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Magnetofluorescent carbon dots derived from crab shell for targeted dual-modality bioimaging and drug delivery Yueh-Yun Yao, Gangaraju Gedda, Wubshet Mekonnen Girma, Chia-Liang Yen, Yong-Chien Ling, and Jia-Yaw Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01599 • Publication Date (Web): 07 Apr 2017 Downloaded from http://pubs.acs.org on April 10, 2017

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ACS Applied Materials & Interfaces

Magnetofluorescent Carbon Dots Derived from Crab Shell for Targeted Dual-Modality Bioimaging and Drug Delivery Yueh-Yun Yao,ab Gangaraju Gedda,a Wubshet Mekonnen Girma,a Chia-Liang Yen,b YongChien Ling,b* Jia-Yaw Changa*

a. Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, 10607, Taiwan, Republic of China b. Department of Chemistry, National Tsing Hua University, Hsinchu, 30013, Taiwan, Republic of China

*Corresponding authors: Jia-Yaw Chang and Yong-Chien Ling Department of Chemical Engineering, National Taiwan University of Science and Technology, 43, Section 4, Keelung Road, Taipei, 10607, Taiwan, Republic of China E-mail: [email protected]; [email protected] Tel.: +886-2-27303636 Fax: +886-2-27376644

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ABSTRACT: We propose a one-pot microwave-assisted pyrolysis method for fabrication of magnetofluorescent carbon quantum dots (MFCQDs) using a combination of waste crab shell and three different transition-metal ions, Gd3+, Mn2+, and Eu3+, referred to as Gd@CQDs, Mn@CQDs, and Eu@CQDs, respectively. Chitin from waste crab shell acted not only as a carbon source but also as a chelating ligand to form complexes with transition-metal ions. Gd@CQDs exhibited a high r1 relaxivity of 4.78 mM–1 s–1 and a low r2/r1 ratio of 1.33, suggesting that they show excellent potential as a T1 contrast agent. Mn@CQDs and Eu@CQDs showed high r2 relaxivity values of 140.7 mM−1 s−1 and 28.32 mM−1 s−1, respectively, suggesting their potential for use as T2 contrast agents. Further conjugation of Gd@CQDs with folic acid enabled specific targeting to folate receptor–positive HeLa cells, as confirmed via in vitro magnetic resonance and fluorescence imaging. Doxorubicin (DOX) was selected as a model drug for conjugation with FA-Gd@CQDs. The as-prepared nanocomposites showed significantly higher cytotoxicity towards HeLa cells than free DOX. No apparent cytotoxicity was observed in vivo (zebrafish embryos) or in vitro (cell viability), suggesting that MFCQDs are potential for development as diagnostic probes or theranostic agents.

KEYWORDS: crab shell, drug carrier, fluorescence imaging, magnetic resonance imaging, magnetofluorescent

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1. INTRODUCTION Functional nanomaterials with magnetic and fluorescence properties have attracted considerable attention owing to their applications in magnetic resonance (MR) and fluorescence imaging. The combination of MR and fluorescence imaging offers a powerful set of complementary techniques. MR imaging provides high spatial resolution and excellent tissue penetration, whereas fluorescence imaging provides high sensitivity and ease of visualization in microscopic tissue examination. Therefore, intensive effort has been invested in the development of various magnetofluorescent (MF) nanomaterials for MR and fluorescence imaging.1-2 However, most of these MF nanomaterials are synthesized using organic solvents, and further surface modifications are necessary to make them water soluble, and these modifications are time-consuming and quench the fluorescence intensity. In particular, the biomedical applications of Cd-based nanomaterials are limited by their intrinsic cytotoxicity. Therefore, direct aqueous synthesis of MF nanomaterials with excellent water solubility, good biological compatibility, superior stability, and high quantum yield is highly desirable, as these materials could be applied for early diagnosis of many diseases. In the past decade, carbon quantum dots (CQDs) have been explored for use in fabrication of in vitro fluorescence imaging agents, owing to their excellent fluorescence efficiency, high biocompatibility, robust chemical inertness, superior water solubility, and easy surface modification.3-4 Thus, CQDs are considered to be the next generation of fluorescent nanomaterials, replacing traditional semiconductor nanocrystals. Recently, several groups have reported that Gd3+ ions can be incorporated into or conjugated with CQDs using chemical reagents as carbon sources, leading to formation of MF CQDs (referred to as MFCQDs hereafter).5-8 For example, Xu et al. reported the synthesis of Gd-doped CQDs via one-pot hydrothermal treatment of a mixture of citrate acid, ethanediamine, and GdCl3.5 Gong et al. reported that the use of a mixture of diethylene glycol, Gd2O3, and hydrochloric acid allowed direct synthesis of MFCQDs.6 The obtained MFCQDs showed a quantum yield of 5.4% and an r1 relaxivity of 11.356 mM−1 s−1. Shi et al. have published a method for synthesis of MFCQDs with a quantum yield of 5.2% via covalent conjugation of Gd3+ and CQDs.7 To minimize the environmental impact of preparation of CQDs, much effort has been made to integrate green chemistry principles, not only making the final products greener, but minimizing the quantity of chemical reagents used for synthesis. Numerous studies have reported that various alternative carbon sources, such as biomass and waste materials, can be


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employed in the preparation of fluorescent CQDs.9-20 However, to the best of our knowledge, no procedure has been developed for fabrication of MFCQDs from waste materials. Despite the significant progress in QD synthesis, the procedure remains complex. Crab shell is a promising waste material because it is a major source of raw material for the commercial production of chitin, a linear polysaccharide composed of (1-4)-linked 2-acetamido-2-deoxyb-D-glucopyranose units. Chitin and its derivatives are widely used in various biomedical applications including tissue engineering, wound healing, drug delivery, gene delivery, and cancer diagnosis due to its biocompatibility, low toxicity, and biodegradability.21-22 Furthermore, chitin-based materials are excellent chelating agents, and are used in detection of various metal ions, due to the presence of hydroxyl and acetamido functional groups in their chemical structure.23-26 Most published methods for the preparation of chitin from waste crab shell involve time-consuming and complicated procedures, such as deproteinization and demineralization steps that require the use of strong acids, limiting their potential applications.27-30 Therefore, the motivation of this work is to develop a facile, effective, and reliable one-pot method for synthesis of water-soluble nanomaterials with magnetic and fluorescence properties from waste crab shell. Herein, we report the first use of waste crab shell for the preparation of three types of MFCQDs with both magnetic and fluorescent properties, using a microwave-assisted approach. Different heteroatoms, such as Gd3+, Mn2+, or Eu3+ transition-metal ions, can be incorporated into the carbon matrix; the resulting products are referred to as Gd@CQDs, Mn@CQDs, and Eu@CQDs, respectively. These transition-metal ions were chosen as dopants because Gd3+, Eu3+, and Mn2+ possess seven, six, and five unpaired electrons, respectively, and thus have relatively high electronic spin that can create a strong local magnetic field in response to an applied external field. Gd3+ dopants made CQD serving as a T1 contrast agent, whereas the incorporation of Eu3+ or Mn2+ dopant into a CQD made it effective as a T2 contrast agent. To the best of our knowledge, the present work is the first attempt to explore the synthesis of MFCQDs using a combination of waste materials and transition-metal dopants. Further conjugation of the MFCQDs with folic acid enabled specific targeting to folate receptor-positive HeLa cells, as confirmed via in vitro MR and fluorescence imaging. Integrated with anticancer drugs (doxorubicin, DOX), this system also provides high potential for application to drug delivery. In vitro and in vivo studies confirmed the high biocompatibility and low toxicity of MFCQDs, confirming their attractiveness for future clinical applications.


