CN117414524A - 一种载药丝胶微针的制备方法及其应用 - Google Patents
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Abstract
本发明公开了一种载药丝胶微针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)多功能丝胶蛋白的制备;(2)改性透明质酸的制备:将透明质酸与氟代氨基苯硼酸FPBA反应生成改性透明质酸,冻干;(3)载药丝胶微针的制备:将多功能丝胶蛋白与改性透明质酸混合均匀后,再向其中加入药物溶液,混合均匀后,倒入微针模具中,调节pH使其原位固化成胶,脱模,制备得到载药丝胶微针。本发明通过制备兼具响应糖尿病创面微环境降解、清除活性氧、产氧以及促进无瘢痕愈合的载药丝胶微针,主要解决了创面瘢痕生成影响皮肤正常功能、现有氧载体无法深入创面内部、现有产氧材料的局限性、糖尿病创面迁延不愈等问题。
Description
技术领域
本发明涉及医用生物复合材料技术领域,具体涉及一种载药丝胶微针的制备方法及其应用。
背景技术
据统计,目前全球糖尿病患者超过4.6亿人,预计到2045年将会达到7亿。在糖尿病患者中,约19-34%的患者患有创面迁延不愈。虽然糖尿病皮肤创面愈合不良的确切机制尚不清楚,但目前已有研究证明,氧化应激、血管生成障碍、促炎细胞因子表达增加和细菌感染等在其中发挥重要作用,其中氧化应激引起的高活性氧水平被认为是糖尿病创面迁延不愈的主要原因之一。目前,临床上主要用抗生素、生长因子等进行治疗,虽然有一定的效果,但也存在一些不良问题,如耐药性、副作用等。
众所周知,氧气广泛参与创面修复过程,如细胞增殖、细菌防御、血管生成和胶原合成,是创面成功愈合的先决条件。而在糖尿病创面中,免疫细胞在炎症阶段消耗了大量的氧气,同时受损的血管阻碍了氧气的传递,导致创面周围的缺氧环境。由于缺氧作用,创面氧合不足,细胞增殖(如成纤维细胞、角质形成细胞和内皮细胞)和促进愈合的生长因子(如VEGF)的产生受到抑制,进而阻碍了创面愈合。
此外,创面重塑阶段细胞外基质(ECM)产生和降解的不平衡导致瘢痕形成,影响正常皮肤超微结构的恢复及正常组织的功能。
丝胶是蚕丝的主要成分之一,是包裹在丝素纤维表层的一种天然大分子蛋白。在传统蚕丝工业中,大量丝胶在缫丝过程中随着工业废水排放到江河湖泊,其生物医学价值一直被人们所忽略。近年来,在组织工程领域,丝胶因具有优越的成胶性能、天然的细胞黏附性、稳定的天然荧光特性以及高生物相容性,被广泛应用于皮肤组织、血管组织、骨组织损伤、神经损伤等多种组织再生及修复。
创面微环境中过量的活性氧被认为是产生氧气和缓解缺氧的重要物质。多种纳米酶通过催化过氧化氢产生氧用于糖尿病创面愈合。然而,纳米酶的金属活性中心赋予了它潜在的生物毒性。含有具有光合作用能力的植物(如蓝藻细菌)的创面敷料也能在光下产生氧气,但在没有光的情况下产生的副产品(二氧化碳)将不可避免地抑制创面愈合。另外,现有的氧载体大多只与创面表层接触,一定程度上限制了它们在创面愈合中的实际性能。
因此,如何逆转功能失调的氧化还原微环境,缓解创面乏氧、抑制创面愈合过程中瘢痕形成、进一步提高糖尿病创面治疗效果是目前亟待解决的问题。
发明内容
本发明提供的一种载药丝胶微针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。通过制备兼具响应糖尿病创面微环境降解、清除活性氧、产氧以及促进无瘢痕愈合的载药丝胶微针,主要解决了创面瘢痕生成影响皮肤正常功能、现有氧载体无法深入创面内部、现有产氧材料的局限性、糖尿病创面迁延不愈等问题。
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,本发明公开了一种载药丝胶微针的制备方法,包括如下步骤:
(1)多功能丝胶蛋白的制备:将丝胶蛋白粉末溶于二甲亚砜溶液中,与丁二酸酐反应生成乙酰化丝胶蛋白,透析、冷冻干燥后,将得到的乙酰化丝胶蛋白粉末溶于4-吗啉乙磺酸缓冲液,与多巴胺发生酰胺缩合反应,透析,冻干后获得多功能丝胶蛋白;
