Microfluidic Nano‐Assembly of Red‐Blood‐Cell (RBC) Lipids and Components for Engineering Extracellular Vesicles

A central challenge in nanomedicine is the scalable and precise engineering of nanocarriers that are simultaneously tunable, immune-compatible, and capable of encapsulating diverse bioactive cargos. Extracellular vesicles (EVs), including native red blood cell-derived EVs (RBCEVs), offer biocompatibility but remain limited by heterogeneous composition, low yield, poor scalability, and limited control in cargo loading. Here, we introduce a bottom-up strategy for developing engineered red blood cell extracellular vesicles (eRBCEVs) through microfluidic diffusional mixing of purified RBC lipids and components. Parametric optimization through multi-physics simulation enabled predictive control over flow rates, lipid concentration, and channel geometry. eRBCEVs exhibited comparable encapsulation efficiency across molecular cargos of distinct sizes and properties, including oligonucleotides, gold nanoparticles, hemoglobin, erythrocruorin (∼3.6 MDa), and full adeno-associated virus (AAV ∼25 nm) particles, validated through cryo-EM, chemical mapping, and high-resolution TIRFM. Surface conjugation with CD47 peptide was established for comparison with RBCEVs; the bio-conjugations with α-PD-L1 antibodies yielded functionalized vesicles with uptake in PD-L1-positive human tumor organoids. In vivo biodistribution and pharmacokinetic analysis in mice demonstrated sustained circulation and tissue distribution in the liver, spleen, and lungs. Neutrophil activation and macrophage uptake assays confirmed minimal immunogenicity compared to conventional particles and free-protein controls. Collectively, our platform enables the scalable synthesis of customizable eRBCEVs that preserve RBC lipid features, support cargo flexibility, and potential for scale-up. This work establishes eRBCEVs as a next-generation therapeutic platform for programmable, patient-specific delivery in gene therapy, immunomodulation, and translational nanomedicine.