CN113616805B - 一种壳聚糖水凝胶及其制备方法和应用 - Google Patents
一种壳聚糖水凝胶及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种壳聚糖水凝胶及其制备方法和应用,所述壳聚糖水凝胶的制备方法包括以下步骤:(1)将壳聚糖溶于酸溶液中,之后与抗炎剂混合,循环冷冻得到初试水凝胶;(2)将步骤(1)得到的初试水凝胶置于1‑(3‑二甲氨基丙基)‑3‑乙基碳二亚胺盐酸盐和N‑羟基琥珀酰亚胺的2‑(N‑吗啉代)乙磺酸混合溶液中孵育,之后经碱溶液处理、去离子水水洗得到所述壳聚糖水凝胶。本发明提供的壳聚糖水凝胶抗炎效果好,皮肤组织损伤修复效果好。
Description
技术领域
本发明属于医用材料领域,具体涉及一种壳聚糖水凝胶及其制备方法和应用,尤其涉及一种抗炎效果好的壳聚糖水凝胶及其制备方法和应用。
背景技术
水凝胶是一种介于固态和液态并具有三维空间的亲水聚合物和水的聚集体。水凝胶具有良好的机械性能、生物相容性和生物可降解性,其湿润而柔软的表面和充盈液体的内部环境有利于细胞粘附、增殖和分化,易于使用特定生物分子进行表面修饰如负载药物、生长因子,这些特性使其成为皮肤组织修复的理想生物材料。此外,在食品工业领域,水凝胶可有效控制风味物质的释放、降低包装内食物的水分活度,减缓微生物滋生等。目前关于壳聚糖水凝胶用于皮肤组织修复已有研究,但并不能解决皮肤组织损伤后的二次炎症问题。
CN105664239B公开了一种自粘附皮肤修复水凝胶的制备方法。首先对天然壳聚糖进行季胺化改性,使其具有优异的杀菌性能。其次,采用去溶剂法制备包埋有促皮肤再生的生长因子的BSA纳米颗粒。再次,对天然明胶材料接枝多巴胺。最后,将季胺化修饰的壳聚糖、包裹生长因子的BSA纳米颗粒、及多巴胺接枝的明胶通过交联形成水凝胶材料,该材料具有粘附性、抗菌性及促进皮肤伤口愈合的多重作用,从而用于皮肤修复。
CN109381738A公开了一种壳聚糖基水凝胶及其制备方法和应用。所述的壳聚糖基水凝胶以壳聚糖或其衍生物为原料,主链接枝邻苯二羟基基团化合物,通过交联剂交联制备而成。所述水凝胶的制备方法:首先在壳聚糖或其衍生物上接枝邻苯二羟基基团,使产物具有湿态粘合性;进一步的,通过交联反应,形成具有足够机械强度及粘弹性的水凝胶材料。该水凝胶材料具有湿态粘合性、止血和促伤口愈合功能,在组织工程和皮肤修复方面具有广泛的应用前景。
由于目前的壳聚糖水凝胶存在无法解决皮肤组织损伤后的二次炎症问题。因此,如何提供一种抗炎效果好的壳聚糖水凝胶,成为了亟待解决的问题。
发明内容
针对现有技术的不足,本发明的目的在于提供一种壳聚糖水凝胶及其制备方法和应用,尤其提供一种抗炎效果好的壳聚糖水凝胶及其制备方法和应用。本发明提供的壳聚糖水凝胶抗炎效果好,皮肤组织损伤修复效果好。
为达到此发明目的,本发明采用以下技术方案:
第一方面,本发明提供了一种壳聚糖水凝胶的制备方法,所述壳聚糖水凝胶的制备方法包括以下步骤:
(1)将壳聚糖溶于酸溶液中,之后与抗炎剂混合,循环冷冻得到初试水凝胶;
(2)将步骤(1)得到的初试水凝胶置于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的2-(N-吗啉代)乙磺酸混合溶液中孵育,之后经碱溶液处理、去离子水水洗得到所述壳聚糖水凝胶。
上述壳聚糖水凝胶通过将壳聚糖与抗炎剂结合,利用酰胺化反应将二者连接,使形成的水凝胶具有孔隙结构,能保证抗炎剂在递送过程中持续释放,使得所述壳聚糖水凝胶能够减轻皮肤组织损伤后二次炎症反应,具有优秀的皮肤组织损伤修复的效果。
优选地,步骤(1)所述酸包括乙酸。
优选地,步骤(1)所述酸的体积分数为0.8~1.2%。
优选地,步骤(1)所述壳聚糖的质量为酸溶液质量的2.5~3.5%。
