A highly stable and biocompatible optical bioimaging nanoprobe based on carbon nanospheres

Abstract

In this report, a facile one-step synthesis strategy has been developed for producing fluorescent carbon nanospheres (CNs) using lactobionic acid (LBA) as a precursor. The resulting CNs are highly stable in aqueous solution with an average size of 120 nm. The obtained CNs contained large amounts of –OH and –COOH on their surfaces which will facilitate further functionalization for more additional usage. In addition, carbon nanospheres possess an intrinsic fluorescence property so they can be used for fluorescence imaging. Our CNs exhibited remarkable photoluminescence properties and low cytotoxicity which can be used to label living cells with high efficiency, suggesting potential applications in biolabeling and bioimaging. In brief, we developed novel fluorescent carbon nanospheres with high stability, biocompatibility and labeling efficiency for cell imaging, as well as abundant active groups on their surface for further modifications.

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1. Introduction

Cancer is one of the main threats to human health, and the annual incidence of cancer worldwide has increased with an average rate ranging from 3% to 5% within the last 30 years.¹ It is predicted that the number of cancer patients worldwide will increase from 12.7 million in 2008 to 22.2 million in 2030 in the coming decades,¹ but what’s more frightening is that most patients are diagnosed at a later or advanced stage, resulting in a poor prognosis. The early detection of cancer is crucial for improving the long-term survival of patients, since cancer cells can be easily and completely removed at an early stage. However, early detection of cancer remains challenging using current diagnostic techniques, including computed X-ray tomography (CT),^2,3 magnetic resonance imaging (MRI),^4,5 ultrasound examination (US),^6,7 and positron emission tomography (PET).^8–10 Compared with these imaging approaches, optical imaging based on luminescent dyes has recently been found to be of great promise for cancer diagnosis at an early or even pre-syndrome stage, due to their advantages of high sensitivity, real-time high resolution, noninvasiveness and molecular or cellular resolution. However, conventional luminescent dyes often suffer from poor photostability, fast photobleaching, aggregation, degradation, undesired biodistribution or susceptible clearance by the RES system making them unfit for optical bioimaging.

The rapid development of nanotechnology brings a promising opportunity for the resolution of these obstacles by developing novel nanoscale fluorescent probes. Compared with small molecular dyes, nanoparticles will preferentially accumulate in the leaky vasculature of tumor tissues through the enhanced permeability and retention effect (EPR).^11,12 In addition, nanoparticles have other advantages such as high optical performance, longer retention in blood circulation, pathological site targeting, and multimodality.^13–16 Up to now, several types of nanometer scaled probes have been used in optical bioimaging including nanodiamonds,¹⁷ upconversion nanoparticles (UCNPs),^18–20 semiconductor quantum dots (QDs),^21–23 carbon dots (CDs)^24–26 or nanomaterials additionally attached to fluorescent tags.^27,28 Among these fluorescent nanoprobes, CDs have attracted increasing attention in recent years because of their excellent stability, green synthesis route, high biocompatibility and low cytotoxicity compared to QDs and organic fluorophores. For example, Wang’s group report a simple, low-cost, one-step microwave-assisted route to fabricate water-soluble luminescent C-dots, and used them as a new biocompatible fluorescent tag.²⁹ Pang’s group also prepared a series of C-dots exhibiting various colored fluorescence, which have the potential for application in multicolor imaging.³⁰ However, there are still some problems to be solved for CDs. One problem is that the size of the reported CDs is too small, which is not suitable for long term imaging as these small CDs will be easily captured by the reticuloendothelial system after systemic delivery.^31–33 Another is the lack of active sites (hydroxyl, amino, carboxyl, etc.) on the CDs’ surface makes them inoperable for further conjugation with other biomolecules, leading to their low labeling efficiency.

In this work, we report that carbon nanospheres derived from lactobionic acid (LBA) undergo thermal polycondensation to overcome the above shortcomings. LBA is an aldonic acid obtained from the oxidation of lactose, with numerous potential applications as an ingredient in food and pharmaceutical products because of its antioxidant, chelating, humectant and biocompatible properties.^34–36 Compared with F127, LBA is rich in –COOH and –OH groups which would further facilitate functionalization of the obtained nanospheres. These advantages mean LBA might be a promising source for the fabrication of fluorescent carbon probes. Remarkably, these carbon nanospheres fabricated by LBA are intrinsically fluorescent and are readily dispersible in water without any prior surface modification. Therefore, these carbon nanospheres could serve as potential fluorescent probes for cell imaging.

