Introduction

Traumatic brain injury (TBI) is one of the leading causes of disability and mortality worldwide1. TBI involves mechanical damage on the brain and triggers a wide range of cortical network abnormalities including the death of functional neurons and the damage of the synaptic circuit2. Currently, there are no effective therapies for the repair of TBI as brain tissues have a very limited capacity to produce new neurons in response to injury3. The use of neural stem cell (NSC) therapy shows potential in rebuilding neuronal circuits and restoring brain tissues by replacing damaged neurons with newly formed neurons4,5. While transplantation of NSCs shows a certain level of success in animal TBI models6, the therapeutic performance of this approach is considerably restricted due to the difficulties in directing the transplanted cells to differentiate into functional neurons within the injured microenvironments7,8. Several neurotrophins, hormones, and small molecule drugs have been proposed for directing the neural differentiation of NSCs9,10. Their application in vivo, however, confronts several challenges, including delivery difficulties, rapid degradation, and uncontrolled distribution11.

Pulsed electrical stimulation represents a potent strategy for augmenting neurogenesis and treating neurodegenerative disease12,13. It has been reported that electrical stimulation can promote the neural differentiation of stem cells through activating cytomembrane receptors, ion transport channels, or intracellular communication12,14. However, conventional electrical stimulation involves the implantation of electrodes and wires connected to the external power input device, which may lead to additional risks of injury, inflammation, and infection15. More importantly, it is difficult to inject the traditional electrodes alongside the stem cells and precisely apply the electrical stimulation to individual cells. Wireless electrical stimulation based on the electromagnetic induction16 or piezoelectric effect17 has received considerable attention in the fields of neural regeneration and neural modulation as it can avoid the risks associated with the traditional electrodes and wires. For example, piezoelectric materials capable of generating wireless electrical stimulation under ultrasound have been used for treatment of the Parkinson’s disease and SCI18,19. Nonetheless, none of the reported wireless stimulation can stimulate transplanted stem cells individually20; such individualized electrical stimulation is very important for effective treatment because stem cells are in a state of dynamic migration post-injection and non-individualized stimulation may electrically harm non-target cells.

In this work, to overcome this hurdle, we propose to use the graphene nanosheets as nanopatches to wrap the membrane of NSCs to in situ generate electric signals to stimulate the neuronal differentiation of NSCs individually for effective TBI therapy (Fig. 1). In this way, even when NSCs are migrating, they are always associated with the stimulating material, enabling us to electrically stimulate NSCs on demand. Namely, thanks to electromagnetic induction, the nanopatches can generate periodical pulse electrical signals that are immediately applied onto the target cells wrapped by them when they are exposed to a periodic magnetic field (Fig. 1a). The amplitude and frequency of the electrical stimulation can be controlled by varying the rotation speed of the magnetic field. The nanopatches, once modified with a layer of laminin, can tightly wrap the cell membranes. The electric signals in situ generated on the surface of NSCs enhance neuronal differentiation of NSCs and the mature of differentiated neurons (Fig. 1b). We then use the nanopatch-wrapped NSCs to replace the conventional un-wrapped NSCs in treating TBI mice. After injected into the lesion site, these wrapped NSCs can be effectively differentiated into new functional neurons under the periodic magnetic field, leading to the improvement in the repair of injured brain tissues, along with substantial amelioration of the behavioral and cognitive deficit ability of TBI mice (Fig. 1c).

Fig. 1: Schematic showing the wireless nanopatch-enabled stimulation for promoting the differentiation of NSCs and TBI repair.
Fig. 1: Schematic showing the wireless nanopatch-enabled stimulation for promoting the differentiation of NSCs and TBI repair.The alternative text for this image may have been generated using AI.
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a Schematic showing the generation of induced wirelessly electrical signal on a nanopatch made of a graphene nanosheet under the periodic magnetic field. b Schematic showing the process of wrapping the membrane of NSCs by the wireless electrical nanopatches and the differentiation of the nanopatch-wrapped NSCs into neurons under rotating magnetic field. c Schematic showing implantation of the nanopatch-wrapped NSCs into the dura mater encephalic of the injured brain to promote the repair of TBI under the rotating magnetic field.

Results

Synthesis of the wireless electrical nanopatches and their anchoring onto the NSCs

The graphene nanopatches were fabricated through electrochemical exfoliation of bulk graphite in an aqueous solution according to a reported protocol21. Supplementary Fig. 1a shows the scheme of the electrochemical exfoliation process. The atomic force microscopy (AFM) image in Fig. 2a shows that the graphene nanosheets had a size of 1.5–5 μm and the AFM image in Supplementary Fig. 2 demonstrates that they had a height of 2.3 nm, indicating that the graphene nanosheets were composed of 2–3 layers of graphene22. Fig. 2b shows the Raman spectroscopy of the as-obtained graphene nanosheets. The peaks at 1330 cm−1, 1578 cm−1, and 2668 cm−1 were attributed to the characteristic D, G, and 2D bands of graphene, respectively. For graphene, the G band is caused by in-plane vibrations of sp2 carbon atoms while the D band is involved in structural defects and disordering. The intensity ratio (ID/IG) of the D and G bands of the as-obtained graphene nanosheets was 0.33, indicating that the graphene nanosheets had a good graphene crustal structure with fewer defects, which would provide good conductivity. The intensity ratio (I2D/IG) of the 2D and G bands was 0.67, suggesting that the graphene nanosheets were multi-layered, which was consistent with the AFM results.

Fig. 2: Characterization of the wireless electrical nanopatches.
Fig. 2: Characterization of the wireless electrical nanopatches.The alternative text for this image may have been generated using AI.
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a AFM image of graphene nanosheets and the corresponding size statistics. b Raman spectra of graphene nanosheets. c COMSOL simulation showing the generated electrical field on a graphene nanosheet under a rotating magnetic field. d Analysis of the induced electrical signals of a graphene nanosheet under the magnetic field at different stages in a representative cycle. e Voltage generated on the graphene film made of graphene nanosheets when placed under a rotating magnetic field at rotation speeds of 600 rpm. f Fluorescence images of NSCs after being wrapped with graphene nanosheets for 3 h and cultured for two days. The graphene nanosheets were labeled with a dye to give red fluorescence. The cell membrane was pre-stained with PKH67 into green. The nuclei were stained into blue by Hoechst. g SEM image of NSCs after being individually wrapped with graphene nanosheets. h Viability of NSCs after being wrapped with graphene nanosheets at concentrations of 0, 30, 60, and 90 μg mL−1 for 1, 2 and 3 days. Data represent three independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (nsp>0.05 compared to the 0 μg mL−1 group). i Proliferation of nanopatch-wrapped and unwrapped NSCs in the absence or present of a 600-rpm magnetic field for 1, 2, and 3 days. Data represent three independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (nsp>0.05 compared to the Ctrl group).

The graphene nanosheets were then explored as wireless electrical nanopatches for generating electrical stimulation under changing magnetic fields. COMSOL Multiphysics was used to simulate the generated induced voltage on an individual graphene nanosheet by using the electromagnetic-rotating magnetic machinery module. Figure 2c shows the simulated results of a graphene nanosheet with a size of 5 μm under a magnet (160 mT) at a rotating speed of 600 rpm. The generated maximal voltage reached 6.3 × 10−3 mV, which was consistent with the value calculated based on the law of electromagnetic induction. Moreover, as the position of the magnet relative to the graphene nanosheet changed, the induced voltage of graphene nanosheets was changed correspondingly, showing a periodical pulse-typed electrical signal (Fig. 2d). We fabricated graphene film by stacking the graphene nanosheets and then tested the generated voltage of the graphene film under a rotating magnetic field by the oscilloscope. As shown in Fig. 2e and Supplementary Fig. 3, when the magnet with a magnetic field intensity of 160 mT was rotated at a speed of 0, 100, 200, 300, 400, 500, and 600 rpm, the corresponding voltages generated on the graphene film were 0, 0.2, 0.4, 0.7, 1.1, 1.3, and 1.8 mV, respectively. Clearly, the amplitude and frequency of the electrical signals can be controlled by varying the rotation speed of the magnetic field. The above results demonstrate the feasibility of using the graphene nanosheet as the nanopatches for individualized electrical stimulation of NSCs.

