Multipurpose Solutions and Contact Lens: Modulation ...

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assessed after direct contact of multipurpose solutions (MPS) with conjunctival cells .... placed directly in the center on monolayer of conjunctival cells in culture ...

MS NO: ICO201506

BASIC INVESTIGATION

Multipurpose Solutions and Contact Lens: Modulation of Cytotoxicity and Apoptosis on the Ocular Surface Me´lody Dutot, PhD,*† Elisa Reveneau, MS,* Thierry Pauloin, PharmD, PhD,* Roxane Fagon, MS,† Caroline Tanter, PharmD,† Jean-Michel Warnet, PhD, PharmD,* and Patrice Rat, PhD, PharmD*

Purpose: We evaluated (1) 4 multipurpose lens care solutions and 3 contact lenses (soft and rigid) for cytotoxicity according to ISO 10993-5 standard (medical device biocompatibility) and (2) the protective effects of a marine cationic solution and hyaluronic acid.

Methods: Low water soft lens, high water soft lens, and rigid lens were laid on a conjunctival cell line after being soaked in multipurpose solution (Optifree Express, Renu, Solocare Aqua, or Menicare Plus). Cell morphology was microscopically observed, and cell viability was evaluated using the neutral red test. Apoptosis was assessed after direct contact of multipurpose solutions (MPS) with conjunctival cells using fluorescence microscopy and flow cytometry. The ability of a controlled ionization marine solution and hyaluronic acid to prevent multipurpose solution’s cytotoxicity was finally evaluated. Results: Contact lens soaked in the MPS induced cell morphology alterations and loss of cell viability. Rinsing the lens with the marine solution improved cell viability and preincubating cells with hyaluronic acid inhibited apoptosis. Conclusions: MPS can be damaging for the ocular surface cells. We proposed to rinse the lens with a marine solution before insertion of the lens on the cornea to wash away the multipurpose solution and to use hyaluronic acid to protect the ocular surface cells against apoptosis induced by MPS. Key Words: contact lens, cytotoxicity, apoptosis, controlled ionization marine solution, hyaluronic acid (Cornea 2010;00:000–000)

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ard plastic lenses were first introduced in the 1940s; they were fitted with a soft wax-like substance, which hardens on the eye so that impressions of the eyeballs can be made. Soft lenses appeared in the 1970s.1 In 1983, a way for contact lenses to be made in a continuous wet state was developed. It

Received for publication April 27, 2009; revision received June 23, 2009; accepted Aug 17, 2009. From the *Laboratoire de Toxicologie, Faculte´ des Sciences Pharmaceutiques et Biologiques, Universite´ Paris Descartes, Paris, France; and †Laboratoire Yslab, Quimper, France. Supported by Adebiopharm ER67 (Paris, France). No conflict of interest. Reprints: Dr. Me´lody Dutot, PhD, 4 avenue de l’Observatoire, 75006 Paris, France (e-mail: [email protected]). Copyright Ó 2010 by Lippincott Williams & Wilkins

