Nanozyme-based Robots Swarm with Exciton-Engineered Z-Scheme Heterojunctions for Depth-Resolved Tumour Penetration and Therapy

Nanozyme-based Robots Swarm with Exciton-Engineered Z-Scheme Heterojunctions for Depth-Resolved Tumour Penetration and Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Nanozyme-based Robots Swarm with Exciton-Engineered Z-Scheme Heterojunctions for Depth-Resolved Tumour Penetration and Therapy Zhiguang Wu, Zhengya Yue, Minglu Tang, Shuo Wang, Xiangwei Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6432792/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Synthetic nanorobots have garnered significant attention for precision oncology, while their clinical translation is hindered by inefficient tumour penetration. Catalytic motion, which mainly rely on fuel gradients, always fails in deep tumour due to limited motility. Here, we introduce exciton-dissociation-enhanced nanozyme-based robots (EDNRs) swarm that leverage Z-scheme heterojunctions to achieve the enhanced tumour penetration, combining photocatalytic propulsion with collective hydrodynamic effects. The EDNRs were constructed via Z-scheme heterogenization of photocatalytic FeOCl nanosheets with polyoxometalates, creating a high-valence-band interface that promotes efficient exciton dissociation. This heterostructure conferred enhanced catalytic activity compared to non-heterogenized counterparts. Under near-infrared irradiation, the EDNRs demonstrated superior motility compared with the nanozyme-based robots without Z-scheme heterogenization, attributed to synergistic photocatalytic propulsion and swarm-induced hydrodynamic interactions. In vivo intravenous administration in mice exhibit that the nanozyme-based robot swarm upon NIR irradiation could exert the swarm penetration of tumour tissue and following arrival at the deep tumour. Meanwhile, accompanying with the production reactive oxygen species (ROS), the nanozyme-based robot swarm in deep tumour substantially inhibits the proliferation of tumour. The swarm dynamics of nanozyme-based robot with enhanced exciton dissociation, potentially impacts the realization of catalytic nanorobots toward deep tumour penetration and therapy. Physical sciences/Materials science/Nanoscale materials/Nanoparticles Physical sciences/Engineering/Biomedical engineering Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Synthetic nanorobots have emerged as a transformative platform for precision oncology, offering the potential to navigate complex biological environments and deliver targeted therapies to tumours 1 – 3 . However, their clinical translation remains hindered by inefficient penetration into deep tumour tissues, a challenge rooted in the dense extracellular matrix (ECM), high interstitial fluid pressure (IFP), and limited motility of conventional nanosystems 4 – 6 . Catalytic motion, one of the most promising propulsion strategies for biomedical nanorobots because their actuation without the need of additional complex instruments to generate external fields 7 . Among various catalytic strategies, utilizing the enzymatic based complex as catalysts is emerging as promising approach for the design of nanorobots owing to their usage of endogenous fuels 8 , 9 . Particularly the artificial nanozymes have been demonstrated to incorporate into the nanorobots to accomplish mobility though their catalytic activity or photocatalytic activity with the assistance of NIR Eg, accompanying with the generation of reactive oxygen species (ROSs) using the peroxidase-like activity. Although the nanozyme-based nanorobot have reported therapy effect toward disease such as fungal infection, often fails in solid tumours due to insufficient motility and rapid fuel depletion, confining nanorobots to superficial tumour regions where fuel is scarce 7 , 10 , 11 . Because conventional nanozymes have a limited catalytic capacity, they are susceptible to pH, fuel concentration, and their own local concentration 12 , 13 . Overcoming these barriers requires innovative designs that integrate robust propulsion with spatiotemporal control over collective behavior. Exciton dissociation, key to photocatalytic activity, has been underexplored in nanorobot design. Photocatalytic nanomaterials generate propulsion via light-driven bubble formation or ion gradients, but inefficient exciton recombination limits catalytic efficiency 14 . Z-scheme heterojunctions, mimicking natural photosynthesis, preserve high redox capacity while promoting ultrafast exciton dissociation 15 , 16 . However, their application in tumour-penetrating nanorobots remains unreported, as does the interplay between heterostructure design and swarm dynamics. Here, we introduce exciton-dissociation-enhanced nanozyme-based robots (EDNRs) that leverage Z-scheme heterojunctions to achieve depth-resolved tumour penetration and therapy. The EDNRs are constructed via Z-scheme heterogenization of FeOCl nanosheets with polyoxometalates (POMs), creating a high-valence-band interface that promotes efficient exciton dissociation and charge separation 17 . This design not only enhances photocatalytic activity for reactive oxygen species (ROS) generation but also enables synergistic photocatalytic propulsion and swarm-induced hydrodynamic interactions under near-infrared (NIR) irradiation 18 . By combining autonomous motion with collective behavior, EDNRs overcome the motility limitations of traditional nanorobots, achieving deep tumour penetration and localized ROS-mediated cytotoxicity 19 – 21 . The Z-scheme heterojunction architecture is critical for the performance of EDNRs. Unlike type II heterojunctions, which suffer from reduced redox potentials, the Z-scheme design preserves the high redox capacity of both semiconductors, enabling efficient oxidation of water to hydroxyl radicals (·OH) and reduction of oxygen 22 – 25 . This dual functionality boosts ROS production while minimizing electron-hole recombination, a key factor in enhancing catalytic activity 26 . Additionally, the photocatalytic decomposition of H₂O₂ generates oxygen bubbles, providing thrust for directional motion, while swarm dynamics reduce drag through collective hydrodynamic interactions 27 – 29 . By integrating Z-scheme heterojunctions with swarm dynamics, EDNRs represent a paradigm shift in nanorobotics, offering spatiotemporally controlled therapy and paving the way for clinical translation of synthetic nanomachines in precision oncology 30 – 32 . Results and discussion Structural design of EDNRs via exciton-dissociation mechanism The structural design of EDNRs was validated through energy band analysis. As illustrated in Fig. 2 A, the Z-scheme heterojunction formed by FeOCl nanosheets and polyoxometalates (POMs) nanozyme created a built-in electric field at the interface. Knowing that the valence band (VB) of FeOCl was positioned at 2.90 eV, while the conduction band (CB) of POM-Mo was 0.15 eV 33 , 34 . Upon NIR light irradiation, this unique band alignment facilitated efficient exciton dissociation, enabling directional charge transfer. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) were used to characterize the morphology of as-prepared NRs and EDNRs. The ultrathin layered structure of the EDNRs was evidenced by Fig. 2 B and Fig. S1 , where various nanosheets with size of ~ 200 nm were dispersed. The HRTEM image of EDNRs clearly shows the crystalline structure with a lattice fringer of 0.395 nm (3.95 Å) corresponding to the 020 facet (Fig. 2 C) 17 . To investigate the existence of POM nanozyme in the EDNRs, area energy dispersive X-ray spectroscopy (EDS) mapping of EDNRs and NRs was tested separately (Fig. 2 D and Fig. S2 ). In comparison of Fig. 2 E and S3, the presence of Mo and P elements (corresponding to POM) in EDNRs proves that the distribution of POM components in EDNRs and the exact elements ratio of EDNRs was shown in Fig. S4 (O: P: Cl: Fe: Mo = 65.5: 0.6: 9.1: 19.0: 5.8). Additionally, X-ray diffraction (XRD) analysis was performed to characterize the phase structure of the as-fabricated NRs and EDNRs. The characteristic diffraction peaks located in 11° indexed to the (010) planes were both found in NRs and EDNRs, and the results were good and consistent with the standard JCDPS card of No.01-72-069 corresponding to NRs (Fig. 2 F) 17 . As shown in Fig. 2 G, atomic force microscopy (AFM) was used to observe the morphology and the thickness of EDNRs. The thickness of EDNRs was measured to be about 14 nm and the corresponding 3D image was presented in Fig. S5 . The hydrated particle size of NRs and EDNRs were measured by dynamic light scattering (DLS), demonstrating their potential as nanomedicines (Fig. 2 I). The Zeta potential value changed from 24.1 mV in NRs to -13.0 mV in EDNRs further proving the successful synthesis of EDNRs (Fig. 2 J). Next, the chemical composition and valence changes of NRs and EDNRs were further measured by X-ray photoelectron spectroscopy (XPS). As shown in Fig. 2 K, in addition to the Fe 2p, O 1s, and Cl 2p characteristic peaks observed in both NRs and EDNRs, the Mo 3d characteristic peaks are observed in the EDNRs due to the presence of the POM. Moreover, the characteristic peak of Fe 2p in EDNRs was divided into four main peaks (Fe 2+ 2p 3/2, Fe 3+ 2p 3/2, Fe 2+ 2p 1/2, and Fe 3+ 2p 1/2 ) and four satellite peaks, while Fe 2p in NRs was mainly divided into two peaks (Fe 3+ 2p 3/2, and Fe 3+ 2p 1/2 ) and two satellite peaks without the presence of Fe 2+ peaks (Fig. 2 L and Fig. S6 ). The bigger binding energy of Cl 2p (199.32 eV) in NRs than Cl 2p (198.11 eV) in EDNRs indicated an increase in electron density in EDNRs ( Fig. S7 ). The above results indicated the successful synthesis of NRs and EDNRs, and the interaction in EDNRs affects the charge transfer. Photocatalytic activity and motility analysis of EDNRs According to energy-band theory, EDNRs promote catalytic activity via the built-in electric field of the Z-scheme heterojunction structure. Under NIR irradiation, the band gap bends, leading to the redistribution of surface electrons and holes to specific positions. This unique Z-scheme heterojunction structure effectively enhances exciton dissociation and inhibits the undesirable recombination of electron- hole pairs on the EDNRs surface, enabling the structure to maintain stronger reduction/oxidation potentials of separated electrons on the CB and holes on the VB. Thus, based on the photocatalytic effect, EDNRs enhance the photocatalytic reaction to drive their propulsion (Fig. 3 A). Considered the critical role of the energy-band structure in photocatalytic activity, a prerequisite for efficient therapy, the energy-band structure of EDNRs was further characterized. Firstly, UV-vis diffuse reflectance spectra (UV-vis DRS) were tested to characterize the absorption difference between synthesized NRs and EDNRs, a key factor influencing catalytic activity. Figure 3 B shows that both NRs and EDNRs exhibit long absorption edges, with EDNRs demonstrating stronger absorption in the NIR region. Using the corresponding tangent lines of UV-vis DRS and the Kubelka - Munk equation, the band gaps of POM-Mo⁻ and NRs were calculated to be 2.61 eV and 2.34 eV, respectively (Fig. 3 C). Furthermore, to characterize the heterojunction formation and type, valence-band spectra were used to detect the VB values of NRs and POM - Mo NSs, which were 2.9 eV and 2.49 eV, respectively (Fig. 3 D). Then, using the relationship \(\:{E}_{g}={E}_{VB}-{E}_{CB}\) , the CB values of NRs and POM-Mo⁻ were calculated to be 0.15 eV and 0.29 eV. These band-structure results suggest the formation of either a type II or Z-scheme heterojunction in EDNRs. The catalytic activity of EDNRs is significantly higher than that of NRs, as the VB potential of NRs is insufficiently positive to oxidize H 2 O into ·OH radicals. PL spectroscopy further confirmed the Z-scheme heterojunction formation in EDNRs. The weak emission intensity of EDNRs indicated a higher separation efficiency than NRs, which is inconsistent with the type II mechanism (Fig. 3 E). These findings indicate that holes accumulate at the high VB of POM-Mo, while electrons transfer from POM- Mo to NRs, suggesting a charge-transfer path that adheres more to the Z-scheme mechanism than the type II mechanism. Photocurrent and electrochemical impedance spectrum (EIS) were used to further characterize the photoelectric conversion performance of the unique Z-type heterojunction structure in EDNRs. The photocurrent-response test showed uniform photocurrent densities in both NRs and EDNRs (Fig. 3 F), but EDNRs had significantly stronger photocurrent intensity. The remarkably smaller arc radius of EDNRs than NRs in Nyquist plots, indicating smaller interfacial charge resistance, reveals the promotion of exciton - dissociation behavior due to the Z-scheme heterojunction in EDNRs ( Fig. S8 ), indicating smaller interfacial charge resistance, reveals the promotion of exciton - dissociation behavior due to the Z-scheme heterojunction in EDNRs. As expected, EDNRs with Z-Scheme heterostructure demonstrate exceptional redox properties, effectively separating holes and electrons (e − ) under light irradiation. Therefore, the separating holes (h + ) facilitates direct reactions with H 2 O generating •OH 35 , as well as a cascade