To ensure the generated electrical signals to act on the surface receptors of individual NSCs, the graphene nanosheets were anchored on the membranes of the stem cells by wrapping them. It has been reported that two-dimensional materials with a lateral size in a few micrometers are able to attach to the cell membrane with negligible internalization23,24. To enable graphene nanosheets to wrap the cell membrane, they were modified with a layer of laminin through absorption (Supplementary Fig. 1b). Laminin can attach to the surface of stem cells by binding to the membrane receptors of cells. To verify the position relationship between the nanopatch and the cell membrane, the graphene nanosheets were pre-labeled with a fluorescent dye (Supplementary Fig. 4). As shown in Supplementary Fig. 5, among the 116 counted cells, 80 cells (69%) were attached with the laminin-modified graphene nanopatches. In comparison, for graphene nanopatches without modification of laminin, only 35% of cells were attached to the graphene nanopatches. The fluorescence distribution in Supplementary Fig. 5d shows that the green fluorescence of the graphene was overlapped with the red fluorescence of the membrane, indicating that the graphene nanopatches were wrapped on the cell membrane. Figure 2f shows the fluorescence image of the NSCs after incubated in a suspension of the laminin-modified graphene nanosheets for 3 h, followed by further culture for 2 days, in which the cell membrane, nuclei, and graphene nanosheets were stained into green, blue, and red, respectively. After 2 days culture, the red fluorescence of graphene nanosheets still appeared around the green fluorescence of the membrane, suggesting that the graphene nanosheets were wrapped on the cell membrane. The Scanning electron microscope (SEM) images in Fig. 2g and Supplementary Fig. 6 further confirm that the graphene nanosheets were attached to the cell membrane. As shown in Supplementary Fig. 7, at day 7, most of the graphene nanosheets remained on the surface of the cell membrane, negligibly falling off from the membrane or being internalization by the cells. The above results demonstrate that the graphene nanosheets could serve as nanopatches and keep wrapping NSCs for at least 7 days.

The Live/Dead staining kit and cell counting kit-8 (CCK-8) were used to evaluate the cytotoxicity of nanopatches. Figure 2h shows the CCK-8 results of NSCs after being wrapped with different concentrations of nanopatches (0, 30, 60, and 90 μg mL−1) for 3 days. The results demonstrate that anchoring the nanopatches at the concentration of 0-90 μg mL−1 did not influence the viability of the NSCs. We further evaluated if the rotating magnetic field itself or the changing magnetic field-induced electrical stimulation on the nanopatches would influence cell activity. Figure 2i shows the CCK-8 results of the unwrapped NSCs without a magnetic field (abbreviated as Ctrl), the unwrapped NSCs under a rotating magnetic field (abbreviated as MF only), the nanopatch-wrapped NSCs without a magnetic field (abbreviated as Nanopatch only), and the nanopatch-wrapped NSCs under a rotating magnetic field (abbreviated as Nanopatch+MF). The results show that the magnetic field alone or the magnetic field-induced electrical stimulation on the nanopatches did not reduce the cell viability. The live/Dead staining results in Supplementary Fig. 8 also confirm that over 95% of the cells were kept alive in the four groups, confirming the good biocompatibility of nanopatches to NSCs even under the rotating magnetic field. The above results demonstrate that the nanopatches made of graphene nanosheets had good cytocompatibility to NSCs and the nanopatches-mediated electrical stimulation did not influence the viability of the cells. The good biocompatibility illustrates that graphene nanosheets can be used as nanopatches for individually stimulating the differentiation of NSCs.

Nanopatch-mediated wireless electrical stimulation for promoting neuronal differentiation

The graphene nanosheets were then used as the nanopatches to generate electrical stimulation on NSCs individually and wirelessly under a rotating magnetic field for promoting their neuronal differentiation. The nanopatch-wrapped NSCs were subjected to a periodic magnetic field for 15 min every day. To evaluate if the nanopatch-generated electrical signals could promote the neuronal differentiation of NSCs, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence staining, and Western Blot were used to assess the expression of neuron markers of the NSCs. Beta tubulin III (Tuj1) is a protein found in neuron and is used as a marker for early neurons25. Microtubule-associated protein 2 (MAP2) is another protein involved in neurogenesis and microtubule assembly and is used as a marker for mature neurons26. The protein known as glial fibrillary acid protein (GFAP) is primarily localized in astrocytes, serving as a reliable indicator for the presence of glial cells27. Fig. 3a shows the RT-qPCR results after the NSCs anchored with the nanopatches at concentrations of 30, 60, and 90 μg mL−1, followed by exposure to a periodic magnetic field (160 mT) rotated at 600 rpm for 5 days. The expression of Tuj1 of NSCs anchored with 30, 60, and 90 μg mL−1 of the nanopatches were up-regulated 3.1, 4.6, and 4.1 folds, respectively. The expression of MAP2 of NSCs anchored with 30, 60, and 90 μg mL−1 of the nanopatches were up-regulated 1.73, 3.65, and 4.44 folds, respectively, compared to the control group. The RT-qPCR results show that under a periodic magnetic field, wrapping NSCs with the nanopatches promoted the neuronal differentiation of NSCs and the promotion effect had a direct correlation with the concentration of the nanopatches. However, there were no obvious differences in the expression of the MAP2 and Tuj1 of the stem cells as the concentration of the electrical nanopatches rose from 60 to 90 μg mL−1. Hence, we choose 60 μg mL−1 of nanopatches in the subsequent experiments.

Fig. 3: Individualized wireless electrical stimulation generated on the nanopatches promoted the neuronal differentiation of nanopatch-wrapped NSCs.
Fig. 3: Individualized wireless electrical stimulation generated on the nanopatches promoted the neuronal differentiation of nanopatch-wrapped NSCs.The alternative text for this image may have been generated using AI.
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a RT-qPCR results of NSCs wrapped with the nanopatches at 0, 30, 60, and 90 μg mL−1 and exposed to a 600 rpm rotating magnetic field at day 5. Data represent three independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (nsp>0.05, ***p < 0.001, and ****p < 0.0001 compared to the 0 μg mL−1 group). b RT-qPCR results of NSCs wrapped with 60 μg mL−1 of the nanopatches under a magnetic field rotated at 0, 300, 600, and 900 rpm at day 5. Data represent three independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (*p < 0.05 and ****p < 0.0001 compared to the 0 rpm group). c RT-qPCR results of the NSCs (Ctrl), the NSCs under 600-rpm rotating magnetic field (MF only), the NSCs wrapped with 60 μg mL−1 of nanopatches (Nanopatch only), and the NSCs wrapped with 60 μg mL−1 of nanopatches under a 600-rpm rotating magnetic field (Nanopatch+MF) at day 5. Data represent three independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (nsp>0.05, *p < 0.05, and ****p < 0.0001 compared to the Ctrl group). d The Tuj1, MAP2, and GFAP immunofluorescence images of NSCs at day 5 in Ctrl, MF only, Nanopatch only, and Nanopatch+MF groups. GFAP was labeled in green, while Tuj1 and MAP2 were labeled in red, Nuclei were labeled with DAPI in blue. e Ratios of Tuj1-positive cells and MAP2-positive cells based on immunofluorescence staining results in d. Data represent three independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (nsp>0.05 and ****p < 0.0001 compared to the Ctrl group). f Sholl analysis of the complexity of neurite in differentiated cells based on immunofluorescence staining results in d. Data represent three independent experiments and are presented as the mean ± standard deviation. g Western blot results of GFAP, Tuj1, and MAP2 of NSCs in Ctrl, MF only, Nanopatch only, and Nanopatch+MF groups at day 5. h The analysis of gray values of western blotting results in g. Data represent three independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (nsp>0.05, *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the Ctrl group).

We also fabricated graphene nanosheets with different sizes to evaluate the influence of the graphene nanosheets size on the differentiation of NSCs. Figure 2a and Supplementary Fig. 9 show the AFM images of the graphene nanosheets with a size of 0.4–1.5 μm, 1.5–5 μm, and 5–10 μm, respectively. Supplementary Fig. 10 shows the RT-qPCR results after the NSCs anchored with graphene nanosheets with different sizes, followed by exposure to a periodic magnetic field (160 mT) rotated at 600 rpm for 5 days. The expression of Tuj1 of NSCs anchored with graphene nanosheets with size of 0.4–1.5 μm, 1.5–5 μm, and 5–10 μm, were up-regulated 1.78, 4.23, and 2.3 folds, respectively, compared to the control group while the expression of MAP2 were up-regulated 1.54, 3.7, and 2.4 folds, respectively. The RT-qPCR results show that the graphene nanopatches with the size of 1.5–5 μm had the best performance in promoting the neuronal differentiation of NSCs. This may be because that graphene nanosheets with a size of 0.4–1.5 μm are easy to be endocytosed after attaching to the cell membrane while the number of the graphene nanosheets with a size of 5–10 μm is too small compared to the other two types of graphene nanosheets at the same mass concentration, both of which will reduce the efficiency for stimulating the receptors on the membrane. To this end, we choose the graphene nanopatches at a size of 1.5-5 μm in the subsequent experiments.

