Nile Red Derivative-Modified Nanostructure for Upconversion

Dec 24, 2015 - Nile Red Derivative-Modified Nanostructure for Upconversion Luminescence Sensing and Intracellular Detection of Fe3+ and MR Imaging ...
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Nile Red Derivative-Modified Nanostructure for Upconversion Luminescence Sensing and Intracellular Detection of Fe3+ and MR Imaging Ruoyan Wei,†,# Zuwu Wei,†,# Lining Sun,*,† Jin Z. Zhang,‡ Jinliang Liu,† Xiaoqian Ge,† and Liyi Shi*,† †

Research Center of Nano Science and Technology, and School of Material Science and Engineering, Shanghai University, Shanghai 200444, P. R. China ‡ Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States S Supporting Information *

ABSTRACT: Iron ion (Fe3+) which is the physiologically most abundant and versatile transition metal in biological systems, has been closely related to many certain cancers, metabolism, and dysfunction of organs, such as the liver, heart, and pancreas. In this Research Article, a novel Nile red derivative (NRD) fluorescent probe was synthesized and, in conjunction with polymer-modified core−shell upconversion nanoparticles (UCNPs), demonstrated in the detection of Fe3+ ion with high sensitivity and selectivity. The core−shell UCNPs were surface modified using a synthesized PEGylated amphiphilic polymer (C18PMH-mPEG), and the resulting mPEG modified core−shell UCNPs (mPEG-UCNPs) show good water solubility. The overall Fe3+-responsive upconversion luminescence nanostructure was fabricated by linking the NRD to the mPEG-UCNPs, denoted as mPEG-UCNPs-NRD. In the nanostructure, the core−shell UCNPs, NaYF4:Yb,Er,Tm@NaGdF4, serve as the energy donor while the Fe3+-responsive NRD as the energy acceptor, which leads to efficient luminescence resonance energy transfer (LRET). The mPEG-UCNPs-NRD nanostructure shows high selectivity and sensitivity for detecting Fe3+ in water. In addition, benefited from the good biocompatibility, the nanostructure was successfully applied for detecting Fe3+ in living cells based on upconversion luminescence (UCL) from the UCNPs. Furthermore, the doped Gd3+ ion in the UCNPs endows the mPEG-UCNPs-NRD nanostructure with effective T1 signal enhancement, making it a potential magnetic resonance imaging (MRI) contrast agent. This work demonstrates a simple yet powerful strategy to combine metal ion sensing with multimodal bioimaging based on upconversion luminescence for biomedical applications. KEYWORDS: Nile red derivative (NRD), Upconversion luminescence nanostructure, Fe3+ sensing, Bioimaging, MR imaging

1. INTRODUCTION Various transition metal ions play a key role in a wide range of environmental and biological processes.1−3 Among them, Fe3+ ion, the physiologically most abundant and versatile transition metal in biological systems, is essential for living systems since it is the oxygen carrier in all tissues in the form of hemoglobin and assists to transport electrons as cytochromes. On the other hand, excessive Fe3+ ions within the body can lead to severe diseases such as various cancers, hepatitis, hemochromatosis, and dysfunction of organs, including the liver, heart, and pancreas.4,5 Therefore, intense research efforts have been devoted to the development of chemosensors for Fe3+ ion detection. Optical detection is an important technique used in the field of chemical and biological analysis with great advantages such as highly sensitive and relatively simple protocols.6−10 In addition, most studies have focused on colorimetric and fluorescent probes for metal ions because of their unique © XXXX American Chemical Society

features such as low cost, simple pretreatment and naked-eye mode in a high-throughput fashion.11−15 With high fluorescence quantum yields in a polar solvents and fluorescence at reasonably long wavelengths, Nile red is a new fluorescent compound that has received increasing attention in recent years. Since the fluorescent emission of Nile red exhibits a large bathochromic shift in polar media, it can be used as a probe to indicate the polarity of the environment to which it is attached.16,17 As the fluorescence yield is low, the probe of Nile red or its derivative has been less studied. Up to now, many available fluorescent probes have been developed with a change in fluorescence intensity under the rich microenvironment of Fe3+.18−20 However, to the best of our knowledge, the intracellular probe of Fe3+ ion detection based on Nile red Received: September 28, 2015 Accepted: December 12, 2015

