Molecular Vision 2016; 22:1176-1187
Received 09 June 2016 | Accepted 10 October 2016 | Published 12 October 2016
1Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 3Oklahoma Neuroscience Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 4Advanced Materials Processing Analysis Center, Mechanical Materials Aerospace Eng., Nanosci. and Tech. Ctr., University of Central Florida, Orlando, FL
Correspondence to: Xue Cai: Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, Phone: (405) 271-3692, FAX: (405) 271-8128; email: email@example.com,
Purpose: We have shown that cerium oxide nanoparticles (nanoceria), with unique characteristics and catalytic activities, are retained in the retina for more than 1 year after a single intravitreal injection and can be potentially used for the treatment of a variety of eye diseases. The objective of this study is to determine whether the retention of nanoceria in the eye causes inflammation or adverse side effects.
Methods: Wild-type (C57BL/6J) mice at P30 were intravitreally injected with several concentrations of nanoceria. The health of the photoreceptors was assessed by analyzing the expression of photoreceptor-specific genes, and the retinal structure and function. The effect of nanoceria was investigated by analyzing of the vascular system, the expression of inflammatory cytokines, and cellular infiltration into the eye.
Results: Our data showed that there were no changes in the retinal structure or function, or cytokine gene expression following a single intravitreal injection of nanoceria.
Conclusions: Nanoceria, at doses ranging from 17.2 ng to 1720 ng per eye, do not cause any damage to the retinal structure and function by 30 days post injection. No cellular infiltration and no increases in inflammatory responses were found in the eyes. Our data indicate that nanoceria are safe to use for treatment of a variety of eye diseases.
An ideal therapeutic reagent for ocular disease treatment should be capable of passing the blood–retinal barrier, have prolonged retention in the ocular tissues, be selectively targeted to the expected sites, and have sustained effectiveness for long periods of time with a maximum benefit. This therapeutic reagent should also be safe with minimum damage to the tissue where the reagent is located and without adverse reactions and undesirable side effects. Selectivity, effectiveness, and safety are the three most important characteristics for an ideal pharmaceutical agent . Unfortunately, thus far there are no such ideal drugs, and the currently available and potential drugs for the treatment of diseases need to have the dosage optimized to reduce their side effects.
We have been using cerium oxide nanoparticles (nanoceria) as therapeutics to treat a variety of ocular diseases in animal models. Nanoceria are catalytic antioxidants that mimic superoxide dismutase and catalase and as a new emerging nanomedicine have great advantages over other traditional antioxidants, such as unique physicochemical features of the surface structure for regenerative scavenging of free radicals that decreases repetitive doses. The tiny particle size (3–5 nm in diameter) on the atom-size scale enables nanoceria to easily cross cell and nuclear membranes. For years, we have used nanoceria as therapeutics to treat inherited and light-induced retinal degeneration [2-4] and to inhibit and regress neovascularization in a wet age-related macular degeneration (AMD) mouse model (vldlr−/−) [5,6]. We also showed storage of nanoceria at room temperature for 6 years does not reduce their effectiveness . And nanoceria were shown to regulate the same antioxidative gene network as thioredoxin . All these results indicated that nanoceria exert their function as a near “ideal” drug and can be used for a broad spectrum of diseases. However, data from our laboratory also demonstrated that nanoceria delivered to the rat eye by a single intravitreal injection are retained in the retina for more than 1 year . Due to the slow elimination and clearance of nanoceria from the tissues , safety following long-term retention in the retinas will be a concern for the clinical application of nanoceria.
Although the published data from our laboratory show that nanoceria do not affect the retinal structure and function , there are no data to show whether the long-term presence of nanoceria in the eyes causes inflammation. To investigate the tolerance of ocular tissues and cells for nanoceria, we performed intravitreal injections of nanoceria, with a variety of doses, into P30 wild-type (WT) C57BL/6J mice. Assessment of the toxicity of nanoceria was conducted at post injection (PI) at 7 h and 3, 7, 15, and 30 day. We evaluated the retinal structure and function, photoreceptor-specific gene expression of mRNA and protein levels, and inflammatory responses, including the alteration of the vascular system and cytokine expression.
Wild-type (C57BL/6J) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and used as breeders for the colonies. Animal care and handling were performed according to the guidance of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research (ARVO), and the protocol for this study was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Oklahoma Health Sciences Center.