The influence of the speed at which the magnetic field rotates on the neuronal differentiation of NSCs was further evaluated. Figure 3b shows the RT-qPCR results of NSCs anchored with 60 μg mL−1 of nanopatches and exposed to the magnetic field at a rotating speed of 0, 300, 600, and 900 rpm for 5 days. Compared with cells cultured without the magnetic field, the expression of Tuj1 of NSCs cultured under 300, 600, and 900 rpm of the magnetic field was up-regulated 2.0, 5.6, and 5.9 folds, respectively, while the expression of MAP2 was up-regulated 2.4, 7.7, and 8.1 folds, respectively. The above results show that the higher rotation speed of the magnetic field promoted more expression of neural markers as it could induce and apply a stronger electrical stimulation to the NSCs. Considering the stability of the homemade setup of the periodic magnetic field, 600 rpm was used in the subsequent experiments when the magnetic field is applied.

We subsequently conducted a methodical examination of the impact of electrical stimulation generated on the wireless nanopatches on the neuronal differentiation of NSCs. We evaluated the differentiation of NSCs without the nanopatches nor the rotating magnetic field (abbreviated as Ctrl), the NSCs without nanopatches but with the 600-rpm rotating magnetic field (abbreviated as MF only), the NSCs with 60 μg mL−1 of nanopatches but without rotating magnetic field (abbreviated as Nanopatch only), and the NSCs with 60 μg mL−1 of nanopatches and 600-rpm rotating magnetic field (abbreviated as Nanopatch+MF) by RT-qPCR, immunostaining, and Western Blot. Figure 3c and Supplementary Fig. 11 show the RT-qPCR results at days 5 and 10. The Tuj1, MAP2, and GFAP expression of NSCs in the MF only group did not change significantly when compared to the control group, indicating that the neuronal differentiation of NSCs is not affected by the presence of a magnetic field. The Tuj1 expression of cells in the Nanopatch only group increased 1.9 folds and 3.5 folds at day 5 and 10, respectively, compared to the control group, suggesting that nanopatches themselves could slightly promote the neuronal differentiation of NSCs probably due to the good conductivity of the graphene nanosheets28. In sharp contrast, the Tuj1 expression of NSCs in the Nanopatch+MF group increased by 6.4 folds and 7.3 folds at day 5 and 10 while the expression of MAP2 increased by 5.7 folds and 4.6 folds at day 5 and 10 compared to the Ctrl group, respectively. These results above suggest that the nanopatch-mediated wireless electrical stimulation under the rotating magnetic field promoted neuronal differentiation significantly. More importantly, the expression of GFAP in the Nanopatch+MF group decreased by 0.5 folds and 0.7 folds at day 5 and 10 compared to the Ctrl group, respectively, further demonstrating that the nanopatches under a rotating magnetic field promoted the differentiation of NSCs into neurons over neuroglia.

Figure 3d and Supplementary Fig. 12 show immunofluorescence staining results of NSCs in the groups of Ctrl, MF only, Nanopatch only, and Nanopatch+MF at days 5 and 10. The results show that the NSCs in the Nanopatch+MF group displayed a characteristic shape of neurons and had the highest levels of Tuj1 and MAP2 expression among all groups (Supplementary Fig. 13). We further calculated the percentage of Tuj1- and MAP2- positive cells among all the cells based on the immunofluorescence staining results. As shown in Fig. 3e, the ratio of Tuj1-positive cells in the Nanopatch+MF group was about 43%, while this value in the groups of Ctrl, MF only, and Nanopatch only were 16.3%, 16.7%, and 16.5%, respectively. The ratio of MAP2-positive cells in the Nanopatch+MF group was about 41%, which was also much higher than that in the groups of Ctrl (16.8%), MF only (17.2%), and Nanopatch only (18.5%). These results were consistent with the RT-qPCR results in Fig. 3c, further demonstrating that the nanopatch-mediated electrical stimulation promoted the neuronal differentiation of NSCs. For a more comprehensive assessment of neuronal morphology, we performed Sholl analysis by quantifying the complexity of neuronal processes through tallying the intersections between neurites and concentric circles at specified radii from the center of the soma. As shown in Fig. 3f, the Nanopatch+MF group presents a higher neurite complexity and a larger neurite length than the Ctrl group, indicating that it experienced a higher degree of nerve differentiation. The higher expression of Tuj1 and MAP2 as well as the longer development of neurites in the Nanopatch+MF group were also observed when the culture time was increased to 10 days (Supplementary Figs. 14–16). The protein expressions of Tuj1, MAP2, and GFAP were further demonstrated by Western Blot. The Western Blot results in Fig. 3g and h demonstrate that the Tuj1 and MAP2 expressions of NSCs in the Nanopatch+MF group were the highest while the expression of GFAP was the lowest among all the groups, which was in line with the results obtained from qPCR and immunostaining. Taking together, the above results demonstrate that after NSCs were wrapped by the nanopatches, the wireless electrical stimulation produced by the nanopatches greatly enhanced their neuronal differentiation.

Validation of the neuronal function and the related signaling pathway

To verify the neuronal function of differentiated cells, we used a calcium ion fluorescence probe (Fluo-4 AM) to assess the intracellular level of calcium ions in response to the depolarization (Fig. 4a). Fluo-4 AM exhibits fluorescence after combining with intracellular Ca2+. For neurons, the intracellular calcium ions have a much lower concentration than the extracellular calcium ions. Hence, Fluo-4 AM will only give a weak fluorescence in the neurons. After the addition of Potassium chloride (KCl), the extracellular K+ will cause intracellular electronegativity, activating calcium ion channels to allow the influx of Ca2+ and thus increasing the intracellular fluorescence intensity. After being wrapped with the nanopatches and under a magnetic field for 5 days, the differentiated cells were depolarized with KCl (50 mM). Figure 4b shows the fluorescence image of differentiated cells while Fig. 4c shows the corresponding fluorescence intensity analysis of representative cells. Among the 39 counted cells, 13 cells expressed obvious fluorescence changes upon depolarization, indicating that 33% of the differentiated cells expressed functional voltage-dependent calcium channels on the membrane. In addition to depolarization, the differentiated cells also showed a quick response to the stimulation of neurotransmitters including acetylcholine and dopamine, further demonstrating the neural functions of the differentiated cells (Supplementary Figs. 17 and  18). The aforementioned findings suggest that neurons differentiated through the wireless nanopatch-generated electrical stimulation exhibited the characteristic functionality observed in fully developed neurons.

Fig. 4: Verification of cell signaling pathway under the individualized stimulation by the nanopatch-mediated wireless electrical signals.
Fig. 4: Verification of cell signaling pathway under the individualized stimulation by the nanopatch-mediated wireless electrical signals.The alternative text for this image may have been generated using AI.
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a Schematic showing the evaluation of the activation of calcium ion channels using Fluo-4 AM through depolarization with KCl. b Fluorescence image of differentiated NSCs upon stimulation with KCl (50 mM). The differentiated NSCs were obtained after treatment with the wireless electrical nanopatches and 600 rpm-magnetic field for 5 days. The cells showing changes in fluorescence intensity are indicated by the arrows. c The corresponding fluorescence intensity analysis based on the fluorescence results in b. d Volcanic map of the differentially expressed genes between the NSCs in the Ctrl group and the Nanopatch+MF group. The genes that were upregulated in the Nanopatch+MF group are indicated in red, while the downregulated genes are indicated in blue. e Heatmap showing the differential expressed gene of the NSCs in the Ctrl and Nanopatch+MF groups. Genes that are upregulated in the Nanopatch+MF group are represented in red, while genes that are downregulated are represented in blue. f RT-qPCR results of representative genes expressed differently in e. Data represent three independent experiments and are presented as the mean ± standard deviation. g The KEGG pathway terms of genes that are expressed differentially. h Western bolt results of ERK, P-ERK, and EGR1 of NSCs in Ctrl, MF only, Nanopatch only, and Nanopatch+MF groups at day 5. i The analysis of gray values of western blotting results in h. Data represent three independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (***p < 0.001 compared to the Ctrl group).