A

DOI: 10.1021/acsami.5b09132 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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further purification. YCl3 ·6H2 O (99.99%), YbCl 3·6H 2O (99.99%), ErCl3·6H2O (99.99%), TmCl3·6H2O (99.99%), GdCl3·6H2O (99.99%), 1,6-naphthalenediol, 3-Dimethylaminophenol and Poly(maleic anhydride-alt-1-octadecene) (C18PMH) were purchased from Sigma-Aldrich Co. Ltd. Sodium hydroxide (NaOH, 96%), ammonium fluoride (NH4F, 98%), sodium nitrite (NaNO2), methanol (CH3OH, 99.5%), 1-octadecane (ODE, 90%), N,N-Dimethylformamide (DMF), dichloromethane (CH2Cl2), triethylamine (TEA), poly(ethylene glycol) methyl ether (mPEG-NH2) and 1ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC·HCl) were obtained from Aladdin Company. Oleic acid (OA, 90%) were obtained from Alfa Aesar Ltd. Cyclohexane, chloroform, H2SO4 and dimethyl sulfoxide (DMSO) were of analytical grade and purchased from Sinopharm Chemical Reagent Co. Ltd. Deionized water was used throughout. 2.2. Synthesis of Nile Red Derivative (NRD). 3Dimethylaminophenol (4.2 g) was dissolved in 10 mL of distilled water. The obtained solution was cooled to 0 °C under stirring, and then an aqueous solution of NaNO2 (3.2 g) was added. After 1 h, the H2SO4 solution was added to adjust the pH value until pH 3−4.16,17 The reaction mixture was stirred for 4 h at 0−5 °C. The precipitated solid was filtered, dried under vacuum and identified by 1HNMR. The product, 2nitroso-5-dimethylaminophenol, was used without any further purification. 1H NMR (500 MHz, DMSO) 9.00 (s, 1H), 6.15 (dd, J = 8.0, 2.5 Hz, 1H), 6.09 (t, J = 2.5 Hz, 1H), 6.07 (dd, J = 8.0, 1.5 Hz, 1H), 2.83 (s, 6H). Following, 1,6-naphthalenediol (4.5 g) and 2-nitroso-5dimethylaminophenol (2.5 g) were dissolved in 20 mL of n,n-dimethylformamide and the resulting solution reflux for 5 h.16,17 The solvent was evaporated under reduced pressure and the crude solid purified by column chromatography (silica gel, CH2Cl2/CH3OH: 20/1). 1H NMR (500 MHz, DMSO) δ 10.45 (s, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.90 (d, J = 2.5 Hz, 1H), 7.62 (d, J = 9.0 Hz, 1H), 7.11 (dd, J = 8.6, 2.5 Hz, 1H), 6.84 (dd, J = 9.1, 2.7 Hz, 1H), 6.68 (d, J = 2.7 Hz, 1H), 5.33 (t, J = 4.8 Hz, 1H), 3.11 (s, 6H). 2.3. Synthesis of NaYF4:Yb(20%),Er(1.5%),Tm(0.5%) being Coated by Oleic Acid (OA). NaYF4:Yb,Er,Tm were prepared by a modified solvo-thermal method according to our previous method.40−42 2 mmol of LnCl3 (Ln: 78 mol %Y+20 mol %Yb+1.6 mol %Er+0.4 mol %Tm) in deionized water were added into 100 mL three-neck round-bottom flask, and then the solution was heated to 110 °C to remove the water to get the white solid. After 12 mL OA and 30 mL ODE were added in sequence, the solution was heated to 150 °C to form transparent yellow solution, and then cooled down to 60 °C. Following, 20 mL methanol containing NaOH (5 mmol, 0.2 g) and NH4F (8 mmol, 0.3 g) were added in, stirring at 110 °C to evaporate methanol and water. Finally, the reaction solution was directly heated to 300 °C and maintained at this temperature for 1 h under Argon. After the reaction was completed, raw products was obtained with centrifugation in the adding of acetone, and washed with cyclohexane and acetone twice times. The sample was redispersed in 20 mL of cyclohexane. 2.4. Synthesis of NaYF4:Yb,Er,Tm@NaGdF4 (UCNPs). The synthesis of UCNPs was according to our previous publication with minor modification.43 800 μM GdCl3 was added into 100 mL three-neck round-bottom flask, and heated to 110 °C to evaporate the water. Then 15 mL OA and 30 mL ODE were added to resolve the white power in 150 °C and

derivative (NRD) have not been reported in the open literature to date.21 In addition, most of the reported fluorescent probes exhibit emission under excitation of UV or visible light, which could cause damage to healthy cells. Moreover, most of the Fe3+-selective probes are insoluble in water because of their organic nature.22 Compared to UV and visible light, near-infrared (NIR) light is more desirable and suitable for many biomedical applications because it penetrates deeper into tissues and causes less damage to biosamples.23−25 Upconversion luminescence (UCL) refers to anti-Stokes luminescence resulting from sequential absorption of two or more low energy photons by ladder-like energy levels of the lanthanide dopants in the upconversion nanoparticles (UCNPs).26 Rare-earth doped UCNPs can convert NIR excitation to UV or visible emission with great advantages such as a large anti-Stokes shift (up to several hundred nm), superior photostability, greater penetration depth and no autofluorescence (noise) from biological specimens.27−32 Because our initial NRD optical probe for Fe3+ is based on the excitation of visible light (541 nm) and needs to be dissolved in organic solvents, its bioapplication is limited. To enhance the capability of the NRD fluorescent probe, we utilize UCL detection from UCNPs and couple it with NRD so the excitation for the coupled composite can be from the NIR and the resulting visible UCL can be transferred to the NRD. To date, a series of composite nanoprobes containing UCNPs and organic luminophores have been successfully demonstrated for bioimaging as well as detection of special ions,33,34 molecules35,36 and DNA37 in solution based on the luminescence resonance energy transfer (LRET) process. Generally, UCNPs serve as the energy donors while the organic luminophores are selected as energy acceptors. And most of the reported LRET systems based on UCNPs use Yb3+ and Nd3+ as the sensitizer, and a continuous wave (CW) 980 or 808 nm laser is used as an excitation resource.38,39 Under the continuous excitation with NIR light, the sensitizer Yb3+ or Nd3+ transfers the energy to activator (Er3+, Tm3+, or Ho3+, ect.) to emit the characteristic emission, and appropriate overlap between the narrow UCL emission band and the broad absorption band of the chromophores results in the intensity changes of the UCL emission or UCL resonance energy transfer. Herein, we report a facile and inexpensive synthesis of a novel NRD that can be potentially applied as an excellent indicator in sensing Fe3+, along with several advantages such as high sensitivity and selectivity. Based on the synthesized PEGylated amphiphilic polymer (C18PMH-mPEG), the mPEG modified mPEG-UCNPs with good water solubility were developed. Subsequently, the new Fe3+-responsive UCL nanostructure was fabricated by linking the NRD to the mPEG-UCNPs, denoted as mPEG-UCNPs-NRD. In the nanostructure, the core−shell UCNPs, NaYF4:Yb,Er,Tm@ NaGdF4, serve as the energy donor and the Fe3+-responsive NRD as the energy acceptor, which, in conjunction, leads to the desired LRET. And the final mPEG-UCNPs-NRD nanostructure shows high selectivity and sensitivity for detecting Fe3+ in water. In addition, due to its good water solubility and biocompatibility, the nanostructure was successfully applied to detect Fe3+ in living cell as well as for MR imaging.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Materials. All reagents and chemicals were obtained from commercial sources and used without B

