Bacterial biofilms play a key role in the pathogenesis of major oral diseases. Nanoparticles open new paths for drug delivery in complex structures such as biofilms. This study evaluated the antimicrobial effect of zein nanoparticles containing anacardic acid (AA) extracted from cashew shells of Anacardium occidentale on in vitro Streptococcus mutans biofilm formation and mature biofilms. The minimum inhibitory concentration (MIC), minimum bacterial concentration (MBC), and antibiofilm assays were performed. Streptococcus mutans UA159 biofilms were formed on saliva-coated hydroxyapatite disk for 5 days. To evaluate the preventive effect on biofilm formation, before contact with the inoculum, the disks were immersed once for 2 min in (1) hydroethanolic solution; (2) blank zein nanoparticles; (3) zein nanoparticles containing AA; and (4) 0.12% chlorhexidine gluconate. To determine the effect against mature biofilms, the disks containing 5-day preformed biofilms were further treated using the same procedure. The bacterial viability and dry weight were determined for both assays and used to compare the groups using ANOVA followed by Tukey’s test (p < 0.05). Both MIC and MBC for AA-loaded zein nanoparticles were 0.36 μg/mL. Groups 3 and 4 were very effective in inhibiting S. mutans biofilm formation, as no colony-forming units were detected. In contrast, for mature biofilms, no difference in bacterial viability (p = 0.28) or dry weight (p = 0.09) was found between the treatments. Therefore, the AA-based nanoformulation presented very high inhibitory and bactericidal activities against planktonic S. mutans, and the results indicate a strong antiplaque effect. However, the formulation showed no antimicrobial effect on the established biofilm.
Dental caries is a sugar-dependent biofilm disease, which occurs due to the progressive destruction of the mineral structure of dental tissues, resulting from a complex interaction of microorganisms, host factors, and diet. Without treatment, caries progresses and can affect the mineralized tissues of the teeth to the pulp [1, 2]. It is the most prevalent oral disease worldwide and is considered a major oral health problem, despite unceasing studies and efforts to find preventive measures . It is characterized by the appearance of delineated regions of demineralization in the dental enamel , which can coalesce into a more extensive lesion.
Mechanical removal through brushing and flossing  and the use of antiseptics and antibiotics  are means of preventing biofilm-dependent oral diseases. In dentistry, chlorhexidine has been studied to exhaustion and is currently considered the gold standard antiplaque agent. Nonetheless, it presents some undesirable side effects such as unpleasant taste and the ability to pigment dental structures as well as esthetic restorations . Therefore, the search for new substances that are efficient and do not have as many drawbacks is very desirable. In contrast, antibiotics may lead to the resistance of the oral microbiota, with a consequent reduction in efficacy .
Accordingly, phytochemicals have emerged as an important therapeutic option for the prevention and treatment of biofilm-dependent diseases, presenting several advantages, such as biological properties, low cost, and availability . Furthermore, the World Health Organization (WHO) reiterates that due to poverty and lack of access to medical care, approximately 80% of the world’s population in developing countries depend primarily on plants for their primary health, although there are a limited number of studies regarding their applicability in dentistry .
The cashew tree (Anacardium occidentale) is a tropical tree commonly found in the Brazilian Northeast region . Cashew nut liquid (CNL), obtained from cashew shells, contains ca. 80% anacardic acid (AA), 15% cardol, and a small amount of cardanol [11, 12]. This oleoresin has emerged as a promising commercial product in tropical countries, as it has several biological and non-biological applications, such as in food preservation, paint, cement, and gasoline stabilization . Moreover, AA has demonstrated several biological properties, such as antiinflammatory , antibacterial [15,16,17,18,19], antitumoral [20, 21], antifungal , and antioxidant . It has also been described as a gastro-protective enzyme inhibitor  and has been indicated as a potential agent for use in therapeutic nanomaterials .
The antimicrobial activity of AA has been related to its ability to permeate the lipid bilayer of cell membranes, causing its disruption . The higher the number of double bonds in the side chain, the higher its antibacterial effect . Although AA has already been evaluated against suspensions of S. mutans [16,17,18,19, 27], no studies have yet evaluated its effectiveness against cariogenic biofilms.
