Gold

Research Article pubs.acs.org/journal/ascecg

Energy-Absorbing and Local Plasmonic Nanodiamond/Gold Nanocomposites for Sustained and Enhanced Photoacoustic Imaging Dukhee Lee,†,+ Eun-Joo Park,‡,+ Sang-Eun Lee,§ Seong Hoon Jeong,∥ Jae Young Lee,*,⊥ and Eunah Kang*,† †

School of Chemical Engineering and Material Science, Chung-Ang University, 221 Heukseok-Dong, Dongjak-Gu, Seoul, Korea Biomedical Research Institute, Seoul National University Hospital/Department of Radiology, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Korea § Division of Allergic and Pulmonary Medicine, Department of Internal Medicine, College of Medicine, Chung-Ang University, 221 Heukseok-Dong, Dongjak-Gu, Seoul, Korea ∥ College of Pharmacy, Dongguk University, Gyeonggi, Republic of Korea ⊥ Department of Radiology, Seoul National University Hospital/Department of Radiology, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, 03080, Korea ‡

S Supporting Information *

ABSTRACT: Photoacoustic (PA) imaging is a laser-mediated optical ultrasound-based visualization that allows imaging of optical energy absorbers in deep tissue, offering higher spatial resolution, compared with that of NIR fluorescence. To enhance a gold nanoparticles-based PA agent, carbon crystalline nanodiamonds and gold nanocomposites (NDAuNPs) were synthesized by chemical reduction of a carboxylate nanodiamond and gold precursor. Reduced hydroxyl-terminated nanodiamonds have stable colloidal dispersion and provide a platform where AuNPs are localized on the ND surface with high density. NDAuNP agglutinates were 100 nm in size, and AuNPs with a size distribution of 5−20 nm were chemically conjugated on the ND surface. The surfaceenhanced Raman scattering spectra showed enhanced intensity of NDAuNPs in a concentration-dependent manner. Energy-absorbing nanodiamonds facilitated energy transfer into AuNPs, inducing a local plasmonic effect. The PA signal of NDAuNPs was stronger than that of the AuNPs, as well as the signal maintenance during a prolonged period of laser irradiation. Tissue images of TEM showed that after 2 h irradiation NDAuNPs were maintained without gold degradation, while AuNPs were degraded. The local plasmonic and the energy-absorbing properties of NDAuNPs amplified the PA signal and impeded the degradation of gold without PA signal decay. The NDAuNP nanocomposites may serve as an imaging probe, providing high PA amplitudes. KEYWORDS: Photoacoustic, Gold nanoparticles, Nanodiamond, Nanocomposite

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carbon-based materials.5−9 Gold nanoparticles have been a common choice for contrast and therapeutic agents based on their superior optical properties, biocompatibility, and ease of bioconjugation with biomarkers to create nanosized contrast agents with molecular specificity. As the first generation of a PA imaging agent, gold nanoparticles (AuNPs) and nanorods have been focus of extensive efforts to tune the nanostructure, including Au anisotropy,7 hollow,10,11 nanovesicles,12,13 and bimetal combined architecture,14,15 to generate a strong photoactive thermal or acoustic effect. Though diverse metal composites including Au displayed enhanced surface plasmon resonance, the enhanced photothermal and photoacoustic

INTRODUCTION Photoacoustic (PA) imaging has emerged as an alternative to optical imaging, overcoming the optical diffusion cutoff. The laser light is absorbed onto the image target and converted to an outward thermoacoustic wave that can be detected by an ultrasound transducer and used to reconstruct images.1 PA imaging can provide deeper tissue penetration and high spatial resolution, compared with those of NIR fluorescence imaging. Although, endogenous hemoglobin and melanin are photoacoustically active, a materials-based PA imaging contrast enhancer has been developed to improve biomedical theranostics as a PA multifunctional imaging agent and drug releasing reservoir.2−4 Recently, employed PA-active materials became diverse, including melamine-modified nanoparticles, porphyrin-modified nanoparticles, semiconductive π-conjugating polymers, their hybrid nanocomposites, and hybrids with © 2017 American Chemical Society

Received: June 15, 2017 Revised: July 26, 2017 Published: August 4, 2017 8284

DOI: 10.1021/acssuschemeng.7b01944 ACS Sustainable Chem. Eng. 2017, 5, 8284−8293

Research Article

ACS Sustainable Chemistry & Engineering

NDAuNP nanocomposites enhance PA signal intensity and sustain PA signal via laser irradiation.