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ACS Applied Materials & Interfaces cooled to 60 °C. Next, 5 mL NaYF4:Yb,Er,Tm and 20 mL methanol containing NH4F (0.039 g, 1.05 mmol) and NaOH (0.067 g, 1.68 mmol) were added in, and the solution was heated to 110 °C to remove methanol and cyclohexane for 30 min. The resultant solution reacted in 300 °C under Argon for 1 h. The purification disposal was similar to the method of NaYF4:Yb,Er,Tm mentioned above. The obtained sample (UCNPs) was dispersed in 10 mL of cyclohexane for further application. 2.5. Synthesis of C18PMH-mPEG. In a typical procedure, C18PMH (100 mg, 0.1 mmol) was reacted with mPEG-NH2 (500 mg, 0.2 mmol, 2 kDa) in 20 mL CH2Cl2/TEA (9:1, v:v) for 2 h at room temperature.33,44 Then, EDC·HCl (59 mg, 0.3 mmol) was added and the solution was stirring for another 24 h. The resulting polymer was dialyzed against ultrapure water through a dialysis membrane (MWCO of 12000−14000) for 2 days. Finally, C18PMH-mPEG powders were freeze-dried under vacuum at ambient temperature for 48 h to yield of 0.4772 g. 1 H NMR (500 MHz, CDCl3) δ: 3.7−3.4 (m, br, CH2 of mPEG), 1.3−1.0 (m, CH2 of C18PMH), 0.88 ppm (m, br, CH3 of C18PMH) (see Figure S1, Supporting Information). 2.6. Assembly of mPEG-Functionalized UCNPs (denoted as mPEG-UCNPs). The synthesis of mPEG-UCNPs was according to previously reported method.45 C18PMHmPEG (15 mg) and UCNPs (15 mg) were dispersed separately in chloroform (10 mL) and then mixed together to obtain a homogeneous phase. The mixture was stirred for 2 h at room temperature. After evaporation of chloroform, the yellow solid was redispersed in 10 mL water and the large aggregations were removed through a 0.22 μm drainage membrane filter and stored at 4 °C. 2.7. mPEG-UCNPs Loaded with NRD (denoted as mPEG-UCNPs-NRD). NRD (1 mg, 3.3 mmol) dissolved in DMSO (50 μL) was added into the water solution of mPEGUCNPs (15 mg·mL−1). The solution was then stirred overnight. Redundant NRD was removed by centrifugation at 15000 rpm for 10 min, and the obtained precipitate was washed twice with DMSO and water, respectively, by centrifugation. The as-prepared water-soluble mPEG-UCNPs-NRD was redispersed in water. 2.8. Zeta potential. The surface charge of the UCNPs in cyclohexane, mPEG-NRD and mPEG-UCNPs-NRD in water (1 mg·mL−1) were determined using a Nanoparticle and Potentiometric Analyzer Zetasizer3000HS and PCS analysis software (Malvern Instruments Corporation). 2.9. Characterization. UCNPs were dispersed in cyclohexane or water, and then the solutions were carefully dripped onto the 300-mesh copper grid coated with a lacy carbon film for TEM characterization, which was performed on a JEM2010F low-to-high resolution transmission electron microscope operated at 120 kV. Powder X-ray diffraction (XRD) measurement was performed on a 3 kW D/MAX2200 PC diffractometer at a scanning rate of 8° min−1 with the 2θ from 10° to 90° (Cu Kα radiation, 60 kV, 80 mA). Fourier transform infrared spectra (FT-IR) were on an Avatar 370 instrument with KBr pellet in the spectral range of 4000 to 400 cm−1. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was employed to determine the concentration of Gd3+ on mPEG-UCNPs. Upconversion luminescence spectra were recorded on an Edinburgh LFS-920 fluorescence spectrometer with the excitation of an external 0−2 W adjustable continuous wave laser (980 nm, Connect Fiber Optics, China). Luminescence spectra were also recorded on

