CN115282288A - Ros响应性软骨靶向水凝胶微球及其制备方法和应用 - Google Patents
Ros响应性软骨靶向水凝胶微球及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种ROS响应性软骨靶向水凝胶微球及其制备方法和应用。其中ROS响应性软骨靶向水凝胶微球的制备方法如下:(1)取聚乙二醇‑聚己内酯‑N1‑(4‑硼苄基)‑N3‑(4‑硼苄基)‑N1,N1,N3,N3‑四甲基丙烷‑1,3‑二胺,与小分子激动剂KGN和地塞米松按照重量比10:1:1混合,于有机溶剂中溶解,然后将混合溶液分散在蒸馏水中,室温下透析过夜,得到纳米粒子;(2)将纳米粒子与水凝胶和WYRGRL肽混合,通过微流控装置在紫外光下交联,制备得到复合水凝胶微球。本发明的复合水凝胶微球具有优秀的缓释能力,可实现骨关节炎的长远治疗,另外具有很好地靶向治疗作用和ROS响应性,可明显减轻炎症并促进软骨分化。
Description
技术领域
本发明属于靶向治疗技术领域,具体涉及一种ROS响应性软骨靶向水凝胶微球及其制备方法和应用。
背景技术
骨关节炎(Osteoarthritis,OA)是一种常见的软骨退行性疾病,严重影响着患者的生活质量。国内外科研工作者对OA进行了大量研究,研究结果表明:关节软骨的损伤以及软骨细胞外基质(主要是II型胶原蛋白和蛋白聚糖)的降解不仅与OA的进展相关,还可引起抗炎和促炎通路的紊乱。现有的药物治疗方法虽然能够缓解OA进程,减少炎症反应,但尚无法逆转OA的进展,因此OA终末期患者大多是采用人工关节置换的方法进行治疗。
临床上,针对OA的药物治疗方法往往是通过关节腔内给药,但是常规的给药方式由于难以达到真正的靶向治疗,且药物从关节腔的清除速度较快,导致这种给药方法较难实现长远的治疗效果。因此寻找各种新的给药手段,以期解决药物在关节腔的清除速度过快的问题,以及针对OA的靶向治疗手段,成为了关乎OA治疗的研究热点。
Kartogenin(KGN)是一种小分子激动剂,其具有通过调节CBFβ-RUNX1通路促进干细胞软骨分化,减缓软骨和软骨下骨退变的功能。然而由于KGN具有疏水性,其水溶性并不佳,同时采用关节腔直接注射的方式给药会使得药物从关节腔内快速清除,导致关节内药物递送效率差。因此,针对于KGN的高效给药系统,有待进一步开发。
专利文献CN 114652856 A公开了一种双蛋白递送载体程序性给药系统,该给药系统能够实现疏水性药物的超长效释放,以KGN为例,现有技术中主流的高分子聚合物纳米封装给药系统,一般在7~15天,最长在28天就达到平台期,而双蛋白递送载体程序性给药系统在60天时仍在持续释放KGN、且释放量尚未达到50%。但当该双蛋白递送载体程序性给药系统用于同时负载地塞米松和KGN时,仅能够实现地塞米松的快速释放和KGN的长效缓释,无法对两种药物均实现很好的缓释效果。
除了药物治疗方式外,活性氧(ROS)在OA进展中也起到了重要作用,大量的ROS可通过损伤DNA和修饰脂质,诱导软骨细胞外基质的广泛降解,且对关节内组织细胞产生毒性。因此,清除细胞内高水平的ROS可进一步增强OA的治疗效果。寻找能够高效清除ROS的给药体系将会在药物治疗基础上更有利于OA的治疗。
专利文献CN 114146191 A公开了一种基于II型胶原靶向肽的有机无机杂化金纳米颗粒,该纳米颗粒内核为金纳米颗粒,金纳米颗粒表面包覆致密的聚多巴胺包覆层,在聚多巴胺表面接枝有II型胶原靶向肽。该纳米颗粒可以靶向关节软骨基质中的II型胶原,通过骨关节局部注射,可以穿过II型胶原网络并与关节软骨基质结合。纳米颗粒表面聚多巴胺上的醌基可以有效的清除广泛的自由基,有效的抑制细胞活性氧(ROS)和活性氮(RNS)在软骨基质细胞中的表达水平,促进抗氧化酶的活性,从而产生了优异的治疗缓解骨关节炎症和软骨保护作用。虽然该纳米颗粒能够一定程度上抑制ROS的表达,但其仍不具有ROS响应性,导致该药物载体在清除ROS以及使药物在ROS响应下释放的效果较差,对OA的治疗效果有待进一步提升。
水凝胶因其优异的生物相容性和良好的药物缓释能力,被广泛应用于组织工程,并作为药物载体进行给药,能够起到较好的缓释作用,以实现长期的治疗效果。然而未被特殊加工的水凝胶因其体积较大,几乎无法实现微创治疗。此外,水凝胶因其形状不均匀,在注射过程中可出现较大且不均匀的注射压力,可能会损害正常组织。因此对水凝胶进行修饰,并用于负载药物,实现效果的高效稳定负载和缓释给药,在一定程度上解决了关节腔给药无法长期滞留的问题。然而,由于水凝胶不具有靶向性,导致药物利用度仍然不高,如何实现水凝胶载药体系的软骨靶向能力,以期进一步提高OA的靶向治疗效果,成为面临的一项新的问题。
