A Bimetallic Silver–Neodymium Theranostic Nanoparticle with

After tightly plugging with cotton-wool, the flasks were exposed to microwave irradiation (Samsung, South Korea) at a constant power of 100 W for 300 ...
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A Bimetallic Silver−Neodymium Theranostic Nanoparticle with Multimodal NIR/MRI/CT Imaging and Combined Chemophotothermal Therapy Sandeep K. Mishra and S. Kannan* Centre for Nanoscience and Technology, Pondicherry University, Puducherry 605 014, India S Supporting Information *

ABSTRACT: An engineered metallic nanostructure is an excellent candidate for “theranosis” of cancer, having intrinsic properties of multimodal imaging and therapy. Toward this target, the development of silver−neodymium bimetallic nanoparticles (Ag−Nd BNPs) via microwave-assisted polyol synthesis is presented. The resultant Ag−Nd BNPs exhibit good monodispersity with average size of 10 nm, fluorescence in the near-infrared (NIR) region, and magnetic properties. The Ag−Nd BNPs also validate MRI, CT, and NIR trimodal imaging ability and enunciate valuable temperature response upon irradiation under a NIR laser. Aided by chitosan functionalization on the surface, the Ag−Nd BNPs deliver good biocompatibility and also promote the loading of paclitaxel, an anticancer drug. Isothermal titration calorimetry affirms the combination of strong binding affinity of drug and high loading efficiency of 7 drug molecules per nanoparticle. Moreover, Ag−Nd BNPs also illustrate a highly efficient photothermal effect in PBS. Therefore, the synergistic effects of paclitaxel and the photothermal effect make BNPs excellent “combined therapeutic agents”, and also give them the important ability to destroy cancer cells in vitro at very low dose in comparison to single therapy. Thus, the Ag−Nd BNPs unveil a combination of MRI/CT/NIR imaging and chemo-photothermal therapy that ensures accurate diagnosis at an early stage and comprehensive eradication of tumor cells without affecting healthy cells. and therapy of cancer.9 A Au−Fe nanoalloy has been reported as an excellent candidate for multimodal MRI-CT imaging.8,10 The combination of lanthanides and NPs to give novel hybrid NPs has gained great momentum in recent years.11 Bimetallic lanthanide bioprobes are reported for applications in cellular imaging, cancerous cell detection, and DNA tagging due to the unique luminescence and magnetic properties of lanthanides.12,13 A recent report emphasized the synthesis of chitosan (CS)-capped Ag−Dy BNPs conjugated with doxorubicin as a theranostic device for MRI/CT/NIR multimodal imaging and drug delivery to cancer cells.14 Silver nanoparticles (AgNPs) are especially attractive in biomedicine due to their size-dependent photophysical properties and antibacterial activities.15,16 Polymer-coated AgNPs deliver biocompatibility and luminescence features that favor its theranostic applications for cancer treatment.17,18 Due to high electron density, AgNPs display X-ray attenuation intensity comparable to omnipaque with iodine concentration similar to silver, which makes it interesting to introduce CT contrast properties in BNPs.19 Moreover, structurally designed AgNPs are also reported as effective photothermal transducers for in vitro cancer cell therapy.20

1. INTRODUCTION Application of nanoparticles (NPs) in biomedicine gains more prominence with exclusive emphasis on drug delivery and imaging.1 In cancer nanomedicine, dual-functional NPs with high drug loading capacity and multimodal imaging expedite early detection and targeted therapy, a phenomenon termed as “nanotheranostics”.2,3 Various modern techniques, inclusive of ultrasonography, magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and optical imaging, are used for the detection of cancer. Conversely, each technique experiences detection limits in terms of sensitivity, resolution, and penetration depth.4 Now, optical imaging in the near-infrared (NIR) region is gaining popularity due to its low tissue autofluorescence and deep tissue penetration in this region.5 NIR imaging is highly sensitive but suffers from poor resolution, while MRI and CT scans offer good resolution and tissue penetration, with the demerit of poor sensitivity.6,7 A device with multimodal contrast abilities in NIR, MRI, and CT imaging would be invaluable for early detection of cancer. Recent advances in constant refinement and optimization of the engineered NPs accord the sophistication to tailor and predesign NPs for cancer theranosis. A smartly architected bimetallic nanostructure is considered a promising prospect to assemble unique properties in a single entity.8 Ag−Au bimetallic nanoparticles (BNPs) are relished for detection © XXXX American Chemical Society

Received: August 15, 2017

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DOI: 10.1021/acs.inorgchem.7b02103 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

oligomers (CSo) were collected. Then, CSo was dissolved in water of pH 8 to make a stock solution of 1 mg/mL for further synthesis. Molecular Weight Determination of CSo. Under γ irradiation, glycosidic bonds of CS scission to shorten the chain without any chemical change which finally result in the reduction of CS molecular weight (MW). The average MW of CSo was determined through viscosity (η) measurements using a capillary viscometer (Ubbelohde Capillary Viscometer type 531/10 I) at 25 °C. A stock solution of the CSo was prepared by 100 mg CSo in 10 mL acetate buffer (0.15 M ammonium acetate and 0.2 M acetic acid), and that was diluted in various concentrations for η measurement by the instrument generated graphical methods. The η of CS solution was used to determine the average viscosimetric MW (Mv) from the Mark−Houwink’s equation (eq 1)29

