CN117659521A - 一种胞外多糖基水凝胶及其制备方法和应用 - Google Patents
一种胞外多糖基水凝胶及其制备方法和应用 Download PDFInfo
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- CN117659521A CN117659521A CN202311641685.3A CN202311641685A CN117659521A CN 117659521 A CN117659521 A CN 117659521A CN 202311641685 A CN202311641685 A CN 202311641685A CN 117659521 A CN117659521 A CN 117659521A
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- extracellular polysaccharide
- polysaccharide
- bacillus
- bailii
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Classifications
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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Abstract
本发明公开了一种胞外多糖基水凝胶,用改性后的贝莱斯芽孢杆菌胞外多糖和改性后的透明质酸钠为原料,构建水凝胶体系;所述的贝莱斯芽孢杆菌胞外多糖采用多巴胺改性,所述的透明质酸钠用甲基丙烯酸酐改性;以及给出了胞外多糖水凝胶的制备方法和应用。本发明使用改性后的贝莱斯芽孢杆菌胞外多糖构建水凝胶网络,该水凝胶机械性能良好,具有优良的生物相容性,对皮肤伤口愈合有较好的促进作用;进一步地本发明胞外多糖基水凝胶易于制备,合成方法简单,生产成本低。
Description
技术领域
本发明涉及生物医药材料领域,具体涉及一种胞外多糖基水凝胶及其制备方法和应用。
背景技术
皮肤是人体中表面积最大的器官,它是保护内部组织免受机械损伤、微生物感染、紫外线辐射和极端温度的关键结构。这使得其极易受到损伤,对个体患者和医疗保健经济都有重大影响。在日常生活中皮肤很容易受到各种损伤形成伤口,一旦皮肤受损,就为有害细菌侵入活组织提供了机会,从而打破皮肤微生物系统的平衡,导致伤口感染,严重的甚至是组织损伤。而涉及真皮甚至更深组织损伤的伤口难以愈合,且可能会出现严重的疤痕。
水凝胶因其温和的加工条件和结合许多生物活性剂的能力,为伤口敷料提供了巨大的优势。天然材料,如多糖和蛋白质,是用于制造皮肤模拟水凝胶的理想材料。作为最主要的天然聚合物之一,多糖具有良好的生物相容性,并且具有丰富的官能团,如羟基、羧基、胺基团,可进行多种化学改性,并具有较高的保水能力。纤维素、壳聚糖、淀粉、海藻酸钠、卡拉胶、透明质酸、肝素和右旋糖酐已被广泛探索用于伤口敷料应用。胞外多糖是微生物在生长与代谢过程中分泌到细胞壁外、易与菌体分离、分泌到环境中的水溶性多糖。由于胞外多糖分子结构和官能团的差异,可以产生具有不同特性的水凝胶,甚至可以通过自组装现象产生纳米颗粒。除了具有生物相容性和可生物降解的特性外,胞外多糖还具有高度亲水性,通常在水中形成假塑性溶液,这些流变特性使其成为医疗、化妆品、食品等领域的优良候选材料。
