Antibiotic gold: tethering of antimicrobial peptides to gold nanoparticles maintains conformational flexibility of peptides and improves trypsin susceptibility

Abstract

Peptide-coated nanoparticles are valuable tools for diverse biological applications, such as drug delivery, molecular recognition, and antimicrobial action. The functionalization of pre-fabricated nanoparticles with free peptides in solution is inefficient either due to aggregation of the particles or due to the poor ligand exchange reaction. Here, we present a one-pot synthesis for preparing gold nanoparticles with a homogeneous distribution that are covered in situ with cationic peptides in a site-selective manner via Cys-residue at the N-terminus. Five representative peptides were selected, which are known to perturb cellular membranes and exert their antimicrobial and/or cell penetrating activity by folding into amphiphilic α-helical structures. When tethered to the nanoparticles at a single site, all peptides were found to switch their conformation from unordered state (in aqueous buffers) to their functionally relevant α-helical conformation in the presence of model membranes, as shown by circular dichroism spectroscopy. The conjugated peptides also maintained the same antibacterial activity as in the free form. Most importantly, when tethered to the gold nanoparticles the peptides showed an enormous increase in stability against trypsin digestion compared to the free forms, leading to a dramatic improvement of their lifetimes and activities. These findings suggest that site-selective surface tethering of peptides to gold nanoparticles has several advantages: (i) it does not prevent the peptides from folding into their biologically active conformation, (ii) such conjugation protects the peptides against protease digestion, and (iii) this way it is possible to prepare stable, water soluble antimicrobial nanoparticles as promising antibacterial agents.

Metal‐Based Nanomaterials in Biomedical Applications: Antimicrobial Activity and Cytotoxicity Aspects

“…The conjugated antimicrobial peptides not only retain their antibacterial activity as in the free form, but also show a dramatic increase in the stability against trypsin digestion, resulting in excellent improvement in their functional life and antibacterial activities. [ 234 ]…”

Towards Robust Delivery of Antimicrobial Peptides to Combat Bacterial Resistance

“…This presents a simple method for conjugation within aqueous buffer. Site-specific conjugation using cysteine is particularly useful in lysine-containing AMPs, as their amine groups can interact with the AuNP and potentially affect activity by holding the peptide close to the AuNP core, thereby preventing sufficient access to bacterial membranes [108]. Conjugation to AuNPs has been shown to markedly enhance the proteolytic stability of AMPs against trypsin while minimally affecting their in vitro activity, thereby improving their active lifetimes [108].…”

“…Site-specific conjugation using cysteine is particularly useful in lysine-containing AMPs, as their amine groups can interact with the AuNP and potentially affect activity by holding the peptide close to the AuNP core, thereby preventing sufficient access to bacterial membranes [108]. Conjugation to AuNPs has been shown to markedly enhance the proteolytic stability of AMPs against trypsin while minimally affecting their in vitro activity, thereby improving their active lifetimes [108]. Furthermore, Chowdhury et al found that AuNP-AMPs displayed good biocompatibility both in vitro and in vivo and were able to internalize into epithelial and macrophage cells without toxicity to destroy internalized Salmonella pathogens, a feat also mirrored in an in vivo mouse model [109].…”

Treating Polymicrobial Infections in Chronic Diabetic Wounds

“…Attachment of doxorubicin to Au-NPs via a pH-sensitive linker allows for the site-directed release of the drug inside tumor cells that have an acidic environment (75). Wadhwani et al functionalized gold NPs with cationic AMPs and showed that the conjugated peptides are biologically active and more resistant to degradation by proteases than free peptides (76).…”