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2. EXPERIMENTAL SECTION 2.1 Chemicals Folic acid (>98%) was obtained from TCI. N-hydroxy sulfosuccinimide sodium salt (Sulfo-NHS, 97%), ethyl (dimethyl aminopropyl) carbodiimide (EDC, 99%), europium (III) chloride (EuCl3, 99.9%), and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS, 97%) were obtained from Alfa-Aesar. DOX (>99%) was obtained from Fusol Materials. Gadolinium (III) chloride (GdCl3, 99.9%) was obtained from Acros Organics. Manganese (II) chloride (MnCl2, 99.9%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 97.5%), and acetic acid (99.8%) were obtained from Sigma-Aldrich. NucView Green nucleic acid stain (NucView Green) was purchased from GeneCopoeia. Crab shells of Portunus sanguinolentus, commonly known as the three-spotted crab, were obtained from a local supermarket. 2.2. Synthesis of Gd@CQDs, Mn@CQDs, and Eu@CQDs Crab shells were washed with water to remove flesh and other impurities, and then dried at 60°C for 10 h. Subsequently, the dried crab shells were ground into a fine powder using an agate mortar and pestle. The fine powder (0.5 g) was dissolved in a 1% acetic acid solution (20 mL), followed by stirring for 12 h at room temperature. The upper layer of the solution (15 mL) was transferred into a glass vessel and GdCl3 (0.1 g) was added, and then heated using a single-mode microwave reactor (Anton Paar Monowave 300) at 220°C for 10 min. The solution was cooled to room temperature and then filtered through a 0.22-µm filter membrane to remove large particles. Finally, the products were purified by centrifugation (1K MWCO, Macrosep Advance Centrifugal Device) at 2000 rpm for 60 min, to obtain CQDs doped with Gd ions, referred to as Gd@CQDs. CQDs doped with Mn or Eu ions, termed Mn@CQDs or Eu@CQDs, were prepared using a similar process, by replacing GdCl3 with MnCl2 or EuCl3, respectively, at the same molar concentration. 2.3. Conjugation of Gd@CQDs with folic acid To activate the carboxylic group of Gd@CQDs, EDC (15 mg) and sulfo-NHS (25 mg) in 1 mL of MES buffer were added to 2 mL of Gd@CQD solution (15 mg/mL) and then stirred in the dark at room temperature for 40 min. Subsequently, 1 mL of MES solution containing folic acid (2.26 × 10-3 mmol) was added to the activated Gd@CQD solution, and the mixture was stirred gently at room temperature in the dark. After 24 h, a Float-A-Lyzer G2 dialysis device (MWCO 500–1000 Da) was used to remove unreacted materials, and the final product was obtained.


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2.4. Cell culture and cell viability test Human embryo lung (HeLung) normal cell, human cervical cancer cell (HeLa), and human liver carcinoma cells (HepG2) were grown in Minimum Essential Medium Eagle (MEME) supplemented with 10% fetal bovine serum, 1% antibiotic/antimycotic, 1% L-glutamine, 1% sodium pyruvate, and 1% non-essential amino acids. The cells were cultured at 37°C in a 5% CO2 humidified incubator. Cell viability was examined using the MTT colorimetric assay kit. Cells were seeded into the wells of a 12-well plate (~5 × 104/well) and preincubated for 24 h. The culture medium was replaced with 100 µL of fresh medium containing different concentrations of MFCQDs (0 to 1000 µg/mL), and the cells were incubated for another 24 h. Subsequently, 1 mL of a MTT aqueous solution (500 µg/mL in PBS, 1 mL per well) was added to each well and the cells were incubated at 37°C in a 5% CO2 humidified incubator for another 4 h. The culture medium in the 12-well plates was discarded, and 1 mL of dimethyl sulfoxide was added to each well to dissolve the formazan crystals. The absorbance of each well was recorded at 570 nm using a microplate reader (BioTek Powerwave XS, Winooski, VT, USA). The experiment was performed three times under identical conditions. The following equation was used to calculate cell viability (%):

cell viability (%) =

optical density of treated sample ×100% optical density of control sample

2.5. In vitro fluorescence imaging Cells were seeded in 6-well plates at a density of ~5 × 104 cells/well for 24 h. Then, the medium was aspirated and 0.5 mL of fresh culture medium containing MFCQDs (500 µg/mL) was added to the wells for 24 h. After incubation, the cells were washed with phosphate-buffered saline (PBS) again and then fixed with 2 mL of 75% alcohol for 10 min. Nuclear staining was carried out by incubating the fixed cells with NucView (2 mL, 0.05 µg/mL in PBS) for 17 min. The cells were then washed three times with 10 mM PBS. Fluorescence images of the cells were visualized with a Leica TCS SP2 confocal laser scanning microscope. 2.6. In vitro targeted MR imaging For T1-weighted MR imaging of in vitro cells, cells were seeded into a 50-mL serum bottle containing 5 mL of medium, and incubated for 24 h. The medium was replaced with fresh medium (5 mL) containing different concentrations of MFCQDs and incubated for another 24 h. The cells were rinsed with PBS, trypsinized, centrifuged, resuspended in 200 µL PBS, and placed into 0.2-mL tubes for MR imaging. MR imaging was performed using a


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Bruker Med Spec 7T with a birdcage head coil. 2.7. Drug loading and release Typically, 2 mg of MFCQDs and 0.1 mg of DOX were incubated in 2 mL PBS for 24 h in the dark at room temperature. The products were collected by centrifugation through a Vivaspin ultracentrifugal filter (3 kDa MWCO) at 3,000 rpm for 15 min to remove any unloaded DOX. The supernatant and the washing solution were collected, and the concentration of loaded DOX was quantified by absorption spectroscopy at 490 nm, relative to a standard DOX calibration curve. The loading efficiency of DOX was expressed as

loading efficiency (%) =

mass of DOX-loaded MFCQDs ×100% mass of DOX in feed

The DOX release study was conducted by adding DOX-loaded MFCQDs to PBS at 37°C at various pH values. After various time intervals, the DOX-loaded MFCQDs were centrifuged through a Vivaspin ultracentrifugal filter (3 kDa MWCO) at 3,000 rpm for 15 min, and the released DOX was collected. The DOX release efficiency was expressed as:

releasing efficiency (%) =

mass of released DOX ×100% mass of DOX-loaded MFCQDs

2.8. Zebrafish culture and embryonic toxicity of CQDs Wild-type and AB strains of male and female zebrafish (Danio rerio) were used for all in vivo experiments. Their use was reviewed and permitted by the zebrafish core facility center, National Tsing Hua University, Taiwan. The zebrafish were raised in an aquarium at 28°C under controlled 10 h/13 h light/dark conditions. All animal experiments were performed in compliance with the Animal Management Rules at National Tsing Hua University. For zebrafish mating, a ratio of one female to two male zebrafish was maintained in a tank. Ten fertilized eggs in each group were treated individually with different concentrations of samples. The developmental status of zebrafish embryos and larvae were analyzed at various time points, in hours, post fertilization (hpf) using an Olympus IX73 microscope equipped with a digital camera. The percentage of surviving embryos was determined by comparison of the number of living embryos with the number of embryos at 96 hpf. The percentage of zebrafish hatching was determined by comparison of the number of hatched embryos with the number of embryos at 96 hpf. All experiments were repeated three times. Each group contained ten embryos per condition and all experiments were performed in triplicate. 2.9. Characterization X-ray diffraction (XRD) patterns were obtained using a Bruker D8 Discover X-ray diffractometer. Transmission electron microscopy (TEM) characterization was performed on


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a FEI Tecnai G2 F20 microscope (Philips, Amsterdam, Holland) at an accelerating voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) measurements were performed using an ESCALAB 250 photoelectron spectrometer (Thermo VG Scientific) with Al Kα (1486 eV) as the X-ray excitation source. Elemental composition was measured by inductively coupled plasma optical emission spectroscopy (ICP-AES) using a Horiba JY2000−2 spectrometer. Fourier transform infrared (FT-IR) spectra were obtained using a Nicolet 5700 FT-IR spectrometer. UV−visible and fluorescence spectra were acquired on a JASCO V-630 and Fluorolog-3 spectrophotometer, respectively. Electron spin resonance (EPR) experiments were performed using a Bruker Elexsys E-580 spectrometer. Fluorescent quantum yield was calculated as previously described, using quinine sulfate (quantum yield = 95%) as a reference standard.31 All MR studies were carried out on a 7T Bruker BioSpec 70/30 imaging spectrometer. Imaging parameters of T1-weighted MR imaging were as follows: field of view = 25 × 25 cm2, slice thickness 1.0 mm, matrix size = 256 × 256, and repetition time/echo time (TR/TE) = 750/11 ms. Imaging parameters of T2-weighted MR imaging were as follows: field of view = 25 × 25 cm2, slice thickness 1.0 mm, matrix size = 256 × 256, flip angle = 180°, and TR/TE = 6000/11 ms. Longitudinal relaxation rates were measured using static TE (11 ms) and variable TR (100, 150, 300, 500, 750, 1000, 1500, 3000, 6000, and 10 000 ms) values. Transverse relaxation rates were measured using static TR (6000 ms) and variable TE (11, 22, 33, 44, 55, 66, 77, and 88 ms) values. 2.10 Statistical analysis Analysis of variance (ANOVA) and a standard t-test were used for statistical evaluation, and differences were considered significant at P < 0.05.


