434868 Protein Encapsulation for Delivery to Erythrocytes

Monday, November 9, 2015: 1:50 PM
151D/E (Salt Palace Convention Center)
Nicholas Flynn1, Rangika Hikkaduwa Koralege1, Kaustuv Sahoo2, «. ÷zge Topal3, Sravanthi Vupputuri1, Liyi Geng4, Stephen Brimijoin4, Steven Hartson5, Ashish Ranjan2, Jing Pope2, Carey Pope2 and Josh Ramsey1, (1)School of Chemical Engineering, Oklahoma State University, Stillwater, OK, (2)Department of Physiological Sciences, Oklahoma State University, Stillwater, OK, (3)School of Mechanical and Aerospace Engineering, Oklahoma State University, Stillwater, OK, (4)Department of Pharmacology, Mayo Clinic, Rochester, MN, (5)Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK

Therapeutic proteins are promising candidates for the treatment of a wide variety of diseases. Although proteins have great potential as therapeutics, they are often limited by short circulation half-lives. One approach to overcome this challenge is to encapsulate and target a protein to long-circulating erythrocytes. Toward this goal, a grafted copolymer, composed of poly-L-lysine (PLL) and polyethylene glycol (PEG), was used to encapsulate a model protein, bovine serum albumin (BSA), which was then targeted to erythrocytes using an ERY 1 peptide ligand.

The aim of the present study was to identify conditions for best encapsulating the protein and to target the resulting nanoparticles to erythrocytes. A library of nine grafted copolymers was synthesized using 4-15 kDa, 15-30 kDa, or 30-70 kDa PLL and PEG:PLL grafting ratios of 2, 10, or 20.  BSA was encapsulated with the copolymers through electrostatic self-assembly of the protein and the grafted copolymer. The resulting nanoparticles were stabilized by cross-linking PLL-g-PEG with glutaraldehyde, and the nanoparticles were functionalized with the ERY 1 peptide for targeting to erythrocytes.

SDS-PAGE gel retardation assays confirmed BSA could be efficiently encapsulated. For several copolymers, nearly complete encapsulation of the protein was achieved, and encapsulation of BSA into nanoparticles was found to be strongly dependent on the molecular weight of the PLL backbone and the copolymer to protein mass ratio. 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 40-100 nm. The BSA retained catalytic activity toward p-nitrophenyl ester, despite encapsulation and exposure to glutaraldehyde. Additionally, the nanoparticles were found to be stable against polyanions and protease degradation. Confocal microscopy and flow cytometry demonstrated that the ERY 1 peptide enabled enhanced binding of the nanoparticles to erythrocytes under both static conditions and within a physiological flow loop circulating whole blood.  Under conditions meant to simulate the circulatory system, approximately 15% of erythrocytes had detectable levels of nanoparticles after 4 hours in circulation. The results of this study demonstrated that therapeutic proteins can be encapsulated within polymer nanoparticles in an active state and delivered to red blood cells circulating in a physiological flow loop.

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See more of this Session: Drug Delivery II
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division