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喜树碱类药物的难溶性限制了其在临床上的广泛应用。为了增强其水溶性，研究人员开发了多种喜树碱类药物制剂，如前药、脂质体、胶束、高分子纳米粒等，但这些制剂分别在不同程度上存在载药率低、靶向性差或毒副作用大等问题。针对这些问题，本论文选择天然的肿瘤细胞膜和红细胞膜为载体，设计构建了仿生羟基喜树碱（HCPT）纳米晶体给药体系，用于恶性肿瘤的治疗。本论文的主要内容如下：1. 构建了包覆肿瘤细胞膜的喜树碱纳米晶体系，并在细胞水平上对其特异肿瘤靶向性及肿瘤杀伤效果进行了系统评价。首先通过软模板诱导法制得喜树碱纳米晶体（NCs），在此基础上，通过分子间作用力在纳米晶体表面吸附光敏剂吲哚菁绿（ICG），之后以物理挤压的方式在纳米晶体表面包裹上4T1细胞膜（CM），成功制备包覆4T1细胞膜且装载光敏剂ICG的喜树碱纳米晶体（NCs/ICG/CM）。与对照组相比，包覆4T1细胞膜的NCs/ICG/CM由于具有同源靶向性，在4T1细胞中的摄取量显著增加。另外，NCs/ICG/CM能够在激光照射下产生高热效应，促进纳米晶体中HCPT分子的快速释放，最终NCs/ICG/CM在高热效应和化疗药物的综合作用下展现了理想的肿瘤细胞杀伤效果。2. 构建了两种三阴性乳腺癌小鼠模型，并综合评价了NCs/ICG/CM的抗肿瘤效果及毒副作用。与单独纳米晶体组相比，NC/ICG/CM能够显著延长药物在小鼠体内的循环时间；在主动靶向的协助下，能够有效增加药物在肿瘤部位的蓄积量；在进一步的近红外激光照射下，能够明显提升小鼠肿瘤部位的温度，最终展现更加优异的抗肿瘤及抗肿瘤转移效果，同时显著延长了小鼠生存时间。此外，NCs/ICG/CM还能显著降低组织毒性和血液毒性等副作用，展现出了良好的生物安全性。3. 构建了嵌合喜树碱纳米晶的红细胞小体体系，并在2D和3D细胞水平上对其进行了系统评价。首先采用低渗法在红细胞小体（RBC）内装载HCPT钠盐溶液，随后利用HCPT的“碱溶酸沉”特性，通过在体系中通入CO2，使HCPT以红细胞为微反应器进行限域结晶，进而获得嵌合喜树碱纳米晶的红细胞小体RBC@HCPT。在2D细胞水平上，RBC@HCPT能够逃避巨噬细胞的清除，展现出良好的隐形效果，而在4T1细胞中的摄取量明显提高，肿瘤细胞杀伤效果显著增强。在3D细胞水平上，RBC@HCPT表现出极强的肿瘤细胞球渗透能力，并对3 D细胞球的增殖生长有明显的抑制效果。4. 利用小鼠肿瘤模型对RBC@HCPT体系进行了体内分布、抗肿瘤效果及毒副作用三方面的考察。与商品化制剂HCPT-Na组相比，RBC@HCPT明显延长体内循环时间，并实现了在肿瘤部位的高效富集，在3种荷瘤小鼠模型（4T1乳腺癌模型、原位肝癌模型以及人源乳腺癌小鼠异种移植模型）中均取得更优的治疗效果，使得小鼠的生存时间得到明显延长。其中在小鼠的4T1乳腺癌模型中，RBC@HCPT还可抑制肿瘤的肺转移及骨转移。另外，RBC@HCPT的组织毒性、血液毒性以及综合毒性均低于HCPT-Na组，具有更小的毒副作用。 综上所述，本论文选取HCPT作为模型药物，构建了两种仿生型喜树碱纳米晶体体系，针对性地解决了传统喜树碱制剂存在的水溶性差、靶向性差和毒副作用大等问题，为今后开发新型喜树碱纳米剂型提供了新的思路。;The insolubility of camptothecin and its derivatives has limited their wide applications in clinic. In order to enhance their water solubility, researchers have developed many camptothecin-based formulations, such as prodrug, liposome, micelle, polymer nanoparticles, but these formulations generally have showed low drug loading efficiency, poor tumor targeting and serious side effects. To solve these problems, this thesis designed and constructed two biomimetic hydroxycamptothecin nanocrystalline drug delivery systems using natural tumor cell membrane and erythrocyte cell membrane as carriers for of malignant tumor treatment. The main contents of this thesis are as follows:1. Tumor cell membrane-based hydrocamptothecin nanocrystals were constructed and their specific tumor targeting and tumor killing effects at the cellular level were systematically evaluated. First, hydrocamptothecin nanocrystals (NCs) were prepared through the soft template induced method. Based on that, photosensitizer Indocyanine green (ICG) was absorbed to the prepared nanocrystals through intermolecular interactions. Then, cancer cell membrane fragments (CM) were decorated by physical extrusion to obtain cancer cell membrane coated and ICG loaded hydrocamptothecin nanocrystals (NCs/ICG/CM). Compared with other HCPT formulations, greater amount of HCPT uptake in 4T1 cells was achieved in NCs/ICG/CM group with the assistance of homologous targeting. In addition, NCs/ICG/CM could produce hyperthermia effect under laser irradiation, which induced the quick release of HCPT molecules from nanocrystals. Finally, under the combined chemo-photothermal effects, NCs/ICG/CM could trigger ideal tumor cell killing effect.2. Two triple-negative breast cancer mouse models were constructed, and the antitumor and side effects of NCs/ICG/CM were comprehensively evaluated. Compared with nanocrystal alone, NC/ICG/CM could significantly extend the in vivo circulation time of drug, effectively increase the intratumor HCPT concentration due to active targeting, and obviously improve the temperature of the tumors upon near infrared laser irradiation. With these advantages, NC/ICG/CM exhibited more effective tumor suppression and metastasis prevention, and greatly prolonged the survival time of mice. Besides, NCs/ICG/CM could significantly reduce side effects, such as tissue toxicity and blood toxicity, showing good biosafety.3. A hydrocamptothecin nanocrystal-embedded erythrocyte vesicle system was constructed and systematically evaluated at 2D and 3D cell levels. First, HCPT sodium salt solution was loaded into RBC vesicles in the low osmotic pressure. Subsequently, by virtue of the ‘dissolve in alkaline solution and precipitate in acidic solution’ property of HCPT, HCPT was crystallized in a confined way using RBC as a micro-reactor after injecting CO2, and finally hydrocamptothecin nanocrystal-embedded erythrocyte vesicle (RBC@HCPT) was obtained. The results showed that at the 2D cell level, RBC@HCPT could evade the clearance of macrophages, performing a good stealth effect. Meanwhile, for RBC@HCPT, HCPT uptake in 4T1 cells was significantly increased, and tumor cell inhibition was greatly improved. At 3D cell level, RBC@HCPT showed an extremely strong penetration ability of tumor cell spheroids and had a significant inhibitory effect on their proliferation and growth.4. The in vivo distribution, antitumor and side effects of RBC@HCPT were investigated by mouse tumor models. Compared with the commercial formulation HCPT-Na, RBC@HCPT showed longer circulation time in vivo and more drug accumulation at tumor sites. Then, it achieved a better therapeutic effect and also extended the survival time of mice in three tumor-bearing mouse model (including 4T1 breast tumor model, orthotopic liver tumor model and patient-derived tumor xenografts model). RBC@HCPT could also inhibit lung and bone metastasis in 4T1 breast tumor model. In addition, RBC@HCPT was superior to HCPT-Na group in terms of tissue toxicity, blood toxicity and comprehensive toxicity, exhibiting low level of side effects. In summary, this thesis selected HCPT as the model drug to construct two biomimetic camptothecin nanocrystal systems, which specifically solved the problems of low water solubility, poor targeting and serious toxicity and side effects of traditional camptothecin formulations and provided a new idea for the development of new camptothecin nano-formulations in the future.
|张莉君. 喜树碱仿生晶体剂型的构建与抗肿瘤应用[D]. 中国科学院大学,2019.|
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