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meant that there would be fewer distortions in the manufacturing process; lenses could be made with high precision and repeatability. Disposable lenses were introduced in 1987.1 Today, there are basically 2 categories of contact lenses based on the materials used to manufacture them: gas permeable and soft contact lenses. The first ones are made from rigid plastic materials permeable to oxygen. The second ones are responsible in making contact lenses very popular because the adaptation period is shorter for these lenses when compared with the rigid lenses. In 2006, in France, rigid contact lens turnover was about 12 million euros and soft contact lens turnover reached almost 167 million euros (www.syffoc.fr). More than 24 million people wear contact lenses in the United States alone. Soft lenses are generally sold as 2-week or 1month lenses. The cost for the lenses along with the boiler was very high; therefore, manufacturers developed new solutions to facilitate the use of lenses. Among contact lens cleaning solutions, multipurpose cleaning solutions represent about 70% of cleaning lens solution’s turnover. Multipurpose solutions (MPS) comprise an aqueous liquid medium, a nonoxidative antimicrobial component, a surfactant to remove deposit material from the lens (mainly poloxamer or poloxamine), a buffer component, preferably a phosphate buffer component to maintain the pH of the solution within a physiologically range, a viscosity-inducing component, preferably hydroxypropylmethylcellulose (HPMC), to increase or enhance effectiveness in removing deposit materials, and a tonicity component (sodium chloride or a combination of sodium chloride and potassium chloride). The present compositions also include a chelating agent, ethylenediaminetetraacetic acid (EDTA). The nonoxidative antimicrobial agent is generally selected from biguanides, biguanide polymers (mainly polyhexamethylene biguanide, PHMB), or quaternary ammoniums salts used in ophthalmic applications such as Polyquaternium-1 (or Polyquad). The concentration range varies from 0.1 ppm (0.00001%) to about 3 ppm or less than 5 ppm (0.0005%). Biguanides and quaternary ammoniums are preservatives, but preservatives are known to be toxic and apoptotic to the ocular surface.2–4 In a former study, we observed important cytotoxic effects of different MPS on human conjunctival cells.5 We showed that after short incubation times (from 15 minutes to 3 hours), MPS induced necrosis with oxidative stress, mitochondrial alterations, and P2X7 cell death receptor activation. Our results were in accordance with previous in vitro findings,6 and a study showed the apoptotic effect of some MPS on corneal cells.7 One of the manufacturers’ arguments to avoid this cytotoxicity www.corneajrnl.com |

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is to affirm that the high molecular weight of antimicrobial agents (PHMB and Polyquad) used in these solutions prevents them to be adsorbed on the lens. The aim of this study was double: first, to study this hypothesis, we performed a protocol using contact lenses (soft and rigid) soaked in several MPS that we laid on human conjunctival cells. Then, we evaluated the effects of some MPS on chromatin condensation (early apoptotic stage) and DNA fragmentation (late apoptotic stage). Second, we proposed 2 alternatives: (1) to avoid MPS cytotoxicity with a supplemental contact lens rinse step using a controlled ionization marine unpreserved solution and (2) to protect ocular surface cells with a hyaluronic acid–marine solution mixture. The marine solution was chosen according to our previous findings that showed its ability to decrease P2X7 receptor activation (which is induced by MPS) and to rinse ocular cells after chemical burn in a better way than NaCl 0.9%.8,9 In a previous study, hyaluronic acid showed cytoprotective effects against preservative cytotoxicity.10 Because active principles in MPS are preservatives, we studied the protective effects of hyaluronic acid against MPS cytotoxicity.

MATERIALS AND METHODS Reagents and Products Reagents for cell culture were purchased from Eurobio (Les Ulis, France). Two kinds of soft lenses were tested: Pure Vision, low water 36% in balafilcon A [Federal Drug Administration (FDA) group III], from Bausch & Lomb and Precision UV, high water 74% in vasurfilcon A (FDA group II), from CIBA Vision. Rigid lenses were provided by Ocellus (Cre´gy le`s Meaux, France). Optifree Express (Alcon, Hu¨nenberg, Switzerland), Renu (Bausch & Lomb Incorporated, Rochester, NY), Solocare Aqua (CIBA Vision Corporation, Duluth, GA), and Menicare Plus (Menicon Europe, Clichy, France) were, respectively, referred to as MPS-a, MPSb, MPS-c, and MPS-1 in the Results section. Composition of these MPS is summarized in Table 1. Controlled ionization marine solution (Lacrymer) (containing chlorine, potassium, sodium, magnesium, sulfur, and calcium) and hyaluronic acid powder were obtained from Yslab (Quimper, France) and Soliance (Pomacle, France), respectively.

Cell Culture Established cell lines are prefered and shall be obtained from recognized repositories. The Wong Kilbourne, derivative of Chang human conjunctival epithelial cell line (WKD), is the only conjunctival cell line available in international cell banks (ATCC CCL-20.2). Besides, culture of the WKD cells does not require supplemental factors such as insulin or cholera toxin that could interfere in toxicological studies. The cells were cultured under standard conditions (moist atmosphere of 5% CO2 at 37°C) in Dulbecco’s minimum essential medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 50 IU/mL penicillin/ streptomycin. Confluent cultures were detached by trypsin incubation, and the cells were seeded into 6-well culture plates. Cultures were kept at 37°C for 24 hours before any exposure, to let the cells attach to the plate and reach confluency.

