CN118126401A - 一种天然糖肽水凝胶的制备方法 - Google Patents
一种天然糖肽水凝胶的制备方法 Download PDFInfo
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- CN118126401A CN118126401A CN202410222029.8A CN202410222029A CN118126401A CN 118126401 A CN118126401 A CN 118126401A CN 202410222029 A CN202410222029 A CN 202410222029A CN 118126401 A CN118126401 A CN 118126401A
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- bletilla striata
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Abstract
一种天然糖肽水凝胶的制备方法,包括以下步骤:S1:将芍药苷和聚乳酸/羟基乙酸共聚物‑双硫键‑聚乙二醇溶于溶剂中,经过超声分散,得到胶束MIC@Pae;S2:将没食子酸溶于溶剂中,加入1-乙基-(3‑二甲基氨基丙基)碳酰二亚胺和N‑羟基琥珀酰亚胺进行搅拌,再加入聚赖氨酸反应,经过透析冻干,得到没食子酸接枝的聚赖氨酸固体;S3:将高碘酸钠和乙二醇加入白芨多糖溶液中反应,经过透析、冷冻干燥得到氧化白芨多糖粉末;S4:将氧化白芨多糖溶液和没食子酸接枝的聚赖氨酸溶液混合,得到氧化白芨多糖水凝胶,将胶束MIC@Pae加入氧化白芨多糖水凝胶混合,得到OBPG&MP水凝胶;本发明的制备方法获得的水凝胶具有提供湿润的愈合环境以及促进伤口愈合的作用。
Description
技术领域
本发明属于生物医学材料领域,具体涉及一种天然糖肽水凝胶的制备方法。
背景技术
慢性伤口的传统治疗方法比如清创、负压伤口治疗和人工皮肤,价格昂贵,并且会造成组织损伤,长期效果不稳定。
生理性伤口愈合包括止血、炎症、增殖和重塑阶段,但慢性伤口常因微环境不利而受阻,表现为氧化应激、免疫细胞功能障碍和炎症因子过度释放。这破坏了愈合阶段的顺序,导致细菌感染、血管生成受损和炎症延长。清除细菌感染、减少氧化应激和抑制炎症是关键,而抗生素的过度使用可能导致细菌产生耐药性。金属及其化合物被视为抗生素的潜在替代品,但受限于生物降解性和细胞相容性。抗菌肽(AMPs)作为有希望的替代方案,可有效对抗微生物感染和免疫细胞功能障碍。聚赖氨酸(PLY)是一种FDA认证的天然抗菌肽,对革兰氏阴性和阳性细菌都具有广泛的抗菌特性。在慢性伤口中,活性氧(ROS)的积累可加剧氧化应激,损害组织再生和血管生成。因此,有效管理细菌感染和消除活性氧是促进慢性伤口愈合的关键因素。其中巨噬细胞在协调免疫防御和组织修复中起到关键作用,在初始阶段,M1巨噬细胞清除病原体和组织碎片,同时释放促炎细胞因子,如TNF-a和IL-6,吸引其他炎症细胞到损伤部位并启动愈合过程。随着修复的进行,M2巨噬细胞占主导地位,分泌IL-10、TGF-β1、VEGF、PDGF等细胞因子,抑制炎症反应,刺激ECM和新血管形成。但是持续高水平的促炎细胞因子会损害慢性伤口中巨噬细胞的侵袭、聚集和表型转化能力,最终导致M1巨噬细胞持续浸润,进一步加重炎症和血管病变,形成恶性循环。
微血管再生的过程,是伤口愈合的另一个关键因素,通过增强必需营养物质和氧气的输送来促进组织修复,慢性伤口微血管完整性受损阻碍了生理血管再生过程的迅速启动,会导致局部血管化减少,因此,如何调节巨噬细胞M2的极化,促进新生血管形成,也是加速慢性伤口愈合的关键。