effects were sustained for a limited time period. The PA signal amplitude weakened as gold particles were degraded upon continuous pulse laser irradiation.7,14 Moreover, at high laser power, the large bubble formation and its sudden disruption occurring by local thermal expansion are not appropriate for cells since imaging and therapy should be selective. Uncontrolled disruption of gold NPs might restrain sustainable PA activity.16 To advance the sustained PA and photothermal effect, several studies have reported that graphene as an energyabsorbing carbon material enhanced the photothermal effect and amplified the PA signal. Versatile nanostructural forms include gold nanorods on a templated carbon nanotube,16 AuNPs distributed on a submicrometer-scaled graphene sheet,17 AuNP clusters coated with reduced graphene,18 enveloped gold nanorods with reduced graphene,7 and silicacoated AuNPs.19 Considering the graphene-enhanced plasmon resonance of gold nanoparticles, there is still room to investigate a controlled gold nanostructure with energyabsorbing carbon materials for laser-responsive photothermal and PA activity.20 Specifically, nanodiamonds that have an sp3-carbon diamond core and a reconstructed sp2-carbon surface layer with a particle diameter of 4−5 nm are known as energy-absorbing carbonbased materials.21 Crystalline carbon with hybrid orbitals, a reconstructed sp2 carbon surface, and dangling surface functional groups exhibit unusual activity, leaving plenty of room to exploit multiple functions. Its surface-modified carboxyl end group provides colloidal dispersion stability in aqueous environments and enhanced permeability even at the endothelial cell layer of blood vessels, as well as chemical designer functionalities.22,23 Nanodiamond/bimetallic composites have been investigated as a catalytic material source.24−26 The enhanced thermal conductivity of nanodiamond/nickel composites27 and nanodiamond nanofluid28 was reported, which is a critical characteristic to determine the laser-reactive signals of a photoacoustic or photothermal effect. Fluorescence nanodiamond with an N-vacancy site has its own photoacoustic properties, though the acquirement of an N-vacancy fluorescence nanodiamond does not have easy access.29 Together with diverse material-based functional nanodiamonds, new sources of nanodiamonds have also been extensively investigated for biomedical applications, clarifying the biocompatibility cellular uptake and toxicity in vivo.30−35 By combining diamond and gold, the potential possibility of photoacoustic emission and thermal effect has been previously shown.35,36 Specifically, this study focused on the approach to significantly improve and sustain photoacoustic imaging by creating a nanocomposite of gold nanoparticles and nanodiamonds. The conventional strategy of designing plasmonic photoacoustic contrast agents usually involves maximizing the optical absorption cross section of nanoparticles by manipulating their size and shape and choosing the peak position and the relative amplitude of the absorption. The surfactant-free nanodiamond/gold nanocomposites were synthesized to investigate the maximized plasmonic photoacoustic effects. The reduced ND surface into hydroxyl groups and deposited seed growth of gold on the ND provide stability in aqueous dispersions, which is required for biomedical applications in vivo. Newly investigated photoacoustic properties of ND/AuNP nanocomposites were examined to determine whether

