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Back to Journal »International Journal of Nanomedicine» Volume 15

Antibacterial activity of novel biocomposite chitosan/graphite based on zinc grafted mesoporous silica nanoparticles

Author Jamshidi D, Sazegar MR 

The 2020 volume will be published on February 7, 2020: 15 pages 871-883

DOI https://doi.org/10.2147/IJN.S234043

Single anonymous peer review

Editor approved for publication: Dr. Thomas J Webster

Donya Jamshidi, Mohammad Reza Sazegar, Department of Chemistry, Islamic Azad University, Tehran, Iran Communications: Mohammad Reza Sazegar, Department of Chemistry, Islamic Azad University, North Tehran, Hakimiyeh, Tehran, Iran Phone +98-9381199151 Email [Email protection ] Introduction: A new type of biocomposite chitosan/graphite based on zinc grafted mesoporous silica nanoparticles (CGZM-bio) was synthesized, and the antibacterial activity of this compound and Zn-MSN nanoparticles was studied. Method: CGZM-bio biocomposite material is synthesized under ultraviolet radiation using sol-gel and post-synthesis methods. The samples were characterized using FTIR, XRD, SEM, and nitrogen adsorption and desorption. Antibacterial activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) after 18 hours at 310 K. Results: Suspension samples of Zn-MSN and CGZM-bio (2-100 μg.mL) − 1) It has antibacterial activity against Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentration (MIC) values ​​of the Zn-MSN and CGZM-bio samples against E. coli are 10 and 5 μg.mL-1, respectively, while the MIC values ​​of the two nanomaterials against Staphylococcus aureus are 10 μg.mL-1 Discussion: The antibacterial activity of these materials is due to the generation of free radical oxygen, such as •OH, H2O2 and O2 2- by generating electron-hole pairs during ultraviolet radiation, which can damage bacterial cells. These nanomaterials can be used in biomedicine. The device acts as an antibacterial agent. Keywords: Zn-MSN, Staphylococcus aureus, Escherichia coli, antibacterial activity, ultraviolet radiation, electron-hole pair

One of the serious risks that long-term destructive diseases pose to human life is bacterial infection, which is the fourth cause of death in the world. Scientific applications such as medicine and pharmacy. The effect of 2,3 MNPs and MONPs is attributed to their small size and high surface area to volume ratio, which provides suitable conditions for the interaction of MNPs and MONPs with bacterial membranes. 4,5 Studies have found that the bactericidal activity of metal species is related to its valence. 5 Researchers have shown that the higher the valence, the stronger and more active the antibacterial effect. 6,7 MONPs including silver, titanium, bismuth and zinc oxide nanoparticles have shown antibacterial effects against Gram-positive and negative bacteria. 8-11 These materials can control the growth of bacteria.

For a long time, silver nanoparticles have been considered as antimicrobial materials 12-14, and they have been developed in many applications in various fields such as medicine, 15 antibacterial water filters, 16 and cosmetics. 17 Among all nanomaterials, these nanoparticles have shown the greatest antibacterial activity. 18-20 Recently, composite materials containing metal atoms dispersed on a silica carrier have been attracted due to their high antibacterial activity. 21 According to scientists, silver-silica and silver-silica-polymer nanocomposites have high antibacterial activity, and these nanocomposites have been used in wound dressings and catheter coatings. 1,22 In addition, several antibacterial activities of ZnO nanoparticles have been reported in recent years. 5-7 In addition, zinc oxide is a low-priced semiconductor with simple preparation process, biocompatibility and high activity photocatalyst. 7 These nanomaterials show remarkable antibacterial properties. A wide range of bacteria types. These nanomaterials have been fixed, impregnated or inserted into other materials (such as natural or man-made polymers) to enhance their bactericidal properties. twenty three

