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Intraocular application of gold nanodisks optically tuned for optical coherence tomography: inhibitory effect on retinal neovascularization without unbearable toxicity
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 13 (2017) 1901 – 1911
Original Article
nanomedjournal.com
Intraocular application of gold nanodisks optically tuned for optical coherence tomography: inhibitory effect on retinal neovascularization without unbearable toxicity Hyun Beom Song, MD a, b, 1 , Jung-Sub Wi, PhD c, 1 , Dong Hyun Jo, MD a, b , Jin Hyoung Kim, PhD a , Sang-Won Lee, PhD c , Tae Geol Lee, PhD c,⁎, Jeong Hun Kim, MD, PhD a, b, d,⁎⁎ a
Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea b Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Republic of Korea c Center for Nano-Bio Measurement, World Class Laboratory, Korea Research Institute of Standards and Science, Daejeon, Republic of Korea d Department of Ophthalmology, College of Medicine, Seoul National University, Seoul, Republic of Korea Received 17 October 2016; accepted 26 March 2017
Abstract Bare gold nanospheres have been shown to have anti-angiogenic effects but are optically unfavorable because their resonant wavelength lies in the visible spectrum. Here, we design gold nanodisks with a higher scattering capability than gold nanorods and with a resonant wavelength at near-infrared region – the area where the source of light utilized by optical coherence tomography (OCT) lies. With a physical synthesis system, we then fabricate 160-nm-sized gold nanodisks exhibiting resonant wavelength at 830 nm. The synthesized nanoparticles were successfully visualized in in vivo OCT at concentrations as low as 1 pM. After demonstrating their binding ability to vascular endothelial growth factor (VEGF), we show that they suppress VEGF-induced migration of endothelial cells. Finally, we demonstrate that intravitreally injected gold nanodisks attenuate neovascularization of oxygen-induced retinopathy in mice, in a dose dependent manner, such that they are cleared from the vitreous within 2 weeks without histologic or electrophysiologic toxicity. © 2017 Elsevier Inc. All rights reserved. Key words: Gold nanodisk; Near-infrared; Optical coherence tomography; Intraocular application; Angiogenesis inhibitor; Retinal neovascularization
Nanoparticles have been widely investigated in the field of imaging as well as treatment. Moreover, advances in nanotechnology have allowed the fabrication of nanoparticles with both diagnostic and therapeutic capabilities by mounting therapeutic functions onto nanoparticles utilized as imaging agents. 1 The reverse approach is considered to be undesirable because mounting diagnostic functions can negatively affect the
pre-existing therapeutic functions of the nanoparticles. However, the self-therapeutic properties 2–4 of bare gold nanoparticles (GNPs) combined with the optical properties of gold with its strong surface plasmon resonance could cater to both approaches. In this study, we designed bare GNPs with self-therapeutic properties to include favorable optical properties for ocular imaging.
Conflict of interest: None of the authors have any competing interests. This study was supported by the Pioneer Research Program of NRF/MEST (2012-0009544); the Bio & Medical Technology Development Program of the NRF funded by the Korean government; MSIP (NRF-2015M3A9E6028949); research grant from NRF/MEST, Republic of Korea (2014M3A7B6027946); and the Bio & Medical Technology Development Program of the NRF funded by the Ministry of Science, ICT & Future Planning (NRF-2015M3A9D7029894). ⁎Correspondence to: T.G. Lee, Center for Nano-Bio Convergence, World Class Laboratory, Korea Research Institute of Standards and Science, Daejeon 305-340, Republic of Korea. ⁎⁎Correspondence to: J.H. Kim, Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Biomedical Research Institute, Seoul National University Hospital, and Department of Ophthalmology, College of Medicine, Seoul National University, 28 Yongon-dong Chongno-gu, Seoul 110-744, Republic of Korea. E-mail addresses: [email protected] (T.G. Lee), [email protected] (J.H. Kim). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.nano.2017.03.016 1549-9634/© 2017 Elsevier Inc. All rights reserved. Please cite this article as: Song HB, et al, Intraocular application of gold nanodisks optically tuned for optical coherence tomography: inhibitory effect on retinal neovascularization without unbearable toxicity. Nanomedicine: NBM 2017;13:1901-1911, http://dx.doi.org/10.1016/j.nano.2017.03.016
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To design the appropriate GNPs, the shape, size and surface charge were considered. We have previously suggested the importance of these physicochemical properties of nanoparticles in determining the therapeutic outcome, 5 but they also play important roles in the optical properties, self-therapeutic properties and safety of the GNPs. The optical properties arise from the strong surface plasmon resonance of gold which helps to enhance the scattering intensity. 6 The scattering intensities and plasmon wavelength maximum are influenced by the shape and size of the nanoparticles. 7 The plasmon wavelength maximum of the gold nanospheres lies from 520 nm to 550 nm – a range that is too limited to be applicable for in vivo imaging. Among nanoparticles that are optically tunable by changing the size or aspect ratio, gold nanodisks (GNDs) are optically more favorable than gold nanorods (GNRs) because the latter possess directional and polarization dependencies. 8 In other words, GNDs can produce reliable signals regardless of the incident angle or polarized directions of the light source. The size and surface charge can influence the self-therapeutic activities of the GNPs. Bare GNPs are widely considered to be therapeutic agents that suppress angiogenesis because they inhibit in vitro vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation and in vivo VEGF-induced angiogenesis. 9 The mechanism behind the anti-angiogenic effect lies in their binding affinity to VEGFs, 9,10 and further evaluation has revealed that large GNPs bind more readily to VEGFs while inhibiting phosphorylation of VEGF receptors and endothelial cell proliferation at a lower concentration than do small GNPs. 10 However, because coating the surface with different charges masks the inhibitory effect of the GNPs on VEGF function, 10 larger GNPs with naked surfaces are recommended for greater effectiveness. The size, shape and surface can affect the toxicity of the GNPs; the toxicity is linked to cellular uptake levels and coating, 11 while in turn, the cellular uptake of GNPs correspond to their size and shape. 5 For spherical GNPs, cellular uptake is highest at 50 nm and decreases as their size increases. 12 When compared to spherical GNPs, GNRs showed reduced cellular uptake that further decreased with a higher aspect ratio. 12 And, because nanodisks also rarely enter cells and do not induce reactive oxygen species, apoptosis or cell cycle perturbation, 13 in this paper, we utilized anisotropic nanoparticles such as nanorods or nanodisks, as opposed to spherical nanoparticles. In terms of coating, top-down synthesis and a bare surface can avoid unwanted chemical coating on the surface. Furthermore, the surface charge is important for diffusion in biological fluids, and gold's advantages in terms of toxicity are its biocompatibility and immunological inertness. After considering the factors that influence optical properties, anti-angiogenic activities and toxicity, we decided on large GNDs with bare surfaces for further evaluation. First, we simulated the optical properties of GNDs of various diameters; after considering the scattering properties and ranges of their resonant wavelengths, we selected 160-nm-sized GNDs to use in the eye. After the physical synthesis of the GNDs, we applied them to the eye and observed strong signals in optical coherence tomography (OCT). An evaluation of the anti-angiogenic effect of the fabricated GNDs demonstrated that GNDs could inhibit in vitro angiogenesis without cellular toxicity and could inhibit retinal neovascularization without significant toxicity when applied to a mouse oxygen-induced retinopathy model.
Methods Electromagnetic simulation Three-dimensional finite-difference time-domain software (Lumerical FDTD Solution 8.9) was utilized to perform electromagnetic simulations. Under illumination from the top with a linearly polarized plane wave, the scattering and absorption cross sections were calculated on particles in a uniform dielectric medium (n = 1.33), with the simulation grid set to a 1.0 nm cubic. Nanoparticle fabrication and characterization The nanoparticles were prepared by top-down synthesis as described previously 14,15 and in Supplementary Methods. The morphology of the gold nanodisks was observed via scanning electron microscopy (SEM, FEI XL30 Sirion) after drying the aliquots of stock solution on a Si substrate. The images were analyzed with the imageJ program (NIH) to measure the average diameter. The zeta potential of the gold nanodisks in distilled water was evaluated with a zeta potential analyzer (Zetasizer Nano-Z, Malvern, UK). Mono-dispersed nanodisks of various concentrations (1, 3 or 10 pM) were prepared in EBM with 0.1% FBS. After incubation for 24 h, UV–Vis measurements (UV-2600, Shimadzu, Kyoto, Japan) were conducted. Changes in absorbance and shift in λmax were evaluated. Optical coherence tomography After the mice were anesthetized and their pupils were dilated, the eyes were positioned under the previously described spectral domain OCT system. 16 The system utilized a superluminescence diode with a center wavelength at 849 nm. After the retinal scans were taken, 3D images were reconstructed from the cross-sectional B-scan images. Then, the same 3D volumes that corresponded to the vitreous cavity were projected to the x-y plane; we then produced a 2D projection image to compare the concentrations of particles in the vitreous. All images were processed with Amira software (FEI, Hillsboro, USA). Wound migration assay Wound migration assay was performed as in our previous descriptions. 17 Human retinal microvascular endothelial cells (HRMECs) were seeded on gelatin-coated 35-mm dishes and incubated to achieve more than 90% confluence. After overnight starvation with basal medium containing 0.1% FBS, the cell layers were wounded with a pipette tip. Then, the medium was replaced with 20 ng/mL VEGF (Sigma–Aldrich, St. Louis, USA) or GNDs at concentrations of 1 pM or 3 pM. The image of the wound was obtained under a light microscope immediately after the scratch to set the reference line. The medium was removed 12 h later and the cells were fixed with absolute methanol and stained with Giemsa's solution (BDH Laboratory Supplies, London, United Kingdom). Migration was quantified by counting the migrated cells across the reference line under a light microscope at ×100 (Leica, Wetzlar, Germany).
