Tilton
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Group

Research
Carnegie Mellon University
Department of Chemical Engineering
Department of Biomedical Engineering
Center for Complex Fluids Engineering
Center for Environmental Implications of Nanotechnology
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Adsorption of Proteins and PEGylated Proteins

Covalently grafting PEG chains to protein therapeutics has been demonstrated in the literature to improve pharmaceutical efficacy by significantly extending protein circulation times in the body.  In collaboration with Professor Todd Przybycien, we have investigated the effects of PEG grafts on the extent, kinetics and reversibility of protein adsorption to solid surfaces. These include model chromatographic materials as may be used in the manufacture of PEGylated protein therapeutics and biodegradable poly(lactide-co-glycolide) (PLG) materials that are used for sustained release of proteins.  Whereas the protein-repellant nature of PEG has led to its recognition as a biologically “stealthy” polymer, PEG is surface active on many types of surfaces. From a fundamental perspective, this makes PEG-protein conjugates an intriguing adsorption system in that it can be considered as a complex block copolymer with two or more surface active blocks.  Under some conditions, the conjugate can be adsorbed to a surface via the protein “block” while under other conditions, the PEG block(s) anchor the conjugate.  We have found that on PLG surfaces, PEGylation decreases the extent of ribonuclease A (a protein with anti-tumoral effect) adsorption relative to the unmodified protein via an excluded area effect on the surface.  The diminished adsorption correlates with more complete release, and improved retention of biological activity of ribonuclease A, from PLG microspheres. See Daly et al. “Adsorption of poly(ethylene glycol)-modified ribonuclease A to a poly(lactide-co-glycolide) surface,”  Biotechnology and Bioengineering 90, 856-868 (2005)

On silica surfaces, steric interactions among PEG grafts on lysozyme distoPEGylated protein adsorption isothermsrt the shape of the adsorption isotherm and flip the preferred orientation of the protein relative to the surface.  PEG grafts also increase the reversibility of adsorption (even though PEG homopolymer adsorption and unconjugated lysozyme adsorption are both effectively irreversible on silica). They inhibit the lateral aggregation of lysozyme, thereby reducing a prevalent mode of surface-induced denaturation of protein therapeutics.  The complexity of protein adsorption phenomena mandates the use of multiple experimental probes in parallel. The conclusions described here were based on several novel variants of total internal reflection fluorescence (TIRF) spectroscopy, optical reflectometry or ellipsometry, AFM colloidal force measurements, electrokinetics measurements and neutron reflectometry. See for example Daly et al. “Adsorption of poly(ethylene glycol) modified lysozyme to silica,” Langmuir 21, 1328-1337 (2005) and Pai et al. “Coverage-dependent morphology of PEGylated lysozyme layers adsorbed on silica,” Journal of Colloid and Interface Science 370, 170-175 (2012).

Adsorption isotherms on silica for lysozyme (squares), monoPEG-lysozyme (triangles) and diPEG-lysozyme (circles).


Representative Publications

S. Daly, T.M. Przybycien, R.D. Tilton, “Coverage Dependent Orientation of Lysozyme Adsorbed on Silica,” Langmuir 19, 3848-3857 (2003). DOI: 10.1021/la026690x

S. Daly, T.M. Przybycien, and R.D. Tilton, “Adsorption of poly(ethylene glycol) modified lysozyme to silica,” Langmuir 21, 1328-1337 (2005). DOI: 10.1021/la048316y

S.M. Daly, T.M. Przybycien and R.D. Tilton, “Adsorption of poly(ethylene glycol)-modified ribonuclease A to a poly(lactide-co-glycolide) surface,” Biotechnology and Bioengineering 90, 856-868 (2005). DOI: 10.1002/bit.20481

S.M. Daly, T.M Przybycien, R.D. Tilton, “Aggregation of lysozyme and of poly(ethylene glycol)-modified lysozyme after adsorption to silica,” Colloids and Surfaces B: Biointerfaces 57, 81-88  (2007). DOI: 10.1016/j.colsurfb.2007.01.007

A. Kusumo, L. Bombalski, Q. Lin, K. Matyjaszewski, J.W. Schneider, R.D. Tilton, “High capacity, charge-selective protein uptake by polyelectrolyte brushes,” Langmuir 23, 4448-4454 (2007). DOI: 10.1021/la063660b

S.S. Pai, T.M. Przybycien, R.D. Tilton, “Protein PEGylation attenuates adsorption and aggregation on a negatively charged and moderately hydrophobic polymer surface,” Langmuir 26, 18231–18238 (2010). DOI: 10.1021/la102709y

S.S. Pai, B. Hammouda, K. Hong, D.C. Pozzo, T.M. Przybycien, R.D. Tilton,The conformation of the poly(ethylene glycol) chain in mono-PEGylated lysozyme and mono-PEGylated human growth hormone,” Bioconjugate Chemistry 22, 2317-2323 (2011). DOI: 10.1021/bc2003583

S.S. Pai, F. Heinrich, A. Canady, T.M. Przybycien, R.D. Tilton, “Coverage-dependent morphology of PEGylated lysozyme layers adsorbed on silica,” Journal of Colloid and Interface Science 370, 170-175 (2012). DOI:10.1016/j.jcis.2011.12.065