目前,中药生物活性提取物在治疗慢性伤口方面受到广泛关注,从中草药中提取的各种提取物在促进慢性伤口愈合方面具有多种治疗优势,由于中草药中含有黄酮类、脂肪酸、生物碱和多酚类物质,这些物质具有抗菌、抗氧化、抗炎、免疫调节和促进血管生成的特性,白芨多糖(Bletilla striata多糖,BSP)是从传统中药白芨的干块茎中提取的天然葡甘露聚糖,主要成分为d-甘露糖和d-葡萄糖。新的研究表明,BSP具有抗氧化和抗炎活性,促进血管再生和胶原沉积在损伤部位。芍药是一种传统中药,通常用于治疗各种免疫系统疾病,包括系统性红斑狼疮、类风湿关节炎、哮喘和牛皮癣。芍药苷(Pae)是从芍药根中提取的一种单萜苷,具有笼状的胸骨骨架。大量实验表明,Pae可激活JAK/STAT信号级联,抑制Lps诱导的M1巨噬细胞极化,促进M1巨噬细胞向M2表型转变。
迄今为止,各种基于生物材料的药物载体系统已经被开发出来,以促进慢性伤口的愈合,包括微针、海绵、水凝胶、气凝胶等。水凝胶因其独特的孔隙度、可调节的机械性能、易于功能修饰和良好的生物相容性而被认为是最有前途的伤口敷料。水凝胶的溶胀特性使其具有良好的保湿能力和吸收创面渗出液的能力,为皮肤修复创造了良好的微环境。此外,基于动态共价键构建的水凝胶具有良好的可注射性和自愈性,使其能够填充不规则的伤口,并在整个应用过程中保持完整性。慢性伤口的微环境是复杂多变的,需要在愈合过程的不同阶段进行特定的治疗。因此,生物活性分子的响应性和时空递送对于慢性伤口的愈合同样重要。并且传统方式治疗慢性伤口的价格昂贵,且效果不理想,因此,急需一种具有良好抗菌活性,自愈性和生物相容性,并且成本较低的方法来处理慢性伤口。
发明内容
针对现有技术,本发明提供一种天然糖肽水凝胶的制备方法。
本发明采用的技术方案如下;
S1:将芍药苷和聚乳酸/羟基乙酸共聚物-双硫键-聚乙二醇溶于溶剂中,经过超声分散,得到胶束MIC@Pae;
S2:将没食子酸溶于溶剂中,加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和N-羟基琥珀酰亚胺进行搅拌,再加入聚赖氨酸反应,经过透析冻干,得到没食子酸接枝的聚赖氨酸固体;
S3:将高碘酸钠和乙二醇加入白芨多糖溶液中反应,经过透析、冷冻干燥得到氧化白芨多糖粉末;
S4:将氧化白芨多糖溶液和没食子酸接枝的聚赖氨酸溶液混合,得到氧化白芨多糖水凝胶,将胶束MIC@Pae加入氧化白芨多糖水凝胶混合,得到OBPG&MP水凝胶。
进一步的,所述S1中芍药苷和聚乳酸/羟基乙酸共聚物-双硫键-聚乙二醇的质量比为1:5~10。
进一步的,所述S2中没食子酸、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺、N-羟基琥珀酰亚胺和聚赖氨酸的摩尔比为1:1:0.3~1,搅拌时间为0.5~2h,透析时间为3~5天。
进一步的,所述S3中的白芨多糖和高碘酸钠质量比为1:1~0.3,反应时间为6h,透析时间为5天。
进一步的,所述S4中氧化白芨多糖溶液和没食子酸接枝的聚赖氨酸溶液的体积比为1:1,胶束MIC@Pae加入氧化白芨多糖水凝胶的体积比为1:100,混合时间为1~12h。
一种天然糖肽水凝胶,所述天然糖肽水凝胶为均匀且内部相互连通的多孔结构。
一种天然糖肽水凝胶的应用,所述水凝胶在制备治疗慢性伤口的药物中应用。
技术效果
(1)OBPG&MP水凝胶可以作为一种三维含水支架来达到支撑伤口和提供湿润的愈合环境,并促进伤口愈合。
(2)通过OBSP和PLY-GA结合使用得到的水凝胶具有良好的孔隙率利于搭载药物;OBPG&MP的可注射性利于临床应用还可以延长水凝胶的使用寿命;OBPG&MP还具有的pH响应性特性有利于药物智能控释。
附图说明
图1为本发明实施例1中天然糖肽OBPG&MP水凝胶在慢性伤口愈合中的制备与应用示意图。
图2为本发明实施例1中MIC@Pae,PLY-GA和OBSP的理化表征示意图。
图3为本发明实施例1中天然糖肽类水凝胶OBPG&MP的成胶性能及理化表征示意图
图4为本发明实施例1中OBPG&MP水凝胶的生物相容性和抗氧化能力表征示意图。
图5为本发明实施例1中OBPG&MP水凝胶的抗菌能力示意图。
图6为本发明实施例1中OBPG&MP水凝胶的促血管生成活性示意图。
图7为本发明实施例1中OBPG&MP水凝胶促进慢性伤口愈合能力示意图。
图8为本发明实施例1中OBPG&MP水凝胶促进慢性伤口愈合的作用效果图。
具体实施方式
