CN113018503B - 一种抗菌和促进伤口愈合的纳孔石墨烯/硝酸纤维素膜及其制备方法 - Google Patents

一种抗菌和促进伤口愈合的纳孔石墨烯/硝酸纤维素膜及其制备方法 Download PDF

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CN113018503B
CN113018503B CN202110317052.1A CN202110317052A CN113018503B CN 113018503 B CN113018503 B CN 113018503B CN 202110317052 A CN202110317052 A CN 202110317052A CN 113018503 B CN113018503 B CN 113018503B
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邱洪灯
杜少博
李湛
陈佳
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Lanzhou Institute of Chemical Physics LICP of CAS
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Abstract

本发明公开了一种具有抗菌和促进伤口愈合功能的纳孔石墨烯/硝酸纤维素膜的制备方法，是将纳孔石墨烯超声分散乙醇中，加入硝酸纤维素溶液超声并混合均匀得混合溶液，然后将混合溶液滴加到培养皿中，自然风干成膜即得。该膜具有良好的韧性与生物相容性，具备广谱抗菌性能和促进伤口愈合的能力，同时具有很好的水蒸气透过率，在创伤伤口敷料方面具临床应用潜力。

Description

一种抗菌和促进伤口愈合的纳孔石墨烯/硝酸纤维素膜及其 制备方法
技术领域
本发明涉及一种纳孔石墨烯复合材料，具体涉及一种具有广谱抗菌性能和促进伤口愈合功能的纳孔石墨烯/硝酸纤维素膜的制备方法，属于复合材料技术领域、抗菌材料和创伤伤口敷料。
背景技术
皮肤伤口治疗对人体健康至关重要，开发性能优良的新型伤口敷料一直是技术热点。目前，硝酸纤维素膜因其生物相容性良好，以及具有多孔网状结构可防止有害细菌进入伤口以避免伤口感染而被用于创面愈合。然而，硝酸纤维素膜表面孔的孔径太小而影响其水蒸气透过率将导致伤口分泌物聚集，且其抗菌性能有待提高。这些缺点使单一的硝酸纤维素膜在抗菌和促进伤口愈合领域的应用受到了限制。目前在临床应用中，为了提高创面敷料抗菌性能，通常在其中添加抗生素或非抗生素抗菌纳米材料。但抗生素耐药性问题与其造成的水土环境污染问题日益严重。
氧化石墨烯因其相对低廉的成本和较低的生物和环境毒性，正迅速成为一类新型非抗生素抗菌材料。然而，氧化石墨烯由于其强π-π共轭作用在溶液中易堆叠与团聚，因此仍不能有效改善硝酸纤维素膜的气体透过性而促进伤口快速愈合。
纳孔石墨烯是氧化石墨烯的衍生物，其片层具有独特纳米级孔隙。它作为非抗生素纳米抗菌材料含有大量sp 3 碳，比氧化石墨烯具有更优异的抗菌性能，且其片层携带的纳米孔及它具有的高比表面积和传质能力，可显著提升硝酸纤维素膜透气性。以此为基础，本发明在技术方案中提出了一种抗菌和促进伤口愈合的纳孔石墨烯/硝酸纤维素膜的制备方法。
发明内容
一、纳孔石墨烯/硝酸纤维素膜的制备
将纳孔石墨烯超声分散乙醇中，加入硝酸纤维素溶液（即火棉胶，硝酸纤维素的质量百分数为4~8%），超声并混合均匀得混合溶液，然后将混合溶液滴加到培养皿中，自然风干成膜即得。
纳孔石墨烯在混合溶液中的含量为0.001~0.006g/mL；硝酸纤维素溶液在混合溶液中的体积百分比为70%~90%。
纳孔石墨烯的制备参见CN 107619040 B：局部燃烧法快速制备多孔石墨烯的方法。具体为：将氧化石墨烯分散液与硝酸锌溶液磁力搅拌混合均匀；混合溶液中，氧化石墨烯的含量为1~6 g/L，硝酸锌的含量为100~1000 g/L；混合液用滤纸抽滤，将滤纸及截留混合物烘箱55℃干燥，然后于酒精灯点燃并充分焚烧；用10%盐酸反复洗涤焚烧产物后用蒸馏水洗涤至pH中性，离心收集纳孔石墨烯。
二、纳孔石墨烯/硝酸纤维素复合膜的性能
下面以实施例1制备的纳孔石墨烯/硝酸纤维素膜为例，说明本发明制备的纳孔石墨烯/硝酸纤维素膜的形貌及性能。
图1为实施例1所制备的纳孔石墨烯/硝酸纤维素膜表面（a）、截面（b）的扫描电镜图。可以看出，膜表面有纳孔石墨烯裸露，截面为疏松的层状结构。将膜对折4次后再展开，可以看出展开后的膜完好无损，表明其可弯曲折叠且具有良好的韧性（见图2）。
为测试膜的抗菌性能，将裁剪至大小合适的膜置于盖玻片上，后将处于对数生长期的革兰氏阴性菌大肠杆菌和革兰氏阳性菌金黄色葡萄球菌分别用PBS稀释至OD600 nm值为0.05，滴50μL稀释后的菌液于膜上，而后在其上再覆盖一片盖玻片，37°C培养2 h，后用2 mLPBS将膜与盖玻片彻底清洗，而后滴50μL清洗液于固体培养基并涂布均匀，37°C过夜培养后观察菌落数。图3为本实施例1与不含纳孔石墨烯的硝酸纤维素膜（对比例）的抗菌效果图。可以看出，本发明所制备的纳孔石墨烯/硝酸纤维素复合膜具有更优异的广谱抗菌性能。
为测试膜的细胞相容性，将裁剪至0.3 cm×0.3 cm的复合膜与人皮肤成纤维细胞分别共培养1天、2天和4天，用MTT法检测细胞活力。图4为本实施例和不含纳孔石墨烯的硝酸纤维素膜（对比例）的细胞相容性结果图。可以看出，本发明所制得的膜均具有良好的细胞相容性。
为测试纳孔石墨烯/硝酸纤维素膜的促进伤口愈合功能，将成年雄性昆明系小鼠麻醉后背部剔毛，75%乙醇擦拭待手术部位后，使用无菌手术剪在背部剪出0.5 cm×0.5 cm创口以制造全层皮肤缺损创面模型，创口分别用无菌纱布（空白对照）、不含纳孔石墨烯的硝酸纤维素膜（对比例）和本发明所制备的纳孔石墨烯/硝酸纤维素复合膜覆盖，并分别于第0天（手术结束时）、第3天与第7天进行观察并拍照记录。如图5、6所示，对比空白对照组，纳孔石墨烯/硝酸纤维素膜具有明显促进伤口愈合的功能。
为了测试纳孔石墨烯/硝酸纤维素复合膜的水蒸气透过率，将本发明制备的纳孔石墨烯/硝酸纤维素复合膜和不含纳孔石墨烯的硝酸纤维素膜（对比例）覆盖于盛有蒸馏水的离心管管口，并将管口密封。在30℃恒温干燥箱中放置24小时检测膜的水蒸气透过率。测试结果表明，硝酸纤维素复合膜的水蒸气透过率为：717.74 ± 5.77 (g·m−2·day−1)，而本发明实施例制备的孔石墨烯/硝酸纤维素复合膜的水蒸气透过率为：1007.94 ± 3.35(g·m−2·day−1)，较对比例提升率了40.43%。说明纳孔石墨烯的添加显著提升了硝酸纤维素膜的水蒸气透过率。
大量实验证明，本发明制备的纳孔石墨烯/硝酸纤维素复合膜均具有良好的韧性与生物相容性，具备广谱抗菌性能和促进伤口愈合的能力，在创伤伤口敷料方面具临床应用潜力。
附图说明
图1为本发明实施例1所制备的纳孔石墨烯/硝酸纤维素膜表面（a）与截面（b）的扫描电镜图。
图2为将膜对折4次再展开后的形貌图。
图3为本发明实施例1和对比例抑制大肠杆菌和金黄色葡萄球菌效果图。
图4为本发明实施例1和对比例与人皮肤成纤维细胞共培养1天、2天和4天的细胞相容性结果图。
图5为本发明实施例1和对比例促进小鼠皮肤伤口愈合效果图。
图6为本发明实施例1和对比例促进小鼠皮肤伤口愈合统计图。
具体实施方式
下面通过具体实施例对本发明纳孔石墨烯/硝酸纤维素膜的制备、性能及其应用效果做进一步说明。
实施例1
（1）将氧化石墨烯分散液与硝酸锌溶液混合，使前者终浓度为4g/L，后者终浓度为800 g/L，磁力搅拌混合均匀，混合液用滤纸抽滤，而后将滤纸及截留混合物烘箱55℃干燥，后于酒精灯点燃并充分焚烧。用10%盐酸反复洗涤焚烧产物后用蒸馏水洗涤至pH中性，离心收集纳孔石墨烯；
（2）取步骤（1）制备的纳孔石墨烯0.003g，加入至250 μL乙醇中，超声分散后加入750 μL硝酸纤维素溶液（火棉胶；含量：w/% 4-8%），继续超声并混合均匀，得混合溶液；
（3）将步骤（2）制得的混合溶液滴加至35mL培养皿，自然风干成膜，制得纳孔石墨烯/硝酸纤维素膜，纳孔石墨烯的含量为0.003 g/mL。该复合膜的水蒸气透过率为：1007.94± 3.35 (g·m−2·day−1)。且具有良好的韧性与生物相容性，具备广谱抗菌性能和促进伤口愈合的能力。
实施例2
（1）将氧化石墨烯分散液与硝酸锌溶液混合，使前者终浓度为6g/L，后者终浓度为800 g/L，磁力搅拌混合均匀，混合液用滤纸抽滤，而后将滤纸及截留混合物烘箱55℃干燥，后于酒精灯点燃并充分焚烧。用10%盐酸反复洗涤焚烧产物后用蒸馏水洗涤至pH中性，离心收集纳孔石墨烯；
（2）取步骤（1）制备的纳孔石墨烯0.001g，加入至250 μL乙醇中，超声分散后加入750 μL硝酸纤维素溶液（火棉胶；含量：w/%4-8%）继续超声并混合均匀，得混合溶液；
（3）将步骤（2）制得的混合溶液滴加至35mL培养皿，自然风干成膜，制得纳孔石墨烯/硝酸纤维素膜，其中纳孔石墨烯的含量为0.001g/mL。该复合膜具有良好的韧性与生物相容性，具备广谱抗菌性能和促进伤口愈合的能力。
实施例3
（1）将氧化石墨烯分散液与硝酸锌溶液混合，使前者终浓度为4g/L，后者终浓度为600 g/L，磁力搅拌混合均匀，混合液用滤纸抽滤，而后将滤纸及截留混合物烘箱55℃干燥，后于酒精灯点燃并充分焚烧。用10%盐酸反复洗涤焚烧产物后用蒸馏水洗涤至pH中性，离心收集纳孔石墨烯；
（2）取步骤（1）制备的纳孔石墨烯0.003g，加入至200 μL乙醇中，超声分散后加入800 μL硝酸纤维素溶液（火棉胶；含量：w/%4-8%）继续超声并混合均匀，得混合溶液；
（3）将步骤（2）制得的混合溶液滴加至35mL培养皿，自然风干成膜，制得纳孔石墨烯/硝酸纤维素膜，纳孔石墨烯的含量为0.003g/mL。该复合膜具有良好的韧性与生物相容性，具备广谱抗菌性能和促进伤口愈合的能力。
实施例4
（1）将氧化石墨烯分散液与硝酸锌溶液混合，使前者终浓度为4 g/L，后者终浓度为1000 g/L，磁力搅拌混合均匀，混合液用滤纸抽滤，而后将滤纸及截留混合物烘箱55℃干燥，后于酒精灯点燃并充分焚烧。用10%盐酸反复洗涤焚烧产物后用蒸馏水洗涤至pH中性，离心收集纳孔石墨烯；
（2）取步骤（1）制备的纳孔石墨烯0.006g，加入至150 μL乙醇中，超声分散后加入850 μL硝酸纤维素溶液（火棉胶；含量：w/% 4-8%）继续超声并混合均匀，得混合溶液；
（3）将步骤（2）制得的混合溶液滴加至35mL培养皿，自然风干成膜，制得纳孔石墨烯/硝酸纤维素膜，纳孔石墨烯的含量为0.006g/mL。该复合膜具有良好的韧性与生物相容性，具备广谱抗菌性能和促进伤口愈合的能力。

Claims (2)

1.一种具有抗菌和促进伤口愈合功能的纳孔石墨烯/硝酸纤维素膜的制备方法，是将纳孔石墨烯超声分散乙醇中，加入硝酸纤维素溶液，超声并混合均匀得混合溶液，然后将混合溶液滴加到培养皿中，自然风干成膜即得；
所述纳孔石墨烯的制备方法为：将氧化石墨烯分散液与硝酸锌溶液磁力搅拌混合均匀；混合液用滤纸抽滤，将滤纸及截留混合物烘箱55℃干燥，然后于酒精灯点燃并充分焚烧；用10%盐酸反复洗涤焚烧产物后用蒸馏水洗涤至pH中性，离心收集纳孔石墨烯；纳孔石墨烯在混合溶液中的含量为0.001~0.006g/mL；
所述硝酸纤维素溶液中硝酸纤维素的质量百分数为4-8%，在混合溶液中的体积百分比为70%~90%。
2.如权利要求1所述一种具有抗菌和促进伤口愈合功能的纳孔石墨烯/硝酸纤维素膜的制备方法，其特征在于：纳孔石墨烯制备方法的氧化石墨烯分散液与硝酸锌溶液混合液中，氧化石墨烯的含量为1~6 g/L，硝酸锌的含量为100~1000 g/L。
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