Article pubs.acs.org/Langmuir
An Effective Polymer Cross-Linking Strategy To Obtain Stable Dispersions of Upconverting NaYF4 Nanoparticles in Buffers and Biological Growth Media for Biolabeling Applications Guicheng Jiang,†,‡ Jothirmayanantham Pichaandi,‡ Noah J. J. Johnson,‡ Robert D. Burke,§ and Frank C. J. M. van Veggel*,‡ †
Department of Physics, University of Science and Technology of China, Hefei 230026, China Department of Chemistry, University of Victoria, P.O. Box 3065, Victoria, British Columbia, Canada V8W 3V6 § Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055, Victoria, British Columbia, Canada V8W 3P6 ‡
S Supporting Information *
ABSTRACT: Ligands on the nanoparticle surface provide steric stabilization, resulting in good dispersion stability. However, because of their highly dynamic nature, they can be replaced irreversibly in buffers and biological medium, leading to poor colloidal stability. To overcome this, we report a simple and effective cross-linking methodology to transfer oleate-stabilized upconverting NaYF4 core/shell nanoparticles (UCNPs) from hydrophobic to aqueous phase, with long-term dispersion stability in buffers and biological medium. Amphiphilic poly(maleic anhydride-alt-1-octadecene) (PMAO) modified with and without poly(ethylene glycol) (PEG) was used to intercalate with the surface oleates, enabling the transfer of the UCNPs to water. The PMAO units on the phase transferred UCNPs were then successfully cross-linked using bis(hexamethylene)triamine (BHMT). The primary advantage of cross-linking of PMAO by BHMT is that it improves the stability of the UCNPs in water, physiological saline buffers, and biological growth media and in a wide range of pH values when compared to un-cross-linked PMAO. The cross-linked PMAO−BHMT coated UCNPs were found to be stable in water for more than 2 months and in physiological saline buffers for weeks, substantiating the effectiveness of cross-linking in providing high dispersion stability. The PMAO−BHMT cross-linked UCNPs were extensively characterized using various techniques providing supporting evidence for the cross-linking process. These UCNPs were found to be stable in serum supplemented growth medium (37 °C) for more than 2 days. Utilizing this, we demonstrate the uptake of cross-linked UCNPs by LNCaP cells (human prostate cancer cell line), showing their utility as biolabels.
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INTRODUCTION Lanthanide (Yb3+, Tm3+, Er3+) doped upconverting nanoparticles (UCNPs) have garnered a lot of attention in the past decade because of their potential applications in security labels, LEDs, photovoltaics, and lasers, with biolabeling currently being the focal point of many research groups.1−4 Unlike downconversion where we observe visible and near-infrared (NIR) light when excited with ultraviolet−visible (UV−vis) light,5−7 upconversion is capable of emitting ultraviolet, visible, and NIR light when excited with a NIR light source with photons of lower energy. UCNPs have a lot of potential advantages compared to current bioimaging agents such as fluorescent proteins,8 organic dyes,9 metal nanoparticles (gold, silver),10,11 and quantum dots (QDs)12 because of their very low phototoxicity,13 extremely stable photocycle, and very minimal autofluorescence.14,15 They also exhibit long lifetimes, narrow emission spectra, and large anti-Stokes shifts and require very low excitation power densities (5−10 W/cm2) to emit photons.14 Moreover, the emission wavelengths can be chosen to be in the transparent region (700−1100 nm) of © 2012 American Chemical Society
biological tissue by varying the lanthanide dopants and their doping concentration in the matrix.16,17 In particular, the β-phase NaYF4 nanoparticles doped with Yb3+ and Er3+(Tm3+) ions are known to be the most efficient upconverting phosphors.18 Recent advances in wet chemical synthesis have demonstrated uniform β-NaYF4:Yb3+, Er3+ (Tm3+) UCNPs with high reproducibility and shape control.19−21 These high quality UCNPs are synthesized in high boiling organic solvents, resulting in UCNPs stabilized with hydrophobic oleate ligands dispersible in apolar organic solvents. To utilize these UCNPs in biological applications, the surface of these hydrophobic UCNPs has to be made hydrophilic while simultaneously generating reactive functional groups which could further be used for conjugation to different molecules depending on the application. There are several strategies to modify the surface functionality of hydrophobic Received: October 13, 2011 Revised: January 16, 2012 Published: January 17, 2012 3239
