BAPN release profile and directional release

PLGA scaffolds (5 mm × 5 mm) were weighed and placed in 1.0 mL of phosphate-buffered saline (PBS) and incubated at 37°C. PBS was collected and replaced with fresh PBS at specified timepoints. The collected PBS was stored at −20°C to determine cumulative BAPN release using high-performance liquid chromatography (HPLC).

For HPLC, a gradient flow (0.5 mL/min) of 60% H2O and 40% methanol was used as the eluent with a C18 column. BAPN peaks were observed at a retention time of 1.80 min using a UV detector (220 nm). The concentration of BAPN was calculated based on a standard calibration curve of the peak integrations. Percent release was calculated as the ratio of the mass of BAPN in the supernatant to the initial mass of BAPN in the PLGA scaffold.

Directional release was determined using a Franz Cell diffusion apparatus (PermeGear, Hellertown, PA). Methylene blue was used as a model compound due to its hydrophilicity and similar molecular weight. Bilayer scaffolds (1 cm × 1 cm) were flipped on either the perivascular (PLA) or periadventitial (PLGA) side and placed across the orifice of the receptor chamber before clamping the donor chamber. Both chambers were filled with PBS, and the PBS from the donor chamber was collected replaced at specified timepoints. The methylene blue concentration was measure using a plate reader at 560 nm. Monolayer scaffolds flipped on either side were used as controls.