CN115624538B - 仿生型巨噬细胞膜包覆纳米配位聚合物及制备方法与其在肝脏缺血再灌注损伤中的应用 - Google Patents
仿生型巨噬细胞膜包覆纳米配位聚合物及制备方法与其在肝脏缺血再灌注损伤中的应用 Download PDFInfo
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Abstract
本发明公开了仿生型巨噬细胞膜包覆纳米配位聚合物及制备方法与其在肝脏缺血再灌注损伤中的应用。该仿生型巨噬细胞膜包覆纳米配位聚合物为涂覆有巨噬细胞膜的由地塞米松磷酸钠和铁离子通过配位作用构建的纳米配位聚合物。该仿生型巨噬细胞膜包覆纳米配位聚合物为器官移植的缺血再灌注损伤治疗提供了一种新的治疗选择。
Description
技术领域
本发明涉及基于细胞膜的仿生纳米技术,尤其涉及仿生型巨噬细胞膜包覆纳米配位聚合物及其制备方法与在肝脏缺血再灌注损伤中的应用。
背景技术
缺血再灌注损伤(IRI)是器官移植及肝脏手术中不可避免的并发症,IRI的发生严重制约着器官移植共体力的扩展,影响术后器官功能的恢复甚至导致器官早期功能恢复延迟。然而,目前对于IRI的发生并没有确切的治疗策略。糖皮质激素(GCs)是临床广泛应用的器官移植围手术期药物,具有良好的抗炎、抗氧化和抗排斥作用,但是由于GC受体在全身的广泛分布,大剂量或者长期的使用会带来严重的副作用或并发症,极大的限制了它的应用。所以若能改善GCs的药物分布,减轻其副作用的产生,将有效增加GCs的治疗效果和临床应用。利用纳米技术可以有效改善药物的药代动力学和靶向性,为移植相关免疫治疗提供了一种新的方向和策略。基于细胞膜的仿生纳米技术是近年新兴的纳米工程技术,这项技术可以利用天然来源的细胞膜改造人工合成的纳米材料,不仅可以改善小分子药物的生物利用率,还可赋予纳米粒子源细胞来源的部分功能。
发明内容
本发明的一个目的是提供一种仿生型巨噬细胞膜包覆纳米配位聚合物。
本发明所提供的仿生型巨噬细胞膜包覆纳米配位聚合物,命名为dNCPs@MM,为涂覆有巨噬细胞膜的由地塞米松磷酸钠(DEXp)和铁离子通过配位作用构建的纳米配位聚合物(dNCPs)。
在一个实施方案中,所述地塞米松磷酸钠和所述铁离子的摩尔比为5:1。
在一个实施方案中,所述的仿生型巨噬细胞膜包覆纳米配位聚合物的平均粒径为107.6±9.7nm,ζ电位为-42.89mV。
在一个实施方案中,所述的仿生型巨噬细胞膜包覆纳米配位聚合物的膜表面含有CD11b蛋白、F4/80蛋白以及CD68蛋白。
上述仿生型巨噬细胞膜包覆纳米配位聚合物可用于预防或治疗肝脏的缺血再灌注损伤。
上述仿生型巨噬细胞膜包覆纳米配位聚合物能够抑制NF-κB信号通路激活。
上述仿生型巨噬细胞膜包覆纳米配位聚合物可用于抑制肝脏的缺血再灌注损伤诱导的细胞凋亡发生。
上述仿生型巨噬细胞膜包覆纳米配位聚合物可用于抑制肝脏的缺血再灌注损伤诱导的炎症反应。
上述仿生型巨噬细胞膜包覆纳米配位聚合物可用于抑制肝移植相关免疫排斥反应。
本发明的另一个目的是提供一种用于制备仿生型巨噬细胞膜包覆纳米配位聚合物的方法,所述方法包括:
将含有三价Fe离子的溶液逐滴添加到含有地塞米松磷酸钠的水溶液中在室温下搅拌形成纳米配位聚合物;
将所述纳米配位聚合物添加到含有巨噬细胞膜囊泡的缓冲液中,震荡处理,然后将所得混合物通过多孔膜挤出,得到所述的仿生型巨噬细胞膜包覆纳米配位聚合物。
本发明的仿生型巨噬细胞膜包覆纳米配位聚合物的生物相容性高,炎症的酸性环境下可以响应释放,保护组织免受IRI后的损伤和抑制移植相关免疫排斥反应。
附图说明
