Fluorescent Nanoparticles with Tissue-Dependent ... - ACS Publications

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Fluorescent Nanoparticles with Tissue-Dependent Affinity for Live Zebrafish Imaging Deepak Kumar Khajuria,†,§ Vijay Bhooshan Kumar,‡,§ David Karasik,*,† and Aharon Gedanken*,‡ †

The Musculoskeletal Genetics Laboratory, Faculty of Medicine in the Galilee, The Musculoskeletal Genetics Laboratory, Bar-Ilan University, Safed 1311502, Israel ‡ Department of Chemistry, Bar-Ilan Institute for Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 5290002, Israel S Supporting Information *

ABSTRACT: Carbon quantum dots (CDs) are widely investigated because of their low toxicity, outstanding water solubility, and high biocompatibility. Specifically, fluorescent CDs have attracted ever-increasing interest. However, so far, only a few studies have focused on assessing the fluorescence of nitrogen-doped CDs (N@CDs) during in vivo exposure. Here, we describe a strategy for low-cost, one-pot synthesis of N@CDs. The low toxicity and suitability of the N@CDs for fluorescence imaging are validated using zebrafish (ZF) as a model. Strong fluorescence emission from ZF embryos and larvae confirms the distribution of N@CDs in ZF. The retention of N@CDs is very stable, long lasting, and with no detectable toxicity. The presence of a strong fluorescence at the yolk sac, especially in the vicinity of the intestine, suggests that a high content of N@CDs entered the digestive system. This indicates that N@CDs may have potential imaging applications in elucidating different aspects of lipoprotein and nutritional biology, in a ZF yolk lipid transport and metabolism model. On the other hand, the presence of a strong selective fluorescence at the eyes and melanophore strips at the trunk and tail region of ZF larvae suggests that N@CDs has a high melanin-binding affinity. These observations support a novel and revolutionary use of N@CDs as highly specific bioagents for eye and skin imaging and diagnosis of defects in them. N@CDs are known for their multifunctional applications as highly specific bioagents for various biomedical applications because of their exceptional biocompatibility, photostability, and selective affinity. These characteristics were validated in the developmental ZF model. KEYWORDS: zebrafish, fluorescence, embryo, larvae, live imaging, nanoparticles concentrations.10−12 Hydrothermal synthesis is one of the commonly used methods for synthesizing undoped and doped CDs.13,14 CDs can also be synthesized by microwaves15 and sonochemistry.16,17 Remarkably, the characteristic properties of CDs depend to some extent on the raw material (such as amino acid, sugar, protein, carbohydrates, juice, milk, glucose, etc.) used in their hydrothermal synthesis. The synthesis of nitrogendoped carbon material, such as nanotubes or nanoparticles, is well established in the literature. However, quantum yield and photostability of the previously reported N@CDs are relatively low for long-term biological processes,18,19 with the challenge of keeping their ability to function without aggregation or precipitation. In this study, we produced low-cost, one-pot hydrothermally synthesized N@CDs. This synthesis procedure does not require organic solvents and results in a quantum yield of 44%, which is higher than all previously reported CDs (12− 30%)16 of the same category.

1. INTRODUCTION Nanoscale materials are emerging as a new area of research that shows enormous potential for momentous impact on the frontiers of various branches of physical, biological, and clinical science. Nanomaterials have been used in fluorescent cell imaging,1 disease analysis, drug targeting, and delivery applications.2,3 Among the advanced nanomaterials used in fundamental and applied research, carbon nanomaterials are one of the cheapest and most abundant in our surroundings.4 Recently, carbon light-emitting (fluorescent) nanoparticles, namely, carbon quantum dots (CDs), have attracted the attention of the scientific community because of their unique optical and electronic properties. Moreover, the CDs have low toxicity, outstanding water solubility, and high biocompatibility.5 The fluorescence of CDs can be improved by nitrogen doping, and such nitrogen-doped CDs (N@CDs) were already tested in fluorescent ratiometric pH sensing,6 in vivo bioimaging, drug delivery, and photocatalysis.7−9 Moreover, several reports have confirmed that N@CDs prepared from clean sources are nontoxic to living organisms, with no significant change in their vitalities even when used in different © XXXX American Chemical Society

