胺丁酸或马莱酰亚胺丙酸的摩尔比为1：1-100；所述多糖与1-乙基-(3-二甲基氨基丙基)碳酰二亚胺的摩尔比为1：1-10；所述多糖与4-二甲氨基吡啶的摩尔比为1：1-10。
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