Dithranol

In the dithranol inflammation model, a solution of 0.3% of 1,8,9-Anthracenetriol (dithranol) (Sigma-Aldrich, Merk Life Science S.r.l Milano, Italy) in acetone (w/v) was applied on the skin surface immediately after mounting the sample in the bioreactor chamber [22 (link)]. The time points chosen for analyzing the inflammatory effects were 1 h, 3 h, 6 h and 24 h. The treatment was not prolonged over 24 h because this is the classical dithranol therapy; after that, healthy skin surrounding the treated area shows irritation signs [21 (link)]; accordingly, preliminary tests on our skin explants showed that, at the concentration chosen for our study, dithranol induced severe tissue damage after 24 h.
In the substance P-inflammation model, the neuropeptide (Bachem AG, Bubendorf, Switzerland) was added to the medium at a concentration of 10 μM [25 (link)]. Analyses were performed not only at 24 h but also at 48 h and 72 h to monitor the inflammatory process at the classical pharmacokinetic time points used to test therapeutic treatments. Moreover, it is known that the effects of substance P administration in vitro may last for a long time, e.g., [67 (link),68 (link)].
The control (untreated) samples were maintained in the bioreactor under the same experimental conditions but avoiding dithranol or substance P treatment and were analyzed at the same time points.

Polyethylene glycol (PEG, M_w ∼ 1500 Da) was supplied by Fluka Chemie
GmbH (Buchs, Switzerland). Polypropylene glycol (PPG, M_w ∼ 1010 Da, M_w/M_n 1.04) was purchased from PSS Polymer Standards
Service GmbH (Mainz, Germany). Polytetrahydrofuran of M_w ∼ 1000 Da and M_w ∼
1400 Da (denoted as PTHF1000 and PTHF1400, respectively) was obtained
from Royal DSM N.V. (Heerlen, The Netherlands). Nylon-6 was purchased
from Scientific Polymer Products Inc. (M_w ∼ 25,000 Da, Oregon, USA). Polystyrene (PS, M_w ∼ 1300 Da, M_w/M_n 1.10) was purchased from Thermo Fisher Scientific
GmbH (Kandel, Germany). Polybutylene terephthalate (PBT, M_w ∼ 16,150 Da). ULC-MS-grade methanol (MeOH), ethanol
(EtOH), and n-hexane were acquired from Biosolve
B.V. (Valkenswaard, The Netherlands). Tetrahydrofuran (THF) and hexafluoroisopropanol
(HFIP, ≥99% purity) were supplied by Sigma-Aldrich Chemie B.V.
(Zwijndrecht, The Netherlands). All organic solvents
were used without further purification. 2,5-Dihydroxybenzoic acid
(DHB, 98% purity), dithranol (DT, ≥90% purity), 2′,4′,6′-trihydroxyacetophenone
monohydrate (THAP, ≥99.5% purity), silver trifluoroacetate
salt (AgTFA, 98% purity), and trifluoroacetic acid (TFA, ≥99%
purity) were purchased from Sigma-Aldrich.