As a useful natural hydrophilic polymer matrix, chitosan has recently been widely used due to its excellent chemical and biological properties. 24,25 Chitosan is prepared from chitin, which is easily extracted from the shells of crustaceans, such as fungi, crabs, insects, prawns and other crustaceans. 26 This natural polymer shows no Toxic, biodegradable, biocompatibility, bioactivity and antibacterial properties make it suitable for medicinal chemistry. 27-29 Due to the presence of amine groups in its structure, chitosan is a weak base, so it has solubility in dilute acid solutions. This NH2 group is converted into a water-soluble protonated form of NH3+30. Now, chitosan has been Extensive research for antibacterial and antifungal materials in different forms of solutions, films and composite materials. 27 In addition, the antibacterial properties of chitosan vary with fabric samples. This activity of chitosan is due to the interaction between the lone pair of electrons on the amino nitrogen atom and the microbial membrane. 28 Therefore, it is obvious that this interaction can be easily carried out between chitosan and microorganisms, because it is combined with DNA to form glycosaminoglycans and proteins, resulting in an increase in antibacterial effect. 29 Since these interactions are weak, when chitosan is used alone, the antibacterial properties are weak. 28,31-33

In the past ten years, several chitosan composite materials have been synthesized for use as fungicides. 34,35 Various types of materials have been used to form composite materials with chitosan, such as carbon nanotubes (CNT), graphene oxide, and graphite. Among the many carbon types, graphite has special characteristics such as large surface area, high porosity, and good electrical conductivity. 36 Graphite has recently attracted attention as a viable and inexpensive filler in composite structures. 37 If graphite can be used, these excellent properties may be suitable for nano-scale use as thin nanosheets. 38 Pure graphite in the form of small thin layers has been studied as a substrate to improve the physical and chemical properties of nanomaterials. 39,40 Metal-carbon composites show antibacterial activity against Escherichia coli. 41 Free from the dispersion of carbon substrates such as graphene, carbon nanotubes and graphite, there are problems, so the use of these materials as antibacterial agents is limited. In addition, direct contact with carbon materials can cause cell damage, which in turn leads to cell death. 42 In order to improve the antibacterial properties of graphene and carbon nanotubes, functionalization with metal nanoparticles has been reported. 42, 43

Mesoporous silica nanoparticles (MSN) with ordered porous structure, high surface area, large pore size and volume can be used as the main body of MNP, so that the metal sites can be better dispersed on the surface of MSN. 44,45 Using MSN as the main metal nanoparticle is an interesting suggestion that can improve antibacterial efficiency. 46,47

In this study, biocomposites of chitosan and graphite were synthesized based on zinc-modified MSN, and their effects on Gram-positive and Gram-negative microorganisms such as Staphylococcus aureus (S. aureus) and the large intestine were studied. The antibacterial activity of E.coli. The main goal of this work is to combine the antibacterial properties of Zn-MSN and chitosan with graphite to design biocompatible antibacterial biomaterials.

Chitosan and graphite were purchased from Kimiya Azma and Black Diamond Companies in Iran, respectively. All other chemicals, including cetyltrimethylammonium bromide (CTAB), tetraethylorthosilicate (TEOS), zinc nitrate, and ammonia solution were purchased from Merck.

Under alkaline conditions, using the cationic surfactant cetyltrimethylammonium bromide (CTAB) as the template and tetraethylorthosilicate (TEOS) as the silicon source, the mesoporous bismuth was synthesized by the sol-gel method. Silicon oxide nanoparticles (MSN). Dissolve 44 cetyltrimethylammonium bromide (CTAB, 1.24 g) in double distilled water (250 g), and then add ethanol (55 mL) and ammonia solution (10.5 mL, 28%). After vigorously stirring at 298 K for about 20 minutes, tetraethyl orthosilicate (TEOS, 2.7 mL) was added to the mixture. The resulting mixture was stirred for another 2 hours at 298 K and allowed to stand under ultraviolet radiation (8 W) at the same temperature for 24 hours. The molar composition of the gel used to synthesize pure MSN is 1.1 TEOS: 0.7 CTAB: 16 Ammonia: 91 Ethanol: 1053 H2O. The samples were then collected by centrifugation at 20,000 rpm for 20 minutes, washed 3 times with deionized water and absolute ethanol, and dried at 383 K for 8 hours before calcination. Then, the surfactant was removed by calcining MSN (1 g) in air at 823 K for 3 hours. Zinc-doped MSN (denoted as Zn-MSN NPs) is prepared by doping zinc into pure MSN. In this reaction, zinc nitrate (Merck) was used as the precursor of zinc, and the initial molar ratio of Si/Zn was 10. The Zn-MSN sample zinc nitrate (0.13 g) was synthesized by adding MSN (1 g) to an aqueous solution (50 mL) for 12 hours at 353 K, then centrifuged and dried overnight at 383 K, then in air at 823 K Then calcined at a heating rate of 1 K min-1 for 4 hours.