(2)改性透明质酸的制备:将透明质酸溶于双蒸水,在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC和1-羟基苯并三唑HOBt的存在下,与氟代氨基苯硼酸FPBA反应生成改性透明质酸,冻干;
(3)载药丝胶微针的制备:将多功能丝胶蛋白与改性透明质酸混合均匀后,再向其中加入药物溶液,混合均匀后,倒入微针模具中,调节pH使其原位固化成胶,脱模,制备得到载药丝胶微针。
在本发明的较佳实施方式中,步骤(1)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(1)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,在惰性气氛保护及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,与多巴胺发生酰胺缩合反应,且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(1)中,透析时,透析袋截留分子量为3500Da。
在本发明的较佳实施方式中,步骤(2)中,透明质酸以3~8mg/mL的浓度溶于双蒸水,且透明质酸与氟代氨基苯硼酸FPBA的摩尔比为0.5:1~5:1。
在本发明的较佳实施方式中,步骤(2)中,反应时的pH为4.5~6.0。
在本发明的较佳实施方式中,步骤(3)中,先分别将改性透明质酸以2%~3%重量体积比w/v的浓度溶于双蒸水,多功能丝胶蛋白以20%~35%重量体积比w/v的浓度溶于双蒸水后,再将改性透明质酸水溶液与多功能丝胶蛋白水溶液按15:1~6:1的体积比混合。
在本发明的较佳实施方式中,步骤(3)中,所述药物溶液选自维替泊芬的二甲亚砜溶液,所述维替泊芬的二甲亚砜溶液浓度为1~2.5mg/mL,原位固化成胶时的pH为7.4~8.0。
在本发明的较佳实施方式中,步骤(3)中,改性透明质酸水溶液与多功能丝胶蛋白水溶液二者的混合物和药物溶液按40:1~10:1的体积比混合。
第二方面,本发明公开了一种由第一方面所述的制备方法制备得到的载药丝胶微针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:
本发明制备得到的载药丝胶微针(HFSVM)是由多功能丝胶蛋白(SDA)和改性透明质酸(HA-FPBA)通过硼酸酯键交联并装载维替泊芬形成,实现了以丝胶作为原料制备糖尿病创面愈合材料;制备得到的载药丝胶微针可以模拟过氧乙酸的构建和快速自分解功能,利用多功能丝胶蛋白作为乙酰基供体快速清除多余的活性氧,缓解缺氧,并通过葡萄糖响应的硼酸酯键响应高血糖状态释放YAP抑制剂维替泊芬,具有优异的活性氧清除性能、响应糖尿病创面微环境降解并基于活性氧触发产氧性能、促进血管生成及细胞迁移和促糖尿病创面无瘢痕愈合性能,能抑制瘢痕形成,加速糖尿病创面愈合;
本发明提供了的载药丝胶微针的制备方法,简单、环保;
本发明制备得到的载药丝胶微针(HFSVM)通过在体外及体内测试其促修复性能,发现该材料能有效促进糖尿病创面的修复,同时具有良好的生物相容性。
附图说明
图1为载药丝胶微针的制作示意图及表征展示图;
图2为载药丝胶微针的葡萄糖响应性能展示图;
图3为丝胶微针的体外活性氧清除性能展示图;
图4为丝胶微针的细胞保护及体外促迁移、促血管生成性能示意图;
图5为各处理组促糖尿病创面愈合展示图;
图6为各处理组创面组织指标表达对比图;
图7为丝胶微针与对照组的mRNA表达对比图;
图8为载药丝胶微针的测序结果展示图;
图9为丝胶微针体外生物相容性展示图;
图10为载药丝胶微针体内生物相容性展示图。
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
(1)丝胶的提取
