Chemical and spatial analysis of protein loaded PLGA microspheres for drug delivery applications.

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.

Towards the understanding and prediction of material changes during micronisation using atomic force microscopy.

In this study we aim to explore the potential links between the mechanical properties, micronisation behaviour and surface energy of carbamazepine polymorphs using atomic force microscopy (AFM) measurements of material properties at the nanoscale. Carbamazepine Forms I, II and III were prepared and confirmed using X-ray powder diffraction (XRPD). AFM measurements of indentation hardness, Young's modulus and surface energy were made on the starting material. In addition, the surface energy was measured immediately after micronisation and after storage for four weeks. Carbamazepine polymorphs could be ranked by Young's modulus and hardness. Surface energy measurements showed an increase after micronisation in all cases, and a varying relaxation after storage for four weeks. Form I showed a smaller particle size distribution, indicating more complete micronisation. A promising correlation was observed between the hardness/Young's modulus ratio and the micronisation behaviour, in terms of particle size reduction and surface energy change. The results show potential for the predictive capacity of such an approach, and help to provide a greater understanding of material behaviour and properties during micronisation.

Measuring and visualizing single molecular interactions in biology.

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.