Effects of a Novel Magnetic Nanomaterial on Oral Biofilms

Abstract

Dental caries is one of the most common oral chronic infectious diseases, and novel antibacterial materials must be developed to control plaque and inhibit formation of dental caries. Combining magnetic nanomaterials with antibacterial agents to decrease the formation of bacterial biofilm has been a hot topic in the biomedical field. The present study developed a novel magnetic nanomaterial chemically combined with dimethylaminododecyl methacrylate (DMADDM) and initially investigated its inhibiting effects on biofilms by using traditional caries-related bacteria and saliva flora models. The novel magnetic nanomaterials successfully loaded DMADDM according to thermogravimetric analysis, Fourier transform infrared spectroscopy, x-ray diffraction, vibrating sample magnetometry, scanning electron microscopy, and transmission electron microscopy results. Further, the novel nanoparticle Fe₃O₄@SiO₂@DMADDM with concentration of 8 mg/mL could effectively reduce Streptococcus mutans biofilm and decrease the production of lactic acid. The 16S rDNA sequencing revealed that Fe₃O₄@SiO₂@DMADDM could depress the proportion of caries-related bacteria in saliva-derived biofilm, such as Streptococcus, Veillonella, and Neisseria. Therefore, Fe₃O₄@SiO₂@DMADDM is a novel effective antibacterial magnetic nanomaterial and has clinical potential in plaque control and dental caries prevention.

Introduction

Dental caries is one of the most common oral chronic infectious diseases, and it is the main cause of oral pain and dental hard tissue defects.¹ According to the Fourth National Oral Health Survey in China, the prevalence of dental caries is still high. For example, the caries prevalence rate in 5 year old children was 71.9%, 89.0% for 35-44 year group, 95.6% for 55-64 olds, and 98.0% for 65-74 year olds. Dental caries affects all ages throughout the life cycle and is the most common noncommunicable disease in the world. It causes a significant individual, social, and economic burden globally.² Therefore, more and more attention has been paid to the prevention and management of dental caries. Dental caries develops through a complex interaction over time between acid-producing bacteria and fermentable carbohydrate, along with many host factors including teeth and saliva. Bacteria are known to be the main source of this chronic oral disease.³ Dental caries is considered to begin with the disruption of oral microecological homeostasis, which might be associated with the existence of the “core microbiome” in dental caries.⁴ Streptococcus mutans is one of the core microbiotas in the development of dental caries.⁵ Thus, novel antibacterial materials must be developed to combat dental caries.

Dimethylaminododecyl methacrylate (DMADDM) has been widely investigated as a favourable antibacterial agent.⁶ Previous studies have shown that dental materials containing DMADDM can inhibit oral infectious diseases, such as dental caries.⁷ However, researchers are still trying to develop novel strategies to enhance the anticaries effect of antibacterial agents including DMADDM. Some studies have combined antimicrobials and remineralised agents, such as nanoparticles of amorphous calcium phosphate, nanoparticles of calcium phosphate, and poly (amidoamine) dendrimer,⁸ whilst other studies have combined magnetic nanomaterials with antibacterial agents to decrease planktonic bacteria and biofilms.9, 10

Magnetic nanomaterials are a class of magnetic-related materials arranged in the order of nanometers The most commonly used magnetic nanomaterials are metal alloys such as iron, cobalt, titanium, nickel and iron oxide, and ferrioxide,¹¹ and iron oxide nanoparticles (usually Fe₃O₄ and Fe₂O₃ nanoparticles) have become a hot topic in the biomedical field because of their low toxicity.12, 13 Magnetic nanoparticles have also been used in the treatment of some infectious oral diseases, such as dental caries and periodontitis.^10,14, 15, 16 Magnetic nanomaterials behave as superparamagnetics and are able to produce a good magnetic response to an external magnetic field, which enable them to manipulate particle motion in the magnetic field.¹⁷

Previous researchers have proven that the joint application of magnetic nanomaterials and antibiotics can effectively enhance the bactericidal effect of original antibacterial agents.¹⁸ For example, magnetic nanomaterials were loaded to chlorhexidine, forming a functional chlorhexidine sphere, which can effectively kill Porphyromonas gingivalis and reduce the minimum bactericidal concentration and cytotoxicity of chlorhexidine.⁹ Thus, the aim of this study was to develop a novel magnetic nanomaterial chemically combined with DMADDM and investigate its effects on inhibiting biofilms of cariogenic bacteria and salivary flora.

