Targeted Stealth Polymer Capsules Encapsulating Ln3+-Doped

Article pubs.acs.org/journal/abseba

Targeted Stealth Polymer Capsules Encapsulating Ln3+-Doped LaVO4 Nanoparticles for Bioimaging Applications Jaishree Jeyaraman,†,‡ Akansha Shukla,†,‡ and Sri Sivakumar*,†,‡,#,§ †

Department of Chemical Engineering, ‡Centre for Environmental Science and Engineering, #Material Science Programme, and DST Thematic Unit of Excellence on Nanoscience and Soft Nanotechnology, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208016, India §

S Supporting Information *

ABSTRACT: We report synthesis of targeted PEGylated polymer capsules encapsulating Stoke’s shift and upconverting LaVO4 nanoparticles by following a unique approach for bioimaging applications. First, LaVO4:Ln3+@silica (Ln3+ = Tb3+, Eu3+, and Yb3+/Er3+) core−shell nanoparticles are prepared by sol−gel method followed by layer-by-layer assembly of polymers and PEGylation over core−shell particles. Second, removal of silica core facilitates the trapping of LaVO4:Ln3+ nanoparticles inside the PEGylated polymer capsules. Finally, capsules are surface modified with antibodies to target cancer cells. The nanoparticles-loaded polymer capsules are found to be internalized and biocompatible with various cells (e.g., HeLa, A498, H460, MCF-7, Schwann, L929, and IC-21) suggesting their applicability in different types of cells. In addition, the capsules modified with antibodies show more specific uptake suggesting their targeting ability by 3-fold for MCF-7 and 10-fold for H460 cancer cells. Moreover, the nanoparticle-loaded polymer capsules were internalized by HeLa cells via macropinocytosis mechanism. We observed localized bright Stoke’s shift (Tb3+ ions, λex = 488 nm) and upconversion (Er3+ ions, λex = 980 nm) green fluorescence from cells suggesting their potential use as targeted bioimaging agents. KEYWORDS: LaVO4, PEGylated polymer capsules, cell uptake mechanism, upconversion lanthanide ions, Ln3+-doped nanoparticles, bioimaging To use them in bioimaging applications, the Ln3+-doped nanoparticles should also possess the following properties such as high water dispersibility, biocompatibility, stealthy nature and preferably targeting ability. These nanoparticles can be applied either as nonencapsulated nanoparticles (free nanoparticles) or encapsulated in a carrier (e.g., polymer capsule and liposomes).6,31,39 Though nonencapsulated nanoparticles show promising results, they offer the following challenges: (1) need of surface modification steps (e.g., conversion of hydrophobic to hydrophilic ones by ligand exchange, silica coating, polymer coating, micelles, etc.),23,28,31,40 (2) optical properties may get quenched because of the presence of surface hydroxyl groups (e.g., hydrothermal and microwave methods),5,41 and (3) poor water dispersibility due to polydispersed bigger size particles (e.g sol−gel approach).42 In addition, it is essential to study their uptake mechanisms individually because they enter into cells via different endocytic pathways because of the difference in the surface functionalities (e.g., surface charge, functionalities, shape, and size).43 These challenges can be circum-

1. INTRODUCTION Optical/fluorescence imaging is an inevitable tool to visualize organelle level changes taking place in a diseased tissue for example, cancerous cells or tumor. This imaging technique utilizes an agent or a material which produces fluorescence when excited with light of a different wavelength. Different fluorescence imaging agents such as organic dyes (e.g., fluoresceins and rhodamines),1 quantum dots (e.g., CdSe and PbSe),2−4 and lanthanide ion (Ln3+)-doped nanoparticles (e.g., LnVO4, NaLnF4, Ln2O3, and LnPO4)5 are generally used in bioimaging in which organic dyes suffer from photobleaching and broad emission whereas quantum dots exhibit toxicity.1,6,7 In this regard, lanthanide-doped nanoparticles have gained popularity because lanthanide ions possess the following unique advantages: (1) wide range of excitation (UV to NIR)8−11 and emission wavelengths in which NIR excitation region (700− 1300 nm) minimizes the autofluorescence12−14 and scattering of light,6,15−22 (2) enhanced photostability23,24 due to parity forbidden transitions occurring within 4f shell shielded by 5s and 5p orbitals,19,25−27 (3) large Stoke’s shift,7,21,28−32 (4) increased lifetimes (milliseconds),21,33,34 and (5) upconversion emission (a multiphoton process in which lower-energy photons are converted into higher energy photons).22,35−38 © XXXX American Chemical Society

