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Interactions of Mucins with Biopolymers and Drug Delivery Particles

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Interactions of Mucins with Biopolymers and Drug Delivery Particles INTERACTIONS OF MUCINS WITH BIOPOLYMERS AND DRUG DELIVERY PARTICLES Malmö University Health and Society Doctoral Dissertations 2008:2 © Olof Svensson 2008 ISBN 978-91-7104-212-5 ISSN 1653-5383 Holmbergs, Malmö 2008 OLOF SVENSSON INTERACTIONS OF MUCINS WITH BIOPOLYMERS AND DRUG DELIVERY PARTICLES Malmö University, 2008 The Faculty of Health and Society To my family CONTENTS ABSTRACT ................................................................................ 10 LIST OF PAPERS ......................................................................... 12 INTRODUCTION ......................................................................... 14 Background and aim ............................................................................ 14 The mucous gel and mucins ................................................................... 16 Polyelectrolyte multilayers ...................................................................... 22 MATERIALS AND METHODS ......................................................... 27 Proteins and polymers ............................................................................ 27 Surfaces .............................................................................................. 30 Ellipsometry ......................................................................................... 31 Particle electrophoresis ........................................................................... 40 Atomic force microscopy ........................................................................ 41 Electrochemistry ................................................................................... 42 RESULTS AND DISCUSSION ......................................................... 44 Layer-by-layer film formation with mucin ................................................... 44 Interactions between drug delivery particles and mucin .............................. 58 SUMMARY AND CONCLUDING REMARKS ..................................... 65 POPULÄRVETENSKAPLIG SAMMANFATTNING ................................ 67 ACKNOWLEDGEMENT ................................................................ 70 REFERENCES .............................................................................. 71 APPENDIX ................................................................................. 81 ABSTRACT The main components in the mucous gels apart from water are mucins, which are proteins with high molecular weights and an abundance of negatively charged oligosaccharide side chains. The aim of the investigations was to char- acterize interactions between mucins and other proteins that are present in the mucous gel, and also between mucins and components used in pharmaceutical formulations. More specifically, the main objectives were (I) to investigate the possibility to assemble multilayer films with mucins and oppositely charged polymers or proteins on solid substrates; (II) to evaluate mucoadhesive proper- ties of drug delivery particles by examination of their interactions with mucins. The construction of multilayer films was performed on silica and hydropho- bized silica surfaces by alternate adsorption, and the adsorbed amount and thickness of the films were measured in situ by time resolved ellipsometry. It was demonstrated that films could be assembled using mucins in combination with both chitosan and lactoperoxidase. The build-up was characterized by ad- sorption and redissolution processes, and the extent of redissolution could be explained by taking the charge densities and concentrations of the components into account. It was also demonstrated that the nature of the substrate can be crucial for the possibilities to assemble multilayer films, and from the results it may be concluded that a high amount of mucin in the first step is important for successful layer-by-layer assembly. Furthermore, it was demonstrated that lac- toperoxidase is catalytically active when adsorbed to mucin layers, and it may thereby exert its antimicrobial action. 