Antisense vicR-Loaded Dendritic Mesoporous Silica Nanoparticles Regulate the Biofilm Organization and Cariogenicity of Streptococcus mutans

Introduction

Dental caries accounts for the highest incidence of oral disease, causing a significant disease burden in many countries.¹ The leading etiological agent of dental caries is Streptococcus mutans (S. mutans), which can form biofilms on solid surfaces such as the enamel, tooth root surfaces, or dental implants.^2,3 Biofilms are three-dimensional structures consisting of microbes and an extracellular matrix. The exopolysaccharides (EPS) produced by the interactions of glucosyltransferases (Gtfs) and fructosyltransferases (Ftfs) with sucrose in S. mutans are the main components and the essential virulence factors of the dental caries.^4–6 The EPS matrix enhances the adhesion of microorganisms, promotes the biofilm formation of three-dimensional structures to impede the penetration of antibiotic substances into the biofilm, and plays a role as a source of energy.^4,5,7 Therefore, reducing the synthesis of the EPS matrix could decrease the virulence of S. mutans, lowering the incidence of caries.

Two-component signal transduction systems (TCSTS) could modulate gene expression in response to external environmental changes.⁸ There are thirty TCSTS in S. mutans based on their genomic sequence.⁹ The VicRK system, one of the TCSTS, consists of a membrane-bound sensor (VicK) and a cytoplasmic response regulator (VicR). It has been reported that VicR regulated the expression of genes that are associated with EPS synthesis.^8,10,11 However, the inability to construct vicR deletion mutants revealed that the vicR gene was also crucial to the viability of S. mutans.⁸ Previous reports indicated that antisense (AS) substances could inhibit the transcription and translation of the related genes.^12–14 An AS RNA could bind to complementary mRNA via Watson-Crick base pairing and suppress its transcription by inducing a steric blockage and/or RNase degradation.^15–17 Our previous studies have shown that an antisense vicR RNA (ASvicR) could be complementary to the vicR mRNA through base pairing, forming an ASvicR-vicR duplex structure and hindering the expression of the vicR gene.¹⁸ In addition, ASvicR inhibited EPS synthesis, bacterial growth, and cariogenicity of S. mutans, indicating that ASvicR could be considered a potential strategy for caries prevention.^18,19 We have constructed a recombinant plasmid containing the ASvicR gene (plasmid-ASvicR) by linking the ASvicR sequence to the pDL278 plasmid.¹⁸ However, the plasmids are supposed to be protected from enzymatic degradation and avoid electrostatic repulsion from the cell surface to endow the recombinant plasmid with higher transformation efficiency.^17,20,21 Hence, it is necessary to explore an appropriate vector that can protect recombinant plasmids and promote their transformation.

Various strategies have been demonstrated to be nucleic acid carriers, including viruses,²² lipids,²³ peptides,²⁴ cationic polymers,²⁵ and inorganic nanoparticles.^26,27 Despite their high transformation efficiency, the biocompatibility and safety of viral vectors remain controversial.^21,26,28 In addition, the immunogenicity of liposomes, the poor delivery efficiency of peptides, and the cytotoxicity of cationic polymers still require further studies to determine whether these materials are suitable as gene delivery vectors.^20,29,30 Among the inorganic nanoparticles, mesoporous silica nanoparticles (MSNs) exhibit excellent potentials as drug and gene delivery vectors due to their favorable biocompatibility, chemical stability, large surface area, and simple synthesis process.^31–34 Furthermore, silica has been classified as generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA) and is commonly used as a food additive as well as in the cosmetic and pharmaceutical industries.³⁵ However, the pore sizes of several conventional MSNs are too small to accommodate and protect macromolecule such as nucleic acids and proteins.^36–38 Improvements in the synthesis methods have resulted in the emergence of different morphologies (spherical, sheet, worm-like, rod-like, fibrous, dendritic, etc) and architectures (solid, hollow, core-shell, Janus, yolk-shell, etc.) in MSNs.^39,40 In recent years, dendritic mesoporous silica nanoparticles (DMSNs) with center-radial oriented pore structures have aroused great interest due to their higher pore permeability, larger pore volume, and better accessibility to the particle inner surface.^41–44 There are several reports about MSNs acting as gene carriers in different cells. However, for bacteria, MSNs are mainly utilized to load traditional antibiotics, metallic ions, proteins, and plant extracts,^45–49 which react on a wide range of bacteria, leading to drug resistance and dysbacteriosis. To load the plasmid-ASvicR and act on the bacteria, the DMSNs should have a positively charged surface, a larger pore size, and a smaller particle size.

Based on the facts mentioned above, we hypothesized that dendritic mesoporous silica nanoparticles could be vectors of plasmid-ASvicR to target the transcriptions and translations of vicR genes to reduce the EPS synthesis in the biofilm and cariogenicity of S. mutans. The delivery system is supposed to have the excellent ability to carry and protect plasmids. The novel plasmid-loaded DMSNs that could target S. mutans show potential as a method of dental caries management.