Materials and methods

Synthesis of novel magnetic nanomaterials

Synthesis of Fe₃O₄@SiO₂-Br nanoparticles

SiO₂ has been widely used in the medical field because of its excellent chemical stability, high temperature resistance, biocompatibility, and absorbability. Previous studies have shown that Fe₃O₄ can chemically combine with SiO₂.[19] The solvothermal method was used to synthesise Fe₃O₄ nanoparticles according to previous studies.²⁰ Briefly, the Fe₃O₄@SiO₂-Br nanoparticles were prepared by multistep modification to bind the ATRP initiator.²¹ After treatment with hydrogen chloride (0.1 M), the Fe₃O₄ nanoparticles were added into the mixed solvent containing ethanol (24 mL) and deionised water (8 mL). Then, 0.5 mL of ammonia (28%) and 0.5 mL of tetraethyl orthosilicate (TEOS) were added under sonication and stirred vigorously for 4 hours at room temperature. The Fe₃O₄@SiO₂ nanoparticles were obtained by magnetic separation and washed with ethanol and deionised water several times.²¹

3-aminopropyltriethoxysilane (APTES; 0.4 mL) was added into Fe₃O₄@SiO₂ suspension (9 mL of ethanol) under stirring for 6 hours at room temperature, and sequentially triethanolamine (TEA; 0.4 mL) was added and stirred for 18 hours. TEA (1.8 mL) was added into Fe₃O₄@SiO₂-NH₂ suspension (20 mL of dry dichloromethane). After cooling to 0 °C, dry dichloromethane (8 mL) containing 2-bromoisobutyryl bromide (1.8 mL) was dropwise added under the protection of N₂ and stirred for 1 hour. Finally, the Fe₃O₄@SiO₂-Br nanoparticles were obtained by magnetic separation; washed with dichloromethane, acetone, ethanol, and deionised water several times; and redispersed in deionised water for subsequent use.

Synthesis of Fe₃O₄@SiO₂@DMADDM nanoparticles

DMADDM was synthesised as described in previous studies.²² The Fe₃O₄@SiO₂-Br nanoparticles were made with 1.0 mL of deionised water/isopropyl alcohol (IPA) mixed solvent (7:3) and 20.0 mg tripropylene glycol methyl ether acetate (TPMA) and 7.6 mg CuBr₂; 3.5 mL of deionised water/IPA mixed solvent (7:3) with 600.0 mg DMADDM was injected separately into a branch bottle under the protection of N₂. Then, the Fe₃O₄@SiO₂@DMADDM nanoparticles were obtained by magnetic separation; washed with isopropanol, ethanol, and deionised water several times; and redispersed in deionised water for subsequent use.

Antimicrobial effect of novel magnetic nanomaterials

Culture of S mutans

S mutans UA 159 was obtained from the American Type Culture Collection and was routinely cultured in brain heart infusion broth (BD) at 37 °C aerobically (95% air/5% CO₂). The inoculum was adjusted to 10⁸ colony-forming unit counts (CFU/mL) based on the OD₆₀₀nm vs CFU/mL graph. Then, 100 µL S mutans was inoculated in 10 mL of culture medium with 1% sucrose to get a final concentration for subsequent use.

CFU counts of S mutans biofilm

S mutans with a concentration of 10⁶ CFU/mL was inoculated and cultured in 96-well microtiter plates for 24 hours to form biofilms. Then, the biofilms were gently washed 3 times with phosphate buffer saline (PBS) and then treated with Fe₃O₄@SiO₂@DMADDM nanoparticles (100 µL each well, with magnetic field) for 10 minutes. According to the pre-experimental results, the material gradient concentration was set to 64, 32, 16, 8, and 4 mg/mL, and 4 replicates were set in each group. Control groups were treated with deionised water under the same conditions. The biofilms were gently washed 3 times with PBS after removal of materials; then 200 µL sterile PBS was added into each well and biofilms were scraped to obtain suspensions. The suspensions were serially diluted and spread on brain heart infusion plates. After 48 hours’ incubation at 37 °C in 5% CO₂, the colony number was counted and CFU counts were determined. One-way analysis of variance was performed to detect the significant effects of the variables. Tukey multiple comparison test was used, with a P value of .05.