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3. RESULTS AND DISCUSSION Compared to traditional convective heating, microwave-assisted heating provides more rapid and homogeneous heating, which is important in reactions with multiple precursors. The reactivities of the multiple precursors must be simultaneously controlled through rapid, uniform heating and pressure. Scheme 1 illustrates the microwave-assisted hydrothermal synthesis of MFCQDs using crab shell as a carbon source, transition-metal ions as doping elements, and water as a solvent containing a low concentration of acetic acid. Crab shell exhibits a hierarchical organization consisting of abundant chitin-protein fibers, which are aligned in an antiparallel manner. Acetic acid was used as the decalcification agent, to remove most of the calcium carbonate from the crab shells. During the reaction period, acetic acid also promoted decomposition of the crab shell into chitin. Chitin contains abundant functional groups, including hydroxyl and acetamido groups, which act as chelating ligands to form complexes with transition-metal ions. Hydrothermal carbonization and heteroatom doping occur simultaneously, generating MFCQDs during the microwave-assisted hydrothermal reaction.

Decalcification

Hydrothermal carbonization

Crab shell

1% Acetic acid Anton Paar microwave 220 °C,10 min

MFCQDs

Addition of GdCl3, MnCl2, or EuCl3 chitin

Scheme 1. Schematic illustration of preparation of MFCQDs using a microwave-assisted hydrothermal approach

3.1. Material and optical characterization of MFCQDs The morphology of the as-synthesized Gd@CQDs, Mn@CQDs, and Eu@CQDs was investigated with TEM. As illustrated in Figure 1a, the TEM images showed quasi-spherical


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particles. Analysis of the particles in the TEM images (Figures 1a–1c), indicated that the average diameter of the Gd@CQDs, Mn@CQDs, and Eu@CQDs was 4.0 ± 0.7, 4.5 ± 1.0, and 4.2 ± 0.6 nm, respectively. A closer examination of the TEM images revealed that the MFCQDs are highly crystalline, with a distance of 0.212 nm between the two adjacent lattice fringes, for the (100) plane of graphitic carbon, as shown in the insets of Figures 1a–1c. High-resolution of TEM images of Gd@CQDs, Mn@CQDs, and Eu@CQDs were displayed in Figure S1. EDS analysis of the three types of MFCQDs (Figures 1d–1f) revealed the presence of Gd, Mn, and Eu in the Gd@CQDs, Mn@CQDs, and Eu@CQDs, respectively.

(d)

(e)

(f)

Figure 1. TEM images and EDS spectra of (a, d) Gd@CQDs, (b, e) Mn@CQDs, and (c, f) Eu@CQDs. The insets show the corresponding high-resolution TEM images of MFCQDs. Scale bars = 4 nm.


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The optical properties of the MFCQDs were measured by UV-vis absorption and fluorescence spectroscopy. For comparison, we also investigated the optical properties of CQDs not doped with transition-metal ions. The CQDs and MFCQDs all exhibited similar absorption spectra profiles, with two peaks at 235 nm and 270 nm, a minor peak at approximately 350 nm, and a tail extending into the visible range, as shown in Figure 2a. The peaks centered at 235 nm and 275 nm were attributed to π → π* transitions of aromatic rings and n → π* transitions of C=O bonds, respectively, consistent with previous reports.32 The minor peak at 350 nm was probably due to the trapping of excited state energy from the surface states.33 The maximum emission wavelength of the CQDs and the MFCQDs was 425 nm at an excitation wavelength of 350 nm, as displayed in Figure 2b. The fluorescent quantum yield (QY) of the MFCQDs was estimated to be 19.84 % for Gd@CQDs, 12.86 % for Mn@CQDs, and 14.97 % for Eu@CQDs, using quinine sulfate (QY = 54% in 0.1 M H2SO4) as a reference. Similar to most CQDs, the PL spectra of the CQDs and MFCQDs all exhibited typical excitation wavelength-dependent characteristics, with maximum emission peak red-shifts in the 320 to 380 nm range as the excitation wavelength varied from 330 nm to 400 nm, as shown in Figures 2c–2f. The exact emission origin of the CQDs is still under debate and several possible explanations have been suggested: (1) bandgap transitions in conjugated π-domains, (2) defects and surface states, and (3) surface groups.34,35 In previous studies, it has been reported that Eu3+ dopants, with f–f intra-orbital electronic transitions, lead to the narrow-band PL in doped nanoparticles.36-38 Nevertheless, there was no additional emission peak for Eu@CQDs, probably due to different coordination number and spatial distribution in host materials.


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(a)

CQD Gd@CQD Mn@CQD Eu@CQD

(b)

300

400 500 Wavelength (nm)

PL Intensity (a.u.)


(e)

300

700

330 340 350 360 370 380 390 400

(c)

300

600

nm nm nm nm nm nm nm nm

600

330 340 350 360 370 380 390 400


nm nm nm nm nm nm nm nm

600

300


600

(d)

330 nm 340 nm 350 nm 360 nm 370 nm 380 nm 390 nm 400 nm

PL Intensity (a.u.)

200

300

400

500 Wavelength (nm)

600

(f)

300

700

330 nm 340 nm 350 nm 360 nm 370 nm 380 nm 390 nm 400 nm

PL Intensity (a.u.)

Absorbance (a.u)

PL Intensity (a.u.)

CQD Gd@CQD Mn@CQD Eu@CQD

PL Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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400

500 Wavelength (nm)

600

700

Figure 2. (a) UV–Vis absorption and (b) fluorescence spectra of CQDs, Gd@CQDs, Mn@CQDs, and Eu@CQDs. Excitation wavelength = 370 nm; a.u. indicates arbitrary units. Excitation-dependent PL emission spectra of (c) CQD, (d) Gd@CQDs, (e) Mn@CQDs, and (f) Eu@CQDs. The successful formation of Gd@CQDs, Mn@CQDs, and Eu@CQDs was further investigated by XPS analysis. The XPS results indicated that the MFCQDs are mainly composed of C, O, and N, combined with their respective magnetic heteroatoms (Gd, Mn, or Eu), as shown in Figures 3a–3c. The XPS spectra of C 1s from Gd@CQDs, Mn@CQDs, and Eu@CQDs could be deconvoluted into several peaks (dashed lines), corresponding to C–C (graphite), C-OH, C=O, and COOH, as shown in Figures 3d–3f. The presence of Gd ions in Gd@CQDs was confirmed by the Gd 4d signal, with a binding energy value of 162.4 eV, as shown in Figure 3g. The Gd 4d signal appeared at a higher binding energy than usually


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reported (~140 eV) for Gd3+, probably due to different coordination binding.39-40 The Mn 2p XPS spectrum shown in Figure 3h for Mn@CQDs shows the binding energy of Mn 2p3/2 and Mn 2p1/2 as 640.95 eV and 652.95 eV, respectively, consistent with previous reports.41 This indicates the presence of Mn2+ ions in Mn@CQDs. The Eu 3d XPS spectrum of the Eu@CQDs in Figure 3i shows four deconvoluted peaks at 1123.6, 1153.9, 1134.9, and 1163.9 eV, which can be attributed to the binding energies of Eu2+ 3d5/2, Eu3+ 3d5/2, Eu2+ 3d3/2, and Eu3+ 3d3/2, respectively, suggesting that both Eu2+ and Eu3+ ions were present in the Eu@CQDs.

Intensity (a.u.)

Intensity (a.u.)

C

Mn@CQD

N Ca

(b)

O

C

Eu@CQD Intensity (a.u.)

(a)

Gd@CQD O

Mn Ca N

(c)

O

Ca C Eu

N

Gd 400

600

800

1000

1200

0

200

400

600

800

1000

0

200

400

Intensity (a.u.)