称取10g干燥的蚕茧(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。
在蚕茧碎片中加入400mL的0.02mol/L Na2CO3水溶液,在100oC煮沸条件下搅拌1小时,使丝胶溶解,得到丝胶溶液。
4000rpm离心5分钟去除丝胶溶液中的杂质成分,收集上清液,装入截留分子量3500Da的透析袋,在双蒸水中透析3天,获得澄清丝胶溶液,冻干得到丝胶粉末。
(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。
(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。
透析过程与步骤2)中的乙酰化丝胶蛋白相同。冷冻干燥后,得到多功能丝胶蛋白粉末。
实施例2:改性透明质酸的制备
称取100mg(0.25mM)透明质酸(HA)粉末溶于20mL双蒸水中,充分溶解,加入48mg(0.25mM)氟代氨基苯硼酸(FPBA)倒入圆底烧瓶中搅拌。
将48mg(0.25mM)EDC粉末与34mg(0.25mM)HOBt混合溶于200μL二甲亚砜溶液中,再倒入上述溶液中。
滴加1M NaOH溶液少许调节至pH 5.0,常温下充分搅拌48小时。
48小时后用双蒸水在截留分子量为3500Da的透析袋中透析3天,冷冻干燥后得到氟代氨基苯硼酸修饰的改性透明质酸(HA-FPBA)。
实施例3:丝胶微针的制备
将实施例1制备得到的多功能丝胶蛋白(SDA)以30%(w/v)的浓度溶于双蒸水,实施例2制备得到的改性透明质酸以2.5%(w/v)的浓度溶于双蒸水;
将4.5mL 2.5%(w/v)的改性透明质酸(HA-FPBA)与500μL 30%(w/v)的多功能丝胶蛋白(SDA)混合均匀,取300μL混合物溶液倒入直径14mm的PDMS微针模具中,并加入80μL0.1M NaOH,4000rpm离心5分钟,抽真空5分钟,再4000rpm离心5分钟,静置30分钟;
配置8%(w/v)高分子透明质酸的水溶液,缓慢加入500μL 8%(w/v)高分子透明质酸作为基底;
通风条件下自然晾干2天,脱模取出即得到改性透明质酸(HA-FPBA)与多功能丝胶蛋白(SDA)交联制作的丝胶微针(HFSM)。
实施例4:载药丝胶微针的制备
将维替泊芬以2mg/mL的浓度溶解于二甲亚砜溶液中;
将4.5mL 2.5%(w/v)的改性透明质酸(HA-FPBA)与500μL 30%(w/v)的多功能丝胶蛋白(SDA)混合均匀,取300μL混合物与15μL 2mg/mL维替泊芬溶液充分混匀后倒入直径14mm的PDMS微针模具中,并加入80μL 0.1M NaOH,4000rpm离心5分钟,抽真空5分钟,再4000rpm离心5分钟,静置30分钟;
配置8%(w/v)高分子透明质酸的水溶液,缓慢加入500μL 8%(w/v)高分子透明质酸作为基底;
通风条件下自然晾干2天,脱模取出即得到改性透明质酸(HA-FPBA)与多功能丝胶蛋白(SDA)交联并搭载了药物维替泊芬的载药丝胶微针(HFSVM)。
实施例5丝胶微针表征检测
图1中A为载药丝胶微针制作示意图,具体制备过程见实施例1。
载药丝胶微针的针由装载维替泊芬的葡萄糖响应性水凝胶构建,该水凝胶由多功能丝胶蛋白(SDA)和改性透明质酸(HA-FPBA)的混合物在pH 7.4条件下原位固化制备。
如图1中B所示,两块单独的由多功能丝胶蛋白(SDA)和改性透明质酸(HA-FPBA)交联形成的水凝胶(HFSH)可以整合到一起,且能够粘附在猪皮上,表明其具有良好的自愈合和组织粘附能力。
如图1中C所示,在振荡应变扫描模式下,HFSH的存储模量(G’)也显著高于损耗模量(G”),表明微针具有良好的弹性性能和机械强度。
图1中D为载药丝胶微针(HFSVM)的实物照片展示图,微针贴片底部呈正圆形,直径为14mm,包含37个针尖,呈圆锥形针,基部宽度为1.25mm,高度为2mm(图1中E)。由于丝胶的天然荧光特性,在561nm(红色)激发光下可以直接观察到图1F所示的载药丝胶微针(HFSVM)。
