Polyethersulfone membrane

The in vitro release assays were conducted assuring sink conditions. Modified Franz cells, equipped with a polyethersulfone membrane (Sigma-Aldrich) and with a diffusion area of 1.77 cm² were used in the assays. A Microette (Hanson Research, USA) was used. The receptor compartment was filled with 7.0 mL of a receptor solution composed of 0.1 M phosphate buffer and ethanol (50:50 v/v), pH 5.5. 1 mL of the formulations was used, as allowed by the Franz cell.
The acceptor solution was constantly agitated at 300 rpm using mini-magnetic agitators. The temperature was maintained at 37 ± 2 °C by utilizing a circulating heating bath in the jacketed cells.
The evaluation of the release of cymene and myrcene from the nanoemulsions was performed at specific time intervals: 30 min and 1, 2, 4, 6, 8, 12, and 24 h. Each measurement was repeated six times to ensure reliability. The released compounds were quantified by high-performance liquid chromatography, following a previously validated method.

Human platelet TGF-β1 (R&D Systems, Abingdon, UK) was reconstituted in sterile 4 mM hydrochloric acid (HCl; VWR International Ltd, Lutterworth, UK) and 0.1% bovine serum albumin (BSA; PAA Laboratories GmbH, Pasching, Austria) that had been filter-sterilised through polyethersulfone membrane with 0.2 μm pore size (Sigma-Aldrich, Gillingham, UK). A stock solution of 10 ng/μl TGF-β1 was stored at − 80 °C until use. A vial of MS-SAFE protease and phosphatase inhibitors (Sigma-Aldrich) was dissolved into 2 ml 10 × RIPA lysis and extraction buffer (Sigma-Aldrich) and stored at − 20 °C as a stock solution. 1 × RIPA buffer was used to lyse cells and extract proteins. Bicinchoninic acid (BCA) protein assay (Thermo-Fisher Scientific, Paisley, UK) was used to measure protein concentration of cell lysates. Total collagen contents were measured indirectly through the measurement of hydroxyproline using the QuickZyme Total Collagen Assay (QuickZyme, Leiden, The Netherlands). Soluble collagens in conditioned media were colourimetrically detected by the Sircol soluble collagen assay (Biocolor Ltd., County Antrim, UK). Information of ELISA kits used in this study is listed in Supplementary table 9.

FMD vaccines were prepared with (i) PEG-precipitated and (ii) chloroform-treated FMD vaccine antigens. The inactivated viral culture supernatant was obtained by the method described in Section 2.1, and the following procedures were performed. For the first group, the supernatant was treated with 7.5% (w/v) PEG 6000 (Sigma-Aldrich), stirred overnight at 4 °C, and centrifuged at 10,000× g for 30 min. After the supernatant was removed, the pellet was resuspended in tris–KCl buffer (pH 7.6) and adjusted to a concentration of 15 μg/mL. For the second group, the supernatant was mixed with 10% (v/v) chloroform by inverting for 5 min, and centrifuged at approximately 3000× g for 15 min. The aqueous layer on the top of the organic solvent layer was harvested and concentrated to a final concentration of 15 μg/mL by an ultrafiltration device fitted with a polyethersulfone membrane with a MWCO (molecular weight cutoff) of 300 kDa (Millipore, Billerica, MA, USA). Saponin (Sigma-Aldrich) and aluminum hydroxide gel (General Chemical, Parsippany, NJ, USA) were added to these two types of antigens, and then the ISA 206 VG adjuvant (Seppic, Paris, France) pre-warmed at 30 °C was added in a ratio of 1:1. After the mixtures were blocked from light and incubated at 20 °C for 1 h in a water bath, they were stored at 4 °C until use.