Biolayer interferometry-based detection of engineered extracellular vesicles binding to type I collagen

Engineered extracellular vesicles (EVs) have garnered significant attention due to their potential serving as both therapeutic agents and drug delivery vehicles. Real-time monitoring of engineered EVs-target interactions constitutes a critical need for advancing EVs-based therapeutics. Here, we present a novel biolayer interferometry (BLI)-based analytical platform for detecting dynamic interactions between collagen-binding domain (CBD)-engineered EVs and type I collagen (Col I). A stable CD63-CBD fusion protein-expressing mouse tendon cell line (designated TT-D6s^CBD cells) was initially established using a lentiviral transduction system. Engineered EVs (TT-D6s^CBDEVs) were subsequently isolated through optimized differential ultracentrifugation. Col I was covalently immobilized on AR2G biosensors for detecting EVs binding using BLI technology. Quantitative analysis revealed that TT-D6s^CBDEVs-treated biosensors demonstrated significantly enhanced BLI signals versus control group (TT-D6s^VecEVs), along with time-dependent signal accumulation. Correlative microscopy validation-including scanning electron microscopy (SEM) and confocal microscopy imaging-confirmed the enhanced EVs binding capacity in the test group compared to controls. This work establishes a paradigmatic methodology for characterizing binding kinetics between engineered EVs and their molecular targets, offering critical technical support for advancing precision EVs-based therapeutics.