Abstract
In the elderly population, dysregulated cellular behaviour during the healing process impacts tissue regeneration after injury. Early in the regeneration process, pro-inflammatory macrophages contribute to immune imbalance, while in later stages, senescent stem cells reduce regenerative capacity. Here we demonstrate that nicotinamide adenine dinucleotide (NAD+) can reprogramme both types of dysfunctional cell. We developed a spatiotemporal-adaptive nanotherapeutic system for the delivery of NAD+ into selected cells during different phases of tissue repair. By replenishing intracellular NAD+ pools, this system reshapes the multicellular regeneration niche, by metabolically rewiring pro-inflammatory macrophages towards a pro-repair phenotype during the early phase, and enhancing the differentiation capacity of senescent stem cells at later stages. This strategy effectively restored impaired bone regeneration in osteoporotic mice and accelerated skin wound healing. Our work presents a spatiotemporal-adaptive nanomedicine platform that bridges cell metabolism, nanomedicine and regeneration therapy.
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Data availability
The main data supporting the results in this study are available within the paper and its Supplementary Information. RNA-seq data generated for this study are deposited in the NCBI Gene Expression Omnibus under accession GSE283189. The publicly available single-cell transcriptomic dataset analysed in this study can be accessed from the Genome Sequence Archive (GSA CRA010641). The raw datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank F. Chen, Zhejiang University, for the technical assistance on SEM, and L. Wu at the Center of Cryo-Electron Microscopy, Zhejiang University, for the technical assistance on cryo-TEM. This work was supported by the National Natural Science Foundation of China (grant numbers U22A20282 and 82472471), the ‘Pioneer’ and ‘Leading Goose’ R&D Program of Zhejiang Province (grant number 2025C02162) to X.F., the Natural Science Foundation of Zhejiang Province (grant number LMS25B010001) to Y.Z., the National Natural Science Foundation of China (grant number 22435006), the Key R&D Program of Zhejiang (grant number 2024C03207), the Fundamental Research Funds for the Central Universities (grant number 2024FZZX02-01-04) to Z.L., the National Natural Science Foundation of China (grant number 82301760) to P.Q. and the Natural Science Foundation of Zhejiang Province (grant number LQ23H150004) to C.Z.
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Contributions
K.L., Z.L., Y.Z. and X.F. designed the project. K.L., L. Zhao, S.Z., L. Zheng, Z.Z., S.W., J.C., W.X., W.W., H.Y., C.S., P.Q., C.Z., W.F. and J.Z. performed the experiments and collected the data. K.L. performed the visualization. K.L., Z.L., Y.Z. and X.F. wrote the original paper. K.L., L. Zhao, Z.L. and Y.Z. revised the paper. S.F., Z.L., R.T., Y.Z. and X.F. supervised the project. K.L. and L. Zhao contributed equally to this project.
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Nature Nanotechnology thanks Xiaoyuan Chen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Characterization of the spatiotemporal-adaptive nanotherapeutic system.
a, b, Diameter distribution in PBS (10 mM, pH 7.4) and representative cryo-TEM images showing nanoparticle morphology of ZIF-8 (a) and NZ (b). Scale bars, 100 nm. Representative of three independent experiments. c, FTIR spectra of ZIF-8, NZ, and free NAD+. a.u., arbitrary units. Representative of three independent experiments. d, X-ray diffraction patterns of ZIF-8 and NZ. Representative of three independent experiments. e, Representative western blots validating cell membrane purification; β-actin (cytoplasmic marker), Na+-K+-ATPase (membrane-specific marker). Representative images from three independent experiments. f, Representative fluorescence images demonstrating membrane fusion of BMDM (DiO, green) and BMSC (DiD, magenta) membranes. Scale bar, 50 μm. Fluorescence colocalization shown at right. Representative of five independent experiments. g, Diameter distribution in PBS and cryo-TEM images of HM. Scale bar, 100 nm. Representative of three independent experiments. h, Representative fluorescence images of DiI-labelled HM. Scale bars, 10 μm. Representative of three independent experiments. i, Cryo-TEM images of GHM. Scale bar, 100 nm. Representative of three independent experiments. j, Western blots demonstrating NZM successfully coated with hybrid membranes. CD68, F4/80 markers for BMDM membranes; CD90, CD105 for BMSC membranes. Representative images from three experiments. k, Cell viability quantification in LPS-stimulated BMDMs (LPS, 100 ng ml−1) treated with free NAD+ (10 µM) or equivalent dose of NPs. n = 5 independent samples. l, Cell viability quantification in H2O2-mediated BMSCs (200 µM H2O2) treated with free NAD+ (10 µM) or equivalent dose of NPs. n = 3 independent samples. m, Flow cytometry analysis of relative cell proportions in early and late stages of bone and skin injury microenvironments. n = 3 independent mice. n, o, Representative flow cytometry plots and MFI quantification of Cy5-labelled NZ uptake during inflammatory (n; NIH3T3:MSC:M1 MΦ = 4:1:3) and repair phases (o; NIH3T3:MSC:M1 MΦ = 4:3:1). n = 3 independent samples. p, Representative flow cytometry plots (left) and MFI quantification (right) showing NZM targeting in co-culture simulating non-injured tissue (NIH3T3:MSC:M1 MΦ = 10:1:1). n = 4 independent samples. Data presented as mean ± s.d. (k, l, m, n, o, p). P values were calculated using using one-way ANOVA followed by Tukey’s two-sided post hoc multiple-comparison test (k, l, n, o, p).
