Improvement of dispersion stability and characterization of upconversion nanophosphors covalently modified with PEG as a fluorescence bioimaging probe
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Upconverting (UC) phosphors (UCPs) are ceramic materials doped with rare earth ions. These materials can absorb and upconvert infrared (IR) radiation to emit visible light by the stepwise excitation among discrete energy levels of the rare earth ions. UCPs are potentially useful reagents for use in bioimaging since the use of low energy photons avoids photo-toxicity. The use of UCP nanoparticles as bioimaging probes requires surface modifications in an effort to improve dispersion stability in aqueous milieu. In this study, we covalently attached poly(ethylene glycol) (PEG) to the surface of Er-doped Y2O3 nanoparticles and firstly demonstrated that PEG covalently bound to the Y2O3 surface markedly improved dispersion stability in water. UC emission of PEG-modified Er–Y2O3 nanoparticles excited with IR light was successfully observed. We also showed that PEG-modified Er–Y2O3 nanoparticles exhibit no cell-toxicity. These observations lend strong support to the potential use of PEG-modified UCP nanoparticles as bioimaging tools.
KeywordsY2O3 Dispersion Stability Yttrium Oxide Upconversion Emission Discrete Energy Level
Bioimaging is a technique that can be employed to assist in the visualization of biological phenomena both in vivo and in vitro, and represents one of the key technologies in the field of biomedical research. Fluorescence microscopic observation of tissues has received particular attention as an essential tool in the areas of medical prevention, diagnosis and cure through the investigation of biological phenomena. However, current imaging methodologies utilizing organic dyes or fluorescent proteins as probes remain problematic. The period during which observations are made is limited due to the bleaching of fluorescent probes . Their use in biological tissues is also restricted due to limited light penetration depth associated with strong scattering of the excitation light of short wavelength . Furthermore, short wavelength excitation with high quantum energy results in tissue photo-toxicity. Although the use of quantum dots might solve the first problem , the latter two concerns remain outstanding even with the usage of quantum dots .
Fluorescence imaging utilizing near-infrared (NIR) excitation is expected to have a major impact on biomedical imaging since the NIR (800–1,500 nm) is located within the so-called biological window, where the absorption of light is comparably lower than that in other wavelength regions [5, 6]. Another advantage is that infrared (IR) light penetrates deeper into tissues given its lower scattering nature resulting from its longer wavelength.
Recently, upconverting (UC) phosphors (UCPs) have been used for bioimaging [7, 8, 9, 10]. UC phosphors are ceramic materials in which rare earth ions are embedded in an inorganic host. The materials can absorb IR radiation and upconvert it to emit visible light by the stepwise excitation among discrete energy levels of the rare earth ions . For example, yttrium oxide (Y2O3) works as a good host matrix for several atomic % of erbium (Er2O3), which is known to show upconversion emission at 550 nm (green) and 660 nm (red) following excitation at 980 nm [12, 13].
In order to fabricate UC bioimaging probes emitting visible light following NIR excitation, it is important to prevent aggregation of UCP molecules in aqueous solutions. To this end, surface modification using biocompatible polymers, such as poly(ethylene glycol) (PEG) would be useful, since PEG has been successfully used to improve the dispersion stability of small particles. Examples include improvements in the dispersion stability of gold nanoparticles and cellulose microcrystals by steric repulsion effects of the tethered PEG strands [14, 15, 16]. Previously, we reported on the PEG-based surface modification of Er-doped Y2O3 (Er–Y2O3) nanoparticles using electrostatic interactions [17, 18]. In this study, we covalently attached PEG to the surface of Er–Y2O3 nanoparticles and demonstrated that PEG modification drastically improved dispersion stability in aqueous milieu. The PEG-modified Er–Y2O3 nanoparticles were demonstrated to show upconversion emission. We also examined the cell toxicity associated with the use of PEG-modified Er–Y2O3 nanoparticles in an effort to assess the potential use of these particles as bioimaging probes.
Materials and methods
Preparation of UCP nanoparticles
Yttrium oxide (Y2O3) nanoparticles with size range from 30 to 60 nm were synthesized using an enzymatic decomposition method as follows. Forty mmol/L Y(NO3)3 (99.99% purity of Y(NO3)3 · 6H2O, Kanto Chemicals, Tokyo, Japan) and 4 mmol/L Er(NO3)3 (>99% purity of Er(NO3)3 · 5H2O, Kojundo Chemical Laboratory, Saitama, Japan) were dissolved in a solution containing 400 mmol/L Urea (99.0% purity, Kanto Chemicals), to make the nominal molar ratio of Y:Er to be 90:10. After addition of 62.5 nmol/L urease (Wako, Osaka, Japan), the solution was stirred at 25 °C for 1 h. The YCO3(OH) precursors were precipitated by decomposition of the urea into precipitants, carbonic acid and ammonia. Several centrifugal washes of the precursors were then performed using distilled water. The Er–Y2O3 nanoparticles were generated by calcinating the hydroxycarbonate precursors at 900 °C for 1 h.
