CN117467153A - 一种多功能丝胶蛋白的制备方法及其应用 - Google Patents
一种多功能丝胶蛋白的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种多功能丝胶蛋白的制备方法及其应用,涉及医用生物复合材料技术领域。包括如下步骤:(1)丝胶蛋白的提取:将剪碎的蚕茧与碳酸钠溶液在加热条件下搅拌,提取出丝胶,除杂,透析,冻干,得到丝胶蛋白粉末;(2)乙酰化丝胶蛋白的制备:将丝胶蛋白粉末溶于二甲亚砜溶液中,然后加入丁二酸酐,搅拌反应,将反应液透析,干燥,得到含有乙酰基的乙酰化丝胶蛋白粉末;(3)多功能丝胶蛋白的制备:将乙酰化丝胶蛋白粉末溶于4‑吗啉乙磺酸缓冲液中,然后加入多巴胺,通过酰胺缩合反应,得到反应液,将反应液透析,干燥,制备得到多功能丝胶蛋白粉末。本发明制备方法简单、环保,可以实现以丝胶作为原料制备糖尿病皮肤创面愈合材料。
Description
技术领域
本发明涉及医用生物复合材料技术领域,具体涉及一种多功能丝胶蛋白的制备方法及其应用。
背景技术
在糖尿病患者中,约19-34%的患者患有创面愈合不良或迁延不愈,且糖尿病足是世界上非创伤性截肢的主要原因之一,因此提高糖尿病创面愈合率极为重要。目前,创面愈合分为止血、炎症、增殖和重塑4个阶段,其中炎症阶段被认为是糖尿病伤口愈合过程中最失调的阶段,由于糖酵解代谢紊乱,诱导高血糖敏感细胞(例如内皮细胞)和血糖不敏感细胞(巨噬细胞)产生过量ROS,引起促炎和抗炎细胞/因子失衡,阻碍伤口闭合。血管生成和再上皮化是增殖和重塑阶段的关键行为,由于周围血管疾病的发生,糖尿病创面供氧不足,同时,在炎症阶段,伤口部位的免疫细胞会消耗大量氧气,进一步加剧缺氧环境。长期缺氧以及高糖微环境的慢性创面通过抑制细胞增殖(如成纤维细胞、角质形成细胞和内皮细胞),限制促愈合生长因子(如VEGF)产生,阻碍血管生成、ECM形成和再上皮化。因此,逆转失调的炎症环境,缓解乏氧微环境是促进糖尿病愈合的关键。
然而,面前仍无没有理想的材料可以同时克服所有这些挑战。因此,迫切需要设计构建一种新的治疗平台来解决上述问题。
发明内容
本发明提供的一种多功能丝胶蛋白的制备方法及其应用,旨在解决上述背景技术中存在的问题。
为了实现上述技术目的,本发明主要采用如下技术方案:
第一方面,本发明公开了一种多功能丝胶蛋白的制备方法,包括如下步骤:
(1)丝胶蛋白的提取:将剪碎的蚕茧与碳酸钠溶液在加热条件下搅拌,提取出丝胶,除杂,透析,冻干,得到丝胶蛋白粉末;
(2)乙酰化丝胶蛋白的制备:将步骤(1)得到的丝胶蛋白粉末溶于二甲亚砜溶液中,然后加入丁二酸酐,搅拌反应,将反应液透析,干燥,得到含有乙酰基的乙酰化丝胶蛋白粉末;
(3)多功能丝胶蛋白的制备:将步骤(2)得到的乙酰化丝胶蛋白粉末溶于4-吗啉乙磺酸缓冲液中,然后加入多巴胺,通过酰胺缩合反应,得到反应液,将反应液透析,干燥,制备得到多功能丝胶蛋白粉末。
丝胶是蚕丝的主要成分,是包裹在丝素纤维表层的一种天然大分子蛋白,含有17-18种氨基酸,具有良好的生物性能如低免疫原性、生物可降解性、抗氧化性、细胞黏附性等,已用于皮肤伤口愈合,心脏功能恢复,神经修复和其他软组织再生。
过氧乙酸是一种常见的消毒成份,经活性含氧化合物(如过氧化氢)与乙酰基供体反应生成,易分解产生氧气。因此,利用丝胶蛋白上丰富的氨基构建乙酰基供体,进而捕获创面环境中的过量活性氧,可模拟过氧乙酸的产氧功能,“一石二鸟”清除活性氧同时缓解局部乏氧环境。