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buffers. Herein, we report a method in which the polymer poly(maleic anhydride-alt-1-octadecene) (PMAO) (modified with and without PEG) is coated over UCNPs using the hydrophobic interaction between the octadecene chain of the polymer and the oleate ligand on the surface of the nanoparticles, followed by cross-linking the anhydride rings of the PMAO with bis(hexamethylene)triamine (BHMT), as illustrated in Scheme 1. There are three major reasons for
nanoparticles and transfer them to the hydrophilic phase. Some of them are (i) encapsulation of the nanoparticles with silica,22,23 (ii) ligand oxidation reaction, 24 (iii) ligand exchange,15,16,22,25 (iv) intercalation using amphiphilic polymers,26,27 and (v) cross-linked polymer coating.28−30 Encapsulation by amorphous silica shell is an efficient approach, in which the nanoparticles are encapsulated by a silica shell through a reverse microemulsion process enabling their transfer to hydrophilic phase. Even though silica coating makes the nanoparticles very stable in physiological buffers, the inability to remove completely the excess surfactants results in low colloidal stability over time, making silica coating rather unattractive.22 In the case of ligand oxidation reaction, the hydrophobic nanoparticles are transferred to water by directly oxidizing the oleic acid ligands present on the surface with the Lemieux−von Rudloff reagent. However, this method suffers from long reaction times and low yield.31 The ligand exchange process results in complete or partial replacement of the hydrophobic surfactant molecules on the nanoparticles by hydrophilic molecules or polymers. Although the ligand exchange strategy is simple for making water-dispersible nanoparticles, they suffer from poor colloidal stability under physiological conditions due to weak interactions of surface ligands with nanoparticles.32 When amphiphilic polymers are used for coating the nanoparticles, the hydrophobic part of the polymer intercalates into the interstitial spaces between the oleate ligands present on the surface of the nanoparticles due to hydrophobic interactions, while the hydrophilic part extends out resulting in water-dispersible nanoparticles.33−36 Our group has recently reported an intercalation process using PEG-oleate to disperse the nanoparticles in water.37 The intercalation process results in the nanoparticles forming a very stable dispersion in water for several days. However, when dispersed in physiological buffers (phosphate, tris, and borate buffers), they are stable for some hours, after which the nanoparticles start to settle down. This is due to the fact that the ligands due to their mobile nature get detached from the surface of the nanoparticles. This irreversible detachment is caused by the presence of salts in buffers which show very high affinity toward lanthanide ions on the surface of the nanoparticle. Recently, cross-linked polymer coatings on the surface of nanoparticles have been developed for quantum dots and iron oxide nanoparticles with different polymers such as polyacrylates,28 polyimidazoles,29 polystyrene-block-poly(acrylic acid),30 dendrimers,38 and poly(maleic anhydride-alt-1-octadecene).33,39 These polymers utilize the hydrophobic interaction between them and the surface ligands on the nanoparticles to wrap the nanoparticles, after which the polymer is cross-linked using a cross-linking agent. The cross-linking strategy would make sure that the surface ligands are not dynamic, which should result in excellent colloidal stability in water as well as in buffers, at different pH values, and when used in vivo.40 In general, only stability in water dispersions is reported and occasionally in pH buffers. To our knowledge, stability in serum-supplemented growth media has not been reported, except in one case.16 Serum-supplemented growth media are arguably a better mimick for the actual environment in in-vitro and in-vivo studies (e.g., cell studies and after injection into a vein of a small animal, respectively). Progress on the surface modification of UCNPs to get stable dispersions in water has widely been reported as mentioned above, yet there is still no generalized procedure to obtain longterm stability in biological growth medium and physiological
Scheme 1. Illustration of Cross-Linked Polymer Stabilized Upconverting NPs (UCNPs−PMAO−BHMT)
choosing the amphiphilic polymer PMAO: (1) the anhydride rings can be cross-linked, (2) functionalizing the polymer with e.g. PEG-amine makes it biocompatible, and (3) the octadecene chains in PMAO can intercalate with the oleate chains present on the surface of the nanoparticles. These hydrophilic shell cross-linked UCNPs exhibit very high stability at different pH values, physiological buffers, and biological growth media. Finally, we show that these cross-linked UCNPs can potentially be used in biolabeling applications, by using them to label human prostate cancer cells (LNCaP).
