Excitation Wavelength Independent Carbon-Decorated Ferrite

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Excitation Wavelength Independent Carbon-Decorated Ferrite Nanodots for Multimodal Diagnosis and Stimuli Responsive Therapy Palani Sharmiladevi,† Najim Akhtar,‡ Viswanathan Haribabu,† Koyeli Girigoswami,† Surojit Chattopadhyay,‡ and Agnishwar Girigoswami*,† †

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Faculty of Allied Health Sciences, Chettinad Academy of Research & Education (CARE), Chettinad Hospital and Research Institute (CHRI), Kelambakkam, Chennai 603 103, India ‡ Institute of Biophotonics, National Yang-Ming University, Taipei 112, Taiwan ABSTRACT: The combination of superparamagnetism and excitation independency have been packed into carbondecorated ferrite nanodots (CDs@MNFs) for the introduction of a cost-effective and less-toxic multimodal contrast agent in fluorescence/MR imaging to replace conventional heavy metal containing Gd-DOTA. The label-free surface engineered ferrite nanodots are capable of generating twin T1 (longitudinal) and T2 (transverse) weighted magnetic resonance (MR) along with fluorescence emission. The calculated molar relaxivities and molar radiant efficiency obtained from in vitro and in vivo studies are the indication of its multimodal efficacy in medical imaging compared to the conventional contrast agents. The cellular internalization of nanodots was established by confocal microscopy and flow cytometric assay, whereas the hemolysis and cell viability assays support their appreciable toxicity. Furthermore, the surface chemistry due to the presence of −COOH was utilized to attach the anticancer agent, doxorubicin (−NH2) making it an external stimuli responsive drug delivery vehicle for the treatment of cancer. Given the ease of fabrication, negligible toxicity, and significant contrast enhancement with stimuli responsive drug release kinetics CDs@MNFs prove to be a potential, costeffective multimodal imaging agent which could be used for theragnosis. KEYWORDS: theragnosis, ferrite, nanodots, multimodal imaging, drug delivery, twin-relaxivity

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INTRODUCTION The emergence of noninvasive diagnostics made it possible to gain insights into the once unfathomable pathological features of the tissues and organ systems. Even though various noninvasive procedures have evolved to serve this purpose, the inbuilt ionizing radiation posed a risk for applications in vivo. Hence, the use of techniques based on magnetic contrast agents and optical emissive probes have been much sought.1 These techniques can elevate the differentiating degree of the diseased tissues from the normal ones preferably when combined together.2 Over the years, plentiful research has been done to explore the applications of nanoparticles in molecular imaging and several types of research have yielded fruitful results along with commercialization.3 As discussed earlier, to bring up the two techniques (i.e., magnetic and optical) together, magneto-fluorescent nanohybrids have been fabricated and investigated. Various nanoparticles used for this purpose are semiconductor quantum dots, SPIONS, Gd-based nanoconjugates, polymeric nanoparticles, silica nanoparticles etc. MRI is the most widely preferred technique among the many molecular imaging techniques because of the noninvasive nature, higher spatial, and temporal resolution, and the lack of exposure to ionizing radiation. Typically, two types of contrast © XXXX American Chemical Society

agents (CAs), T1 or positive contrast and T2 or negative contrast agents, are used to perform MR imaging. The CAs’ potentiality to bring about a change in the relaxation time of the T1 and T2 is denoted as relaxivity (r1 and r2) expressed in terms of mM−1 s−1. The ratio of r2/r1 value determines the contrasting ability of the material to act as either T1 (1−3) or T2 (>10) contrast agent. The intermediate values between 3 and 10 denotes the dual contrast (T1 and T2) ability of the contrast agent.4 Gadolinium-based CAs with seven unpaired valence electrons continued to be the most clinically preferred T1 contrast agent.5 Superparamagnetic Iron Oxide Nanoparticles (SPIONS) are quite contrary to the Gd-based CAs in the perspective of toxicity. Although the SPIONs are FDA approved clinically used CAs, T2MR Images are more prone to susceptible artifacts. To circumvent the misinterpretations dual contrast acquisitions are most preferred. Recent findings proclaimed that doping of paramagnetic ions into spinnel ferrites at specific molar ratio can produce twin relaxivity, T1 and T2 in MR Imaging.6 The higher magnetization and relaxivity values have immensely promoted the Received: January 15, 2019 Accepted: March 1, 2019

