CN115584034B - 一种用于伤口修复的可注射水凝胶材料及其制备方法 - Google Patents
一种用于伤口修复的可注射水凝胶材料及其制备方法 Download PDFInfo
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- CN115584034B CN115584034B CN202211140539.8A CN202211140539A CN115584034B CN 115584034 B CN115584034 B CN 115584034B CN 202211140539 A CN202211140539 A CN 202211140539A CN 115584034 B CN115584034 B CN 115584034B
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Abstract
本发明属于创伤修复材料技术领域,具体涉及一种用于伤口修复的可注射水凝胶材料及其制备方法。本发明公开的壳聚糖‑绿原酸‑氧化透明质酸‑去铁胺(CCOD)复合水凝胶材料具有显著的活性氧清除效果,表现出了较好的抗氧化能力,并且还能促进细胞迁移和血管再生,同时可以有效抑制细菌的增殖,起到抗感染、抗炎和抗菌的作用;糖尿病感染伤口模型实验表明该水凝胶材料可以促进伤口快速愈合,效果显著,而且还能去除掉伤口部位感染的细菌,减轻炎症。此外,本发明中的水凝胶材料主要成分来源于天然物质,安全性好,具有良好的生物相容性、可吸收性等优点;且将其制备成可注射水凝胶的方法简单,适用于各种伤口。
Description
技术领域
本发明属于创伤修复材料技术领域,具体涉及一种用于伤口修复的可注射水凝胶材料及其制备方法。
背景技术
严重糖尿病患者常发生足部溃疡,临床上称为糖尿病足溃疡。这种类型的足部伤口愈合困难,且因为足部缺乏血管,自由基积聚,会产生二次损伤。同时,慢性伤口由于长期暴露,也会导致组织微环境极度紊乱,容易滋生细菌,引发感染,甚至形成细菌生物膜。细菌的存在会进一步引发伤口的炎症和溃疡,形成一个恶性循环。因而,现有的慢性伤口不能简单地用敷料包扎,受影响的区域往往需要清创和消毒。在临床实践中,口服或注射抗生素是治疗感染的主要方法,然而,耐药菌的发展使得人们认识到需要寻找其他不导致超级细菌的抗菌途径,并减少药物在体内的循环,以减少药物积累。因此,对于慢性伤口,需要研发新型敷料,在对感染部位进行抑菌、消毒的同时还要能促进伤口的愈合。
目前,生物医学材料已经成为一种很有前途的替代材料。这些生物医用敷料通常是由生物友好型材料制成的,并配有促进伤口愈合的药物和抗生素,因而比常规敷料更能快速促进伤口愈合。因为这些生物医用敷料通常可作为类似细胞外基质的支架,从而提供机械和空间支持,并传递生物信号,调节和指导组织修复,进而加速伤口愈合。但是,现有的生物医用敷料在应用于慢性创伤修复时,仍然存在用药副作用大、组织再生能力一般、创伤修复效果有限等缺陷。因此,针对慢性创面愈合困难的问题,有必要构建新的细胞外基质结构,形成有效的抗菌药物体系,从而实现进一步的个性化治疗。
发明内容
为了克服上述现有技术的不足,本发明提出了一种可注射水凝胶材料的制备方法,制备所得的水凝胶材料具有显著创伤修复效果、促进上皮和微小血管再生、抑制瘢痕形成的可注射水凝胶,该水凝胶适用于糖尿病伤口等慢性创伤的修复中。
为了实现上述目的,本发明所采用的技术方案是:
本发明提供了一种可注射水凝胶材料的制备方法,所述方法包括以下步骤:
