(194g) Permeation Analysis of Large Molecules to the Surface of Protein-Conjugates with High-Density Polymer Coats

Protein-polymer conjugation has become an area of increasing interest for a wide range of applications, from therapeutics, diagnostics and food industries to functional coating preparations. Conjugation of polymers to proteins enables manipulation of protein properties, leading to conjugates with enhanced bioactivity, increased stability and solubility, as well as imparting added functionality through the use of stimuli responsive polymers. Although many protein-polymer conjugates have been synthesized and used for therapeutics, the understanding of permeability of molecules through the polymer chains towards the surface of the protein is still not well understood. The molecular sieving of polymers is an important criterion that affects the efficacy of the conjugate. The enzyme-polymer conjugates used in therapy should repel the proteins from immune system and different types of proteases, while allowing their substrate to reach the active site.

Herein, we have focused on determining the permeability of molecules towards the protein surface as a step forward in understanding the role of polymer chain length and grafting density on a surface of a protein in changing the permeability and the diffusion rate of the molecules. We have used atom transfer radical polymerization (ATRP) to generate well-defined, high-density polymer conjugates.

We synthesized Avidin-polymer conjugates with varying polymer lengths and grafting densities and systematically characterized the diffusion of molecules towards the protein surface by exploiting the high binding affinity of Biotin toward Avidin protein. For molecular permeation studies, Lysozyme, Horse Radish Peroxidase and Bovine Serum Albumin (hydrodynamic sizes of 3.8, 5.7 and 7.0 nm respectively) were selected based on their sizes and biotinylated with Biotin-PEG-NHS. Another substrate that was used to test molecular sieving is Biotin-PEG with different PEG size 550-30000 Da.

Binding of the different substrates was investigated next using a spectrophotometric assay for avidin and biotin based on 2-(4-hydroxyphenylazo) benzoic acid (HABA displacement. Binding kinetics of biotin and other biotinylated substrates to avidin were studied using a stopped-flow system observing the change in tryptophan fluorescence. Experimental data from both equilibrium binding and binding kinetics showed that the permeation and diffusion rate of the molecules is primarily affected by polymer grafting density and not polymer chain length.