图1示出了仿生型巨噬细胞膜包覆纳米配位聚合物dNCPs@MM的表征。(A、B)dNCPs@MM的透射电镜照片,比例尺分别为200nm、50nm。(C)DLS测量的dNCPs和dNCPs@MM的水合粒径分布。(D)通过DLS测量dNCPs和dNCPs@MM的ζ电位。
图2示出了dNCPs@MM膜表面蛋白分析。(A)SDS-PAGE总蛋白分析。(B)WB分析dNCPs@MM中巨噬细胞膜特征蛋白。(1:巨噬细胞;2:dNCPs;3:dNCPs@MM;4:MM。)
图3示出了肝脏IRI治疗后的H&E染色和血清生化肝功能指标检测。(A)各组血清中AST和ALT水平。AST和ALT水平较低说明肝功能较好。(B)各组肝组织的H&E染色。比例尺:100μm。
图4示出了各组肝脏样本再灌注后的凋亡水平检测。(A)各组具有代表性的肝脏切片TUNEL荧光染色结果,(B、C)Western blot检测Cleaved caspase-3、Bax和Bcl-2蛋白表达水平。比例尺:100μm。
图5示出了各组肝脏IRI肝脏内NF-κB信号通路相关蛋白的表达。(A)Western blot检测结果,(B)抑制NF-κB信号通路激活的能力。
具体实施方式
本发明首先通过地塞米松磷酸钠(DEXp)和铁离子的配位作用构建了的纳米配位聚合物(dNCPs),并提取巨噬细胞膜涂覆dNCPs纳米粒子,合成了一种新型仿生型巨噬细胞膜包覆纳米配位聚合物(dNCPs@MM)。
缺血再灌注损伤(IRI)是影响移植术后受体并发症和死亡率的重要因素之一。本发明用C57BL/6小鼠成功地建立了肝脏IRI动物模型。通过缺血前预先给小鼠静脉注射仿生型巨噬细胞膜包覆纳米配位聚合物dNCPs@MM,发现dNCPs@MM能够显著降低肝脏IRI后血清AST、ALT水平,减少肝脏坏死面积。dNCPs@MM纳米粒子还可以抑制IRI诱导的细胞凋亡发生。此外,dNCPs@MM还可以通过使NF-κB信号通路失活,抑制IRI诱导的炎症反应。
这些结果表明,利用巨噬细胞膜包裹DEXp和铁离子组装的纳米颗粒dNCPs,得到了一种形貌稳定的新型仿生型巨噬细胞膜包覆纳米配位聚合物。这种仿生型巨噬细胞膜包覆纳米配位聚合物的生物相容性高,利用肝脏组织对纳米药物的“自靶向”效应,使得纳米级别药物可以滞留在肝脏组织中发挥治疗效果,从而更能够保护组织免受IRI后的损伤从而减轻移植相关免疫排斥反应。为器官移植的抗炎、抗排斥治疗提供一种新的治疗策略。
实施例
仿生型巨噬细胞膜包覆纳米配位聚合物dNCPs@MM的合成:
(1)dNCPs纳米粒子的合成:将FeCl3·6H2O(5.4mg,0.02mmol)溶解在10mL去离子水中,然后逐滴加入到含有DEXp(51.6mg,0.1mmol)水溶液中,并在室温下磁力搅拌12小时,可以肉眼观察到反应溶液逐渐变浑浊并呈浅灰色。通过在4℃以12000rpm下离心30分钟收集产物,并用去离子水洗涤3次。最后,将制备的金属离子-有机药物纳米粒子冻干,并用精密电子天平称重确定其产率(产率:49%)。
(2)巨噬细胞膜(Macrophage membrane,MM)的提取及MM囊泡的制备:
1)巨噬细胞在含有10%FBS和1%青链霉素的1640培养基中培养。
2)通过刮刀刮取、离心获得细胞,用PBS洗涤3遍。
3)使用4℃的含有1%蛋白酶抑制剂的Tris-Mg缓冲液重悬,并使用匀浆器研磨细胞悬液50次,充分研磨细胞,离心机4℃,3200rpm离心5分钟,取上清液;
4)沉淀再次用4℃含有1%蛋白酶抑制剂的Tris-Mg缓冲液重悬,匀浆器研磨细胞悬液50次,充分研磨,离心机4℃,3200rpm离心5分钟,取上清液;
5)将之前2)和3)步骤中的上清液混合,离心机4℃,20000rpm离心20分钟,去上清液;
6)上清液离心4℃,100000rpm离心70分钟,弃上清液,沉淀重悬,4℃,100000rpm离心30-60分钟,弃去上清,沉淀重悬,获得细胞膜悬液;
7)Avanti微型挤出器挤压,最后获得细胞膜沉淀并用PBS重悬。使用Mini挤出器悬液反复挤过0.4μm的聚碳酸酯多孔膜(重复15次)得到MM囊泡。
(3)dNCPs纳米粒子的细胞膜包覆:将含有dNCPs(2mg)的水溶液缓慢加入含有MM囊泡(从107个细胞中提取)的200μL PBS溶液。先将混合溶液在100W超声震荡仪中处理2分钟,随后使用Mini挤出机将所得混合物通过聚碳酸酯多孔膜(孔径:0.4μm)挤出20次。最终产物在4℃下以15000rpm离心30分钟得到纯化的dNCPs@MM,并用去离子水洗涤3遍。纯化的仿生型巨噬细胞膜包覆纳米配位聚合物dNCPs@MM重新分散在PBS中以供后续实验。
表征:
(1)仿生型巨噬细胞膜包覆纳米配位聚合物dNCPs@MM的电镜、粒径以及ζ电位分析:
将合成后的dNCPs和dNCPs@MM分别滴在铜网上,待干燥后,在120kV透射电子显微镜(Transmission Electron Microscope,TEM)下观察其粒径及形貌。通过TEM的结果可以清晰地看到细胞膜涂层纳米粒子的额外外层(图1A和图1B)。
采用动态光散射(Dynamic light scattering,DLS)测定纳米粒子的水合粒径尺寸并进行Zeta电势能分析以确定纳米粒子的表面电荷。
随后采用DLS检测,结果也发现dNCPs的平均粒径在进行细胞膜包覆后从(92.2±8.3nm)变成(107.6±9.7nm)(图1C),而ζ电位从-27.41mV降至-42.89mV(图1D),证实了纳米粒子的成功包覆。
(2)仿生型巨噬细胞膜包覆纳米配位聚合物dNCPs@MM的膜蛋白分析:
使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行膜总蛋白分析。使用RIPA裂解液获得MM囊泡和dNCPs@MM中的总蛋白,使用BCA蛋白试剂盒定量,与上样缓冲液混合并煮沸。将准备好的dNCPs@MM、MM囊泡和dNCPs共同上样,在120V下电泳2小时,使用考马斯亮蓝染色试剂盒染色,随后在乙酸中脱色过夜。
采用免疫印迹法(Western blot)检测巨噬细胞、巨噬细胞膜(MacrophageMembrane,MM)和dNCPs@MM的特异性表面标志物。
1)蛋白提取:取1mL裂解液,加入蛋白酶抑制剂,使抑制剂终浓度为1mM。按照每1×106个细胞加100uL的裂解液的比例,分别在巨噬细胞、MM、dNCPs和dNCPs@MM中加入对应量的裂解液,在冰上放置5-10分钟充分裂解;
2)蛋白定量:采用BCA法进行蛋白定量。以牛血清蛋白(BSA)做标准曲线,在酶标仪下检测不同组的吸光度,并计算对应的蛋白浓度。将所得到的蛋白和1×聚丙烯酰胺(SDS)加样缓冲液混匀,100℃金属浴5分钟;
3)配胶:根据目蛋白的分子量大小,选择6%的分离胶。配置好下层分离胶和上层浓缩胶后,插入梳子,留置上样孔;
3)电泳:将配置好的胶放入电泳仪,加入1×电泳缓冲液,注意检查有无漏液。取出梳子,加样,每孔蛋白上样量20-40μg,电泳80V,20分钟,分离胶110V,30分钟;
4)转膜:配置1×转膜缓冲液,提前浸泡转膜用仪器。取出胶,切去上层浓缩胶和下层溴酚蓝部分。按顺序组装转膜装置:白板、黑网、滤纸、聚偏二氟乙烯(PVDF)膜、胶、滤纸、黑网、黑板。100V,电泳60-100分钟,整个过程要在冰中进行;
5)封闭:用TBST缓冲液封闭1小时;
6)一抗孵育:一抗稀释液稀释抗体(F4/80、CD11b、CD68,稀释倍数:1:1000),将PVDF膜与稀释的抗体共同在4℃下孵育过夜;
7)二抗孵育:一抗孵育过夜后,TBST缓冲液洗涤三次,每次10分钟,加入相应的二抗,室温孵育2小时。再次用TBST洗涤三次,每次10分钟;
显影发光:使用Meilunbio飞克特超敏ECL发光检测试剂盒检测响应条带曝光强度并拍照留存。
为了验证所制备的dNCPs@MM纳米粒子是否成功包覆MM,我们使用SDS-PAGE和WB免疫印迹分析dNCPs@MM纳米粒子中的蛋白含量。从SDS-PAGE测定的结果(图2A)中可以看出,制备的dNCPs@MM与MM中的蛋白含量基本类似。与此同时,我们还使用WB检测了dNCPs@MM上巨噬细胞膜表面的特征性蛋白来佐证纳米粒子制备的成功(图2B)。根据WB的结果,可以发现,dNCPs@MM上保留了CD11b、F4/80以及CD68蛋白,同时内参蛋白几乎没有表达。以上结果均可以说明在使用共挤压法制备的纳米粒子时基本保留了细胞膜上大多数的蛋白。
效果验证:
(1)仿生型巨噬细胞膜包覆纳米配位聚合物dNCPs@MM减轻肝脏IRI:
小鼠肝脏70%缺血再灌注损伤模型的建立
(1)小鼠术前禁食不禁水8小时,戊巴比妥(50mg/kg)腹腔注射麻醉;
(2)平卧位固定小鼠,腹部备皮,使用75%乙醇消毒3遍;
(3)沿腹部正中逐层开腹,用棉签小心暴露第一肝门,显微外科剪离断肝胃韧带,小心分离出门静脉和肝动脉;
(4)使用无损伤血管夹在肝中、肝右叶间隙处夹闭血管,见肝左叶及中叶颜色由鲜红变为白色说明造模成功。将小鼠置于37℃恒温台上保温,止血钳临时关闭腹腔;
(5)持续缺血90分钟后,取走血管夹,观察肝脏颜色由白色重新恢复鲜红,表面再灌注成功;
(6)使用3-0不可吸收外科缝线逐层关腹,术后将小鼠置于恒温台等待其清醒,遂将其放回消毒后的鼠笼继续饲养;
(7)对照组不做血管夹闭处理,其他操作相同;
(8)再灌注12小时后,麻醉小鼠,摘眼球取血,留取缺血处理后的肝左叶组织留作病理标本;
(9)标本室温静置1小时后于4℃下以3500rpm离心15分钟,吸取上层血清用于检测,剩余血清-80℃冰箱冻存以备后续检测。
动物分组
将25只C57BL/6小鼠随机分为五个数量相等的组:
(1)第一组(Sham组):术前1小时,于小鼠尾静脉注射200μL的生理盐水,开腹后不做缺血处理,其他操作相同;
(2)第二组(IRI组):术前1小时,于小鼠尾静脉注射等剂量生理盐水(200μL),随后建立肝脏部分IRI模型;
(3)第三组(DEXp组):术前1小时,于小鼠尾静脉注射1mg/kg的DEXp生理盐水溶液(200μL),随后建立小鼠肝脏部分IRI模型;
(4)第四组(dNCPs组):术前1小时,小鼠尾静脉注射等效剂量为1mg/kg的dNCPs纳米粒子溶液(200μL),随后建立肝脏部分IRI模型;
(5)第五组(dNCPs@MM组):术前1小时,小鼠尾静脉注射等效剂量为1mg/kg的dNCPs@MM纳米粒子溶液(200μL),随后建立肝脏部分IRI模型;
在再灌注后第12小时,麻醉小鼠获取血液样本,随后脱颈处死小鼠,并解剖小鼠获取肝脏左叶标本,待后续的研究处理和分析。
肝功能检测
将获取的血清样品使用PBS缓冲液进行稀释(稀释倍数根据具体情况而定);将所需检测项目谷丙转氨酶(Alanine aminotransferase,ALT)和谷草转氨酶(Aspartateaminotransferase,AST)的干式生化检测试剂卡放入全自动生化分析仪,吸取10μL稀释后的血清样品滴入试剂中央芯片内进行检测,每个样本至少重复检测3次。
如图3A所示,给予生理盐水的假手术组小鼠(Sham组)ALT和AST水平均处于正常范围。肝脏缺血再灌注后小鼠血清中的AST和ALT值均出现了显著的升高,在分别给予仿生型巨噬细胞膜包覆纳米配位聚合物dNCPs@MM和地塞米松磷酸钠(DEXp)药物治疗后,ALT和AST值出现明显的降低,表明dNCPs@MM和DEXp减轻了肝组织的损伤,都具有整体治疗效果。进一步对各组肝组织进行H&E染色,以提供更为直接的病理学证据。如图3B中所示,在生理盐水处理的IRI组小鼠的肝脏切片中出现了大面积的细胞坏死和肝脏结构损伤,可以发现明显的肝细胞溶解、坏死和淤血。然而,在dNCPs@MM组中,仅观察到轻微的肝组织损伤。这些结果都证明了未经治疗的IRI小鼠出现严重的肝损伤,而通过DEXp、dNCPs和dNCPs@MM治疗都可以有效预防IRI后的肝脏损伤。同时,根据肝功能和病理结果,还可以发现,dNCPs@MM相较于游离DEXp和dNCPs表现出更为优异的治疗效果。
(2)仿生型巨噬细胞膜包覆纳米配位聚合物dNCPs@MM减轻肝脏IRI后的细胞凋亡水平:凋亡是肝脏IRI后常见的病理表现,尤其发生在炎症反应和氧化应激之后。为了进一步评估仿生型巨噬细胞膜包覆纳米配位聚合物dNCPs@MM对小鼠肝脏IRI的保护作用,我们对肝脏IRI后细胞凋亡水平进行了详细的研究。我们通过Western blot(实验步骤同前)对各组再灌注后肝脏样本中相关凋亡蛋白的表达进行了检测,发现dNCPs@MM组中抑制凋亡发生的Bcl-2蛋白表达明显增加,而促凋亡蛋白Cleaved caspase-3和Bax表达水平降低(图4A、B)。所以这些结果表明,dNCPs@MM可以显著减轻肝脏IRI后的细胞凋亡水平。
(3)仿生型巨噬细胞膜包覆纳米配位聚合物dNCPs@MM显著抑制肝IRI后NF-κB通路的活化
研究表明,DEXp的强大抗炎能力很大部分是通过抑制NF-κB通路的活化,从而抑制炎性细胞因子的表达和分泌。为了进一步验证dNCPs@MM对分子机制的影响,我们采用Western blot检测了各组肝脏IRI肝脏内NF-κB信号通路相关蛋白的表达。如图5A、B所示,dNCPs@MM比游离DEXp表现出更强的抑制NF-κB信号通路激活的能力。
Claims (7)
1.一种仿生型巨噬细胞膜包覆纳米配位聚合物,其特征在于,所述仿生型巨噬细胞膜包覆纳米配位聚合物为涂覆有巨噬细胞膜的由地塞米松磷酸钠和铁离子通过配位作用构建的纳米配位聚合物;
其中,所述的仿生型巨噬细胞膜包覆纳米配位聚合物的膜表面含有CD11b蛋白、F4/80蛋白以及CD68蛋白;
所述地塞米松磷酸钠和所述铁离子的摩尔比为5:1。
2.根据权利要求1所述的仿生型巨噬细胞膜包覆纳米配位聚合物,其特征在于,所述的仿生型巨噬细胞膜包覆纳米配位聚合物的平均粒径为107.6±9.7nm,ζ电位为-42.89mV。
3.权利要求1至2中任一项所述的仿生型巨噬细胞膜包覆纳米配位聚合物在制备用于预防或治疗肝脏的缺血再灌注损伤的药物中的应用。
4.根据权利要求3所述的应用,其特征在于,所述仿生型巨噬细胞膜包覆纳米配位聚合物抑制NF-κB信号通路激活。
5.权利要求1至2中任一项所述的仿生型巨噬细胞膜包覆纳米配位聚合物在制备用于抑制肝脏的缺血再灌注损伤诱导的细胞凋亡发生的药物中的应用。
6.权利要求1至2中任一项所述的仿生型巨噬细胞膜包覆纳米配位聚合物在制备用于抑制肝脏的缺血再灌注损伤诱导的炎症反应的药物中的应用。
7.一种用于制备权利要求1-2中任一项所述的仿生型巨噬细胞膜包覆纳米配位聚合物的方法,包括:
将含有三价Fe离子的溶液逐滴添加到含有地塞米松磷酸钠的水溶液中在室温下搅拌形成纳米配位聚合物;将所述纳米配位聚合物添加到含有巨噬细胞膜囊泡的缓冲液中,震荡处理,然后将所得混合物通过多孔膜挤出,得到所述的仿生型巨噬细胞膜包覆纳米配位聚合物。
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