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標題: | 發展pH響應型藥物傳遞系統及具光活化性質之苯胺芥前藥應用於選擇性烷化去氧核醣核酸 Developments of pH-Responive Drug Delivery System and Photoactivated Aniline Mustard Prodrugs Aiming to Selectively Alkylate DNA |
作者: | Chi-Hao Shiang 項祈豪 |
指導教授: | 陳昭岑(Chao-Tsen CHEN) |
關鍵字: | 藥物傳遞系統,酸性響應釋放藥物,比例螢光顯影,光活化化學療法,去氧核醣核酸烷化, drugs delivery systems,pH-responsive drug release,ratiometric fluorescence readout,photoactivated chemotherapy,DNA alkylating agent, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 發展有效降低脫靶毒性的化學療法,解決抗癌藥物造成的副作用為目前癌症治療的重要目標。刺激響應型前藥能藉癌細胞過度表達的生物指標分子或透過施予光、熱等外源性刺激,造成藥物釋放或活性調控,以增加藥物的選擇性。本研究設計並合成含酸敏感腙鍵之前藥,以點擊反應將前藥連接聚合物,合成聚合物3HF-hzPTXP-PEG-P,並以其自組裝形成酸性響應之藥物傳遞系統。設計中引入3-羥基黃酮作為螢光團,希望藉其所處環境不同時的藍綠比例螢光變化達到同時治療及診斷的效果。
從螢光光譜、動態光散射光譜以及穿透式電子顯微鏡的結果可發現,3HF-hzPTXP-PEG-P由於聚合物的親水性質不足,造成聚合物聚集並析出。增加親水重複單元比例的聚合物3HF-hzPTXP-PEG-P1則能形成穩定的微胞,並可見ESIPT的螢光放出。然而在酸性溶液中,聚合物在脫去藥物後親疏水性質變化不足,而有聚集析出造成螢光下降的現象。增加藥物承載量的3HF-hzPTXP-PEG-P2,由於載體無法包覆過多的前藥,無法在pH 7.4形成微胞。當此奈米載體在酸性環境時藥物被釋放,造成親疏水性質改變,隨著時間增加能看到ESIPT的螢光緩慢下降而ICT的螢光上升。未來的實驗目標為找到適當的聚合物親疏水比例以及藥物承載量,使微胞只在酸性環境下崩解,並具比例螢光消長性質。 除上述利用內源性刺激原發展之藥物傳遞系統外,在複雜的生物體內藥物與生物指標受體進行專一性的配對較為困難。利用光活化化學療法可在空間和時間上控制藥物的活性達到選擇性地針對患部治療。因此以二噻吩乙烯作為光調控開關,在兩端修飾苯胺氮芥基團及吡啶鎓官能基,合成具近紅外吸收波長且具水溶性的Py-F6DTE-Mus-c (Py-c)、NAEPy-F6DTE-Mus-c (NAE-c) 和SPy-F6DTE-Mus-c (SPy-c) 分子。合環狀態分子中,氮芥基團中氮原子的電子可經未定域化至帶正電荷的強拉電子基吡啶鎓環上,使氮原子電子密度下降而降低氮芥基團與DNA進行烷化的能力。照射近紅外光使分子轉為開環形式後,氮芥基團不再受強拉電性的吡啶鎓影響,恢復與DNA進行烷基化的能力。 由化合物在緩衝溶液中的吸收光譜可知,SPy-c比NAE-c有更佳的水溶性,而Py-c則由於水溶性不佳,可能不適合以此化合物發展為藥物。透過凝膠電泳實驗可比較分子在照光前後對DNA烷基化能力的差異。結果顯示NAE-c在照射近紅外光活化後,提高了DNA單加合物的產率;而SPy-c即使提高當量,在照光活化後也難與DNA進行烷基化。由動態光散射光譜及穿透式電子顯微鏡可發現SPy-c在水中形成粒徑小於100 nm之微胞,此時氮芥基團被包覆於內,加上微胞表面帶負電荷可能與帶負電之DNA磷酸鹽骨架相斥而難以與DNA反應。以上研究結果顯示NAE-c相較SPy-c更有成為光活化藥物的潛力。然而從細胞毒性實驗結果來看,NAE-o、NAE-c與經近紅外光活化之NAE-c對MCF-7的毒性差異不大,有中度的細胞毒性,可能仍受限於其水溶性不夠好而未能累積於細胞內,因此未來仍須增進此系列化合物進入細胞的能力。 Minimizing off-target toxicity chemotherapy in order to resolve the side effect caused by the anticancer drugs has drawn a lot of research attentions. Stimulated-responsive produgs could release anticancer drugs or modulate the drug activity by the overexpressing biomarkers or exogenous stimulant such as irradiation or heat, so that the selectivity toward the cacncer cells can be achieved. Herein, a pH-sensitive prodrugs containing hydrazone bond was synthesized and attached to the biodegradable polymer backbones via the click reaction. The obtained polymers 3HF-hzPTXP-PEG-P can self-assemble to micelles. Furthermore, the ratiometric environmentally sensitive fluorophore 3-hydroxyflavone (3-HF) could change the emission wavelength from the excited state intramolecular proton transfer (ESIPT) to the excited state intramolecular charge transfer (ESICT) when micelles collapse to attain theranostic effects. Based on fluorescence spectroscopy, dynamic light scattering spectroscopy (DLS), and transmission electron microscope (TEM) images, it was found that 3HF-hzPTXP-PEG-P would precipitate from the buffer solution due to the insufficient of hydrophilic segments in the polymer. 