水凝胶是一种新型的生物材料,因其具有良好的生物相容性、可调控的物理化学性质以及较高的生物活性而备受关注。然而,研究表明,现有的一些水凝胶容易引起免疫排斥反应,这主要是由于其化学成分、物理性质和微结构等方面与人体组织的相容性存在差异所导致的。免疫排斥反应不仅可能导致水凝胶在体内的降解和吸收过程受到阻碍,还可能导致组织炎症反应和免疫系统的激活,从而对患者的健康造成不利影响。因此,为了进一步提高水凝胶的生物相容性,需要在材料的设计、制备和应用等方面进行深入的研究和优化。
发明内容
为解决上述技术问题,本发明提供一种胞外多糖基水凝胶及其制备方法和应用,以改性后的贝莱斯芽孢杆菌胞外多糖为原料制备水凝胶,旨在得到一种有良好的机械性能和生物相容性的水凝胶。将其应用于皮肤全层损伤模型中,可显著促进皮肤附属器官的形成,从而加速伤口愈合。
为实现上述目的,本发明提供的技术方案如下:
一种胞外多糖基水凝胶,用改性后的贝莱斯芽孢杆菌胞外多糖和改性后的透明质酸钠为原料,构建水凝胶体系;所述的贝莱斯芽孢杆菌胞外多糖采用多巴胺改性,所述的透明质酸钠用甲基丙烯酸酐改性。
进一步地,所述贝莱斯芽孢杆菌胞外多糖的改性为磷酸盐缓冲液和贝莱斯芽孢杆菌胞外多糖(M76-EPS),充分搅拌溶解,得到混合溶液,然后将碳二亚胺(EDC),N-羟基琥珀酰亚胺(NHS)溶解在上述混合溶液中,充分搅拌并溶解,调节pH至5.5,持续搅拌30min;再加入盐酸多巴胺(DA),充分搅拌溶解,持续搅拌反应4h,然后透析,冷冻干燥后得到多巴胺改性的贝莱斯芽孢杆菌胞外多糖(76DA)。
进一步地,所述磷酸盐缓冲液的体积为50mL(pH值为5.5);所述贝莱斯芽孢杆菌胞外多糖(M76-EPS)用量为500mg,碳二亚胺(EDC)、N-羟基琥珀酰亚胺(NHS)以及盐酸多巴胺(DA)的添加量均为2.5mmol;所述使用规格为8000-14000Da的透析袋,用去离子水透析48h。
进一步地,所述贝莱斯芽孢杆菌胞外多糖的提取方法,包含以下操作步骤:
(1)菌种活化:将保藏号为GDMCC NO:61384且保藏于4℃冰箱中的Bacillusvelezensis M76T11B在MRS平板上进行三区划线活化菌株,37℃恒温条件下,静置培养48h后挑取生长状态良好的单菌落接种至MRS肉汤中,37℃、150rpm培养16h;
(2)扩大培养:将在MRS肉汤中培养后的菌株以5%(v/v)接种量接种到1000mLMRS-S液体培养基中,在37℃、150rpm条件下摇床培养48h,即得发酵液;
(3)粗多糖的沉淀:取发酵液经4℃、10,000×g离心20min去除菌体沉淀,取上清液浓缩到原体积1/5加入4倍体积的4℃体积浓度95%乙醇至终浓度为80%,搅拌均匀,4℃静置过夜沉淀多糖,在4℃、10,000×g离心20min获得多糖沉淀;
(4)脱蛋白:将多糖沉淀溶于去离子水中,加入80%(m/v)的三氯乙酸至终浓度4%(m/v),搅拌均匀4℃静置10h后在4℃下10,000×g离心20min弃去蛋白沉淀,收集上清液;
(5)多糖的二次醇沉:将上清液浓缩,加入4倍体积的95%乙醇至终浓度为80%,在4℃下静置过夜以沉淀多糖,过滤,收集多糖沉淀,将多糖沉淀重新溶解于去离子水中,装入透析袋(截留分子量8000-14000Da)透析48h,每隔8h换水,将透析后的糖溶液冷冻干燥,即得贝莱斯芽孢杆菌胞外多糖(M76-EPS)。
进一步地,所述透明质酸钠的改性为将透明质酸钠(HA)溶于去离子水中,调节溶液的pH值为8,加入甲基丙烯酸酐(MA),在室温下搅拌12h,再通过乙醇(体积浓度80%)沉淀得到HAMA粗产物,并用无水乙醇洗涤三次以去除剩余的甲基丙烯酸酐和反应过程中产生的甲基丙烯酸,然后透析48h,冷冻干燥得到HAMA,即改性后的透明质酸钠。
