Nanoparticle formulations for new cytostatic agents against glioblastomas [Elektronische Ressource] / by Telli Hekmatara

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• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Nanoparticle Formulations for New
Cytostatic Agents against Glioblastomas




A Thesis
Submitted to the
Department of Pharmaceutical Technology
at the
Goethe University
Frankfurt am Main, Germany
by
Telli Hekmatara



In Partial Fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
(Pharmaceutics)



Frankfurt am Main, 2008
(D30) Vom Fachbereich Biochemie, Chemie und Pharmazie
der Goethe-Universität Frankfurt am Main als Dissertation angenommen.

















Dekan: ....................................................................................................Prof. Dr. Harald Schwalbe

1. Gutachter: ................................................................................................Prof. Dr. Jörg Kreuter
2. Gutachter: ................................................................................................Prof. Dr. Jochen Klein

Datum der Disputation:......................................................................................................................






To my parent Dr. M. H. Hekmatara and Manijeh Rabbani  Table of Contents 
Abbreviations ..................................................................................................... 7

1 Zusammenfassung ........................................................................................ 10

2 Theory: Drug delivery to the brain by nanoparticles ............................... 16 
2.1 Structure of the blood-brain barrier ......................................................................... 16 
2.1.1 Physical barrier: Tight junctions ............................................................................ 16 
2.1.2 Neurovascular units/cells associated to the BBB ................................................... 17 
2.1.3 Metabolic barriers: Enzymes ................................................................................. 18 
2.2 Transport across the blood-brain barrier ................................................................ 19 
2.2.1 Cell migration ........................................................................................................ 19 
2.2.2 Passive diffusion .................................................................................................... 19 
2.2.3 Carrier-mediated efflux .......................................................................................... 20 
2.2.4 Carrier-mediated influx 20 
2.2.5 Vesicular transport ................................................................................................. 20 
2.2.5.1 Absorptive-mediated transcytosis (AMT) ...................................................... 21 
2.2.5.2 Receptor-mediated transcytosis (RMT) .......................................................... 21 
2.2.6 The role of tight junctions ...................................................................................... 21 
2.3 Drug delivery to the CNS ........................................................................................... 22 
2.3.1 Craniotomy-based drug delivery ............................................................................ 23 
2.3.2 Disruption of the BBB 23 
2.3.3 Chemical modification of the drug ........................................................................ 24 
2.3.4 Inhibition of efflux mechanisms 24 
2.4 Nanocarriers for brain delivery ................................................................................. 25 
2.4.1 Biodistribution of the nanoparticles to the normal brain ....................................... 25 
2.4.2 Distribution of the nanoparticles under the pathological condition of the brain ... 27 
2.4.3 Brain delivery with PBCA nanoparticles coated with polysorbate 80 .................. 28 
2.5 Pharmacological activity of the nanoparticle-bound drugs .................................... 29 
2.5.1 Neuroactive agents ................................................................................................. 29 
2.5.2 Doxorubicin ........................................................................................................... 31 
2.5.3 Mechanisms of drug delivery to the brain by nanoparticles .................................. 33 
2.6 Toxicological aspects ................................................................................................... 43
   
1 
   Table of Contents 

3 Materials and Methods ................................................................................. 46 
3.1 Poly(butyl cyanoacrylate) nanoparticles................................................................... 46 
3.1.1 Doxorubicin-loaded PBCA nanoparticles .............................................................. 47 
3.1.2 Preparation of drug-loaded PBCA nanoparticles in the presence of organic
solvents ........................................................................................................................... 48 
3.1.3 Characterisation of nanoparticles ........................................................................... 48 
3.1.3.1 Determination of the particle size and the zeta potential ................................ 49 
3.1.3.2 Analytical ultracentrifugation (ANUC) .......................................................... 50 
3.1.3.3 Scanning Electron Microscopy (SEM) ........................................................... 51 
3.1.3.4 Atomic Force Microscopy (AFM) .................................................................. 51 
3.1.3.5 Evaluation of the loading capacity of the nanoparticles ................................. 52 
3.1.3.5.1 Doxorubicin assay .................................................................................... 53 
3.1.3.5.2 Loperamide assay..................................................................................... 54 
3.1.3.5.3 Paclitaxel assay ........................................................................................ 55 
3.1.3.6 Determination of polymer yield ...................................................................... 55 
3.1.3.7 Determination of residual organic solvents .................................................... 57 
3.1.4 Coating of the nanoparticles with surfactants ........................................................ 58 
3.1.5 In vivo experiments ................................................................................................ 59 
3.1.5.1 Tail-flick test ................................................................................................... 59 
3.1.5.1.1 Loperamide-loaded PBCA nanoparticles for tail-flick test ..................... 60 
3.1.5.2 Chemotherapy of 101/8 rat glioblastoma using doxorubicin formulations .... 60 
3.1.5.2.1 Intracranial implantation of rat glioblastoma ........................................... 60 
3.1.5.2.2 Doxorubicin formulations and treatment regimen ................................... 61 
3.1.5.2.3 Histological analysis ................................................................................ 61 
3.1.5.2.4 Immunohistochemical analysis ................................................................ 62 
3.1.5.2.5 Measurement of tumour size .................................................................... 63 
3.1.5.2.6 Analysis of proliferation .......................................................................... 63 
3.1.5.2.7 Analysis of vessel density ........................................................................ 63 
3.1.5.2.8 Analysis of necrosis ................................................................................. 64 
3.1.5.2.9 Analysis of GFAP expression of glioma cells ......................................... 64 
3.1.5.2.10 Analysis of microvascular proliferation ................................................. 64 
   
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   Table of Contents 

3.1.5.2.11 Analysis of VEGF expression of glioma cells ....................................... 64 
3.1.5.2.12 Statistical analysis .................................................................................. 64 
3.1.5.3 Chemotherapy of 101/8 rat glioblastoma using paclitaxel formulations ........ 65 
3.2 Human serum albumin nanoparticles ....................................................................... 66 
3.2.1 Nanoparticle preparation ........................................................................................ 67 
3.2.1.1 Yield determination of the HSA nanoparticles ............................................... 67 
3.2.1.2 Preparation of sulfhydryl-reactive HSA nanoparticles ................................... 67 
3.2.1.3 Purification of apolipoproteins A-I and B-100 68 
3.2.1.4 Thiolation of apolipoproteins .......................................................................... 68 
3.2.1.5 Modification of the nanoparticles by thiolated apolipop