Introduction
Metabolic diseases, long considered of minor importance, are now taking place as the main threats to human health. DM is the most common non-communicable disease worldwide and the fourth to fifth cause of death in developed countries, and also now in developing countries. The global number of people with DM will increase from 220 million to 300 million people by 2025 (Tripathi et al., 2006).
Chronic complications of diabetes mellitus affect many organ systems and are responsible for most morbidity and mortality. Chronic complications can be divided in vascular and non-vascular, a serious example of these complications is the important pressure zones for DM, since an untreated injury in these areas can lead to diabetic foot, others like burns or varicose ulcers (Figure 1) are also relevant because chronic wounds promote a high colonization of microorganisms (Baczako et al., 2019; Armstrong et al., 2023).
Regarding bacterial infections and inflammation, it poses another severe threat to human health and the social economy in developed countries. In addition, the existence of super-bacteria and the increasingly severe phenomenon of antibiotic resistance highlight the development of new antibacterial agents (Fang et al., 2023).
Staphylococcus aureus is the most relevant pathogenic species within its genus, since it is mainly responsible for causing various infections such as skin infections, pneumonia, endocarditis and osteomyelitis. S. aureus has a high level of antibiotic resistance and is a common cause of infections in hospitals and the community (Ahmad-Mansour et al., 2021); It is also one of the most common bacteria found in chronic wounds.
Instead, Pseudomonas aeruginosa is a common, Gram-negative environmental organism due to its natural resistance to antibiotics and the ability to form biofilms. Infection with this pathogen can cause severe therapeutic problems like respiratory infections, urinary tract infections, wound infections, blood infections, eye infections, among others (Mielko et al., 2019), principally in chronic wounds and especially in pressure ulcers and burns.
Although the use of topical antibiotics has been the frequently used treatment to control the colonization and proliferation of these pathogens in wounds, treatment with silver has revolutionized infection care due to its beneficial effects. However, it has been shown that treatments containing only the silver ion can alter healing, causing cosmetic abnormalities in the skin. Virulence factors and biofilms constitute attractive targets for the prevention of infections caused by multidrug-resistant bacteria (Kim et al., 2019; Reese et al., 2022).
Metallic nanoparticles of silver (AgNps), gold (AuNPs), zinc oxide (ZnO NPs), copper and iron oxides, among others, have great potential in the pharmaceutical industry since these materials can be synthesized and modified in a relatively simple way (Chávez-Sandoval et al., 2021; Medina-Solano et al., 2024).
AgNPs have many advantages, such as the quantum confinement of electronic movement in semiconductor nanoparticles, surface plasmon resonance in some metallic particles or super magnetism in magnetic nanomaterials. Although several theories have been formulated about the antimicrobial mechanism of silver nanoparticles, their bacteriostatic effect is related to various biological processes. These processes include the generation of reactive oxygen species (ROS) and the induction of oxidative stress (OS) through enzyme inactivation. One of the key mechanisms of action is the denaturation of disulfide bonds present in bacterial proteins, which ultimately leads to cell death. These properties confer great benefits to the use of nanoparticles compared to current commercial drugs.
In this work we prepared a hydrogel with a concentration of 1000 μg/mL of AgNPs, its antimicrobial activity was evaluated and compared against a commercial cream containing silver sulfadiazine in S. Aureus and P. aeruginosa, our results suggest that the combination of gels and AgNPs displays the great potential to manage superbug-infected trauma.
Materials and methods
All materials used in this work were reagent grade and all tests were done in triplicate.
Synthesis and characterization of AgNPs
The method described by Chávez-Sandoval et al. (2021) was used, whit some modifications as described below.
Briefly: Sodium citrate was used as the reducing agent. JT Baker brand silver nitrate was used as the precursor.
Once the nanoparticles were obtained, they were stored at 4 ºC for characterization and subsequent use.
