A graphene quantum dot photodynamic therapy agent with high singlet oxygen generation

Abstract

Clinical applications of current photodynamic therapy (PDT) agents are often limited by their low singlet oxygen ((1)O2) quantum yields, as well as by photobleaching and poor biocompatibility. Here we present a new PDT agent based on graphene quantum dots (GQDs) that can produce (1)O2 via a multistate sensitization process, resulting in a quantum yield of ~1.3, the highest reported for PDT agents. The GQDs also exhibit a broad absorption band spanning the UV region and the entire visible region and a strong deep-red emission. Through in vitro and in vivo studies, we demonstrate that GQDs can be used as PDT agents, simultaneously allowing imaging and providing a highly efficient cancer therapy. The present work may lead to a new generation of carbon-based nanomaterial PDT agents with overall performance superior to conventional agents in terms of (1)O2 quantum yield, water dispersibility, photo- and pH-stability, and biocompatibility.

Figure 1. Characterization of GQDs.

(a) TEM images. Scale bar, 20 nm. (b) HRTEM images. Scale bar, 2 nm. (c) XPS survey spectrum. (d) Deconvolution of high-resolution C1s XPS spectra.

Figure 2. Photophysical and photochemical properties of GQDs.

(a) Normalized UV–vis absorption and emission spectra (λ_ex=500 nm) of the GQDs dispersed in water at room temperature. The inserts show a photograph and fluorescence image of the GQD solution under UV light (365 nm). (b) Fluorescence decay curve (black line) of GQDs recorded at 680 nm with an excitation of 488 nm. Red line: the instrument noise; blue line: fitting of the fluorescence decay curve. Fit=A+B₁exp(−t/τ₁)+B₂exp(−t/τ₂)+B₃exp(−t/τ₃; τ₁=0.27 ns, τ₂=1.10 ns, τ₃=7.52 ns). (c) A comparison of the photostabilities of the GQDs, CdTe QDs and PpIX. All of the samples were continuously irradiated using a 500-W xenon lamp. A₀ and A are the absorbance of the samples at 470 nm before and after irradiation, respectively. After 75 min of irradiation, no obvious decline was observed in the absorbance of the GQDs, while the absorbance of PpIX and the CdTe QDs decreased below 78% of their initial value. (d) The ESR signals of ¹O₂ (up) and other ROS (down) obtained upon irradiation of GQDs for 8 min in the presence of 2,2,6,6-Tetramethylpiperidine and 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide, respectively. (e) The normalized absorbance of Na₂-ADPA at 378 nm as a function of irradiation time in the presence of GQDs and RB. (f) The ¹O₂ emissions at ~1,280 nm induced by the GQDs and RB in a CH₃CN-D₂O mixture solution (v/v=15/1) under excitation with a 532-nm laser.

Figure 3. In vitro imaging and PDT.

(a) Confocal fluorescent microscopy image of HeLa cells labelled with GQDs (0.4 μM). Scale bar, 20 μm. (b) Time-dependent confocal bright field and (c) corresponding fluorescence images of HeLa cells incubated with GQDs (0.4 μM) and Hoechst 33342 (1.8 μM) after irradiation with 405 and 633 nm lasers. Scale bar, 50 μm. Dose-dependent PDT effects of the cell viability of HeLa cells: (d) GQDs in the concentration range 0.036–1.8 μM and (e) PpIX in the concentration range 0.36–18 μM.

Figure 4. In vivo imaging and PDT.

(a) Bright-field image and (b) red-fluorescence image after subcutaneous injection of GQDs in different areas. The excitation wavelength was 502–540 nm, and the collected fluorescence channel was 695–775 nm. (c) Photographs of mice after various treatments on the 1st, 9th, 17th and 25th day. (PDT: GQDs+light irradiation; C1: GQDs only; C2: light irradiation only.) (d) Time-dependent tumour growth curves (n=5) after different treatments. P<0.05 for each group.

Figure 5. Multistate sensitization mechanism.

(a) Schematic illustration of the ¹O₂ generation mechanisms by conventional PDT agents (left) and GQDs (right). (b) Fluorescence intensity of GQDs at 680 nm versus the O₂ concentration in solution. (c) The dependence of the ¹O₂ quantum yield (Q_Δ) on the fluorescence intensity ratio at 680 nm (F/F⁰).