Materials and Methods

Materials

Cetyltrimethylammonium chloride (CTAC), triethanolamine (TEA), tetraethyl orthosilicate (TEOS), cyclohexane, and 3-aminopropyltriethoxysilane (APTES) were purchased from Macklin (Shanghai, China). Hydrochloric acid (37%) and anhydrous ethanol were purchased from Kelong (Chengdu, China). EndoFree Maxi Plasmid Kit was purchased from Tiangen (Beijing, China). Brain Heart Infusion (BHI) medium was purchased from Oxiod (USA). Alexa Fluor^® 647 and SYTO™ 9 dyes were purchased from Life Tech (USA). MasterPure™ RNA purification kit was purchased from Epicentre (USA). DNase I, PrimeScript™ RT reagent kit with gDNA Eraser, and TB Green^® Premix Ex Taq™ II were purchased from Takara (Japan). Cell counting kit-8 (CCK-8) was purchased from Dojindo (Japan). Live/dead cell double staining kit (Calcein-AM/PI) was purchased from Solarbio (Beijing, China). DMEM, FBS, trypsin, and PBS were purchased from ThermoFisher (USA). Keyes2000# cariogenic fodder was purchased from Dossy (Chengdu, China).

Synthesis of DMSNs

DMSNs were synthesized according to previous literature with some modifications.^42,50,51 Six gram of CTAC and 0.18 g of TEA were added to 60 mL of ultrapure water. Then, the solutions were stirred (300 rpm) in a 100-mL round-bottom flask at 60°C for 1 hour. Next, 20 mL of TEOS in cyclohexane (20 v/v%) was carefully added to the water-CTAC-TEA solution and kept at 60°C in an oil magnetic stirrer for 10 hours. The products were collected by centrifugation, washed with ethanol three times, added to 100 mL of 1 v/v% hydrochloric acid ethanol solution, and refluxed at 80°C for 12 hours twice. The final products were washed with ethanol and ultrapure water three times and lyophilized for further investigations.

Surface Amination of DMSNs with APTES

DMSNs were modified to acquire a positively charged surface to load negatively charged plasmids.^52,53 In the present study, the amino groups were introduced using 3-aminopropyltriethoxysilane (APTES) through a post-grafting method to obtain positively charged DMSNs.^37,54 To prepare amino-DMSNs (DMSNs-NH₂), 500 mg of DMSNs was dispersed in 100 mL of ethanol by ultrasonication. Then, 1.5mL of APTES was added to the suspension and the mixtures were refluxed at 80°C for 24 h. The final products were washed with ethanol and ultrapure water three times and lyophilized for the following experiments.

Characterization

Transmission electron microscopy (TEM) (Tecnai G2 F20 S-TWIN; FEI, USA) was used to observe the structure of DMSNs-NH₂, and ImageJ software (National Institute of Mental Health, USA) was used to obtain the diameter distribution of the particles. Nitrogen adsorption-desorption measurements were conducted with a specific surface area analyzer (Kubo-X1000; Builder, China) to evaluate the surface areas, pore size distributions, and pore volumes. Zeta potentials were analyzed with a Zetasizer Nano ZS instrument (Malvern, UK), and the measurements were repeated three times. Energy-dispersive spectrometry (EDS) mapping (JEM-F200_TFEG, Japan), and Fourier transform infrared (FTIR) spectroscopy (Thermo Fisher Nicolet Is5, USA) were also conducted to characterize the DMSNs-NH₂.

Binding of Plasmid-ASvicR to DMSN-NH₂ (DMSNs-NH₂-ASvicR)

The recombinant plasmids containing the ASvicR consequence were obtained from E. coli using an EndoFree Maxi Plasmid Kit, and the concentrations of plasmids were measured with NanoDrop One^c (Thermo Fisher, USA). DMSNs-NH₂ was suspended in deionized water and prepared into several solutions of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 μg/μL, respectively. Then, 1 μg of plasmid-ASvicR was dispersed in 10 μL of DMSNs-NH₂ solutions of the different concentrations, in which the DMSNs-NH₂ was mixed with plasmid-ASvicR by mass at 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 ratios. After shaking (200 rpm) at room temperature for 1 h, the mixtures were used to perform gel electrophoresis by loading onto 1% agarose gel at 100V for 30 minutes. The resulting products were observed using a Bio-Rad electrophoresis system (Bio-Rad Laboratories, USA).

To quantify the amount of plasmid-ASvicR absorbed on DMSNs-NH₂, 6 μg of plasmid-ASvicR was dispersed in 60 μL of DMSNs-NH₂ solutions with different concentrations (as mentioned above). After shaking (200rpm) at room temperature for 1 h, the mixtures were centrifuged at 12,000 rpm for 10 min, and the concentration of plasmid-ASvicR in the supernatant was analyzed by NanoDrop One^c. The amount of plasmid-ASvicR adsorbed on DMSNs-NH₂ was calculated by subtracting the content in the supernatants from the initial concentration.

In addition, the DMSNs-NH₂-ASvicR solution (the weight ratio of DMSNs-NH₂/plasmid-ASvicR=80) was prepared as mentioned above. Then the solution was centrifuged at 12,000 rpm for 10 min to obtain DMSNs-NH₂-ASvicR precipitate. The precipitate was lyophilized and stored at room temperature for the following experiments. To further prove that plasmids-ASvicR had been absorbed by the DMSNs-NH₂, the lyophilized powder of DMSNs-NH₂-ASvicR was dispersed in deionized water and used for Zeta potential test, EDS-mapping, and FTIR spectroscopy.