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EXPERIMENTAL SECTION
Materials. All chemicals were used as received without further purification. LnCl3·6H2O (Ln = Y, Yb, and Tm, 99.99%), oleic acid (OA, 90%), 1-octadecene (ODE, 90%), bis(hexamethylene)triamine (BHMT), octylamine (OTA), poly(maleic anhydride-alt-1-octadecene) (PMAO, average Mn 30 000−50 000), chloroform, methanol, ammonium fluoride (99.99+%), and sodium hydroxide (≥97%) were all purchased from Sigma-Aldrich. PEG-amine (2000) was from Huntsman Chemicals. Synthesis of NaYF4:30 mol % Yb3+, 0.5 mol % Tm3+ and NaYF4:20 mol % Yb3+, 2 mol % Er3+ Core Nanoparticles. The synthesis of UCNPs is similar to the previously reported protocol with some minor modifications.19 YCl3·6H2O (421.6 mg, 1.39 mmol), YbCl3·6H2O (232.5 mg, 0.6 mmol), and TmCl3·6H2O (3.8 mg, 0.01 mmol) were mixed in a 100 mL three-neck round-bottom flask with 12 mL of oleic acid and 30 mL of octadecene. This mixture was heated at 130 °C under vacuum for 40 min to obtain an optically clear solution and then cooled to room temperature. A methanol (∼20 mL) solution containing NaOH (0.2 g, 5 mmol) and NH4F (296.4 mg, 8 mmol) was added dropwise into the three-necked flask. The mixed solution was stirred for 40 min at room temperature followed by the slow evaporation of methanol. Once methanol was completely evaporated, the reaction temperature was increased to 100−120 °C. Subsequently, the reaction mixture was rapidly heated (15 °C/min) to 300 °C under an argon flow and was maintained at that temperature for 1 h. The solution was cooled down to room temperature, and the nanoparticles were subsequently precipitated in anhydrous ethanol. The UCNPs were then washed with ethanol (30 mL) for three times. The obtained NaYF4:30 mol % Yb3+, 0.5 mol % Tm3+ core nanoparticles were 3240
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Cell Imaging by Fluorescence Microscopy. Imaging of the nanoparticle uptake by LNCaP cells was done using a Leica DM 6000B microscope with the excitation at 980 nm using a JDS Uniphase laser diode (device type 63−00342) coupled to a 105 μm core fiber. A Leica 50X 0.5 NA long working distance lens as objective was used to image the cells. The power density was ∼5 W/cm2 in front of the lens. The green fluorescence from the UCNPs was collected using a Hamamatu digital camera C47420−80−12AG. A band-pass filter (527/30 nm) from Leica was employed to filter the scattered excitation light. The nucleus of the live cells were stained with 4′,6diamidino-2-phenylindole (DAPI) before imaging. Images of cells using DAPI were obtained with a 40X 0.75 NA Leica lens, with excitation (360/40 nm) and emission band (470/40 nm) pass filters (Leica). The DAPI images were obtained with 358 nm (UV light) excitation and the emission was collected at 460 nm (blue light). Characterization. X-ray Diffraction (XRD). XRD patterns were obtained using a Rigaku Miniflex X-ray diffractometer with a Cr source (Kα, λ = 2.2890 Å) operating at 30 kV and 15 mA. The XRD spectra were collected with a step size of 0.05° (2θ) and scanning speed of 1° /min. The peaks at 45° and 67° 2θ were used to calculate the size of the nanoparticles using Scherrer’s equation. Transmission Electron Microscopy (TEM). TEM images of nanoparticles were obtained using a JEOL JEM-1400 transmission electron microscope operating at 80 kV equipped with a chargecoupled device (CCD) camera. The dilute dispersion of nanoparticles in hexanes (oleate-coated) or in water (polymer-coated) was drop-cast on copper grids and then dried in air at room temperature. The average size of the UCNPs was obtained by measuring a group of 50 particles from the image. A very small percentage of small nanoparticles were observed in TEM, which has been neglected in the measurement of the size and size dispersion of the nanoparticles. Energy Dispersive X-ray Spectroscopy (EDS). EDS analysis was done using a Hitachi S-4800 field emission scanning electron microscope at 20 kV with a resolution of 102 eV. The nanoparticle dispersion was dried under air to obtain a white powder and tagged on to the substrate using a double-sided carbon tape and mounted onto the sample holder for analysis. Three measurements were done on each sample to determine the average and standard deviation. Luminescence Measurements. Upconversion emission spectra were obtained using an Edinburgh Instruments FLS920 fluorimeter. A 980 nm JDS Uniphase laser diode (device type 63-00342) coupled to a 105 μm core fiber was used as the excitation source. The emission spectra were collected using a red-sensitive Peltier-cooled Hamamatsu R955 PMT. A long band-pass filter (810 nm) was used on the diode laser to block unwanted wavelengths from the laser, and a 900 nm short band-pass filter was employed before the monochromator to remove any scattered excitation light. All dispersions were measured in a 1 cm path length quartz cuvette, and the emission curves obtained were corrected for the detector sensitivity. Thermogravimetric Analysis Measurement (TGA). TGA was performed on Q600 SDT simultaneous DSC−TGA in the temperature range from room temperature to 600 °C at a heating rate of 4 °C/min. Nuclear Magnetic Resonance (NMR). NMR was recorded on a Bruker 300 MHz system. Samples were dispersed in chloroform-d (Sigma-Aldrich). Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectra were acquired from a Perkin-Elmer FTIR spectrometer 1000 with a resolution of 2 cm−1 and averaged over four scans. The samples were dried and pelletized with KBr. The ratio of sample to KBr was roughly 1 to 10. Zeta-Potential. Zeta-potential analysis to determine the surface charge on the cross-linked nanoparticles in tris buffered saline was performed using a ZetaPALS zeta-potential analyzer (Brookhaven Instruments Corp.) equipped with a 15 mW solid-state laser (658 nm). Three runs of 30 cycles each were performed to determine the reproducibility and standard deviation of the measured zeta-potential values.