Received: May 9, 2016 Accepted: July 5, 2016

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DOI: 10.1021/acsbiomaterials.6b00252 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Dulbecco’s modified eagle’s medium (DMEM), trypsin- EDTA, penicillin-streptomycin antibiotic, (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), bisBenzimide (Hoechst), FITC-phalloidin (fluorescein isothiocyanate-phalloidin), Rhodamine B isothiocyanate (RITC), RPMI-1640, human serum endocytic inhibitors (filipin-III, genistein, cytochalasin-D, rottlerin, chlorpromazine, and nocodazole), and gelatin (from cold water fish skin) were obtained from Sigma-Aldrich and used without further purification. Fetal bovine serum was purchased from Gibco, Cy 5.0 CellMask deep red plasma membrane stain (ex 649 nm/em 666 nm), TRITC CellMask orange plasma membrane stain (ex 554 nm/em 567 nm) were purchased from Invitrogen, India. Dimethyl sulfoxide (DMSO) and formaldehyde were obtained from Merck chemicals, India. Mouse (monoclonal) antihuman p185 HER-2 FITC-conjugate and epidermal growth factor receptor (EGFR) antibodies were obtained from invitrogen, India. 2.2. Synthesis of Polymer-Encapsulated LaVO4:Ln3+ Nanoparticles. Silica particles were prepared by base-catalyzed Stöber’s process58 with the hydrolysis of tetraethylorthosilicate (4.7 mL) using ammonia (54.4 mL) in a solution of water (2 mL) and ethanol (50 mL). The particles were separated centrifugally by washing with deionized water followed by ethanol to remove the excess of reactants and heated at 80 °C overnight. The size of silica has been controlled by slight modification in Stö ber’s synthesis.58 For coating of LaVO4:Eu3+ or Tb3+ shell @silica core, 5 mol % of Eu3+ or Tb3+ was doped in LaVO4 matrix using protocol carried out by Yu et al.59 Stoichiometric weights of LaNO3·xH2O, Eu(NO3)3·5H2O or Tb(NO3)3·5H2O, and NaVO3 were added to a mixture of 1:7 ratio of water to ethanol and kept under magnetic stirring. Citric acid was used as chelating agent for metal ions with a molar ratio of 2:1 and polyethylene glycol (PEG) was added as a cross-linking agent with the concentrations of 0.08 g/mL. The solution was stirred for 1 h and silica particles were added. The suspension was further stirred for 3 h and the coated silica particles were separated by centrifugation. The samples were dried at 80 °C overnight and annealed at 500 °C for 3 h in air at a heating rate of 4 °C/min. For preparation of upconversion nanoparticles coated on silica, 20% Yb3+ and 2% Er3+ were doped on LaVO4 matrix. The similar procedure was followed and the sample was annealed at 800 °C for 16 h. Polymer coating on core−shell particles were done using layer-by-layer (LbL) assembly method. PEI (1 mg/ mL) was added to the particles and kept in rotospin for 15 min. This adsorption step was followed by washing the samples with 0.5 M NaCl and centrifuging it at 1000 rcf for 3 min for removal of the excess of polymer present in supernatant. Subsequently, 7 polyelectrolyte layers were made with poly(sodium 4-styrene-sulfonate) (PSS) (1 mg/mL) and poly(allylamine hydrochloride) (PAH) (1 mg/mL) to form 8 layers with alternate positive and negative charge. Finally, removal of silica core was done with buffer oxide etchant 0.75 M HF: 4 M NH4F (Caution: HF is very toxic and should be handled with all precautions mentioned in Material Safety Data Sheet). Centrifugation was performed at 4500 rcf for 3 min followed by 5 times washing with deionized water to separate the polymer capsules and finally dispersed in 1 mL of deionized water. For charge-dependent cell uptake studies, LbL was also performed with PEI and PSS as outer layers. RITC-PAH tagged PSS/PAH assembled core−shell particles are also prepared to fabricate capsules (RITC-PAH was formed by labeling PAH with RITC).60 2.3. PEG Modification of LbL Assembled LaVO4:Tb3+@silica. For preparing PEG-coated polymer capsules, 10 mg of LbL assembled LaVO4:Tb3+@silica with PSS as outer layer was incubated with Bisamine-(PEG) (1 mg/mL) for 24 h. Excess Bisamine-(PEG) was removed by centrifugation and washing with deionized water. Silica core removal was carried out using buffer oxide etchant 0.75 M HF: 4 M NH4F (Caution: HF is very toxic and should be handled with all precautions mentioned in Material Safety Data Sheet). Centrifugation was performed at 4500 rcf for 3 min followed by 5 times washing with deionized water to separate the polymer capsules. 2.4. Antibody Modification of PEGylated Polymer Capsules. Antibody modification on the surface of PEGylated polymer capsules encapsulating LaVO4:Tb3+ or Eu3+ was carried out using EDC-NHS coupling.61 PEGylated capsules (1 × 107 capsules) were dispersed in

