Extracellular vesicles (EVs) are biologically derived nanoparticles secreted by cells under physiological and pathological conditions, naturally transporting proteins, lipids, and nucleic acids between cells. Their inherent biocompatibility and ability to cross biological barriers make them attractive candidates for drug delivery. Among them, red blood cell-derived EVs (RBCEVs) are especially promising due to their immunological stealth and lack of nuclear content. However, conventional RBCEVs derived through endogenous biogenesis suffer from heterogeneity in size and composition, limited encapsulation efficiency, and poor scalability—critical barriers for their clinical translation. This study addresses these limitations by developing a bottom-up, microfluidic-based approach to engineer red blood cell extracellular vesicles (eRBCEVs) with tunable, reproducible, and functional properties.
Methods
A microfluidics-enabled platform was designed for the bottom-up synthesis of eRBCEVs using purified human RBC membrane extracts. We employed multiphysics simulation-guided optimization to determine flow rate ratios (FRRs), lipid concentrations, and microchannel geometries for enhanced chaotic mixing and efficient lipid self-assembly. This enabled formation of vesicles with precisely controlled diameters (80–200 nm) and low polydispersity (<0.2). Characterization was conducted using transmission electron microscopy (TEM), cryo-transmission electron microscopy (cryo-TEM), nanoparticle tracking analysis (NTA), zeta potential measurements, and total internal reflection fluorescence microscopy (TIRFM). Molecular cargos including human hemoglobins (hHb) and oligonucleotides were loaded in situ during vesicle formation. Surface modification was achieved using strain-promoted azide–alkyne click chemistry to conjugate anti-PD-L1 nanobodies to eRBCEVs. Organoid uptake studies were performed in PD-L1–positive (MDA-MB-231) and PD-L1–low (MCF7) breast cancer spheroids to evaluate targeting specificity. In vivo biodistribution and pharmacokinetics were assessed in BALB/c mice injected with DiR-labeled eRBCEVs and conjugates via IVIS imaging at 4 and 24 hours post-injection. Immune evasion was evaluated via neutrophil activation assays and morphological assessments.
Results
Microfluidic synthesis yielded highly monodisperse vesicles with consistent size distributions influenced by both FRR and lipid concentration, as verified through ANOVA and Tukey-Kramer statistical tests. Herringbone devices enhanced mixing efficiency compared to flow-focusing device and resulted in unimodal populations with near-neutral zeta potential, ideal for systemic circulation. Encapsulation of molecular cargos was robust: hHb showed encapsulation efficiencies >70%, confirmed by NTA and TEM. Oligonucleotides achieved >55% co-localization under TIRFM, confirming stable internalization and preservation of vesicle integrity. Surface-functionalized eRBCEVs demonstrated ~60% conjugation efficiency with anti-PD-L1 nanobodies. In PD-L1–expressing MDA-MB-231 organoids, functionalized eRBCEVs showed markedly higher uptake compared to MCF7, confirming targeted delivery and immune checkpoint relevance. In vivo imaging showed primary accumulation in the liver and spleen, consistent with uptake by the mononuclear phagocyte system. Notably, lungs exhibited significant signal at both early and late time points, likely reflecting native erythrocyte transit through the pulmonary vasculature thereby suggesting potential for pulmonary therapeutics. Pharmacokinetic analysis indicated peak circulation between 2 and 4 hours, with conjugated vesicles demonstrating marginally extended half-life. Compared to cationic lipid nanoparticles (cLNPs), eRBCEVs elicited significantly lower neutrophil activation, attributed to the biomimetic composition of RBC-derived lipids and absence of immunogenic proteins thereby highlighting their suitability for repeated administration.
Conclusion
This study presents a microfluidics-based toolkit for engineering eRBCEVs with customizable physicochemical properties, tunable size, and efficient therapeutic payload encapsulation. The platform’s ability to deliver molecular cargos like hemoglobins and oligonucleotides, while enabling precise PD-L1-targeted delivery, establishes it as a promising candidate for cancer immunotherapy, gene modulation, and oxygen delivery applications. The observed lung tropism further opens possibilities for respiratory therapies. Future directions include translation toward GMP-grade production, and exploration of disease-specific targeting. Lastly, the platform offers a highly adaptable, immune-compatible foundation for developing next-generation nanocarriers.
Keywords: Extracellular Vesicles, Drug Delivery, Microfluidics