CN113069592A - 一种控释抗菌复合水凝胶及其制备方法与应用 - Google Patents
一种控释抗菌复合水凝胶及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种控释抗菌复合水凝胶,其包括水凝胶基体、以及负载于所述水凝胶基体中的金属多酚胶囊;所述水凝胶基体由甲基丙烯酸化透明质酸与甲基丙烯酸酯改性丝素蛋白交联形成,所述金属多酚胶囊包括如下组分:表没食子儿茶素‑3‑没食子酸酯和铜离子。本发明将EGCG和铜离子形成Cu‑EGCG配位网络,制得金属多酚胶囊并将其负载到水凝胶中,实现了EGCG和铜离子的持续缓释释放,在促进伤口血管生成的同时,抑制伤口发炎,提高了EGCG的生物利用度,降低了铜离子的毒性,更利于慢性伤口的愈合。同时,HAMA与Sil‑MA的复合交联可形成双网络结构的水凝胶,不仅具有良好的生物相容性,保持了聚合物的原始性能,而且还具有更好的机械性能,更符合伤口敷料的应用要求。
Description
技术领域
本发明属于医用生物材料技术领域,具体涉及一种控释抗菌复合水凝胶及其制备方法与应用。
背景技术
皮肤在保持动态平衡、以及防止微生物和化学物质的入侵方面起着至关重要的作用,是防止人体受到伤害、感染和脱水的第一道防线。但是,愈合能力有限的皮肤也极易受到诸如烧伤、青肿等创伤的伤害。通常,皮肤再生的优选选择是自体皮肤移植,但是供体皮肤的供不应求和在供体部位出现不可避免的新伤口限制了其广泛应用。因此,如今,伤口敷料已成为皮肤修复的主流疗法,以应对每年高的皮肤损伤发生率。
性能优良的伤口敷料应含有促进组织再生的成分,同时还需具备抗炎、抗菌和促血管化的成分。在各种类型的伤口敷料中,水凝胶具有独特的优势,例如保持潮湿的伤口环境,加速创口愈合,因而与其他材料相比,水凝胶被认为是最有希望的候选者。相比于生物惰性合成聚合物,基于天然聚合物的水凝胶具有令人满意的生物相容性和生物活性,受到越来越多的关注。透明质酸(HA)是一种天然聚合物,也是具有高保水能力和弹性的多糖,对伤口愈合有促进作用,是粘弹性组织的重要组成部分。但是,HA的机械强度较差,对细胞不具备粘性,由此阻碍了其在生物医学领域的应用。
发明内容
为解决上述现有技术中存在的不足之处,本发明的目的在于提供一种控释抗菌复合水凝胶及其制备方法与应用。
为达到其目的,本发明所采用的技术方案为:
一种控释抗菌复合水凝胶,其包括水凝胶基体、以及负载于所述水凝胶基体中的金属多酚胶囊;所述水凝胶基体由甲基丙烯酸化透明质酸与甲基丙烯酸酯改性丝素蛋白交联形成,所述金属多酚胶囊包括如下组分:表没食子儿茶素-3-没食子酸酯和铜离子。
表没食子儿茶素-3-没食子酸酯(EGCG)是绿茶中含量最丰富的多酚化合物,具有多种药理特性,包括杀菌、抗炎、抗氧化、抗衰老、促血管生成和抗癌作用。但是,若将EGCG直接负载到水凝胶中,会导致EGCG的生物利用度较低和代谢过快,不利于慢性伤口的愈合。此外,铜离子可有效促进血管生成和诱导VEGF的分泌,然而,铜引起毒性风险的增加限制了其广泛应用。
本发明将EGCG和铜离子形成配位网络,以金属多酚胶囊的形式负载到水凝胶中,实现了EGCG和铜离子的持续缓释释放,将EGCG精确地递送至伤口部位并以控释方式释放多酚物质,提高了EGCG的生物利用度,并降低了铜离子的毒性。而且,EGCG与铜离子联用,不仅可促进伤口血管生成,还能抑制炎症发生,更利于加速创口愈合。
