Surface Modification of Silicone Intraocular Implants To Inhibit Cell

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Biomacromolecules 2005, 6, 2630-2637

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Surface Modification of Silicone Intraocular Implants To Inhibit Cell Proliferation Paolo Yammine, Graciela Pavon-Djavid, Gerard Helary,* and Veronique Migonney Laboratoire des Biomate´ riaux et Polyme` res de Spe´ cialite´ , UMR 7052, Universite´ Paris 13, Avenue Jean Baptiste Cle´ ment, 93 430 Villetaneuse, France Received April 11, 2005

Photo-cross-linkable polymers bearing cinnamic, sulfonate, and carboxylate groups were synthesized by radical polymerization leading to randomly distributed copolymers. These polymers were used to coat silicone intraocular lenses in order to reduce posterior capsule opacification, also named “secondary cataract”. We previously demonstrated that polymers containing both carboxylate and sulfonate groups inhibit cell proliferation, and formulations with the ratio R ) COO-/(COO- + SO3-) equal to 0.64 provided the highest inhibitory effect. Ionic polymers with this formulation were synthesized to contain a monomer with pendant siloxane groups in order to get compatibility with the silicone matrix of the intraocular lenses. Anchorage of the ionic polymer at the surface of the silicone implant was achieved by a cycloaddition reaction of the photosensitive groups according to two options. These modified silicone surfaces grafted onto intraocular lenses were shown to inhibit cell proliferation to 60%. Introduction Posterior capsule opacification (PCO) or “secondary cataract” is a major problem associated with cataract surgery and intraocular lens (IOL) implantation. The incidence of this phenomenon remains as high as 50% after two years of follow-up. Considering the large number of cataract surgeries, PCO may generate important medical, social, and economic problems. The decrease of visual acuity is assumed to be provoked by the proliferation of remaining lens epithelial cells, both onto the IOL and on the inner face of the capsular bag.1-3 During the last ten years, improvements in surgical techniques4 as well as in IOL design5-7 have shown progress in delaying the migration and proliferation of these cells, but PCO remains a challenging problem. One of the most important parameters in successful IOL application is the biocompatibility of the polymer used to manufacture the implant. Biocompatibility of various polymers, such as poly(methyl methacrylate),8-10 silicone,11,12 and hydrogels,13,14 has been examined. IOLs with high hydrophobic or hydrophilic surface properties have been shown to induce decreases in cell adhesion and proliferation.15-17 Preparation of such materials has been achieved by different methods, including polymerization or copolymerization of appropriate monomers (hydrophilic or hydrophobic),18-20 plasma treatment,21,22 and deposition of hydrophilic or hydrophobic coatings.20,23,24 However, until now, modified IOLs have not been successful in preventing in vivo cell adhesion and proliferation. This is believed to arise from a hostile host response toward the foreign body IOL consisting of nonspecific * Correspondence should be addressed to Gerard He´lary, Laboratoire des Biomate´riaux et Polyme`res de Spe´cialite´, Universite´ Paris 13, Avenue Jean Baptiste Cle´ment, 93430 Villetaneuse. [email protected].

protein adsorption, inflammatory reaction, dedifferentiation of remnant or regenerated epithelial cells into fibroblasts, cell migration, and proliferation. Several groups have targeted functionalization of surfaces with biological molecules in order to control cell adhesion, signaling, and function.25,26 Another proposed way to prevent PCO consists of controlling cell proliferation by orienting the intracellular signaling through the control of adhesive proteins/cell receptor interaction. Recently, we have shown that poly(methyl methacrylate) (PMMA)-based copolymers bearing randomly distributed sulfonate and carboxylate groups inhibit fibroblast and epithelial cell proliferation.18,27 The highest inhibiting properties, ∼70%, were observed for random copolymers bearing equivalent amounts of sulfonate and carboxylate groups. The aim of this work was to synthesize a bioactive polymer bearing sulfonate and carboxylate groups randomly distributed and to graft it on silicone intraocular lenses. Indeed, silicone is a good candidate for IOL application because of its high flexibility, allowing folding and minimal incision for implant insertion. Moreover, silicone implants present good optical properties, oxygen permeability, and chemical stability. Appropriate copolymers for grafting onto silicone implants have to present appropriate physicochemical properties such as optical transparency and chemical compatibility with the silicone matrix. These two requirements led us to choose tris(trimethylsiloxy)methacryloxy propyl silane (TTMPS), a monomer with a silicone pendant group, which has been used to prepare rigid contact lenses.28 Because polymers with pendant photosensitive groups such as cinnamoyl groups have been described as undergoing cross-linking upon UV irradiation,29-32 we selected a photocross-linking reaction to bind the bioactive copolymer at the surface of the silicone implant. To obtain a network at the surface of silicone lenses (Figure 1), we introduced a