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DOI: 10.1021/acsabm.9b00039 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

Jasco (FP-3800) fluorimeter was used to obtain the fluorescence spectra. Rigaku X-ray diffractometer was used to perform the crystal structural study. The magnitude of magnetism was calculated from Lakeshore VSM. Functional group analysis of the synthesized nanostructures was done using Bruker-Alpha FTIR spectrometer and Bruker Raman spectrometer. Malvern Nano-Zs particle size analyzer was employed to determine the particle size distribution and surface zeta potential through dynamic light scattering principle. The fluorescence lifetime of the samples was measured with Fluoro Cube Lifetime System from HORIBA Jobin-yvon and the fitting was done using time correlated single photon counting (TCSPC) method. The X-ray photoelectron spectra (XPS) were performed on VG Multilab 2000-Thermo Scientific, USA, K-alpha. Phantom and animal MR Imaging was done with GE signa HDxT 1.5 T MRI scanner and fluorescence animal imaging was done using IVIS-Lumina LT, PerkinElmer small animal imaging system at 37 °C. Hemolysis Assay. Hemolysis assay was performed following the procedure reported in the literature with slight modification.15 In brief, fresh human blood stabilized with EDTA was centrifuged at 1500 rpm for 15 min and the supernatant was removed. Further, the pellet was washed with PBS for 5 min to remove the serum completely. The obtained Red Blood Cells (RBCs) were diluted ten times with PBS. From the diluted suspension of RBCs 0.1 mL was added into a 1.5 mL tube containing 0.9 mL water (as positive control), and 0.9 mL PBS (as negative control), and 0.9 mL PBS containing CDs@MNFs with varied concentrations (0.05−0.4 mg/ mL), respectively. After incubating the samples for 2 h at 37 °C, the samples were centrifuged at 12000 rpm for 1 min. Later the absorbance of the supernatants was measured at 541 nm and the percentage of hemolysis was calculated by using the formula given below:

consideration of Manganese Ferrite Nanoparticles (MNFs) as MR CAs. The ease of synthesis and the outstanding chemical stability of MNFs instigated several research groups to work on with MNFs based CAs. However, the inclusion of the Mn ions again raises the question of the toxicity.7 Out of the numerous coating approaches opted for addressing the issue of toxicity, biocompatible materials play a crucial role in significant toxicity reduction thereby ensuring the stability of the particles in in vivo environments.8 The coating of the MNFs could possibly hinder the interaction between the MNFs and the water proton. Therefore, it becomes essential for the selected coating material to be highly water-dispersible with the allowance of the water proton to access the MNFs core. As a new member of the carbonaceous family, fluorescent carbon as well as carbon nanodots (CDs) have gained intense significance in the past decade.9,10 CDs possess exceptional characteristic features such as tunable photoluminescent behavior, enhanced water solubility, superior chemical, and photostability. Despite the several advantages possessed by CDs, the in vivo applications are limited to a certain extent as CDs suffer from poor quantum yield. Later it was found that the issue can be resolved by the doping of hetero nitrogen atoms into the CDs.11 As the doping increased, the quantum yield of CDs gained its popularity back and henceforth it continues to be an assured fluorescent nanomaterial. Regardless of the controversy over the origin of CDs photoluminescence, the participation of the surface traps in the regulation of radiative transition has been agreed to a larger extent.12 Recently, Li and coauthors clearly depicted the role of surface traps in determining the photoluminescent behavior of the CDs.13 Proper passivation of the surface leads to excitation independent behavior, while the contrary leads to excitation dependent behavior of the CDs. Organic compounds composed of Nitrogen atoms can efficiently act as a surface passivating agent.13 Hence, Urea was chosen as a precursor of CDs thereby performing the dual role as a passivating agent along with quantum yield enhancer. Eventually, CDs become the ideal candidate to be decorated over the ferrite cores as CDs are highly porous in nature with excellent water solubility and negligible toxicity in vivo.14 Herein, we report a novel onepot hydrothermal strategy to synthesize carbon decorated ferrite cores (CDs@MNFs) to deliver remarkable enhancement in twin relaxivity MR and optical images.