Cytotoxicity Assay of the Lens Soaked in Multipurpose Solution A lens case contains 5 mL of lens care solution. For 20 cycling of lenses, the lens will be exposed to a total of 100 mL of lens care solution. To substitute 20 cycling of lenses, 100 mL was chosen. A soaking time of 96 hours was chosen to reach a plateau level of total accumulation on the lens. After the soaking time of 96 hours, the lens was either placed directly in the center on monolayer of conjunctival cells in culture medium (protocol 1, according to ISO 10993-5 standard11) or rinsed with controlled ionization marine solution (Lacrymer) and exposed to this solution (100 mL) for 24 supplemental hours (protocol 2). The cells were exposed to the lens for 24 hours at 37°C. After exposure, the cell morphology was evaluated microscopically. We focused our attention on the cells around the lens. Then, a neutral red (NR) test was performed to evaluate cell viability.

Cell Viability Assay Using the NR Test Membrane integrity, closely correlated with cell viability, was evaluated with NR using fluorometric detection. NR was used at 50 mg/mL in accordance with the validated protocol.12 Two hundred microliters per well of NR solution were added to living cells, and the microplate was incubated for 3 hours at 37°C. The cells were washed in phosphatebuffered saline (PBS), and the dye was extracted from the intact viable cells with a solution of acetic acid and ethanol.

TABLE 1. Composition of Multipurpose Solutions

Quaternary ammonium (antimicrobial agent) (Polyquad) Myristamidopropyl dimethylamine (antimicrobial agent) (Aldox) PHMB (antimicrobial agent) Poloxamer (surfactant agent) Poloxamine (surfactant agent) HPMC

Optifree Express (Alcon) MPS-a

Renu (Bausch & Lomb) MPS-b

Solocare Aqua (CIBA Vision) MPS-c

Menicare Plus (Menicon) MPS-1

0.001%







0.0005%







— — Unknown concentration —

0.00005% — 1% —

0.0001% Unknown concentration — —

0.0005% 0.5% — 0.275%

HPMC, hydroxypropylmethylcellulose.

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The plate was agitated on a microplate shaker for 30 minutes. The NR fluorescence was then measured (lex = 535 nm; lem = 600 nm).

Apoptosis Assay of the MPS by Direct Contact: Chromatin Condensation Evaluation Hoechst 33342 was used to evaluate chromatin condensation in cells with propidium iodide (PI) to discriminate necrotic cells. The UV fluorescent probe Hoechst 33342 (excitation, 360 nm; emission, 450 nm) enters living cells and apoptotic cells, whereas PI enters necrotic cells much faster than Hoechst 33342. The cells were exposed for 30 minutes to a 10 mg/L Hoechst 33342 solution containing 1 mL of PI (1 mg/mL). They were then observed with inverted fluorescence microscopy (DMIRB; Leica, Heidelberg, Germany) and photographed (numeric Coolpix 5000; Nikon, Tokyo, Japan).

Apoptosis Assay of the MPS by Direct Contact: DNA Fragmentation Evaluation Two protocols were performed: The cells were incubated with MPS for 30 minutes, followed by a 24-hour incubation time in culture medium supplemented with 2.5% fetal bovine serum. The cells were incubated with a hyaluronic acid/marine solution mixture (0.2% wt/vol) for 20 minutes; hyaluronic acid/marine solution was removed, and the cells were incubated with MPS for 30 minutes, followed by a 24-hour incubation time in culture medium supplemented with 2.5% fetal bovine serum. The cells were gently detached and collected after 10 minutes of incubation in 0.5 mM EDTA. Then, they were washed in PBS and suspended in 1 mL of PBS. The cells were fixed with a 1% paraformaldehyde solution for 24 hours. Finally, the cells were incubated for 5 minutes with saponin, washed with PBS, and incubated for 3 minutes with 50 mg/mL of PI before flow cytometry analysis (FC500; Beckman Coulter, Minneapolis, MN). Use of PI that is capable of binding and labeling DNA makes it possible to obtain a rapid and precise evaluation of cellular DNA content by flow cytometric analysis and subsequent identification of hypodiploid cells.