DNase I Digestion Assay

The DMSNs-NH₂ mixed with plasmid-ASvicR was prepared by mass ratios of 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 and incubated with 1 μL of DNase I. After incubation, half of the mixtures were used for 1% agarose gel electrophoresis, as mentioned above. The other half of the mixtures were added to 500-μM EDTA to stop the digestion, and then heparin (4 mg/mL) was added to release the plasmids.^37,55 After shaking at 50°C and 200 rpm for 1 h, the mixture was centrifuged, and the supernatant was used for 1% agarose gel electrophoresis.

Transformation of DMSNs-NH₂-ASvicR

The plasmid-ASvicR contains the antisense vicR sequence, the spectinomycin-resistant sequence, and the gene encoding the green fluorescent protein (GFP). DMSNs-NH₂ was mixed with plasmid-ASvicR at a mass ratio of 80, and the mixture was stored in a refrigerator at 4°C for subsequent experiments. We conducted antibiotic plate screening assay and GFP expression assay to prove that the plasmid-ASvicR loaded on DMSNs-NH₂ can enter S. mutans and be expressed.

A single colony of S. mutans UA159 was selected and cultured overnight in a fresh Brain Heart Infusion (BHI) medium at 37°C (80% N₂, 10% CO₂, and 10% H₂). On the one hand, the bacterial suspensions were diluted in 10 mL of fresh BHI medium at a volume ratio of 1:20 and cultured for 2–3 h. After adjusting the optical density at 600nm (OD600) to 0.4, 500 μL of bacterial suspensions were cultured in the presence of deionized water, plasmid-ASvicR, and DMSNs-NH₂-ASvicR (containing 200 ng of plasmids-ASvicR) for 30 min, 3 h, 6 h, 12 h, and 24 h, respectively. Then, the mixed solutions were spread onto the BHI solid medium with spectinomycin (Spe, 1mg/mL). On the other hand, the bacterial suspensions cultured overnight were diluted in 10 mL of BHI medium containing 1% sucrose (BHIS) at a volume ratio of 1:10. Then, 2 mL of the diluted liquid was added to the confocal dish, followed by adding deionized water, pDL278 (the plasmid without GFP and antisense vicR sequence), DMSNs-NH₂, plasmids-ASvicR, and DMSNs-NHs-ASvicR, respectively. The mixed solutions were cultured for 3 h, and the cultures were evaluated under a confocal laser scanning microscope to confirm the expression of GFP. In addition, the average fluorescence intensity of each group was analyzed using ImageJ, and a relative quantitative result was obtained.

Biofilm Formation Assay

The bacteria suspensions of S. mutans UA159 in the mid-log phase (OD₆₀₀=0.5) were diluted in BHIS at a volume ratio of 1:100 for the following experiments. Taking into account the effective time of DMSNs-NH₂ and simulating people’s usual oral cleaning habits (gargling or brushing teeth three times during the day, no cleaning behavior at night during sleep), we designed a 12-hour group and a 24-hour group in the biofilm formation experiment. There were five subgroups in each group: (1) “UA159 + sterile deionized water” was designed as a blank control group; (2) “UA159 + pDL278” was designed to exclude the influence of the plasmid without vicR sequence on biofilm formation; (3) “UA159 + plasmid-ASvicR” was designed to explore the impact of the naked plasmid-ASvicR on biofilm formation; (4) “UA159 + DMSNs-NH₂” was designed to investigate whether the nanoparticles themselves impacted biofilm formation; (5) “UA159 + DMSNs-NH₂-ASvicR” was designed to verify the effect of plasmid-ASvicR loaded on DMSNs-NH₂ on biofilm formation.

Concerning scanning electron microscopy (SEM), 2 mL of bacterial suspensions was added to each well of the 12-well plate containing circular glass slides and cultured at 37°C (80% N₂, 10% CO₂, and 10% H₂) for 12 or 24 h. In the 12-hour group, sterile deionized water, naked pDL278, naked plasmid-ASvicR, DMSNs-NH_2, or DMSNs-NH₂-ASvicR (containing 800 ng of plasmids-ASvicR) was added every 4h. In the 24-hour group, the treatment for the first 12 hours was the same as the “12-hour group”, with no treatment for the next 12 hours. After 12 h or 24 h, the biofilms were washed with PBS three times and fixed with 2.5% glutaraldehyde in the dark for 4 h. Next, the serial concentrations of ethanol solutions were used to dehydrate biofilms in gradient. The final biofilms were observed using SEM (Inspect Hillsboro, USA).

Concerning confocal laser scanning microscopy (CLSM), bacterial solutions were added to sterile circular glass slides in 12-well plates, and 1-μM of Alexa Fluor^® 647 was added to label the biofilm EPS in situ. After 12 or 24 hours co-culture, the medium was removed, and each sample was washed with sterile PBS three times. Next, 50 μL of SYTO™ 9 dye diluted by deionized water at a 1:100 ratio was added to label the bacteria for 15 min. Next, the biofilm architectures were measured by CLSM (Olympus, Japan), and Z sections at 0.8-μM intervals were used to record the thickness of the biofilms. Finally, Imaris 7.0 software (Bitplane, Switzerland) was used to reconstruct three-dimensional images of the biofilms and analyze the biomass of EPS and bacterial cells.

