24. LYSOZYME CAN BE CHEMICALLY BOUND TO TITANIUM: RESULTS OF A WET-CHEMICAL FUNCTIONALIZATION TECHNIQUE

Abstract

Purpose: Component loosening is a leading cause of joint replacement failure. Modifying titanium surfaces with chemically bound functional proteins, such as bone morphogenetic protein (BMP), can efficiently strengthen the interface between prosthesis and bone. A prototype system was developed by using gold nanoparticles (AuNPs) to bridge lysozyme (compositionally similar to BMP) and titanium.

Method: For reference, lysozyme-conjugated gold nanoparticles (Lys-AuNPs) were prepared in solution via two different pathways:

1. gold compound was reduced in the presence of lysozyme to form Lys-AuNPs or

2. citrate-stabilized AuNPs were functionalized with mercaptopropionic acid (MPA) to produce carboxylic acid terminated AuNPs which were mixed with lysozyme.

Both solutions were characterized with transmission electron microscopy, ultraviolet-visible spectroscopy, circular dichroism spectroscopy (CD), and enzymatic assays. Next, AuNPs were prepared on 99.5% titanium foil discs (n=32) through electroless deposition. Deposition parameters were modified to create two groups of discs with different average diameters of AuNPs, measured by scanning electron microscopy. Some discs from both groups also underwent treatment with MPA. All discs were treated with lysozyme and the adsorbed amounts and activities of lysozyme were examined with micro BCA and enzymatic assays.

Results: Lysozyme and AuNPs can be conjugated in solution via two different pathways. CD results showed a significant change in the secondary structure of the lysozyme and decrease in enzymatic activity when directly conjugated to AuNPs; however, little change in secondary structure and enzymatic activity was observed for the lysozyme with MPA functionalized AuNPs. For the AuNPs on the titanium discs, SEM showed that the two groups had significantly different average AuNP diameters. Bioactive lysozyme was immobilized onto the discs and the results suggested that discs with the largest AuNPs treated with MPA had higher adsorption and activity of lysozyme.

Conclusion: A wet-chemical technique may be used to bind lysozyme to titanium via gold nanoparticles. Additionally, it was possible to control the size of the AuNPs on titanium which provides a good platform for further functionalisation with thiol molecules such as MPA. This technique holds promise for binding more functional molecules to surgical implants, hence creating “smart” implants that react to their local environment.