FGF receptor-mediated gene delivery using ligands coupled to PEI-β-CyD

Abstract

A novel vector with high gene delivery efficiency and special cell-targeting ability was developed using a good strategy that utilized low-molecular-weight polyethylenimine (PEI; molecular weight: 600 KDa [PEI600]) crosslinked to β-cyclodextrin (β-CyD) via a facile synthetic route. Fibroblast growth factor receptors (FGFRs) are highly expressed in a variety of human cancer cells and are potential targets for cancer therapy. In this paper, CY11 peptides, which have been proven to combine especially with FGFRs on cell membranes were coupled to PEI-β-CyD using N-succinimidyl-3-(2-pyridyldithio) propionate as a linker. The ratios of PEI600, β-CyD, and peptide were calculated based on proton integral values obtained from the (1)H-NMR spectra of the resulting products. Electron microscope observations showed that CY11-PEI-β-CyD can efficiently condense plasmid DNA (pDNA) into nanoparticles of about 200 nm, and MTT assays suggested the decreased toxicity of the polymer. Experiments on gene delivery efficiency in vitro showed that CY11-PEI-β-CyD/pDNA polyplexes had significantly greater transgene activities than PEI-β-CyD/pDNA in the COS-7 and HepG2 cells, which positively expressed FGFR, whereas no such effect was observed in the PC-3 cells, which negatively expressed FGFR. Our current research indicated that the synthesized nonviral vector shows improved gene delivery efficiency and targeting specificity in FGFR-positive cells.

Figure 1

¹H-NMR analysis of (a) CY11, (b) PEI-β-CyD, and (c) CY11-PEI-β-CyD. The peaks at 2.5–3.2 ppm are assigned to the protons of PEI600 in (b) and (c). At 0.8–1.3 and 6.7–7.6 ppm, the new peaks are assigned to the protons of the amino acids of CY11 in (a) and (c).

Figure 2

Agarose gel electrophoresis of 0.5 μg DNA complexed with (a) PEI-β-CyD and (b) CY11-PEI-β-CyD at N/P ratios from 1 to 7.

Figure 3

Transmission electron micrograph of the CY11-PEI-β-CyD/DNA polyplex. The polyplex is spherical with a diameter of about 200 nm.

Figure 4

The cytotoxicity of PEI600, PEI-β-CyD, CY11-PEI-β-CyD, and PEI25KDa against (a) COS-7, (b) HepG2, and (c) PC-3 cells. The cells were treated with polymer of different concentrations for 4 h in a serum-containing medium. Cell viability was determined using MTT assays and expressed as percentages of the control. When the concentration of the polymer was <20 nmol/mL, the toxicities of the polymers were similar (P > 0.05).

Figure 5

The gene delivery efficiencies of PEI600, PEI-β-CyD, CY11-PEI-β-CyD, and PEI25KDa with different N/P ratios in (a) COS-7, (b) HepG2, and (c) PC-3 cells. PEI25KDa at an N/P ratio of 10 shows the highest gene delivery efficiency. Data were shown as mean ± SD, (*P < 0.05 as compared with other samples in the same group). The green fluorescence emitted by green proteins expressed after the transfer of CY11-PEI-β-CyD/pEGFP at an N/P ratio of 25 to cells: (d) COS-7, (e) HepG2, and (f) PC-3 cells. A fluorescence microscope is used. Fluorescence is more evident in COS-7 (FGFR-positive) and HepG2 (FGFR-positive) cells than in PC-3 (FGFR-negative) ones.