本文關(guān)鍵詞： 聚膦腈 囊泡 阿霉素 氯喹 耐藥 抗癌治療 藥物共載　出處：《浙江大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：納米技術(shù)的迅速發(fā)展極大地推動(dòng)了納米載藥系統(tǒng)在癌癥治療中的應(yīng)用。近年來,一類被稱為聚合物囊泡的新穎納米自組裝體吸引了人們的注意。相較于自組裝膠束僅能裝載疏水性藥物,聚合物囊泡以類似脂質(zhì)體的結(jié)構(gòu)特點(diǎn),不僅能裝載疏水性藥物也能裝載親水性藥物,甚至能共載親疏水性兩種藥物,極具開發(fā)潛力。本文選取聚膦腈作為聚合物主鏈,以分子量2000的聚乙二醇單甲醚(mPEG)為親水性側(cè)鏈和對(duì)氨基苯甲酸乙酯(EAB)為疏水性側(cè)基進(jìn)行修飾,通過mPEG/EAB投料比調(diào)節(jié),合成一系列PEG接枝率不同的兩親性接枝聚膦腈(PEP),又對(duì)聚合物結(jié)構(gòu)進(jìn)行核磁共振(NMR)、傅里葉紅外光譜(FT-IR)和凝膠滲透色譜(GPC)表征。采用普通透析法即可簡(jiǎn)便制得該系列聚合物的納米自組裝體。通過TEM和cryo-TEM觀察,PEG接枝率為0.89形成膠束,0.86形成過渡態(tài),0.79至0.67形成囊泡。為考察PEP裝載親疏水性藥物的能力,本文分別以水溶性化療藥物鹽酸阿霉素(DOX·HCl)及其脫鹽產(chǎn)物疏水性阿霉素(DOX)作為模型藥物,采用透析法裝載,結(jié)果表明聚合物中疏水基團(tuán)EAB接枝率上升將促進(jìn)PEP的藥物裝載。尤其,PEP-5裝載DOX·HCl或DOX均能獲得高載藥量(LC)和包封率(EE)。其中,載DOX·HCl的PEP-5系統(tǒng)(PEP-5-DH) LC為16.23%,載DOX的PEP-5系統(tǒng)(PEP-5-D)LC為15.55%,兩者對(duì)應(yīng)EE均高于90%,PEP-5-DH為97.30%,PEP-5-D為93.30%。經(jīng)2D 1H-1H NOESY檢測(cè),證明載體與藥物間存在極強(qiáng)分子間作用力,有利于載藥。藥物釋放實(shí)驗(yàn)表明,DOX·HCl從PEP載藥系統(tǒng)的釋放較DOX快,且與pH條件有關(guān)。MCF-7細(xì)胞毒性實(shí)驗(yàn)發(fā)現(xiàn),載體本身幾乎無毒。PEP-5-DH與游離DOX·HCl毒性相近,而PEP-5-D,由于藥物水溶性不佳釋放緩慢,導(dǎo)致細(xì)胞毒性減弱。由PEG修飾形成的納米自組裝體被廣泛報(bào)道具有增強(qiáng)滲透與滯留(EPR)效應(yīng)相關(guān)的腫瘤被動(dòng)靶向性,能降低化療藥物毒副作用,因此本課題構(gòu)建了MCF-7乳腺癌荷瘤裸鼠模型,對(duì)PEP載藥系統(tǒng)進(jìn)行體內(nèi)評(píng)價(jià)。結(jié)果表明,PEP載體、藥物親水性及給藥方案均對(duì)藥效產(chǎn)生不同影響。實(shí)驗(yàn)過程中,PEP載藥的劑型相比游離DOX·HCl毒性明顯減小。藥效方面,雖然2mg/kg每天1次連續(xù)3天方式的給藥總量比5mg/kg每3天給1次共3次的方式更低,但2mg/kg對(duì)應(yīng)組的療效更佳。采用相同的給藥方式,PEP-5-DH的抑瘤效果明顯優(yōu)于PEP-5-D,已與游離DOX·HCl相當(dāng),但安全性卻較游離DOX·HCl顯著提高,達(dá)到了降低化療藥物毒副作用的同時(shí),不犧牲藥物療效的目的。為克服MCF-7/Adr腫瘤細(xì)胞對(duì)阿霉素的耐藥性,本文還利用PEP自組裝囊泡的獨(dú)特結(jié)構(gòu)共載DOX·HCl或DOX和氯喹(CQ)。CQ為一種傳統(tǒng)抗瘧藥物,但也被報(bào)道具有抗腫瘤藥物增敏作用,已在臨床試驗(yàn)中進(jìn)行研究。與之相關(guān)的機(jī)制可能是抑制耐藥蛋白的外排,也可能與促癌干細(xì)胞(CSC)凋亡相關(guān)。細(xì)胞毒性實(shí)驗(yàn)發(fā)現(xiàn)阿霉素和CQ聯(lián)合用藥可顯著逆轉(zhuǎn)耐藥,通過Chou-Talalay聯(lián)合指數(shù)法計(jì)算證實(shí)兩者具有協(xié)同作用且隨CQ比例的增加而增強(qiáng)。為了更好的地模擬體內(nèi)環(huán)境,本文構(gòu)建了耐藥性MCF-7/Adr細(xì)胞3D腫瘤球模型,并采用激光共聚焦(CLSM)斷層掃描及掃描電鏡(SEM)對(duì)各試驗(yàn)組的腫瘤球內(nèi)滲透劑抑瘤效果進(jìn)行評(píng)價(jià)。相比游離DOX·HCl只能停留在腫瘤球表面,CQ和DOX·HCl聯(lián)合用藥能更好地滲透進(jìn)入腫瘤深部,滲透效果最好的是按DOX·HCl和CQ以1：1比例共載的PEP制劑(PEP-DHC-1),能完全滲透進(jìn)入腫瘤深部,且給藥7天后,腫瘤球明顯瓦解。流式凋亡檢測(cè)證實(shí)PEP-DHC-1處理的腫瘤球凋亡率也最高,提示該載藥自組裝體有望在動(dòng)物體內(nèi)逆轉(zhuǎn)耐藥,提高阿霉素的抗腫瘤療效。總之,PEP作為一種新型納米自組裝藥物載體,具有高效裝載親水性或疏水性藥物能力,可在抗腫瘤治療方面,保證安全性并提高藥效,值得進(jìn)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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943;R96
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本文編號(hào)：1445523