Synthesis of a highly fluorescence nitrogen-doped carbon quantum dots bioimaging probe and its in vivo clearance and printing applications

Abstract

Highly fluorescent, broad range pH and ionic-stable N-doped carbon quantum dots (N-CQDs) were successfully synthesized and their chemical structure and fluorescence mechanism were characterized. Mono-dispersed N-CQDs averaging 4.5 nm in diameter were achieved in a quantum yield (QY) of 74.16%. These N-CQDs showed great potential in biomedical and optical imaging applications both in vivo and in vitro. Biodistribution, retention, toxicity, and pharmacokinetics profiles are mandatory in their potential clinical applications. Herein, the biodistribution, clearance, and toxicity of our invariable N-CQDs species were systematically investigated over 7 days in mice. Most of the N-CQDs were cleared at 6 days post-injection without any accumulation in any vital organs or tissues. Moreover, the high fluorescence N-CQDs were demonstrated to be effective as printing inks for multicolour patterns. This study suggests that N-CQDs may be an attractive alternative to metal quantum dots for secure biomedical and other industrial applications.

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Introduction

Recently nanomaterials have become very interesting research tools for bioimaging, clinical diagnosis, bionic devices and tissue engineering.^1–7 Semiconductor quantum dots (QDs) exhibit several unique photoelectric properties, such as size, wavelength-dependent luminescence and low photo-bleaching,^8–10 due to their strong quantum confinement effects. QDs-based imaging integrates these properties to improve fluorescence imaging with merits including real-time use, accuracy, and in vivo observations with high sensitivity, rapid response, low cost, and no need for radiation. On the other hand, researchers revealed the using of metal-based QDs (M-QDs) are involved with long-term toxicity and affect the environment with heavy metals.^11,12 Obtaining M-QDs is a complex procedure and their storage is also difficult preventing aggregation and chemical changes.¹³ Normally, carbon QDs (CQDs) contain a large amount of carbon with relatively less oxygen, hydrogen and nitrogen causing them to be nontoxic and a safer nanomaterial than heavy metal QDs.^14,15 With a favourable size of less than 10 nm diameters,¹⁶ high fluorescence, photostability,^17,18 and good storage can be observed.¹⁷ In carbon dots the oxygen and hydrogen forms hydroxyl and carboxyl groups, which facilitate the functionalization and improve the hydrophilicity.¹⁶ Thus, CQDs are becoming a desirable alternative to M-QDs when taking their low toxicity and unique photophysical and chemical properties into consideration. Many complex synthetic methods, such as laser ablation, electrochemical oxidation, high temperature calcinations and solvo-thermal and microwave-assisted pyrolysis, have been used to prepare CQDs to date.¹⁹ However, two issues that need to be addressed for the effective synthesis of cost-effective CQDs still remain: the size non-uniformity and the time consuming fabrication process.²⁰

Doping CQDs with other non-metallic components is beneficial for adjusting the structure and composition and making good fluorescence inks for printing.^21,22 N-doped CDs have been found to efficiently induce charge delocalization and enhance performance in bio-imaging and catalysis with N atoms as dopants.²³ However, many scientific issues with C-QDs still await further investigation and large scale pharmaceutical applications are challenging in this field. Herein, we used agarose in combination with ethylenediamine passivated agents, to successfully prepare nitrogen-doped carbon quantum dots (N-CQDs) with enhanced fluorescence, high yield (74.16%), a simple procedure, a low cost, and high biocompatibility, and tested their imaging application in the important model organism, mice. To gain comprehensive clinical acceptance of these N-CQDs as a bioimaging agent, we need to investigate toxicity, biodistribution, clearance of nanoparticles in vivo, pharmacologic profiles,^24,25 routes of administration, biodistribution patterns, and dosage. These are all important considerations that are only beginning to be addressed in detail.^26–29

Therefore, we demonstrated a multi-parametric in vivo study, clearance quantities, rates and toxicity determination of N-doped CQDs over short term (up to 7 days) time points. We observed that most of the N-doped CQDs were cleared before 7 days post-injection in mice without any major accumulation in any vital organs or tissues.

Experimental section

Synthesis of carbon nanoparticles

N-doped carbon quantum dots (N-CQDs) were synthesized by hydrothermal treatment. Carbon source material agarose (2.5 g) and amine source 1 mL ethylenediamine (EDA) were added in 30 mL of water and mixed well with stirring and then the mixture was transferred into an 80 mL Teflon-lined stainless steel autoclave and heated at a constant temperature of 200 °C for between 1 and 6 h (2 °C min⁻¹) in separate sets. After the reaction is over, the autoclave was cooled naturally. The product, which was brown-black and transparent, was subjected to dialysis to obtain the N-doped carbon quantum dots (N-CQDs). The product yield was ca. 6–74% for different time conditions listed in Table S2.†

Fluorescent N-doped CQDs for printing. We used normal non-fluorescence paper as a drawing paper. Aqueous N-doped CQDs ink was loaded into an empty ink pen (concentrations 0.01 mg mL⁻¹ to 1 mg mL⁻¹). The desired words or images were drawn onto a piece of “paper” by this ink pen. The fluorescence images were obtained under a hand held UV lamp.

Cellular toxicity test. Breast cancer cell line (MCF-7) cells (1 × 10⁴ cells per well) were cultured first for 24 h in an incubator (37 °C, 5% CO₂) and for another 24 h after the culture medium was replaced with 100 μL of Dulbecco's modified Eagle's medium (DMEM) containing the N-doped CQDs at different doses (mg mL⁻¹). Then, 20 μL of 5 mg mL⁻¹ MTT solution was added to every cell well. The cells were further incubated for 4 h, followed by removal of the culture medium with MTT and then 100 μL of DMSO was added. The resulting mixture was shaken for 10 min at room temperature. The optical density (OD) of the mixture was measured at 490 nm. The cell viability was estimated according to the equation as follows:
where OD_control was obtained in the absence of N-CQDs and OD_treated obtained in the presence of N-CQDs.

Cellular imaging. The MCF-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin. 0.1 mg mL⁻¹ concentrated solution of N-doped CQDs was prepared in DI water. After the well dispersion, an aliquot (typically 100 μL) of the suspension was added to culture plate, then incubated at 37 °C in a 5% CO₂ incubator for 24 h. Prior to fixation of the cells on the slide for inspection with a fluorescence microscope, the excess N-doped CQDs were removed by washing 3 times with PBS. Bioimaging was undertaken on a Zeiss inverted fluorescence microscope (Carl Zeiss vert. A1 microscope, Carl Zeiss Micro imaging GmbH, 07740, Jena, Germany).

Organs imaging. The organs were harvested and cut into small pieces, fixed on glass slides and then directly subjected to microscopy study in the fluorescence microscope.

Pharmacokinetics of N-doped CQDs. Eight week old male mice (n = 30) were housed in pairs in a standard 12 h light/dark cycle with food and water, ad libitum. The N-doped CQDs dose given was 5 mg kg⁻¹ and intravenously injected into mice (n = 27). At 1 hour, 4 hours, 12 hours and daily post-injection, the animals (n = 3 day⁻¹) were sacrificed. Blood samples were obtained via retro-orbital blood collection. The following organs and tissues, including liver, spleen, kidney, lung and muscle, were collected, weighed and dissected. The urine and faeces were continuously collected from mice housed in the metabolic cage. N-doped CQDs clearance was observed under fluorescence microscope. All animal experiments reported herein were carried out according to a protocol approved by IIT Roorkee Ethical or Animal Care Committee.

Quantum yields (QY) measurements. Quinine sulphate (0.1 M H₂SO₄ as solvent; QY = 0.54) was chosen as standard. The QY of N-doped CQDs (in water) was determined by the slope method¹ by reference to quinine sulphate: comparing the integrated photoluminescence intensity and the absorbance values (several values, less than 0.1 at excitation wavelength, gave the curve) of the samples with those of the reference.

We then used the equation:

φ_x = φ_st(K_x/K_st)(η_x/η_st)²

where φ is the QY, K is the slope determined by the curves and η is the refractive index. The subscript “st” refers to the standards and “x” to the unknown samples. For these aqueous solutions, η_x/η_st = 1.

Statistical analysis. All data presented in this study are averages ± standard error of experiments repeated at least three times. A paired student's t-test was performed.

Materials characterization

High-resolution transmission electron microscopy (HRTEM) was carried out with a JEM-2100F microscope operating at 200 kV for characterizing the size and shape of the N-doped CQDs. AFM images were recorded in the tapping mode with a Nanoscope-III, a scanning probe microscope from Digital Instruments, under ambient conditions with partial wafer of NanoSensors Point probes (type S3G3T6-8L224) wherein other settings were integral gain 0.5, proportional gain 0.7 and scanning rate 1 Hz. Fluorescence spectroscopy was performed with a CARY Eclipse 5, 5 fluorescence spectrophotometer and fluorescence spectra were measured using a 4 mL glass cuvette.

UV-vis absorption spectra were obtained using a CARY 50 Conc UV-vis spectrophotometer. IR spectra were obtained on a Nicolet Nexus Aligent 1100 series FT-IR spectrometer. The fluorescent images were taken at the fluorescence microscope (Carl Zeiss vert. A1 microscope, Carl Zeiss Micro imaging GmbH, 07740, Jena, Germany) using different band pass filtered 461 nm, 560 nm and 633 nm excitations and 5×, 10×, 20P× objectives. X-ray photoelectron spectroscopy (XPS) was investigated using an ESCALAB 250 spectrometer with a monochromatic X-ray source and Al K_α excitation (1486.6 eV).

Results and discussion

The synthetic procedure is illustrated in Fig. 1a. The reaction was conducted by first condensing agarose and ethylenediamine, whereupon they formed polymer-like carbon quantum dots (CQDs), which were then carbonized to form the CQDs. The morphology and particle size of the CQDs, examined by TEM and AFM and shown in Fig. 1b and c, reveal that the CQDs are uniform and regularly spherical in shape with an average diameter of about 4.5 nm without aggregation. The corresponding particle size measure (Fig. 1d) indicates that CQDs have a relatively narrow size distribution between 2 and 6 nm. The inset in Fig. 1b, a typical HRTEM image of CQDs, shows the lattice spacing of 0.19 nm, which corresponds to the (102) planes of graphitic carbon.³⁰

Fig. 1 (a) Schematic of N-CQDs formation by hydrothermal treatment; (b) low and high magnified (inset) TEM images of N-CQDs; (c) AFM images of N-CQDS; (d) particle size distribution of N-CQDs; (e) XPS spectrum of N-CQDs. High-resolution XPS spectra of (f) C 1s, (g) N 1s and (h) O 1s.

XPS measurements were performed to identify the effective incorporation of nitrogen and surface functional groups of CQDs. From a survey scan of the XPS spectrum in Fig. 1e, three distinct peaks centred at 285.7 eV, 399.05 eV, and 532.1 eV can be observed, corresponding to C 1s, N 1s, and O 1s where the particle is composed of carbon (C 1s 51.73)%, nitrogen (N 1s 12.79%) and oxygen (O 1s 35.8%) (see ESI Table S1†).

In detail, the C 1s spectrum (Fig. 1f) displays three distinct peaks at 284.5 eV, 285.8 eV, and 287.6 eV, which are attributed to C–C, C–N, and C=O, respectively.^31,32 The N 1s spectrum has two typical peaks at 399.2 (pyridinic N)³² and 401.0 eV (pyrrolic N).³³ Fig. 1g indicates that nitrogen is present in a π–π conjugated system wherein two p-electrons are present in the system of the prepared CQDs.²³ This illustrates that the CQDs were successfully doped with nitrogen atoms. Fig. 1h revealed that the O 1s peak can be resolved into two components centered at 530.8 and 532.1 eV, representing the presence of the C=O and C–OH/C–O–C groups.^34,35 The XPS results indicate that the surface of the CQDs is functionalized by multiple oxygen- and nitrogen-containing groups from the reaction between agarose and ethylenediamine. Fig. 2a shows the UV-vis spectra; the peak was focused on 272 nm in an aqueous solution of N-doped CQDs, wherein the fluorescence spectra of N-doped CQDs has optimal excitation and emission wavelengths at 390 nm and 474.54 nm, respectively. Fig. 2a inset represents the aqueous N-doped CQDs in a 4 mL cuvette under a white and UV lamp. Excitation-dependent fluorescence behavior was observed, which is common in fluorescent carbon materials (Fig. 2b).^36–40 This behavior contributes to the surface state affecting the band gap of N-doped CQDs. The surface state is analogous to a molecular state, whereas the size effect is a result of quantum dimensions, both of which contribute to the complexity of the excited states of N-doped CQDs.⁴¹ ESI Fig. S1a† exhibits the multicolour fluorescence images of N-doped CQDs under microscopy and fortunately we applied this excitation-dependent fluorescence character to acquire multicolour imaging of cancer cells (see ESI Fig. S1b†).

Fig. 2 (a) UV-vis absorption (blue line) and fluorescence emission (red line) of N-CQDs, wherein excitation is 390 nm and emission 494 nm and insets show images of N-CQDs in aquatic media under white and hand-held UV lamp, (b) excitation-dependent emission spectra of aqueous N-CQDs solution (0.01 mg mL⁻¹), (c) FTIR spectrum of N-CQDs and (d) XRD pattern of N-CQDs.

Moreover, the surface groups were also investigated by FTIR analysis of the N-doped CQDs. The FTIR spectrum in Fig. 2c shows the characteristic absorption bands at 3403 cm⁻¹ corresponding to the stretching vibrations of O–H and N–H.⁴² The absorption bands at 1639 cm⁻¹ and 1496 cm⁻¹ are due to the C–O stretching vibrations and C–N stretching vibrations, respectively. The existence of carboxylic groups can be clearly proven by the peak at 2087 cm⁻¹ corresponding to C=O and the peak at 1042 cm⁻¹, attributed to C–O, suggest the partial oxidation of CQD surfaces.⁴³ Furthermore, the band centered at 1395 cm⁻¹ is assigned to the asymmetric stretching vibrations of the C–O–C bonds.⁴⁴ It can be concluded from the results of XPS and FTIR analysis that the hydrothermal degradation of agarose in the presence of EDA offered the N-doped CQDs with hydrophilic groups, such as –COOH and –OH, which are beneficial for the improvement of aqueous solubility for potential biochemical applications, including drug delivery and detection.

The XRD patterns of the N-doped CQDs (Fig. 2d) also displayed a broad (002) peak centered at 24.20°. The corresponding interlayer spacing 0.42 nm is also attributed to highly disordered carbon atoms.⁴⁵

Furthermore, the fluorescence stability of N-doped CQDs to the effects of the ionic strength and pH of the solutions was investigated. There was no change in fluorescence intensity or peak characteristics at different ionic strengths (ESI Fig. S2†), which is significant because it is necessary for N-doped CQDs to be used in the presence of physical salt concentrations in practical applications. Another interesting phenomenon is the pH-dependent fluorescence intensity behavior (ESI Fig. S3†). Fluorescence intensities decreased in a solution of high pH 14 or low pH 1, but remain constant in a solution of pH 2–13. This result may be a consequence of the chemical structure of N-doped CQDs, which have graphitic lattices. The emission control of the surface state/molecule state was not affected by surrounding factors such as solvent pH. At a pH that is very low or very high, molecular groups are strongly affected.^39,46 Therefore, in our case, it was found that redispersion of dry sample in water and other solvent without any aggregation, has great significance in preservation and transportation.

The time-conditioning synthetic method can also be used to prepare different N-doped CQDs (ESI Table S2†). During these experiments, we found time is very important for the formation of high QY N-doped CQDs. 6 hours reaction produced a QY of 74%, whereas 1 h gave only 6% (using quinine sulphate as the reference) (ESI Fig. S4†). This is the best quantum yield performance according to the best of our knowledge (see ESI Table S3†). ESI Fig. S5 and S6† represent the comparative studies of excitation-dependent fluorescence spectra and open eye fluorescence intensity under white and hand-held UV lamp, respectively.

To make use of the high QY, the N-doped C-QDs were employed as ink for printing patterns on normal non-fluorescing paper. Colourless aqueous solutions of N-doped CQDs were loaded in ink pens. The N-doped CQDs concentration used was from 0.01 mg mL⁻¹ to 1 mg mL⁻¹ and gave strong enough fluorescence under a hand-held UV lamp. Fluorescing images and words were distinctly visible (Fig. 3a–g). The fluorescence intensity was increased with increasing concentration of N-doped CQDs used as the printing ink. By using this property, multicolour printing images were obtained (Fig. 3d, e, and f). Moreover, the printed patterns retained their stability after 1.6 years in a laboratory ambient environment, which is beneficial for practical applications (Fig. 3g). An MTT assay showed that the N-doped CQDs possess low cytotoxicity, and thus could act as an excellent bioimaging agent (see ESI Fig. S7†). After incubating with N-doped CQDs in a culture solution, human breast cancer MCF-7 cells showed high intensity fluorescence, and the excitation-dependent fluorescence resulted in multicolour imaging under different excitation wavelengths (see ESI Fig. S1b†).

Fig. 3 Printed patterns obtained by N-CQDs ink (illuminated by a portable UV lamp). (a–c) Different concentrations of N-CQDs ink using letter and graphic pattern, (d and e) different concentrations of N-CQDs ink obtains different intensities in this pattern, (f) different concentrations of N-CQDs ink used to obtain different colour letters, and (g) image of printing after 1.6 years exposure to laboratory ambient environment.

High stability of N-doped CQDs in body fluid is crucial for their further in vivo applications. Fig. 4a–c shows the pre-injection and post-injection whole body bioimaging of mice. After 12 hours post-injection it gave strong fluorescence intensity over the whole body (Fig. 4b); however, after 24 hours post-injection little of the body fluorescence intensity still remained (Fig. 4c). This data clearly suggest that N-doped CQDs were stable in the body fluid. After one hour of post injection, highly vascular organs and tissue imaging was studied under fluorescence microscopy. Results show the multicolour imaging under different excitation wavelengths (Fig. 4d). This proved that our N-doped CQDs easily crossed the organ epithelial layer by a nano-leaking process.^47–49 This fantastic feature also facilitates our in vivo biodistribution and clearance studies. Herein, we chose only two month old male mice as the animal model. The N-doped CQDs (final dose ∼5 mg kg⁻¹) were injected intraperitoneally into the mice (27 mice). The mice were sacrificed (n = 3day⁻¹) daily after post-injection. The presence/absence of N-doped CQDs in major organs and tissues was determined by fluorescence microscopy to monitor the fluorescence intensity level. The biodistribution data were presented in Fig. 5a. In the first 48 hours post–injection, the liver was the dominant organ for N-doped CQDs accumulation, followed by the kidney. Lung, muscle and urinary sac showed relatively low fluorescence intensity meaning low concentrations of N-doped CQDs accumulating. By 6 days post-injection, no accumulation shown in any studied organs and tissues. The kidney is the main organ for foreign particle clearance from the blood and we also observed a drastic decrease of fluorescence intensity in blood and excretory materials as N-doped CQDs were completely cleared through urine and faeces within 6 days of post-injection (Fig. 5b–d). ESI Fig. S8† shows the actual fluorescence images of blood, faeces and urine.

Fig. 4 Whole body fluorescence images of nude mice at different times of injection: (a) pre-injection, (b) 12 hours post-injection, (c) 24 hours post-injection. (d) Fluorescence microscopy images of different organs and tissues under different band pass filters wherein organs and tissues collected after 1 hour from post-injected mice. Scale bar is 50 μm.

Fig. 5 (a) Distributions of N-CQDs probe in different organs and tissues of nude mice determined at 1 to 7 days post-injection. Post-injection clearance profile of N-CQDs at different times from 1 to 7 days through (b) blood, (c) faeces and (d) urine.

Conclusions

In summary, high quantum yields (ca. 74.16%) of N-doped CQDs were obtained and their chemical structure, fluorescence mechanism, toxicity, bioimaging application, biodistribution and clearance were studied successfully. N-doped CQDs show high ionic strength and stability under long range pH (2–13), which will be very important for pharmaceutical applications. Quicker clearance efficiency without accumulation in organs and tissues was also observed, whereas other metal nanomaterials cause organ damage due to their long-term accumulation (90 days or more) in organs and tissues.^47–51 Finally, we can conclude that our durable N-doped CQDs are harmless and nontoxic in biomedical and bioimaging fields. Moreover, they could potentially be used in the printing industry as N-doped CQDs were successfully applied as printing inks for multicolour patterns.

Acknowledgements

Tapas K. Mandal and Nragish Parvin would like to thank SERB, DST, (Project File no- SR/FT/LS-76/2012) and DBT Government of India for providing financial support respectively. Also thanks to “Chinese Academy of Science postdoctoral and visiting scholar for developing country programme” for financial support.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra25402g

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