(342b) Understanding the Effects of Protein Conjugation with Poly(ethylene glycol) on Protein Adsorption for Therapeutic Protein Drug Delivery

Over the last two decades, proteins have become an important class of drug molecules. Most of these protein drugs are used for life-threatening and seriously debilitating diseases such as diabetes, cancer, and hepatitis. The high activity and specificity of proteins compared with the more conventional, low molecular weight drugs often allows for better treatment of these diseases [1]. Sustained concentrations of the protein may be achieved by incorporating the drug into an implantable or injectable, biodegradable delivery system, such as FDA approved, biodegradable poly(lactide-co-glycolide) (PLG) microspheres. These polymers have an excellent record of biocompatibility and are known to degrade to toxicologically acceptable metabolites. Water soluble protein molecules have been encapsulated within PLG microspheres using a method based on a (water-in-oil)-in-water, (W/O)/W, multiple emulsion technique [2].

However, protein delivery from PLG microspheres has not been fully realized due to the difficulty in maintaining bioactivity when proteins are encapsulated. Protein conformation is very sensitive to local environment, particularly to the damaging effects of protein-surface interactions. Protein adsorption to interfaces limits the performance of sustained protein release from biodegradable depots [3]. During the encapsulation process, protein is exposed to an oil/water interface, where adsorption may cause denaturation or aggregation. During release, protein is exposed to a solid PLG/liquid interface as the microspheres undergo bulk erosion. Protein adsorption to this interface has been implicated as a critical factor leading to incomplete protein release and loss in bioactivity [2]. In order to achieve the full promise of sustained protein release from biodegradable microspheres, adsorption to these interfaces must be understood and minimized.

The covalent attachment of poly(ethylene glycol) (PEG) to proteins, PEGylation, has been shown to increase the in vivo half life of circulating proteins and decrease immune response [4,5]. Our research investigates whether protein PEGylation can also reduce the damaging effects of protein adsorption and thereby increase the extent of protein release from PLG microspheres in an active form. Such knowledge can aid in the design of therapeutic protein drug delivery devices. Furthermore, protein adsorption studies are necessary in order to establish a basis for the prediction, generalization, and control of protein behavior on surfaces.

We employed a total internal reflection fluorescence technique to explore the extent of lysozyme adsorption at the solid PLG/liquid interface and, in particular, to determine how covalent grafting of poly(ethylene glycol) (PEG) to lysozyme affects adsorption. Adsorption results showed that PEGylation significantly decreased the amount of lysozyme adsorption onto PLG surfaces. Additionally, the surface coverage values led to a possible explanation of the orientation of the PEGylated lysozyme versus unmodified lysozyme upon adsorption onto PLG.

References

[1] Van de Weert, M.; Jorgensen, L.; Moeller, E. H.; Frokjaer, S. Expert Opinion on Drug Delivery 2005, 2, 1029.

[2] Arai, T; Norde, W. Colloids and Surfaces 1990, 51, 1.

[3] Crotts, G.; Sah, H.; Park, T. G. Journal of Controlled Release 1997, 47, 101-111.

[4] Harris, J. M.; Martin, N. E.; Modi, M. Clinical Pharmacokinetics 2001, 40, 539.

[5] Michaelis, M.; Cinatl, J.; et al. Anti-Cancer Drugs 2002, 13, 149-154.