- Email: [email protected]
Alkylphosphocholines for intraocular lens coating
LABORATORY SCIENCE
Alkylphosphocholines for intraocular lens coating Kirsten H. Eibl, MD, FEBO, Christian Wertheimer, Marcus Kernt, MD, Armin Wolf, MD, FEBO, Daniel Kook, MD, FEBO, Christos Haritoglou, MD, Anselm Kampik, MD, FEBO
PURPOSE: To assess the effect of alkylphosphocholine (APC)-coated intraocular lenses (IOLs) on human lens epithelial cell (LEC) proliferation in vitro and the corneal biocompatibility of APC in an organ culture model of human donor eyes. SETTING: Research Laboratory for Experimental Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. DESIGN: Experimental study. METHODS: Six foldable IOLs differing in optic material were incubated with APC, washed in phosphate-buffered saline, and dried overnight. Intraocular lenses of the same lot served as uncoated controls. Each rehydrated IOL was placed in 1 well of a 24-well plate containing proliferating human LECs. Cell survival was tested by the tetrazolium-dye reduction assay 5 days later. Biocompatibility of the human corneal endothelium was assessed by a 24-hour incubation of human donor corneas with different concentrations of APC before the live/dead assay was performed. RESULTS: Human LEC proliferation was attenuated by APC-coated IOLs (PZ.001). Coated hydrophilic acrylic IOLs were more effective inhibitors of human LEC proliferation than coated hydrophobic acrylic or silicone IOLs (PZ.001). Alkylphosphocholines were well tolerated by the corneal endothelium in an organ culture model of human donor corneas up to a concentration of 1 mM (n Z 12). CONCLUSIONS: Results show that APCs are suitable agents for IOL coating without linker molecules. Coated IOLs can inhibit human LEC proliferation and were well tolerated by human donor corneas in organ culture. Financial Disclosure: Dr. Eibl-Lindner holds the patent for “Intraocular lenses treated with alkylphosphocholines for pharmacological aftercataract prophylaxis,” international application no. PCT/EP2010/051490. No other author has a financial or proprietary interest in any material or method mentioned. J Cataract Refract Surg 2013; 39:438–445 Q 2012 ASCRS and ESCRS
Today, cataract surgery has become lens refractive surgery, and long-term complications such as posterior capsule opacification (PCO) are unacceptable to patients and surgeons.1,2 Nevertheless, the incidence of PCO is high, with more than 40% of eyes developing it 10 years after cataract surgery with hydrophobic acrylic intraocular lens (IOL) implantation.3 Neodymium:YAG capsulotomy is the treatment of choice for PCO; however, this places a significant financial burden on society4 and complications can be severe.5–7 In addition, future cataract surgery will focus not only on vision restoration but also on presbyopia correction through small-incision cataract surgery with foldable IOL implantation. However, microincision hydrophilic acrylate IOLs are associated with poorer PCO performance8 because the square-edged design 438
Q 2012 ASCRS and ESCRS Published by Elsevier Inc.
of the IOL cannot be manufactured as precisely as in hydrophobic acrylic IOLs because of the material properties.9 At the same time, long-term clinical observations10–12 suggest that the sharp-edged design of the optic margin is the only factor protecting the patient from PCO formation. The development of coated IOLs for PCO prevention has been the topic of numerous studies.13–15 The idea of using the IOL itself as a polymeric drugdelivery device for the prevention of postoperative complications is intriguing in its simplicity. Several coating agents, such as rapamycin14 and heparin,15 have been proposed. Rapamycin was proven effective after lens extraction in rabbit eyes with a follow-up of more than 6 months.14 However, rapamycin depends on a linker molecule, namely a solution of polyglycolic 0886-3350/$ - see front matter http://dx.doi.org/10.1016/j.jcrs.2012.09.028
LABORATORY SCIENCE: ALKYLPHOSPHOCHOLINES FOR IOL COATING
lactic acid (PGLA) and chloroform, for adherence to the IOL surface. However, in the study with rapamycin-coated IOLs,14 the tested IOLs were made of poly(methyl methacrylate), a material that is rarely used in current cataract surgery.16 Another compound that has been applied clinically as an IOL coating agent for PCO prevention is heparin. Although heparin-coated IOLs were well tolerated by the 99 patients with complicated cataract in 1 study,15 there was no difference in the PCO rate between heparincoated hydrophilic acrylic IOLs and the uncoated hydrophobic acrylate controls 12 months after surgery. The ideal coating agent should be characterized by the following: biocompatibility in the anterior and posterior segment of the eye, IOL loading without linker molecules, and inhibition of human lens epithelial cell (LEC) proliferation. Alkylphosphocholines (APCs) may meet these criteria because their chemical structure is amphiphilic with hydrophobic and hydrophilic properties present in 1 compound. They are synthesized as small molecules based on natural membrane phospholipids. As amphiphilic particles, they may be able to accumulate in the water compartment of hydrophilic IOLs. This represents a new concept of IOL coating that might better be referred to as IOL soaking or loading.17 At present, APCs are applied clinically outside the field of ophthalmology for their antitumor (Miltex)18 and antiparasitic (Impavido)19 properties. Furthermore, they are known for their good biocompatibility.20 This applies to secondgeneration APCs in particular, which are characterized by a carbon chain of more than 16 C-atoms and a cis double bond in the central position of the alkyl chain (N-APC: 18 C-atoms; APC: 22 C-atoms). Previous in vitro and in vivo studies21,22 have shown that APC can inhibit human ocular cell proliferation at nontoxic concentrations.
Submitted: July 9, 2012. Final revision submitted: September 26, 2012. Accepted: September 26, 2012. From the Department of Ophthalmology, Ludwig-MaximiliansUniversity, Munich, Germany. Katja Obholzer provided expert technical assistance. Presented in part at the World Congress of Ophthalmology, Berlin, Germany, June 2010, and the XXVIII Congress of the European Society of Cataract & Refractive Surgeons, Paris, France, September 2010. Corresponding author: Kirsten H. Eibl-Lindner, MD, FEBO, University Eye Hospital, Ludwig-Maximilians-University of Munich, Mathildenstrasse 8, D-80336 Munich, Germany. E-mail: kirsten. [email protected].
