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ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2006 Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 46 Linear and Branched Chitosan Oligomers as Delivery Systems for pDNA and siRNA In Vitro and In Vivo MOHAMED MAHMOUD ISSA ISSN 1651-6192 ISBN 91-554-6747-4 urn:nbn:se:uu:diva-7376 !" # #$$% $&'( ) " ) ) *"" +, ) *" !" / 0". 1 . #$$%. 2 " " 3 4 ) 56 756 . 6 . %. 8% . . 145 &9((9%889. 1 " " " " " " ) / ) ) " ) ) )) 9 . * "9 ) " /" "" /" " /"" / " ) /" ) . !") " ) "9 ) " " /" " " ) " " 9 " ) /9) / /" " +" - ) +56 756- ) . 56 ) " " ) "" /" " ) "" ) " " +*01- ) " )) 9 . ) " " " " ) ) )) ) " : . 1 56 " / ; ) "9 /" 756 " )) . , " ) " ) " ) ) 56 ) /" " . 1 " " 9 . ! " 56 756 " " # # $% &'(# # )*+&, # < " " 1 #$$% 1445 %(9%&# 145 &9((9%889 ''''98 8% +"'==.:.=>?''''98 8%- “You are not given aught of knowledge but a little” Holy Qur’an: 17:85. To my family Papers discussed The thesis is based on the following papers, which will be referred to by their Roman numerals: I Köping-Höggård M, Vårum KM, Issa M, Danielsen S, Christen- sen BE, Stokke BT, Artursson P. Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers. Gene Ther. (2004)11(19): 1441-52. II Issa M, Köping-Höggård M, Tømmeraas K, Vårum KM, Chris- tensen BE, Strand SP, Artursson P. Targeted gene delivery with trisaccharide-substituted chitosan oligomers in vitro and after lung administration in vivo. J. Control. Rel. (2006) 115 (1): 103- 112. III Issa M, Strand SP, Vårum KM, Artursson P. Chitosan oligomers as siRNA delivery systems in vitro. In Manuscript. IV Köping-Höggård M * , Issa MM * , Köhler T, Tronde A, Vårum KM, Artursson P. A miniaturized nebulization catheter for im- proved gene delivery to the mouse lung, J Gene Med. 7(9) (2005): 1215-22. * shared first authorship. Also published: Mohamed M. Issa, Magnus Köping-Höggård and Per Artursson. Chitosan and the mucosal delivery of biotechnology drugs. Drug Discovery Today: Technologies (2005), 2 (1) 1-6. Reprints were made with permission from the publishers. Contents Introduction 11 Nucleic acids and oligonucleotides as potential pharmaceutical products 11 Gene delivery systems 14 Viral gene delivery systems 14 Non-viral gene delivery systems 14 Naked (unformulated) nucleic acids 15 Barriers to non-viral gene delivery 15 Pharmaceutical barriers 15 Extracellular barriers 16 Cell-surface and intracellular barriers 17 Bacterial gene delivery (bactofection) and alternative gene therapy (AGT) 19 Physical methods of non-viral gene delivery 20 Chemical methods of non-viral gene delivery 20 Lipids 21 Polymers 22 Strategies to improve the in vitro/in vivo efficiency of non-viral gene delivery: Structure-activity relationship 25 Molecular weight reduction 25 Positive charge shielding 26 Active targeting 26 In vivo toxicity of polyplexes 26 Chitosan chemical structure 27 Properties of chitosan 28 Physicochemical properties 28 Biological properties 29 General applications of chitosan in drug delivery 29 Chitosan as a delivery system for proteins and peptide drugs 29 Chitosan as non-viral gene delivery system 30 Aims 32 Materials and methods 33 Nucleic acids 33 Polycations 33 Cells 34 Formulation of complexes 34 Size and morphology of the complexes 35 Physical and enzyme stability 35 In vitro studies 35 Transfection experiments 35 Cellular uptake of chitosan complexes 36 Cellular toxicity (Intracellular dehydrogenase activity) 37 In vivo studies 37 Luciferase gene expression 37 Distribution pattern of gene expression in the mouse lung 37 Toxicological evaluations 37 Statistics 38 Results and discussion 39 Optimised linear chitosan oligomers as non-viral gene delivery systems (Paper I) 39 Characterisation of DP n 18 polyplexes in vitro and in vivo 39 Characterisation of polyplexes based on oligomer fractions isolated from DP n 18 in vitro and in vivo 41 Intracellular release of pDNA from chitosan oligomer-based polyplexes 43 Trisaccharide-substituted chitosan oligomers as non-viral gene delivery systems (Paper II) 44 Structure-activity relationship of polyplexes based on trisaccharide- substituted chitosan oligomers (TCO). 45 Impact of trisaccharide substitution on chitosan oligomer-based polyplexes 45 Chitosan oligomers as siRNA delivery systems in vitro (Paper III) 50 In vitro siRNA delivery under conditions previously optimised for pDNA 50 Structure-activity relationship of chitosan oligomer-based siRNA complexes 52 Improved aerosol gene delivery to the mouse lung in vivo (Paper IV) 56 Physical stability and in vitro transfection efficiency following nebulisation with the NCD 57 In vivo nebulisation with the NCD VS intratracheal instillation 58 Summary and conclusions 60 Acknowledgements 62 References 64 Abbreviations A-A-M 2-acetamido-2-deoxy-D-glucopyranosyl-E-(1-4)-2- acetamido-2-deoxy- D-glucopyranosyl-E-(1-4)-2,5- anhydro- D-mannofuranose AGT Alternative gene therapy AUC Area under the curve CMV Cytomegalovirus promoter Da Dalton D A Degree of acetylation DNA Deoxynucleic acid DP Degree of polymerisation DP n Number-average degree of polymerisation DS Degree of substitution FA Fraction of acetylated units GFP Green fluorescent protein GlcNAc N-acetylglucosamine GMP Good manufacturing practice HSPG Heparan sulphate proteoglycans Luc Luciferase reporter mRNA Messenger ribonucleic acid M w Molecular weight NCD Nebulisation catheter device NLS Nuclear localisation signal NPC Nuclear pore complex ODN Oligodeoxynucleic acid PBS Phosphate-buffered saline pDNA Plasmid DNA PEI Polyethyleneimine PLL Poly-L-lysine RNA Ribonucleic acid RNAi RNA interference siRNA Small interfering RNA TCO Trisaccharide-substituted chitosan oligomers UPC Ultrapure chitosan [...]