10 The evaluation of mucoadhesive properties of drug delivery particles was per- formed with lipid nanoparticles stabilized by a poly(ethylene oxide) based polymer and with particles modified by chitosan. Both types of model particles (unmodified and chitosan modified) were investigated by measuring their ad- sorption to mucin-coated silica surfaces by ellipsometry. It was shown that the binding of unmodified particles to mucin-coated silica surfaces was weak and pH-dependent. Based on the pH and electrolyte dependence of the adsorption, it was proposed that the interaction is mediated by hydrogen bonding between protonated carboxyl groups in the mucin molecule and oxygen atoms in poly(ethylene oxide). Chitosan modified particles, on the other hand, showed a substantial and strong binding to mucin-coated surfaces, which can probably be attributed to interactions between amino groups in chitosan and negatively charged groups in the mucin layer. The findings from the present investigations are in agreement with previous reports on the interaction of mucins with poly(ethylene oxide) and chitosan. It can therefore be concluded that the meth- odology applied is useful for evaluating mucoadhesive properties of nanoparti- cles. 11 LIST OF PAPERS I. Layer-by-layer assembly of mucin and chitosan - Influence of surface properties, concentration and type of mucin. Olof Svensson, Liselott Lindh, Marité Cárdenas and Thomas Arnebrant, Journal of Colloid and Interface Science 2006, 299(2), 608-16. II. The salivary mucin MUC5B and lactoperoxidase can be used for layer- by-layer film formation. Liselott Lindh, Ida Svendsen, Olof Svensson, Marité Cárdenas and Thomas Arnebrant, Journal of Colloid and Inter- face Science 2007, 310(1), 74-82. III. Activity of lactoperoxidase when adsorbed on protein layers. Karolina Haberska, Olof Svensson, Sergey Shleev, Liselott Lindh, Thomas Arne- brant and Tautgirdas Ruzgas, Manuscript IV. Interactions between drug delivery particles and mucin in solution and at interfaces. Olof Svensson, Krister Thuresson and Thomas Arnebrant, Ac- cepted for publication in Langmuir V. Interactions between chitosan-modified particles and mucin-coated sur- faces. Olof Svensson, Krister Thuresson and Thomas Arnebrant, Manu- script Reprint permission of papers I and II has been granted by Elsevier Inc. and a blanket permission is granted by the American Chemical Society for reprinting of paper IV. 12 Contributions to the publications I performed most of the planning and essentially all experimental work in pa- pers I, IV and V. In addition I did the writing of the manuscripts with support from the co-authors. My contribution to paper II was to perform data analysis, take part in discussions of the results and write parts of the manuscript. I also made minor contributions to the experimental work. In paper III, I was con- tributing to the planning of the experimental work as well as performing most of the ellipsometric measurements. 13 INTRODUCTION Background and aim The mucous gel layer is a highly hydrated protein gel that covers the mucosal surfaces of our body and its general function is to protect the underlying muco- sal tissues from dehydration, mechanical stress and bacterial infections. In hu- mans the average thickness of the mucous gel is estimated to be a few hundred micrometers, and the main component apart from water is a group of glycopro- teins referred to as mucins. This class of high molecular weight glycoproteins is important in many aspects and is for example considered to form the backbone of the gel. As all nutrients and most pharmaceuticals on the market enter our body through the mucous gel, the composition and structure of this gel is of obvious scientific interest. From this perspective my research at Malmö University has been focused on the interactions of mucins with other types of proteins that are naturally present in the mucous gel as well as molecules and assemblies of molecules used in pharmaceutical formulations. The general aim has been to gain a deeper understanding of how molecules present in the native mucous gel can combine to form a three-dimensional network and how mucins interact with pharmaceutical constituents. The main objectives have been: I. To investigate the possibility to form multilayer films with mucins and oppo- sitely charged polymers or proteins on solid substrates. The possibility to meas- ure enzymatic activity of proteins in these structures was also addressed. This work was done with the ambition to create artificial gels that could act as mu- 14 cous models to study the interactions with for example pharmaceutical formu- lations. In addition the assembled films could have interesting lubricating and antiadhesive properties that would be of interest for coatings of contact lenses and dental implants. II. To study the interactions between drug delivery particles and mucin in order to evaluate their mucoadhesive properties and also to understand interactions between mucin and pharmaceutical constituents. Such knowledge is of interest in the area of mucosal drug delivery and the development of novel mucosal drug delivery systems. The Introduction of the thesis consists of a description of the general properties of the mucous gel with emphasis on mucins and their interactions with other mucus components and adsorption to solid surfaces. Also the layer-by-layer as- sembly of oppositely charged polymers or proteins is described focusing on the assembly process and the use of proteins in these structures. In the Materials
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