Quantitative Reverse Transcription Polymerase Chain Reaction

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted to verify the regulatory effect of DMSNs-NH₂-ASvicR on the vicR gene related to EPS synthesis. The 12-hour and 24-hour biofilms of each group (sterile deionized water, naked pDL278, naked plasmid-ASvicR, DMSNs-NH₂, or DMSNs-NH₂-ASvicR) were obtained as mentioned above. After being washed twice with PBS, the biofilms were harvested by a cell scraper and centrifuged (5000 rpm, 4°C, 10 min) to obtain bacterial precipitation. Total RNA was extracted using a MasterPure™ RNA purification kit and dissolved in RNase-free water. The cDNA was prepared according to the instructions of PrimeScript™ RT reagent kit with gDNA Eraser. Then, qRT-PCR was performed using LightCycler^® 480 (Roche, USA), following the instructions of TB Green^® Premix Ex Taq™ II. DNA gyrase subunit A (gyrA) was selected as a reference gene, and the group of “UA159 + deionized water” was used as control. Table 1 lists the corresponding primers. After amplification, each well’s quantification cycle (Cq) value was obtained, and the relative level of vicR mRNA in each group was calculated by the 2^−ΔΔCt method. Western blot was also conducted (see “Western blot” section in the Supplementary materials) to further investigate the productions of VicR proteins.

Table 1 Primer Information of RT-qPCR

Cytotoxicity Assessment of DMSNs-NHs-ASvicR

The cytotoxicity of DMSNs-NH₂-ASvicR was evaluated via cell counting kit-8 (CCK-8) assay and live/dead staining assay. In addition, human gingival fibroblasts (HGFs) were obtained from clinical samples and cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS at 37°C in a 5% CO₂ incubator according to the literature.⁵⁶

For the CCK-8 assay, the cells were seeded in a 96-well cell culture plate overnight at a density of 5000 cells/well before DMSNs-NHs-ASvicR treatment. Then, the cells were added with various concentrations of DMSNs-NH₂-ASvicR (0, 32, 64, and 96 μg/mL of DMSNs-NH₂ and the weight ratio of DMSNs-NH₂/plasmid-ASvicR = 80). After 12 or 24 hours incubation, 10 μL of CCK-8 solution was added to each well. The 96-well plates were then incubated for 3 hours. The absorbance at 450nm was measured by SpectraMaxiD5 (Molecular Devices, USA).

For the live/dead staining assay, the cells were seeded in a 24-well cell culture plate overnight at a density of 15,000 cells/well before DMSNs-NH₂-ASvicR treatment. Then, the cells were added with various concentrations of DMSNs-NH₂-ASvicR (0, 32, 64, and 96 μg/mL of DMSNs-NH₂ and the weight ratio of DMSNs-NH₂/plasmid-ASvicR = 80). After 24 hours of incubation, the working solutions (2-μM Calcein-AM and 4.5-μM PI) and 1×assay buffer were first prepared according to the instructions. Then, the previous DMEM was removed, and 100 μL of staining solution was added to each well. The mixture was mixed and incubated for 15 min. Images of live (green fluorescence) and dead (red fluorescence) cells were obtained under a fluorescence microscope (Leica, Germany).

Animal Experiment

A rat caries model^57,58 was used to study the inhibitory effects of DMSNs-NH₂-ASvicR on the cariogenicity of S. mutans in vivo. Twenty-four Wistar male rats aged 21 days were provided by Dossy Experimental Animals Co., Ltd (Chengdu, China) and randomly assigned to the control group (UA159 + PBS) and experimental groups (UA159 + DMSNs-NH₂, UA159 + plasmid-ASvicR, UA159 + DMSNs-NH₂-ASvicR), with six rats in each group. Before the infection, 0.1% ampicillin sodium was administered for bacteriostatic treatment during 22–27 days of age. After eluting bacteriostatic treatment for one day, the saliva of those rats was collected with sterile cotton swabs and coated on the MSA mediums containing 0.2-U/mL bacitracin to detect the inhibition of endogenous S. mutans. During 28–34 days of age, each rat was infected orally once daily using 200 µL of bacterial suspension containing the UA159 strain. Meanwhile, the control group was treated with 200 µL of PBS, and the experimental groups were treated with 200 µL of DMSNs-NH₂, plasmid-ASvicR, and DMSNs-NH₂-ASvicR, respectively, at 28–41days of age. All the rats were fed with Keyes2000# cariogenic fodder and sterilized water containing 5% sucrose. The rats were weighed once a week and recorded daily for their health statuses. At the rats age of 56 days, they were sacrificed by CO₂ asphyxiation, and their mandibles were removed and dissected into left and right halves. After washing with PBS three times, the mandibular specimens were immersed in 10% formalin solution for 48h. Then the sections were embedded and ground to be observed under a stereomicroscope to determine caries level according to a modified Keyes score.^59,60 The scores for each sample were the sum of the scores for the first and second molars.