下面将结合本发明的实施例1-3和附图1-8,对本发明的技术方案进行清楚、完整的描述,显然所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明的制备方法主要包括以下步骤:
S1;将质量比为1:10的芍药苷和聚乳酸/羟基乙酸共聚物-双硫键-聚乙二醇分别溶于二甲亚砜中,在超声分散下,将溶液滴加到去离子水中,制得胶束MIC@Pae;
S2:将没食子酸溶解于乙醇中,经过磁力搅拌,加入到去离子水中,在没食子酸溶液中加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和N-羟基琥珀酰亚胺进行室温搅拌0.5~2h,再加入聚赖氨酸,充分反应过夜,反应后的混合物经过去离子水透析3~5天,将透析后的溶液冻干,得到没食子酸接枝的聚赖氨酸固体;
S3:将白芨多糖溶于去离子水中,剧烈搅拌制成白芨多糖溶液,加入高碘酸钠,在常温下反应2~12h,加入乙二醇使反应猝灭,然后用去离子水对混合物透析五天,将透析液冷冻干燥,得到氧化白芨多糖粉末;
S4:将氧化白芨多糖和没食子酸接枝的聚赖氨酸分别溶解于去离子水中,分别制备氧化白芨多糖和没食子酸接枝的聚赖氨酸溶液,将氧化白芨多糖和没食子酸接枝的聚赖氨酸溶液混合1~12h,制备OBPG水凝胶,再向OBPG水凝胶中加入胶束MIC@Pae,制备得到OBPG&MP。
本发明制备了一种可以满足慢性伤口不同愈合阶段的适应性刺激反应水凝胶,结合图1所示,氧化白芨多糖(OBSP)与没食子酸接枝的聚赖氨酸(PLY-GA)通过席夫碱键聚合形成OBPG水凝胶。没食子酸(GA)是一种天然的多酚类生物活性分子,具有优异的抗氧化活性,它的羧基使它能够通过酰胺键接枝到PLY的侧链上,合成聚合物PLY-GA,采用两亲性胶束(MIC)PEG-S-S-PLGA来加载芍药苷(Pae),以解决其水溶性差的问题。最后,在凝胶化之前,在OBPG水凝胶中加入Pae负载胶束(MIC@Pae),构建OBPG&MP水凝胶给药体系。该糖肽水凝胶OBPG&MP由天然多糖和抗菌肽组成,具有类似于皮肤细胞外基质(ECM)的糖蛋白成分和纳米纤维结构。该水凝胶具有良好的可注射性、自愈能力、刺激反应性和生物相容性,在细菌感染的酸性伤口环境中,OBPG&MP水凝胶逐渐溶解并释放PLY-GA和OBSP,有效消除细菌感染,缓解氧化应激,为皮肤再生创造良好的微环境。在高ROS水平下,MIC@Pae中的二硫键被破坏,导致被包裹的Pae释放,从而促进巨噬细胞M2极化和新生血管形成,加速皮肤ECM重塑和胶原沉积,最终促进慢性伤口愈合。
原材料中白芨多糖和PLY-GA虽然具有抗菌性,但是很弱,无法满足伤口修复的抗菌条件,BSP有一定的促血管生成活性,这是PLY-GA不具备的,此外通过氧化改性BSP形成OBSP后才能与PLY-GA通过席夫碱键形成水凝胶。OBSP和PLY-GA的联合使用产生了比单独使用更好的效果,因为OBPG&MP水凝胶具有良好的孔隙率利于搭载药物;还具有良好的可注射性可以填满不规则的皮肤缺损,更利于临床应用;以及自愈合性可耐受正常活动导致的敷料破损,延长使用寿命;pH响应性可适应感染伤口微酸的特征,利于药物智能控释;良好的保湿性能和溶胀性能,可以吸收渗液并保持伤口湿润,利于修复。
实施例1
S1:将2mg的芍药苷和聚乳酸/羟基乙酸共聚物-双硫键-聚乙二醇溶于1mL二甲亚砜中,在超声分散下,将溶液滴加到去离子水中。将得到的混合物用0.45μm滤头过滤,去除较大的杂质,制成胶束MIC@Pae;
S2:将2.86gGA溶解于30mL乙醇中,经过磁力搅拌,加入到100mL去离子水中,在GA溶液中加入3.55g 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和2.16g N-羟基琥珀酰亚胺,室温搅拌0.5h,加入4.00g聚赖氨酸,反应过夜,反应混合物经过去离子水透析3天,然后将透析后的溶液冻干,得到没食子酸接枝的聚赖氨酸固体;
S3:将5g白芨多糖溶于100mL去离子水中,剧烈搅拌制成白芨多糖溶液,然后加入3g的高碘酸钠,在常温下搅拌6h,加入3mL乙二醇使反应猝灭。对混合物进行去离子水透析五天。将透析液冷冻干燥,得到氧化白芨多糖粉末;