实施例6载药丝胶微针的葡萄糖响应性能测试
一、氟代氨基苯硼酸(FPBA)-葡萄糖相互作用和氟代氨基苯硼酸(FPBA)-多功能丝胶蛋白(SDA)相互作用分析测试
利用等温滴定量热法(ITC)研究FPBA与葡萄糖的相互作用。实验采用等温量热计MicroCal PEAQ-ITC进行。在每次滴定中,在室温条件下,将FPBA(1mM,40μL)水溶液分别注入葡萄糖水溶液(10mM,200μL)和SDA(50mM,200μL)溶液中,测量结合过程中释放的热量。采用非线性最小二乘法将数据拟合为单点结合模型,并给定了结合亲和度Ka和焓ΔH。
结果如图2中A-B所示,FPBA/葡萄糖结合后的放热反应比FPBA/SDA结合更强。计算得到的FPBA-葡萄糖的结合亲和力(由结合常数反映)和摩尔结合焓(由焓变ΔH反映)分别达到1.83×105M-1和-41.5kcal mol-1均显著高于FPBA-SDA(7.7×104M-1和-1.49kcal mol-1)。葡萄糖与FPBA具有较高的结合亲和力表明葡萄糖与FPBA发生竞争性反应,从而导致硼酸酯键的解离和维替泊芬的释放(图2中C)。
二、载药丝胶微针的药物释放测试
将载药丝胶微针(HFSVM)与葡萄糖溶液(1mg mL-1或4mg mL-1,1mL)在室温下孵育,检测不同时间点溶液中释放的维替泊芬药物浓度。结果如图2中D所示,维替泊芬在20小时内释放完全,且高血糖水平下药物释放更快。
三、载药丝胶微针的葡萄糖响应性测试
将载药丝胶微针(HFSVM)与葡萄糖溶液(1mg mL-1或4mg mL-1,1mL)在室温下孵育。在预定时间点取上清,并用葡萄糖检测试剂盒检测葡萄糖浓度。结果如图2中E所示,与正常血糖水平(1mg mL-1)相比,在高血糖水平下观察到维替泊芬的快速释放,而在正常血糖状态下则出现相对缓慢的释放。此外,图2中F显示在高血糖状态下的基质结合的葡萄糖是在正常血糖状态下结合的葡萄糖的4倍。
实施例7丝胶微针的活性氧清除与产氧能力测试
图3中A-B分别为丝胶微针与过氧化氢溶液反应产氧的示意图及实物图。
一、实验过程
1.将丝胶微针(HFSM)加入10mL 10mM的过氧化氢溶液中,用长针固定住丝胶微针(HFSM),并用2mL石蜡油在上层进行液封,可见丝胶微针(HFSM)周围产生大量氧气气泡。
2.将10mg的丝胶微针(HFSM)分别加入10mL 0.1mM、0.5mM、1.0mM、2.5mM以及5mM的过氧化氢溶液中,并用2mL石蜡油在上层进行液封,用溶氧仪检测30分钟内溶液中的溶解氧浓度变化情况。
3.为研究丝胶微针(HFSM)清除过氧化氢的活性,在室温条件下将10mg的丝胶微针(HFSM)与1mL 1mM的过氧化氢溶液共孵育。使用过氧化氢检测试剂盒配置过氧化氢检测工作液,在预定的时间点收集50μL的上清液,并与50μL的工作液在黑暗中共孵育10分钟,检测上述混合物的荧光强度,并根据标准曲线计算过氧化氢的浓度。
4.将过量丝胶微针(HFSM)加入10mL水溶液中,固定插入溶解氧探头,并在溶液上层用2mL石蜡油进行液封,在如箭头所示时间点加入100μL的10mM过氧化氢溶液至含丝胶微针的水溶液中,检测50分钟内溶解氧浓度的变化。
5.分别采用NBT法和TMB法检测了丝胶微针(HFSM)的超氧阴离子和羟基自由基的清除活性。
二、结果分析
1.如图3中A-B所示,丝胶微针(HFSM)可与过氧化氢反应产生大量氧气气泡。
2.如图3中C所示,在一定浓度范围内,丝胶微针(HFSM)的溶解氧浓度随过氧化氢浓度增加而增加,呈现过氧化氢浓度依赖性。
3.如图3中D所示,丝胶微针(HFSM)可以在4小时内几乎完全清除1mM的过氧化氢。图3中E显示生成的氧量取决于过氧化氢的浓度,它们之间存在明显的线性关系。
4.如图3中F所示,在箭头所示时间点,在丝胶微针(HFSM)水溶液中加入过氧化氢,溶解氧升高,待过氧化氢消耗完毕后,溶液中溶解氧因向四周扩散而略有下降,此时再加入新鲜的过氧化氢后溶解氧又会再度升高,直到丝胶微针(HFSM)中基于多功能丝胶蛋白的乙酰供体耗尽,表明丝胶微针(HFSM)表现出由活性氧(过氧化氢)触发的氧气生成。