LFS-920 excited by 541 nm. UV−vis absorption spectra were measured by using a Shimadzu UV-2501PC ultraviolet−visible spectrometer. 1H NMR spectra were recorded on a Bruker AVANCE/AV 500 MHz spectrometer. All chemical shifts are reported in the standard δ notation of ppm relative to tetramethylsilane (TMS) in CDCl3 or DMSO. Electrospray ionization mass spectra were measured on a Agilent LC-MS (TOF) system. Zeta potentials was measured on a Potentiometric Analyzer Zetasizer3000HS and PCS analysis software (Malvern Instruments Corporation) was used. The cell images were obtained digitally on a Nikon multiple charge-coupled device (CCD). 2.10. Cell Culture. HeLa cells were provided by the Shanghai Institutes for Biological Sciences, SIBS, Chinese Academy of Sciences. HeLa cells were cultured in RPMI 1640 (Roswell Park Memorial Institute’s medium) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin− streptomycin at 37 °C and 5% CO2. 2.11. Cytotoxicity of mPEG-UCNPS-NRD. In vitro cytotoxicity was evaluated by performing methyl thiazolyl tetrazolium (MTT) assays on HeLa cells. Cells were seeded into 96-well cell culture plates at 5 × 104 per well, and were cultured at 37 °C and 5% CO 2 for 24 h; different concentrations of mPEG-UCNPs-NRD (0, 25, 50, 100, 200, 400 μg·mL−1, diluted in water) were added into the wells. The cells were subsequently incubated for another 24 h at 37 °C and 5% CO2. Then, MTT (10 μL, 5 mg mL−1) was added to each well and the plate was incubated for an additional 4 h under the same conditions. Next, 100 μL DMSO was added to dissolve the formazan crystals. The optical density OD 570 value (Abs) of each well was measured by means of a Tecan Infinite M200 monochromator-based multifunction microplate reader. The following formula was used to calculate the cell viability: Cell viability (%) = (mean Abs value of treatment group)/(mean Abs value of control group) × 100%. 2.12. Confocal Laser Scanning Upconversion Luminescence Microscopy (LSUCLM) Imaging in Vitro. Before the experiments, HeLa cells were washed with PBS buffer for three times and then the cells were incubated with 400 μg·mL−1 in RPMI 1640 for 4 h at 37 °C. Experiment to assess Fe3+ uptake was performed over 1 h in the same medium supplemented with 40 μM Fe3+. Confocal LSUCLM cell imaging was then carried out after washing the cells with RPMI 1640 for five times. Cell imaging was performed with an OLYMPUS FV1000 scanning unit. Cells loaded with mPEGUCNPs-NRD were excited by a CW laser at 980 nm (Connet Fiber Optics, China) with the focused power of ∼500 mW. UCL emission of green and red region was collected at 520− 560 and 630−680 nm, respectively. 2.13. Longitudinal Relaxation Time T1 and Relaxivity r1 Measurement in Water. The longitudinal relaxation time T1 and relaxivity r1 of the mPEG-UCNPs-NRD was measured with a magnetic resonance imaging (MRI) instrument with 3.0 T magnetic field. Different concentrations (containing 0−1.5 mM Gd3+) of mPEG-UCNP, mPEG-UCNPs-NRD and mPEGUCNPs-NRD upon addition of different concentration of Fe3+ (0.02, 0.04, 0.06, 0.08, 0.1 mM) dispersed in water were placed in 2 mL plastic centrifuge tubes for T1 measurement. The resulting T1 values were recorded at different concentrations of Gd3+ and the longitudinal relaxivity r1 (1/T1) was deduced by the slope of the fitted regression line. C

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Scheme 1. Schematic Illustration of the Synthesis of Nile Red Derivative (NRD) (1), mPEG-UCNPs-NRD and Their Use for Detecting Fe3+ Based on Change in UCL Emission (2)a

a

The green UCL emission of mPEG-UCNPs-NRD is quenched due to the presence of Fe3+ that has absorption at the corresponding wavelength. Upon addition of Fe3+, the structure of NRD changed, resulting in absorption increased and UCL emission quenched.

3. RESULTS AND DISSCUSSION 3.1. Synthesis and Characterization of the Overall Nanostructure. As shown in Scheme 1, the NRD was

Figure 2. UV−vis absorption spectra (dot lines) of mPEG-UCNPsNRD (1.1 mg·mL−1) and mPEG-UCNPs-NRD upon addition of Fe3+ (2.0 equiv), and luminescence spectra (solid lines, 980 nm, 1.5 W) of mPEG-UCNPs-NRD and mPEG-UCNPs-NRD upon addition of Fe3+.

synthesized from 2-nitroso-5-dimethylaminophenol with excessive 1,6-naphthalenediol. The 3-dimethylaminophenol was used, instead of 3-diethylaminophenol as originally reported, to increase the yield of the product. The structures of the new compounds were confirmed by 1HNMR spectroscopies and MALDI-TOF-MS (as shown in Figure S2−S3, Supporting Information), and the peak at m/z 307.1 in Figure S3 (Supporting Information) was ascribed to NRD (calculated as 306.1). In order to apply the new probe (NRD) for bioapplications, the water-soluble mPEG and NRD-functionalized UCNPs nanostructure was developed, denoted as mPEG-UCNPs-NRD. Typically, the NaYF4 doped with 20% Yb,1.6%Er,0.4%Tm nanoparticles were prepared. For the purpose of enhancing the luminescence of NaYF4:Yb,Er,Tm nanoparticles and affording potential application of the contrast

Figure 1. TEM (A) and HRTEM (B) images of NaYF4:Yb,Er,Tm nanoparticles; TEM (C) and HRTEM (D) of NaYF4:Yb,Er,Tm@ NaGdF4 (UCNPs); HRTEM images of mPEG-UCNPs (E) and mPEG-UCNPs-NRD (F). D

