Self-driven micromotors loaded with photosensitive adhesives and their application in dentin sensitivity

Abstract

Dentin hypersensitivity is primarily caused by the exposure of dentinal tubules due to various factors, so the key to treatment is to effectively seal these exposed tubules. However, traditional dentinal tubule sealants used in clinical practice often fail to adhere securely to the tubule surface when exposed to external stimuli, resulting in a recurrence of sensitivity. In this study, we developed a silicon micromotor that moved autonomously and loaded with silver nanoparticles and a photosensitive adhesive for dentin sensitivity therapy. These micromotors move autonomously to reach deep into the dentin tubules and surface loaded adhesives are solidified under blue light. The compact structure formed by the cross-linking of micromotors effectively seals the dentin tubules from the inside to the outside, and also forms a firm bond between the micromotor and the inner layer of the dentin, thereby improving the sealing effect and providing strong protection. Silver nanoparticles on the surface of micromotors can slowly release silver ions, effectively inhibiting the growth of caries-causing bacteria such as S. mutans, and preventing secondary caries. Our research demonstrates that the closure rate of dentinal tubules after treatment can reach 79.17% with a closure depth of 17.22 μm, while also withstanding various stimuli without detachment. In conclusion, the use of self-propelled micromotors presents a promising new strategy for treating dentin hypersensitivity in clinical settings.

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1. Introduction

When dentin is exposed to various stimuli, patients experience short-term sharp pain during daily activities such as eating, brushing, or drinking, significantly impacting their quality of life.^1–4 Currently, numerous clinical treatments for dentin hypersensitivity exist, including pharmacological interventions and various techniques, such as applying sealants to the dentinal tubules or utilizing lasers to seal the tubules, and so on.^5–7 Among these, the application of Gluma adhesive is the most commonly employed method in clinical practice, demonstrating the best immediate results.⁸ However, these techniques primarily seal the surface of the dentinal tubules and do not penetrate deeply, resulting in only a temporary seal. We found that these conventional materials usually cover the dentin tubule surface rather than penetrate into the tubule, and the sealing deposits can easily fall off the tooth when stimulated by mechanical forces, leading to secondary tooth decay or irreversible damage to the tooth. Therefore, our goal is to develop a drug delivery system that can deliver therapeutic agents deep into the dentin tubules, thereby improving the long-term stability of treatment to prevent secondary dental caries.

In recent years, self-motion micro/nanomotors have received widespread attention due to their good biosecurity and high delivery efficiency.^9–11 A micro/nanomotor is a micro or nanoscale device that is able to move autonomously to a specific location depending on the specific application in the presence of a chemical fuel (i.e. hydrogen peroxide, glucose, urea, etc.) or an externally controlled propulsion mechanism.^12–14 Chemically driven micro–nano motors have garnered significant attention among various types of micro–nano motors due to their strong integration with clinical applications and their ease of use.^15,16 Chemically driven micro–nanomotors can be propelled by the recoil generated from fuel-decomposed bubbles, exemplified by Au and Pt bimetallic nanomotors.¹⁷ In particular, under hydrogen peroxide conditions, the platinum end of the nanomotor catalyzes the decomposition of hydrogen peroxide, resulting in the generation of oxygen bubbles. The released bubbles create a reverse effect, thereby pushing the motor in the opposite direction.¹⁸ Additionally, chemically driven micro–nanomotors can exhibit movement through self-diffusive penetration, but their speed, efficiency, and propulsion are nowhere near as good as bubble-driven systems.^19–21 Furthermore, hydrogen peroxide is widely used in the treatment of oral diseases,²² thus chemically driven micromotors powered by hydrogen peroxide are considered particularly suitable for applications in oral drug delivery. Gluma nanoparticles with a motor can effectively help in preventing secondary caries. Additionally, silver nanoparticles have been demonstrated to possess strong antibacterial properties.²³ The combination of silver nanoparticles with a motor can effectively help in preventing secondary caries.

In this study, we developed a self-driven micromotor (SSN-Ag@Pt-GLM) composed of a silica core, a silver nanoparticle layer, and a platinum and GLM adhesive shell as an active treatment for dentin sensitivity. We used the sol–gel method to prepare a symmetrical spherical silica substrate and then loaded the silver nanoparticle layer, platinum layer, and adhesive layer on the surface of the microspheres using the redox method, asymmetric modification technology, and co-precipitation method. The power of SSN-Ag@Pt-GLM comes from the oxygen bubble generated by the decomposition of hydrogen peroxide on the Pt side. Micromotors have strong autonomous motor ability and move autonomously and efficiently in the oral physiological environment, even resisting the fluid outflow from dentin tubules into the deeper dentin tubules. After the micromotor entered the dentinal tubules, the micromotors are tightly bonded to each other using the GLM adhesive upon blue light irradiation. The GLM layer solidifies and seals the dentinal tubules and slowly releases silver ions with antibacterial properties. The biosafety and sealing effect of the micromotors were further evaluated, and the bacteriostatic effect of silver ions was evaluated using Streptococcus mutans, a typical cariogenic bacterium in the oral cavity. This micromotor, which combines good immediate efficacy, good penetration depth, good stability, good biosafety, and effective prevention of secondary caries, proposes new strategies and methods for the active treatment of dentin sensitivity. The adhesive exhibits excellent performance and sending it into dentin tubules by micromotors will get a more optimal treatment outcome.