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EXPERIMENTAL SECTION

Preparation of NDAuNP Nanocomposites and AuNPs. Acidwashed and highly purified nanodiamonds terminated with carboxyl groups were gifted from Nanoresouces, Inc. (Seoul, Korea). To prepare NDAuNP nanocomposites, AuNP crystalline growth on the nanodiamond surface (ND-COOH) was synthesized by in situ reduction of AuCl3 ions (99%, Strem Chemicals, MA, USA) in the presence of a reducing agent, NaBH4 (98+%, Acros Organics, NH, USA). The ND powder (10 mg) in a 1 wt % ethylene glycol aqueous solution (99.5%, 10 mL in water, Sigma-Aldrich, MO, USA) was sonicated for 10 min until NDs were homogeneously dispersed. The ND suspension was incubated under stirring at 70 °C for 10 min, and then, 20 mg of AuCl3 was added. After 10 min incubation of the NDAuCl3 mixture, 0.1 mL of ice-cooled NaBH4 solution (conc. = 100 mg/mL) was slowly added to the incubated mixture, and the color of the ND-AuCl3 mixture solution instantly changed to dark purple. After vigorous stirring for 10 min, the reactor was cooled to 25 °C, and the mixture was further stirred for 24 h. The produced NDAuNP nanocomposites were centrifuged at 7000 rpm and washed with deionized water five times to remove unbound AuNPs and residual NaBH4. A final stock solution of the NDAuNP nanocomposites was prepared with a concentration of 2.0 mg/mL in DI water. The NDAuNP suspension was maintained stable in water for weeks at 4 °C and used for the characterization. For solid-state characterization, centrifuged NDAuNPs were dried at 80 °C in an incubating oven for 2 h and under vacuum at 50 °C for 24 h. The final soft powder of NDAuNP nanocomposites was obtained (yield 91%). To prepare AuNPs, 10 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB; 99+%, Sigma-Aldrich) was mixed with AuCl3 (100 μL, 10 mg) in a vial per the typical protocol.37 Then, 100 μL ice-cold NaBH4 (10 mg) was added with shaking. After 1 min of shaking, the solution was kept at room temperature for 12 h. The solution turned dark pink immediately after adding NaBH4, indicating particle formation. After removal of CTAB, the AuNPs were further characterized. Physical Characterization of NDs, AuNPs, and NDAuNP Nanocomposites. Crystalline structures of NDs, AuNPs, and NDAuNP nanocomposites were characterized with X-ray diffraction (XRD) (D8-Advance X-ray diffractometer, Bruker Corp., MA, USA) equipped with a Cu Kα radiation source (λ = 0.154 nm, 40 kV, 40 mA) and a high speed LynxEye detector. XRD spectra of NDs, AuNPs, and NDAuNP nanocomposites were recorded over the 2θ ranges from 10° to 80° with a 0.02° step size. The sample was ground to soft powder and placed flat into a sample holder. The UV absorption of NDs, AuNPs, and NDAuNP nanocomposites were measured with a V-670 UV−vis/NIR spectrophotometer (JASCO Corp., Tokyo, Japan) with a synthetic quartz cuvette (1 cm light path, Hellma Analytics, Germany). The conditions were set with a scanning speed of 200 nm/min, data interval of 1 nm, UV/ vis bandwidth of 1.0 nm, and NIR bandwidth of 2.0 nm. A 2 mL siluted NDAuNP dispersion (0.02 mL of NDAuNP nanocomposite stock solution and 1.98 mL of deionized water) was used for the UV measurement ranging from 350 to 900 nm. The hydrodynamic averaged diameter and zeta potential measurements with the 0.1 mg/ mL concentration of NDs, AuNPs, and NDAuNP suspensions were conducted using dynamic light scattering (DLS) (SZ-100, Horiba Ltd., Kyoto, Japan) with five-time repeated measurements at 25 °C. The detector angle of the light scattering was fixed at 90°. A disposable polystyrene cuvette (Sarstedt AG & Co., Germany) and a disposable capillary carbon electrode zeta cell (Horiba Ltd.) were used for the hydrodynamic radius and zeta potential measurements, respectively. The Raman spectra of NDs, AuNPs, and NDAuNP nanocomposites were obtained with a 633 nm He−Ne laser excitation source equipped with a Horiba Jobin Yvon LabRam Aramis spectrometer. The beam intensity was 10μW at the sample surface, and each spectrum was accumulated for 10 s. Methylene blue was used as the Raman reporter 8285


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Figure 1. Field emission transmission electron microscopy (FE-TEM) images of nanodiamonds (a, d), nanodiamond/gold nanocomposites (NDAuNPs) (b, e), and gold nanoparticles (c, f) (scale bars 50 and 5 nm, respectively). Individual crystalline structures of nanodiamond and gold nanoparticles were clearly observed as aligned structures. Size distribution of AuNPs on the ND surface and AuNPs only were measured by counting 130 AuNPs presented in the inlet boxes. were diluted 2-fold (500, 250, 125, 62.5, and 31.3 μg/mL) with growth medium and then added to the cells. After a 24 h treatment, cells were incubated with 40 μL of 2 mg/mL MTT (Duchefa Biochemie, Haarlem, The Netherlands) for 2 h in order to evaluate the cell viability. The supernatant was then removed, and the purple formazan precipitate was solubilized with 100 μL DMSO (Daejung Chemicals, Gyeonggi-do, South Korea) in each well of the 96-well plates. Optical density was measured at 570 nm using a SpectraMax i3x ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

