Photoluminescent graphene quantum dots for in vitro and in vivo bioimaging using long wavelength emission

Abstract

Graphene quantum dots (GQDs), due to their ultrasmall size, excellent optical properties, chemical stability, biocompatibility, anti-photobleaching as well as low toxicity, have been widely used as fluorescent bio-probes. In this study, we used the top-down “nano-cutting” route to prepare fluorescent GQDs. The as-prepared GQDs possessed ca. 4 nm diameter with 0.218 nm crystal lattice constant, and they had outstanding solubility as many oxygen and nitrogen based groups were present on them. The GQDs were exploited in bioimaging in vitro and in vivo. Using rat Schwann cells as the model system, the time-dependent cellular uptake of the GQDs was tested by a fluorescence activated cell sorter (FACS) and confocal laser scanning microscope (CLSM), and it reached the saturation point after 24 h. Benefiting from the excitation-dependent PL of the GQDs, multi-color cell labeling was achieved, and the GQDs were proved to be mainly distributed in the cytoplasm and lysosomes. Furthermore, using long wavelength emission (620 nm), in vivo imaging was realized in nude mice.

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1. Introduction

A wide range of fluorescent carbon nanomaterials exhibit fascinating optical properties and are very promising for low toxicity fluorescent materials.^1,2 The most common carbon nanomaterials are carbon dots (CDs),^3–5 which are mainly comprised of graphene quantum dots (GQDs), carbon nanodots (CNDs), and polymer dots (PDs).⁶ GQDs, labeled as rising fluorescent carbon materials, have drawn more and more attention these past few years.^7–11 The GQDs are also one of the heavy-metal-free fluorescent nanomaterials and are one class of “zero-dimensional” CDs containing an integrated graphene core and connected surface groups.¹² GQDs were widely applied in the fields of bioimaging, biosensing, and drug delivery, as well as bio-diagnosis, owing to their outstanding advantages such as PL stability, low toxicity and good compatibility.¹³ As a result, increasing reports have focused on the bio-based application of GQDs.^9,14–17 In addition, the cellular uptake mechanism and internalization of GQDs began to be investigated in detail.^18,19 Furthermore, the application of the GQDs to bioimaging in vivo using the long wavelength light excitation is highly significant and can decrease the autofluorescence of the living body.²⁰

In this work, the top-down “nano-cutting” route was used to prepare fluorescent GQDs.¹⁴ The applied HNO₃/H₂SO₄ was a solution phase-based scissor, which exfoliated and cut the carbon source, as well as modified the edge with oxygenic/nitrous groups simultaneously.^21–23 The as-prepared GQDs had outstanding solubility as many oxygen and nitrogen based groups were present on them. The GQDs were exploited in bioimaging in vitro and in vivo. Benefiting from the excitation-dependent PL of the GQDs, multi-color cell labeling was achieved using rat Schwann cells as the model system, and the GQDs were proved to be mainly distributed in the cytoplasm and lysosomes. Furthermore, using long wavelength emission, in vivo imaging was realized in nude mice.

2. Experimental detail

2.1. Preparation of GQDs

300 mg graphite powder was dispersed in the mixed acid (containing concentrated HNO₃ (20 mL) and concentrated H₂SO₄ (60 mL)). Then, the reaction system was ultrasonicated for 30 min, followed by placing the solution into a 100 mL round-bottom flask and stirring at 120 °C for 12 h.^14b After the reaction, the solution was diluted by pouring it into 300 mL DI-water, followed by neutralizing the acid with Na₂CO₃. Then, the solution was placed into a refrigerator to remove the Na₂SO₄ and NaNO₃ salts from the solution as much as possible (repeated three times). Aggregates in the solution were then excluded by a filter membrane of 220 nm. Finally, a 3500 dialysis bag was used to further purify the sample.

