CN116731109A - 一种多肽水凝胶的制备方法及其应用 - Google Patents
一种多肽水凝胶的制备方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种多肽水凝胶的制备方法及其应用。该多肽水凝胶的结构如下，由精氨酸和萘乙酸、萘普生和苯丙氨酸和甘氨酸为基本单元构建水凝胶的基本骨架；萘普生作为抗炎药物引入到水凝胶中增加其生物活性，甘氨酸和苯丙氨酸作为亲疏水调节部分，精氨酸和组氨酸作为调节水凝胶的性能，该多肽可通过固相合成，制备工艺成熟易得。本发明的多肽可在水中通过自组装形成超分子水凝胶，可以清除活性氧，抑制TLR4相关基因通路的表达，通过下调NF‑kB，改善炎症环境。本发明的多肽水凝胶还可促进巨噬细胞的极化，通过免疫调节改善创面环境，促进创面愈合，在糖尿病创面修复中具有广阔的应用前景。
Description
技术领域
本发明涉及生物医学及新型药物开发领域，具体地说，是关于一种多肽水凝胶的制备方法及其应用。
背景技术
糖尿病创面是一种较为常见的慢性创面，其诱因较多，包括高血糖、血管病变、神经性病变等，对患者生活和社会医疗系统均造成较大的负担。伤口的复杂性导致缺乏足够的伤口治疗材料，难以满足糖尿病创面的治疗需求。水凝胶由于多孔结构和良好的肿胀性能，保持湿润的环境及功能化修饰，在伤口治疗中具有显著的优势。多肽水凝胶比普通聚合物凝胶具有更好的生物相容性和生物降解性，此外，还包括配体受体识别、刺激反应性自组装和模拟细胞外基质等优势，通过合理设计的多肽序列可以实现多种生物学功能，如诱导巨噬细胞的极化、改善糖尿病创面微环境，实现创面的时空调控。因此，多肽水凝胶在治疗糖尿病创面修复中极具治疗潜力。
在糖尿病致慢性创面中，由于高血糖和AGE影响，巨噬细胞清除凋亡细胞能力降低，难以完成从促炎表型向抗炎表型的转变，这也是慢性创面处M1型巨噬细胞/M2巨噬细胞比增加的原因之一。在糖尿病致慢性创面中，M1型巨噬细胞分泌的促炎性细胞因子如单核细胞趋化蛋白1(MCP-1)和IL-1β募集了更多巨噬细胞到创面部位，并将其极化为M1表型，该过程形成的正反馈循环维持了创面环境长期的高炎症水平。因此，从M1型向M2型巨噬细胞转变的阻碍同样也是糖尿病致慢性创面无法正常愈合的重要因素之一。NF-κB从细胞质移位到细胞核，与DNA特定序列结合，调节炎症细胞因子、参与炎症反应，其也是巨噬细胞由M1向M2极化的一大障碍。研究表明NF-κB通路上游调控基因TLR4基因被沉默，TLR4 mRNA表达量降低，细胞核内游离NF-κBp65含量显著减少，激活NF-κB通路的作用也随之降低。因此通过抑制TLR4对于调控糖尿病的创面的炎症环境具有重要的意义。
中文专利CN109157504A，公开日2018.08.15，公开了一种多肽水凝胶及其制备方法和应用，其制备方法采用固相合成多肽的方法，先将非天然氨基酸依次和树脂连接，再通过剪切、纯化、冻干，得到白色粉末状固体；将白色粉末状固体分散、超声、加热到体系澄清、最后冷却得到自组装的多肽水凝胶。本发明的多肽水凝胶能有效地调控控制药物释放的动力学性能。文献(Liping Zhou,Tong Huo,Wenmin Zhang,Na Han,Yongqiang Wen,PeixunZhang,New techniques and methods for prevention and treatment of symptomatictraumatic neuroma:A systematic review,Frontiers in Neurology,10.3389/fneur.2023.1086806,14,(2023))中通过DNA接枝、聚乙烯亚胺动态交联以及掺杂加热功能的黑磷量子点，制备了一种多功能DNA水凝胶敷，该DNA水凝胶敷料显示出适当的机械性能、自愈能力、可写性和组织粘附性，为治疗糖尿病感染性伤口提供了一种有效、有前景、实用的联合疗法。而目前关于如本发明的一种多肽水凝胶的制备方法及其应用还未见报道。