vented by encapsulating the nanoparticles into a carrier. To this end, we propose a unique sol−gel approach combined with layer-by-layer assembly (LbL) to prepare highly luminescent water dispersible Ln3+-doped LaVO4 nanoparticle-encapsulated polymer capsules for bioimaging applications. LaVO4 matrix has been widely studied by various groups because of low phonon energy (800 cm−1) and efficient energy transfer from vanadate to dopant ion with broad absorption range varying from 260 to 340 nm.39,44−46 The proposed approach comprises the following steps: (1) formation of LaVO4:Ln3+@silica core− shell particles via sol−gel approach and (2) polymer coating via LbL assembly followed by removal of silica core to encapsulate the particles inside the polymer capsule.47−56 The proposed approach has following advantages: (1) Sol−gel method facilitates to the preparation of highly luminescent nanoparticles. (2) Polymer capsules facilitates water dispersibility, biocompatibility, stealth nature due to polyethylene glycol (PEG),57 and possibility of encapsulation of drug along with nanoparticles39 to fabricate potential multifunctional vehicle. (3) This method can be generic to encapsulate various lanthanide-doped nanoparticles (e.g., LaVO4, GdVO4, YVO4, Gd2O3, and Y2O3). (4) Covalent attachment of antibodies over capsules facilitates the targeted imaging. Apart from above advantages, the proposed method possesses a unique advantage of delivering the nanoparticles to cells by a “single uptake mechanism” which is independent of nanoparticles encapsulated inside the capsule. We note that our earlier approach involves in situ synthesis of nanoparticles inside polymer capsules at room temperature which cannot be adapted to encapsulate upconverting nanoparticles requiring higher temperature synthesis conditions.39 Though the current approach seems to be complex, it provides upconversion emission along with essential biological characteristics for imaging applications. Herein, we report development of lanthanide ion (Ln3+ = Eu3+, Tb3+ or Yb3+/Er3+)-doped LaVO4 nanoparticles encapsulated inside PEGylated polymer capsules of sizes ∼500 nm and ∼300 nm using template-based approach. Capsules possessing nanoparticles show good Stoke’s shift and upconversion luminescence in aqueous solution without any ligand on the surface. They were also found to be cyto-compatible and get internalized in various cells including HeLa, A498, H460, MCF7, Schwann, L929, and IC-21. Macropinocytosis is found to be the major mechanism of polymer capsules uptake in cells. It has been observed that Erbb-2/HER-2-modified capsules explicitly target the MCF-7 cancer cells 3-fold more than nonspecific cells and similarly, EGFR modified capsules specifically target the H460 cells by 10 times.

2. MATERIALS AND METHODS 2.1. Materials. Lanthanum nitrate hydrate (LaNO3.xH2O), europium nitrate pentahydrate (Eu(NO3)3·5H2O), ytterbium(III) nitrate pentahydrate (Yb (NO3)3·5H2O), erbium(III) nitrate pentahydrate (Er(NO3)3·5H2O), terbium nitrate pentahydrate (Tb(NO3)3· 5H2O), polyethylene glycol (Mw 10 000), poly(sodium 4-styrenesulfonate) Mw 70 000, poly(allylamine hydrochloride), Mw 56 000, bisamine (polyethylene glycol) Mw 6000, N-hydroxysuccinimide, and (1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide) (EDC) were purchased from Sigma-Aldrich. All the lanthanide salts were 99.9% pure and used without further purification. Polyethylene imine (PEI, Mw 70000 branched, Alfa Aesar) citric acid (Qualigens), liquor ammonia (about 24%, Merck), ammonium fluoride (Merck), sodium metavanadate lobachemie, hydrofluoric acid (40%w/v) sd-fine chem., tetraethylorthosilicate (Fluka). B


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ACS Biomaterials Science & Engineering Scheme 1. Schematic Illustration of the Antibody Modified PEGylated polymer capsules Encapsulating LaVO4:Ln3+ Nanoparticles (scheme is not drawn to scale)

