Comparative study on PD-L1/ALK dual-targeted biomimetic exosomes by two preparation approaches in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) remains a fatal malignancy with a highly immunosuppressive and fibrotic tumor microenvironment, extremely limiting drug penetration and therapeutic efficacy. Simultaneously targeting PD-L1 and ALK might overcome the therapeutic bottleneck of PDAC. In this study, we developed a novel dual-targeted nanoformulation designed to enhance targeting and therapeutic bioactivities in PDAC. Specifically, we utilized a human naïve phage display library to identify humanized monoclonal antibodies with high affinities for PD-L1, followed by constructing the single-chain variable fragments (scFvs) and chemically conjugating to ceritinib-loaded biomimetic exosomes (Cer-BEs) that were prepared via two different methods of ultrasonic extrusion and freeze-thaw. The formulation of PD-L1-scFvs-conjugated Cer-BEs (PD-L1@Cer-BEs) possessed significant binding activity to PD-L1, cell uptake rates and cytotoxicity in high PD-L1 expressed cells, superior tumor targetability and growth inhibitory bioactivities in PDAC. Compared to freeze-thaw, ultrasonic extrusion yielded PD-L1@Cer-BEs with smaller particle sizes, higher homogeneity, superior encapsulation efficiency and stability, enhanced anti-tumor effectiveness in vitro and vivo, indicating ultrasonic extrusion is more suitable for constructing PD-L1@Cer-BEs with desirable physicochemical properties and bioactivity. These findings not only prove the novelty and effectiveness of PD-L1@Cer-BEs targeting PDAC but also underscore the critical role of BE preparation in determining the efficacy of antibody-drug conjugate (ADC)-like targeted BEs.