Biodegradable Gene Carriers Containing Rigid Aromatic Linkage with Enhanced DNA Binding and Cell Uptake

Abstract

The linking and modification of low molecular weight cationic polymers (oligomers) has become an attracted strategy to construct non-viral gene carriers with good transfection efficiency and much reduced cytotoxicity. In this study, PEI 600 Da was linked by biodegradable bridges containing rigid aromatic rings. The introduction of aromatic rings enhanced the DNA-binding ability of the target polymers and also improved the stability of the formed polymer/DNA complexes. The biodegradable property and resulted DNA release were verified by enzyme stimulated gel electrophoresis experiment. These materials have lower molecular weights compared to PEI 25 kDa, but exhibited higher transfection efficiency, especially in the presence of serum. Flow cytometry and confocal laser scanning microscopy results indicate that the polymers with aromatic rings could induce higher cellular uptake. This strategy for the construction of non-viral gene vectors may be applied as an efficient and promising method for gene delivery.

Keywords: gene delivery; non-viral vectors; polyethylenimine; structure–activity relationship.

Scheme 1

Schematic illustration for the synthetic route of target polymers.

Figure 1

Gel retardation assay of polymer/DNA complexes at different w/w ratio. (−) and (+) represent the condition of serum-free or in the presence of 10% serum, respectively. Naked plasmid DNA was used as control.

Figure 2

EB exclusion assay aroused by the addition of polymer.

Figure 3

(A) The stability of polymer/DNA complexes against heparin. The w/w ratio of polymer/DNA was fixed to 0.8; (B) The biodegradable property of the polymers by enzyme stimulated gel electrophoresis experiment. All w/w ratios of polymer/DNA are 0.8. (−): 10 μL of PBS; (+): 10 μL of elastase (10 U/mL).

Figure 4

Characterization of the polyplexes. Mean particle sizes (A) and zeta potentials (B) of the polyplexes at various mass ratios measured by DLS. TEM images of NaM/DNA polyplex (w/w = 4) in water (C) and in water with 10% serum (D).

Figure 5

Cytotoxicity of the polyplexes at various w/w ratios in 7702 (A) and HeLa (B) cells. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. PEI complexes.

Figure 6

GFP expression in 7702 and HeLa cells induced by the polyplexes at the optimal mass ratio. Images were taken by inverted fluorescence microscope.

Figure 7

Luciferase expression of mediated by the polyplexes at various w/w in 7702 (A: without FBS, B: with 10% FBS) and HeLa cells (C: without FBS, D: with 10% FBS). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. PEI complexes.

Figure 8

Cellular uptake of complexes with 10% serum at optimal w/w ratio in 7702 (A) and HeLa (B) cells quantified by flow cytometry. Columns: percentage of Cy5-positive cells; Dots and lines: fluorescence intensity of internalized Cy5-labeled DNA. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. the mean intensity of fluoresscence of PEI complexes; (C) Cellular uptake level of NaM/DNA polyplexes (w/w = 8) in HeLa cells in the presence of various endocytic inhibitors. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control.

Figure 9

CLSM images of HeLa cells transfected with Cy5-labeled DNA by the complexes at the optimal w/w ratio after 4 h.