dispersed in 20 mL of chloroform, of which half was used as the seeding agent for the synthesis of core/shell UCNPs. For the synthesis of the NaYF4:20 mol % Yb3+, 2 mol % Er3+ core nanoparticles the same procedure outlined above for the synthesis of the NaYF4:30 mol % Yb3+, 0.5 mol % Tm3+ core nanoparticles was employed. Synthesis of NaYF4:30 mol % Yb3+, 0.5 mol % Tm3+/NaYF4 and NaYF4:20 mol % Yb3+, 2 mol % Er3+/NaYF4 Core/Shell Nanoparticles. The procedure of core/shell synthesis is also very similar to previously reported synthesis.20 YCl3·6H2O (303.4 mg, 1 mmol) was added to a 100 mL three-neck round-bottom flask containing 6 mL of oleic acid and 15 mL of octadecene. The solution was then heated at 130 °C under vacuum for 40 min. The temperature was brought down to 80 °C under a steady flow of argon, and a dispersion (10 mL) containing NaYF4:30 mol % Yb3+, 0.5 mol % Tm3+ core nanoparticles in chloroform was added. After evaporating the chloroform, the solution was brought to room temperature, after which 10 mL of methanol solution containing NaOH (0.1 g, 2.5 mmol) and NH4F (148.2 mg, 4 mmol) was added dropwise into the reaction mixture. The cloudy solution obtained was stirred for 30 min at room temperature. The mixture was then slowly heated to evaporate the methanol. Once the temperature reached 110 °C, the solution was rapidly heated (15 °C/min) to 300 °C under an argon flow and kept at this temperature for 1.5 h. After cooling to room temperature, the core/shell nanoparticles were separated from the reaction mixture by precipitating them in anhydrous ethanol. The NaYF4:30 mol % Yb3+, 0.5 mol % Tm3+/NaYF4 core/shell UCNPs were washed with ethanol (30 mL) thrice and then dried and dispersed in 10 mL of chloroform. When NaYF4:20 mol % Yb3+, 2 mol % Er3+/NaYF4 core/shell UCNPs were synthesized, the amount of shell material added was 0.9 mmol instead of 1 mmol. Phase Transfer of UCNPs to Water. PMAO (∼300 polymer units per nm2 of nanoparticle surface) was dissolved in chloroform, to which core/shell nanoparticles in chloroform was added. After stirring the solution for 2 h at room temperature, BHMT was added. The molar ratio of PMAO unit to cross-linker (BHMT) was 10:1. The solution was sonicated for 30 min, followed by evaporation of the solvent (chloroform) using a rotary evaporator. The resulting polymer−UCNPs mixture was dispersed in water by adding a few drops of 1 M NaOH, which was further sonicated for 1 h. The solution was filtered through a 0.45 μm pore size filter and subsequently through a 0.22 μm pore size filter. Finally, the polymer-coated nanoparticles (UCNPs−PMAO−BHMT) were isolated by centrifuging at 12 000 rpm (Beckman Coulter Spinchron 15 - rotor F0630) for 30 min and then dispersed in distilled water and various buffer solutions. The synthesis of UCNPs−PMAO−OTA (octylamine) was similar to UCNPs−PMAO−BHMT, where OTA was used instead of BHMT (5:1, PMAO unit: OTA). In the case of UCNPs−PMAO− PEG−BHMT, the nanoparticles were mixed with PMAO and stirred for 2 h, to which PEG-amine (20:1 PEG:PMAO ratio) was added. This mixture was sonicated for 30 min followed by stirring for another 2 h, after which chloroform was evaporated from the mixture using a rotary evaporator. The UCNPs−polymer mixture was dispersed in water, and BHMT (10:1, PMAO unit:BHMT) was added and then sonicated for 30 min. This mixture was then filtered successively through 0.45 and 0.22 μm pore size filters. The nanoparticles were separated from the excess polymer by centrifugation at 12 000 rpm (Beckman Coulter Spinchron 15 - rotor F0630) for 30 min. Cell Culture and UCNP Biolabeling. Human prostate cancer cells (LNCaP) were grown in tissue culture treated dish (100 mm × 20 mm) with 10% fetal bovine serum supplemented RPMI-1640 growth medium. The cells were detached at 60−70% confluency and seeded on a 22 × 22 mm2 glass coverslip placed in a six-well culture plate. The cells were grown for 3 days and then carefully washed with PBS multiple times to remove any unbound cells and debris. To the washed cells, growth medium (2 mL) with cross-linked nanoparticles (core/shell NaYF4:20% Yb3+, 2% Er3+/NaYF4−PMAO−PEG− BHMT, ∼200 μL, sterile filtered from TBS) was added and incubated at 37 °C for 2 h. The cells were carefully washed with PBS multiple times to remove UCNPs that had not been internalized, and the coverslip was inverted and mounted onto a glass slide for imaging. 3241