The APTES-modified Er–Y2O3 (APTES–Er–Y2O3) nanoparticles (20 mg) were suspended in 10 mL of dry- N,N-dimethylformamide (DMF, Junsei Chemical, Tokyo, Japan), to which was added N-Hydroxysuccinimide-PEG (NHS-PEG) (MW = 5000, Sunbright MEPA-50H, NOF Corp., Tokyo, Japan) at different concentrations (1.0 ng/mL or 30 μg/mL) and stirred for 24 h at room temperature. The particles were isolated, washed five times with distilled water by centrifugation, and dried in air at room temperature.
Characterization of PEG-modified UPC nanoparticles
SEM observations were employed using a HITACHI FE-SEM S-4200 (Tokyo, Japan) instrument operated with an acceleration voltage of 10.0 keV and a working distance of 15 mm. PEG-modified APTES-Er–Y2O3 (PEG–Er–Y2O3) nanoparticle suspension was placed onto a silicon wafer and dried at room temperature. The observation magnification was 60,000×.
FT-IR spectra of the PEG–Er–Y2O3 nanoparticles were recorded on a FTIR spectrometer (FTIR-6200, Jasco, Tokyo, Japan) with a resolution of 4 cm−1 and 100 times accumulation using the KBr pellet method. Er–Y2O3 and APTES–Er–Y2O3 nanoparticle spectra were also measured for comparisons.
Thermogravimetric analyses (TGA) were carried out using a SHIMADZU DTG-60 (Kyoto, Japan) instrument at a heating rate of 10 °C/min in dry air for the PEG-Er–Y2O3 nanoparticles (2 mg), which were dried at 150 °C for 10 min in air.
The dispersion stability of the PEG-modified nanoparticles was examined by measuring solution turbidity. PEG–Er–Y2O3 nanoparticles (10 mg) were suspended in 25 mL of distilled water or Tris buffer (10 mM Tris–HCl, pH 7.4, 100 mM NaCl) and then subjected to ultrasonication for 1 min. The turbidity was monitored at 500 nm using a UV spectrometer (Cary, Varian, NC, USA).
UC emission spectra were obtained using the SHIMADZU RF-5000 fluorescence spectrometer upon excitation of 980 nm laser diode (800 mA, 980 nm, L9418-04, Hamamatsu Photonics, Shizuoka, Japan).
Imaging of the PEG–Er–Y2O3 nanoparticles by detecting the UC emission under IR excitation was performed using an inverted microscopy system (IX71, Olympus, Tokyo, Japan). The UCP nanoparticles were illuminated with a continuous-wave laser diode (1,200 mA, 980 nm). The UC emission between 660 and 740 nm was collected with 40× microscope object lens (UPlanFLN, Olympus) through a bandpass emission filter (HQ700/75, Chroma Technology, VT, USA). Images were taken using a CCD camera (MC681SPD-R0B0, Texas Instruments, TX, USA) coupled to an image intensifier (C8600-05, Hamamatsu Photonics).
The cell toxicity associated with the use of PEG–Er–Y2O3 nanoparticles was determined using an MTT Cell Proliferation kit (Roche, Basel, Switzerland) , based on the conversion of tetrazolium salt by mitochondrial dehydrogenase to a formazan product that can be spectrophotometrically measured at 550 nm, according to the manufacturer’s instructions. Briefly, PC12 cells (a clonal line of rat pheochromocytoma) were maintained in RPMI1640 medium with 10% horse serum, 5% fetal bovine serum, penicillin (100 units/mL) and streptomycin (100 μg/mL) in 5% CO2 at 37 °C. Cells were plated in 96-well plates at a density of 50,000 cells/well and grown overnight. Cells were then incubated in 100 μL medium in the absence (control) or presence of PEG–Er–Y2O3 nanoparticles for 24 h. Following this, 10 μL of MTT reagents was added to each well and the cells were incubated for 4 h at 37 °C in a 5% CO2 incubator. The reaction was stopped by the addition of 100 μL of 10% SDS in 10 mM HCl. Plates were read at 550 nm using a microplate reader (SPECTRAmax, Molecular Devises, CA, USA). Each data point derived represents an average of three triplet-well assays.
Results and discussion
Covalent PEG-modification of the surface of Er-doped Y2O3 nanoparticles markedly improved dispersion stability in aqueous milieu, which is essential for applications utilizing bioimaging probes. The upconversion emission and low cell toxicity associated with the use of PEG-modified Y2O3 nanoparticles supports its utility as a bioimaging probe. Further bio-functionalization of PEG-modified Y2O3 nanoparticles using bi-functional block copolymer PEG with heterogeneous ends for specific biotargeting is currently in progress.
The authors thank Prof. Y. Nagasaki (Univ. of Tsukuba, Ibaraki, Japan) for his advice and discussion on the PEG modification of the particles. This work is financially supported by RIKEN and the Ministry of Education, Science, Sports, Culture, and Technology (MEXT) of Japan. K.S. was financially supported by “Development of upconversion nano-particles for bio-nano-photonics” from New Energy and Industrial Technology Development Organization (NEDO) of Japan.
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