Highly Hydrophilic Luminescent Magnetic Mesoporous Carbon

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Highly hydrophilic multifunctional mesoporous carbon nanospheres for control release of anticancer drug and MRI/Optical imaging Smruti R Rout, Rahul K Das, Santoshi Nayak, Sudip K Ghosh, and Sasmita Mohapatra Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b03898 • Publication Date (Web): 21 Jan 2016 Downloaded from http://pubs.acs.org on January 27, 2016

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Highly

hydrophilic

luminescent

magnetic

mesoporous carbon nanospheres for control release of anticancer drug and multimodal imaging Smruti R. Rout,† Rahul K. Das,† Santoshi Nayak,‡Sudip K. Ghosh,,‡ Sasmita Mohapatra*† †

Department of Chemistry, National Institute of Technology, Rourkela, India-769008, E-mail:

[email protected], [email protected] ‡

Department of Biotechnology, Indian Institute of Technology, Kharagpur, India-721302

KEYWORDS: mesoporous carbon, nanotheranostics, T2 contrast agent, anticancer drug, optical imaging, upconversion

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 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 resorcinol-formaldehyde precursor. The mesoporous matrix shows control release of 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-1S-1. The multicolouremission and upconversion luminescence property of our sample are advantageous in

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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 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 otherhand, nanosystems can carry small payloads of anti-cancer 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], upconversion particles [UCNPs], MRI contrast agents, CT contrast agents, etc.) and therapeuticagents (anticancer drugs, DNA, small interfering RNA [siRNA],proteins, hyperthermia-inducing nanoparticles, ROS-generating agents, etc.).10-12


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Mesoporous silica based nanoparticles have been explored as the 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, temperature etc.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 high proton relaxivity allows the visualization of target diseased tissue using T2weighted magnetic resonance 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 magnetic nanoparticle luminescent carbon hybrid nanostructure, there have been very few literatures in this direction. Zhou et al have reported the fabrication and multifunctional investigation of a new type 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 nanotube. Owing to their magnetic property these materials offer good contrast enhancing property in MRI.26,27 Gupta et al have shown novel and facile approach to synthesize a magnetic


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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 for targeted delivery of anticancer drug camptothecin.29 Very recently Chen et al have reported the composition property relationship in 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. 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 resorcinol-formaldehyde polymer (RF) on a CoFe2O4@mSiO2 nanosphere template. We choose RF as a polymer shell because it provides large number of aromatic πelectrons after carbonization for π-π stacking with small aromatic anticancer agent doxorubicin. The hydrophilicity of as synthesized CoFe2O4@mCs was increased by simple thermal activation in oxygen atmosphere at 300ºC without using any harsh chemical for oxidation. The silica template was removed to form mesoporous magnetic carbon nanospheres (CoFe2O4@mC). The


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DLS and zeta potential measurements overtime 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.

EXPERIMENTAL SECTION Chemicals. Fe(acac)3, Co(acac)2, oleic acid, oleylamine, Doxorubicin hydrochloride (DOX.HCl) were obtained from Sigma Aldrich. Tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), resorcinol, formaldehyde solution, 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 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 (30ml) were mixed and stirred for 15 minutes at room temperature and then the total mixture was transferred to a teflon lined stainless steel autoclave for 10h at 190ºC. Then 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 48h.


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Synthesis of cobalt ferrite nanoparticles embedded in mesoporous silica spheres (CoFe2O4@mSiO2). 20 mg 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 micro emulsion was obtained. The resulting mixture was heated at 60ºC 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, 0.15 mL of tetraethyl orthosilicate, were successively added drop wise to the diluted aqueous solution containing the magnetite nanoparticles. The resulting mixture was heated at room temperature for 3 h to ensue 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 100ºC 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


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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 at this temperature 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 2M sodium hydroxide solution. The surface of CoFe2O4@mC was oxidized to -COOH by oxidation of CoFe2O4@mC in air at 300ºC 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 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 Φx = ΦST (mx / mST) (η2x /η2ST ) 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.


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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: = − +

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).

= + 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


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dispersed in buffers with different pH values and shaken at 100 rpm at 37 ºC. 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 concentration of sample (0.5, 1, 5, 10, 20µg per ml) in fresh DMEM without serum for another 24 h. Following treatment, cells were washed with 1XPBS 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 1% antibiotic (penicillin, streptomycin) at 37°C inside the incubator with 5% CO2. After 24h of incubation, media were removed and replaced with fresh DMEM without serum containing sample 1, 2 and 3 separately at concentration 5µg per ml. After incubation for 6h, media was removed and washed with 1XPBS three times. Thereafter, cells were fixed with 2% Paraformaldehyde (PFA) for 15 min at RT followed by washing with 1XPBS and viewed under confocal microscope (Olympus, Japan) with excitation at both 405,488 and 546 nm. DAPI staining for nuclear morphology.


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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 24h of incubation, cells were treated with 5µg per ml of sample in DMEM without serum and further incubated for different time period (2h, 4h, 6h). Then cells were fixed with 2% PFA followed by washing thrice with 1X 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˚A). 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 SQUID-VSM 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 analyser. 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


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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 AlKα excitation source in ESCA-2000 Multilab apparatus (VG microtech). Images of particles were captured under Olympus FV-1000 confocal microscope with laser excitations of 405, 488 and 561 nm. RESULTS AND DISCUSSION Synthesis Scheme1 illustrates the synthetic strategy for CoFe2O4@mC. The mesoporous silica coated CoFe2O4 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, (2) organic soft template for the formation of mesoporous silica spheres. Then after mesoporous carbon was synthesized following 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 presence of ammonia which serves as a precursor for porous carbon. The high-temperature treatment at 600ºC under the N2 atmosphere only serves to carbonise the polymer into carbon. Removal of silica on washing with 5M 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 300ºC in order to remove disordered carbon and also to oxidize their surface with –COOH groups producing highly water soluble CoFe2O4@mC nanopsheres.


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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 (Fig.1). In case of free cobalt ferrite nanoparticles five diffraction 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 = 15º-35º indicating amorphous silicon dioxide phase coated on the surface of magnetic nanoparticles. After polymer modification, broad peak in the range 2θ = 12º-25º originated from 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 of organic polymer. The absence of amorphous range and appearance of new plane at (110)* for graphitic carbon (JCPDS no 82-0505)indicates that 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 0.78º and 1.3º, respectively (Fig. 2), which can be indexed as the typical (110) and (210) reflections of a highly ordered body-centered cubic Im3m mesostructure. The bonding, order, and crystallinity of the materials are studied by Raman spectroscopy (Fig. 3) which reveals 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, and is related to sp2 hybridized


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carbon atom in a graphite layer, which is consistent with the XRD results. The D-band 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 signature of graphitic sp2 materials. The Raman bands at 2950 cm-1 corresponds to the combination mode of the D-band and G-band. SEM and TEM. The morphology of the obtained nanospheres CoFe2O4@mSiO2, CoFe2O4@mSiO2@RF and CoFe2O4@mC was investigated by SEM and TEM. Figure S1 shows representative SEM image of CoFe2O4@mSiO2 which indicate that particles spherical and monodispersed and having 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 100ºC for 24h. 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 600ºC under the N2 atmosphere only serves to carbonise 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 (Fig. 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 (Fig. 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


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contrast outer layer, which are attributed to the CoFe2O4 and carbon components, respectively. In the HRTEM image (Fig. 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 non-aggregated particles with hydrodynamic size 52 nm, PDI

Highly Hydrophilic Luminescent Magnetic Mesoporous Carbon

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