2. Experimental

2.1. Materials

(3-Mercaptopropyl)trimethoxysilane (MPTMS, 97%), ammonia solution (NH₄OH, 25–28%), sliver nitrate solution (AgNO₃, 0.1 mol L⁻¹), and ethanol absolute (C₂H₆O, ≥99%) were purchased from MACKLIN, Shanghai. GLUMA Comfort Bond was purchased from Wehrheim, Germany. Artificial saliva (ASL), Ringer's solution, and Cell Counting Kit-8 (CCK-8) were purchased from Phygene, Fuzhou. Human gingival fibroblasts (HGFs) were purchased from ATCC. The teeth were obtained from the Second Affiliated Hospital of Harbin Medical University and were approved by the Medical Ethics Committee. All laboratory animals were purchased from the animal experiment center of the Second Affiliated Hospital of Harbin Medical University and were approved by the Animal Ethics Committee of the Harbin Medical University, Harbin, China (2024GZRYS-061).

2.2. Synthesis of SSN-Ag@Pt-GLM

According to the referenced method,²⁴ silica microspheres rich in sulfhydryl groups were prepared using the sol–gel technique. In particular, 1 g of MPTMS was added to 100 mL of deionized water, and the pH was adjusted to approximately 11 using NH₄OH. The mixture was stirred in a nitrogen atmosphere at room temperature at a speed of 400 rpm for 48 hours. Following this, a high-speed centrifuge was employed for precipitate separation. The microspheres were then washed three times with water and ethanol, followed by vacuum drying at room temperature to obtain the SiO₂ microspheres.

The SiO₂ microspheres obtained were weighed at 20 mg and then dispersed in a 0.1 mol L⁻¹ silver nitrate solution. The dispersion was continuously stirred at 400 rpm for 1 hour at room temperature, followed by three consecutive washes with water and ethanol. Finally, the resulting SiO₂@Ag NPs powder was stored in an ethanol solution at room temperature. The obtained SiO₂@Ag NPs were evenly dispersed on the glass sheet to form a single layer of particles. Under the pressure of 2.5 mTor, platinum was plated on the surface of the particles using a magnetron vacuum sputtering instrument with a power of 200 W. The Janus particles were carefully collected, washed with water and ethanol three times in turn, and stored in ethanol solution at room temperature. The resulting Janus particles were evenly dispersed on the glass sheet to form a single layer of particles. 0.0205 g of Gluma adhesive was added into 10 mL of anhydrous ethanol, thoroughly mixed, and co-precipitated with the glass sheet for 12 h without avoiding light. The SSN-Ag@Pt-GLM particles were carefully collected and cleaned with water repeatedly 3 times. The obtained SSN-Ag@Pt-GLM particles were stored in an aqueous solution at 4 °C away from light for future use.

2.3. Characterization of particles

Scanning electron microscopy (SEM, HITACHI SU5000) and transmission electron microscopy (TEM, Thermo Fisher Scientific Talos 200X S) were used to observe the morphology of particles, and energy dispersive X-ray spectroscopy (EDS, HITACHI SU5000) was used to determine the distribution of elements. A laser particle size analyzer (Zetasizer Lab, Malvern) was used to measure the mean particle size and ZETA potential. The percentages of elements and atoms were determined using X-ray photoelectron spectroscopy (XPS). The composition of the materials was analyzed through thermogravimetric analysis (TGA). The particles were analyzed by X-ray diffraction (XRD, PANalytical B.V. X’Pert PRO).

2.4. Motion analysis of SSN-Ag@Pt-GLM

An appropriate amount of SSN-Ag@Pt-GLM was dispersed in water. The dispersion and fuel (1 : 1) were added to the surface of the slides together, and the SSN-Ag@Pt-GLM trajectory was observed using an inverted optical microscope (OLYMPUS IX 71). At the same time, a video was recorded at 41 frames per second to observe the trajectory of SSN-Ag@Pt-GLM in different concentrations of fuel (0.5% H₂O₂, 1% H₂O₂, 3% H₂O₂) with the same medium water. SSN-Ag@Pt-GLM was studied at different adhesive thicknesses, with 3% hydrogen peroxide as fuel, and SSN-Ag@Pt-GLM was studied in different media (ASL and Ringer's solution) with 3% H₂O₂ as fuel. ImageJ was used to analyze the movement trajectory of randomly selected 35 particles over 6 s and then draw the time–MSD curve based on previous research.²⁵

2.5. In vitro toxicity test

HGnFs were selected to detect the in vitro cytotoxicity of SSN-Ag@Pt-GLM using the CCK-8 kit. HGFs (100 μL per well) were inoculated in 96-well plates and cultured for 24 h (at 37 °C, 5% CO₂). Then, 10 μL of SSN-Ag@Pt-GLM with different concentrations (1.25 mg mL⁻¹, 2.5 mg mL⁻¹, 5 mg mL⁻¹, 10 mg mL⁻¹) or normal saline were added and incubated at 37 °C for 24 hours, and 10 μL of the CCK-8 solution was added to each well. After incubation for 3 hours, the absorbance (450 nm) of each group was detected. The experiment was repeated 3 times before calculating the cell viability and drawing the image.

2.6. In vivo toxicity test

Female BALB/c mice (4–6 weeks old) were selected to test the toxicity of SSN-Ag@Pt-GLM. 20 mice were randomly divided into two groups and marked as the SSN-Ag@Pt-GLM group and the control group. The mice in the SSN-Ag@Pt-GLM group were given the SSN-Ag@Pt-GLM dispersion via gavage for three consecutive days, and the mice in the control group received normal saline using the same method. After administration, the mice were weighed for 10 consecutive days and then arterial blood was collected for blood routine examination. Mice were sacrificed after collecting blood. The main organs (heart, liver, spleen, lung, and kidney) were collected and subjected to hematoxylin and eosin (H&E) staining.

