NO-driven self-propelled nanomotors with deep biofilm penetration for synergistic therapy of peri-implantitis

Abstract

Phototherapy as an adjuvant treatment for peri-implantitis (PI) still faces multiple challenges in clinical translation, including weak targeting capability of photosensitizers, poor biofilm penetration, and lack of immunomodulatory and osteogenic functions. To address these limitations, a core–shell nanomotor (IHPS) was constructed, whereby a hollow mesoporous polydopamine core encapsulating indocyanine green (ICG) was coated with S-nitrosothiol-modified ε-polylysine. Under near-infrared light excitation, the IHPS utilizes the positive surface charge provided by ε-polylysine to actively target plaque biofilms, and employs the photothermal effect of ICG to trigger burst release of nitric oxide (NO), thereby enhancing its penetration capacity into the deep regions of the biofilm through a self-propelled mechanism. The released NO can react with reactive oxygen species generated by ICG-mediated photodynamic therapy to form peroxynitrite, further synergistically improving antibacterial and biofilm eradication efficacy. Moreover, under physiological conditions, the IHPS enables sustained and slow release of NO, effectively promoting macrophage polarization toward the M2 phenotype to suppress inflammation, and enhancing osteogenic differentiation via activation of the sGC-cGMP-PKG signaling pathway. Ultimately, this approach achieves synergistic antibacterial, immunomodulatory, and bone regeneration effects at the infection site. This study provides a novel multifunctional therapeutic strategy with promising clinical translation potential for the treatment of PI.

Graphical abstract

This study develops a near-infrared-triggered nitric oxide-propelled nanomotor (IHPS) that achieves deep biofilm penetration and exerts synergistic antibacterial, immunomodulatory, and osteogenic effects for comprehensive peri-implantitis therapy.

Highlights

• •An NIR-triggered, NO-propelled nanomotor (IHPS) enables deep bacterial biofilm penetration for enhanced peri-implantitis therapy.
• •It synergizes PTT, PDT, and NO therapy, where NO reacts with PDT-generated ROS to yield highly bactericidal peroxynitrite for effective biofilm eradication.
• •Sustained NO release under physiological conditions promotes M2 macrophage polarization and facilitates osteogenesis via the sGC-cGMP-PKG pathway.

1. Introduction

Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has become an important adjuvant to traditional mechanical debridement and antibiotic therapy for peri-implantitis (PI) due to its minimal invasiveness, rapid efficacy, and broad-spectrum antibacterial activity [[1], [2], [3]]. However, its clinical application faces multiple challenges. Firstly, most available photosensitizers (PSs) lack specific targeting capability towards bacterial biofilms, leading to nonspecific phototoxicity towards surrounding healthy tissues while eliminating pathogenic bacteria [4]. Secondly, the complex morphology of the peri-implant pocket, coupled with the physicochemical barrier formed by extracellular polymeric substances (EPS) in mature biofilms, significantly hinders the penetration of PSs and restricts the diffusion of singlet oxygen and the conduction of thermal effects, thereby compromising the therapeutic outcome of phototherapy [5,6]. Furthermore, existing phototherapeutic strategies are unable to effectively modulate the immune microenvironment imbalance in PI or promote bone regeneration, thus exhibiting clear limitations in achieving synergistic antibacterial-immunomodulatory-osteogenic effects [7]. Therefore, the development of a novel therapeutic strategy capable of functioning synergistically at multiple levels is of great significance for improving the overall efficacy of PI treatment.

Targeted nanoparticles (NPs), as a cutting-edge research direction in the biomedical field [8,9], have garnered significant attention in the treatment of PI due to their excellent targeting capability towards plaque biofilms. Currently, many studies [10,11] employ cationic modification strategies to fabricate NPs with positively charged surfaces, enhancing their recognition and binding ability to negatively charged biofilm components, such as pathogen-associated molecular patterns (PAMPs), thereby improving antibacterial efficacy [12,13]. However, although positively charged materials can adsorb onto the biofilm surface and achieve preliminary penetration via electrostatic interactions, their limited diffusion capacity prevents them from overcoming the physical barrier of the deep matrix [14]. As a result, they only act on the superficial layers of the biofilm, leaving bacteria in the deeper layers viable and able to proliferate, which substantially restricts the full exertion of their bactericidal effect. Therefore, developing innovative delivery strategies capable of overcoming the penetration barriers of biofilms has become a critical direction for advancing the clinical translation of positively charged NPs.

In this context, self-propelled nanomotors offer a novel strategy to overcome the aforementioned penetration limitations. These microdevices can convert various forms of energy (such as chemical or light energy) into mechanical motion, thereby achieving autonomous movement (common propelling gases include O₂, CO₂, H₂, NO, etc) [15,16]. Their self-propulsion characteristic enables nanomotors to actively penetrate biological barriers, demonstrating exceptional infiltration capability particularly within dense bacterial biofilms [17], thus effectively compensating for the limited diffusion depth of conventional positively charged NPs. Leveraging this property, nanomotors can serve as efficient drug delivery systems, carrying antibacterial agents through multiple biological barriers deep into the periodontal pocket, enabling precise and potent antibacterial therapy [18]. Among the various bioactive molecules suitable for delivery via nanomotors, nitric oxide (NO) has attracted considerable attention due to its multifunctional physiological roles. Based on these multifaceted effects, the application of NO-driven nanomotor systems extends far beyond the antibacterial field. In recent years, such nanomotors have demonstrated considerable potential in various biomedical areas, including cancer therapy, cardiovascular disease intervention, and the treatment of neurodegenerative disorders such as Parkinson's disease [[19], [20], [21], [22], [23]]. Unlike conventional antibiotics, NO kills bacteria via unique mechanisms that are less likely to induce drug resistance. At high concentrations, NO can react with reactive oxygen species (ROS) to form peroxynitrite (ONOO⁻), which effectively disrupts biofilm integrity by intensifying lipid peroxidation, damaging bacterial membrane structures, and altering the extracellular polymeric matrix, thereby exerting potent bactericidal effects [24,25]. Beyond its direct antibacterial activity, low concentrations of NO can modulate the immune microenvironment; for instance, it induces a shift in macrophage polarization from the M1 to the M2 phenotype and suppresses the release of pro-inflammatory cytokines, thereby alleviating excessive inflammatory responses [26,27]. Furthermore, studies [28,29] have shown that low concentrations of exogenous NO can activate the soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP)-protein kinase G(PKG) signaling pathway, promoting the proliferation and differentiation of osteoblasts, which plays an important role in mitigating osteoporosis and enhancing bone regeneration. Therefore, NO demonstrates great potential in integrated therapeutic strategies that synergistically combine antibacterial action, immunomodulation, and osteogenic repair.

In this study, we developed an intelligent nanomotor system with photothermal-responsive NO release capability. This system achieves autonomous motion in the PI infection area, significantly enhancing the deep penetration efficiency of the PS while simultaneously modulating the immune microenvironment and promoting bone regeneration. The nanomotor features a core of hollow mesoporous polydopamine encapsulating indocyanine green (ICG@HMPDA). Using S-nitrosothiol (SNO) as the NO donor, which is grafted onto ε-polylysine (ε-PL) via amide bonds, the final ICG@HMPDA@ε-PL-SNO (IHPS) NP is constructed through electrostatic adsorption-based self-assembly with ICG@HMPDA. After being injected into the peri-implant pocket, the NPs first utilize their surface positive charge to actively target the plaque biofilm. Under near-infrared (NIR) light irradiation, ICG-mediated PTT and PDT synergistically exert antibacterial effects. Simultaneously, the localized heat generated by PTT triggers the cleavage of S-NO bonds, leading to substantial NO release and enabling NO-driven deep penetration. Furthermore, the released NO can react with ROS produced during PDT to generate ONOO⁻, further enhancing the biofilm eradication effect. More importantly, under physiological conditions, the slowly and continuously released NO can modulate the polarization of macrophages from the M1 to the M2 phenotype and promote the osteogenic differentiation of bone marrow mesenchymal stem cells via the sGC-cGMP-PKG signaling pathway, thereby synergistically enhancing bone tissue regeneration capacity on the basis of anti-infection therapy. This study provides a potential novel strategy with multi-mechanistic synergistic effects for the clinical treatment of PI.

2. Results and discussion

2.1. Synthesis and characterization of IHPS nanomotors

A multifunctional nanoplatform integrating photothermal therapy (PTT), photodynamic therapy (PDT), and controlled nitric oxide (NO) release capabilities, designated as ICG@HMPDA@ε-PL-SNO (IHPS), was successfully constructed in this study. As illustrated in Fig. 1A, the synthesis primarily involved three critical steps. Initially, hollow mesoporous polydopamine NPs (HMPDA) were formed under alkaline conditions (pH 8.5) via a trimethylbenzene (TMB)-guided emulsion template method, coupled with the self-polymerization of dopamine in the presence of F127. Subsequently, ICG was loaded into the mesoporous channels of HMPDA through hydrophobic interactions and π–π stacking, resulting in the formation of ICG@HMPDA (IH). Ultimately, the final product, IHPS, was obtained by coating the IH surface with SNO-modified ε-polylysine (ε-PL-SNO) via electrostatic adsorption.

Fig. 1.

Synthesis and characterization of IHPS nanomotors. (A) Schematic diagram of the fabrication process for IHPS. (B) TEM images of (i) HMPDA, (ii) IH, (iii) IHPS, and (iv) the surface coating of IHPS (Scale bar: 100 nm). (C) UV–vis spectra of ICG, HMPDA, and IH. (D) UV–vis spectra of ε-PL, SNO-COOH, and ε-PL-SNO. (E) UV–vis spectra of IH, ε-PL-SNO, and IHPS. (F) Hydrodynamic diameter of HMPDA, IH, and IHPS NPs. (G) Zeta potential of HMPDA, IH, and IHPS. (H) Encapsulation efficiency (EE) and drug loading (DL) at varying HMPDA:ICG mass ratios. (I) Hydrodynamic size of IHPS NPs as a function of the IH:ε-PL-SNO mass ratio.(J) The particle size variation of IHPS in different aqueous media over 6 days.

The morphological evolution of the materials at each synthesis stage was systematically characterized by transmission electron microscopy (TEM) (Fig. 1B). Initially, the HMPDA NPs exhibited a well-defined spherical architecture with a distinct hollow cavity and a uniform mesoporous shell (Fig. 1Bi), a structure conducive to high specific surface area and enhanced drug loading capacity. Following ICG loading, the IH NPs retained their integral hollow spherical morphology (Fig. 1Bii); however, the mesoporous channels were occupied by ICG, resulting in a slightly roughened surface, which indicates successful incorporation of ICG into the mesopores without compromising the carrier framework. After coating with ε-PL-SNO, the resulting IHPS NPs maintained a favorable spherical shape with a smooth surface and uniformly encapsulated mesostructure (Fig. 1Biii). High-magnification TEM imaging further revealed a continuous, film-like coating on the outer surface (Fig. 1Biv), confirming the successful modification of the particles with ε-PL-SNO via electrostatic adsorption, which did not induce NP aggregation.