reaction with H 2 O 2 , producing 1 O 2 . To verify this mechanism, the •OH, and 1 O 2 generated in the cascade reaction were detected, respectively. The generation of •OH was detected using 3,3’,5,5’-tetramethyl-benzidine (TMB) as a probe, which was oxidized to blue oxide (ox-TMB) with increasing absorbance at 652 nm (Fig. 3 G). The UV-vis absorption spectrum for the EDNRs + NIR group exhibits a prominent peak at 652 nm, indicating the generation of a substantial amount of •OH (Fig. 3 H). Meanwhile, the increase of its absorption peak with the increase of the concentration further confirms the enhanced •OH generation capacity of EDNRs, which might attribute to the Z-scheme heterojunction structure ( Fig. S9 ). In addition, 1,3-Diphenylisobenzofuran (DPBF) was used as a 1 O 2 probe, which would gradually degrade from yellow to colorless and was illustrated in Fig. 3 I. It was found that EDNRs have superior 1 O 2 generating ability compared to NRs and its absorption peaks were significantly weakened with time, while NIR irradiation further enhanced 1 O 2 generation (Fig. 3 J and S10). As a result of the above results, it can be concluded that EDNRs possesses excellent catalytic properties, in addition to converting H 2 O 2 to 1 O 2, it also reacts directly with H 2 O to form •OH, which might attribute to the enhanced exciton dissociation and inhibition of electron-hole recombination by the Z-scheme heterojunction structure. Subsequently, the motion behavior of the nanorobot was captured using an optical microscope, and its trajectory was subsequently tracked and analyzed using TrackMate (an open Fiji plugin) 21 . Figure 3 K shows the tracking motion trajectories of NRs and EDNRs under the corresponding conditions (with or without NIR irradiation). NRs-POM with NIR irradiation shows a tendency of directional motion compared to the other controls (NRs, NRs + NIR, and EDNRs), and this discrepancy may originate from the enhanced catalytic reaction progress of EDNRs under the NIR irradiation (Fig. 3 K, Supplementary Movie 1 ). Velocity analysis was conducted at fixed time intervals. Following light exposure, the EDNRs group exhibited a clear response, with a significant increase in velocity. In contrast, the NRs group showed no substantial change before or after light exposure, with the slight increase potentially attributed to light-enhanced Brownian motion (Fig. 3 L). Then, the corresponding mean squared displacement (MSD) was also calculated according to the collected motion trajectories (Fig. 3 M) 20 , 36 . The MSD curves versus time interval (Δt) in the EDNRs + NIR group showed a rising parabolic line (representing autonomous motion), while the other control groups displayed a linear growth (often representing Brownian motion). Moreover, the effective diffusion coefficients (De) were also calculated according to the equation ( \(\:{D}_{e}=MSD/4\varDelta\:t\) ), which were also consistent with the above results (Fig. 3 N). Based on the above experimental results and previous reports in the literature 28 , 37 , it can be concluded that EDNRs heterojunction has enhanced catalytic activity and motion behavior performance as expected. Swarm Dynamics of EDNRs upon NIR illumination Based on the detailed exploration of the photocatalytic reaction mechanism presented in the previous section, the valence band holes participate in the oxidation of water, releasing protons (H⁺). This process may create a local gradient field, which in turn generates an electrophoretic flow. This flow exerts a force on the charged EDNRs within the gradient field, driving the collective behavior of the nanorobots ( Fig. S10 ). To verify the dynamics and collective light chemotaxis behavior of the EDNRs in vitro , optical microscopy was used to directly observe the dynamics of the EDNRs under NIR irradiation. The motion behavior of the EDNRs emerged upon turning on the NIR light and evolved from single-particle motion to collective behavior with increasing exposure time ( Supplementary Movie S2 ). By further quantifying the collective behavior based on the ROI values of snapshots taken at different time points (0, 3, 6, and 9 min), light was identified as the key factor driving the emergence of collective behavior of EDNRs (Fig. 4 A). As shown in Fig. 4 B, with the extension of light exposure time, the swarm gradually congregates towards the center of the illuminated area, shifting its distribution from a broad peak to a narrow, high peak. This arises as the rapid motion of individual EDNRs intensifies frequent monomer-to-monomer collisions, and the POM on the nanobot surface enables gradual cluster formation. More interestingly, as the position of the NIR source keeps moving (P1, P2 and P3), the EDNRs swarms also respond to it by following the center of the source, showing a phototropic chemotaxis (Fig. 4 C, D and Supplementary Movies S3 ). In addition, UV-Vis spectroscopy was also used to further quantify the collective and phototactic behavior of the EDNRs swarms. As shown in Fig. 4E , 3 mL of the EDNRs solution was placed in a cuvette, while the top of the cuvette was continuously illuminated with NIR irradiation instead of the bottom in order to exclude the effect of gravity on results. The digital images in Fig. 4F shows that near the bottom of the cuvette, light scattering by EDNRs suspended in solution causes the appearance of distinct light paths when a light beam is present (Tyndall effect), but the intensity of the light path at the bottom does not show a significant change after 100 min of NIR irradiation. However, it is noteworthy that the intensity of the light path near the top of the cuvette increased after 100 min, indicating an increase in the concentration of EDNRs. In contrast, the intensity f the two light paths at the top and bottom of the cuvette did not show any significant change before and after in the no-NIR light irradiation group. Next, we further confirmed this collective behaviors of EDNRs by employing UV-Vis spectroscopy to examine the changes in the solution concentration at the top with increasing irradiation time. Fig. 5G shows that, after 100 min of NIR irradiation, the absorption intensity of the solution at the top of the cuvette increased, which was significantly different from that before irradiation, suggesting that the NIR irradiation induced the clustering of the EDNRs at the top of the cuvette ( Fig. 5H ). Based on the above results, this kind of swarming, positively phototropic EDNRs is expected to achieve better tumour therapeutic effects in vivo , by increasing the enrichment rate and penetration depth. Furthermore, considering that the holes oxidize water to generate ROS while simultaneously producing H⁺, 5(6)-carboxy fluorescein (5(6)-FAM) was used to measure the pH of the photocatalytic region, which produces significant differences in fluorescence intensity at different pHs ( Fig. 3I ). Quantitative analysis of the results revealed a significant difference in fluorescence intensity between the photocatalytic reaction region and the surrounding environment, confirming the generation of a pH gradient field ( Fig. S12 ). In vitro therapeutic efficacy of EDNRs Before evaluating the therapeutic effect, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the biocompatibility of the heterostructural EDNRs. Incubation of normal fibroblasts (L929) with EDNRs for 24 h and 48 h did not result in significant damage, indicating the biosecurity for normal tissues ( Fig. S13 ). Given the excellent biocompatibility observed, we investigated whether the heterostructural EDNRs exhibit effective tumour inhibition in vitro . Initially, as shown in Fig. 5 A, the tumour-killing effect was examined utilizing the live/dead staining assays for HeLa cells. The gradually increasing red channel signaling implies a decreased cell viability of HeLa cancer cells, with the EDNRs + NIR condition, showing as high as 90% red signals ( Fig. S14 ). Based on these results, we qualitatively analyzed the survival of HeLa cells under different conditions. Cell survival was as low as 33.85% within 24 h after treatment with EDNRs (Fig. 5 B). Meanwhile, NIR irradiation also significantly reduced tumour survival, which was consistently in line with previous results (Fig. 5 C). As mentioned earlier, the generation of ROS is a major factor in killing cancer cells. Thus, 2’,7’-dichlorofluorescein diacetate (DCFH-DA) was utilized to evaluate intracellular ROS levels, which would be oxidized by ROS into 2',7'-dichlorofluorescein (DCF) in cells with green fluorescence. The presence of a clear green fluorescent channel in the EDNRs group indicates ROS production (Fig. 5 D). After being treated with the NIR laser irradiation (0.5 mW/cm 2 , 5 min), the average ROS level increased from 15 to 90, demonstrating a greater capacity for ROS generation ( Fig. S15 ). Notably, flow cytometry was utilized to further quantify the effect of different treatment conditions on intracellular ROS levels. As displayed in Fig. 5 E, no significant difference was observed between the intensity values of the ctrl and NIR groups and the slight enhancement in the NRs, NRs + NIR, and EDNRs groups was due to the production of ROS, whereas the ROS level was significantly enhanced by the NRs + NIR treatment. Considering that the depolarization in mitochondrial membrane potential (MMP) is an important feature of mitochondrial dysfunction associated with cell apoptosis. Therefore, MMP was measured using JC-1 as the fluorescent probe under various treatments, and the mechanism was illustrated in detail in Fig. S16 . The green fluorescence signal of the JC-M channel in Fig. 5 F indicated mitochondrial damage, and the detailed intensity of green fluorescence in different treatment groups was presented in Fig. 5 G. Crystal violet staining in cell cloning experiments once again demonstrated the feasibility of this therapeutic strategy, with a reduction in the purple area implying inhibition of HeLa cell colony formation (Fig. 5 H). In addition to superior ROS generation capability, the ability to enhance tumour tissue penetration through photocatalytic propulsion is another key factor determining the therapeutic efficacy. To determine the enhancement of tumour penetration ability by swarm motion, trans-well migration experiments were performed by using FITC-labeled nanomotors. In the trans-well experiment, the upper and lower chambers are separated by a membrane. FITC-labeled nanomotors are only placed in the lower chamber, and cells are seeded in the upper chamber to model the in vivo barrier meet by nanomotor. The experimental results were observed using fluorescence microscopy; the green signal channel represents FITC and the blue signal channel is DPAI. As shown in Fig. 6A , uniform blue signals indicate consistent cell attachment, while varying green signals reflect differences in penetration abilities among different groups. The enhanced green signal in group EDNRs + NIR, compared to the other controls, demonstrates that enhanced motility induces deeper penetration with cellular uptake. Besides, the corresponding MFI in Fig. 6 B is found that the fluorescence intensity of the robot group was seven times higher than that of the other groups, indicating a significant enhancement in penetration ability. After confirming the enhanced permeability across the membrane, three-dimensional multicellular tumour spheres (3D-MTSs) were constructed to further evaluate the deep penetration ability of the nanorobot within the dense collagen fibers of tumour tissues. In the 3D-MTSs model, it was obvious from the optical microscope images that the EDNRs with NIR group showed extensive detachment damage with loose ( Fig. S17 ). Next, FITC-labeled nanorobots were co-incubated with the 3D-MTS model, and confocal fluorescence microscopy was used to observe the depth distribution and evaluate the penetration ability. Using a 20 µm increment, Z-axis sectioning of the 3D-MTS was performed from the bottom. Compared to the control group, significant green fluorescence signals were still observed even at a depth of 120 µm in Robot group (Fig. 6 C). A fluorescence intensity distribution comparison along the X-axis for (depth of 100 µm) clearly shows the difference in penetration depth between the two groups (Fig. 6 D). In Fig. 6 E, slices from the robot group at the same depth exhibited significantly enhanced fluorescence intensity compared to the control group. The decrease in fluorescence intensity between 80 µm and 120 µm may be attributed to the limited fluorescence tissue penetration depth of the confocal microscope. Additionally, based on the average fluorescence intensity of the slices, the robot group exhibited six times higher intensity than the control group (Fig. 6 F). In vivo elucidation of the mechanism of ENDRs swarm- mediated antitumour activity Motivated by the in vitro experimental results, it is necessary to further investigate whether ROS, enhanced permeability, and effective accumulation in vivo can also improve tumour inhibition effects, thereby clarifying the tumour-killing mechanism of EDNRs in vivo . Consistent with the previous discussion, the S180 tumour-bearing KM mice model was selected again to study the in vivo mechanism of EDNRs swarm. To observe the differences in in vivo ROS levels, mice were euthanized 6 h after treatments, and tumour tissues were collected for frozen sectioning and ROS staining. Except for the control group, green fluorescence signals were observed in all other groups, indicating the generation of ROS ( Fig. 7A ). The trend of enhanced green fluorescence also coincided with the in vivo and in vitro tumour inhibition rate growth, fully demonstrating the Z-type heterojunction structure can also promote ROS generation in vivo , thereby increasing the inhibition rate. Then, to further test the penetration ability of the motors in tumour tissues, tumour tissues were collected in vivo and co-incubated with FITC-labeled motors, followed by washing with PBS. Tumour slices at different depths were then collected, and the fluorescence signal intensity in the FITC channel was evaluated using fluorescence microscopy to reflect the penetration depth of the motors ( Fig. 7B ). Compared to the control group, the motors group exhibited more prominent green fluorescence signals, indicating greater tumour accumulation ( Fig. 7C ). Additionally, as the distance from the outer edge of the tissue slices increased, the motors group still showed significant fluorescence intensity. Quantitative analysis of the tissue slices revealed that at a depth of 600 μm, the fluorescence intensity was seven times higher than that of the control group ( Fig. 7D ). Additionally, an inductively coupled plasma emission spectrometer (ICP) was used for more accurate quantification of the motor accumulation efficiency ( Fig. 7E ). Similarly, the accumulation rate in the motors group was significantly higher than that of the control group, reaching a peak around 12 hours. At the same time, the motors group exhibited a longer tumour retention time. Therefore, it can be concluded that the EDNRs therapeutic strategy can effectively inhibit tumours in vivo due to its superior ROS generation capacity, deeper penetration and higher accumulation in tumour tissues. In vivo antitumour performance and biocompatibility of EDNRs Considering the above superior ROS production and deep penetration capacity in vitro, an S180 tumour-bearing KM mice model was established to investigate the antitumour performance in vivo , and the KM mouse was randomly divided into six groups: G1 Ctrl, G2 NIR, G3: NRs, G4: NRs+NIR, G5: EDNRs and G6: EDNRs+NIR. The body weights of the mice and the volume of the tumours were also measured daily, and the mice were euthanized 14 days after various treatments, and the tumours along with the major organs were collected forfuther analysis ( Fig. 8A ). As demonstrated by digital photographs of hormonal mice taken in Fig. 8B , there was a significant ablation of the tumours after EDNRs+NIR treatment. Digital images of tumours collected from the mice were shown in Fig. 8C , and the relative tumour sizes of NIR, NRs, and NRs+NIR treated mice were not significantly different from those of the ctrl group, suggesting minimal efficacy.Compare EDNRs and EDNRs+NIR, both the digital images and weight data clearly show a significant enhancement after NIR irradiation in tumour ablation, almost all tumours from the treated group of mice were ablated, and the average weight of the collected tumours was less than 0.1 g ( Fig. 8D ). Meanwhile, the dynamic tumour volume plots showed a dramatic increase after treatments in the Ctrl group, suggesting minimal antitumour efficacy of NIR, NRs, or NRs+NIR compared to EDNRs+NIR. This can mainly be attributed to the deep penetration and effective accumulation induced by NIR irradiation ( Fig. 8E ). In addition, the body weights of the mice in each group did not differ significantly from those of the ctrl group indicating that EDNRs were not significantly physiologically toxic to the growth of the mice ( Fig. 8F ). The hematoxylin and eosin (H&E) staining images showed that tumour cells in the EDNRs + NIR group were extremely necrotic or apoptotic, while there was no or little change in the number of tumour cells in the other groups ( Fig. 8G ). The H&E staining of the major metabolic organs (liver, kidney, and spleen) in each group of mice did not show any obvious damage, which proved the good biocompatibility, which is consistent with the previous results ( Fig. S18 ). In summary, we have reported a kind of NIR-driven nanomotor swarms with collective and chemotaxis behavior that enable better penetration in solid tumours. This nanomotor robot swarm consists of EDNRs nanosheets with a Z-scheme heterojunction structure, which can efficiently generate ROS to kill tumour cells while moving autonomously and penetrating deeply, thus effectively enhancing tumour catalytic therapy. In vitro and in vivo experiments have shown that this therapeutic strategy can effectively inhibit tumour growth. This work not only effectively overcomes the problem of low penetrating ability of nanomaterials in TME, but also improves the catalytic activity of conventional POM-based nanomaterials by rational designing of valence band structure. Methods Reagents and Chemicals All chemicals in this experiment were used directly and not further purified. H 3 PMo 12 O 40 (≥ 99.0%) and FeCl 3 ·6H 2 O (≥ 99.0%) were purchased from Aladdin Industrial Corporation (Shanghai, China). Calcein acetoxymethyl ester (Calcein AM) and propidium iodide (PI) were provided by the Beyotime Institute of Biotechnology (Shanghai, China). Methyl thiazolyl tetrazolium (MTT), 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 4′,6-diamidino-2-phenylindole (DAPI) and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were provided by Sigma-Aldrich (Shanghai, China). JC-1 staining kit and Crystal Violet Staining Solution were purchased from Beyotime Biotechnology. For the cell culture, Dulbecco's Modified Eagle 5 Medium (DMEM), phosphate-buffered saline (PBS), antibiotic/anti-mycotic solution, and the fetal bovine serum (FBS) were provided by Thermo Fisher Scientific (Beijing, China). The reagents and solvents in this experiment were certified as the analytical grade. Experimental animals The KM mice were purchased from the Second Affiliated Hospital of Harbin Medical University. The process of all animal experiments followed the guidelines and regulations of the Northeast Forestry University Animal Protection Committee (Approval No. 2022040). Synthesis of NRs and EDNRs NRs were obtained by direct calcination of FeCl 3 •6H 2 O powder. A certain mass of yellow lumps of FeCl 3 •6H 2 O was weighed, thoroughly ground, and placed in an alumina crucible. The temperature was increased to 230°C under vacuum in a tube furnace at 5°C/min and maintained for 1 h. The solid was cooled naturally to room temperature to obtain a reddish-brown solid, and the unreacted FeCl 3 • 6H 2 O was washed with acetone several times. The synthesis of EDNRs nanosheets was similar to NRs nanosheets. H 3 PMo 12 O 40 (91 mg, 50 µmol) and 270 mg (1 mmol) of FeCl 3 •6H 2 O were dissolved in 2 mL of deionized water. Stirring was carried out continuously for 30 min at 25°C to allow for thorough dissolution and mixing. It was dried in an oven overnight at 60°C and subsequently ground in an alumina crucible. The mixture was heated to 230°C under vacuum in a tube furnace at 5°C/min and kept for 1 h. The mixture was then heated up to 230°C under vacuum in a tube furnace at 5°C/min and kept for 1 h. The mixture was then dried overnight in an oven at 60°C. 1 O 2 Detection The 1,3-Diphenylisobenzofuran (DPBF) was used as the indicator to detect the generation of 1 O 2, which would degrade from yellow to colorless in the presence of 1 O 2 . 2 mg DBPF powder was dissolved in 1mL DMSO, and then add 100 µl solution to each samples need to be assayed. Finally, the solution was observed using UV-vis spectroscopy and the corresponding absorbance value changes were recorded. •OH Detection The TMB was used as the •OH probe, which would change from colorless to blue in the presence of •OH. 2 mg TMB powder was dissolved in 1mL ethanol, and then 200 µl solution to each sample needed to be assayed. Finally, the solution was observed using UV-vis spectroscopy and the corresponding absorbance value changes were recorded. Electrochemical performance of NRs and EDNRs The three-electrode system was used for the evaluation of the electrochemical performance of as-prepared NRs and EDNRs. The three-electrode system consisted of a Pt sheet and Ag/AgCl electrode as the counter and reference electrodes, respectively, and a F-doped tin oxide (FTO) glasses with a uniformly loaded 20mg sample powder on one side (area 1.0 × 1.0 cm) as the working electrode. The HeLa Cervical Cancer and L929 Cells Culture The HeLa cervical cancer cells and L929 cells were provided by the Chinese Academy of Sciences Cell Bank (Beijing, China). The HeLa cancer cells and L929 cells were cultured in RPMI 1640 medium containing 10% FBS at 37°C in a 5% CO 2 atmosphere. Cell Survival of NRs and EDNRs The MTT method was used to assess the inhibition of NRs and EDNRs in vitro. First, HeLa cell suspensions were cultured overnight in (5 × 10 3 per well) 96-well plates to make them adherent. Then, HeLa cells were co-cultured with different concentrations of EDNRs. 20 µL of MTT (5 mg/mL) was added to each well and incubated for an additional 4 hours. Finally, 150 µL of DMSO was added to the pie in each well after draining and shaking to ensure that the crystals were dissolved. The experimental results were then measured using a microplate reader (MR-96A, Beijing, China). The Live-dead double staining assays were also used to assess cell survival. The double staining reagents consisted of calcein-AM and propidium iodide (PI) reagents were co-incubated with HeLa cells for 0.5 h. The staining results were visualized by confocal laser scanning microscopy (CLSM). Detection of Intracellular ROS Production To further determine the reason for tumour cell death, intracellular ROS generation was examined using DCFH-DA as a ROS probe. DCFH-DA was co-cultured with HeLa cells in 6-well plates for 0.5 h and then visualized by CLSM. Intratumoural ROS oxidized DCFH-DA to green fluorescent DCF, and the intracellular ROS levels were further determined by flow cytometry. Tumour Penetration Study of EDNRs in 3D tumor spheroids For the establishment of 3D tumor spheroids, 1600 HeLa cells (200 µL complete medium) were added tointo U-shaped 96-well plates. On the 7th day, the 3D tumor cell spheres were formed. The Suppression Rate Assessment in vivo The same size KM mice (15 g) were purchased from the Second Affiliated Hospital of Harbin Medical University, and we established the S180 tumour KM mouse model to evaluate its tumour suppression rate in vivo . In order to construct a mouse model of hormonal tumour, S180 cells were resuspended and dispersed, diluted to 1×10 7 /mL with PBS, and injected with 0.1 mL per mouse. When the tumour volume was about 200 mm 3 then the mice were randomly divided into seven groups. And then they were treated accordingly and their body weights and tumour sizes were recorded and euthanised at the end of the treatment or when their volume exceeded 1500 mm 3 . Groups were defined as follows: Control group (G1), Only NIR irradiation (G2), NRs (G3), NRs with NIR irradiation (G4), 5 mg/Kg ENDRs (G5), and 5 mg/Kg EDNRs with NIR (G6) and 10 mg/Kg EDNRs with NIR irradiation (G7). The NIR irradiation condition is 650 nm, 1 W cm − 2 , 10 min. Staining in vivo Freshly collected tumours were embedded with an Optimal cutting temperature compound (OCT) embedding agent, snap-frozen in liquid nitrogen for 10–20 S, and sliced using a frozen sectioning machine to obtain 8 µm tissue sections. Then, as with the in vitro ROS staining, DCFH was used as the ROS probe. Statistical information Unpaired two-tailed Student’s t -test was used to compare statistical significance between two data groups. One-way analysis of variance (ANOVA) with a Bonferroni post hoc test was used to compare three or more groups. Quantitative data were indicated as mean ± S.D. Asterisks were used to represent significant differences (n.s.: no significance, *P < 0.05, **P < 0.01, and ***P < 0.001). Declarations Acknowledgements This work was funded by the National Natural Science Foundation of China (No. 52372264, 21972035, and 52375565), the Heilongjiang Provincial Natural Science Foundation of China (No. LH2023B002), Interdisciplinary Research Foundation of HIT (IR2021112), State Key Laboratory of Robotics (2019-O02), and Supported by the Fundamental Research Funds for the Central Universities (2572023CT11-05). Ethics declarations Competing interests The authors declare no competing interests. Contributions Z.Y. and T.S. performed the experiments and analyzed the results. M.T., S.W., and X.L. assisted with the experiment design and data analyses. Z.Y., T.S., and Z.W., wrote and revised the original draft of the manuscript. T.S., L.S., and Z.W., reviewed and edited the manuscript. Z.Y., T.S., and Z.W., supervised the whole project. All authors discussed the results and commented on the manuscript. References Gao W et al (2023) Deciphering the catalytic mechanism of superoxide dismutase activity of carbon dot nanozyme. Nat Commun 14:160 Huo M, Wang L, Chen Y, Shi J (2017) Tumor-selective catalytic nanomedicine by nanocatalyst delivery. Nat Commun 8:357 Wang D et al (2020) Self-assembled single-atom nanozyme for enhanced photodynamic therapy treatment of tumor. Nat Commun 11:357 Jain RK (2013) Normalizing Tumor Microenvironment to Treat Cancer: Bench to Bedside to Biomarkers. J Clin Oncol 31:2205–2218 Kobayashi H, Watanabe R, Choyke PL (2014) Improving Conventional Enhanced Permeability and Retention (EPR) Effects; What Is the Appropriate Target? Theranostics 4:81–89 Overchuk M, Zheng G (2018) Overcoming obstacles in the tumor microenvironment: Recent advancements in nanoparticle delivery for cancer theranostics. 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Sci Adv 9:eabg3015 Dong R, Ren B, Cai Y (2017) Light-driven micro/namorobots: Mechanisms and performances. Chin Sci Bull 62:152–166 Additional Declarations There is NO Competing Interest. Supplementary Files Videos.zip Supplementary Movie.1: The motion trajectories of NRs, NRs+NIR, EDNRs and EDNRs+NIR (NIR 650 nm). Supplementary Movie.2: The collective swarm motion of the EDNRs. Supplementary Movie.3: The chemotaxis movement of EDNRs swarms. SupplementaryInformation20250412.docx Supplementary Information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6432792","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":444956879,"identity":"d427150c-2e3d-4dfc-92f2-39f18ef58ba2","order_by":0,"name":"Zhiguang Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYDACCcYGKIv5GEMCiD5AvBa2NIaEBKK0wFk8ZgwMxGiRn93c/Jqn5o7dhhs53x48/MEgx3cjgfFzAR4tjHMOtlnzHHuWvOFG7nYDoMOMJW8kMEvPwKOFWSKxzZiH7XCy2Y3cbRJALYkbbiSwMfPg0cIG1vIPpCXnGUhLPUEtPBKJzY952w7bAbWwgbQkGBDSIgG0hXFu3+EE+zPPzCQS0iQMZ5552CyNT4v8jPTHH958O2wv2Z78TPKHjY083/Hkg5/xaQF7B0gkNkBtBWJ45OIEzB+AhD0hVaNgFIyCUTCCAQDl4066SnyFAQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-0570-0757","institution":"Harbin Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"Zhiguang","middleName":"","lastName":"Wu","suffix":""},{"id":444956880,"identity":"3f298544-173b-4248-97e0-bde256735437","order_by":1,"name":"Zhengya Yue","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Zhengya","middleName":"","lastName":"Yue","suffix":""},{"id":444956881,"identity":"17ed77d5-d684-46d1-beb9-88b89bd0eb57","order_by":2,"name":"Minglu Tang","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Minglu","middleName":"","lastName":"Tang","suffix":""},{"id":444956882,"identity":"89f4a815-93b2-435e-a67b-b03935aa49d4","order_by":3,"name":"Shuo Wang","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Wang","suffix":""},{"id":444956883,"identity":"8aabf109-180f-43fc-b3de-aafff71676d4","order_by":4,"name":"Xiangwei Liu","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xiangwei","middleName":"","lastName":"Liu","suffix":""},{"id":444956884,"identity":"4b5667f7-fda1-4500-a3fb-44aa8f871389","order_by":5,"name":"Tiedong Sun","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Tiedong","middleName":"","lastName":"Sun","suffix":""},{"id":444956885,"identity":"413f1d87-a329-47bb-8300-67d822d70909","order_by":6,"name":"Li Shang","email":"","orcid":"https://orcid.org/0000-0003-1575-1934","institution":"Northwestern Polytechnical University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Shang","suffix":""}],"badges":[],"createdAt":"2025-04-12 07:10:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6432792/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6432792/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88211025,"identity":"5749dc9a-0db5-4ac3-8c5b-d1bd0d9e7fe1","added_by":"auto","created_at":"2025-08-04 05:13:13","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":553275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic exciton-dissociation-enhanced nanozyme-based robots (EDNRs) swarm enable depth-resolved tumour penetration and therapy.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e EDNRs leveragingZ-scheme heterojunctions facilitates ultrafast exciton dissociation under NIR irradiation, enhancing catalytic activity for ROS generation. EDNRs assemble into swarms \u003cem\u003evia\u003c/em\u003e synergistic photocatalytic propulsion and hydrodynamic interactions. \u003cstrong\u003e(B)\u003c/strong\u003e Intravenous-injected EDNRs swarms are activated by NIR light, achieving depth-dependent tumour penetration-overcoming interstitial fluid pressure to reach the deep tumour, where ROS production substantially inhibits tumour cell proliferation. This design addresses the challenge of shallow accumulation and deep-penetration failure in nanomedicine.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6432792/v1/a90ea99651da2a232db6a005.jpg"},{"id":88211026,"identity":"56c424c5-e7a4-4a70-9151-03c4b976014b","added_by":"auto","created_at":"2025-08-04 05:13:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":672699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFabrication and structural characterization of EDNRs. (A)\u003c/strong\u003e Schematic illustration of the energy band structure of FeOCl and POM-Mo, formation of the built-in electric field at the Z-scheme heterojunction interface, and NIR-light-induced directional charge transfer in EDNRs. (\u003cstrong\u003eB\u003c/strong\u003e) Transmission electron microscopy (TEM) images of EDNRs. Scale bar, 50 nm. (\u003cstrong\u003eC\u003c/strong\u003e) Corresponding High-resolution TEM (HR-TEM) images of EDNRs. Scale bar, 10 nm.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eD\u003c/strong\u003e) The local area energy dispersive X-ray spectroscopy (EDS) mapping images of EDNRs. Scale bar, 100 nm. (\u003cstrong\u003eE\u003c/strong\u003e) Corresponding EDS spectroscopy of the local area (Fe, O, Cl, P, and Mo elements). (\u003cstrong\u003eF\u003c/strong\u003e) X-ray diffractometry (XRD) patterns of NRs, EDNRs, and the standard card of NRs (FeOCl: S No.01-072-069). (\u003cstrong\u003eG\u003c/strong\u003e) Atomic force microscopy (AFM) image of EDNRs, and (H) corresponding thickness. Scale bar, 100 nm. (\u003cstrong\u003eI\u003c/strong\u003e) Dynamic light scattering (DLS) patterns of NRs and EDNRs. (\u003cstrong\u003eJ\u003c/strong\u003e) Zeta-potential measurement of POM-Mo, NRs and EDNRs. (\u003cstrong\u003eK\u003c/strong\u003e) The X-ray photoelectron spectroscopy (XPS) spectra of NRs and EDNRs, and (\u003cstrong\u003eL\u003c/strong\u003e) High-resolution spectra of Fe 2p in EDNRs and corresponding fitting analysis.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6432792/v1/46306a47af9bfb0851bfb027.jpg"},{"id":88211029,"identity":"36b5b414-57f2-4cb3-a21a-6eb0ba57776b","added_by":"auto","created_at":"2025-08-04 05:13:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":568963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotocatalytic activity and motility analysis of EDNRs. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic illustration of the energy band structure of FeOCl and POM-Mo⁻, formation of the built-in electric field at the Z-scheme heterojunction interface, and NIR-light-induced directional charge transfer in EDNRs. (\u003cstrong\u003eB\u003c/strong\u003e) UV–vis diffuse reflectance spectra (DRS) curves, demonstrating electronic structure changes at the FeOCl/POM-Mo⁻ interface in EDNRs. (\u003cstrong\u003eC-F\u003c/strong\u003e) Plot of (αhv)\u003csup\u003e2\u003c/sup\u003e versus (hv) (C), XPS conduction band (VB) spectra (D), PL spectra (E), and photocurrent response curves (F) of EDNRs and NRs. (\u003cstrong\u003eG and H\u003c/strong\u003e) Schematic of 3,3',5,5'-tetramethylbenzidine (TMB) oxidation by ·OH (G) and corresponding absorbance spectra (H) under NIR irradiation, showing higher ·OH generation in EDNRs. (\u003cstrong\u003eI and J\u003c/strong\u003e) Schematic of ¹O₂-sensitive probe reaction (I) and absorbance changes (J) indicating ¹O₂ production, with EDNRs + NIR group exhibiting prominent ¹O₂ generation. (\u003cstrong\u003eK-N\u003c/strong\u003e) Trajectories (K), velocity analysis over time (L), Mean squared displacement (MSD) curves (M), and Diffusion coefficient statistics (N) of individual NRs, NRs+NIR, EDNRs, and EDNRs+NIR, confirming superior directional motility of EDNRs under NIR.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6432792/v1/147b9984f0a1cb14f569c6c7.jpg"},{"id":88211717,"identity":"068959f3-30db-4738-b063-2e4648efae4d","added_by":"auto","created_at":"2025-08-04 05:37:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":913350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSwarm dynamics of EDNRs under NIR irradiation. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Time-lapse density images showing the collective motion of the EDNRs. Scale bar, 100 μm. (\u003cstrong\u003eB\u003c/strong\u003e) Corresponding pixel intensity analysis of swarm motion over time, reflecting increasing collective motion intensity. Scale bar, 100 μm. (\u003cstrong\u003eC and D\u003c/strong\u003e) Schematic representation (C) and corresponding density images (D) of moving path of EDNRs with the position of the NIR light, transition from dispersed states (P1, P2, P3) to swarm motion (S1, S2, S3). (\u003cstrong\u003eE\u003c/strong\u003e) Illustration of the vertical chemotaxis of nanomotor in a cuvette. (\u003cstrong\u003eF\u003c/strong\u003e) Digital images of the distribution trend of swarm nanomotors in the cuvette before and after 100 min with or without the NIR irradiation. (\u003cstrong\u003eG\u003c/strong\u003e) UV-vis spectra of swarm nanomotors and (\u003cstrong\u003eH\u003c/strong\u003e) corresponding intensity after NIR irradiation for 0-100min. (\u003cstrong\u003eI\u003c/strong\u003e) The confocal microscope image of 5(6)-FAM with EDNRs, showing the generation of ·OH\u003cem\u003e via\u003c/em\u003e the Z-scheme heterostructure-driven reaction. Scale Bars, 50 μm.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6432792/v1/d2fde1c5837790e9556a138b.jpg"},{"id":88211037,"identity":"8a3f5348-63a6-4ec1-b0b9-dd0f48fdb463","added_by":"auto","created_at":"2025-08-04 05:13:14","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":880381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntitumour evaluation of EDNRs \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e\u0026nbsp;(\u003cstrong\u003eA\u003c/strong\u003e) Live/Dead cell staining images (green: live cells; red: dead cells) of six groups (G1–G6). (Green channel: Calcein-AM, Red channel: PI). Scale bar, 200 μm. (\u003cstrong\u003eB\u003c/strong\u003e) Quantitative analysis of relative cell viability after incubation with various concentrations of EDNRs for 12 h with or without NIR. (n = 3; means ± SD). (\u003cstrong\u003eC\u003c/strong\u003e) Concentration-dependent cell viability under different treatments. (n = 3; means ± SD). (\u003cstrong\u003eD\u003c/strong\u003e) ROS detection using the DCFH-DA probe (Blue channel: DAPI, Green channel: DCF), Scale bar, 200 μm. (\u003cstrong\u003eE\u003c/strong\u003e) The flow cytometry analysis of intracellular ROS generation. (\u003cstrong\u003eF\u003c/strong\u003e) Fluorescent images of changes in mitochondrial membrane polarization using JC-1 as the indicator (Red channel: JC-1 aggregates, Green channel: JC-1 monomer. Scale bar, 100 μm. (\u003cstrong\u003eG\u003c/strong\u003e) The relevant ratio of JC-1 aggregates. (n = 3; means ± SD). (\u003cstrong\u003eH\u003c/strong\u003e) Crystal violet staining image of HeLa cells after incubation for 14 days. Groups were defined as follows: PBS as Control group (G1), PBS + NIR irradiation (G2), NRs (G3), NRs with NIR irradiation (G4), ENDRs (G5), and EDNRs with NIR irradiation (G6).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6432792/v1/6f3716c8afc40a90ef9a1642.jpg"},{"id":88211028,"identity":"8fa6c542-3fa7-4a1e-8fd1-56104fcf354a","added_by":"auto","created_at":"2025-08-04 05:13:13","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":80201,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudies of tumour penetration of EDNRs \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (A)\u003c/strong\u003e Trans-well experiments fluorescent microscopy images of cellular uptake (Green channel: FITC, Blue channel: DAPI). Scale bar, 50 μm.\u003cstrong\u003e (B)\u003c/strong\u003e Corresponding quantitative fluorescence intensity (FI) (n = 3; means ± SD). (C) Representative confocal laser scanning microscope (CLSM) 3D MTSs images of tumour penetration at different depths (0 - 120 μm). Scale bar, 100 μm. (D) Representative quantitative fluorescence intensity profile along the X-axis with depth of 120 μm. (E) Normalized FI area analysis along the Z-axis distance. (F) Quantitative comparison of mean FI intensity, confirming the superior penetration capability of EDNRs. (n = 3; means ± SD).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6432792/v1/9eaf89f9b7ae45042da476d0.jpg"},{"id":88211035,"identity":"796414d1-8cd5-4528-8a28-0fa384f8d40b","added_by":"auto","created_at":"2025-08-04 05:13:14","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":641687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e evaluation of the mechanism underlying ENDRs swarm-enhanced antitumour activity. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Fluorescent staining of tumour tissues for ROS (green) and nuclei (DAPI, blue). The G6 group (EDNRs + NIR) exhibited the strongest ROS signal. Scale bar, 200 µm. (\u003cstrong\u003eB\u003c/strong\u003e) 3D Schematic diagram illustrating different slice depths (50 μm, 300 μm, 600 μm) for penetration analysis \u003cem\u003ein vivo.\u003c/em\u003e (\u003cstrong\u003eC \u003c/strong\u003eand\u003cstrong\u003e D\u003c/strong\u003e) Fluorescent images (C) and corresponding fluorescence intensity (D) of tumour tissue sections at different depths. Scale bar, 200 µm. (n = 3; means ± SD). (\u003cstrong\u003eE\u003c/strong\u003e) Iron content analysis over time. EDNRs exhibited higher Fe accumulation, peaking at 12 h, and gradual metabolism, reflecting biocompatibility. Groups were represented as follows: PBS as Control group (G1), PBS + NIR irradiation (G2), NRs (G3), NRs with NIR irradiation (G4), ENDRs (G5), and EDNRs with NIR irradiation (G6). (n = 3; means ± SD).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6432792/v1/a14e00e345df32981a010249.jpg"},{"id":88211720,"identity":"7628325d-6398-4046-b9ed-f890f8eb9570","added_by":"auto","created_at":"2025-08-04 05:37:14","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":811573,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapeutic efficacy and biocompatibility of EDNRs \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic diagram of \u003cem\u003ein vivo \u003c/em\u003eantitumour investigation including tumour inoculation, formation, treatment, and analysis. (\u003cstrong\u003eB\u003c/strong\u003e) Representative photographs of tumour-bearing mice after the different treatments for 14 days. (\u003cstrong\u003eC\u003c/strong\u003e) Excised tumour specimens from each group, with G6 group showing the smallest tumour size (red dashed circle highlights the tumour). (\u003cstrong\u003eD\u003c/strong\u003e) Quantitative tumour weight from different groups, demonstrating G6 had the lowest weight. (n = 3; means ± SD). (\u003cstrong\u003eE and F\u003c/strong\u003e) Tumour volume growth curves (E) and body weight change curves (F) over 14 days upon different groups. (n = 3; means ± SD). \u003cstrong\u003e(G)\u003c/strong\u003e H\u0026amp;E staining of tumour slices in different treatments. Groups were defined as follows: PBS as Control group (G1), PBS + NIR irradiation (G2), NRs (G3), NRs with NIR irradiation (G4), ENDRs (G5), and EDNRs with NIR irradiation (G6).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6432792/v1/7230b4b488296bd4db0714c8.jpg"},{"id":88505331,"identity":"1faa1fbc-21dd-4361-952b-6adab96b5313","added_by":"auto","created_at":"2025-08-07 07:24:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6339115,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6432792/v1/53a1b89e-686d-4247-a3e7-37f7c5ac4a07.pdf"},{"id":88211716,"identity":"d8f16d94-a41f-4df5-8d14-d18cc5096013","added_by":"auto","created_at":"2025-08-04 05:37:13","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":300342,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Movie.1: The motion trajectories of NRs, NRs+NIR, EDNRs and EDNRs+NIR (NIR 650 nm).\u003c/p\u003e\n\u003cp\u003eSupplementary Movie.2: The collective swarm motion of the EDNRs.\u003c/p\u003e\n\u003cp\u003eSupplementary Movie.3: The chemotaxis movement of EDNRs swarms.\u003c/p\u003e","description":"","filename":"Videos.zip","url":"https://assets-eu.researchsquare.com/files/rs-6432792/v1/5a1e908c5f9e6abd6db100b6.zip"},{"id":88211718,"identity":"8d13c422-408d-43fa-b992-7e701c1072c9","added_by":"auto","created_at":"2025-08-04 05:37:14","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4171292,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation20250412.docx","url":"https://assets-eu.researchsquare.com/files/rs-6432792/v1/a03cf20f09cdf2928c58ca00.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Nanozyme-based Robots Swarm with Exciton-Engineered Z-Scheme Heterojunctions for Depth-Resolved Tumour Penetration and Therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSynthetic nanorobots have emerged as a transformative platform for precision oncology, offering the potential to navigate complex biological environments and deliver targeted therapies to tumours\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, their clinical translation remains hindered by inefficient penetration into deep tumour tissues, a challenge rooted in the dense extracellular matrix (ECM), high interstitial fluid pressure (IFP), and limited motility of conventional nanosystems\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Catalytic motion, one of the most promising propulsion strategies for biomedical nanorobots because their actuation without the need of additional complex instruments to generate external fields\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Among various catalytic strategies, utilizing the enzymatic based complex as catalysts is emerging as promising approach for the design of nanorobots owing to their usage of endogenous fuels \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Particularly the artificial nanozymes have been demonstrated to incorporate into the nanorobots to accomplish mobility though their catalytic activity or photocatalytic activity with the assistance of NIR Eg, accompanying with the generation of reactive oxygen species (ROSs) using the peroxidase-like activity.\u003c/p\u003e\u003cp\u003eAlthough the nanozyme-based nanorobot have reported therapy effect toward disease such as fungal infection, often fails in solid tumours due to insufficient motility and rapid fuel depletion, confining nanorobots to superficial tumour regions where fuel is scarce\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Because conventional nanozymes have a limited catalytic capacity, they are susceptible to pH, fuel concentration, and their own local concentration\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Overcoming these barriers requires innovative designs that integrate robust propulsion with spatiotemporal control over collective behavior. Exciton dissociation, key to photocatalytic activity, has been underexplored in nanorobot design. Photocatalytic nanomaterials generate propulsion via light-driven bubble formation or ion gradients, but inefficient exciton recombination limits catalytic efficiency\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Z-scheme heterojunctions, mimicking natural photosynthesis, preserve high redox capacity while promoting ultrafast exciton dissociation\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, their application in tumour-penetrating nanorobots remains unreported, as does the interplay between heterostructure design and swarm dynamics.\u003c/p\u003e\u003cp\u003eHere, we introduce exciton-dissociation-enhanced nanozyme-based robots (EDNRs) that leverage Z-scheme heterojunctions to achieve depth-resolved tumour penetration and therapy. The EDNRs are constructed \u003cem\u003evia\u003c/em\u003e Z-scheme heterogenization of FeOCl nanosheets with polyoxometalates (POMs), creating a high-valence-band interface that promotes efficient exciton dissociation and charge separation\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. This design not only enhances photocatalytic activity for reactive oxygen species (ROS) generation but also enables synergistic photocatalytic propulsion and swarm-induced hydrodynamic interactions under near-infrared (NIR) irradiation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBy combining autonomous motion with collective behavior, EDNRs overcome the motility limitations of traditional nanorobots, achieving deep tumour penetration and localized ROS-mediated cytotoxicity\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe Z-scheme heterojunction architecture is critical for the performance of EDNRs. Unlike type II heterojunctions, which suffer from reduced redox potentials, the Z-scheme design preserves the high redox capacity of both semiconductors, enabling efficient oxidation of water to hydroxyl radicals (\u0026middot;OH) and reduction of oxygen \u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. This dual functionality boosts ROS production while minimizing electron-hole recombination, a key factor in enhancing catalytic activity\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Additionally, the photocatalytic decomposition of H₂O₂ generates oxygen bubbles, providing thrust for directional motion, while swarm dynamics reduce drag through collective hydrodynamic interactions\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. By integrating Z-scheme heterojunctions with swarm dynamics, EDNRs represent a paradigm shift in nanorobotics, offering spatiotemporally controlled therapy and paving the way for clinical translation of synthetic nanomachines in precision oncology\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eStructural design of EDNRs\u003c/strong\u003e \u003cstrong\u003evia\u003c/strong\u003e \u003cstrong\u003eexciton-dissociation mechanism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe structural design of EDNRs was validated through energy band analysis. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, the Z-scheme heterojunction formed by FeOCl nanosheets and polyoxometalates (POMs) nanozyme created a built-in electric field at the interface. Knowing that the valence band (VB) of FeOCl was positioned at 2.90 eV, while the conduction band (CB) of POM-Mo was 0.15 eV\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Upon NIR light irradiation, this unique band alignment facilitated efficient exciton dissociation, enabling directional charge transfer. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) were used to characterize the morphology of as-prepared NRs and EDNRs. The ultrathin layered structure of the EDNRs was evidenced by Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cstrong\u003eFig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e, where various nanosheets with size of ~\u0026thinsp;200 nm were dispersed. The HRTEM image of EDNRs clearly shows the crystalline structure with a lattice fringer of 0.395 nm (3.95 \u0026Aring;) corresponding to the 020 facet (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. To investigate the existence of POM nanozyme in the EDNRs, area energy dispersive X-ray spectroscopy (EDS) mapping of EDNRs and NRs was tested separately (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cstrong\u003eFig. S2\u003c/strong\u003e). In comparison of Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE and S3, the presence of Mo and P elements (corresponding to POM) in EDNRs proves that the distribution of POM components in EDNRs and the exact elements ratio of EDNRs was shown in \u003cstrong\u003eFig. S4\u003c/strong\u003e (O: P: Cl: Fe: Mo\u0026thinsp;=\u0026thinsp;65.5: 0.6: 9.1: 19.0: 5.8). Additionally, X-ray diffraction (XRD) analysis was performed to characterize the phase structure of the as-fabricated NRs and EDNRs. The characteristic diffraction peaks located in 11\u0026deg; indexed to the (010) planes were both found in NRs and EDNRs, and the results were good and consistent with the standard JCDPS card of No.01-72-069 corresponding to NRs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG, atomic force microscopy (AFM) was used to observe the morphology and the thickness of EDNRs. The thickness of EDNRs was measured to be about 14 nm and the corresponding 3D image was presented in \u003cstrong\u003eFig. S5\u003c/strong\u003e. The hydrated particle size of NRs and EDNRs were measured by dynamic light scattering (DLS), demonstrating their potential as nanomedicines (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI). The Zeta potential value changed from 24.1 mV in NRs to -13.0 mV in EDNRs further proving the successful synthesis of EDNRs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Next, the chemical composition and valence changes of NRs and EDNRs were further measured by X-ray photoelectron spectroscopy (XPS). As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eK, in addition to the Fe 2p, O 1s, and Cl 2p characteristic peaks observed in both NRs and EDNRs, the Mo 3d characteristic peaks are observed in the EDNRs due to the presence of the POM. Moreover, the characteristic peak of Fe 2p in EDNRs was divided into four main peaks (Fe\u003csup\u003e2+\u003c/sup\u003e 2p\u003csub\u003e3/2,\u003c/sub\u003e Fe\u003csup\u003e3+\u003c/sup\u003e 2p\u003csub\u003e3/2,\u003c/sub\u003e Fe\u003csup\u003e2+\u003c/sup\u003e 2p\u003csub\u003e1/2,\u003c/sub\u003e and Fe\u003csup\u003e3+\u003c/sup\u003e 2p\u003csub\u003e1/2\u003c/sub\u003e) and four satellite peaks, while Fe 2p in NRs was mainly divided into two peaks (Fe\u003csup\u003e3+\u003c/sup\u003e 2p\u003csub\u003e3/2,\u003c/sub\u003e and Fe\u003csup\u003e3+\u003c/sup\u003e 2p\u003csub\u003e1/2\u003c/sub\u003e) and two satellite peaks without the presence of Fe\u003csup\u003e2+\u003c/sup\u003e peaks (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eL and \u003cstrong\u003eFig. S6\u003c/strong\u003e). The bigger binding energy of Cl 2p (199.32 eV) in NRs than Cl 2p (198.11 eV) in EDNRs indicated an increase in electron density in EDNRs (\u003cstrong\u003eFig. S7\u003c/strong\u003e). The above results indicated the successful synthesis of NRs and EDNRs, and the interaction in EDNRs affects the charge transfer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhotocatalytic activity and motility analysis of EDNRs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to energy-band theory, EDNRs promote catalytic activity via the built-in electric field of the Z-scheme heterojunction structure. Under NIR irradiation, the band gap bends, leading to the redistribution of surface electrons and holes to specific positions. This unique Z-scheme heterojunction structure effectively enhances exciton dissociation and inhibits the undesirable recombination of electron- hole pairs on the EDNRs surface, enabling the structure to maintain stronger reduction/oxidation potentials of separated electrons on the CB and holes on the VB. Thus, based on the photocatalytic effect, EDNRs enhance the photocatalytic reaction to drive their propulsion (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Considered the critical role of the energy-band structure in photocatalytic activity, a prerequisite for efficient therapy, the energy-band structure of EDNRs was further characterized.\u003c/p\u003e\n\u003cp\u003eFirstly, UV-vis diffuse reflectance spectra (UV-vis DRS) were tested to characterize the absorption difference between synthesized NRs and EDNRs, a key factor influencing catalytic activity. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB shows that both NRs and EDNRs exhibit long absorption edges, with EDNRs demonstrating stronger absorption in the NIR region. Using the corresponding tangent lines of UV-vis DRS and the Kubelka - Munk equation, the band gaps of POM-Mo⁻ and NRs were calculated to be 2.61 eV and 2.34 eV, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). Furthermore, to characterize the heterojunction formation and type, valence-band spectra were used to detect the VB values of NRs and POM - Mo NSs, which were 2.9 eV and 2.49 eV, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). Then, using the relationship \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{E}_{g}={E}_{VB}-{E}_{CB}\\)\u003c/span\u003e\u003c/span\u003e, the CB values of NRs and POM-Mo⁻ were calculated to be 0.15 eV and 0.29 eV. These band-structure results suggest the formation of either a type II or Z-scheme heterojunction in EDNRs. The catalytic activity of EDNRs is significantly higher than that of NRs, as the VB potential of NRs is insufficiently positive to oxidize H\u003csub\u003e2\u003c/sub\u003eO into \u0026middot;OH radicals. PL spectroscopy further confirmed the Z-scheme heterojunction formation in EDNRs. The weak emission intensity of EDNRs indicated a higher separation efficiency than NRs, which is inconsistent with the type II mechanism (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). These findings indicate that holes accumulate at the high VB of POM-Mo, while electrons transfer from POM- Mo to NRs, suggesting a charge-transfer path that adheres more to the Z-scheme mechanism than the type II mechanism. Photocurrent and electrochemical impedance spectrum (EIS) were used to further characterize the photoelectric conversion performance of the unique Z-type heterojunction structure in EDNRs. The photocurrent-response test showed uniform photocurrent densities in both NRs and EDNRs (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF), but EDNRs had significantly stronger photocurrent intensity. The remarkably smaller arc radius of EDNRs than NRs in Nyquist plots, indicating smaller interfacial charge resistance, reveals the promotion of exciton - dissociation behavior due to the Z-scheme heterojunction in EDNRs (\u003cstrong\u003eFig. S8\u003c/strong\u003e), indicating smaller interfacial charge resistance, reveals the promotion of exciton - dissociation behavior due to the Z-scheme heterojunction in EDNRs.\u003c/p\u003e\n\u003cp\u003eAs expected, EDNRs with Z-Scheme heterostructure demonstrate exceptional redox properties, effectively separating holes and electrons (e\u003csup\u003e\u0026minus;\u003c/sup\u003e) under light irradiation. Therefore, the separating holes (h\u003csup\u003e+\u003c/sup\u003e) facilitates direct reactions with H\u003csub\u003e2\u003c/sub\u003eO generating \u0026bull;OH\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, as well as a cascade reaction with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, producing \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. To verify this mechanism, the \u0026bull;OH, and \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generated in the cascade reaction were detected, respectively. The generation of \u0026bull;OH was detected using 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethyl-benzidine (TMB) as a probe, which was oxidized to blue oxide (ox-TMB) with increasing absorbance at 652 nm (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG). The UV-vis absorption spectrum for the EDNRs\u0026thinsp;+\u0026thinsp;NIR group exhibits a prominent peak at 652 nm, indicating the generation of a substantial amount of \u0026bull;OH (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH). Meanwhile, the increase of its absorption peak with the increase of the concentration further confirms the enhanced \u0026bull;OH generation capacity of EDNRs, which might attribute to the Z-scheme heterojunction structure (\u003cstrong\u003eFig. S9\u003c/strong\u003e). In addition, 1,3-Diphenylisobenzofuran (DPBF) was used as a \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e probe, which would gradually degrade from yellow to colorless and was illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI. It was found that EDNRs have superior \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generating ability compared to NRs and its absorption peaks were significantly weakened with time, while NIR irradiation further enhanced \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eJ and S10). As a result of the above results, it can be concluded that EDNRs possesses excellent catalytic properties, in addition to converting H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2,\u003c/sub\u003e it also reacts directly with H\u003csub\u003e2\u003c/sub\u003eO to form \u0026bull;OH, which might attribute to the enhanced exciton dissociation and inhibition of electron-hole recombination by the Z-scheme heterojunction structure.\u003c/p\u003e\n\u003cp\u003eSubsequently, the motion behavior of the nanorobot was captured using an optical microscope, and its trajectory was subsequently tracked and analyzed using TrackMate (an open Fiji plugin)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eK shows the tracking motion trajectories of NRs and EDNRs under the corresponding conditions (with or without NIR irradiation). NRs-POM with NIR irradiation shows a tendency of directional motion compared to the other controls (NRs, NRs\u0026thinsp;+\u0026thinsp;NIR, and EDNRs), and this discrepancy may originate from the enhanced catalytic reaction progress of EDNRs under the NIR irradiation (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eK, \u003cstrong\u003eSupplementary Movie 1\u003c/strong\u003e). Velocity analysis was conducted at fixed time intervals. Following light exposure, the EDNRs group exhibited a clear response, with a significant increase in velocity. In contrast, the NRs group showed no substantial change before or after light exposure, with the slight increase potentially attributed to light-enhanced Brownian motion (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eL). Then, the corresponding mean squared displacement (MSD) was also calculated according to the collected motion trajectories (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eM)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The MSD curves versus time interval (\u0026Delta;t) in the EDNRs\u0026thinsp;+\u0026thinsp;NIR group showed a rising parabolic line (representing autonomous motion), while the other control groups displayed a linear growth (often representing Brownian motion). Moreover, the effective diffusion coefficients (De) were also calculated according to the equation (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{D}_{e}=MSD/4\\varDelta\\:t\\)\u003c/span\u003e\u003c/span\u003e), which were also consistent with the above results (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eN). Based on the above experimental results and previous reports in the literature\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, it can be concluded that EDNRs heterojunction has enhanced catalytic activity and motion behavior performance as expected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSwarm Dynamics of EDNRs upon NIR illumination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the detailed exploration of the photocatalytic reaction mechanism presented in the previous section, the valence band holes participate in the oxidation of water, releasing protons (H⁺). This process may create a local gradient field, which in turn generates an electrophoretic flow. This flow exerts a force on the charged EDNRs within the gradient field, driving the collective behavior of the nanorobots (\u003cstrong\u003eFig. S10\u003c/strong\u003e). To verify the dynamics and collective light chemotaxis behavior of the EDNRs in \u003cem\u003evitro\u003c/em\u003e, optical microscopy was used to directly observe the dynamics of the EDNRs under NIR irradiation. The motion behavior of the EDNRs emerged upon turning on the NIR light and evolved from single-particle motion to collective behavior with increasing exposure time (\u003cstrong\u003eSupplementary Movie S2\u003c/strong\u003e). By further quantifying the collective behavior based on the ROI values of snapshots taken at different time points (0, 3, 6, and 9 min), light was identified as the key factor driving the emergence of collective behavior of EDNRs (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, with the extension of light exposure time, the swarm gradually congregates towards the center of the illuminated area, shifting its distribution from a broad peak to a narrow, high peak. This arises as the rapid motion of individual EDNRs intensifies frequent monomer-to-monomer collisions, and the POM on the nanobot surface enables gradual cluster formation. More interestingly, as the position of the NIR source keeps moving (P1, P2 and P3), the EDNRs swarms also respond to it by following the center of the source, showing a phototropic chemotaxis (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, D \u003cstrong\u003eand Supplementary Movies S3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn addition, UV-Vis spectroscopy was also used to further quantify the collective and phototactic behavior of the EDNRs swarms. As shown in \u003cstrong\u003eFig. 4E\u003c/strong\u003e, 3 mL of the EDNRs solution was placed in a cuvette, while the top of the cuvette was continuously illuminated with NIR irradiation instead of the bottom in order to exclude the effect of gravity on results. The digital images in \u003cstrong\u003eFig. 4F\u003c/strong\u003e shows that near the bottom of the cuvette, light scattering by EDNRs suspended in solution causes the appearance of distinct light paths when a light beam is present (Tyndall effect), but the intensity of the light path at the bottom does not show a significant change after 100 min of NIR irradiation. However, it is noteworthy that the intensity of the light path near the top of the cuvette increased after 100 min, indicating an increase in the concentration of EDNRs. In contrast, the intensity f the two light paths at the top and bottom of the cuvette did not show any significant change before and after in the no-NIR light irradiation group. Next, we further confirmed this collective behaviors of EDNRs by employing UV-Vis spectroscopy to examine the changes in the solution concentration at the top with increasing irradiation time.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 5G\u0026nbsp;\u003c/strong\u003eshows that, after 100 min of NIR irradiation, the absorption intensity of the solution at the top of the cuvette increased, which was significantly different from that before irradiation, suggesting that the NIR irradiation induced the clustering of the EDNRs at the top of the cuvette (\u003cstrong\u003eFig. 5H\u003c/strong\u003e). Based on the above results, this kind of swarming, positively phototropic EDNRs is expected to achieve better tumour therapeutic effects \u003cem\u003ein vivo\u003c/em\u003e, by increasing the enrichment rate and penetration depth. Furthermore, considering that the holes oxidize water to generate ROS while simultaneously producing H⁺, 5(6)-carboxy fluorescein (5(6)-FAM) was used to measure the pH of the photocatalytic region, which produces significant differences in fluorescence intensity at different pHs (\u003cstrong\u003eFig. 3I\u003c/strong\u003e). Quantitative analysis of the results revealed a significant difference in fluorescence intensity between the photocatalytic reaction region and the surrounding environment, confirming the generation of a pH gradient field (\u003cstrong\u003eFig. S12\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e \u003cstrong\u003etherapeutic efficacy of EDNRs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore evaluating the therapeutic effect, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the biocompatibility of the heterostructural EDNRs. Incubation of normal fibroblasts (L929) with EDNRs for 24 h and 48 h did not result in significant damage, indicating the biosecurity for normal tissues (\u003cstrong\u003eFig. S13\u003c/strong\u003e). Given the excellent biocompatibility observed, we investigated whether the heterostructural EDNRs exhibit effective tumour inhibition in \u003cem\u003evitro\u003c/em\u003e. Initially, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, the tumour-killing effect was examined utilizing the live/dead staining assays for HeLa cells. The gradually increasing red channel signaling implies a decreased cell viability of HeLa cancer cells, with the EDNRs\u0026thinsp;+\u0026thinsp;NIR condition, showing as high as 90% red signals (\u003cstrong\u003eFig. S14\u003c/strong\u003e). Based on these results, we qualitatively analyzed the survival of HeLa cells under different conditions. Cell survival was as low as 33.85% within 24 h after treatment with EDNRs (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Meanwhile, NIR irradiation also significantly reduced tumour survival, which was consistently in line with previous results (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). As mentioned earlier, the generation of ROS is a major factor in killing cancer cells. Thus, 2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescein diacetate (DCFH-DA) was utilized to evaluate intracellular ROS levels, which would be oxidized by ROS into 2\u0026apos;,7\u0026apos;-dichlorofluorescein (DCF) in cells with green fluorescence. The presence of a clear green fluorescent channel in the EDNRs group indicates ROS production (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). After being treated with the NIR laser irradiation (0.5 mW/cm\u003csup\u003e2\u003c/sup\u003e, 5 min), the average ROS level increased from 15 to 90, demonstrating a greater capacity for ROS generation (\u003cstrong\u003eFig. S15\u003c/strong\u003e). Notably, flow cytometry was utilized to further quantify the effect of different treatment conditions on intracellular ROS levels. As displayed in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE, no significant difference was observed between the intensity values of the ctrl and NIR groups and the slight enhancement in the NRs, NRs\u0026thinsp;+\u0026thinsp;NIR, and EDNRs groups was due to the production of ROS, whereas the ROS level was significantly enhanced by the NRs\u0026thinsp;+\u0026thinsp;NIR treatment. Considering that the depolarization in mitochondrial membrane potential (MMP) is an important feature of mitochondrial dysfunction associated with cell apoptosis. Therefore, MMP was measured using JC-1 as the fluorescent probe under various treatments, and the mechanism was illustrated in detail in \u003cstrong\u003eFig. S16\u003c/strong\u003e. The green fluorescence signal of the JC-M channel in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF indicated mitochondrial damage, and the detailed intensity of green fluorescence in different treatment groups was presented in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG. Crystal violet staining in cell cloning experiments once again demonstrated the feasibility of this therapeutic strategy, with a reduction in the purple area implying inhibition of HeLa cell colony formation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e\n\u003cp\u003eIn addition to superior ROS generation capability, the ability to enhance tumour tissue penetration through photocatalytic propulsion is another key factor determining the therapeutic efficacy. To determine the enhancement of tumour penetration ability by swarm motion, trans-well migration experiments were performed by using FITC-labeled nanomotors. In the trans-well experiment, the upper and lower chambers are separated by a membrane. FITC-labeled nanomotors are only placed in the lower chamber, and cells are seeded in the upper chamber to model the in vivo barrier meet by nanomotor. The experimental results were observed using fluorescence microscopy; the green signal channel represents FITC and the blue signal channel is DPAI. As shown in \u003cstrong\u003eFig. 6A\u003c/strong\u003e, uniform blue signals indicate consistent cell attachment, while varying green signals reflect differences in penetration abilities among different groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe enhanced green signal in group EDNRs\u0026thinsp;+\u0026thinsp;NIR, compared to the other controls, demonstrates that enhanced motility induces deeper penetration with cellular uptake. Besides, the corresponding MFI in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB is found that the fluorescence intensity of the robot group was seven times higher than that of the other groups, indicating a significant enhancement in penetration ability.\u003c/p\u003e\n\u003cp\u003eAfter confirming the enhanced permeability across the membrane, three-dimensional multicellular tumour spheres (3D-MTSs) were constructed to further evaluate the deep penetration ability of the nanorobot within the dense collagen fibers of tumour tissues. In the 3D-MTSs model, it was obvious from the optical microscope images that the EDNRs with NIR group showed extensive detachment damage with loose (\u003cstrong\u003eFig. S17\u003c/strong\u003e). Next, FITC-labeled nanorobots were co-incubated with the 3D-MTS model, and confocal fluorescence microscopy was used to observe the depth distribution and evaluate the penetration ability. Using a 20 \u0026micro;m increment, Z-axis sectioning of the 3D-MTS was performed from the bottom. Compared to the control group, significant green fluorescence signals were still observed even at a depth of 120 \u0026micro;m in Robot group (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). A fluorescence intensity distribution comparison along the X-axis for (depth of 100 \u0026micro;m) clearly shows the difference in penetration depth between the two groups (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). In Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE, slices from the robot group at the same depth exhibited significantly enhanced fluorescence intensity compared to the control group. The decrease in fluorescence intensity between 80 \u0026micro;m and 120 \u0026micro;m may be attributed to the limited fluorescence tissue penetration depth of the confocal microscope. Additionally, based on the average fluorescence intensity of the slices, the robot group exhibited six times higher intensity than the control group (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003eelucidation of the mechanism of ENDRs swarm- mediated antitumour activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMotivated by the in \u003cem\u003evitro\u003c/em\u003e experimental results, it is necessary to further investigate whether ROS, enhanced permeability, and effective accumulation in \u003cem\u003evivo\u003c/em\u003e can also improve tumour inhibition effects, thereby clarifying the tumour-killing mechanism of EDNRs in \u003cem\u003evivo\u003c/em\u003e. Consistent with the previous discussion, the S180 tumour-bearing KM mice model was selected again to study the in \u003cem\u003evivo\u003c/em\u003e mechanism of EDNRs swarm. To observe the differences in in \u003cem\u003evivo\u003c/em\u003e ROS levels, mice were euthanized 6 h after treatments, and tumour tissues were collected for frozen sectioning and ROS staining. Except for the control group, green fluorescence signals were observed in all other groups, indicating the generation of ROS (\u003cstrong\u003eFig. 7A\u003c/strong\u003e). The trend of enhanced green fluorescence also coincided with the in \u003cem\u003evivo\u003c/em\u003e and in \u003cem\u003evitro\u003c/em\u003e tumour inhibition rate growth, fully demonstrating the Z-type heterojunction structure can also promote ROS generation in \u003cem\u003evivo\u003c/em\u003e, thereby increasing the inhibition rate. Then, to further test the penetration ability of the motors in tumour tissues, tumour tissues were collected in vivo and co-incubated with FITC-labeled motors, followed by\u0026nbsp;washing with PBS. Tumour slices at different depths were then collected, and the fluorescence signal intensity in the FITC channel was evaluated using fluorescence microscopy to reflect the penetration depth of the motors (\u003cstrong\u003eFig. 7B\u003c/strong\u003e). Compared to the control group, the motors group exhibited more prominent green fluorescence signals, indicating greater tumour accumulation (\u003cstrong\u003eFig. 7C\u003c/strong\u003e). Additionally, as the distance from the outer edge of the tissue slices increased, the motors group still showed significant fluorescence intensity. Quantitative analysis of the tissue slices revealed that at a depth of 600 \u0026mu;m, the fluorescence intensity was seven times higher than that of the control group (\u003cstrong\u003eFig. 7D\u003c/strong\u003e). Additionally, an inductively coupled plasma emission spectrometer (ICP) was used for more accurate quantification of the motor accumulation efficiency (\u003cstrong\u003eFig. 7E\u003c/strong\u003e). Similarly, the accumulation rate in the motors group was significantly higher than that of the control group, reaching a peak around 12 hours. At the same time, the motors group exhibited a longer tumour retention time. Therefore, it can be concluded that the EDNRs therapeutic strategy can effectively inhibit tumours\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e due to its superior ROS generation capacity, deeper penetration and higher accumulation in tumour tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo antitumour performance and biocompatibility of EDNRs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsidering the above superior ROS production and deep penetration capacity in\u003cem\u003e\u0026nbsp;vitro,\u003c/em\u003e an S180 tumour-bearing KM mice model was established to investigate the antitumour performance in \u003cem\u003evivo\u003c/em\u003e, and the KM mouse was randomly divided into six groups: G1 Ctrl, G2 NIR, G3: NRs, G4: NRs+NIR, G5: EDNRs and G6: EDNRs+NIR. The body weights of the mice and the volume of the tumours were also measured daily, and the mice were euthanized 14 days after various treatments, and the tumours along with the major organs were collected forfuther analysis\u0026nbsp;(\u003cstrong\u003eFig. 8A\u003c/strong\u003e). As demonstrated by digital photographs of hormonal mice taken in \u003cstrong\u003eFig. 8B\u003c/strong\u003e, there was a significant ablation of the tumours after EDNRs+NIR treatment. Digital images of tumours collected from the mice were shown in \u003cstrong\u003eFig. 8C\u003c/strong\u003e, and the relative tumour sizes of NIR, NRs, and NRs+NIR treated mice were not significantly different from those of the ctrl group, suggesting minimal efficacy.Compare EDNRs and EDNRs+NIR, both the digital images and weight data clearly show a significant enhancement after NIR irradiation in tumour ablation, almost all tumours from the treated group of mice were ablated, and the average weight of the collected tumours was less than 0.1 g (\u003cstrong\u003eFig. 8D\u003c/strong\u003e). Meanwhile, the dynamic tumour volume plots showed a dramatic increase after treatments in the Ctrl group, suggesting minimal antitumour efficacy of NIR, NRs, or NRs+NIR compared to EDNRs+NIR. This can mainly be attributed to the deep penetration and effective accumulation induced by NIR irradiation (\u003cstrong\u003eFig. 8E\u003c/strong\u003e). In addition, the body weights of the mice in each group did not differ significantly from those of the ctrl group indicating that EDNRs were not significantly physiologically toxic to the growth of the mice (\u003cstrong\u003eFig. 8F\u003c/strong\u003e). The hematoxylin and eosin (H\u0026amp;E) staining images showed that tumour cells in the EDNRs + NIR group were extremely necrotic or apoptotic, while there was no or little change in the number of tumour cells in the other groups (\u003cstrong\u003eFig. 8G\u003c/strong\u003e). The H\u0026amp;E staining of the major metabolic organs (liver, kidney, and spleen) in each group of mice did not show any obvious damage, which proved the good biocompatibility, which is consistent with the previous results (\u003cstrong\u003eFig. S18\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, we have reported a kind of NIR-driven nanomotor swarms with collective and chemotaxis behavior that enable better penetration in solid tumours. This nanomotor robot swarm consists of EDNRs nanosheets with a Z-scheme heterojunction structure, which can efficiently generate ROS to kill tumour cells while moving autonomously and penetrating deeply, thus effectively enhancing tumour catalytic therapy. In \u003cem\u003evitro\u003c/em\u003e and in \u003cem\u003evivo\u003c/em\u003e experiments have shown that this therapeutic strategy can effectively inhibit tumour growth. This work not only effectively overcomes the problem of low penetrating ability of nanomaterials in TME, but also improves the catalytic activity of conventional POM-based nanomaterials by rational designing of valence band structure.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eReagents and Chemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll chemicals in this experiment were used directly and not further purified. H\u003csub\u003e3\u003c/sub\u003ePMo\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e40\u003c/sub\u003e (\u0026ge;\u0026thinsp;99.0%) and FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO (\u0026ge;\u0026thinsp;99.0%) were purchased from Aladdin Industrial Corporation (Shanghai, China). Calcein acetoxymethyl ester (Calcein AM) and propidium iodide (PI) were provided by the Beyotime Institute of Biotechnology (Shanghai, China). Methyl thiazolyl tetrazolium (MTT), 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) and 2\u0026apos;,7\u0026apos;-dichlorofluorescein diacetate (DCFH-DA) were provided by Sigma-Aldrich (Shanghai, China). JC-1 staining kit and Crystal Violet Staining Solution were purchased from Beyotime Biotechnology. For the cell culture, Dulbecco\u0026apos;s Modified Eagle 5 Medium (DMEM), phosphate-buffered saline (PBS), antibiotic/anti-mycotic solution, and the fetal bovine serum (FBS) were provided by Thermo Fisher Scientific (Beijing, China). The reagents and solvents in this experiment were certified as the analytical grade.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe KM mice were purchased from the Second Affiliated Hospital of Harbin Medical University. The process of all animal experiments followed the guidelines and regulations of the Northeast Forestry University Animal Protection Committee (Approval No. 2022040).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynthesis of NRs and EDNRs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNRs were obtained by direct calcination of FeCl\u003csub\u003e3\u003c/sub\u003e\u0026bull;6H\u003csub\u003e2\u003c/sub\u003eO powder. A certain mass of yellow lumps of FeCl\u003csub\u003e3\u003c/sub\u003e\u0026bull;6H\u003csub\u003e2\u003c/sub\u003eO was weighed, thoroughly ground, and placed in an alumina crucible. The temperature was increased to 230\u0026deg;C under vacuum in a tube furnace at 5\u0026deg;C/min and maintained for 1 h. The solid was cooled naturally to room temperature to obtain a reddish-brown solid, and the unreacted FeCl\u003csub\u003e3\u003c/sub\u003e \u0026bull; 6H\u003csub\u003e2\u003c/sub\u003eO was washed with acetone several times. The synthesis of EDNRs nanosheets was similar to NRs nanosheets. H\u003csub\u003e3\u003c/sub\u003ePMo\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e40\u003c/sub\u003e (91 mg, 50 \u0026micro;mol) and 270 mg (1 mmol) of FeCl\u003csub\u003e3\u003c/sub\u003e\u0026bull;6H\u003csub\u003e2\u003c/sub\u003eO were dissolved in 2 mL of deionized water. Stirring was carried out continuously for 30 min at 25\u0026deg;C to allow for thorough dissolution and mixing. It was dried in an oven overnight at 60\u0026deg;C and subsequently ground in an alumina crucible. The mixture was heated to 230\u0026deg;C under vacuum in a tube furnace at 5\u0026deg;C/min and kept for 1 h. The mixture was then heated up to 230\u0026deg;C under vacuum in a tube furnace at 5\u0026deg;C/min and kept for 1 h. The mixture was then dried overnight in an oven at 60\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u003cstrong\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e \u003cstrong\u003eDetection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 1,3-Diphenylisobenzofuran (DPBF) was used as the indicator to detect the generation of \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2,\u003c/sub\u003e which would degrade from yellow to colorless in the presence of \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. 2 mg DBPF powder was dissolved in 1mL DMSO, and then add 100 \u0026micro;l solution to each samples need to be assayed. Finally, the solution was observed using UV-vis spectroscopy and the corresponding absorbance value changes were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026bull;OH Detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TMB was used as the \u0026bull;OH probe, which would change from colorless to blue in the presence of \u0026bull;OH. 2 mg TMB powder was dissolved in 1mL ethanol, and then 200 \u0026micro;l solution to each sample needed to be assayed. Finally, the solution was observed using UV-vis spectroscopy and the corresponding absorbance value changes were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrochemical performance of NRs and EDNRs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe three-electrode system was used for the evaluation of the electrochemical performance of as-prepared NRs and EDNRs. The three-electrode system consisted of a Pt sheet and Ag/AgCl electrode as the counter and reference electrodes, respectively, and a F-doped tin oxide (FTO) glasses with a uniformly loaded 20mg sample powder on one side (area 1.0 \u0026times; 1.0 cm) as the working electrode.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe HeLa Cervical Cancer and L929 Cells Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HeLa cervical cancer cells and L929 cells were provided by the Chinese Academy of Sciences Cell Bank (Beijing, China). The HeLa cancer cells and L929 cells were cultured in RPMI 1640 medium containing 10% FBS at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Survival of NRs and EDNRs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MTT method was used to assess the inhibition of NRs and EDNRs in vitro. First, HeLa cell suspensions were cultured overnight in (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e per well) 96-well plates to make them adherent. Then, HeLa cells were co-cultured with different concentrations of EDNRs. 20 \u0026micro;L of MTT (5 mg/mL) was added to each well and incubated for an additional 4 hours. Finally, 150 \u0026micro;L of DMSO was added to the pie in each well after draining and shaking to ensure that the crystals were dissolved. The experimental results were then measured using a microplate reader (MR-96A, Beijing, China). The Live-dead double staining assays were also used to assess cell survival. The double staining reagents consisted of calcein-AM and propidium iodide (PI) reagents were co-incubated with HeLa cells for 0.5 h. The staining results were visualized by confocal laser scanning microscopy (CLSM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Intracellular ROS Production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further determine the reason for tumour cell death, intracellular ROS generation was examined using DCFH-DA as a ROS probe. DCFH-DA was co-cultured with HeLa cells in 6-well plates for 0.5 h and then visualized by CLSM. Intratumoural ROS oxidized DCFH-DA to green fluorescent DCF, and the intracellular ROS levels were further determined by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumour Penetration Study of EDNRs in 3D tumor spheroids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the establishment of 3D tumor spheroids, 1600 HeLa cells (200 \u0026micro;L complete medium) were added tointo U-shaped 96-well plates. On the 7th day, the 3D tumor cell spheres were formed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Suppression Rate Assessment\u003c/strong\u003e \u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe same size KM mice (15 g) were purchased from the Second Affiliated Hospital of Harbin Medical University, and we established the S180 tumour KM mouse model to evaluate its tumour suppression rate \u003cem\u003ein vivo\u003c/em\u003e. In order to construct a mouse model of hormonal tumour, S180 cells were resuspended and dispersed, diluted to 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e/mL with PBS, and injected with 0.1 mL per mouse. When the tumour volume was about 200 mm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e then the mice were randomly divided into seven groups. And then they were treated accordingly and their body weights and tumour sizes were recorded and euthanised at the end of the treatment or when their volume exceeded 1500 mm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Groups were defined as follows: Control group (G1), Only NIR irradiation (G2), NRs (G3), NRs with NIR irradiation (G4), 5 mg/Kg ENDRs (G5), and 5 mg/Kg EDNRs with NIR (G6) and 10 mg/Kg EDNRs with NIR irradiation (G7). The NIR irradiation condition is 650 nm, 1 W cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 10 min.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStaining\u003c/strong\u003e \u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFreshly collected tumours were embedded with an Optimal cutting temperature compound (OCT) embedding agent, snap-frozen in liquid nitrogen for 10\u0026ndash;20 S, and sliced using a frozen sectioning machine to obtain 8 \u0026micro;m tissue sections. Then, as with the \u003cem\u003ein vitro\u003c/em\u003e ROS staining, DCFH was used as the ROS probe.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to compare statistical significance between two data groups. One-way analysis of variance (ANOVA) with a Bonferroni post hoc test was used to compare three or more groups. Quantitative data were indicated as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. Asterisks were used to represent significant differences (n.s.: no significance, *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the National Natural Science Foundation of China (No. 52372264, 21972035, and 52375565), the Heilongjiang Provincial Natural Science Foundation of China (No. LH2023B002), Interdisciplinary Research Foundation of HIT (IR2021112), State Key Laboratory of Robotics (2019-O02), and Supported by the Fundamental Research Funds for the Central Universities (2572023CT11-05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.Y. and T.S. performed the experiments and analyzed the results. M.T., S.W., and X.L. assisted with the experiment design and data analyses. Z.Y., T.S., and Z.W., wrote and revised the original draft of the manuscript. T.S., L.S., and Z.W., reviewed and edited the manuscript. Z.Y., T.S., and Z.W., supervised the whole project. All authors discussed the results and commented on the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGao W et al (2023) Deciphering the catalytic mechanism of superoxide dismutase activity of carbon dot nanozyme. Nat Commun 14:160\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuo M, Wang L, Chen Y, Shi J (2017) Tumor-selective catalytic nanomedicine by nanocatalyst delivery. Nat Commun 8:357\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang D et al (2020) Self-assembled single-atom nanozyme for enhanced photodynamic therapy treatment of tumor. 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Chin Sci Bull 62:152\u0026ndash;166\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6432792/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6432792/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSynthetic nanorobots have garnered significant attention for precision oncology, while their clinical translation is hindered by inefficient tumour penetration. Catalytic motion, which mainly rely on fuel gradients, always fails in deep tumour due to limited motility. Here, we introduce exciton-dissociation-enhanced nanozyme-based robots (EDNRs) swarm that leverage Z-scheme heterojunctions to achieve the enhanced tumour penetration, combining photocatalytic propulsion with collective hydrodynamic effects. The EDNRs were constructed \u003cem\u003evia\u003c/em\u003e Z-scheme heterogenization of photocatalytic FeOCl nanosheets with polyoxometalates, creating a high-valence-band interface that promotes efficient exciton dissociation. This heterostructure conferred enhanced catalytic activity compared to non-heterogenized counterparts. Under near-infrared irradiation, the EDNRs demonstrated superior motility compared with the nanozyme-based robots without Z-scheme heterogenization, attributed to synergistic photocatalytic propulsion and swarm-induced hydrodynamic interactions. In vivo intravenous administration in mice exhibit that the nanozyme-based robot swarm upon NIR irradiation could exert the swarm penetration of tumour tissue and following arrival at the deep tumour. Meanwhile, accompanying with the production reactive oxygen species (ROS), the nanozyme-based robot swarm in deep tumour substantially inhibits the proliferation of tumour. The swarm dynamics of nanozyme-based robot with enhanced exciton dissociation, potentially impacts the realization of catalytic nanorobots toward deep tumour penetration and therapy.\u003c/p\u003e","manuscriptTitle":"Nanozyme-based Robots Swarm with Exciton-Engineered Z-Scheme Heterojunctions for Depth-Resolved Tumour Penetration and Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-04 05:13:09","doi":"10.21203/rs.3.rs-6432792/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"11253807-c32a-4536-85f8-50d21b103e23","owner":[],"postedDate":"August 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":47377267,"name":"Physical sciences/Materials science/Nanoscale materials/Nanoparticles"},{"id":47377268,"name":"Physical sciences/Engineering/Biomedical engineering"}],"tags":[],"updatedAt":"2026-02-16T20:25:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-04 05:13:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6432792","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6432792","identity":"rs-6432792","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}