發展具有比例螢光輸出酸性應答型前藥奈米粒子應用於藥物制放

Development of Acid-Responsive Prodrugs Nanoparticles with Fluorescence Ratiometric Readout for Application in Drug Delivery

劉力瑋(Li-Wei Liu)

指導教授 ： 陳昭岑

國立臺灣大學/理學院/化學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201704279

摘要

現今化療藥物如喜樹鹼以及紫杉醇溶解度不佳，導致在標的有效濃度過低，因此將藥物裝載於聚合物奈米載體中，有助於提升水溶性、延長在體內循環時間、提升藥物在標的之濃度等優點。並且利用癌細胞特殊微環境如缺氧、高溫、過度表達之生物標記以及偏酸之環境設計奈米載體，其會在癌細胞釋放藥物，減少對正常細胞之副作用。因此設計並合成兩種酸敏感鍵結型聚合物奈米載體，在3HF-hzPTXPC設計中以腙鍵連接紫杉醇在生物可降解之兩性嵌段聚碳酸酯上作為疏水端，並引入3-羥基黃酮作為螢光團，在酸性條件下腙鍵會被水解使原本疏水性的部分轉換成親水性的聯胺，造成微胞脹大伴隨藥物釋放以及3-羥基黃酮綠到藍之比例螢光輸出變化。在DOX-hzCPTPC設計中，以腙鍵連接具有螢光之喜樹鹼作為疏水端，並在製作微胞同時包入阿黴素，喜樹鹼與阿黴素具有協同作用。此外，在微胞中喜樹鹼與阿黴素距離相近會有FRET機制，但當在酸性條件下微胞脹大，兩藥物分離FRET機制消失，因此可藉由紫紅色到藍色之螢光顏色變化觀測藥物釋放之情形。
以動態光散射光譜、穿透式電子顯微鏡以及螢光光譜分別觀測在生理條件與酸性條件下微胞大小、螢光之變化。在3HF-hzPTXPC設計中，在pH 5.0之下微胞隨時間增加直徑增大1.3倍並有綠到藍的比例螢光輸出，而在pH 7.4之下微胞大小與螢光不隨時間改變。接著利用HPLC定量分析紫杉醇在pH 5.0釋放量是在pH 7.4之下的2.1倍。最後進行細胞實驗以流式細胞儀、共軛焦雷射顯微鏡證明3HF-hzPTXPC有進入到細胞中，以及細胞毒性實驗證明3HF-hzPTXPC相較於紫杉醇可以用較少之藥物量便可殺死癌細胞。以上之定性及定量分析證明設計之3HF-hzPTXPC具有潛力作為兼具診斷及治療雙功能之奈米載體。在DOX-hzCPTPC設計中，在酸性條件下微胞確實有脹大之情形，但在螢光光譜中不論是在酸性條件下微胞脹大或是中性條件下喜樹鹼開環皆會導致FRET機制消失，而有紫紅色到藍色螢光的改變，因此在DOX-hzCPTPC設計中以螢光顏色變化來偵測藥物釋放需重新構思。

關鍵字

酸刺激回應聚碳酸酯 ； 自我組裝 ； 比例螢光輸出 ； 3-羥基黃酮 ； 螢光共振能量轉移 ； 藥物傳遞

並列摘要

Poor bioavailability of clinically used chemotherapy drugs such as Camptothecin (CPT) and Paclitaxel (PTX) having low water solubility and the limited dose at the target sites has been a long standing problem for effective therapy. Polymeric drug delivery system, which can improve the water solubility, prolonging circulation time, increasing the amount of drug at the target sites, is a promising carrier to alleviate the problem encountered in the clinic. Utilizing unique phenomena such as hypoxia, higher temperature, acidic milieu, and overexpressing specific biomarkers of the cancer cells, which proliferate fast and metabolize actively, to design theranostic agents for specifically targeting cancer cells and minimizing the adverse effects on the normal cells is a highly fervent pursuit in recent years. Propelled by these motives, two types of acid-responsive polymeric nanoparticles consisting of biodegradable diblock polycarbonate, hydrazine-linked Camptothecin (CPT)/Paclitaxel (PTX), and fluorophores to serve as theranostic agents for effectively delivering Camptothecin (CPT)/Paclitaxel (PTX) and possible imaging in the cells are designed and fabricated. In response to the low pH milieu (e.g. in lysosomes), the hydrazone bonds are hydrolyzed turning the hydrophobic part into a hydrophilic hydrazine moieties in the 3HF-hzPTXPC design. Subsequently the micelle structure would swell or disassemble with the concomitant release of the loaded drug and the fluorescence color of 3-HF changes from green to blue. In the DOX-hzCPTPC design, hydrazone bonds were used to covalently link the fluorescent drug (CPT) as a hydrophobic part and Doxorubicin (DOX) was encapsulated inside the micelles as well. CPT and DOX are both in the confined space facilitating the FRET. Once the micelles swell or disassemble, CPT and DOX are well separated resulting in the low FRET. Synergistically therapeutic effects are expected to achieve.
The morphology and the fluorescence behaviors of the micelle were studied by DLS, TEM and fluorescence microscopy respectively. In the case of 3HF-hzPTXPC, the results indicated that the micelles increase 1.3 fold in diameter with ratiometric fluorescence readout under acid milieu (pH 5.0). In addition, there were no changes in the size and fluorescence color of the micelles at physiological pH value (pH 7.4). The drug release experiment of micelles was conducted by HPLC analysis. PTX was released 2.1 fold at acid milieu than at physiological pH value. Furthermore, cell uptake study confirmed that the micelles are uptake by the cells. In vitro cytotoxicity analysis showed that the micelles can use fewer drugs to kill the cancer cells than free PTX. Collectively, 3HF-hzPTXPC can be potentially served as theranostic nanoparticle. In the case of DOX-hzCPTPC, the micelles increased 1.4 fold in its diameter under acid milieu. However, there were fluorescence color changes no matter under acid milieu or pH 7.4 due to the ring opening of CPT resulting in the low FRET at pH 7.4. As a result, the design of DOX-hzCPTPC micelles is needed to reconstruct.

並列關鍵字

acid-responsive polycarbonates ； self-assembly ； fluorescence ratiometric readout ； 3-hydroxyflavone ； FRET ； drug delivery

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