January 2017
Volume 58, Issue 1
Open Access
Cornea  |   January 2017
The Effects of Nonporous Silica Nanoparticles on Cultured Human Keratocytes
Author Affiliations & Notes
  • Bora Yim
    Department of Ophthalmology, Dongguk University, Ilsan Hospital, Goyang, South Korea
  • Joo-Hee Park
    Department of Ophthalmology, Dongguk University, Ilsan Hospital, Goyang, South Korea
  • Hyejoong Jeong
    School of Chemical Engineering and Materials Science, Chung-Ang University, Seoul, South Korea
  • Jinkee Hong
    School of Chemical Engineering and Materials Science, Chung-Ang University, Seoul, South Korea
  • Young-Joo Shin
    Department of Ophthalmology, Hallym University College of Medicine, Seoul, South Korea
  • Roy S. Chuck
    Department of Ophthalmology and Visual Sciences, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, United States
  • Choul Yong Park
    Department of Ophthalmology, Dongguk University, Ilsan Hospital, Goyang, South Korea
  • Correspondence: Choul Yong Park, Department of Ophthalmology, Dongguk University, Ilsan Hospital, 814, Siksadong, Ilsan-dong-gu, Goyang, Kyunggido 410-773, South Korea; oph0112@gmail.com
  • Footnotes
     BY and J-HP contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science January 2017, Vol.58, 362-371. doi:10.1167/iovs.16-20603
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      Bora Yim, Joo-Hee Park, Hyejoong Jeong, Jinkee Hong, Young-Joo Shin, Roy S. Chuck, Choul Yong Park; The Effects of Nonporous Silica Nanoparticles on Cultured Human Keratocytes. Invest. Ophthalmol. Vis. Sci. 2017;58(1):362-371. doi: 10.1167/iovs.16-20603.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Silica nanoparticles (SiNPs) are promising carriers for ophthalmic drug delivery. In this study, we investigated the effect of various sizes of nonporous SiNPs on cultured human keratocytes.

Methods: Three different sizes of SiNPs (50, 100, and 150 nm) were manufactured. Primarily cultured human keratocytes were exposed to different concentrations (0, 25, 50, and 100 μg/mL) of three sizes of SiNPs for up to 72 hours. Intracellular reactive oxygen species (ROS) generation, cellular viability, lactate dehydrogenase (LDH) assay, autophagy, vimentin expression, and mammalian target of rapamycin (mTOR) pathway activation were evaluated. Intracellular distribution of SiNPs was evaluated with transmission electron microscopy.

Results: Transmission electron microscopy revealed SiNPs were taken up by keratocytes inside cytoplasmic vacuoles. Neither nuclear entry of SiNPs nor mitochondrial structural damage was observed. Both intracellular ROS generation and LDH level remained unchanged with up to 100 μg/mL SiNP treatment. Cellular viability was not affected by SiNP treatment. Autophagy showed significant dose-dependent activation with 50- and 100-nm SiNPs. However, mTOR activation remained unchanged. Vimentin expression did not show any significant increase with SiNPs.

Conclusions: Our findings suggested that 50-, 100-, and 150-nm SiNPs did not induce significant cytotoxicity in cultured human keratocytes at concentrations up to 100 μg/mL.

