發展1,4-二硫蘇糖醇響應性比例螢光型高分子奈米探針與麩胱甘肽響應性高分子奈米藥物載體

Development of Ratiometric Fluorescent Polymeric Micelles for 1,4-dithiothreitol (DTT) Detection and Glutathione (GSH) Responsive Theranostics Polymeric Drug Carrier

張乃文(Nai-Wen Chang)

指導教授 ： 陳昭岑

國立臺灣大學/理學院/化學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202103698

摘要

基於高分子奈米粒子具有高生物相容性、高生物可降解性、易調控的物理與化學性質，以及高度可修飾性的優點，可藉由裝載上導引、診斷、治療或顯影等不同功能，形成多功能的智慧載體，在近年來成為備受矚目的研究領域。本研究主要發展具有螢光的嵌段式高分子，在水溶液中可自組裝成奈米粒子，根據偵測的分子以及是否裝載藥物區分為兩部份。第一部分針對胱胺酸症發展兼具診斷與顯影功能的1,4-二硫蘇糖醇 (1,4-dithiothreitol, DTT) 響應性比例螢光奈米探針。設計並合成單邊側鏈型3HF-SS-DEG、雙邊側鏈型3HF-2(SS-DEG) 與直鏈型PEG-3HF-SS三種不同結構的兩親性高分子，三者皆具有親水段、質子環境敏感螢光團3-HF，與具雙硫鍵之疏水段，皆可經自組裝形成奈米微胞。經由不斷優化高分子親水性與微胞溶液之製作與組成條件，最後以直鏈型PEG-3HF-SS發展出偵測效果最佳的Micelle-C 。在有DTT存在的環境中，DTT會與PEG-3HF-SS之雙硫鍵進行硫醇-雙硫鍵交換反應而使高分子結構親水性提升，並使Micelle-C微胞膨脹，且伴隨由綠到藍的比例螢光輸出變化，以此偵測環境中DTT之濃度。在常見的生物硫醇中，Micelle-C對DTT有較高的選擇性，且藍綠螢光比值與DTT濃度高度相關，因此適合應用於輔助胱胺酸症治療，以避免DTT用量過高導致中毒。
第二部份則是發展兩款能針對癌症環境中過度表達之麩胱甘肽 (glutathione, GSH) 控制釋放藥物的螢光奈米藥物載體Micelle-click-PTX與Micelle-crosslink-PTX，前者利用共價鍵連接藥物；後者藉由疏水作用力包覆藥物 (圖2)。Micelle-click-PTX利用雙硫鍵共價連接藥物的兩親性高分子PEG-3HF-prodrug自組裝形成，在與GSH作用後會進行雙硫鍵交換反應，並在自我級聯反應後脫去藥物，然而無法將藥物順利從微胞內部釋出。經由穿透式顯微影像，以及高效液相層析分析結果得知Micelle-click-PTX疏水結構過於緻密 。Micelle-crosslink-PTX則是透過具有螢光特性的AO-3-DSF作為交聯劑，利用兩親性高分子PEG-N3自組裝的同時形成交聯性奈米載體，並將藥物包覆在內。此結構設計可降低藥物在運送過程中洩漏的機會。Micelle-crosslink-PTX在GSH的作用下AO-3-DSF斷裂，放出綠色螢光，在兩天內可釋放出約87%的藥物。細胞實驗結果證明Micelle-crosslink-PTX能順利的進入細胞中，並具有良好的顯影與毒殺細胞的效果，具有運用於生物體中探測GSH濃度變化與治療癌症的潛力 。

關鍵字

奈米探針 ； 比例螢光 ； 1,4-二硫蘇糖醇 ； 交聯性藥物載體 ； 麩胱甘肽

並列摘要

Polymeric nanoparticles have attracted considerable interest over recent years due to their high biocompatibility, biodegradability, and flexible structural modifications. By installing various functions such as targeting, diagnosis, therapy, or imaging, multifunctional smart polymeric nanoparticles can be designed and frabricated. This study takes the advantages of polymeric nanoparticles aiming to develop fluorescent block copolymers that can self-assemble to polymeric micelles. Based on the targeted analytes and whether drugs are loaded, the study is divided into two parts. The first part aims to develop a ratiometric 1,4-dithiothreitol (DTT)-responsive fluorescent nanoprobe. Three different structural features of amphiphilic block copolymers containing a hydrophilic segment, a proton-sensitive ratiometric fluorophore 3-HF, and a hydrophobic segment with a disulfide bond were designed and synthesized, denoted by 3HF-SS-DEG (branched diblock copolymer), 3HF-2(SS-DEG) (branched triblock copolymer), and PEG-3HF-SS (linear diblock copolymer). After optimizing the hydrophilicity of the copolymers as well as the micelle fabrication conditions and the composition of the solutions, stable and DTT-responsive nanoprobes can only be obtained by using PEG-3HF-SS copolymers. The resulting self-assembled stable micelle is denote by Micelle-C. In response to DTT, Micelle-C swells and the fluorescent color changes from green to blue, indicating that DTT reacts with the disulfide bonds via the thiol-disulfide exchange, resulting in the increased hydrophilicity. Moreover, Micelle-C displays higher selectivity towards DTT than other common biothiols, and the blue/green emission ratio is highly correlated with the concentration of DTT. Consequently, Micelle-C is suitable for Cystinosis therapy to prevent using excessive dosage of DTT.
The second part describes two types of glutathione (GSH)-responsive fluorescent drug nanocarriers, Micelle-click-PTX and Micelle-crosslink-PTX, which carry drugs either via covalent bond or encapsulation of noncovalent interactions. Micelle-click-PTX is self-assembled by the amphiphilic polymer PEG-3HF-prodrug which covalently links the anticancer drug PTX by the disulfide bonds. After GSH reacts with the disulfide bond, a cascade reaction is triggered and PTX can be released. However, the drugs could not escape from the micelle successfully owing to the high densed hydrophobic part of Micelle-click-PTX confirmed by the TEM images and HPLC analysis. Micelle-crosslink-PTX was fabricated by crosslinking the amphiphilic polymer PEG-N3 via the thiol reactive fluorescent crosslinker AO-3-DSF, and PTX was encapsulated during the self-assembly of PEG-N3. The resulting Micelle-crosslink-PTX can effectively reduce drug leakage during transportation. In response to GSH, the S-S cross-linkage is cleaved and the green fluorescence is emitted. 87% of PTX drugs can be released from Micelle-crosslink-PTX in 2 days. Moreover, Micelle-crosslink-PTX can get into the HeLa cells in which the endogenous GSH concentration is high and displays significant green fluorescence. In vitro cytotoxicity analysis revealed that the polymer itself is not very toxic to the cells. Only the drug loaded Micelle-crosslink-PTX displays high cell toxicity. It can thus be concluded that Micelle-crosslink-PTX has potential to serve as a target-responsive theranostics for cancer treatment.

並列關鍵字

Nanoparticle ； 1,4-Dithiothreitol ； Ratiometric fluorescence ； Glutathione ； crosslinked nanocarrier

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