Low-Molecular Weight Polyethylenimine Modified with Pluronic 123 and RGD- or Chimeric RGD-NLS Peptide: Characteristics and Transfection Efficacy of Their Complexes with Plasmid DNA

Abstract

To solve the problem of transfection efficiency vs. cytotoxicity and tumor-targeting ability when polyethylenimine (PEI) was used as a nonviral gene delivery vector, new degradable PEI polymers were synthesized via cross-linking low-molecular-weight PEI with Pluronic P123 and then further coupled with a targeting peptide R4 (RGD) and a bifunctional R11 (RGD-NLS), which were termed as P123-PEI-R4 and P123-PEI-R11, respectively. Agarose gel electrophoresis showed that both P123-PEI-R4 and P123-PEI-R11 efficaciously condense plasmid DNA at a polymer-to-pDNA w/w ratio of 3.0 and 0.4, respectively. The polyplexes were stable in the presence of serum and could protect plasmid DNA against DNaseI. They had uniform spherical nanoparticles with appropriate sizes around 100-280 nm and zeta-potentials about +40 mV. Furthermore, in vitro experiments showed that these polyplexes had lower cytotoxicity at any concentration compared with PEI 25 kDa, thus giving promise to high transfection efficiency as compared with another P123-PEI derivate conjugated with trifunctional peptide RGD-TAT-NLS (P123-PEI-R18). More importantly, compared with the other polymers, P123-PEI-R11 showed the highest transfection efficiency with relatively lower cytotoxicity at any concentration, indicating that the new synthetic polymer P123-PEI-R11 could be used as a safe and efficient gene deliver vector.

Keywords: RGD; nuclear localization signal; physico-chemical properties; polycations; transfection efficiency.

Figure 1

¹H-NMR spectra of (A) P123-PEI; (B) P123-PEI-R4; and (C) P123-PEI-R11 in deuterium oxide at room temperature.

Figure 2

Determination of the buffering capacity of PEI 25 kDa, P123-PEI-R4, P123-PEI-R11 and P123-PEI-R18 by acid-base titration. Each polymer (6 mg) was first dissolved in 30 mL water, and subsequently set to pH 10.0 using 0.1 M NaOH. Then 25 µL increments of 0.1 M HCl were added, and the pH of the solution was measured with a pH meter after each addition. The solutions were titrated to about pH 3.0 with 0.1 M HCI. The titration curve of water is presented as a control.

Figure 3

Representative TEM images of P123-PEI-R4/DNA (a), P123-PEI-R11/DNA (b) and P123-PEI-R18/DNA (c); (B) Particle size of P123-PEI-R4/DNA, P123-PEI-R11/DNA and P123-PEI-R18/DNA at various polymer-to-pDNA w/w ratios; (C) Zeta potential (mV) of P123-PEI-R4/DNA, P123-PEI-R11/DNA and P123-PEI-R18/DNA at various polymer-to-pDNA w/w ratios.

Figure 4

Agarose gel electrophoresis of the complexes at various polymer-to-pDNA w/w ratios: (A) P123-PEI-4/DNA; (B) P123-PEI-11/DNA; (C) P123-PEI-18/DNA).

Figure 5

(A) Plasmid DNA protection by P123-PEI-R4 (a), P123-PEI-R11 (b) and P123-PEI-R18 (c) from degradation by DNaseI at varying concentrations; (B) Plasmid DNA protection by P123-PEI-R4 (a), P123-PEI-R11 (b) and P123-PEI-R18 (c) from degradation by serum at varying concentrations.

Figure 6

Cytotoxicity of PEI 25 kDa, P123-PEI, P123-PEI-R4, P123-PEI-R11 and P123-PEI-R18 at various concentrations in B16 cell lines using the MTT assay. ** p < 0.01.

Figure 7

(A) Green fluorescent protein (GFP) reporter gene transfection in B16 cells by PEI 25 kDa (a); P123-PEI (b); P123-PEI-R4 (c); P123-PEI-R11 (d) and P123-PEI-R18 (e) in 24-well plate. Note: 100 µL polymer/DNA complex solution containing 2.5 µg plasmid pEGFP-N2 at various weight ratios ranging from 5 to 30 were added to each well; (B) Transfection efficiency of PEI 25 kDa/DNA, P123-PEI/DNA, P123-PEI-R4/DNA, P123-PEI-R11/DNA and P123-PEI-R18/DNA in B16 cells at the polymer/pDNA w/w ratio of 5, 10, 20, and 30 in the 24-well plate. Note: 100 µL polymer/DNA complex solution containing 2.5 µg plasmid pGL3-Control at various weight ratios ranging from 5 to 30 was added to each well.