3.2.2. Mechanical Properties of 3D Printed Rings

To enable successful implantation in the esophagus, the mechanical properties of the FTS-eluting esophageal rings were tested and optimized. In general, a post-fabrication UV or thermal curing process is implemented in 3D printing in order to improve the mechanical properties of 3D printed parts [63–68]. These post-fabrication curing treatments lead to improved compression strength and elastic modulus of the 3D-printed parts [69–71]. The effect of post-fabrication UV curing on the mechanical properties of placebo rings fabricated without (PCL700-DMA) or with diluent (PCL700-DMA/HEMA) was investigated. UV-treated and non-UV-treated rings were compressed to a distance of 10, 20, and 50% of their outer diameter. The load at these percent compressions in Newtons (N) was reported (Table 4). All measurements were done in triplicates. Results showed that using both resins (PCL700-DMA, PCL700-DMA/HEMA), rings exposed to UV treatment post fabrication had significantly higher compression forces at all compression distances compared to rings that did not undergo post-fabrication UV treatment (Table 4).



The Young's modulus measures the resistance of a ring to elastic deformation under a specific load and its ability to recover its original shape once the stress force is removed. Results showed that rings exposed to UV curing post fabrication were stiffer and had a higher Young's modulus (1736.33 ± 34.8 Pa for PCL700-DMA and 1830.9 ± 60.3 Pa for PCL700-DMA/HEMA) compared to 8.61 ± 3.5 Pa (PCL700-DMA) and 10.93 ± 1.2 Pa (PCL700-DMA/HEMA) for rings that did not undergo post-fabrication UV cure (Supplementary Table S1). Collectively, these results demonstrate that UV curing post fabrication significantly improved the mechanical properties of 3D printed rings. The increase in compression force and Young's modulus was attributed to an increase in crosslink density of the polymer matrix upon exposure to UV and formation of a tighter network. This effect was confirmed by determining the gel fraction and percent (%) swelling of the rings in organic and aqueous solvents. Rings that were exposed to a UV curing treatment post fabrication had higher gel fraction and lower percent swelling compared to rings that did not undergo UV curing (Supplementary Table S1) due to higher crosslink density of the polymer matrix as a result of UV exposure. *Polymers* **2021**, *13*, x FOR PEER REVIEW 18 of 26

> To assess the stability of FTS when exposed to post-fabrication UV curing, in vitro release kinetics of fluticasone from UV cured rings was investigated (Figure 2). Results showed that rings exposed to a post-fabrication UV cure had slightly higher burst of FTS in the first 24 h (4%, 1179.9 µg) and higher zero-order release rate (370 µg/day) compared to non-UV cured rings (Table 5). These results showed that UV curing improved the mechanical properties of rings and resulted in higher release rates of FTS in vitro.

**Figure 2.** In vitro release kinetics of FTS from non-UV- and UV-cured rings printed with PCL700-DMA resin formulation. Rings were incubated in PBS at 37 ◦C for 30 days and sample aliquots (1 mL) were collected and analyzed by HPLC analysis. All error bars represent standard deviation for *n* = 3.

**Figure 2.** In vitro release kinetics of FTS from non-UV- and UV-cured rings printed with PCL700-DMA resin formulation. Rings were incubated in PBS at 37 °C for 30 days and sample aliquots (1 mL) were collected and analyzed by HPLC

**Table 5.** Total amount of FTS in rings normalized to the weights of rings (n = 3), release rate of FTS at zero order kinetics

**FTS burst in 24h (%)**

32.11 ± 1.12 2.98 ± 0.24 958.17 ± 74.45

0.41 1179.9 ± 180.8

32.77 ± 1.11 3.59 ±

**FTS burst in 24h (**μ**g)**

**FTS zero order release rate (**μ**g/day**

282.0

370.0

(R2 = 0.99)

(R2 = 0.98)

analysis. All error bars represent standard deviation for *n* = 3.

**(mg)**

**Amount of FTS in rings** 

(μg/day).