Statistical Analysis

Statistical analyses of the experimental results were performed with Prism 8 (GraphPad, USA). The Shapiro–Wilk test was used to analyze the data normality, and the Bartlett method was used to test the homogeneity of variance. If the data of each group were normally distributed and consistent with homogeneity of variance, the one-way ANOVA was used to analyze data. If the data did not exhibit normal distribution or homogeneity of variance, the Kruskal–Wallis method was used to conduct a completely random non-parametric test on the data. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 indicated statistically significant differences. All the experiments were repeated at least three times.

Results

Characterization of DMSNs-NH₂

A transmission electron microscope (TEM) was used to observe the morphologies and dispersion of DMSNs-NH₂, and ImageJ software was used to determine the diameters of nanoparticles. Figure 1A–D shows that DMSNs-NH₂ consisted of dendrimer-like particles with center-radial pore channels. The particles were highly monodispersed, and the average particle diameter was approximately 59 nm. The information of mesopores was obtained using N₂ adsorption-desorption measurements. The N₂ adsorption-desorption curve of DMSNs-NH₂ (Figure 1E) displayed a type-IV isotherm and type-H4 hysteresis loop, illustrating the presences of mesopores. The size distribution of mesopores (Figure 1F) was acquired from N₂ adsorption isotherm via Barrett-Joyner-Halenda (BJH) method, indicating an average pore size of 8.1 nm. In addition, the Brunauer-Emmett-Teller (BET) surface areas of 449.1 m²g⁻¹ and large total pore volumes of 0.8 cm³g⁻¹ were also acquired. Supplementary Figure 1 and Supplementary Table 1 indicate that the DMSNs were successfully aminated. Figure 1G shows that the average potentials of the particles changed from −6.2 mV to 6.3 mV after the modification of positively charged amino groups on DMSNs. However, after loading the plasmid-ASvicR, the average potential of the particles changed to −25 mV. Figure 1H is a schematic diagram of the DMSNs-NH₂ synthesis process.

Figure 1 (A–C) TEM images of DMSNs-NH2. (D) Particle diameter distribution of DMSNs-NH2. (E) N2 adsorption-desorption isotherm of DMSNs-NH2. (F) Pore diameter distribution of DMSNs-NH2. (G) Zeta potentials of the DMSNs, DMSNs-NH2, and DMSNs-NH2-ASvicR in deionized water. (H) The schematic diagram of DMSNs-NH2 synthesis process: the DMSN precursor was synthesized one-pot biphase stratification approach, then the cationic surfactant was removed by the hydrochloric acid ethanol solution to obtain DMSN, and the DMSN is aminated by APTES to obtain DMSN-NH2.

Loading and Protection of Plasmid-ASvicR by DMSNs-NH₂

The ability of DMSNs-NH₂ to load plasmid-ASvicR was confirmed by agarose gel electrophoresis and the loading curve. The bright bands in the lanes in Figure 2A represent the different forms of the plasmid-ASvicR,³⁷ and the brightness in the wells represent the plasmids-ASvicR bound to the DMSNs-NH₂ that could not run out of the wells. As the weight ratio of DMSNs-NH₂/plasmid-ASvicR increased, the brightness in the well increased, while the brightness of the plasmid band in the lane decreased. Table 2 shows the loading data, and Figure 2B is a graph of plasmids loaded by DMSNs-NH₂ with different qualities, and the loading trend of the graph is consistent with Figure 2A. When the mass ratio of DMSNs-NH₂/plasmid-ASvicR was <80, the amount of the plasmid loaded by DMSNs-NH₂ increased with an increase in the mass ratio. When the mass ratio reached 80, the plasmid was loaded 92%, and the loaded plasmid did not increase as the mass ratio continued to increase. Therefore, in the follow-up experiments, the weight ratio of 80 was used as the working ratio.

Table 2 Data of Loading Curve

Figure 2 (A) Agarose gel electrophoresis after different concentrations of DMSNs-NH2 interacted with plasmid-ASvicR. The red arrow indicates open-loop form of the plasmid-ASvicR, and the yellow arrow indicates supercoiled form of the plasmid-ASvicR. (B) The graph of DMSNs-NH2 loading plasmid-ASvicR with different weight ratio. (C) Agarose gel electrophoresis after various weight ratios of DMSNs-NH2/plasmid-ASvicR incubated with 1μL DNase I for 15 minutes. (D) Agarose gel electrophoresis of the plasmid-ASvicR released by heparin after incubation with the DNase I. (E) EDS-mapping images of DMSNs-NH2 and DMSNs-NH2-ASvicR with sacle bar of 100 nm. Si represents silicon in DMSNs-NH2 and P represents phosphorus in plasmids-ASvicR. (F) Schematic diagram of the plasmid-ASvicR loaded and protected by DMSNs-NH2. The negatively charged plasmids-ASvicR were adsorbed in the mesopores of the positively charged DMSNs-NH2, thereby avoiding degradation by DNase I.

The ability of DMSNs-NH₂ to protect plasmid-ASvicR was confirmed by agarose gel electrophoresis. After incubation with DNase I for 15 min, DMSNs-NH₂/plasmid-ASvicR complexes at different weight ratios were used for agarose gel electrophoresis. DNA bands of DMSNs-NH₂/plasmid-ASvicR complexes could be observed at various weight ratios except for the group at the weight ratio of 0, indicating that the naked plasmid-ASvicR was degraded completely by DNase I without the protection of DMSNs-NH₂ (Figure 2C). After treatment with heparin, the plasmid-ASvicR adsorbed on DMSNs-NH₂ were released. Figure 2D shows that most of the plasmids adsorbed on DMSNs-NH₂ were not degraded. The new band appearing between the two bands (a supercoiled and open-loop form of the plasmids) was a linear form that might have been caused by the vortex oscillation during the operation. EDS-mapping was performed in order to observe the distribution of plasmid-ASvicR on DMSNs-NH₂ more intuitively. Figure 2E indicates that plasmid-ASvicR was distributed on the both surface and mesopores of DMSNs-NH₂. However, Supplementary Figure 2 shows that the FTIR of DMSNs-NH₂ and DMSNs-NH₂-ASvicR had no obvious differences, which is probably because there were no chemical bonds formed between DMSNs-NH₂ and plasmid-ASvicR. Figure 2F is a schematic diagram showing that DMSNs-NH₂ could load and protect plasmids from degradation by DNase I.

Transformation of DMSNs-NH₂-ASvicR

Antibiotic plate screening and fluorescence microscopy were used to determine whether DMSNs-NH₂-ASvicR entered S. mutans UA159 and was expressed successfully. As shown in Figure 3A, colonies grew on the plates after being coated with the bacterial solution containing plasmid-ASvicR or DMSN-NH₂-ASvicR, which had been cultured for 30 min, 3 h, and 6 h, respectively. However, there were no colonies on the plates coated with a bacterial solution cultured for 12 h and 24 h. It has been shown that UA159 and DMSNs-NH₂ have no fluorescence, and pDL278 cannot make UA159 express green fluorescence. However, plasmid-ASvicR and DMSNs-NH₂-ASvicR can both enter the bacteria, expressing green fluorescent protein (GFP) successfully (Figure 3B). In addition, Figure 3B and C show that the DMSNs-NH₂-ASvicR group expressed a higher intensity of green fluorescence than the naked plasmid-ASvicR group (P < 0.01).

Figure 3 (A) UA159 were cultured with deionized water, plasmid-ASvicR, DMSNs-NH2-ASvicR respectively for 30 min, 3h, 6h, 12h, and 24h, the mixed solution was coated onto the BHI solid medium added with spectinomycin, and the colony growth was observed after 48h of culture. (B) The expression of GFP in UA159 added with deionized water, pDL278 (the plasmid without ASvicR sequence and GFP), DMSNs-NH2, plasmid-ASvicR and DMSNs-NH2-ASvicR. (C) The fluorescence intensity of the group treated with plasmid-ASvicR and DMSNs-NH2-ASvicR; **P < 0.01, n = 3.

Effect of DMSNs-NH₂-ASvicR on Biofilm Formation of UA159

SEM and CLSM were used to evaluate the effects of DMSNs-NH₂-ASvicR on biofilm formation of S. mutans UA159. Figure 4 shows the SEM images of biofilms’ morphologies of different groups. Compared with the control group, the extracellular matrix of the 12-hour biofilms in the DMSNs-NH₂-ASvicR group lacked a three-dimensional structure of network crosslinking and bacteria were arranged loosely without morphological abnormality in S. mutans itself. The extracellular matrix of the plasmid-ASvicR and the DMSNs-NH₂ groups was reduced to some extent, but not obviously. However, the biofilms of the pDL278 group and DMSNs-NH₂ group show no significant differences from the control group. The 24-hour biofilms were generally denser than 12-hour biofilms. Even so, the biofilms of the DMSNs-NH₂-ASvicR group still lacked a dense structure. It could also be seen from the 24-hour biofilms of the DMSNs-NH₂-ASvicR group that nanoparticles clustered around S. mutans, and some nanoparticles even directly contacted the surface of S. mutans, which may be beneficial to plasmid transformation. The CLSM images of the 12-hour (Figure 5A) and 24-hour (Figure 5D) biofilms showed that EPS in the DMSNs-NH₂-ASvicR group decreased visibly, consistent with the SEM observations. The EPS biomass and EPS/bacteria ratio in the DMSNs-NH₂-ASvicR group were significantly lower than those in the control group (P < 0.0001). However, there was no significant difference in bacterial biomass (Figure 5B, C, E and F). Meanwhile, EPS biomass and EPS/bacteria ratio in the plasmid-ASvicR group also decreased but not as significantly as in the DMSNs-NH₂-ASvicR group.

Figure 4 Scanning electron micrograph of biofilms in which S. mutans UA159 were treated with deionized water, pDL278, plasmid-ASvicR, DMSNs-NH2, and DMSNs-NH2-ASvicR thrice and developed in brain heart infusion (BHI) supplemented with 1% sucrose for 12 hours and 24 hours. The partial enlarged view of the 24-hour biofilm in the DMSNs-NH2-ASvicR group shows that nanoparticles clustered around S. mutans, and some nanoparticles even directly contacted the surface of Streptococcus mutans.

