Palladium chloride (59–60%) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China), hydrochloric acid (AR), hydrogen peroxide (30%) was purchased from Guangzhou brand reagent company (Guangzhou, China), oleic acid, oleylamine, benzyl alcohol (99%), absolute ethanol, and sucrose were obtained from Aladdin Biochemical Technology Co. (Shanghai, China). Fluo-4 AM, Hoechst 33342, Trypan Blue Solution (0.4%), HEPES buffer solution (pH = 7.4), PBS buffer solution (pH = 7.4) was bought from Beyotime Biotechnology (Shanghai, China). Roswell Park Memorial Institute (RPMI-1640) cell media were bought from Gibco. Jurkat T cells were donated by Southern Medical University. All other chemical reagents used in this experiment were analytically pure without further purification. Purified deionized water was prepared by the Milli-Q Plus system (Millipore, United States).

Pd nanospikes were firstly synthesized by hydrothermal method. In a typical procedure, 0.2 mmol PdCl₂ (dissolved in 1M HCl), 80.0 ml benzyl alcohol, 4.0 ml oleylamine (OAm) and 4.0 ml oleic acid (OA) were mixed in a 100 ml beaker and stirred at room temperature for 30 min. Then, it was transferred to a stainless teel autoclave lined with a polytetrafluoroethylene container, and reacted at 120°C for 8 h. Naturally cool to room temperature, centrifuge at 10,000 rpm to remove the surfactant, wash the precipitate with ethanol 3 times, and then dry it under vacuum at 40°C. Subsequently, an aqueous solution (∼200 mg/ml) containing Pd nanoparticles was dropped onto the glass slide by a spin coater to form a monolayer, using an ion sputter coater to sputter gold (∼3 nm, 50s) were used to produce Pd/Au Janus surface. Then, it was collected by ultrasound and resuspended in PBS, washed three times, and dispersed in PBS buffer solution for further use.

The optical videos were recorded by A Nikon Ti2-A inverted optical microscope with a ×40 objective. Dilute Pd/Au nanomotors to the appropriate concentration with PBS buffer and add H₂O₂ solution with different concentrations (0.00, 0.05, 0.10, 0.50% wt, add 10 µL in 1 ml PBS dispersion solution, the final concentration is 0.00, 0.165, 0.33, 1.65mM, respectively) in a petri-dish and record the movement videos up to 10 s at 10 fps, respectively. Then, the moving tracking trajectories and the speed of nanomotors (at least 15) were analyzed by ImageJ.

The mean square displacements (MSD) curves were fitted by the following Eq. 1 (Arque et al., 2019; Tang et al., 2020): M S D ( Δ t ) = < ( x i ( t + Δ t ) − x i ( t ) ) 2 >, where i = 2 refers to two-dimensional analysis.

Extract the slope of the MSD curves, and then calculate the long-term diffusion coefficient (D_L) according to the formula 2 (Wang et al., 2019; You et al., 2019): M S D = 4 D L Δ t

Jurkat T cells were cultured in Roswell Park Memorial Institute (RPMI-1640) cell media supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin/streptomycin (1%, v/v) in an incubator at 37°C with an atmosphere with 5% CO₂. For calcium imaging, transfer the cells to a serum-free cell culture medium and starve them for 2 h. Then, incubate with 5 μM Fluo-4 AM in serum-free cell culture medium at 37°C for 30 min, and then stain with Hoechst 33342 for 10 min.

Resuspended the Jurkat T cells loaded with Fluo-4 AM with the DEP cell medium and seeded into the 96-well plates with a density of about 1× 10⁴ cells per well. Incubated the cells with Pd/Au nanomotors (∼20 μg/ml) in the presence or absence of H₂O₂ (add 2 µL 0.05%wt in 200 µL dispersion, final concentration is 0.165 mM) and only H₂O₂ were investigated, respectively. Then, the green fluorescence of Fluo 4 was captured by A Nikon Ti2-A fluorescence microscopy (Intensity:100%; Exposure time:100 ms; Gain:1.0X). Corresponding fluorescence intensity was calculated with the Image J software according to the previous research (Sheffield 2007).

The cytotoxicity of nanomotors was assessed by a standard CCK-8 method. NIH3T3 cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS, 10%,v/v) and penicillin/streptomycin (1%, v/v) at 37°C in a 5% CO₂ incubator. NIH3T3 cells were seeded into a 96-well plate at a concentration of 1 ×10⁴ cells/well in 100 μL medium in triplicate for 24 h. Then, the medium was replaced by H₂O₂, Pd/Au NPs, Pd/Au NMs with different concentrations. 2 h later, 10 μL/well Cell Counting Kit-8 (CCK-8) was added after washing with PBS twice, allowing cells to continuously culture for 30min in the culture incubator. Absorbance at 450 nm was then measured in a BioTek SynergyHTX microplate reader. Live/dead staining has further assessed the biocompatibility of Pd/Au nanomotors by cell viability. The Jurkat T cells were seeded in a 96-well plate and incubated with nanomotors for 2 h. Subsequently, the treated cells were stained with Calcein-AM (green)/PI(red) for 30 min. The staining results of cells were observed under an inverted fluorescence microscope.

The morphology of the tested nanoparticles was captured with a transmission electron microscope (JEM-1400 Plus, JEOL) and scanning electron microscope (SEM) images were recorded on Field-Emission-SEM (Zeiss GeminiSEM500, Germany). Energy-dispersive X-ray spectroscopies (EDX) were obtained by elemental analysis with an EDX analyzer installed on a Field-Emission-SEM (Zeiss GeminiSEM500, Germany) at an accelerating voltage of 15kV. Nikon Ti2-A Inversion Fluorescence Microscope was utilized to track the motion of nanomotors, cell morphology, and fluorescence images. A Multi-Mode Reader (BioTek SynergyHTX, United States)was used to detect the absorbance of the CCK-8 assay.

To synthesize Pd nanoparticles with dendritic structure, oleic acid (OA) and oleylamine (OAm) were used as surfactants/templates, and benzyl alcohol play as the solvent and reducing agent. First, OA and OAm were dissolved in benzyl alcohol to form spherical micelles having a dendritic peripheral structure. Then PdCl^4- ions were slowly reduced and deposited on the interior of the micelles and on the dendrites. Finally, the Pd nanoparticles with special morphology were obtained by removing the OA/OAm templates with ethanol washing. As depicted in Figures 2A,2B, TEM images, the as-prepared Pd nanoparticles have a 3D dendritic structure, which consists of spherical structures with diameters of about 215 ± 50 nm (Figure 2E) and dendrites with an average length of 40 ± 17 nm (Figure 2F) that are densely dispersed on the surface of the nanospheres.

For the formation of Pd/Au Janus structures, a layer of the spiky Pd nanoparticles was uniformly dispersed onto a glass slide, followed by an asymmetrical coating of the nanospheres by sputtering with a thin (5 nm) Au layer. The SEM images are shown in Figures 2C,D further confirms its unique structure. According to its corresponding energy-dispersive X-ray spectroscopy (EDX) (Figure 2H), demonstrating the presence of Pd and Au about 64%, 17%, respectively. In addition, Figure 2G is clearly shown that the Au element is distributed on surface of the Pd nanospheres. These results demonstrate the successful preparation of spiky-like nanomotors, indicating its geometrical asymmetry ensures an asymmetrical generation of forces.

After confirming the asymmetric structure of the nanomotor, its self-propelling ability was recorded by an inverted optical microscope and analyzed by the manual tracking plugin of ImageJ software according to previous reports. Owing to the Janus structure of Pd NPs coated with Au nano-sized thin, an effective propulsion driving force was generated through the asymmetric catalytic decomposition reaction of the hydrogen peroxide, as illustrated in Figure 3A. The optical tracking images are shown in Figure 3B (captured from Supplementary Video S2), illustrating the obvious autonomous propulsion of motors in an H₂O₂ solution. Varying the fuel concentrations, changing motion was observed, as the typical tracking trajectories displayed in Figures 3C–F (acquired from Supplementary Videos S1–S4). In the absence of fuel, the motion trajectory of the motor showed a typical random walk and localized within a small area behavior as a result of Brownian motion. As the fuel concentration increases, the catalytic nanomotors display relatively efficient propulsion. Obtained the coordinates of the moving trajectories and quantified the corresponding velocity of the motor by ImageJ on the basis of the optical tracking videos, results are depicted in Figure 3G. It is obvious that the velocity of the motor can be adjusted by the concentration of H₂O₂, the average velocity of the motor is 4.16 μm s⁻¹ and 4.64 μm s⁻¹at H₂O₂ concentrations of 0.165 and 0.33 mM, respectively. When the H₂O₂ concentration is up to1.65 mM, the nanomotors conducted self-driving motion with a speed of 5.77 μm s⁻¹, which is 1.95 times higher than that of performing Brownian motion (2.95 μm s⁻¹). Further, according to the X-Y coordinates of the trajectories, obtained from the optical videos using ImageJ software, the mean square displacements (MSD) of Pd/Au nanomotors along with different concentrations were investigate (You et al., 2019; Tang et al., 2020). As shown in Figure 2H, the plots of MSD versus time interval (t) reveal that the slope of MSD plots tends to increase with the concentration of the fuel. Subsequently, fitted the plots and estimated the effective diffusion coefficient (D_L) according to formula 2. The corresponding results are shown in Figure 2I where the diffusion coefficient shows a significant increase from 0.26 ± 0.18 μm² s⁻¹ (without fuel) to 0.83 ± 0.51 μm² s⁻¹ (in the presence of 0.165 mM H₂O₂) and with a maximum enhancement of 5 times to 1.69 ± 0.58 μm² s⁻¹ (1.65 mM H₂O₂), exhibiting the fuel concentration dependence of enhanced diffusion motion. These results showed that the Pd/Au nanomotor system could achieve enhanced motion in the presence of hydrogen peroxide even under a relatively low concentration, providing the possibility for subsequent application in the bioenvironment.

To address whether the moving Pd/Au nanomotors could optimize the activation of the immune cells via mechanotransduction, we monitored the performance to activate Jurkat T cells, which express high levels of endogenous mechanical sensitive ion channel (Liu and Ganguly, 2019). The Jurkat cell line has been used to model and characterize signaling events in T cell activation (TCA), a critical process in an effective adaptive immune response (Abraham and Weiss, 2004). To prove that spiky nanomotors can achieve high-efficiency movement in biologically relevant media, the self-driving ability of the nanomotors has been observed in the dielectrophoresis (DEP)cell culture medium (Supplementary Video S5). Note that the DEP medium is a non-ionic iso-osmotic solution with a small dielectric shielding effect, so the effect of the background solution on the motor movement efficiency can be eliminated to a greater extent (Marino et al., 2015). Considering high concentration of hydrogen peroxide is cytotoxic, so we chose a relatively low concentration of 0.165 mM that does not harm the cells and also has a relatively high speed of motion as the driving fuel (Supplementary Figure S8) (Kuang et al., 2011; Yu et al., 2021). As shown in Supplementary Figure S1 (Supplementary Material), in the presence of 0.165 mM hydrogen peroxide, the nanomotor can approach the cells or move continuously in the culture medium. Then, to quantify T cell activation, we measured the concentration fluctuation of intracellular Ca²⁺ ions over the duration of the operation of the nanomotors. It has been verified that professional mechanical sensitive ion channels (especially Piezo 1 ion channels) are calcium ion-permeable and the activation of such channels is accompanied by an influx of Ca²⁺ into the cell cytosol from the extracellular environment (Pottosin et al., 2015; Ranade et al., 2015; Liu et al., 2018; Atcha et al., 2021). Also, a rapid increase of calcium ion concentration in the cytoplasm is a common mark of T cell activation, this suggested that mechanical-mediated T cell activation could be reflected via the dynamic change of calcium flux (Dirar et al., 2020). In our experiments, the influx of Ca²⁺ was measured by a calcium-sensitive dye, Fluo-4, which was loaded into the cell cytosol. The fluorescence emission of Fluo-4 increases proportionally to intracellular [Ca²⁺], so it provides a direct and quantitative readout of the T cell activation (Figure 4A). Considering the oscillation and flow of the entire solution system caused by the movement of the autocatalytic motor, the state of the whole cell was observed. The images recorded in Figures 4B–G illustrate the effect of the moving nanospikes on the overall cells (t = 0 min, t = 50 min) and then Image J analysis was used to obtain the fluctuation results of calcium ion concentration. It displays that the mean values of the Ca²⁺ fluorescence intensity gradually increased, and reaches the maximum value at t = 50 min (Figure 4H). Compare their fluorescence intensity at t = 50 min, the intensity of the former is 1.45 ± 0.19 times that of the resting T cells (t = 0 min) (Figure 4I). In the same time interval, resting T cells only containing Fluo-4 exhibited a 7.12 ± 7.13% increase in fluorescence (Supplementary Figure S3). To exclude the influence of chemical fuel hydrogen peroxide on the state of cells, calcium ions were tested under the same conditions, as depicted in Figure 4J, and an evaluation of the corresponding results implies that the interference of low concentration hydrogen peroxide solution (0.165 mM) can be ignored (an increase of 4.08 ± 1.34%). Moreover, to explore the performance of the unique self-driving character of the nanomotor on the activation efficiency, we incubated the spiky-like nanoparticles with Jurkat T cells without adding hydrogen peroxide as fuel and examined calcium fluorescent intensity after the same time interval (Figure 4J). Compared with movable nanomotors, static nanoparticles influence T cell activation with an amplitude (1.18 ± 0.07 fold) smaller than the dynamic state (1.45 ± 0.19 fold). In addition to the effective self-driving force of the dynamic motor, the dynamic behavior of the nanomotor in the cell microenvironment induces fluid flows, generating distinct flow fields surrounding the cell is also an important physical factor, according to reports in the literature (Syeda et al., 2015; Liu et al., 2018). Working synergistically with the inherent nature of the motor to enhance the forces exerted on the cell during activation. Sustained acts for a period of time, mechanosensitive ion channels on T cells percept these physical stimuli and mediate an intracellular calcium response, which contributes to the activation of Jurkat T cells in the overall environment.

The self-propulsion of the nanomotors endows itself as an effective force. To calculate how large the force is, we based on a theory for self-propelled particles, involving the long-time translational diffusion coefficient reported by ten Hagen et al. (2011) (ten Hagen et al., 2015). Such aspherical particles with two rotational degrees of freedom, the absolute value of the effective driving force can be evaluated by the following formula 3: F = 3 k B T 2 r 2 ( D L D τ − 1 ) where k_B is the Boltzmann constant, T the absolute temperature, r the radius of the nanoparticles, and D_L, D_τ is the long-time and translational diffusion coefficient, respectively.

In our cases, the active motion of the self-propelled Janus nanomotors was investigated by mean square displacement (MSD) analysis (Figure 2I). In the absence of fuel, Pd/Au nanomotors perform Brownian motion, the value of D_L is equal to the translational diffusion coefficient Dτ (Campbell and Ebbens, 2013; Ma X. et al., 2015; You et al., 2019). As illustrated in Figure 2I, the value of Dτ was about 0.26 ± 0.18 μm² s⁻¹, at room temperature of 25°C. At 0.165 mM H₂O₂, the diffusion coefficient is increased by 2.13 times compared to that without fuel. Taken together, we can estimate the effective driving force generated by a single nanomotor with the decomposition of 0.165 mM hydrogen peroxide is about 120.94 ± 79.6 fN, which is approximate to that calculated by DLS results (Supplementary Figure S6).

In addition, the dynamic behavior of multiple nanomotors in the cellular microenvironment causes rapid local fluid convection, generating pressure and shear stresses around the cell, thereby synergizing the forces of multiple nanomotors and enhancing the expression of mechanical signals to a certain extent (Takatori et al., 2014). Mechanosensitive ion channels, particularly the Piezo1 ion channel used as a physical stimulus sensor, have recently been shown to be abundantly expressed on T cells (Liu and Ganguly, 2019). The mechanical force or shear stress produced by the advancing nanospike is captured by such biosensors and benefits ion influx or efflux, which is a prerequisite for subsequent T cell activation (Syeda et al., 2015; Liu et al., 2018).