The presence of bacterial resistance to antibiotics is a very important issue and the search of new alternatives is necessary. In this work, a combination of chitosan gel with silver or gold nanoparticles was prepared and characterized using thermal, rheology, bactericide, and biocompatibility analyses. ESEM images were also taken to visualize the incorporation of the nanoparticles into the gel matrix. Thermal analysis showed a better thermal stability in the chitosan-gold nanoparticles gels compared to the chitosan-silver nanoparticles gels. Rheology analyses showed that the viscosity of the gels decreased when velocity increased and there were differences in viscosity when silver and gold nanoparticles concentrations change. ESEM images showed the presence of agglomerates of silver and gold nanoparticles into the gel matrix with a good distribution; in some cases the formation of microstructures was found. Bactericide results show that these materials present an antibacterial activity against
The development of bacterial resistance to antibiotics has become an important health problem; it is common in isolates from healthy persons and from patients with community acquired infections in developing countries. Resistance is increasing, particularly to first line, inexpensive, and broad-spectrum antibiotics but also to the new drugs; therefore the search of new alternatives is necessary [
In recent years, the application of nanoparticles (NPs) in various fields has expanded considerably. NPs have been successfully applied in the medical and pharmaceutical fields for the delivery of therapeutic agents, in chronic disease diagnostics, and in biomedical sensors [
An important factor to take into consideration when nanoparticles are going to be used is the way they are transported to the site of action; for this, several methods like films, nanocapsules, foams, and gels have been used [
In the current work a chitosan gel was prepared and mixed with silver and gold nanoparticles; different concentrations of the components were used in order to evaluate the changes in the properties of the polymer-nanoparticles combination. These polymer gels were characterized using differential scanning calorimetry (DSC), rheometry test (RT), and environmental scanning electron microscopy (ESEM). The antimicrobial activity of the gels was tested on four types of both gram-positive and gram-negative bacteria, including clinical isolates of
Chitosan (high molecular weight, 85% DD, Sigma-Aldrich), auric tetrachloride (HAuCl4, 99.9%, Sigma-Aldrich), glycerol (C3H8O3, 99.5%, Sigma-Aldrich), gallic acid (C7H6O5, Sigma-Aldrich), silver nitrate (AgNO3, CTR Scientific), acetic acid (C2H4O2, 99.7%, J.T. Baker), and sodium hydroxide (NaOH, 98.2%, Baker) were used as received. Brain heart infusion and bactoagar for the microbiological analyses were purchased from BD DIFCO and used without previous treatment.
Silver nanoparticles: 0.169 g of silver nitrate was dissolved in 100 mL of deionized water; then we added 0.1 g of gallic acid previously dissolved in 10 mL of deionized water and immediately after the pH was adjusted to 11 using a 1.0 M NaOH solution [
Gold nanoparticles: 100 mL of a 0.001 M gold solution was prepared from HAuCl4 and deionized water. To this solution, 10 mL of deionized water containing 0.001 mol of gallic acid was added under magnetic stirring and then the pH was adjusted to 10 using a 0.1 M NaOH solution [
The silver and gold nanoparticles were characterized, to know particle size, with dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern) operating with a He-Ne laser at a wavelength of 633 nm and a detection angle of 90°; all samples were analyzed for 60 s at 25°C and, to know morphology, with transmission electron microscopy (TEM, JEOL JEM-1230) at an accelerating voltage of 100 kV.
Chitosan gels with a concentration of 2, 4, and 6 vol% of acetic acid were prepared with the following procedure: 1.75 g of chitosan and 0.2 mL of glycerol were added to 49.8 mL of a 2, 4, or 6 vol% acetic acid solution in deionized water. This mixture was stirred at 1000 rpm at room temperature during 5 minutes until the gel was formed.
Concentrations of the different reagents used to prepare the gels.