Lactic acid production by S mutans biofilm

S mutans with concentration of 10⁶ CFU/mL was inoculated and cultured in 48-well microtiter plates for 24 hours to form biofilms; then, the process was as described earlier. After being washed twice with PBS, the biofilms were immersed in 1.5 mL buffered peptone water (Sigma-Aldrich) supplemented with 0.2% sucrose and incubated at 37 °C in 5% CO₂ for 2 hours (n = 4). The lactate concentrations in buffered peptone water were determined using a lactic acid detection kit.

Scanning electron microscopy (SEM) analysis of S mutans biofilm

S mutans with a concentration of 10⁶ CFU/mL was inoculated and cultured in 24-well microtiter plates for 24 hours to form biofilms, and then the process was as described in "CFU counts of S mutans biofilm" section The biofilms were gently washed twice with PBS after removal of materials, fixed with glutaraldehyde (2.5%) overnight at 4 °C, washed 3 times with PBS, dehydrated using a series of ethanol rinses (30%, 50%, 70%, 80%, 85%, 90%, 95%, and 100%), immersed for 10 minutes in 100% ethanol, and dried in a desiccator.

Saliva collection and biofilm development

This study was authorised by the Ethical Committee of West China School of Stomatology, Sichuan University (Chengdu, China, WCHSIRB-D-2020-386). The human unstimulated saliva was collected from 10 healthy adult donors who had natural dentition without periodontitis or active caries and who had not used antibiotics in the last 3 months, as described previously.²³ Donors were asked not to brush teeth for 24 hours and abstain from food/drink intake for 2 hours prior to donating saliva. The saliva was pooled and diluted 2-fold with sterile 50% glycerol. Then the saliva was stored at −80 °C.

Every sterile disc was placed into a well of the polystyrene 24-well flat-bottomed microtiter plate, after which 1.5 mL Salmonella-Shigella (SHI) medium was added, as previously described.²⁴ The saliva–glycerol stock was seeded (1:50 final dilution) into plates and incubated at 37 °C for 48 hours under anaerobic conditions (90% N₂, 5% CO₂, 5% H₂). The medium was refreshed every 24 hours, and PBS was used to rinse the biofilms to remove loose bacteria before immersion into fresh medium.

16S rDNA sequencing

The saliva-derived biofilm samples were subjected to microbial diversity sequencing (Majorbio), in which the total DNA was isolated, amplified, and sequenced according to their standard procedures. Microbial DNA was extracted from the saliva-derived biofilms using the FastDNA Spin Kit for Soil (MP Biomedicals) according to the manufacturer's protocols. DNA concentration was assessed by a NanoDrop Spectrophotometer (Thermo Fisher Scientific), and quality was determined by agarose gel electrophoresis. Using 515F-907R barcoded primers, the variable region 4 and 5 (V4-V5, 5′-GTGCCAGCMGCCGCGG-3′, 5′-CCGTCAATTCMTTTRAGTTT-3′) of bacterial 16S rDNA was amplified by polymerase chain reaction (PCR). PCR was performed in triplicate 20 µL mixture containing 4 µL of 5 × Fast Pfu Buffer, 2 µL of 2.5 mM dNTPs, 0.8 µL of each primer (5 µM), 0.4 µL of Fast Pfu Polymerase (TransGen), and 10 ng of template DNA. The amplicons were then extracted from 2% agarose gels and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences) and quantified by Quantus Fluorometer (Promega). Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq System per instructions. The raw readings were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP092150).

