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CN107325319B - 一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法及应用 - Google Patents

一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法及应用 Download PDF

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CN107325319B
CN107325319B CN201710570180.0A CN201710570180A CN107325319B CN 107325319 B CN107325319 B CN 107325319B CN 201710570180 A CN201710570180 A CN 201710570180A CN 107325319 B CN107325319 B CN 107325319B
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polyvinylidene fluoride
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刘梦龙
吴军
罗高兴
贺伟峰
邓君
龚雅利
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First Affiliated Hospital of TMMU
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Abstract

本发明公开了一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法及应用,所述制备方法包括:1)制备混悬液,将硝酸银分散溶解在N,N‑二甲基甲酰胺中,再加入聚偏氟乙烯,进行磁力搅拌;2)制备铸膜液,将混悬液置于摇床中,恒温反应;将反应后的混悬液置于真空烘箱中静置脱泡;3)成膜,将铸膜液由浸没沉淀相转化法得到固化薄膜;4)清洗;5)干燥,将清洗后的固化薄膜置于真空烘箱中干燥,得到多孔聚偏氟乙烯复合纳米银薄膜。制得的多孔聚偏氟乙烯复合纳米银薄膜用于制造人工皮肤,具有良好的抗菌性能,无明显生物毒性,制备方法工艺简单,耗时少,成本低。

Description

一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法及应用
技术领域
本发明涉及生物材料应用领域,具体涉及一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法及应用。
背景技术
皮肤是人体最重要的天然屏障,可以保护体内脏器,防止致病微生物的入侵及体液丢失等。创伤、烧伤、糖尿病溃疡等原因造成的皮肤创面,在愈合过程中极易受到致病微生物的侵袭而引起创面感染、化脓,导致创面延迟愈合甚至不愈合。因此,一种理想的人工皮肤应当具备良好的抗菌性能,一方面能够持续保护创面不受外界致病微生物的侵袭,另一方面也能杀灭已经定植于创面的细菌防止感染进一步播散,最终促进创面愈合,恢复皮肤的完整功能。
临床常用的抗菌制剂为抗生素,但是近年来由于抗生素的滥用,导致细菌耐药问题不断加剧,并由此催生了多种多重耐药菌,如耐甲氧西林金黄色葡萄球菌,鲍曼不动杆菌,铜绿假单胞菌等,严重威胁人类健康。所以,寻找能够替代抗生素的抗菌制剂十分必要。研究证实,纳米银不易受体内外因素的影响,可以很好地避免抗生素耐药问题。纳米银主要通过破坏细菌胞膜、DNA以及干扰细菌呼吸链酶的活性来杀灭细菌。它的粒径小,比表面积大,对革兰阴性菌和革兰阳性菌,均具有良好的杀菌作用;同时,纳米银还具有抗炎止痛、促进创面愈合的作用。
