The Synthesis and Characterization of Peptide-Based Carriers for Gene Delivery and Peptide Therapeutics

Daniel Coles (2010). The Synthesis and Characterization of Peptide-Based Carriers for Gene Delivery and Peptide Therapeutics PhD Thesis, School of Chemistry and Molecular Biosciences, The University of Queensland.

       
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Author Daniel Coles
Thesis Title The Synthesis and Characterization of Peptide-Based Carriers for Gene Delivery and Peptide Therapeutics
School, Centre or Institute School of Chemistry and Molecular Biosciences
Institution The University of Queensland
Publication date 2010-03
Thesis type PhD Thesis
Total pages 149
Total colour pages 6
Total black and white pages 143
Subjects 06 Biological Sciences
Abstract/Summary Gene therapy is a promising approach for the treatment of genetic diseases. Currently, there are no gene therapy drugs available on the market. There are many therapeutic genes available for treating various diseases but they are not delivered effectively to the target site. The size, negative charge and susceptibility of DNA to degradation can be overcome by using a gene carrier. Current gene carriers are divided into two groups, viral and non-viral. Viral carriers are very efficient but they can induce an immune response. Non-viral carriers are less efficient and sometimes toxic. The optimisation of gene carriers is an ongoing endeavour for researchers. We have designed gene carriers to overcome the barriers associated with gene delivery. These barriers include DNA stability, the cell membrane, endosomal membrane, nuclear membrane and dissociation of DNA/carrier complex. Therefore, our gene carrier design included a cell-penetrating peptide (CPP) TAT, a nuclear localization signal (NLS) peptide, dendritic polylysine and lipoamino acids (LAAs). The carriers were synthesized by solid phase peptide synthesis using Boc chemistry. They were purified by preparative RP-HPLC and characterized by mass spectrometry and analytical RP-HPLC. The carriers’ ability to form a complex with DNA was assessed by gel shift assay, dynamic light scattering (DLS) and zeta potential (zetasizer), isothermal titration calorimetry (ITC) and transmission electron microscopy (TEM). Cell uptake and gene expression were analysed by our collaborators. The carriers were successfully synthesized and were shown to form complexes with plasmid DNA. The gel shift showed a shift of the band to the well of the gel compared to free DNA. The sizes of the complexes were 60-100 nm according to DLS and TEM measurements. The zeta potential of the particles was positive when an excess of carrier was used. ITC experiments revealed that the ionic interaction between the carrier and DNA was strong and increased with the carriers possessing more positive charges. The cell uptake was high when TAT was used in combination with NLS and/or polylysine. Also, the inclusion of LAAs increased cell uptake in Jurkat cells. Gene expression was low for all carriers without the application of chloroquine, a lysosomotropic agent. The TAT peptide was synthesized with different polycationic dendrimers to determine the optimum type, composition and size of dendrimer. We found that a polylysine dendrimer with eight positive charges provided the best results. A different CPP, penetratin, was used to see if it employed a different mechanism of cell uptake and therefore increase gene expression. However, these carriers did not significantly improve gene expression because the complexes were trapped inside the endosome. Carriers incorporating a substitute for chloroquine, the N-terminal of the influenza virus haemagglutinin HA2, were synthesized to increase endosomal escape and therefore, gene expression. An improvement in gene expression only occurred when using high ratios of carrier (20:1) but still the gene expression was low and increased upon the application of chloroquine. An anticancer peptide, shepherdin, was enhanced by adding penetratin and a LAA to improve its cell uptake and to lower the concentration of administration required for a therapeutic effect. The inclusion of a LAA did improve shepherdin’s anticancer ability. However, the negative control peptide coupled with a LAA was also toxic to cancer cells. A LAA conjugated with thymine was assessed for its ability to hydrogen bond to single-stranded DNA and characterized by diffusion ordered NMR spectroscopy (DOSY), ITC and TEM. It was found that the LAA-thymine compound binds to DNA at a ratio of 1.7:1 (LAA-thymine:DNA). Additionally, a control experiment titrating LAA-thymine into a solution of adenine showed a ratio of 0.9:1. A selection of peptide vaccines were analysed by DOSY, DLS and mathematical expressions to determine their particle size and to compare the three methods used. DOSY yielded results that were closer to the expected size compared to DLS measurements. Also, the smaller the peptide, the more accurate the measurements were when compared to the expected size generated from mathematical models. Our investigations proved that combinations of CPPs, NLS peptides, charged dendrimers, fusogenic peptides and lipids can be applied for successful gene delivery.
Keyword gene delivery
non-viral carriers
cell-penetrating peptides
lipid
dendrimer
peptide therapeutics
Additional Notes 24, 96, 100, 101, 113, 125

 
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Created: Tue, 23 Nov 2010, 10:47:27 EST by Mr Daniel Coles on behalf of Library - Information Access Service