5.如图3中G-J所示,除过氧化氢外,丝胶微针(HFSM)对羟基自由基的清除率超过30%,对超氧阴离子的清除率超过65%。
实施例8丝胶微针的体外抗氧化应激能力测试
将小鼠单核巨噬细胞(RAW264.7)(5×104个/孔)接种于24孔板中,在37℃细胞培养箱中培养24小时,然后将磷酸盐缓冲液(PBS)、过氧化氢(1mM)、过氧化氢+丝胶微针(HFSM)加入到培养基中,孵育1小时。弃去培养基后,将DCFH-DA探针(10μM,200μL/孔)加入24孔板中,与细胞一起孵育30分钟。然后,弃去染液,用磷酸盐缓冲液(PBS)洗涤后用胰酶消化细胞。然后用磷酸盐缓冲液(PBS)重悬细胞,用流式细胞术检测细胞内活性氧水平。此外,为了更直观地了解细胞内活性氧水平,在另一经同样处理的24孔板中加入Hoechst染料以追踪细胞核,然后在荧光显微镜下观察DCFH-DA阳性(绿色)的细胞。结果如图4中A-B所示,丝胶微针(HFSM)处理组DCFH-DA阳性(绿色)的细胞明显减少(图4中A-B),表明其具有优异的活性氧清除能力,可以使细胞免受氧化应激损伤。
将RAW264.7(5×104个/孔)接种于24孔板中,在37℃细胞培养箱中培养24小时,然后将磷酸盐缓冲液(PBS)、过氧化氢(1mM)、过氧化氢+丝胶微针(HFSM)加入到培养基中,孵育1小时,利用trizol提取各组细胞RNA,使用逆转录试剂盒(HiScript IIQ RT SuperMixfor qPCR)合成cDNA。采用AceQ qPCR SYBR Green Master Mix试剂进行实时荧光定量PCR。结果如图4中C-D所示,丝胶微针(HFSM)的加入有效抑制了过氧化氢诱导的细胞促炎因子IL-6及TNF-α的上调。
实施例9丝胶微针的体外促细胞迁移和血管生成能力测试
一、细胞内氧气的检测
[Ru(dpp3)]Cl2探针被广泛应用于细胞胞内氧气含量的检测。在此,我们额外使用了亚硫酸钠(一种除氧剂,缩写为SS)来研究胞内氧气产生情况。HUVEC(1×105/孔)在6孔板中培养,在乏氧条件下(5%二氧化碳,1%氧气)培养24小时。首先加入[Ru(dpp3)]Cl2(10μM),与HUVEC孵育4小时。然后,分别加入丝胶微针(HFSM)(含或不加入过氧化氢)、丝胶微针联合亚硫酸钠(HFSM/SS)(含或不含过氧化氢)和亚硫酸钠(SS),并与HUVEC一起孵育4小时。去除额外的[Ru(dpp3)]Cl2并用PBS洗涤后,在488nm激发下,在610nm下观察细胞内荧光图像。结果如图4中E-F所示,在乏氧条件下,丝胶微针(HFSM)大大猝灭了[Ru(dpp3)]Cl2的荧光,表明其刺激了细胞内氧气的产生,而添加过氧化氢与否对其胞内氧含量无显著差异,这可能与HUVEC本身胞内活性氧就很强有关。与之形成鲜明对比的是,丝胶微针联合亚硫酸钠(HFSM/SS)在过氧化氢的存在下猝灭了氧气的产生,从而导致[Ru(dpp3)]Cl2没有明显的荧光变化。这些结果表明,丝胶微针(HFSM)能有效地促进氧气的生成。
二、丝胶微针的体外促细胞迁移能力测试
为检测丝胶微针(HFSM)促进细胞迁移的能力,将HUVEC以5×104/孔的密度在24孔板中培养。在细胞密度超过90%后,用10μL的尖端进行划痕,并在显微镜的白光条件下拍照,记录为0小时。然后加入含丝胶微针(HFSM)、丝胶微针联合亚硫酸钠(HFSM/SS)或亚硫酸钠(SS)的无血清培养基,并以单独的无血清培养基组作为对照。然后将HUVEC培养24小时并在显微镜下拍照,用imageJ软件分析24小时后的细胞迁移率。图4中G-H的结果表明,丝胶微针(HFSM)可以有效促进细胞迁移,相反,经过丝胶微针联合亚硫酸钠(HFSM/SS)(生成的氧气被迅速猝灭)后,未观察到明显的HUVEC迁移,这证实了丝胶微针(HFSM)通过产生氧气显著促进了细胞迁移。
三、丝胶微针的体外促血管生成能力测试