10.1021/bm058010l CCC: $30.25 © 2005 American Chemical Society Published on Web 08/16/2005

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Figure 1. UV photoreticulation of pendant cinnamic groups linked to macromolecular chains.

photosensitive monomer unit, cinnamoyl ethyl methacrylate, in the chains of the bioactive polymers to be grafted. Sulfonate and carboxylate groups capable of inducing controlled interactions with adhesive protein were introduced in the macromolecular chains via sodium styrene sulfonate and methacrylic acid monomers. Radical polymerization of the four monomers provided for a random distribution along the macromolecular chains. In the present paper, we demonstrated adequate photografting of this polymer onto silicone lens and targeted inhibition of cell proliferation. Materials and Methods Materials. Tris(trimethylsiloxy)methacryloxy propyl silane (TTMPS) and methacrylic acid (MA) are commercial products from Aldrich. TTMPS was used as received without any purification, and MA was purified by distillation under vacuum before use. Cinnamoyl ethyl methacrylate (CEM) was prepared by equimolecular reaction of hydroxyl ethyl methacrylate (19.5 mmol) and cinnamoyl chloride (19.5 mmol) in 70 mL of dry ethyl ether in the presence of pyridine (4.5 mL) under argon. The reaction was carried out at 4 °C. Pyridinium salt resulting from the reaction of pyridine and hydrochloric acid was eliminated by filtration. The reaction solution was washed with 10% sodium hydroxide solution until total disappearance of cinnamic acid was detected by gas chromatography (Perkin-Elmer 8500). Sodium styrene sulfonate (NaSS) (Fluka) was purified by recrystallization in methanol and dried under vacuum. Sodium counterion was displaced by dimethyloctylammonium in order to allow the monomer solubilization in dioxane, which is a common solvent of the three other monomers. The reaction was achieved in two steps at room temperature: (1) acidification reaction of 24.2 mmol of NaSS with 24.2 mmol of hydrochloric acid in 70 mL THF; and (2) neutralization of sulfonic acid by dimethyloctylamine. NaCl generated during the first step was eliminated by filtration. Crystallization of dimethyloctylammonium styrene sulfonate (DOASS) was achieved by cooling the solution at 0 °C. After filtration, ammonium salt was dried under vacuum. Chemical structures of the monomers are presented in Figure 2. The thermal initiator 2,2-azobis(isobutyronitrile) (AIBN) from Aldrich was purified by recrystallization in methanol and kept under an argon atmosphere. Dioxane (Aldrich) was purified by distillation from CaH2 prior to the polymerization reaction. Copolymerization and Copolymer Characterization. Copolymers of different molar compositions in monomers

Figure 2. Chemical structures of monomers.

TTMPS, CEM, DOASS, and MA (total concentration 0.2 mol/L) were synthesized in dioxane at 70 °C under argon using AIBN as initiator (1% molar with respect to the total monomer concentration). Copolymerization reactions were stopped at about 70% conversion versus TTMPS, as determined by gas chromatography. Copolymers were then washed in water. Copolymer compositions were determined by 1H NMR in CDCl3 using a Brucker AC 200 spectrometer. Molar weights of the copolymers were determined by size exclusion chromatography in tetrahydrofuran (THF) on a Waters apparatus equipped with a refractive index detector and microstyragel columns. Carboxyl group content was determined by acidimetric titration performed with an automatic titrator TT100/TT300 from Tacussel/Radiometer. Titration consisted of a potentiometric reaction in homogeneous phase performed by slow addition (every 100 s or equilibrium) of small amounts (5 × 10-3 mL) of aqueous sodium hydroxide solution. About 50 mg of copolymer was dissolved in 37 mL of dioxane prior to the addition of 13 mL of water. After acidification of the solution by 0.1 M hydrochloric acid aqueous solution, the titration was carried out by addition of 0.1 M aqueous sodium hydroxide solution. In addition, acidimetric titration of poly(methacrylic acid) (PMA) was achieved in a 70/30 (v/v) dioxane/water mixture and in water as control, to determine the apparent pKa value of MA units in the copolymers. Photo-Cross-Linking and Preparation of Copolymer Lens-Shaped Films. The biological properties of the copolymers were assessed on lens-shaped polymer films. Copolymers were dissolved in dioxane at approximately 200 g/L. To avoid bubble formation in the films, solutions were degassed under vacuum for 1 min. The solutions were then poured into 14-mm-diameter molds to obtain lens-shaped films. Samples were dried at 70 °C under vacuum for 2 days. Copolymer films were UV-irradiated for selected times using a “MINI CURE” apparatus from PRIMARC Company equipped with two middle pressure 1200-W mercury lamps. Lenses were extensively washed with 0.1 M hydrochloric acid aqueous solution followed by washes with 0.1 M sodium

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Figure 3. Chemical structure of cinnamoyloxydimethylvinylsilane COMDVS.

