Ivis lumina xr imaging system

After TCIs, mice were monitored weekly using caliper and fluorescence measurements, similar as described [28 (link)]. Once human 143B osteosarcoma cells established measureable primary tumors (unambigous mCherry signal and a volume greater than 25 mm³), drug treatment was started. mCherry tumor fluorescence was measured using an IVIS Lumina XR imaging system (Caliper Life Sciences, Inc., USA) and quantified with Living Image v3.1 software (Xenogen Corporation, USA).

The morphologies of BPQDs were recorded with a transmission electron microscope (TEM) with a working voltage of 200 kV (JEOL, JEM-2100, Japan). The TEM imaging of Apt-bioinspired MVs and energy dispersive X-ray analysis were performed using a field-emission TEM (Talos F200S). The SEM image of Apt-bioinspired MVs was obtained on a field-emission scanning electron microscope (Zeiss Merlin Compact). Atomic force microscope (AFM, Veeco, NanoMan) was performed using Bruker Multimode 8 operated in tapping mode. The NIR laser-induced heat conversion curves and photos were obtained with the FLIR A35 infrared thermal imaging camera (USA). UV–vis absorbance spectra were recorded on a UV–vis absorbance spectrometer (UV-2550, Shimadzu, Japan). The cell viability was tested on a confocal laser scanning microscope (FV1200, Olympus, Japan). The binding of aptamer with cells was evaluated by an Olympus D71 fluorescence microscope (Olympus, Japan). The tissue distribution was evaluated on an IVIS Lumina XR Imaging System (Caliper, America). Microcomputed tomography (μ-CT) reconstruction was recorded by the μ-CT imaging system (μ-CT50, Bruker Skyscan1172, Germany). The histomorphological analysis was carried out on an Olympus DP72 microscope (Olympus, Japan).

All chemicals and solvents used in syntheses were purchased from Sigma- Aldrich or Fisher Scientific and used without further purification. NQO1 was purchased from Sigma-Aldrich (D1515). NQO1 siRNA (h) (sc-37139) and NQO1 (A180) (sc-32793) antibodies were purchased from Santa Cruz. All cell lines were obtained from ATCC (American type cell culture collection) The ¹H, ¹³C NMR spectra were recorded on a Bruker-Avance 400 MHz Spectrometer. Chemical shifts (δ) are reported in ppm. ESI mass spectra were recorded on AB sciex QTRAP 5500 mass spectrometer. High-performance liquid chromatography (HPLC) was performed on an Agilent HPLC instrument. Peaks in NMR spectra are listed as singlet (s), doublet (d), triplet (t), or multiplet (m), and coupling constants (J) are reported in hertz (Hz). Fluorescence spectra were recorded on a Hitachi F-2500 Fluorescence spectrophotometer in a 10 mm standard cell with both excitation and emission slit widths of 10 nm. The incubation of NQO1 with NIR-ASM in the presence of NADH was carried out on a shaker at 37 °C. IVIS Lumina XR Imaging system (Caliper Life Sciences, Inc.) was used for the in vivo imaging. The BD LSRFortessa™ cell analyzer was used for the flow cytometric analysis. A Nikon multiphoton microscope equipped with NIS-Elements C acquisition and analysis software was used for the fluorescence microscopy.

As the reference described [26 (link)], mice received 150 mg/kg D-Luciferin (Caliper Life Science Part Number #119222) intraperitoneally, were anesthetized with 2.5-3.5% isoflurane, and imaged after 15 min, which is the peak of maximum bioluminescence, with an IVIS Lumina XR imaging system (Caliper, USA). The whole body was imaged in a supine gesture. Bioluminescence intensity is expressed as photons per second (p/s). Then the mice were returned to their cages where they awake quickly.

On day 50, plain radiographs of the hind paws were obtained using an IVIS Lumina XR Imaging System (Caliper, MA, USA). Then the bone and muscle were separated into two parts and fixed in 10% neutral phosphate-buffered formalin respectively. 24 h later, the muscles were used to detect the expression levels of IL-6, IL-1β and IL-10 with immunohistochemical staining as previously [13 (link)]; 7 days later, the bone specimens were decalcified in mixed acid solution (8% hydrochloric acid, v/v; 5% acetic acid, v/v; 10% salicylic acid, m/v) for 2–3 weeks. The decalcified bones were used for histopathological evaluation on the joint injury with hematoxylin & eosin staining and proteoglycan assessment on cartilage degeneration with Safranin O staining as previously [13 (link), 14 (link)].