Highly Hydrophilic Luminescent Magnetic Mesoporous Carbon

Jan 21, 2016 - Judicious combination of fluorescence and magnetic properties along with ample drug loading capacity and control release property remai...
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Highly Hydrophilic Luminescent Magnetic Mesoporous Carbon Nanospheres for Controlled Release of Anticancer Drug and Multimodal Imaging Sasmita Mohapatra,*,† Smruti R. Rout,† Rahul K. Das,† Santoshi Nayak,‡ and Sudip K. Ghosh‡ †

Department of Chemistry, National Institute of Technology, Rourkela, India 769008 Department of Biotechnology, Indian Institute of Technology, Kharagpur, India 721302

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S Supporting Information *

ABSTRACT: Judicious combination of fluorescence and magnetic properties along with ample drug loading capacity and control release property remains a key challenge in the design of nanotheranostic agents. This paper reports the synthesis of highly hydrophilic optically traceable mesoporous carbon nanospheres which can sustain payloads of the anticancer drug doxorubicin and T2 contrast agent such as cobalt ferrite nanoparticles. The luminescent magnetic hybrid system has been prepared on a mesoporous silica template using a resorcinol-formaldehyde precursor. The mesoporous matrix shows controlled release of the aromatic drug doxorubicin due to disruption of supramolecular π−π interaction at acidic pH. The particles show MR contrast behavior by affecting the proton relaxation with transverse relaxivity (r2) 380 mM−1 S−1. The multicolored emission and upconversion luminescence property of our sample are advantageous in bioimaging. In vitro cell experiments shows that the hybrid nanoparticles are endocyted by the tumor cells through passive targeting. The pH-responsive release of doxorubicin presents chemotherapeutic inhibition of cell growth through induction of apoptosis.

INTRODUCTION Development of intelligent stimuli responsive theranostic system that can detect and treat cancer at its early stage is an emerging area in the field of nanobiotechnology.1 In this regard synthesis of multifunctional particulate drug delivery agents combined with bimodal MRI/optical bioimaging detection is significant.2,3 Particularly, composite nanoparticles combining fluorescence and magnetic components have received much attention because they ally the high sensitivity of the fluorescence phenomenon to the high spatial resolution of MRI.4−7 For example, the strong superparamagnetism of magnetic nanoparticles allows the visualization of target diseased tissue in any part of the body using T2-weighted magnetic resonance imaging.8 Additionally, the quantum confinement effect exhibited by semiconductor quantum dots allows ultrasensitive and multiplexed fluorescence imaging both in vitro and in vivo, providing new tools to understand cellular processes related to cancer development.9 On the other hand, nanosystems can carry small payloads of anticancer drugs and deliver them directly to a tumor. These multiple components can be combined perceptively into a single matrix system to administer therapeutic and diagnostic functions in a single dose. In this regard mesoporous silica is extensively investigated as a matrix system which can sustain a number of payloads like imaging agents (e.g., organic dyes, quantum dots [QDs], © 2016 American Chemical Society

upconversion particles [UCNPs], MRI contrast agents, CT contrast agents, etc.) and therapeutic agents (anticancer drugs, DNA, small interfering RNA [siRNA], proteins, hyperthermiainducing nanoparticles, ROS-generating agents, etc.).10−12 Mesoporous silica based nanoparticles have been explored as stimuli-responsive nanosystems using different capping strategies such as varieties of nanovalves, thermosensitive polymers to block the pores. The opening of mesopores and subsequent release of therapeutic cargoes are finely controlled by external stimuli such as pH and temperature.13−15 However, this tedious post-synthetic capping, incorporation of therapeutic cargoes and surface modification processes, limit the industrial translation of the protocol. Alternatively, carbon nanomaterials such as carbon nanotubes,16 fullerene,17 graphene oxide,18 and carbon dots19 are leading theranostic materials due to their larger surface area and low toxicity. The intrinsic luminescent property of these materials has been unanimously explored in bioimaging and monitoring curative responses.20 At the same time, high anisotropy of nanosized cobalt ferrite nanoparticles resulting in high proton relaxivity allows the visualization of target diseased tissue using T2-weighted magnetic resonance Received: October 21, 2015 Revised: January 12, 2016 Published: January 21, 2016 1611

