含EAB基团的两亲接枝聚膦腈载药自组装体的构建及抗肿瘤作用评价

发布时间：2018-01-19 21:02

  本文关键词： 聚膦腈 囊泡 阿霉素 氯喹 耐药 抗癌治疗 药物共载　出处：《浙江大学》2014年博士论文　论文类型：学位论文

【摘要】：纳米技术的迅速发展极大地推动了纳米载药系统在癌症治疗中的应用。近年来,一类被称为聚合物囊泡的新颖纳米自组装体吸引了人们的注意。相较于自组装胶束仅能装载疏水性药物,聚合物囊泡以类似脂质体的结构特点,不仅能装载疏水性药物也能装载亲水性药物,甚至能共载亲疏水性两种药物,极具开发潜力。本文选取聚膦腈作为聚合物主链,以分子量2000的聚乙二醇单甲醚(mPEG)为亲水性侧链和对氨基苯甲酸乙酯(EAB)为疏水性侧基进行修饰,通过mPEG/EAB投料比调节,合成一系列PEG接枝率不同的两亲性接枝聚膦腈(PEP),又对聚合物结构进行核磁共振(NMR)、傅里叶红外光谱(FT-IR)和凝胶渗透色谱(GPC)表征。采用普通透析法即可简便制得该系列聚合物的纳米自组装体。通过TEM和cryo-TEM观察,PEG接枝率为0.89形成胶束,0.86形成过渡态,0.79至0.67形成囊泡。为考察PEP装载亲疏水性药物的能力,本文分别以水溶性化疗药物盐酸阿霉素(DOX·HCl)及其脱盐产物疏水性阿霉素(DOX)作为模型药物,采用透析法装载,结果表明聚合物中疏水基团EAB接枝率上升将促进PEP的药物装载。尤其,PEP-5装载DOX·HCl或DOX均能获得高载药量(LC)和包封率(EE)。其中,载DOX·HCl的PEP-5系统(PEP-5-DH) LC为16.23%,载DOX的PEP-5系统(PEP-5-D)LC为15.55%,两者对应EE均高于90%,PEP-5-DH为97.30%,PEP-5-D为93.30%。经2D 1H-1H NOESY检测,证明载体与药物间存在极强分子间作用力,有利于载药。药物释放实验表明,DOX·HCl从PEP载药系统的释放较DOX快,且与pH条件有关。MCF-7细胞毒性实验发现,载体本身几乎无毒。PEP-5-DH与游离DOX·HCl毒性相近,而PEP-5-D,由于药物水溶性不佳释放缓慢,导致细胞毒性减弱。由PEG修饰形成的纳米自组装体被广泛报道具有增强渗透与滞留(EPR)效应相关的肿瘤被动靶向性,能降低化疗药物毒副作用,因此本课题构建了MCF-7乳腺癌荷瘤裸鼠模型,对PEP载药系统进行体内评价。结果表明,PEP载体、药物亲水性及给药方案均对药效产生不同影响。实验过程中,PEP载药的剂型相比游离DOX·HCl毒性明显减小。药效方面,虽然2mg/kg每天1次连续3天方式的给药总量比5mg/kg每3天给1次共3次的方式更低,但2mg/kg对应组的疗效更佳。采用相同的给药方式,PEP-5-DH的抑瘤效果明显优于PEP-5-D,已与游离DOX·HCl相当,但安全性却较游离DOX·HCl显著提高,达到了降低化疗药物毒副作用的同时,不牺牲药物疗效的目的。为克服MCF-7/Adr肿瘤细胞对阿霉素的耐药性,本文还利用PEP自组装囊泡的独特结构共载DOX·HCl或DOX和氯喹(CQ)。CQ为一种传统抗疟药物,但也被报道具有抗肿瘤药物增敏作用,已在临床试验中进行研究。与之相关的机制可能是抑制耐药蛋白的外排,也可能与促癌干细胞(CSC)凋亡相关。细胞毒性实验发现阿霉素和CQ联合用药可显著逆转耐药,通过Chou-Talalay联合指数法计算证实两者具有协同作用且随CQ比例的增加而增强。为了更好的地模拟体内环境,本文构建了耐药性MCF-7/Adr细胞3D肿瘤球模型,并采用激光共聚焦(CLSM)断层扫描及扫描电镜(SEM)对各试验组的肿瘤球内渗透剂抑瘤效果进行评价。相比游离DOX·HCl只能停留在肿瘤球表面,CQ和DOX·HCl联合用药能更好地渗透进入肿瘤深部,渗透效果最好的是按DOX·HCl和CQ以1：1比例共载的PEP制剂(PEP-DHC-1),能完全渗透进入肿瘤深部,且给药7天后,肿瘤球明显瓦解。流式凋亡检测证实PEP-DHC-1处理的肿瘤球凋亡率也最高,提示该载药自组装体有望在动物体内逆转耐药,提高阿霉素的抗肿瘤疗效。总之,PEP作为一种新型纳米自组装药物载体,具有高效装载亲水性或疏水性药物能力,可在抗肿瘤治疗方面,保证安全性并提高药效,值得进行深入研究。
[Abstract]:The rapid development of nanotechnology has greatly promoted the application of nano drug delivery system in the treatment of cancer. In recent years, a new class called nano polymer vesicles self-assembly has attracted much attention. Compared with the self-assembled micelles can only be loaded with hydrophobic drug, polymer vesicles to similar structural features of liposomes that not only can contain hydrophobic drugs can also load hydrophilic drugs, even to a total of two kinds of drug loaded Pro hydrophobic, great development potential. This paper selects polyphosphazene as polymer backbone, polyethylene glycol monomethyl ether with a molecular weight of 2000 (mPEG) as the hydrophilic side chain and parathesin (EAB) as hydrophobic of the side group modified by adjusting the feed ratio of mPEG/EAB, the synthesis of a series of PEG grafting rate of two different amphiphilic graft polyphosphazenes (PEP), and the polymer structure of nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR) and gel Permeation chromatography (GPC) respectively. The series is conveniently prepared nano polymer using ordinary dialysis self-assembly. Through TEM and cryo-TEM observation, PEG grafting rate was 0.89, the formation of micelles, formed 0.86 transition states, 0.79 to 0.67. In order to investigate the formation of vesicles PEP hydrophobic drug loading capacity, this paper respectively water soluble drug doxorubicin hydrochloride (DOX - HCl) and desalination product hydrophobic doxorubicin (DOX) as a model drug loading by dialysis method, results show that the polymer hydrophobic groups of EAB grafted rate will promote PEP drug loading. In particular, PEP-5 HCl or DOX DOX loading can obtain high drug loading (LC) and entrapment efficiency (EE). Among them, the PEP-5 DOX HCl system (PEP-5-DH) LC 16.23%, PEP-5 system DOX (PEP-5-D) LC is 15.55%, which corresponds to EE were higher than 90%, PEP-5-DH is 97.30%, PEP-5-D is 93.30%. by 2D 1H-1H NOESY detection, carrier proof Between the drug and the existence of strong intermolecular forces, in favor of carrying drugs. Drug release experiment showed that DOX and HCl from the PEP drug carrier system release faster than DOX and pH, and the conditions of the.MCF-7 cell toxicity test, the carrier itself almost non-toxic.PEP-5-DH and free DOX - HCl and PEP-5-D, due to similar toxicity, drug water the poor solubility is slowly released, causing cell toxicity decreased. Nano PEG modified formed by self-assembly is widely reported to have enhanced permeability and retention (EPR) effect of passive tumor targeting, can reduce the side-effect of chemotherapy, so we established the nude mice model of breast cancer MCF-7, in vivo evaluation the PEP drug carrier system. The results show that the PEP carrier, hydrophilic drug and dosage regimen of pharmacodynamics have different effects. During the experiment, PEP drug formulations compared to free DOX HCl toxicity was significantly reduced. The drug effect, while 2mg /kg 1 times a day for 3 consecutive days the total dose 5mg/kg every 3 days to less than 1 times 3 times, but the curative effect is better. The corresponding 2mg/kg group with the same dose, PEP-5-DH inhibitory effect was significantly higher than that of PEP-5-D, and free DOX HCl, but the security is free DOX HCl increased significantly, reduce chemotherapy side effects at the same time, without sacrificing efficacy. In order to overcome the resistance of MCF-7/Adr cells to adriamycin, we also use the PEP DOX - HCl or DOX and chloroquine since the unique structure of assembled vesicle (CQ) is a kind of traditional anti.CQ antimalarial drugs, but has also been reported to have radiosensitizing effect of antitumor drug, has been studied in clinical trials. The mechanisms involved may be resistant to inhibition of protein efflux, and may promote cancer stem cells (CSC) apoptosis. The cytotoxicity of adriamycin combined with CQ and found The drug can significantly reverse the drug resistance, the Chou-Talalay combination index method proves that they have synergistic effect and increase with the proportion of CQ increased. In order to better simulate the in vivo environment, this paper constructs the drug resistance of MCF-7/Adr cell 3D tumor model, and using confocal laser tomography (CLSM) and scanning electron microscopy (SEM) to evaluate the agent the inhibitory effect of tumor infiltration in each experimental group compared with the ball. The free DOX HCl can only stay in the tumor sphere surface, CQ and DOX - HCl combination can better penetrate into the tumor deep penetration, PEP was the best preparation by DOX - HCl and CQ 1:1 in the proportion of total load (PEP-DHC-1), can completely penetrate deep into the tumor, and after 7 days of treatment, the tumor spheres were disintegrated. Flow cytometry apoptosis detection confirmed tumor apoptosis of PEP-DHC-1 ball processing rate is the highest, suggesting that the drug loaded self-assembly is expected in the animal in vivo reversal of drug resistance, To improve the anti-tumor effect of doxorubicin. In short, PEP as a new nano self assembled drug carrier has high capacity to load hydrophilic or hydrophobic drugs, and it can ensure safety and improve drug efficacy in anti-tumor treatment. It is worth further research.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943;R96
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本文编号：1445523