To gain insight into the underlying mechanisms that contribute to neuronal differentiation of NSCs induced by the nanopatch-mediated electrical stimulation, we examined the transcriptomic changes in NSCs between the Ctrl and Nanopatch+MF groups by RNA sequencing analysis (RNA-seq). The differentially expressed genes (DEGs) are shown in Fig. 4d. Among the DEGs, we found that the genes involved in the NSCs differentiation were changed significantly. As shown in Fig. 4e, for cells anchored with the nanopatches and treated with the magnetic field, the genes capable of promoting neuronal differentiation, such as EGR1, Neuregulin 1 (Nrg1), Wnt2, sonic hedgehog (Shh), and so on, were upregulated, while gliogenesis-related genes such as hes family BHLH transcription factor 5 (Hes5), G protein-coupled receptor 17 (Gpr17), sex determining region Y-box transcription factor 8 (Sox8), and sex-determining region Y-box transcription factor 10 (Sox10) were downregulated, compared to the Ctrl group. We then used RT-qPCR to further evaluate the expression levels of upregulated and downregulated DEGs (Fig. 4f). Among the above genes, the early growth response protein 1 (EGR1) showed the most significant increase in expression, which was 20.2 folds compared to the Ctrl group. As a strong early neurogenic transcription factor, EGR1 has been reported to be identified as a mediator in neuronal differentiations29. Based on the differential gene expression analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed. As shown in Fig. 4g, the DEGs were enriched in the pathways associated with neural differentiation including Axon guidance and Glutamatergic synapse, as well as the electrical stimulation-related pathways including the mitogen-activated protein kinase (MAPK) signaling pathway, ECM-receptor interaction, and Calcium signaling pathway. These results were consistent with previous reports that electrical stimulation can activate the MAPK signaling pathway to promote neural differentiation30. More importantly, EGR1 is usually involved in the MAPK/Extracellular regulated protein kinases (ERK) cascade signaling pathway in regulating the cell fate31. Considering the up-regulated expression of EGR1 and its critical role in the MAPK/ERK pathways, we selected EGR1 as the target gene for evaluating the pathways involved in the nanopatch-induced neuronal differentiation using Western Blot (Fig. 4h). As shown in Fig. 4i, the expressions of EGR1 and phosphorylation of ERK (P-ERK) in the Graphene+MF group was increased 2.6 folds and 1.8 folds, respectively, indicating that the ERK signaling pathway in the Nanopatch+MF group was activated and promoted the expression of EGR1. The above results suggest that the wireless nanopatch-generated stimulation promoted neuronal differentiation through the activation of the MAPK/ERK signaling pathway and EGR1.

Nanopatch-mediated wireless electrical stimulation for TBI therapy

The above results indicate that wireless electrical stimulation produced on the nanopatches promoted the neuronal differentiation of NSCs wrapped by them. The nanopatch-wrapped NSCs were then injected into the TBI mice for the repair of brain tissues. Figure 5a shows the schematic illustration of using the nanopatch-wrapped NSCs for the TBI treatment. After 3 days of adaptive training, the mice received stainless-steel hit injury to create the TBI model, followed by transplantation of the nanopatch-wrapped NSCs or unwrapped NSCs into the lesion site. Then a 600-rpm rotating magnetic field was applied to the lesion site 30 minutes per day for 14 days. Mice who underwent identical procedures without stainless-steel hit injuries were used as control (Sham).

Fig. 5: Behavioral analysis of TBI mice after treatment with nanopatch-wrapped NSCs that are individually stimulated by a rotating magnetic field.
Fig. 5: Behavioral analysis of TBI mice after treatment with nanopatch-wrapped NSCs that are individually stimulated by a rotating magnetic field.The alternative text for this image may have been generated using AI.
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a Schematic illustration showing the use of the nanopatch-mediated wireless electrical stimulation for the treatment of TBI mice. The NSCs were wrapped by the nanopatches and implanted into the lesion site together with the NSCs. A rotating magnetic field was then applied 30 min a day from outside the body to individually stimulate the implanted NSCs electrically. b Images showing the path taken during the orientation navigation trails and spatial exploration trials of MWM of the mice in Sham, TBI, TBI+NSCs, TBI+NSCs+MF, TBI+NSCs+Nanopatch, and TBI+NSCs+Nanopatch+MF groups at day 28. c Frequency of platform crossing examined during the probe trial of the mice in Sham, TBI, TBI+NSCs, TBI+NSCs+MF, TBI+NSCs+Nanopatch, and TBI+NSCs+Nanopatch+MF at day 28 based on the images in b. Data represent nine independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, and ****p < 0.0001 compared to the TBI group). d Time spent in the target quadrant in the probe trial of the mice in Sham, TBI, TBI+NSCs, TBI+NSCs+MF, TBI+NSCs+Nanopatch, and TBI+NSCs+Nanopatch+MF at day 28 based on the images in b. Data represent nine independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, and ****p < 0.0001 compared to the TBI group). e NORT performances of the mice in Sham, TBI, TBI+NSCs, TBI+NSCs+MF, TBI+NSCs+Nanopatch, and TBI+NSCs+Nanopatch+MF at day 28. Data represent nine independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (*p < 0.05, ***p < 0.001, and ****p < 0.0001 compared to the TBI group). f Evaluation of motor coordination and balance of the mice in Sham, TBI, TBI+NSCs, TBI+NSCs+MF, TBI+NSCs+Nanopatch, and TBI+NSCs+Nanopatch+MF at day 28. Data represent nine independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the TBI group).

Mice with TBI exhibit common behavioral abnormalities as a result of impaired motor skills and memory. To evaluate the recovery of the TBI, we assessed the motor and memory abilities of the mice 28 days post-surgery by behavioral tests including the rotarod, novel object recognition test (NORT), and the morris water maze (MWM) test. Figure 5b shows the trajectory of mice after receiving different treatments. The movements of TBI mice implanted with NSCs anchored with the nanopatch and treated with the magnetic field (TBI+NSCs+Nanopatch+MF group) in the orientation navigation trials were effective and effortless. In the spatial exploration trials, the trajectory of these mice was kept around the hidden platform, which was similar to the Sham group, indicating the recovery of TBI. The frequency of cross in the hidden platform in the TBI+NSCs+Nanopatch+MF group was 5.3 while these values were 1.5, 3.3, 3.5, and 3.4, respectively, for the TBI mice (TBI), TBI mice implanted with unwrapped NSCs (TBI+NSCs), TBI mice implanted with the unwrapped NSCs but treated with the magnetic field (TBI+NSCs+MF), and TBI mice implanted with the nanopatch-wrapped NSCs but without treatment with the magnetic field (TBI+NSCs+Nanopatch) (Fig. 5c). The length of time in the section of the concealed platform in the TBI+NSCs+Nanopatch+MF group was 2.7 folds compared to the TBI group (Fig. 5d). The statistical results for the frequency of cross in the hidden platform and the length of time in the section of the concealed platform show that the mice in the TBI+NSCs+Nanopatch+MF group presented superior memory skills compared to other TBI mice including those implanted with unwrapped NSCs. The mice in the TBI+NSCs+Graphene+MF group also showed significant concern for novel objects in the NORT test (Fig. 5e). The exploration time of these mice was 2 folds and 1.4 folds, in contrast to the TBI group or TBI+NSCs group, suggesting the cognitive abilities related to learning and memory in mice were improved after treatment with the nanopatch-wrapped NSCs that enabled individualized electrical stimulation under a rotating magnetic field. Furthermore, the rotarod test shows that the time of latency to fall in the TBI+NSCs+Graphene+MF group was 2.7 folds longer than the TBI mice, indicating an improvement in motor function (Fig. 5f). The above behavioral test results demonstrate that transplantation of the nanopatch-wrapped NSCs into the TBI mice under the 600-rpm rotating magnetic field improved the motor and memory recovery of the mice.