C=O(287.64 eV)

C-OH(285.92 eV) ←

COOH(288.24 eV) C-OH(285.05 eV)

←

292

296

300

←

(g) Intensity (a.u.)

150

160

170

290

Mn 2+ 2p 3/2

Intensity (a.u.) 140

285

← C=O(285.11 eV)

295

280

Binding energy (eV)

Binding energy (eV)

Gd 4d

280

180

630

Binding energy (eV)

(h)

Mn 2+ 2p 1/2

640 650 Binding energy (eV)

284

288

292

Binding energy (eV)

660

(i)

Eu 2+ 3d 5/2 Intensity (a.u.)

288

275

1200

→ C-C(284.39 eV)

COOH(288.89 eV)

C=O(287.69 eV) 284

1000

(f)

→

C-C(284.53 eV)

→

C-OH(285.5 eV)

COOH(289.21 eV)

280

800

(e)

C-C(284.72 eV)

276

600

Binding energy (eV)

(d) Intensity (a.u.)

1200

Binding energy (eV)

Binding energy (eV)

→

200

Intensity (a.u.)

0

→

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1100

Eu 3+ 3d 5/2 Eu 2+ 3d 3/2 Eu 3+ 3d 3/2

1120

1140

1160

1180

1200

Binding energy (eV)

Figure 3. XPS survey spectra of (a) Gd@CQDs, (b) Mn@CQDs, and (c) Eu@CQDs. C1s high-resolution XPS spectra of (d) Gd@CQDs, (e) Mn@CQDs, and (f) Eu@CQDs. (g) Gd 4d high-resolution XPS spectrum of Gd@CQDs. (h) Mn high-resolution XPS spectrum of Mn@CQDs. (i) Eu 3d high-resolution XPS spectrum of Eu@CQDs. Black circles correspond to raw data, blue lines correspond to deconvoluted spectra, and the red lines correspond to the summation of the deconvoluted spectra. 3.3. Stability of MFCQDs in solution


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A crucial parameter for biomedical application of nanomaterials is stability in physiological conditions. Therefore, the stability of the Gd@CQDs, Mn@CQDs, and Eu@CQDs was evaluated using optical imaging and fluorescence intensity analysis. First, we studied the stability of Gd@CQDs dispersed in aqueous solutions with pH values ranging from 2 to 11. As shown in Figure 4a, the Gd@CQDs were highly stable, with no obvious variation in emission intensity at the different pH values after 72 h. Next, we systematically investigated the influence of salt concentration on the fluorescent properties and colloidal stability of Gd@CQDs in aqueous solution. Figure 4b shows that the ionic strength had negligible effect on the fluorescence intensity of Gd@CQDs at the selected concentrations. Likewise, the relative changes in the fluorescence intensity of Mn@CQDs and Eu@CQDs in aqueous solutions at various NaCl concentrations and pH values were similar to those of Gd@CQDs, as shown in Figures S2 and S3. The above results suggested that the as-prepared MFCQDs show good stability at various ionic strengths and pH values, making them suitable for biomedical applications in vivo. (a)

NaCl: 0 M 1.2

2M

0h

Relative Intensity

1

0h

0.8

3 h 72 h 6 h 3h

0.6 0.00 M 0.15 M 0.30 M 0.60 M 1.20 M 2.00 M

0.4 0.2 0 0

(b) 1.2

6 12 18 24 30 36 42 48 54 60 66 72 pH 3 Time (h)

3h 72 h

pH 12

0h

1 Relative Intensity

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0.8 pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 pH 11

0.6 0.4 0.2 0 0

6

3h

72 h

12 18 24 30 36 42 48 54 60 66 72 Time (h)

Figure 4. Effect of (a) NaCl concentration and (b) pH on the emission intensities of Gd@CQDs in aqueous solution for different time periods. The left panels show relative emission intensities (I/I0), and the corresponding fluorescent images under UV excitation are shown in the right panels


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3.4. Evaluation of the MR properties of MFCQDs The incorporation of different transition-metal ions into the CQDs was confirmed via EPR, which permits determination of the local environment features of the dopant in the host, as shown in Figure 5. Figure 5a shows the EPR signal of Gd@CQDs with different Landé g factors, including g1 value = 2.00, g2 value = 2.48, g3 value = 2.80, g4 value = 3.00, g5 value = 6.00, and g6 value = 6.30. The hyperfine splitting observed and the g values calculated are consistent with previous reports of Gd doping.42-43 The EPR spectrum of Mn@CQDs, shown in Figure 5b, displays a typical six-line structure with hyperfine splitting of approximately 5.90 mT, due to electron-nuclear hyperfine interactions in isolated Mn2+ ions (nuclear spin I = 5/2). As shown in Figure 5c, the Eu@CQDs exhibited a broad and very weak EPR signal at g value = 2.055, confirming the incorporation of Eu ions in the CQD host. This broad signal may be attributed to electron spin–spin interactions of clustered Eu ions. As expected, the control sample (CQDs) without transition-metal dopants showed no EPR signal (Figure 5d). Intensity (a.u.) Intensity (a.u.) Intensity (a.u.) Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


g6

g5

(a)

g4 g3 g2 g1

(b)

(c)

(d)

0

100

200 300 400 500 Magnetic field (mT)

600

700

Figure 5. EPR spectra of (a) Gd@CQDs, (b) Mn@CQDs, (c) Eu@CQDs, and (d) CQDs, respectively, measured at 10 K. To verify the suitability of the three MFCQDs for use as MR contrast agents, T1- and T2weighted MR images, as well as relaxation rate, were scanned using the 7T MR system. For comparison, a commercial MR reagent (Gd-DTPA) was used as a control. The different


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metal concentrations in the solution were determined by ICP-AES. The T1-weighted MR images shown in Figures 6a and 6b indicate that Gd@CQDs and Gd-DTPA induced a positive contrast that brightened as the concentration of Gd3+ ions increased, while the signal intensity of the T2-weighted MR images decreased as the concentration of Gd3+ ions increased, for both samples. Similarly, as the concentration of Mn2+ or Eu3+ ions increased, the brighter intensities in T1-weighted MR image increased and the darker intensities in T2weighted MR image increased, as shown in Figures 6c and 6d. It is worth noting that Mn@CQDs displayed stronger hypointense contrast than the same concentrations of the other samples in T2-weighted images, suggesting that Mn@CQDs is a T2-weighted dominant MR contrast agent.

Figure 6. T1- and T2-weighted spin-echo images of (a) Gd@CQDs, (b) Gd-DTPA, (c) Mn@CQDs, and (d) Eu@CQDs at various concentrations of transition-metal ions. Water proton longitudinal (1/T1) and transverse (1/T2) relaxation rates of (e) Gd@CQDs and GdDTPA, (f) Mn@CQDs, and (g) Eu@CQDs as a function of different concentrations of metal ions using a 7.0 T MRI scanner. To quantitatively investigate the capacity of the CQDs to enhance MR imaging, relaxivity constants (r1 and r2) were calculated from the slopes of the linear regression fits from the plots of longitudinal (1/T1) and transverse (1/T2) relaxation rates versus metal ion


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concentrations (e.g., Gd3+, Mn2+, and Eu3+), as shown in Figures 6e-6g. Table 1 presents a summary of the relaxivity constants for three MFCQDs and the Gd-DTPA complex. The r1 values of the Gd@CQDs, Mn@CQDs, and Eu@CQDs were 11.04 mM−1 s−1, 7.43 mM−1 s−1, and 2.65 mM−1 s−1, respectively. The r1 value of Gd@CQDs was almost 2.6-fold higher than that of Gd-DTPA (4.30 s−1 mM−1) under the same conditions. The result could be attributed to greater coordination of water molecules with unpaired electrons of Gd3+ in [email protected] However, the total hydration position of Gd-DTPA is one electron, because its other six electrons are coordinated by chelates, producing a low relaxivity constant. The r2 values were calculated to be 14.69 mM−1 s−1, 140.7 mM−1 s−1, and 28.32 mM−1 s−1 for Gd@CQDs, Mn@CQDs, and Eu@CQDs, respectively, suggesting that Mn@CQDs are better suited for negative contrast enhancement than Gd@CQDs and Eu@CQDs. The r2/r1 ratio is known to be a key factor to evaluate whether a given contrast agent has potential to be a T1 (positive) or a T2 (negative) contrast agent. In general, an r2/r1 ratio in the range of 1–2 suggests potential as a positive contrast agent, while an r2/r1 ratio higher than 10 suggests potential as a negative contrast agent.45 The r2/r1 ratio of Gd@CQDs was 1.33, suggesting that this MFCQD has excellent potential as a T1 contrast agent. As shown in Table 1, the r2/r1 ratios of Mn@CQDs and Eu@CQDs were calculated to be 18.94 and 10.68, respectively, suggesting that both are candidates for T2 contrast agents. Relaxation times for three MFCQDs were measured at different time interval. As shown in Figure S4, relaxation times of three MFCQDs exhibit negligible change after incubation time of 72 hr and 96 hr. It suggests that less transition-metal ions were released from MFCQDs or no significant particle aggregation is observed in the obtained solution.46 To validate the structure stability of three MFCQDs, the leakage of free metal ions was measured after three MFCQDs were incubated in PBS buffer at pH 7.4 after four days. Subsequently the resulting solution was centrifuged to collect the supernatant, and the leakage amounts of free metal ions from three MFCQDs were measured by ICP-AES. The result shows the leakage percentage of free metal ions is less than 0.6%, which indicated the good stability of the as-prepared MFCQDs.

