Fig. 3: A2-APM effectively crosses BTB.

a Schematic illustration of the in vitro BTB model using a transwell system to evaluate the penetration capability of A2-APM across the endothelial monolayer. Quantities of aPD-L1 were determined in the supernatant, bEnd.3 cells, filtrate, and GL261 cells. b Quantification of the aPD-L1 distribution in the chamber after incubation with free aPD-L1, APM and A2-APM for 4 h (n = 3 biologically independent samples). c, d Fluorescence imaging and signal intensities at tumor sites at 1, 2, 4, 6, 8, 12, 24, 48 and 72 h after i.v. injection of GL261 tumor bearing-mice with saline, aPD-L1, APM and A2-APM (n = 5). e, f Representative ex vivo fluorescence images and semiquantitative biodistribution of Cy7.5-aPD-L1 in brains collected from mice injected with saline, free aPD-L1, APM, and A2-APM at 90 min post i.v. injection (saline and free aPD-L1 treated mice, n = 4; APM and A2-APM treated mice, n = 5). g, h Representative ex vivo fluorescence images and semiquantitat ion of biodistribution of Cy7.5-aPD-L1 in main organs collected from mice injected with saline, free aPD-L1, APM, and A2-APM at 90 min post i.v. injection (saline and free aPD-L1, n = 4; APM and A2-APM, n = 5). i Representative histological images showing A2-APM accumulation in the TME of GL261 glioma. n = 3. Scale bar = 100 µm. j, k Biodistribution analysis of A2-APM-Alexa647 at 90 min after a single intravenous dose as assessed by flow cytometry of T cells (CD45⁺CD3⁺), NK cells (CD45⁺NK1.1⁺), microglia (CD45⁺CD11b^intermediate), monocyte-derived myeloid cells (CD45⁺CD11b^highLy6C⁺) and macrophages (CD45⁺CD11b^high F4/80⁺) in the tumor-bearing hemisphere, contralateral hemisphere and blood. A2-APM-Alexa647 treated mice, n = 5 and saline-treated mice, n = 3. All statistics are expressed as mean ± SD. Statistical significance was calculated by one-way ANOVA with Fisher’s LSD test in (b), (d) and (f), and two-sided t-test in (k). BioRender.com was used to create (a). Source data are provided as a Source Data file.