Polypropylene tube

For the intramolecular oxidation
of hLF peptides, peptides were dissolved to 5 mM in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES; Cat No. H4034, Sigma-Aldrich, St. Louis, MO, USA) buffer,
at pH 8.0. For intermolecular disulfide bond formation, peptides were
dissolved in 50 mM HEPES at pH 9.0 at a final peptide concentration
of 50 mM. After dissolution, the pH was adjusted with 0.1 M NaOH.
In either case, 30 μL of peptide solution was added to polypropylene
tubes (Greiner Bio-One, Kremsmünster, Austria, Cat No. 673210)
and incubated in a T300 thermocycler (Biometra, Göttingen,
Germany) for 2 h at 37 °C for the formation of intramolecular
disulfide bonds (mono-hLF), and for 24 h at 37 °C for the formation
of intermolecular disulfide bonds (poly hLF). Samples were diluted
in 20 mM citrate buffer at pH 5.0 or MQ water to stabilize the formed
disulfide bonds.

An aliquot from pyrogen-free water and Tween was used to elute cloacal swabs (N = 144) collected for the analysis on the fecal microbiome, and a small volume (1 mL) was also used for the analysis of endotoxins. Samples were thawed at room temperature and then transferred to 15-mL polypropylene tubes (Greiner Bio-One B.V., Etten Leur, Netherlands) and agitated for 1 h at an end-over-end roller at room temperature. The samples were then centrifuged for 15 min at 1,000 × g, and the supernatant was aliquoted and stored frozen at −20°C. Analyses were conducted in glass tubes, rendered pyrogen-free by heating for 4 h at 200°C. Endotoxin content in the samples was analyzed by testing the samples in a 1:1,000 dilution with pyrogen-free water (B. Braun medical B.V., Oss, Netherlands) in a kinetic chromogenic Limulus amebocyte lysate (LAL) assay (Lonza, Verviers, Belgium), as described previously and in accordance with recommendations by Spaan et al. (2008) (link). Endotoxin content was expressed as endotoxin units per ml (EU/mL).

Elements were determined in the samples after mineralization with nitric acid using ICP-MS. Briefly, the liquid samples (∼500 mg weighed to 0.1 mg) were placed in 12 ml quartz vessels, 1 ml subboiled nitric acid was added and the samples were placed in the autoclave (UltraCLAVE IV, EMLS, Leutkirch, Germany). Then the autoclave was pressurized with argon to 40 bars and the samples heated in 45 min to a temperature of 250 °C and kept at this temperature for 45 min. After cooling the samples were transferred into 15 ml polypropylene tubes (Greiner Bio-One). Zn (determined at m/z 66) and Ag (determined at m/z 107) were determined with ICP-MS (Agilent 7500ce, Agilent Technologies, Waldbronn, Germany) after appropriate dilution. The accuracy of the results was validated with the certified reference material 1640a (trace elements in water, NIST, Gaithersburg, ML, USA).

For EV production and harvest, THP-1 cells were collected in 50 ml polypropylene tubes (Greiner Bio One, Frickenhausen, Germany, #227261) by 10 minutes of centrifugation at 200 rcf and 20°C in a swinging bucket Rotanta 460 R centrifuge (Hettich, Tuttlingen, Germany). Next, they were seeded at a density of 1×10⁶ cells/ml in a T75 cm² tissue culture treated cell culture flask (Greiner Bio One, #658170) in 15 ml of RPMI 1460 medium with GlutaMAX supplemented with 2% EV-depleted FBS (Gibco, #A2720801), and stimulated with 100 ng/ml lipopolysaccharide (LPS) from E. coli serotype 055:B5 (Sigma-Aldrich, St. Louis, USA, #L2880). After 24 hours of incubation at 37°C, 5% CO₂ and 95% humidity, cells and the conditioned cell medium were collected.

The in vitro VHCl release test I has been used to investigate the release properties of inserts (prepared by means of a syringe of 2 mm in diameter and 4 mm in length) in the freshly prepared simulated tear fluid (NaHCO₃ 0.218 g/100.0 mL, NaCl 0.678 g/100.0 mL, CaCl₂ × 2H₂O 0.0084 g/100.0 mL, KCl 0.138 g/100.0 mL, Milli-Q^® water up to 100.0 mL; pH = 7.4) [39 (link)], simulating the administration in the eye topically. The trials have been done in triplicate, at 32 ± 0.5 °C, using 1.5 mL polypropylene tubes (Greiner Bio One GmbH, Frickenhausen, Germany) filled with 1 mL of the release medium and an insert (10–20 mg), shaken at 300 rpm by the Eppendorf Comfort thermomixer (Eppendorf AG, Hamburg, Germany).
The release pattern was investigated at the time point: 0, 5, 10, 30, 60, 120, 180, 360, 600, 1440, 2880 min, centrifuged (Centrifuge Eppendorf 5804R, at 24 °C, 14,000 rpm, 10 min) and analyzed using the later described HPLC method.
The Higuchi diffusion equation was used to check if the release pattern could be described by diffusion:
where Q is the amount of the drug released in time t per unit area A, D is the diffusion coefficient of the drug molecules, C is the initial concentration of the drug, and C_s is the solubility of the drug in the matrix media [40 (link)].