Design,Preparation And Therapeutic Effects Of HD5-Derived Antibiotics On Bacterial Infection After Irradiation In Mice

Exposure to high-dose irradiation attenuate the immunity of human body,which make it susceptible to many kinds of pathogens,then the infection of pathgens aggravate the damage of irradiation.Although antibiotics can alleviate the after-irradiation infection,due to the abuse of antibiotics in recent years,increasing numbers of drug-resistant bacteria have emerged,which is detrimental to irradiated patients with reduced immunity.Antimicrobial peptides(AMP)are cationic peptides with broad spectrum microbicidal activities against pathogenic microbes,which including bacteria,protozoa,fungi and viruses.They are produced by tissues and cells of plants,insects and animals,and are important parts of the innate immune system.Human defensin 5(HD5)is secreted in Paneth cells of the small intestine.It belongs to ? defensin and with broadest spectrum antibacterial activity among the six ? defensins.Thus,it is a promising candidate to treat irradiation-related infection.Because the antimicrobial activity of HD5 is limited in complex humoral environment,we have designed T7E21R-HD5 based on mutations in adaptive evolutionary loci.T7E21R-HD5 is an atypical dimer with improved antibacterial activity and stability,and it is less prone to inducing antibiotic resistence.Thus,it appears to be an excellent candidate for the development of antimicrobial agents.Intestinal-derived antimicrobial peptides are ideal antibiotic substitutes for intestinal infections.To deliver the peptide into intestine and make it controlled release in the intestine is the key problems to solove.Here,we used a mesoporous silica nanoparticle(MSN)as the peptide carrier.Encapsulated by succinylated casein,it can be released controllable in intestine.In addition,according to the study of structure-effect relationship,we designed a linear antimicrobial peptide with alanine and arginine mutations.The linear antimicrobial peptide has stronger antimicrobial activity both in vitro and in vivo,which further promoted the pharmaceutical development of T7E21R-HD5.Here are the main results and conclusions:1.MSN prepared by thermal solvent method was with uniform particle size and good dispersion.T7E21R-HD5 was successfully loaded by electrostatic adsorption and the loading ratio of T7E21R-HD5 could reach up to 70%.The successful synthesis of MSN@T7E21R-HD5 was identified by changing of pore size and specific surface area detection,Acid urea-polyacrylamide gel electrophoresis,energy dispersive X-ray spectrometry and mass spectrometry.2.Minimum Inhibitory Concentration(MIC)Detection and Laser Confocal Analysis showed that MSN@T7E21R-HD5 was more potent than T7E21R-HD5 in killing MDR E.coli and MRSA.Corresponding to it,MSN@T7E21R-HD5 could increase the depolarization rate of bacterial membrane.3.MSN@T7E21R-HD5 destroyed the inner and outer membranes of bacteria in a concentration-dependent manner,and the effect was significantly stronger than that of T7E21R-HD5.Laser confocal and scanning electron microscopy showed that MSN@T7E21R-HD5 killed bacteria by an adsorption-aggregation-penetration-fragmention step.With the same concentration of T7E21R-HD5,the mechanical damage of MSN@T7E21R-HD5 to bacterial membrane was more obvious than that of T7E21R-HD5.4.After surface modification with succinylated casein(SCN),the particle size of nanoparticles increased.Potentiometric detection,urea acetate polyacrylamide gel electrophoresis and transmission electron microscopy confirmed the successful package of SCN.SCN modification significantly reduced the release of T7E21R-HD5 in strong acid environment and thus reduced the loss of T7E21R-HD5 in gastric acid environment.Under the catalysis of trypsin,controlled release of MSN@T7E21R-HD5 in the intestine can be achieved by SCN encapsulation.5.MSN@T7E21R-HD5@SCN has low hemolysis and cytotoxicity in vitro and low toxicity in vivo.Compared with the control group,oral administration of MSN@T7E21R-HD5@SCN could significantly lower the colonization of MDR E.coli in the intestinal cavity,and alleviate inflammation induced by drug-resistant Escherichia coli(down-regulation of inflammatory factors' transcription and expression);Oral administration of MSN@T7E21R-HD5@SCN can alleviate irradiation-caused endogenous infection.Besides mitigation,it can also reduce the incidence of bacteremia and the content of endotoxin in mice blood.6.The linear peptide T7E21R-HD5-d3 can be obtained by reserving the C-terminal active region of T7E21R-HD5 and replacing cysteine with alanine,replacing non-hydrophobic and non-positively charged amino acids with arginine.The derivative peptide exhibits irregular curl and helix structure in aqueous solution and lipid environment respectively.It can be prepared by solid-phase synthesis method.7.T7E21R-HD5-d3 was a potent bactericide,The MIC of T7E21R-HD5-d3 against Acinetobacter baumannii was 2.5?g/mL.T7E21R-HD5-d3 has superior biocompatibility.Compared with the control group,T7E21R-HD5-d3 could improve the survival rate of mice with Irradiation-Pulmonary infection,significantly lower the colonization of resistant bacteria8.Results of biofilm interferometry,scanning electron microscopy and NPN uptake experiments suggest that: the ability of T7E21R-HD5-d3 to bind the outer membrane protein OMPA of Acinetobacter baumannii and destroy the outer membrane of bacteria was significantly stronger than that of T7E21R-HD5.In addition,sublethal dose of T7E21R-HD5-d3 can penetrate into the cytoplasm of MDRAb and combine with 50 S ribosomal protein,interfere with ribosomal protein synthesis function and inhibit bacterial growth.