Drug delivery of oligonucleotides at the blood brain barrier [Elektronische Ressource] : a therapeutic strategy for inflammatory diseases of the central nervous system / Berit Osburg

Berit Osburg-Bickel

Sujets

• Gesundheit

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2003
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

From the Institute of Normal and Pathological Physiology
Philipps-University Marburg
Faculty of Medicine
Germany
Director: Prof. Dr. Dr. J. Daut

in collaboration with


School of Pharmacy, Texas Tech University Health Sciences Center,
Department of Pharmaceutical Sciences,
Amarillo, Texas, U.S.A.
Dean: A. A. Nelson, R.Ph., Ph.D.






Drug Delivery of Oligonucleotides at the Blood-Brain Barrier:
a Therapeutic Strategy for Inflammatory Diseases
of the Central Nervous System



Inaugural-Dissertation for attaining the degree
of Doctor of Human Biology (Dr. rer. physiol.)
submitted to the Faculty of Medicine
Philipps University Marburg






Berit Osburg
of Erfurt/Germany




Marburg 2003


























Accepted by the Faculty of Medicine,
Philipps University Marburg on May 27, 2003
Printed with the Faculty’s permission

Dean: Prof. Dr. B. Maisch
Referent: Prof. Dr. K. Voigt
Co-referent: Prof. Dr. K. Heeg






































To Uli and Jördis





Table of Contents

Page

1. Introduction 1

1.1. Drug delivery strategies through the blood-brain barrier 1
1.1.1. Small molecules 3
1.1.2. Carrier-mediated transport of drugs 3
1.1.3. Nanoparticles and Liposomes 4
1.1.4. Peptide-based neuropharmaceuticals 5
1.1.5. Antisense drugs 5
1.2. Receptor-mediated delivery of “chimeric peptides” 7
1.2.1. “Chimeric peptide” strategies 7
1.2.2. The transferrin receptor system 9
1.3. The transcription factor NF- κB and its regulation via
inflammatory stimulation 10
1.4. NF- κB decoys as pharmacological tools and potential drugs 14
1.5. Characterization of Polyethylenimine 16
1.5.1. Role of PEI and its advantage over viral delivery strategies 16
1.5.2. Structure and synthesis 16
1.5.3. DNA condensation and particle size 17
1.5.4. Cellular uptake and intracellular trafficking 18
1.5.5. In vivo gene delivery 18
1.5.6. Modification of PEI 19
1.5.7. LMW-PEI as preferred polymer 20
1.6. Multiple Sclerosis – an inflammatory disease of the central
nervous system 21
1.6.1. Multiple Sclerosis as an autoimmune disorder 21
1.6.2. Pathophysiological changes at the BBB under
inflammatory conditions and MS 21
1.7. Objective of this work 23

2. Materials and Methods 24

2.1 Materials 24
2.1.1. Instruments 24
2.1.2. Chemicals 25
2.1.3. Enzymes
2.1.4. Buffers and Solutions 26
2.1.5. Media 30
2.1.6. Primers and Oligodeoxynucleotides 32
2.1.7. DNA markers 33
2.1.8. Bacteria and Plasmids 33
2.1.9. Animals
2.2. Methods
2.2.1. Characterization of a 8D3-SA vector complex 34
2.2.1.1. 8D3 hybridoma culture 34
2.2.1.2. Synthesis of 8D3-SA 34
2.2.1.3. i.v. pharmacokinetics 36
2.2.1.4. Capillary depletion
2.2.1.5. Cell culture of bEnd5 cells 37
2.2.1.6. Binding and internalization experiments 37
2.2.1.7. Immunohistochemistry
2.2.2. Physico-chemical properties of bioPEGPEI/ODN or
8D3SAbioPEGPEI/ODN 38
2.2.2.1. Hybridization of transcription factor decoys 38
2.2.2.2. HABA (2(4’-hydroxyazobenzene)benzoic acid) assay 38
2.2.2.3. Complex formation 38
2.2.2.4. Polyacrylamide gel electrophoresis (PAGE) 38
2.2.2.5. Retardation assay 39
2.2.2.6. Binding characteristics of 8D3-SA to its ligand bioPEGPEI/NF- κB 39
2.2.2.7. Interaction of FITC-NF- κB decoys with bEnd5 cells 39
2.2.2.8. Particle sizing 39
2.2.2.9. Stability tests 40
2.2.3. Stimulation experiments with LPS or TNF α and inhibition of
activation 40 2.2.3.1. Extraction of nuclear fragments and NF- κB gel shift assay 41
2.2.3.2. mRNA isolation 42
2.2.3.3. RT-PCR 42
2.2.3.4. pGEM-T vector cloning 42
2.2.3.5. Northern blotting - denaturating formaldehyde gel and
hybridization 43
2.2.4. i. v. pharmacokinetics of bioPEGPEI/NF- κB and
8D3SA-bioPEGPEI/NF- κB 44

3. Results 45

3.1. Synthesis and characterization of a vector for brain delivery
in the mouse 45
3.1.1. Production and purification of hybridoma grown 8D3 45
3.1.2. Coupling of the 8D3 antibody to recombinant streptavidin 45
3.1.3. Pharmacokinetics and brain uptake of 8D3 and 8D3-SA after i.v.
administration 46
3.1.4. Binding and uptake studies with 8D3 and 8D3-SA using the
bEnd5 brain endothelial cell line 48
3.2. Polyethylenimine as carrier for oligonucleotides 50
3.2.1. Synthesis and characterization of low molecular weight
biotinylated PEGPEI 50
3.2.2. Complex formation and Retardation assays 51
3.2.3. Binding characteristics of 8D3-SA to its ligand bioPEGPEI/NF- κB 53
3.2.4. Vector-mediated increase in cellular uptake 56
3.2.5. Interaction of FITC-NF- κB decoys with bEnd5 cells 57
3.2.6. Particle sizing 58
3.2.7. TCA (Trichloro acetic acid) precipitation and ultrafiltration 61
3.3. Investigation of inflammatory markers influenced by activation
of NF- κB 63
3.3.1. NF- κB gel shift assays 63
3.3.2. Northern blots for quantification of gene expression related to
inflamation 5 3.3.3. Inhibition of VCAM-1 expression after NF- κB decoy treatment 68
3.4. i.v. pharmacokinetics of bioPEGPEI/NF- κB and
8D3SA-bioPEGPEI/NF- κB 73

4. Discussion 75

4.1. 8D3-SA as vector for drug delivery 75
4.2. Physico-chemical characteristics of the bioPEGPEI/ODN
complex and cell uptake 77
4.3. NF- κB gel shift assays for analysis of activation pattern after
stimulation with LPS/TNF α or LPS 81
4.4. mRNA expression of inflammatory markers after LPS, TNF α,
and LPS/TNF α stimulation 84
4.5. Effect of NF- κB decoy on the expression of VCAM-1 85
4.6. Pharmacokinetic characteristics of bioPEGPEI/NF- κB 86


5. Sumary 87
6. Zusammenfassung 89
7. Biblography 91
8. Appendix
Academic teachers
Publications
Acknowledgement List of abbreviations used in this script

AEC aminoethylcarbazol
APC antigen presenting cell
Atm atmosphere
ATP adenosine tri phosphate
AUC area under the curve
AZT azidothymidine
BBB blood-brain barrier
BCA bicinchoninic acid
BDNF brain derived nerve growth factor
BFGF basic fibroblast
bioPEGPEI biotinylated PEGPEI
bp base pairs
BSA bovine serum albumin
cAMP cyclic adenosine monophosphate
CD cluster of differentiation
cDNA complementary DNA
Ci Curie
CNS central nervous system
COX-2 cyclooxygenase-2
cpm counts per minute
°C degree Celsius
Da Dalton
DALDA Tyr-D-Arg-Phe-Lys-NH2
dATP desoxy adenosine tri phosphate
dCTP cytosine thph
dGTP guanine tri phosphate
DHP dihydropyridine
DMEM Dulbecco’s minimal essential medium
dNTP nucleotide tri phosphate
dpm decays per minute
dTTP desoxy thymidine tri phosphate
DMSO dimethylsulfoxide DNA desoxy ribonucleic acid
ds double stranded
DTT dithiothreitol
EAE Experimental Autoimmune Encephalomyelitis
E. coli Escherichia coli
EDTA ethylene diamine tetra acetate
EGF epithelial growth factor
ELAM-1 endothelial leukocyte adhesion molecule-1
EMSA electrophoretic mobility shift assay
et al. et alii
FCS fetal cf serum
FPF folate-PEG-folate
FPLC fast protein liquid chromatography
g gram
GAPDH glycerinaldehydephosphodehydrogenase
GLUT glucose transporter
h hour
HABA 2(4’-hydroxyazobenzene)benzoic acid
HBSS Hank’s buffered saline
HIV human immunodeficiency virus
HMW high molecular weight
HPLC pressure liquid chromatography
ID injectd ose
IGF insulin like growth factor
IgG immuno globulin G
I κB nhibitory κB
IL terlukin
iNOS inducible itric oxide synthetase
IPTG isopropyl β D thiogalactoside
i.v. intravenous
l liter
kb kilo base Da il Dlton
LB medium Luria-Bertani medium LDH lactate dehydrogenase
LDL low density lipoprotein
LFA-1 lymphocyte function associated antigen-1
LMW molecular weight
LNAA large neutral amino acid
LPS