at a desired concentration to investigate surface-enhanced Raman effects of AuNPs and NDAuNPs nanocomposites. Morphology of NDs, AuNPs, and NDAuNP nanocomposites was observed using field emission transmission electron microscopy (FE-TEM, 200kV, JEM2100f, JEOL, Tokyo, Japan). The diluted dispersion was dropped on the lacy carbon grid and dried overnight at room temperature. Measurement of Photoacoustic Activity. PA and ultrasound (US) images were acquired with a high frequency US and PA imaging system (Vevo2100 LAZR, FUJIFILM VisualSonics, Inc., Ontario, Canada) equipped with a linear array transducer (LZ-550, 32−55 MHz center frequency linear array with integrated light source). The nanosecond laser pulses were aligned with integrated fiber-optic transducers. As NDAuNP nanocomposites absorbed the light, acoustic pressure waves were generated and detected by 256 sensitive piezoelectric elements. The transmitted US signal was similarly received and used to acquire microscopic US images. The spatial area of the photoacoustic images was 15 mm (width) by 10 mm (depth). A phantom mold casted with 5% agar gel or a silicone thin tube (iXAK, R300−0151, ID 1.5 mm, OD 2 mm) was used to load NDAuNPs, NDs, and AuNPs dispersion samples. Clear gel was centrifuged to remove air bubbles and was added on the mold to link with the PA probe. The PA spectra were obtained from 680 to 830 nm, with a PA signal gain of 40 dB. All laser energies were applied below the American National Standards Institute (ANSI) safe exposure level for human skin. PA images were analyzed using postprocessing software tools (FUJIFILM VisualSonics, Inc.). Cell Culture and Cell Viability Assay. C2C12 (mouse myoblast cell line) and A549 (adenocarcinomic human alveolar basal epithelial cell line) were obtained from the American Type Culture Collection (ATCC). Cells were maintained at 37 °C in a humidified incubator under 5% CO2 in a complete growth medium: Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, GE Healthcare, Little Chalfont, UK) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 2 mM L-glutamine, and 1% penicillin/ streptomycin antibiotics (Life Technologies). Cells were seeded in each well of 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) and settled overnight. NDAuNPs, ND-COOH, and control

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RESULTS AND DISCUSSION The chemical structures of NDs, AuNPs, and NDAuNP nanocomposites were investigated using X-ray photoelectron spectroscopy (XPS), as shown in Figure S1. The C 1s spectra were deconvoluted to prove the complex formation of the AuNPs and oxygenated carbonic components on the ND surface (Figure S1a, S1d). The COOH peak at 289.0 eV was decayed after the reduction and AuNP deposition.38 The disappearance of the COOH peak on NDAuNPs indicates that the carboxyl-terminated ND surfaces were exposed to the reduction, resulting in a hydroxylated surface, as were Au3+ ions, resulting in deposition of AuNPs on the ND surface. The ionic complex with the Au3+ ion−carboxyl group on the ND graphitic shell layer might provide nucleation sites for the growth of AuNPs. After reduction and composite formation of NDAuNPs, the binding energy of the sp2 C−C peak was shifted from 283.3 eV to a higher binding energy of 283.8 eV. The shift to a higher binding energy generally represents that the electron-rich AuNPs on the ND surface facilitate the electron transfer into the graphitic shell layer of the ND, resulting in the increased electron mobility between the ND and metal interface.39,40 The electron mobility contributes to the localized plasmonic effect around AuNPs on ND agglutinates, which can further enhance the photoacoustic signal. 8286


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Figure 2. Physicochemical characterization of NDAuNPs: (a) hydrodynamic size and zeta potentials, (b) UV absorbance, and (c) comparative XRD patterns.

