Investigations into the characterization and thioether-based immobilization of antimicrobial peptides NK-2, arenicin and their derivatives

Abstract

The misuse and overuse of antibiotics have led to a rise in antimicrobial resistance, which is now considered a global health emergency (Kyvik et al., 2023). This problem is further worsened by the ability of bacteria to form biofilms, which make them 10–1000 times more resistant to antibiotics than planktonic bacteria (Kyvik et al., 2023). Biofilm formation on medical implants such as heart valves, catheters, contact lenses, joint prostheses, intrauterine devices, and dental units can cause persistent urinary tract and bloodstream infections. In many cases, the only effective treatment is removal of the infected implant, which can be a major burden for patients (Costerton, Montanaro, and Arciola, 2005). Immobilization of antimicrobial peptides (AMPs) onto solid surfaces has therefore become a promising strategy to prevent biomaterial-associated infections, as medical implants and prosthetic devices are often colonized by biofilm-forming bacteria that are resistant to conventional antibiotic treatment (Riool et al., 2017). Prior to immobilization, the peptides were characterized using SDS-PAGE and RP-HPLC. In total, seven peptides were investigated in this study: NK-2 and its derivatives NK27, ALK-33C, and N33-ALK, as well as Ar-1 (arenicin) and its derivatives C/S-Ar-1 and R/K-Ar-1. For peptide immobilization, one core immobilization strategy was applied, which was developed and optimized based on previous Bachelor and Master theses conducted by Sara Jashari and Marina Lesniewski. In the present thesis, this immobilization approach was further expanded, and four different immobilization combinations were investigated. In addition, trypsin digestion and UV/VIS-spectrophotometry were employed as complementary analytical methods. The results demonstrate that all seven peptides were successfully characterized using SDSPAGE and RP-HPLC. Regarding the immobilization, two out of the four tested combinations showed progressive results compared to previous studies, with indications of successful peptide immobilization observed in both SDS-PAGE and RP-HPLC results. However, further verification is required, including microdilution assays to determine minimum inhibitory concentration (MIC) values and activity studies of the peptides before and after immobilization. Future studies may address alternative immobilization strategies beyond the current thioetherbased approach, such as Schiff base formation using AminoLink® resins (Lapetina and Gil- Henn, 2017) or covalent immobilization on gold surfaces (Wadhwani et al., 2017).