Non-Viral Delivery of Nucleic Acids and Image-Guided Assessment of in vivo Performance of Multifunctional Nanomedicines

In this thesis, new and multifunctional non-viral vectors for delivery of nucleic acids were synthesized and characterized concerning biophysicochemical parameters. Chapter 1 introduces into the field of nanomedicine which advanced drug delivery systems can be numbered among. Chapter 2 describes t...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
1. Verfasser: Merkel, Olivia Monika
Beteiligte: Kissel, Thomas (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2009
Schlagworte:
Online Zugang:PDF-Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung:In this thesis, new and multifunctional non-viral vectors for delivery of nucleic acids were synthesized and characterized concerning biophysicochemical parameters. Chapter 1 introduces into the field of nanomedicine which advanced drug delivery systems can be numbered among. Chapter 2 describes the synthesis of 5 different PEI-based integrin alpha-v-beta-3-targeting conjugates by attaching a novel small molecule in two different coupling routes. Their ability to condense pDNA into nano-sized complexes, concerning their surface charge, biocompatibility, transfection efficiency, and finally specificity were investigated. Highly specific and efficient transgene expression was observed in integrin-overexpressing MeWo cells, where the most efficient conjugate lead to 26-fold higher luciferase expression than the PEG-PEI the conjugate was based on. In Chapter 3, a novel family of dendrimers for gene delivery is presented. The synthesis of 8 new modifications in the periphery of generation 2 rigid piperazine-connected triazine dendrimers is described as well as the periphery-induced impact on biophysicochemical parameters of self-assembled “dendriplexes” from pDNA and dendrimer. Transfection efficiencies of the differently substituted triazine dendrimers was assessed leading to the result that a certain amount of primary amines or guanidinium groups among the end groups is most important to maintain good transfection efficiency, while alkylation also enhanced transgene expression. Chapter 4 involved further synthetic progress concerning the structure of triazine dendrimers. Design criteria for this two-dimensional study were to investigate both the influence of generation and of core structure on in vitro parameters. Therefore, generation 1, 2 and 3 of the rigid triazine-piperazine structures were compared with each other. But also more flexible generation 2 cores in a “bow-tie” structure, and ethylene glycol chain-connected flexible structure were synthesized and compared with the rigid core dendrimers. At comparably low toxicity, the highly flexible generation 2 dendrimer achieved higher transfection efficiencies than both PEI and SuperFect®, which is a commercially available generation 4 fractured PAMAM dendrimer. Chapter 5 deals with siRNA delivery in vivo. This chapter describes a fast and efficient method to radiolabel and purify siRNA, and also shows results of in vivo application and SPECT imaging. Invasively and non-invasively acquired pharmacokinetic parameters and distributional data are compared for free siRNA and PEI-complexed siRNA which showed well known accumulation in the liver. In Chapter 6, the method optimized in the chapter before was applied to radiolabel siRNA for an extensive comparative study on in vivo stability, pharmacokinetics and biodistribution of differently PEGylated PEI/siRNA complexes. In order to investigate stability of these complexes and to distinguish between possibly different distribution profiles of vector and load, each component was separately labeled and pharmacokinetic and distributional profiles were assessed for each vector and load invasively and by SPECT imaging. Additionally, Fluorescence Fluctuation Spectroscopy was applied to quantify stability under in vivo conditions. Due to the rather poor pharmacokinetic profiles of intravenously administered (PEG-)PEI/siRNA polyplexes, Chapter 7 investigates suitability of the same complexes for intratracheal application. Therefore, in stability of the complexes, in vivo residence time in the lung, biocompatibility and knock down efficiency in transgenic mice was tested. PEI-complexed siRNA did not experience any benefit compared with free siRNA concerning lung residence time or washout. PEG-PEI/siRNA complexes, on the other hand seemed to be stable after intratracheal application and to exhibit sustained release of siRNA which allowed for significant down-regulation of EGFP in the lung five days after treatment of Actin-EGFP-expressing mice. Biocompatibiliy was tested, and only six of 22 chemo- and cytokines were found to be increased in the BALF. Chapter 8 deals with off-target effects. After unaccountable toxic effects of PEG-PEI were observed in a stably luciferase-expressing cell line, which was supposed to serve as a screening model, the reasons for these effects were systematically investigated. Membrane toxicity was investigated but found not to be the cause. An array experiment for investigation of gene up- and down-regulation allowed presuming that activation of CD30 regulated certain pro-apoptotic pathways involving NFkB in this p53-deficient cell line. In other cell lines, NFkB did not cause severe cell death but activation of CMV-promoter driven luciferase expression. Only the establishment of a luciferase expressing cell line where the reproter gene was independent of a CMV-promoter allowed for specific RNAi without major off-target effects.
Umfang:278 Seiten
DOI:10.17192/z2009.0472