The structures of key intermediates and the final product during the synthesis were systematically characterized by ultraviolet–visible (UV–vis) absorption spectroscopy. HMPDA exhibited a distinct characteristic absorption peak near 190 nm (Fig. 1C). This wavelength shows a blue shift compared to the characteristic absorption peak of conventional polydopamine [30], which is primarily attributed to the significant enhancement of incident light scattering by the mesoporous and hollow structure of HMPDA. After loading ICG, a pronounced absorption peak appears at 850 nm in the spectrum. This peak position is red-shifted compared to the characteristic absorption peak of free ICG (780 nm), which is presumably due to the π-π stacking interaction between ICG molecules and the HMPDA carrier. This result confirms the successful loading of ICG onto the HMPDA carrier. To verify the feasibility of S-nitrosation modification, the intermediate 3-(S-nitrosocysteamine) propanoic acid (SNO-COOH) was synthesized. Its UV–vis spectrum displayed a characteristic absorption peak at 320 nm (Fig. S2A), corresponding to the S-NO group, indicating the successful conversion of the thiol group into the S-nitroso functionality. Furthermore, SNO-COOH was grafted onto ε-PL via an EDC/NHS-catalyzed amidation reaction. The UV–vis spectrum of the resulting product, ε-PL-SNO, still retained a distinct characteristic absorption peak at 320 nm (Fig. 1D), demonstrating that the structural integrity of the S-NO functional group was successfully maintained throughout the amidation reaction without decomposition, thereby confirming the successful synthesis of ε-PL-SNO. Finally, characterization of the final product IHPS revealed characteristic absorption peaks at both 320 nm and 850 nm in its UV–vis spectrum (Fig. 1E), corresponding to the S-NO bond in the ε-PL-SNO component and the loaded ICG, respectively. This result clearly confirms the successful modification of ε-PL-SNO onto the IH surface.

The hydrodynamic diameters of the NPs were systematically characterized by dynamic light scattering (DLS) (Fig. 1F). The results indicated that the average hydrodynamic diameters of HMPDA, IH, and IHPS were 196.9 ± 2.6 nm, 257.0 ± 13.8 nm, and 212.1 ± 1.3 nm, respectively. Following ICG loading, the NP size increased significantly from 196.9 ± 2.6 nm to 257.0 ± 13.8 nm, suggesting the successful incorporation of ICG molecules into the mesoporous channels of HMPDA, rather than mere surface adsorption. This size increase confirms the effective drug-loading function of the mesoporous structure and implies potential π–π stacking interactions between ICG and the polydopamine framework. This result corroborates the inference from UV–vis spectroscopy, collectively supporting the effective loading of ICG into the HMPDA carrier via π–π stacking. After modification with ε-PL-SNO, the size of IHPS decreased compared to that of IH. This change can be attributed to the steric hindrance and electrostatic stabilization imparted by the ε-PL-SNO coating layer, which effectively inhibits further NP aggregation, thereby significantly enhancing dispersion stability. Concurrently, the clarity of the IHPS sample solution was markedly improved (Fig. S2B), visually reflecting its enhanced aqueous solubility. Zeta potential analysis (Fig. 1G) further confirmed the successful surface modification of the NPs. The zeta potential of HMPDA was −19.8 ± 0.2 mV. After ICG loading (IH), the potential shifted negatively to −27.1 ± 2.5 mV. In contrast, after modification with ε-PL-SNO, the potential reversed to a positive value of +24.1 ± 1.8 mV. This reversal from negative to positive directly indicates the successful coating of ε-PL-SNO on the IH surface, driven by the protonation of the primary amino groups in the ε-PL molecular chains under the experimental pH conditions. The highly positive surface charge lays the foundation for strong electrostatic interactions with negatively charged bacterial biofilms.

To optimize the composition ratio of the NPs, the effect of the mass ratio of HMPDA to ICG on the encapsulation efficiency (EE) and drug loading (DL) was systematically investigated (Fig. 1H). First, a standard curve was established based on the UV–vis absorption spectrum of an ICG standard solution (Fig. S2C–D). Within the concentration range of 0–30 μg/mL, the absorbance showed a good linear relationship with the ICG concentration (R² > 0.999), providing a reliable basis for accurate drug quantification. When the HMPDA-to-ICG mass ratio was increased from 1:0.25 to 1:0.5, the EE remained in the range of 60.2 %–65.8 %, while the DL increased significantly from 17.4 % to 26.7 %. However, when the amount of ICG feed was further increased, the EE dropped sharply to 12.4 %, and the DL only increased slightly to 29.5 %. These results indicate that an excessive ICG feed exceeds the loading capacity of the mesoporous structure of the HMPDA carrier, leading to the loss of a substantial amount of unencapsulated drug. Consequently, the optimal mass ratio was determined to be 1:0.5, as this proportion achieves a desirable drug load while maintaining a high encapsulation efficiency. Subsequently, the mass ratio of IH to ε-PL-SNO was adjusted to optimize the surface modification process (Fig. 1I). Size analysis revealed that at a mass ratio of 1:1, the particle size of IHPS decreased significantly. The size remained stable within the range of 1:1 to 1:16. However, when the mass ratio exceeded 1:16, the particle size increased noticeably. Therefore, the ratio of 1:16 was selected as the optimal feed ratio. This ratio enables the achievement of the smallest particle size while maximizing the amount of ε-PL-SNO modification, thereby enhancing the NO generation capacity and yielding the final NPs with excellent dispersity and performance.

The colloidal stability and photostability of IHPS were subsequently evaluated. Tests were conducted in PBS, DMEM (supplemented with 10 % FBS), and artificial saliva. The results demonstrated negligible changes in the hydrodynamic diameter of IHPS over 6 days in all three media, confirming its excellent colloidal stability. The polydispersity index (PDI) values were approximately 0.29, suggesting a relatively good dispersion state of the IHPS nanomotors (Fig. 1J). The slight increase observed in DMEM and artificial saliva can be attributed to the adsorption of protein molecules from the media onto the NP surface. Furthermore, the photostability of free ICG was compared with that of ICG encapsulated within IHPS using UV–vis spectroscopy. Throughout the experimental period, the absorbance of free ICG decreased rapidly, with its relative absorbance dropping to only 30.2 % by day 6 (Fig. S2E), reflecting the inherent photolability of ICG. In contrast, the absorbance of IHPS decreased more gradually under identical conditions, retaining 80.9 % of its initial value on day 6 (Fig. S2F). These results conclusively demonstrate that encapsulating ICG within the HMPDA core of IHPS significantly enhances its photostability. This improvement is primarily ascribed to the confinement effect provided by HMPDA, which effectively suppresses the photodegradation and molecular aggregation of ICG.

2.2. ROS generation, photothermal properties, NO release, and nanomotor effect of IHPS

The IHPS nanomotor is a multifunctional platform capable of synergistic therapy under NIR irradiation (Fig. 2A). This platform integrates three core therapeutic modalities: PDT, PTT, and NO therapy. To systematically evaluate its multimodal therapeutic potential, the ROS generation capability, photothermal conversion performance, NO release kinetics, and self-propulsion capabilities of IHPS NPs were thoroughly characterized.

Fig. 2.

Functional characterization of IHPS nanomotors. (A) Schematic diagram of synergistic therapeutic modalities triggered by NIR irradiation. (B) UV–vis spectra of DPBF hydrolysis induced by IHPS NPs under NIR irradiation. (C) Relative UV absorption of DPBF at 420 nm in different solutions (HMPDA, IH, and IHPS NPs) under NIR irradiation. (D) Thermal images of various NPs under NIR irradiation and (E) photothermal properties of different NPs. (F) Photothermal response characteristics of IHPS NPs at different concentrations under NIR irradiation. (G) Photothermal performance of IHPS NPs under NIR laser irradiation at different power densities. (H) Evaluation of photothermal stability of free ICG and IHPS under cyclic NIR irradiation. (I) NO release profiles of IHPS at different concentrations. (J) NO release profiles of IHPS controlled by NIR irradiation. (K) Comparison of NO release from HPS and IHPS NPs. (L) Sustained NO release behavior of IHPS under simulated physiological temperature (37 °C). (M) Optical tracking analysis of motion trajectories for different NPs. (N) Quantitative analysis of the mean squared displacement (MSD), (O) diffusion coefficient, and (P) instantaneous velocity of NPs under various experimental conditions. Data are presented as mean ± SD (n = 3) (∗P < 0.05, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001).

To evaluate the ROS generation capability of the IHPS nanomotors—a property primarily derived from the loaded photosensitizer ICG—the singlet oxygen (¹O₂) production of HMPDA, IH, and IHPS NPs was comparatively analyzed using 1,3-diphenylisobenzofuran (DPBF) as a specific probe for ¹O₂ under 808 nm laser irradiation (1.0 W/cm²). The generation efficiency of ¹O₂ was quantified by monitoring the decrease in the characteristic absorption peak of DPBF at 420 nm. The results indicated that the absorbance of the HMPDA group showed no significant change after laser irradiation (Fig. S2G), demonstrating its inability to generate ROS photodynamically. In contrast, the DPBF absorption peak decreased markedly in the IH group after irradiation (Fig. S2H), confirming that ICG retained its photodynamic activity after being loaded into the HMPDA carrier. Similarly, the IHPS group also exhibited a significant reduction in the absorption peak following laser irradiation (Fig. 2B), verifying its efficient ROS generation. Quantitative comparison further revealed (Fig. 2C) that after 10 min of laser irradiation, the DPBF absorption decreased by 97 % and 77 % for the IH and IHPS groups, respectively, indicating excellent ROS generation capability for both. It is noteworthy that the efficiency of the IHPS group was slightly lower than that of the IH group, which may be attributed to the outer ε-PL-SNO coating partially impeding oxygen diffusion toward the core and reducing the utilization efficiency of the excitation light.

To evaluate the photothermal performance of the NPs, PBS, HMPDA, ICG, IH, and IHPS samples were irradiated with an 808 nm laser (1 W/cm²) for 10 min, and the temperature changes were recorded (Fig. 2E). The results showed that free ICG, as a photothermal agent, exhibited a significant photothermal effect, with its temperature increasing from 25 °C to 51 °C; whereas HMPDA showed only a mild temperature rise, reaching 35.5 °C after 10 min. The IH group demonstrated the most pronounced temperature increase, reaching 52.8 °C, which was further elevated compared to free ICG, indicating that loading ICG into the HMPDA carrier effectively enhanced the photothermal conversion performance. The IHPS group also exhibited excellent photothermal effects, with the temperature rising to 49.6 °C, only slightly lower than the IH group, confirming that surface modification with ε-PL-SNO did not significantly affect the photothermal effect, thereby providing an important foundation for subsequent light-controlled NO release. Thermal images captured by an infrared thermal camera (Fig. 2D) further visually displayed the temperature distribution of the different samples during laser irradiation. Furthermore, the photothermal effect of IHPS showed concentration and power dependencies (Fig. 2F, G) and maintained excellent photothermal stability over three heating-cooling cycles (Fig. 2H), whereas free ICG showed significant attenuation. These results collectively indicate that loading ICG into the HMPDA carrier and further modifying it with ε-PL-SNO to form IHPS can effectively improve the photothermal stability and overall PTT performance of ICG.

