文献标题：Aminated fullerene-based nanoplatform enables synergistic VEGFR2-targeted anti-angiogenesis and tumor immunotherapy
发表期刊：Bioact Mater. (IF=20.3)
DOI：https://doi.org/10.1016/j.bioactmat.2026.03.016
使用 Absin 产品：ECL化学发光检测试剂盒（货号：abs920）

Fig. 1. Aminated fullerene exhibits potent anti-angiogenic activity. (a) Schematic workflow for screening aminated fullerene derivatives. Synthesized compounds were evaluated in a HUVEC tube formation assay. TAPC emerged as the lead anti-angiogenic candidate and was further validated using the CAM assay and DSWC model. (b) Representative images of capillary-like networks formed by HUVEC cells after treatment. Scale bar, 100 μm. (c-d) Quantification of total tube length in HUVEC cells. (e) CAM images 72 h post-topical treatment with TAPC (0.125-1 mM), bevacizumab (positive control), or PBS (control). Circular regions of interest (ROIs) denote avascular zones for vascular quantification Scale bar, 1 mm. (f) Quantified vascularized area within CAM ROIs, n = 5. (g) Intravital fluorescence imaging of tumor vasculature in the DSWC model post-intravenous TAPC injection. Red cycles indicate progressive microvessel rupture and hemorrhage. Scale bars, 1000 μm. Data are presented as mean ± SEM. Statistical analyses were performed using one-way ANOVA with Tukey's post hoc test, ∗∗∗∗p < 0.0001.

Fig. 3. TAPC interacts with VEGFR2 and modulates downstream signaling. (a) Cell viability assay of MC38 cells treated with increasing concentrations of TAPC. (b) Immunoblot analysis of VEGFR2 and key regulators of the PI3K–AKT signaling pathway (PI3K, AKT, and STAT3) in MC38 cells treated with PEG-PO or TAPC (5 and 10 μM). β-Actin was used as a loading control. (c) Pull-down assay of VEGFR2 from MC38 cell lysates using biotinylated TAPC, beads-only sample served as control. (d) Confocal IF imaging of MC38 cells incubated with Cy5.5-labeled TAPC and stained for VEGFR2, nuclei counterstained with DAPI. Scale bars: 20 μm. (e) BLI analysis of TAPC binding to recombinant VEGFR2 using serial concentrations (100, 66.7, 44.4, 29.6, 19.8, 13.2, and 8.8 μM). (f) Molecular dynamics simulations showing predicted protein–ligand complexes (top) and binding pocket visualizations (bottom) of VEGFR2 with TAPC, NDMPFI, MBAMF, and TPFE. (g) Binding free energy calculations of these complexes, including van der Waals, electrostatic, solvation, and total energy components. (h) Extracellular acidification rate (ECAR) of MC38 cells treated with control (0 μM), TAPC (2.5 μM), or TAPC (10 μM), with sequential addition of glucose, oligomycin, and 2-deoxyglucose (2-DG). (i) Quantification of glycolysis and glycolytic capacity in MC38 cells treated with control (0 μM), TAPC (2.5 μM), or TAPC (10 μM) (n = 8). Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Tukey's multiple comparisons test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 4. Characterization and cellular uptake of TAPC@CNPs. (a) Schematic illustration of TAPC@CNP fabrication. (b) Photographs of nanoparticle suspensions of CNPs and TAPC@CNPs. (c) TEM image of TAPC@CNPs. Scale bar: 100 nm. (d) DLS hydrodynamic size distribution of TAPC@CNPs. (e) Zeta potential measurement of PLGA, TAPC, TAPC-PLGA, cell membranes and TAPC@CNPs. (f) SDS-PAGE/Coomassie staining of TAPC@CNPs and MC38 cell membranes. (g) Hydrodynamic size of TAPC@CNPs measured by DLS after incubation in 10% serum at 37 °C over time. (h) Hydrodynamic size of TAPC@CNPs measured by DLS during storage at 4 °C over the indicated days. (i) Confocal fluorescence images of MC38 cells incubated with Cy5.5-labeled TAPC@CNPs (red) for 6 h at 37 °C, nuclei were counterstained with DAPI (blue). The dashed line indicates the cell boundary. Scale bar: 10 μm. (j) Flow cytometry quantification of cellular uptake of Cy5.5-labeled TAPC@CNPs in MC38 cells after 6 h incubation, presented as MFI. (k) Release profile of TAPC-Cy5.5@CNPs at pH 7.4 and pH 6.0 over time, expressed as release percentage. Data are mean ± SEM. Statistical analysis by unpaired two-tailed t-test, ∗∗∗p < 0.001.

Fig. 6. Biodistribution and pharmacokinetic analysis of TAPC@CNPs. (a) Ex vivo fluorescence imaging of major organs and tumors collected at 24 h, 48 h, 4 days, and 7 days after intravenous injection of free Cy5.5 or Cy5.5-labeled TAPC@CNPs (n = 3). Organs are displayed from left to right in the following order: heart, liver, spleen, lung, kidney, and tumor. (b) Quantification of fluorescence intensity in spleen at the indicated time points. (c) Quantification of fluorescence intensity in tumors at the indicated time points. (d) Pharmacokinetic profile of Cy5.5-labeled TAPC@CNPs based on serum fluorescence intensity measured at different time points after intravenous injection. The data were fitted using a one-phase exponential decay model, and the calculated circulation half-life is shown. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparisons test, ∗∗p < 0.01.

Fig. 5. In vivo anti-tumor and anti-angiogenic effects of TAPC@CNPs. (a) Schematic illustration of the therapeutic study in Balb/c mice bearing subcutaneous MC38 tumors (n = 7). (b) Body weights of mice during treatment. (c) Photographs of excised tumors collected at endpoint. (d) Tumor growth curves during treatment. Tumor volume was calculated using the formula (length × width2)/2. (e) Tumor weights measured at endpoint. (f) Immunoblot analysis of VEGFR2 expression in tumor lysates from different treatment groups, β-actin was used as a reference protein. (g) IHC staining of CD31 in tumor sections from different treatment groups. Scale bar, 100 μm. (h) H&E staining of major organs (heart, liver, spleen, lung, kidney) and tumor tissues. (i) Serum ALT and AST levels measured at endpoint. Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparisons test, ns indicates not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig. 7. Immune cell profiling in lymph nodes, spleen, and tumors following TAPC@CNP treatment. (a) Representative flow cytometry plots showing CD4 and Foxp3 expression in lymph node cells from Control and TAPC@CNP-treated groups. (b) Quantification of the percentage of CD4+Foxp3+ regulatory T cells among CD4+ T cells in lymph nodes from different treatment groups. (c) Quantification of the percentage of CD4+ T cells in spleen from different treatment groups. (d) IF staining of tumor sections for CD3+ T cells (red), CD4+ T cells (green), and nuclei (DAPI, blue) in different treatment groups. Scale bar, 200 μm. (e) Quantification of CD8+ T cells as a percentage of tumor-infiltrating lymphocytes (TILs) from Control and TAPC@CNP-treated groups. (f) Quantification of CD206 expression as a percentage of CD45+CD11b+ myeloid cells in tumor samples. (g) Quantification of CD69 expression in CD8+ T cells. (h) Representative flow cytometry plots showing intracellular IFNγ expression in CD8+ T cells from tumor samples. (i) Quantification of IFNγ+ CD8+ T cells from Control and TAPC@CNP-treated groups. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparisons test or unpaired two-tailed Student's t-test, ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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