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RESULTS AND DISCUSSION Synthesis and Characterization. NaYF4 upconverting nanoparticles (UCNPs) doped with Yb3+ and Er3+ or Tm3+ ions were synthesized following previous reports with some slight modifications.19 These core UCNPs have lower luminescence efficiency due to quenching of the emitting surface ions by ligands and solvents. To improve the luminescence efficiency, an undoped shell of NaYF4 was grown over the core UCNPs which shields them from ligand and solvent effects. The assynthesized core and core/shell UCNPs are of pure hexagonal phase NaYF4, which is confirmed from the well-matched XRD patterns (Figure 1) with the standard reference pattern of β-
in comparison with core UCNPs. The intensity for the core/ shell UCNPs are 60 and 17 times higher for the blue (473 nm) and NIR (803 nm) emissions, respectively, as compared to the core UCNPs. The comparison of core and core/shell UCNPs was done for the same weight percent of nanoparticles. This results in the fact that the number of core particles is larger than the number of core/shell NPs. Because of this, the increase in luminescence intensity of core/shell UCNPs is thus an underestimation. Based on the upconversion pump efficiency formula I ∝ Pn (n ≥ 2),42 where I stands for the upconversion intensity, P stands for the excitation pump power, and n is the number of absorbed photons, the 473 and 803 nm emissions are three-photon and two-photon processes, respectively (Figure S2). The core NaYF4:20% Yb3+, 2% Er3+ and core/ shell NaYF4:20% Yb3+, 2% Er3+/NaYF4 UCNPs when excited with 980 nm show upconverted green and red emission from Er3+ ions, as shown in Figure 4. The emissions from Er3+ ions for 520, 540, and 654 nm are assigned to 2H11/2 → 4I15/2, 4S3/2 → 4I15/2, and 4F9/2 → 4I15/2 transitions, respectively.16,22,41 The emission intensity of core/shell UNCPs is higher by a factor of 27 and 48 times for the green and red emissions, respectively, relative to the core nanoparticles. From the power dependence curve for green and red emission, both the 540 and 654 nm emissions are two-photon processes (Figure S3). The increase in the luminescence intensity for core/shell UCNPs compared to core UCNPs is attributed to the presence of a shell which protects the dopants in the core, especially those near the surface, from quenching arising from solvents and surface ligands. The reduction in quenching improves the overall upconversion quantum yield of the UCNPs.26,43−46 Furthermore, our group has shown strong evidence for the formation of core/shell lanthanide nanoparticles using the aforementioned synthesis procedure,47,48 and we conclude the same is true for these core/shell UCNPs. Characterization of Water-Dispersible UCNPs. The asprepared hydrophobic core/shell UCNPs were transferred to hydrophilic phase after coating them with poly(maleic anhydride-alt-1-octadecene) (PMAO) modified with and without amine-functionalized methyl ether poly(ethylene glycol) (m-PEG amine) and subsequently cross-linking the amphiphilic polymer shell using bis(hexamethylene)triamine (BHMT) abbreviated as UCNPs−PMAO−BHMT in the text. The octadecene chains present in the alternating copolymer PMAO intercalate with the oleate ligands present on the surface of the UCNPs due to hydrophobic interactions while the anhydride rings present in the polymer are exposed to the solvent. Subsequently, PMAO coated onto the UCNPs were cross-linked by reacting the anhydride rings in the PMAO chains with BHMT. The opening of the anhydride rings using BHMT results in carboxylic acid groups, which make the nanoparticle surface hydrophilic. This renders the polymercoated UCNPs dispersible in water and makes them viable for biological applications. This method of dispersing UCNPs in water using intercalation and cross-linking process is similar to the ones reported in the literature for QDs and FexOy.33,36,39 The intercalation between oleates and the octadecene chain was confirmed by performing 1H NMR on the polymer PMAO and UCNPs−PMAO−BHMT in chloroform-d (Figure S4). The peaks at δ = 5.2−5.4 ppm are assigned to −CHCH− from oleate ligand on the nanoparticle surface.49,50 This indicates that the oleate ligands are still present on the surface of the UCNPs along with the coated polymer. To obtain a clear dispersion of PMAO−BHMT coated UCNPs in water, a few
Figure 1. XRD patterns of (A) NaYF4:30% Yb3+, 0.5% Tm3+; (B) NaYF4:30% Yb3+, 0.5% Tm3+/NaYF4; (C) NaYF4:20% Yb3+, 2% Er3+; (D) NaYF4:20% Yb3+, 2% Er3+/NaYF4; and (E) β-NaYF4 standard reference pattern.