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The purpose of this study was to evaluate whether APCs are suitable coating agents for foldable IOLs of different optic material currently used for cataract surgery and whether these coated IOLs can inhibit human LEC proliferation in vitro. In addition, the biocompatibility of APC was assessed on corneal endothelial cells in an organ culture model of human donor eyes. MATERIALS AND METHODS Human Lens Epithelial Cell Culture The human LEC line HLE-B3 was obtained from ATCC, Rockville, Maryland, USA, and cultured on tissue culture flasks and Nunc plates (Thermo Fisher Scientific, Inc.) in Eagle’s modified essential medium (Biochrom AG) supplemented with 20% fetal calf serum, 50 IU penicillin/mL, and 50 mg streptomycin/mL at 37 C in an atmosphere of 5% carbon dioxide. HLE-B3 cells are a well-established in vitro model for PCO research.23–25 The medium was changed every third day. Versene with 2.5% trypsin (Invitrogen-Gibco, Life Technologies Corp.) was used for subculturing cells on confluence. Cellular growth was observed daily under a Leica phase-contrast microscope. To determine the proliferation characteristics of the cell line, growth was measured by counting cells using a Neubauer chamber and an automated cell counter (CASY 1, Roche Innovatis AG) at different time points. Maximum proliferation was detected at 48 to 72 hours (data not shown).
Alkylphosphocholines Alkylphosphocholines (APC and N-APC) were of analytical grade. They were synthesized and provided by Hansjoerg Eibl, PhD, Max-Planck-Institute for Biophysical Chemistry, Goettingen Germany.21 Alkylphosphocholines (APC and N-APC) were dissolved in phosphate-buffered saline (PBS) and stored at 4 C. A stock solution was prepared to obtain a final concentration of 10 mM in PBS. This solution was used as the coating solution in further experiments. Equal volumes of PBS without addition of APC or N-APC were used for the control IOL of the same lot.
Intraocular Lens Coating with Alkylphosphocholines Six foldable IOLs were selected for coating with N-APC or APC without linker molecules, such as PGLA. The IOLs included in the study are part of everyday cataract surgery and reflect the current clinical situation for this in vitro study. The IOLs differed in optic material (Table 1). The optic diameter of all IOLs tested was 6.0 to 6.5 mm. All IOLs had a square-edged design as indicated by the manufacturer. Four IOLs of the same lot were used for each experiment. Using a 12-well plate, 2 IOLs were coated separately in 500 mL APC coating solution for 12 hours at 4 C as described previously.17 In brief, APCs as amphiphilic compounds are able to form a monomolecular film on acrylic IOLs. Because they can permeate through cell membranes and can pass the blood–brain barrier, it can be postulated that they will diffuse into the capsular bag after IOL implantation. The other 2 IOLs were incubated in PBS and served as controls. Consecutively, all IOLs were allowed to dry for 4 hours at 4 C, followed by rehydration with PBS for 1 hour under standard cell-culture conditions. The IOLs were controlled for transparency under a phase-contrast microscope (Axioplan 100, Carl Zeiss Meditec AG).
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LABORATORY SCIENCE: ALKYLPHOSPHOCHOLINES FOR IOL COATING
Table 1. Characteristics of the 6 IOL types assessed for IOL coating with N-APC/APC.
IOL
Material
Water Content (% Vol.)
APC/N-APC
P Value*
% Reduction in Cell Number of Control
95% CI
APC N-APC APC N-APC APC N-APC APC N-APC APC N-APC APC N-APC
.001 .34 .001 .18 .001 .001 .001 .001 .001 .001 .001 .004
22 2 12 4 79 74 78 74 77 38 23 19
16, 28 3, 8 6, 19 2, 11 53, 105 49, 101 69, 88 65, 83 68, 87 28, 47 11, 35 7, 31
1
Hydrophobic
0
2
Hydrophobic
0
3 4
Hydrophilic; hydrophobic surface Hydrophilic
28
5
Hydrophilic
26
6
Silicone
25
0
APC Z alkylphosphocholine; CI Z confidence interval; IOL Z intraocular lens; Vol. Z volume *Analysis of variance
Cell Proliferation Assay A tetrazolium dye-reduction assay (MTT, 3-[4,5 dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide) was used to determine cell survival. HLE-B3 cells (500 mL/well at a density of 2 105 cells/well) were seeded in 12-well plates for 48 to 72 hours after the serum concentration was gradually reduced to 3%. Human LECs were then exposed to 1 IOL per well coated with N-APC or APC for 5 days under standard cell-culture conditions (n Z 2). Intraocular lenses of the same lot without coating served as controls (n Z 2). A cell-culture weight was placed on top of each IOL to ensure a proper position of the IOL on the cell-culture plate. The MTT test was performed as described by Mosmann26 with some modifications. After removal of the cell-culture weight, the IOL, and the cell-culture medium, the cells were washed with PBS and the MTT solution was added. The cells were incubated at 37 C for 30 minutes. After 3 washes with PBS, the insoluble formazan crystals were dissolved in dimethyl sulfoxide. The optical density was determined using a microplate reader at 550 nm (Molecular Probes, Life Technologies Corp.). Results were expressed as the mean percentage of the control. Experiments were performed in duplicate (n Z 4) and repeated 6 times (24 IOLs per IOL type). For the 6 IOLs, 72 IOLs were assessed after N-APC or APC coating (144 IOLs in total).
Human Donor Cornea Organ Culture Model Biocompatibility of alkylphosphocholines was assessed in an organ model of human donor corneas. The research was compliant with the Declaration of Helsinki and approved by the local ethics committee. Because APCs might diffuse from the IOL surface into the anterior and/or posterior chamber (in case of capsule rupture), it is a sensible and straightforward method to assess corneal biocompatibility on human donor corneal endothelium after direct exposure to the substance. To assess corneal biocompatibility of APCs, human donor eyes that did not qualify for corneal transplantation for serologic reasons were incubated with APC at 3 concentrations (2 per concentration; 6 pairs in total; concentrations tested: 0.1 mM, 1 mM, and 10 mM). Of each
pair of corneas, 1 was exposed to APC; the fellow eye was used as a control. Both corneas were incubated for 24 hours under standard cell-culture conditions. Care was taken that the same amount of PBS (solvent) was added to each cornea. To assess corneal endothelial cell biocompatibility, a 2-color fluorescence assay (Live/Dead Assay, Invitrogen) was performed. With this assay, the DNA in fragmented nuclei of dead cells is colored red by membrane impermeable propidium iodide when excited by light of 488 nm wavelength, whereas the DNA in all cells is colored blue by membrane permeable Hoechst 33342 dye when excited at 350 nm. After incubation, cells were washed with PBS and incubated with 2.0 mg/mL propidium iodide and 1.0 mg/mL Hoechst 33342 for 20 minutes at 37 C. Subsequently, cells were analyzed with an epifluorescence microscope (Leica DMR). For each cornea, 4 representative areas of the corneal endothelium were selected and photodocumented digitally (Leica Image Capturer). Subsequently, labeled nuclei were counted in fluorescence photomicrographs. Dead cells (red) were expressed as the percentage of total nuclei in the field (blue). The data are based on counts in 3 experiments performed in duplicate.
