Equilibrium study of copper absorption to different types of soft contact lens

To measure binding affinity of copper, one of the heavy metals in particulate matter (PM) was applied to soft contact lenses made of two different materials because contact lenses are readily exposed to PM. Copper binding to ionized silicon hydrogel lens yielded an equilibrium association constant Ka,eq value of 14.03 μM without color change of lens, compared to that of 19.16 μM for copper binding to de-ionized hydrogel lenses with color change of lens. The results indicated that the color change of lens is not consistent with the concentration of cooper deposition on lens, and copper bound relatively stronger in ionized silicon hydrogel lens than in de-ionized hydrogel lens. Therefore, the continuous exposure of contact lenses to high PM levels might lead to heavy metal deposition on the lens, which would be detrimental to ocular health.


Introduction
Particular matter (PM), known as particle pollution, is a complex mixture of extremely small particles including acids, organic chemicals, metals and dust, and liquid droplets [1]. PM levels have increased over the past decades in Asia [2,3].
Exposure to PM affects both the lungs and heart, resulting in asthma, irritation of the airways, irregular heartbeat, heart attacks, and cancers [4,5]. PM is also associated with ocular toxicity [6,7]. Exposure to PM causes ocular irritation, discomfort and unstable vision in severe cases [8,9].
Copper is of increasing interest as an environmental pollutant because it is one of the heavy metals [10] and is a transition metal component thought to be particularly harmful as it has the potential to produce reactive oxygen species causing disease over time [11]. Even though contact lenses are commonly used to correct vision, little is known about the direct influence of copper or metals in PM on contact lenses. The most of metal absorption studies have been worked in the field of material sciences and environmental engineering. They focused on investigating metal uptake ability of absorbent materials including hydrogel lens material [12][13][14]. So, there is no indication of exact metal binding affinity on lens. Also, the biological relevance studies for contact lenses have been mainly subjected to not a metal deposition but protein and lipids absorption [15,16].
In this study, the absorption of copper to both ionized and non-ionized hydrogel lenses was directly examined using UV-Vis spectrophotometer. We firstly reported a simple method of measuring the binding of copper to lenses and then characterized the binding affinity of copper on lenses.

Contact lens
Daily disposable contact lenses, Biotrue ONEDAY (Bausch ? Lomb) and 30-day contact lenses, PureVision2 (Bausch ? Lomb) were used. The most important parameters of the lenses are summarized in Table 1.

Copper absorption
Contact lenses were soaked three times in ultra-trace elemental analysis-grade water (Fisher Scientific) for 10 min each to remove residual lens solution. Then, contact lenses were incubated with different concentrations of copper (II) sulfate (Sigma-Aldrich) solution; 5 ml of 10, 25, 50, 100, 150 and 200 lM on 6-well cell-culture plate. The plate containing lens and copper solution was gently shaken on agitator. Then, the color changes of contact lenses were observed, and absorbance of copper solution was monitored.

Equilibrium analysis of copper binding to lenses
Aqueous copper solutions exhibit maximum absorbance at 200 nm [17]. Absorption spectra of copper solution were monitored at 200 nm using a Sinco spectrophotometer. The absorption spectra of copper solution were measured before and after soaking the contact lenses when the system had reached equilibrium. Equilibrium binding of copper was checked by recording absorbance of copper solution having lenses at 200 nm every 3 min for initial 30 min and then every 10 min for last 30 min. It was believed that the copper had reached to equilibrium because the absorbance was constant after 3 min till 1 h. The magnitude of copper binding on lenses was calculated based on the spectral changes at 200 nm; copper solution without lenses minus copper solution with lenses. The experimental procedure was the same for all measurement points (10, 25, 50, 100, 150 and 200 lM). These data were analyzed using Eq. 1 [18] in which Y is the bound copper concentration on lenses corresponding to the absorbance change of copper solution at 200 nm, Y max is the maximum copper-binding concentration, and K a,eq is the apparent K a value obtained from the equilibrium measurements. Equation 1 is a modified Michaelis-Menten or Langmuir equation to explain generic adsorption of bimolecular species into surfaces Results and discussion

