Freezone 4.5 plus

Five replicates of E. faecalis OG1RF wild type, EFC3C and EFC3Py were grown for 12 h in BHI at 37 °C. Overnight suspensions were centrifuged at 9000 g for 2 min and the resultant cell pellets of all three E. faecalis strains were freeze dried for 12 h at 80 °C using a Freezone 4.5 plus (Labconco, USA), weighed and then subjected to the Bligh and Dyer method of whole cell lipid extraction. Fatty acid analyses were carried out by the Identification Service of DSMZ, Braunschweig, Germany.

The three extracts were individually dried in a rotary evaporator and the drying completed by lyophilization for 24 h in a Labconco Freezone 4.5 plus instrument. The methanol sample was then suspended in 500 µL methanol-d4 and the dichloromethane and hexane extracts directly in CDCl₃. The NMR experiments were performed on a Fourier 300 Bruker^® 9.4 Tesla (300 MHz for hydrogen frequency and 75.48 MHz for carbon frequency) spectrometer equipped with a 5 mm internal diameter BBI probe, with reverse detection and field gradient coils in the coordinate. The chemical shifts were determined relative to TMS as the internal standard and are expressed as δ values (ppm), with coupling constants reported in Hz. The spectra were acquired for the CDCl₃ solutions using 5 mm quartz tubes at 303 K. For all analyses, 64 scans were used. NMR data acquisition and processing were performed using Bruker TopSpin™ software.

The ELRs were^{27 (link)} dissolved in 10% (v/v) Ethanol (1015, Solveco) in PBS at a concentration of 64.28 mg/ml for the HE5-C and 35.71 mg/ml for the HRGD6-N3 and left at 4 °C overnight. MilliQ water (Millipore Reference, Merck) was sprayed using a spray bottle on top of liquid nitrogen and sieved through strainers with mesh sizes 500, 200, and 100 µm (Pluristrainer, 43-50500-03, 43-50200-03, 43-50100-51, pluriSelect Life Science) to collect ice crystals of desired size. Ice crystals were added to a mold (using a syringe (1 ml syringe, 329654, BD Bioscience)). Ice crystal and dissolved ELR were left in a cryostat (HM500M, Microm) at −5 °C to equilibrate. The HE5-C and HRGD6-N3 ELRs were mixed at a volume ratio of 1:1 by pipetting using cold tips, then this mixture was added to an equal volume of ice crystals and incubated at −5 °C for 15 min to fully form the cross-linkage, and then moved to −80 °C overnight. The samples were extracted from the mold and the edges were removed while being kept cold, and then samples were freeze dried (Freezone 4.5 Plus, Labconco) overnight. Hydrogels were made by dissolving each ELR in PBS at the same concentrations as for cryogels and mixed at a 1:1 volume ratio using cold tips. The mixture was pipetted into the desired molds and incubated at 4 °C for 15 min followed by 10 min at 37 °C to complete hydrogel formation.

The solvents used in the analytical and preparative systems were MilliQ water (Solvent A) and acetonitrile (Solvent B), both modified with 0.1% TFA. The crude and purified peptides were analyzed on an ACQUITY UPLC H-class system with a photodiode array (PDA) detector (Waters, Milford, MA, USA) equipped with an ACQUITY CEH C18 UPLC column (Waters, 2.1 × 50 mm, 1.7 µm). A gradient of 0–50% Solvent B over 30 min with a flow rate of 1 mL/min was used, and detection was set at 200–500 nm. The crude peptides were purified to >95% on a XSelect CSH C18 OBD prep column (19 × 250 mm, 5 µm; Waters, Milford, MA, USA) installed in an AutoPurificayion System (Waters). A default gradient of 10–40% Solvent B over 30 min with a flow rate of 20 mL/min was used, but it was adjusted as required. The detection was set at 200–500 nm. The molecular weight of the peptides was confirmed on the Xevo G2 Q-TOF with ACQUITY UPLC I-Class system (Waters). The purified peptides were then freeze-dried (Labconco FreeZone 4.5 Plus, Kansas City, MO, USA) as TFA-salts and stored at −20 °C until further use.

Briefly, 4 mg of Fitc-Dextran was added to 500 µL ddH₂O and added dropwise to PLGA solution (100 mg of PLGA dissolved in 4 mL DCM). The Fitc-dextran/PLGA solution was then sonicated for 60 s at 80% amplitude using Fisher scientific ultrasonic homogeniser CL-18 to form a w/o emulsion. The w/o emulsion was added dropwise to a 1.25% PVA solution (15 mL) and sonicated for 2 min at 80% amplitude to form a w/o/w emulsion. The w/o/w emulsion was placed onto a magnetic stirrer overnight in the dark at RT to allow DCM evaporation. The emulsion was centrifuged at 18,809× g at 4 °C for 30 min using Sigma^® centrifuge 3–30 K. The supernatant was aspirated and stored at −20 °C for encapsulation efficiency analysis. The pellet was washed thrice with ddH₂O and resuspended in 5 mL ddH₂O, and placed in −80 °C for 2 h. Once frozen, the NP were placed into Labconco Freezone 4.5 Plus freezedryer, for 48 h. NP were stored at −20 °C until sample sizing and PDI analysis.