A Bioorthogonal Strategy for Preparing BMP4-Associated HempDerived Extracellular Vesicle Preparations: Comparative Evaluation of Reaction Conditions and Physicochemical Characterization

Plant-derived extracellular vesicle (PEV) preparations are promising bio-derived platforms for therapeutic cargo association, although controlled modification of heterogeneous PEV preparations remains challenging. Here, a proof-of-concept evaluation of a two-component dibenzocyclooctyne (DBCO)/azide workflow was performed for preparing bone morphogenetic protein 4 (BMP4)-associated hemp-derived extracellular vesicle (HEV) preparations under mild, strain promoted azide–alkyne cycloaddition-compatible conditions. Purified native HEV preparations were characterized by nanoparticle tracking analysis and transmission electron microscopy, supporting predominantly nanoscale particle populations with vesicle-like morphology. Fluorescamine accessible primary amines were quantified to define nominal DBCO-reagent-to-amine feed conditions, and DBCO-responsive behavior was compared using a 9-(azidomethyl) anthracene (Anth-N3) fluorescence assay. At a nominal feed ratio of 4.5, apparent DBCO-responsive conversion metrics were 13.0 ± 0.64% at pH 7.4 and 18.3 ± 2.74% at pH 8.5, whereas exploratory higher-feed conditions (~30–50) produced values around 12%. Following incubation of DBCO-treated HEVs with azide reagent-treated BMP4 and purification, enzyme-linked immunosorbent assay (ELISA) showed particle-normalized BMP4 association of approximately 1.60 and 6.66 pg per 109 particles at pH 7.4 and 8.5, respectively, compared with approximately 0.04–0.60 pg per 109 particles for exploratory high-feed preparations. Nano-flow cytometry further detected fluorescence associated with fluorescein-labeled BMP4 within membrane-dye-positive particle populations. For the Anth-N3-responsive and particle-normalized ELISA readouts, the lower-feed pH 8.5 condition was most favorable among those evaluated. recovered materials are conservatively described as BMP4-associated HEV preparations because the analytical workflow was comparative rather than bond-specific. These findings support continued development and more comprehensive validation of bioorthogonal protein-association strategies for PEV preparations.