| 摘要: | 聚乙烯亞胺(Polyethyleneimine，PEI)除了是一帶有高正電荷密度的高分子外，也因其具有不同質子化程度的胺基，因而能藉由高正電性有效的以胞飲作用傳送基因質體進入細胞，並協助基因質體逃脫核內體(Endosome)，而達到良好的轉染效率，因而堪稱新一代的非病毒型之基因載體。然而由於PEI之高正電密度易導致血栓或凝血作用的產生，反而限制了其在臨床的應用。為了降低PEI的血栓或凝血作用，本研究將高生物相容性的聚乙二醇甲基丙烯酸酯(Poly(ethylene glycol) methyl ether methacrylate，PEGMA)以Michael addition的方式接枝於PEI上，並調控PEGMA在PEI上的接枝數目(PEGMA/PEI) 比為2, 4, 6, 10, 13, 18。首先我們發現PEGMA/PEI小於10時，PEI-g-PEGMA與質體DNA的複合物具有高結合穩定性且其表面電荷仍為正電。此外若PEGMA/PEI大於4以上則具有低溶血性，這意味著PEGMA/PEI之接枝數目比在4到10時之間為基因轉染之最佳比例。另一方面，我們藉由調控PEGMA/PEI與質體DNA複合物的重量百分比(2, 5, 10, 40 wt%)來找尋最佳化基因轉染的條件，結果發現若PEGMA在PEI上的接枝數目比增加時，由於PEGMA的保護作用，會使得轉染效率的區間會趨往高複合物的重量百分比，但轉染效率並未隨之增加。由我們的實驗結果發現最佳轉染效率是在PEGMA/PEI之接枝數目比在4時，且PEGMA/PEI與質體DNA複合物的重量比在2時，這個條件下的轉染效率比以PEI轉染更高且細胞毒性也低於PEI。
本研究進一步將PEI做不同程度之四級胺化，其四級胺化程度為19.8%、21.8%及26.8%，並期望能藉由調整PEI上之不同質子化胺基程度及N/P比，來降低PEI對細胞之毒性和最佳之轉染濃度。我們藉由調控四級胺化之PEI(QPEI)與質體DNA複合物的電荷比(N/P=5, 10, 30, 35, 40, 70)來找尋最佳化之基因轉染條件。由膠體電泳實驗發現QPEI之包覆能力並未比PEI強，在動態雷射光散射粒徑儀(Dynamic Light Scattering, DLS) 和滴定實驗，發現四級胺化後，由於四級胺之質子化能力降低，導致緩衝能力降低。粒徑大小也由於正電性較強，導致粒徑較PEI大。但在轉染實驗及細胞毒性之檢測，可發現QPEI並沒有什麼細胞毒性在N/P比70以下且四級胺化程度為26.8%在N/P比為40之螢光圖下發現其轉染與PEI相當。
Polyethyleneimine(PEI), a polymer with highly positive charged density at physiological condition, has been proposed to be an effectively gene delivery carrier for the translocation of plasmid DNA in cell via endocytosis pathway. According to the proton sponge hypothesis, PEI could facilitate the gene to escape from early endosome resulting in high transfection efficiency. However, the higher cytotoxicity and lower blood compatibility of PEI limit its clinical applications
in particular, PEI would cause the blood coagulation and thrombus. Therefore, we conjugated the biocompatible polymer, poly(ethylene glycol) methyl ether methacrylate(PEGMA), with branched PEI (25 KDa) to reduce the mentioned drawbacks of PEI through the Michael addition method. The PEGMA grafting densities were 2, 4, 6, 10, 13, and 18. The measurements of Zeta potential and gel retardation assay revealed that the stable PEI-PEGMA-DNA complexes (weight ratio above 2) with positive charge are formed as the grafting density less than 10, but the stability of complexes decrease as the grafting density up to 13, even 18. Besides, it exhibited low hemolysis if PEGMA/PEI was more than 4. Otherwise, we found that the PEI-PEGMA-DNA complexes exhibit lower toxicity than PEI-DNA complexes, but the PEI-PEGMA-DNA complexes still have cytotoxicity as the weight ratio of PEI-PEGMA-DNA complexes increase. Furthermore, the transfection experiments of EGFP expression showed that effective transfection locates within the different intervals of the weight ratio of PEI-PEGMA-DNA complexes for different grafting ratio of PEGMA in PEI, and the minimum weight ratio of PEI-PEGMA-DNA complexes for transfection would increase with the increase of grafting ratio of PEGMA in PEI. In this study, we have proposed that the optimal transfection efficiency is under the condition of the grafting ratio of PEGMA/PEI being 4 and the weight ratio of PEI-PEGMA-DNA complexes is 2. Consequently, we developed a polymer-based vector with lower cytotoxicity for gene transfection, and the optimal operation windows are also presented.
Furthermore, we investigated the different degrees of quaternization in PEI whose degrees are 19.8%, 21.8%, and 26.8%, respectively. By tuning the composition of different protonated amines and N/P ratio, we intended to decrease the cytotoxicity of PEI and obtain the optimized N/P ratio for gene transfection. On the other hand, we regulated the charge ratio of quaternized PEI (QPEI) over plasmid DNA ( N/P=5, 10, 30, 35, 40, 70) to achieve the optimized gene transfection. The result of gel retardation showed that the stability of QPEI is less than PEI. From the dynamic light scattering (DLS) measurement, we found that the particle diameter of QPEI-DNA complex is larger than that of PEI due to its stronger positive charge. Besides, the buffering effect in QPEI-DNA complex titration decreases owing to the decrease in the protonation of quaternary amine. Nevertheless, QPEI exhibits little cytotoxicity at the N/P ratio less than 70. Under the N/P ratio equaling 40 and 26.8% of the quaternization degree, the transfection efficiency of QPEI is equivalent to PEI from gene transfection and cytotoxicity measurement. |