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hemolysis (%) =

A sample − A negative control A positive control − A negative control

Cell Viability Assessment. The cytotoxicity of the CDs@MNFs was assessed by MTT assay on B16F10 melanoma cells using standard protocol.16 Cells were maintained in DMEM with 10% FBS and 1% antibiotic solution at 37 °C, 5% CO2 incubator with humidified atmosphere. 104 cells were plated in each well of a 96 well plate and incubated for 24 h. Treatments with different concentration of CDs@MNFs were done and further incubated for 24 h. MTT (5 mg/mL) was added to each well and incubated in dark at CO2 incubator for further 4 h until the formazan crystals are formed. The formazan crystals were then dissolved with DMSO and absorbance was measured at 570 nm. The percentage of cell viability is calculated according to Girigoswami et al.16 Confocal Microscopy and Flow Cytometric Assay. Intracellular distribution of CDs@MNFs was determined in B16F10 melanoma cells using Confocal Laser Scanning Microscope (Zeiss LSM880 with AiryScan, Carl Zeiss Microscopy GmbH, Germany). One ×104 cells/well were seeded on coverslips for 24h followed by treatment with CDs@MNFs. Then the cells were washed with PBS and stained using DAPI (blue). Similarly, cells were attached in 12 well plates for the flow cytometric assay. After 24h incubation, cells were treated with CDs@ MNFs and incubated for another 24h for the internalazitaion. The cells were then washed with PBS followed by detachment using Trypsin-EDTA, and finally resuspended in PBS for introduction into the CytoFLEX A (Beckman Coulter, A00−1−1102, USA). The instrument was equipped with 488 nm laser. Dot plots and fluorescence data were obtained. The fluorescence histograms were generated using CytExpert Software (Beckman Coulter, USA). In Vitro and in Vivo MR Imaging. The increasing concentrations of synthesized CDs@MNFs were added in water phantoms in a 24 well plate at room temperature.17 The phantoms were imaged under a 1.5 T GE Signa HDxT MRI scanner with both T1- and T2-weighted imaging protocols. TR = 3000 ms; TE = 14 ms; FOV = 24 × 24 and variable TI = 400−2000 ms were fixed for T1 weighted FLAIR imaging sequence to obtain slices of 2 mm thickness. T2 weighted

MATERIALS AND METHODS

Ferric chloride hexahydrate (FeCl3.6H2O, 97%), Manganese chloride tetrahydrate (MnCl2.4H2O, 99%), Urea (CH4N2O), and Citric acid (C6H8O7)were obtained from Sigma and were used without any further purification. The double distilled autoclaved water has been used in the entire experiments. Synthesis of Carbon-Nanodot-Coated Ferrite (CDs@MNFs). For the synthesis of CDs@MNFs, one-pot hydrothermal synthesis method was employed. Briefly, 0.1 M MnCl2.4H2O and 0.2 M FeCl3.6H2O were dissolved in 50 mL of distilled water to maintain 1:2 molar ratios. To this 1.5 g urea and 3 g citric acid were added as molecular precursors for CDs. The reaction mixture was transferred to a Teflon lined stainless steel autoclave and kept at 160 °C for 12 h. The reactor was allowed to come down to room temperature and centrifuged at 10,000 rpm for 10 min to remove unreacted components. Then the dispersion was dialyzed against water through a 1 kDa dialysis membrane. Finally the resultant dispersion was dried in vacuum overnight at 75 °C. The powdered sample was used for the further study. Sample Characterization. The UV−vis absorption spectra of the samples were recorded with Shimadzu UV-1800 spectrophotometer. B

