Self-Assembling Nucleopeptides Exhibiting Strong Antimicrobial Activity against Multidrug-Resistant Clinically Isolated Strains and In Vitro Wound Healing Compatibility

To combat the emerging threat of antimicrobial resistance (AMR), in this study, two amphiphilic nucleopeptides (NPs) were synthesized by conjugating the nucleobase thymine with peptide amphiphiles. These compounds were fully characterized using various analytical techniques. Notably, both nucleopeptides formed hydrogels in milli-Q water at neutral pH (pH 6.9). X-ray diffraction further confirmed antiparallel β-sheet-like structures, along with aromatic π-π stacking and hydrogen-bonding (H-bonding) interactions between the thymine moieties in the gel phase. Field emission gun transmission electron microscopy revealed a nanofibrillar network structure in these self-assembled peptides. A significant feature of these peptide supramolecular self-assemblies is their potent antimicrobial activity against both types of bacteria, such as Gram-positive and Gram-negative standard American Type Culture Collection (ATCC) bacteria, including <i>Bacillus subtilis</i>, <i>Escherichia coli</i>, and multidrug-resistant clinically isolated ATCC strains such as methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), <i>Klebsiella pneumoniae</i>, and <i>Pseudomonas aeruginosa</i>. Among these, both peptides demonstrated remarkable inhibition of MRSA (MIC: 15.92-16.86 μM) and <i>K. pneumoniae</i> (MIC: 8.8-50 μM), highlighting their potential as antimicrobial agents against deadly multidrug-resistant (MDR) bacteria. Additionally, these peptide assemblies were found to be highly biocompatible, as demonstrated by MTT assays on HEK-293 cells, showing IC₅₀ values in the range of 0.5-1.1 mM. In an in vitro wound healing assay using HeLa cells, fluorescence microscopy confirmed that treatment with these nucleopeptides did not disrupt the cell or mitochondrial membranes in HEK-293 cells. This work presents two nucleopeptides with broad-spectrum antimicrobial efficacy against MDR strains and demonstrates high biocompatibility, supporting their potential use as antimicrobial agents.