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Surface-Engineered Multifunctional Eu:Gd2O3 Nanoplates for Targeted and pH-Responsive Drug Delivery and Imaging Applications Arindam Saha,*,† Subas Chandra Mohanta,† Kashmiri Deka,‡ Pritam Deb,‡ and Parukuttyamma Sujatha Devi*,† †

Sensor and Actuator Division, CSIR-Central Glass and Ceramic Research Institute, Kolkata 700032, India Department of Physics, Tezpur University, Tezpur 784028, India

‡

S Supporting Information *

ABSTRACT: In this paper, we report the synthesis of surfaceengineered multifunctional Eu:Gd2O3 triangular nanoplates with small size and uniform shape via a high-temperature solvothermal technique. Surface engineering has been performed by a one-step polyacrylate coating, followed by controlled conjugation chemistry. This creates the desired number of surface functional groups that can be used to attach folic acid as a targeting ligand on the nanoparticle surface. To specifically deliver the drug molecules in the nucleus, the folate density on the nanoparticle surface has been kept low. We have also modified the drug molecules with terminal double bond and ester linkage for the easy conjugation of nanoparticles. The nanoparticle surface was further modified with free thiols to specifically attach the modified drug molecules with a pH-responsive feature. High drug loading has been encountered for both hydrophilic drug daunorubicin (∼69% loading) and hydrophobic drug curcumin (∼75% loading) with excellent pH-responsive drug release. These nanoparticles have also been used as imaging probes in fluorescence imaging. Some preliminary experiments to evaluate their application in magnetic resonance imaging have also been explored. A detailed fluorescence imaging study has confirmed the efficient delivery of drugs to the nuclei of cancer cells with a high cytotoxic effect. Synthesized surface-engineered nanomaterials having small hydrodynamic size, excellent colloidal stability, and high drug-loading capacity, along with targeted and pH-responsive delivery of dual drugs to the cancer cells, will be potential nanobiomaterials for various biomedical applications. KEYWORDS: multifunctional, controlled, targeted, imaging, cytotoxicity, drug delivery, MRI

1. INTRODUCTION

controlled surface functionality for the specific targeting and delivery of cargos. Doped nanomaterials are most attractive among various single-phase nanomaterials developed for biomedical applications.25−28 In the recent past, lanthanide-doped nanomaterials have emerged as a new class of imaging materials that can overcome the shortcomings of conventional imaging probes.26 Conventionally, organic dye molecules and semiconductor quantum dots have been widely used as bioimaging probes, although they suffer from photobleaching (dye molecules), photoblinking, size-dependent emission properties, and severe toxicity.10−12 In the recent past, carbon nanoparticles,29,30 graphene quantum dots,31,32 silicon nanoparticles,33 plasmonic nanoparticle clusters, etc.,34,35 have been developed as alternative imaging probes. However, their extensive use is

Multifunctional nanomaterials with integrated surface functionality for responsive and targeted drug delivery along with multimodal imaging capability have immense importance in biomedical applications.1−3 In the past decade, there has been a huge advancement in this area in terms of controlled synthesis techniques,4,5 surface engineering6−9 and biomedical applications including noninvasive imaging of cell,8,10 tissue,3,11 specific organ12,13 or whole body,14 controlled and targeted drug delivery,15 and biosensors.16,17 Most of these multifunctional nanoparticles are of either the core−shell type18−20 or the composite type21−23 with a few examples of heterodimers.24 Such particles lead to larger size, poor colloidal stability, uncontrolled surface properties, and often sacrificial loss of nanoparticle properties. Presently, the major challenge in this direction is to develop a single-phase multifunctional nanomaterial with small size and viable colloidal stability that can accommodate more than one imaging modality along with © XXXX American Chemical Society

Received: October 8, 2016 Accepted: January 3, 2017

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DOI: 10.1021/acsami.6b12804 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces limited because of uncontrolled shape and size, uncontrolled emission properties, small Stokes shift, and photoblinking properties. Lanthanide-doped nanomaterials, on the other hand, possess large Stokes shift, size-independent strong optical properties (emission depends on the dopant ion), high chemical and photochemical stability, and low toxicity.25,27,36−38 Lanthanide ions like europium (Eu), terbium (Tb), ytterbium (Yb), and erbium (Er) or a mixture of ions have been used as dopants in a suitable host matrix for tunable emission properties.25,27,36−40 Gd2O3 nanomaterials have been found to be excellent host materials to dope lanthanide ions, especially Eu3+, because of their similar atomic properties, thermal stability, chemical durability, low phonon energy, and both up-conversion and down-conversion efficiency.25,27,37−40 Eu3+-doped Gd2O3 exhibits an intense red emission and has been found to be suitable for biomedical applications. The emission from Eu3+ is associated with f−f electronic transitions, which is independent of the shape and size and is distributed between 570 and 720 nm. Moreover, Gd3+ is a well-known and clinically approved T1 magnetic resonance imaging (MRI) contrast agent because it contains seven unpaired 4f electrons, which provide a large electron magnetic moment.27,37,41,42 Because these electrons only yield an S state (8S7/2), they can proficiently induce a longitudinal relaxation of water protons. In recent times, the development of drug-delivery vehicles has been focused on targeted delivery to nullify toxic side effects, responsive drug release to avoid affecting normal cells, and reduced dose and integrated imaging modality to track the drug molecules.43−45 Various nanoformulations have been explored for targeted drug-delivery applications including liposomes,46 solid lipid nanoparticles,47 dendrimers,48 polymer nanoparticles,49 and mesoporous nanomaterials.45 The major disadvantages of these systems are large hydrodynamic size, poor colloidal stability, uncontrolled surface functionalization, and a lack of specific targeting. Moreover, in most of the cases, nanosystems do not provide any integrated imaging modality to track their nature inside the cells or tissues. Although hydrophilic drug delivery is most common and tremendously explored using various nanosystems, the delivery of hydrophobic drug molecules is rather difficult and is less explored.28,50 Suitably surface-engineered nanovehicles have been projected as superior alternatives to address these drawbacks in the field of drug delivery. The poor water solubility of hydrophobic drugs makes them less popular in nanoparticle-based drug delivery. Although multifunctional Eu3+:Gd2O3 has shown promise in biomedical applications, its extensive use in drug delivery with simultaneous imaging capability is limited because of poor size and shape control, large colloidal size, and a lack of surface engineering25,27,37,38,51−58 (Table S1). For biomedical applications, controlled surface engineering plays a pivotal role along with its narrow size and shape distribution and colloidal stability. Previously, we have shown that nanoparticle multivalency dictates the cellular uptake mechanism, and thus it can control the rate of uptake as well as the fate of nanoparticles inside cells.59 Thus, proper control of the surface functional groups is essential without compromising the nanoparticle properties. Furthermore, the attachment of targeting ligands and drug molecules to the nanoparticle surface is critical, and separate surface chemistries must be adopted for each conjugation. Extensive surface functionalization of Eu3+:Gd2O3 nanoparticles is less explored mostly because of strong phonon-coupling-induced quenching of the Eu3+