Statistical Analysis Each experiment was performed 3 independent times. The mean values for each concentration were analyzed by 1-way analysis of variance followed by the Dunnett test (Sigma Stat 2.0; HighText Interactive, Chicago, IL), and the level of significance was fixed at 0.05.

Animals All procedures in this study conformed to the Guiding Principles in the Use of Animals in Toxicology. Animal care and experimentation complied with the European Council Guidelines. The license for experimental studies on living animals and agreement of animal facilities number is A75-0602 (Direction of Veterinary Services, Paris Police Department, France). Eight-week-old male New Zealand rabbits (Cegav, Saint Mars d’Egrenne, France) were used in the study. Each q 2010 Lippincott Williams & Wilkins

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animal was placed in an individual cage and was kept in standard laboratory conditions (20°C, 12 hours/12 hours light/dark cycle), fed ad libitum on the standard laboratory diet, and had free access to water.

Ocular Tolerance: Draize Test Controlled ionization marine solution (Lacrymer) and hyaluronic acid/marine solution mixture (0.2% wt/vol) were tested on rabbits for their ability to induce ocular irritation. 0.1 mL of solutions was instilled into the conjunctival bag, and the upper and lower eyelids were gently held together for 10 seconds. The opposite eye of each animal served as an untreated control. At hour 1, day 1, day 2, and day 6 after marine solution (Lacrymer) or hyaluronic acid/marine solution mixture treatment, rabbits’ eyes were examined for ocular irritation and scored according to a weighted scale for grading the severity of ocular lesions from the Draize test. We evaluated the degree of redness, chemosis, and tearing of the conjunctiva; the degree and the area of cornea opacity; and the increased prominence of the folds and congestion of the iris. The possible maximum total score was 110 (conjunctiva = 20, cornea = 80, iris = 10). Three animals were used for each treatment.

RESULTS Low Water 36% Soft Contact Lenses When the conjunctival cells were incubated with the low water soft lens (no soaking in the MPS), there were no more cells under the lens (Fig. 1A). Around the lens, the cell morphology was normal: the cells were adherent and cell–cell junctions were observed. When the conjunctival cells were incubated with the low water soft lens soaked in MPSa (Optifree Express), the cell morphology was highly altered around the lens: the cells were refringent, nonadherent, and had a round shape. When the conjunctival cells were incubated with the low water soft lens soaked in MPS-b (Renu), the cell morphology was normal around the lens. When the conjunctival cells were incubated with the low water soft lens soaked in MPS-c (Solocare Aqua), the cell morphology was mostly normal around the lens. Some cells were nonadherent and had a round shape. Figure 1B showed that cell viability was significantly and strongly altered when the cells had been in contact with the soft lens soaked in MPS-a (38% of control). With MPS-b and -c, cell viability was also significantly decreased but to a lesser extent (75% and 62% of control, respectively) compared with cell control. MPS-b was not different from soft lens alone. When the lens was rinsed with the controlled ionization marine solution and soaked in the same solution for 24 hours, cell viability was considerably increased: NR signals observed with the 3 MPS we tested were close to cell control signal (90%, 102%, and 96%, not statistically different).

High Water 74% Soft Contact Lenses When the conjunctival cells were incubated with the high water soft lens (no soaking in the MPS), there were no more cells under the lens (Fig. 2A). Around the lens, the cell morphology was normal: the cells were adherent and cell–cell junctions were www.corneajrnl.com |

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FIGURE 1. A, Effect of low water soft lens soaked in MPS-a (Optifree Express), -b (Renu), and -c (Solocare) on conjunctival cell viability. Photographs are representative of 3 independent experiments. B, Effect of low water soft lens soaked in MPS-a (Optifree Express), -b (Renu), and -c (Solocare) on conjunctival cells morphology. ***P , 0.001; **P , 0.005 compared with cell control; §P , 0.001 compared with lens.

observed. When the conjunctival cells were incubated with the high water soft lens soaked in MPS-a, the cell morphology was highly altered around the lens: the cells were refringent, nonadherent, and had a round shape. When the conjunctival cells were incubated with the high water soft lens soaked in MPS-b, the cell morphology was mostly normal around the lens, but some cells were refringent. When the conjunctival cells were incubated with the high water soft lens soaked in MPS-c, the cell morphology was normal around the lens.

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Figure 2B showed that cell viability was significantly and strongly altered when the cells had been in contact with the soft lens soaked in MPS-a (33% of control). With MPS-b, cell viability was also significantly decreased but to a lesser extent (58% of control) compared with cell control. With MPS-c, cell viability was slightly lower than those of control (85% vs 100%) but not different from soft lens alone. When the lens was rinsed with the controlled ionization marine solution and soaked in the same solution for 24 hours, cell q 2010 Lippincott Williams & Wilkins

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FIGURE 2. A, Effect of high water soft lens soaked in MPS-a (Optifree Express), -b (Renu), and -c (Solocare) on conjunctival cell morphology. Photographs are representative of 3 independent experiments. B, Effect of high water soft lens soaked in MPSa (Optifree Express), -b (Renu), and -c (Solocare) on conjunctival cell viability. ***P , 0.001; **P , 0.005; *P , 0.01 compared with cell control; §P , 0.001 compared with lens.

viability was considerably increased: NR signals observed with the 3 MPS we tested were close to cell control signal (not statistically different).

Rigid Contact Lenses When the conjunctival cells were incubated with the rigid lens (no soaking in the MPS), there were no more cells q 2010 Lippincott Williams & Wilkins

under the lens (Fig. 3A). Around the lens, the cell morphology was somewhat normal: the cells were adherent and cell–cell junctions were observed. Some cells were refringent. When the conjunctival cells were incubated with the rigid lens soaked in MPS-1 (Menicare Plus), the cell morphology was altered around the lens: some cells were refringent, nonadherent, and had a round shape. The other cells tended to aggregate into www.corneajrnl.com |

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FIGURE 3. A, Effect of rigid lens soaked in MPS-1 (Menicare Plus) on conjunctival cell morphology. Photographs are representative of 3 independent experiments. B, Effect of rigid lens soaked in MPS-1 (Menicare Plus) on conjunctival cell viability. Lens was cleaned with MPS-1 or cleaned with MPS-1 and rinsed with marine solution. **P , 0.005; *P , 0.01 compared with cell control; §P , 0.001 compared with lens.

islets. When the cells were incubated with the rigid lens soaked in the MPS-1 and rinsed with the marine solution, more cell– cell junctions were observed than previously. Figure 3B showed that cell viability was significantly altered when the cells had been in contact with the soft lens soaked in MPS-1 (71% of control). When the lens was rinsed with the controlled ionization marine solution and soaked in the same solution for 24 hours, cell viability increased from 71% to 88% of control.

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MPS and Apoptosis Reversible Apoptosis: Chromatin Condensation Cells with bright blue fragmented nuclei showing condensed chromatin were identified as apoptotic cells. Continuous 15-minute exposure of conjunctival cells to the 3 MPS we tested substantially enhanced the number of bright fragmented nuclei that showed condensed chromatin, which is typical for cells undergoing apoptosis (Fig. 4A). Optifree q 2010 Lippincott Williams & Wilkins

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Express and Menicare Plus were the solutions that induced chromatin condensation the most. As chromatin condensation can be reversible in some cases, we performed the analysis of DNA fragmentation, which is irreversible.

