Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 3 Particle Transcytosis Across the Human Intestinal Epithelium Model Development and Target Identification for Improved Drug Delivery ELISABET GULLBERG ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6192 UPPSALA ISBN 91-554-6145-X 2005 urn:nbn:se:uu:diva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ill pappa List of Papers The thesis is based on the following papers, which will be referred to by their Roman numerals: I Gullberg, E., Leonard, M., Karlsson, J., Hopkins, A.M., Brayden, D., Baird, AW., Artursson, P. Expression of specific markers and particle transport in a new human intestinal M-cell model. Biochem Biophys. Res. Commun. (2000) 279, 808-813. Reprinted by permission. © 2000 Elsevier Science. II Gullberg, E., Velin Keita, Å., Salim, SY., Andersson, M., Cald- well, K., Söderholm, JD., Artursson, P. Increased CD9 and E1- integrin expression in human follicle associated epithelium is linked with selective transport into human Peyer´s patches. Submitted III Gullberg, E., Ragnarsson, EGE., des Rieux, A., Artursson, P. TNF-D, TGF-E1 and LTĮ1/ȕ2 induces particle uptake and aug- ments B-cell-induced particle transcytosis in intestinal epithelial cells. Manuscript IV Ragnarsson, EGE., Gullberg, E., Schoultz, I., Magnusson, K.-E., Söderholm, J., Artursson, P. Yersinia Pseudotuberculosis induces E1-integrin expression and particle transcytosis in human intesti- nal epithelial Caco-2 cells. Manuscript Contents 1. Introduction.................................................................................................9 1.1. The intestine........................................................................................9 1.1.1. M-cells.......................................................................................11 1.2. Mechanisms of intestinal uptake and transport .................................12 1.2.1. Mechanisms of endocytic uptake...............................................13 1.2.2. Transcytosis ...............................................................................14 1.3. Factors influencing intestinal particle uptake and transport..............15 1.3.1. Properties of the intestinal barrier..............................................15 1.3.2. Properties of the administered particle ......................................17 1.4. Methods of studying intestinal particle uptake and transport............19 1.4.1. In vivo studies ............................................................................20 1.4.2. In vitro studies on intestinal tissue.............................................20 1.4.3. Cell culture models....................................................................20 1.5. Concluding remarks ..........................................................................21 2. Aims of the thesis......................................................................................22 3. Methods ....................................................................................................23 3.1. Establishment of a model FAE..........................................................23 3.1.1. Choice of Caco-2 cell clone/line................................................24 3.1.2. Choice of B-cells .......................................................................24 3.1.3. Choice of co-culture conditions.................................................25 3.2. Particle uptake and transport studies.................................................25 3.2.1. Cell culture experiments............................................................25 3.2.2. Ussing chamber studies on intestinal tissue...............................25 3.2.3. Analysis of particle uptake ........................................................26 3.2.4. Analysis of particle transport.....................................................26 3.2.5. Assessment of integrity and viability.........................................26 3.3. Gene expression studies ....................................................................28 3.3.1. cDNA-array analysis .................................................................28 3.3.2. Interpretation..............................................................................28 3.4. Protein expression studies .................................................................29 3.4.1. Cell-based ELISA......................................................................30 3.4.2. Immunofluorescence..................................................................30 4. Results and discussion ..............................................................................31 4.1. Characteristics of the model FAE in comparison with human FAE .31 4.1.1. Characteristics of the induced particle transport........................32 4.2. Identification of target structures in human FAE..............................33 4.2.1. Cell surface carbohydrates.........................................................33 4.2.2. Cell adhesion proteins................................................................33 4.3. Exploiting identified targets in human FAE......................................37 4.3.1. Transport of RGD-coated particles across the model FAE .......38 4.3.2. Selective transport of RGD-coated particles into human Peyer´s patches ....................................................................................38 4.4. Soluble factors as mediators of particle uptake and transport...........39 4.4.1. Gene expression profile of Raji B-cells that induce epithelial particle transport ..................................................................................39 4.4.2. Effects of B-cell-secreted proteins on cell monolayer integrity and particle uptake...............................................................................40 4.4.3. Effects of B-cell-secreted proteins on particle transport............42 4.5. Influence of enteric bacteria on particle uptake and transport...........44 4.5.1. Effects of Yersinia Pseudotuberculosis on cell monolayer integrity and particle uptake ................................................................45 4.5.2. Effects of Yersinia Pseudotuberculosis on particle transport....46 5. Summary and conclusions ........................................................................48 Acknowledgements.......................................................................................50 References.....................................................................................................52 Abbreviations AP Alkaline Phosphatase BMP Bone morphogenic protein cDNA Complementary DNA CK cytokeratin ELISA Enzyme-linked immunosorbent assay FAE Follicle-associated epithelium FCS Fetal Calf Serum HBSS Hank’s Balanced Salt Solution IFN-J Interferon gamma IL Interleukin IRF-1 Interferon regulatory factor 1 LT Lymphotoxin Papp Apparent permeability coefficient RANTES T-cell expressed and secreted RT-PCR Reverse transcription polymerase chain reaction SD Standard deviation TDGF Thyroid derived growth factor TER Transepithelial Electrical Resistance TGF-ȕ Transforming growth factor beta TNF-Į Tumor Necrosis factor alpha VE Villus epithelium 1. Introduction Oral delivery of proteins using micro-particulate carriers has been under investigation for several decades, with emphasis on the formulation of anti- gens for mucosal vaccination.1, 2 The development of new peptide- and nu- cleic acid-based drugs has attracted further interest in particulate carriers for drug delivery across the intestinal epithelium.3 Drugs encapsulated in micro-particles have a low bioavailability.4 There- fore, the most appropriate, and important, application for these carrier sys- tems is the administration of mucosal vaccines. Indeed, oral delivery of anti- gens is by far the most effective method of inducing gastrointestinal mucosal immune responses.2 Furthermore, the benefits of needleless immunization in third world countries would be substantial.5 However, it is being increasingly questioned whether encapsulated anti- gens can be taken up in sufficient quantities to achieve adequate immune responses.6 While early studies in rodents and rabbits showed that up to 30% of orally administered particles were taken up across the intestinal epithe- lium,7, 8 9 more recent studies indicate that the uptake is typically less than 1% of the given dose.6 Although little is known of the extent of particle up- take in the human intestine, the moderate effects on the immune response after particulate delivery of antigens in man10-12 suggest