To elucidate the NO release behavior of the IHPS nanomotors, the NIR-triggered release mechanism was systematically investigated: under 808 nm laser irradiation, the photothermal effect synergistically generated by the core ICG and HMPDA triggers the cleavage of the S–NO bond in the surface-modified SNO molecules, thereby enabling light-controlled NO release. The released NO was quantified indirectly by detecting nitrite (NO₂⁻) using the Griess method, and its concentration was calculated based on a standard curve (Fig. S2I). The results showed that the amount of NO released from IHPS increased significantly with rising NP concentration, demonstrating a favorable concentration dependence (Fig. 2I). Light-controlled release experiments further indicated (Fig. 2J) that NO was rapidly released within 2 min of laser irradiation; upon cessation of irradiation, a small amount of continued NO generation was detected within the initial 2 min due to residual heat in the system, after which release nearly ceased between 4 and 6 min. Re-irradiation successfully triggered the release process again, and this cycle remained stable over multiple repetitions, proving that IHPS possesses excellent light-controlled NO release capability and cycling stability. To clarify the enhancing effect of ICG on NO release, the release behaviors of HPS and IHPS at the same concentration were compared (Fig. 2K). The amount of NO released from IHPS was significantly higher than that from HPS, because the loading of ICG substantially enhanced the photothermal conversion efficiency of the NP, providing more sufficient energy for the decomposition of SNO. Furthermore, under simulated body temperature conditions (37 °C), IHPS sustained a slow release of NO, with a cumulative release amount reaching 8.53 μM over 72 h (Fig. 2L), indicating its potential for long-acting NO therapy in the physiological environment and offering the possibility for sustained regulation of the immune microenvironment and promotion of tissue repair in vivo.

To evaluate the autonomous motion capability of the IHPS nanomotors under NIR irradiation, their movement trajectories were tracked via fluorescence microscopy, and relevant motion parameters were analyzed. The motion trajectory results (Fig. 2M) showed that IH NPs exhibited typical Brownian motion characteristics both with and without 808 nm laser irradiation (1 W/cm²), indicating the absence of gas-driven propulsion in the absence of ε-PL-SNO modification. In contrast, the IHPS nanomotors displayed significantly extended movement trajectories under laser irradiation. It is noteworthy that the IHPS NPs still possessed a certain degree of autonomous motion capability even without light irradiation, consistent with their inherent slow-release NO capacity. Further mean square displacement (MSD) analysis (Fig. 2N) revealed that under laser irradiation, the slope of the MSD curve for IHPS increased significantly, confirming a marked enhancement in its diffusion capacity due to directed propulsion. Without irradiation, the MSD slope of IHPS remained higher than that of the IH group, again verifying the autonomous motion contribution from the baseline NO release. Quantitative comparison of the diffusion coefficient (Fig. 2O) showed that under irradiation, the diffusion coefficient of IHPS reached 0.76 μm²/s, approximately 3.3 times higher than that of the IH group under the same conditions and about twice that of the IHPS group without irradiation, highlighting the synergistic enhancement of motion capability via photothermally triggered burst NO release. Furthermore, instantaneous velocity tracking results (Fig. 2P) demonstrated that the average velocity of IHPS under continuous irradiation was 3.2 μm/s, significantly higher than that of the non-irradiated IH group (1.0 μm/s), confirming its favorable motion stability.

2.3. Superior bacterial selectivity and biofilm penetration capability of IHPS nanomotors

Bacterial surfaces typically carry a net negative charge under physiological conditions, primarily due to the dissociation of phosphate groups in the teichoic acids of Gram-positive bacterial cell walls and functional groups such as phosphate and carboxyl groups in the lipopolysaccharide (LPS) of Gram-negative bacterial outer membranes [31]. In contrast, mammalian cell membranes are electrically neutral or only weakly charged at physiological pH. This difference provides a theoretical basis for electrostatic interaction-based targeted antibacterial strategies. The designed IHPS nanomotors, endowed with a strong positive surface charge via coating with SNO-modified ε-PL, can specifically target pathogenic bacteria like Porphyromonas gingivalis (P. g) through electrostatic adsorption, while minimizing nonspecific binding to mammalian cells due to the charge disparity. Furthermore, the positive charge facilitates initial attachment and penetration of the nanomotors into the biofilm. Under NIR laser irradiation, the decomposition of SNO in IHPS triggers burst release of NO. The resulting propulsive force further drives the nanomotors to penetrate the dense biofilm structure, effectively reaching deeper layers and thereby achieving efficient eradication of embedded bacteria (Fig. 3A).

Fig. 3.

Selective adhesion and biofilm penetration capability of IHPS nanomotors. (A) Schematic diagram of the targeted adsorption and penetration of IHPS into bacterial biofilms. (B, C) TEM images of P. g treated with IH and IHPS (Scale bar: 1 μm), with insets showing magnified views of the corresponding regions (Scale bar: 500 nm). (D) Zeta potentials of P. g and HGFs after incubation with various concentrations of IHPS for 30 min. (E) Flow cytometry analysis of (i) Nile red-stained P. g and (ii) Hoechst 33342-stained HGFs after 30 min co-culture with IHPS. (F) (i) Schematic diagram of biofilm penetration mechanisms for IH, IHPS, and IHPS + L; (ii) 3D CLSM and z-stack images of P. g biofilms after treatment with IH, IHPS, and IHPS + L at indicated time points (Overview scale bar: 100 μm; Z-stack scale bar: 30 μm); (iii) Quantitative analysis of relative fluorescence intensity. Data are presented as mean ± SD (n = 3) (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

2.3.1. Evaluation of selective binding to bacteria and cells

PI is an infectious disease affecting the soft and hard tissues surrounding dental implants, commonly associated with Gram-negative pathogenic bacteria such as P. g and Fusobacterium nucleatum. This study utilized negatively charged P. g as a model bacterium to evaluate the bacterial targeting capability of the IHPS nanomotors. As shown in Fig. 3B and C, a significant interaction was observed between the IHPS nanomotors and P. g: some nanomotors adhered closely to the bacterial surface, while others penetrated further into the bacterial interior. In contrast, the negatively charged IH NPs showed only minimal adhesion to the bacterial surface, with the majority remaining in a dispersed state. This indicates that the positive surface modification significantly enhances the bacterial binding and internalization capacity of IHPS.

Based on the near-neutral charge of mammalian cell membranes under physiological conditions, we hypothesized that IHPS preferentially targets bacteria via electrostatic interactions. To verify this hypothesis, the zeta potential changes on the bacterial surface were measured after co-incubating P. g with increasing concentrations of IHPS for 30 min. As the concentration of IHPS increased, the surface charge of P. g shifted from −14.63 mV to −8.60 mV, indicating that a substantial number of positively charged nanomotors bound to the bacterial surface via electrostatic attraction, thereby neutralizing its negative charge. Under the same conditions, when co-incubated with human oral fibroblasts, the cell surface zeta potential only exhibited a minor increase from −2.15 mV to 0.49 mV (Fig. 3D). The magnitude of this change was significantly lower than that observed in the bacterial group, demonstrating the pronounced binding selectivity of IHPS towards bacteria. Furthermore, the cellular uptake of the IHPS was quantitatively assessed using flow cytometry. Nile red-labeled P. g and Hoechst-labeled human oral fibroblasts were respectively co-incubated with IHPS. The results revealed that the uptake of IHPS by P. g was approximately 8.2 times higher than that by fibroblasts (Fig. 3E), indicating that IHPS is more readily internalized by bacteria than by mammalian cells.

In summary, the surface positive charge enables IHPS nanomotors to achieve specific bacterial targeting, thereby reducing nonspecific retention in normal cells. This targeting capability enhances antibacterial efficacy while simultaneously minimizing potential toxicity to host cells.

2.3.2. Evaluation of biofilm penetration and dispersal capability

Bacterial biofilms are structured aggregates formed by microbial communities and their secreted EPS. The intricate three-dimensional spatial architecture of biofilms can severely impede the penetration and uniform diffusion of therapeutic agents, such as PSs, thereby significantly compromising the efficacy of PDT and PTT. Biofilms effectively obstruct the penetration of exogenous agents and enhance bacterial tolerance through multiple mechanisms, including their dense physical barrier, internal enzymatic degradation, and metabolic heterogeneity [32]. Consequently, achieving effective drug delivery to the deep regions of biofilms is a critical step for enhancing antibacterial efficacy and overcoming current therapeutic limitations.

To systematically evaluate the penetration and accumulation behavior of IHPS within mature biofilms, a P. g biofilm model was employed. IH, IHPS, and IHPS + L were respectively co-incubated with the biofilms for 20, 40, and 60 min (Fig. 3Fi). After washing away unbound NPs, the biofilms were entirely stained with DMAO, and the spatiotemporal distribution of the NPs was observed using confocal laser scanning microscopy (CLSM) (Fig. 3Fii). The results indicated that the penetration depth and accumulation within the biofilms increased over time for all three groups, but with significant differences among them: The IH group exhibited localized attachment with shallow and uneven overall penetration; the negative surface charge of IH NPs caused electrostatic repulsion with the negatively charged bacterial biofilm, severely limiting their deep accumulation. In contrast, the IHPS group, benefiting from the positive charge introduced by ε-PL modification, demonstrated enhanced adsorption to negatively charged components in the biofilm matrix, leading to superior penetration performance. Notably, the IHPS + L group under NIR laser irradiation displayed uniform and extensive blue fluorescence signals, with penetration nearly reaching the entire depth of the biofilm. The fluorescence intensity of the IHPS + L group was approximately 1.5 times that of the IHPS group, indicating that the burst release of NO gas effectively propelled the nanomotors across the physical barriers of the biofilm, enabling active and deep penetration. Statistical analysis further confirmed the significant advantage of the IHPS + L group in terms of penetration depth (Fig. 3Fiii). This demonstrates that the synergistic effect of photothermal drive and gas propulsion can effectively overcome the limitation of insufficient penetration of traditional PSs within biofilms, offering a novel strategy for eradicating embedded bacterial populations deep within biofilms.

2.4. Superior antibacterial and anti-biofilm performance of IHPS nanomotors

Recent advances NO-releasing nanomaterials demonstrate their considerable potential for combating bacterial infections and biofilms, with several studies highlighting improved biofilm penetration, controlled NO delivery against drug-resistant pathogens, and innovative release mechanisms [[33], [34], [35]]. To address the critical challenges of bacterial colonization and biofilm formation in PI treatment, this study focused on evaluating the efficacy of IHPS nanomotors against planktonic bacteria, mature biofilms, and biofilm formation processes. Through a multifaceted analysis of their capabilities in bacterial capture and eradication, biofilm disruption, and formation inhibition, we comprehensively demonstrated their excellent antibacterial and anti-biofilm properties. Furthermore, the intrinsic relationship between these properties and effective PI treatment was elucidated.

2.4.1. Capture and killing effect on planktonic bacteria

To evaluate the ability of IHPS nanomotors to capture and kill planktonic bacteria, the plate counting method was initially employed to analyze the antibacterial effect of different concentrations of IHPS nanomotors (subjected to 5-min NIR laser irradiation and 24-h co-incubation) against P. g. The results demonstrated that at an IHPS concentration of 100 μg/mL, the bactericidal rate against planktonic P. g exceeded 60 %. When the concentration was increased to 200 μg/mL, complete eradication of the bacteria was achieved (Fig. 4Ai, 4C). Transmission electron microscopy images further revealed that as the concentration increased, more IHPS nanomotors interacted with the bacterial cell membrane or penetrated into the bacterial interior, leading to progressive structural disintegration of the bacteria (Fig. 4Aii).

Fig. 4.