DOI: 10.1021/acsabm.9b00039 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 1. (A) X-ray diffraction pattern of synthesized CDs@MNFs using hydrothermal method in support of crystallinity. (B) FTIR spectra of CDs@MNFs. (C) Raman spectra of CDs@MNFs to confirm the carbon-coated ferrite dots synthesis. (D) Hysteresis loop of CDs@MNFs at room temperature for the assessment of superparamagnetism. (E) Broad absorption spectra of CDs@MNFs and F) Particle size distribution showing the major scattering peak at 142 nm. turbo spin echo sequence was used to obtain slices of 2 mm thickness carried out with variable TE = 10−100 ms where TR = 10000 ms; FOV = 24 × 24 and an echo train length = 12. Similar parameters were applied to image intravenously injected CDs@MNFs mice after getting the clearance from Institutional Animal Ethics Committee (IAEC), CHRI. In Vitro and in Vivo Fluorescence Imaging. The fluorescence imaging was done by IVIS animal imaging system for both phantom and mice imaging. Phantom imaging was conducted with varying concentrations (0.22 to 5.59 mM) of CDs@MNFs taken in a 96-well plate. 460 nm optical filter was used to acquire the fluorescence images and the obtained results were extended further to determine in vivo properties in mice following IAEC approved protocol. Drug Loading to CDs@MNFs and Release Kinetics. Doxorubicin (DOX), an anticancer drug was loaded to the CDs@ MNFs to analyze the release kinetics of the drug as a therapeutic agent. This loading was carried out by adding 10 mg/mL DOX to an aqueous solution containing 10 mg/mL CDs@MNFs under constant stirring overnight in dark light conditions. Unbound DOX was removed by dialyzing the final formulation against distilled water. The release kinetics were studied spectrophotometrically (λ = 480 nm) in vitro in two different pH of 7.4 and 6.2 following standard protocols.18

tometer is elucidated in Figure 1B. The Fe−O bond vibration at 560 cm−1 confirms the formation of metal oxide. The absorption bands at 3042 and 3140 cm−1 corresponds to the aromatic C−H stretching vibrations. The absorption bands at 1552 and 1613 cm−1 are assigned to amine N−H bending. Further, the absorption bands at 1113 and 1254 cm−1 represent amine C−N stretching. Thus, FTIR spectra wellconfirmed the decoration of CDs over the ferrite cores. Figure 1C describes the Raman spectrum of CDs@MNFs and the significant A1g band at 607 cm−1 corresponds to the vibrational stretching of the tetrahedral units. The T2g bands at 553 and 437 cm−1 represent the octahedral sites. Two broad peaks at 1344 and 1541 cm−1 attributed to the D-band and G-band, respectively, for carbon dots. The intensity ratio of the disordered D-band and the crystalline G-band (ID/IG) is around 0.81, thus indicating the similar structure to graphite. To validate the hypotheses of utilizing the synthesized CDs@MNFs for MR imaging, the magnetic nature of the samples is of utmost importance. The magnetic nature of the CDs@MNFs was evaluated by VSM measurements. The absence of the hysteresis loop depicted the superparamagnetic nature of the CDs@MNFs, which is highly valuable for performing the role of contrast agents (Figure 1D). The absorption spectrum of the synthesized CDs@MNFs is shown in Figure 1E. The broad absorption peak centered at 457 nm is attributed to the formation of CDs in the ferrofluid. It is wellestablished that neither urea nor citric acid can absorb at the given wavelength.20 Particle size analysis of the CDs@MNFs showed that the particles are uniformly distributed with the hydrodynamic diameter of 142 nm (Figure 1F). The zeta potential value of −53.6 mV showed that the CDs@MNFs are stable colloidal suspensions and are well dispersed in water due

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RESULTS AND DISCUSSION The patterns of the XRD spectrum revealed the successful synthesis of CDs@MNFs (Figure 1A). The peak centered at 23.02° is substantial as it corresponds to the characteristic feature of carbon atoms. The broad peak at colloidal XRD represents that the carbon atoms present in the CDs@MNFs are amorphous in nature.19 The cubic nature of the MNFs is evident from the peaks which are inconsistent with the Braggs reflections of the planes, respectively. The XRD peaks are also in good accordance with the standard XRD pattern of the MNFs (JCPDS card no 74−2403). The surface chemistry of the synthesized CDs@MNFs recorded with FTIR spectrophoC

DOI: 10.1021/acsabm.9b00039 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 2. (A) FESEM image, (B) TEM image, and (C) EDX-based elemental analysis for CDs@MNFs.

