(188cn) Antimicrobial Actitvity and Citotoxicity of Buforin II Immobilized on Magnetite Nanoparticles

Over the past few years, a limited number of topical formulations have been developed against skin bacterial infections, which usually consist of excipients and antibiotics. Unfortunately, some strains, such as Staphylococcus aureus, have developed resistance to such treatments particularly within clinical setups. For this reason, much effort is invested nowadays towards incorporating peptide molecules into preparations with increased antimicrobial activity for an increasing number of biological and biomedical applications. One of such molecules is the antimicrobial peptide Buforin II. Despite its activity against bacteria, Buforin II is prone to proteolytic degradation, which ultimately leads to shorter lifetimes in vivo. To overcome this issue, we explored the immobilization of Buforin II on magnetite nanoparticles, which were prepared via co-precipitation of Iron Chlorides. Immobilization was conducted after silanation, which rendered free amine groups on the nanoparticles’ surface. Buforin II molecules were conjugated with the aid of glutaraldehyde to the N-terminal by forming imine bonds. As synthesized and silanized magnetite size distribution in distilled water at 1 mg/mL was estimated via DLS (Zeta-Sizer Nano). immobilization was verified with the aid of FTIR analysis, SDS-PAGE and BCA assay. Hydrophobicity, instability, half-life in vivo and in vitro, net charge, and Boman index were predicted in silico aided by the antimicrobial peptide database (http://aps.unmc.edu/AP/main.php). As-synthesized magnetite nanoparticles exhibited an average diameter of 120 ±62.7 nm, which was maintained after silanation. Successful conjugation of Buforin II was determined by looking for the absorption bands of the silanation agent and the peptide. Accordingly, vibrational bands at 3000 cm^-1 and ~1600 cm^-1 were identified and correlated to the terminal -NH₂ group of the silanation reagent APTES. Additionally, after conjugation an absorption band at about 1650 cm^-1 was identified and correlated to the amide I’ band of Buforin II. The in silico analysis estimated a positive charge of 6 and low percentage of hydrophobicity (33%) for Buforin II. Also, calculated an in vitro half-life of ~7 hours while that in vivo was superior to 20 hours. Under real conditions; however, much shorter half-life values are expected as reported for similar peptide molecules after in vivo and in vitro testing. We are currently conducting cytotoxicity activity tests on Vero cells via LDH using successive dilutions starting at 100 of immobilized peptides for 24 and 48 hours. Additionally, we are testing antimicrobial activity against Staphylococcus aureus and Staphylococcus aureus MRSA by successive dilutions starting at 100 for 20 hours. Strains were selected due ubiquitous presence on the skin surface and resistance to the antibiotic methicillin, respectively.