400 nmr spectrometer

Substrate (CYBA or CUBA) and PBS buffer (100mM, pH 7.4) were incubated with BChE for 30 min at 37°C. Then cold acetonitrile was added into the incubations to terminate the reaction. After centrifugation, a preparative HPLC system equipped with a reversed-phase column was used to purify the supernatant. The purified metabolites (TBO and TBBO) were dissolved in dimethylsulfoxide-d₆ for structural analysis using Bruker 400 NMR spectrometer, with a ¹H NMR (600 MHz) and ¹³C NMR (150 MHz) recording. Meanwhile, the CYBA metabolites were centrifuged at 20,000 g for 15 min at 4 °C. Subsequently, the supernatant metabolites were all characterized by UHPLC-MS/MS.

The ¹H NMR and ¹³C NMR spectra of Compounds 1–6 in deuterated chloroform (CDCl₃) were measured by the Bruker 400 NMR spectrometer at 400 MHz, and the values were explained in relation to the remaining non-deuterated solvent signal. The unit of measurement for coupling constants was Hertz (Hz). Chemical shifts were quantified in ppm.

${}^1\text{H}$ and ${}^{13}\text{C}$ NMR spectra were recorded using Bruker 400 NMR spectrometer using ${\text{CDCl}}_3$ as solvent. FE-SEM images and EDS analyses were recorded using Zeiss Merlin compact Microscope and Oxford instruments, respectively by drop casting the nanoparticles sample on carbon tape. HRTEM images were acquired using JEOL JEM 2100 electron microscope. Powder XRD analyses were carried out using a Bruker D8 Advance Diffractometer (Bruker AXS) with Cu ${\text{K}}_{\mathrm{\alpha }}$ radiation ( $\mathrm{\lambda }=1.54$ Å) over a $2\mathrm{\theta }$ range of ${10}^{\circ }-{110}^{\circ }$ with a scanning rate of ${40}^{\circ }/\text{min}$ . The samples for powder XRD was prepared by making a thin film of nanocomposites on glass slide.

All commercial chemicals were of reagent grade from VWR (Radnor, PA), Aldrich (St. Louis, MO), or Oakwood Chemicals (Estill, SC), and were used without further purification unless otherwise indicated. ¹H and 13C spectra were obtained on a Bruker 400 NMR spectrometer at 400 and 100 MHz, respectively, in deuterated solvent with TMS (δ = 0.00 ppm) or deuterated solvent as internal reference. For all reactions, analytical grade solvent was used. Anhydrous solvents were used for all moisture-sensitive reactions. The Mass Spectrometry Facilities at Georgia State University obtained high-resolution mass spectra on a Waters Micromass Q-TOF (ESI) instrument.

The ¹H NMR spectra of compounds 1–5 in deuterated chloroform (CDCl₃) were measured at 400 MHz using a Bruker 400 NMR spectrometer, and the values are presented in relation to the residual non-deuterated solvent signal. The coupling constants are given in Hertz (Hz). Chemical shifts are quantified in δppm.

Malvern Z90 Zetasizer equipped with a 633 nm laser and an avalanche photodiode detector was used to characterize the hydrodynamic size. ¹H NMR spectra were recorded using a Bruker 400 NMR spectrometer. Transmission Electron Microscopy (TEM) images were recorded on a FEI Tecnai 20 (type Sphera). Scanning electron microscopy (SEM) images were characterized by FEI Quanta 200 3D FEG. Ultraviolet-visible Spectroscopy (UV/Vis) absorbance was recorded on a Jasco V-650 UV/Vis spectrometer. The O₂ concentration was determined using a portable meter MultiLine® Multi 3510 IDS. Microplate reader (Safire², TECAN) was employed for the CCK-8 assay. Fluorescence images were observed and captured by QuantaMaster-40 fluorescence S-3 spectrophotometer and Leica TCS SP5X. A Becton Dickinson FACScan flow cytometer was used to determine the cellular uptake. The photoacoustic images were captured by an in vivo photoacoustic system (MSOT inVision, iThera Medical). The in vivo circulation behavior was recorded by an in vivo imaging system (PerkinElmer IVIS Lumina Series III). The ultrasound imaging was performed on a Mindray resona 7. FUS treatment was conducted using Intelect® Mobile Ultrasound. Tumor position temperature changes were collected by a thermal imager (FLIR A300, IRS Systems Inc.) coupled with an infrared camera.