Polyethyleneimine-lipid conjugate-based pH-sensitive micellar carrier for gene delivery

Abstract

A low molecular weight polyethyleneimine (PEI 1.8 kDa) was modified with dioleoylphosphatidylethanolamine (PE) to form the PEI-PE conjugate investigated as a transfection vector. The optimized PEI-PE/pDNA complexes at an N/P ratio of 16 had a particle size of 225 nm, a surface charge of +31 mV, and protected the pDNA from the action of DNase I. The PEI-PE conjugate had a critical micelle concentration (CMC) of about 34 μg/ml and exhibited no toxicity compared to a high molecular weight PEI (PEI 25 kDa) as tested with B16-F10 melanoma cells. The B16-F10 cells transfected with PEI-PE/pEGFP complexes showed protein expression levels higher than with PEI-1.8 or PEI-25 vectors. Complexes prepared with YOYO 1-labeled pEGFP confirmed the enhanced delivery of the plasmid with PEI-PE compared to PEI-1.8 and PEI-25. The PEI-PE/pDNA complexes were also mixed with various amounts of micelle-forming material, polyethylene glycol (PEG)-PE to improve biocompatibility. The resulting particles exhibited a neutral surface charge, resistance to salt-induced aggregation, and good transfection activity in the presence of serum in complete media. The use of the low-pH-degradable PEG-hydrazone-PE produced particles with transfection activity sensitive to changes in pH consistent with the relatively acidic tumor environment.

Fig. 1

HPLC analysis of the pH-sensitive PEG₂₀₀₀-Hz-PE micelles after incubation at pH 7.4 or 5.0 at 37 °C for 3 h.

Fig. 2

Agarose gel electrophoresis of: A) PEI-PE/DNA complexes in comparison to PEI-1.8/DNA complexes at various N/P ratios; (B) complexes after treatment with DNAase. 1: naked DNA, 2: PEI-1.8/DNA (N/P 16), 3: PEI-PE/DNA (N/P 16).

Fig. 3

Effect of PEG₂₀₀₀-PE on size and zeta potential of PEI-PE/DNA complexes. 1: PEI-PE N/P 16, and PEI-PE N/P 16 with 2: PEG 0.25, 3: PEG 0.5, 4: PEG 1, 5: PEG 2, 6: PEG 5.

Fig. 4

In vitro cytotoxicity of various tested polymers towards B16F10 cells.

Fig. 5

Cellular uptake of DNA-Y from different complexes (PEI1.8 N/P 16, PEI 25 N/P 4, PEI-PE N/P 16) by B16F10 cells after 4h. A) Histogram analysis, and B) Mean fluorescence intensity (MFI) of B16F10 cells after incubation with different formulations. *p < 0.05.

Fig. 6

Confocal microscopy images of cellular interaction of different complexes with B16F10 cell after 4 h incubation. A) Free DNA-Y, and DNA-Y complexes with B) PEI-1.8 (N/P 16); C) PEI-25 (N/P 4); and D) PEI-PE (N/P 16).

Fig. 7

In vitro transfection with: A) PEI-1.8 and PEI-25 complexes; B) PEI-PE and PEI-25 complexes with (+S) and without serum (−S); C) complexes with varying amounts of PEG₂₀₀₀-PE, with (+S) and without serum (−S), 1: PEI-PE N/P 16, and PEI-PE N/P 16 with 2: PEG 0.25, 3: PEG 0.5, 4: PEG 1, 5: PEG 2, and 6: PEG 5; D) complexes with varying amounts of PEG₂₀₀₀-Hz-PE at pH 7.4 or 5.0. 1: PEI-PE N/P 16, and PEI-PE N/P 16 with 2: PEG-Hz 0.5, 3: PEG-Hz 1, 4: PEG-Hz 2.