DOI: 10.1021/acs.langmuir.5b03898 Langmuir 2016, 32, 1611−1620


Langmuir imaging.21 The combination of these two classes of nanomaterials would lead to a successful integration of the properties that present advanced features in medical applications. In spite of such beneficial advantages of the magnetic nanoparticle luminescent carbon hybrid nanostructure, there have been very few reports in the literature in this direction. Zhou et al. have reported the fabrication and multifunctional investigation of a new type of iron oxide and fluorescent carbon dot integrated nanoparticles which combine the magnetic properties with fluorescent properties, photothermal conversion ability, and drug carrier function.22,23 Subsequently, a variety of fluorescent carbon and magnetic hybrid nanostructures have been reported by the same research group validating their multifunctional medical applications.24,25 Binaco et al. have also reported coupling of magnetic iron oxide nanoparticles on the wall of carbon nanotubes. Owing to their magnetic property, these materials offer a good contrast enhancing property in MRI.26,27 Gupta et al. have shown a novel and facile approach to synthesize a magnetic nanoparticle (iron oxide)-doped carbogenic nanocomposite (IO−CNC) for magnetic resonance (MR)/fluorescence imaging applications.28 Recently, our research group has published fabrication of luminescent magnetic mesoporous silica nanoparticles decorated with highly fluorescent carbon quantum dots which have been successfully used in multifunctional applications such as a T2 contrast agent in MRI, fluorophore in luminescence imaging, and targeted delivery of the anticancer drug camptothecin.29 Very recently Chen et al. reported the composition−property relationship in a multifunctional hollow magnetic mesoporous carbon nanosystem for pH-responsive magnetic resonance imaging and on demand drug release.30 Although the above-mentioned reports present the fabrication of intelligent inorganic theranostic nanosystems, facile synthesis of monodisperse magnetic carbon spheres with high surface area and ample drug loading capacity, a size below 200 nm, good proton relaxometric properties, monodispersity, and excellent water solubility is a challenge and needs urgent attention. In the present study, we have developed highly luminescent magnetic hybrid nanoparticles by employing superparamagnetic CoFe2O4 as the magnetic component and mesoporous carbon as the luminescent component. The relaxometric property of CoFe2O4 is well illustrated in our previous report on delivery of multiple anticancer drugs using cobalt ferrite encapsulated hollow mesoporous silica nanospheres.31 In this paper mesoporous carbon has been synthesized by carbonization of a resorcinol-formaldehyde polymer (RF) on a CoFe2O4@ mSiO2 nanosphere template. We choose RF as a polymer shell because it provides a large number of aromatic π-electrons after carbonization for π−π stacking with the small aromatic anticancer agent doxorubicin. The hydrophilicity of as synthesized CoFe2O4@mCs was increased by simple thermal activation in oxygen atmosphere at 300 °C without using any harsh chemical for oxidation. The silica template was removed to form mesoporous magnetic carbon nanospheres (CoFe2O4@mC). The DLS and zeta potential measurements over time ensure excellent stability of the nanoparticles in physiological medium. The in vitro cytotoxicity of the drug carrier as well as intracellular uptake and cell apoptosis have been evaluated in HeLa cells.

Aldrich. Tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), resorcinol, formaldehyde solution, and ammonium nitrate were purchased from Spectrochem India. All other reagents and solvents were used without further purification. Millipore water (18.2 M Ω cm) was used throughout the experiment. Synthesis of Monodisperse Hydrophobic CoFe2O4 Nanoparticles. CoFe2O4 nanoparticles were prepared by the solvothermal method. Briefly, Fe(acac)3 (706 mg, 2 mmol), Co(acac)2 (257 mg, 1 mmol), 1,2 dodecanediol (2.023 g, 10 mmol), oleic acid (1.9 mL, 6 mmol) oleylamine (1.97 mL, 6 mmol), and benzyl ether (30 mL) were mixed and stirred for 15 min at room temperature and then the total mixture was transferred to a Teflon lined stainless steel autoclave for 10 h at 190 °C. The as-synthesized mixture was allowed to cool to room temperature and CoFe2O4 nanoparticles were precipitated by adding ethanol. The precipitate was collected by magnetic separator and dried in vacuum for 48 h. Synthesis of Cobalt Ferrite Nanoparticles Embedded in Mesoporous Silica Spheres (CoFe2O4@mSiO2). Twenty milligrams of oleic acid stabilized monodisperse magnetite nanoparticles dispersed in 1.5 mL of chloroform was added to a 20 mL of aqueous solution containing 0.4 g of CTAB (1.1 mmol). After vigorous stirring, a homogeneous oil-in-water microemulsion was obtained. The resulting mixture was heated at 60 °C for 10 min to evaporate the chloroform part, which generated aqueous phase of the dispersed nanoparticles. Then 20 mL of this aqueous solution was diluted with 200 mL of water. After that, 1.5 mL of NH4OH solution and 0.15 mL of tetraethyl orthosilicate were successively added dropwise to the diluted aqueous solution containing the magnetite nanoparticles. The resulting mixture was heated at room temperature for 3 h to ensure complete polymerization of silica. Then the particles were washed three times with ethanol to remove the unreacted species and dispersed in 20 mL of ethanol. CTAB was removed from the matrix by dispersing the as-synthesized nanoparticles in a solution of 160 mg of ammonium nitrate and 60 mL of 95% ethanol and heating the mixture at 60 °C for 15 min. Nanoparticles were then washed with ethanol and separated using the magnetic separator (DynaMag-2, Invitrogen). Synthesis of CoFe2O4@mSiO2@RF and CoFe2O4@mC. For polymer coating, 0.08 g of as obtained CoFe2O4@mSiO2 was homogeneously dispersed in deionized water (10 mL). 0.035 g of resorcinol, 2.82 mL of ethanol, and 0.01 mL of ammonia were stirred at 35 °C for 30 min to form a uniform dispersion. Then, 0.05 mL of formalin was added to the above solution. After 6 h, the total mixture was heated at 100 °C for another 24 h. Then the entire mixture was cooled to room temperature overnight without stirring. CoFe2O4@ mSiO2@RF was collected by centrifugation and then repeatedly washed with water and ethanol. The obtained CoFe2O4@mSiO2@RF was heated at 5 °C min−1 from room temperature to 150 °C and kept at this temperature for 1 h under a nitrogen flow. The temperature was again raised at 5 °C min−1 to 600 °C and maintained for 2 h. CoFe2O4@mSiO2@mC nanoparticles were obtained after the product was cooled to room temperature. After that, mesoporous magnetic carbon nanospheres (CoFe2O4@mC) were formed by etching silicon dioxide with 2 M sodium hydroxide solution. The surface of CoFe 2 O 4 @mC was oxidized to −COOH by oxidation of CoFe2O4@mC in air at 300 °C for 1 h.32 After thermal treatment, particles were dispersed in aqueous medium, which is necessary for bio applications. Calculation of Fluorescence Quantum Yield. The quantum yield (Φ) of the CoFe2O4@mC was calculated using quinine sulfate as a reference. For calculation of quantum yield, five concentrations of each compound were made, all of which had absorbance less than 0.1, at 360 nm. Quinine sulfate (literature Φ = 0.54) was dissolved in 0.1 M H2SO4 (refractive index (η) of 1.33) while the nanoparticles were taken in water (η = 1.33). Their fluorescence spectra were recorded at the same excitation of 360 nm. Then by comparing the integrated photoluminescence intensities (excited at 360 nm) and the absorbance values (at 360 nm) of the sample with the references quinine sulfate, quantum yield was determined. The quantum yield was calculated using the following equation