进一步地,所述透明酸钠(HA)与去离子水按照质量比1:100混合;加入4.8mL甲基丙烯酸酐;然后使用规格为8000-14000Da的透析袋,用去离子水透析48h。
如上所述胞外多糖基水凝胶的制备方法为将改性后的透明质酸钠(HAMA)和改性后的贝莱斯芽孢杆菌胞外多糖(76DA)溶于LAP溶液中,405nm光交联30s得胞外多糖基水凝胶(HAMA-76DA水凝胶)。
进一步地,HAMA、贝莱斯芽孢杆菌胞外多糖(76DA)和蓝光引发剂LAP溶液之间的质量比为1:2:100;所述LAP溶液质量浓度为0.25%;即将50mg HAMA和100mg 76DA溶于5mL浓度为质量浓度为0.25%的LAP溶液中。
如上所述胞外多糖基水凝胶在促进皮肤伤口愈合药物方面的应用。
与现有技术相比,本发明的有益效果:
本发明使用改性后的贝莱斯芽孢杆菌胞外多糖构建水凝胶网络,该水凝胶机械性能良好,具有优良的生物相容性,对皮肤伤口愈合有较好的促进作用;进一步地本发明胞外多糖基水凝胶易于制备,合成方法简单,生产成本低。
保藏信息说明
Bacillus velezensis M76T11B于2020年12月23日保藏于广东省微生物菌种保藏中心,保藏号为GDMCC NO:61384。
附图说明
图1是制备所得水凝胶扫描电镜图(SEM);其中图1左图为本发明实施例5制备所得HAMA水凝胶,图1右图为本发明实施例4制备所得胞外多糖基水凝胶(HAMA-76DA)。
图2为水凝胶细胞毒性图;HAMA-76DA为本发明实施例4制备所得胞外多糖基水凝胶;HAMA指本发明实施例5制备所得HAMA水凝胶。
图3为水凝胶血液相容性;HAMA-76DA为本发明实施例4制备所得胞外多糖基水凝胶;HAMA指本发明实施例5制备所得HAMA水凝胶;Trition为阳性对照组,PBS为阴性对照
图4为不同时间大鼠伤口图片(左)及愈合率(右);HAMA-76DA为本发明实施例4制备所得胞外多糖基水凝胶;HAMA指本发明实施例5制备所得HAMA水凝胶;Control为对照组。
图5为不同时间各组伤口HE染色图;HAMA-76DA为本发明实施例4制备所得胞外多糖基水凝胶;HAMA指本发明实施例5制备所得HAMA水凝胶;Control为对照组。
具体实施方式
下面结合附图具体实施方式进行详细描述,但应当理解本发明的保护范围并不受具体实施方式的限制。实施例中采用的原料、试剂若无特殊说明,皆为市售所得。实施例中使用的菌种:
贝莱斯芽孢杆菌M76T11B(Bacillus velezensis M76T11B)为本课题组自主筛选所得,于2020年12月23日保藏于广东省微生物菌种保藏中心,保藏地址为广州市先烈中路100号大院9号楼5楼,保藏号为GDMCC NO:61384。
实施例中使用的各培养基组成:
1L MRS固体培养基的组成是:蛋白胨10g,牛肉膏10g,酵母粉10g,吐温80 1mL,磷酸氢二钾2g,乙酸钠5g,柠檬酸三铵2g,硫酸镁0.2g,硫酸锰0.05g,葡萄糖10g,琼脂15g。
1L MRS液体培养基的组成是:蛋白胨10g,牛肉膏10g,酵母粉10g,吐温80 1mL,磷酸氢二钾2g,乙酸钠5g,柠檬酸三铵2g,硫酸镁0.2g,硫酸锰0.05g,葡萄糖10g。
1L MRS-S液体培养基的组成是:蛋白胨10g、牛肉膏5g、酵母粉4g、葡萄糖5g、蔗糖50g、磷酸氢二钾2g、柠檬酸三铵2g、乙酸钠5g、硫酸镁0.2g、吐温80 1g、硫酸锰0.05g。
实施例1
贝莱斯芽孢杆菌胞外多糖的提取方法,操作步骤如下:
(1)菌种活化:将保藏号为GDMCC NO:61384且保藏于4℃冰箱中的Bacillusvelezensis M76T11B在MRS平板上进行三区划线活化菌株,37℃恒温条件下,静置培养48h后挑取生长状态良好的单菌落接种至MRS肉汤中,37℃、150rpm培养16h;
(2)扩大培养:将在MRS肉汤中培养后的菌株以5%(v/v)接种量接种到1000mLMRS-S液体培养基中,在37℃、150rpm条件下摇床培养48h,即得发酵液;
(3)粗多糖的沉淀:取发酵液经4℃、10,000×g离心20min去除菌体沉淀,取上清液浓缩到原体积1/5,然后加入体积浓度95%乙醇至终浓度为80%(v/v),搅拌均匀,4℃静置过夜沉淀多糖,在4℃、10,000×g离心20min获得多糖沉淀;
(4)脱蛋白:将多糖沉淀溶于去离子水中,加入80%(m/v)的三氯乙酸至终浓度4%(m/v),搅拌均匀4℃静置10h后在4℃下10,000×g离心20min弃去蛋白沉淀,收集上清液;
(5)多糖的二次醇沉:将上清液浓缩,加入浓度95%乙醇至终浓度为80%(v/v),在4℃下静置过夜以沉淀多糖,过滤,收集多糖沉淀,将多糖沉淀重新溶解于去离子水中,装入透析袋(截留分子量8000-14000Da)透析48h,每隔8h换水,将透析后的糖溶液冷冻干燥,即得贝莱斯芽孢杆菌胞外多糖(M76-EPS)。
实施例2
贝莱斯芽孢杆菌胞外多糖的改性
量取50mL磷酸盐缓冲液(pH值为5.5),加入500mg实施例1制备所得贝莱斯芽孢杆菌胞外多糖(M76-EPS),充分搅拌溶解,得到混合溶液。然后称量碳二亚胺(EDC)2.5mmol,N-羟基琥珀酰亚胺(NHS)2.5mmol溶解在上述混合溶液中,充分搅拌并溶解,调节pH至5.5,持续搅拌30min。再加入盐酸多巴胺(DA)2.5mmol,充分搅拌溶解,持续搅拌反应4h。然后使用规格为8000-14000Da的透析袋,用去离子水透析48h,冷冻干燥得到多巴胺改性的贝莱斯芽孢杆菌胞外多糖(76DA)。
实施例3
透明质酸钠的改性
将1g透明质酸钠(HA)溶于100mL去离子水中,调节溶液的pH值为8,加入4.8mL甲基丙烯酸酐(MA),在室温下搅拌12h,再通过乙醇(体积浓度80%)沉淀得到HAMA粗产物,并用无水乙醇洗涤三次以去除剩余的甲基丙烯酸和甲基丙烯酸酐,然后使用规格为8000-14000Da的透析袋,用去离子水透析48h,冷冻干燥得到HAMA,即改性后的甲基丙烯酰化透明质酸钠。
实施例4
胞外多糖基水凝胶的制备:将50mg实施例3制备所得HAMA和100mg实施例2制备所得76DA溶于5mL(重约5g)浓度为0.25%(w/v)的LAP溶液中(LAP溶液是蓝光引发剂),405nm光交联30s得HAMA-76DA水凝胶,即为胞外多糖基水凝胶(HAMA-76DA)。
实施例5
水凝胶的制备:将50mg实施例3制备所得HAMA溶于5mL浓度为0.25%(w/v)的LAP溶液中,405nm光交联30s得HAMA水凝胶。
性能测定
1、水凝胶扫描电镜观察
将实施例4制备所得胞外多糖基水凝胶(HAMA-76DA)浸入液氮冷冻5min,冷冻干燥48h,然后喷金,置于扫描电镜上观察水凝胶的微观结构。实施例5制备所得水凝胶(HAMA)也进行上述同样操作。
结果如图1所示,SEM表征结果显示,两种水凝胶表现出典型的三维网状结构,而实施例4的HAMA-76DA则具有更加紧密的三维结构,这是由于贝莱斯芽孢杆菌胞外多糖与透明质酸的交联,说明成功合成了所需材料。
2、水凝胶的细胞毒性测定
