Understanding the mechanism of action of endosomolytic polyamidoamines by studying their physico-chemical properties.

Khayat, Zeena. 2006. Understanding the mechanism of action of endosomolytic polyamidoamines by studying their physico-chemical properties. PhD Thesis, Cardiff University.

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Abstract

Polyamidoamines PAAs are a family of synthetic, water-soluble, linear polymers that are synthesised by hydrogen-transfer polyaddition of aliphatic amines or bis-secondary amines to bisacrylamides (reviewed by Ferruti et al. 2002). Over the last 15 years, PAAs been developed as drug carriers and as pH-responsive polymers for protein and gene delivery (Richardson et al., 2001). The latter, called endosomolytic PAAs, can disrupt membranes at low pH, but the delivery of genes and proteins is still poorly efficient. The precise mode of action is still poorly understood, therefore the main aim of this work was to examine the physico-chemical properties of PAAs in order to better define their mechanism of membrane permeabilisation to allow design of more efficient chemical structures. First PAA ISA1 (23 k g/mol) and ISA23 (52 - 67 k g/mol) were synthesised and were characterised using H-NMR, GPC and acid-base titration. In agreement with past studies, ISA23.HC1 demonstrated a pH-dependant haemolytic activity. The solution conformation of ISA23.HC1 was then investigated using small-angle neutron scattering (SANS). ISA23.HC1 possessed a Gaussian coil like-shape in solution at all pHs (pH 2 to 14) and polymer concentrations examined (10 mg/ml to 50 mg/ml). However as the pH decreased the radius of gyration increased to a maximum (Rg ~8 nm) at ~pH 3. Surface tension, electron paramagnetic resonance (EPR) and SANS were then used to investigate the interaction of ISA23.HC1 with simple micelles as model surfaces. Both pH and micelle composition played an important role in the strength of interaction seen. For SDS micelles, three different responses were observed, strong interaction at pH < 4.5, weak interaction at pH 4.5 - 6.5, and no interaction at pH > 7. However, ISA23.HC1 did not appear to interact with more biologically relevant lyso-PC surfactants at pHs 7.4 - 5.5. EPR experiments showed that micelle fluidity was effected when a PAA-micelle interaction occurred, and SANS reinforced this conclusion. Finally, liposomes were used as a more complex model membrane. Liposomes were prepared to mimic three phospholipids composition of plasma, endosomal and lysosomal membranes. The liposomes were stable over 2 days at pH 7.4, 5.5 and 4, but not at pH 3. SANS was used to study polymer-liposomes interaction, and the Schultz polydisperse 3 shell sphere model was used to fit the liposomal (alone) SANS scattering data. The radius of the plasma, endosome and lysosome liposomes was ~ 50 nm. When liposomes were incubated in the presence of ISA23.HC1, it was difficult to interpret the scattering data. However, it was clear that at pH 4 and 5.5 scattering of model liposomes alters in the presence of PAA indicating possible interaction. Using a contrast approach the scattering data for ISA23.HC1 in the presence of model liposomes could be extracted. This was again fitted using Gaussian coil model. Generally, the polymer increased in size in the presence of liposomes, at pHs where there was an interaction. However, these are only preliminary experiments, and there is a need for further mathematical modelling. In conclusion, this project emphasise the importance of combining the different disciplines to fully understand a system that can be the first step in finding a way to cure devastating genetic diseases.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Pharmacy
Subjects:	R Medicine > RS Pharmacy and materia medica
ISBN:	9781303174155
Date of First Compliant Deposit:	30 March 2016
Last Modified:	12 Feb 2016 23:11
URI:	https://orca.cardiff.ac.uk/id/eprint/54271

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