by Chan Chi-kin. / Bibliography: leaves 95-99 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1986

構建安全、高效、可控的基因傳輸載體是當前人類基因治療中亟待解決的問題。儘管人工合成的高分子型非病毒載體在安全性上優於病毒載體，它們通常不具備必要的基因轉染效率。當前，由於缺乏對聚合物載體細胞內傳輸機制的深入認識，研究人員尚未建立起有關聚合物分子物理化學性質與其基因傳輸效率的有效聯繫，從而難以進一步提高聚合物載體在體外和體內的基因轉染效率。 / 為實現有效的基因轉染，陽離子型聚合物載體的首要任務是將負電性的DNA長鏈壓縮成為納米級別的複合物。由於聚乙烯亞胺（PEI）是當前最有效的陽離子型聚合物基因載體，我們首先以PEI為模型，運用鐳射光散射及凝膠電泳等方法重新對DNA分子與PEI載體在溶劑中的複合過程進行了跟蹤表徵。研究發現，DNA分子在較低的“PEI氮元素：DNA磷元素“（N:P）比值下即能被PEI分子完全壓縮，形成納米級別、表面帶有微弱正電的複合物，且這一結果不因所選用的PEI分子尺寸、拓撲結構和複合溶劑而有所不同； 另一方面，DNA/PEI複合物僅在較高的N:P比值下（N:P ≥ 10）才具備理想的基因轉染效率。上述結果表明，1）在N:P ~ 10的DNA/PEI複合溶液中，PEI分子以“複合PEI“及“遊離PEI“(~70%）兩種形式存在；2) 相比於複合PEI，遊離PEI對基因傳輸起著至關重要的作用。無論在轉染數小時前或數小時後加入，遊離PEI均能顯著提高複合物的體外基因轉染效率。 / 在上述研究的基礎上，我們分別考察了不同分子鏈尺寸及拓撲結構對複合PEI及遊離PEI轉染性能的影響。研究表明，游離PEI的分子鏈長對DNA/PEI複合物的基因傳輸效率有著至關重要的影響。長鏈遊離PEI（~25K）對轉染的促進效果是短鏈游離PEI的數百倍。另一方面，長鏈遊離PEI的分子拓撲結構（線型、支化）對其轉染性能沒有顯著影響，但短鏈線型遊離PEI的轉染性能通常是短鏈支化游離PEI的10倍。上述結果表明，複合PEI主要在DNA分子的壓縮與保護方面發揮作用，而游離PEI則是幫助DNA/PEI複合物在細胞內跨過層層障礙的關鍵。 / 為了闡明游離的陽離子聚合物鏈在基因傳輸中的作用，我們開發並採用了一種基於鐳射共聚焦顯微鏡的三維圖像觀測處理方法，定量比較了在不同游離PEI存在下，DNA/PEI複合物在細胞內的攝入總量及其在溶酶體中的分佈。細胞內吞動力學顯示，長鏈游離PEI在一定程度上提高了DNA/PEI複合物的攝入速率，但游離PEI的主要貢獻在於促進複合物的細胞內傳輸。在clathrin介導的傳輸路徑中，游離PEI對基因轉染的促進作用與其阻止複合物陷入溶酶體的能力密切相關。研究表明，在無游離PEI或只有短鏈支化游離PEI存在的情況下，細胞內複合物陷入溶酶體的比例隨加入時間的延長而持續上升，並在加入6小時後升至40%。另一方面，在短鏈線形或長鏈游離PEI的协助下，複合物陷入溶酶體的比例則低至15%。值得注意的是，利用特異性藥物關閉溶酶體上的質子泵後，遊離PEI的效率僅有部分降低，說明被廣為接受的質子海綿效應在這一過程中並未起到主導和決定性作用。 / 游離PEI對細胞內基因傳輸的促進作用在caveolae介導的傳輸路徑中同樣有所體現。在僅有少量游離PEI存在的情況下（N:P = 4），caveolae介導的傳輸路徑一旦被切斷，複合物的基因傳輸將被完全抑制；在同樣條件下，如果clathrin介導的傳輸路徑被切斷，複合物的基因轉染效率则被提高近3倍。上述結果說明，在低N:P下(≤ 4)，大多數沿clathrin路徑傳輸的複合物最终被限制在酸性溶酶體中，只有沿caveolae路徑傳輸的複合物才能表現出一定的基因轉染效率。另外，在有足夠游離PEI存在的情況下（N:P = 7），如果分別抑制clathrin和caveolae介導的傳輸路徑，複合物的轉染效率僅被降低2倍。上述結果綜合說明，具有適宜分子鏈長(15-20 nm)的游離PEI能夠促進基因的細胞內傳輸，其作用機制主要為1）通過遮罩嵌於細胞內膜的信號蛋白，延遲或阻止複合物所在的早期內吞囊泡與細胞內晚期內涵溶酶體的融合；2）通過破壞早期內吞囊泡、早期內涵體或小窩體（caveosome）的磷脂雙分子膜，促使陷入其中的複合物釋放到細胞質中進行後續的基因傳輸。 / The development of safe, efficient and controllable gene-delivery vectors has become a bottleneck to human gene therapy. Synthetic polymeric vectors, although safer than their viral counterparts, generally do not possess the required efficacy. Currently, the exact mechanisms of how these polymeric vectors navigate to pass each intracellular gene-delivery obstacle (“slit“) and their particular physical/chemical properties that contribute to the efficient intracellular trafficking remain largely unknown, making it rather difficult to further improve the efficacy of non-viral polymeric vectors in vitro and in vivo. / The complexation and condensation of long anionic DNA with cationic polymer chains into small aggregates (~10²-nm) is the first and necessary step in the development of polymeric vectors. Our revisit of the complexation between DNA and polyethylenimine (PEI), one of the most efficient cationic polymeric vectors, by using a combination of laser light scattering and gel electrophoresis demonstrates that nearly all the DNA chains are complexed with PEI to form polyplexes when the molar ratio of nitrogen from polymer to phosphate from DNA (N:P) reaches ~3, irrespective of the chain length of PEI and solvent used. However, a high in-vitro gene transfection efficiency is only achieved when N:P ≥ 10. Putting these two facts together, it has been concluded that 1) each solution mixture with a higher N:P ratio actually contains two kinds of cationic chains: bound to DNA and free