Polyvinyl butyral PVB was supplied by Solutia. The active parts of the functionalized fluids to produce the printable antibacterial devices was synthesized using two different process. The formation of the silver nanoparticles was monitored using UV-Vis spectroscopy. The images were analyzed by a software published in [ 45 ]. The ecotoxicity of PVB as a fluid component and AC, as printing support substrate, were analyzed by exposure with Artemia franciscana. For the hatching of A. After hatching, the artemias were placed on a well microtitre plate 5 individuals per well and each well with 1 mL of the previously prepared saline solution.
AC was taken in a layer disc form of 6 mm diameter. The assay was controlled by two targets in which five individuals were placed. The survival and mortality of Artemia franciscana was evaluated at 24 hours, as described in [ 46 ]. In both cases, the printing support was AC. For each fluid, the drop formation, which is function of the viscosity and surface tension, was optimized varying the electric pulse waveform of the printer. These waveforms, very important mainly for Non-Newtonian fluids, allows the droplets to be ejected properly from the nozzle, carrying the Ag-NPs.
The voltage variation Vxt to be applied in each PZT actuator was obtained for each fluid with the help of embedded drop watcher and fiducial cameras. Using the Waveform Editor tool embedded in the printer software and the voltage and frequency parameters obtained empirically from a standard fluid, modifications were made to the waveform shape, which influences the formation, shape, and volume of the produced droplets. In order to determine the parameters, the following steps were performed:. The waveform changes were tracked with the DropWatcher tool camera, while the print parameters were determined from the Drop Spacing tool: to adjust the distance between the center of two adjacent drops, the angle of the print head was adjusted, which influences the resolution of the print.
The drying time between layer was set to 5 min. Devices were produced with different number of layers for each fluid. The inocula were adjusted to 0.
The mixture was autoclaved and placed in Petri boxes. The bacteria were sown in the Petri dish boxes with a swab over the entire surface, on which the discs of printed devices 5,15 and 30 layers were placed. The discs were prepared for this assay using a standard metal perforating punch. As target control, sterile AC discs were used. To insert the Ag-NPs, a well has been made in the center of the Petri dish, drilling the middle and removing it with the help of a cork drill.
The diffusion coefficient of Ag-NPs in the M-H-agar medium was calculated by measuring the apparent dynamic viscosity of the culture medium in the following concentrations: M-H 0.
Polymeric Materials with Antimicrobial Activity (RSC Publishing)
The data were then extrapolated to determine the apparent viscosity at the concentration M-H 6. Finally, the measured inhibition zone was related to the minimum inhibition zone A min expected in relation to the diffusion coefficient calculated in 24 h of bacterial incubation, as indicated in the following relation: herein mm 2. Survival of Artemia franciscana : statistical analysis was performed using an ANOVA variance analysis of a 2x3 factorial design 6 treatments to evaluate the combination of two types of levels: material type PVB and AC and concentrations of each 0.
In both analyses the combination of bacterial type E. For each experimental measurement, average value and standard deviation were calculated for the inhibition zone using a millimeter ruler. The presence of silver nanoparticles was confirmed both in the chemical synthesis method with a maximum absorption peak at nm and in the microwave irradiation method with a peak at nm see Fig 1A.
This indicates an approximate size for spherical silver nanoparticles between 35 to 50 nm [ 47 ]. This conclusion is based on the size dependence of the optical property of the plasmon resonance of silver nanoparticles [ 48 ].
The frequency of the surface plasmon resonance over the silver nanoparticles depends on the nanoparticle shape and size, as well as the dielectric functions of the host matrix and nanoparticle. Also, the formation of nanoparticles aggregates or clusters may displace the absorption band up to the infrared, not observed in the present work [ 49 ]. The hydrodynamic diameters of the nanoparticles were determined using DLS.
In cases of polydisperse size distribution of nanoparticles, the greater the dispersity of the sample, the greater the probability of unsuccessful data from DLS technique. The average diameter was found to be From Fig 2 , diffracted peaks were observed at They correspond to the set of planes and of the crystalline structure of silver FCC. For Ag-NPs M , the diffraction angles As shown in the diffractogram of Fig 2 , one may notice that, regardless of the nanoparticle preparation technique microwave or chemical , the ratio between the Bragg peaks intensities from the metallic silver and the scattering band associated to the amorphous part of the material, do not changes, although the intensity ratio between the peaks related to and planes have its ratio intensities changed, probably due to a possible preferential orientation of the powders during the preparation of the samples.
Using an analysis software developed in our group [ 45 ], an average diameter of For Ag-NPs M an average diameter of This result is different from the DLS measurements, as the refraction index of the formed complex is unknown and due to the fact that the sample distribution is not perfectly monodisperse [ 3 ].
Prepared from microwave synthesis A and B , and from chemical synthesis C and D. The white bar scale is 50 nm. To verify this experimental error rate, A. Therefore, comparing the survival rate of control individuals with respect to individuals exposed to PVB and AC, we conclude that there is no influence of these polymers on the survival of A. To produce the two printed devices, the fluids droplets ejected through the nozzles were optimized applying a specific electric pulse waveform to the PZT actuators.
A detailed protocol can be found in [ 54 ]. In order to evaluate the efficacy of the printed antibacterial devices, initially the bactericidal effect of silver nanoparticles without PVB and before printing was observed for Ag-NPs M. After the production of the printed devices, the bactericidal effect was analyzed using the method of diffusion in agar with Kirby—Bauer discs, where the inhibition zone generated by the devices of Ag-NPs Q and Ag-NPs M in both bacteria was evidenced.
The same process of measurements was performed for S. All the inhibition zones measurements are summarized in Table 2.
Original Research ARTICLE
By the previous results, one can conclude that the cell wall thickness of the Gram-positive bacteria affects the action of the devices, showing less effect in S. This is also confirmed by other research groups, as published by Malegowd et al. Furthermore, it was measured an apparent minimum inhibition zone for Ag-NPs Q of 0. These experiments show clearly the antibacterial effect of the obtained devices. However, the antibacterial mechanism of Ag-NPs is not clearly defined.
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There are several theories in the current literature. Ag-NPs can anchor to the cell membrane, causing damage and leakage of intracellular material. The theory mentions that the formation of free radicals by Ag-NPs is one of the causes of cell death since they have the ability to generate pores in the membrane [ 56 ].
Another theory considers that the release of silver ions from nanoparticles damages the integrity and permeability of the membrane, and that these ions can react with functional protein molecules and DNA, interfering with DNA metabolism and replication, originating the cell death [ 25 ]. Considering these various theories, we consider the schematic diagram for explaining the antibacterial mechanism presented here in the nanocomposite system, see Fig 8. The bacteria interact with the composite and in presence of water a process of creating silver ion is generated, thus leading to the antibacterial effect.
The mechanism presented in Fig 8 is related to metallic pure silver nanoparticles, that releases ionic silver in aqueous solution, as proposed here, but, using the same procedure proposed in the present paper, it is also possible to print new hybrid structures, as those recently proposed by Assis et Al. The new class of spherical hybrid nanoparticle presents a fold improvement of antibacterial performance and may be fully compatible with the presented DoD printing process.
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The result of the ANOVA, carried out in the survival of Artemia franciscana , showed that there is no influence of the polymer type user in fluid and substrate , since the p value obtained for the type and the concentration of each polymer is 0. Moreover, these values are greater than 0. Two antibacterial devices printed using DoD technology were obtained.
The first, with silver nanoparticles obtained by chemical process and the second one, with silver nanoparticles produced under microwave irradiation, in a PVB composite. We show that both devices have a bactericidal effect against S. The substrate acetate cellulose and the polymer used in the fluid PVB were subjected to an ecotoxic test, in which it was determined that both polymers do not generate any type of toxicity, according to the biomodel used.
The results indicate that the best antibacterial characteristics of the printed device is obtained for the polymer compound with silver nanoparticles synthesized by microwave irradiation and considering the highest number of printing layers. In addition, the inclusion of silver nanoparticles into the polymer matrix avoid any aggregation and so the possibility of a reduction of its antibacterial activity.
To ensure the effect on a long time, an interleaved or simultaneous release of silver and inhibitors of proteins produced by Gram-negative bacteria could be produced in the next future. Therefore, this kind of device could be used for industrial applications as a printable antibacterial device produced by drop-on-demand inkjet technology. The authors are especially grateful to Prof. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
Abstract Silver nanoparticles Ag-NPs are known for their efficient bactericidal activity and are widely used in industry. Introduction Silver has been used for thousands of years as an antimicrobial and antibacterial agent, up to the antibiotic era which started 90 years ago [ 1 — 2 ].
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