2.7. In vitro dentin tubule blocking experiment

We prepared isolated dentin discs according to the reported method,^26–28 and freshly extracted maxillary third molars aged 20–40 years old were selected, disinfected after scraping the soft tissue, soaked in normal saline, and stored at 4 °C. The excess dental tissue was removed using a grinding wheel and a 2 mm thick dentin disc was prepared. The discs were stored at 4 °C in normal saline after EDTA etching.

The dentin tubule is filled with dentin fluid, which is constantly drained due to the presence of pressure in the pulp cavity. To construct the dentin pressure model, Ringer's fluid was used to simulate the dentin tubule fluid, and the pressure of the pulp cavity was simulated by hydraulic pressure according to previous studies.^29–32 Each dentin disc was fixed on the polymer cover and sealed on all sides with wax to make sure no liquid flowed except across the surface of the dentin disc. Pulp cavity pressure was controlled by adjusting the distance between the fluid level and the dentin disc to 27 cm height. Prior to our experiments, each fixed dentin disc was soaked in Ringer's solution and vacuumed for 20 min to make sure that the disc was filled with liquid.

Take an appropriate amount of SSN-Ag@Pt-GLM or SSN@Ag and disperse in water, respectively. 10 μL of dispersion and fuel were added to the dentin disc pressure model and kept for 30 min as a record of 1 treatment. Then remove the dentin disc, gently brush with a toothbrush 50 times, rinse alternately with ethanol and water and all the treatments were repeated 3 times. Taking 3 dentin discs in each group, capturing SEM images of the SSN-Ag@Pt-GLM group and SSN-Ag@Pt-GLM group after 1 or 3 treatments, 10 images were randomly taken from each dentin disc. If a single dentin tubule is blocked by more than 50%, it is recorded as a blocking success. As long as particles enter the dentin tubule, it should be recorded as an entry. Count each image and calculate the blocking rate and entry rate, then determine average values.

2.8. Dentin disk performance test after treatment

According to the reported methods,⁴ ultrasonic, tape stripping, acid resistance, and micro-dentin hardness tests were used to study the stability of dentin disk surface materials after treatment. The sealing effect of the dentin disk was observed after 3 min of treatment with intense ultrasonic waves (50 kHz, 200 W). The commercial 3 M tape was used to peel the dentin disk, and the sealing effect of the disk was observed after three repeated peelings. The sealing effect of the dentin disk after treatment was observed after 30 min of treatment with a citric acid solution with pH = 2.0. The sample was polished moderately and then micro-etched, and the scratches on its surface were calculated via scanning electron microscopy. The results were compared.

2.9. Study on the antibacterial effect of SSN-Ag@Pt-GLM

To study the release ability of silver ions and platinum from TSP-Ag@Pt-GLM micromotors in dentinal tubular fluid, Ringer's solution (simulated dentinal tubular fluid) was used as a solvent at 37 °C in a dark environment. The same volume of TSP-Ag@Pt-GLM micromotors was mixed with 10 ml of Ringer's solution and the supernatant was collected after 0, 3, 6, 12, 24, 48 and 72 hours. All samples obtained were diluted 1000 times and analyzed by ICP to determine the concentration of free silver ions and platinum ions in the samples. The concentrations of silver and platinum ions were calibrated using commercial SPEX Certified Company inorganic standards prior to analysis. The above experimental results were statistically plotted and analyzed.

According to previous reports,³³ the Gram-positive bacterium Streptococcus mutans was selected as the representative, and the antibacterial activity of SSN-Ag@Pt-GLM was verified through an antibacterial ring experiment. 100 μL of water, SSN@Ag and SSN-Ag@Pt-GLM water-dispersed droplets with appropriate concentrations were added to blank drug-sensitive tablets with a diameter of 6 mm, dried at 37 °C for 24 h, and disinfected under ultraviolet light for 30 min. A 50 μL suspension of 10⁻⁶ CFU was coated on solid medium, the drug-sensitive tablet was placed in the center of the medium, and the size of the antibacterial ring was observed after incubation in an anaerobic environment for 16 h, and the average value was repeated 3 times.

An appropriate amount of medium was added into the 10⁻⁶ CFU suspensions of the three groups, and the medium dispersions of SSN@Ag and SSN-Ag@Pt-GLM were co-cultured in an anaerobic environment, respectively. Suspensions with different contact times of 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, and 24 h were respectively taken for the absorbance test, and the values at OD630 were repeated three times to draw the OD value curve. Dentin discs and glass plates were incubated with 10⁻⁶ CFU suspension solution for 36 h, and PI and calcein AM were used for staining. The dead or alive status of bacteria was observed under a fluorescence microscope and photographs were recorded to observe the changes in mortality. Dentin plates and glass plates were incubated with 10⁻⁶ CFU suspension solution for 36 h and fixed, and gradient dehydration was carried out. The treated samples were dried and sprayed with gold, and then observed using a scanning electron microscope (SEM).

2.10. Statistical analysis

All data are expressed as means ± standard deviations (S.D.). A one-way analysis of variance followed by the t-test was applied to all statistical comparisons.

