Pattrick, N.G.; (2003) Direct and indirect evidence for the endosomolytic properties of poly(amidoamine)s. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Many potential therapeutics (such as genes, antisense and protein drugs) have their targets in a variety of intracellular compartments and thus it is essential to develop intracytoplasmic delivery vectors to mediate this process, in particular, escape of the therapeutic from endosomal vesicles to the cytoplasm. It is in this context that amphoteric, biocompatible, biodegradable poly(amidoamine) (PAA) polymers were studied. These polymers are able to display pH-dependent conformational changes in response to pH, thus displaying membrane-permeabilising properties at endosomal but not physiological pH. In this thesis different methods were used to demonstrate their endosomolytic properties. The ability of PAAs to mediate transfection of the plasmid pSV-β-galactosidase in HepG2 cells provided first direct evidence for their endosomolytic ability. For the first time it was shown that the PAAs termed ISA 1 and 4 were able to mediate delivery of the non-permeant toxins ricin A-chain (RTA) and gelonin to the cytoplasm of B16F10 cells. Toxin cytosolic delivery was indicated by a decrease in the IC50 value from > 1.8mg/mL to < 0.65mg/mL. To investigate the intracellular trafficking of PAAs alone and in combination with gelonin, fluorescent-labelled PAAs and gelonin were prepared for fluorescence microscopy. Uptake studies showed that the intracellular fate of the PAA and toxin differed according to the PAA structure used. Direct evidence for PAA-mediated intracellular membrane disrupting capability was investigated using isolated rat liver lysosomes as a model membrane system. After intravenous (i.v.) administration of ISA 1 (50mg/kg), nearly a 3-fold increase in the lysosomal enzyme N-acetyl-β-D-glucosaminidase (NAGase) was observed compared to the control. Morphologically ISA 1-containing lysosomes appeared less densely stained compared to the control. Finally, an ISA 1-gelonin construct was examined for endosomolytic ability. However conjugation of gelonin alleviated the pH-dependent membrane rupturing ability of the parent PAA. In conclusion, evidence for the endosomolytic properties of PAAs was demonstrated. However in order for a successful PAA-conjugate to be developed, it is important that the activity of PAAs and/or the drug is maintained upon conjugation.

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