MES buffer (500 mM, pH 6.1, 1 mL) followed by addition of EDC (50 mg/mL) and NHS (10 mg/mL) in a 1:2 (v/v) ratio to polymer capsules and incubated for 30 min at room temperature. Polymer capsules were centrifuged and resuspended in MES buffer (9 mL, 50 mM) containing HER-2 or EGFR antibody (10 μg/mL) (pH 6.1) and incubated overnight with proper mixing. The antibody modified polymer capsules were separated by centrifugation at 4500 rcf for 5 min and unconjugated antibody was removed by multiple washing steps. 2.5. Cell Lines. For in vitro studies, the following cell lines were purchased from National Centre for Cell Science Pune, which is a national repository of cell lines in India: HeLa cells (human cervical adenocarcinoma cell line), A498 cells (kidney cancer), H460 cells(lung cancer), IC-21 (macrophages), MCF-7 (breast cancer), L929 cells (fibroblast). Schwann cells were obtained from American Type Culture Collection (ATCC). 2.6. In Vitro Cytotoxicity Assay. For in vitro studies, cells were cultured in DMEM medium containing heat inactivated FBS (10% v/ v) and penicillin-streptomycin (1% v/v), grown in incubator having humidified atmosphere containing 5% CO 2 at 37 °C. The biocompatibility of PEGylated polymer encapsulated LaVO4:Tb3+ capsules were evaluated by MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide).39 Cells (1 × 104) were seeded in clear bottom 96-well plate. After 6 h of incubation, capsules (25, 50, and 75 capsules/cell) dispersed in media were added and in “control” wells media was added without capsules (i.e., only cells). The cells were incubated with capsules for 17 h at 37 °C, 5% CO2 humidified incubator. After incubation the culture medium was removed, MTT (0.5 mg/mL) dissolved in fresh basal media was added to each well, and the plates were reincubated for 4 h. After incubation, media containing MTT was removed and DMSO (300 μL) was added to each well. The appearance of blue color solution in the wells was measured using a UV−visible microplate reader at 570 nm to determine optical density value. All the assays were done in triplicates and repeated 5 times. 2.7. Cell Uptake Studies. To analyze cellular uptake, we seeded cells (1 × 104) on 13 mm glass coverslip coated with 0.2% gelatin in a 24-well plate. Once the cells get attached, polymer capsules were added and incubated for 17 h, and the treatment ratio was 25 capsules (nanoparticles-loaded) per seeded cell. After incubation, the respective media was discarded and cells were washed thrice with PBS (300 μL) to remove unbound capsules. Cells were fixed with 4% formaldehyde for 20 min followed by staining for actin cytoskeleton and nucleus of

the cells with FITC-Phalloidin (green)/deep red plasma membrane dye/cell mask orange membrane dye and/or hoechst (blue), respectively. A monolayer of cells was washed thrice with PBS (300 μL) and mounted in a glass slide to be observed under confocal laser scanning microscopy.39 2.8. Cell Pathway Mechanism Study. The cell pathway mechanism study was carried out on HeLa (1 × 104) cells. Cells were seeded on 13 mm glass coverslip coated with 0.2% gelatin in a 24-well plate. Six chemical inhibitors were used, including filipin (1 μg/mL), chlorpromazine (10 μg/mL), cytochalasin D (5 μM), genistein (100 μM), nocodazole (20 μM), and rottlerin (25 μg/ mL). The inhibitors were preincubated with seeded cells for 30 min (nocodazole, chlorpromazine) and 1 h (remaining inhibitors), respectively. Inhibitors were removed and capsules (25 capsules/ cell) were incubated with cells for 4 h. Cells were fixed with 4% formaldehyde for 20 min followed by staining with cell mask orange membrane dye or deep red plasma membrane dye and nucleus was stained with hoechst (blue). Monolayer of cells was washed thrice with PBS (300 μL) and mounted in glass slide to be observed under confocal laser scanning microscopy. For flow cytometry studies, similar protocol was followed and instead of fixing the cells, they were trypsinized using trypsin-EDTA and resuspended in media.39 2.9. Characterization. Photoluminescence measurements were done with Edinburgh Instruments Fluorescence spectrometer (FLSP 920) equipped with 450W Xe arc lamp as source for emission spectra and microflash lamp for lifetime decay curve analysis. All the upconversion emission spectra were measured using 980 nm continuous wave (CW) laser coupled with fiber (power density: 90 mW/cm2). The morphology of the samples was acquired from SUPRA Series ultra high resolution field emission-scanning electron microscope FE-SEM (SEM). The formation of core−shell structures were visualized from transmission electron microscope (TEM) images obtained using FEI Technai Twin microscope with high contrast and resolution at 20 kV to 120 kV. LbL assembly on core−shell particles were analyzed for zeta potential values using Delsa Nano C zeta sizer (Beckman Coulter, Inc., USA). The thickness of the polymer layers was obtained using Asylum research atomic force microscopy (AFM).62,63 The X-ray diffraction (XRD) pattern was acquired with PANalytical’s (X’Pert3 Powder) system. The concentration of lanthanum in the capsules was found by Thermo Scientific XSERIES2 inductively coupled plasma-mass spectrometry (ICP-MS). Thermo Scientific Multiskan UV−vis spectrophotometer was used for measuring absorbance for MTT assay. The experiments have been C


