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Development and evaluation of immobilised anti-inflammatory peptide surfaces

Taylor, Paul J (2018) Development and evaluation of immobilised anti-inflammatory peptide surfaces. PhD thesis, University of Sheffield.

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PhD Thesis Paul J Taylor FINAL.docx
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Abstract

As modern technology and medicine develops, implantable medical devices are increasingly utilised for improving the quality of life. The immune system responds to these implantable materials initiating an inflammatory response. In some cases, chronic inflammation will ultimately result in device failure due to the immune system affecting the material integrity of the implant device. Generally, non-steroidal anti-inflammatory drugs (NSAIDs) and steroids are used to limit this effect but they are not specific in treating this type of inflammation. α-Melanocyte Stimulating Hormone (α-MSH) is a natural and potent anti-inflammatory peptide hormone with a short peptide sequence that can be synthesised and used for therapeutic applications. The peptide is effective in attenuating inflammation and nanomolar concentrations of peptide is adequate to diminish inflammation. In previous work, a truncated version of α-MSH has been immobilised onto glass coverslips using resorcinarenes as surface attachment molecules. These surface coatings were demonstrated to reduce inflammation in a variety of cells when stimulated in inflammatory conditions, with the therapeutic activity equivalent to a 1 nM solution of α-MSH. Resorcinarenes attach onto surfaces to form self-assembled monolayers (SAMs). However, the surface attachment of resorcinarene is reversible ultimately leading to the desorption of bioactive surface molecules from the surface. Additionally, resorcinarene chemical functionalisation strategies for conjugation with bioactive molecules is limited due to the stability of the molecule. To understand in more detail the activity of immobilised α-MSH, a second-generation surface attachment coating that can be patterned was designed. The aim of this work is to use these surfaces to pattern α-MSH across the surface to observe, compare, and evaluate its anti-inflammatory effects. Aryl azide conjugated organosilanes were chosen as they covalently bind to the surface forming chemically robust surface systems. These aryl azide functionalised SAMs can be patterned using UV light, and amines and peptides were successfully immobilised and patterned using photolithographic techniques. This work has demonstrated that surface patterning did not enhance nor diminish the anti-inflammatory properties of the surface coating when compared to a surface fully functionalised with α-MSH. However, when α-MSH on the surfaces was fluorescently labelled, it was shown that peptide is released from the surface in the presence of cells under the inflammatory conditions. In the absence of the inflammatory conditions, the peptides remained attached to the surfaces. It is suggested that the inflammatory conditions induce the cells to release peptide from the surface, possibly through peptidase enzymes. It is likely that a carbamate linkage in the hexaethylene glycol tether, serving as a molecular spacer, that is the cleavage site. The labelling studies also identified that the lysine residue is essential for anti-inflammatory activity, as the labelled peptides did not show any anti-inflammatory activity. The findings of the work have identified the mechanism behind the anti-inflammatory properties of these surface coatings. The surface coating acts as an anti-inflammatory prodrug that in inflammatory conditions where the change in chemistry, from the presence of released protease enzymes from inflamed cells, are capable of releasing the peptide from the coating. The work highlights an important finding for future clinical studies based on these coatings offering a therapeutic prodrug surface coating to attenuate inflammation. This coating system can also be adapted to address other diseases that require a prodrug release strategy. Due to the versatility of the surface coating system, alternative bioactive substrates was also explored. Carbon monoxide releasing molecules (CORMs) are inorganic molecules that act as a synthetic source for CO. Literature has shown that therapeutic amounts of CO can also attenuate inflammation through the MAPK pathway that also has parallel therapeutic properties akin to α-MSH. The overall goal is to take advantage of the surface coating system to pattern both α-MSH and CORMs onto a surface to act a dual functional bioactive coating. A preliminary investigation into immobilising CORMs onto surfaces revealed that insufficient CO was released to have a therapeutic effect. However, if the mass of CORM at the surface can be amplified, this may provide a strategy to create anti-inflammatory surfaces that contain two complementary activities.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > Chemistry (Sheffield)
Depositing User: Mr Paul J Taylor
Date Deposited: 27 Mar 2018 14:49
Last Modified: 27 Mar 2018 14:49
URI: http://etheses.whiterose.ac.uk/id/eprint/19634

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