Microwave Synthesis of Chitosan Capped Silver ... - ACS Publications

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Microwave Synthesis of Chitosan Capped Silver−Dysprosium Bimetallic Nanoparticles: A Potential Nanotheranosis Device Sandeep K. Mishra and S. Kannan* Centre for Nanoscience and Technology, Pondicherry University, Puducherry-605 014, India S Supporting Information *

ABSTRACT: Accurate imaging of the structural and functional state of biological targets is a critical task. To amend paucities associated with individual imaging, there is high interest to develop a multifunctional theranostic devices for cancer diagnosis and therapy. Herein, chitosan coated silver/dysprosium bimetallic nanoparticles (BNPs) were synthesized through a green chemistry route and characterization results inferred that the BNPs are crystalline, spherical, and of size ∼10 nm. Highangle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and X-ray photoelectron spectroscopy (XPS) confirm the reduced metallic states of Ag and Dy in nanoparticles. These BNPs demonstrate high emission in a second near-infrared (NIR-II, 1000−1400 nm) biological window on excitation at 808 nm. Moreover, magnetization and magnetic resonance imaging (MRI) studies perceive the inherent paramagnetic features of Dy component that displays dark T2 contrast and high relaxivity. Due to high X-ray attenuation effect, BNPs exhibit better Hounsfield unit (HU) value than the reported contrast agents. BNPs unveil good biocompatibility and also express sturdy therapeutic effect in HeLa cells when tethered with doxorubicin.

1. INTRODUCTION In biomedicine, combined applications in multimodal imaging, targeted therapy, and drug delivery makes their uses more attractive in comparison to the molecular drugs and contrast agents.1 Modern clinical techniques like magnetic resonance imaging (MRI), ultrasonography, positron emission tomography, contrast enhanced computed tomography (CT), and optical imaging are employed to gather structural and functional information on a specific site of the body part. However, each technique has its own constraint in sensitivity, resolution and penetration depth that limits their application in early diagnosis. The integration of these imaging facilities is essential for early and accurate diagnosis, which is not possible by a single technique.2,3 In this context, development of multimodal imaging contrast agents would be a promising approach to overcome these limitations. Among all imaging techniques, MRI is exclusively used for soft tissue resolution and CT prevalently for hard tissue/anatomical imaging. Near infrared (NIR) imaging has become increasingly popular due to low tissue autofluorescence and absorption in the NIR region.4 NIR imaging possess the merit of high sensitivity and demerit of poor resolution. In contrast, MRI and CT scanning offer excellent resolution and tissue penetration, however, with poor sensitivity.5,6 In this context, there is a necessity of new multimodal imaging agents that paramount in combining NIR, MRI and CT contrast abilities. The best possible approach could be the development of a single device in the form of © XXXX American Chemical Society

nanoparticles (NPs) that possess the ability to fetch all the mandatory characteristics in a single entity. In recent times, the application of silver nanoparticles (AgNPs) has increased exponentially in biomedical research due to its flexible physicochemical properties and significantly low cytotoxicity after polymer capping.7,8 Biocompatibility and luminescence of polymer capped AgNPs are found valuable in theranostic applications of cancer treatment.9,10 Despite low atomic number of silver than iodine, AgNPs with size < 23.2 nm exhibit approximately similar X-ray attenuation intensity to omnipaque with iodine concentration similar to silver.11 Nanoparticles (NPs) as MRI contrast agents are exclusively pursued due to their favorable features of higher water proton relaxivities, reluctance in diffusion across blood capillaries and longer circulation time than molecular chelates. Till date, many NPs as MRI contrast agents, based on Fe2O3/Fe3O4,12 MnO,13 Gd2O3,14 and Dy2O315 are investigated. Fe2O3/Fe3O4 suffers from fast renal clearance that depends on its size distribution and Gd2O3 is well-known for its high cellular toxicity. Many works are reported in the development of NPs based MRI/CT contrast agents that focus on functionalization of NPs with lanthanide chelates, especially Gd.16 However, this approach has drawbacks of limited functionalization, low payload of Received: September 19, 2016 Revised: December 2, 2016

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DOI: 10.1021/acs.langmuir.6b03438 Langmuir XXXX, XXX, XXX−XXX

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weight (MW). The average MW of gamma irradiated CS was determined through viscosity measurements. A capillary viscometer (Ubbelohde Capillary Viscometer type 531/10 I) was used to determine the viscosity (η) of CS solutions at 25 °C. The initial CS solution of 10 mg/mL was prepared in acetate buffer (0.15 M ammonium acetate and 0.2 M acetic acid) with various dilutions. The η of CS solution was determined by the graphical method, generated through instrument. The η of CS solution was used to determine the average viscosimetric MW (Mv) from the Mark−Houwink equation (eq 1)30

