Herein, we demonstrate that GMP polymer showed a little cytotoxicity and enhanced transfection efficiency, cellular uptake and maintained the phenotypic expression of human AD-MSCs. The results suggest that GMP polymer has excellent potential to be used in in vivo gene delivery.
3. Results and Discussion
3.1 Synthesis of GMP polymer
Very recently, Lee et al. reported that glycol chitosan-methyl acrylate-polyethylenimine (GMP) polymer showed potential for gene carrier to cell types such cancer cells . The synthesis of GMP polymer was confirmed by 1H-nuclear magnetic resonance (NMR) spectroscopy (the Supporting information, Figure S1). According to the 1H NMR spectroscopy of GMP polymer, both glycol chitosan and PEI 800 Da grafting were achieved.
3.2 Polyplex formation assay for the synthesized GMP polymer and pDNA
The potential of the synthesized GMP polymer as a cationic polymer efficiently forming polyplexes with pDNA is an important factor for efficient gene delivery. To further confirm the previous findings regarding polyplex formation with pDNA, the PicoGreen assay was performed. The fluorescent signal of PicoGreen was significantly enhanced when it strongly interacted with double-stranded DNA (dsDNA). The complex of GC, PEI 800 Da, and GMP polymer were mixed with pDNA at several weight ratios. As shown in Figure 1, complete pDNA condensing occurred at the weigh ratios of 1 for each of the PEI 800 Da and GMP polymer. However, GC/pDNA did not form complete pDNA packing even at a high weight ratio. Therefore, the GMP/pDNA completely interacted at the weight ratio of 1. These results suggest that GMP polymer can be suitable for efficient gene delivery in vitro.
3.3 Cytotoxicity studies
In the previous reports, the high ...
... middle of paper ...
... weight ratios. As shown in Figure 3A, after 24-h post-transfection, compared to PEI25KD at a weight ratio of 1:1, the gene expression level of GMP polymer increased with increasing weight ratios. However, GMP polymer showed enhanced transfection efficiency.
Furthermore, cell viability assays were performed concurrently to the transfection assay. As shown in Figure 3B, the cell viability was dependent on the weight ratio. PEI25KD showed very high cytotoxic effects even at a ratio of 1. In contrast, GC, PEI 800 Da, and GMP polymer showed high cell viability, particularly for the highest concentrations. Based on these results, a weight ratio of 8 was used for further experiments. The results suggest that cancer cell line as well as human AD-MSCs could have improved gene delivery efficiency owing to the facilitation of the endosomal escape by the proton sponge effect.
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