Enhanced near-infrared photoacoustic imaging of silica-coated rare-earth doped nanoparticles

Materials Science and Engineering C 70 (2017) 340–346

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Short communication

Enhanced near-infrared photoacoustic imaging of silica-coated rare-earth doped nanoparticles Yang Sheng a,f,1, Lun-De Liao b,c,1, Aishwarya Bandla c,d, Yu-Hang Liu c,e, Jun Yuan c, Nitish Thakor c,d,e, Mei Chee Tan a,⁎ a

Engineering Product Development, Singapore University of Technology and Design, 8 Somapah Road, Singapore 487372, Singapore Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, 35 Keyan Rd., Zhunan Town, Miaoli County 35053, Taiwan, ROC Singapore Institute for Neurotechnology (SINAPSE), National University of Singapore, 28 Medical Drive, #05-COR, Singapore 117456, Singapore d Department of Biomedical Engineering, National University of Singapore, 21 Lower Kent Ridge Rd, Singapore 119077, Singapore e Department of Electrical and Computer Engineering, National University of Singapore, 21 Lower Kent Ridge Rd, Singapore 119077, Singapore f School of Materials Science and Engineering, Changzhou University, Changzhou, Jiangsu 213164, PR China b c

a r t i c l e

i n f o

Article history: Received 9 July 2016 Received in revised form 14 August 2016 Accepted 6 September 2016 Available online 7 September 2016 Keywords: Rare-earth Nanoparticles Silica Near-infrared Fluorescence Photoacoustic

a b s t r a c t Near-infrared photoacoustic (PA) imaging is an emerging diagnostic technology that utilizes the tissue transparent window to achieve improved contrast and spatial resolution for deep tissue imaging. In this study, we investigated the enhancement effect of the SiO2 shell on the PA property of our core/shell rare-earth nanoparticles (REs) consisting of an active rare-earth doped core of NaYF4:Yb,Er (REDNPs) and an undoped NaYF4 shell. We observed that the PA signal amplitude increased with SiO2 shell thickness. Although the SiO2 shell caused an observed decrease in the integrated fluorescence intensity due to the dilution effect, fluorescence quenching of the rare earth emitting ions within the REDNPs cores was successfully prevented by the undoped NaYF4 shell. Therefore, our multilayer structure consisting of an active core with successive functional layers was demonstrated to be an effective design for dual-modal fluorescence and PA imaging probes with improved PA property. The result from this work addresses a critical need for the development of dual-modal contrast agent that advances deep tissue imaging with high resolution and signal-to-noise ratio. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Near-infrared photoacoustic (PA) imaging is a minimally-invasive imaging modality that utilizes the tissue transparent window so as to provide high contrast and good spatial resolution for deep tissue imaging [1,2]. PA imaging possesses the advantages of both optical and ultrasonic technologies by detecting the acoustic signal generated via the non-radiative decay of the light absorber [3]. The diagnostic depth of PA imaging can go beyond the limitations of traditional near-infrared fluorescence imaging, to up to ~4 cm [4], due to the propagation characteristic of ultrasonic waves in tissues. To improve the PA imaging contrast by PA signal enhancement, exogenous probes such as gold nanostructures [5–7] and carbon nanotubes [8,9] have been studied. However, the fluorescence emissions from these materials (e.g., carbon nanotubes) are much weaker compared to well established fluorescent probes such as quantum dots ⁎ Corresponding author. E-mail address: [email protected] (M.C. Tan). 1 Yang Sheng and Lun-De Liao are co-first authors of this work.

http://dx.doi.org/10.1016/j.msec.2016.09.018 0928-4931/© 2016 Elsevier B.V. All rights reserved.

(QDs) and rare-earth doped nanoparticles (REDNPs) [10,11]. Consequently, these probes are not suitable for developing an optical-based dual-modal fluorescence and PA imaging platform. Optical-based dual-modal imaging is advantageous due to a couple of reasons: Firstly, a cooperative synergy of the strengths from both modalities provides: (1) the capability of deep tissue imaging with high spatial resolution from PA imaging, and (2) the capability of rapid visualization from fluorescence imaging. Secondly, an optical-based system is relatively safe, portable and cost-effective since no ionizing radiation source is used. QDs such as CdSe/ZnS have been reported for the purpose of multimodal PA imaging [12]. However, the presence of highly toxic element of Cd limits the application of these QDs in translational clinical applications [13]. In previous studies we have shown that the rare-earth doped particles (both micro- and nano-sized), which have relatively low cytotoxicity, were excellent candidates as contrast agents for dual-modal fluorescence and PA imaging [14,15]. REDNPs have been demonstrated to be amongst the most promising shortwave infrared (SWIR) imaging probes with their tunable emissions within the second near-infrared window which increases the fluorescence imaging depth [11,16]. The