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Figure 1. (a) HRTEM image of LaVO4:Tb3+@silica, (b) TEM image of PEGylated polymer coated LaVO4:Tb3+@silica (∼500 nm), (c) TEM image of PEGylated polymer capsules (∼500 nm) encapsulating LaVO4:Tb3+ nanoparticles (inset images, top, SAED pattern; and bottom, FFT pattern of LaVO4:Tb3+ nanoparticles) (d) TEM image of PEGylated polymer capsules (∼300 nm) encapsulating LaVO4:Tb3+ nanoparticles (inset images: top, SEM image of PEGylated capsules (∼500 nm) encapsulating LaVO4:Tb3+ nanoparticles; and bottom, HRTEM image of LaVO4:Tb3+ nanoparticles showing lattice fringes). carried out 5 times and difference in data points were calculated using standard deviation and standard error. Internalization of polymer encapsulated nanoparticles in cells were confirmed by images from confocal laser scanning microscopy (CLSM) Carl Zeiss LSM 710 and Carl Zeiss LSM 780 equipped with Chameleon multiphoton laser (1.3 W). Flow cytometry was carried out using Partec flow cytometer equipped with 488 nm laser (software: FloMax). It was used for quantification of capsules and analyzing its cellular uptake. Green emission from Tb3+ ions (encapsulated in polymer capsules) was exploited for the quantification of capsules. In addition, forward and side scattering data were obtained for quantifying the internalization of capsules, as the cellular uptake leads to changes in size and granularity of cells.39,43,64

LaVO4@silica core−shell particles are prepared for the current study. (3) LaVO4:Ln3+@silica core−shell particles have been annealed at higher temperature to obtain LaVO4 monoclinic phase and to remove OH groups present on the surface of LaVO4 nanoparticles. (4) LaVO4:Ln3+@silica core−shell particles showing Stoke’s shift fluorescence (Eu3+and Tb3+) are annealed at 500 °C, whereas core−shell particles doped with upconverting ion (Yb3+/Er3+) were annealed at 800 °C to obtain brighter upconversion emission. (5) LbL assembly has been carried out using PEI, PSS and PAH as polyelectrolytes where PEI is used as first layer, followed by alternate deposition of PSS and PAH forming eight layers. (6) To achieve the structural integrity for polymer capsules, we need to form eight polymer layers, which also prevent the leaching of cargo (nanoparticles) from the capsules before reaching target site. (7) PEGylation has been carried out with PEG-Bisamine over PSS layer by electrostatic interaction between sulfonate of PSS and amine group of PEG-Bisamine. (8) After the removal of silica, the polymer layers stick together to retain as the capsule because of stronger electrostatic interaction between PEI and PSS than PEI with the LaVO4 surface. In addition, LaVO4 shell possesses weak mechanical strength leading to breaking of LaVO 4 shell into smaller particles, which have been encapsulated inside the polymer. (9) The size of capsules is controlled by the size of silica, which matches with earlier reports.65 We have performed electron microscopy studies to confirm the loading of LaVO4 nanoparticles inside the polymer capsules. Contrast difference in the TEM image (Figure S1) clearly suggests the formation of LaVO4 shell with thickness of

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization. Scheme 1 illustrates the schematic representation of the synthesis of antibody modified PEGylated polymer capsules encapsulating LaVO4:Ln3+ nanoparticles involving five steps methodology: (a) fabrication of LaVO4:Ln3+ over silica template to form a core− shell structure by sol−gel method,59 (b) LbL assembly of polyelectrolytes over core−shell particles, (c) PEGylation over LbL assembly, (d) removal of silica core to encapsulate LaVO4:Ln3+ nanoparticles inside PEGylated polymer capsules, and (e) surface modification of capsules with antibody (e.g., Erbb2/HER2, EGFR) for targeting purpose. We wish to note the following points with respect to the current fabrication protocol. (1) Silica has been chosen as sacrificial template because it can be annealed at higher temperature which is required for synthesis of highly luminescent LaVO4 nanoparticles. (2) Various Ln3+ (Eu3+, Tb3+, Yb3+/Er3+)-doped D


