Arote Rohidas B, Hwang Soon-Kyung, Yoo Mi-Kyong, Jere Dhananjay, Jiang Hu-Lin, Kim You-Kyoung, Choi Yun-Jai, Nah Jae-Woon, Cho Myung-Haing, Cho Chong-Su
Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea.
J Gene Med. 2008 Nov;10(11):1223-35. doi: 10.1002/jgm.1252.
Polyethylenimine (PEI) vectors are widely used in gene delivery because of their high transfection efficiency owing to a unique proton sponge effect. An increase in molecular weight increases transfection efficiency, but simultaneously results in increased toxicity. Therefore, the design and synthesis of new degradable gene delivery carriers having high transfection efficiencies and reduced cytotoxicity are necessary.
In the present study degradable poly(ester amines) (PEAs) based on glycerol dimethacrylate (GDM) and low molecular weight branched polyethylenimine (LMW-PEI) were synthesized in anhydrous methanol at 60 degrees C following Michael addition reaction. The transfection efficiencies of the synthesized PEA/DNA complexes were evaluated using three different cell lines (HeLa, HepG2 and 293T cells) in vitro.
PEAs with zeta potential in the range of 30-55 mV (at physiological pH) condensed plasmid DNA into nanosized particles (<150 nm) suitable for intracellular delivery. The PEAs degraded in a controlled fashion (t(1/2) of approximately 9-10 days). Compared with PEI 25K, the PEAs showed significantly lower cytotoxicity in three different cells. The PEAs demonstrated much higher transfection efficiency compared to conventional PEI 25K and Lipofectamine. The PEA synthesized using a 1 : 4 mole ratio of GDM to PEI [GDM/PEI-1.2 (1:4)] showed the highest transfection efficiency in HepG2 cells. Significantly higher pEGFP-N(2) reporter gene expression in 293T cells was achieved using these PEAs. The hyperosmotic effect of PEAs was demonstrated by the reduction in packed cell volume (PCV). The GDM/PEI-1.2 (1:4) showed comparable reduction in PCV with respect to glycerol in 293T cells. The effect of bafilomycin A(1) on transfection efficiency of PEAs on 293T cells indicated its endosomal buffering capacity.
We hypothesized that the higher transfection efficiency of PEAs was the synergistic effect arising from hyperosmotic glycerol and endosomal buffering capacity of PEAs resulting from the presence of a glycerol backbone and PEI amine groups, respectively.
聚乙烯亚胺（PEI）载体因其独特的质子海绵效应导致高转染效率而被广泛用于基因递送。分子量的增加会提高转染效率，但同时也会导致毒性增加。因此，设计和合成具有高转染效率且细胞毒性降低的新型可降解基因递送载体是必要的。
在本研究中，基于甘油二甲基丙烯酸酯（GDM）和低分子量支链聚乙烯亚胺（LMW-PEI）的可降解聚（酯胺）（PEA）在无水甲醇中于60℃通过迈克尔加成反应合成。使用三种不同的细胞系（HeLa、HepG2和293T细胞）在体外评估合成的PEA/DNA复合物的转染效率。
ζ电位在30 - 55 mV范围内（在生理pH下）的PEA将质粒DNA浓缩成适合细胞内递送的纳米颗粒（<150 nm）。PEA以可控方式降解（半衰期约为9 - 10天）。与PEI 25K相比，PEA在三种不同细胞中显示出显著更低的细胞毒性。与传统的PEI 25K和脂质体转染试剂相比，PEA表现出更高的转染效率。使用GDM与PEI摩尔比为1 : 4合成的PEA [GDM/PEI-1.2 (1:4)]在HepG2细胞中显示出最高的转染效率。使用这些PEA在293T细胞中实现了显著更高的pEGFP-N₂报告基因表达。PEA的高渗效应通过红细胞压积（PCV）的降低得以证明。在293T细胞中，GDM/PEI-1.2 (1:4)显示出与甘油相当的PCV降低。巴弗洛霉素A₁对PEA在293T细胞中转染效率的影响表明了其内涵体缓冲能力。
我们推测PEA较高的转染效率是分别由甘油主链和PEI胺基的存在导致的高渗甘油和PEA内涵体缓冲能力产生的协同效应。
