Cottonseed oil

Rats were acclimatized for 1 week, followed by bilateral orchiectomies to prevent the influence of endogenous testosterone. After 1 week, rats were divided into five groups: NC, BPH, HX200, HX300, and Fina (n = 5 per group). Prostatic hyperplasia was induced in four groups (BPH, HX200, HX300, Fina) by subcutaneous injection of 3 mg/kg of testosterone propionate (TP) (Tokyo Chemical Industry, Tokyo, Japan) dissolved in cottonseed oil (Sigma-Aldrich, St. Louis, MO, USA) every three days. The NC group received only cottonseed oil in order to provide similar subcutaneous injection conditions in all groups. During the induction of prostate hyperplasia, rats orally received respective reagents on a daily basis for 4 weeks. The HX200 group and HX300 group were orally administrated 200 mg/kg of HX109 or 300 mg/kg of HX109. The Fina group was orally administrated 5 mg/kg of finasteride as a positive control. The NC group and BPH group were orally administrated distilled water as a vehicle. Body weight was measured once a week during the experiment. After 4 weeks, rats were sacrificed, and prostates were immediately removed and weighed.

Metarhizium acridum (ARSEF-324, obtained from the Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (ARSEF), Ithaca, NY) was cultured on Potato Dextrose Agar; maintained at 28 °C; and kept under continuous light. Suspensions of fungal conidia in cottonseed oil were prepared to a concentration of 3.75 × 10⁷ per ml^{13 (link), 17 (link)}. Ten milliliters of cottonseed oil (Sigma-Aldrich, Dorset, UK) was added to 7-day old fungal cultures. Conidia were dislodged using a sterile spreader and the suspension was transferred to a sterile 25-ml bottle. The suspension was then filtered through four layers of sterile muslin to remove mycelia and clumps of conidia, centrifuged at 3000 rpm for 3 min, and then placed in a sonicating water bath at 15 °C for 5 min. The concentration of the conidial suspension was determined using a Neubauer haemocytometer and adjusted as required^{13 (link), 17 (link)}. Adult locusts were inoculated with 2 μl of fungal suspension (~75,000 conidia) under the pronotal shield using a micro-syringe (VICI®, Baton Rouge, Louisiana, USA). Controls were treated with 2 μl of cottonseed oil alone.

World Health Organization (WHO) recommended oral supplementation at 30–60 mg of ATRA for infant children 6–59 months of age [17 ]. The dosage of ATRA adjuvant chemotherapy for APL was 45 mg/m² [23 (link)]. As reported, the median lethal dose (LD₅₀) of ATRA in Swiss mice was 31 mg/kg by intraperitoneal injection [34 (link)], and the equivalent dose ratio for human and mice was 1:0.0026 based on the default weights of humans and mice (70 kg and 20 g) as well as their body surface areas. Considering the above facts along with the bioavailability of different administering routes, we determined that the intraperitoneal injection doses of ATRA were 1, 5, 10, 20, 40, 60, and 80 mg/kg. In addition, ATRA was divided into four doses according to its clinical application: low (1 mg/kg), medium (5 or 10 mg/kg), high (20 mg/kg), and toxic dose (40, 60, or 80 mg/kg). The ATRA powder (Sigma, St. Louis, MO, USA. R2625) was dissolved in dimethyl sulfoxide (DMSO), further diluted in cottonseed oil (Sigma, St. Louis, MO, USA. C7767) at the appropriate concentrations, and then stored at 4 °C. Mice were intraperitoneally injected with 0.2 mL ATRA solution once a day for 10 days, while the control group was injected with the same volume of cottonseed oil.

Multilayer extruded commercial-grade polymer films (thickness ≈ 0.1 mm) obtained from Bemis Company, Inc. (Neenah, WI) were used as substrates for infusion. Most commonly, the top and bottom layers of the films were comprised of ULDPE (thickness ≈ 10 μm), while the intermediate layers were a proprietary combination of highly impermeable polymers. For oil impregnation within the ULDPE polymer matrix, cottonseed oil (Sigma Aldrich) was used. Impregnation with oil was achieved using a motorized drawdown coater (ChemInstruments, EC-100) with the smallest size coating rod (size 0). First, 100–200 μL of cottonseed oil (ρ = 925 kg/m³) was pipetted on the leading edge of the polymer film. Second, the motorized rod spread the oil uniformly across the top face of the film. After waiting for a few seconds to allow for the oil to impregnate the ULDPE, any excess oil atop the film was firmly wiped away with absorbent wipes (Kim wipes). For the control case where the oil did not impregnate the ULDPE, the same process was repeated but with 10 cSt silicone oil (Sigma Aldirch) instead of cottonseed oil. Other polymer films that were also used included medium density polyethylene (MDPE), cyclic olefin copolymer (COC), polypropylene (PP), or polyethylene terapthalate (PET) as the top layer.

Fluconazole (FLZ) was received as a gift from Hasco-Lek S.A. (Wroclaw, Poland). Isopropyl myristate, castor oil, olive oil, peanut oil, cottonseed oil, sesame oil, Pluronic F127 (PLU), Tween 80 (T80), Tween 20 (T20), Kolliphor EL (KOL), polyethylene glycol 200 (PEG 200), and Span 80 were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Oleic acid (OA) and propylene glycol (PG) were obtained from Chempur (Piekary Slaskie, Poland). High-performance liquid chromatography (HPLC) grade methanol was purchased from Honeywell Specialty Chemicals (Seelze, Germany). Deuterium oxide was purchased from Armar Chemicals (Leipzig, Germany). All chemicals used in this study were of analytical grade.
Four Candida strains were involved in this study from American Type Culture Collection (Manassas, VA, USA) from the laboratory collection of the Department of Pharmaceutical Microbiology and Parasitology at the Wroclaw Medical University (C. albicans ATCC 90028, C. parapsilosis ATCC 90018, C. glabrata ATCC 90030, C. tropicalis ATCC 750). The strains were stored in trypticase soy broth with the addition of 15% glycerol and kept at −80 °C.