Bioinformatics and statistical analysis

Raw FASTQ files were demultiplexed and quality-filtered by QIIME (version 1.9.1). Operational taxonomic units (OTUs) were clustered with a 98.5% similarity cutoff based on UPARSE (version 7.1). The taxonomy of each 16S rRNA gene sequence was analysed by the Ribosomal Database Project Classifier (http://rdp.cme.msu.edu/) against the Human Oral Microbiome Database with a confidence threshold of 70%.²⁵ Alpha diversity index (Shannon index) calculations were performed using mothur v.1.30.2. Phylogenetic beta diversity was determined based on the represented sequences of OTUs. Principal component analysis was conducted according to the distance matrices determined by the represented sequences of OTUs for each sample. Mann–Whitney U tests were performed to detect the significant effects of the variables at a P value of .05. For statistical analyses, SPSS version 21.0 (SPSS Inc.) software was used.

Results

Material characterisation of nanoparticles

Fe₃O₄@SiO₂@DMADDM nanoparticles were successfully developed as illustrated in Figure 1A. Thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), vibrating sample magnetometry (VSM), and x-ray diffraction (XRD; Fig. 1B–E) were performed to test Fe₃O₄ nanoparticles, Fe₃O₄@SiO₂ nanoparticles, Fe₃O₄@SiO₂-NH₂ nanoparticles, Fe₃O₄@SiO₂-Br nanoparticles, and Fe₃O₄@SiO₂@DMADDM nanoparticles.

Fig. 1.

Material characterisation of Fe3O4@SiO2@DMADDM. A, Schematic diagram of the preparation of Fe₃O₄@SiO₂@DMADDM. B, Thermogravimetric analysis image showed that the masses of Fe₃O₄@SiO₂-NH₂ nanoparticles and Fe₃O₄@SiO₂@DMADDM nanoparticles were reduced by about 14% and 16%, respectively, indicating the dense grafting of DMADDM. C, Fourier transform infrared spectroscopy image of Fe₃O₄@SiO₂@DMADDM. The N–H bond at 1642 cm⁻¹ from 3-aminopropyltriethoxysilane (APTES) and Si–O bond at 1091 cm⁻¹ confirmed the successful modification of APTES and tetraethyl orthosilicate. The C–N peak of DMADDM is found at 1549 cm⁻¹, which proves that Fe₃O₄@SiO₂@DMADDM has been developed successfully. D, Vibrating sample magnetometry image showed all samples exhibited superpara magnetism. E, X-ray diffraction image implied that all products maintained magnetic crystal structure.

To explore the composition differences of synthetic materials, the composition of Fe₃O₄@SiO₂@DMADDM nanoparticles was determined by TGA. The mass of Fe₃O₄@SiO₂-NH₂ nanoparticles and Fe₃O₄@SiO₂@DMADDM nanoparticles was reduced by about 14% and 16%, respectively, indicating the dense grafting of DMADDM (Fig. 2B). To observe the characteristic peak spectra of each component, the Fe–O bond vibration peak was obvious at 600 cm⁻¹, the N–H bond characteristic peak from APTES can be seen at 1642 cm⁻¹, and the Si–O bond vibration peak from TEOS can be seen at 1091 cm⁻¹. The above peaks confirm the successful modification of APTES and TEOS and the synthesis of Fe₃O₄@SiO₂-NH₂. The C–N peak of DMADDM is found at 1549 cm⁻¹, proving that Fe₃O₄@SiO₂@DMADDM was developed successfully (Fig. 2C). Then, in order to investigate whether the structure of Fe₃O₄ particles in the final product was destroyed, the magnetic properties of the Fe₃O₄@SiO₂@DMADDM nanoparticles were examined by VSM. All the samples exhibited the original supermagnetism of Fe₃O₄ (Fig. 2D). The saturation magnetisation of Fe₃O₄@SiO₂@DMADDM nanoparticles was about 36 emu/g and could be quickly separated from the solution within 10 seconds using a magnet. The XRD (Fig. 2E) measurement shows that Fe₃O₄, and Fe₃O₄@SiO₂@DMADDM exhibited the same diffraction peaks at 30 ° (220), 35 ° (311), 43 ° (400), 53 ° (422), 56 ° (511), and 62 ° (400) in XRD patterns. These results implied that all products maintained magnetic crystal structure, which made Fe₃O₄@SiO₂@DMADDM the potential for targeted sterilisation under magnetic responsiveness.

