CN115300664A - 基于壳聚糖与多聚磷酸钠的喷涂式止血膜 - Google Patents

基于壳聚糖与多聚磷酸钠的喷涂式止血膜 Download PDF

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CN115300664A
CN115300664A CN202210788372.XA CN202210788372A CN115300664A CN 115300664 A CN115300664 A CN 115300664A CN 202210788372 A CN202210788372 A CN 202210788372A CN 115300664 A CN115300664 A CN 115300664A
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刘承琨
王小强
刘畅
路伟
石壮
黄方
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China University of Petroleum East China
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Abstract

本发明公开了一种负载组织因子纳米颗粒的壳聚糖和多聚磷酸钠分层喷涂式止血膜，包括以下步骤：(1)将壳聚糖在pH为4.3的乙酸溶液中搅拌16h,多聚磷酸钠在pH为8.3的Tris‑HCl中溶解；(2)将DOPC：DOPS为3：2的组织因子脂质体加入步骤(1)中的壳聚糖溶液中搅拌1h；(3)分别通过直径0.8mm与0.5mm的喷枪将壳聚糖与多聚磷酸钠溶液形成喷雾，按照壳聚糖：多聚磷酸钠体积比为10：1，分层喷涂两种材料制备层状止血膜；(4)可将步骤(3)中得到的层状止血膜置于37℃的烘箱中1h以去除表面多余水分。通过利用壳聚糖可以富集红细胞和血小板以及多聚磷酸钠可以活化凝血因子的特性，采用分层式喷涂使壳聚糖和多聚磷酸钠雾化结合，形成均匀且致密的止血膜结构，该止血膜不仅具有良好的止血效果，并且具有可塑型的特点，可以充分满足复杂的伤口环境对于止血材料形式的需求。