同时,甲基丙烯酸化透明质酸(HAMA)与甲基丙烯酸酯改性丝素蛋白(Sil-MA)的复合交联可形成双网络结构的水凝胶,不仅具有良好的生物相容性,保持了聚合物(即HAMA和Sil-MA)的原始性能,而且还具有更好的机械性能,更符合伤口敷料的应用要求。
本发明还提供了一种所述控释抗菌复合水凝胶的制备方法,包括如下步骤:将甲基丙烯酸化透明质酸、甲基丙烯酸酯改性丝素蛋白、金属多酚胶囊和光引发剂加入去离子水中溶解,然后经光交联固化制得所述控释抗菌复合水凝胶。
优选地,所述控释抗菌复合水凝胶的制备方法中,按去离子水的体积计,甲基丙烯酸化透明质酸的添加量为0.5w/v%~2w/v%,更优选为1w/v%。
优选地,所述控释抗菌复合水凝胶的制备方法中,按去离子水的体积计,甲基丙烯酸酯改性丝素蛋白的添加量为4w/v%~12w/v%,更优选为8w/v%。
优选地,所述控释抗菌复合水凝胶的制备方法中,按去离子水的体积计,金属多酚胶囊的添加量为0.00125w/v%~0.05w/v%,更优选为0.025w/v%。
优选地,所述控释抗菌复合水凝胶的制备方法中,按去离子水的体积计,光引发剂的添加量为0.01w/v%~0.5w/v%,更优选为0.1w/v%。
优选地,所述控释抗菌复合水凝胶的制备方法中,光交联反应条件为紫外光照射10~120s,优选为30s。
优选地,所述金属多酚胶囊的制备方法,包括如下步骤:将表没食子儿茶素-3-没食子酸酯、铜盐溶液和CaCO3颗粒混合,得到浆液,将浆液进行涡旋处理后,加入洗涤液洗除多余的表没食子儿茶素-3-没食子酸酯和铜盐,最后获得表面包裹有膜层的CaCO3颗粒,所述膜层由表没食子儿茶素-3-没食子酸酯与铜离子配位键合形成;重复上述包覆过程,即可获得表面包裹有至少一层所述膜层的CaCO3颗粒;最后,将表面包裹有膜层的CaCO3颗粒浸入EDTA溶液中,去除CaCO3颗粒,获得金属多酚胶囊。
优选地,所述金属多酚胶囊的制备方法中,表没食子儿茶素-3-没食子酸酯的加入量为1~10mg,铜盐溶液的加入量为0.5mL,铜盐溶液的浓度为12~48mM,CaCO3颗粒的加入量为16~20mg。
优选地,所述洗涤液为MOPS缓冲溶液。
优选地,所述甲基丙烯酸化透明质酸的制备方法,包括如下步骤:将透明质酸完全溶于水中,然后加入甲基丙烯酸酐,调节pH值至8~9,常温下反应4~12h,透析,冷冻干燥,得到甲基丙烯酸化透明质酸。
优选地,所述甲基丙烯酸化透明质酸的制备方法中,按质量体积比计,透明质酸:甲基丙烯酸酐=2g:1~3mL,更优选为2g:1.6mL。
优选地,所述甲基丙烯酸化透明质酸的制备方法中,透析截留的分子量为3500Da。
优选地,所述甲基丙烯酸酯改性丝素蛋白的制备方法,包括如下步骤:将甲基丙烯酸缩水甘油酯加入丝素蛋白溶液中,进行接枝反应,反应完成后,离心,取上清液,过滤,透析,冷冻干燥,得到甲基丙烯酸酯改性丝素蛋白。
优选地,所述甲基丙烯酸酯改性丝素蛋白的制备方法中,透析截留的分子量为12~14kDa。
本发明还提供了所述控释抗菌复合水凝胶在医用敷料中的应用。
与现有技术相比,本发明的有益效果为:本发明将EGCG和铜离子形成Cu-EGCG配位网络,制得金属多酚胶囊并将其负载到甲基丙烯酸化透明质酸与甲基丙烯酸酯改性丝素蛋白交联形成的复合水凝胶中,实现了EGCG和铜离子的持续缓释释放,在促进伤口血管生成的同时,抑制伤口发炎,提高了EGCG的生物利用度,降低了铜离子的毒性,更利于慢性伤口的愈合。同时,HAMA与Sil-MA的复合交联可形成双网络结构的水凝胶,不仅具有良好的生物相容性,保持了聚合物的原始性能,而且还具有更好的机械性能,更符合伤口敷料的应用要求。
附图说明
图1为实施例2制备的HAMA聚合物的核磁氢谱图;