S1、将绿原酸溶解于有机溶剂中,再加入1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺进行活化反应,反应后将该体系与壳聚糖溶液混合,避光反应后,经透析、浓缩得到壳聚糖-绿原酸浓缩液;
S2、将透明质酸溶解于水中,再加入高碘酸钠,避光反应后经透析、浓缩得到氧化透明质酸;
S3、将壳聚糖-绿原酸浓缩液与氧化透明质酸混合,再加入去铁胺,静置成胶后得到可注射复合水凝胶。
优选地,步骤S1中,所述壳聚糖、绿原酸、1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺的摩尔比为5-10:1-3:1-3:1-3。更优选的,所述壳聚糖、绿原酸、1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺的摩尔比为10:1:1:1。
优选地,步骤S3中,所述壳聚糖-绿原酸浓缩液与氧化透明质酸的体积比为3-5:1,所述去铁胺的添加量为50-100μg/mL。更优选的,所述壳聚糖-绿原酸浓缩液与氧化透明质酸的体积比为3:1,所述去铁胺的添加量为50μg/mL。在该浓度范围,所述水凝胶材料的创伤修复效果更好。
优选地,所述氧化透明质酸在混合前,先制成浓度为10%-20%的氧化透明质酸溶液。
优选地,步骤S3中,所述透明质酸和高碘酸钠的摩尔比为12:1。
优选地,步骤S1中,所述活化反应为4℃反应1-3小时。
优选地,步骤S1、S2中,所述避光反应为室温下避光反应20-30小时。
优选地,步骤S1中的透析采用14kDa的透析袋,在纯水中透析5天,每天换一次水;步骤S2中的透析采用8kDa的透析袋,在纯水中透析5天,每天换一次水。
优选地,所述壳聚糖为脱乙酰度不小于98%的壳聚糖。
优选地,所述壳聚糖溶液的配制方法为:将壳聚糖溶于含1%乙酸的水中,所述壳聚糖与水的料液比为1-2g/100mL。
本发明还提供了采用上述制备方法制备得到的可注射水凝胶材料。
本发明还提供了采用上述的可注射水凝胶材料在制备创伤修复医用材料中的应用。
采用本发明方法制备得到的可注射水凝胶,在物理结构上,该水凝胶具有可注射性和自愈性,适用于各种伤口,易于管理。在化学组成上,包括壳聚糖、绿原酸、氧化透明质酸和去铁胺,其中水凝胶中的壳聚糖是一种生物可降解的多糖,具有良好的生物相容性和抑菌功能;绿原酸具有抗炎、抗氧化、抗菌等生物活性,体内外实验均证实可抑制金黄色葡萄球菌和大肠杆菌;去铁胺具有良好的血管生成作用和抗氧化能力,适合于缺血组织的修复;将绿原酸接枝到壳聚糖上,可以实现绿原酸的长期释放。可见,将壳聚糖-绿原酸和氧化透明质酸相结合,并负载去铁胺构建的水凝胶CCOD具有可注射性,且具有抗氧化、抗炎、抑菌、促进血管再生和上皮修复的功能,在糖尿病慢性伤口等慢性创伤的修复中具有极佳的应用前景。
优选地,所述创伤包括糖尿病慢性伤口;所述糖尿病为Ⅰ型糖尿病。
与现有技术相比,本发明的有益效果是:
本发明公开了一种可注射水凝胶材料的制备方法,制得的壳聚糖-绿原酸-氧化透明质酸-去铁胺(CCOD)复合水凝胶材料具有显著的活性氧清除效果,表现出了较好的抗氧化能力,并且还能促进细胞迁移和血管再生,同时可以有效抑制细菌的增殖,起到抗感染、抗炎和抗菌的作用;糖尿病感染伤口模型实验表明该水凝胶材料可以促进伤口快速愈合,14天就能使伤口完全愈合,效果显著,而且还能去除掉伤口部位感染的细菌,减轻炎症。此外,本发明中的水凝胶材料主要成分来源于天然物质,如壳聚糖来源于甲壳,绿原酸来源于咖啡,安全性好,具有良好的生物相容性、可吸收性等优点;且将其制备成可注射水凝胶的方法简单,适用于各种伤口,克服了现有慢性创伤修复用药副作用大、组织再生能力一般、创伤修复效果有限等不足。
附图说明
图1为CS-CGA处理组、CCOD处理组以及对照组的细菌活性涂布平板图;
图2为CS-CGA处理组和CCOD处理组的细胞内活性氧清除效果图(比例尺为100μm);
图3为CS-CGA处理组、CCOD处理组以及对照组的SD大鼠糖尿病慢性伤口愈合情况图;
图4为CS-CGA处理组、CCOD处理组以及对照组的SD大鼠在感染细菌后糖尿病伤口的愈合面积图;
图5为CS-CGA处理组、CCOD处理组和对照组在第5天时,感染细菌的糖尿病伤口创面组织的H&E染色和Masson染色结果图(比例尺分别为200μm和100μm)。
图6为CS-CGA处理组、CCOD处理组和对照组在第14天时,糖尿病慢性伤口创面组织的血管内皮生长因子(VEGF)、低氧诱导因子(HIF-1α)和血小板-内皮细胞黏附因子(CD31)的免疫组织化学(Immunohistochemical staining)染色图(比例尺为100μm)。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例中的实验方法,如无特殊说明,均为常规方法,下述实施例中所用的试验材料,如无特殊说明,均为可通过常规的商业途径购买得到。
实施例1一种可注射水凝胶材料及其制备方法
(1)壳聚糖-绿原酸材料(CS-CGA)的制备:
将1.6g脱乙酰度为98%的壳聚糖溶于100mL含1%乙酸的超纯水中,搅拌至完全溶解,备用;将354mg绿原酸粉末溶解于10mL无水乙醇中,先加入191mg 1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC HCL),10min后再加入115mg N-羟基琥珀酰亚胺(NHS)进行活化,4℃反应30分钟后将该体系加入到上述的壳聚糖溶液中,室温下避光反应24小时。反应完成后,将产物转移到14kDa的透析袋中,在纯水中透析5天,每天换一次水。透析完成后以3000rpm/min的转速离心,收集上清并置于25℃的恒温干燥箱里浓缩7天,得到CS-CGA浓缩液。
(2)氧化透明质酸(OHA)的制备:
将10g透明质酸溶解于超纯水中,并定容至100mL,再加入417mg高碘酸钠,室温下避光反应24小时,反应结束后加入过量乙二醇终止反应。反应完成后,将产物转移到8kDa的透析袋中,在纯水中透析5天,每天换一次水。透析完成后离心、冻干得到氧化透明质酸。
(3)可注射水凝胶材料(CCOD)的制备:
将氧化透明质酸溶于超纯水中配成质量分数为15%的溶液,然后将3mL壳聚糖-绿原酸浓缩液与1mL 15%氧化透明质酸溶液混合,再加入200μg去铁胺,静置30秒后成胶,成胶后装入到注射器中,即得到可注射复合水凝胶。
实验例1细菌活性检测实验
(1)实验方法:
各取100μL的金黄色葡萄球菌菌液和大肠杆菌菌液于20mL的LB培养基中,置于摇床中37℃活化12h,将得到的菌液稀释至OD500值为0.5,即细菌浓度为1×108CFU/mL。然后将1mL CS-GA和CCOD分别加入4mL稀释了100倍的金黄色葡萄球菌(S.aureus)和大肠杆菌(E.coli)的菌悬液中,在37℃摇床中以150rpm/min的转速培养4h。然后,取菌液用0.9%的生理盐水稀释10倍,再将100μL稀释后的悬液涂抹在琼脂平板上,并在37℃恒温箱中培养12h,最后计数各组的菌落数。以未经处理的细菌菌落数作为对照。
(2)实验结果:
结果参见图1,由图可见,CS-CGA和CCOD均具有一定的细菌清除能力,可以有效抑制金黄色葡萄球菌和大肠杆菌的增殖,从而抑制菌落的形成,达到抗菌抗炎的效果。其中CCOD对细菌的活性影响更明显,尤其是对金黄色葡萄球菌而言,CS-CGA和CCOD基本都能完全抑制该菌的活性。
实验例2细胞内活性氧的清除实验
(1)实验方法:
将3T3细胞接种在24孔板中,加入含血清的DMEM培养基培养至贴壁,去除旧培养基,分别加入100μL CS-CGA和CCOD,再加入H2O2溶液(100μM),继续培养24h;阳性对照组则加入含血清培养基和H2O2溶液(100μM),阴性对照组加入含血清培养基和与H2O2溶液等体积的PBS溶液;24h后去除旧培养基,用PBS溶液清洗细胞2遍,每孔加入300μLDCFH-DA探针孵育20min,孵育后去除探针溶液,再用PBS溶液清洗细胞2遍,把孔板放置于倒置荧光显微镜下观察荧光强度。
2、实验结果:
结果参见图2,由图可见,阴性对照组的细胞在没有任何刺激的情况下,细胞内ROS水平较低;阳性对照组经过过氧化氢溶液(100μM)预处理后,细胞内ROS水平显著增加,在图中观察到强烈的荧光。而经过CS-CGA和CCOD预处理后,荧光强度明显降低;并且,值得注意的是,当用CCOD预处理细胞时,细胞中的荧光强度很低,即细胞内ROS水平很低。
上述结果表明,CCOD水凝胶材料可以清除细胞内的活性氧,具有良好的细胞内抗氧化活性。
实验例3对创面修复速度的影响
1、实验方法:
(1)糖尿病造模:选取体重180~220g的雄性SD(Sprague Dawley)大鼠作为研究对象。将2.1g柠檬酸和2.94g柠檬酸钠分别溶于100mL生理盐水中,然后将柠檬酸溶液和柠檬酸钠溶液按照1:1.32的体积比混合,并调pH为4.5,得到缓冲液。取链脲佐菌素(STZ)溶于缓冲液中,配成10mg/mL的溶液,再用STZ溶液对大鼠进行腹腔注射,注射量为链脲佐菌素/大鼠体重=50mg/kg。一周后,测量大鼠空腹血糖,取血糖值在16.7-30mmol/L范围的大鼠进行后续实验。
(2)糖尿病慢性伤口造模:将所有糖尿病大鼠随机分为3组,给所有大鼠背部剃毛,先通过腹腔注射2%戊巴比妥钠(50mg/kg)进行麻醉,再于大鼠背部全层切除直径为1.5cm的圆形皮肤;
(3)细菌感染的糖尿病伤口造模:在糖尿病慢性伤口造模的基础上,在伤口部位涂抹100μL OD500值为0.5金黄色葡萄球菌和大肠杆菌的混合菌液。
(4)创口上药:在对照组伤口处滴加PBS溶液,实验组伤口处分别施加约200μL CS-CGA浓缩液和CCOD水凝胶,每次滴加的PBS溶液和CS-CGA浓缩液体积相同,每三天换一次药,并拍照记录伤口变化情况。
(5)组织切片:取治疗14天后的糖尿病慢性伤口的创面皮肤组织进行VEGF(血管内皮生长因子)、CD31(内皮细胞黏附分子)和HIF-1α(缺氧诱导因子-1α)免疫组化染色,取治疗5天后的细菌感染伤口的创面皮肤进行苏木精-伊红染色(H&E staining)和马松染色(Masson staining),在显微镜下拍照观察。
2、实验结果:
实验结果参见图3-6,由图3可见,CCOD水凝胶材料可以显著促进创面愈合,经过CS-CGA和CCOD处理后,大鼠的糖尿病慢性伤口的创面闭合面积均大于对照组。其中,经CCOD水凝胶处理后的实验组大鼠创面在第14天即完全闭合,而对照组创面仍然明显存在;CS-CGA处理的实验组大鼠创面虽然未完全愈合,但其面积也均明显小于对照组。说明本发明的CS-CGA和CCOD材料可加速创面愈合,而CCOD水凝胶材料由于包含去铁胺(DFO),促愈合效果更佳。
由图4可见,在治疗的第5天,经CCOD处理后的细菌感染伤口的创面闭合率为64%,而对照组创面闭合率仅为41%;而且,经CCOD处理后的大鼠伤口状况发展良好,未发现明显炎症反应,但对照组还存在明显的炎症。
