cytotoxicity of rigid gas-permeable lens care solutions

RESEARCH PAPER

Cytotoxicity of rigid gas-permeable lens care solutions

Clin Exp Optom 2013; 96: 467–471 DOI:10.1111/cxo.12039

Camus Kar Man Choy* BSc(Hons) PhDPauline Cho* BOptom PhD FAAOMaureen V Boost† BSc(Hons) MSc DPhil* School of Optometry and† Department of HealthTechnology and Informatics, The Hong KongPolytechnic University, Hong Kong SAR, ChinaE-mail: [email protected]

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 solutionson human corneal epithelial cells and assessed the proliferation rate at different levels of cellmembrane damage.Methods: The human corneal epithelial cells were exposed to multipurpose solutionscontaining 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 andmembrane integrity were assessed by flow cytometry. Effects of exposure to concentrationsof 10 to 40 per cent multipurpose solutions for 12 hours on the metabolic rate of humancorneal epithelial cells were assessed by 3-(4-,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) cell proliferation assay. Recovery rates were assessed after re-culturefor 96 hours at 37°C.Results: MPS-A exposure caused the highest percentage of early and late necrotic cells forall 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 percent in MPS-B or MPS-C, were in late necrotic stage. After 12 hours of exposure, cell activitywas 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 theeffects 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 multipur-pose solutions for rigid gas-permeable lenses. This was apparent in both increased levels ofnecrotic cells on initial exposure and reductions in viability after prolonged exposures atlower dilutions. Multipurpose solutions containing PHMB as a preservative, while notcausing 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 torinse lenses with saline before insertion and use artificial tears for rewetting.

Submitted: 9 August 2012Revised: 9 October 2012Accepted for publication: 18 October 2012

Keywords: cornea, cytotoxicity, human corneal epithelium, multipurpose solutions, rigid gas-permeable lens solutions

Contact lens solutions have an importantrole in reducing the risk of microbial infec-tion in contact lens wearers. Such solutionsmust be effective in killing microorgan-isms contaminating contact lenses withoutcausing damage to the cornea. Severalstudies have been published concerning thecorneal toxicity of soft contact lens solutionsand have reported that some formulationscause corneal staining in vivo and effects oncell cultures, indicating cytotoxicity.1–6 Fewerstudies have investigated potentially harmfuleffects of rigid gas-permeable lens care solu-tions and even less have examined recentformulations.3,7,8 Although soft contact lens

wear predominates, rigid gas-permeablelenses remain an alternative for the correc-tion of vision, especially for patients withcorneal disorders, such as keratoconus.Orthokeratology, which is increasing inpopularity for myopic control in children,also uses specially designed rigid gas-permeable lenses.9–12 As orthokeratology ispredominantly used in children, the safetyand effectiveness of rigid gas-permeablesolutions is particularly important.13,14

As rigid gas-permeable lens materials donot absorb solutions15 and hence, they willnot leach out into the eye during use, higherconcentrations of disinfecting agents are

usually employed in rigid gas-permeablelens solutions than in those for soft lenses.Traditional rigid gas-permeable disinfectingformulations have concentrations of pre-servatives up to five times higher than thosein soft contact lens solutions. Our previousstudies have shown that multipurpose solu-tions can cause both necrotic and apoptoticdamage to corneal epithelial cells.5,16

Current multipurpose solutions for rigidgas-permeable lenses are recommended forrinsing, cleaning and disinfection. Althoughrigid gas-permeable lens materials do notabsorb solution during storage, residualsolution will be introduced into the eye

C L I N I C A L A N D E X P E R I M E N T A L

OPTOMETRY

© 2013 The Authors Clinical and Experimental Optometry 96.5 September 2013

Clinical and Experimental Optometry © 2013 Optometrists Association Australia 467

mailto:[email protected]

during lens insertion. Blinking and dilutionby tears will rapidly reduce the concentra-tion of the active agent(s) but in overnightorthokeratology, tear circulation is reduced,allowing prolonged solution exposure whilethe eyes are closed.17–19 The situation can beexacerbated if wearers use their solution asa wetting agent to increase the comfort ofrigid gas-permeable lens wear.20

A range of disinfecting agents has beenused in rigid gas-permeable lens solutions,including thimerosal, chlorhexidine andbenzalkonium chloride but newer formula-tions mainly contain polyhexamethylenebiguanide (PHMB) as a preservative. Cur-rently, solutions from only one manufac-turer retain chlorhexidine.

Our previous studies demonstrated thatdirect exposure to multipurpose solutionscontaining 0.0001% PHMB for 15 minutescaused cell membrane damage to 30 percent of human epithelial cells,5 althoughother researchers have shown no signifi-cant adverse ocular reaction with PHMB

at this concentration.5,6,21 Further studiesare needed to determine the cytotoxicityof higher concentrations of PHMB used inrigid gas-permeable lens multipurpose solu-tions This study determined and comparedthe cytotoxic effects of three commerciallyavailable rigid gas-permeable lens multipur-pose solutions on human corneal cells. Theproliferation rate at different levels of cellmembrane damage was also assessed.

METHODS

The cytotoxicity was investigated for threerigid gas-permeable lens multipurposesolutions, namely, Boston Simplus Multi-purpose Solution (MPS-A) (Bausch &Lomb Ltd, Rochester, NY, USA), MeniCarePlus (MPS-B) (Menicon Co Ltd, Nagoya,Japan) and Total Care 1™ (MPS-C) (AbbottLaboratories Inc, Abbott Park, IL, USA),

commonly used for orthokeratology inHong Kong (Table 1).22

Cytotoxicity was assessed by the effects ofthe multipurpose solutions on a humancorneal epithelial cell (HCEC) line (ATCCCRL-11135). Cultures were prepared aspreviously described.5 Briefly, HCEC wereseeded into 24-well precoated cell cultureplates and cultured in supplementedkeratinocyte-serum free medium (Gibco17005-042; Gibco/Invitrogen, Carlsbad,CA, USA) at 37°C in a humidified, CO2-controlled (five per cent) incubator, untilthe formation of a healthy confluent monol-ayer was observed in the wells. Cell integrityafter one, five and 10 minutes of multipur-pose solutions exposures was determined byflow cytometry and using Annexin V-FITC/7-AAD kit (Beckman Coulter Inc, Brea, CA,USA). Annexin binds to phosphatidyl serinein the cell membrane of apotoptic cells, in

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Figure 1. Cytotoxic effects following 10 minutes exposure to (A) Dulbecco’s phosphatebuffered saline, (B) multipurpose solution A (MPS-A) containing 0.003 per cent chlo-rhexidine gluconate and 0.0005 per cent polyaminopropyl biguanide, (C) MPS-B con-taining 0.0005 per cent polyhexamethylene biguanide and (D) MPS-C containing 0.0001per cent polyhexamethylene biguanide.B1: amount of 7-AAD stained cells—early necrosis, B2: amount of Annexin V-FITC(fluorescein isothiocyanate) + 7-AAD (7-amino-actinomycin D) stained cells—latenecrosis, B3: amount of non-stained cells—healthy cells, B4: amount of Annexin V-FITCstained cells—apoptosis.FL1: 525 nm Band Pass filter to detect green staining with FITC, FL4: 675 nm Band Passfilter to detect deep red staining with AAD.

Preparation Active agent

MPS-A PoloxamineBoric acidHydroxypropylmethylcelluloseChlorhexidine gluconate(0.003%)Polyaminopropyl biguanide(0.0005%)

MPS-B HypromelloseEtheylenediaminetetraaceticacidPolyhydric alcoholPoloxamerePolyhexamethylene biguanide(0.0005%)

MPS-C HypromelloseEtheylenediaminetetraaceticacidTromethamineTyloxapolPolyhexamethylene biguanide(0.0001%)

Table 1. Major ingredients in multipur-pose contact lens solutions (MPS) tested inthis study (source of information: manu-facturers’ brochures or instruction leaflets)

Cytotoxicity of rigid gas-permeable lens care solutions Choy, Cho and Boost

Clinical and Experimental Optometry 96.5 September 2013 © 2013 The Authors

468 Clinical and Experimental Optometry © 2013 Optometrists Association Australia

which the phosphatidyl serine is transferredto the extracellular side of the membrane.Its presence is detected by the additionof conjugated fluorescein isothiocyanate(FITC). 7-AAD (7-amino-actinomycin D)stains the guanine-cytosine pairs of DNA. Inlate stage apoptosis or necrosis, the cellmembrane integrity is lost, allowing dyesto enter the cell. 7-AAD is a DNA specificviability dye. Use of both dyes allows thedistinction between early apotosis in whichcells are only stained by Annexin V-FITC,and late stage apoptosis/necrosis whenstaining with both dyes occurs. Effects ofdiluted solutions on cell metabolism wereassessed by exposing cells to 10 to 40 per centof the multipurpose solutions for 12 hoursand determining activity using 3-(4-,5-Di-methylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) cell proliferation assay. Inaddition, cell proliferation after 96 hoursof re-culture was determined after expo-sure to the contact lens solutions. Follow-ing removal of culture media and washing

with Dulbecco’s phosphate buffered saline(DPBS), the metabolic rate of recoveringcells was assessed by Vybrant MTT cell pro-liferation assay kit (V-13154) (Gibco/Invi-trogen), following standard procedures.5

Effects on cells were compared with thoseof control cultures exposed to DPBS. Allprocedures were performed as previouslydescribed.5 Assays were performed in tripli-cate and repeated on three occasions. Themean and standard deviation (SD) of theaverage score for each experimental condi-tion was calculated. The data from the cellu-lar analysis were verified as being normallydistributed by one-sample Kolmogorov-Smirnov D test before performance of para-metric tests for analysis. Repeated measuresanalysis of variance (ANOVA) was used toinvestigate the differences in the percentageof healthy cells, apoptotic cells and necroticcells and the differences in the absorbanceof MTT between the control group andthe three treatment groups. A post-hoc test(Tukey-Kramer Multiple Comparisons Test)

was performed if a significant difference(p < 0.05) was detected in ANOVA.

RESULTS

Over 95 per cent of cells remained healthywhen epithelial cells were exposed to DPBS(control group) for up to 10 minutes, withless then one per cent in an apoptopic stage(stained with Annexin V-FITC), less than fiveper cent in early necrotic stage (stained with7-AAD) and less than two per cent in latenecrotic stage (stained with both). Exposureto MPS-A resulted in the highest percentageof early and late necrotic cells for each expo-sure time and these percentages were signifi-cantly higher than those for the other twomultipurpose solutions tested (p < 0.0001).After 10 minutes, around 40 per cent ofthe MPS-A exposed cells but less than fiveper cent of the MPS-B or MPS-C exposedcells, were in late necrotic stage (Figure 1).Although exposure to MPS-B and MPS-Calso resulted in statistically significantincreases in percentages of 7-AAD-stainedcells compared with cells in DPBS (p <0.005), these differences were small and onlyfound after 10 minutes exposure. Table 2shows a summary of the results.

After 12 hours of exposure, the MTT assayshowed a significant dose-response decreaseof 500 nm absorbance only for MPS-Atreated cells (p > 0.05). Absorbance at differ-ent concentrations of each multipurposesolution after 96 hours of re-culture showedsome reduction in viability attributable toPHMB but the effects of MPS-A were moreextreme and in the 30 and 40 per cent MPS-Atreated groups, nil absorbance was noted,indicating that the treated cells were non-viable (Figure 2).

DISCUSSION

Flow cytometric assay clearly demonstratedthat exposure to the three commerciallyavailable rigid gas-permeable multipurposesolutions resulted in different levels ofcytotoxic damage to corneal epithelial cells.Ten minutes of exposure to chlorhexidine-containing MPS (MPS-A) caused more than50 per cent of cells to display membranedamage (early and late necrosis). Althoughexposures to MPS-B and MPS-C for 10minutes resulted in fewer cells with mem-brane damage (less than 15 per cent),mainly at the early necrotic stage, our previ-ous studies showed that such damaged cells

Exposuretime inminutes

No staining(No evidence

of celldamage)

AnnexinV-FITC

staining(Apoptotic

cells)

7-AAD staining(Cells in early

necrosis)

AnnexinV-FITC +

7-AAD staining(Cells in

late necrosis)

DPBS(Control)

1 96.3 � 0.2 <1.0% 2.4 � 0.4 0.8 � 0.65 95.9 � 0.6 <1.0% 1.2 � 1.1 1.8 � 0.4

10 94.9 � 0.6 <1.0% 3.3 � 1.4 1.5 � 1.7

MPS-A 1 77.9 � 0.9F <1.0% 16.6 � 1.7* 5.3 � 1.1*5 59.2 � 1.3F <1.0% 28.3 � 5.8* 12.3 � 4.6*

10 41.4 � 0.7F <1.0% 18.5 � 4.5* 36.2 � 3.0*

MPS-B 1 94.9 � 1.2 <1.0% 1.9 � 1.0 2.3 � 1.35 92.6 � 1.9 <1.0% 5.1 � 3.2 2.0 � 1.1

10 85.3 � 0.5# <1.0% 10.0 � 1.2Y 3.9 � 0.8

MPS-C 1 96.2 � 0.9 <1.0% 1.9 � 1.0 1.3 � 0.65 94.0 � 2.0 <1.0% 4.0 � 2.8 1.5 � 1.0

10 87.7 � 0.7# <1.0% 8.5 � 2.2Y 2.4 � 0.3

MPS-A—multipurpose solution with 0.003% chlorhexidine gluconate and 0.0005% polyamino-propyl biguanideMPS-B—multipurpose solution with 0.0005% polyhexamethylene biguanideMPS-C—multipurpose solution with 0.0001% polyhexamethylene biguanideDPBS—Dulbecco’s phosphate buffered saline (as control)FSignificantly lower than those in DPBS, MPS-B and MPS-C at each exposure time (p < 0.005).*Significantly higher than those in DPBS, MPS-B and MPS-C at each exposure time (p < 0.005).#Significantly lower than those in DPBS at this specific exposure time (p < 0.001).YSignificantly higher than those in DPBS at this specific exposure time (p < 0.001).

Table 2. Percentages of cells displaying cytotoxic effects after exposure to test solutionsusing flow cytometry (mean and SD; n = 9)




cannot recover even after two hours ofre-culturing with cells progressing to latestage necrosis.5 In this study, the effects ofmultipurpose solutions on cell proliferationas determined by MTT revealed that expo-sure to MPS-A, MPS-B or MPS-C led to asignificant reduction of cell growth after 96hours of re-culture, compared to the DPBScontrol. This cytotoxic effect was dose-related and exposure to more than 30 percent MPS-A resulted in loss of cell viability.Although effects on cell proliferation afterMPS-B and MPS-C exposures were limited,they may adversely affect wound healingability of the cornea as well as routine mitoticactivity.

Multipurpose solutions for rigid gas-permeable lenses have better efficacy againststandard organisms than those for soft lensesbut this is attributable to somewhat higherconcentrations of preservatives in these for-mulations.23 The presence of PHMB, even at

0.0001 per cent in soft lens multipurposesolutions has been associated with cytoto-xicity on the basis of corneal staining.16,24,25

Biguanide-based antimicrobial agentscontain cationic active sites that can bind tomicrobial surfaces and lyse their cellularmembranes by electrostatic interaction.26,27

Our earlier study5 showed that cells becamenecrotic following exposure to soft lens MPScontaining 0.0001 per cent PHMB for 15minutes. Although the PHMB concentra-tion in MPS-B was five times higher than thatin MPS-C, necrotic effects were only slightlyhigher for MPS-B. The current findingsfor PHMB-based solutions for rigid gas-permeable lenses are in agreement with ourearlier study on multipurpose solutions forsoft lenses in that, following exposures, lessthan 10 per cent of cells displayed cell mem-brane damage;5 however, for MPS-A, bothnecrosis and effects on cell proliferationwere significantly higher than observed

for the other two solutions (Table 2 andFigure 2). This suggests that the presence ofchlorhexidine may significantly increasesolution toxicity; however, the differencemay also be attributable to variations inother constituents of the solutions.28

Studies on solution cytotoxicity shouldinvestigate the effects of the whole solutionformulation, including surfactants, viscosityand chelating agents as well as the ionicbalance; however, patients can only choosemultipurpose solutions from currently avail-able formulations. Although individual con-stituents may affect cytotoxicity,29 it is theoverall effect of the solution as marketedthat has to be assessed.

Although most studies have only addres-sed soft lens multipurpose solution cytotox-icity on the grounds that rigid gas-permeablelens materials do not absorb solution,15 mul-tipurpose solutions for rigid gas-permeablelenses can contact the cornea on lens inser-tion, especially if they are used to rewet thelens during lens wear.20 As our results haveshown that exposure to these multipurposesolutions, in particular those containingchlorhexidine, can lead to cytotoxic effects,rinsing soaked lenses with saline beforeinsertion and using a unit-dose of artificialtears as a rewetting agent may be prudent.This is especially important for the over-night use of orthokeratology lenses asthe exposure time to the multipurpose solu-tions may be prolonged under closed eyeconditions.11,13,30

In conclusion, this study showed thatexposure to the multipurpose solutionstested has potential for cytotoxicity, espe-cially in the solution containing chlorhe-xidine. Rinsing soaked lenses with salinebefore re-insertion may be advisable.

ACKNOWLEDGMENTSThis work was financially supported byresearch grants J-BB7P and GYD-48 fromThe Hong Kong Polytechnic University.

The authors have no proprietary or com-mercial interest in any of the ophthalmicsolutions used in this study.

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10 20 30 40

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cytotoxicity of rigid gas-permeable lens care solutions

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