Surface-engineered heparin- and methoxy poly(ethylene glycol)-modified gelatin microspheres for enhanced...

Extracellular vesicles (EVs) are promising liquid biopsy biomarkers, but their practical use is limited by isolation methods that are labor-intensive, costly, or susceptible to nonspecific protein co-isolation. Here, we report surface-engineered gelatin microspheres (GMs) functionalized with heparin (Hep) and methoxy-polyethylene glycol (mPEG) for affinity-based EV enrichment with reduced background protein adsorption. Structural and interfacial characterization confirmed successful functionalization and tunable surface properties. Quantitative optimization identified mPEG2000/Hep-GMs as the preferred formulation, showing reduced nonspecific protein adsorption at pH 6.0. Using PANC-1 conditioned medium, mPEG/Hep-GMs achieved the highest particle-to-protein ratio among the tested groups (6.31 × 10⁷ particles/μg) while maintaining recovery of EV-sized particles. In exploratory plasma experiments using pooled clinical samples (0.5 mL), mPEG/Hep-GMs consistently yielded higher particle-to-protein ratios than Hep-GMs. Proteomic analysis further showed increased ExoCarta-associated protein proportions and reduced high-abundance plasma background protein signals, including albumin, immunoglobulins, transferrin, and apolipoprotein A1. Targeted multiple reaction monitoring showed stronger CD63 and CD81 signals in pancreatic cancer plasma-derived fractions than in non-cancer controls, and both Hep-GMs and mPEG/Hep-GMs outperformed a commercial precipitation-based EV isolation kit in EV-associated marker detection. The workflow requires approximately 30 min of hands-on operation, supports parallel processing, and avoids ultracentrifugation or antibody-based capture. These results demonstrate that mPEG/heparin -functionalized gelatin microspheres provide a simple and scalable platform for EV enrichment and downstream molecular analysis.