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SWIR fluorescence can be tuned from 1000 to 1600 nm by changing the doping elements so as to achieve a maximum imaging depth of ~1 cm [11]. For PA imaging probes, we have found that the PA signal amplitude generally increased with the size of REDNPs [17], and that the coating of an undoped NaYF4 shell further enhanced both fluorescence intensity and PA amplitude [15]. However, we have also observed that the PA signal amplitudes of these nano-sized REDNPs (0.14–0.37 a.u.) were much lower when compared to their micro-sized counterparts (0.22– 0.91 a.u.) under the same experimental conditions. The design of nanomaterials with the core-shell architecture has emerged as a useful strategy towards improving the detected signal sensitivity [18–22]. For instance, Fe@Au and Ag@Au core-shell nanoparticles have shown potential in enhancing the detection sensitivity of Au nanoparticles for biosensor applications [23–25]. In our preliminary work where we used the SiO2 shell to improve the biocompatibility and colloidal stability of our REDNPs in aqueous conditions, we have found that the SiO2-coated REDNPs also showed significant PA amplitude enhancement. The observed enhancement was attributed to the increased phonon modes and phonon energy with the introduction of the SiO2 coating [15]. However, the SiO2 coating had also simultaneously gave rise to a deleterious effect where the SWIR fluorescence intensity was reduced due to the interactions between the rare-earth emitting ions and quenching chemical groups such as the hydroxyl (\\OH) groups. Using an undoped NaYF4 shell was demonstrated to enhance the fluorescence of REDNPs by preventing undesired interactions between the rare-earth emitting ions and quenching chemical groups [26]. Therefore, we aim to design and synthesize heterostructures with both NaYF4 and SiO2 layers to create imaging probes that exhibit a simultaneous enhancement in both fluorescence and PA intensities. In this study, we report a method to synthesize a multilayer structure of NaYF4:Yb,Er/NaYF4/SiO2 (REs/SiO2) nanoparticles by growing a SiO2 shell of well-controlled thickness around the core/shell rare-earth nanoparticles (REs) that have a NaYF4:Yb,Er (REDNPs cores) - NaYF4 (undoped shell) structure. The undoped NaYF4 shell around the REDNPs must be sufficiently thick (at least ~1.5 to 2 nm) to prevent the undesired quenching from the \\OH groups of SiO2 shell [27]. In this work, we studied the effects of the SiO2 shell thickness on the PA signal amplitude and fluorescence intensity of the REs. The increase in the number of phonon modes and phonon energy with the introduction of SiO2 would most likely facilitate heat dissipation leading to a significantly enhanced PA signal amplitude [28]. We have also acquired in vivo PA images using these REs/SiO2 nanoparticles to evaluate the feasibility and effectiveness of these multilayer structures in identifying the cortical superior sagittal sinus (SSS) blood vessel.

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2.2. Synthesis of β-NaYF4:Yb,Er core (REDNPs) and core/shell NaYF4:Yb,Er/ NaYF4 nanoparticles (REs) Typically, to synthesize core β-NaYF4:Yb,Er nanoparticles (REDNPs), a mixture of 1.5 mmol CF3COONa, 0.78 mmol (CF3COO)3Y, 0.2 mmol (CF3COO)3Yb, and 0.02 mmol (CF3COO)3Er was dissolved in 2 mL ODE with 4.4 mL OA and 3.5 mL OM at 120 °C for 30 min until a clear solution was formed. The mixture was subsequently heated to 330 °C, stirred for 1 h, and then cooled to room temperature. Nanocrystals in the solution were precipitated by addition of excess ethanol (∼40 mL), followed by centrifugation, redispersion, and washing (yield: ~ 120 mg). An undoped NaYF4 shell was coated by adding a mixture containing CF3COONa (1.5 mmol), (CF3COO)3Y (1 mmol), OA (3 mL) and OM (4 mL) to the reaction solution after keeping at 330 °C for 1 h. The reaction was maintained at 330 °C for another 30 min before cooling to room temperature (yield: ~ 260 mg). As reported in our previous work, the thickness of undoped NaYF4 shell was estimated based on the difference in average particle sizes for the core nanoparticles and core/shell nanoparticles [15]. 2.3. Synthesis of silica coated REs (REs/SiO2) Silica shell was coated by adopting previous reported method with some modifications [29]. In general, Igepal CO-520 (6 mL) as surfactant was dispersed in cyclohexane (108 mL) by stirring and ultrasonication. A solution of REs in cyclohexane (60 mg, 12 mL) was added. The resulting mixture was vigorously stirred, and ammonium hydroxide (0.96 mL, 28%) was added to form a transparent reverse microemulsion. Finally, TEOS (0.12 mL, 0.48 mL, and 0.72 mL dissolved in 4.8 mL cyclohexane for preparing ~5.5 nm, ~11.0 nm, and ~16.8 nm SiO2 shell) was added, and the reaction was continued for 24 h. The REs/SiO2 nanoparticles were collected by centrifugation and then washed with ethanol. The thickness of SiO2 shell is obtained by the direct observation and measurement of the SiO2 shell thickness from the electron micrographs. 2.4. Synthesis of PMAO-coated REs (REs/PMAO) The REs/PMAO nanoparticles were prepared by adopting a previously reported method [30]. Typically, 10 mg REs dispersed in 2 mL chloroform was mixed with 10 mL chloroform solution containing 100 mg PMAO, followed by sonication for 20 min. The mixture was dried by blowing N2 gas, followed with vacuum drying for 12 h. The resultant dry product was mixed with 8 mL NaOH solution (0.1 M) and maintained in an ultrasonic bath until a clear aqueous solution was formed. The REs/PMAO nanoparticles were obtained by centrifugation and redispersed in water.

