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. 2024 Feb 24:11:rbae013.
doi: 10.1093/rb/rbae013. eCollection 2024.

Polyethylenimine-modified graphene quantum dots promote endothelial cell proliferation

Qirong Xu  1   2 , Chen Li  1   2 , Xiangyan Meng  3 , Xinghong Duo  1   2 , Yakai Feng  4   5
Affiliations

Polyethylenimine-modified graphene quantum dots promote endothelial cell proliferation

Qirong Xu et al. Regen Biomater. .

Abstract

Endothelial cell proliferation plays an important role in angiogenesis and treatment of related diseases. The aim of this study was to evaluate the effect of polyethylenimine (PEI)-modified graphene quantum dots (GQDs) gene vectors on endothelial cell proliferation. The GQDs-cationic polymer gene vectors were synthesized by amidation reaction, and used to deliver pZNF580 gene to Human umbilical vein endothelial cells (HUVECs) for promoting their proliferation. The chemical modification of GQDs can adjust gene vectors' surface properties and charge distribution, thereby enhancing their interaction with gene molecules, which could effectively compress the pZNF580 gene. The CCK-8 assay showed that the cell viability was higher than 80% at higher vector concentration (40 μg/mL), demonstrating that the GQDs-cationic polymer gene vectors and their gene complex nanoparticles (NPs) having low cytotoxicity. The results of the live/dead cell double staining assay were consistent with those of the CCK-8 assay, in which the cell viability of the A-GQDs/pZNF580 (94.38 ± 6.39%), C-GQDs-PEI- polylactic acid-co-polyacetic acid (PLGA)/pZNF580 (98.65 ± 6.60%) and N-GQDs-PEI-PLGA/pZNF580 (90.08 ± 1.60%) groups was significantly higher than that of the Lipofectamine 2000/pZNF580 (71.98 ± 3.53%) positive treatment group. The results of transfection and western blot experiments showed that the vector significantly enhanced the delivery of plasmid to HUVECs and increased the expression of pZNF580 in HUVECs. In addition, the gene NPs better promote endothelial cell migration and proliferation. The cell migration rate and proliferation ability of C-GQDs-PEI-PLGA/pZNF580 and N-GQDs-PEI-PLGA/pZNF580 treatment groups were higher than those of Lipofectamine 2000/pDNA treatment group. Modified GQDs possess the potential to serve as efficient gene carriers. They tightly bind gene molecules through charge and other non-covalent interactions, significantly improving the efficiency of gene delivery and ensuring the smooth release of genes within the cell. This innovative strategy provides a powerful means to promote endothelial cell proliferation.

Keywords: ZNF580 gene; endothelial cell; graphene quantum dots; polyethylenimine; proliferation.

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Figures

None
Graphical abstract
Figure 1.
Figure 1.
FTIR (A), average particle size and zeta potential distributions (B) and TEM images (C) of A-GQDs, C-GQDs-PEI, C-GQDs-PEI-PLGA and N-GQDs-PEI-PLGA.
Figure 2.
Figure 2.
Steady-state fluorescence spectra of A-GQDs, C-GQDs-PEI, C-GQDs-PEI-PLGA and N-GQDs-PEI-PLGA.
Figure 3.
Figure 3.
Agarose gel electrophoregrams of different NPs at different weight ratios (A); the relative cell viability of HUVECs with different gene vectors and NPs for 48 h by CCK-8 assay (B); (a) A-GQDs/pZNF580 treatment group, (a′) A-GQDs treatment group, (b) C-GQDs-PEI/pZNF580 treatment group, (b′) C-GQDs-PEI treatment group, (c) C-GQDs-PEI-PLGA/pZNF580 treatment group, (c′) C-GQDs-PEI-PLGA treatment group, (d) N-GQDs-PEI-PLGA/pZNF580 treatment group, (d′) N-GQDs-PEI-PLGA-treated group, (e) Lipofectamine 200 0/pZNF580 treatment group, (e′) Lipofectamine 2000 treatment group. The relative cell viability of HUVECs with different gene vectors and NPs for 48 h by live/dead cell double staining assay (C) and relative cell viability of HUVECs with different composite gene vectors and NPs for 48 h by Image J (D). (a) pZNF580, (b) A-GQDs-PEG-CAG/pZN F580, (c) C-GQDs-PEI-PEG-CAG/pZNF580, (d) C-GQDs-PEI-PLGA-PEG-CAG/p ZNF580, (e) N-GQDs-PEI-PLGA-PEG-CAG/pZNF580, (f) Lipofectamine 2000/pZ NF580. (Mean ± SEM, n = 3, *P < 0.05 VS a, #P < 0.05 VS F).
Figure 4.
Figure 4.
Fluorescence plots (A) and transfection efficiency plots (B) after transfection with different NPs at 24 h. Western blot of ZNF580 protein expression in HUVECs cells transfected with NPs (C) and protein expression efficiency plots by Image J software (D) (wNPs/wpZNF580 = 3). (a) pZNF580 treatment group, (b) A-GQDs/pZNF580 treatment group, (c) C-GQDs-PEI/pZNF580 treatment group, (D) C-GQDs-PEI-PLGA/pZNF580 treatment group, (e) N-GQDs-PEI-PLGA/pZNF580 treatment group, (f) Lipofectamine 2000/pZNF580 treatment group. (Mean ± SEM, n = 3, *P < 0.05 VS a, #P < 0.05 VS F).
Figure 5.
Figure 5.
The migration process of HUVECs cells at different time points (A) and the relative percentage of cell migration area at 24 h calculated by Image J (B). Proliferation of HUVECs by NPs at 24 h determined by EdU doping exp eriment (C) and the total number of orange and blue fluorescence was statistically analyzed by Image J (D). (a) pZNF580 treatment group, (b) A-GQDs/pZNF580 treatment group, (c) C-GQDs-PEI/pZNF580 treatment group, (d) C-GQDs-PEI-PLGA/pZNF580 treatment group, (e) N-GQDs-PEI-PLGA/pZN F580 treatment group, (f) Lipofectamine 2000/pZNF580 treatment group. (Mean ± SEM, n = 3, *P < 0.05 VS a, #P < 0.05 VS F).
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