3. Results and discussion

3.1. Synthesis and characterization of SSN-Ag@Pt-GLM

SSN-Ag@Pt-GLM was synthesized using the sol–gel method, REDOX method, magnetron sputtering method, and coprecipitation method (Fig. 1a). The scanning electron microscopy (SEM) image (Fig. 1b) reveals that SSN-Ag@Pt-GLM is spherical and has an obvious asymmetric structure. The energy dispersive spectroscopy (EDS) analysis, also shown in Fig. 1b, indicates a high concentration of platinum in the upper right region of the particle, confirming the successful deposition of platinum. Additionally, a single crystal calibration of Ag was performed using selective area electron diffraction (SAED) by TEM and the transmission electron microscopy (TEM) image (Fig. 1c) illustrates that the surface of the particles is densely covered with numerous silver nanoparticles, each approximately 5 nm in size, with local particles exhibiting distinct characteristic lattice structures when magnified. In addition, EDS images of transmission electron microscopy (TEM) (Fig. 1c) also showed that silver was deposited on the surface of the particle, platinum was deposited on the lower right side of the particle, and carbon was deposited on the upper left corner of the particle, confirming the successful synthesis of the motor. Additionally, the transmission electron microscopy (TEM) image (Fig. 1d) illustrates that the surface of the particles is densely covered with numerous silver nanoparticles, each approximately 5 nm in size, with local particles exhibiting distinct characteristic lattice structures when magnified. The infrared spectral analysis image (Fig. 1e) shows that the silica microspheres prepared using the sol–gel method have a strong sulfhydryl characteristic peak at 2558 cm⁻¹, which confirms the presence of a large number of sulfhydryl groups on the surface and it is a vital foundation for further in situ reduction of silver nanoparticles. Fig. 1f shows the particle size distribution of SSN-Ag@Pt-GLM. It shows a normal distribution trend and the widest distribution at 980 nm. Combined with SEM images, the sample morphology is uniform, and the particle size is stable. XPS analysis images (Fig. 1g) show characteristic peaks of elements (O, Ag, C, Si, and Pt) in SSN, SSN-Ag, SSN-Ag@Pt and SSN-Ag@Pt-GLM, in which the Pt and Ag elements of SSN-Ag@Pt-GLM are diluted due to the introduction of adhesives, resulting in a decrease in characteristic peaks. TGA analysis images are shown in Fig. 1h. The products in all stages showed a downward trend. There are two rapid weight loss stages in SSN, which is due to the volatilization and decomposition of the crystalline water and sulfhydryl groups. The curve of SSN-Ag's decline rate showed a downward trend, and the weight loss step became less obvious. This is due to the increasing proportion of heat-stable components. The overall trend of the SSN-Ag@Pt curve was like that observed before modifications, and the proportion of the final residual product was slightly increased with the addition of heat-stable components. The curve of SSN-Ag@Pt-GLM showed rapid decline stages, and the proportion of residual products decreased. This is due to the addition of GLM, which increases the proportion of organic matter, leading to rapid decomposition upon heating. XRD analysis images (Fig. 1i) show that silver characteristic peaks appear at 38 and 44 (PDF#00-004-0783), and platinum characteristic peaks appear at 40, 46, and 67 (PDF#00-004-0802). Fig. 1j shows the analysis image of the particle zeta potential change during the preparation process. The zeta potential distribution curves of the preparation process are shown in Fig. S1 (ESI†). The zeta potential of SSN is −25.09 mV, SSN@Ag is −18.78 mV, SSN-Ag@Pt is −7.905 mV, and SSN-Ag@Pt-GLM is −11.382 mV, and it can be observed that the potential of the particle surface changes after each reaction step. These results prove that we have successfully prepared SSN-Ag@Pt-GLM.

Fig. 1 Preparation and characterization of the SSN-Ag@Pt-GLM. (a) Preparation of the process flow diagram. (b) SEM images of SSN-Ag@Pt-GLM and EDX images of element distribution of Ag, Pt, and C. (c) TEM SAED image of SSN-Ag@Pt-GLM and its surface lattice. (d) TEM images of SSN-Ag@Pt-GLM and EDX images of element distribution of Ag, Pt, and C. (e) FTIR analysis of SSN, SSN-Ag, SSN-Ag@Pt and SSN-Ag@Pt-GLM. (f) Average size distribution of SSN-Ag@Pt-GLM. (g) XPS analysis of SSN, SSN-Ag, SSN-Ag@Pt and SSN-Ag@Pt-GLM. (h) TGA analysis of SSN, SSN-Ag, SSN-Ag@Pt and SSN-Ag@Pt-GLM. (i) XRD analysis of SSN-Ag@Pt-GLM. (j) ZETA potential of SSN-Ag@Pt-GLM.

3.2. Motion analysis

The motion unit of SSN-Ag@Pt-GLM is the Pt layer, which catalyzes the decomposition of hydrogen peroxide to produce oxygen bubbles. Then the recoil pushes micromotors in the opposite direction producing a self-propelled motion. In particular, the motion trajectory of SSN-Ag@Pt-GLM in different media was observed to study the motor performance. Fig. 2a shows a model diagram of the motion mechanism in which SSN-Ag@Pt-GLM's self-propelled motion occurs. The Pt layer on the surface of SSN-Ag@Pt-GLM acts as a catalyst to promote the decomposition of hydrogen peroxide, and the O₂ generated by the decomposition of hydrogen peroxide acts as the driving force to promote SSN-Ag@Pt-GLM to move in the opposite direction of the generation of O₂, thus realizing the self-driven motor motion.

Fig. 2 Motion analysis of SSN-Ag@Pt-GLM. (a) SSN-Ag@Pt-GLM motion simulation diagram. (b) Motion trajectories of SSN-Ag@Pt-GLM in different media. (c) MSD analysis of SSN-Ag@Pt-GLM motion in different media. (d) Velocity analysis of SSN-Ag@Pt-GLM in different media.

