Study On The Bactericidal Effect And Mechanism Of New Nano Antibiotics Based On Human Alpha Defensin 5

Part1 constrction and characterization of human a-defensin 5 modified nano antibioticObjectives:Human alpha defensin 5(HD5)is a cationic antibacterial peptide produced and secreted by Paneth cells in the small intestine,which is composed of 32 amino acids.It obtains bactericidal activity against a variaty of pathogens,such as Escherichia coli and Staphylococcus aureus.But its bactericidal effect is easily interfered by many factors such as NaCl,and the biological stability as a polypeptide is poor,which is easily degraded by enzyme.Drug modification based on nanotechnology has become a hot spot of recent research.Therefore,this part of study is devoted to the nano-transformation of HD5,and the size,potential and stability of the modified nanoparticles were researched.Methods:The molecular structure of HD5 varients was designed,which increased hydrophobicity by myristoylation at the N-or C-terminus of HD5.In order to avoid the influence of myristic acid on HD5 strcture,glycine was added as a linker between HD5 and myristic acid.In addition,as the absence of free amino group at the C-terminus of HD5,an additional lysine is attached to its C-terminus for myristoylation as myristic acid could attached to its ?-amino group.The N-or C-terminus modified peptides were named as myr-HD5 and HD5-myr respectively.They were synthesized by the method of solid phase synthesis,separated and puricity verified by high performance liquid chromatography(HPLC),and their successful synthesis were verified by mass spectrometry(MS).The synthesized peptides were dissolved in water,and the presence of nanoparticles and the diameter of the particles were detected by dynamic light scattering.The Zeta potential of the particles was analyzed.Transmission electron microscopy(TEM)was used to further verify the presence of nanoparticles and the diameter of the particles.The modification of nanoparticles has been proved to increase the stability of the material,as the clustered molecules collectively creat steric hindrance to shield one another from hydrolysis by proteolytic enzymes.Therefore,the stability of the reformed body and HD5 in the water,the DMEM medium and the serum was further observed.Results:The purity of HD5 by solid phase synthesis was 99.6%and its molecular weight was 3582.4 Da,which is in accordance with the theoretical molecular weight of 3582.1 Da.The purity of myr-HD5 was 97%and the molecular weight was 3849.6 Da,which is in accordance with the theoretical molecular weight of 3849.5 Da.The purity of HD5-myr was 98.5%and the molecular weight was 3976.7 Da,which is in accordance with the theoretical molecular weight of 3976.7 Da.The hydrodynamic diameter of myr-HD5 in DI water was 80.2 + 2.2 nm,and for HD5-myr,it was 56 + 8.4 nm by the method of dynamic light scattering.The polydispersity index(polydispersity index,PDI)of the particles was narrow.The Zeta potential of myr-HD5 was 44.9 + 3.8 mV and for HD5-myr,it was 21.8 + 4.3 mV,which proved that both these two kinds of nanoparticles were stable in the solution.TEM observation further confirmed successful formation of nanoparticles by HD5-myr and myr-HD5,while HD5 could not form nanoparticles.These nanoparticles were stable in water,DMEM and NaCl for at least 24 hours.In aqueous solution,HD5 and HD5-myr could be stable for at least 48 h proved by HPLC.In DMEM,both remained stable at least 48 h.In the mice serum,HD5 was easily degraded and its stable existence time was less than 24 h.And HD5-myr was stable for at least 48 h.Conclusions:The attachment of myristic acid on HD5 molecules can effectively form nanoparticles,and the formation of nanoparticles can effectively enhance the stability in serum.Part 2 Study on the bactericidal effect of human alpha defensin 5 derived nano antibiotics in vitro and its toxicityObjectives:In view of the successful transformation of HD5 into nanoparticle,we further explore its bactericidal effect on clinically frequent pathogenic bacteria.Methods:Several strains that patients were frequently suffered were choosed as the tested strains,including Escherichia coli(E.coli),Staphylococcus aureus(S.aureus),methicillin resistant Staphylococcus aureus(MRSA),Klebsiella pneumoniae(K.pneumoniae),Pseudomonas aeruginosa(P.aeruginosa)and Bauman Acinetobacter(A.baumannii).Using a virtual colony count assay method,bactericidal activity between the parent HD5 and modified nanobiotics