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ACS Biomaterials Science & Engineering ∼90 nm over silica (∼500 nm). Lattice fringes in HRTEM image (Figure 1a) further confirms the formation of crystalline LaVO4 shell. Further, contrast in the TEM image (Figure 1b) of LbL assembled core−shell particles (size ∼500 nm) suggest the formation of polymer layers (denoted by arrow). LbL assembly was further confirmed by zeta potential measurements (Figure S2), which show alternative positive and negative charges. Bare LaVO4:Tb3+@silica core−shell particles possess negative charge whereas first layer possess positive charge due to formation of PEI. Consecutive layers possess alternative charges which supports assembly of PSS (-vely charged) and PAH (+vely charged) layers. We have also attempted to prepare polymer capsules with 6 layers, SEM image (Figure S3) clearly indicates the presence of broken polymer network along with nanoparticles supporting the need of 8 polymer layers. Further, we performed AFM analysis of polymer capsules (8 PSS/PAH and one PEG layer) for measuring thickness of polymer layers. Figure S4 clearly suggests that the thickness of polymer layers is ∼33 nm. Therefore, it can be concluded that thickness of each layer could be ∼2 nm which matches with earlier report.62,63,66 The entrapment of nanoparticles inside PEGylated polymer capsules and removal of silica core were confirmed by TEM images (Figure 1c, d). Figure 1c suggest the formation of LaVO4 nanoparticles (size ∼45 nm) inside the polymer capsules (size ∼500 nm), in addition, selected area electron diffraction (SAED) pattern (top inset) and fast Fourier transform (FFT) pattern (bottom inset) confirm their polycrystalline nature. Figure 1d shows the TEM image of polymer capsules (size ∼300 nm), which also supports the encapsulation of nanoparticles. SEM image (Figure 1d: top inset) shows the surface morphology of polymer capsules, which suggest the absence of nanoparticles on the surface of capsules as observed in our previous report.39 We note that aggregation appearing in SEM and TEM images may be attributed to drying effect during sample preparation. The XRD pattern of LaVO4 nanoparticles matches with monoclinic phase (Figure S5). A small peak shift is observed toward higher angle which may be due to higher doping concentration.67 HRTEM image (Figure 1d, bottom inset) suggests that the calculated dspacing (0.33 nm) matches with (002) plane of LaVO4 monoclinic phase. Figure S6 shows the FTIR spectrum of LbL-assembled LaVO4:Tb3+@silica particles suggesting the presence of Si−O−Si bond at 1100 cm−1, which is absent after silica core removal. This suggests the removal of silica template. The amount of LaVO4 loaded inside the capsules is quantified as ∼58 μg/1 × 107 capsules using ICP-MS. 3.2. Luminescence Properties. Emission spectra of PEGylated polymer-encapsulated LaVO4:Yb3+/Er3+ nanoparticles dispersed in water show sharp peaks at 515, 545, and 660 nm corresponding to 2H11/2 → 4I15/2, 4S3/2 → 4I15/2 and 4F9/2 → 4I15/2 transition levels of Er3+ ions, excited with 980 nm continuous wave laser (Figure 2). Excitation of PEGylated capsules encapsulating LaVO4:Eu3+ nanoparticles at 280 nm wavelength, shows emission spectrum (Figure 3) with 5D0-7F2 transition (614 nm) of Eu3+ attributing to noninversion symmetry of the ion in LaVO4 matrix. Emission spectrum of polymer encapsulated LaVO4:Tb3+ nanoparticles at 280 nm excitation shows prominent emission peaks of Tb3+ ions at 545 nm, 585 and 620 nm (Figure 3). Further, our luminescence lifetime data of encapsulated LaVO4:Eu3+ nanoparticles (∼815 μs) and bare nanoparticles (∼823 μs) is similar (Figure S7a, c). In case of surface modification on LaVO4 nanostructures, after the core removal, the lifetime data is expected to decrease

Figure 2. Upconversion photoluminescence spectra of LaVO4:Yb3+/ Er3+, nanoparticles encapsulated in PEGylated polymer capsule (∼500 nm) dispersed in water (λex = 980 nm continuous wave laser, (*) represents artifact during measurement).

Figure 3. Photoluminescence spectra of PEGylated polymer capsules dispersed in water encapsulating (a) LaVO4:Eu3+ and (b) LaVO4:Tb3+ nanoparticles (λex = 280 nm wavelength, excitation source: xenon lamp).

sharply because of the presence of organic groups.37 This suggests that surface modification has not happened over LaVO4 after the core removal. Luminescence lifetime of PEGylated polymer capsules encapsulating LaVO4-doped Tb3+ ions are found to be 1.5 ms suggesting the doping of Tb3+ ions in LaVO4 matrix (Figure S7b).7,26,39,46,68 3.3. In Vitro Studies. For an imaging vehicle to be effectively applied in biological systems, it has to be checked for its biocompatibility, internalization in cells, elucidation of uptake (endocytic) mechanism and finally specific targeting ability at in vitro level. Biocompatibility was tested using MTT assay in various cancer cells (e.g., HeLa (human cervical adenocarcinoma cells), A498 cells (human kidney cancer cells), H460 cells (human lung cancer), MCF-7 (human breast cancer), schwann cells (noncancerous neuronal cells), L929 cells (mouse fibroblast), and primary cells (IC-21 (mouse peritoneal macrophages)). Figure 4a demonstrates the cytocompatibility of PEG-modified polymer capsule-encapsulated E


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Figure 4. MTT assay plots of PEGylated polymer capsules encapsulating LaVO4:Tb3+ nanoparticles incubated with cells for 17 h: (a) HeLa cells (capsules size ∼500 nm) with different number of capsules/cell, (b) ∼500 nm size capsules (50 capsules/cell), and (c) ∼300 nm capsules (50 capsules/cell) with different types of cells.