2. Experimental

2.5. Materials characterization

2.1. Materials

The size and morphology of the synthesized REs were characterized by transmission electron microscopy (TEM) using JEOL 2010 microscope at an acceleration voltage of 200 kV. Samples were prepared by adding a sample solution onto the carbon grids followed by drying in an ambient environment. Powder XRD patterns were measured using the D8 ADVANCE ECO powder diffractometer (Bruker AXS Inc., Madison, WI) with CuKα (λ = 1.541 Ǻ) source operating at 40 V and 25 mA with a step size of 0.02° and duration of 0.5 s. The sample was prepared by pressing the dry powder on glass slide and was then placed on the holder. The Fourier Transform-Infrared (FT-IR) spectra were recorded using Bruker ALPHA spectrometer fitted with the single reflection attenuated total reflection module (Bruker Optik GmbH, Ettlingen, Germany). Data was obtained from pressed powder samples (without potassium bromide) and 64 scans were averaged with a resolution of 4 cm−1 at room temperature. The dynamic light scattering (DLS) measurement and zeta potential of REs/SiO2 nanoparticles dispersed in water

All chemicals were purchased from manufacturers and used as obtained without any further purification. Lanthanide oxides (99.99%, Y2O3, Yb2O3, Er2O3), sodium trifluoroacetate (98.0%, CF3COONa), 1octandecene (97%, ODE), oleylamine (70%, OM), oleic acid (90%, OA), cyclohexane (99.5%), tetraethyl orthosilicate (98%, TEOS), Igepal CO520, 1-hexanol (98%), ammonium hydroxide (28%), and poly(maleic anhydride-alt-1-octadecene) (PMAO, MW = 30,000–50,000) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO). Chloroform (99.99%) was purchased from Aik Moh Chemicals Inc. Sodium hydroxide (97.0%) was purchased from Sigma-Aldrich. Lanthanide trifluoroacetates ((CF3COO)3Ln) were prepared by dissolving the respective lanthanide oxides in trifluoroacetic acid (CF3COOH) in a glass bottle in an oven (Memmert Gmbh, Schwabach, Germany) at 80 °C overnight until all solids were dissolved and a clear solution was obtained.

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was measured using a Particle Sizer and Zeta Potential Analyzer (Brookhaven Nanobrook Omni, Brookhaven Instruments Corporation) at room temperature (~25 °C). Steady state and time-resolved spectra were measured using Edinburgh Instruments spectrometer (FLS980, Edinburgh Instruments, Livingston, U.K.) equipped with PMT (Hamamatsu R928P, Hamamatsu Photonics K.K., Japan) and NIR-PMT (Hamamatsu H1033A-75, Hamamatsu Photonics K.K., Japan) upon excitation with a 975 nm continuous wave laser (CNI MDL-III-975, Changchun New Industries Optoelectronics Tech. Co. Ltd., China). To measure steady state spectra, dry powder samples were packed in demountable Spectrosil far UV quartz Type 20 cells (Starna Cells, Inc., Atascadero, CA) with 0.5 mm path lengths for emission collection. The power of the 975 nm CW laser was set to ~0.53 mW/mm2. The optical path for all photoluminescence measurements was kept constant. All fluorescence measurements were measured three times and the average curves were shown in this study. To measure the time-resolved fluorescence spectrum, the excitation source was modulated using an electronic pulse modulator to obtain excitation pule at a pulse duration of 20 μs with a repetition rate of 10 Hz. The obtained spectra were fitted and calculated using Origin 8® with the equations shown in the Results section. 2.6. PA microscopy (PAM) and PA signal analysis In this study, a customized dark-field confocal PAM system was used to image the contrast changes with 32 × 61 μm spatial resolution. An optical parametric oscillator (OPO) pumped by a frequency-tripled Nd:YAG Q-switched laser was employed to provide ~ 4 ns laser pulses at a repetition rate of 10 Hz. 975 nm wavelength laser pulses were

