Photostable and Biocompatible Fluorescent Silicon Nanoparticles

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Photostable and Biocompatible Fluorescent Silicon Nanoparticles-Based Theranostic Probes for Simultaneous Imaging and Treatment of Ocular Neovascularization Miaomiao Tang, Xiaoyuan Ji, Hua Xu, Lu Zhang, Airui Jiang, Bin Song, Yuanyuan Su, and Yao He Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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Analytical Chemistry of tumor growth.30 On the other hand, recent years have witnessed giant achievement in the fabrication of ultrasmall (< 10 nm) fluorescent silicon nanoparticles (SiNPs) featuring ultrahigh photostability and negligible toxicity, which are extremely suitable for real-time and long-term bioimaging applications.31-34 Several studies have demonstrated that, under singlecell level, the SiNPs-labeled cancer cells could preserve strong and stable fluorescence of SiNPs under 180-min continuous irradiation in laser-scanning fluorescent confocal microscopy; in marked contrast, the fluorescent intensity of the cells stained by organic dye (e.g., fluorescein isothiocyanate, FITC) rapidly quenched within 10 min.35-37 Furthermore, fluorescent SiNPs were employed as high-quality drug carriers for optical imaging-guided in vivo cancer therapy.38 In this case, the fluorescent SiNPs nanocarriers simultaneously featured bright and stable fluorescence (photoluminescence quantum yield (PLQY):~25%), as well as adjustable drug-loading capacity. Due to the high photostability of SiNPs, such SiNPs-based drug carriers were highly efficacious for simultaneous tumor imaging and chemotherapy. It is worthwhile noting that, despite these exciting progresses, there currently exists scanty information presenting the use of fluorescent SiNPs for the treatment of ocular neovascularization.

um. Rat retinal precursor cells (R28) and Human U87MG glioblastoma cancer (U87MG) cells were cultured in LDMEM. The above medium were supplemented with 10% FBS and 1% penicillin/streptomycin. Human retinal microvascular endothelial cells (HRMECs) were cultured in ECM containing 5% FBS, 1% penicillin/streptomycin and 1% endothelial cell growth supplement (ECGS). Both cellular lines were cultured at 37 °C in a 5% CO2 incubator with humidified atmosphere. For MTT assay, the above cells were cultured in 96-well plates at 5×103/well for 24 h. Afterwards, different concentrations of SiNPs (0, 70, 140, 280 and 560 µg/mL), c(RGDyC) peptides (0, 7, 14, 28 and 56 µg/mL) and SiNPs-RGD (0, 70, 140, 280 and 560 µg/mL, based on SiNPs) were added and incubated for another 24 h, respectively. For each well, cells were incubated with 20 µL stock 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL, purchased from Sigma-Aldrich) for another 4 h and 10% acidified sodium dodecyl sulfate (10% SDS) was added to lyse the cells. For hypoxic group, HRMECs were incubated at 37 °C with 1% CO2 during the whole process. Finally, the absorbance of MTT assay was measured by microplate reader (Bio-Rad 680, U.S.A.) at a wavelength of 570 nm.

Inspired by the above-mentioned achievement, we herein introduce a kind of SiNPs-based theranostic agents, which could realize fluorescence imaging of angiogenic blood vessels and suppression of ocular neovascularization. The high binding affinity of cyclo-(Arg-Gly-Asp-D-Tyr-Cys) (c(RGDyC))-conjugated SiNPs (SiNPs-RGD) to human retinal microvascular endothelial cells (HRMECs) tube formation allows specific detection of angiogenic endothelial cells in vitro. Of particular significance, we reveal that the resultant SiNPs-RGD show obvious anti-angiogenic ability in vitro, and are high-efficacy for inhibiting wound healing migration, transwell migration, transwell invasion, and tube formation. Such resultant SiNPs-RGD is thus further demonstrated as high-performance theranostic agents, especially suitable for labeling angiogenic blood vessels and neovascularization inhibition in a synchronous manner.

In Vitro Cellular Labeling in Tube Formation. 120 µL of the Basement Membrane Matrix (8-12 µg/mL, purchased from Corning Incorporated) was placed in a 24-well plate and incubated for 30 min under 37 °C to form a gel. Afterwards, HRMECs (8×103/well) were added to the 24-well plate and incubated for 8 h. SiNPs (0.5 mg/mL), SiNPs-RGD (0.5 mg/mL, based on SiNPs) or SiNPs-RGD (0.5 mg/mL, based on SiNPs) mixed with 1 μM of c(RGDyC) (i.e., blocking) were then added and co-incubated with HRMECs for 30 min. After washing with 1×PBS three times, 6 µL 1,1′-dioctadecyl3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) solution (1 mM) was added in the well, incubating for another 30 min. Finally, the labeled cells were washed with 1×PBS three times and fixed on slide glasses with fluoromount (Sigma, F4680). The samples were examined using a confocal microscope (Leica, TCS-SP5 II). SiNPs and DiD were excited by 405 and 633 nm, while the emission windows for them were 420-480 nm and 644-663 nm, respectively.

EXPERIMENTAL SECTION Preparation of SiNPs and SiNPs-RGD. The water soluble SiNPs were synthesized based on the previously reported photochemical method.36 To prepare maleimide-activated SiNPs, 100 µL of SiNPs solution (30 mg/mL, pH = 7.5) was mixed with 100 µL of 4-(N-Maleimidomethyl) cyclohexane-1carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC, 50 mM, pH = 7.5, purchased from Thermo Scientific) solution for 2-h incubation at 25 °C. To remove the residual Sulfo-SMCC, the maleimide-activated SiNPs were purified by ultra-filtration (10 kDa) twice with phosphate buffer (50 mM, pH = 6.8, at 6500 rpm for 15 min). Afterwards, the SiNPs-RGD was obtained by reacting 100 µL of cyclo(Arg-Gly-Asp-D-Tyr-Cys) (c-(RGDyC), 50 mM, pH = 6.8, purchased from Apeptide Co., Ltd.) peptides with purified maleimide-activated SiNPs solution for 12 h at 4 °C. Finally, the resultant SiNPs-RGD was purified by ultra-filtration (10 kDa, at 6500 rpm for 15 min) six times (Figure S3).

Wound Healing Assay. To study the effect of SiNPs-RGD on HRMECs migration, wound healing assay was performed.39 In brief, HRMECs dispersed in 12-well plates at 1.5×105/well were incubated for 24 h under 37 °C (5% CO2). Cells were wounded by scraping and the debris was cleaned by gentle washing with 1×PBS three times after 12-h serum starvation. Different concentrations of pure SiNPs (i.e., 280 and 560 µg/mL), c(RGDyC) peptides (i.e., 28 and 56 µg/mL), and SiNPs-RGD (i.e., 280 and 560 µg/mL, based on SiNPs) were added to the medium containing only 2% FBS, respectively. Images of the scratches were taken immediately before and after incubation (i.e., 0, 6, 10, and 14 h) by a microscope. Transwell Migration and Transwell Invasion Assays. Transwell assays were carried out through the previously reported method.40 In details, HRMECs were seeded into per transwell chamber at 2×106 cells and incubated with pure SiNPs (560 µg/mL), c(RGDyC) peptides (56 µg/mL), and SiNPs-RGD (560 µg/mL, based on SiNPs) in medium containing only 0.1% FBS, respectively. The lower chamber contained 5% FBS as chemoattractant. After 24-h incubation,

Cell Culture and MTT Assay. Human breast cancer MCF7 cells were cultured in RPMI-1640 medium. Acute retinal pigment epithelial cells (ARPE-19) were grown in F12 medi-

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HRMECs were fixed with 4% paraformaldehyde for 40 min and stained by haematine for 30 min. In the next, cotton buds were used to remove the non-migrating/invading HRMECs on the upper surface. HRMECs on the basal surface of the membrane were visualized with a microscope. Photographs of three random regions from three repeated wells were acquired and the number of cells was measured using ImageJ software. For transwell invasion assay, cells were seeded into per transwell chamber coated with 120 µL of the Basement Membrane Matrix (1.6-2.4 µg/mL) at 2×106 cells, and then treated as above.

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mg/mL), c(RGDyC) (3.5 mg/mL), and SiNPs-RGD (35 mg/mL, based on SiNPs), respectively. After treatment, corneas were examined, and photographs were taken under the slitlamp microscope on day 1, 7, 8, 10, 12, and 14. Images were used to measure the length of neovascular blood vessels. RESULTS AND DISCUSSION Characterizations of the resultant SiNPs-RGD. The water-dispersed SiNPs are prepared based on the previously reported photochemical method.36 To obtain c(RGDyC)-tagged SiNPs (SiNPs-RGD), two steps are required. Firstly, the asprepared SiNPs are incubated with Sulfo-SMCC to prepare maleimide-activated SiNPs. Secondly, the SiNPs-RGD is obtained by reacting the c(RGDyC) peptides with maleimideactivated SiNPs solution (please see corresponding characterizations (Figure S1-S6) in supporting information). Figure 1a and Figure 1b display the transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images of SiNPs (Figure 1a) and SiNPs-RGD (Figure 1b), appearing as spherical morphology and good monodispersity. Moreover, typical lattice patterns of a single SiNP and SiNP-RGD demonstrate excellent crystallinity, ascribing to (220) lattice planes of silicon with ∼0.19 nm spacing (enlarged HRTEM images of a single SiNP and SiNP-RGD insets in Figure 1a and Figure 1b). By calculating more than 200 particles in the TEM images, the size distributions of SiNPs and SiNPs-RGD are measured as ~3.21 ± 0.59 nm (Figure S1) and ~3.9 ± 0.78 nm (Figure 1c), respectively. It is worth pointing out that, SiNPs and SiNPsRGD show similar sizes owing to the hard discrimination of low contrast of c(RGDyC) peptides measured by TEM.34 However, the dynamic light scattering (DLS) value of the SiNPs-RGD increases to ∼7.5 nm (Figure 1d) obviously, much larger than that of pure SiNPs (~3.7 nm, Figure 1d). Both SiNPs and the resultant SiNPs-RGD show similar profiles of UV-vis absorbance and PL spectra (Figure 1e). In comparison to that of pure SiNPs, the fluorescence intensity of the SiNP-RGD is enhanced to some extent, which is due to the surface modification of aromatic electron-rich systemcontaining organic groups.34 Figure 1f displays the photographs of the pure SiNPs and SiNPs-RGD aqueous samples in ambient environment (left) and under UV irradiation (right). The Fourier transform infrared spectroscopy (FTIR) absorption is further performed to give additional evidence to the successful RGD modification (Figure S7).

Tube Formation Assay. The anti-angiogenic ability of SiNPs-RGD in vitro was evaluated based on an established tube formation assay.41 Briefly, 120 µL of the Basement Membrane Matrix (8-12 µg/mL) was placed in a 24-well plate and incubated for 30 min to form a gel. Afterwards, HRMECs (8×103/well) were added to the 24-well plate and incubated for 4 h. Different concentrations of pure SiNPs (i.e., 280 and 560 µg/mL), c(RGDyC) peptides (i.e., 28 and 56 µg/mL), and SiNPs-RGD (i.e., 280 and 560 µg/mL, based on SiNPs) were then added to the medium, respectively. After 20-h incubation, photographs of three random regions from three repeated wells were acquired by a microscope. The number of junctions and meshes were assessed using ImageJ software. An Alkali-Burn Corneal Neovascularization Model in the Mouse. The mice were firstly anesthetized by intraperitoneal injection of 75 µL 1% pentobarbital sodium and then the corneas were treated with a piece of NaOH (1 M) soaked filter paper to stimulate angiogenesis. After 30-s stimulation, the residual NaOH was washed away by 1×PBS (pH = 7.4) immediately. In Vivo Corneal Neovascularization Labeling. Mice treated with alkali after 7 d were used in this experiment and were injected intravenously with 100 µL SiNPs (30 mg/mL) and 100 µL SiNPs-RGD (30 mg/mL, based on SiNPs), respectively. The eyes were taken out immediately after 4-h circulation time and fixed in 4% paraformaldehyde for at least 4 h at 4 °C. With the aid of a stereomicroscope, the anterior portion of the cornea was cut from the posterior portion with a surgical scissor. For immunofluorescence staining, the cornea was permeabalized for 10 min in 0.25% Triton X-100 and then blocked for 30 min in 1% BSA. The cornea was next stained by incubating in 200 µL of 1% Alexa Fluor 568 conjugatedisolectin IB4 (purchased from Thermo Scientific) solution overnight. The next day, the cornea was washed three times in 1×PBS before proceeding. The cornea was carefully made as four incisions from periphery towards the center and fixed on slide glasses with fluoromount (Sigma, F4680). Corneal neovascularization images were examined using a confocal microscope (Leica, TCS-SP5 II). The excitation wavelength and corresponding emission window for SiNPs were set as 405 nm and 420-480 nm, respectively. For IB4, 633 nm excitation with detection window of 644-663 nm were set. For quantitative assessment of colocalization of green (SiNPs-RGD) and red (Alexa Fluor 568 conjugated-isolectin IB4) signals, the “colocalization finder” plugin of the ImageJ software was employed for calculating the Person’s coefficient (Rr). In Vivo Suppression of Corneal Neovascularization. Mice treated with alkali after 7 d were used in this experiment and were randomly divided into four groups, which were injected intravenously with 150 µL of saline, pure SiNPs (35

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Analytical Chemistry

Figure 1. Characterizations of SiNPs and SiNPs-RGD. TEM images of (a) SiNPs and (b) SiNPs-RGD. Insets in (a) and (b) present the enlarged HRTEM images of a single SiNP and SiNP-RGD. (c) The TEM diameter distribution of SiNPs-RGD. (d) DLS histogram of SiNPs and SiNPs-RGD. (e) UV-vis absorbance and PL spectra of SiNPs and SiNPs-RGD. (f) Photographs of pure SiNPs and SiNPs-RGD aqueous solution under ambient light (left) or 365 nm UV irradiation (right).

Figure 2. Fluorescence images of HREMCs in tube formation. (a) A schematic diagram of the processing procedure. HRMECs are incubated with pure (b) SiNPs or (d and f) SiNPs-RGD for 30 min at 37 °C, followed by treatment with DiD membrane tracker for 30 min. (f) “Blocking” denotes coincubation with 1 µM of c(RGDyC) peptides. Scale bar = 50 µm. Fluorescence intensity profiles with ROI (white line in b, d, and f) of SiNPs/SiNPs-RGD (green) and DiD (red) in (b) SiNPs-, (d and f) SiNPs-RGD-treated cells.

In vitro cellular labeling in tube formation. To verify the binding affinity of the as-prepared SiNPs-RGD, membrane labeling of U87MG cells (high integrin αvβ3 expression) and MCF-7 cells (low integrin αvβ3 expression) is carried out (Figure S8). Feeble fluorescence signals of SiNPs-RGD are observed from MCF-7 cells (Figure S8b), which is in contrast to obvious fluorescence signals detected in SiNPs-RGD-treated U87MG cells (Figure S8h), indicating high affinity of the asprepared SiNPs-RGD. In the next step, we test the biological activity of SiNPs-RGD in labeling angiogenic endothelial cells. It is well established that integrin αvβ3 is strongly expressed during angiogenesis.42,43 To simulate the formation of new blood vessels in vitro, HRMECs are induced to differentiate and generate tube-like structures when cultured on Basement Membrane Matrix (Figure 2a).44 To label integrin αvβ3, HRMECs are incubated with SiNPs (0.5 mg/mL), SiNPs-RGD (0.5 mg/mL, based on SiNPs) or SiNPs-RGD (0.5 mg/mL, based on SiNPs) mixed with 1 µM of c(RGDyC) (i.e., blocking) for 30 min, respectively. The DiD membrane tracker is used for co-localization in our experiment. As shown in Figure 2b and Figure 2c, little fluorescence signals are detected when cells are incubated with pure SiNPs, indicating there is no non-specific binding of SiNPs to HRMECs. Comparatively, obvious fluorescence signals are detected when HRMECs are incubated with SiNPs-RGD (Figure 2d). The green signals of SiNPs-RGD and the red signals of DiD clearly display integrin αvβ3-specific binding affinity of SiNPs-RGD, which is confirmed by the line scanning profiles (Figure 2e). Moreover, decreased fluorescence signals are observed in HRMECs blocked with the c(RGDyC) peptides (Figure 2f and Figure 2g). Collectively, these data suggest that the SiNPs-RGD possesses high specificity for labeling angiogenic endothelial cells in vitro.

Cytotoxicity assay. Numerous studies have presented that antibodies blocking or immunoconjugates targeting integrin αvβ3 could suppress angiogenesis by inducing the apoptosis of angiogenic blood vessels.45-47 We therefore reason that the resultant SiNPs-RGD might exhibit anti-angiogenic ability to a certain extent. To confirm this hypothesis, cytotoxicity effect of pure SiNPs, c(RGDyC) peptides and SiNPs-RGD are systematically tested using an established MTT assay. Under normoxic conditions, cell viabilities of HRMECs incubated with pure SiNPs, c(RGDyC) peptides and SiNPs-RGD all maintain above 85% (Figure 3a), indicating feeble toxicity to non-angiogenic endothelial cells. Alternatively, as exhibited in Figure 3b, when HRMECs are exposed to hypoxic condition to ensure that integrin αvβ3 is expressed at high levels on cell surface, the cell viability of SiNPs-treated HRMECs is decreased which may contribute to attenuation of VEGF-related signaling pathways.48,49 In addition, concentration-dependent cell death is observed when HRMECs and U87MG cells (Figure S9) are treated with the SiNPs-RGD. Moreover, the cell viability of SiNPs-RGD-treated HRMECs (e.g., ~60% cell viability is observed at 560 µg/mL SiNPs-RGD) is slightly lower than those (e.g., ~75% or ~80% cell viability is observed at 560 µg/mL SiNPs or 560 µg/mL c(RGDyC) peptides, respectively) of pure SiNPs and c(RGDyC) peptides since the specific binding mediated by integrin αvβ3. The cell viabilities of ARPE-19 (Figure 3c) and R28 (Figure 3d) maintain nearly 100%, implying that SiNPs have little or no toxic effect on other kinds of ocular cell lines. Of note, cell viabilities of ARPE-19 cells (Figure 3c) and R28 cells (Figure 3d) with low integrin αvβ3 expression remain almost 100% owing to the low binding affinity of few c(RGDyC) peptides or SiNPs-RGD to these cells. These obtained results demonstrate that the SiNPs-

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together, the presented results imply that the SiNPs-RGD displays anti-angiogenic effects in vitro.

RGD enables selective inhibition of angiogenic endothelial cells in vitro.

Figure 3. Cytotoxicity effect of pure SiNPs, c(RGDyC) peptides and SiNPs-RGD on different ocular cells. In vitro cellular viabilities of (a) HRMECs, (b) hypoxic HRMECs, (c) ARPE-19 cells, and (d) R28 cells. Cells are incubated with pure SiNPs, SiNPs-RGD, or c(RGDyC) peptides for 24 h, respectively. Each bar stands for mean ± SD of three independent experiments. The analysis of variance is completed using a one-way ANOVA. Asterisks (** and *) indicate p < 0.01 and p < 0.05, respectively.

Figure 4. In vitro anti-angiogenic ability of pure SiNPs (560 µg/mL), c(RGDyC) peptides (56 µg/mL), and SiNPs-RGD (560 µg/mL, based on SiNPs). (a) Wound healing assays of HRMECs after incubated with pure SiNPs, c(RGDyC) peptides, and SiNPs-RGD for 14 h. (b) Quantitative analysis represents the width of remaining open wound calculated in relation to time 0. (c) Transwell migration and invasion assays of HRMECs after incubated with pure SiNPs, c(RGDyC) peptides, and SiNPs-RGD for 24 h. (d) Quantitative analysis represents the number of cells. (e) Representative images of tubelike structures of HRMECs are acquired at 24 h. (f) Quantitative analysis of tube formation assay characterizing mesh numbers and junction numbers. Scale bars = 200 µm. Each bar represents the mean ± SEM of three independent experiments. The analysis of variance is completed using a one-way ANOVA. Asterisks (***) indicate p < 0.001

The SiNPs-RGD suppresses wound healing migration, transwell migration, tranwell invasion, and tube formation of HRMECs. In the next step, we evaluate the migratory response of HRMECs to pure SiNPs, c(RGDyC) peptides, and SiNPs-RGD with different incubation time, since the migration of endothelial cell is a critical step for angiogenesis. Corresponding results are presented in Figure 4, Figure S10, and Figure S11. Representative photographs are acquired at 0 and 14 h after the formation of wounds in the culture of HRMECs (Figure 4a). Figure 4b presents that SiNPs-, c(RGDyC) peptides-, and SiNPs-RGD-treated HRMECs attenuate wound closures (~44% closure, ~43% closure, and ~35% closure, respectively) after 14-h incubation. Meanwhile, transwell migration and invasion assays are performed here to assess the contribution of pure SiNPs, c(RGDyC) peptides, and SiNPsRGD in HRMECs migration and invasion. Photographs of representative fields are acquired at 24 h (Figure 4c). As shown in Figure 4d, SiNPs-, c(RGDyC) peptides-, and SiNPsRGD-treated HRMECs suppress migration in transwell migration assays (by 40%, 50%, and 31%, respectively) as well as invasion in transwell invasion assays (by 50%, 41%, and 38%, respectively). To further test our hypothesis that SiNPs-RGD inhibits angiogenesis, we perform tube formation assays with pure SiNPs, c(RGDyC) peptides, and SiNPs-RGD after 20-h incubation (Figure 4 and Figure S12). Capillary tube formations are recorded (Figure 4e) and quantitatively analyzed (Figure 4f) at 24 h. In comparison with the untreated HRMECs, the treatment of HRMECs with SiNPs-RGD (560 µg/mL, based on SiNPs) exhibits a significant (***P < 0.001) reduction both in the number of meshes and junctions. Taken

In vivo angiogenic blood vessels labeling. Previous studies demonstrate that RGD peptides are able to specifically target integrin αvβ3 highly overexpressed in angiogenic vessels.42,43 Meanwhile, RGD peptides could induce apoptosis of the proliferative angiogenic vascular cells, thus inhibiting ocular neovascularization.45-47 Therefore, we deduce that SiNPs-RGD could simultaneously realize fluorescence imaging of angiogenic blood vessels and suppression of ocular neovascularization. To verify this hypothesis, we first study the specificity and stability of the prepared SiNPs-RGD for in vivo angiogenic blood vessels imaging. Mouse corneal angiogenesis, known as a classic model, is generally used to evaluate vascular formation, stability and remodeling.50 As shown in Figure 5a, the mouse cornea is firstly treated with a piece of NaOH soaked filter paper to stimulate the formation of new blood vessels, followed by intravenous injection of a SiNP/SiNPs-RGD solution. Figure 5b presents the progression of corneal neovascularization after alkali-burn injury, which begins to occur on day 1 and advances greatly on day 7.51 In the cornea of mouse injected with pure SiNPs, little to no fluorescence signals (Figure 5c) are observed, indicating no non-specific binding of pure SiNPs to angiogenic blood vessels. For SiNPs-RGDtreated mouse, fluorescence signals are clearly detected (Figure 5c). To confirm the localization of SiNPs-RGD in corneal

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Analytical Chemistry neovascularization, counterstaining experiments with Alexa Fluor 568 conjugated-isolectin IB4 (a standard marker for endothelial cells labeling) are performed (Figure 5c) and they show a similar distribution pattern. Moreover, Person’s correlation coefficient (Rr) is used for analyzing the coincidence degree of green fluorescence (SiNPs-RGD) and red fluorescence (Alexa Fluor 568 conjugated-isolectin IB4).38 The Rr value is calculated as ~ 0.73, indicating good colocalization between SiNPs-RGD and angiogenic blood vessels. These findings clearly indicate that SiNPs-RGD indeed features significant binding ability to angiogenic blood vessels in the cornea, making it possible to selectively detect angiogenic blood vessels in vivo.

on day 14. However, the blood-vessel lengths are efficiently suppressed in SiNPs-RGD-treated mice and reach 3.23 ± 0.60 mm on day 14, indicating superior efficacy of corneal neovascularization treatment.

Figure 6. Inhibition of alkali burn-induced corneal neovascularization by physiological saline, pure SiNPs, c(RGDyC) peptides, and SiNPs-RGD. (a) A schematic diagram of the processing procedure. (b) Representative slit-lamp photographs of mice corneas treated with alkali and further intravenous injected with physiological saline, pure SiNPs, c(RGDyC), and SiNPs-RGD. Scale bar = 2 mm. (c) Quantitative analysis of vessel lengths of the corresponding treatment groups. Each bar stands for mean ± SD of three independent experiments. Herein, “mean” stands for the average value of three independent experiments. Biodistribution and biosafety investigation of SiNPsRGD. For potential clinical application, it is essential to investigate the clearance routes and biosafety of the SiNPs-RGD. Major organs (i.e., liver, heart, lung, kidney and spleen) are collected at various time (0.5 h, 4 h, 6 h, 3 d, and 7 d) after systematic administration of 100 µL SiNPs (14 mg/mL) and 100 µL SiNPs-RGD (14 mg/mL, based on SiNPs), and fluorescence images are immediately captured by a IVIS Lumina III in vivo fluorescence imaging system (PerkinElmer) (excited and detected wavelength: 460 nm and 520-570 nm). As shown in Figure 7a, SiNPs are mainly accumulated in the liver and a small part is distributed in kidney. After 7 d, no significant signal is detected in any organs, revealing that these ultrasmall particles (3-10 nm) are cleared out of the body. Figure 7b shows that the body weight of mice treated with physiological saline, SiNPs, c(RGDyC) peptides, and SiNPs-RGD exhibits no obvious difference. Serum biochemistry is carried out to assess the biosafety of SiNPs-RGD. Blood is collected from physiological saline-, SiNPs-, c(RGDyC) peptides-, and SiNPs-RGD-treated mice at 28 d (The former three groups are set as controls). Representative indicators (i.e., alanine transaminase (ALT), aspartate transaminase (AST), albumin (ALB), alkaline phosphatase (ALP), total protein (TP), globulin (GLOB), albumin/globulin (A/G), creatinine (CRE) and UREA) of kidney and liver function, and hepatocellular injury, are similar to those of control animals (Figure 7c). Histological analysis of major organs (i.e., liver, heart, lung, kidney and spleen) is further conducted to evaluate the biosafety of the SiNPs-RGD in vivo. As shown in Figure 7d, no apparent abnormality of major organs is observed after the mice treated with SiNPs, c(RGDyC) peptides, and SiNPs-RGD at 28 d. Moreover, no apparent abnormality (e.g., body shape, action,

Figure 5. The binding ability of SiNPs/SiNPs-RGD to angiogenic blood vessels in the cornea of mice. (a) A schematic diagram of the processing procedure. (b) Representative images of corneal neovascularization after alkali-burn injury. Scale bar = 5 mm. (c) Fluorescence images of angiogenic blood vessels in the corneas with pure SiNPs (upper) and SiNPs-RGD (lower) upon intravenous injection after 4-h circulation. Scale bar = 25 µm.

The intravenous injection of SiNPs-RGD inhibits alkali burn-induced corneal neovascularization. We further investigate the high anti-angiogenic activity of SiNPs-RGD in vivo. As shown in Figure 6a, intravenous injections of physiological saline, pure SiNPs, c(RGDyC) peptides, and SiNPs-RGD are performed on day 7, and the measurement of vessel length is carried out on day 7, 8, 10, 12, and 14. Figure 6b presents the representative photographs of mice corneal neovascularization captured by slit-lamp. For the 3 groups of physiological saline-, pure SiNPs- and c(RGDyC) peptides-treated corneal neovascularization, similar tissue morphologies are observed (e.g., an obvious and progressive development of the vascular network is observed). In contrast, the SiNPs-RGD-treated group shows obvious suppression during 7-d post injection, indicating distinct and stable suppression of blood-vessel lengths. Corresponding quantitative results of inhibition of vessel lengths are shown in Figure 6c. For physiological saline-, pure SiNPs-, and c(RGDyC)-treated mice, average blood-vessel lengths reach 7.3 ± 0.95, 4.8 ± 1.18, and 4.9 ± 0.70 mm, respectively,

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dietary status, etc.) is observed during the whole process (Figure S13). These above results suggest the feeble toxicity of the resultant SiNPs-RGD.

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The Supporting Information is available free of charge on the ACS Publications website at DOI:... the size distribution of SiNPs (Figure S1), UV-vis absorbance spectra of c(RGDyC) with different concentrations and concentration vs. absorbance at 275 nm of c(RGDyC) peptides (Figure S2), UV-vis absorbance spectra of the SiNP-RGD filtered solutions (Figure S3), the pH effects on the fluorescence intensity of SiNPs-RGD (Figure S4), the storage stability of SiNPs-RGD (Figure S5), the XPS spectrum of SiNPs-RGD (Figure S6), FTIR characterization data (Figure S7), selective labeling (Figure S8) and killing effect (Figure S9) of SiNPs-RGD in cancer cells, wound healing assays treated by 560 µg/mL SiNPs-RGD (Figure S10) and 280 µg/mL SiNPs-RGD (Figure S11), tube formation by SiNPs-RGD (280 µg/mL, based on SiNPs) treatments (Figure S12), images of BALB/c mice (Figure S13).

AUTHOR INFORMATION Corresponding Author [email protected]

Author Contributions ‡These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We appreciate financial support from the National Basic Research Program of China (973 Program, 2013CB934400), NSFC (61361160412, 31400860, and 81500733), Natural Science Foundation of Jiangsu Province, China (BK20150292), and Projects Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), 111 project as well as Collaborative Innovation Center of Suzhou Nano Science and Technology (NANO-CIC).

Figure 7. Biodistribution and biosafety analysis after intravenous injection of nanoparticles. (a) Ex vivo images of isolated organs of mice at 0 h, 0.5 h, 4 h, 6 h, 3 d, and 7 d. (b) The weight change of mice. (c) Serum biochemistry analysis of mice on day 28. (n=3) (d) Histological analysis of heart, liver, spleen, lung and kidney on day 28 after intravenous injections of physiological saline, SiNPs, c(RGDyC) peptides, and SiNPs-RGD. Scale bar = 100 µm.

REFERENCES (1) Delplace, V.; Payne, S.; Shoichet, M. J. Control Release 2015, 219, 652-668. (2) Renaud, R. C.; Xuereb, H. Nat. Rev. Drug Discov. 2003, 2, 425426. (3) Rama, N.; Dubrac, A.; Mathivet, T.; Chárthaigh, R. A. N.; Genet, G.; Cristofaro, B.; Pibouin-Fragner, L.; Ma, L.; Eichmann, A.; Chédotal, A. Nat. Med. 2015, 21, 483-491.

CONCLUSIONS

(4) Chopdar, A.; Chakravarthy, U.; Verma, D. BMJ-Brit. Med. J. 2003, 326, 485-488.

In summary, we herein introduce the functionalized SiNPsRGD as a new kind of theranostic probes for the combined imaging and treatment of ocular neovascularization. The high binding affinity of SiNPs-RGD to human retinal microvascular endothelial cells tube formation enables specific detection of angiogenic endothelial cells in vitro. Meanwhile, wound healing migration, transwell migration, transwell invasion, and tube formation assays prove that the as-prepared SiNPs-RGD presents high-efficacious anti-angiogenic ability in vitro. Taking advantage of these unique merits, the as-prepared SiNPsRGD is further employed for concurrent angiogenic blood vessels imaging and treatment of corneal neovascularization. These results indicate that the photostable and biocompatible SiNPs-based theranostic probes are highly promising for simultaneous diagnosis and treatment of neovascular eye diseases.

(5) Pollinger, K.; Hennig, R.; Ohlmann, A.; Fuchshofer, R.; Wenzel, R.; Breunig, M.; Tessmar, J.; Tamm, E. R.; Goepferich, A. Proc. Natl. Acad. Sci. USA 2013, 110, 6115-6120. (6) Calcutt, N. A.; Cooper, M. E.; Kern, T. S.; Schmidt, A. M. Nat. Rev. Drug Discov. 2009, 8, 417-430. (7) Ambati, J.; Atkinson, J. P.; Gelfand, B. D. Nat. Rev. Immunol. 2013, 13, 438-451. (8) Jia, Y.; Bailey, S. T.; Hwang, T. S.; McClintic, S. M.; Gao, S. S.; Pennesi, M. E.; Flaxel, C. J.; Lauer, A. K.; Wilson, D. J.; Hornegger, J.; Fujimoto, J. G.; Huang, D. Proc. Natl. Acad. Sci. USA 2015, 112, E2395-E2402. (9) Wilde, C.; Patel, M.; Lakshmanan, A.; Amankwah, R.; DharMunshi, S.; Amoaku, W. Eye 2015, 29, 602-610. (10) Ng, E. W. M.; Shima, D. T.; Calias, P.; Cunningham Jr., E. T.; Guyer, D. R.; Adamis, A. P. Nat. Rev. Drug Discov. 2006, 5, 123-132.

ASSOCIATED CONTENT

(11) Lai, P. X.; Chen, C. W.; Wei, S. C.; Lin, T. Y.; Jian, H. J.; Lai, I. P. J.; Mao, J. Y.; Hsu, P. H.; Lin, H. J.; Tzou, W. S.; Chen, S. Y.; Harroun, S. G.; Lai, J. Y.; Huang, C. C. Biomaterials 2016, 109, 12-22.

Supporting Information

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Page 7 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry (12) Ferrara, N.; Adamis, A. P. Nat. Rev. Drug Discov. 2016, 15, 385403.

(31) Zhou, Y.; Zhang, Y.; Zhong, Y.; Fu, R.; Wu, S.; Wang, Q.; Wang, H.; Su, Y.; Zhang, H.; He, Y. Nano Res. 2017, 1-11.

(13) Luo, L.; Zhang, X.; Hirano, Y.; Tyagi, P.; Barabás, P.; Uehara, H.; Miya, T. R.; Singh, N.; Archer, B.; Qazi, Y.; Jackman, K.; Das, S. K.; Olsen, T.; Chennamaneni, S. R.; Stagg, B. C.; Ahmed, F.; Emerson, L.; Zygmunt, K.; Whitaker, R.; Mamalis, C.; Huang, W.; Gao, G.; Srinivas, S. P.; Krizaj, D.; Baffi, J.; Ambati, J.; Kompella, U. B.; Ambati, B. K. ACS Nano 2013, 7, 3264-3275.

(32) Pang, J.; Su, Y.; Zhong, Y.; Peng, F.; Song, B.; He, Y. Nano Res. 2016, 9, 3027–3037. (33) Wu, S.; Zhong, Y.; Zhou, Y.; Song, B.; Chu, B.; Ji, X.; Wu, Y.; Su, Y.; He, Y. J. Am. Chem. Soc. 2015, 137, 14726–14732. (34) Song, C.; Zhong, Y.; Jiang, X.; Peng, F.; Lu, Y.; Ji, X.; Su, Y.; He, Y. Anal. Chem. 2015, 87, 6718-6723.

(14) Salehi-Had, H.; Roh, M. I.; Giani, A.; Hisatomi, T.; Nakao, S.; Kim, I. K.; Gragoudas, E. S.; Vawas, D.; Guccione, S.; Miller, J. W. Plos One, 2011, 6, e18864.

(35) Zhong, Y.; Peng, F.; Bao, F.; Wang, S.; Ji, X.; Yang, L.; Su, Y.; Lee, S. T.; He, Y. J. Am. Chem. Soc. 2013, 135, 8350–8356. (36) Zhong, Y.; Sun, X.; Wang, S.; Peng, F.; Bao, F.; Su, Y.; Li, Y.; Lee, S. T.; He, Y. ACS Nano 2015, 9, 5958–5967.

(15) Liu, J. N.; Bu, W. B.; Shi, J. L. Acc. Chem. Res. 2015, 48, 17971805.

(37) Zhong, Y.; Peng, F.; Wei, X.; Zhou, Y.; Wang, J.; Jiang, X.; Su, Y.; Su, S.; Lee, S. T.; He, Y. Angew. Chem., Int. Ed. 2012, 51, 8485–8489.

(16) Kunjachan, S.; Ehling, J.; Storm, G.; Kiessling, F.; Lammers, T. Chem. Rev. 2015, 115, 10907-10937.

(38) Ji, X.; Peng, F.; Zhong, Y.; Su, Y.; Jiang, X.; Song, C.; Yang, L.; Chu, B.; Lee, S. T.; He, Y. Adv. Mater. 2015, 27, 1029-1034.

(17) Wang, C. F.; Sarparanta, M. P.; Mäkilä, E. M.; Hyvönen, M. L. K.; Laakkonen, P. M.; Salonen, J. J.; Hirvonen, J. T.; Airaksinen, A. J.; Santos, H. A. Biomaterials 2015, 48, 108-118.

(39) Tay, C. Y.; Cai, P.; Setyawati, M. I.; Fang, W.; Tan, L. P.; Hong, C. H. L.; Chen, X.; Leong, D. T. Nano Lett. 2013, 14, 83-88.

(18) Zhang, W.; Wang, Y.; Sun, X.; Wang, W.; Chen, L. Nanoscale 2014, 6, 14514-14522.

(40) Setyawati, M. I.; Tay, C. Y.; Bay, B. H.; Leong, D. T. ACS Nano 2017, 11, 5020-5030.

(19) Hu, J. J.; Liu, L. H.; Li, Z. Y.; Zhuo, R. X.; Zhang, X. Z. J. Mater. Chem. B 2016, 4, 1932-1940.

(41) Setyawati, M. I.; Leong, D. T. ACS Appl. Mater. Inter. 2017, 9, 6690-6703.

(20) Song, X.; Han, X.; Yu, F.; Zhang, X.; Chen, L.; Lv, C. Theranostics 2018, 8, 2217-2228.

(42) Desgrosellier, J. S.; Cheresh, D. A. Nat. Rev. Cancer 2010 10, 922.

(21) Yu, L.; Chen, Y.; Wu, M.; Cai, X.; Yao, H.; Zhang, L.; Chen, H.; Shi, J. J. Am. Chem. Soc. 2016, 138, 9881-9894.

(43) Kim, H. A.; Nam, K.; Kim, S. W. Biomaterials 2014, 35, 75437552.

(22) Yong, Y.; Cheng, X.; Bao, T.; Zu, M.; Yan, L.; Yin, W.; Ge, C.; Wang, D.; Gu, Z.; Zhao, Y. ACS Nano 2015, 9, 12451-12463.

(44) Arnaoutova, I.; Kleinman, H. K. Nat. Protoc. 2010 5, 628-635. (45) Brooks, P. C.; Clark, R. A.; Cheresh, D. A. Science 1994, 264, 569-571.

(23) Wang, Y.; Chen, L.; Liu, P. Chem. Eur. J. 2012, 18, 5935-5943. (24) Meng, H. M.; Lu, L.; Zhao, X. H.; Chen, Z.; Zhao, Z.; Yang, C.; Zhang, X. B.; Tan, W. Anal. Chem. 2015, 87, 4448-4454.

(46) Maubant, S.; Saint-Dizier, D.; Boutillon, M.; Perron-Sierra, F.; Casara, P. J.; Hickman, J. A.; Tucker, G. C.; Obberghen-Schilling, E. V. Blood 2006, 108, 3035-3044.

(25) Gao, M.; Yu, F.; Lv, C.; Choo, J.; Chen, L. Chem. Soc. Rev. 2017, 46, 2237-2271.

(47) Brooks, P. C.; Montgomery, A. M. P.; Rosenfeld, M.; Reisfeld, R. A.; Hu, T.; Klier, G.; Cheresh, D. A. Cell 1994, 79, 1157-1164.

(26) Tay, C. Y.; Setyawati, M. I.; Leong, D. T. ACS Nano 2017, 11, 2764-2772.

(48) Yi, H.; Wang, Z.; Li, X.; Yin, M.; Wang, L.; Aldalbahi, A.; ElSayed, N. N.; Wang, H.; Chen, N. Fan, C.; Song, H. Theranostics 2016, 6, 1810-1820.

(27) Setyawati, M. I.; Tay, C. Y.; Chia, S. L.; Goh, S. L.; Fang, W.; Neo, M. J.; Chong, H. C.; Tan, S. M.; Loo, S. C. J.; Ng, K. W.; Xie, J. P.; Ong, C. N.; Tan, N. S.; Leong, D. T. Nat. Commun. 2013, 4, 1673.

(49) Jo, D. H.; Kim, J. H.; Son, J. G.; Piao, Y.; Lee, T. G.; Kim, J. H. Nano Res. 2014, 7, 844-852.

(28) Kang, C.; Gwon, S.; Song, C.; Kang, P. M.; Park, S. C.; Jeon, J.; Hwang, D. W.; Lee, D. ACS Nano 2017, 11, 6194−6203

(50) Seo, S.; Singh, H. P.; Lacal, P. M.; Sasman, A.; Fatima, A.; Liu, T.; Schultz, K. M.; Losordo, D. W.; Lehmann, O. J.; Kume, T. Proc. Natl. Acad. Sci. USA 2012, 109, 2015-2020.

(29) Sun, Z.; Huang, P.; Tong, G.; Lin, J.; Jin, A.; Rong, P.; Zhu, L.; Nie, L.; Niu, G.; Cao, F.; Chen, X. Nanoscale 2013, 5, 6857-6866. (30) Lv, G.; Guo, W.; Zhang, W.; Zhang, T.; Li, S.; Chen, S.; Eltahan, A. S.; Wang, D.; Wang, Y.; Zhang, J.; Wang, P. C.; Chang, J.; Liang, X. J. ACS Nano 2016, 10, 9637-9645.

(51) Anderson, C.; Zhou, Q.; Wang, S. Jove-J. Vis. Exp. 2014, 86, e51159.

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