The most common anticaries agents (liquids and varnishes) are well accepted by patients, although their retention on the dental surface is poor, leading to limited efficacy . As such, nanometric materials such as nanoparticles with different compositions have been increasingly studied for use as carriers, improving not only their mechanical properties but also modified to achieve the desired size, morphology, and applicability [29, 30]. Nanoparticles from corn protein zein are biodegradable and have a relatively low cost . For instance, the encapsulation of AA provided its stabilization and enhanced its esthetic characteristics. Hence, this study aimed to evaluate the bactericidal and antibiofilm effects of zein nanoparticles containing AA extracted from cashew shells of A. occidentale against S. mutans biofilms in vitro.
Materials and methods
AA was obtained and purified from the oleoresin extracted by cold solvent from dried cashew shells of A. occidentale according to the method proposed by Trevisan et al. (2006)  and purity-checked using 1H (NMR).
The nanoparticles were prepared by nanoprecipitation  of zein at 0.07% (w/v). Nanoprecipitation is an economical, green, low-energy, and scale-up reproducible method [30, 33], which allows small and homogeneous droplets to be obtained by increasing the surface area through disruption in the semipolar solvent containing the polymer and the aqueous interphase . AA was incorporated at 9.4 μg/mL, which was the maximum concentration attained. Briefly, zein was dissolved in 70% (v/v) ethanol, which was thereafter diluted with ultra-pure water to promote nanoparticle formation. Separately, AA was solubilized in 96% ethanol and the preformed nanoparticles were added dropwise under constant stirring until complete homogenization , resulting in AA-loaded zein nanoparticles (ZAa). Blank nanoparticles were prepared in the same manner, except for the presence of the drug (ZB). The size, polydispersity index (pdI), zeta potential (ζ), and pH for the nanoparticles were ZAa (381.6 nm, 0.067, − 15.9 mV, and 4.95) and ZB (376.5 ± 3.951, 0.137, + 6.56 mV, and 5.67).
These formulations were previously tested in our group and it demonstrated good biopharmaceutical properties, such as long-term stability (90 days), as a monodisperse system under storage conditions (room temperature and refrigerator), physiological pH, esthetic white color/lack of coloring on an enamel surface, and easy handling, making it feasible for clinical use .
Determination of the minimum inhibitory concentration and minimum bactericidal concentration
Preparation of the inoculum
The microorganism used in this study was S. mutans UA159. The inoculum was prepared in tryptone soy broth (TSB) (Difco Detroit, MI, USA) containing 1% glucose. The suspension was incubated at 37 °C in a 5% CO2 partial atmosphere (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 18 h and then diluted in sterile broth to obtain a suspension with 106 colony-forming units (CFU)/mL .
The following treatments were used in the assays:
Test group: culture broth + inoculum + ZAa ([AA] = 93–0.022 μg/mL)
Negative control group 1: culture broth + inoculum + ZB
Negative control group 2: culture broth + inoculum + sterile saline solution 0.89%
Turbidity control: culture broth + ZAa ([AA] = 93–0.022 μg/mL); inoculum-free group
Positive control group: culture broth + inoculum + chlorhexidine gluconate 0.12% (Colgate-Palmolive®, São Bernardo do Campo, SP, Brazil)
Growth group: culture broth + inoculum
Sterility control group: culture broth.
For the experimental groups, 100 μL of culture broth + 10 μL of the inoculum + 100 μL of the solution tested were distributed in 96-well cell culture plates. The plates were incubated for 24 h in a bacteriological oven with 5% CO2 at 37 °C. After this period, the microbial growth was inspected visually and the absorbance was measured at λ = 490 nm using a spectrophotometer (Bio-Tek, Winooski, Vermont, USA). Determination of the minimum inhibitory concentration (MIC) was considered the lowest concentration capable of inhibiting S. mutans growth, ascertained by naked eye inspection (absence of visible turbidity) and absorbance reading.
To obtain the minimum bactericidal concentration (MBC), 25 μL of the content of the MIC well and the two dilutions immediately above were collected and seeded using a Drigalski loop over brain heart infusion agar plates (BHI) (Difco, Detroit, MI, USA). The assay was performed in triplicate. After incubation for 24 h at 37 °C and 5% CO2, the colonies were counted. The concentration that determined a microbial growth of less than 0.1% of the initial inoculum was considered the MBC, the lowest concentration of the drug tested (AA-loaded zein nanoparticles) with a lethal action on S. mutans .