used for PA wave excitation, which was delivered by a 1 mm multimode fiber. The fiber tip was coaxially aligned into the customized light path including a convex lens, an axicon, a plexiglass mirror, forming darkfield illumination. A large numerical-aperture, wideband 50-MHz ultrasonic transducer was employed for the efficient collection of PA signals. The focal point of this ultrasonic transducer was confocal with the darkfield microscopy. The PA signals received by this ultrasonic transducer were pre-amplified by a low-noise amplifier (noise Fig. 1.2 dB, gain 55 dB), then digitized and sampled by a computer-based 14-bit analog to digital (A/D) card at a 200-MHz sampling rate for data storage. To compensate variations caused by laser energy instability, a photodiode was used to monitor fluctuations of optical signals. The scanning step size was 10 μm for each B-scan of this PAM system. PA wave excitation was elicited by a single wavelength (λ975) and for evaluating the effectiveness of REs/SiO2 in PA signal. It was assumed that the received PA signal was proportional to the thickness of amorphous SiO2 at λ975. PA cross-sectional B-scan imaging in polyethylene tubing (in vitro) and specific cortical regions (in vivo) were conducted. A pixel with strong PA signal (at least three times greater than the background signal) is defined as the nanoparticle pixel. The PA amplitude was calculated using the total nanoparticle pixel count of a selected cross-sectional area. Please refer to our previous studies for detailed PA signal analysis [31,32]. 2.7. PA measurements for nanoparticles Samples for in vitro PA imaging and signal measurements were obtained by dispersing the nanoparticles in ethanol. Concentration of 0.05 mmol/mL (~ 10 mg/mL; concentration based on the mass of REs

Fig. 1. TEM images of (a) REs with an average size of 24.7 ± 3.5 nm and SiO2-coated REs with SiO2 shell thickness of (b) 5.5 ± 0.7 nm (REs/SiO2-5), 11.0 ± 1.2 nm (REs/SiO2-11), and 16.8 ± 1.3 nm (REs/SiO2-17).