Table 1. Summary of relaxation parameters of three MFCQDs and Gd-DTPA at room temperature using a 7 T MRI scanner.

Sample

r1 ( mM−1 s−1)

r2 ( mM−1 s−1)

r2/r1


17


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Gd@CQDs

11.04

14.69

1.33

Gd-DTPA

4.30

4.42

1.01

Mn@CQDs

7.43

140.7

18.94

Eu@CQDs

2.65

28.32

10.68

3.5 Conjugation of Gd@CQDs with folic acid Gd@CQDs were selected for further investigation of the potential of MFCQDs as dualmodality bioimaging agents. We used folate-receptor targeting to study the capability of Gd@CQDs for target-specific detection of cancer. Folic acid–labeled Gd@CQDs (termed FA-Gd@CQDs) were prepared by activation of the Gd@CQDs, using EDC and sulfo-NHS as crosslinking reagents, followed by coupling of folic acid to the Gd@CQDs via the formation of an amide linker between the amine group in folic acid and the carboxyl groups in Gd@CQDs. Folic acid is a water-soluble form of vitamin B with a high targeting affinity to folate receptors (Kd = 10-10 M), which are 38-kDa glycosyl-phosphatidyl-inositol–anchored glycoproteins that are overexpressed in many carcinomas, such as ovarian, lung, and uterine tumors, but are rarely found in healthy tissues.47 The optical properties of folic acid, Gd@CQDs, and FA-Gd@CQDs were determined by UV-visible absorption and FT-IR spectroscopy. As shown in Figure 7a, folic acid has characteristic absorption peaks at 280 nm and 370 nm, corresponding to π → π* and n → π* transitions, respectively, in the pterin rings of folic acid. Compared to Gd@CQDs, an additional intense absorption peak characteristic of folic acid was observed at 266 nm for FAGd@CQDs, confirming conjugation of folic acid. As shown in Figure 7b, free folic acid shows IR absorption peaks at 3545, 3323, 3107, 2923, 2852, 1695, 1606, 1485, 1390, 1193, 1071, and 716 cm–1 1. The peaks above 3000 cm-1 are due to -OH and N-H stretching, and the peaks appearing at 2923 and 2852 cm–1 can be assigned to C-H vibration bands.2 The band at 1695 cm–1 is the C=O stretching vibration of the carbonyl group. The peak at 1606 cm–1 is the bending mode of N-H vibration, and the bands at 1485 cm–1 and 1390 cm–1 can be attributed to phenyl rings and –CH3 symmetrical deformation, respectively. Further, the peaks at 1193 and 1071 cm–1 indicate C-O stretching. The peak at 716 cm–1 indicates =C-H bending vibration. The FT-IR spectrum of Gd@CQD


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without folic acid modification displayed a peak at 3400 cm–1 indicating stretching vibrations of N-H groups. The peaks at 1541 and 1427 cm–1 indicate stretching vibrations of C-C and CH2, respectively. After conjugation of folic acid, the broad band at 3400 cm–1 in Gd@CQDs was shifted to 3282 cm-1 in FA-Gd@CQDs, which can be assigned to the –OH and –NH2 functional groups. The peaks of FA-Gd@CQDs at 2923 and 2852 cm–1, attributed to C-H stretching of folic acid, shifted to 2974 and 2731 cm–1, whereas no peaks were observed for Gd@CQDs. In addition, the peaks indicating C=O stretching of the carbonyl group and N-H bending vibrations shifted to 1658 and 1569 cm–1, respectively. Thus, the UV-Vis absorption and FT-IR results confirmed that folic acid conjugation with Gd@CQDs was successful. (a)

200

300

400 500 600 Wavelength (nm)

700

(b)

Figure 7. (a) UV–Vis absorption and (b) FTIR spectra of folic acid, Gd@CQDs, and FAGd@CQDs


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3.6. In vitro and in vivo biocompatibility of FA-Gd@CQDs The in vitro cytotoxicity of FA-Gd@CQDs was investigated by conducting MTT assays against HeLa, HepG2, and HeLung cells. As shown in Figure 8a, incubation with various concentrations of NPs (10–1000 µg/mL) for 24 h had no obvious effects on the viability of the three cell types. Thus, FA-Gd@CQDs show low cytotoxicity, facilitating future biological applications. The zebrafish (Danio rerio) is considered to be an excellent animal model for evaluating the in vivo biocompatibility and bioimaging potential of nanomaterials. 5,48,49 Zebrafish offer several advantages, such as rapid embryonic development within 120 h post-fertilization (hpf), easy maintenance, high optical transparency for tissue visualization, and high similarity to the human genome.50,51 Herein, the in vivo biocompatibility of FA-Gd@CQDs was evaluated using the embryonic development of zebrafish, including phenotypic development, embryonic survival rate, and hatching interference. The embryonic developmental stages of a blank control, not treated with FA-Gd@CQDs, are shown in Figure 8b; it exhibited normal development from cleavage (3 hpf), segmentation (24 hpf), hatching stages (48 hpf), to a fully developed larva (96 hpf). Zebrafish embryos incubated with FA-Gd@CQDs (20, 50, 100, 200, and 1000 µg/mL) showed phenotypic development similar to the blank control, as displayed in Figure S5. The embryonic survival and hatching rate of zebrafish embryos treated with FAGd@CQDs are displayed in Figure 8b. The survival rate was calculated as the number of embryos capable of becoming zebrafish larvae at 96 hpf over all embryos, while the hatching rates were calculated as the number of completely hatched embryos at 96 hpf over all embryos. The blank control, without FA-Gd@CQDs, showed a 100% survival rate at 96 hpf. After exposure to different concentrations of FA-Gd@CQDs, zebrafish embryos showed no mortality, even at the highest concentration of 1000 µg/mL. Additionally, the results shown in Figure 8b indicate that no hatching delay was observed for FA-Gd@CQDs at concentrations of 0–1000 µg/mL. These results of the in vitro and in vivo cytotoxicity tests confirmed the biocompatibility of FA-Gd@CQDs.


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120

HeLung cell Hela cell HepG2 cell

(a) Cell viability (%)

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0

Concentration (µg/mL)

Figure 8. (a) MTT assay of HeLa, HepG2, and HeLung cell viability after incubation with FA-Gd@CQDs at different concentrations (20, 50, 100, 200, 500, and 1000 µg/mL) for 24 h at 37°C. HeLa and HepG2 cells incubated with PBS were used as a control (0 µg/mL). The error bars indicate mean square standard deviations (n = 3). (b) Hatching and survival rates of zebrafish embryos incubated with FA-Gd@CQDs. All tests were repeated in triplicate in three independent experiments.

