*4.2. Peripheral-Simulating Bioreactor*

The peripheral-simulating bioreactor was designed to shorten and twist two harvest porcine carotid arteries subjected to pulsatile flow conditions (Figure 1A). The overall system (46 × 19 × 19 cm) was designed to fit inside of a standard CO2 incubator by arranging the arteries in a parallel configuration. The system utilizes one stepper motor per artery for rotational motion and one stepper motor for the translational motion of both arteries. Custom connectors were machined to mount the arteries to the stepper motors. The motion of the stepper motors was measured using rotary encoders (CUI AMT11, Tualatin, OR, USA) mounted on the shaft of each motor. An Arduino microcontroller with two motor shields was used to control the motors along with an LCD keypad module to provide an intuitive user experience, displaying time and cycles remaining for each test and providing physical inputs to start, stop, input artery length and the duration of testing.

The carotid arteries, positioned within the vascular-simulating bioreactor, were harvested from large pigs (250–350 lbs.) from a local abattoir and transferred in sterile PBS with 1% antibiotic-antimitotic (Gibco, Grand Island, NY, USA). The arteries were then rinsed in sterile PBS in a culture hood and trimmed. Eight-cm-long segments were cut and tied with sutures onto fittings within the *ex vivo* setup. The circulating medium consisted of the system made up of Dulbecco's modified eagle's medium containing 10% fetal bovine serum and 1% antibiotic–antimycotic.

### *4.3. Ex Vivo DCB Testing and Arterial Time Drug Concentration*

Prior to any vascular motion (twisting and shortening), all arteries were subjected to pulsatile flow for 1 h, as defined by a custom LabVIEW program as previously described [42] The pressure was monitored via a pressure catheter transducer (Millar Instruments, Houston, TX). Flow was monitored by an ultrasonic flow meter. Following this pre-conditioning phase, the vessel diameter was measured by ultrasound (Figure 1F,G). Harvested arteries were then treated by either the KOS–paclitaxel-coated balloon or a commercially available DCB (In.PACT Admiral DCB, Medtronic, Santa Rosa, CA, USA). The delivery pressure of the DCB was determined by the manufacturers' specification at a 10–20% overstretch. At timepoints of one hour and three-days, flow was ceased, and the treated portion of the vessel was removed. Excised vessels were flash frozen, stored at −80 ◦C and shipped on dry ice to iC42 Clinical Research and Development (Aurora, CO, USA) for the quantification of arterial paclitaxel. Quantification of arterial paclitaxel levels was performed using a validated high-performance liquid

chromatography (HPLC)-electrospray ionization- tandem mass spectrometry assay (LC-MS/MS) [44–46]. In brief, the LC-MS/MS system was a series 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA) linked to a Sciex 5000 triple-stage quadrupole mass spectrometer (MS/MS, Sciex, Concord, ON, USA) via a turbo-flow electrospray ionization source. The artery tissue samples were homogenized using an electric wand homogenizer (VWR 200, VWR International, Radnor, PA, USA) after the addition of 1 mL of phosphate buffer. Eight hundred (800) μL of 0.2 M ZnSO4 30% water/70% methanol *v*/*v* protein precipitation solution containing the internal standard (paclitaxel-D5, 10 ng/mL) was added. Samples were vortexed for 5 min, centrifuged (16,000, 4 ◦C, 15 min) and transferred into glass HPLC vials. Study samples were diluted as necessary for detector signals to fall within the dynamic MS/MS detector range. One hundred (100) μL of the samples was injected onto a 4.6 × 12.5 mm extraction column (Eclipse XDB C8, 5 μm particle size, Agilent Technologies, Palo Alto, CA, USA). Samples were washed with a mobile phase of 15% methanol and 85% 0.1% formic acid using a flow of 3 mL/min. The temperature for the extraction column was 65 ◦C. After 1 min, the switching valve was activated and the analytes were eluted in the backflush mode from the extraction column onto a 150 × 4.6 mm analytical column (Zorbax XDB C8, 3.5 μm particle size, Agilent). The analytes were eluted from the analytical column using a gradient of methanol/acetonitrile (1/1 *v*/*v*) plus 0.1% formic acid (solvent B) and 0.1% formic acid in HPLC grade water (solvent A). The MS/MS was run in the positive multi-reaction mode and the following ion transitions were monitored: *m*/*z* = 876.6 [M + Na]+ → 308.2 (paclitaxel) and *m*/*z* = 881.6 [M + Na]+ → 313.1 (the internal standard paclitaxel-D5). Paclitaxel tissue concentrations were calculated based on paclitaxel/paclitaxel-D5 peak area ratios using a quadratic regression equation with 1/x weighting. The range of reliable response was 0.5–100 ng/mL tissue homogenate. Inter-day imprecision was less than 15% and accuracy was within 85–115% of the nominal concentrations. There were no significant matrix interferences, carry-over or matrix effects. For more details, please see the aforementioned publications [42,44,45].

### *4.4. Rabbit Injury Model*

This study was approved by the Institutional Animal Care and Use Committee and conformed to the position of the American Heart Association on use of animals in research. The experimental preparation of the animal model has been previously reported [42,46]. Under fluoroscopic guidance, eight anesthetized adult male New Zealand White rabbits underwent endothelial denudation of both iliac arteries using an angioplasty balloon catheter (3.0 × 8 mm). Subsequently, arteries were treated by either KOS–paclitaxel (3.0 × 15 mm), KOS-only balloon (3.0 × 15 mm), paclitaxel-only balloon (3.0 × 15 mm), or an uncoated balloon (3.0 × 15 mm) at a delivery pressure of 8 atm for two minutes. Anti-platelet therapy consisted of aspirin (40 mg/day) given orally 24 h before catheterization, with continued dosing throughout the in-life-phase of the study, while single-dose intra-arterial heparin (150 IU/kg) and lidocaine were administered at the time of catheterization. The animals survived for 7 days and subsequent histological evaluations were performed.