Antibacterial and antibiofilm activity of IHPS nanomotors against P. gingivalis. (A, B) (i) Colony-forming unit (CFU) images and (ii) representative TEM images of P. gingivalis after treatment with IHPS at various concentrations or different treatments (Scale bar: 1 μm). (C, D) Quantitative analysis of P. gingivalis viability following IHPS at various concentrations or different treatments. (E–J) Evaluation of established biofilms: (E) 3D live/dead CLSM images (Scale bar: 100 μm), (F) crystal violet staining, (G) dead/live cell ratio, (H) mean biofilm thickness, (I) photographic images, and (J) corresponding quantification. (K–P) Evaluation of forming biofilms: (K) 3D live/dead CLSM images (Scale bar: 100 μm), (L) crystal violet staining, (M) dead/live cell ratio, (N) mean biofilm thickness, (O) photographic images, and (P) corresponding quantification. Data are presented as mean ± SD (n = 3) (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

At an IHPS concentration of 200 μg/mL, the antibacterial performance was systematically compared by establishing control, IH + L, HPS + L, IHPS, and IHPS + L groups (Fig. 4Bi). The results demonstrated that the IH + L group exhibited stronger bactericidal effects compared to the HPS + L and IHPS groups, primarily attributable to the synergistic antibacterial effect of ICG-mediated PTT and PDT under NIR laser irradiation. The antibacterial efficacy of the IHPS + L group was significantly superior to that of the IH + L group, indicating that the combined effect of positively charged targeted adsorption and the propulsion of NO nanomotors enhanced the penetration capability of IHPS within the plaque biofilm. Furthermore, the substantial amount of ROS generated by ICG during PDT reacted with NO to produce highly cytotoxic ONOO⁻, thereby amplifying the bactericidal effect. TEM results further revealed that all treatment groups, except the control, caused damage to bacterial cell structures, with the most severe destruction observed in the IHPS + L group (Fig. 4Bii, 4D). In summary, the antibacterial mechanism of IHPS under NIR irradiation stems from the synergistic interaction between phototherapy and NO therapy. On one hand, ICG instantaneously generates a large amount of ROS upon 808 nm laser excitation, directly killing bacteria. On the other hand, the photothermal effect triggers the cleavage of the S-NO bond, releasing NO. This not only propels the NPs deeper into the bacterial structures but also allows NO to react with ROS, generating ONOO⁻, thereby achieving synergistic bactericidal action.

To assess the integrity and extent of damage to the bacterial cell membrane, a nucleic acid leakage assay was further conducted. Damage to the cell membrane leads to the leakage of intracellular nucleic acids (such as DNA and RNA) into the surrounding environment, subsequently causing bacterial death. As shown in Fig. S3A, after NIR laser irradiation, the OD260 value of the IHPS + L group increased significantly and continued to rise even after irradiation ceased. A similar trend was observed in the protein leakage assay (Fig. S3B), confirming that IHPS, under the combined antibacterial action of phototherapy and NO, not only disrupts the bacterial membrane structure but also induces significant lysis of bacterial cells. Furthermore, to investigate the stability of the antibacterial performance of IHPS nanomotors, IHPS was incubated in PBS for 7 days, after which its antibacterial efficacy against P. g, Staphylococcus aureus (S. aureus), and Escherichia coli (E. coli) was re-evaluated. The results showed no significant difference in antibacterial activity compared to non-incubated IHPS (Fig. S4A–D), indicating that IHPS maintains good antibacterial stability under in vitro conditions, providing an experimental basis for its potential long-term application.

2.4.2. Evaluation of mature biofilm eradication and biofilm formation inhibition

Eradicating established mature biofilms and inhibiting their reformation are critical steps in treating PI. Bacterial biofilm formation is a complex process, primarily involving five stages: reversible attachment, irreversible attachment, microcolony formation, biofilm maturation, and bacterial dispersal/detachment. Bacteria within the biofilm exhibit extremely high drug resistance due to the barrier function of EPS, metabolic heterogeneity, quorum sensing, and other factors [[36], [37], [38]].This study confirms that IHPS nanomotors, upon NIR laser triggering, can effectively eradicate mature biofilms and suppress their subsequent formation.

To evaluate the eradication capability of IHPS against mature biofilms, a P. g mature biofilm model was analyzed. As shown in Fig. 4E–H and S5A, the IH + L group exhibited certain anti-biofilm activity mediated by the photothermal and photodynamic effects of ICG. However, over 40 % of viable bacteria remained within the biofilm, and the biofilm thickness was only slightly reduced, reflecting the limitations of conventional phototherapy in penetrating dense biofilm structures. In contrast, the anti-biofilm efficacy of the IHPS + L group was significantly enhanced, showing a 1.46-fold improvement in eradication efficiency compared to the IH + L group, with the biofilm thickness being only 70.33 % of the latter. These results clearly demonstrate that the combination of gas propulsion and chemodynamic effects mediated by NO in IHPS significantly enhances the synergistic destructive effect of PTT and PDT on biofilms. Plate counting results further confirmed that IHPS + L treatment effectively killed bacteria within the biofilm, reducing the colony count by 8.92-fold compared to the control group (Fig. 4I, J). Furthermore, parallel experiments using S. aureus and E. coli biofilm models demonstrated that IHPS nanomotors possess broad-spectrum eradication capability against biofilms formed by different bacterial species (Fig. S6), indicating the universality of its mechanism of action.

In clinical practice, biofilm recolonization and regrowth are major factors leading to the recurrence of PI. Therefore, this study further evaluated the inhibitory effect of IHPS nanomotors on biofilm formation. Through Live/Dead staining and plate counting analysis (Fig. 4K-P, S5B), it was found that after treatment with IHPS, the thickness of the newly formed biofilm was significantly reduced, and the ratio of dead to live bacteria was higher than that in the mature biofilm treatment group. This is likely because the structure of the early-stage biofilm is relatively loose and more porous, facilitating more effective penetration and diffusion of the nanomotors into the biofilm interior via positive charge adsorption and NO propulsion, thereby enhancing their antibacterial effect. The results indicate that IHPS nanomotors can not only eradicate established biofilms but also effectively inhibit bacterial reaggregation and colonization by interfering with the early developmental stages of biofilms, providing a dual strategy for preventing the recurrence of PI.

2.5. Biocompatibility and biosafety evaluation of IHPS nanomotors

Biocompatibility and biosafety constitute the fundamental basis for the biomedical application of nanomaterials. This study systematically investigated the biosafety of IHPS through in vitro cytocompatibility assays, hemocompatibility evaluation, and in vivo systemic safety assessment. The results demonstrated that IHPS exhibits favorable biocompatibility under various experimental conditions, providing a reliable basis for its subsequent therapeutic research and clinical translation.

2.5.1. In vitro cytocompatibility assessment

To evaluate the in vitro biocompatibility of IHPS nanomotors and the biosafety of near-infrared (NIR) irradiation, the cell viability of L929 fibroblasts, RAW264.7 macrophages and bone marrow mesenchymal stem cells (BMSCs) was measured using the CCK-8 assay after 2 days of co-incubation. The results indicated that all three cell types maintained high viability following treatment with different concentrations of IHPS. Even at a high concentration of 400 μg/mL, cell viability still exceeded 80 % (Fig. S7A), indicating that IHPS nanomotors induced no significant cytotoxicity across a wide concentration range.

Regarding hemocompatibility, the hemolysis assay results indicated that no significant hemolysis occurred in any of the IHPS treatment groups across the tested concentrations. Quantitative analysis confirmed that the hemolysis rate remained below 5 % (Fig. S7B). This result demonstrates that the IHPS nanomotors possess favorable hemocompatibility, supporting their potential use in blood-contacting environments. Based on these in vitro safety data, a concentration of 200 μg/mL was selected as the therapeutic concentration for subsequent experiments.

To further evaluate the long-term cytocompatibility of IHPS, the cell status was dynamically monitored over a 5-day period using Live/Dead staining (Fig. S7C, D). The results showed that both the control group and the IHPS-treated group maintained high cell viability throughout the culture period, with cell density demonstrating a consistent increasing trend. Although the cell density in the IHPS group was slightly lower than that in the control group, the sustained proliferative behavior indicated that the material did not elicit significant cytotoxic effects or growth inhibition, suggesting its potential for long-term biocompatibility.

2.5.2. In vivo systemic safety evaluation

Given the intended administration route of IHPS nanomotors via local injection into the peri-implant sulcus, their potential exposure pathways include direct contact with oral mucosa, ingestion via swallowing, and absorption into the circulatory system through gingival crevicular fluid. To comprehensively simulate these exposure scenarios, this study employed two administration methods in a rat model: daily oral gavage (simulating ingestion via swallowing) and tail vein injection (simulating systemic circulation exposure). After one week of continuous administration, histological analysis of major organs was performed using H&E staining (Fig. S7E).

It is important to emphasize that the therapeutic dose used in this experiment was significantly higher than the conventional therapeutic dose, aimed at thoroughly evaluating its safety margin. Histopathological analysis revealed no significant pathological alterations or abnormal tissue structures in vital organs, including the heart, liver, spleen, lungs, and kidneys. This result indicates that even under repeated administration conditions, the IHPS did not induce systemic toxicity or organ damage, demonstrating excellent in vivo safety.

Integrating the results from in vitro cytocompatibility, hemocompatibility, and in vivo systemic toxicity evaluations, the IHPS nanomotors demonstrated favorable biocompatibility and biosafety across a broad concentration range and under repeated administration conditions, thereby establishing a reliable safety foundation for their further application in the treatment of PI.

While our short-term (7-day) data demonstrate excellent biosafety of IHPS nanomotors, the chronic nature of PI necessitates consideration of long-term biocompatibility. The IHPS platform comprises biomaterials with favorable degradation profiles: HMPDA undergoes enzymatic hydrolysis to non-toxic metabolites, ε-PL is proteolytically degraded to lysine, and ICG is cleared hepatobillarily. These pathways suggest low potential for long-term accumulation or toxicity. Future studies should focus on chronic exposure models to validate biodegradation kinetics and metabolic fate over extended periods, ensuring clinical translation for chronic conditions like PI.

2.6. IHPS nanomotors remodel the PI immune microenvironment by regulating macrophage polarization

Remodeling the local immune microenvironment is a core aspect of treating PI. Macrophages, as key components of the innate immune system, are highly plastic and can polarize into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes in response to microenvironmental signals, playing a critical role in the progression and resolution of inflammation. M1 macrophages typically highly express inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), and are involved in host defense; however, their overactivation can exacerbate tissue damage and inflammatory responses. In contrast, M2 macrophages highly express arginase-1 (ARG-1), Mannose Receptor C-Type 1(CD206), and anti-inflammatory cytokines (such as IL-10), primarily participating in inflammation resolution, tissue repair, and wound healing [39,40]. Therefore, precisely modulating the direction of macrophage polarization has become an important strategy for treating PI. The ability of IHPS nanomotors to regulate macrophage polarization via NO release is of significant importance for re-establishing immune homeostasis around implants (Fig. 5A).

Fig. 5.

Anti-inflammatory effects of IHPS nanomotors on macrophage polarization. (A) Schematic diagram of the immunomodulatory mechanism. (B) Relative mRNA expression levels of M1 macrophage markers (IL-6, TNF-α, iNOS) after various treatments. (C) Relative mRNA expression levels of M2 macrophage markers (IL-4, CD206, ARG-1). (D) Representative immunofluorescence staining of iNOS and CD206; nuclei were stained with DAPI (Scale bar: 20 μm). (E, F) Quantitative analysis of the fluorescence intensity of iNOS and CD206. (G) Flow cytometry analysis of the surface expression of M1 marker CD86 and M2 marker CD206. Data are presented as mean ± SD (n = 3) (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001).

To evaluate the effect of IHPS on macrophage polarization, an in vitro M1 macrophage model was first established using LPS induction. qRT-PCR results (Fig. 5B, C) showed that compared to the control group, LPS stimulation significantly upregulated the gene expression of M1 markers (iNOS, IL-6, TNF-α). In contrast, the IHPS + L group significantly reversed this trend, simultaneously upregulating the expression of M2 markers (ARG-1, CD206) and the anti-inflammatory cytokine IL-4. These results preliminarily confirm that IHPS, upon NIR laser excitation, can effectively inhibit M1 polarization and promote M2 polarization.