图2为实施例3制备的Sil-MA聚合物的核磁氢谱图;
图3为实施例1制备的Cu-EGCG的扫描电镜图;
图4为对比例2、对比例5和实施例5和制备的水凝胶的扫描电镜图;其中,A为对比例2制备的水凝胶的扫描电镜图,B为对比例5制备的水凝胶的扫描电镜图,C为实施例5制备的水凝胶的扫描电镜图;
图5为对比例2、对比例4、对比例5、对比例6和实施例5制备的水凝胶的溶胀性能图;
图6为实施例5制备的水凝胶的铜离子的释放性能曲线图;
图7为实施例5制备的水凝胶的EGCG的释放性能曲线图;
图8为对比例5、实施例4~6制备的水凝胶的细胞存活率统计图。
具体实施方式
下面通过具体实施例对本发明作进一步的说明,其目的在于帮助更好地理解本发明的内容,具体包括金属多酚胶囊(简称Cu-EGCG)、甲基丙烯酸化透明质酸(简称HAMA)、甲基丙烯酸酯改性丝素蛋白(简称Sil-MA)、以及控释抗菌复合水凝胶(简称HAMA/Sil-MA/Cu-EGCG水凝胶)的制备方法。但所描述的实施例仅仅是本发明的部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。除非另外指出,否则所有试剂都是商业获得的。
实施例1
金属多酚胶囊(Cu-EGCG)的制备方法,步骤如下:
首先制备浓度分别为20mM的CaCl2储备溶液和Na2CO3储备溶液,将PSS以1mg/mL的浓度分别溶解在上述两种储备溶液中。在剧烈搅拌下,将10mL含Na2CO3的储备溶液快速添加到10mL含CaCl2的储备溶液中,连续搅拌30秒,然后通过离心收集固体颗粒,并用去离子水洗涤3次,得到CaCO3颗粒。
向上述制备的16mg CaCO3颗粒中添加5mg EGCG和0.5mL浓度为24mM的CuCl2水溶液,得到浆液,将浆液涡旋10s,然后加入5mL MOPS缓冲溶液(100mM,pH 8.0),洗除多余的EGCG和CuCl2,过滤后获得表面包裹有一层膜层的CaCO3颗粒,该膜层由EGCG与铜离子形成Cu-EGCG配位网络而得。之后,重复上述包裹过程,即可获得表面包裹有三层膜层的CaCO3颗粒。最后,将颗粒浸入EDTA溶液(200mM,pH=8)中,重复两次,每次3min,去除CaCO3颗粒,从而获得Cu-EGCG胶囊。
实际上,上述制备方法中EGCG的添加量可以是1~10mg,CuCl2的浓度可以是12~48mM,膜层可以包裹2~5层,以上条件下均可满足本发明的性能要求。
实施例2
甲基丙烯酸化透明质酸(HAMA)的制备方法,步骤如下:
称取2g透明质酸溶解在100mL的蒸馏水中,待透明质酸完全溶解后,加入1.6mL的甲基丙烯酸酐,然后用浓度为1mol/L的氢氧化钠溶液调节反应体系的pH至8.5,然后常温下反应8h,在透析袋(截留分子量:3500Da)中用蒸馏水透析3~5日,最后冷冻干燥,得到HAMA。
实际上,上述制备方法中甲基丙烯酸酐的添加量可以是1~3mL,反应体系的pH值可以控制在8~9,反应时间可以是4~12h,以上条件下均可满足本发明的性能要求。
实施例3
甲基丙烯酸酯改性丝素蛋白(Sil-MA)的制备方法,步骤如下:
将去除蚕蛹的蚕茧放入1L浓度为0.05M的Na2CO3溶液中,于100℃煮沸20分钟,重复3次,以完全除去丝胶,然后用蒸馏水洗涤数次,60℃烘干以备下一步使用。
取20g上述脱胶后的蚕茧,溶于60℃的100mL浓度为9.3M的溴化锂(LiBr)溶液中1h。在用溴化锂溶解丝素蛋白(SF)后,立即将12mL甲基丙烯酸缩水甘油酯(GMA)引入混合溶液中,在60℃下以300rpm的速度反应6h,然后将所得溶液以8000r/min离心5min,取上清,用神奇滤布过滤,在透析袋(截留分子量:12~14kDa)中用蒸馏水透析3~5日,最后冷冻干燥,得到Sil-MA。
