V Silicone derivatives for contact lenses

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chains were functionalized with 4-hydroxybutyric acid, sodium sait. ... 3 phosphate buffer saline (PBS), trypsin-EDTA solution, and antibiotics were purchased.
V / Bîomaier. Sci. Polymer EJn. Vol. 0. No. 0. pp- 1-0 11995) C VSP 1995.

2* / V

Silicone derivatives for contact lenses: Functîonalization, chemical characterization, and cell compatibility assessment V. MIGONKEY1*, M. D. LACROIX 1 , B. D. RATNER2 and M. JOZEFOWICZ1 'Laboratoire de Recherches sur les Macromoiécules, CNRS URA 502, Institut Galilée, Université Paris-Nord, 93430 Villeîaneuse, "Center for Bioengineering and Department of Chemical Engineering, BF-10, University of Washington, Seattle, WA 98195, USA Received 30 April 1994; accepted 12 December 1994. Abstract—Epoxy ring-opening functionalîzation of polvmers at random sites along chains with various chemical groups has been demonstrated. The reaction is performed in an aqueous solution under mild conditions in order to minimize dégradation of thé macromolecuiar chains. Siîïcone lenses made of copoiymers with epoxy sîde chains were functionalized with 4-hydroxybutyric acid, sodium sait. The carboxylated silicone derivarives were characterized by ESCA and radiotracers. A mean vaiue of 30% réaction yîeid was conchided, based upon data from both methods; nevenheless, thé laner can be improved up to 50% or more if thé conditions of préparation of thé epoxydized silicone lenses are optimized. Derivatized silicones were coated in thé wells of culture plates to evaluate thé cell compatibility of thèse new poiymers with a fibroblast cell line (McCoy's}. No ceiiular toxicity was observed. Key words: Siîicone derivatives; surface characterization, cell compatibility.

INTRODUCTION

A complication that continues to trouble contact lens wearers is thé déposition of a thick protein laver from tears f 1-10]. Depending on thé lens materials. thé composition of thé protein deposit differs. However, in every case, it results in degraded lens properties as weil as wearer discomfort, a decrease of gaseous exchange between thé tears and thé comea, and thé increased potential for comeal ulcération leading to infection. This 'incompatibility' of current lenses with thé ocular environment makes continuous wear impracticai or dangerous. Therefore, there is a need for new biomateriais with improved ocular biocompatibility. Silicone elastomers are potentially valuable candidates as materials for new contact lenses because of their high gas permeabih'ty. Unfortunately, a characteristic property of silicone rubber is its hydrophobicity [11]. This can lead to poor compatibility with thé eye tissues and tears. Many investigators hâve explored methods to improve silicone poiymers by rendering them hydrophilic (for example, see [12-15]. Hère, we explore thé random functionalîzation of silicones by chemical reaction. Random functionalization of epoxydized poiymers with various chemical groups by opening epoxy rings was previously described [16].

* To whom correspondence should be addressed.

V Migonney et al. MATERIALS AND METHODS

Materials The lenses were prepared by thé cross-linking of thé two chains, A and B,

CH3

CH 3

,

ÇH3

*

-feiO— , —SiO4I I CH=CH2 CH3

Chain A

CH3 , -SiO- ,

CH,

1

H

Chain B

-SiC496); thé culture médium (Dulbecco's Modified Eagle Médium. DMEM), fêtai calf sérum (FCS)

