Chloroform

Gamborg B-5 basal medium, Murashige and Skoog (MS) medium (Murashige and Skoog, 1962 (link)), yeast extract beef broth (YEB), Agargellan™, and cefotaxime were obtained from PhytoTechnology Laboratories (Lenexa, KS, USA). YE powder was obtained from HiMedia (Maharashtra, India). MeJA (1 g/mL in water), CHI (from shrimp shells), vanillin, and acetosyringone were purchased from Sigma-Aldrich (St. Louis, MO, USA). SA was procured from Ajax Fine Chem (Sydney, Australia). The friedelin analytical standard was obtained from Extrasynthese (Genay, France), and the epifriedelanol analytical standard was acquired from Biopurify (Sichuan, China). Glacial acetic acid, sulfuric acid, toluene, dichloromethane, and ethyl acetate were obtained from Merck (Darmstadt, Germany). Chloroform was procured from Labscan (Bangkok, Thailand). For DNA isolation and amplification, a DNAsecure Plant Kit (Tiangen, Beijing, China), Taq Phusion polymerase, and GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA) were used, and the primers were synthesized by Macrogen (Seoul, Korea).

High purity synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], sodium salt (DOPG) was purchased from NOF (Tokyo, Japan) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, sodium salt (DOPS) was purchased from Avanti Polar Lipids Inc (Sigma-Aldrich, Budapest, Hungary). Liposomes were prepared by using the lipid thin film hydration technique. Lipids were dissolved in chloroform (LabScan, Budapest, Hungary) containing 50% vol methanol (Reanal, Budapest, Hungary), which was then evaporated using a rotary evaporator. The resulting lipid film was kept in a vacuum for at least 8 h to remove residual traces of solvent. The dried lipid film was hydrated with the assay buffer. After repeated heating (37 °C) and cooling (−196 °C) steps (at least 10 times), the solutions were extruded through polycarbonate filters with 100 nm pore size (at least 11 times) using a LIPEX extruder (Northern Lipids Inc., Burnaby, BC, Canada). Final lipid concentration was 13 mM. For mimicking mammalian, bacterial, and cancer cell membranes, pure DOPC, DOPC/DOPG (80/20 n/n%), and DOPC/DOPS (80/20 n/n%) referred to as PC, PC/PG, and PC/PS, respectively, were used thoroughly in the study.

All chemicals used were of analytical and ultra-pure LC-MS grade quality, and organic solvents mostly included chloroform (Labscan, Poland), methanol (Labscan, Poland) and acetone (Associated Chemical Enterprises, South Africa). All equipment was sterilised prior to use and cell treatment was carried out under sterile conditions.

Lead and cadmium standard solutions (TraceCERT^® 1000 mg/L) for AAS were purchased from Supelco (Bellefonte, PA, USA). Concentrated nitric acid (65%), ammonium solution (25%) and diammonium hydrogen citrate were purchased from Merck (Darmstadt, Germany). Chloroform was purchased from LabScan (Bangkok, Thailand). Ammonium-1-pyrrolidine dithiocarbamate, polyethylene, polyethylene terephthalate, polypropylene, polystyrene and polyvinyl chloride were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Solvents including hexane (95%), chloroform (HPLC grade), methanol (HLPC grade), and acetonitrile (HPLC grade) were obtained from LAB-SCAN (Gliwice, Poland) and absolute ethanol, sodium carbonate (Na₂CO₃), and sulfuric acid (H₂SO₄) from Panreac (Barcelona, Spain). Potassium hydroxide (KOH), ascorbic acid, 2,6-Di-tert-butyl-p-cresol (BHT), bovine serum albumin (BSA), acetylacetone, p-dimethylaminebenzaldehyde, HCl (37%), phenol, hexadecane, ergosterol (95%), D-glucose, D-glucosamine hydrochloride, FAME (fatty acid methyl ester), and gallic acid were purchased from Sigma–Aldrich (Madrid, Spain) as well as acarbose and α-glucosidase from Saccharomyces Cerevisae and α-amylase from the porcine pancreas. All reagents and solvents were of analytical grade.

HPMC (Onimax Co. Ltd., Bangkok, Thailand), PVP (Dai-ichi Kogyo Seiyaku, Kyoto, Japan), MLT (Shanghai Chemical, Shanghai, China), sodium chloride (Carlo Erba, Milano, Italy), sorbitan monostearate (Span60), cholesterol, P407, chitosan from shrimp shells, mucin from porcine stomach, succinic anhydride, glutaric anhydride and sodium hydride (60% in oil) (Sigma-Aldrich, St. Louis, MO, USA), dimethylformamide, methanol, chloroform, n-hexane, hydrochloric acid, ethyl acetate and dichloromethane (Labscan, Bankok, Thailand), and normal saline solution (NSS, A.N.B. Laboratories, Bankok, Thailand) were used as received.

Morphine hydrochloride was purchased from Alcaliber Laboratories. Hydrogenated soy phosphatidylcholine (HSPC) and monomethoxy polyethyleneglycol 2000-distearoyl phosphatidyl-ethanolamine (mPEG-DSPE) were a kind gift from Lipoid K.G. (Ludwigshafen, Germany). Cholesterol (CHOL) was purchased from Avanti Polar Lipid, Inc. (Alabama, USA), ethanol from Scharlau (Barcelona, Spain), chloroform from Lab Scan (Sowinskiego, Poland), methanol and ammonium sulfate from Merck KGaA (Darmstadt, Germany), and diafiltration cassette from Thermo Scientific (IL, USA). Dialysis membrane (with MW cutoff of 12,000–14,000 Da), desoxycholate, tert-butanol, sodium acetate, and all other chemicals used in this work were purchased from Sigma-Aldrich (Madrid, Spain).