U 2810 spectrophotometer

Dauno-MNC, HA/DNR complex or free DNR were transferred to Float-A-Lyzer dialysis tubes with a molecular-weight cutoff of 3.5–5 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA). The amount of DNR per tube was fixed to 20 µg. The dialysis tubes were immersed in 5 mL of 10 mM sodium citrate buffer (pH 5.5) or 10 mM PBS (pH 7.4) and then incubated at 37 °C on an orbital shaker at 50 rpm with Parafilm sealing to minimize water evaporation. At selected time points, 1 mL of the release fraction was transferred to a quartz cuvette and its absorbance at 480 nm was measured on a Hitachi U-2810 spectrophotometer. After each measurement, the release fraction was returned back to the dialysis tube. The extent of DNR release was determined by comparing the absorbance of nanocomplex samples at 480 nm with those of a series of DNR standard solutions (0.2–10 µg mL⁻¹).

The purified compounds were prepared at a concentration of 1 mg/mL in MeOH for the measurement of optical rotation, UV spectra, and IR spectra. An optical rotation [α]_D of the compound suspension was measured using a P-2200 polarimeter (JASCO, Tokyo, Japan). UV spectra of each compound were recorded with a U-2810 spectrophotometer (Hitachi High-Tech Science Co., Tokyo, Japan), and IR spectra (ATR) were measured using a FT–IR 4600 (JASCO, Tokyo, Japan). The isolated compounds were dissolved in chloroform-d (CDCl₃) or methanol-d₄ (CD₃OD) for NMR analyses. NMR spectra of each compound were obtained on a JNM ECP500 NMR spectrometer (JEOL, Tokyo, Japan) with 500 MHz for ¹H NMR and 125 MHz for ¹³C NMR. Chemical shifts (ppm) of CDCl₃ (δ_H 7.26, δ_C 77.0) and CD₃OD (δ_H 3.30, δ_C 49.0) were used as references. The accurate mass and molecular formulas of the isolated compounds were established by liquid chromatography–mass spectrometry (LC–MS) analyses. Spectra of electron ionization mass spectrometry (EI–MS) were analyzed using a JMS-AX505 HA spectrometer (JEOL, Tokyo, Japan), while the spectra of electrospray ionization mass spectrometry (ESI–MS) were obtained by a JMS-T100LP spectrometer (JEOL, Tokyo, Japan) equipped with an Agilent1100 HPLC system (Agilent, CA, USA).

L. bulgaricus and S. thermophilus were cultured anaerobically in de Man Rogosa Sharpe broth (Becton Dickinson, Cockeysville, MD, USA) and M17 broth (Becton Dickinson, Cockeysville, MD, USA) supplemented with 1% lactose, respectively, at 37 °C for 18 h. For in vitro cytokine production assays, bacteria were washed twice with phosphate-buffered saline (PBS; pH 7.4) and suspended in PBS so that the optical density at 600 nm was 2.0 using U-2810 spectrophotometer (Hitachi, Tokyo, Japan). Then, bacteria were heat-killed at 75 °C for 1 h.
All bacteria used in this study are the property of Meiji Co., Ltd.

Sora-MNC-1 was transferred to Float-A-Lyzer dialysis tubes with a molecular weight cutoff of 3.5–5 kDa (Spectrum Laboratories, USA). The amount of sorafenib per tube was fixed to 7.5 µg. The dialysis tubes were immersed in total 5 mL of 10 mM PBS (pH 7.4) without or with either 100 mM urea or 0.1% Tween-20, and subsequently incubated at 37 °C on an orbital shaker at 50 rpm with Parafilm sealing to minimize water evaporation. At selected time points, 1 mL of the release fraction was transferred to a quartz cuvette and its UV-visible spectrum was acquired on a Hitachi U-2810 spectrophotometer. After each measurement, the release fraction was returned back to the dialysis tube. The extent of drug release was determined by measuring the absorbance of sorafenib at 265 nm.