CN114230808A - 一种以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法 - Google Patents
一种以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法 Download PDFInfo
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Abstract
本发明公开了一种以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法，包括：配制氨基聚乙二醇溶液和邻苯二酚溶液；将氨基聚乙二醇溶液、邻苯二酚溶液、壳聚糖、卡波姆、三乙醇胺、羟乙基纤维素、尼泊金甲酯钠、魔芋葡甘聚糖和H2O2混合后，调节pH，搅拌，加压超声，得到抗菌凝胶。本发明的以氨基聚乙二醇为凝胶基质的抗菌凝胶对金黄色葡萄球菌和大肠杆菌具有非常好的抑制作用，此外本发明的抗菌凝胶材料的不会抑制细胞活性，具有优良的应用前景。
Description
技术领域
本发明涉及医用敷料技术领域，具体涉及一种以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法。
背景技术
在处理病人伤口时，需要在伤口上覆盖敷料，防止细菌侵入和防止水分损失。传统的敷料如纱布、棉垫等对创面虽有保护作用，但止血效果不满意，没有保湿作用，一般认为对创面愈合没有促进作用。而添加有抗菌剂的凝胶类敷料能克服传统敷料的一些不足，在医药领域被广泛的使用。凝胶被广泛应用于治疗外科创伤、皮肤炎症、妇科炎症、直肠炎症。许多凝胶类敷料或药物，含有的抗菌成分有很大的副作用。
抗菌凝胶敷料是一种强吸水能力的三维网状结构高分子胶状物质，且可促进伤口愈合，是一种性能优异的新型创伤敷料。壳聚糖、透明质酸、海藻酸钠、胶原等天然聚合物是制备水凝胶敷料的常用原料，这些天然聚合物大多具有一定的抗菌活性，可赋予水凝胶抗菌性能，但是现有技术中的水凝胶制备过程较为复杂，工艺控制难度较高，制备得到的水凝胶抗菌效果差，因此需要一种制备方法较为简单，同时还具有较好的抗菌效果的医用凝胶材料。
发明内容
本发明的一个目的是解决至少上述问题和/或缺陷，并提供至少后面将说明的优点。
为了实现根据本发明的这些目的和其它优点，提供了一种以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法，其特征在于，包括以下步骤：
步骤一、将氨基聚乙二醇加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为1～3g/mL的氨基聚乙二醇溶液；将邻苯二酚加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为20～30mg/mL的邻苯二酚溶液；
步骤二、按重量份，将10～12份氨基聚乙二醇溶液、5～8份邻苯二酚溶液、1.5～2.5份壳聚糖、0.1～0.3份卡波姆、三乙醇胺0.5～1份、0.1～0.2份羟乙基纤维素、0.2～0.3份尼泊金甲酯钠、0.05～0.1份魔芋葡甘聚糖和0.1～0.2份质量分数为30％的H2O2混合后，调节pH至8.5～9，并以1500～2000r/min的速度搅拌5～10min，然后加压超声30～60min，得到抗菌凝胶。
优选的是，其特征在于，所述加压超声的压力为0.5～0.8MPa；超声频率为60～75KHz，超声功率为200W～300W。
优选的是，所述壳聚糖替换为改性壳聚糖，其制备方法为：按重量份，将12～16份壳聚糖、4～6份氨基酸、3～5份1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1～2份N-羟基琥珀酰亚胺加入超临界二氧化碳反应器中，通入二氧化碳，在温度为40～60℃，压力为12～25MPa下搅拌反应3～5h，然后以1～2MPa/min的速度泄压，将泄压后的物料加入微波超声波一体化反应器中，同时加入加入10～12份质量分数为20～30％的硝酸锌溶液，同时开启微波和超声波进行协同处理60～90min，过滤，洗涤，干燥，得到改性壳聚糖。
优选的是，所述氨基酸为甘氨酸、缬氨酸、苯丙氨酸、精氨酸、谷氨酸、天冬酰胺、谷氨酰胺、色氨酸中的一种或者几种。
优选的是，所述微波的功率为200～350W；超声波功率为400～600W，超声频率为40～60KHz；处理温度为40～60℃。