Keratocytes are the specialized fibroblasts residing in corneal stroma.1 Their role is to maintain corneal transparency by synthesizing corneal crystallins, keratin sulfate, and well-organized collagen fibers. Therefore, the disturbance of keratocyte homeostasis may lead to corneal opacity and eventual visual loss.2,3 Keratocytes are known to undergo apoptosis against various external stimuli such as mechanical injury and inflammation. On the other hand, keratocytes can be activated after injury and transform into myofibroblasts, which produce abnormal collagen fibers observed in corneal scar tissue.3 
Amorphous silica nanoparticles (SiNPs) are commonly used as additives in cosmetics, printer toners, packaging, and imaging.4 In addition, SiNPs are actively investigated as promising nanocarrier systems for drug delivery to various human tissues.4 The stable chemical structure, large surface to volume ratios, ease of surface modification, and tolerable biodegradability of SiNPs further increase their attractiveness for biologic applications.4 A recent observation that small sizes (5–50 nm) of SiNPs are permeable into the cornea also suggests their possible use as an ophthalmic drug delivery system.5 However, possible cytotoxicity is the most serious issue concerning SiNPs. It is known that SiNPs can induce biological effects and cellular toxicity depending on SiNP size and concentration and cell types.6,7 In previous reports, different cell types have shown different patterns of SiNP-induced toxicity. When considering the vital roles of keratocytes in the maintenance of corneal transparency, significant cytotoxicity of SiNPs to keratocytes may result in the loss of corneal transparency. However, to date, the effect of SiNPs on keratocytes has not been fully studied, although active research on SiNP-based ocular drug delivery is fully anticipated. 
Herein, monodisperse nonporous SiNPs with diameters of 50, 100, and 150 nm were used to investigate how particle sizes and concentrations affect the biological activities of human keratocytes. Specifically, the effects of different sizes and concentrations of SiNPs on critical biological responses including cellular viability, reactive oxygen species (ROS) generation, and autophagy were evaluated. In addition, the effect of SiNPs on an upstream cellular proliferative pathway, the mammalian target of rapamycin (mTOR) pathway, was investigated. 
Materials and Methods
Nanoparticle Synthesis and Characterization
The SiNPs (sizes: 50, 100, and 150 nm) were prepared using the Stöber synthesis method.8 To synthesize 50 nm SiNP, 2 mL ammonia (NH4OH, 28%; Junsei, Tokyo, Japan) and 50 mL ethyl alcohol (EtOH, anhydrous, 99.5%; Daejung, Kyeonggi, Korea) were first mixed. Then 1 mL tetraethylorthosilicate (TEOS; Samchun, Gyunggi, Korea) was added; 100- and 150-nm SiNPs were prepared using equal molar ratios of ingredients. Next, 1.5 mL TEOS was added to the as-prepared 3-mL solution of ammonia in 50 mL ethyl alcohol. Smaller-sized SiNPs could be produced by quickly adding TEOS while stirring the solution. Afterward, the solutions were stirred for 12 hours under ambient conditions (25°C, 1 atm). The prepared SiNPs were washed three times with EtOH using centrifugation (10,000 rpm, 15 minutes). The final SiNP precipitates were dispersed in distilled water. 
The surface charge of the prepared SiNPs was measured by ζ potential measurement (SZ-100; Horiba, Kyoto, Japan) in distilled water and Dulbecco's PBS at room temperature (25°C). To analyze surface charges during cell culture, each SiNP was measured after incubation for 24 hours in cell culture media (DMEM/F12 supplemented with 10% FBS) at 37°C. The size and distribution were analyzed by scanning electron microscopy (SEM) (SIGMA; Carl Zeiss Meditec, Jena, Germany) images and ImageJ (National Institutes of Health, Bethesda, MD, USA) software. The dispersity of the nanoparticles was defined as the coefficient of variation [dispersity (%) = σ / d × 100, where σ is the SD and d is the mean size].9 
Cell Culture
The primary culture of human keratocytes was performed. Descemet's membrane and epithelium were removed using forceps and an ophthalmic knife, and stroma was minced in a laminar flow hood. Midstroma and posterior stroma explants were then suspended in culture medium and cultured in 24-well plates. The corneal stroma was sliced into quarters and digested overnight with 2.0 mg/mL collagenase (Roche, Basel, Switzerland) and 0.5 mg/mL hyaluronidase (Worthington Biochemicals, Lakewood, NJ, USA) in DMEM at 37°C. Isolated cells were washed in DMEM and cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS; Gibco-Invitrogen, Grand Island, NY, USA). The cells were cultured on tissue culture-treated plastic at 4 × 104 cells/cm2. After reaching confluency, cells were harvested and resuspended in culture medium. The cells were plated in 75-cm2 tissue flasks and then maintained at 37°C in 5% CO2 and 95% air. Culture medium was changed every 3 days, and the cells were passaged using 0.25% Trypsin-EDTA (Gibco BRL, Carlsbad, CA, USA). Passage number ≤7 was used in this paper. 
Treatment of SiNPs
The sizes of 50-, 100-, and 150-nm SiNPs were confirmed using an SEM. The stock solutions of SiNPs were 10 mg/mL in DPBS (Gibco), and all particles were sonicated for 30 minutes before mixing into culture media. 
Electron Microscopy and Ultrastructural Analysis
For transmission electron microscopic (TEM) observations, keratocytes were treated with three sizes of SiNPs (100 μg/mL) for 24 hours and then fixed in 3.7% paraformaldehyde (Sigma-Aldrich Corp., St. Louis, MO, USA) and 2.5% glutaraldehyde (Sigma-Aldrich Corp.) in 0.1 M phosphate buffer (PB; pH 7.6) overnight. After washing in 0.1 M PB, keratocytes were fixed with 1% osmium tetroxide (OsO4) in the same buffer for 1 hour. The cells were then dehydrated with a series of the graded EtOH (Merck, Kenilworth, NJ, USA). The cells were embedded in epoxy embedding medium (Sigma-Aldrich Corp.), and then polymerization was performed at 60°C for 3 days. Ultrathin sections (60–70 nm) were obtained by ultramicrotome (Leica Ultracut UCT; Leica, Wetzlar, Germany). Ultrathin sections collected on grids (200 mesh) were examined under the TEM (JEM-1010; JEOL, Tokyo, Japan) operating at 60 kV, and images were recorded by CCD camera (SC1000; Gatan, Warrendale, PA, USA). Length on the electron micrograph was measured using GMS software (Gatan). The normal control was incubated only in corneal basal medium for 24 hours, and the positive control for autophagy was treated with 50 μM chloroquine diphosphate for 24 hours. 
Measurement of ROS