Figure 3. XPS spectra of (A) CDs@MNFs and (B) Fe 2p, (C) Mn 2p, (D) O 1s, and (E) C 1s of CDs@MNFs.

to the abundance of surface terminal amine groups. This study affirmed that the decoration of CDs over the ferrite cores has dramatically increased the water dispersibility of the ferrites, which otherwise would not have been possible for the ferrites to achieve alone. The surface morphology and crystal structure with the presence of elements were confirmed by FESEM and TEM (Figure 2). Scanning electron micrograph (JEOL JSM-7600F, Japan) shows the uniform distribution of the synthesized CDs@MNFs and the TEM (Philips JEM-2000EX) image confirms the core−shell nanostructure. The magnetic core is in the center and the CDs forms the shell around. The elemental analysis by EDX shows 7.5 and 3.8 wt % Fe and Mn respectively along with 78.4% carbon. Therefore, electron microgram confirms the synthesis of uniformly distributed CDs having manganese and iron in the center as ferrite. The structure and chemical compositions were further investigated by XPS spectroscopy. The full range XPS spectrum showed the presence of Fe, Mn, C, O, and N at their respective binding energies (Figure 3). The photoelectron lines at 724.12 and 710.2 eV binding energies are attributed to Fe 2p, whereas lines at 652.82 and 641.5 eV binding energies are ascribed to Mn 2p. The characteristic peak at 530.1 eV in O 1s spectrum confirms the existence of metal oxide as Fe−O and Mn−O, which is present in

MnFe2O4. The peak at 532.3 eV may be caused due to the metal−O−C bond formed in CDs@MNFs and at 531 eV attributed to CO. The high-resolution C 1s spectrum consists of three main peaks arising from CO (287.91 eV), C−N/C−O (285.49 eV) and C−C (284.37 eV) also support the formation of CDs@MNFs. The obtained results are in good conformity with the FTIR, Raman spectra and EDX. Steady-State and Time-Resolved Fluorescence. The recorded steady-state fluorescence spectra of the CDs@MNFs are given in Figure 4A. It was observed that the particle exhibits intriguing dual emissive behavior. The fluorescence emission was found to be independent of the excitation wavelength due to the abundance of amine functional groups on the CDs surface.13 Urea, primarily added to the reaction mixture to dope the nitrogen atom has passivated the surface traps, thereby making the CDs exhibit excitation independent behavior (Figure 4B). Moreover, the milder reaction temperature plays a prime role in tuning the photoluminescence, as temperatures higher than 200 °C will cause the amino-groups to abandon the surface. The emission spectra exhibited a peak at 540 nm with a shoulder at 490 nm. This behavior was attributed to originate from the different emission centers or surface states of CDs. These surface states could be explained as so-called “traps” and these are generated from different surface functional groups as it is observed in the XPS D

DOI: 10.1021/acsabm.9b00039 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 4. (A) Steady-state fluorescence emission and excitation spectra. (B) Excitation independent fluorescence emission. (C) Time-resolved fluorescence spectra of CDs@MNFs with λex/λem= 460/490 nm. (D) Time resolved fluorescence spectra of CDs@MNFs with λex/λem= 460/540 nm.

Table 1. Photoluminescent Properties of the Synthesized CDs@MNFs λex/ λem [nm]

τ1 [ns]

a1

τ2 [ns]

a2

τ3 [ns]

a3

χ2

460/490 460/540

2.3875 2.4272

0.04 0.31

6.2760 6.3268

0.02 0.54

0.3753 0.4391

0.94 0.15

0.99 1.12

Figure 5. (A) Phantom MR images of CDs@MNFs in comparison with Gd-DOTA: (a) T1-weighted (b) T2-weighted. (B) Linear plots of concentration-dependent relaxivities for Gd-DOTA and CDs@MNFs to calculate molar relaxivities (r2 and r1).

spectra.21,22 Fluorescence decay profile of the CDs@MNFs was analyzed by time-correlated single photon counting (TCSPC) method and the data were fitted with the triple exponential fitting (Figure 4C and 4D). The excitation wavelength was fixed at 460 nm and the emission was measured at two different emissions 490 and 540 nm. The particles were found to emit three different fluorescence decays with similar values of lifetime, but differing in respect to the contribution values (Table 1). The variation of the

contribution values was due to the quantum confinement effect. The CDs@MNFs was also prepared in triplicates following the same synthesis strategy to determine the effect of size over the optical features (data not shown). Even though there was a slight variation in the hydrodynamic diameter, the optical features of the CDs@MNFs remained the same. Phantom MRI. The capability of the CDs@MNFs to bring about the relaxation change in water protons was evaluated by phantom MR imaging with respect to the standard E

