INTERACTION AND ACTIVITY STUDIES OF BIOACTIVE PEPTIDES, IMMOBILIZED WITH ALUMIN NANOPARTICLES, FOR BIOTECHNOLOGICAL APPLICATIONS

Currently, global health problems related to bacterial resistance is responsible for more than 1.2 million deaths worldwide (HWO, 2019). Antimicrobial peptides are promising for the development of new therapeutic agents. One of the focuses of this work is the functionalization of alumina nanoparticles (NP) with the antimicrobial peptide Lunatin-1 for use in bone implant materials. This peptide is composed by 13 amino acid residues (FIGGLLKTLTSFF-NH2), originally isolated from the venom of the Hadruroides lunatus scorpion active against Gram-negative (E. coli and P. fluorescens), and Gram-positive (S. agalactiae, S. bovis, S. uberis and S. aureus) bacteria strains. Furthermore, Lunatin-1 also showed activity against human breast cancer and leukemia cells.
In this work the Lunatin-1 and two analogs EAAA-Lun-1 and Lun-1-AAAE were synthetized by SFPS. The analogs were covalently bound to the alumina NP through the glutamic acid side-chain in the N-terminal (NP-EAAA-Lun-1) and in the C-terminal (Lun-1-AAAE-NP) regions. The peptides were characterized by mass spectrometry and NP-peptides by ssNMR and Zeta Potential. The changes in the zeta potential throughout the functionalization of the conjugated nanoparticle provide evidence that the synthesis steps were successful and, therefore, both analogs were functionalized in on the metallic oxides surface. The values obtained for the NP-EAAA-Lun-1 and Lun-1-AAAE-NP were approximately -10 mV. 13C spectra of ssNMR were recorded for each functionalization step of NP syntheses and for the NP-peptides. While no chemical shifts were observed for NP and NP-OH, characteristic signals of the methylene groups are present in the NP-NH2 spectrum. Chemical shift of the side chains carbons of the peptides between 50-100 ppm observed in the both NP-peptide spectra. Chemical shift of 74.2 ppm for NP-EAAA-Lun-1 and 74.5 for Lun-1-AAAE-NP were attributed to the Cδ of the glutamic acid side chain. Both analyzes suggest successful synthesis and functionalization of NP-peptides. Antibacterial studies were carried out and showed lower MIC values for Lun-1-AAAE-NP when compared with NP-EAAA-Lun-1, for E. coli (48 and 128,5 µmol.L -1, respectively), P. aeruginosa (32,1 µmol.L -1 and ND) and S. aureus (16,0 and 64,2 µmol.L -1).
The membrane interaction of the NP-peptides was investigated by Differential Scanning Calorimetry. Whereas changes were identified in the thermotropic profile of the DMPC/DMPG bilayers for Lun-1-AAAE-NP at concentrations from 25 µM, significant effect in the thermotropic profile for NP-EAAA-Lun-1 only above 100 µM. For the NP-peptide bound in the C-terminal portion an enlargement in the main peak followed by a greater loss of symmetry is noted in comparison with NP-EAAA-Lun-1 in the of 100 µM concentration, indicating the higher insertion of the peptide molecules on the membrane. These results indicate that the N-terminal region of the Lunatine-1 is essential to retain the membrane interaction and, consequently, the antimicrobial activity when bound to the nanoparticle.