Sample | %wt Ag | %wt chitosan | %vol acetic acid | %wt Au |
---|---|---|---|---|
Ag1Ch3.5 | 0.1 | 3.5 | 4 | — |
Ag05Ch3.5 | 0.05 | 3.5 | 4 | — |
Ag02Ch3.5 | 0.025 | 3.5 | 4 | — |
Ag1Ch1.7 | 0.1 | 1.75 | 4 | — |
Ag1Ch.875 | 0.1 | 0.875 | 4 | — |
Au02Ch3.5 | — | 3.5 | 4 | 0.02 |
Au01Ch3.5 | — | 3.5 | 4 | 0.01 |
Au005Ch3.5 | — | 3.5 | 4 | 0.005 |
Au02Ch1.7 | — | 1.75 | 4 | 0.02 |
Au02Ch.875 | — | 0.875 | 4 | 0.02 |
Ch2 | — | 3.5 | 2 | — |
Ch4 | — | 3.5 | 4 | — |
Ch6 | — | 3.5 | 6 | — |
The gels were characterized using differential scanning calorimetry (DSC), rheometry test (RT), and environmental scanning electron microscopy (ESEM). A DSC calorimeter (Waters DSC 500) was used with a constant heating rate of 20°C/min from room temperature to 1000°C under nitrogen atmosphere. The rheometry test was performed with a U.S. 200, Paar Physica Rheometer equipped with a Peltier temperature control. The determination was carried using a cone-plate geometry (MK31, 50 mm in diameter, 1° tilt, and 0.49-micron truncation). An aliquot of the sample was deposited at the base of the plate to further secure the upper cone. Determining viscosity was carried out at a temperature of 25°C; the shear rate increased from 50 rpm to 250 rpm. The distribution of the nanoparticles in the gels was observed using an environmental scanning electron microscope (ESEM Quanta 200, FEI) and no special preparation of the sample was needed for this observation.
The following gram-negative microorganism was evaluated:
For the antimicrobial assays the bacterial strains were grown overnight in BHI broth at 37°C. The bacterial concentration was standardized to an optical density of 0.08 at 600 nm (approximately
For the well diffusion assay BHI solid media were prepared and sterilized at 121°C for 20 min. Approximately 20 mL of the sterilized media was poured in petri dishes (
The materials chosen for this test were those with the highest content of silver or gold nanoparticles because it is well known that, for a given material, at the highest concentration, the material could have the most toxic effect. The protocol for this study was conducted under the test guidelines of the Organization for Economic Cooperation and Development (OECD 1995) applying good laboratory practices (GLP) and approved by the investigation committee of the master’s degree in advanced clinical dentistry. 15 healthy Wistar rats (230 g) were divided into three groups (
For the experiment, each rat was anesthetized with an intraperitoneal injection of ketamine (8–12 mg/Kg) and xylazine (30–50 mg/Kg) and shaved in the dorsum area to perform a 1 cm incision. In each incision 200
After the wound was epithelialized (28 days) a full-thickness biopsy was taken. The samples were stored in 10% formaldehyde for 48 h prior to inclusion in paraffin. After that, each histological sample was stained using hematoxylin-eosin and Masson’s trichrome staining methods. The qualitative analysis was done with a light microscope (Olympus, 100X magnification).
For the bactericide evaluation, all of the experimental results are expressed as mean ± standard deviation (SD) of three determinations. Student
In general terms the hydrogels present several characteristics like high water vapor and oxygen permeability, as well as mechanical properties that resemble physiological soft tissues [
AgNP and AuNP were synthetized according to methods previously published [
(a) Ag nanoparticles and (b) Au nanoparticles.
(a) Au gels, (b) chitosan gels, and (c) Ag gels. Gels are presented in syringes just for this image; it does not correspond to a specific intended application.
TGA analyses for chitosan gel without nanoparticles and chitosan gel with AgNPs have been previously reported [
TGA and DSC results of all the synthesized gels.
Label | Exothermic peak (°C) | First weight loss (%)* | Second weight loss (%)** | Residues (%)† |
---|---|---|---|---|
Ag1Ch3.5 | 116.88 | 93.01 (40–200) | 4.55 | 0.475 |
Ag05Ch3.5 | 125.94 | 89.19 (50–175) | 8.71 | 1.504 |
Ag02Ch3.5 | 112.50 | 93.37 (30–147) | 2.948 | 1.569 |
Ag1Ch1.7 | 123.50 | 95.50 (30–200) | 1.386 | 2.338 |
Ag1Ch.875 | 122.40 | 96.88 (30–200) | 1.511 | 0.55 |
Au02Ch3.5 | 144.81 | 78.20 (30–200) | 12.06 | 7.82 |
Au01Ch3.5 | 133.63 | 86.34 (30–200) | 6.54 | 5.904 |
Au005Ch3.5 | 112.50 | 93 (30–200) | 4.90 | 0.542 |
Au02Ch1.7 | 123.23 | 95.50 (30–200) | 1.38 | 0.77 |
Au02Ch.875 | 132.57 | 91.85 (30–200) | 5.52 | 0.569 |
Ch2 | 136.92 | 63.38 (30–200) | 26.25 | 8.07 |
Ch4 | 109 | 81.26 (30–200) | 2.27 | 15.46 |
Ch6 | 112.23 | 89.50 (30–200) | 6.279 | 1.91 |
Ch-gallic acid | 103.18 | 93.39 (20–200) | 2.76 | 0.92 |
TGA and DSC analysis of selected samples. (a) Au02Ch3.5 and (b) Ch-gallic acid gel.