Statistical Analysis All results were expressed as the percentage of the control (control was set as 100%), and the mean was taken for each experimental group. The error was expressed as the standard error of the mean. To discriminate between 2 univariate groups, the Wilcoxon-Mann-Whitney test was applied. To differentiate whether the IOL coating with APC/N-APC or the IOL material itself had the larger effect on the total variance of results, a factor analysis was performed and the total explained variance was calculated. For multivariate analysis, analysis of variance with a least-significant-difference post hoc test was calculated. In addition, a factor analysis using the explained total variance was performed to discriminate between the effect of APC/N-APC exposure and the effect of the material. All statistical testing was performed using SPSS software (versions 17.0 and 19.0, SPSS, Inc.). For all analyses, a P value less than 0.05 was considered significant with a 95% confidence interval (CI).
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Figure 1. Cell number (mean G SD) after incubation of the human LECs with an N-APC– or APC-coated IOL over 5 days as determined by the tetrazolium-dye reduction assay. The results for the 6 foldable IOLs used for coating (Table 1) are shown. All coated IOLs vary significantly from the control except those marked with an * (IOL 1 and IOL 2 with N-APC coating) (APC Z alkylphosphocholine; Co Z control; IOL Z intraocular lens).
Figure 2. Effect of IOL material. Cell number (mean G SD) after incubation of the human LECs with a coated IOL over 5 days as determined by the tetrazolium-dye reduction assay. All coated IOLs varied significantly from the control (PZ.001) except the one marked with an * (hydrophobic IOLs coated with N-APC [PZ.43]) (APC Z alkylphosphocholine; Co Z control; H-Phil Z hydrophilic; H-Phob Z hydrophobic; IOL Z intraocular lens; Silic Z silicone).
RESULTS
N-APC: PZ.43, mean difference in % control, 3%, 95% CI, 5%-12%). The APC/N-APC treatment explained 50% of the variance in cell number, and the IOL material explained 50%. This indicates a strong interaction between APC/N and APC and the optic material during the coating process and qualifies hydrophilic IOLs as a delivery system for this compound. Intraocular lens 3 (hydrophilic acrylate with hydrophobic surface) was effective in controlling cell proliferation after coating with APC and N-APC, although N-APC was not effective on pure hydrophobic acrylates (Table 1 and Figures 1 and 2). Alkylphosphocholine was tolerated well by human corneal endothelial cells up to a maximum concentration of 1 mM over 24 hours (Figure 4). N-APC was not part of this assay due to the limited availability of human donor corneas and its inferiority to APC in coating hydrophobic IOLs. Thus, the APC with the better efficiency was selected, showing cell death at the maximum concentration of 10 mM.
Foldable hydrophobic acrylic, silicone, and hydrophilic acrylic IOLs were successfully coated with N-APC and APC without linker molecules such as PGLA (Table 1 and Figures 1 to 3). Alkylphosphocholinecoated IOLs reduced human LEC proliferation significantly (Figures 1 and 2) and remained transparent (Figure 3). Over all measurements, both alkylphosphocholines (APC and N-APC) attenuated cell proliferation after coating compared with the uncoated control IOLs of the same lot (APC: PZ.001, mean difference, 50% of control; 95% CI, 40%-60%; N-APC: PZ.001, mean difference, 37% of control; 95% CI, 28%-47%) (Figure 1). N-APC and APC varied significantly in the effect on growth inhibition compared with the control (PZ.03; mean difference, 13% of control; 95% CI, 1%-24%), whereas APC seemed slightly more efficient. For hydrophobic acrylic IOLs, the N-APC coating did not reduce cell proliferation significantly (IOL 1: PZ.34; IOL 2: PZ.18) (Figures 1 and 2). Regarding the optic material assessed, alkylphosphocholine coating with APC or N-APC inhibited cell growth significantly (control – hydrophobic: PZ.01, mean difference in % control, 9%, 95% CI, 2%-16%; control – hydrophilic: PZ.001, mean difference in % control, 70%, 95% CI, 65%-76%; control – silicone: PZ.001, mean difference in % control, 21%, 95% CI, 12%-30%). From these data it can be derived that hydrophilic IOLs coated with alkylphosphocholines had the largest effect on cell proliferation whereas N-APC-coated hydrophobic acrylic IOLs had the smallest effect (control – hydrophobic
DISCUSSION Posterior capsule opacification degrades visual function by reducing visual acuity and by increasing intraocular light scatter and straylight.26,27 The IOL material is an important factor influencing the optical quality of vision and the accuracy of the sharpedged design during manufacture.28,29 Hydrophilic IOLs were shown to display more forward scatter than hydrophobic IOLs.29 Moreover, they are associated with poorer PCO performance.8 Thus, IOL
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LABORATORY SCIENCE: ALKYLPHOSPHOCHOLINES FOR IOL COATING
Figure 3. Effect of APC-coated hydrophilic acrylic IOL on human LEC proliferation in cell culture compared with an uncoated control IOL of the same lot after 2 days (left) and 5 days (right) in cell culture. Above: Control IOL. Below: Alkylphosphocholinecoated hydrophilic acrylic IOL (APC Z alkylphosphocholine; IOL Z intraocular lens) (phase contrast microscope; original magnification 100).