myr-HD5 and HD5-myr was compared.Single colony of bacteria was culture for 4-6 hours to its log phase.After washing with phosphate buffer(pH=7.4),they were co-cultured with different concentrations of antibacterial peptides for 2 hrs.Their OD value were dynamically monitored after the addition of culture medium.Their bactericidal activity was calculated by the time they spend to reach a critical OD value.In addition,the influences of sodium chloride and serum on their bactericidal effect were detected.To detect toxicity,mice erythrocytes and murine macrophages were selected as the test cells.The liposomal amphotericin B was used as control,and their hemolytic toxicity were detected.In additon,the effect of nanobiotics on the cell viability and phagocytosis function of RAW 264.7 was studied by MTT assay and gentmycin protection assay.In addition,the mice were given intraperitoneal injection of nanbiotics,and its toxicity in vivo was detected by observing the dynamic changes of the weight of mice.Results:HD5-myr nanobiotic have a strong killing effect for all tested strains,for example,the number of E.coli decreased by 7 orders of magnitude(the number of bacteria decreased to 1 in the control group of 10 million),the number of S.aureus decreased by 4 orders of magnitude,the number of MRSA and A.baumannii decreased by 5 orders of magnitude,the P.aeruginosa decreased by 6 orders of magnitude,the number of K.pneumoniae decreased by 9 orders of magnitude.While bactericidal effect of HD5 is negligible.Their difference was statistically significant.my-HD5 nanobiotic could effectively kill gram positive S.aureus and MRSA,but its effect was negaliable for gram negative K.pneumoniae,P.aeruginosa,A.baumanni and E.coli,even weaker than HD5.Based on this result,the effects of different concentrations of NaCl on their bactericidal effect were further examined.In NaCl Solution,the bactericidal effects of both HD5 and HD5-myr were decreased.In detail,the bactericidal effect of HD5 was not detected,while HD5-myr still had a strong effect on killing E.coli.In the NaCl solution of 150 mM,25?g/ml of HD5-myr could still decrease the number of E.coli by 3 orders of magnitude.Further detection of their bactericidal effect in the serum showed that 50 ?g/ml of HD5-myr still retained strong bactericidal effect in the presence of 50%of the mice serum.It reduced the number of E.coli by 5 orders of magnitude and reduced the number of MRSA by 3 orders of magnitude.The hemolytic activity of HD5,myr-HD5 and HD5-myr were relatively low.For example,all the hemolytic rate of HD5,myr-HD5 and HD5-myr at 400 ?g/ml were lower than 5%,while it was more than 30%for liposomal amphotericin B at the same concentration.The toxicity of HD5-myr on murine macrophage was negligible tested by MTT assay.For example,at its 100 ?g/ml concentration,the activity of RAW264.7 cells was more than 90%,while the cell survival rate of liposomal amphotericin B group was merely 40%.In vivo experiments further confirmed safety of HD5-myr,as the mice had no significant changes in weight after intraperitoneal injection of 60 ?g HD5-myr.Conclusions:Bactericidal effect was significantly enhanced after modification at the C-terminus of HD5 with a myristic acid.The N-terminal modification enhanced its bactericidal effect against gram positive bacteria while weakened the acitivity against gram negative bacteria.The toxicity of HD5-myr was low.Part 3 Study on the therapeutic effect of the HD5-myr nano antibiotic derived from human alpha defensin 5 in a mouse sepsis model and a mouse skin infection modelObjectives:The modified nano antibiotic HD5-myr has good bactericidal effects in vitro and a good safety in vitro and in vivo.In this part,a E.coli infection induced sepsis model and a skin MRS A infection model were constructed to verify the effect of the nano antibiotics HD5-myr in vivo.Methods:Mice were intraperitoneally injected with lethal dose of E.coli to establish the sepsis model.Then they were treated with HD5(10 ?g per mouse),HD5-myr(10 ?g and 20 ?g per mouse)or saline.The survival rate of the four groups was observed.Another mouse sepsis model was built by given a lethal dose of E.coli.They were treated with HD5(20?g per mouse)or HD5-myr(20 ?g per mouse).Their liver,kidney,spleen,lung,blood and peritoneal lavage fluid were harvested at 7 hours after treatment to detect the bacterial load.In addition,HE staining and pathological scores of the liver,kidney and lung were performed to evaluate the therapeutic