专利文献CN 112618571 A公开了一种治疗骨科疾病的可注射水凝胶微球,其是以水凝胶微球作为基质,将具有增强生物反应活性的物质掺入水凝胶微球中,采用微流控技术和光化学交联反应制备得到功能化的水凝胶微球,其中生物反应活性物质包括负载有KGN的脂质体、富勒醇纳米晶体等。该水凝胶微球能够有效清除ROS,为间充质干细胞的存活创造良好的微环境,促进间充质干细胞的成骨分化;同时还能提高药物负载的稳定性,实现药物的控制释放,起到治疗和修复骨科疾病的作用。但是该水凝胶微球不具备高效的ROS响应性和软骨靶向性。
专利文献CN 114042147 A公开了一种靶向调控线粒体呼吸链的微纳水凝胶微球,该方法通过微流控技术制备透明质酸微球,并将SS-31肽和Wyrgrl肽修饰的负载白藜芦醇的长循环脂质体通过非共价键连接在微球纳米网络内,从而构建出靶向调控细胞MRC功能的微纳水凝胶微球系统。该水凝胶微球系统具有高效的细胞摄取效率和线粒体靶向性,能显著提高MRC功能,减少质子泄漏,保护线粒体,下调ROS的表达,促进软骨细胞外基质的生成。同时该系统在大鼠骨关节炎模型中可以有效的减缓骨性关节炎的进展。然而该系统中药物的释放速率在第25天时已经释放了接近70%,药物缓释效果有待进一步提高,另一方面,由于该系统不具备ROS响应性,该系统的抗炎效果以及对关节软骨的修复效果有待进一步提升。
因此,如何制备一种具有高效ROS响应性和软骨靶向性的水凝胶微球,以期解决现有水凝胶给药体系在ROS响应性上的不足和不具备软骨靶向性的问题,以及进一步提升药物的缓释性,以期更长效的发挥对骨关节炎的长远治疗作用,成为亟待解决的技术问题。
发明内容
本发明的目的就是为了解决上述技术问题,从而提供一种ROS响应性软骨靶向水凝胶微球及其制备方法和应用。本发明的技术目的在于,一方面解决现有水凝胶给药体系对于药物的缓释效果仍有待进一步提高,以期更长远的实现OA的治疗;另一方面解决现有水凝胶给药体系难以达到真正的软骨靶向治疗,以及水凝胶体系不具备高效的ROS响应性能的问题,进一步实现OA的靶向和高效治疗,显著减轻炎症反应,促进软骨分化和实现有效修复软骨损伤。
本发明的目的之一是提供一种ROS响应性软骨靶向水凝胶微球的制备方法,包括以下步骤:
(1)取聚乙二醇-聚己内酯-N1-(4-硼苄基)-N3-(4-硼苄基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺,与KGN和地塞米松按照质量比10:1:1混合,于有机溶剂中溶解,然后将混合溶液分散在蒸馏水中,室温下透析过夜,得到纳米粒子;
(2)将步骤(1)所得纳米粒子与水凝胶溶液和WYRGRL肽混合,通过微流控装置在紫外光下交联,制备得到复合水凝胶微球。
本发明提供的上述方法,是受到蜜蜂顺着花香采蜜这一独特生物学现象启发,试图构建一种可精准靶向软骨并高效引起ROS反应的水凝胶,显著减轻炎症。本发明首先利用共聚物之间的静电吸附作用,通过聚乙二醇-聚己内酯-N1-(4-硼苄基)-N3-(4-硼苄基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺与Dex和KGN共同构建得到了一种ROS响应性纳米粒子。经实验证明,该纳米粒子为球形,同时在H2O2处理下可发生解聚,使得纳米粒子的结构发生变化,表现出了很好的ROS响应性。
另一方面,本发明通过微流控和紫外光交联技术,在上述纳米粒子的基础上进一步制备得到了一种单分散、尺寸均匀的可注射水凝胶微球。实验结果发现,由于水凝胶网络以及响应性纳米粒子的双重阻碍作用,该可注射水凝胶微球具有更优秀的药物缓释能力,水凝胶负载的药物KGN和Dex均可实现很好的缓释效果,在超过30天后,药物KGN和Dex的释放效率均不超过40%,实现了骨关节炎的长远治疗。另外,由于锚定在水凝胶基质中的WYRGRL肽具有软骨靶向能力,这些复合水凝胶微球可以像蜜蜂采蜜一样实现很好地靶向治疗作用。从可注射水凝胶微球中扩散出来的响应性纳米粒子,可与ROS反应消耗细胞内大量ROS,引起双重药物的ROS响应性释放,进一步减轻炎症并促进软骨分化,在OA的恢复和治疗上表现出极优的效果。基于上述优势,本发明提供的像蜜蜂一样的可注射复合水凝胶微球在OA治疗中具有很好的应用前景。