聚偏氟乙烯具有优良的机械性能、化学稳定性及热稳定性。同时无毒、无害、无致畸作用,具有良好的生物相容性。聚偏氟乙烯价格低廉,且容易批量加工生产。因此,它是一种优良的构建人工皮肤的支架材料。
目前,用于制备纳米银的化学还原法操作复杂,耗时耗力。此外,如何将纳米银有效地与聚偏氟乙烯复合来制备抗菌薄膜也面临一定挑战。
发明内容
本发明的目的是提供一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法及应用,其操作简单易行,制得的薄膜厚度可控,生物相容性好,抗菌效果佳。
本发明所述的多孔聚偏氟乙烯复合纳米银薄膜的制备方法,包括如下步骤:
1)制备混悬液:将硝酸银分散溶解在N,N-二甲基甲酰胺中,再加入聚偏氟乙烯,在温度为40-80℃和搅拌速度为1000-1500rpm的条件下磁力搅拌10-60min,得到混悬液,所述混悬液中硝酸银的浓度为0.1~1wt%,聚偏氟乙烯的浓度为5~20wt%;
2)制备铸膜液:将步骤1)得到的混悬液置于摇床中,在温度为25—60℃和转速为50—150rpm的条件下反应12—72h,再在温度为30—50℃的条件下静置脱泡1—6h,得到铸膜液;
3)成膜:将步骤2)得到的铸膜液倒入模具中,控制铸膜液的厚度为100—1000μm,再将含有厚度为100—1000μm的铸膜液的模具置于去离子水中静置10—60min,得到固化薄膜;
4)清洗:将步骤3)得到的固化薄膜先后用乙醇和去离子水清洗;
5)干燥:将步骤4)清洗后的固化薄膜置于真空烘箱中,在温度为30—50℃的条件下干燥8—16h,得到多孔聚偏氟乙烯复合纳米银薄膜,所述薄膜的孔径为0.5—2.5μm。
进一步,所述步骤1)中混悬液中硝酸银的浓度为0.425wt%,聚偏氟乙烯的浓度为10wt%。
进一步,所述步骤1)中在温度为60℃和搅拌速度1200rpm的条件下磁力搅拌30min。
进一步,所述步骤2)中在温度为37℃和转速为100rpm的条件下恒温反应24h,在温度为40℃的条件下静置脱泡1h。
进一步,所述步骤3)中模具的材质为玻璃、聚四氟乙烯或聚酯。
进一步,所述步骤3)中控制铸膜液的厚度为500μm,再将含有厚度为500μm的铸膜液的模具置于去离子水中静置30min,得到固化薄膜。
进一步,所述步骤5)中在温度为40℃的条件下干燥12h。
一种多孔聚偏氟乙烯复合纳米银薄膜在制备用于治疗人的皮肤缺损的人工皮肤或外科敷料中的用途。
进一步,所述皮肤缺损是皮肤全层缺损或皮肤感染创面。
对本发明的聚偏氟乙烯复合纳米银薄膜进行以下性能测试。
1.扫描电镜观察。
参见图1,采用扫描电子显微镜在不同倍数下观察制得的聚偏氟乙烯复合纳米银薄膜的表面结构。使用Image-Pro Plus 6.0软件,计算出聚偏氟乙烯复合纳米银薄膜表面孔洞的孔径为0.5—1.5μm。
2.透射电镜观察。
参见图2,采用透射电子显微镜观察制得的聚偏氟乙烯复合纳米银薄膜中纳米银的形态及大小。观察可知,纳米银在聚偏氟乙烯中无团聚现象,分散均匀;通过Image J软件测量纳米银颗粒的直径为1—5nm。
本发明的有益效果是:
1、本发明采用N,N-二甲基甲酰胺作为溶解剂和还原剂,将硝酸银和聚偏氟乙烯分散溶解于N,N-二甲基甲酰胺中,在搅拌速度为1000-1500rpm的条件下磁力搅拌,能够在聚偏氟乙烯中原位生成纳米银,纳米银颗粒直径为1—5nm;通过剧烈的磁力搅拌,无需添加致孔剂进行超声分散,即可实现纳米银在聚偏氟乙烯中的分散,无团聚现象,将纳米银的制备和纳米银与聚偏氟乙烯的复合一步完成。
