Cytotoxicities and wound healing effects of contact lens multipurpose solution on human corneal epithelial cell.

CLINICAL RELEVANCE: Contact lens multipurpose solutions (MPSs) contain several components that have the potential to cause corneal epithelial cell toxicity. Evaluating the components and the toxic effect of MPS should be considered for effective eye care. BACKGROUND: The cytotoxic and wound healing effects of five commercially available MPSs on human corneal epithelial cells (HCECs) are is investigated. METHODS: The following commercially available MPSs were used: Queen's PLURISOL®, Frenz®, Boston SIMPLUS®, DL+PLUS EYE® (DL), and NEW YORK DEFINE® (NY). The proliferation of HCECs exposed to each MPS for 1, 6, and 24 h and the cytotoxicity of these solutions were analyzed using methyl thiazolyl tetrazolium-based colorimetric and lactate dehydrogenase leakage assays, respectively. The cellular morphology was evaluated by inverted phase-contrast and electron microscopy. A scratch-wound assay was performed to measure wound widths 24 h after confluent HCEC monolayers were scratch-wounded. RESULTS: The tested MPS had a time-dependent inhibitory effect on HCEC proliferation and cytotoxicity, significantly at 24 h after exposure (p< 0.05 in all MPSs). HCECs exposed to MPS detached from the bottom of the culture dishes, showed degenerative changes such as loss of microvilli, cytoplasmic vacuole formation and nuclear condensation, and decreased wound healing, compared to the controls (p< 0.001 in Boston, DL and NY). Among the tested MPS, DL and NY were more cytotoxic and showed less wound healing. CONCLUSION: MPS has a toxic effect on HCECs, which is dependent on the concentration of the disinfecting component. Since the components that constitute the MPS are absorbed and retained in the lens, cautious scrutiny of the concentration and attention to lens cleaning are warranted to mitigate the related cytotoxicity.

Evaluation of in vitro effect of selected contact lens solutions conjugated with nanoparticles in terms of preventive approach to public health risk generated by Acanthamoeba strains.

INTRODUCTION: Various Acanthamoeba species are free-living organisms widely distributed in the human environment. Amphizoic amoebae as facultative parasites may cause vision-threatening eye disease - Acanthamoeba keratitis, mostly among contact lens wearers. As the number of cases is increasing, and applied therapy often unsuccessful, proper hygienic measures and effective contact lenses disinfection are crucial for the prevention of this disease. Available contact lens solutions are not fully effective against amphizoic amoebae; there is a need to enhance their disinfecting activity to prevent amoebic infections. The use of developing nanotechnology methods already applied with success in the prevention, diagnostic and therapy of other infectious diseases might be helpful regarding amoebic keratitis. This study assesses the in vitro effect of selected contact lens solutions conjugated with nanoparticles against Acanthamoeba trophozoites. MATERIAL AND METHODS: Three selected contact lens solutions conjugated with silver and gold nanoparticles in concentration of 0.25-2.5 ppm were used in vitro against the axenically cultured ATCC 30010 type Acanthamoeba castellanii strain. The anti-amoebic efficacy was examined based on the oxido-reduction of AlamarBlue. The cytotoxicity tests based on the measurement of lactate dehydrogenase (LDH) activity were performed using a fibroblast HS-5 cell line. RESULTS: Enhancement of the anti-amoebic activity of contact lens solutions conjugated with selected nanoparticles expressed in the dose dependent amoebic growth inhibition with a low cytotoxicity profile was observed. CONCLUSIONS: Results of the study showed that conjugation of selected contact lens solutions with silver nanoparticles might be a promising approach to prevent Acanthamoeba keratitis among contact lens users.

Chloroquine as a possible disinfection adjunct of disinfection solutions against Acanthamoeba.

Acanthamoeba keratitis is commonly encountered by contact lens wearers. Contact lens solution plays an important role in the safe use of contact lenses. The most popular products for disinfecting lenses are multipurpose disinfecting solutions (MPDS). However, almost all MPDS retailed in Korea are ineffective in killing Acanthamoeba. The objective of this study was to determine the possibility of using autophagy inhibitor chloroquine as a disinfecting agent to improve the amoebicidal activity of MPDS against Acanthamoeba, especially the cyst. Amoebicidal effects of eight different MPDSs combined with chloroquine (CQ), an autophagy inhibitor, and their cytotoxicities to human corneal epithelium cells were determined. Almost all MPDS showed strong amoebicidal effect on trophozoites after 8 h of exposure. However, they showed inadequate amoebicidal effect on cysts even after 24 h of exposure. MPDSs combined with 100 µM CQ increased their amoebicidal effects on immature cyst by inhibiting formation of mature cysts. Incubation with 100 µM CQ for 30 min did not have cytotoxicity to human corneal epithelial cells.

Efficacy of Korean Multipurpose Contact Lens Disinfecting Solutions against Acanthamoeba castellanii.

Acanthamoeba keratitis has been increasing in recent years. Main risk factors are contact lens wear and their cleaning solutions. Most contact lens wearers use multipurpose disinfecting solutions (MPDS) for cleansing and disinfecting microorganisms because of its convenience. We determined amoebicidal effects of MPDS made in Korea and their cytotoxicity on human corneal epithelium cells. Fifteen commercial MPDS (A to O) were tested for their amoebicidal effects on Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique. Among them, 7 kinds of MPDS showed little or no amoebicidal effects for 24 hr exposure. Solutions A, B, G, H, L, and O showed positive amoebicidal effects, and solutions M and N killed almost all trophozoites and cysts after 24 hr exposure. However, 50%-N solution showed 56% cytotoxicity on human corneal epithelial cells within 4 hr exposure, and 50%-O solution also showed 62% cytotoxicity on human cells within 4 hr exposure. Solution A did not show any cytotoxicity on human cells. These results revealed that most MPDS made in Korea were ineffective to kill Acanthamoeba. The solutions having amoebicidal activity also showed high levels of cytotoxicity on human corneal epithelial cells. New formulations for improved MPDS that are amoebicidal but safe for host cells are needed to prevent Acanthamoeba keratitis.

Cytotoxicity of rigid gas-permeable lens care solutions.

PURPOSE: Reports on cytotoxic effects of rigid gas-permeable lens multipurpose solutions, which remain important because of increasing popularity of orthokeratology, are limited. This study determined cytotoxic effects of rigid gas-permeable lens multipurpose solutions on human corneal epithelial cells and assessed the proliferation rate at different levels of cell membrane damage. METHODS: The human corneal epithelial cells were exposed to multipurpose solutions containing chlorhexidine gluconate (0.003%) and polyaminopropyl biguanide (PHMB) (0.0005%) (MPS-A), PHMB (0.0005%) (MPS-B) and PHMB (0.0001%) (MPS-C) for one, five and 10 minutes. Following staining with Annexin V-FITC/7-AAD, cell viability and membrane integrity were assessed by flow cytometry. Effects of exposure to concentrations of 10 to 40 per cent multipurpose solutions for 12 hours on the metabolic rate of human corneal epithelial cells were assessed by 3-(4-,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. Recovery rates were assessed after re-culture for 96 hours at 37°C. RESULTS: MPS-A exposure caused the highest percentage of early and late necrotic cells for all exposure times and was significantly higher than other multipurpose solutions (p < 0.0001). After 10 minutes exposure, almost 40 per cent of cells in MPS-A but less than five per cent in MPS-B or MPS-C, were in late necrotic stage. After 12 hours of exposure, cell activity was significantly reduced in a dose-response manner for MPS-A treated cells only (p > 0.05). After 96 hours of re-culture, all exposed cells showed some reduction in viability but the effects of exposure to 30 and 40 per cent MPS-A resulted in loss of viability. CONCLUSION: The presence of chlorhexidine appeared to increase cytotoxicity of multipurpose solutions for rigid gas-permeable lenses. This was apparent in both increased levels of necrotic cells on initial exposure and reductions in viability after prolonged exposures at lower dilutions. Multipurpose solutions containing PHMB as a preservative, while not causing acute cytotoxicity, did affect cell viability following exposure to diluted solutions. This indicated it is inadvisable to expose the cornea to multipurpose solutions but rather to rinse lenses with saline before insertion and use artificial tears for rewetting.

Ocular cytotoxicity evaluation of medical devices such as contact lens solutions and benefits of a rinse step in cleaning procedure.

Contact lens care solutions are known to have toxic effects on the ocular surface. The ISO 10993-5 standard describes test methods to assess the cytotoxicity of medical devices, but it needs some improvements to discriminate contact lens care multipurpose solutions. First we evaluated the biological hazards associated with the use of ophthalmic solutions, running a collaborative study with the French medical agency to propose adapted tools to study contact lens care solutions' ocular cytotoxicity (human cell line, short incubation times, and no dilution of solutions to test). Then we took into account the potential risk of these ophthalmic solutions adsorbed on contact lenses and released on the ocular surface, highlighting the addition of a rinse step with unpreserved marine solution in the contact lens cleaning procedure to avoid side effects of contact lens care solutions.

Cytotoxicity and effects on metabolism of contact lens care solutions on human corneal epithelium cells.

PURPOSE: The aim was to determine the cytotoxic effects of three multipurpose solutions (MPS) on human corneal epithelial cells (HCEC) and to assess the metabolic rates of recovering cells at different levels of cell membrane damage. METHOD: The effects of one to 15 minutes exposure to multipurpose solutions containing polyquaternium-1 (MPS-A), polyaminopropyl biguanide (MPS-B) and polyhexanide (MPS-C) on HCEC were determined. Recovery rates at different levels of cell membrane damage were assessed after re-culture for two hours at 37°C. Cell viability and membrane integrity were assessed using Annexin V-FITC/7-AAD staining and flow cytometry. Effects of concentrations of 10 to 40 per cent multipurpose solutions on the metabolic rate of recovering HCEC were assessed using a Vybrant MTT cell proliferation assay kit. RESULTS: The highest percentage of late necrotic cells resulted after exposure to MPS-A compared with other solutions and the control (p < 0.001). The percentages of early necrotic cells after 10 and 15 minutes of soaking in MPS-B were significantly higher than the control and other multipurpose solutions (p < 0.001). Although MPS-C exposure also resulted in statistically significant higher percentages of early necrotic cells than the control (p < 0.005), these differences were small. No recovery was noted when HCEC treated with multipurpose solutions were re-cultured, with numbers of dead cells in MPS-B-treated cultures increasing fourfold. The MTT assay showed significant dose-response decreases of 500 nm absorbance for all MPS-treated cells. In 40 per cent MPS-A-treated HCEC, lack of activity indicated the cells were non-viable. CONCLUSIONS: Multipurpose solutions induced varying levels of irreversible tissue sensitivity reactions, with MPS-A showing the greatest effects. The solutions damaged cell integrity and reduced metabolic rates suggesting delayed healing ability. The formulations of multipurpose solutions need to balance antimicrobial effectiveness with low cytotoxicity, which might not be currently possible to achieve. In light of our results, we suggest that contact lens wearers should be advised to rinse the soaked lenses with saline before lens insertion.

Effect of contact lens material on cytotoxicity potential of multipurpose solutions using human corneal epithelial cells.

PURPOSE: Multipurpose solutions (MPS) are used daily to clean and disinfect silicone hydrogel (SiHy) contact lenses. This in vitro study was undertaken to identify the potential for interaction between MPS, SiHy surface treatments, and lens materials, which may lead to changes in the response of human corneal epithelial cells (HCEC) to MPS-soaked lenses. METHODS: The MPS tested were renu fresh (formerly known as ReNu MultiPlus; ReNu), OptiFree Express (OFX), OptiFree RepleniSH, SoloCare Aqua, and Complete Moisture Plus. The SiHy materials evaluated were lotrafilcon A, lotrafilcon B, comfilcon A, galyfilcon A, and balafilcon A (BA). MPS-soaked lenses were placed on top of adherent HCEC. The effect of MPS dilutions (0.1 to 10% final concentration in medium) was also characterized. Cell viability, adhesion phenotype and caspase activation were studied after 24-h cell exposure. OFX released from lenses was determined using UV absorbance. RESULTS: A significant reduction in viability (between 30 to 50%) was observed with cells exposed to lenses soaked in ReNu and OFX. A significant downregulation of α(3) and ß(1) integrins, with integrin expression ranging from 60% to 75% of control (cells with no lens), was also observed with OFX and ReNu-soaked lenses. With the exception of BA, all other lenses soaked in OFX resulted in significant caspase activation, whereby over 18% of cells stained positive for caspases. Minimal caspase activation was observed in cells exposed to ReNu and Solo soaked lenses. For both OFX and ReNu, exposing cells to at least a 5% dilution had a significant effect on viability and integrin expression. While Complete and Solo did not lead to reduction in viability, cells exposed to a 10% dilution showed reduced integrin expression down to less than 70% of control value. Comparing cell response to diluted MPS solutions and various MPS-soaked lenses showed that it is not possible to reliably use cell response to MPS dilution alone to assess MPS biocompatibility. CONCLUSIONS: Our results demonstrate that the reaction of HCEC to MPS are affected by the type of lenses the MPS is released from and may potentially be influenced by the surface treatment (or lack of it) of SiHy materials.

[Toxicity of a new moistening agent and preservative in vitro]. / Toxizität neuer Benetzungs- und Konservierungsmittel in vitro.

PURPOSE: The use of preservatives such as benzalkonium chloride (BAC) usually increases the toxicity of pharmaceutical tear substitutes. HP-guar has been recently introduced as a new artificial tear substitute and includes the preservative Polyquad (0.001%), which is considered to be non-toxic. We therefore examined the effect of preserved (cetrimide 0.01%) and unpreserved HPMC (hydroxypropylmethyl cellulose) and HP-guar in dose and time-response experiments in a human corneal and conjunctival epithelial cell culture model. METHODS: Immortalized human conjunctival and corneal epithelial cells were cultured in 96-well plates at 37 degrees C with 5% CO(2) and exposed to the test solutions. The ATP content was quantified by means of a luminescence-based ATP assay, intracellular esterase activity by double fluorescent viability staining (calcein AM/ethidium homodimer D-1) and cell migration by a colony dispersion assay. All experiments were performed in triplicate and repeated at least once. The significance of differences was determined with an unpaired two-sided t-test. RESULTS: HPMC with preservative severely reduced the ATP content at all concentrations tested. Unpreserved HPMC, however, showed an inhibition of ATP production only at 100% and good esterase activity. HP-guar with and without preservative were found to reduce ATP activity more than unpreserved HPMC, but the unpreserved solution was found to reduce cellular ATP levels significantly more than the preserved solution. CONCLUSIONS: The new preservative Polyquad induced significantly less cytotoxicity than cetrimide. However, even unpreserved HP-guar can induce cytotoxicity in vitro, while unpreserved HPMC remains a good alternative tear substitute with low cytotoxicity.

Efficacy of contact lens storage solutions against trophozoite and cyst of Acanthamoeba castellanii strain 1BU and their cytotoxic potential on corneal cells.

Acanthamoeba is a free-living amoeba causing a potentially blinding infection of the cornea. Contact lens wearers are most at risk and account for about 95% of cases. We investigated the in vitro effectiveness of 10 contact lens solutions against Acanthamoeba castellanii and their cytotoxicity on corneal cells in vitro. Contact lens solutions were evaluated for their amoebicidal activities using an inverted light microscope. To determine of their cytotoxicity on corneal cells, agar diffusion tests were performed. According to the results obtained from the tests, AVIZOR Aqua Soft Comfort and Elegance(R) showed the best amoebicidal effect on A. castellanii trophozoites. Cysts were still viable after overnight (8 h) exposure. In the case of the cyctotoxic acitivities, All In One Light, Astek, SOLO-Care Aqua, Maxima, and Horien showed no cytotoxicity on the corneal cells. ReNu MultiPlus, AVIZOR Aqua Soft Comfort, Carrera, and Elegance showed mild cytotoxicity on the corneal cells. Plurisol.M presented moderate cytotoxicity on the corneal cells. All commercial solutions examined in this study are the lack of efficacy against A. castellanii. Improvement or development of new contact lens disinfecting systems by the manufacturers is needed to prevent Acanthamoeba keratitis.

A fluorometric study of relative ocular lens cytosensitivity to multipurpose contact lens solutions using the resazurin assay method.

This study investigated the relative ocular lens cytosensitivity to three multipurpose contact lens solutions using lens culture and resazurin microplate assay method. Comparison in the cell viability levels of the cultured bovine lenses as influenced by 3h experimental exposure to three contact lens multipurpose solutions (OPTI-FREE Express, Alcon; ReNu MultiPlus, Bausch & Lomb; and COMPLETE Comfort Plus, Allergan) was performed. The pre- and post-exposure fluorescence emission levels of the lenses were obtained, and findings were compared to baseline and control measurements. The solutions demonstrated varying levels of sensitivity (P<0.1), exhibiting non-significant to significant reversible disruption of the lenses'cell viability levels as revealed by the fluorescence intensity levels in the following ranked order (COMPLETE< ReNu< OPTI-FREE solutions). The findings show that OPTI-FREE Express and ReNu MultiPlus solutions exhibited more cytosensitive effect compared to COMPLETE Comfort Plus. These results suggest that the resazurin assay together with lens culture would be a reliable system for in vitro investigation of the effect of contact lens solutions and other ophthalmic formulations on ocular tissues.