称量实施例4和实施例5中的两种水凝胶各10mg、20mg、50mg、100mg分别浸于1mLDMEM培养基,24h后1000rpm离心5min,取上清过0.22um有机滤膜得到浓度分别为10mg/mL、20mg/mL、50mg/mL、100mg/mL的水凝胶培养基浸出液。
将5×104个L929细胞接种在96孔板中,待细胞长至密度为80%左右时,更换100uL无血清培养基培养24h。然后将无血清培养基更换为上述的水凝胶培养基浸出液100uL,培养24小时后加入10uL 5mg/ml的噻唑蓝(MTT,终浓度为0.5mg/mL)孵育4h。最后,加入200ulDMSO震荡5min后于490nm处测量每孔吸光度,每组设6个平行。按以下公式计算细胞存活率:
细胞存活率=(对照组-空白组)/(实验组-空白组)×100%
其中对照组为未加水凝胶培养基浸出液的,用正常DMEM培养的细胞孔。空白组为未加入细胞,含DMEM培养基和MTT的孔。结果如图2所示,在100mg/mL和50mg/mL的浓度下,实施例5的HAMA和实施例4的HAMA-76DA水凝胶表现出微弱的细胞毒性,细胞存活率在70%-80%之间,但当浓度低于20mg/L时,两种水凝胶表现出较好的细胞相容性。L929细胞存活率菌大于90%。综上所述,本发明制备所得胞外多糖基水凝胶无明显细胞毒性,生物相容性较好。
3、水凝胶的溶血性能测定
取SD大鼠血500μL,在4℃、3500rpm下离心5min。下层红细胞再用PBS离心3次,用5mL PBS重新悬浮。然后,将250mg实施例4、5制备所得水凝胶与1mL红细胞在室温下在试管中混合4h,并设阴性对照(PBS)和阳性对照(Triton),计算溶血程度。所有样品在3500rpm下离心5min,取上清液用微量平板阅读器在570nm处测定吸光度。结果如图3所示,阳性对照组Trition表现出较强的溶血能力,而水凝胶组的溶血能力较弱,与阴性对照组PBS无显著区别。表明实施例4和实施例5制备所得水凝胶具有优良的血液相容性。
4、水凝胶在伤口愈合中的应用
将8周龄雄性SD大鼠随机分为4组,每组4只,剔除大鼠背部毛发,在背部切出一个直径约10mm的全层皮肤伤口模型。按表1的分组情况分别处理大鼠伤口,以无菌生理盐水为对照,缠绕纱布,每天更换样品,并拍照记录伤口愈合情况。在第4、9、14天时,每组分别处死4只大鼠,取伤口处全层皮肤固定、包埋、HE染色。图4为不同时间大鼠伤口图片(左)及愈合率(右)。结果显示第8天,各组创面面积均缩小,实施例4的HAMA-76DA组伤口闭合率超过60%,显著高于对照组。在第12和第14天,实施例4的HAMA-76DA组表皮组织变得光滑,伤口愈合效果显著优于对照组,也优于实施例5的HAMA。
表1大鼠皮肤伤口不同处理分组
组别 | 处理方式 |
对照(Control) | 无菌生理盐水处理 |
HAMA | 水凝胶HAMA覆盖伤口 |
HAMA-76DA | 水凝胶HAMA-76DA覆盖伤口 |
图5为不同时间各组伤口的HE染色图像,在第4天时,HAMA-76DA组伤口表现出的一定程度的上皮化。第9天时,HAMA-76DA组出现了较多的新生毛囊和毛细血管。第14天时,HAMA-76DA组毛囊发育完全,皮肤结构完整,伤口基本愈合。图中所示的伤口面积大小、伤口愈合率随时间的变化和伤口HE染色结果,都进一步证明的HAMA-76DA水凝胶具有优良的伤口愈合功效。
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式,并且很显然,根据上述教导,可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用,从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
