Polymer microspheres for controlled release of therapeutic protein from within an implantable scaffold were produced and analysed using complimentary techniques to probe the surface and bulk chemistry of the microspheres. Time of Flight - Secondary Ion Mass Spectrometry (ToF-SIMS) surface analysis revealed a thin discontinuous film of polyvinyl alcohol (PVA) surfactant (circa 4.5 nm thick) at the surface which was readily removed under sputtering with C(60). Atomic Force Microscopy (AFM) imaging of microspheres before and after sputtering confirmed that the PVA layer was removed after sputtering revealing poly(lactic-co-glycolic) acid(PLGA). Scanning electron microscopy showed the spheres to be smooth with some shallow and generally circular depressions, often with pores in their central region. The occurrence of the protein at the surface was limited to areas surrounding these surface pores. This surface protein distribution is believed to be related to a burst release of the protein on dissolution. Analysis of the bulk properties of the microspheres by confocal Raman mapping revealed the 3D distribution of the protein showing large voids within the pores. Protein was found to be adsorbed at the interface with the PLGA oil phase following deposition on evaporation of the solvent. Protein was also observed concentrated within pores measuring approximately 2 μm across. The presence of protein in large voids and concentrated pores was further scrutinised by ToF-SIMS of sectioned microspheres. This paper demonstrates that important information for optimisation of such complex bioformulations, including an understanding of the release profile can be revealed by complementary surface and bulk analysis allowing optimisation of the therapeutic effect of such formulations.

In recent years, considerable attention has focused upon the biological applications of the atomic force microscope (AFM), and in particular in its ability to explore biomolecular interaction events at the single molecule level. Such measurements can provide considerable advantages, as they remove the data averaging inherent in other biophysical/biochemical approaches that record measurements over large ensembles of molecules. To this end AFM has been used for both the high-resolution imaging of a range of individual biological molecules and their complexes, and to record interaction forces between single interacting molecules. In a recently initiated project we have begun to utilize these approaches to explore the interactions of a range of biologically important peptides with model and cell membrane surfaces. In this review, the potential value of AFM for the investigation of a range of biomolecular interaction events will be discussed, but highlighting in particular its potential for the study of interactions of peptides/proteins with biological membranes.

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The effect of the presequence peptide of cytochrome c oxidase subunit IV (p25) on supported phospholipid bilayers (SPBs) was visualized using atomic force microscopy (AFM). The presequence was found to cause the complete disruption of supported bilayers containing neutral lipids. At relatively low concentrations of presequence, the peptide was found to bind to the membrane, coalescing to form microdomains within the liquid-crystalline bilayer that were located predominantly at bilayer-mica boundaries. Further increases in peptide concentration resulted in the formation of holes within the SPB that were spanned by an interpenetrating network of narrower regions of the bilayer, which, at higher applied peptide concentrations, were observed to disappear through a budding process, ultimately leading to the formation of spherical structures at yet higher peptide concentrations. Within this paper, the impact the presequence has upon the structure and order of the membrane is discussed, as is the potential implication of this apparent solubilization process on the translocation of cytochrome c oxidase into the inner mitochondrial membrane.

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