(556b) Self-Assembled Collagen-Mimetic Triple Helices with Antimicrobial Peptide Amphiphiles As Novel Antibacterial Agents

The emergence of bacterial drug-resistance to antibiotics has caused significant difficulty for the treatment against infectious diseases. Bacteria can develop resistance within a few years after a new antibiotic is introduced. For instance, methicillin was first introduced into clinical use in 1959, which was rapidly followed by the emergence of methicillin-resistant Staphylococcus aureus (MRSA) reported for the first time in 1960. Moreover, MRSA are able to continuously develop a resistance to a broad range of antibiotics. It was reported that MRSA caused at least 80,000 invasive infections in the United States alone, and 53 million people worldwide may be colonized with MRSA. Therefore, the objective of this study is to design and develop novel self-assembling antimicrobial peptide amphiphiles to combat the antibiotic-resistant S. aureus.

Cationic antimicrobial peptides (AMPs) are naturally occurring short peptides that can penetrate across bacterial cell membranes and inhibit the development of bacterial drug-resistance. The antibacterial property of AMPs can be facilitated by their charge and secondary conformations. First, unlike the commonly used antibiotics, cationic AMPs can disrupt the bacterial cell membrane by electrostatic attachment and insertion, and these non-specific interactions can thus reduce the development of drug-resistance as the entire bacterial cell membrane is the main target site of most AMPs. Secondly, the amphiphilicity of AMPs, which represents the peptide, contains both hydrophilic charged domains and hydrophobic non-polar domains, can organize the AMPs into secondary conformations (e.g. β-sheet and α-helix) and facilitate the insertion of AMPs into bacterial cell membranes. Furthermore, since the collagen adhesion domains on the membrane of S. aureus have been shown to have a high affinity to the collagen-like triple helical structure, cooperation of a self-coiling collagen-mimetic peptide sequence with AMPs can be a novel and promising approach to promote the accumulation of AMPs on S. aureus, further enhancing the antibacterial efficacy of the AMPs.

2. Materials and Methods

Two types of self-assembled supramolecular nanostructures were successfully prepared in aqueous solution in this study: the liquid extracellular matrix-like (LEM) nanofibers and the amphiphilic Cardin antimicrobial (ACA) nanorods. Each of these two nanostructures was constituted by rationally designed amphiphilic peptide building blocks, which was driven by hydrophobic interactions and β-sheet stacking. The hydrophobic interactions were attributed to the aliphatic tail group conjugated to the N-terminus of the peptide amphiphiles, and the β-sheet stacking could direct the self-assembled structures into cylindrical shapes that facilitate the insertion into bacterial membranes.

In water, the self-assembled LEM nanofibers and ACA nanorods presented two different bioactive properties on the surface. The (Gly-Pro-Hyp)₆ repeating sequences of LEM nanofibers formed a collagen-mimetic triple helix, whereas the Cardin antimicrobial motifs (Ala-Lys-Lys-Arg-Ala)₂ were exposed on the outer surface of the ACA nanorods. More importantly, as the peptide amphiphiles of LEM nanofibers and ACA nanorods contained the same hydrophobic domain and β-sheet forming domain, these two peptide amphiphiles were combined into hybrid nanofibers by mixing LEM nanofibers and ACA nanorods at equal molars. These hybrid nanofibers then co-assembled via β-sheet stacking in the process of self-assembly, carrying both collagen-mimetic groups and Cardin antimicrobial motifs. Followed by the preparation of the self-assembled supramolecules, the morphology and size of the self-assembled structures were characterized by a transmission electron microscope (TEM) and dynamic light scattering (DLS) spectroscopy.

In bacterial assays, Gram-positive S. aureus (ATCC#25923) and MRSA (ATCC#43360) were incubated in 0.3% tryptic soy broth (TSB) media containing the self-assembled nanoscale supramolecules of peptide amphiphiles at concentrations from 20 to 100 µM at 37°C, and the growth curves were monitored by the optical density of a wavelength at 562 nm (O.D. at 562 nm) for 20 h. Also, to evaluate bactericidal effects, the self-assembled supramolecules of peptide amphiphiles at concentrations from 20 to 100 µM in 0.3% TSB were used to treat S. aureus and MRSA for 4 h in 37°C, and the viability of bacteria were quantified by colony forming units per ml (CFU/ml) after treatment. The viability of bacteria was evaluated by: [(CFU of treated sample)/ (CFU of untreated control sample)]*100%. All experiments were run in triplicate and repeated at least two times (N�2) to demonstrate significance.

 

3. Results and Discussion

The TEM characterization showed successful self-assembly of the peptide amphiphiles discussed above. The self-assembled LEM nanofibers were about 10 nm in diameter and microns in length; the ACA nanorods had similar diameters but were shorter in length to about 100 nm, which probably resulted from the high cationic charge of the Cardin antimicrobial motifs. The hybrid nanofibers, which were combined by the LEM nanofibers and ACA nanorods, also exhibited diameters to about 10 nm and had a highly aggregated network structure, and the length of these nanofibers ranged from about 200 nm to 500 nm. The results of DLS measurements showed that although the hydrodynamic diameter of the LEM nanofibers (603 nm) was much larger than that of ACA nanorods (17 nm), the hybrid nanofibers had uniform hydrodynamic diameters (26 nm), indicating the successful co-assembly of the two peptide amphiphiles.

In bacterial assays, the ACA nanorods significantly inhibited the growth of both S. aureus and MRSA, as well as had high bactericidal effects towards these two bacterial strains. The 20 h bacterial growth curve of bacteria showed that the ACA nanorods at a concentration of 100 µM delayed the lag phase of S. aureus and MRSA to 18 h, whereas the untreated bacteria entered the exponential growth phase after the lag phase within 3 h. Also, the ACA nanorods at concentrations higher than 80 µM reduced the survival rate of both S. aureus and MRSA to as low as 10% compared with the untreated control sample. The results demonstrated for the first time that the self-assembled ACA nanorods had promising antibacterial properties against Gram-positive S. aureus as well as MRSA, suggesting that these self-assembling peptide amphiphiles could combat bacterial drug-resistance. In our ongoing studies, the antibacterial effects of the hybrid nanofibers in cooperation with a collagen-mimetic group, will be further elucidated.