Description

基于壳聚糖与多聚磷酸钠的喷涂式止血膜
技术领域
本发明涉及一种负载组织因子纳米颗粒的壳聚糖和多聚磷酸钠分层喷涂式止血膜，主要应用于术中或术后的伤口止血处理。
背景技术
急性出血死亡是军事冲突、交通事故和外科手术等方面的主要问题。外科手术过程中或手术后的伤口破裂易导致出血失控，传统的止血方法是在伤口处手动加压或使用加压设备，如可吸收的塞子和缝线。理想的止血材料应该具有安全、高效、方便、经济等特点，但这些特点仍然具有挑战性，大多数止血材料不可能同时实现。
当前已经开发了基于不同种类、不同形式的生物聚合物止血材料，包括改性纱布、喷雾、流体止血剂和止血膜等形式。止血材料形式多样，但都无法同时满足可塑型、可降解以及主动止血的需求。如专利CN202111514386公开了一种流体胶原蛋白止血材料的研制方法；专利CN202210221448公开了一种快速止血喷雾及其制备方法；专利CN202210197243公开了一种含氟共聚物抗菌止血材料及其制备方法和应用。如上止血材料可以在一定程度上满足不同形式伤口需求，但是无法满足临床中复杂的伤口环境以及体内应用必需的可降解性，因此为了充分满足各类手术伤口形式，采用分层喷涂的方式制备止血膜以及采用的可降解材料和生物活性止血颗粒可以为材料的体内应用环境提供基本保障。
壳聚糖作为甲壳素脱乙酰产物，具有可降解性、无毒性、抑菌等多种生理功能，其自身带正电荷可以与人体的红细胞和血小板之间发生静电作用，利于富集血液中的血小板和红细胞。多聚磷酸盐是广泛存在于整个生物学领域的高度阴离子的线性无机磷酸盐聚合物。血小板释放的多聚磷酸盐能够激活接触通路，促进凝血因子Ⅴ活化，增强纤维蛋白凝块结构，促进凝血酶激活凝血因子XI，具有显著促进止血、血栓形成和消除炎症的能力。通过分层喷涂壳聚糖与多聚磷酸钠，利用两者之间的静电相互作用，可以有效结合形成致密的封堵膜。
发明内容
本发明的目的在于构建一种可释放生物活性纳米颗粒的止血膜，该体系是通过壳聚糖与多聚磷酸钠分层喷涂制备的膜体系。本发明提供了一种由所述止血膜为手术阶段伤口管理的方法。
为了实现上述技术方案，本发明涉及的负载生物活性纳米颗粒的壳聚糖与多聚磷酸钠喷涂式止血膜的制备方法，具体包括以下步骤：
(1)将壳聚糖在pH为4.3的乙酸溶液中搅拌16h,多聚磷酸钠在pH为8.3的Tris-HCl中溶解。
(2)将DOPC：DOPS为3：2的组织因子脂质体加入步骤(1)中的壳聚糖溶液中搅拌1h。
(3)分别通过直径0.8mm与0.5mm的喷枪将壳聚糖与多聚磷酸钠溶液形成喷雾，按照壳聚糖：多聚磷酸钠体积比为10：1，分层喷涂两种材料制备层状止血膜。
(4)可将步骤(3)中得到的层状止血膜置于37℃的烘箱中1h以去除表面多余水分。
本发明涉及的步骤(1)壳聚糖分子量为15W、30W、50W,多聚磷酸钠的聚合度为100-1000。
本发明涉及的步骤(2)中磷脂囊泡用的是DOPC和DOPS，其比例为3：2和7：3。
本发明涉及的步骤(3)中喷枪直径可选0.8和0.5，但不限于这两种。
与现有技术相比，本发明具有以下优点：(1)成本低，制备简单，操作容易，易大规模生产和保存；(2)止血膜生物相容性良好，可降解且无细胞毒性；(3)壳聚糖带正电荷可吸引红细胞和血小板聚集且多聚磷酸钠可以激活人体凝血因子；(4)分层喷涂的制备方法对于任何复杂的伤口环境都十分契合，不会受到材料形状的局限。
附图说明：
附图1为本发明涉及的实施例1SEM外貌结构图；
附图2为本发明涉及的实施例1生物活性纳米颗粒释放动力学研究；
附图3为本发明涉及的实施例1止血膜溶胀特性；
附图4为本发明涉及的实施例1止血膜体外模拟降解；
附图5为本发明涉及的实施例1体外凝血与活体实验。
附图6为本发明涉及的实施例1细胞毒性实验。
发明的具体实施方式
下面通过具体实施例和说明书附图对本发明做进一步说明。
实施例1：
(1)止血膜SEM表征
通过所述制备步骤得到止血膜，冻干后在SEM电镜下对其表面和截面进行观察。如附图1中所示，可以明显区别分层止血膜的各层级结构，其内部具有明显的孔隙结构。
(2)生物活性纳米颗粒释放动力学研究
取10mL不同分子量壳聚糖溶液，加入150uL组织因子脂质体搅拌均匀后，与多聚磷酸钠10：1在直径10cm的培养皿中分层喷涂成膜，加入8mL模拟体液(pH＝7.4)。室温下，在不同时间点取模拟体液(200uL)，在515nm(λex＝495nm)处记录TF脂质体的荧光强度，用于TF定量。每次测量所吸取的模拟体液需放回培养皿中，以保持其总体积不变。以不同分子量壳聚糖喷涂胶在24h时的荧光强度作为最大释放量，释放分数计算为各个时间点取样的荧光强度/最大释放荧光强度。如附图2所示，分子量为15W的壳聚糖膜释放速度最快，随着分子量增大，释放速度变缓慢。
(3)止血膜溶胀特性
在室温下将体积比为10：1的壳聚糖溶液与多聚磷酸钠分层喷涂在直径10cm圆形培养皿中，截取1×1cm的薄膜，冻干后置于5mL模拟体液中浸泡，10h内每2h进行一次取样去除多余液体，对冻干薄膜进行称重。Wf为湿重，Wo为干重。
Swelling degree(％)＝[(Wf-Wo)/Wo]×100
如附图3所示，每组从左至右为2h取点，可以看出冻干后的止血膜的最大溶胀可以是自身质量的16倍，体现了止血膜较好的溶胀特性。
(4)止血膜体外模拟降解
将冻干后的不同分子量的壳聚糖薄膜置于含有1.5ug/mL溶菌酶的5mL模拟体液(pH7.4)中，在37℃、90rpm下进行降解。选择溶菌酶的浓度与人血清中的浓度相对应。溶菌酶溶液每天更新，以确保酶的持续活性。在预定时间(0、3、7、12、17天)从模拟体液中取出样品，冻干后并称重。通过重量损失计算体外降解程度：
Weight loss(％)＝[(Wo-Wt)/Wo]×100
W0是降解试验前壳聚糖薄膜的干重，Wt是预定时间t时壳聚糖薄膜的干重。
如附图4所示，分子量越低的壳聚糖膜其具备更快的体外降解速度，最快可在两周内完成降解。
(5)体外凝血
血液：新西兰大耳兔新鲜心脏血液、拮抗剂：0.02775g氯化钙加入10mL超纯水中，混合均匀。使用体积比例为拮抗剂：血液1：2。
10mL不同分子量的壳聚糖溶液中，加入150uL的磷脂化组织因子搅拌均匀，在直径10cm的培养皿中与多聚磷酸钠按照10：1分层喷涂成胶，截取1×1cm的止血膜置于2mL的离心管中，加入500uL拮抗后血液，在37℃下孵育，测量凝血时间。
(6)细胞毒性实验
将NIH-3T3细胞与Dulbecco改良的Eagle's培养基(添加了10％的牛犊血清)在37℃的湿度为5％的CO2湿度培养箱中培养。将止血膜预孵育过夜的细胞培养基用于培养NIH-3T3细胞，培养24小时后将细胞用Tris-HCl缓冲液(pH＝7.4)洗涤两次，然后在37℃下浸入含有Calcein-AM和PI的Tris-HCl缓冲液中30分钟。接下来，将细胞用Tris-HCl缓冲液洗涤3次，并使用荧光显微镜(Leica，DMI3000B)拍摄图像以评估细胞生存力。如附图6所示，图片中未展现出明显的红色荧光，可以表明该止血膜具有较好的生物相容性。
实施例2：
本实施例与实施例1除步骤(1)中，壳聚糖分子量为3W、5W、10W；步骤(3)中壳聚糖：多聚磷酸钠体积比为20：1，其他步骤均相同。
测试表明实施例2制备的止血膜在上述表征中与实施例1外貌特征类似之外，其他特性均具有一定的差异，但均具有良好的止血效果。

