Microfluidic-engineered red blood cell EVs (eRBCEVs) enable scalable, tunable cargo delivery with low immunogenicity.

Developing effective nanocarriers for drug delivery faces challenges in achieving tunability, immune-compatibility, and broad cargo encapsulation at scale. While native extracellular vesicles (EVs), particularly red blood cell-derived EVs (RBCEVs), offer intrinsic biocompatibility, their clinical utility is hampered by heterogeneous composition, low yield, and limited control over cargo loading. This research addresses the critical need for a scalable, precisely engineered nanocarrier platform that overcomes these limitations, paving the way for advanced therapeutic applications in areas like gene therapy and immunomodulation.

Researchers engineered red blood cell extracellular vesicles (eRBCEVs) via microfluidic diffusional mixing of purified RBC lipids and components. Multi-physics simulation guided parametric optimization for predictive control of flow rates, lipid concentration, and channel geometry. Encapsulation efficiency was assessed across diverse cargos: oligonucleotides, gold nanoparticles, hemoglobin, erythrocruorin (~3.6 MDa), and full adeno-associated virus (AAV ~25 nm). Validation used cryo-EM, chemical mapping, and high-resolution TIRFM. Surface conjugation with CD47 peptide and α-PD-L1 antibodies was performed. In vivo biodistribution and pharmacokinetic analyses in mice, plus neutrophil activation and macrophage uptake assays, completed the study.

eRBCEVs exhibited comparable encapsulation efficiency across a wide range of molecular cargos, from oligonucleotides to large AAV particles (~25 nm) and erythrocruorin (~3.6 MDa), demonstrating cargo flexibility. Surface conjugation with CD47 peptide was successfully established, showing comparability to native RBCEVs. > Functionalized eRBCEVs with α-PD-L1 antibodies demonstrated specific uptake in PD-L1-positive human tumor organoids, highlighting their potential for targeted delivery. In vivo studies in mice revealed sustained circulation and broad tissue distribution, including the liver, spleen, and lungs. Furthermore, neutrophil activation and macrophage uptake assays confirmed minimal immunogenicity for eRBCEVs when compared to conventional particles and free-protein controls, suggesting enhanced immune compatibility.