Beeswax

Chloroform (99%), toluene (99%), carnauba
wax, beeswax, and spherical hydrophilic SiO₂ nanoparticles
of different sizes (11 nm, 90 nm, 260 nm, 4 μm, 8 μm,
20 μm) were purchased from Sigma-Aldrich. Ethanol (99%), paraffin
wax, and acetone (99%) were obtained from Merck. These chemicals were
used as received. Glass slides (2.6 cm × 7.6 cm) were bought
from Isolab Inc. The L929 (murine fibroblast) cell line was purchased
from the American Type Culture Collection (ATCC, Manassas, VA), cultured
in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Chemical
Company, St. Louis) supplemented with 10% fetal bovine serum (Gibco
BRL), 1% penicillin/streptomycin (Sigma-Aldrich, Germany), and 1%
glutamine (Gibco, U.K.) at 37 °C in a humidified atmosphere of
5% CO₂.

Magnesium ribbon (99.5%+),
sodium chloride (99.0%+), agar (for microbiology), phenolphthalein
indicator, tin(II) chloride (98%+), tin foil (99.9%+, 0.127 mm thick),
and beeswax were purchased from Sigma-Aldrich. Calcium chloride dihydrate
(99%+), calcium d-gluconate monohydrate (98%+), calcium sulfate
dihydrate (98.0%+), and quercetin dihydrate (97%+) were acquired from
Alfa Aesar. Sodium hydroxide concentrate (0.1 M in water) was purchased
from Fluka. Tin wire (96.5% tin, with 3.0% silver, and 0.5% copper)
was obtained from RS Components Ltd. Glycerol (99.0%+) was purchased
from Merck. Gold wires (99.99%+) were acquired from 8853 SPA. Polydimethylsiloxane
(PDMS), Sylgard 184, was purchased from Sigma-Aldrich. Apple, melon,
grapes, and red cabbage were sourced from local supermarkets. The
water used in all of the experiments was Milli-Q water.

The following materials were used as received, without further purification: petroleum jelly (Unilever, London, UK), medium-chain triglycerides (Sasol Germany GmbH, Hamburg, Germany), marketed body emulsion containing “free” BP-3 (Neutrogena Co., Los Angeles, CA, USA), carbomer 940 (Lubrizol Corp., Wickliffe, OH, USA), poloxamer 188 (BASF, Ludwigshafen, Germany). BP-3, nonivamide, acetic acid, formic acid, ethylenediamine tetraacetic acid (EDTA) sodium, methyl paraben, mineral oil, sodium hydroxide, and beeswax were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Acetonitrile, water, and methanol (all HPLC grade) were purchased from Avantor (Radnor, PA, USA).

E2 pellets were prepared as previously described 18 . Briefly, 3.9 g of beeswax (Sigma-Aldrich, St. Louis, MO) was placed into a 20-mL glass sample vial (Thermo Fisher Scientific Inc., Waltham, MA) and melted on the hot plate at 65°C. Then 100-mg β-Estradiol (Sigma-Aldrich) was added to the melted wax and mixed using a sterile 7-mm magnetic stir bar until the mixture turned clear. A sheet of aluminum foil was placed on top of the dry ice and the wax/β–Estradiol mixture was dropped onto the foil using a preheated glass Pasteur pipet to create a pellet. Each pellet was made from three drops of the wax/β–Estradiol mixture, resulting in ∼30 mg per pellet. Pellets were stored at 4°C for further study.

Gelatin from porcine skin (type A), poly (vinyl alcohol), Span 80 and beeswax were purchased from Sigma-Aldrich (Poznan, Poland). Polylactide was supplied from NatureWorks (Minnetonka, MN, USA). Sodium alginate was obtained from BÜCHI Labortechnik AG (Flawil, Switzerland). Glycerol was purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland). Cottonseed oil was supplied by Alston Nova, S.L. (Barcelona, Spain). The hydroglycolic Calendula officinalis flower extract (propylene glycol/water (80:20)) was obtained from Provital S.A. (Barcelona, Spain). All used chemicals were of analytical grade.

Chitosan (origin: crab shells), beeswax (yellow soft), gallic acid (MW: 170.12), Folin-Ciocalteu (2 N), glycerol (MW: 92.09), tween 20 (conductivity: 50 µS/cm), acetic acid (≥99.5%) and NaOCl (4–4.99% available chlorine) were purchased form Sigma Aldrich, USA. Fresh Aloe vera plants were harvested from a local nursery in Faisalabad, Pakistan.

S-(+)-ibuprofen (dexibuprofen/DXI) was purchased from Amadis Chemical^® (Hangzhou, Zhejiang, China). Beeswax, Tween^® 80 (polysorbate 80) and ortho-phosphoric acid (85%) were purchased from Sigma-Aldrich (Madrid, Spain). Miglyol 812 (caprylic/capric triglycerides) was purchased from Roig Farma SA (Terrassa, Spain). Sodium citrate tri-base and urea were purchased from Montplet & Esteban SA (Barcelona, Spain). Methanol (99.8%) was a product of Fisher Scientific (Geel, Belgium). All other reagents including ethanol (99.8%, PanReac AppliChem, Barcelona, Spain) were of analytical grade. Millipore^® Milli^® Q system (Darmstadt, Germany) purified water was used across all experiments and formulations.