Enzyme-responsive hydrogels: Development, characterisation and on-demand modulation of protease activity

Obenza Otero, Rebeca Lucia (2020) Enzyme-responsive hydrogels: Development, characterisation and on-demand modulation of protease activity. PhD thesis, University of Nottingham.

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Abstract

Bioresponsive materials that are able to release their cargoes in response to disease-specific cues are of great interest for developing targeted therapeutics due to their potential to limit drug release to its site of action, thus minimising side effects. The use of proteases as triggers for biomaterial response is particularly attractive as upregulated activity of proteases, such as elastase, is related to several pathological states such as tissue destruction associated with chronic wounds and respiratory diseases. As elastase also possesses important functions that are crucial for maintaining a healthy status, full enzyme suppression is not desirable. Instead modulation of elastase activity to restore the natural protease/inhibitor balance that favours restoration of tissue integrity would be advantageous.

Feedback-controlled modulation of elastase in response to the level of enzyme activity could be achieved by incorporating inhibitors and elastase-sensitive components in the material. This thesis aims to develop peptide-crosslinked PEG hydrogels able to release the elastase inhibitor alpha-1-antitrypsin (AAT) in response to the level of elastase in its environment, mimicking the body’s natural strategy for balancing protease levels to restore tissue integrity. To achieve this, hydrogels were formed by crosslinking of thiol-terminated elastase-sensitive peptides with multi-arm thiol-reactive polymers. Initially the hydrogel crosslinking kinetics, mesh size, mechanical properties and ability to physically entrap molecules were optimised using a model short dithiol. Then, an elastase-responsive peptide sequence with suitable solubility was incorporated into hydrogels via thiol-crosslinking chemistries. Elastase-responsive hydrogels were shown to release a model dextran upon incubation with elastase, while elastase non-responsive gels (fabricated with a scrambled peptide) were unaffected by the enzyme. This confirmed that the responsive release was dictated by the peptide sequence. Further confirmation of the hydrogel’s selective responsiveness was achieved by incubation with a non-targeted enzyme, Matrix Metalloproteinase-2, which did not increase macromolecule release.

The ability of AAT encapsulated in elastase-responsive hydrogels to inhibit elastase was demonstrated, as characterised by monitoring the release of a model probe and measuring elastase activity in the milieu. Scrambled-peptide crosslinked gels were used as passive release control, and modulated elastase activity to a lower extent than the responsive hydrogels. Finally, the effects of hydrogels in cells and their ability to promote a beneficial effect on cells through elastase activity modulation was studied in vitro. In vitro tests confirmed hydrogels did not affect epithelial cell viability and AAT-loaded gels were able to modulate elastase activity in the presence of cells and improved elastase-hindered epithelial repair.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Zelzer, Mischa
Mantovani, Giuseppe
Chan, Weng
Subjects: Q Science > QP Physiology
Faculties/Schools: UK Campuses > Faculty of Science > School of Pharmacy
Item ID: 59688
Depositing User: Obenza Otero, Rebeca
Date Deposited: 16 Jul 2020 04:40
Last Modified: 16 Jul 2020 04:40
URI: http://eprints.nottingham.ac.uk/id/eprint/59688

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