To observe the recovery of the injured brain tissue, the mice were euthanized, and brain tissues were harvested at 28 days post-surgery. Figure 6a shows digital pictures of the brains of the mice under different treatments. The mice after TBI treatment exhibited significant tissue loss. Compared to the TBI mice in other groups, the mice transplanted with NSCs anchored with the nanopatches and treated with the magnetic field (TBI+NSCs+Nanopatch+MF) showed the most complete tissue. In the TBI+NSCs+Nanopatch+MF group, the volume of tissue loss in cortical lesions was reduced by 3.5 folds compared with the TBI group (Fig. 6b). Figure 6c shows the Nissl staining of the sectioned tissues from mice brain. Compared to the Sham group, the tissue cavities were significantly increased and the density of Nissl was significantly decreased in the TBI group due to the injury of the brain tissue. Implantation of unwrapped NSCs led to a slight reduction in the cavities and a slight increase in Nissl density. However, implantation of the wrapped NSCs and exposure to the rotating magnetic field significantly reduced the cavities and improved the Nissl intensity. The above results indicate that wireless electrical stimulation generated on the nanopatches wrapping individual NSCs could significantly enhance the recovery of the injured brain tissue in the NSC-based therapy.

Fig. 6: Brain tissue repair of TBI mice after treatment with nanopatch-wrapped NSCs that are individually stimulated by a rotating magnetic field.
Fig. 6: Brain tissue repair of TBI mice after treatment with nanopatch-wrapped NSCs that are individually stimulated by a rotating magnetic field.The alternative text for this image may have been generated using AI.
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a Digital pictures of the mice brain in Sham, TBI, TBI+NSCs, TBI+NSCs+MF, TBI+NSCs+Nanopatch, and TBI+NSCs+Nanopatch+MF groups at day 28. b Percentage of tissue loss in injured site obtained by referring to the pictures in a. Data represent six independent experiments and are presented as the mean ± standard deviation, as analyzed by a one-way ANOVA, with Tukey’s multiple comparisons test (***p < 0.001 compared to the TBI group). c Nissl staining of sectioned brain tissues of the mice in Sham, TBI, TBI+NSCs, TBI+NSCs+MF, TBI+NSCs+Nanopatch, and TBI+NSCs+Nanopatch+MF groups at day 28. d Immunofluorescence images of DCX of sectioned brain tissues of the mice in Sham, TBI, TBI+NSCs, TBI+NSCs+MF, TBI+NSCs+Nanopatch, and TBI+NSCs+Nanopatch+MF groups at day 28. DCX was stained into green. The cell membrane was pre-stained with PKH 26 to red. The nuclei were labeled with DAPI. e Immunofluorescence images of NeuN of the sectioned brain tissues of the mice in Sham, TBI, TBI+NSCs, TBI+NSCs+MF, TBI+NSCs+Nanopatch, and TBI+NSCs+Nanopatch+MF groups at day 28. NeuN was stained into green. The cell membrane was pre-stained with PKH 26 to red. The nuclei were stained into blue by DAPI.

To further confirm the performance of the nanopatch-wrapped NSCs in the recovery of the TBI, we investigated the formation of newborn neurons (DCX-positive) and mature neurons (NeuN-positive) in the injured area of the cortex by immunofluorescence staining. The transplanted NSCs were pre-stained with PKH26 to give red fluorescence. As shown in Fig. 6d, Supplementary Fig. 19, Fig. 6e, and Supplementary Fig. 20, few DCX- and NeuN-positive neurons coincided with PKH26-stained NSCs at the lesion sites in the TBI+NSCs group and TBI+NSCs+MF group, indicating most of the implanted stem cells did not differentiate into neurons. In the TBI+NSCs+Nanopatch group, a little more DCX- and NeuN-positive cells merged with the PKH26-stained NSCs, indicating that nanopatches themselves slightly promoted the neuronal differentiation of the transplanted NSCs, which was consistent to the in vitro results (Fig. 3c) and previous report30. In sharp contrast, in the TBI+NSCs+Nanopatch+MF group, the mice showed the highest degree of merge of the DCX- and NeuN-positive cells with the PKH26-stained NSCs. The above results indicate that the nanopatch-enabled individualized electrical stimulation could enhance the differentiation of implanted stem cells in the lesion site, promoting the recovery of TBI.

To verify the biodegradability and biocompatibility of graphene in vivo, the brain tissues were harvested at day 30 post-surgery. The Raman results in Supplementary Fig. 21 show that graphene nanosheets with a typical Raman spectrum pattern of graphene were found in the bran tissues at day 30 post-surgery, indicating that graphene nanopatches did not degrade significantly in vivo within 30 days. The immunofluorescence staining and RT-qPCR results in Supplementary Fig. 22a and b show that inflammation and glial activation in the TBI+NSCs, TBI+NSCs+MF, TBI+NSCs+Nanopatch, and TBI+NSCs+Nanopatch+MF groups were reduced compared with the TBI group at day 30. Among all the groups, TBI+NSCs+Nanopatch+MF group showed the least inflammation and glial activation probably due to the enhanced neuronal differentiation of NSCs in this group. The TBI+NSCs+Nanopatch shows similar inflammation and glial activation in the injury site compared to the TBI+NSCs group, indicating that graphene nanosheets did not cause inflammation and glial activation in vivo. In addition, the graphene nanopatches also did not cause the M1 activation of BV2 microglia cells in vitro (Supplementary Fig. 22c). The above results demonstrate that although the graphene nanopatches did not degrade significantly in vivo within 30 days, they had a good biocompatibility in vivo, which ensures the future translation of the wireless electrical nanopatches for treatment of TBI.

Discussion

Traumatic brain injury remains a substantial medical challenge due to its limited regenerative capacity and long-term cognitive and motor deficits in affected individuals. NSCs have shown promise in treating TBI as they possess the potential to differentiate into functional neurons, replacing damaged ones and rebuilding neuronal circuits7. However, when implanted, NSCs often differentiate into astrocytes in the injured microenvironment, limiting their therapeutic effect on TBI. Strategies involving neurotrophins, hormones, small molecule drugs, or their combinations are effective for inducing neuronal differentiation in vitro but face limitations such as easy degradation, uncontrolled distribution, and delivery issues for in vivo application10,11,12. Neural network activity primarily manifests as electrical activity32. Previous studies have revealed the potential of pulsed electrical stimulation to regulate NSC differentiation using an electroactive material. However, applying electrical stimulation generally requires surgery to implant the electrode or the stimulator, which will induce inflammation and infection. More importantly, the transplanted cells are in a state of dynamic migration in a 3-dimensional environment. Previous reported electrical stimulators are fixed in the brain tissue and could not move together with the dynamically migrating cells and stimulating them effectively33. This study tackled this daunting challenge by always allowing the NSCs to be associated with the stimulating material (the nanopatches) through wrapping individual NSCs with the nanopatches.

COMSOL simulation and theoretical calculations indicated that the electric field intensity on the nanopatches reached 1.26 mV mm−1, sufficient to activate membrane receptors34. It has been reported that electric fields with a intensity of 0.3–1 mV mm−1 can induce electrophysiological reaction35, and electric fields of 0.2-1 mV mm−1 can activate neuronal activity in primates36, rodents37,38, and humans39,40. Thus, the electrical field intensity generated on the nanopatches should be able to stimulate the NSCs. We modified a layer of laminin on their surface before the nanopatches are used to wrap the cell membrane. More importantly, the size (2–5 μm wide) and the morphology enabled the nanopatches to avoid cell internalization22,23, allowing them to wrap the cell membrane for over 7 days, which would ensure a long-term and stable stimulation to the stem cells.

Biocompatibility tests confirmed that the wireless electrical nanopatches exhibited good compatibility with NSCs after wrapping them. A lab-made setup, consisting of a rotating neodymium-iron-boron permanent magnet connected to an agitator motor, was used to provide the changing magnetic field to drive the nanopatches to generate electrical stimulation for enhancing the neuronal differentiation of NSCs. Through evaluation of the expression of neuron-associated genes and proteins, we confirmed that the nanopatch-mediated electrical stimulation resulted in 41% of NSCs differentiating into functional neurons, compared to 16.3% without nanopatches within only 5 days. We also verified that the differentiated cells showed typical functions of neurons, including the ability to respond to the depolarization of KCl and the stimulation of neurotransmitters (e.g., acetylcholine and dopamine). Furthermore, we demonstrated that the MAPK/ERK signaling pathway and its key associated protein EGR1 were activated during nanopatch-mediated wireless electrical stimulation.

