Study On The Synthesis And Antibacterial Activity Of Cobalt-Metal Organic Framework

1 BackgroundDiseases caused by bacterial infections are one of the global public health problems.According to statistics,millions of people die every year due to bacterial infections.Antibiotics are the traditional method of treating infectious bacteria,but the abuse of antibiotics has led to increasing bacterial resistance and even the emergence of"superbugs".Pseudomonas aeruginosa is an obligate aerobic gram-negative bacterium and one of the common opportunistic pathogens of nosocomial infection.It can cause serious infections such as respiratory tract,urinary tract,burn wound and bacteremia.The increasing drug resistance of Pseudomonas aeruginosa has brought difficulties to clinical treatment.Nanomimetic enzymes,a class of nanomaterials with similar reaction mechanisms to natural enzymes,have attracted great attention in antibacterial therapy.In particular,functional nanoparticles with peroxidase-like properties can convert hydrogen peroxide（H₂O₂）into hydroxyl radicals（·OH）,which are more toxic to bacteria,thereby killing bacteria.Due to the facile aggregation of nanoparticles,the structure and morphology of nanomaterials are key factors affecting their performance.To develop better antibacterial agents,it is often necessary to maximize the enzyme-like activity of nanomaterials by customizing the morphology and composition of nanomaterials.In this study,Cobalt（Co）and TCPP（Fe）,two nanomaterials with peroxidase activity,were combined to synergize the peroxidase-like activities of the two to synthesize a Co-metal_-organic framework composites（Co-MOF）which can effectively kill bacteria at low concentration H₂O₂.2 Materials and methods2.1 Reagent preparationAcetic acid-acetate buffer solution（Buffer）,3,3’,5,5’-tetramethylbiphenyl acid solution（TMB）,hydrogen peroxide,lysogeny broth（LB）liquid medium,LB solid medium,physiological saline and phosphate buffered saline（PBS）buffer were prepared accordingly.2.2 Preparation of Co-MOF15.0 mg of Co（NO₃）₂·6H₂O（0.05 mol）,4.0 mg of pyrazine（0.05 mol）and100.0 mg of polyvinylpyrrolidone（PVP）were dissolved in a mixed solution of 60m L（Dimethyl formide）DMF and ethanol（V:V=3:1）,stirred evenly and 12 m L was taken for use.4.4 mg tetracyanoclopentane（TCPP）（0.005 mol）was dissolved in 8 m L mixed solution of DMF and ethanol（V:V=3:1）,and added in a drop wise to the above mixed solution under stirring.The solution was sonicated for 10minutes,placed in the autoclave and heated to 80°C for 24 hours.Then the reactant was taken out and centrifuged at 20℃,8000 r/min for 10 minutes,the obtained substance was washed twice with ethanol and then re-dispersed in 2 m L ethanol.2.3 Verification of peroxidase activity of Co-MOFThe peroxidase activity of Co-MOF was investigated using H₂O₂-TMB catalytic oxidation model.The absorption spectrum of the reaction system in the range of 550 nm-750 nm was measured by UV-Vis spectrophotometer.2.4 Characterization of materialsMorphological characteristics of the synthesized MOF were studied by field emission scanning electron microscope and field emission transmission electron microscope.The Energy Dispersive X-ray Spectroscopy was used for elemental analysis of Co-MOF.2.5 Steps of killing Pseudomonas aeruginosa in vitro（1）The pure strain of Pseudomonas aeruginosa was purchased from Wuhan Science Research Institute,China.（2）Configure H₂O₂and Co-MOF gradient concentrations,bacterial liquid optical density（OD）and adjust the bacterial concentration to 10⁸ CFU/m L.（3）The Volume ratio of bacterial solution reagent of each group:（1）Pure bacterial solution control group:Take 100μL bacterial solution and add 900μL normal saline（i.e.1 m L 10⁸normal saline bacterial solution）（2）H₂O₂control group:100μL bacterial solution+800μL normal saline+100μL H₂O₂（3）MOF control group:100μL bacterial solution+800μL normal saline+100μL Co-MOF（4）Experimental group:100μL bacterial solution+700μL normal saline+100μL Co-MOF+100μL H₂O₂Placed in an air-bath shaking incubator for sterilization for 2 hours at a speed of 200 r/min and a temperature of 37°C.（4）Colony plate culture experimen by coating plate method was used to study growth inhibition.