- 學位論文
光感物質共軛樹枝狀高分子以促進細胞內基因傳輸
Enhancing intracellular DNA delivery by photosensitizers conjugated with PAMAM dendrimers
摘要
近年來，樹枝狀高分子(dendrimer)的應用受到廣泛的討論，尤其是在藥物傳輸及基因轉殖上，其中很常被使用到的一種為Polyamidoamine (PAMAM) dendrimer，它的特色在於：(1)結構穩定，顆粒小 (相較於liposome，micell等藥物載體而言)；(2)易透過不同代數的製備來控制大小與構形；(3)近球型結構，且周圍具有許多胺基(amine groups)；(4)高親水性；(5)良好的基因載體。於是我們便可透過它來改變藥物的親水性，以及利用它周圍大量胺基來攜帶藥物或基因。 許多具有光動力療效的光感藥物為疏水性，因此在臨床使用上很難以靜脈注射的方式來給藥。在本研究當中，我們將光感藥物TAMCPP共軛結合第四代PAMAM樹枝狀高分子(PAMAM dendrimer G4)，以期望增加光感藥物的親水性。並且希望透過光感藥物與PAMAM樹枝狀高分子的結合，將光化學內化作用photochemical internalization (PCI)的概念整合到樹枝狀高分子，透過光誘導的方式提昇原來高分子載體的基因轉殖能力。本研究透過化學反應來合成 PAMAM dendrimer G4-TAMCPP (G4-TAMCPP)分子，並利用化學方法分析其基本性質。使用螢光顯微鏡及流式細胞儀探討細胞攝入情形。細胞光毒殺效果則利用430nm光源來引動及分析，再透過此光源的誘導來評估其基因轉殖效率。 由實驗結果得知，所製備的G4-TAMCPP的可見光吸收峰值為423nm，親水性很高，平均粒徑約為60~80nm。由流式細胞儀定量細胞攝入量顯示結合樹枝狀高分子後的光感藥物較原光感藥物攝入量為高，因此等光感藥物濃度餵食下G4-TAMCPP對細胞光毒殺效果亦較TAMCPP為佳。在基因轉殖試驗當中，顯示光照後G4-TAMCPP效果較暗狀態高許多，亦較等量PAMAM G4分子效果佳。 綜合上述，本研究建構了光感藥物與樹枝狀高分子結合的平台，也證明了樹枝狀高分子對疏水性光感藥物應用性的提升，以及整合PCI概念到樹枝狀高分子基因載體的可行性。
並列摘要
Dendrimers as drug or carriers attract highly concern in recent years, and one of the most popular dendrimers is Polyamidoamine (PAMAM) dendrimers. They characterize as : (1)more stable and smaller size (compared with other drug carriers, such as liposomes and micelles) ; (2)easily-controlled size and conformation ; (3) well-difined globular shape and high density of peripheral aminal groups ; (4)high hydrophilicity ; (5)possess good gene transfer capability. Therefore, we could increase the hydrophilicity of drugs by conjugating with PAMAM dendrimers and take advantage of peripheral aminal groups to delivery drusg or genes. Many photosensitizers are phydrophobic, and it would decrease their utility in clinic. In this study, we conjugated photosensitizers (TAMCPP) with PAMAM G4 dendriers to expect to increase the hydrophilicity. Meanwhile, we hoped that the conjugates could integrate the photochemical internalization (PCI) into PAMAM dendrimers to perform light-induced gene transfection. After these PAMAM dendrimer G4-TAMCPP conjugates (G4-TAMCPP) were synthesized, we used different methods to analysis their properties including partition coefficients, thin layer chromatography (TLC) analysis, particle size, morphology and UV-Vis absorption spectrum. Moreover, we analyzed and quantified the uptake of G4-TAMCPP conjugates in cells, compared the PDT effect with free TAMCPP molecules (with 430nm light source), and evaluated their light-induced DNA transfection efficacy. The G4-TAMCPP conjugate had UV-Vis absorption peak at 423nm, revealed high hydrophilicity (low partition coefficients), and their particle size were about 60~80nm. As shown in the quantitative results of flow cytometry, the cellular uptake of G4-TAMCPP conjugates was higher than free TAMCPP molecules at the incubation time 6-48 hours, and resulted in higher PDT effects for G4-TAMCPP than TAMCPP. In DNA transfection experiments, the G4-TAMCPP conjugate revealed DNA transfection efficacy at light-induced condition than dark condition or free PAMAM G4 condition. To sum up, here we constructed a platform of dendrimer-photosensitizer system, proved the possibility and application of hydrophobic photosensitizers modificated with dendrimer and integrated PCI concept to dendrimer gene carriers.
參考文獻
延伸閱讀
- 賴鈺森（2015）。光降解自組裝樹枝狀分子在基因傳遞之樹枝狀複合體形成與光觸發DNA釋放〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/CSMU.2015.00201
- Hsu, W. H. (2012). 結締組織生長因子誘導多功能性幹細胞基因並促進間質上皮轉化效應 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2012.02815
- 林怡文（2017）。Mef2c轉錄因子調控漿狀樹突細胞發育〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU201703495
- 伍函辰（2013）。Oral Specific Promoter Plasmids DNA Delivery by PEGylated Liposomes in Brain and Spinal Cord〔碩士論文，臺北醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0007-2907201323371400
- Yang, P. J. (2020). Construction of intracellular asymmetry and functional differentiation in Escherichia coli [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU202002426