Study Of The Antibacterial Activity And The Underlying Mechanisms Of Amino-terminated Poly(amidoamine) Dendrimers

AIMThe bacterial resistance, primarily caused by abuse of antibiotics, has been one ofthe most intractable challenges for human being. Many infectious diseases,previously easy to treat, are becoming more and more difficult to cure. Infectiousdiseases often cause high mortality and many complications. More seriousely,resistant strains tend to resistant to multi-drugs, and bacteria resistant to allantibiotics (pan resistant bacteria) have emerged and human being may have nocure for the situation once they infect this type of bacteria. Faced with the rapidspread of drug-resistant bacteria, the increased mortality caused by infectiousdisease, governments in different countries have made efforts to control the harmcaused by infection of drug-resistant bacteria, and the development of newstrategies and new drugs to effectively control drug-resistant bacterial infectionshave become imperative aim of research for scientists all over the world.The main problem of antibiotics used in clinic is inevitably inducing bacterialresistance. In addition, most of new antibiotics are chemically modified from thefew scaffolds of antibiotic classes discovered half a century ago, and in fact fewmajor classes of antibiotics were introduced in the past half of century. It takes about10year to develop a new kind of antibiotic, while it only takes about1to2year or even several months to induce bacterial resistance. Therefore, the researchof novel kinds of antibacterial agents, especially those with scaffold structuresand antibacterial mechanisms different from the traditional antibiotics, is morethan ever needed.Polyamidoamine dendrimer (PAMAM) is a class of spheroid nanoscaledmacromolecules that possess a particular architecture constituted of three distinctdomains, including a central core, branches emanating from the core and manyidentical terminal functional groups. Dendrimers have broad applications incontrolled and targeted drug/gene delivery systems, polyvalentnanopharmaceuticals, et al. Recent studies reported that G3.0or highergenerations of PAMAM-NH2have prominent antibacterial activities to P.aeruginosa and S. aureus. Compared with the existing types of antibiotics andantimicriobial agents, PAMAM-NH2is a novel class of antimicrobial agents andthe mechanisms may be related to the number of terminal amino groups.Currently, the synthesis methods of PAMAM-NH2are mature, and a series ofG1.0-G10.0PAMAM-NH2are commercially available. The nanoscale, number ofterminated groups, shapes and molecular architecture are changed according tothe generation of PAMAMs. However, whether PAMAM-NH2would be a novelkind of antibacterial agent is not known. Several critical issues should be clarifiedto resolve this issue:â—‹1Is there a association between the generation andantibacterial activity of PAMAM-NH2?â—‹2How about the antibacterial spectrumof PAMAM-NH2?â—‹3How about the in vivo toxicity of PAMAM-NH2？○4Isthere action differences of PAMAM-NH2to mammalian cells and bacteria?â—‹5Whether PAMAM-NH2would exert therapeutic effects on infectious animalmodels?â—‹6Would PAMAM-NH2induce bacterial resistance?â—‹7What is the mechanism of antibacterial activity of PAMAM-NH2?â—‹8How to improve thetargeting potential and reduce the toxicity of PAMAM-NH2? A series ofexperiments were carried out to answer the above questions, explore themechanisms of antibacterial activity of PAMAM-NH2and systemicially evaluatewhether PAMAM-NH2would be used as novel antibacterial agents.METHODS1. In vitro antibacterial activity of PAMAM-NH2The minimum inhibitory concentration (MIC) and minimum bactericidalconcentration (MBC) values of G1.0-G4.0PAMAM-NH2against17bacterialstrains including6resistant strains.were determined; the growth inhibitory effectsof G1.0-G4.0PAMAM-NH2on E.coli MG1655and vancomycin intermediateresistant S. aureus Mu50were evaluated; the killing effects of G1.0-G4.0PAMAM-NH2on methicillin-resistant S. aureus (MRSA) and Escherichia colistrains producing extended-spectrum beta lactamase (ESBLs-EC), weredetermined by examining the numbers of colony-forming units (CFU) in colonyforming assays in plates.2. In vitro toxicity of G1.0-G4.0PAMAM-NH2to normal human stomachmucous membrane epithelial cell lines GES-1In vitro toxicity of G1.0-G4.0PAMAM-NH2to GES-1cells were evaluated byMTT assays.3. Induction of Resistance of G2.0PAMAM-NH2MIC values of G2.0PAMAM-NH2were determined for S. aureus (ATCC29213)and ESBLs-EC (ATCC35218) for15continuous days, then the relative MICvalue was calculated from the ratio of the MIC obtained for the fifteenthsubculture to that obtained for the first-time exposure.4. The inhibitory effects of G2.0-G4.0PAMAM-NH2on biofilm formation of ESBLs-EC and MRSAESBLs-EC and MRSA were cultured with20μg/mL of G2.0, G3.0or G4.0PAMAM-NH2for7days, then silver staining was used to detect the effects ofPAMAM on bioflim formation of ESBLs-EC and MRSA on the glass.; After thebiofilm formation, the biofilm were treated with G2.0PAMAM with differentconcentration for24h, then the antibacterial effects of G2.0was determined byexamining CFU in colony forming assays in plates.5. In vivo acute toxicity of G2.0PAMAM-NH2to BALB/c miceThe LD50of G2.0PAMAM-NH2to BALB/c mice by intraperitoneal injectionwas determined by Spearman-Karber method. HE staining were performed toinvestigate the toxicity of G2.0PAMAM-NH2to organs of mice.6. Therapeutic effects of G2.0PAMAM-NH2on septicemia AnimalsESBLs-EC induced sepsis model of BALB/c mice, MRSA and ESBLs-ECinduced sepsis model of BALB/c mice were established, and the effects of10mg/kg,20mg/kg, and10+10mg/kg G2.0PAMAM-NH2(10mg/kg was given12h before bacterial challenge, and another10mg/kg was given0.5h after bacterialchallenge) by intraperitoneal injection on survival time and rates of mice wasobserved; the effect of G2.0on the growth inhibition of ESBLs-EC in BALB/cmouse blood was observed by measuring the CFU; the protective effect of theG2.0on the lungs and livers of BALB/c mice was observed by HE staining.7. Study on the functional mechanism of G2.0PAMAM-NH2The change of surface morphology and the ultrastructure changes of ESBLs-ECwere observed by scanning electron microscopy (SEM) and transmission electronmicroscopy (TEM) at different time (30,120,300min) after treating with6MICconcentrations of G2.0PAMAM-NH2.8. Synthesis, characterization and evaluation of LED209-PAMAM G3.0 LED209is a newly discovered, potent, non-toxic and inhibitor of QseC receptorpresent on most of Gram-negative bacteria membrane, which can effectivelyinhibit the expression of virulence genes by inhibiting the QseC. To furtherimprove the targeted antibacterial effect of PAMAM, LED209was used tomodify G3.0PAMAM, aiming at getting a new antimicrobial molecule which hasantibacteria-targeted activity and lower toxicity.(1) Synthesis, characterization and evaluation of LED209-carboxylderivativesLED209carboxyl derivatives of para and meta position of benzene ring(para-LED209-COOH and meta-LED209-COOH) were synthesized, andcharacterized by1H NMR and mass spectrum (MS) and the purity weredetermined by high performance liquid chromatography (HPLC); the growthinhibitory effects of para/meta-LED209-COOH on Enterohemorrhagic E.coli(EHEC) were evaluated in vitro; the effects of para/meta-LED209-COOH on thekey virulence gene expression of EHEC by inbibiting QseC receptor were testedto evaluate their activity difference by PCR methods.