Nano silver used in alcohol-free mouthwash

Researchers have developed an effective and non-irritating mouthwash that is alcohol-free and has a low concentration of silver nanoparticles (SNPs) to be used to prevent oral cavity infections in cancer patients. immunodeficiency. We investigated the antibacterial effects of silver nanoparticles (SNPs) in the range (50–0.024 µg/ml) and 3% ethanol (30,000 µg/ml) in mouthwash. The antibacterial effects of the two treatments were studied by performing tests on microorganisms such as Streptococcus mutans, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa , Candida albicans and measuring the MIC and MBC (MFC) values. of SNPs for the microorganisms mentioned. These values ​​for SNPs range from (0.78–3.12) and (1.56–12.5 µg/ml, respectively). The results showed that nanosilver in the MIC and lower concentrations killed all the microorganisms used. No difference was found between the antibacterial effects of the SNP-containing ethanol-free mouthwash and the SNP- and ethanol-containing mouthwash (30,000 µg/ml). SNP has high antibacterial activity at low concentrations and it can be a good substitute for ethanol (30,000 µg/ml) because ethanol is also irritating, especially for sensitive or inflamed mucous membranes.

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In immunocompromised patients, the oral cavity is a common habitat for several multidrug-resistant bacteria and fungi that can cause infection. In these patients, oral candidiasis is very common [ ]. Over the past few decades, the number of immunocompromised patients has increased, partly due to the increase in the number of bone marrow and solid organ transplants, the increasing number of patients requiring care. severity, and the use of chemotherapy and radiation therapy [ ]. In addition, candida stomatitis is also a complication noted in elderly denture users, especially in the absence of oral hygiene. [ ].

Epstein et al found that oropharyngeal colonization of Candida species is common in hematopoietic cell transplant recipients, despite topical and systemic antifungal prophylaxis [ 15 ] . The prevalence of fungal microorganisms such as Candida species causing blood-borne infections in hospitals in the United States or the United Kingdom is high. Although Candida species are part of the common oral flora in 25–50% of healthy individuals, they are often referred to as local asymptomatic [ ].

Within 5 years after seroconversion, up to 26% of HIV-positive patients develop oral candidiasis, which is also seen in 12–100% of cancer patients undergoing chemotherapy, according to an analysis published from 15 studies, in 76% of patients receiving bone marrow treatment. transplants and up to 77% of asthmatics are carefully monitored using inhaled corticosteroids [ ]. Silver and its derivatives are the oldest antibacterial agents in traditional medicine [ 19]. Ag-containing compounds (silver ions) are not suitable as cosmetic preservatives because they gradually precipitate in cosmetic and cosmetic products. This precipitation reduces the antibacterial effect of silver. Ag nanoparticles are stable in solution and their antibacterial properties can be maintained for a long time and can therefore be used as a preservative in cosmetic and cosmetic products. [  ].Nano silver releases positively charged silver ions. These ions can bind to DNA and proteins, because these molecules contain negatively charged compounds, such as phosphorus and sulfur. Furthermore, Ag ions can adhere to the surface of microorganisms causing disruption in cell wall integrity. [ ]. In this study, the concentration of nanosilver was lower than that of ethanol ..

The aim of the research is to develop an effective antibacterial alcohol-free mouthwash containing SNPs that are non-irritating and will be useful for the oral care of immunocompromised patients, such as cancer patients, HIV patients, etc. We tested the solution against  Candida albicans , a common cause of fungal infections in cancer patients, as well as against  Pseudomonas aeruginosa  and  Staphylococcus aureus , all of which are common sources of infection in immunocompromised patients. Translate. In addition, we used this solution against the bacteria Streptococcus mutans, which causes tooth decay.

2. Materials and methods

2.1 Nano silver

SNP colloidal solution was obtained from Nano Nasb Pars Company (Tehran, Iran). The nano silver concentration was 4,000 µg/ml. Transmission electron microscopy (TEM) was used to determine the shape and size of the SNP.

2.2 Preparation of microorganisms

In this study,  Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027 were used as Gram-negative bacteria, Staphylococcus aureus ATCC 6538, Streptococcus mutans 25175 as Gram-positive bacteria and  Candida albicans ATCC 190231 as yeast. Bacterial strains were inoculated on the surface of TSA and  Candida albicans  cultured on sabouraud dextrose agar (SDA) and then incubated at 37 °C (degrees C.) for 24 h. NaCl solution (0.9%) was used to prepare the microbial suspension. During this suspension, the final microbial density was 10 8 CFU/mL. It is used as an inoculant[ ]. The inoculum density for each strain was confirmed by bacterial enumeration by serial dilution and colony counting as well as the optical density (OD) of the microorganism suspension adjusted by spectrophotometer to zero. ,1 at 600 nm.