A suspension of Zn-MSN (1 g) in distilled water (100 mL) was sonicated for 30 minutes. In a separate container, sonicate chitosan (0.2 g), pure graphite (0.1 g), and distilled water (100 mL) in a separate container for 30 minutes. The chitosan-graphite mixture was slowly added to the Zn-MSN suspension, and the pH of the mixture was adjusted to 8-9 by adding a small amount of NaOH (0.1 M). Then stir for 30 minutes in a 343 K water bath under ultraviolet radiation. The precipitate was washed with distilled water (2 × 40 mL) and dried in a vacuum oven at 333 K.

The effects of Zn-MSN nanoparticles and chitosan/graphite/Zn-MSN biocomposites on two pathogenic strains of Gram-negative bacteria Escherichia coli (E.coli) and Staphylococcus aureus (Staphylococcus aureus) were tested. Antibacterial activity-positive bacteria, using agar plate diffusion method. The antibacterial activity is measured by the paper disc diffusion method with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). In this method, the samples were cultured in Muller Hinton (MH) and nutrient agar at pH 7.3 on a Petri dish prepared using E. coli and Staphylococcus aureus growth medium. Use a sterile glass rod to spray the microorganisms evenly on the top of the plate. Allow the microorganisms to dry for 10 minutes. Then, drop test solutions of samples of different concentrations into the disc. Incubate the medium at 310 K for 18 hours. After that, prepare a sample of Mc-Farland standard suspension of No. 0.5, which is equal to 1.5 × 108 colony forming units/mL (CFU.mL-1). The standard dilution micro method is used to test the antibacterial effectiveness of MH petri dishes. 48

Several aqueous dispersions of Zn-MSN nanoparticles and CGZM-bio samples at different concentrations were prepared from the initial aqueous dispersion samples. In order to obtain a uniform distribution, the nutrient MH medium is heated to 323 K. In the next step, 10 mL of each sample solution was added to a petri dish containing 30 mL of nutrient MH medium. Keep a total volume of 40 mL in each petri dish, and harden the solution with MH after 15 minutes. Then, on each sample, 100 µL of microbial suspension was added and spread on the surface of the MH medium including the above samples. The petri dishes were incubated in a shaking incubator (150 rpm) at 310 K for 18 hours to grow microorganisms. In the absence of Zn-MSN NP or CGZM biocomposite materials, compare the results with a standard petri dish for the growth strength test of each microorganism. The quantitative measurement of the bactericidal activity of Zn-MSN and CGZM-bio is based on the CFU growth number of the lower concentration (104 CFU) bacterial suspension in the MH medium of different concentrations of Zn-MSN and CGZM-bio samples. At the same time, Compare all tests with those of the control sample. All experiments were performed on Zn-MSN nanoparticles and biocomposites three times under aseptic conditions, and the results were averaged. According to the equation, the quantitative antibacterial activity is calculated based on the reduction rate (percentage) of microorganisms. 1. As follows: (1)

Where R is the percentage reduction rate, ni is the number of bacterial colonies in the test petri dishes of the control sample, and nf is the number of bacterial colonies in these petri dishes after treatment with Zn-MSN and CGZM-bio. According to the European Antimicrobial Susceptibility Testing Committee (EUCAST) standard, the disc diffusion method and MH are used to study the in vitro antibacterial activity of samples by determining the size of the inhibition zone in millimeters (mm). 49

The antibacterial effects of Zn-MSN and CGZM-bio on two pathogenic strains of Escherichia coli and Staphylococcus aureus were studied. The initial concentration of the Zn-MSN nanoparticle and CGZM-bio culture was a 0.5 Mc-Farland standard suspension of the two microorganisms, which was determined by testing using an agar dish. The paper discs coated with Zn-MSN nanoparticles and CGZM-bio (the disc sample size is 6 mm) were sterilized by dripping ethanol onto these materials and aging for 10 minutes, and then placed on the MH inoculated with 1 mL of microorganisms On the surface cultural. The petri dishes were inoculated at 310 K for 18 hours. The size of the inhibition zone of the disc was measured to determine the antibacterial activity of each sample. Each sample uses the average of three test measurements. In this study, some disks were also used, including amikacin (AN) and erythromycin (E) standard antibiotics placed on the surface of MH.