Received: April 3, 2017 Accepted: May 15, 2017 Published: May 15, 2017 A

DOI: 10.1021/acsami.7b04668 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Procedures of soaking ZF embryos in N@CDs and fluorescence imaging. (A, B) Soaking ZF embryos in N@CD solutions with different concentrations: 0, 0.75, 1.5, 3, and 6 mg mL−1. Bright-field (upper) and fluorescence (lower) images of the ZF embryos: (C) control and (D) after soaking in 6 mg mL−1 of N@CDs. The scale bar is 2.0 mm. of N@CDs was analyzed using a fluorometer (Cary Eclipse, Varian GmbH, Germany). UV−vis analysis of the N@CDs was performed using a spectrophotometer (Varian Cary 100). The morphology and the crystalline properties of the N@CDs were analyzed by highresolution transmission electron microscopy (HR-TEM, JEOL 2100). X-ray photoelectron spectroscopy (XPS) and Raman analysis were performed by lyophilization after drying the N@CDs. The XPS analysis was carried out using an ESCALAB 250 spectrometer with a monochromatic X-ray source of Al Kα excitation (1486.6 eV). Raman spectra of powdered N@CDs were analyzed with a Renishaw inVia Raman microscope equipped with RL785 and RL830 Class 3B wavelength-stabilized diode lasers with a Leica DM2500 M (Leica Microsystems, Wetzlar, Germany) material analysis microscope. The dynamic light scattering (DLS) measurements of an aqueous N@CD solution were performed on a ZetaSizer Nano ZS instrument (Malvern Instruments Ltd, Worcestershire, U.K.). 2.3. ZF Maintenance and Embryo Harvesting. ZF were maintained according to the approved guidelines of the Institutional Animal Care and Use Committee of Bar-Ilan University, Israel. They were cultured in 3 L aquaria with recycled water (approximate salinity: 450−500 μs cm−1 with NaCl and NaHCO3) and pH 7.0−7.2 (10% fresh deionized water added daily) at 28 °C with a 10/14 h dark/light cycle. Adult ZF were fed with 3 mL of freshly hatched live brine shrimp (Artemia nauplii) grown in the lab (Ocean Nutrition, Salt Lake City, UT) in 3 L of water and fish feed TetraMin (Tetra, Blacksburg, VA) at 8:30 and 16:00 daily. For spawning, one adult male and two female ZF were chosen and placed at opposite sides of a small (1 L) breeding tank (Aquazone, Kfar Sava, Israel) separated by a tank divider, at 16:30 of the previous day of the experiment. On the next day, the tank divider was removed at 8:30 and the water level was lowered. After 1 h, ZF embryos were collected from the breeding tank and transferred to Petri dishes (50 eggs/dish) containing 40 mL of fresh methylene blue-rich E3 (embryo) medium. All unfertilized embryos and debris were removed under observation by an inverted modular routine stereo microscope (Leica M80; Leica, Leica Microsystems, Heerbrugg, Switzerland)). During all of the procedures, the ZF embryos and the E3 medium were kept at 28 °C, either in an incubator or in a climatized room at 28 °C, with a 10/14 h dark/light cycle. 2.4. Soaking of ZF Embryos in N@CDs. N@CD solutions with concentrations of 0, 0.75, 1.5, 3, and 6 mg mL−1 were prepared by dissolving appropriate amounts of N@CDs in the E3 medium. A small amount (5 mL) of each of these N@CD solutions was added to separate wells of a six-well cell-culture plate, as shown in Figure 1A. In five wells of the six-well cell-culture plate, 30 ZF embryos (0.5 hpf (hours post fertilization)) were soaked in the E3 medium containing different concentrations of N@CDs (Figure 1B). The ZF embryos (0.5 hpf) were used at the one-cell stage to ensure that the N@CDs are permeated into the embryos and dispersed throughout the ZF cytoplasm. After 2.5 h, the ZF embryos were washed three times with the E3 medium and then placed on a glass slide with a small amount of the same medium. This glass slide was viewed through a fluorescent