Generation of intracellular ROS was detected using a Fluorometric Intracellular Ros Kit (catalog number: MAK144; Sigma-Aldrich Corp.). Keratocytes were cultured at 3 ×103 cells per well in a 96-black well plate and treated with each size of SiNP at different concentrations (0, 25, 50, and 100 μg/mL) for 24, 48, and 72 hours. Following incubation, cells were assayed for the measurement of intracellular ROS following the manufacturer's protocol. Briefly, 100 μL master reaction mix was put into the well plate, and the plate was incubated at 5% CO2 and 37°C for 30 minutes. Finally, fluorescence was measured at 540-nm excitation and 570-nm emission. A standard curve was generated by using different concentrations (0–20 μM) of H2O2. The measured fluorescence intensity of the experimental samples (various concentration of SiNPs addition) and negative control (no SiNPs addition) were translated into a standard curve. Reactive oxygen species in various concentrations of SiNP solutions was represented as relative percentage compared with the negative control (no SiNP addition). 
Cell Viability Assay
The cell viability assay was performed using a cell counting kit reagent (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol. Briefly, keratocytes were cultured at 3 × 103 cells per well in a 96-well plate and incubated for 24 hours. Following the adherence of cells, 50-, 100-, and 150-nm SiNPs were added to the culture media for 24, 48, and 72 hours, dose-dependently at 0, 25, 50, and 100 μg/mL. The wells with no SiNP addition and the wells with dimethyl sulfoxide (DMSO) addition were used as negative and positive controls, respectively. After the appropriate incubation, 10 μL CCK-8 solution was added to each cultured well, and the absorbance was measured at 450 nm after 2-hour incubation of keratocytes with the reagent. Cell viability in various SiNP solutions was represented as relative percentage compared with negative control. 
Lactate Dehydrogenase Assay
Cellular death via membrane damage was measured using a LDH cytotoxicity detection kit (Takara Bio, Inc., Shiga, Japan). The experimental procedure was performed following the manufacturer's protocol. Briefly, keratocytes were cultured at 3 ×103 cells per well in a 96-well plate and incubated for 24 and 48 hours. Following the adherence of cells, 50-, 100-, and 150-nm SiNPs were applied to cells for 48 hours, dose-dependently, at 0, 25, 50, and 100 μg/mL. The wells with no SiNP addition and the wells with 1% Triton X-100 addition were used as negative and positive controls, respectively. Following the incubation of cells, all supernatants were transferred into the new 96-well plate, and the reaction mixture was added and incubated for 20 minutes at room temperature. Absorbance was measured at 490 nm. 
Western Blot Analysis
All SiNPs treated keratocytes were lysed in ice-cold radio immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS) for 30 minutes. The debris were removed by centrifugation at 16,000g for 1 minute. Equal amounts (20 μg) of total cell protein were separated by SDS-PAGE and transferred to the polyvinvlyidene difluoride (PVDF) membrane. After blocking with 5% BSA in TTBS buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-LC3A/B (1:1000; catalog number: 12741; Cell Signaling, Beverly, MA, USA), rabbit anti–phospho-mTOR (1:1000; catalog number: 5536; Cell Signaling), rabbit anti-mTOR (1:1000; catalog number: 2983; Cell Signaling), rabbit anti-vimentin (1:1000; catalog number: MAB3400; Merck Millipore, Guyancourt, France), and mouse anti–β-actin (1:10,000; catalog number: sc-47778; Santa Cruz, Biotechnology, Dallas, TX, USA). The membranes were incubated with peroxidase-conjugated secondary antibody for 1 hour at room temperature. Blots were developed using an enhanced chemiluminescence kit (catalog number: RPN2232; GE Healthcare, Buckinghamshire, UK) and visualized using a Fujifilm Image Reader LAS-3000 (Fujifilm, Tokyo, Japan). Each experiment was repeated at least three times, and the densitometric analysis was performed using the Multi Gauge V3.0 (Fujifilm Life Science, Tokyo, Japan). 
Immunocytochemistry
Keratocytes were seeded at a density of 2 × 104 cells/mL and grown on 4-well Lab-Tek chamber slides (Nalgene Nunc International, Penfield, NY, USA), and 0, 25, 50, and 100 μg/mL SiNPs were treated for 24 hours. Cells were fixed with 3.7% paraformaldehyde for 10 minutes at room temperature, and permeabilization was carried out using 0.1% Triton X-100 for 5 minutes at room temperature. Following the washing steps with DPBS, cells were blocked using 1% BSA in DPBS for 30 minutes at room temperature. The chamber slides were incubated overnight at 4°C with rabbit polyclonal anti-LC3B (0.5 μg/mL; catalog number: L10382; Molecular Probes, ThermoFisher Scientific, Inc., Waltham, MA, USA) or rabbit anti-vimentin (1:1000; catalog number: MAB3400; Merck Millipore). The chamber slides were then washed with DPBS and incubated with Alexa 488–conjugated donkey anti-rabbit antibody (1:1000; catalog number: A21206; Molecular Probes) or Alexa 488–conjugated donkey anti-mouse antibody (1:1000; catalog number: A21202; Molecular Probes) for 2 hours at room temperature. Staining for F-actin was carried out using tetramethylrhodamine isothiocyanate–conjugated phalloidin (1 μg/mL; Sigma-Aldrich Corp.). Counterstaining of cell nuclei was carried out using 4′,6-diamidino-2′-phenylindole (DAPI; catalog number: P36931; Molecular Probes) with mounting solution. Slides were viewed using a fluorescence microscope. 
In Vivo Corneal Effect of Topically Applied SiNPs
To investigate the in vivo corneal effect of SiNPs, we used 10 Sprague-Dawley rats (7 weeks old; five males and five females). Animals were treated in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The rats were treated with topical SiNP solution (100-nm SiNPs, 10 mg/mL concentration mixed with PBS) of one drop every 6 hours for 4 weeks to the right eyes. Corneal photographs of the right eyes were taken at baseline and then weekly. Corneal transparency was evaluated based on these photographs. 
Statistical Analysis
Data are presented as mean ± SE, and statistical significance was determined by 1-way ANOVA followed by the Dunnett's multiple comparison test. P < 0.05 was regarded as significant using GraphPad Prism Ver. 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). 
Results
Characterization of SiNPs
The morphologies of each SiNP were observed by SEM, and size distribution graphs were obtained from the micrographs (Fig. 1). The numerical data of mean size and dispersity are summarized in the Table. The mean sizes of SiNPs are 50.68, 102.81, and 149.41 nm according to the size distribution graphs in Figure 1. Dispersity of nanoparticles is determined on the basis of the coefficient of variation. Nanoparticles with less than a 5% coefficient of variation are defined as monodisperse nanoparticles. To identify the stability of SiNPs in different aqueous solutions, we investigated the ζ potential. In distilled water, SiNPs have good stability and dispersion, with higher ζ potential over −50 mV. On the contrary, SiNPs dispersed in DPBS showed a lower ζ potential close to a neutral charge. This is because the negative charge of SiNPs is offset by various salts in DPBS. After 24 hours of incubation in the medium, SiNPs are closer to neutral surface charge due to protein absorption.10 From this result, we could predict that the charges of the SiNPs are almost neutral in cell culture media and are prone to agglomerate together in cell culture 
Figure 1
 