Basic tests for the study of antimicrobial activity
Minimum inhibitory concentration (MIC)
This test measures antimicrobial susceptibility by determining the smallest amount of an agent, which is generally an antibiotic, capable of completely inhibiting the growth of a given bacterial strain.
It is ideal for non-demanding growth bacteria such as S. aureus and P. aeruginosa. In addition, it is a method that allows determining the inhibitory activity and the bactericidal activity (Andrews et al., 2001).
Formulation and preparation of the hydrogel
The AgNPs have antimicrobial activity by themselves, however, they have a low binding affinity, so it is recommended to use hydrogels as an effective vehicle to improve this antimicrobial activity, since in combination with the appropriate polymers, their antimicrobial activity can be enhanced. The gel produced belongs to the classification of “hydrogel” which is a three-dimensional network composed of flexible polymer chains that absorb a large amount of water, although insoluble, they swell in the presence of it, significantly increasing its volume while maintaining its shape until reaching the physicochemical balance (Gao et al., 2016).
In this work the hydrogel was prepared according to the methodology of Algin et al. (2020) and adding silver nanoparticles, according to the AgNPs concentrations obtained from the MIC test, for each microorganism.
Once the hydrogel was obtained, it was compared against a commercial cream using the Kirbi-Bauer test.
Kirby-Bauer test
The Kirby-Bauer test was performed to compare the antimicrobial activity of the hydrogel, made from silver nanoparticles, against the antimicrobial activity of a commercial silver sulfadiazine cream, widely used to prevent and treat infections in wounds and burns (Reese et al., 2022). The mechanism of action of sulfadiazine cream is based on the combination of sulfadiazine, which acts as an inhibitor of folic acid synthesis, and silver ions, which interfere with the function of bacterial enzymes and proteins (De Francesco et al., 2022).
It is important to mention that the commercial cream is formulated with a higher proportion of silver ions, according to its secondary packaging, it contains 280 mg of silver sulfadiazine for each g of the final formulation. Thus a 1:100 dilution was made accordingly that its silver concentration was comparable to the hydrogel prepared.
This test consists of insertion paper discs impregnated with antimicrobial agents on the surface of agar previously inoculated with the bacteria in our case S. aureus and P. aeruginosa.
The antimicrobial agent (AgNPs or commercial cream) contained in the disks diffuses through the agar, and as it moves away from the disk, its concentration decreases logarithmically, thus generating a gradient of the antimicrobial agent in the agar surrounding the disk (Yin et al., 2023).
Although there is still no standardized data regarding the inhibition diameter zones using silver nanoparticles, the sensitivity determination of the AgNPs was carried out by comparing their diameter with other antibiotics that are commonly used against Staphylococcus aureus and Pseudomonas aeruginosa (Tables 1 and 2).
Table 1
Determination of antibiotic sensitivity and resistance by the disk method for Staphylococcus aureus.
[i] Source: Prepared by the authors based on data from American Society for Microbiology (2009).
Table 2
Determination of antibiotic sensitivity and resistance by the disk method for Pseudomonas aeruginosa.
[i] Source: Prepared by the authors based on data from American Society for Microbiology (2009).
Results and discussion
Synthesis and characterization of AgNPs
We obtained a yellow colloid characteristic of the silver nanoparticles (Medina-Solano et al., 2024), and they were characterized by using a Perkin Elmer spectrophotometer, model Lambda XLS, from a wave sweeping in a range of 276 to 700 nm, obtaining a maximum lambda of 429 nm with an absorbance of 0.311 a.u. (Figure 2). The appearance of absorption bands in the spectrum around wavelengths from 400 nm to 450 nm indicates the presence of AgNPs, the maximum lambda at these wavelengths is due to resonance absorption of surface plasmons.
Figure 2
A) Yellow colloid characteristic of AgNPs. B) UV-Vis characterization, the maximum lambda was 429 nm.
Source: Author’s elaboration.