S4:将氧化白芨多糖和聚赖氨酸溶解于去离子水中,形成浓度为10%w/v和5%w/v的溶液,将氧化白芨多糖溶液和没食子酸接枝的聚赖氨酸溶液等体积比快速混合,制备氧化白芨多糖水凝胶,将100μL的浓缩MIC@Pae滴入10mL氧化白芨多糖水凝胶中制备OBPG&MP水凝胶。
结合图2所示,其中2a为PEG-S-S-PLGA的H NMR谱,2b为MIC,Pae,MIC@Pae的紫外可见光谱,2c和2d为MIC,MIC@Pae,MIC@Pae的平均尺寸和ζ-电位(在加入1mm H2O2的情况下),2e和2f为MIC@Pae在生理或ros丰富条件下的代表性TEM图像(标尺为50nm),2g和2h为ROS孵育后MIC@Pae的大小分布及PDI值的变化。如图2a所示。在3.6ppm和1.2ppm的峰分别为PEG片段上的亚甲基和PLGA片段上的甲基。3.1ppm和2.7ppm的峰为PEG-SS-PLGA上的亚甲基(与二硫键相连),这些特征峰在确证了PEG-S-S-PLGA的结构。利用PEG-S-S-PLGA包封疏水药物Pae。疏水药物Pae可通过疏水相互作用被包裹成胶束,促进疏水药物Pae与亲水性水凝胶的结合,实现Pae的缓释。Pae、MIC(PEG-S-S-PLGA)和MIC@Pae的紫外光谱如图2b所示。在MIC@Pae中观察到Pae的特征吸光度峰(242nm),表明Pae被成功封装到ROS响应胶束PEG-SS-PLGA中。此外,通过粒径势分析即图2c、2d可以观察到,加载Pae后,MIC@Pae的Zeta电位在增加;相比之下,对平均粒径没有明显的影响。如图2c和2d所示,在1mM H2O2(ROS)中孵育6h后,MIC@Pae的平均尺寸从105.6nm略微减小到96.0nm,MIC@Pae的Zeta电位从-18.1mV增加到-8.4mV。经ROS处理后,MIC@Pae的形态发生塌陷,由球形变为不规则形状即图2f。同时,在尺寸分布上出现了新的曲线即图2g所示,并且MIC@Pae的PDI值从0.182增加到0.378,此外,在ROS条件下,MIC@Pae的PDI值随着暴露时间的延长而增加,这些结果也证明了ROS响应胶束MIC@Pae的成功制备。
结合图3所示,可以得到OBPG&MP水凝胶的理化表征结果,3a为OBPG水凝胶的凝胶化,3b和3c为OBPG和OBPG&MP水凝胶的代表性SEM图像,3d为OBPG&MP水凝胶的EDS元素图,3e为OBPG&MP水凝胶的可注射性、自愈性和响应能力的示意图,3f为OBPG&MP水凝胶可注射性示意图,3g为OBPG&MP水凝胶自愈能力示意图,3h和3i为水凝胶的流变特性示意图,3k为OBPG&MP水凝胶的pH响应性示意图,3l为不同环境下OBPG&MP水凝胶的Pae释放动力学示意图,3m和3n为OBPG&MP水凝胶的溶胀和降解特性。
OBPG水凝胶是通过在室温下简单混合等体积的OBSP溶液和PLY-GA溶液获得的即图3a。冻干OBPG水凝胶横截面的SEM观察和元素图显示出均匀的多孔结构即图3b,利于载药。随后,在凝胶化之前将浓缩的MIC@Pae溶液添加到OBPG水凝胶中,形成OBPG&MP药物递送系统。从冻干OBPG&MP的SEM图像即图3c来看,实施MIC@Pae后,孔结构没有明显变化。放大后,还可以明显看出MIC@Pae均匀分布在水凝胶支架的骨架上即图3c。此外,元素图谱和EDS分析还检测到C、N、O和S元素的存在,进一步证实了OBPG&MP水凝胶中均质结构的形成即图3d。
由于席夫碱键的动态可逆性和pH敏感性,如图3e所示,所得的OBPG&MP水凝胶表现出优异的可注射性、自修复能力和响应性。如图3f所示,可以使用注射器连续注射OBPG水凝胶,形成不同字母,表明了水凝胶优良的可注射性。如图3g所示,用于评估OBPG&MP水凝胶自愈能力的宏观愈合测试表明破裂的水凝胶在室温下无缝愈合,没有任何裂纹。如图3h~j所示,水凝胶的流变数据也表明该水凝胶具有良好的力学性能及自愈合性能。如图3k所示,由于席夫碱键的pH敏感性,所得OBPG&MP水凝胶表现出pH响应行为。如图3l所示,OBPG&MP水凝胶在pH值较低的环境中崩解得更广泛。随后的药物释放行为也表明酸性条件下Pae释放显著增加。此外,由于形成MIC@Pae的ROS响应二硫键,水凝胶在ROS的存在下Pae的释放率较对照组显著增加,药物释放特性表明pH和ROS可以作为智能触发器来控制OBPG&MP水凝胶中Pae的释放。如图3m~n所示,此水凝胶在生理模拟条件下具有良好的溶胀和降解行为,这有利于吸收更多的伤口渗出液,在7天内表现出逐渐降解,有助于轻松去除敷料,同时保护新再生的组织免受敷料更换带来的进一步伤害。
