CN113577275A - 一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备 - Google Patents
一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备 Download PDFInfo
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Abstract
本发明公开了一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备方法,其特征在于:所述给药载体为Asp8[H40‑TPZ/IR780@(RBC‑H)]NPs,制备流程如下:通过水包油乳液溶剂扩散法制备H40‑PEG负载的TPZ/IR780纳米粒子;将RBC细胞膜和WSU‑HN6细胞膜混合得到RBC‑H混合膜溶液;通过挤压法将RBC‑H融合的混合膜涂覆在H40‑TPZ/IR780NPs上;使用天冬氨酸低聚肽Asp8对混合膜进行修饰。该用于骨破坏癌症的双靶向纳米仿生给药载体的制备方法,制得双靶向仿生纳米颗粒Asp8[H40‑TPZ/IR780@(RBC‑H)],集成了骨靶向、肿瘤定位和免疫逃逸能力,可以作为一个高效的多靶点药物递送平台,在骨破坏的位置实现抗癌精确治疗。
Description
技术领域
本发明涉及抗癌药物相关技术领域,具体为一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备。
背景技术
颌骨因其特殊的解剖关系,是口腔恶性肿瘤最常破坏的器官,骨破坏已成为口腔鳞状细胞癌(OSCC)最常见的并发症之一,骨破坏极有可能引起OSCC的复发,一系列严重的并发症,如病理性骨折、骨痛等,会大大降低患者的生活质量,甚至影响生存,虽然手术和化疗经常被用来治疗OSCC的骨破坏,但普通化疗药物由于缺乏肿瘤特异性靶向性和骨组织的渗透性差,化疗在临床上的应用一直受到限制,因此,开发用于骨癌双靶向给药平台是非常必要的,一种既能针对骨的破坏性微环境又能针对OSCC细胞的联合治疗方法,将比单一的治疗方案提供更好的治疗效果。
生物医学纳米粒子的快速发展使得在肿瘤组织内靶向传递、持续和控制释放治疗性化合物成为可能,因此,纳米颗粒作为药物输送载体在癌症诊断和治疗领域受到广泛关注,尽管纳米颗粒已被广泛用于设计各种载体以提高疗效,但纳米医学治疗平台与临床治疗的目标之间仍有一段距离,其仍存在一些问题,需要提高靶向能力,如对应提高口腔鳞状细胞癌对应同源靶向,提高骨病变部位的骨靶向能力,需要逃避免疫识别和清除延长循环时间等,因此,有必要引入更多的功能来加强对骨病变部位的抗癌治疗。
针对上述问题,在原抗癌药物给药载体的基础上进行创新设计。
发明内容
本发明的目的在于提供一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备,以解决上述背景技术中提出普通骨病变抗癌药物靶向能力不足,纳米级药物载体在体内的循环寿命较短的问题。
为实现上述目的,本发明提供如下技术方案:一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备,所述给药载体为Asp8[H40-TPZ/IR780@(RBC-H)]NPs;
其中Asp8为天冬氨酸;
H40-TPZ/IR780为聚酯H40-聚乙二醇(H40-PEG)负载的抗癌药物提拉帕胺TPZ和光热剂IR780碘化物纳米颗粒;
RBC-H为头颈部鳞状细胞癌WSU-HN6细胞(H)和红细胞(RBC)混合膜。
制备流程如下:
a、通过水包油乳液溶剂扩散法制备H40-PEG负载的TPZ/IR780纳米粒子;
b、将RBC细胞膜和WSU-HN6细胞膜混合得到RBC-H混合膜溶液;