(2) The synthesis, characterization and evaluation of LED209-PAMAM G3.0Meta-LED209-PAMAM G3.0was synthesized, and characterized by1H NMRand13C NMR, and the structure composition was determined by elementalanalysis; the MIC values of meta-LED209-PAMAM G3.0for12bacterial strainsincluding3resistant strains were determined to assess the change of antibacterialactivity after modification; the effects of meta-LED209-PAMAM G3.0on the keyvirulence gene expression of EHEC by inbibiting QseC receptor were tested toevaluate the activity difference after modification; MTT assay was used toevaluate the toxicity of meta-LED209-PAMAM G3.0to GES-1cells in vitroï¼›G3.0PAMAM-FITC and G3.0FITC-PAMAM-LED209were synthesized and the content of FITC was detected by UV-visible spectophotometric titration;EHEC and SW480were treated with FITC labled G3.0and G3.0-LED209respectively, then the images were collected by fluorescence microscope atdifferent time.RESULTS1. The antibacterial activity of G1.0-G4.0PAMAM-NH2in vitro.MIC results showed that G1.0-G4.0PAMAM-NH2had a broad antibacterialspectrum against almost all Gram-positve and Gram-negative bacteria strains,including sensitive and resistant strains, and the order antimicrobial activity wasas follows: G2.0(MIC0.78-12.5μg/mL)≥G3.0≈G4.0(MIC1.56-25μg/mL)>>G1.0(MIC≥50μg/mL). The MBC values of G2.0against strains tested were2-or4-fold MIC. The inhibitory effect of G1.0-G4.0PAMAM-NH2on the growthof E.coli MG1655was concentration-dependent, with G1.0exhibiting the leastefficacy and G2.0exhibiting the highest efficacy at the same concentration.Analysis of the kill curves demonstrated that12μM of G2.0-G4.0PAMAM-NH2showed the significant bactericidal activity to ESBLs-EC (50CFU/mL afterincubation with G2.0for2h) and MRSA (50CFU/mL after incubation with G2.0for6h), however60μM of G1.0showed almost no effect.2. The cytotoxicity of G1.0-G4.0PAMAM-NH2to GES-1cellsThe MTT results indicated that the cytotoxicity of PAMAM-NH2was dependenton the generation and the concentration. G1.0showed the least toxicity to cellseven at the highest concentration of1024μg/mL (P>0.05), and G2.0and G3.0atlower concentration than102.4μg/mL also showed no toxicity (P>0.05).However, G4.0exhibited obvious cytotoxicity even at lower concentration of0.512μg/mL (P<0.05).3. The induction of drug resistance of G2.0PAMAM-NH2 After15successive subcultures, the relative MIC values of G2.0PAMAM-NH2,erythromycin, oxacillin and ceftazidime for S.aureus are1,64,8and8respectively. The relative MIC values of G2.0, Levofloxacin, ampicillin andceftazidime for ESBLs-EC are1,64,32å'Œ8. All these results indicated G2.0didnot induce bacterial resistance like other antibiotics.4. The inhibitory effects of G2.0-G4.0PAMAM-NH2on biofilm formationof ESBLs-EC and MRSASilver staining results showed that20μg/mL of G2.0, G3.0or G4.0PAMAM-NH2could inhibit the bioflim formation of ESBLs-EC and MRSA onthe glass.; but the bacteria in the biofilm were resistant to both G2.0andLevofloxacin.5. Acute toxicity of G2.0PAMAM-NH2to BALB/c miceThe in vivo acute toxicity of G2.0PAMAM-NH2to BALB/c mice byintraperitoneal injection demonstrated that the average confidence interval ofLD50of G2.0was84.04±21.72mg/kg. HE staining results suggested thatmicro-thrombus were formed in all kinds of organs (heart, liver, spleen, lung andkidney) of BALB/c mice1h after treating with180mg/kg G2.0, andintravascular coagulation and bleeding were also observed. Among these organs,the damage of liver and kidney was the most serious but the injury of lung wasmild with little thrombus.6. Therapeutic effects of G2.0PAMAM-NH2on sepsis model of BALB/cmiceIn the experiments of BALB/c sepsis model infected by ESBLs-EC,20mg/kgand10+10mg/kg G2.0PAMAM-NH2treatment significantly improved theanimal survival rates to41.67%(P<0.05vs. control and ampicillin–treated group)and100%(P<0.001vs. control and ampicillin–treated group), reduced the bacterial titers markedly (P<0.05and P<0.01vs. control group) and inhibited thedamage caused by infection to lung and livers of mice. The similar results wereobserved in another sepsis model induced by ESBLs-EC and MRSA,20mg/kgand10+10mg/kg G2.0PAMAM-NH2treatment improved the survival rates ofanimal to40%(P=0.03vs. control and P=0.15vs. ampicillin–treated group) and60%(P=0.003vs. control and P=0.014vs. ampicillin–treated group),respectively.7. The antibacterial mechanism of G2.0PAMAM-NH2The SEM images collected at different time points after treatment with G2.0PAMAM-NH2demonstrated a marked change in bacterial morphologies,including shrinking, cracks and holes formed