Morphology of SiNPs was observed by SEM: (A) 50, (B) 100, and (C) 150 nm. Size distribution graphs corresponding to above SEM images are shown: (D) 50, (E) 100, and (F) 150 nm. Reprinted from Park J-H, Jeong H, Hong J, et al. The effect of silica nanoparticles on human corneal epithelial cells. Sci Rep. 2016;6:37762, licensed under a Creative Commons Attribution 4.0 International License.
Figure 1
 
Morphology of SiNPs was observed by SEM: (A) 50, (B) 100, and (C) 150 nm. Size distribution graphs corresponding to above SEM images are shown: (D) 50, (E) 100, and (F) 150 nm. Reprinted from Park J-H, Jeong H, Hong J, et al. The effect of silica nanoparticles on human corneal epithelial cells. Sci Rep. 2016;6:37762, licensed under a Creative Commons Attribution 4.0 International License.
Table
 
Size and ζ Potential of Silica Nanoparticles (at Concentration of 1 mg/mL) Investigated in This Study
Table
 
Size and ζ Potential of Silica Nanoparticles (at Concentration of 1 mg/mL) Investigated in This Study
Intracellular Distribution of SiNPs
Cultured keratocytes showed high vimentin expression, as expected (Fig. 2). Internalization and intracellular distribution of SiNPs in keratocytes were evaluated by TEM. SiNPs were localized primarily in cytoplasmic vesicles (including some autophagosomes) (Fig. 3). There were no SiNPs observed inside either the nucleus or mitochondria. Neither mitochondrial damage nor nuclear membrane damage was observed (Fig. 3). 
Figure 2
 
Phase contrast microscopic appearance of cultured human keratocytes and vimentin expression. (A) A phase contrast microscopic image of cultured human keratocytes. (B) Vimentin expression of keratocytes was stained green, whereas the nucleus was stained blue.
Figure 2
 
Phase contrast microscopic appearance of cultured human keratocytes and vimentin expression. (A) A phase contrast microscopic image of cultured human keratocytes. (B) Vimentin expression of keratocytes was stained green, whereas the nucleus was stained blue.
Figure 3
 
Cellular uptake of SiNPs in keratocytes evaluated by TEM. Keratocytes were cultured with various sizes of SiNPs for 24 hours (AC). The SiNPs (black spheres) were mainly accumulated in cytoplasmic vesicles (insets), whereas no SiNPs were observed in a negative control (D). No nuclear entry of SiNPs was observed. Mitochondria remained intact with no visible damage (black arrows in insets): (A) 50-nm SiNPs added (100 μg/mL); (B) 100-nm SiNPs added (100 μg/mL); (C) 150-nm SiNPs added (100 μg/mL); and (D) negative control with no SiNPs.
Figure 3
 
Cellular uptake of SiNPs in keratocytes evaluated by TEM. Keratocytes were cultured with various sizes of SiNPs for 24 hours (AC). The SiNPs (black spheres) were mainly accumulated in cytoplasmic vesicles (insets), whereas no SiNPs were observed in a negative control (D). No nuclear entry of SiNPs was observed. Mitochondria remained intact with no visible damage (black arrows in insets): (A) 50-nm SiNPs added (100 μg/mL); (B) 100-nm SiNPs added (100 μg/mL); (C) 150-nm SiNPs added (100 μg/mL); and (D) negative control with no SiNPs.
Reactive Oxygen Species Generation, LDH, and Cellular Viability Assay
All sizes of SiNPs did not significantly increase intracellular ROS levels of keratocytes at concentrations up to 100 μg/mL (Figs. 4AC). Keratocytes viability was not changed significantly with treatment by SiNPs (Figs. 4DF). In addition, LDH, which is released with cell membrane damage, showed no increase with treatment by SiNPs (Figs. 4G, 4H). 
Figure 4
 
Intracellular ROS generation assay, cellular viability assay, and LDH assay. (AC) Intracellular ROS generation shows no significant change after SiNPs addition for 24, 48, and 72 hours. (DF) Cellular viability assay using CCK-8 kit shows increasing trend of viability with SiNPs addition for 24, 48, and 72 hours. However, no statistical significance was found. (G, H) Lactate dehydrogenase assay showed no significant increase after SiNP addition for 24 and 48 hours. Lactate dehydrogenase decreased significantly with SiNPs of 50 nm at 25 μg/mL concentration at 48 hours. Triplicates of each treatment group were used in each independent experiment. Values are the mean ± SEM from three independent experiments. *P < 0.05.
Figure 4
 