c、通过挤压法将RBC-H融合的混合膜涂覆在H40-TPZ/IR780NPs上;
d、使用天冬氨酸低聚肽Asp8对混合膜进行修饰。
采用上述技术方案,通过内芯纳米颗粒负载药物,混合膜提高免疫逃逸性和癌症靶向能力,配合Asp8修饰提高骨质靶向能力,拿到双靶向仿生纳米颗粒的效果,有助于骨质破坏病灶癌细胞的生长抑制。
优选的,所述流程a具体为将H40-PEG和IR780溶解在的氯仿中,将TPZ溶解在去离子水中;
两种溶液混合后,在超声处理下进行自组装,在冰浴中超声处理1小时后,成功合成H40-TPZ/IR780NPs,通过离心法收集棕色沉淀物;
接下来,用去离子水清洗3次NPs纳米颗粒,以便进一步分析;
整个流程a反应过程是避光的。
采用上述技术方案,通过H40-PEG对IR780和TPZ进行包裹,实现两种药物的共同装载。
优选的,所述流程b具体为将RBC细胞膜和WSU-HN6细胞膜混合,混合物在冰浴中超声处理后静置。
采用上述技术方案,通过混合膜处理使其重视具有RBC细胞和WSU-HN6细胞的特性,有助于药物逃脱免疫同时便于靶向引导直达病灶。
优选的,所述流程c具体为将RBC-H混合膜溶液加入到H40-PEGNPs或H40-TPZ/IR780NPs溶液中,然后将混合物通过聚碳酸酯多孔膜挤出,形成尺寸小于200nm的混合膜包覆NPs。
最后,通过离心得到仿生NPs,用PBS缓冲液洗涤,并重新悬浮在缓冲液中。
采用上述技术方案,通过混合膜包覆挤出,除了使纳米载体具有混合细胞特性的同时,纳米级挤出处理可保持载体的小尺寸结构避免被RES识别和清除,还能增强对肿瘤组织的被动靶向性。
优选的,所述TPZ和IR780为别为模型中的抗肿瘤和光热治疗药物,且TPZ和IR780分别为亲水性药物和疏水性药物,通过水包油乳液溶剂扩散技术共同装载到纳米腔内。
采用上述技术方案,使用TPZ和IR780作为抗肿瘤和光热治疗药物,通过其纳米腔载体的Asp8提供良好的骨质靶向性配合外部激光照射治疗可便于提高对骨质破坏病灶处的准确治疗效果。
优选的,所述RBC细胞膜的获取流程为:
将样本血液在离心处理,以去除血清;
收集到的红细胞沉淀物用冷的磷酸盐缓冲盐水清洗,以去除残留的浆液;
之后,为了低渗裂解红细胞,在试管中加入含有PBS稀释液,重悬红细胞;
随后,将裂解液离心处理,仔细去除上清液;
在冷PBS中经过重复三次洗涤-离心循环,得到粉红色沉淀的红细胞膜碎片;
采用上述技术方案,获取红细胞膜用于提供给纳米颗粒包覆混合膜免疫逃逸特性,减少巨噬细胞对纳米颗粒的细胞摄取。
优选的,所述WSU-HN6细胞膜的获取流程为:
首先将WSU-HN6细胞放在细胞培养皿中,含有完整的DMEM,在37℃和含有5%CO2的湿润环境中培养;
48小时后,用细胞橡胶刮刀轻轻地收集细胞,用冷PBS洗涤两次后,离心收集癌细胞。用含有1mMPMSF的膜蛋白提取试剂来重悬收获的癌细胞;
随后,将悬浮液在冰浴中冷却后,在冰浴中超声处理;
之后,离心以去除未破碎的细胞或细胞碎片;
然后,小心地移出上层,再以14000×g的速度进一步离心处理;
最后,将得到的WSU-HN6细胞膜碎片保存在-80℃,以便进一步利用。
采用上述技术方案,获取癌症细胞膜片段用于提供给纳米颗粒包覆混合膜对应病灶的肿瘤定位能力。
与现有技术相比,本发明的有益效果是:该用于骨破坏癌症的双靶向纳米仿生给药载体的制备,
1、通过融合红细胞和WSU-HN6细胞膜构建RBC-H混合外壳的方式,伪装到TPZ和IR780负载的超支化聚合物纳米粒子(Asp8[H40-TPZ/IR780@(RBC-H)]NPs)上,用于治疗骨破坏部位,对仿生NPs进行了大幅度的表征,同时继承了红细胞和WSU-HN6细胞的特征功能,利用红细胞膜的蛋白标志物抑制巨噬细胞的吞噬作用,明显提高了免疫逃逸能力,WSU-HN6细胞膜涂层可以增强OSCC细胞的同源靶向能力,突出的同质靶向癌和免疫回避能力使Asp8[H40-TPZ/IR780@(RBC-H)]OSCC的化疗表现出色;