从图4中所示可知OBPG&MP水凝胶的生物相容性和抗氧化能力表征结果,4a~c为L929和HUVECs与水凝胶提取物孵育的活/死染色CCK-8测定图,4d为水凝胶的血液相容性示意图,4e为脂多糖(LPS)激活L929划痕愈合率与水凝胶提取物孵育示意图,4f为OBPG&M清除ROS的示意图,4g~i为水凝胶清除ROS(OH、O2 -和H2O2)能力示意图,4j~k为DCFH-DA指示剂检测的水凝胶细胞内ROS清除能力(标尺为50μm)。生物相容性是生物材料与组织和血液直接接触的基本特征。通过细胞毒性和溶血试验评价OBPG&MP水凝胶的体外生物相容性。小鼠成纤维细胞(L929)和人脐静脉内皮细胞(HUVECs)与各种水凝胶提取物共培养3天后进行细胞毒性试验,然后进行活/死染色和CCK-8测定。如图4a所示,死的细胞在图4a中圆圈内,荧光成像显示大多数细胞在3天内持续增殖,保持其典型的纺锤形形态。与对照组相比,红色标记的死亡细胞数量较少,差异无统计学意义,表明细胞处于正常增殖状态。CCK-8实验结果也表明,OBPG&MP水凝胶对L929和HUVEC细胞没有明显的细胞毒作用,如图4b~c所示,孵育3天后细胞存活率高于95%。通过体外溶血实验进一步研究OBPG水凝胶的血液相容性。如图4d所示,所有样品的宏观图像均未观察到明显的溶血现象。OBPG&MP水凝胶符合生物材料生物相容性检测标准,具有良好的血液相容性,溶血率小于5%,无溶血风险。细胞迁移过程在伤口愈合中起着至关重要的作用。采用细胞划痕法检测OBPG&MP水凝胶对细胞迁移的影响。采用LPS激活的L929体外模拟创面感染和炎症损伤。图4e所示的结果表明,LPS的加入实质上阻碍了L929的迁移。相比之下,OBPG&MP组在促进细胞迁移方面表现出显著的潜力,24小时内细胞愈合率为61.8%。这一比例明显高于阳性对照组(15.7%),如图4f所示,可能是由于OBPG&MP水凝胶中OBSP、GA和MIC@Pae的抗氧化能力。
炎症部位产生过多的ROS,导致氧化应激,损伤结构蛋白、磷脂膜、酶和DNA,导致ECM降解和细胞死亡,严重阻碍伤口愈合。因此,抗氧化水凝胶可以通过去除活性氧来加速伤口愈合。为验证OBPG&MP水凝胶的抗氧化活性,系统评估了OBPG&MP水凝胶清除常见活性氧的能力,包括羟基自由基(·OH)、超氧阴离子自由基(O2 -)和H2O2。如图4g~i所示,OBPG&MP水凝胶能有效清除·OH、O2 -和H2O2,清除率分别为68.1%、74.6%和71.8%。此外,空白水凝胶和MIC@Pae均表现出清除ROS的能力。空白水凝胶的活性氧清除作用主要归因于酚类物质GA结构中的羟基,可以通过氢原子转移或扩展的电子离域来破坏自由基链。此外,BSP即白芨多糖中存在的活性糖苷键可能导致电子或氢供体清除ROS。MIC@Pae清除ROS的能力主要归功于二硫键的氧化还原敏感性和中草药提取物Pae的抗氧化活性。进一步地,研究了OBPG&MP水凝胶在模拟病理氧化微环境(100μm H2O2)下对成纤维细胞对ROS抗性的保护作用。ROS指标2′,7′-二氯荧光素二醋酸酯(DCFH-DA)用于监测细胞内ROS水平的变化。如图4j~k所示,与OBPG&MP水凝胶共培养的L929细胞的绿色荧光强度较阳性对照组明显降低。这表明OBPG&MP水凝胶有效地减轻了细胞内的氧化应激。总之,OBPG&MP水凝胶在去除溶液和细胞内环境中各种类型的ROS方面表现出了多功能性,提供了保护细胞免受氧化损伤的能力。