Claims (6)

1.一种负载组织因子纳米颗粒的壳聚糖和多聚磷酸钠分层喷涂式止血膜的制备方法，其特征在于，具体包括以下步骤：
(1)将壳聚糖在pH为4.3的乙酸溶液中搅拌16h,多聚磷酸钠在pH为8.3的Tris-HCl中溶解。
(2)将DOPC：DOPS为3：2的组织因子脂质体加入步骤(1)中的壳聚糖溶液中搅拌1h。
(3)分别通过直径0.8mm与0.5mm的喷枪将壳聚糖与多聚磷酸钠溶液形成喷雾，按照壳聚糖：多聚磷酸钠体积比为10：1，分层喷涂两种材料制备层状止血膜。
(4)可将步骤(3)中得到的层状止血膜置于37℃的烘箱中1h以去除表面多余水分。
2.根据权利要求1所述的负载组织因子纳米颗粒的壳聚糖和多聚磷酸钠分层喷涂式止血膜的制备方法，其特征在于，壳聚糖分子量可以为15W、30W、50W，但不限于这几种。
3.根据权利要求2所述的负载组织因子纳米颗粒的壳聚糖和多聚磷酸钠分层喷涂式止血膜的制备方法，其特征在于，多聚磷酸钠的聚合度在100-1000间。
4.根据权利要求3所述的负载组织因子纳米颗粒的壳聚糖和多聚磷酸钠分层喷涂式止血膜的制备方法，其特征在于，步骤(2)中磷脂比例可以为3：2或7：3。
5.根据权利要求4所述的负载组织因子纳米颗粒的壳聚糖和多聚磷酸钠分层喷涂式止血膜的制备方法，其特征在于，步骤(3)中喷枪直径可以为0.8和0.5，壳聚糖与多聚磷酸钠体积比为10：1或20：1，但不限于这几种。
6.根据权利要求1-5任意项所述的负载组织因子纳米颗粒的壳聚糖和多聚磷酸钠分层喷涂式止血膜的制备方法，其特征在于，该止血膜可用于临床手术阶段止血，伤口处生理管理。
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