Drug release studies

Passive release. Seven individual vials containing DECON particles (1 mg/ml) diluted in 1 ml of MEM were incubated at 37°C for up to 7 days. Every day, a single vial was removed and centrifuged at 14,000g for 15 min. The supernatant (2 μl) was used to analyze the ACV concentration using a UV spectrophotometer, as mentioned above. MEM was used as blank for these studies.

Triggered release. For active drug release studies, purified virus (sucrose gradient purification) and cell debris (50 million HCEs sonicated in 1 ml of MEM) were used. Increasing concentrations of purified virus and cell debris were added to DECON (1 mg/ml) in MEM and incubated for a period of 15 min at 37°C before the mixtures were spun at 14,000g, and the supernatants were analyzed using the UV spectrophotometer for the presence of ACV. In parallel, DECON (1 mg/ml in MEM) was heated to 90°C using a preheated heat block for a period of 5 min before the samples were centrifuged and the supernatant was analyzed for ACV. In addition, DECON (1 mg/ml in MEM) was sonicated using a probe sonicator (Fisher Scientific, USA) at 30% amplitude for 40 s separated by a 5-s pause. The sample was then centrifuged at 14,000g before the supernatant was analyzed for ACV using a UV spectrophotometer.

Sustained release. A total of 5 × 10⁴ PFU (plaque-forming units) of purified virus (18) or equivalent cell debris was added to 100 μl of DECON (1 mg/ml in MEM) and incubated at 37°C for a period of 24 hours. At set time intervals, 2 μl of supernatant from the vial was removed and analyzed for the concentration of ACV using a UV spectrophotometer.