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Erythrocytes have been extensively investigated as drug delivery vehicles. However, many existing methods for loading therapeutic biomolecules into erythrocytes perturb the cell membrane, resulting in a loss of erythrocyte structural integrity and recognition by the host immune system. To overcome this challenge, we developed a novel nanoparticle carrier for transporting proteins into intact erythrocytes. We designed and characterized a nanoparticle where a poly-L-lysine-grafted-poly(ethylene) glycol copolymer was used to encapsulate a model protein, bovine serum albumin. The nanoparticle was functionalized with either an erythrocyte targeting peptide ERY1, the cell-penetrating peptide low molecular weight protamine, or a combination of the two peptides. The three resulting constructs, ENP, LNP, and ELNP, had a core/shell structure and, depending on functionalization, were approximately 30–90 nm in diameter and had negative surface charge. Esterase-like activity of the encapsulated protein was not affected by encapsulation. Under physiological conditions, nanoparticles caused no significant hemolysis and were resistant to dissociation by surfactants. The three nanoparticle constructs were evaluated using confocal microscopy and flow cytometry for their ability to bind and internalize within intact erythrocytes. LNPs showed significant binding and internalization into intact erythrocytes, with ~90% of erythrocytes containing particles after 24 h of co-incubation. Internalization occurred without adversely affecting erythrocyte membrane structure. Together, these results demonstrate that low molecular weight protamine–functionalized nanoparticles may serve as a universal platform to deliver protein therapeutics into erythrocytes. [ABSTRACT FROM AUTHOR]

Purpose: Nanoparticle (NP) attachment to biocompatible secondary carriers such as red blood cell (RBC) can prolong blood residence time of drug molecules and help create next-generation nanotherapeutics. However, little is known about the impact of RBC-targeted NPs on erythrocyte function. Methods: The objectives of this study were to develop and characterize in vitro a novel poly-L-lysine (PLL) and polyethylene glycol (PEG) copolymer-based NP containing fluorescent-tagged bovine serum albumin (BSA), and conjugated with ERY1, a 12 amino acid peptide with high affinity for the RBC membrane protein glycophorin A (ENP). Results: Confocal and flow cytometry data suggest that ENPs efficiently and irreversibly bind to RBC, with approximately 70% of erythrocytes bound after 24 h in a physiologic flow loop model compared to 10% binding of NPs without ERY1. Under these conditions, synthesized ENPs were not toxic to the RBCs. The rheological parameters at the applied shear. (0-15 Pa) were not influenced by ENP attachment to the RBCs. However, at high concentration, the strong affinity of ENPs to the glycophorin-A reduced the deformability of the RBC. Conclusions: ENPs can be efficiently attached to the RBCs without adversely affecting cellular function, and this may potentially enhance circulatory half-life of drug molecules. [ABSTRACT FROM AUTHOR]