β glucan

Thermogravimetric analyses were performed using a Mettler Toledo TGA1 XP1 thermogravimetric analyzer by heating the samples having the weight of 8–10 mg were inserted in an alumina crucible with a perforated lid at the temperature from 25 °C to 800 °C by the heating rate of 20 °C/min under N₂ atmospheric condition with a mass flow rate of 100 mL/min. A blank performed with an empty crucible together with a perforated lid was subtracted from the curves [[37] (link), [38] (link), [39] (link), [40] (link), [41] (link), [42] (link), [43] (link)]. Standards of β-glucan and chitin (both from Sigma Aldrich) were analyzed in the same conditions, allowing the comparison with the fungal mass loss at different temperatures. Thermogravimetric measurements were analyzed together with their first derivative (DTGA) to better estimate the temperature of the decomposition peaks (relative maximum of DTGA) and to locate the start/end of the decomposition (zeroing or flattening of DTGA).

Gemcitabine hydrochloride, MW 299.66, β-glucan, (hydroxypropyl)methyl cellulose (HPMC), ethylcellulose (with 48% ethoxy content) (EC), acetic acid, 3-(4, 5-dimethyl-thiazol-2-yl)-2, and 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louise, MO, USA). Methylcellulose LR was purchased from Chem-Supply (Gillman, South Australia). Chitosan (MW 150,000, DD 70%), was purchased from Comwin Fine Chemicals Co. (Changzhou, China). Methylcellulose (MC) was purchased from ICN Biomedical, Inc. (Santa Ana, CA, USA). Polypropylene glycol was purchased from Midwest Pharmaceutics (Hawke’s Bay, New Zealand). Dimethyl sulfoxide (DMSO) was purchased from Labpartner (Shanghai, China). The 4T1breast cancer cell line was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). The Roswell Park Memorial Institute (RPMI) medium 1640 basic was purchased from Gibco (Grand Island, NE, USA), and fetal calf serum was purchased from Hyclone (Logan, UT, USA). Penicillin–streptomycin–glutamine and nonessential amino acids were purchased from Life Technology (Grand Island, NY, USA). All other chemicals used were of reagent grade. Milli-Q water was obtained through reverse osmosis using a Millipak^® system (Millipore, Burlington, MA, USA, 0.22 µm).

β-glucan derived from Laminaria digitata was purchased from Sigma Chemical Co. (St. Louis, MO, USA; L-9634). This glucan has the molecular form β-(1,3;1,6)-D-glucan with a molecular weight of 5.85 kDa, and it is soluble in neutral water [12] (link). Stock solutions of 6 mg/ml β-glucan were aseptically prepared. The concentration of β-glucan that was used in the assays was 3 mg/ml, a concentration that does not affect the viability of neutrophils and induces a neutrophil response. The test of cell viability was determined by means of trypan blue, with 95%±10 viability. This concentration was based on a dose-response curve that used 0.5, 1.0, 3.0, 5.0, and 10 mg/ml (data not shown).

Monocytes stimulated with 5 µg/mL β-glucan or not were treated with oxyresveratrol alone or encapsulated in PLGA nanoparticles, or with corresponding amounts of bare PLGA particles for 18 h, and then the O₂⁻ release was estimated by cytochrome c reduction. Briefly, after cell culture for the required time the medium of each well was replaced with HBSS pH 7.4, containing 80 μM ferricytochrome c type III (Sigma-Aldrich, St. Louis, MO, USA), with or without 5 μg/mL of β-glucan (Sigma-Aldrich, St. Louis, MO, USA). Cytocrome c reduction was evaluated at 550 nm by using an automated microplate reader (Bioteck^® Instruments Inc., Winooski, VT, USA).

For in vivo studies, neonatal mice at postnatal days 3–4 were injected intraperitoneally (i.p.) with 0.5 mg of β-glucan (Sigma) in 50 μl of PBS, and i.p. injection of PBS alone was performed as control according to a previous study [15 (link)]. For in vitro study, lin⁻ cells isolated from bone marrow cells of neonatal mice were cultured in RPMI 1640 medium (BI) supplemented with 10% fetal bovine serum, 20 ng/ml GM-CSF (Peprotech), and 50 μM 2-mercaptoethanol, with or without β-glucan (5, 25, or 50 μg/ml). Media were half changed on day 3, and cells were analyzed by flow cytometry on day 5.

Mastic gum, carrageenan, xanthan gum, guar gum, gum tragacanth, locust bean gum, and β-glucan were purchased from Sigma Aldrich (Saint Louis, MO, USA). Extracts from these items were prepared according to the procedures described by Vojdani [27 ]. Ten grams of each gum was extracted in 500 mL of buffer pH 4.6 by mixing them for 8 hours at 25°C on a magnetic stirrer. The solution was centrifuged at 20,000g, and supernatant was removed and concentrated by a factor of 10 using an Amicon filter. The protein concentration was measured using a kit provided by Bio-Rad (Hercules, CA, USA). All extracts were aliquoted and stored frozen at −20°C until used. Different gum extracts were dissolved in 0.1 M PBS. These antigens were diluted 1 : 50 in 0.1 M carbonate buffer pH 9.2 and 100 μL of each gum antigen.