S. mutans biofilm assays
Preparation of the inoculum
S. mutans (UA 159) was also used as an inoculum to promote biofilm formation. For this purpose, the bacteria were first seeded in blood agar for 48 h at 37 °C in a 5% CO2 atmosphere. Subsequently, 5–10 colonies were transferred to TSB (Difco, Detroit, MI, USA) and yeast extract media supplemented with 1% glucose, and thereafter incubated for 18 h in a 5% CO2 atmosphere at 37 °C.
Acquired salivary pellicle formation
Human saliva was collected from a healthy unidentified donor by stimulation with flexible film (Parafilm M, Pechiney Safety Laboratory Products and Equipment, USA). Adsorption buffer (50 mM KCl, 1.0 mM KPO4, 1.0 mM CaCl2, 0.1 mM MgCl2, pH = 6.5) was added to the saliva together with phenylmethylsulfonyl fluoride (PMSF) in the proportion: 1% PMSF + 48.5% saliva + 48.5% adsorption buffer. This solution was clarified by centrifugation at 8500 rpm for 10 min at 4 °C and filtered with a 0.22-μm pore size cellulose acetate filter (Stericup, Millipore, USA) . After these procedures, the filtered solution was distributed in 12 or 24-well plates (2 mL per well). Subsequently, the HA disks (Clarkson Chromatography Products Inc., South Williamsport, PA, USA) (HA) were immersed in the solutions to stimulate the formation of the acquired pellicle. This set (plate + filtered contents + HA disks) was placed for 1 h in an orbital shaker with a 5% CO2 atmosphere at 37 °C.
Biofilm formation and treatment of the disks
To evaluate the effect of the treatments on the inhibition of biofilm formation, after 1 h of contact with the salivary pellicle, the HA disks were transferred to the wells containing the test and control solutions for 2 min (three disks per group in each experiment repeated 3 times):
Group 1: vehicle (35% hydroethanolic solution);
Group 2: blank zein nanoparticles;
Group 3: anacardic acid–loaded zein nanoparticles ([AA] = 9.375 μg/mL);
Group 4: chlorhexidine gluconate 0.12%
Subsequently, the disks were transferred to a new 24-well plate containing 2 mL of TSB sterile broth supplemented with leaven extract containing 1% sucrose (previously inoculated with the bacterial culture) obtained from 100 μL of bacterial inoculum and 45 mL of TSB + yeast extract media with 1% sucrose. They were incubated for 24 h to allow biofilm formation in a 5% CO2 atmosphere at 37 °C. For five consecutive days, the disks were transferred to a new plate containing sterile broth to avoid saturation.
For the mature biofilm analysis, the saliva-coated HA disks were initially incubated with TSB + yeast extract media with 1% sucrose containing S. mutans inoculum in 24-well plates to form biofilms during 120 h, with the culture medium changed every 24 h . After 5 days, the HA disks were treated using the same protocol and treatments described above, except being immersed for 2 min (12 disks for each occasion and three disks per group).
Finally, for the analysis of the effect on the inhibition of S. mutans biofilms, the disks were immersed in the formulations in a single moment (2 min), immediately after salivary pellicle formation. For the analysis of the effect on mature S. mutans biofilms, the disks were immersed in the formulations in a single moment (2 min), only on the 5th day of biofilm formation.
The biofilm was analyzed using bacterial viability and dry weight determination. The biofilm present on the surfaces of the HA disks was collected by scraping on the 5th day and dispersed in 1 mL of saline solution. This dispersion was sonicated in sterile polypropylene microtubes using 3 × 15 s of 7 W pulses (Fisher Scientific, Sonic Dismembrator Model 100, USA) to disperse the cells properly.
For the analysis of bacterial viability, the suspensions containing the homogenized biofilm were diluted from 10−1–10−7. Subsequently, an aliquot of each dilution was seeded in BHI agar and incubated in 5% CO2 at 37 °C for 48 h.
To determine the dry weight, 600 μL of cold ethanol (− 20 °C) was added to 200 μL of the biofilm suspension. The resulting precipitate was centrifuged (10,000×g for 10 min at 4 °C). The supernatant was discarded, and the precipitate was washed with cold ethanol and then dried until constant weight in a desiccator containing silica . Subsequently, the ratio of colony-forming units per mg of biofilm (CFU/mg) of each treatment replicate was individually calculated.
The results of the biofilm assays were transformed to log10, and the mean and standard deviation of each group were calculated. Data (bacterial viability and dry weight) were normality checked with the Kolmogorov-Smirnov test and submitted to ANOVA followed by Tukey’s post-test. The significance level was set at 5% (p < 0.05). The statistical program used was GraphPad Prism 5.0.
