設計及合成生物可降解之比率螢光輸出型高分子奈米粒子並應用於偵測過氧化氫及酯解酶之探討

Biodegradable Polymeric Nanoparticles Disassemble in Response to Hydrogen Peroxide and Lipase with Fluorescence Ratiometric Readout

傅映樺(Ying-Hwa Fu)

指導教授 ： 陳昭岑

國立臺灣大學/理學院/化學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.11122

摘要

兩性嵌段共聚碳酸酯在生醫方面的用於治療與診斷是非常具有潛力的材料。聚碳酸酯具有生物低毒性、高度生物可相容性、生物可降解性以及在生物可降解前保有良好的機械性質。目前許多棘手的疾病，例如阿茲海默症與帕金森氏症，都與活性氧原子過度表現有關，發展偵測活性氧原子的材料是目前發展蓬勃的領域。我們利用聚碳酸酯這種高度生物相容性及可降解的的特質，發展出一系列兩性嵌段共聚碳酸酯(MTC-MPA-3HF)包含對活性氧原子(Reactive oxygen species)有回應的官能基，並修飾上對環境親疏水敏感的螢光團(3-羥基黃酮)作為顯影之用，並嘗試將其作為藥物載體。我們以環碳酸酯作為單體並修飾上末端烯及硼酯分別作為偵測臭氧與過氧化氫的反應官能基，同時也構成聚合物疏水嵌段的部分。親水嵌段的部分以修飾上乙二醇甲醚的環碳酸酯作為親水嵌段的單體。再以具有3-羥基黃酮結構的雙醇作為開環聚合的引發劑。在路易士酸(TU)及路易士鹼(DBU)的催化下作開環聚合，合成的兩段嵌段共聚碳酸酯可以在水相環境中自我組裝成奈米粒子，在環境中有臭氧或是過氧化氫的存在下，會與對臭氧或是過氧化氫有回應的官能基作用，使疏水段轉為親水段而使整個奈米結構崩散。原先包覆在粒子中心的3-羥基黃酮會從疏水環境轉成親水因而會有綠色轉藍色螢光的比例螢光表現。
從螢光表現來看，設計對臭氧有回應的兩性嵌段共聚碳酸酯(MTC-MPA-3HF-O3)再有臭氧的環境中並沒有預期的綠轉藍的變化，只有發現螢光驟熄。從動態光散射以及穿透式電子顯微鏡觀察，也沒有明顯的大小與外觀轉變。而對過氧化氫兩性嵌段共聚碳酸酯(MTC-MPA-3HF-H2O2)一共設計兩種硼酯作為反應官能基: 脂肪硼酯和芳香硼酯。修飾有此兩種硼酯的兩性嵌段共聚碳酸酯，都具有明顯的綠轉藍的比例螢光表現。從芳香硼酯的例子在加入過氧化氫之後，自動態光散射與穿透式電子顯微鏡都有觀察到結構崩散的現象。而在脂肪硼酯的例子則是觀察到有過氧化氫的環境下，奈米粒子會從著時間漲大而非預期的崩散。此外聚碳酸酯的結構具有很多可能被酯解酶水解的結構，從一系列酯解酶的篩選，發現只有自南極念珠菌提煉的酯解酶有最好的比例螢光表現。

關鍵字

對外在刺激有回應的高分子聚合物 ； 自我組裝 ； 3-羥基黃酮 ； 兩性嵌段高分子 ； 聚碳酸酯

並列摘要

Amphiphilic block polycarbonates have received significant attention in designing a variety of therapeutics and diagnosis owing to their biocompatibility, and tunable mechanical properties. Herein we demonstrate a biodegradable amphiphilic polycarbonate (MTC-MPA-3HF) bearing ROS-reactive pendent groups as a new biomaterial to sense ROS in physiological environment. Before the treatment of stimuli, MTC-MPA-3HF assembles into a stable nanoparticle in aqueous solution resulting ESIPT-dominated green fluorescence in hydrophobic milieu, whereas MTC-MPA-3HF may disassemble upon the treatment of ozone- or hydrogen peroxide emitting ESICT-dominated blue fluorescence (Figure A). Correspondingly, two polymeric structures decorating with different reactive triggers in more hydrophobic block to sense ozone and hydrogen peroxide were designed and synthesized. O3-responsive polymer (MTC-MPA-3HF-O3) employs MTC-Obutene to serve as the ozone-reactive moiety as well as to confer the required hydrophobicity, while H2O2-responsive polymer (MTC-MPA-3HF-H2O2) having MTC-O3dCHBE or MTC-OArBE to serve as the H2O2-reactive group (Figure B). Both polymers feature the same hydrophilic unit, MTC-OmDEG, and the environmentally-sensitive bis-MPA-3HF fluorophore as an initiator.
The fluorescence behaviors and the morphology of the particles were studied by fluorescence spectroscopy and transmission electron microscope, respectively, before and after the treatment of stimuli. The results showed that in the case of MTC-MPA-3HF-O3, ozone seems to react with bis-MPA-3HF much faster than MTC-Obutene resulting in fluorescence quenching without manifesting size change. However, MTC-MPA-3HF-O3 displayed an excellent specificity to the lipase from Candida Antarctica. Either the skeleton or the pendent of the polymer was presumably hydrolyzed to give the fluorescence color change, rendering the application of MTC-MPA-3HF-O3 in antibacterial therapy quite feasible. In the case of MTC-MPA-3HF-H2O2 with alkyl boronic ester, nanoparticles became 500-fold larger with ratiometric fluorescence readout upon reacting with hydrogen peroxide. It is worthwhile to note that replacing alkyl boronic ester with aryl boronic ester gave even better ratiometric fluorescence signals.
In conclusion, we have successfully developed a series of biodegradable amphiphilic polycarbonates bearing ozone- or hydrogen peroxide-reactive groups that may disassemble upon exposure to each targeted stimuli with concomitant fluorescence color change. Furthermore, taking advantages of ester-rich MTC-MPA-3HF, it is feasible to detect the bacterial lipase by monitoring the ratiometric fluorescent readout. These materials can be potentially used in delivering therapeutics or serve as diagnostic agents.

並列關鍵字

stimuli-responsive polymers ； self-assembly ； 3-hydroxyflvaone ； amphiphilic block copolymers ； polycarbonate

參考文獻

2. Sung, Y. M.; Hsu, F. C.; Chen, C. T.; Su, W. F.; Chen, Y. F. Sol Energ Mat Sol C. 2012, 98, 103.

3. Bader, H.; Hoigne, J. Water Res. 1981, 15 , 449.

5. Dharia, J. R.; Johnson, K. F.; Schlenoff, J. B. Macromolecules 1994, 27, 5167.

8. Stuart, M. A. C.; Huck, W. T. S.; Genzer, J.; Muller, M.; Ober, C.; Stamm, M.; Sukhorukov, G. B.; Szleifer, I.; Tsukruk, V. V.; Urban, M.; Winnik, F.; Zauscher, S.; Luzinov, I.; Minko, S. Nat. Mater. 2010, 9, 101.

10. Lehn, J. M. Angew. Chem. Int. Ed. 1990, 29, 1304.