Synthesis of radioafterglow nanoprobes for deep-tissue imaging of hydrogen peroxide

Afterglow imaging eliminates the need for real-time excitation light, thereby greatly reducing tissue autofluorescence to achieve highly sensitive bioimaging. However, the clinical translation of existing photoafterglow and sonoafterglow probes developed so far is hampered by their limited tissue penetration. To address this, we recently developed radioafterglow nanoprobes (RANPs) on the basis of a cascade X-ray energy-converting mechanism for cancer-specific imaging and image-guided surgery. RANPs are constructed by co-assembling radioabsorbers, radiosensitizers and radioafterglow substrates into the amphiphilic polymers using the film rehydration method. This approach is rapid (<10 min), offers precise control over the concentration of encapsulated compounds and is highly scalable. Upon X-ray irradiation, RANPs exhibit near-infrared radioafterglow emission (peaked at 788 nm), with long half-life (~4.8 min) and high brightness. These properties enable an excitation depth of the RANPs up to 15 cm, three times deeper than that of photoafterglow. Here we further develop a tumor-specific RANP for precision molecular imaging that activates its radioafterglow signal specifically in the presence of hydrogen peroxide, a key biomarker of the tumor microenvironment. This design enables the ultrasensitive detection (signal-to-background ratio of 169) and surgical resection of small tumors (1 mm³) under an X-ray dosage 20 times lower than that required for inorganic materials. This protocol is suitable for users with expertise in chemistry, biology and materials science. The typical timeline is 1-2 weeks for nanoprobe construction and characterization, 1-2 weeks for cell assays and 3-4 weeks for animal experiments.