多功能丝胶蛋白是在丝胶蛋白的基础上,对其进行官能团修饰,从而增加了其乙酰基供体含量,使其有望成为新型产氧前体分子。一方面,丝胶蛋白本身的优越性能已被证实可以促进皮肤组织的再生修复;另一方面,丝胶蛋白在进行修饰多功能后,成为含有丰富乙酰基的供体材料,也可响应糖尿病创面的高活性氧水平,产生氧气进一步促进创面修复再生。
在本发明的较佳实施方式中,步骤(1)中,10g剪碎的蚕茧与浓度为0.02mol/L的碳酸钠溶液混合后,在100℃条件下搅拌,提取丝胶。
在本发明的较佳实施方式中,步骤(1)、步骤(2)和步骤(3)中,透析时,透析袋截留分子量为3500Da。
进一步的,步骤(1)中,透析袋在双蒸水中透析3天;步骤(2)和步骤(3)中,透析袋先在0.1M磷酸缓冲液中透析2小时,然后在0.05M磷酸缓冲液中透析2小时,再在0.01M磷酸缓冲液中透析2小时,最后在双蒸水中透析3天。
在本发明的较佳实施方式中,步骤(2)中,丝胶蛋白粉末以0.02g/mL的浓度溶于二甲亚砜溶液中,且丝胶蛋白粉末与丁二酸酐的质量比为1:1~10:1。
在本发明的较佳实施方式中,步骤(3)中,乙酰化丝胶蛋白粉末以0.011g/mL的浓度溶于pH为4.5~6.5的4-吗啉乙磺酸缓冲液中。
在本发明的较佳实施方式中,步骤(3)中,乙酰化丝胶蛋白粉末与多巴胺的反应,在1-乙基-(3-二甲基氨基丙基)碳酰二亚胺与N-羟基琥珀酰亚胺存在条件下进行。
在本发明的较佳实施方式中,步骤(3)中,乙酰化丝胶蛋白粉末与多巴胺的质量比10:1~3:1。
在本发明的较佳实施方式中,步骤(3)中,乙酰化丝胶蛋白与多巴胺的反应在惰性气氛保护条件下进行。
第二方面,本发明公开了一种由第一方面所述制备方法制备得到的多功能丝胶蛋白在制备糖尿病创面愈合材料中的应用。
与现有技术相比,本发明具有如下有益效果:
本发明提供的多功能丝胶蛋白的制备方法简单、环保,制备得到的多功能丝胶蛋白可以实现以丝胶作为原料制备糖尿病皮肤创面愈合材料;且多功能丝胶蛋白具有优异的清除活性氧及与响应糖尿病创面高活性氧水平产氧性能和促进糖尿病创面修复再生性能。
附图说明
图1为各处理组采用溶氧仪检测溶液中溶解氧浓度的操作示意图;
图2为各处理组的产氧性能展示图;其中,A为溶解氧浓度;B为产氧外观图;C、D为多功能丝胶蛋白SDA与过氧化氢的反应产物的离子流图及质谱图;E、F为乙酸标准品的粒子流图及质谱图;
图3为各处理组促糖尿病创面愈合展示图;其中,A为操作流程图;B、C为各处理组小鼠伤口愈合率对比图;D为各处理组小鼠体重大小对比图;E-G为小鼠伤口愈合率及愈合后的皮肤质量对比图;H为各处理组小鼠创面组织切片H&E染色对比图;
图4为各处理组创面组织指标表达对比图;
图5为多功能丝胶蛋白生物相容性展示图;其中,A-D为多功能丝胶蛋白体外生物相容性测试结果图;E为多功能丝胶蛋白体内生物相容性测试结果图。
具体实施方式
下面结合附图对本发明做进一步说明
实施例1多功能丝胶蛋白的制备
步骤1)丝胶蛋白的提取
1.称取10g干燥蚕茧(家蚕蚕茧(白玉、皓月),棹蚕茧(A.mylitta)或蓖麻蚕茧等),将其剪成碎片;
2.在蚕茧碎片中加入400mL的0.02mol/L碳酸钠溶液,置于恒温水浴锅在100oC条件下搅拌1小时,使丝胶溶解,得到丝胶溶液;
3.将得到的丝胶溶液转入50mL离心管中,4000rpm离心5分钟去除丝胶溶液中的杂质成分,得到上清液;
