Linear poly(amidoamine) (PAA) cationic polymers complexed with DNA have potential as non-viral nucleic acid delivery systems. However, they have poor in vivo biodistribution and a tendency to aggregate. PEGylation polymers are a potential solution for these problems but previous work shows that they do not always form compact complexes. This might be overcome by using mixtures of homopolymer and PEGylated copolymer to form complexes. However, the complexes are unstable in the presence of serum. Therefore, the aim of this study was to improve stability and to allow release of DNA by incorporating a novel cross-linking system. A disulphide-based cross-linker was suggested for this work. Formulation and screening studies for the best homopolymer to copolymer ratio of cross-linked (XL) DNA delivery systems in comparison to non-cross-linked (non- XL) complexes were investigated. The formation of cross-linked complexes was carried out using mixtures of homopolymer (HP) and triblock copolymer (CP), based on the most promising P AA candidate from these aforementioned studies. In this study, we have used three different molecular weights of PEG (655, 1700 and 4600 Da). HP:CP at 1: 1 ratio proved to have the best collective physicochemical characteristics among all of the investigated ratios. The findings from biological evaluation of PEG655-PAA systems revealed that transfection efficiency of cross-linked and non-cross-linked complexes at all HP:CP ratios was similar to PAA in serum-free medium. Transfection of cross- linked complexes at 1: 1 HP:CP ratio did not show a significant difference in serum-free and serum-supplemented media, the transfection of all other formulations, lipofectAMINE and P AA were remarkably reduced in the presence of serum. The results also showed the ability of PEG 1700-P AA-XL complexes to improve the transfection 100 times relative to non-cross-linked complexes. Again, no significant difference could be observed for the gene expression of PEG 1700- P AA systems at 1: 1 HP:CP ratio, whereas the transfection of other systems dramatically reduced in the presence of 10% serum. The same pattern was seen with PEG4600-PAA systems. In vivo assessment of the cross-linked complexes showed that PEG655-PAA-XL complexes provided strikingly higher luciferase activity in the liver, whereas PEG4600-PAA-XL complexes showed the best transfection in non-reticuloendothelial system organs expected to benefit from a longer circulation time. PEG 1700-P AA-XL complexes showed similar transfection in all organs. Therefore, the disulphide-based cross-link was validated as a suitable DNA delivery system for in vivo application.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:588065 |
| Date | January 2012 |
| Creators | Aljaeid, Bader Mubarak |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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