Figure 5 (A and D) Double labeling image and three-dimensional visualization of EPS (red) and bacteria (green) in S. mutans biofilms after treated with deionized water, pDL278, plasmid-ASvicR, DMSNs-NH2, and DMSNs-NH2-ASvicR thrice respectively and developed in brain heart infusion (BHI) supplemented with 1% sucrose for 12 hours and 24 hours. (B and E) Quantitative data of bacterial and EPS biomass; **P < 0.01, ***P < 0.001, and ****P < 0.0001, n = 6. (C and F) Volume ratio of the exopolysaccharide (EPS) matrix to the bacterial biomass in biofilms.

Regulation of vicR Gene Expression in Streptococcus mutans

RT-qPCR was performed on mRNA extracted from 12-hour and 24-hour biofilms to demonstrate the regulatory effect of DMSNs-NH₂ on the target gene vicR. As shown in Figure 6A, the pDL278 and DMSNs-NH₂ groups show no effect on the expression of vicR. Nevertheless, the relative quantity of vicR was down-regulated by 0.74 times (P < 0.0001) in the 12-hour biofilms and 0.83 times (P < 0.0001) in 24-hour biofilms of the DMSNs-NH₂-ASvicR group. In addition, the level of vicR was also down-regulated by 0.91 times (P < 0.01) in the 12-hour biofilms of the plasmid-ASvicR group, with no significant difference in 24-hour biofilms compared to the control group. Supplementary Figure 3 is the result of Western blot of 24-hour biofilms, which is consistent with result of RT-qPCR. Figure 6B displays the mechanism of DMSNs-NH₂-ASvicR regulating biofilm formation of biofilms and the expression of vicR in S. mutans.

Figure 6 (A) qRT-PCR analysis of the vicR expression using gyrA as a reference gene and the group “UA159+deionized water” as control; **P < 0.01 and ****P < 0.0001, n = 4. (B) The working model of DMSNs-NH2-ASvicR entering S. mutans and regulating biofilm formation.

Cytotoxicity Assessment of DMSNs-NH₂-ASvicR

In vitro cytotoxicity of DMSNs-NH₂-ASvicR was evaluated by CCK-8 assay and live/dead staining assays. The fluorescence micrograph of live/dead staining assay showed that most HGFs were viable (green fluorescence) without morphological abnormality after being treated with different concentrations of DMSNs-NH₂-ASvicR, with very few dead cells (red fluorescence) similar to the control group (Figure 7A). As shown in Figure 7B, the influence of DMSNs-NH₂-ASvicR on absorbance at 450nm was excluded. DMSNs-NH₂-ASvicR had no adverse effects on the viability of HGFs after 12 and 24 hours of incubation, even at a concentration of 96 μg/μL (Figure 7C).

Figure 7 (A) Live/dead staining of Human gingival fibroblasts (HGFs) after 24 hours of incubation with different concentrations of DMSNs-NH2-ASvicR. (B) Excluding the influence of DMSNs-NH2-ASvicR on absorbance. (C) CCK-8 assay of human gingival fibroblasts (HGFs) treated by different concentrations of DMSNs-NH2-ASvicR; n = 4.

DMSNs-NH₂-ASvicR Decreased the Cariogenicity of S. mutans in vivo

A rat caries model was used to investigate the effects of DMSNs-NH₂-ASvicR on the cariogenicity of S. mutans in vivo. Figure 8 presents the stereomicroscopic images and Keys scoring results of rat mandibular molars. “E” scored 1 point, indicating that caries only involved enamel. “Ds” scored 2 points, meaning that caries involved no more than 1/4 of the dentin thickness. “Dm” scored 3 points, implying that caries involved 1/4 to 3/4 of dentin thickness. “Dx” scored 4 points, indicating that caries involved more than 3/4 of dentin thickness. The DMSNs-NH₂-ASvicR group had no apparent caries or only a few shallow carious cavities, and the total Keys score was significantly lower than the control group (P < 0.01, Figure 8B). There was also a decrease in caries in the plasmid-ASvicR group, however, with no statistical significance.

Figure 8 (A) Stereoscopic microscopy of pit fissure caries lesions on mandibular molars of rats. (B) Total Keys scores of caries on the first and second mandibular molars of rats. **P < 0.01, n = 10.

Discussion

It is well established that larger pore volume and surface area of nanoparticles provide more spaces for delivering biomacromolecules.^38,61 Studies have shown that mesoporous silica nanoparticles with smaller sizes could induce higher cell uptake.^54,62,63 In the present study, the bacteria were much smaller than cells; thus, nanoparticles with sufficiently small sizes were required. Therefore, we synthesized DMSNs-NH₂, with morphology similar to that reported in the literatures.^42,50,51 In addition, the particles were smaller in diameter (~59 nm), because we reduced the reaction time according to the literature.⁵⁰ The DMSNs-NH₂ has a large pore size of 8.1 nm, a surface area of 449.1 m²g⁻¹, and a pore volume of 0.8 cm³g⁻¹, which are beneficial for loading and protecting plasmid DNA and increasing the delivery efficiency.^38,54,64 It is worth mentioning that according to literature, mesoporous silica nanoparticles with a hollow architecture showed higher loading efficiency than those with the same mesoporous morphology but no hollow architecture.^44,65 This reminds us that we can add the hollow architecture based on DMSNs-NH₂ in future research, which might be more helpful in improving the efficiency of loading and transferring the plasmid.

The positive potential is essential to ensure the ability of DMSNs-NH₂ to encapsulate and deliver negatively charged plasmid-ASvicR.⁵² However, the average zeta potential of DMSNs-NH₂ synthesized in the present study was only 6.3 mV, which is lower than that reported in the literature.^37,54,66 Therefore, the efficiency of DMSNs-NH₂ loading plasmid-ASvicR was not high enough; thus, a higher mass ratio (DMSNs-NH₂/plasmid-ASvicR=80) is required to load plasmid-ASvicR completely. Hence, it is one of the goals in our future work to improve the amination method to increase loading efficiency. The ability of DMSNs-NH₂ to load and protect plasmid-ASvicR has also been demonstrated. When DMSNs-NH₂ was mixed with plasmid-ASvicR by mass at a ratio of 80, the plasmid-ASvicR could be loaded 92% and most plasmids could be protected from degradation.

It has been reported that bacteria can take up extracellular free DNA naturally.^67–69 The transformation assays in the present study indicated that the naked plasmid-ASvicR could enter S. mutans UA159. Furthermore, the plasmids loaded on the DMSNs-NH₂ could successfully enter S. mutans, express related genes, and exhibit higher transformation efficiency than naked plasmids. The possible reasons are as follows: (1) DMSNs-NH₂ increased the stability of the plasmids in the bacterial solution;⁵⁴ (2) DMSNs-NH₂ loaded with plasmid-ASvicR was prone to aggregation and adsorption by bacteria, which increased the chance of plasmid contacting the bacteria, promoting the entry of plasmids into the bacteria. However, the exact mechanism remains unknown, which is one of the limitations of the present study, necessitating further investigations. There were no colonies in the 12-hour and 24-hour group, perhaps because ASvicR entered S. mutans and interfered with the expression of vicR, which has been reported to be critical for viability,^8,19 thereby reducing their survival advantage. These findings imply that DMSNs-NH₂-ASvicR can enter the bacteria and affect the expression of related genes, but the effective time is short. That is why we treated the bacteria with samples every 4 hours in the subsequent biofilm formation assay.

Antibiotics have been used to fight bacterial infections for several decades. However, most antibiotics used to treat bacterial infections have several side effects and induce drug resistance.^70–72 Therefore, it is necessary to develop highly effective antibacterial agents to address this threatening resistance crisis. The results of biofilm formation assays and RT-qPCR indicate that pDL278 and DMSNs-NH₂ did not impact the cariogenicity of S. mutans compared to the blank control. However, DMSNs-NH₂-ASvicR could down-regulate the expression of vicR and reduce the productions of EPS by S. mutans, affecting the morphology and structure of the biofilm. Interestingly, although DMSNs-NH₂-ASvicR affected the biofilms, it did not change the morphology and quantity of bacteria itself, which might be conducive to maintaining the homeostasis of the microecological flora. The CLSM results showed that EPS of the plasmid-ASvicR group was also decreased, with no significant changes in the biofilm structure under SEM. One possible explanation was that although the naked plasmid-ASvicR could enter S. mutans and affect the synthesis of EPS, the transformation efficiency of the naked plasmid-ASvicR entering S. mutans was too limited to cause significant changes in biofilm morphology. Both DMSNs-NH₂-ASvicR and plasmid-ASvicR showed lower regulatory effects on the biofilm formation after 24 h, indicating that the frequency of treatment should be increased or a controlled release system should be used to achieve long-term ideal biofilm inhibitory effects.

For biomedical applications, the delivery system should possess functional performance and excellent biocompatibility.⁷³ The cytotoxicity experiments indicated that DMSNs-NH₂-ASvicR had no cytotoxicity, providing a basis for the subsequent application in vivo. The results of Keys scores showed that DMSNs-NH₂-ASvicR could reduce caries incidence in rats significantly, indicating that DMSNs-NH₂ could effectively protect the plasmid-ASvicR from degradation and facilitate its entry into the bacteria in the rat’s oral cavity. There was also a decrease in caries with no statistical significance in the plasmid-ASvicR group compared with the blank control. It was speculated that some plasmid-ASvicR could enter the bacteria before the plasmids were degraded by nuclease in the rat’s mouth. Preliminarily, the results indicated the regulatory effect of DMSNs-NH₂-ASvicR on the cariogenicity of S. mutans in vivo, laying a foundation for further studies. Nevertheless, there are some limitations in the animal experiment, necessitating more concentration gradients and longer research time.

Conclusion

DMSNs-NH₂ synthesized in the present study has been verified to possess the capacity of delivering plasmid-ASvicR and protecting them from degradation. With the protection of DMSNs-NH₂, plasmid-ASvicR could enter S. mutans and down-regulate the expression of the vicR gene, reducing the synthesis of EPS and affecting the formation of biofilm. In vivo experiments confirmed that DMSNS-NH₂-ASvicR could reduce the cariogenicity of S. mutans preliminarily. Nevertheless, the specific mechanisms of DMSNs-NH₂-ASvicR in regulating the S. mutans should be further explored. It is expected that DMSNs-NH₂-ASvicR can undergo clinical transformation to become a substance for dental caries prevention in the future.