hydroxide aqueous solution in order to replace the ammonium group by sodium counterion. Lenses were then sequentially washed with 1.5 and 0.15 M aqueous sodium chloride solution and water to remove leachable materials. Sterilization of the samples was performed by 15-min UV light exposition on each side. Prior to biological tests, lenses were equilibrated at physiological pH by at least three rinses in sterile phosphate-buffered saline solution (PBS). Lenses were placed in Costar 24-well microtest plates and maintained at the bottom of the wells with poly(tetrafluoroethylene) inserts. Samples were incubated in cell culture medium (DMEM) supplemented with 10% of fetal calf serum (FCS). Coating of IOLs with Bioactive Copolymer. Two routes to coat silicone implants with bioactive copolymers were pursued as follows: •Monolayer Option. Lenses were immersed in a 25 g/L dioxane copolymer solution for 1 min. After drying, lenses were UV-irradiated for 1 min on each side. •Bilayer Option. A first layer of silicone-containing photosensitive groups was deposited on the IOL followed by a dip-coating with the bioactive copolymer. This was achieved in three steps: (a) Preparation of the photosensitive molecule to be grafted on silicone; (b) grafting of the photosensitive molecule by hydrosilylation reaction on silicone chains at the surface of IOLs; (c) dip-coating of the modified lenses in the bioactive copolymer. (a) The photosensitive molecule cinnamoyloxymethyldimethylvinylsilane (COMDVS) (Figure 3) was prepared by reaction of chloromethyldimethylvinylsilane (CMDMVS) with sodium cinnamate. The latter molecule was prepared by reaction of cinnamic acid (0.17 mol) with sodium ethanolate (0.17 mol) in 220 mL of methanol. CMDMVS (14.8 mmol) was reacted at 120 °C in N-methyl pyrrolidone (25 mL) with sodium cinnamate under argon. After 5 h of reaction, the solution was filtered in order to eliminate NaCl. The solvent was removed under reduced pressure, and the product was diluted in diethyl ether, washed with an aqueous ammonium chloride solution, and finally washed with water. After evaporation of diethyl ether, COMDVS was purified by distillation under vacuum. (b) Silicone IOLs were immersed in a solution of dioxane containing poly(dimethylsiloxane-co-hydrogenomethylsiloxane) PHDMS (53.3 g/L), R,ω-divinylpoly(dimethylsiloxane) PDMS (266.7 g/L), and COMDVS (6.7 g/L). PHDMS and PDMS are commercial products from Rhoˆne-Poulenc Company (Rhodorsil RTV 141) containing a hydrosilylation catalyst. After drying, lenses were heated at 80 °C for 24 h to allow the addition reaction of silane PHDMS functions to the terminal double bonds of PDMS and COMDVS. (c) The last step is to immerse the coated lenses in dioxane solution at different concentrations of the bioactive polymer.

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After drying under vacuum, lenses are UV-irradiated on each face. •Blend option consisted of immersing the lenses in a solution of 3 mL of dioxane with the following components: PDMS (266.7 g/L), PHDMS (53.3 g/L), COMDVS (6.7 g/L), and various amounts of bioactive copolymers. After 24 h drying at 80 °C, lenses are UV-irradiated on each side. Cell Culture and Analyses. Human McCoy fibroblast cell line (CRL-1696) was obtained from American Type Culture Collection. Cells were cultured in DMEM supplemented with 10% FCS and 1% penicillin-streptomycin at 37 °C under a humidified atmosphere in 5% CO2. Two techniques were used to follow the kinetics of the cell proliferation: •5-Bromo-2′-desoxy Uridin (BrdU) Incorporation. McCoy fibroblast cells were cultured on bioactive copolymer-coated and control lenses. Cells (50 000) in 2 mL of culture medium were seeded per well and incubated at 37 °C for 6 days. BrdU incorporation was performed using a commercial kit from La Roche. Briefly, cells were pulsed with 50 µL per well of BrdU solution (1/100 dilution in serum-free medium). After incubation at 37 °C for 2 h, the medium was removed, and 250 µL of “fixdenate” was left to react for 30 min at room temperature. Samples were incubated in antibodies against BrdU, and substrate was incubated for 5 min at room temperature. The reaction was stopped by the addition of 50 µL of 1 M sulfuric acid, and the resulting absorbance was measured within 5 min at 450 nm. •Cell Number Counting. McCoy fibroblast cells were cultured in the same way as described above. Cells were detached with 500 µL of trypsin/EDTA at 37 °C for 3 min. Cell numbers were counted each day for 10 days using a Coulter Counter ZM. Cell viability was routinely checked by the Trypan blue dye exclusion assay. Results and Discussion Synthesis of the Copolymers. Copolymers were synthesized by radical copolymerization of TTMPS, MA, CEM, and DOASS in dioxane. The ionic monomer contents of the copolymers were maintained around 15% for the following reasons: (1) to avoid unacceptably high swelling of the copolymers in physiological medium, (2) at such ionic monomers contents, important inhibition of cell proliferation (up to 70%) was observed in the case of PMMA-based copolymers,18 (3) at higher ionic contents, copolymers were not transparent, and (4) high TTMPS content was required for chemical compatibility with the silicone lenses. The optimal required CEM monomer concentration allowing a good curing process and coating of the IOLs surface was determined by UV irradiation of films of poly(TTPMSco-CEM) with two chemical compositions of TTPMS/ CEM: 90/10 and 80/20. Copolymerizations of TTMPS and CEM were conducted using a TTMPS initial concentration equal to 0.2 mol/L in order to avoid the observed gel formation at higher concentrations (>0.4 mol/L). This was demonstrated by following the kinetics of TTMPS homopolymerization and by plotting kp/kt1/2 versus initial TTMPS

Silicon Intraocular Implants

Figure 4. Variation of kp/kt1/2 vs initial concentration of TTMPS.

Figure 5. Evolution of the band at 285 nm characteristic of cinnamic group as function of irradiation time.

concentrations. As shown in Figure 4, kp/kt1/2 increases for [TTMPS]0 higher than 0.5 mol/L. Molar compositions of both synthesized copolymers (poly(TTPMS-co-CEM)) were determined to be 90.7/9.3 and 83.7/16.3. The optimal irradiation time was determined by kinetics of disappearance of the cinnamic double bonds. A thin film of the two copolymers was cast on quartz plates to allow UV detection of the double bonds at 280 nm. The photochemical behavior of the two polymers represented by UV spectra shows a complete disappearance of the UV peak at 280 nm within 2 min (Figure 5). According to the literature, the main photoreaction of solid-state photosensitive polymers is the [2+2] photocycloaddition,33,34 causing photocrosslinking. To ensure that the [2+2] photocycloaddition is an intermolecular reaction producing a network (Figure 1), lenses of poly(TTPMS-co-CEM) for the two chemical compositions (TTMPS/CEM of 90.7/9.3 and 83.7/16.3) were UV-irradiated on each side for 1 min. Lenses were then left in dioxane, a good solvent of the copolymer, for one week under stirring. Regardless of chemical composition, lens shapes were not modified, and no weight loss was observed. For the synthesis of the bioactive polymers, we chose a CEM feed of approximately 10% molar, because for this molar percentage, the intermolecular reactions are sufficient to obtain a good network. According to the literature,18,27,35 the random copolymerization of sulfonated and carboxylated monomers leads to the formation of active sites along the macromolecular chains. The number and distribution of theses sites, which depend on the molar ratio of carboxylate units on total ionic units (R ) COO-/(COO- + SO3-), are the key parameters controlling adhesive protein adsorption and conformation and cell adhesion and proliferation. Recently, we determined the reactivity ratios of TTMPS, MA, and DOASS monomers

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and showed that the monomer units are randomly distributed along the macromolecular chains and that the compositions of the different copolymers are homogeneous.36 However, in the case of a terpolymerization of TTMPS, MA, and DOASS with a monomer feed of 85/10/5, the probability of formation of TTMPS-MA-TTMPS and TTMPS-DOASSTTMPS triads is quite high and is estimated to be 8.5% and 3.7%, respectively, for a conversion rate less than 70%. At higher conversion rates, the incorporation of TTMPS in the terpolymer chains dramatically increased, whereas the MA and DOASS incorporation decreased. Therefore, copolymerizations of the four monomers (TTMPS, CEM, MA, and DOASS) were carried out at conversion versus TTMPS close to 70%. Quaterpolymers were synthesized with monomer feeds in TTMPS, CEM, and ionic monomers of respectively 75, 10, and 15 mol %. Within the range of 15% in ionic groups, different initial feeds in ionic monomers were tested as reported in Table 1. Because of the difficulty in finding a nonsolvent, quaterpolymers were only washed with water in order to remove nonpolymerized ionic monomers and water-soluble chains. After drying under vacuum, chemical compositions of the quaterpolymers were determined by 1H NMR in deuterated dioxane. Chemical Characterization by 1H NMR and Acidimetric Titration AT. The analysis of 1H NMR spectra exhibiting characteristic peaks of TTMPS (SisCH3) at 0.06 ppm, CEM (OCOsCH dCHs phenyl) at 6.45 ppm, and two peaks at 2.8 and 2.9 ppm of DOASS ((CH3)2sN+sCH2-) allowed the determination of the quaterpolymer chemical compositions. For MA, the peak related to the acid proton at 12 ppm was not detected. The other signals of MA protons (5 protons), in the region between 0.5 and 2 ppm, are overlapped by proton signals from TTMPS (7 protons), CEM (5 protons), and DOASS (18 protons). The total intensity of the signals due to the protons of the three monomers (TTMPS, CEM, DOASS) in the domain 0.5-2 ppm was easily calculated from the intensity of the signals at 0.06, 6.45, and 2.8-2.9 ppm of TTMPS, CEM, and DOASS, respectively. By difference, we can calculate the integral intensity due to the 5 protons of MA and then finally the chemical compositions of the quaterpolymers. However, the accuracy of the chemical compositions determined by NMR remains quite low mainly because of the low MA content in the quaterpolymers chains. For this reason, COOH content of the polymers bearing MA units were titrated by AT in 75/25 dioxane/water solution. The chemical compositions of the synthesized polymers determined by both NMR and AT showed good agreement but sometimes differ from the monomer feed (Table 1). For example, the ionic group content of QP8 is twice as low when compared to the monomer feed. This decrease in ionic content is probably due to the washing of the copolymers at the end of polymerization, since chains rich in ionic groups would have been eliminated. Recently, we demonstrated that, during terpolymerization of TTMPS, MA, and DOASS, the two latter monomers are inserted more rapidly in the macromolecular chains than TTMPS.36 This means that some composition heterogeneity of the chains is likely, but the

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Table 1. Chemical Composition of Copolymers Determined by H NMR and Acidimetric Titration (AT)

reference

monomer feed TTMPS/CEM/MA/DOASS (mol %)

by NMR (mol %)

by AT (mol %)

R ) COO-/(COO- + SO3-)

QP0 QP1 QP2 QP3 QP4 QP5 QP6 QP7 QP8 QP9 QP10

75/15/0/10 75/10/2/13 75/10/3/12 75/10/5/10 75/10/6/9 75/10/7.5/7.5 75/10/10/5 75/10/11/4 75/10/12/3 75/10/14/1 65/20/15/0

77.6/12.4/0/10.0 85.0/8.5/2.9/3.6 80.4/10.7/2.2/6.7 77.5/9.3/8.8/4.4 77.0/9.2/7.2/6.6 80.8/10.5/4.8/3.9 75.7/10.2/10.2/4.0 77.1/9.9/9.9/2.5 86.4/7.4/4.9/1.3 71.0/8.0/20.2/0.8 63.0/20.8/16.2/0

85.2/8.3/3.1/3.4 80.7/10.7/1.8/6.8 78.7/9.4/7.2/4.7 77.0/9.2/6.9/6.9 78.6/10.2/7.3/3.9 77.7/10.3/8.0/4.0 78.5/10.6/8.4/2.5 86.5/7.0/5.3/1.2 71.6/7.9/19.8/0.7 63.4/20.2/16.4/0

0 0.46 0.23 0.64 0.51 0.61 0.69 0.78 0.79 0.97 1

experimental compositions

deviation is not too important for conversion less than 70% as mentioned earlier. However, in many cases, the average experimental compositions calculated from compositions determined by 1H NMR and AT are enough close to initial feed to consider that the composition chains are homogeneous. For each polymer, the ratio R ) COO-/(COO- + SO3-) was calculated (Table 1); this is the relevant parameter which gives the characteristics of the chemical compositions of the chains in ionic groups. Photoreticulation. To check that the irradiation time of 1 min was sufficient to obtain a network with the required mechanical properties, a diluted solution of a quaterpolymer (QP3) was prepared in dioxane (10 g/L) and poured onto a quartz plate to obtain a thin film of QP3. UV spectra recorded after one minute of UV irradiation showed the total disappearance of the band at 286 nm, attributed to the cinnamic double bond as previously observed with a film made of copolymer TTMPS/CEM 90/10 (Figure 5). Lenses were prepared from solutions of the different quaterpolymers in dioxane (200 g/L). After drying, lenses were irradiated for 1 min on each face. Lenses were then immersed at room temperature in water or in dioxane under stirring. After one week, the weight loss reached 3.5% in water and 7% in dioxane, a very good solvent of quaterpolymers. These results confirm that a 10% content in CEM and an irradiation time of 1 min are enough to obtain a good cross-linking of the polymer chains. Evaluation of Biological Properties. Because biological experiments were carried out at physiological pH of 7.4, it was important to evaluate the percentage of dissociated COO- groups at this pH. The apparent pKa values of carboxylic groups in the polymers were determined by acidimetric titration in dioxane/water solutions. The mean pKa value of the quaterpolymers was found equal to 8.6 ( 0.5. Apparent pKa values of MA in a dioxane/water mixture and in water were respectively found to equal pKa dioxane ≈ 8.5 and pKa H2O ≈ 5.5. By extrapolation, pKa values of quaterpolymer carboxylic groups from MA units was approximated to be 5.7 ( 0.2. Therefore, at pH 7.3, carboxylic acid groups were estimated to be 97% charged. Kinetics of the cell proliferation on polymer samples were quantified via BrdU incorporation during DNA synthesis. In addition, the number of cells at day 6 on quaterpolymers QP3 and QP7 was determined as a complementary assay.

Figure 6. Inhibition percentages of cell proliferation determined by BrdU and cell counting methods.

Figure 7. Influence of the carboxylate/sulfonate composition of the quaterpolymers on the inhibition of the cell proliferation.

Cell numbers were measured at day 6 in culture, because cells have reached a confluent monolayer. Commercial silicone lenses (COR16) were used as a polymer control. The inhibition of the cell proliferation by TTMPS-based polymers was expressed as inhibition percentage and calculated as follows: % inh ) [(Ncor16 - Ni)/Ncor16] × 100 with Ncor16 and Ni corresponding to cell numbers at day 6 on silicone lenses (COR16) and on quaterpolymers, respectively. Cell inhibition values are reported in Figure 6. Both methods are in excellent agreement and indicate that BrdU incorporation is appropriate for the analysis of cell proliferation in these materials. Cell inhibition percentages were plotted against R ) COO-/(COO-) + (SO3-) (Figure 7). Results show that a maximum value of the cell proliferation inhibition equaling 56 ( 2% was observed for R ) 0.64,

Silicon Intraocular Implants

Figure 8. Vital fluorescence staining micrograph of cells cultured on QP3 and QP7 which are inhibiting and noninhibiting polymers of cell proliferation, respectively.

whereas polymers presenting one ionic group (QP0 and QP10) did not exhibit any inhibitory properties. A similar result was recently observed in the case of a PMMA-based polymer with a percentage of cell proliferation inhibition reaching 70% for R ) 0.59.18 The observed cell inhibitory effect exhibited by polymers bearing sulfonate and carboxylate groups and presenting a precise R value has been suggested to correspond to the presence of appropriate active sites along the macromolecular chains formed during the radical polymerization process. Active sites are composed of styrene sulfonate and methacrylic acid units in the proportion around 1:1 separated by an unknown number of TTMPS units. A possible explanation for the lower proliferation rates on QP3 is a polymer-dependent cytotoxic effect. To examine this possibility, cells cultured on the different quaterpolymers were stained with orange acridin (vital staining) at day 6 of

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proliferation and observed by fluorescence microscopy (Figure 8). Viability staining showed equivalent viability levels among polymer formulations. Moreover, cell number is smaller on QP3 as compared to the silicone control lens, confirming the inhibitory effect of quaterpolymers bearing both sulfonate and carboxylate groups. The morphology of cells was examined after the staining of actin with phalloidin. Images of cell morphology at different times of the adhesion and spreading process (3, 24, and 48 h) onto QP3 and QP7 are presented in Figure 9. Cells seeded on QP7 (noninhibiting polymer) spread out very quickly, and the cytoskeleton (actin fibers) was assembled within 3 h. In contrast, cells attached to QP3 (inhibiting polymer) did not spread and remain round even after 48 h. Therefore, the extent of cell spreading on these polymers is inversely related to cell inhibition proliferation. Coating of Silicone Lenses. To evaluate the ability to graft the quaterpolymers onto IOL lenses, three approaches were pursued using QP3, the highest cell inhibitory polymers, as follows: •Monolayer Option. Silicone lenses were dipcoated in QP3 solution, dried, and UV-irradiated for 1 min on each side. It was hypothesized that the TTMPS pendant silicone groups could favor compatibility with the silicone matrix and lead to a stable coating of the lens surface. After 24 h of immersion in dioxane or water, insoluble parts detached from the lenses were observed in the solution, especially in dioxane. This probably means that a QP3 network was

Figure 9. Images of stained cells with phalloidin adherent to QP3 and QP7, respectively, as inhibiting and noninhibiting polymers of cell proliferation.

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Figure 10. Representation of the bilayer option.

Figure 11. Infrared spectra of coatings of QP3 and siliconecontaining photosensitive groups: (A) before UV irradiation, (B) after 1 min of UV irradiation.

formed by photodimerization of cinnamic groups without anchorage to the silicone matrix. •Bilayer Option. To covalently bind the QP3 network to the silicone matrix, an intermediate layer made of silicone functionalized by photosensitive groups was first coated on silicone prior to QP3 coating. We hypothesized that one part of the QP3 photosensitive groups would react with those of the intermediate layer, while the others would form a network (Figure 10). To test this hypothesis, the presence of silane and cinnamic functional groups after each step were followed by IR. A dioxane solution of PHDMS, PDMS, and COMDVS was deposited on a KBr plate, solvent eliminated, and 2-h cured at 80 °C to allow network formation by addition of PHDMS silane functions onto terminal double bonds of PDMS and COMDVS. IR spectrum (Figure 11) shows the presence of the characteristic peak of cinnamic groups at 1638 cm-1, while the characteristic peak of silane functions at 2120 cm-1 has almost disappeared, confirming that the hydrosilylation reaction occurred. On this photosensitive silicone layer, QP3 was deposited, solvent was eliminated,

Yammine et al.

Figure 12. Inhibition percentages of cell proliferation for bilayer and blend options.

and the sample was UV-irradiated. The corresponding IR spectrum shows the complete disappearance of the cinnamic groups at 1638 cm-1 (Figure 11). No weight loss of the bilayer silicone was observed after dioxane washing, confirming the formation of a network. Silicone IOLs were coated following this bilayer process. In the second step, three concentrations of QP3 dioxane solution (10, 50, and 100 g/L) were assessed. After drying, the lenses were UV-irradiated for 1 min on each side. At concentrations higher than 50 g/L, lenses were slightly opaque. Coated lenses were washed in water and dioxane for 24 h under stirring, and weight loss percents were estimated at 5% in water and 8% in dioxane. •Blend. To create an interpenetrated network in one step at the surface of the implants, silicone lenses were incubated in a dioxane solution containing PHDMS, PDMS, COMDVS, and QP3 at 2.5%, 5%, and 10% (w/w). After drying, coated lenses were cured at 80 °C in order to allow the addition reaction of PHDMS silane groups on the double bonds of PDMS and COMDVS, leading to the formation of a network involving QP3 chains. Lenses were then UVirradiated to induce intermolecular cycloaddition reactions between photosensitive groups of QP3 and cycloaddition reactions between photosensitive groups of QP3 and COMDVS linked to the silicone matrix. Coated lenses were washed in water and dioxane for one week. No weight loss was detected, indicating that QP3 chains bearing sulfonate and carboxylate groups are wellanchored to the IOL silicone matrix. It is noteworthy that lenses prepared with QP3 concentrations higher than 5% (w/ w) were slightly opaque. To check that the ionic groups are at the surface of the lenses in aqueous medium, contact angles with water were determined by the technique of the deposited drop on coated and uncoated lenses. Values of 116° ( 3° and 87° ( 4° were found on uncoated and coated silicone lenses, respectively, for either the bilayer or blend method. The sensitive decrease in contact angles confirms the presence of ionicgroup-functionalized QP3 macromolecular chains at the surface of the coated IOLs. Cell Proliferation Inhibition on Modified IOLs. To demonstrate the bioactivity of the grafted QP3 layer, inhibition of cell proliferation was assessed (Figure 12). Inhibition percentages were calculated by comparison to the cell proliferation on the silicone lens control reference COR16, which corresponds to 100% of cell proliferation. For both grafting methods and two coating concentrations, cell inhibi-

Silicon Intraocular Implants

tion was significantly lower than on control lenses (p < 0.05), reaching values as low as 60%. Modified IOL with low QP3 contents (10 g/L or 2.5%) remain transparent and are able to inhibit cell proliferation up to 60% as compared to uncoated IOLs. Conclusion Statistic silicone-based copolymers bearing carboxylate and sulfonate groups inhibited fibroblast proliferation in vitro to 60% compared to controls. The inhibitory effect correlates with the composition of the copolymer and is maximum for R values equaling 0.64 with R ) [COO-]/[(COO-) + (SO3-)]. These statistic quaterpolymer macromolecular chains were stably attached to the surface of silicone IOLs via photo-cross-linking involving (1) photosensitive groups linked at the surface of the IOL silicone matrix and pendant photosensitive groups on silicone-based copolymers and (2) formation of an interpenetrated network. For both approaches, coated silicone IOLs exhibited inhibition of cell proliferation to 60% compared to nonmodified silicone IOLs. These functionalized IOLs were recently implanted in rabbit eyes to show their ability to eradicate PCO. References and Notes (1) Apple, D. J.; Solomon, K. D.; Tetz, M. R.; Assia, E. I.; Holland, E. Y.; Legler, U. F.; Tsai, J. C.; Castenada, V. E.; Hoggatt, J. P.; Kostick, A. M. SurV. Ophthalmol. 1992, 37, 73-116. (2) Duncan, G.; Wormstone, I. M.; Davies, P. D. Br. J. Ophthalmol. 1997, 81, 818-823. (3) Oshika, T.; Nagata, T.; Ishii, Y. Br. J. Ophthalmol. 1998, 82, 549553. (4) Peng, Q.; Apple, D. J.; Visessook, N.; Werner, L.; Pandey, S. K.; Escobar-Gomez, M.; Schoderbek, R.; Guindi, A. J. Cataract RefractiVe Surg. 2000, 26, 188-197. (5) Nishi, O.; Nishi, K. J. Cataract RefractiVe Surg. 1999, 25, 521526. (6) Assia, E. I.; Blumenthal, M.; Apple, D. J. J. Cataract RefractiVe Surg. 1999, 25, 347-356. (7) Nishi, O.; Nishi, K.; Mano, C.; Ichihara, M.; Honda, T. Ophthalmic Surg. Lasers 1998, 29, 119-125. (8) Humphry, R. C.; Ball, S. P.; Brammall, J. E.; Conn, S. J.; Rich, W. J. Eye 1991, 5, 66-69. (9) Power, W. J.; Neylan, D.; Collum, L. M. J. Cataract RefractiVe Surg. 1994, 20, 440-445. (10) Werner, L. P.; Legeais, J. M. J. Fr. Ophthalmol. 1998, 21, 515524.