2、通过纳米级聚碳酸酯多孔膜挤出,形成尺寸小于200nm的混合膜包覆纳米颗粒,加上纳米颗粒外修饰的Asp8,丰富的天冬氨酸组成的骨钙素和骨蓬素对骨组织有极强的结合能力,通过Asp8装饰的内部的纳米颗粒同样被赋予了骨靶向能力,可以精确地将抗癌药物递送到骨病灶部位,小尺寸也是避免被免疫识别和清除的另一个重要原因,此外,它还能增强对肿瘤组织的被动靶向性,集成了骨靶向、肿瘤定位和免疫逃逸能力,可以作为一个高效的多靶点药物递送平台,在骨破坏的位置实现抗癌精确治疗。
附图说明
图1为本发明制备流程示意图;
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
请参阅图1,本发明提供一种技术方案:一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备,给药载体为Asp8[H40-TPZ/IR780@(RBC-H)]NPs;
其中Asp8为天冬氨酸;
H40-TPZ/IR780为聚酯H40-聚乙二醇(H40-PEG)负载的抗癌药物提拉帕胺TPZ和光热剂IR780碘化物纳米颗粒;
RBC-H为头颈部鳞状细胞癌WSU-HN6细胞(H)和红细胞(RBC)混合膜。
制备流程如下:
a、通过水包油乳液溶剂扩散法制备H40-PEG负载的TPZ/IR780纳米粒子;
选择TPZ和IR780作为抗肿瘤和光热治疗药物模型,疏水性药物(IR780)和亲水性药物(TPZ)可以通过水包油乳液溶剂扩散技术共同装载到纳米腔内;
将H40-PEG(25mg)和IR780(2.5mg)溶解在5mL的氯仿中,将TPZ溶解在5mL的去离子水中;
两种溶液混合后,在超声处理下进行自组装。在冰浴中超声处理1小时后,成功合成了H40-TPZ/IR780NPs。通过离心法收集棕色沉淀物;
接下来,用去离子水清洗3次NPs,以便进一步分析;
整个反应过程是避光的;
通过H40-PEG对IR780和TPZ进行包裹,实现两种药物的共同装载。
b、将RBC细胞膜和WSU-HN6细胞膜混合得到RBC-H混合膜溶液;
RBC细胞膜和WSU-HN6细胞膜以1:1的膜重量比混合,混合物在60W的功率水平下超声处理5分钟,在冰浴中超声处理1分钟,静置2分钟;
通过混合膜处理使其重视具有RBC细胞和WSU-HN6细胞的特性,有助于药物逃脱免疫同时便于靶向引导直达病灶。
c、通过挤压法将RBC-H融合的混合膜涂覆在H40-TPZ/IR780NPs上;
将超声处理过的RBC-H混合膜溶液加入到H40-PEGNPs或H40-TPZ/IR780NPs溶液中,然后将混合物依次通过1μm、400nm和200nm的聚碳酸酯多孔膜挤出,形成尺寸小于200nm的混合膜包覆NPs;
最后,通过离心(10000rpm,5min,4℃)得到仿生NPs,用PBS(0.01M,pH=7.4)洗涤三次,并重新悬浮在缓冲液中;
通过混合膜包覆挤出,除了使纳米载体具有混合细胞特性的同时,纳米级挤出处理可保持载体的小尺寸结构避免被RES识别和清除,还能增强对肿瘤组织的被动靶向性。
d、使用天冬氨酸低聚肽Asp8对混合膜进行修饰。
通过内芯纳米颗粒负载药物,混合膜提高免疫逃逸性和癌症靶向能力,配合Asp8修饰提高骨质靶向能力,拿到双靶向仿生纳米颗粒的效果,有助于骨质破坏病灶癌细胞的生长抑制。
红血球细胞膜碎片的制备:
血液在4℃下以3000rpm离心5分钟,以去除血清。
收集到的红细胞沉淀物用冷的磷酸盐缓冲盐水(PBS0.01M,pH=7.4)清洗三次,以去除残留的浆液。
之后,为了低渗裂解红细胞,在试管中加入含有0.2mMEDTA的1/4×PBS(pH=7.4)稀释液,在4℃下重悬红细胞1小时。
