A. Rafati, et al., Journal of Controlled Release, 138(1) (2009) 40 - 44 S. Ravi, et al., Indian Journal of Pharmaceuti

1. Visualising surfactant and protein distribution within PLGA microspheres for controlled drug release A. Rafati1, A. Boussahel1, A.G. Shard2, K.M. Shakesheff1, P.T. Whiteside3, S. Rigby-Singleton3,C.J. Roberts1, X. Chen1, D.J. Scurr1, M.R. Alexander1, M.C. Davies1 1University of Nottingham, School of Pharmacy, Nottingham, UK 2 Analytical Sciences Division, National Physical Laboratory, Teddington, UK 3Molecular Profiles Ltd., Nottingham, UK Introduction • The surfactant layer was analysed with AFM before and after sputtering with C602+ primary ions in order to determine the thickness of the surfactant film and to confirm it is removed by sputtering, which also confirms it is not a surface feature of PLGA (Figure 2). • AFM shows the PVA layer is approximately 4 nm in thickness. The surface sensitivity of ToF-SIMS is vital for our ability to clearly image the surfactant and protein in this system. • Lysozyme is found at the surface as a thin layer before sputtering by ToF-SIMS. After we have sputtered we note it is removed from the area where sputtering is affective apart from within a surface pore created during the microemulsion fabrication process. • W/O/W microspheres were ultramicrotomed in order to analyse their internal structure with ToF-SIMS (Figure 3). a) • Poly (lactic-co-glycolic acid) (PLGA) microspheres for controlled delivery of protein drugs are investigated in depth to visualise the surfactant and drug distribution, in order to understand how the fabrication affects the distribution. • In order to rationalise drug release from such microspheres it is important to investigate the surface and bulk properties (1). b) a) b) c) 50 µm 50 µm Methods 50 µm 50 µm • A water in oil in water (W/O/W) double emulsion described previously (2) was undertaken to produce PLGA microspheres measuring approximately 100 µm in diameter, encapsulating lysozyme as a model of a protein therapeutic. The surfactant used to stabilise the emulsion is poly vinyl alcohol (PVA) (87-89% hydrolysed). Figure 4.a) Stack of confocal Raman images representing lysozyme in red and PLGA in green in 3D. b) Raman map of a 40 µm area found 26 µm in from the surface. c) Interpolated 3D representation of drug distribution Results and Discussion • Confocal Raman shows protein adsorbed to the PLGA interface in large pores with void space within or concentrated within small pores. The size of the pores determined by Raman correlating well with those found by ToF-SIMS imaging of the sectioned microspheres. • The SEM shows the spherical structure of the W/O/W microspheres including some surface pores and deformations indicating sub surface pores (Figure 1a.) • Multivariate curve resolution (MCR, Figure 1b) was used to identify peaks describing the chemical components contained within the various surface phases which were used to reconstruct the ToF-SIMS images in Figure 1c. • ToF-SIMS analysis of the surface of the microspheres shows a discontinuous PVA surfactant layer (Figure 1c). We also note the presence of protein surrounding what appears to be a surface pore. Figure 2. a)ToF-SIMS overlain image showing lysozyme (red), PLGA (green) and PVA (blue) and below a corresponding AFM image showing the topography of an 8 µm area of the microspheres before sputtering and b) after sputtering. Conclusions • We have used complimentary techniques to show for the first time, discontinuous PVA surfactant and localised protein at the surface of microspheres and that the PVA films are in the region of 4 nm in thickness. • The chemical specificity and sampling depth of ToF-SIMS allows the surface chemistry to be determined at high resolution. • Confocal Raman is shown to provide powerful 3D chemical images showing the distribution of drug in this optically transparent system. • A complex multicomponent double emulsion system has been characterised with complementary techniques improving our understanding of how the fabrication process affects structure and ultimately function. LysozymeIndicating b) a) Lysozyme PLGA Overlay PLGAIndicating 50 µm 50 µm 50 µm Acknowledgements PLGA PVA Lysozyme Overlay We thank the BBSRC, NPL and the East Midlands Development Agency for funding and Molecular Profiles Ltd. for the use of the Raman and their expertise. 50 µm 50 µm 50 µm 50 µm 50 µm 50 µm 50 µm 50 µm c) References Figure 3. ToF-SIMS of a sectioned microsphere showing lysozyme (CNO-), PLGA (C3H3O2-, C3H5O2-, C3H3O3- &C3H5O3-), an overlay showing lysozyme (red), PLGA (green) and PVA (C2H3O2- blue) . A. Rafati, et al., Journal of Controlled Release, 138(1) (2009) 40 - 44 S. Ravi, et al., Indian Journal of Pharmaceutical Sciences, 70(3) (2008) 303 - 309 Figure 1. a) Representative SEM image of the surface of the W/O/W microspheres. b) MCRscores image and corresponding loadings plot for a microsphere, the loadings plot shows the peaks which influence the scores image most. c) ToF-SIMS imaging showing PLGA, PVA, lysozyme and an overlay of all three components. • Lysozyme is found to be encapsulated within pores in the microsphere bulk. The pores range from 2 – 18 µm in diameter. PVA is noted in one pore where lysozyme is not detected. Detailed analysis of bulk distribution utilised confocal Raman mapping (Figure 4). Contact: paxar2@nottingham.ac.uk