(262c) Does Poly(ethylene glycol) Conjugation Protect Proteins From Conformational Change During Emulsion-Based Microsphere Encapsulation?

Due to their high activity and specificity, proteins are prime candidates for use as therapeutic drugs. Since proteins cannot be administered orally, alternative delivery strategies are required. The typical route is via injection, which leads to fluctuations in the concentration of drug in serum and often is associated with poor patient compliance. To improve efficacy in chronic therapies, protein drugs would benefit from sustained release delivery. One potential candidate would be a biodegradable poly(lactide-co-glycolide) (PLG) depot which often takes the form of subcutaneously injected microspheres.  Despite its benefits, this delivery method presents its own challenges, namely that proteins tend to adsorb to the interfaces generated during the formation of and release from microspheres.  Adsorption can be irreversible and result in protein denaturation, aggregation and, ultimately, bioactivity loss.  The covalent attachment of poly(ethylene glycol) (PEG) chains to the protein (“PEGylation”) has been shown to mitigate adsorption effects in addition to imbuing conjugates with several other useful benefits.  PEGylated proteins tend to better preserve bioactivity during sustained release and to experience more complete release from PLG microspheres. Prior research has shown that PEGylation significantly modulates protein adsorption isotherms, adsorption reversibility and surface-induced aggregation at the solid PLG/aqueous interface, which is relevant to the release from PLG depots.  The current work focuses on adsorption at the oil/water interface, which is relevant to possible protein damage during the emulsion-based microsphere formation process.  Understanding how PEGylation affects protein adsorption to the oil/water interface formed during microsphere preparation will facilitate improved formulations for PLG depot-based protein therapeutics.  To this end, two model proteins, ribonuclease A (RNase A) and apo-α-lactalbumin (ALA), were grafted with 20kDa PEG chains.  RNase A and ALA differ substantially in their thermodynamic stability and hydrophobicity. Circular dichroism and fluorescence spectroscopy were used to elucidate how PEGylation alters the conformational effects of protein exposure to the oil/water interface during emulsification. RNase A suffered little conformational alteration during oil/water interface exposure under emulsification conditions, while ALA experienced significant changes in tertiary structure.  PEGylation proved to have no protective effect against conformational change at the oil/water interface for either protein. Differences in emulsification-induced conformational change are being interpreted with the aid of ellipsometry measurements of the extent of adsorption and interfacial tension measurements for the oil/water interface, for both PEGylated and unmodified proteins.  The tentative conclusion is that the benefit of protein PEGylation in terms of enhanced bioactivity preservation and increased extent of release from PLG microspheres arises more from its effects on adsorption to the solid PLG/aqueous interface than to protection during microsphere encapsulation.