进一步的是,步骤(1)中所述聚乙二醇-聚己内酯-N1-(4-硼苄基)-N3-(4-硼苄基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺的合成方法如下:以4-(溴甲基)苯基硼酸和N1,N1,N3,N3-四甲基丙烷-1,3-二胺为原料,溶于N,N-二甲基甲酰胺后,升温至反应温度进行充分反应,待反应完全后,将所得产物与聚乙二醇-聚己内酯共聚物发生取代反应,即得。
进一步的是,所述4-(溴甲基)苯基硼酸与N1,N1,N3,N3-四甲基丙烷-1,3-二胺的摩尔比为2:1。
进一步的是,升温反应的条件为升温至60℃反应24小时。
进一步的是,步骤(1)中所述有机溶剂为二甲亚砜。
进一步的是,步骤(2)所述水凝胶溶液为甲基丙烯酸化明胶水凝胶溶液,其是由明胶与甲基丙烯酸酐反应而得。
进一步的是,步骤(2)中采用微流控装置制备复合水凝胶微球的具体操作为:将水凝胶溶液和光引发剂按照10:1的重量比混合,超声溶解于PBS中,然后与WYRGRL肽混合,作为水相;将5%司盘80添加到矿物油中作为油相,将水相和油相在微流控装置中共同流动,产生的水凝胶微液滴经紫外线照射后发生交联,收集交联后的水凝胶微球。
进一步的是,所述水凝胶溶液与WYRGRL肽的重量比为37.5:1。
本发明的目的之二是提供一种由上述方法制备得到的ROS响应性软骨靶向水凝胶微球,该水凝胶微球为可注射水凝胶微球。
本发明的目的之三是提供如上所述的ROS响应性软骨靶向水凝胶微球的应用,其是将该ROS响应性软骨靶向水凝胶微球制备成可靶向治疗骨关节炎的药物。
本发明的有益效果如下:
本发明受蜜蜂顺着花香采集花蜜现象的启发,制备得到了一种封装有WYRGRL肽、KGN、Dex以及纳米粒子的可注射水凝胶微球,该可注射水凝胶微球表现出高效的ROS响应性和软骨靶向性,能够很好实现长远的OA治疗作用。实验结果表明,可注射水凝胶微球不仅可特异性靶向II型胶原,而且具有良好的ROS清除能力以及药物的ROS响应性释放能力,可与ROS反应消耗细胞内大量ROS,引起双重药物的ROS响应性释放,进一步减轻炎症并促进软骨分化。此外,可注射水凝胶微球在体内滞留时间较长,双重药物均能够得到很好的缓释效果,可长时期促进体内受损软骨的修复,符合骨修复所需时间长的特点。因此,本发明为骨关节炎的治疗提供了一种新的策略。
附图说明
图1为实施例中纳米粒子的组装、复合HM的制备以及复合HM治疗OA的示意图;(a)纳米粒子KGN/Dex-TSPBA的制备;(b)KGN/Dex-TSPBA@WHM的制备;(c)KGN/Dex-TSPBA@WHM治疗OA的作用机制。
图2为纳米粒子KGN/Dex-TSPBA的表征;(a)KGN/Dex-TSPBA的电镜图;(b)与H2O2共孵育后KGN/Dex-TSPBA的电镜图;(c)KGN/Dex-TSPBA的粒径分布图;(d)与H2O2共孵育后KGN/Dex-TSPBA的粒径分布图;(e)游离KGN、游离Dex和KD/TM的吸光度;(f)与H2O2共孵育后KGN/Dex-TSPBA的紫外吸收图谱。
图3为水凝胶微球的表征;(a)KGN/Dex-TSPBA@WHM的显微图像;(b)HM的电镜图;(c)KGN/Dex-TSPBA@WHM的电镜图;(d)32d游离KGN、KGN/Dex-TSPBA和KGN/Dex-TSPBA@WHM中KGN的释放曲线;(e)32d游离Dex、KGN/Dex-TSPBA和KGN/Dex-TSPBA@WHM中Dex的释放曲线;(f)24h游离KGN、KGN/Dex-TSPBA和KGN/Dex-TSPBA@WHM中KGN的释放曲线;(g)24h游离Dex、KGN/Dex-TSPBA和KGN/Dex-TSPBA@WHM中Dex的释放曲线。
图4为水凝胶微球的体外细胞毒性和细胞摄取;(a)对照组、HM组和KGN/Dex-TSPBA@WHM组第1、2和3天的活/死染色结果;(b)对照组、HM组和KGN/Dex-TSPBA@WHM组第1、2和3天的MTT结果;(c)KGN/Dex-TSPBA@HM或KGN/Dex-TSPBA@WHM处理的ATDC5细胞的荧光显微镜图像;(d)细胞摄取的图像的定量分析。