Irreversible Apoptosis: DNA Fragmentation Flow cytometric analysis measuring PI staining of DNA (Fig. 4B) demonstrated that after a 24-hour recovery time, MPS-a (for soft contact lenses) and -1 (for rigid contact lenses)

Modulation of Multipurpose Solutions Cytotoxicity

induced significant increases in DNA fragmentation on the conjunctival epithelial cell line (mean fluorescence 40% and 65%) compared with control cells (mean fluorescence 2.7%). Results with MPS-b (mean fluorescence 2.7%) were not significantly different from results obtained with cell control. Cells preincubated with hyaluronic acid before a 30-minute incubation time with MPS-a or 21 showed a significant decrease in DNA fragmentation (mean fluorescence 4.2% and 31.1%, respectively) compared with control cells. When the

FIGURE 4. A, Chromatin condensation evaluation. Conjunctival cells were incubated with phosphate buffer (A), Optifree Express (B), Renu (C), or Menicare Plus (D) for 15 minutes. Nuclei showing condensed chromatin appeared white, whereas nuclei of living cells appeared gray. Photographs are representative of 3 independent experiments. B, DNA fragmentation quantification. Conjunctival cells were preincubated with PBS or hyaluronic acid (HA) and then incubated with MPS-a (Optifree Express), -b (Renu), or -1 (Menicare Plus) for 30 minutes. ***P , 0.001. q 2010 Lippincott Williams & Wilkins

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cells were preincubated with hyaluronic acid, MPS-a–induced DNA fragmentation was inhibited.

In Vivo Assessment of Ocular Tolerance Results of the Draize test revealed no ocular irritation after treatment with rinse marine solution or marine solution– hyaluronic acid mixture (Table 2). These 2 solutions are well tolerated.

DISCUSSION MPS represent more than 70% of contact lens cleaning solutions sales in France (www.syffoc.fr) and remain the most prescribed care regimens with all soft lenses in the whole world13 because of their benefits of convenience, simplicity, and disinfection properties for part-time wear. These solutions are meant to disinfect, preserve, and rinse contact lenses. However, solution–lens interactions resulting from the uptake of preservatives from MPS into the lens during overnight disinfection and subsequent release into the eye can result in clinically meaningful levels of superficial punctate corneal staining for the wearer.14 Adsorption of care product preservative or other solution ingredients by the lens may affect the ocular biocompatibility of the lens. The chemical composition, water content, and the ionic nature of contact lenses dictate the uptake and release of various exogenous chemicals. Thus, we tested 2 types of soft contact lens and 1 rigid contact lens to assess the lens–solution interactions. First, the behavior of cells in contact with lenses was observed (no soaking in MPS). NR test results show that low water soft lenses seem to be more cytotoxic than high water soft lenses. This may be because of the contact lens material; the low water soft lens we tested is made of ionic polymer (FDA group III), whereas the high water soft lens we tested is made of nonionic polymer (FDA group II). The ionic nature of the material could alter cell viability because of interactions with cell membrane charged components. Besides, soft lenses soak up water, and water allows the eye to breathe through the contact lens. Therefore, the more water the lens soaks up then the easier it is for the oxygen to pass through it. Second, the behavior of cells in contact with lenses that have been soaked in the tested MPS was observed. According to our experiments, there were differences in cell morphology between the cells that were in contact with the soaked lens and the lens alone: lenses soaked in MPS led to cell death and cell dystrophies (round and small refringent cells). We can conclude that MPS impregnated the

TABLE 2. Ocular Tolerance Using the Draize Test Treatment Time

Marine Solution (Lacrymer)

Matine Solution–Hyaluronic Acid Mixture (0.2%)

1h Day 1 Day 2 Day 6

0 0 0 0

0 0 0 0

Two solutions were tested on rabbits’ eyes: the rinse solution Lacrymer and the mixture Lacrymer and hyaluronic acid. A score ‘‘0’’ indicates no ocular irritation.