The color of the AgNPs can vary mainly due to the method used, as well as the concentration of the reducing agent, in this work we use sodium citrate as a reducing agent. Sodium citrate also acts as a stabilizer and pH mediator. The AgNPs thus obtained were kept refrigerated at a temperature of between 2-7 ºC until their characterization.
Transmission electron microscopy (TEM) characterization
The TEM analysis allowed us to observe that the AgNPs synthesized in this work are spherical with a variable size from 5 to 100 nm (Figure 3). Spherical, polymorphic AgNPs of different sizes were observed. The variation in the size of the AgNPs can be attributed to the temperature, since, in general, the size of the nanoparticles increases with the reaction time.
We observed some agglomeration, so it is advisable to use other stabilizing agents as well as surfactants, which can protect the nanoparticles and prevent their agglomeration.
Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration test was carried out to know the lowest concentration at which silver nanoparticles inhibited the growth of Staphylococcus aureus and Pseudomonas aeruginosa, to determine the concentration of AgNPs in the hydrogel formulation.
The concentration of the AgNPs in each tube is described in Table 3.
A total of 11 concentrations were used.
Table 3
Concentration of AgNPs used for the MIC test.
| Tube | AgNPs Concentration ( μg / mL ) |
|---|---|
| 1 | 1000.00 |
| 2 | 500.00 |
| 3 | 250.00 |
| 4 | 125.00 |
| 5 | 62.50 |
| 6 | 31.25 |
| 7 | 16.62 |
| 8 | 7.81 |
| 9 | 3.90 |
| 10 | 1.95 |
| 11 | 0.97 |
Staphylococcus aureus
As seen in Figure 4, the minimum concentration at which there is inhibition of Staphylococcus aureus corresponds to 1000 μg/mL section number 1, and tube 1 in Table 3.
Pseudomonas aeruginosa
In the case of P. aeruginosa, in Figure 5, the minimum inhibitory concentration was 250 μg/mL, section 3, corresponding to tube 3 in Table 3.
Figure 5
A) Battery tubes with different concentrations of AgNPs. B) Replating each concentration of AgNPs used.
Source: Author’s elaboration.
Thus, it seems that the AgNPs synthesized in this work are more effective against P. aeruginosa than against S. aureus.
It is important to mention that the MIC test depends on the antibacterial activity of the AgNPs and this in turn is related to the synthesis method used, including reducing and stabilizing agents, since for cell disruption to be effective the concentration of the AgNPs, the charge in their surface, its morphology and especially its size, it has been reported that the smaller the AgNPs are, from 5 nm to 10 nm, the greater their antimicrobial effect (Nie et al., 2023; Medina-Solano et al., 2024).
Furthermore, there is no standardized data for the MIC test using silver nanoparticles and given that other protocols evaluate AgNPs with different characteristics, synthesized by various methods, a comparison cannot be made between the values obtained and those reported by previous research. Likewise, there is also no standardized MIC test data for antibiotics, since the articles reviewed works with different conditions and concluded that antibiotic therapy does not completely eradicate the bacteria Staphylococcus aureus and Pseudomonas aeruginosa, which could be because of the prescription of antibiotics in developing countries where the use of antibiotics is not yet well controlled (Sulis et al., 2022). This has led to the evolution of bacteria to resist antibiotics even at high concentrations, which is why AgNPs hydrogel are an excellent therapeutic alternative.
Formulation and preparation of the hydrogel
In accordance with the Official Mexican Standard NOM-073-SSA1-2015, stability of drugs and medications, as well as herbal remedies, in section 7.5.1, the conditions are stipulated according to the type of stability study, whether accelerated or intermediate condition, or long-term with the objective of demonstrating the useful life of the new drug (Table 4).
Table 4
General study conditions for new medication.
These tests serve as quality control to ensure the effectiveness and safety of the medications. According to the results obtained, it is observed that our hydrogel maintains its pH, appearance, color and smell for 30 days. The hydrogel must have a pH of 7.0 and this pH must be maintained throughout its useful life since a lower or higher value can cause undesirable side effects such as irritation, pain, burning or itching since the purpose of the hydrogel is to provide a mechanical barrier, that is, it remains on the application surface, avoiding colonization of the wound by opportunistic microorganisms.