As we can see from Table
According to our results, the addition of Ag and Au nanoparticles to a chitosan gel produces a higher thermal stability compared to gels without nanoparticles (it is reflected on the temperature of the endothermic peak); for example, sample Ch4 has an endothermic peak at 109°C and sample Au02Ch3.5 (the sample with the highest Au content) has an endothermic peak at 144.81°C; these results agree with a previous report where a chitosan gel with iron oxide nanoparticles was prepared [
Gels consistency is a very important feature for the manipulation of the product and is related to its viscosity. Part of our work was focused on finding the gel with the best manipulation characteristics without affecting its bactericide properties. We observed a change in the viscosity values when the chitosan quantity decreased making the gels less viscous in all cases. The viscosity test was performed to evaluate the change in properties of the synthesized gels depending on the concentration of the components. Due to the low viscosity of gels without nanoparticles, they were not analyzed and high and low concentrations of the metal nanoparticles and chitosan were evaluated and results are shown in Figure
Viscosity test of the gels with a 4 vol% of acetic acid and with (a) AgNPs and (b) AuNPs.
Nascimento et al. prepared a chitosan gel with silver sulfadiazine and reported that the presence of silver sulfadiazine increased the viscosity of the material and an increase of the shear rate decreases the viscosity; although the silver compound is different to ours the results are similar, confirming that viscosity is strongly dependent on the chitosan concentration [
Once inside the chitosan matrix, nanoparticles agglomerate but the ESEM images (Figures
ESEM images of chitosan gels with AgNPs. (a) Ag1Ch3.5; (b) Ag05Ch3.5; (c) Ag02Ch3.5; (d) Ag1Ch1.7; and (e) Ag1Ch.875.
ESEM images of chitosan gels with AuNPs. (a) Au02Ch3.5; (b) Au01Ch3.5; (c) Au005Ch3.5; (d) Au02Ch1.75; and (e) Au02Ch.875.
This behavior could be a good characteristic because it seems that nanoparticles are not strongly linked to the gel matrix and can migrate from the gel to the action site. Hsu et al. [
Metal nanoparticles have shown important antibacterial activity likely due to both their size and high surface to volume ratio; those characteristics allow them to interact with bacterial membranes producing the biocide effect by a combination of mechanisms. Bacterial walls are different depending on the thickness of their peptidoglycan layer; this characteristic classifies bacteria into gram-negative and gram-positive groups [
The antibacterial activity of the gels was tested using BHI agar plates inoculated with different bacteria; this is a common method already reported elsewhere [
Average (in mm) of the different inhibition zones obtained by the gels tested in each bacterium.
