Isopropanol

The viability of cells was evaluated after using the tested compounds according to ISO 10993 part 5 Annex C–MTT assay. Both cell lines were seeded into 96-well plates at a density of 10,000 cells per well. The cells were incubated overnight to allow them to adhere to the surface of the well. The non-adherent cells were removed with supernatant and freshly prepared tested compounds were added for 24 or 48 h. After that time, the MTT solution was prepared at a 1 mg/mL concentration with PBS (Sigma-Aldrich, St. Louis, MO, USA, cat. no.: M2003-1G; Lonza; cat. no.: BE17-516F). The supernatant was removed and MTT solution was added for 2 h at 37 °C in 5% CO₂ with 95% humidity. Then, the solution was gently removed and the violet crystal was dissolved in isopropanol (Pol-Aura, Olsztyn, Poland, cat.no.: 67-63-0) for 30 min by shaking. Finally, the absorbance was measured at 570 nm using a VariuScan microplate reader.

Chitosan produced from Aspergillus niger (Deacetylation degree (DD) = 92%, determined by FT-IR method; Mw = 890,000 g/mol; and 100–300 cps), fluorescein, L-aspartic, and L-glutamic acid were purchased from PolAura, Dywity, Poland. Propylene glycol, isopropanol, methanol (HPLC grade), (CH₃COO)₂Zn∙2H₂O, CaCl₂·2H₂O, MgSO₄, KCl, KOH, ethanol (96%, 99.9%), KHPO₄, Na₂HPO₄, NaHCO₃, NaCl, HCl, NaOH, mouse L929 (commercial cell line from mouse C3H/An connective tissue for research purpose only, The European Collection of Authenticated Cell Cultures (ECACC)), Dulbecco’s Modified Eagle Medium (DMEM) with glucose content cell culture medium, antibiotics (streptomycin/penicillin), fetal bovine serum (FBS), phosphate buffer solution (PBS), trypsin with EDTA, and Triton-X-100 were purchased from Sigma Aldrich, Poznań, Poland. For the study, pure cannabidiol of HPLC confirmed purity was used, purchased from Dobre Konopie, Mysłowice, Poland.

CLT was purchased from Pol-Aura (Różanowo, Poland). Castor oil was supplied from Microfarm (Zabierzów, Poland). Rapeseed oil was purchased from Bunge Polska Sp. z o.o. (Kruszwica, Poland). In addition, 90% Lactic acid, Poloxamer 188 (Pluronic F-68) was purchased from Pol-Aura (Różanowo, Poland). Poloxamer 407 (Pluronic F-127), poly(ethylene glycol) 200 (PEG200), sorbitan monooleate (Span 80), and polyoxyethylenesorbitan monooleate (Tween 80) were purchased from Sigma-Aldrich (St. Louis, MO, USA or Steinheim, Germany). Propylene glycol was supplied by Firma Chempur (Piekary Śląskie, Poland).
Acetonitrile for HPLC (ACN) was purchased from S.Witko (Łódź, Poland). Acetone, methanol, and potassium phosphate monobasic (KH₂PO₄) were obtained from Chempur (Piekary Śląskie, Poland). Isopropanol was purchased from Pol-Aura (Różanowo, Poland). Milli-Q water (<0.05 µS/cm), used in batches manufacturing and all experiments, was self-produced from Hydrolab Ultra UV (Hydrolab Sp z o.o., Straszyn, Poland). All the chemicals and reagents used in this study were analytical grade.
Market reference, i.e., 1% CLT cream, MycoHydralin (Bayer Healthcare, Loos, France), was purchased in a local retail pharmacy in France.