Minimum inhibitory concentration and minimum bactericidal concentration values
The MIC and MBC values are listed in Table 1. It is noteworthy that the lethal concentration (MBC) was almost 500× smaller for the test group (AA nanoparticles) than for the negative control group 1 (blank zein nanoparticles) against S. mutans.
The ZAa tested in group 3 were very effective in inhibiting the formation of S. mutans biofilm over HA disks, as no colonies were detected after 5 days of stimulated biofilm formation, similar to what was found in group 4, treated with 0.12% chlorhexidine gluconate. In contrast, it was evident the biofilm formation in groups 1 (6.44 ± 0.15) and 2 (6.53 ± 0.23) did not present any significant difference regarding the bacterial viability between them (p = 0.8124) (Fig. 1). Regarding the dry weight results in the analysis of biofilm formation, no significant difference was found between groups 3 and 4 (p = 0.9996) and groups 1 and 2 (p = 0.9999). In addition, the minor biofilm weight formed in group 3 was significantly lower than that in groups 1 (p = 0.0015) and 2 (p = 0.0013) (Fig. 2).
The potential antiplaque effect of the AA-based nanoformulation, the most active constituent of CNL, against planktonic and S. mutans biofilms was observed. However, no antimicrobial effect over the pre-established biofilm was found.
This study is the first to verify the antimicrobial effect of this nanotechnology-based formulation (zein nanoparticles containing anacardic acid) against S. mutans biofilms. The choice of the biofilm model to complement the planktonic suspension was because biofilms are extremely complex and structured communities of microorganisms adhered to a solid surface and are considerably more resistant. They agglomerate within a three-dimensional matrix of extracellular polysaccharides, lipids, proteins, and nucleic acids. They improve and promote adhesion and coadjustment of microorganisms, act as energy reserves, affect the diffusion of substances into and out of the biofilm (including drugs), and aid in concentrating metal ions and other physiological nutrients in this microenvironment [5, 38,39,40]. The choice of S. mutans is based on its association with the onset and development of carious lesions, especially those that are more aggressive, although it is not always the most abundant strain found [4, 41, 42].
Some authors have already tested the activity of AA against suspensions of S. mutans using different in vitro methodologies [15,16,17,18,19, 43]. The growth inhibitory activity of the cashew tree trunk and bark (A. occidentale) extracts against cariogenic bacteria (Streptococcus mitis, S. mutans, S. oralis, S. salivarius, S. sanguinis, and S. sobrinus) was demonstrated against all microorganisms tested, whereas S. mutans and S. mitis were the most sensitive . AA obtained from Amphipterygium adstringens also demonstrated antimicrobial activity against S. mutans and Porphyromonas gingivalis .
The activities of CNL and AA on biofilms of other microorganisms have also been observed in other studies. The antimicrobial activity of A. occidentale leaf extract against Enterococcus faecalis, Staphylococcus aureus, S. mutans, Escherichia coli, and Candida albicans, as well as its possible action on biofilm suppression, was found in all tested microorganisms. Furthermore, both chlorhexidine and Anacardium extract significantly reduced biofilm development. The authors suggested that such effects can be attributed to the phytochemicals found in the extract . Silicone catheters impregnated with different AA concentrations (0.002–0.25%) showed no colonization/formation of S. aureus biofilms over their surface .
It was suggested by some authors that the antimicrobial effect of AA against S. mutans might not be potent enough to have practical applications, and further studies should be conducted to increase this antimicrobial effect . The results of the MIC and MBC were the same for both, 0.36 μg/mL, which classify the AA nanoparticles in a group of strong antimicrobial activity, according to the classification proposed by Aligiannis et al. (2001)  (strong MIC < 500 μg/mL, moderate MIC between 600 and 1500 μg/mL, and low MIC > 1600 μg/mL).
Antimicrobial optimization of AA, when incorporated into the zein nanoparticles, was observed when comparing the MICs from this study with the MICs between 0.78 and 6.25 μg/mL found in other studies [15,16,17]. Anand et al. (2015)  found a MIC of 78.12 μg/mL and MBC of 156.25 μg/mL, which were 200–400 times higher than those obtained in the present study.
The use of nanotechnology in dentistry has been receiving increasing attention in recent years, since it has a wide range of applications, including pharmaceuticals. In this study, nanoencapsulated AA was demonstrated to be an effective agent against the formation of dental biofilms. The incorporation of nanomaterials can modify the optical, chemical, electrical, and mechanical properties of substances . Regarding AA, a caustic, brown, viscous, and fat-soluble phytochemical with little aggregated value became white, fluid, water-dispersed, and esthetically appropriate for use in the oral cavity. The small diameter of nanoparticles increases not only its mechanical properties but also its antimicrobial action due to its high surface-volume ratio, causing damage to the cell membranes. Its great advantage and novelty over conventional antimicrobials is the mechanism it uses to deliver the drug [31, 46]. In this study, the nanoencapsulated AA at a very low dose prevented S. mutans biofilm formation for 5 days after a single 2-min application.
Nonetheless, neither zein nanoparticles loaded with AA (9.375 μg/mL) nor 0.12% chlorhexidine gluconate had antimicrobial effects on a 5-day preformed biofilm. First-line targets of CHX (at lower concentrations) are cytoplasmic membrane integrity as well as the function of membrane-bound enzymes, while secondary effects (at higher concentrations) are cytoplasmic leakage, and ultimately, coagulation and precipitation of intracellular constituents such as proteins and nucleic acids . Previous studies have evaluated the antimicrobial activity of chlorhexidine in established biofilms. S. mutans viability (in a 2-day biofilm) was reduced by 4–5 logs with chlorhexidine treatment (at 50 μg/mL) for 1 h . Re et al. 2019  showed that chlorhexidine was able to reduce a 4-day established S. mutans biofilm, although the concentration and chlorhexidine-biofilm exposure time were not reported.
A. occidentale has great potential to inhibit bacterial proliferation. This activity was associated with a high concentration of flavonoids, tannins, and alkaloids present in the extracts [50, 51]. Although a systematic review reported that several studies have shown the potential of A. occidentale to prevent oral bacterial biofilm proliferation , there is only one study that examined the activity of AA over a preformed biofilm. Preformed biofilms were incubated with increasing concentrations of AAs. Fluorescence microscopic images demonstrated an increased S. aureus biofilm dispersion with increased concentrations of AA. The biofilm dispersion was 40, 76, 80, and 99.96% when the preformed biofilms were incubated with 0.002, 0.01, 0.05, and 0.25% AA solutions, respectively, . It is noteworthy that the composition of extracellular polysaccharide (EPS) in the biofilms of S. aureus and S. mutans is different .
The limited activity over the 5-day mature biofilm may be related to the polysaccharide presence. The biofilm matrix is a complex mixture of EPS, extracellular DNA, proteins, and lipoteichoic acid (Gram-positive), which is responsible for the strength of the matrix . The production of the matrix has long been known to facilitate the survival of cells. Within the matrix, EPS plays a critical role in enmeshing microbial cells and providing a three-dimensional diffusion-limiting, protection against antimicrobials, and the microbial cells can become highly acidogenic or aciduric . Therefore, none of the treatments managed to diffuse into this microstructure at an adequate concentration to act against the established bacteria.
A limitation of the formulation is its high concentration of ethanol (35%) used to disperse the nanoparticles, although it would not limit its clinical use, especially under clinical surveillance in patients with limited hygiene and more propensity to develop cariogenic biofilms. Nevertheless, further preclinical studies are needed to evaluate the oral tissue toxicity of ZAa before promoting this formulation as a clinical alternative to prevent cariogenic oral biofilms.
The results showed strong bactericidal activity and the potential antiplaque effect of zein nanoparticles containing anacardic acid. This nanoformulation showed a long-lasting inhibitory activity against biofilm formation in a Streptococcus mutans biofilm model. Further studies should be conducted to analyze the microbiological effect of this formulation at different concentrations and/or in combination with other substances as well as testing against multispecies biofilms to consider it a novel antiplaque agent for dental use.
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The authors thank the Federal University of Ceará (Fortaleza, Brazil) for the infrastructure used in this research. Sousa, FFO thank FAPEAP/CNPq for the financial support.
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Lima, R.A., de Souza, S.L.X., Lima, L.A. et al. Antimicrobial effect of anacardic acid–loaded zein nanoparticles loaded on Streptococcus mutans biofilms. Braz J Microbiol (2020). https://doi.org/10.1007/s42770-020-00320-2
- Anacardium occidentale
- Streptococcus mutans