Acetone

CpTi (grade 2; Kobe Steel, Kobe, Japan) and yttria-stabilized TZP (TZ-3YB-E; Tosoh, Tokyo, Japan) were used. Both CpTi and TZP specimens had a diameter of 13 mm and thickness of 0.5 mm. For polishing of specimens, a polishing machine (Ecomet 3; Buehler, Lake Bluff, IL, USA) was used. First, the CpTi specimens were ground using silicon carbide paper (Aqra; SANKEI, Tokyo, Japan) down to 1,200 grit. Then, they were polished using 3-μm diamond pastes (MetaDi Monocrystalline Suspension 3-μm; Buehler, Lake Bluff, IL, USA) followed by 0.06-μm colloidal silica (MetaDi Monocrystalline Suspension 0.06-μm; Buehler, Lake Bluff, IL, USA). The TZP specimens were ground progressively with 70- and 45-μm diamond disks (DGD 70-μm and DGD 45-μm; Buehler, Lake Bluff, IL, USA). Then, they were finely polished using 9- and 3-μm diamond pastes (MetaDi Monocrystalline Suspension 9-μm and 3-μm; Buehler, Lake Bluff, IL, USA) followed by 0.06-μm colloidal silica. CpTi and TZP specimens were cleaned ultrasonically (Ultrasonic cleaner; Aiwa, Saitama, Japan) and underwent further cleaning with Ethyl acetate (Ethyl acetate; Wako, Osaka, Japan), Acetone (Acetone; Wako, Osaka, Japan) and distilled water cleaning. Subsequently, the specimens were sterilized in an autoclave (121 °C, 15 min) (HICLAVE HG-80; HIRAYAMA, Saitama, Japan). For at least 24 h until use, they were kept in dry conditions.

Acetone (>99%, FUJIFILM Wako Pure Chemical, Osaka, Japan), ethanol (>99.5%, FUJIFILM Wako Pure Chemical, Osaka, Japan), and pure water (Direct-Q, UV3, Merck, Hessen, Germany) were used to wash the silicon wafer and microspheres. A 2 wt% aqueous solution of polyvinyl alcohol (PVA; GL-03; M_w ~20,000 g/mol; 87–89% hydrolyzed; Mitsubishi Chemical, Tokyo, Japan) having a viscosity of 1.75 mPa s and a density of 1.00 g/cm³ was prepared for the continuous phase. A 1 wt% solution was prepared by adding 2-hydroxy-2-methylpropiophenone (Darocur 1173, BASF, Tokyo, Japan) to 1,6-hexanediol diacrylate (HDDA; Shin-Nakamura Kagaku, Tokyo, Japan) having a viscosity of 6.35 mPa s and a density of 1.02 g/cm³ for the photocurable dispersed phase.

Cellulose acetate (average Mn = ≈50,000) was purchased from Sigma-Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). Sodium hydroxide (NaOH, 97%), diethyl ether (99%), and silver nitrate (AgNO₃, 99.8%) were acquired from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Acetone (99.5%) and N,N-dimethylformamide (DMF, 99.8%) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sodium borohydride (NaBH₄, 95%) was obtained from BDH chemicals Ltd. (Poole, UK). Orange essential oil was extracted from the peel of Vietnam’s sweet orange (citrus sinensis) and purchased from local company. All chemicals were used as received without further purification. Bacillus subtilis ATTC 6633 and Escherichia coli ATCC 25922 were obtained from Microbiologics, Inc (Saint Cloud, MN, USA).

Fluorescent-labeled JCM5805 were prepared as follows: 30 mg JCM5805 / 1 ml FITC solution [0.1 mg FITC isomer 1 (Sigma) / ml 0.1 M NaHCO₃ buffer, pH 9.0] was incubated for 60 min at 25°C and then washed three times with PBS. To investigate phagocytosis by CD11c⁺ cells, C57BL/6J mice were orally administered FITC-labeled JCM5805 (30 mg/mouse), sacrificed anaesthetically 8 hr later, and their Peyer’s patches (PP) were excised. PP cells were frozen in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA) and sliced into 6 μm sections. The sections were fixed in cold acetone (Wako) for 10 min at −20°C. Then the slides were washed with PBS containing 1% BSA and stained with affinity purified anti-CD11c (Thermo) for 2 hr at room temperature. The slides were washed again with PBS and stained with anti-rat IgG labeled with Alexa Fluor 546 (Invitrogen) for 30 min at room temperature. The slides were washed again with PBS and mounted with Fluoromount (Diagnostic Biosystems). Fluorescence microscopy was performed using an Olympus BX60.

NOB was synthesized by an established method^{50 (link)} and provided by the Ushio Chemix Corp. (Shizuoka, Japan). Geranic acid (85%), choline bicarbonate (~80% in water), dimethyl sulfoxide-d₆ (99.5 atom %D), chloroform-d (99.8 atom % D), and phosphate buffered saline tablets were purchased from Merck KGaA (Darmstadt, Germany). Acetone, 4% paraformaldehyde phosphate buffer solution, formic acid, and ethanol were purchased from Wako Pure Chemical Industries (Osaka, Japan). Diethylene glycol ethyl methyl ether was purchased from Tokyo Chemical Industries (Tokyo, Japan). Porcine skin (Yucatan Micropig) was purchased from Charles River Laboratories International (Yokohama, Japan). All other chemicals were of the highest grade commercially available, and all solutions were prepared in deionized and distilled water.

Ethanol (Wako), acetonitrile (Wako), mEthanol (Wako), sulfuric acid (Wako), acetone (Wako), trifluoroacetate (Tokyo Chemical Industry), dimethyl sulfoxide (Wako), MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide), LPS and Griess reagent were all purchased from Sigma (St. Louis, MO, USA). An ELx 800 Universal Microplate Reader (BIO-TEK), VD-250R Freeze Dryer (TAITEC), US-105 Sonicator (SND), 5420 Centrifuge (IMOTO), R-300 Rotavapor (BUCHI), V-300 Vacuum Pump (BUCHI), High Performance Flash Chromatography (HPFC) system (Biotage AB), Medium Pressure Liquid Chromatography (MPLC) system (EPCLC, Yamazen), Preparative High Performance Liquid Chromatography (PHPLC) system (EPCLC, Yamazen), 1220 Infinity LC (Agilent Technologies), NMR spectrometer (Bruker DRX-600; Bruker Daltonics, Billerica MA, USA), Quadrupole time-of-flight (qTOF) mass spectrometer (Agilent Technologies, USA) and JASCO DIP-370 polarimeter (JASCO, Tokyo, Japan) were used.