（5）Bacterial liquid culture experiment was usedto assess the the growth trend of the bacteria.Bacterial measurements were plotted as growth curves.2.6 Optimization of sterilization conditions2.6.1 Effect of Co-MOF concentration on antibacterial effect50μg/m L,100μg/m L,150μg/m L,200μg/m L,250μg/m L and 300μg/m L of the synthesized Co-MOF were added to the 250μmol/L H₂O₂ 10⁸ CFU/m L bacterial solution,respectively.Pure bacterial solution control group,H₂O₂control group and Co-MOF control group were set.2.6.2 The effect of H₂O₂ concentration on the antibacterial effect250μg/m L of the synthesized Co-MOF with excellent bactericidal effect were added to the 10⁸ CFU/m L bacterial solution of H₂O₂ at 100μmol/L,150μmol/L,200μmol/L,250μmol/L,300μmol/L and 350μmol/L respectively,and placed in an air bath oscillation incubator for sterilization for 2 h.Pure bacterial solution control group,H₂O₂control group and Co-MOF control group were set.2.6.3 Effect of sterilization time on antibacterial effectThe combination of Co-MOF and H₂O₂concentration with excellent bactericidal effect was used to explore the effect of bactericidal time on the antibacterial effect for different durations 0.5-3 hours.2.7 The 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-2H-tetrazolium bromide（MTT）assay to test the toxicity of Co-MOFThe cytotoxicity of the synthesized Co-MOF was tested using MTT assay on the human umbilical vein endothelial cells（HUVEC）.The absorbance was measured with a micro plate reader at a wavelength of 600 nm,and the results were expressed as the percentage of experimental viable cells relative to the control.2.8 St atisti cal anal ysisThe results of statistical analysis were plotted using Origin 2021 software,and all experiments were repeated three times.IBM SPSS 21.0 was used for statistical analysis,and the comparison between the mean of various samples showed that the data followed normal distribution and had uniform variance,with significance levelα=0.05.When the P value obtained was less than 0.05,the difference was significant.3 Results3.1 Verification of peroxidase activity of Co-MOFThere was no oxidation peak in the control group in the range of 550 nm-750nm;there was an oxidation peak in the experimental group,and the maximum absorption peak appeared at 650 nm.At the same time,it could be observed with the naked eye that the color of the experimental group system changed from light yellow to green,while the control group system solution remained unchanged（light yellow）.3.2 Morphological and structural characteristicsThe synthesized Co-MOF were flake-like structures with uneven surfaces,but not typical pores.The basic skeleton of Co-MOF was formed by element C,and the distribution of elements C,N,O,Co and Fe was uniform.3.3 Effect of Co-MOF concentration gradient against Pseudomonas aeruginosa3.3.1 Plate culture of Co-MOF concentration gradient against Pseudomonas aeruginosaThe effect of Co-MOF dosage on Pseudomonas aeruginosa colony formation was investigated by bacterial plate culture experiment.From the results obtained by the automatic colony counter on the plate it can be seen that,as compared with the pure bacterial solution control group,the Co-MOF group was able to inhibit the visible growth of Pseudomonas aeruginosa at a minimum concentration（MIC）of100μg/m L.When the Co-MOF group concentration was 250μg/m L,the anti-pseudomonas aeruginosa effect was significant;the survival rate was about 6%,almost achieving the bactericidal effect.The differences between the control group and the experimental groups of each Co-MOF gradient concentration were analyzed by Analysis of variance,and the differences in the number of colonies between the experimental groups of 100μg/m L and higher gradient concentrations compared with the control group were statistically significant（P<0.05）by two-by-two comparison.3.3.2 Liquid culture of Co-MOF with concentration gradient against Pseudomonas aeruginosaThe effect of Co-MOF on the growth of Pseudomonas aeruginosa was investigated through liquid culture experiment as well.The growth curve results of the Co-MOF experimental group showed that the pure bacterial liquid group began to grow after 4 h,while the bacteria containing 50μg/m L Co-MOF began to grow after 7 h.The bacteria containing 100μg/m L,150μg/m L,200μg/m L,250μg/m L and 300μg/m L Co-MOF began to grow after 9 h.Analysis of variance showed that there were differences between the control group and the experimental groups with each Co-MOF gradient concentration,and the differences in the number of bacteria between the experimental groups with 100μg/m L and higher gradient concentrations compared to the control group were statistically significant（P<0.05）after two-by-two comparison.Overall,the experimental group containing 250μg/m L Co-MOF showed obvious inhibition compared with the pure bacterial solution group.3.4 Effect of H₂O₂ concentration gradient