聚乙烯亞胺接枝幾丁聚醣混摻硫酸化軟骨素作為基因傳輸載體之評估

Polyethyleneimine grafted Chitosan blending Chondroitin sulfate as a non-viral gene vector

虞翔(Yu, Siang)

指導教授 ： 朱一民

國立清華大學/工學院/化學工程學系/碩士(2014年)

摘要

本研究的目的是探討將不同重量比例的帶負電高分子聚合物硫酸化軟骨素 (Chondroitin sulfate, CS) 混摻聚乙烯亞胺 (Polyethyleneimine, PEI average MW = 600) 接枝幾丁聚醣 (Chitosan, CHI) 而形成的高分子 (PEI600-g-CHI) 和質體DNA (pDNA) 形成之三元混合物PEI600-g-CHI/pDNA/CS奈米粒子並將其用於基因傳輸載體之可行性評估。

現今將陽離子高分子 (cationic polymer) 用於基因載體 (gene vector) 已被大量的研究。但已有文獻指出帶正電的高分子因其具有很高的電荷密度 (charge density) 而與基因有過強的結合能力，導致載體將基因攜帶進入到細胞內部時，基因無法被載體所釋放。且過高的正電荷密度會影響帶負電的細胞膜表面，造成陽離子高分子材料對細胞具有毒殺性，對細胞造成損害使得基因表現效率變差。而目前有許多的方法來改善陽離子高分子的缺點，導入帶負電的高分子為其中之一。此方法可有效的將陽離子高分子的電性中和，降低其電荷密度，在不影響其承載性能下，降低對細胞的傷害，增加其基因傳遞效果與基因表現效率。

在本研究中所使用的陽離子高分子為聚乙烯亞胺接枝幾丁聚醣 (PEI600-g-CHI)，此高分子結合了聚乙烯亞胺與幾丁聚醣的的優點並同時改善兩者的缺點。而帶負電的物質則選用了硫酸化軟骨素 (CS) ，此物質具有生物相容性與生物可以降解性，並被廣泛的使用於美容與醫療產品中。

加入硫酸化軟骨素可能會影響PEI600-g-CHI與帶負電DNA兩者之間的靜電作用力，導致PEI600-g-CHI與DNA的結合能力下降，使得DNA無法完整的被材料包覆與保護，造成DNA進入細胞內部時被溶酶體 (lysosome) 分解，最終造成轉染效率低落。為了證實加入帶負電的CS仍然不影響PEI600-g-CHI與DNA的結合能力，進行膠體電泳測試；實驗結果顯示出在加入各種不同重量比例的CS情況下，並無任何的pDNA受到靜電作用力的影響而被釋放出來，表示加入的CS並不影響PEI600-g-CHI 包覆基因的能力。在細胞毒性測試方面我們使用了人類胚胎腎細胞 (HEK 293T cell line) 為模型，並以MTT方法測試其對於細胞的毒性；由實驗結果得到無論CS與PEI600-g-CHI的濃度為何，其細胞毒殺性皆不明顯，細胞存活率皆接近百分之百，表示材料對細胞的存活率並沒有顯著的影響。

在粒子粒徑 (Particle size) 與表面電位 (Zeta potential) 測試中，在少量CS加入的比例下，PEI600-g-CHI/pDNA/CS形成之三元混合物粒子粒徑大小小於PEI600-g-CHI與DNA形成的二元混合物，而粒子的表面電位在少量的CS加入下變得較為穩定，並不會出現表面電位不均勻的現象。混合物粒子的外觀形態由穿透式電子顯微鏡 (TEM) 觀察之，在CS對DNA的重量比 (w/w) 為0.2及0.4時，混合物粒子具有比較小的直徑且粒子形態接近球型，是較容易經由細胞的內吞作用 (endocytosis) 機制進入到細胞質的。

在材料的基因傳遞效率與細胞進行轉染步驟之後所分泌的螢光蛋白表現量實驗中，我們將材料包覆帶有綠色螢光片段的基因 (pEGFP-N1) 並使用人類胚胎腎細胞 (HEK 293T cell line) 作為基因轉染表達的細胞。當材料將基因成功送入細胞核進行轉錄與轉譯步驟之後，因為經由載體傳輸送入的質體基因帶有能產生綠色螢光蛋白的基因序列，細胞會製造出綠色螢光蛋白 (Green Fluorescent Protein，GFP)。基因表現效率則以螢光蛋白分泌量的多寡確認之。在本實驗中依照轉染進行的時間不同分為兩個不同的組別，兩組的細胞轉染進行的時間分別為6個小時及24個小時，並在轉染步驟開始後的第72小時取樣並在螢光顯微鏡 (Fluorescent microscope) 下觀察並拍照紀錄綠色螢光蛋白分泌的情形；再以螢光分析儀 (fluorescence plate reader) 測量分泌出的綠色螢光蛋白的螢光值。在轉染步驟進行的時間為24小時之組別，添加CS重量比為0.2的組別，其螢光蛋白表現量有顯著的提升。

