Toward High-Efficient Red Emissive Carbon Dots - ACS Publications

Nov 10, 2016 - preparation of high-efficient red emissive CDs (R-CDs) is reported, and ..... well with nucleoli, the densest region in nucleus (see mo...
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Toward High-Efficient Red Emissive Carbon Dots: Facile Preparation, Unique Properties, and Applications as Multifunctional Theranostic Agents Shan Sun,†,‡ Ling Zhang,† Kai Jiang,† Aiguo Wu,† and Hengwei Lin*,† †

Key Laboratory of Additive Manufacturing Materials of Zhejiang Province & Ningbo Institute of Materials Technology & Engineering (NIMTE), Chinese Academy of Sciences (CAS), Ningbo 315201, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: The achievement of high-efficient pure red emissive carbon dots (CDs) is still a great challenge as well as one of the most critical issues that hinders widespread applications of CDs. Herein, a facile approach for the preparation of high-efficient red emissive CDs (R-CDs) is reported, and they exhibit numerous unique features including pure red emission (λmax ≈ 640 nm), respectable quantum yield (22.9%), low cytotoxicity, two-photon excited fluorescence (TPEF), and high photothermal conversion efficiency (43.9% under irradiation of 671 nm laser). Moreover, the chemical composition and photophysical properties of the R-CDs are detailed characterized and analyzed, and from which their photoluminescence mechanism is proposed. Interestingly, the R-CDs are found to particularly light up RNA-rich nucleolus both in one-photon and two-photon modes as well as show excellent counterstain compatibilities with other classical subcellular dyes. The localization of the R-CDs in nucleolus is supported by ribonuclease digestion testing, and the stronger emission is further verified to be due to an accumulation process. In addition, the R-CDs are confirmed to be facilely conjugated with fluorescein isothiocyanate (FITC) and then bring it into living cells, which reveals their potentials to perform as carriers for delivery of drugs that cannot (or hardly) enter into living cells directly. Finally, the R-CDs are shown to be excellent in photothermal cancer therapy in vitro due to their high photothermal conversion efficiency. This study represents not only a facile method for the preparation of high-efficient R-CDs, but also opens many possibilities for applications, such as in biomedicine (multifunctional theranostic agents) and emitting/display devices, thanks to their unique and superior properties.

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INTRODUCTION As a new class of photoluminescent (PL) nanomaterials, carbon dots (CDs) have attracted considerable attention in recent years due to numerous merits such as superior optical properties (e.g., tunable emission, high photostability, and two-photon excited fluorescence (TPEF)), water dispersibility, excellent biocompatibility, and facile preparation and modification.1−5 Consequently, CDs have been demonstrated in many promising applications including sensing,6−10 theranostics,11−18 bioimaging,19−23 and optoelectronic devices.24−28 Although many kinds of CDs had been reported so far, most of them show intense emission only at blue- to green-light regions under excitation with ultraviolet- or blue-light.1−28 Such drawbacks restrict their further applications, particularly in the fields of biomedicine and optoelectronic devices, because of the well-known blue autofluorescence of biological matrix and severe photodamage of ultraviolet excitation light, and meanwhile, red phosphor is one of the indispensable components for fabricating full-color emitting and display devices. Therefore, it is highly significant to achieve high© 2016 American Chemical Society

efficient red-light emissive CDs that can be excited with longwavelength lights as well. To the best of our knowledge, however, only a handful reports have reached this aim, but certain critical problems still exist including very low quantum yields (QYs), not pure red emission, or requiring complicated synthesis/separation procedures.11,29−32 For instance, Ge et al. prepared red emissive CDs using polythiophene derivatives as carbonaceous source, but their approach needs harsh conditions to synthesize the starting materials, and meanwhile the prepared CDs show very low PL QYs (e.g., 2.3%).11 Several groups reported nice work for preparing CDs with longwavelength or tunable emissions, but their λmax of PL mostly only reached to the orange- or orange−red-light region (λmax < 620 nm).19,30,31 Recently, Xiong’s group achieved relative high QY of red emitting CDs; however, these CDs are only one of the many components from “great patience” of chromatogReceived: September 1, 2016 Revised: October 22, 2016 Published: November 10, 2016 8659

DOI: 10.1021/acs.chemmater.6b03695 Chem. Mater. 2016, 28, 8659−8668

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decomposed products) must take part in the formation of the R-CDs. Subsequently, the as-prepared R-CDs are characterized with transmission electron microscopy (TEM) and atomic force microscopy (AFM) to confirm their nature of nanoparticles. As shown in Figure 1, panel B, the TEM image of the R-CDs displays size distribution between 2.5 and 5.5 nm with an average diameter about 4.0 nm. High-resolution TEM (HRTEM) shows a lattice spacing of 0.21 nm (inset of Figure 1B), which is attributed to the (100) facet of graphite and suggests the R-CDs containing graphite-like structures. AFM image reveals that the heights of the R-CDs are about 3.5−4.5 nm (Figure 1C), which are in good consistence with the TEM observation. Thus, we infer that the R-CDs are monodispersed nanospheres with an average size of about 4.0 nm. To determine the surface states and compositions of the RCDs, Fourier transform infrared (FT-IR), X-ray photoelectron spectroscopy (XPS), and Raman measurements are performed. In the FT-IR spectrum (Figure 1D), peaks at 1645, 1596, and 1350 cm−1 are attributed to CN/CO, CC, and C−N stretching vibrations, respectively.33−35 The peaks at 1211 and 1100 cm−1 can be ascribed to the asymmetric and symmetric stretching vibrations of C−O−C.36 The peaks between 2850 and 2960 cm−1 are arising from C−H stretching vibrations of methyl/methylene. The broad absorption bands from 3150− 3400 cm−1 indicate the presence of amino (−NH2) and hydroxyl (−OH) functional groups on the surface of the RCDs. The XPS surveys support these FT-IR analyses. For instance, three major elements (i.e., C, O, and N with molar ratio 1.0:0.45:0.11) are identified from the wide scan XPS spectrum (Figure S1A). The high resolution XPS C 1s can be deconvoluted into five binding energies (i.e., 284.7 eV (CC/ C−C), 285.4 eV (C−N), 286.3 eV (C−O), 287.8 eV (CN/ CO), and 288.8 eV (−COOH)) (Figure 1E).37 In the highresolution XPS N 1s, two fitting peaks at 399.85 and 400.8 eV are corresponding to the pyrrolic-like N and graphitic-like/ amino N, respectively (Figure S1B).38 The two fitting peaks of O 1s XPS at 531.6 and 532.35 eV are attributed to CO and C−OH/C−O−C bonds (Figure S1C). Moreover, the Raman measurement also confirms the above analysis. As shown in Figure S2, the Raman spectrum shows two dominating bands at about 1630 and 1350 cm−1, which indicate the presence of the sp2 carbon networks (G band) and the disorder or defects in the graphitized structure (D band). Besides, some minor peaks at about 1551, 1484, 1263−1296, 1100, and 963 cm−1 are also observed in this Raman spectrum, which could be attributed to NN, C−O−C, C−C, pyrrole-ring breathing, and pyridinering breathing Raman signals, respectively.39 By combining all these characterization data and analyses, the R-CDs could be tentatively considered to be composed of nanoscaled graphitelike skeleton/core, on which they are covered with N and O containing heterocycle chemical structures and abundant amino and hydroxyl functional groups. Optical Properties and PL Mechanism of the R-CDs. The UV−vis absorption, PL excitation, and emission of the RCDs are primarily examined to evaluate their optical properties. As shown in Figure 2, panel A, the R-CDs display an extraordinarily broad absorption (nearly crossing the entire UV−vis region) with a dominating band at λmax ≈ 550 nm. Figure 2, panel B represents the PL emission of the R-CDs under different excitation wavelengths, which exhibit emission λmax nearly constant with the excitation wavelengths varying from 340−580 nm (the optimal excitation and emission being at about 540 and 640 nm, respectively). Such an excitation-

raphy separation, and consequently the preparation efficiency would be low.32 Thus, it is still highly desirable to develop new and facile methods for synthesizing high-efficient pure red emitting CDs. Herein, a facile one-step microwave approach for the preparation of high-efficient red emissive CDs (R-CDs) is reported, of which exhibit optimal excitation and pure red emission wavelengths at about 540 and 640 nm, respectively. The as-prepared R-CDs are further confirmed to be not only low cytotoxicity, respectable QY (22.9%), and high photostability, but more importantly excellent TPEF feature, ease of conjugation, and high photothermal conversion efficiency (43.9% under irradiation with a 671 nm NIR laser). Moreover, the R-CDs are confirmed to be able to selectively stain RNArich nucleolus, carry fluorescein isothiocyanate (FITC) into living cells, and be used for photothermal cancer therapy in vitro. These findings demonstrate that the as-prepared R-CDs could be potentially employed as multifunctional theranostic agents for nucleolus imaging (both in one-photon and twophoton modes), drugs/genes delivery, and photothermal therapy (PTT).

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RESULTS AND DISCUSSION Preparation and Characterization of the R-CDs. The RCDs can be facilely prepared through microwave-assisted heating of citric acid formamide solution followed by a simple purification process (Figure 1A, and also refer to the Experimental Section for details). It is worthy to note herein that formamide plays a key role for the successful preparation of R-CDs (other solvents including water, ethanol, and DMF do not work). This finding indicates that formamide (or its

Figure 1. Synthesis and characterizations of the R-CDs. (A) Schematic illustration of the preparation of the R-CDs. (B) TEM images (insets showing HR-TEM and particles size distribution) of the R-CDs. (C) AFM image (inset showing height profiles analysis) of the R-CDs. (D) FT-IR spectrum of the R-CDs. (E) XPS C 1s spectrum and the deconvoluted results of the R-CDs. 8660

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reported for red emissive CDs so far. Subsequently, two photon excitation fluorescence (TPEF) of the R-CDs was investigated with femtosecond (fs) pulse laser.44,45 As shown in Figure S6, intense emission (λmax ≈ 650 nm) was observed from the RCDs under wavelengths of the fs pulse laser varying from 800− 1000 nm and with ∼850 nm being the optimum. The similar emissions between TPEF and one-photon PL indicate that they are arisen from the same excited state. Moreover, the emission intensity of the R-CDs was found to linearly increase with the square of laser power (the optimal 850 nm fs pulse laser being used, Figure 2C,D), which demonstrated a nature of the TPEF process.46 Finally, photostabilities of the R-CDs were measured, and the results are shown in Figure S7, from which one can see that the R-CDs are highly stable under continuous irradiation for 1 h. Properties in Cell Culture Medium and in Vitro Cytotoxicity of the R-CDs. Before potential applications of the R-CDs as theranostic agents were explored, their properties in cell culture medium and cytotoxicity were first investigated. As shown in Figure S8, the R-CDs are observed forming and keeping as a clear dispersion solution even after standing for 48 h, possessing a narrow size distribution between about 4 and 15 nm (from DLS, and with an average diameter of 6.8 nm), and possessing negative charges on their surface (zeta potential, −38.1 mV). In addition, their cytotoxicity in vitro was evaluated by the standard 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay using MCF-7 and HeLa cells as models. As shown in Figure S9, approximately 90% and 100% cell viabilities are observed for MCF-7 and HeLa cells, respectively, after 24 h incubation with the R-CDs at their concentrations ranging from 0−200 μg mL−1. These results indicate that the R-CDs are well stable in cell culture medium and low cytotoxicity even at a relatively high concentration up to 200 μg mL−1, which is much higher than the needed concentration for cellular imaging (i.e., 40 μg mL−1, see below). Living Cells Imaging Property of the R-CDs. The superior optical properties (i.e., high photostability, red-light emission, and TPEF) and extremely low cytotoxicity of the RCDs inspired us to investigate their applications in bioimaging (MCF-7 and HeLa cells being taken as models). Typically, the cell samples were incubated for 4 h with the R-CDs (40 μg mL−1) before imaging by confocal fluorescence microscopy. As shown in Figure 3 (for MCF-7 cells) and Figure S10 (for HeLa

Figure 2. Optical properties of the R-CDs. (A) UV−vis absorption (black line), PL excitation (Ex) (λem = 640 nm, green line), and emission (Em) (λex = 540 nm, red line) spectra. (B) PL emission spectra with different excitation wavelengths. (C) TPEF spectra with different laser powers by a femtosecond pulse laser (850 nm). (D) Relationship between the TPEF intensity and the square of laser power.

independent PL feature of CDs had also been observed in many other reports and frequently attributed to the surface states/defects emission.32,36,40−42 To make a further proof for this assumption, a broad range of pH (from 2−12) effects to the optical properties of the R-CDs are investigated. As shown in Figure S3, all the UV−vis absorption, PL excitation, and emission are found significantly altered with pH. The high pH sensitivity of the optical properties of the R-CDs implies their surface state would be mostly responsible for their PL emission. Importantly, the fluorescence excitation spectrum (at λem = 640 nm) of the R-CDs is observed to be well overlapping with their major absorption band, which indicates that the corresponding structure of this absorption should be mainly responsible for the red emission of the R-CDs. According to the knowledge of the literature and our own study,32,37,40,43 the intense absorption band of the R-CDs at λmax ≈ 550 nm could be tentatively ascribed to the n → π* transition of π systems containing C−N/CN or C−O structure, but more in-depth studies are still required to clarify this issue. Since the n → π* transition features a blue-shift of absorption with the increase of dielectric constant of the solvent, solvents effects were investigated. As shown in Figure S4, manifest blue shifts of this absorption (i.e., λmax ≈ 550) occurred with dielectric constant of solvents increasing from CH2Cl2 to H2O, which demonstrated its nature of n → π* transition.36 To further understand the PL properties of the R-CDs, their time-resolved PL spectrum was measured (under excitation at 588 nm) in solution, and an averaged lifetime of 0.68 ns was determined based on the fittings of the PL decay curve (Figure S5 and Table S1). A lifetime on the ns level implies the singlet state nature of emission of the R-CDs. Besides, although a biexponential fitting has to be applied herein, one of the components shared as high as 91.56% (Table S1), which indicated that only one radiative transition channel is mostly responsible for the PL emission of the R-CDs. Moreover, QY of the R-CDs was determined to be 22.9% (in methanol) and 16.2% (in water) under the optimal excitation wavelength (i.e., 540 nm) using an absolute quantum efficiency measurement system (Table S2), which is among the highest value that

Figure 3. Cells imaging properties of the R-CDs in living MCF-7 cells. (A) One-photon confocal fluorescent images. (B) Quantitative figure of selected MCF-7 cells (λex = 543 nm, λem = 550−750 nm, scale bar = 20 μm). 8661

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(3D) imaging of living cells was implemented.54 Before the experiment, MCF-7 living cells are counterstained with the RCDs and Hoechst to obtain clear comparison. As presented in Figure S12, nucleoli are seen significantly lighting up with red emission by the R-CDs, and meanwhile nuclei emit blue fluorescence by Hoechst. Moreover, a series of the xy sections and yz and xz cross-section images of MCF-7 cells are completely visualized through 3D reconstruction, which indicate the R-CDs being localized inside the nucleolus rather than on its surface. Subsequently, the origin of lighting up nucleoli by the R-CDs is examined. In general, there are two possibilities: (i) the R-CDs distributing evenly throughout the entire cell, but the enhanced fluorescence being generated due to their interaction with nucleoli; and (ii) the R-CDs specifically accumulating in the nucleoli, and the more intense emission being due to a locally high concentration of materials. Since RNA is a main component of nucleoli, fluorescence titrations of the R-CDs with RNA were carried out. As shown in Figure S13, fluorescence intensity of the R-CDs is observed only increasing (∼20%) slightly even with the addition of 25fold excess RNA. This finding suggests that the interaction between the R-CDs and RNA does not significantly affect their intensity of emission. Thus, we infer that an accumulation of the R-CDs in nucleoli might be a primary reason for the observed stronger emission. TPEF Living Cells Imaging of the R-CDs. To eliminate autofluorescence from biological matrix, TPEF imaging technique has been broadly applied, which usually uses NIR light as excitation source and thus provides many benefits including large penetration depth, minimal photodamage, and long observation time.55,56 Because of possessing superior TPEF feature, the two-photon bioimaging capability of the RCDs was examined. As shown in Figure 5, bright TPEF images

cells), the R-CDs were found to be able to penetrate into cells and display brightly red fluorescence under irradiation with a laser of 543 nm. More interestingly, it is observed that the RCDs were able to further pass through the nuclear pore complex of MCF-7 and HeLa cells effectively and localize within the nuclear region, notably in some round areas (Figures 3A and S7A). Through comparing the fluorescence and brightfield images (see the right two images in Figure 3A), the particularly bright fluorescence spots are observed to coincide well with nucleoli, the densest region in nucleus (see more evidence in the next section).47 Quantification of the fluorescence intensity profiles of the R-CDs treated living cells revealed a high signal intensity ratio (i.e., more than fivefold, Figures 3B and S7B) for the nucleoli (region 1 and 3) compared to the cytoplasm (region 2). Such a high fluorescence intensity ratio of nucleolus and cytoplasm suggests a particularly staining nucleolus with the R-CDs. Confirmation of Nucleolus Imaging and Staining Features. Since the nucleolus is a central site to take part in the synthesis, process, and assembly of rRNAs,48,49 the achievement of nucleolus imaging in living cells plays a vital role in the studies of nucleolar (or RNA’s) roles in cellular activities.50−53 Thus, further evidence and more properties of nucleolus staining by the R-CDs are investigated. Because of the nucleolus being a RNA-rich region, the observed nucleolus staining implies that the R-CDs have higher affinity to RNA than DNA and other substances in cytoplasm. To further confirm the facts of nucleolus staining by the R-CDs, ribonuclease (RNase) digestion tests were performed because RNase is an enzyme that can only hydrolyze RNA in cells while having no apparent influence on DNA. As shown in Figures 4

Figure 4. Confocal fluorescent images of the R-CDs and Hoechst without (i.e., control) and with the RNase treatment of MCF-7 cells (Hoechst, λex = 405 nm, λem = 420−500 nm; R-CDs, λex = 543 nm, λem = 550−750 nm; scale bar = 20 μm).

Figure 5. TPEF nucleolus imaging in living cells. Two-photon confocal fluorescent images of (A) living MCF-7 cells and (B) living HeLa cells, and corresponding quantitative figure along the green line (λex = 850 nm fs pulse laser, λem = 600−700 nm, scale bar = 20 μm).

and S11, the bright fluorescence spots disappeared in both MCF-7 and HeLa cells if they were first incubated with the RNase instead of redistributing into the entire nucleus. However, no obvious effect on the staining results of Hoechst was found after digestion treatment with RNase since Hoechst prefers to stain DNA rather than RNA. These RNase digestion testing results reconfirm that the R-CDs have higher affinity to RNA-rich nucleolus in the complicated interior environment of cells, and their selectivity is good enough for identifying nucleolus from nucleus. To determine if the R-CDs are localizing inside the nucleoli rather than just covering on their surfaces, a three-dimensional

of the MCF-7 and HeLa cells can be observed under irradiation of 850 nm fs pulse laser, and more importantly, stronger emissions are also found localizing in the nucleoli regions, which are similar as the one-photon fluorescent images. Quantification of the fluorescence intensity profiles further verified this phenomenon. These results demonstrate great potentials of the R-CDs in deep-tissue imaging and detection by TPEF imaging technique. Counterstain Compatibility of the R-CDs. For cell imaging, it is significant to achieve multitarget staining 8662

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Figure 6. Counterstain compatibility of the R-CDs in living and fixed cells. (A) Fluorescent images of living MCF-7 and HeLa cells after incubation with R-CDs and Hoechst. (B) Fluorescent images of fixed MCF-7 cells after counterstain with FITC-phalloidine, Hoechst, and R-CDs (Hoechst, λex = 405 nm, λem = 420−500 nm; FITC-phalloidine, λex = 488 nm, λem = 500−560 nm; the R-CDs, λex = 543 nm, λem = 550−750 nm; scale bar = 20 μm).

Figure 7. Confocal fluorescent images of (A) RCDs−FITC, (B) FITC, and (C) control of R-CDs in MCF-7 cells. The images b, f, and j are collected in the range of 550−750 nm (R-CDs channel) under excitation of 543 nm. The images c, g, and k are collected in the range of 493−540 nm (FITC channel) under excitation of 488 nm. The images d, h, and l are bright-field. Image a is an overlay of images b−d; image e is the overlay of images f−h; and image i is the overlay of images j−l.

simultaneously through introducing different probes with various light-emitting ranges.57,58 Good compatibility of nucleolus tracker with other classical cellular dyes would be helpful for investigating the relationship among different organelles or biomolecules. Figure 6, panel A represents the counterstain results of the R-CDs and the commercial nucleus tracker Hoechst in living cells. Bright red fluorescence from the nucleoli is easily recognized from the blue nucleus region (i.e., emission from Hoechst) either in MCF-7 or HeLa cells.

Besides, counterstain of the R-CDs with cell membrane tracker FITC-phalloidine and Hoechst in fixed MCF-7 cells are also investigated. It is very interesting to see from the merged image that all three organelles (i.e., cell membrane, nucleus, and nucleolus) can be clearly distinguished with different colors (Figure 6B). These observations demonstrate that the R-CDs are not only able to be employed as excellent nucleolus trackers, but also suitable for costaining imaging of both living and fixed cells. 8663

DOI: 10.1021/acs.chemmater.6b03695 Chem. Mater. 2016, 28, 8659−8668

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Figure 8. Photothermal performance of the R-CDs. (A) Temperature elevation of pure water, culture medium, and the R-CDs aqueous dispersions with different concentrations as a function of laser irradiation time for 10 min. (B) Plot of temperature change (ΔT) of 10 min laser irradiation versus concentrations of the R-CDs. (C) Photothermal effects of the R-CDs water dispersion (50 μg mL−1, 1.0 mL) for 10 min irradiation. The inset is a photograph of the R-CDs aqueous dispersion after laser irradiation. (D) Plot of cooling time versus −ln θ (i.e., the negative natural logarithm of the temperature driving force obtained from the cooling stage in Figure 6C). (E) The viability of MCF-7 cells incubated with different concentrations of the R-CDs (0−200 μg mL−1) before and after laser irradiation for 10 min. (F) The viability of MCF-7 cells incubated with 200 μg mL−1 of the R-CDs under various irradiation time. C1 represents a control group of MCF-7 cells without the R-CDs addition and laser irradiation, and C2 represents a control group without the R-CDs addition but with 10 min laser irradiation.

addition of the R-CDs from 20−200 μg mL−1 (Figure 8B). These results demonstrate that the R-CDs can efficiently and rapidly convert the laser energy into heat. Next, temperature changes of the R-CDs water dispersion (50 μg mL−1, 1.0 mL) as a function of time were recorded, first with continuous irradiation until reaching a steady-state temperature, and then by turning off the laser until the temperature going back to the room temperature (Figure 8C). By combining the fitting data obtained from the cooling stage in Figure 8, panels C and D and the literatures’ method,59,60 the photothermal conversion efficiency (η) of the R-CDs is calculated to be 43.9% (see Experimental Section for details). Moreover, photothermal stability of the R-CDs was also investigated (2.5 W·cm−2 of 671 nm laser for 2 h). As shown in Figure S16, all the UV−vis absorption, PL excitation and emission, and TEM characterizations are observed to be only slightly changed, but the FT-IR is altered a little bit more. These results demonstrate that the functional groups on surface of the R-CDs somewhat change after such a long-term irradiation, but their optical properties and morphology can still keep stable. The high η value of the R-CDs inspired us to further examine their photothermal cytotoxicity on cancer cells via MTT assay (MCF-7 cells being taken as an example). The cytotoxicity experiments have shown that the survival of MCF-7 cells is nearly no effect in the presence of the R-CDs up to 200 μg mL−1 without laser irradiation (Figure S9). Upon laser irradiation for 10 min, however, viabilities of the MCF-7 cells are found significantly decreasing with the concentration of the R-CDs increasing from 20 to 200 μg mL−1 (Figure 8E). Notably, nearly 90% and 100% of the MCF-7 cells are killed with the presence of 100 and 200 μg mL−1 of the R-CDs, respectively. Moreover, cell viabilities of MCF-7 cells incubated with 200 μg mL−1 of the R-CDs under different irradiation times were investigated. As shown in Figure 8, panel F, time as short as 3 min of irradiation could induce the cancer cell mortality rate reaching approximate 80%, and nearly 100% with only 6 min of irradiation. These results indicate that the as-

Potential Carriers for Drugs Delivery. Given covering abundant amino and hydroxyl functional groups on the surfaces of R-CDs, their potentials of serving as carriers for drugs delivery are preliminarily investigated through conjugating fluorescein isothiocyanate (FITC) as a model (see details of preparation in Experimental Section).36 FT-IR characterization confirms the successfully coupling FITC onto the R-CDs (named RCDs−FITC, Figure S14), that is, disappearance of the characteristic peak of FITC at 2067 cm−1 (i.e., NCS) and accompanied by decrease of peak intensity at 3190 cm−1 (i.e., − NH2) of the R-CDs. Subsequently, the FITC loading capacity was determined to be 18.5 mg per gram of the R-CDs using a fluorometric method by referencing to the literature (Figure S15).36 Finally, the RCDs−FITC conjugate was incubated with MCF-7 cells and then subjected to confocal imaging. As shown in Figure 7, panels b and c, both red (from R-CDs) and green (from fluorescein) emissions are clearly observed. However, the free FITC are found to hardly enter into living cells (Figure 7e−h). As a control, the fluorescence image of R-CDs in FITC channel is also performed, which displays no obvious signal (Figure 7k). These results demonstrate that the R-CDs can bring FITC into living cells effectively and thus could be potentially serve as carrier for delivery of drugs/genes that cannot (or hardly) enter into living cells directly. Photothermal Property of the R-CDs and their PTT Performance. Given the unique NIR absorption feature (i.e., 650−800 nm) and special nucleolus localization in tumor cells, the R-CDs are proposed to behave as a potential PTT agent for cancers. First of all, photothermal conversion capability of the R-CDs is evaluated. As performed in Figure 8, panel A, the temperature elevation of the R-CDs water dispersions with different concentrations (0−200 μg mL−1) is measured under continuous irradiation (671 nm laser, 2.5 W cm−2, the same below) for 10 min. Pure water and medium for cell cultivation are taken as controls, and their temperatures only increased by 1.3 and 1.4 °C, respectively. In contrast, the temperatures increased by 19.5−60.4 °C after 10 min irradiation with the 8664

