Fig 2: Schematic illustration of the NAcp@CD47 nanocapsule for dual delivery of anti-CD47 antibodies and STING agonists for GBM immunotherapy by repolarization of macrophages and promotion of phagocytosis. (A) The process of constructing the FAPá responsive prodrug nanocapsule: anti-CD47 antibodies and CDG are co-encapsulated in NAcp@CD47 using MPC, APM and FAP-α-responsive peptide crosslinker in situ polymerization nanocapsule. (B) The mechanism of NAcp@CD47 polarization of microglia and macrophages against GBM. After intravenous injection, NAcp@CD47 can be delivered to the CNS crossing the BBB mediated by MPC, release anti-CD47 antibodies and CDG after FAP-α enzymatic degradation in the GBM microenvironment to block the phagocytosis checkpoint CD47-SIRPα and promote the production of IFNs via the STING signaling pathway. Consequently, the reprogrammed microglia (or macrophages) enhances the phagocytosis of cancer cells. Similarly, IFNs facilitate increased infiltration of immune cells and increase immunogenicity to convert “cold” tumors into “hot” tumors.

Fig 3: NAcp@CD47-mediated phagocytosis in vitro. (A-B) Representative fluorescence images of phagocytosis assays, in which peritoneal macrophage (A) and brain microglia (B) from eGFP transgenic mice displayed green were co-cultured with GL261 GBM cells labelled with Hoechst 33342 (blue) for 30 min in the presence of NAcp@CD47, CD47 antibody or IgG control. Scale bar, 200 ìm. Experiments were repeated three times. (C-D) Representative flow cytometric analysis images (C) and relative quantification (D) of the phagocytosis of GL261 GBM cells by peritoneal macrophage and brain microglia in the presence of NAcp@CD47, CD47 antibody or IgG control. Data are presented as mean ± SD (n = 3). Phagocytosis or phagocytized cancer cells were quantified as the percentage of double-positive cells. ***P < 0.001; ns, not significant.

Fig 4: Characterization of NAcp@CD47. (A) TEM images of NAcp@CD47 (scale bar, 200 nm). (B) The average hydrodynamic size of NAcp@CD47 determined using dynamic light scattering (DLS). (C) TEM images of NAcp@CD47 after FAP-α enzymatic degradation. (D) Cell viability of GL261 glioblastoma cells and primary mouse astrocytes after incubation with various concentrations of NAcp for 24 or 48 h (n = 3, mean ± SD). (E-F) Cumulative release profiles of aCD47 (E) or CDG (F) from the NAcp@CD47 dispersed in artificial cerebrospinal fluid (ACSF) or PBS (25 ìg/mL) containing FBS with or without FAP-α enzyme at 37℃ as determined by high performance liquid chromatography (HPLC). (G) Schematic of the in vitro BBB model. (H) Average fluorescence signal of GL261 GBM cells in the in vitro BBB model after 4 h of incubation with NAcp@CD47-APC, Lipo@CD47-APC, or aCD47-APC with or without nAChR inhibitor. (I) Average fluorescence signal of GL261 GBM cells in the in vitro BBB model after 0 to 4 h of incubation with NAcp@CD47-APC, Lipo@CD47-APC, or aCD47-APC. (J) Ex vivo fluorescence imaging of brains from mice after injection of NAcp@CD47-APC, Lipo@CD47-APC, or aCD47-APC. Excitation/emission wavelengths 488/507 nm for GFP and excitation/emission wavelengths 633/670 nm for APC revealed a colocalization of GBM and NAcp@CD47 (scale bar, 2 mm). (K) In vivo fluorescence imaging of mice at indicated time points after intravenously injected with NAcp@CD47-APC, Lipo@CD47-APC, or aCD47-APC. (L-M) Quantitative analysis of in vivo (L) and ex vivo (M) fluorescence efficiency of NAcp@CD47 and control groups in the brain. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig 5: NAcp@CD47-mediated antitumor immune response in in vivo orthotopic syngeneic models for GBM. (A) Immunofluorescence analysis of frozen sections stained with IBA1 (red) in GBM tumors and peri-tumoral tissues in different groups. Scale bar, 200 mm. (B, D) iNOS, CD47, ARG1, CD8, and IBA1 protein expression levels in GBM tumors and peri-tumoral tissues of groups treated with NAcp@CD47 and aCD47, as determined by western blotting (B) and relative quantification (D). (C, E) Representative fluorescence microscopic images of frozen sections stained with TUNEL, iNOS, CD47, ARG1, CD8, CD47 and FAP-α in different groups (C) and relative quantification of the staining intensity (E). (F) The levels of IFN-β in tumor tissues and peri-tumoral tissues by ELISA. Scale bar, 200 ìm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.