We then implanted the NSCs anchored with the wireless electrical nanopatches to the lesion site in TBI mice. Since the nanopatches are always associated with the NSCs even when they are dynamically migrating post transplantation, the electrical stimulation is always applied directly to the individual NSCs under a rotating magnetic field placed outside the animal body. As a result, the nanopatch-mediated wireless electrical stimulation promoted the differentiation of these individual nanopatch-wrapped NSCs to be differentiated into new neurons in vivo. Hence, compared to the unwrapped NSCs, the mice treated with the nanopatch-wrapped NSCs showed significant improvement in brain tissue repair as well as the recovery of the behavioral and cognitive abilities under a periodic magnetic field. The regulation of stem cell differentiation towards mature neurons holds significant implications beyond the context of TBI. The wireless electrical nanopatch-based technology should also have promising applications in conditions such as stroke, spinal cord injury, and other neurodegenerative diseases.

It is worth mentioning that although it has been reported that the changing magnetic field itself could electrically stimulate the neuronal differentiation of stem cells, the stimulation either requires a long period time (4 h/d) or large magnetic field strength (0.83 T). Under such intensive stimulations, the ratio of Tuj1-positive cells was only increased 1.6 folds41,42,43. In contrast, the wireless electrical nanopatches we proposed only require a short period of time (15 min/d) at a low magnetic field strength (0.16 T) and the ratio of Tuj1-positive cells was increased 2.6 folds. More importantly, directly using high intensity of changing magnetic field will apply electrical stimulation to all tissues and cells exposed to the magnetic field, which may cause side effects. In comparison, our wireless electrical nanopatches were wrapped on the membrane of stem cells, which could precisely stimulate the implanted cells individually. To this end, the wireless electrical nanopatches are more effective and reliable for promoting neuronal differentiation of injected stem cells for treatment of TBI and other neurodegenerative diseases.

In summary, we have prepared a type of wireless electrical nanopatches that could wrap the cell membranes and generate wireless electrical stimulation to enhance the differentiation of NSCs into functional neurons. Transplantation of the wrapped NSCs enhanced the repair of the injured brain tissue under a changing magnetic field because the stimulating material is always wrapping and thus stimulating the NSCs individually even when the NSCs are migrating post transplantation. The wireless electrical nanopatches hold promise in stem cell-based therapy for nerve injuries and neurodegenerative diseases. It is important to note that our current setup for supplying the changing magnetic field could only provide a maximum magnetic field of 160 mT with a maximal frequency of 15 Hz due to the limitation of the permanent magnet and rotating motor. In future experiments, alternative setups, such as transcranial magnetic stimulation equipment, may be used in conjunction with wireless electrical nanopatches.

Methods

Materials

Bulk graphite was purchased from XFNano (China). Calcein AM and propidium iodide (PI) were obtained from Sigma-Aldrich (USA). Neurobasal medium, glutaMAXTM−1, and Bmur27 supplement (B27) were obtained from Gibco (USA). Fetal bovine serum (FBS) and BV2 cell specialized medium were purchased from Procell (China). Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were obtained from PeproTech (USA). Fluo-4 AM and BCA Protein Assay Kit were purchased from Beyotime (China). Bovine serum albumin (BSA), PBS, paraformaldehyde, Triton X-100, Rhodamine Phalloidin, RIPA lysis buffer, and ECL plus reagents were obtained from Solaribio (China). The primary antibodies targeting Tuj1, MAP2, GFAP, and MBP, as well as the secondary antibodies were sourced from Abcam (UK). SARS-CoV-2 protein and Anti-sars-cov-2 monoclonal antibody were purchased from Bioantibody (China). CCK-8, DAPI, and penicillin-streptomycin were purchased from Biosharp (China). Trizol reagent was obtained from Cwbio (China). The RT-qPCR primers for Tuj1, MAP2, GFAP, Iba1, and iNOS were purchased from Biosune (China).

Synthesis and characterization of graphene nanosheets

The graphene nanosheets were fabricated through electrochemical exfoliation of bulk graphite in a two-electrode system. The working electrode, counter electrode, and electrolyte in this experiment were bulk graphite, platinum foil, and 0.1 M sulfuric acid, respectively. The electrochemical exfoliation was performed at +10 V under the magnetic stirring for 10 min. After filtering through a mixed cellulose ester membrane with a pore size of 0.22 μm, the graphene nanosheets were collected and washed with deionized (DI) water 3 times. Afterward, the samples were dispersed in 100 mL of DI water under sonication to form a suspension of graphene nanosheets. After the suspension stood for 48 h, the supernatant containing dispersed graphene nanosheets was collected. The suspension was centrifuged at 6000 rpm to remove the supernatant. The precipitated graphene is collected and redispersed for future use. The AFM (Icon, Bruker, USA) and Raman spectrometer (LabRAM HR Evolution, France) were used to analyze the morphology and structure of graphene nanosheets.

Synthesis and characterization of graphene nanosheets with different sizes

The graphene nanosheets were fabricated through electrochemical exfoliation. The samples were dispersed in 100 mL of DI water under sonication to form a suspension of graphene nanosheets. Different sizes of graphene nanosheets were obtained through centrifugation of the graphene nanosheet suspension at 112 and 4025 g sequentially. The precipitation and supernatant graphene nanosheets were collected after each centrifugation. The AFM (Icon, Bruker, USA) was used to analyze the morphology and size of graphene nanosheets.

Wirelessly electrical stimulation setup

The rotating magnetic field was generated by the Neodymium-iron-boron permanent magnet connected to an agitator motor at different rotation speeds. Two magnets were placed on the agitator and rotated along with the motor shaft to generate a periodic magnetic field. A plastic bracket with a distance of 0.8 cm from the magnet was installed above the magnet to support the culture plate. The magnetic field strength was measured using the CH-1800 gaussmeter (Reanow, China) at a distance of 0.8 cm above the magnet.

Calculation of motional electromotive force

The induced voltage of an individual graphene nanosheet was determined using the principles of electromagnetic laws:

$$\varepsilon={BVL}$$
(1)

where ε is the induced voltage, B is the magnetic field intensity, V is the relative velocity of the magnetic field, and L is the length of graphene nanosheets.

For a graphene nanosheet with a length of 5 μm (L = 5 μm) placed in magnetic field of 160 mT (B = 160 mT) rotating at a 600 rpm (V = 6.6 m s−1), the generated maximal voltage is 6.3 × 10−3 mV. The electric field intensity is equal to 1.26 V m−1.

COMSOL simulation of the electrical signals generated on a graphene nanosheet

COMSOL Multiphysics software was used to simulate finite element analysis (FEA) with perfect matching layer (PML) as a boundary condition. Electromagnetic-rotating magnetic machinery (RMM) module was used in COMSOL Multiphysics® (COMSOL Inc.). The material properties of graphene nanosheets were used in reference to the built-in material library. In this model, the graphene nanosheets were simplified as a stack of seven prisms with side lengths of 2–5 μm. A cylindrical magnet with a magnetic field strength of 160 mT was placed 1 cm away from the graphene nanosheets. It was then rotated at a speed of 600 rpm around its central axis in order to intersect the magnetic induction lines and produce an electromotive force. The graphene nanosheets were divided into small cells along their boundaries, with a maximum cell size of 3.5 nm and a minimum cell size of 0.24 nm.

Measurement of the electrical signals generated on the graphene film

The graphene film with a diameter of 1 cm was obtained by draining 5 mL of graphene nanosheets dispersion (1 mg mL−1) on a filter. Subsequently, an ITO film was covered on the filter, followed by applying 1 MPa of pressure. The graphene nanosheets on the filter was then attached to the ITO film. The oscilloscope (Keysight infiniivision dsox3034t, USA) was used to detect the voltage of the graphene film at a distance of 0.8 cm from the magnet. The measurements were taken at various rotating speeds of 0, 100, 200, 300, 400, 500, and 600 rpm.

NSCs culture

The NSCs were obtained in 12–14 days gestation mouse embryos (C57/BL6 strain) (Beijing Vital River Laboratory Animal Technology, China) according to the previous protocol10. The proliferation medium was the neurobasal medium, B27 supplement (2%), 20 ng mL−1 of mEGF, 20 ng mL−1 of mbFGF, GlutaMAXTM−1 (1%), and penicillin/streptomycin (1%). The NSCs were kept in a controlled environment with a humidity level and 5% CO2 concentration at 37 °C. The differentiation medium was the neurobasal medium with B27 supplement (2%), FBS (1%), glutaMAXTM−1 (1%), and penicillin/streptomycin (1%).

Anchoring the graphene nanosheets to NSCs

To facilitate the observation under fluorescence microscopy, the graphene nanosheets were pre-labeled with a fluorescence dye through the antigen-antibody reaction. The antigen-antibody attached to the graphene nanosheet could reduce the quenching of the fluorescence of the dye44. Briefly, 2 μL of the commercial recombinant SARS-CoV-2 protein (1 mg mL−1) was added into the 1 mL of an aqueous suspension of graphene nanosheets (1 mg mL−1), followed by stirring for 2 h. Afterwards, the samples were washed three times with DI water through centrifugation at 21913 g and then incubated with 2 μg mL−1 of primary antibody (Anti-SARS-CoV-2 monoclonal antibody) at 4 oC overnight. The samples were then dispersed in a solution containing a fluorescent secondary antibody at a concentration of 2 μg mL−1. After incubation for 2 h at room temperature, the fluorescently labeled graphene nanosheets were obtained after washing with water twice through centrifugation (21913 g). Subsequently, 2 μL of laminin (1 mg mL−1) was added to 1 mL of the suspension of graphene (1 mg mL−1) and incubated at 37 oC for 3 h. The laminin coated graphene nanosheets were used as the wireless electrical nanopatches to wrap the cells and apply the stimulation.

The NSCs were also pre-labeled with the cell membrane dye (PKH67) and nuclei dye (Hoechst). Afterwards, 120 μL of fluorescence-labeled graphene nanosheets (1 mg mL−1) were added to 2 mL of the cell suspension containing 2 × 106 of cells for incubation (3 h). Afterwards, the un-anchored graphene nanosheets were removed through centrifugation at 72 g for 3 min. The nanopatch-wrapped NSCs were re-dispersed in PBS for observation under the Laser confocal microscope (TCS SP8 confocal microscope, Leica).

For SEM imaging, the nanopatch-wrapped cells were treated with 2.5% glutaraldehyde at room temperature for 30 min, followed by dehydration with different concentrations (30%, 50%, 70%, 80%, 90%, 95%, 100%) ethanol through centrifugation (72 g). The samples were freeze-dried using a lyophilizer before observed under SEM (ZEISS Gemini SEM 500).

Cell viability test

For Live/Dead staining, 5 × 105 of wrapped or unwrapped NSCs were seeded in 24-well plates. To apply the wirelessly electrical stimulation, the cells were exposed to the changing magnetic field provided by the permanent magnet (160 mT) on a rotator (600 rpm) at a distance of 0.8 cm for 15 min every day. The cells without magnetic field treatment served as control. 300 μL of neurobasal medium with calcein-AM (0.5 μM) and propidium iodide (PI, 3 μM) was added, after culturing for 48 h. Subsequently, the samples were incubated at 37 °C for 30 min. The CLSM (Zeiss Co, Germany) was used to observe the samples. The software Image J was used to quantify live and dead cells in three selected images from each group, in order to determine the survival rate. For CCK-8 test, 1 × 105 of wrapped or unwrapped NSCs were cultured in the 96-well culture plates. To apply the wireless electrical stimulation, the cells were exposed to the changing magnetic field for 15 min every day, for 1, 2, and 3 days, respectively. The cells without the magnetic field treatment served as control. Afterwards, 10 μL of CCK-8 solution was added into each well, and then incubated for 1 h at 37 oC. The microplate reader (Multiscan MK3, Thermo, USA) was used at the absorption wavelength of 450 nm to measure the concentration of formazan dye. Three independent replicates were performed for each data point.

Immunofluorescence staining

5×105 of wrapped or unwrapped NSCs were seeded in 24-well plates. To apply the wireless electrical stimulation, the cells were exposed to the changing magnetic field for 15 min every day, for 5 and 10 days, respectively. The cells without magnetic field treatment served as a control. After washing three times with PBS, the NSCs were immobilized with paraformaldehyde (4%) for 20 min at room temperature. The samples were treated with Triton X-100 (0.1%) for 10 min to permeabilization, followed by blocking with bovine serum albumin (BSA, 1%) for 30 min at room temperature. Subsequently, the cells were subjected to incubation with primary antibodies (Tuj1, Nestin, MAP2, and GFAP) that were diluted at specific ratios (800 times for Tuj1, Nestin, and MAP2, and 1000 times for GFAP) in a solution containing 1% BSA. This incubation took place overnight at a temperature of 4 oC. Subsequently, a secondary antibody (diluted 1000 times) was applied to the cells for 1 hour at room temperature. Following this, the cells were washed three times with PBS and then stained with a concentration of 1 μg mL−1 of DAPI for a period of 5 min in order to label the nuclei. Leica SP8 STED was used to observe the samples. We employed Image J for a semi-quantitative analysis of the fluorescence intensity associated with Tuj1 and MAP2 proteins.

Cytoskeleton staining

5 × 105 of wrapped or unwrapped NSCs were seeded in 24-well plates. To apply the wireless electrical stimulation, the cells were exposed to the changing magnetic field for 15 min every day. The wireless electrical stimulation lasted for 5 days. The cells without the magnetic field treatment served as a control. After washing three times with PBS, the NSCs were immobilized with paraformaldehyde (4%) for 20 min at room temperature. Subsequently, the samples were treated with Triton X−100 (0.1%) for 10 min to permeabilization, followed by blocking with bovine serum albumin (BSA, 1%) for 30 min. Following this, the samples were exposed to rhodamine-labeled phalloidin (diluted 1000 times) for 1 hour. Subsequently, the samples were treated with 1 μg mL−1 of DAPI for 5 min. CLSM was used to observe the samples.

RT-qPCR

1 × 106 of wrapped or unwrapped NSCs were seeded in 12-well plates. To apply the wireless electrical stimulation, the cells were exposed to the changing magnetic field for 15 min every day, for 5 and 10 days, respectively. The cells without the magnetic field treatment served as a control. The NSCs were subjected to Trizol reagent for extracting total RNA. The Q-5000 spectrophotometer (Quawell, Q-5000, America) was used to assess the concentration and purity of the extracted RNA. The RT-qPCR analysis was conducted using the LightCyler@96 SW 1.1 software to evaluate the levels of the gene expression, the housekeeping gene, actin, as well as the four genes Tuj1, GFAP, and MAP2 (refer to Supplementary Table. 1). The levels of gene expression for the target gene were standardized by normalizing them to the expression of actin. Each data point was obtained from three parallel replicates.

2 × 105 of wrapped or unwrapped BV2 were seeded in 12-well plates. The cells without the nanopatches wrapped served as a control. The RT-qPCR analysis was conducted using the LightCyler@96 SW 1.1 software to evaluate the levels of the gene expression, the housekeeping gene, actin, as well as the four genes IL-1β and iNOS (refer to Supplementary Table. 1). The levels of gene expression for the target gene were standardized by normalizing them to the expression of actin. Each data point was obtained from three parallel replicates.

Western blot

2 × 106 of wrapped or unwrapped NSCs were seeded in 6-well plates. To apply the wireless electrical stimulation, the cells were exposed to the changing magnetic field for 15 min every day, for 5 days. The cells without the magnetic field treatment served as a control. Cellular total protein extraction was performed using the radioimmunoprecipitation assay (RIPA) lysis buffer. We utilized the BCA protein assay kit to determine the protein concentration. The protein samples were subsequently subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and thereafter, the proteins were transferred from the gel to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with skim milk powder at room temperature for 1 h and then incubated with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. The primary antibodies were diluted 1000 times for MAP2, CaMKII, P-CaMkII, CREB, P-CREB, ERK, P-ERK, and EGR1, 5000 times for GAPDH and Tuj1, 5000 times for GFAP. The samples underwent the treatment with ECL plus reagents and observed using the chemiluminescence system (Vazyme, China). The level of the proteins was semi-quantitatively analyzed using the Image J software.

Calcium imaging

5 × 105 of wrapped or unwrapped NSCs were seeded in 24-well plates. To administer wireless electrical stimulation, the cells were subjected to a changing magnetic field for a duration of 15 minutes daily, over the course of 5 consecutive days. After three washes with PBS, the cells were exposed to a prepared solution of Fluo-4 AM (500 μL, 2 μg mL−1) for 10 min at 37 °C. Following this, the samples underwent three additional PBS washes and were then subjected to incubation with KCl (50 mM) to depolarize the differentiated cells. Changes in fluorescence were observed and analyzed using CLSM and the Image J. Moreover, to assess the presence of neurotransmitter receptors in the differentiated cells, various neurotransmitters (acetylcholine at a concentration of 90.8 mg mL−1, γ-aminobutyric acid at 51.6 mg mL−1, L-glutamic acid at 73 mg mL−1, and dopamine at 94.8 mg mL−1) were used to stimulate the cells, and the resulting fluorescence changes were observed using CLSM and analyzed with the Image J.

RNA sequencing analysis

2 × 106 of wrapped or unwrapped NSCs were seeded in 24-well plates. To apply the wireless electrical stimulation, the cells were exposed to the changing magnetic field for 15 min every day, for 5 days. The cells without the wireless electrical nanopatches and magnetic field treatment served as a control. The NSCs were subjected to Trizol reagent to extract the complete RNA. The RNA-seq analysis was performed using a BGISEQ-500 instrument from Beijing Genomic Institution (www.genomics.org.cn, BGI). Each group was represented by three independent replicates.

TBI mouse experiments

A mouse traumatic brain injury model was established by following the previous protocol45. Briefly, adult male C57BL6/J mice (22–25 g, n = 60) provided by Beijing Vital River Laboratory Animal Technology were randomly divided into 6 groups: Sham, TBI, TBI+NSCs, TBI+NSCs+MF, TBI+NSCs+Nanopatch, and TBI+NSCs+Nanopatch+MF groups. Each group was replicated nine times in parallel.

The mice were anesthetized with isoflurane, then fixed on the standard brain stereotactic instrument (68507, RWD, China). A piece of skull over the right parietal cortex was excised with a drill, exposing a dural operating window with a diameter of about 3 mm (2.0 mm posterior from bregma and 2.0 mm lateral to the sagittal suture). For the Sham group, mice underwent identical procedures without stainless-steel hit injury. For the TBI group, a 40 g of stainless-steel containing a 2.5-mm diameter tip was released from a height of 20 cm to hit the cortex with a depth of 3.0 mm (68093, RWD, China), followed by injecting PBS (2 μL) into the injury cortex. For the TBI+NSCs group, 2 μL of medium containing 2 × 105 of NSCs were injected into the injury cortex. Cell viability and concentration were quantified by trypan blue on Countstar BioTech before transplantation. For the TBI+NSCs+MF group, 2 μL of medium containing 2 × 105 of NSCs were injected into the injury cortex, followed by treatment with the changing magnetic field. For the TBI+NSCs+Nanopatch group, 2 μL of medium containing 2 × 105 of the wrapped NSCs were injected into the injury cortex. For the TBI+NSCs+Nanopatch+MF group, 2 μL of medium containing 2 × 105 of the wrapped NSCs were injected into the injury cortex, followed by treatment with the changing magnetic field. The changing magnetic field was applied through exposing the injured contes to a permanent magnet (160 mT) on a rotator (600 rpm) at a distance of 0.8 cm for 30 min every day for 14 days.

Morris water maze test

Spatial learning and memory ability were evaluated by the Morris water maze (MWM) test, which consisted of a circular pool (120 cm diameter, 40 cm height) filled with water. The tank was divided into four quadrants with a round platform hidden 1 cm beneath the surface of the water in one of the quadrants and indicated by prominent extra maze cues. The mice were given a maximum of 60 sec to locate the platform during each trial. If a mouse could not locate the platform within the specified time limit, it was gently guided to the platform. Subsequently, all mice were granted a 30-second period on the platform. The mice were introduced into random quadrants and given 1 min to locate the platform, with 4 such trials taking place each day for a span of 5 days. On day 6, a probe trial was conducted after removing the platform. The research team analyzed the swimming path chosen to reach the platform, the duration spent in the designated target quadrant, and the time taken to traverse the platform.

Novel object recognition test

The experiments were performed as described previously46. Memory retention was assessed by the Novel object recognition test. The test included three stages: habituation, training, and test. The equipment included a square area measuring 35 cm by 35 cm, with sidewalls that were 25 cm in height. In the training stage, two objects that looked exactly the same were positioned at the left and right corners. Mice were given the freedom to explore for a duration of 10 min in the field. For the testing stage, one familiar object was changed to the other novel object, and the time spent with the novel object was recorded in 10 min. The discriminating index was calculated using the following equation:

$${Di}=\frac{{Tn}}{{Tn}+{Tf}}\times 100\%$$
(2)

where Di is the discriminating index, Tn is the time spent with the novel object, and Tf is the time spent with the familiar object.

Rotarod test

To evaluate the recovery of the motor function of the TBI mice, the rotarod test was performed. Before testing, animals underwent three days of adaptation training in the rotarod apparatus. During the testing, mice were positioned on a rotating rod, and the apparatus speed was progressively increased from 4 r/min to 40 r/min over the course of 60 sec. Subsequently, this speed was maintained for 5 min. The latency to fall off the rotating rod was recorded three times for repeated testing and the average was calculated as the results. Each group was replicated nine times in parallel.

Nissl staining and Immunofluorescence staining

At 28 days post-surgery, mice were deeply anesthetized, euthanized, and subsequently perfused with physiological saline followed by fixing with paraformaldehyde for 0.5 h. The brain tissue at the injury site was collected approximately 5 mm from the lesion site. The collected tissue was embedded in paraffin and cut into 5 μm sections using a paraffin microtome. For Nissl staining, the sections were deparaffinized, washed in water, and then stained with 0.5% Nissl staining solution for 3 min. The sections were then dehydrated, cleared with xylene, and mounted, followed by observation using an inverted microscope. For immunofluorescence staining, the sections were dewaxed, followed by performing antigen repair. Afterwards, the sections were incubated overnight at 4 oC with primary antibodies (diluted by 100 times for DCX, NeuN, Iba1, and GFAP respectively). After three PBS washes, the tissue sections were subjected to incubation with secondary antibodies at room temperature for 1 h before being stained with DAPI. The CLSM was used to observe the samples. The levels of DCX and NeuN proteins were analyzed semi-quantitatively by assessing the fluorescence intensities with the Image J software.

Biodegradation of graphene nanosheets in brain tissue

In order to assess the degradation of graphene in vivo, 2 μL of PBS containing 12 μg of graphene was injected into mice brain. The coordinates of brain injection are anteroposterior (AP): −1.7 mm, mediolateral (ML): ± 1.5 mm, and dorsoventral (DV): −2.3 mm relative to the bregma. At 30-days post-surgery, mice were deeply anesthetized, euthanized, and subsequently perfused with physiological saline followed by 4% paraformaldehyde for 0.5 h. The brain tissue was embedded in paraffin and cut into 5 μm sections using a paraffin microtome. The sections were deparaffinized and washed in water for the Raman test using a Raman spectrometer (LabRAM HR Evolution, France).

Lesion volume

Cortical lesion volume was measured at day 28 after the TBI surgery. At day 28, the mice were deeply anesthetized and euthanized. The brain tissue at the injury site was collected approximately 5 mm from the lesion site. The collected tissues were continuously coronally sectioned into slices (50 μm thick and approximately 300 μm apart), and subsequently perfused with physiological saline, followed by treatment with 4% paraformaldehyde at room temperature for 30 min. The sections were stained with cresyl violet for histological structure identification and quantified using the software ImageJ.

RT-qPCR of the brain tissue

At 28 days post-surgery, mice were deeply anesthetized, euthanized. The brain tissue at the injury site was collected and subjected to Trizol reagent for extracting total RNA. The Q-5000 spectrophotometer (Quawell, Q-5000, America) was used to assess the concentration and purity of the extracted RNA. The RT-qPCR analysis was conducted using the LightCyler@96 SW 1.1 software to evaluate the levels of the gene expression, the housekeeping gene, actin, as well as the four genes IL-1β and iNOS (refer to Supplementary Table. 1). The levels of gene expression for the target gene were standardized by normalizing them to the expression of actin. Each data point was obtained from three parallel replicates.

Statistical analysis and reproducibility

The data were presented as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software by one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test. Statistical significance was accepted at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. The above information as well as the number of replicates and animals were indicated in each figure legends. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.