4.将上清液转入分子截留量为3500Da的透析袋中,于双蒸水中透析3天,慢速搅拌,每隔6小时换一次水;
5.将丝胶溶液转入50mL离心管中,置于液氮中速冻5分钟后放入冻干机,冻干后得到丝胶粉末,放入-20℃冰箱保存备用。
步骤2)乙酰化丝胶蛋白的制备
将步骤1)中得到的丝胶蛋白粉末以0.02g/mL的浓度溶于二甲亚砜溶液中,加入丁二酸酐溶液(丝胶粉末与丁二酸酐的质量比为2:1)在室温下搅拌5小时。
将上述溶液装入截留分子量3500Da的透析袋,在磷酸缓冲液(PB)溶液(0.1M)透析2小时,然后透析液改为PB溶液(0.05M)透析2小时,PB溶液(0.01M)透析2小时,再在双蒸水中透析3天,冷冻干燥后得到丁二酸酐修饰的乙酰化丝胶蛋白。
步骤3)多功能丝胶蛋白的制备
称取步骤2)中的乙酰化丝胶蛋白粉末1g、EDC(89.7mg)、NHS(53.8mg)和多巴胺(135mg)溶于4-吗啉乙磺酸缓冲液(0.1M,pH 6.0、90mL)中,置于三通瓶中(一端持续通入氮气,另一端出气)于室温及避光条件下搅拌24小时,得到反应液,将反应液透析。
其中,透析过程与步骤2)中的乙酰化丝胶蛋白相同。再经冷冻干燥后,得到多功能丝胶蛋白粉末。
实施例2多功能丝胶蛋白的产氧能力检测
一、实验过程
1、如图1所示,将100μL的1mg/mL的丝胶蛋白溶液(Ser)、乙酰化丝胶蛋白溶液(SSA)、多功能丝胶蛋白溶液(SDA)及多巴胺(Dopamine)溶液分别加入10mL 10mM的过氧化氢溶液中,将溶氧仪探头伸入溶液中,并用2mL石蜡油在上层进行液封,用溶氧仪检测60分钟内溶液中的溶解氧浓度变化情况。
2、将200μL的1mg/mL的乙酰化丝胶蛋白溶液(SSA)及多功能丝胶蛋白溶液(SDA)分别加入10mL 10mM的过氧化氢溶液中,并用2mL石蜡油在上层进行液封,拍摄反应前、反应后1min、3min、5min、10min、20min的产氧实物照片。
3、将20mg的多功能丝胶蛋白(SDA)与2mL 5mM的过氧化氢溶液混合反应1小时后的溶液进行液相色谱与串联质谱联用(LC-MS)分析,并以标准化乙酸(CH3COOH)作为对照。
4、将100μL的1mg/mL的丝胶蛋白(Ser)、牛血清白蛋白(BSA)、卵清蛋白(OVA)及溶菌酶(LZM)溶液分别加入10mL 10mM的过氧化氢溶液中,将溶氧仪探头伸入溶液中,并用2mL石蜡油在上层进行液封,用溶氧仪检测60分钟内溶液中的溶解氧浓度变化情况。
二、结果分析
1.如图2中A所示,丝胶蛋白溶液(Ser)本身及官能化修饰后的乙酰化丝胶蛋白溶液(SSA)、多功能丝胶蛋白溶液(SDA)均可与过氧化氢反应产氧,而多巴胺本身并无与过氧化氢反应产氧的能力,且在对丝胶进行官能化修饰后,其溶解氧浓度有较大提升,推测其原因是对丝胶蛋白进行修饰后,其乙酰基供体含量增加,与过氧化氢反应并产氧的能力也得到有效提升。
2.如图2中B所示,乙酰化丝胶蛋白溶液(SSA)及多功能丝胶蛋白溶液(SDA)均可与过氧化氢反应产氧,可见在20min内,积聚在液封处的氧气气泡越来越多,表明乙酰化丝胶蛋白溶液(SSA)及多功能丝胶蛋白溶液(SDA)均具有优越的产氧能力,且多功能丝胶蛋白溶液(SDA)产氧能力更佳。
3.如图2中C-D所示,多功能丝胶蛋白(SDA)与过氧化氢的反应产物与乙酸标准品相比,液相色谱的出峰时间均在2-4min,且质谱的结构一致,表明其反应产物中有乙酸的存在。
实施例3多功能丝胶蛋白的促糖尿病创面愈合能力测试
一、实验过程
如图3中A所示,使用1mL注射器给体重均一(20-25g)的雄性Balb/c小鼠按每1g体重0.12mg的剂量腹腔注射链脲佐菌素(STZ),每周一次,连续三周。3周后,用罗氏血糖仪测量血糖值,以血糖值超过16.7mmoL/L的小鼠为建模成功。小鼠按体重大小均一分为4组。在对小鼠进行脱毛、碘伏消毒和戊巴比妥钠麻醉后,在其背部创建一个10mm全层伤口。立即将灭菌后的磷酸盐缓冲液(PBS)、亚硫酸钠(一种除氧剂,缩写为SS)、多功能丝胶蛋白(SDA)、多功能丝胶蛋白联合亚硫酸钠(SDA/SS)均匀滴加至伤口上。术后每两天给小鼠称重并拍照,直到第14天安乐死小鼠,并取切口周围的组织皮肤,用4%多聚甲醛对其进行固定,并进行组织石蜡包埋及切片,进行H&E、马松及免疫荧光染色,并对其结果进行统计学分析;同时取小鼠的心、肝、脾、肺、肾进行4%多聚甲醛固定、石蜡组织包埋、切片及H&E染色,以进行生物安全性分析。
二、结果分析
1.如图3中B-C所示,多功能丝胶蛋白处理组(SDA)的小鼠创面愈合最快,伤口愈合率与对照组显著高于对照组;图3中D显示,各处理组小鼠体重大小无明显差异;图3中E-G显示,多功能丝胶蛋白处理组(SDA)创面愈合质量较好,有毛囊生成,胶原沉积率也最高,明显优于对照组;而在联合使用除氧剂亚硫酸钠(SDA/SS)后,小鼠伤口愈合率及愈合后的皮肤质量均有所降低,显示出多功能丝胶蛋白用于创面愈合的优越性,也证实了氧气对于创面愈合的有益作用。
2.如图3中H所示,多功能丝胶蛋白处理组(SDA)的小鼠创面组织切片中H&E染色中较之对照组可见更为明显的新生血管生成。
3.如图4中A-F所示,与对照组相比,多功能丝胶蛋白处理组(SDA)的小鼠创面组织CD31和VEGF的表达均升高,抗炎因子IL-10的表达升高,促炎因子IL-6的表达降低。
实施例4多功能丝胶蛋白体外生物相容性测试
一、实验过程
1.用2mL含有10%FBS的DMEM培养基分别于6孔板中培养小鼠成纤维细胞(L929)及人脐静脉内皮细胞(HUVEC)105个/孔24小时。
2.24小时后,弃去原培养基,更换为含有0.5mg/mL的多功能丝胶蛋白溶液(SDA)的新鲜DMEM培养基,在37℃细胞培养箱中共培养24小时,并以加磷酸盐缓冲液(PBS)的新鲜培养基组作为对照。
3.24小时后,弃去原培养基,用PBS清洗一遍,每孔加入含1μL钙黄绿素乙酰氧基甲酯(Calcein-AM)及1μL碘化丙啶(PI)的1mL缓冲液,37℃避光孵育30分钟后弃去染液,用PBS清洗后用荧光显微镜拍摄细胞活死染色图像。
4.用100μL含有10%FBS的DMEM培养基分别于96孔板中培养小鼠成纤维细胞(L929)及人脐静脉内皮细胞(HUVEC)10000个/孔24小时,以不加细胞作为空白背景;
5.24小时后各孔分别加入100μL含有0.5mg/mL多功能丝胶蛋白溶液(SDA)的新鲜DMEM培养基,以不加材料作为空白对照;
6.24小时,弃去原上清,配置含0.5mg/mL MTT的不含FBS的DMEM培养基后每孔加入100μL试剂,37℃孵育4小时后,弃尽上清,每孔加入150μL二甲亚砜溶液,将96孔板置于37℃摇床摇10min后于490nm处检测吸光度。
二、结果分析
如图5中A-D图所示,多功能丝胶蛋白组(SDA)与PBS组相比,小鼠成纤维细胞(L929)及人脐静脉内皮细胞(HUVEC)仍能维持很好的活性,表明多功能丝胶蛋白(SDA)不具有细胞毒性。
实施例5多功能丝胶蛋白体内生物相容性测试
一、实验过程
在小鼠安乐死后,取各处理组小鼠的心、肝、脾、肺、肾用4%多聚甲醛进行固定,然后进行石蜡组织包埋切片并进行H&E染色,在显微镜下拍摄其组织切片H&E染色结果。
二、结果分析
如图5中E图所示,多功能丝胶蛋白处理组(SDA)的小鼠的心、肝、脾、肺、肾等主要脏器的HE染色切片与对照组处理相比,未见明显病理异常,表明多功能丝胶蛋白(SDA)具有良好的体内生物相容性。
本发明通过实验测试该多功能丝胶蛋白的产氧性能及促糖尿病创面修复性能,发现该材料具有较好的清除活性氧及与响应糖尿病创面高活性氧水平产氧性能,可有效促进糖尿病创面的修复,同时具有良好的生物相容性。
最后应说明的是:以上仅为本发明的优选实例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种多功能丝胶蛋白的制备方法,其特征在于,包括如下步骤:
(1)丝胶蛋白的提取:将剪碎的蚕茧与碳酸钠溶液在加热条件下搅拌,提取出丝胶,除杂,透析,冻干,得到丝胶蛋白粉末;
(2)乙酰化丝胶蛋白的制备:将步骤(1)得到的丝胶蛋白粉末溶于二甲亚砜溶液中,然后加入丁二酸酐,搅拌反应,将反应液透析,干燥,得到含有乙酰基的乙酰化丝胶蛋白粉末;
(3)多功能丝胶蛋白的制备:将步骤(2)得到的乙酰化丝胶蛋白粉末溶于4-吗啉乙磺酸缓冲液中,然后加入多巴胺,通过酰胺缩合反应,得到反应液,将反应液透析,干燥,制备得到多功能丝胶蛋白粉末。
2.根据权利要求1所述的多功能丝胶蛋白的制备方法,其特征在于:步骤(1)中,10g剪碎的蚕茧与浓度为0.02mol/L的碳酸钠溶液混合后,在100℃条件下搅拌,提取丝胶。
3.根据权利要求1所述的多功能丝胶蛋白的制备方法,其特征在于:步骤(1)、步骤(2)和步骤(3)中,透析时,透析袋截留分子量为3500Da。
4.根据权利要求3所述的多功能丝胶蛋白的制备方法,其特征在于:步骤(1)中,透析袋在双蒸水中透析3天;步骤(2)和步骤(3)中,透析袋先在0.1M磷酸缓冲液中透析2小时,然后在0.05M磷酸缓冲液中透析2小时,再在0.01M磷酸缓冲液中透析2小时,最后在双蒸水中透析3天。
5.根据权利要求1所述的多功能丝胶蛋白的制备方法,其特征在于:步骤(2)中,丝胶蛋白粉末以0.02g/mL的浓度溶于二甲亚砜溶液中,且丝胶蛋白粉末与丁二酸酐的质量比为1:1~10:1。
6.根据权利要求1所述的多功能丝胶蛋白的制备方法,其特征在于:步骤(3)中,乙酰化丝胶蛋白粉末以0.011g/mL的浓度溶于pH为4.5~6.5的4-吗啉乙磺酸缓冲液中。
7.根据权利要求1所述的多功能丝胶蛋白的制备方法,其特征在于:步骤(3)中,乙酰化丝胶蛋白粉末与多巴胺的反应,在1-乙基-(3-二甲基氨基丙基)碳酰二亚胺与N-羟基琥珀酰亚胺存在条件下进行。
8.根据权利要求1所述的多功能丝胶蛋白的制备方法,其特征在于:步骤(3)中,乙酰化丝胶蛋白粉末与多巴胺的质量比10:1~3:1。
9.根据权利要求1所述的多功能丝胶蛋白的制备方法,其特征在于:步骤(3)中,乙酰化丝胶蛋白与多巴胺的反应在惰性气氛保护条件下进行。
10.由权利要求1-9任一项所述制备方法制备得到的多功能丝胶蛋白在制备糖尿病创面愈合材料中的应用。
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