Tetrahydrofuran

Solvents for peptide synthesis and tetrahydrofuran for PySeSePy synthesis were purchased from Fisher Scientific (Pittsburgh, PA). Fmoc-Cys(Acm)-OH, 2-chlorotrityl chloride resin SS (100–200 mesh), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) for solid-phase synthesis were purchased from Advanced ChemTech (Louisville, KY). Fmoc-Cys(Mob)-OH was purchased from NovaBiochem (Burlington, MA). All other Fmoc-amino acids were purchased from RSsynthesis (Louisville, KY). L-Ascorbic acid (sodium salt), Triisopropylsilane (98%) and thioanisole (99%) scavengers, as well as selenium powder (200 mesh), 2-bromopyridyine (99%), and sodium borohydride for the synthesis of PySeSePy, were purchased from Acros Organics (Pittsburgh, PA). Guanylin was purchased from CPC Scientific (Sunnyvale, CA). All other chemicals were purchased from either Sigma-Aldrich (Milwaukee, WI) or Fisher Scientific (Pittsburgh, PA). The HPLC system is from Shimadzu with a Symmetry® C18 −5 mm column from Waters Corp. (Milford, MA) (4.6 × 150 mm). Mass spectral analysis was performed on an Applied Biosystems QTrap 4000 hybrid triple-quadrupole/linear ion trap liquid chromatograph-mass spectrometer (SciEx, Framingham, MA).

Nanofiber and microfiber PCL scaffolds were created by electrospinning using a custom made apparatus. PCL (80 kDa, Sigma-Aldrich, St. Louis, MO) was dissolved at 40°C overnight in a 1:1 mixture of N,N-dimethylformamide and tetrahydrofuran (Fisher Scientific, Pittsburgh, PA) at a concentration of 11.5% and 22% w/v for nanofibers and microfibers, respectively. 0.06% w/v sodium chloride was added to the 11.5% solution to increase conductivity and improve homogeneity of fiber diameter. The polymer solution was drawn into a 20 ml syringe connected to a stainless steel needle. A syringe pump (Harvard Apparatus, Holliston, MA) was used to deliver the solution at a constant rate of 2.0 ml/hr. Microfibers were electrospun using a 12-inch, 18G blunt-ended needle charged to 8 kV with a high voltage power supply (Gamma High Voltage Research Inc., Ormond, FL) at a distance of 23 cm from the collector. For nanofibrous scaffolds, a 4-inch, 22G blunt-ended needle was charged to 13.5 kV at a distance of 15 cm from the collector. The collector was a custom-made grounded aluminum mandrel rotating at 0.75 m/s. Additionally, aluminum shields were placed on either side of the mandrel and plate and charged to either 10 kV or 2 kV for nanofibers and microfibers, respectively, in order to guide the fibers onto the grounded surface.

Nanofibrous scaffolds composed of poly(ε-caprolactone) (PCL, 80 kDa) were created via electrospinning (5–7). Briefly, PCL was dissolved in equal parts dimethylformamide and tetrahydrofuran (Fisher Chemical) at 40°C (14.3% w/v) and driven through an 18G needle charged to +12kV (Gamma High Voltage Research Inc.) at 2 mL/hr. The needle was situated ~150 mm from the collection mandrel. Separate collection mandrels were designed to collect fibers along the long-axis of a rotating cylinder or the end of a rotating disk (~10 m/s surface velocity) to collect fibers with uniform alignment in a single direction (linearly aligned) or circumferentially aligned, respectively (Fisher, et al., 2013 (link)). Mats of ~250µm thickness were formed, and from these, individual scaffolds (5 mm in width × 40 mm in length) were isolated (Fig. 1A). From linearly aligned mats, scaffolds were produced such that the long axis was parallel (0°) or perpendicular (90°) to the fiber direction. Additional scaffolds were produced such that the midpoint of the long-axis of the scaffolds was tangent to the radial direction of the fibrous mat, capturing fibers aligned in a circumferential manner (C), as previously described (Fisher, et al., 2013 (link)).