2、本发明通过浸没沉淀相转化法制备多孔聚偏氟乙烯复合纳米银薄膜,通过N,N-二甲基甲酰胺与去离子水的相互作用,瞬时进行液液分相,最终得到特征性的含有空隙的包腔状结构,制备出孔径为0.5—1.5μm的多孔聚偏氟乙烯复合纳米银薄膜,若孔径过大则会降低薄膜的韧性,并且易受细菌的侵袭,若孔径过小则会降低薄膜的透气性,影响其在人工皮肤中或外科敷料中的应用。
3、本发明的制备过程中无废气、废液产生,对环境友好,且操作简单易行,成本低。
4、通过浸没沉淀相转化法所制备的聚合物膜通常具有多种结构,聚合物的选择、聚合物的浓度、溶剂/非溶剂体系的选择、铸膜液的组成和凝胶浴的组成对膜的结构和性能影响较大,本发明结合特定的原料配比和制备工艺,制备出孔径为0.5—1.5μm的多孔聚偏氟乙烯复合纳米银薄膜,其厚度可控,生物相容性好,抗菌效果佳,能够用于临床耐药菌感染创面的治疗。
附图说明
图1是本发明制备的聚偏氟乙烯复合纳米银薄膜不同倍数下的扫描电子显微镜照片;
图2是本发明制备的聚偏氟乙烯复合纳米银薄膜的透射电子显微镜照片;
图3是制备的多孔聚偏氟乙烯复合纳米银薄膜的细胞活性对比柱状图;
图4是制备的多孔聚偏氟乙烯复合纳米银薄膜的抗菌性能检测实验的吸光度对比柱状图;
图5是制备的多孔聚偏氟乙烯复合纳米银薄膜的动物实验的创面愈合率对比柱状图;
图6是制备的多孔聚偏氟乙烯复合纳米银薄膜的动物实验的创面愈合时间对比柱状图。
具体实施方式
下面结合具体实施例对本发明作详细说明。
实施例一:一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法,包括如下步骤:
1)制备混悬液:将硝酸银分散溶解在N,N-二甲基甲酰胺中,再加入聚偏氟乙烯,在温度为60℃和搅拌速度为1200rpm的条件下磁力搅拌30min,得到混悬液,所述混悬液中硝酸银的浓度为0.425wt%,聚偏氟乙烯的浓度为10wt%;
2)制备铸膜液:将步骤1)得到的混悬液置于摇床中,在温度为37℃和转速为100rpm的条件下反应24h,再在温度为40℃的条件下静置脱泡1h,得到铸膜液;
3)成膜:将步骤2)得到的铸膜液倒入模具中,控制铸膜液的厚度为500μm,再将含有厚度为500μm的铸膜液的模具置于去离子水中静置30min,得到固化薄膜;
4)清洗:将步骤3)得到的固化薄膜先后用乙醇和去离子水清洗;
5)干燥:将步骤4)清洗后的固化薄膜置于真空烘箱中,在温度为40℃的条件下干燥12h,得到多孔聚偏氟乙烯复合纳米银薄膜,所述薄膜的孔径为0.5—2.5μm。
实施例二:一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法,包括如下步骤:
1)制备混悬液:将硝酸银分散溶解在N,N-二甲基甲酰胺中,再加入聚偏氟乙烯,在温度为40℃和搅拌速度为1000rpm的条件下磁力搅拌10min,得到混悬液,所述混悬液中硝酸银的浓度为0.1wt%,聚偏氟乙烯的浓度为5wt%;
2)制备铸膜液:将步骤1)得到的混悬液置于摇床中,在温度为25℃和转速为50rpm下恒温反应12h,在温度为30℃的条件下静置脱泡1h,得到铸膜液;
3)成膜:将步骤2)得到的铸膜液倒入模具中,控制铸膜液的厚度为100μm,再将含有厚度为100μm的铸膜液的模具置于去离子水中静置10min,得到固化薄膜;
4)清洗:将步骤3)得到的固化薄膜先后用乙醇和去离子水清洗;
5)干燥:将步骤4)清洗后的固化薄膜置于真空烘箱中,在温度为30℃的条件下干燥8h,得到多孔聚偏氟乙烯复合纳米银薄膜,所述薄膜的孔径为0.5—2.5μm。
实施例三:一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法,包括如下步骤:
1)制备混悬液:将硝酸银分散溶解在N,N-二甲基甲酰胺中,再加入聚偏氟乙烯,在温度为80℃和搅拌速度为1500rpm的条件下磁力搅拌60min,得到混悬液,所述混悬液中硝酸银的浓度为1wt%,聚偏氟乙烯的浓度为20wt%;
2)制备铸膜液:将步骤1)得到的混悬液置于摇床中,在温度为30℃和转速为150rpm下恒温反应72h,再在温度为50℃的条件下静置脱泡6h,得到铸膜液;
3)成膜:将步骤2)得到的铸膜液倒入模具中,控制铸膜液的厚度为1000μm,再将含有厚度为1000μm的铸膜液的模具置于去离子水中静置60min,得到固化薄膜;
4)清洗:将步骤3)得到的固化薄膜先后用乙醇和去离子水清洗;
5)干燥:将步骤4)清洗后的固化薄膜置于真空烘箱中,在温度为50℃的条件下干燥16h,得到多孔聚偏氟乙烯复合纳米银薄膜,所述薄膜的孔径为0.5—2.5μm。
实施例四:一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法,包括如下步骤:
1)制备混悬液:将硝酸银分散溶解在N,N-二甲基甲酰胺中,再加入聚偏氟乙烯,在温度为50℃和搅拌速度为1100rpm的条件下磁力搅拌20min,得到混悬液,所述混悬液中硝酸银的浓度为0.2wt%,聚偏氟乙烯的浓度为7wt%;
2)制备铸膜液:将步骤1)得到的混悬液置于摇床中,在温度为45℃和转速为70rpm下恒温反应48h,再在温度为35℃的条件下静置脱泡2h,得到铸膜液;
3)成膜:将步骤2)得到的铸膜液倒入模具中,控制铸膜液的厚度为300μm,再将含有厚度为300μm的铸膜液的模具置于去离子水中静置40min,得到固化薄膜;
4)清洗:将步骤3)得到的固化薄膜先后用乙醇和去离子水清洗;
5)干燥:将步骤4)清洗后的固化薄膜置于真空烘箱中,在温度为35℃的条件下干燥10h,得到多孔聚偏氟乙烯复合纳米银薄膜,所述薄膜的孔径为0.5—2.5μm。
实施例五:一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法,包括如下步骤:
1)制备混悬液:将硝酸银分散溶解在N,N-二甲基甲酰胺中,再加入聚偏氟乙烯,在温度为70℃和搅拌速度为1400rpm的条件下磁力搅拌40min,得到混悬液,所述混悬液中硝酸银的浓度为0.7wt%,聚偏氟乙烯的浓度为15wt%;
2)制备铸膜液:将步骤1)得到的混悬液置于摇床中,在温度为50℃和转速为120rpm下恒温反应36h,再在温度为45℃的条件下静置脱泡4h,得到铸膜液;
3)成膜:将步骤2)得到的铸膜液倒入模具中,控制铸膜液的厚度为700μm,再将含有厚度为700μm的铸膜液的模具置于去离子水中静置50min,得到固化薄膜;
4)清洗:将步骤3)得到的固化薄膜先后用乙醇和去离子水清洗;
5)干燥:将步骤4)清洗后的固化薄膜置于真空烘箱中,在温度为45℃的条件下干燥14h,得到多孔聚偏氟乙烯复合纳米银薄膜,所述薄膜的孔径为0.5—2.5μm。
实施例六:为检验制备的用于人工皮肤的多孔聚偏氟乙烯复合纳米银薄膜的细胞活性,进行如下实验。
1)将由实施例一制得的样本裁剪成1×1cm大小的薄膜片,浸泡于75%乙醇溶液中消毒30min,然后用磷酸盐缓冲液漂洗3次;
2)将步骤1)处理后的薄膜片加入1mL含10%胎牛血清的1640培养液,在温度为37℃的条件下浸提24h,制得浸提液;
3)取对数生长期人表皮细胞系HaCaT细胞,以密度为3000个/孔接种至96孔板培养,24小时细胞贴壁生长后去除原培养基;
4)实验分组:
实验组:向步骤3)培养的HaCaT细胞按100μL/孔加入步骤2)制得的浸提液;
对照组:向步骤3)培养的HaCaT细胞加入等量的含10% 胎牛血清的1640培养基;实验组和对照组均复种4孔;
空白组:不含细胞,只加入含10% 胎牛血清的1640培养基;
5)培养三天,每天均进行细胞增殖检测实验,弃去原有的浸提液或培养基,用磷酸盐缓冲液分别对实验组及对照组的细胞洗涤两次,然后每孔加入100μL新的培养基和10μLCCK8试剂,所述CCK8试剂购自上海碧云天公司,在温度为37℃的条件下孵育2h后,用酶标仪在450nm波长处检测每孔的吸光度,计算实验组的细胞活性,计算公式为:(实验组吸光度—空白组吸光度)/ (对照组吸光度—空白组吸光度)×100%,根据计算结果绘制柱状图。
结果:参见图3,在接种后的第一天、第二天和第三天,实验组的细胞活性均在95%以上,与对照组的细胞活性相比无明显差异,提示制备的多孔聚偏氟乙烯复合纳米银薄膜无明显细胞毒性,安全性较高。
实施例七:为检验制备的用于人工皮肤的多孔聚偏氟乙烯复合纳米银薄膜对耐药菌的抗菌活性,进行如下实验:
1)实验分组:实验组:实施例一制得的多孔聚偏氟乙烯复合纳米银薄膜,对照组:浓度为1mg/mL的链霉素,浓度为10μM的四环素,浓度为10μM的头孢他啶,空白组:不加入任何试剂。
2)将由实施例一制得的样本裁剪成1×1cm大小的薄膜片,浸泡于75%乙醇溶液中消毒30min,然后用磷酸盐缓冲液漂洗3次;
3)取对数生长期多重耐药鲍曼不动杆菌,所述多重耐药鲍曼不动杆菌由第三军医大学西南医院烧伤科微生物室提供,用LB培养基稀释至酶标仪在600nm波长处检测吸光度为0.07的菌液,将稀释后的菌液按500μL/孔加入24孔板中;
4)分别向24孔板的每孔菌液中加入步骤2)制得的薄膜片、浓度为1mg/mL的链霉素、浓度为10μM的四环素和浓度为10μM的头孢他啶,再将24孔板置于温度为37℃和转速为50rpm的摇床中反应24h;
5)收取24孔板,每孔吸取100μL菌液至96孔板中,用酶标仪在600nm波长处检测吸光度值并记录,绘制柱状图。
结果:参见图4,实验组较空白组及对照组的吸光度值明显偏低,提示其能显著抑制鲍曼不动杆菌的生长,且效果明显好于抗生素,故多孔聚偏氟乙烯复合纳米银薄膜能够有效抑制耐药菌的生长,具有良好的抗菌效果。
实施例八:为检验制备的用于人工皮肤的多孔聚偏氟乙烯复合纳米银薄膜对体内细菌感染的抑制及对创面愈合的影响,进行如下实验:
1) BALB/C小鼠(购自第三军医大学实验动物中心)全层皮肤缺损创面模型的制备:小鼠予以1%戊巴比妥钠腹腔注射(100mg/kg,0.01ml/g)麻醉后,固定于俯卧位并予以背部术区备皮。而后用75%乙醇棉球消毒术区,用打孔器于背部两侧各造一直径为6mm的全层皮肤缺损创面,立即对创面进行拍照记录,该创面面积作为初始创面面积。
2)样本涂菌准备:将由实施例一制得的多孔聚偏氟乙烯复合纳米银薄膜及凡士林纱布裁剪成0.8×0.8cm大小,正反两面均用紫外线灭菌30min,取10μL稀释为0.5个标准麦氏单位的鲍曼不动杆菌菌液,涂布于样本表面,自然风干10min后备用。
3)实验组:将多孔聚偏氟乙烯复合纳米银薄膜涂菌面覆盖创面,并用负压吸引膜固定;对照组:将凡士林纱布涂菌面覆盖于创面,并用负压吸引膜固定;空白组:只用负压吸引膜贴附创面。取30只小鼠,随机分配10只小鼠到每组进行实验。
4)术后于第3天、第7天从三组中分别取5只小鼠打开创面拍照记录,观察创面愈合情况。小鼠于第7天后采用断颈法处死。剩余的15只小鼠(每组各剩余5只)在第八天去除固定的负压吸引膜,每日观察创面,待创面已完全上皮化即为创面完全愈合,记录创面完全愈合所需时间。
5)利用Image Pro Plus 6.0软件对第3天和第7天各组创面照片进行分析,测量剩余创面面积,即创缘所包围的面积,并按下式计算创面愈合率:
创面愈合率=(初始创面面积-剩余创面面积)/初始创面面积×100%;
6)采用单因素方差分析法分别对第3天和第7天三组创面愈合率及创面完全愈合所需时间进行统计学分析。
结果:多孔聚偏氟乙烯复合纳米银薄膜覆盖创面在第3天和第7天均未见红肿及脓液渗出,与空白组一致,而凡士林纱布组创面可见大量脓液渗出,创缘红肿。参见图5,各时间点创面愈合率的比较结果显示多孔聚偏氟乙烯复合纳米银薄膜较凡士林纱布组均明显较高,与无细菌感染的空白组无显著性差异,提示多孔聚偏氟乙烯复合纳米银薄膜可以有效防止耐药鲍曼不动杆菌的侵袭感染,维持创面正常的愈合微环境。参见图6,创面愈合时间的比较结果显示:多孔聚偏氟乙烯复合纳米银薄膜组较凡士林纱布组愈合时间明显缩短,提示多孔聚偏氟乙烯复合纳米银薄膜能防止细菌感染对创面愈合的不良影响。
以上列举的仅是本发明的具体实施例。显然,本发明不限于上述实施例,还可以有许多的操作组合。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有情形,均应当认为是本发明的保护范围。

Claims (9)

1.一种多孔聚偏氟乙烯复合纳米银薄膜的制备方法,其特征在于包括如下步骤:
1)制备混悬液:将硝酸银分散溶解在N,N-二甲基甲酰胺中,再加入聚偏氟乙烯,在温度为40-80℃和搅拌速度为1000-1500rpm的条件下磁力搅拌10-60min,得到混悬液,所述混悬液中硝酸银的浓度为0.1~1wt%,聚偏氟乙烯的浓度为5~20wt%;
2)制备铸膜液:将步骤1)得到的混悬液置于摇床中,在温度为25—60℃和转速为50—150rpm的条件下反应12—72h,再在温度为30—50℃的条件下静置脱泡1—6h,得到铸膜液;
3)成膜:将步骤2)得到的铸膜液倒入模具中,控制铸膜液的厚度为100—1000μm,再将含有厚度为100—1000μm的铸膜液的模具置于去离子水中静置10—60min,得到固化薄膜;
4)清洗:将步骤3)得到的固化薄膜先后用乙醇和去离子水清洗;
5)干燥:将步骤4)清洗后的固化薄膜置于真空烘箱中,在温度为30—50℃的条件下干燥8—16h,得到多孔聚偏氟乙烯复合纳米银薄膜,所述薄膜的孔径为0.5—2.5μm。
2.根据权利要求1所述的多孔聚偏氟乙烯复合纳米银薄膜的制备方法,其特征在于:所述步骤1)中混悬液中硝酸银的浓度为0.425wt%,聚偏氟乙烯的浓度为10wt%。
3.根据权利要求1或2所述的多孔聚偏氟乙烯复合纳米银薄膜的制备方法,其特征在于:所述步骤1)中在温度为60℃和搅拌速度1200rpm的条件下磁力搅拌30min。
4.根据权利要求1或2所述的多孔聚偏氟乙烯复合纳米银薄膜的制备方法,其特征在于:所述步骤2)中在温度为37℃和转速为100rpm的条件下恒温反应24h,在温度为40℃的条件下静置脱泡1h。
5.根据权利要求1或2所述的多孔聚偏氟乙烯复合纳米银薄膜的制备方法,其特征在于:所述步骤3)中模具的材质为玻璃、聚四氟乙烯或聚酯。
6.根据权利要求1或2所述的多孔聚偏氟乙烯复合纳米银薄膜的制备方法,其特征在于:所述步骤3)中控制铸膜液的厚度为500μm,再将含有厚度为500μm的铸膜液的模具置于去离子水中静置30min,得到固化薄膜。
7.根据权利要求1或2所述的多孔聚偏氟乙烯复合纳米银薄膜的制备方法,其特征在于:所述步骤5)中在温度为40℃的条件下干燥12h。
8.由权利要求1—7任一条所述的方法制得的多孔聚偏氟乙烯复合纳米银薄膜在制备用于治疗人的皮肤缺损的人工皮肤或外科敷料中的用途。
9.根据权利要求8所述的用途,所述皮肤缺损是皮肤全层缺损或皮肤感染创面。
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