3.7. In vitro targeted fluorescence and MR imaging of FA-Gd@CQDs To examine the feasibility of FA-Gd@CQDs for targeted cellular imaging, folate receptorpositive HeLa cells and folate receptor-deficient HepG2 cells were incubated with FAGd@CQDs. The bright-field images shown in Figures 9a and 9e demonstrate the morphological integrity of HeLa and HepGa cells. Figures 9b and 9f show the green fluorescence signal from nuclei stained with NucView. In the fluorescence images in Figures 9c and 9g, the blue fluorescence signal was more intense in receptor-positive HeLa cells treated with FA-Gd@CQDs than in receptor-deficient HepG2 cells treated with FAGd@CQDs, confirming targeting of the folic acid on the Gd@CQD surface. As shown in Figures 9d and 9h, the blue fluorescence signals were mostly localized in the cytoplasm. To verify uptake of FA-Gd@CQDs through folate receptor-dependent targeting, competitive


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inhibition studies were carried out by addition of excess free folic acid to the culture medium prior to incubating cells with FA-Gd@CQDs. The confocal microscopy images in Figure S6 revealed that only weak blue fluorescence could be observed, indicating that few FAGd@CQDs entered HeLa cells, as the folate receptors on the cell membrane were competitively bound with free folic acid. HeLa cells were also incubated with Gd@CQDs, which lacked folic acid conjugation. Figure S7 shows that only weak blue fluorescence was observed in the HeLa cells, indicating that folic acid plays an important role in efficient intracellular delivery to folate receptor-positive tumor cells. These findings confirmed that FA-Gd@CQDs were specifically internalized into HeLa cells via folate receptor-mediated endocytosis. To further evaluated the specific cell-targeting ability of the FA-Gd@CQDs using cellular MR contrast, HeLa and HepG2 cells were incubated with FA-Gd@CQDs at different concentrations. At the same concentration, T1-weighted MR images of FA-Gd@CQD-treated HeLa cells showed a brighter MR signal than FA-Gd@CQD-treated HepG2 cells (Figure 9i). No obvious variation in MR contrast signal was observed in FA-Gd@CQD-treated HepG2 cell pellets (Figure 9j). Confocal and MR imaging provided evidence that FA-Gd@CQDs are suitable for simultaneous MR and optical dual mode imaging, which could provide complementary information and more accurate diagnosis. HepG2

HeLa (a)

(c)

(i)

Brightfield

FA-Gd@CQDs

(b)

(d)

NucView

Overlay

(e)

(g)

Brightfield

FA-Gd@CQDs

(f)

(h)

NucView

Overlay

(j)

Figure 9. Fluorescence images of (a–d) HeLa and (e–h) HepG2 cells incubated with FA-


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Gd@CQDs, fixed and stained with NucView agent. (a and e) Bright-field transmission images, (b and f) NucView fluorescence images, (c and g) FA-Gd@CQDs fluorescence images, and (d and h) the overlay image of fluorescence images. Scale bars = 40 µm. T1weighted (i) HeLa and (j) HepG2 cellular MR images of FA-Gd@CQDs at various concentrations. 3.8 Drug release study and cytotoxicity assay To further explore the potential therapeutic applications of FA-Gd@CQDs, DOX was selected as a model drug for conjugation with FA-Gd@CQDs via π–π stacking and hydrophobic interactions. The product is referred to as FA-Gd@CQDs/DOX. The concentration of loaded- and released- DOX was calculated by absorption measurements using calibration plots of DOX (Figure S8) made before sample measurements. The loading efficiency of DOX in FA-Gd@CQDs/DOX was 74.5 ± 7.9%. The cumulative release of DOX from FA-Gd@CQDs/DOX at different pH values is shown in Figure 10a. DOX release was found to show pH-dependent release kinetics. During the first 24 h, 24.74 ± 6.27%, 30.33 ± 8.46%, and 40.93 ± 3.96% of DOX was released from FA-Gd@CQDs at pH 5.0, 6.0, and 7.4, respectively. After 72 h, cumulative drug release from FA-Gd@CQDs/DOX was determined to be only 34.81 ± 1.46% and 41.32 ± 2.09% at pH 6.0 and 7.4, respectively. However, the relative DOX release percentage of FA-Gd@CQDs/DOX at pH 5.0 after 72 h was 62.54 ± 1.03%. The higher release rate of DOX in low-pH medium may be explained by protonation of the amino group in the DOX resulting in higher solubility, weakening the interaction with the hydrophobic graphene surface of CQDs.52,53 This characteristic has practical significance for clinical cancer therapy because most tumor tissues possess low extracellular pH values, as do intracellular endosome/lysosomes (near 5).54 To evaluate the therapeutic efficiency, we investigated the cell growth inhibition of free DOX and FAGd@CQDs/DOX in HeLa cells via MTT assays (Figure 10b). FA-Gd@CQDs/DOX showed greater cell growth inhibition towards HeLa cells than free DOX, at an equivalent concentration. The half maximal inhibitory concentration (IC50) value of FAGd@CQDs/DOX against HeLa cells was found to be ~2.5 mg/mL, whereas the IC50 value of free DOX was ~5 mg/mL. Thus, FA-Gd@CQDs/DOX more effectively kills HeLa cells than free DOX at the same drug concentration, most probably due to folic acid receptor-mediated endocytosis.


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(a) 80

pH 5.0 pH 6.0 pH 7.4

70 Cumulative release (%)

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60 50 40 30 20 10 0 0

12

24

36 48 Time(hr)

60

72

(b)

Figure 10. (a) Drug release profiles of FA-Gd@CQDs/DOX in aqueous phase at different pH values (7.4, 6.0, or 5.0). (b) Cytotoxicity studies of HeLa cells incubated with FAGd@CQDs/DOX and free DOX at different concentrations for 24 h. Error bars represent standard deviations (n = 3). * and ** indicate significant differences at the same drug concentration at P < 0.05 and P < 0.01, respectively.


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4. CONCLUSIONS In conclusion, we report a facile microwave-assisted approach for the synthesis of MFCQDs within a short reaction time of 10 min, using waste crab shell and transition-metal ions. Using different transition-metal ions, we produced Gd@CQDs, Mn@CQDs, and Eu@CQDs that featured excellent aqueous dispersibility, high stability, intense fluorescence, and excellent MR response. The Gd@CQDs exhibited a much stronger T1-weighted MR effect than commercial contrast agents (Gd-DTPA), whereas the obtained Mn@CQDs and Eu@CQDs both exhibited a negatively enhanced T2-weighted MR effect. Furthermore, covalent conjugation of folic acid to Gd@CQDs enabled specific targeting to cancer cells overexpressing folate receptor, as determined by confocal microscopy and MR imaging studies. As-prepared FA-Gd@CQDs/DOX exhibited greater cytotoxicity against HeLa cells than free DOX, suggesting that it is a promising candidate for drug delivery vehicle. In vivo (zebrafish embryos) and in vitro (cell viability) studies revealed that FA-Gd@CQDs show low cytotoxicity and favorable biocompatibility up to 1000 µg/mL. The simple, environmentally friendly method described here, which involves direct aqueous synthesis, rapid and efficient heating, reuse of waste materials, and no need for harmful chemicals, may be extended as a general method to synthesize CQDs, with addition of unique features by doping with different heteroatoms.


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Acknowledgments This work was supported by the Ministry of Science and Technology of the Republic of China under Contract No. MOST 105-2119-M-011-002 and MOST 104-2113-M-007-008MY3. We thank the 7T Animal MRI Core Lab of the Neurobiology and Cognitive Science Center, National Taiwan University for provision of technical support and facilities. Human embryo lung cell was a generous gift from Virology Laboratory, Department of Laboratory Medicine, National Taiwan University Hospital.

Conflict of interest The authors declare no conflict of interest.

Supporting information Effect of NaCl and pH on emission intensities of Mn@CQDs & Eu@CQDs, high-resolution TEM images and relaxation stability of MFCQDs, optical images of developmental stages of zebrafish embryos, fluorescence images of HeLa cells preincubated with free folic acid and FA-Gd@CQDs. This information is available free of charge via the Internet at http://pubs.acs.org/.