Chemicals. Fe(acac)3, Co(acac)2, oleic acid, oleylamine, and doxorubicin hydrochloride (DOX.HCl) were obtained from Sigma1612

DOI: 10.1021/acs.langmuir.5b03898 Langmuir 2016, 32, 1611−1620


Langmuir Scheme 1. Schematic Presentation of Synthesis of CoFe2O4@mC Nanospheres

1% antibiotic (penicillin, streptomycin) at 37 °C inside the incubator with 5% CO2. After 24 h of incubation, media were removed and replaced with fresh DMEM without serum containing sample 1, 2, and 3 separately at concentration 5 μg per mL. After incubation for 6 h, media was removed and washed with 1×PBS three times. Thereafter, cells were fixed with 2% paraformaldehyde (PFA) for 15 min at RT followed by washing with 1×PBS and viewed under confocal microscope (Olympus, Japan) with excitation at both 405, 488, and 546 nm. DAPI Staining for Nuclear Morphology. HeLa cells were seeded into 24 well plates and incubated in 400 μL DMEM containing 10% FBS and 1% antibiotic (penicillin, streptomycin) at 37 °C inside the incubator with 5% CO2. After 24 h of incubation, cells were treated with 5 μg per mL of sample in DMEM without serum and further incubated for different time periods (2, 4, and 6 h). Then, cells were fixed with 2% PFA followed by washing thrice with 1×PBS. To observe the nuclear changes inside the cell, 0.5 μg per mL of DAPI was added to each well. Cells were examined under confocal microscope (Olympus, Japan) with excitation at 405 nm. Characterizations. The phase formation and crystallographic state of the material were investigated by an Expert Pro Phillips X-ray diffractometer. Low angle powder X-ray diffraction (XRD) analysis was carried out with a Philips PW3040/00 diffractometer (operating at 40 kV and 30 mA), using a Cu Kα radiation (λ = 1.54°). The Raman spectrum of as-prepared samples was recorded at ambient temperature (Ranishaw Viarelex). The morphology and microstructure were analyzed by scanning electron microscope (Hitachi COM-S-4200) and high resolution transmission electron microscopy (JEOL 3010, Japan) operated at 300 kV. The magnetic properties of CoFe2O4 and CoFe2O4@mCN nanocomposites were determined using a SQUIDVSM instrument (Evercool SQUID VSM DC Magnetometer) at 25.0 ± 0.5 °C. The fluorescence emission spectra were obtained on a Horiba Fluoromax 4 spectrophotometer at excitation energies ranging from 320 to 500 nm, and 634 to 908 nm to verify up-conversion fluorescence. Nitrogen adsorption/desorption isotherms were measured at a liquid nitrogen temperature (77 K) using a Quantachrome surface area analyzer. The specific surface areas and total pore volume were calculated by the Brunauer−Emmett−Teller (BET) and BJH methods, respectively. Mean hydrodynamic sizes were measured by laser light scattering using a particle size analyzer (Nano ZS 90, Malvern). The presence of surface functional group was investigated through FTIR spectra measured by a Thermo Nicolet Nexux FTIR model 870 spectrometer with the KBr pellet technique ranging from 400 to 4000 cm−1. Surface composition of the CoFe2O4@mCN was investigated by XPS using Al Kα excitation source in ESCA-2000 Multilab apparatus (VG microtech). Images of particles were captured

