Renally Clearable Ultraminiature Chain‐Like Gold Nanoparticle Clusters for Multimodal Molecular Imaging of Choroidal Neovascularization

Currently, available gold nanoparticles (GNPs) typically accumulate in the liver and spleen, leading to concerns for their long‐term biosafety. To address this long‐standing problem, ultraminiature chain‐like gold nanoparticle clusters (GNCs) are developed. Via self‐assembly of 7–8 nm GNP monomers, GNCs provide redshifted optical absorption and scattering contrast in the near‐infrared window. After disassembly, GNCs turn back to GNPs with a size smaller than the renal glomerular filtration size cutoff, allowing their excretion via urine. A one‐month longitudinal study in a rabbit eye model demonstrates that GNCs facilitate multimodal molecular imaging of choroidal neovascularization (CNV) in vivo, non‐invasively, with excellent sensitivity and spatial resolution. GNCs targeting αvβ3 integrins enhance photoacoustic and optical coherence tomography (OCT) signals from CNV by 25.3‐fold and 150%, respectively. With excellent biosafety and biocompatibility demonstrated, GNCs render a first‐of‐its‐kind nanoplatform for biomedical imaging.


Introduction
Advanced non-invasive photoacoustic microscopy (PAM) and optical coherence tomography (OCT) imaging have attracted significant attention as promising techniques to evaluate disease DOI: 10.1002/adma.202302069 pathogenesis, such as cancer and angiogenesis, and treatments, such as stem cell therapy. [1][2][3][4][5][6][7][8] The PAM and OCT signals are generated from the optical absorption and the optical scattering properties of the biological tissues, respectively, which can provide both anatomical and functional information. Most living organism tissues show low endogenous absorption and scattering of light in the red and near-infrared (R/NIR) spectrum (i.e., 600-1200 nm) which serves as an "optical window" for PAM and OCTbased molecular imaging powered by exogenous contrast agents.
Currently, there are several R/NIR PAM and OCT photosensitizers, including genetically encoded chromophores, [1] small-molecular NIR dye (indocyanine green (ICG), compound of designation near-infrared 7 (CDnir7), near-infrared dyes (IRDye800, ATTO740), silicon 2,3naphthalocyanine bi(trihexylsilyloxide) (SiNc), semiconductor and organic nanofluorophores in the second near-infrared (NIR-II) window [9,10] as well as nanostructures made from carbon (e.g., carbon nanotubes, graphene-based nanomaterials and nanodiamonds), and gold (e.g., sphere, stars, tripods, plates, rods, and prisms). [11][12][13][14] Although some of the small molecular R/NIR dyes (e.g., ICG) are approved by the US Food and Drug Administration (FDA) for clinical utilization, these imaging materials have limited photostability and surface functionalization for active targeting. Gold nanoparticles (GNPs) show great potential as an emerging class of contrast agents in PAM and OCT imaging due to their excellent biocompatibility, low cytotoxicity, size-/shape-dependent superb optical absorption, and scattering up to five orders of magnitude higher than those of traditional organic dyes, easy method of fabrication, and rich surface chemistry for functionalization. Despite significant progress in the last two decades in developing GNPs as contrast agents for molecular biomedical imaging, in vivo utility of conventional spherical GNPs in PAM imaging is restricted as a result of the overlapping of their localized surface plasmon resonance (LSPR) absorption peak (≈520 nm) with that of hemoglobin.
As a first step toward overcoming such limitations, we recently developed a chain-like gold nanoparticle cluster (GNC) via a method of self-assembly of 20-nm GNPs fabricated by www.advancedsciencenews.com www.advmat.de physical method using pulsed laser ablation (PLA). [5,6,15] The PLA technique was developed to assist with biomedical translation since it is a pure method lacking the toxic chemicals used in wet-chemical synthesis. PLA utilizes sterile deionized water and produces stable, nontoxic, and capping-agent-free GNP monomers with high purity. This is different from chemical manners that comprise toxic precursors, such as reducing substances and stabilizers. With a narrow gap (≈1.0 nm) between adjacent GNPs in a chain, these GNC demonstrate a desired, redshifted plasmonic absorption peak (longitudinal SPR) at the optical wavelength of 650 nm. In the experiments using multiple clinically relevant models of age-related macular degeneration (AMD) with the new development of blood vessels termed choroidal neovascularization (CNV) in large animals (rabbits), we have demonstrated that these GNC have excellent targeting efficiency, contrast enhancement, biocompatibility, and photostability.
When utilized as a novel contrast material for PAM and OCT multimodal molecular imaging, the angiogenesis-targeting GNC achieved a 17-fold enhancement in PA signal, facilitating highly sensitive detection and visualization of CNV in rabbit eyes in vivo. [5] However, after intravenous administration, the majority of these GNC accumulate in the liver and spleen, leading to concerns about their long-term biosafety. This problem is common and shared by a variety of designs of GNPs with sizes >10 nm. Mitchell et al. have reported that GNPs with a size <10 nm are rapidly eliminated by the kidneys. [16] To address this problem, we have successfully developed a first-of-its-kind ultraminiature GNC composed of 7-8 nm spherical GNPs. Because the sizes of individual spherical GNPs forming the chains are smaller than the effective renal glomerular filtration size cutoff (6-10 nm), these ultraminiature GNC, while offering optical contrast enhancement in the red regime, can be renally excreted.
It is worth emphasizing that various types of renal clearable nanoparticles (NPs) and nanoclusters (NCs) have been developed since the pioneering work by Choi et al. on renal clearable quantum dots in 2007. [17] Especially, in 2011, Chen et al. first reported the high-efficiency renal clearance of glutathione-coated gold NCs with core sizes of 1-3 nm. [18] Due to their efficient renal clearance, these gold NCs have become one of the most extensively studied inorganic renal clearable NPs/NCs over the last decade. In comparison to gold NCs, the novelty of the renal clearable ultraminiature chain-like gold NP clusters (GNCs) demonstrated in our work lies in their unique shape, enhanced optical properties, improved performance, multifunctionality, and sustainability.
GNCs are elongated structures made up of multiple gold NPs linked together in a linear arrangement with a length in the range of tens of nanometers, whereas gold NCs, typically consisting of several to tens of gold atoms, are spherical with a size on the order of a few nanometers. The shape of gold NPs can affect their optical and electronic properties. Due to the plasmonic coupling between adjacent NPs, the elongated shape of GNCs gives them enhanced optical absorption and scattering properties, which are not present in gold NCs, although, their small size leads to unique properties like quantum confinement effects and size-dependent fluorescence. As described in our work, the enhanced optical properties of GNCs enable them to be used as contrast agents for creating highly sensitive multimodal photoacoustic microscopy (PAM) and optical coherence tomography (OCT) imaging. In contrast, gold NCs, exhibiting discrete absorption bands in the visible region, are not well-suited for these applications. Moreover, the peak wavelength of the enhanced optical absorption and scattering exhibited by GNCs is tunable and can be extended into the near-infrared (IR) region by manipulating their lengths. [15] This distinctive feature makes GNCs a superior choice for biomedical imaging due to the deep penetration depth of near-IR photons.
Another advantage of GNCs is their easy and controllable surface functionalization property. In our previous publication, we showed that GNCs fabricated in this study allow versatile surface functionalization to achieve the conjugation of multiple types of ligands with predetermined amounts due to the cappingagent-free surface of the gold NP monomers used in the formation of the GNCs. [19][20][21] This unique property enables to overcome the long-standing challenge of developing nanomaterialbased probes for in vivo multimodal molecular imaging that demands excellent colloidal stability, outstanding biocompatibility, and highly effective targeting capabilities, in which precise quantitative control over the surface modification of multiple types of functional ligands is necessary to accomplish these goals. Unlike GNCs, gold NCs are typically coated with a compact layer of organic ligands to prevent aggregation or oxidation. However, this coating inhibits the use of the rich gold chemistry for facile binding of functional biomolecules, making it difficult to achieve precise surface modification.
Finally, the GNCs are synthesized using a novel and environmentally friendly method called pulsed laser ablation (PLA). PLA has been chosen as the preferred method because it is a completely green synthesis that produces colloidal stable and capping-agent-free gold NP monomers directly in deionized (DI) water. Nevertheless, gold NCs are generally synthesized using wet-chemical methods. These methods involve chemical precursors, reducing agents, as well as stabilizing/capping ligands, all of which could have negative environmental impacts.
Besides the benefit of largely improved biosafety, these ultraminiature GNCs generate stronger PA signals than their absorption-matched counterparts due to the fact that PA signal amplitude is proportional to the surface-to-volume ratio of the NPs. [12] To validate the ultraminiature GNCs as a multimodal imaging contrast agent and to demonstrate their urinary excretion, we performed in vivo longitudinal PA and OCT molecular retinal imaging in two clinically relevant rabbit models of CNV diseases: laser-induced retinal vein occlusion (RVO) and subretinal injection of vascular endothelial growth factor (VEGF) and Matrigel. To achieve specific targeting to the angiogenesis in the choroid, the ultraminiature GNCs, after fabrication, were conjugated with linear arginine-glycine-aspartate (RGD) peptides (i.e., GNC-RGD). They were then administered to the rabbits by intravenous injection route to examine their performance in molecular imaging of CNV, as well as their biosafety.

Fabrication and Characterization of Red-Light Absorbing Ultraminiature GNC
In our development of the PLA method for the fabrication of GNPs, we observed that the size of the generated GNP www.advancedsciencenews.com www.advmat.de monomers could be reduced by producing them in DI water containing a micromolar (μm) concentration of NaCl. Spherical GNP monomers with a mean diameter of 7-8 nm utilized in the formation of ultraminiature GNCs for the present studies were fabricated by femtosecond PLA of a gold target in 100 μm NaCl aqueous solution as shown in Figure 1a. Following the fabrication of these GNP monomers, an established method described in our previous publications [5] was used to self-assemble them into redlight absorbing GNC (Figure 1b). Briefly, this self-assembly was implemented by transforming the surface of 7-8 nm spherical GNP monomers with pentapeptides having an amino acid sequence Cys-Ala-Leu-Asn-Asn (CALNN) and small molecules, cysteamine. The colloidal solution of GNP monomers was sequentially mixed with CALNN peptides and cysteamine. The mixture was maintained at room temperature for ≈24 h to 72 h until its color changed from pink-red to blue. The absorption spectrum of the mixture solution was determined to confirm the successful self-assembly of GNPs to ultraminiature GNCs. To prevent the formation of a protein corona on the surface of the ultraminiature GNCs in vivo, we modified them with poly(ethylene glycol) (PEG), a well-known and effective method for avoiding protein adsorption in vivo because of the fact that PEG moieties on the NP surface create steric hindrance for protein adsorption [22,23] and the high resistance of the obtained GNCs to protein binding was confirmed via in-vitro test using an aqueous solution of bovine serum albumin (BSA) with mass concentration as high as 5 mg mL −1 ( Figure S1a, Supporting Information). For purposes of in vivo targeting of angiogenesis and fluorescence imaging, the ultraminiature GNCs were then conjugated with Arg-Gly-Asp (RGD) peptides and near-infrared ICG dyes (Figure 1b). The morphologies of a single ultraminiature GNC (top) and a large GNC (bottom) composed of 20 nm diameter GNPs were visualized by transmission electron microscopy (TEM) (Figure 1c), where the chain structure is formed by 3-6 individual GNPs. The synthesized ultraminiature GNC had an optical absorption peak that shifted from 520 nm to 610 nm ( Figure 1d) when compared with that of the GNP monomers. As shown in the TEM images (Figure 1c), the sizes of the ultraminiature GNCs are ≈30.0 ± 2.1 nm (length) by 7.8 ± 1.1 nm (width). In addition, their hydrodynamic particle size distribution was determined via dynamic light scattering analysis which revealed an average size of 55.3 nm with a polydispersity index of 0.3 ( Figure 1e). Furthermore, zeta potential of the synthesized ultraminiature GNCs was measured to be ≈−41 ± 2 mV, which allowed to achieve excellent colloidal stability demonstrated by no obvious change in the absorption spectra over time for up to two months ( Figure S1b, Supporting Information).
Finally, the loading efficiency of ICG and the amount of RGD peptide on the surface of ultraminiature GNCs were determined using a method involving DLS size measurement described in our previous papers. [19,21,24] It was concluded that the loading efficiency of ICG is ≈100% (or 25 ICG molecules per GNC) because the amount of ICG initially added is below that necessary for saturating the surface of GNCs and there were ≈200 RGD peptides on the surface of individual ultraminiature GNCs. Since the number of ICG molecules contained within individual GNCs was very small and did not change the peak absorption of GNC after conjugation ( Figure S1c, Supporting Information), except for fluorescence imaging, the signal for the other two imaging modalities (PAM and OCT) reported in this study was generated directly from the GNCs.
As a reference for comparing the photoacoustic performance and the photostability of the GNC, we also fabricated GNC composed of 20-nm GNPs (named large GNC henceforth). For the synthesized large GNCs, their average size was ≈64 nm (length) by 20 nm (width), as shown in the TEM images (Figure 1c), and their redshifted longitudinal SPR was controlled to be ≈650 nm, for matching to that of the ultraminiature GNC as much as possible.

Targeting Efficiency, In Vitro Photostability, and PA Performance of the Ultraminiature GNC
We first examined the targeting efficiency of the ultraminiature GNC. RGD peptides and ICG dyes were capped on the surface of the synthesized GNC ( Figure 1b). The presence of RGD and ICG were confirmed by FT-IR analysis ( Figure S1d). In our previous study, we have shown that RGD peptides have a great ability to target newly developed retinal vessels. [5,25,26] ICG is approved by the FDA for use in ophthalmology and is thus a great fluorophore candidate for in vivo molecular imaging. [27] The targeting specificity of the GNC was examined on human retinal pigment epithelial (ARPE-19) cells with differentiated properties, human umbilical Vein Endothelial Cells (HUVEC), and HeLa cells with and without RGD conjugation using laser scanning confocal microscopy ( Figure 1f; Figure S2, Supporting Information). The results show that GNC (red color) accumulated in the cytoplasm (yellow color) of the cells, confirming the targeting specificity of RGD-conjugated GNC. The internalized ultraminiature GNC inside the cells could also be visualized using PA microscopy (PAM) imaging (Figure 1g,h), which illustrates that our custom-built multimodal PAM and OCT system ( Figure S3, Supporting Information) has a great capability to detect the accumulation of ultraminiature GNC inside single cells with excellent contrast and resolution ( Figure 1h).
To evaluate the potential decay of PA signals caused by photodamage of the ultraminiature GNC under nanosecond pulsed laser irradiation, as seen by changes in their UV-vis absorption spectrum, colloidal solutions of both ultraminiature and large GNC were exposed to 75 000 laser pulses at various laser fluences (i.e., 0.005 to 0.04 mJ cm −2 ). The relative PA signal amplitudes at each fluence were recorded and the absorption spectra of the ultraminiature GNC suspended solutions post-pulsed laser irradiation were also measured (see Note S3, Supporting Information). At the highest laser fluences of 0.04 mJ cm −2 , PA signals of both the large GNC and the ultraminiature GNC were stable and showed no obvious changes ( Figure 1i). The fluctuation of the PA signal amplitude was primarily caused by the intensity variation of the laser system operating at 650 nm. To determine the laser threshold capable of disassembling GNC into GNP monomers, we increased the laser fluence to 8 and 10 mJ cm −2 . Working at 10 mJ cm −2 , we found that the PA signal amplitude reduced ≈3.9fold for the ultraminiature GNC and 6.7-fold for the large GNC after 6000 pulses (Figure 1j). The decay of the PA signal amplitude could be due to the disassembly of the ultraminiature GNC since during the laser irradiation of solutions containing the ultraminiature GNC, the color of solutions gradually changed from www.advancedsciencenews.com www.advmat.de blue to pink-blue for the ultraminiature GNC and to pink-red for the large GNC ( Figure 1k). The disassembly of the GNC were further examined by the observed optical absorption spectra of the samples after laser illumination as shown in Figure 1l-o. The results show that the peak absorption of the large GNC have shifted from 650 nm to 520 nm after being treated by 1200 pulses of 650nm laser beam at both 8 and 10 mJ cm −2 (Figure 1l,n). In contrast, the peak absorption of the ultraminiature GNC was only moderately changed after being irradiated by 1200 laser pulses. Even after being illuminated by 6000 laser pulses at 10 mJ cm −2 , the ultraminiature GNC still kept a relatively broad optical absorption spectrum (Figure 1m,o). This confirms that ultraminiature GNC have better photostability compared to the large GNC.
To compare PA and OCT signals generated by the ultraminiature and the large GNC, we acquired PAM and OCT images for samples, made from silicon tubes and glass tubes, containing either the ultraminiature or the large GNC at two different mass concentrations and for control sample filled with saline (Figure 2a-d). The PAM images show that the ultraminiature GNC provides higher contrast enhancement than the large GNC ( Figure 2a), whereas OCT image contrast of the sample filled with the large GNC is higher than that of the ultraminiature GNC ( Figure 2c). The PAM signal of the ultraminiature GNC exhibits 3.03-fold and 1.99-fold higher than that of the large GNC at a mass concentration of 100 g mL −1 (PA signal = 84.4 ± 1.4 (a.u.) for the ultraminiature GNC and 27.9 ± 2.6 (a.u.) for the large GNC) and 250 g mL −1 (PA signal = 238.2 ± 8.5 (a.u.) for the ultraminiature GNC and 119.7 ± 6.4 (a.u.) for the large GNC), respectively ( Figure 2b). Compared to the background, the ultraminiature GNC at 250 g mL −1 enhanced the PA signal ≈118.2fold (PA signal = 0.2 ± 0.1 (a.u.) for background). By quantification of the OCT signal extracted from the targeted areas on the OCT images, the OCT intensity of the ultraminiature GNC is 1.65-fold lower than that of the large GNC at 100 g mL −1 (OCT signal = 79.5 ± 9.4 (a.u.) for the ultraminiature GNC vs OCT signal = 167.3 ± 5.7 (a.u.) for the large GNC). However, there was not significant difference between these two samples at a higher mass concentration of 250 g mL −1 (OCT signal = 182.5 ± 2.3 (a.u.) for the ultraminiature GNC vs OCT signal = 207.4 ± 8.5 (a.u.) for the large GNC) as shown in Figure 2d. The observed stronger PA signal of ultraminiature GNC than that of large GNC can be explained by that PA signal is proportional to the surfaceto-volume ratio of the NPs, a mechanism reported by Gambhir and Emelianov, [ 12 while large GNC scatter more light, leading to induced stronger OCT signal than that of ultraminiature GNC. We further examined the OCT signal of the ultraminiature GNC at different concentrations. We found that the OCT intensities were linearly dependent on the concentration of both the ultraminiature and the large GNC. The minimal detectable concentration is 1 pg mL −1 (Figure 2e-f).

Biocompatibility, Disassembly Ability, and Renal Excretion Analysis,
We first evaluated the potential toxicity of the ultraminiature GNC in vitro and in vivo. Cytotoxicity analysis shows that the ultraminiature GNC induced a mild decrease in cell viability on HUVECs, HeLa cells, and ARPE-19 cells. The cell viability is >80% after being treated with 500 μg mL −1 of the ultraminiature GNC for 48 h (Figure 3a,c; Figure S4a, Supporting Information). To observe any potential of photothermal effect which could cause cell death after pulsed laser irradiation, MTT assay analysis of cells was performed after they were incubated with the ultraminiature GNC followed by laser irradiation at various fluences (i.e., 0.0025 to 0.08 mJ cm −2 ). A quantitative cell viability assay demonstrated a mild decrease in the viable cell population, and the percentage of live cells was ≈80% at 48 h post laser irradiation (Figure 3b,d; Figure S4b, Supporting Information).
The cytotoxicity was further investigated using flow cytometry to determine the percentage of apoptotic cells caused by internalized ultraminiature GNC with and without laser illumination ( Figure 3e). The results show that ultraminiature GNC induced mild apoptosis in the cells after treatment even after an incubation time of 48 h. Compared to the control, the population of viable cells is not significantly changed for the groups treated with ultraminiature GNC (live = 92.05% for control vs 91.12% and 90.07% for GNC at 24 h and 48 h). The percentage of the apoptotic cell population is only ≈1-2% higher than that of the control group (apoptosis = 1.58% for control vs 3.64% and 4.01% for GNC at 24 and 48 h). In addition, the results also illustrate that incubation with ultraminiature GNC followed by laser irradiation of 0.01 and 0.02 mJ cm −2 only induced minimal toxicity to the cells. Biodistribution of GNC in organs shows that some of them still accumulate in the spleen and kidney (Figure 3f) although the ultraminiature GNC can possibly escape from the RES system and be cleared from the body via the renal system as shown in Figure 3l. However, the amount of the ultraminiature GNC in the liver and kidney was significantly lower than that of the large GNC. We then investigated the disassembly of the ultraminiature GNC in liver tissues and found that small amounts of GNPs were observed on the TEM image of the tissue treated with ultraminiature GNC (Figure 3g). In contrast, a large amount of GNPs was accumulated in the cytoplasm of the group treated with large d) UV-vis absorption spectra of 7-8 nm diameter GNP monomers and ultraminiature GNC-RGD. e) Dynamic light scattering (DLS) data showing the particle distribution of GNP monomers and GNC-RGD measured in an aqueous solution. The inset images show the colloidal solutions of 7-8 nm CNP monomers (red color) and ultraminiature GNCs (blue color). f) Confocal microscopy images of: fi-fiii) HUVEC, ARPE-19, and HeLa cells incubated with 100 μg mL −1 of non-targeting GNC and fiv-fvi) GNC-RGD for 24 h. Yellow fluorescence reveals the location of the lysosome stained by the lysotracker. The blue color represents the morphology of the cell's nuclei marked by DAPI. Green fluorescence shows the cell's actin stained by phalloidin. The red color displays the internalized targeting ultraminiature GNCs in lysosomes stained by ICG dye. g,h) PAM images of ARPE-19 cells incubated with nontargeting GNCs (g) and with targeting GNCs (h). The orange color specifies the internalized GNCs uptake by cells. i) Comparison of PA signals obtained from ultraminiature GNCs (7-8 nm) and large GNCs (20 nm) under laser illumination at 75000 pulses and fluence of 0.04 mJ cm −2 . j) Threshold damage of ultraminiature and large GNCs. PA signals from large GNCs were significantly reduced. k) Photograph of samples before and after laser irradiation. Large GNCs almost disassembled under 6000 pulses laser illumination of 10 mJ cm −2 (pink color) while a fraction of ultraminiature GNC disassembled (blue-pink color), confirmed by the suspension solution color shift from blue to pink. l-o) UV-vis absorption spectra of large and ultraminiature GNCs after laser illumination at different exposure pulses (1200, 3000, and 6000 pulses) and laser fluences (8 and 10 mJ cm −2 ).   19 and HUVEC cells after treatment with ultraminiature ICG-GNC-RGD at 100 μg mL −1 followed by laser illumination at different laser fluences (i.e., 0.01-0.08 mJ cm −2 ). e) Flow cytometry analysis. f) Biodistribution of GNC in different organs. Data are shown as mean and standard deviation (SD) (N = 3, *p < 0.05, **p < 0.01, and ***p < 0.001). g-i) Transmission electron microscopy (TEM) images of rabbit's liver after administration of ultraminiature GNC (g), large GNC (h), and 60 nm-diameter GNPmonomers (i) at day 28 post-injection. The large GNCs and 60 nm-diameter GNP monomers were found in the cytoplasm GNC and 60-nm GNPs (Figure 3h,i). This illustrates that the ultraminiature GNC can disassemble and thereby enhance clearance from the liver at 1-month post-treatment. The efficient disassembly of ultraminiature GNC inside live cells was further confirmed by TEM Figure 3j,k). The results demonstrate that ultraminiature GNCs were accumulated in the cytoplasm (Figure 3j) and broken into 7-8 nm GNP monomers at day 7 post-treatment ( Figure 3k).
The renal excretion ability of the ultraminiature GNC was evaluated and compared with the large GNC and GNP monomers in living rabbits. The ultraminiature and the large GNC and large GNP monomers were conjugated with RGD peptides and ICG dyes and intravenously administrated into the rabbits at a volume of 400 μL (5 mg mL −1 ). The collected urine solutions were imaged with a fluorescent imaging system for both pre-and postinjection at different time points (Figure 3l). No fluorescent signal was observed for the large GNC and GNP monomer samples at any time after injection, whereas for the ultraminiature GNC, the fluorescent signal reaches the maximum at 2 h post-injection before gradually reducing over time (Figure 3m). The amounts of GNC in the urine at different time points ranging from 2 h to 4 days were measured by inductively coupled plasma mass spectrometry (ICP-MS) technique and estimated to be 3.3 × 10 4 ppb at 2 h post-injection. The renal excretion of the ultraminiature GNCs was observed for up to 7 days (Figure 3n). To further evaluate the distribution of GNC in urine as a result of renal clearance, we implemented an extra experiment by euthanizing the animal at 6 hrs post I.V. injection of GNC at a volume of 400 μL (5 mg mL −1 ). The organs and urine were harvested for ICP-MS and UV-vis spectrometer analysis. The ICP-MS data ( Figure S5a, Supporting Information) demonstrates that most GNCs were detected in urine samples at higher rates than that in other organs, illustrating that the administered GNCs were able to be cleared from the body via the urine pathway. We also found that the accumulation of GNCs in kidneys is quite low. This can be explained by the short retention period of GNCs in kidneys as a result of their 1D linear structures. This is consistent with the results obtained by Ruggiero et al. who reported enhanced renal excretion of rod-shaped nanostructures due to flow-induced orientation, which caused a renal clearance of 65% of the injected dose within 20 min. [28,29] In addition, large molecules/particles were filtered faster than small ones because the smaller ones are physically retained by the stationary phase for a longer period. [30] The absorption spectrum of urine was further characterized and plotted in Figure S5b, Supporting Information. There is an absorption peak observed at 550 nm which is associated with the peak absorption of gold nanoparticles (GNPs) generated after the disassembly of GNCs. This result confirms the distribution of GNPs in urine which is consistent with ICP-MS data and fluorescent images (Figure 3l). To assess blood circulation half-life (t 1/2 ) of the ultraminiature and the large GNC, blood was drawn at 15 min, 2 h, 4 h, 1 day, 2 days, 3 days, and 7 days post-injection, and the amount of GNC was determined using ICP-MS as shown in Figure 3o. The data shows that the large GNCs have a circulation half-life of 0.5 h whereas the ultraminiature GNCs have a significantly extended circulation half-life of ≈4 h.
In vivo biosafety was carefully examined using different methods. The body weight of the treated animal was monitored at different time points for up to 1 month (Figure 4a). The data shows that all treated animal weights gradually increased, confirming nanoparticles induce minimal toxicity at the tested concentration. We further performed liver and kidney function tests (LFTs and KFTs) on mice and rabbits. We first tested the dose effect of ultraminiature GNC in mice by administrating them at different concentrations from 2.5 to 20.0 mg kg −1 and followed up for 30 days (N = 3 per group). Both LFTs and KFTs show that blood urea nitrogen (BUN), alkaline phosphatase (ALP), and alanine amino transferase (ALT) at all tested levels were normal (Table S1, Supporting Information), illustrating no severe damage to liver and kidney function or systemic toxicity. In addition, LFTs and KFTs were also performed on rabbits post-administration. There were no adverse changes in ALT, BUN, and ALP, indicating that ultraminiature GNC is biocompatible with the animal at low concentrations (Table S2, Supporting Information). To determine the threshold of the GNC that causes a toxic effect on the kidney and liver, we administrated ultraminiature GNC and large GNC at high concentrations of 2200 and 8800 mg kg −1 , respectively, and followed the animals for 7 days (N = 3 per group). We found that the body weight of the treated animal rapidly reduced and exhibited a 35% loss post-treatment (Figure 4b). In addition, the level of ALT, BUN, and ALP was higher than the normal range (Table S3, Supporting Information). This indicates that the LFTs and KFTs of the animals could be affected by GNC at a high concentration. We further performed histological and TUNEL assay analyses on the treated tissues to examine whether administered GNC had long-term toxicity. The cellular morphology fragmentation and tissue structure damage were not observed on the hematoxylin and eosin images achieved from the control and treated groups at low concentrations (Figure 4c,h). In contrast, there was evidence of tissue structural changes observed in the liver, kidney, lung, spleen, and heart samples treated with a high concentration of large GNC (Figure 4d). Minimal tissue damage was found on the liver and spleen treated with a high concentration of ultraminiature GNC (Figure 4e). Minimal evidence of cells undergoing apoptosis was detected on TUNEL images obtained from samples treated with ultraminiature GNC at both high and low concentrations (Figure 4g,i). But significant numbers of apoptotic cells were found in the samples treated with high concentrations of large GNC (Figure 4f). Figure 4j shows the positive control of spleen tissues.  , but all other groups showed stable body weight. c-e) Biosafety analysis of the treated organs at the highest concentration (8800 mg kg −1 ) using H&E staining: c) control, d) large GNC, and e) ultraminiature GNC, and f,g) TUNEL assay of large GNC (f) and ultraminiature GNC (g) group. The green color indicates the apoptosis cells caused by the accumulation of GNPs. h,i) Biosafety analysis of the treated rabbit's organs using H&E staining (h) and TUNEL assay (i). j) Positive control of spleen tissues. No evidence of the apoptosis cells was observed on the treated tissues with a low concentration of ultraminiature GNC, confirming that the ultraminiature GNC had greatly biocompatible and induced minimal toxicity to the animal. Blue fluorescence shows the cells' nuclei.

Efficient Molecular Targeting of Laser-Induced Choroidal Neovascularization
To assess the feasibility of our ultraminiature GNC as multimodality PA and OCT molecular imaging contrast agents, we carried out multimodality PAM and OCT imaging by applying a clinically relevant AMD disease model in rabbits, named choroidal neovascularization (CNV), as shown in Figure 5a. The CNV models were created by laser-induced retinal vein occlusion (RVO) with Rose Bengal (Note S6, Supporting Information). We allowed the newly developed CNV to stabilize at day 28 after RVO and day 7 after VEGF administration. Then, four groups of rabbit models were formed (RVO = 2 groups and VEGF injection = 2 groups) with each group containing three rabbits. Four groups with ei-ther RVO or VEGF injection received an intravenous injection (I.V.) of non-targeted ultraminiature GNC or targeted ultraminiature GNC via RGD functionalization, respectively, through the marginal ear vein with the same injection amount of 400 μL at a mass concentration of 5 mg mL −1 . Longitudinal color fundus, fluorescence, PAM, and OCT imaging were obtained for both preinjection and for up to 28 days post-injection. Color imaging and fluorescence angiography (FA) were used to monitor the development of CNV (Figure 5b Figure S6, Supporting Information), allowing easy observation and distinguishing CNV from the adjacent healthy retinal and choroidal vessels as shown in Figure 5f. The location of CNV was longitudinally observed up to 21 days post-injection with great contrast and then the PA signal amplitude decreased and was almost resolved by day 28 post-administration. Selected co-registered three-dimensional PAM rendering obtained at 578 and 650 nm demonstrate better morphology of choroidal vessels (CVs), capillaries, retinal vessels (RVs), and CNV in 3D with high resolution (Figure 5h; Videos S1 and S2, Supporting Information). The margin and degree of CNV were significantly distinguishable from the surrounding vessels. PAM depth view along XZ depicts the location of CNV lies between RVs and CVs (Figure 5i) and is co-registered well with 2D OCT image (Figure 5j). In contrast, for rabbits injected with nontargeted GNC, the PA signal was very low and almost undetected at the optical wavelength of 650 nm post-nanoparticle administration owing to a lack of binding of GNC onto CNV (Figure 6).
The PA signals reached a maximum at 48 h post-injection of targeted GNC and then gradually decreased over time (Figure 5l). When compared to pre-injection, we observed a 25.3-fold increase in PA signal post-injection of targeted ultraminiature GNC (PA signal = 1.20 ± 0.01 (a.u.) for pre-administration vs 30.5 ± 1.2 (a.u.) for 48 h after administration; p = 0.001). Remarkably, the PA signal obtained from using RGD-conjugated ultraminiature GNC was significantly higher than that obtained from using RGD-conjugated large GNC (Figure 5m). Compared to RGDconjugated large GNC, RGD-conjugated ultraminiature GNC exhibited a 1.5-fold increase in PA signal (p = 0.001). In addition, the ultraminiature GNC provided a strong PA signal up to 21 days post-injection, while large GNC only produced a strong PA signal within 7 days post-injection, [5] gold nanorods (GNRs) targeted CNV up to 9 days, [25] and gold nanostars (GNS) targeted CNV for up to 7 days. [26] These results confirm the better performance of the ultraminiature GNC in the context of enhancing PA signal and allowing prolonged longitudinal observation time compared to that of large GNC, GNR, and GNS. In addition, the ultraminiature GNCs have great photostability. There was no significant signal decay seen in the PAM images obtained at three different scans (Figure 7a). Spectroscopic PAM image (Figure 7b) exhibits that the CNV lesion was clearly detected and distinguished at wavelengths from 610 to 670 nm. The quantitative measurement of PAM signals at ROIs extracted from PAM images (Figure 7c) is consistent with the UV-vis absorption spectrum shown in Figure 1d.
Cross-sectional 2D OCT images were also obtained for both pre-and post-injection at different time points (day 1, 2, 3, 5, 7, 14, 21, and 28) as shown in Figure 8 and Figure S7 (Supporting Information). CNV were clearly observed on the 3D volumetric OCT images obtained at 24 h post-injection (Figure 8b) showing higher contrast than that of the one obtained pre-injection ( Figure 8a). The CNV lesion was clearly found on the 3D image post-injection. Cross-sectional 2D OCT image (Figure 8b1) and the further magnified one (Figure 8b2) display the depth location of CNV in the subretinal space with higher contrast in comparison with the pre-injection one (Figure 8a1). To better identify the margin and position of CNV, PAM images were co-registered with OCT images on the same imaging plane and displayed in Figure 8c,c1. The position of CNV were noticeably observed and discriminated from the surrounding vasculature. These results illustrate the great capability of multimodal PAM and OCT platforms for precisely identifying newly developed microvasculature. Quantitative OCT signal intensity shows that the highest OCT signal peaked at 24-72 h post-administration. Afterward, this signal progressively reduced. Although the ultraminiature GNC generated a strong OCT signal post-injection, allowing easy visualization of CNV in the subretinal space, the OCT signal was only moderately increased by 51%. Compared to large GNC, OCT signal generated by ultraminiature GNC was ≈20% lower, which could be attributed to their lower scattering efficiency. This result is consistent with the in vitro result (Figure 2a).

In Vivo GNC Targeting CNV after Subretinal Injection of Matrigel and VEGF
To evaluate the targeting efficiency of the synthesized ultraminiature GNC for visualization of different ocular diseases, we further performed administration of the ultraminiature GNC in a rabbit model with subretinal administration of a mixture of Matrigel and VEGF-165 (Figure 9a). CNV generated from 3-7 days post Matrigel and VEGF injection was monitored by color fundus photography ( Figure 5b) and indocyanine green angiography (ICGA) (Figure 9c-e). Color, early phase, and middle phase ICGA pictures clearly display architecture of retinal and choroidal vessels whereas the late phase ICGA image presents the CNV lesion due to leakage of ICG dye (Figure 9e).  Figure S8 (Supporting Information). We found a minimal PA signal in the 650-nm PAM image pre-injection. On the other hand, PA signal was significantly increased in the 650 nm PAM images obtained after treatment. Interestingly, the PA signal persisted up to 21 days post-injection ( Figure 9g) and then nearly resolved by day 28. 3D PAM images generated at 578 and 650 nm were coregistered to facilitate visualization of the CNV lesion from the background signal from the surrounding CVs (Figure 9h; Video S3, Supporting Information).
The 3D OCT image (Figure 9j) acquired after administration of ultraminiature GNC also clearly exhibits the distribution of the GNC at the CNV lesion (white dotted circle), while minimal OCT signal was found in the 3D OCT image pre-injection

Discussion
The current study demonstrates the successful development and utility of ultraminiature GNC as a contrast agent for multimodal PAM and OCT imaging. These results are significant because they demonstrate a platform technology for biomedical molecu-lar imaging. Benefiting from its unique design, the ultraminiature GNC has the optical absorption peak in the red and NIR window and is able to be renally excreted to improve biosafety. As shown in this study, the ultraminiature GNC has largely reduced accumulation in the liver and spleen when compared to large GNCs and large GNPs with other designs. This study also shows that the formulation of the ultraminiature GNC has great photostability and biocompatibility when tested in vivo in a rabbit model. Compared to pre-injection, the ultraminiature GNC achieved a 25.3-fold enhancement in PA contrast and a 150% enhancement in OCT contrast in a rabbit CNV model. Another advantage of synthesizing the ultraminiature GNCs is that the GNC demonstrates the great binding capability to v 3 integrins which are overexpressed in CNV in two different clinically relevant rabbit models using the physical method (RVO model) and chemical method (VEGF and Matrigel injection). Although the mechanism of CNV development is different between these models, the RVO model triggers the release of various growth factors (e.g., VEGF) via the hypoxic effect, and VEGF is delivered directly into the subretinal space via the subretinal injection route, the ultraminiature GNCs demonstrated high targeting efficiency and could aid in the visualization of both the position and the boundaries of CNV in 3D, permitting early diagnosis and therapy.
The study also conducted a comparison between large GNP monomers, large GNC, and ultraminiature GNC. The ultraminiature GNC had an absorption peak similar to that of large GNC but generated an increased PA signal due to their increased surface areas. The particles showed absorption primarily in the red (≈610 nm) optical window which might have ability to shift the optical absorption wavelength to NIR window (≈650 nm) due to the accumulation of GNC at CNV, resulting in increased PA signals and helps to distinguish CNV from the surrounding microvasculature, where there was a very low intrinsic PA signal from the background tissue such as hemoglobin (absorption coefficient: μa 650 = 1.98 cm −1 ). The smaller size was of interest to this study because larger GNPs have been shown to accumulate in the liver and spleen, which raises long-term toxicity concerns. [31] Current knowledge about the effect of NP size indicates that smaller size could penetrate the blood-brain and blood-retinal barriers. [32] However, this concern is alleviated by the biocompatibility of GNPs and the kidneys' ability to clear compounds <8 nm in size, [33] which would indicate the com-pound could be efficiently excreted while maintaining an ideal residence time for image acquisition.
The toxicities related to the size of GNPs have been studied recently by several researchers. [34,35] Notably, Chen et al. discussed the size-dependent biological response of GNPs. They found that some larger sizes GNPs (8 to 37 nm) induced sickness in rats, while those in the range of 3 to 5 nm did not exhibit sickness or lethality. [35] Similarly, a study by Chou et al. reported size reduction in GNPs was associated with reduced long-term and short-term toxicity. [36] This evidence demonstrates that ultraminiature GNC are ideal because they can be efficiently cleared over time, reducing long-term toxicity, while maintaining sufficient residence time to ensure they can be effective contrast agents. Their relatively higher molar mass in comparison to organic fluorochromes like ICG and Prussian blue allows them residence in CNV over time, providing strong PA signal amplitude at 2 h post-administration, and subsequently can be cleared over the next 28 days through urine. However, there were some GNPs detected in the lespleen and liver at day 28 post-injection (Figure 3f). This might be caused by the accumulation of some large GNC that could not clear from the urine. These GNPs might need additional time to disassemble into smaller GNP monomers in or-der to be cleared from the liver and spleen tissue through hepatic clearance, and lymphatic clearance. [33] We also observed that the liver and renal functions of the animal were normal with no evidence of systemic toxicity when the animal received less or regular dose of the injection. On the other hand, severe toxicity was . The potential toxicity of large GNC might be caused by size-dependent toxicity, reactive oxygen species (ROS) production, and immune response. Large GNCs (>200 nm) may have a greater tendency to accumulate in certain tissues or organs such as the spleen and liver and may also be more difficult for the body to clear, leading to potential toxicity. [20,25,26,37,38] The large size of the GNCs may also affect their interactions with biological molecules, such as proteins or cell membranes, which could lead to toxicity. The higher concentrations of GNCs the greater the surface area to volume ratio of these particles causing their higher chemical reactivity and interaction of their surfaces with biological systems. This interaction could potentially lead to increased production of ROS, which can cause oxidative stress, inflammation, and consequent damage to the proteins, cell membrane, and tissues. [39][40][41] Large metal nanoparticles may be recognized as foreign and trigger an immune response, which could lead to acute inflammation of the lung, liver, and systemic damage. [42,43] As demonstrated by this study, the ultraminiature GNCs have the benefits of good in vivo stability in the short term, good photostability, and no cellular toxicity. The chains were gradually disassembled and cleared by 28 days post-administration. The study on imaging quality showed that the ultraminiature GNC allowed for effective PA imaging of CNV for 21 days post-injection. The PA signal enhancement in CNV over time was consistent, and the relatively larger change in PA signal enhancement due to the clearance of GNC was detectable only after 7 days post-injection. This duration is beneficial because it permits repeated imaging without the requirement of multiple injections. One shortcoming of the smaller size observed in the ultraminiature GNC is a slightly decreased OCT signal. However, this can be overcome by performing overlay images of PAM and OCT to better define CNV boundaries. The smaller GNC showed robust absorption efficiency in the red window, allowing their utility in imaging CNV using PAM at 650 nm. This wavelength is ideal since hemoglobin produces almost no PA signal at the optical wavelength of 650 nm, which enables differentiation of the biodistribution of photosensitizer materials from the native microvasculature with an injection of only 400 uL ultraminiature GNC at 5 mg mL −1 .