However, it is necessary to measure its viscosity, extensibility and extrusion for at least 6 months to evaluate its adhesion and extensibility on the skin, as well as determine its stability and expiration date. However, these tests go beyond the scope of this work.
Kirby-Bauer test
With Kirby-Bauer test we observe that after 48 hours there is antimicrobial effectiveness of both preparations against P. aeruginosa, however, a greater inhibition is observed by the AgNPs hydrogel that obtained an inhibition zone of up to 20 mm, with an average of 15 mm, against the sulfadiazine cream, whose resulting halo was 10 mm (Table 2), demonstrating that the antibacterial action of the AgNPs hydrogel is greater than that of the commercial cream (Figure 6).
During the analysis of the antimicrobial effectiveness test of the hydrogel and the commercial cream against Staphylococcus aureus, we observe that both products showed relatively low inhibition halos, however, it is notable that the hydrogel presented a slightly larger halo (13 mm) compared to cream (10 mm) (Figure 6 and Table 5).
Figure 6
Antibiogram result of the Kirby-Bauer test for microorganisms studied, A) S. aureus, and, B) P. aeruginosa both treatment with AgNPs hydrogel and commercial cream.
Source: Author’s elaboration.
Table 5
The average diameter of the haloes generated for Staphylococcus aureus and Pseudomonas aeruginosa.
| Inoculum | Average diameter (mm) | |
|---|---|---|
| AgNPs hydrogel | Commercial cream | |
| Staphylococcus aureus | 13.00 | 10.00 |
| Pseudomonas aeruginosa | 15.00 | 10.00 |
Although we mention that our results are not comparable with results reported with antibiotics, the largest inhibition zone observed for the hydrogel against Pseudomonas aeruginosa was 15 mm, which could be considered like the values reported as sensitive for antibiotics such as gentamicin and tobramycin (≥15 mm).
On the other hand, the inhibition of Staphylococcus aureus was considerably lower, similar only with antibiotics such as oxacillin (Tables 1-2).
The improved results of the AgNP hydrogel compared to the commercial cream may be because the silver nanoparticles integrate more efficiently into cells, inhibit microorganisms, and facilitate healing.
Conclusions
Silver nanoparticles were synthesized that showed a maximum lambda of 429 nm by UV-Vis spectrometry.
Transmission electron microscopy (TEM) revealed that the synthesized silver nanoparticles have a spherical morphology, with different sizes and some agglomeration.
A hydrogel was formulated with the silver nanoparticles obtained, which complies with the quality control of pH and stability after 30 days, according to Mexican regulations.
The hydrogel demonstrated higher antimicrobial effectiveness compared to commercial silver sulfadiazine cream at the same concentration, against Staphylococcus aureus and Pseudomonas aeruginosa.
Acknowledgments
Blanca Estela Chávez Sandoval was awarded a grant from the National System of Researchers (SNI-Conahcyt) for 2022-2024 CVU 249131.
To Dr. Gerardo Zúñiga Bermúdez for his invaluable support in the Laboratory of Biological Variation and Evolution of the ENCB-IPN.
To Dra. Aura Chávez Recio for her support in the clinic and comments.
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Notes
[1] This article was published in preprint mode, with the following DOI: https://doi.org/10.21203/rs.3.rs-4838660/v
Author contributions
Blanca Estela Chávez Sandoval: Conception and design of the article. Methodological development, writing of the original draft and review, and final editing of the text.
Alejandra Saraí Espinosa Vega and Grecia Nuñez Tahuilán: Data mining, analysis and interpretation.
María de Lourdes Moreno Ribera and María Isabel García-Ventura: Methodological development, data mining, analysis and interpretation.
José Abraham Balderas: Review and final editing of the text.
Conflict of interest The authors declare that there is no conflict of interest of any kind.