结合图5所示,可以得到OBPG&MP水凝胶的抗菌能力,5a为OBPG&MP水凝胶抗菌机理示意图,5b~c为水凝胶的抑菌率示意图,5d为不同组LB板细菌菌落图,5e为水凝胶共培养的细菌的活/死染色图(标尺:50μm),5f~g为不同组细菌的代表性SEM和TEM图像(标尺:200nm,1μm)。PLY-GA的抗菌机制依赖于与微生物细胞表面的静电相互作用。带正电荷的PLY-GA分子可以穿透带负电荷的细菌细胞壁,导致外膜畸变和细胞质分布异常。因此,如图5a所示,细胞膜失去了对底物的选择性,导致自溶和死亡。通过多种技术评估了OBPG&MP水凝胶的抗菌潜力。
首先,采用光密度法计算OBPG&MP水凝胶的抑菌率;如图5b~c所示,OBPG&MP水凝胶对两种细菌表现出优异的抗菌活性,对金黄色葡萄球菌和铜绿假单胞菌的抗菌率分别为97.2%和97.8%。采用琼脂平板培养法检测水凝胶的抗菌性能。如图5d所示,与对照组(control)相比,OBPG&MP水凝胶成功地抑制了细菌的生长,导致琼脂板上的菌落最小。此外,活/死细菌活力测定的图像如图5e所示。在OBPG&MP组的荧光显微镜图像中,可以观察到大量的死亡细菌。相比之下,在空白对照组中,大多数是活菌。最后,如图5f~g所示,利用扫描电子显微镜(SEM)和扫描电镜(SEM)对细菌膜的微观结构进行了研究,研究水凝胶的抗菌机理透射电镜技术。SEM图像显示,空白组金黄色葡萄球菌和铜绿假单胞菌分别为规则的球形和杆状菌,膜结构完整清晰。与OBPG&MP水凝胶共孵育后,金黄色葡萄球菌和铜绿假单胞菌膜表面的生理结构被破坏。呈现不同程度的凹陷和破裂,并有内容物渗出。这是因为细菌膜带负电荷,而富含-NH2的水凝胶在水中质子化带正电荷,使膜结构与细菌膜接触后发生卷曲,突破静电相互作用。同样,通过分析OBPG&MP水凝胶组的TEM图像,也可以观察到金黄色葡萄球菌和铜绿假单胞菌的细菌膜破裂和内容物渗漏。这些结果表明,OBPG&MP水凝胶具有良好的抗菌活性,可以促进感染部位的伤口愈合。
结合图6所示,可以得到OBPG&MP水凝胶的促血管生成能力的结果,6a为水凝胶的血管生成机制示意图,6b为不同组中生长因子VEGF和HIF-1ɑ的代表性免疫荧光染色图像,6c~e为WB和PCR检测不同组中VEGF和HIF-1α的表达示意图,6f~h为不同组中的代表性管形成图像以及量化管长度和密度。
Pae即芍药苷是芍药中发现的主要活性成分,具有增强血管生长因子分泌、促进内皮细胞增殖、迁移和血管形成的能力。同时,如图6所示,BSP可以通过上调VEGF表达和促进细胞迁移来促进伤口愈合。在伤口愈合过程中,血管生成与血管内皮细胞分泌VEGF、HIF-1α等生长因子密切相关。经过48小时的不同处理后,采用不同方法评估HUVEC中VEGF和HIF-1α的表达水平。如图6b所示,免疫荧光成像显示,与对照组相比,OBPG&MP组的VEGF和HIF-1α水平显著升高。如图6c~e所示,OBPG&MP组中的PCR和WB分析进一步证实了这些表达地显著上调。此外,如图6f所示,管形成测定测量了水凝胶对HUVEC血管化的影响,如图6g和6h所示,管密度和总管长度的量化提供了对血管网络发育的见解。相对于空白样本,用OBPG&MP水凝胶培养的HUVEC显示出增强的新血管形成能力,形成更多的互联和细长的管。这些结果表明OBPG&MP水凝胶可显得增强血管生成因子的表达并促进新血管形成,可能有助于慢性伤口的血管再生和内皮修复。
结合图7和图8所示,可得OBPG&MP水凝胶促进慢性伤口愈合的作用,7a为处理程序示意图,8b为不同处理后伤口愈合痕迹的代表性图像示意图(标尺为5mm),7c为不同治疗的伤口收缩率示意图,8d和7e为水凝胶的体内抗菌能力示意图,8f'为各组DHE的代表性免疫荧光染色图像示意图(标尺为100μm),7g~h为第14天各组再生皮肤创面边缘、毛囊和胶原沉积的定量测定图,8j~k为HE和Masson染色对再生皮肤进行组织学评价图(标尺为2mm和200μm)。体外研究表明,OBPG&MP水凝胶具有良好的生物相容性、抗菌性能、清除ROS能力、免疫调节功能和促进血管生成能力。为了评估水凝胶在体内促进慢性伤口愈合的有效性,采用金黄色葡萄球菌感染的糖尿病大鼠皮肤缺损模型,以及采用链脲佐菌素(STZ)有效诱导糖尿病大鼠模型,随后进行每日血糖监测,直到血糖水平持续超过16.7mmol/L。然后,制作12毫米的全层皮肤病变,并用金黄色葡萄球菌使大鼠伤口处感染,以创建糖尿病感染伤口的模型。通过一种标准敷料Hydrosorb作为对照,在指定的时间间隔内评估各种治疗的愈合效果。