2.3 Prepare mouthwash solution

In this study, mouthwash was obtained from Tolid Daru Company (Tehran, Iran). We have prepared an alcohol-free mouthwash. It is prepared from 0.6% propylene glycol, 0.2% sodium fluoride, 0.03% menthol and 0.0004% sodium saccharin. We used this mouthwash as a control (To compare the antibacterial effects of ethanol with nano silver, we removed 0.1% sodium benzoate from the guide.). Instructions and materials for the preparation of this mouthwash were also obtained from Tolid Daru Company. The ethanol concentration in the initial mouthwash was 30,000 µg/ml (3%) and we compared this concentration with the SNP.

2.4 Measure minimum inhibitory and bactericidal (fungicidal) concentrations (MIC and MBC, MFC)

MICs and MBCs (MFCs for yeast) were determined using the tube macrodilution method, whereby 13 tubes contained 1 ml broth with serial dilutions of SNPs (within the concentration range of 50–0.19 µg/ml) were inoculated with the test strains (final cell density was 10 6 CFU/mL) and incubated at 37 °C for 24 h. The lowest concentration of silver nanoparticles (SNPs), which exhibits growth inhibition (visible to the naked eye) is considered the minimum inhibitory concentration. The minimum bactericidal (fungicidal) concentration recorded was the lowest concentration of nano silver showing no growth on the agar plate after in situ inoculation and incubation for 24 h. Trials were performed with the appropriate controls (no-inoculation and SNP-free media) [ 38]. In this study, an initial mouthwash concentration of ethanol (30,000 µg/ml) was used.

2.5 Study on antibacterial properties of silver nanoparticles and ethanol

Challenge test [  ], [  ] was used to compare the antibacterial effects of nano silver and ethanol. In this method, we provide three different mouthwash samples such as a non-antibacterial mouthwash and a mouthwash containing one of the treatments (SNP at MIC and two lower concentrations or ethanol). 30,000 µg/ml) for each microorganism tested. 1 ml of microbial suspension containing 10 8 Then, CFU microorganisms were inoculated with 100 ml of each sample, 1 ml of each sample was removed at defined intervals (0, 2, 5 and 10 min) and were Transfer to TSA plate after appropriate dilution (clear dilution is required to remove residual SNP). These plates were incubated at 37 °C for 24 h. The review is carried out in two issues. Finally the number of colonies on the plates was counted.

3. Result

3.1 Features of SNP

The shape and size of the SNPs were determined by transmission electron microscopy. The TEM image has been illustrated in Figure 1. This figure shows the size of the SNPs ranges from 10–40 nm, they are also spherical.

Microscopic image of silver nanoparticles in transmission electron microscopy
Microscopic image of silver nanoparticles in transmission electron microscopy


3.2 Minimum inhibitory and bactericidal (fungicidal) concentration

The MIC and MBC (MFC) values of the SNPs for microorganisms are summarized in Table 1. It is clear from Table 1 that nanosilver has shown high antibacterial activity against all the microorganisms used. The MIC and MBC values of the SNPs for the two Gram-positive bacteria ( S. mutans  and  S. aureus ) were different. The most susceptible and resistant microorganisms are  C. albicans  and  S. aureus .

Bảng 1 Giá trị MIC và MBC (µg / ml) của nano bạc †
Table 1
MIC and MBC (µg/ml) values of nanosilver †

3.3 Study on preservation properties of nano silver and ethanol

The results showed that mouthwash containing SNP at MIC and two lower concentrations was completely effective within 0–5 min but mouthwash containing SNP 0.4 µg/ml completely eradicated  S. aureus  in 10 minutes. In this study, the antibacterial effects of an ethanol-free mouthwash containing SNPs (various concentrations) and a mouthwash containing two treatments (SNPs with different concentrations and 30,000 g/ml ethanol). ) was the same for all microorganisms (data not available). SNPs (different concentrations) kill all microorganisms faster than ethanol. The results on the antibacterial effects of SNPs (different concentrations) and ethanol (30,000 µg/ml) over a period of time (0–10 min), are presented in Table 2, Table 3, Table 4, Table 5, Table 6.