were 5.3 nm. The distribution of AuNPs only also showed more narrow distribution (inlet box of Figure 1c). Because gold seed and growth was randomly formed on the carboxylate nanodiamond surface by fast reduction, AuNP on ND with relatively broad distribution was produced, compared to bare AuNPs. Magnified images of AuNPs on ND showed gold crystalline structures with a 0.24 nm distance and aligned crystalline distance angle of 80°, indicating that AuNPs were formed with a (111) facet structure via fast reduction. As a control, AuNPs were also prepared using a seed growth method.37 Surfactant-free NDAuNP nanocomposites showed similar size distribution and structure to that of AuNPs prepared by CTAB surfactant-mediated seed and growth methods (Figure 1c). Physical properties of NDs, AuNPs, and NDAuNPs were characterized by hydrodynamic diameter, zeta potentials, and crystalline structure (Figure 2). The sizes for NDs, AuNPs, and NDAuNPs in aqueous dispersion were measured as 56 ± 10, 72 ± 5, and 105 ± 8 nm, respectively (Figure 2a) and distributions are presented in Figure S2. Though nanodiamond/gold composites do not have complete spherical forms, the usual decay of the autocorrelation function was used and showed the coincidence with geometrical size seen by TEM. Hydrodynamic diameters of NDs and NDAuNPs measured by DLS were coincident, while the hydrodynamic diameter of AuNP presents a bigger size than geometric diameter as seen in TEM. Since the CTAB-mediated surfactant layer of AuNPs was not seen due to a contrast limit of TEM and because the positive charge of CTAP on gold might induce mild aggregation of AuNPs, the bigger hydrodynamic diameter of AuNPs was observed. The zeta potentials for NDs, AuNPs, and NDAuNPs were −49.3 ± 3.1, + 57.1 ± 13.3, and −48.1 ± 2.0 mV, respectively (Figure 2b). UV peak absorbance was 530 nm both for AuNPs and NDAuNPs. The UV absorbance of NDAuNP nanocomposites was broadened, compared with that of AuNPs, while its peak position was not changed. AuNPs possessing similar optical properties to NDAuNPs were used as a control for further PA signal activity. The X-ray diffraction pattern of AuNPs and NDAuNPs showed diffraction peaks with four sharp characteristics at 2θ = 38.1, 44.4, 64.7, and 77.7 (Figure 2c). These polycrystalline gold peaks were, respectively, indexed as the (111), (200), (220), and (311) lattice planes of gold crystals with the standard face-centered cubic phase of metallic gold.42,43 As a control, the diffraction peaks of NDs showed at one dominant peak at 2θ = 44.0 and 76.0, indexed as the (111) and (220) lattice planes of the nanodiamond crystalline structure. The relatively broad peak of ND is probably due to the graphitic-like shell around the ND surface that exists with a different lattice distance. The Au (111) peak showed the

The O 1s peak for NDs and NDAuNPs were deconvoluted to 531.5 eV (CO) and 532.9 eV (C−O−C/C−OH) (Figure S1b, S1e). The O 1s peak of NDs also showed shoulder due to the peak attributed to the carboxylic group at 531.5 eV. The NDAuNPs binding energy at 531.5 eV presented weak intensity, and the peak at 532.9 eV was strengthened, compared with that of the carboxyl group-rich NDs. This result clarified that carboxyl groups on the ND surface were changed into a hydroxyl group as the NDAuNP nanocomposite was formed under the process of gold reduction. The binding energy survey of AuNPs and NDAuNPs was characterized by the doublet of two spin−orbit components. The binding energy of AuNP and NDAuNPs was attributed to the peaks of Au 4f7/2 and 4f5/2. Au 4f peaks appeared at 83.4 and 86.9 eV, providing evidence of AuNP formation (Figure S1c). The low binding energy of the respective Au 4f7/2 and 4f5/2 peaks at 83.4 and 86.9 eV was attributed to the Au0 metallic state of AuNPs. Higher energy binding for AuNPs at 84.6 and 88.2 eV indicates the presence of the Au+ ionic oxidative state. A higher phase ratio of metallic Au0 to oxidative Au+ ion (83.4 to 84.6 eV) of NDAuNPs was observed, compared with that of AuNPs, indicating that the metallic state of the AuNPs was more distributed on the ND surface platform (Figure S1f). The results indicate that NDAuNPs existed in a much more stable form than AuNPs only. The hypothesis to explain the coexistence of metallic Au0 and oxidative Au+ ion suggested that a positive Au+ ion with low electron density provides electron mobility to the adjacent site.41 NDAuNP composites include both gold metallic atoms and ion on gold islands of ND agglutinates. This local nanoscaled distribution of AuNPs on the ND suggests that the ND substrate provides a holding platform of Au+ ions, together with crystalline Au metallic particles, further affecting the plasmonic effect and the photoacoustic activity of NDAuNP composites. The morphology of the NDAuNP nanocomposites was observed by TEM images. Nanodiamonds with sizes of 4−5 nm existed as agglutinates, not individual particles. The agglutinated ND-COOH was well distributed with a size of 50 nm and an irregular shape, which allows its use as a substrate for smaller AuNPs (Figure 1a). In the magnified images, the nanodiamond crystalline structure was observed with a carbon distance of 0.2 nm. After gold deposition on the ND surface, the size of the NDAuNP composite increased to 104.8 nm. The geometric size of AuNPs on the ND surface and AuNPs only were measured by counting 130 AuNPs from TEM images, and its distribution is presented in the inlet box. The AuNP size on the ND surface ranged from 2 to 27 nm, showing homogeneous distribution (inlet box of Figure 1b). The size of AuNPs on the ND surface was 7.6 nm, while the diameters of AuNPs only 8287


