A Smart Nanovector for Cancer Targeted Drug Delivery Based on Graphene Quantum Dots

Abstract

Graphene quantum dots (GQD), the new generation members of graphene-family, have shown promising applications in anticancer therapy. In this study, we report the synthesis of a fluorescent and biocompatible nanovector, based on GQD, for the targeted delivery of an anticancer drug with benzofuran structure (BFG) and bearing the targeting ligand riboflavin (RF, vitamin B2). The highly water-dispersible nanoparticles, synthesized from multi-walled carbon nanotubes (MWCNT) by prolonged acidic treatment, were linked covalently to the drug by means of a cleavable PEG linker while the targeting ligand RF was conjugated to the GQD by π⁻π interaction using a pyrene linker. The cytotoxic effect of the synthesized drug delivery system (DDS) GQD-PEG-BFG@Pyr-RF was tested on three cancer cell lines and this effect was compared with that exerted by the same nanovector lacking the RF ligand (GQD-PEG-BFG) or the anticancer drug (GQD@Pyr-RF). The results of biological tests underlined the low cytotoxicity of the GQD sample and the cytotoxic activity of the DDS against the investigated cancer cell lines with a higher or similar potency to that exerted by the BFG alone, thus opening new possibilities for the use of this drug or other anticancer agents endowed of cytotoxicity and serious side effects.

Keywords: anticancer therapy; drug delivery systems; graphene quantum dots.

Figure 1

Cancer targeted drug delivery system (DDS) based on graphene quantum dots (GQD).

Figure 2

(a) Representative TEM image of the as-prepared GQD; (b) high-resolution TEM image showing the crystal structure of an individual GQD;(c) size distribution of GQD derived from TEM images; (d) Raman spectra of GQD and MWCNT; (e) volume-weighted size distribution of GQD dispersions in deionized water; (f) isoelectric titration graph of the GQD sample in the pH range of 6–8 (evaluated as dependence of zeta potential on the pH of the medium for aqueous solutions of GQD).

Scheme 1

Synthesis of GQD-PEG-BFG. Reagents and conditions: (a) Boc-NH-PEG-NH₂, EDC⋅HCl, HOBt, ETA, DMAP, DMF, 4 d, r.t., then, HCl 4 M, dioxane, 1 h, r.t.; (b) BFG, KOtBu, air. H₂O/THF, 1 h, r.t.

Figure 3

(a) FTIR spectra of GQD, GQD-PEG, GQD-PEG-BFG, PEG-NH₂, and BFG; (b) TGA curves for GQD, GQD-PEG, and GQD-PEG-BFG. The experiments were performed under an argon atmosphere.

Scheme 2

Synthesis of GQD@Pyr-RF and GQD-PEG-BFG@Pyr-RF. Reagents and conditions: (a) Riboflavin, EDC·HCl, ETA, DMAP, HOBt, CH₂Cl₂,1 h, r.t., then 1-pyrene butyric acid, 5 d, r.t.; (b) GQD-PEG-BFG or GQD, Pyr-RF, PBS (pH 7.4), 5 d, r.t.

Figure 4

PL spectra of GQD (black line), GQD@Pyr-RF (green line), GQD-PEG-BFG@Pyr-RF (blue line), and Pyr-RF (red line) in water, at the excitation wavelength of 360 nm. The samples were tested at a concentration of 100 ng/mL.

Figure 5

Raman spectra of GQD (curve a) and of the DDS GQD-PEG-BFG@Pyr-RF (curve b) samples. Lorentzian peaks (green lines) are used to reproduce the Raman spectrum.

Figure 6

(a) AFM image (scan area 2 µm × 2 µm) of the GQD sample. (b) AFM image (scan area 2 µm × 2 µm) of the GQD-PEG-BFG@Pyr-RF sample.

Figure 7

(a) Volume-weighted size distribution of GQD-PEG-BFG sample; (b) zeta potential measurement of GQD-PEG-BFG sample; (c) volume-weighted size distribution of GQD-PEG-BFG@Pyr-RF sample; (d) zeta potential measurement of GQD-PEG-BFG@Pyr-RF sample. All the experiments were performed in PBS solutions at pH 7.4.

Figure 8

Cytotoxic effect of the synthesized drug delivery nanosystems in (a) laryngeal cancer cells HEp-2, (b) human lung epithelial cancer cell line A549, and (c) the human colorectal adenocarcinoma cell line HT-29. The results, obtained in cells treated for 24 h, are reported as percentages of dead cells (treated/untreated cultures). The concentrations of 10, 25, 50, 100, and 200 µg mL⁻¹, shown at the top of the graphs, are those of GQD-PEG-BFG@Pyr-RF, corresponding to 2.1, 5.25, 10.5, 21, and 42 μg mL⁻¹ doses of BGF assayed in the positive controls. For the GQD-PEG-BFG, the assayed doses were calculated to assess the same drug concentrations while for GQD, GQD-PEG, and GQD@Pyr-RF, the examined doses were 10, 25, 50, 100, and 200 μg mL⁻¹. Each value represents the mean (±SD) of the experiments made in triplicate for each cell line. In each graph, the corresponding values of IC₅₀ are reported below.