1260 system

Column chromatography was carried out using silica gel (3.0 × 30 cm, Qingdao Marine, China), CHP20P (3.0 × 25 cm, Mitsubishi, Japan), octadecylsilyl silica (ODS) (YMC, Japan) and Sephadex LH-20 (1.5 × 180 cm, GE Healthcare, Sweden). Semi-preparative high performance liquid chromatography (HPLC) was conducted on a 1260 system (Agilent, USA) equipped with an XDB-C₁₈ column (9.4 × 250 mm, 5 μm, Agilent, USA). Ultra violet (UV) spectra were determined using a 241 spectrophotometer (Perkin Elmer, USA). High resolution electrosparay ionization mass spectrum (HR-ESIMS) spectra were recorded on a 6545 Q-TOF instrument (Agilent, USA). Nuclear magnetic resonance (NMR) spectra were acquired using an AV-600 instrument (Bruker, Germany). Electronic circular dichroism (ECD) spectra were obtained using a MOS-450 instrument (Bio-Logic, France). The solvents used in column chromatography were analytical grade (Tianjin Hengxing, China), the solvents used in HPLC, HR-ESIMS, ECD and activity assays were chromatographic grade (Sigma-Aldrich, USA), and the solvent used in NMR was methanol-d₄ (Cambridge Isotope Laboratories, USA).

CLAE-DAD analysis was performed as described by [48 (link),49 (link)]. An Agilent 1260 system equipped with a Zorbax C18 column (150 mm × 4.6 mm × 3.5 µm) at 45 °C was used. A gradient of 0.1% acetic acid (AcOH) and acetonitrile (CH₃CN) was employed as the mobile phase, and the start composition was 10% CH₃CN (0–6 min), followed by an increase to 15% (6–7 min), remaining isocratic for 15 min, followed by an increase to 50% (22–32 min) and to 100% (32–42 min), and then kept in isocratic conditions for an additional 8 min. The solvent flow rate was set at 1.0 mL/min, and the injection volume for analysis was 10 µL at a concentration of 2 mg/mL. The chromatogram was processed with λ = 352 nm, and compound suggestions were based on the analysis of absorption-UV−visible spectra and through a comparison of retention times with standards.

EVs were pre-treated for HPLC measurement using the methods reported by Pascucci et al.^{20 (link)} Briefly, EV samples were added to equal volumes of 0.6 N perchloric acid, mixed and centrifuged at 200×g at 4 °C for 10 min, followed by filtration and injection into HPLC. An Agilent 1260 system combined with diode-array detection (DAD) was used for HPLC analysis of these samples. An Agilent C18 column (5 µm, 250 mm × 4.6 mm) with an HPLC guard cartridge system (Phenomenex, SecurityGuard) was used for HPLC analysis. The mobile phases consisted of acetonitrile (A) and 20 mmol/l KH₂PO₄ with pH adjusted to 3 with phosphoric acid, (B) using an isocratic elution program of 37% A for 0–30 min. The flow rate was 1. 0 ml/min, and column temperature was set to 30 °C. The DAD detector was set at 210 nm.

Absorption spectra of nanoparticles were measured by Lambda 950 spectrometer. Fluorescence spectra of nanoparticles were measured by Fluorolog 3-TCSPC spectrometer. 1H NMR of intermediates was measured on Bruker Avance II 300 MHz NMR spectrometer. DLS and zeta potential profiles were measured on Malvern Nano-ZS Particle Sizer. TEM images were captured by JEOL JEM 1400 transmission microscope (accelerating voltage 100 kV). Photothermal temperature of solution and tumor was measured by FLIR thermal camera (T420). Laser source (1064 nm) was purchased from Shanghai Connet Fiber Optics. HPLC was performed on an Agilent 1260 system with ACN (0.1% of trifluoroacetic acid) and H₂O (0.1% of trifluoroacetic acid) as the eluent. LCMS spectra were measured on Thermo Finnigan Polaris Q quadrupole ion trap mass spectrometer equipped with a standard electrospray ionization (ESI) source. Flow cytometry was measured on Fortessa X20 (BD Biosciences). Confocal fluorescence images were captured on Zeiss LSM 800 confocal laser scanning microscope. In vivo NIR fluorescence imaging was performed on IVIS imaging system (PerkinElmer). Tumor and organ tissues were dissected on a cryostat (Leica). H&E images of tissue sections were captured on ECLIPSE 80i microscope (Nikon). NMR spectra were analyzed using Mestre Nova LITE v5.2.5-4119 software (Mestre lab Research S.L.).

The analysis was performed on 0.5 g of a faecal sample, which was then homogenized in 5 mL of water for 5 min. The pH was acidified to pH = 3 with 5M HCl and further samples were centrifuged for 20 min. The obtained samples were analyzed by gas chromatography with a flame ionization detector (FID). SCFA analysis included: acetic acid (C2: 0), propionic acid (C3: 0), isobutyric acid (C4: 0 i), butyric acid (C4: 0 n), isovaleric acid (C5: 0 i), and valeric acid (C5: 0 n). The analysis was performed using an Agilent Technologies 1260 System ( Santa Clara, USA) gas chromatograph on a DB-FFAP column, 30 m × 0.53 mm × 0.5 µm. Hydrogen was supplied as carrier gas at a flow rate of 14.4 mL/min. The starting temperature was 100 °C. It was held for 0.5 min and then raised to 180 °C at a rate of 8 °C/min and held for 1 min. The temperature was then increased to 200 °C at a rate of 20 °C/min and held at 200 °C for 5 min. The fatty acids were identified by comparing their retention times with commercially available standards.