Intracellular ROS generation assay, cellular viability assay, and LDH assay. (AC) Intracellular ROS generation shows no significant change after SiNPs addition for 24, 48, and 72 hours. (DF) Cellular viability assay using CCK-8 kit shows increasing trend of viability with SiNPs addition for 24, 48, and 72 hours. However, no statistical significance was found. (G, H) Lactate dehydrogenase assay showed no significant increase after SiNP addition for 24 and 48 hours. Lactate dehydrogenase decreased significantly with SiNPs of 50 nm at 25 μg/mL concentration at 48 hours. Triplicates of each treatment group were used in each independent experiment. Values are the mean ± SEM from three independent experiments. *P < 0.05.
Cellular Autophagy
We investigated the effect of SiNPs on the cellular autophagy system using the signal alteration of LC3A/B, the autophagy marker (Figs. 5A, 5B). With the activation of autophagy, the LC3A/B II form increases relative to the LC3A/B I form, and SiNPs of 50 and 100 nm triggered significant expression of LC3A/B II proteins. The increased ratio of activated LC3A/B was more prominent at a high concentration (100 μg/mL) of SiNPs. However, the expression of LC3A/B II proteins by SiNPs with 150-nm size did not reach statistical significance. Increased LC3B proteins in cytoplasm with the SiNPs addition were also demonstrated by immunocytochemistry (Fig. 5C). Autophagosome formation was activated to remove SiNPs in keratocytes (Fig. 5D). 
Figure 5
 
The effect of SiNPs on keratocytes' autophagy. (A) The expression levels for the autophagy signal and LC3A/B proteins were measured in keratocytes treated with SiNPs for 24 hours by Western blot analysis. The inactive I form is 16 kDa, and the active II form is 14 kDa. (B) Densitometric analyses of Western blots showed increased expression of the II form with higher concentration of SiNPs (50 and 100 nm) added. Values (mean ± SEM) are expressed as a percentage of the control and were obtained from three independent experiments; each independent experiment was performed in triplicate (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Immunocytochemical staining with LC3B antibody revealed the increased autophagy in keratocytes with 100 μg/mL 50-nm SiNP (b), 100-nm SiNP (c), and 150-nm SiNP (d) addition. White arrows indicate the increased LC3B staining (green). DAPI-stained nucleus with blue and red represented F-actin. Negative control is keratocytes with no SiNP addition (a). (D) Transmission electron microscopy revealed some SiNPs inside amphisomes (AS), endosomes (ES), and lysosomes (LS), which were usually formed during the process of authophagosomes (white arrowheads in ad). (a) Positive control of autophagosomes (white arrowheads) induced by incubation with 50 μM chloroquine diphosphate for 24 hours in keratocytes.
Figure 5
 
The effect of SiNPs on keratocytes' autophagy. (A) The expression levels for the autophagy signal and LC3A/B proteins were measured in keratocytes treated with SiNPs for 24 hours by Western blot analysis. The inactive I form is 16 kDa, and the active II form is 14 kDa. (B) Densitometric analyses of Western blots showed increased expression of the II form with higher concentration of SiNPs (50 and 100 nm) added. Values (mean ± SEM) are expressed as a percentage of the control and were obtained from three independent experiments; each independent experiment was performed in triplicate (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Immunocytochemical staining with LC3B antibody revealed the increased autophagy in keratocytes with 100 μg/mL 50-nm SiNP (b), 100-nm SiNP (c), and 150-nm SiNP (d) addition. White arrows indicate the increased LC3B staining (green). DAPI-stained nucleus with blue and red represented F-actin. Negative control is keratocytes with no SiNP addition (a). (D) Transmission electron microscopy revealed some SiNPs inside amphisomes (AS), endosomes (ES), and lysosomes (LS), which were usually formed during the process of authophagosomes (white arrowheads in ad). (a) Positive control of autophagosomes (white arrowheads) induced by incubation with 50 μM chloroquine diphosphate for 24 hours in keratocytes.
Mammalian Target of Rapamycin Pathway Activation
Silica nanoparticles induced no significant change in the mTOR pathway of keratocytes. We measured the expression level of phosphorylated mTOR (p-mTOR) and mTOR (Fig. 6). The expression of p-mTOR increased compared with the normal control, but it failed to attain statistical significance. This suggests that SiNPs used in this study did not inhibit one of the most important cell survival pathways such as mTor pathway. Which is consistent with the results of our cellular viability assay. 
Figure 6
 
Effect of SiNPs on mTOR signaling. The expression levels of phosphorylated mTOR (p-mTOR) and mTOR detected by Western blot analysis (A) and relative densitometry (B) were calculated as a percentage of the control, and all values (mean ± SEM) were obtained from three independent experiments; each independent experiment was performed in triplicate. The mTOR signal activation was not significantly changed with the treatment of SiNPs at concentrations up to 100 μg/mL.
Figure 6
 
Effect of SiNPs on mTOR signaling. The expression levels of phosphorylated mTOR (p-mTOR) and mTOR detected by Western blot analysis (A) and relative densitometry (B) were calculated as a percentage of the control, and all values (mean ± SEM) were obtained from three independent experiments; each independent experiment was performed in triplicate. The mTOR signal activation was not significantly changed with the treatment of SiNPs at concentrations up to 100 μg/mL.
Vimentin Expression
The vimentin expression level was investigated to predict myofibroblastic change of keratocytes by SiNP addition. Both the size and concentration of SiNPs had no significant effect on vimentin expression (Fig. 7). 
Figure 7
 
Western blot analysis for vimentin expression with SiNPs. Vimentin expression levels were not changed significantly with SiNP addition in cultured human keratocytes. Each panel has a picture of a representative Western blot and a graph showing the relative densitometry compared with the negative control (no addition of SiNPs): (A) 50-nm SiNPs; (B) 100-nm SiNPs, and (C) 150-nm SiNPs.
Figure 7
 