NaYF4. The TEM images show that the particles are of uniform size and shape with the size for the core UCNPs doped with Yb3+ and Tm3+ ions being 19 ± 1 nm and the core/shell being 32.5 ± 3.5 nm (Figure 2A,B). The size of the core and core/ shell UCNPs doped with Yb3+ and Er3+ is 17 ± 1 and 22.5 ± 1.4 nm, respectively (Figure 2C,D). These average sizes calculated from the TEM images (Figure S1c,d) corroborate with that of the sizes calculated from the XRD peaks using the Scherrer equation, as shown in Table 1. The presence of a very small percentage of tiny nanoparticles in TEM is very likely due to the separate nucleation which occurs during the core/shell synthesis. The amount of doping levels in the core NaYF4 UCNPs determined using energy dispersive X-ray spectroscopy (EDS) (see Supporting Information Figure S1) confirms that the doping in the UCNPs is similar to that of the initial reaction mixture, as shown in Table 2. The upconversion luminescence measurements of core NaYF4:30% Yb3+, 0.5% Tm3+ and core/shell NaYF4:30% Yb3+, 0.5% Tm3+/NaYF4 UCNPs resulted in a blue emission (473 nm) coupled with a relatively strong NIR emission (803 nm) under 980 nm excitation, as shown in Figure 3. The emission peaks at 449, 473, 646, and 803 nm come from intra4f transitions of Tm3+ ions involving 1D2 → 3F4, 1G4 → 3H6, 1 G4 → 3F4, and 3H4 → 3H6, respectively.16,41 We also observed an increase in the luminescence intensity for core/shell UCNPs 3242
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Figure 2. TEM images of (A) NaYF4:30% Yb3+, 0.5% Tm3+; (B) NaYF4:30% Yb3+, 0.5% Tm3+/NaYF4; (C) NaYF4:20% Yb3+, 2% Er3+; and (D) NaYF4:20% Yb3+, 2% Er3+/NaYF4 nanoparticles.
Table 1. Sizes of Nanoparticles from TEM and XRD TEM size (nm)
XRD size (nm)
19 ± 1 32.5 ± 3.5
18 20 × 24
17 ± 1 22.5 ± 1.4
16 21
nanoparticle NaYF4:30% Yb3+, 0.5% Tm3+ core NaYF4:30% Yb3+, 0.5% Tm3+/NaYF4 core/ shell NaYF4:20% Yb3+, 2% Er3+ core NaYF4:20% Yb3+, 2% Er3+/NaYF4 core/shell
drops of 0.1 M NaOH were added. The addition of NaOH deprotonates the surface carboxylic acid groups and may have opened any remaining unreacted anhydride groups which resulted in making the polymer easily water dispersible. PMAO modified with PEG amine dispersed readily in water due to the presence of ∼20 PEG units per PMAO chain. The presence of PEG chain in the polymer-coated UCNPs also makes the UCNPs biocompatible for in-vivo and in-vitro studies. When the cross-linking of the polymer was done in the organic phase, most of the UCNPs were lost during filtration, suggesting a high degree of interparticle cross-linking, resulting in large aggregates. Conversely, when the cross-linking of the polymer was done after transferring the UCNPs to water, the loss of UCNPs due to aggregation was very minimal. Furthermore, the presence of PEG in the PMAO polymer chain also helped in drastically reducing the aggregation and interparticle crosslinking due to its chain being well solvated in water. The TEM images (Figure 5) show that the particles are well separated
Figure 3. Upconversion emission spectra of core NaYF4:30% Yb3+, 0.5% Tm3+ and core/shell NaYF4:30% Yb3+, 0.5% Tm3+/NaYF4 nanoparticles in chloroform under 980 nm diode laser excitation (156 W/cm2).
after the surface modification, and the dispersion was stable with no observable settling/precipitation of the UCNPs for several months. In addition, thermogravimetric analysis (TGA) showed that the cross-linked polymer PMAO−BHMT thermally decomposed at a higher temperature than the
Table 2. EDS of Core Nanoparticlesa nanoparticle 3+
3+
NaYF4:30% Yb , 0.5% Tm NaYF4:20% Yb3+, 2% Er3+ a
Y3+ (%)
Yb3+ (%)
Tm3+ (%)
76.52 ± 0.24 83.23 ± 1.27
22.98 ± 0.33 15.23 ± 1.13
0.53 ± 0.03
Er3+ (%) 1.53 ± 0.13
The percentage refers to the atomic mole percent of the Ln3+ ions. 3243
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Figure 4. Upconversion emission spectra of core NaYF4:20% Yb3+, 2% Er3+ and core/shell NaYF4:20% Yb3+, 2% Er3+/NaYF4 nanoparticles in chloroform under 980 nm diode laser excitation (156 W/cm2).