關鍵字

聚乙烯亞胺 ； 硫酸化軟骨素 ； 幾丁聚醣 ； 基因轉染 ； 三元混合物

並列摘要

The purpose of this study is to investigate the gene delivery efficacy of a non-viral gene vector by blending an anionic polymer chondroitin sulfate (CS) with a cationic polymer which is composed of polyethyleneimine (PEI, average molecular weight = 600) and chitosan (CHI). These two polymers are linked together via a PEG linker (Poly(ethylene glycol) diglycidyl ether, EX-810) by chemical bonds written as polyethyleneimine grafted chitosan (PEI600-g-CHI).

The use of cationic polymers as gene vector has been widely researched and reported in recent years. However, positively charged polymers could have strong electrostatic force to bind negatively charged DNA. This property of cationic polymers makes DNA unable to be released from polymer/DNA polyplexes, and the high charge density of cationic polymer could damage cell membrane. These all lead to lower transfection efficiency.

In this study, different amounts of CS (compared to DNA) were blended with PEI600-g-CHI and plasmid DNA (pDNA) to form ternary PEI600-g-CHI/pDNA/CS polyplexes. To know whether the adding of negatively charged polymer CS would reduce the binding strength between PEI600-g-CHI and pDNA or not, gel electrophoresis was done and no plasmid DNA was released due to the addition of CS. Results showed that the adding of anionic polymer does not affect the binding ability of PEI600-g-CHI/pDNA polyplexes.

Particle size and zeta potential measurements of the polyplexes were also performed. With adding low weight ratios of CS to pDNA, the PEI600-g-CHI/pDNA/CS nanoparticle compacted pDNA tightly leading the size smaller than PEI600-g-CHI/pDNA nanoparticle. The particle size of the polyplexes is suitable for cellular uptake. Zeta potential measurements showed that the surface charge of polyplexes was stabilized while low weight ratio of CS added. The morphology of polyplexes was observed by transmission electron microscope (TEM). At low weight ratios of CS to pDNA, nanoparticles have diameters about 140 nm and their shapes are spherical.

Cell viability is also a major concern about a good gene vector. Cytotoxicity assay toward human embryonic kidney cell line (HEK 293T cell line) was assayed by MTT method. These tests showed that there was no significant cytotoxicity for binary PEI600-g-CHI/pDNA and ternary PEI600-g-CHI/pDNA/CS polyplexes. Cell viability at any concentration is above ninety percent.

In the experiment of transfection efficiency, a green fluorescent protein-encoding gene (pEGFP-N1) was used to be transfected into HEK 293T cells. If gene can be successfully delivered into cell nucleus, it would produce green fluorescent protein (GFP) through steps of transcription and translation. The transfection efficiency is determined by the amount of green fluorescent protein produced. There were two experimental groups with different time of transfection step; the 6th and 24th hour groups. At the 72th hour after transfection step, samples of GFP expression were observed under fluorescence microscope. Then the intensity of expressed GFP was measured by the fluorescence plate reader. Results showed that ternary PEI600-g-CHI/pDNA/CS polyplexes at 0.2 weight ratio of CS to pDNA expresses the largest fluorescence intensity of GFP. The fluorescence intensity is nearly twice as large as the PEI600-g-CHI polyplexes.

並列關鍵字

Chondroitin sulfate (CS) ； Polyethyleneimine (PEI) ； chitosan (CHI) ； gene delivery, transfection ； ternary polyplexes

參考文獻

[1] D. Stone, "Novel viral vector systems for gene therapy," Viruses, vol. 2, pp. 1002-7, Apr 2010.

[2] S. A. Rosenberg, P. Aebersold, K. Cornetta, A. Kasid, R. A. Morgan, R. Moen, et al., "Gene-Transfer into Humans - Immunotherapy of Patients with Advanced Melanoma, Using Tumor-Infiltrating Lymphocytes Modified by Retroviral Gene Transduction," New England Journal of Medicine, vol. 323, pp. 570-578, Aug 30 1990.

[4] W. F. Anderson, "Human gene therapy," Nature, vol. 392, pp. 25-30, Apr 30 1998.

[5] N. Nayerossadat, T. Maedeh, and P. A. Ali, "Viral and nonviral delivery systems for gene delivery," Adv Biomed Res, vol. 1, p. 27, 2012.

[6] C. Shi, Y. Zhu, X. Ran, M. Wang, Y. Su, and T. Cheng, "Therapeutic potential of chitosan and its derivatives in regenerative medicine," J Surg Res, vol. 133, pp. 185-92, Jun 15 2006.