CN113995891B - 一种自更新水合润滑载药水凝胶微球及其制备方法与应用 - Google Patents
一种自更新水合润滑载药水凝胶微球及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种自更新水合润滑载药水凝胶微球及其制备方法与应用。本发明通过非共价相互作用将甲基丙烯酸化透明质酸和装载雷帕霉素的阳离子脂质体进行结合,进一步通过微流控技术和光引发的自由基聚合反应,获得了一种单分散、粒径均一、兼具“自更新”润滑功能和维持细胞稳态作用的脂基‑水凝胶微球。该微球具有良好的生物相容性,能够包载脂质体形成“储存库”,并在表面发生磨损的情况下,不断暴露出包裹于内部的脂质体,形成“自更新”的水合润滑层,同时作为“滚动轴承”起到润滑效果。从微球中释放出的阳离子脂质体能够靶向结合在软骨表面释放药物,还能够明显缓解关节软骨的磨损,抑制软骨组织的退变和骨赘形成,延缓骨关节炎的发展。
Description
技术领域
本发明属于可注射载药水凝胶技术领域,具体涉及一种自更新水合润滑载药水凝胶微球及其制备方法与应用。
背景技术
实践证明,良好的润滑对生物摩擦界面(如关节软骨、眼睑与眼球等)的正常运作至关重要,然而,由创伤或疾病引起的组织结构和细胞功能的改变,常常会导致润滑失效,从而引发一系列的并发症(如骨关节炎、干眼症等)。因此,改善生物摩擦界面的润滑情况,同时利用药物维持细胞稳态,能够更好的治疗局部病变。
水凝胶微球是由亲水性聚合物交联制成的微米级水凝胶材质的球体颗粒,具有良好的生物相容性和微创可注射性,能够利用“滚动轴承”效应将滑动摩擦转变为滚动摩擦,从而达到降低摩擦的目的,因此被常被用来改善生物润滑。良好的表面润滑性能对于轴承滚动至关重要。
水合润滑层是带电基团与水分子之间相互吸引而形成的具有一定厚度的水化层,可以承受压力而不被挤出并在剪切下维持流体的性质,能够使摩擦界面的摩擦系数降得很低。因此,在水凝胶微球表面形成稳定的水合润滑层将有望拓展水凝胶微球在生物润滑领域的应用前景。
通过将带有两性离子基团的聚合物/囊泡(如聚合物刷、磷脂脂质体等)结合在微球表面,能够形成稳定的水合润滑层,从而发挥润滑作用。Liu等将PSPMK聚合物刷接枝在PNIPAAm水凝胶微球表面,发现能够通过水合润滑作用降低摩擦。本发明的研究者将pSBMA聚合物刷接枝在GelMA水凝胶微球表面,发现其润滑效果明显高于单纯的GelMA微球。Zheng等人发现,将DOPC脂质体与丝素蛋白微球表面进行结合能够通过水合润滑作用改善微球滚动,从而降低摩擦。然而,通过表面结合的方式形成的水合润滑层对磨擦的耐受性低,在摩擦过程中容易遭到破坏,而裸露出来的微球表面由于润滑层得不到更新和补充,将会明显影响微球的润滑性能。因此,如何对水凝胶微球表面的水合润滑层进行更新和补充是当下急需突破的技术瓶颈。
关节软骨能够利用表面与透明质酸(HA)结合的脂质形成稳定的水合润滑层,并利用关节液里的脂质进行更新和补充。受到关节润滑的启发,Sorkin等将摩擦界面置于脂质体悬液(脂质体储存库)中,发现能够在摩擦表面形成稳定的润滑层,并在摩擦过程中进行修复更新。然而,相比于利用外源性“脂质体储存库”进行润滑层的更新,通过构建“脂质体储存库”并直接应用于界面润滑,使其在摩擦过程中进行润滑层的自我更新,将具有更广阔的应用场景。
因此,若将脂质体分散在HA水凝胶微球中形成“脂质体储存库”,则能够在微球表面形成“自更新”的水合润滑层,这将大大提高水凝胶微球对摩擦的耐受性,进而在关节等需要长期发生相对摩擦的生物界面中提供稳定持续的润滑效果。然而该方面的研究尚未开展。
发明内容
本发明的目的就是为了解决上述技术问题,而提供一种自更新水合润滑载药水凝胶微球及其制备方法与应用,本发明提供的水凝胶微球具有良好的生物相容性,能够包载脂质体形成“储存库”,并在表面发生磨损的情况下,不断暴露出包裹于内部的脂质体,形成“自更新”的水合润滑层,同时作为“滚动轴承”起到润滑效果。
本发明的目的之一是提供一种自更新水合润滑载药水凝胶微球的制备方法,其包括以下步骤:
(1)甲基丙烯酸化透明质酸的制备
将透明质酸与甲基丙烯酸酐在碱性条件下反应,得到甲基丙烯酸化透明质酸;
(2)脂质体的制备
