CN116440287A - 抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体及其制备方法和应用 - Google Patents
抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体及其制备方法和应用 Download PDFInfo
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Abstract
抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体及其制备方法和应用,属于药物制剂新辅料和新剂型领域,将小分子化疗药物10‑羟基喜树碱和巴诺蒽醌共载于外泌体中,再通仿生矿化策略,将aTIM‑4与矿化颗粒磷酸钙有机结合,得到仿生矿化外泌体。在肿瘤微环境的酸性响应下,磷酸钙外壳溶解,外泌体内核进入肿瘤细胞,诱导细胞死亡,产生凋亡小体,程序性的递送药物,将巴诺蒽醌递送到肿瘤深处,同时释放出的aTIM‑4抑制了肿瘤相关巨噬细胞的胞葬作用,放大了基于凋亡小体的药物程序性渗透。本发明为解决纳米药物在肿瘤渗透瓶颈提供了新的策略和更多的选择,满足了临床中对高效化疗制剂的迫切需求。
Description
技术领域
本发明属于药物制剂新辅料和新剂型领域,涉及抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体及其制备方法和应用,具体涉及一种同时载有10-羟基喜树碱,低氧激活前药巴诺蒽醌和抑制胞葬的单克隆抗体aTIM-4的仿生矿化外泌体及其构建方法,以及其在药物递送中的应用。
背景技术
癌症的发病率和致死率逐年增加,严重危害了我国人民的生命健康。各种用于肿瘤治疗的新型纳米制剂已成为当下的研究热点,如利用外泌体作为药物载体构建的仿生递药系统,或者经由无机矿化修饰的具有良好生物相容性、长血液循环时间的功能型矿化纳米平台。然而,由于实体瘤内部致密的细胞外基质、由外向内不断升高的组织间静水压,以及紧密排列的肿瘤细胞,经由血管进入肿瘤组织的纳米药物往往被限制在血管周围,难以对肿瘤进行全面的杀伤,降低了其临床治疗效果。因此,如何提高纳米药物对肿瘤的渗透能力仍是临床上亟待解决的难题。
细胞凋亡后,细胞膜皱缩内陷,对细胞质进行切割,形成了内含核碎片、细胞器以及大分子蛋白等的凋亡小体。以往研究表明,小分子化疗药物诱导肿瘤细胞凋亡后,细胞内剩余的药物可以被储存在凋亡小体中,被相邻的肿瘤细胞摄取后发挥细胞毒性作用,像“剥洋葱”一样,一层又一层不断地杀伤肿瘤细胞,向肿瘤深处渗透。然而,肿瘤部位富集的巨噬细胞同样可以发挥胞葬作用,迅速识别并清除产生的凋亡小体,限制了凋亡小体在细胞间的传递,无法取得好的抗肿瘤效果。同时基于凋亡小体的药物渗透机制尚未清晰,不同药物模型在凋亡小体中的递送效率差异有待考察。这些问题严重制约了基于凋亡小体的药物肿瘤深部渗透纳米技术的发展。
发明内容
本发明所解决的技术问题是将肿瘤不同空间位置起效的两种小分子化疗药物共载于仿生外泌体中,并在仿生外泌体表面通过生物矿化的方式,覆盖载有抑制胞葬的单克隆抗体的矿化外壳,制得抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体。所制备的仿生矿化外泌体纳米粒稳定性好、循环时间长、抑制胞葬的效果显著、对肿瘤组织的程序性渗透能力强,验证了基于凋亡小体的药物渗透机制,体现了本发明制剂在肿瘤治疗方面的优越性。
本发明将抑制胞葬的单克隆抗体和肿瘤不同空间位置起效的两种小分子化疗药物共载于仿生矿化外泌体中,研究三者联合使用对药物在肿瘤组织中的渗透以及药效学作用的影响,并探究基于凋亡小体的药物渗透机制,为提高药物肿瘤渗透能力提供新的策略并指导基于凋亡小体的实现肿瘤深部渗透的药物设计,加速了纳米药物的临床转化。
