CN111569139A - 一种负载血小板裂解液的自支撑自组装多层膜及其应用 - Google Patents
一种负载血小板裂解液的自支撑自组装多层膜及其应用 Download PDFInfo
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Abstract
本发明的第一方面是提供一种负载血小板裂解液的自支撑自组装多层膜，包括血小板裂解液；以及负载血小板裂解液的自支撑自组装多层膜。发明首次把血小板裂解液和自支撑多层膜相结合并用于创面修复领域，自支撑多层膜和血小板裂解液是比较完美的搭配，两者均来源和制备简便，通过膜直接吸附血小板裂解液可以避免一些化学交联带来的蛋白构象的改变，另外随着膜的缓慢降解，血小板裂解液也可以避免直接应用导致生长因子突释的弊端；另外通过调控膜的交联程度可以准确而控制释放(降解)的时间，因此两者结合用于创面做敷料很有可行性。
Description
技术领域
本发明涉及创面修复领域，尤其涉及一种负载血小板裂解液的自支撑自组装多层膜及其应用。
背景技术
由烧伤、创伤和慢性疾病引起的大面积创面的修复仍然是临床上的一大挑战。由于自体皮肤可供移植的来源有限，碰到大面积全层皮肤创伤的患者，常常需要人工皮肤或一些生物活性创面敷料的介入。然而目前临床应用的皮肤材料价格高昂，因此兼顾有效性和成本的皮肤材料亟待开发。病理生理学上，创面愈合过程是多方面协同促进的结果，包括细胞增殖，血管生成和细胞外基质(ECM)的沉积。因此，理想的皮肤替代品不仅需要易于制备、成本可控，而且能够为创面提供良好的微环境以助于重建正常的血液供应。
在本发明中，我们通过pH的调控的方法通过交替沉积聚-左旋-赖氨酸(PLL)和透明质酸(HA)构建了指数增长型的层层自组装(LBL)的聚电解质多层膜(PEMs)，以交联剂调控其降解，并通过渗透和静电吸附的方式将其与血小板裂解液(PL)结合在一起，而后对PEMs进行表征并评估其吸附和释放生长因子的能力。通过评估其在体外的增殖，促迁移和促血管生成作用以及在大鼠全层皮肤缺损模型中检查其创面愈合潜力，来评估复合材料PEMs的生物活性。
发明内容
本发明的目的是针对现有技术中的不足，提供一种负载血小板裂解液的自支撑自组装多层膜及其应用。
为实现上述目的，本发明采取的技术方案是：
本发明的第一方面是提供一种负载血小板裂解液的自支撑自组装多层膜，包括血小板裂解液；以及负载血小板裂解液的自支撑自组装多层膜。
优选地，所述自支撑自组装多层膜是通过层层自组装技术，在基底材料上由静电吸引交替沉积构建的。
优选地，所述自支撑自组装多层膜中，分别选取多聚赖氨酸和透明质酸作为带正负电荷的高分子材料，所述自支撑自组装多层膜构建中最后层的选取是由计算机模拟方法实现。
优选地，所述自支撑自组装多层膜为多聚赖氨酸-(透明质酸-多聚赖氨酸)n-透明质酸膜。
优选地，所述自支撑自组装多层膜中5≤n≤10。
进一步优选地，所述支撑自组装多层膜中n＝7。
优选地，所述基底材料为聚四氟乙烯。
优选地，所述负载血小板裂解液的自支撑自组装多层膜采用如下方法制备：
S1、选择聚四氟乙烯作为基底材料，并分别在乙醇、丙酮和水中分别超声处理15分钟，然后使用氮气流干燥；
S2、将干燥后的基底材料交替浸入1-5mg/mL，pH为9-10的多聚赖氨酸溶液和1-5mg/mL，pH为2.5-3.5的透明质酸溶液中，并交替在相应pH值的水中漂洗，吹干直至获得5-10个双层，且末层为透明质酸层的自支撑自组装多层膜；
S3、将S2所得的自支撑自组装多层膜在含有9-13mg/mL的N-羟基磺基琥珀酰亚胺和25-35mg/mL的1-乙基-3-[3-二甲基氨基丙基]碳二亚胺盐酸盐的组合溶液中于0-4℃孵育过夜进行交联；
S4、将S3交联后的自支撑自组装多层膜浸入五倍浓缩血小板裂解液中过夜，以负载血小板裂解液，然后用水冲洗3-5次，并冷冻干燥4-8小时。
进一步优选地，所述负载血小板裂解液的自支撑自组装多层膜采用如下方法制备：
S1、选择聚聚四氟乙烯作为基底材料，并分别在乙醇、丙酮和水中分别超声处理15分钟，然后使用氮气流干燥；
S2、将干燥后的基底材料交替浸入1mg/mL，pH为9.5的多聚赖氨酸溶液和3mg/mL，pH为2.9的透明质酸溶液中，并交替在相应pH值的水中漂洗，吹干直至获得8个双层，且末层为透明质酸层的自支撑自组装多层膜；
S3、将S2所得的自支撑自组装多层膜在含有11mg/mL的N-羟基磺基琥珀酰亚胺和30mg/mL的1-乙基-3-[3-二甲基氨基丙基]碳二亚胺盐酸盐的组合溶液中于4℃孵育过夜进行交联；
S4、将S3交联后的自支撑自组装多层膜浸入五倍浓缩的血小板裂解液中过夜，以负载血小板裂解液，然后用水冲洗3次，并冷冻干燥4小时。
优选地，所述血小板裂解液的制备步骤包括：
S4-1、通过两次离心法从40位健康供体每位的50ml静脉血中收集富血小板血浆；
S4-2、将富血小板血浆(PRP)进行三次循环冻融以破坏血小板膜，从而释放生长因子；
S4-3、将S4-2中所获得的液体离心去除沉淀后，获得血小板裂解液的上清液并进行冷冻干燥，并用原溶液量五分之一的生理盐水进行复溶，从而得到五倍浓缩的血小板裂解液。
优选地，所述两次离心法的转速和时间为依次160g，10min和250g，15min。
本发明的第二方面是提供一种如前所述负载血小板裂解液的自支撑自组装多层膜在创面修复领域中的应用。
本发明采用以上技术方案，与现有技术相比，具有如下技术效果：
本发明首次把血小板裂解液和自支撑多层膜相结合并用于创面修复领域，自支撑多层膜和血小板裂解液是比较完美的搭配，两者均来源和制备简便，通过膜直接吸附血小板裂解液可以避免一些化学交联带来的蛋白构象的改变，另外随着膜的缓慢降解，血小板裂解液也可以避免直接应用导致生长因子突释的弊端；另外通过调控膜的交联程度可以准确而控制释放(降解)的时间，因此两者结合用于创面做敷料很有可行性。
本发明自支撑多层膜的构建上是正负电荷分子A-B-A-B-……不断循环的过程，如此就不清楚，到底让A做最后一层好，还是B做最后一层好，本发明通过计算机分子模拟A和B也就是多聚赖氨酸和透明质酸与血小板裂解液中主要的生长因子(PDGF，TGF，bFGF)的亲和力，算出透明质酸做最后一层对三种生长因子能有更好的亲和力，这是一个方法学上的创新，首次用模拟来代替材料的筛选。
附图说明
图1为本发明自支撑自组装多层膜的创面愈合百分比结果；
图2为本发明自支撑自组装多层膜的胶原纤维沉积率结果；
图3为本发明自支撑自组装多层膜的血流灌注体积结果；
图4为本发明自支撑自组装多层膜的血管体积结果；
图5为本发明自支撑自组装多层膜的血管数量结果。
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作出创造性劳动的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。
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。
下面结合附图和具体实施例对本发明作进一步说明，但不作为本发明的限定。
实施例1