The crystallinity of the catalyst was measured with a Bruker Advance D8 X-ray powder diffractometer, and Cu Kα (l = 1.5418 Å) radiation was used as a diffracted monochromatic beam of 40 kV and 40 mA. The nitrogen physical adsorption analysis was performed on Quantachrome Autosorb-1 at 77 K. Before measurement, the sample was evacuated at 573 K for 3 hours. The bulk Si/Zn molar ratio of 10 was determined by Bruker S4 Explorer X-ray fluorescence spectroscopy (XRF), using Rh as the anode target material, and operating at 20 mA and 50 kV. XRF analysis shows that the Si/Zn ratio of the 10-frame is actually 9.3.

Use Agilent Carry 640 FTIR spectrometer for Fourier Transform Infrared (FTIR) measurement. The morphology and average particle size of the catalyst were estimated by scanning electron microscopy (SEM). A scanning electron microscope equipped with an energy dispersive X-ray spectrometer (EDX) was performed on the SEM (JEOL JSM-6701 F) to observe the morphology and obtain the elemental analysis of the catalyst. Before observation by SEM-EDX, the sample was coated with platinum using a sputtering instrument.

The pure MSN was synthesized by the sol-gel method and the Zn-MSN sample was prepared by the post-synthesis technique. Figure 1 shows the XRD patterns of MSN, Zn-MSN and CGZM-bio samples with a Si/Zn molar ratio of 10. Pure MSN shows four diffraction peaks, including a strong peak at 100 and three small peaks 110, 200, and 210 at low angles of 2.2–5.6° 2θ. All these diffraction peaks indicate the existence of a 2D hexagonal (p6mm) structure with a d100 spacing of approximately 3.7 nm. As the order of the mesoporous structure is reduced, the incorporation of zinc atoms into MSN reduces the strong peak of 100. 50 The introduction of chitosan and graphite into Zn-MSN nanoparticles greatly reduced this strong peak, thus completely changing the mesoporous structure to a less ordered structure. The small shift of the peak position from 2.35° in MSN to 2.20° in Zn-MSN can be explained by the substitution of plausible Si atoms by zinc atoms with larger atomic radius, which leads to a decrease in the interplanar spacing of MSN (Figure 1A) The high-angle XRD patterns of MSN, Zn-MSN-10 and CGZM-bio samples at 10-80° are shown in Figure 1B. X-ray microanalysis of these materials indicated the presence of zinc atoms in their structure. The diffraction peaks corresponding to the 001, 101, and 102 planes indicate the presence of ZnO phase in these nanostructures. 51 Figure 1 XRD diagram: (A) the low degree of MSN, Zn-MSN and CGZM-bio at 0-10°; (B) the height of Zn-MSN and CGZM-bio at 10-80°.

Figure 1 XRD pattern: (A) MSN, Zn-MSN and CGZM-bio at a low degree of 0-10°; (B) Zn-MSN and CGZM-bio at a height of 10-80°.

The diffraction pattern also shows that the Zn-MSN sample has good crystallinity. Due to the replacement of atoms between the exchangeable layers, the intercalation of zinc ions in the interlayer spacing of MSN leads to an increase in the basic spacing. The presence of amorphous structure also reflects the presence of chitosan and graphite in the MSN framework, as shown in the diffraction pattern of Figure 2B. Figure 2 (A) Nitrogen adsorption isotherms and (B) pore size distribution of MSN, Zn-MSN NPs and CGZM-bio samples.

Figure 2 (A) Nitrogen adsorption isotherms and (B) pore size distribution of MSN, Zn-MSN NPs and CGZM-bio samples.

