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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Multi-component peptide-based carriers for gene delivery

Shu Yang Unknown Date (has links)
The feasibility of most gene therapy strategies depends on the efficient delivery of DNA to target cells and tissues. Current gene delivery carriers can be divided into two classes: viral and non-viral delivery systems. Although the viral carriers are highly efficient due to their invasive nature, safety concerns may restrict their application in clinical settings. Synthetic non-viral carriers attract increasing attention because they are less toxic and allow readily modification. Non-viral carrier mediated gene delivery involves several processes. They must condense DNA into small particles, allow membrane penetration and protect DNA from extracellular and intracellular degradative enzymes. In the present study, a small library of carriers containing various combinations of cell penetrating peptide TAT, SV40 large T protein nuclear localisation signal (NLS) and cationic dendrimer of 7 lysine residues (DEN) was synthesised and tested for their ability to deliver DNA to mammalian cells. We evaluated the contribution of each component as well as the combination of the components on DNA condensation, uptake and gene expression. It was found that all carriers condensed DNA and protected DNA from DNase degradation. We showed that the TAT peptide was essential, but not sufficient, for uptake of exogenous DNA. The addition of either NLS or DEN significantly enhanced uptake. The most efficient carrier contained all three components (DEN-NLS-TAT). The carriers were able to deliver DNA in the presence of serum and were non-toxic to cells at up to 30 μM. However, for those peptides that facilitated DNA uptake, the complexes were targeted to intracellular compartments that required a fusogenic agent, such as chloroquine, before gene expression was observed. Modifications were introduced to the initial carrier library in order to circumvent the chloroquine dependence. The addition of cell penetrating peptide penetratin, virus derived fusogenic peptide or lipoamino acid C12 enhanced either DNA uptake or endosomal release. However, none of the modified carriers were able to produce high level transgene expression in the absence of chloroquine. We also found that the carriers containing lipid components were able to deliver DNA to T-lymphocytes derived cells, which are usually resistant to transfection. However, the toxicity of the lipid-based carriers needs to be reduced before further application. We also evaluated the function of chloroquine as a gene expression enhancer. We demonstrated that chloroquine did not enhance expression solely by promoting endosomal release. This was supported by the fact that fusogenic peptide and endosomal disruptive reagents (bafilomycin A1 and monensin) did not improve gene expression. Other properties of chloroquine, such as DNA protection and transcription enhancement, may also contribute to gene expression. We characterised the uptake mechanism of DEN-NLS-TAT in HeLa cell lines. We found that the uptake of DEN-NLS-TAT/DNA complex in HeLa cell line was mainly via receptor-mediated endocytosis and caveolae endocytosis. Moreover, various intracellular processes, such as intact cytoskeleton and microtubule network, tyrosine and PI 3 kinase activity, and membrane cholesterol were also required for the uptake of the carrier/DNA complex. In conclusion, the results from the present study demonstrated that multi-component peptide-based carriers are versatile carriers for the delivery of plasmid DNA in human cells. The results have improved our understanding of the role of chloroquine as a widely used gene expression enhancer which may be useful in the future improvement of non-viral gene delivery carriers. A strategy to overcome the dependence on chloroquine for gene expression or reduce the toxicity of chloroquine will be necessary for further in vivo applications. The current carrier library may also be used to delivery other cargos such as siRNA or protein to human cells.
2

Propriétés structurales et associations en solution des dendrimères polyamidoamine (Pamam) / Structural properties of pamam dendrimers and their interactions in solution

Zerrad, Louiza 13 January 2010 (has links)
Nouveaux polymères avec une structure arborescente unique, les dendrimères suscitent un grand intérêt auprès des chercheurs de tous les domaines scientifiques confondus et spécialement auprès des biologistes. Théoriquement synthétisés de façon à ce qu’ils soient parfaitement mono disperses en taille et en masse et de forme sphérique, les dendrimères PAMAM ont des propriétés structurales qui pourraient donner de bons résultats lors de leur utilisation comme vecteurs de médicaments ou d’acides nucléiques.De récentes études biologiques ont montré que les dendrimères PAMAM pouvaient transfecter un grand nombre de cellules de différente nature. Les propriétés structurales et physicochimiques du dendrimère joueraient un rôle essentiel dans l’efficacité de cette transfection et pouvoir prédire ces propriétés permettraient de mieux contrôler le comportement de ces«vecteurs-médicaments» dans l’organisme / In the context of securing clinical gene transfer, new strategies are developed with the creationof synthetic gene vectors based on cationic polymers to replace commonly used viral vectors ingene therapy. PAMAM dendrimers are highly branched macromolecules with controlled nearmonodisperse three-dimensional architecture emanating from a central core. Polymer growthstarts from a central core molecule and growth occurs in an outward direction by a series ofpolymerisation reactions. Hence, precise control over size can be achieved by the extent ofpolymerisation, starting from a few nanometers. Cavities in the core structure and folding of thebranches create cages and channels. The surface groups of dendrimers are amenable tomodification and can be tailored for specific applications. Therapeutic and diagnostic agents areusually attached to surface groups on dendrimers by chemical modification.Recent biological studies have shown that PAMAM dendrimers could transfect a large numberof cells of different nature. The structural properties and physico-chemical properties ofdendrimer play a key role in the efficiency of transfection and to predict these properties wouldbetter control the behavior of these "drug delivery systems in the body.

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