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MB did not appear at 450 and 1620 cm−1. When the same 10−4 and 10−5 M concentrations of MB were doped on the AuNPs and NDAuNPs, the surface-enhanced Raman scattering showed clear amplified intensity at 450 and 1620 cm−1. When the MB concentration was increased from 10−5 to 10−4 M, the enhanced intensity ratios I450/I520 and I1620/I520 increased for both AuNPs and NDAuNPs. In particular, SERS of NDAuNPs was significantly enhanced, showing increased intensity ratios of 13.6 (I450/I520) and 9.3 (I450/I520) at the 10−4 M MB concentration, compared with that of AuNPs. Intensity ratios at (I450/I520) and (I450/I520) were significantly increased by 20.4 and 33.6 times, respectively, compared with those of AuNP at the MB concentration of 10−4 M. This result indicates that the ND substrate absorbed the light energy and diffused heat into gold nanoparticles, augmenting the local plasmonic effects. Chemical conjugation on ND and localized form of gold ion on AuNPs might provide the developed SERS effect compared with AuNPs alone. Moreover, locally distributed AuNPs on ND substrates offer high local AuNP concentrations in dispersion, generating boosted SERS. Spectral photoacoustic activity of AuNPs and NDAuNPs was characterized as the laser was tuned from 680 to 980 nm. After the sample solution (100 μL) was injected into chicken breast muscle and the transducer was focused on the loci, the images were acquired at the spectral PA mode every 3 nm (Figure 4d). When a PA spectrum was read for a selected image area, the peak intensity of the spectrum appeared at 701 or 705 nm for AuNPs and NDAuNPs, respectively (Figure 4a). As the laser wavelength moved to the NIR region, PA activity significantly decayed for both AuNPs and NDAuNPs because the UV light absorption peak mainly occurred at 530 nm. Notably, PA intensity of NDAuNPs was decayed more gradually compared with that of AuNPs. Spectral intensity of NDAuNPs also showed constantly higher PA intensity in the NIR range from 700 to 950 nm. For a quantitative optical PA indicator, PA signal intensity was measured as the concentration of AuNP and NDAuNP dispersion increased (0.01−1 mg/mL) (Figure 4b). The PA intensity of both NDAuNPs and AuNPs linearly increased with the concentration. Particularly, the PA signal of NDAuNPs was sustained as IOD 22.9% at the low concentration of 0.02 mg/mL, while AuNPs at the same concentration showed background noise level. PA intensity of NDAuNPs was higher in the measured concentration range (from 0.01 to 1 mg/mL) than that of AuNPs. This result is coincident with the Raman spectra, showing enhanced local plasmonic effects of NDAuNPs. The composite with nanodiamond and AuNPs enhanced the PA-active signal, probably due to enhanced local plasmonic effects. Energy-absorbing nanodiamond might transfer energy and heat onto chemically conjugated AuNPs. The oxidate state of gold ions adjacent to metallic Au may also facilitate energy transfer from nanodiamond to gold. Moreover, localized distribution of AuNPs on the ND agglutinate substrate resulted in amplified PA signal intensity due to local high concentration of AuNPs, compared with that of dispersed AuNPs with the same concentration in solution. The visualized PA-active images are shown for NDAuNPs and AuNPs injected into the chicken breast muscle in Figure 4c. The PA intensity of both NDAuNPs and AuNPs (0.5 mg/ mL, 100 μL) injected into chicken breast muscle was monitored under pulsed laser exposure for 80 min (Figure 5). The PA spectra of AuNPs over tuned laser wavelengths were significantly decayed after 80 min of laser irradiation.

highest intensity compared with that of the Au (200) and Au (220) peaks because the major Au crystalline structure is attributed to the Au (111) facet. Crystalline AuNPs on an ND substrate were formed by the process of fast nucleation−growth of Au3+ ions on the carboxylate ND substrate surface using the strong reducing agent NaBH4. The noisy background between 20° and 30° and strong background at a range below 15° are also indicative of the presence of amorphous carbon in the ND powder.44 In this study, nanodiamond with pure carbon crystalline without an N vacancy site was used for the AuNP nanocomposite. Nanodiamond with an N-vacancy site within a crystalline structure has properties of fluorescence and photoluminescence. Individual fluorescence nanodiamonds have relatively large sizes from 100 to 200 nm, compared to normal nanodiamond with a 5 nm size in agglutinate form. Nanodiamonds dependent on surface functionality, size, and the presence of an N-vacancy site would have, respectively, unique optical properties and chemical functionality, resulting in versatility of the nanocomposite. Though intrinsic elements of carbon nanodiamond and gold are the same, resulting applications and properties can depend on the characteristic nanodiamonds and methods of complex formation, such as fluorescence nanodiamond with N-vacancy site/AuNPs complexes constructed via electrostatic interaction or simple protein absorption of DNA36 or bovine serum protein.45 In this study, carboxylate nanodiamond with a high purity functional substrate for AuNPs produced nanocomposites, using strong reductant NaBH4, where AuNPs were seeded and grown on ND surfaces. Surface-enhanced Raman scattering spectra of AuNPs and NDAuNPs were obtained for the concentrations of 10−4 and 10−5 M of methylene blue (MB) as a Raman reporter (Figure 3). The characteristic peak of MB powder appeared at 450 and 1620 cm−1 indicating C−N−C skeletal bending and C−C stretching, respectively.46 The effect of SERS was analyzed as intensity ratios of I450/I520 and I1620/I520, where intensity at 520 cm−1 was assigned to the silicone substrate as a control. At the 10−4 and 10−5 M concentrations of MB, the Raman intensity of

Figure 3. Surface-enhanced Raman spectra of methylene blue, AuNPs, and NDAuNPs. Methylene blue was used as a Raman reporter and was mixed with NDAuNPs and AuNPs in DI water (1 mg/mL) at various concentrations, allowing adsorption of MB onto AuNPs and NDAuNPs. 8288


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Figure 4. Photoacoustic intensity of NDAuNPs and AuNPs at various concentrations. (a) Schematic of nanodiamond/gold nanocomposites and local plasmonic enhanced PA activity. (b) Photoacoustic spectra from laser wavelength of 680 to 980 nm. (c) Photoacoustic intensity of optical density analyzed with acquired images of various concentrations from 1 to 0.01 mg/mL. NDAuNPs and AuNPs (100 μL) were injected into chicken breast muscle. (d) Acquired PA images of NDAuNPs and AuNPs at different concentrations.

Figure 5. Photoacoustic intensity of NDAuNPs and AuNPs before and after laser irradiation. (a) Photoacoustic spectra of NDAuNPs and AuNPs before and after 80 min of laser irradiation. The laser was scanned from 680 to 980 nm. (b) Acquired PA images of NDAuNPs and AuNPs at the respective times. (c) Photoacoustic intensity of optical density analyzed from images acquired as the time variance of laser exposure. NDAuNPs and AuNPs (100 μL each) were injected into chicken breast muscle. PA intensity of NDAuNPs was sustained without decay of PA signals under 80 min of laser irradiation.

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Figure 6. Local distribution of AuNPs and NDAuNPs nanocomposites injected into chicken breast muscle tissue. TEM images of before laser irradiation (a, c) and after 80 min laser irradiation (b, d) of AuNPs (a, b) and NDAuNPs (c, d) in chicken breast muscle cells with 0.5 μm and 100 nm scale bar. After laser irradiation for 80 min, the particle boundary of AuNPs within tissue was obscure, indicating degradation of AuNPs.