图5为KGN/Dex-TSPBA@WHM对细胞的影响;(a)对照组、IL-1β、IL-1β+HM和IL-1β+KGN/Dex-TSPBA@WHM组的DHE染色结果;(b)DHE染色的图像的定量分析;经PBS、IL-1β、IL-1β+HM、IL-1β+TSPBA@WHM、IL-1β+KGN/Dex-M@WHM和IL-1β+KGN/Dex-TSPBA@WHM处理后ATDC5细胞分泌的(c)TNF-α和(d)IL-6的水平;(e)II型胶原表达定量分析;(f)对照组、HM和KGN/Dex-TSPBA@WHM组的免疫荧光染色结果。
图6为KGN/Dex-TSPBA@WHM的体内软骨靶向能力和治疗效果;(a)大鼠注射KGN/Dex-TSPBA@HM和KGN/Dex-TSPBA@WHM后特定时间点的IVIS图像;(b)KGN/Dex-TSPBA@HM和KGN/Dex-TSPBA@WHM于不同时间点的荧光强度;(c)各组关节间隙的相对宽度;(d)各组的X射线图像。
图7为KGN/Dex-TSPBA@WHM缓解OA模型中OA的进展;(a)各组组织病理学及免疫组化图像;(b)各组关节软骨的OARSI评分;(c)各组的相对GAG表达;(d)各组II型胶原阳性细胞的定量分析;(e)各组I聚集蛋白聚糖阳性细胞的定量分析。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例对本发明进行具体描述,有必要指出的是,以下实施例仅仅用于对本发明进行解释和说明,并不用于限定本发明。本领域技术人员根据上述发明内容所做出的一些非本质的改进和调整,仍属于本发明的保护范围。
实施例1
一、实验方法
1、纳米粒子的合成
取KGN(Kartogenin,1mg)和Dex(地塞米松,1mg)以及10mg聚乙二醇-聚己内酯-N1-(4-硼苄基)-N3-(4-硼苄基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺,超声溶解于400μL二甲亚砜中,然后将混合溶液超声分散在9mL蒸馏水中5min,最后在室温下透析过夜,得到纯化后的纳米粒子,记为KGN/Dex-TSPBA。其制备路径如图1中a分图。
作为对比方案一,以PEG-PCL(聚乙二醇-聚己内酯)作为药物递送载体封装KGN和Dex,制备得到纳米粒子,记为KGN/Dex-M。
作为对比方案二,以聚乙二醇-聚己内酯-N1-(4-硼苄基)-N3-(4-硼苄基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺,未负载任何药物得到的纳米粒子,记为TSPBA。该纳米粒子的合成方法如下:以4-(溴甲基)苯基硼酸和N1,N1,N3,N3-四甲基丙烷-1,3-二胺按照摩尔比2:1混合作为原料,溶于N,N-二甲基甲酰胺后,升温至60℃反应24小时,将所得产物与聚乙二醇-聚己内酯共聚物按照摩尔比1:4发生取代反应,得到聚乙二醇-聚己内酯-N1-(4-硼苄基)-N3-(4-硼苄基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺,结构式见图1中a分图所示的Polymer。
2、甲基丙烯酸明胶水凝胶(GelMA)的合成
将20g明胶在60℃下均匀分散于200mL磷酸盐缓冲盐水(PBS)中,再将16mL甲基丙烯酸酐(MA)以0.25mL/min的速度缓慢加入溶液中,2h后,加入200mL PBS终止反应,反应终止15min后,将溶液分装于透析袋(MWCO 3500)中透析过夜,然后,将上清液离心15分钟,透析纯化,3天后,将上清液通过0.22微米滤膜过滤,收集滤液并在-80℃下储存过夜,冻干3天后得到产物GelMA。
3、复合水凝胶微球的合成
HM水凝胶微球的制备(微流控法):将150mg GelMA和15mg光引发剂2-羟基-4-(2-羟乙氧基)-2-甲基苯丙酮超声溶解于3mL PBS用作水相,然后将5%Span 80添加到矿物油中用作油相。水相及油相在微流控芯片中共同流动,管道中的水凝胶微液滴经紫外线照射后发生交联,收集交联后的水凝胶微球,记为HM,利用75%乙醇反复洗涤。
复合HM水凝胶微球的制备:按照上述HM的制备方法,将不同纳米粒子(即TSPBA、KGN/Dex-M或KGN/Dex-TSPBA)和4mg WYRGRL肽溶于水相中,其余步骤与HM的制备方法一致,得到不同纳米粒子对应的复合HM水凝胶微球,分别记为:TSPBA@WHM、KGN/Dex-M@WHM和KGN/Dex-TSPBA@WHM。
KGN/Dex-TSPBA@WHM的制备路径如图1中b分图。
4、纳米粒子及微球表征
利用透射电子显微镜(TEM,Tecnai-G2)表征纳米粒子KGN/Dex-TSPBA的形态,并利用酶标仪(Multiskan GO)和马尔文粒径电位分析仪(ZetasizerNano ZSE,Malvern)分别测量其紫外吸光度和粒径分布。
通过H2O2响应性验证纳米粒子KGN/Dex-TSPBA的ROS响应性,具体步骤为:将样品与0.5%H2O2工作溶液混合并孵育2小时,通过TEM观察混合液的形貌,利用酶标仪与马尔文粒径电位分析仪分别测定其紫外吸光度及粒径分布。
通过明场显微镜观察HM水凝胶微球和复合HM水凝胶微球的形态,通过扫描电子显微镜(SEM,HITACHI SU8010)观察冻干后的HM和复合HM微球的形态。
5、药物体外释放及微球降解实验
通过酶标仪评估游离KGN、游离Dex、KGN/Dex-TSPBA和KGN/Dex-TSPBA@WHM的药物释放过程,具体步骤如下:
取1mL KGN/Dex-TSPBA@WHM溶液封装于透析袋(MWCO8000-14000),将透析袋浸入5mLPBS后置于37℃、80rpm的恒温震荡培养箱中,于每个时间点,收集接收液并加入新鲜的PBS,最后,于每个时间点各取1mL接收液,利用酶标仪测定其药物浓度。
通过以下公式计算药物释放百分比(%):
药物释放量(%)=(释放药物量)/(载药总量)×100%
将HM和复合HM分散于Ⅱ型胶原酶/PBS溶液中模拟其生理环境下的降解过程,具体方法为:将5mg HM和5mg复合HM分别悬浮于1mLⅡ型胶原/PBS溶液(2μg/mL)中,于37℃及60rpm条件下孵育,于各时间点,将样品冻干并测量HM和复合HM的剩余质量,最后分别与其初始质量进行比较。
6、活死染色
将ATDC5细胞以每孔1×106个细胞的密度接种于6孔板中,6孔板中各加入HM或KGN/Dex-TSPBA@WHM共孵育于37℃、5%CO2的条件下,实验第1、2和3天,分别将细胞与活/死染料共孵育20分钟,通过荧光显微镜观察细胞形态,用PBS处理的细胞用作对照组。
7、MTT实验
将ATDC5细胞以每孔5×103个细胞的密度接种于96孔板上,细胞接受如6中的上述干预后,分别于第1、2和3天通过MTT检测各组细胞活性。
8、细胞摄取及靶向实验
由于WYRGRL肽的负载,纳米载体的软骨靶向效率会有所提高。为进一步进行荧光定位,将负载ICG的纳米粒子封装于复合HM中(即KGN/Dex-TSPBA@HM和KGN/Dex-TSPBA@WHM),将ATDC5细胞接种于6孔板上,并培养过夜,与上述KGN/Dex-TSPBA@HM和KGN/Dex-TSPBA@WHM各孵育24h后,PBS洗涤ATDC5细胞3次,并与Hoechst 33342共孵育,孵育10min后,洗涤载玻片,于荧光显微镜下成像,通过Image J软件对图像进行分析。
9、细胞内ROS清除实验
利用二氢乙锭(DHE)对细胞内ROS进行染色,ATDC5细胞以每孔1×106个细胞的密度接种于6孔板上过夜,将细胞与IL-1β(10ng/mL)共孵育24h,用于模拟细胞内炎症。然后分别接受PBS、HM和KGN/Dex-TSPBA@WHM的干预,洗涤细胞并利用DHE染色30分钟,染色后的细胞洗涤后于荧光显微镜下观察,利用Image J软件对图像进行分析。
10、体外促软骨分化实验
人骨髓间充质干细胞(BMSCs)以1×104个细胞/mL的密度接种于24孔板上,过夜培养,然后分别与PBS、HM和KGN/Dex-TSPBA@WHM共孵育过夜,采用4%多聚甲醛固定BMSCs15min,0.1%Triton X-100透化20min,5%牛血清白蛋白/磷酸盐缓冲盐水(BSA/PBS)封闭BMSCs 30分钟,BMSCs与抗II型胶原抗体于4℃染色过夜,并利用PBST洗涤。将各组BMSCs与山羊抗兔IgG H&L共孵育1h,接着分别与4,6-二脒基-2-苯吲哚二乳酸(DAPI)孵育5min及鬼笔环肽孵育30min,最后于荧光显微镜下观察细胞切片,利用Image J软件评估II型胶原蛋白的表达。
11、炎症因子的测定
如前所述,构建细胞内炎症环境,将细胞分别与HM、TSPBA@WHM、KGN/Dex-M@WHM和KGN/Dex-TSPBA@WHM共孵育24h,收集上清液并利用ELISA试剂盒进行定量,以期定量各组ATDC5细胞内TNF-α和IL-6的水平。
12、大鼠骨关节炎模型的建立及分组
所有动物实验均按照嘉兴学院医学院动物实验伦理委员会批准的方案进行。经水合氯醛腹腔麻醉后,于8周龄雄性Sprague-Dawley(SD)大鼠关节腔注射2.5mg碘乙酸单钠(MIA)以期诱导大鼠骨关节炎模型。
造模3天后,将大鼠随机分为6组:1)对照组:未进行任何治疗的健康大鼠;2)PBS组:OA大鼠关节腔内只注射PBS;3)HM组:经HM治疗的OA大鼠;4)TMW@WHM组:关节腔注射TSPBA@WHM的OA大鼠;5)KGN/Dex-M@WHM组:关节腔内注射KGN/Dex-M@WHM的OA大鼠;6)KGN/Dex-TSPBA@WHM组:经KGN/Dex-TSPBA@WHM处理的OA大鼠。
所有大鼠每周均局部注射上述样品(50μL),持续5周。
13、小动物活体成像
为进一步评估体内软骨靶向特性,利用ICG进行荧光定位,并采用IVIS Lumina II系统观察KGN/Dex-TSPBA@HM或KGN/Dex-TSPBA@WHM的滞留时间,关节腔注射上述复合HM后,通过IVIS Lumina II系统定期扫描大鼠,检测ICG的荧光。
14、影像学评估
治疗五周后,对大鼠进行X射线评估,测量各关节侧位片关节间隙相对宽度。
15、组织病理学和免疫组织化学评估
处死所有大鼠,分离膝关节组织并将其包埋于石蜡,利用苏木精伊红(H&E)染色和番红O-快绿染色。根据国际骨关节炎研究协会(OARSI)标准对各组的番红O-快绿切片进行分级,该标准可量化关节表面病变的深度和程度,并通过Image J软件定量各组的番红O-快绿的切片糖胺聚糖(GAG)含量。切片与0.4%胃蛋白酶孵育,利用3%H2O2和1%BSA对其进行封闭。切片与抗II型胶原抗体和抗聚集蛋白聚糖抗体于4℃处理过夜,于室温下与二抗共孵育1小时。经二氨基联苯胺(DAB)底物系统显色,切片于荧光显微镜下成像。最后,利用ImageJ软件对阳性染色细胞进行量化。
16、统计学方法
所有数据均以均数±标准差(SD)形式,数据采用SPSS 25.0软件进行分析。对不同治疗组之间的差异进行t检验(*P<0.05,**P<0.01,***P<0.001)。
二、实验结果
1、KGN/Dex-TSPBA纳米材料的制备及表征
按上述步骤,先制备得到聚乙二醇-聚己内酯-N1-(4-硼苄基)-N3-(4-硼苄基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺,然后通过与Dex和KGN之间的静电吸附,成功制备得到了ROS响应性纳米粒子,并用1H NMR评估其化学结构。图2中a分图的电镜图表明球形纳米粒子KGN/Dex-TSPBA构建成功,其大小为66.32±10.32nm。
将球形纳米粒子KGN/Dex-TSPBA经H2O2工作液处理后,几乎无球形结构,表明纳米粒子的解聚(图2中b分图)。动态光散射(DLS)结果所示,KGN/Dex-TSPBA的粒径从123.9nm增大至146.1nm,与H2O2工作液共孵育后,多分散指数(PDI)从0.24变为0.36(图2中c分图和d分图)。DLS结果的变化表明了纳米粒子结构的变化,进一步证实了纳米粒子的ROS响应性。
此外,游离KGN和游离Dex均有其最大吸收峰,KGN/Dex-TSPBA在相同药物浓度下呈现出新的特征峰(图2中e分图),同样表明双药递送纳米粒子的成功构建。在经H2O2工作液干预后,特征峰消失并被杂峰取代(图2中f分图),表明纳米粒子结构发生了变化,再次证明了其ROS响应性。
2、HM和KGN/Dex-TSPBA的制备及表征
按照上述方法成功制备了HM和复合HM,并通过高效液相色谱(HPLC)和质谱仪(MS)对其进行表征。明场显微镜下观察HM和KGN/Dex-TSPBA@WHM的粒径分别为15.98±0.28μm和18.25±0.34μm(图3中a分图)。此外,通过SEM对冻干HM及KGN/Dex-TSPBA@WHM进行表征,可以看出,冻干的HM呈光滑的球形,无裂纹(图3中b分图),而冻干的KGN/Dex-TSPBA@WHM呈粗糙的球形(图3中c分图)。
3、体外释放及降解实验
为评估KGN/Dex-TSPBA@WHM的缓释能力,分别研究游离KGN、游离Dex、KGN/Dex-TSPBA及KGN/Dex-TSPBA@WHM于PBS中的药物释放过程。
如图3中d分图和e分图所示,KGN/Dex-TSPBA@WHM中的KGN和Dex于32d内均表现出良好的持续释放。所有曲线均显示6h内两种药物的快速释放(图3中f分图和g分图),随之出现相对缓慢释放期(图3中d分图和e分图),两组游离药物均于24h内完全释放。由于KGN/Dex-TSPBA锚定于HM基质,其释放的KGN从约62%下降至28%(图3中d分图),凝胶化后,KGN/Dex-TSPBA释放的Dex从约47%下降至30%(图3中e分图)。上述结果表明,KGN/Dex-TSPBA@WHM组的药物释放结果可能由于纳米粒子和水凝胶基质的双重阻碍作用,KGN/Dex-TSPBA@WHM组有着更优异的药物缓释能力。
随后,为研究水凝胶微球的降解过程,将HM和KGN/Dex-TSPBA@WHM分别浸没于Ⅱ型胶原酶/PBS溶液中,发现HM和KGN/Dex-TSPBA@WHM在28天均降解了约80%,表明其具有较好的生物降解性。
4、KGN/Dex-TSPBA@WHM的生物相容性
通过活死染色法与MTT实验评估HM及KGN/Dex-TSPBA@WHM对软骨细胞的体外细胞毒性。如图4中a分图所示,绝大多数ATDC5细胞均保持良好的细胞活性,并且在三天的培养过程中,各组细胞密度均不断增加。图4中b分图表明ATDC5细胞数量随时间增加,且组间没有显著差异。总之,活死染色实验和MTT实验的结果均表明KD/TMW@MS具有良好的生物相容性。
5、体外摄取实验
通过利用ICG示踪纳米粒子的细胞摄取活动,检测了KGN/Dex-TSPBA@HM及KGN/Dex-TSPBA@WHM的24h细胞摄取活动,以期间接验证材料的靶向能力。如图4中c分图所示,从水凝胶基质释放的KGN/Dex-TSPBA均分布在细胞核周围,此外,图4中d分图显示,KGN/Dex-TSPBA@WHM组的红色荧光强于KGN/Dex-TSPBA@HM组,组间存在统计学差异,表明KGN/Dex-TSPBA@WHM具有良好的靶向治疗潜力。
6、体外ROS清除及抗炎能力评估
过量的ROS诱导软骨细胞凋亡并引起炎症反应,继之导致软骨损伤。因此,IL-1β用于模拟细胞内炎症环境,而DHE用作监测细胞内ROS水平的染色液。用IL-1β处理后,ATDC5细胞呈鲜红色荧光,提示细胞内高水平ROS,这与HM组的结果一致(图5中a分图)。相比之下,KGN/Dex-TSPBA@WHM导致ROS水平显著降低(图5中b分图),验证了其可有效得减轻氧化应激。
通过ELISA试剂盒进一步评估KGN/Dex-TSPBA@WHM抗炎作用,TNF-α及IL-6是典型的炎症因子,可作为ATDC5细胞内的炎症指标。图5中c分图和d分图的高水平IL-6和TNF-α表明产生强烈的炎症反应,而在复合HM组中IL-6和TNF-α均有或多或少的降低,其中与TSPBA@WHM组和KGN/Dex-M@WHM组相比,KGN/Dex-TSPBA@WHM组具有最强的抗炎作用,且复合HM组之间具有统计学差异。总之,上述结果均表明KGN/Dex-TSPBA@WHM可有效地降低细胞内ROS水平并减轻ATDC5细胞的炎症水平,表明KGN/Dex-TSPBA@WHM可有效治疗OA。
7、体外促软骨分化能力评估
为评估KGN/Dex-TSPBA@WHM对BMSCs成软骨分化的影响,以II型胶原的表达作为成软骨分化的指标。如免疫荧光染色所示,KGN/Dex-TSPBA@WHM组II型胶原的表达明显高于其余组(图5中e分图和f分图),表明KGN/Dex-TSPBA@WHM可有效促进BMSCs的软骨分化。
8、小动物活体成像图像的评估
为验证KGN/Dex-TSPBA@WHM体内的靶向能力,利用小动物活体成像检测HM的滞留时间。注射8天后,KGN/Dex-TSPBA@HM组荧光消失,KGN/Dex-TSPBA@WHM组仍可检测到荧光,表明KGN/Dex-TSPBA@WHM于关节腔内可滞留更长的时间(图6中a分图)。如图6中b分图所示,经KGN/Dex-TSPBA@HM处理的大鼠于注射后24h内显示更高的荧光强度,而经KGN/Dex-TSPBA@WHM处理的大鼠注射48h后较KGN/Dex-TSPBA@HM组显示更高的荧光强度。上述结果表明:KGN/Dex-TSPBA@WHM在体内具有良好的靶向治疗潜力,这与如前所述的体外研究结果一致。
9、体外OA治疗效果
鉴于KGN/Dex-TSPBA@WHM在体外良好的治疗潜力,通过体内实验进一步评估KGN/Dex-TSPBA@WHM的治疗作用。大鼠随机分为6组,其中5组分别于OA诱导3天后局部注射50μLPBS、HM、TSPBA@WHM、KGN/Dex-M@WHM及KGN/Dex-TSPBA@WHM,对照组不做任何处理。治疗五周后,利用X射线评估关节形态(图6中c分图和d分图)。PBS组和HM组中显示严重的软骨及软骨下骨损缺,提示两组均存在显著的OA进展。各组复合HM对OA进展均有不同程度的抑制作用,其中KGN/Dex-TSPBA@WHM可有效减缓OA的进展。此外,上述结果于关节间隙相对宽度的分析中得到进一步证实(图6中c分图)。与对照组相比,PBS组及HM组关节间隙相对宽度明显增加,表明PBS组和HM组软骨破坏严重。同时KGN/Dex-TSPBA@WHM组的关节间隙宽度明显减小,表明KGN/Dex-TSPBA@WHM对OA的治疗效果最佳。
为进一步评估体内治疗潜力,分别对各组进行组织学和免疫组织化学评估。H&E染色及番红O-快绿染色结果(图7中a分图)均显示PBS组及HM组明显侵蚀和不规则软骨表面,而经KGN/Dex-TSPBA@WHM处理的大鼠与对照组无明显差异。此外,基于番红O-快绿染色结果,与PBS组及HM组相比,复合HM组的OARSI评分均降低,而KGN/Dex-TSPBA@WHM组呈现最佳的评分(图7中b分图),这可能是因为KGN/Dex-TSPBA@WHM具有ROS清除以及促软骨分化的综合作用。此外,与其他组相比,KGN/Dex-TSPBA@WHM组糖胺聚糖的相对含量最高(图7中c分图),这表明KGN/Dex-TSPBA@WHM具有极佳的维持软骨基质的能力。
随后,通过免疫组织化学评估各组II型胶原和聚集蛋白聚糖的表达。与对照组相比,所有复合HM组中II型胶原的表达均降低(图7中a分图和d分图)。其中,KGN/Dex-TSPBA@WHM组II型胶原的表达最高。随后,进一步评估聚集蛋白聚糖的表达(图7中a分图和e分图),其结果与II型胶原的表达相同。PBS及HM组II型胶原及聚集蛋白聚糖的表达均显著减少,表明两组均出现较明显的OA进展。经KGN/Dex-TSPBA@WHM治疗,大鼠OA明显减轻。总之,上述结果表明:KGN/Dex-TSPBA@WHM在体内有着良好的治疗效果,有助于OA治疗。
Claims (10)
1.一种ROS响应性软骨靶向水凝胶微球的制备方法,其特征在于,包括以下步骤:
(1)取聚乙二醇-聚己内酯-N1-(4-硼苄基)-N3-(4-硼苄基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺,与小分子激动剂KGN和地塞米松按照重量比10:1:1混合,于有机溶剂中溶解,然后将混合溶液分散在蒸馏水中,室温下透析过夜,得到纳米粒子;
(2)将步骤(1)所得纳米粒子与水凝胶溶液和WYRGRL肽混合,通过微流控装置在紫外光下交联,制备得到复合水凝胶微球。
2.根据权利要求1所述的制备方法,其特征在于,步骤(1)中所述聚乙二醇-聚己内酯-N1-(4-硼苄基)-N3-(4-硼苄基)-N1,N1,N3,N3-四甲基丙烷-1,3-二胺的合成方法如下:以4-(溴甲基)苯基硼酸和N1,N1,N3,N3-四甲基丙烷-1,3-二胺为原料,溶于N,N-二甲基甲酰胺后,升温至反应温度进行充分反应,待反应完全后,将所得产物与聚乙二醇-聚己内酯共聚物发生取代反应,即得。
3.根据权利要求2所述的制备方法,其特征在于,所述4-(溴甲基)苯基硼酸与N1,N1,N3,N3-四甲基丙烷-1,3-二胺的摩尔比为2:1。
4.根据权利要求2所述的制备方法,其特征在于,升温反应的条件为升温至60℃反应24小时。
5.根据权利要求1所述的制备方法,其特征在于,步骤(1)中所述有机溶剂为二甲亚砜。
6.根据权利要求1所述的制备方法,其特征在于,步骤(2)所述水凝胶溶液为甲基丙烯酸化明胶水凝胶溶液,其是由明胶与甲基丙烯酸酐反应而得。
7.根据权利要求1所述的制备方法,其特征在于,步骤(2)中采用微流控装置制备复合水凝胶微球的具体操作为:将水凝胶溶液和光引发剂按照10:1的重量比混合,超声溶解于PBS中,然后与WYRGRL肽混合,作为水相;将5%司盘80添加到矿物油中作为油相,将水相和油相在微流控装置中共同流动,产生的水凝胶微液滴经紫外线照射后发生交联,收集交联后的水凝胶微球。
8.根据权利要求7所述的制备方法,其特征在于,所述水凝胶溶液与WYRGRL肽的重量比为37.5:1。
9.一种由权利要求1-8任一项所述方法制备得到的ROS响应性软骨靶向水凝胶微球,其特征在于,所述水凝胶微球为可注射水凝胶微球。
10.权利要求9所述的ROS响应性软骨靶向水凝胶微球的应用,其特征在于,是将该ROS响应性软骨靶向水凝胶微球制备成可靶向治疗骨关节炎的药物。
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