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Figure 3. Absorption spectra of mPEG-UCNPs-NRD with addition of Fe3+ with different concentrations (A) and the absorbance intensity (at 551 nm) as a function of Fe3+ concentration (0−30 μM) (B).

mPEG-UCNPs-NRD (1.1 mg·mL−1) was determined using the luminescence spectra and calculated to be approximately 1.1 wt % (see Figure S7. Figure 2 shows the UV−vis absorption and photoluminescence (PL) spectra of mPEG-UCNPs-NRD and mPEG-UCNPs-NRD upon addition of Fe3+. The NRD in mPEG-UCNPs-NRD shows weak absorption in the range of 400−800 nm in the absence of Fe3+. Upon addition of Fe3+, the absorption intensity of mPEG-UCNPs-NRD increases and there is a small color change in the first absorption band. This is attributed to chemical complexation between Fe3+ and NRD in mPEG-UCNPs-NRD that contains a phenol unit. The saturated-coordinate number of the Fe3+ complex was deduced to be five, because Fe3+ itself belongs to spin−orbit coupling interaction for a d5 configuration in a trigonal ligand-field.47 Under CW 980 nm excitation, the UCNPs in mPEG-UCNPsNRD show five bands at 475 and 800 nm (attributed to the G4 → 3H6 and 3H4 → 3H6 transitions of Tm3+, respectively), 521 nm, 540 and 654 nm (attributed to the 2H11/2 → 4I15/2, 4S3/2 → 4 I15/2 and 4F9/2 → 4I15/2 transitions of Er3+, respectively). Upon addition of Fe3+, the UCL intensity of mPEG-UCNPs-NRD decreases, since energy transfers from the UCNPs to NRD is expected and increased absorption of mPEG-UCNPs-NRD would decrease the UCL. The results suggest that the mPEGUCNPs-NRD nanostructure is sensitive to Fe3+ and the UCLLRET between UCNPs and NRD allows detection of Fe3+ ion. In addition, the energy transfer efficiency between mPEGUCNPs and NRD was investigated from the upconversion emission intensities in 530−560 nm (green region) and 640− 680 nm (red region) by the following equation,48

agents for MRI, a similar lattice constants NaGdF4 shell was grown on the NaYF4:Yb,Er,Tm core, resulting in the core−shell structured UCNPs. Then, the hydrophobic UCNPs were transferred into hydrophilic by using the synthesized C18PMHmPEG, denoted as mPEG-UCNPs. Finally, the hydrophobic NRD was assembled to the mPEG-UCNPs because of the hydrophobic interactions with the hydrophobic C18 chain of C18PMH-mPEG, resulting in the hydrophilic mPEG-UCNPsNRD nanostructure. The morphology and structure of NaYF4:Yb,Er,Tm and NaYF4:Yb,Er,Tm@NaGdF4 (UCNPs), mPEG-UCNPs and mPEG-UCNPs-NRD were characterized by TEM and HRTEM (see Figure 1). As can been seen, after the NaGdF4 shell was coated onto the surface of the NaYF4:Yb,Er,Tm nanoparticles, the average size of core−shell UCNPs increases to ∼41 nm from ∼28 nm of the core (Figure 1A−1D). After modification with C18PMH-mPEG and loading with NRD, the mPEG-UCNPs and mPEG-UCNPs-NRD still keep the spherical structure and show good monodispersity (Figure 1E and 1F). In addition, dynamic light scattering (DLS) measurements show that the mPEG-UCNPs have an average diameter of 118 nm (Figure S4), and the narrow diameter distribution indicates that there is no obvious agglomeration of the nanostructures. In addition, X-ray diffraction (XRD) patterns of UCNPs and mPEG-UCNPs are displayed in Figure S5, and all the diffraction peaks can be well indexed to the standard hexagonal phase of NaYF4 (JCPDS no. 16-0334), which was also confirmed by HR-TEM images shown in Figure 1B and 1D.46 Fourier-transform infrared (FT-IR) spectra of UCNPs, mPEG-UCNPs, NRD, and mPEG-UCNPs-NRD are displayed in Figure S6. For UCNPs, the peaks at 2927 and 2858 cm−1 are assigned to the asymmetric and symmetric stretch vibrations of methylene (−CH2−) in the long alkyl chain of OA, and the band at 1557 cm−1 is attributed to (−CO−), indicating that OA was coated on the surface of the UCNPs.25 For the curve of mPEG-UCNPs, the new peak at 1109 cm−1 is attributed to −C−O− in mPEG, which suggests the formation of mPEGUCNPs. In comparison with mPEG-UCNPs and NRD, mPEGUCNPs-NRD shows some new peaks in the range of 1400− 1600 cm−1, attributed to CN and benzene, which indicates the successful loading of NRD in the mPEG-UCNPs-NRD. In addition, the luminescence spectra of NRD with different concentrations were measured in CH2Cl2, as along with the luminescence intensity at 605 nm as a function of NRD concentration, as shown in Figure S7). The NRD content in

E = 1 − UCL/UCL0

where E denotes the energy transfer efficiency, and UCL and UCL0 are the integrated emission intensities of 530−560 nm band (or 640−680 nm) of mPEG-UCNPs-NRD with addition of Fe3+ and no Fe3+, respectively. After addition of 2.0 equiv of Fe3+, the UCL intensity of mPEG-UCNPs-NRD decreased dramatically in the wavelength range of 400−800 nm (see Figure 2), due to the increased overlap between the emission spectrum of the energy donor (mPEG-UCNPs) and the absorption spectrum of energy acceptor (NRD). Using the equation above and the corresponding UCL spectra of mPEGUCNPs-NRD, the energy transfer efficiency of green UCL and red UCL were determined to be 90.1% and 87.7%, respectively. 3.2. Optical Response of Fe3+. The sensing ability of NRD for Fe3+ was demonstrated by fluorescence emission E

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after adding Fe3+, respectively). At the beginning (0−2.0 equiv), as the concentration of Fe3+ increases, the (F0−F) increases rapidly with a good linear relationship. At higher concentrations of Fe3+, the luminescence intensity (F0−F) reaches a plateau, which is likely caused by saturation of the chelating sites with Fe3+ binding.49−51 Generally, a continuous-wavelength (CW) laser (980 nm, 1− 103 W cm−2) can be used to induce Yb3+-activated (976 nm, 2 F7/2−2F5/2) upconversion emissions, attributed to its highly efficient conversion of NIR photons to visible or UV photons. However, a simultaneous two-photon absorption process in organic dyes (such as Nile Red) requires pulsed laser excitation (106−109 W·cm−2), with no intermediate energy levels involved.52,53 In our case, the 980 nm continuous-wavelength (CW) laser was used to excite the mPEG-UNCPs-NRD nanostructures for sensing of Fe3+, which should not result in two-photon absorption of NRD. Therefore, whether the value of the two-photon absorption cross-section of NRD changes or not with Fe3+ does not affect the sensing process studied in this work. The sensing ability of mPEG-UCNPs-NRD nanostructure to Fe3+ was investigated in water under excitation of 980 nm laser. Figure 3 shows the absorption spectra of mPEG-UCNPs-NRD with the addition of Fe3+ at different concentrations and the absorption intensity (at the 551 nm peak position) as a function of Fe3+ concentration (0−30 μM). Upon addition of Fe3+ ion, the absorption intensity of mPEG-UCNPs-NRD gradually increases with a good linear relation. With excitation at 980 nm, the UCL spectra of mPEG-UCNPs-NRD were also studied as a function of Fe3+ concentration (Figure 4). With the addition of Fe3+ ion, the emission intensities of green UCL, red UCL and NIR UCL all gradually decrease, which is attributed to the increased spectral overlap between the absorption of NRD and UCL emission of the UCNPs in the mPEG-UCNPsNRD nanostructure. The UCL emission intensity at 540 nm decreased linearly with the amount of Fe3+ from 0 to 30 μM and the detection limit was calculated to be 89.6 nM (Figure 4B).10 For the red emission, there was also a linear decrease of the intensity at 654 nm and the detection limit of the mPEGUCNPs-NRD was estimated to be 106.2 nM (see Figure S9). In addition, the mPEG-UCNPs-NRD nanostructure could be dispersed easily in water, resulting in a purple color in daylight and, under excitation of 980 nm, emitting green UCL. With Fe3+ (2.0 equiv), the color of the system changed to lavender and nearly no UCL signal was observed (see Figure 4C). Along with the sensitivity requirement, high specificity is crucial for most sensing applications, especially in real sample detections.54,55 To investigate the specificity of the mPEGUCNPs-NRD, the UCL spectral and color response of mPEGUCNPs-NRD to different ions in water solutions were investigated, including 10 equiv of Hg2+, Fe2+, Cr3+, Al3+, Co2+, Mn2+, Ba2+, Zn2+, Ca2+, Mg2+, Ni2+, K+, Na+, and NH4+, 5 equiv of Cu2+, and 2 equiv of Fe3+. As shown in Figure 5A, only the addition of Fe3+ ion can lead to the luminescence decrease of mPEG-UCNPs-NRD, while other ions do not influence the luminescence. The mPEG-UCNPs-NRD solution exhibits significant color change (from purple to lavender) with the addition of Fe3+, while it maintains its original color with the addition of other metal ions (see Figure 5B), indicating that only Fe3+ have strong interaction with the NRD in mPEGUCNPs-NRD. Therefore, the nanostructure is highly selective for Fe3+ detection.

Figure 4. UCL spectra of mPEG-UCNPs-NRD with increasing the concentration of Fe3+ in water (λex = 980 nm, 1.5 W) (A), the emission intensity changes (monitored at 540 nm) against the concentration of Fe3+ (0−70 μM), UCL0 and UCL are the UCL intensity of mPEG-UCNPs-NRD and mPEG-UCNPs-NRD upon addition of Fe3+, respectively (λex = 980 nm) (B), color change and UCL response (under excitation of 980 nm) of mPEG-UCNPs-NRD upon addition of Fe3+ in water (C).

spectroscopy. The fluorescence emission spectra of the NRD upon titration with Fe3+ are shown in Figure S8. In the absence of Fe3+, the NRD probe emitted pink fluorescence with an intense emission band centered at 605 nm. With the addition of Fe3+, the emission intensity of NRD decreased. From Figure S8, it can be seen that the NRD was highly sensitive to Fe3+ and the fluorescence intensity at 605 nm decreased substantially as the concentration of Fe3+ increased. The reaction between NRD and Fe3+ could happen within 1 s (a color change from pink to blue can be observed with the addition of Fe3+ in Figure S8C), which shows that the probe is fast in responding to Fe3+ and can be applied in the detection of Fe3+ in real time. Figure S8B shows the plot of (F0−F) against the concentrations of Fe3+ (F0 and F represent the emission intensity of the probe before and F

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Figure 5. UCL spectra (A, λex = 980 nm, 1.5 W) and the photographs of mPEG-UCNPs-NRD in water upon addition of 2 equiv of Fe3+, 5 equiv of Cu2+ or 10 equiv of other various ions.

Figure 6. Metal-ion competition experiments of mPEG-UCNPs-NRD toward different metal ions in water. The multicolour bars represent the UCL intensity (λem = 540 nm) of a solution of mPEG-UCNPsNRD and 5 equiv of other metal ions of interest, respectively. The red bars show the UCL intensity after the subsequent addition of 2 equiv of Fe3+ to the solution mentioned above.

Generally, for practical detections in complex environments, interaction between different components can occur, and thus competition experiments are necessary to evaluate the capability of a detection system.56−58 Such experiments were performed by adding Fe3+ ions to solutions of mPEG-UCNPsNRD in the presence of other metal ions, which include Hg2+, Cu2+, Fe2+, Cr3+, Al3+, Mn2+, Ba2+, Zn2+, Ca2+, Ag+, Mg2+, Ni2+, K+, Na+, and NH4+. The studies were conducted with presence of 2 equiv of Fe3+ in mPEG-UCNPs-NRD solution to induced UCL quenching after adding 5 equiv of interferential metal ions. As shown in Figure 6, the UCL intensities at 540 nm were quenched distinctly when 2 equiv of Fe3+ was added to the

Figure 7. T1-Weighted and color-mapped MR images for various Gd3+ concentrations of mPEG-UCNPs-NRD (A). Relaxation rate r1 (1/T1) against the different Gd3+ concentrations of mPEG-UCNPs-NRD (B).

solutions containing mPEG-UCNPs-NRD and other different metal ions of interest, indicating that the competitive ions show no obvious interference to Fe3+ detection, that is, sensing of Fe3+ by mPEG-UCNPs-NRD was hardly affected by these commonly coexistent ions. The results of UCL analysis above demonstrated that the mPEG-UCNPs-NRD nanostructure can be a highly sensitive and selective UCL sensor for Fe3+. G

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intensity of the nanostructure but also affords it with the ability of T1-weight magnetic resonance (MR) imaging. The T1 values were measured by using a 3.0 T MRI scanner, and the concentration of Gd3+ was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). As shown in Figure 7A, a positive enhancement for MR signal was observed as the concentration of Gd3+ concentration increased in colormapped images based on T1 values. Figure 7B shows that the T1-weighted MR signal of the mPEG-UCNPs-NRD increases linearly with an increase of Gd3+ ion concentration. The linear fitting of the experimental data confirms the longitudinal relaxivity value (r1) to be 1.908 mM−1·S1−, which is high in comparison with NaGdF4 nanomaterials encapsulated with other polymer or shells.60,61 This is because the mPEG shell allows better access of water molecules to NaGdF 4. Furthermore, as shown in Figure S12 the longitudinal relaxivity value (r1) of mPEG-UCNPs (before loading of NRD), was measured to be 2.756 mM−1·S1−, which is higher than that of mPEG-UCNPs-NRD. This further demonstrates that the NRD was loaded into the shell of mPEG. In addition, the measurements of MR imaging of mPEG-UCNPs-NRD upon addition of different concentrations of Fe3+ ion (0.02, 0.04, 0.06, 0.08, 0.1 mM) were carried out. The MR imaging results of mPEG-UCNPs-NRD upon addition of different concentrations of Fe3+ are very similar, of which the result of mPEGUCNPs-NRD with addition of 0.1 mM Fe3+ ion was displayed in Figure S13. The result shows the potential of mPEGUCNPs-NRD as a T1 contrast agent under the high Fe3+ concentration (0.1 mM), which could serve as a MR T2 contrast agent according to Karagoz’s publication.62 3.4. Cytotoxicity Assessment. To evaluate the potential bioapplication of mPEG-UCNPs-NRD nanostructure in living cells, cytotoxicity was investigated by a standard methyl thiazolyl tetrazolium (MTT) assay (see Figure 8). The HeLa cells and MCF-7 cells were incubated with mPEG-UCNPsNRD with different concentrations ranging from 0 to 400 μg· mL−1 for 24 h. The viabilities of HeLa cells and MCF-7 cells were over 95% even after incubation of mPEG-UCNPs-NRD with an highest concentration (400 μg·mL−1) for 24 h. The data indicate a relatively low cytotoxicity of mPEG-UCNPsNRD in different types of cells, making it potentially useful for imaging in vitro and in vivo.40,41 3.5. Detection Fe3+ in Living Cells. On the basis of the good photostability and good biocompatibility of mPEG-

Figure 8. In vitro cell viabilities of HeLa cells and MCF-7 cells incubated with mPEG-UCNPs-NRD at different concentrations (0, 25, 50, 100, 200, 400 μg·mL−1) for 24 h.