We have studied and analyzed the motion ability of SSN-Ag@Pt-GLM in different media. We first studied the motion of SSN-Ag@Pt-GLM in 0.5% H₂O₂, 1% H₂O₂, 3% H₂O₂, 3% H₂O₂ + Ringer's fluid and 3% H₂O₂ + artificial saliva, respectively. Fig. 2b shows the motion path in different media, and it can be observed that the length of the motion path of the motor increases with the increase of H₂O₂ concentration. It is worth noting that when Ringer's solution and artificial saliva are added to 3% H₂O₂, the length of the motor trajectory is also shortened due to the increase in the viscosity of the liquid. The viscosity of artificial saliva is greater than that of Ringer's solution, and the motor's motion trajectory is also shorter.

The MSD (mean azimuth shift) analysis of the motor in different media confirms the motor's autonomous motion ability. In the absence of H₂O₂ (i.e. fuel), the mean azimuth shift of the motor is linear in time, so we can judge that it is in Brownian motion, that is, no autonomous motion occurs, which is the same result as previously reported.^34–36 When the motor is added to the H₂O₂ environment, the mean azimuth shift of the motor presents a parabolic distribution in time, further confirming the autonomous movement of the motor, and the speed of the movement increases with the increase of the fuel concentration (Fig. 2d). In the case of 3% H₂O₂ as fuel, the MSD curve presents the most obvious parabolic distribution. It's worth noting that the curves of 0. 5% and 1% H₂O₂ are smoother than 3%, but the curve distribution can still indicate the autonomous movement of the motor in it. According to the research results, the motor has the fastest motion speed in 3% hydrogen peroxide, so we chose 3% hydrogen peroxide as the fuel for the subsequent experiment.

To ensure that SSN-Ag@Pt-GLM can still occur in the complex oral environment of self-propulsion movement, we used artificial saliva and Ringer's fluid to simulate motor movement in saliva and dentin tubule fluid. In the mixture of these two solutions and 3% H₂O₂, the motor can still maintain autonomous motion and maintain a certain motion rate (Fig. 2c and d). Since the viscosity of artificial saliva is greater than that of Ringer's fluid, the motor moves faster in Ringer's fluid than in artificial saliva. Under the condition of no external stimulation, the dentin tubule fluid maintained a slow outflow rate of 0.35 μL cm⁻² per minute.³⁷ We assumed that the diameter of each dentin tubule was 3 μm. After calculation (v = Q/S, Q is the flow rate, S is the cross-sectional area), the fluid flow rate in a single dentin tubule was 1.28 μm s⁻¹, much lower than the flow rate measured in Ringer's fluid (0.73 μm s⁻¹ from MSD). Therefore, we hypothesized that SSN-Ag@Pt-GLM can resist the outflow of dentin tubule fluid into the dentin tubule.

3.3. SSN-Ag@Pt-GLM motor biosafety test

Because after treatment, SSN-Ag@Pt-GLM will stay in the dentin tubules and block them, so we verified the biosafety of SSN-Ag@Pt-GLM. Human gingival fibroblasts (HGnFs) were selected to verify the effect of the motor on cell activity due to the contact of the motor with gingival tissue during treatment. First, we verified the in vitro safety of SSN-Ag@Pt-GLM. As shown in Fig. 3a, we calculated the cell viability of the cells after they were co-incubated with 1.25 mg mL⁻¹, 2.5 mg L⁻¹, 5 mg mL⁻¹, and 10 mg L⁻¹ SSN-Ag@Pt-GLM for 24 h. The cell activity in coculture for 24 hours was 95.64%, 112.48%, 113.45%, and 115.80% (Table S1, ESI†). There was no significant difference in cell viability between the experimental group and control group (P > 0.05).

Fig. 3 SSN-Ag@Pt-GLM motor biosafety test. (a) Cytotoxicity tests at different concentrations of SSN-Ag@Pt-GLM at different times (ns: no statistical difference; P > 0.05, *: statistical difference, P < 0.05; **: extremely significant statistical difference, P < 0.01). (b) and (c) The blood routine indexes of mice in the control group and SSN-Ag@Pt-GLM group: leukocyte, lymphocyte, monocyte, percentage of neutrophil, hemoglobin, erythrocyte, hematopoietic volume, and platelets, which were compared with SSN-Ag@Pt-GLM. (d) Body weight change of mice in 13 days in the control group and SSN-Ag@Pt-GLM group. (e) Comparison of HE staining in vital organs of mice between the control group and SSN-Ag@Pt-GLM group.

Then, we verified the in vivo safety of SSN-Ag@Pt-GLM. Twenty female BALB/c mice were selected separately for verification. The experimental mice were given 5 mg mL⁻¹ SSN-Ag@Pt-GLM by gavage. After the completion of drug administration, we collected arterial blood of mice for routine blood testing, and the test results are shown in Fig. 3b and c. We conducted the statistical analysis of the results, and there was no significant difference between the experimental group and the control group, which was not statistically significant (P > 0.05). At the same time, we also recorded the weight changes of mice before and after administration. As shown in Fig. 3d, no significant weight changes were observed in both groups of mice, indicating that SSN-Ag@Pt-GLM was not associated with acute toxicity. Finally, we collected the vital organs of the two groups of mice for H&E staining, and the results are shown in Fig. 3e. Compared with the control group, the vital organs of the experimental group showed no significant histological changes. The above results all proved that 5 mg mL⁻¹ SSN-Ag@Pt-GLM had sufficient biosafety and could be used in the following studies.

