CN114886869A - 一种基于巨噬细胞的超声递送系统及其构建方法与应用 - Google Patents
一种基于巨噬细胞的超声递送系统及其构建方法与应用 Download PDFInfo
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Abstract
本发明属于生物材料制备技术领域,具体涉及一种基于巨噬细胞的超声递送系统及其构建方法与应用。本发明将负载声敏剂的四氧化三铁纳米粒作为初级载药载体,利用巨噬细胞的吞噬功能负载纳米药物,制备基于巨噬细胞的超声激活型二级递送系统。该系统具备良好的靶向性、高载药量、超声激活以及保留自身活性、表型与功能的特征,利用“特洛伊木马”效应在静脉注射后可以有效靶向到炎症目标区域。同时,在给予炎症目标区域超声波辐射后,能有效触发声敏药物产生强毒性自由基实现声动力治疗,起到对RA部位浸润的炎性细胞及增生滑膜细胞的彻底清除的目的,从根本上治疗类风湿关节炎。
Description
技术领域
本发明属于生物材料制备技术领域,具体涉及一种基于巨噬细胞的超声递送系统及其构建方法与应用。
背景技术
类风湿关节炎(RA)是一种以侵蚀性关节炎为主要特征的自身免疫性疾病,其病理基础是滑膜炎;初期表现为手、足等小关节晨僵、肿胀、疼痛,后期可发展为关节畸形并致残。目前,RA的治疗主要集中于炎症反应阻断和免疫抑制,尚无针对浸润/驻留炎性细胞和增生滑膜细胞(FLSs)的治疗策略,免疫抑制剂虽有一定抑制效果,但由于毒副作用大、靶向性差,治疗效果有限。因此,亟需一种针对浸润/驻留炎性细胞和增生FLSs的精准递送及治疗系统,以改善RA的治疗效果。
精准递送及治疗系统应具有良好靶向性,可将药物精准递送至RA病变部位。尽管目前已经报道多种主动和被动靶向载体,但其在特异性方面仍需进一步加强。研究表明,纳米药物静脉注射进入血管后需要跨越多重生理屏障,特别是内皮网状系统(RES)的清除,导致纳米药物在炎症病变区域的分布极少。因此,由于RA部位的环境的复杂性与机体自身的保护机制,设计可精准靶向RA部位进行治疗的纳米载体极为困难,亟需一种新的靶向策略。
近年来,基于细胞的仿生递送策略已引起人们的关注,因为它们在药物递送系统中具有更好的优势。生物工程载体通常从亲代细胞继承特定的本能能力,或提供同源靶向能力;良好的生物相容性;作为源自活生物体的内源成分,在体内具有高安全性等。这些优势正在成为它们用作药物载体的绝佳条件。目前研究较多的生物工程载体有免疫细胞、干细胞和红细胞等。在RA发病过程中,炎性细胞包括巨噬细胞(Mφ)、T细胞和B细胞等免疫细胞的浸润是重要特征之一。普遍认为,这些浸润的炎症细胞来自于外周血,增生的FLSs可通过释放趋化因子将免疫细胞等募集到炎症部位。这其中,Mφ是被募集的细胞中较为重要的一类,也是TNF和IL-1β最主要的来源。向炎症部位的募集,表明Mφ对RA具有良好的主动靶向性,尽管基于巨噬细胞的药物递送载体已有较多报道,但是目前尚未有对Mφ这一主动靶向载体的SDT进行系统的研究,近而为RA新型治疗方法做出有益尝试的报道。
发明内容
为了解决现有技术存在的不足,本发明的目的是提供一种基于巨噬细胞的超声递送系统及其构建方法与应用。该递送系统具有高载药量和主动靶向性,克服巨噬细胞作为递送载体运载纳米药物时易受到细胞毒性影响,载药量低以及可控释药不显著等缺陷,实现高载药量和超声触发释药。同时,在RA部位给予超声照射后,药物能快速从载体中释放,并触发SDT,有效杀伤恶性增生的滑膜细胞与浸润的炎性细胞,最终实现药物RA部位精准递送。
本发明的目的可以通过以下技术方案来实现:
本发明提供了一种基于巨噬细胞的超声激活型递送系统。所述的递送系统以巨噬细胞作为药物靶向递送载体,通过主动吞噬方式载入四氧化三铁纳米粒子和声敏剂形成的纳米粒子,实现炎症部位特异性的药物递送。
在其中的一些实例中,所述的巨噬细胞包括鼠源巨噬细胞,腹腔来源巨噬细胞或骨髓来源巨噬细胞。
优选的,所述鼠源巨噬细胞为RAW264.7。
在其中的一些实例中,所述的声敏剂为原卟啉IX(PPIX),竹红菌素B,血卟啉单甲醚,ATX-70或血卟啉。优选的,声敏剂为原卟啉IX。
本发明还提供了一种基于巨噬细胞的超声激活型递送系统的制备方法,包括以下步骤:
(1)将声敏剂溶于二甲基亚砜中作为有机相,四氧化三铁纳米粒子溶于去离子水中作为水相,两相混合避光搅拌过夜;离心至上清无色,收集沉淀得到纳米载体;
(2)将巨噬细胞接种与纳米载体共同孵育,巨噬细胞主动吞噬纳米载体后洗涤,收集载药巨噬细胞得到基于巨噬细胞的超声激活型递送系统。
进一步地,步骤(1)中所述的声敏剂为原卟啉IX,竹红菌素B,血卟啉单甲醚,ATX-70或血卟啉中的一种或多种。
步骤(2)中所述的巨噬细胞为鼠源巨噬细胞,腹腔来源巨噬细胞或骨髓来源巨噬细胞。
优选的,所述鼠源巨噬细胞为RAW264.7。
步骤(1)中所述有机相与水相的浓度比为3~5:1;所述有机相与水相的体积比为1:10~15。
步骤(1)中所述离心的速率为13000~20000 rpm,离心的时间为10~30分钟。
步骤(1)中所述纳米载体中声敏剂的浓度为30~50µg·mL-1。
步骤(2)所述孵育的时间为4~6 h。
本发明还提供了上述基于巨噬细胞的超声激活型递送系统在制备治疗类风湿性关节炎的靶向药物中的应用。
本发明还提供了一种用于治疗类风湿性关节炎的药物和/或试剂,所述药物和/或试剂包括载药巨噬细胞。
与现有技术相比,本发明的有益效果是:
本发明将负载声敏剂的四氧化三铁纳米粒作为初级载药载体,利用巨噬细胞的吞噬功能负载纳米药物,制备基于巨噬细胞的超声激活型二级递送系统。该系统具备良好的靶向性、高载药量、超声激活以及保留自身活性、表型与功能的特征,利用“特洛伊木马”效应在静脉注射后可以有效靶向到炎症目标区域。同时,在给予炎症目标区域超声波辐射后,能有效触发声敏药物产生强毒性自由基实现声动力治疗(SDT),起到对类风湿关节炎(RA)部位浸润的炎性细胞及增生滑膜细胞的彻底清除的目的,从根本上治疗RA。本发明以四氧化三铁纳米粒子作为纳米载体,具有良好的生物相容性,其表面的介孔结构易于负载大量药物。同时,所用声敏剂易于化学修饰,无明显毒副作用,代谢快等优点;通过声敏剂介导的声动力治疗,实现声控药物释放,有效提高生物安全性、降低毒副作用,对机体正常组织损伤小。
附图说明
图1是制备的Fe3O4纳米载体的透射电镜图;
图2是PPIX,Fe3O4纳米载体和Fe3O4-PPIX纳米粒子的红外图谱对比图;
图3是Fe3O4-PPIX纳米粒子在pH 7.4和pH 5.0介质中PPIX的累计释放图;
图4是孵育后的巨噬细胞内PPIX负载情况对比图,**p < 0.01;
图5是巨噬细胞对游离PPIX和Fe3O4-PPIX纳米粒子的PPIX摄取情况对比图;图6是IL-1β、IL-6和TNF-α因子水平变化结果图;
图7是Fe3O4-PPIX纳米粒子对巨噬细胞的毒性结果图;
图8是载药巨噬细胞Fe3O4-PPIX@M的活死细胞染色图;
图9是载药巨噬细胞在含有FLSs生长介质中的迁移能力图;
图10是载药巨噬细胞在超声条件下的药物释放及细胞毒性图;左图是药物释放图;右图是FLSs毒性图;
图11是载药巨噬细胞的活体成像图;
图12是对CIA小鼠的RA抑制结果曲线图;
图13是关节组织恶性增生滑膜细胞和浸润炎性细胞的消除情况的H&E染色结果图;
图 14是滑膜组织恶性增生滑膜细胞和病理学血管的消除情况的H&E染色结果图。
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施例。虽然附图中显示了本公开的示例性实施例,然而应当理解,可以以各种形式实现本公开而不应被这里阐述的实施例所限制。相反,提供这些实施例是为了能够更透彻地理解本公开,并且能够将本公开的范围完整的传达给本领域的技术人员。
实施例1:Fe3O4-PPIX纳米粒子的制备
Fe3O4纳米粒的制备:将0.333 g PEG 4000、0.63 g FeCl3∙6H2O (4 mM)和0.466 g柠檬酸三钠(1.6 mM)溶解在乙二醇(47 mL)中得到混合溶液;将上述混合溶液在室温(RT)下搅拌至澄清;添加4.640 g NaOAc (32 mM),室温下剧烈搅拌1小时后,将溶液转移到容量为70 mL的不锈钢高压反应釜中200℃下反应10小时;将得到的黑色产物分别用乙醇和去离子水洗涤得到Fe3O4纳米粒。图1是制备的Fe3O4纳米粒的透射电镜图;如图1所示,所制得Fe3O4纳米粒的粒径约为70 nm,尺寸分布均一。
Fe3O4-PPIX纳米粒子的制备:将声敏剂原卟啉IX(PPIX)溶于二甲基亚砜(DMSO)中配成浓度为6 mg·mL-1的有机相,上述制备的Fe3O4纳米粒溶于去离子水中作为浓度为2mg·mL-1的水相,两相按体积比为1:10混合,避光搅拌过夜; 13000rpm离心,离心30分钟至上清无色,收集沉淀得到超声可激活声敏剂的纳米载体Fe3O4-PPIX纳米粒子。
可选的是,所述声敏剂包括原卟啉IX,竹红菌素B,血卟啉单甲醚,ATX-70或血卟啉,为了便于对照分析,以下实施例中均以制备的Fe3O4-PPIX纳米粒子作为纳米载体。
实施例2:红外光谱图
分别将原卟啉IX(PPIX)粉末、Fe3O4纳米粒和Fe3O4-PPIX纳米粒子与溴化钾(KBr)晶体按质量比1:100进行混合,研磨均匀后压片制备样品。使用傅立叶红外光谱仪检测4000-500 cm-1波长范围内的吸收峰。
图2是PPIX,Fe3O4纳米粒和Fe3O4-PPIX纳米粒子的红外图谱对比图;由图2所示,在Fe3O4-PPIX纳米粒子中能够观察到PPIX和Fe3O4纳米粒的特征吸收峰,说明Fe3O4-PPIX纳米粒子的成功制备。
实施例3:体外累计释药
在本实施例中分别对不同pH环境(pH为5.0和7.4)中进行体外释放实验。将两组含有相同PPIX含量(1 mg•mL-1)的Fe3O4-PPIX纳米粒子分别分散在3 mL pH值为 5.0和7.4的PBS缓冲液中,一组在1 MHz,2 W/cm2超声(US)处理后置于37 °C的摇床中,作为实验组;另一组未经超声处理作为对照。在不同时间点(即0.5、1、2、4、6、8、12和24 h)离心收集3 mL上清液,再补充3 mL新鲜PBS溶液,放回摇床。用紫外-可见分光光度计测试上清液中的PPIX浓度,根据载药量计算出PPIX的累积释放率。
图3是Fe3O4-PPIX纳米粒子在pH 7.4和pH 5.0介质中PPIX的累计释放图;如图3所示,在不同pH值的PBS缓冲液中实验组的药物释放量均显著高于对照组。可见,超声破坏了Fe3O4-PPIX纳米粒子之间的分子间作用力,导致药物释放速度较快且最终释放量较高。
实施例4:细胞载体载药量测定
将巨噬细胞(RAW264.7,1×105)接种在6孔板中培养24 h后,去除原培养基(RPMI1640,购自赛默飞世尔生物化学制品有限公司)后分别与Fe3O4-PPIX纳米粒子、游离PPIX(PPIX浓度:40 µg•mL-1)在新鲜培养基中共孵育以观察不同孵育时间(0.5、1、2、4 h)、不同孵育浓度(10、20、40、80 µg•mL-1)对巨噬细胞负载量的影响。Fe3O4-PPIX纳米粒子、游离PPIX与巨噬细胞共孵育后,用PBS洗涤3次,细胞刮铲将细胞刮落离心收集细胞后得到载药巨噬细胞。使用王水溶解细胞中的铁纳米粒子,然后通过电感耦合等离子体发射光谱仪(ICP-OES)检测细胞中的铁元素含量,以及检测上清中PPIX的量计算游离PPIX组的细胞负载量。以空白巨噬细胞作为对照,通过纳米粒子的载药量进行计算每个细胞中的PPIX吸收。
图4是孵育后的巨噬细胞内PPIX负载情况对比图,**p < 0.01。其中,左图为不同孵育时间的负载情况图,右图为不同浓度的负载情况图;如图4所示,随着孵育时间延长和孵育浓度的增大,负载量随之增大,细胞中PPIX含量存在时间与浓度依赖性。当处于20-80µg•mL-1的浓度范围时,具有较好的负载量;负载Fe3O4-PPIX纳米粒子的载药区间达到25-35pg•cell-1,远大于负载游离药物PPIX的8-10 pg•cell-1;尤其当浓度在40 µg•mL-1时,负载量达到31.7 pg•cell-1,具有最有效的负载量。因此,相比于负载游离药物(Free PPIX),负载纳米粒子可以大大增加药物的负载量。
实施例5:纳米药物的体外细胞摄取
将RAW264.7细胞以3×105每孔的密度接种于预先放置有20 mm盖玻片的6孔板中,培养过夜后去除原培养基,加入1 mL含有游离PPIX或Fe3O4-PPIX纳米粒子(以PPIX浓度为20µg•mL-1计)的新鲜培养基,分别孵育1 h和4 h后用无菌PBS洗去残留PPIX或Fe3O4-PPIX纳米粒子。再分别用1 mL浓度为10 µg•mL-1的Hoechst33342和1 µM的溶酶体绿色荧光探针(LysoGreen)分别孵育15 min和30 min。无菌PBS洗涤数次,用4%的多聚甲醛固定30 min,PBS洗涤后取出盖玻片,用50%甘油封片后用激光共聚焦显微镜获取荧光图像。
图5是巨噬细胞对游离PPIX和Fe3O4-PPIX纳米粒子的PPIX摄取情况对比图;由图5可见,巨噬细胞对纳米药物的摄取存在时间依赖性,对PPIX具有更大的药物摄取量;另外,可以看出纳米药物通过溶酶体途径进入细胞,而PPIX的大量摄取有利于提高声动力治疗(SDT)效果。
实施例6:纳米药物的负载对RAW264.细胞表型影响
RAW264.7细胞以3×105每孔的密度接种在6孔板中培养过夜,每孔加入1 mL含有PPIX浓度为40 µg•mL-1的Fe3O4-PPIX纳米粒子的新鲜培养基,分别在孵育0 h,4 h和24 h时,收集培养基离心取上清后,通过酶联免疫吸附试验(ELISA)检测巨噬细胞在不同条件下分泌的白细胞介素1β(IL-1β)、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)等细胞因子的含量。以0 h组为100%计算相对细胞因子水平。
图6是IL-1β、IL-6和TNF-α因子水平变化结果图;如图6可见,三种细胞因子水平均无显著性变化,在4 h时有略微变化可以归因于细胞因子在正常范围内的波动,而在24 h趋于稳定显示无明显变化,表明纳米药物不会诱导巨噬细胞M1极化。
实施例7:纳米药物对RAW264.7细胞的毒性考察
将RAW264.7细胞以每孔1×105的密度接种于单个6孔板中培养24 h。每孔中加入1mL含有不同浓度(0、10、20、40、80 µg•mL-1)Fe3O4-PPIX纳米粒子的新鲜培养基代替原培养基,共孵育4 h。用无菌PBS冲洗数次去除残余纳米粒子,用超声治疗仪超声5 min(1 MHz,1W/cm2)作为超声组,不做任何处理的为对照组;继续培养20 h,每孔中加入1 mL的MTT甲臜溶液(1 mg•mL-1),继续培养4 h后移除培养基,每孔加入1 mL的二甲基亚砜(DMSO),轻微震荡单孔板以溶解紫色甲臜后转移到96孔板,每个实验设置6个复孔。使用酶标仪(λ=490 nm)测量每孔的吸光度(OD值),根据下式计算细胞存活率。
细胞存活率(%)=(实验组OD/对照组OD)×100%
图7是Fe3O4-PPIX纳米粒子对巨噬细胞的毒性结果图;如图7可见,当未超声触发时Fe3O4-PPIX纳米粒子在PPIX浓度高达80 µg•mL-1时仍未观察到明显毒性(细胞存活率大于85%),说明Fe3O4-PPIX纳米粒子是一种具有良好生物相容性的纳米材料,可作为初级载体。而使用超声触发Fe3O4-PPIX纳米粒子后,Fe3O4-PPIX纳米粒子浓度大于20 µg•mL-1即显示出明显的细胞毒性(细胞存活率低于50%);可见,超声激活了Fe3O4-PPIX纳米粒子中的PPIX,生成大量ROS,对巨噬细胞产生杀伤作用。证实Fe3O4-PPIX纳米粒子是一种良好的初级载体,可以实现RA部位的精准定向可控毒性。
实施例8:基于巨噬细胞的超声激活型递送系统的制备及活性考察
将RAW264.7细胞以3×105每孔的密度接种于预先放置有20 mm盖玻片的6孔板中,去除原培养基后加入Fe3O4-PPIX纳米粒子在新鲜培养基中共孵育4~6h,用PBS洗涤3次,细胞刮铲将细胞刮落离心收集细胞后得到Fe3O4-PPIX@M载药巨噬细胞药物;以正常的巨噬细胞(UM)做空白对照,按照活/死细胞染色试剂盒上的操作步骤,依次用Calcein-AM和PI染色,PBS冲洗3次后用50%的甘油封片,使用激光共聚焦显微镜获取图像。
图8是载药巨噬细胞Fe3O4-PPIX@M的活死细胞染色图;图中,左为UM,右为Fe3O4-PPIX@M;由图8可见,载药巨噬细胞几乎都是存活的(绿色荧光),与正常的巨噬细胞对照组相当,表明载药后基本不影响细胞活性。
实施例9:巨噬细胞载体的体外炎症趋向性考察
将实施例8制备的载药巨噬细胞的细胞刮下来,离心(1000 r•min-1,5 min),用无血清的RPMI 1640培养基重悬(密度:1×104个mL-1),然后分别吸取1 mL细胞悬液加入transwell上室,下室加入含有滑膜细胞的无血清RPMI 1640完全培养基用来模拟RA部位的炎症环境。在5% CO2,37℃的恒温细胞培养箱中培养24 h后,用棉签擦去上室细胞,用4%的多聚甲醛固定30 min,0.1%的结晶紫染色20 min,用PBS浸泡冲洗干净染液,在倒置电子显微镜下观察。以RAW264.7细胞作为对照。图9是载药巨噬细胞在含有FLSs生长介质中的迁移能力图;图中,左为UM,右为Fe3O4-PPIX@M;如图9可见,巨噬细胞本身具有较强的炎症取向能力,且在载药后其趋化能力基本不受影响。
实施例10:超声激活的载药巨噬细胞对FLSs的细胞毒性考察
将接种于6孔板中(5×106)的RAW264.7细胞与Fe3O4-PPIX纳米粒子共孵育得到载药巨噬细胞;超声5 min(1 MHz,1 W/cm2)后,分别在不同时间点(6 h、12 h、24 h、48 h)收集上清液,通过紫外分光光度计检测上清中PPIX含量,计算载药巨噬细胞的累计释药。收集的细胞上清液,分别在对应时间加入含有滑膜细胞的培养基(RPMI1640)中(1×105),孵育4h后,避光超声5 min(1 MHz,1 W/cm2),继续培养20 h,每孔中加入1 mL的MTT溶液(1 mg•mL-1),继续培养4 h后,移除培养基后每孔加入1 mL的二甲基亚砜(DMSO),轻微震荡单孔板,待紫色甲臜溶解后转移到96孔板,每个实验设置6个复孔。使用酶标仪(λ=490 nm)测量每孔的吸光度(OD值),计算细胞存活率。
图10是载药巨噬细胞在超声条件下的药物释放及细胞毒性图;左图是药物释放图;右图是FLSs毒性图;如图10可见,在超声触发后的6 h药物存在一个快速释放期,12 h的释放量超过50%,48 h药物释放接近80%,可见,超声激活巨噬细胞显著提高了药物利用效率。而对FLSs毒性分析可见,在12 h时显示出显著增加的细胞毒性,说明药物累计达到一定浓度才会显示出明显毒性作用,体现出超声激活载药巨噬细胞对FLSs具有优异的SDT作用。
实施例11:细胞载体体内同步靶向RA的能力考察
使用牛Ⅱ型胶原诱导小鼠类风湿性关节炎(CIA)。具体方法如下:将牛Ⅱ型胶原蛋白溶于0.1 mol•L-1的乙酸配置成2 mg•mL-1的浓度,与等体积完全弗氏佐剂混合,在冰水浴搅拌制成乳剂。在ICR小鼠右后爪足底皮下注射50 µL乳剂,第14天再次注射相同部位用于加强免疫。在第28天显示出最大程度炎症症状。得到CIA模型小鼠。将实施例8制备的载药巨噬细胞(Fe3O4-PPIX@M)和Fe3O4-PPIX纳米粒子(Fe3O4-PPIX NPs)通过尾静脉注射到CIA模型小鼠体内,分别在注射后2 h、6 h、12 h、24 h和48 h处死小鼠,取心、肝、脾、肺、肾和关节组织进行成像,研究上述组织中在小鼠体内的分布及滞留情况。(剂量:2 mg•kg-1,以PPIX浓度计),图11是载药巨噬细胞的活体成像图;如图11所示,在各个检测时间点,超声激活后Fe3O4-PPIX@M组在RA中的荧光强度均高于Fe3O4-PPIX纳米粒子组,并显示出更加持久的荧光。以上结果表明,相比于传统的纳米药物,制备的载药巨噬细胞可更加高效、同步靶向到RA区域。
实施例12:细胞载体体内抑制RA的能力考察
按实施例11的方法制备CIA模型小鼠,将其随机分为6组(每组n=5);分为生理盐水组(Control),US组,Fe3O4-PPIX组,Fe3O4-PPIX+US组, Fe3O4-PPIX@M组,Fe3O4-PPIX@M+US组(剂量:5 mg•kg-1,以PPIX浓度计,0.2 mL)。US组在静脉注射6 h和12 h后用超声照射5 min(1 MHz,2 W/cm2)。在所有小鼠每周两次治疗两周的治疗过程中,使用游标卡尺测量治疗爪的厚度直到第21天处死小鼠。治疗结束后评价治疗效果。
图12是对CIA小鼠的RA抑制结果曲线图;如图12所示, 21天后Fe3O4-PPIX@M+US治疗的爪厚度变化最显著,厚度为4.07 cm,高于Fe3O4-PPIX+US组的4.80 cm,与其他对比组具有更为明显的厚度变化效果。结果表明,Fe3O4-PPIX@M具有优越的靶向作用和显著的SDT效果。解剖类风湿性关节炎小鼠,收集器官(心、肝、脾、肺和肾)和关节组织,用10%福尔马林固定,并对这些器官组织进行H&E染色验证治疗效果。
图13是关节组织恶性增生滑膜细胞和浸润炎性细胞的消除情况的H&E染色结果图;图 14是滑膜组织恶性增生滑膜细胞和病理学血管的消除情况的H&E染色结果图;如图13、14所示,与其它治疗组相比,在Fe3O4-PPIX@M治疗组中可以观察到明显的滑膜细胞消除、炎性症状消退和病理性血管减少。这些结果表明,Fe3O4-PPIX@M组比Fe3O4-PPIX纳米粒子组具有最好的治疗效果。主要是由于巨噬细胞优异的RA靶向性,显著提高了药物的利用率,通过SDT有效杀伤恶性增殖的FLSs。
综上所述,超声介导的Fe3O4-PPIX@M具有良好的靶向性、生物相容性和较高的生物安全性。因此,Fe3O4-PPIX@M作为治疗类风湿关节炎的试剂,对提高类风湿关节炎治疗效果具有重要意义。
本发明的保护内容不局限于以上实施例。在不背离本发明构思的精神和范围下,本领域技术人员能够想到的变化和优点都被包括在本发明中,并且以所附的权利要求书为保护范围。
Claims (10)
1.一种基于巨噬细胞的超声激活型递送系统,其特征在于,所述的递送系统以巨噬细胞作为药物靶向递送载体,通过主动吞噬方式载入四氧化三铁纳米粒子和声敏剂形成的纳米粒子,实现炎症部位特异性的药物递送。
2.根据权利要求1所述的所述的超声激活型递送系统,其特征在于,所述的巨噬细胞包括鼠源巨噬细胞,腹腔来源巨噬细胞或骨髓来源巨噬细胞。
3.根据权利要求1所述的所述的超声激活型递送系统,其特征在于,所述的声敏剂为原卟啉IX,竹红菌素B,血卟啉单甲醚,ATX-70或血卟啉。
4.一种基于巨噬细胞的超声激活型递送系统的制备方法,其特征在于,包括以下步骤:
(1)将声敏剂溶于二甲基亚砜中作为有机相,四氧化三铁纳米粒子溶于去离子水中作为水相,两相混合避光搅拌;离心至上清无色,收集沉淀得到纳米载体;
(2)将巨噬细胞接种与纳米载体共同孵育,巨噬细胞主动吞噬纳米载体后洗涤,收集载药巨噬细胞得到基于巨噬细胞的超声激活型递送系统。
5.根据权利要求4所述的制备方法,其特征在于,步骤(1)中所述的声敏剂为原卟啉IX,竹红菌素B,血卟啉单甲醚,ATX-70或血卟啉中的一种或多种。
6.根据权利要求4所述的制备方法,其特征在于,步骤(1)中所述有机相与水相的浓度比为3~5:1;所述有机相与水相的体积比为1:10~15。
7.根据权利要求4所述的制备方法,其特征在于,步骤(1)中所述搅拌时间为大于6h,所述纳米载体中声敏剂的浓度为30~50µg·mL-1。
8.根据权利要求4所述的制备方法,其特征在于,步骤(1)中所述离心的速率为13000~20000 rpm,离心的时间为10~30分钟。
9.根据权利要求4所述的制备方法,其特征在于,步骤(2)中所述的巨噬细胞为鼠源巨噬细胞,腹腔来源巨噬细胞或骨髓来源巨噬细胞;所述孵育的时间为4~6 h。
10.权利要求1所述基于巨噬细胞的超声激活型递送系统在制备治疗类风湿性关节炎的靶向药物中的应用。
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