Biomacromolecules, Vol. 6, No. 5, 2005 2637 (11) Wang, M. C.; Woung, L. C. J. Cataract RefractiVe Surg. 2000, 26, 56-61. (12) Werner, L. P.; Legeais, J. M. J. Fr. Ophthalmol. 1999, 22, 492501. (13) Amon, M.; Menapace, R. J. Cataract RefractiVe Surg. 1991, 17, 774779. (14) Ravalico, G.; Baccara, F.; Lovisato, A.; Tognetto, D. Ophthalmology 1997, 104, 1084-1091. (15) Cunanan, C. M.; Tarbaux, N. M.; Knight, P. M. J. Cataract RefractiVe Surg. 1991, 17, 767-773. (16) Absolom, D. R.; Thomson, C.; Hawthorn, L. A.; Neumann, A. W. J. Biomed. Mater. Res. 1988, 22, 215-229. (17) Werner, L.; Legeais, J. M.; Nagel, M. D.; Renard, G. J. Biomed. Mater. Res. 1999, 46, 347-354. (18) El Kadhali, F.; Pavon-Djavid, G.; Helary, G.; Migonney, V. Biomacromolecules 2002, 3, 51-56. (19) Packard, R. B.; Garner, A.; Arnott, E. J. Br. J. Ophthalmol. 1981, 65, 585-587. (20) Legeais, J. M.; Werner, L. P.; Legeay, G.; Briat, B.; Renard, G. J. Cataract RefractiVe Surg. 1998, 24, 371-379. (21) Hettlich, H. J.; Otterbach, F.; Mittermayer, C.; Kaufmann, R.; Klee, D. Biomaterials 1991, 12, 521-524. (22) Eloy, R.; Parrat, D.; Duc, T. M.; Legeay, G.; Bechetoille, A. J. Cataract RefractiVe Surg. 1993, 19, 364-370. (23) Lloyd, A. W.; Dropcova, S. A.; Faragher, R. G.; Gard, P. R.; Hanlon, G. W.; Mikhalvosky, S. V.; Ollif, C. J.; Denyer, S. P.; Letko, E.; Filipec, M. J. J. Mater. Sci.: Mater. Med. 1999, 10, 621-627. (24) Werner, L.; Legeais, J. M.; Nagel, M. D.; Renard, G. J. Biomed. Mater. Res. 1999, 46, 347-354. (25) Pande, M.; Shah, S. M.; Spalton, D. J. J. Cataract RefractiVe Surg. 1995, 21, 326-330. (26) Miyake, K.; Maekubo, K.; Gravagna, P.; Tayot, J. L. Eur. J. Implant. Ref. Surg. 1991, 3, 99-102. (27) Latz, C.; Pavon-Djavid, G.; Helary, G.; Evans, M. D.; Migonney, V. Biomacromolecules 2003, 4, 766-771. (28) Tsutsumi, N.; Nishikawa, Y.; Nagata, T. K. Polymer 1992, 33, 209211. (29) Reddy, A. V. R.; Madheswari, D.; Subramanian, K. Eur. Polym. J. 1996, 32, 417-422. (30) Coqueret, X.; El Achari, A.; Hajaiej, A.; Lablache-Combier, A.; Loucheux, C.; Randrianarisoa, L. Makromol. Chem. 1991, 192, 1517-1534. (31) Egerton, P. L.; Reiser, A. Photogr. Sci. Eng. 1979, 23, 144-150. (32) Rehab, A. Eur. Polym. J. 1998, 34, 1845-1855. (33) Egerton, P. L.; Trigg, J.; Hyde, E. M.; Reiser, A. Macromolecules 1981, 14, 100-104. (34) Guru Row, T. N.; Ramachandra Swamy, H.; Ravi Acharya, K.; Ramamurthy, V.; Venkatesan, K.; Rao, C. N. R. Tetrahedron Lett. 1983, 24, 3263-3266. (35) Najab-Benhayoun, M.; Serne, H.; Jozefowicz, M.; Fisher, A. M.; Brisson, C.; Sultan, Y. J. Biomed. Mater. Res. 1993, 27, 511-520. (36) Helary, G.; Migonney, V.; Belleney, J.; Heinrich, L. Eur. Polym. J. 2000, 36, 2365-2369.

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