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標題: | 轉運體與受體標靶修飾之幾丁聚醣奈米顆粒應用於質體DNA遞送之潛力 Potential of Transporter and Receptor Ligand-Modified Chitosan Nanoparticles for Plasmid DNA Delivery |
其他標題: | Potential of Transporter and Receptor Ligand-Modified Chitosan Nanoparticles for Plasmid DNA Delivery |
作者: | 林雋言 Jiun Yann Lim |
指導教授: | 林文貞 Wen-Jen Lin |
關鍵字: | 幾丁聚醣奈米粒子,核酸遞送,LAT1 轉運蛋白,FOLR1 受體,癌細胞轉染, chitosan-base NPs,nucleic acid delivery,LAT1 transporter,FOLR1 receptor,cancer cells transfection, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 核酸藥物是一類新型的生物製劑,在治療多種癌症方面有著臨床潛力。然而,強負電荷,親水性以及對於核酸降解酶的高度敏感限制了其臨床應用。奈米粒子(NPs)遞輸系統有望為上述的缺點帶來改進。儘管奈米粒子擁有被動靶向作用,但主動靶向作用有助於通過一些生物屏障並增加藥物在細胞内的濃度。
幾丁聚醣是一種有幾丁質中萃取出的陽離子聚合物。幾丁聚醣因爲其生物降解性以及良好的生物相容性而常作爲奈米粒子研究中的主要材料。幾丁聚醣上的官能基可以接枝上配體以實現核酸藥物的主動靶向遞輸。奈米載體通過細胞膜的能力對於轉染的效率至關重要。受體介導胞吞作用(Receptor-mediated endocytosis, RME)是靶向遞輸的主要途徑之一。近年來轉運蛋白介導胞吞作用(Transporter-mediated endocytosis, TME)被提出並研究作爲奈米粒子攝取的替代途徑。 在這項研究中,我們通過EDC/NHS偶聯反應將苯丙氨酸(LAT1 轉運蛋白的配體)、葉酸(FOLR1 受體的配體)或 PEG(非靶向配體對照組)接枝到幾丁聚醣上以提高靶向與癌細胞的轉染效率。實驗使用離子凝膠法在交聯劑三磷酸鈉(TPP)的作用下製備奈米粒子。聚合物與TPP重量比2.5以及聚合物與TPP莫耳比5.5所製備之奈米粒子用於癌細胞轉染的評估。配體接枝之幾丁聚醣聚合物用於製備配體修飾之奈米粒子。 通過動態光散射(Dynamic light scattering, DLS)分析,所製備的奈米粒子直徑皆小於200奈米且分佈較密集(PDI<0.3)。表面電位值也顯示正電荷,説明質體DNA被完全包覆在劑型中。凝膠阻滯分析結果也證實了奈米粒子的形成,其穩定性以及對於質體DNA的保護作用。 癌細胞轉染實驗顯示所製備之奈米粒子具有劑量依賴性。當質體DNA的濃度從10 µg/mL 增加到 30 µg/mL 時,EGFP 陽性細胞群的百分比與 gMFI 值都有顯著的增加。EGFP蛋白相對表現量結果顯示CPhe2 與CFA2奈米粒子相較於CPhe1與CFA1奈米粒子在A549人類非小細胞肺癌細胞,bEnd.3小鼠腦微血管內皮細胞,U87-MG惡性神經膠質細胞瘤以及HCT 116人類結腸直腸癌細胞中有著1.3-6.3倍的變化。 苯丙氨酸的靶向效率在 U87-MG 細胞中最高,其次是 A549、bEnd.3 和 HCT116 細胞。葉酸的靶向效率與其在細胞中的表達相關,其中 FOLR1 受體的表達按 A549、bEnd.3、U87-MG 和 HCT116 細胞的依序增加。儘管 LAT1 轉運蛋白的基因表現量高於 FOLR1 受體,但FA接枝之奈米粒子有著相對更好的結果。實驗結果説明RME相對與TME為更佳的靶向遞輸途徑。 Nucleic acid therapeutics is a class of biologics drugs that exhibit great clinical potential in the treatment of a variety of cancers. Yet some unfavorable properties including strong negative charge, hydrophilic nature, and highly sensitive to nuclease degradation of nucleic acid therapeutics restrain its clinical applications. The nanoparticles (NPs) delivery system may be a key to the solution. Despite the passive targeting effect of NPs enhancing cellular uptake, active targeting may help in passing through biological barriers and increased drug accumulation in the human body. Chitosan is a natural cationic biopolymer derived from chitin with renowned biodegradable and biocompatible properties it is often used in studies as the main composition of NPs. Chitosan with various functional groups can be functionalized by a ligand to obtain desired targeted delivery of nucleic acid therapeutics. The entry of cargo-loaded NPs through the cellular membrane is critical for achieving high transfection efficiency. Receptor-mediated endocytosis (RME) is one of the main pathways to achieve targeting delivery. In recent years, transporter-mediated endocytosis (TME) has been proposed and studied as an alternative pathway for NPs cellular uptake. In this study, we conjugated phenylalanine (ligand of LAT1 transporter), folic acid (ligand of FOLR1 receptor), and PEG (non-targeting control ligand) onto the chitosan backbone via EDC/NHS coupling method to improve the targeting and transfection efficiency in cancer cells. Ionic gelation method with sodium triphosphate (TPP) as a crosslinking agent was employed to prepare NPs. Chitosan-based NPs with a weight ratio of 2.5 and a molar ratio of 5.5 of polymer-to-TPP was selected for cancer cell transfection evaluation. Ligand-modified chitosan was used to prepare ligand-modified NPs. Through dynamic light scattering (DLS) analysis, most of the NPs established possessed a size <200 nm with narrow distribution (PDI < 0.3). The positive values of zeta potential indicate full encapsulation of pDNA in the NPs. The electrophoretic mobility shift assay confirms the formation, stability, and protective effect of NPs. The transfection study showed that the prepared NPs possess a dose-dependent manner. The percentage of EGFP positive cell population and gMFI values increase when the concentration of pDNA increase from 10 µg/mL to 30 µg/mL. The relative EGFP expression results showed that CPhe2 and CFA2 NPs have 1.3-6.3 folds change compared to CPhe1 and CFA1 NPs in A549 human lung carcinoma, bEnd.3 mouse brain endothelioma, U87-MG human glioblastoma, and HCT116 human colorectal carcinoma. The targeting efficiency of phenylalanine is highest in U87-MG cells, followed by A549, bEnd.3, and HCT116 cells. The targeting efficiency of folic acid is correlated to its expression in cells where the expression of FOLR1 receptor increases in the order of A549, bEnd.3, U87-MG and HCT116 cells. Although LAT1 transporter has higher gene expression than FOLR1 receptor, FA-conjugated NPs showed better performance in our study, suggesting that RME pathway may have more potential for the targeting delivery of NPs. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/83296 |
DOI: | 10.6342/NTU202300602 |
全文授權: | 同意授權(限校園內公開) |
電子全文公開日期: | 2028-02-20 |
顯示於系所單位: | 藥學系 |
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