本实施例提供了一种负载血小板裂解液的自支撑自组装多层膜的制备方法。
由分子模拟方法确定自支撑自组装多层膜的最后层。HA和PLL重复单元的结构式由ChemBioDraw软件绘制，由ChemBio3D转换为3D格式。从PDB网站(https://www.rcsb.org/)下载PDGF-BB(PDB ID：4QCI)，TGF-β1(PDB ID：1KLC)和bFGF(PDB ID：1CVS)的3D结构式，并用PyMOL软件进行了优化。根据公开的方法，使用AutoDock Vina软件进行了分子对接模拟和随后的结合亲和力计算。通过PyMOL生成高分子重复单元对每种蛋白质具有最低结合能时的对接复合物构象的图像，并生成高分子和周围氨基酸残基之间形成的极性键，并挑选出此类构象用于进一步的分子动力学分析。用Ligplot+软件显示分子和蛋白间的氢键和疏水键。
通过使用GROMACS软件进行进一步的分子动力学模拟(MD)，使用GROMOS96 43A1力场和SPC/E分子水模型生成蛋白分子的力场参数和拓扑文件。使用PRODRG软件生成HA和PLL的力场参数和拓扑文件。使用一个立方体的盒子进行模拟，并用氯离子或钠离子进行电荷的中和。氢原子的键长由SHAKE算法固定，使用粒子网格法(PME)计算静电相互作用。对复合物进行能量最小化，及压力和温度的平衡。最后，在1个大气压和80.3℉下进行了20ns的动力学模拟，过程中每100ps获取一次坐标记录，并最终通过计算坐标均方根偏差(RMSD)来体现所形成的大分子和蛋白复合体的结构稳定性。结果证实HA与相应生长因子之间形成的复合体结构稳定性优于PLL与生长因子所形成的复合体结构。
选择聚四氟乙烯作为基底材料，并分别在乙醇、丙酮和水中依次超声清洗15分钟，然后使用氮气吹干；将干燥后的基底材料交替浸入1mg/mL，pH9.5的多聚赖氨酸溶液和3mg/mL，pH2.9的透明质酸溶液中，并交替在相应pH值的双蒸水中漂洗，吹干直至获得8个双层且末层为透明质酸层的自支撑自组装多层膜，即EDC0(未交联)；将所述自支撑自组装多层膜在含有11mg/mL的N-羟基磺基琥珀酰亚胺和30mg/mL的1-乙基-3-[3-二甲基氨基丙基]碳二亚胺盐酸盐的组合溶液中于4℃孵育过夜，以获得交联的自支撑自组装多层膜，即EDC30(交联)。
血小板裂解液的制备：提取PRP，随后通过液氮冻融法获得浓缩的PL。这项研究是根据《赫尔辛基宣言》的原则进行的，并且由上海交通大学附属的第六人民医院独立伦理委员会(中国上海)批准了该研究方案用于收集样本并将其用于科学实验。每个参与者都签署了知情同意书。通过两次离心法从40位健康成人的50ml静脉血中收集富血小板血浆(依次160g，10min和250g，15min)；将富血小板血浆进行三次循环的液氮-37℃水浴冻融以破坏血小板膜，从而释放其胞内的生长因子，以获得血小板裂解液；将获得的血小板裂解液进行冷冻干燥，并用原溶液量五分之一的生理盐水进行复溶，从而得到五倍浓缩的血小板裂解液。
将所述自支撑自组装多层膜浸入五倍浓缩的血小板裂解液中过夜，使血小板裂解液富集于膜之中，然后用去离子水冲洗3次，并冷冻干燥4小时，即得负载血小板裂解液的自支撑自组装多层膜，即EDC0@PL和EDC30@PL。
检测实施例
本实施例共使用25只成年SD大鼠(年龄＝2个月，体重＝250±15g，每组n＝5)。该实验得到上海交通大学附属上海市第六人民医院动物实验伦理委员会的批准。在实验的每个阶段都严格遵守了相关的准则。在实验前，用等离子束辐射对各组敷料进行灭菌。腹腔注射戊巴比妥钠(2.5％，30mg/kg)对大鼠进行麻醉。随后使用碘伏对手术部位进行消毒后，由熟练的外科医生在大鼠背侧沿脊柱旁开1cm处，全层切取一个标准的圆形缺损(直径＝20mm)。然后，将由实施例1制备的EDC0，EDC30，EDC0@PL和EDC30@PL四组敷料分别放置在皮肤缺损处。用纱布覆盖创口并由针线固定，对照组则只用纱布覆盖而没有敷料的填入。
1.1不同敷料处理的缺损皮肤的创面闭合和组织学分析：
实验结果如图1所示，为了研究负载血小板裂解液的自支撑自组装多层膜在体内的作用，在本实施例中构建了标准的大鼠全层皮肤缺损模型(20mm)，并且在整个实验过程中各组均未观察到任何不良反应。大体观图像显示，所有膜敷料均具有良好的亲水性，可以很好地覆盖在创面处并保持创面干燥。此外，记录了在0、3、7和14天采用不同治疗方法后创面愈合的过程。尽管所有五个组的创面大小均随着时间的推移而减小，但EDC0@PL组的创面大小在3天内比其他组小。而在7天和14天中，EDC30@PL组也显示出相当高的愈合率。同时，与体外研究不同，在第14天，单独的EDC0和EDC30组与未治疗组相比也表现出一定的促修复作用，表明未负载PL的多层膜敷料也有助于创面的愈合。在愈合的早期(3天和7天)，未交联EDC0@PL的多层膜因降解较快(早期可以释放更多的生长因子)比EDC30@PL能够更好地修复创面。
收取各组创面组织进行H&E染色，可以进一步评估创面的愈合质量。根据H&E染色结果，7天时，EDC0@PL和EDC30@PL处理的创面相比其他三组有更多新生上皮组织形成。到第14天时，EDC30@PL组新生上皮最多，其后依次是EDC0@PL，EDC0和EDC30以及对照组。
1.2对创面组织行Masson三色染色评估其胶原沉积情况：
结果如图2所示，与未治疗的对照组相比，EDC0和EDC30膜，尤其是负载PL的EDC0和EDC30膜处理的创面在7天时就能显示出波浪状胶原纤维沉积在缺损区域。14天时，用PL负载的膜处理的创面呈现出大量胶原蛋白纤维，且有序排列，其与正常皮肤的胶原排列相似。这表明PL能促进上皮再生和胶原蛋白重塑。此外，14天时，在EDC30@PL治疗的创面组织内还发现了一些毛囊和皮脂腺，这进一步表明交联后的膜(EDC30)搭载PL，通过对PL的长效控释，能获得更好的治疗效果。
1.3评估通过不同敷料处理的创面中的血流和功能性血管的形成：
如图3-5所示，使用激光多普勒成像系统对创面血流进行评估，使用MoorLDIReview软件对血流结果进行定量。负载PL的膜处理组对比其他三组，其创面处血供更好。EDC0@PL组在早期(3和7天)血流供应最丰富。然而，除EDC30@PL组外，所有其他组在术后第14天均显示出血液灌注减少，表明交联膜通过长效控释PL能持续的促进创面区域的血流供应。此外，在14天时，对血管灌注组织行微CT扫描分析，其结果显示，载有PL的膜，尤其是EDC30@PL组具有更多的新生血管形成。
综上所述，尽管富血小板血浆(PRP)及其衍生物已经在临床实践中被广泛使用，但仍然存在生长因子突释，原位降解快，组织在位性差等局限性。本发明通过LBL法构建的自支撑自组装多层膜来搭载PL能有效解决其临床应用的局限性。该膜对GFs富集显示出显著的优势。负载有PL的敷料在促进皮肤创面愈合方面表现出巨大潜力。同时，通过控制交联度，可以精确调节PL的控制释放行为。
以上所述仅为本发明较佳的实施例，并非因此限制本发明的实施方式及保护范围，对于本领域技术人员而言，应当能够意识到凡运用本发明说明书及图示内容所作出的等同替换和显而易见的变化所得到的方案，均应当包含在本发明的保护范围内。
Claims (10)
1.一种负载血小板裂解液的自支撑自组装多层膜，其特征在于，包括血小板裂解液；以及负载血小板裂解液的自支撑自组装多层膜。
2.根据权利要求1所述负载血小板裂解液的自支撑自组装多层膜，其特征在于，所述自支撑自组装多层膜是通过层层自组装技术，在基底材料上由静电吸引交替沉积构建的。
3.根据权利要求2所述负载血小板裂解液的自支撑自组装多层膜，其特征在于，所述自支撑自组装多层膜中，分别选取多聚赖氨酸和透明质酸作为带正负电荷的高分子材料，所述自支撑自组装多层膜构建中最后层的选取是由计算机模拟方法实现。
4.根据权利要求3所述负载血小板裂解液的自支撑自组装多层膜，其特征在于，所述自支撑自组装多层膜为多聚赖氨酸-(透明质酸-多聚赖氨酸)n-透明质酸膜。
5.根据权利要求4所述负载血小板裂解液的自支撑自组装多层膜，其特征在于，所述自支撑自组装多层膜中5≤n≤10。
6.根据权利要求2所述负载血小板裂解液的自支撑自组装多层膜，其特征在于，所述基底材料为聚四氟乙烯。
7.根据权利要求1所述负载血小板裂解液的自支撑自组装多层膜，其特征在于，所述负载血小板裂解液的自支撑自组装多层膜采用如下方法制备：
S1、选择聚四氟乙烯为基底材料，并分别在乙醇、丙酮和水中分别超声处理15分钟，然后使用氮气流干燥；
S2、将干燥后的基底材料交替浸入1-5mg/mL，pH为9-10的多聚赖氨酸溶液和1-5mg/mL，pH为2.5-3.5的透明质酸溶液中，并交替在相应pH值的水中漂洗，吹干直至获得5-10个双层，且末层为透明质酸层的自支撑自组装多层膜；
S3、将S2所得的自支撑自组装多层膜在含有9-13mg/mL的N-羟基磺基琥珀酰亚胺和25-35mg/mL的1-乙基-3-[3-二甲基氨基丙基]碳二亚胺盐酸盐的组合溶液中于0-4℃孵育过夜进行交联；
S4、将S3交联后的自支撑自组装多层膜浸入五倍浓缩血小板裂解液中过夜，以负载血小板裂解液，然后用水冲洗3-5次，并冷冻干燥4-8小时，即得负载血小板裂解液的自支撑自组装多层膜。
8.根据权利要求7所述负载血小板裂解液的自支撑自组装多层膜，其特征在于，所述血小板裂解液的制备步骤包括：
S4-1、通过两次离心法从40位健康供体每位的50ml静脉血中收集富血小板血浆；
S4-2、将富血小板血浆进行三次循环冻融以破坏血小板膜，从而释放生长因子；
S4-3、将S4-2中所获得的液体离心去除沉淀后，获得血小板裂解液的上清液并进行冷冻干燥，并用原溶液量五分之一的生理盐水进行复溶，从而得到五倍浓缩的血小板裂解液。
9.根据权利要求7所述负载血小板裂解液自支撑自组装多层膜，其特征在于，所述两次离心法的转速和时间为依次160g，10min和250g，15min。
10.一种如权利要求1-9任一项所述负载血小板裂解液的自支撑自组装多层膜在创面修复领域中的应用。
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| CN112107733A (zh) * | 2020-09-14 | 2020-12-22 | 上海市第六人民医院 | 一种注射型负载血小板裂解液的温敏水凝胶-高分散纳米颗粒体系及其应用 |
| CN112107733B (zh) * | 2020-09-14 | 2024-02-23 | 上海市第六人民医院 | 一种注射型负载血小板裂解液的温敏水凝胶-高分散纳米颗粒体系及其应用 |
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