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Cell viability assay Cell viability was evaluated by WST-1 assay using the EZ-Cytox Cell Viability Assay kit (Itsbio, Seoul, Korea) according to the manufacturer's instructions. Briefly, HRMECs were plated in 96-well plates at a density of 6 × 10 3 cells per well. After overnight incubation, the cells were treated with GNDs at various concentrations (10 0, 10 1,10 2,10 3 or 10 4 nanoparticles per cell) and incubated for 48 h. After evaluation under a light microscope, EZ-Cytox reagents were added to each well. After 2 h of additional incubation, the plates were analyzed by the microplate reader to measure the absorbance at 450 nm. Measurement of binding between nanodisks and VEGF Two different concentrations of nanodisks (1 or 3 pM) were incubated in phenol free EBM (Lonza) supplemented with VEGF (20 ng/mL) and 0.1% FBS. After incubation overnight, centrifugation was performed to separate the nanodisk-bound VEGF. Then, the concentrations of unbound VEGF were measured in the supernatant using a VEGF Human ELISA Kit (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. Animal C57BL/6 J mice obtained from Central Lab Animals Incorporation (Seoul, Korea) were kept in alternate dark–light cycles of 12 h at room temperature and served with food and water ad libitum. The care, use, and interventions were approved by the Institutional Animal Care and Use Committee of Seoul National University and were in agreement with the ARVO statement for the use of animals in ophthalmic and vision research.
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a magnification of ×40. The avascular area and neovascularization were measured with Adobe Photoshop CS6 (San Jose, USA) as described elsewhere. 18 In vivo toxicity evaluations: Histology, TUNEL assay and electroretinogram To assess in vivo toxicity, 6-week-old C57BL/6 J mice were intravitreally injected with 1 μL of DW (n = 6) or 10 pM solution of GNDs (n = 6). Five weeks after the injection, the electroretinogram (ERG) was recorded using the UTAS Visual Electrodiagnostic System (LKC Technologies, Gaithersburg, USA). The detailed protocol is described in Supplementary Methods. The animals were then sacrificed and, and the eyes were enucleated, fixed in 4% paraformaldehyde overnight and embedded in paraffin. Sagittal sections of 4-μm-thicknesses were prepared, stained with hematoxylin and eosin and evaluated under a light microscope (Ni-U; Nikon). As previously described, the ratio of A (the retinal thickness from the internal limiting membrane to the inner nuclear layer) to B (the retinal thickness from the internal limiting membrane to the outer nuclear layer) was measured and compared with the control group. 19,20 For the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, at least 10 sections from each eye (at least five from each side of the optic nerve) were stained with the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics, Indianapolis, USA) according to the manufacturer's instructions. The sections were then observed with a fluorescence microscope (Ni-U; Nikon) at a magnification of ×400 and TUNEL-positive cells were counted per section. Statistics
Oxygen-induced retinopathy Oxygen-induced retinopathy was induced as described previously. 3 Briefly, newborn mice were randomly assigned to experimental (n = 18) and control (n = 12) groups. At postnatal day 7 (P7), the mice were exposed to hyperoxia (75 ± 0.5% oxygen) for 5 days from P7 to P12 and then returned to normoxia (room air). The oxygen concentration level was measured twice daily with an oxygen analyzer (Miniox I; Bertocchi srl Elettromedicali, Cremona, Italy). To assess the anti-angiogenic properties of the GNDs, the pups were injected intravitreally with 0.5 μL of DW or 1 pM and 3 pM solutions of GNDs at P14. The mice were sacrificed by inhalation of CO2 at P17 to evaluate retinal neovascularization and toxicity. In another set of mice (n = 12), oxygen-induced retinopathy was induced and the pups were injected intravitreally with DW or GNDs in the same manner as mentioned above. At P15, one day after the injection, the mice were sacrificed and vitreous-retina complexes were prepared for the measurement of mouse VEGF using an ELISA kit (R&D systems, Minneapolis, USA). Assessment of retinal neovascularization After fixation in 4% paraformaldehyde for 1.5 h, the enucleated eyes were dissected, and the isolated retinas were subjected to immunostaining with the anti isolectin-B4 antibody (GSA-IB4; Invitrogen) and flat-mounted. The images were obtained using a fluorescence microscope (Ni-U; Nikon, Tokyo, Japan) at
The differences between the groups were evaluated with the Mann–Whitney U-test using SPSS 19.0 (SPSS Inc., Chicago, USA). P-values less than 0.05 were considered to be statistically significant.
Results Design of optimal gold nanoparticles for ocular imaging We first simulated the optical properties of the GNDs to make fine adjustments to the design of the GNDs to make them suitable for ocular imaging. Electromagnetic simulations of 20 nm thick GNDs with diameters from 40 nm to 200 nm revealed a shift in the resonant wavelength to the infrared region, enabling the imaging of deeper tissues due to better penetration. 21 In addition, an increased diameter caused scattering to also increase (Figure 1, A), so that these larger GNDs could be detected at lower concentrations, unlike smaller GNDs. Unfortunately, large GNDs with resonant wavelengths larger than 900 nm are difficult to practically apply because light in the region can be absorbed by the abundance of water 22 throughout the vitreous that covers the retina, a common target of imaging in ophthalmology. Wavelengths adjacent to 787 nm also need to be avoided because the light source could produce autofluorescence of the melanin pigment in the retinal pigment epithelium. 23 Furthermore, a widely used imaging method in ophthalmology, OCT, employs a broadband source of
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Figure 1. Finite-difference time-domain simulation of gold nanoparticles. (A) 20-nm-thick single gold nanodisks with diameters of 40, 80, 120, 160, and 200 nm were evaluated. The refractive index of the medium was assumed to be 1.33. Calculated cross sections of absorption, scattering and extinction were plotted according to the disk diameters and corresponding resonant wavelengths. (B) Gold nanoparticles resonating at near-infrared wavelengths were evaluated: sphere with diameter of 200 nm, disk with diameter of 160 nm and thickness of 20 nm, and rod with length of 70 nm and diameter of 20 nm.
light centered at wavelengths between 800 nm and 850 nm, the region where the resonant wavelength of GNDs with diameters of 160 nm lies. Taking into consideration these factors, in this study we used 160 nm GNDs with a resonant wavelength at 830 nm. To compare the optical characteristics according to the dimensions of the plasmon nanostructures, we simulated GNPs with the same resonant wavelength at 830 nm but of different shapes (Figure 1, B). As the backscattered light produced signals in OCT, the scattering cross sections were then compared. Higher scattering was observed for gold nanospheres (diameter 200 nm) and GNDs (diameter 160 nm, thickness 20 nm) than for GNRs (length 70 nm, diameter 20 nm). Most of the extinction cross sections consisted of scattering cross sections in gold nanospheres and GNDs and absorption cross sections in the GNRs. Although the scattering cross section of GNR grew as its geometrical size increased, as shown in Supplementary Figure 3, light-absorption is more dominant than the scattering for GNRs of typical sizes and shapes. 7,24,25 The simulated 200-nm-sized gold nanospheres were about 10 times heavier than the 160-nm-sized GNDs, so less mass concentration was required to produce comparable signals in OCT when the particles were manipulated into the shape of disks. Preparation and characterization of the gold nanodisks To prepare monodisperse bare GNDs, a top-down synthesis described previously 14,26 was applied (Figure 2, A). This physical synthesis allowed a great deal of flexibility in designing the nanoparticles, so GNDs of various diameters could be prepared simply by adjusting the etching times (Supplementary Table 1). We used an etching time of 40 s to prepare GNDs with diameters of 162 ± 3.3 nm and heights of 20 nm. All the particles were uniform in shape and size according to SEM (Figure 2, B). The zeta potential was estimated to be −23.4 ± 9.3 mV, which is comparable to bare gold nanoparticles. 27 When these charged particles were injected into the vitreous, the anionic particles diffused more easily than the cationic particles 28
because the overall anionic charge of the vitreous can disturb the diffusion of the cationic particles. 29 Hence, a bare surface is not only more conducive to an anti-angiogenic effect 10 but also allows for better diffusion in the eyes. The aggregation status was also evaluated at various concentrations since aggregation of gold nanoparticles can reduce the surface area of the VEGF binding sites. 10 Even after GNDs with concentrations of up to 10 pM were incubated for 24 h in an environment where further in vitro experiments were conducted, UV–Vis measurements did not show any of the changes in absorbance patterns and resonant wavelengths that aggregation of particles can make (Figure 2, C). 30 Optical properties of the gold nanodisks The synthesized nanoparticles were visualized in the OCT system we previously described. 16 To compare their signal intensities, gold nanospheres and GNDs with the same resonant wavelength at 830 nm were prepared in aqueous solutions. As simulated in Figure 1, B, the same number of particles produced almost similar signal intensities in OCT (Figure 3, A). On the other hand, a solution of GNDs containing about 50 ppm of gold produced a much stronger signal than gold nanospheres with the same mass concentration of gold (Figure 3, A). The results were consistent with the data from the simulation above. Mice vitreous bodies were then imaged after intravitreal injections of GNDs with diameters of 160 nm. Eyes injected with distilled water or 0.1 pM of GNDs showed a minimal OCT signal, but those injected with 1 pM of GNDs produced a significant OCT signal. Intravitreal injections of 10 pM of GNDs produced a much higher signal in both cross-sectional and projectional OCT images (Figure 3, B and Videos 1–4). Ability of gold nanodisks to bind to VEGF and inhibit in vitro angiogenesis without cellular toxicity Before the in vivo application of GNDs on the retinal neovascularization model of a mouse, we evaluated the degree to
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Figure 2. Preparation and characterization of GNDs. (A) Schematic illustration of the overall process in this study: nanoimprint lithography (NIL) using a truncated nanocone array, O2 plasma etching of residual PMMA, wet chemical dissolution of PMGI, vacuum deposition of gold, gold lift-off, nanodisk release, and centrifugation. (B) Scanning electron microscopy images of GNDs fabricated with an etching time of 40 sec. (C) UV–Vis measurements after 24 h incubation of nanodisks at concentrations of 1 pM (red), 3 pM (green) and 10 pM (blue) in EBM with 0.1% FBS. Scale bar =1 μm.
which the GNDs bind to VEGF, a key regulator of angiogenesis that induces the proliferation and migration of endothelial cells, which contributes to the formation of new blood vessels. 31 The GND concentration levels used to produce the anti-angiogenic effect were based on our previous work, which found that 20-nm-sized nanospheres demonstrated anti-angiogenic effects both in vitro and in vivo at concentrations of 1 μM 3 and at even lower concentrations of 16.64 pM. 20 Smaller spheres with diameters of 5 nm inhibited VEGF-induced proliferation and phosphorylation of VEGF receptors at 1 nM where they were able to bind to approximately 8 ng/mL of VEGF. 10 To determine the minimal concentration level that could exert an anti-angiogenic effect for 160-nm-sized nanodisks, we calculated that a surface area, 640 times larger than the 5-nm-sized nanospheres, yielded 1.56 pM as a comparable concentration level for the GNDs. As a result, we began our investigation into the anti-angiogenic effect of synthesized GNDs at concentrations of 1 pM and 3 pM. These levels lie above the minimal detectable concentration level, according to Figure 3, A. After determining the concentrations to be used, we used an ELISA test to determine the concentration of bound VEGF by measuring the unbound VEGF remaining in the solution after
separation from the nanoparticles as previously described. 10 1 pM of GNDs were bound to 2.26 ± 0.13 ng/mL of VEGF and 3 pM of GNDs were bound to 5.44 ± 0.42 ng/mL of VEGF (Figure 4, A). Using these concentrations, in vitro angiogenesis assays were performed to evaluate the anti-angiogenic effect of the GNDs. Previously, we demonstrated that various nanoparticles inhibit VEGF-induced migration of endothelial cells. 3,20 As shown in Figure 4, B and C, GNDs inhibited VEGF-induced migration of HRMECs at a concentration of 3 pM but were less effective at 1 pM. At a concentration of 3 pM, which accounts for about 240 particles per cell seeded, the cells showed no aberrant changes in morphology that would indicate the toxic effects of the GNDs. At higher concentrations up to 10 4 per cell, the GNDs did not decrease the viability of HRMECs even when the incubation time was extended to 48 h (Figure 4, D). Ability of gold nanodisks to inhibit in vivo angiogenesis without histologic and electrophysiologic toxicity We further evaluated the effect of GNDs on angiogenesis in vivo. We previously demonstrated that intravitreally injected
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Figure 3. Optical properties of the GNDs in optical coherence tomography (OCT). (A) Cross-sectional OCT images were obtained from a 1 pM solution of 200-nm-sized gold nanospheres (left), a 1 pM solution of 160-nm-sized GNDs (center) and a 10.4 pM solution of 160-nm-sized GNDs (right), which correspond to the mass concentration of a 1 pM solution of 200-nm-sized gold nanospheres. (B) Cross-sectional OCT images (upper) were obtained from the eye of a mouse immediately after an intravitreal injection of distilled water and a solution of 160-nm-sized GNDs (0.1, 1, 10 pM). The images were processed to produce projectional images (lower) from the marked area between the red dotted lines. GNDs: gold nanodisks.
nanoparticles could inhibit neovascularization in the mouse model of oxygen-induced retinopathy. 3,20 Local administration can reduce the minimum concentration required to produce an effect. 20 When the concentrations evaluated above were tested on the oxygen-induced retinopathy model, the GNDs inhibited neovascularization at concentrations of both 1 pM and 3 pM in a dose dependent manner (Figure 5, A). Further quantitative analysis revealed a decreased area of neovascular tufts also in a dose dependent manner (Figure 5, B) while leaving a vascular area unchanged (Figure 5, C). The binding capabilities of GNDs were evaluated by measuring unbound VEGF in the vitreous-retina complex at P15, one day after the injection. Intravitreally administered GNDs decreased unbound VEGF in the vitreous and retina to 82.9% at 3 pM compared to intravitreally injected DW (Figure 5, D). To evaluate unwanted toxicity in vivo, histologic observation, TUNEL assay and ERG recording were performed five weeks after the intravitreal injection of nanoparticles at 10 pM. Even
after allowing for a sufficient time to manifest any toxic effects, 32 histologic observation revealed no signs of inflammation, and integrity was unaffected as demonstrated by the ratios between the partial thicknesses of the retinal layers (Figure 5, D and Supplementary Figure 1, A). The TUNEL assay revealed that apoptotic activity throughout the retina was unaffected by the administration of GNDs (Figure 5, E and Supplementary Figure 1, B). Furthermore, ERG amplitudes for a-waves and b-waves showed little difference in GND-injected eyes compared to controls (Figure 5, F). In vivo clearance of gold nanodisks Although we demonstrated the in vivo safety of GNDs by histologic and electrophysiologic evaluation, there were still some concerns regarding the clearance of the GNDs. Therefore, the same concentration of GNDs as used for in vivo toxicity evaluation was injected into mice vitreous bodies, after which the
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Figure 4. Ability of gold nanodisks to bind to VEGF and to inhibit in vitro angiogenesis without cellular toxicity. (A) After overnight incubation with GNDs and VEGF (20 ng/mL) in EBM with 0.1% FBS, unbound VEGF was measured with ELISA. (B, C) Wound migration assays were performed on HRMECs. GNDs at concentrations of 1 pM or 3 pM were treated with VEGF (20 ng/mL) for 12 h, and migrated cells that moved beyond the reference line were quantitatively analyzed at ×200 magnification. (D) HRMECs were treated with GNDs at various concentrations (10 0, 10 1,10 2,10 3 or 10 4 nanoparticles per cell) and incubated for 48 h. Cell viability was evaluated by WST-1 assay. The figures were selected to represent 3 independent experiments. Each bar indicates the mean ± SEM of 3 independent experiments. GNDs: gold nanodisks. Scale bar =200 μm; **, P b 0.01.
clearance was evaluated by OCT because of its wide availability and sensitivity in detecting GNDs with diameters of 160 nm. As early as 6 h after the injection, GNDs were dispersed throughout the vitreous and were gradually cleared out (Figure 6). At day 7, most of the GNDs were not detected in the vitreous but GNDs at the hyaloid canal still remained. At day 14, GNDs were not detectable in the vitreous. There were no signs of accumulation on the inner limiting membranes at all time points.
Discussion We evaluated GNDs that are optically favorable for use as a contrast agent for retinal imaging. Among the GNDs with various diameters that could be finely tuned by adjusting the etching time, GNDs with a resonant wavelength at 830 nm were selected. When intravitreally applied, they produced strong signals in OCT and could attenuate retinal neovascularization without toxicity. Our intravitreally injected GNDs were detectable under OCT at a concentration as low as 1 pM. When the concentration was compared with studies using intracamerally injected GNRs with a peak absorbance wavelength of 850 nm, our particles were detected at about 100 times lower concentration than the GNRs, which produced a detectable signal at 120 pM concentration level. 33 Although a direct comparison between studies with
different OCT systems and administration sites is limited, these in vivo results are consistent with our simulation data that the GNDs have superior scattering capabilities than the GNRs. We applied a comparable concentration of nanodisks to bind VEGF according to the calculation based on the surface area, but they failed to show comparable binding activity toward the VEGF even at double the originally calculated concentration, 3 pM. One possible explanation comes from the radius of curvature of the GNPs that can affect the loading density on the GNPs, 34 as GNPs with a small radius of curvature exhibit a more extensive coverage than those with a large radius of curvature. 34 Our GNDs consisted of two large planar surfaces at the top and bottom and a small side surface with a radius of curvature of 80 nm, so the loading densities are as low as those of a planar gold surface. This can also partly explain our previous data demonstrating a smaller inhibitory effect of larger nanospheres on VEGF-mediated angiogenesis. 4 We demonstrated the in vivo anti-angiogenic effects of the GNDs by using the oxygen-induced retinopathy model. When mice are exposed to normoxia, the retina becomes exposed to relative hypoxia, which leads to increased expression of VEGF in the retina and vitreous 35. The VEGF is secreted and mediates angiogenesis, increasing the area of neovascular tufts that protrude into the vitreous. Despite the inhibitory effect on retinal neovascularization, there was a small difference in unbound VEGF within the vitreous-retina complexes (Figure 5, D).
