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Ultrasound-activated piezoelectric nanostickers for neural stem cell therapy of traumatic brain injury

Nature Materials volume 24, pages 1137–1150 (2025)Cite this article

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Abstract

Traumatic brain injury (TBI) is associated with life-threatening and permanent disabilities. Given the limited capacity of neurons to regenerate, effective treatments for TBI are lacking. Neural stem cells (NSCs) can differentiate into fully functioning neurons and thus hold promise for TBI treatment. Nonetheless, NSC differentiation and proliferation are slow and inefficient. Studies have shown that piezoelectric stimulation is capable of promoting the differentiation and proliferation of NSCs. Here, we describe barium titanate–reduced graphene oxide (BTO/rGO) hybrid piezoelectric nanostickers that promote NSC proliferation and differentiation. These hybrid nanostickers attach to NSC membranes, serving as long-term generators of piezoelectric potentials upon ultrasound stimulation. BTO/rGO nanostickers promote rapid neuronal differentiation and maturation by activating the voltage-gated calcium channel/Ca2+/calmodulin-dependent protein kinase II/cAMP response element-binding protein pathways. Transplantation of NSCs with BTO/rGO nanostickers into the injured brain region of rats with TBI substantially repairs brain tissue and effectively restores physiological functions after 28 d following 5-min ultrasound irradiation every 2 d. These results demonstrate the potential of the combination of NSCs and BTO/rGO nanostickers for TBI treatment.

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Fig. 1: Characterization of BTO nanoparticles and their effects on NSCs.
Fig. 2: Characterization and biocompatibility of BTO/rGO nanostickers.
Fig. 3: BTO/rGO piezoelectric nanostickers promote the expression of neuron-related biomarkers.
Fig. 4: BTO/rGO piezoelectric nanostickers accelerate neuronal maturation and synapse formation.
Fig. 5: BTO/rGO piezoelectric nanostickers activate VGCC and induce intracellular signal transduction.
Fig. 6: NSC-based therapy accelerates recovery of neurological function and tissue regeneration.

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Data availability

The data that support the findings are available within the main text and the Supplementary Information and can be obtained from the corresponding authors upon request. The RNA-Seq experiment data are available in the Gene Expression Omnibus (GEO) with the accession code GSE285483 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). Source data are provided with this paper.

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Acknowledgements

We would like to thank the STI 2030-Major Projects (2021ZD0201600 (H.L.)), the National Natural Science Foundation of China (T2321004 (F.Y.), 52202351 (J.Q.), 82273195 (G.L.), 82072776 (G.L.)), the Natural Science Foundation of Shandong Province (ZR2022ZD20 (J.Q.), 2022HWYQ-052 (J.Q.), ZR2021LSW025 (G.L.)), the Fundamental Research Funds for the Central Universities (2022JC019 (J.Q.)) and the Taishan Scholarship in Shandong Provinces (tspd20210322 (G.L.), tsqn20221102 (J.Q.)) for the support of our work.

Author information

Author notes

  1. These authors contributed equally: Wenhan Wang, Keyi Li.

Authors and Affiliations

  1. State Key Laboratory of Crystal Materials, Shandong University, Jinan, China

    Wenhan Wang, Keyi Li, Wenjun Ma, Yiwei Li, Feng Liu, Ying Kong, Liang Wang, Yuanhua Sang, Hong Liu & Jichuan Qiu

  2. Department of Neurosurgery, Qilu Hospital, Cheeloo College of Medicine and Institute of Brain and Brain-Inspired Science, Shandong University, Jinan, China

    Wenhan Wang & Gang Li

  3. The Key Laboratory of Infection and Immunity of Shandong Province, Department of Pharmacology, School of Basic Medical Sciences, Shandong University, Jinan, China

    Fan Yi

  4. Institute for Advanced Interdisciplinary Research (iAIR), University of Jinan, Jinan, China

    Hong Liu

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Contributions

H.L., W.W. and J.Q. conceived and designed the experiments. W.W. and K.L. performed the experiments and made analyses; W.M. and Y.L. assisted in conducting animal experiments. F.L., Y.K. and L.W. participated in the discussion. Y.S., F.Y., G.L., J.Q. and H.L. provided the contributed reagents/materials/analysis tools. W.W. and K.L. wrote the manuscript with inputs from all authors.

Corresponding authors

Correspondence to Fan Yi, Yuanhua Sang, Gang Li, Hong Liu or Jichuan Qiu.

Ethics declarations

Competing interests

J.Q., W.W., K.L., W.M., Y.L., L.W., Y.S. and H.L. have filed a patent (Piezoelectric composite nanostickers for regulation of the differentiation of neural stem cells) related to this paper. F.L., Y.K., F.Y. and G.L. declare no competing interests.

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Nature Materials thanks Xiangzhong Chen, Gianni Ciofani and Jin Nam for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Schematic of the effects of BTO/rGO nanostickers in vitro and in vivo.

(a) BTO/rGO nanostickers facilitated the differentiation of NSCs into functional neurons and prevented damage by ROS during ultrasound treatment. (b) The application of BTO/rGO nanostickers in NSC-based therapy for TBI. Figure elements have been created with Figdraw.com.

Extended Data Fig. 2 Influence of BTO nanoparticles on NSCs and PC12 cells under ultrasound irradiation.

(a–b) mRNA levels of Tuj1 and MAP2 in NSCs after 5 days. (c) Western blot of Tuj1 and MAP2 levels in NSCs after 5 days and (d) associated analyses. (e–f) mRNA levels of Tuj1 and MAP2 in PC12 cells after 5 days. (g) Western blot of Tuj1 and MAP2 levels in PC12 cells after 5 days and (h) associated analyses. (i) Images from the live/dead assay (green: live cell; red: dead cell) and (j) analysis of the survival rate of NSCs after 3 days. (k) Images from the live/dead assay (green: live cell; red: dead cell) and (l) analysis of the survival rate of PC12 cells after 3 days. (m) CCK-8 assay results for NSCs. (n) CCK-8 assay results for PC12 cells. (o) EdU proliferation assay results for NSCs (green: proliferating cells; blue: Hoechst). (p) The proliferation ability of NSCs was assessed via the EdU proliferation assay. (q) EdU proliferation assay results for PC12 cells (green: proliferating cells; blue: Hoechst). (r) The proliferation ability of PC12 cells was assessed via the EdU proliferation assay. The data in a, b, d-f, h, j, l-n, p and r are shown as mean ± s.d., n = 3 independent biological samples for a, b, d-f, h, j, l, p and r; n = 5 independent biological samples for the data in m and n. Statistical analyses were performed via one-way ANOVA with Dunnett’s multiple comparisons tests (NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Source data

Extended Data Fig. 3 Comparison of tolerance for ROS between NSCs and PC12 cells.

(a) ROS levels in NSCs after different treatments. (b) Statistical analysis of ROS levels in NSCs. (c) ROS levels in PC12 cells after different treatments. (d) Statistical analysis of ROS levels in PC12 cells. (e) Images from the live/dead assay of NSCs after incubation with various concentrations of H2O2 for 3 days (green: live cells; red: dead cells). (f) Survival rate of NSCs incubated with various concentrations of H2O2 for 3 days. (g) Images from the live/dead assay of PC12 cells after incubation with various concentrations of H2O2 for 3 days (green: live cells; red: dead cells). (h) Survival rate of PC12 cells incubated with various concentrations of H2O2 for 3 days. The data in b, d, f and h are shown as mean ± s.d., n = 3 independent biological samples. Statistical analyses were performed via one-way ANOVA with Dunnett’s multiple comparisons tests (NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Source data

Extended Data Fig. 4 Changes in the membrane potential of NSCs upon treatment with ultrasound-activated BTO/rGO nanostickers.

(a) Schematic showing the measurement of changes in the membrane potential of NSCs upon stimulation with BTO/rGO nanostickers under ultrasound irradiation through MEA. (b) Representative optical image of an NSC adhered to the MEA electrode. A BTO/rGO nanosticker is attached to the NSC. (c) Electrical signal generated by the NSC adhered to the MEA electrode under ultrasound irradiation. The arrow indicates the moment of time when the ultrasound was applied. (d) Signal generated by the NSC with the BTO/rGO nanosticker attached under ultrasound irradiation. The right is enlarged figure of partial signal. The arrow indicates the moment of time when the ultrasound was applied. (e) Schematic showing the examination of the membrane potential of NSCs through a fast-response cell membrane potential probe. (f) Representative fluorescence images of the membrane potential in the NSC group and the corresponding change in fluorescence intensity. (g) Representative fluorescence images of the membrane potential in the NSC + US group and the corresponding change in fluorescence intensity. (h) Representative fluorescence images of the membrane potential in the NSC + BTO/rGO+US group and the corresponding change in fluorescence intensity; the inset shows the fluorescence signal between 22 s and 24 s. The micrographs in f, g and h were obtained from three independent experiments. Figure elements in a,e have been created with Figdraw.com.

Extended Data Fig. 5 Production efficiency of intracellular and extracellular ROS and their influence on NSCs.

(a) Measurement of ROS levels in NSCs after incubation with BTO nanoparticles and treatment with ultrasound at different powers and (b) associated analysis of the ROS level data. (c) ROS levels in NSCs after incubation with BTO/rGO nanostickers and treatment with ultrasound at different powers and (d) associated analysis of the ROS level data. (e) Images from the live/dead assay and (f) analysis of the survival rate of NSCs after incubation with BTO nanoparticles and treatment with ultrasound at different powers (green: live cells; red: dead cells). (g) Images from the live/dead assay and (h) analysis of the survival rate of NSCs after incubation with BTO/rGO nanostickers and treatment with ultrasound at different powers (green: live cells; red: dead cells). (i–j) mRNA levels of Tuj1 and MAP2 in NSCs that were incubated with BTO nanoparticles and treated with ultrasound at different powers. (k–l) mRNA levels of Tuj1 and MAP2 in NSCs that were incubated with BTO/rGO nanostickers and treated with ultrasound at different powers. The data in b, d, f, and h-l are shown as mean ± s.d., n = 3 independent biological samples. Statistical analyses were performed via one-way ANOVA with Dunnett’s multiple comparisons tests (NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Source data

Extended Data Fig. 6 Differences in genes expression and gene enrichment analysis.

(a) Volcanic map of differentially expressed genes (BTO/rGO+US group vs. TCP group) (DEGs: differentially expressed genes; red dots: upregulated genes; green dots: downregulated genes). (b) Heatmap of gene expression among the four groups. (c) KEGG pathway enrichment analysis of the differentially expressed genes (BTO/rGO+US group vs. TCP group). (d) GO enrichment analysis (biological process terms) of the differentially expressed genes (BTO/rGO+US group vs. TCP group). (e) GO enrichment analysis (cellular component terms) of the differentially expressed genes (BTO/rGO+US group vs. TCP group). (f) GO enrichment analysis (molecular function terms) of the differentially expressed genes (BTO/rGO+US group vs. TCP group).

Extended Data Fig. 7 P-KN93 inhibits intracellular signal transduction induced by BTO/rGO + US treatment.

(a) Schematic of intracellular signal transduction after P-KN93 treatment. (b) Western blot data derived from three independent experiments and (c) associated analyses of Tuj1 and MAP2 levels in NSCs treated with or without P-KN93. (d) Western blot data derived from three independent experiments and (e) associated analysis of P-CREB, CREB and BDNF levels in NSCs treated with or without P-KN93. (f) Immunofluorescence staining for P-CREB in NSCs treated with or without P-KN93 (red: F-actin; green: P-CREB; blue: DAPI). (g) Statistical analysis of the fluorescence intensity of P-CREB. (h) Immunofluorescence staining for BDNF in NSCs treated with or without P-KN93 (red: F-actin; green: BDNF; blue: DAPI). (i) Statistical analysis of the fluorescence intensity of BDNF. (j) Measurement of the amount of BDNF secreted by NSCs treated with or without P-KN93 by ELISA. The data in c, e, g, i and j are shown as mean ± s.d., n = 3 independent biological samples. Statistical analyses were performed via one-way ANOVA with Dunnett’s multiple comparisons tests (NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Figure elements in a have been created with Figdraw.com.

Source data

Extended Data Fig. 8 BTO/rGO piezoelectric nanostickers promote the neuronal differentiation of NSCs in vivo.

(a) Schematic of the in vivo experiments. (b) Representative image of surgery for NSC injection. (c) Representative image of the brain after 7 days of treatment. (d–e) Representative images of immunofluorescence images staining Tuj1/GFAP and MAP2/GFAP in brain sections from one of three rats in each group on day 7 (red: GFAP; green: MAP2 or Tuj1; blue: DAPI). (f) mRNA levels of Tuj1 and MAP2 in the four groups. (g) Western blot of Tuj1 and MAP2 levels in the four groups and (h) associated analyses. The data in f and h are shown as mean ± s.d., n = 3 independent biological samples. Statistical analyses were performed via one-way ANOVA with Dunnett’s multiple comparisons tests (NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Figure elements in a have been created with Figdraw.com.

Source data

Extended Data Fig. 9 Repair of brain tissue in experimental rats.

(a) Representative images of brains after 28 days of treatment. (b) Representative panoramic scans and locally enlarged images of HE staining in the brains. (c) Representative images of Nissl staining of the brains. (d-f) Representative immunofluorescence images of brain sections from rats that received different treatments for 28 days (green: NeuN, MAP2, and Tuj1; red: red: PKH26-labeled NSCs; blue: DAPI). Each image in b-f is from one of six rats per group.

Supplementary information

Supplementary Information

Legends for Supplementary Videos 1–10, Supplementary Methods, Figs. 1–13, Tables 1–5 and unprocessed blots for Supplementary Figs. 2c and 5b,f.

Reporting Summary

Supplementary Video 1

Representative video of cell membrane potential in individual NSC incubated with BTO/rGO nanostickers when treated with ultrasound.

Supplementary Video 2

Representative video of cell membrane potential in individual NSC.

Supplementary Video 3

Representative video of cell membrane potential in individual NSC when treated with ultrasound.

Supplementary Video 4

Representative video of calcium imaging in BTO/rGO + US group when stimulated with acetylcholine.

Supplementary Video 5

Representative video of calcium imaging in TCP group when treated with acetylcholine.

Supplementary Video 6

Representative video of calcium imaging in BTO/rGO group when treated with acetylcholine.

Supplementary Video 7

Representative video of calcium imaging in US group when treated with acetylcholine.

Supplementary Video 8

Representative video of calcium imaging in individual NSC incubated with BTO/rGO nanostickers when treated with ultrasound.

Supplementary Video 9

Representative video of calcium imaging in individual NSC.

Supplementary Video 10

Representative video of calcium imaging in individual NSC when treated with ultrasound.

Supplementary Video 11

Representative video of beam balance test in Sham, TBI and TBI + NSCs + BTO/rGO + US group. The video is accelerated by a factor of 4.

Supplementary Data 1

Source Data for Supplementary Fig. 2.

Supplementary Data 2

Source Data for Supplementary Fig. 5.

Source data

Source Data Fig. 1

Statistical source data for Fig. 1.

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Statistical source data for Fig. 2.

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Statistical source data for Fig. 3.

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Statistical source data for Fig. 4.

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Statistical source data for Fig. 5.

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Statistical source data for Fig. 6.

Source Data Fig. 1

Unprocessed western blots for Fig. 1.

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Unprocessed western blots for Fig. 3.

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Unprocessed western blots for Fig. 4.

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Unprocessed western blots for Fig. 5.

Source Data Extended Data Fig. 2

Statistical source data for Extended Data Fig. 2.

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Statistical source data for Extended Data Fig. 3.

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Statistical source data for Extended Data Fig. 5.

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Statistical source data for Extended Data Fig. 7.

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Statistical source data for Extended Data Fig. 8.

Source Data Extended Data Fig. 2

Unprocessed western blots for Extended Data Fig. 2.

Source Data Extended Data Fig. 7

Unprocessed western blots for Extended Data Fig. 7.

Source Data Extended Data Fig. 8

Unprocessed western blots for Extended Data Fig. 8.

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Wang, W., Li, K., Ma, W. et al. Ultrasound-activated piezoelectric nanostickers for neural stem cell therapy of traumatic brain injury. Nat. Mater. 24, 1137–1150 (2025). https://doi.org/10.1038/s41563-025-02214-w

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