emission. This phonon coupling arises because of interaction between surface Eu3+ ions and functional moieties like −OH, −NH2, etc. Thus, core doping is preferred instead of surface doping from an application point of view because it ends up with fewer surface Eu3+ ions for phonon coupling.25 Another important factor is the size and size distribution of nanoparticles. Eu3+:Gd2O3 nanoparticles have been synthesized by various methods including coprecipitation,38,57 polyol,37,56 spray pyrolysis,51,52 hydrothermal,53 sol−gel,25 solvothermal,60,61 etc. Most of these methods produce nanoparticles with uncontrolled size and shape, wide size distribution, and poor crystallinity. Among these methods, the solvothermal method, which includes breaking down a precursor salt at high temperature in a noncoordinating solvent, is interesting because it can precisely control the shape and size of the nanoparticles along with its size distribution. Although this method has been applied for the synthesis of various oxide and semiconductor nanoparticles to control their shape and size, its application in generating Eu3+:Gd2O3 nanoparticles is scarce.60,61 Here, we report the synthesis, characterization, and application of surface-engineered multifunctional Eu3+:Gd2O3 triangular nanoplates for pH-responsive targeted drug delivery and imaging. We have synthesized Eu3+:Gd2O3 nanoparticles by thermolysis of oleate salts at high temperature in a noncoordinating solvent. The as-prepared hydrophobic triangular nanoplates were small in size (∼20 nm) and exhibited narrow size distribution. These nanoparticles were then converted into hydrophilic nanoparticles via one-step polyacrylate coating. This polymerization step allows us to precisely control the surface functionality of the nanoparticles by varying the monomer ratio. The surface-engineered doped nanoparticles were then attached with folic acid via 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide coupling in a controlled manner to specifically target the cancer cells. Both hydrophilic drug daunorubicin and hydrophobic drug curcumin have been tested for drug-delivery applications. Modified drug molecules have been attached to the nanoparticles via ester linkage, which exhibited efficient pH-responsive release characteristics due to acid-induced ester hydrolysis. Moreover, we have also studied the multimodal imaging capability of these nanoparticles. Fluorescence imaging was performed on HeLa and KB cancer cells, which are well-known to contain overexpressed folate receptors.62−65 A cellular imaging study confirms drug internalization inside the cell. We have done the proton relaxivity studies of these nanoparticles and demonstrated its potential as a T1 MRI contrast agent. These surface-engineered nanoparticles are highly emissive and small in size, having excellent colloidal stability suitable for pH-responsive targeted drug delivery and imaging.

2. EXPERIMENTAL SECTION Materials and Methods. Europium(III) nitrate pentahydrate, gadolinium(III) nitrate hexahydrate, oleic acid, 1-octadecene (ODE), N-(3-aminopropyl)methacrylamide hydrochloride, acrylic acid, poly(ethylene glycol) methacrylate, bis[2-(methacryloyloxy)ethyl] phosphate, tetramethylethylenediamine, Igepal, ammonium persulfate (APS), folic acid, N-hydroxysuccinamide (NHS), poly(ethylene glycol) (PEG; acid-terminated), N,N′-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), fluorescamine, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), curcumin, daunorubicin hydrochloride (DAUN), cysteamine, crotonyl chloride, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cellulose membrane (MWCO ∼ 12000 Da), and a Roswell Park Memorial Institute (RPMI) medium were obtained from SigmaB

DOI: 10.1021/acsami.6b12804 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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distilled DCM in an airtight vial. A total of ∼5 μL of TEA was charged into the vial and stirred for 30 min. Next, ∼5 μL of crotonyl chloride in 1 mL of distilled DCM was injected into the vial, and the reaction was continued for 5 h at 0 °C. Finally, the solution was dried by a rotavapor, and the yellow solid was collected. In the reaction, curcumin was taken in excess compared to crotonyl chloride. For daunorubicin modification, ∼5 mg of DAUN was mixed with ∼10 μL of TEA and ∼50 μL of crotonyl chloride in distilled DCM, and the reaction was carried out using a protocol similar to that of curcumin. Controlled Conjugation of Folate-NHS to the PolymerCoated Nanoplates. For controlled folate conjugation with Eu:Gd2O3 nanoplates, we adopted a competitive conjugation chemistry.8 For this purpose, we varied the folate-NHS and PEGNHS ratio to prepare low folate and high folate-modified nanoplates. In brief, separate solutions of folate-NHS and PEG-NHS were prepared with a 0.05 M concentration in DMF. Next, ∼1.5 mL of a nanoplate solution was mixed with the above solutions in 1:9 and 9:1 ratios and then stirred overnight. Finally, the solution was dialyzed (cellulose membrane MWCO ∼ 12000 Da) against basic water and fresh water, respectively. Cysteamine Conjugation with Polymer-Coated Nanoplates. Cysteamine was conjugated to the folate-modified nanoplates via EDC coupling chemistry. The carboxylic acid groups of the nanoplates were first activated with EDC. For this purpose, ∼1.6 mL of the nanoplate was mixed with a ∼100 μL EDC solution (1 mg mL−1 concentration) and stirred for 30 min. Next, a ∼100 μL NHS solution (1 mg mL−1 concentration) was mixed and further stirred for 1 h. Finally, a ∼200 μL cysteamine solution (∼0.05 M concentration) was added to the nanoplate solution and stirred overnight. Finally, the solution was dialyzed (cellulose membrane MWCO ∼ 12000 Da) against fresh water and stored for further use. Quantum Yield Measurement. For quantum yield measurement, we excited both the hydrophobic and hydrophilic Eu:Gd 2 O 3 nanoplates at 470 nm and compared them with fluorescein as a standard dye (95% quantum yield). We have collected the absorbance of each sample by keeping the maximum optical density (OD) value between 0.01 and 0.1, and subsequently their emission spectra were collected. By comparing the OD values and the area of the emission curve between the sample and standard, we evaluated the quantum yield values. Fluorescamine Test. To confirm the presence of a primary amine on the nanoparticle surface, a fluorescamine test was performed. In brief, 1 mL of polyacrylate-coated Eu:Gd2O3 nanoplates was mixed with a freshly prepared fluorescamine solution (2 mg mL−1) in acetone. The emission spectra were then measured by exciting at 400 nm. DTNB Test. To confirm the presence of free thiols on the nanoparticle surface in the sample, a DTNB test was performed. A DTNB solution was first made in DMSO (4 mg mL−1). Then it was diluted to a 0.1 M Tris/HCl (pH 7.5) buffer to a final concentration of 0.1 mM. Next, 50 μL of functionalized Eu:Gd2O3 nanoplates was mixed with 950 μL of a DTNB solution and incubated for 5 min. Finally, it was observed by UV−visible spectroscopy at 412 nm. Drug Loading and Release Study. For drug loading, surfaceengineered nanoplates were mixed with both of the modified drug solutions separately. Typically, 5 mL of a nanoplate solution was mixed with 500 μL of modified drug solutions (250 μg mL−1 concentration) and stirred overnight. Finally, the drug-loaded sample was dialyzed for 4−6 h by repeated replacement with fresh water. For a drug release study, small portions of drug-loaded samples (typically 200 μL) were taken in a dialyzed membrane and dialyzed against buffer solutions of pH 4.7 (citrate buffer) and pH 7.4 [phosphate-buffered saline (PBS) buffer] for varying time periods, typically from 30 min to 72 h. Drug released in the buffer medium was collected and measured via photoluminescence spectroscopy. Cell Imaging Study. For cell imaging, HeLa and KB cells were cultured in a 24-well plate using a folate-free RPMI medium separately. After the desired confluency was attained, cells were treated with drugloaded Eu:Gd2O3 nanoplate solutions and incubated for 2 h at 37 °C and 5% CO2 atmosphere. After incubation, the cells were washed with

