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The Effect of Silica Nanoparticles on Human Corneal Epithelial Cells

View Article: PubMed Central - PubMed

ABSTRACT

Ocular drug delivery is an interesting field in current research. Silica nanoparticles (SiNPs) are promising drug carriers for ophthalmic drug delivery. However, little is known about the toxicity of SiNPs on ocular surface cells such as human corneal epithelial cells (HCECs). In this study, we evaluated the cytotoxicity induced by 50, 100 and 150 nm sizes of SiNPs on cultured HCECs for up to 48 hours. SiNPs were up-taken by HCECs inside cytoplasmic vacuoles. Cellular reactive oxygen species generation was mildly elevated, dose dependently, with SiNPs, but no significant decrease of cellular viability was observed up to concentrations of 100 μg/ml for three different sized SiNPs. Western blot assays revealed that both cellular autophagy and mammalian target of rapamycin (mTOR) pathways were activated with the addition of SiNPs. Our findings suggested that 50, 100 and 150 nm sized SiNPs did not induce significant cytotoxicity in cultured HCECs.

No MeSH data available.


The effect of SiNPs on HCECs’ autophagy.(A) LC3A/B conversion in HCEC treated with SiNPs for 24 h. The expression levels for the autophagy signal, LC3A/B proteins, were measured by Western blot analysis. The inactive I form is 16 kDa and the active II form is 14 kDa. Densitometric analyses of western blots showed the increased expression of II form with higher concentration of SiNPs 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). (B) Immunocytochemical staining with LC3B antibody revealed the increased autophagy in HCECs with 100 μg/mL of 50 nm SiNP (b), 100 nm SiNP (c) and 150 nm SiNP (d) addition. White arrowheads indicated the cells with increased LC3B staining (green). DAPI stained nucleus with blue and orange represented F-actin. Negative control is HCECs with no SiNPs addition (a). (C) Transmission electron microscope (TEM) revealed some SiNPs inside amphisomes (AS), endosome (ES), and lysosome (LS) (white arrowheads in b, c and d). a: positive control of autophagosomes (white arrowheads) induced by incubation with 50 μM-chloroquine diphosphate for 24 h in HCECs.
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f5: The effect of SiNPs on HCECs’ autophagy.(A) LC3A/B conversion in HCEC treated with SiNPs for 24 h. The expression levels for the autophagy signal, LC3A/B proteins, were measured by Western blot analysis. The inactive I form is 16 kDa and the active II form is 14 kDa. Densitometric analyses of western blots showed the increased expression of II form with higher concentration of SiNPs 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). (B) Immunocytochemical staining with LC3B antibody revealed the increased autophagy in HCECs with 100 μg/mL of 50 nm SiNP (b), 100 nm SiNP (c) and 150 nm SiNP (d) addition. White arrowheads indicated the cells with increased LC3B staining (green). DAPI stained nucleus with blue and orange represented F-actin. Negative control is HCECs with no SiNPs addition (a). (C) Transmission electron microscope (TEM) revealed some SiNPs inside amphisomes (AS), endosome (ES), and lysosome (LS) (white arrowheads in b, c and d). a: positive control of autophagosomes (white arrowheads) induced by incubation with 50 μM-chloroquine diphosphate for 24 h in HCECs.

Mentions: We investigated the effect of SiNPs on the cellular autophagy system using the signal alteration of LC3A/B, the autophagy marker (Fig. 5A). With the activation of autophagy, LC3A/B II form increases relative to LC3A/B I form. All three sizes of SiNPs triggered significant expression of LC3A/B II proteins. The increased ratio of activated LC3A/B were more prominent with a high concentration (100 μg/mL) of SiNPs stimuli and reached up to 1.35 fold (50 nm SiNPs), 1.62 fold (100 nm SiNPs) and 1.63 fold (150 nm SiNPs). Increased LC3B proteins in cytoplasm with SiNPs addition were also demonstrated by immunocytochemistry. (Fig. 5B) Some of SiNPs were captured inside endosomes or amphisomes (Fig. 5C) and some were found in autophagosomes and lysosomes, which were characterized by double membranous vacuoles.