Fig. 2.

Microscopic characteristics of nanoparticle materials. A, Scanning electron microscopy (SEM) image of Fe₃O₄. B, SEM image of Fe₃O₄@SiO₂-NH₂. C, SEM image of Fe₃O₄@SiO₂@DMADDM. D, Transmission electron microscopy (TEM) image of Fe₃O₄. E, TEM image of Fe₃O₄@SiO₂-NH_2. The red arrow indicates a transparent shell around the Fe₃O₄ nanoparticles. F, TEM image of Fe₃O₄@SiO₂@DMADDM. The size of as-prepared nanoparticles was 200 to 300 nm. Scale bar, 200 nm.

SEM and transmission electron microscopy (TEM) observation of nanoparticles

SEM and TEM were performed to characterise the morphology and microstructure of the materials (Fig. 2). Images showed that Fe₃O₄ nanoparticles, Fe₃O₄@SiO₂-NH₂ nanoparticles, and Fe₃O₄@SiO₂@DMADDM nanoparticles had a narrow size of distribution and were uniformly spherical. A shell was clearly observed around Fe₃O₄ nanoparticles after coating with SiO₂. DMADDM layers were not obvious due to the low contrast amongst SiO₂. The size of as-prepared nanoparticles was 200 to 300 nm.

The inhibitory effect of Fe₃O₄@SiO₂@DMADDM nanoparticles on the biofilm of S mutans

To verify whether the newly developed magnetic nanomaterials had antibacterial properties, the appropriate conditions for the antibacterial effects of materials were explored at the beginning of the experiment. Under the magnetic field, Fe₃O₄@SiO₂@DMADDM had an inhibitory effect on the biofilm formation of S mutans (P < .05), but there was no difference between the control group and the condition without the magnetic field (P > .1; Fig. 3A). Therefore, all subsequent experiments were carried out under the magnetic field. According to CFU counts, the antibacterial effect of Fe₃O₄@SiO₂@DMADDM was concentration-dependent, and the bactericidal rate rose with increases in concentration until 16 mg/mL (P < .05; Fig. 3B–C). In the occurrence and development of chronic oral diseases, the acid production of S mutans is important; thus, to investigate the effect of different concentrations of Fe₃O₄@SiO₂@DMADDM on the acid production of S mutans under the action of the magnetic field, lactic acid was evaluated. Findings revealed that 8 mg/mL of the novel nanoparticles could effectively reduce the lactic acid production of S mutans (P < .05; Fig. 3D). At the same time, we observed the morphologic differences of S mutans biofilm treated with different concentrations of Fe₃O₄@SiO₂@DMADDM under the action of the magnetic field. SEM images showed that higher concentrations removed much more S mutans biofilm that lower concentrations did (Fig. 3E). The measurement (mg/mL) was set according to the weight of Fe₃O₄ in the progress of nanoparticle fabrication.

Fig. 3.

Colony-forming unit counts and lactic acid production by Streptococcus mutans biofilm. A, Inhibitory effect of Fe₃O₄@SiO₂@DMADDM on S mutans biofilm. The figure shows the inhibitory effect on the biofilm of S mutans biofilm between DDW and Fe₃O₄@SiO₂@DMADDM (10 mg/mL) with or without the magnetic field. B and C, Bactericidal rate of S mutans biofilm treated with different concentrations of Fe₃O₄@SiO₂@DMADDM. D, Effects of different concentrations of Fe₃O₄@SiO₂@DMADDM on lactic acid production by S mutans. The figure showed S mutans biofilm treated with different concentration of Fe₃O₄@SiO₂@DMADDM, control group with DDW (mean [SD]; n = 4; ns, P > .1; *P < .05). E, SEM analysis of S mutans biofilm (n = 4). The red arrows indicate S mutans, whilst the blue arrows indicate Fe₃O₄@SiO₂@DMADDM. SEM images showed that higher concentrations removed much more S mutans biofilm. S mutans was inoculated in 10 mL of culture medium with 1% sucrose.

The microbial community of saliva-derived biofilms

16S rDNA sequencing showed that Fe₃O₄@SiO₂@DMADDM could depress the proportion of caries-related bacteria in salivary biofilm, such as Streptococcus, Veillonella, and Neisseria. Fe₃O₄@SiO₂@DMADDM at concentrations of 16 mg/mL or greater might influence microbial diversity (Fig. 4A). In addition, the principal coordinates analysis showed that the distribution of the five groups of biofilms was quite different (Fig. 4B). Streptococcus was the dominant bacteria in SHI culture (Fig. 4C); thus, we analysed the influence and interaction of other vulnerable genera after removing Streptococcus.

Fig. 4.

16S rDNA sequencing analysis of saliva-derived biofilms. A, The alpha diversity of was measured using the Shannon index. B, Principal coordinates analysis score plot. The flora within the same group tended to be distributed in a close range, indicating that the samples were representative and the differences within the group were small. C, The percentage of community abundance at the genus level indicated Streptococcus as the dominant bacteria in SHI culture (n = 4; *P < .05).

When the concentration was greater than 16 mg/mL, Fe₃O₄@SiO₂@DMADDM reduced the proportion of Neisseria, Granulicatella, Gemella, Haemophilus, and other harmful bacteria (Fig. 5A). Moreover, Fe₃O₄@SiO₂@DMADDM at a low concentration (8 mg/mL) could inhibit Veillonella (Fig. 5B), which were closely related to dental caries, pulpitis, and periapical diseases; higher concentrations had stronger inhibitory effects on Neisseria (Fig. 5C). Meanwhile, a higher concentration of Fe₃O₄@SiO₂@DMADDM (32 mg/mL) could increase the proportion of beneficial flora such as Lactobacillus and Lactococcus (Fig. 5B–C).²⁶ These results suggest that a high concentration (64 mg/mL) of Fe₃O₄@SiO₂@DMADDM may maintain the homeostasis of the oral microenvironment by regulating the decrease in the proportion of caries-associated bacteria in the salivary flora.

Fig. 5.

Analysis of bacterial genera after treatment of saliva biofilm with different concentrations of Fe₃O₄@SiO₂@DMADDM. A, The percentage of community abundance at the genus level after removing Streptococcus.B, Pairwise comparison of Granulicatella, Gemella, Veillonella, Lactococcus, and Lactobacillus in salivary flora treated at different concentrations. C, Pairwise comparison of Neisseria, Lactococcus, and Lactobacillus in salivary flora treated at different concentrations (n = 4; *P < .05).

Discussion

In the present study, TGA, FTIR, XRD, VSM, SEM, and TEM findings showed that DMADDM was, first, chemically combined with magnetic nanomaterials and synthesised Fe₃O₄@SiO₂@DMADDM nanoparticles. Then, its effects on inhibiting biofilm formation by using traditional caries-related bacteria and saliva flora were investigated. S mutans is typically used in the research of the aetiology and prevention of dental caries.²⁷ It is believed that S mutans relies mainly on adhesion, acid production, and acid resistance to cause caries.²⁸ Many studies have shown that S mutans can produce lactic acid under the condition of sufficient sugar sources, and when the pH of the dental microenvironment decreases below the threshold for enamel and dentine demineralisation and remineralisation (usually 5.5), caries occurs and progresses. At the same time, S mutans can enhance its resistance to host defense.²⁹ Thus, the occurrence and development of dental caries may be prevented by employing strategies including reducing acid production and improving the acidic environment of biofilms.

The novel Fe₃O₄@SiO₂@DMADDM nanoparticles in the present experiment may remove bacterial biofilms and inhibit their acid production. On the one hand, Fe₃O₄@SiO₂@DMADDM may rely on the guidance of the magnetic field to remove the biofilm mechanically. It is believed that positively charged metal nanoparticles may be attracted to the negatively charged surface of bacterial cells by static electricity, resulting in bacteria death.³⁰ On the other hand, our material contains the cationic fungicide DMADDM,³¹ which effectively inhibited the acid production of the Gram-positive bacteria S mutans. Therefore, Fe₃O₄@SiO₂@DMADDM could play a high-performance antibacterial effect under the guidance of the magnetic field. It was also found that the antibacterial effect of Fe₃O₄@SiO₂@DMADDM is concentration-dependent, which is consistent with the results of previous studies.³² Fe₃O₄@SiO₂@DMADDM may effectively inhibit the production of lactic acid, even at lower concentrations. The result is a low exposure risk of DMADDM, with high concentrations having cytotoxic effects on human cells.³¹ Furthermore, metal nanoparticles have less bacterial drug resistance than antibiotics, which indicates low toxicity and good biocompatibility with human cells.³³

The oral cavity is a complex microecological environment with more than 700 microbes.³⁴ The most common microbiome are bacteria, including Veillonella, Actinobacteria, Streptococcus, and Neisseria.³⁵ The colonisation of the human oral microbiome occurs mainly on the teeth, restorations, and mucosal surfaces, thus forming plaque biofilms, with substantial evidence that this is the main cause of infectious oral disease.³⁶ Oral saliva contains most of the bacteria in plaque biofilms, so saliva-derived biofilm is often used to simulate the oral microenvironment and study the aetiology and prevention of oral plaque-related diseases, including dental caries. 16S rRNA encoding genes are present in all bacterial genomes and are the most useful, most commonly used molecular marker in the classification of bacterial systems. Researchers can directly analyse microbial gene sequences by using 16S rDNA sequencing technology to determine information about sample species abundance, population structure, and community comparison in a specific environment.³⁷ Therefore, in this experiment, we chose the saliva-derived biofilm model in vitro to test its anti-biofilm effect, as well as the single-species biofilm model. Then, the changes in flora following Fe₃O₄@SiO₂@DMADDM treatments were observed using 16S rDNA sequencing technology. Through diversity and intergroup differences analysis, microbial diversity decreased as Fe₃O₄@SiO₂@DMADDM concentrations increased (8 to 16 mg/mL); the abundance in diversity recovered when the concentration continued to rise (to 64 mg/mL). Further analysis showed that a low concentration (8 mg/mL) of Fe₃O₄@SiO₂@DMADDM could inhibit Veillonella, Granulicatella and Neisseria. Veillonella can assist S mutans adhesion and decompose lactic acid, so Veillonella is considered a caries-related factor.³⁸ Some studies showed that an abundance of Streptococcus and Granulicatella could be detected in early childhood caries plaque;³⁹ and others have shown that Neisseria is more prevalent in caries than in healthy individuals.⁴⁰ Taken together, these findings indicate that Granulicatella and Neisseria were associated with dental decay. Results of the present experiment show that Fe₃O₄@SiO₂@DMADDM not only retains its original antibacterial effect but also reduced the proportion of caries-related genera (eg, Streptococcus, Veillonella, Granulicatella, and Neisseria) in salivary biofilms. However, further research is needed to elucidate the mechanism of this effect.

All results proved that Fe₃O₄@SiO₂@DMADDM nanoparticles had inhibitory effects on biofilm formation by using a single-species biofilm and saliva-derived biofilm model. This novel magnetic material not only retains the high-performance antibacterial effect of DMADDM but also possesses the characteristics of magnetic materials that can mechanically remove biofilm by relying on magnetic field guidance. This approach is promising in the creation of dental materials and can be incorporated into cavity disinfectants or anticavity gel for clinical use. However, some questions remain to be answered, such as the ideal concentration of DMADDM.

Conclusions

The present study first synthesised Fe₃O₄@SiO₂@DMADDM nanoparticles, which had excellent effects in inhibiting biofilm formation under the guidance of the magnetic field. Further, Fe₃O₄@SiO₂@DMADDM was found to effectively remove S mutans biofilm, inhibit lactic acid production, and reduce the proportion of caries-related bacteria in saliva-derived biofilm. Thus, the novel nanoparticle may provide an innovative strategy to enhance the anticaries effect of antibacterial agents, but more experiments are required to prove its efficiency.

Declaration of competing interest

None disclosed.