Ribeiro, Suzie Jesus; (2006) A study of DNA/dendron nanoparticles for genetic immunisation against anthrax. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A series of cationic polylysine dendrons of diverse lipophilicity were synthesised and characterised. These cationic dendrons were selected to have the properties required for complexation with DNA. The dendrons series used in this thesis all have branched asymmetrical polylysine dendritic head group attached to a central core containing none or maximum of three lipidic chains of varying chain lengths. Dendrons interact with and condense DNA producing a complex "dendriplex" with a small residual negative surface charge, and enhance cellular internalization of DNA, in part protecting the DNA from degradation. The colloidal properties of the dendriplexes were measured. These included size, morphology and zeta potential as a function of charge ratio of dendron to DNA (the N/P ratio) accomplished by standard dynamic light scattering and transmission electron microscopy. The relative strengths of DNA/dendron interactions were determined by ethidium bromide efficiency dye displacement together with assessment of the release of the DNA when challenged with anionic compounds. A double emulsion method was employed to encapsulate the dendriplexes in poly-lactide-co-glycolide (PLGA) particles, characterised by the same biophysical methods as for the dendriplex formulations. The DNA was radiolabelled prior to formulation of PLGA-dendriplexes using a nick translation kit with 32S catalyzed DNA polymerase I, to introduce radioactive labelled nucleotides into DNA to allow the measurement of encapsulation efficiency, release and in vitro DNA uptake studies. The preliminary studies of both dendriplexes and PLGA-dendriplexes demonstrate their potential application as a genetic vaccine. Both particulate systems encapsulated protective antigen (PA), one of the protein components required for specific immunity to anthrax. Two types of plasmid DNA were developed one encoding PA 83 cloned into the eukaryotic expression plasmid pSecTag 2B (7.3kbp) and a control plasmid without PA 83. In vitro assays were conducted analysing expression of PA in cells. The assays verified that complexation with dendron aids the stability and uptake of PA DNA. In vivo immunisation with naked PA DNA even with multiple dosing did not induce sufficient antibody response even after secondary boosting post primary i.m immunisation, whereas, both dendriplexes and PLGA particles produced strong anti-PA antibody responses. However, they did not provide protection against lethal toxin challenge in the toxin neutralisation studies. To our knowledge this is the first in vivo study using dendriplexes and encapsulated dendriplexes as a vaccine against anthrax. Further work needs to be conducted to investigate what levels of antibodies are needed to protect humans against anthrax after vaccination.

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