随后,将裂解液在4℃下以1.3×104r/min的速度离心5分钟,仔细去除上清液。
在冷PBS中经过三次洗涤-离心循环,得到粉红色沉淀的红细胞膜碎片。得到的红细胞膜保存在-80℃以备进一步使用
获取红细胞膜用于提供给纳米颗粒包覆混合膜免疫逃逸特性,减少巨噬细胞对纳米颗粒的细胞摄取。
制备癌症细胞膜片段:
首先将WSU-HN6细胞放在直径为15cm的细胞培养皿中,含有完整的DMEM(10%(v/v)FBS,1%青霉素和链霉素),在37℃和含有5%CO2的湿润环境中培养。
48小时后,用细胞橡胶刮刀轻轻地收集细胞。在4℃下用冷PBS洗涤两次后,在4℃下以600×g离心5分钟收集癌细胞。用含有1mMPMSF的膜蛋白提取试剂A来重悬收获的癌细胞。
随后,将悬浮液在冰浴中冷却30分钟。30分钟后,在冰浴中超声处理1分钟(脉冲:开启时间2秒,关闭时间3秒)(功率:13W)。
之后,在4℃下以700×g离心10分钟,以去除未破碎的细胞或细胞碎片。
然后,小心地移出上层,在4℃下以14000×g的速度进一步离心0.5小时。
最后,将得到的WSU-HN6细胞膜碎片保存在-80℃,以便进一步利用。
获取癌症细胞膜片段用于提供给纳米颗粒包覆混合膜对应病灶的肿瘤定位能力。
为了评估各种TPZ/IR780负载的纳米颗粒在骨破坏模型中的抗癌效果。本研究选择右下颌骨破坏的小鼠模型作为体内的实验对象。
将总共2.0×106个WSU-HN6细胞注射到雌性BALB/c裸鼠(4-6周龄)的右下颌骨区域。
1周后,肿瘤形成率为100%,然后,将所有小鼠随机分为7组(每组5只),包括:
(1)正常盐水;
(2)正常盐水+激光;
(3)TPZ+激光;
(4)Asp8[H40-PEG@(RBC-H)]NPs+激光;
(5)H40-PEG加载IR780NPs+激光;
(6)[H40-IR780/TPZ@(RBC-H)]NPs+激光;
(7)Asp8[H40-IR780/TPZ@(RBC-H)]NPs+激光。
这些组的所有小鼠都通过尾部静脉注射100μLPBS或各种制剂,TPZ的剂量为1.5mg/Kg,IR780的剂量为1.6mg/Kg。其中,PBS组的小鼠作为阴性对照组。
给药的第一天被指定为第0天。并每天测量小鼠的肿瘤体积和体重。24小时后,激光组的小鼠被808nm波长的激光照射(1.0W/cm2,5分钟)。此外,在第2、4、6、8、10、12、14、16天重复治疗和激光照射。
在骨破坏模型建立成功后,与正常生理盐水组和正常生理盐水加激光组(对照组)相比,TPZ和Asp8[H40-PEG@(RBC-H)]在激光照射下都没有表现出明显的抗癌效果,H40-TPZ/IR780@(RBC-H)]的癌细胞生长抑制效率优于H40-PEG@(RBC-H)]。NPs在激光照射下比H40-PEG加载IR780NPs的效果更好。这表明H40-PEG负载的IR780裸纳米粒子可以被RES迅速清除,与上述所有组别相比,Asp8[H40-TPZ/IR780@(RBC-H)]NPs在激光照射下显示出最出色的肿瘤生长抑制效果。而这要归功于混合细胞膜涂层和仿生NPs的Asp8骨靶向配体,Asp8[H40-TPZ/IR780@(RBC-H)]与激光照射组的平均肿瘤重量比对照组轻约3倍。此外,双靶向生物仿生NPs出色的致癌治疗效率也证明了TPZ的化疗效果可以通过基于IR780的光热治疗得到协同增强。因此,上述结果表明Asp8[H40-TPZ/IR780@(RBC-H)]NPs可以精确定位骨破坏部位以发挥抗癌作用。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (7)