Label | Reference strains | Clinical isolates | ||||||
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Ag1Ch3.5 | 16.81 ± 0.2 | 10.58 ± 9.31 | 17.45 ± 1.71 | 14.67 ± 0.14 | 8.68 ± 0.65 | 10.39 ± 3.2 | 8.51 ± 0.3 | 9.29 ± 3.46 |
Ag05Ch3.5 | 15.16 ± 1.60 | 14.68 ± 1.16 | 19.31 ± 0.60 | 14.32 ± 1.09 | 5.89 ± 5.22 | 5.15 ± 4.46 | 9.04 ± 3.1 | 9.38 ± 4.33 |
Ag02Ch3.5 | 14.36 ± 0.62 | 12.29 ± 4.52 | 16.76 ± 3.20 | 14.75 ± 0.12 | 8.68 ± 2.4 | 5.05 ± 4.37 | 5.35 ± 4.64 | 5.68 ± 9.84 |
Ag1Ch1.7 | 16.65 ± 0.98 | 15.58 ± 1.64 | 19.23 ± 3.45 | 15.41 ± 0.12 | 9.24 ± 2.85 | 11.88 ± 3.1 | 9.68 ± 3.58 | 11.55 ± 4.28 |
Ag1Ch.875 | 16.87 ± 2.71 | 18.17 ± 1.55 | 17.32 ± 1.44 | 15.84 ± 0.40 | 9.63 ± 2.4 | 11.16 ± 5.7 | 10.2 ± 3.5 | 13.08 ± 4.57 |
Au02Ch3.5 | 17.03 ± 2.39 | 12.38 ± 4.36 | 17.4 ± 0.76 | 14.23 ± 0.57 | 9.15 ± 2.52 | 8.56 ± 0.77 | 7.99 ± 0.57 | 9.57 ± 0.9 |
Au01Ch3.5 | 14.43 ± 0.50 | 9.12 ± 7.91 | 17.91 ± 2.3 | 13.17 ± 0.35 | 7.07 ± 6.31 | 6.06 ± 5.27 | 7.93 ± 0.58 | 11.67 ± 3.87 |
Au005Ch3.5 | 15.49 ± 0.28 | 11.95 ± 4.45 | 16.23 ± 0.58 | 14.44 ± 0.43 | 4.04 ± 6.99 | 2.67 ± 4.62 | 7.88 ± 0.5 | 6.79 ± 6.29 |
Au02Ch1.7 | 15.16 ± 0.48 | 15.34 ± 3.43 | 17.88 ± 0.87 | 15.05 ± 0.10 | 11.08 ± 3.2 | 9.54 ± 1.58 | 7.58 ± 0.21 | 11.94 ± 0.59 |
Au02Ch.875 | 15.96 ± 0.74 | 14.62 ± 4.13 | 17.48 ± 0.80 | 16.29 ± 0.84 | 10.54 ± 2.6 | 10.03 ± 0.1 | 7.63 ± 0.48 | 10.72 ± 0.66 |
Ch2 | 9.97 ± 1.02 | 0 | 11.73 ± 1.49 | 9.09 ± 1.22 | 0 | 0 | 0 | 0 |
Ch4 | 8.41 ± 1.24 | 0 | 12.06 ± 1 | 11.02 ± 0.18 | 0 | 0 | 0 | 0 |
Ch6 | 19.06 ± 1.63 | 18.37 ± 0.33 | 21.1 ± 2.55 | 17.1 ± 0.93 | 6.95 ± 6.02 | 13.59 ± 1.4 | 11.4 ± 2.08 | 14.07 ± 1.65 |
Metal nanoparticles like gold and silver have been explored for their broad-spectrum antimicrobial activity [
Samples with Ag and Au nanoparticles showed a bactericide effect against both standard and clinical strains: the sample with the highest inhibition halo was Ag05Ch3.5 against
Other aspects to take into consideration are the difference between bacteria given the change in membrane structure and the difference between clinical isolate and reference strains [
In the biocompatibility
In general, the rats did not show behavioral changes. The three groups of rats presented a normal healing at 28 days in the wound site as shown in Figure
The images represent the healed wounds performed in the dorsum of the rats after 28 days with their respective histological analysis observed at 1000X. (a) 3.5% chitosan gel with 0.1% silver nanoparticles. (b) 3.5% chitosan gel with 0.02% gold nanoparticles. (c) 3.5% chitosan gel without nanoparticles and 4% acetic acid concentration.
Chitosan gels with silver and gold nanoparticles were prepared with different amounts of the reagents in order to evaluate the resulting characteristics given by the change in concentrations. The gels presented a better thermal stability due to the combination with the metal nanoparticles. When the chitosan concentration decreased the consistency of the gels was better and so their manipulation. In the ESEM images the formation of cubes and dendrites was present in the case of AgNPs gels. In the antimicrobial test the best results were obtained with the reference strains; meanwhile the smaller inhibition zones were obtained with the clinical isolates of
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was partially supported by Consejo Nacional de Ciencia y Tecnología (CONACYT Grant CB-169020), Programa de Mejoramiento del Profesorado (PROMEP), and Fondo de Apoyo a la Investigación (FAI) of Universidad Autónoma de San Luis Potosí (UASLP). C. Sámano-Valencia would like to thank CONACYT for its support with the Scholarship no. 297778.