Zebrafish (ZF) is an organism emerging as a model of choice to evaluate biomaterials from various aspects, such as nanotoxicity, imaging, and gene therapy. This organism is easy to use because of its low maintenance cost, optical transparency, and significant similarity to the human genome.20,21 Previously, ZF as a model have been used to investigate a wide range of metal nanoparticles, carbon-based nanoparticles (graphene, graphene oxide, carbon nanotubes), and polymers.22−25 The ZF body is transparent during early embryonic development and hence it is easy to observe the in vivo transport, ratiometric fluorescence probe,26 toxicity and biocompatibility of various nanoparticles, and real-time drug screening at this stage.27−29 Kang et al. reported a useful application of CDs in a ZF model for studying the toxicity, transport mechanism of several test compounds, and drug screening.30 Earlier, Ma et al. demonstrated the practical application of iridium, which can be used as a selective-affinity luminescent probe for the visualization of intracellular zinc in a living ZF model.31 Recently, Mao et al. have demonstrated an iridium chemosensor for visualizing cysteine in a living ZF model, indicating the significance of ZF as a potential model for in vivo imaging applications.32 Here, we report fluorescence imaging of live ZF embryos and larvae using N@CDs as a probe. When live ZF embryos are soaked in a N@CD solution, the N@CDs easily entered the embryos across the chorion and the germ ring around the yolk sac. The presence of N@CDs in the ZF model is detected by the fluorescence emitted by them, which also validates the potential of using N@CDs as probes in experimental research. Moreover, the N@CDs retained in the yolk sac, eyes, and the longitudinal melanophore strip, at the trunk and tail region, are very stable and long lasting, with no detectable toxicity.

2. EXPERIMENTAL SECTION 2.1. Chemicals Required. Quinine sulfate, bovine serum albumin (BSA), and tricaine methane sulfonate (MS222) were purchased from Sigma-Aldrich, Israel. Double-distilled water (DDW, 18.3 MU) was used in all of the experiments. 2.2. N@CD Synthesis and Characterization. Water-soluble N@ CDs were synthesized using our modified hydrothermal method described previously.13 Briefly, a homogeneous BSA aqueous solution was prepared by dissolving 1.25 g of BSA (66.5 kD) in 250 mL of DDW. This solution was then transferred to a 100 mL Teflon-lined autoclave and heated at 190 °C for 6 h in an oven. Afterward, the reaction was quenched by cooling the autoclave in water. A large carbide slag was discarded from the product solution by centrifugation. The pale yellow-brown aqueous solution of N@CDs was evaluated by physicochemical techniques and subsequently exploited for bioimaging of the ZF embryo and larvae. The fluorescence of the aqueous solution B


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Figure 2. Procedure of soaking ZF larvae in N@CD solution and acquiring images. (A, B) Culturing 6−10 ZF larvae (72 hpf) in N@CD solution. (C) Placing the anesthetized ZF larvae on a glass slide with a small amount of MS222 solution, in which the larvae are immersed. (D) Acquiring bright-field (upper) and fluorescence (lower) images of ZF larva soaked in 6 mg mL−1 N@CDs. The scale bar is 1.6 mm. stereo microscope (Leica M165 FC, Leica Microsystems, Heerbrugg, Switzerland) (Figure 1C,D). The bright-field and fluorescence images of the ZF embryos soaked in N@CD solutions with different concentrations were captured at 6, 24, and 48 hpf. 2.5. Soaking of ZF Larvae in N@CDs. For this experiment, ZF embryos (24 hpf) were incubated in 0.003 wt % 1-phenyl-2-thiourea (PTU)-E3 medium to abolish pigmentation and allow better visualization until the larvae hatched (∼72 hpf). After 72 h, 2 mL samples of PTU-E3 solution containing N@CDs with different concentrations were prepared and 6−10 live ZF larvae were placed in five wells of the 24-well cell-culture plate (Figure 2B). These ZF larvae were incubated for 5 h at 28 °C. After 5 h, the PTU-E3 culture solution containing N@CDs was removed and replaced by E3 medium. After washing three times with the E3 medium, the ZF larvae from each of the five wells were anesthetized in 160 μg/mL MS222 solution and then placed on a glass slide while being immersed in the solution. The glass slide was placed under a fluorescent stereo microscope for imaging (Figure 2C,D). The bright-field and fluorescence images of the ZF larvae with N@CDs at different concentrations were captured.

Figure 3C confirm the presence of carbon, nitrogen, and oxygen on the surface of the N@CD particles. In addition, we analyzed the Raman spectroscopy of the N@CDs to investigate the graphitic nature of carbon. This analysis revealed the presence of functional groups on the surface of N@CDs. Figure 3B illustrates the Raman spectra of N@CDs, with two major bands, one at the D-band at about 1346 cm−1, attributed to the presence of sp3 carbon defects, and another at the G-band at about 1566 cm−1, attributed to the carbon G-band as well as the stretching vibration of CC bonds (sp2). These results indicate the presence of graphitic carbons in the N@CDs. The HR-TEM images showed spherical N@CD particles with relatively narrow size distribution (Figure 3D,E). The average size of the N@CDs was about ∼5 nm, which is in agreement with the findings of our previous study. 13 Furthermore, the particle size of N@CDs confirmed by DLS was found to be 5−14 nm (Figure S1), which is in reasonable agreement with the TEM analysis (3−8 nm). The optical pictures of N@CDs were collected under sunlight and UV light at pHs 3.5, 5.0, 7.0, and 10.0 (Figure S1). The fluorescence emission spectra of N@CDs were collected at the excitation of 390 nm and various pHs (Figure S2), and the results showed that N@CDs were photo-stable over a wide range of pH (3.5− 10.0). The XPS spectra of N@CDs (Figure 3C) have three major bands at around 288.66, 398.99, and 532.61 eV, which correspond to C1s, N1s, and O1s, respectively. The XPS analysis revealed that the nitrogen present in the N@CD samples originated from the amino acid present in BSA. The synthesis mechanism of N@CDs from BSA aqueous solution involves several steps, such as fragmentation, formation of hydronium ion, polymerization, aromatization, and carbonization. The hydrothermal reaction (>170 °C) causes fragmentation of the BSA polymer, which leads to the formation of a small-chain oligomer of the amino acid. The possible mechanism for the formation of N@CDs is shown in

3. RESULTS AND DISCUSSION 3.1. Characterization of N@CDs. The obtained transparent aqueous supernatant solution containing N@CDs (Figure 3A, inset) was pale-yellow in daylight and showed blue-green emission under UV light. The characterization of N@CDs has been previously described.13 The biocompatible N@CDs were analyzed for their fluorescence properties at different excitation wavelengths (λex = 330, 350, 370, 390, 410, 430, 450, and 470 nm). The fluorescence emission spectra of the N@CDs showed narrow bands, with the maximum emission centered between 450 and 490 nm (Figure 3A). The maximum fluorescence emission intensity was detected at the excitation wavelength of 390 nm, revealing the maximumintensity emission peak at 462 nm, implying a relatively narrow size distribution of the particles. The XPS images shown in C


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Figure 3. Characterization of N@CDs. (A) Fluorescence spectra of N@CD aqueous solution, with no detection of precipitation/aggregation. (B) Raman spectra. (C) XPS full-scan spectrum of N@CDs. (D) Transmission electron microscopy (TEM) images of a cluster of N@CDs. (E) Higherresolution image of the N@CDs. Inset: the crystal lattice of a single particle.

Scheme 1. In contrast to the previous study,13 the proposed mechanism is considered to be temperature dependent. When the solution temperature reached about 190 °C, BSA amide and hydronium ions were formed via the reaction between two fragments (small oligopeptide) of the BSA molecules. Further heating initiated the condensation of the oligopeptide by

dehydration and dehydrogenation of BSA. We previously reported that the aqueous dispersion of N@CDs was transparent in daylight and remained stable up to several weeks. Upon excitation under a 365 nm UV lamp (Figure 3A, inset), the N@CDs emitted a strong blue luminescence and the quantum yield was approximately 44%.13 D


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ACS Applied Materials & Interfaces Scheme 1. Possible Mechanism for the Formation of N@CDs

Figure 4. Bright-field and fluorescence images of ZF embryos and larvae after soaking in 6 mg mL−1 N@CD solution for 2.5 h at different time intervals: (A, A″) 6; (B, B″) 24; and (C, C″) 48 hpf. A, B, and C are the control groups. The scale bar is 2.0 mm.

