(408d) Lysozyme Transport in p-HEMA Hydrogels

Protein binding in hydrogels impacts their performance in several biomedical applications including contact lenses. The tear fluid contains several proteins that can diffuse into the contact lenses leading to a number of problems including discomfort, reduced visual acuity, dryness and reduced lens life. Protein binding to lenses may also facilitate bacterial adhesion and subsequent infections of the eye, as well as inflammation and adverse immunological responses. The talk is aimed to obtain a detailed quantitative understanding of lysozyme uptake in p-HEMA hydrogels cross-linked and copolymerized with EGDMA (Tefilcon lenses). Lysozyme, which is the most abundant tear fluid protein, is a small compact globular protein with a slightly ellipsoidal shape (45 х 30 х 30 Å). Pure p-HEMA hydrogels have a pore size of approximately 95 Å; therefore, it is likely that lysozyme could diffuse into the pores of pure p-HEMA and is also expected to adsorb on the polymer chains in the gel. In particular, we aim to determine the diffusivity of lysozyme in the hydrogel and its binding isotherm. The key and unique aspect of our approach is to measure the uptake dynamics for gels of several different thicknesses. The diffusion time scale increases with the square of the thickness. The p-HEMA hydrogels of thicknesses 18, 30 and 50μm were synthesized by free-radical polymerization with photoinitiation with crosslinker ethelene glycol dimethacrylate (EGDMA). Lysozyme solutions were prepared at various concentrations (0.1, 0.2, 0.5, 0.8, 1.2, 1.5, and 3.0 mg/ml) using Dulbecco's phosphate buffered saline (PBS), pH 7.4.The gels were soaked in 3.5 ml of a lysozyme-PBS solution until equilibrium was reached. The lysozyme concentration in the fluid was monitored by measuring the absorbance spectrum of the solution over the wavelength range 252-321 nm with a UV?vis spectrophotometer. Once equilibrium was reached in the lysozyme uptake experiments, the lysozyme solution was removed and replaced with 3.5 ml of PBS, marking the beginning of the release experiments. The lysozyme concentration in the PBS was analyzed in the same way as in lysozyme uptake experiments. Uptake experiments were also performed on gels with very high degree of crosslinking Analysis of results reveals that the transport process is limited by diffusion of protein. The facts that values of partition coefficient (K) are significantly high and 1/K is linear in equilibrium fluid concentration clearly suggest that protein is getting adsorbed on the polymer chains. The release experiments showed that the protein adsorption was reversible. Uptake was negligible for gels with very high degree of crosslinking indicating that lysozyme does diffuse in the gel for smaller cross-linking and that surface adsorption is negligible. A lysozyme transport model with Fickian diffusion was used and the transient concentration profiles were fitted to the model to obtain protein diffusivity in the polymer. Adsorption coefficients were found using a Langmuir binding isotherm. The experimental data for adsorption fitted to the model with reliable parameters. The model validity was proven by predicting the release experiments without any additional parameters. The results of this study clearly show that a large fraction of the protein in the gel is adsorbed to the polymer. The mathematical model developed here characterizes lysozyme transport across these gels. The model developed could assist in the development of hydrogel contact lens materials that exhibit minimal protein binding and in designing cleaning regimens for enzyme based protein removal from the lenses It is also noted that while this talk focuses on a specific type of hydrogel and protein, the model provided here is expected to be applicable to other protein-hydrogel systems. Thus, the results and methods described here could be very useful in understanding the mechanisms and address issues related to protein binding in several other bio-materials.