Fig. 6

Characterization and in vivo validation of the Pep-Cu_5.4O@H151 cascade-targeting nanotherapeutic. a Schematic of cascade targeting and peptide sequences: Ionizing radiation disrupts BBB integrity, enabling BBB penetration and subsequent MMP-9-responsive activation at neuroinflammatory lesions. MMP-9 cleaves the shielding peptide to expose targeting motifs for activated M1 microglia. The BBB peptide sequence is PPWWYYYLVVAA-LGLPG-YEETKFNNRKGRSGGYFF, and the MG1 peptide sequence is PPWWYYYLVVAA-CHHSSSARC. The peptide sequences were designed in ChemDraw software, and the scheme image was created in BioRender. Shang, Y. (2025) https://BioRender.com/h2 5oed1 (agreement number: DV28JKG5ZS). b Z-potential measurements confirming surface charge modification. c Drug-loading efficiency at various precursor ratios. d pH-dependent drug release profiles (pH = 6.5, 7.4). e TEM image of Pep-Cu_5.4O@H151. f–i ROS scavenging capacity: Hydroxyl radical (•OH, f), hydrogen peroxide (H₂O₂, g), superoxide anion (O₂⁻, h), and total ROS elimination (i). j Whole-body fluorescence imaging showing time-dependent brain accumulation after intravenous injection in mice. k, l Ex vivo brain imaging (k) and quantitative ROI analysis (l, n = 3 mice; one-way ANOVA). m, n Immunofluorescence image of a brain section showing the colocalization of Cy5 (red) with iba1⁺ microglia (green) (m), and the results were quantified (n, one-way ANOVA). All the statistical data are presented as the means ± SEMs. Scale bar: 100 nm (e); 20 μm (m)