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Figure 5. Ability of GNDs to inhibit in vivo angiogenesis in the oxygen-induced retinopathy model without toxicity. (A) After intravitreal injection of 0.5 μL of DW or 1 pM and 3pM solution of GNDs at P14, the retina was isolated at P17 and evaluated after immunostaining with anti isolectin-B4 antibody. (B, C) The in vivo anti-angiogenic effect of GNDs was quantitatively analyzed by measuring the neovascular tufts and the avascular area. (D) After intravitreal injection of 0.5 μL of DW or 1 pM and 3pM solution of GNDs at P14, the vitreous and retina were collected at P15 and unbound VEGF was evaluated with ELISA. (E) Five weeks after the intravitreal injection of GNDs at a concentration of 10 pM, the eyes were enucleated, fixed and sectioned. Histologic integrity was evaluated by measuring the A/B ratio, the ratio of A (the retinal thickness from the internal limiting membrane to the inner nuclear layer) to B (the retinal thickness from the internal limiting membrane to the outer nuclear layer). (F) TUNEL assay was performed and cells exhibiting positive staining were counted as apoptotic cells. (G) Electroretinograms were recorded at 5 weeks after the intravitreal injection of GNDs and analyzed to measure a-waves and b-waves. The figures were selected to represent 6 independent experiments. Each bar indicates the mean ± SEM of 6 independent experiments. Scale bar =500 μm; ***, P b 0.001; **, P b 0.01.
According to the distribution data (Figure 6), most of the GNDs differentially remained in the vitreous at day 1, where they were able to bind to vitreous VEGF. Technically inevitable analysis of vitreous-retina complex as a whole probably underestimated the differences made by GNDs and resulted in a small difference in the VEGF concentration despite a substantial decrease in retinal neovascularization by the GNDs. We evaluated in vivo toxicity by intravitreally injecting 10 pM of GNDs. The number of nanodisks that we injected
corresponds to about 3 × 10 6 particles. According to a study on retinal cell populations in the C57 mouse, the ganglion cell layer, the most superficial and least populated layer, has a density of ~8200 cells/mm 2. 36 Even assuming that all the particles became concentrated on ~300 cells, which corresponds to the retinal surface area of 0.036 mm 2, they would not have induced toxicity, according to our in vitro experiments. Considering that the total surface area of the whole mounted retina is 16.5 mm 2, a much higher concentration could be tolerated. The mouse retina
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Figure 6. In vivo clearance of gold nanodisks. After intravitreal injections of 160-nm-sized GNDs at a concentration of 10 pM, the eyes of mice were evaluated at 6 h, 2 days, 7 days, and 14 days. Obtained cross-sectional OCT images (upper) were processed to produce projectional images (lower) from the marked area between the red dotted lines. GNDs: gold nanodisks. The figures were selected to represent 5 independent experiments.
would be a very safe place to apply our GNDs at concentrations above therapeutic range levels. Clearance is another safety concern, as non-degradable nanoparticles that are not cleared from the eye could accumulate and cause long-term toxicity. There are two main mechanisms of clearance for particles intravitreally administered: the anterior elimination pathway via aqueous flow to the trabecular meshwork and the posterior elimination pathway via retina to choroid due to hydrostatic and osmotic pressure gradients in the posterior segments. 37 Nanoparticles take both pathways while large particles take mainly the anterior elimination pathway and remain longer in the eye. 38,39 The clearance is known to be associated with size and surface properties. 38–40 A study with intravitreally administered particles of different sizes demonstrated that 50-nm-sized nanoparticles were localized throughout the vitreous and especially on the adjacent surface of retina and lens 1 day after the intravitreal injection, then decreased by 1 week and became undetectable either in the eye or other organs after 5 weeks, while 4-μm-sized particles were persistent in the eye even after 5 weeks. 39 Nanoparticles with diameters around 200 nm were administered intravitreally and showed different distribution according to the surface properties. 40 Particles with positive zeta potential were trapped in the vitreous or accumulated on the inner limiting membrane at 72 h after injection. Particles with negative zeta potential similar to our gold nanodisks were distributed to the whole retinal layer as early as 6 h after injection and markedly diminished at 72 h after injection. In this study, a follow-up evaluation by OCT after intravitreal injection of GNDs revealed diffuse distribution throughout the vitreous as early as 6 h and gradual clearance by 2 weeks. Although gold is known to be biocompatible and immunologically inert, the possibility, however small, of undetectably remaining gold causing harmful effects needed further evaluation. Our histologic and electrophysiologic evaluation at 5 weeks revealed no evidence of toxicities in retinal integrity and function.
Despite anti-angiogenic activity and safety mentioned above, there were limitations in specificity and potency. The interaction between gold nanoparticles and VEGF can be explained by electrostatic interactions of negatively charged gold nanoparticles with positively charged thiol and amine functionalities of VEGF. 41 This could give limited specificity of gold nanoparticles to discriminate VEGF165 with heparin-binding domain from VEGF121 without heparin-binding domain 42 or from epidermal growth factor, a non-heparin-binding growth factor. 10 However, the potency of this interaction is not as high as the potency of the anti-VEGF antibody that is specifically designed for binding VEGF. According to studies regarding the dissociation constant, 43 gold nanoparticles have dissociation constants in the range of 10 −5 to 10 −9 in interactions with various proteins. This is much greater than the dissociation constant of bevacizumab and ranibizumab for VEGF, which is 3.51 × 10 −11 and 2.06 × 10 −11, respectively. 44 In the presence of a constant amount of anti-VEGF antibodies coated on the ELISA kit, different concentrations of GNDs were incubated with a known amount of VEGF165, after which VEGF bound to the antibodies was measured by ELISA. This competition ELISA resulted in inhibition of binding up to 10% as concentration of GNDs increased to 10 pM (Supplementary Figure 2). The limitations in specificity and potency are barriers to overcome in the following studies. Attaching antibodies specific for VEGF on the surface can be one option to enhance specificity and potency while preserving the optical properties as others have reported using antibody-conjugated gold nanorods for immunostaining and evaluating under OCT. 45 In this study, we demonstrated a process of designing optically favorable nanoparticles for intraocular application and proved their optical properties, effectiveness and safety in vivo. A physical synthesis that allowed a great deal of freedom in designing the nanoparticles facilitated the fabrication of nanodisks with a variety of optical properties. After considering the environment where the nanodisks were to be applied, the
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desired one was selected, and the VEGF binding ability of the nanodisks at selected concentrations was evaluated. This was followed by their application in in vitro angiogenesis assays. After demonstrating their anti-angiogenic effect and safety in vitro, we applied them to retinal neovascularization and showed their anti-angiogenic effect and safety in vivo. Although there are limitations in potency, the anti-angiogenic effect can offer additional benefits for intraocular application where suppression of neovascularization is critical to maintain visual integrity. 46 Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2017.03.016.