Silicone derivatives for contact lenses

3

phosphate buffer saline (PBS), trypsin-EDTA solution, and antibiotics were purchased from Gibco. Culture microtest plates (24 wells) were purchased from Costar; they were used coated with epoxydized siiicone and silicone derivatives or uncoated. ESCA experiments and analyses were performed at thé NESAC/BIO laboratory, University of Washington, Seattle, WA. USA on a Surface Science Instruments X-probe instrument. An Al Kalf2 X-ray source and a low energy flood gun to control sample charging were used. Typical pressures during analysis were 10~8-10~9 Torr. Methods Tilraiion of thé epoxy groups. Reaction of HC1, 14C Benzylamine on epoxydized silicone CL. Five epoxydized CL (200 mg representing about 5 microequivalent of epoxy) were incubated with 4 ml distilled water and 1 ml 14C solution of benzyiamine hydrochioride plus 0.36 g (2.5 meq) cold benzylamine hydrochioride and 0.10 g sodium hydroxide (2.5 meq); îhe suspension was magnetically stirred at reflux for about 20 h, or times varying from 5 min to 20 h for thé study of thé kinetics of thé reaction. Extensive washings with distilled water were performed before /?-counting thé contact lenses. This experiment was repeated at least twenty times in order to détermine thé epoxy content of thé epoxydized silicone CL; thé control consisted of this reaction performed on silicone CL without epoxy groups. When used as a method to quantify thé yield of thé opening reaction of epoxy by hydroxybutyric acid, thé reaction was performed five times. /?-counting. Five 14C benzylamine treated CL were eut and swelled for 1 h with 1 ml of dichloremethane in a counting vial before thé addition of 5 ml of aqueous counting scintillant solution (ACS). The biank vial was prepared with 1 ml of dichloromethane and 5 ml of ACS. The spécifie activity of thé 14C benzylamine was 870 000 dpm g ~ ' . Préparation of îhe modified contact lenses Reaction of 4-Hydroxybutyric acid on thé epoxydized silicone contact lenses. Five epoxydized CL (40 mg per lens) at 24 microequivalent epoxy per gram were incubated with 80 ml distilled water at room température before thé introduction of 8 g 4-hydroxybutyric acid, sodium sait. (63 meq) and 0.5 g of sodium hydroxide (12.5 meq); thé suspension was stirred for about 24 h. Extensive washings with distilled water were performed before drying under vacuum at 4Q°C. Reaction of sodium hydroxide on epoxydized siiicone CL. The same réaction as described just above was performed using thé same conditions except that thé hydroxyacid was omitted, Characterizanon by ESCA. The elemental composition and thé bonding states of thé uppermost layer (50-100 À) of chemically treated and unmodified epoxydized silicone lenses surface were determined using électron spectroscopy for chemical analysis (ESCA). Wide scan spectra (0-1000 eV) were taken to idenrify ail thé éléments, then thé C \ spectra in 270—290 eV range were measured at a higher resolution to obtain information on carbon bonding states, At a high resolution, deconvolution of thé main peak into différent subpeaks representing différent molecular environments was performed. By resoivîng thèse subpeaks from a compiex curve enveiope using tabulated bînding energy shifts, it was possible to establîsh thé chemical concentrations of various species and

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y Migonney et al.

functional groups within thé surface layer. The Cl s hydrocarbon peak was assigned to 285 eV and used as a binding energy référence. McCoy fibroblast cell culture. Aliquots of McCoy fibroblasts were stored frozen; they were cultured in 25 cm2 tissue culture flasks with DMEM médium supplemented with 10% of FCS and 1% of antibiotics, at 37°C, under a humidified atmosphère in a CO2 (5%) incubator (ASSAB T304 GF) Cell growth. McCoy fibroblast cells were cultured in: commercial 24-well microtest plates; 24-well microtest plates coated with epoxydized silicone; 24-weil microtest plates coated with epoxydized silicone that had been 4-hydroxybutyric acid-treated. Numerous washings of thé coated plates were performed with aqueous sodium chloride solutions (1.5. 1, and 0.5 M) and water, in order to remove leachable materiais. For ail thé plates, incubations with ethanol. PBS, and FCS were performed before thé cell culture. Usually, 30 000 cells in 500//l culture médium were seeded per well. The kinetics of thé cell growth was studied until confluence. Before counting, cells were detached from thé well with 250 a\A at 37°C for 5 min; thé enzyme was inhibited by adding 750 ^1 complète médium; thé collected cell suspension was ten diluted with ISOTON in a counting vial. Cell numbers were determined using a Coulter Counter ZM. A trypan blue dye exclusion assay was performed to check cell viability. RESULTS AND DISCUSSION

Réaction of 14C Benzylamine on epoxydized silicone The opening reaction of thé epoxy groups of thé silicone lenses by benzylamine was achieved in aqueous solution under reflux..in thé présence of 14C labeied benzylamine according to thé procédure previously described [16]. This reaction leads to thé formation of thé aminohydroxy derivative according to thé following scheme; CH,

CH, |

NaOH fr» «AV

walcr, reflux

oI

S J —O

vAAAr

fTH^

' ~3

This resuft was demonstrated with epoxydized polystyrène derivatives by four methods: IR spectroscopy (thé epoxy bands at 1250 and 905 cmT ' disappeared, whereas simultaneously thé characteristic amino substituted band at 743 cm ~ ' appearedl. elementai analysis. acidimétrie titration. and /7-counting. Ail thé methods of characterizatlon gave thé same resuîts; small différences between thé resuîts given by each methods were in thé range of accuracy for each one.

Silicone dérivâmes for contact lenses

For weakly epoxydized silicone lenses. thé more convenient method of characterization and measuring thé extent of reaction was /?-counting. Standard curves permitted thé détermination of benzylamine substitution. Moreover, as established formerly in thé case of epoxy polystyrène derivatives [16], thé reaction of aminé with epoxy is stoichiometric and quantitative. Therefore, this method allows thé détermination of thé epoxy content of any epoxydized insoluble sample, even at a low degree of epoxydation, with an acceptable accuracy and sensitivity since thé covaiently bound radioactivity is directly proportional to thé epoxy content of thé initial matehal. This method was systematically applied to characterize thé epoxy content of thé CL. Numerous epoxydized silicone CL from différent batches — about 100 lenses — were assessed and thé average value of thé epoxy content of CL was found to be 25.7 ± 3.9 //eq g~ '. A control was performed, where epoxy titration of unepoxydized silicone lenses gave a mean value of epoxy equal to 0.48 ± 0.35/ieqg~ ! . Kinetics of thé benzylamine opening reaction of epoxydized CL: 'évaluation ofthe reactivity of thé epoxy \ kinetic study ofthe epoxy ring-opening reaction by benzlamine was performed. varying thé reaction time from 5 min to 16 h. The plot ofthe amount of benzylamine per grain of CL against time (see Fig. 1 ) suggests two plateaus at about 7 weq g ~ ' and 25 fieq g , The first plateau may be reached at about 5 min and lasts about 30 min to 1 h. The second plateau is reached after 2 h of reaction. Based on thèse results, we postutate that thé heterogeneous reaction is kinetically diffusion controlled, i.e. thé diffusion ofthe reagent within thé lens is thé slow step and thé chemical reaction is faster. This implies that thé first plateau in Fig, 1 corresponds to thé reaction that occurs at thé interface whereas thé second plateau corresponds to completion ofthe réaction within thé lens. It afso suggests that thé cross-link density may be différent in thé surface zone and throughout thé bulk. If thé cross-link density were high in thé interior of thé spécimen, a reaction rate plateau might be seen initially. In order to check thé aboyé hypothesis. thé ring-opening reaction ofthe epoxy groups by 14C benzylamine was performed on cylinders of différent thicknesses, respectively 2-12 mm, made of epoxydized silicone. The reaction was performed in aqueous solution 50

S

30 h

20

2

10 £_

3

5

10

15

20

(hourl

Figure 1. Influence of thé time ot thé benzyiamine epoxy ring-opening reaction on ihe benzyiamme cornent 01 thé contact lenses.

V Migonney et al. Table 1.

Benzylamine titraiion of thé epoxy groups: différent thickness silicone ienses Thickness (mm) 30 min titraiion 2.5 5.5 7.5 10 12 2 h titration 3 6 7.5 10.5 12

Epoxy groups (/*eq g ~ ' )

Epoxy groups l^eq cm ~2 )

1 .45 ± 0.27 1.14 ±0.04 1.13 ±0.08 1.12 ±0.08 1.18 ±0.14 mean value = 1.21 ±0.12

0.048 ± 0.004 0.044 ± 0.009 0.032 = 0.008 0.032 r 0.001 0.030 r 0.007 mean value = 0.037 ± 0.007

5.75 ± 0.23

0.175 0.145

4.85 ± 0.26 4.12 ±0.12 4.45 ± 0.48

4.62 ± 0.53

0.135 0.121 0.135

under reflux at two reaction times: 30 min and 2 h. The results are presented in Table 1. They suggest that thé thicker thé cylinder, thé lower thé level of epoxy titrated (through 14C benzylamine reaction). This is consistent with our hypothesis that thé réaction occurred irom thé surface to thé core and was limited by thé diffusion of thé reagent. Moreover, within thirty minutes of benzylamine reaction, thé results are identical for ail thé cylinders (see Table 1) corresponding to thé titration of thé available epoxy groups within thé surface. In order to more clearly study this reaction, reaction times shorter than 30 min should be studied since most of thé reaction was close to compietion even at thé 30 min time. Based upon this observation, it is possible to estimate thé ddsity of thé epoxy groups. Averaging ail thé values of epoxy titrated within 30 mm, it corresponded to 0.038//eqcm~ 2 . Stability of thé epoxy groups in aqueous solution In order to pro ve that thé opening of thé epoxy rings of silicone CL cannot occur with water or sodium hydroxide aqueous solutions, différent assays were performed. First, epoxydîzed silicone Ienses were mcubated with water under reflux for 18 h. Second, thé reaction of sodium hydroxide aqueous solution with thé epoxy groups of thé CL was also performed under reflux for thé same réaction time. The epoxy groups of thé Ienses were titrated by benzylamine after thèse treatments, and thé results gave a mean value of thé epoxy content of25.5 ± 3.6,ueqg~' for thé sodium hydroxide-treated epoxy Ienses and 25 ueq g~ ' for thé water-treated ones. The resullts show that thé epoxy content of thé Ienses was thé same before and after thèse procédures. Thus, neither water nor sodium hydroxide aqueous solutions can hydrolyze thé epoxy groups of thé silicone CL. Ring-opening reaction of epoxy groups with 4-hydroxybutyric acid. sodium sait The ring-opening reaction of thé epoxy groups from thé silicone ienses was performed with 4-hydroxybutyric acid. sodium sait in water under reflux in thé présence of sodium hydroxide. Based upon our previous work [ ! 6], we postulated that thé reaction led to thé corresponding alcohol ether acid derivative. it is noteworthy îhat thé silicone derivative obtained after this chemical treatment retained ils optical transparency.

Silicone derivatives for contact lenses

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Characterization of thé 4-hydroxybutric acid-treated lenses was first performed by 14C benzylamine titration of thé unreacted epoxy groups as described in thé above paragraph. A kinetics study of thé ring-opening reaction of thé silicone CL epoxy groups by 4-hydroxybutyric acid was performed by varying thé reaction time from 5 min to 16 h. After each experiment, thé 14C benzylamine titration of thé unreacted epoxy groups permitted thé calculation of thé yield of thé ring-opening by thé alcohol acid. The complète set of results are presented in Table 2; thé statistical calcufation of epoxy content after reaction with hydroxybutyric acid for ail thé lenses (approximately 100) was performed and thé mean value of thé epoxy content was found to be 18.8 ± 6.4 weq g ~. Nevertheless, if typical results from one particular batch of lenses, prepared under optimal process conditions, are plotted (Fig. 2), it appears that for thèse CL, a decrease of thé unreacted epoxy against time to about half of thé initial level is observed. This value, which was invariant after 2 h of reaction, represented a yield of about 30% of thé alcohol ether derivative. It is significant that changes in thé parameters of thé processing of thé lenses may induce gréât variation in thé kinetics. Indeed. in some cases, only very slow reaction could occur, while in others, thé reaction is fast. This may be due to différences in thé structure of thé CL. Variancies in thé densities of thé silicone networks from thé différent batches may induce big différences in thé swelling of thé materials in solvent and in thé efficiency of thé reaction. Surface characterization of thé 4-hydroxybutyric acid-treated lenses was performed by ESCA. The high resolution Cls spectra of epoxydized silicone lenses, untreated and chemicaily treated, are presented (Fig. 3a, b). The subpeak at 285.0 eV is représentative of carbon bonded by hydrogen and other carbon atoms; thé two other subpeaks at, respectively, 286.5 and 288.8 eV correspond to thé ether bonds from epoxy groups and thé carbonyl C = 0) from thé substituted carboxylic acid. As thé area under each subpeak is related to thé percentage of thé bonds, it was then possible to calculate surface molecular compositions for thé treated and thé untreated lens. For thé epoxydized lens (untreated), Table 2. Kinetics of 4-hydroxybutyric acid reaction with epoxy groups 01 silicone lenses Reaction time with 4-hydroxybutync acid

//eq of epoxy per gram of lens

Batch 1 corresponding m optimal conditions of process 5 min 17.4 ± 2.5 15 min 11.7 ± 1.5 30min 16-5 = 3.2 lh 9.8 ±1.35 2h 13.3 ± 2.0 6h 13.4 ±1.9 IOU 14.0 ± 1.8 14 h 10.6 ±0.95 16h 13.0 ± I.S Batch 2 30 mm 2h 15 h 24 h Linses rrom différent batches 24 h

26.5 ± 3.5 26.5 ± 3-7 25.8 ±3.6 18.5 ±3.7

V Migonney et al.

90

"ET

a.

>* x O 10

14

18

time (hours) Figure 2. Influences of thé 4-hydroxybutyric acid reaction tîme on thé unreacted epoxy content of thé contact lenses. Présentée are typical results for one batch of epoxydized silicone contact lenses prepared under optimal processing conditions.

two primary subpeaks could be observed and attributed to C — C and C — O bonds; their calculated areas represented respectively 90.5 and 7.6% of thé C bonds. Thèse two peaks wou-ld be expected for a material comprised of a poly(dimethyl siloxane)-like polymer with epoxy groups. A small C = O component (1.9%, see inset in Fig. 3a) was aiso noted. The elemental composition (atomic %) of thé untreated lens was 50.1% C, 25.1% Si, and 24.8% O. No other éléments were found by ESCA analysis. For thé Cls spectrum of thé 4-hydroxybutyric acid-treated lens, three subpeaks attributed to C — C, C — O, and C = O were found with areas of 83, 11.3, and 5.5%. The 5.5% corresponding to thé carboxylic acid substitution représenta approximately one third of (C—O + C=O). From this, we concluded that thé yield of thé 4-hydroxybutyric acid reaction with epoxy was around 30%. This resuit corroborated thé above resuit obtained by thé radiolabel method. However, with such low levels of epoxy groups, thé détermination of thé subpeaks and their areas will be sensitive to small variations in sampie préparation and data acquisition. The elemental composition (at %) of thé chemically treated Sens found by ESCA was 52.3% C, 20.5% Si, 23.4% O, 1.2% Na, 0.9% CI. and 1.7% N. thé présence of nitrogen and chlorine in thèse 4-hydroxybutyric acid-treated lenses îs not readily explained and must be investigated further. The accuracy of thé ESCA results can be complicated by surface localization of small amounts of poly(dimethyl siloxane)-rich oligomers. Thèse highly surface active compounds, that may even be formed (albeit in low quantities) by X-ray exposure during thé ESCA analysis, will tend to reduce thé size of thé signais indicative of carbons bound to oxygen species (higher binding energy peaks). The effect of thèse low molecular weight chain fragments is mitigated by thé thorough extractions that occured during thé reaction steps. From thé high signal-to-noise spectra, it is apparent that thé 4-hydroxybutyric acidtreated lenses hâve greater amounts of thé binding energy components, qualitatively consistent with thé reaction we propose. Based on thé studies of thé surface reactivity of thé epoxydized lenses, it appears that neither water nor sodium hydroxide could hydrolyze thé epoxy. Moreover. thé previous siudy on epoxydized polystyrène resms [16] showed thé higher epoxy group reactivity to thé alcohol functional groups than to thé carboxyl acid functionality in mild aqueous

Stlicone derivotives for contact lenses

9

conditions. For thèse reasons, thé proposed scheme for thé reaction of 4-hydroxybutyric acid on epoxydized silicone contact lenses is; i—O- •*>* +

UD—a ij- (a i,) .-rcami

NaOH

ww—Si—O—t+ff, boil

AV»—Si-O (CH,)3

O l 0*1

oraj

i O

A*

OXNa

Ce// culture Cell growth studies were performed on commercial tissue culture polystyrène well plates, untreated epoxy-silicone coated plates and 4-hydroxybutyric acid-silicone treated plates.



4000

295

300

290

285

Blnding Enargy (eV)

(b)

5.5% 11.3% 83 Q%

295

290

385

fJIntflng ari»rgy

aVl

Figure 3. ESCA C 1s spectra for contact lenses: [ai unreacted. epoxycized silicone eiastomer; and (b) -i-iiydroxybutyric acid reacied with thé epoxydized silicone material.