Table 1 shows the physical properties of MSN, Zn-MSN and CGZM-bio samples. The surface area is reduced from 892 m2g-1 in MSN to 475 m2g-1 in Zn-MSN, which is due to the loading of Zn species into pure MSN. Therefore, it reduces the pore volume and pore size of Zn-MSN. This result may be due to the fact that zinc atoms blocked some pores, which caused the pore size to change from 3.41 nm in MSN to 3.36 nm in ZN-MSN, and also changed the pore volume from 0.72 cm3g−1 in MSN to 0.43 cm3g−1 in. Zn-MSN.45 Table 1 Physical characteristics of MSN, Zn-MSN NPs and CGZM-Bio samples

Table 1 Physical characteristics of MSN, Zn-MSN NPs and CGZM-Bio samples

The introduction of chitosan and graphite into mesoporous Zn-MSN nanoparticles completely changed the structure of microporous materials. It is believed that chitosan and graphite completely blocked most of the pores, greatly reducing the pore surface area of ​​CGZM-bio to 13 m2g-1 and increasing the pore size to 36.6 nm. These results clearly show that this biocomposite has a dense structure and wide pores, and the SEM image confirms its morphology (Figure 3). Figure 3 SEM and TEM images of (A, D) MSN, (B, E) Zn-MSN NPs, (C, F) CGZM-bio samples.

Figure 3 SEM and TEM images of (A, D) MSN, (B, E) Zn-MSN NPs, (C, F) CGZM-bio samples.

Figures 2A and B show the nitrogen adsorption isotherms and corresponding pore size distributions of MSN, Zn-MSN NPs, and CGZM-bio samples calculated by the NLDFT method. The nitrogen adsorption isotherm patterns of these nanomaterials show type IV isotherms of pure and zinc-modified MSN, which indicates the existence of mesoporous nanomaterials with cylindrical pores, and shows the type III isotherm of CGZM-bio, which is attributed to Microporous structure (Figure 2A). The high resolution of the CGZM-bio sample is observed in the square, which clearly shows the microporous structure with additional framework structure. The isotherms of MSN and Zn-MSN show the bending characteristics of capillary condensation when the relative pressure (P/P0) is about 0.1-0.3, which indicates the existence of a porous structure with a small and uniform mesoporous structure. 52 Relative pressures of about 0.9-1.0 in high Zn-MSN indicate the presence of external framework porosity, which is clear evidence that the mesoporous structure is not so ordered due to the loading of zinc atoms into the pure mesoporous silica material. For the CGZM-bio composite, a slow increase in relative pressure from 0 to 0.9 was observed, which was attributed to the microporous structure, while the sharp increase in P/P0 near 0.9-1.0 indicated the presence of additional framework structures in the biocomposite. Figure 2B shows the pore size distribution of MSN, Zn-MSN NPs and CGZM-bio. The results showed that the incorporation of chitosan, graphite and zinc atoms almost blocked the pores of the biocomposite material, resulting in the formation of multiple pore diameters in the composite frame.

Use SEM and TEM images to observe the morphology of MSN, Zn-MSN and CGZM-bio, as shown in Figure 3. The results show that MSN and Zn-MSN have uniform spherical particles with a diameter of 70-130 nm, and their EDX elements. The change of CGZM-bio's surface structure is also obvious from its SEM image, as shown in Figure 3C. For CGZM-bio, large spherical layered features with a size of more than 200 nm were also observed. The rough surface of the biocomposite material indicates that the Zn-MSN nanoparticles have been assembled on the chitosan and graphite layer, resulting in the formation of a large spherical layered structure. The TEM images of MSN, Zn-MSN and CGZM-bio are shown in Figure 3 (DF). The mesoporous framework of MSN was confirmed by TEM (Figure 3D), showing a regular and parallel pore structure. The TEM image of the 53 Zn-MSN sample (Figure 3E) shows that the material particles have a regular array of mesoporous structures. The Zn atoms are well dispersed in the silica matrix. The TEM (Figure 3F) of the CGZM-bio sample shows the spherical monolithic particles in the sample. The framework silica mesopores are wormhole-like in the whole particle. No significant long-range order was detected in the pore structure. 54

EDX analysis (shown in Figure 4) shows the presence of silicon, zinc, and oxygen in Zn-MSN nanomaterials. Figure 4 Energy dispersive X-ray spectrum of Zn-MSN sample.

