以乙二醇幾丁聚醣、氧化鐵與聚吡咯製備而成之奈米團 簇應用於協同性化學動力及光熱療法

Glycol chitosan/iron oxide/polypyrrole nanoclusters for precise chemodynamic/photothermal synergistic therapy

曲丹尼(Edgar Daniel Quinones Pardo)

指導教授 ： 游佳欣

國立臺灣大學/工學院/化學工程學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101326

摘要

新出現的奈米生醫科技被證實在治療癌症等難以治療的疾病上有好的效果，其中最有潛力的一項技術便是使用光熱療法(photothermal therapy)來靶向癌組織，同時搭配癌細胞的內在機制在治療前弱化癌細胞，改善過往光熱療法需要長時間高溫照射的缺點。本研究利用乙二醇殼聚醣聚吡咯氧化鐵奈米團簇(Glycol Chitosan Polypyrrole Iron Oxide nanoclusters)作為一創新材料，此奈米團簇以乙二醇殼聚醣與氧化鐵包覆核心的聚吡咯，外圍的乙二醇殼聚醣具有透過pH值調控達到靶向癌組織之功能，聚吡咯作為光熱療法的載體，氧化鐵則可搭配活性氧化物質(reactive oxygen species)驅動的化學動力療法，透過光熱與化學動力療法的結合，來滅活熱休克蛋白(heat shock proteins)，達到治療的效果。在此研究中，我們在乙二醇殼聚醣的酸性溶液下，使用六水氯化鐵（𝐹𝑒𝐶𝑙3∙6𝐻2𝑂）和氯化鐵（II）四水合物(𝐹𝑒𝐶𝑙2∙4𝐻2𝑂)作為氧化劑。將吡咯(pyrrole)聚合成聚吡咯，形成乙二醇殼聚醣聚吡咯奈米團簇（GCP NC's），隨後使用吡咯聚合中殘留的鐵離子原位合成氧化鐵 (Fe3O4)。利用氧化鐵的陰離子性質和乙二醇殼聚醣的陽離子性質來自組裝行成奈米團簇。此載體運用了乙二醇殼聚醣pH改變時會改變表面電位的特性，來使載體對癌細胞的酸性微環境具有靶向能力，同時，聚吡咯在近紅外光線照射下的吸熱能力確保奈米載體可達到高溫作為熱療，氧化鐵通過芬頓反應(Fenton reaction)在細胞中產生活性氧化物質，引起氧化損傷並啟動化學動力學治療，從而使熱休克蛋白失活。癌細胞中活性氧化物質的產生是透過2ʹ,7ʹ-二氯熒光素二乙酸酯 (DCFH-DA)的染色與測量螢光強度所得，而熱休克蛋白是使用熱休克蛋白抗體染色後使用螢光顯微鏡測量而得，我們同時使用細胞存活率分析(MTT assay)，此載體對子宮頸癌細胞株 (HeLa) 和正常細胞小鼠成纖維細胞株(L929)的細胞毒性，在癌細胞中的實驗結果中，細胞活性顯著降低，而對正常的細胞影響較小，我們更在細胞實驗中將細胞核透與奈米團簇染色，來映證此載體具有靶向之功能。

關鍵字

癌細胞 ； 奈米團簇 ； 活性氧化物質 ； 芬頓反應 ； 光熱效應 ； pH 響應

並列摘要

Arising novel nanotechnologies have been shown to be exceptional in the treatment of difficult to tackle diseases such as cancer. One of the most promising technics for treatment is the use of photothermal therapy (PTT) to target cancer tissue while at the same time using the intrinsic mechanism of cancer cells to weaken the cells ahead of the treatment reducing the need of excessively high temperatures being applied for a long time. The current study presents Glycol Chitosan Polypyrrole Iron Oxide nanoclusters (GCPI NC´s) as an innovative material for the synergetic treatment of reactive oxygen species (ROS)-driven chemodynamic therapy (CDT) to inactivated HSPs with photothermal-hyperthermia achieved through the development of pH-targeting glycol chitosan/iron oxide enclosed core polypyrrole nanoclusters (GCPI NCs). In the current study Pyrrole was polymerized into Polypyrrole (Ppy) in the presence of an acidic solution of Glycol Chitosan (GC) using iron (III) chloride Hexahydrate (〖FeCl〗_3∙6H_2 O) and Iron (II) chloride tetrahydrate (〖FeCl〗_2∙4H_2 O) as oxidaxing agents. To form Glycol Chitosan Polypyrrole nanoclusters (GCP NC´s). Followed by the in situ synthesis of Iron oxide (Fe3O4) using the residual iron ions from the polymerization of Pyrrole. Taking advantage of the anionic nature of Iron Oxide and cationic nature of Glycol Chitosan to self-assemble the nanoclusters. The nanoclusters take advantage of the pH responsiveness nature of the Glycol Chitosan to enhance the targeting ability of the nanoclusters in the acidic microenvironment of the cancer cells. Similarly, the NIR heat absorbance capabilities of Polypyrrole ensure that the nanoparticle reaches temperatures were hyperthermia affects the cells. While the Iron Oxide generates ROS via Fenton reaction in the cells bringing about oxidative damage and initiates the chemodynamic therapy which in turn inactivate the heat shock proteins. The production of ROS in cancer cells was measured by dyeing with 2ʹ,7ʹ-Dichlorofluorescin Diacetate (DCFH-DA) and the fluorescence intensity was measured. While HSP´s were measured using a fluorescence microscope after applying HSP antibody dye. The effectiveness of the treatment was evaluated in vitro by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to measure the cytotoxicity in both Henrietta Lacks cell line (HeLa) and Normal Mouse fibroblast cell line (L929). Significant reduction in cell viability was exhibited after the treatment in HeLa cells and minor effect on non-cancer L929 cells. Cell experiments also displayed the targeting ability of the nanoclusters to cancer cells by fluorescence microscopy in addition of DAPI dyeing of the cells and Cyanine5 (Cy5) dyeing of the nanoclusters.

並列關鍵字

Cancer cells ； nanoclusters ； ROS ； Fenton reaction ； photo thermal ； pH-responsive

參考文獻

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