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16. Lee S-W, Kang H, Park JH, Lee TG, Lee ES, Lee JY. Ultrahighresolution spectral domain optical coherence tomography based on a linear-wavenumber spectrometer. J Opt Soc Korea 2015;19:55-62. 17. Song HB, Jun H-O, Kim JH, Fruttiger M, Kim JH. Suppression of transient receptor potential canonical channel 4 inhibits vascular endothelial growth factor-induced retinal neovascularization. Cell Calcium 2015;57:101-8. 18. Connor KM, Krah NM, Dennison RJ, Aderman CM, Chen J, Guerin KI, et al. Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis. Nat Protoc 2009;4:1565-73. 19. Kim JH, Kim C, Kim JH, Lee BJ, Yu YS, Park KH, et al. Absence of intravitreal bevacizumab-induced neuronal toxicity in the retina. Neurotoxicology 2008;29:1131-5. 20. Jo DH, Kim JH, Son JG, Song NW, Kim YI, Yu YS, et al. Anti-angiogenic effect of bare titanium dioxide nanoparticles on pathologic neovascularization without unbearable toxicity. Nanomedicine 2014;10:1109-17. 21. Wang L, Jacques SL, Zheng L. MCML–Monte Carlo modeling of light transport in multi-layered tissues. Comput Methods Programs Biomed 1995;47:131-46. 22. Weissleder R. A clearer vision for in vivo imaging. Nat Biotechnol 2001;19:316. 23. Kellner U, Kellner S, Weber BH, Fiebig B, Weinitz S, Ruether K. Lipofuscin- and melanin-related fundus autofluorescence visualize different retinal pigment epithelial alterations in patients with retinitis pigmentosa. Eye (Lond) 2009;23:1349-59. 24. SoRelle ED, Liba O, Hussain Z, Gambhir M, de la Zerda A. Biofunctionalization of large gold Nanorods realizes ultrahigh-sensitivity optical imaging agents. Langmuir 2015;31:12339-47. 25. Ye X, Zheng C, Chen J, Gao Y, Murray CB. Using binary surfactant mixtures to simultaneously improve the dimensional tunability and monodispersity in the seeded growth of gold nanorods. Nano Lett 2013;13:765-71. 26. Hu W, Zhang M, Wilson RJ, Koh AL, Wi J-S, Tang M, et al. Fabrication of planar, layered nanoparticles using tri-layer resist templates. Nanotechnology 2011;22:185302. 27. Giesbers M, Kleijn JM, Stuart MAC. The electrical double layer on gold probed by electrokinetic and surface force measurements. J Colloid Interface Sci 2002;248:88-95. 28. Kim H, Robinson SB, Csaky KG. Investigating the movement of intravitreal human serum albumin nanoparticles in the vitreous and retina. Pharm Res 2009;26:329-37. 29. Mains J, Wilson CG. The vitreous humor as a barrier to nanoparticle distribution. J Ocul Pharmacol Ther 2012;29:143-50. 30. Norman TJ, Grant CD, Magana D, Zhang JZ, Liu J, Cao D, et al. Near infrared optical absorption of gold nanoparticle aggregates. J Phys Chem B 2002;106:7005-12. 31. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669-76. 32. Prow TW, Bhutto I, Kim SY, Grebe R, Merges C, McLeod DS, et al. Ocular nanoparticle toxicity and transfection of the retina and retinal pigment epithelium. Nanomedicine 2008;4:340-9. 33. de la Zerda A, Prabhulkar S, Perez VL, Ruggeri M, Paranjape AS, Habte F, et al. Optical coherence contrast imaging using gold nanorods in living mice eyes. Clin Experiment Ophthalmol 2015;43:358-66. 34. Hill HD, Millstone JE, Banholzer MJ, Mirkin CA. The role radius of curvature plays in thiolated oligonucleotide loading on gold nanoparticles. ACS Nano 2009;3:418-24. 35. Coleman RJ, Beharry KD, Brock RS, Abad-Santos P, Abad-Santos M, Modanlou HD. Effects of brief, clustered versus dispersed hypoxic episodes on systemic and ocular growth factors in a rat model of oxygeninduced retinopathy. Pediatr Res 2008;64:50-5. 36. Jeon CJ, Strettoi E, Masland RH. The major cell populations of the mouse retina. J Neurosci 1998;18:8936-46. 37. Edelhauser HF, Rowe-Rendleman CL, Robinson MR, Dawson DG, Chader GJ, Grossniklaus HE, et al. Ophthalmic drug delivery systems
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Original Article
nanomedjournal.com
Intraocular application of gold nanodisks optically tuned for optical coherence tomography: inhibitory effect on retinal neovascularization without unbearable toxicity Hyun Beom Song, MD a, b, 1 , Jung-Sub Wi, PhD c, 1 , Dong Hyun Jo, MD a, b , Jin Hyoung Kim, PhD a , Sang-Won Lee, PhD c , Tae Geol Lee, PhD c,⁎, Jeong Hun Kim, MD, PhD a, b, d,⁎⁎ a
Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea b Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Republic of Korea c Center for Nano-Bio Measurement, World Class Laboratory, Korea Research Institute of Standards and Science, Daejeon, Republic of Korea d Department of Ophthalmology, College of Medicine, Seoul National University, Seoul, Republic of Korea Received 17 October 2016; accepted 26 March 2017
Abstract Bare gold nanospheres have been shown to have anti-angiogenic effects but are optically unfavorable because their resonant wavelength lies in the visible spectrum. Here, we design gold nanodisks with a higher scattering capability than gold nanorods and with a resonant wavelength at near-infrared region – the area where the source of light utilized by optical coherence tomography (OCT) lies. With a physical synthesis system, we then fabricate 160-nm-sized gold nanodisks exhibiting resonant wavelength at 830 nm. The synthesized nanoparticles were successfully visualized in in vivo OCT at concentrations as low as 1 pM. After demonstrating their binding ability to vascular endothelial growth factor (VEGF), we show that they suppress VEGF-induced migration of endothelial cells. Finally, we demonstrate that intravitreally injected gold nanodisks attenuate neovascularization of oxygen-induced retinopathy in mice, in a dose dependent manner, such that they are cleared from the vitreous within 2 weeks without histologic or electrophysiologic toxicity. © 2017 Elsevier Inc. All rights reserved. Key words: Gold nanodisk; Near-infrared; Optical coherence tomography; Intraocular application; Angiogenesis inhibitor; Retinal neovascularization
Nanoparticles have been widely investigated in the field of imaging as well as treatment. Moreover, advances in nanotechnology have allowed the fabrication of nanoparticles with both diagnostic and therapeutic capabilities by mounting therapeutic functions onto nanoparticles utilized as imaging agents. 1 The reverse approach is considered to be undesirable because mounting diagnostic functions can negatively affect the
pre-existing therapeutic functions of the nanoparticles. However, the self-therapeutic properties 2–4 of bare gold nanoparticles (GNPs) combined with the optical properties of gold with its strong surface plasmon resonance could cater to both approaches. In this study, we designed bare GNPs with self-therapeutic properties to include favorable optical properties for ocular imaging.
Conflict of interest: None of the authors have any competing interests. This study was supported by the Pioneer Research Program of NRF/MEST (2012-0009544); the Bio & Medical Technology Development Program of the NRF funded by the Korean government; MSIP (NRF-2015M3A9E6028949); research grant from NRF/MEST, Republic of Korea (2014M3A7B6027946); and the Bio & Medical Technology Development Program of the NRF funded by the Ministry of Science, ICT & Future Planning (NRF-2015M3A9D7029894). ⁎Correspondence to: T.G. Lee, Center for Nano-Bio Convergence, World Class Laboratory, Korea Research Institute of Standards and Science, Daejeon 305-340, Republic of Korea. ⁎⁎Correspondence to: J.H. Kim, Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Biomedical Research Institute, Seoul National University Hospital, and Department of Ophthalmology, College of Medicine, Seoul National University, 28 Yongon-dong Chongno-gu, Seoul 110-744, Republic of Korea. E-mail addresses: [email protected] (T.G. Lee), [email protected] (J.H. Kim). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.nano.2017.03.016 1549-9634/© 2017 Elsevier Inc. All rights reserved. Please cite this article as: Song HB, et al, Intraocular application of gold nanodisks optically tuned for optical coherence tomography: inhibitory effect on retinal neovascularization without unbearable toxicity. Nanomedicine: NBM 2017;13:1901-1911, http://dx.doi.org/10.1016/j.nano.2017.03.016
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To design the appropriate GNPs, the shape, size and surface charge were considered. We have previously suggested the importance of these physicochemical properties of nanoparticles in determining the therapeutic outcome, 5 but they also play important roles in the optical properties, self-therapeutic properties and safety of the GNPs. The optical properties arise from the strong surface plasmon resonance of gold which helps to enhance the scattering intensity. 6 The scattering intensities and plasmon wavelength maximum are influenced by the shape and size of the nanoparticles. 7 The plasmon wavelength maximum of the gold nanospheres lies from 520 nm to 550 nm – a range that is too limited to be applicable for in vivo imaging. Among nanoparticles that are optically tunable by changing the size or aspect ratio, gold nanodisks (GNDs) are optically more favorable than gold nanorods (GNRs) because the latter possess directional and polarization dependencies. 8 In other words, GNDs can produce reliable signals regardless of the incident angle or polarized directions of the light source. The size and surface charge can influence the self-therapeutic activities of the GNPs. Bare GNPs are widely considered to be therapeutic agents that suppress angiogenesis because they inhibit in vitro vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation and in vivo VEGF-induced angiogenesis. 9 The mechanism behind the anti-angiogenic effect lies in their binding affinity to VEGFs, 9,10 and further evaluation has revealed that large GNPs bind more readily to VEGFs while inhibiting phosphorylation of VEGF receptors and endothelial cell proliferation at a lower concentration than do small GNPs. 10 However, because coating the surface with different charges masks the inhibitory effect of the GNPs on VEGF function, 10 larger GNPs with naked surfaces are recommended for greater effectiveness. The size, shape and surface can affect the toxicity of the GNPs; the toxicity is linked to cellular uptake levels and coating, 11 while in turn, the cellular uptake of GNPs correspond to their size and shape. 5 For spherical GNPs, cellular uptake is highest at 50 nm and decreases as their size increases. 12 When compared to spherical GNPs, GNRs showed reduced cellular uptake that further decreased with a higher aspect ratio. 12 And, because nanodisks also rarely enter cells and do not induce reactive oxygen species, apoptosis or cell cycle perturbation, 13 in this paper, we utilized anisotropic nanoparticles such as nanorods or nanodisks, as opposed to spherical nanoparticles. In terms of coating, top-down synthesis and a bare surface can avoid unwanted chemical coating on the surface. Furthermore, the surface charge is important for diffusion in biological fluids, and gold's advantages in terms of toxicity are its biocompatibility and immunological inertness. After considering the factors that influence optical properties, anti-angiogenic activities and toxicity, we decided on large GNDs with bare surfaces for further evaluation. First, we simulated the optical properties of GNDs of various diameters; after considering the scattering properties and ranges of their resonant wavelengths, we selected 160-nm-sized GNDs to use in the eye. After the physical synthesis of the GNDs, we applied them to the eye and observed strong signals in optical coherence tomography (OCT). An evaluation of the anti-angiogenic effect of the fabricated GNDs demonstrated that GNDs could inhibit in vitro angiogenesis without cellular toxicity and could inhibit retinal neovascularization without significant toxicity when applied to a mouse oxygen-induced retinopathy model.
