**2. Results**

### *2.1. Ex Vivo Results*

The non-coated angioplasty balloons were successfully coated with the KOS–paclitaxel mixture (Figure 1D,E). To determine the impact of vascular deformation on DCB retention, both the KOS–paclitaxel DCB and the commercially available DCB were tested under only physiological pulsatile conditions (no twisting or shortening) and physiological pulsatile conditions with vascular deformation conditions. The pulsatile flow conditions consisted of pressures ranging from 70 to 120 mmHg with a mean flow rate of 120 mL/min at 60 beats per minute. The vascular deformation conditions consisted of the artery shortening 10% in the axial direction and twisting of the artery at 15◦/cm. The frequency of the artery twisting and shortening was 0.05 Hz (3 cycles/min). All DCBs were inserted through a 6 Fr sheath into the closed-circulatory system under the physiological pulsatile conditions. The treated sections of the artery were marked during inflation of the DCBs. It is noted that vascular deformation (twisting and shortening) occurred following 4 h of physiological pulsatile conditions of DCB deployment. At timepoints of 1 h and 3 days, the treated section of the arteries were removed and analyzed for arterial tissue paclitaxel concentration (Figure 2). There was a reduction in arterial paclitaxel levels from 1 h to 3 days post-treatment for both the KOS–paclitaxel and the commercially available DCB under physiological pulsatile conditions (3 days—pulse only: KOS-PXL: 17.56 ± 7.19 ng/mg vs. commercial DCB: 24.44 ± 27.03, *p* = 0.96, Table 1). However, under pulsatile and vascular deformation, paclitaxel was significantly retained within the treated artery (3 days—pulse and vascular deformation: KOS-PXL: 14.89 ± 4.12 ng/mg vs. commercial DCB: 0.60 ± 0.26, *p* = 0.018).

**Figure 1.** Schematic illustration of the novel peripheral simulating bioreactor system. (**A**) Two servos (a and b) provide axial deformation and twisting, respectively. Servo (a) moves the harvested arteries (c) forward and backward in a linear motion. Servo b rotates the artery by degrees. The three-way values (d and e) are used to introduce flow and pressure to the artery. The artery is surrounded by matrigel, mimicking external tissue, providing support during vascular movement (insert, black arrow). (**B**,**C**) Harvested arteries under pulsatile (no deformation) and pulsatile conditions with peripheral deformation. (**D**,**E**) Gross photos of an uncoated balloon and a keratose–paclitaxel coated balloon. (**F**,**G**) Diameter measurements performed by ultrasound on the harvested artery.

**Figure 2.** Paclitaxel levels of the drug-coated balloon (DCB) and the keratose–paclitaxel (KOS-PXL)-coated balloon arterial segments undergoing pulsatile flow conditions versus pulsatile flow conditions with vascular motion.

**Table 1.** *Ex vivo* arterial drug concentration measurements of treated arteries.

