One third of drugs in development are water insoluble, and one half of all drugs fail in trials because of poor pharmacokinetics. Block copolymer micelles are nanosized particles that can solubilize hydrophobic drugs and coincidentally alter their kinetics in vitro and in vivo. In using such micelles it is important that the drug is not lost due to premature vehicle disassembly and that its effectiveness is not diminished by unfavorable distribution of the micelles. Disintegration of micelles depends on a number of environmental conditions, with consequent variation in drug release rates; these rates, however, are always faster than those which occur by simple diffusion from intact micelles. When these thesis studies began no data was available on integrity of micelles in cells and in vivo, and in vitro studies of distribution of micelles were scarce. This thesis begins by providing a detailed account of the distribution of model block copolymer micelles in live cells using fluorescent-labeled polymer and confocal microscopy. Next, the evidence for endocytosis of micelles, and their effectiveness in delivering a model micelle-incorporated agent anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) is presented. The thesis ends with an account of the disruption of integrity of micelles in vitro and in vivo, made possible by a novel application of a fluorogenic-based assessment that enables the monitoring of the integrity of micelles under complex biological conditions. / Block copolymer micelles were made from Food and Drug Administration approved polymers poly(caprolactone) and poly(ethylene oxide). In vitro studies were done in cell lines (PC12, NIH/3T3, T24) and primary cultures (human islets of Langerhans). In vivo studies were done in hairless SKH-1 mice. The in vitro studies demonstrated the uptake of micelles by all investigated cells. The micelles were confined to the cytoplasmic compartment, with no detectable nuclear localization. Detailed studies in PC12 cells revealed co-localization of micelles with several organelle selective dyes (mitochondria, Golgi apparatus, endoplasinic reticulum). Pharmacological manipulations (sucrose, FK506, wortmannin, horseradish peroxidase, methyl-beta-cyclodextrin) revealed an endocytotic uptake of micelles and suggested a significant contribution of clathrin-mediated endocytosis. The effectiveness of c-Jun NH2-terminal kinases (JNK)---micelle-incorporated inhibitor, SP600125, was demonstrated in human islets of Langerhans challenged with cytokines. Micelle-incorporated SP600125 prevented the cytokine induced cell death of islets at a lower starting concentration (1.5 $M in the medium) compared to free drug (20 muM). / Lastly, a graded loss of integrity of micelles was demonstrated in media with increasing biological complexity, in cells, and, as a proof of principle, in vivo. Taken together, the results presented in this thesis suggest (i) a need for assessment of the integrity of micellar drug carriers under biological conditions to further improve the micelle-based drug delivery systems, (ii) the identification of the contribution of micelles with disrupted integrities to the internalization and effectiveness of micelle-incorporated drugs, (iii) an assessment of cellular retention and degradation of internalized polymers. That the pharmacokinetics of micelle-incorporated drugs reported in recent clinical trials showed only modest improvements over non-micelle delivered agents may at least in part be due to disruption of micelle integrity. Additional studies are needed to understand exactly where the delivery system goes, when and where is the drug released, and how recent advances in vehicle design and targeting can be harnessed to overcome the present challenges and realize the full potential of polymeric micelles.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.102168 |
| Date | January 2005 |
| Creators | Savić, Radoslav. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Pharmacology & Therapeutics.) |
| Rights | © Radoslav Savić, 2005 |
| Relation | alephsysno: 002326492, proquestno: AAINR25248, Theses scanned by UMI/ProQuest. |
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