Aldrich and used as received. Absolute ethanol, cyclohexane, hexane, chloroform, diethyl ether, triethylamine (TEA), and N,N-dimethylformamide (DMF) were obtained from Merck (Germany) and used as received. Dimethyl sulfoxide (DMSO) and dichloromethane (DCM) were received from Merck and distilled before use. Preparation of Gadolinium(III) Oleate and Europium(III) Oleate. The oleate complexes were prepared using a previously reported method.28 In brief, 1 mmol of Gd(NO3)3 (∼345 mg) or Eu(NO3)3 (∼430 mg) and 4 mmol of sodium oleate (∼1.2 g) were mixed with 2 mL of water, 13 mL of ethanol, and 15 mL of hexane. The mixture was stirred vigorously at 80 °C for 4 h. After the reaction, the hexane layer was collected in a separatory funnel and washed with water several times to remove unreacted sodium oleate and salts. Finally, hexane was evaporated, and the powder of europium oleate and gadolinium oleate was collected for further use. Synthesis of Nanoplates. The synthesis was carried out in a typical three-necked flask system attached with a Schlenk line. In brief, 0.3 mmol of an oleate complex (x mol % europium oleate and 100 − x mol % gadolinium oleate, where x = 2, 5, 10, and 15) was mixed with 0.3 mmol of oleic acid (∼95 μL) and ∼10 mL of ODE in a threenecked flask. The mixture was purged with nitrogen gas under vigorous stirring for 30 min, followed by heating to 300 °C. The reaction was retained at this condition for 3 h, followed by cooling to room temperature. The obtained sample was washed with acetone for the removal of ODE. Next, it was washed twice with hexane/ethanol by a redispersion/precipitation method. The final product was dispersed in cyclohexane for further use. The above reaction was carried out at different conditions for optimization. The reaction time was varied (1, 2, and 4 h), keeping the other reaction parameters constant (x = 5). In an alternate hot injection method, europium oleate was injected into the gadolinium oleate/oleic acid mixture in ODE at 300 °C, keeping the other reaction parameter fixed (x = 5). We also performed the reaction without adding any oleic acid. Polymer Coating of the Nanoplates. Hydrophobic Eu:Gd2O3 nanoparticles were transformed into hydrophilic nanoparticles by adopting previously reported method with some modification.6,8 A total of 2 mL of a hydrophobic nanoparticle (5 mg mL−1) was mixed with 100 μL of an aqueous solution of N-(3-aminopropyl)methacrylamide hydrochloride (∼5 mg), 100 μL of an aqueous solution of acrylic acid (∼4 μL), 100 μL of an aqueous solution of poly(ethylene glycol) methacrylate (36 μL), 100 μL of an aqueous solution of a phosphate cross-linker (bis[2-(methacryloyloxy)ethyl] phosphate; 6 μL), and 100 μL of a base (tetramethylethylenediamine) to prepare a 10 mL reverse micelle solution with Igepal/cyclohexane. The optically clear solution was purged with nitrogen gas under stirring conditions, followed by the initiation of polymerization by adding 100 μL of an aqueous solution of APS (3 mg). The reaction was carried out for 1 h and finally precipitated by adding absolute ethanol. The precipitate obtained was washed thoroughly with chloroform and ethanol to remove any unreacted monomer or particle. Next, the nanoparticles dispersed in water were further purified by precipitation with solid Na2HPO4. Finally, the waterdispersed nanoparticles were thoroughly dialyzed against fresh water by a cellulose membrane (MWCO ∼ 12000 Da). Folate-NHS and PEG-NHS Preparation. Folate-NHS was prepared using a previously reported method.28 It was prepared by DCC coupling chemistry. In brief, folic acid (∼30 mg), DCC (1 mg mL−1), and NHS (1 mg mL−1) (DCC:NHS = 1:1) were mixed in dry DMSO and stirred overnight. At the end, folate-NHS was extracted in diethyl ether, and finally the solvent was evaporated to obtain folateNHS as a dark-brown solid. For PEG-NHS synthesis, acid-terminated PEG (PEG-COOH; ∼10 mg) was first dissolved in dry DCM and then mixed with EDC (1 mg mL−1) and NHS (1 mg mL−1) (1:1 ratio). PEG-COOH was taken in slightly large excess compared to NHS for total consumption of the NHS used. The reaction was continued for 24 h, and finally the solvent was evaporated to collect the product. Drug Modification with Crotonyl Chloride. Curcumin and DAUN were modified with crotonyl chloride.66 For curcumin modification, ∼10 mg of curcumin was dissolved in ∼3 mL of C

DOI: 10.1021/acsami.6b12804 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Synthesis of Eu:Gd2O3 Triangular Nanoplates and One-Step Polyacrylate Coating To Develop Hydrophilic Eu:Gd2O3 Triangular Nanoplates

(PerkinElmer). The T1 and T2 relaxation times of the sample were measured with a Bruker Minispec (mq 60) TD-NMR instrument (60 MHz and 1.41 T) at 37 °C. The application that determines T1 relaxation times by applying inversion−recovery pulse sequences was employed for the T1 measurements. Similarly, the application that determines T2 relaxation times by applying Carr−Purcell−Meiboom− Gill pulse sequences was employed for the T2 measurements. The relaxivities (r1 and r2) were determined from the slopes of concentration-dependent 1/T1 and 1/T2 changes.

a PBS buffer twice, followed by the addition of fresh RPMI media. The labeled cells were then stained with Hoechst by 10 min of incubation for nucleus staining, followed by washing with a PBS buffer, and finally observed under a fluorescence microscope. For a quantification study, cells were treated with ∼100 μL of trypsin/ethylenediaminetetraacetic acid, and the detached cells were collected by centrifugation. Typically, 0.1−0.2 million cells were dispersed in PBS (pH 7.4) and used for flow cytometry [fluorescence-assisted cell sorting (FACS)], inductively coupled plasma (ICP), and photoluminescence studies. For, ICP studies, cells were digested with 10% suprapure HNO3 prior to measurement. Cytotoxicity Study. A cytotoxicity study was performed by wellknown MTT assay. HeLa cells were first cultured in a 24-well plate in folate-free RPMI media. The cultured cells were treated with drugloaded samples and samples without drugs of varying concentrations. The cells were incubated for 24 h at 37 °C and 5% CO2 atmosphere. Next, the cells were treated with 50 μL of a MTT solution (∼5 mg mL−1) and further incubated for 3 h. The formazon crystals thus formed were carefully separated and dissolved in a water/DMF (1:1) mixture with sodium lauryl sulfate. The cytotoxicity was evaluated by measuring the absorbance of each well of the plate at 570 nm and comparing it with the control cell cytotoxicity. The cytotoxicity study was also performed with free drugs for comparison. Proton Relaxivity Study. A proton relaxivity study was performed by dispersing the functionalized, drug-loaded nanomaterials in water at different concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mM) of Gd3+. Instrumentation. X-ray diffraction (XRD) analysis was performed on an X’Pert Pro MPD X-ray diffractometer (PANalytical) system using Cu Kα radiation (λ = 1.5406 Å) at a 2θ scanning rate of 2° min−1 between 10° and 80°. A transmission electron microscopy (TEM) study was performed on a Tecnai G2 30ST (FEI) highresolution transmission electron microscope operating at a voltage of 300 kV. The particle size and ζ potential were measured in a Horiba (SZ-100) analyzer. The photoluminescence properties of the nanoparticles were studied on a steady-state spectrofluorometer (QM-40, Photon Technology International, Pvt) using a 150 W xenon lamp. UV−visible absorbance spectroscopy was performed on a UV− visible−near-IR spectrophotometer (Shimadzu UV-3600). X-ray photoemission spectroscopy (XPS) measurements were performed on a PHI 5000 Versa probe II scanning XPS microprobe (ULVACPHI, U.S.). The measurements were monitored at room temperature and at a base pressure of better than 6 × 10−10 mbar. All of the spectra were acquired with monochromatic Al Kα radiation (hν = 1486.6 eV) with a total resolution of about 0.7 eV and a beam size of 100 μm. Fourier transform infrared (FTIR) spectra were performed between 4000 and 400 cm−1 on a Nicolet 380 FTIR spectrometer (Thermo Scientific) using KBr pellets. Magnetic measurement was performed using a Cryogenic Physical Property Measurement System. Differential interference contrast microscopy images and fluorescence images of the cells were obtained by an Olympus IX81 microscope using ImagePro Plus, version 7.0, software. MTT assay was carried out in a Synergy Mx monochromator-based multimode microplate reader (BioTek). Flow cytometry was performed on a BD Accuri C6 flow cytometer. An ICP study was carried out on an Optima 2100DV ICP-AES instrument