Figure 4 Energy dispersive X-ray spectrum of Zn-MSN sample.

Figure 5 shows the FTIR spectra of MSN, Zn-MSN NP and CGZM-bio samples. It is observed that the silanol (Si-OH) groups of MSN and zinc-modified MSN are broadband in the range of 3100-3700 cm-1. These broad peaks are attributed to the non-acidic silanol groups located on the outer surface of the mesoporous structure. 55 The small spike in the 3405 cm-1 region indicates the presence of amine groups in the biocomposite. In addition, due to the presence of chitosan and graphite species in the biological composite structure, the two sharp bands at 2923 and 2852 cm-1 are clear evidence of CH stretching. The peak near 1636 cm-1 corresponds to the amide structure of chitosan. 56 Since the silanol group reacts with the Zn atom to form Si-O-Zn, the grafting of zinc into the MSN structure will form a sharper band 45 MSN and Zn-MSN d100 spacing or pore size at 3445 cm-1. There is no significant difference in the band intensity in this area (3445 cm-1), which indicates that their primary particle sizes are equivalent. 57 The FTIR spectral peaks of the two vibrations located in the region of 465 and 1070 cm-1 are related to the Si-O stretching frequency in the mesoporous silica material. These peaks correspond to the O-Si-O and Si-O-Si bonds. respectively. The absorption peak at 796 cm-1 is attributed to the Si-O-Si bending vibration in the sample structure. 58 Figure 5 FTIR spectra of MSN, Zn-MSN NPs and CGZM-bio in the 400-4000 cm- region 1.

Figure 5 FTIR spectra of MSN, Zn-MSN NPs and CGZM-bio in the range of 400–4000 cm-1.

The proposed structure of CGZM-bio is shown in Scheme 1. This shows that Zn-MSN is connected to chitosan through the electrostatic band between the amine group of chitosan and the Zn atom of Zn-MSN nanoparticles, and there are other Zn grafted MSN between graphite molecules and chitosan. Si-O-Si tape of nanomaterials. Scheme 1 The proposed structure of CGZM-biocomposite.

Scheme 1 The proposed structure of CGZM-biocomposite.

The antibacterial activity of Zn-MSN nanoparticles and CGZM-bio suspension against Escherichia coli and Staphylococcus aureus was studied by disc diffusion in aqueous MH agar, and their MIC and MBC were measured. MBC is the lowest concentration (µg mL-1) of the material that kills more than 99% of existing microorganisms. MIC is the concentration at which the solution becomes turbid. Lower MIC corresponds to higher antibacterial efficiency. 59 In terms of MIC, we found that there is an inverse relationship between particle size and activity. In order to qualitatively evaluate the antibacterial activity of the material, 1.5 × 108 CFU bacterial suspension was used to inseminate MH discs containing different concentrations of nanoparticles and biocomposite materials. The results showed that under different concentrations of Zn-MSN nanoparticles and CGZM-bio at 5, 10, and 100 µg mL-1, bacterial growth on the MH plate was observed after 18 hours. The control sample used a biocomposite material in a solution of 0 µg mL-1 Zn-MSN and 1% acetic acid, and the microorganisms were fully grown after 18 hours at 310 K. The results show the MIC values ​​of Zn-MSN and CGZM-bio for E. Escherichia coli are 10 µg.mL-1 and 5 µg.mL-1, respectively, and the concentration of the two nanomaterials against Staphylococcus aureus is 10 µg.mL- 1. The E. coli 60 measured by the MIC value of the control sample is about 900 µg.mL-1, and the Staphylococcus aureus is about 1550 µg.mL-1. 61 The results show that there is no bacterial growth at the MIC value and above, so these concentrations can Effectively inhibit the growth of bacteria.

According to published results, chitosan and graphite have weak antibacterial activity when used alone. 32,62 The mechanism of biocomposites is to inhibit bacterial growth, possibly by forming cationic parts on zinc atoms and amine groups, and interacting with anionic parts on the surface of microbial cells, and possibly destroying bacteria by disrupting the exchange with the culture medium Growth. 61 The inhibitory mechanism of Zn-MSN nanoparticles on bacterial growth may be through the interaction between zinc sites and bacterial membranes.

Recently, N. Padmavathy and R. Vijayaraghavan (2008) have demonstrated the qualitative antibacterial effect of 12 nm and 45 nm ZnO nanoparticle suspensions on E. coli in Luria Bertani (LB) broth through disk diffusion. They reported that at 310 K.61,63 Sharifian-Esfahni et al. (2017) synthesized 80 µg.mL-1 to quantitatively measure E. coli, and synthesized chitosan-modified iron oxide (SPIO/CTS) nanocomposite Material to improve the antibacterial activity against Escherichia coli and Staphylococcus aureus. The results show that the MIC value of SPIO/CTS nanocomposites for E. coli is 45 µg.mL-1. 64 In another study, Hu et al. (2017) prepared a zinc oxide-based nanocomposite, in which Silver nano-encapsulated in polyvinylpyrrolidone and polycaprolactone (PVP/PCL) nanofibers improve the antibacterial effect on Staphylococcus aureus and Escherichia coli under MH broth medium. The results show that this nanocomposite suspension can inhibit the growth of bacteria against these two microorganisms. 65 This enhanced antibacterial strength may be due to the enhanced stability of biomaterials in aqueous media, because the chitosan/graphite composite material protects the Zn-MSN from the aggregated nanoparticles. The biological composite material has significant antibacterial activity against Escherichia coli at a low concentration, and can be used as an ideal nanomaterial to play a long-term effect in the green industry.

Amikacin (AN) (30 µg.disk-1) and erythromycin (E) (15 µg.disk-1) as two standard antibiotics for comparison of the zone of inhibition diameter (IZD) test, Zn-MSN The contents of Escherichia coli and Staphylococcus aureus on NPs and CGZM-bio MH plates were 5, 10 and 100 µg.mL-1, respectively. For the Zn-MSN NPs and CGZM-bio samples with a concentration of 10 µg.disk-1, the average diameter of the IZD to E. coli around the two samples was observed to be 21 ± 1 mm. For Zn-MSN NPs and CGZM-bio, at a concentration of 10 µg.disk-1, the results were 22 ± 1 mm and 24 ± 1 mm, respectively.

The results of the antibacterial activity of the samples indicate that the chitosan/graphite organic layer related to the biocomposite may be the reason for the stronger activity of the biocomposite, because the Zn-MSN NPs are better dispersed on the surface of the biocomposite. Chitosan/graphite part. No IZD against the two bacteria was observed around the control sample using this disc. Table 2 shows the results of the two samples of Zn-MSN NPs and CGZM-bio against the two bacteria of Escherichia coli and Staphylococcus aureus. Table 2 IZD, MIC and MBC of Zn-MSN NPs and CGZM-Bio samples

Table 2 IZD, MIC and MBC of Zn-MSN NPs and CGZM-Bio samples

There are several factors that can cause the bactericidal effect of Zn-MSN NPs and CGZM-bio. The mechanism by which antibacterial agents and antibiotics act through oxidative stress generated by reactive oxygen species (ROS). 66 Park and his colleagues have explained that the generation of ROS is responsible for the antibacterial activity of metals. 67

In this study, zinc oxide was the source of ROS that caused Staphylococcus aureus and Escherichia coli to inhibit the formation of ROS. The active mechanism of Zn-MSN NPs and biocomposites is based on the oxygen species produced by these materials, which can cause damage to microorganisms. 68 Highly active substances are formed, such as •OH, H2O2 and O22-. Because the Zn-MSN part can be activated by ultraviolet radiation by generating electron-hole pairs (e-h+) to dissociate H2O molecules into OH- and H+. The dissolved oxygen molecules form superoxide radical anions (•O2−), which react with H+ to form HO2•radicals, and collide with electrons to form hydrogen peroxide anions of HO2−. These anions react with H+ and produce H2O2 molecules, which penetrate the bacterial membrane and kill microorganisms. 69

Superoxide and hydroxyl radicals are negatively charged and cannot penetrate into the membrane of bacteria and can only contact the outer surface of microorganisms. However, hydrogen peroxide can penetrate the bacterial membrane. The explanation of the antibacterial effect of 70 Zn-MSN NPs may be based on the rough surface of the nanoparticles due to their surface defects. 71 This surface roughness provides mechanical damage to the membrane. Escherichia coli.

In this study, for E. coli and Staphylococcus aureus, the minimum inhibitor concentration (MIC) of Zn-MSN and CGZM-bio nanomaterials ranged from 2 to 100 (μg/mL). According to the results obtained, the concentrations of CGZM-bio and Zn-MSN of 5 and 10 µg/mL show the smallest inhibitors against E. coli microorganisms, while the minimum inhibitor concentrations of the two nanomaterials against Staphylococcus aureus are both 10 µg /mL. The MIC and MBC results of the two samples of Zn-MSN and CGZM-bio against Escherichia coli and Staphylococcus aureus are roughly the same.

Therefore, sample suspensions with Zn-MSN and CGZM-bio concentrations lower than 10 and 5 µg.mL-1 respectively have lower antibacterial activities against Escherichia coli and Staphylococcus aureus. This may be due to the presence of low zinc content to generate the aforementioned free radical species. The growth of Escherichia coli at a lower concentration of 5 µg.mL-1 around the disc including biocomposites and the growth of Staphylococcus aureus at a lower concentration of 10 µg.mL-1 around the Zn-MSN disc can be proved. The direct relationship between bacterial activity and zinc concentration is shown. On the other hand, zinc is not toxic at these low concentrations because this metal is an essential cofactor in many cellular processes. Therefore, the amount of bacterial colonies produced is lower than the MIC, indicating that Zn species is a supplement that promotes the metabolic properties of low-concentration bacteria.

Figure 6 shows the photos of the antibacterial activity of CGZM-bio and Zn-MSN samples against Escherichia coli and Staphylococcus aureus. Figure 6 (A) 5 µg/mL CGZM-bio, (B) 10 µg/mL Zn-MSN and 10 µg/mL Staphylococcus aureus antibacterial test (C) Figure 6) CGZM-bio, and (D) Zn -MSN.

Figure 6 (A) 5 µg/mL CGZM-bio, (B) 10 µg/mL Zn-MSN and 10 µg/mL Staphylococcus aureus antibacterial test (C) Figure 6) CGZM-bio, and (D) Zn -MSN.

The new Zn-MSN NPs and CGZM-bio nanomaterials were prepared using ultraviolet radiation, and their antibacterial activity was studied. The biocomposite nanomaterial is synthesized by combining the inorganic part (Zn-MSN) with chitosan/graphite as the organic part. 5. The colloidal solutions of Zn-MSN nanoparticles and CGZM-bio at 10 and 100 µg mL-1 showed antibacterial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (E. coli) microorganisms on MH In all three concentrations, the dish was placed at 310 K for 18 hours. The MIC values ​​of Zn-MSN NPs and CGZM-bio to E. coli are 10 µg.mL-1 and 5 µg.mL-1, respectively, and the MIC values ​​of the two nanomaterials to Staphylococcus aureus are 10 µg.mL-1. The MBC values ​​of Zn-MSN and CGZM-bio against Escherichia coli and Staphylococcus aureus are almost the same as those MIC values.

For the samples of Zn-MSN NPs and CGZM-bio with a concentration of 10 µg.disk-1, the diameter of the zone of inhibition (IZD) around the samples was observed to have an average diameter of 21±1 mm for E. coli. For Zn-MSN NPs and CGZM-bio, similar observations for Staphylococcus aureus were 22±1 mm and 24±1 mm, respectively, at a concentration of 10 µg.disk-1. The antibacterial properties of Zn-MSN NPs and biocomposite materials are based on the fact that these materials produce oxygen species, such as •OH, H2O2 and O22-, which cause damage to microorganisms. Therefore, these nanomaterials may be suitable for antibacterial applications in biomedical devices.

We thank the Department of Chemistry of Islamic Azad University for all support for this research.

The authors report no conflicts of interest in this work.

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