The rise of multidrug-resistant pathogens necessitates the development of new antibiotics with innovative mechanisms. Traditional drug design methods, initially relying on modifying natural products and later incorporating principles of organic chemistry, have advanced to encompass medicinal chemistry, molecular biology, and quantum chemistry. Biophysical approaches offer a promising avenue, enabling deeper understanding of drug mechanisms. Recent research showcases successful integration of existing functional units to enhance antimicrobial activity, such as the combination of β-hairpin and colistin. Self-assembled nanostructures also present opportunities for combined antimicrobial and other therapeutic effects. This study takes a different approach, designing specialized architectures from melittin (Mel), a natural antimicrobial peptide, and polyethylene glycol (PEG), a clinically approved agent, aiming to create potent and low-toxicity antibacterial complexes. Melittin's amphiphilic α-helical structure when bound to cell membranes is leveraged, and different PEG molecular weights and architectures are explored to modulate the antibacterial properties.

The introduction cites several studies highlighting previous approaches to drug design and antimicrobial development. These include structural transformation of natural active ingredients, modifications based on organic chemistry principles, and more modern approaches integrating medicinal chemistry, molecular biology, and quantum chemistry. The review also mentions the integration of β-hairpin and colistin to combat colistin-resistant pathogens and various self-assembled protein- or peptide-based hybrid nanostructures, showcasing previous efforts to improve antimicrobial efficacy through combining functionalities or designing novel architectures. The literature review sets the stage by demonstrating existing approaches and highlighting the novel aspect of this study's architectural design.

The study involved the synthesis of PEG-Mel complexes through covalent conjugation of Mel peptides to the active sites of PEG molecules with varying molecular weights and architectures (detailed in Supplementary Information). Characterization techniques included dynamic light scattering (DLS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), UV-Vis absorption spectroscopy, and Fourier transform infrared spectroscopy (FT-IR). Antimicrobial activity was assessed using minimal inhibitory concentration (MIC) tests and time-dynamic bactericidal assays against Escherichia coli and Staphylococcus aureus. A dynamic giant unilamellar vesicle (GUV) leakage assay, using DOPC and DOPG lipids, was performed to study the mechanism of action. Acute toxicity in mice was evaluated by intraperitoneal injection of PEG12k-1*Mel and Mel, followed by measuring ALT and creatinine levels and histological examination of liver and kidney tissues. Molecular dynamics (MD) simulations using the Gromacs 5.1.4 package with CG force fields were used to further investigate the interaction of the complex with lipid membranes. Statistical analysis involved ANOVA using OriginPro 9.0.

The PEG12k-1*Mel complex demonstrated superior antimicrobial activity compared to pristine melittin and other tested antibiotics. Scanning electron microscopy (SEM) images showed significant membrane damage in bacteria treated with PEG12k-1*Mel. MIC values for PEG12k-1*Mel against E. coli and S. aureus were significantly lower than those of pure Mel and the control antibiotics polymyxin B and fusidic acid. The complex also exhibited greatly reduced in vivo cytotoxicity and acute toxicity compared to Mel. The GUV leakage assays indicated a lipid-specific mode of action, suggesting membrane disruption as the primary mechanism. Molecular dynamics simulations supported the experimental findings and provided further insights into the interaction between the complex and the bacterial membrane.

The significantly enhanced antimicrobial activity and reduced toxicity of the PEG12k-1*Mel complex compared to pristine Mel highlight the importance of architectural design in improving the efficacy of antimicrobial peptides. The lipid-specific mode of action and the observed membrane disruption suggest a potential mechanism of action different from traditional antibiotics. The results demonstrate a promising strategy for designing highly efficient and low-toxicity antimicrobial drugs by leveraging the architectural construction of readily available components. The findings contribute to the ongoing effort to develop novel therapeutics to combat multidrug-resistant pathogens.

This study successfully demonstrated that the architectural design of antimicrobial complexes, using Mel and PEG building blocks, can significantly enhance their antimicrobial activity while reducing toxicity. The PEG12k-1*Mel complex shows promising potential as a novel antimicrobial agent. Future research could focus on optimizing the architecture of the complex, exploring other PEG lengths and modifications, and investigating its efficacy against a broader range of pathogens in vivo.

The study primarily focused on in vitro and limited in vivo testing. Further in vivo studies with larger sample sizes are necessary to fully evaluate the safety and efficacy of the PEG12k-1*Mel complex. The mechanism of action was primarily investigated through GUV leakage assays and MD simulations; more detailed mechanistic studies, such as those involving cellular uptake and intracellular targets, might provide further insights.