3. RESULTS Synthesis of Eu:Gd2O3 Triangular Nanoplates. The doped triangular nanoplates were synthesized via a hightemperature colloidal synthesis by decomposing the metal precursors at 300 °C. In this method, europium and gadolinium oleate were separately synthesized as metal precursors. These precursors were then mixed with oleic acid in a 1:1 molar ratio along with the noncoordinating solvent ODE (Scheme 1). Oleic acid acts as a capping ligand in the nanoparticle formation. The mixture was heated at 300 °C for 3 h under an inert atmosphere. High temperature initiates decomposition of the oleate complexes with a change in the solution color from light yellow to brown. In this synthesis technique, we varied the europium doping concentration to 2%, 5%, 10%, and 15% by varying the initial mole ratio of europium and gadolinium oleate complexes (Table S2). The doping level was optimized to 5% because it revealed a highest quantum yield as well as intense emission properties, as evidenced from its normalized spectra (Table S2 and Figure S1). To optimize the reaction conditions, we varied the reaction time from 1 to 4 h and optimized the nanoplate formation to 3 h of reaction time (Figure S2). It was observed that for shorter reaction time (1−2 h) small and thin rod-shaped nanoparticles were formed with lamellar distribution. For longer reaction time (4 h), broken triangular-shaped nanoparticles were found. We also carried out the optimized reaction by a hot injection method to study the position of the dopant ions in the Gd2O3 matrix. In this method, the degassed europium oleate complex (5 mol %) in hot ODE was injected at 300 °C into a gadolinium oleate and oleic acid mixture (1:1) with ODE in a three-necked flask, and the reaction was carried out for 3 h. The nanoparticles were purified via a typical precipitation/redispersion method based on the change in the solvent polarity. First acetone was added to the ODE solution to completely precipitate the nanoparticles, followed by redispersion in hexane. It was reprecipitated with ethanol and further dispersed in hexane. This precipitation/redispersion was carried out two more times to remove any free surfactants or unreacted precursors and finally dispersed in cyclohexane. D

DOI: 10.1021/acsami.6b12804 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. XRD pattern of as-synthesized Eu:Gd2O3 triangular nanoplates, which matches with the cubic phase of Gd2O3 (JCPDS 12-797) (a). TEM images of as-synthesized Eu:Gd2O3 triangular nanoplates under different magnifications (b and c), SAED pattern of as-synthesized Eu:Gd2O3 triangular nanoplates exhibiting crystal planes of 222, 400, and 440 (d), HRTEM image of Eu:Gd2O3 triangular nanoplates showing a lattice spacing of 3.2 Å, which closely matches that of the 222 crystal plane (e), and EDAX study of the Eu:Gd2O3 triangular nanoplates, confirming the presence of both europium and gadolinium in the sample (f). From quantitative analysis, the europium content was found to be ∼5.5 atom %.

Figure 2. XPS analysis of as-synthesized Eu:Gd2O3 triangular nanoplates. The survey graph showing the presence of carbon, oxygen, gadolinium, and europium in the sample (a), detailed analysis of C 1s spectra showing a strong peak at ∼284.5 eV corresponding to (−C−C−)n and a small peak at ∼288 eV due to the carbonate ions (−CO2−) (b), O 1s spectra revealing two peaks after deconvolution, at ∼529.5 and 531.4 eV, corresponding to the oxides of Gd2O3 and carbonate ions, respectively (c), Gd 3d spectra exhibiting a strong peak at ∼1186.9 eV due to Gd 3d5/2 and a weak peak at ∼1219.7 eV responsible for Gd 3d3/2 (d), Eu 3d spectra showing a very weak signature of Eu 3d5/2 at ∼1134.7 eV (e), and in the 4d spectra of gadolinium and europium, two peaks at ∼142.4 and ∼147 eV due to Gd 4d5/2 and Gd 4d3/2 but a small signature of Eu 4d5/2 observed at ∼135.3 eV (f).

Characterization of Eu:Gd2O3 Triangular Nanoplates. The XRD pattern of the purified sample exhibited four peaks at 2θ = 20.067°, 28.56°, 33.25°, and 47.53° corresponding to 211, 222, 400, and 440 crystal planes, respectively, of a cubic Gd2O3 structure with a calculated lattice parameter a = 10.8 Å (Figure 1a). These peaks match with the JCPDS 12-797 file of Gd2O3. The 400 peak shifted a little toward a higher 2θ value possibly

because of the internal strain generated during europium doping, which is due to close similarities of the ionic radii of Gd3+ (107.8 pm) and Eu3+ (108.7 pm).67 The absence of any other peak in the XRD pattern confirms that the material is phase-pure and no separate Eu2O3 has formed. From TEM analysis, it was observed that small nanotriangles formed and are ∼20 nm in size (Figure 1b−f). The nanotriangles show a E

DOI: 10.1021/acsami.6b12804 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 3. UV−visible absorbance spectra of as-synthesized Eu:Gd2O3 triangular nanoplates showing a strong peak at ∼260 nm arising because of charge transfer between O2− of Gd2O3 and Eu3+ (a). The inset shows photoluminescence excitation spectra, keeping the emission fixed at 617 nm. Characteristic peaks are observed at 398 and 470 nm. To collect the emission spectra, we used 398 and 470 nm excitation. Two strong peaks were recorded at ∼617 and ∼629 nm, responsible for the 5D0−7F2 transition (b). The inset shows the digital image of the nanoplates in cyclohexane under normal light (transparent solution) and UV light, exhibiting red emission (365 nm). Magnetic separation of these nanoparticles in the presence of a bar magnet was also shown under UV light. The lifetimes of these nanoparticles were measured with an average radiative lifetime of 1.6 ms for as-synthesized Eu:Gd2O3 triangular nanoplates [(iii) and (iv) represent decay and fit, respectively], which decreases to 0.9 ms after functionalization [(i) and (ii) represent decay and fit, respectively] (c). Magnetic measurement of these nanoparticles and a M−H curve exhibiting paramagnetic behavior at room temperature (d). The volume magnetic susceptibility (χv) was calculated as ∼2.35 × 10−5 for as-synthesized Eu:Gd2O3 triangular nanoplates (i) and decreases to ∼1.56 × 10−5 after functionalization (ii).

a result of charge transfer between O2− of Gd2O3 and Eu3+. To better understand the absorptions, we performed photoluminescence excitation spectra, keeping the emission fixed at 617 nm. Two strong peaks were observed at ∼398 nm due to the 7F0−5L6 transition and at ∼470 nm due to the 7F0−5D2 transition in Eu3+53,56 (Figure 3a). To collect the emission spectra, we used 398 and 470 nm excitations because 260 nm excitation falls under the short UV range and is detrimental for tissues and cells. So, for application purposes, we chose 398 and 470 nm excitation wavelengths. Both excitations exhibited similar types of signatures in the emission spectra, although the relative intensities of the peaks varied. Two strong peaks were recorded at ∼617 and ∼629 nm, responsible for the 5D0−7F2 transition in Eu3+. Two weak peaks were observed at ∼594 and ∼710 nm, originating from the 5 D 0 − 7 F 1 and 5 D 0 − 7 F 4 transitions, respectively53,56 (Figure 3b). The inset of Figure 3b demonstrates the digital image of the nanoplates in cyclohexane under normal and UV light (365 nm). Under UV light, the particle dispersion exhibited an intense red emission. Magnetic separation of these nanoparticles in the presence of a bar magnet also appeared after the addition of acetone to the cyclohexane solution of nanoparticles under UV light. The lifetime of these nanoparticles was measured, and it demonstrated typical phosphorescence properties of the 5d electrons of europium with an average radiative lifetime of 1.6 ms (Figure 3c). The quantum yield of these nanoparticles was calculated by exciting at 470 nm and comparing with a fluorescein dye as the standard. The as-synthesized hydrophobic nanoparticles exhibit ∼23% quantum yield for 5% europium doping. Other doping concentrations exhibit comparatively low quantum yields (Table S2). Magnetic