图7a显示了治疗过程的细节。结合图8b所示,在特定时间点,不同治疗组创面宏观图像,并绘制创面愈合痕迹,直观展示治疗效果。在整个治疗过程中,OBPG&MP水凝胶组的伤口愈合效率明显优于其他组。如图7c所示,治疗7天后,对照组创面收缩率仅为36.2%,而OBPG&MP水凝胶组创面收缩率为75.2%。此外,在第14天,OBPG&MP水凝胶组创面几乎完全愈合,创面收缩率为98.1%,超过了Hydrosorb、OBPG和MIC@Pae组,后者的收缩率为75.3%。分别为87.9%和77.2%。对照组创面收缩率仅为63.0%。
还可以消除细菌感染,缓解氧化应激,可改善慢性创面部位的不良微环境特征,为皮肤再生创造有利条件。因此,进而对OBPG&MP水凝胶的抗菌和抗氧化能力进行研究。因为控制感染是慢性伤口愈合的先决条件,所以采用平板培养法对伤口组织上残留的细菌进行了3天的评估,以评估OBPG&MP水凝胶的体内抗菌性能。如图8d和7e所示,与对照组、Hydrosorb组和MIC@Pae组相比,OBPG和OBPG@MP水凝胶组平板上的细菌菌落明显减少,表明OBPG水凝胶具有固有的抗菌活性,可以有效地清除慢性创面微环境中的细菌。双氢乙啶(DHE)染色可以反映体内ROS水平。如图8f所示,在第7天和第14天收集再生组织,制成冷冻切片,用DHE染色评估OBPG&MP水凝胶的抗氧化能力。正如预期的那样,可以观察到应用水凝胶后荧光强度显著下降,表明水凝胶具有特殊的抗氧化属性。HE和Masson染色对不同愈合阶段再生皮肤的炎症和胶原沉积进行组织学评价。如图7g和8j所示,HE染色结果与各组创面收缩趋势一致。第7天,与其他组相比,OBPG&MP组创面边缘明显收缩,炎症细胞浸润减少。第14天,对照组出现明显炎症,炎症细胞大量浸润。如图8k所示,相比之下,OBPG&MP水凝胶组实现了完全的再上皮化,显示出很少的炎症细胞,成纤维细胞增殖,新生血管,毛囊,优越的结缔组织和表皮结构。如图7h~i所示,Masson染色显示,与其他组相比,水凝胶组表皮层透明,表皮下真皮附着物比例更高,胶原沉积致密而有组织。通过研究表明,OBPG&MP水凝胶具有加速伤口收缩、再上皮化和胶原沉积来促进慢性伤口修复的功能。
实施例2
S1:将2mg的芍药苷和聚乳酸/羟基乙酸共聚物-双硫键-聚乙二醇溶于5mL二甲亚砜中,在超声分散下,将溶液滴加到去离子水中。将得到的混合物用0.45μm滤头过滤,去除较大的杂质,制成胶束MIC@Pae;
S2:将2.86gGA溶解于50mL乙醇中,经过磁力搅拌,加入到200mL去离子水中,在GA溶液中加入3.55g 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和2.16g N-羟基琥珀酰亚胺,室温搅拌2h,加入4.00g聚赖氨酸,反应过夜,反应混合物经过去离子水透析5天,然后将透析后的溶液冻干,得到没食子酸接枝的聚赖氨酸固体;
S3:将5g白芨多糖溶于200mL去离子水中,剧烈搅拌制成白芨多糖溶液,然后加入3g的高碘酸钠,在常温下搅拌6h,加入3mL乙二醇使反应猝灭。对混合物进行去离子水透析五天。将透析液冷冻干燥,得到氧化白芨多糖粉末;
S4:将氧化白芨多糖和聚赖氨酸溶解于去离子水中,形成浓度为10%w/v和5%w/v的溶液,将氧化白芨多糖溶液和没食子酸接枝的聚赖氨酸溶液等体积比快速混合,制备氧化白芨多糖水凝胶,将100μL的浓缩MIC@Pae滴入10mL氧化白芨多糖水凝胶中制备OBPG&MP水凝胶。
实施例3
S1:将2mg的芍药苷和聚乳酸/羟基乙酸共聚物-双硫键-聚乙二醇溶于3mL二甲亚砜中,在超声分散下,将溶液滴加到去离子水中。将得到的混合物用0.45μm滤头过滤,去除较大的杂质,制成胶束MIC@Pae;