Figure 6. FTIR of samples (A) BHMT, (B) PMAO, (C) UCNPs− PMAO−BHMT (10:1, PMAO unit: BHMT), and (D) UCNPs− PMAO−BHMT (2:1, PMAO unit: BHMT).
polymer PMAO (Figure S5), indicating that the cross-linking process was successful, resulting in high dispersion stability of UCNPs.51 The cross-linking process was further substantiated using Fourier transform infrared spectroscopy (FTIR), and the spectrum is shown in Figure 6. The peaks at 1852 and 1780 cm−1 in Figure 6B correspond to the anhydride rings of the PMAO,36,52 and these two peaks are absent in Figure 6C,D, implying that the anhydride rings had reacted with BHMT, thereby cross-linking PMAO. The peak at 1711 cm−1 in Figure 6C,D is attributed to the COOH groups present in the polymer. The peaks at 1568 and 1401 cm−1 are assigned to the stretching mode of the RCOO− group.53 The C−H bonds of CH2 groups present in the polymer chain can be identified from the peaks at 2920, 2850, 1468, and 720 cm−1.49 The peaks at 3356, 3277, and 1595 cm−1 are attributed to the N−H bonds present in free amine groups of BHMT (Figure 6A),53 and those peaks are absent when reacted with PMAO (Figure 6C,D). In Figure 6C,D the new peak at 1640 cm−1 corresponds to the absorption of the amide groups.52 The absence of free amine groups and the presence of amide groups as observed in the FTIR spectra indicate that the cross-linking process was indeed successful. The zeta-potential measurement of the UCNPs−PMAO−BHMT in tris-buffered saline (TBS) showed a negative potential (−12.92 ± 1.93 mV) which is due to the carboxylate group as the anhydride rings were opened by BHMT and through the deprotonation by NaOH. This
negative value from zeta-potential further substantiates the successful cross-linking of the polymer shell. The upconversion luminescence spectra of water-dispersible core/shell UCNPs are shown in Figure 7 and Figure S6. The presence of undoped shell and the hydrophobic ligands (oleates and octadecene intercalation) around the UCNPs effectively suppress the quenching by water. Stability of Water-Dispersible UCNPs in Buffers, Serum Supplemented Growth Medium, and at Different pH Values. If the UCNPs−PMAO−BHMT have very poor stability when dispersed in buffers and serum supplemented growth medium, their usefulness for cellular imaging and invivo bioimaging is reduced. Moreover, the stability of these UCNPs in a biological growth medium is of paramount importance to understand whether these UCNPs will be stable during in-vivo studies using small animals. So, it is imperative that the nanoparticles are stable in buffers and serum for biological applications. UCNPs coated with un-cross-linked PMAO (modified with octylamine) precipitated quickly (few hours) in phosphate-buffered saline (PBS, pH ∼7.4) and started to settle down slowly in tris-buffered saline (TBS, pH ∼ 7.6) and sodium borate-buffered saline (SBB, pH ∼ 8.6) after a few hours. This is because of the fact that the ligands coated on UCNPs are generally very dynamic, resulting in poor stability of the UCNPs in all the three aforementioned buffers and
Figure 5. TEM images of (A) NaYF4:30% Yb3+, 0.5% Tm3+/NaYF4−PMAO−BHMT and (B) NaYF4:20% Yb3+, 2% Er3+/NaYF4−PMAO−BHMT nanoparticles. 3244
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Figure 7. Upconversion emission spectrum of core/shell nanoparticles (NaYF4:20% Yb3+, 2% Er3+/NaYF4−PMAO−BHMT) in water (∼1 mg/mL). Figure 9. Core/shell nanoparticles (NaYF4:20% Yb3+, 2% Er3+/ NaYF4−PMAO−BHMT) dispersed in water at different pH from 3 to 13 and serum supplemented cell growth medium and respective images under 980 nm excitation (bottom).
biological medium. In contrast, the UCNPs−PMAO−BHMT (0.3 mL, ∼1 mg/mL) in water when dispersed in the three buffers were stable even after 10 days with no precipitation observed, as shown in Figure 8. Figure 8A shows the UCNPs
nature of the ligands has been suppressed due to cross-linking of the ligand shell around the UCNPs. Furthermore, it is clear that the green to red ration has not changed to any extent, confirming the high stability in buffers. When ∼0.1 mL (∼0.2 mg/mL) of UCNPs−PMAO−BHMT was dispersed in 1 mL of serum supplemented cell growth media, the dispersion was stable for 48 h at 37 °C, after which the UNCPs started to settle slowly. The stability when stored at 4 °C was more than a week, suggesting that the serum proteins in the growth media do not decrease the dispersion stability of cross-linked UCNPs at low temperature. Furthermore, UCNPs−PMAO−BHMT was stable in water over a wide range of pH (3−13), as shown in Figure 9. There is a yellowish nature to the emitted light we observe in Figures 8 and 9 which comes from the combination of green and red light from the UCNPs (Figure 8 spectra). We cannot clearly see the red emission because our eyes are more sensitive to green than red. The UCNPs coated with PMAO−PEG−BHMT had a similar stability as the unmodified PMAO in all the three physiological buffers (TBS, PBS, and SBB). Cell Imaging. To demonstrate the utility of cross-linked PMAO-modified UCNPs in biolabeling, the UCNPs were incubated with human prostate cancer cells (LNCaP). The uptake of the UCNPs by the cancer cells was examined with a Leica compound epifluorescence microscope after multiple PBS washes to remove UCNPs that had not been internalized. Cells were incubated in DAPI, a nuclear fluorescent probe, and the coverslip with the live cells was transferred to a glass slide before mounting on the microscope. Clearly visible, upconverted green emission was observed from the cells with 980 nm excitation. In Figure 10B, we can easily identify four cells. The upconverted emission was observed predominantly as granules in the cytoplasm, which is most clear in overlaid images of DAPI emission (blue nucleus stain) and the UCNPs (green) (Figure 10D). A lower magnification image shows a large proportion of the LNCaP cells labeled with UCNPs (Figure S7), indicating the effectiveness of the cellular uptake of these
Figure 8. Upconversion emission spectra of core/shell nanoparticles (NaYF4:20% Yb3+, 2% Er3+/NaYF4−PMAO−BHMT) upon 980 nm excitation in (A) water (1 day old sample), (B) tris-buffered saline (TBS), (C) sodium borate buffer (SBB), and (D) phosphate-buffered saline (PBS). The buffer dispersions were 10 days old by the time the spectra were measured. Inset: UCNPs−PMAO−BHMT dispersions under 980 nm excitation.
