Doxorubicin-Conjugated Bimetallic SilverâGadolinium Nanoalloy for

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX-XXX

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Doxorubicin-Conjugated Bimetallic Silver−Gadolinium Nanoalloy for Multimodal MRI-CT-Optical Imaging and pH-Responsive Drug Release Sandeep K. Mishra and S. Kannan* Centre for Nanoscience and Technology, Pondicherry University, R. V. Nagar, Kalapet, Puducherry 605 014, India S Supporting Information *

ABSTRACT: Monodispersed chitosan-capped bimetallic nanoparticles (BNPs) of AgGd have been synthesized for “cancer theranosis” through environmentally benign microwave-assisted polyol synthesis. In the present article, we report the one-pot synthesis of AgGd BNPs with varied Ag:Gd molar ratios via coreduction of Ag+ and Gd3+ ions. Studies reveal a well-dispersed AgGd BNPs of average size 12 nm and also the presence of face-centered cubic (FCC) Ag and cubic Gd components within the individual crystal. Chitosan (CS) a biopolymer, as a capping agent facilitates the bioconjugation of doxorubicin (Dox), a potential anticancer drug on the surface of BNPs. The Dox was covalently conjugated onto the BNPs through pH-sensitive hydrazone linkage with CS. In vitro study reveals negligible drug release at physiological pH while release rate accelerates in acidic medium. The mutual properties of the host metals in AgGd BNPs at the nanoscale offer concurrent magnetic resonance imaging (MRI) and computed tomography (CT) contrast performances. Moreover, the paramagnetic behavior inherited by Gd in BNPs demonstrates both T1 and T2 contrast ability. BNPs ensures biocompatibility and also express sturdy therapeutic effects in HeLa cells when conjugated with Dox. KEYWORDS: chitosan, bimetallic nanoparticles, MRI, CT, drug delivery

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antibacterial activities.10 On the contrary, the biologically functionalized AgNPs are also synthesized in various shapes and sizes to tailor the desired optoelectronic properties.11 Biocompatibility and luminescence features of polymer-coated AgNPs is a welcome prospect in theranostic applications for cancer treatment.12,13 Despite the lower atomic number of silver than iodine, AgNPs exhibit almost comparable X-ray attenuation intensity to omnipaque with iodine concentration similar to silver.14 Gadolinium (Gd) is a well-known MRI contrast agent (CA) which shortens the longitudinal (T1) relaxation time of surrounding water protons.15 The seven unpaired 4f electrons (8S7/2) of Gd3+ possess the ability to facilitate a large electron magnetic moment. Current generation Gd-based small molecular MRI contrast agents fall off to a large extent due to its diffusion along normal epithelia, and thus get rapid eliminated from the blood circulation. Recently, there has been a demand to develop more sensitive and advanced T1 MRI contrast agents apart from Gd3+ chelates. In this context, NPs based on Gd2O3, GdPO4, and GdF3 have been reported.16−18 Most of these NPs showed larger r1 values than the Gd(III)

INTRODUCTION Over the past two decades, formulation of nanostructured materials with desired properties have become an enthralling field to address issues of early diagnosis and targeted cancer therapy.1 In cancer nanomedicines, embedding both diagnostic and therapeutic abilities in a single entity is an interesting approach to reduce patient complications and early detection, and is designated as “nanotheranostics”.2,3 A smartly architected bimetallic nanostructures provide an opportunity to assemble unique properties in a single entity.4,5 Advances in modern clinical imaging techniques have made rapid strides in clinical diagnosis; however, with severe constraints to gain comprehensive information from a single technique.5,6 Among them, computed tomography (CT) is employed as a diagnostic tool for anatomical imaging whereas magnetic resonance imaging (MRI) is prevalent for soft tissue resolution. Nanochemistry architects the ability to integrate multiple imaging techniques by introducing nanoparticles (NPs) that exhibit intrinsic abilities for multimodal imaging. Over recent years, noble metal NPs are exclusively investigated as potential candidates for bioimaging and drug delivery owing to their chemical stability and size dependent optoelectronic properties.7−9 Silver nanoparticles (AgNPs) are currently incorporated in many medical products in large scale due to their exclusive size dependent optical properties and broad spectrum © XXXX American Chemical Society

