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2015|16 Annual Report Fraunhofer IGB

82 1 50 μm Label-free analysis for formulations of active ingredients One important aspect in the development of medicinal prod- ucts is the uptake of the formulated active ingredients via the mucous membranes, for example in the gastrointestinal tract. aser scanning confocal fluorescence microscopy is the stan- dard method used to investigate these ingredients in dissected cross-sections of tissue. Prior labelling of the components in the formulation with fluorescent molecules is generally re uired for this purpose These dyes can significantly change the transport properties of the formulation through the tissue compared with the actual, non-labelled formulation. There is thus great demand for label-free analytical methods. At Fraunhofer IGB, confocal Raman microscopy and infrared microscopy are used as spectroscopic methods with a high spatial resolution that require no molecular probes. The research pro ect Platform for efficient epithelial transport for pharmaceutical applications using innovative particulate carrier systems” (PeTrA), funded by the German Federal Ministry of Education and Research, has conducted transport studies with newly developed active ingredient formulations in a variety of in vitro tissue models. The distribution of the poly- mer nanoparticles in these models has then been investigated with methods including infrared microscopy. Measurement techniques and sample preparation Sample preparation is particularly important for the successful conduct of measurement, in addition to the selection of suit- able equipment and measurement techniques (transmission, reflection or attenuated total reflection TR nalyses were carried out using the infrared microscope Hyperion 3000 (Bruker) in the example described here. An FPA (focal plane array) detector with 64 x 64 “pixels” was used to produce the images. Each pixel that is recorded contains a full infrared spectrum, with each image therefore containing 4096 indi- vidual spectra. The biological samples are intestinal tissue models, based on Caco-2 cells on a biological collagen matrix perfused with the polymer particle formulation in a bioreactor. Cross-sections of 20 µm were prepared using a cryomicrotome and transferred to a calcium fluoride window that is permeable for infrared Tissue models and cross-sections were produced by Prof. Dr. Heike Walles’ research group at the Institute of Tissue Engi- neering and Regenerative Medicine, University of Würzburg. Infrared spectra were recorded in transmission on an area measuring approx. 170 x 170 µm² using the FPA detector, resulting in a theoretical lateral resolution of approx. 2.7 µm. ANALYSIS OF PARTICLE DISTRIBUTION IN TISSUE MODELS USING IR MICROSCOPY Michaela Müller, Monika Riedl PHARMACY 2 transmission wavenumber in cm–1 2000 1000 150 μm 20001000