magnetic centers, and unusual leaching of chelates. Moreover, the highest magnetic moment among all lanthanides has made Dysprosium (Dy) the most effective T2 contrast agent.17 Furthermore, the dual advantages of both absorption and fluorescence emission in NIR region has made Dy a promising candidate in designing hybrid NPs. Chitosan (CS) is a biopolymer offers noteworthy features in biomedicine such as nontoxicity, biocompatibility, and cellular adhesion.18 Chitosan coated nanoparticles are reported to have preferred cellular uptake via overcoming the endothelial cell barrier. 19 Chitosan coated AgNPs are reported as a biocompatible device for selective photothermal cancer therapy.20 Thus, a CS coated NPs could be a better candidate for bioimaging and drug delivery due to its natural biocompatibility and the abundant amine group for targeting and tethering ability. In recent years bimetallic nanoparticles (BNPs) have drawn considerable attention because of their unique possessions with respect to monometallic systems.21−23 Silver−gold BNPs are exclusively reported for specific detection and therapy of cancer.24 BNPs of europium and gold (Eu−Au) has been successfully synthesized through bioreduction using plant extract.25 A surge of interest in bimetallic lanthanide bioprobes is currently triggered due to unique luminescence and magnetic properties for applications in cellular imaging, cancerous cell detection and DNA tagging.26−28 Herein, we report the first example of BNPs as NIR/MRI/ CT imaging nanoprobe through coreduction of Dy3+ and Ag+ using glycerol as reducing agent. Unlike multistep synthesis with hazardous reagents and conventional heating methods, the present study reports the following distinguished features: (a) integration of NIR/MRI/CT imaging functions in a one-step synthesis, (b) use of environmentally benign microwave (MW) energy source to yield stable monodispersed NPs, (c) use of glycerol and chitosan (CS) as a corresponding reducing and capping agent to avoid harmful chemicals that refrain biological applications, and (d) the presence of ample NH2 groups in CS facilitates conjugation of doxorubicin for therapeutic applications.

η = KM v α

(1)

where K and α are constants for a particular polymer−solvent− temperature system. Reported values of K and α are 9.66 × 10−5 dm3/ g and 0.742 for chitosan−water solution in acetate buffer at 25 °C. The η of CS solution reduced from 1560.5 mL/g for pristine to 46.4 mL/g in the case of irradiated solution at a dose of 100 kGy. Correspondingly, the viscosity-average MW of CS decreased from 190 to 12 kDa at a dose of 100 kGy. 2.2.1.2. Chemical Characterization of CSo. Fourier transform infrared (FTIR) was used to determine chemical features of irradiated chitosan (CSo) in comparison to the pristine CS. FTIR analysis was carried out in transmission mode using a FTIR spectrophotometer (PerkinElmer) in the spectral range from 400 to 4000 cm−1. Both the irradiated CS and pristine CS indicated good similarities in the characteristics peak positions of amides I, II, and III at 1656, 1594, and 1256 cm−1 respectively (Figure S5). 2.2.2. Synthesis of Chitosan Coated Bimetallic Nanoparticles. One pot synthesis of CS capped BNPs was carried out using microwave assisted modified polyol reduction method. In brief, fixed aliquots of stock CSo was added to 30% glycerol solution in order to yield a final concentration of 0.2 mg/mL in Erlenmeyer flasks. To this mixture, 2.50, 3.75, 4.50, and 5.00 mL from AgNO3 stock solution (50 mM) and 2.50, 1.25, 0.50, and 0.00 mL from Dy(NO3)3 stock solution (50 mM) were added to maintain a final volume of 10 mL each, and these solution mixtures were coded as Ag0.5Dy0.5, Ag0.75Dy0.25 Ag0.9Dy0.1, and AgNPs, respectively. The pH of all solutions were maintained at 8.0 ± 0.3 by adding the required amounts of ammonia and the flasks were tightly plugged with cotton wool and were subjected to microwave irradiation (Samsung, South Korea) for 300 s at a constant power of 100 W. Upon this treatment, the solution color turned from transparent to brownish yellow. After the completion of reaction, the solutions were immediately cooled in an ice bath and centrifuged at 12 000 rpm for 40 min with added ethanol. Then, samples were washed with 100%, 50%, and 30% ethanol solution and finally twice with distilled water. 2.3. Characterizations. UV−vis spectra analysis was carried out using spectrophotometer (PerkinElmer Lambda 650 S) in the range of 200−1200 nm using a quartz cell of path length 1 cm. Luminescence spectrum was recorded using a photoluminescence spectrophotometer (HORIBA Jobin Yvon Fluorolog FL3-11). The crystalline phase and purity of material was analyzed using a powder X-ray diffractometer (RIGAKU, ULTIMA IV, Japan) with Cu Kα radiation (λ = 1.5406 Å) produced at 40 kV and 30 mA to scan the diffraction angles (2θ) between 5° and 90° with a step size of 0.02° 2θ/s. XRD measurements were also performed after heating the samples at 800 °C under nitrogen atmosphere (residues after TGA analysis). JCPDS standards were used to confirm crystalline purity of constituent metals. 2.3.1. Percent of Lattice Parameter Mismatch (%Δa). The %Δa is determined by using equation given below (eq 2):

2. EXPERIMENTAL SECTION 2.1. Materials. Unless otherwise stated, reagents were procured from Sigma-Aldrich (India) and were used without further purification. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS), liquid ammonia (25%), and sodium hydroxide (NaOH) were acquired from Hi-media, India. Deionized water was used for all synthesis and mentioned as water throughout. 2.2. Method of Synthesis. 2.2.1. Synthesis of Water-Soluble Chitosan Oligomer (CSo) and Its Characterization. For one pot synthesis of chitosan (CS) capped Ag/Dy bimetallic nanoparticles (BNPs), CS was degraded in oligomeric composition that is soluble in water at pH 8−9. Aqueous solution of CS in 1% acetic acid was irradiated by gamma (γ) radiation according to the protocol reported elsewhere.29 Briefly, 2g CS was dissolved in 100 mL of 1% acetic solution and the solution was irradiated by Co-60 γ radiation (Gamma chamber 5000, Atomic Energy Regulatory (AERB), India) with 100 kGy dose at 4 kGy/h exposer rate. An adequate amount of 2 M NaOH was added to the irradiated CS solution to maintain pH ∼ 9 and filtered through a 0.22 μm Nylon-66 hydrophilic syringe membrane (Hi-media, India). The filtered solution was lyophilized at −80 °C for 24 h, and finally, 1 mg/mL stock solution of this purified short chain chitosan oligomer (CSo) was prepared by dissolving in water of pH 8. 2.2.1.1. Molecular Weight Determination of CSo. The irradiation under γ rays scissions glycosidic bonds of CS without affecting its functional groups, and that results in the reduction of CS molecular

%Δa =

2(a1 − a 2) × 100 (a1 + a 2)

(2)

where a1 and a2 correspond to lattice parameters of the metal ions. The lattice parameters of BNPs were determined by employing the Rietveld method using GSAS-EXPGUI software package to explore the effect of increasing Dy concentration in solid solution of Dy and Ag. For the analysis through Rietveld refinement, an average of three X-ray scans was recorded for each sample. B

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Langmuir Thermal characterization of pure Ag0.5Dy0.5 sample along with the doxorubicin loaded Ag0.5Dy0.5 were evaluated through differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) in nitrogen atmosphere at a heating rate of 10 °C min−1 (TA Instrument Q600 SDT). X-ray photoelectron spectroscopy (XPS, Axis Ultra) analysis was carried out for surface chemical analysis in an ultrahigh vacuum environment (1.9 × 10−9 mbar) using Al Kα anode (1486.6 eV). Pass energy of 40 eV for survey scan and 30 eV for high resolution scan have been used during acquisition of the XPS spectra. Each scan was repeated three times to reduce the signal-to-noise ratio. The size and morphology of NPs were demonstrated by transmission electron microscopy (FEI Tecnai G2 T30, Netherlands). The particle size analysis of NPs was carried out at 25 °C using a Malvern Nano S Zetasizer (UK) with a backscattering angle of 90° and a “He laser” of wavelength 632.8 nm using 3 mL disposable plastic cuvettes. Cuvettes were cleaned using compressed air and loaded underneath a fume hood using a 0.22 μm syringe filter (Hi-media) to minimize dust interference. Size measurements were computed by collecting at least 12 runs with varied counting rates between 400 and 600 kilo counts per second (kcps). The elemental composition and distribution in NPs was examined through high-angle annular darkfield scanning transmission electron microscopy (HAADF-STEM) mapping (FEI Tecnai G2 T30, Netherlands). Elemental mapping was also carried out using field emission scanning electron microscopy (FE-SEM) coupled with electron-dispersive X-ray spectroscopy (EDS; JEOL, JSM-7100F, Japan). Compositional analysis of the Ag and Dy was carried out with the aid of ICP-AES (Arcos, Spectro, GERMANY). Magnetic properties of BNPs were analyzed using vibrating sample magnetometer (Lake Shore: 7404). 2.4. In Vitro Magnetic Resonance Imaging (MRI). In vitro T2 weighted MRI imaging was carried out on 1.5T Siemens Magnetom Essenza utilizing Siemens’ Syngo software (Siemens Medical Systems), in conjunction with a 32 channel head coil. Phantoms with five dilutions of Ag0.5Dy0.5 equivalent to 2.5, 2.0, 1.0, 0.5, and 0.25 mM Dy concentration in PBS were taken in 5 mL tubes. To simulate in vivo condition, similar dilutions of Ag0.5Dy0.5 were also taken in 10 mg/mL fetal bovine serum (FBS) in PBS. Control phantoms consisting of PBS and 10 mg/mL FBS alone were concurrently imaged. The following parameters were adopted: a repetition time (TR) = 5000 ms, the field of view (FOV) = 3 × 3 cm2, base resolution =256 × 256. The echo time (TE) was varied from 22 to 352 ms with a difference of 22 ms resulting in 18 images with varied T2 weighting. For each sample, the signal intensity on magnitude images was averaged within regions of interest (ROIs) and plotted TE for T2 decay curves. Data were then fitted to the following monoexponential functions:

T2 = A(1 − e(−TR/T2))

coupling occurs by removal of H2O molecule between amino, hydroxyl and carboxyl group. To attach carboxylic residues on Dox, succinic anhydride was employed to react with Dox that converts the amine group of Dox to carboxylic acid by conjugating succinic acid residues. The resulted succinyl doxorubicin (SDox) was then introduced to CS via amide bond formation mediated by EDC and NHS.31 Briefly, synthesis of SDox was carried out by dissolving 50 mg of Dox-HCl in 5 mL of dry acetonitrile and 80 μL of triethylamine followed by the addition of 100 mg of succinic anhydride, and the resultant mixture was stirred overnight at 4 °C in dark conditions. Finally, ethyl acetate was added and the residual solution was extracted after lowering the pH using 1 M HCl. Ethyl acetate was evaporated by using a rotary evaporator to acquire SDox. For covalent conjugation of SDox to CS capped Ag/Dy bimetallic nanoparticles (BNPs), 100 mg of BNPs and 100 mg of SDox were dissolved in 50 mL of acetate buffer (pH = 6.5) containing EDC (200 mg) and NHS (60 mg), and the final solution was stirred overnight in the dark at 4 °C. Free SDox was removed by centrifugation at 12 000 rpm for 15 min. The supernatant was discarded and the precipitate (Ag0.5Dy0.5-Dox) was washed thrice with PBS. The Ag0.5Dy0.5-Dox was resuspended by sonication to form a homogeneous clear solution and stored at 4 °C. To evaluate the Dox loading efficiency, the supernatant was collected and the residual Dox content was determined using the calibration curve of Dox standard solutions by UV−vis measurement at 485 nm. 2.7. Cell Culture and Cell Viability Assay. Human cervical cancer cells (HeLa cells) were procured from National Centre for Cell Science, India, and the cells were maintained and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic streptomycin at a constant temperature of 37 °C and 5% CO2 with 95% humidity. Cultured HeLa cells were utilized to test in vitro cytotoxicity of Ag0.5Dy0.5 BNPs and cell apoptosis effect of Ag0.5Dy0.5-Dox. A stock solution of 10 mM MTT was prepared in PBS and preserved at −8 °C. Briefly, 1.2 × 104 cells/well were seeded in 96-well plates in DMEM medium and incubated overnight in the growth conditions as described above. After overnight incubation, the medium was replaced with the medium containing Ag0.5Dy0.5, Ag0.5Dy0.5-Dox and medium control. After a predefined incubation time, the medium of each well containing a particular agent was removed. For cell viability measurement, the stock MTT solution was thawed and diluted as 1:10 with complete medium. Then 120 μL of MTT containing medium was added to each well and further incubated for 4 h. Finally, the supernatant was removed and 160 μL of DMSO was added to each well to solubilize the formazan crystals. The absorbance of the solutions was measured at 570 nm to determine the optical density (OD) values. The cell viability was calculated as follows:

(3) −1

The obtained T2 values were converted into R2 [1/T2 (s )] relaxation rates and finally, R2 values were plotted against the concentration of Dy, and relaxivities r2 (s−1 mM−1) were obtained as the slope of the resulting linear plots using the following equations:

R 2 = r2[C ] + R *2

cell viability (%) =

(4)

ODtreated × 100 ODcontrol

where ODtreated is the optical density recorded for the cells treated by a particular agent and ODcontrol is for the cells without any treatments. The data is given as mean ± standard deviation (SD) based on three independent measurements. 2.8. Live/Dead Cell Staining. Live−dead assays were carried out using the Live/Dead Viability/Cytotoxicity Assay Kit (ThermoFisher Scientific, CNo. L3224) after 24 h incubation. As described above, 1.2 × 104 cells/well were seeded in 96-well plates in DMEM medium and incubated overnight in the growth conditions. After overnight incubation, the medium was replaced with the medium containing 50 μg/mL Ag0.5Dy0.5, 50 μg/mL Ag0.5Dy0.5-Dox and medium control, and allowed for 24 h incubation. Finally, cells were stained with Live/ Dead assay according to manufacturer’s guidelines. The green fluorescence of live cells was due to the uptake and hydrolysis of calcein AM, while the red fluorescence of dead cell nuclei was due to ethidium homodimer-1. Background levels are low due to nonfluorescence state of both dyes prior to cell interaction.

where R2* is relaxation rate of control phantoms and [C] is the concentration of Dy in mM. 2.5. In Vitro Computed Tomography (CT). Five different mass concentrations of Ag0.5Dy0.5 nanoparticles equivalent to 0.067, 0.134, 0.268, 0.536, and 0.670 mg/mL in PBS and PBS solution as control were taken in 5 mL tubes for phantom test. CT images along with Hounsfield units (HU) values were acquired using a GE/CT e High Speed Spiral CT. The following parameters were adopted to acquire images: field of view (FOV) = 53 mm × 151 mm; effective pixel size = 3.0 cm; rotation steps = 160; 120 kVp, 160 mA; exposure time = 150 ms/rotation. The images of phantom CT analysis were analyzed with Kodak Molecular Imaging Software. 2.6. Drug Tethering. Carbodiimide chemistry was utilized to conjugate doxorubicin (Dox) with CS on the surface of BNPs using coupling reagents such as 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). EDC-NHS C

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Scheme 1. Schematic Presentation of Synthesis Mechanism of CS Capped Ag/Dy Bimetallic Nanoparticles and Its Application in Multimodal Imaging

Figure 1. TEM analysis: (A) TEM image and (B) high resolution TEM image representing crystal fringes of both Ag and Dy. (C) SAED pattern reflecting rings of Ag (111, 222) and Dy (111, 222). (D) Particle size distribution, HAADF-STEM mapping of nanoparticles presenting; (E) particles image; elemental distribution of (F) only Ag; (G) only Dy; (H) combined distribution mapping of Ag and Dy. (I) Elemental line spectra for the same area. White scale bar denotes = 10 nm, if not specified.

3. RESULTS AND DISCUSSION

and capping agents, respectively. An environmentally benign approach was executed by using nonconventional microwave (MW) energy source because of quick and uniform heating with low energy consumption and higher yield of stable

3.1. General Synthetic Route. In this novel one pot synthesis, AgNO3 and Dy(NO3)3 precursors were reduced by a polyol coreduction method using glycerol and CS as reducing D

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Table 1. Combined Data on Elemental Analysis by ICP-AES, Particle Size Analysis by XRD, TEM, and Hydrodynamic Size by DLS and Magnetic Properties precursor (wt %)

ICP-AES (wt %)

avg size (nm)

magnetic properties

sample code

Ag

Dy

Ag

Dy

XRD

DLS

TEM

coercivity (G)

magnetization (emu/g)

retentivity (emu/g)

Ag0.5Dy0.5 Ag0.75Dy0.25 Ag0.9Dy0.1

40.00 66.67 85.71

60.00 33.33 14.28

37.48 69.28 88.32

62.51 30.72 11.68

11.30 14.60 16.70

26.30 23.70 24.50

11.73

40.23 77.08 98.57

0.962 0.508 0.164

4.61 × 10−3 4.97 × 10−3 2.80 × 10−3

Figure 2. (A) XRD pattern of Ag0.5Dy0.5 bimetallic nanoparticles (BNPs) as prepared and at 800 °C (after TGA) under nitrogen atmosphere. (B) TGA-DSC curves of BNPs as prepared and after drug loading.

monodispersed nanoparticles.32 On microwave heating, a transparent solution of AgNO3 and Dy(NO3)3 in glycerol turns brownish yellow which indicates the formation of Ag/Dy bimetallic nanoparticles (BNPs) (Scheme 1). Three different molar ratios of [Ag+]:[Dy3+] designated as 9:1, 3:1, and 1:1 finally produced BNPs that are coded as Ag0.9Dy0.1, Ag0.75Dy0.25, and Ag0.5Dy0.5 respectively. Meticulous observation revealed that increasing Dy3+ concentration with respect to constant concentration of Ag+ requires more time for reduction on constant heating at 90 °C (100 W MW). Interestingly, reduction of Dy3+ follows nucleation and growth of Ag(0). So, at the first instance thermodynamically driven reduction of Ag+ produces Ag(0). Instantaneous generation of the Ag(0) nucleation center facilitates the Dy3+ reduction and finally coreduction of Ag+ and Dy3+ produces BNPs. So, the increased reduction time with increasing Dy(NO3)3 concentration is understood. Thus, without Ag+, no significant change in color of reaction medium with Dy3+ is observed on prolonged heating. In this case, reaction kinetics becomes sluggish because of the absence of Ag(0) nucleation sites in solution, which is crucial to catalyze Dy3+ reduction. 3.2. Morphological Characterization and Elemental Distribution. The TEM images of BNPs with high Dy content (Ag0.5Dy0.5) are illustrated in Figure 1. HR-TEM confirmed spherical nanostructure with an average diameter of ∼10 nm and also the presence of both Ag and Dy metals in NPs with their corresponding lattice fringes (Figure 1B). The SAED pattern of Ag0.5Dy0.5 suggested the polycrystalline nature of the bimetallic system. The SAED pattern (Figure 1C) of the representative sets shows the presence of diffraction rings for both metals simultaneously in a single system, which thus successfully explains the homogeneous distribution of Ag and Dy in the polycrystalline system. The hydrodynamic size (DH) of BNPs determined through DLS implies the absence of any significant deviations in the DH of BNPs with respect to the varied Dy content. The formation of size controlled BNPs through the opted synthetic route is enunciated by the determined polydispersity index value < 0.35. Meanwhile, DH

values (Figure S1) of Ag0.5Dy0.5 (26 nm), Ag0.75Dy0.25 (24 nm), and Ag0.9Dy0.1 (24 nm) are measured comparably larger than the size determined from TEM analysis, which is because of the hydrated CS capping in solution state. To ensure the reduction process and distribution of both constituent Ag and Dy, elemental mapping was performed using HAADF-STEM (Figure 1E−H) and EDS using FE-SEM (Figure S2). The HAADF-STEM area mapping, line mapping (Figure 1I), and EDS area mapping warranted homogeneous distribution of Ag and Dy, which transpired due to simultaneous coreduction process. EDS of Ag0.5Dy0.5 revealed Ag:Dy ratio as 51.83:48.17 (∼1:1), which is consistent with the corresponding concentration of precursors. Quantitative estimation by ICP-AES also demonstrates the consistency of Ag:Dy ratio in all three systems with their corresponding precursor concentrations (Table 1). 3.3. Crystal Structure and Phase Analysis. The XRD reflections of representative Ag/Dy bimetallic systems ensured the formation of face centered cubic (FCC) Ag and cubic Dy which exhibited good coherence with their corresponding standard (Figures S3 and 2A). The XRD pattern of Ag0.5Dy0.5 recorded at 800 °C under nitrogen atmosphere (after TGA) displayed intense reflections that correspond to the Ag and Dy presence (Figure 2A). It is reported that during heating of alkaline aqueous solution of Dy3+ salts, Dy(OH)3 is readily formed and thereafter it transforms to Dy2O3 during calcination.33 The absence of any X-ray reflections that pertains to Dy(OH)3 implies the fact that Dy underwent simultaneous reduction along with Ag to yield BNPs. Broad X-ray reflections that correspond to Dy(0) is detected due to the formation of ultrasmall Dy crystals in single nanoparticle. The size mismatch between Ag and Dy (Ag = 408 pm, Dy = 518 pm) approximates to 24%. Homogenous nucleation of second component is more prompt in comparison to heterogeneous nucleation when the lattice mismatch is considerably large (>14%).34 Thus, the reaction product is a homogeneous mixture of FCC Ag and cubic Dy in BNPs. This has been confirmed by HR-TEM images that presents lattice fringes of E

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Figure 3. Surface analysis for chemical composition (A) XPS survey spectra and high resolution spectra of (B) Dy 4f, 5p, (C) Dy 4d, (D) Ag 3d, (E) O 1s, and (F) C 1s of BNPs.

Figure 4. (A) Absorption spectra of AgNPs and Ag/Dy bimetallic nanoparticles in visible region. (B) Absorption spectra in near-infrared (NIR) region. (C) NIR emission spectra after excitation at λex = 808 nm.

eV for Ag2+.35,36 The core level photoemission of Dy 4d electrons displays doublet, which after deconvolution exhibited peaks of 160.3 eV for 4d5/2 and 163.5 eV for 4d3/2, with a shift from 154.0 eV binding energy of 4d electrons of Dy3+ ions. The core level Dy 4d binding energy in Dy2O3 corresponds to the standard oxidation state of Dy3+ ions (156.0 eV).37,38 The Dy 4f and Dy 5p binding energy peaks also reflected similar trend of shift from 4.8 and 16.1 eV of Dy3+ ions to 13.0 and 30.25 eV of bimetallic system, respectively.39 Thus, XPS results reveals the reduced Dy(0) state in Ag/Dy bimetallic system. The C 1s spectrum of AgNCs was deconvoluted with three binding energies at 287.1, 288.7, and 290.6 eV assigned to hydroxyl (−C−OH) amide (N−CO) and carbonyl (−CO) groups, respectively, which is in good agreement with C 1s peaks of CS.40,41 Further FTIR spectrum was recorded to confirm the capping of CS on BNPs (Figure S5). The characteristic N−H bending of amines determined at 1594 cm−1 for pure CS showed a

both constituent metals. This lattice mismatch creates strain in the bimetallic system, which is followed by the lattice expansion as a function of Dy content (Figure S4). Broadening of the XRD peak position suggests small grain size and crystal grain sizes of Ag for the three compositions were determined as 11.3 ± 1, 14.6 ± 2, and 16.7 ± 2 nm for Ag0.5Dy0.5, Ag0.75Dy0.25, and Ag0.9Dy0.1, respectively from the Scherer’s relation. 3.4. Surface Characterization and Oxidation State of Ag and Dy in BNPs. XPS was executed to determine the exact oxidation states of constituent metals in Ag/Dy systems and capping ability of CS (Figure 3). The wide XPS spectra of Ag0.5Dy0.5 depicts carbon (C 1s), oxygen (O 1s), dysprosium (4f, 4d, 5p), and silver (Ag 3d). The XPS spectra exhibit 3d5/2 and 3d3/2 peaks of Ag at 372.9 and 379.0 eV, respectively with a shifting of 6 eV from Ag+, thus confirming the uncharged state of Ag in Ag/Dy bimetallic system. Reported binding energies for the Ag 3d5/2 peak range from 368.1 to 369.0 eV for neutral Ag, from 367.3 to 367.6 for Ag+ ions, and from 367.8 to 368.0 F

DOI: 10.1021/acs.langmuir.6b03438 Langmuir XXXX, XXX, XXX−XXX

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display significant signal attenuation on increasing the concentration of Dy from 0.25 to 2.5 mM (Figure 6A and B). The relaxation rates (1/T2) exhibit a linear trend with the Dy concentration in both cases. The transversal relaxivity (r2) value is estimated as 7.26 s−1 mM−1 for PBS and 4.43 s−1 mM−1 for FBS solutions of BNPs. This reduction of r2 values in FBS solution is understood by the protein adsorption on the surface of BNPs. However, the recorded low r2 value of BNPs in FBS solution is still found higher than the r2 values reported in case of Dy containing contrast agents.49 The results established the prospect of these BNPs as T2 weighted clinical MRI contrast agents. 3.7. In Vitro CT Analysis. The feasibility as an X-ray contrast agent was tested by preparing different concentrations of Ag0.5Dy0.5 in PBS solution along with PBS as a control. The obtained phantom images portray gray to bright transformation with simultaneous increase in the BNPs concentration (Figure 6C). By setting the Hounsfield unit (HU) value of PBS solution as zero, the HU values of Ag0.5Dy0.5 indicated a progressive upsurge with increasing BNPs concentrations (Figure 6D). The CT signal intensity of Ag0.5Dy0.5 approximates to 46 HU at a mass concentration of 0.50 mg/mL, which has been found much higher than the HU values for conventional iodinated material, barium sulfate or metal nanoparticles.50,51 The high HU value obtained from the present investigation is mainly due to the high X-ray attenuation coefficient of Dy and smaller size of BNPs than threshold (23.2 nm) within which Ag have similar X-ray attenuation coefficients as iodine in omnipaque.11 3.8. Drug Tethered BNPs and MTT Assay. Doxorubicin (Dox) tethered on gold nanoparticles are reported to facilitate efficient intracellular drug delivery to overcome drug resistance of cancer cells.52 Here, we tethered Dox on synthesized CS capped Ag/Dy BNPs to formulate a novel theranostic agent with capabilities of multimodal imaging and drug delivery (Figure 7A). The optical absorbance and TGA-DSC data substantiates the Dox loading efficiency of Ag0.5Dy0.5 that has been determined as 12.5 ± 4% (Figure 2B). To evaluate the biological compatibility of Ag0.5Dy0.5 and apoptotic effect of Dox tethered BNPs (Ag0.5Dy0.5-Dox), MTT assay was carried out on a human cervical cancer (HeLa) cells and the results are presented in Figure 7B and C, respectively. The Ag0.5Dy0.5 ensures good biocompatibility by the virtue of its negligible cytotoxicity. Ag0.5Dy0.5-Dox revealed better therapeutic efficiency in HeLa cells even at low concentration of 10 μg/mL. The apoptotic effect of Ag0.5Dy0.5-Dox was found dose dependent with half maximal inhibitory concentration (IC50) equal to ∼42 μg/mL. According to MTT assays, BNPs showed negligible toxicity up to 400 μg/mL for 6 h and 24 h incubation studies. However, apoptotic effect of Dox tethered BNPs were obvious even for 10 μg/mL and thereafter a minor increase in the concentration led to the exponential destruction of cell populations. Live/dead cell viability assays were performed by the incubation of cells with 50 μg/mL each of Ag0.5Dy0.5 and Ag0.5Dy0.5-Dox. The results confirm the absence of cytotoxicity by BNPs and high apoptotic effect of Dox tethered BNPs (Figure 5C−E) by the corresponding green and red fluorescence for live and dead cells. The better therapeutic efficiency of Ag0.5Dy0.5-Dox could also be explained on the antiproliferative ability of AgNPs as reported by Sanpui et al.53

strong shift toward lower wavenumber with broad band in BNPs.42 On the other hand, the CO stretching (1655 cm−1) of amide-I group did not indicate any significant changes.42,43 However, 1020 and 1070 cm−1 peaks that corresponds to the presence of primary (C2-OH) and secondary (C3-OH) alcohols indicated significant change with narrowed band and shift toward lower wavenumber.42,43 Hence, FTIR signifies the effective coordination of −OH and −NH2 groups of CS with BNPs. 3.5. Absorption and Emission Characterizations. The absorption spectrum of AgNPs and BNPs recorded from 200 to 1200 nm (Figure 4A and B) substantiated the characteristic absorption of constituents. In visible region, the absorption spectrum expresses the characteristic AgNPs band at 400 nm in all the samples with a narrow red shift. This red shift is due to the reduced electron density on the Ag surface. The extended absorption spectra (800−1200 nm) demonstrates the characteristic nature of Dy in NIR region with the observed electronic transitions 6F5/2, (6F7/2−6H5/2), and (6F9/2−6H7/2) at 808, 895, and 1098 nm, respectively.44 The enhancement in Dy concentration resulted in the accomplishment of a well refined spectra. It has been specified that NIR light possesses the ability to penetrate deep into the tissue than visible light and this phenomenon has been recommended for optical imaging.45 Thus, the absorption ability of these BNPs in first biological window (700−950 nm) could be a favorable prospect for applications in hyperthermia and laser phototherapy.46 The selection of 808 nm excitation wavelength to determine the emission nature of the BNPs yields a strong NIR emission in 1000−1500 nm range (Figure 4C). NIR fluorescence in the second biological window (1000−1350 nm) could be a promising facet for deep tissue imaging.47,48 Thus, the BNPs with NIR fluorescence characteristics could be an ideal fluorophores for in vivo imaging. 3.6. In Vitro MRI Analysis. The discernible coercivity and remanence values are measured from hysteresis curves recorded at room temperature, indicating a paramagnetic behavior of BNPs (Figure 5, Table 1). The saturation magnetization (at 1.2

Figure 5. VSM analysis of bimetallic nanoparticles (BNPs) and AgNPs.

T) gradually increases from 0.16 to 0.96 emu g−1 as a function of Dy contents in BNPs. Owing to the high paramagnetism, T2 weighted MR images of Ag0.5Dy0.5 at different Dy concentrations in PBS are recorded at 1.5 T. To simulate in vivo condition, phantoms were also prepared in 10 mg/mL fetal bovine serum (FBS) in PBS. The obtained phantom images G

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Figure 6. (A) T2 weighted MR phantom images of BNPs in PBS and in 10 mg/mL FBS. (B) Relaxivity (1/T2) plotted against varying Dy concentrations. (C) CT images of the phantoms with reference to PBS control. (D) mass concentration vs HU plot showing linear dependence.

Figure 7. (A) Schematic presentation of doxorubicin (Dox) tethering on CS functionalized Ag/Dy bimetallic nanoparticles. (B) Viability of HeLa cells after 6 h and (C) 24 h treatment with Ag0.5Dy0.5 and Ag0.5Dy0.5-Dox. Live/dead cell staining with treatment of (D) PBS (control), (E) 50 μg/ mL Ag0.5Dy0.5, and (F) 50 μg/mL Ag0.5Dy0.5-Dox after 24 h incubation. Live cells are stained into green and dead/apoptosis cells are stained into red. Values in figures are expressed as the means ± standard deviation (SD) based on three independent measurements.

4. CONCLUSION

been determined that the proposed synthetic technique could efficiently produce BNPs with an average particle size of 10 nm with low polydispersity. It is also instituted that the reduction of Dy3+ is dependent on Ag nucleation. Besides the size and structure, the BNPs also display unique characteristics for

To conclude, the Ag/Dy bimetallic nanoparticles (BNPs) were successfully prepared through microwave assisted polyol synthesis with their respective assorted concentrations. It has H

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applications in multimodal imaging. The absorption features in first biological window and emission characteristics in second biological window signifies the corresponding application of BNPs in hyperthermia and laser phototherapy as well as cell level in vivo imaging. The high CT value (for 0.50 mg/mL, HU = 46) unveiled by BNPs is vital for solid tumor imaging. The T2 weighted MRI contrast ability of BNPs validate good stability in simulated in vivo condition. Cytotoxicity studies authorize good biocompatibility and effectiveness of BNPs as a drug carrier with high apoptotic effect of Dox tethered BNPs. Owing to the impeccable features, the synthesized BNPs could be a promising candidate in theranosis for early diagnosis and treatment of cancer.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b03438. Hydrodynamic size of bimetallic nanoparticles by DLS; EDS (FE-SEM) elemental mappings of the bimetallic nanoparticles; XRD reflections of bimetallic nanoparticles along with standard JCPDS data of Ag and Dy; lattice parameters (in Å) and unit cell volume (in Å3) of hybrid nanoparticle refined from XRD reflections; FTIR spectrum of chitosan (CS), short chain chitosan oligomers (CSo), and bimetallic nanoparticles Ag0.5Dy0.5; vertical CT image of varying concentration of Ag0.5Dy0.5 in PBS reflects the water solubility of nanoparticles (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 0091-413-2654973. ORCID

S. Kannan: 0000-0003-2285-4907 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial assistance received from Department of Science and Technology, DST [Reference: SB/FT/CS-101/2012(SR)], India is acknowledged. The facilities availed from Central Instrumentation Facility (CIF) of Pondicherry University is also acknowledged. The characterization facilities availed from IIT Bombay and IISc Bangalore under INUP which is sponsored by DeitY, MCIT, Government of India is gratefully acknowledged. Mr. Biji M. Cheriyan, Radiographer, Tellacherry co-operative hospital is highly acknowledged for providing assistance in MRI and CT scanning.

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REFERENCES

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