1.一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备方法,其特征在于:所述给药载体为Asp8[H40-TPZ/IR780@(RBC-H)]NPs;
其中Asp8为天冬氨酸;
H40-TPZ/IR780为聚酯H40-聚乙二醇(H40-PEG)负载的抗癌药物提拉帕胺TPZ和光热剂IR780碘化物纳米颗粒;
RBC-H为头颈部鳞状细胞癌WSU-HN6细胞(H)和红细胞(RBC)混合膜。
制备流程如下:
a、通过水包油乳液溶剂扩散法制备H40-PEG负载的TPZ/IR780纳米粒子;
b、将RBC细胞膜和WSU-HN6细胞膜混合得到RBC-H混合膜溶液;
c、通过挤压法将RBC-H融合的混合膜涂覆在H40-TPZ/IR780NPs上;
d、使用天冬氨酸低聚肽Asp8对混合膜进行修饰。
2.根据权利要求1所述的一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备方法,其特征在于:所述流程a具体为将H40-PEG和IR780溶解在的氯仿中,将TPZ溶解在去离子水中;
两种溶液混合后,在超声处理下进行自组装,在冰浴中超声处理1小时后,成功合成H40-TPZ/IR780NPs,通过离心法收集棕色沉淀物;
接下来,用去离子水清洗3次NPs纳米颗粒,以便进一步分析;
整个流程a反应过程是避光的。
3.根据权利要求1所述的一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备方法,其特征在于:所述流程b具体为将RBC细胞膜和WSU-HN6细胞膜混合,混合物在冰浴中超声处理后静置。
4.根据权利要求1所述的一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备方法,其特征在于:所述流程c具体为将RBC-H混合膜溶液加入到H40-PEGNPs或H40-TPZ/IR780NPs溶液中,然后将混合物通过聚碳酸酯多孔膜挤出,形成尺寸小于200nm的混合膜包覆NPs。
最后,通过离心得到仿生NPs,用PBS缓冲液洗涤,并重新悬浮在缓冲液中。
5.根据权利要求2所述的一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备方法,其特征在于:所述TPZ和IR780为别为模型中的抗肿瘤和光热治疗药物,且TPZ和IR780分别为亲水性药物和疏水性药物,通过水包油乳液溶剂扩散技术共同装载到纳米腔内。
6.根据权利要求3所述的一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备方法,其特征在于:所述RBC细胞膜的获取流程为:
将样本血液在离心处理,以去除血清;
收集到的红细胞沉淀物用冷的磷酸盐缓冲盐水清洗,以去除残留的浆液;
之后,为了低渗裂解红细胞,在试管中加入含有PBS稀释液,重悬红细胞;
随后,将裂解液离心处理,仔细去除上清液;
在冷PBS中经过重复三次洗涤-离心循环,得到粉红色沉淀的红细胞膜碎片。
7.根据权利要求3所述的一种用于骨破坏癌症的双靶向纳米仿生给药载体的制备方法,其特征在于:所述WSU-HN6细胞膜的获取流程为:
首先将WSU-HN6细胞放在细胞培养皿中,含有完整的DMEM,在37℃和含有5%CO2的湿润环境中培养;
48小时后,用细胞橡胶刮刀轻轻地收集细胞,用冷PBS洗涤两次后,离心收集癌细胞。用含有1mMPMSF的膜蛋白提取试剂来重悬收获的癌细胞;
随后,将悬浮液在冰浴中冷却后,在冰浴中超声处理;
之后,离心以去除未破碎的细胞或细胞碎片;
然后,小心地移出上层,再以14000×g的速度进一步离心处理;
最后,将得到的WSU-HN6细胞膜碎片保存在-80℃,以便进一步利用。
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