clearly defined shape and uniform size distribution, as evidenced from Figure 1b,c. The selective area electron diffraction (SAED) pattern confirms the crystal planes obtained from the XRD spectra. Three well-distinguished ring patterns corresponding to the 222, 400, and 440 planes are observed in the SAED pattern. High-resolution TEM (HRTEM) shows well-defined crystal planes with a lattice spacing of ∼3.2 Å, which closely matches that of the 222 crystal plane (Figure 1e). Elemental analysis of the sample results in ∼5.5 atom % of europium, which is close to the starting concentration of europium (Figure 1f). To further characterize the material, we performed a detailed XPS study (Figure 2). The C 1s spectra show a strong peak at ∼284.5 eV, which corresponds to the (−C−C−)n obtained from the long-chain fatty acids present in the capping ligands. A small peak at ∼288 eV is observed due to the carbonate ions (−CO2−) of the oleate capping ligands. O 1s spectra reveal two peaks after deconvolution, at ∼529.5 and 531.4 eV, which correspond to oxides from Gd2O3 and carbonate ions, respectively. In Gd 3d spectra, two peaks are found. The strong peak at ∼1186.9 eV is responsible for Gd 3d5/2, and the weak peak at ∼1219.7 eV is responsible for Gd 3d3/2. The Eu 3d spectra show a very weak signature of Eu 3d5/2 at ∼1134.7 eV. A similar type of signature is observed in the 4d spectra of gadolinium and europium. The two peaks at ∼142.4 and ∼147 eV are due to Gd 4d5/2 and Gd 4d3/2, whereas a small signature of Eu 4d5/2 is observed at ∼135.3 eV. The XPS data confirm the presence of only 3+ oxidation states of europium in the synthesized sample. The optical properties of the nanoparticles were studied by absorption and emission spectroscopy (Figure 3a−c). In the absorption spectra, there is a strong peak at ∼260 nm arising as F

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Scheme 2. Scheme for the Controlled Surface Functionalization of Eu:Gd2O3 Triangular Nanoplates along with Its Drug Conjugation and pH-Responsive Release (a) and Strategy for Drug Modification (b)a

a

Both curcumin and DAUN were modified with crotonyl chloride.

formed, salt induced precipitation of the nanoparticles was performed, followed by extensive dialysis. Targeting ligands and drug molecules were attached to these coated nanoparticles via a competitive conjugation method following separate surface chemistry59 (Scheme 2a). We attached folic acid as a targeting ligand to target the cancer cells specifically. This is because cancer cells are known to possess overexpressed folate receptors. To control the folic acid density on the nanoparticle surface, we performed competitive conjugation chemistry by varying the molar ratio of folic acid and PEG-COOH. Primary amines on the nanoparticle surface were first confirmed by a fluorescamine test, where the primary amines react selectively, resulting in green fluorescence (Figure S3). Next, both folic acid and PEG-COOH were first converted to their NHS derivatives via well-known DCC coupling chemistry.28 For competitive conjugation, we have used two different ratios of folate-NHS and PEG-NHS, 9:1 and 1:9. Higher folate concentration generates particles with high folate density (Eu:Gd2O3-HF) on the nanoparticle surface, and subsequently lower folate concentration gives particles with low folate density (Eu:Gd2O3-LF). Folate variation has been reflected in the photoluminescence spectra of the respective samples at 370 nm excitation, which show blue emission from folic acid (Figure S4). Coupling chemistry has been performed by a specific linkage between primary amines on the nanoparticle surface and NHS groups of the ligands. PEG-

measurement of these nanoparticles was performed, and the M−H curve exhibited paramagnetic behavior at room temperature. This paramagnetism is typical of a Gd2O3 host because of its seven unpaired parallel f electrons. The volume magnetic susceptibility (χv) was calculated as ∼2.35 × 10−5 (Figure 3d). Surface Engineering of Eu:Gd2O3 Triangular Nanoplates. To execute surface engineering, the hydrophobic nanoparticles were first converted into hydrophilic nanoparticles via a single-step controlled polyacrylate coating6,8 (Scheme 2). In this step, nanoparticles in cyclohexane were mixed with three different monomers: amine, acid, and PEG monomer with 1:4:5 molar ratio. This step controls the density of the available amine and/or acid groups on the nanoparticle surface. Both monomers also served the purpose of surface anchoring groups for further functionalization. The monomer mixtures in water were mixed with nanoparticles in cyclohexane via reverse-micelle formation. The reaction was initiated using an APS initiator under basic conditions and an inert atmosphere. After the desired time period (typically 1 h), polymerization was quenched by adding absolute ethanol. Ethanol, being polar, ruptured the reverse micelle, and the hydrophilic nanoparticles thus formed precipitated. The nanoparticles were washed several times with ethanol and chloroform and finally dialyzed against fresh water to remove unreacted monomers. To further remove any free polymer thus G

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Figure 4. TEM image of functionalized Eu:Gd2O3 triangular nanoplates that retain their shape and size (a). Hydrodynamic diameter of the functionalized particles showing ∼45−75 nm hydrodynamic size (b). UV−visible (c) and photoluminescence (d) spectra of functionalized Eu:Gd2O3 triangular nanoplates retaining their optical properties. FTIR characterization performed to understand the surface functional groups of the functionalized Eu:Gd2O3 triangular nanoplates (e). The characteristic peaks of functionalized nanoparticles were observed in FTIR.

NHS serves two purposes; first it acts as a control ligand in competitive coupling chemistry, and second it enriches the nanoparticles with PEG groups, which increase the biocompatibility of the nanoparticles. Next, the acid groups on the nanoparticle surface were converted to thiol-terminated groups by attaching cysteamine via standard EDC coupling chemistry. This coupling provides thiol-terminated nanoparticles. Free thiols on the nanoparticle surface were confirmed via a DTNB test (Figure S5). In each step of the functionalization, the sample was purified via extensive dialysis against fresh water. Characterization of Surface-Engineered Eu:Gd2O3 Triangular Nanoplates. The functionalized doped nanoparticles retain their shape and size, as observed from TEM (Figure 4a). A dynamic light scattering (DLS) study was performed to obtain the hydrodynamic diameter, and it was found that these particles exhibit ∼45−75 nm DLS size (Figure 4b). The DLS size accounts for polymer coating and surface

functionalization of the nanoparticles. ζ-potential measurement results in a −12 mV surface charge at neutral pH. This nearly neutral surface charge is crucial for biomedical applications to avoid nonspecific interactions during cellular internalization. The optical properties of the samples were similar to those of as-synthesized particles, although their emission intensities decreased because of strong phonon interaction of surface Eu3+ and functional groups (−OH, −NH2, etc.; Figure 4c,d). A quantum yield of ∼12% was obtained for these surfacefunctionalized nanoparticles. The radiative lifetime was also decreased to 0.9 ms. The decrease in the lifetime and quantum yield suggests nonradiative energy transfer arising from phonon coupling. A FTIR spectroscopy study was performed to understand the surface functionality of the nanoparticles (Figure 4e). A broad peak at ∼3420 cm−1 is due to −OH stretching of the PEG functionality. Two sharp peaks at ∼2925 and 2875 cm−1 are H

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Figure 5. DLS size distribution of functionalized Eu:Gd2O3 triangular nanoplates at pH 7.4 recorded initially (a), after 7 days (b), after 30 days (c), and after 90 days (d). The average hydrodynamic diameter remained in the range of 50−100 nm even after 3 months. The insets of parts a and c show the digital images of functionalized Eu:Gd2O3 triangular nanoplates in three different pH conditions (4.7, 7.4, and 10) and RPMI media under normal (colorless solutions) and UV (red solutions) light under 365 nm excitation.