1、将HUVEC(2×105个/孔)接种于6孔板中,在37℃细胞培养箱中培养24小时,然后替换为含PBS、丝胶微针(HFSM)、丝胶微针联合亚硫酸钠(HFSM/SS)或亚硫酸钠(SS)的新鲜DMEM培养基,并进一步培育24小时。24小时后,用RIPA裂解液(添加蛋白酶抑制剂和磷酸酶抑制剂)提取细胞蛋白,经BCA法定量,SDS-PAGE聚丙烯酰胺凝胶电泳分离。经过转膜和封闭后,将膜按分子量切成条带,与一抗孵育。然后,在TBST(TBS+0.05%Tween 20)洗涤和二抗孵育后对条带进行曝光。最后,用Image J软件定量蛋白表达。结果如图4中I-J所示,丝胶微针(HFSM)显著促进了VEGF蛋白的表达,而在丝胶微针联合亚硫酸钠(HFSM/SS)处理后,其表达有所降低。
2、将HUVEC(2×105个/孔)接种于6孔板中,在细胞培养箱中培养24小时,然后替换为含PBS、丝胶微针(HFSM)、丝胶微针联合亚硫酸钠(HFSM/SS)或亚硫酸钠(SS)的新鲜DMEM培养基,并进一步培育24小时。24小时后,利用trizol提取细胞胞内RNA,使用逆转录试剂盒(HiScript IIQ RT SuperMix for qPCR)合成cDNA,使用AceQ qPCR SYBR Green MasterMix试剂进行实时荧光定量PCR。结果如图4中K所示,丝胶微针(HFSM)上调了细胞的VEGF的表达水平。
实施例10丝胶微针及载药丝胶微针的促糖尿病创面愈合能力测试
一、实验过程
如图5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,将血糖值超过16.7mmoL/L的小鼠视为建模成功。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全层创面。立即将灭菌后的磷酸盐缓冲液(PBS)、丝胶微针(HFSM)、维替泊芬(V)、载药丝胶水凝胶(HFSVH)、载药丝胶微针(HFSVM)均匀置于创面上。术后每两天给小鼠称重并拍照,直到第14天安乐死小鼠,并取创面周围的组织皮肤,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。
同时,取部分磷酸盐缓冲液(PBS)和丝胶微针(HFSM)处理组小鼠的创面皮肤,剪成碎片,加入trizol后进行组织匀浆,取上清提取RNA。使用逆转录试剂盒(HiScript IIQ RTSuperMix for qPCR)合成cDNA,使用AceQ qPCR SYBR Green Master Mix试剂进行实时荧光定量PCR分析。
二、结果分析
1.如图5中B-C所示,丝胶微针(HFSM)及载药丝胶微针(HFSVM)处理组的小鼠创面愈合最快,创面愈合率与对照组显著高于对照组;图6中D显示,各处理组小鼠体重大小无明显差异;图5中E-G显示,丝胶微针(HFSM)加速了创面愈合,而载药丝胶微针(HFSVM)处理组小鼠创面愈合最快,创面中有较多毛囊生成,胶原沉积率也最高,明显优于对照组。
2.如图5中H所示,丝胶微针(HFSM)及载药丝胶微针(HFSVM)处理组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6中A-E所示,与对照组相比,丝胶微针(HFSM)及载药丝胶微针(HFSVM)的小鼠创面皮肤CD31和VEGF的表达均升高,且丝胶微针(HFSM)处理组较之对照组抗炎因子IL-10的表达升高,促炎因子IL-6的表达降低。
4.如图6中F-H所示,维替泊芬的使用有效抑制了YAP蛋白的表达,载药丝胶微针(HFSVM)较之丝胶微针(HFSM)组小鼠创面YAP蛋白表达明显降低,有利于创面的无瘢痕愈合。
5.如图7所示,丝胶微针(HFSM)处理组小鼠较之对照组,促血管生成因子(VEGFA、PDGFβ)表达升高,TNFα、IL-6等炎症因子表达降低,而IL-4、IL-10、TGF-β等抗炎因子的表达,表明丝胶微针(HFSM)可以减轻创面的氧化应激,促进创面血管生成。
实施例11:载药丝胶微针的RNA-seq测试试验
一、实验过程
将雄性Balb/c小鼠(6-8周)分为两组(n=3),分别为PBS组和载药丝胶微针(HFSVM)处理组,在第14天取糖尿病小鼠的创面,立即用液氮冷冻,直到从创面组织中提取总RNA。RNA定量和鉴定、转录组测序、文库制备、聚类和测序以及数据分析在诺禾致源技术有限公司进行。采用标准提取方法从伤口组织中提取RNA,并使用生物分析仪2100系统(安捷伦科技公司)的RNA nano 6000检测试剂盒对RNA样本进行严格控制,以确保RNA的完整性。文库的构建和测序过程主要包括以下七个步骤:总RNA鉴定、mRNA富集、双链cDNA合成、末端修复、poly-A&适配器添加、片段选择和PCR、文库质量评估和illumina测序。为了保证数据分析的质量和可靠性,对原始数据进行过滤,以获得干净的数据。
二、结果分析
为了进一步了解载药丝胶微针(HFSVM)的促再生机制,我们在第14天收集了接受治疗的小鼠的皮肤组织进行转录组学分析。图8中A的主成分分析(PCA)中,载药丝胶微针(HFSVM)处理的小鼠表现出与PBS处理的小鼠显著不同的基因表达模式。图9中B的火山图显示PBS组和载药丝胶微针(HFSVM)组之间存在显著的差异表达基因,其中1360个基因被载药丝胶微针(HFSVM)上调。图8中C的GO功能富集分析显示,载药丝胶微针(HFSVM)处理正向调控MAPK和ERK级联,显著上调伤口愈合相关的基因,包括血管生成、细胞迁移、胶原沉积和细胞外基质形成。KEGG信号通路分析表明,与HO-1表达激活相关的PI3K-Akt信号通路与载药丝胶微针(HFSVM)的促伤口愈合机制高度相关(图8中D)。此外,图8中E-F表明载药丝胶微针(HFSVM)还显著上调了血管生成相关基因(Vegfd、Nrp1、Tgfbr2、Ephb4)和VEGF相关基因(Sulf1,C5ar1)。在载药丝胶微针(HFSVM)处理后,细胞迁移相关基因(Pdgfra、Tgfbr2、Prox1)、细胞外基质形成相关基因也显著增加(图8中G-H)。同样,我们分析了伤口愈合相关基因在伤口组织中的表达,发现Vegfd、HO-1、Pdgfra、Pdgfrb、Tgfbr2、和Ephb4(图8I-M)也显著上调。综上,载药丝胶微针(HFSVM)激活了MAPK-ERK和PI3K-Akt信号通路,促进了创面皮肤组织中的血管生成和细胞迁移,从而加速了糖尿病创面的愈合过程。
实施例12丝胶微针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。24小时后,弃去原培养基,更换为含有丝胶微针(HFSM)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。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1mLmL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。
2、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;24小时后各孔分别加入100μL含有丝胶微针(HFSM)的新鲜DMEM培养基,以加磷酸盐缓冲液(PBS)的新鲜培养基组作为对照;24小时后,弃去原上清,每孔加入100μL含0.5mg/mLmL MTT的不含FBS的DMEM培养基后,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10分钟后在酶标仪上于490nm处检测吸光度。
3、用2mLmL含有10%FBS的DMEM培养基分别于6孔板中培养小鼠人脐静脉内皮细胞(HUVEC)2x104个/孔24小时。24小时后,弃去原培养基,更换为含有丝胶微针(HFSM)的新鲜DMEM培养基,在37℃细胞培养箱中共培养1天、2天、3天,并以加磷酸盐缓冲液(PBS)的新鲜培养基组作为对照。分别在第1、2、3天弃去原培养基,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,并用Image J软件统计细胞存活率。
二、结果分析
如图9中A-F图所示,丝胶微针(HFSM)组与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及良好的生长速率,表明丝胶微针(HFSM)组不具有细胞毒性。
实施例10载药丝胶微针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10所示,丝胶微针(HFSM)及载药丝胶微针处理组(HFSVM)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,表明丝胶微针(HFSM)及载药丝胶微针(HFSVM)具有良好的体内生物相容性。
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)
1.一种载药丝胶微针的制备方法,其特征在于,包括如下步骤:
(1)多功能丝胶蛋白的制备:将丝胶蛋白粉末溶于二甲亚砜溶液中,与丁二酸酐反应生成乙酰化丝胶蛋白,透析、冷冻干燥后,将得到的乙酰化丝胶蛋白粉末溶于4-吗啉乙磺酸缓冲液,与多巴胺发生酰胺缩合反应,透析,冻干后获得多功能丝胶蛋白;
(2)改性透明质酸的制备:将透明质酸溶于双蒸水,在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC和1-羟基苯并三唑HOBt的存在下,与氟代氨基苯硼酸FPBA反应生成改性透明质酸,冻干;
(3)载药丝胶微针的制备:将多功能丝胶蛋白与改性透明质酸混合均匀后,再向其中加入药物溶液,混合均匀后,倒入微针模具中,调节pH使其原位固化成胶,脱模,制备得到载药丝胶微针。
2.根据权利要求1所述的载药丝胶微针的制备方法,其特征在于,步骤(1)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。
3.根据权利要求1所述的载药丝胶微针的制备方法,其特征在于,步骤(1)中,乙酰化丝胶蛋白粉末溶于pH为4.5~6.5的4-吗啉乙磺酸缓冲液中,在惰性气氛保护及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,与多巴胺发生酰胺缩合反应,且乙酰化丝胶蛋白粉末与多巴胺的质量比10:1~3:1。
4.根据权利要求1所述的载药丝胶微针的制备方法,其特征在于,步骤(1)中,透析时,透析袋截留分子量为3500Da。
5.根据权利要求1所述的载药丝胶微针的制备方法,其特征在于,步骤(2)中,透明质酸以3~8mg/mL的浓度溶于双蒸水,且透明质酸与氟代氨基苯硼酸FPBA的摩尔比为0.5:1~5:1。
6.根据权利要求1所述的载药丝胶微针的制备方法,其特征在于,步骤(2)中,反应时的pH为4.5~6.0。
7.根据权利要求1所述的载药丝胶微针的制备方法,其特征在于,步骤(3)中,先分别将改性透明质酸以2%~3%重量体积比w/v的浓度溶于双蒸水,多功能丝胶蛋白以20%~35%重量体积比w/v的浓度溶于双蒸水后,再将改性透明质酸水溶液与多功能丝胶蛋白水溶液按15:1~6:1的体积比混合。
8.根据权利要求1所述的载药丝胶微针的制备方法,其特征在于,步骤(3)中,所述药物溶液选自维替泊芬的二甲亚砜溶液,所述维替泊芬的二甲亚砜溶液浓度为1~2.5mg/mL,原位固化成胶时的pH为7.4~8.0。
9.根据权利要求1所述的载药丝胶微针的制备方法,其特征在于,步骤(3)中,改性透明质酸水溶液与多功能丝胶蛋白水溶液二者的混合物和药物溶液按40:1~10:1的体积比混合。
10.由1-9任一项所述的制备方法制备得到的载药丝胶微针在制备糖尿病创面愈合材料中的应用。
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