against Pseudomonas aeruginosa3.4.1 Plate culture of H₂O₂concentration gradient against Pseudomonas aeruginosaThe effect of H₂O₂ concentration on Pseudomonas aeruginosa colony formation was investigated by bacterial plate culture experiment.From the results of automatic colony counter on the plate showed that when H₂O₂ concentration was 250μmol/L,the anti-pseudomonas aeruginosa effect was significant,the survival rate was about 6%,almost achieving the bactericidal effect.Analysis of variance was used to analyze the differences between the control group and each H₂O₂ gradient concentration experimental group,and the differences in the number of colonies were statistically significant（P<0.05）when comparing the experimental group with the control group at 100μmol/L and above gradient concentrations after a two-by-two comparison.When the concentration of H₂O₂experimental group was 250μmol/L,the anti-Pseudomonas aeruginosa effect was significant and reached the bactericidal effect.3.4.2 Liquid medium culture of H₂O₂concentration gradient against Pseudomonas aeruginosaThe effect of H₂O₂concentration on the growth of Pseudomonas aeruginosa was also investigated through liquid culture experiment.From the growth curve results of H₂O₂ experimental group,showed that the pure bacteria liquid group and the H₂O₂ control group began to grow after 4 hours,while the bacteria containing different concentrations of H₂O₂ began to grow after 7 hours.Analysis of variance showed that there were differences between the control group and the experimental groups for each H₂O₂ gradient concentration,and the differences in bacterial numbers were statistically significant（P<0.05）between the experimental groups with gradient concentrations of 100μmol/L and above compared with the control group after a two-by-two comparison.As a whole,Pseudomonas aeruginosa containing 250μmol/L H₂O₂was significantly inhibited compared with the pure liquid control group.3.5 Bactericidal time gradient against Pseudomonas aeruginosaThe effect of sterilization time on Pseudomonas aeruginosa colony formation was observed through bacterial plate culture experiment.The results obtained by the automatic colony counter showed that,the maximum inhibition was reached at2.5 hours where the bacteria survival was almost 0%.Analysis of variance was used to analyze the differences between the control group and the experimental groups for each bactericidal gradient time,and after a two-by-two comparison,the differences in the number of bacteria were statistically significant（P<0.05）in the experimental group with 1.5 h and above gradient time compared to the control group.Overall,it can be seen that the experimental group containing 2.5 h almost reached the bactericidal effect compared to the pure bacterial solution group.3.6 MTT toxicity test resultsThe experimental results showed that low concentration（0μg/m L-250μg/m L）of Co-MOF basically did not have a significant damage on human umbilical vein endothelial cells,while the maximum concentration of 300μg/m L Co-MOF damaged the human cells.Anova was used for analysis.The differences between the control group and each Co-MOF gradient concentration experimental group were analyzed by Analysis of variance,and the differences in the survival rate of human umbilical vein endothelial cells in the experimental group with Co-MOF gradient concentrations of 250μg/m L and below compared with the control group were not statistically significant（P>0.05）,and the survival rate of human umbilical vein endothelial cells in the experimental group with 300μg/m L The difference between the experimental group and the control group was statistically significant（P<0.05）.4 Discussion4.1 Analysis of synthesis methods of Co-MOF nanocompositesIn the present experiments,Co-TCPP（Fe）nanoflakes were successfully prepared using a solvothermal method in which one or more precursors are dissolved in a non-aqueous solvent,and the reaction occurs under liquid-phase or supercritical conditions where the reactants are dispersed in solution and become more reactive and the products are slowly produced.The process is relatively simple and easy to control,and the volatilization of toxic substances can be effectively prevented in a closed system.In this study,four precursors,namely Co（NO₃）₂·6H₂O,TCPP（Fe）,pyrazine and PVP,were dissolved in a mixed solution of DMF and ethanol,and the synthesis process did not involve extremely high temperature and did not require much operation,which was safe and simple.Moreover,the MOF material,i.e.,TCPP（Fe）,is an existing reagent,and only the chemical reduction of Co（NO₃）₂·6H₂O is required to further modify the MOF to enhance its peroxidase-like activity.In addition to the use of chemical reagents for this purpose,a large body of literature shows that the use of pure biomaterials or extracts from biological resources such as plants,bacteria,fungi and algae as reducing agents has emerged as a cost-effective and eco-friendly platform for the synthesis of nanoparticles with enhanced biocompatibility,better stability and higher antibacterial functionality.Essentially,the green synthesis of metal nanoparticles is a redox reaction due to the reducing power of cellular or extracellular components such as proteins,carbohydrates,organic acids,phenols and other metabolites such as individual metabolites or groups of metabolites that provide electrons to metal cations,resulting in the formation of zero-charge and nanoscale metal forms.The intermediate functional groups for the synthesis of nanoparticles are alcohols,aldehydes,amines,carboxyl,ketones,hydroxyl,and sulfhydryl groups,so almost any biological compound that provides these functional groups can be used to convert metal ions into nanoparticles.However,some compounds,such as terpenoids,flavonoids,various heterocycles,polyphenols,reducing sugars,and ascorbic acid are directly involved in the synthesis,while others,such as proteins,act as stabilizers and form an organic coating known as a halo.Notably,a prominent drawback of green synthesis is the inhomogeneous size and shape of nanoparticles in the reaction medium.In this way,the high polydispersity of nanoparticles can affect biological and pharmaceutical effects,especially under physiological conditions.Therefore,more research is needed to improve this obstacle,such as using other physical and chemical methods.Although this study was not explored further,it can provide us with new ideas for the synthesis of materials in the future.4.2 Analysis of characterization results of Co-MOF nanocompositesTo explore the morphological characteristics of the composites,Co-MOF was characterized by field emission scanning electron microscopy and field emission transmission electron microscopy,and elemental analysis of Co-MOF was carried out by energy dispersive X-ray energy spectrometry.The synthesized Co-MOF has a lamellar structure with a heterogeneous surface and more atypical pores,increasing its specific surface area and releasing effective metal ions and reactive oxygen species.the basic backbone of Co-MOF consists of C elements with uniform distribution of C,N,O,Co and Fe elements,indicating that the Co nanoparticles can be uniformly modified on the MOF with stable and homogeneous properties.4.3 Analysis of peroxidase activity of Co-MOF nanocompositesCompared with natural enzymes,artificial enzymes have the advantages of excellent catalytic activity,high stability and low cost.Co-MOF is characterized by the synergistic enhanced peroxidase activity of the material,compared with single Co nanoparticles or TCPP（Fe）,Co-MOF has a synergistic dual mimetic enzyme activity,which greatly enhances its peroxidase-like activity.Co-MOF can catalyze the peroxidase oxidation of the substrate TMB by H₂O₂,and the oxidation peak of TMB at 650 nm appears as an indicator,which is green in aqueous solution.This provides a sensitive colorimetric detection of H₂O₂,which has significant potential for practical applications.On the other hand,Co-MOF converts H₂O₂ to toxic·OH,which can be detected by fluorescence experiments in the next experiments,such as the conversion of the non-fluorescent compound terephthalic acid（TA）to the highly fluorescent product 2-hydroxyterephthalic acid,which shows a characteristic fluorescence signal at 435 nm,confirming the production of·OH to or by electron paramagnetic resonance.The production of·OH was further investigated by electron paramagnetic resonance spectroscopy.4.4 Optimization analysis of sterilization conditions of Co-MOF nanocompositesThe prepared Co-TCPP nano composites have peroxidase like activity,which can convert H₂O₂ into·OH which is more toxic to bacteria,so as to kill bacteria.In this study,we first determined the approximate concentration of Co-MOF and H₂O₂ through pre experiment,and then further determined and optimized the experimental conditions.We mainly evaluated the antibacterial experimental results through plate culture and growth curve.Firstly,by optimizing the dosage conditions of Co-MOF from the plate culture,we can see that when the concentration of Co-MOF was 100μg/m L,there was a visible growth inhibition of Pseudomonas aeruginosa,when the concentration of Co-MOF was 250μg/m L,the bactericidal effect was significant and when the concentration of Co-MOF-H₂O₂was 250μg/m L-250μmol/L the bactericidal effect is almost achieved.But also the antibacterial effect was prominent with time,hence showing both concentration and time contributed to the antibacterial effect of this material and the same trend could be seen from the growth curve.Then the toxicity of Co-MOF was tested by MTT method.Low concentration（0μg/m L-250μg/m L）Co-MOF basically did not have a significant damage to human umbilical vein endothelial cells,and high concentration of 300μg/m L Co-MOF seriously damaged the human umbilical vein endothelial cells and reduced their viability up to 45%.Moreover,the higher concentration gradient of H₂O₂ can be toxic to the cells,therefore,it is acceptable that 250μmol/L H₂O₂ was selected and exhibited a stronger antibacterial effect.Finally,250μg/m L Co-MOF and 250μmol/L H₂O₂ were selected to study the effect of bactericidal time gradient against Pseudomonas aeruginosa.The bactericidal effect was increasing with time and the complete bactericidal effect was achieved in 2.5 h.4.5 Analysis of sterilization mechanism of Co-MOF nanocompositesNano enzymes with peroxidase like activity can produce toxic reactive oxygen species at low concentration,even without hydrogen peroxide,completely avoiding the toxicity of H₂O₂.However,the utilization of some nano enzymes still needs the assistance of H₂O₂enhance their peroxidase like activity.An important reason is that their enzyme-like activity is not sufficient to generate sufficient concentrations of reactive oxygen species to kill the bacteria.Therefore,in order to develop better antibacterial nano materials,it is necessary to maximize their enzyme like activities by adjusting the morphology and composition of nano materials.Apart from releasing reactive oxygen species,metal nanoparticles that release metal cations can be adsorbed on the bacterial surface and cause cell wall damage by forming pits,furthermore,the electrostatic interaction between the positive charge of metal ions and the negative charge of the cell membrane can affect outflow and influx of bacterial biomaterials,which ultimately kills the bacteria.Some of these mechanisms were out of the scope of this experimental study.Moreover,the antibacterial study of this Co-MOF can be extended to other bacteria to observe its activity against them.4.6 Innovative analysisIn this study,Co-MOF,a new material with peroxidase-like activity,was synthesized by a simple method,and the bactericidal conditions were optimized with Pseudomonas aeruginosa as the target bacterium,which has good bactericidal effect at low concentrations of H₂O₂and Co-MOF,providing another antibacterial compound for the highly pathogenic multi-drug resistant P.aeruginosa.4.7 Limitations analysisThe experiment evaluated only one gram negative bacteria（P.aeruginosa）among the wide range of Enterococcus faecium,Staphylococcus aureus,Klebsiellapneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa,and Enterobacterspp group that has been repeatedly reported to be resistant against the conventional antibiotics.Another limitation of the proposed experiment is that it was centred only on in vitro bactericidal effect of Co-TCPP（Fe）-MOF.Furthermore,due to lack of some tool and apparatus such as inductively coupled plasma-optical emission spectrometry and electron spin resonance spectroscopy limited the scope of our experimental study.5 Conclusion（1）In this study,Co-TCPP（Fe）nanomaterials were synthesized by solvothermal method with Co（NO₃）₂·6H₂O,TCPP and other reagents.The morphology and composition of Co-MOF were analyzed by SEM,TEM and EDS.The results showed that the nanomaterials with flake structure and uneven surface were successfully prepared.The main element composition were:C,N,O,Co and Fe in an even distribution manner.（2）The study of H₂O₂-TMB catalytic oxidation model showed that Co-MOF had excellent peroxidase activity.On this basis,the synthesized Co-MOF had good antibacterial activity against Pseudomonas aeruginosa.（3）The cytotoxicity experiment on the human umbilical vein endothelial cells showed that the nanoparticles had no side effects on healthy cells in the low concentration range.After optimizing a series of antibacterial conditions,the effective concentration range of 100μg/m L-250μg/m L for Co-MOF was determined,which provides a theoretical basis for the practical application of this nano material.