CN115120573B - 含肼屈嗪的高效抗肿瘤纳米载药体系及其制备方法 - Google Patents
含肼屈嗪的高效抗肿瘤纳米载药体系及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种含肼屈嗪的高效抗肿瘤纳米载药体系(H‑NPs),所述载药体系是以通过ππ键结合后的肼屈嗪与阿霉素作为疏水核心,以葡聚糖作为亲水外壳。本发明的含肼屈嗪的高效抗肿瘤纳米载药体系可通过扩张肿瘤血管和调节TME,从而增加肿瘤部位的血管通透性、血液灌注、改善纳米颗粒在肿瘤内的富集和渗透、减少肿瘤乏氧,增加肿瘤对化疗药物阿霉素的敏感度,显著提高其抗肿瘤效果。
Description
技术领域
本发明属于生物医用材料技术领域,特别涉及一种pH响应性和靶向肿瘤的含肼屈嗪的高效抗肿瘤纳米载药体系。
背景技术
据《2017中国肿瘤登记年报》报道癌症已成为我国居民的第一致死病因。目前,各种新型肿瘤治疗手段虽已兴起很多年,但基于小分子药物(阿霉素、喜树碱等)的化疗仍然是临床上治疗肿瘤的主要手段。这些小分子药物都存在着水溶性或结构稳定性差、药代动力学和生物分布不理想等问题,造成有效药物在肿瘤组织的富集浓度低,同时大部分药物分布到患者健康组织器官中并对其造成严重的毒副作用,这降低了患者的生存质量和免疫能力,从而限制了药物的临床使用剂量、治疗的连续性,最终导致了化疗对患者总体上毒副作用太大、无法达到预期的治疗效果的现状。
纳米粒子递送药物系统为癌症治疗带来了巨大的希望。由于肿瘤部位增强的通透性和滞留效应(EPR),基于纳米粒子递送药物的化疗可以大大延长药物分子在人体的血液循环时间,从而增强纳米粒子在肿瘤部位的滞留和富集,另外纳米粒子很难从致密的正常毛细血管中渗出,减少了药物在正常组织中的分布,并大大降低了化疗药物脱靶造成的全身毒性。与传统小分子化疗药物相比,这些优势可以减少药物带给患者的副作用,提高癌症患者的药物耐受剂量。例如,盐酸阿霉素脂质体-DOXIL降低了阿霉素在患者心脏中的富集从而避免了阿霉素对心脏产生的致命毒性。近年来,盐酸阿霉素脂质体-DOXIL、紫杉醇-白蛋白纳米粒ABRAXANE等纳米制剂已在临床应用,另有很多个高分子纳米药物正处在临床试验的各个阶段。但是与游离原药相比,临床研究结果表明目前临床应用的纳米药物大部分仅仅能够降低药物带给患者的毒副作用,仅有为数不多的纳米粒子递送药物系统表现出很好治疗肿瘤效果。这也导致许多纳米药物在临床试验中最终的失败。因此,进一步设计和制备出提高治疗肿瘤效果的纳米药物是目前药物输送领域亟待解决的问题。
通过进一步的临床研究发现,其主要原因是:肿瘤靶向纳米药物的体内输送过程是一个从血液循环系统(Circulation)向肿瘤组织内蓄积(Accumulation)、渗透(Penetration)、细胞内吞(Internalization)、胞内药物释放(Release)的五步"CAPIR级联"过程。每步都有高的效率是纳米药物获得高的治疗肿瘤疗效的关键。国内外学者在长循环 C 、提高细胞内吞 I 与胞内释放 R 等方面做了大量工作,但纳米药物在肿瘤部位的富集 A 和肿瘤组织内的渗透 P 仍是瓶颈。这是因为:1)曲折的脉管系统是肿瘤微环境(TME)的特点,是纳米颗粒递送的主要障碍。肿瘤部位曲折的脉管系统使肿瘤部位血管变得狭窄,血流量减少,减少了纳米颗粒在肿瘤部位的富集。2)此外,升高的间质液压力(IFP)和异常的细胞外基质(ECM)形成了第二道物理屏障。因为纳米药物的尺寸在几个纳米至100多纳米之间,扩散速率与尺寸成反比,所以这种大尺寸的纳米药物的扩散能力远远小于小分子,进一步削弱了纳米颗粒在肿瘤部位的渗透,导致纳米颗粒仅在肿瘤脉管系统周围区域积累。因此,纳米颗粒通常无法到达远离血管系统的深层肿瘤细胞。