Contact lenses were prepared from lotrafilcon B silicone-acrylate prepolymers (Ciba Vision, Johns Creek, GA) using standard lens forms. Lens formation was initiated by addition of a photosensitizer and irradiation under UV light for 1 minute. Newly formed lenses were removed from forms by soaking in an isopropanol water mixture. Dry weights of lenses were 18.8 ± 0.21 mg. To incorporate CSA-120 covalently into lenses, the ceragenin was dissolved in the prepolymer solution at 1.25 or 2.5 weight percent (dry lens weight) before irradiation. To ensure that only covalently attached CSA-120 remained in newly-formed lenses, they were soaked in isopropanol, which swelled the lenses and solubilized unattached ceragenin. CSA-120 was incorporated into lenses at 2.50% and 1.25% relative to the dry mass of the lenses. Lenses containing 2.50% CSA-120 experienced a phase separation of the polymeric materials, which made the lenses partially opaque. Lenses with 1.25% CSA-120 retained their clarity, and this loading was used for further experiments in which the ceragenin was attached covalently to the lenses. To verify that antibacterial activity was due to covalently attached CSA-120 and not residual unattached ceragenin, lenses were soaked in 10% tryptic soy broth (TSB) in PBS for 24 hours at 37°C. The lenses were removed from solution, and the resulting solutions were inoculated with Staphylococcus aureus (106 CFU). Control growth medium was prepared with 10% TSB in PBS, and was inoculated with the same number of bacteria. Samples were incubated for 24 hours, then aliquots were serially diluted and plated on TSB agar. After 24 hours, colonies were counted to determine the number of CFUs present in the test and control samples. To incorporate ceragenins noncovalently into lenses, the ceragenins were dissolved in the prepolymer solution at 1 weight percent (dry lens weight) before irradiation. After removal from lens forms, lenses were stored in PBS (0.5 mL). Elution of CSAs from lenses during removal from lens forms and storage was measured by liquid chromatograph–mass spectrometry (LC/MS), and was estimated as less than 20%.