Study On The Neutralizing Endotoxin Effect Of New Nano Antibiotics Based On Human Alpha Defensin 5

Objectives:Pervious study demonstrated that the newly generated nanobiotics C-terminally myristoylated human a-defensin 5(HD5-myr)compared with its parent human a-defensin 5(HD5),self-assembled into nanomicelles in aqueous solution and exhibited improved resistance to hydrolysis and stability properties.Compared with HD5,HD5-myr showed significantly enhanced broad-spectrum bactericidal activity both in vitro and in vivo and rescued mice from E.coli-induced sepsis with a favorable safety profile.This study aims to verify the new role of HD5-myr in sepsis by neutralizing endotoxins and further investigate the possible mechanisms involved,so as to provide theoretical evidence for clinical practice of the new-generation nanobiotics.Methods:The new nanobiotic HD5-myr was chemically synthesized via solid-phase peptide synthesis(SPPS).All peptides were separated and purified by high performance liquid chromatography(HPLC)and verified by mass spectrometry(MS).The nanomicelle structures were observed by transmission electron microscope(TEM)and the nanoparticle diameters were measured by dynamic light scattering(DLS).Fluorescence labeled HD5,HD5-myr and E.coli were used to analyse the combination of HD5-myr with E.coli by laser scanning confocal microscope and observe the bacterial morohological changes when co-cultured with different peptides by scanning electron microscope(SEM).Experiments in vivo and in vitro were carried out to verify whether HD5-myr can improve inflammation and organ injury in lipopolysaccharide(LPS)-induce systemic inflammatory response.Combination of LPS with HD5-myr was observed via TEM and secondary structure changes of HD5-myr following LPS challenge were analysed by performing circular dichroism(CD).Results:The purity of HD5 and HD5-myr was 99.6%and 98.5%,with a molecular weight of 3582.4 Dalton(Da)and 3976.7 Da,respectively.HD5-myr molecules formed into nanomicelles in aqueous solution according to TEM.HD5-myr alleviated inflammatory responses and improved acute lung injury in LPS-induced mice sepsis compared with HD5.Diameters of particles increased after the mixture of LPS and HD5-myr,and nano hybrid micelles are formed,but the secondary strucutres of HD5-myr did not change.Conclusions:The newly generated nanobiotics HD5-myr derived from HD5,can combined with LPS and neutralize endotoxin,independent with the change of HD5-myr secondary structures.