发明内容
本发明的目的是，针对现有技术中的不足，提供一种抑制TLR4活性自组装肽，多肽水凝胶的制备方法和应用。
一方面，提供了一种自组装多肽，所述的多肽序列为(Naproxen)-FGGRGGHG GGG-(3-Aminobenzeneboronic acid)(Remark:Naproxen is萘普生,3-Amin-obenzeneboronicacid is 3-氨基苯硼酸)。
第二方面，提供了一种多肽水凝胶，所述的多肽凝胶包括上述的自组装多肽。
第三方面，提供了上述多肽水凝胶的制备方法，包括以下步骤：
S1称量所述的自组装冻干粉末于容器中；
S2加入去离子水溶液，混匀；
S3将混匀后的溶液，于超声条件下溶解；
S4将溶解后的溶液在室温下静置10分钟以上可形成具有粘附性的水凝胶。
作为一个优选例，所述的步骤S1中，使用的是多肽冻干粉末，纯度≥95％。
更优选地，所述的步骤S2中，使用的是去离子水。
更优选地，所述的步骤S3中，使用的温度为室温，超声条件为100w，30s。
更优选地，所述步骤S4中，使用的多肽浓度为30mg/mL。
第四方面，提供了一种上述多肽水凝胶在制备治疗伤口愈合药物中的应用。
作为一个优选例，所述伤口为糖尿病慢性伤口。
更优选地，所述多肽水凝胶是通过抑制TLR4促进巨噬细胞极化以促进糖尿病创面的修复。
本发明优点在于：
本发明的多肽水凝胶讲过各项检测指标，水凝胶是纤维状互相缠绕。体外实验表明，多肽水凝胶具有良好的生物相溶性，如细胞相容性和血液相容性，并能够促进人脐静脉内皮细胞(HUVEC)的细胞迁移，并通过调节TLR4通路，降低炎症通路NF-kB的炎症表达，改善炎症反应。体内实验表明，与普通的辅料相比，该多肽水凝胶可显著促进糖尿病大鼠慢性创面伤口的愈合。该水凝胶具有一定的延展性和粘附性及抑制TLR4促进巨噬细胞的极化，改善抗炎效果，这些特征使得该水凝胶作为糖尿病创面辅料具有很大的临床转化潜力。
附图说明
图1为多肽水凝胶的倒置实验照片。
图2为多肽水凝胶的透射电镜图。
图3为多肽水凝胶的成胶前后的红外光谱图。
图4-6为多肽水凝胶的细胞毒性实验图。
图7为多肽水凝胶的血液相容性实验图。
图8为多肽水凝胶的促进细胞迁移图。
图9为多肽水凝胶的迁移率统计图。
图10为多肽水凝胶的活死细胞染色图和活性氧清除图。
图11为多肽水凝胶的促进巨噬细胞极化图。
图12为多肽水凝胶的免疫印迹。
图13为考察巨噬细胞raw264.7的极化，流式细胞图。
图14为多肽水凝胶促进糖尿病大鼠背部创面愈合图。
图15为多肽水凝胶促进糖尿病创面愈合率统计图。
具体实施方式
下面结合具体实例，进一步阐述本发明，这些实例仅用说明本发明而用于限制本发明的使用范围。应理解，这些实例仅用于说明本发明而不用于限制本发明的应用范围。下面实施例中未注明具体实验条件的方法，通常按照常规条件或按照产品说明书上所提供的条件。下面实施例中所用的材料、试剂等如无特殊说明,均可从商业途径得到。以下结合技术方案和附图详细说明本发明的具体实施方式。
实施例中的多肽水凝胶的结构如下所示，由精氨酸和萘乙酸、萘普生和苯丙氨酸和甘氨酸为基本单元构建水凝胶的基本骨架：
实例1多肽水凝胶的制备及形貌结构表征
多肽水凝胶的制备：称取5mg的多肽冻干粉末(Naproxen)-FGGRGGHG GGG-(3-Aminobenzeneboronic acid)(Remark：Naproxen is萘普生,3-Amin-obenzeneboronicacid is 3-氨基苯硼酸)，加入0.5mg的超纯水，超声溶解后，静置10分钟，即可得到多肽水凝胶，如图1所示，倒置小瓶证明凝胶的形成。