2.2. Characterization

High-resolution transmission electron microscopy (HTEM) images were recorded using a JEOL JEM-2100F. Fluorescence spectroscopy was performed with a Shimadzu RF-5301 PC spectrophotometer. UV-vis absorption spectra were obtained using a Shimadzu 3100 UV-vis spectrophotometer. IR spectra were obtained using a Nicolet AVATAR 360 FT-IR spectrophotometer. X-ray photoelectron spectroscopy (XPS) was investigated using an ESCALAB 250 spectrometer with a mono X-ray source of Al Kα excitation (1486.6 eV). Binding energy calibration was based on C1s at 284.6 eV.

2.3. Bio-based experimental section

Materials. Fetal bovine serum (FBS), phosphate buffer solution (PBS), Dulbecco's Modified Eagle's Medium (DMEM), penicillin/streptomycin, trypsin/EDTA, anhydrous dimethyl sulfoxide, and lysosome-tracker were obtained from Life Technologies. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Chloral hydrate was purchased from Sinopharm Chemical Reagent Co. (Shanghai, China).

Cytotoxicity. The RSC96 cell line was obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China) and cultured in the DMEM supplemented with 10% FBS, 100 U mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin at 37 °C in a humidified atmosphere with 5% CO₂. Briefly, RSC96 cells were seeded in a 96-wellplate at 1 × 10⁴ cells per well in 200 μL culture medium under 37 °C and 5% CO₂. After 24 h of incubation, different concentrations of GQDs (5, 10, 15, 20, 25, 35, 50, 70, 100, 150, 200, 300 μg mL⁻¹) were added, and the cultures were incubated for another 24 h at 37 °C. Then, the GQDs dispersed in each well were replaced by 180 μL of serum free medium (SFM) and 20 μL of MTT (5 mg mL⁻¹). The cells were further incubated for 3 h; the supernatant was removed by inverting the plates to decant the liquid; and 200 μL per well DMSO was added to dissolve the crystals remaining at the bottom of the plate. The absorbance was measured at 490 nm using a Synergy HT microplate reader (Bio-Tek, Winooski, VT). Cell viability was expressed as a percentage relative to the untreated cell, which served as the control.

Time-dependent cellular uptake. RSC96 cells (5 × 10⁵ cells per well) were seeded in 6-well plates and incubated for 24 h. GQDs (20 μg mL⁻¹) were added and incubated for different times (0.5, 1, 6, 24, 48 h). Subsequently, the cells were washed 3 times with PBS, trypsinized, harvested, centrifuged, and resuspended in 200 μL of PBS. Finally, the MFI of 10 000 cells at each time point was measured using a fluorescence activated cell sorter (FACS), Aria II flow cytometer with 488 nm laser and three channels (FITC, PE and Texas Red).

In vitro fluorescence imaging. RSC96 cells (5 × 10⁴ cells per dish) were seeded in a 35 mm glass-bottom dish and incubated for 24 h. GQDs (200 μg mL⁻¹) were added and incubated for 6 h. After that, the cells were rinsed 3 times with PBS and 1 mL of PBS was added for scanning. Live cell imaging was applied here to avoid the auto-fluorescence of the fixative paraformaldehyde, and scanning was performed at different excitation/emission wavelengths, including 405/425–525 nm, 488/500–600 nm, 515/530–630 nm, 559/570–670 nm, and 635/650–750 nm, with a FV1000 Olympus IX81 CLSM (Osaka, Japan) using a 60× oil-immersion objective lens. Moreover, cells were double labeled with LysoTracker red and GQDs to observe the accumulation of GQDs in lysosomes (propidium iodide (PI) and LysoTracker red were used to label the nucleus and lysosomes, respectively).

In vivo fluorescence imaging. Male BALB/c nude mice with the age of 5 weeks were purchased from the Laboratory Animal Center of Jilin University and were housed in the standard facility. Mice were subcutaneously injected with GQDs (1 mg mL⁻¹, 100 μL) on their back after being anesthetized by intraperitoneal injection of 4% chloral hydrate. The mice were imaged using the IVIS spectrum imaging system (Perkin Elmer) with varied excitation light wavelengths from 465 nm to 500 nm and detected emission light wavelengths from 580 nm to 620 nm. The most suitable excitation/emission wavelength was 465/620 nm.

2.4. Live subjects

The experiment was performed with approval from the Animal Care and Ethics Committee of Jilin University in China. All experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

3. Results and discussion

The GQDs were prepared by a modified “nano-cutting” method from graphite powder and HNO₃/H₂SO₄, as shown in Fig. 1a. The average diameter of the prepared GQDs was ca. 3.93 nm, with crystal lattice constant of ca. 0.218 nm, which was attributed to the (100) lattice plane of graphene (Fig. 1b–d).²⁴ In the FTIR analysis of the GQDs, stretching vibrations of C–OH at 3406 cm⁻¹ and C–H at 2914 cm⁻¹ and 2858 cm⁻¹, as well as bending vibrations of N–H at 1570 cm⁻¹ and a vibrational absorption band of C=O at 1624 cm⁻¹, were all observed (Fig. S1†).²⁵ In addition, surface groups were also investigated by XPS analysis (Fig. S2†). The C1s analysis revealed three different types of carbon: graphitic or aliphatic carbon (C=C/C–C), oxygenated carbon and nitrous carbon, and the nitrous carbon contained pyridine-like, pyrrole and nitric nitrogen. The percentages of the different groups are listed in the ESI.† All these oxygen and nitrogen based chemical groups endow the GQDs with outstanding solubility (over 20 mg mL⁻¹ in aqueous solution), which will be very important for their practical applications.²⁶

Fig. 1 (a) The synthetic scheme for GQDs and (b and c) TEM images of GQDs at different amplifications. (d) The diameter distribution of GQDs determined by the TEM.

The optical properties of the GQDs were further investigated. In the UV-Vis spectra, there were evident absorption peaks from the UV to blue region (Fig. 2a), which contained the π–π*, n–π* and surface state transitions.^27,28 In the fluorescence spectra, the GQDs possess the optimal emission wavelengths at ca. 530 nm (465 nm excitation) and showed green-yellow color under a hand-held UV lamp (Fig. 2b and the inset of Fig. 2a). The PL center of the GQDs was suggested to be the surface state,^14b,29,30 which was formed by the hybridization structure of edge groups and the connected graphene core, and the efficient edge groups for green emission were mainly carboxyl and amide.^14b,31 Due to the wide size and surface chemical group distributions, the GQDs possessed excitation-dependent PL. Fortunately, the excitation-dependent PL behaviors can be applied in long wavelength for biological imaging fields.

Fig. 2 The optical properties of the GQDs. (a) UV/Vis absorption spectra of the GQDs; insets show images of GQDs in aqueous solutions under sunlight and UV light. (b) The PL spectra of GQDs in aqueous solutions. (c) The photo-stability of the GQDs under portable UV lamp. (d) The pH-dependent PL behavior of the GQDs aqueous solution.

In addition, the GQDs possessed high photo-stability (Fig. 2c). After 20 hours UV exposure (hand-held UV lamp), the PL intensity and the peak shape were nearly unchanged. The intensities of green emissions decrease in the solutions of both high and low pH value (Fig. 3d), but remained stable in the range of 4–10. This indicated that the pH-dependent behaviors at specific ranges may contribute to the protonation/deprotonation of carboxyl groups, which affects the PL center of the green emission.^31–33

Fig. 3 (a) The cell cytotoxicity of GQDs determined by MTT. (b) The time-dependent cellular uptake of GQDs by fluorescence activated cell sorter (FACS). The values are expressed as mean ± SD (n = 3). (c–h) Time-dependent uptake of GQDs was observed by CLSM. The scale bar is 10 μm.

The as-prepared GQDs possessed stable PL, which indicates huge potential for bio-based applications.^19,34 The rat Schwann cells (RSC96) were chosen as the model system to investigate the cellular toxicity and uptake of the GQDs, and the results showed that the GQDs possessed low cell toxicity.³⁵ At the concentration of GQDs ranging from 10 to 100 μg mL⁻¹, the cell viability remained over 90% (Fig. 3a), and the cell viability still remained over 60% with even when 300 μg mL⁻¹ was added. The low cytotoxicity is one of the most important characteristics of the GQDs and was consistent with previous results.^9,13 Furthermore, the time-dependent cellular uptake of the GQDs was investigated, and Fig. 3b shows the linear increase in the total intracellular amount of GQDs with incubation time in RSC96 cells. The uptake of GQDs reached the saturation point after 24 h. Time-dependent uptake of the GQDs was also observed by confocal laser scanning microscopy (CLSM). From Fig. 3c–h, partial green emission was observed in the cells after 1 h culturing with GQDs, and it reached the saturation point at 24 h of culturing. Long uptake time (48 h) resulted in the same emission intensity.

Due to the excitation dependent behaviors of the GQDs, they can be exploited to bio-label at different wavelengths.³⁶ Fig. 4a–e show the bioimaging results of the GQDs by CLSM at tunable excitation wavelengths. Subsequently, we investigated the intracellular distribution of GQDs by co-localizing with cell organelle specific dyes. Propidium iodide (PI) and LysoTracker red were used to label the nucleus and lysosomes, respectively (Fig. S3†). As a result, the extensive co-localization of GQDs with the LysoTracker was observed, which suggested that endocytosed GQDs could partially transport to lysosomes (Fig. 4f).³⁷ The other GQDs were distributed in the cytoplasm. However, it should be noted that the organic dyes can easily adsorb onto GQD, so the co-localization might also be a false appearance. Energy dependent uptake behavior was also observed in the large polymer dots,^37,38 which indicated that the size of the nanoparticles was very important for cell uptake and internalization.

Fig. 4 The bioimaging of GQDs in vitro in RSC96 cells. (a–e) The multi-color bioimaging of GQDs (by different excitation wavelengths, 405, 488, 515, 559 and 635 nm, respectively) determined by CLSM. The scale bar is 5 μm. (f) GQDs co-localized with cellular organelle specific dyes: LysoTracker was used to label the lysosomes, and the GQDs were mainly distributed in lysosomes. The scale bar is 5 μm.

Furthermore, because the GQDs possess excitation-dependent PL, they can give deep-red emission, which is very important for decreasing the influence of body autofluorescence.^39–41 A nude mouse was subcutaneously injected with GQDs for in vivo imaging.¹⁸ The mouse was anesthetized and imaged in vivo with an optical imaging system. Two excitations including blue and green light with center wavelengths at 465 and 500 nm, respectively, were applied during in vivo imaging (Fig. S4† and Fig. 5). The detected wavelength was tuned from 580 nm to 600 nm, and we found that Ex/Em = 465/620 nm showed the best bioimaging result with little autofluorescence background and increased signal-to-noise ratio.⁴²

Fig. 5 (a and b) In vivo imaging of GQDs in BALB/c mice. Left mouse is saline-injected and right mouse is GQDs-injected, (a) is bright field and (b) is visible light excitation (Ex/Em = 465/620 nm).

4. Conclusions

In conclusion, a top-down route to GQDs was developed based on acid-assisted cutting methods. The as-prepared GQDs possessed ca. 4 nm diameter with 0.218 nm crystal lattice constant, and they had outstanding solubility as many oxygen and nitrogen based groups were present on them. The GQDs were exploited in bioimaging in vitro and in vivo. Benefiting from the excitation-dependent PL of the GQDs, multi-color cell labeling was achieved using rat Schwann cells as the model system, and the GQDs were proved to be mainly distributed in cytoplasm and lysosomes. Furthermore, using long wavelength emission, the in vivo imaging was realized in nude mice. In the future study, we will focus on the GQDs with multi-functional properties, such as specific target, efficient bio-label and diagnosis.⁴³

Acknowledgements

The authors thank Yu Fu from our lab for the useful comments for this work. This work was supported by the National Science Foundation of China (Grand no. 51373065, 21221063, 81171145, 81371363), the National Basic Research Program of China (973 Program, Grant no. 2012CB933800), and the Specialized Research Fund for the Doctoral Program of Higher Education (no. 20130061130010).

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Footnotes

† Electronic supplementary information (ESI) available: FT-IR spectra and XPS analysis of GQDs, as well as in vivo imaging of GQDs in BALB/C mice at different excitation and emission wavelength. See DOI: 10.1039/c5ra02961a

‡ S. Zhu and Nan Zhou contributed equally to this work.

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