coating with the aim of PCO prevention could be an approach to avoid these complications in future cataract surgery. Current research supports this concept because it has been shown that cell adhesion on the IOL surface was attenuated 1 month after implantation of a pharmacologically modified IOL in the rabbit eye.30 In this study, we selected APCs, small molecules with amphiphilic properties, as new coating agents and evaluated their in vitro efficiency on human LEC proliferation and their safety in an organ model of human donor corneas. We found that APCs are suitable coating agents for IOLs of all materials and are independent of linker compounds, such as PGLA.13 Coated IOLs were transparent and controlled human LEC proliferation in cell culture over several days. However, the IOL material seems to strongly affect the ability to coat with APC or N-APC. The maximum antiproliferative effect as a percentage of the control was achieved by APC-coated hydrophilic acrylic IOLs. It can be assumed that APCs can integrate into the water compartment of the hydrophilic IOL material because their amphiphilic molecular structure forms a monomolecular film on the IOL surface. This applies even to hydrophilic acrylic IOLs with hydrophobic surface properties (IOL 3). After coating, the performance of IOL type 3 was very similar to that of pure hydrophilic acrylates (IOLs 4 and 5) and not like the performance of hydrophobic acrylic IOLs. The N-APC coating elucidates this nicely because N-APC was not successful in inhibiting proliferation when used as a coating on hydrophobic acrylic IOLs (IOLs 1 and 2) but was effective when applied to IOL 3. A possible explanation for this might be
a slight difference in the chemical structure between APC und N-APC. N-APC has a carbon chain that is 4 carbon atoms shorter than APC, which makes N-APC more hydrophilic than APC. This seems to be a disadvantage because N-APC was not as effective as APC in LEC proliferation inhibition. It can be hypothesized that because of its increased hydrophilic properties, N-APC does not adhere to the IOL surface as effectively and is cleared faster from the IOL surface than APC. The hydrophilic IOLs used in this study had a water content of 25% to 28%. This makes APCs a valuable tool for PCO prophylaxis for this type of IOL in particular. Other pharmacologic substances are too hydrophilic, such as mitomycin-C (MMC) and 5-fluorouracil, or too hydrophobic, such as Triton X-100, to be considered IOL coating agents without linker molecules. Also, in previous studies,31,32 MMC and Triton X-100 were dependent on the Perfect Capsule System (Milvella Ltd.) to prevent anterior chamber toxicity. Nevertheless, human LECs were not completely eradicated after incubation with APC-coated or N-APC–coated IOLs. At the same time, it is questionable whether complete eradication should be the aim of PCO prevention. A capsular bag devoid of cells might be unstable, resulting in IOL decentration or even dislocation. In addition, compounds that achieve clarity of the capsular bag, such as actinomycin D when combined with cycloheximide and dispersed in hyaluronic acid, induced significant corneal toxicity with opacification in a primate model of PCO after lens refilling.33 Thus, complete eradication of PCO might not be the ultimate goal of PCO prophylaxis but rather a modification of the cellular response. In addition to the antiproliferative effect on human LECs, other
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higher than the half maximum inhibitory concentration determined for this compound on human LECs (0.1 mM).24 Human donor corneas are a valuable model for assessing biocompatibility in the anterior segment. This reflects the rise in the quality of ex vivo experimental approaches in current ophthalmologic research, such as the capsular bag model described by Dawes et al.34 In addition, we previously found good biocompatibility of alkylphosphocholines after intravitreal injection in the rabbit eye and rat eye.22,35 This is an important aspect for future clinical considerations because release of the molecule by diffusion or after rupture of the capsular bag cannot be completely avoided. WHAT WAS KNOWN Human LEC proliferation is the target of numerous pharmacologic approaches to prevent PCO. However, corneal biocompatibility of effective substances is often compromised and a hurdle to further clinical assessment. The IOL itself has been suggested as a drug-delivery system for pharmacologic substances. The influence of different IOL materials during the coating process has not been evaluated. WHAT THIS PAPER ADDS In this in vitro study, APCs were effective as IOL coating agents in the prevention of human LEC proliferation. Alkylphosphocholines were well tolerated by the corneal endothelium after assessment in an organ culture model of human donor corneas. Hydrophilic acrylic IOLs with a hydrophobic surface behaved like pure hydrophilic IOLs in cell culture. Hydrophilic IOLs might serve as a future drug-delivery system for APCs with the aim of PCO prevention.
Figure 4. Biocompatibility of APC after incubation of the corneal endothelium of a human donor cornea over 24 hours under standard cell-culture conditions (APC Z alkylphosphocholine; PBS Z phosphate-buffered saline).
factors such as the biocompatibility and physicochemical properties of the coating agent must be considered before clinical application as an IOL coating agent should be pursued. In this study, we found that APC concentrations of 1 mM were well tolerated by human corneal endothelial cells over 24 hours in an organ model of human donor corneas. This concentration is significantly
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multicenter trial of 6% miltefosine solution, a topical chemotherapy in cutaneous metastases from breast cancer. J Clin Oncol 2001; 19:4150–4159. Available at: http://jco.ascopubs.org/ content/19/21/4150.full.pdf. Accessed October 20, 2012 Sundar S, Jha TK, Thakur CP, Engel J, Sindermann H, Fischer C, Junge K, Bryceson A, Berman J. Oral miltefosine for Indian visceral leishmaniasis. N Engl J Med 2002; 347:1739–1746. Available at: http://www.nejm.org/doi/pdf/10. 1056/NEJMoa021556. Accessed October 20, 2012 Bagley RG, Kurtzberg L, Rouleau C, Yao M, Teicher BA. Erufosine, an alkylphosphocholine, with differential toxicity to human cancer cells and bone marrow cells. Cancer Chemother Pharmacol 2011; 68:1537–1546 Eibl KH, Banas B, Schoenfeld C-L, May CA, Neubauer AS, Priglinger S, Kampik A, Welge-Lussen U. Alkylphosphocholines inhibit proliferation of human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2003; 44:3556–3561. Available at: http://www.iovs.org/content/44/8/3556.full.pdf. Accessed October 20, 2012 Eibl KH, Lewis GP, Betts K, Linberg KA, Gandorfer A, Kampik A, Fisher SK. The effect of alkylphosphocholines in intraretinal proliferation initiated by experimental retinal detachment. Invest Ophthalmol Vis Sci 2007; 48:1305–1311. Available at: http:// www.iovs.org/content/48/3/1305.full.pdf. Accessed October 20, 2012 Choi J, Park SY, Joo C- K. Hepatocyte growth factor induces proliferation of lens epithelial cells through activation of ERK1/2 and JNK/SAPK. Invest Ophthalmol Vis Sci 2004; 45:2696–2704. Available at: http://www.iovs.org/content/45/8/ 2696.full.pdf. Accessed October 20, 2012 Eibl KH, Liegl R, Kernt M, Priglinger S, Kampik A. Alkylphosphocholines as a potential pharmacologic prophylaxis for posterior capsule opacification. J Cataract Refract Surg 2009; 35:900–905 Li J, Tang X, Chen X. Comparative effects of TGF-ß2/Smad2 and TGF-ß2/Smad3 signaling pathways on proliferation, migration, and extracellular matrix production in a human lens cell line. Exp Eye Res 2011; 92:173–179 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65:55–56. Available at: http://download.bioon.com.cn/upload/month_1001/20100118_ 33466306dce52f3e38a8VzE5OT8o1Xt4.attach.pdf. Accessed October 20, 2012 van Bree MCJ, van der Meulen IJE, Franssen L, Coppens JE, Reus NJ, Zijlmans BLM, van den Berg TJTP. Imaging of forward light-scatter by opacified posterior capsules isolated from pseudophakic donor eyes. Invest Ophthalmol Vis Sci 2011; 52:5587– 5597. Available at: http://www.iovs.org/content/52/8/5587.full. pdf. Accessed October 20, 2012 A, Canut MI, Montenegro GA, Marvan P, Dexl A, Pico Grabnes G, Barraquer RI, Michael R. Posterior capsule opacification assessment and factors that influence visual quality after posterior capsulotomy. Am J Ophthalmol 2010; 150:248–253 Nanavaty MA, Spalton DJ, Boyce JF. Influence of different acrylic intraocular lens materials on optical quality of vision in pseudophakic eyes. J Cataract Refract Surg 2011; 37:1230–1238 Ishikawa N, Miyamoto T, Okada Y, Saika S. Cell adhesion on explanted intraocular lenses. Part 2: experimental study of a surface-modified IOL in rabbits. J Cataract Refract Surg 2011; 37:1339–1342 Kim S-Y, Kim J-H, Choi J-S, Joo C- K. Comparison of posterior capsule opacification in rabbits receiving either mitomycin-C or distilled water for sealed-capsule irrigation during cataract surgery. Clin Exp Ophthalmol 2007; 35:755–758