其中,酸的体积分数可以是0.8%、0.9%、1%、1.1%或1.2%等,壳聚糖的质量可以是酸溶液质量的2.5%、2.6%、2.7%、2.8%、2.9%、3%、3.1%、3.2%、3.3%、3.4%或3.5%等,但不限于以上所列举的数值,上述数值范围内其他未列举的数值同样适用。
优选地,步骤(1)所述抗炎剂包括N-乙酰神经氨酸、聚唾液酸或原儿茶酸中任意一种或至少两种的组合,例如N-乙酰神经氨酸和聚唾液酸的组合、聚唾液酸和原儿茶酸的组合或N-乙酰神经氨酸和原儿茶酸的组合等,但不限于以上所列举的组合,上述组合范围内其他未列举的组合同样适用。
上述抗炎剂的选择提高了所述壳聚糖水凝胶的抗炎效果,促进了皮肤损伤组织的修复。
优选地,步骤(1)所述抗炎剂包括N-乙酰神经氨酸、聚唾液酸或原儿茶酸中至少两种的组合。
优选地,步骤(1)所述抗炎剂为N-乙酰神经氨酸和聚唾液酸的组合。
上述特定抗炎剂进一步提高了所述壳聚糖水凝胶的抗炎效果,促进了皮肤损伤组织的修复。
优选地,N-乙酰神经氨酸和聚唾液酸的质量比为(1-3):(1-3),其中N-乙酰神经氨酸的份数可以是1、2或3等,聚唾液酸的份数可以是1、2或3等,,但不限于以上所列举的数值,上述数值范围内其他未列举的数值同样适用。
优选地,步骤(1)所述壳聚糖的单体与抗炎剂的摩尔比为(9~11):1。
优选地,步骤(1)所述循环冷冻的次数为至少3次。
优选地,步骤(1)所述循环冷冻的冷冻温度为-22~-18℃,单次时间为11-13h。
优选地,步骤(1)所述循环冷冻的解冻温度为23-27℃,单次时间为11-13h。
优选地,步骤(2)所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)的2-(N-吗啉代)乙磺酸(MES)混合溶液的pH为5.5-6.5。
优选地,步骤(2)所述孵育的时间为11-13h。
其中,壳聚糖的单体与抗炎剂的摩尔比可以是9:1、9.5:1、10:1、10.5:1或11:1等,循环冷冻的次数可以是3次、4次或5次等,冷冻温度可以是-22℃、-21℃、-20℃、-19℃或-18℃等,单次时间可以是11h、11.5h、12h、12.5h或13h等,解冻温度可以是23℃、24℃、25℃、26℃或27℃等,单次时间可以是11h、11.5h、12h、12.5h或13h等,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的2-(N-吗啉代)乙磺酸混合溶液的pH可以是5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4或6.5等,孵育的时间可以是11h、11.5h、12h、12.5h或13h等,但不限于以上所列举的数值,上述数值范围内其他未列举的数值同样适用。
第二方面,本发明提供了如上所述的壳聚糖水凝胶的制备方法制备得到的壳聚糖水凝胶。
第三方面,本发明还提供了如上所述的壳聚糖水凝胶在制备皮肤组织修复材料中的应用。
相对于现有技术,本发明具有以下有益效果:
本发明提供了一种壳聚糖水凝胶,通过将壳聚糖与抗炎剂结合,利用酰胺化反应将二者连接,使形成的水凝胶具有孔隙结构,能保证抗炎剂在递送过程中持续释放,使得所述壳聚糖水凝胶能够减轻皮肤组织损伤后二次炎症反应,具有优秀的皮肤组织损伤修复的效果和抗炎效果,NO细胞炎症因子含量低至70.10±1.22μM以下;同时通过选择特定抗炎剂,进一步提高了所述壳聚糖水凝胶的抗炎效果,促进了皮肤损伤组织的修复。
附图说明
图1是实施例1提供的壳聚糖水凝胶的电镜照片;
图2是实施例1提供的壳聚糖水凝胶的红外光谱图。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。
以下示例中,聚唾液酸购自于武汉中科光谷绿色生物技术有限公司。
实施例1
本实施例提供了一种壳聚糖水凝胶,制备方法如下:
(1)将壳聚糖溶于体积分数1%的乙酸溶液(质量比3:100)中,之后与N-乙酰神经氨酸和聚唾液酸(质量比1:1)混合(摩尔比:壳聚糖单体:(N-乙酰神经氨酸和聚唾液酸)=10:1),循环冷冻(冷冻温度-20℃,时间12h,解冻温度25℃,时间12h)3次得到初试水凝胶;
(2)将步骤(1)得到的初试水凝胶置于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的2-(N-吗啉代)乙磺酸混合溶液(摩尔比为1:1:10,调节pH为6)中孵育12h,之后经1M氢氧化钠溶液淋洗得到所述壳聚糖水凝胶,其电镜照片如图1所示,红外光谱如图2所示。图2中出现在1641cm-1处和1567cm-1对应着-CO-NH-的伸缩振动吸收峰,并且在1637cm-1还出现了弯曲振动峰,证实了N-乙酰神经氨酸、聚唾液酸与壳聚糖的相互作用。
实施例2
本实施例提供了一种壳聚糖水凝胶,制备方法如下:
(1)将壳聚糖溶于体积分数0.8%的乙酸溶液(质量比2.5:100)中,之后与N-乙酰神经氨酸混合(摩尔比:壳聚糖单体:N-乙酰神经氨酸=9:1),循环冷冻(冷冻温度-20℃,时间12h,解冻温度25℃,时间12h)4次得到初试水凝胶;
(2)将步骤(1)得到的初试水凝胶置于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的2-(N-吗啉代)乙磺酸混合溶液(摩尔比为1:1:10,调节pH为5.5)中孵育11h,之后经1M氢氧化钠溶液淋洗得到所述壳聚糖水凝胶。
实施例3
本实施例提供了一种壳聚糖水凝胶,制备方法如下:
(1)将壳聚糖溶于体积分数1.2%的乙酸溶液(质量比3.5:100)中,之后与原儿茶酸混合(摩尔比:壳聚糖单体:原儿茶酸=11:1),循环冷冻(冷冻温度-20℃,时间12h,解冻温度25℃,时间12h)4次得到初试水凝胶;
(2)将步骤(1)得到的初试水凝胶置于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的2-(N-吗啉代)乙磺酸混合溶液(摩尔比为1:1:10,调节pH为5.5)中孵育11h,之后经1M氢氧化钠溶液淋洗得到所述壳聚糖水凝胶。
实施例4
本实施例提供了一种壳聚糖水凝胶,制备方法中除将步骤(1)中聚唾液酸替换为等量的N-乙酰神经氨酸外,其余与实施例1一致。
实施例5
本实施例提供了一种壳聚糖水凝胶,制备方法中除将步骤(1)中N-乙酰神经氨酸替换为等量的聚唾液酸外,其余与实施例1一致。
实施例6
本实施例提供了一种壳聚糖水凝胶,制备方法中除将步骤(1)中N-乙酰神经氨酸和聚唾液酸替换为等量的原儿茶酸外,其余与实施例1一致。
实施例7
本实施例提供了一种壳聚糖水凝胶,制备方法中除将步骤(1)中N-乙酰神经氨酸替换为等量的原儿茶酸外,其余与实施例1一致。
实施例8
本实施例提供了一种壳聚糖水凝胶,制备方法中除将步骤(1)中聚唾液酸替换为等量的原儿茶酸外,其余与实施例1一致。
皮肤损伤修复测试:
对实施例1-8提供的壳聚糖水凝胶进行皮肤损伤修复测试,测试方法如下:(1)小鼠皮肤缺损创面模型建立:BALB/c雄性小鼠(由安徽医科大学动物实验中心(许可证号:SYXK(皖)2017-006)提供)予以腹腔注射质量分数1%戊巴比妥纳溶液麻醉,用电动理发刀小心去除小鼠背部毛发后经温水浸润的棉球清洗背部皮肤,再用体积分数75%乙醇棉球对小鼠背部皮肤进行消毒处理。待乙醇完全挥发后,应用打孔器于背部左右两侧各制造一0.6cm直径的全层皮肤缺损创面,将直径为0.6cm的标准圆盘贴附于创面旁边,数码相机拍照记录以计算初始创面面积;(2)给药处理:将实施例1-8提供的壳聚糖水凝胶(1×1cm)分别置于体积分数75%乙醇中消毒30分钟后,用PBS清洗5遍,用无菌滤纸片吸干水分后,分别贴附于滴加菌液后的创面表面,并且覆盖粘性贴膜予以固定(每组5只小鼠);(3)小鼠皮肤缺损创面完全愈合时间测定:造创后每日观察各组小鼠创面愈合情况,以创面完全上皮化为愈合标准,记录各创面完全愈合所需天数,测试结果如下:
组别 | 伤口愈合所需天数 | 组别 | 伤口愈合所需天数 |
实施例1 | 10.5 | 实施例5 | 16.7 |
实施例2 | 16.0 | 实施例6 | 16.5 |
实施例3 | 15.8 | 实施例7 | 13.2 |
实施例4 | 17.0 | 实施例8 | 14.3 |
以上数据显示,本发明提供的壳聚糖水凝胶具有优秀的皮肤损伤修复效果;比较实施例1、4-8可以发现,本发明通过采用抗炎剂的组合提高了所述壳聚糖水凝胶的皮肤损伤修复效果,同时采用特定抗炎剂的组合进一步提高了所述壳聚糖水凝胶的皮肤损伤修复效果。
抗炎效果测试:
以NO细胞炎症因此为模型,取RAW264.7细胞培养至对数生长期,消化、重悬并调整细胞浓度至5×105细胞/mL,与96孔板中接种细胞悬液100μL/孔,在含5%CO2的培养箱中37℃孵育过夜,移去培养基,每孔加入200μL含脂多糖的培养基刺激炎症因子产生,测试组分别加入实施例1-8提供的壳聚糖水凝胶20μg,继续在培养箱中孵育48h,收集上清液,于492nm处测定吸光度,利用NO工作曲线计算NO细胞炎症因子含量,结果如下:
以上数据显示,本发明提供的壳聚糖水凝胶具有优秀的抗炎效果;比较实施例1、4-8可以发现,本发明通过采用抗炎剂的组合提高了所述壳聚糖水凝胶的抗炎效果,同时采用特定抗炎剂的组合进一步提高了所述壳聚糖水凝胶的抗炎效果。
申请人声明,本发明通过上述实施例来说明本发明的壳聚糖水凝胶及其制备方法和应用,但本发明并不局限于上述实施例,即不意味着本发明必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
Claims (10)
1.一种壳聚糖水凝胶的制备方法,其特征在于,所述壳聚糖水凝胶的制备方法包括以下步骤:
(1)将壳聚糖溶于酸溶液中,之后与抗炎剂混合,循环冷冻得到初试水凝胶;
(2)将步骤(1)得到的初试水凝胶置于1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的2-(N-吗啉代)乙磺酸混合溶液中孵育,之后经碱溶液处理、去离子水水洗得到所述壳聚糖水凝胶;
其中,步骤(1)所述抗炎剂为N-乙酰神经氨酸和聚唾液酸的组合;
步骤(1)所述循环冷冻的冷冻温度为-22~-18℃,单次时间为11-13h;
步骤(1)所述循环冷冻的解冻温度为23-27℃,单次时间为11-13h。
2.根据权利要求1所述的壳聚糖水凝胶的制备方法,其特征在于,步骤(1)所述酸包括乙酸。
3.根据权利要求1所述的壳聚糖水凝胶的制备方法,其特征在于,步骤(1)所述酸的体积分数为0.8~1.2%。
4.根据权利要求1所述的壳聚糖水凝胶的制备方法,其特征在于,步骤(1)所述壳聚糖的质量为酸溶液质量的2.5~3.5%。
5.根据权利要求1所述的壳聚糖水凝胶的制备方法,其特征在于,步骤(1)所述壳聚糖的单体与抗炎剂的摩尔比为(9~11):1。
6.根据权利要求1所述的壳聚糖水凝胶的制备方法,其特征在于,步骤(1)所述循环冷冻的次数为至少3次。
7.根据权利要求1所述的壳聚糖水凝胶的制备方法,其特征在于,步骤(2)所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的2-(N-吗啉代)乙磺酸混合溶液的pH为5.5-6.5。
8.根据权利要求1所述的壳聚糖水凝胶的制备方法,其特征在于,步骤(2)所述孵育的时间为11-13h。
9.一种根据权利要求1-8中任一项所述的壳聚糖水凝胶的制备方法制备得到的壳聚糖水凝胶。
10.一种根据权利要求9所述的壳聚糖水凝胶在制备皮肤组织修复材料中的应用。
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