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A self-directed Trojanbot-enzymatic nanobot in neutrobot for active target therapy of glioblastoma

Gao, Yuanyuan, Mao, Meng, Li, Yue, Xuan, Mingjun, Wu, Yingjie, He, Qiang·2025·Nature Communications
DOI 10.1038/s41467-025-60422-zPMC PMC12144173
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Fig. 1 Schematic illustration of the enzymatic nanobot-in-neutrobot system, inspired by the “Trojan Horse” strategy, which leverages neutrophil phagocytosis, chemotactic movement, BBB crossing, and the response of CatNbots to H2O2 in the TME to enhance the tumor penetration of DOX, thereby achieving targeted drug delivery to GBM.
Fig. 1 Schematic illustration of the enzymatic nanobot-in-neutrobot system, inspired by the “Trojan Horse” strategy, which leverages neutrophil phagocytosis, chemotactic movement, BBB crossing, and the response of CatNbots to H2O2 in the TME to enhance the tumor penetration of DOX, thereby achieving targeted drug delivery to GBM.CC BY-NC-ND · PubMed Central
Fig. 2 Preparation and characterization of Trojanbots. a Schematic outlining the fabrication of the Trojanbots. b TEM images of GeNPs. Inset: Magnified TEM image of GeNPs. c TEM images of EMV@GeNPs. Inset: Magnified TEM image of EMV@GeNPs. d TEM image of CatNbots. e The mapping spectrum of CatNbot, Fe element indicates that catalase is successfully connected to EMV@GeNPs with random asymmetry. f DLS analyzes the particle diameter of GeNPs, EMV@GeNPs, and CatNbots. g Zeta potential values of GeNPs, EMVs, EMV@GeNPs, and CatNbots (n = 3). The Zeta analysis represented data obtained from three independent experiments. h LPS content of GeNPs, EMVs, EMV@GeNPs, and CatNbots (n = 3). The LPS content analysis represented data from three independent experiments. i Changes in the dissolved O2 level of the reaction medium treated with CatNbots under different pH conditions represent the enzymatic activity of CatNbots. j Movie snapshot capturing neutrophils undergoing phagocytosis of CatNbots. The trajectory of a representative neutrophil was depicted in green, while the typical neutrophil and CatNbot were marked in purple and yellow, respectively. k Quantitative analysis of neutrophil phagocytosis efficiency (n = 3). l The confocal laser scanning microscopy (CLSM) image of a Trojanbot. The nucleus of the neutrophil was stained with Hoechst 33342 (blue); the cell membrane of the neutrophil was stained with Dio (green), and the fluorescence of CatNbot was red. Data are presented as mean ± SD of three independent experiments (g, h, k). P-values are determined by a two-tailed Student’s t-test in (k). Source data are provided as a Source Data file.
Fig. 2 Preparation and characterization of Trojanbots. a Schematic outlining the fabrication of the Trojanbots. b TEM images of GeNPs. Inset: Magnified TEM image of GeNPs. c TEM images of EMV@GeNPs. Inset: Magnified TEM image of EMV@GeNPs. d TEM image of CatNbots. e The mapping spectrum of CatNbot, Fe element indicates that catalase is successfully connected to EMV@GeNPs with random asymmetry. f DLS analyzes the particle diameter of GeNPs, EMV@GeNPs, and CatNbots. g Zeta potential values of GeNPs, EMVs, EMV@GeNPs, and CatNbots (n = 3). The Zeta analysis represented data obtained from three independent experiments. h LPS content of GeNPs, EMVs, EMV@GeNPs, and CatNbots (n = 3). The LPS content analysis represented data from three independent experiments. i Changes in the dissolved O2 level of the reaction medium treated with CatNbots under different pH conditions represent the enzymatic activity of CatNbots. j Movie snapshot capturing neutrophils undergoing phagocytosis of CatNbots. The trajectory of a representative neutrophil was depicted in green, while the typical neutrophil and CatNbot were marked in purple and yellow, respectively. k Quantitative analysis of neutrophil phagocytosis efficiency (n = 3). l The confocal laser scanning microscopy (CLSM) image of a Trojanbot. The nucleus of the neutrophil was stained with Hoechst 33342 (blue); the cell membrane of the neutrophil was stained with Dio (green), and the fluorescence of CatNbot was red. Data are presented as mean ± SD of three independent experiments (g, h, k). P-values are determined by a two-tailed Student’s t-test in (k). Source data are provided as a Source Data file.CC BY-NC-ND · PubMed Central
Fig. 3 Chemotactic motion of Trojanbots and the release of CatNbots. a Schematic illustration of the chemotactic motion of Trojanbots and the release of CatNbots under PMA stimulation, and the catalytic propulsion of CatNbots. b Time-lapse images of the chemotactic motion of Trojanbots along a chemokine gradient. c Trajectories (n = 7) and d direction distribution (n = 7) of Trojanbots in response to a fMLP gradient (10 pM µm−1). The duration of chemotactic motion is 10 min. e Dependence of chemotaxis velocity of neutrophils and Trojanbots on the chemokine gradient of fMLP (n = 3). f Dependence of CI (ratio of distance to path length) of Trojanbots on the chemokine gradient of fMLP (n = 6). The center line of the box plots represents the median, the box bounds indicate the 25th and 75th percentiles and the whiskers extend to the minimum and maximum values within the 1.5× interquartile range (IQR). Individual data points are overlaid. No outliers are present. g TAD of Trojanbots under chemokine gradient (n = 600). h TAD at 0° of Trojanbots under different gradients of fMLP (n = 6). The center line of the box plots represents the median, the box bounds indicate the 25th and 75th percentiles and the whiskers extend to the minimum and maximum values within 1.5× IQR. Individual data points are overlaid. No outliers are present. i Time-lapse images of CatNbots releasing process under PMA stimulation. The resting Trojanbots (i) were activated by PMA stimulation (ii), and the as-released CatNbots (iii) exhibited self-propelled motion in H2O2 solution (iv). j The effect of different concentrations of PMA on the release rate of Trojanbots (n = 3). k Dependence of Trojanbot release percentage on time with or without PMA (100 nM) stimulation. (n = 3). l CLSM images of CatNbots released from Trojanbots with (i) and without 100 nM PMA (h) after 8 h of in vitro culturing. The CatNbots were loaded with Cou6 (green), and the released DNA segments were stained with PI (red). m Representative tracking trajectories of the CatNbots (n = 20) in H2O2 solutions with different concentrations (0, 2, 4, 6, 8 µM) within 1 s. n MSD analysis (Δt = 1 s, n = 20) of CatNbots at different H2O2 concentrations (0, 2, 4, 6, and 8 µM). o The effective diffusion coefficient (Deff) (left) and self-propelled velocity of CatNbots (right) at different H2O2 concentrations (0, 2, 4, 6, and 8 µM) based on MSD. (n = 20). p The instantaneous velocity of CatNbots at different H2O2 concentrations (0, 2, 4, 6, and 8 µM) (n = 20). Data are presented as mean ± SD of three independent experiments (e, j, k), six independent experiments (f, g, h), seven independent experiments (c, d), and 20 independent experiments (o, p). Source data are provided as a Source Data file.
Fig. 3 Chemotactic motion of Trojanbots and the release of CatNbots. a Schematic illustration of the chemotactic motion of Trojanbots and the release of CatNbots under PMA stimulation, and the catalytic propulsion of CatNbots. b Time-lapse images of the chemotactic motion of Trojanbots along a chemokine gradient. c Trajectories (n = 7) and d direction distribution (n = 7) of Trojanbots in response to a fMLP gradient (10 pM µm−1). The duration of chemotactic motion is 10 min. e Dependence of chemotaxis velocity of neutrophils and Trojanbots on the chemokine gradient of fMLP (n = 3). f Dependence of CI (ratio of distance to path length) of Trojanbots on the chemokine gradient of fMLP (n = 6). The center line of the box plots represents the median, the box bounds indicate the 25th and 75th percentiles and the whiskers extend to the minimum and maximum values within the 1.5× interquartile range (IQR). Individual data points are overlaid. No outliers are present. g TAD of Trojanbots under chemokine gradient (n = 600). h TAD at 0° of Trojanbots under different gradients of fMLP (n = 6). The center line of the box plots represents the median, the box bounds indicate the 25th and 75th percentiles and the whiskers extend to the minimum and maximum values within 1.5× IQR. Individual data points are overlaid. No outliers are present. i Time-lapse images of CatNbots releasing process under PMA stimulation. The resting Trojanbots (i) were activated by PMA stimulation (ii), and the as-released CatNbots (iii) exhibited self-propelled motion in H2O2 solution (iv). j The effect of different concentrations of PMA on the release rate of Trojanbots (n = 3). k Dependence of Trojanbot release percentage on time with or without PMA (100 nM) stimulation. (n = 3). l CLSM images of CatNbots released from Trojanbots with (i) and without 100 nM PMA (h) after 8 h of in vitro culturing. The CatNbots were loaded with Cou6 (green), and the released DNA segments were stained with PI (red). m Representative tracking trajectories of the CatNbots (n = 20) in H2O2 solutions with different concentrations (0, 2, 4, 6, 8 µM) within 1 s. n MSD analysis (Δt = 1 s, n = 20) of CatNbots at different H2O2 concentrations (0, 2, 4, 6, and 8 µM). o The effective diffusion coefficient (Deff) (left) and self-propelled velocity of CatNbots (right) at different H2O2 concentrations (0, 2, 4, 6, and 8 µM) based on MSD. (n = 20). p The instantaneous velocity of CatNbots at different H2O2 concentrations (0, 2, 4, 6, and 8 µM) (n = 20). Data are presented as mean ± SD of three independent experiments (e, j, k), six independent experiments (f, g, h), seven independent experiments (c, d), and 20 independent experiments (o, p). Source data are provided as a Source Data file.CC BY-NC-ND · PubMed Central
Fig. 4 BBB penetration ability of Trojanbots in vitro. a Schematic illustration of the in vitro BBB model used to evaluate the potential BBB penetration abilities of GeNPs, CatNbots, Neutrophils, and Trojanbots, both in the presence and absence of fMLP. b–d CLSM images of the bEnd.3 cell layer (b), BBB layer (c), and GL261 cells (d). In the images, bEnd.3 cells were labeled with Dio, while GeNPs, CatNbots, Neutrophils, and Trojanbots were labeled with Dil. The nuclei of the GL261 cells were stained with Hoechst 33342 (blue). e–g The fluorescence statistics of the bEnd.3 monolayer, BBB layer, and GL261 cells layer, respectively (n = 3) (exact P value of e: GeNP vs. Trojanbot + fMLP P = 1.51E−8; exact P value of f: GeNP vs. Trojanbot + fMLP P = 2E−10, CatNbot vs. Trojanbot + fMLP P = 3E−10, Neutrophil vs. Trojanbot + fMLP P = 9.666E−7, Trojanbot vs. Trojanbot + fMLP P = 1.17148E−5; exact P value of g: GeNP vs. Trojanbot + fMLP P = 4.2E−9, CatNbot vs. Trojanbot + fMLP P = 1.25964E−5.) h The penetration ratio of different groups for 6 h (n = 3). (exact P value: GeNP vs. Trojanbot + fMLP P = 1.484E−7, CatNbot vs. Trojanbot + fMLP P = 1.731E−7, Trojanbot vs. Trojanbot + fMLP P = 1.484E−7). i–l The time-dependent penetration ratios of GeNPs (i), CatNbots (j), Neutrophils (k), and Trojanbots (l) with and without fMLP (10 nM, n = 3). Data are presented as mean ± SD of three independent experiments (e–l). P-values are determined by one-way ANOVA and Tukey multiple comparisons tests in (e–h). Source data are provided as a Source Data file.
Fig. 4 BBB penetration ability of Trojanbots in vitro. a Schematic illustration of the in vitro BBB model used to evaluate the potential BBB penetration abilities of GeNPs, CatNbots, Neutrophils, and Trojanbots, both in the presence and absence of fMLP. b–d CLSM images of the bEnd.3 cell layer (b), BBB layer (c), and GL261 cells (d). In the images, bEnd.3 cells were labeled with Dio, while GeNPs, CatNbots, Neutrophils, and Trojanbots were labeled with Dil. The nuclei of the GL261 cells were stained with Hoechst 33342 (blue). e–g The fluorescence statistics of the bEnd.3 monolayer, BBB layer, and GL261 cells layer, respectively (n = 3) (exact P value of e: GeNP vs. Trojanbot + fMLP P = 1.51E−8; exact P value of f: GeNP vs. Trojanbot + fMLP P = 2E−10, CatNbot vs. Trojanbot + fMLP P = 3E−10, Neutrophil vs. Trojanbot + fMLP P = 9.666E−7, Trojanbot vs. Trojanbot + fMLP P = 1.17148E−5; exact P value of g: GeNP vs. Trojanbot + fMLP P = 4.2E−9, CatNbot vs. Trojanbot + fMLP P = 1.25964E−5.) h The penetration ratio of different groups for 6 h (n = 3). (exact P value: GeNP vs. Trojanbot + fMLP P = 1.484E−7, CatNbot vs. Trojanbot + fMLP P = 1.731E−7, Trojanbot vs. Trojanbot + fMLP P = 1.484E−7). i–l The time-dependent penetration ratios of GeNPs (i), CatNbots (j), Neutrophils (k), and Trojanbots (l) with and without fMLP (10 nM, n = 3). Data are presented as mean ± SD of three independent experiments (e–l). P-values are determined by one-way ANOVA and Tukey multiple comparisons tests in (e–h). Source data are provided as a Source Data file.CC BY-NC-ND · PubMed Central
Fig. 5 Deep penetration of CatNbots into the tumor. a Schematic diagram of the chemotaxis of CatNbots toward tumor tissue and the internalization by tumor cells. b Schematic of chemotactic motion of CatNbots along the concentration gradient of the H2O2. c Fluorescent images of the different chambers. d The corresponding fluorescence quantification values at different time points (n = 3). e In vitro penetration results in 3D tumor spheroids. The bright-field images and Z-stack CLSM images were taken from the top to the equatorial plane in 160 µm thickness of 3D cellular spheroids after treatment with GeNPs, EMV@GeNPs, and CatNbots for 4 h. f The corresponding fluorescent spectra and g the mean fluorescence intensity of the GL261 tumor spheroids treated with different samples (n = 3) (exact P value: GeNP vs. CatNbot P = 8.16414E−5). h Tumor spheroid morphology and TUNEL apoptosis assay of GL261 3D spheroids treated with various groups for 3 days. Spheroid slices, 160 µm thick, were subjected to the TUNEL assay, with positive cells identified using CLSM imaging. i Volume statistics of tumor spheroids before and after treatment across different experimental groups. j Cytotoxicity assessment of GeNPs, EMV@GeNPs, and CatNbots on 3D tumor spheroids (n = 3). k Cellular uptake CLSM images for different samples (Green: labeled with Cou6, Red: cell membranes stained with Dil, Blue: cell nuclei stained with Hoechst 33342). l The corresponding fluorescence semi-quantitative analysis (n = 3) (exact P values: GeNP vs. CatNbot P = 3.77E−7, EMV@GeNP vs. CatNbot P = 1.2024E−6). m Cytotoxicity of GeNPs, EMV@GeNPs, and CatNbots on GL261 cell lines in a 2D culture (n = 3). n Mechanism diagram of CatNbots regulating the TME. o Typical CLSM images of intracellular H2O2 (labeled with the H2O2 fluorescent probe, ROSGreenTM H2O2 Probe) in cancer cells after CatNbots treatment for 12 h (n = 3). p Relative H2O2 level in cancer cells after treatment with different samples for 12 h (n = 3) (exact P-value: 3 h Control vs. CatNbot P = 1.51132E−5, 3 h GeNP vs. CatNbot P = 3.32681E−5, 9 h Control vs. CatNbot P = 3.9146E−6, 9 h GeNP vs. CatNbot P = 2.219E−6, 9 h EMV@GeNP vs. CatNbot P = 3.617E−7, 12 h Control vs. CatNbot P = 3.18E−8, 12 h GeNP vs. CatNbot P = 2.9E−8, 12 h EMV@GeNP vs. CatNbot P = 1.72E−8). Data are presented as mean ± SD of three independent experiments (d, g, j, l, m, p). P-values are determined by one-way ANOVA and Tukey multiple comparisons tests in (g, l, p). Source data are provided as a Source Data file.
Fig. 5 Deep penetration of CatNbots into the tumor. a Schematic diagram of the chemotaxis of CatNbots toward tumor tissue and the internalization by tumor cells. b Schematic of chemotactic motion of CatNbots along the concentration gradient of the H2O2. c Fluorescent images of the different chambers. d The corresponding fluorescence quantification values at different time points (n = 3). e In vitro penetration results in 3D tumor spheroids. The bright-field images and Z-stack CLSM images were taken from the top to the equatorial plane in 160 µm thickness of 3D cellular spheroids after treatment with GeNPs, EMV@GeNPs, and CatNbots for 4 h. f The corresponding fluorescent spectra and g the mean fluorescence intensity of the GL261 tumor spheroids treated with different samples (n = 3) (exact P value: GeNP vs. CatNbot P = 8.16414E−5). h Tumor spheroid morphology and TUNEL apoptosis assay of GL261 3D spheroids treated with various groups for 3 days. Spheroid slices, 160 µm thick, were subjected to the TUNEL assay, with positive cells identified using CLSM imaging. i Volume statistics of tumor spheroids before and after treatment across different experimental groups. j Cytotoxicity assessment of GeNPs, EMV@GeNPs, and CatNbots on 3D tumor spheroids (n = 3). k Cellular uptake CLSM images for different samples (Green: labeled with Cou6, Red: cell membranes stained with Dil, Blue: cell nuclei stained with Hoechst 33342). l The corresponding fluorescence semi-quantitative analysis (n = 3) (exact P values: GeNP vs. CatNbot P = 3.77E−7, EMV@GeNP vs. CatNbot P = 1.2024E−6). m Cytotoxicity of GeNPs, EMV@GeNPs, and CatNbots on GL261 cell lines in a 2D culture (n = 3). n Mechanism diagram of CatNbots regulating the TME. o Typical CLSM images of intracellular H2O2 (labeled with the H2O2 fluorescent probe, ROSGreenTM H2O2 Probe) in cancer cells after CatNbots treatment for 12 h (n = 3). p Relative H2O2 level in cancer cells after treatment with different samples for 12 h (n = 3) (exact P-value: 3 h Control vs. CatNbot P = 1.51132E−5, 3 h GeNP vs. CatNbot P = 3.32681E−5, 9 h Control vs. CatNbot P = 3.9146E−6, 9 h GeNP vs. CatNbot P = 2.219E−6, 9 h EMV@GeNP vs. CatNbot P = 3.617E−7, 12 h Control vs. CatNbot P = 3.18E−8, 12 h GeNP vs. CatNbot P = 2.9E−8, 12 h EMV@GeNP vs. CatNbot P = 1.72E−8). Data are presented as mean ± SD of three independent experiments (d, g, j, l, m, p). P-values are determined by one-way ANOVA and Tukey multiple comparisons tests in (g, l, p). Source data are provided as a Source Data file.CC BY-NC-ND · PubMed Central
Fig. 6 In vivo distribution of Trojanbots. a The schematic representation of the construction of the brain glioma model. b In vivo fluorescence images of orthotopic GL261-Luc mouse models with GBM tumors following a single tail vein injection of potassium fluorescein (n = 3 mice per group). c Fluorescence intensity measurements of glioma mouse brains treated with various therapeutic agents (n = 3 mice per group). d Fluorescent images (IVIS imaging) of the brain and major organs, taken 48 h after injection (n = 3 mice per group). e Quantitative ROI analysis of DiR signals in the brain and organs (n = 3 mice per group) (exact P value: Spleen CatNbot vs. Trojanbot P = 5.89774E−5, Lung EMV@GeNP vs. Trojanbot P = 3.86669E−5, Brain EMV@GeNP vs. Trojanbot P = 3.98E−8, Brain CatNbot vs. Trojanbot P = 1.084E−7, NE@EDGN vs. Trojanbot P = 1.62003E−5). f Fluorescence images (IVIS imaging) of brain slices from four groups of mice, labeled with DiR (8 h post tail vein injection). g Quantitative analysis of the fluorescence images (IVIS imaging) of brain slices from the four groups (n = 3 mice per group). (exact P value: EMV@GeNP vs. Trojanbot P = 6.055E−7). h Representative fluorescence images of glioma tissue slices from GBM mice. In the images, EMV@GeNPs, CatNbots, EMV@GeNPs within NE@EDGN, and CatNbots within Trojanbots were labeled with FITC (green), while DOX exhibits intrinsic autofluorescence properties (red). The nuclei of the GL261 cells were stained with Hoechst 33342 (blue). Data are presented as mean ± SD of three independent experiments (e, g). P-values are determined by one-way ANOVA and Tukey multiple comparison tests in (e, g). Source data are provided as a Source Data file.
Fig. 6 In vivo distribution of Trojanbots. a The schematic representation of the construction of the brain glioma model. b In vivo fluorescence images of orthotopic GL261-Luc mouse models with GBM tumors following a single tail vein injection of potassium fluorescein (n = 3 mice per group). c Fluorescence intensity measurements of glioma mouse brains treated with various therapeutic agents (n = 3 mice per group). d Fluorescent images (IVIS imaging) of the brain and major organs, taken 48 h after injection (n = 3 mice per group). e Quantitative ROI analysis of DiR signals in the brain and organs (n = 3 mice per group) (exact P value: Spleen CatNbot vs. Trojanbot P = 5.89774E−5, Lung EMV@GeNP vs. Trojanbot P = 3.86669E−5, Brain EMV@GeNP vs. Trojanbot P = 3.98E−8, Brain CatNbot vs. Trojanbot P = 1.084E−7, NE@EDGN vs. Trojanbot P = 1.62003E−5). f Fluorescence images (IVIS imaging) of brain slices from four groups of mice, labeled with DiR (8 h post tail vein injection). g Quantitative analysis of the fluorescence images (IVIS imaging) of brain slices from the four groups (n = 3 mice per group). (exact P value: EMV@GeNP vs. Trojanbot P = 6.055E−7). h Representative fluorescence images of glioma tissue slices from GBM mice. In the images, EMV@GeNPs, CatNbots, EMV@GeNPs within NE@EDGN, and CatNbots within Trojanbots were labeled with FITC (green), while DOX exhibits intrinsic autofluorescence properties (red). The nuclei of the GL261 cells were stained with Hoechst 33342 (blue). Data are presented as mean ± SD of three independent experiments (e, g). P-values are determined by one-way ANOVA and Tukey multiple comparison tests in (e, g). Source data are provided as a Source Data file.CC BY-NC-ND · PubMed Central
Fig. 7 In vivo antitumor activity of Trojanbots in glioma-bearing mice. a The schematic diagram of intravenous injection treatment. b Time-course bioluminescence images of orthotopic GL261 tumor-bearing mice treated with PBS, EMV@GeNPs, CatNbots, NE@EDGNs, and Trojanbots (n = 5 mice per group). c Quantified luminescence levels of mice using the Lumina IVIS III system (n = 5 mice per group). d Body weight profiles (n = 5 mice per group). e Survival curves of mice (n = 5 mice per group) (exact P value: PBS vs. Trojanbots, P = 0.0003, EMV @ GeNP vs. Trojanbot, P = 0.0005, CatNbot vs. Trojanbot, P = 0.0022, NE @ EDGN vs. Trojanbot, P = 0.2517). f H&E images of the whole brains excised from the representative mice in treatments as described above, Histological analysis using the TUNEL (Green: apoptotic cells; Blue: Hoechst-stained cell nuclei) and Ki67 assay (Green: proliferating cells; Blue: Hoechst-stained cell nuclei), CD31, HIF-1α, and N-cadherin. The histological analyses were representative of data from three mice. g–k The fluorescence statistics derived from the histological analyses shown in (f) (n = 3 mice per group) (exact P value of h: PBS vs. Trojanbot P = 5.65E−8, exact P value of j: PBS vs. Trojanbot P = 6.91E−8, EMV@GeNP vs. Trojanbot P = 1.258E−7, exact P value of k: PBS vs. Trojanbot P = 9.00532E−5). l Representative flow cytometry results showing different groups of CD45+CD3+T cells in tumor tissues of GBM. For the gating strategy for T-cell analysis, refer to Supplementary Fig. 35. m Quantitative analysis of CD3+CD8+ cytotoxic Helper T cells (n = 3 mice per group). n Quantitative analysis of CD3+CD4+ Helper T cells (n = 3 mice per group). Data are presented as mean ± SD of three independent experiments (g–k, m, n) and five independent experiments (c–e). P-values are determined by one-way ANOVA and Tukey multiple comparisons tests in (c, e, g–k, m, n). Source data are provided as a Source Data file.
Fig. 7 In vivo antitumor activity of Trojanbots in glioma-bearing mice. a The schematic diagram of intravenous injection treatment. b Time-course bioluminescence images of orthotopic GL261 tumor-bearing mice treated with PBS, EMV@GeNPs, CatNbots, NE@EDGNs, and Trojanbots (n = 5 mice per group). c Quantified luminescence levels of mice using the Lumina IVIS III system (n = 5 mice per group). d Body weight profiles (n = 5 mice per group). e Survival curves of mice (n = 5 mice per group) (exact P value: PBS vs. Trojanbots, P = 0.0003, EMV @ GeNP vs. Trojanbot, P = 0.0005, CatNbot vs. Trojanbot, P = 0.0022, NE @ EDGN vs. Trojanbot, P = 0.2517). f H&E images of the whole brains excised from the representative mice in treatments as described above, Histological analysis using the TUNEL (Green: apoptotic cells; Blue: Hoechst-stained cell nuclei) and Ki67 assay (Green: proliferating cells; Blue: Hoechst-stained cell nuclei), CD31, HIF-1α, and N-cadherin. The histological analyses were representative of data from three mice. g–k The fluorescence statistics derived from the histological analyses shown in (f) (n = 3 mice per group) (exact P value of h: PBS vs. Trojanbot P = 5.65E−8, exact P value of j: PBS vs. Trojanbot P = 6.91E−8, EMV@GeNP vs. Trojanbot P = 1.258E−7, exact P value of k: PBS vs. Trojanbot P = 9.00532E−5). l Representative flow cytometry results showing different groups of CD45+CD3+T cells in tumor tissues of GBM. For the gating strategy for T-cell analysis, refer to Supplementary Fig. 35. m Quantitative analysis of CD3+CD8+ cytotoxic Helper T cells (n = 3 mice per group). n Quantitative analysis of CD3+CD4+ Helper T cells (n = 3 mice per group). Data are presented as mean ± SD of three independent experiments (g–k, m, n) and five independent experiments (c–e). P-values are determined by one-way ANOVA and Tukey multiple comparisons tests in (c, e, g–k, m, n). Source data are provided as a Source Data file.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Gao, Y., Mao, M., Li, Y., Xuan, M., Wu, Y., & He, Q.
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-06-06
Volume
16
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-025-60422-z
PMC
PMC12144173
Cited by
42
References
2
Language
en
Licence
Funders
  • National Natural Science Foundation of China (22193033, U23A20342)
  • the Key R&D Program of Heilongjiang Province, China
  • China Postdoctoral Science Foundation (2024M764205)
  • the Postdoctoral Fellowship Program of CPSF

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