The investigation further supports the use of multimodal OCT and PAM for the detection of neovascularization in the sub-RPE. Many studies and clinical practices have investigated solely using OCT or OCTA. These methods are important for the visualization of healthy vasculature and conditions like RVO. However, these methods rely on backscattering light into target tissues, which limits their penetration depth. [44] This study verified the hybrid biomedical imaging method of integrated PAM and OCT for imaging of CNV over time, benefiting from the noninvasive, 3-D imaging, and high spatial and temporal resolution nature of the PAM and OCT imaging modalities. [45] Through this integration, OCT provides valuable structural and functional information, [46] while PAM provides pertinent functional and molecular details. [47][48][49] While this system provides enhanced imaging of retinal neovascularization with high contrast and resolution, it still needs refinement prior to clinical translation. One of the current limitations is the lengthy image acquisition time, which is limited by the 1 kHz of pulse repetition rate of the optical parametric oscillator (OPO) laser used for PAM. The current image acquisition time is roughly 65 s which could be significantly reduced by employing a high-speed laser system. Future investigation evaluating the clearance of GNC with various sizes, shapes, and formulations is also indicated. In addition, a more comprehensive understanding of GNC clearance can be performed in a future study to better understand the molecular pathology of GNCs.
In conclusion, this study validates the capability of biocompatible and photostable ultraminiature GNC as an excellent photosensitizer for PAM and OCT molecular imaging of a CNV rabbit model. The ultraminiature GNC exhibits great optical properties such as absorption and scattering efficiencies in the redshifted spectral range ≈610 nm. Also, these GNC are photostable even after thousands of pulses of laser irradiation. Furthermore, they demonstrate excellent renal excretion which results in reduced long-term toxicity, while maintaining a relatively long residence in CNV. The in vivo data illustrates that PAM signal was enhanced up to 25.3-fold and OCT intensities were enhanced by 150% in comparison to pre-injection. Even at doses as low as 2 mg, these contrast enhancements permitted visualization of the position and margin of CNV with high sensitivity and high spatial resolution. The combination of the ultraminiature GNC and multimodal PAM and OCT yields high-contrast, 3D volumetric imaging of CNV anatomy with sub-micrometer resolution.

Experimental Section
Targeting Ultraminiature GNC Labeled with ICG Dye for Retinal Molecular Imaging: RGD functionalized ultraminiature GNC labeled with ICG dye (ICG@GNC-RGD) were fabricated as an imaging photosensitizer to improve high-resolution multimodality fluorescence, PAM, and OCT imaging for evaluation of clinically relevant disease models of CNV. Briefly, we first produced primary colloidal GNP spheres (7 -8 nm) to be utilized for the synthesis of ultraminiature GNC via a laser-based method by ablating a massive fine gold sample (99.99%) in deionized water (18 MΩ cm) with a femtosecond pulsed laser beam, as described in the previous publication [19] and Notes S1-S3 (Supporting Information). In addition, it was worth mentioning that the GNPs fabricated by the pulsed laser ablation (PLA) technique were naturally negatively charged without requiring capping ligands and stabilizing agents for retaining their colloidal stability and avoiding aggregation. This unique countenance allowed versatile surface modifications, [19] which were employed in this study to synthesize ultraminiature GNC.
The self-assembly processing of dispersed GNPs into ultraminiature GNCs was implemented by sequentially mixing the dispersed GNPs solution with CALNN peptides and followed by cysteamine. In a typical process, 0.75 mL CALNN solution at a concentration of 200 μm was added to a 50 mL sample of GNPs (7-8 nm) having an optical density (OD) of 1.0 at 512 nm. This mixture was maintained for 2 h at room temperature to allow the binding of CALNN ligands onto the surface of GNPs via gold-sulfur bonds. Afterward, the mixture CALNN-GNPs were further conjugated with cysteamine molecules by adding 1.0 mL cysteamine at a concentration of 200 μm. The mixed solution was kept undisturbed until observing a change of color from pink to blue. The raw ultraminiature GNCs were then labeled with NIR ICG dye and functionalized with RGD peptides for CNV targeting. Briefly, a 50 mL colloidal solution of ultraminiature GNC with OD 1 at 610 nm was partially PEGylated by adding 0.1 mL PEG thiol-terminated (PEG-SH) with 2000 g mol −1 molar mass (PEG 2k-SH) with a concentration of 100 μm and kept undisturbed at room temperature for 2 h to enable sufficient conjugation of PEG 2k-SH onto the ultraminiature GNC. After the reaction, partially PE-Gylated ultraminiature GNC were labeled with ICG dye by the addition of 0.1 mL aqueous solution of ICG-PEG 2k-SH at a concentration of 100 μm. This mixture was left unperturbed for 2 h to allow ICG labeling (ICG-GNC). In the end, 1.0 mL RGD solution at a concentration of 200 μm was then added to the ICG-GNC. The solution was then left for two additional hours at room temperature to allow conjugation of RGD onto unoccupied sites of the ICG-GNC. The resultant solution was diluted by adding 50 mL of 4 mm borate buffer (pH 8.2) containing 8 mg mL −1 BSA and then was transferred into two 50 mL centrifugal tubes for spinning down at 500 g for 2 h to a pellet. The final OD of the colloidal solution of PEGylated and RGD functionalized ultraminiature GNC labeled with ICG (ICG-GNC-RGD) was then adjusted to ≈100 at 610 nm through resuspension of the pellet with borate buffer (4 mm, 8.2 pH) containing 4 mg mL −1 BSA after removal of the supernatant.
Photophysical Properties of the RGD-Conjugated Ultraminiature GNC (GNC-RGD) and ICG-labeled GNC-RGD (ICG-GNC-RGD): TEM images of colloidal GNPs and the ultraminiature GNCs were obtained using JEOL 2010F operated at 200 kV. The maximum plasmonic absorption peak of GNPs and the ultraminiature GNCs were determined from the optical wavelengths of 350 to 800 nm through the use of a spectrophotometer (UV-3600, Shimadzu Corp., Japan). Surface zeta potential values and hydrodynamic nanoparticle size distribution were determined by a Zetasizer Nano ZS90 equipment (Malvern Instruments, Malvern, UK). The IR spectra of GNP monomer, CALNN peptide, GNC, thiolated PEG 2000, and PE-Gylated GNC were evaluated by an FTIR spectrometer (PerkinElmer Inc., Waltham, MA, USA).
The colloidal stability of the ultraminiature GNC was evaluated by measuring the plasmonic absorption spectrum of the ultraminiature GNC sample incubated in serum and followed up for 1 month. Zeta potential values and particle size of the sample were also acquired. The potential redshift of NPs was also characterized in the biological environment (Notes S1 and S3, Supporting Information). Photostability of the synthesized NPs was determined by measuring the plasmonic absorption spectrum of the samples after being exposed to nanosecond pulsed laser (650 nm and 75 000 pulses) at different fluences: 0.005, 0.01, 0.02, and 0.04 mJ cm −2 and higher fluence of 8 and 10 mJ cm −2 (Note S5, Supporting Information).
In Vivo Renal Clearance of GNC-RGD and Half-Life Circulation: To examine the renal clearance of the synthesized ultraminiature GNC-RGD in living rabbits, analysis was performed including fluorescence imaging and inductively coupled plasma mass spectrometer (ICP-MS) analysis. Three different GNP samples (disperse GNPs with a core diameter of 60 nm, GNC composed of 20 nm diameter GNP spheres, and ultraminiature GNC composed of 7-8 nm diameter GNP spheres) with the same concentration of 5 mg mL −1 were intravenously administered to the rabbits at a dose of 400 μL. Urine solutions were obtained from 2 h to day 7 post-injection. 5 mL of urine was added to an assay well on a 24-well tissue culture plate. Fluorescence images were captured at an NIR wavelength of 800 nm with an exposure time of 1 ms using Azure 600 bioimaging system (Dublin, CA, USA). The fluorescent signal intensity was then measured using region of interest (ROI) analysis. The kinetics of GNC in urine was examined. The urine samples were then diluted in aqua regia solution (mixed nitric acid and hydrochloric acid with a ratio of 1:3) to determine the amount of GNPs in those samples using wet dissolve protocol as described in previous studies [5,6,25] (Note S4, Supporting Information. The dissolved samples were mixed with 4 mL of D.I. water for the measurement using an inductively coupled plasma mass spectrometer (ICP-MS) (Nexion 2000S, Perkin-Elmer, MA, USA).
The circulation time of GNPs in blood was also characterized using wet ICP-MS method. Briefly, 400 μL of blood samples were collected from the injected animal described above at different time points: 15 min, 45 min, 1,2,4,8,16,24,48,72 h, day 4, 5, and 7. The collected blood samples were processed the same as the protocol applied for urine samples and the amount of GNPs in blood were determined by ICP-MS method.
In Vivo Biodistribution of the Ultraminiature GNC-RGD and Cytotoxicity Analysis: In vivo toxicity was tested on three groups of rabbits (N = 3). These animals were intravenously injected with 400 μL of ultraminiature GNC, large GNC, and the PEGylated 20 nm diameter GNPs at the same concentration of 5 mg mL −1 via the marginal ear vein. The body weight of all animals including the control group was acquired daily for up to 1 month. After 1 month, 400 μL of blood samples were taken from all the animals. The samples were then centrifuged at 3 000 rpm for 4 min to isolate serum from red blood cells. The collected serum was used for mini chemistry panel analysis to validate the kidney and liver functions of the treated animal. Afterward, all the rabbits were sacrificed, and the organs (liver, kidney, lung, heart, and spleen) and the eyeballs were isolated. These organ samples were sectioned into 2 g and kept at −20°C to determine the amount of GNPs by ICP-MS (Note S4, Supporting Information). The remained samples were fixed in a 10% formaldehyde solution. The eye tissue was processed for histological, immunohistochemistry, and TUNEL assay analyses (Note S4, Supporting Information).
The potential toxicity of the GNC was further evaluated on mice. Five groups of mice (n = 3 per group) received tail vein injections of ultraminiature GNC and large GNC at different concentrations (0 (control), 5, 10, 20, 40, 2200, and 8800 mg kg −1 ). The body weight of the treated mice was measured daily for up to 7 days. Then, mice were sacrificed. Blood and organs were harvested for LFTs, KFTs, and histological analysis (Note S4, Supporting Information).
Multimodal PAM and OCT Retinal Molecular Imaging System: A specialized, custom-modified, noninvasive multimodality PAM and OCT imaging platform was exhibited in Note S7 and Figure S3a (Supporting Information). [47,50] To generate the PA signal, nanosecond laser pulses (3-5 ns and pulse repetition rate of 1 kHz) from an Nd:YAG laser (NT-242, Ekspla, Lithuania) were used to excite the tissues. The optical wavelength of the excitation light from the OPO could be turned from 405 nm to 2600 nm to match the maximum absorption peak of the sample to achieve the optimal PA signal. The excitation light was filtered and controlled to establish a circular beam of 2 mm before illuminating the sample. For in vivo experiments, a laser fluence of 0.04 mJ cm −2 was selected to excite the sample. Spectroscopy PAM was implemented by tuning the optical wavelength from 500 nm to 700 nm under the same excitation laser fluences. The generated PA signal was detected by an ultrasonic transducer with a center frequency of 27 MHz (Optosonic Inc., Arcadia, CA, USA). [51] The PAM system had a lateral resolution of 4.1 μm and an axial resolution of 37.0 μm. The preamplifier was used to augment the generated PA signal (AU-1647, gain 57 dB, L3 Narda-MITEQ, Hauppauge, NY, USA). The PA signal were then digitized by a DAQ card (PX1500-4, Signatec Inc., Newport Beach, CA). Amira 6.0 software was used to render 3D images and videos (Amira, FEI, Fisher Sci, USA).
Customized high-resolution OCT imaging was established from a Ganymede-II-HR OCT device (Thorlabs, Newton, NJ, USA) as reported in the previous studies. [47,50] The OCT system was combined with PAM system to achieve a multimodality imaging platform. To do so, the OCT light source was coaxially integrated with the excitation light of the PAM system. OCT system used two superluminescent diodes (SLDs) with central frequencies of 846 and 932 nm to illuminate the tissues. The OCT device has a lateral resolution of 3.8 μm and an axial resolution of 4.0 μm. The scanning area was 5.5 (length) × 5.5 (width) × 1.8 mm (depth) with a resolution of 512 × 512 × 1024 pixels.
In Vivo GNC-RGD Targeting of Choroidal Neovascularization: All the animal experiments were implemented in accordance with experimental the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) Statement and the approved protocols by the Institutional Animal Care and Use Committee (IACUC) at the University of Michigan (PRO000010388). Healthy New Zealand White rabbits both genders at age of 3-4 months old and weighing 2.4-2.8 kg were utilized in this report. Choroidal neovascularization (CNV) in the rabbit model was created by two different methods: subretinal injection of Matrigel and human vascular endothelial growth factor (VEGF-165, Shenandoah Biotechnology,