Immunofluorescence staining and flow cytometry analysis provided more direct evidence supporting the above findings. As shown in Fig. 5D-F, the LPS group exhibited significantly higher iNOS fluorescence intensity compared to the control group, while IHPS + L treatment markedly reduced iNOS expression. Furthermore, the CD206 fluorescence intensity in the IHPS group was significantly higher than that in the LPS group and the IHPS + L treatment group, suggesting that the kinetics of NO release play a critical regulatory role in macrophage polarization behavior: under physiological temperature, the slow and sustained release of NO by IHPS is more conducive to promoting M2 polarization [41]. In contrast, although the photothermal effect triggered by laser irradiation accelerates NO release, the burst release of a large amount of NO within a short period may exceed the physiological regulatory capacity of the local microenvironment, thereby impeding the sustained progression of M2 polarization. Concurrently, the excessive ROS generated by the ICG-mediated photodynamic effect in the IHPS + L group may further exacerbate oxidative stress in the peri-implant tissues, potentially interfering with the stability of M2 polarization to some extent. Flow cytometry results further confirmed that IHPS + L treatment significantly increased the proportion of CD206-positive cells and decreased the proportion of CD86-positive cells (Fig. 5G, S8), consistent with the aforementioned findings.

In summary, the IHPS nanomotors effectively induce macrophage polarization from the M1 to the M2 phenotype through photothermally triggered NO release and subsequent signaling pathway regulation, thereby reversing the immune imbalance in the PI microenvironment. The underlying mechanism involves not only the direct immunomodulatory effects of NO, but, more importantly, the dynamic coordination between its release kinetics and the local microenvironment. This multifaceted "antibacterial–anti-inflammatory–reparative" regulatory mechanism creates a favorable immune microenvironment for subsequent osteogenic repair, highlighting the comprehensive advantages of IHPS as an intelligent nanomotor for treating complex infectious diseases.

2.7. Exceptional in vitro osteogenic capability of IHPS nanomotors

Successful bone defect repair not only depends on the establishment of an antibacterial and anti-inflammatory microenvironment but also critically relies on subsequent osteogenic promotion. To systematically evaluate the ability of IHPS to induce bone regeneration under inflammatory conditions, this study conducted a multifaceted systematic analysis of the osteogenic differentiation process of BMSCs in an LPS-induced inflammatory microenvironment. Utilizing various techniques, including alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining, qPCR, Western blot, and immunofluorescence, the study aimed to comprehensively elucidate the mechanism and efficacy of the osteogenic-promoting effects of IHPS under inflammatory conditions.

2.7.1. Multidimensional analysis based on ALP/ARS staining and qPCR/WB/IF

ALP is a key early marker of osteogenic differentiation, and its activity level directly reflects the early differentiation capacity of osteoblasts. ALP staining and quantitative analysis results (Fig. 6A, B) showed that the IH + L group exhibited the weakest ALP activity, even lower than the LPS group. This result indicates that under NIR irradiation, the excessive ROS produced by ICG in the IH group exacerbated oxidative stress in the PI microenvironment. As this group lacked NO release capability, it not only failed to alleviate the inflammatory suppression of the osteogenic process but may also have exerted certain negative effects on the cells' intrinsic osteogenic activity. In contrast, the IHPS group without NIR irradiation demonstrated the strongest ALP activity and staining intensity, significantly higher than the IHPS + L group. We speculate that under conditions without NIR irradiation, this group achieves sustained, slow release of NO at body temperature. This release pattern, on one hand, effectively reverses the inflammation induced by LPS, and on the other hand, maintains the optimal bioactive concentration window required for promoting osteogenic differentiation, thereby significantly enhancing ALP activity. Although the IHPS + L group possesses both ICG and an NO donor, under NIR irradiation, the photodynamic antibacterial effect mediated by ICG generates a large amount of ROS, increasing the oxidative stress level in the PI microenvironment, which may interfere to some extent with the osteogenic-promoting effect of NO. Consequently, the ALP activity in this group was weaker than that in the IHPS and HPS + L groups. ARS staining was used to assess late-stage extracellular matrix mineralization capacity (Fig. 6C). The results showed that the IH + L group had the fewest mineralized nodules, while the IHPS group without NIR irradiation formed the most and most intensely stained calcium nodules, indicating its superior performance in promoting matrix mineralization. Quantitative analysis results were consistent with these observations (Fig. 6D). Combined with the ALP activity assay, these findings fully demonstrate that the sustained, slow release of NO significantly enhances the entire process of osteogenic differentiation.

Fig. 6.

Osteogenic differentiation evaluation of BMSCs in vitro. (A, B) ALP staining and ALP activity quantification on day 7 (Scale bar: 500 μm). (C, D) ARS staining and quantification of mineralization on day 21 (Scale bar: 500 μm). (E–G) Relative mRNA expression levels of RUNX2, OPN, and OCN. (H) Western blot analysis of RUNX2, OPN, and OCN protein expression. (I–L) Immunofluorescence images and quantitative analysis of RUNX2, OPN, and OCN (Scale bar: 100 μm). Data are presented as mean ± SD (n = 3) (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001).

Given the critical roles of RUNX2, OPN, and OCN in bone matrix synthesis and osteogenic regulation, we further analyzed their expression at both the gene and protein levels. qRT-PCR results (Fig. 6E–G) showed that LPS treatment significantly suppressed the expression of osteogenesis-related genes (RUNX2, OPN, and OCN), whereas IHPS treatment markedly reversed this trend. Among the groups, the IHPS group exhibited the highest mRNA expression levels of all three genes, further confirming that the sustained release of NO effectively promotes the transcription of osteogenesis-related genes. Western blot analysis (Fig. 6H) revealed that the protein expression levels of RUNX2, OPN, and OCN were highest in the IHPS group, a trend further validated by quantitative analysis (Fig. S9A–C). Immunofluorescence staining results (Fig. 6I-L) demonstrated that the fluorescence signal intensity in the IHPS group was significantly stronger than in the other groups, indicating its superior capability in promoting the expression of osteogenesis-specific proteins.

These multidimensional evaluations consistently demonstrate that the IHPS nanomotors exhibit excellent osteogenic-promoting capacity in an inflammatory environment, with the non-NIR-irradiated IHPS group showing the most outstanding performance. This group significantly outperformed other treatment groups in early ALP activity, late-stage mineralization nodule formation, and the transcription and protein expression of osteogenic genes. Compared to the transient high-concentration NO release triggered by NIR, the sustained release mode achieved by the IHPS group is more conducive to maintaining the local bioactive concentration window required for osteogenic differentiation. This stable NO supply not only enables synergistic regulation of the inflammatory microenvironment but also allows for the continuous activation of downstream osteogenesis-related signaling pathways.

2.7.2. Validation of the sGC-cGMP-PKG signaling pathway activation mechanism

NO, a key signaling molecule produced by the catalysis of L-arginine by nitric oxide synthase, plays multiple regulatory roles in bone metabolism. NO acts not only as an intracellular messenger involved in the proliferation and differentiation of osteoblasts but also regulates osteoclast activity through intercellular communication, collectively maintaining bone remodeling balance. During bone regeneration, elevated local NO levels can initiate downstream signaling cascades. The classical pathway is as follows: NO activates sGC, promotes the generation of cGMP, which in turn activates cGMP-dependent PKG, ultimately driving the expression of osteogenesis-related genes and promoting bone formation. Based on the central role of NO in bone metabolism and the established regulatory mechanism of this pathway, this study further investigated whether the osteogenic-promoting effect of the IHPS nanomotor system is mediated by the NO-sGC-cGMP-PKG signaling pathway (Fig. 7A).

Fig. 7.

IHPS enhances osteogenic differentiation through the sGC/cGMP/PKG pathway. (A) Schematic diagram of the mechanism involving sGC/cGMP/PKG activation by IHPS. (B–D) Western blot and quantitative analysis of sGC and PKG protein expression. (E–G) Immunofluorescence images and quantitative analysis of sGC and PKG (Scale bar: 100 μm). Data are presented as mean ± SD (n = 3) (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001).

To validate the role of this pathway in IHPS-mediated osteogenic differentiation, the protein expression levels of sGC and PKG were first assessed by Western blot analysis (Fig. 7B–D). The results showed that compared with the control group, the expression of both sGC and PKG was significantly downregulated in the LPS-stimulated group. In contrast, treatment with the HPS and IHPS groups significantly reversed this trend, with the IHPS group exhibiting the highest protein expression levels of sGC and PKG, consistent with the expression trends of osteogenic markers. These findings suggest that the sustained release of NO from the IHPS nanomotors effectively activates the sGC-cGMP-PKG signaling pathway. To further verify the activation of this pathway at the cellular level, the localization and quantitative analysis of sGC and PKG were performed using immunofluorescence staining (Fig. 7E–G). The results demonstrated that the fluorescence signal intensities of both sGC and PKG in the IHPS group were significantly stronger than those in the other groups, indicating abundant expression and a high state of activation of these proteins within the cells. Quantitative analysis further confirmed the significant advantage of the IHPS group in activating the sGC-cGMP-PKG pathway, with fluorescence intensities 2.16-fold and 1.85-fold higher than those in the LPS group, respectively. These consistent experimental results indicate that the IHPS, through the sustained and slow release of NO at body temperature, effectively activate the sGC-cGMP-PKG signaling pathway, thereby significantly enhancing the expression of osteogenesis-related markers and ultimately promoting bone repair and regeneration.

2.8. In vivo evaluation of the antibacterial, anti-inflammatory, and osteogenic properties of IHPS nanomotors

To systematically evaluate the comprehensive therapeutic efficacy of IHPS nanomotors in the complex microenvironment of PI, this study established a rat PI model to thoroughly investigate their in vivo antibacterial, anti-inflammatory, and osteogenic capabilities (Fig. 8A). The experimental procedure involved extracting the maxillary first molars of rats and placing implants, followed by ligating a silk suture around the implant neck and inoculating with P. g four weeks later to induce PI. Successful modeling was confirmed by the presence of typical inflammatory signs in the peri-implant tissues, including swelling, dark redness, bleeding on probing, and plaque accumulation. The in vivo photothermal performance of IHPS was assessed using infrared thermography (Fig. S10A, B). The results indicated that after 8 min of NIR irradiation, the local temperature in the IHPS group increased from 38 °C to 45 °C, whereas no significant change was observed in the normal saline group. This temperature increase confirms the excellent photothermal conversion capability of IHPS, laying the foundation for subsequent PTT and controlled NO release.

Fig. 8.

In vivo evaluation of therapeutic effects on PI. (A) Schematic diagram of PI model induction and treatment regimen. (B) (i) Photographs of the oral cavity in PI model rats after different treatments (Scale bar: 2 mm); (ii) Representative bacterial colonies isolated from peri-implant tissue; (iii) In vivo fluorescence images of ROS levels at the implant site. (C) Micro-CT 3D reconstruction and sectional views of implant sites (Scale bar: 1 mm). (D) Quantitative analysis of ROS fluorescence intensity. (E–G) Quantitative statistics of alveolar bone for different parameters. Data are presented as mean ± SD (n = 3) (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001).

After two weeks of treatment, intraoral photographs revealed severe gingival recession and exposed implant threads in the PI group, whereas the gingival tissue morphology in the IHPS + L group was similar to that of the healthy control group (Fig. 8Bi). CFU counts demonstrated that the bacterial load in the plaque surrounding the implants in the IHPS + L group was lower than that in the IH + L, HPS + L, and other treatment groups (Fig. 8Bii, S10C), highlighting its potent antibacterial effect achieved through the synergistic mechanisms of PDT, PTT, and NO therapy. To evaluate the regulatory effect of IHPS on ROS levels, in vivo imaging was performed using the DCFH-DA fluorescent probe. The PI group exhibited high-intensity fluorescence signals, indicating significantly elevated ROS levels in the inflammatory microenvironment. All treatment groups suppressed ROS generation to varying degrees, with efficacy in the following order: IHPS + L > IH + L > HPS + L > IHPS (Fig. 8Biii). Quantitative analysis confirmed that the ROS level in the IHPS + L group was comparable to that of the healthy control group, demonstrating its ability to effectively alleviate oxidative stress (Fig. 8D). This effect stems from the direct eradication of bacterial biofilms by ICG-mediated PTT/PDT, which significantly reduces the excessive ROS production triggered by persistent bacterial infection and subsequent inflammatory responses at the source. Furthermore, the photothermal effect promotes the cleavage of the S-NO bond, releasing NO, which can react with ROS to form ONOO⁻. This reaction not only enhances the antibacterial efficacy but also further consumes excess ROS, leading to a significant mitigation of oxidative stress and creating a favorable microenvironment for osteogenic repair.

To systematically evaluate the improvement effect of different treatment groups on PI-related bone resorption, the rat maxillae were scanned using Micro-CT, followed by image analysis and three-dimensional reconstruction (Fig. 8C, S10D). Quantitative analysis results indicated that, compared to the PI group, all treatment groups exhibited varying degrees of reduction in bone resorption (Fig. 8E). Among them, the three-dimensional reconstructed Micro-CT images of the IHPS + L group showed nearly complete repair of the bone defect area, with continuous trabecular structure and significantly increased density. Its bone volume fraction (BV/TV, Fig. 8F) and trabecular number (Tb.N, Fig. 8G) were significantly higher than those of the other groups, demonstrating the best osteogenic capability. This advantage stems from the synergistic therapy activated under NIR irradiation—ICG-mediated phototherapy combined with NO-mediated bacterial biofilm eradication—while the slow release of NO at body temperature not only induces macrophage polarization towards the M2 phenotype but also promotes osteogenic differentiation by activating the sGC-cGMP-PKG signaling pathway, thereby synergistically optimizing the immune microenvironment and bone repair process.

In contrast, the IH + L group, owing to the significant reduction in biofilm load achieved by its PTT/PDT antibacterial effects, laid the foundation for subsequent osteogenesis. Consequently, its bone regeneration outcome was superior to that of the HPS + L and IHPS groups. Although the HPS + L group possessed NO-release capability, the absence of ICG-mediated antibacterial effects prevented it from achieving NIR-triggered efficient NO release and deep tissue penetration, resulting in weaker antibacterial and osteogenic performance. Micro-CT images of this group revealed evident residual bone defects and sparse trabecular structures. The IHPS group, which did not receive NIR irradiation, failed to activate photothermal and photodynamic effects, leading to a lack of early antibacterial intervention and allowing the biofilm to persistently disrupt the osteogenic microenvironment. Despite its capacity for slow NO release, it remained ineffective in repairing the bone defects. Micro-CT indicated the lowest degree of bone repair in this group, with the bone resorption area still being extensive. These results underscore that, within the complex in vivo infection environment, effective antibacterial action is a necessary prerequisite for successful bone regeneration, forming a stark contrast to the in vitro mechanism which relies solely on the direct osteogenic regulation by NO.

In terms of histological and molecular phenotypic evaluations, H&E and Masson staining revealed massive inflammatory cell infiltration and severe degradation of collagen fibers in the PI group. In contrast, the IHPS + L group showed only minimal inflammatory cell infiltration, with intact collagen fiber structure and dense staining, closely resembling the healthy control group in morphology. Immunofluorescence detection of key markers (Fig. 9A) demonstrated high expression of the pro-inflammatory cytokine IL-6 in the PI group and low expression in the IHPS + L group, while the anti-inflammatory phenotype ARG-1 was highly expressed in the IHPS + L group. The fluorescence intensities of osteogenic markers RUNX2, OPN, and OCN were significantly stronger in the IHPS + L group compared to other groups. Quantitative analysis (Fig. 9B) further confirmed that the IHPS + L group had the lowest number of inflammatory cells and the highest collagen volume fraction (Fig. 9C, D), the lowest expression of IL-6 and the highest expression of ARG-1 (Fig. 9E, F), alongside the strongest expression of the osteogenic markers RUNX2, OPN, and OCN (Fig. 9G–I). These results molecularly corroborate that IHPS + L can induce macrophage polarization towards the M2 phenotype, creating an anti-inflammatory immune microenvironment. The enhanced osteogenic differentiation in this anti-inflammatory environment directly explains why the IHPS + L group exhibited the best bone regeneration outcome in the Micro-CT analysis, highlighting the synergistic advantage of combining anti-inflammatory and osteogenic therapy.

Fig. 9.

Therapeutic evaluation of PI via histological and immunofluorescence analysis. (A, B) Representative images of H&E staining, Masson staining, and immunofluorescence images (IL-6, ARG-1, RUNX2, OPN and OCN) under different treatments (Scale bar: 100 μm) as well as schematic diagram of histological and immunofluorescence analysis. (C, D) Quantitative analysis of number of immune cells and collagen volume fraction. (E–H) Relative fluorescence intensities of IL-6, ARG-1, RUNX2, OPN and OCN. Data are presented as mean ± SD (n = 3) (∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001).

In summary, under NIR triggering, the IHPS nanomotors demonstrate outstanding comprehensive therapeutic performance in a PI model through the synergistic "antibacterial–anti-inflammatory–osteogenic" therapy mediated by PTT, PDT and NO therapy. This provides new strategies and insights for the clinical treatment of infectious bone defects, highlighting the significant advantages of intelligent responsive nanomotors in achieving multi-mechanism synergistic therapy within complex infectious environments.

3. Conclusion

Based on a multi-mechanism synergistic therapeutic strategy, this study successfully constructed a photothermally responsive NO-releasing nanomotor (IHPS). The core advantages of this system are primarily manifested in three aspects. First, the IHPS integrates initial electrostatic targeting with NIR-triggered burst release of NO, achieving a transition from static anchoring to autonomous propulsion, which significantly enhances the penetration capability into dense biofilms and overcomes the permeation limitations of traditional cationic nanomaterials. Second, under NIR irradiation, the IHPS realizes a synergistic effect of PTT, PDT and NO therapy. The reaction between NO and ROS generated by PDT produces highly cytotoxic ONOO⁻, thereby achieving efficient eradication of biofilms. Finally, leveraging the multifunctional regulatory properties of NO, the IHPS further modulates macrophage polarization towards the M2 phenotype on the basis of its antibacterial action and promotes osteogenic differentiation via the sGC-cGMP-PKG pathway, achieving integrated therapy for immune microenvironment regulation and bone regeneration. This intelligent nanomotor, which integrates active penetration, efficient antibacterial activity, and immunomodulatory/osteogenic regulation, provides a highly promising novel strategy for the clinical treatment of PI.

4. Materials and methods

4.1. Materials

All chemical reagents, including dopamine hydrochloride (DA), F127, 1,3,5-trimethylbenzene (TMB), tris(hydroxymethyl)aminomethane (Tris), ethanol, acetone, indocyanine green (ICG), 3-mercaptopropanoic acid, tert-butyl nitrite (TBN), ε-poly-L-lysine (ε-PL), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 1,3-diphenylisobenzofuran (DPBF), LPS, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), crystal violet and Nile red were supplied by Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).The Bacterial Live/Dead Staining Kit (DMAO/PI), nitric oxide assay kit, BCA protein assay kit, osteoblast mineralized nodule staining kit (Alizarin Red S method), and DAPI staining solution were all purchased from Beyotime Biotechnology (Shanghai, China). Hoechst 33342, Cell Counting Kit-8 (CCK-8) and the alkaline phosphatase (ALP) color development kit were provided by Meilun Biotechnology Co., Ltd. (Dalian, China). Antibodies specific for iNOS, CD86, CD206, OCN, OPN, PKG1, sGC, Runx2, and β-actin were obtained from Boster Biological Technology Co., Ltd. (Wuhan, China).

4.2. Characterizations

The hydrodynamic size distribution and zeta potential of the hydrated nanoparticles (NPs) were determined using dynamic light scattering (DLS, Nano ZS90, UK). Their morphology was characterized by transmission electron microscopy (TEM, JEM2010, Japan). The ultraviolet–visible (UV–vis) spectra of intermediate and final products were recorded on a UV–vis spectrophotometer (PerkinElmer LAMBDA 950, USA). The CCK-8 assay was conducted using a microplate reader (Sunrise, USA) to evaluate cell viability. Biofilms were visualized for live/dead staining, intracellular reactive oxygen species (ROS) generation, and immunofluorescence experiments under a confocal laser scanning microscope (CLSM, Leica, Germany). Macrophage polarization was assessed via flow cytometry (Accuri C6, USA). The movement trajectories of nanomotors were recorded using a fluorescence microscope (IX83, Olympus, Japan). Western blot analysis was performed using the Bio-Rad imaging system (ChemiDoc XRS, Germany).

4.3. Methods

4.3.1. Synthesis of IHPS nanomotors

4.3.1.1. Synthesis of HMPDA

HMPDA NPs were synthesized via a template method based on the self-oxidation polymerization of dopamine. Briefly, 25 mL of ethanol was mixed with 25 mL of deionized water. Then, 0.30 g of F127 and 500 μL of TMB were added to the mixture and stirred for 2 h to form the template. Subsequently, 15 mg of dopamine hydrochloride and 90 mg of Tris were added, and the solution was stirred in the dark for 24 h. The black precipitate was collected by centrifugation at 14,000 rpm for 15 min. The precipitate was washed three times with an ethanol/acetone mixture (2:1, v/v) to remove the template, and the final product, HMPDA NPs, was collected again by centrifugation at 14,000 rpm for 15 min. The obtained HMPDA NPs were lyophilized and stored at 4 °C for subsequent experiments.

4.3.1.2. Synthesis of IH

After the synthesis of HMPDA NPs, ICG was loaded into the HMPDA NPs to form IH NPs. First, pre-synthesized and purified HMPDA NPs (1 mg) were dispersed in 4 mL of deionized water and sonicated to obtain a homogeneous dispersion. Then, different masses of ICG (0.25, 0.5, 1, 2, 3 mg) were added to the dispersion, resulting in HMPDA-to-ICG mass ratios (w/w) of 1:0.25, 1:0.5, 1:1, 1:2, and 1:3, respectively. The mixture was stirred at room temperature in the dark for 24 h to ensure sufficient diffusion and adsorption of ICG molecules into the mesopores and hollow cavity of the HMPDA NPs. Finally, the IH NPs precipitate was collected by centrifugation at 14,000 rpm for 15 min and washed three times with deionized water. The product was lyophilized and stored at 4 °C for subsequent use.

4.3.1.3. Synthesis of ε-PL-SNO

The synthesis began with the preparation of SNO-COOH. An excess of TBN (971.55 mg, 9.42 mmol) and SH-COOH (200 mg, 1.88 mmol) were mixed in 20 mL of deionized water. The reaction proceeded under a nitrogen atmosphere, in an ice bath, and protected from light, with stirring for 6 h. Subsequently, the mixture was subjected to vacuum treatment for 15 min to remove unreacted TBN, yielding the SNO-COOH solution. The SNO-COOH was then purified by freeze-drying. Next, the purified SNO-COOH (101.25 mg, 0.75 mmol) was dissolved in 10 mL of deionized water. To this solution, EDC (718.13 mg, 3.75 mmol) and NHS (172.5 mg, 1.5 mmol) were added and stirred for 30 min to activate the carboxyl groups. Meanwhile, ε-PL (100 mg, 0.025 mmol) was dissolved in 2 mL of deionized water. This ε-PL solution was then added dropwise to the activated SNO-COOH solution. The reaction mixture was stirred for 6 h in an ice bath and protected from light. The final product, ε-PL-SNO, was purified by dialysis, followed by freeze-drying, and stored at 4 °C for subsequent use (Fig. S1).

4.3.2. Preparation of IHPS nanomotors

IHPS Nanomotors were formed via electrostatic adsorption. Briefly, 1 mg of pre-synthesized IH NPs was dissolved in 4 mL of deionized water. Subsequently, different masses of ε-PL-SNO (1, 2, 4, 8, 16, 32 mg) were dissolved in 4 mL of deionized water to achieve IH-to-ε-PL-SNO mass ratios of 1:0, 1:1, 1:2, 1:4, 1:8, 1:16, 1:32 and 1:40 (w/w), respectively. The ε-PL-SNO solutions were then separately added dropwise to the IH NPs solution. The mixture was stirred in an ice bath protected from light for 4 h to allow self-assembly via electrostatic interactions, forming the IHPS Nanomotors. Finally, the reaction solution was purified by centrifugation (14,000 rpm, 15 min) to remove unreacted components, yielding IHPS Nanomotors with different mass ratios. The products were freeze-dried and stored in the dark at 4 °C for subsequent use.

4.3.3. Evaluation of EE and DL of IHPS nanomotors

To assess the loading efficacy of ICG, the EE and DL of IHPS Nanomotors were determined. The unencapsulated free ICG was separated from the ICG-loaded IHPS Nanomotors using an ultrafiltration centrifugation method. The concentration of free ICG in the supernatant was quantified by UV–vis spectrophotometry, based on a standard curve generated from pure ICG.

The EE and DL were then calculated using the following equations:

EE (%) = (Weight of drug encapsulated in NPs)/(Total weight of drug used in feeding) × 100 %

DL (%) = (Weight of drug encapsulated in NPs)/(Total weight of drug-loaded NPs) × 100 %

4.3.4. Assessment of IHPS nanomotors stability

The hydrodynamic stability and photostability of the IHPS Nanomotors were systematically evaluated. First, the variation in particle size was monitored over a 6-day period using DLS after incubating the NPs at 37 °C in three different physiological media: PBS, DMEM supplemented with 10 % serum, and artificial saliva. Additionally, the photostability of ICG within the IHPS Nanomotors was investigated by measuring the absorbance changes in PBS (pH 7.4) over 6 days, with free ICG serving as a control. Briefly, a free ICG solution was prepared in PBS at a concentration of 10 μg/mL. Simultaneously, the synthesized IHPS Nanomotors were dispersed in an equal volume of PBS, ensuring the final concentration of ICG was equivalent to that of the free ICG group. All sample solutions were incubated in a constant-temperature chamber, and the characteristic absorbance of each sample was measured daily for six consecutive days using a UV–vis spectrophotometer, without exposure to any additional light source beyond the necessary spectroscopic measurement.

4.3.5. In vitro ROS generation and photothermal performance evaluation

The ability of various NPs (HMPDA, IH, and IHPS) to generate ROS, specifically singlet oxygen (¹O₂), under laser irradiation was assessed using DPBF as a chemical probe. DPBF was introduced into dispersions of the NPs, which were then irradiated with an 808 nm laser for 10 min. The decrease in the characteristic absorption peak of DPBF at 420 nm was monitored using a UV–vis spectrophotometer. Prior to testing, all samples were saturated with oxygen via sonication, and the liquid surface was sealed with liquid paraffin to prevent oxygen dissipation. Absorbance readings were recorded at 1-min intervals during the irradiation period. The photothermal performance was evaluated by exposing 1 mL of dispersions of PBS (control), HMPDA, ICG, IH, and IHPS Nanomotors to an 808 nm near-infrared laser at a power density of 1 W/cm² for 10 min. The temperature of the solutions was recorded every 2 min using a thermocouple probe, and thermal images were simultaneously captured with an infrared thermal camera. To investigate the concentration-dependent PTT effect, IHPS Nanomotors were prepared at various concentrations (25, 50, 100, and 200 μg/mL) and irradiated under identical conditions, with PBS as a blank control. Furthermore, the influence of laser power on the PTT conversion efficiency was systematically examined at a fixed IHPS concentration. To compare the photothermal stability, IHPS and free ICG were subjected to three on/off cycles of 808 nm laser irradiation (10 min on followed by natural cooling).

4.3.6. Measurement of NO generation and release capacity

The NO generation and release performance was measured using a nitric oxide assay kit. The released NO is oxidized to form nitrate, which subsequently reacts with Griess reagent to produce an azo compound with a characteristic absorption peak at 540 nm. The absorbance at 540 nm was measured using a microplate reader. The concentration of NO (μM) reported in all experiments represents the final concentration in the aqueous release medium (i.e., the solution volume in which the nanoparticles were dispersed), as calculated based on a standard curve generated from sodium nitrite (NaNO₂). To assess the effect of material concentration on NO release, IHPS Nanomotors solutions at 25, 50, 100, and 200 μg/mL were prepared, and the amount of NO released after 5 min of 808 nm laser irradiation was quantified. To evaluate the controllable release capability, a 200 μg/mL IHPS Nanomotors solution was divided into two groups: one received two cycles of 808 nm laser irradiation (2 min per cycle), while the other was kept in the dark, and the NO release kinetics of both groups were dynamically monitored. To assess the influence of the PTT effect on NO release, solutions of HPS and IHPS at the same concentration (200 μg/mL) were prepared, and the amount of NO released after 5 min of 808 nm laser irradiation was recorded and compared. Additionally, to evaluate the sustained release profile of the material at physiological temperature, a 200 μg/mL IHPS Nanomotors solution was incubated in a 37 °C constant-temperature incubator for 3 days, with daily sampling to measure the cumulative NO release.

4.3.7. Recording and analysis of IHPS nanomotors' motion

The motion behavior of IHPS nanomotors was observed using a fluorescence microscope, and their trajectories were recorded over 10 s. During the experiment, different NPs (IH, IH + L, IHPS, and IHPS + L) were irradiated vertically with an 808 nm NIR laser. Motion videos of the nanomotors were captured using the microscope's built-in imaging system. The movement trajectories were then manually tracked using an ImageJ plugin to obtain coordinate sequences and instantaneous velocities. Based on the trajectory data, the MSD curves and diffusion coefficients were calculated using open-source code in MATLAB. The resulting data were exported, and graphs were plotted using Prism software.

4.3.8. Evaluation of the bacterial targeting ability of IHPS nanomotors

To evaluate the bacterial targeting ability of IHPS Nanomotors, the following experimental procedure was conducted. First, HGFs cells were stained with Hoechst 33342 and P. g bacteria were stained with Nile red, each for 20 min, followed by washing with PBS. HGFs cells and P. g were adjusted to concentrations of 10⁵ cells/mL and 10⁷ CFU/mL, respectively, mixed in PBS, and co-incubated with IHPS Nanomotors for 30 min. After incubation, the mixture was centrifuged at 3000 r/min for 3 min and washed three times with PBS to thoroughly remove unbound NPs. The final pellet was collected, resuspended, and subjected to quantitative analysis using flow cytometry. To further observe the interaction between IHPS Nanomotors and bacteria, P. g bacteria were co-incubated with IHPS Nanomotors for 30 min. The bacteria were then collected by centrifugation and fixed with glutaraldehyde for 30 min. The binding of NPs to bacteria was finally analyzed using TEM.

4.3.9. Evaluating the biofilm penetration capability of IHPS nanomotors

This study aimed to assess the penetration capability of IHPS Nanomotors into plaque biofilms formed by P. g. Initially, the bacterial suspension was adjusted to a concentration of 10⁸ CFU/mL and inoculated into confocal dishes containing Brain Heart Infusion (BHI) broth. To meet the growth requirements of P. g, the BHI medium was supplemented with 5 μg/mL hemin and 1 μg/mL menadione. The cultures were then incubated under anaerobic conditions (85 % N₂, 10 % H₂, 5 % CO₂) at 37 °C for 3 days to allow the formation of mature biofilms. To analyze the penetration performance of the NPs, the prepared IH and IHPS Nanomotors were divided into three treatment groups: IH group, IHPS group, and IHPS + L group (with 808 nm laser irradiation at a power density of 1 W/cm²). For each group, 1 mL of NPs solution at the same concentration was co-incubated with the biofilms for time gradients of 20, 40, and 60 min. After incubation, the biofilms were washed three times with PBS buffer to remove unbound particles thoroughly. Subsequently, the biofilms were stained with DMAO in the dark for 20 min. The penetration depth of the NPs within the biofilms was ultimately observed and recorded using CLSM.

4.3.10. Evaluation of the antibacterial and anti-biofilm activity of IHPS nanomotors

4.3.10.1. Assessment of antibacterial activity against free bacteria

To evaluate the antibacterial activity of IHPS Nanomotors against free P. g, bacterial suspensions (10⁶ CFU/mL) were mixed with various concentrations of IHPS Nanomotors. The mixtures were irradiated with an 808 nm laser at a power density of 1 W/cm² for 5 min. Subsequently, the samples were incubated anaerobically at 37 °C for 24 h. After incubation, the bacterial solutions were serially diluted, plated uniformly on blood agar plates, and further cultured under identical anaerobic conditions. The resulting colonies were finally counted to determine the antibacterial efficacy. To analyze bacterial morphology and the interaction between bacteria and IHPS Nanomotors, the co-incubated bacterial suspensions were collected and centrifuged at 3000 rpm for 3 min. The pellets were washed three times with PBS buffer to remove unbound NPs. The bacterial precipitates were then resuspended and fixed with 2.5 % glutaraldehyde solution, and ultimately observed and analyzed using TEM.

For a systematic evaluation of the antibacterial performance of different NPs, five experimental groups were established: Control, IH + L, HPS + L, IHPS, and IHPS + L. The colony counting method on blood agar plates was employed after anaerobic cultivation to compare the antibacterial effects among groups. Furthermore, TEM was used to observe the interactions between bacteria and NPs, helping to elucidate the antibacterial mechanisms.

To assess the impact of NPs on bacterial membrane integrity, a suspension of P. g was adjusted to a concentration of 10⁸ CFU/mL and treated with different NPs under respective conditions. Supernatant samples were collected at various time points (every 10 min), centrifuged, and appropriately diluted. The extent of nucleic acid leakage was analyzed by measuring the absorbance at 260 nm, while the level of protein leakage in the supernatant was determined using a BCA protein assay kit.

4.3.10.2. Evaluation of bacterial biofilm eradication capability

To systematically evaluate the biofilm eradication efficacy of different NPs, five groups were set up: Control, IH + L, HPS + L, IHPS, and IHPS + L. Both immature and mature biofilms were cultured and then treated with the respective NPs, with some groups receiving additional 808 nm laser irradiation (power density: 1 W/cm², time: 5 min). Subsequently, the biofilms were ultrasonically dispersed, serially diluted, and 100 μL of the bacterial suspension was spread evenly on blood agar plates. These plates were incubated anaerobically at 37 °C for 7 days, and the survival status of the biofilm bacteria was assessed by colony counting. After biofilm cultivation, the samples were gently rinsed with PBS to remove loosely attached and planktonic bacteria. Then, a Bacterial Live/Dead Fluorescent Staining Kit was used according to the manufacturer's instructions to prepare the working solution. The biofilms were stained in the dark for 20 min. After staining, three-dimensional image acquisition and biofilm thickness quantification were performed using CLSM to analyze the viability and spatial distribution of bacteria within the biofilm. To quantitatively assess the residual biomass of bacterial biofilms under different treatment conditions, Crystal Violet staining was employed. Firstly, the biofilm samples from each group were fixed for 15 min, followed by three washes with PBS to thoroughly remove the fixative, and air-dried at room temperature. Then, 1 mg/mL Crystal Violet staining solution was added to stain for 15 min. Unbound dye was removed by thorough washing with PBS. Finally, the Crystal Violet bound to the biofilm was dissolved using 95 % ethanol, and the absorbance at 590 nm was measured with a microplate reader to quantitatively evaluate the biofilm biomass.

4.3.11. Evaluation of biocompatibility and biosafety of IHPS nanomotors

4.3.11.1. In vitro cytotoxicity assay

The impact of IHPS nanomotors combined with near-infrared irradiation (NIR, power density: 1.0 W/cm²) on the viability of L929, RAW264.7, and BMSCs was evaluated using the CCK-8 assay. Briefly, the cells were seeded into 96-well plates. The experimental groups were treated with culture medium containing varying concentrations of IHPS nanomotors, and a separate NIR-only group (exposed to irradiation without nanomotors) was included. After 24 h of incubation, the medium was replaced with fresh medium containing the CCK-8 reagent. Following an appropriate incubation period, the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader. Cell viability was calculated to determine cellular activity.

4.3.11.2. Hemolysis assay

The hemolysis assay was performed to evaluate the hemocompatibility of the IHPS Nanomotors. RBCs were isolated from whole blood via centrifugation and washed with PBS to prepare an RBC suspension at a specific concentration. Positive control (deionized water) and negative control (PBS) groups were established. The RBC suspension was co-incubated with different concentrations of IHPS Nanomotors for 2 h. After centrifugation, the absorbance of the supernatant was measured at 576 nm, and the hemolysis ratio was calculated.

4.3.11.3. Live/dead cell staining

To further evaluate the effect of IHPS Nanomotors on cell viability, live/dead cell staining was performed on treated L929 cells. After being treated with 200 μg/mL IHPS Nanomotors for 1, 3, and 5 days, the cells were stained with calcein-AM and PI under light-protected conditions for 30 min. The stained cells were then observed and imaged using CLSM. Cells treated with PBS served as the control group.

4.3.11.4. In vivo biosafety assessment

For the in vivo biosafety evaluation, nine 6-week-old male Sprague-Dawley (SD) rats were randomly divided into three groups (n = 3): a control group, an intragastric injection group, and an intravenous injection group. After continuous treatment for one week, the animals were euthanized. Major organs, including the heart, liver, spleen, lungs, and kidneys, were harvested for histological examination to comprehensively assess the in vivo safety profile of the NPs.

4.3.12. Evaluation of anti-inflammatory effects and macrophage phenotype polarization

4.3.12.1. Establishment of inflammatory model and treatment groups

RAW 264.7 cells were seeded into 6-well plates at a density of 2 × 10⁵ cells per well and cultured for 24 h to allow adhesion. Except for the control group, all other groups were stimulated with 1 μg/mL P. g-derived LPS for 3 h to establish the inflammatory model. After replacing the medium, the following treatments were applied: the control group received normal culture medium; the LPS group received no further treatment; the IH + L group was treated with IH NPs followed by 808 nm laser irradiation (1 W/cm², 5 min); the HPS + L group received HPS NPs with irradiation; the IHPS group was treated with IHPS Nanomotors alone; and the IHPS + L group received IHPS Nanomotors combined with laser irradiation. All groups were subsequently cultured for an additional 24 h before further analysis.

4.3.12.2. Analysis of M1/M2 marker gene expression by RT-qPCR

The mRNA expression levels of M1 markers (IL-6, TNF-α, iNOS) and M2 markers (IL-4, CD206, ARG-1) were detected using RT-qPCR. β-Actin was used as the internal reference gene, and the relative expression of each gene was calculated using the 2^−ΔΔCt method. The experiment was independently repeated three times, and the primer sequences used are detailed in the Supplementary Table S1.

4.3.12.3. Detection of M1/M2 marker protein expression by immunofluorescence staining

To evaluate the impact of IHPS Nanomotors on macrophage polarization, the expression of M1/M2 marker proteins was assessed by immunofluorescence staining. After the respective treatments, cells were fixed with 4 % paraformaldehyde, permeabilized with 0.25 % Triton X-100, and blocked with serum. The cells were then incubated overnight at 4 °C with specific primary antibodies (anti-iNOS for M1, anti-CD206 for M2), followed by incubation with appropriate fluorescently labeled secondary antibodies. Nuclei were counterstained with DAPI. Images were captured using a CLSM to analyze protein expression and localization.

4.3.12.4. Analysis of macrophage surface markers by flow cytometry

For further analysis of M1/M2 macrophage polarization markers CD86 and CD206 expression by flow cytometry, RAW 264.7 cells were seeded and the inflammatory model was constructed. After treatment, the cells were collected, stained with CD86 and CD206 antibodies in the dark at room temperature for 15 min, washed three times with PBS, resuspended, and then analyzed by flow cytometry.

4.3.13. Evaluation of osteogenic differentiation in BMSCs

BMSCs were seeded in 12-well plates at a density of 1 × 10⁵ cells per well and cultured until they reached approximately 80 % confluency. The culture medium was then replaced with osteogenic induction medium, which was refreshed every three days. Except for the control group, all groups were first stimulated with 1 μg/mL P. g-derived LPS for 3 h to establish an inflammatory microenvironment. Following LPS stimulation and medium replacement, the groups received the respective treatments as defined in the inflammatory model section.

4.3.13.1. Assessment of osteogenic differentiation capacity

After the respective treatments, osteogenic induction was continued for 2 weeks. ALP activity, an early marker of osteogenic differentiation, was assessed after one week of induction using an ALP staining kit. To evaluate the formation of a mineralized matrix, a separate set of BMSCs was induced for 3 weeks under the same grouping conditions. The cells were then fixed and stained with Alizarin Red S solution to visualize calcium nodules. For quantitative analysis, the bound Alizarin Red S was dissolved with 10 % acetic acid, and the absorbance was measured at 562 nm using a microplate reader to semi-quantify the degree of mineralization.

4.3.13.2. Analysis of osteogenesis-related gene expression

To analyze the changes in the expression of key osteogenic genes under different treatment conditions, total RNA was extracted, and the mRNA expression levels of osteogenic marker genes, including RUNX2, OPN and OCN, were detected by RT-qPCR. β-Actin was used as the internal reference gene for normalization. The relative expression level of each target gene was calculated using the 2^−ΔΔCt method. The experiment was independently repeated three times, and the primer sequences used are detailed in the Supplementary Table S2.

4.3.13.3. Protein expression analysis via immunofluorescence staining

To analyze the impact of IHPS Nanomotors on the expression and localization of key proteins involved in osteogenic differentiation and related signaling pathways (including RUNX2, OPN, OCN, sGC, and PKG), immunofluorescence staining was performed. After the respective treatments, cells were fixed with 4 % paraformaldehyde, permeabilized with Triton X-100, and blocked with serum to prevent non-specific binding. The cells were then incubated overnight at 4 °C with specific primary antibodies against the target proteins. After washing, the cells were incubated with appropriate fluorescently labeled secondary antibodies. Nuclei were counterstained with DAPI. Finally, images were captured using a CLSM to analyze the protein expression levels and their subcellular localization.

4.3.13.4. Western blot assay

For Western blot analysis, total proteins were extracted using RIPA lysis buffer containing 100 μM PMSF, and protein concentrations were determined by the BCA method. Equal amounts of protein samples were separated by SDS-PAGE electrophoresis and transferred onto PVDF membranes. The membranes were blocked at room temperature for 2 h with 5 % BSA in TBST, followed by incubation with specific primary antibodies (including anti-Runx2, anti-OPN, anti-OCN, anti-sGC, anti-PKG, and anti-β-actin antibodies) at 4 °C overnight. After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1.5 h. Protein signals were visualized using an ECL detection reagent, and images were captured with a Bio-Rad imaging system. Band intensity was quantified with ImageJ software, normalized to β-actin as the internal reference.

4.3.14. Evaluation of the in vivo antibacterial, anti-inflammatory, and osteogenic capabilities of IHPS nanomotors

To assess the antibacterial, anti-inflammatory, and osteogenic efficacy of IHPS Nanomotors in vivo, a rat model of PI was established and therapeutic interventions were conducted. All animal experiments were approved by the Ethics Committee of the Affiliated Hospital of Qingdao University. Six-week-old male Sprague-Dawley rats (purchased from Beijing Haifukang Biotechnology Co., Ltd.) were randomly divided into six groups: Control, PI, IH + L, HPS + L, IHPS, and IHPS + L. Following extraction of the right maxillary first molar, an implant was immediately placed. After 4 weeks, osseointegration was confirmed. Except for the control group, PI was induced in all other groups by ligating the implant neck with a silk suture for 2 weeks and inoculating with P. g. After model establishment, the IH + L, HPS + L, and IHPS + L groups received additional intervention with 808 nm NIR laser irradiation (1 W/cm², 5 min). Two weeks post-treatment, gingival crevicular fluid and soft debris around the implant were collected using sterile swabs, eluted with PBS, serially diluted, and plated on blood agar plates for colony counting.

To further analyze local oxidative stress levels, ROS expression in peri-implant tissues was detected by in vivo fluorescence imaging. After anesthetizing the rats with sodium pentobarbital, the ROS fluorescent probe DCFH-DA (1.8 mg/kg) was injected intravenously. Thirty minutes later, fluorescence signals were captured using an IVIS® imaging system (PerkinElmer; excitation 525 nm/emission 495 nm). After the experiment, the animals were euthanized, and the right maxilla samples were collected for micro-CT scanning and immunohistochemical analysis.

When evaluating the degree of peri-implant bone resorption (Fig. S10D), the line connecting the buccal and lingual alveolar crests of the tooth distal to the implant served as the measurement baseline (indicated by a blue reference line). The height of bone loss on the buccal and lingual sides of the implant was measured and recorded as Value A and Value B, respectively. The arithmetic mean of these two values was calculated as the final bone resorption height for quantitative analysis.

4.3.15. Statistical analysis

Experimental data are presented as the mean ± standard deviation. Statistical analysis for between-group comparisons was performed using independent samples t-test and one-way analysis of variance (ANOVA). The significance levels were defined as follows: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

CRediT authorship contribution statement

Xin Zhan: Writing – original draft, Data curation. Ying Li: Investigation, Data curation. Chenglin Yang: Writing – original draft. Wenping Zhang: Writing – original draft. Zeyu Han: Investigation. Yimeng Li: Investigation. Qiutong Meng: Investigation. Yuanyong Feng: Writing – review & editing. Youcheng Yu: Writing – review & editing, Supervision, Funding acquisition. Baodong Zhao: Writing – review & editing, Supervision. Fan Li: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare no competing interests.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.