3HF-hzPTXP-PEG-P1 with the increased hydrophilicity could form stable micelles with ESIPT emission. Under the acidic condition, after releasing the steric hinder drugs, the polymer would precipitate and result in fluorescence quenching. The lack of appreciable changes from hydrophobicity to hydrophilicity can be accounted for the observation. 3HF-hzPTXP-PEG-P2 contaning more prodrugs however could not form micelles in pH 7.4 buffer, perhaps the cavity of the nanoparticles is too small to encapsulate the prodrugs. In low pH milieu, the steric hindered drugs could be released, so these polymers could form micelles with ratiometric fluorescence emission. To attain the ESICT emission increase accompanied with the IPT decrease when the micelles disassembling, the ratio of hydrophilic-to-hydrophobic segments and the amount of prodrugs loding still need to be optimized. Owing to the complicated physiological environment, releasing the prodrugs by the specific biomarkers is rather challenging. The photoactivated chemotherapy could overcome this problem to achieve drug selectivity by high spatiotemporal control over the activity of the drugs. Propelled by the motives, the water solubile and near infrared light-activated Py-F6DTE-Mus-c (Py-c), NAEPy-F6DTE-Mus-c (NAE-c) and SPy-F6DTE-Mus-c (SPy-c) consisting of photo-switchable dithienylethene (DTE) core and aniline mustard, DNA alkylator, were design and synthesized. The elctron-withdrawing pyridinium group attached to the DTE core is aimed to mask the activity of the nitrogen mustard by reducing the electron density via the electron delocalization from nitrogen mustard to the pyridinium moiety. After irradiation of near infrared light (NIR), photoisomerization leads to the interception of the resonance, and the activity of DNA alkylator is thus recovered. From the result of UV-vis absorption spectra, the water solubility of SPy-c is improved and better than NAE-c, whereas the solubility of Py-c was too low to serve as a drug. The denatuerd gel electrophoresis analysis was used to investigate the ability to alkylate double-strand DNA and the selectivity of SPy-c and NAE-c upon the irradiation of NIR. The results showed that NAE-c could alkylate more efficiently upon the irradiation of NIR, whereas SPy-c showed extremely low alkylation ability toward DNA, even if the addition of high equivalents of SPy-c to DNA. The results of DLS and TEM indicate that SPy-c forms micelles with negative Zeta potential on the surface, and the alkylator pointed inward resulting poor alktlation and repulsion between the negative charges of DNA and micelles. Cytotoxicity assays demonstrated that NAE-o, NAE-c and NIR-activated NAE-c displayed moderate cytotoxicity toward MCF-7. The poor water solubility might be the reason. The ability of these photoactivated nitrogen mutard agents to enter the cells remains to be solved. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17154 |
DOI: | 10.6342/NTU202002588 |
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顯示於系所單位: | 化學系 |
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