3500 mwco

The release profiles of drug from NERs containing fasudil were evaluated using dialysis cassettes (Slide-A-Lyzer, 3500 MWCO, 0.1–0.5 ml, Thermo-Scientific, Waltham, MA) as reported previously (18 (link)). Briefly, the cassettes were first hydrated for 30–60 min with PBS (pH 7.4) and NERs (500 μl) were loaded carefully using a syringe without puncturing the dialysis membrane. To assess whether dialysis cassettes serve as barriers to drug release, we used plain fasudil as control in this set of experiment. Cassettes were then immersed in a beaker containing 100 ml PBS and placed inside an incubator with preset temperature (37°C) along with moderate stirring. At predetermined time intervals, samples were withdrawn from the beaker and the media was immediately replenished with an equal volume of fresh PBS. The amount of drug released was estimated by a UV spectrophotometer as described above.

Silk solution was loaded into cellulose dialysis tubing (3500 MWCO, Thermo Scientific, Rockford, IL) for 48 hours in a fume hood to increase the concentration to 10–12 w/v %. Silk was placed in a CentriVap concentrator (Labconco, Kansas City, MO) to achieve a final concentration of 20–25 w/v %. Cylindrical wax molds (Machinablewax.com, Inc., Traverse City, MI) with 6 wells, 3.30 cm in height and 0.76 cm diameter were parafilm sealed to a 1mm thick wafer bottom (0.76 diameter) (Sigma-Aldrich, St. Louis, MO). Concentrated silk solution was loaded into wax mold wells and submerged in 100 v/v % methanol for 24 h to render insoluble silk gel cylinders. Cylinders were ejected with a plastic punch, dried for 1 h at room temperature. To prevent warping, cylinders were placed back into respective wells and dried in a fume hood for 48 h. Final silk cylinders were machined into desired ear tube dimensions and measured with scanning electron microscopy (SEM) and ImageJ analysis. Non-drug coated tubes were autoclaved with a dry cycle of 25/15 min for steam and drying. Ethylene oxide (ETO) sterilization for drug-coated tubes was achieved with 100 % ETO atmosphere at 55 ºC for 3 h and placed in a hood for 12 h pre-implantation.