Methods Electromagnetic simulation Three-dimensional finite-difference time-domain software (Lumerical FDTD Solution 8.9) was utilized to perform electromagnetic simulations. Under illumination from the top with a linearly polarized plane wave, the scattering and absorption cross sections were calculated on particles in a uniform dielectric medium (n = 1.33), with the simulation grid set to a 1.0 nm cubic. Nanoparticle fabrication and characterization The nanoparticles were prepared by top-down synthesis as described previously 14,15 and in Supplementary Methods. The morphology of the gold nanodisks was observed via scanning electron microscopy (SEM, FEI XL30 Sirion) after drying the aliquots of stock solution on a Si substrate. The images were analyzed with the imageJ program (NIH) to measure the average diameter. The zeta potential of the gold nanodisks in distilled water was evaluated with a zeta potential analyzer (Zetasizer Nano-Z, Malvern, UK). Mono-dispersed nanodisks of various concentrations (1, 3 or 10 pM) were prepared in EBM with 0.1% FBS. After incubation for 24 h, UV–Vis measurements (UV-2600, Shimadzu, Kyoto, Japan) were conducted. Changes in absorbance and shift in λmax were evaluated. Optical coherence tomography After the mice were anesthetized and their pupils were dilated, the eyes were positioned under the previously described spectral domain OCT system. 16 The system utilized a superluminescence diode with a center wavelength at 849 nm. After the retinal scans were taken, 3D images were reconstructed from the cross-sectional B-scan images. Then, the same 3D volumes that corresponded to the vitreous cavity were projected to the x-y plane; we then produced a 2D projection image to compare the concentrations of particles in the vitreous. All images were processed with Amira software (FEI, Hillsboro, USA). Wound migration assay Wound migration assay was performed as in our previous descriptions. 17 Human retinal microvascular endothelial cells (HRMECs) were seeded on gelatin-coated 35-mm dishes and incubated to achieve more than 90% confluence. After overnight starvation with basal medium containing 0.1% FBS, the cell layers were wounded with a pipette tip. Then, the medium was replaced with 20 ng/mL VEGF (Sigma–Aldrich, St. Louis, USA) or GNDs at concentrations of 1 pM or 3 pM. The image of the wound was obtained under a light microscope immediately after the scratch to set the reference line. The medium was removed 12 h later and the cells were fixed with absolute methanol and stained with Giemsa's solution (BDH Laboratory Supplies, London, United Kingdom). Migration was quantified by counting the migrated cells across the reference line under a light microscope at ×100 (Leica, Wetzlar, Germany).
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Cell viability assay Cell viability was evaluated by WST-1 assay using the EZ-Cytox Cell Viability Assay kit (Itsbio, Seoul, Korea) according to the manufacturer's instructions. Briefly, HRMECs were plated in 96-well plates at a density of 6 × 10 3 cells per well. After overnight incubation, the cells were treated with GNDs at various concentrations (10 0, 10 1,10 2,10 3 or 10 4 nanoparticles per cell) and incubated for 48 h. After evaluation under a light microscope, EZ-Cytox reagents were added to each well. After 2 h of additional incubation, the plates were analyzed by the microplate reader to measure the absorbance at 450 nm. Measurement of binding between nanodisks and VEGF Two different concentrations of nanodisks (1 or 3 pM) were incubated in phenol free EBM (Lonza) supplemented with VEGF (20 ng/mL) and 0.1% FBS. After incubation overnight, centrifugation was performed to separate the nanodisk-bound VEGF. Then, the concentrations of unbound VEGF were measured in the supernatant using a VEGF Human ELISA Kit (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. Animal C57BL/6 J mice obtained from Central Lab Animals Incorporation (Seoul, Korea) were kept in alternate dark–light cycles of 12 h at room temperature and served with food and water ad libitum. The care, use, and interventions were approved by the Institutional Animal Care and Use Committee of Seoul National University and were in agreement with the ARVO statement for the use of animals in ophthalmic and vision research.
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a magnification of ×40. The avascular area and neovascularization were measured with Adobe Photoshop CS6 (San Jose, USA) as described elsewhere. 18 In vivo toxicity evaluations: Histology, TUNEL assay and electroretinogram To assess in vivo toxicity, 6-week-old C57BL/6 J mice were intravitreally injected with 1 μL of DW (n = 6) or 10 pM solution of GNDs (n = 6). Five weeks after the injection, the electroretinogram (ERG) was recorded using the UTAS Visual Electrodiagnostic System (LKC Technologies, Gaithersburg, USA). The detailed protocol is described in Supplementary Methods. The animals were then sacrificed and, and the eyes were enucleated, fixed in 4% paraformaldehyde overnight and embedded in paraffin. Sagittal sections of 4-μm-thicknesses were prepared, stained with hematoxylin and eosin and evaluated under a light microscope (Ni-U; Nikon). As previously described, the ratio of A (the retinal thickness from the internal limiting membrane to the inner nuclear layer) to B (the retinal thickness from the internal limiting membrane to the outer nuclear layer) was measured and compared with the control group. 19,20 For the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, at least 10 sections from each eye (at least five from each side of the optic nerve) were stained with the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics, Indianapolis, USA) according to the manufacturer's instructions. The sections were then observed with a fluorescence microscope (Ni-U; Nikon) at a magnification of ×400 and TUNEL-positive cells were counted per section. Statistics
Oxygen-induced retinopathy Oxygen-induced retinopathy was induced as described previously. 3 Briefly, newborn mice were randomly assigned to experimental (n = 18) and control (n = 12) groups. At postnatal day 7 (P7), the mice were exposed to hyperoxia (75 ± 0.5% oxygen) for 5 days from P7 to P12 and then returned to normoxia (room air). The oxygen concentration level was measured twice daily with an oxygen analyzer (Miniox I; Bertocchi srl Elettromedicali, Cremona, Italy). To assess the anti-angiogenic properties of the GNDs, the pups were injected intravitreally with 0.5 μL of DW or 1 pM and 3 pM solutions of GNDs at P14. The mice were sacrificed by inhalation of CO2 at P17 to evaluate retinal neovascularization and toxicity. In another set of mice (n = 12), oxygen-induced retinopathy was induced and the pups were injected intravitreally with DW or GNDs in the same manner as mentioned above. At P15, one day after the injection, the mice were sacrificed and vitreous-retina complexes were prepared for the measurement of mouse VEGF using an ELISA kit (R&D systems, Minneapolis, USA). Assessment of retinal neovascularization After fixation in 4% paraformaldehyde for 1.5 h, the enucleated eyes were dissected, and the isolated retinas were subjected to immunostaining with the anti isolectin-B4 antibody (GSA-IB4; Invitrogen) and flat-mounted. The images were obtained using a fluorescence microscope (Ni-U; Nikon, Tokyo, Japan) at
The differences between the groups were evaluated with the Mann–Whitney U-test using SPSS 19.0 (SPSS Inc., Chicago, USA). P-values less than 0.05 were considered to be statistically significant.
Results Design of optimal gold nanoparticles for ocular imaging We first simulated the optical properties of the GNDs to make fine adjustments to the design of the GNDs to make them suitable for ocular imaging. Electromagnetic simulations of 20 nm thick GNDs with diameters from 40 nm to 200 nm revealed a shift in the resonant wavelength to the infrared region, enabling the imaging of deeper tissues due to better penetration. 21 In addition, an increased diameter caused scattering to also increase (Figure 1, A), so that these larger GNDs could be detected at lower concentrations, unlike smaller GNDs. Unfortunately, large GNDs with resonant wavelengths larger than 900 nm are difficult to practically apply because light in the region can be absorbed by the abundance of water 22 throughout the vitreous that covers the retina, a common target of imaging in ophthalmology. Wavelengths adjacent to 787 nm also need to be avoided because the light source could produce autofluorescence of the melanin pigment in the retinal pigment epithelium. 23 Furthermore, a widely used imaging method in ophthalmology, OCT, employs a broadband source of
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Figure 1. Finite-difference time-domain simulation of gold nanoparticles. (A) 20-nm-thick single gold nanodisks with diameters of 40, 80, 120, 160, and 200 nm were evaluated. The refractive index of the medium was assumed to be 1.33. Calculated cross sections of absorption, scattering and extinction were plotted according to the disk diameters and corresponding resonant wavelengths. (B) Gold nanoparticles resonating at near-infrared wavelengths were evaluated: sphere with diameter of 200 nm, disk with diameter of 160 nm and thickness of 20 nm, and rod with length of 70 nm and diameter of 20 nm.
light centered at wavelengths between 800 nm and 850 nm, the region where the resonant wavelength of GNDs with diameters of 160 nm lies. Taking into consideration these factors, in this study we used 160 nm GNDs with a resonant wavelength at 830 nm. To compare the optical characteristics according to the dimensions of the plasmon nanostructures, we simulated GNPs with the same resonant wavelength at 830 nm but of different shapes (Figure 1, B). As the backscattered light produced signals in OCT, the scattering cross sections were then compared. Higher scattering was observed for gold nanospheres (diameter 200 nm) and GNDs (diameter 160 nm, thickness 20 nm) than for GNRs (length 70 nm, diameter 20 nm). Most of the extinction cross sections consisted of scattering cross sections in gold nanospheres and GNDs and absorption cross sections in the GNRs. Although the scattering cross section of GNR grew as its geometrical size increased, as shown in Supplementary Figure 3, light-absorption is more dominant than the scattering for GNRs of typical sizes and shapes. 7,24,25 The simulated 200-nm-sized gold nanospheres were about 10 times heavier than the 160-nm-sized GNDs, so less mass concentration was required to produce comparable signals in OCT when the particles were manipulated into the shape of disks. Preparation and characterization of the gold nanodisks To prepare monodisperse bare GNDs, a top-down synthesis described previously 14,26 was applied (Figure 2, A). This physical synthesis allowed a great deal of flexibility in designing the nanoparticles, so GNDs of various diameters could be prepared simply by adjusting the etching times (Supplementary Table 1). We used an etching time of 40 s to prepare GNDs with diameters of 162 ± 3.3 nm and heights of 20 nm. All the particles were uniform in shape and size according to SEM (Figure 2, B). The zeta potential was estimated to be −23.4 ± 9.3 mV, which is comparable to bare gold nanoparticles. 27 When these charged particles were injected into the vitreous, the anionic particles diffused more easily than the cationic particles 28
because the overall anionic charge of the vitreous can disturb the diffusion of the cationic particles. 29 Hence, a bare surface is not only more conducive to an anti-angiogenic effect 10 but also allows for better diffusion in the eyes. The aggregation status was also evaluated at various concentrations since aggregation of gold nanoparticles can reduce the surface area of the VEGF binding sites. 10 Even after GNDs with concentrations of up to 10 pM were incubated for 24 h in an environment where further in vitro experiments were conducted, UV–Vis measurements did not show any of the changes in absorbance patterns and resonant wavelengths that aggregation of particles can make (Figure 2, C). 30 Optical properties of the gold nanodisks The synthesized nanoparticles were visualized in the OCT system we previously described. 16 To compare their signal intensities, gold nanospheres and GNDs with the same resonant wavelength at 830 nm were prepared in aqueous solutions. As simulated in Figure 1, B, the same number of particles produced almost similar signal intensities in OCT (Figure 3, A). On the other hand, a solution of GNDs containing about 50 ppm of gold produced a much stronger signal than gold nanospheres with the same mass concentration of gold (Figure 3, A). The results were consistent with the data from the simulation above. Mice vitreous bodies were then imaged after intravitreal injections of GNDs with diameters of 160 nm. Eyes injected with distilled water or 0.1 pM of GNDs showed a minimal OCT signal, but those injected with 1 pM of GNDs produced a significant OCT signal. Intravitreal injections of 10 pM of GNDs produced a much higher signal in both cross-sectional and projectional OCT images (Figure 3, B and Videos 1–4). Ability of gold nanodisks to bind to VEGF and inhibit in vitro angiogenesis without cellular toxicity Before the in vivo application of GNDs on the retinal neovascularization model of a mouse, we evaluated the degree to
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Figure 2. Preparation and characterization of GNDs. (A) Schematic illustration of the overall process in this study: nanoimprint lithography (NIL) using a truncated nanocone array, O2 plasma etching of residual PMMA, wet chemical dissolution of PMGI, vacuum deposition of gold, gold lift-off, nanodisk release, and centrifugation. (B) Scanning electron microscopy images of GNDs fabricated with an etching time of 40 sec. (C) UV–Vis measurements after 24 h incubation of nanodisks at concentrations of 1 pM (red), 3 pM (green) and 10 pM (blue) in EBM with 0.1% FBS. Scale bar =1 μm.
which the GNDs bind to VEGF, a key regulator of angiogenesis that induces the proliferation and migration of endothelial cells, which contributes to the formation of new blood vessels. 31 The GND concentration levels used to produce the anti-angiogenic effect were based on our previous work, which found that 20-nm-sized nanospheres demonstrated anti-angiogenic effects both in vitro and in vivo at concentrations of 1 μM 3 and at even lower concentrations of 16.64 pM. 20 Smaller spheres with diameters of 5 nm inhibited VEGF-induced proliferation and phosphorylation of VEGF receptors at 1 nM where they were able to bind to approximately 8 ng/mL of VEGF. 10 To determine the minimal concentration level that could exert an anti-angiogenic effect for 160-nm-sized nanodisks, we calculated that a surface area, 640 times larger than the 5-nm-sized nanospheres, yielded 1.56 pM as a comparable concentration level for the GNDs. As a result, we began our investigation into the anti-angiogenic effect of synthesized GNDs at concentrations of 1 pM and 3 pM. These levels lie above the minimal detectable concentration level, according to Figure 3, A. After determining the concentrations to be used, we used an ELISA test to determine the concentration of bound VEGF by measuring the unbound VEGF remaining in the solution after
separation from the nanoparticles as previously described. 10 1 pM of GNDs were bound to 2.26 ± 0.13 ng/mL of VEGF and 3 pM of GNDs were bound to 5.44 ± 0.42 ng/mL of VEGF (Figure 4, A). Using these concentrations, in vitro angiogenesis assays were performed to evaluate the anti-angiogenic effect of the GNDs. Previously, we demonstrated that various nanoparticles inhibit VEGF-induced migration of endothelial cells. 3,20 As shown in Figure 4, B and C, GNDs inhibited VEGF-induced migration of HRMECs at a concentration of 3 pM but were less effective at 1 pM. At a concentration of 3 pM, which accounts for about 240 particles per cell seeded, the cells showed no aberrant changes in morphology that would indicate the toxic effects of the GNDs. At higher concentrations up to 10 4 per cell, the GNDs did not decrease the viability of HRMECs even when the incubation time was extended to 48 h (Figure 4, D). Ability of gold nanodisks to inhibit in vivo angiogenesis without histologic and electrophysiologic toxicity We further evaluated the effect of GNDs on angiogenesis in vivo. We previously demonstrated that intravitreally injected
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Figure 3. Optical properties of the GNDs in optical coherence tomography (OCT). (A) Cross-sectional OCT images were obtained from a 1 pM solution of 200-nm-sized gold nanospheres (left), a 1 pM solution of 160-nm-sized GNDs (center) and a 10.4 pM solution of 160-nm-sized GNDs (right), which correspond to the mass concentration of a 1 pM solution of 200-nm-sized gold nanospheres. (B) Cross-sectional OCT images (upper) were obtained from the eye of a mouse immediately after an intravitreal injection of distilled water and a solution of 160-nm-sized GNDs (0.1, 1, 10 pM). The images were processed to produce projectional images (lower) from the marked area between the red dotted lines. GNDs: gold nanodisks.
nanoparticles could inhibit neovascularization in the mouse model of oxygen-induced retinopathy. 3,20 Local administration can reduce the minimum concentration required to produce an effect. 20 When the concentrations evaluated above were tested on the oxygen-induced retinopathy model, the GNDs inhibited neovascularization at concentrations of both 1 pM and 3 pM in a dose dependent manner (Figure 5, A). Further quantitative analysis revealed a decreased area of neovascular tufts also in a dose dependent manner (Figure 5, B) while leaving a vascular area unchanged (Figure 5, C). The binding capabilities of GNDs were evaluated by measuring unbound VEGF in the vitreous-retina complex at P15, one day after the injection. Intravitreally administered GNDs decreased unbound VEGF in the vitreous and retina to 82.9% at 3 pM compared to intravitreally injected DW (Figure 5, D). To evaluate unwanted toxicity in vivo, histologic observation, TUNEL assay and ERG recording were performed five weeks after the intravitreal injection of nanoparticles at 10 pM. Even
after allowing for a sufficient time to manifest any toxic effects, 32 histologic observation revealed no signs of inflammation, and integrity was unaffected as demonstrated by the ratios between the partial thicknesses of the retinal layers (Figure 5, D and Supplementary Figure 1, A). The TUNEL assay revealed that apoptotic activity throughout the retina was unaffected by the administration of GNDs (Figure 5, E and Supplementary Figure 1, B). Furthermore, ERG amplitudes for a-waves and b-waves showed little difference in GND-injected eyes compared to controls (Figure 5, F). In vivo clearance of gold nanodisks Although we demonstrated the in vivo safety of GNDs by histologic and electrophysiologic evaluation, there were still some concerns regarding the clearance of the GNDs. Therefore, the same concentration of GNDs as used for in vivo toxicity evaluation was injected into mice vitreous bodies, after which the
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Figure 4. Ability of gold nanodisks to bind to VEGF and to inhibit in vitro angiogenesis without cellular toxicity. (A) After overnight incubation with GNDs and VEGF (20 ng/mL) in EBM with 0.1% FBS, unbound VEGF was measured with ELISA. (B, C) Wound migration assays were performed on HRMECs. GNDs at concentrations of 1 pM or 3 pM were treated with VEGF (20 ng/mL) for 12 h, and migrated cells that moved beyond the reference line were quantitatively analyzed at ×200 magnification. (D) HRMECs were treated with GNDs at various concentrations (10 0, 10 1,10 2,10 3 or 10 4 nanoparticles per cell) and incubated for 48 h. Cell viability was evaluated by WST-1 assay. The figures were selected to represent 3 independent experiments. Each bar indicates the mean ± SEM of 3 independent experiments. GNDs: gold nanodisks. Scale bar =200 μm; **, P b 0.01.
clearance was evaluated by OCT because of its wide availability and sensitivity in detecting GNDs with diameters of 160 nm. As early as 6 h after the injection, GNDs were dispersed throughout the vitreous and were gradually cleared out (Figure 6). At day 7, most of the GNDs were not detected in the vitreous but GNDs at the hyaloid canal still remained. At day 14, GNDs were not detectable in the vitreous. There were no signs of accumulation on the inner limiting membranes at all time points.
Discussion We evaluated GNDs that are optically favorable for use as a contrast agent for retinal imaging. Among the GNDs with various diameters that could be finely tuned by adjusting the etching time, GNDs with a resonant wavelength at 830 nm were selected. When intravitreally applied, they produced strong signals in OCT and could attenuate retinal neovascularization without toxicity. Our intravitreally injected GNDs were detectable under OCT at a concentration as low as 1 pM. When the concentration was compared with studies using intracamerally injected GNRs with a peak absorbance wavelength of 850 nm, our particles were detected at about 100 times lower concentration than the GNRs, which produced a detectable signal at 120 pM concentration level. 33 Although a direct comparison between studies with
different OCT systems and administration sites is limited, these in vivo results are consistent with our simulation data that the GNDs have superior scattering capabilities than the GNRs. We applied a comparable concentration of nanodisks to bind VEGF according to the calculation based on the surface area, but they failed to show comparable binding activity toward the VEGF even at double the originally calculated concentration, 3 pM. One possible explanation comes from the radius of curvature of the GNPs that can affect the loading density on the GNPs, 34 as GNPs with a small radius of curvature exhibit a more extensive coverage than those with a large radius of curvature. 34 Our GNDs consisted of two large planar surfaces at the top and bottom and a small side surface with a radius of curvature of 80 nm, so the loading densities are as low as those of a planar gold surface. This can also partly explain our previous data demonstrating a smaller inhibitory effect of larger nanospheres on VEGF-mediated angiogenesis. 4 We demonstrated the in vivo anti-angiogenic effects of the GNDs by using the oxygen-induced retinopathy model. When mice are exposed to normoxia, the retina becomes exposed to relative hypoxia, which leads to increased expression of VEGF in the retina and vitreous 35. The VEGF is secreted and mediates angiogenesis, increasing the area of neovascular tufts that protrude into the vitreous. Despite the inhibitory effect on retinal neovascularization, there was a small difference in unbound VEGF within the vitreous-retina complexes (Figure 5, D).
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Figure 5. Ability of GNDs to inhibit in vivo angiogenesis in the oxygen-induced retinopathy model without toxicity. (A) After intravitreal injection of 0.5 μL of DW or 1 pM and 3pM solution of GNDs at P14, the retina was isolated at P17 and evaluated after immunostaining with anti isolectin-B4 antibody. (B, C) The in vivo anti-angiogenic effect of GNDs was quantitatively analyzed by measuring the neovascular tufts and the avascular area. (D) After intravitreal injection of 0.5 μL of DW or 1 pM and 3pM solution of GNDs at P14, the vitreous and retina were collected at P15 and unbound VEGF was evaluated with ELISA. (E) Five weeks after the intravitreal injection of GNDs at a concentration of 10 pM, the eyes were enucleated, fixed and sectioned. Histologic integrity was evaluated by measuring the A/B ratio, the ratio of A (the retinal thickness from the internal limiting membrane to the inner nuclear layer) to B (the retinal thickness from the internal limiting membrane to the outer nuclear layer). (F) TUNEL assay was performed and cells exhibiting positive staining were counted as apoptotic cells. (G) Electroretinograms were recorded at 5 weeks after the intravitreal injection of GNDs and analyzed to measure a-waves and b-waves. The figures were selected to represent 6 independent experiments. Each bar indicates the mean ± SEM of 6 independent experiments. Scale bar =500 μm; ***, P b 0.001; **, P b 0.01.
According to the distribution data (Figure 6), most of the GNDs differentially remained in the vitreous at day 1, where they were able to bind to vitreous VEGF. Technically inevitable analysis of vitreous-retina complex as a whole probably underestimated the differences made by GNDs and resulted in a small difference in the VEGF concentration despite a substantial decrease in retinal neovascularization by the GNDs. We evaluated in vivo toxicity by intravitreally injecting 10 pM of GNDs. The number of nanodisks that we injected
corresponds to about 3 × 10 6 particles. According to a study on retinal cell populations in the C57 mouse, the ganglion cell layer, the most superficial and least populated layer, has a density of ~8200 cells/mm 2. 36 Even assuming that all the particles became concentrated on ~300 cells, which corresponds to the retinal surface area of 0.036 mm 2, they would not have induced toxicity, according to our in vitro experiments. Considering that the total surface area of the whole mounted retina is 16.5 mm 2, a much higher concentration could be tolerated. The mouse retina
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Figure 6. In vivo clearance of gold nanodisks. After intravitreal injections of 160-nm-sized GNDs at a concentration of 10 pM, the eyes of mice were evaluated at 6 h, 2 days, 7 days, and 14 days. Obtained cross-sectional OCT images (upper) were processed to produce projectional images (lower) from the marked area between the red dotted lines. GNDs: gold nanodisks. The figures were selected to represent 5 independent experiments.
would be a very safe place to apply our GNDs at concentrations above therapeutic range levels. Clearance is another safety concern, as non-degradable nanoparticles that are not cleared from the eye could accumulate and cause long-term toxicity. There are two main mechanisms of clearance for particles intravitreally administered: the anterior elimination pathway via aqueous flow to the trabecular meshwork and the posterior elimination pathway via retina to choroid due to hydrostatic and osmotic pressure gradients in the posterior segments. 37 Nanoparticles take both pathways while large particles take mainly the anterior elimination pathway and remain longer in the eye. 38,39 The clearance is known to be associated with size and surface properties. 38–40 A study with intravitreally administered particles of different sizes demonstrated that 50-nm-sized nanoparticles were localized throughout the vitreous and especially on the adjacent surface of retina and lens 1 day after the intravitreal injection, then decreased by 1 week and became undetectable either in the eye or other organs after 5 weeks, while 4-μm-sized particles were persistent in the eye even after 5 weeks. 39 Nanoparticles with diameters around 200 nm were administered intravitreally and showed different distribution according to the surface properties. 40 Particles with positive zeta potential were trapped in the vitreous or accumulated on the inner limiting membrane at 72 h after injection. Particles with negative zeta potential similar to our gold nanodisks were distributed to the whole retinal layer as early as 6 h after injection and markedly diminished at 72 h after injection. In this study, a follow-up evaluation by OCT after intravitreal injection of GNDs revealed diffuse distribution throughout the vitreous as early as 6 h and gradual clearance by 2 weeks. Although gold is known to be biocompatible and immunologically inert, the possibility, however small, of undetectably remaining gold causing harmful effects needed further evaluation. Our histologic and electrophysiologic evaluation at 5 weeks revealed no evidence of toxicities in retinal integrity and function.
Despite anti-angiogenic activity and safety mentioned above, there were limitations in specificity and potency. The interaction between gold nanoparticles and VEGF can be explained by electrostatic interactions of negatively charged gold nanoparticles with positively charged thiol and amine functionalities of VEGF. 41 This could give limited specificity of gold nanoparticles to discriminate VEGF165 with heparin-binding domain from VEGF121 without heparin-binding domain 42 or from epidermal growth factor, a non-heparin-binding growth factor. 10 However, the potency of this interaction is not as high as the potency of the anti-VEGF antibody that is specifically designed for binding VEGF. According to studies regarding the dissociation constant, 43 gold nanoparticles have dissociation constants in the range of 10 −5 to 10 −9 in interactions with various proteins. This is much greater than the dissociation constant of bevacizumab and ranibizumab for VEGF, which is 3.51 × 10 −11 and 2.06 × 10 −11, respectively. 44 In the presence of a constant amount of anti-VEGF antibodies coated on the ELISA kit, different concentrations of GNDs were incubated with a known amount of VEGF165, after which VEGF bound to the antibodies was measured by ELISA. This competition ELISA resulted in inhibition of binding up to 10% as concentration of GNDs increased to 10 pM (Supplementary Figure 2). The limitations in specificity and potency are barriers to overcome in the following studies. Attaching antibodies specific for VEGF on the surface can be one option to enhance specificity and potency while preserving the optical properties as others have reported using antibody-conjugated gold nanorods for immunostaining and evaluating under OCT. 45 In this study, we demonstrated a process of designing optically favorable nanoparticles for intraocular application and proved their optical properties, effectiveness and safety in vivo. A physical synthesis that allowed a great deal of freedom in designing the nanoparticles facilitated the fabrication of nanodisks with a variety of optical properties. After considering the environment where the nanodisks were to be applied, the
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desired one was selected, and the VEGF binding ability of the nanodisks at selected concentrations was evaluated. This was followed by their application in in vitro angiogenesis assays. After demonstrating their anti-angiogenic effect and safety in vitro, we applied them to retinal neovascularization and showed their anti-angiogenic effect and safety in vivo. Although there are limitations in potency, the anti-angiogenic effect can offer additional benefits for intraocular application where suppression of neovascularization is critical to maintain visual integrity. 46 Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2017.03.016.
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