Bảng 2. So sánh hiệu lực kháng khuẩn của SNP † (MIC và hai nồng độ thấp hơn) và Ethanol (30.000 µg / ml) trong khoảng thời gian (0–10 phút) đối với E. coli
Table 2. Comparison of antibacterial efficacy of SNP † (MIC and two lower concentrations) and Ethanol (30,000 µg/ml) over time (0–10 min) against E. coli


Bảng 3. Khả năng SNP † (MIC và hai nồng độ thấp hơn) và ethanol (30.000 µg / ml) trong khoảng thời gian (0–10 phút) đối với P. aeruginosa
Table 3. SNP potency † (MIC and two lower concentrations) and ethanol (30,000 µg/ml) over time (0–10 min) against P. aeruginosa


Bảng 4 So sánh hiệu lực kháng khuẩn của SNP † (MIC và hai nồng độ thấp hơn) và ethanol (30.000 µg / ml) trong khoảng thời gian (0–10 phút) đối với S. aureus
Table 4
Comparison of antibacterial potency of SNP † (MIC and two lower concentrations) and ethanol (30,000 µg/ml) over a period of time (0–10 min) against S. aureus


Bảng 5 So sánh hiệu lực kháng khuẩn của nano bạc † (MIC và hai nồng độ thấp hơn) và ethanol (30.000 µg / ml) trong khoảng thời gian (0-10 phút) đối với S. mutans
Table 5
Comparison of antibacterial efficacy of nanosilver † (MIC and two lower concentrations) and ethanol (30,000 µg/ml) over a period of time (0-10 min) against S. mutans


Bảng 6 So sánh hiệu lực kháng khuẩn của nano bạc † (MIC và hai nồng độ thấp hơn) và Ethanol (30.000 µg / ml) trong khoảng thời gian (0–10 phút) đối với C. albicans
Table 6
Comparison of antibacterial efficacy of nanosilver † (MIC and two lower concentrations) and Ethanol (30,000 µg/ml) over a period of time (0–10 min) against C. albicans

