硫酸軟骨素修飾包覆依託泊苷之膽固醇脂微粒及其在小鼠大腸直腸癌對抗藥性治療之研究分析

Cholesterol-based micelles coated with chondroitin sulfate for etoposide delivery to overcome drug resistance in a mouse colon cancer model

朱逸翃(I-Hung Chu)

指導教授 ： 張富雄

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601923

摘要

化學治療是目前最主要治療癌症的方法，但長時間使用藥物下會產生抗藥性降低治療的效果，像是改變藥物的標靶位置使藥物攝取量降低、改變DNA修補系統及細胞凋亡的機制讓癌細胞生存率大幅提升，亦或者是細胞膜上運送蛋白質會將藥物打出細胞體外使它無法毒殺細胞，這些皆是造成該現象的原因，然而藥物輸出蛋白中又以 P醣蛋白(P-gp) 研究最為廣泛，且許多藥物皆是它的受質，例如: Doxorubicin、Etoposide等。現今隨著奈米科技進步，發現能把抗癌藥物包覆進奈米載體中，並透過胞吞作用將藥物運輸至細胞中來避免P-gp之作用，使大量藥物累積在細胞內以達到毒殺效果。奈米載體除了能透過高滲透長滯留(EPR) 效應被動地進入到腫瘤所在的位置釋放出來外，還能修飾上主動標靶物質來增加載體專一性避免攻擊到正常的組織對身體產生副作用，像是玻尿酸能辨認到在多數腫瘤都大量表現的CD44蛋白質受體，因此當載體到達作用位置時會利用胞吞作用進入到細胞本體釋放藥物以達到克服抗藥性的目的，跟玻尿酸相同糖胺聚糖家族的硫酸軟骨素，也能辨認到腫瘤上CD44蛋白質受體，但差異之處在於硫酸軟骨素多了一個硫酸基能避免跟血液中蛋白質作用以提高在血液當中循環的時間，目前並沒有將硫酸軟骨素修飾過後之脂微粒來當作克服抗藥性的方法，因此本研究的目的是將脂微粒與硫酸軟骨素修飾過後之脂微粒做比較來觀察它是否更適合當作一個藥物傳遞系統。本實驗利用以膽固醇為基礎的正價脂質製作出帶正電之脂微粒後，再加入固定莫耳比例的硫酸軟骨素或玻尿酸，運用正負電相吸引原理來製備出被修飾的脂微粒，接著用粒徑分析儀來比較兩者奈米粒子大小，於體外實驗使用螢光顯微鏡、流式細胞儀、及細胞毒殺實驗來分析兩者的吞噬效率、標靶作用和毒性效果，最後在活體上進行腫瘤的治療，從以上實驗結果得知硫酸軟骨素修飾過之脂微粒有較小的粒徑大小有助於高滲透長滯留 (EPR) 效應進入到腫瘤組織當中，此外它的吞噬效率更好能使更多藥物進入到細胞內達到較佳的毒殺效果，在活體方面能大幅降低腫瘤的體積。本研究運用能主動標靶的硫酸軟骨素修飾到帶正價之脂微粒上，去克服由P-gp該運送蛋白質所造成的抗藥性，藉此來分析在體外及活體內治療抗藥性的效果，期望未來能當作應用於活體抗藥性治療之參考。

關鍵字

硫酸軟骨素 ； 脂微粒 ； 癌症

並列摘要

Chemotherapy is a major cancer treatment, but this treatment could be impeded by cellular mechanisms such as altered DNA repair, altered drug targets, and drug efflux pumps. One of the possibilities that cancer cell becoming drug resistance is the overexpression of P-glycoprotein (P-gp) , which is the plasma membrane protein encode by mdr1 gene and acts as an energy-dependent efflux transporter. It can pump out the antitumor drug to reduce intracellular drug accumulation and cause less drug efficiency. There are many ways to overcome the drug resistance such as combining the drug and siRNA which knock down the mdr1 gene. Among these strategies, nanoparticle is preferably and receiving great attention for treatment of drug resistance cancers. Because the nanoparticles can passive target the tumor site through the enhanced permeability and retention (EPR) effect accumulating the drug to kill them, but it still have some problems to apply it in reality. For example, it absences the selectivity to targeting the tumor and leads low antitumor activity and several side effects. Therefore, using various targeting molecules such as folic acid, biotin, hyaluronic acid to develop the active tumor-targeting delivery system becomes a novel treatment. In previously studies, pointing out hyaluronic acid (HA) can increase the antitumor activity owing to it specifically binds to CD44 receptor which is overexpressed in tumor. The other studies notice that the chondroitin sulfate (CS) also can bind to CD44 receptor and it may inhibit undesirable interactions with plasma proteins while circulating in the body due to its sulfate group. Based on these studies, the purpose of this work is comparing every properties of drug-loaded micelle coated with HA or CS to discover which one has a more potential to overcome the drug resistance in colon cancer. In this work, the colon cancer cell lines CT26/WT and CT26/MDR were used as a model and the amount of mdr1a gene of both cell lines was confirmed by PCR assay. The cholesterol based micelles named GCC was good in carrying poorly water-soluble anticancer agent so we used these micelles as a carrier to overcome drug resistance. Then, the etoposide-loaded GCC micelles coated with CS were analyzed with the zetasizer. After confirming these physical properties, flow cytometry, fluorescence imaging, and cell viability analysis were used to observe the different cell uptake rate, cytotoxicity effect and targeting efficiency between the two of them. These results proved that the CS-coated micelles not only had a smaller size to enhance the EPR effect but also had a higher cytotoxicity to CT26/MDR owing to the binding ability of CD44 receptor. Most important of all, CS-coated micelles could truly decrease the volume of tumor in vivo. In this study, we find that CS-coated micelles can overcome drug resistance caused by drug efflux pumps, P-glycoprotein (P-gp), in cancer cell. Then, we use a lot of experiments to analyze their properties in vitro and in vivo. Eventually, we hope this research can provide new applications of nanomaterial for multiple drug resistance treatment.

並列關鍵字

chondroitin sulfate ； micelle ； cancer

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