ORCID Wubshet Mekonnen Girma: 0000-0003-3370-6731 Yong-Chien Ling: 0000-0002-9903-8062 Jia-Yaw Chang: 0000-0002-4172-6612


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References Lee, D. E.; Koo, H.; Sun, I. C.; Ryu, J. H.; Kim, K.; Kwon, I. C. Multifunctional (1) Nanoparticles for Multimodal Imaging and Theragnosis. Chem. Soc. Rev. 2012, 41, 26562672. (2) Kunjachan, S.; Ehling, J.; Storm, G.; Kiessling, F.; Lammers, T. Noninvasive Imaging of Nanomedicines and Nanotheranostics: Principles, Progress, and Prospects. Chem. Rev. 2015, 115, 10907-10937. (3) Miao, P.; Han, K.; Tang, Y.; Wang, B.; Lin, T.; Cheng, W. Recent Advances in Carbon Nanodots: Synthesis, Properties and Biomedical Applications. Nanoscale 2015, 7, 1586-1595. (4) Lim, S. Y.; Shen, W.; Gao, Z. Q. Carbon Quantum Dots and Their Applications. Chem. Soc. Rev. 2015, 44, 362-381. (5) Xu, Y.; Jia, X. H.; Yin, X. B.; He, X. W.; Zhang, Y. K. Carbon Quantum Dot Stabilized Gadolinium Nanoprobe Prepared via a One-Pot Hydrothermal Approach for Magnetic Resonance and Fluorescence Dual-Modality Bioimaging. Anal. Chem. 2014, 86, 12122-12129. (6) Gong, N. Q.; Wang, H.; Li, S.; Deng, Y. L.; Chen, X. A.; Ye, L.; Gu, W. MicrowaveAssisted Polyol Synthesis of Gadolinium-Doped Green Luminescent Carbon Dots as a Bimodal Nanoprobe. Langmuir 2014, 30, 10933-10939. (7) Shi, Y. P.; Pan, Y.; Zhong, J.; Yang, J.; Zheng, J. H.; Cheng, J. L.; Song, R.; Yi, C. Q. Facile Synthesis of Gadolinium (III) Chelates Functionalized Carbon Quantum Dots for Fluorescence and Magnetic Resonance Dual-Modal Bioimaging. Carbon 2015, 93, 742-750. (8) Chiu, S. H.; Gedda, G.; Girma, W. M.; Chen, J. K.; Ling, Y. C.; Ghule, A. V.; Ou, K. L.; Chang, J. Y. Rapid Fabrication of Carbon Quantum Dots as Multifunctional Nanovehicles for Dual-Modal Targeted Imaging and Chemotherapy. Acta Biomater. 2016, 46, 151-164. (9) Chen, D.; Lawton, D.; Thompson, M. R.; Liu, Q. Biocomposites Reinforced with Cellulose Nanocrystals Derived from Potato Peel Waste. Carbohydr. Polym. 2012, 90, 709716. (10) Wang, J. P.; Sahu, S.; Sonkar, S. K.; Tackett, K. N.; Sun, K. W.; Liu, Y. M.; Maimaiti, H.; Anilkumar, P.; Sun, Y. P. Versatility with Carbon Dots-from Overcooked BBQ to Brightly Fluorescent Agents and Photocatalysts. RSC Adv. 2013, 3, 15604-15607. (11) Zhu, L. L.; Yin, Y. J.; Wang, C. F.; Chen, S. Plant Leaf-derived Fluorescent Carbon Dots for Sensing, Patterning and Coding. J. Mater. Chem. C 2013, 1 (32), 4925-4932. (12) Qin, X. Y.; Lu, W. B.; Asiri, A. M.; Al-Youbi, A. O.; Sun, X. P. Microwave-Assisted Rapid Green Synthesis of Photoluminescent Carbon Nanodots from Flour and Their Applications for Sensitive and Selective Detection of Mercury(II) Ions. Sens. Actuators BChem. 2013, 184, 156-162. (13) Prasannan, A.; Imae, T. One-Pot Synthesis of Fluorescent Carbon Dots from Orange Waste Peels. Ind. Eng. Chem. Res. 2013, 52, 15673-15678. (14) Tan, X. W.; Romainor, A. N. B.; Chin, S. F.; Ng, S. M. Carbon Dots Production via Pyrolysis of Sago Waste as Potential Probe for Metal Ions Sensing. J. Anal. Appl. Pyrol. 2014, 105, 157-165. (15) Park, S. Y.; Lee, H. U.; Park, E. S.; Lee, S. C.; Lee, J. W.; Jeong, S. W.; Kim, C. H.; Lee, Y. C.; Huh, Y. S.; Lee, J. Photoluminescent Green Carbon Nanodots from Food-WasteDerived Sources: Large-Scale Synthesis, Properties, and Biomedical Applications. ACS Appl. Mater. Interfaces 2014, 6, 3365-3370. (16) Yang, X. M.; Zhuo, Y.; Zhu, S. S.; Luo, Y. W.; Feng, Y. J.; Dou, Y. Novel and Green Synthesis of High-Fluorescent Carbon Dots Originated from Honey for Sensing and Imaging. Biosens. Bioelectron. 2014, 60, 292-298. (17) Liu, Y. S.; Zhao, Y. A.; Zhang, Y. Y. One-Step Green Synthesized Fluorescent


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 31

Carbon Nanodots from Bamboo Leaves for Copper(II) Ion Detection. Sens. Actuators BChem. 2014, 196, 647-652. (18) Hu, Y. P.; Yang, J.; Tian, J. W.; Jia, L.; Yu, J. S. Waste Frying Oil as a Precursor for One-Step Synthesis of Sulfur-Doped Carbon Dots with pH-Sensitive Photoluminescence. Carbon 2014, 77, 775-782. (19) Barbosa, C. D. D. S.; Correa, J. R.; Medeiros, G. A.; Barreto, G.; Magalhaes, K. G.; de Oliveira, A. L.; Spencer, J.; Rodrigues, M. O.; Neto, B. A. D. Carbon Dots (C-dots) from Cow Manure with Impressive Subcellular Selectivity Tuned by Simple Chemical Modification. Chem. 2015, 21, 5055-5060. (20) Essner, J. B.; Laber, C. H.; Ravula, S.; Polo-Parada, L.; Baker, G. A. Pee-Dots: Biocompatible Fluorescent Carbon Dots Derived from the Upcycling of Urine. Green Chem. 2016, 18, 243-250. (21) Jayakumar, R.; Menon, D.; Manzoor, K.; Nair, S. V.; Tamura, H. Biomedical Applications of Chitin and Chitosan based Nanomaterials-A Short Review. Carbohydr. Polym. 2010, 82, 227-232. (22) Hirano, S. Chitin Biotechnology Applications. Biotechnol. Annu. Rev. 1996, 2, 237258. (23) Kartal, S. N.; Imamura, Y. Removal of Copper, Chromium, and Arsenic from CCATreated Wood onto Chitin and Chitosan. Bioresour. Technol. 2005, 96 (3), 389-392. (24) Vijayaraghavan, K.; Palanivelu, K.; Velan, M. Biosorption of Copper(II) and Cobalt(II) from Aqueous Solutions by Crab Shell Particles. Bioresour. Technol. 2006, 97, 1411-1419. (25) Vijayaraghavan, K.; Winnie, H. Y. N.; Balasubramanian, R. Biosorption Characteristics of Crab Shell Particles for the Removal of Manganese(II) and Zinc(II) from Aqueous Solutions. Desalination 2011, 266, 195-200. (26) Pinto, P. X.; Al-Abed, S. R.; Reisman, D. J. Biosorption of Heavy Metals from Mining Influenced Water onto Chitin Products. Chem. Eng. J. 2011, 166, 1002-1009. (27) Setoguchi, T.; Kato, T.; Yamamoto, K.; Kadokawa, J. Facile Production of Chitin from Crab Shells using Ionic Liquid and Citric Acid. Int. J. Biol. Macromol. 2012, 50, 861864. (28) Hajji, S.; Ghorbel-Bellaaj, O.; Younes, I.; Jellouli, K.; Nasri, M. Chitin Extraction from Crab Shells by Bacillus Bacteria. Biological Activities of Fermented Crab Supernatants. Int. J. Biol. Macromol. 2015, 79, 167-173. (29) Zargar, V.; Asghari, M.; Dashti, A. A Review on Chitin and Chitosan Polymers: Structure, Chemistry, Solubility, Derivatives, and Applications. ChemBioEng Rev. 2015, 2, 204-226. (30) Ifuku, S.; Urakami, T.; Izawa, H.; Morimoto, M.; Saimoto, H. Preparation of a Protein-Chitin Nanofiber Complex from Crab Shells and Its Application as a Reinforcement Filler or Substrate for Biomineralization. RSC Adv. 2015, 5, 64196-64201. (31) Wang, C. X.; Xu, Z. Z.; Cheng, H.; Lin, H. H.; Humphrey, M. G.; Zhang, C. A Hydrothermal Route to Water-Stable Luminescent Carbon Dots as Nanosensors for pH and Temperature. Carbon 2015, 82, 87-95. (32) Dong, Y. Q.; Pang, H. C.; Yang, H. B.; Guo, C. X.; Shao, J. W.; Chi, Y. W.; Li, C. M.; Yu, T. Carbon-Based Dots Co-doped with Nitrogen and Sulfur for High Quantum Yield and Excitation-Independent Emission. Angew. Chem. Int. Ed. 2013, 52, 7800-7804. (33) Ding, H.; Yu, S. B.; Wei, J. S.; Xiong, H. M. Full-Color Light-Emitting Carbon Dots with a Surface-State-Controlled Luminescence Mechanism. ACS Nano 2016, 10 (1), 484491. (34) Yuana, F.; Lia, S.; Fana, Z.; Mengb, X.; Fana, L.; Yang, S. Shining Carbon Dots: Synthesis and Biomedical and Optoelectronic Applications. Nano Today 2016, 11, 565–586.