In addition, the stability of mPGE-UCNPs-NRD was assessed for a period of more than one month. There was no obvious dissociation or aggregation of the nanostructure in water. In particular, the photoluminescence intensity maintained more than 95% of initial value, when measured over a period of 42 days (see Figure S10). This shows that the mPEGUCNPs-NRD nanostructure has good colloidal stability and photostability in water, which are important for applications such as bioimaging. In addition, it was reported that the electroneutral and electropositive nanoparticles may be more effective for bioimaging, because they are taken up to a greater extent by proliferating cells.59 The surface potential measurements were carried out to support the fact that mPEG-UCNPsNRD can enter the living cells, as shown in the Figure S11. The zeta potential of UCNPs, mPEG-UCNPs and mPEG-UCNPsNRD was measured to be +27.8, −22.6 and +3.2 mV, respectively. It shows that the zeta potential of UCNPs with a value of +27.8 mV was strongly influenced by the presence of mPEG (with − COOH in the hydrophilic chain), and as a result it is negative (−22.6 mV) for mPEG-UCNPs. After the loading of NRD, the final mPGE-UCNPs-NRD nanostructure is electropositive, which is benefited to bioimaging. 3.3. MR Imaging of mPEG-UCNPs. In the mPGEUCNPs-NRD nanostructure, the NaYF4:Yb,Er,Tm core was coated by NaGdF4 shell, which not only improves the UCL

Figure 9. Confocal UCL images in living HeLa cells (A−E) and 40 μM Fe3+-pretreated HeLa cells (F−J) incubated with 400 μg·mL−1 mPEGUCNP-NRD for 4 h at 37 °C. UCL Emission was collected by a green UCL channel at 520−560 nm (B and G) and a red channel at 600−700 nm (C and H), λex = 980 nm, 500 mW. H

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ACS Applied Materials & Interfaces UCNPs-NRD and its high specificity and selectivity to Fe3+, laser scanning upconversion luminescence microscopy (LSUCLM) experiments under CW excitation at 980 nm were carried out to demonstrate potential application of mPEG-UCNPs-NRD in bioimaging of intracellular Fe3+. As shown in Figure 9, HeLa cells displayed a strong UCL emission at 520−560 nm (green region) and 630−680 nm (red region) after being incubated with mPEG-UCNPs-NRD (400 μg mL−1) for 4 h at 37 °C. When the cells were supplied with 40 μM Fe3+ in the growth medium for 20 min at 37 °C and then incubated with mPEG-UCNPs-NRD under the same conditions, both the green and red UCL channels in the intracellular region decreased (Figure 9G and 9H). From the merged images of green channel and red channel (Figure 9D and Figure 9I) and color mapped images (Figure 9E and Figure 9J), it can be further confirmed that the UCL intensity decreases as expected. From the brightfield measurements with or without addition of Fe3+, it was noted that the cells were viable throughout the imaging experiments. Furthermore, the merged confocal luminescence imaging of green and red channel collected as a series along the Z optical axis (Z-stack) confirmed that the UCL signals of the mPEG-UCNPs-NRD were almost localized in the cytosol region rather than staining the membrane surface (Figure S14). They were internalized into the cells possibly by endocytosis.63 This study demonstrates that the mPEG-UCNPs-NRD nanostructure could be used for detecting the intracellular Fe3+. To the best of our knowledge, the nanoprobe being applied for detecting the intracellular Fe3+ based on LRET has not been reported in the open literature to date.64

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions #

R.W. and Z.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the financial support from the National Natural Science Foundation of China (Grant No.s 21231004, 21571125), Innovation Program of Shanghai Municipal Education Commission (13ZZ073), the Science and Technology Commission of Shanghai Municipality (13NM1401100, 13NM1401101, 14520722200), and Shanghai Rising-Star Program (14QA1401800). Prof. Jin Z. Zhang is grateful to the US NSF (ECCS-0823921) and the BES Division of the US DOE (DE-FG02-ER46232) for financial support. We are also grateful to Instrumental Analysis & Research Center of Shanghai University.

4. CONCLUSIONS In summary, we have synthesized a novel NRD and demonstrated it as an excellent indicator for sensing Fe3+. When used in conjunction with mPEG-modified UCNPs, the new mPEG-UCNPs-NRD nanostructure has been successfully used for Fe3+ sensing in aqueous solution and living cells as well as MR imaging. To the best of our knowledge, this is the first example of a NRD-modified UCL nanostructure for both sensing and bioimaging of Fe3+. In the overall mPEG-UCNPsNRD nanostructure, the NaYF4:Yb,Er,Tm@NaGdF4 UCNPs serve as the energy donor while the Fe3+-responsive NRD as the energy acceptor, which results in LRET with high efficiencies based on green and red UCL. The obtained nanostructure shows high selectivity and specificity for detecting Fe3+ in water with a detection limit of 89.6 nM (green UCL as signal). Good photostability and biocompatibility make the mPEG-UCNPs-NRD nanostructure ideal for bioimaging of intracellular Fe3+ based on UCL. In addition, the effective T1-weighted MRI enhancement indicates that mPEG functionalization facilitates binding of water molecules to Gd3+ ions in the NaGdF4 layer, making the mPEG-UCNPs-NRD nanostructure a potential MRI contrast agent. In all, this work demonstrates a new approach for metal ions sensing as well as multimodal bioimaging based on a rationally designed multifunctional nanostructure.

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H NMR spectrum, MALDI-TOF-MS spectrum, DLS, XRD patterns, FT-IR spectra, NRD concentration of mPEG-UCNPs-NRD determined using the luminescence spectra, optical responses of Nile red derivative, emission intensity changes, emission spectra of mPEGUCNPs-NRD, zeta potential of UCNPs, mPEG-UCNPs, and mPEG-UCNPs-NRD, T1-weighted and colormapped MR images for various Gd3+ concentrations of mPEG-UCNPs and mPEG-UCNPs-NRD, and laser scanning confocal upconversion luminescence imaging (PDF)

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REFERENCES

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DOI: 10.1021/acsami.5b09132 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.5b09132 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX