466655 Internalization of Nanoparticles Functionalized with Low Molecular Weight Protamine into Erythrocytes
Toward this goal, we introduced a novel method for transporting nanoparticles (NPs) into intact erythrocytes by combining the advantages of both cell-penetrating and targeting peptides. The method employs an ERY1 (WMVLPWLPGTLDGGSGCR) peptide that has a high affinity for the erythrocyte membrane protein glycophorin A and a low molecular weight protamine (LMWP) peptide that has been shown to transport proteins and small molecules into erythrocytes. These peptides are conjugated to a NP formed between fluorescently-labeled bovine serum albumin and a grafted copolymer of poly(ethylene) glycol and poly-L-lysine.
The formation of nanoparticles was observed using SEM, TEM, and dynamic light scattering. The nanoparticles had a core/shell structure and their size ranged from approximately 20-100 nm. Confocal microscopy and flow cytometry data confirmed that uptake of LMWP functionalized NPs was significantly higher (approximately > 3-5 fold) than that of other NPs at 6 h. Under these conditions, synthesized NPs were not toxic to the erythrocytes and no significant hemolysis was observed. Catalytic activity of the encapsulated protein was not negatively affected upon encapsulation or crosslinking. In conclusion, NPs were efficiently internalized into RBCs without adversely affecting cellular function and this method has the advantage over existing methods in that it does not appear to alter the structural integrity of the cell. The above approach of utilizing a surface modified NP architecture to improve internalization into erythrocytes may serve as a universal platform to deliver therapeutic proteins/drugs into RBCs in future. Furthermore, this nanoparticle system capable of entering red blood cells and extending circulation time has enormously broad potential, including improved treatment of blood-specific diseases such as malaria as well as treatment of acute lymphoblastic leukemia.