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32. Maloof AJ, Pandey SK, Neilson G, Milverton EJ. Selective death of lens epithelial cells using demineralized water and Triton X-100 with PerfectCapsule sealed capsule irrigation; a histological study in rabbit eyes. Arch Ophthalmol 2005; 123:1378–1384. Available at: http://archopht.jamanetwork.com/data/Journals/ OPHTH/9946/els40030.pdf. Accessed October 20, 2012 33. Koopmans SA, Terwee T, van Kooten TG. Prevention of capsular opacification after accommodative lens refilling surgery in rabbits. Biomaterials 2011; 32:5743–5755 34. Dawes LJ, Illingworth CD, Wormstone IM. A fully human in vitro capsular bag model to permit intraocular lens evaluation. Invest Ophthalmol Vis Sci 2012; 53:23–29. Available at: http://www. iovs.org/content/53/1/23.full.pdf. Accessed October 20, 2012
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35. Schuettauf F, Eibl KH, Thaler S, Shinoda K, Rejdak R, May CA, Blatsios G, Welge-Lussen U. Toxicity study of erucylphosphocholine in a rat model. Curr Eye Res 2005; 30:813–820
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First author: Kirsten H. Eibl, MD, FEBO Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany
Alkylphosphocholines for intraocular lens coating Kirsten H. Eibl, MD, FEBO, Christian Wertheimer, Marcus Kernt, MD, Armin Wolf, MD, FEBO, Daniel Kook, MD, FEBO, Christos Haritoglou, MD, Anselm Kampik, MD, FEBO
PURPOSE: To assess the effect of alkylphosphocholine (APC)-coated intraocular lenses (IOLs) on human lens epithelial cell (LEC) proliferation in vitro and the corneal biocompatibility of APC in an organ culture model of human donor eyes. SETTING: Research Laboratory for Experimental Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. DESIGN: Experimental study. METHODS: Six foldable IOLs differing in optic material were incubated with APC, washed in phosphate-buffered saline, and dried overnight. Intraocular lenses of the same lot served as uncoated controls. Each rehydrated IOL was placed in 1 well of a 24-well plate containing proliferating human LECs. Cell survival was tested by the tetrazolium-dye reduction assay 5 days later. Biocompatibility of the human corneal endothelium was assessed by a 24-hour incubation of human donor corneas with different concentrations of APC before the live/dead assay was performed. RESULTS: Human LEC proliferation was attenuated by APC-coated IOLs (PZ.001). Coated hydrophilic acrylic IOLs were more effective inhibitors of human LEC proliferation than coated hydrophobic acrylic or silicone IOLs (PZ.001). Alkylphosphocholines were well tolerated by the corneal endothelium in an organ culture model of human donor corneas up to a concentration of 1 mM (n Z 12). CONCLUSIONS: Results show that APCs are suitable agents for IOL coating without linker molecules. Coated IOLs can inhibit human LEC proliferation and were well tolerated by human donor corneas in organ culture. Financial Disclosure: Dr. Eibl-Lindner holds the patent for “Intraocular lenses treated with alkylphosphocholines for pharmacological aftercataract prophylaxis,” international application no. PCT/EP2010/051490. No other author has a financial or proprietary interest in any material or method mentioned. J Cataract Refract Surg 2013; 39:438–445 Q 2012 ASCRS and ESCRS
Today, cataract surgery has become lens refractive surgery, and long-term complications such as posterior capsule opacification (PCO) are unacceptable to patients and surgeons.1,2 Nevertheless, the incidence of PCO is high, with more than 40% of eyes developing it 10 years after cataract surgery with hydrophobic acrylic intraocular lens (IOL) implantation.3 Neodymium:YAG capsulotomy is the treatment of choice for PCO; however, this places a significant financial burden on society4 and complications can be severe.5–7 In addition, future cataract surgery will focus not only on vision restoration but also on presbyopia correction through small-incision cataract surgery with foldable IOL implantation. However, microincision hydrophilic acrylate IOLs are associated with poorer PCO performance8 because the square-edged design 438
Q 2012 ASCRS and ESCRS Published by Elsevier Inc.
of the IOL cannot be manufactured as precisely as in hydrophobic acrylic IOLs because of the material properties.9 At the same time, long-term clinical observations10–12 suggest that the sharp-edged design of the optic margin is the only factor protecting the patient from PCO formation. The development of coated IOLs for PCO prevention has been the topic of numerous studies.13–15 The idea of using the IOL itself as a polymeric drugdelivery device for the prevention of postoperative complications is intriguing in its simplicity. Several coating agents, such as rapamycin14 and heparin,15 have been proposed. Rapamycin was proven effective after lens extraction in rabbit eyes with a follow-up of more than 6 months.14 However, rapamycin depends on a linker molecule, namely a solution of polyglycolic 0886-3350/$ - see front matter http://dx.doi.org/10.1016/j.jcrs.2012.09.028
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lactic acid (PGLA) and chloroform, for adherence to the IOL surface. However, in the study with rapamycin-coated IOLs,14 the tested IOLs were made of poly(methyl methacrylate), a material that is rarely used in current cataract surgery.16 Another compound that has been applied clinically as an IOL coating agent for PCO prevention is heparin. Although heparin-coated IOLs were well tolerated by the 99 patients with complicated cataract in 1 study,15 there was no difference in the PCO rate between heparincoated hydrophilic acrylic IOLs and the uncoated hydrophobic acrylate controls 12 months after surgery. The ideal coating agent should be characterized by the following: biocompatibility in the anterior and posterior segment of the eye, IOL loading without linker molecules, and inhibition of human lens epithelial cell (LEC) proliferation. Alkylphosphocholines (APCs) may meet these criteria because their chemical structure is amphiphilic with hydrophobic and hydrophilic properties present in 1 compound. They are synthesized as small molecules based on natural membrane phospholipids. As amphiphilic particles, they may be able to accumulate in the water compartment of hydrophilic IOLs. This represents a new concept of IOL coating that might better be referred to as IOL soaking or loading.17 At present, APCs are applied clinically outside the field of ophthalmology for their antitumor (Miltex)18 and antiparasitic (Impavido)19 properties. Furthermore, they are known for their good biocompatibility.20 This applies to secondgeneration APCs in particular, which are characterized by a carbon chain of more than 16 C-atoms and a cis double bond in the central position of the alkyl chain (N-APC: 18 C-atoms; APC: 22 C-atoms). Previous in vitro and in vivo studies21,22 have shown that APC can inhibit human ocular cell proliferation at nontoxic concentrations.