4. Discussion

Numerous investigative studies have documented the inhibition of plaque growth and reduction of bacterial acid formation using antibacterial agents added to mouthwash or toothpaste preparations [ 32 ]. According to their chemical characteristics, commercially available mouthwashes contain cationic, anionic and non-ionic active ingredients which, to a greater or lesser extent, alter bacterial membrane function. Among cationic agents, chlorhexidine and some divalent metal ions such as Cu +2 , Zn +2 , and Sn +2  are the most widely used [ 9 ]. It has been shown that  Streptococcus mutans  cannot obtain the nutrients necessary for its survival and reproduction [35 ]. It is possible that ferric iron can alter the function of the cell membrane and also the activity of enzymes in the cell [ 16 ]. Modification of various zinc salts and its derivatives with a novel formulation is useful for controlling oral plaque and gingival bleeding [ 33 ]. Dobl and Nossek in 1990 [ 10 ] showed that 0.2% and 0.4% zinc chloride mouthwash had antibacterial activity against dental plaque, especially Streptococcus microflora [ 32]. To investigate the antibacterial activity of the SNP solution against microorganisms, we measure the MIC and MBC (MFC), then a challenge test was performed to compare the antibacterial effects of 30,000 µg/ml ethanol and nano silver (various concentrations). Eldridge et al. 1998 [ 14] found no difference between commercial alcohol-based 0.12% chlorhexidine and alcohol-free 0.12% chlorhexidine through both in vitro and in vivo studies. The commercially available mouthwash tested in this study contained 30,000 µg/ml alcohol. Mouthwash containing alcohol, is contraindicated in patients particularly with mucositis and other immunodeficiency [ 36 ]. Winn and colleagues found a relationship between oral cancer and alcoholic mouthwashes. Oral cancer risk increased 40-60%, after adjusting for other risk factors, such as tobacco and alcohol intake [ 39 ]. Therefore, it is important to avoid using alcohol-based mouthwash. Gram-negative bacteria, such as  Klebsiella pneumoniae  and Pseudomonas aeruginosa , and Gram-positive bacteria, such as  Methicillin-resistant Staphylococcus aureus  commonly residing in the oral cavity of hospitalized patients [ 25 ], have emerged as the cause of inflammation hospital lung. This has prompted the search for preventive and therapeutic measures to minimize oral and respiratory penetration by simple use of broad-spectrum antiseptic mouthwash prior to operation or prior to placement. endotracheal [ 2 ].
In this study, nanosilver killed both Gram-negative bacteria investigated faster than S. aureus despite the fact that the MIC of nanosilver for  S. aureus  was lower than that of  E. coli , with a wide range between MIC and MBC of SNP for  S. aureus ( MIC = 1.56, MBC = 12.5). These results are similar to those reported by Jain et al. [ 21 ], they reported that Gram-negative bacteria such as  E. coli  and  P. aeruginosa  were killed at the MIC (6.25 µg/ml) SNP faster than Gram-positive bacteria such as  S. aureus  and  Bacillus subtilis at the MIC (12 (12). ,5 µg/ml) of the SNP also failed to determine the MBC value because the SNP was found to be bacteriostatic even at the highest concentration available for testing, i.e. 50 µg/ml. Although the MICs of nanosilver-stable PVP for  S. aureus  and  E. coli  are 5 µg/ml and 10 µg/ml, respectively; all surviving cells of  E.coli  were inhibited by 10 and 20 µg/ml SNP solutions faster than  S. aureus [ 8 ]. Previous studies have shown that  S. aureus  is less sensitive to SNPs than  E. coli  and  P. aeruginosa [ 31]. It was determined that the antibacterial effect of SNPs also depends on the type of microorganism strain. Different strains of  E. coli , Bacillus subtilis  and  S. aureus  showed differences in SNP-established antimicrobial effects [ 34 ]. Others also reported similar results [ 21 ]. They show that the MIC value and time required to achieve the SNP inhibitory effect for  S. aureus  are different from that of  S. epidermis . Our results corroborate these findings because the MIC and MBC values ​​of SNPs for both Gram-positive bacteria ( S. mutans  and  S. aureus ) are different. S. mutans  are killed faster than S. aureus with a mouthwash containing SNPs with MICs and two lower concentrations. This is an important result, because very low concentrations of SNPs can be used to prevent tooth decay in mouthwash but it requires more experiments. Hernandez-Sierra showed that the MIC and MBC for silver nanoparticles (Average size of nanoparticles is 25 nm.) are 4.86 µg/ml and 6.25 µg/ml. They reported that silver nanoparticles inhibited  S. mutans  at lower concentrations than gold or zinc nanoparticles [ 18 ]. In previous studies, copper and zinc were used as an antibacterial factor in mouthwash [ 6 ]. Burguera-Pascu et al. used zinc salt as washing solution and they reported high efficiency of Zn salt on  S. mutans [6 ].
Mouthwashes containing very low zinc compounds have shown a high antibacterial effect against strains of Streptococcus in the mouth [10 ]. We used a very effective SNP colloidal solution for  C. albicans . The MIC and MBC values ​​of the SNPs for  C. allon bi  were 0.78 and 1.56 µg/ml, respectively. Test-tube studies showed that the tested mouthwash was alcohol-free and contained a reduced concentration of SNPs that were shown to be effective in inhibiting bacterial and candida activity. These values ​​are comparable to those obtained by Kokura et al. [ 22 ]. They reported that the SNP 1 µg/ml showed sufficient antibacterial efficacy against mixed bacteria ( S. aureus , E. coli , P. aeroginusa ) and mixed fungi ( Candida albicans , Aspergillus niger , Penicillium citrium , Aureobasidium) pullulans) in addition, previous studies showed that hybrid silver nanoparticles (3–7 nm in size) loaded on SiO2 nanoparticles inhibited a wide range of standard fungi at a concentration of 1 g/ml [ 30 ]. It has been established that the antibacterial effect of nanosilver depends on the size of the particles. The 25 nm SNP has a lower MIC than the 25 nm SNP. The MIC of this SNP (25 nm) is 1.69–13.5 µg/ml [29 ]. The MIC results obtained from the 25 nm particles are comparable with the results obtained in the present study, when using the SNP suspension containing 10–40 nm particles, the MIC and MBC values ​​obtained in the study Ours is also close to the value reported by Jain et al. [ 21]. They used a suspension of SNPs containing 7–20 nm particles that had MICs between 6.25 and 12.5 µg/ml. In another study, the 24 nm SNP had an MIC between 1.2 and 1.7 µg/mL against clinical isolates such as  E. coli ,  S. aureus  and  P. aeroginusa [ 24 ].
In conclusion, our study shows that SNPs have high antibacterial properties against  C. albicans and other common bacteria and the new mouthwash (SNP-containing and alcohol-free) can be used as a water Convenient alternative mouthwash for immunocompromised cancer patients and preoperative patients at high risk of nosocomial pneumonia. Low nanosilver concentration can minimize unpleasant taste and also reduce toxicity. Moreover, it does not contain alcohol, so it does not cause irritation and is comfortable to use for patients with sensitive or inflamed mucous membranes. However, the biological and environmental effects of the SNP must be further investigated before the SNP can be considered for addition to a commercially available antibacterial mouthwash.

Reference: Silver nanoparticles as active ingredient used for alcohol-free mouthwash