Ivis lumina 3 fluorescence imaging system

As an in vivo hypoxia model, BALB/c mice with CT26 tumors were employed. The CS release from hypoxia-responsive COFs (TAP) was measured using a FRET-based technique, with azo-free COFs (TBP) serving as a control. The FRET pair cy5/cy7 was loaded into TAP and TBP at a ratio of 1:1:10 (cy5/cy7/COFs, w/w/w) using a manner identical to that used for CS encapsulation. The concentration of cy5/cy7 in both COFs was measured fluorescently. The mice were given either cy5/cy7@TAP or cy5/cy7@TBP (12.5 μL, 20 mg mL⁻¹) intratumorally. At predetermined time points post COFs dosing (0, 2, and 4 h), the mice were scanned using an IVIS® Lumina III fluorescence imaging system (PerkinElmer). The excitation wavelength of cy5 was 640 nm; the emission wavelengths were 670–730 nm (cy5) and 730–790 nm (cy7), respectively. The in vivo FRET ratio was calculated by dividing the emissive fluorescence intensity at 730–790 nm against that at 670–730 nm.