and the size distribution shifts to ∼55−95 nm due to protonation of the surface acid groups, resulting in decreased stability. However, it shows good colloidal stability, and after 1 month, the DLS size remains at ∼60−120 nm (Figure S6). In basic pH (pH 10), the size distribution gets narrower because of ionization of the surface acid groups, and even after 3 months, the colloidal stability does not change much (∼50−90 nm; Figure S7). We also checked the colloidal stability of drugloaded Eu:Gd2O3-LF and Eu:Gd2O3-HF at physiological pH. In the former case, excellent colloidal stability was obtained even after 3 months with little size broadening (∼60−120 nm after 3 months), whereas in the later case, agglomeration was noticed after 1 month (∼60−120 nm after 1 month) because of the high concentration of hydrophobic folic acid on the nanoparticle surface (Figures S8 and S9). Colloidal stability in the cell culture medium is extremely important for imaging and drug-delivery applications. Particles should not agglomerate in cell culture media during the biomedical studies. To confirm this, we performed the colloidal stability of drug-loaded Eu:Gd2O3-LF and Eu:Gd2O3-HF in RPMI media (Figures S10 and S11). Excellent colloidal stability of these nanoparticles was found in cell culture media with little or no aggregation compared to pH 7.4. The emission properties of the nanoparticles are also retained in this media (Figure 5a,c). Drug Loading and Release Study. For the drug-loading studies, we tested two different drug molecules, DAUN as a model hydrophilic drug molecule and curcumin as a model hydrophobic drug molecule. These drug molecules are well studied, and the course of action is well-known. For responsive delivery purposes, we first modified the drug molecules with crotonyl chloride, which incorporates ester linkage in the drug molecules with terminal double bonds (Scheme 2b). Modified drug molecules were characterized via NMR and highresolution mass spectroscopy (Figure S12). These modified

due to C−H asymmetric and symmetric stretching, respectively. A sharp peak at ∼1734 cm−1 arises due to CO stretching of the acid functional groups. A peak at ∼1612 cm−1 arises because of N−H deformation of the primary amine functionality. A strong peak at ∼1513 cm−1 is due to C−N and amide II stretching of the primary amine groups. Symmetric −CO2− stretching of the acid groups is observed at ∼1390 cm−1. Asymmetric and symmetric C−O−C stretching of PEG molecules are observed at ∼1251 and ∼1125 cm −1 , respectively. C−P rocking vibration and PO stretching of the phosphate cross-linker are obtained at ∼955 and ∼556 cm−1, respectively. The coated particles also exhibit paramagnetic properties, as evidenced from the M−H curve, although their magnetic susceptibility decreases to 1.56 × 10−5 because of the incorporation of an organic mass (Figure 3d). Colloidal Stability of Surface-Engineered Eu:Gd2O3 Triangular Nanoplates. Colloidal stability is one of the key factors for biomedical applications. We performed colloidal stability of the nanoparticles in three different pH conditions for 3 months and also in a cell culture medium to check the feasibility of using these nanoparticles in drug-delivery applications (Figures 5 and S6−S11). The polymer-coated nanoparticles show exceptional colloidal stability in physiological pH. Even after 1 month, the size distribution obtained from the DLS study did not change much (∼50−80 nm). After 3 months, it showed a little broadening in the size distribution, although it remained within ∼100 nm (∼50−100 nm) hydrodynamic diameter (Figure 5a−d). It also retains its excellent emission property even after months, as evidenced from the digital images. The insets of Figure 5a,c show the digital images of functionalized Eu:Gd2O3 triangular nanoplates in three different pH conditions (4.7, 7.4, and 10) and RPMI media under normal and UV light (365 nm excitation). In acidic pH (pH 4.7), the particles show minor agglomeration I

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Figure 6. UV−visible spectra showing curcumin loading on functionalized Eu:Gd2O3 triangular nanoplates (a). Enhanced absorbance at ∼420 nm confirming the curcumin loading on the nanoplates. Inset showing digital images of curcumin-loaded samples under normal light and 365 nm excitation. The percent of curcumin release with time at pH 4.7 and 7.4 calculated from photoluminescence spectroscopy (b). Higher release observed at lower pH compared to physiological pH. Daunorubicin loading on functionalized Eu:Gd2O3 triangular nanoplates also confirmed from the enhanced absorbance at ∼480 nm for drug-loaded samples (c). Inset showing the digital images of drug-loaded samples under normal light and 365 nm excitation. Daunorubicin release profile at pH 4.7 and 7.4 (d). More drug release was encountered at lower pH compared to physiological pH. Error bars indicate the standard deviation obtained after collecting the data for five different experiments.

observed that DAUN-loaded samples release almost 55% drug in acidic pH, whereas in neutral pH, the release was ∼10%. It was also observed that, within 30 h, most of the drugs (∼45%) had been released in acidic pH and then remains almost constant. For curcumin, in acidic pH release has been found ∼80% in 72 h which was reasonably higher than in neutral pH (∼12%). Here it has been observed that ∼75% drugs released within 40 h for acidic pH and then remained constant. This responsive release was due to ester hydrolysis at acidic pH, which was substantially slow under neutral conditions. Multimodal Imaging and Toxicity Study. The multimodal imaging capability of these nanoparticles was studied by MRI and fluorescence imaging. The MRI capability of these nanoparticles was studied via proton relaxation studies by a time-dependent NMR relaxometer (Figure 7). We have investigated both the T1 and T2 relaxation time as a function of the gadolinium ion concentration. From the slope of the plot, the relaxivity (r1 and r2) was obtained. The T1 relaxivity (r1) was obtained as ∼33.2 mM−1 s−1, which was comparable to or even higher than that of the clinically approved samples (Magnevist, r1 = 4.7 mM−1 s−1). The r2/r1 value was found to be ∼1.51, which is low enough to exploit these nanoparticles as efficient T1 contrast agents in MRI.27,37,58 Fluorescence imaging was performed in two different cancer cell lines, HeLa and KB. Both cells are well-known to possess overexpressed folate receptors, and thus folate-functionalized nanoparticles can specifically target these cells. Cells were incubated for 2 h with surface-engineered and drug-loaded samples in folate-free RPMI media, followed by washing with a PBS buffer. Finally, the cells were observed under a fluorescence microscope (Figures 8 and S16). Nuclei of the cells were stained with Hoechst to observe localization of the

drug molecules were conjugated with thiol-terminated nanoparticles via thiol−ene coupling chemistry under mild basic conditions (pH 8.0). After vigorous stirring for 24 h, the drugloaded sample was dialyzed extensively to get rid of the free drug molecules. UV−visible and photoluminescence spectroscopy studies confirmed the drug attachment on the nanoparticles (Figures 6 and S13). The nanoparticle properties remain unaltered, as evidenced from the spectroscopic studies. The digital images of the drug-loaded samples show typical emissions from drug molecules (Figure 6). Curcumin-loaded samples exhibit yellowish emission due to the presence of green-emitting curcumin molecules as well as red-emitting europium-doped nanoparticles, whereas daunorubicin-loaded nanoparticles exhibit red emission. The loading efficiency was calculated from UV−visible spectroscopy (Figure S14). The amount of loaded drug was calculated by subtracting the absorbance value of bare Eu:Gd2O3 from drug-loaded Eu:Gd2O3 at 420 nm for curcumin and at 480 nm for daunorubicin, respectively. Loaded drugs were then compared with the added drugs after the appropriate dilution using the equation % loading = (IL/IT) × 100, where IL is the amount of loaded drug and IT is the amount of added drug after proper dilution. The loading efficiency was found to be ∼69% for DAUN (∼15 μg mg−1 of nanoparticles) and ∼75% for curcumin (∼20 μg mg−1 of nanoparticles). A release study was performed for 72 h in acidic (pH 4.7) and neutral (pH 7.4) pH for both drug-loaded samples (Figures 6 and S15). Small amounts of drug-loaded samples were dialyzed against acidic or neutral pH under the appropriate conditions for varying time periods. Released drug was collected by concentrating the dialysis medium and monitored via emission spectroscopy of the drug molecules. It was J

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folate-modified bare nanoparticles do not exhibit any toxicity after 24 h of incubation, and almost 90% cells remain viable. Curcumin-loaded Eu:Gd2O3-LF nanoparticles exhibited ∼30% cell viability, whereas curcumin-loaded Eu:Gd2O3-HF nanoparticles exhibited ∼45% cell viability. This discrepancy in the cell viability was observed for both drugs (curcumin and daunorubicin). The amount of drug was kept constant in both cases. Thus, effective cell death was realized in Eu:Gd2O3-LF nanoparticles. We used different particle concentrations to study the cytotoxic effect of these nanoparticles. For comparison, we used a control cell line (CHO) that was cancer negative and devoid of any folate receptors. The CHO did not show any appreciable cell death when treated with drug-loaded samples.

