Physical and chemical modification of medical metal alloys to enhance surface functionalisation for antimicrobial peptide immobilisation

Zare, Mohadeseh (2025). Physical and chemical modification of medical metal alloys to enhance surface functionalisation for antimicrobial peptide immobilisation. University of Birmingham. Ph.D.

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Abstract

The rise of antibiotic-resistant bacterial infections has become a significant concern in implant failure. A promising strategy to combat peri-implant infections involves the use of antimicrobial peptides (AMPs), which exhibit broad-spectrum activity against Gram-positive and Gram-negative bacteria, fungi, and viruses, while also demonstrating a low propensity for inducing bacterial resistance. Recently, various strategies have been applied for the chemical and physical immobilisation of AMPs on surfaces. These approaches vary in terms of binding strength, modularity, stability, and complexity. While physical binding methods often result in weak immobilisation and the potential detachment of bioactive molecules, chemical integration of AMPs into biomaterials provides a more robust immobilisation, ensuring that the peptides remain stable and optimally oriented on the surfaces. However, chemical integration is a complex process involving multiple steps that can impact overall efficiency. Among the numerous techniques available for chemical immobilisation, plasma nitriding has gained attention for its time- and energy-efficient process, as well as the use of nontoxic materials. The primary objective of this study was to develop innovative antimicrobial surfaces for titanium alloys by incorporating AMPs and utilising advanced surface engineering techniques. In this study KR-12 peptide has been employed as an AMP derived from the human cathelicidin LL-37, known for its broad-spectrum activity against bacteria by disrupting microbial membranes. Titanium implants were functionalised with the KR-12 peptide using two approaches: titanium surface plasma treatment and silanisation, both aimed at generating amine groups on the surface to facilitate subsequent reactions with KR-12. Additionally, the study investigated how different linking arms -Polyethylene Glycol (PEG) and human-elastin-like-polypeptide (HELP)- affect the antimicrobial activity of the peptides through comprehensive surface characterisations, biological assays, and antimicrobial evaluations.
In the initial phase of this study, plasma treatment was utilised to further immobilise the KR-12 peptide on titanium implant surfaces, and polyethylene-glycol (PEG) was employed as a linker to impart antifouling properties and enhance peptide mobility, thereby improving antimicrobial efficacy. Plasma treatment was chosen for its ability to introduce functional groups, streamline chemical reactions, and reduce processing time. The primary amine groups (NH2) generated during plasma treatment could form strong attachments to titanium surfaces, serving as anchors for immobilising bioactive agents. In this study, our first objective was to optimise plasma treatment parameters to achieve surface-bound amine group levels comparable to those obtained through chemical techniques like silanisation. X-ray photoelectron spectroscopy (XPS) analysis revealed that the distribution and density of primary amine groups could be finely tuned by adjusting plasma parameters, reaching a maximum of 9.18% without altering the titanium surface morphology or bulk properties.
Following the generation of primary amine groups, PEG and KR-12 were immobilised on the surface at specific concentrations. The antimicrobial activity of these coatings against four pathogenic bacterial strains demonstrated over 90% inhibition of bacterial adhesion and colonisation. The enhanced activity was due to the improved orientation of linker-grafted AMPs compared to directly immobilised peptides. Also, the plasma-treated surfaces were proven to enhance cell proliferation and adhesion. These results underscored the potential of combining plasma treatment with the covalent bonding of AMPs, exploiting both the antimicrobial properties of the AMPs and the cell-promoting effects of the plasma-treated surface.
In the second phase of the study, silanisation was employed to introduce primary amine groups onto the surface, serving as anchor points for the immobilisation of the KR-12. This study introduced an enzyme-responsive antimicrobial coating for orthopaedic devices, utilising KR-12 in conjunction with Human Elastin-Like Polypeptide (HELP) as a biomimetic, stimuli-responsive linker that emulates the ECM. Upon implantation, these tailored interfaces interacted with the innate immune response, leading to the release of elastase. The biodegradation of the HELP biopolymer by elastase facilitated the controlled release of KR-12. The antimicrobial activity of the KR-12-HELP-functionalised titanium surface was evaluated against four pathogenic bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa), demonstrating an inhibition of bacterial adhesion and colonisation exceeding 91.86% for all tested strains. When compared to a titanium surface functionalised directly with KR-12 without the HELP linker, the antimicrobial efficacy was significantly enhanced. This improvement was due to the increased mobility of KR-12 when coupled with HELP and its stimuli-responsive controlled release.
Plasma treatment demonstrated superior efficiency, introducing 9.18% primary amine groups on titanium surfaces compared to 4.67% with silanisation. Both methods achieved comparable peptide immobilisation levels (59.67 nmol and 61.34 nmol respectively), although plasma treatment offered additional advantages, such as enhanced surface energy, improved protein adsorption, reduced toxicity, and lower costs. Plasma-treated surfaces maintained biocompatibility, promoting cell adhesion and proliferation without altering surface morphology.
This study highlights plasma treatment as a cost-effective and efficient alternative to silanisation, particularly when combined with advanced linker systems such as PEG and HELP. The integration of these methods offers a promising pathway for developing next-generation antimicrobial coatings for orthopaedic implants, enhancing both bacterial resistance and tissue integration.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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