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obtained directly from synthesis) was obtained for both core/shell REs and REs/SiO2. To evaluate the PA properties of our REs/SiO2, PA signals of our REs/SiO2 injected into a ~ 15 cm polyethylene tubing (0.38 × 1.09 mm, Scientific Commodities, Inc., Lake Havasu City, Arizona) were measured using the confocal PAM system upon excitation at 975 nm. Afterwards, the tubing was positioned at depth of the transducer's focus, i.e., the depth of 9 mm with respect to the transducer in water tank. The system was maintained in a 25 °C water bath throughout the experiment. 2.8. In vivo PA cortical SSS blood vessel imaging All experimental procedures used in this study were approved by the Institutional Animal Care and Use Committee of the National University of Singapore. Six male Sprague Dawley rats weighing 250 to 300 g (InVivos Pte Ltd., Singapore) each, were used for in vivo PA imaging. The animals were housed at a constant temperature and humidity with free access to food and water before the procedures. The animals were maintained anesthetized with pentobarbital (50 mg/kg bolus and 15 mg/kg/h maintenance, intraperitoneal) throughout the experiments. The animals' body temperature was regulated at 37 ± 0.5 °C by a self-regulating thermal plate (TCAT-2 Temperature Controller, Physitemp Instruments, Inc., New Jersey, USA). The anesthetized rats were mounted on a customized acrylic stereotaxic head holder. A scalp incision was made to expose the bregma landmark. A high-speed drill was employed to fashion a bilateral cranial window of size approximately 8 (horizontal) × 3 (vertical) mm, with care taken not to damage the dura during the craniotomy. The anteroposterior distance between the bregma and the interaural line was surveyed directly and was then used to position the animal's head in the PAM system without additional surgery in following experiments. Further, the designed particles were infused via the tail vein as an aqueous solution (150 μL) of REs/SiO217 (10 mg/mL). None of the animals suffered any ill effects as a result of the REs/SiO2-17 administration. 3. Results and discussions 3.1. Physical characterizations of REs and REs/SiO2 REs were synthesized with a core/shell structure consisting of a rareearth doped core (NaYF4:Yb,Er) and undoped shell (NaYF4). As shown in the TEM image (see Fig. 1a), the obtained REs have an average size of 24.7 ± 3.5 nm. Using the Scherrer equation [33] (see Supplementary Information Fig. S1), the estimated grain size of REs is 20.8 ± 0.5 nm and is comparable to the particle size observed from TEM, which indicates that these as-prepared REs are most likely single crystals. The core size of NaYF4:Yb,Er is ~11 nm based on our previous work [15], and verified later based on its fluorescence lifetime values. Therefore, the ~ 6 nm shell of undoped NaYF4 that was coated around the emitting active cores would be sufficient to prevent the quenching of the rare earth emitting centers from the surrounding impurities, such as \\OH and CH2 groups. The SiO2 shell was subsequently coated on the surface of REs using the reverse microemulsion technique. The SiO2 coating offers two main benefits: (1) SiO2 improves the biocompatibility by reducing toxicity as well as enhancing particle hydrophilicity which facilitates colloidal stability in aqueous systems; [34] (2) SiO2 enables further functionalization with targeting moieties (e.g., antibodies, peptides) [35]. By changing the ratio between the TEOS and REs, uniform SiO2 shell thicknesses of 5.5 ± 0.7 nm (REs/SiO2-5), 11.0 ± 1.2 nm (REs/ SiO2-11), and 16.8 ± 1.3 nm (REs/SiO2-17) were obtained (see Fig. 1b–c). The crystal structure of the REs and REs/SiO2 were characterized using XRD (see Supplementary Information Fig. S1), where both samples exhibited identical peaks which were consistent with the characteristic diffraction peaks of hexagonal phase NaYF4 (β-NaYF4). The XRD pattern for REs/SiO2 nanoparticles showed an additional small

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hump between 2θ = 20° to 30°, which was associated with the presence of the amorphous SiO2 shell. The presence of the SiO2 shell was also validated using FT-IR (see Supplementary Information Fig. S2), where the observed absorption band at 1064 cm−1 was attributed to the Si\\O\\Si stretching vibration mode. 3.2. Fluorescence properties of REs and REs/SiO2 nanoparticles upon excitation at 975 nm To evaluate the influence of the SiO 2 shell on the fluorescence properties of the synthesized REs/SiO 2 nanoparticles, the steady state and the time resolved fluorescence spectra were measured. The characteristic fluorescence emission peaks at 540 nm, 660 nm, and 1530 nm of Er doped materials, which correspond to 2 H 11/2 ( 4 S 3/2 ) → 4 I 15/2 , 4 F 9/2 → 4 I 15/2 , and 4 I 13/2 → 4 I 15/2 , were observed as shown in Fig. 2a [17,36]. The fluorescence intensity for all emissions was generally observed to decrease as the SiO2 shell thickness increased. Since the visible upconversion fluorescence at 540 and 660 nm occurs via nonlinear optical processes, it will be more complex to evaluate the shells effects on the radiative and non-radiative pathways that govern these emissions [17]. Consequently, the downshifted 1530 nm SWIR fluorescence (a linear optical process) was used to study the correlation of the fluorescence and PA properties. As shown in Fig. 2b and Table 1, the SWIR emission at 1530 nm for REs is ~ 3.6 times higher than those coated with a 5 nm SiO2 shell, and ~ 5.8 times higher than those coated with a 17 nm SiO2 shell. The decrease in the SWIR emission intensity may be attributed to a few possibilities including the higher phonon energy and presence of\\OH groups of the SiO2 coating leading to increased non-radiative transitions through the multi-phonon relaxation pathways, or the dilution effect with the addition of a non-optically active shell. However, our FT-IR results showed that the \\OH concentration within the SiO2 shell was very low (see Supplementary Information Fig. S2). In addition, based on our estimates, the thickness of the undoped NaYF4 should be sufficient to prevent surface quenching effects. Therefore, it is unlikely that the decrease in intensity is due to fluorescence quenching by SiO2. Consequently, one possible explanation for the observed reduction in fluorescence intensity is the dilution of the emission intensity from the REs due to the introduction of a non-optically active SiO2 matrix. It should be noted here that since the REs in the dry powder form were packed in demountable cells of a fixed path length, the steady fluorescence intensities were measured from the same volume of samples under the same excitation conditions. As the SiO2 shell thickness increased, the volume fraction (vol%) of fluorescence emitting REs was reduced, leading to a smaller amount of fluorescence being collected by the detector under steady state conditions. To evaluate the dilution effect from the SiO2 shell, the measured integrated SWIR intensity was compared with the calculated SWIR intensity estimated based on the volume fraction of optically-active REs (see Table 2). With the increase in SiO2 shell thickness, the volume fraction of REs was decreased significantly leading to the decrease in calculated fluorescence intensities. The marginal difference between the measured and converted intensity values was most likely due to the differences in packing density, as well as particle size and shape distribution. Therefore, the decrease in measured fluorescence intensity was mostly due to the dilution effect from the addition of SiO2. To further understand the effects of SiO2 on the fluorescence properties, the fluorescence decay time was measured. Unlike the steady state fluorescence measurements which are dependent on the path length and sample concentration that is measured as well as the conditions of the excitation source, the decay time value is an intrinsic characteristic of the sample that does not change with the measurement condition or sample concentration. The downshifted SWIR emission at 1530 nm representing the 4I13/2 → 4I15/2 transition of Er was studied as this emission is a linear optical process [17]. Fig. 2c shows the characteristic timeresolved fluorescence spectra of REs and REs/SiO2 nanoparticles for the

