CN116942889B - 一种止血防粘连多肽水凝胶的制备方法 - Google Patents
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Abstract
本发明提供了一种止血防粘连多肽水凝胶的制备方法,本发明多肽自组装水凝胶的凝血率高达99.11±0.66%,具备优良的止血性能。同时,采用D型氨基酸替换并不会引起止血性能的显著下降。本发明多肽自组装水凝胶也具备优良的防粘连功效,可适用于术后伤口的治疗。本发明采取的制备方法,体系简单、反应迅速、不需要额外添加辅助成胶因子。本发明制备得到的自组装水凝胶具备良好的止血防粘连活性,具备较大的临床应用前景。本发明采取的制备方法,体系简单、反应迅速、不需要额外添加辅助成胶因子,具备较大的临床应用前景。
Description
技术领域
本发明涉及生物医药领域,特别涉及一种止血防粘连多肽水凝胶的制备方法。
背景技术
传统的止血材料,如脱脂棉、纱布等经过灭菌后在创伤处进行压迫性止血,以使用历史长久、应用广泛、价格低廉以及使用方便等优点为人们所熟悉。但是,传统型止血材料只是对创面起简单的物理保护作用,在应对动脉大量出血的紧急情况时并不能起到及时控制出血量的作用,而且,脱脂棉以及纱布容易粘连创面,后期处理相对比较繁琐,同时会引起伤者的疼痛感。水凝胶是指水溶性聚合物分子间通过离子键、共价键、氢键等物理或化学方式交联形成三维网状结构的体系,具有强度可调、形状可塑以及可设计功能化的优点。根据不同应用水凝胶可以被制备成多种形式,包括平板、微粒、纳米颗粒、涂层和薄膜等。基于以上特性,水凝胶在食品、组织工程和医用敷料等诸多领域发挥关键作用。水凝胶敷料相比于传统敷料具有更优异的生物功能性,如与创面结合良好、保湿透气、方便更换、可吸收渗液以及可负载各类药物等诸多优点。有研究表明,湿性愈合己经被确立为各类迁移不愈的创面的理想治疗方式,保持创面的湿润具有重要意义。在水凝胶敷料提供的水润环境中受损部位的新生上皮成型速度加快、患处疼痛感减弱、愈合周期缩短、创面修复效果显著提高。临床上己经有了产品化的水凝胶敷料用于糖尿病足创面、老年人褥疮以及烧伤烫伤等难愈合创面的治疗。
但是,现阶段,制备止血防粘连多肽水凝胶仍是目前行业研发的重点方向,如何通过肽分子的改造和设计开发出高效率止血和防粘连的水凝胶材料也是一个难题。
发明内容
针对现有技术所存在的技术问题,本发明提供了一种止血防粘连多肽水凝胶的制备方法。本发明多肽自组装水凝胶具备优良的止血性能、防粘连功效,可适用于术后伤口的治疗。并且,本发明采取的制备方法,体系简单、反应迅速、不需要额外添加辅助成胶因子。本发明制备得到的自组装水凝胶具备良好的止血防粘连活性,具备较大的临床应用前景。
本发明的目的之一在于提供一种止血防粘连多肽水凝胶,其特征在于,所述多肽自组装水凝胶的氨基酸选自LHis-LHis-LHis-LHis-LHis-LHis-LHis-LHis、DHis-DHis-DHis-DHis-DHis-DHis-DHis-DHis、LArg-LHis-LHis-LHis-LHis-LHis-LHis-LHis、LHis-LHis-LHis-LArg-LHis-LHis-LHis-LHis、LHis-LHis-LHis-LHis-LHis-LHis-LHis-LArg、LArg-LHis-LHis-LArg-LHis-LHis-LHis-LHis、LArg-LHis-LHis-LArg-LHis-LHis-LHis-LArg一种或多种。
优选地,所述止血防粘连多肽水凝胶的氨基酸是选自LHis-LHis-LHis-LHis-LHis-LHis-LHis-LHis或DHis-DHis-DHis-DHis-DHis-DHis-DHis-DHis。
本发明的另一目的在于提供了一种止血防粘连多肽水凝胶的制备方法,其特征在于,所述方法包括如下步骤:
1)称取多肽粉末,并将其加入盐溶液中,同时调整体系pH至溶液澄清;
2)将步骤1)的反应体系静置,即可得到多肽自组装水凝胶。
优选地,步骤1)中的盐溶液是选自钙盐;
进一步优选地,所述钙盐选自氯化钙、碳酸钙、磷酸氢二钙、醋酸钙、柠檬酸钙、乳酸钙、葡萄糖酸钙的水溶液;
优选地,步骤1)中多肽的使用剂量为1-10mg/mL;
优选地,步骤1)中盐溶液的浓度为2-10mM;
优选地,所述合成方法的温度控制在25-37℃之间;
进一步地,本发明提供的一种止血防粘连多肽水凝胶的制备方法的详细合成步骤如下所示:
1)称量多肽粉末10mg于5mL Ep管中,向Ep管中加入2mL 5mM CaCl2水溶液,并逐级滴加0.1M氢氧化钠至Ep管溶液澄清;
2)滴加过程同时进行涡旋混匀处理,经2min静置即可得到多肽自组装水凝胶。
本发明的另一目的在于提供了一种止血防粘连多肽水凝胶在制备止血防粘连药物中的用途;
优选地,所述止血防粘连药物还包含至少一种药学上可接受的药用载体和/或辅料。
进一步优选地,本发明的所述止血防粘连药物为片剂、胶囊、糖衣片剂、粒剂、滴剂、喷雾剂、冲洗剂、漱口剂、用于皮肤表面的油膏和药贴、以及用于注射的无菌溶液中的至少一种剂型。
进一步优选地,本发明的水凝胶可以直接对创面进行冲洗、喷涂、湿敷或覆盖,制作成方便使用的喷雾剂,直接喷敷于创面形成保护膜,能瞬间止血、保持创面湿润,创造利于上皮细胞的生长和愈合的低氧环境,加速伤口愈合。
本发明的优点如下:本发明多肽自组装水凝胶的凝血率高达99.11±0.66%,具备优良的止血性能。同时,采用D型氨基酸替换并不会引起止血性能的显著下降。此外,本发明多肽自组装水凝胶也具备优良的防粘连功效,可适用于术后伤口的治疗。并且,本发明采取的制备方法,体系简单、反应迅速、不需要额外添加辅助成胶因子。本发明制备得到的自组装水凝胶具备良好的止血防粘连活性,具备较大的临床应用前景。
附图说明
图1.多肽自组装水凝胶的止血效果评价。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明,以使本领域的技术人员更加清楚地理解本发明。
以下各实施例,仅用于说明本发明,并不用来限制本发明的范围。基于本发明中的具体实施例,本领域普通技术人员在没有做出创造性劳动的情况下,所获得的其他所有实施例,都属于本发明的保护范围。
在本发明实施例中,若无特殊说明,所有原料组分均为本领域技术人员熟知的市售产品;在本发明实施例中,若未具体指明,所用的技术手段均为本领域技术人员所熟知的常规手段。
实施例1多肽自组装水凝胶的制备
一种多肽自组装水凝胶的制备方法,其包括如下步骤:称量LHis-LHis-LHis-LHis-LHis-LHis-LHis-LHis粉末10mg于5mL Ep管中,向Ep管中加入2mL 5mM CaCl2水溶液,并逐级滴加0.1M氢氧化钠至Ep管溶液澄清,滴加过程同时进行涡旋混匀处理,经2min静置即可得到多肽自组装水凝胶。
并通过控制变量法,对上述多肽自组装水凝胶制备方法中的多肽使用浓度(1mg/mL、5mg/mL、10mg/mL)、温度(25℃、30℃、37℃)、CaCl2水溶液浓度(2mM、5mM、10mM)进行优化分析其对水凝胶形成的影响。
结果显示:本发明先固定反应温度为37℃和CaCl2水溶液浓度为5mM,伴随着多肽使用浓度的增加,液体的粘度也逐渐加大,当其浓度达到5mg/mL时,足以在该体系下快速形成水凝胶,考虑到水凝胶的实际应用,本发明选择了5mg/mL的多肽。紧接着,本发明在固定多肽浓度为5mg/mL和CaCl2水溶液浓度为5mM时,分析了不同温度下的水凝胶自组装情况。结果显示在25℃环境下,该多肽体系呈现溶液状态,无法形成水凝胶;随着温度升至30℃时,该多肽体系需经过液处理才能由溶液状态转变为水凝胶状态,不利于水凝胶的试剂应用;当升温至37℃时,该多肽体系只需2min静置即可形成水凝胶。最后在固定多肽浓度为5mg/mL和反应温度为37℃下,对CaCl2水溶液浓度为5mM进行优化调整,结果显示,2mM CaCl2水溶液下,多肽需要5h才能自组装形成水凝胶;当CaCl2水溶液浓度提高至5mM下,该多肽体系经2min静置即可形成水凝胶;当CaCl2水溶液浓度提高至10mM下,该多肽体系形成水凝胶的时间并未明显缩短。基于此,综合考虑上述因素,本发明采用的多肽自组装水凝胶体系中多肽的使用浓度为5mg/mL,反应温度为37℃,CaCl2水溶液浓度为5mM。
实施例2多肽自组装水凝胶止血效果评价
为进一步评价多肽自组装水凝胶的止血效果,发明人还合成了D型氨基酸替换和不同的精氨酸替换的多肽自组装水凝胶作为对比例,具体的氨基酸序列如下所述:
对比例1:DHis-DHis-DHis-DHis-DHis-DHis-DHis-DHis;
对比例2:LArg-LHis-LHis-LHis-LHis-LHis-LHis-LHis;
对比例3:LHis-LHis-LHis-LArg-LHis-LHis-LHis-LHis;
对比例4:LHis-LHis-LHis-LHis-LHis-LHis-LHis-LArg;
对比例5:LArg-LHis-LHis-LArg-LHis-LHis-LHis-LHis;
对比例6:LArg-LHis-LHis-LArg-LHis-LHis-LHis-LArg。
本发明进一步通过凝血率(BCR)测定来评价本发明所述水凝胶的止血活性。首先0.5mL新鲜小鼠血液滴加入上述1mL不同组的多肽自组装水凝胶材料中直至血液被完全吸收,将其放置于恒温培养箱中静置5min后,将5mL纯水缓慢沿着管壁加入,在加入过程中避免剧烈晃动,在恒温培养箱中放置20min后,未被多肽自组装水凝胶材料吸收的血红细胞会被溶解在纯水中,将溶液部分在500rpm/min的条件下离心5min。取上清液利用紫外分光光度计检测540nm处的吸收值。并采用0.5mL血液溶解于5mL纯水作为阳性对照,根据如下公式计算凝血率BCR。
结果如图1所示:本研究中我们利用凝血率来评价不同多肽自组装水凝胶材料的止血性能。本发明实施例1的凝血率达到了99.11±0.66%,对比例1的凝血率达到了95.82±1.17%。可见,采用D型氨基酸替换会引起该多肽水凝胶的止血性能稍有下降,但整体仍保持较高的止血性能。相反,当采用精氨酸进行部分取代时,多肽水凝胶的止血性能下降比较显著,尤其是对比例6为主,其凝血率仅剩69.25±0.93%。因此,为保持较好的止血性能,尽量选择实施例1和对比例1中公开的多肽水凝胶材料。
实施例3多肽自组装水凝胶防粘连效果评价
本发明进一步构建大鼠盲肠磨损-腹壁缺损模型来评价多肽自组装水凝胶材料的防粘连效果。具体是,将80只雄性SD大鼠随机分为8组,分别对照组、实施例1和对比例1-6,每组10只。首先,麻醉大鼠,剃除腹部毛发,在腹部切口暴露盲肠,用无菌纱布摩擦盲肠100次,使盲肠表面出现点状出血后放回腹腔。然后,选取盲肠所在腹腔位置的一侧腹壁,用手术刀刮擦腹壁表面形成腹膜损伤。然后用缝合线将盲肠与对应腹壁固定,使两处的创面完全贴合,在创面上分别使用本发明实施例和对比例的多肽自组装水凝胶材料,对照组不做任何处理。经术后7天,观察大鼠腹腔粘连程度并依据标准粘连评分系统对粘连程度进行分级评价。标准粘连评分系统:无粘连,0分;出现1处薄膜状粘连,1分;出现多于1处薄粘连,2分;出现厚点粘连,3分;出现厚跖连或多于1处厚点粘连,4分;出现非常厚的血管化或多于1处跖连,5分。
统计结果如下表1所示:在第一次手术7天后,对照组大鼠均出现了十分严重的粘连现象,平均粘连分数高达4.9分。实施例1和对比例1组大鼠基本没有出现粘连,其平均粘连分数相对较低,评分仅有0.1/0.2,进一步证实采用D型氨基酸替换后对其粘连效果影响不大。然而,采用精氨酸取代后,对比例1-6的粘连程度呈现严重的恶化现象,平均粘连评分也呈现加大趋势。尤其以,对比例6的效果最差,10只大鼠中有5只出现5分黏连,其余5只大鼠出现4分黏连,平均黏连分数高达4.5分。
表1防粘连效果评价
在此有必要指出的是,以上实施例仅限于对本发明的技术方案做进一步的阐述和说明,并不是对本发明的技术方案的进一步的限制,本发明的方法仅为较佳的实施方案,并非用于限定本发明的保护范围。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种止血防粘连多肽水凝胶,其特征在于,所述多肽自组装水凝胶的氨基酸选自LHis-LHis-LHis-LHis-LHis-LHis-LHis-LHis、DHis-DHis-DHis-DHis-DHis-DHis-DHis-DHis一种或多种;
其中,所述的止血防粘连多肽水凝胶的制备方法,其特征在于,所述方法包括如下步骤:
1)称取多肽粉末,并将其加入浓度为2-10mM盐溶液中,同时调整体系pH至溶液澄清;其中,所述盐溶液是选自氯化钙、碳酸钙、磷酸氢二钙、醋酸钙、柠檬酸钙、乳酸钙、葡萄糖酸钙的水溶液;所述多肽粉末的使用剂量为1-10mg/mL;
2)将步骤1)的反应体系静置,即可得到多肽自组装水凝胶;
其中,制备方法的温度控制在25-37℃之间。
2.权利要求1所述的止血防粘连多肽水凝胶的制备方法,其特征在于,所述方法包括如下步骤:
1)称取多肽粉末,并将其加入浓度为2-10mM盐溶液中,同时调整体系pH至溶液澄清;其中,所述盐溶液是选自氯化钙、碳酸钙、磷酸氢二钙、醋酸钙、柠檬酸钙、乳酸钙、葡萄糖酸钙的水溶液;所述多肽粉末的使用剂量为1-10mg/mL;
2)将步骤1)的反应体系静置,即可得到多肽自组装水凝胶;
其中,制备方法的温度控制在25-37℃之间。
3.如权利要求2所述的制备方法,其特征在于,所述盐溶液选自氯化钙的水溶液。
4.权利要求1所述的多肽自组装水凝胶在制备止血防粘连药物中的用途。
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