多肽水凝胶的(TEM)形貌表征：按照上述制备的多肽水凝胶，摇匀后，取出10μL滴于200目的碳支持膜上，待干燥后用1％磷钨酸溶液染色约30s，用超纯水清洗3次，干燥后用于TEM测试。如图2，该多肽水凝胶由致密的纳米纤维相互缠绕形成。
多肽水凝胶的红外光谱测定：上述方法制备的水凝胶，-50℃下冷冻干燥得到水凝胶冻干粉末，1：20加入溴化钾压片，测定红外光谱，将其与多肽的冻干粉末对照得到图3的红外光谱，凝胶状态下的红外光谱吸收波长红移，说明凝胶状态下形成分子间的氢键作用。
实例2水凝胶对细胞生长的影响
按照实施例1中的水凝胶配制方法制备浓度20μg/mL，15μg/mL，10μg/mL，5μg/mL的多肽水凝胶溶液，将上述不同浓度水凝胶加入96孔板中，紫外下光照过夜测定细胞毒性。
水凝胶的细胞毒性：采用小鼠上皮样成纤维细胞L929细胞和人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells，HUVEC)来考察水凝胶的细胞毒性和细胞的增殖。将制备好的水凝胶溶液在60℃下灭菌12h，混合后倒入无菌培养皿中，在CO2培养箱中培养，使用10％胎牛血清和双抗的(DMEM)作为完全生长培养基。将L929细胞以20000细胞/孔密度接种于96孔板中培养24h后将水凝胶盘加入孔中。培养1、3、5d后，将细胞培养基换成100μL新鲜DMEM培养基，加入10％(v/v)CCK-8试剂(Beyotime，中国)，37℃培养2h后，用450nm酶标仪(TECAN，瑞士)定量细胞活力24h后采用LIVE/DEAD活/死细胞试剂盒法评估细胞在水凝胶下的增殖和活力(如图4-6)。此外，Live/Dead试剂盒由钙黄素(绿色荧光)和碘化丙啶(红色荧光)组成，用于测定L929细胞的活力。此外，荧光显微镜测定荧光图像(如图10)。
用划痕实验测定多肽水凝胶对细胞迁移的影响：首先，细胞接种在24孔板上。当细胞融合理想时，用200μL的针尖在孔的整个中心划伤，然后用PBS冲洗孔。然后，细胞用多肽水凝胶提取液孵育，并定期观察伤口闭合情况并跟踪长达12小时。采用ImageJ软件对创面区域进行量化，根据迁移图(图8)来计算迁移率(图9)。
水凝胶的血液相容性：红细胞通过离心(1000rpm)从小鼠血液中分离10分钟，所得红细胞用Tris缓冲液洗涤3次，稀释至终浓度5％(v/v)。水凝胶(500μL)和红细胞原液(500μL)加入24孔微孔板中，然后在培养箱中37℃摇1h，摇速为150rpm。随后，将微孔板孔内内容物离心(1000rpm)10分钟，上清液(100μL)放入96孔微孔板中。用酶标仪(MolecularDevices)测定上清液在540nm处的吸光度。阳性对照为Triton x-100，阴性对照为Trisbuffer。根据溶血率的公式计算溶血率：Hemolysis(％)＝[(Ap-Ab)/(At-Ab)]×100％其中Ap为实验组吸光度值，At为Triton x-100阳性对照吸光度值，Ab为Tris缓冲液吸光度值(图7)。
免疫印迹：RAW264.7蛋白通过RIPA裂解缓冲液分离，BCA蛋白测定试剂盒定量，SDS电泳分离，转移到聚偏氟乙烯膜。然后，PVDF膜与一抗、抗TLR4、抗NF-kB和抗Actin在4℃共孵育过夜。然后将PVDF膜浸泡在5％的脱脂牛奶中1h，然后用二抗在室温下孵育2h。最后用TBST洗涤3次，在增强化学发光检测系统中观察印迹(图12)。
体外巨噬细胞调节：通过LPS激动剂刺激细胞的炎症反应，之后加入凝胶孵育，通过免疫荧光，流式细胞仪的抗炎效果。用100ng/mL LPS和20ng/mL IFN-γ刺激RAW264.724h诱导M1极化，用20ng/mL IL-4刺激24h诱导M2极化。