本发明至少包括以下有益效果：本发明的以氨基聚乙二醇为凝胶基质的抗菌凝胶对金黄色葡萄球菌和大肠杆菌具有非常好的抑制作用，此外本发明的抗菌凝胶材料的不会抑制细胞活性，具有优良的应用前景。
本发明的其它优点、目标和特征将部分通过下面的说明体现，部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。
附图说明：
图1为实施例4制备的抗菌凝胶冷冻干燥后的SEM图(500X)；
图2为实施例4制备的抗菌凝胶冷冻干燥后的SEM图(5KX)；
图3为实施例4制备的抗菌凝胶冷冻干燥后的SEM图(1KX)；
图4为实施例4制备的抗菌凝胶冷冻干燥后的SEM图(20KX)；
图5为实施例4的抗菌凝胶冷冻干燥后再水合，测量得到的溶胀比曲线；
图6为实施例4制备的抗菌凝胶的大肠杆菌抑菌圈试验；
图7为实施例4制备的抗菌凝胶的金黄色葡萄球菌抑菌圈试验；
图8为实施例2和4制备的抗菌凝胶的细胞活性检测细泡存活率图；
图9为对照组生理盐水的动物烧伤抗菌验证SEM扫描图(大肠杆菌)；
图10为对照组生理盐水的动物烧伤抗菌验证SEM扫描图(大肠杆菌)；
图11为实施例4制备的抗菌凝胶的动物烧伤抗菌验证SEM扫描图(大肠杆菌)；
图12为实施例4制备的抗菌凝胶的动物烧伤抗菌验证SEM扫描图(大肠杆菌)；
图13为对照组生理盐水的动物烧伤抗菌验证SEM扫描图(金黄色葡萄球菌)；
图14为对照组生理盐水的动物烧伤抗菌验证SEM扫描图(金黄色葡萄球菌)；
图15为实施例4制备的抗菌凝胶的动物烧伤抗菌验证SEM扫描图(金黄色葡萄球菌)；
图16为实施例4制备的抗菌凝胶的动物烧伤抗菌验证SEM扫描图(金黄色葡萄球菌)。
具体实施方式：
下面结合附图对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。
应当理解，本文所使用的诸如“具有”、“包含”以及“包括”术语并不配出一个或多个其它元件或其组合的存在或添加。
对实施例1～4得到的抗菌凝胶依据标准GB15979-2002《一次性使用卫生用品卫生标准》进行抗菌性能测试，测试用菌为大肠杆菌、金黄色葡萄球菌。
实施例1：
一种以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法，包括以下步骤：
步骤一、将氨基聚乙二醇加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为2.4g/mL的氨基聚乙二醇溶液；将邻苯二酚加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为26.4mg/mL的邻苯二酚溶液；
步骤二、将10g氨基聚乙二醇溶液、5g邻苯二酚溶液、1.5g壳聚糖、0.1g卡波姆、三乙醇胺0.5g、0.1g羟乙基纤维素、0.2g尼泊金甲酯钠、0.05g魔芋葡甘聚糖和0.1g质量分数为30％的H2O2混合后，调节pH至8.5，并以1500r/min的速度搅拌5min，然后加压超声60min，得到抗菌凝胶；所述加压超声的压力为0.6MPa；超声频率为60KHz，超声功率为200W；
经测试，本实施例中得到的抗菌凝胶对大肠杆菌、金黄色葡萄球菌的抑菌率分别为97.5％、97.7％。
实施例2：
一种以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法，包括以下步骤：
步骤一、将氨基聚乙二醇加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为2.4g/mL的氨基聚乙二醇溶液；将邻苯二酚加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为26.4mg/mL的邻苯二酚溶液；
步骤二、将12g氨基聚乙二醇溶液、8g邻苯二酚溶液、2g壳聚糖、0.3g卡波姆、三乙醇胺0.8g、0.1g羟乙基纤维素、0.3g尼泊金甲酯钠、0.1g魔芋葡甘聚糖和0.2g质量分数为30％的H2O2混合后，调节pH至9，并以2000r/min的速度搅拌5min，然后加压超声60min，得到抗菌凝胶；所述加压超声的压力为0.8MPa；超声频率为60KHz，超声功率为200W；
经测试，本实施例中得到的抗菌凝胶对大肠杆菌、金黄色葡萄球菌的抑菌率分别为97.3％、97.7％。
实施例3：
一种以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法，包括以下步骤：