3.4. In vitro dentin blockage test

According to the previous reports,^29,31 the dentin tubules were filled with dentin tubule fluid and were continuously drained due to the internal pressure of the pulp cavity. Therefore, we designed a hydraulic model and used Ringer's fluid to simulate the continuous outflow of dentin tubule fluid. Fig. 4b reveals the dentin tubule pressure model. We used EDTA acid-etched dentin disks to construct dentin sensitivity models. Fig. 4c and e show the SEM images of the transverse and longitudinal sections of the dentin tubules in the dentin sensitivity model. The model was treated with SSN-Ag@GLM plus 3% H₂O₂ and SSN-Ag@Pt-GLM plus 3% H₂O₂ for one or three cycles, respectively.

Fig. 4 In vitro dentin blockage test of SSN-Ag@Pt-GLM. (a) Schematic diagram of simulated occluding dentin tubules SSN-Ag@Pt-GLM in vitro. (b) Diagram of the hydraulic device for simulating dentin tubule outflow. (c) Cross-sectional SEM images of multiple dentin tubules before treatment. (d) Post-treatment cross-sectional SEM images of multiple dentinal tubules. (e) SEM images of longitudinal sections of multiple dentin tubules before treatment. (f) SEM images of longitudinal sections of multiple dentin tubules after treatment. (g) Statistical images of entry and blocking rates after 1 or 3 treatments using TSP@Ag-GLM and SSN-Ag@Pt-GLM. (ns: no statistical difference; P > 0.05, *: statistical difference, P < 0.05; **: extremely significant statistical difference, P < 0.01) (h) and (i) SEM images of a single dentin tubule before and after treatment. (j) and (k) SEM images of longitudinal sections of single dentin tubules before and after treatment (above is before treatment, below is after treatment for 3 times).

Then, we studied occlusions of the dentin disk after 1 or 3 treatments with SSN-Ag@GLM and SSN-Ag@Pt-GLM, respectively. Fig. S2a and b (ESI†) show the transverse and longitudinal SEM images of the dentin disc after the first treatment. Due to the low concentration of the drug used in the first treatment, the dentin tubules in both groups remained largely open after treatment with the drug. However, compared with the SSN-Ag@Pt-GLM treatment group, the blank rate of the SSN-Ag@GLM group was higher, and most particles were located around the tubules but did not enter the interior of the tubules. These results all indicated that SSN-Ag@Pt-GLM could actively enter the interior of the tubules. Fig. S2c and d (ESI†) show the transverse and longitudinal section SEM images of the dentin discs after three treatments. Compared with the SSN-Ag@GLM group, almost all dentin tubules in the SSN-Ag@Pt-GLM group were full of SSN-Ag@Pt-GLM particles, and few particles were located around the tubules.

For a more intuitive observation of the blockage of the dentin disk, we calculated the occlusion rate of the dentin tubule after 1 and 3 treatments, respectively (Fig. 4g). After 1 treatment, the closure rate in the SSN-Ag@Pt-GLM group (16.05%) was higher than that in the SSN-Ag@GLM group (2.05%) and 7.8 times that in the SSN-Ag@GLM group, but there was no significant difference between the groups (P < 0.05). The admission rate of the SSN-Ag@Pt-GLM group (76.98%) was higher than that of the SSN-Ag@GLM group (27.29%), and 2.8 times that of the SSN-Ag@GLM group. There were significant differences between the groups and within groups (P < 0.05). Due to the small number of particles, the effect of sealing is not ideal. When the treatment times were increased to 3 times, the closure rate was apparently increased, with the closure rate in the SSN-Ag@Pt-GLM group being 79.17%, compared to 28.98% in the SSN-Ag@GLM group, with the closure rate in the SSN-Ag@Pt-GLM group being 2.7 times that of the SSN-Ag@GLM group. The results were different between and within the groups. The difference was statistically significant (P < 0.01). The admission rate of the SSN-Ag@Pt-GLM group (94.63%) was higher than that of the SSN-Ag@GLM group (59.15%), and 1.6 times that of the SSN-Ag@GLM group. There were significant differences between and within the groups (P < 0.01). These results show that the motor has the ability of autonomous movement and can reverse into the dentin tubule under the condition of outflow of the dentin tubule and has a good sealing effect.

Subsequently, we conducted an entry depth analysis on five longitudinal profile images of dentin tubules after three treatments and obtained the following results: the average entry depth of the motor could reach 54.35 μm, as shown in Fig. 4k, and the motor could enter the interior of the dentin tubules and achieve a good blocking effect.

3.5. Dentin disk hardness test after treatment

We investigated the adhesion stability of SSN-Ag@Pt-GLM on the dentin disk through tape, ultrasound and acid stripping experiments. Similarly, we also studied the physical properties of dentin discs before and after treatment with SSN-Ag@Pt-GLM using a dentin microhardness test. After the commercial 3 M tape stripping experiment, it was found that most of SSN-Ag@Pt-GLM was still firmly attached to the dentin disc (Fig. 5a and b), and its adhesion strength was higher than 0.47 N mm⁻¹. We also carried out intensive ultrasonic treatment (40 Hz, 200 W) on the dentin discs after three treatments, and most of the SSN-Ag@Pt-GLM remained still firmly attached to the dentin discs (Fig. 5c and d). In addition, we treated the dentin discs after three treatments with citric acid (PH = 2.0, 30 min) in a simulated acidic environment as well. SSN-Ag@Pt-GLM remained stable after treatment, and no dentin tubules were found to reopen (Fig. 5e and f).