2. Experimental

2.1. Materials

LBA was purchased from Alfa Aesar. Cell Counting Kit (CCK-8) was obtained from Dojindo laboratories. Penicillin–streptomycin, fetal bovine serum (FBS), and RPMI-1640 medium were purchased from Gibco BRL. All these materials were used as received without further purification. DAPI was purchased from Sigma-Aldrich. Ultrapure water was used throughout.

2.2. Synthesis of carbon nanospheres

The carbon nanospheres were prepared by a one-step hydrothermal method. Briefly, 0.7 g of LBA was dissolved in 35 mL of ultrapure water and stirred at room temperature for about 2 h. Then, the solution was transferred to a Teflon-equipped stainless-steel autoclave and reacted at 200 °C for 2 h. After cooling to room temperature, the mixture was centrifuged at 10 000 rpm for 15 min to remove the aggregated particles. The supernatant was further centrifuged at 20 000 rpm for another 15 min, and the precipitates were washed twice again to obtain the product. The yield in grams of nanoparticles with respect to the starting LBA is about 2.9%. The purified carbon nanospheres were dispersed in ultrapure water and stored at 4 °C for further usage.

2.3. Characterization

Transmission electron microscopy (TEM) was performed using a Tecnai F20 transmission electron microscope with an accelerating voltage of 200 kV. Dynamic light scattering (DLS) experiments were carried out at 25 °C using a NanoZS (Malvern Instruments, UK) with a detection angle of 173°, and a 3 mW He–Ne laser operating at a wavelength of 633 nm. The polydispersity index (PDI) value was acquired from the analysis of the correlation functions using cumulants analysis. Luminescence spectra were acquired using an Agilent Cary Eclipse fluorescence spectrometer. Laser-confocal scanning luminescence imaging of HepG-2 cells was executed using a Zeiss LSM780 confocal microscope with 405 nm laser excitation for the CNs.

2.4. Quantum yield measurements

The quantum yield (QY_s) of the CNs can be calculated from the following equation: QY_s = QY_r(I_sA_rn_s²)/(I_rA_sn_r²). The s and r refer to the CN sample and reference, respectively. The reference is quinine sulfate dissolved in 0.1 M H₂SO₄. I represents the emission intensity at 360 nm excitation. And A is the UV-vis absorption intensity at 360 nm. n presents the refractive index with 1.33 as the default for both quinine sulfate and the CN solvent.³⁷ The UV-vis absorption and FL emission spectra at 360 nm excitation for the CNs and reference (QY_r = 0.54) were recorded.

2.5. Cytotoxicity assay

The in vitro cytotoxicity was studied by the Cell Counting Kit (CCK-8) assay. In detail, HepG-2 cells were seeded in a 96-well cell-culture plate at a density of 10⁴ (100 μL) cells per well and incubated for 24 h. Then, the medium was replaced with 100 μL fresh medium containing various concentrations of CNs. After 24 or 48 h incubation, the medium was removed. Subsequently, 100 μL fresh medium and 10 μL CCK-8 were added and incubated for another 2 h. The absorbance was measured using a Bio-Rad Model-680 microplate reader at a wavelength of 450 nm. The following formula was applied to calculate the viability of cell growth: cell viability (%) = (OD_sample − OD_blank)/(OD_control − OD_blank) × 100%.³⁸ The OD_sample and OD_control are the absorbance values of the experimental cells (as indicated) and the control cells (without incubation with CNs), respectively. The OD_blank represents the absorbance of CCK-8 itself at 450 nm detected using the SpectraMax M5 microplate reader. All experiments were performed in quadruplicate. Results are shown as mean ± standard deviation (SD).³⁹

2.6. Laser scanning luminescence imaging

HepG-2 cells were plated on a 35 mm glass-bottom Petri dish and allowed to adhere for 24 h.^40,41 After washing with PBS, the cells were plated in a serum-free medium containing 200 μg mL⁻¹ CNs for 3 h at 37 °C under a 5% CO₂ atmosphere, and then washed with PBS extensively to remove excess nanoparticles. Laser scanning fluorescence imaging of HepG-2 cells was carried using a laser scanning luminescence microscope at a laser excitation of 405 nm.

3. Results and discussion

3.1. Synthesis and characterization of carbon nanospheres

The carbon nanospheres were synthesized using a thermal polycondensation reaction of LBA in a Teflon-equipped stainless-steel autoclave at 200 °C. The formation of the carbon nanospheres can be determined using TEM. As shown in Fig. 1A, the obtained nanoparticles have a uniform spherical morphology with a size of approximately 120 nm. The average hydrodynamic size of the carbon nanospheres was 130 ± 15 nm as confirmed from the DLS experiments (Fig. 1B), and is consistent with the TEM results. In addition, the polydispersity index (PDI) was measured to be 0.012, which suggested a relatively well-dispersed distribution of these nanoparticles in water. To investigate whether there are any active functional groups of LBA on the surface of the carbon nanospheres after thermal polycondensation, Fourier-transform infrared (FTIR) spectra of free LBA and carbon nanospheres were recorded (Fig. 2). As for free LBA, the characteristic peaks at 3400 cm⁻¹ and 1710 cm⁻¹ were attributed to the –OH stretching vibration and –C=O stretching vibration from –COOH, respectively. However, in addition to these two typical peaks, the carbon nanospheres showed a new strong peak at 1750 cm⁻¹, corresponding to the ester (–O–C=O) stretching vibration. These results indicate that the obtained carbon nanospheres still have active groups (–OH and –COOH) even after undergoing the thermal polycondensation process, which will facilitate further functionalization for additional usage. Meanwhile the ester group on the carbon nanospheres demonstrates the occurrence of an esterification reaction between the LBA molecules at high temperature and pressure.

Fig. 1 TEM images of the CNs dispersed in water (A); size distribution of the CNs measured using DLS (B).

Fig. 2 FT-IR spectra of LBA (black) and the CNs (red) measured using an FTIR spectrometer.

We further examined the UV-vis absorption and fluorescence emission of the CNs, and the results are shown in Fig. 3. The aqueous solution of CNs showed a broad absorption spectrum ranging from 200–600 nm, with a maximum absorbance platform at 225–325 nm. However, free LBA showed a sharp decrease in absorption from 200 to 350 nm, and there was no absorbance above 350 nm. When excited with a UV-wavelength laser (350 nm), the CNs displayed strong photoluminescence between 400 and 480 nm (Fig. 3b) with a maximum peak at 436 nm, which exhibited a blue color under a hand-held UV lamp (inset). In addition to the fluorescence emission, other photophysical features of the CNs, such as excitation spectra, excited state lifetime and photoluminescence quantum yield have also been investigated. As shown in Fig. S1A,† at the emission wavelength of 436 nm, the CNs showed a characteristic excitation peak at 350 nm. The fluorescence lifetime of the CNs at 436 nm was measured to be 1.58 ns (Fig. S1B†) and the quantum yield of the CNs was determined to be 4.1%. Furthermore, the aqueous solution of the CNs showed excitation-independent FL behavior (Fig. S2†). When the excitation wavelength is changed from 280 to 405 nm, the corresponding FL emission peaks for the CNs do not show significant shifts. This behavior is in accordance with that of organic fluorescent dyes whose emission wavelength is only related to the energy transition from a singly excited state to the ground state, but cannot be affected by external excitation. However, most quantum dots have multi-energy levels associated with various surface states formed by distinct functional groups on the surface, which lead to the excitation-dependent PL behavior.^37,42

Fig. 3 UV-vis absorption spectra of LBA and the CNs in aqueous solution (A); fluorescence emission spectra of the CNs in aqueous solution, inset: photographs of the N-doped CNs in aqueous solution when exposed to daylight and 365 nm UV irradiation (B).

Photostability is very important for any fluorescent material designed for bioimaging. We first studied the luminescence properties of CNs in buffer solutions with various pH values. As shown in Fig. 4A and S3,† the FL intensity of the CNs peaked at 436 nm remained constant as the pH of the solution ranged from 3 to 11, making them a promising candidate for bioimaging. However, when the pH value went above 10 or below 3, the CNs’ FL intensity decreased rapidly, which might be ascribed to the dissociation of the CNs as their ester bond underwent hydrolysis at this extreme condition. The photostability of the CNs was further studied from the aspect of the photobleaching effect from UV exposure. In this experiment, we selected DAPI, a common cell-nuclear counterstain dye, due to its similar excitation wavelength and also its CN-like blue fluorescence. As shown in Fig. 4B and S4,† DAPI exhibited a quick decrease of FL intensity at its maximum peak of 480 nm under UV laser irradiation, and dropped to 55% of its original intensity with only 1 h of irradiation, suggesting the fast photobleaching of this organic dye. Compared with DAPI, there is almost no bleaching of the CNs (98% of original intensity) after 1 h of UV laser irradiation, and there was still 70% of the FL intensity at its maximum peak of 436 nm even after 8 h of continuous UV exposure. These results demonstrate that the CNs have a relatively high photostability which would be extremely suitable for their use in bioimaging.

Fig. 4 Dependence of fluorescence intensity (FL) on excitation time for the CNs (436 nm) and DAPI (480 nm) (λ_ex = 350 or 405 nm) (A); effects of pH on the FL intensity of the CNs at 436 nm (λ_ex = 350 nm) (B).

3.2. Cytotoxicity assay

Non-toxicity or low toxicity is a key criterion for the biomedical application of any nanoparticles. Before examining the cell imaging ability, the cytotoxicity of the CNs was assessed on HepG-2 and NIH3T3 cells using a Cell Counting Kit (CCK8). The HepG-2 or NIH3T3 cells were incubated with the CNs with different concentrations ranging from 0 to 400 μg mL⁻¹ for 24 h or 48 h, and the cell viability results are shown in Fig. 4. The CNs showed a very low cytotoxic effect on the HepG-2 and NIH3T3 cells; even at a high dose of 400 μg mL⁻¹, these two cell lines both remained more than 80% viable after 48 h of incubation (Fig. 5).

Fig. 5 In vitro cell viability of HepG-2 (A) and NIH3T3 (B) cells incubated with the CNs at different concentrations for 24 h (black) and 48 h (red).

3.3. Luminescence imaging of HepG-2 cells in vitro

As stated above, the as-prepared CNs show superior luminescence properties, low cell toxicity, and high photostability, and these characteristics inspired us to estimate the capacity of the CNs for cell imaging. The HepG-2 cells were incubated with RPMI 1640 medium containing 200 μg mL⁻¹ CNs and the imaging performance was examined using confocal fluorescence microscopy. As shown in Fig. 6, compared with the control group, the HepG-2 cells labeled with the CNs exhibited blue fluorescence under 405 nm light excitation, confirming the high cellular uptake and labeling efficiency of the CNs; therefore, HepG-2 cells can be clearly imaged and no cell damage was observed under the microscope. To further investigate the influence of the addition of conjugated biomolecules on the labeling efficacy, glycine (a model biomolecule) was conjugated onto the CNs through the EDC/NHS coupling method. As shown in Fig. S5,† the CNs’ hydrodynamic diameter increased from 130 nm to 150 nm, after conjugation with glycine molecules. However, their cellular luminescence had nearly the same intensity as the CNs, indicating that a small biomolecule like glycine, has almost no influence on the CNs’ labeling efficacy, Fig. S6.† All these features suggest that the CNs prepared from LBA can serve as a promising candidate for cell bioimaging.

Fig. 6 Confocal imaging of HepG-2 cells incubated with the CNs with a concentration of 200 μg mL⁻¹ for 3 h at 37 °C.

4. Conclusion

We have synthesized fluorescent carbon nanospheres using lactobionic acid as a precursor through a one-step hydrothermal method. The average size of the resulting CNs is about 120 ± 10 nm. The large number of hydrophilic groups on the surface, such as –OH and –COOH, make these nanospheres possess outstanding aqueous dispersibility, as well as easily modifiable with other functional groups for additional usage. In addition, the obtained carbon nanospheres exhibited strong photoluminescence and excellent photostability. Based on the favorable biocompatibility and low cytotoxicity as verified by performing CCK-8 assays, the CNs were internalized into HepG-2 cells as cell-imaging agents showing bright blue fluorescence upon UV light excitation. These results reveal that this kind of material could be used in cell imaging, and also could be potentially used in the drug delivery, biosensor or other biomedical fields after undergoing further functionalization.

Acknowledgements

This work is supported by the key clinical specialty discipline construction program of Fujian, P. R. C.; the Natural Science Foundation of China (Grant No. 61575044); the specialized Science and Technology Key Project of Fujian Province (Grant No. 2013YZ0002-3); the Science and Technology Infrastructure Construction Program of Fujian Province (Grant No. 2014Y2005); the scientific innovation project of Fujian provincial Health and Family Planning Commission (Grant No. 2014-CX-32); the Natural Science Foundation of Fujian Province (Grant No. 2015J05175); the Scientific Foundation of Fuzhou City (Grant No. 2014-S-139-6, Grant No. 2015-S-143-8, Grant No. 2015-S-143-11 and Grant No. 2015-S-143-15).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra02754g

‡ Postal Address: Shangshan Road 8, Fuzhou 350007, Fujian Province, P. R. China, Tel.: +86591-83438080, Fax: +86591-83465373.

§ Postal Address: Xihong Road 312, Fuzhou 350025, Fujian Province, P. R. China, Tel.: +86591-83705927, Fax: +86591-83705927.

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