28

Page 29 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(35) Wang, Y.; Hu, A. Carbon Quantum Dots: Synthesis, Properties and Applications. J. Mater. Chem. C 2014, 2, 6921-6939 (36) Liu, Z.; Liu, X.; Yuan, Q.; Dong, K.; Jiang, L.; Li, Z.; Ren, J.; Qu, X. Hybrid Mesoporous Gadolinium Oxide Nanorods: a Platform for Multimodal Imaging and Enhanced Insoluble Anticancer Drug Delivery with Low Systemic Toxicity. J. Mater. Chem. 2012, 22, 14982-14990. (37) Hu, X.; Wang, M.; Miao, F.; Ma, J.; Shen, H.; Jia, N. Regulation of Multifunctional Mesoporous Core-shell Nanoparticles with Luminescence and Magnetic Properties for Biomedical Applications. J. Mater. Chem. B 2014, 2, 2265-2275. (38) Luo, N.; Yang, C.; Tian, X.; Xiao, J.; Liu, J.; Chen, F.; Zhang, D.; Xu, D.; Zhang, Y.; Yang, G.; Chen, D.; Li, L. A General top-down Approach to Synthesize Rare Earth DopedGd2O3 Nanocrystals as Dualmodal Contrast Agents. J. Mater. Chem. B 2014, 2, 5891-5897. (39) Zhang, G. L.; Du, R. H.; Zhang, L. L.; Cai, D. Q.; Sun, X.; Zhou, Y.; Zhou, J.; Qian, J. C.; Zhong, K.; Zheng, K.; Kaigler, D.; Liu, W. Q.; Zhang, X.; Zou, D. H.; Wu, Z. Y. Gadolinium-Doped Iron Oxide Nanoprobe as Multifunctional Bioimaging Agent and Drug Delivery System. Adv. Funct. Mater. 2015, 25, 6101-6111. (40) Yu, C. Y.; Xuan, T. T.; Chen, Y. W.; Zhao, Z. J.; Liu, X. X.; Lian, G. H.; Li, H. L. Gadolinium-Doped Carbon Dots with High Quantum Yield as an Effective Fluorescence and Magnetic Resonance Bimodal Imaging Probe. J. Alloy Compd. 2016, 688, 611-619. (41) Huang, C. C.; Khu, N. H.; Yeh, C. S. The Characteristics of Sub 10 nm Manganese Oxide T1 Contrast Agents of Different Nanostructured Morphologies. Biomaterials 2010, 31, 4073-4078. (42) Komban, R.; Klare, J. P.; Voss, B.; Nordmann, J.; Steinhoff, H. J.; Haase, M. An Electron Paramagnetic Resonance Spectroscopic Investigation on the Growth Mechanism of NaYF4:Gd Nanocrystals. Angew. Chem., Int. Ed. 2012, 51, 6506-6510. (43) Chang, J. Y.; Chen, G. R.; Li, J. D. Synthesis of Magnetofluorescence Gd-Doped CuInS2/ZnS Quantum Dots with Enhanced Longitudinal Relaxivity. Phys. Chem. Chem. Phys. 2016, 18 , 7132-7140. (44) Liu, J.; Tian, X. M.; Luo, N. Q.; Yang, C.; Xiao, J.; Shao, Y. Z.; Chen, X. M.; Yang, G. W.; Chen, D. H.; Li, L. Sub-10 nm Monoclinic Gd2O3:Eu3+ Nanoparticles as Dual-Modal Nanoprobes for Magnetic Resonance and Fluorescence Imaging. Langmuir 2014, 30 , 1300513013. (45) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Gadolinium(III) Chelates as MRI Contrast Agents: Structure, Dynamics, and Applications. Chem. Rev. 1999, 99 , 22932352. (46) Zhang, J.; Hao, G.; Yao, C.; Hu, S.; Hu, C.; Zhang, B. Paramagnetic Albumin Decorated CuInS2/ZnS QDs for CD133+ Glioma Bimodal MR/Fluorescence Targeted Imaging. J. Mater. Chem. B 2016, 4, 4110–4118. (47) Low, P. S.; Henne, W. A.; Doorneweerd, D. D. Discovery and Development of FolicAcid-Based Receptor Targeting for Imaging and Therapy of Cancer and Inflammatory Diseases. Acc. Chem. Res. 2008, 41, 120-129. (48) Liu, T. P.; Wu, S. H.; Chen, Y. P.; Chou, C. M.; Chen, C. T. Biosafety Evaluations of Well-Dispersed Mesoporous Silica Nanoparticles: Towards in vivo-Relevant Conditions. Nanoscale 2015, 7, 6471-6480. (49) Yuan, Y.; Sun, H. B.; Ge, S. C.; Wang, M. J.; Zhao, H. X.; Wang, L.; An, L. N.; Zhang, J.; Zhang, H. F.; Hu, B.; Wang, J. F.; Liang, G. L. Controlled Intracellular SelfAssembly and Disassembly of F-19 Nanoparticles for MR Imaging of Caspase 3/7 in Zebrafish. ACS Nano 2015, 9, 761-768. (50) Karlstrom, R. O.; Tyurina, O. V.; Kawakami, A.; Nishioka, N.; Talbot, W. S.; Sasaki, H.; Schier, A. F. Genetic Analysis of Zebrafish gli1 and gli2 Reveals Divergent


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 31

Requirements for gli Genes in Vertebrate Development. Development 2003, 130, 1549-1564. (51) Kane, D. A.; Hammerschmidt, M.; Mullins, M. C.; Maischein, H. M.; Brand, M.; vanEeden, F. J. M.; FurutaniSeiki, M.; Granato, M.; Haffter, P.; Heisenberg, C. P.; Jiang, Y. J.; Kelsh, R. N.; Odenthal, J.; Warga, R. M.; NussleinVolhard, C. The Zebrafish Epiboly Mutants. Development 1996, 123, 47-55. (52) Liu, Z.; Sun, X. M.; Nakayama-Ratchford, N.; Dai, H. J. Supramolecular Chemistry on Water-Soluble Carbon Nanotubes for Drug Loading and Delivery. ACS Nano 2007, 1, 5056. (53) Huang, C. L.; Huang, C. C.; Mai, F. D.; Yen, C. L.; Tzing, S. H.; Hsieh, H. T.; Lingd, Y. C.; Chang, J. Y. Application of Paramagnetic Graphene Quantum Dots as a Platform for Simultaneous Dual-Modality Bioimaging and Tumor-Targeted Drug Delivery. J. Mater. Chem. B 2015, 3, 651-664. (54) Rosenholm, J. M.; Peuhu, E.; Eriksson, J. E.; Sahlgren, C.; Linden, M. Targeted Intracellular Delivery of Hydrophobic Agents using Mesoporous Hybrid Silica Nanoparticles as Carrier Systems. Nano Lett. 2009, 9, 3308-3311.


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Addition of GdCl3, MnCl2, or EuCl3 Crab shell

Hydrothermal carbonization MFCQDs

microwave O

OH O

O HO O

CH3

220 °C,10 min

NH

O

NH

CH3

O OH

MR imaging n

chitin


Optical imaging

Magnetofluorescent Carbon Dots Derived from ... - ACS Publications

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