in the solution (~70%); and 2) it is those free PEI chains that actually promote the gene transfection no matter whether they exist (are added) many hours before or after the administration of the polyplexes (N:P = 3). / Further, effects of the length and topology of both the bound and free polycationic chains on the gene transfection were respectively studied. Notably, both short (~2K) and long (~25K) PEI chains are capable of condensing DNA completely at N:P ~ 3 but long free chains are ~10²-fold more effective in enhancing the gene transfection, indicating that the length of free chains plays a vital role in the gene transfection. It is also interesting to note that for long free PEIs, the chain topology has nearly no effect on the transfection efficiency; but for short PEIs, linear free chains are ~10-fold more effective than branched ones. These results illustrate that bound PEI chains mainly play a role in DNA condensation and protection, while it is those polycationic chains free in the solution mixture that should get our attention. / To address how free polycationic chains with a proper length/topology promote the gene transfection, we quantitatively compared the cellular internalization and lysosomal distribution of polyplexes in the presence of different free PEIs by using a confocal image-assisted three-dimensionally integrated quantification (CIQ) method. Cellular uptake kinetics reveals that long free PEI chains boost the uptake rate, but their major contribution is in the intracellular space. In the clathrin-mediated (CME) pathway, the efficacy of free cationic chains is tightly correlated to their ability in preventing the entrapment of polyplexes into the late endosomes/lysosomes. Namely, without free PEI chains or with short branched ones, the fraction of polyplexes inside lysosomes keeps escalating and reaches ~40% in the first 6 h; whereas with short linear or long free chains, the percentage of polyplexes trapped into lysosomes is reduced to less than ~15%. Notably, the well-accepted “proton sponge“ effect plays a certain, but not dominant and decisive, role here because the shut-down of proton pump only partially attenuates the gene transfection efficiency. / In addition to the CME pathway, the efficacy of long cationic free PEI chains in promoting the intracellular trafficking is also revealed in the caveolae-mediated pathway. At N:P = 4, with limited amount of free PEIs, the gene transfection was almost completely abolished when the caveolae-mediated pathway was blocked, whereas the gene transfection efficiency was enhanced by ~3 times when the CME pathway was inhibited. This indicates that at lower N:P ratios (≤ 4) , most of the polyplexes are destined to the acidic lysosomes in the CME pathway, and only the caveolae-dependent route leads to an effective gene transfection. On the other hand, at N:P = 7, with sufficient amount of long free PEIs, the inhibition of either the CME or caveolae route decreased the transfection efficiency only by ~2 times. Collectively, our study found that free cationic PEI chain with a proper length (1520 nm) are able to facilitate the intracellular trafficking, presumably via 1) blocking the signal proteins on the inner cell membrane so that the endolysosomes development is prevented or slowed down; and 2) compromising/disrupting the membrane of the virginal endocytic vesicles, early endosomes or caveosomes so that polyplexes entrapped inside can escape into the cytosol. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yue, Yanan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 109-112). / Abstract also in Chinese. / i / iii / v / viii / ix / Chapter CHAPTER 1 --- Introduction and background --- p.1 / Chapter 1.1 --- Non-viral vectors made of commercial and specially designed polymers --- p.2 / Chapter 1.1.1 --- Polyethylenimine (PEI) --- p.3 / Chapter 1.1.2 --- Poly[2-(dimethylamino) ethyl methacrylate](PDMAEMA) --- p.7 / Chapter 1.1.3 --- Cyclodextrin-based polymers --- p.8 / Chapter 1.1.4 --- p-H-sensitive, membrane-disruptive polymers --- p.11 / Chapter 1.2 --- Important remaining issues --- p.14 / Chapter 1.2.1 --- Role of cationic chains free in solution mixtures of DNA and polymer --- p.15 / Chapter 1.2.2 --- Endocytosis pathway on intracellular fate of polyplex --- p.16 / Chapter 1.2.3 --- The “proton sponge“ effect --- p.18 / Chapter 1.2.4 --- Nuclear localization and unloading of DNA --- p.21 / Chapter 1.3 --- Objective and main achievements --- p.24 / Chapter 1.4 --- References and notes --- p.27 / Chapter CHAPTER 2 --- Revisit complexation between DNA and polyethylenimine-Effect of uncomplexed chains free in the solution mixture on gene transfection --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and methods --- p.34 / Chapter 2.3 --- Results and discussion --- p.39 / Chapter 2.4 --- Conclusion --- p.53 / Chapter 2.5 --- References and notes --- p.55 / Chapter CHAPTER 3 --- Revisit complexation between DNA and polyethylenimine-Effect of length of free polycationic chains on gene transfection --- p.58 / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and methods --- p.60 / Chapter 3.3 --- Results and discussion --- p.65 / Chapter 3.4 --- Conclusion --- p.80 / Chapter 3.5 --- References and notes --- p.82 / Chapter CHAPTER 4 --- Quantitative comparison of endocytosis and intracellular trafficking of DNA/polymer complexes in the absence/presence of free polycationic chains --- p.85 / Chapter 4.1 --- Introduction --- p.85 / Chapter 4.2 --- Materials and methods --- p.87 / Chapter 4.3 --- Results and discussion --- p.90 / Chapter 4.3.1 --- Quantitative comparison of intracellular trafficking of polyplexes in the absence/presence of free PEI chains in clathrin-mediated pathway --- p.90 / Chapter 4.3.2 --- Effect of endocytosis pathway on the intracellular fate of polyplexes --- p.101 / Chapter 4.4 --- Conclusion --- p.104 / Chapter 4.5 --- Future research and perspective on development of non-viral polymeric vectors --- p.106 / Chapter 4.6 --- References and notes --- p.109 / 112

No description available.

Thesis (PhDAppliedScience)--University of South Australia, 2003.

Thesis (PhDAppliedScience)--University of South Australia, 2003.

Thesis (Ph. D.)--University of Canterbury, 2008. / Typescript (photocopy). "First submitted June 2007. Final version January 2008." Includes bibliographical references. Also available via the World Wide Web.

Thesis (MSc)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

Dissertation (PhD)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Thesis (MSc)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Thesis (M.S.)--Rochester Institute of Technology, 2006. / Typescript. Includes bibliographical references (leaves 68-71).