Methyl β cyclodextrin me β cd

Quercetin (Que, MW: 302.24 g/mol), Methyl-β-cyclodextrin (Me-β-CD, MW: 1310 g/mol) and Hydroxypropyl-β-cyclodextrin (HP-β-CD, MW: 1460 g/mol) were purchased from Sigma-Aldrich (St. Louis, MO, USA), Fluka Chemika (Buchs, Switzerland) and Ashland (Covington, KY, USA), respectively. Naringenin was supplied from Alfa Aesar (Ward Hill, MA, USA). Mannitol (Ph. Eur.) was obtained from Lisapharma S.p.A. (Erba (CO), Italy), and soybean lecithin (Lipoid^® S45) was obtained from Lipoid AG (Steinhausen, Switzerland). Potassium dihydrogen phosphate and sodium acetate were acquired from Merck KGaA (Darmstadt, Germany), while sodium hydroxide (1 mol/L) was acquired from Chem-Lab NV (Zedelgem, Belgium). Glacial acetic acid was purchased from PanReac AppliChem (Chicago, IL, USA). sodium acetate buffer solution (pH 5.0) was prepared using sodium acetate and Glacial acetic acid in HPLC-grade water. The β-glucuronidase enzyme solution (3000 units/mL) was freshly prepared using the sodium acetate buffer. HPLC-grade solvents (water, methanol, acetonitrile) and reagents were obtained from Fischer Scientific (Pittsburgh, PA, USA). Triple-deionized water purchased from Fischer Scientific was used for all preparations.

Two hundred fifty μg/mL TNF-α and 12 mg/mL CHP were mixed, sterilized by filtration, and incubated at 37°C for 5 d. During the incubation, CHP encapsulated TNF-α molecules to form nanoparticles. Unencapsulated TNF-α was removed by size-exclusion chromatography with a PD-10 column (GE Healthcare, Fairfield, CT). The size of the particles was determined by dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). To estimate the amount of TNF-α encapsulated, nanoparticles were treated with 100 mg/mL methyl-β-cyclodextrin (Me-β-CD, Sigma-Aldrich Co., Saint Louis, MO) at 37°C for 2 h to disrupt the particle structure and release TNF-α, as described [21 (link)]. The amount was determined by an enzyme-linked immunosorbent assay (ELISA) system described below.