本发明所述的抑制胞葬的单克隆抗体是结合巨噬细胞表面TIM-4受体的单克隆抗体aTIM-4,可显著抑制巨噬细胞的胞葬作用;所述的肿瘤不同空间位置起效的两种小分子化疗药物是指疏水的、对于全部肿瘤区域有细胞毒性的小分子药物,如10-羟基喜树碱(HCPT);以及亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药,如巴诺蒽醌(AQ4N);所述的仿生外泌体是指与肿瘤细胞同源的外泌体,如4T1小鼠乳腺癌细胞分泌的外泌体;所述的矿化外壳是指由无机离子构成的在肿瘤酸性微环境中降解的坚硬外壳,如磷酸钙外壳。所述抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体的各组分按重量百分比含量为:矿化外壳占44%-56%,仿生外泌体占24%-38%,亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药占7%-10%,疏水的、对于全部肿瘤区域有细胞毒性的小分子药物占1%-3%,aTIM-4占3%-5%。各组分优选的重量比为:矿化外壳:仿生外泌体:亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药:疏水的、对于全部肿瘤区域有细胞毒性的小分子药物:aTIM-4=2.28mg:1mg:0.4mg:0.14mg:0.2mg。
所述的磷酸钙由氯化钙(CaCl2)和磷酸氢二钠(Na2HPO4)制得。按重量比为氯化钙:磷酸氢二钠=(1-4):(2-6),优选为重量比1mg:1.28mg。
本发明还提供了所述抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体的制备方法:收集培养小鼠癌细胞的无血清细胞培养液,通过多次离心先后除去肿瘤细胞、死细胞和细胞碎片,使用含有蛋白酶抑制剂的PBS重悬后,通过超速离心去除上清液,得到空白外泌体(exosomes)。将疏水的、对于全部肿瘤区域有细胞毒性的小分子药物和亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药分别溶解在有机溶剂和去离子水中,与制得的空白外泌体混合。在冰浴下进行超声。超声完成后,将制得的共载有疏水的、对于全部肿瘤区域有细胞毒性的小分子药物和亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药的外泌体放入恒温培养箱里孵育,恢复外泌体膜的完整性。然后,搅拌分散分泌体,逐滴加入含有抑制胞葬的单克隆抗体aTIM-4和CaCl2的水溶液,待搅拌均匀后,加入Na2HPO4的水溶液,继续搅拌,得到仿生矿化外泌体。
上述抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体的制备方法,其中:
所述疏水的、对于全部肿瘤区域有细胞毒性的小分子药物,优选HCPT;亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药,优选AQ4N。
所述有机溶剂为甲醇、无水乙醇、二甲基亚砜中的一种,优选二甲基亚砜。有机溶剂溶解疏水的、对于全部肿瘤区域有细胞毒性的小分子药物;去离子水溶解亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药、aTIM-4、CaCl2、Na2HPO4。
所述在冰浴下进行超声,其超声功率为100W-200W,优选180W。
本发明还提供上述抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体在注射给药、口服给药或局部给药系统中的应用。
本发明具有以下有益效果:
(1)制备了粒径均匀的同时载有aTIM-4、HCPT和AQ4N的磷酸钙外壳覆盖的仿生矿化外泌体制剂,制备方法简单高效,稳定性好,实现了小分子药物和单克隆抗体高效共递送。
(2)研究了磷酸钙外壳在肿瘤微环境的响应解体,aTIM-4对肿瘤相关巨噬细胞的胞葬抑制作用,两个不同药物模型HCPT和AQ4N在基于凋亡小体的细胞间递送效率,仿生矿化外泌体在肿瘤组织中的渗透,以及对肿瘤生长的抑制作用。进行了仿生矿化外泌体的制剂表征、酸响应药物释放行为检测、凋亡小体的提取以及其邻位效应实验、凋亡小体传递药物过程中的药物含量变化分析、单抗的胞葬抑制作用探究、肿瘤球实验,以及动物体内的药效学考察。结果表明,仿生矿化外泌体粒径均一、稳定性好,可以在酸性环境解体,通过抑制胞葬作用,放大凋亡小体递送药物过程中的分选行为,实现了肿瘤深处的程序性渗透以及肿瘤生长的完全抑制。为解决纳米药物在肿瘤渗透瓶颈提供了新的策略和更多的选择,满足了临床中对高效化疗制剂的迫切需求。
附图说明
图1为本发明实施例1小分子药物共载外泌体制备实验图。
A:药物比例筛选。
B:AH-Exos的透射电镜显微镜图。
图2为本发明实施例2中仿生矿化外泌体的特性实验图。
A:AH-Exos@CPM的粒径、电位图。
B:AH-Exos@CPM的透射电镜图。
C:AH-Exos@CPM的元素能谱分析图。
D:AH-Exos@CPM、AH-Exos和Exosomes的SDS-PAGE检测图。
E:AH-Exos@CPM、AH-Exos的紫外-可见分光光度计和荧光光谱仪扫描图。
图3为本发明实施例3中仿生矿化外泌体的体外释放检测图。
A:钙离子在不同pH条件下的释放动力学结果图,***p<0.001。
B:aTIM-4在不同pH条件下的释放动力学结果图,***p<0.001。
C:HCPT在不同pH条件下的释放动力学结果图。
D:AQ4N在不同pH条件下的释放动力学结果图。
图4为本发明实施例4中仿生矿化外泌体的胶体稳定性和放置稳定性图。
图5为本发明实施例5中仿生矿化外泌体的体外细胞摄取实验图。
A:AQ4N溶液、HCPT溶液、AH-Exos、AH-Exos@CPM、AH-Exos@CPM(pH 6.5)处理12h的4T1细胞的共聚焦荧光图像。
B:各制剂处理12h的4T1细胞的流式细胞术测量图,***p<0.001,****p<0.0001。
C:各制剂处理6h的4T1细胞的共聚焦荧光图像。
D:各制剂处理6h的4T1细胞的流式细胞术测量图。
图6为本发明实施例6中仿生矿化外泌体的4T1细胞毒性实验图。
A:常氧情况下AQ4N溶液、HCPT溶液、AQ4N与HCPT混合溶液、AH-Exos、AH-Exos@CPM,以及AH-Exos@CPM(pH 6.5)处理48h的4T1细胞存活率。
B:低氧情况下各制剂处理48h的4T1细胞存活率。
图7为本发明实施例7中凋亡小体的提取与表征图。
A:从AH-Exos@CPM(pH 6.5)组诱导的细胞凋亡中,提取出的凋亡小体的透射电镜图。
B:凋亡小体的特征性蛋白western blot检测图。
C:凋亡小体的共聚焦荧光图和4T1肿瘤的摄取荧光图像。
D:凋亡小体对4T1细胞的细胞毒性图。
图8为本发明实施例8中基于凋亡小体的邻位效应探究图。
A:邻位效应探究方法示意图。
B:各个板上4T1细胞的共聚焦荧光图像。
C:AH-Exos@CPM(pH 6.5)处理组中,凋亡小体(i-ii)和凋亡小体(ii-iii)中药物的共聚焦荧光图
D:AH-Exos@CPM(pH 6.5)处理组中,在细胞(i-I)、凋亡小体(i-ii),以及细胞(ii-I)中AQ4N的含量变化图。
E:在细胞(i-I)、凋亡小体(i-ii),以及细胞(ii-I)中HCPT的含量变化图。
F:在细胞(i-I)、凋亡小体(i-ii),以及细胞(ii-I)中AQ4N与HCPT的总摩尔比,****p<0.0001。
G:AH-Exos@CPM(pH 6.5)处理组中,i-I和i-A细胞里AQ4N相对于AQ4的含量变化图。
H:i-I和i-A细胞里凋亡标志性蛋白western blot检测图。
图9为本发明实施例9中抑制胞葬提升凋亡小体渗透能力考察图。
A:探究aTIM-4抑制巨噬细胞对凋亡小体的清除的流式细胞术测量图。
B:肿瘤球实验中通过共聚焦显微镜观察AQ4N溶液、HCPT溶液、AQ4N与HCPT混合溶液、AH-Exos和AH-Exos@CPM的渗透情况。
C:肿瘤球中AQ4N和HCPT的荧光强度定量图。
图10为本发明实施例10中仿生矿化外泌体的血浆药物浓度-时间曲线图
图11为本发明实施例11中仿生矿化外泌体的体内组织分布图。
A:AQ4N溶液、AH-Exos和AH-Exos@CPM在小鼠体内不同时间的荧光分布图。
B:注射不同制剂24h后,小鼠主要器官(心、肝、脾、肺、肾、肿瘤)的离体荧光分布图。
C:注射不同制剂24h后,小鼠主要器官的离体荧光强度半定量图,***p<0.001,****p<0.0001。
图12为本发明实施例11中通过共聚焦显微镜观察AQ4N溶液、HCPT溶液、AQ4N与HCPT混合溶液、AH-Exos和AH-Exos@CPM在小鼠肿瘤模型中的渗透情况。
图13为本发明实施例13中仿生矿化外泌体的药效学考察图。
A:生理盐水、HCPT溶液、AQ4N溶液、HCPT与AQ4N混合溶液、AH-Exos,以及AH-Exos@CPM尾静脉注射后的4T1肿瘤体积大小变化图,**p<0.01,***p<0.001。
B:生理盐水、HCPT溶液、AQ4N溶液、HCPT与AQ4N混合溶液、AH-Exos,以及AH-Exos@CPM尾静脉注射后的4T1荷瘤小鼠体重变化图。
C:不同类型的制剂治疗后,解剖离体的4T1肿瘤照片。
D:不同类型的制剂治疗后,解剖离体的4T1肿瘤重量,**p<0.01,****p<0.0001。
E:不同类型的制剂治疗后,4T1荷瘤小鼠的肝功能指标。
F:不同类型的制剂治疗后,4T1荷瘤小鼠的肾功能指标。
G:不同类型的制剂治疗后,解剖离体的4T1肿瘤H&E染色图、TUNEL染色图、Ki67染色图
H:不同类型的制剂治疗后,4T1荷瘤小鼠心、肝、脾、肺、肾的H&E染色图。
具体实施方式
实施例1:小分子药物共载外泌体的制备
采用超速离心法提取外泌体。首先用含10%除外泌体的胎牛血清的细胞培养液培养细胞,收集细胞上清液,在300g转速下,离心10min,去除漂浮细胞,再在15,000g转速下,离心30min,去除细胞碎片,再次用100,000g转速离心120min,得到纯净的外泌体。获得的外泌体用PBS溶解后,通过0.22μm的针式滤器过滤除菌,并通过BCA蛋白定量试剂盒对外泌体进行定量以及分装,-80℃冻存备用。所有操作均在4℃下进行。将不同比例的HCPT和AQ4N分别溶解在14μl的二甲基亚砜和100μl的去离子水中,与膜蛋白含量为1mg的外泌体混合。在冰浴下用探头进行超声,超声条件是180W,工作2s,间隙4s,总工作时间为2min。超声完成后,将制得的共载有HCPT和AQ4N的外泌体(AH-Exos)放入37℃恒温培养箱里孵育1h,恢复外泌体膜的完整性。用马尔文激光粒度分析仪测定外泌体的粒径和电位。取1ml制剂加入孔隙大小为10kDa的超滤离心管离心,用紫外-可见分光光度计和荧光光谱仪测定AQ4N和HCPT的包封效率。计算公式如下:
制剂包封率(%)=(被包载的药物质量)/(总的药物质量)*100%
实验结果如图1所示,当AQ4N和HCPT的摩尔量比为2:1时具有最优的粒径和适中的包封效率,且在透射电镜下呈盘状囊泡形。
实施例2:仿生矿化外泌体的特性实验
在4℃下用磁力搅拌器分散实施例1中制得的共载有HCPT和AQ4N的外泌体(AH-Exos),逐滴加入含有0.2mg aTIM-4和1mg CaCl2的水溶液,待搅拌均匀后,加入等体积的含有1.28mg Na2HPO4的水溶液,继续搅拌2h,得到仿生矿化外泌体(AH-Exos@CPM),采用马尔文激光粒度分析仪,检测其粒径、电位,使用TEM透射电镜观察AH-Exos@CPM的形貌特征,如图2A、2B所示,纳米粒子的形貌由圆盘状变为核壳状,粒径和电位也发生了改变,初步证明了仿生矿化外泌体的成功制备。
接着,采用元素能谱分析对仿生矿化外泌体表面元素进行半定量分析,可以清晰的看到磷酸钙以及aTIM-4的特征元素Ca、P、O、S均匀的分散在粒子表面(图2C),同时通过SDS-PAGE法检测制剂表面的特征蛋白,发现AH-Exos和AH-Exos@CPM均表现出外泌体特征蛋白,同时矿化外泌体中还表现出aTIM-4的特征蛋白(图2D),用多功能酶标仪对AH-Exos@CPM进行光谱扫描,检测出其同时含有AQ4N和HCPT的特征峰(图2E)。利用ELISA试剂盒,测aTIM-4的制剂包封效率为87.3±7.3%
实施例3:对AH-Exos@CPM的体外释放实验
取1ml AH-Exos@CPM置于20ml不同pH的PBS中(pH=7.4和pH=6.5)孵育,并在37℃恒温水浴振荡器中模拟释放,在预定时间点(0,0.5,1,2,4,8,12,24h)取出混合释放液,在13,000g转速下,离心10min,取上清夜,分别用钙离子浓度测定仪和ELISA试剂盒监测其中的钙离子和aTIM-4的浓度。结果如图3所示,12h的时间内,在pH=6.5的环境下有63.7±3.4%的钙离子和33.5±0.9%的aTIM-4从AH-Exos@CPM中释放出来,而在pH=7.4的中性环境中仅有不到20%的钙离子和aTIM-4释放,证明了磷酸钙的肿瘤微环境响应性(图3A、3B)。
另取1ml AH-Exos@CPM置于20ml不同pH的PBS(pH 7.4,pH 6.5,pH 5.0)中,在预先设置好的时间点,取出混合释放液,离心,通过多功能酶标仪,测定溶液中AQ4N和HCPT的释放含量,发现其也表现出酸性环境下的响应释放(图3C、3D)。
实施例4:AH-Exos@CPM的稳定性检测
将AH-Exos@CPM与PBS(pH 7.4)(1:20,v/v)混合,置于37℃恒温振荡器中孵育,于0,2,4,6,8,12,18,24h使用动态光散射法测定粒径,以此评价其胶体稳定性。此外,还将其置于4℃中储存,考察7天内的粒径变化情况。结果如图4所示,仿生矿化的稳定性较好,粒径未发生较大变化。
实施例5:AH-Exos@CPM的体外摄取实验
将4T1细胞(1×105个细胞/孔)接种于12孔板中,置于37℃二氧化碳细胞培养箱内贴壁培养24h。弃去旧的培养液,加入含有AQ4N溶液、HCPT溶液、AH-Exos纳米粒、AH-Exos@CPM纳米粒(HCPT的量为10μM)的pH 7.4或者pH 6.5的培养基,置于37℃二氧化碳细胞培养箱中分别孵育6h和12h。孵育结束后,弃去含药培养液,加入冰冷的PBS(pH7.4)清洗3次,终止细胞摄取过程。细胞用4%多聚甲醛固定10min,并在37℃用Hoechst 33342对细胞核染色10min,取出爬片,置于滴有防淬灭封片液的载玻片上。将载玻片取出于共聚焦显微镜下观察细胞内的红色和绿色药物荧光。对于细胞摄取的流式定量分析,用24孔板进行培养,给药后,用0.05%胰酶将细胞消化下来,离心收集细胞,将其均匀分散于PBS(pH 7.4)后,用流式细胞仪定量分析细胞摄取情况。如图5所示,外泌体因其自身同源性更容易被肿瘤细胞摄取,表现出最高的摄取效率,AH-Exos@CPM的磷酸钙外壳在pH 6.5的条件下溶解,也暴露出外泌体内核,有着和AH-Exos相似的摄取特性。
实施例6:AH-Exos的细胞毒性实验
将4T1细胞(2000个细胞/孔)接种于96孔板中,置于37℃二氧化碳细胞培养箱内,在常氧条件下,贴壁培养12h,而后在常氧条件下或加入低氧产气袋的低氧培养盒中继续培养12h。弃去旧的培养液,加入系列梯度浓度(HCPT:20nM,50nM,100nM,200nM,500nM;AQ4N:40nM,100nM,200nM,400nM,1000nM)的含有AQ4N溶液、HCPT溶液、AH-Exos纳米粒、AH-Exos@CPM纳米粒的pH 7.4或pH 6.5培养基,继续于常氧条件下或加入低氧产气袋的低氧培养盒中孵育48h。每孔加入0.5mg/ml的MTT孵育4h,弃掉培养基后加入200μl二甲基亚砜,振荡10min,用多功能酶标仪测量490nm波长处的吸光度,如图6所示,AQ4N在常氧下几乎没有细胞毒性,在低氧下可以有效的杀死细胞,HCPT在常氧低氧下均有显著的细胞毒性,因此将HCPT与AQ4N联用,可以全面杀伤肿瘤细胞。
实施例7:凋亡小体的提取与表征
在4T1细胞与AH-Exos@CPM(HCPT的量为20μM)在pH 6.5的环境下孵育12h后,弃去旧的培养液,清洗细胞,更换为新的空白培养基,继续培养24h。将培养基收集起来,使用低温离心机,在1500g转速下离心20min收集凋亡小体(ApoBDs)。使用粒径仪分析凋亡小体的粒径大小以及zeta电位值,并使用透射电镜观察凋亡小体的形貌,在共聚焦显微镜下观察凋亡小体内部的药物荧光,如图7所示,凋亡小体中红色和绿色荧光重叠,表明凋亡小体可以作为药物递送载体,装载肿瘤细胞凋亡后胞内的剩余药物。将收集的凋亡小体加到培养有4T1细胞的20mm玻璃底培养皿中,孵育24h。加入冰冷的PBS(pH 7.4)清洗3次,并用4%多聚甲醛固定10min,然后用Hoechst 33342对细胞核染色10min,取出爬片,置于滴有防淬灭封片液的载玻片上,在显微镜下观察,可以明显观察到肿瘤细胞对凋亡小体的摄取。通过MTT法测定凋亡小体对肿瘤细胞的毒性,发现凋亡小体对肿瘤细胞仍有显著的杀伤作用。
实施例8:基于凋亡小体的邻位效应机制探究
如图8所示,将4T1细胞(4×105个细胞/孔)接种于6孔板(i)中,置于37℃二氧化碳细胞培养箱内贴壁培养24h。弃去旧的培养液,加入含有AQ4N溶液、HCPT溶液、AQ4N与HCPT混合溶液剂、AH-Exos纳米粒、AH-Exos@CPM纳米粒(HCPT的量为20μM)的pH 7.4或者pH6.5培养基,置于37℃二氧化碳细胞培养箱中孵育12h得到细胞(i-I)。孵育结束后,弃去含药培养基,用空白培养基继续孵育12h,让细胞充分凋亡,得到细胞(i-A)。孵育结束后,提取细胞清液中的凋亡小体,与6孔板(ii)的空白细胞共培养,重复上述过程,得到板(iii)上的细胞。将6孔板上的细胞(i-I、ii-I、iii-I)用4%多聚甲醛固定10min,并用Hoechst33342对细胞核进行染色,在共聚焦显微镜下观察细胞内的药物荧光。在AH-Exos和AH-Exos@CPM组中,板(ii)和板(iii)上的细胞均可以观察到明显的荧光,且代表AQ4N的红色荧光强度相对于代表HCPT的绿色荧光越来越强,呈现出程序性递送现象,而在AQ4N溶液组、HCPT溶液组、AQ4N与HCPT混合溶液组的板ii和板iii中几乎观察不到荧光存在。将AH-Exos@CPM纳米粒诱导产生的凋亡小体收集,均匀分散于PBS(pH 7.4)中,通过5次冻融循环破碎细胞,使用孔隙为3kDa的超滤离心管将游离型药物分离,通过高效液相色谱和多功能酶标仪测定AQ4N和HCPT的游离型药物量在总药物中所占比值,我们发现游离型的HCPT所占比例越来越少,而游离AQ4N所占比例基本不变,AQ4N相对于HCPT的量随着递送过程不断升高。另外收集AH-Exos@CPM组中处于i-I以及i-A阶段的细胞,破碎细胞后用HPLC检测胞内AQ4N以及AQ4N的还原细胞毒性药物AQ4的含量,并利用western blot检测两种细胞内凋亡标志蛋白γH2AX和cleaved caspase-3的含量,观察到凋亡细胞内AQ4N仍维持自身状态并未转化为毒性药物AQ4,引起细胞凋亡并产生凋亡小体并促进药物渗透的是不断消耗的HCPT。
实施例9:抑制胞葬提升凋亡小体渗透能力考察
从小鼠中提取骨髓源的巨噬细胞,用20ng/ml的小鼠M-CSF,在37℃培养箱中培养7天,得到成熟的骨髓源巨噬细胞(BMDMs)。用白细胞介素-4极化细胞,得到M2型的巨噬细胞(M2Φ),利用western blot检测细胞膜蛋白aTIM-4的分布。加入AH-Exos诱导的凋亡小体、AH-Exos诱导的凋亡小体与aTIM-4的混合溶液,共同培养4h,用PBS吹下细胞后,收集细胞,用流式细胞仪进行荧光定量分析,如图9所示,巨噬细胞对凋亡小体有着很强的摄取能力,但是这种摄取可以被aTIM-4阻断。将1×104个4T1细胞与5×103个BMDM细胞分散到15μl的细胞培养基中,接种于含有琼脂糖的96孔板中每2天换新的培养液,第6天加入含有AQ4N溶液、HCPT溶液、AQ4N与HCPT混合溶液剂、AH-Exos纳米粒、AH-Exos@CPM纳米粒的pH 7.4和pH 6.5培养液,置于37℃二氧化碳细胞培养箱中孵育24h。而后弃去旧的培养液,清洗细胞球,于共聚焦显微镜下通过Z轴扫描,以40μm扫描间隔观察药物渗透情况,因为aTIM-4对巨噬细胞胞葬的抑制作用,AH-Exos@CPM有着最远距离的渗透,并且有着明显的程序性渗透。对于肿瘤球生长抑制实验,细胞球每2天更换一次含上述药物的培养基,在第6天,通过光学显微镜拍照记录,并计算肿瘤球体积,同样的,具有最强渗透能力的仿生矿化外泌体有着最强的肿瘤抑制作用。
实施例10:AH-Exos@CPM的药动学研究
将雄性SD大鼠(220-250g)随机分组,通过尾静脉注射给予HCPT溶液、AH-Exos纳米粒、AH-Exos@CPM纳米粒,HCPT等效给药剂量为2mg/kg。在预定的时间点(0.03,0.083,0.25,0.5,1,2,4,8,12,24h)进行大鼠眼底静脉丛采血置于肝素化的EP管内,将血样离心获得血浆。离心、沉淀蛋白后取上清液加入到96孔黑板中,采用多功能酶标仪测定HCPT的血浆浓度,如图10所示,HCPT溶液剂在血液中被快速清除,而外泌体包载的或者经过矿化的仿生外泌体可以显著延长HCPT在血液中的半衰期,这是由于矿化的坚硬的磷酸钙外壳可以有效的阻挡血液的剪切力,维持纳米粒子的稳定。
实施例11:AH-Exos@CPM的组织分布探究
将生长状态良好的4T1细胞经0.05%胰酶消化后,重新均匀分散于PBS(pH 7.4)中,浓度为5×107个/ml。取100μl上述细胞悬液注射于雌性BALB/c小鼠右后侧腰背部皮下。待小鼠乳腺癌(4T1)异位瘤小鼠模型的肿瘤体积长至约300mm3时,通过尾静脉注射给予AQ4N溶液、AH-Exos纳米粒、AH-Exos@CPM纳米粒,AQ4N等效给药剂量为8mg/kg。在2、4、8、12和24h静脉注射后,将小鼠麻醉并使用小动物成像系统进行活体成像。注射24h后处死小鼠,收集心、肝、脾、肺、肾、肿瘤等器官,进行离体组织荧光成像,如图11所示,溶液剂AQ4N迅速被排除体内,且没有在肿瘤部位蓄积,而外泌体包载的或者经过矿化的仿生外泌体制剂可以特异性的长时间的蓄积在肿瘤部位,这与纳米制剂的药动结果相一致。
实施例12:AH-Exos@CPM的体内渗透检测
将生长状态良好、处于对数期的4T1肿瘤细胞用0.05%胰酶消化后,用新鲜的RPMI1640细胞培养液终止消化,离心并收集细胞,用PBS(pH 7.4)均匀分散成浓度为2×107cells/mL,置于冰盒中。取100μl混悬均匀的4T1细胞接种于小鼠右后侧腰背部皮下。当瘤体积长至500mm3左右时,通过尾静脉注射AQ4N溶液、HCPT溶液、AQ4N与HCPT的混合溶液、AH-Exos纳米粒、AH-Exos@CPM纳米粒,HCPT的等效给药剂量为2.8mg/kg,48h后,将小鼠处死,收集肿瘤,制作肿瘤冷冻切片,用DAPI对细胞核进行染色,通过共聚焦显微镜观察药物荧光,如图12所示,AH-Exos@CPM表现出最强的肿瘤渗透能力,这是由于纳米粒较强的肿瘤富集能力,以及aTIM-4对胞葬的抑制进一步放大了凋亡小体的渗透效率。
实施例13:AH-Exos@CPM的药效学检测
建立Balb/c小鼠(4-5周周龄,平均体重18-22g)皮下异位乳腺癌荷瘤模型,待荷瘤鼠身上的肿瘤体积长至100mm3左右时记为第0天,将其随机分成6组,每组5只。在第0,2,4,6,8天经尾尾静脉注射生理盐水、AQ4N溶液、HCPT溶液、AQ4N与HCPT的混合溶液、AH-Exos纳米粒、AH-Exos@CPM纳米粒,HCPT等效给药剂量为2.8mg/kg。开始给药后,每天测量荷瘤鼠的肿瘤大小和体重,绘制体重随时间的变化曲线,评价给药后荷瘤鼠的体重变化。在第12天处死小鼠,摘掉小鼠眼球取血,离心、分离获得血清,检测血清中天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、血尿素氮(BUN)和肌酸酐(CREA)的量,作为评价肝肾功能的指标。将所有荷瘤鼠处死后,获得心、肝、脾、肺、肾用4%多聚甲醛固定,用H&E染色评价组织形态学。同时剥离肿瘤组织,拍照并对其称重,将称重后的肿瘤组织用4%多聚甲醛进行固定,并进行肿瘤组织切片。根据试剂盒公司提供的切片染色操作指南,将石蜡切片用二甲苯和系列浓度的乙醇溶液进行脱蜡,使用TUNEL凋亡检测试剂盒和Ki67细胞增殖检测试剂盒对肿瘤组织切片进行染色,并用共聚焦显微镜进行拍照。结果如图13所示,注射溶液剂的小鼠的肿瘤增长迅速,仅次于注射生理盐水的小鼠,这是由溶液剂在体内的快速清除导致的。因为快速的肿瘤部位蓄积,以及最强的肿瘤渗透能力,仿生矿化外泌体产生了最好的肿瘤治疗效果,几乎完全抑制了肿瘤的生长,另外H&E染色、Ki 67染色、以及TUNEL染色的结果表明,AH-Exos@CPM可以引起广泛的细胞凋亡、抑制了细胞的增殖,但是具有良好的安全性。除HCPT溶液剂、AQ4N和HCPT混合溶液剂处理的小鼠体重有所波动,其他组别的小鼠体重均没有明显的减轻,所有组别小鼠的肝肾功能和主要器官的H&E染色均没有明显的异常。
Claims (10)
1.一种抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体,其特征在于,通过将肿瘤不同空间位置起效的两种小分子化疗药物共载于仿生外泌体中,并在仿生外泌体表面通过生物矿化的方式,覆盖载有抑制胞葬的单克隆抗体的矿化外壳制得。
2.根据权利要求1所述的抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体,其特征在于,所述的抑制胞葬的单克隆抗体是结合巨噬细胞表面TIM-4受体的单克隆抗体aTIM-4;所述的肿瘤不同空间位置起效的两种小分子化疗药物是指疏水的、对于全部肿瘤区域有细胞毒性的小分子药物,以及亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药;所述的仿生外泌体是指与肿瘤细胞同源的外泌体;所述的矿化外壳是指由无机离子构成的在肿瘤酸性微环境中降解的坚硬外壳。
3.根据权利要求2所述的抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体,其特征在于,所述的疏水的、对于全部肿瘤区域有细胞毒性的小分子药物是指10-羟基喜树碱;所述的亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药是指巴诺蒽醌;所述的仿生外泌体是指4T1小鼠乳腺癌细胞分泌的外泌体;所述的矿化外壳是指磷酸钙外壳。
4.根据权利要求1或2所述的抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体,其特征在于,所述抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体的各组分按重量百分比含量为:矿化外壳占44%-56%,仿生外泌体占24%-38%,亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药占7%-10%,疏水的、对于全部肿瘤区域有细胞毒性的小分子药物占1%-3%,aTIM-4占3%-5%。
5.根据权利要求3所述的抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体,其特征在于,所述的磷酸钙由氯化钙和磷酸氢二钠制得,按重量比为氯化钙:磷酸氢二钠=(1-4):(2-6)。
6.根据权利要求4所述的抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体,其特征在于,所述抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体的各组分按重量比为:矿化外壳:仿生外泌体:亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药:疏水的、对于全部肿瘤区域有细胞毒性的小分子药物:aTIM-4=2.28mg:1mg:0.4mg:0.14mg:0.2mg。
7.一种权利要求1或2所述的抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体的制备方法,其特征在于,包括以下步骤:
收集培养小鼠癌细胞的无血清细胞培养液,通过多次离心先后除去肿瘤细胞、死细胞和细胞碎片,使用含有蛋白酶抑制剂的PBS重悬后,通过超速离心去除上清液,得到空白外泌体;将疏水的、对于全部肿瘤区域有细胞毒性的小分子药物和亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药分别溶解在有机溶剂和去离子水中,与制得的空白外泌体混合;在冰浴下进行超声,超声完成后,将制得的共载有疏水的、对于全部肿瘤区域有细胞毒性的小分子药物和亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药的外泌体放入恒温培养箱里孵育,恢复外泌体膜的完整性,然后,搅拌分散分泌体,逐滴加入含有抑制胞葬的单克隆抗体aTIM-4和CaCl2的水溶液,待搅拌均匀后,加入Na2HPO4的水溶液,继续搅拌,得到仿生矿化外泌体。
8.根据权利要求7所述的抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体的制备方法,其特征在于,所述疏水的、对于全部肿瘤区域有细胞毒性的小分子药物是指10-羟基喜树碱;亲水的、只对肿瘤内部缺氧部位有细胞毒性的低氧激活前药是指巴诺蒽醌;所述有机溶剂为甲醇、无水乙醇、二甲基亚砜中的一种;所述在冰浴下进行超声,其超声功率为100W-200W。
9.根据权利要求8所述的抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体的制备方法,其特征在于,所述有机溶剂为二甲基亚砜;所述在冰浴下进行超声,其超声功率为180W。
10.一种权利要求1或2所述抑制胞葬的肿瘤程序性药物渗透仿生矿化外泌体在注射给药、口服给药或局部给药系统中的应用。
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