DOI: 10.1021/acsabm.9b00039 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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Figure 6. In vivo T1- and T2-weighted MR images with pre- and postinjection of CDs@MNFs in mice.

a clear picture of the major organs such as lungs, kidneys, spleen, liver, and heart. In contrast, the contrast-administered images clearly revealed the aforementioned organs in both T1and T2-weighted imaging immediately after the administration of the contrast agent (Figure 6). This contrast-enhanced differentiation of the organs from the surrounding tissues is due to the interaction of the water protons with the magnetic core. To study the clearance profile of the administered particles, the mouse was imaged at 1, 6, and 24 h. It was observed that 1 h post injection, the particles were retained in the system and the organs remained clearly visible in the T1weighted and T2 -weighted images. After 6 h, the particles were seen to be cleared from the system with a decrease in the signal intensity. After 24 h, steep reduction in the signal intensity of the images proved that the particles have been cleared from the system. Optical Imaging. To illustrate the optical capability of CDs@MNFs to function as fluorescence imaging probes, CDs@MNFs at varied concentrations were imaged under the small animal imaging system. Figure 7A demonstrated the efficiency of CDs@MNFs to perform as an optical fluorophore. The fluorescence intensities of the CDs@MNFs were found to increase accordingly with the concentration. All the fluorescence images were normalized to photons per second per sq. centimeter per steradian (p/sec/cm2/sr) after removing the background signals. The linear increment in the intensity with increasing concentrations of CDs@MNFs was observed. Figure 7B shows the graphical representation of average radiant efficiency with the corresponding CDs@MNFs concentrations and the slope represents the average radiant efficiency, which was found to be 4.551 × 107 p/sec/cm2/sr per mM. In vivo mice imaging was also performed for the same

commercially marketed gadolinium-DOTA chelated contrast agent (Gd-DOTA). For phantom MR Imaging concentrations of the Gd-DOTA and CDs@MNFs were altered and taken in a 24-well plate. Utilizing the imaging protocols mentioned in the literature,6 the 24-well plate was imaged under the MR scanner. Images analyzed with the Centricity software demonstrated that the intensities of the images distinctly increased in T1 weighted imaging and gradually decreased in T2 weighted imaging parallel with the increasing concentration of CDs@MNFs (Figure 5). In order to figure out the contrasting ability of the contrast agents, longitudinal (r1 = 1/ T1) and transverse (r2 = 1/T2) relaxivities are highly valuable. The relaxivity values were computed by obtaining T1 FLAIR images with variable TI (400−2000 ms) and T2 TSE images with variable TE (10−100 ms). The graphs have been plotted for longitudinal and transverse relaxivities against the increasing concentrations to evaluate both the molar relaxivities. The r1 and r2 molar relaxivities for Gd-DOTA were found to be 3.08 ± 0.112 mM−1 s−1 and 3.98 ± 0.08 mM−1 s−1, respectively, whereas the same for CDs@MNFs were 10.56 ± 0.02 mM−1 s−1 and 55 ± 0.05 mM−1 s−1, respectively. The ratio of both the molar relaxivities (r2/r1) value was found to be 5.208, which was in the range of 10 > r2/ r1 > 3, indicting their potential nature as T1 and T2 weighted twin mode MR contrast agent. In Vivo MR Imaging. To further validate the twin mode contrasting ability of the synthesized CDs@MNFs in vivo, we intravenously injected the synthesized CDs@MNFs into a swiss albino mouse and imaged it under 1.5 T MR scanner. Mouse images were acquired before and after intravenous administration of the contrast agent. The image acquired before the administration of the contrast agent did not display F