Claims (9)
1.一种胞外多糖基水凝胶,其特征在于:用改性后的贝莱斯芽孢杆菌胞外多糖和改性后的透明质酸钠为原料,构建水凝胶体系;所述的贝莱斯芽孢杆菌胞外多糖采用多巴胺改性,所述的透明质酸钠用甲基丙烯酸酐改性。
2.根据权利要求1所述胞外多糖基水凝胶,其特征在于:所述贝莱斯芽孢杆菌胞外多糖的改性为磷酸盐缓冲液和贝莱斯芽孢杆菌胞外多糖,充分搅拌溶解,得到混合溶液,然后将碳二亚胺,N-羟基琥珀酰亚胺溶解在上述混合溶液中,充分搅拌并溶解,调节pH至5.5,持续搅拌;再加入盐酸多巴胺,充分搅拌溶解,持续搅拌反应4h,然后透析,冷冻干燥后得到多巴胺改性的贝莱斯芽孢杆菌胞外多糖。
3.根据权利要求2所述胞外多糖基水凝胶,其特征在于:所述磷酸盐缓冲液的体积为50mL;所述贝莱斯芽孢杆菌胞外多糖用量为500mg,碳二亚胺、N-羟基琥珀酰亚胺以及盐酸多巴胺的添加量均为2.5mmol;所述使用规格为8000-14000Da的透析袋,用去离子水透析48h。
4.根据权利要2所述胞外多糖基水凝胶,其特征在于,所述贝莱斯芽孢杆菌胞外多糖的提取方法,包含以下操作步骤:
(1)菌种活化:将保藏号为GDMCC NO:61384的Bacillus velezensis M76T11B在MRS平板上活化菌株,37℃恒温条件下,静置培养48h后挑取生长状态良好的单菌落接种至MRS肉汤中,37℃、150rpm培养16h;
(2)扩大培养:将在MRS肉汤中培养后的菌株以5%接种量接种到1000mL MRS-S液体培养基中,在37℃、150rpm条件下摇床培养48h,即得发酵液;
(3)粗多糖的沉淀:取发酵液经4℃、10,000×g离心20min去除菌体沉淀,取上清液浓缩到原体积1/5加入4倍体积的4℃体积浓度95%乙醇至终浓度为80%,搅拌均匀,4℃静置过夜沉淀多糖,在4℃、10,000×g离心20min获得多糖沉淀;
(4)脱蛋白:将多糖沉淀溶于水中,加入80%的三氯乙酸至终浓度4%,搅拌均匀4℃静置10h后在4℃下10,000×g离心20min弃去蛋白沉淀,收集上清液;
(5)多糖的二次醇沉:将上清液浓缩,加入4倍体积的95%乙醇至终浓度为80%,在4℃下静置过夜以沉淀多糖,过滤,收集多糖沉淀,将多糖沉淀重新溶解于水中,装入透析袋透析48h,每隔8h换水,将透析后的糖溶液冷冻干燥,即得贝莱斯芽孢杆菌胞外多糖。
5.根据权利要1所述胞外多糖基水凝胶,其特征在于:所述透明质酸钠的改性为将透明质酸钠溶于水中,调节溶液的pH值为8,加入甲基丙烯酸酐,在室温下搅拌,再通过乙醇沉淀得到HAMA粗产物,并用无水乙醇洗涤,然后透析,冷冻干燥得到HAMA,即改性后的透明质酸钠。
6.根据权利要5所述胞外多糖基水凝胶,其特征在于:所述透明酸钠与水按照质量比1:100混合;加入4.8mL甲基丙烯酸酐;然后使用规格为8000-14000Da的透析袋,用水透析48h。
7.如权利要求1-6任一所述胞外多糖基水凝胶的制备方法,其特征在于:将改性后的透明质酸钠和改性后的贝莱斯芽孢杆菌胞外多糖溶于的LAP溶液中,405nm光交联得胞外多糖基水凝胶。
8.根据权利要求7所述胞外多糖基水凝胶的制备方法,其特征在于:HAMA、贝莱斯芽孢杆菌胞外多糖(76DA)和LAP溶液之间的质量比为1:2:100;所述LAP溶液的质量浓度为0.25%。
9.如权利要求1-6或8任一所述胞外多糖基水凝胶在促进皮肤伤口愈合药物方面的应用。
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