Intravitreal injection was performed as previously reported [11,12]. Briefly, WT mice at P30 were anesthetized by intraperitoneal injection with ketamine (85 mg/kg) and xylazine (14 mg/kg; Henry Schein Animal Health, Dublin, OH). A puncture was made in the sclera just below the cornea with a 30 gauge needle; a 33 gauge needle attached to a Hamilton syringe was then inserted into the puncture, and 1 µl of saline or 1 µl of nanoceria in saline at the following concentrations, 0.1 mM (17.2 ng), 0.3 mM (51.6 ng), 1 mM (172 ng), 3 mM (516 ng), and 10 mM (1720 ng), were injected into the vitreous. After fully recovering from the anesthesia, the mice were returned to their original cages and maintained under the standard conditions. Age-matched mice were used as uninjected controls.
Micron IV fundoscopy (Phoenix Research Labs, Pleasanton, CA) was performed, and evaluation of the fundus and neovascularization was the same as we previously reported . The mice at the PI7 and PI30 day were anesthetized with ketamine and xylazine, the eyes were dilated, and the whiskers were trimmed. One drop of 2.5% Goniotaire (hypromellose, Altaire pharmaceuticals, Inc., Aquebogue, NY) was applied to the surface of the cornea. The mouse was placed on the bed of the Micron IV system, and the position of the eye and the objective of the funduscope were adjusted until the fundus was clearly seen. After a fundus image was taken, the mice were intraperitoneally injected with 40 µl of 5% AK-Fluor (Alcon, Fort Worth, TX). Then additional images were taken at 2 min and 4 min after injection using StreamPix software and fluorescein isothiocyanate (FITC) filters.
Full-field electroretinography (ERG) was performed on the mice according to the procedure reported previously . Briefly, mice at the PI30 day were dark adapted overnight, the eyes of the fully anesthetized mice were dilated, the whiskers were trimmed, and rod ERG was recorded by stimulating with a light flash of 600 cds/m2 intensity. Cone responses were recorded by stimulating with a light flash of an intensity of 1000 cds/m2 five times after 5 min of adaption to the light intensity of 100 cds/m2.
Three to eight retinas from each group were collected and kept in TRIzol (Invitrogen, Carlsbad, CA) at −80 °C. Total RNA isolation, cDNA synthesis, and quantitative RT-PCR (qRT-PCR) were performed the same as previously reported . Ten nanograms of cDNA was used in a 25 µl reaction volume for qRT–PCR. Primers for tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and macrophage migration inhibitory factor (MIF) were the same as previously reported . The primers used for IL-1β were the forward primer: 5′-GGG CCT CAA AGG AAA GAA TC and the reverse primer: 5′-TAC CAG TTG GGG AAC TCT GCA. Calculation of the relative expression level of the target genes against the housekeeping gene (GAPDH) was the same as we previously reported . Data shown are fold changes. PCR array assay using the “mouse common cytokines” array plates and retinas at the PI7 day was performed according to the instructions from SABiosciences. Data were analyzed with the array plate software (SABiosciences) and are shown as fold changes in the nanoceria-injected and saline-injected mice compared to the uninjected WT mice (CeO2/WT and saline/WT) with the p value indicated.
The immunohistochemistry procedure was the same as we previously published [2,5]. Briefly, paraffin sections were dewaxed and hydrated through a series of ethanol solutions, and then blocked with 5% bovine serum albumin (BSA). The slides were incubated with anti-rhodopsin antibody (1D4, 1:2,000, generous gift from Dr. Robert Molday, University of Columbia, Vancouver, Canada) and rabbit anti-M-opsin (1:500, Millipore) for 2 h at room temperature. After three washes, secondary antibody of Alexa-Flour 488 conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) was applied. The slides were coverslipped with mounting medium containing 4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Inc., Burlingame, CA). Observation and image capture were performed using a Nikon Eclipse 800 fluorescence microscope (Tokyo, Japan) with proper filters.