Submitted: July 9, 2012. Final revision submitted: September 26, 2012. Accepted: September 26, 2012. From the Department of Ophthalmology, Ludwig-MaximiliansUniversity, Munich, Germany. Katja Obholzer provided expert technical assistance. Presented in part at the World Congress of Ophthalmology, Berlin, Germany, June 2010, and the XXVIII Congress of the European Society of Cataract & Refractive Surgeons, Paris, France, September 2010. Corresponding author: Kirsten H. Eibl-Lindner, MD, FEBO, University Eye Hospital, Ludwig-Maximilians-University of Munich, Mathildenstrasse 8, D-80336 Munich, Germany. E-mail: kirsten. [email protected].
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The purpose of this study was to evaluate whether APCs are suitable coating agents for foldable IOLs of different optic material currently used for cataract surgery and whether these coated IOLs can inhibit human LEC proliferation in vitro. In addition, the biocompatibility of APC was assessed on corneal endothelial cells in an organ culture model of human donor eyes. MATERIALS AND METHODS Human Lens Epithelial Cell Culture The human LEC line HLE-B3 was obtained from ATCC, Rockville, Maryland, USA, and cultured on tissue culture flasks and Nunc plates (Thermo Fisher Scientific, Inc.) in Eagle’s modified essential medium (Biochrom AG) supplemented with 20% fetal calf serum, 50 IU penicillin/mL, and 50 mg streptomycin/mL at 37 C in an atmosphere of 5% carbon dioxide. HLE-B3 cells are a well-established in vitro model for PCO research.23–25 The medium was changed every third day. Versene with 2.5% trypsin (Invitrogen-Gibco, Life Technologies Corp.) was used for subculturing cells on confluence. Cellular growth was observed daily under a Leica phase-contrast microscope. To determine the proliferation characteristics of the cell line, growth was measured by counting cells using a Neubauer chamber and an automated cell counter (CASY 1, Roche Innovatis AG) at different time points. Maximum proliferation was detected at 48 to 72 hours (data not shown).
Alkylphosphocholines Alkylphosphocholines (APC and N-APC) were of analytical grade. They were synthesized and provided by Hansjoerg Eibl, PhD, Max-Planck-Institute for Biophysical Chemistry, Goettingen Germany.21 Alkylphosphocholines (APC and N-APC) were dissolved in phosphate-buffered saline (PBS) and stored at 4 C. A stock solution was prepared to obtain a final concentration of 10 mM in PBS. This solution was used as the coating solution in further experiments. Equal volumes of PBS without addition of APC or N-APC were used for the control IOL of the same lot.
Intraocular Lens Coating with Alkylphosphocholines Six foldable IOLs were selected for coating with N-APC or APC without linker molecules, such as PGLA. The IOLs included in the study are part of everyday cataract surgery and reflect the current clinical situation for this in vitro study. The IOLs differed in optic material (Table 1). The optic diameter of all IOLs tested was 6.0 to 6.5 mm. All IOLs had a square-edged design as indicated by the manufacturer. Four IOLs of the same lot were used for each experiment. Using a 12-well plate, 2 IOLs were coated separately in 500 mL APC coating solution for 12 hours at 4 C as described previously.17 In brief, APCs as amphiphilic compounds are able to form a monomolecular film on acrylic IOLs. Because they can permeate through cell membranes and can pass the blood–brain barrier, it can be postulated that they will diffuse into the capsular bag after IOL implantation. The other 2 IOLs were incubated in PBS and served as controls. Consecutively, all IOLs were allowed to dry for 4 hours at 4 C, followed by rehydration with PBS for 1 hour under standard cell-culture conditions. The IOLs were controlled for transparency under a phase-contrast microscope (Axioplan 100, Carl Zeiss Meditec AG).
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Table 1. Characteristics of the 6 IOL types assessed for IOL coating with N-APC/APC.
IOL
Material
Water Content (% Vol.)
APC/N-APC
P Value*
% Reduction in Cell Number of Control
95% CI
APC N-APC APC N-APC APC N-APC APC N-APC APC N-APC APC N-APC
.001 .34 .001 .18 .001 .001 .001 .001 .001 .001 .001 .004
22 2 12 4 79 74 78 74 77 38 23 19
16, 28 3, 8 6, 19 2, 11 53, 105 49, 101 69, 88 65, 83 68, 87 28, 47 11, 35 7, 31
1
Hydrophobic
0
2
Hydrophobic
0
3 4
Hydrophilic; hydrophobic surface Hydrophilic
28
5
Hydrophilic
26
6
Silicone
25
0
APC Z alkylphosphocholine; CI Z confidence interval; IOL Z intraocular lens; Vol. Z volume *Analysis of variance
Cell Proliferation Assay A tetrazolium dye-reduction assay (MTT, 3-[4,5 dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide) was used to determine cell survival. HLE-B3 cells (500 mL/well at a density of 2 105 cells/well) were seeded in 12-well plates for 48 to 72 hours after the serum concentration was gradually reduced to 3%. Human LECs were then exposed to 1 IOL per well coated with N-APC or APC for 5 days under standard cell-culture conditions (n Z 2). Intraocular lenses of the same lot without coating served as controls (n Z 2). A cell-culture weight was placed on top of each IOL to ensure a proper position of the IOL on the cell-culture plate. The MTT test was performed as described by Mosmann26 with some modifications. After removal of the cell-culture weight, the IOL, and the cell-culture medium, the cells were washed with PBS and the MTT solution was added. The cells were incubated at 37 C for 30 minutes. After 3 washes with PBS, the insoluble formazan crystals were dissolved in dimethyl sulfoxide. The optical density was determined using a microplate reader at 550 nm (Molecular Probes, Life Technologies Corp.). Results were expressed as the mean percentage of the control. Experiments were performed in duplicate (n Z 4) and repeated 6 times (24 IOLs per IOL type). For the 6 IOLs, 72 IOLs were assessed after N-APC or APC coating (144 IOLs in total).
Human Donor Cornea Organ Culture Model Biocompatibility of alkylphosphocholines was assessed in an organ model of human donor corneas. The research was compliant with the Declaration of Helsinki and approved by the local ethics committee. Because APCs might diffuse from the IOL surface into the anterior and/or posterior chamber (in case of capsule rupture), it is a sensible and straightforward method to assess corneal biocompatibility on human donor corneal endothelium after direct exposure to the substance. To assess corneal biocompatibility of APCs, human donor eyes that did not qualify for corneal transplantation for serologic reasons were incubated with APC at 3 concentrations (2 per concentration; 6 pairs in total; concentrations tested: 0.1 mM, 1 mM, and 10 mM). Of each
pair of corneas, 1 was exposed to APC; the fellow eye was used as a control. Both corneas were incubated for 24 hours under standard cell-culture conditions. Care was taken that the same amount of PBS (solvent) was added to each cornea. To assess corneal endothelial cell biocompatibility, a 2-color fluorescence assay (Live/Dead Assay, Invitrogen) was performed. With this assay, the DNA in fragmented nuclei of dead cells is colored red by membrane impermeable propidium iodide when excited by light of 488 nm wavelength, whereas the DNA in all cells is colored blue by membrane permeable Hoechst 33342 dye when excited at 350 nm. After incubation, cells were washed with PBS and incubated with 2.0 mg/mL propidium iodide and 1.0 mg/mL Hoechst 33342 for 20 minutes at 37 C. Subsequently, cells were analyzed with an epifluorescence microscope (Leica DMR). For each cornea, 4 representative areas of the corneal endothelium were selected and photodocumented digitally (Leica Image Capturer). Subsequently, labeled nuclei were counted in fluorescence photomicrographs. Dead cells (red) were expressed as the percentage of total nuclei in the field (blue). The data are based on counts in 3 experiments performed in duplicate.
Statistical Analysis All results were expressed as the percentage of the control (control was set as 100%), and the mean was taken for each experimental group. The error was expressed as the standard error of the mean. To discriminate between 2 univariate groups, the Wilcoxon-Mann-Whitney test was applied. To differentiate whether the IOL coating with APC/N-APC or the IOL material itself had the larger effect on the total variance of results, a factor analysis was performed and the total explained variance was calculated. For multivariate analysis, analysis of variance with a least-significant-difference post hoc test was calculated. In addition, a factor analysis using the explained total variance was performed to discriminate between the effect of APC/N-APC exposure and the effect of the material. All statistical testing was performed using SPSS software (versions 17.0 and 19.0, SPSS, Inc.). For all analyses, a P value less than 0.05 was considered significant with a 95% confidence interval (CI).
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Figure 1. Cell number (mean G SD) after incubation of the human LECs with an N-APC– or APC-coated IOL over 5 days as determined by the tetrazolium-dye reduction assay. The results for the 6 foldable IOLs used for coating (Table 1) are shown. All coated IOLs vary significantly from the control except those marked with an * (IOL 1 and IOL 2 with N-APC coating) (APC Z alkylphosphocholine; Co Z control; IOL Z intraocular lens).
Figure 2. Effect of IOL material. Cell number (mean G SD) after incubation of the human LECs with a coated IOL over 5 days as determined by the tetrazolium-dye reduction assay. All coated IOLs varied significantly from the control (PZ.001) except the one marked with an * (hydrophobic IOLs coated with N-APC [PZ.43]) (APC Z alkylphosphocholine; Co Z control; H-Phil Z hydrophilic; H-Phob Z hydrophobic; IOL Z intraocular lens; Silic Z silicone).
RESULTS
N-APC: PZ.43, mean difference in % control, 3%, 95% CI, 5%-12%). The APC/N-APC treatment explained 50% of the variance in cell number, and the IOL material explained 50%. This indicates a strong interaction between APC/N and APC and the optic material during the coating process and qualifies hydrophilic IOLs as a delivery system for this compound. Intraocular lens 3 (hydrophilic acrylate with hydrophobic surface) was effective in controlling cell proliferation after coating with APC and N-APC, although N-APC was not effective on pure hydrophobic acrylates (Table 1 and Figures 1 and 2). Alkylphosphocholine was tolerated well by human corneal endothelial cells up to a maximum concentration of 1 mM over 24 hours (Figure 4). N-APC was not part of this assay due to the limited availability of human donor corneas and its inferiority to APC in coating hydrophobic IOLs. Thus, the APC with the better efficiency was selected, showing cell death at the maximum concentration of 10 mM.
Foldable hydrophobic acrylic, silicone, and hydrophilic acrylic IOLs were successfully coated with N-APC and APC without linker molecules such as PGLA (Table 1 and Figures 1 to 3). Alkylphosphocholinecoated IOLs reduced human LEC proliferation significantly (Figures 1 and 2) and remained transparent (Figure 3). Over all measurements, both alkylphosphocholines (APC and N-APC) attenuated cell proliferation after coating compared with the uncoated control IOLs of the same lot (APC: PZ.001, mean difference, 50% of control; 95% CI, 40%-60%; N-APC: PZ.001, mean difference, 37% of control; 95% CI, 28%-47%) (Figure 1). N-APC and APC varied significantly in the effect on growth inhibition compared with the control (PZ.03; mean difference, 13% of control; 95% CI, 1%-24%), whereas APC seemed slightly more efficient. For hydrophobic acrylic IOLs, the N-APC coating did not reduce cell proliferation significantly (IOL 1: PZ.34; IOL 2: PZ.18) (Figures 1 and 2). Regarding the optic material assessed, alkylphosphocholine coating with APC or N-APC inhibited cell growth significantly (control – hydrophobic: PZ.01, mean difference in % control, 9%, 95% CI, 2%-16%; control – hydrophilic: PZ.001, mean difference in % control, 70%, 95% CI, 65%-76%; control – silicone: PZ.001, mean difference in % control, 21%, 95% CI, 12%-30%). From these data it can be derived that hydrophilic IOLs coated with alkylphosphocholines had the largest effect on cell proliferation whereas N-APC-coated hydrophobic acrylic IOLs had the smallest effect (control – hydrophobic
DISCUSSION Posterior capsule opacification degrades visual function by reducing visual acuity and by increasing intraocular light scatter and straylight.26,27 The IOL material is an important factor influencing the optical quality of vision and the accuracy of the sharpedged design during manufacture.28,29 Hydrophilic IOLs were shown to display more forward scatter than hydrophobic IOLs.29 Moreover, they are associated with poorer PCO performance.8 Thus, IOL
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Figure 3. Effect of APC-coated hydrophilic acrylic IOL on human LEC proliferation in cell culture compared with an uncoated control IOL of the same lot after 2 days (left) and 5 days (right) in cell culture. Above: Control IOL. Below: Alkylphosphocholinecoated hydrophilic acrylic IOL (APC Z alkylphosphocholine; IOL Z intraocular lens) (phase contrast microscope; original magnification 100).
