Introduction: Improved delivery systems are needed for intracellular delivery of sensitive biologics such as plasmid DNA. Cationic polymers have been developed for this end due to their safety and facile chemistry to tailor their properties. These polymers complex with DNA via electrostatic interactions, and condense them into compact nano-sized polyplexes. Chemical modification of low molecular weight PEIs with hydrophobic groups improves delivery due enhanced cellular uptake and unpacking efficacy of the polyplexes. Unlike bulky lipids, the impact of small hydrophobes on cationic polymers for DNA delivery has not been explored in detail; it is possible that small hydrophobes might be superior substituents due to better grafting reactions and unique properties. In this study, we modified 1.2 kDa PEIs using small hydrophobe propionate (PrA; CH2-CH3) and extensively explored the features of resultant DNA polyplexes and delivery efficiency.
Materials and Methods: The PrA grafted 1.2 PEI (PEI-PrA; Figure 1) was synthesized via N-acylation and characterized through 1H-NMR spectroscopy[1]. DNA binding efficacy of PEI1.2-PrAs were investigated through agarose gel electrophoresis and size-surface charge through dynamic light scattering[2]. Cellular toxicity, DNA uptake (with Cy3-labeleld DNA) and transfection efficacy of PEI1.2-PrA/DNA complexes was studied in breast cancer cells MDA-231 and MCF-7 as a function of PrA substitution amount, post transfection time and polymer:DNA ratio for complex formation. Green fluorescent protein (GFP) transfection was assessed by flow cytometry.
Results and Discussions: Substitution efficacy of PrA onto 1.2 PEI was increased with feed ratio (PrA/PEI) with highest grafting of 1.6 PrA/PEI. DNA binding efficacy of PEI-PrA was decreased in proportion with PrA substitution as a consequences of –NH2 consumption and/or steric hindrances. Hydrodynamic size of polyplexes was identical irrespective to PrA substitution, but surface charge initially increased with PrA substitution and later decreased at high PrA substitutions. Cellular toxicity of the polymers was increased with PrA substitution but the polymers still displayed less toxicity compared to 25 kDa PEI (PEI25). GFP transfection efficiency in both MDA-231 and MCF-7 was significantly increased with optimal PrA substitution (0.5-1 PrA/PEI) while polymers with the highest substitution and parent polymer were ineffective (Figure 2). These results paralleled the results obtained from cellular uptake of plasmid DNA. Importantly, transfection efficiency of PEI-PrA1 (PrA/PEI = 0.76) was higher than long chain lipid (C=18) grafted 1.2 PEI and comparable to PEI25. In addition, PEI-PrA1 showed higher transfection than PEI 25 at day 7 and 14, showing a relatively stable transfection in MDA-231 cells.
Conclusions: Substitution of small hydrophobe PrA onto PEI1.2 enhanced transfection efficiency in breast cancer cells, but excess substitution (>1.2 PrA/PEI) was detrimental, emphasizing the importance of balancing polymer hydrophobicity for effective gene delivery. Thus, integration of small hydrophobe onto low molecular weight PEIs seems as effective as large lipids.
This study was supported by an NCPRM (NSERC CREATE program for regenerative medicine)
References:
[1] KC R., Landry B, Aliabadi HM, Lavasanifar A, Uludag H. Lipid substitution on low molecular weight (0.6-2.0 kDa) polyethylenimine leads to a higher zeta potential of plasmid DNA and enhances transgene expression. Acta Biomater. 2011;7:2209-17
[2] Neamnark A, Suwantong O, KC R., Hsu CY, Supaphol P, Uludag H. Aliphatic lipid substitution on 2 kDa polyethylenimine improves plasmid delivery and transgene expression. Mol Pharm. 2009;6:1798-815.