dispersed in water after 1 day. The rest of the pictures in Figure 8 show dispersions in buffers after 10 days. The UCNP dispersions in buffers did not show aggregation, which can be concluded from the fact that the emitted light seen in Figures 8 and 9 does not show any significant amount of scatter. In principle, a TEM image of the UCNPs would confirm this. First of all, a TEM image is a measurement of a dry sample. More importantly, obtaining TEM image of UCNPs dispersed in saline buffers is not possible due to crystallization of the NaCl salt (at 150 mM!) on the grid when preparing the sample on the TEM grid. The improved stability shows that the dynamic 3245
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Figure 10. LNCaP cell imaging (A) bright-field differential interference Contrast image, (B) the same field with 980 nm excitation, (C) DAPI excitation (nucleus stain), and (D) overlay of upconverted emission and DAPI. Core/shell NaYF4:20% Yb, 2% Er/NaYF4 coated with PMAO− PEG−BHMT was used for imaging the cells.
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particles. The inhomogeneous green intensity observed in cells can be attributed to the uneven uptake of the nanoparticles by the cells. In a 10 s, time-lapse sequence of images prepared as a movie stack, the intracellular motion of UCNPs is revealed. This demonstrates that the UCNPs could potentially be used for live cell imaging (movie in the Supporting Information). It should be possible to attach tumor specific antibodies to the cross-linked PMAO-coated UNCPs, as they have multiple carboxylic acid moieties on their surface, which will be explored for targeted delivery.
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ASSOCIATED CONTENT
S Supporting Information *
Figure S1a,b: EDS spectra of core nanoparticles; Figure S1c,d: size distribution of the core and core-shell nanoparticles; Figure S2 and S3: power dependence graph for green, red, blue, and 800 nm emissions; Figure S4: H1 NMR spectra for the polymer PMAO and the cross-linked polymer coated UCNPs; Figure S5: TGA of cross-linked and un-cross-linked polymer; Figure S6: upconversion spectrum of NaYF4:30% Yb3+, 0.5% Tm3+/ NaYF4−PMAO−BHMT nanoparticles in water; Figure S7: lower magnification images of the human prostate cancer (LnCaP) cells (bright-field, green emission obtained under 980 nm excitation and overlay of both); movie showing a 10 s timelapse sequence of intracellular movement of particles within live cells. This material is available free of charge via the Internet at http://pubs.acs.org.
CONCLUSIONS
Core/shell NaYF4:Yb3+, Er3+(Tm3+)/NaYF4 UCNPs were successfully transferred to aqueous phase by coating a crosslinked PMAO polymer. The aqueous dispersion of the modified UCNPs was stable for months with no precipitation of UCNPs observed. At various pH values, in different physiological buffers, and in serum supplemented cell growth medium the cross-linked core/shell nanoparticles were very stable for several days. In contrast, the un-cross-linked sample was not stable in buffers and biological medium, substantiating the effectiveness of the proposed cross-linking strategy for longterm dispersion stability which is necessary for biological applications. The cross-linking process suppresses the dynamic nature of the ligands, thereby improving the stability of the UCNPs in various biological media. The cross-linked UCNPs were successfully internalized into human prostate cancer cells (LNCaP), which substantiates the utility of these PMAO− BHMT−UCNPs in biolabeling applications.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the Natural Science and Engineering Research Council (NSERC), the Canada Foundation for Innovation (CFI), and the British Columbia Knowledge Development Fund (BCKDF) of Canada. G.J. thanks the China Scholarship Council (CSC). We also acknowledge Dr. X. 3246
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