Effects of PEGylated and Quaternized Polyethylenimines on Gene Transfection Efficiency

• Main Authors: Yu-Chi Huang, 黃于齊
• Other Authors: Wen-Yih Chen
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/09411422678502580373

碩士 === 國立中央大學 === 化學工程與材料工程學系 === 101 === 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.