Eye enucleation, fixation, sectioning, and staining were the same as previously reported [2,5]. Representative retinal images from three to eight eyes stained with hematoxylin and eosin (H&E) were taken at 0.96 mm from the optic nerve head (ONH) at the superior hemisphere using Nikon Eclipse 800 microscopy under 20X and 40X objectives. For morphometric and quantitative histological analysis of the outer nuclear layer (ONL) thickness, blinded to each groups, five images were taken under 60X at a distance of every 0.32 mm from each side of the retina section starting from the ONH. The data are shown as mean ± standard error of the mean (SEM).
Three to five individual eyecups, without the lens and cornea, from each group were homogenized, centrifuged, and 50 µg of the soluble protein were loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel. The proteins were detected with the following primary antibodies: mouse anti-IL-1β (1:1,000, Millipore), rabbit anti-IL-6 (Proteintech, 1:1000), anti-TNF-α (1:1,000, Millipore) and anti-MIF (1:1000, Santa Cruz), mouse anti-rhodopsin (1D4, 1:4,000), rabbit anti-M-opsin (1:1,000, Millipore), goat anti-S-opsin (1:1,000, Santa Cruz), and rabbit anti-caspase 3 (1:1,000, Cell Signaling Technology). After stripping, the same membranes were probed with rabbit anti-actin-HRP (Horseradish peroxidase conjugate; 1:1,000, Cell Signaling Technology) or rabbit anti-GAPDH (1:2,500, Abcam). Development of bands, image capture, and the densitometric analysis of the bands were the same as we previously reported .
The unpaired Student t test for two group comparison or one-way ANOVA with the Bonferroni post hoc test for multiple comparisons was used to analyze the difference between groups. A p value of less than 0.05 was considered statistically significant and was indicated in each figure.
In vivo observation of the overall appearance and movement of the eyeballs demonstrated that the injected eyes were normal sized without redness, with a clear cornea, normal iris response to light, and normal eyeball movement. These findings indicated that there were no ocular abnormalities in the injected eyes.
To evaluate the overall morphological changes in the retinas and any loss of photoreceptors, we performed histological and quantitative histological analysis on the H&E-stained retinal sections at the PI7 h and the PI3, PI7, PI15, and PI30 day. As shown in Figure 1, distinct and normal retinal layers were apparent. The eyes injected with various concentrations of nanoceria have 12–13 nuclear rows in the ONL of the retina which is the same number as in the uninjected controls, indicating that there is no alteration of the retinal structure in the injected eyes at any of the time points examined. Morphometric analysis of the ONL thickness across the entire retinas of each group demonstrated that no statistically significant difference in the ONL thickness was seen among the groups at all time points we tested. This finding demonstrated that nanoceria retention in the retina does not result in any damage to the normal tissue or to the photoreceptor cells. In addition, we did not see any retinal detachment at any of the time points.
To assess retinal health, western blots were performed at the PI7 h (Figure 2) and the PI30 day (Appendix 1) to test photoreceptor-specific gene expression. The data showed that the protein levels of rhodopsin, M-opsin, and S-opsin were similar to those of the uninjected and the saline-injected controls. We also performed immunohistochemistry on the paraffin sections of the eyes to evaluate the localization and distribution of these proteins in the retinas. The data showed that rhodopsin, M-opsin, and S-opsin were properly localized in the photoreceptor cells in all groups. We also tested caspase 3 expression levels with western blot and demonstrated that the expression of caspase 3 within the group was similar (Figure 2B). These data indicated that nanoceria, even when present at high dosage, do not cause photoreceptor injury or mislocalization of these proteins.
Full-field ERG was performed at PI30 day to assess retinal function by evaluating the responses of rods and cones to light stimulation. The ERG data showed that there were no statistically significant changes in the ERG amplitudes among the different groups at each time point tested (Figure 3), indicating that retention of nanoceria in the eye does not affect normal retinal function.
Infection induces innate and acute inflammation or chronic inflammatory responses, which involve activation and migration of various immune cells (macrophages, microglia, and neutrophils) to the site of inflammation and persistent release of proinflammatory cytokines [14,15]. To test the effects of nanoceria retention in the retina on the expression of proinflammatory cytokine genes, PCR array assays were performed using the “mouse common cytokines” array plates and the retinas from the uninjected, saline-injected, and 1 mM nanoceria-injected eyes at the PI7 day. A total of 89 genes were surveyed, and only 11 were upregulated (Bmp1, Bmp6, Fgf10, Il1a, Il1f5, Il1f9, Tnfrsf11b, Tnfsf9) or downregulated (Bmp2, Ctf1, Inhba; Table 1). However, these genes were also similarly upregulated or downregulated following saline injection, suggesting that the increases in the expression of these genes were most likely caused by the injury resulting from the injection procedure itself or from the saline in which the nanoceria were suspended. To determine whether any acute inflammatory responses were triggered by nanoceria, the expression of the mRNAs of the proinflammatory cytokines, TNF-α, IL-1β, IL-6, and MIF, was analyzed with qRT-PCR at an early time point (PI7 h). Compared to the higher levels of expression in the lipopolysaccharide (LPS)-induced inflammatory cytokines, none of the concentrations of nanoceria induced alterations in the expression of these cytokines compared to the uninjected controls (Figure 4A-D). Western blot assay at the PI7 h (Figure 4E-H) and the PI30 day (Appendix 2) demonstrated that the protein levels of TNF-α, IL-1β, IL-6, and MIF were similar among the groups injected with various concentrations of nanoceria versus the uninjected mice with no statistically significant differences seen.
Acute and chronic inflammation causes increased vascular permeability and neovascularization . To observe whether nanoceria in the eyes cause any abnormalities in the fundus appearance and organization of the retinal vascular system, we performed fundoscopy and fluorescein angiography using the eyes at the PI7 day and the PI30 day. Figure 5 shows that the patterns of the blood veins were similar among all the groups, and the fundus in all the eyes, either uninjected or injected, exhibited a normal appearance without noticeable flecks or spots. Fluorescein angiographic observation showed a well-organized vasculature without abnormal blood vessels and leakage in the uninjected mice, compared to the typical neovascularization in a wet AMD mouse model, the very low density lipoprotein receptor knockout (vldlr−/−) mouse, in which numerous hyper-fluorescence spots were seen and fluorescein leakage increased with time (the far right panel). In all mice injected with either saline or various doses of nanoceria, a clear and neat, uniform vascular structure and pattern were seen at these time points.
Inflammation also causes cellular infiltration into the vitreous. To investigate this, we performed histological analysis on the H&E-stained slides at five time points, with 0.5 µg of LPS in 1 µl of saline-injected eyes as a positive control . Compared to the LPS-induced massive anterior segment and vitreous infiltration, we did not find any cells inside the vitreous of the eyes injected with nanoceria or saline (Figure 6).
Nanomaterials have attracted much attention over the past two decades. Because of their small sizes (usually less than 100 nm), surface structures, and unique physicochemical features, nanomaterials easily pass through membranes and are taken up into cells . We and our colleagues have published a series of papers demonstrating the antioxidant properties of nanoceria in scavenging reactive oxygen species (ROS) and nitric oxide species (NOS), the enhancement of cellular survival, and the inhibition of apoptosis in numerous tissues and cells in vivo and in vitro [18,19]. In the eye, we showed that a single intravitreal injection of nanoceria produced sustained therapeutic effects in several mouse models of ocular diseases [2-6]. These results indicate the potential clinical benefit of nanoceria. It has been reported that intravitreal injection of many drugs (and biologic products) at a high dose usually produces ocular inflammation . Although data from our laboratory showed that nanoceria at 1 mM did not alter the retinal structure and function in albino rats , obtaining acceptance of nanoceria as a therapeutic agent remains challenging , especially as the safety of the long-term retention of nanoceria at a maximum dose in the eye is largely unknown.
The toxicity of nanoceria has been assessed in a variety of cells and tissues, including the human neuroblastoma cell line (IMR32) , human lung cells [23,24], cultured human lung cancer cells , wild-type rats , human gastric cancer cells , and human hepatoma SMMC-7721 cells . Interestingly, the data showed that nanosized CeO2 are more toxic than microparticles . A similar report indicated that accumulation of nanoceria-caused toxicity is correlated with increased exposure time , and in a dose- and time-dependent manner that resulted from lipid peroxidation and cell membrane damage , DNA damage and apoptosis , inflammation , oxidative stress, and activation of the mitogen-activated protein kinase (MAPK) pathways . In contrast to these reports, data documenting the protective functions of nanoceria have been reported by many research groups, and in a variety of cells and tissues with varied doses. Data showed that nanoceria decrease by 70% ischemia-induced 3-nitrotyrosine (indicator of protein damage) in a mouse model of cerebral ischemia , protect human tumor monocytes (U937 cells) against TNF-α and cycloheximide-induced alteration of calcium signals, ROS production, and apoptosis , and prevent apoptosis in primary cortical brain cultures . Nanoceria attenuated the systemic inflammatory response associated with peritonitis and significantly improved survival rates , and selectively protected normal human cells, but not cancer cells, from ultraviolet (UV) radiation . In another report, treatment with nanoceria (100 µg/ml) for 48 h did not cause growth or morphological changes in human lens epithelial cells, but exposure to a lower dosage (10 µg/ml) for a longer period of time (72 h) can harm cells . Published data from our group and associated colleagues showed that pretreatment with varying concentrations of nanoceria protects normal cells from radiation-induced damage [36,37]. Similarly, nanoceria prevented tumor growth and invasion  and showed antiangiogenic properties , neuroprotection , cardioprotection , and anti-inflammatory activity [40,41] in multiple tissues or organs. Most importantly, our formulated nanoceria have been reported to be distributed in multiple organs and tissues of CD1 mice via various means of administration and do not cause overt toxicity or pathology, and no significant immune response was detected . We think the differences observed in beneficial versus negative effects of nanoceria by various laboratories are primarily due to differences in the formulations, particle sizes, surface charges, concentrations, distribution and location of nanoceria inside the cells, the age of the animals at treatment, or the experimental conditions in general.
Currently, treatment of eye diseases caused by endogenous intraocular changes involves delivery of therapeutic agents (proteins, DNAs, and cells) into the back of the eye by subretinal or intravitreal injections that by themselves may cause infection and inflammation. In this paper, we experimentally demonstrated that nanoceria, at all doses used, are well tolerated by the ocular cells and do not induce detectable damage to the retinal health as determined with qRT-PCR, western blots, histology, and ERG. There were no changes in the levels of photoreceptor-specific proteins, distribution of visual pigments, retinal morphology, and response to the light. These results demonstrated that nanoceria exert their protective function without negative effects on retinal cells.
As nanoceria do not cause structural and functional alterations in the retina, we were especially interested in determining whether the retention of nanoceria in the eye was associated with increased expression of inflammatory cytokines. Foreign materials in the eye usually cause acute or chronic inflammatory responses , such as elevated cytokine gene expression within several hours or several days after the onset of inflammation. This is followed by cellular infiltration, immune cell activation, and migration to the sites of inflammation . In this study, we performed a PCR array of the common mouse cytokines to assay 89 cytokines at the PI7 day. Of these cytokines, only 11 were either upregulated or downregulated by the nanoceria treatment, but they were similarly affected by saline injection alone (Table 1). Inflammation-associated cytokines, including TNF-α, IL-1β, IL-6, etc., are mainly produced by macrophages and monocytes at inflammatory sites  in response to acute and chronic inflammation. MIF, a multifunctional and ubiquitously expressed protein, is an upstream regulator of inflammatory-immune processes and is expressed at the site of inflammation where MIF primarily modulates macrophage and T cell function for host defense. qRT-PCR and western blots of IL-6, IL-1β, TNF-α, and MIF at PI7 h demonstrated that, compared to the high expression levels of these genes in the LPS-induced positive controls, there was no elevation of these proteins compared to the untreated group. Our previous study demonstrated that the retention of nanoceria in the retina for months after a single intravitreal injection did not alter the retinal structure and function . Our current study suggests that there is no cytotoxicity of nanoceria to the retinal tissues and provides further, and direct, evidence that nanoceria could be a special therapeutic agent for the treatment of ocular diseases.
The authors thank the personnel at the animal, imaging and molecular modules of the Vision Research Core Facility at the Oklahoma University Health Sciences Center. This work was supported in part by NIH NEI P30 EY021725, R21EY018306, R01EY018724 and R01EY022111, National Science Foundation: CBET-0708172. Dr. Xue Cai (firstname.lastname@example.org) and Dr. James F. McGinnis (email@example.com) are co-corresponding authors for this paper. Conflict of Interest and Financial Disclosure Statements: University of Central Florida and University of Oklahoma Health Sciences Center hold patents with James F. McGinnis and Sudipta Seal listed as inventors.