IntroductionOrthopedic implant-associated biofilm infections (IABIs) are the leading cause of surgical failure in orthopedic procedures and pose a significant, ongoing challenge in clinical practice1,2,3. Unfortunately, IABIs are refractory to elimination and show a high risk of relapse rate because of dense microbial biofilms formed on the implant, which call for large amounts of antibiotics and multiple revision surgeries, bringing tremendous physical and mental torture to patients4,5. Moreover, common pathogens such as Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), which reside within biofilm shelters, exhibit robust resistance to conventional antibiotics and innate host immunity responses6,7. Additionally, the highly acidic, hypoxic, and nutrient-depleted biofilm microenvironment (BME) compromises local immunological competence and promotes the polarization of pro-inflammatory immune cells toward anti-inflammatory phenotypes, further complicating antibiofilm therapeutic strategies8,9,10. Therefore, developing antibiotic-free treatments that can effectively disrupt biofilms while reprogramming immune cells holds great promise for achieving improved therapeutic outcomes for IABIs with minimal risk of drug resistance.Recently, a variety of nanomaterials that simultaneously exhibit antimicrobial properties and immunomodulatory effects have been investigated11,12. Copper serves as an essential cofactor for all living organisms, playing a critical role in numerous biochemical processes. However, when its concentration exceeds the threshold regulated by evolutionarily conserved homeostatic mechanisms, copper becomes toxic and disrupts bacterial metabolism13,14. Specifically, the accumulation of copper within cellular structures interacts with lipid-acylated components of the tricarboxylic acid cycle (TCA), initiating cuproptosis in cells and cuproptosis-like death in bacteria, thereby providing insights into copper-based therapeutic nanomaterials15,16,17,18. Moreover, copper ions can generate reactive oxygen species (ROS) via a Fenton-like reaction upon exposure to BME. This process enhances macrophage immune function by facilitating chemotaxis toward planktonic bacteria escaping from disintegrating biofilms through phagocytosis and the release of pro-inflammatory cytokines19. Consequently, due to their direct antimicrobial capabilities coupled with their excellent reprogramming effect on immune cells, copper ion-based treatments are considered promising candidates for antibiotic-free treatment options in the post-antibiotic era20. Copper peroxide (CP) nanodots represent emerging candidates as copper ion donors. Under acidic conditions, these CP nanodots decompose into copper(II) along with hydrogen peroxide (H2O2), which subsequently generates hydroxyl radicals (•OH)21,22. These radicals contribute to disrupting bacterial membrane integrity, thereby enhancing copper uptake while promoting cuproptosis18,19.However, the efficacy of copper ions is significantly influenced by intracellular copper accumulation within bacteria, primarily due to two key factors: (1) The dense biofilms formed on implants limit the penetration efficiency required for delivering adequate quantities of copper ions23,24. (2) Bacteria have developed multiple defense mechanisms, including enhanced efflux systems that expel excess copper and the production of substances that bind available free copper, thereby reducing the internal concentrations capable of inducing cuproptosis-like death25,26. Consequently, ensuring effective delivery methods that promote widespread distribution throughout existing mature biofilm remains a critical challenge in IABIs management. Current research primarily focuses on utilizing ROS-generating or hyperthermia-inducing nanocarriers to enhance biofilm permeability and promote the passive bioavailability of relevant ions. However, these innovations still exhibit inadequate biofilm infiltration, particularly in terms of the autonomous diffusion of copper ions within the biofilm. Recently, nanomotors with autonomous movement capabilities have garnered increasing attention due to their exceptional ability to penetrate barriers and deliver drugs effectively27,28,29,30. Notably, near-infrared (NIR) laser-propelled nanomotors show great potential for overcoming complex physiological barriers, owing to their fuel-free nature and stable motion characteristics, making them promising for a wide range of applications31,32. Ji et al. demonstrated that NIR laser-propelled nanomotors exhibited a 3.8-fold higher dermal penetration efficiency compared to passive nanomaterials33. Our group also verified that NIR laser-propelled nanomotors achieved a 14.6-fold enhancement in autonomous mucus penetration29. Encouraged by these remarkable autonomous movement properties, self-thermophoretic nanomotors propelled by NIR laser irradiation represent a promising strategy for efficiently traversing the biofilm matrix barrier and promoting intracellular copper overload to combat IABIs.Herein, we introduce a BME-responsive self-thermophoretic Janus bisphere nanomotor (Motor@CP) to enhance the eradication of orthopedic IABIs (Fig. 1). Initially, mesoporous silicon nanoparticle (MSN) is utilized as a substrate, while gold nanoparticle (AuNP) serves as a photothermal structure to construct the Janus bisphere nanostructure (Motor). Subsequently, CP nanodots are encapsulated within the MSN to form Motor@CP (Fig. 1a). The NIR-propelled Motor@CP exhibits significant autonomous movement through self-thermophoretic propulsion, effectively penetrating dense biofilms in different bacterial species and extensively delivering CP within biofilms. Notably, the Motor@CP explosively decomposes into copper(II) and H2O2 in an acidic BME (pH ~ 5.5), subsequently generating •OH via a Fenton-like reaction. Due to its autonomous motion and self-supplying H2O2 generation, Motor@CP + NIR substantially disrupts the extracellular DNA (eDNA) component within biofilms, thereby significantly altering their integrity and permeability and facilitating enhanced penetration by Motor@CP. In vitro experiments demonstrate that Motor@CP + NIR increases the intracellular uptake of copper ions in S. aureus and Escherichia coli (E. coli) by 3.4- and 4.1-fold, respectively, while disrupting TCA cycle and inducing extensive cuproptosis-like bacterial death. Additionally, Motor@CP markedly influences infiltrating macrophages, promoting their repolarization toward the pro-inflammatory M1 phenotype and enhancing the antimicrobial immune response (Fig. 1b). Collectively, this approach presents a promising antibiotic-free alternative involving amplified copper ion interference and macrophage reprogramming to eradicate refractory orthopedic IABIs.Fig. 1: Design strategy for BME-responsive self-thermophoretic nanomotor to treat refractory IABIs.a Construction of the Janus bisphere nanostructure Motor@CP. b The NIR-propelled Motor@CP exhibits remarkable biofilm penetration and significant autonomous movement within biofilms, further leveraging amplified copper ion interference and macrophage reprogramming to eradicate refractory orthopedic IABIs.Full size imageResultsIn vitro antibiofilm efficacy of copper ions and characterization of CP and Motor@CPCopper, a historically established antimicrobial metal exhibiting dose-dependent antibacterial properties and cytotoxicity, has been extensively utilized for controlling various pathogenic microorganisms34,35,36. Studies have shown that the optimal concentration range of copper ions for antibacterial activity is 0.63 μg ml−1 to 6.3 μg ml−1, which can achieve an antibacterial effect of over 90% and maintain a cell proliferation rate of over 80%37. However, it remains unclear whether this concentration range can effectively maintain bactericidal activity against dense bacterial biofilms. We systematically evaluated the antibacterial efficacy of the conventional copper ion donor, cupric chloride dihydrate (CuCl2·2H2O), against both planktonic forms and biofilms of S. aureus and MRSA. The concentrations of CuCl2·2H2O used in the experiment were 2, 4, and 8 μg ml−1, corresponding to copper ion concentrations of 0.746, 1.491, and 2.982 μg ml−1, respectively. These values were within the concentration range reported in previous studies. The antibacterial and antibiofilm efficacy of CuCl2·2H2O in vitro was evaluated using OD600 measurements (Fig. 2a), colony counts on spread plates (Fig. 2b and Supplementary Fig. 1), crystal violet staining (Fig. 2c and Supplementary Fig. 2) and inductively coupled plasma optical emission spectrometry (ICP-OES) (Fig. 2d). At a CuCl2·2H2O concentration of 2 μg ml−1, 90% of bacterial populations were effectively eradicated (Fig. 2b), which is consistent with previously reported minimum inhibitory concentration (MIC) of copper ions for S. aureus (0.63 μg ml−1)18. However, no significant increase in either copper content within biofilms or biofilm eradication was observed following CuCl2·2H2O treatment in the aforementioned investigations. These findings indicate that while copper ions within the safe concentration range demonstrate effective dose-dependent antibacterial activity against planktonic bacteria; however, they show limited efficacy against biofilms, likely due to the dense extracellular polymeric substance (EPS) of biofilms38.Fig. 2: In vitro antibiofilm activities of two copper ion donors against S. aureus, planktonic MRSA, and biofilms, as well as the characterization of CP and Motor@CP.a The OD600 values of S. aureus, MRSA planktons, and biofilms were measured after incubation with different concentrations of CuCl2·2H2O. Data are presented as mean ± s.d. (n = 3 biologically independent samples). b The viability of S. aureus, MRSA planktons, and biofilms was assessed at varying concentrations of CuCl2·2H2O using SPM. Data are presented as mean ± s.d. (n = 3 biologically independent samples). c Quantification of the biomass of S. aureus and MRSA biofilms with different concentrations of CuCl2·2H2O by crystal violet assay. Data are presented as mean ± s.d. (n = 3 biologically independent samples). d Intracellular copper concentrations were determined via ICP-OES. Data are presented as mean ± s.d. (n = 3 biologically independent samples). e Schematic diagram of Fenton-like catalytic kinetics of CP. f The hydrodynamic diameter of CP and its representative TEM image. g KMnO4 assay was performed to detect peroxide groups. h TMB assay was used to assess •OH generation in PBS at pH 5.5, 6.5, and 7.4. i Cu 2p XPS spectra of CP nanodots and CuO NPs. j Cu ions release rates of CP in different PBS (pH 7.4, 6.5, and 5.5). Data are presented as the mean ± s.d. (n = 3 biologically independent samples). k pH values of the pellet and supernatant from S. aureus, MRSA planktonic culture systems, and biofilm medium were recorded. Data are presented as mean ± s.d. (n = 3 biologically independent samples). l Schematic illustration of an autonomously moving nanomotor for biofilm penetration to increase the action of copper ions. m TEM and HRTEM images of Motor@CP. n Element mapping images of Motor@CP. o, p Hydrodynamic diameter distribution and zeta potential values of CP, MSN, Au, Motor, and Motor@CP. Data are presented as mean ± s.d. (n = 3 biologically independent samples). q, r Survey XPS spectra of Motor and Motor@CP. Statistical significance was assessed using two-way ANOVA with Tukey’s multiple comparison test.Full size imageRecently, nanoscale metal peroxides comprising metal ions and peroxo groups, have emerged as effective sources of H2O2 for antibacterial applications22,39. Among the Fenton-like metal peroxide enzymes, CP nanodots uniquely provide H2O2 and Fenton-type copper(II), enabling self-supplying chemodynamic therapy (Fig. 2e). The utilization of metal peroxides significantly enhances the efficacy of therapies that rely on ROS. Transmission Electron Microscopy (TEM) image and size distribution analysis confirmed the successful synthesis of CP nanodots, which exhibited a remarkably uniform size of approximately 5 nm (Fig. 2f). A colorimetric assay demonstrated that H2O2 released from CP caused the discoloration of KMnO4 (from purple to colorless) via a redox reaction (Fig. 2g). Similarly, the capability of CP to generate •OH radicals was evidenced by an absorption peak at 650 nm, corresponding to the oxidation of TMB and the resulting color change (Fig. 2h). X-ray photoelectron spectroscopy (XPS) analysis was conducted on CP and CP + NIR to evaluate the valence state changes of copper ions in CP. The results showed that the Cu 2p XPS spectra of CP featured two primary peaks at 932.8 and 953.2 eV, along with satellite peaks at 943.2 and 961.8 eV (Fig. 2i). For CP + NIR, the Cu 2p XPS spectra exhibited two main peaks at 933.1 and 953.4 eV, accompanied by satellite peaks at 943.3 and 962.1 eV. These findings confirm that the copper in CP nanodots remains in the +2 valence state, indicating that NIR irradiation does not alter the valence state of copper ions in CP (Supplementary Fig. 3). The release of copper ions from CP was systematically monitored over 24 h in phosphate-buffered saline (PBS) at varying pH levels (7.4, 6.5, and 5.5). The results clearly demonstrated the acid-responsive nature of copper ion release by CP (Fig. 2j). To more accurately assess the self-supplying H2O2 capacity of CP, the H2O2 level was continuously monitored over a 24 h period. The results revealed that CP is capable of generating sustained H2O2 production (achieving 69.9 ± 4.3% within 12 h), thereby creating favorable conditions for enhancing the Fenton-like reaction and ensuring sufficient ROS generation (Supplementary Fig. 4). Additionally, the chemical properties of the BME were analyzed to determine optimal conditions for CP-BME interactions. The biofilm pellets in S. aureus and MRSA cultures exhibited a low pH (Fig. 2k), creating an acidic environment conducive to the generation of H2O2, copper(II), and toxic •OH by CP nanodots.Subsequently, the aforementioned routine antibiofilm experiments were conducted in S. aureus and MRSA biofilms. The results demonstrated that CP outperformed CuCl2·2H2O in biofilm eradication (Supplementary Figs. 5–7). ICP-OES analysis further confirmed that CP treatment resulted in an increase in copper content within both S. aureus and MRSA biofilms (Supplementary Fig. 7d), attributed to CP’s self-supplying H2O2 capacity, which enhances ROS production and interferes with copper ion uptake and efflux channels. Compared with traditional copper ion donor, these findings highlight the potential of CP to promote intracellular copper accumulation in biofilm infections. The in vitro cytotoxicity of various concentrations of CP nanodots on L929 cells, RAW 264.7 cells, and HUVEC cells was evaluated using the CCK-8 assay. It was determined that a concentration of 4 μg ml−1 represents the optimal antibiofilm concentration while exhibiting negligible cytotoxic effects on normal cells (Supplementary Fig. 8a–c). Although CP is capable of providing copper ions within an appropriate range; its passive nanoparticle action mode exhibits limited penetration efficacy against dense biofilms, necessitating further investigation into strategies to enhance its biofilm-penetrating capability.Owing to the superior and controllable autonomous motion, nanomotors hold promise in overcoming dense biofilm barriers, diffusing extensively within biofilms, and maximizing the antibiofilm effect through effective delivery of copper ions within a safe concentration range (Fig. 2l). In this study, MSNs were chosen as the nanomotor carrier due to their high loading capacity and biocompatibility, while AuNPs were utilized as propulsion components for their outstanding photothermal conversion efficiency. TEM images revealed that MSNs exhibited monodispersity with central radial structures, and AuNPs displayed similarly spherical shapes with smooth surfaces (Supplementary Figs. 9 and 10). Nitrogen absorption-desorption tests and Brunauer-Emmett-Teller analysis indicated an average pore size of 10.1 nm for MSNs, confirming the feasibility of loading CP nanodots into MSNs (Supplementary Fig. 11a, b). The Pickering emulsion method was employed to incorporate MSNs into paraffin wax, demonstrating a promising technique for the formation of Janus particles40. Scanning electron microscope (SEM) images showed a monolayer of MSNs anchored on paraffin wax (Supplementary Fig. 12). To achieve precise fabrication of Janus bisphere structures, various mass ratios of MSN to AuNPs (1:1, 1:1.5, and 1:2) were tested to identify the optimal combination. TEM images confirmed the successful attachment of AuNPs to one side of MSNs, forming the optimal Janus bisphere MSN-Au (Motor) structure at a mass ratio of 1:1.5 (Supplementary Fig. 13). Our preparation method ensured excellent uniformity of the Janus bisphere Motor (Supplementary Fig. 14). Subsequently, CP nanodots were encapsulated within the nanomotor to fabricate the Motor@CP structure. High-resolution transmission electron microscopy (HRTEM) images revealed that MSN exhibited a regular aperture structure before CP loading, however, this regular structure was no longer observable after CP encapsulation (Fig. 3m and Supplementary Fig. 15). Elemental mapping images confirmed the successful synthesis of the Motor@CP Janus bisphere, characterized by a large and uniform distribution of copper elements on the MSN side (Fig. 3n). DLS measurements were then performed on various nanomaterials, including MSN, AuNPs, Motor, and Motor@CP, to evaluate their size distributions. The hydrodynamic sizes of all sample was similar, with the addition of CP causing a slight increase in particle size compared to the Motor alone (Fig. 3o). Zeta potential measurements further corroborated the successful formation of Motor@CP. The zeta potential values for MSN and AuNPs were −31.9 ± 2.0 mV and −12.8 ± 0.9 mV, respectively. In contrast, the negative surface charge of Motor and Motor@CP decreased to −22.3 ± 0.6 mV and −11.4 ± 1.0 mV, respectively (Fig. 3p). Additionally, XPS detection was conducted to evaluate the Motor and Motor @CP (Fig. 3q, r). Compared with the spectrum of pristine Motor, the survey spectrum showed an obvious characteristic peak of Cu 2p, while the high-resolution spectrum of Cu 2p showed two deconvolved peaks at 932.9 and 952.5 eV, confirming the successful formation of Motor@CP (Supplementary Fig. 16). The self-supplying H2O2 capability of Motor@CP was further investigated. The release of H2O2 was continuously monitored over a 24 h period, and the results demonstrated that Motor@CP could sustainably produce H2O2, achieving a release rate of 69.4 ± 5.2% within 12 h (Supplementary Fig. 17). Electron spin resonance (ESR) analysis also confirmed the ability of Motor@CP to generate •OH radicals in an acidic environment (Supplementary Fig. 18). Collectively, these results demonstrate successful loading of CP. The high nanoparticle-loading capacity of MSN enabled the CP-loading efficiency of the Janus bisphere nanomotors to reach 12.5 ± 0.7%.Fig. 3: In vitro movement and biofilm penetration performance of Motor@CP.a Schematic illustration of the movement of NIR laser-driven Motor@CP. b Temperature elevation of Motor@CP in PBS (pH 7.4) with different NIR laser power (0, 0.5, 1.0, and 1.5 W cm−2). c Representative tracking trajectories of Motor@CP in PBS (pH 7.4) with different NIR laser power (0, 0.5, 1.0, and 1.5 W cm−2) over 3 s. d–f Velocity, MSD curve, and corresponding Deff of Motor@CP in PBS (pH 7.4) under different NIR laser power. Data are presented as mean ± s.d. (n = 3 biologically independent samples). g Temperature elevation of Motor@CP in PBS (pH 5.5) with different NIR laser power (0, 0.5, 1.0, and 1.5 W cm−2). h Representative tracking trajectories of Motor@CP in PBS (pH 5.5) with different NIR laser power (0, 0.5, 1.0, and 1.5 W cm−2) over 3 s. i–k Velocity, MSD curve, and Deff of Motor@CP in PBS (pH 5.5) under different NIR laser power. Data are presented as mean ± s.d. (n = 3 biologically independent samples). l Cu ions release rates of CP, Motor@CP, and Motor@CP + NIR in PBS (pH 5.5). Data are presented as mean ± s.d. (n = 3 biologically independent samples). m Schematic diagram of transwell assay composed of S. aureus biofilm. Created in BioRender. Liuliang He (2025) (https://BioRender.com/ov97w82). n The ratio of Motor@CP penetrated into basal chamber after different time (1, 3, 5, and 10 min) of NIR laser irradiation in apical chamber. Data are presented as mean ± s.d. (n = 3 biologically independent samples). o The ratio of nanoparticles penetrated into basal chamber after 5 min of incubation in apical chamber. Data are presented as mean ± s.d. (n = 3 biologically independent samples). p 3D CLSM images and corresponding z-stack fluorescent images of nanoparticles penetrating S. aureus biofilm for 5 min. Green: live bacteria, Red: rhodamine B labeled nanoparticles. These experiments (g–k, m–o) were covered by a slice of 2.0 mm-thick pork ham. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparison test.Full size imageMovement performance and biofilm penetration behavior of Motor@CPLeveraging asymmetric absorption of NIR light, our Janus bisphere nanomotors Motor@CP were capable of generating self-thermophoretic propulsion (Fig. 3a). Initially, we monitored temperature changes in aqueous solutions containing Motor@CP under varying NIR laser irradiation intensities. The results demonstrated that the solution temperature increased proportionally with the intensity of the NIR laser, achieving a photothermal conversion efficiency of ~30.6% (Fig. 3b and Supplementary Fig. 19). Representative tracking trajectories of Motor@CP in PBS solution (pH 7.4) were observed at different NIR laser power intensities (Fig. 3c and Supplementary Fig. 20). Notably, the movement distance of Motor@CP progressively increased as the NIR laser power was elevated. Specifically, under no NIR laser irradiation (0 W cm−2), the motion was irregular, under low-intensity irradiation (0.5 W cm−2), the motion appeared relatively random (Supplementary Movie 1). In contrast, Motor@CP with the Janus bisphere structure exhibited more directional movement under higher NIR laser powers (1.0 and 1.5 W cm−2). The velocities of Motor@CP under different NIR laser irradiations (0.5, 1.0, and 1.5 W cm−2) were 4.2 ± 0.9 μm s−1, 8.3 ± 0.9 μm s−1, and 11.1 ± 0.8 μm s−1, respectively (Fig. 3d). Additionally, as the laser power increased, both the mean square displacement (MSD) and the corresponding effective diffusion coefficient (Deff) of Motor@CP showed significant increases (Fig. 3e, f).To mimic subcutaneous acidic biofilm microenvironments, we further evaluated the motor performance in acidic PBS (pH 5.5) covered by a 2.0 mm-thick slice of pork ham41,42. The temperature increase of Motor@CP was proportional to the increase in NIR laser power (Fig. 3g). Representative tracking trajectories under varying NIR laser powers were shown in Fig. 3h, Supplementary Fig. 21, and Supplementary Movie 2. As the NIR laser power increased in acidic solution (0.5, 1.0, and 1.5 W cm−2), the velocity of Motor@CP was caculated as 4.8 ± 0.4 μm s−1, 8.7 ± 0.6 μm s−1, and 11.6 ± 0.8 μm s−1, respectively (Fig. 3i), and the corresponding MSD and Deff also exhibited significant increases (Fig. 3j, k). This superior self-thermophoretic capability may facilitate more efficient penetration into biofilms for copper ions delivery. The release behavior of copper ions from CP after loading was evaluated using ICP-OES. Within 24 h in PBS (pH 5.5), the copper ion release ratios of CP, Motor@CP and Motor@CP + NIR (4 μg ml−1, expressed in terms of CP) were 70.1 ± 2.2%, 66.3 ± 3.4% and 72.6 ± 3.0% respectively (Fig. 3l), with actual copper ion concentrations of 1.362 ± 0.043, 1.287 ± 0.067 and 1.412 ± 0.052 ug ml−1 respectively (Supplementary Fig. 22). This implies that Motor@CP + NIR achieves a more efficient antibiofilm effect through autonomous nanomotor movement and ROS generation at lower copper ion concentrations. Efficient nanoparticle penetration through the dense protective layer of biofilms is critical for disrupting biofilm integrity and effectively eliminating bacteria via copper ion release. Therefore, this study evaluated the penetration properties into biofilms of different nanoparticles by co-incubating them with biofilms. Prior to this, the biocompatibility of the samples was assessed through hemolysis and cytotoxicity assays conducted in vitro. The hemolysis ratio for all samples was below 5%, comparable to the negative control (PBS solution), indicating excellent hemocompatibility (Supplementary Fig. 23). Additionally, we assessed in vitro biocompatibility under varying concentration and NIR laser power intensities using the CCK-8 assay. After 24 h co-culture with L929 cells, RAW 264.7 cells, and HUVEC cells, Motor@CP + NIR demonstrated excellent biocompatibility at the concentration (4 μg ml−1, expressed in terms of CP) (Supplementary Fig. 24). In subsequent experiments, we employed a NIR laser power of 1.0 W cm−2, as this power level enabled Motor@CP to exhibit superior motility performance while exerting negligible cytotoxic effects (Supplementary Fig. 25).Subsequently, to simulate realistic conditions of subcutaneous biofilm infections, the transwell system was employed to quantify the penetration capability (Fig. 3m and Supplementary Fig. 26). S. aureus biofilm was established in the apical chamber, and rhodamine B-labeled fluorescent nanoparticles were introduced into this chamber, with a 2.0 mm thick pork ham slice covering the surface. The penetration efficiency of Motor@CP through the biofilm was evaluated under varying durations of NIR laser irradiation. Prolonged laser exposure significantly enhanced the penetration efficacy of Motor@CP (Fig. 3n), achieving near-optimal efficiency (>90%) after 5 min of irradiation. Additionally, irradiation for 10 min demonstrated no significant increase in penetration efficiency, while prolonged irradiation may result in undesirable tissue heat damage. Therefore, we selected a laser irradiation time of 5 min in the whole experiment. Furthermore, the biofilm penetration efficiencies of different nanoparticles were compared by measuring the fluorescence intensity in the basal chamber after 5 min of laser irradiation. The results demonstrated that the biofilm penetration ratio of Motor@CP + NIR was 14.2-fold higher than that of the CP group (Fig. 3o), highlighting the exceptional potential of Motor@CP + NIR with autonomous motion in penetrating biofilms. 3D confocal laser scanning microscopy (CLSM) was further performed following the incubation of rhodamine B-labeled nanoparticles with S. aureus and E. coli biofilms. The results demonstrated that Motor@CP + NIR exhibited robust penetration into both S. aureus and E. coli biofilms (Fig. 3p and Supplementary Figs. 27 and 28). In contrast, negligible penetration was observed in the CP group alone. These findings suggest that the self-thermophoretic propulsion of Motor@CP + NIR significantly enhanced biofilm penetration and exhibited broad-spectrum applicability to different biofilms.In vitro antibiofilm activity of Motor@CPRepresentatives of Gram-positive (S. aureus and MRSA) and Gram-negative (E. coli) bacteria were selected as model biofilm-forming organisms to investigate the in vitro antibiofilm activity of Motor@CP. These bacteria were cultured for 3 days to establish stable biofilms, followed by different treatments, including Control (untreated), CP, Motor, Motor@CP, and Motor@CP + NIR, to validate their therapeutic effects on biofilm eradication. After a 12 h co-incubation period with established biofilms, the Motor@CP + NIR group exhibited a significant reduction in OD600 values for S. aureus, MRSA, and E. coli biofilms (Supplementary Fig. 29a–c). Crystal violet staining revealed that while the Control and Motor groups maintained intact biofilm structures, the CP and Motor@CP treatment groups showed biofilms with partial structural damage but largely preserved viability (Fig. 4a–c and Supplementary Fig. 30a, b). Spread plate method (SPM) analysis further confirmed a marked decrease in bacterial presence within the biofilm in the Motor@CP + NIR treatment group, and colony-forming unit (CFU) analysis demonstrated that the Motor@CP + NIR treatment achieved a 99.9% bacterial clearance rate, indicating superior inhibition of bacterial growth and biofilm formation (Fig. 4d–f and Supplementary Figs. 31a, b). This clearance rate was significantly higher compared to any other treatment groups, confirming that Motor@CP + NIR provided the most potent disruption of the biofilm. 3D CLSM images of S. aureus and MRSA biofilms were utilized to assess the extent of biofilm disruption. In the Motor@CP + NIR group, a pronounced disruption of the biofilm architecture was observed, characterized by intense red fluorescence, which strongly indicated a high degree of bacterial cell death. This finding underscores the efficacy of the treatment in compromising the biofilm structure (Fig. 4g). Furthermore, SEM images (Fig. 4h and Supplementary Fig. 32) revealed dense bacterial colonies with intact morphology in the Control, CP, Motor, and Motor@CP groups. Conversely, bacteria within the biofilms treated with Motor@CP + NIR exhibited predominantly shriveled, distorted, or fully lysed cells, thereby confirming the treatment’s effectiveness in biofilm eradication. The photothermal effect of AuNPs was investigated separately, and the results indicated that at a power of 1.0 W cm−2, the photothermal effect of AuNPs alone on the biofilms was negligible (Supplementary Figs. 33–35). Collectively, these results suggest that single-ion therapy (CP or Motor@CP groups) exhibits moderate antibiofilm activity, while the Motor@CP + NIR group demonstrates superior efficacy in eliminating biofilms in vitro through the utilization of self-thermophoretic nanomotors.Fig. 4: In vitro antibiofilm activity of Motor@CP.a Representative crystal violet staining images of S. aureus and MRSA biofilms in the Control, CP, Motor, Motor@CP, and Motor@CP + NIR treatment groups. b, c Quantitative analysis of the biomass of S. aureus and MRSA biofilms after different treatments. Data are presented as mean ± s.d. (n = 3 biologically independent samples). d Representative images of colonies derived from S. aureus and MRSA biofilms in the aforementioned treatment groups. e, f CFU counts for S. aureus and MRSA biofilms after different treatments was determined using SPM. Data are presented as mean ± s.d. (n = 3 biologically independent samples). g 3D CLSM images of S. aureus and MRSA biofilms stained with SYTO9 (green: live bacteria) and PI (red: dead bacteria) to visualize biofilm disruption. h SEM images of S. aureus and MRSA biofilms following treatment with different groups. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparison test.Full size imageAntibacterial mechanism of Motor@CPBio-TEM was employed to investigate the ultrastructure and intracellular copper ion uptake of S. aureus and E. coli following diverse treatment. The S. aureus and E. coli exposed to Motor@CP + NIR exhibited more pronounced structural disruptions. Further elemental mapping analysis revealed a significantly higher intracellular copper concentration in the Motor@CP + NIR group compared to the CP group, indicating enhanced copper ion internalization (Fig. 5a and Supplementary Fig. 36). To quantify this observation, ICP-OES analysis was performed. The results demonstrated that the intracellular copper levels of S. aureus and E. coli treated with Motor@CP + NIR increased significantly by 3.4- and 4.1-fold respectively, confirming substantial copper ion influx (Fig. 5b and Supplementary Fig. 37). This enhanced copper uptake can be attributed to the synergistic effect of autonomous movement and ROS generation, leading to increased membrane permeability (Fig. 5c and Supplementary Figs. 38–40). Extracellular DNA (eDNA) plays a critical role in the formation, maturation and structural stability of biofilms43,44. Through SYTOX green nucleic acid staining of eDNA in S. aureus and E. coli biofilms, the results indicated that Motor@CP + NIR group exhibited superior eDNA degradation within the biofilm. This is due to the autonomous movement enabling penetration of the biofilm structure and simultaneous delivery of CP to disrupt the eDNA component (Supplementary Figs. 41 and 42). Additionally, protein leakage assays and O-nitrophenyl β-D-galactopyranoside (ONPG) hydrolysis assays were conducted to evaluate bacterial content leakage, providing further evidence for the damaged membrane integrity (Fig. 5d, e and Supplementary Fig. 43a, b). These findings strongly support the conclusion that Motor@CP + NIR treatment significantly compromises the intergrity and alters the permeability of S. aureus and E. coli biofilm and bacterial membranes, resulting in evident cytoplasmic leakage.Fig. 5: In vitro antibacterial mechanism of Motor@CP.a Microstructural analysis and element mapping of copper ions in S. aureus using Bio-TEM. b Intracellular copper ions concentration within S. aureus biofilm measured by ICP-OES. Data are presented as the mean ± s.d. (n = 3 biologically independent samples). c Flow cytometry results of ROS levels in S. aureus biofilm measured by DCFH-DA. d Protein leakage of S. aureus biofilm after various treatments calculated by BCA assay. Data are presented as the mean ± s.d. (n = 3 biologically independent samples). e Membrane permeability of S. aureus biofilm after various treatments evaluated with ONPG hydrolysis assay. Data are presented as the mean ± s.d. (n = 3 biologically independent samples). f Volcano plot depicting the DEGs in the comparison of Motor@CP + NIR versus CP is presented. Statistical significance was determined using a two-tailed t-test. Genes with an adjusted P