实际上,上述制备丝素蛋白溶液时,煮沸时间可以为10~60min;在制备Sil-MA时,GMA的加入量可以为8~20mL,反应时间可以是4~12h,离心条件可以为3000~10000r/min离心3~8min,以上条件下均可满足本发明的性能要求。
实施例4
控释抗菌复合水凝胶(HAMA/Sil-MA/Cu-EGCG水凝胶)的制备方法,步骤如下:
将0.02g实施例2制备的HAMA、0.16g实施例3制备的Sil-MA、25μg实施例1制备的Cu-EGCG和0.002g LAP光引发剂溶于2mL去离子水中,紫外光照30s,即得HAMA/Sil-MA/Cu-EGCG水凝胶。
实施例5
控释抗菌复合水凝胶(HAMA/Sil-MA/Cu-EGCG水凝胶)的制备方法,步骤如下:
将0.02g实施例2制备的HAMA、0.16g实施例3制备的Sil-MA、50μg实施例1制备的Cu-EGCG和0.002g LAP光引发剂溶于2mL去离子水中,紫外光照30s,即得HAMA/Sil-MA/Cu-EGCG水凝胶。
实施例6
控释抗菌复合水凝胶(HAMA/Sil-MA/Cu-EGCG水凝胶)的制备方法,步骤如下:
将0.02g实施例2制备的HAMA、0.16g实施例3制备的Sil-MA、100μg实施例1制备的Cu-EGCG和0.002g LAP光引发剂溶于2mL去离子水中,紫外光照30s,即得HAMA/Sil-MA/Cu-EGCG水凝胶。
对比例1
HAMA水凝胶的制备方法,步骤如下:
将0.01g实施例2制备的HAMA和0.002g LAP光引发剂溶于2mL去离子水中,紫外光照30s,即得HAMA水凝胶。
对比例2
HAMA水凝胶的制备方法,步骤如下:
将0.02g实施例2制备的HAMA和0.002g LAP光引发剂溶于2mL去离子水中,紫外光照30s,即得HAMA水凝胶。
对比例3
HAMA水凝胶的制备方法,步骤如下:
将0.04g实施例2制备的HAMA和0.002g LAP光引发剂溶于2mL去离子水中,紫外光照30s,即得HAMA水凝胶。
对比例4
HAMA/Sil-MA水凝胶的制备方法,步骤如下:
将0.02g实施例2制备的HAMA、0.08g实施例3制备的Sil-MA和0.002g LAP光引发剂溶于2mL去离子水中,紫外光照30s,即得HAMA/Sil-MA水凝胶。
对比例5
HAMA/Sil-MA水凝胶的制备方法,步骤如下:
将0.02g实施例2制备的HAMA、0.16g实施例3制备的Sil-MA和0.002g LAP光引发剂溶于2mL去离子水中,紫外光照30s,即得HAMA/Sil-MA水凝胶。
对比例6
HAMA/Sil-MA水凝胶的制备方法,步骤如下:
将0.02g实施例2制备的HAMA、0.24g实施例3制备的Sil-MA和0.002g LAP光引发剂溶于2mL去离子水中,紫外光照30s,即得HAMA/Sil-MA水凝胶。
试验测试
一、核磁检测
使用核磁共振光谱仪对实施例2制备的HAMA和实施例3制备的Sil-MA进行了1H-NMR谱图测定。
从图1可知,甲基丙烯酸基团的特征共振(δ=5.7ppm和6.2ppm),证实了实施例2制备的HAMA成功合成。从图2可知,甲基丙烯酸酯乙烯基的特征共振(δ=5.7ppm和6.1ppm),证实了实施例3制备的Sil-MA成功合成。
二、扫描电子显微镜(SEM)
分别将实施例1制备的Cu-EGCG胶囊,实施例5、对比例2和对比例5制备的水凝胶经冷冻干燥后放在专用样品盘上,经喷金后置入环境扫描电子显微镜,观察样品表面形貌。
从图3可知,实施例1制备的Cu-EGCG是不规则球形颗粒,表面光滑,平均直径约为100nm。
从图4可知,对比例2(图中A)、对比例5(图中B)和实施例5(图中C)制备的水凝胶所获得的SEM图像显示了所有样品的互连且均匀的多孔微观结构。这种相互连接的多孔结构有利于伤口愈合,因为它可以促进伤口缺损中营养物质和细胞废物的传输,以及细胞的增殖。此外,能明显看出对比例5和实施例5制备的水凝胶孔径相对于对比例2小,可能是由于Sil-MA的加入,增大了材料的交联密度,进而减小了材料的孔径。总体而言,这种多孔网络结构可以吸收多余的渗出物,加速氧气和水蒸气的自由传递,这适用于伤口敷料的应用。
三、水凝胶的溶胀性能
将对比例2、对比例4、对比例5、对比例6和实施例5制备的水凝胶置于0.1M PBS(pH=7.4)中于37℃进行测试。相隔一定的时间范围取出测试样品,表面水分通过称量纸擦拭。记录样品的初始体重(W0)和不同时间溶胀体重(Wt)。使用以下公式计算溶胀度:
用于生物医学的水凝胶,尤其是在伤口愈合的情况下,应在伤口部位保持相当潮湿的环境,并具有去除多余伤口渗出液以改善愈合的能力。图5为上述对比例2、对比例4、对比例5、对比例6和实施例5的五种水凝胶在37℃PBS中浸泡24小时后的溶胀特性。由于在透明质酸和丝素蛋白主链上存在易于水合的亲水基团,因此获得的结果表明,制备的水凝胶的溶胀度为1.3~1.6。同时可以看出,随着Sil-MA浓度的增加,光交联水凝胶的溶胀度逐渐降低,这也表明水凝胶中第二次交联的形成。而且,Cu-EGCG的加入对水凝胶的的溶胀度影响不大。
四、压缩性能测试
使用万能试验机测试样品的压缩弹性模量。将制备的等尺寸水凝胶(高度为6mm,直径为12mm的圆柱形状)在压缩率最大为60%的情况下进行压缩,压缩速率为0.05mm/s。
对于水凝胶伤口敷料而言,理想的机械性能是必不可少的,因为水凝胶需要足够的机械强度来维持其结构完整性并承受周围组织施加的力。如表1所示,对比例1~3的纯HAMA水凝胶中,对比例2~3的压缩性能明显大于对比例1,但是对比例3的水凝胶过于粘稠,不利于注射,因此以对比例2的HAMA浓度作为最优选择。比较对比例2和对比例4~6还可看出,在水凝胶中引入Sil-MA能显着改善水凝胶的机械性能。具体而言,与纯HAMA水凝胶相比,HAMA与Sil-MA的复合水凝胶由于具有较高的交联密度,因而压缩弹性模量显着提高。抗压强度从3.5±0.1kPa(对比例2制备的HAMA水凝胶)提高到48.3±2.9kPa(对比例5制备的HAMA/Sil-MA水凝胶),增加了约13倍,同时对比例5水凝胶的强度与皮肤正常组织的强度接近,更适合伤口愈合敷料的应用。此外实施例4、实施例5和实施例6制备的水凝胶与对比例5制备的HAMA/Sil-MA水凝胶的压缩性能无明显差异,证明Cu-EGCG的加入不会影响水凝胶的力学性能。
表1水凝胶的压缩强度
组别 | 压缩强度(kPa) |
对比例1 | 1.2±0.1 |
对比例2 | 3.5±0.1 |
对比例3 | 4.1±0.3 |
对比例4 | 27.2±2.6 |
对比例5 | 48.3±2.9 |
对比例6 | 76.5±4.8 |
实施例4 | 48.1±3.4 |
实施例5 | 48.6±3.0 |
实施例6 | 48.4±2.7 |
五、铜离子和EGCG释放性能
将实施例5制备的水凝胶放入20mL磷酸盐缓冲液中,置于恒温摇床中,温度为37℃,转速为100rpm。在不同的时间点,从PBS溶液中取出每个样品1mL,并用等量的新鲜PBS代替。用电感耦合等离子体质谱(ICP-MS)用于检测负载Cu-EGCG的水凝胶中铜离子的含量,计算累计释放速率。用紫外分光光度计用于检测负载Cu-EGCG的水凝胶中EGCG的含量,计算累计释放速率。
为了测定负载Cu-EGCG的实施例5制备的水凝胶对铜离子和EGCG的缓释能力,采用ICP-MS技术和紫外光谱检测技术分别测试了释放铜离子和EGCG的浓度,进而得到铜离子和EGCG的累计释放曲线。从图6和图7可以看出,该负载Cu-EGCG的水凝胶在14天内展现出铜离子和EGCG的持续释放。在PBS下,仅30%的铜离子和10%EGCG在24h内释放,之后水凝胶中铜离子和EGCG呈现逐渐缓慢释放的趋势,并且能持续释放14天以上。因此,本发明以Cu-EGCG负载于水凝胶中,有效解决了水凝胶直接负载铜离子时存在细胞毒性大、或直接负载EGCG时存在EGCG容易被氧化的技术问题。
六、抗菌性能
金黄色葡萄球菌和大肠杆菌是典型的革兰氏阳性和革兰氏阴性细菌。简而言之,将固态LB琼脂平板上的金黄色葡萄球菌和大肠杆菌的单菌落在固定温度(37℃)轻轻摇动(75rpm)下浸入40mL新鲜LB溶液中24h。然后将获得的细菌悬浮液稀释至105CFU/mL。将实施例4、实施例5和实施例6水凝胶添加到2mL细菌悬浮液中,以对比例5制备的水凝胶为对照组。然后将悬浮液在37℃下以75rpm的摇动速度温育。24h孵育后,计算菌落数并使用以下方程式评估抗菌率:
抗菌率(%)=(对照组细菌数-实验组细菌数)/对照组细菌数×100
由于许多基于生物材料的治疗失败案例都是由细菌感染引起的,因此具有抑菌作用的生物材料目前在临床应用中越来越受到关注,尤其是伤口敷料。如表2所示,与水凝胶直接接触24小时后,在HAMA/Sil-MA/Cu-EGCG水凝胶(即实施例4~6)处理组的琼脂平板上存活的大肠杆菌和金黄色葡萄球菌的存活率显着低于HAMA/Sil-MA对照组(即对比例5)。另外细菌生长的抑制率是Cu-EGCG浓度依赖性的。特别是,当Cu-EGCG浓度高达0.0025%时,大肠杆菌和金黄色葡萄球菌几乎都受到抑制。HAMA/Sil-MA/Cu-EGCG水凝胶之所以具有如此优异的抗菌能力,归因于Cu离子和EGCG的协同抗菌能力。
表2水凝胶的抗菌率
组别 | 大肠杆菌抗菌率(%) | 金黄色葡萄球菌抗菌率(%) |
实施例4 | 96.5 | 93.1 |
实施例5 | 100 | 99.5 |
实施例6 | 100 | 99.8 |
七、水凝胶生物相容性测试
将接种于96孔板(5×103个/孔)的3T3成纤维细胞分别与100μL不同水凝胶浸提液孵育24h、48h和72h。以不加任何样品只添加100μL细胞悬液为空白对照组。在指定的时间点,每孔加入100μL浓度为0.5mg/mL的MTT液,继续孵育4h。取出培养液,加入100μL二甲基亚砜溶解,以空白培养基为阴性对照组。用酶标仪测量570nm处的吸光度,根据公式计算细胞存活率。将对比例5、实施例4~6所制备的水凝胶进行生物相容性测试,测试结果如图8所示。
为了用作伤口敷料,生物相容的水凝胶是生物医学应用中的先决条件。本研究选择了NIH-3T3成纤维细胞,因为它们在伤口愈合中起着至关重要的作用。通过CCK-8测定法对细胞增殖进行的定量分析也证实了水凝胶的生物相容性。细胞毒性如图8所示,对比例5所制备水凝胶的细胞存活率高于空白对照组,说明本发明所用的天然聚合物HAMA和Sil-MA具有良好的生物相容性。而实施例6所制备水凝胶的细胞存活率低于对比例5所制备水凝胶,这是由于Cu-EGCG浓度过高所致。同时,实施例5所制备水凝胶的细胞存活率最高,这是由于水凝胶释放出EGCG,EGCG的儿茶酚基团可以通过与咪唑或硫醇在细胞膜上的相互作用而与成纤维细胞形成牢固的接触,能够促进细胞的增殖和迁移。因此,实施例5中的Cu-EGCG加入量为最佳的Cu-EGCG加入浓度。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
1.一种控释抗菌复合水凝胶,其特征在于,包括水凝胶基体、以及负载于所述水凝胶基体中的金属多酚胶囊;所述水凝胶基体由甲基丙烯酸化透明质酸与甲基丙烯酸酯改性丝素蛋白交联形成,所述金属多酚胶囊包括如下组分:表没食子儿茶素-3-没食子酸酯和铜离子。
2.一种如权利要求1所述的控释抗菌复合水凝胶的制备方法,其特征在于,包括如下步骤:将甲基丙烯酸化透明质酸、甲基丙烯酸酯改性丝素蛋白、金属多酚胶囊和光引发剂加入去离子水中溶解,然后经光交联固化制得所述控释抗菌复合水凝胶。
3.如权利要求2所述的控释抗菌复合水凝胶的制备方法,其特征在于,按去离子水的体积计,所述甲基丙烯酸化透明质酸的添加量为0.5w/v%~2w/v%,所述甲基丙烯酸酯改性丝素蛋白的添加量为4w/v%~12w/v%,所述金属多酚胶囊的添加量为0.00125w/v%~0.05w/v%,所述光引发剂的添加量为0.01w/v%~0.5w/v%。
4.如权利要求2所述的控释抗菌复合水凝胶的制备方法,其特征在于,所述光交联反应条件为紫外光照射10~120s。
5.如权利要求2所述的控释抗菌复合水凝胶的制备方法,其特征在于,所述金属多酚胶囊的制备方法,包括如下步骤:将表没食子儿茶素-3-没食子酸酯、铜盐溶液和CaCO3颗粒混合,得到浆液,将浆液进行涡旋处理后,加入洗涤液洗除多余的表没食子儿茶素-3-没食子酸酯和铜盐,最后获得表面包裹有膜层的CaCO3颗粒,所述膜层由表没食子儿茶素-3-没食子酸酯与铜离子配位键合形成;重复上述包覆过程,即可获得表面包裹有至少一层所述膜层的CaCO3颗粒;最后,将表面包裹有膜层的CaCO3颗粒浸入EDTA溶液中,去除CaCO3颗粒,获得金属多酚胶囊。
6.如权利要求5所述的控释抗菌复合水凝胶的制备方法,其特征在于,所述金属多酚胶囊的制备方法中,表没食子儿茶素-3-没食子酸酯的加入量为1~10mg,铜盐溶液的加入量为0.5mL,铜盐溶液的浓度为12~48mM,CaCO3颗粒的加入量为16~20mg。
7.如权利要求2所述的控释抗菌复合水凝胶的制备方法,其特征在于,所述甲基丙烯酸化透明质酸的制备方法,包括如下步骤:将透明质酸完全溶于水中,然后加入甲基丙烯酸酐,调节pH值至8~9,常温下反应4~12h,透析,冷冻干燥,得到甲基丙烯酸化透明质酸。
8.如权利要求7所述的控释抗菌复合水凝胶的制备方法,其特征在于,所述甲基丙烯酸化透明质酸的制备方法中,按质量体积比计,透明质酸:甲基丙烯酸酐=2g:1~3mL。
9.如权利要求2所述的控释抗菌复合水凝胶的制备方法,其特征在于,所述甲基丙烯酸酯改性丝素蛋白的制备方法,包括如下步骤:将甲基丙烯酸缩水甘油酯加入丝素蛋白溶液中,进行接枝反应,反应完成后,离心,取上清液,过滤,透析,冷冻干燥,得到甲基丙烯酸酯改性丝素蛋白。
10.如权利要求2所述的控释抗菌复合水凝胶在医用敷料中的应用。
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