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Fig. 2. (a) Steady state fluorescence spectra of REs and REs/SiO2 with varied SiO2 shell thickness. The spectra were measured using continuous wave 975 nm laser at a power density of ~0.53 mW/mm2 for all samples. (b) Plot of integrated intensity of SWIR emission intensity of REs and REs/SiO2 nanoparticles. (c) Time-resolved fluorescence spectra of REs and REs/SiO2 nanoparticles corresponding to the 4I13/2 → 4I15/2 transition of Er3+ (1530 nm emission). (d) Plot of fitted decay time of REs and REs/SiO2 nanoparticles with different SiO2 shell thickness. The values of decay time τave obtained from Eq. (2) are summarized in Table 1. The error bars are given by fitting using Origin 8®, which represents the deviation of the fitted value from the measured data.

1530 nm emission. All decay curves were fitted to a double exponential equation as follows: Iðt Þ ¼ A1  exp

    t t − þ A2  exp − τ1 τ1

ð1Þ

where I(t) is the decaying intensity at the maximum of the emission band 1530 nm, A1 and A2 are the exponential pre-factors and τ1 and τ2 are the fitted decay times. The average decay time constant τave can be determined using the following equation [27], τave ¼

A1 τ21 þ A2 τ22 A1 τ 1 þ A2 τ 2

ð2Þ

The fitting of all spectra were completed using Origin 8®, and the fitted average decay time of REs and REs/SiO2 are plotted in Fig. 2d. The values of integrated SWIR intensity and decay time with errors are summarized in Table 1. Fluorescence decay time characterizes the contribution of non-radiative relaxation from a specific excited state. With the coating of an

Table 1 Summary of integrated fluorescence intensity and decay time at 1530 nm emission for REs and REs/SiO2 nanoparticles. Samples

SWIR intensity (a.u.)

Decay time (ms)

Core NaYF4:Yb,Er REs REs/SiO2-5 REs/SiO2-11 REs/SiO2-17 REs/PMAO

– 4.71 × 108 1.33 × 108 1.17 × 108 0.77 × 108 1.3 × 108

1.27 ± 0.02 5.32 ± 0.02 6.50 ± 0.03 5.91 ± 0.03 6.04 ± 0.03 5.96 ± 0.02

undoped NaYF4 layer around the REDNPs, the decay time was increased from 1.27 to 5.32 ms mostly due to reduced surface quenching, which is consistent with our previous result [15]. As shown in Fig. 2d and Table 1, the coating of a 5.5 nm SiO2 shell leads to a slight increase in the decay time of REs from ~5.32 to ~6.50 ms. The coating of a thicker SiO2 shell of 11 and 17 nm leads to a slight decrease in decay time to ~ 5.91 and 6.04 ms. It should be noted that despite the slight variation, the decay time of the SiO2-coated REs remained higher than the REs. The overall slight increase in fluorescence decay time for all SiO2 coated samples indicated that the quenching effect from surface defects and impurities was successfully prevented by the presence of the undoped NaYF4 shell surrounding the emitting centers within the active core of the REDNPs. In addition, since the decay time was increased after SiO2 coating, the observed decrease in fluorescence intensity (see Fig. 2a) was not likely due to quenching by the \\OH groups or the higher phonon energy of SiO2 shell. The slight increase in fluorescence decay time was mostly likely due to the increased phonon modes of upon coating with SiO2. It has been reported that with increasing particle size, the number of phonon modes generally increases. The increase in number of phonon modes would probably also favor the phonon-assisted Table 2 Comparison between calculated intensity and converted intensity based on REs' volume fraction (vol%).a Samples

SiO2 shell thickness (nm)

Vol% Intensity calculated of REs from spectra (a.u.)

REs REs/SiO2-5 REs/SiO2-11 REs/SiO2-17

0 5.5 11.0 16.8

100 33 15 8

a

4.71 × 108 1.33 × 108 1.17 × 108 0.77 × 108

Intensity calculated based on vol% (a.u.)a 4.71 × 108 1.58 × 108 0.71 × 108 0.37 × 108

Refer to the supplementary information for the calculation of these values.

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energy transfer pathways leading to higher decay times for SiO2 coated REs [37]. The slight reduction in decay time for REs/SiO2-11 and REs/ SiO2-17 compared to REs/SiO2-5 was most likely due to the higher \\OH amounts with increased shell thickness. Chemical groups such as the\\OH group are known to effectively quench infrared emissions [17,38].

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cross section of vessel was enhanced after 10 mins post injection. Further studies on improving the image contrast is needed, which is beyond the current scope of this work.

3.4. Fluorescence and PA properties of PMAO-coated REs upon excitation at 975 nm

3.3. Influence of SiO2 shell on the PA properties upon excitation at 975 nm To demonstrate the influence of the SiO2 shell on the in vitro PA properties of the synthesized REs/SiO2 nanoparticles, the PA amplitudes of REs and RE/SiO2 dispersed in ethanol were measured and plotted in Fig. 3a. Considering the higher viscosity of REs/SiO2 nanoparticles which led to difficulty during injection of the nanoparticles into the micro tubing for in vitro PA signal measurement, the concentrations of all samples including REs and REs/SiO2 were fixed at 0.05 mmol/mL (~10 mg/mL, concentration based on REs). The results showed that at this low concentration, no PA signals from REs were detected. The undetectable signal was consistent with our previous study [15], where a signal amplitude of only 0.16 a.u. was obtained for a concentration at 0.2 mmol/mL which was 4 times higher than the concentration used here. However, the PA signal amplitude increased significantly to 0.18, 0.31 and 0.52 a.u. after coating with ~5.5, ~11.0 and 16.8 nm SiO2 shells, respectively, while all other experimental parameters were kept the same. The increase in PA amplitude was attributed to the high phonon modes of the amorphous SiO2. The PA signal amplitude was influenced by the heat transfer rate between the nanoparticle and the solvent, where a faster heat dissipation generally resulted in a higher PA signal amplitude [39]. Heat is generally transferred by phonons (i.e., lattice vibration waves) and electrons. For non-conductors, the heat is mainly carried by the acoustic phonons [28]. The higher number of phonon modes as a consequence of the addition of a SiO2 shell resulted in an increase in the acoustic phonon modes, which increased the heat dissipation rate and therefore resulted in the enhanced PA signal amplitude [37]. Sample REs/SiO2-17 was used as an example for demonstration of in vivo PA imaging of rat's cortical SSS blood vessel. The DLS results (see Supplementary Fig. S3) shows that the hydrodynamic diameter for REs/SiO2-17 is ~82 nm, which is consistent with the size observed earlier from the TEM images (see Fig. 1). The zeta potential of these SiO2coated nanoparticles was −71.6 mV which was attributed to the negatively charged surface OH groups. Consequently, the colloidal stability of REs/SiO2 nanoparticles would be improved due to the electrostatic repulsion between particles. As shown in Fig. S4, the contrast of the

To verify that the PA amplitude was enhanced due to the higher number of phonon modes with the increased particle size using the SiO2 coating, the REs were also coated with a polymer coating of poly(maleic anhydride-alt-1-octadecene) (PMAO) [30]. Fig. 4a shows the TEM image of monodisperse PMAO coated REs (REs/PMAO) with no severe aggregation since large agglomerates were not observed. The average size of the REs/PMAO was measured to be 25.8 ± 2.9 nm, where the polymer shell was estimated to be 4 to 5 nm (see Fig. 3a inset). The FT-IR spectrum also verifies the successful coating of PMAO (see Supplementary Information Fig. S2). The presence of C\\H stretch vibrations (2800 and 2950 cm− 1) and O\\H stretch vibration (i.e. 3200–3600 cm−1) that were characteristic of PMAO were also observed for REs/PMAO. The integrated SWIR fluorescence intensity for REs/PMAO was ~ 1.30 × 108 a.u., which was similar to that for REs/SiO2-5 (1.33 × 108 a.u.). The fluorescence decay time for the 1530 nm emission was ~5.96 ms (comparable to ~5.32 ms of REs), which further verified that the quenching was effectively suppressed by the presence of the undoped NaYF4 coating around REDNPs. The decreased fluorescence intensity for REs/PMAO was most likely due to the dilution effect. However, no PA signal was detected for the REs/PMAO samples under the same experimental conditions with identical REs concentration (0.05 mmol/ mL). The absence of PA signal enhancement suggested that the phonon characteristics (e.g., number of modes and propagation characteristics) of the PMAO coating were different from that of the SiO2 coating. A possible explanation is that there is no significant change in the inorganic particle size when a soft polymer coating is used. Therefore, this would lead to no changes in the number of phonon modes (or heat dissipation) and PA signal amplitudes using the PMAO coating. In contrast, the increase in the number of phonon modes from the inorganic particle size increase using a SiO2 coating led to the observed PA enhancement. Further work to understand the heat dissipation and phonon characteristics of a soft polymer coating is currently underway. It should be noted that although the exact mechanism remains unclear, the result from the PMAO-coated REs highlights the importance and effectiveness of the SiO2 layer in enhancing the PA signal amplitudes.

Fig. 3. (a) SiO2 shell thickness dependent PA amplitudes of REs/SiO2 nanoparticles with SiO2 thicknesses of 5.5 ± 0.7 nm (REs/SiO2-5), 11.0 ± 1.2 nm (REs/SiO2-11), and 16.8 ± 1.3 nm (REs/SiO2-17). (b) Corresponding PA images of REs/SiO2 samples. The PA amplitudes are REs/SiO2-5: 0.18, REs/SiO2-11: 0.31, REs/SiO2-17: 0.52. All measurements were performed using a 50-MHz dark field confocal PAM system with ~4 ns laser pulses (975 nm) at a pulse repetition rate of 10 Hz. The concentration of all samples was 0.05 mmol/mL.

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Fig. 4. (a) TEM image of water-soluble REs/PMAO nanoparticles with core REs size of 25.8 ± 2.9 nm. The inset shows a 4–5 nm polymer shell outside the REs. (b) Steady state fluorescence spectra of REs, REs/PMAO and REs/SiO2-5 nanoparticles. (c) Time-resolved luminescence spectra corresponding to the 1530 nm emission of REs, REs/PMAO and REs/SiO2-5.

4. Summary In this work, we report on the synthesis of a multilayered imaging contrast agent consisting of REs (NaYF4:Yb,Er/NaYF4) and an amorphous SiO2 shell. Due to the dilution effect from the introduction of a non-optically-active SiO2, we observed a decrease in the integrated fluorescence intensity. Based on the fluorescence decay time measurements which characterize the intrinsic behavior of the as-synthesized REs, we found that fluorescence quenching was effectively suppressed by the presence of the undoped NaYF4 shell. As the SiO2 shell thickness increased, the PA signal amplitude was increased accordingly. The PA signal enhancement was attributed to the higher number of phonon modes due to the particle size increase with the addition of a SiO2 shell. The higher number of phonon modes would likely lead to the increased heat dissipation from the contrast agents to the surroundings as well. Therefore, the SiO2 coating around the REs with the undoped NaYF4 shell provided a facile method to enhance the PA property of a fluorescence imaging probe. From the in vivo PA imaging using these SiO2-coated REs, we demonstrated their effectiveness to enhance the contrast of the cortical SSS blood vessel. Acknowledgements MC Tan and Y Sheng would like to gratefully acknowledge the funding support from the SUTD-MIT International Design Center (Grant number IDSF1200105), and Ministry of Education (MOE), Singapore. MC Tan and Y Sheng would also like to thank Dr. XY Zhao for obtaining the TEM images. LD Liao, A Bandla, YH Liu, J Yuan and N Thakor would like to thank The Agency for Science, Technology and Research (A*STAR), Singapore for their support under grant number R719-003-100-305 and Ministry of Education, Singapore for their support under the Tier II grant MOE2014-T2-2-145. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2016.09.018. References [1] H.F. Zhang, K. Maslov, G. Stoica, L.V. Wang, Nat. Biotech. 24 (2006) 848–851. [2] Y. Zhang, H. Hong, W. Cai, Cold Spring Harb. Protoc. 2011 (2011) 065508. [3] L.V. Wang, S. Hu, Science 335 (2012) 1458–1462.

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