Design, synthesis and preclinical evaluation of small-molecule FAP-targeted photosensitizers for photodynamic therapy.

Fibroblast activation protein (FAP)-expressing cancer-associated fibroblasts are an attractive target for spatially controlled photodynamic therapy (PDT) of pancreatic ductal adenocarcinoma (PDAC). Here, we report the design, synthesis and preclinical evaluation of two FAP-targeted small molecules for application in PDT. Both compounds combine a FAP-binding motif based on FAPI-04, a DOTA chelator for indium-111 radiolabeling, and IRDye700DX as photosensitizer, but differ in the linker used for photosensitizer attachment. PhoFAP-01 was prepared via a DBCO-based SPAAC strategy, whereas PhoFAP-02 contains a compact amide linkage. Both small molecules retained FAP-mediated binding to 3T3-FAP cells and induced light-dependent cytotoxicity after 690 nm irradiation. Linker chemistry had a strong effect on performance: PhoFAP-02 showed higher apparent FAP affinity than PhoFAP-01 (IC50 28 nM versus 76 nM) and displayed improved phototoxic selectivity, with no detectable dark toxicity or light-induced toxicity in FAP-negative cells. In mice bearing orthotopic syngeneic PDAC299 tumors, 111In-labeled PhoFAP-02 reached 6.6 ± 1.1%ID/g tumor uptake at 1 h post-injection and accumulated in FAP-rich stromal regions, as shown by autoradiography and FAP immunohistochemistry. These findings identify PhoFAP-02 as a promising small-molecule photosensitizer for FAP-targeted PDT and highlight the importance of linker design for preserving target binding and phototoxic selectivity. Future studies should evaluate therapeutic efficacy after light activation in vivo.