CN113754903B - 一种用于皮肤修复的双交联透明质酸/壳聚糖复合水凝胶的制备方法 - Google Patents
一种用于皮肤修复的双交联透明质酸/壳聚糖复合水凝胶的制备方法 Download PDFInfo
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- CN113754903B CN113754903B CN202111060377.2A CN202111060377A CN113754903B CN 113754903 B CN113754903 B CN 113754903B CN 202111060377 A CN202111060377 A CN 202111060377A CN 113754903 B CN113754903 B CN 113754903B
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Abstract
本发明公开了一种用于皮肤修复的双交联透明质酸/壳聚糖复合水凝胶的制备方法,将含叠氮化改性的壳聚糖、邻硝基苄基类光扳机分子修饰的透明质酸的预聚液于紫外照射下发生光交联,即得。通常光交联反应需要使用光引发剂,光引发剂具有一定的细胞毒性,本发明所述的叠氮化壳聚糖,光照后成胶迅速且不需要加入任何光引发剂,该水凝胶具有良好的机械性能,组织粘附性和生物相容性,且有利于加速皮肤伤口愈合的过程。
Description
技术领域
本发明属于医用生物材料技术领域,具体涉及一种用于皮肤修复的双交联透明质酸/壳聚糖复合水凝胶的制备方法。
背景技术
人体皮肤是人体抵抗外界环境的重要器官,当其大规模损伤时会导致严重的残疾甚至死亡。水凝胶可以在伤口界面处保持湿润的环境、吸收体液、渗透氧气,营养物质和其他水溶性代谢产物,还有利于细胞粘附,增殖,细胞因子,营养物和运输代谢废物。因此,水凝胶在组织修复和再生中具有重要作用。在再生医学和修复医学中,生物材料与组织的整合可以提供稳定的生物固定,降低感染的风险,并促进愈合过程。目前,在伤口上进行原位胶凝的方法可以准确贴合不规则形状的组织缺损。良好的伤口敷料还需要水凝胶与组织表面之间有紧密的,最好是化学键合的界面。
透明质酸(HA)是广泛分布在体内唯一的非硫酸化聚葡萄糖胺,它是一种无毒,可生物降解且具有生物相容性的天然聚合物,已被用于制造生物医学领域的可注射水凝胶,它是由D-葡萄糖醛酸和N-乙酰葡糖胺组成的双糖单位糖胺聚糖。它作为细胞外基质的主要成分,在皮肤伤口愈合过程的多个阶段有着关键性的作用。研究表明,HA可有利于伤口愈合过程中内皮细胞的迁移,增殖和分化,并促进血管生成和炎症调节。因此,本发明提供了一种用于皮肤修复的双交联透明质酸/壳聚糖复合水凝胶的制备方法。
发明内容
发明目的:本发明所要解决的技术问题是针对现有技术的不足,提供一种双交联透明质酸/壳聚糖复合水凝胶及其制备方法。
本发明还要解决的技术问题是提供上述双交联透明质酸/壳聚糖复合水凝胶的应用。
发明思路:本发明中所述双交联水凝胶由邻硝基苄基类光扳机分子修饰的透明质酸(HA-NB)和叠氮化的壳聚糖(CMC-AZ)通过光照后壳聚糖上的叠氮发生偶氮交联,形成第一层网络,HA-NB上的光生醛基与CMC-AZ上的氨基发生希夫碱反应,形成第二层网络,从而极大的增强了胶体的力学,且胶体与组织界面处,HA-NB的光生醛基与组织上的氨基反应,能增强胶体在组织上的粘附力。
为了解决上述第一个技术问题,本发明公开了一种双交联透明质酸/壳聚糖复合水凝胶的制备方法,将含叠氮化改性的壳聚糖(CMC-AZ)、邻硝基苄基类光扳机分子修饰的透明质酸(HA-NB)的预聚液于紫外照射下发生光交联,即得双交联透明质酸/壳聚糖复合水凝胶。
其中,所述叠氮化改性的壳聚糖可以按照现有技术其他方法制备,也可以按照下述方法制备,包括如下步骤:
(1)将4-叠氮苯甲酸、N-羟基琥珀酰亚胺与N,N'-二环己基碳二亚胺反应得到叠氮活化酯;
(2)将壳聚糖与所得叠氮活化酯反应,即得叠氮化改性的壳聚糖。
其中,所述邻硝基苄基类光扳机分子修饰的透明质酸可以按照现有技术其他方法制备,也可以按照下述方法制备,包括如下步骤:
(i)将硝酸与硼氢化钠、香草醛、4-溴丁酸甲酯、乙二胺等反应得到邻硝基苄基类光扳机分子;
(ii)通过1-羟基苯并三唑(HOBT)/1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)将所得邻硝基苄基类光扳机分子接枝到透明质酸上,即得邻硝基苄基类光扳机分子修饰的透明质酸。
其中,所述叠氮化改性的壳聚糖和邻硝基苄基类光扳机分子修饰的透明质酸的质量比为(3-5):(1-3),优选为(3-5):(2-3),进一步优选为5:3。
其中,所述预聚液的溶剂为水。
其中,所述预聚液中,叠氮化改性的壳聚糖的质量浓度为3%-5%。
其中,所述预聚液中,邻硝基苄基类光扳机分子修饰的透明质酸的质量浓度为1%-3%,优选为2%-3%。
优选地,所述预聚液还包括抗炎药,优选为阿莫西林。
其中,所述预聚液中,抗炎药的质量浓度为0.05%-0.35%,优选为0.2%。
其中,所述紫外照射的时间为1-3min。
其中,所述紫外照射的波长为365nm。
其中,所述紫外照射的功率为0.5-1.5mW/cm2。
上述方法制备得到的水凝胶也在本发明的保护范围之内。
其中,所述水凝胶使用的工艺参数为:反应温度为室温,固化时间为1-3min。
上述过程中,所述壳聚糖包括但不限于羧甲基壳聚糖。
为了解决上述第二个技术问题,本发明公开了上述水凝胶在制备创面闭合材料中的应用。
有益效果:与现有技术相比,本发明具有如下优势:
1、本发明的双交联水凝胶,成胶时间快,机械性能、粘附性能和细胞相容性良好且有利于皮肤伤口愈合。
2、希夫碱反应迅速且难以控制,但本发明所述的邻硝基苄基类光扳机分子修饰的透明质酸本不含醛基,只有在紫外光照后才能生成醛基,与壳聚糖或者组织上的氨基发生希夫碱反应,交联可控且能增强组织粘附力。
3、通常光交联反应需要使用光引发剂,光引发剂具有一定的细胞毒性,本发明所述的叠氮化壳聚糖,光照后成胶迅速且不需要加入任何光引发剂,具有良好的生物相容性,且可提高水凝胶机械性能。
4、本发明选用透明质酸和壳聚糖为水凝胶原料,两种原料通过简单改性可发生交联作用,且制备简单,原料并已实现商业化。因此,其选用及对此类凝胶方法的建立、推广和促进其在组织工程和再生医学中的应用具有非常重要的价值。
5、本发明所述的邻硝基苄基类光扳机分子修饰的透明质酸可在紫外光照后生成醛基,与壳聚糖或者组织上的氨基发生希夫碱反应,不需要加入任何交联剂。另外叠氮官能团在紫外光照后产生的氮烯基团也可与组织上的氨基发生夺氢反应。两种光交联反应迅速且能增强组织粘附力。
附图说明
下面结合附图和具体实施方式对本发明做更进一步的具体说明,本发明的上述和/或其他方面的优点将会变得更加清楚。
图1为改性壳聚糖和改性海藻酸钠的傅里叶红外光谱图。
图2为单交联水凝胶和双交联水凝胶的压缩应力-应变曲线。
图3为单交联水凝胶和双交联水凝胶的组织粘附性曲线图。
图4为3T3细胞在水凝胶上的细胞毒性。
图5为水凝胶处理的小鼠背部伤口的图片和定量分析。
图6为水凝胶处理的小鼠伤口H&E染色图片以及肉芽组织厚度分析。
具体实施方式
下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。
实施例1
1)将香草醛(8.9g)、4-溴丁酸甲酯(9.89g)和碳酸钾(10.2g)溶解在40ml N,N-二甲基甲酰胺中,并室温反应16h。再将反应液倒入200ml冰水中,滤出沉淀后水洗3次,将沉淀溶解在二氯甲烷中,用无水硫酸镁干燥6h,抽滤后旋蒸得固体粉末。将上步产物溶解于140ml硝酸中,并在0℃反应3h。将反应液倒入400ml冰水中,滤出沉淀后水洗3次,将沉淀溶解在二氯甲烷中,用无水硫酸镁干燥6h,抽滤后旋蒸得黄色固体粉末。将上步产物(7.7g)和硼氢化钠(1.5g)溶解于100ml乙醇/四氢呋喃(1:1)混合液中,在0℃下反应3h。反应结束后旋蒸除去所有溶剂,加入50mg甲苯磺酸,和产物一起溶解在甲醇中,室温反应过夜。真空除去所有溶剂,将产物溶解在水和二氯中,取有机层。再将水层用二氯萃取两次,合并有机层后用无水硫酸镁干燥6h,过滤后旋蒸至黄色固体粉末。最后用薄层层析硅胶柱纯化,得到产物。将上部产物(0.5g)与乙二胺(1.1ml)一起溶解在甲醇(50ml)中,50℃回流反应4天,真空蒸发溶剂后用乙酸乙酯沉淀,滤出沉淀后用乙酸乙酯洗3次,最后放置真空烘箱干燥过夜,得黄色固体产物邻硝基苄基类光扳机分子(NB)。将1g透明质酸,0.55gNB,0.38g 1-羟基苯并三唑完全溶解在100ml去离子水中,将pH调至4.5-5,再将EDC(200mg)加入反应液中,室温下反应48h后装入透析袋(MW=3500),用DI透析2天后冷冻干燥得到邻硝基苄基类光扳机分子修饰的透明质酸(HA-NB)。
2)将2g 4-叠氮苯甲酸、1.4g N-羟基琥珀酰亚胺和1.9ml N,N'-二环己基碳二亚胺完全溶解于40ml 1,4二氧六环中,室温下避光反应12h,反应结束后抽滤除去沉淀副产物,再用二氧六环多次冲洗沉淀,合并下清液后旋蒸后得白色固体粉末NHS-AZ。再将1g羧甲基壳聚糖搅拌溶解于40ml的碳酸氢钠溶液中,再将0.6g NHS-AZ溶解于40ml二甲基亚砜中,避光将其缓慢滴加至羧甲基壳聚糖溶液中,室温反应72h后装入透析袋(MW=3500),用DI透析2天后冷冻干燥得到叠氮化的羧甲基壳聚糖(CMC-AZ)。
3)用KBr压片法制备样品,并用傅立叶变换红外光谱仪在4000-500cm-1范围内进行扫描,得到HA、HA-NB和CMC、CMC-AZ的红外光谱。
通过FT-IR分析进一步证实了聚合物的化学结构。图1(左)显示HA和HA-NB的FT-IR光谱。与HA的光谱相比,在HA-NB中观察到酰胺II和III的吸收峰出现在1112cm-1和1400cm-1处,表明NB已成功接枝到HA上。CMC和CMC-AZ的FT-IR光谱如图1(右)所示。检测到由叠氮基团引起的2129cm-1处的新吸收峰,证明了CMC-AZ的成功合成。
实施例2
(1)将实施例1中制备的CMC-AZ溶于水中,配成的预聚液,CMC-AZ的质量百分比为5%,在玻璃样品瓶中室温(25℃)放置在波长365nm、功率1mW/cm2的紫外光照射2min发生光交联得到单网络水凝胶CMC-AZ。
(2)将实施例1中制备的HA-NB、实施例1中制备的CMC溶于水中,配成的预聚液,HA-NB的质量百分比为2%,CMC的质量百分比为5%,在玻璃样品瓶中室温(25℃)放置在波长365nm、功率1mW/cm2的紫外光照射2min发生光交联得到单网络水凝胶HA-NB/CMC-1。
(3)将实施例1中制备的HA-NB、实施例1中制备的CMC溶于水中,配成的预聚液,HA-NB的质量百分比为3%,CMC的质量百分比为5%,在玻璃样品瓶中室温(25℃)放置在波长365nm、功率1mW/cm2的紫外光照射2min发生光交联得到单网络水凝胶HA-NB/CMC-2。
(4)将实施例1中制备的HA-NB、实施例1中制备的CMC-AZ溶于水中,配成的预聚液,HA-NB的质量百分比为2%,CMC-AZ的质量百分比为5%,在玻璃样品瓶中室温(25℃)放置在波长365nm、功率1mW/cm2的紫外光照射2min发生光交联得到双网络水凝胶HA-NB/CMC-AZ-1。
(5)将实施例1中制备的HA-NB、实施例1中制备的CMC-AZ溶于水中,配成的预聚液,HA-NB的质量百分比为3%,CMC-AZ的质量百分比为5%,在玻璃样品瓶中室温(25℃)放置在波长365nm、功率1mW/cm2的紫外光照射2min发生光交联得到双网络水凝胶HA-NB/CMC-AZ-2。
(6)分别记录单/双网络水凝胶的直径和厚度之后,将水凝胶放置在下板上,并以5mm/min的应变速率由上板压缩,分析所有样品以获得应力-应变曲线。见图2为水凝胶的压缩应力-应变曲线,其可以反映出水凝胶的力学性能;其中,双交联水凝胶CMC-AZ/HA-NB-1和CMC-AZ/HA-NB-2相较于单交联水凝胶CMC-AZ、CMC/HA-NB-1和CMC/HA-NB-2表现出较高的断裂应力,这可能是因为双网络的引入提高了水凝胶的交联密度,从而提高了水凝胶的机械性能。其中,CMC-AZ/HA-NB-2水凝胶的断裂应力最高,达到了0.72MPa,这可以得出随着HA-NB浓度的增加,水凝胶网络内部交联位点增多,水凝胶的机械强度进一步增强。
实施例3
(1)将实施例2中制备的CMC-AZ,HA-NB/CMC-1,HA-NB/CMC-2单网络水凝胶和HA-NB/CMC-AZ-1,HA-NB/CMC-AZ-2双网络水凝胶应用于本实施例。
(2)用CA胶将猪皮粘合到硬质聚对苯二甲酸乙二醇酯(PET)膜(1cm×3cm)上。然后,将水凝胶溶液注入到猪皮表面(1cm×1cm)上,并通过UV照射使水凝胶聚合。最后,用CA将另一张PET膜粘合到水凝胶上。之所以使用该实验装置,是因为猪皮对紫外线不是透明的,并且如果在两面都使用这些膜则不会形成水凝胶。使用万能试验机施加单向张力,同时记录力和伸展。加载速率保持恒定在1mm/min。所有测量重复三次。
如图3所示,水凝胶的剪切强度随着交联密度的增加而显着增加。CMC-AZ/HA-NB-2水凝胶的粘合强度达到最大值513kPa,远高于其他任何样品,一方面是因为水凝胶内部交联密度大,机械性能强,另一方面也因为水凝胶中HA-NB含量较高,因此醛基含量较高,可以更好的和组织上的氨基反应,形成紧密的连接。
实施例4
(1)将实施例2中制备的CMC-AZ,HA-NB/CMC-1,HA-NB/CMC-2单网络水凝胶和HA-NB/CMC-AZ-1,HA-NB/CMC-AZ-2双网络水凝胶应用于本实施例。
(2)将灭菌的水凝胶前体溶液添加到24孔板中并通过UV处理凝胶化。在完全培养基中于37℃孵育24小时后,将3T3细胞以2000个细胞/孔的密度接种在96孔板中的水凝胶上,并在CO2孵育箱中培养。孵育设计的时间(1、2和3天)后,将10μL CCK-8试剂添加到每个孔中,再孵育40分钟。用酶标仪在450nm处测量每个孔的吸光度。相对细胞活力通过下式计算。计算方法为:相对细胞活力(%)=(样品组吸光度-空白组吸光度)/(对照组吸光度-空白组吸光度)×100%。其中,1天的对照组组的吸光度作为对照(100%)。其中,对照组是空白孔板加细胞。
为了评估水凝胶的生物相容性,我们将3T3细胞在水凝胶上直接培养。然后进行CCK-8溶液细胞活力测定。如图4所示,与对照组相比,水凝胶组培养的细胞存活率均高于90%并且在第2,3天生长良好,这表明水凝胶具有良好的生物相容性和较低的细胞毒性。
实施例5
(1)将实施例2中制备的HA-NB/CMC-2单网络水凝胶和HA-NB/CMC-AZ-2双网络水凝胶应用于本实施例。
(2)将实施例1中制备的HA-NB、实施例1中制备的CMC-AZ和阿莫西林溶于水中,配成的预聚液,HA-NB的质量百分比为3%,CMC-AZ的质量百分比为5%,阿莫西林的质量百分比为0.2%,在玻璃样品瓶中室温(25℃)反应3min得到双网络水凝胶D-HA-NB/CMC-AZ。
(3)将40只ICR雄性小鼠(20g)分成五组,并且所有小鼠在手术前适应1周。通过腹膜内注射4%水合氯醛(0.1mL/10g)麻醉小鼠,并在尾巴和背部之间的背部区域剃毛。在每只ICR小鼠的背部形成了两个全层皮肤缺陷(圆形,直径8毫米)。用医用酒精消毒后,使用创可贴(不含药物)和纱布将水凝胶固定在伤口上。商业产品(3M水胶体敷料)用作阳性对照,生理盐水(NS)用作阴性对照。在第3、7和14天,记录伤口的大小和图像,并通过等式计算伤口闭合率,伤口收缩率%=[区域(0天)-区域(n天)]/区域(0天)×100%。其中,n代表日期,例如第3、7、14天。
如图5所示,在第3天时,所有组的伤口均无明显渗出液,伤口周围干燥无明显红肿现象,且伤口面积均有一定程度的减少。使用水凝胶D-HA-NB/CMC-AZ组的伤口愈合最好,伤口愈合率高达75.1%。第7天,各组伤口面积进一步明显减少。经过14天的治疗,NS组和商品组的小伤口仍未完全愈合(NS组,商品组的伤口面积约9%和2%),而所有其他水凝胶组的伤口均已闭合。但是,在D-HA-NB/CMC-AZ水凝胶处理的组中可以看出伤口部位皮肤颜色接近于旁边正常组织,无明显瘢痕凸起,创面愈合区域有大量新生毛发长出来,并且其在14天的伤口愈合过程中速度最快,这可能一方面是因为水凝胶与伤口位置黏附紧密,保持有利于伤口愈合的良好环境,同时阿莫西林具有抗菌抗炎的能力,是水凝胶具有优异的清创能力。
(4)在第3、7和14天收集了再生皮肤进行苏木精和伊红(H&E)。图6为H&E染色结果。第3天时可以看到明显的皮肤表皮缺损。所有伤口都出现轻度急性炎症反应,在D-HA-NB/CMC-AZ组在伤口中显示出较弱的炎症细胞浸润。第7天,D-HA-NB/CMC-AZ处理后的伤口形成了完整的表皮层,表皮和真皮分界明显。此外,D-HA-NB/CMC-AZ组中显示出一些毛囊和血管等的皮肤附件。在第14天,水凝胶处理后的伤口区域明显小于商品敷料组。此外,对比其他水凝胶组,D-HA-NB/CMC-AZ组的上皮和结缔组织更加规整,新生血管和毛囊的数量也更多。如图6所示的第14天时的肉芽组织厚度量化分析,HA-NB/CMC-AZ和D-HA-NB/CMC-AZ水凝胶组的肉芽组织厚度分别为1036.78μm和1159.76μm,与NS组和商品化产品之间存在明显差异。D-HA-NB/CMC-AZ水凝胶能够通过促进肉芽组织增厚和新生血管生成来加速伤口愈合。在炎症初期,由于D-HA-NB/CMC-AZ水凝胶中包载的阿莫西林起到抗炎作用因此炎症反应相较于其他组更弱,也更能有效地改善伤口修复,在各组中表现出最佳的伤口愈合效果。
本发明提供了一种用于皮肤修复的双交联透明质酸/壳聚糖复合水凝胶的制备方法的思路及方法,具体实现该技术方案的方法和途径很多,以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
Claims (7)
1.一种双交联透明质酸/壳聚糖复合水凝胶的制备方法,其特征在于,将含叠氮化改性的壳聚糖、邻硝基苄基类光扳机分子修饰的透明质酸的预聚液于紫外照射下发生光交联,即得双交联透明质酸/壳聚糖复合水凝胶;
所述叠氮化改性的壳聚糖和邻硝基苄基类光扳机分子修饰的透明质酸的质量比为5:3;
其中,所述叠氮化改性的壳聚糖的制备方法包括如下步骤:
(1)将4-叠氮苯甲酸、N-羟基琥珀酰亚胺与N,N'-二环己基碳二亚胺反应得到叠氮活化酯;
(2)将壳聚糖与所得叠氮活化酯反应,即得叠氮化改性的壳聚糖;
其中,所述邻硝基苄基类光扳机分子修饰的透明质酸的制备方法包括如下步骤:
(i)将硝酸与硼氢化钠、香草醛、4-溴丁酸甲酯、乙二胺反应得到邻硝基苄基类光扳机分子;
(ii)通过1-羟基苯并三唑和1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐将所得邻硝基苄基类光扳机分子接枝到透明质酸上,即得邻硝基苄基类光扳机分子修饰的透明质酸。
2.根据权利要求1所述制备方法,其特征在于,所述预聚液的溶剂为水。
3.根据权利要求1所述制备方法,其特征在于,所述预聚液中,叠氮化改性的壳聚糖的质量浓度为3%-5%。
4.根据权利要求1所述制备方法,其特征在于,所述紫外照射的波长为365 nm。
5.根据权利要求1所述制备方法,其特征在于,所述紫外照射的功率为0.5-1.5 mW/cm2。
6.根据权利要求1所述制备方法,其特征在于,所述紫外照射的时间为1-3 min。
7.权利要求1-6中任意一项所述方法制备得到的水凝胶在制备创面闭合材料中的应用。
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