... of chitosan oligomer-based pDNA formulations in vitro and after lung administration in vivo by coupling a targeting moiety (a trisaccharide branch) to the chitosan backbone (Paper II) Investigate the potential of chitosan oligomers as delivery systems for siRNA in vitro, and to study the effect of controlling chitosan structural variables and formulation parameters on the gene silencing activity of siRNA. .. oligomers) as delivery systems for nucleic acids (pDNA and siRNA) in vitro and after lung administration in vivo In addition, the concept of receptor targeting was exploited to further enhance the gene delivery efficiency of chitosan oligomer-based polyplexes Finally, improved methodology for the administration of gene formulations to the lung in vivo was explored 31 Aims The overall aim of the thesis was... gel-forming properties and propensity for degradation, chitosan- based controlled and targeted delivery systems have also been developed and examined [159] Several reports have confirmed the absorption enhancing characteristics of chitosan formulations following nasal administration of vaccines, peptides, proteins and low Mw drugs such as morphine [172-174] In addition to the nasal route, chitosan has... and 4 % agarose in 40 mM TAE buffer were used as previously described for pDNA and siRNA complexes, respectively [54] Protection of complexed pDNA and siRNA against enzyme degradation was studied after incubating the complexes with DNase I and RNase A, respectively as described previously The integrity of pDNA and siRNA was investigated by incubating the complexes with the polyanion heparin (5 mg/ml)... method for in vivo targeting involves hydrodynamic injection, where the naked nucleic acids are injected in large volume under high pressure into the tail vein of animal models This technique has proved to be efficient for pDNA and siRNA targeted delivery (passive targeting) mainly to the liver and, to a lesser extent to the skeletal muscles [88,89] However, as for nuclear microinjection, hydrodynamic injection... be stable following intravenous injection in vivo [104] The improved transfection efficiency of lipoplexes over naked pDNA is attributed to their resistance to enzymatic degradation, improved cellular uptake and efficient endosomal release Besides their efficiency as pDNA delivery systems, lipid-based formulations were reported as efficient delivery systems for siRNA in vitro and in vivo [105-107] Although... protein delivery, the most promising in vivo results have been obtained in vaccine research The first significant result was obtained in mice after oral administration of chitosan nanoparticles carrying plasmid pDNA encoding a peanut allergen [186] The treatment resulted in induced tolerance to peanut allergy, which was related to allergen-specific secretory Immunoglobulin A and serum Immunoglobulin... Iqbal et al [188] In 2005, nanoparticles of chitosan complexed to pDNA coding for siRNA against the RSV NS1 protein were intranasally delivered to mice, resulting in a substantial decrease in virus titres in the lungs and decreased inflammation and airway reactivity [189] The significance of these results is related to the current lack of effective prophylaxis against RSV infection in the lower respiratory... nasal route, chitosan has been investigated as a vehicle for ocular and peroral drug delivery in order to prolong contact time and improve drug absorption [175,176] Chitosan as a delivery system for proteins and peptide drugs Since Illum et al [177] reported in 1994 that high Mw chitosan solutions significantly increased the transport of insulin across the nasal mucosa of rats and sheep, several human studies... facilitating the transport of macromolecular drugs through well organised epithelia [184] Chitosan as non-viral gene delivery system The use of chitosan in gene delivery was described for the first time in 1995 [185] Since then, slow but steady progress has been made, resulting in chitosan now being placed among the most effective non-viral gene delivery systems for mucosal application As for chitosan- mediated . Uppsala Dissertations from the Faculty of Pharmacy 46 Linear and Branched Chitosan Oligomers as Delivery Systems for pDNA and siRNA In Vitro and In Vivo MOHAMED MAHMOUD ISSA ISSN 1651-6192 ISBN 91-554-6747-4 urn:nbn:se:uu:diva-7376 . substitution on chitosan oligomer-based polyplexes 45 Chitosan oligomers as siRNA delivery systems in vitro (Paper III) 50 In vitro siRNA delivery under conditions previously optimised for pDNA 50 Structure-activity. 37 Statistics 38 Results and discussion 39 Optimised linear chitosan oligomers as non-viral gene delivery systems (Paper I) 39 Characterisation of DP n 18 polyplexes in vitro and in vivo 39 Characterisation