Facile Synthesis of N-Doped Graphene Quantum Dots as Novel Transfection Agents for mRNA and pDNA

Abstract

In the wake of the coronavirus disease 2019 (COVID-19) pandemic, global pharmaceutical companies have developed vaccines for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Some have adopted lipid nanoparticles (LNPs) or viral vectors to deliver the genes associated with the spike protein of SARS-CoV-2 for vaccination. This strategy of vaccination by delivering genes to express viral proteins has been successfully applied to the mRNA vaccines for COVID-19, and is also applicable to gene therapy. However, conventional transfection agents such as LNPs and viral vectors are not yet sufficient to satisfy the levels of safety, stability, and efficiency required for the clinical applications of gene therapy. In this study, we synthesized N-doped graphene quantum dots (NGQDs) for the transfection of various genes, including messenger ribonucleic acids (mRNAs) and plasmid deoxyribonucleic acids (pDNAs). The positively charged NGQDs successfully formed electrostatic complexes with negatively charged mRNAs and pDNAs, and resulted in the efficient delivery and transfection of the genes into target cells. The transfection efficiency of NGQDs is found to be comparable to that of commercially available LNPs. Considering their outstanding stability even at room temperature as well as their low toxicity, NGQDs are expected to be novel universal gene delivery platforms that can outperform LNPs and viral vectors.

Keywords: gene delivery; graphene quantum dots; mRNA; pDNA; transfection.

Figure 1

(a) TEM images of NGQDs (scale bar = 20 nm). (b) Size distribution of NGQDs. (c) zeta potentials of PEI, PEI + citric acid and NGQDs.

Figure 2

(a) FT-IR spectrum, (b,c) XPS spectra (C1s, N1s), and (d) Raman spectrum of NGQDs.

Figure 3

(a) UV-visible absorption spectrum, (b) optical and fluorescence (λ_Ex = 365 nm) images, and (c) emission spectrum of NGQDs (λ_Ex = 360 nm).

Figure 4

Loading capacity of NGQDs to (a) mRNA and (b) pDNA. (c) Relative cell viability of NGQDs.

Figure 5

Fluorescence microscopy images of HeLa cells after 24 h transfection with (a) control (1 × PBS), (b) mRNA only, (c) Lipofectamine only, (d) NGQDs only, (e) Lipofectamine + mRNA complex, and (f) NGQDs + mRNA complex. All scale bars are 200 μm.

Figure 6

Flow cytometry analysis of HeLa cells after 24 h transfection with each group; (a) control, (b) mRNA only, (c) Lipofectamine only, (d) NGQDs only, (e) Lipofectamine + mRNA complex, (f) NGQDs + mRNA complex groups. (g) mRNA transfection efficiency of each group.

Figure 7

Fluorescence microscopy images of HeLa cells after 24 h transfection with (a) control, (b) pDNA only, (c) Lipofectamine only, (d) NGQDs only, (e) Lipofectamine + pDNA complex, and (f) NGQDs + pDNA complex. All scale bars are 200 μm.

Figure 8

Flow cytometry analysis of HeLa cells after 24 h transfection with each group; (a) control, (b) pDNA only, (c) Lipofectamine only, (d) NGQDs only, (e) Lipofectamine + pDNA complex, (f) NGQDs + pDNA complex groups. (g) pDNA transfection efficiency of each group.