466655 Internalization of Nanoparticles Functionalized with Low Molecular Weight Protamine into Erythrocytes

Thursday, November 17, 2016: 5:03 PM
Continental 6 (Hilton San Francisco Union Square)
Rangika Hikkaduwa Koralege1, Kaustuv Sahoo2, Nicholas Flynn1, Steven Hartson3, Jing Pope2, Ashish Ranjan2, Carey Pope2 and Joshua Ramsey1, (1)School of Chemical Engineering, Oklahoma State University, Stillwater, OK, (2)Department of Physiological Sciences, Oklahoma State University, Stillwater, OK, (3)Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK

Erythrocytes have been exploited extensively for their potential applications as delivery vehicles due to their remarkable biocompatibility, biodegradability and prolonged life-span in circulation. The mature human erythrocyte is a terminally differentiated cell that does not possess an endocytic uptake mechanism. As a result, the efficient migration of bioactive molecular cargos such as large macromolecular drugs, genetic material, or proteins through the plasma membrane of erythrocytes remains as a major challenge for intracellular delivery applications. A variety of techniques have been successfully developed to entrap proteins, drugs, and enzymes within erythrocytes. The majority of these techniques, however, require disruption of the cell membrane to create large pores or perturbations through which biomolecules can diffuse. These techniques include chemical methods such as drug-induced endocytosis, mechanical methods such as hypotonic osmosis/dialysis, and electrical methods such as electroporation. The main disadvantages associated with these methods are the loss of structural integrity and cellular components of the cells due to weakened cell membranes. As a consequence, these modified erythrocytes are easily recognized by the host immune system. To achieve the benefits associated with erythrocytes as a drug delivery vehicle it is vital to maintain complete structural and functional integrity of 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.

Extended Abstract: File Not Uploaded
See more of this Session: Drug Delivery II
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division