Among lanthanides, neodymium has received attraction in the design of multimodal imaging devices due to its unique optical properties, such as absorption in the NIR region, and narrow and long-range visible or NIR emission, allied with strong magnetic properties.21,22 For time-resolved detection, Nd-based materials provide significant signal-to-noise ratio enrichment in comparison to common organic fluorophores.23,24 Under excitation at 808 nm, Nd emits within the NIR (750−1200 nm) region, which facilitates imaging and sensing of sub-tissue-located tumors.21,22,24,25 Besides, with long-range absorption ability in the visible−NIR region, Nd is considered a favorable prospect for photothermal therapy (PTT). PTT is advantageous especially as the primary mode of tumor eradication or in combination with other treatments, and as a consequence, the incorporation of Nd in a bimetallic system is anticipated to be a viable approach to design a theranostic NP. Despite the advantages, gradual ion release and surface contact of metallic NPs induce toxic effects in human cells, and hence a biocompatible polymer coating on NPs is considered as a promising approach to reduce systemic toxicity. Chitosan, a biopolymer, offers remarkable characteristics of nontoxicity, biocompatibility, and cellular adhesion.26,27 A recent report highlights the application of CS-coated AgNPs in photodynamic therapy of cancer.20 Thus, a CS-capped NP could be a better candidate for bioimaging and drug delivery due to its natural biocompatibility; also, the excess amine groups in CS facilitates targeting and promotes conjugation with ligands. Chemotherapy, an approach used in cancer treatment, is generally associated with severe systemic side effects. Paclitaxel (Pax), a natural chemotherapeutic agent, has widespread application for cancer therapy; however, it has a shortcoming of dose limitation due to low water solubility. Thus, alternative approaches in the form of targeted drug delivery or combined treatments are desirable to overcome these complications. “Combined therapy” of chemotherapy along with PTT offers synergistic consequences to destroy tumor cells while the normal cells remain unaffected.28 The present study aims at the one-pot, green synthesis of silver−neodymium bimetallic nanoparticles (Ag−Nd BNPs) coated with biocompatible polymer CS via a polyol reduction method using microwave energy. The study also elaborates the NIR, MRI, and CT multimodal contrast ability, and Pax drug loading and release, along with combined chemo-phototherapetutic ability of Ag−Nd BNPs.

η = KMvα

(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 CS−water solution in acetate buffer at 25 °C. The η of pristine CS solution was calculated as 1593.0 mL/g, which reduces to 48.9 mL/g in case of irradiated CSo solution at a dose of 100 kGy. Consequently, the measured viscosity-average MW of CSo reduced to 11.6 kDa from its pristine CS of viscosity-average MW 190 kDa. Chemical Characterization of CSo. The chemical stability of CSo in comparison to the pristine CS was confirmed from FT-IR spectra. FTIR analysis was carried out in transmission mode using a FT-IR spectrophotometer (PerkinElmer, USA) in the spectral range from 400 to 4000 cm−1. The characteristic peak positions of amidea I, II, and III at 1656, 1592, and 1256 cm−1, respectively, were found remarkably similar in both CSo and CS, which confirms the chemical stability of CS after γ irradiation (Figure S4). 2.2.2. Microwave-Assisted One-Pot Synthesis of ChitosanDecorated Ag−Nd Bimetallic Nanoparticles. To avoid trace impurities of health hazardous reducing agents, 30% v/V glycerol solution was used as a reducing agent. One-pot microwave synthesis of CS decorated Ag−Nd BNPs were carried out by modified polyol reduction method with glycerol solution as reducing agent and 0.2 mg/mL CSo as a capping agent in Erlenmeyer flasks. To the CSo and glycerol 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 Nd(NO3)3 stock solution (50 mM) were added to make a final volume 10 mL of each. These solution mixtures were respectively coded as Ag0.5Nd0.5, Ag0.75Nd0.25, Ag0.9Nd0.1, and AgNPs. An adequate amount of ammonia was added to maintain the pH of solutions 8.0 ± 0.3. After tightly plugging with cotton-wool, the flasks were exposed to microwave irradiation (Samsung, South Korea) at a constant power of 100 W for 300 s. Upon irradiation in microwave, the transparent solutions turned into brownish yellow color and were immediately cooled in an ice bath. Finally, the solutions were centrifuged at 16 000 rpm for 30 min with ethanol additions. The samples were then washed with 100, 50, and 30% ethanol solution and finally twice with distilled water. 2.3. Characterization Techniques. UV−vis−NIR spectra have been recorded using a spectrophotometer (PerkinElmer Lambda 650 S, USA) in the range of 200−1200 nm using a quartz cell of path length 1 cm. Photoluminescence spectra were collected using a photoluminescence spectrophotometer (HORIBA Jobin Yvon Fluorolog FL3-11, USA). X-ray diffraction reflections were recorded to determine the crystalline phases of the synthesized materials 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 per second. XRD reflections were also recorded after heating samples up to 800 °C under nitrogen atmosphere to confirm the crystalline purity of samples after polymer degradation. The XRD reflections were compared with the standard JCPDS data of respective metals to confirm the crystalline purity of constituent metals. Percent Lattice Parameter Mismatch (%Δa). %Δa is determined by using the following equation,

2. EXPERIMENTAL SECTION 2.1. Materials. All the reagents were procured from Sigma-Aldrich (India) and used without further purification, if not specified. Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM), liquid ammonia (25%), and sodium hydroxide (NaOH) were procured from Hi-media, India. Deionized water was used for all syntheses and has been mentioned as water throughout. 2.2. Method of Synthesis. 2.2.1. Synthesis of Water-Soluble Chitosan Oligomer and Its Characterization. To obtain short chain water (pH ≥ 7) soluble oligomers, low-molecular-weight chitosan (CS) was further degraded as reported elsewhere.14 Briefly, 2 g of CS was dissolved in 100 mL of 1% acetic solution and then irradiated by Co-60 gamma (γ) radiation (Gamma chamber 5000, Atomic Energy Regulatory Board (AERB), INDIA) with 100 kGy dose exposed at rate of 4 kGy/h. Adequate amount of NaOH (2M) was added in the γ irradiated solution of CS to raise the pH ∼9. Then solution was filtered through a Nylon-66 hydrophilic syringe membrane (Hi-media, India) of 0.22 μm to remove the residual large particles. The filtered solution was lyophilized at −80 °C for 24 h, and degraded chitosan B

DOI: 10.1021/acs.inorgchem.7b02103 Inorg. Chem. XXXX, XXX, XXX−XXX

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2(a1 − a 2) × 100 (a1 + a 2)

pixel size = 3.0 mm; 120 kVp, 160 mA; field of view (FOV) = 53 mm × 151 mm; rotation steps = 180; exposure time = 150 ms/rotation. The images of phantom CT analysis were analyzed with Kodak Molecular Imaging Software. 2.7. Preparation of Paclitaxel (Pax)-Loaded Ag−Nd BNPs, Drug Loading Efficiency, and Drug Release. For the preparation of Pax-loaded Ag−Nd BNPs, 5 mg/mL Ag0.5Nd0.5 BNPs was prepared in PBS solution (pH 7.4), to which 5 mg of Pax in ethanol was introduced in dropwise manner to ensure BNP to Pax mass ratio as 1, and further subjected to constant stirring for 2 h. The resultant solution was filtered (0.22 μm syringe filter) to remove any aggregations and centrifuged at 10 000 rpm for 15 min. Supernatant was collected, and pellets were washed thrice with PBS to eliminate loosely bounded Pax. Finally, drug-loaded BNPs (BNPs-Pax) was suspended in PBS and stored at 4 °C. The drug loading amount was calculated by recording the optical density (OD) at 227 nm of supernatant and the OD of initial drug.

(2)

where a1 and a2 correspond to lattice parameters of the metal ions. Rietveld analysis was employed to evaluate the effect of increasing concentration of Nd on the lattice parameters and cell volume of BNPs using GSAS-EXPGUI software package. Oxidation states of constituent elementals of bimetallic system was assessed through multitechnique X-ray photoelectron spectroscopy (XPS, Axis Ultra, UK) in an ultrahigh-vacuum environment (1.9 × 10−9 mbar) using Al Kα anode (1486.6 eV) with a pass energy of 40 eV for survey scan and 30 eV for high-resolution scan. To reduce the signal-to-noise ratio, each scan was repeated thrice, and data were analyzed using CasaXPS software. Transmission electron microscope (FEI Tecnai G2 T30, Netherlands) was utilized to determine the size and morphology of NPs. The hydrodynamic size of NPs was demonstrated using a Malvern Nano S Zetasizer, UK, at 25 °C with a backscattering angle of 90° and a “He laser” of wavelength 632.8 nm using 3 mL disposable plastic cuvettes. Sample preparation for Zettasizer analysis was carried out underneath a fume hood using a 0.22 μm syringe filter to minimize dust interference. Elemental mapping was carried out at nanoscale using high-angle annular darkfield scanning TEM (HAADF-STEM) mapping simultaneously with TEM analysis and at microscale using electron-dispersive X-ray spectroscopy (EDS) (Jeol, JSM-7100F, Japan) to determine the elemental detection and distribution. Elemental composition was also evaluated using inductively coupled plasma atomic emission spectroscopy (ICP-AES, Arcos, Spectro, Germany). Magnetic properties of BNPs were evaluated using vibrating sample magnetometer (Lake Shore: 7404, USA). 2.4. Colloidal Stability of Ag−Nd BNPs. The colloidal stability of BNPs were analyzed in water, PBS, and cell culture media (DMEM + 10% FBS) to confirm the stability of BNPs in colloidal state. A known concentration of Ag0.5Nd0.5 BNPs was prepared in water, PBS, and DMEM media and stored at 4 °C. At predetermined time intervals, 5 mL solution was collected, and hydrodynamic size was recorded for 30 days study period. 2.5. In Vitro Magnetic Resonance Imaging. In vitro T1-weighted MRI imaging was carried out on 1.5 T Siemens Magnetom Essenza utilizing Siemens’ Syngo software (Siemens Medical Systems), in conjunction with a 32 channel head coil. A known amount of Ag0.5Nd0.5 BNPs was suspended in PBS to prepare a stock solution, and the Nd concentration in the solution was measured using ICPAES. Phantoms were prepared with five dilutions of Ag0.5Nd0.5 BNPs equivalent to 2.5, 2.0, 1.0, 0.5, and 0.25 mM Nd concentration in PBS in 5 mL tubes through serial dilution of the stock solution. Similar dilutions of Ag0.5Nd0.5 BNPs were also taken in 10 mg/mL fetal bovine serum (FBS) in PBS to simulate in vivo condition. Control phantoms of PBS and 10 mg/mL FBS alone were prepared for comparison. The following parameters were adopted: the field of view (FOV) = 1 × 1 cm2, a repetition time (TR) = 20 ms, base resolution = 256 × 256, and echo time (TE) = 5 ms. Data were then fitted to the following monoexponential function:

T1 = A(1 − e(−TR / T1))

drug loading efficiency(%) =

(5) The drug release kinetics of BNPs-Pax was carried out by suspending 5 mg BNPs-Pax in 10 mL PBS (pH 7.4) containing 0.2% (w/v) CTAB. The solution was taken into a dialysis bag (MWCO = 12 kDa) and immersed in 40 mL PBS containing 0.2% (w/v) CTAB. CTAB was added to increase the solubility of Pax in PBS. The solution was incubated at 37 °C for 100 h, and 2 mL of medium was withdrawn at periodical time intervals. The OD of withdrawn samples were recorded at 227 nm on “ELISA reader” (VersaMax Elisa Microplate Reader) and added back into the medium.