Changes in contact lens color
Biotrue ONEDAY lenses changed color after copper absorption from aqueous copper solution (Fig. 1A). The lenses turned blue after exposure to 10 and 25 lM copper solution and greener at higher concentrations. At 200 lM, the Biotrue ONEDAY lenses turned completely green. Interestingly, PureVision2 lenses did not change color upon incubation in the copper solutions. The incubation time was extended to exclude any effect of absorption time. After 1 h of incubation, the color of lenses was unchanged irrespective of the copper concentration (Fig. 1B).
Original FDA group II hydrogel contact lenses are composed of 2-hydroxy-ethyl methacrylate (polyHEMA) crosslinked with ethylene glycol dimethacrylate. They are copolymerized with N-vinylpyrrolidone to improve their water content [19] by forming hydrogen bonds with two molecules of water. The surface of group II hydrogel contact lenses is de-ionized to reduce the negative charge preventing to bind positively charged proteins, but it might not completely de-ionized. It was indicated that the uptake of cation drug was directly bound to the anionic ligands in the de-ionized polyHEMA gel [20]. As the same reason, treatment of Biotrue ONEDAY lenses with copper solution results in Cu 2? ion binding to the partially negatively charged surface due to the hydroxyl group of HEMA, which leads to a color change. The greater the quantity of Cu 2? ion bound to the lens, the darker is the color of the lens.
Purevison2 belongs to FDA group V-Cm. Its monomer includes tris (trimethylsiloxy) silylpropyl vinyl carbamate (TPVC) [21]. The TPVC molecule contains a hydrophobic silicone linked to a hydrophilic vinyl carbamate group, resulting in marked polarity. The surface of FDA group V-Cm lenses is non-ionic, but the vinyl carbamate group of TPVC is in its ionized form at pH 6-8. Copper sulfate is normally ionized in water to form the Cu 2? ion resulting in over pH 6 solution (data not shown). FDA group V-Cm lenses have an ionic surface at pH 6, and the vinyl carbamate group of TPVC in silicone hydrogel lenses provides a greater negative charge than the hydroxyl group of HEMA in FDA group II lenses. Therefore, the PureVision2 lens theoretically might take up a greater quantity of Cu 2? ion leading to a darker color than Biotrue ONEDAY lenses. However, the color of PureVision2 lenses was not changed. It suggests that the bound copper may be status of Cu 1? rather than Cu 2? . Binding Cu 2? ions on lenses should be reduced by the vinyl carbamate group of TPVC material because the vinyl carbamate group functions as an electron donator [22] causing a loss of Cu 2? color.

Equilibrium analysis of copper binding
The stoichiometric ratio between copper and absorbance is evaluated in Fig. 2. The plot of absorbance versus mole fraction of copper showed a linear regression (R 2 = 0.99).
Comparison of the absorption spectra of copper before and after soaking of Biotrue ONEDAY lenses in copper solution revealed that the absorbance was significantly reduced. The difference in the absorption spectra corresponded to the copper concentration deposited on lenses. The same pattern was observed for PureVision2 lenses with more marked changes resulting in more copper bound to the PureVision2 lenses. It indicates that color change of copper deposition on lenses is not a critical factor to judge copper binding on lens.
The equilibrium binding of copper to lenses was determined by the fit of bound copper concentration on lenses to Eq. 1. A bound copper amount was calculated by linear regression fit in Fig. 2. It yielded an equilibrium association constant (K a,eq ) of 19.16 ± 3.42 lM and maximum magnitude of copper binding (Y max ) of 20.94 ± 0.89 lM for Biotrue ONEDAY lenses (Fig. 3A), and K a,eq of 14.03 ± 1.63 lM and Y max of 31.43 ± 0.77 lM for PureVision2 lenses (Fig. 3B). K a,eq value of PureVision2 is significantly smaller than that of Biotrue ONEDAY. These results reveal that the copper-binding affinity of PureVision2 lenses is greater than that of Biotrue ONEDAY lenses.  Normal concentration of copper in atmospheric PM, \1 lM, was not able to be tested due to copper detection limit by UV-Vis spectrophotometer. However, it should be considered that lenses are usually worn more than 1 day. If lenses are exposed to PM over 1 day, copper deposition should be accumulated day by day. In Fig. 3, it exhibits copper absorption on lens even at 10 lM of copper solution within 3 min and saturation behavior over 100 lM of copper, suggesting that copper might irreversibly bind on lenses very fast and continuously bind up to 100 lM of copper environment. Therefore, long-term exposure of contact lenses to high levels in PM might lead to accumulation of heavy metal on lenses, which would be detrimental to ocular health. Thus, the health implications of PM with respect to binding to the lens surface and toxicity to the eye warrant further investigation.