A Biodegradable Polyethylenimine-Based Vector Modified by Trifunctional Peptide R18 for Enhancing Gene Transfection Efficiency In Vivo

Abstract

Lack of capacity to cross the nucleus membrane seems to be one of the main reasons for the lower transfection efficiency of gene vectors observed in vivo study than in vitro. To solve this problem, a new non-viral gene vector was designed. First, a degradable polyethylenimine (PEI) derivate was synthesized by crosslinking low-molecular-weight (LMW) PEI with N-octyl-N-quaternary chitosan (OTMCS), and then adopting a designed trifunctional peptide (RGDC-TAT-NLS) with good tumor targeting, cell uptake and nucleus transport capabilities to modify OTMCS-PEI. The new gene vector was termed as OTMCS-PEI-R18 and characterized in terms of its chemical structure and biophysical parameters. Gene transfection efficiency and nucleus transport mechanism of this vector were also evaluated. The polymer showed controlled degradation and remarkable buffer capabilities with the particle size around 100-300 nm and the zeta potential ranged from 5 mV to 40 mV. Agraose gel electrophoresis showed that OTMCS-PEI-R18 could effectively condensed plasmid DNA at a ratio of 1.0. Besides, the polymer was stable in the presence of sodium heparin and could resist digestion by DNase I at a concentration of 63U DNase I/DNA. OTMCS-PEI-R18 also showed much lower cytotoxicity and better transfection rates compared to polymers OTMCS-PEI-R13, OTMCS-PEI and PEI 25 KDa in vitro and in vivo. Furthermore, OTMCS-PEI-R18/DNA complexes could accumulate in the nucleus well soon and not rely on mitosis absolutely due to the newly incorporated ligand peptide NLS with the specific nuclear delivery pathway indicating that the gene delivery system OTMCS-PEI-R18 could reinforce gene transfection efficiency in vivo.

Fig 1. Synthesis and putative structure of OTMCS-PEI-R18.

Fig 2

¹H NMR spectra of (A) OTMCS-PEI and OTMCS-PEI-R18.

Fig 3

MALDI-TOF mass spectra of (A) OTMCS-PEI and (B) OTMCS-PEI-R18.

Fig 4. Determination of buffering capacity of the OTMCS-PEI-R18 polymer by acid-base titration.

Solution containing the polymer (0.2 mg/mL) was adjusted to pH 10.0, and then titrated with HCl from 10.0 to 3.0.

Fig 5. Particle size and Zeta potential of OTMCS-PEI-R18/DNA at various w/w ratios.

The average diameter gradually decreased (A) and the Zeta potential gradually increased (B) as the ratio of conjugated DNA increased.

Fig 6. Agarose gel electrophoresis of plasmid DNA and OTMCS-PEI-R18 /DNA complexes.

Agarose gel electrophoresis of OTMCS-PEI-R18-h/DNA(A) and OTMCS-PEI-R18-l/DNA(B) complexes at various w/w ratios, respectively. (C) Protection of plasmid DNA from degradation by DNase I at different concentrations of 0, 3, 6, 9, 18, 27, 36, 45, 54 and 63 U DNase I/μg DNA. (D) Protection of plasmid DNA from dissociation by sodium heparin at varying concentrations of 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 μg/mL.

Fig 7. The cytotoxicity of OTMCS-PEI-R18.

(A) Degradation of OTMCS-PEI-R18. (B) Cytotoxicity of OTMCS-PEI-R18 and other PEI derivates at various concentrations in Hela cell line using the MTT assay. OTMCS-PEI-R13, OTMCS-PEI and PEI 25 KDa were used as controls.

Fig 8. Gene transfection efficiency of OTMCS-PEI-R18/DNA polyplexes.

(A) Representative fluorescence images for transfection in Hela cells using OTMCS-PEI-R18 at w/w ratios of 5, 10, 20, and 30. (B) Transfection efficiency of OTMCS-PEI-R18/DNA complexes in Hela cell line at w/w ratios of 5, 10, 20, and 30.

Fig 9. The intracellular distribution of complexes OTMCS-PEI-R18/DNA.

(A) Fluorescent confocal microscopic images of Hela cells treated with FITC-labeled OTMCS-PEI-R18 polyplexes for 0.5 h, 1 h and 2 h, where the FITC-labeled OTMCS-PEI-R18 was shown in green, and the HE stained nuclear was shown in blue. (B) Inhibitory effect of different concentrations of Paclitaxel (PTX), Colchicine (Col) and Acrylamide on transfection efficiency of OTMCS-PEI-R18/DNA complex in Hela cells at w/w ratio of 30. The RLU/mg protein in control cells was set to 100%.

Fig 10. Transfection efficiency of pGL3-Control as a reporter gene in mice.