免疫荧光和流式细胞仪考察巨噬细胞极化：100ng/mL LPS和20ng/mL在培养液中加入IFN-γ24h，引起全身炎症微环境。
免疫荧光染色：4％多聚甲醛/0.5％Triton/10％BSA固定/渗透/封闭巨噬细胞；一抗CD206多克隆抗体(1:2000,Abcam)孵育，随后是Alexa Fluor 488山羊抗兔IgG二抗(1:2000)。然后用DAPI反染细胞核，用共聚焦显微镜观察(图11)。使用Image J软件统计CD206阳性细胞数。CD86(1:2000)用同样的方法染色。此外，流式细胞术评估了多肽水凝胶对巨噬细胞极化状态的影响；2×105RAW 264.7细胞接种于6孔板中。接种1天后，LPS组在培养基中加入200ng/mL LPS，试验组加入多肽水凝胶。培养2天后，用刮板收集RAW 264.7细胞，1000rpm离心5分钟。再悬浮和1％BSA封闭后，细胞与CD86(M1 marker)和CD206(M2 marker)孵育30分钟。然后，在BD FACS-Calibur细胞仪上测试细胞悬液。数据采用FlowJo X 10.2软件进行分析(图13)。
实例3水凝胶的体内糖尿病大鼠创面愈合的治疗效果检测
促进糖尿病大鼠慢性伤口愈合的方法：本研究所使用的动物为SPF级雄性SD大鼠(190±10g)。用链脲菌素(streptozocin，STZ)单次腹腔注射65mg/kg诱导1型糖尿病大鼠。给药1周后血糖超过16.7mmol/L，判定为糖尿病。STZ是一种DNA烷基化试剂，可以通过GLUT2葡萄糖转运蛋白选择性地积聚在胰岛β细胞中，其结构中的亚硝脲基部分可以在短时间内破坏胰岛β细胞，从而诱发糖尿病。选择一定数量的大鼠于STZ诱导高血糖后3周建立糖尿病创面模型。用穿孔活检仪在大鼠背部皮肤上制备直径10mm的全厚度缺损。用生理盐水冲洗创面，每个伤口用无菌纱布覆盖，用弹性胶布固定。将水凝胶原位注射创面部位，在1、3、7、11天使用数码相机记录创面，用Image J软件计算创面。根据创面的愈合速度来评价水凝胶的促修复效果(图14-15)。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (10)
1.一种自组装多肽，其特征在于，所述的多肽序列为(Naproxen)-FGGRGGHGGGG-(3-Aminobenzeneboronicacid)(Remark:Naproxenis萘普生,3-Amin-obenzeneboronicacidis3-氨基苯硼酸)。
2.一种多肽水凝胶，其特征在于，所述的多肽凝胶包括权利要求书1中所述的自组装多肽。
3.权利要求2所述多肽凝胶的制备方法，其特征在于，包括如下步骤：
S1称量所述的自组装冻干粉末于容器中；
S2加入去离子水溶液，混匀；
S3将混匀后的溶液，于超声条件下溶解；
S4将溶解后的溶液在室温下静置10分钟以上可形成具有粘附性的水凝胶。
4.根据权利要求书3所制备的方法，其特征在于，所述的步骤S1中，使用的是多肽冻干粉末，纯度≥95％。
5.根据权利要求书3所制备的方法，其特征在于，所述的步骤S2中，使用的是去离子水。
6.根据权利要求书3所制备的方法，其特征在于，所述的步骤S3中，使用的温度为室温，超声条件为100w，30s。
7.根据权利要求3所述的制备方法，其特征在于，所述步骤S4中，使用的多肽浓度为30mg/mL。
8.权利要求2所述的多肽水凝胶在制备治疗伤口愈合药物中的应用。
9.根据权利要求8所述的应用，其特征在于，所述伤口为糖尿病慢性伤口。
10.根据权利要求8所述的应用，其特征在于，所述多肽水凝胶是通过抑制TLR4促进巨噬细胞极化以促进糖尿病创面的修复。
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