Extended Data Fig. 2 The effects of NZM in aged MSC.
a, Representative fluorescence images and quantification of free NAD+ (10 µM) or NP uptake by aged MSCs. n = 4 independent samples. Scale bar, 20 μm. b, Representative flow cytometry plots and quantification of ROS-induced apoptosis across treatments, indicating apoptotic cell populations. n = 3 independent samples. c, d, Representative flow cytometry plots (c) and MFI quantification (d) of senescence-associated β-galactosidase activity in aged MSCs post-NZM treatment (10 µM NAD+). Cells were pretreated with Bafilomycin A prior to detecting β-gal activity using C12FDG. n = 3 independent samples. e, Gene expression quantification of cell-cycle and inflammatory secretome markers. n = 3 independent samples. f, Flow cytometry analysis showing proportion (left) and quantification (right) of ROS-positive aged MSCs after indicated treatments. n = 3 independent samples. g, Flow cytometry plots with MitoTracker Green and MFI quantification illustrating mitochondrial mass changes in aged MSCs post-NZM treatment (10 µM NAD+). n = 5 independent samples. h, Representative western blots of mitochondrial respiratory chain proteins following NZM treatment (10 µM NAD+). Images represent three independent experiments. i, Quantification of protein levels (SIRT1, PGC1α, mtTFA, NRF1, NRF2, γH2AX, p16, p21, p53), normalized to β-actin, in aged MSCs post-NZM treatment. n = 3 independent samples. j, Representative immunofluorescence images and quantification of prohibitin (PHB) in aged MSCs following NZM treatment (10 µM NAD+). n = 4 independent samples. Scale bar, 50 μm. k, PCA of gene expression in aged MSCs with or without NZM treatment; three independent replicates shown. l, Heatmap of mitochondrial respiratory chain-related genes, indicating enhanced oxidative phosphorylation and improved mitochondrial function in aged MSCs after NZM treatment. n = 3 independent samples. m, Heatmap depicting expression of UPRmt and mitophagy-related genes, highlighting potential compensatory pathways for mitochondrial quality control and stress adaptation induced by NZM. n = 3 independent samples. Data presented as mean ± s.d. (a, b, d, e, f, g, j, i). P values were calculated using using one-way ANOVA followed by Tukey’s two-sided post hoc multiple-comparison test (a, b, d, e, f, g, j, i).
Extended Data Fig. 3 Systematically generate a regeneration niche by cutting off synergistic effects.
a, Schematic representation of the interactions between senescent stem cells and inflammatory macrophages. Created in BioRender. Liang, K. (2025) https://BioRender.com/ou25z6p. b, Schematic illustration of the systemic remodeling of the regenerative niche following NZM treatment. Created in BioRender. Liang, K. (2025) https://BioRender.com/g9snj9v. c, Schematic diagram of the experimental design of the paracrine effects of senescent MSCs. Created in BioRender. Liang, K. (2025) https://BioRender.com/8i38cqy. d, Relative mRNA expression of Il1β, Nos2, Arg1 and Cd206 in macrophages determined by qRT-PCR. n = 4 biologically independent samples. e, f, Representative microscopy images (e) and quantification (f) of iNOS and ARG1 in macrophages. n = 4 biologically independent samples. Scale bars, 50 μm. g, Levels of cytokines secreted by macrophages determined by ELISA. n = 4 biologically independent samples. For panels d–g, the groups represent cells in the Transwell chamber. The Control group represents young MSCs in the Transwell chamber, the Aged group consists of senescent MSCs, and the Aged+NZM group represents NZM-treated senescent MSCs. h, Schematic diagram of the experimental design of the paracrine effects of macrophages. Created in BioRender. Liang, K. (2025) https://BioRender.com/a2j1cv8. Macrophages were stimulated with LPS (100 ng ml−1) and treated with or without NZM (10 µM, based on NAD+) for 24 h. The culture supernatants were then collected and used to treat MSCs for 7 days. i, j, Representative microscopy images (i) and quantification (j) of SA-β-Gal staining in MSCs. Scale bars, 100 μm. k, Relative mRNA expression of Cdkn1a, Cdkn2a, p53, Il6 and Il18 in MSCs determined by qRT-PCR. n = 4 biologically independent samples. For panels i–k, the group labels indicate the specific interventions applied to macrophages. Data are presented as mean ± s.d. (d, f, g, j, k). P values were calculated using using one-way ANOVA followed by Tukey’s two-sided post hoc multiple-comparison test (d, f, g, j, k).
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Liang, K., Zhao, L., Zhang, S. et al. Spatiotemporal-adaptive nanotherapeutics promote post-injury regeneration in ageing through metabolic modulation. Nat. Nanotechnol. (2025). https://doi.org/10.1038/s41565-025-02017-9
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DOI: https://doi.org/10.1038/s41565-025-02017-9