Osmolarity‐Directed Encapsulation of Size‐Tuned Nanoparticles into Red Blood Cells

Abstract Red blood cells (RBCs) have emerged as promising carriers for therapeutic and diagnostic agents due to their long circulation time, biocompatibility, and immune‐evasive properties. Hypotonic encapsulation is the most widely employed technique; however, the correlation between nanoparticle sizes and the hypotonic osmolarities required for efficient encapsulation remains unclear. In this study, the size‐dependent osmolarity requirements governing nanoparticles into RBCs are investigated. Using monodisperse gold nanoparticles as model systems, the optimal osmolarity correlated with nanoparticle diameter is identified as 150 mOsm for particles ≤33 nm, 100 mOsm for particles 66–91 nm, and 50 mOsm for particles ≈133 nm. These conditions maximized encapsulation efficiency while maintaining RBC membrane integrity and preserving the expression of key surface proteins, including CD47. The applicability of this approach is further validated using nanoparticles of diverse compositions and zeta potentials. In vitro assays demonstrated minimal hemolysis and significantly reduced macrophage uptake across all formulations. Complementary in vivo imaging reveals prolonged systemic circulation and biodistribution profiles that closely resemble those of native RBCs. This study establishes a standardized, size‐adaptive hypotonic encapsulation protocol, offering a versatile and scalable platform for engineering RBC‐based carriers with broad translational potential in nanomedicine.