(∼500 nm size) LaVO4:Tb3+ nanoparticles for concentrations up to 75 capsules per cell in HeLa cells. MTT assay was also performed in all the above-mentioned cell lines for both ∼500 nm (Figure 4b) and ∼300 nm (Figure 4c) size polymer capsules respectively, and the results indicate cell line independent biocompatibility (Figures S8 and S9). We note that the upconversion nanoparticles encapsulated polymer capsules also possess biocompatibility suggesting the compatibility of different dopants (Figure S9g). For cell uptake studies,

either PEGylated polymer capsules encapsulating LaVO4:Yb3+/ Er3+ or Tb3+ was fabricated, which exhibits green fluorescence, or RITC-PAH-tagged PSS/PAH capsules were prepared, which showed red emission. Cell uptake studies were carried out by incubating the PEGylated polymer capsules encapsulating LaVO4:Tb3+ nanoparticles (both ∼500 nm and ∼300 nm, respectively) with a concentration of 25 capsules per cell in the previously mentioned cells to study the internalization of polymer capsules which shows green emission arising from F


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Figure 5. CLSM images representing internalization of PEGylated polymer capsules (∼300 nm, 25 capsules/cell) encapsulating LaVO4:Tb3+ nanoparticles with different types of cells. Cell membrane was stained with Cy5 dye. Scale bar =20 μm.

Tb3+-doped nanoparticles confirming the internalization of capsules (Figure 5, Figure S10). Figure S11 shows the red emission arising from RITC-labeled PAH layer, which clearly proves the structural integrity of polymer layers. From the biocompatibility and uptake of different cells it can be concluded that these capsules can be employed as imaging vehicles in variety of cancerous/noncancerous cells. Further, quantification of HeLa cells internalizing the polymer capsules encapsulating LaVO4:Tb3+ nanoparticles after 2 h have been calculated using flow cytometry. We deduced the percentage of cell internalization using flow cytometry software (FloMax). Figure S12 shows the plot between forward scattering (FSC) and side scattering (SSC) in which Q3 belongs to bare cells whereas Q2 belongs to cells internalized with nanoparticlesencapsulated polymer capsules. It has been found that 64% of polymer capsules encapsulating LaVO4:Tb3+ nanoparticles were internalized in HeLa cells after 2 h incubation. This has been further confirmed by the control experiment of bare HeLa cells which does not show any population of cells in Q2 (Figure S12). We have also quantified the internalization percentage of both ∼300 nm and ∼500 nm size capsules in HeLa cell (after 6 h incubation) by plotting FSC Vs FL1 (green emission from Tb3+). Further, Figure S13 suggests that internalization is efficient in the case of ∼300 nm size polymer capsules compared to ∼500 nm sizes. Next, charge-dependent cell uptake study has been performed in HeLa cells to analyze the effect of surface charge of polymer capsules. In case of positively charged PAH, high efficiency of capsule internalization was observed in the confocal microscopy (red emission of RITC-labeled capsules), which can be attributed to the fact that cell membrane is negatively charged. The uptake of PEGylated polymer capsules is less compared to capsules possessing PAH as outer layer because of neutral charge and stealth nature of PEG.69 It is noted that for imaging purposes, the capsules can be even associated with the cells instead of internalization. In contrast to PAH/PEI capsules, negatively charged capsules having PSS as outer layer showed lesser uptake because of the negative charge of plasma membrane (Figure S14). We note that capsules with highly positively

charged PEI as outer layer exhibited some toxicity as reported in previous studies.70,71 Elucidation of endocytic pathway through which the polymer capsules get internalized in HeLa cells has been studied to find the interaction of capsules with cells. Two different capsule surfaces were taken for study of endocytosis mechanism: PEGylated capsules and non-PEGylated PSS/PAH capsules. Cells were preincubated with inhibitors to block a specific pathway. Six inhibitors were used to study the endocytic pathway which includes filipin-III and genistein (cavaloe mediated),72 cytochalasin-D (actin polymerization),72 rottlerin (macropinocytosis),73 chlorpromazine (clathrin mediated),72 and nocodazole (microtubule mediated intracellular uptake).43 In the case of PEGylated polymer capsules, it can be described from the Figure S15 that the internalization of capsules is less in the case of cytochalasin D and nocodazole (graph in Figure 6a represents capsules internalized per 50 HeLa cells). Because these two chemical inhibitors are related to actin polymerization-based macropinocytosis pathway, the capsules are evidently following macropinocytosis mechanism. A detailed flow cytometry based analysis (Figure S16a) shows individual FSC Vs green emission from nanoparticles of endocytic inhibitors. Graphical representation (Figure S16b) derived from flow cytometry data also corroborates with the confocal microscopy based observation. From confocal microscopy images (Figure S17), it can be interpreted that internalization of PSS/PAH capsules encapsulating LaVO4:Tb3+ nanoparticles (RITC-labeled) are observed in all cases except rottlerin as inhibitor where negligible number of particles were found to be internalized (Figure 6b demonstrates the numerical value of number of internalized capsules per 100 cells with minimal uptake in rottlerin inhibited pathway). This clearly delineates that capsules were mainly following macropinocytosis mechanism, because the uptake depends on outer surface of polymer capsules and not on the encapsulated nanoparticles leading to a nanoparticle independent uptake mechanism. MTT assay plot of HeLa cells incubated with endocytic pathway inhibitors for 1 h with the treatment concentration the same as that used in cell G


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Figure 7. Internalization of antibody decorated PEGylated polymer capsules encapsulating LaVO4:Tb3+ nanoparticles with different cells: (a) HER-2 (specific antibody for MCF7) decorated capsules and (b) EGFR (specific antibody for H460) decorated capsules. Plots represent the percentage of capsules internalized within cells.

