*3.2. Fluticasone-Eluting Esophageal 3D Printed Rings*

Fluticasone-loaded esophageal rings were fabricated using biocompatible resins. PCL is commonly used for biomedical applications owing to its biocompatibility and robust mechanical properties [56–59]. To fabricate esophageal rings PCL was chemically functionalized to introduce methacrylate groups and enable photopolymerization cross-linking during the 3D printing process via radical-mediated cross-linking reactions. PCL-diol was successfully functionalized with methacrylate groups to produce PCL dimethacrylate (PCL-DMA) using a previously reported method (Supplementary Scheme S1) [51]. The structure of PCL-DMA was confirmed with nuclear magnetic resonance (NMR) analysis by the presence of peaks at δ5.8 ppm (1H) and δ6.1 ppm (1H), corresponding to the vinyl protons and a peak δ1.5 ppm corresponding to methyl groups (3H) of the methacrylate moiety (Supplementary Figure S3).

Fluticasone (FTS) was formulated in the rings via a pre-loading or post-loading process. In the pre-loading process, FTS was dissolved in a liquid resin prior to 3D printing fabrication. The resin formulation composed of a photoinitiator (TPO), UV absorber (BLS), and PCL700-DMA added at predetermined ratios (Table 2) [51]. The mole percent (%) of the various resin components was calculated relative to the methacrylate groups. The resin components were mixed and stirred overnight in an amber glass bottle at room temperature (RT). Resin viscosity in the range of 200 to 600 cP was required in order to allow fabrication accuracy and part shape fidelity using the 3D printing process [60,61]. For photopolymerization-based 3D printing, a light reactive diluent is usually added to reduce the viscosity of the resin [59,62]. Resin formulations used to fabricate the rings had viscosities in the aforementioned printable range. The viscosities of resin formulations were 490 cP and 350 cP for PCL700-DMA resin without diluent and PCL700-DMA with a diluent (HEMA) respectively.


**Table 2.** Composition of resin formulations (PCL700-DMA) and (PCL700-DMA/HEMA) used to 3D print rings.

The saturation solubility of FTS in the PCL700-DMA and PCL700-DMA/HEMA resin formulation was determined by HPLC analysis and quantified at 14.62 ± 0.82 mg/g and 12.91 ± 0.09 mg/g, respectively. To ensure FTS was homogenously dissolved within the resin, sample aliquots (*n* = 4) were collected from different areas in the solution and analyzed by HPLC. The FTS-resin formulation was deemed homogenous if the average concentration of FTS in the sample aliquots (*n* = 4) had a standard deviation ≤5%. The final resin formulations contained 1.4% *w/w* FTS in the resin. FTS-loaded rings were fabricated using 3D printing as described above and the amount of FTS loaded in 3D-printed rings was determined by incubating rings in acetonitrile (ACN, 50 mL) at 37 ◦C for 24 h to extract FTS and quantifying FTS concentration in ACN by HPLC analysis.

The stability of FTS in the resin formulations (PCL700-DMA and PCL700-DMA/HEMA) was determined under three storage conditions of varying temperature and relative humidity (RH) (25 ◦C, 25 ◦C/60% RH, 40 ◦C/75%RH) over 6 months by quantifying FTS concentration in the resin by HPLC analysis. Results showed that FTS was stable under all three storage conditions for up to 6 months (Supplementary Figure S7).

3.2.1. In Vitro Release Studies

In order to achieve the target drug release rate of 1 mg/day over 30 days, rings were loaded with 30 mg of FTS (Figure 1A). The average amount of drug loaded in the rings was 32.11 ± 1.21 mg as determined by drug extraction and HPLC analysis. Results showed FTS had a minimum burst release of ~3% (958 µg) in the first 24 h followed by zero-order kinetics at 282 µg/day over 30 days (Figure 1B, Table 3). The daily drug release from these rings was lower than the target drug release (1 mg/day). An important advantage of utilizing 3D printing is the ability to use a variety of photopolymerizable resins and ring designs. The rate of diffusion and drug release can be controlled by changing the crosslink density of the resin as well as the chemical structure and properties of the polymer. Hydroxyethylmethacrylate (HEMA) was added as a hydrophilic chain extending diluent to study the effect of crosslink density of resin formulation on the release kinetics of FTS (Table 2).

**Table 3.** Total amount of FTS in rings normalized to the weights of rings (n = 3), release rate of FTS at zero order kinetics (µg/day).


Results showed that FTS exhibited a minimum burst release in the first 24 h (~4.5%, 1.31 mg) and slower zero-order kinetics with 207.0 µg/day over 30 days compared to 282 µg/day obtained with FTS rings fabricated without HEMA (Table 3). The slower release kinetics when HEMA was included as a diluent in the resin was attributed to the presence of hydrogen bonding between the hydroxyl groups of HEMA and the carboxyl groups in FTS (Supplementary Figure S8). The hydrogen bonding resulted in a tighter association between the drug and the polymeric network of the resin formulation and thus slower release rate in vitro. Collectively, these results showed that FTS release kinetics could be fine-tuned by changing the resin composition.
