CN109260508A - 一种具有促进皮肤成纤维细胞增殖功能性的聚乙烯醇/海藻酸钠水凝胶的制备方法 - Google Patents
一种具有促进皮肤成纤维细胞增殖功能性的聚乙烯醇/海藻酸钠水凝胶的制备方法 Download PDFInfo
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Abstract
本发明属于材料技术领域。具体公开了一种具有促进皮肤成纤维细胞增殖功能性的聚乙烯醇/海藻酸钠水凝胶的制备方法。本发明采用聚乙烯醇(PVA)和海藻酸钠(SA)为原材料,并将药物5‑羟甲基糠醛载入,通过两步法交联:反复冷冻‑解冻及浸泡于氯化钙溶液中;得到由聚乙烯醇和海藻酸钠制得而成具有缓释作用及促进皮肤成纤维细胞增殖的水凝胶。本发明的载药水凝胶能够很好地模拟人体皮肤细胞外基质(ECM)的环境,具有优异的生物相容性,对人体无害,提示对皮肤创面修复有促进愈合的作用,同时有作为理想的敷料的潜能。
Description
技术领域
本发明属于材料技术领域。涉及一种具有促进皮肤成纤维细胞增殖功能性的聚乙烯醇/海藻酸钠水凝胶的制备方法。
背景技术
5-羟甲基糠醛,是常见杂环美拉德反应的产物,主要通过果糖的酸催化热脱水生产并用作热处理产品中的调味物质。它具有很多优良的药理作用,如抗炎、抗氧化活性,防止急性低压缺氧抑制酒精性肝脏的氧化损伤和心脏保护作用等。
聚乙烯醇(PVA)作为一种人工合成高分子材料,是由聚醋酸乙烯酯经碱催化醇解而得,合成方便、价格便宜、对人体安全,具有良好的生物相容性,具有较好的缓释功能,是一种极具潜力的优良药用辅料。通过PVA制得的水凝胶有多种交联方法,可根据目的选取最适合自己的交联方法。本发明采用反复冷冻-解冻方法进行交联,本方法无毒方便,不使用化学试剂,对人体或细胞安全。
海藻酸钠(SA)是一种从褐海藻中分离得到的天然多糖,微溶于水且不容于大部分有机溶剂;在室温下对二价离子具有较好的亲和力,即在二价离子水溶液中能够快速的经过交联反应形成凝胶。利用这一特性,本发明采取氯化钙溶液对海藻酸钠进行交联得到凝胶。而海藻酸钠具有凝胶速率很难控制、结构不均一的特点,为解决这一难题,可以通过改性或者用其他体系来实现对凝胶速率的控制。
因此,本发明通过PVA和海藻酸钠两者混合,能够充分利用两者的优势,同时弥补各自的不足之处,同时将具有促进皮肤成纤维细胞增殖的药物5-羟甲基糠醛载入水凝胶中,以达到长效促进细胞增殖的作用,这种载药水凝胶可作为伤口敷料及有促进伤口愈合的潜能。
发明内容
本发明的目的在于提供一种方便有效的交联方法,具有缓释作用的水凝胶的制备方法。
本发明的技术方案如下。
一种具有促进皮肤成纤维细胞增殖功能性的聚乙烯醇/海藻酸钠水凝胶的制备方法,包括如下步骤:
(1)通过配制质量百分比浓度6-10 %聚乙烯醇(PVA)和质量百分比浓度1.5-3 %海藻酸钠(SA)高分子溶液,然后将PVA/SA按照体积比1-4:1均匀混合,通过磁力搅拌器搅拌过夜,静置脱泡得到均匀无色透明的溶液;
(2)在步骤(1)所得溶液中加入100-150 μg/mL的5-羟甲基糠醛(5-HMF),继续进行磁力搅拌,静置脱泡得到均匀透明的载药高分子溶液;
(3)将步骤(2)所得高分子溶液转移至模具内,在-18~-20 ℃进行反复冻融2-4个循环,使用质量百分比浓度1-3 %氯化钙(CaCl2)溶液进行交联,得到载药水凝胶;
(4)将步骤(3)得到的载药水凝胶放至4 ℃冰箱里保存或者经真空冷冻干燥制成水凝胶海绵后放在干燥箱里备用;
(5)将制得载药水凝胶经紫外灭菌后,将人皮肤成纤维细胞悬液种植于水凝胶上,在37℃ 5 %二氧化碳培养箱里培养24 h和48 h后,用CCK8试剂盒检测细胞增殖情况。
上述方法中,所述反复冷冻-解冻的具体步骤为:将放置有高分子溶液的模具放至在-18~-20 ℃冰箱中冷冻18-24 h后,放置在室温解冻2-4 h,如此重复2-4次。
本发明的所用的药物具有促进皮肤成纤维细胞增殖的作用,聚乙烯醇和海藻酸钠具有良好的生物相容性,水凝胶对药物具有缓释作用,保证药物长效发挥作用又能很好地模拟人体皮肤细胞外基质环境。
与现有技术相比,本发明具有如下的技术效果:
本发明制备工艺简单,得到的载药纳米水凝胶具有良好的生物相容性,载有促进皮肤成纤维细胞增殖的药物,能使药效得以持久,对皮肤成纤维细胞有增殖作用;采用两步交联法制得的水凝胶含水量大,机械性能好,生物相容性优异,扩大了其使用范围,能够作为理想的伤口敷料。
附图说明
图1为本发明实施实例1制备的聚乙烯醇/海藻酸纳米水凝胶的扫描电镜图。
图2为本发明实施实例1制备的载药PVA/海藻酸纳米水凝胶不同时间段促进皮肤成纤维细胞增殖的表达效果。
图3为本发明实施实例1制备的载药PVA/海藻酸纳米水凝胶药物累积释放曲线。
图4为本发明实施实例2制备的聚乙烯醇/海藻酸纳米水凝胶的扫描电镜图。
图5为本发明实施实例3制备的聚乙烯醇/海藻酸纳米水凝胶的扫描电镜图。
具体实施方式
实施例1
1、称取8 g聚乙烯醇溶于100 mL蒸馏水中,在90 ℃下用磁力搅拌器加热使其溶解,称取2 g海藻酸钠100 ml蒸馏水中,在80 ℃下用磁力搅拌器加热使其溶解;
2、将搅拌均匀的聚乙烯醇和海藻酸钠溶液按体积比PVA:SA=2:1混合均匀,继续用磁力搅拌器搅拌直至得到透明均一溶液;
3、按照100 μg/mL 的5-羟甲基糠醛(5-HMF)加入到混合好的溶液中,继续在磁力搅拌器中搅拌;
4、将混合均匀的溶液转移至相对应的模具中,放至在-20 ℃冰箱中冷冻20 h后,放置在室温解冻4 h,如此重复3次
5、将经过反复冷冻-解冻的水凝胶用2 %氯化钙溶液浸泡交联,得到交联后的水凝胶,然后用蒸馏水冲洗,洗去多余的氯化钙溶液,得到载药聚乙烯醇/海藻酸钠水凝胶;
图1为聚乙烯醇/海藻酸钠水凝胶=2:1混合的扫描电镜图片,从图中可以看出,与图4、5相比,随着海藻酸钠含量的增加,孔径也逐渐增大。
将大小均一的载药水凝胶放置96孔板中,经紫外线照射后将人皮肤成纤维细胞培养在水凝胶上,在37 ℃ 5 %二氧化碳培养箱里分别培养24 h和48 h后,用CCK8试剂检测细胞增殖情况。
测试药物累积释放率;将载药水凝胶浸泡在5 mL PBS溶液中,在不同的的时间点取样3 mL,然后补充3 mL新的PBS溶液,然后再OD=298 nm处测试吸光度,记录并绘制药物累积释放曲线,如图3所示。
实施例2
1、称取8 g聚乙烯醇溶于100 mL蒸馏水中,在90 ℃下用磁力搅拌器加热使其溶解,称取2 g海藻酸钠100 ml蒸馏水中,在80 ℃下用磁力搅拌器加热使其溶解;
2、将搅拌均匀的聚乙烯醇和海藻酸钠溶液按体积比PVA:SA=1:1混合均匀,继续用磁力搅拌器搅拌直至得到透明均一溶液;
3、按照100 μg/mL 的5-羟甲基糠醛(5-HMF)加入到混合好的溶液中,继续在磁力搅拌器中搅拌;
4、将混合均匀的溶液转移至相对应的模具中,放至在-20 ℃冰箱中冷冻20 h后,放置在室温解冻4 h,如此重复3次
5、将经过反复冷冻-解冻的水凝胶用2 %氯化钙溶液浸泡交联,得到交联后的水凝胶,然后用蒸馏水冲洗,洗去多余的氯化钙溶液,得到载药聚乙烯醇/海藻酸钠水凝胶;
图4为聚乙烯醇/海藻酸钠水凝胶=1:1混合的扫描电镜图片,从图中可以看出,与图1、5相比较,随着海藻酸钠含量的增加,孔径也逐渐增大。
实施例3
1、称取8 g聚乙烯醇溶于100 mL蒸馏水中,在90 ℃下用磁力搅拌器加热使其溶解,称取2 g海藻酸钠100 ml蒸馏水中,在80 ℃下用磁力搅拌器加热使其溶解;
2、将搅拌均匀的聚乙烯醇和海藻酸钠溶液按体积比PVA:SA=3:1混合均匀,继续用磁力搅拌器搅拌直至得到透明均一溶液;
3、按照100 μg/mL 的5-羟甲基糠醛(5-HMF)加入到混合好的溶液中,继续在磁力搅拌器中搅拌;
4、将混合均匀的溶液转移至相对应的模具中,放至在-20 ℃冰箱中冷冻20 h后,放置在室温解冻4 h,如此重复3次
5、将经过反复冷冻-解冻的水凝胶用2 %氯化钙溶液浸泡交联,得到交联后的水凝胶,然后用蒸馏水冲洗,洗去多余的氯化钙溶液,得到载药聚乙烯醇/海藻酸钠水凝胶;
图5为聚乙烯醇/海藻酸钠水凝胶=3:1混合的扫描电镜图片,从图中可以看出,与图1、4相比较,随着海藻酸钠含量的增加,孔径也逐渐增大。
Claims (2)
1.一种具有促进皮肤成纤维细胞增殖功能性的聚乙烯醇/海藻酸钠水凝胶的制备方法,其特征在于,包括如下步骤:
(1)通过配制质量百分比浓度6-10 %聚乙烯醇(PVA)和质量百分比浓度1.5-3 %海藻酸钠(SA)高分子溶液,然后将PVA/SA按照体积比1-4:1均匀混合,通过磁力搅拌器搅拌过夜,静置脱泡得到均匀无色透明的溶液;
(2)在步骤(1)所得溶液中加入100-150 μg/mL的5-羟甲基糠醛(5-HMF),继续进行磁力搅拌,静置脱泡得到均匀透明的载药高分子溶液;
(3)将步骤(2)所得高分子溶液转移至模具内,在-18~-20 ℃进行反复冻融2-4个循环,使用质量百分比浓度1-3 %氯化钙(CaCl2)溶液进行交联,得到载药水凝胶;
(4)将步骤(3)得到的载药水凝胶放至4 ℃冰箱里保存或者经真空冷冻干燥制成水凝胶海绵后放在干燥箱里备用。
2.根据权利要求1所述具有促进皮肤成纤维细胞增殖功能性的聚乙烯醇/海藻酸钠水凝胶的制备方法,其特征在于,所述反复冷冻-解冻的具体步骤为:将放置有高分子溶液的模具放至在-18~-20 ℃冰箱中冷冻18-24 h后,放置在室温解冻2-4 h,如此重复2-4次。
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