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lens during soaking time and diffused on the cells, inducing morphology alterations and loss of cell viability. High– molecular weight preservatives (quaternary ammoniums or PHMB), contained in MPS, are able to be adsorbed by the lens. This idea contradicts the manufacturers’ postulate according to which new generation preservatives are not hazardous for the ocular surface. Optifree Express induced the most cell alterations in both types of soft lenses, compared with Renu and Solocare Aqua, as previously described.15 A small amount of MPS carried over by the lens could produce the cytotoxic effect rather than the diffusion of solutions from the lenses. Nevertheless, Renu was cytotoxic with high water soft lens but not with low water soft lens. If the cytotoxic effect we observed was just because of a small amount of residual MPS on the lens, the cytotoxic effect would have been the same with both low and high water lenses. Renu was probably more adsorbed on high water lens and more released on conjunctival cells. One hypothesis could be that PHMB (nonionic preservative) contained in Renu has more affinity for nonionic lens materials. However, this seems unlikely because Solocare Aqua contains a higher concentration of PHMB than Renu, and yet it is more cytotoxic with low water soft lens. Besides, the size of PHMB is not communicated by Renu and Solocare Aqua manufacturers and could play a role in cytotoxicity. Preservatives, harmful for the ocular surface,2–4,16 are probably at the origin of the cytotoxic effects we observed. Nevertheless, preservatives may not be the only responsible for MPS cytotoxicity. The concentrations of poloxamines and poloxamers can reach higher levels than preservatives concentrations in MPS. Poloxamers and poloxamines have been less studied in toxicology, but they represent a risk too because of their surfactant properties. Poloxamers and poloxamines are used in ophthalmology as ocular delivery system to increase drug transport across corneal membrane and increased ocular retention time.17 Therefore, in MPS, they may amplify preservatives toxicity. Concerning rigid lenses, we tested 1 multipurpose solution that altered cell morphology and cell viability too. These results may be surprising because aqueous MPS are not supposed to be adsorbed by rigid lenses because of their nonporous nature. In all cases (low and high water content soft lenses snd rigid lenses), both cell morphology and cell viability were improved when a rinse step with unpreserved solution was added to the protocol. Washing of the lenses with the sterile marine solution probably washed away the hydrophilic chemicals (such as preservatives) adsorbed on the lenses from the lens care solution. This cationic marine solution was chosen because of its effectiveness in rinsing chemicals away in a model of ocular burn and in modulating P2X7 cell death receptor activation.8,9 The rise in the use of MPS has been accompanied by substantially more reports of solution-related complications, in particular corneal staining and inflammatory infiltrates. Most of MPS manufacturers recommend rinsing the lenses with the multipurpose solution before insertion of the lens. Therefore, multipurpose solution can be in direct contact with the ocular surface. Chromatin condensation, an early q 2010 Lippincott Williams & Wilkins

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apoptotic stage, was induced by Optifree Express, Menicare Plus, and in a lesser extent Renu after a short incubation time (15 minutes), which is in accordance with previous studies.18 DNA fragmentation, a late apoptotic stage, was induced by Optifree Express and Menicare Plus but not by Renu. The initial chromatin condensation can be a reversible step in the early stage of apoptosis. The results we observed suggest that chromatin condensation induced by Renu is reversible and does not lead to apoptosis, contrary to the other MPS we tested. DNA fragmentation was inhibited or at least decreased when the cells were preincubated with hyaluronic acid– marine solution mixture before multipurpose solution incubation. We previously showed that hyaluronic acid was an effective protective agent against DNA fragmentation induced by benzalkonium chloride, the quaternary ammonium currently used the most in eyedrops.10 The results we observed with Optifree Express, which also contains a quaternary ammonium, are in good correlation with these previous findings. Moreover, hyaluronic acid–marine solution mixture decreased DNA fragmentation induced by Menicare Plus, which does not contain any quaternary ammonium but PHMB. Consequently, hyaluronic acid–marine solution mixture could be proposed as cytoprotective eyedrops to contact lens wearers. Furthermore, our in vivo experiments confirmed the innocuity of this solution for the ocular surface. Studies using reconstructed tridimensional human corneal epithelium could complete our experiments.19 According to our in vitro results, MPS can be damaging for the ocular surface cells. In the present study, we can propose 2 alternatives: (1) to rinse the lens with an unpreserved marine solution to wash away the multipurpose solution and (2) to use a solution of hyaluronic acid to protect the ocular surface cells against MPS cytotoxicity.

REFERENCES 1. Hard Contact Lenses. The College of Optometrists. Available at: http:// www.college-optometrists.org. Accessed June 17, 2009. 2. Liang H, Baudouin C, Pauly A, et al. Conjunctival and corneal reactions in rabbits following short- and repeated exposure to preservative-free tafluprost, commercially available latanoprost and 0.02% benzalkonium chloride. Br J Ophthalmol. 2008;92:1275–1282.

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3. Xiong C, Chen D, Liu J, et al. A rabbit dry eye model induced by topical medication of a preservative benzalkonium chloride. Invest Ophthalmol Vis Sci. 2008;49:1850–1856. 4. Dutot M, Pouzaud F, Larosche I, et al. Fluoroquinolone eye drop-induced cytotoxicity: role of preservative in P2X7 cell death receptor activation and apoptosis. Invest Ophthalmol Vis Sci. 2006;47:2812–2819. 5. Dutot M, Warnet JM, Baudouin C, et al. Cytotoxicity of contact lens multipurpose solutions: role of oxidative stress, mitochondrial activity and P2X7 cell death receptor activation. Eur J Pharm Sci. 2008;33:138–145. 6. Oriowo MO. A fluorometric study of relative ocular lens cytosensitivity to multipurpose contact lens solutions using the resazurin assay method. Toxicol In Vitro. 2006;20:1548–1554. 7. Chuang EY, Li DQ, Bian F, et al. Effects of contact lens multipurpose solutions on human corneal epithelial survival and barrier function. Eye Contact Lens. 2008;34:281–286. 8. Said T, Dutot M, Labbe´ A, et al. Ocular burn: rinsing and healing with ionic marine solutions and vegetable oils. Ophthalmologica. 2009;223:52–59. 9. Dutot M, Liang H, Pauloin T, et al. Effects of toxic cellular stresses and divalent cations on the human P2X7 cell death receptor. Mol Vis. 2008;14: 889–897. 10. Pauloin T, Dutot M, Warnet JM, et al. In vitro modulation of preservative toxicity: high molecular weight hyaluronan decreases apoptosis and oxidative stress induced by benzalkonium chloride. Eur J Pharm Sci. 2008;34:263–273. 11. ISO 10993-5. Biological Evaluation of Medical Devices. Part 5: Tests for Cytotoxicity: In Vitro Methods. Geneva, Switzerland: International Organization for Standardization; 2006. 12. Borenfreund E, Puerner JA. A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). Methods Cell Sci. 1985;9:7. 13. Morgan PB, Efron N, Woods CA. International contact lens prescribing in 2005. Cont Lens Spectr. 2006;21:135–139. 14. Dassanayake NL, Garofalo RJ, Carey C, et al. Correlating biocide uptake and release profiles with corneal staining and subjective symptoms. Invest Ophthalmol Vis Sci. 2005;46. E-abstract 915. 15. McCanna DJ, Harrington KL, Driot JY, et al. Use of a human corneal epithelial cell line for screening the safety of contact lens care solutions in vitro. Eye Contact Lens. 2008;34:6–12. 16. Garofalo RJ, Dassanayake N, Carey C, et al. Corneal staining and subjective symptoms with multipurpose solutions as a function of time. Eye Contact Lens. 2005;31:166–174. 17. Gupta H, Jain S, Mathur R, et al. Sustained ocular drug delivery from a temperature and pH triggered novel in situ gel system. Drug Deliv. 2007; 14:507–515. 18. Dutot M, Paillet H, Chaumeil C, et al. Severe ocular infections with contact lens: role of multipurpose solutions. Eye. 2009;26:470–476. 19. Pauly A, Meloni M, Brignole-Baudouin F, et al. Multiple endpoint analysis of the 3D-reconstituted corneal epithelium (SkinEthic) after treatment with benzalkonium chloride: modified MTT procedure and new markers for the early detection of toxic damage. Invest Ophthalmol Vis Sci. 2009; 50:1644–1652.

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