DOI: 10.1021/acs.chemmater.6b03695 Chem. Mater. 2016, 28, 8659−8668

Article

Chemistry of Materials

Zeiss confocal laser fluorescence microcope (LSM-710, Germany). The photothermal performance of the R-CDs was carried out by 671 nm NIR laser device (Changchun New Industries Optoelectronics Technology Co. Ltd., China), and the temperature change was recorded by a photothermal imaging system (Ti400, Fluke, USA). Preparation of the R-CDs. In a typical synthesis process, 2.8 g of citric acid was dissolved in 50 mL of formamide to form a transparent solution. The above solution was transferred into a Teflon-line autoclave, sealed, and placed in a microwave chemical reactor for heating at 160 °C (400 W) for 1 h and 120 °C (400 W) for another hour. After the autoclave cooled to room temperature naturally, the obtained product solution was precipitated by the addition of 250 mL of acetone overnight in a refrigerator (−20 °C). The precipitation was then washed with acetone and 10% methanol/acetone three times (50 mL for each washing step), respectively. Then the residue was redispersed in methanol and filtered by 0.22 μm membrane filter to remove large particles. After removal of methanol using rotary evaporating (40 °C) and further dried in vacuum oven, the R-CDs were finally obtained as a dark red powder. Preparation of the RCDs−FITC Conjugate. Fluorescein isothiocyanate (FITC, 2.0 mg) and R-CDs (10 mg) were dissolved in 20 mL of anhydrous methanol in a flask. Then 20 μL of the triethylamine was added into the solution. The reaction mixture was stirred for 24 h at room temperature, the obtained mixture was fivefold diluted with 0.1 M NaHCO3 and purified through dialysis with 1000 Da of cutoff molecular weight (first with 0.1 M NaHCO3 for 2 days to remove the unreacted FITC, and then deionized water was used for another 2 days to remove NaHCO3). Finally, the RCDs− FITC conjugate can be obtained by rotary evaporating off the water (60 °C) and further dried in vacuum oven. Cell Culture and Staining. Human breast cancer cell line (MCF7) and human cervical cancer cell line (HeLa) were cultured in culture medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) in a 5% CO2 incubator at 37 °C. The RCDs were dissolved in ultrapure water that contains 1% DMSO at a concentration of 40 μg mL−1; Hoechst 33258 and FITC-phalloidin were prepared as 5.0 μg mL−1 and 10 μg mL−1, respectively. For all cells staining experiments, cells were seeded in glass coverslips with an original density of 4 × 104 cells per mL and allowed to adhere overnight. For living cell staining experiments, cells were stained with the R-CDs (40 μg mL−1) that dispersed in culture medium for 4 h at 37 °C and then rinsed with clean PBS twice before imaging. For fixed cell staining experiments, cultured cells grown on glass coverslips were pretreated in accordance with the following procedures: first, cells were fixed by 4% paraformaldehyde for 30 min; second, these samples were permeabilized by 0.2% Triton X-100 for 5 min and then enclosed by 0.1% BSA at ambient temperature. The prefixed cells were stained with the R-CDs (40 μg mL−1) for 1 h before imaging. For cells counterstain experiments, living cells were cocultured with 40 μg mL−1 of the R-CDs in culture medium for 4 h, and then the same sample was stained with 5.0 μg mL−1 Hoechst 33258 for another 20 min before imaging. Prefixed cells experienced the similar procedures, except the incubation time with the R-CDs changed to 1 h. For RNase digest experiments, the prefixed cells were divided into two sets. Samples of 1.0 mL of PBS (pH 7.4, as control experiment) buffer solution and 1.0 mL of RNase (50 μg mL−1) were added into these two sets, respectively, and then two sets of cells were incubated at 37 °C in 5% CO2 for 4 h. After being rinsed with PBS twice, all cells were stained with 40 μg mL−1 of the R-CDs for 1 h. At last, these samples were imaged with fluorescence microscopy after being rinsed with PBS again. MTT Assay for the Cell Cytotoxicity. To evaluate biocompatibility of the R-CDs, samples were carried out with MCF-7 and HeLa cells by the standard MTT assay in vitro cytotoxicity studies. Generally, 100 μL of cells was seeded in a 96-well plate at a density of 1 × 105 cells per mL and allowed to adhere overnight. After cultured in a 5% CO2 incubator at 37 °C for 24 h, the culture medium was discarded, and cells were then treated with the other 100 μL of culture

prepared nucleolus-targeting R-CDs hold promising potentials as an effective PTT agent for cancer therapy.

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CONCLUSION In summary, a very facile method for the preparation of highefficient red emissive CDs with high QY of 22.9% is reported in this study. The as-prepared R-CDs also hold many other attractive characteristics such as pure red emission, low cytotoxicity, high photostability, superior TPEF, broad absorption, and high photothermal conversion efficiency. Through chemical composition and photophysical characterization and analysis, a possible PL mechanism of the R-CDs is proposed. Interestingly, the R-CDs are found to be particularly light up RNA-rich nucleolus through an accumulation process as well as show excellent counterstain compatibilities with other classical subcellular dyes for both living and fixed cells. Moreover, the R-CDs are shown to have ease of conjugation with FITC and then bring it into living cells, which indicate their potentials of serving as carriers for delivery of drugs/genes that cannot (or hardly) enter into living cells directly. Finally, the R-CDs are found to be an outstanding candidate for photothermal cancer therapy in vitro. Notably, this work is regarded as a preliminary step for the preparation and applications of the R-CDs, and further studies including clarification of their mechanisms of PL emission and nucleolus accumulation, performances as multifunctional theranostic agent in vivo, and red phosphor in fabricating optoelectronic devices are still ongoing.

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EXPERIMENTAL SECTION

Materials and Characterizations. All chemicals are from commercial sources and are of analytical grade. Citric acid, formamide, acetone, methanol, and 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) were acquired from Sinopharm Chemical Reagent Co., Ltd. Bovine serum albumin (BSA) and dimethyl sulfoxide (DMSO) were purchased from Aladdin Industrial Inc. (Shanghai). Triton X-100 was purchased from Suolaibao BioTechnology Co., Ltd. (Shanghai). Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI1640) medium were obtained from Thermo Fisher Scientific Inc. FITC, Hoechst 33258, and FITC-phalloidine were purchased from Invitrogen, Carlsbad, CA, USA. Ribonuclease A (RNase) was purchased from Sigma (USA). Ribonucleic acid from Baker’s Yeast (RNA) was purchased from Sangon Biotech Co., Ltd. (Shanghai). R-CDs were synthesized by using MDS-6G microwave chemical reactor (SMART, Shanghai SINEO Microwave Chemistry Technology), and the characterizations of R-CDs were performed by the following instruments. UV−vis absorption spectra were recorded on a PERSEE T10CS UV−vis spectrophotometer. Fluorescent emission and excitation spectra were measured on a Hitachi F-4600 spectrophotometer at ambient conditions. Two photon excitation fluorescence (TPEF) was carried out using a femtosecond pulsed laser (Coherent Inc., USA) connecting an optical-multichannel analyzer system (Spectrapro-300i, ARC). Fourier transform infrared (FT-IR) spectra were obtained on a Nicolet 6700 FT-IR spectrometer. Transmission electron microscopy (TEM) and high-resolution TEM (HR-TEM) observations were performed on a Tecnai F20 microscope. Atomic force microscopy (AFM) measurements were carried out with Veeco Dimension 3100 V. X-ray photoelectron spectroscopy (XPS) was performed on an AXIS UL TRA DLD spectrograph with Al/Kα as the source. Raman spectra were obtained using Renishaw inVia-reflex spectroscopy (Renishaw, UK). Quantum yields were measured on a QE-2100 quantum efficiency measurement system (Otsuka Electronics, Japan). One-photon cellular images were performed with a Leica confocal laser fluorescence microscope (TSC SPS II, Germany). Two-photon cellular images were taken by the 8665

DOI: 10.1021/acs.chemmater.6b03695 Chem. Mater. 2016, 28, 8659−8668

Chemistry of Materials medium that contained various concentrations of the R-CDs (0−200 μg mL−1) for another 24 h. Five replicate wells were used for every control and tested concentrations. At the end of the incubation, 10 μL of MTT (5.0 mg mL−1 in PBS) was added into each well for an additional coculture of 4 h. Then the growth medium was removed, and 100 μL of DMSO was added into each well to dissolve the colored fromazan. Finally, optical densities of these samples were recorded by a microplate reader (Imark 168−1130, Biorad, USA) at a wavelength of 550 nm. Measurement of Photothermal Performance. To measure the photothermal performance of the R-CDs, 1.0 mL R-CDs aqueous dispersion with different concentrations (0−200 μg mL−1) was introduced in a cuvette. The 671 nm laser at a power density of 2.5 W cm−2 was employed to deliver perpendicularly through the cuvette and irradiated for 600 s. The temperature was monitored every 10 s by the digital photothermal imaging system. MTT Assay for Photothermal Cytotoxicity. For quantitative evaluation of photothermal cytotoxicity of the R-CDs, MCF-7 cells with a density of 104 cells per well were seeded in the 96-well plate for 24 h. Then the culture medium was replaced by the R-CDs dispersions with different concentrations (0−200 μg mL−1), and the cells were further incubated for 4 h. Thereafter, the cells were treated with or without the irradiation of 671 nm laser (2.5 W·cm−2) for 10 min and then incubated for another 24 h. To investigate the effects of different irradiation times, 200 μg mL−1 of the R-CDs was incubated with MCF-7 cells for 4 h. The cells were then irradiated by 671 nm laser for various times (e.g., 3, 6, and 9 min). After 24 h incubation, the viability of MCF-7 cells was measured by the MTT assay. Calculation of the Photothremal Conversion Efficiency (η). The η was measured by recording the temperature change of the RCDs (50 μg mL−1, 1.0 mL) as a function of time under continuous irradiation of 671 nm laser (2.5 W cm−2) until a steady-state temperature was reached. Then η was calculated by the following equation:

η=

ACKNOWLEDGMENTS

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REFERENCES

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hA(Tmax − Tsurr) − Q s I(1 − 10−Aλ)

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.6b03695. XPS, Raman, pH and solvent effects, PL lifetime, TPEF, photostability, cytotoxicity test, extra imaging images, size histograms and distributions, loading capacity, QYs, FTIR after conjugating FITC, stability of the R-CDs under laser irradiation (PDF)

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The authors acknowledge the Natural Science Foundation of China (21277149), Zhejiang Provincial Natural Science Foundation of China (LR13B050001), and the Ningbo Science and Technology Bureau (2014B82010 and 2016C50009) for supporting this work. The authors would also like to sincerely thank Prof. Qing Huang at NIMTE for allowing us to access the fluorescence spectrophotometer in the lab and Prof. Chunxiang Xu and Dr. Junfeng Lu in the State Key Laboratory of Bioelectronics and School of Electronic Science and Engineering, Southeast University for their assistance in the measurements of TPEF using femtosecond pulse laser.

Where the parameters are defined: h is the heat transfer coefficient, A is the surface area of the sample cuvette, Tmax is the steady-state temperature, Tsurr is the ambient temperature, Qs is the heat associated with the light absorbance of the solution, I is the incident laser power, and Aλ is the absorbance of the R-CDs solution at a wavelength of 671 nm. The detailed calculation steps were referred to in refs 59 and 60.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-574-86685130. Fax: +86-574-86685163. ORCID

Aiguo Wu: 0000-0001-7200-8923 Notes

The authors declare no competing financial interest. 8666

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