Biomimetic Phage Mimicking Antimicrobial Nanoparticles for Antibiotic Free, Bactericidal Action Against the Multi-Drug Resistant ESKAPE Class of Pathogens

<b>Statement of Purpose: </b>We are losing in an antibiotic arms race with bacteria due to the emergence and global spread of new antibiotic resistance mechanisms.^1,2 At current rates of antibiotics discovery and development we will eventually lose to antibiotic-resistant strains, and by 2050, antibiotics resistant strains of bacteria will kill more patients per year than all cancers combined. Therefore, there is a desperate need to look for methods beyond antibiotics to kill harmful bacteria and slow the rise of resistant bacterial pathogens. This work describes an antibiotic-independent, structure-based antimicrobial system that is effective for topological applications (e.g., skin infections) while also effectively disrupting the bacterial populations at the surface of implant materials that are responsible for recurrent infections.<br/><b>Methods: </b>We investigated a new class of core-shell (SiO2@Au@Ag) nanoparticles with a silica core,⁴ a discontinuous shell of silver-alloyed gold nanospheres, and with or without cysteine bearing antimicrobial peptides³ (ANPs, pep@ANPs). The spacing of the silver-alloyed gold nanospheres was designed to mimic the spacing of pikes on bacteriophages such as PRD1.⁵ The peptides were designed from antimicrobial peptides secreted by the Asiatic grass frog (Rana <i>chensinensis</i>).³ The silica core diameter was either 65 nm or 130 nm, and the nanoparticles were effective in the solution phase or the immobilized phase<b>. </b>Four bacterial species isolated from clinical infections were chosen as test organisms: <i>Staphylococcus aureus </i>USA300, <i>Pseudomonas aeruginosa </i>FRD1, <i>Enterococcus faecalis</i>, and <i>Corynebacterium striatum</i>.⁶ Dose-dependent bacterial growth curves were measured on plate readers, bacterial viability was assessed using CFU assays, and fluorescent LIVE/DEAD assays, ANPs, and pep@ANPs biocompatibility to human cells (HaCaT) were assessed using ethidium homodimer permeabilization assay.<br/><b>Results: </b>Pep@ANPs immobilized on implant-grade steel coupons achieved a 100% kill rate of bacteria that encountered the implant surface<b>. </b>ANPs and pep@ANPs were completely biocompatible with model skin cells (HaCaT keratinocytes) thus confirming their suitability for topological applications<b>. </b>The phage structure mimicking, peptide-free ANPs killed ~65% of all 4 bacterial species. The antimicrobial peptide immobilized on the antibiotic-free, phage-mimicking nanoparticles (pep@ANPs) demonstrated a &gt; 99% bacterial kill rate against all four infectious bacterial strains at a peptide concentration on pep@ANPs that was ~10X lower than the concentration of free peptides required to achieve similar results.³ Interestingly, the pep@ANPs and ANPs showed a silver dose-dependent antibacterial effect as well.<br/><b>Conclusion: </b>We successfully mimicked the nanoarchitecture of antimicrobial viruses (Phages),^7, and clearly demonstrated a nanostructure-dependent antimicrobial effect that will be a viable alternative to traditional antibiotics. Capping antimicrobial peptides to the silver alloyed gold nanospheres demonstrated material savings advantage by controlling the steric presentation of the peptide and by reducing the concentration of peptides required to kill &gt;99% of bacteria by 1 order of magnitude. The 100% bacterial killing rate achieved by surface-immobilized pep@ANPs immobilized is a good indicator for their application as antimicrobial coatings on implant surfaces. Ongoing research is validating these results in an <i>in vivo </i>skin infection model.<br/><b>Reference</b>: <b>[1] </b>Ventola, C.L. P T 40, 277-83 (2015). <b>[2]</b>Zaman,S.B. et al. Cureus 9, e1403 (2017). <b>[3]</b>Dong,W Sci Rep 7, 40228 (2017).<b> [4] </b>Hopf et al., Nanoscale Adv., 2019,1, 4812-4826 <b>[5] </b>Benson, S.D., Cell 98, 825-33 (1999). <b>[6] </b>Penes, N.O. et al. Rom J Morphol Embryol 58, 909-922 (2017). <b>[7] </b>Abedon, S.T., Front Microbiol 8,981 (2017).