4. DISCUSSION In this work, our target was to develop a surface-engineered multifunctional nanosystem for targeted drug delivery with a stimuli-responsive feature and the capability for multimodal imaging. As has been reported by many investigators, the shape, size, surface charge, and surface functionality of the nanoparticles play a pivotal role in biomedical applications, both in vitro and in vivo.6−8,59,68−70 As described earlier, we chose europium(III)-doped gadolinium oxide nanoparticles with sizes in the nanoregime and triangular platelike shape for our studies. Nanoplates have advantages over spherical particles because of their higher accumulation tendency near leaky blood vessels commonly found in metastatic cancers.68,69 To synthesize this small (∼20 nm) nanoplate, a one-pot high-temperature solvothermal technique was employed. We also observed that, without the addition of oleic acid, no nanoparticle was formed (Figure S19). One major disadvantage of these Eu:Gd2O3 nanoparticles is the quenching of fluorescence during functionalization (the quantum yield decreases from 23% to 12% after functionalization), resulting from phonon coupling. In an alternative method, europium oleate was injected at 300 °C, keeping the other reaction parameters the same. Both nanoparticles, obtained by standard and alternative methods, were etched using dilute HCl, and the gadolinium/europium ratio was monitored via ICP. The nanoparticles obtained in the alternative method exhibited considerable fluorescence quenching after functionalization (∼1% quantum yield). ICP measurements confirmed that, in this method, most of the Eu3+ ions remained on the nanoparticle surface, thus experiencing strong phonon coupling. In the optimized method, the ICP study confirmed that only a few Eu3+ ions remained on the surface, thus suffering comparatively low phonon coupling and suitable for functionalization and biomedical applications (Figure S20). In this case, both gadolinium oleate and europium oleate decompose simultaneously, resulting in bulk doping of Eu3+ rather than surface doping. We adopted one-step polyacrylate coating to transform the nanoparticles into hydrophilic ones. The advantages of this coating approach are (1) small hydrodynamic diameter, (2) excellent colloidal stability, (3) preservation of the nanoparticle properties, and (4) on-demand control of multiple surface functional groups. In this one-step polyacrylate coating, hydrophilic monomers and hydrophobic nanoparticles are dispersed in a reverse micelle made by igepal and cyclohexane. The reverse micelle helps to initiate the reaction in a single phase. All of the monomers used are separately encapsulated in reverse micelles before being added to the reaction flask. This ensures minimum cross-talking between the monomers.

Figure 7. Proton relaxivity study of functionalized Eu:Gd2O3 triangular nanoplates by a time-dependent NMR relaxometer. The black curve fitting represents the T1 relaxation rate as a function of the gadolinium concentration. From the slope of the curve, the relaxivity (r1) was obtained as 33.2 mM−1 s−1. The red curve is for the T2 relaxation rate, which gives r2 relaxivity. Low r2/r1 (1.51) and high r1 values suggest strong T1 contrast agents in the MRI study. Relative standard deviations for each measurement were found between 0.1% and 0.5%.

nanoparticles inside cellular compartments. We observed that Eu:Gd2O3-LF nanoparticles mostly localize in the nucleus or in the perinuclear region, whereas Eu:Gd2O3-HF nanoparticles are distributed throughout the cell and enter into the lysosome, with very few targeting the nucleus. In Figure 8, the results of fluorescence imaging on KB cells are demonstrated. It is clearly observed that, in the case of Eu:Gd2O3-HF, nanoparticles are distributed throughout the cell and exhibit higher internalization tendency. Curcumin delivery was also studied in this regard. Under blue excitation, green emission from curcumin is observed, and after merging with the nanoparticle emission, it is clearly seen that drug molecules and nanoparticles are colocalized, as evidenced from the yellow emission (Figure 8a−f). However, in the case of Eu:Gd2O3-LF, although internalization is low, the nanoparticle is located mostly in the perinuclear region or in the nucleus, as evidenced from the merged image (Figure 8g−j). Here, green emission from the drug molecules is also obtained from the perinuclear region, as observed in the merged image (Figure 8k−l). This result thus confirms the ability of the nanoparticles as drug-delivery vehicles. Similar results were obtained from HeLa cells also (Figure S16). Cellular internalization was confirmed by FACS, emission study, and ICP analysis of the targeted cells (Figure S17). For a control study, we used nanoparticles without folate and treated in HeLa cells to confirm the specific targeting capability of the nanoparticles. In another control, we used a CHO cell as the folate negative normal cell line. Because it does not possess any overexpressed folate receptor, no internalization was observed (Figure S18). To confirm folate-induced specific targeting, in one experiment we pretreated the KB cells with free folic acid, followed by treatment with folic acid modified Eu:Gd2O3. Because the folate receptors were blocked by free folic acid, no imaging was observed. It is obvious that Eu:Gd2O3-LF nanoparticles will carry more drugs to the nucleus or to the perinuclear region and would cause effective cell death, as compared to Eu:Gd2O3-HF. This was confirmed from a cytotoxicity study (Figure 9). Here, the K

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Figure 8. Cellular imaging of KB cells with curcumin-loaded Eu:Gd2O3-HF (a−f) and Eu:Gd2O3-LF (g−l) nanoparticles. Bright-field (a and g), fluorescence (b, c, e, h, i, and k), and merged (d, f, j, and l) images confirm the uptake of the drug-loaded samples on KB cells. Hoechst staining was performed to identify the cell nucleus and observed under UV excitation under a microscope (b and h). Red emission from the samples obtained by green excitation (c and i). Green emission from curcumin observed under blue excitation (e and k). Merged images of the samples and nucleus (d and j) confirming perinuclear localization of the Eu:Gd2O3-LF samples. Merged images with drugs confirming drug delivery to the cellular compartments by these nanoparticles, as evidenced from the yellow emission of merged images (f and l). Scale bar = 50 μm.

Figure 9. Cytotoxicity assay (MTT assay) of curcumin- and daunorubicin- loaded Eu:Gd2O3-HF and Eu:Gd2O3-LF nanoparticles performed on HeLa cells with varying drug concentrations. Eu:Gd2O3-HF nanoparticles without drugs do not show any appreciable cytotoxicity. Curcumin-loaded Eu:Gd2O3-LF show ∼15% higher cell death compared to Eu:Gd2O3-HF nanoparticles (a). These samples do not affect normal cell lines, as evidenced from CHO cells (green bars) treated with drug-loaded Eu:Gd2O3-HF samples. A similar trend is observed in daunorubicin loaded samples (b). Varying drug concentrations under study are a 125 μg mL−1 sample with 2.5 μg mL−1 curcumin (or 1.875 μg mL−1 daunorubicin) (1), a 250 μg mL−1 sample with 5 μg mL−1 curcumin (or 3.75 μg mL−1 daunorubicin) (2), a 375 μg mL−1 sample with 7.5 μg mL−1 curcumin (or 5.625 μg mL−1 daunorubicin) (3), and a 500 μg mL−1 sample with 10 μg mL−1 curcumin (or 7.5 μg mL−1 daunorubicin) (4). The error bars in blue indicate the standard deviations of each measurement after the data were collected from three sets of experiments.

