What Is the Cause of Toxicity of Silicone Oil?

Abstract

Purpose: To investigate the toxicity of the low-molecular-weight components (LMWCs) in ophthalmic silicone oils (SilOils) on retinal cell lines.

Methods: The toxicity of six types of LMWCs were studied and compared with conventional SilOil 1000 cSt. In vitro cytotoxic tests of LMWCs, in both liquid and emulsified forms, on three retinal cell lines (Müller cells (rMC-1), photoreceptor cells (661W) and retinal pigment epithelial cells (ARPE-19)) were conducted using a transwell cell culturing system. The morphology and viability of cells were assessed by light microscopy and Cell Counting Kit-8 (CCK-8) assay at different time points (6, 24 and 72 h). The ARPE-19 apoptotic pathway was investigated by Mitochondrial Membrane Potential/Annexin V Apoptosis Kit at different time points (6, 24 and 72 h).

Results: Apart from dodecamethylpentasiloxane (L5), all liquid LMWCs showed varying degrees of acute cytotoxicity on retinal cell lines within 72 h. Emulsified LMWCs showed comparable cytotoxicity with liquid LMWCs on retinal cell lines. Cyclic LMWCs, octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5) had significantly higher cytotoxicity when compared with their linear counterparts decamethyltetrasiloxane (L4) and L5 with similar molecular formula. Using ARPE-19 cells as an example, we showed that LMWCs induce the apoptosis of retinal cells.

Conclusions: Most LMWCs, in both liquid and emulsified forms, can induce acute cytotoxicity. In addition, cyclic LMWCs are suspected to have higher cytotoxicity than their linear counterparts. Therefore, LMWCs are suspected to be the main cause of the long-term toxicity of ophthalmic SilOil, due to their toxicity and propensity to cause ophthalmic SilOil to emulsify. The amount of LMWCs should be considered as the paramount parameter when referring to the quality of SilOil.

Keywords: Müller cell (rMC-1); emulsification; low-molecular-weight component (LMWC); photoreceptor cell (661W); polydimethylsiloxane; retinal toxicity.

Figure 1

SilOil emulsion generated by high-speed homogenisation: (a) the number of emulsified droplets (relative to the number of droplets on Day 1) of various types of LMWC within 72 h after generation; (b) the representative emulsified droplets distribution profile of L4 with highly overlapped curves within 72 h after generation; (c) the representative morphology of emulsified droplets under fluorescence microscope. Scale bar = 50 μm. The results in (a–c) are representative of those from 3 independent experiments with 5 replicates each.

Figure 2

Cytotoxicity of liquid LMWCs. Cell viability of (a) Müller cells (rMC-1); (b) photoreceptor cells (661W); and (c) retinal pigment epithelial cells (ARPE-19) after incubation with various types of LMWCs at different time points. n = 6, * p < 0.05, vs. media; # p < 0.05, vs. SilOil 1000 (one-way ANOVA followed by Bonferroni test). Scale bar = 50 μm. The results in (a–c) are representative of those from 3 independent experiments with 2 replicates each.

Figure 3

Cell morphology after incubation with liquid LMWCs. Representative images of (a) Müller cells (rMC-1); (b) photoreceptor cells (661W); and (c) retinal pigment epithelial cells (ARPE-19) after incubation with various types of LMWCs at different time points. Morphologies of (d) Müller cells (rMC-1), (e) photoreceptor cells (661W) and (f) retinal pigment epithelial cells (ARPE-19) after incubation with control groups including culture and SilOil 1000 for 6, 24 and 72 h. The results in (a–f) are representative of those from 3 independent experiments with 2 replicates each.

Figure 4

Cytotoxicity of emulsified LMWCs. Cell viability of (a) Müller cells (rMC-1); (b) photoreceptor cells (661W); and (c) retinal pigment epithelial cells (ARPE-19) after incubation with various types of emulsions at different time points. n = 6, * p < 0.05, vs. media; # p < 0.05, vs. SilOil 1000 (one-way ANOVA followed by Bonferroni test). Scale bar = 50 μm. The results in (a–c) are representative of those from 3 independent experiments with 2 replicates each.

Figure 5

Cell morphology after incubation with emulsified LMWCs. Representative images of (a) Müller cells (rMC-1); (b) photoreceptor cells (661W); and (c) retinal pigment epithelial cells (ARPE-19) after incubation with various types of emulsions at different time points. Morphologies of (d) Müller cells (rMC-1); (e) photoreceptor cells (661W); and (f) retinal pigment epithelial cells (ARPE-19) after incubation with control groups including culture media (media) and emulsion of SilOil 1000 for 6, 24 and 72 h. The results in (a–f) are representative of those from 3 independent experiments with 2 replicates each.

Figure 6

Fluorescent microscopic images of the ARPE-19 cells after incubating with liquid LMWCs and control groups at (a) 6 h, (b) 24 h and (c) 72 h. Cells in red and green represent living and dead cells, respectively. The results in (a–c) are representative of those from 1 independent experiment with 2 replicates each.

Figure 7

Fluorescent microscopic images of the ARPE-19 cells after incubating with emulsified LMWCs and control groups at (a) 6 h, (b) 24 h and (c) 72 h. Cells in red and green represent living and dead cells, respectively. The results in (a–c) are representative of those from 1 independent experiment with 2 replicates each.