将氢化大豆磷脂酰胆碱、胆固醇、十八胺和雷帕霉素溶解在有机溶剂中,采用膜分散法制备得到装载有雷帕霉素的阳离子脂质体;
(3)脂质体-水凝胶微球的制备
将步骤(1)所得甲基丙烯酸化水透明质酸和步骤(2)所得脂质体与光引发剂进行混合,采用微流控装置制备微液滴,然后在紫外光下交联,得到自更新水合润滑载药水凝胶微球。
本发明通过电荷偶极相互作用将甲基丙烯酸化透明质酸(HAMA)和装载雷帕霉素(RAPA)的阳离子HSPC脂质体进行结合,进一步通过微流控技术和光引发的自由基聚合反应,获得了一种单分散、粒径均一、兼具“自更新”润滑功能和维持细胞稳态作用的脂基-水凝胶微球。该微球具有良好的生物相容性,能够包载脂质体形成“储存库”,并在表面发生磨损的情况下,不断暴露出包裹于内部的脂质体,形成“自更新”的水合润滑层,同时作为“滚动轴承”起到润滑效果。除此之外,从微球中释放出来的阳离子脂质体还能通过静电相互作用靶向结合在带负电的软骨表面,进而释放药物。
进一步的是,步骤(1)中所述透明质酸与甲基丙烯酸酐和摩尔比为1:2.6。
进一步的是,步骤(1)中所述透明质酸的分子量为Mw=74kDa。
进一步的是,步骤(1)中所述反应的条件为在冰浴下搅拌反应24小时,pH为8.0。
进一步的是,步骤(2)中所述氢化大豆磷脂酰胆碱、胆固醇、十八胺和雷帕霉素的质量比为40:10:4:6。
进一步的是,步骤(2)中所述有机溶剂为氯仿。
进一步的是,步骤(2)中所述膜分散法的步骤为:将反应混合物在60℃下反应30分钟,所得产物干燥后进行水合,超声20分钟,得到分散的多层脂质体,然后用膜过滤器挤压脂质体,即得。
进一步的是,步骤(3)中所述甲基丙烯酸化透明质酸、脂质体和光引发剂的质量比为40:6:4。
本发明的目的之二是提供一种自更新水合润滑载药水凝胶微球,所述水凝胶微球是在电荷偶极相互作用下将甲基丙烯酸化透明质酸和装载雷帕霉素的阳离子脂质体进行结合,通过微流控技术和光引发的自由基聚合反应,得到具有“自更新”润滑功能和维持细胞稳态作用的脂基-水凝胶微球。
本发明的目的之三是提供上述自更新水合润滑载药水凝胶微球的应用,其是将该水凝胶微球用于作为治疗骨关节炎的药物载体。
本发明的有益效果如下:
(1)本发明提供了一种单分散、粒径均一、兼具“自更新”润滑功能和维持细胞稳态作用的脂基-水凝胶微球。该微球具有良好的生物相容性,能够包载脂质体形成“储存库”,并在表面发生磨损的情况下,不断暴露出包裹于内部的脂质体,形成“自更新”的水合润滑层,同时作为“滚动轴承”起到润滑效果。
(2)本发明的水凝胶微球能够释放阳离子脂质体,其能够通过静电相互作用靶向结合在带负电的软骨表面,进而释放药物。
(3)体外实验表明,本发明的水凝胶微球可以通过缓慢持续释放自噬激活剂(RAPA)来特异性上调氧化应激损伤的软骨细胞的自噬水平,并下调基质金属蛋白酶13(MMP13)等表达,从而维持软骨细胞稳态。体内实验表明,本发明的脂基-水凝胶微球以微创的方式注入大鼠创伤性骨关节炎的模型中,发现能够明显缓解关节软骨的磨损,抑制软骨组织的退变和骨赘形成,延缓骨关节炎的发展。
附图说明
图1为HAMA的1HNMR谱图。
图2为脂质体和Lipo@HMs的表征图:(A)脂质体的TEM图;(B)脂质体的Zeta电位图;(C)脂质体的粒径分布图;(D)Lipo@HMs的光镜图;(E)Lipo@HMs的粒径分布图;(F)Lipo@HMs的SEM图;(G)Dil标记的Lipo@HMs的共聚焦图。
图3为软骨切片与Dil标记的脂质体孵育图。
图4为Lipo@HMs的润滑性能表征:(A)UMT-3的照片和模式图;(B)新制备的Lipo@HMs的COF-时间曲线;(C)磨损的Lipo@HMs的SEM图;(D)PBS、HMs和Lipo@HMs的COF-时间曲线和COF柱状图;(E)不锈钢板磨损痕迹的明场图像。
图5为Lipo@HMs的降解、载药和释放性能,RAPA@Lipo@HMs的生物相容性表征:(A)Lipo@HMs的降解曲线;(B)延缓降解过程的机理示意图;(C)RAPA在脂质体和Lipo@HMs中的包封率;(D)脂质体和Lipo@HMs的对RAPA的药物释放曲线;(E)在1、2和3天内的活(绿色)/死亡(红色)荧光结果图;(F)活/死染色分析总结的活细胞计数;(G)CCK-8检测不同分组对软骨细胞的细胞毒性。
图6为RAPA@Lipo@HMs维持细胞内稳态结果图:(A)DCF测定细胞内ROS生成;(B)TUNEL染色法检测细胞凋亡;(C)用活/死染色测定细胞死亡;(D)基于DCF荧光强度定量分析ROS表达;(E)基于TUNEL荧光强度定量分析细胞凋亡率;(F)活/死染色法测定的死细胞百分比;(G)第1、3、5天CCK-8结果。(#和*分别表示与对照组和空白组相比P<0.05)。
图7为RAPA@Lipo@HMs增强自噬,促进合成代谢,抑制分解代谢结果图:(A)LC3B蛋白的代表性免疫荧光图像;(B)DAPI和LC3B荧光定量;(C)MMP13蛋白的代表性免疫荧光图像;(D)DAPI和MMP13荧光定量;(E)RT-PCR结果显示Col2、LC3B、ATG5和MMP13的水平。(#和*分别表示与对照组和空白组相比P<0.05)。
图8为RAPA@Lipo@HMs减少关节间隙狭窄和骨赘形成结果图:(A)膝关节AP位和LAT位视图的代表性x线图像;(B)具有代表性的MicroCT图像在膝关节的AP和LAT视图;(C)从AP图像测量的相对关节间隙;(D)从LAT图像测量的相对关节间隙;(E)通过MicroCT图像测量相对骨赘体积。(#、$和*分别表示与对照组、RAPA@Lipo@HM和空白组相比P<0.05)。
图9为组织学染色结果图:(A)HE染色的代表性图像;(B)甲苯胺蓝染色的代表性图像;(C)番红O-固绿染色的代表性图像;(D)关节软骨的Mankin组织学评分。(#、$和*分别表示与对照组、RAPA@Lipo@HM和空白组相比P<0.05)。
图10为免疫组织化学染色结果图:(A)Col2蛋白免疫组化染色代表图像;(B)聚集蛋白聚糖蛋白免疫组化染色代表图像;(C)Col2相对表达的定量分析;(D)聚集蛋白聚糖相对表达的定量分析。(#、$和*分别表示与对照组、RAPA@Lipo@HM和空白组相比P<0.05)。
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。
一、实施方法例
(一)甲基丙烯酸化透明质酸(HAMA)的合成
将2wt%透明质酸(HA,MW=74kDa;华熙生物科技股份有限公司,中国)与甲基丙烯酸酐(2.6倍摩尔过量,阿拉丁,中国)加入去离子水中,在pH=8.0下反应,将反应液在冰浴中连续搅拌24小时,透析3天后将纯化后的产物冻干,得到白色粉末。HAMA的取代度由1HNMR(600MHz)测定。
(二)脂质体的制备和表征
氢化大豆磷脂酰胆碱(HSPC)购自上海艾伟拓医药科技有限公司(中国),采用薄膜分散法制备脂质体。具体方法如下:将HSPC、胆固醇(麦克林,中国)、十八胺(阿拉丁,中国)和雷帕霉素(RAPA,麦克林,中国)(40:10:4:6,质量比)溶解在氯仿中,并在60℃下旋蒸30分钟。然后将形成的脂质膜用去离子水水合,超声处理20分钟后过膜获得分散的多层脂质体。在透射电镜(TEM)下观察脂质体的形态。利用动态光散射法(DLS)测量脂质体粒径、多分散指数(PDI)和Zeta电位。为了验证阳离子脂质体的软骨靶向能力,从猪膝关节中获取软骨切片,并与Dil(碧云天,中国)标记的脂质体在37℃下孵育1小时。在去除脂质体并用PBS洗涤切片后,通过激光扫描共聚焦显微镜(LSCM,蔡司,德国)观察。
(三)含脂质体的水凝胶微球(Lipo@HMs)的制备和表征
将5wt%HAMA-脂质体-光引发剂混合物(40:6:4,w/w/w)在含有95wt%石蜡油和5wt%司班80的微流控油中乳化,在微流控装置的流聚交汇处形成预凝胶液滴。将产生的液滴通过紫外线照射交联。之后,依次用丙酮和去离子水清洗交联的水凝胶微球,并在使用前保存在去离子水中。在光学显微镜下观察Lipo@HMs的形态和大小。通过扫描电镜(SEM)观察冻干后Lipo@HMs的表面形貌。为了证实脂质体已成功地嵌入到微球中,在激光共聚焦显微镜(LSCM)下观察了微球内的Dil标记的脂质体。
(四)摩擦学试验
本研究使用UMT-3多功能摩擦磨损测试仪进行摩擦学试验。所有试验均采用8mm聚乙烯(PE)球体(弹性模量:1GPa;泊松比:0.4)作为上摩擦面和不锈钢板(弹性模量:194GPa;泊松比:0.3)作为下摩擦面,振荡幅度和频率分别为2mm和1Hz。为了模拟关节腔的压强(高达25MPa),我们施加1N的载荷(相当于25.68MPa)。首先,我们将1mL的Lipo@HMs(1mg/mL)滴在下摩擦表面上,并进行3600秒的测试。实验结束后,收集Lipo@HMs用于SEM观察和接下来的摩擦学测试。在相同的实验条件下,对PBS、不含脂质体的水凝胶微球(HMs)和收集到的Lipo@HMs的润滑性能进行测试和比较,测试时间为600秒。每次试验后,在光学显微镜下观察各组不锈钢板的磨损情况。
(五)降解测试
将30mg的Lipo@HMs/HMs浸入1mL含有5mg透明质酸酶(北京莱博科技有限公司,中国)的PBS(pH=7.4)中,然后在37℃和80rpm下搅拌,每隔2天更换并补充新的透明质酸酶溶液。在预定时间点,测量样品的残余重量并与其初始重量进行比较。
(六)药物包封和释放
使用UV-5100紫外可见分光光度计(Metash,China)测量RAPA的包封效率。利用透析法绘制RAPA的释放曲线,具体如下:将含有RAPA的Lipo@HMs(RAPA@Lipo@HMs)/脂质体(RAPA@Lipos)装入透析袋中,在37℃下浸入PBS中并以80rpm搅拌直至药物释放完成。在特定时间点,收集释放介质用于UV分析并替换为等体积的PBS。
(七)细胞生物相容性
永生化人软骨细胞系C-28/I2购自华拓生物科技有限公司(编号HTX2308),并用于体外实验研究。为了确定水凝胶微球的细胞相容性,我们将C-28/I2细胞接种在Transwell下室(0.4μm孔,美国康宁),并将微球接种在上室。共培养1天、2天和3天后,进行活/死染色试验以区分活细胞和死细胞,并使用细胞计数试剂盒-8(CCK-8)试验评估细胞增殖。对于活/死染色测定,将细胞与钙黄绿素-AM/碘化丙啶(碧云天,中国)一起孵育30分钟,并使用荧光显微镜进行检查。对于CCK-8测定,将CCK-8溶液(碧云天,中国)添加到培养基中2小时后,使用酶标仪测试吸光度。
(八)骨关节炎细胞模型
为了模拟创伤后骨关节炎,我们将C-28/I2细胞接种在Transwell下室(0.4μm孔)上,并暴露于200μM过氧化氢(Sigma,美国)。为了确定微球对细胞稳态的治疗效果,我们在Transwell上室加入PBS、Lipo@HMs或RAPA@Lipo@HMs。未经H2O2处理的C-28/I2细胞用作对照。
(九)ROS检测
使用ROS检测试剂盒(碧云天,中国)测定ROS的生成。在孵育48小时后,在37℃下用10μM二氯二氢荧光素二乙酸酯(DCFH-DA)将细胞染色20分钟,然后使用荧光显微镜观察。
(十)细胞活力检测
使用TUNEL染色试剂盒(Beyotime,China)检测细胞凋亡水平。方法如下:在孵育48小时后,将细胞固定在4%多聚甲醛(赛维尔,中国)中30分钟,然后用透化溶液(P0097,赛维尔,中国)孵育5分钟,将细胞与TUNEL染色溶液(赛维尔,中国)一起孵育1小时,之后,将细胞用DAPI(赛维尔,中国)染色并在荧光显微镜下观察。为了评估活/死细胞比率和细胞增殖,按照上述相同程序进行活/死染色和CCK-8测定。
(十一)mRNA表达检测
在孵育48小时后,对细胞进行RT-PCR检测mRNA表达。方法如下:从C-28/I2细胞中分离总RNA,并使用RevertAid First Strand cDNA Synthesis Kit(赛默飞,美国)进行逆转录。之后,使用ABI 7300实时PCR系统(ABI,美国)进行RT-PCR。Col2、LC3B、ATG5、MMP13和GADPH的引物序列见表1。使用比较CT方法(ΔΔCT方法)计算相对mRNA表达。
表1用于实时PCR的引物序列
(十二)免疫荧光染色
通过免疫荧光染色测定C-28/I2细胞中LC3B和MMP13的蛋白质水平。具体方法如下:细胞用4%多聚甲醛固定15分钟后用2%BSA处理30分钟以减少背景,然后在4℃下用兔抗LC3B/MMP13一抗(赛维尔,中国)将细胞标记过夜,然后与Cy3偶联的山羊抗兔IgG(赛维尔,中国)孵育50分钟。细胞核用DAPI(赛维尔,中国)染色10分钟。在LSCM下观察样品。
(十三)骨关节炎大鼠模型
经重庆医科大学第一附属医院研究伦理委员会批准进行动物实验。雄性SD大鼠(12周龄)随机分为假手术组(5只)和骨关节炎组(25只)。骨关节炎组大鼠在麻醉后进行膝关节前交叉韧带横断和内侧半月板切除术(ACLT+MMx)。术后1周,将骨关节炎大鼠进一步随机分为5个亚组(每组5只),关节内分别注射PBS、HMs、RAPA@Lipos、Lipo@HMs和RAPA@Lipo@HMs,每隔四周重复一次。
(十四)放射学评估
术后8周,使用Faxitron X射线机(Faxitron X射线,美国)在10秒和32kV技术下获得大鼠膝关节的X射线图像。在前后位(AP)和侧位(LAT)中测量关节间隙宽度。为了进一步检查,采集膝关节并进行离体显微CT分析(SkyScan 1172,比利时)。
(十五)组织学和免疫组织化学检查
手术后8周,对离体膝关节进行多聚甲醛固定、脱钙、石蜡包埋,然后以5μm的厚度切片。对于组织学分析,矢状面用HE、番红O-固绿和甲苯胺蓝染色。膝关节的病理状况由两名观察者使用改良的Mankin评分系统进行评估。对于免疫组织化学染色,将切片与兔多克隆抗Col2/聚集蛋白聚糖(赛维尔,中国)抗体在4℃下孵育过夜,然后与二抗孵育1小时。之后,用DAB底物对石蜡切片进行染色。使用Image J软件量化Col2和聚集蛋白聚糖的相对表达量。
(十六)统计分析
使用SPSS软件进行统计分析。采用单因素方差分析和Tukey事后分析检验各组之间的差异,显着性水平为p=0.05。
二、实验结果例
1、“自更新”润滑载药水凝胶微球的制备与表征
本发明选用透明质酸作为构成“自更新”润滑载药水凝胶微球的主体材料。透明质酸作为细胞外基质的主要成分之一,具有良好的生物相容性,能够通过电荷偶极相互作用与脂质体结合,形成脂质体“储存库”,并暴露出其亲水的磷酸胆碱头部,从而形成稳定的水合润滑层。为了赋予透明质酸光固化交联的特性,需要对透明质酸进行丙烯酸改性。通过氢核磁共振(1H-NMR)结果显示,甲基丙烯酸酐已成功接枝在了透明质酸的分子链上,其取代度为52.91%(如图1)。
通过TEM观察,利用薄膜分散法制备的脂质体呈多层椭圆囊状结构(如图2中的A部分)。通过动态光散射激光粒度仪(DLS)对脂质体进行测定,显示该脂质体的Zeta电位为(47.4±17.4)mV(图2中的B部分),这种正电性质将有助于脂质体通过静电相互作用与软骨表面带负电的糖胺聚糖(GAG)相结合,从而起到软骨靶向的作用。
然而,软骨表面存在着大量的Ⅱ型胶原,它们相互交联,形成致密的三维网格(孔隙:50-200nm),将阻碍脂质体与GAG的结合。因此,只有对脂质体的粒径进行控制,才能实现更好的软骨靶向。在本发明中,脂质体的平均粒径为(102.3±35.2)nm,多分散系数(PDI)为0.132(图2中的C部分),表明该脂质体具有良好的分散性,同时脂质体较小的粒径使其更容易与GAG结合。进一步将Dil(一种亲脂性的荧光染料)标记的脂质体与软骨切片进行孵育,并通过共聚焦显微镜进行观察,发现红色点状荧光在软骨切片表面广泛分布(如图3),提示该阳离子脂质体能够靶向结合在软骨表面。
本实施例中,具有“自更新”润滑功能的脂基-水凝胶微球是利用微流控装置使水相(HAMA和HSPC脂质体)和油相(石蜡油和司班80)在交汇处形成油包水的液滴,再通过紫外光交联制备而成。通过光镜观察,利用微流控技术制备的脂基-水凝胶微球分散良好、粒径均一、并且形态完整(图2中的D部分),这一特征有助于微球进行滚动润滑。通过粒径检测显示,微球的粒径呈窄的单峰分布,平均粒径为(208.36±7.37)μm(图2中的E部分)。
为了观察脂基-水凝胶微球的表面形貌,进一步利用SEM对冻干的微球进行观察。如图2中的F部分所示,微球呈明显的多孔结构,由于微球在干燥过程中会发生失水收缩,其粒径明显小于分散在水中的微球。值得注意的是,在微球表面仅观察到极少量的脂质体,这可能是因为在微球制备过程中,大部分的脂质体被包裹在了微球内部,并且结合在最表层的脂质体由于缺乏微球的保护,容易在洗球的过程中遭到破坏并被去除。为了证明脂质体已成功包裹在了水凝胶微球内,进一步利用Dil对脂质体进行标记,并通过共聚焦显微镜进行观察,结果显示红色点状荧光在微球内呈散状分布,提示脂质体已成功包裹在了水凝胶微球内(图2中的G部分)。
2、脂基-水凝胶微球的“自更新”润滑能力测定
在本实施例中,摩擦实验通过多功能微摩擦磨损试验机(UMT-3)进行。
为了观察脂基-水凝胶微球润滑的整个过程,首先对脂基-水凝胶微球进行了长达3600s的摩擦实验(图4中的A部分)。如图4中的B部分所示,整个润滑过程大致可以分为4个阶段:1、第一阶段(灰色区域;0-300s)的摩擦系数呈现出缓慢下降的趋势,提示微球能在摩擦过程中进行自我更新,并不断暴露出更多的脂质体形成水合润滑层;2、第二阶段(蓝色区域;300-1200s)的摩擦系数大致稳定在了0.03左右,提示微球已经暴露出足够多的脂质体,正在发挥稳定的润滑作用;3、第三阶段(紫色区域;1200-2700s)的摩擦系数出现缓慢的上升,提示微球在不断往复摩擦的过程中逐渐被挤出摩擦区域;4、第四阶段(绿色区域;2700-3600s)的摩擦系数呈现快速上升的趋势,提示摩擦界面的水分正在不断减少,摩擦方式在逐渐向着干摩擦进行转变。
另一方面,与本实验中的持续接触摩擦不同,下肢步态在行走过程中呈周期性变化。在下肢摆动阶段,上下关节面会出现短暂的分离,这使得微球和关节液能够重新回到摩擦区域,因此微球在关节内的润滑过程主要包括前两个阶段。
为了验证脂基-水凝胶微球的“自更新”润滑能力,首先将上述实验用到的脂基-水凝胶微球进行冻干,并在SEM下进行观察。与刚制备好的脂基-水凝胶微球不同,经历了摩擦实验的微球表面暴露出了较多的脂质体(图4中的C部分),提示微球能够在表面发生磨损的情况下,不断暴露出包裹于内部的脂质体,从而形成“自更新”的水合润滑层。接下来,在相同条件下对PBS、单纯水凝胶微球和上述表面暴露出脂质体的水凝胶微球进行了摩擦实验。如图4中的D部分所示,利用PBS进行水润滑所得到的摩擦系数为0.06,而单纯水凝胶微球通过“滚动轴承”效应能够将摩擦系数降低至0.04。与之前的摩擦实验不同,已经暴露出足够多脂质体的水凝胶微球并没有出现第一阶段的过程,而是直接将摩擦系数降低并稳定在了0.03。进一步将三组摩擦实验用到的不锈钢板放在光镜下进行观察比对,发现脂基-水凝胶微球组所造成的划痕明显浅于PBS和单纯水凝胶微球组(图4中的E部分)。
综上,这些结果证明水凝胶微球能够包裹脂质体形成“脂质体储存库”,并在表面形成“自更新”的水合润滑层,进而在摩擦过程中发挥稳定的润滑效果。
3、“自更新”润滑载药水凝胶微球的降解测定
对于生物润滑材料来说,较慢的降解可以延长材料在生物摩擦界面的停留时间,从而发挥更加持久的润滑作用。降解实验结果显示,在透明质酸酶的作用下,相较于脂基-水凝胶微球,单纯水凝胶微球呈现出相对较快的降解趋势,并在第42天时降解完全。脂基-水凝胶微球在前7天呈现出与单纯水凝胶微球相似的降解趋势,之后逐渐减慢,并在第63天时几乎完全降解(图5中的A部分),这种两阶段式的降解趋势可能是因为微球早期的降解是一个自外向内的过程,而脂基-水凝胶微球能够在降解过程中逐渐暴露出包裹于其中的脂质体,并在表面形成“自更新”的水合润滑层。该水合层对透明质酸酶有一定的屏障作用,能够延缓微球的降解过程,进而延长微球润滑作用的时间(图5中的B部分)。
4、“自更新”润滑水凝胶微球对药物的负载和释放
雷帕霉素(RAPA)是一种脂溶性的小分子药物,存在溶解性差、治疗窗口窄、在关节腔内易被清除的问题。将RAPA装载在阳离子脂质体内,能够增加药物的溶解性,并提高对软骨的靶向性,而将阳离子脂质体包裹于水凝胶微球中,可以进一步提高该体系的稳定性,有利于局部递送。
本实施例的药物负载实验结果显示,单纯脂质体和脂基-水凝胶微球对RAPA的负载效率分别为(87.55±6.49)%和(67.39±4.06)%(图5中的C部分),这种负载效率的差异可能是由在微球制备过程中药物渗漏以及部分脂质体被去除所导致。在后续的释放实验中,脂基-水凝胶微球在前3天呈现出相对较快的释放趋势,之后逐渐减慢,在第28天时几乎完全释放。然而,与脂基-水凝胶微球的释放特性不同,单纯脂质体在第3天的累计释放率便超过了80%,并在第14天释放完全(图5中的D部分)。这种截然不同的释放特性显示将脂质体包裹于水凝胶微球中形成“储存库”能够保护脂质体,提高其稳定性,从而达到长效缓释的目的。
5、“自更新”润滑载药水凝胶微球的生物相容性检测
“自更新”润滑载药水凝胶微球作为可注射的生物润滑剂,需要具备良好的生物相容性。为此,本实施例利用活死染色和CCK-8试验来检测微球对细胞的影响。
活死染色结果显示,绝大多数细胞在这3天的培养过程中存活,组间比较没有统计学差异(图5中的E和F部分)。与活死染色的结果相一致,CCK-8试验结果显示,在这3天的培养过程中,各组细胞均持续增殖,组间比较没有统计学差异(图5中的G部分)。
综上,这些结果证明“自更新”润滑载药水凝胶微球具有良好的生物相容性,能够作为生物润滑剂来改善生物摩擦界面的润滑情况。
6、“自更新”润滑载药水凝胶微球维持细胞稳态
已有研究表明,软骨在受到损伤时会产生大量的ROS,而ROS过度产生可以通过超过氧化、蛋白质羰基化以及DNA损伤来损害软骨细胞功能。自噬是一种细胞分解代谢机制,能够通过去除功能失调的细胞器和大分子来维持细胞内稳态。通过提高自噬水平可以减少ROS的产生,从而缓解氧化应激对细胞造成的损伤。
在本发明中,通过DCFH-DA荧光探针来检测细胞内的ROS水平,结果显示RAPA@Lipo@MG组的ROS水平明显低于Lipo@MG组和空白组(图6中的A和D部分),提示RAPA@Lipo@MG能够通过释放自噬激活剂(RAPA)来提高细胞自噬水平,从而减少ROS的产生。ROS的升高是诱导细胞凋亡的关键因素之一。本发明通过TUNEL染色来检测细胞凋亡,结果显示RAPA@Lipo@MG组凋亡细胞明显少于Lipo@MG组和空白组(图6中的B和E部分),提示RAPA@Lipo@MG能够通过释放RAPA来维持细胞稳态,从而减少细胞凋亡。进一步通过活死染色和CCK-8来检测RAPA@Lipo@MG对细胞存活和增殖的影响,发现RAPA@Lipo@MG组的死细胞比例明显低于Lipo@MG组和空白组,同时细胞的增殖速率明显高于Lipo@MG组和空白组(图6中的C,F和G部分),提示RAPA@Lipo@MG能够通过释放RAPA来提高氧化应激损伤的细胞存活率,并促进其增殖。
Ⅱ型胶原作为透明软骨细胞外基质中的重要成分,是软骨细胞增殖的标志。作为自噬的关键蛋白,LC3B和ATG5的表达与自噬活性水平密切相关。而MMP13作为II型胶原蛋白裂解的关键酶,在骨关节炎软骨基质降解中起主要作用。为了验证RAPA@Lipo@MG对软骨细胞的自噬水平以及合成、分解代谢活动的影响,本发明利用RT-PCR来检测Col2、LC3B、ATG5以及MMP13的基因表达水平。结果显示,RAPA@Lipo@MG组的Col2、LC3B以及ATG5的基因表达水平明显高于Lipo@MG组和空白组,而MMP13的基因表达水平明显低于Lipo@MG组和空白组(图7中的E部分)。
进一步利用免疫荧光来检测LC3B和MMP13的蛋白表达水平,发现其结果与RT-PCR的结果相一致,RAPA@Lipo@MG组中的LC3B蛋白的表达量明显高于Lipo@MG组和空白组(图7中的A和B部分),而MMP13蛋白的表达量明显低于Lipo@MG组和空白组(图7中的C和D部分)。
这些结果提示RAPA@Lipo@MG能够通过释放RAPA来提高软骨细胞的自噬水平,促进软骨细胞外基质的分泌,并减少MMP13的生成。
7、“自更新”润滑载药水凝胶微球对骨关节炎的治疗效果评估
为了验证“自更新”润滑载药水凝胶微球能否缓解骨关节炎软骨的磨损,并抑制软骨组织的退变,本发明采用内侧半月板切除加前交叉韧带切断的方法建立了大鼠创伤性骨关节炎模型,并通过关节腔注射微球来进行干预,通过体内实验评估“自更新”润滑载药水凝胶微球的作用。骨关节炎常常具有较为明显的X线表现,其中关节间隙可以在一定程度上反应关节软骨的磨损情况,因此,本发明利用X线对造模后8周的大鼠膝关节进行了观察。结果显示,相较于假手术组,单纯造模组(PBS组)的关节间隙明显变窄,提示手术造模成功,并出现了明显的软骨损伤。而由于注射了“自更新”润滑载药水凝胶微球,RAPA@Lipo@MG组的软骨损伤得到了显著改善,关节间隙明显高于PBS组,与假手术组比较无统计学差异(图8中的A,C和D部分)。
骨赘作为关节损伤的一种代偿表现,是骨关节炎的另一特征性表现。在本实施例中,利用MicroCT对各组的骨赘量进行测定,发现在PBS组有大量的骨赘形成,而在假手术组并未发现有明显骨赘形成。相较之下,RAPA@Lipo@MG组的关节损伤得到了显著改善,其骨赘形成量明显低于PBS组(图8中的B和E部分)。
除此之外,还对造模后8周的膝关节标本进行了HE染色、番红-固绿染色、甲苯胺蓝染色以及免疫组化检测。结果显示,假手术组的关节软骨完整光滑,细胞分布均匀,Ⅱ型胶原和聚蛋白聚糖在细胞外周染色明显。而PBS组的关节软骨则磨损严重、软骨层变薄、细胞数目变少、Ⅱ型胶原和聚蛋白聚糖的表达下降,Mankin评分明显低于假手术组。相比之下,RAPA@Lipo@MG组的关节软骨较为完整光滑,细胞分布较均匀,细胞外周有一定的Ⅱ型胶原和聚蛋白聚糖的表达,Mankin评分明显高于PBS组,与假手术组比较无统计学差异(图9和图10)。
综上,这些结果证明“自更新”润滑载药水凝胶微球能够利用“自更新”的水合润滑层和“滚动轴承”效应发挥润滑作用,同时释放脂质体靶向软骨,通过缓慢释放RAPA来维持软骨细胞稳态,从而达到减少关节软骨磨损、抑制软骨退变的目的,进而延缓骨关节炎的发展。
Claims (6)
1.一种自更新水合润滑载药水凝胶微球的制备方法,其特征在于,包括以下步骤:
(1)将透明质酸与甲基丙烯酸酐按照摩尔比为1:2.6,在碱性条件下反应,得到甲基丙烯酸化透明质酸;
(2)将氢化大豆磷脂酰胆碱、胆固醇、十八胺和雷帕霉素溶解在有机溶剂中,采用膜分散法制备得到装载有雷帕霉素的阳离子脂质体;所述氢化大豆磷脂酰胆碱、胆固醇、十八胺和雷帕霉素的质量比为40:10:4:6;所述膜分散法的步骤为:将反应混合物在60℃下反应30分钟,所得产物干燥后进行水合,超声20分钟,得到分散的多层脂质体,然后用膜过滤器挤压脂质体,即得;
(3)将步骤(1)所得甲基丙烯酸化透明质酸和步骤(2)所得脂质体与光引发剂混合,采用微流控装置制备微液滴,然后在紫外光下交联,得到所述自更新水合润滑载药水凝胶微球;所述甲基丙烯酸化透明质酸、脂质体和光引发剂的质量比为40:6:4。
2.根据权利要求1所述的制备方法,其特征在于,步骤(1)中所述透明质酸的分子量为Mw=74kDa。
3.根据权利要求1所述的制备方法,其特征在于,步骤(1)中所述反应的条件为在冰浴下搅拌反应24小时,pH为8.0。
4.根据权利要求1所述的制备方法,其特征在于,步骤(2)中所述有机溶剂为氯仿。
5.一种自更新水合润滑载药水凝胶微球,其特征在于,是由权利要求1-4任一项所述方法制备得到。
6.如权利要求1-4任一项所述方法制备得到的自更新水合润滑载药水凝胶微球或权利要求5所述的自更新水合润滑载药水凝胶微球的应用,其特征在于,是将该水凝胶微球用于作为治疗骨关节炎的药物载体。
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CA2703807A1 (en) * | 2010-05-12 | 2011-11-12 | Molly S. Shoichet | Enhanced stability of inverse thermal gelling composite hydrogels |
CN106466299A (zh) * | 2015-08-19 | 2017-03-01 | 上海本素医药科技有限公司 | 以人参皂苷为膜材的空白脂质体、其制备方法及应用 |
CN109833298A (zh) * | 2017-11-29 | 2019-06-04 | 厦门本素药业有限公司 | 以人参皂苷衍生物为膜材的新型空白脂质体、其制备方法及应用 |
CN109880151A (zh) * | 2019-02-21 | 2019-06-14 | 上海市伤骨科研究所 | 一种水凝胶多孔微球的制备方法与多孔支架材料 |
WO2020181393A1 (en) * | 2019-03-13 | 2020-09-17 | Susmita Bose | Enzyme-degradable hydrogel for delivery of a payload |
CN112618571A (zh) * | 2020-09-30 | 2021-04-09 | 上海市伤骨科研究所 | 治疗骨科疾病的可注射水凝胶微球及其制备方法与应用 |
CN113398283A (zh) * | 2021-06-18 | 2021-09-17 | 上海市伤骨科研究所 | 一种基于生物膜耦合的可吸入雾化微球及制备方法与应用 |
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