coating with the aim of PCO prevention could be an approach to avoid these complications in future cataract surgery. Current research supports this concept because it has been shown that cell adhesion on the IOL surface was attenuated 1 month after implantation of a pharmacologically modified IOL in the rabbit eye.30 In this study, we selected APCs, small molecules with amphiphilic properties, as new coating agents and evaluated their in vitro efficiency on human LEC proliferation and their safety in an organ model of human donor corneas. We found that APCs are suitable coating agents for IOLs of all materials and are independent of linker compounds, such as PGLA.13 Coated IOLs were transparent and controlled human LEC proliferation in cell culture over several days. However, the IOL material seems to strongly affect the ability to coat with APC or N-APC. The maximum antiproliferative effect as a percentage of the control was achieved by APC-coated hydrophilic acrylic IOLs. It can be assumed that APCs can integrate into the water compartment of the hydrophilic IOL material because their amphiphilic molecular structure forms a monomolecular film on the IOL surface. This applies even to hydrophilic acrylic IOLs with hydrophobic surface properties (IOL 3). After coating, the performance of IOL type 3 was very similar to that of pure hydrophilic acrylates (IOLs 4 and 5) and not like the performance of hydrophobic acrylic IOLs. The N-APC coating elucidates this nicely because N-APC was not successful in inhibiting proliferation when used as a coating on hydrophobic acrylic IOLs (IOLs 1 and 2) but was effective when applied to IOL 3. A possible explanation for this might be
a slight difference in the chemical structure between APC und N-APC. N-APC has a carbon chain that is 4 carbon atoms shorter than APC, which makes N-APC more hydrophilic than APC. This seems to be a disadvantage because N-APC was not as effective as APC in LEC proliferation inhibition. It can be hypothesized that because of its increased hydrophilic properties, N-APC does not adhere to the IOL surface as effectively and is cleared faster from the IOL surface than APC. The hydrophilic IOLs used in this study had a water content of 25% to 28%. This makes APCs a valuable tool for PCO prophylaxis for this type of IOL in particular. Other pharmacologic substances are too hydrophilic, such as mitomycin-C (MMC) and 5-fluorouracil, or too hydrophobic, such as Triton X-100, to be considered IOL coating agents without linker molecules. Also, in previous studies,31,32 MMC and Triton X-100 were dependent on the Perfect Capsule System (Milvella Ltd.) to prevent anterior chamber toxicity. Nevertheless, human LECs were not completely eradicated after incubation with APC-coated or N-APC–coated IOLs. At the same time, it is questionable whether complete eradication should be the aim of PCO prevention. A capsular bag devoid of cells might be unstable, resulting in IOL decentration or even dislocation. In addition, compounds that achieve clarity of the capsular bag, such as actinomycin D when combined with cycloheximide and dispersed in hyaluronic acid, induced significant corneal toxicity with opacification in a primate model of PCO after lens refilling.33 Thus, complete eradication of PCO might not be the ultimate goal of PCO prophylaxis but rather a modification of the cellular response. In addition to the antiproliferative effect on human LECs, other
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higher than the half maximum inhibitory concentration determined for this compound on human LECs (0.1 mM).24 Human donor corneas are a valuable model for assessing biocompatibility in the anterior segment. This reflects the rise in the quality of ex vivo experimental approaches in current ophthalmologic research, such as the capsular bag model described by Dawes et al.34 In addition, we previously found good biocompatibility of alkylphosphocholines after intravitreal injection in the rabbit eye and rat eye.22,35 This is an important aspect for future clinical considerations because release of the molecule by diffusion or after rupture of the capsular bag cannot be completely avoided. WHAT WAS KNOWN Human LEC proliferation is the target of numerous pharmacologic approaches to prevent PCO. However, corneal biocompatibility of effective substances is often compromised and a hurdle to further clinical assessment. The IOL itself has been suggested as a drug-delivery system for pharmacologic substances. The influence of different IOL materials during the coating process has not been evaluated. WHAT THIS PAPER ADDS In this in vitro study, APCs were effective as IOL coating agents in the prevention of human LEC proliferation. Alkylphosphocholines were well tolerated by the corneal endothelium after assessment in an organ culture model of human donor corneas. Hydrophilic acrylic IOLs with a hydrophobic surface behaved like pure hydrophilic IOLs in cell culture. Hydrophilic IOLs might serve as a future drug-delivery system for APCs with the aim of PCO prevention.
Figure 4. Biocompatibility of APC after incubation of the corneal endothelium of a human donor cornea over 24 hours under standard cell-culture conditions (APC Z alkylphosphocholine; PBS Z phosphate-buffered saline).
factors such as the biocompatibility and physicochemical properties of the coating agent must be considered before clinical application as an IOL coating agent should be pursued. In this study, we found that APC concentrations of 1 mM were well tolerated by human corneal endothelial cells over 24 hours in an organ model of human donor corneas. This concentration is significantly
REFERENCES 1. Apple DJ, Solomon KD, Tetz MR, Assia EI, Holland EY, Legler UFC, Tsai JC, Castaneda VE, Hoggatt JP, Kostick AMP. Posterior capsule opacification. Surv Ophthalmol 1992; 37: 73–116 2. Mastropasqua L, Toto L, Falconino G, Nubile M, Carpineto P, Ciancaglini M, Di Nicola M, Ballone E. Longterm results of 1 CUÒ accomodative intraocular lens implantation: 2-year followup study. Acta Ophthalmol Scand 2007; 85:409–414. Available at: http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0420.2006. 00866.x/pdf. Accessed October 20, 2012 3. Vock L, Menapace R, Stifter E, Georgopoulos M, Sacu S, €hl W. Posterior capsule opacification and neodymium:YAG Bu laser capsulotomy rates with a round-edged silicone and a sharp-edged hydrophobic acrylic intraocular lens 10 years after surgery. J Cataract Refract Surg 2009; 35:459–465
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First author: Kirsten H. Eibl, MD, FEBO Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany