Simultaneous Gene Delivery and Tracking through Preparation of Photo-Luminescent Nanoparticles Based on Graphene Quantum Dots and Chimeric Peptides

Abstract

Designing suitable nano-carriers for simultaneous gene delivery and tracking is in the research priorities of the molecular medicine. Non-toxic graphene quantum dots (GQDs) with two different (green and red) emission colors are synthesized by Hummer's method and characterized by UV-Vis, Photoluminescence (PL), Fourier Transform Infrared (FTIR) and Raman spectroscopies, Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The GQDs are conjugated with MPG-2H1 chimeric peptide and plasmid DNA (pDNA) by non-covalent interactions. Following conjugation, the average diameter of the prepared GQDs increased from 80 nm to 280 nm in complex structure, and the ζ-potential of the complex increased (from -36.87 to -2.56 mV). High transfection efficiency of the nano-carrier and results of confocal microscopy demonstrated that our construct can be considered as a nontoxic carrier with dual functions for gene delivery and nuclear targeting.

Figure 1

Schematic depiction of assembling MPG-2H1/pDNA/GQDs complexes through noncovalent interactions, transfection into the cells and excitation.

Figure 2

UV-Vis absorption spectrum (A) FTIR spectrum (B) Raman spectrum (C) and photoluminescence spectrums (D and E) of GQDs.

Figure 3

AFM topography image of GQDs (A), the bottom image shows height distribution of GQDs. SEM image and size distribution analysis of GQDs (B). Separation of two populations of free-GQDs by electrophoresis on 1% agarose gel (C).

Figure 4

The coomassie blue stained purified peptide in 15% SDS-PAGE (MW.14 kDa) (A). Native acrylamide gel retardation (PAGE 15%) assay of MPG-2H1, MPG-2H1/pDNA and MPG-2H1/pDNA/GQDs complex (B). Cropped agarose gel electrogram (1%) pDNA retardation assay (C). ζ-potential of MPG-2H1 peptide, GQDs, and MPG-2H1/pDNA/GQDs complex 3 (D), error bars denote the standard deviation.

Figure 5

TEM image of the negatively stained targeted MPG-2H1/pDNA complex (A) GQDs (B) and MPG-2H1/pDNA/GQDs complex (C).

Figure 6

Emission spectrum of GQDs, complex 1, 2 and 3 (excitation at 400 nm).

Figure 7

Stability assay of GQDs and complexes at different time intervals (24, 48 and 72 h).

Figure 8

Cell viability assays of GQDs (A) and peptide (B) for 48 h. C shows control cells incubated in cell culture medium without GQDs or peptides.

Figure 9

Fluorescence microscopy images of transfected cells with complex 1 (A), complex 3 (B), GQDs (C) and control cells (D) are shown in 4 different panel. In each panel: left top is excited by 469 nm and detected in 525 emission wavelength, right top is excited by 585 nm and detected in 647 emission wavelength, left bottom shows bright field and overlay is indicated in right bottom.

Figure 10

Confocal microscopy images of transfected cells with complex 3 (A) and GQDs (B). Blue color show DAPI nucleir stain, green and red show GQDs. Four images of each part are: excited by 408 nm for detecting DAPI nucleir stain (left top), excited by 457 nm for detecting green GQDs (right top), excited by 642 nm for detecting red GQDs (left bottom) and overlay (right bottom).

Figure 11

Transfection of psiCHEK plasmid harboring luciferase gene using different complexes of MPG-2H1/pDNA/GQDs and MPG-2H1 peptide/pDNA. Luciferase activity is indicator of transfection efficiency.

Figure 12

Effect of chloroquine on transfection efficiency of psiCHEK plasmid harboring luciferase gene using different complexes of MPG-2H1/pDNA/GQDs and MPG-2H1 peptide/pDNA. Luciferase activity is indicator of transfection efficiency.

Figure 13

Fluorimetric assay of transfected cells with complexes 1 to 5, GQDs, MPG-2H1/pDNA complex and control cells using scanning area mode.