Fig. 5 Dentin disk performance test after treatment. (a) and (b) Typical SEM images of typical dentin discs before and after 3 treatments, followed by 3 M Scotch tape (repeated 3 times) for the peel experiment. (c) and (d) Typical SEM images of the dentin disc after 3 treatments before and after 3 minutes of ultrasound in a water bath (40 Hz, 200 W). (e) and (f) Typical SEM images of dentin discs after 3 treatments soaked in the citric acid solution (PH = 2.0) for 30 minutes. (g) and (h) Typical SEM image of the dentin disc with scratches under 49.94 N (t = 10 s) before and after 3 treatments. (i) The statistical chart of scratches.

To gain more insight, we studied the hardness changes of the dentin disk before and after the treatment. The surface of the dentin disk was properly polished and fixed, and the surface was microengraved respectively under the same conditions (F = 49.04 N, t = 10 s), and then SEM images were taken to observe the size of surface scratches. It can be observed from Fig. 5g and h that the scratches on the surface of the dentin disk after treatment are significantly smaller than those of the untreated one. To quantify the change of microhardness, we used the following formula,

to calculate and analyze specific results, where HV is the Vickers hardness, F is the test force in Newtons (N), and d (μm) represents the arithmetic mean of the two diagonal lines d1 and d2. The Vickers hardness (42.72 N, no dimensional unit) of the dentin disk after treatment with SSN-Ag@Pt-GLM 0.42 N, GPa unit was much higher than the Vickers hardness of the dentin disk before treatment (31.99 N, no dimensional unit. 0.31 N, GPa unit), and 1.35 times of the latter, and there was a significant difference between the groups and the difference was statistically significant (p < 0.05). The above results showed that SSN-Ag@Pt-GLM could be firmly attached to the dentin disk and strengthen and improve the hardness of the dentin disk.

3.6. Antibacterial experiment in vitro

The antibacterial effect of SSN-Ag@Pt-GLM comes from Ag⁺, so the Ag⁺-release ability of SSN-Ag@Pt-GLM in artificial saliva was first investigated. SSN-Ag@Pt-GLM was incubated in Ringer's fluid at 37°, and then the supernatant was collected at a specific time to measure the Ag⁺ content by inductively coupled plasma. The Ag⁺ in SSN-Ag@Pt-GLM can be released slowly in artificial saliva, as shown in Fig. 6a. With the increase of time, Ag⁺ in the supernatant continued to increase, significantly increasing at 4–8 hours, and then slowly increasing in the time following. To exclude the influence of the power unit on the antibacterial action of SSN-Ag@Pt-GLM, we also tested the release ability of platinum ions. These results are shown in Fig. S3 (ESI†), and there is almost no platinum ion release, which is attributed to the chemical stability of platinum. Due to the coating of the adhesive on the surface of the motor, Ag⁺ can be released within 12 h to achieve the effect of slow release, to maintain a long-term bacteriostatic effect. The fully released silver ion content of each therapeutic dose of SSN-Ag@Pt-GLM is about 0.029 mg, which is much lower than the daily intake of silver ions in normal adults (70 kg) given in EPA's National Center for Environmental Assessment reported (Silver; CASRN 7440-22-4): 0.98 mg. So the treatment is very safe.

Fig. 6 In vitro antibacterial activity of SSN-Ag@Pt-GLM. (a) SSN-Ag@Pt-GLM in vitro silver ion release rate. (b) TPY AGAR medium showing inhibition rings of S. mutans in the 5 mg mL⁻¹ blank control group, SSN@Ag group, and SSN-Ag@Pt-GLM group. (c) OD growth curves of S. mutans in the 5 mg mL⁻¹ blank control group, SSN@Ag group, and SSN-Ag@Pt-GLM group. (d) Live/dead staining images of S. mutans biofilm cultured for 36 h with the 5 mg mL⁻¹ blank control group, SSN@Ag group and SSN-Ag@Pt-GLM group. (e) SEM images of S. mutans biofilms on glass plates(left) and dentin disc(right) after co-culture with the 5 mg mL⁻¹ blank control group, SSN@Ag group and SSN-Ag@Pt-GLM, respectively, for 36 h.

Furthermore, the antibacterial action of SSN-Ag@Pt-GLM was studied by model bacteria in vitro. The results of the antibacterial ring showed (Fig. 6b) that the antibacterial ring of the SSN-Ag@Pt-GLM group and SSN@Ag group was more clear and effective against the standard strains of S. mutans than the blank control group, and the diameters of the antibacterial rings were (1.015 ± 0.28) mm, (1.102 ± 0.102) mm, and (0.647 ± 0.019) mm. Analysis of variance showed that compared with the control group, the SSN@Ag group had a higher bacteriostatic effect on S. mutans standard strains, and the result was statistically significant (P < 0.05), while the SSN-Ag@Pt-GLM group had no statistically significant bacteriostatic effect on S. mutans standard strains (P > 0.05).

After the culture of S. mutans, the concentration of bacteria was adjusted to 10⁻⁶. An equal amount of blank TSB medium was taken, and the TSB medium was co-cultured with 5 mg mL⁻¹ SSN@Ag and SSN-Ag@Pt-GLM suspension and bacterial suspension. OD630 values of 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, and 24 h were respectively measured, and the average value was repeated three times in each group to evaluate the time-dependent inhibitory effects of 5 mg mL⁻¹ SSN@Ag and SSN-Ag@Pt-GLM on the growth of S. mutans. As shown in Fig. 6c, with the change of time, the OD value of bacteria in the control group showed a significant increase trend. The OD curve of the SSN@Ag group displayed a significant decreasing trend over time. The OD curve of year-time change in the SSN-Ag@Pt-GLM group revealed a significant decreasing trend. These results indicated that SSN-Ag@Pt-GLM had a significant inhibitory effect on the growth of S. mutans.

Subsequently, we verified the inhibitory effect of SSN-Ag@Pt-GLM on bacterial biofilms, and the results are shown in Fig. 6d. Compared with the control, SSN-Ag and SSN-Ag@Pt-GLM showed obvious bacteriostasis, and large areas of bacteria showed a death red stain, based on the autonomous motor ability of SSN-Ag@Pt-GLM, and the biofilm area in SSN-Ag@Pt-GLM was smaller and the antibacterial action was better. The activity of the biofilm treated with SSN@Ag was significantly decreased. The number of viable bacteria in the biofilm treated with SSN-Ag@Pt-GLM was similar to that treated with SSN@Ag. The SEM images in Fig. 6e show that compared with the blank control group, the biofilm structures treated by SSN@Ag and SSN-Ag@Pt-GLM were significantly damaged. These results may indicate that SSN-Ag@Pt-GLM has an inhibitory effect on S. mutans biofilms.

The Pt layer on the surface of SSN-Ag@Pt-GLM can act as a catalyst to accelerate the decomposition of hydrogen peroxide, provide a powerful force for SSN-Ag@Pt-GLM movement, and enable the motor to resist the outflow of dentin tubules and enter the interior of dentin tubules against the direction of the outflow of dentin tubules. In addition, after blue light irradiation, the GLM adhesion layer on the SSN-Ag@Pt-GLM surface can achieve a tight bond between the motors and between the motors and the inner wall of the dentin tubule. The SSN-Ag@Pt-GLM used in this study is of an appropriate concentration and has good biosafety. Through testing, SSN-Ag@Pt-GLM has an excellent sealing rate and entry rate in the pressure model. According to the analysis of the longitudinal profile of the dentin disk, the deepest blocking depth of SSN-Ag@Pt-GLM can reach 50 μm. After treatment, SSN-Ag@Pt-GLM is difficult to detangle from the dentin tubule, so the dentin disc can resist mechanical stimulation and acid corrosion, making it difficult for the dentin tubule to be reopened. At the same time, the silver nanoparticles loaded on the SSN-Ag@Pt-GLM surface have considerable antibacterial ability, which can prevent secondary dental caries.

4. Conclusions

In this study, an effective treatment strategy for dentin sensitivity was proposed, which was based on a movable motor loaded with dental adhesives and prepared via the REDOX reaction and vacuum ion sputtering technology. According to SEM, SSN-Ag@Pt-GLM is a Janus structure with an average size of 980 nm. The Pt layer on the SSN-Ag@Pt-GLM surface acts as a catalyst to promote the decomposition of hydrogen peroxide and produce oxygen bubbles, thus realizing the self-driven motion of the motor, which also realizes the self-driven motion of the motor in the high-viscosity liquid. SSN-Ag@Pt-GLM can move at a speed of 1.28 μm s⁻¹ in Ringer's fluid, which enables the motor to resist the outflow of dentin tubule fluid and successfully enter the dentin tubule and obtain a good sealing rate and entry rate. After ultraviolet curing, the motor can enter the dentin tubule at a depth of more than 30 μm and resist mechanical stimulation to maintain long-term efficacy. Moreover, silver nanoparticles on the surface of the motor have a considerable antibacterial effect, which can prevent secondary caries to a certain extent. In addition, previous studies have suggested that silver nanoparticles can denature the proteins inside the dentin tubules to better seal the dentin tubules.³⁸

Although the traditional blocking drugs can achieve a good curative effect, their long-term curative effect is poor because of the dentin structure and oral physiological environment. In addition, exposure of dentin tubules may also cause aggregation of biofilms, leading to secondary dental caries. In contrast, SSN-Ag@Pt-GLM uses an inside-out blocking strategy to improve long-term efficacy while introducing an inorganic nanocompound antibacterial strategy to reduce secondary caries.³⁴ In addition, antibacterial photodynamic therapy showed strong biofilm ablation performance,³⁵ which provided us with ideas for further research. In future study, we will develop an integrated therapy combining light-guided biofilm ablation and dentin sealing. Using SSN-Ag@Pt-GLM as a simple and non-invasive treatment strategy for dentin sensitivity, it is expected to bring benefits to patients undergoing radiation therapy and those with other exposed dentin diseases such as abnormal mineralization in the future.³⁶

Author contributions

Q. Z., N. H., Y. W., and Z. J. designed the experiments and analyzed the experimental results. Q. Z., Y. Z., and W. W performed the experiments in this study. N. H., Y. W., and Z. J. supervised the project. N. H., Y. W., and Z. J. provided feedback on the results. Q. Z. wrote the manuscript with the input of all the authors listed.

Data availability

The data presented in the documents are available upon reasonable request.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported by the National Nature Science Foundation of China (Grant No. 51972087, 22172044, and U23A20342), the new round of the “Double First-class” discipline Collaborative innovation Achievement project in 2023 of Heilongjiang Province (Grant No. LJGXCG2023-067), the Key R&D Program of Heilongjiang Province, China (Grant No. 2022ZX02C23), the Heilongjiang Provincial Natural Science Foundation of China (Grant No. YQ2022B005), and the HMU Marshal Initiative Funding (Grant No. HMUMIF-21006).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb02361g

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