S2:将2.86gGA溶解于40mL乙醇中,经过磁力搅拌,加入到150mL去离子水中,在GA溶液中加入3.55g 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和2.16g N-羟基琥珀酰亚胺,室温搅拌1h,加入4.00g聚赖氨酸,反应过夜,反应混合物经过去离子水透析5天,然后将透析后的溶液冻干,得到没食子酸接枝的聚赖氨酸固体;
S3:将5g白芨多糖溶于150mL去离子水中,剧烈搅拌制成白芨多糖溶液,然后加入3g的高碘酸钠,在常温下搅拌6h,加入3mL乙二醇使反应猝灭。对混合物进行去离子水透析五天。将透析液冷冻干燥,得到氧化白芨多糖粉末;
S4:将氧化白芨多糖和聚赖氨酸溶解于去离子水中,形成浓度为10%w/v和5%w/v的溶液,将氧化白芨多糖溶液和没食子酸接枝的聚赖氨酸溶液等体积比快速混合,制备氧化白芨多糖水凝胶,将100μL的浓缩MIC@Pae滴入10mL氧化白芨多糖水凝胶中制备OBPG&MP水凝胶。
本发明所使用的制备方法制作的水凝胶,克服了单独OBSP和PLY-GA组分的溶液无法形成水凝胶的问题,并且OBSP和PLY-GA组分的溶液流动性较强、黏度较低,单独OBSP和PLY-GA组分的溶液在离心管倒立后即流动到底部。将OBSP和PLY-GA组分的溶液混合制得的水凝胶,BSP和PLY-GA组分的溶液混合后由于成胶导致流动性下降,黏度增强,使得在混合后制备的OBPG在离心管倒立后不会流动到底部,形成了水凝胶,可有利于达到支撑伤口和提供湿润的愈合环境促进伤口愈合的作用。
水凝胶OBPG&MP可促进巨噬细胞M2极化,刺激血管生成。水凝胶OBPG&MP模拟了细胞外基质(ECM)的糖蛋白纤维结构,具有优异的可注射性、自愈性和生物相容性。水凝胶OBPG&MP响应性地适应慢性伤口的炎症微环境,顺序释放治疗药物,以消除细菌感染,中和活性氧(ROS),调节巨噬细胞极化,抑制炎症,促进血管再生和ECM重塑,在伤口愈合的炎症、增殖和重塑阶段发挥关键作用。水凝胶OBPG&MP在调节创面微环境、促进慢性创面皮肤组织再生和重塑方面的作用。
Claims (7)
1.一种天然糖肽水凝胶的制备方法,其特征在于,包括以下步骤:
S1:将芍药苷和聚乳酸/羟基乙酸共聚物-双硫键-聚乙二醇溶于溶剂中,经过超声分散,得到胶束MIC@Pae;
S2:将没食子酸溶于溶剂中,加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和N-羟基琥珀酰亚胺进行搅拌,再加入聚赖氨酸反应,经过透析冻干,得到没食子酸接枝的聚赖氨酸固体;
S3:将高碘酸钠和乙二醇加入白芨多糖溶液中反应,经过透析、冷冻干燥得到氧化白芨多糖粉末;
S4:将氧化白芨多糖溶液和没食子酸接枝的聚赖氨酸溶液混合,得到氧化白芨多糖水凝胶,将胶束MIC@Pae加入氧化白芨多糖水凝胶混合,得到OBPG&MP水凝胶。
2.如权利要求1所述的一种天然糖肽水凝胶的制备方法,其特征在于,所述S1中芍药苷和聚乳酸/羟基乙酸共聚物-双硫键-聚乙二醇的质量比为1:5~10。
3.如权利要求1所述的一种天然糖肽水凝胶的制备方法,其特征在于,所述S2中没食子酸、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺、N-羟基琥珀酰亚胺和聚赖氨酸的摩尔比为1:1:0.3~1,搅拌时间为0.5~2h,透析时间为3~5天。
4.如权利要求1所述的一种天然糖肽水凝胶的制备方法,其特征在于,所述S3中的白芨多糖和高碘酸钠质量比为1:1~0.3,反应时间为6h,透析时间为5天。
5.如权利要求1所述的一种天然糖肽水凝胶的制备方法,其特征在于,所述S4中氧化白芨多糖溶液和没食子酸接枝的聚赖氨酸溶液的体积比为1:1,胶束MIC@Pae加入氧化白芨多糖水凝胶的体积比为1:100,混合时间为1~12h。
6.如权利要求1~5任一所述制备方法得到的一种天然糖肽水凝胶,其特征在于,所述天然糖肽水凝胶为均匀且内部相互连通的多孔结构。
7.如权利要求6所述的一种天然糖肽水凝胶的应用,其特征在于,所述水凝胶在制备治疗慢性伤口的药物中应用。
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