(652f) Using Solid-State NMR As a Means to Quantify Protein Integration in Hydrogel Contact Lens Materials

Contact lenses are a ubiquitous biomaterial, with over 140 million users worldwide, and the majority of wearers choose hydrogel contact lenses to meet their vision correction needs. These lenses are placed in the tear-film of the user and are subject to a variety of proteins of varying size and charge that may diffuse into the hydrogel. Methods to quantify the amount of protein integrated within the hydrogel currently require either (1) efficient extraction of the protein from the hydrogel before quantification (e.g. by high performance liquid chromatography (HPLC)) or (2) use of a radio-labeled or fluorescently tagged protein to quantify the amount of protein within the hydrogel in situ.

We chose lysozyme as a model tear-film protein in this work due to its high concentration in the tear-film (approximately 2 mg/mL), small size relative to other tear-film proteins (14.4 kDa), and cationic nature (pI = 11.4) in the tear-film (pH = 7.4), which enable the protein to be integrated into an anionic, high water (58%) hydrogel, such as etafilcon A (i.e. ACUVUE® MOIST, Johnson & Johnson Vision Care, Inc.). In this work, we demonstrate the use of solid state cross-polarization magic angle spinning ¹³C nuclear magnetic resonance (CPMAS NMR) to quantify lysozyme integration within etafilcon A, without the need to alter or tag the materials.

After a 72 hour incubation of etafilcon A in lysozyme (2 mg/mL in phosphate buffered saline) at 37°C, lenses were flash frozen and lyophilized prior to CPMAS NMR. We determined the lysozyme to contribute 9-11 wt% of the carbon mass within the dry etafilcon A material (3.6-4.4 carbon wt% within the hydrated material). This determination resulted from a direct spectral subtraction of the clean hydrogel spectrum from the lysozyme-fouled hydrogel spectrum. The difference spectrum was a close match to the pure lysozyme spectrum, which was integrated and divided by the area of the fouled hydrogel spectrum. This quantity of lysozyme is in close agreement with other in vitro quantification methods.

The use of CPMAS NMR has proven to be an efficient method for quantifying tear-film component integration in contact lenses and enables simultaneous inspection of all the molecular carbon contributions in the material. Additional applications of this technique include measuring ex vivo contact lenses for several types of tear-film deposits and using isotope-labeled tear-film components in vitro for compounds with a lower affinity for integration in hydrogels.