由图5可见,CS-CGA和CCOD材料可促进细菌感染伤口更完整的上皮形成,使得胶原纤维聚集、排列紧密,还可以促进皮肤附属物毛囊的形成,其中CCOD的效果最好。由H&E和Masson染色可见,与对照组相比,CS-CGA和CCOD处理后的肉芽组织程度较高,血管分布丰富,而且细菌分布和炎症细胞较少。而对照组可见散落着大面积的细菌和炎症细胞,说明感染没有得到控制。
由图6可见,CS-CGA和CCOD材料可促使糖尿病慢性伤口更丰富的微小血管形成,血管标志物CD31凸显了血管横截面的外型,其中CCOD的效果更好,通过计数可知CCOD处理后的组织具有更多数量的微小血管。VEGF和HIF-1α染色区域及强度明显大于对照组,说明CCOD处理后的组织具有更强的血管生成潜力。以上结果表明本发明的CCOD水凝胶材料具有显著的血管生成促进作用。
综上所述,本发明制备的壳聚糖-绿原酸-氧化透明质酸-去铁胺(CCOD)复合可注射水凝胶材料具有良好的生物相容性、可吸收性。所制备的可注射水凝胶含有丰富的营养成分,可以为参与伤口修复的细胞提供养分支持,同时具有良好的抗氧化能力,优秀的抗菌能力和促血管再生能力,不仅可以加速创面愈合,还具有抑制瘢痕形成和抗炎的作用。可见,壳聚糖-绿原酸和氧化透明质酸结合去铁胺制成创伤修复材料后,非常适合用于克服糖尿病慢性伤口以及细菌感染伤口修复过程中存在的一系列困难,应用前景良好。
以上对本发明的实施方式作了详细说明,但本发明不限于所描述的实施方式。对于本领域的技术人员而言,在不脱离本发明原理和精神的情况下,对这些实施方式进行多种变化、修改、替换和变型,仍落入本发明的保护范围内。
Claims (8)
1.一种可注射水凝胶材料的制备方法,其特征在于,包括以下步骤:
S1、将绿原酸溶解于有机溶剂中,再加入1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺进行活化反应,反应后将该体系与壳聚糖溶液混合,避光反应后,经透析、浓缩得到壳聚糖-绿原酸浓缩液;
S2、将透明质酸溶解于水中,再加入高碘酸钠,避光反应后经透析、浓缩得到氧化透明质酸;
S3、将壳聚糖-绿原酸浓缩液与氧化透明质酸混合,再加入去铁胺,所述去铁胺的添加量为50-100μg/mL,静置成胶后得到可注射复合水凝胶;所述氧化透明质酸在混合前,先制成浓度为10%-20%的氧化透明质酸溶液。
2.根据权利要求1所述的一种可注射水凝胶材料的制备方法,其特征在于,步骤S1中,所述壳聚糖、绿原酸、1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐和N-羟基琥珀酰亚胺的摩尔比为5-10:1-3:1-3:1-3。
3.根据权利要求1所述的一种可注射水凝胶材料的制备方法,其特征在于,步骤S2中,所述透明质酸和高碘酸钠的摩尔比为12:1。
4.根据权利要求1所述的一种可注射水凝胶材料的制备方法,其特征在于,步骤S1中,所述活化反应为4℃反应1-3小时。
5.根据权利要求1所述的一种可注射水凝胶材料的制备方法,其特征在于,步骤S1、S2中,所述避光反应为室温下避光反应20-30小时。
6.采用权利要求1-5任一项所述的制备方法制备得到的可注射水凝胶材料。
7.权利要求6所述的可注射水凝胶材料在制备创伤修复医用材料中的应用。
8.根据权利要求7所述的应用,其特征在于,所述创伤包括糖尿病慢性伤口。
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