肿瘤还原微环境响应的透明质酸胶束抗肿瘤研究

发布时间：2018-02-28 05:21

本文关键词： 透明质酸紫杉醇二硫键 CD44受体肿瘤治疗选择性释放　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：近年来药物传递系统(Drug Delivery System, DDS)受到广泛关注,因为与传统制剂相比,DDS可以减少药物毒副作用,提高药物治疗效果,提升药物稳定性,提高药物的生物利用度(bioavailability)。目前已发展建立了多种类型的新型药物递送系统,包扩脂质体(liposome),胶束(micelle)和纳米粒(nanoparticle)等。但同时DDS也面临缺少主动靶能力和在病灶部位有效释放活性药物等一系列的问题。肿瘤与正常组织之间存在天然的氧化还原生理差异,即肿瘤细胞内微环境中的还原型谷胱甘肽(glutathione, GSH)浓度远高于正常组织(100-1000倍)。利用此种特性,本研究构建一种能主动靶向肿瘤细胞CD44受体和在肿瘤高GSH微环境中触发释放的透明质酸维生素E琥珀酸酯衍生物,以期实现DDS主动靶向病灶部位和选择性释放药物的目的。以亲水性的透明质酸(Hyaluronic acid, HA)为骨架,以维生素E琥珀酸酯(D-α-Tocopherol acid Succinate, α-TOS)为疏水链段,以具有二硫键的胱胺(氧化还原敏感型)和已二胺(非氧化还原敏感型)为桥链,通过酰胺化反应,合成了氧化还原敏感型和非氧化还原敏感型透明质酸维生素E琥珀酸酯衍生物HA-SS-TOS (HSST)和HA-CC-TOS (HCCT),二者临界胶束浓度分别为48.0 μg/mL和64.9μg/mL,具有良好的胶束形成能力。HSST和HCCT衍生物胶束的粒径为114±11.6 nm和108±16.2 nm,电位为-20.9±4.3 mV和-18.2±4.2 mV。HSST和HCCT都能对紫杉醇(PTX)进行有效包载,最高包封率和载药量分别为88.3%,27.2%和89.7%,28.5%(w/w)。HSST在高GSH环境下快速释放药物,显示出典型的氧化还原敏感性。以人卵巢癌细胞SKOV3为模型细胞,HSST和HCCT在SKOV3(细胞内高GSH浓度)和丁硫氛酸—亚讽亚胺(Buthionine sulfoximine, BSO)预处理的SKOV3(细胞内低GSH浓度)细胞中的摄取情况无明显差异。HSST能选择性在胞内高GSH浓度的肿瘤细胞中快速释放药物,而在低GSH肿瘤细胞中释放缓慢。联合使用PTX与TOS可以增强PTX的抗肿瘤效果,起到协同抗肿瘤的作用。由于比HCCT释药更快,HSST体现出更强的肿瘤细胞增殖抑制率和选择性杀伤肿瘤细胞的能力。采用免疫印迹技术(Western blot)和免疫荧光染色(Immunofluorescence technique)考察了CD44受体在SKOV3, A549, S180和CT26细胞组织上的表达,结果显示SKOV3, S180和CT26细胞组织为CD44受体高表达,而A549为低CD44受体表达,在此基础上构建了SKOV3/A549双侧皮下异位肿瘤小鼠模型。同时成功构建了S180单侧皮下异位肿瘤小鼠模型,S180双侧皮下异位肿瘤小鼠模型,CT26单侧皮下异位肿瘤小鼠模型和CT26-Luc原位结肠癌肿瘤小鼠模型。系统地考察HSST胶束在上述肿瘤模型中CD44受体靶向和分布情况,以及胶束的体内释放情况。结果表明透明质酸胶束在肿瘤中的累积是通过CD44受体介导,具有特异性靶向肿瘤的能力；而其在高GSH肿瘤中快速释放,说明透明质酸胶束具有选择性释放药物的能力。以S180单侧皮下异位肿瘤小鼠和CT26-Luc原位结肠癌小鼠为动物模型,紫杉醇载药透明质酸胶束对肿瘤的抑制效果呈现剂量依赖,载药胶束具有明显的抑制肿瘤生长和转移扩散的作用。
[Abstract]:In recent years, drug delivery system (Drug Delivery System, DDS) received extensive attention, because compared with the traditional formulation, DDS can reduce the side effects of drugs, improve the therapeutic effect of drugs, enhance the stability of the drug and improve the bioavailability of drugs (bioavailability). Currently has developed many types of new drug delivery system, package expansion of liposomes (liposome), (micelle) and micellar nanoparticles (nanoparticle). But at the same time, DDS is also facing a lack of initiative and ability in target lesion effectively release the active drug and a series of problems. There is a natural redox physiological difference between tumor and normal tissue, the tumor microenvironment in cell type glutathione (glutathione, GSH) was much higher than that in normal tissue (100-1000 times). The use of this property, this study constructs a targeting tumor cells and CD44 receptor in tumor micro GSH Hyaluronic acid vitamin E succinate derivatives in triggering the release of the release of the drug to the lesion site and selectivity in order to achieve the DDS target. The hyaluronic acid hydrophilic (Hyaluronic acid HA) as a skeleton, with vitamin E succinate (D- -Tocopherol acid Succinate alpha, alpha -TOS) as hydrophobic chain in section, cysteamine had two disulfide (redox sensitive) and has two amine (non redox sensitive) as chain bridge, through amidation reaction, redox sensitive and non sensitive oxidation reduction synthesis of hyaluronic acid vitamin E succinate derivatives of HA-SS-TOS (HSST) and HA-CC-TOS (HCCT two), the critical micelle concentration were 48 g/mL and 64.9 g/mL, with good micelle forming ability of.HSST and derivatives of HCCT micelle size were 114 + 11.6 and 108 + 16.2 nm nm + 4.3 mV potential of -20.9 and -18.2 + 4.2 mV.HSST and HCCT The paclitaxel (PTX) effectively loaded, the highest encapsulation efficiency and drug loading were 88.3%, 27.2% and 89.7%, 28.5% (w/w).HSST quick release drugs in high GSH environment, showing typical redox sensitivity. In human ovarian cancer cells SKOV3 as model cell, HSST and HCCT in SKOV3 (high intracellular GSH concentration) and butyl sulfur atmosphere acid arylmerghylene imine (Buthionine sulfoximine, BSO a) pretreated SKOV3 (low intracellular GSH concentration) uptake in cells had no significant difference in.HSST can selectively quickly release the drug in cells with high GSH concentration in tumor cells, and tumor cells in low GSH in the slow release. The combined use of PTX and TOS can enhance the antitumor effect of PTX plays a synergistic antitumor effect. The drug release ratio HCCT faster, HSST shows the inhibition rate and selectivity of tumor cell proliferation and stronger killing tumor cells by Western blot technology ability. (Western blot) and immunofluorescence staining (Immunofluorescence technique) on CD44 receptor in SKOV3, A549, and CT26 expression of S180 cells, the results showed that SKOV3, S180 and CT26 cells for CD44 receptor expression and A549 low expression of CD44 receptor, on the basis of the construction of SKOV3/A549 bilateral subcutaneous ectopic tumor a mouse model of S180 was constructed successfully. At the same time, unilateral subcutaneous ectopic tumor model in mice, S180 bilateral subcutaneous ectopic tumor model in mice, CT26 unilateral subcutaneous ectopic tumor mice and CT26-Luc in colon cancer mouse model system to study HSST micelles CD44 in the tumor model receptor targeting and distribution, as well as the release of micelles in vivo. The results showed that the accumulation of hyaluronic acid micelles in tumors is mediated by CD44 receptor, with specific tumor targeting ability; and the high GSH in tumor Quick release that hyaluronic acid micelle has the ability to selectively release drugs. S180 unilateral subcutaneous ectopic tumor in mice and CT26-Luc mice as the animal model of colon carcinoma in situ, tumor inhibitory effect of paclitaxel hyaluronan micelles showed dose-dependent, micelle has obvious inhibition of tumor growth and metastasis diffusion effect.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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