**Rings** 

PCL700-DMA

PCL700-DMA

(non-UV)

(UV)


**Table 5.** Total amount of FTS in rings normalized to the weights of rings (n = 3), release rate of FTS at zero order kinetics (µg/day).

3.2.3. Effect of Drug Incorporation Process

Fabrication of drug-loaded rings can be done by either incorporating drug(s) in the initial resin formulation (termed pre-loading, Figure 1A) or by adding drug(s) to a 3D printed ring post fabrication (termed post-loading, Figure 3A). We sought out to investigate the effect of FTS incorporation step on in vitro release kinetics from rings. A number of solvents were screened to determine a suitable solvent for post-loading FTS, and acetone was selected based on high solubility of FTS and ease of removal. Placebo rings were post-loaded with FTS by incubating them in a near saturated solution of FTS in acetone (20 mg/mL; 50 mL) at RT for 24 h. *Polymers* **2021**, *13*, x FOR PEER REVIEW 20 of 26

**Figure 3.** Effect of drug incorporation process onto drug release kinetics of rings printed with the PCL700-DMA resin formulation. (**A**) A pictorial representation of post-loading of FTS into 3D printed placebo ring. (**B**) In vitro release kinetics of FTS from pre-loaded (non-UV- and UV-cured rings) and post-loaded (non-UV- and UV-cured rings) printed using the PCL700-DMA resin formulation. Rings were incubated in PBS at 37 °C for 30 days and sample aliquots (1 mL) were collected **Figure 3.** Effect of drug incorporation process onto drug release kinetics of rings printed with the PCL700-DMA resin formulation. (**A**) A pictorial representation of post-loading of FTS into 3D printed placebo ring. (**B**) In vitro release kinetics of FTS from pre-loaded (non-UV- and UV-cured rings) and post-loaded (non-UV- and UV-cured rings) printed using the PCL700-DMA resin formulation. Rings were incubated in PBS at 37 ◦C for 30 days and sample aliquots (1 mL) were collected and analyzed by HPLC analysis. All error bars represent standard deviation for *n* = 3.

**FTS burst in 24h (%)**

UV) 6.84 7.94 ± 0.50 550.0 ± 1.0 90.0 (R2 = 0.99)

Pre-loaded (UV) 11.70 3.12 ± 0.13 450.0 ± 7 0.1 237 (R2 = 0.978)

Pre-loaded (non-UV) 7.13 3.99 ± 0.11 280.0 ± 70.0 50.0 (R2 = 0.99)

**FTS burst in 24h (**μ**g)**

**FTS zero order release rate (**μ**g/day**

and analyzed by HPLC analysis. All error bars represent standard deviation for *n* = 3.

**FTS loading per ring** 

**(mg/g)**

(μg/day).

**Rings**

Post-loaded (non-

Results demonstrated that rings post-loaded with FTS exhibited ~2-fold increase in burst release within the first 24 h (~8%, 550 µg) compared to rings pre-loaded with FTS (~4%, 280 µg). Rings post-loaded with FTS also exhibited greater zero-order release kinetics over 30 days, with 90 µg/day compared to 50 µg/day for pre-loaded rings (Figure 3B, Table 6). These results demonstrate the ability to fine-tune drug release kinetics using different drug loading processes.

**Table 6.** Total amount of FTS in rings normalized to the weights of rings (n = 3), release rate of FTS at zero order kinetics (µg/day).


Similar to the pre-loaded rings, the effect of post-fabrication UV curing on fluticasone release rate in post-loaded rings was investigated. 3D-printed placebo rings were UV cured using the method described above and subsequently incubated in a near saturated solution of fluticasone in acetone at RT for 24 h. Results showed that post-loaded UV-cured rings exhibited a lower burst release of FTS in the first 24 h (~3%, 420 µg) but faster zero order release rates ~270 µg/day over 30 days compared to non-UV cured post-loaded rings (Table 6). For both drug loading strategies, fluticasone exhibited a sustained zero-order release profile over 30 days, demonstrating the potential use of 3D printed rings as a long-acting esophageal drug delivery device.