Western blot analysis for vimentin expression with SiNPs. Vimentin expression levels were not changed significantly with SiNP addition in cultured human keratocytes. Each panel has a picture of a representative Western blot and a graph showing the relative densitometry compared with the negative control (no addition of SiNPs): (A) 50-nm SiNPs; (B) 100-nm SiNPs, and (C) 150-nm SiNPs.
In Vivo Corneal Effect of Topical SiNPs
Corneal transparency was well maintained in all studied rats for 4 weeks. No abnormal corneal lesions such as corneal neovascularization, signs of active inflammation, or focal opacity were observed (Fig. 8). 
Figure 8
 
In vivo corneal effect of topical SiNP application. Corneas were imaged at baseline and then every week up to 4 weeks. Both corneal transparency and limbal vessels were maintained normal.
Figure 8
 
In vivo corneal effect of topical SiNP application. Corneas were imaged at baseline and then every week up to 4 weeks. Both corneal transparency and limbal vessels were maintained normal.
Discussion
In this study, we found that monodisperse nonporous SiNPs of 50, 100, and 150 nm induced no significant cytotoxicity in cultured human keratocytes up to 48 hours and at a concentration of up to 100 μg/mL. Cellular ROS, autophagy, and the mTOR pathway remained intact with a relatively high concentration of SiNPs. 
Ocular drug delivery is one of the most challenging routes of administration of therapeutic agents to the human body.11 For efficient intraocular drug delivery, nanoparticle-based ocular drug delivery has been actively investigated in cornea including gold nanoparticles, carbon nanoparticles, lipid nanoparticle, chitosan nanoparticle, liquid crystalline nanoparticle, and albumin nanoparticles.1217 The reports of successful drug delivery by these various nanocarriers have inspired future development of nano-based eye drops for clinical use. 
Silica nanoparticles are some of the most promising nanocarrier systems for potential ocular drug delivery. However, the two most important prerequisites of effective ophthalmic drugs are safety and efficient corneal penetration.18 As mentioned earlier, the safety of SiNPs has always been a challenge for clinical application. It is well known that the crystalline form of silica is toxic to alveolar cells and can induce irreversible silicosis of lung.19,20 However, amorphous silica such as nonporous silica particles is considered relatively biologically safe due to its biodegradability.21 Recently, Kim et al.22 reported the relative safety of 20- and 100-nm SiNPs after high-dose oral intake (2000 mg/kg) for 90 days in rats. In addition, topical skin exposure to 20-nm SiNPs for 90 days induced no significant toxicity.23 However, there are negative reports suggesting significant nanotoxicity induced by SiNPs.2428 The debates regarding SiNPs nanotoxicity are still ongoing. The nanotoxicity of SiNPs seems to be dependent on size, dose, and cell types.7,29 In addition, the size of the silica nanoparticle seems to be important in interactions between the nanoparticle and the cell membrane.30 Larger sizes of SiNPs (>100 nm) can induce significant distortion of cell membrane and eventual cell rupture, whereas smaller-sized SNPs can penetrate cell membrane without membrane rupture. 
Although active investigations of SiNPs in various human diseases have been undertaken recently, few studies exist of SiNPs as an ocular delivery carrier.31,32 Recently, one study demonstrated that SiNPs had no direct cytotoxicity on retinal endothelial cells and retinal neuronal tissue.31 Another study reported the antiangiogenic effect of SiNPs in a corneal neovascularization model.32 However, nanotoxicity of SiNPs on corneal cells has not been fully studied, although corneal cells are the first cells encountered when SiNPs are topically administered for ocular therapy. 
Verifying the safety of SiNPs on cultured human keratocytes is important for the future development of a SiNPs-based topical ocular delivery system. Keratocytes are important cells necessary to the maintenance of corneal transparency.1 Therefore, any potential medication developed for ophthalmic use should be screened for keratocytes toxicity. In this study, we observed stable intracellular ROS generation and mild increase of autophagy by SiNPs in keratocytes. Excessive ROS generation and autophagy are well-known cellular stress responses. Although intracellular ROS remained stable in our study, the increase of autophagy is consistent with previous reports that SiNPs can induce cellular stress and sometimes lead to cell death.3336 However, the increased autophagy in keratocytes by SiNPs did not result in decreased cell viability in our study. 
The safety of SiNPs on cultured keratocytes was further verified by the intact mTOR pathway. It has been known that the mTOR pathway is the key regulator of cell survival. mTOR activation leads to cellular protein synthesis and cell division. However, when the apoptotic pathway is activated by excessive cellular stress, mTOR is usually inactivated. In addition, mTOR is also a negative regulator of autophagy. Autophagy is a natural cellular process to clean up unnecessary and dysfunctional cellular components for recycling.37 It helps cells overcome external stress and survive in a harsh environment. However, autophagy can also be regulated by various pathways independent of mTOR.3841 Uncoupling between mTOR and autophagy was found in our study. Our data suggest that increased autophagy activation and unchanged mTOR activation can coexist in keratocytes with intracellular SiNP accumulation. We hypothesize that the activation of cellular survival machinery was maintained with cleaning up of intracellular debris (including SiNPs) by activated autophagy. 
Various stimuli can transform keratocytes into myofibroblasts, which produce disorganized extracellular matrix and corneal opacity.2,3 With myofibroblast transformation, vimentin expression significantly increases in keratocytes. However, our finding that vimentin expression was not affected by SiNPs also suggests the tolerability of human keratocytes to SiNPs treatment. 
Our study verified the safety issue of SiNPs in human keratocytes and may increase the potential of SiNPs as a candidate platform for topical ocular drug delivery. One of the promising fields of topical SiNPs application is nitric oxide delivery. SiNPs can be successfully combined with various nitric oxide donors to become a controlled delivery platform for nitric oxide to target tissue.42,43 As previously reported, nitric oxide releasing silica nanoparticles can be useful in controlling intraocular pressure.44,45 Corneal wound healing modulation is another possibility for nitric oxide releasing SiNPs.46 
There are several limitations to our study. First of all, we tested the concentration of SiNPs up to 100 μg/mL. It is possible that a further increase of SiNP concentration may induce significant cellular damage. Another limitation is that we tested only three different sizes of SiNPs. There is a possibility that smaller or larger sizes of SiNPs can induce different changes in keratocytes. In addition, uptake of SiNPs by cells may eventually lead to perturbation of intracellular mechanisms even though no acute cytotoxicity is observed. Therefore, close observation for an extended period of time is necessary to further verify the safety of SiNPs. The lack of molecular analysis using harvested animal tissue can be another limitation of our study. Although sufficient safety was verified in vitro and in vivo through gross imaging analysis, further molecular studies using animal tissue can enhance safety confirmation of SiNPs on the ocular surface. 
In conclusion, we found SiNPs of sizes 50, 100, and 150 nm in cultured keratocytes up to 72 hours to be safe. Cellular uptake of SiNPs was localized to cytoplasm and inside autophagosomes. Adding SiNPs to the culture medium enhanced cellular autophagy pathways. Cellular ROS generation, mTOR pathway, and viability were not affected. Our findings suggest that SiNPs of 50-, 100-, and 150-nm sizes did not induce a deleterious effect on keratocytes in the tested concentrations. 
Acknowledgments
Supported in part by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (Grant HI-15C1653). 
Disclosure: B. Yim, None; J.-H. Park, None; H. Jeong, None; J. Hong, None; Y.-J. Shin, None; R.S. Chuck, None; C.Y. Park, None 
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Figure 1
 