The Effect of Silica Nanoparticles on Human Corneal Epithelial Cells
The effect of SiNPs on HCECs’ autophagy.(A) LC3A/B conversion in HCEC treated with SiNPs for 24 h. The expression levels for the autophagy signal, LC3A/B proteins, were measured by Western blot analysis. The inactive I form is 16 kDa and the active II form is 14 kDa. Densitometric analyses of western blots showed the increased expression of II form with higher concentration of SiNPs 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). (B) Immunocytochemical staining with LC3B antibody revealed the increased autophagy in HCECs with 100 μg/mL of 50 nm SiNP (b), 100 nm SiNP (c) and 150 nm SiNP (d) addition. White arrowheads indicated the cells with increased LC3B staining (green). DAPI stained nucleus with blue and orange represented F-actin. Negative control is HCECs with no SiNPs addition (a). (C) Transmission electron microscope (TEM) revealed some SiNPs inside amphisomes (AS), endosome (ES), and lysosome (LS) (white arrowheads in b, c and d). a: positive control of autophagosomes (white arrowheads) induced by incubation with 50 μM-chloroquine diphosphate for 24 h in HCECs.
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Related In: Results  -  Collection

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f5: The effect of SiNPs on HCECs’ autophagy.(A) LC3A/B conversion in HCEC treated with SiNPs for 24 h. The expression levels for the autophagy signal, LC3A/B proteins, were measured by Western blot analysis. The inactive I form is 16 kDa and the active II form is 14 kDa. Densitometric analyses of western blots showed the increased expression of II form with higher concentration of SiNPs 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). (B) Immunocytochemical staining with LC3B antibody revealed the increased autophagy in HCECs with 100 μg/mL of 50 nm SiNP (b), 100 nm SiNP (c) and 150 nm SiNP (d) addition. White arrowheads indicated the cells with increased LC3B staining (green). DAPI stained nucleus with blue and orange represented F-actin. Negative control is HCECs with no SiNPs addition (a). (C) Transmission electron microscope (TEM) revealed some SiNPs inside amphisomes (AS), endosome (ES), and lysosome (LS) (white arrowheads in b, c and d). a: positive control of autophagosomes (white arrowheads) induced by incubation with 50 μM-chloroquine diphosphate for 24 h in HCECs.
Mentions: We investigated the effect of SiNPs on the cellular autophagy system using the signal alteration of LC3A/B, the autophagy marker (Fig. 5A). With the activation of autophagy, LC3A/B II form increases relative to LC3A/B I form. All three sizes of SiNPs triggered significant expression of LC3A/B II proteins. The increased ratio of activated LC3A/B were more prominent with a high concentration (100 μg/mL) of SiNPs stimuli and reached up to 1.35 fold (50 nm SiNPs), 1.62 fold (100 nm SiNPs) and 1.63 fold (150 nm SiNPs). Increased LC3B proteins in cytoplasm with SiNPs addition were also demonstrated by immunocytochemistry. (Fig. 5B) Some of SiNPs were captured inside endosomes or amphisomes (Fig. 5C) and some were found in autophagosomes and lysosomes, which were characterized by double membranous vacuoles.

View Article: PubMed Central - PubMed

ABSTRACT

Ocular drug delivery is an interesting field in current research. Silica nanoparticles (SiNPs) are promising drug carriers for ophthalmic drug delivery. However, little is known about the toxicity of SiNPs on ocular surface cells such as human corneal epithelial cells (HCECs). In this study, we evaluated the cytotoxicity induced by 50, 100 and 150&thinsp;nm sizes of SiNPs on cultured HCECs for up to 48&thinsp;hours. SiNPs were up-taken by HCECs inside cytoplasmic vacuoles. Cellular reactive oxygen species generation was mildly elevated, dose dependently, with SiNPs, but no significant decrease of cellular viability was observed up to concentrations of 100&thinsp;&mu;g/ml for three different sized SiNPs. Western blot assays revealed that both cellular autophagy and mammalian target of rapamycin (mTOR) pathways were activated with the addition of SiNPs. Our findings suggested that 50, 100 and 150&thinsp;nm sized SiNPs did not induce significant cytotoxicity in cultured HCECs.

No MeSH data available.