玻尿酸修飾包覆依託泊苷之脂微粒及其對抗藥性腫瘤治療之分析研究

Abstract

以抗癌藥物治療腫瘤一段時間後，往往會有多重抗藥性(multidrug resistance; MDR)的產生，以至於抗癌藥物無法有效地對腫瘤進行治療。而最常導致MDR的產生即是腫瘤細胞之細胞膜上大量表現藥物運輸蛋白(drug transporter)，其中又以p-glycoprotein (P-gp)最常見。P-gp位於細胞膜表面，可以將其受質排出細胞外，避免受質停留於細胞內，對細胞造成毒殺效果。而P-gp有廣泛的受質專一性，許多抗癌藥物亦是其受質，例如：Doxorubicin、Etoposide等。 以脂微粒作為藥物的載體，用於治療抗藥性腫瘤被認為是一個具有潛力的治療方法，因其可以透過內吞作用 (endocytosis)，有效的將藥物運輸至細胞中，避免P-gp之作用，並有利於增加藥物在細胞中的含量以達到毒殺效果。此外，以玻尿酸 (hyaluronic acid)修飾脂微粒可以減少非專一性之結合、增加循環效果，且玻尿酸為CD44 受體之配位體，可用於做為標靶治療。因此，本研究利用玻尿酸修飾正價脂微粒並包覆抗癌藥物，分析其對腫瘤之治療效果。 本實驗利用膽固醇為基礎的正價脂質 (GEC-Chol)，與膽固醇 (Chol)以不同比例混和，製備膽固醇基礎之脂微粒 (GCC)。接著脂微粒以不同濃度之玻尿酸進行修飾。實驗結果發現，GEC-Chol : Chol之molar ratio為1 : 3，且脂微粒與玻尿酸之濃度比為22.5 : 1時，是脂微粒與玻尿酸混和之最佳比例。而利用流式細胞儀分析細胞對脂微粒及玻尿酸脂微粒之吞噬效果，其結果顯示，細胞對玻尿酸脂微粒吞噬效果較差，表示玻尿酸脂微粒有助於減少非專一性之結合。另一方面，玻尿酸脂微粒包覆抗癌藥物組與未被包覆之藥物組相比，不論在活體外或活體內，皆顯著的增加治療抗藥性腫瘤之效果。 本研究探討玻尿酸包覆脂微粒及其對抗藥性腫瘤治療效果之分析，期望未來可以應用於活體治療，且作為往後用於活體抗藥性腫瘤治療之參考，並改善正價奈米材料應用上的不足，運用在更廣泛之用途。

Chemotherapy is one of the major cancer treatments, but this treatment could be impeded by cellular mechanisms such as multidrug resistance (MDR). The most common attribute to 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. P-gp has the wide range of substrate specificity, for example, Doxorubicin, Etoposide; it can pump out the antitumor drug to reduce intracellular drug accumulation and compromise drug efficacy. Previous study indicates that nanoparticles, with the endocytosis mechanism of cell uptake, have potential to overcome drug resistance and prevent P-gp from pumping out. On the other hand, micelles modified with hyaluronic acid could reduce non-specific binding and improve half life in blood circulation. Furthermore, hyaluronic acid as ligand of CD44 receptor on general tumor cells surface also could be strategy of targeting therapy for cancers. Consequently, we used hyaluronic acid modified micellar etoposide to study the drug resistance tumor. In this study, the major lipid, GEC-Chol which is a kind of cationic cholesterol-based lipid was fabricated, and mixed with different ratio of cholesterol to prepare cholesterol-based nanoparticles (GCC). Then, modified with hyaluronic acid in different concentration. The results indicate that, GEC-Chol : Chol as 1 : 3, and micelles : hyaluronic acid as 22.5 : 1, is the most perfect ratio to study. To reveal the cellular uptake of hyaluronic acid modified micelles, flow cytometry was used to analyze the changes in amount of fluorescence in cells and analysis statistical results. The results shows that, hyaluronic acid modified micelles have less affinity with tumor cells than GCC, so it is a potential strategy to bring down the non-specific binding. Moreover, the cytotoxicity of hyaluronic acid modified micellar etoposide has more efficiency compared to free drug whatever in vivo or in vitro. The findings of this study have important implications for understanding effectiveness of hyaluronic acid modified micelles targeting to drug resistant tumor cells. It is expected that the hyaluronic acid modified micelles will be used widely in the future, and can improve the inadequate in nanomaterial applications for cancer therapy.