Φx = ΦST(m x /mST)(η2 x /η2 ST) where Φ is the quantum yield, m is slope, η is the refractive index of the solvent, ST is the standard, and X is the sample. Relaxometric Measurements. The relaxation time (T2) and transverse relaxivity (r2) of the nanoparticle were measured with varying iron concentration (0.015−0.075 mM) using a clinical MRI scanner (MAGNETOM Symphony, SIEMENS) at a magnetic field of 1.5 T. T2-weighted images were obtained with a spin echo multisection pulse sequence having fixed repetition time (TR) of 4000 ms with various echo times (TE) ranging from 105 to 291 (105, 116, 128, 139, 151, 163, 174, 186, 198, 291). The spatial resolution parameters were as follows: field of view (FOV) = 300 × 300 mm2, matrix = 358 × 358, slice thickness = 4.0 mm. The MRI signal intensity (SI) was measured using in-built software. T2 values were obtained by plotting the SI of each sample over a range of TE values. T2 relaxation times were then calculated by fitting a first-order exponential decay curve to the plot. The fitting equation can be expressed as

SI = S0e − TE/T2 + B where SI is the signal intensity, TE is the echo time, S0 is the amplitude, and B is the offset. The relaxivity value r2 is determined from the slope of the linear plots of relaxation rate R2 (1/T2, s−1) against Fe concentrations (mM).

R 2 = R 20 + r2[Fe] Loading and Release of DOX. CoFe2O4@mCNs (20 mg) and DOX (10 mg) were added to 15 mL of water. The mixture was stirred at room temperature for 48 h. The drug loading capacity was measured by UV/vis spectrophotometry with different time intervals. To verify drug release, CoFe2O4@mCNs were dispersed in buffers with different pH values and shaken at 100 rpm at 37 °C. The supernatant was collected by magnetic separation for quantitative analysis by UV/vis spectrophotometry. In Vitro Cytotoxicity. HeLa cells (3 × 103 cells) were seeded into 96-well plates and incubated in 200 μL DMEM containing 10% FBS for 24 h (37 °C, 5% CO2). After incubation, cells were treated with different concentrations of sample (0.5, 1, 5, 10, 20 μg per mL) in fresh DMEM without serum for another 24 h. Following treatment, cells were washed with 1×PBS, and 100 μL of MTT solution (1 mg per mL in PBS buffer) was added to each well. After 3 h incubation, MTT solution was removed and replaced with 200 μL of DMSO. Absorbance was read at 570 nm using microplate reader (Thermo Scientific Multiskan Spectrum, USA). Experiment was performed at least three times independently. Intracellular Uptake. HeLa cells (3 × 104) were seeded into 24 well plates and incubated in 400 μL DMEM containing 10% FBS and 1613

DOI: 10.1021/acs.langmuir.5b03898 Langmuir 2016, 32, 1611−1620


Langmuir under Olympus FV-1000 confocal microscope with laser excitations of 405, 488, and 561 nm.

of organic polymer. The absence of amorphous range and appearance of new plane at (110)* for graphitic carbon (JCPDS no 82−0505) indicates that the silicon dioxide template has been completely removed after etching. The low-angle X-ray diffraction pattern of mC nanospheres exhibited two resolved diffraction peaks at 2θ values of 0.78° and 1.3°, respectively (Figure 2), which can be indexed as the typical (110) and (210) reflections of a highly ordered bodycentered cubic Im3m mesostructure.

RESULTS AND DISCUSSION Synthesis. Scheme 1 illustrates the synthetic strategy for CoFe2 O4 @mC. The mesoporous silica coated CoFe2 O4 nanoparticles were synthesized using CTAB as a surfactant where it plays dual role such as (1) for the transfer of hydrophobic CoFe2O4 to aqueous phase and (2) organic soft template for the formation of mesoporous silica spheres. Then, after mesoporous carbon was synthesized following the nanocasting technique using CoFe2O4@mSiO2 as a hard template.33 The pores of the mesoporous silica can be filled up with resorcinol and formaldehyde by capillary force, and in situ polymerization takes place in the presence of ammonia which serves as a precursor for porous carbon. The hightemperature treatment at 600 °C under the N2 atmosphere only serves to carbonize the polymer into carbon. Removal of silica on washing with 5 M NaOH gives highly porous magnetic CoFe2O4@mC nanospheres which are sparingly dispersible in water. These hydrophobic CoFe2O4@mC nanospheres were purified by thermal treatment in air at 300 °C in order to remove disordered carbon and also to oxidize their surface with −COOH groups producing highly water-soluble CoFe2O4@ mC nanopsheres. Investigation of Phase by XRD and Raman Spectroscopy. The phase purity of CoFe2O4, CoFe2O4@mSiO2, CoFe2O4@mSiO2@RF, and CoFe2O4@mC particles were investigated through powder X-ray diffraction analysis (Figure 1). In the case of free cobalt ferrite nanoparticles five diffraction

Figure 2. Low angle X-ray diffraction pattern of CoFe2O4@mC nanoparticles.

The bonding, order, and crystallinity of the materials are studied by Raman spectroscopy (Figure 3) which reveals

Figure 3. Raman spectrum of CoFe2O4@mC nanoparticles.

disordered graphitic materials, as suggested by the two Raman modes. The peak at 1594 cm−1 (G-band) corresponds to an E2g mode of hexagonal graphite and is related to the vibration of sp2 hybridized carbon atoms in a graphite layer. This means that the magnetic mesoporous carbon spheres are composed of graphitic carbon, related to sp2 hybridized carbon atom in a graphite layer, which is consistent with the XRD results. The Dband at about 1360 cm−1 is associated with the vibration of carbon atoms with dangling bonds in the plane termination of disordered graphite. Due to this graphite structure with defects aromatic molecules can strongly interact with the basal plane of graphite via π−π supramolecular stacking. The peak at 2702 cm−1 corresponds to the 2D band of the Raman spectrum, which shows a signature of graphitic sp2 materials. The Raman bands at 2950 cm−1 correspond to the combination mode of the D-band and G-band. SEM and TEM. The morphology of the obtained nanospheres CoFe 2 O 4 @mSiO 2 , CoFe 2 O 4 @mSiO 2 @RF, and CoFe2O4@mC was investigated by SEM and TEM. Figure S1

Figure 1. XRD patterns of (a) CoFe2O4, (b) CoFe2O4@mSiO2, (c) CoFe2O4@mSiO2@RF, and (d) CoFe2O4@mC.

peaks were indexed at 30°, 35.40°, 43.16°, 57.01°, and 62.63°, which correspond to reflection of planes of (220), (311), (400), (511), and (440) indicating the inverse spinel structure of CoFe2O4 (JCPDS no 22−1086). The crystallite size was also calculated by using Debye Scherer equation and it was found to be 9.2 nm. CoFe2O4@mSiO2 did not show any characteristics peaks within 2θ = 15−35° indicating amorphous silicon dioxide phase coated on the surface of magnetic nanoparticles. After polymer modification, a broad peak in the range 2θ = 12−25° originated from the amorphous phase of RF component which overlaps with the amorphous mesoporous silica range. After carbonization the peaks corresponding to cobalt ferrite nanoparticles were intensified and showed the decomposition 1614

DOI: 10.1021/acs.langmuir.5b03898 Langmuir 2016, 32, 1611−1620



Figure 4. (a) FESEM images ofCoFe2O4@mSiO2@RF, (b) CoFe2O4@mC, (c) TEM image of CoFe2O4@mC, (d) HRTEM image of CoFe2O4@ mC showing lattice fringes of CoFe2O4 [111] and carbon particles [002].

with acid group CoFe2O4@mC-COOH and CoFe2O4@mC@ DOX was found to be 71 and 100 nm with narrow size distributions. The stability of drug adsorbed CoFe2O4@mC@ DOX was examined by measuring hydrodynamic size with time using dynamic light scattering (Figure S2b) in PBS, which shows that there is slight increase in hydrodynamic size after 7 days in aqueous dispersion. This observation implies that such stable drug adsorbed particles can be circulated in bloodstream for a long period minimizing the rapid clearance of particles by macrophages. In the case of CoFe2O4@mC, the surface charge varied from 40 mV at lower pH (2) to −11.2 mV at pH (11.2). After thermal activation the surface charge increases significantly in the negative direction with an increase in pH, indicating the presence of −COOH groups on the surface (Figure 5). Nitrogen adsorption−desorption curves of CoFe2O4@mSiO2 and CoFe2O4@mC are presented in Figure 6 (data in Table 1). Both curves present type IV isotherm which is typical of mesoporous materials. The BET surface area of the as-synthesized CoFe2O4@mSiO2 and CoFe2O4@mC was found to be 925 m2/g and 542 m2/g, respectively. In the case of CoFe2O4@mSiO2 nanoparticles, the mesopores were formed due to the removal of soft template CTAB and aggregation of spherical particles. After polymer coating, the surface area of CoFe2O4@mSiO2 nanoparticles decreases to 236 m2/g due to blockage of pores. In the case of CoFe2O4@ mC, the surface area appreciably increases due to the removal of hard template silicon dioxide in the etching process, which indicates the opening of pores. BJH shows a narrow pore size distribution. The size of the pores in case of CoFe2O4@mC is 10 nm, which is greater than that of mesoporous silica template (8 nm). Such an increase in pore size and disorder is obvious in

shows a representative SEM image of CoFe2O4@mSiO2 which indicates that particles are spherical and monodispersed and have diameter in the range of 60−65 nm. Figure 4a shows the FESEM image of CoFe2O4@mSiO2 recovered after polymerization of resorcinol and formaldehyde at 100 °C for 24 h. It is clear from the image that the polymerization has taken place inside the pore channels without forming an outer layer over mSiO2. The high-temperature treatment at 600 °C under the N2 atmosphere only serves to carbonize the polymer into carbon. The FESEM image of CoFe2O4@mC shows that the magnetic carbon nanoparticles which are spherical in nature and with a mean average size 120 nm (Figure 4b). The morphology of the particles does not undergo a transformation after carbonization at high temperature. The lowest size of spherical magnetic carbon particle as reported by other researchers is 300 nm.30 On the contrary, we have synthesized magnetic carbon samples with diameter less than 150 nm which is perfect for drug delivery applications.34 From the TEM image (Figure 4c), the size of CoFe2O4@mC is found to be 120 nm and the particles are porous in nature. The nanospheres are composed of a dark-contrast inner layer and a light-contrast outer layer, which are attributed to the CoFe2O4 and carbon components, respectively. In the HRTEM image (Figure 4d), the lattice fringes of carbon particles and CoFe2O4 were observed. Measurement of hydrodynamic size after each step of modification of magnetic nanocomposites is presented in Figure S2a. CoFe2O4@mSiO2 shows the presence of stable nonaggregated particles with hydrodynamic size 52 nm, PDI < 0.2. After polymer modification the particle size was found to be 70 nm, PDI < 0.3. The size of final product, magnetic mesoporous carbon nanoparticles after surface modification 1615

DOI: 10.1021/acs.langmuir.5b03898 Langmuir 2016, 32, 1611−1620



deconvoluted into four single peaks which correspond to C−C (284.98 eV), C−O (285.97), CC (284 eV), and CO (289.2 eV) functional groups (Figure 7a). The O 1s region

Figure 5. Comparison of surface charge of CoFe2O4@mC particles (a) after and (b) before thermal activation.

Figure 7. (a) High resolution scans of C 1s and (b) O 1s.

shows binding energies corresponding to four types of oxygens such as CO (530.4 eV), C−C−O (531.4), O−H (532 eV), CO/COOH (533.3 eV) (Figure 7b). The peak appearing at 284 eV indicates the presence of aromatic unsaturated carbon containing π electrons, which are responsible for the adsorption of anticancer drug doxorubicin by π−π stacking. The peaks at 533.3 eV of O 1s and 289.2 eV of C 1s validate the generation of carboxylic group on the surface of magnetic mesoporous carbon nanoparticles after thermal activation in oxygen atmosphere. Also, high resolution scans of Co 2p3/2 and Co 2p1/2 electrons show binding energy at 779 and 795.3 eV associated with corresponding shakeups at 785 and 801 eV. In addition to this, the asymmetric nature of the peak is ascribed to Co2+ present in octahedral/tetrahedral sites (Figure S3b). Fe 2p region shows two peaks at 713.5 and 727.6 eV which is consistent with Fe 2p binding energy for CoFe2O4 nanoparticles (Figure S3c). Fluorescence and Magnetic Properties. The aqueous solution of − COOH functionalized CoFe2O4@mC is yellow and transparent in day light but changes to intense green under UV excitation. The absorption in the UV region is attributed to the presence of aromatic π orbitals. At a fixed excitation of λex = 360 nm, CoFe2O4@mC shows an emission peak at 500 nm (Figure 8). It has been reported in the literature that if the fluorescent material comes in contact with magnetic iron oxide, most of the fluorescence is quenched because of nonradiative energy transfer.35 However, in our case, no significant fluorescence quenching was observed possibly owing to the synthesis of the material via nanocasting method which produces carbon particles as a negative replica of a silica

Figure 6. (a) N2 adsorption−desorption isotherms and (b) pore size distribution of CoFe2O4@mSiO2 and CoFe2O4@mC particles.

Table 1. N2 Adsorption Desorption Data of CoFe2O4@ mSiO2, CoFe2O4@mSiO2@RF, and CoFe2O4@mC Nanoparticles sample

BET surface area (m2/g)

pore size (nm)

CoFe2O4@mSiO2 CoFe2O4@mSiO2@RF CoFe2O4@mC

925 236 542

8 4.5 10

the case of mesoporous carbons prepared by the nanocasting method.35 However, the unique structures having large surface area and pore size are advantageous for transportation and encapsulation of guest molecules. X-ray photoelectron spectra were further used to confirm the surface composition of CoFe2O4@mC. The peak in the survey spectrum at 280−290, 529−538, and 705−730 eV corresponds to the binding energy of C 1s, O 1s, Co 2p, and Fe 2p electrons (Figure S3a). The high resolution scan of C 1s can be 1616

DOI: 10.1021/acs.langmuir.5b03898 Langmuir 2016, 32, 1611−1620



Figure 9. (a) Fluorescence intensity−UV absorbance of CoFe2O4@ mC particles. (b) Fluorescence images of CoFe2O4@mC particles at λ = 405, 488, and 561 nm. Figure 8. (a) UV absorption (black) and PL emission spectra(λex = 360 nm) (red) of CoFe2O4@mC particles, Inset: optical images under daylight (left) and UV light (right). (b) Excitation dependent emission spectra of CoFe2O4@mC.

no reduction in luminescent intensity even after prolonged exposure. All these requisites make the developed CoFe2O4@ mC nanospheres a suitable optical imaging agent. The magnetization curves at room temperature for both samples CoFe2O4@mSiO2@RF and CoFe2O4@mC do not show any hysteresis and hence are superparamagnetic at room temperature (Figure S7). The saturation magnetization values were found to be 20 and 51 e.m.u./g at high magnetic field (2T). The low Ms value of CoFe2O4@mSiO2@RF is attributed to the presence of thick inert material silicon dioxide and a polymer which is coated on the surface of cobalt ferrite nanoparticles. The substantial increase in saturation magnetization of CoFe2O4@mC has been attained after removal of nonmagnetic SiO2 in the etching process. High saturation magnetization of CoFe2O4@mC along with superparamagnetism is highly desirable for application in magnetically guidable drug delivery and as a contrast-enhancing agent in MRI. Hence, CoFe2O4@mC would be a suitable material for the above applications. Relaxometric Measurements. As an initial investigation toward potential imaging functionality during chemotherapy, the visibility of CoFe2O4@mC@DOX was tested in water, PBS, and minimal essential medium (MEM) by MRI scanner (1.5 T). As shown in Figure 10 with an increase in concentration of particles in the phantom solution, the signal intensity decreased, indicating that the magnetic mesoporous carbon spheres have generated magnetic resonance contrast on the transverse (T2) proton relaxation time weighted sequences due to the dipolar interaction of magnetic moments between the nanoparticles and proton in the water.22 The T2 relaxation time was inversely proportional to the particle concentration, as expected. The transverse relaxivity (r2) of the drug DOX adsorbed particles is found to be 380 mM−1 S−1, resulting in better negative contrast effect than commercially available dextran coated ferrite nanoparticles such as feridex (120 mM−1 S−1), combidex (65 mM−1 S−1), and CLIO-tat (62 mM−1 S−1).

template.36 The distance from magnetic particle to carbon is not appropriate for a nonradiative energy transfer. Furthermore, the unique property of excitation dependent photoluminescence (PL) was observed in the case of CoFe2O4@mC, consistent with carbon quantum dots already reported from our laboratory37 and also other researchers.38 Luminescence of the as-prepared magnetic mesoporous carbon nanoparticles was studied at different excitation wavelengths, ranging from 300 to 500 nm (Figure 8b). In addition to conventional downconversion, these carbon dots show conversion luminescence (600 to 900 nm). The mechanisms of luminescence and upconversion luminescence have been illustrated in Figure S5. It is notable that upconversion fluorescence in NIR region opens the possibility for application in NIR bioimaging and phototherapy. Similar to carbon dots, the excitation dependent PL in magnetic mesoporous carbon may be ascribed to the presence of surface states.39 As evident from FTIR (Figure S4) and XPS there are several functional groups like CO, −COOH, and so forth present on the surface which creates a series of emissive traps between π and π* orbitals of mesoporous carbon. PL intensity of the synthesized sample remains unchanged over pH 4.5 to 8 (Figure S6a). The measurement of fluorescence intensity with time shows that mesoporous carbon does not show photobleaching for hours (Figure S6b). The PL quantum yield was found to be 9.5% which is appreciably high as compared to other magnetic carbon samples (Figure 9a). Laser confocal microscopy images show that CoFe2O4@mC are excellent imaging agents. The excitation dependent luminescence gives rise to several visible advantages in imaging. When the laser excitation was changed to 405, 488, and 561 nm, blue, green, and red colors were observed, respectively (Figure 9b). In addition to this there was 1617

DOI: 10.1021/acs.langmuir.5b03898 Langmuir 2016, 32, 1611−1620



respectively. Such a pH-responsive drug release behavior was based on the unique supramolecular π−π stacking between the carbonaceous framework and aromatic DOX molecules, which could be interfered with and broken in a mild acidic environment.40 Importantly, such a special noncovalent π−π stacking could also be broken by the ionized state of doxorubicin. So, such DOX-loaded fluorescent magnetic CoFe2O4@mC can release drug in an intelligent on-demand manner. Calculation of Drug Loading Content and Encapsulation Efficiency. Drug loading content (%) Weight of the drug in nanoparticles = × 100 Weight of the nanoparticles 6.023 = × 100 20 = 30.12%

Encapsulation efficiency (%) Weight of the drug in nanoparticles = × 100 Weight of the feeding drug 6.023 = × 100 10

Figure 10. (a) 1/T2 with metal ion concentration. (b) MR phantom images with CoFe2O4@mC particles in different medium.

DOX Loading and pH Dependent Release. To examine the doxorubicin (DOX) loading and release using CoFe2O4@ mC carrier, DOX was loaded into this matrix and the DOX loaded particles are presented by CoFe2O4@mC@DOX. The loading efficiency of DOX was measured to be as high as 86.1% due to the high BET surface area and pore volume of the prepared CoFe2O4@mC particles. Before drug loading to the matrix, free DOX exhibited high fluorescence intensity, while the fluorescence of extracted supernatant decreased significantly after DOX was bound to CoFe2O4@mC (Figure S8). The carbonaceous composition interacts with aromatic DOX through supramolecular π−π stacking, thus decreasing the concentration of DOX in the supernatant. In vitro drug release experiments were carried out within buffer solutions with different pH (7.4, 6.0, and 4.6). Figure 11 shows the DOX

= 60%

The drug loading content and encapsulation efficiency of DOX were found to be 30.12% and 60%, respectively. Intracellular Uptake and Imaging. The satisfactory biocompatibility of CoFe2O4@mC nanospheres along with excellent luminescent properties makes it a reliable bioimaging agent. Figure 12 shows the fluorescence images of HeLa cells incubated with DOX loaded CoFe2O4@mC particles revealing that the CoFe2O4@mC particles can easily be internalized within and label the HeLa cells. Further, the mC nanospheres are accumulated and distributed uniformly in the cytosol, which indicates that the CoFe2O4@mC can be well endocytosed by

Figure 11. Cumulative DOX release from CoFe2O4@mC@DOX with time at different pH.

release profile of the CoFe2O4@mC@DOX with variable pH in PBS at 37 °C, which shows initial burst release up to 40% in the first 10 h which slowly increases up 70% with 100 h at pH 4. The acidic release medium was used to mimic the essential tumor acidic microenvironment. The amount of DOX released from the matrix over 10 h at neutral pH condition was only 5.5%. However, 10% and 40% release percentages could be achieved within 10 h in pH 5.5 and pH 4 buffer solutions,

Figure 12. HeLa cells incubated with CoFe2O4@mC@DOX particles. Cell images at (a) bright field and excitation (b) 360 nm, (c) 380 nm, and (d) 480 nm. 1618

DOI: 10.1021/acs.langmuir.5b03898 Langmuir 2016, 32, 1611−1620


Langmuir living HeLa cells, favoring the fluorescence imaging of the whole cells and drug delivery into the cytoplasm. To further investigate the multicolor emission of our particle under varied excitations, we varied the excitation wavelengths. For fluorescence excitation set at 360 nm, intense blue color was observed. When excitation varies to 380 and 480 nm, green and light blue color were observed (Figure 12), respectively. In addition to this, there was no significant loss in fluorescence intensity even after excitation for a prolonged time. All these indicate that our designed CoFe2O4@mC nanospheres can be used as a potential substitute to organic dyes in fluorescence imaging. Cytotoxicity. To confirm whether the released DOX was pharmacologically active, we investigated the cytotoxic effect of the CoFe2O4@mC, DOX, and CoFe2O4@mC@DOX in HeLa cells. From Figure 13, it is observed that CoFe2O4@mC did not

and saturation magnetization of CoFe2O4, the hybrid sphere shows high transverse relaxivity as 380 mM−1 S−1. Due to surface modification with carboxylic acid groups the nanoparticles are highly stable in aqueous buffer. The extended conjugated double bonds present in the carbon matrix promote high adsorption of doxorubicin by supramolecular π−π stacking which is sensitive to low pH in the tumor microenvironment.The dual optical and magnetic properties of the mesoporous spheres may be utilized in advanced imaging technologies to track the curative responses. Such a uniquely designed intelligent nanosystem can be further explored as a theranostic agent for administration of other hydrophobic aromatic drugs having complicated structure.


S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b03898. SEM, particle size distribution, XPS survey, FTIR, photoluminesence, DAPI fluoresence, and calculation of drug loading content and encapsulation efficiency (PDF)


Corresponding Author

*E-mail: [email protected]. Fax: +91-661-2462651. Tel: +91-661-2462661. Author Contributions

All authors have given approval to the final version of the manuscript.

Figure 13. Cell viability by MTT assay.


The authors declare no competing financial interest.

induce any significant change in the proliferation with a concentration up to 20 μg/mL with respect to the control, suggesting the absence of toxicity of the CoFe2O4@mC. The DOX loaded particles (CoFe2O4@mC@DOX) exhibit significantly higher cytotoxicity than free DOX, especially at relatively low DOX concentrations. This may be attributed to the easier uptake of the CoFe2O4@mC@DOX via endocytosis by HeLa cells as compared to the passive diffusion of free drug molecules which is believed to be highly important in minimizing the toxic side effects of free drugs to normal tissues/cells. The IC50 values of plain DOX and CoFe2O4@mC@DOX were found to be 10 μg/mL and 5 μg/mL. Further, DOX is known to induce apoptosis in cancer cells. Hence we intended to study whether the pH-dependent release of DOX in tumor cell would result in a similar cell fate. Hence, we investigated the nuclear fragmentation which is a hallmark of apoptosis with the cells treated with MTX loaded particles. With an increasing dose of the CoFe2O4@mC@DOX nanoparticles, an increase in the formation of fragmented nuclei containing condensed nuclear material was observed in confocal microscopy after nuclear staining with DAPI. In addition to this, treating the HeLa cells with 2 μg of CoFe2O4@mC@DOX particles also increases apoptosis in a time dependent manner (Figures S9, S10).

ACKNOWLEDGMENTS Authors are thankful to Dr. Meghray Majhi, Department of Radiodiagnostics, Ispat General Hospital, Rourkela, India for MRI experiments. This work is financially supported by BRNS, DAE, Govt. of India (ref: 2013/37C/8/BRNS/393) and DST Nanomission, Govt. of India (ref: SR/NM/NS-27/2012).


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SUMMARY In summary, simple and inexpensive magnetic mesoporous carbon-based multimodal theranostic nanoagent bestowed with magnetic targeting, magnetic resonance contrast behavior, both downconversion and upconversion fluorescence imaging, high loading, and controlled release of the anticancer drug doxorubicin have been developed. Due to larger anisotropy 1619

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DOI: 10.1021/acs.langmuir.5b03898 Langmuir 2016, 32, 1611−1620