3.2. Live ZF Imaging. CDs have been widely investigated for their catalysis and photoelectric characteristics, such as fluorescence and electrochemiluminescence.33 The most appealing characteristic of N@CDs is their application in in vivo imaging. In our previous study, we reported that N@CDs not only enter the cell membrane but may also enter the cell nucleus by penetrating the nuclear pore of a mammalian cell. We further showed that N@CDs can bind with cells more efficiently and more specifically than CDs.13 Because of the presence of NH2, COOH, CO, and COH, N@CDs can interact specifically with various types of biological molecules and receptors.5,34 Here, we employ a ZF model that, among vertebrates, represents short generation time, rapid embryonic development, and optical transparency for in vivo imaging, as well as shared anatomical and physiological characteristics.35,36 Currently, for in vivo imaging, the animals are required to be fixed with aldehyde and undergo injection with dyes or genetic constructs, whereas here, the ZF embryo

and larvae are simply soaked in N@CDs and observed by a fluorescence microscope in real time. Our in vivo study indicates that N@CDs possess both high biocompatibility and selective fluorescence imaging in ZF developmental model.11 3.2.1. Biological Toxicity and Biocompatibility of N@CDs. Viabilities of more than 75% were observed for the ZF embryo and larvae incubated with 3 and 6 mg mL−1 N@CDs, and it was 85−90% for N@CD concentrations lower than 3.0 mg mL−1. Most strikingly, no malformation was observed in the ZF embryo and larvae incubated with N@CD solutions at various concentrations (0.75, 1.5, 3, and 6 mg mL−1), illustrating their low toxicity to the living fish. These results also confirm the good biocompatibility of the as-synthesized N@CDs after soaking. These data also agree with the findings of our previous study, wherein we demonstrated the nontoxic effect of the N@ CDs in human osteosarcoma cells.13 3.2.2. Soaking of ZF Embryos in N@CDs. As shown in Figures 1D and 4A, ZF embryos at 3 and 6 hpf exposed to 6 mg E


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Figure 5. Bright-field (upper) and fluorescence (lower) images of (A) ZF whole bodies, (B) head and yolk sac, and (C) tail of ZF larvae (78 hpf) after soaking for 6 h in 3 and 6 mg mL−1 N@CD solutions. Enlarged images show (B) eye, lens, yolk sac, and intestine and (C) melanophore strips in the tail. The scale bar is 1.6 mm for (A) and 600 μm for (B, C). IB, intestinal bulb; YS, yolk sac; MS, melanophore strip; EY, eye; and LN, lens.

mL−1 N@CD solution for 2.5 and 5 h showed strong fluorescence, which indicates that, because of their smaller size, N@CD nanoparticles easily and spontaneously enter ZF embryos across the chorion and the germ ring around the yolk sac during soaking. As reported previously,37 the pore size of the ZF embryo chorion is 0.17 μm, which is much larger than the size of N@CDs (∼5 nm). Therefore, N@CDs can easily penetrate ZF embryos through the pores of the chorion. It is noteworthy that ZF embryos exposed to 6 mg mL−1 N@CD solution at 24 hpf showed strong fluorescence, selectively in the yolk sac, trunk, and tail region, indicating the tissue-dependent affinity of N@CDs. The N@CD fluorescence emission in other parts of the ZF embryo at 24 hpf is very weak (Figure 4B). We assume that this redistribution of N@CDs is due to prolonged incubation time and that, therefore, some amount of N@CDs was removed from the embryos by the digestive system.

Similarly, at 48 hpf, when the embryos hatched into larvae, the fluorescence emission of N@CDs in ZF larvae was primarily in the yolk sac. Furthermore, fluorescence was observed in the head (mainly eyeball) and on the longitudinal melanophore strip, at the rear part of the body (Figure 4C). These observations confirm that N@CDs have no toxic effect on embryo development because of their stable fluorescence emission and biocompatibility. 3.2.3. Soaking of ZF Larvae in N@CDs. As shown in Figure 5, ZF larvae exposed to 3 and 6 mg mL−1 N@CD solutions showed strong fluorescence, selectively in the yolk sac, eyes, and the longitudinal melanophore strip, at the rear part, indicating the tissue-dependent affinity of the N@CDs. The fluorescence intensities of the ZF larvae exposed to 3 and 6 mg mL−1 N@CDs were almost the same, indicating their concentration-independent behavior. In contrast, no detectable F


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Figure 6. Enlarged fluorescence images of ZF (A) eye, (B) yolk sac, and (C) dorsal and ventral melanophore strips in the tail (78 hpf) after soaking for 6 h in 6 mg mL−1 N@CD solution. IB, intestinal bulb; PI, proximal intestine; MI, middle intestine; DI, distal intestine; MS, melanophore strip.

Scheme 2. Possible Mechanism for the Binding of Melanin and N@CDs

fluorescence was detected in the ZF larvae exposed to 0.75 and 1.5 mg mL−1 N@CD solutions (data not shown). Because of their small size and tremendous water solubility, the N@CDs can easily penetrate the ZF larvae, mainly by swallowing and skin absorption.38 The ZF larvae tolerated the N@CDs very well and retained fluorescence for up to 48 h post soaking. To study how the larvae retained N@CD fluorescence, the 5 dpf larvae were first soaked in 6 mg mL−1 N@CD solution for 5 h. After 2 days, the fluorescent sites in the ZF larvae were found to be the same as those just after soaking (Figure S3). These observations reveal that the fluorescence of the N@CDs was strongly reserved after soaking, indicating that the N@CDs continue to circulate in the ZF larvae. Also this result agrees with the findings of our previous study, wherein we demonstrated the long-lasting effect of N@CDs when applied for imaging of human osteosarcoma cells.13 The presence of strong fluorescence at the yolk sac, especially in the vicinity of the intestine, suggests that a high content of N@CDs has entered the digestive system (Figures 5B and 6B). Therefore, the absorption, distribution, metabolism, and excretion of the N@CDs are proposed on the basis of their distribution. The N@CDs enter the yolk sac and are partially excreted through the gut. There are many similarities between ZF and mammalian lipid metabolisms.39 Therefore, N@CDs may have potential imaging applications in elucidating different aspects of lipoprotein and nutritional biology, in a ZF yolk lipid transport and metabolism model. There are three main types of pigment cells in ZF: melanophores (black), iridophores (silvery or blue), and xanthophores (yellow). The melanophore strips in the ZF larvae can be traced easily. Furthermore, the ZF larvae contain additional population of pigment cells, called the retinal pigment epithelium (RPE), which contains melanin.40 In this study, we used PTU to inhibit melanization. However, as reported previously, we observed that this approach is not very effective, as the ZF larvae still showed melanization in the RPE

(Figure 5B) and also a longitudinal melanophore strip was seen at the trunk and tail region, as shown in Figure 5B,C. The selective fluorescence of the ZF larva eyes, where the lens can be easily identified within the eyeball, clearly showed that the N@CDs can easily cross the blood−ocular barrier (Figures 5B and 6A). In contrast, the lack of fluorescence in the ZF larva head shows that the N@CDs could not enter the blood−brain barrier. The ocular development in ZF is similar to that of other vertebrates.41 Therefore, N@CDs may have potential applications in eye-related imaging. Furthermore, N@CDs are selectively bound to the longitudinal melanophore strips at the trunk and tail region of the ZF larvae (Figures 5C and 6C). The melanocytes in ZF are similar to those in other vertebrates, developed from multipotent neural crest cells.42 We assume that this selective accumulation is due to the binding of the N@CDs to the melanin present inside the pigmented cells (melanocytes) in the eyes and the melanophore strips at the trunk and tail region of the ZF larvae. As shown in Scheme 2, we hypothesized that the binding of N@CDs to melanin is due to the strong interaction between the functional groups of melanin and N@ CDs. Therefore, the investigation of various skin diseases may benefit from the development of novel melanin-targeting N@ CDs.

4. CONCLUSIONS In summary, we successfully engineered N@CDs using a new approach. We have shown that this new and revolutionary use of multifunctional N@CDs as highly specific bioagents for various biomedical applications, because of their exceptional biocompatibility, photostability, and selective affinity, is confirmed by the developmental ZF model. This study of the selective transfer and distribution of N@CDs in a ZF developmental model can promote their clinical applications. G


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b04668. The supporting information contains DLS measurements, pH-dependent fluorescence, and bioimaging of ZF. DLS, pH-dependent fluorescence, and bioimaging of ZF (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (D.K.). *E-mail: [email protected] (A.G.). ORCID

Deepak Kumar Khajuria: 0000-0002-3737-8504 Aharon Gedanken: 0000-0002-1243-2957 Author Contributions §

V.B.K. and D.K.K. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful to Ortal Lidor-Shalev of the Department of Chemistry, Bar-Ilan University, Israel, for her help with the TEM measurements.

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ABBREVIATIONS N@CDs, nitrogen-doped carbon dots BSA, bovine serum albumin FWHM, full width at half-maximum HR-TEM, high-resolution transmission electron microscopy XPS, X-ray photoelectron spectroscopy UV-vis, Ultraviolet-visible

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REFERENCES

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Fluorescent Nanoparticles with Tissue-Dependent ... - ACS Publications

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