The cytotoxicity of ND-COOH and NDAuNPs was analyzed using C2C12 mouse myoblast cells and A549 carcinoma cells (Figure 7). The cell viability assay showed that nanodiamond/ gold nanocomposites caused limited toxicity at a high concentration of 250 μg/mL and no cytotoxicity at a low concentration below 62.5 μg/mL. Below the concentration of 125 μg/mL, both C2C12 mouse myoblast cells and A549 carcinoma cells showed cell viability over 80%. The result proves that a high concentration of ND/gold nanocomposites (125 μg/mL) causes low cytotoxicity at the cellular level. Cytotoxicity of nanodiamonds has been demonstrated to insight the potential biomedical usage in previous studies.31,47 Though intrinsic materials, nanodiamonds are substantially similar, and the cytotoxicity of nanodiamonds can be dependent on the purification, size-determining milling process, and surface-dangling functionality. The NDAuNP nanocomposites used in this study was synthesized with surfactant-free methods, indicating that surface functional groups of NDAuNPs were highly hydroxylated by strong reduction and thus nontoxic even at the cellular level. Overall, the ND/AuNP nanocomposites provide sufficient enhanced and sustained longevity of PA signals and images in the chicken breast muscle. Low cell cytotoxicity of ND/AuNP nanocomposites to 125 μg/mL also count potentials in biomedical application of inorganic composites. PA imaging in vivo applications that can image NIR regions require strong differentiable signals within 1 cm shallow tissue depth. Moreover, a PA-reliable signal is required in intramuscular, intratumoral, and intravenous administration under blood circulation. ND/AuNP nanocomposites for PA imaging further need to be evaluated for targeted specific or nonspecific accumulation in vivo.

Photoacoustic intensity of AuNPs after 80 min of laser irradiation was close to the intensity of the background (Figure 5c). It is well known that gold nanoparticles emit heat by laser irradiation and are subsequently degraded, allowing utilization for photothermal therapy and photoacoustic imaging.14 In contrast, PA intensity of NDAuNPs was sustained and slightly increased even after 80 min of laser irradiation. From the PA images, the PA signal active area was slightly increased at 80 min, compared with that of the PA images before irradiation (Figure 5b). PA spectra recorded from 680 to 970 nm showed slightly increased PA intensity over all wavelengths during 80 min of laser irradiation (Figure 5a). The continuous laser irradiation of NDAuNPs caused enhanced signal intensity and increased the signal emitting area, resulting in consistently amplified and sustained PA intensity. This result indicated that ND nanocomposites with AuNPs provided energy absorption and retarded AuNP degradation. Moreover, energy transition into AuNPs offered more effective thermal expansion to amplify PA intensity, coinciding with the results of surface-enhanced Raman scattering. TEM images of tissue showed the morphology of AuNPs and NDAuNPs injected into chicken breast muscle before and after 80 min of laser exposure (Figure 6). The AuNPs were distributed within tissue in aggregates of 100−200 nm because the injection to bloodless tissue makes local distribution of AuNP, not individual AuNP. After laser irradiation, direct visualization of AuNP degradation in crystalline structure of high magnification could not be observed after laser irradiation since the preclinical PA instrument was set to the level safe exposure of human skin and laser power cannot be controlled. However, it is speculated that reduced PA signal during laser exposure for 80 min prolonged time and undetermined boundary of AuNPs in TEM images were resulted from the degradation of AuNPs. In contrast, the morphology of NDAuNPs was not distinctly changed before and after 80 min laser exposure. Dark, irregular forms of NDAuNPs presented in both images before and after laser irradiation without recognizable morphological change.

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CONCLUSION Gold nanoparticles and nanorods have been utilized as the first generation of PA imaging agents and still offer the advantages of inertness and instant signal in vivo. Our study proposed that 8290



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01944. Information as mentioned in the text. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Jae Young. Tel: 82-2-2072-3073. E-mail: [email protected]. *Eunah Kang. Tel: 82)-2-820-6684. E-mail: [email protected]. kr. ORCID

Eunah Kang: 0000-0001-9847-1246 Author Contributions +

Dukhee Lee and Eun-Joo Park contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1C1A2A01053307).

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Figure 7. Cell viability of NDAuNPs and NDs. C2C12 myoblast and A549 lung carcinoma cells were used to examine cytotoxicity of NDAuNPs and NDs. The concentration of NDAuNP and ND-COOH were diluted from 250 to 15.6 μg/mL, and cells were exposed to nanocomposites for 24 h. The same volume of PBS was used as the control.

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