Another advantage of the reverse micelle is that the probability of any reaction between the monomers is less compared to the interaction with the hydrophobic nanoparticles. Because the

reverse micelle is dynamic in nature, hydrophobic nanoparticles initially undergo ligand exchange between the monomers and the long-chain surface ligands on the hydrophobic nanoparticle L

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Moreover, because the drug molecules are small, once inside the cell, they have a higher tendency to diffuse out of the cells or localize in the lysosome. We also observed that free drug cannot distinguish between cancer and normal cells. It is wellknown that cancer cells and tissues exhibit acidic pH in their leaky blood vessel because of a high rate of metabolism. Thus, site-specific drug release is necessary to avoid toxic effects toward the normal cells. We modified the drug molecules with terminal double bond and ester linkage to attach with nanoparticles. This provides pH-responsive release because ester hydrolysis is fast in acidic pH and negligible in normal pH. Thiol-terminated nanoparticles conjugated with the drug molecules via selective thiol−ene coupling chemistry. Higher loading of drug molecules (75% for curcumin and 69% for daunorubicin) can be explained by the higher number of surface thiol groups on the nanoparticle and efficient thiol−ene coupling chemistry. We compared the therapeutic efficiency (drug loading, release, responsive behavior, etc.) of a few simple and cost-effective systems (Table S3). The comparison reveals the large size of nanosystems with rare examples of simultaneous multimodal imaging and responsive drug delivery. Moreover, hydrophobic drug delivery is scarce using these nanosystems. In our system, drug-loaded nanoparticles exhibit excellent colloidal stability as well as retain emission and magnetic properties. These surface-engineered nanoparticles serve several advantages like (1) target-specific delivery, (2) pH-responsive delivery, (3) excellent colloidal stability, and (4) multimodal imaging (both fluorescence and MRI) capability. In drug delivery, both drugs exhibited slow and sustained delivery for 40 h in acidic pH. Sustained delivery is preferred to minimize the dose issue without compromising the drug efficacy. In physiological pH, almost no drugs were found to release. This helps to defend the normal cells from the cytotoxic effects of the anticancer drug molecules.

surface. In the presence of an initiator, polymerization thus initiates from the surface of the nanoparticles. This results in polymer-coated nanoparticles with the desired surface functional groups at the end of the reaction.6,8,21 To obtain controlled surface functional groups, the amine and acid monomer ratio was varied, keeping the PEG monomer ratio fixed. In our optimized conditions, we kept 10 mol % amine of the total moles of monomers used. This lowers the number of toxic surface amine groups as well as provides sufficient surface amine groups for further functionalization. The feasibility of the existence of both amine and acid groups on the nanoparticle surface can be confirmed from a fluorescamine test and ζpotential measurements. The presence of primary amines can be confirmed from the fluorescamine test. Fluorescamine is a nonemissive reagent that can specifically react with the primary amines to generate strong green emission. We further measured the ζ potential in different pH conditions. We found that, in acidic pH, the ζ potential is slightly positive (+10 mV). At pH 7.4, the ζ potential becomes slightly negative (−12 mV), and in basic pH, it is highly negative (−30 mV). These ζ-potential results ensure a higher number of surface −COOH groups compared to amine groups. This amine/acid ratio also maintains a near-zwitterionic surface charge of the nanoparticles, crucial to avoid nonspecific interactions by creating a balance between the cationic surface amine groups and the anionic surface carboxylate groups. PEG serves the purpose for an extended enhanced permeability and retention effect in blood as well as avoids nonspecific interactions and increases the biocompatibility. This has been confirmed from a colloidal stability study in cell culture media (RPMI). It has been previously reported that the density of targeting ligands on the nanoparticle surface can alter the targeting efficiency and mechanism of endocytosis.59 Lower surface folate groups help nanoparticles to preferentially target the nucleus or perinuclear regions of cancer cells. In a drug-delivery approach, it is highly desirable to deliver the drugs in the nucleus because drugs’ action is based on interaction with cellular DNA present in the nucleus. Thus, we deliberately modified the nanoparticle surface with low folic acid by competitive conjugation chemistry. Lower folate serves many purposes: (1) effective targeting to the perinuclear region with minimum loss in lysosome, (2) superior colloidal stability, and (3) preservation of the high emission intensity of the nanoparticles. The results obtained clearly demonstrate selective targeting of drug molecules to the nucleus or perinuclear regions, leading to higher cell death. Curcumin-loaded Eu:Gd2O3-HF nanoparticles exhibited higher internalization, as confirmed from FACS, ICP, and photoluminescence studies (Figure S17). Thus, it was expected to cause more cell death. Surprisingly, the obtained trend is just the opposite. Curcumin-loaded Eu:Gd2O3-LF nanoparticles cause higher cell death compared to curcumin-loaded Eu:Gd2O3-HF nanoparticles (∼10−15% higher). This can be explained by the selective targeting of drug-loaded Eu:Gd2O3-LF nanoparticles to the nucleus or perinuclear region. However, drug-loaded Eu:Gd2O3-HF nanoparticles mostly reach the lysosome and thus carry less drug toward the nucleus. The same trend was observed for daunorubicin-loaded nanoparticles, as confirmed from cytotoxicity measurements. We have also tested the effects of free drug on HeLa and CHO cells (Figure S21). Much less cytotoxic effect was found for free drugs compared to drug-loaded nanoparticles. This is expected because the internalization of drugs into cells is difficult because of its defense mechanism.

5. CONCLUSION In conclusion, we synthesized multifunctional Eu:Gd2O3 nanoplates with triangular shape and small size via a solvothermal method exhibiting strong red emission as well as paramagnetic properties. These surface-engineered nanoparticles via polymer coating demonstrated small hydrodynamic diameter, excellent colloidal stability, zwitterionic surface charge, and controlled surface anchoring groups. The surfaceengineered nanoparticles were modified with varying amounts of folic acid and attached with drug molecules via pHresponsive linkage. These nanoparticles exhibited efficient drug loading for both hydrophilic drug daunorubicin (∼69% loading) and hydrophobic drug curcumin (∼75% loading). In addition, these drug-loaded nanoparticles exhibited excellent pH-responsive release of drug molecules. Because of their strong red emission, the particles were used as bioimaging probes, and because of their paramagnetic properties, they have shown potential as T1 contrast agents in MRI. Synthesized surface-engineered nanomaterials having small hydrodynamic size, excellent colloidal stability, and high drug-loading capacity, along with targeted and pH-responsive delivery of dual drugs to the cancer cells, will be potential nanobiomaterials for various biomedical applications.

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DOI: 10.1021/acsami.6b12804 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

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Experimental methods, tables of comparisons of existing work (Tables S1 and S3), more TEM images (Figures S2 and S19), DLS measurement to study the colloidal stability under different conditions (Figures S6−S11), more cellular imaging (Figures S16 and S18), FACS (Figure S17), ICP results (Figures S17 and S20), cytotoxicity results (Figure S21), etc. (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: 91-33-2483 8082. Fax: 91-33-2473 0957. *E-mail: [email protected]. Tel: 91-33-2483 8082. Fax: 91-33-2473 0957. ORCID

Parukuttyamma Sujatha Devi: 0000-0002-6224-7821 Author Contributions

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.S. acknowledges the Council of Scientific and Industrial Research (CSIR), India, for providing a Nehru Science PostDoctoral Fellowship. P.S.D. and S.C.M. acknowledge the CSIR for financial support through the network project BIOCERAM, ESC 0103. Authors thank Dr. N. R. Jana, Indian Association for the Cultivation of Science, for his kind assistance in cellular imaging experiments.

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