CN115382000A - 一种具有抗菌涂层的敷料及其制备方法 - Google Patents
一种具有抗菌涂层的敷料及其制备方法 Download PDFInfo
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Abstract
本发明提供一种具有抗菌涂层的敷料及其制备方法，涉及生物医用材料技术领域，以抗菌性壳聚糖和羧甲基纤维素为涂层主体材料，通过静电作用力驱动的层层自组装成膜技术，利用抗菌性苦参碱为功能药物，构建了基于抗菌性壳聚糖/羧甲基纤维素复合药物涂层的聚氨酯敷料。进而通过分子间化学交联改变了聚合物内部结构，进一步提升涂层的机械稳定性，抑菌实验表明，由于壳聚糖和苦参碱的协同抗菌作用，与单药组相比，复合药物涂层的抑菌效果增强了1倍。复合药物涂层对金黄色葡萄球菌和大肠杆菌展现出明显的抑制生长作用，生长抑制率分别为35.7％和53.8％。
Description
技术领域
本发明涉及生物医用材料技术领域，具体涉及一种具有抗菌涂层的敷料及其制备方法。
背景技术
生物医用材料被广泛地应用于人造组织、创伤辅料、医疗器械、药物缓释等领域，生 物医用材料使用过程中发生的医源性感染严重威胁人类健康，对于医疗器械和医用敷料而 言，在其表面构建功能性涂层能有效地的防治细菌感染。因此，研究人员一直致力于开发 多种表面抑菌涂层，常用的制备方法包括引发聚合物毛刷法、水凝胶法和自组装法等。根 据功能和作用机制的不同，表面抑菌涂层分为抗黏附抑菌、接触式抑菌以及智能抑菌涂层。 其中，作为自组装方式之一的层层自组装技术在涂层制备方面更具优势，该技术是通过分 子间作用力驱动，包括静电相互作用、主客体相互作用和生物特异性相互作用等，在模板 表面层层交替沉积形成完整涂层的一种技术。LbL具有工艺简便，适用范围广的优点，所使 用的基底或成膜材料具有灵活性，这些因素导致LbL技术已广泛应用于药物传递系统、生 物传感器、生物医学设备、表面改性和细菌感染预防等方面。
LbL技术需要具有生物相容性和生物降解性的聚电解质，天然高分子聚电解质正好满足 这一条件。常用的天然高分子聚电解质有壳聚糖(CHI)、海藻酸盐、透明质酸、鱼精蛋白、 纤维素及其衍生物。羧甲基纤维素钠(CMC)是天然纤维素经过化学改性后的产物，具有亲 水性、生物黏附性、成膜性和pH敏感性等优点，在复合材料、医药载体、药物缓释等领域 具有广阔的应用前景。壳聚糖是天然多糖甲壳素脱乙酰基的产物，可降解性、无毒性和抗 菌性等特质使其广泛应用于医用敷料，人造组织材料和抗菌剂等众多领域。
发明内容
本发明的目的在于提供一种具有抗菌涂层的敷料及其制备方法，利用静电吸附作用将 CHI/CMC复合药物涂层制备到聚氨酯(PU)敷料上。通过化学交联改变CHI/CMC涂层的内部 结构，增加其稳定性从而将抗菌中药小分子苦参碱(Mat)结合到涂层上，并提升聚氨酯敷 料的机械性能。该载药交联涂层能抑制大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus) 的生长，结合壳聚糖潜在的抗菌性能，对细菌的抑制作用进一步增强，基于抗菌性壳聚糖/ 羧甲基纤维素复合药物涂层的聚氨酯敷料有望作为创面敷料应用于临床。
为了实现上述目的，本发明采用的技术方案如下：
本发明第一个方面公开了一种抗菌涂层，所述抗菌涂层是以抗菌性壳聚糖和羧甲基纤 维素为主体材料通过层层自组装成膜技术制成的。
本发明第二个方面公开了利用所述抗菌涂层制成的敷料，以聚氨酯为基底，将抗菌涂 层组装在所述聚氨酯上。
本发明第三个方面公开了所述敷料的具体制备方法：S1、分别将壳聚糖(CHI)溶解在 乙酸溶液中形成CHI溶液，将羧甲基纤维素钠(CMC)溶解在去离子水中形成CMC溶液，备用；
S2、将聚氨酯基底交替浸入CHI溶液和CMC溶液中在聚氨酯基底表面形成CHI/CMC涂 层；
S3、利用交联剂对CHI/CMC涂层进行化学交联处理得到交联产物；
S4、再将S3制备的交联产物浸入苦参碱溶液浸泡1-1.5h，去离子水清洗后再干燥得到 敷料。
优选地，所述步骤S1中乙酸溶液质量分数为2-3wt％。
更优选地，所述步骤S1的CHI溶液中壳聚糖含量为0.5-1.5mg/mL，CMC溶液中羧甲基 纤维素钠的含量为0.5-1.5mg/mL。
较佳地，所述步骤S1的CHI溶液和CMC溶液的pH为3.5-4.5，优选pH＝4。
优选地，所述步骤S2中基底在CHI溶液和CMC溶液的浸入时间为8-12min。
更优选地，所述步骤S2中基底从CHI溶液和CMC溶液中取出后用去离子水洗涤3-5次。
本发明相对于现有技术具有如下的显著优点及效果：
(1)本发明公开的CHI/CMC涂层，通过化学交联手段改变其内部结构，提升涂层 稳定性，交联后的CHI/CMC涂层涂层成功制备到聚氨酯敷料上，赋予其良好的力学性 能，与PETG底板相比，交联PETG-(CHI/CMC)10涂层的断裂时间和标准应变均提升了 25.1％。
(2)本发明的敷料具有优良的药物装载能力，交联CHI/CMC涂层修饰的聚氨酯敷料对苦参碱的装载量为330.0μg/cm2，装载苦参碱后的复合药物涂层对大肠杆菌和金黄 色葡萄球菌的抑制率分别为53.8％和35.7％，基于壳聚糖/羧甲基纤维素复合药物涂层 改性的聚氨酯敷料显示出作为创伤敷料的潜力。
附图说明
图1为本发明基于壳聚糖和羧甲基纤维素复合药物涂层的聚氨酯敷料制备示意图；
图2A为本发明CHI/CMC涂层的UV-VIS全光谱吸光值曲线图；图2B为本发明CHI/CMC涂层的紫外吸收峰值曲线图；
图3A为实施例2中(CHI/CMC)25涂层的SEM图像；图3B为交联(CHI/CMC)25涂层的SEM图像；图3C为聚氨酯基底(CHI/CMC)10涂层的SEM图像；图3D为聚 氨酯基底交联(CHI/CMC)10涂层的SEM图像；
图4为CHI、CMC、CHI/CMC涂层和交联CHI/CMC涂层的红外光谱图；
图5A为(CHI/CMC)25涂层的静态水滴接触角；图5B为交联(CHI/CMC)25涂层的静态水滴 接触角；
图6A为药物累积释放曲线图；图6B为药物累积释放百分比曲线图；
图7为载药交联(CHI/CMC)25涂层对E.coli和S.aureus的抑制率的柱形图。
具体实施方式
下面结合实施例和附图对本发明的具体实施方式做进一步的详细描述。以下实施例和 附图用于说明本发明，但不用来限制本发明的范围。
仪器与试剂：
Regulus8100型扫描电子显微镜(SEM，日本Hitachi公司)；SpectrumTwoDTGS型傅里叶变换红外光谱仪(FT-IR，英国PerkinElmer公司)；DR6000型紫外可见分光光度计 (UV-VIS，美国HACH公司)；SDC-100型接触角测角仪(东莞SINDIN晟鼎精密有限公司)； Proline型电子万能试验机(德国Zwick公司)；MultiskanSky型酶标仪(美国Thermo公 司)。
壳聚糖(中等相对分子质量，取代度75％-85％)、羧甲基纤维素(相对分子质量为250000) 均购自美国Sigma公司；苦参碱(≥98％)购自上海麦克林生化科技有限公司；2-吗啉乙磺 酸(MES，≥99％)、50％戊二醛购自上海Adamas公司；磷酸盐缓冲液(PBS)购自美国Gibco 公司；LB肉汤、琼脂粉购自青岛高科技工业园海博生物技术有限公司；大肠杆菌(CMCC44102) 购自北京索莱宝科技有限公司；金黄色葡萄球菌(ATCC43300)购自宁波泰斯拓生物技术有 限责任公司；聚氨酯敷料基底购自于青岛海氏海诺集团有限公司；1-乙基-(3-二甲基氨基 丙基)碳酰二亚胺盐酸盐(EDC)、N-羟基琥珀酰亚胺(NHS)均购自国药集团化学试剂有限 公司。
敷料的制备方案：
首先，将硅片、载玻片和聚氨酯敷料基底在无水乙醇中浸泡清洗，去离子水冲洗后用 氮吹仪吹干备用。用等离子体处理洁净的基底，使其表面带负电荷。1mg/mL的CHI在2％乙 酸溶液中溶解，1mg/mL的CMC在去离子水中溶解，搅拌过夜，形成均一溶液。将上述聚电解质溶液的pH均调整为4。使用CHI作为带正电荷的聚合物，CMC作为带负电荷的聚合物， 利用LBL技术在基底上制备CHI/CMC涂层。将基底浸入CHI溶液中1min，取出后用去离子 水洗涤3次，以去除基底表面未牢固吸附的CHI分子。然后，再将基底浸泡在CMC溶液中 10min，去离子水漂洗。重复以上操作n次，得到了不同层数的(CHI/CMC)n涂层(n＝双层膜 的数量)。最后，用N2干燥保存备用。
使用EDC/NHS/MES交联剂作为涂层化学交联的第1步。将带有CHI/CMC涂层的基底浸 在含有0.2mol/LEDC和5mmol/LNHS的0.05mol/L的MES缓冲液中20min，在1×PBS缓冲液中浸泡10min。利用2.5％戊二醛溶液作为第2交联剂，涂层与其额外交联处理40min。交联完成后用去离子水冲洗3次。
实施例1：复合涂层的厚度表征
采用UV-VIS观测载玻片基底层层自组装过程中涂层的厚度变化，波长扫描范围为190-900nm，扫速为2nm。
使用层层自组装技术制备了(CHI/CMC)25涂层。CHI和CMC在不同pH值下的电离度不同， 调整pH为4更有利于CHI和CMC的电离。CHI的—NH2基团被电离成NH3 +而带正电荷，CMC的—COOH基团被电离成—COO-而带负电荷。在负电荷修饰基底表面的情况下，LbL组装开始于通过静电作用沉积1个带正电荷的CHI层，然后是1个负电荷的CMC层。为了直观反映 多层膜的成功沉积，使用UV-VIS测量涂层的吸光度。涂层在300nm附近处的紫外吸收峰值 越大，则涂层越厚。图2的增长曲线显示涂层的紫外吸收随着自组装层数的增加而增加， 增厚曲线无明显的不规则性，表明了LBL自组装涂层的逐层成功沉积。
实施例2：复合涂层的SEM表面形貌
采用SEM观察不同基底涂层及化学交联涂层的表面形貌；测试之前，喷金处理2min， 放大倍数为1×104倍。
性质不同的天然高分子材料层层自组装形成不同结构的涂层，涂层的表面也呈现出有 区别的形貌。图3的SEM图像显示，(CHI/CMC)25涂层是具有小孔隙的致密粗糙结构，化学 交联改变自组装涂层的表面形貌，用于洗涤EDC/NHS/MES交联剂的PBS盐被分散于高分子 材料的分子链之间，部分影响了其电荷强度，涂层经交联膨胀后，从而确保了孔隙可以形 成的区域。交联后的(CHI/CMC)25涂层表面的孔隙度更高，最外层表面局部扩张断裂，从而 出现细小缝隙，涂层转变为疏松的粗糙结构。在不同基底上制备的功能性涂层具有不同的 表征和性能。聚电解质在聚氨酯泡沫上堆积成膜，聚氨酯基底上的交联涂层更具颗粒感， 表面不平整；表面形貌的改变是主要是由于分子间交联形成的额外化学键、内部分子结构 变化和涂层内空间结构改变的结果。
实施例3：复合涂层的FT-IR表征
采用FT-IR研究了聚合物和涂层的官能团特征，扫描范围为700-4000cm-1，扫描次数为 32次。
从图3的FT-IR光谱中可以看出，CHI和CMC的光谱清晰地反映了这2种高分子材料相 应的特征峰。CHI的红外光谱图中，3500cm-1是O—H和N—H的伸缩振动吸收峰，1346和1152cm-1处为C—N的伸缩振动峰。在CMC的红外光谱图中，2926cm-1处是C—H的伸缩振动，1598cm-1附近是C—O伸缩振动峰。另外，(CHI/CMC)25涂层在3400cm-1附近有2个明显的峰，主要是由于O—H和N—H信号峰重叠，在1640cm-1主要是C＝O—O峰，这些结果表明了多层 膜的成功组装。化学交联涂层的FT-IR光谱观察到2个明显的分离峰，分别在1680cm-1(O＝C)和1645cm-1(N—H)处对应酰胺键，显示成功交联。
实施例4：复合涂层的接触角
为了评估薄膜的亲水性，利用接触角测角仪测量了化学交联前后涂层的静态接触角。 使用座滴法分别测量样品的左接触角和右接触角，取其平均值，测试时滴加的液滴体积为 5L。
液体在固体涂层表面上的水滴接触角WCA，是衡量表面润湿性能的重要参数。若接触角 小于90°，代表是亲水性表面，液体较易润湿涂层表面。接触角越小，表示润湿性越好。表面粗糙度的改变会影响涂层表面的亲水性。根据图5的接触角测量结果，经过化学交联，(CHI/CMC)25涂层的静态接触角由43°降低为3°，形成超亲水状态，可形成水合层，抵御 微生物的聚集粘附。涂层中CHI和CMC具有亲水性官能团—NH2和—COOH，在对亲水表面进 行化学交联时，形成新的酰胺键使得涂层的亲水性增强。同时，影响亲水性的另一个重要 因素是粗糙度，化学交联形成更为粗糙的涂层表面结构，在粗糙的表面下，交联涂层的浸 润性更强。
实施例5：复合涂层的机械性能
参考国标GB/T1040-2006，使用电子万能试验机测试纯PETG板和交联PETG-(CHI/CMC)10的力学性能，在室温下以预载5N，拉伸速度1.5mm/min进行测试，拉伸至断裂。
通常，涂层的机械性能取决于成膜材料的相对分子质量和含量，以及它们之间的相互 作用。高分子聚合物准备的涂层具有较好的强度，刚度和柔韧性。抗拉强度和断后延伸率 是反映涂层力学性能的两个重要特性。聚(对苯二甲酸乙二醇酯-1,4-环己二烯二亚甲基对 苯二甲酸酯)(PETG)板和交联PETG-(CHI/CMC)10的力学性能如表1所示。测试结果表明， 与单纯PETG板基底相比，在PETG板上制备交联(CHI/CMC)10涂层后，涂层的断裂时间、标准应变、断后延伸率、最大失效载荷和抗拉强度都有不同程度的增加。交联PETG-(CHI/CMC)10的断裂时间由纯PETG板的87.3min增加到109.2min，标准应变从130.9mm增加到163.7mm， 抗拉强度也从45.8MPa升高到48.1Mpa，表明CHI/CMC涂层的存在可以显著改善基底的力学 性能。
表1化学交联涂层的力学性能
实施例6：涂层和涂层修饰聚氨酯敷料的药物释放性能
将硅片基底交联(CHI/CMC)25涂层、空白聚氨酯敷料和聚氨酯基底交联(CHI/CMC)10涂层 在1mg/mL的苦参碱溶液中浸泡1h，用去离子水将表面残留的苦参碱去除。然后置于10mL 的1×PBS缓冲液(pH＝7.4,37℃)中，于37℃的环境下进行药物释放，间隔一段时间，定 时吸取1mL的释药溶液储存到离心管中，然后补足等量的1×PBS缓冲液。利用紫外可见分 光光度计测定离心管中释药溶液在210nm处的吸光度，根据苦参碱的药物浓度-吸光度标准 曲线计算出单位面积载药涂层的累积释药量。
采用扩散加载的方式，将苦参碱载入不同基底的交联涂层中，测定其药物释放量。化 学交联大幅提升涂层在载入碱性药物时的稳定性，药物载释过程中涂层不会发生崩解或溶 蚀现象。图6是在生理条件下的药物释放谱图，硅片基底交联(CHI/CMC)25涂层的累积药物 释放量为27.8μg/cm2，空白聚氨酯基底的载药量为285.2μg/cm2，聚氨酯基底交联(CHI/CMC)10涂层的累积药物释放量为330.0μg/cm2。化学交联不仅提高了涂层的稳定性，还扩大了涂层的孔隙，交联后的涂层结构更有利于药物小分子的进入，药物可以加载到更深的内部。相较于空白聚氨酯基底，聚氨酯基底(CHI/CMC)10交联涂层的载药量增加了 44.8μg/cm2，该增加量高于硅片基底(CHI/CMC)25交联涂层的载药量，表明在聚氨酯基底上 修饰交联涂层能够提升其载药量。与硅片基底(CHI/CMC)25交联涂层相比，聚氨酯基底 (CHI/CMC)10交联涂层的载药量提升了近11倍，聚氨酯基底表面存在大量的原始孔洞，具有 极强的药物装载能力，使得载药量大幅提升。同时，硅片基底(CHI/CMC)25涂层的释药百分 比在0.5h后就趋近于100％，空白聚氨酯和聚氨酯基底(CHI/CMC)10交联涂层的药物释放量 在1h时才达到90％左右，表明聚氨酯基底可减缓药物的释放速度，可能是由聚氨酯基底表 面的多孔膜结构增加了药物从聚氨酯内部向外扩散的阻力而造成。