drug release (%) =

free drug released × 100 loaded drug conconcentration

(6)

2.8. Thermodynamics Interaction of Nanoparticles and Drug. The accurate binding affinity of drug molecules with NPs is evaluated through isothermal titration calorimetry analysis using a Nano-ITC (TA Instruments, USA). Among the available techniques to measure the affinity of two molecules, ITC is the most accurate technique, which not only measure the binding affinity but also the important thermodynamic parameters, namely enthalpy change (ΔH) and entropy change (ΔS). For Nano-ITC analysis, separate solutions of Pax and Ag−Nd BNPs (Ag0.5Nd0.5) were prepared in PBS (pH = 7.4) with 0.25 M CTAB, and experiment was carried out at 25 °C. CTAB was used to increase the solubility of hydrophobic drug in water. PBS was degassed thoroughly before sample preparation to remove air bubbles. Reference cell was filled with 150 μL of Ag−Nd BNPs (0.05 μM concentration). In injection syringe, 50 mL of drug solution (1 μM concentration) was loaded. In a single experiment, 24 injections of 50 μL of drug were titrated in 150 μL of NPs solution with 300 s time intervals between consecutive titrations. The heat of dilution of drug was subtracted from interaction profile data, and then data were fitted into independent model using Nanoanalyze software. Dissociation constant (Kd), affinity constant (Ka), ΔH, ΔS, number of drug molecules binding per nanoparticles (n), and Gibb’s free energy (ΔG) were calculated. 2.9. Photothermal Effect. To assess the photothermal ability of Ag−Nd BNPs, a series of concentrations (5 to 200 μg/mL) of Ag0.5Nd0.5 BNPs in PBS were irradiated with an 808 nm laser diode for 10 min at a power density of 2 W/cm2, and temperature variations were determined at regular time intervals using thermocouple. To evaluate the photothermal ability of BNPs after cellular internalization, HeLa cells were incubated with 50 μg/mL BNPs in DMEM overnight and then harvested by centrifugation, and finally washed thrice with PBS. The solutions of blank cells and BNPs-treated cells in PBS, each with 105 cells/mL in PBS, were irradiated under 808 nm laser at a power density of 2W/cm2 in an incubator at 37 °C, and temperature rise was recorded at different time points up to 10 min.

(3) −1

The obtained T1 was converted into R1 [1/T1 (s )] relaxation rate. Finally, R1 values were plotted against the concentration of the contrast agent, and relaxivity r1 (s−1 mM−1) was measured as the slope of the resulting linear plots using the following equation:

R1 = r1[C] + R1*

(OD227 of initial drug) − (OD227 of supernatant) × 100 OD227 of initial drug

(4)

where R1* is the relaxation rate of control phantoms and [C] concentration of Nd in mM. 2.6. Computerized Tomography (CT) Imaging. Phantom of different mass concentrations of Ag−Nd BNPs were prepared in PBS equivalent to 0.07, 0.13, 0.26, 0.51, and 0.65 mg/mL in PBS, and PBS solution as control 5 mL tubes. 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: effective C

DOI: 10.1021/acs.inorgchem.7b02103 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 1. Characterization of Ag0.5Nd0.5 BNPs. (A) TEM analysis: (A1) TEM image with size distribution histogram; (A2) TEM image represents clear image of nanoparticles; (A3) high-resolution TEM image represents crystal fringes of both Ag and Nd; (A4) SAED pattern reflects diffraction rings of Ag and Nd. (B) High-angle annular dark-field scanning TEM (HAADF-STEM) mapping of nanoparticles present nanoscale (50 nm scale) level uniform elemental distribution: (B1) selected STEM image, (B2) Ag, (B3) Nd, and (B4) overlapped image of Ag and Nd. (C) Energy-dispersive X-ray spectroscopy (EDS) elemental mapping shows microscale (10 μm scale) level distribution in uniform manner: (C1) mapped area, (C2) carbon, (C3) Ag, and (C4) Nd. (D) Elemental line spectra. 2.10. Cell Viability of BNPs and Synergistic Chemo-photothermal Therapeutic Ability of BNPs-Pax. For in vitro cellular studies, human cervical cancer cells (HeLa cells) were cultured and maintained 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. HeLa cells were placed in the 96-well plate with 104 cells/well and cultured overnight, and then the medium was replaced with medium containing Ag0.5Nd0.5, Ag0.5Nd0.5-Dox, and medium control with serial concentrations of 5, 10, 25, 50, 100, and 200 μg/ mL. After incubation for next 2 h, cells were irradiated under 808 nm laser of power density 2.0 W/cm2 for 2 min, followed by incubation for next 24 h. The MTT reagent was added, followed by 4 h incubation. Finally, the supernatant was removed, and 200 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 OD values. The cell viability was calculated as follows:

cell viability (%) =

ODtreated × 100 ODcontrol

(7)

where ODtreated is 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. Live/dead assays were accomplished after 24 h incubation using the Live/Dead Viability/Cytotoxicity Assay Kit (ThermoFisher Scientific, CNo. L3224) according to manufacturer’s guidelines. Live cells stained green due to the uptake and hydrolysis of calcein AM, while the nuclei of dead cells were labeled the red due to D

DOI: 10.1021/acs.inorgchem.7b02103 Inorg. Chem. XXXX, XXX, XXX−XXX

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Table 1. Combined Data on Elemental Analysis by ICP-AES, Particle Size Analysis by XRD and TEM, and Hydrodynamic Size by DLS with Polydispersity Index (PDI)a precursor (wt %)

a

ICP-AES (wt %)

size (nm)

sample

Ag

Nd

Ag

Nd

crystallite

hydrodynamic

PDI

Ag0.5Nd0.5 Ag0.75Nd0.25 Ag0.9Nd0.1

42.85 69.24 87.09

57.15 30.76 12.91

47.42 69.97 92.59

52.58 30.06 7.41

12.00 ± 2.50 9.80 ± 3.65 10.20 ± 2.80

30.50 ± 5.0 26.50 ± 6.0 28.80 ± 5.5

0.30 ± 0.05 0.25 ± 0.05 0.34 ± 0.05

Data are presented with standard deviation (± SD) of three independent results.

ethidium homodimer-1. Background levels are low due to nonfluorescent state of both dyes prior to cell interaction. 2.11. Statistical Analysis. All data were presented as mean ± SD (standard deviation). Statistical analysis of data was carried out by ANOVA or Student’s t test and considered significantly different if p ≤ 0.05.

3.2. Morphological Characterization and Elemental Distribution. Figure 1 presents the morphological features and elemental distribution of BNPs. Electron microscopic image ratify the uniform spherical NPs with an average diameter of ∼10 nm (Figure 1 A1,A2). The high resolution TEM (HRTEM) image confirms the presence of both Ag and Nd metals in NPs with their analogous lattice fringes (Figure 1A3). The selected area electron diffraction (SEAD) pattern (Figure 1A4) signifies the presence of diffraction planes for the constituent metals in bimetallic system, which thus epitomizes the homogeneous distribution of Ag and Nd crystals in single entity. The impeccable role of Nd content on the size of BNPs is confirmed by the virtue of the respective hydrodynamic sizes of Ag0.5Nd0.5, Ag0.75Nd0.25, and Ag0.9Nd0.1 determined as 30, 26, and 28 nm with polydispersity index 14%). 35 As a consequence, the homogeneous nucleation of both the metals result in the formation of nanoalloy made of FCC Ag and hexagonal Nd in BNPs. The lattice mismatch between both the metals creates strain and as a consequence induces lattice expansion as a function of Nd content (Figure S2). The amorphous nature of spherical BNPs determined through XRD also garners support from TEM analysis, which show poor diffraction ring for Nd (Figure 1 A4). The HR-TEM image of single particle reflects the random distribution of Ag and Nd nanocrystals with their corresponding interplanar spacing, which is due to the rapid crystallization of amorphous Nd on exposure to the high energy electron beams (Figure 1 A3).33

Figure 3. Photoemission spectra of BNPs: (A) survey spectra, and high-resolution spectra of (B) Ag 3d (C) Nd 3d. F

DOI: 10.1021/acs.inorgchem.7b02103 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 4. Absorption spectra of AgNPs, Ag−Nd BNPs along with Nd3+ precursor (A) full range spectrum from 300−1100 nm; (B) spectrum in near-infrared range from 750 to 950 nm exhibit similarities in NIR absorption of BNPs with characteristic absorption of Nd3+ ions.

peaks characteristic of Nd corroborate the successful synthesis of Ag−Nd BNPs. The characteristic AgNPs band at 400 nm shows red shift with increasing concentration of Nd, which is due to the reduced electron density on the Ag surfaces.14 The NIR absorption spectrum of BNPs exhibit a broad intense band between 785 and 810 nm, with two broad peaks centered at 795 and 805 nm corresponding to 4I9/2 → 4F5/2 and 4I9/2 → 2 F9/2 electronic transitions that are distinctive of Nd precursor (Figure 4B).48−50 Other characteristic peaks of Nd precursors are not clearly apparent in BNPs. Similarities in the absorption peaks of BNPs in NIR with Nd precursor are obvious because the transition electron of Nd belong to the well-shielded inner f-orbitals.49−51 The absorption spectra demonstrate characteristic Nd absorption with electronic transition from ground level 4 I9/2 (4f3) electrons of Nd to different high energy levels as depicted in Figure 4A. The broadening of the absorption peaks of BNPs is possibly instigated from the nanosized crystal distribution of Nd in BNPs.49 Owing to the deep penetration properties of NIR light into the tissue than visible light, the materials with NIR absorption are strongly recommended for in vivo optical imaging.52 Thereby the absorption ability of Ag− Nd BNPs in first biological window (700−950 nm) could be a favorable prospect for hyperthermia and laser phototherapy applications.53 3.6. NIR Imaging Ability in Second Biological Window. NPs with the simultaneous ability of excitation and emission in the near-infrared (NIR-to-NIR) region are auspicious for in vivo optical imaging since NIR (750−1600 nm) light ensures deeper penetration with low absorption and scattering by biological tissues.54 In NIR region, two optically transparent region for biological tissues are 750−850 nm (first biological window, NIR-I) and 1000−1400 nm (second biological window, NIRII).22,55 NIR-II provides more transparent path for the deep penetration of light. Absorption spectra confirms the capability of Ag−Nd BNPs to absorb light in NIR-I region around 808 nm.55 The emission ability of Ag−Nd BNPs in NIR-II region due to the characteristic Nd is measured by selecting the excitation wavelength of 808 nm. On excitation with 808 nm, Ag−Nd BNPs exhibit three main emissions around 910, 1050, and 1330 nm, corresponds to 4F3/2 → 4I9/2, 4F3/2 → 4I11/2, and 4 F3/2 → 4I13/2, which are similar to Nd3+ ions (Figure 5).50,56,57 The emission intensity is high for 4F3/2 → 4I11/2 transition that falls in NIR-II region, a promising feature of Ag−Nd BNPs for in vivo imaging applications.25,58 The intensity of emission

Figure 5. Emission spectra of Ag−Nd BNPs on excitation with 808 nm, reveals emission ability in NIR (750−1600 nm) region, with strong emission in the region of second biological window which is more transparent for deep tissue penetration.

around 1050 nm declines on increasing Nd concentration in BNPs due to quenching effect.59 3.7. Magnetic Properties and MRI Contrast Ability. The magnetic properties of Ag−Nd BNPs were measured using VSM at room temperature (Figure 6A, Table 2). Magnetic measurements reveal superparamagnetic nature of all Ag−Nd BNPs at room temperature. Generally, Nd and its compounds display ferromagnetism below 20 °C and paramagnetism at room temperature.60 By the variations of Nd content in bimetallic composition, increasing magnetic moment has been demonstrated with respect to the increasing Nd in BNPs. The magnetic behavior of Nd depends on the surrounding atoms; antiferromagnetic behavior of Nd3+ ion-doped glasses and paramagnetic behavior of Nd3+-doped aluminate are reported.61,62 Owing to the magnetic properties of BNPs, the longitudinal (1/T1) relaxation rates are exclusively measured due to high longitudinal relaxivity of Nd compounds, as a function of varied concentrations of BNPs in PBS and FBS.63 To evaluate the effectiveness of the Ag−Nd BNPs as a MRI contrast agent, images were gathered by the dispersion of Ag−Nd BNPs in PBS and FBS with gradual increasing concentrations of Nd. The results demonstrate the enhanced brightness in 1/T1 map (Figure 6B) and increment in relaxation rates (Figure 6C) as a function of Nd concentration. As a preliminary effort to diagnose the in vivo biostability of BNPs as a MRI contrast agent, longitudinal (1/T1) relaxation of Ag−Nd BNPs is G

DOI: 10.1021/acs.inorgchem.7b02103 Inorg. Chem. XXXX, XXX, XXX−XXX

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MRI contrast ability of Ag−Nd BNPs due to drug loading. The relaxivity values of Ag−Nd BNPs are comparatively better than those reported for Gd based NPs (Gd2O3 or GdPO4) as MRI contrast agents, which presents r1 relaxivity data between 5.0 to 9.6 s−1 mM−1 at analogous conditions.65,66 Nevertheless, the longitudinal relaxation rate along with other synergistic features make Ag−Nd BNPs a promising candidate for in vivo imaging and imaging guided therapy. The Ag−Nd BNPs appear to be a novel material as a positive contrast agent in conjunction with other imaging abilities. 3.8. CT Contrast Ability. The X-ray contrast ability of Ag− Nd BNPs were evaluated from concentration gradient phantoms of BNPs in PBS (Figure 7) scanned at 120 kV.

Figure 7. X-ray attenuation ability of Ag−Nd BNPs. (A) CT images of the phantoms with reference to PBS control. (B) Attenuation values (HU) vs mass concentration plot showing linear dependence of CT contrast ability.

Figure 6. (A) VSM spectrum reveals superparamagnetic behavior of Ag−Nd BNPs. (B) T1-weighted MR phantom images of Ag−Nd BNPs in PBS and FBS. (C) Longitudinal relaxivity (1/T1) plot against increasing Nd concentrations.

The X-ray attenuation ability of Ag−Nd BNPs displays a linear increment in the brightness with increasing mass concentrations of BNPs (Figure 7A). The attenuation values (Hounsfield unit, HU) of phantoms are measured in respect to PBS control (setting HU = zero for PBS). The HU values Ag−Nd BNPs exhibit a linear trend with increasing BNPs mass concentrations (Figure 7B). The CT signal intensity for Ag0.5Nd0.5 is ∼40 HU for a concentration of 0.50 mg/mL. The high attenuation value of Ag−Nd BNPs make it a better CT contrast agents in comparison to the conventional iodinated material or barium sulfate. Hence, in lieu of the conventional CT contrast agent like iodinated materials, barium sulfate which are toxic, the use of Ag−Nd BNPs with high HU complementary with MRI contrast ability could be advantageous and cost-effective. The HU value for omnipaue at 4 mg/ mL is reported as 208.3 HU.67 The high HU value of Ag−Nd BNPs could be attributed to the high atomic number and electron density of Nd (60, 7.01 g/cm3) than iodine (53, 4.93 g/cm3) and also high electron density of Ag (10.49 g/cm3), cumulative effect to enhance the X-ray attenuation property of Ag−Nd BNPs.68 3.9. Drug Loading Efficiency, Drug Release Kinetics, and Binding Affinity. The Pax loading efficiency of Ag−Nd BNPs is determined as 9 ± 2% (n = 3). The pharmacokinetics study of BNPs-Pax has been carried out in PBS containing CTAB to enhance the solubility of Pax in water. The release

Table 2. Magnetic Properties of BNPs Determined through VSM Analysis at Applied Field of 10 000 G (1 T) magnetic properties sample Ag0.5Nd0.5 Ag0.75Nd0.25 Ag0.9Nd0.1

coercivity (G) 157.94 217.45 218.40

magnetization (emu/ g) −3

99.06 × 10 41.02 × 10−3 14.46 × 10−3

retentivity (emu/g) 5.04 × 10−3 6.16 × 10−3 1.52 × 10−3

reviewed after incubation in fetal bovine serum (FBS) at physiological pH (7.4) and temperature (37 °C) for 24 h. Basically, free magnetic ions possess high relaxivity than NPs, however, after in vivo protein corona formation and/or agglomeration, NPs express sharp decline in relaxivity.64 To avoid such artifact, MRI study is also carried out after incubation in FBS, a plasma protein. The results illustrate a marginally reduced relaxivities after incubation in FBS, and thus ensure the in vivo stability of BNPs. The r1 relaxivity values are determined as 9.7 and 6.4 s−1 mM−1 for Ag−Nd BNPs in PBS and FBS solutions respectively at 1.5 T. The relaxivity data of drug-loaded Ag−Nd BNPs is determined as 9.3 ± 0.2 s−1 mM−1 (n = 3), a value that is marginally less than the unloaded Ag−Nd BNPs. This infers the negligible effect on magnetic and H

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Figure 8. Isothermal titration calorimetric analysis (Nano-ITC) of drug and nanoparticle interaction: (A) heat change with increasing number of injections, (B) enthalpy change per mole of paclitaxel plotted against the concentration of BNPs in titration cell after deducting the heat of dilution, (C) thermodynamic parameters extracted from Nano-ITC analysis, and (D) drug release kinetics.

kinetics reveals a sustained release of Pax with 38 ± 3% for 100 h after initial burst release (Figure 8D). Drug release from BNPs depends on interactions between Pax molecules and CS, which is mainly governed by the hydrophobic force. The interaction of Pax with BNPs surface is also expected to play an important role in sustained release of drug. To study the interaction between Pax and BNPs, calorimetric analysis was carried out using a Nano-ITC. Binding affinity of Pax in CScoated Ag−Nd BNPs was evaluated through ITC analysis in terms of the enthalpy (ΔH) and entropy (ΔS) changes and affinity constant (Ka). The titration of Pax to Ag−Nd BNPs expresses a progressive upsurge in exothermic heat with the increasing number of injections (Figure 8A). The heat evolved during interaction of hydrophobic drug molecules with BNPs yields negative ΔH, which implies a strong interaction of drug with BNPs. The integrated data of the ΔH per mole of Pax are plotted against the concentration of BNPs in titration cell after deducting the heat of dilution (Figure 8B). This ΔH is mainly due to the absorption of Pax molecules on CS-coated BNPs and partially due to the electrostatic interaction of polar groups of Pax with BNPs.69 Low dissociation constant (Kd) confirms strong affinity of Pax with BNPs (Figure 8C). The number of Pax molecules binding per NPs is calculated as 7, which endorses the high loading efficiency of CS-coated BNPs. The ΔG value of −21 kJ/mol indicates the feasibility of Pax absorption in BNPs and authorizes strong binding affinity of Pax at physiological condition, a major prerequisite to reduce systemic side effect of drug due to premature release. The binding affinity (Ka) calculated as 4.89 × 103 M−1, implies slow

drug release at physiological condition and thus clearly endorses the ability of Ag−Nd BNPs for drug delivery to the target site. The hydrodynamic size of Ag0.5Nd0.5 BNPs has increased to 42.0 ± 6.0 nm after drug loading in comparison to the 30.50 ± 5.0 nm size of unloaded BNPs. Similarly, increase in the PDI of the drug-loaded BNPs has also been determined as 0.36 in comparison to the 0.30 PDI of the unloaded BNPs. This upsurge in the hydrodynamic size and polydispersity of BNPs on drug loading is mainly due to the partial aggregation of BNPs, which might have caused by the reduced surface potential. 3.10. Photothermal Effect of BNPs and in Vitro Photothermal Studies. Owing to the absorption and emission properties of Ag−Nd BNPs in NIR region, the photothermal ability was examined by exposing various concentrations of Ag−Nd BNPs to an 808 nm laser (Figure 9A). At a concentration of Ag−Nd BNPs above 50 μg/mL, 2 min short period NIR irradiation intends to raise the temperature of solution >40 °C, a criteria necessitated for thermal ablation of tumor cells. The rise in temperature is rapid during the first 5−6 min and thereafter the temperature of solutions attains a plateau. The rate of temperature rise and the final temperature at any time point directly correlates with the concentration of BNPs. The solution devoid of BNPs exhibits negligible change in temperature on laser irradiation. The temperature of the 50 μg/mL BNPs solution exhibits sufficient rise in temperature within a short span of 2 min laser irradiation, a major benchmark for the photothermal tumor cell ablation. I

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Figure 9. (A) Photothermal effect of Ag−Nd BNPs examined by exposing various concentrations under 808 nm NIR laser at a power density of 2 W/cm2 up to 10 min. (B) Temperature plot of HeLa cells treated with 50 μg/mL solution of Ag−Nd BNPs irradiated under 808 nm laser at a power density of 2 W/cm2 up to 10 min. (C) HeLa cell viability treated with BNPs (Ag0.5Nd0.5), BNPs + Laser, BNPs-Pax, and BNPs-Pax + Laser under different concentrations. (D) Fluorescence images of treated cells (scale bar = 100 μm). Data are shown as the means ± standard error of the means, with statistical significance indicated as *p < 0.05 and **p < 0.001.

cell viability up to 60% for 200 μg/mL BNPs concentration, which directly correlates with the photothermal ability of BNPs. When cells were treated with the BNPs-Pax, the cell viability showed exponential reduction with increasing BNPs-Pax concentration. 50 μg/mL BNPs-Pax treatment induced 45% cell death while photothermal treatment of 2 min laser irradiation results in >85% cell death for analogous concentration of BNPs-Pax (p < 0.001). “Combined chemophototherapy” reveals synergistic effect of Pax and thermal ablation as 80% cell death is observed for 25 μg/mL BNPs-Pax. On the contrary, any single therapy either chemo or photo exhibits negligible therapeutic effect as only 10−20% cell death is witnessed. To achieve therapeutic effect through single therapy, comparatively high dose of ∼150 μg/mL of BNPs-Pax for chemotherapy, and high dose along with longer irradiation time for phototherapy is needed. The combined therapeutic ability of BNPs-Pax is advantageous for in vivo applications where minimal concentration of anticancer drug is sufficient for optimal therapy that avoids the chance of systemic side effect and drug-resistance of cancer cells. More accurate cell viability assay was accompanied by using Live/Dead cell staining technique, which segregate viable cells in green and dead cells in red. The ethidium homodimer-1 penetrates cells with a compromise in plasma membrane and subsequently interacts with DNA, whereupon the dead or dying cells stains in bright red color. The calcein AM is membrane permeable dye, readily penetrate cell membrane and get hydrolyzed, which labels the cells in green color. Live/Dead fluorescence staining further corroborates the excellent

In vitro photothermal effect on tumor cell was appraised by incubating HeLa cells with BNPs overnight and then collected in vials (Figure 9B). The solutions of blank cells and BNPstreated cells in PBS, each with 105 cells/mL in 200 μL tubes were irradiated under 808 nm laser. The BNPs-treated cells show sharp rise in temperature that exceed the thermal ablation temperature within 1 min, and this inference clearly validates the application of BNPs in cancer PTT. 3.11. Cell Viability and Chemo-photothermal Therapeutic Effect. Cytotoxicity is a major concern for in vivo application of metallic NPs. For PTT of cancer, biocompatibility of photothermal transducers is highly desirable and therefore healthy tissues at the vicinity of tumor site should be least affected. The cytotoxicity of BNPs (Ag0.5Nd0.5) assessed on HeLa cells for 200 μg/mL unveils cell viability above 85% after 24 h incubation (Figure 9C). Therefore, the CS-coated Ag−Nd BNPs has not shown any obvious cytotoxicity to HeLa cells. The cell density marginally gets reduced as the concentration of BNPs exceeds 50 μg/mL, possibly due to the inhibition of cytokinesis in cells infected with high NPs content. However, Pax-loaded Ag−Nd BNPs exert cytotoxicity in various degrees. The therapeutic ability of BNPs (Ag0.5Nd0.5) under NIR laser irradiation (BNPs + laser), paxlitaxel-loaded BNPs (BNPs-Pax) and combined therapy of BNPs-Pax under NIR laser irradiation was assessed on HeLa cells (Figure 9C). The cell viability plots as a function of BNPs concentrations unveil the existence of 85% viable cells even after 24 h incubation up to 200 μg/mL. Nevertheless, while on NIR laser irradiation for 2 min, the BNPs treated cells showed reduced J

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MCIT, Government of India, are gratefully acknowledged. Dr. Ramadas Krishna, Assistant Professor, and Ms. T. Pragna Lakshmi, Research Scholar, Department of Bioinformatics, Pondicherry University, are gratefully acknowledged for their support in Nano-ITC characterization and analysis.

therapeutic efficacy of combined chemo-photothermal therapy in comparison to the individual chemotherapy or photothermal therapy and also elicit negligible cytotoxicity of BNPs (Figure 9D).

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4. CONCLUSION CS-coated Ag−Nd BNPs are successfully synthesized through microwave-assisted polyol synthesis for application in theranostics. The CS-coated BNPs exhibit excellent monodispersity with average size of 10 nm, fluorescence in the NIR region, and weak ferromagnetic properties. Due to intrinsic magnetoplasmic properties, BNPs corroborate longitudinal relaxivity for MRI contrast ability and fluorescence in the second biological window. Due to the high X-ray attenuation coefficient of Nd and ultrasmall crystal size of Ag, BNPs exhibit better CT contrast ability than the reported values for its counterparts. The loading efficiency of paclitaxel drug is determined as 7 drug molecules per nanoparticle, and the assessment of thermodynamic parameters confirms the strong binding affinity of drug with BNPs. Phototheramal efficacy of BNPs reveals a dosedependent temperature rise in short duration. In vitro studies establish that “combined chemo-phototherapy” possesses the ability to absolutely destroy cancer cells with a single dose of 50 μg/mL BNPs-Pax +2 min NIR laser irradiation. Ag−Nd BNPs show the ability of guided “chemo-phototherapy” for safe and effective cancer theranosis. The experimental results from the investigation imply that the multifunctional nanoparticles combined with NIR/MRI/CT imaging and drug delivery may ensure advanced application for concurrent diagnosis and treatment of cancer. The adopted synthesis technique is also attractive because a multifunctional nanoparticle can be primed in “one pot” with no toxic residues. Future work will be dedicated to evaluating this material for magnetic field guided therapy and multimodal imaging for precise diagnosis in vivo.

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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.inorgchem.7b02103. XRD reflections, lattice parameters, photoemission spectra, and FTIR spectra of BNPs, and DLS analysis of colloidal stability (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

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

Sandeep K. Mishra: 0000-0002-1016-0206 S. Kannan: 0000-0003-2285-4907 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial assistance received from the Department of Science and Technology−Science and Engineering Research Board (Reference: EMR/2015/002200 dated 20.01.2016) India is acknowledged. The use of facilities at the 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, K

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DOI: 10.1021/acs.inorgchem.7b02103 Inorg. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.inorgchem.7b02103 Inorg. Chem. XXXX, XXX, XXX−XXX