on the surfaces. The longerincubation time, the more damaged bacterial cells were observed. TEMdemonstrated that ESBLs-EC cell treated with G2.0PAMAM-NH2for2h wasseriously damaged, including pycnosis, necrosis, the formation of bleb-like gapsbetween the cell membrane and cell cytoplasm, and the leakage of intracellularcontent.8. The synthesis, characterization and evaluation of LED209-PAMAM G3.0(1) The synthesis, characterization and evaluation of LED209-carboxylderivativesThe structures of meta/para-LED209-COOH were identified aftercharacterization by MS and1H NMR, and the two products purity were greaterthan95%, indicating in line with the experimental requirements; the growthcurves suggested that10-100μM meta-or para-LED209-COOH had no effect onthe proliferation of EHEC in vitro; PCR assay found that the effect of NE onactivation of QseC receptor could be antagonized by1μM of meta-orpara-LED209-COOH, and the expression of key virulence genes of EHEC, such as stx2A, flhD, fliC and ler, were inhibited significantly. In addition, theantagonistic activity of meta-LED209-COOH was higher thanpara-LED209-COOH.(2) The synthesis, characterization and evaluation of LED209-PAMAM G3.0It was confirmed that meta-LED209-COOH had been successfully conjugated toPAMAM G3.0surfaces after characterization by1H NMR and13C NMR, and thestructure composition was calculated as meta-LED209-PAMAM（n=1.89）accordingto the results of elemental analysis, which means every PAMAM G3.0molecularwere connected with1.89LED209moleculars. The MIC test showed that G3.0meta-LED209-PAMAM had strong antibacterial activity against all strains butS.typhimurium and ESBLs-KP tested, and MIC values were1-or2-fold MICvalues for PAMAM G3.0. PCR assay found that the effect of NE on activationof QseC receptor could be antagonized by10μM of meta-LED209-PAMAMG3.0, and the expression of key virulence genes, such as stx2A, flhD, fliC and ler,were inhibited significantly. MTT assay results showed that the toxicity ofmeta-LED209-PAMAM G3.0to GES-1, SW480and MC3T3cells was muchlower than that of unmodified G3.0PAMAM (P <0.05); the concentrations ofG3.0PAMAM-FITC and G3.0FITC-PAMAM-LED209were3.15and3.08mMï¼›the results of fluorescence microscope showed that LED209modified G3.0PAMAM could improve the affinity to EHEC cells, but lower the affinity toSW480cells, compared with unmodified PAMAM G3.0.CONCLUSION1. The antibacterial potency of G1.0-G4.0PAMAM-NH2is related to thegeneration, and the order of antibacterial activity is G2.0≥G3.0≈G4.0>>G1.0.2. The cytotoxicity of G1.0-G4.0PAMAM-NH2to GES-1are positivelycorrelated with the generation, and the order of cytotoxicity is G1.0<G2.0< G3.0<G4.0.3. It was confirmed that G2.0-G4.0PAMAM-NH2have broad-spectrumantimicrobial activity and also exhibited strong inhibitory effects onmulti-resistant strains, such as MRSA, vancomycin intermediate resistantS.aureus Mu50, ESBLs-EC and MDR-EC.4. It was confirmed that G2.0-G4.0PAMAM-NH2could inhibit the biofilmformation of ESBLs-EC and MRSA, but the bacteria in the biofilm wereresistant to G2.0PAMAM.5. It was confirmed that ESBLs-EC and S.aureus ATCC29213do not developresistance to PAMAM-NH2G2.0.6. The damage intensity of PAMAM to mammalian cells and bacterial cells isdifferent and both are related to the generation. This selectivity differencemay be due to the different volume of cells of different types, which meansthat the charge density relative to eukaryotic cells is much less than thecharge density relative to the bacteria at the same concentration of G2.0.7. It was confirmed that10+10mg/kg PAMAM G2.0could significantlyincrease the survival rates of BALB/c mice infected by ESBLs-EC or MRSA,and exhibited the excellent therapeutic effects on the sepitic animal models.8. The modified PAMAM G3.0with LED209(meta-LED209-PAMAM(n=1.89))show much lower cytotoxicity, much higher affinity to EHEC and similarantimicrobial potency compared with PAMAM G3.0, while it can inhibit theexpression of the key virulence genes of EHEC by inbibiting QseC receptor.