实施例7：自组装涂层的抑菌性能研究
分别选取革兰氏阴性菌大肠杆菌和革兰氏阳性菌金黄色葡萄球菌作为实验对象，采用 细菌悬液法测试涂层的抑菌率。细菌复苏活化，待其生长到指数期时，将菌液稀释约至1 ×105CFU/mL的浓度。将带有涂层和载药涂层的硅片样品正反两面均放在紫外灯下照射灭菌30min备用。96孔板每孔中加入100μL的菌液和等体积的LB肉汤，设置阴性对照组(仅加 入200μL的LB肉汤)和阳性对照组(仅加入100μL菌液和100μL的LB肉汤)。然后，将 灭菌的带有涂层的硅片样品、带有载药涂层的硅片样品和等量游离苦参碱添加到96孔板中， 放于37℃培养箱中静置培养。24h后用酶标仪测定OD600，按照公式(1)计算细菌抑菌率(BR)。
式中，ODs为样品组的吸光度值，ODp为阳性对照组的吸光度值，ODn为阴性对照组的吸 光度值。
为了评估涂层的抑菌性能，以大肠杆菌和金黄色葡萄球菌作为代表性菌。涂层对大肠 杆菌、金黄色葡萄球菌的抑菌率如图7所示。结果表明，实验组的涂层和载药涂层对大肠 杆菌和金黄色葡萄球菌均有一定的抑制作用。CHI/CMC涂层与苦参碱单药的抑菌率大致相同， 苦参碱是通过抑制细菌早期生物被膜生成而产生抑菌作用。壳聚糖则是通过质子化的氨基 与细菌表面带负电的物质产生相互作用，从而破坏细菌的细胞壁抑制其生长，并且壳聚糖 对革兰氏阴性菌和革兰氏阳性菌细胞壁的破坏方式不同，导致CHI/CMC膜对大肠杆菌和金 黄色葡萄球菌的抑制率有所差别。载药CHI/CMC涂层对对大肠杆菌和金黄色葡萄球菌的抑 制率分别为53.8％和35.7％。与涂层、等量的游离苦参碱组对比，载药涂层对细菌具有更强 的抑菌作用，体现出了壳聚糖和苦参碱的共同抑菌效果。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制， 其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均 应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (8)
1.一种抗菌涂层，其特征在于，该涂层是以抗菌性壳聚糖和羧甲基纤维素为主体材料通过层层自组装成膜技术制成的。
2.一种利用权利要求1所述抗菌涂层制成的敷料，其特征在于，以聚氨酯为基底，将抗菌涂层组装在所述聚氨酯上。
3.根据权利要求2所述的一种敷料，其特征在于，具体制备方法如下：
S1、分别将壳聚糖(CHI)溶解在乙酸溶液中形成CHI溶液，将羧甲基纤维素钠(CMC)溶解在去离子水中形成CMC溶液，备用；
S2、将聚氨酯基底交替浸入CHI溶液和CMC溶液中在聚氨酯基底表面形成CHI/CMC涂层；
S3、利用交联剂对CHI/CMC涂层进行化学交联处理得到交联产物；
S4、再将S3制备的交联产物浸入苦参碱溶液浸泡1-1.5h，去离子水清洗后再干燥得到敷料。
4.根据权利要求3所述的一种敷料的制备方法，其特征在于，所述步骤S1中乙酸溶液质量分数为2-3wt％。
5.根据权利要求3所述的一种敷料的制备方法，其特征在于，所述步骤S1的CHI溶液中壳聚糖含量为0.5-1.5mg/mL，CMC溶液中羧甲基纤维素钠的含量为0.5-1.5mg/mL。
6.根据权利要求3所述的一种敷料的制备方法，其特征在于，所述步骤S1的CHI溶液和CMC溶液的pH为3.5-4.5，优选pH＝4。
7.根据权利要求3所述的一种敷料的制备方法，其特征在于，所述步骤S2中基底在CHI溶液和CMC溶液的浸入时间为8-12min。
8.根据权利要求3所述的一种敷料的制备方法，其特征在于，所述步骤S2中基底从CHI溶液和CMC溶液中取出后用去离子水洗涤3-5次。
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