Figure 6. Representation of internalization of polymer capsules encapsulating LaVO4:Tb3+ nanoparticles in HeLa cells, preincubated with different endocytic pathway inhibitors (a) PEGylated PSS/PAH and (b) PSS/PAH polymer capsules.

uptake pathway study was performed to understand the cytotoxicity behavior of the inhibitors (Figure S18). To illustrate the targeting capability of capsules, we carried out antibody modification with HER-2/Erbb-2 (antibody specific for MCF-7)74 and EGFR (antibody specific for H460)75 using EDC-NHS coupling reaction. Fluorescence microscopy image of Figure S19 shows the green emission from HER2-FITC conjugated PEGylated polymer capsules indicating their modification with antibody. HeLa cells were incubated with capsules for analysis as negative control because HeLa is not specific to both the antibodies. MTT assay studies (Figure S20) with MCF7, H460, and HeLa cells suggest that antibody modification did not induce any cytotoxicity. Flow cytometry analysis demonstrate the specificity of antibody conjugated polymer capsules toward cancer cells with higher percentage of uptake in specific cancer cells compared to nonspecific cells (Figures S21 and S22). Figure 7 demonstrates the specific targeting of cancer cells using antibody conjugated capsules where Erbb-2/HER-2 modified capsules clearly target the MCF-7 cancer cells (Figure 7a) 3-fold and, EGFR modified capsules specifically target the H460 cells by 10 times (Figure 7b) more than nonspecific HeLa cells. We note that representative confocal microscopy images of internalization studies with antibody conjugated polymer capsules in MCF-7 and H460 cells is given in Figure S23. To demonstrate their upconversion imaging capability, we performed uptake imaging studies with polymer capsules encapsulated with LaVO4:Yb3+/Er3+ nanoparticles. Figure 8 clearly shows the presence of green emission arising from Er3+ ions through excitation with 980 nm wavelength using

Figure 8. CLSM image of upconverting PEGylated polymer capsules (∼300 nm) encapsualting LaVO4:Yb3+/Er3+ nanoparticles internalized in A498 cells (image was acquired using 980 nm laser excitation). Cell membrane was stained red using Cy5 dye. Scale bar = 20 μm.

multiphoton laser. Further, it suggests that polymer capsules encapsulating LaVO4:Yb3+/Er3+ nanoparticles are internalized in A498 and the encapsulation process does not affect the green emission. Furthermore, LaVO4:Yb3+/Er3+ nanoparticles present inside the capsules have not been affected/degraded during the internalization process (it is to be noted that these images were taken after a 17 h incubation period). We have also observed the emission of Er3+and Tb3+ ions through 488 nm laser excitation from HeLa cells (Figure S24). H


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4. CONCLUSIONS We report the encapsulation of Stoke’s shift and upconverting LaVO4:Ln3+ nanoparticles in PEGylated polymer capsules fabricated using LbL assembly. These capsules were biocompatible and internalized in a range of cells including e.g. HeLa, A498, H460, MCF-7, Schwann, L929, and IC-21 suggesting the cell line independent biocompatibility and uptake. Further, antibody-modified PEGylated polymer capsules encapsulated with nanoparticles show enhanced specific uptake suggesting their targeting ability by 3-fold for MCF-7 and 10-fold for H460 cancer cells. Endocytic cell pathway mechanism studies suggest that the capsules were internalized by macropinocytosis. Localized bright green upconversion emission from cells internalized with LaVO4:Yb3+/Er3+ nanoparticles suggest their potential application in targeted fluorescence bioimaging of cancer cells.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00252. TEM image of core−shell particle, zeta potential measurements, AFM of polymer capsules, FTIR spectra, lifetime curve of PEGylated polymer capsules, MTT assay plots, CLSM images, flow cytometry analysis of uptake mechanism of PEGylated capsules, MTT assay of inhibitors, fluorescence microscope image, MTT assay of antibody conjugated capsules, flow cytometry plots (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +91-512-2597697. Fax: 91-5122590104. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly acknowledge the grants from Nanomission, Department of Science and Technology (DST), Department of Biotechnology (DBT), and UK-India Education and Research Initiative (UKIERI). We also acknowledge Dr. Ashok Kumar, Department of Biological sciences and Bioengineering, IIT Kanpur for providing his lab facilities for conducting preliminary cell culture experiments. We also thank Dr. Andrew Jackson and Prof. Poulam Patel, Academic Unit of Oncology, University of Nottingham-UK for their fruitful discussions.

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REFERENCES

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