Received: July 18, 2017 Accepted: October 26, 2017

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DOI: 10.1021/acsbiomaterials.7b00498 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering chelates, depending on their particle size.16−18 NPs-based MRI contrast agents rarely diffuse across blood capillaries and show longer circulation time. Moreover, NPs can cross cellular barriers, which encompass to design novel AgGd BNPs. There are numerous reports on the development of NPs-based MRI/ CT contrast agents that focus on the modification of NPs with Gd3+ chelates.19,20 Nonetheless, this approach possess the shortcomings of limited functionalization, low payload of magnetic centers and unusual leaching of chelates. In this context, a bimetallic system with nanocrystals of both Ag and Gd could be a novel approach to design a multimodal contrast agent. Chitosan (CS) offers remarkable characteristics of nontoxicity, biocompatibility and cellular adhesion.21,22 Preferred cellular uptake of CS coated NPs via overcoming the endothelial cell barrier has been reported.23 A biocompatible device of CS coated AgNPs is reported for photothermal therapy of cancer.24 Thus, a CS capped nanoparticle could be a better candidate for bioimaging and drug delivery due to its natural biocompatibility, and the abundant amine groups that could facilitate targeting and conjugation of ligands. Doxorubicin (Dox) is a leading potent anticancer drug, used against diverse cancer types. Dox is poorly water-soluble because of its hydrophobic nature, and cause severe toxicity to healthy tissues during direct injection. Covalent conjugation of Dox to CScoated NPs is anticipated to reduce the chance of systemic side effect due to premature drug release. Moreover, because the pH values of extracellular matrix (ECM) and endosomes of tumor cells are often acidic and the hydrazone linkage hydrolyses in acidic condition, the conjugation of Dox with NPs through hydrazone linkage could facilitate precise therapeutic effect without severe side effects. In recent years, bimetallic nanoparticles (BNPs) elucidate special attention because of their unique properties in comparison to respective monometallic systems.25−27 Silver− gold BNPs are exclusively reported for specific detection and therapy of cancer.25,28 Successful synthesis of europium and gold (Eu−Au) BNPs through bioreduction using plant extract has been reported.29 Au−Fe nanoalloy has been reported as an excellent candidate for multimodal MRI-CT-SERS imaging.4,30 Because of unique luminescence and magnetic properties, much interest has been devoted on bimetallic lanthanide bioprobes for applications in cellular imaging, cancerous cell detection and DNA tagging.31−33 A recent report emphasize the synthesis of Ag−Dy BNPs as a theranostic device for MRI-CT-NIR multimodal imaging and drug delivery to cancer cells.9 Most of the available methods to synthesize NPs are based on the use of harmful chemicals, either as reducing agents or as stabilizing agents. Furthermore, the use of reducing agents or surfactants also intend to complicate the synthesis and purification processes, and thus restrict their use in biological applications.34 Herein, we report one-pot synthesis of CScapped Ag−Gd bimetallic nanoparticles (BNPs) by polyol coreduction of AgNO3 and Gd(NO3)3 via green chemistry, and bioconjugation of doxorubicin a potent cancer drug, with CScapped AgGd to fabricate a cancer theranostic device.

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was procured from Hi-media, India. Deionized water was used for all syntheses and has been mentioned as water throughout. Method of Synthesis. Synthesis of Water-Soluble Chitosan Oligomer and Its Characterization. Chitosan (CS) was further degraded to find a short chain oligomeric compositions, which should be soluble in water of 8−9 pH. Chitosan (2g) was dissolved in 100 mL of 1% acetic solution, and then the solution was irradiated by Co-60 gamma (γ) radiation (Gamma chamber 5000, Atomic Energy Regulatory Board (AERB), INDIA) with 100 kGy dose at 4 kGy/h exposure rate.35 The degraded chitosan sample was added with an adequate amount of 2 M NaOH to make the pH ∼ 9 and was then filtered through a Nylon-66 hydrophilic syringe membrane (Hi-media, India) of 0.22 μm. The filtered solution was lyophilized at −80 °C for 24 h. This purified chitosan oligomer (CSo) was then dissolved in water of pH 8 to prepare a stock solution of 1 mg/mL. Molecular Weight Determination of CSo. The γ irradiation scissions glycosidic bonds of CS without affecting any chemical change which results in reduction in molecular weight (MW) of CS. The average MW of γ irradiated CS was determined through viscosity (η) measurements using a capillary viscometer (Ubbelohde Capillary Viscometer type 531/10 I) at 25 °C. The degraded chitosan (CSo) was dissolved in acetate buffer (0.15 M ammonium acetate and 0.2 M acetic acid) to make a stock solution of 10 mg/mL, and was diluted in various concentrations. The η of CSo solution was determined 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)36

η = KM vα

(1)

Where K and α are constants for a particular polymer−solventtemperature 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 pristine CS solution was measured as 1558.0 mL/g, which reduced to 52.2 mL/g in the case of irradiated CSo solution at a dose of 100 kGy. Consequently, the measured viscosity-average MW of degraded chitosan (CSo) reduced to 12 kDa from its pristine CS of viscosity-average MW 190 kDa. Chemical Characterization of CSo. FT-IR spectra was recorded to confirm chemical similarities of irradiated chitosan (CSo) with the pristine CS. 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. Both, the CS and irradiated CSo have showed remarkable similarities in the characteristics peak positions of amide I, II, and III at 1656, 1592, and 1256 cm−1 respectively (Figure S6). Microwave Synthesis of Chitosan Coated Bimetallic Nanoparticles. As per our concerns about biological applications, 30% v/V glycerol was used as a reducing agent. One-pot synthesis of CS capped AgGd bimetallic nanoparticles (BNPs) were carried out through microwave assisted polyol synthesis, by taking 30% glycerol solution in water followed by the addition of 0.2 mg/mL CSo, as a capping agent in Erlenmeyer flasks. To this CSo and glycerol mixture were added 2.50, 3.75, 4.50, and 5.00 mL of AgNO3 stock solution (50 mM) and 2.50, 1.25, 0.50, and 0.00 mL of Gd(NO3)3 stock solution (50 mM) to maintain a final volume of 10 mL each. These solution mixtures were respectively coded as Ag0.5Gd0.5, Ag0.75Gd0.25 Ag0.9Gd0.1 and AgNPs. Required amounts of ammonia were added to maintain the pH of solutions 8.0 ± 0.3. 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 microwave irradiation, the transparent solutions turned brownish yellow color, and become darker as a function of enhanced Gd content. Finally, the solutions were immediately cooled in an ice bath after the completion of reaction, and were centrifuged at 12000 g for 40 min with the addition of ethanol. Samples were then washed with 100, 50, and 30% ethanol solution, and finally twice with distilled water. For washing, the centrifuged BNPs were dissolved in respective volume ratios of ethanol and water, and were centrifuged at 12 000 g for 40 min to collect and the process was repeated until the BNPs were collected in water. Characterization. UV−vis spectra were collected using spectrophotometer (PerkinElmer Lambda 650 S, USA) in the range of 200−

EXPERIMENTAL SECTION

Materials. All the reagents were obtained 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) B


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ACS Biomaterials Science & Engineering 800 nm using a quartz cell of path length 1 cm. Luminescence measurements were carried out using a photoluminescence spectrophotometer (HORIBA Jobin Yvon Fluorolog - FL3−11, USA). The band-pass for excitation and emission was set as 3 nm. Picosecond-resolved fluorescence decay transients were measured with 60 ps instrument response function (IRF). For all samples, the excitation/emission wavelengths were set at 350/500 nm. The crystalline phase and purity of materials were 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 per second. XRD reflections were also recorded after heating samples up to 800 °C under nitrogen atmosphere to confirm the crystalline purity of samples. Crystalline purity of constituent metals were confirmed by comparing recorded XRD data with the standard JCPDS data of metals. Percent lattice parameter mismatch (%Δa) is determined using the equation given below:

also prepared in 10 mg/mL fetal bovine serum (FBS) in order to simulate in vivo condition and preliminary tests were performed to endorse the stability of BNPs. Control phantoms consisting of PBS and 10 mg/mL FBS alone were concurrently imaged. The following parameters were adopted for T1 measurement: a repetition time (TR) = 20 ms; echo time (TE) = 5 ms; base resolution = 256 × 256; field of view (FOV) = 1 × 1 cm2. The following parameters were adopted for T2 measurement: TR = 3000 ms, TE = 22; FOV = 1 × 1 cm2; base resolution = 256 × 256. For each sample, the signal intensity on magnitude images was averaged within regions of interest (ROIs) and plotted against TR for T1 recovery curves or TE for T2 decay curves. Data were then fitted to the following monoexponential functions: T1 = A(1 − e(−TR / T1))

T2 = A(1 − e(−TR / T2))

(2) (3) −1

The obtained T1 and T2 values were converted into R1 [1/T1 (s )] and R2[1/T2 (s−1)] relaxation rates. Finally, R1 and R2 values were plotted against the concentration of the corresponding contrast agent, and relaxivities r1 and r2 (s−1 mM−1) were obtained as the slope of the resulting linear plots using the following equations

2(a1 − a 2) %Δa = 100 (a1 + a 2) Where a1 and a2 correspond to lattice parameters of the metal crystals. The lattice parameters and cell volume of bimetallic nanoparticles were determined by employing the Rietveld method using GSASEXPGUI software package to explore the effect of increasing concentration of Gd in solid solutions of Gd and Ag. For the analysis through Rietveld refinement, an average of three X-ray scans was recorded for each sample. Thermal properties of both the pure Ag0.5Gd0.5 and the one bioconjugated with doxorubicin drug were assessed 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). Multitechnique X-ray Photoelectron Spectroscopy (XPS, Axis Ultra) was utilized 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 3 times to reduce the signal-to-noise ratio. CasaXPS software was used to analyze XPS data. The size and morphology of nanoparticles were determined by the transmission electron microscope (FEI Tecnai G2 T30, Netherlands). The particle size analysis of nanoparticles 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 of dust 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 performed with counting rates varying between 400 and 800 kilo counts per second (kcps). The elemental detection and distribution of nanoparticles was analyzed using highangle annular dark-field scanning TEM (HAADF-STEM) mapping simultaneously with TEM analysis and using field-emission scanning electron microscope (FE-SEM), along with elemental mapping by electron-dispersive X-ray spectroscopy (EDS) (JEOL, JSM-7100F, Japan). Compositional analyses of the Ag and Gd were done with the aid of inductively coupled plasma atomic emission spectroscopy (ICPAES, Arcos, Spectro, Germany). Magnetic properties of bimetallic nanoparticles were assessed using vibrating sample magnetometer (Lake Shore: 7404, USA). In Vitro MRI. In vitro longitudinal (1/T1) and transverse (1/T2) relaxation rates were measured on 1.5T Siemens Magnetom Essenza and MR images were obtained using Siemens’ Syngo software (Siemens Medical Systems), in conjunction with a 32 channel head coil at a constant temperature of 25 °C in a vertical 1.5 T magnetic field. Stock solutions of Ag0.5Gd0.5 in PBS and FBS were prepared, and Gd concentration was determined using ICP-AES. Phantoms of Ag0.5Gd0.5 were prepared from stock solution through serial dilutions with equivalent Gd concentration in the order of 2.5, 2.0, 1.0, 0.5, and 0.25 mM Gd in PBS. Phantoms with similar Gd concentrations were

R1 = r1[C] + R1*

(4)

R 2 = r2[C] + R 2*

(5)

Where R1* and R2* are relaxation rates of control phantoms and [C] concentration of Gd in mM. In Vitro CT Scan. Various mass concentrations of Ag0.5Gd0.5 nanoparticles were prepared in PBS equivalent to 0.067, 0.134, 0.268, 0.536, and 0.670 mg/mL in PBS, and PBS solution as control and were taken in a 5 mL tube 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: effective 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. Biocojugation of Doxorubicin with CS -Coated BNPs and in Vitro Drug Release. Bioconjugation of Doxorubicin (Dox) with CS on the surface of bimetallic nanoparticles (BNPs) can be done by using coupling reagents, such as 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). EDC-NHS promotes coupling by removal of H2O molecule between amino, hydroxyl and carboxyl groups. Succinic anhydride was employed to react with and convert the amine group of doxorubicin to carboxylic residues, i.e., succinic acid residues. The resulting succinyl doxorubicin (SDox) was then covalently conjugated with chitosan via amide bond formation interceded by EDC and NHS.37 Briefly, SDox was synthesized by dissolving 10 mg Dox-HCl in 5 mL dry acetonitrile and 100 μL triethylamine followed by the addition of 100 mg succinic anhydride. The mixture was kept constantly stirred overnight at 4 °C in the dark. Ethyl acetate was added to extract residual solution after lowering the pH using 1 M HCl and, ethyl acetate was evaporated by a rotary evaporator to obtain SDox. For covalent conjugation of SDox to CS capped AgGd BNPs, 10 mg of SDox and 10 mg of BNPs were dissolved in 10 mL of acetate buffer (pH 6.5) that contained EDC (25 mg) and NHS (20 mg), and the solution was stirred overnight at 4 °C, in the dark. Thereafter, solution was centrifuged at 12 000 rpm for 15 min to remove free SDox, and the precipitate (Ag0.5Gd0.5-Dox) was washed thrice with PBS. Dox conjugated BNPs was freeze-dried at −80 °C and collected as a powder. 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. The pH-dependent Dox release from conjugated nanoparticles has been evaluated in pH 7.4, 6.5, and 5.0 PBS at 37 °C. Briefly, 5 mg of freeze-dried Ag0.5Gd0.5-Dox was suspended in 10 mL PBS and C


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ACS Biomaterials Science & Engineering Scheme 1. Schematic Depiction of Synthesis Mechanism and Bioconjugation of Druga

a

Panel A represents the synthesis mechanism of chitosan (CS)-capped AgGd bimetallic nanoparticles (BNPs); panel B shows the bioconjugation of doxorubicin on the surface of BNPs and its pH-dependent drug release.

transferred into a dialysis bag of 3500 Da molecular weight cutoff, and then dialysis bag was immersed in 40 mL of PBS solution of predetermined pH. The complete setup was incubated at 37 °C. At different time points, 2 mL of release media was collected and replaced with an equal volume of fresh media. The amount of DOX released was determined by recording optical density (OD) at 485 nm. The cumulative drug release was determined by measuring the amount of Dox conjugated in BNPs. A 5 mg of freeze-dried Ag0.5Gd0.5-Dox was dissolved in 10 mL of 0.2 M HCl and after 12 h the amount of Dox conjugated in BNPs was determined by recording the OD of solution after centrifugation at 8000 rpm for 30 min. The cumulative drug release % was calculated by following equation: drug release (%) =

free DOX released in supernatant 100 loaded DOX concentration

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 Cell Staining. Live/dead cell assays were performed after 24h incubation using the Live/Dead Viability/Cytotoxicity Assay Kit (ThermoFisher Scientific, CNo. L3224). As done earlier, 10 000 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 50 μg/mL Ag0.5Gd0.5, 50 μg/mL Ag0.5Gd0.5-Dox, and medium control, incubated for 24 h, and finally stained with Live/ Dead assay kit according to manufacturer’s guidelines. Live cells fluoresced green due to the uptake and hydrolysis of calcein AM, whereas the nuclei of dead cells were labeled the red due to ethidium homodimer-1. Background levels are low because of nonfluorescent state of both dyes prior to cell interaction.

(6)

Cell Culture. Human cervical cancer cells (HeLa cells) were purchased from National Centre for Cell Science, India. The 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. In Vitro Cell Viability Assay. Cultured HeLa cell line was utilized to test in vitro biocompatibility of Ag0.5Gd0.5 BNPs and cell apoptosis effect of Ag0.5Gd0.5-Dox. A stock solution of 10 mM MTT was prepared in PBS and preserved at −8 °C. Briefly, 10,000 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 Ag0.5Gd0.5, Ag0.5Gd0.5-Dox and medium control. After predetermined incubation time, the medium of each well containing a particular agent was removed. For cell viability measurement, MTT containing medium of 120 μL 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 OD values. The cell viability was calculated as follows

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RESULTS AND DISCUSSION General Synthetic Route. In a typical synthesis, a transparent solution of AgNO3 and Gd(NO3)3 in 30% glycerol and CS turns brownish yellow on heating under MW, which indicates the formation of AgGd BNPs. Three different molar ratios of [Ag+]:[Gd3+] are deliberately selected as 9:1, 3:1 and 1:1 for BNP synthesis and coded as Ag0.9Gd0.1, Ag0.75Gd0.25, and Ag0.5Gd0.5 respectively. Microwave (MW) energy source was used for its salient features of quick and uniform heating with low energy consumption and the ability to yield high concentration of stable monodispersed nanoparticles.38 On increasing the concentration of Gd3+ with respect to Ag+, reduction time was found increasing exponentially on constant heating at 90 °C (100 W MW) (Figure S1). Because of the thermodynamically feasible reduction of Ag+, reduction of Gd3+ D


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ACS Biomaterials Science & Engineering

Figure 1. (A) Absorption spectra showing red shift and quenching of the Ag surface plasmon resonance with increasing Gd concentration in solution and (B) photoluminescence emission spectra of BNPs on excitation at 350 nm and (C) life decay on excitation and emission set (λex/λem) of 350/ 500. The digital photographs of AgNPs and BNPs solution taken under (D) daylight and (E) UV lamp. Designations are [1] AgNPs, [2] Ag0.9Gd0.1, [3] Ag0.75Gd0.25, [4] Ag0.5Gd0.5, [5] Gd*, and [6] 350 μg/mL solution of Ag0.5Gd0.5 cell culture media (DMEM media).

analysis reveals the influence of Gd presence to enhance the half-life decay, which is almost twice the life decay of AgNPs. The half-life decay of AgNPs, Ag0.9Gd0.1, Ag0.75Gd0.25, and Ag0.5Gd0.5 was determined as 3.62, 6.98, 6.39, and 6.65 μs respectively. The increased life decay indicates a better suitability of BNPs for optical imaging especially for cellular imaging. Colloidal solutions of AgNPs and BNPs were illuminated under UV light (Figure 1E). Ag0.5Gd0.5 solution (350 μg/mL) in cell culture media (DMEM media) was also illuminated to confirm the stability of BNPs (Figure 1E[6]). The UV exposed images of NPs solution clearly reflect the reduced brightness of solutions as a function of enhancement in Gd concentration and there was no luminescence from Gd* solution (100% Gd). Morphology and Composition Assessment. The TEM images of Ag0.5Gd0.5 BNPs reveal an average particle size of ∼12 nm (Figure 2A5). The HRTEM image in a broader range reflects the presence of both metals Ag and Gd in NPs with their corresponding well-defined lattice spacing (Figure 2A3). The polycrystalline nature of the bimetallic system was confirmed by the selected area electron diffraction (SAED) pattern. The SAED pattern (Figure 2A4) of the representative sets shows the concurrent presence of planes for both metals in a single system, which thus reveals the homogeneous distribution of Ag and Gd in the polycrystalline system. The detected lattice fringes possess d-spacing of 0.240 and 0.312 nm, which corresponds to the 111 planes of FCC Ag and cubic Gd respectively. The hydrodynamic size (DH) of BNPs are demonstrated as 24.7, 23.2, and 22.5 nm for Ag0.5Gd0.5, Ag0.75Gd0.25, and Ag0.9Gd0.1 respectively, which are larger than the size determined using TEM, this is solely due to the hydrated CS capping (Figure S2, Table 1). Increasing Gd concentration did not show any sign of impact on DH and has produced NPs of a smaller DH with polydispersity index 14%).47 Thus, the coreduction product is a homogeneous mixture of FCC Ag and cubic Gd in BNPs. This lattice mismatch generates strain in the bimetallic system, which is followed by the lattice expansion as a function of Gd content (Figure S5). The broad XRD peaks suggest small grain size, and crystal grain sizes of Ag was determined from the Scherrer equation as 12.6 ± 1, 13.6 ± 2, and 16.7 ± 2 nm for Ag0.5Gd0.5, Ag0.75Gd0.25, and Ag0.9Gd0.1, respectively. Surface Characterization and Oxidation State of Ag and Gd in BNPs. X-ray photoelectron spectroscopy (XPS) was executed to determine the exact oxidation states of constituent metals in AgGd bimetallic systems and capping ability of CS (Figures 4). Figure 4A presents the survey XPS spectra of Ag0.5Gd0.5, which depicts carbon (C 1s), oxygen (O 1s), gadolinium (Gd 3d, 4d) 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 reduced metallic state of Ag in AgGd bimetallic system (Figure 4B). 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 eV for Ag2+.48,49 The core level photoemission of Gd 3d electrons exhibit doublets at 1188.7 eV for 3d5/2 and 1220.9 eV for 3d3/2, with a shift from 1186.1 eV for 3d5/2 and 1218.0 eV for 3d3/2 of Gd3+ ions (Figure 4C).50,51 The core level Gd3d binding energy in Gd2O3 corresponds to the standard oxidation state of Gd3+ ions. The Gd 4d binding energy peaks also reflect a similar trend of shift from 141.2 eV of Gd3+ to 153.6 eV in bimetallic system (Figure 4D).52,53 Thus, XPS results reveal the reduced Gd(0) state in AgGd bimetallic system and the reaction system prevents the oxidation of Gd.50,52,53 The C1s spectrum was deconvoluted in three bands centered at 287.1, 288.7, and 290.6 eV assigned to hydroxyl (−C−OH) amide (N−CO) and carbonyl (−CO) groups respectively, being in good agreement with C1s peaks of CS (Figure 4E).54,55 The BNPs were coated by biocompatible and hydrophilic short chain CS to avoid metallic toxicity. The coating ability of CS was confirmed by FTIR transmission spectrum (Figure S6). The characteristic peak of CS at 1594 cm−1 (N−H bending of amines) showed a strong shift toward lower wavenumber in CS-coated BNPs.56 On the other hand, the CO stretching (1655 cm−1) of amide-I group have not showed any significant shifts.56,57 However, peaks correspond to the presence of primary (C2-OH) and secondary (C6-OH) alcohols at 1020 and 1070 cm−1 respectively, shown shift toward lower wavenumber with narrowed band.56,57 Hence, FTIR signifies

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

endorses the paramagnetic behavior of these water-soluble BNPs, which is a prerequisite for a MRI contrast agent. The magnetic moment values of these BNPs are in direct proportion to the Gd content and are measured as 0.462, 0.391, and 0.158 emu/g for Ag0.5Gd0.5, Ag0.75Gd0.25 and Ag0.9Gd0.1, respectively, up to 1.2 T applied magnetic field (Table 2). Because of isotropic electronic ground state 8S7/2 Table 2. Magnetic Properties of BNPs Determined through VSM Analysis magnetic properties sample code

coercivity (g)

magnetization (emu/g)

retentivity (emu/g)

Ag0.5Gd0.5 Ag0.75Gd0.25 Ag0.9Gd0.1

65.18 93.08 98.90

0.462 0.391 0.158

3.93 × 10−3 4.97 × 10−3 4.38 × 10−3

and 7 unpaired electrons in the f-orbital of Gd, BNPs exhibited high magnetic moment, leading to apparent effects on both longitudinal and transverse proton relaxation at low applied magnetic field.52 Because of the positive signal enhancement ability, Gdcontaining materials have been exclusively used as a potential MR imaging contrast agents. Ag0.5Gd0.5 has been subjected to MRI analysis in the present investigation. The longitudinal (1/ T1) and transverse (1/T2) relaxation rates have been measured as a function of varied concentrations of BNPs in PBS and the results display a well-correlated linear relationship (Figure 6A, C, respectively). The recorded 1/T1 and 1/T2 map images (Figure 6B, D respectively) have showed a clear dosedependent change in contrast that is due to the increasing water proton relaxation. Consistently, with increasing concentration of BNPs, the T1-weighted images clearly exposed enhancement in the positive contrast and the brightness became more prominent. On the contrary, T2-weighted images revealed enhancement in the negative contrast with simultaneous reduction in brightness. The r1 and r2 values were determined to be 19.6 and 23.0 s−1 mM−1 from the slopes of the 1/T1 (R1) and 1/T2 (R2) plots versus Gd concentration, H


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Figure 6. (A) Relaxivity (1/T1) plotted against varying Gd concentrations (B) T1 weighted MR phantom images of BNPs in PBS and FBS. (C) Relaxivity (1/T2) plotted against varying Gd concentrations (D) T2 weighted MR phantom images of BNPs in PBS and FBS. (E) mass concentration (total metal mass of Ag + Gd) vs HU plot showing linear dependence. (F) CT images of the phantoms with reference to PBS control.

The BNPs seem to satisfy the conditions to a great extent as a positive contrast agent. Preliminary biostability study of BNPs has been carried out by reviewing MRI contrast abilities in fetal bovine serum (FBS) at physiological pH (7.4) and temperature (37 °C) after 24 h incubation. Basically, free Gd3+ ions comparably possess higher relaxivity than Gd based NPs, which generally induce sharp decline in relaxivity due to its drawback of high agglomeration.65−67 The results showed a slight reduction in relaxivities after incubation in FBS and this indicates the stability of BNPs in FBS at physiological conditions. Nevertheless, a slight decline in the relaxivities of BNPs is attributed to possible protein adsorption on its surface. In Vitro CT Analysis. To assess the feasibility of BNPs as an X-ray contrast agent, different concentrations of Ag0.5Gd0.5 in PBS solutions along with PBS as a control were prepared and scanned at 120 kV (Figure 6E, F). CT signal intensity display a linear enhancement with the increase in mass concentrations of BNPs (total mass of Ag + Gd), leading to a brighter image (Figure 6F). By setting the attenuation values (Hounsfield unit,

respectively at 1.5 T (Figure 6A, C, respectively). The relaxivity values of BNPs were found comparably higher than the reported data on Gd based NPs.58−61 The r1 for Gd-loaded liposome is reported as 6.4 s−1 mM−1, which is 3-fold lower than the r1 of AgGd BNPs.59 The r1 (∼9 s−1 mM−1) and r2 (∼11 s−1 mM−1) values of Gd2O3 NPs is also lower than that of AgGd BNPs.60,61 The most studied Gd3+ chelate functionalized gold NPs (AuNPs) have also expressed r1 (13 to 16 s−1 mM−1) values comparatively lower than AgGd BNPs.62,63 Thus, it is deduced that Gd3+ presence on the surface of AgGd BNPs induces the longitudinal relaxation of the water proton, which consequently accelerates the relaxation of the water proton, providing a large r1 value to AgGd BNPs than Gd chelated NPs. The high values of both relaxivities emphasize the potential use of BNPs as a T1- and/or T2- MRI contrast agent. Nevertheless, the r2/r1 ratio is estimated to be 1.17. Generally, a contrast agent with large r1 and a r2/r1 ratio close to unit is considered as a highly sensitive T1 MRI contrast agent.64 However, a high dose of AgGd BNPs might be used to record T2 weighed MRI.” I


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observed to be slow, with an initial ∼5% release during first hour and only ∼17% release in 24 h. This result endorses that the hydrazone linkage between Dox and BNPs is intact at physiological condition, which is a prerequisite to prevent the premature release of drug from carrier. Conversely, the Dox release accelerates on increasing acidic conditions, with ∼20% and 46% drug release determined in the first hour at pH 6.5 and 5.0, respectively. The drug release rate is much faster in acidic conditions, with ∼95% and ∼70% Dox has been discerned within 24 h at pH 5.0 and 6.5, respectively. This result infers that the Dox release from Ag0.5Gd0.5-Dox is governed by the intrinsic hydrolysis of the hydrazone linkage between the Dox and the BNPs in an acidic environment.69,70 The pHdependent drug release is interesting specially for targeted delivery of anticancer drug to the tumor.69,70 Moreover, drug release kinetics of Dox from Ag0.5Gd0.5-Dox endorse that the most Dox conjugated with BNPs will remain intact in the plasma (pH 7.4) when injected, and thereby will greatly reduce the chance of systemic side effects to the healthy tissues. However, faster Dox release will occur after endocytosis of Dox-conjugated BNPs in tumor, because of much lower pH (4.5 to 6.5) than physiological pH (7.4). Therefore, Doxconjugated BNPs is expected to improve the efficiency of targeted cancer therapy. Cytotoxicity Assay and Therapeutic Effect. MTT cell viability assay was used to evaluate the biocompatibility of Ag0.5Gd0.5 and apoptotic ability of Ag0.5Gd0.5-Dox on human cervical cancer (HeLa) cells (Figures 8A, B). Results reveal a dose and time-dependent induction of cytotoxicity and apoptotic effect over 24 h exposures of BNPs and drug conjugated BNPs, respectively. The MTT results expressed significantly low cytotoxicity even for a high dose of 400 μg/mL BNPs exposure to HeLa cells. The apoptotic effect of Ag0.5Gd0.5-Dox was found to be dose-dependent with IC50 equal to ∼52 μg/mL. The results demonstrate that the apoptosis of cell increases exponentially on increasing the dose of Dox conjugated BNPs. This observation confirms that BNPs viably cross the cell membrane and internalizes in endosomes where the bond between Dox and BNPs is hydrolyzed due to acidic environment. Live/dead cell viability assays corresponding to MTT data were carried out by incubation with Ag0.5Gd0.5 and Ag0.5Gd0.5-Dox based on IC50 of Ag0.5Gd0.5-Dox. The outcome supported the negligible toxicity demonstrated by BNPs and high apoptotic effect of Dox conjugated BNPs (Figure 8C−E) that corroborates with the respective green and red fluorescence for live and dead cells. The better therapeutic efficiency of Ag0.5Gd0.5-Dox is also explained by the antiproliferative ability of high AgNP concentrations as reported.71

HU) of PBS solution as zero, the HU values of Ag0.5Gd0.5 demonstrated a linear increase with increasing mass concentrations (Figure 6E). At a concentration of 0.50 mg/mL, the CT signal intensity for Ag0.5Gd0.5 was approximately 45 HU, which has been found much higher than the already reported HU values for conventional iodinated material, barium sulfate or metal nanoparticles.65,66 The high HU value of BNPs is mainly due to the cumulative effect of high X-ray attenuation coefficient of Gd and Ag. Biocojugation of Doxorubicin with CS-Coated BNPs and in Vitro Drug Release. For efficient intracellular drug delivery to avoid premature release and overcome the drug resistance of cancer cells, doxorubicin (Dox) conjugated gold NPs have been reported.68 Here, we bioconjugated Dox on the synthesized CS-capped AgGd BNPs to formulate a novel theranostic agent with multimodal imaging and pH-dependent drug delivery ability (Scheme 1B). The Dox has been functionalized with succinic acid to introduce a −COOH group that act as a spacer to attach hydrazine. The hydrazine linker is then coupled with − COOH group of SDox and amine group of CS on the surface of BNPs through a well-known carbodiimide chemistry using EDC and NHS. The conjugation of Dox through succinyl-hydrazone is confirmed by recording OD of SDox in supernatant before and after conjugation. The Dox conjugation efficiency is measured by digesting hydrazine linkage in acidic medium. The freeze-dried Ag0.5Gd0.5-Dox is treated with 0.2 M HCl for 12 h to hydrolyze the hydrozone linkage between the Dox and BNPs, and OD has been recorded at 485 nm. The Dox conjugating efficiency for Ag0.5Gd0.5 is determined as 11.2 ± 4% that corroborates with TGA-DSC (Figure 3B). The drug releasing kinetics of Ag0.5Gd0.5-Dox has been investigated at simulated physiological condition (pH 7.4) and also under simulated tumor ECM acidic conditions (pH 5.0 and 6.5) at 37 °C to evaluate the feasibility of Ag0.5Gd0.5Dox as a drug delivery device. The drug release kinetics of Ag0.5Gd0.5-Dox clearly reveal pH dependent Dox release from BNPs (Figure 7). The Dox release at physiological pH is

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CONCLUSION To conclude, successful synthesis of chitosan-capped AgGd BNPs is achieved through microwave-assisted polyol synthesis. The TEM-HAADF studies revealed average nanoparticle size of 12 nm with uniform distribution of the constituent metals. The reduction of Gd3+ in mild reducing conditions is facilitated by the catalytic activity of nucleated Ag(0). Besides the uniformity in size and structure, the AgGd BNPs display unique characteristics for multimodal imaging applications. The paramagnetic features of BNPs is ensured by Gd presence and as a consequence displays effective T1 (r1 = 19.6 s−1 mM−1) and T2 (r2 = 23.0 s−1 mM−1) weighted MRI contrast abilities. BNPs also display stability in serum protein and express

Figure 7. In vitro drug release kinetics of Dox conjugated AgGd BNPs at pH 7.4, 6.5, and 5.0 reveals pH-dependent drug release due to hydrolysis of hydrazone linkage between Dox and BNPs in acidic condition. Values are expressed as the means ± standard deviation (SD) based on three independent measurements.. J


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Figure 8. In vitro cell toxicity against HeLa cells based on MTT assay (A) after 6 and (B) after 24 h treatment of Ag0.5Gd0.5 and Ag0.5Gd0.5-Dox. Live/dead cell staining with treatment of (C) PBS (control), (D) 50 μg/mL Ag0.5Gd0.5 and (E) 50 μg/mL Ag0.5Gd0.5-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..

Notes

negligible changes in T1 and T2 contrast ability after 24 h incubation. BNPs exhibits comparatively higher CT contrast ability than the reported values (for 0.50 mg/mL, HU = 45). The drug release of BNPs into tumors is pH dependent with negligible release at physiological condition and fast release at acidic pH. The biocompatibility of BNPs is ensured from the cytotoxicity studies, and also strong apoptotic effect of drug conjugated BNPs is confirmed. The results from the investigation confirm the ability of BNPs for simultaneous applications in multimodal imaging, drug delivery and also a promising prospect in theranosis for early diagnosis and treatment of cancer.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial assistance received from DST-SERB [Reference: EMR/2015/002200 dated 20.01.2016] 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, are gratefully acknowledged. Mr. Biji M. Cheriyan, Radiographer, Tellacherry Co-operative Hospital is highly acknowledged for providing assistance in MRI and CT scanning.

ASSOCIATED CONTENT

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00498. Microwave irradiation time required for bimetallic nanoparticle (BNPs) synthesis vs mol % of Gd in AgGd bimetallic composition, hydrodynamic size of bimetallic nanoparticles by DLS, energy-dispersive X-ray spectroscopy (EDS) elemental mapping showing uniform distribution of Ag and Gd at microscale, and XRD reflections of bimetallic nanoparticles along with standard JCPDS data of Ag and Gd (PDF)

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Corresponding Author

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

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


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