3)肿瘤中间质细胞、细胞间质基质和成纤维细胞(TAFs)形成了第三道物理屏障。在大多数肿瘤中,细胞间质基质屏障限制了大分子药物和纳米颗粒在肿瘤内的扩散,具有细胞间质增生的肿瘤通常具备含有大量的间质细胞的特征,其分泌的纤维连接蛋白和胶原蛋白会导致高的、渗透压力。与此同时,间质细胞也是纳米颗粒间质穿透的障碍。肿瘤相关TAFs作为基质细胞的主要组成部分,是肿瘤中最丰富的细胞,尤其是在胰腺癌和乳腺癌中。TAFs通过重塑ECM、分泌生长因子和细胞因子以及抑制免疫反应来促进肿瘤的生长、侵袭和转移。
因此,提高纳米药物在肿瘤部位的大量富集和在肿瘤组织中的渗透能力,使其很好地完成整个CAPIR五步输送过程是提高纳米药物抗肿瘤疗效的关键。针对这些问题,科学家们进行了大量的探索工作。例如,血管紧张素抑制剂氯沙坦被报道可减少间质胶原,从而改善肿瘤内部的氧渗透和增加纳米颗粒药物的输送。异常TME能帮助癌细胞逃避免疫系统,并增加其侵袭和转移潜能,而血管重构策略可以抑制血液成分外渗从而降低肿瘤组织间液压力,促进了纳米颗粒在肿瘤部位的输送。提出使用血管生成抑制剂可以使肿瘤血管系统正常化,改善血液灌注,减少肿瘤缺氧,增加药物在肿瘤部位的富集。抗血管生成策略通过恢复肿瘤血管来重建肿瘤压力梯度的结构完整性和功能,从而能有效促进纳米颗粒在肿瘤部位的输送。血管舒张药物也可增加血液灌注、血管密度、渗漏、扩张,从而提高化疗效能。同时,针对ECM或重构血管的治疗策略也能促进药物和纳米粒子的外渗和渗透。例如,通过胶原酶(EMC-调节酶)、赖氨酸氧化酶抑制抗体、透明质酸酶或TGF-β抑制剂降低肿瘤间质成分,可增加大分子在肿瘤里的通透性和增强纳米颗粒的穿透性,从而增强化疗效果。
发明内容
针对上述背景技术中存在的缺陷,本发明通过H-NPs扩张肿瘤血管和调节TME,从而增加肿瘤部位的血管通透性、血液灌注、改善纳米颗粒在肿瘤内的富集和渗透、减少肿瘤乏氧,增加肿瘤对化疗药物阿霉素的敏感度,显著提高其抗肿瘤效果。
本发明采用以下技术方案实现:
一种含肼屈嗪的高效抗肿瘤纳米载药体系,所述的载药体系以通过ππ键结合后的肼屈嗪(HDZ)与阿霉素(DOX)作为疏水核心,以葡聚糖作为亲水外壳。
本发明还公开了一种含肼屈嗪的高效抗肿瘤纳米载药体系的制备方法,包括以下步骤:
1)制备Dex-hyd-DOX/cRGD:
首先,制备具有叠氮端基的阿霉素,然后通过click点击化学法将具有叠氮端基的阿霉素和cRGD多肽与炔基修饰的葡聚糖反应形成Dex-hyd-DOX/cRGD;所述的cRGD为含有RGD序列的环八肽。所述RGD序列由精氨酸、甘氨酸和天冬氨酸组成。
2)制备载药体系
将Dex-hyd-DOX/cRGD与HDZ加入超纯水中,600 r/min搅拌溶解后进行超声处理,使用间断超声法,边超声边搅拌,直至溶液变成红褐色,停止超声;继续搅拌直至得到红色溶液,然后用透析袋(MWCO : 3500 Da)透析一天左右(24-30 h),即可得到所述含肼屈嗪的高效抗肿瘤纳米载药体系。
上述技术方案中,进一步地,步骤1)中,所述具有叠氮端基的阿霉素、cRGD与炔基修饰的葡聚糖的摩尔比为2.51 :1 :4.58。所述的Dex-hyd-DOX/cRGD与HDZ的质量比为10-13.3 :1此时每条Dex链上结合的DOX数量平均为1.1个,cRGD的数量平均是0.6个,这样既可以保证能够制备得到合适的纳米载药体系,又可以保证尽可能多的包载HDZ。因为如果阿霉素包载太多,阿霉素与HDZ结合形成的疏水性核心太大,会使得纳米粒子的粒径变大,纳米粒子的疏水性太强,容易形成聚集。
进一步地,所述间断超声法中,超声的参数具体为:超声电压为120 V,超声频率为20 KHz,每间隔5秒超声20秒。
本发明的有益效果为:
本发明首先制备出具有pH响应性和靶向肿瘤的cRGD多肽葡聚糖-阿霉素前药(Dex-hyd-DOX/cRGD),然后通过ππ键将HDZ与阿霉素结合形成疏水核心,葡聚糖为亲水外壳的纳米粒子(H-NPs)。H-NPs不仅可以通过扩张血管增加纳米粒子在肿瘤部位的富集,而且可以调节肿瘤微环境,增强化疗的治疗肿瘤的效果。H-NPs可以延长HDZ在生物体内血液循环时间,通过EPR效应增强HDZ在肿瘤部位的富集,并且还可以通过H-NPs表面的cRGD多肽使H-NPs主动靶向富集到肿瘤部位,减少HDZ脱靶对人体造成的毒副作用。另一方面,H-NPs在肿瘤微酸环境下释放出包载的HDZ来扩张肿瘤血管和调节TME,从而可进一步增强药物在肿瘤部位的富集与渗透。且本发明中由于HDZ和DOX通过ππ结合使得二者结合力更强,有效克服了被包载的肼屈嗪在血液中容易泄露这一缺陷(具体可参考以下文献:cRGD-ModifiedBenzimidazole-based pH-Responsive Nanoparticles for Enhanced Tumor TargetedDoxorubicin Delivery),从而可以使其在肿瘤部位释放并对肿瘤微环境进行有效改善。
附图说明
图1为Dex-hyd-cRGD和Dex-hyd-DOX/cRGD的1H NMR;
图2中(A)H-NPs的TEM与DLS图;(B)H-NPs的稳定性测试;(C)H-NPs体外释放曲线;
图3中(A)HDZ对MBA-MD-231、HUVEC和4T1细胞的毒性;D-NPs、C-NPs、H-NPs和DOX对4T1(B)、MBA-MD-231(C)和HUVEC(D)细胞的毒性;
图4中(A)游离阿霉素和H-NPs的血液循环时间;(B)在24 h和48 h时,游离阿霉素、D-NPs、H-NPs在小鼠体内的生物分布;
图5为C-NPs、C-NPs/HDZ和H-NPs在生物体内的分布;
图6为注射药物48 h后4T1肿瘤中TME的变化:(A)纤连蛋白;(B)肿瘤血管(C)α平滑肌肌动蛋白;(D)免疫T细胞(CD3);(E)阿霉素的渗透;
图7为制备Dex-hyd-DOX/cRGD的化学流程图。
具体实施方式
以下结合附图和具体实施例对本发明进行进一步说明。
实施例1 一种含肼屈嗪的高效抗肿瘤纳米载药体系,具体的制备过程如下:
葡聚糖炔丙基碳酸酯(Dex-C≡C)的合成
实验开始前将反应所需玻璃器皿提前放入烘箱烘干。称取14.34 g(88.5 mmol)N,N'-羰基二咪唑(CDI)加入到在圆底烧瓶中,随后倒入50 mL二氯甲烷并充分搅拌均匀。然后称取3.61 g(51.6 mmol)3-丁炔-1-醇加入到恒压滴液漏斗,加入30 mL二氯甲烷对其进行溶解。将恒压滴液漏斗中溶剂逐滴滴加到圆底烧瓶,并在室温下反应1 h。用饱和氯化钠水溶液萃取反应液3次,收集有机相,用无水Na2SO4干燥一晚,过滤。最后通过旋蒸除去二氯甲烷溶剂,得到粗产品炔基丁基羰基咪唑。
称取2 g(0.05 mmol)葡聚糖加入到支管烧瓶中,加入20 mL DMSO溶解。称取0.317g(1.5 mmol)炔丁基羰基咪唑,用8 mL DMSO溶解,并将溶液转移到恒压滴液漏斗中,然后缓慢滴加到炔丁基羰基咪唑溶液中,在70 ℃条件下,反应24 h。产物溶液用ddH2O透析2天以通过纤维素管状膜(MWCO 2000 Da)除去未反应的炔基丁基羰基咪唑和DMSO。最后冷冻干燥得到炔基修饰的葡聚糖(Dex-C≡C)。(1.464 g,产率:63.2%)
腙键修饰的并含有氮端基团的阿霉素(DOX-hyd-N3)的制备
实验开始前将反应所需玻璃器皿提前放入120 ℃烘箱烘干。称取6-叠氮己酰肼102.4 mg(6.02 × 10−1 mmol)和DOX·HCl 118.8 mg(2.04 × 10−1 mmol)加入到圆底烧瓶中,量取30 mL无水甲醇加入使其溶解,向其滴入一滴冰醋酸,随后称取113.8 mg无水硫酸钠加入其中,在瓶口处接上蛇形冷凝管(水冷凝),在60 ℃条件下避光反应24 h。反应结束后,通过旋蒸将反应溶液浓缩至6 mL,随后加入30 mL无水乙醚并充分混合,离心得到深红色固体粗产物,最后通过真空干燥获得DOX-hyd-N3产物。然后将粗产物再次通过无水乙醚洗涤,直至除尽未反应完的6-叠氮己酰肼,最终得到最终纯净的DOX-hyd-N3产物(110.0mg,产率:73.3%)。
Dex-hyd-DOX/cRGD的合成:
阿霉素-葡聚糖前药(Dex-hyd-DOX/cRGD)通过DOX-hyd-N3、cRGD-PEG2000-N3和Dex-C≡C进行“click”点击化学反应制备(如图7)。
首先,在氮气气氛下,称取9.79 mg(0.0682 mmol)CuBr2和23.66 mg(0.1365mmol)N,N,N' ,N''', N'''-五甲基二乙烯三胺(PMDETA)溶解于6 mL DMSO中反应10 min;然后,分别加入0.2 g(0.0374 mmol)Dex-C≡C和0.02 g(0.0149 mmol)cRGD-PEG2000-N3,将混合物溶液在室温搅拌8 h;接着加入50 mg(0.0683 mmol)DOX-hyd-N3,再搅拌8 h;反应结束后,将混合溶液用DMSO透析12 h以去除未反应的DOX-hyd-N3和cRGD-PEG2000-N3,然后用超纯水透析48 h以去除DMSO。最后通过冷冻干燥最后得到红色固体产物Dex-hyd-DOX/cRGD(0.232 g,产率:85.4%)。
H-NPs抗肿瘤纳米载药体系的构建
16 mg Dex-hyd-DOX/cRGD和1.6 mg HDZ溶解在2 mL超纯水中,适度搅拌后进行10min超声处理,使用间断超声法(超声波细胞破碎仪型号为:KC-250W,超声电压:120 V,超声频率:20 KHz,间隔5秒,超声20秒),边超声边搅拌,直至溶液变成红褐色,停止超声。最后溶液再搅拌4 h,溶液变为红色,然后用透析袋(MWCO : 3500 Da)透析一天,最终得到红色溶液即为Dex-hyd-DOX/cRGD与HDZ质量比为10 :1的H-NPs。
实施例2
与实施例1的区别在于,H-NPs抗肿瘤纳米载药体系的制备具体如下:
16 mg Dex-hyd-DOX/cRGD和1.4 mg HDZ溶解在2 mL超纯水中,适度搅拌后进行10min超声处理,使用间断超声法(超声波细胞破碎仪型号为:KC-250W,超声电压:120 V,超声频率:20 KHz,间隔5秒,超声20秒),边超声边搅拌,直至溶液变成红褐色,停止超声。最后溶液再搅拌4 h,溶液变为红色然后用透析袋(MWCO : 3500 Da)透析28h,最终得到红色溶液即为Dex-hyd-DOX/cRGD与HDZ质量比为11.4 :1的H-NPs。
实施例3
与实施例1的区别在于,H-NPs抗肿瘤纳米载药体系的制备具体如下:
16 mg Dex-hyd-DOX/cRGD和1.2 mg HDZ溶解在2 mL超纯水中,适度搅拌后进行10min超声处理,使用间断超声法(超声波细胞破碎仪型号为:KC-250W,超声电压:120 V,超声频率:20 KHz,间隔5秒,超声20秒),边超声边搅拌,直至溶液变成红褐色,停止超声。最后溶液再搅拌4 h,然后用透析袋(MWCO : 3500 Da)透析30h,最终得到红色溶液即为Dex-hyd-DOX/cRGD与HDZ质量比为13.3 :1的H-NPs。
对实施例1中合成的Dex-hyd-DOX/cRGD和H-NPs进行表征:
1)Dex-hyd-DOX/cRGD的表征
使用1H NMR分析了Dex-hyd-cRGD和Dex-hyd-DOX/cRGD的化学结构。如图1所示,可以看到Dex-hyd-cRGD和Dex-hyd-DOX/cRGD结构上的质子均能找到相应的化学位移,证明Dex-hyd-DOX/cRGD的顺利合成。使用BCA法测试聚合物Dex-hyd-cRGD中cRGD质量百分比为9.5%。使用紫外光谱法测试聚合物Dex-hyd-DOX/cRGD中DOX的含量为11.2%。Dex(葡聚糖,平均分子量5000),Dex-hyd-DOX/cRGD中:当Dex为1条链时,DOX数量平均为1.1个,cRGD的数量平均是0.6个,这个比例既可以保证能够制备得到合适的纳米载药体系,又可以保证尽可能多的包载HDZ,因为如果阿霉素包载太多,阿霉素与HDZ结合形成的疏水性核心太大,会使得纳米粒子的粒径变大,纳米粒子的疏水性太强,容易形成聚集。
)H-NPs的表征
Dex-hyd-DOX/cRGD与HDZ共同组装成以DOX和HDZ为疏水核心的H-NPs纳米粒子。
作为两亲性共聚物,Dex-hyd-DOX和Dex-hyd-DOX/cRGD共聚物在水溶液中自组装成D-NPs和C-NPs。
通过动态光散射仪测定纳米颗粒的粒径、粒径分布和电位。其结果如表1所示。值得注意的三种纳米粒子的粒径没有存在很大的差异。但是它们的所带电荷存在很大的差异。D-NPs带有很强的负电性,而C-NPs和H-NPs具有很弱的正电性。使用透射电镜(TEM)观察H-NPs形貌。如图2中(A)所示,H-NPs外貌是圆形球状。进一步研究了H-NPs纳米粒子在3种条件下(Milli-Q water, phosphate buffered saline (PBS), and fetal bovine serum(FBS))的稳定性。如图2中(B)所示,在2天内监测时间内,H-NPs在PBS和FBS中的平均直径基本保持不变,而在水溶液中体积发生了变大现象。说明制备的H-NPs在生物血液中向目标组织输送过程中具有良好的稳定性。
表1. Dex-C≡C和三种纳米粒子的表征
H-NPs的体外药物释放
将H-NPs溶液置于三种不同的缓冲溶液(pH 7.4,pH 6.0和pH 5.0)中,37℃恒温振荡条件下研究其DOX的释放行为。由于肿瘤细胞中的弱酸环境,使腙键易断裂,因此,Dex-hyd-DOX分子中的DOX在酸性条件下可以被释放出来,其累积释放结果如图2中(C)。随着pH值的降低,DOX的释放速率加快,表明H-NPs具有良好的pH响应性。因此,H-NPs可以在肿瘤细胞弱酸性环境中达到智能控释的效果。因为肼屈嗪与阿霉素以ππ键形成疏水性的内核,所以当阿霉素释放出的时候,肼屈嗪也会被一起释放出来。
细胞毒性
在细胞层面上,首先测试HDZ对细胞的毒性。将不同浓度的HDZ与小鼠乳腺癌细胞(4T1)、人乳腺癌细胞(MBA-MD-231)和血管上皮细胞(HUVEC)共孵育48小时,实验结果如图3中(A)所示,可以看出HDZ对三种细胞的毒性都不是很高。然后,后续做了D-NPs、C-NPs、H-NPs、DOX与4T1、MBA-MD-231和HUVEC细胞的细胞毒性。实验结果如图3中(B)、(C)、(D)所示,从图中可以看出,相比于D-NPs,C-NPs对4T1和MBA-MD-231细胞具有更强的细胞毒性,而二者对HUVEC的细胞毒性相差很小,这是因为C-NPs表面具有靶向4T1和MBA-MD-231肿瘤细胞表面过度表达的整合素αvβ3的多肽cRGD,可以使更多的C-NPs纳米粒子进入肿瘤细胞,提高其细胞杀伤能力。而HUVEC细胞因为其表面没有过度表达的整合素αvβ3,所以D-NPs和C-NPs对其细胞毒性相差很小。而具有靶向的H-NPs的毒性比D-NPs和C-NPs的毒性都强,说明HDZ和阿霉素的联合具有更好的联合杀伤肿瘤细胞效果。
血液循环和生物分布
一般情况下,粒径在200 nm以下的纳米粒子在生物体血液中具有更长的血液循环时间,为了验证H-NPs在血液中是否具有更长的血液循环时间,H-NPs和游离阿霉素溶液通过尾静脉注射进BALB / c小鼠体内(高剂量7.5 mg kg−1)。H-NPs和游离阿霉素在小鼠血液中不同时间的%ID/g值如图4中(A)所示,可以发现,与游离药物相比,H-NPs具有更长的血液循环时间,增加药物在肿瘤部位的积累。
此外,为了检测游离阿霉素、D-NPs和H-NPs纳米颗粒在肿瘤和其他器官中的分布差别,在静脉注射不同药物后不同时间间隔后处死4T1荷瘤小鼠,取出小鼠各器官进行组织研磨和萃取,最后进行荧光分析。实验结果如图4中(B)所示,从图中可以看出相比于游离的阿霉素,D-NPs和H-NPs纳米颗粒在小鼠各个器官和肿瘤中富集率比较高,分别是游离阿霉素的~1.52和~1.81倍(24 h)。随着时间的延长,在48 h时,D-NPs和H-NPs在肿瘤部位的富集还有一定的增加,而游离的阿霉素却在减少(是游离的阿霉素的~1.92和~2.21倍)。从上边的细胞毒性实验和血液循环实验可以推断出这是因为相比于游离阿霉素,D-NPs和H-NPs纳米颗粒在血液中具有更长的血液循环能力而且不容易被细胞清除。而且相比于没有接靶向多肽的D-NPs,具有靶向的H-NPs在肿瘤部位具有更好的肿瘤富集效果。
增强肿瘤中纳米颗粒的积累
HDZ导致肿瘤血管扩张和TAFs的减少可以为纳米颗粒在肿瘤部位的富集和药物在肿瘤部位的渗透创造有利条件。在实际临床中,大部分癌症都是到癌症晚期才被检测到,所以本发明使用肿瘤体积大约300 mm3的荷瘤小鼠进行试验,模拟实际临床癌症患者的真实状况。三组荷有乳腺癌肿瘤模型(肿瘤体积大约300 mm3)的小鼠被连续两天给与三种不同治疗方案:H-NPs,C-NPs/HDZ,C-NPs(HDZ:7.5 mg/kg,DOX:7.5 mg/kg)。然后在不同设定时间点取出小鼠的各个组织进行分析,来探究不同情况下HDZ对纳米粒子在肿瘤的积累、渗透和分布情况(图5)。通过小动物活体成像仪检测到DOX的信号,计算出在24 h时H-NPs组的DOX富集量分别是C-NPs/HDZ和C-NPs的~1.04和~1.30倍(图5 中(B))。在48 h时,H-NPs和C-NPs/HDZ组DOX在肿瘤部位的富集量还相比于C-NPs组还有所增加,H-NPs组分别是C-NPs/HDZ和C-NPs的~1.10和~1.36倍。并且可以看到,相比于C-NPs组,C-NPs/HDZ组也具有更高的DOX富集量。这说明游离的HDZ也可以增强C-NPs在肿瘤部位的富集。而包载HDZ的H-NPs可以使HDZ在肿瘤部位大量富集,HDZ又大大的增强H-NPs在肿瘤部位的富集效应,从而能够形成相辅相成的效果。通过对不同时间点的小鼠器官组织进行研磨,然后通过荧光测试法对其进行分析,得到相似的实验结果。发明人推测其原因是H-NPs在肿瘤酸性微环境下释放出包载的HDZ来增强肿瘤部位的血管舒张和修复血管系统,进而增强了H-NPs在肿瘤部位的富集。
调节TME
小鼠成像实验结束后,将肿瘤进行组织切片和免疫荧光染色,对肿瘤微环境中纤连蛋白数量、肿瘤血管、α平滑肌肌动蛋白数量、免疫T细胞(CD3)数量、阿霉素分布进行相应的检测,观察H-NPs对肿瘤微环境的影响。测试结果如图6所示。使用x5倍放大倍数对纤连蛋白进行观察,这样可以方便观察其在整个肿瘤的分布状况。相比于C-NPs组,C-NPs/HDZ组的纤连蛋白得到了相应的减少,令人惊讶的是H-NPs组在肿瘤部位纤连蛋白几乎全部消失了。并且α平滑肌肌动蛋白数量也发生了相似的数量变化。对肿瘤血管检测可以看出相比于C-NPs组,C-NPs/HDZ组和H-NPs组的血管都得到扩张,而且H-NPs组的血管的扩张效果比C-NPs/HDZ组好。相比于C-NPs组和C-NPs/HDZ组H-NPs的治疗导致肿瘤中CD3+ T细胞浸润非常明显的增加。最后检测DOX在肿瘤部位的渗透,相比于C-NPs组,C-NPs/HDZ组的DOX以血管为中心具有一定的渗透,而H-NPs组具有非常明显的DOX的渗透,DOX可以达到离血管很远的距离。这些实验结果表明H-NPs可以很好的调节肿瘤微环境,促进纳米药物在肿瘤部位的富集与渗透,也预示着H-NPs对肿瘤具有很好的治疗效果。
Claims (5)
1.一种含肼屈嗪的高效抗肿瘤纳米载药体系,其特征在于,所述载药体系是Dex-hyd-DOX/cRGD与肼屈嗪共同组装成以阿霉素和肼屈嗪为疏水核心的H-NPs纳米粒子;所述的Dex-hyd-DOX/cRGD为具有pH响应性和靶向肿瘤的cRGD多肽葡聚糖-阿霉素前药。
2.如权利要求1所述的含肼屈嗪的高效抗肿瘤纳米载药体系的制备方法,其特征在于,具体步骤如下:
1)制备Dex-hyd-DOX/cRGD:
首先,制备具有叠氮端基的阿霉素,然后通过click点击化学法将具有叠氮端基的阿霉素和cRGD与炔基修饰的葡聚糖反应形成Dex-hyd-DOX/cRGD;
2)制备载药体系
将Dex-hyd-DOX/cRGD和HDZ溶解在超纯水中,然后使用间断超声法,边超声边搅拌,直至溶液变成红褐色,停止超声;继续搅拌直至得到红色溶液,然后用透析袋MWCO : 3500 Da透析24-30 h,即得到所述含肼屈嗪的高效抗肿瘤纳米载药体系。
3.根据权利要求2所述的含肼屈嗪的高效抗肿瘤纳米载药体系的制备方法,其特征在于,步骤1)中,所述具有叠氮端基的阿霉素、cRGD与炔基修饰的葡聚糖的摩尔比为2.51 :1:4.58。
4.根据权利要求2所述的含肼屈嗪的高效抗肿瘤纳米载药体系的制备方法,其特征在于,步骤2)中,所述的Dex-hyd-DOX/cRGD与HDZ的质量比为10-13.3:1。
5.根据权利要求2所述的含肼屈嗪的高效抗肿瘤纳米载药体系的制备方法,其特征在于,步骤2)中,所述的间断超声法中,超声的参数具体为:超声电压为120 V,超声频率为20KHz,每间隔5秒超声20秒。
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US9421194B2 (en) * | 2010-04-05 | 2016-08-23 | Rutgers, The State University Of New Jersey | Lung targeting dual drug delivery system |
CN103301472A (zh) * | 2013-04-28 | 2013-09-18 | 中国药科大学 | 生物体病灶部位特异性释药的两亲性多糖-抗肿瘤药物偶联物及其药物组合物的制备和应用 |
CN104523598B (zh) * | 2014-12-16 | 2017-11-14 | 中国科学院长春应用化学研究所 | 葡聚糖/阿霉素键合药纳米粒及其制备方法 |
CN112516329B (zh) * | 2020-12-28 | 2024-02-20 | 南京宁丹新药技术有限公司 | 基于高分子前药的自组装组合药物载体及其应用 |
CN114099705B (zh) * | 2021-11-18 | 2023-06-13 | 浙江大学杭州国际科创中心 | 一种基于肼屈嗪改善肿瘤微环境的纳米药物及其制备和应用 |
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