effect.A mouse skin infection model was established and was treated with 12 ?g of HD5 or HD5-myr.The therapeutic effects were compared with the control group by observing the bacterial load on the skin surface and in skin deeper tissues at 24 hours after treatment.Results:The minimus lethal dose of intraperitoneal injection of E.coli was 7×1.6 CFU per mouse.At this dose,100%of the mice died within 24 hours.The 10 ?g HD5 treatment could increase the survival rate to 30%,but there was no statistically significant difference.The treatment of 10 ?g HD5-myr could increase the survival rate to 70%,the difference is statistically significant(**,p<0.01),and further increase the dose of HD5-myr to 20?g could further improve the survival rate to 90%(***,p<0.001),which proved that HD5-myr has obvious therapeutic effect on the sepsis model of mice infected with E.coli.Further using mice E.coli abdominal infection sepsis model,treating with 10 ?g HD5 or HD5-myr to observe the organ pathological appearance at 8 hours after treatment.The HD5-myr group was close to the normal mouse organ characterized with pink lung and red liver,while the HD5 group was similar to the non-drug treatment group,the liver showed congestion,the lungs showed hemorrhage and congestion.Another mice E.coli abdominal infection sepsis model were treated with HD5 or HD5-myr(20 ?g per mouse)and the liver,kidney,lung,spleen,blood and peritoneal lavage fluid were harest to observe the organ bacterial burden and injury.Both HD5-myr and HD5 treatment significantly reduced the bacterial burden and organ injury,the pathological score was statistically difference when compared with the control group.What's more,effect of the HD5-myr treatment group was significantly better than that in the HD5 treatment group.In the mouse skin MRS A infection model,12 ?g HD5-myr compared with the control group significantly reduced the bacterial burden on the skin surface and in the skin deeper tissues(***p<0.01),while HD5 had no effect at the same dose.Conclusions:HD5-myr can significantly improve the survival rate of mice with sepsis induced by intraperitoneal injection of E.coli by reducing bacterial burden and organ damage,and HD5-myr can effectively reduce the bacterial burden in mouse skin infection model.Part 4 Study on the bactericidal mechanism of HD5-myr nano antibioticsObjectives:HD5-myr has good bactericidal effect both in vivo and in vivo.We further studied the mechanism of its bactericidal activity.Methods:TAMRA labeled HD5 and HD5-myr were designed and synthesized.They were named TAMRA-HD5 and TAMRA-HD5-myr respectively.After incubation with E.coli expressing enhanced green fluorescent protein(EGFP)for 10 minutes,their binding with bacteria were observed using a laser confocal microscope.The E.coli expressing EGFP was intravenously injected to mice,and immediately after its injection,TAMRA-HD5-myr was injected intravenously.The binding of peptides and bacteria in vivo was observed by two-photon microscopy.HD5,myr-HD5 and HD5-myr were incubated with E.coli or MRSA for 2 hrs,and the morphological changes of the bacteria were observed by scanning electron microscopy(SEM)to study the bactericidal mechanism.Results:TAMRA-HD5-myr has a stronger combination with bacteria than TAMRA-HD5 after co-incubation for 10 minutes,which proved that HD5-myr has a good ability to target bacteria.After intravenous injection of E.coli expressing EGFP and TAMRA-HD5-myr,their co-location indicated that HD5-myr can effectively target bacteria in vivo.Using SEM,we observed morphologic changes of HD5,myr-HD5 and HD5-myr treated MRSA and E.coli.The degree of bacterial surface morphologic changes was positively correlated with the ability of peptides to kill bacteria.For example,the bactericidal ability against E.coli is HD5-myr>HD5>myr-HD5.It could be seen after treating with HD5,there were many vesicles on the surface of E.coli,which were all over its surface.While the surface morphologic changes of myr-HD5 were not obvious.Notably,after HD5-myr treatment,many holes were formed on the surface of E.coli.These holes were relatively uniform and the edges were irregular.For MRSA,the surface got rough after HD5 treatment,and both myr-HD5 and HD5-myr got the surface of the bacteria rougher than HD5.Conclusions:HD5-myr can be effectively bind to bacteria.Unlike the original HD5,which kills E.coli by destroying the outer membrane,HD5-myr destroies the whole cell membrane and cell wall which changes the bactericidal mechanism.