Morphology of SiNPs was observed by SEM: (A) 50, (B) 100, and (C) 150 nm. Size distribution graphs corresponding to above SEM images are shown: (D) 50, (E) 100, and (F) 150 nm. Reprinted from Park J-H, Jeong H, Hong J, et al. The effect of silica nanoparticles on human corneal epithelial cells. Sci Rep. 2016;6:37762, licensed under a Creative Commons Attribution 4.0 International License.
Figure 1
 
Morphology of SiNPs was observed by SEM: (A) 50, (B) 100, and (C) 150 nm. Size distribution graphs corresponding to above SEM images are shown: (D) 50, (E) 100, and (F) 150 nm. Reprinted from Park J-H, Jeong H, Hong J, et al. The effect of silica nanoparticles on human corneal epithelial cells. Sci Rep. 2016;6:37762, licensed under a Creative Commons Attribution 4.0 International License.
Figure 2
 
Phase contrast microscopic appearance of cultured human keratocytes and vimentin expression. (A) A phase contrast microscopic image of cultured human keratocytes. (B) Vimentin expression of keratocytes was stained green, whereas the nucleus was stained blue.
Figure 2
 
Phase contrast microscopic appearance of cultured human keratocytes and vimentin expression. (A) A phase contrast microscopic image of cultured human keratocytes. (B) Vimentin expression of keratocytes was stained green, whereas the nucleus was stained blue.
Figure 3
 
Cellular uptake of SiNPs in keratocytes evaluated by TEM. Keratocytes were cultured with various sizes of SiNPs for 24 hours (AC). The SiNPs (black spheres) were mainly accumulated in cytoplasmic vesicles (insets), whereas no SiNPs were observed in a negative control (D). No nuclear entry of SiNPs was observed. Mitochondria remained intact with no visible damage (black arrows in insets): (A) 50-nm SiNPs added (100 μg/mL); (B) 100-nm SiNPs added (100 μg/mL); (C) 150-nm SiNPs added (100 μg/mL); and (D) negative control with no SiNPs.
Figure 3
 
Cellular uptake of SiNPs in keratocytes evaluated by TEM. Keratocytes were cultured with various sizes of SiNPs for 24 hours (AC). The SiNPs (black spheres) were mainly accumulated in cytoplasmic vesicles (insets), whereas no SiNPs were observed in a negative control (D). No nuclear entry of SiNPs was observed. Mitochondria remained intact with no visible damage (black arrows in insets): (A) 50-nm SiNPs added (100 μg/mL); (B) 100-nm SiNPs added (100 μg/mL); (C) 150-nm SiNPs added (100 μg/mL); and (D) negative control with no SiNPs.
Figure 4
 
Intracellular ROS generation assay, cellular viability assay, and LDH assay. (AC) Intracellular ROS generation shows no significant change after SiNPs addition for 24, 48, and 72 hours. (DF) Cellular viability assay using CCK-8 kit shows increasing trend of viability with SiNPs addition for 24, 48, and 72 hours. However, no statistical significance was found. (G, H) Lactate dehydrogenase assay showed no significant increase after SiNP addition for 24 and 48 hours. Lactate dehydrogenase decreased significantly with SiNPs of 50 nm at 25 μg/mL concentration at 48 hours. Triplicates of each treatment group were used in each independent experiment. Values are the mean ± SEM from three independent experiments. *P < 0.05.
Figure 4
 
Intracellular ROS generation assay, cellular viability assay, and LDH assay. (AC) Intracellular ROS generation shows no significant change after SiNPs addition for 24, 48, and 72 hours. (DF) Cellular viability assay using CCK-8 kit shows increasing trend of viability with SiNPs addition for 24, 48, and 72 hours. However, no statistical significance was found. (G, H) Lactate dehydrogenase assay showed no significant increase after SiNP addition for 24 and 48 hours. Lactate dehydrogenase decreased significantly with SiNPs of 50 nm at 25 μg/mL concentration at 48 hours. Triplicates of each treatment group were used in each independent experiment. Values are the mean ± SEM from three independent experiments. *P < 0.05.
Figure 5
 
The effect of SiNPs on keratocytes' autophagy. (A) The expression levels for the autophagy signal and LC3A/B proteins were measured in keratocytes treated with SiNPs for 24 hours by Western blot analysis. The inactive I form is 16 kDa, and the active II form is 14 kDa. (B) Densitometric analyses of Western blots showed increased expression of the II form with higher concentration of SiNPs (50 and 100 nm) added. Values (mean ± SEM) are expressed as a percentage of the control and were obtained from three independent experiments; each independent experiment was performed in triplicate (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Immunocytochemical staining with LC3B antibody revealed the increased autophagy in keratocytes with 100 μg/mL 50-nm SiNP (b), 100-nm SiNP (c), and 150-nm SiNP (d) addition. White arrows indicate the increased LC3B staining (green). DAPI-stained nucleus with blue and red represented F-actin. Negative control is keratocytes with no SiNP addition (a). (D) Transmission electron microscopy revealed some SiNPs inside amphisomes (AS), endosomes (ES), and lysosomes (LS), which were usually formed during the process of authophagosomes (white arrowheads in ad). (a) Positive control of autophagosomes (white arrowheads) induced by incubation with 50 μM chloroquine diphosphate for 24 hours in keratocytes.
Figure 5
 
The effect of SiNPs on keratocytes' autophagy. (A) The expression levels for the autophagy signal and LC3A/B proteins were measured in keratocytes treated with SiNPs for 24 hours by Western blot analysis. The inactive I form is 16 kDa, and the active II form is 14 kDa. (B) Densitometric analyses of Western blots showed increased expression of the II form with higher concentration of SiNPs (50 and 100 nm) added. Values (mean ± SEM) are expressed as a percentage of the control and were obtained from three independent experiments; each independent experiment was performed in triplicate (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Immunocytochemical staining with LC3B antibody revealed the increased autophagy in keratocytes with 100 μg/mL 50-nm SiNP (b), 100-nm SiNP (c), and 150-nm SiNP (d) addition. White arrows indicate the increased LC3B staining (green). DAPI-stained nucleus with blue and red represented F-actin. Negative control is keratocytes with no SiNP addition (a). (D) Transmission electron microscopy revealed some SiNPs inside amphisomes (AS), endosomes (ES), and lysosomes (LS), which were usually formed during the process of authophagosomes (white arrowheads in ad). (a) Positive control of autophagosomes (white arrowheads) induced by incubation with 50 μM chloroquine diphosphate for 24 hours in keratocytes.
Figure 6
 
Effect of SiNPs on mTOR signaling. The expression levels of phosphorylated mTOR (p-mTOR) and mTOR detected by Western blot analysis (A) and relative densitometry (B) were calculated as a percentage of the control, and all values (mean ± SEM) were obtained from three independent experiments; each independent experiment was performed in triplicate. The mTOR signal activation was not significantly changed with the treatment of SiNPs at concentrations up to 100 μg/mL.
Figure 6
 
Effect of SiNPs on mTOR signaling. The expression levels of phosphorylated mTOR (p-mTOR) and mTOR detected by Western blot analysis (A) and relative densitometry (B) were calculated as a percentage of the control, and all values (mean ± SEM) were obtained from three independent experiments; each independent experiment was performed in triplicate. The mTOR signal activation was not significantly changed with the treatment of SiNPs at concentrations up to 100 μg/mL.
Figure 7
 
Western blot analysis for vimentin expression with SiNPs. Vimentin expression levels were not changed significantly with SiNP addition in cultured human keratocytes. Each panel has a picture of a representative Western blot and a graph showing the relative densitometry compared with the negative control (no addition of SiNPs): (A) 50-nm SiNPs; (B) 100-nm SiNPs, and (C) 150-nm SiNPs.
Figure 7
 
Western blot analysis for vimentin expression with SiNPs. Vimentin expression levels were not changed significantly with SiNP addition in cultured human keratocytes. Each panel has a picture of a representative Western blot and a graph showing the relative densitometry compared with the negative control (no addition of SiNPs): (A) 50-nm SiNPs; (B) 100-nm SiNPs, and (C) 150-nm SiNPs.
Figure 8
 
In vivo corneal effect of topical SiNP application. Corneas were imaged at baseline and then every week up to 4 weeks. Both corneal transparency and limbal vessels were maintained normal.
Figure 8
 
In vivo corneal effect of topical SiNP application. Corneas were imaged at baseline and then every week up to 4 weeks. Both corneal transparency and limbal vessels were maintained normal.
Table
 
Size and ζ Potential of Silica Nanoparticles (at Concentration of 1 mg/mL) Investigated in This Study
Table
 
Size and ζ Potential of Silica Nanoparticles (at Concentration of 1 mg/mL) Investigated in This Study
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