步骤一、将氨基聚乙二醇加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为2.4g/mL的氨基聚乙二醇溶液；将邻苯二酚加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为26.4mg/mL的邻苯二酚溶液；
步骤二、将10g氨基聚乙二醇溶液、5g邻苯二酚溶液、1.5g改性壳聚糖、0.1g卡波姆、三乙醇胺0.5g、0.1g羟乙基纤维素、0.2g尼泊金甲酯钠、0.05g魔芋葡甘聚糖和0.1g质量分数为30％的H2O2混合后，调节pH至8.5，并以1500r/min的速度搅拌5min，然后加压超声60min，得到抗菌凝胶；所述加压超声的压力为0.6MPa；超声频率为60KHz，超声功率为200W；
所述壳聚糖替换为改性壳聚糖，其制备方法为：按重量份，将13g壳聚糖、4g甘氨酸、3g 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1g N-羟基琥珀酰亚胺加入超临界二氧化碳反应器中，通入二氧化碳，在温度为60℃，压力为20MPa下搅拌反应3h，然后以1MPa/min的速度泄压，将泄压后的物料加入微波超声波一体化反应器中，同时加入加入10g质量分数为20％的硝酸锌溶液，同时开启微波和超声波进行协同处理60min，过滤，洗涤，干燥，得到改性壳聚糖；所述微波的功率为300W；超声波功率为500W，超声频率为45KHz；处理温度为55℃；
经测试，本实施例中得到的抗菌凝胶对大肠杆菌、金黄色葡萄球菌的抑菌率分别为99.5％、99.8％。
实施例4：
一种以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法，包括以下步骤：
步骤一、将氨基聚乙二醇加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为2.4g/mL的氨基聚乙二醇溶液；将邻苯二酚加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为26.4mg/mL的邻苯二酚溶液；
步骤二、将12g氨基聚乙二醇溶液、8g邻苯二酚溶液、2g改性壳聚糖、0.3g卡波姆、三乙醇胺0.8g、0.1g羟乙基纤维素、0.3g尼泊金甲酯钠、0.1g魔芋葡甘聚糖和0.2g质量分数为30％的H2O2混合后，调节pH至9，并以2000r/min的速度搅拌5min，然后加压超声60min，得到抗菌凝胶；所述加压超声的压力为0.8MPa；超声频率为60KHz，超声功率为200W；
所述壳聚糖替换为改性壳聚糖，其制备方法为：按重量份，将15g壳聚糖、5g精氨酸、4g 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1g N-羟基琥珀酰亚胺加入超临界二氧化碳反应器中，通入二氧化碳，在温度为60℃，压力为20MPa下搅拌反应3h，然后以1MPa/min的速度泄压，将泄压后的物料加入微波超声波一体化反应器中，同时加入加入12g质量分数为25％的硝酸锌溶液，同时开启微波和超声波进行协同处理60min，过滤，洗涤，干燥，得到改性壳聚糖；所述微波的功率为300W；超声波功率为500W，超声频率为45KHz；处理温度为50℃；
经测试，本实施例中得到的抗菌凝胶对大肠杆菌、金黄色葡萄球菌的抑菌率分别为99.6％、99.8％。
图1～4为实施例4制备的抗菌凝胶冷冻干燥后的SEM图；
图5为实施例4的抗菌凝胶冷冻干燥后再水合，测量得到的溶胀比，在再水合后的几个时间点(2、4、6、8、10、12和14小时)测量水凝胶的湿重；水凝胶的溶胀比使用以下等式计算:(Ws-Wi)/Wi×100％，其中Ws表示在每个时间点溶胀水凝胶的重量；
图6为实施例4制备的抗菌凝胶的大肠杆菌抑菌圈试验；首先，将处于对数生长期中期的50uL细菌(大肠杆菌)悬浮液铺在LB琼脂平板的表面上；然后，将孵育有水凝胶(抗菌凝胶)药敏纸片(直径＝0.5厘米)(图6中b和c)和对照空白药敏纸片(直径＝0.5厘米)(图6中b和c)放在琼脂板上，并在37℃孵育过夜24h拍照，图6中b和c形成抑菌圈的为水凝胶(抗菌凝胶)药敏纸片，未形成抑菌圈的为对照空白药敏纸片；图6中a未放置药敏纸片；从图6中b和c可以得到抑菌圈大小为11.097±1.855mm；
图7为实施例4制备的抗菌凝胶的金黄色葡萄球菌抑菌圈试验；首先，将处于对数生长期中期的50uL细菌(金黄色葡萄球菌)悬浮液铺在LB琼脂平板的表面上；然后，将孵育有水凝胶(抗菌凝胶)药敏纸片(直径＝0.5厘米)(图7中b和c)和对照空白药敏纸片(直径＝0.5厘米)(图7中b和c)放在琼脂板上，并在37℃孵育过夜24h拍照，图7中b和c形成抑菌圈的为水凝胶(抗菌凝胶)药敏纸片，未形成抑菌圈的为对照空白药敏纸片；图7中a未放置药敏纸片；从图7中b和c可以得到抑菌圈大小为14.749±1.221mm；
图8为实施例2和4制备的抗菌凝胶的细胞活性检测：96孔板，L929细胞0.5万/孔中，14小时后，分别加入含抗菌凝胶培养液，24/48h后加入cck8试剂，加入含10％cck8培养液培养3h后进行酶标仪检测得出OD值，确定凝胶对细胞活性的影响；其中图8中对照为不加抗菌凝胶得到的结果；
图9～16为实施例6制备的抗菌凝胶的动物烧伤抗菌验证；6-8周龄SD大鼠；老鼠可以自由饮水，并且可以自由饮水；在实验之前，通过以每千克体重30毫克的剂量腹膜内注射10％水合氯醛来麻醉小鼠；用直径为1.5的黄铜圆筒在麻醉小鼠的剃过的背上造成部分厚度的烧伤，该圆筒在90℃的水浴中加热10分钟，并在小鼠皮肤上按压6秒钟；每组至少使用6只小鼠；将含有1×108CFU/毫升大肠杆菌(图9～12)或金黄色葡萄球菌(图13～16)的10微升等份细菌悬液移至烧伤创面；10分钟后，将水凝胶(抗菌凝胶)(直径＝1.5cm)涂于烧伤创面，以相同大小的对照(生理盐水)组作为对照组；3天后，收集伤口部位的皮肤组织样本进行SEM扫描(其中图9～10、图13～14为生理盐水对照的结果；图11～12、图15～16为涂覆抗菌凝胶的结果)。
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本发明的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节和这里示出与描述的图例。
Claims (5)
1.一种以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法，其特征在于，包括以下步骤：
步骤一、将氨基聚乙二醇加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为1～3g/mL的氨基聚乙二醇溶液；将邻苯二酚加入到10mmol/L的Tris-Hcl缓冲液中，配制浓度为20～30mg/mL的邻苯二酚溶液；
步骤二、按重量份，将10～12份氨基聚乙二醇溶液、5～8份邻苯二酚溶液、1.5～2.5份壳聚糖、0.1～0.3份卡波姆、三乙醇胺0.5～1份、0.1～0.2份羟乙基纤维素、0.2～0.3份尼泊金甲酯钠、0.05～0.1份魔芋葡甘聚糖和0.1～0.2份质量分数为30％的H2O2混合后，调节pH至8.5～9，并以1500～2000r/min的速度搅拌5～10min，然后加压超声30～60min，得到抗菌凝胶。
2.如权利要求1所述的以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法，其特征在于，所述加压超声的压力为0.5～0.8MPa；超声频率为60～75KHz，超声功率为200W～300W。
3.如权利要求1所述的以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法，其特征在于，所述壳聚糖替换为改性壳聚糖，其制备方法为：按重量份，将12～16份壳聚糖、4～6份氨基酸、3～5份1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1～2份N-羟基琥珀酰亚胺加入超临界二氧化碳反应器中，通入二氧化碳，在温度为40～60℃，压力为12～25MPa下搅拌反应3～5h，然后以1～2MPa/min的速度泄压，将泄压后的物料加入微波超声波一体化反应器中，同时加入加入10～12份质量分数为20～30％的硝酸锌溶液，同时开启微波和超声波进行协同处理60～90min，过滤，洗涤，干燥，得到改性壳聚糖。
4.如权利要求3所述的以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法，其特征在于，所述氨基酸为甘氨酸、缬氨酸、苯丙氨酸、精氨酸、谷氨酸、天冬酰胺、谷氨酰胺、色氨酸中的一种或者几种。
5.如权利要求3所述的以氨基聚乙二醇为凝胶基质的抗菌凝胶的制备方法，其特征在于，所述微波的功率为200～350W；超声波功率为400～600W，超声频率为40～60KHz；处理温度为40～60℃。
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