DOI: 10.1021/acsabm.9b00039 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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seen to be cleared from the system suggesting that the particles posed negligible toxicity and moderate clearance rate which is positive for the consideration of biomedical contrast. This study shows the inherent biodistribution nature of the synthesized CDs@MNFs, thereby promoting the use of the particles for in vivo applications. Internalization. Confocal microscopy was done to observe the cellular uptake of CDs@MNFs with respect to DAPI stained nucleus. Figure 8A shows the DAPI stained (blue) nucleus of B16F10 melanoma cells whereas, the green fluorescence generated from the internalized particles were prominent in Figure 8B upon excitation at 488 nm. Figure 8C depicts the merged fluorescence images of both DAPI and CDs@MNFs and Figure 8D shows the bright field images of the same. Herewith the study confirmed the efficient internalization of particles in the cells after 24 h of incubation. For further confirmation flow cytometry was conducted for CDs@MNFs treated B16F10 melanoma cells. The signals obtained from the forward scattering and the side scattering were plotted with dots for untreated and treated cells in Figure 8E, G respectively. The treated cells showed a distinct change in the side scattering which signify the optical inhomogeneity due to the presence of CDs@MNFs in the cells which was missing in the untreated one. The PC7 window is showing the fluorescence generated from the treated cells (Figure 8H) which is highly intense compared to the untreated one (Figure 8F). The negligibly small fluorescence observed in an untreated one might be due to autofluorescence. The entire flow cytometric study confirms the particles’ internalization inside the cells. Hemocompatibility and Toxicity Assessment. The primary goal of investigating the hemocompatibility of the nanoparticles is to administer the synthesized CDs@MNFs for in vivo applications. The hemocompatibility of the synthesized nanoparticles was evaluated by hemolysis assay to determine the effect of the nanoparticle on RBCs. From Figure 9A, it is

Figure 7. IVIS-based fluorescence imaging. (A) Phantom with increasing concentrations of CDs@MNFs. (B) Average radiant efficiency of CDs@MNFs. (C) In vivo mice imaging with CDs@ MNFs, immediate postinjection (left), 1 h postinjection (middle), and 2 h postinjection (right).

and the results were in coherence with the phantom results. The exemplification of the fluorescence ability of CDs@MNFs in in vivo was demonstrated in a mouse system. 1.72 mM of CDs@MNFs was intravenously administered to swiss albino mice. Immediately after the injection, strong fluorescence signals were visualized from the organs, such as liver, lungs, and kidneys which confirm the rapid accumulation of the administered particles (Figure 7C). At 1 h postinjection, the signal intensity was found to be decreased in the organs mentioned earlier representing the gradual clearance of the particles from the organs. At 2 h postinjection, the particle was

Figure 8. Confocal microscopic images of B16F10 melanoma cells: (A) DAPI excited at 405 nm, (B) CDs@MNF-treated cells excited at 488 nm, (C) merged fluorescence images of both DAPI and CDs@MNFs, and (D) bright-field images of merged DAPI and CDs@MNFs. Flowcytometric analysis of B16F10: (E) dot plots for untreated control, (F) fluorescence histograms from PC7 gated areas for control, (G) dot plots for CDs@ MNFs treated cells, and (H) fluorescence histograms from PC7-gated areas for CDs@MNF-treated cells. G

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Figure 9. (A) Hemolytic assay with positive and negative controls for CDs@MNFs (inset: samples incubated with variable CDs@MNFs concentrations). (B) Comparison of viability of melanoma cells treated with CDs@MNFs and MNFs of varying concentrations.

obvious that the CDs@MNFs showed no detrimental effects on the RBCs similar to the negative control (PBS). The experiment was conducted spectrophotometrically with variable concentrations of 0.05, 0.1, 0.2, 0.3, and 0.4 mg/mL of CDs@MNFs and percent hemolysis obtained were 1.02, 1.01, 1.11, 1.85, and 1.92, respectively (as marked 1−5 in Figure 9A along with positive and negative control). The results clearly showed no apparent hemolysis with the maximum hemolysis activity of ∼2% in the maximum concentration. The ASTM E252408 standard value (Standard test method for analysis of hemolytic products of nanoparticles), has delimited the use of nanoparticles and is permissible only if the hemolytic activity is below 5%. The obtained hemolysis percentage of the CDs@MNFs is in good agreement with the standard value, thereby highlighting the excellent hemocompatible nature of the CDs@MNFs. In vitro cytotoxicity assessment of the synthesized CDs@ MNFs was evaluated by MTT assay on B16F10 melanoma cells. The results showed that the manganese ferrite nanoparticles coated with CDs (CDs@MNFs) showed better cell viability than the uncoated manganese ferrite nanoparticles (MNFs) (Figure 9B). Hence, it was concluded that the toxic effects of manganese ions have been reduced to a significant effect by the carbon coating and the synthesized CDs@MNFs are biocompatible in nature. pH-Responsive Drug Release Kinetics. The DOX loading on to CDs@MNFs was due to the electrostatic interaction which is influenced by hydrogen bonds. This interaction is possible due to the carboxyl groups present at the CDs@MNFs and −NH2 groups of DOX.23,24 The loading efficiency was quantified spectrophotometrically and was found to be 51.2%. The drug release kinetics profiling shown in Figure 10, illustrates that the cumulative percentage of drug release of DOX from the formulation at pH 7.4 and 6.2 which was calculated to be 58.87 and 88.91%m respectively. This shows that the DOX release is pH dependent and is found to be higher when the pH is acidic. Hence, it is concrete that the DOX is released in response to an acidic pH noted in the cancer environment, due to loss of electrostatic interactions.

Figure 10. pH dependence of cumulative percent DOX release kinetics from DOX-loaded CDs@MNFs at variable pH over 12 h time duration.

the surface of the MNFs markedly enhanced the twin mode contrast in MR Imaging. In addition to MR contrast enhancement, the inbuilt fluorescence ability of the CDs gave excellent high-intensity optical images, thereby making the CDs@MNFs a multimodal imaging agent. The hemocompatibility and toxicity studies ensured that the CDs@MNF is safe to administer. The pH-responsive DOX release kinetics proved CDs@MNFs as a potential nanocarrier for anticancer drugs.

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AUTHOR INFORMATION

Corresponding Author

*Email: [email protected]. FAX: +91 044 4741 1011. Tel: +91 9445 268 615. ORCID

Surojit Chattopadhyay: 0000-0002-1384-3379 Agnishwar Girigoswami: 0000-0003-0475-2544 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Council of Scientific and Industrial Research (CSIR), INDIA is acknowledged for financial assistance (01(2868)/17/EMR-II dated 02/05/2017). P.S. thanks CARE for fellowship. Authors acknowledge Ms. Pragya Pallavi for her help in preparation of images.

CONCLUSION In summary, we have illustrated a novel one-pot hydrothermal method to fabricate CDs@MNFs, which demonstrated excellent photostability, water solubility, and excitation wavelength independent PL properties. The decoration of CDs over H

DOI: 10.1021/acsabm.9b00039 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

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(19) Li, J.-Y.; Liu, Y.; Shu, Q.-W.; Liang, J.-M.; Zhang, F.; Chen, X.P.; Deng, X.-Y.; Swihart, M. T.; Tan, K.-J. One-Pot Hydrothermal Synthesis of Carbon Dots with Efficient Up-and Down-Converted Photoluminescence for the Sensitive Detection of Morin in a DualReadout Assay. Langmuir 2017, 33 (4), 1043−1050. (20) Qu, S.; Wang, X.; Lu, Q.; Liu, X.; Wang, L. A Biocompatible Fluorescent Ink Based on Water Soluble Luminescent Carbon Nanodots. Angew. Chem. 2012, 124 (49), 12381−12384. (21) Dhenadhayalan, N.; Lin, K.-C.; Suresh, R.; Ramamurthy, P. Unravelling the Multiple Emissive States in Citric-Acid-Derived Carbon Dots. J. Phys. Chem. C 2016, 120 (2), 1252−1261. (22) Mohan, R.; Drbohlavova, J.; Hubalek, J. J. Dual Band Emission in Carbon Dots. Chem. Phys. Lett. 2018, 692, 196−201. (23) Zeng, Q.; Shao, D.; He, X.; Ren, Z.; Ji, W.; Shan, C.; Qu, S.; Li, J.; Chen, L.; Li, Q. J. J. o. M. C. B. Carbon dots as a trackable drug delivery carrier for localized cancer therapy in vivo. J. Mater. Chem. B 2016, 4 (30), 5119−5126. (24) Kong, T.; Hao, L.; Wei, Y.; Cai, X.; Zhu, B. J. C.P. Doxorubicin conjugated carbon dots as a drug delivery system for human breast cancer therapy. Cell Proliferation 2018, 51 (5), No. e12488.

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DOI: 10.1021/acsabm.9b00039 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX