This study proposes the synthesis of the conjugate WLBU2-AgNP starting with a modified version of WLBU2 (hereafter called WLBU2C) (wheel: CRRWVRRVRRWVRRVVRVVRRWVRR). The development of this peptide is theoretically proposed by assessing the structure of WLBU2C through I-TASSER (iterative threading assembly refinement) simulation and an alpha helical diagram (Figure 1 and Figure 2) to retain amphipathicity and secondary structure [15-17]. Synthesis should be performed with the standard Fmoc procedure, after which the product should be purified with reversed-phase high-performance liquid chromatography using a C18 Vydac column as the stationary phase and be confirmed with mass spectrometry. The secondary structure can be evaluated with circular dichroism (CD) in the presence of phosphate buffer with saline (PBS) for an aqueous environment or 30% trifluoroethanol for a membrane mimetic environment [8]. The addition of cysteine as the conjugation point between the proposed antimicrobial peptide and AgNP has been theorized through review of prior studies in which cysteine group conjugation provided enhanced binding and stability with increased activity against Klebsiella pneumoniae [18,19].

This study proposes the synthesis of WLBU2C conjugated to AgNP (hereafter called WLBU2C-AgNP) by means of a photochemical method, as similarly described for LL37@AgNP by Vignoni et al [14]. The method specifically adapted for this research involves the use of deoxygenated silver nitrate (AgNO₃), Igracure 2959 as a photo initiator, and WLBU2C in sliding scale concentrations from 0 to 100 μM to test for the optimal concentration [20]. UVA lamps equivalent to Luzchem CCPV4 photoreactors should be used at 25°C, and the reaction can be monitored with UV-visible absorption spectroscopy. Based on the literature, the surface plasmon band is expected to be centered at around a wavelength of 395 to 425 nm [14]. According to a review of the above studies, it is expected that the absorption will increase in the UV-visible spectrum until all the Ag⁺ molecules are reduced.

After synthesis and purification with a dialyzer of appropriate size, transmission electron microscopy and dynamic light scattering (DLS) should confirm the presence of a larger DLS particle size due to the binding of the proteins around the nanoparticles. The secondary structure can be evaluated by CD, unless the nanoparticles interfere with CD resolution.

Testing of antibacterial activity is suggested against ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). Bacterial killing can be evaluated in the setting of potassium phosphate buffer and PBS by the dilutional assay method, in which WLBU2C-AgNP (0-100 μM) is mixed with bacteria (diluted to 1×10⁶ colony forming units/mL) at 37°C for 60 minutes, and the mixture is then plated and incubated (appropriate conditions and time) [8]. Subsequent analysis may be performed with spectrophotometry at 600 nm [21]. Further information regarding the tests for the minimum inhibitory concentration and minimum bactericidal concentration can be obtained from the Clinical & Laboratory Standards Institute manual for antibacterial susceptibility testing.

Once antimicrobial activity has been assessed, cytotoxicity can be evaluated against human red blood cells and normal cells, such as keratinocytes and fibroblasts, to explore practicality. The cytotoxicity protocol can be derived from the procedures performed by Deslouches et al [22]. Briefly, a red blood cell hemolytic assay can be performed in PBS by changing the WLBU2C-AgNP concentration. Further cytotoxicity can be assessed by culturing keratinocytes and fibroblasts in Dulbecco Eagle’s medium and performing tests with a range of WLBU2C-AgNP concentrations, as well as an MTT assay for metabolic activity.

Anticipated limitations of this study include the short half-life of CAPs and associated cytotoxicity in higher concentrations, which may be counteracted with immobilization of the peptide onto solid surfaces [23]. Silver might be toxic to mammalian cells and the environment [10]. As all proposed approaches are theoretical, there is no guarantee to achieve the expected outcome.

Recently, more research has been reported regarding the combination of antimicrobial peptides with AgNPs, with positive results [18,24]. This proposal provides another idea to efforts for counteracting antimicrobial resistance. It has been hypothesized that the conjugation of a de novo–engineered antimicrobial peptide and AgNP may increase the antibiofilm effect against multidrug-resistant bacteria while retaining selectivity and safety. The present method involving cysteine group modification on the antimicrobial peptide for conjugation with AgNP has to my knowledge not yet been published for de novo–engineered CAPs. De novo–engineered antimicrobial peptides are still undergoing active research to increase their potency while balancing cytotoxicity. The conjugation of improved peptides with AgNPs would provide a second degree of freedom to their functions, hopefully unlocking opportunities to develop more potent antimicrobials. If further studies on this topic are successful, future long-term prospects may include emergency last-line antibiotic therapy and treatment for difficult-to-eradicate bacterial colonization, such as in cystic fibrosis, implantable medical devices, cancer, and immunotherapy [25]. I encourage further studies on this topic to better understand the proposed theories. As I do not anticipate proactively obtaining funding for this idea in the future, I hope this proposal provides inspiration to other researchers.