Nano‐Plumber Reshapes Glymphatic‐Lymphatic System to Sustain Microenvironment Homeostasis and Improve Long‐Term Prognosis after Traumatic Brain Injury

Abstract Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Long‐term changes in the microenvironment of the brain contribute to the degeneration of neurological function following TBI. However, current research focuses primarily on short‐term modulation during the early phases of TBI, not on the critical significance of long‐term homeostasis in the brain microenvironment. Notably, dysfunction of the glymphatic‐lymphatic system results in the accumulation of danger/damage‐associated molecular patterns (DAMPs) in the brain, which is regarded as the leading cause of long‐term microenvironmental disturbances following TBI. Here, a nanostructure, Nano‐plumber, that co‐encapsulates the microenvironment regulator pro‐DHA and the lymphatic‐specific growth factor VEGF‐C is developed, allowing for a sustainable and orderly regulation of the microenvironment to promote long‐term neurological recovery. Nano‐plumber reverses the injury microenvironment by suppressing microglia and astrocytes activation and maintaining reduced activation via enhanced glymphatic‐lymphatic drainage, and significantly improves the neurological function of rodents with TBI. This study demonstrates that glymphatic‐lymphatic system reconstruction is essential for enhancing long‐term prognosis following TBI, and that the Nano‐plumber developed here may serve as a clinically translatable treatment option for TBI.


Figure S4 .
Figure S4.Representative scheme and sequence of plasmid.a) Representative scheme of plasmid simutaneously encoding VEGF-C and EGFP.b) The sequence encoding VEGF-C in the plasmid.

Figure S5 .
Figure S5.Cell-selective uptake of Nano-plumber (n = 3).Data represent mean ± SD of three independent biological replicates.Statistical analyses are performed using oneway ANOVA with Tukey's post hoc test.

Figure S6 .
Figure S6.The anti-inflammatory effects of pro-DHA and Vorinostat on microglial.a, b) Flow cytometry analysis of CD80 + cells in activated BV2 cells with different concentrations of (a) pro-DHA or (b) Vorinostat treatments (n = 3).Data represent mean ± SD of three independent biological replicates.Statistical analyses are performed using one-way ANOVA with Tukey's post hoc test.

Figure S7 .
Figure S7.The modulatory effects of pro-DHA and Vorinostat on microglial phagocytosis capacity.a, b) Flow cytometry analysis of the phagocytic ability of dysfunctional BV2 cells with different concentrations of (a) pro-DHA or (b) Vorinostat treatments (n = 3).Data represent mean ± SD of three independent biological replicates.Statistical analyses are performed using one-way ANOVA with Tukey's post hoc test.

Figure S8 .
Figure S8.Representative images of the expression of Claudin-5 on bEnd.3 cells with different treatments after OGD (red: claudin-5).The experiments were repeated three times independently.

Figure S9 .
Figure S9.Representative images of the expression of ZO-1 on bEnd.3 cells with different treatments after OGD (blue: Hoechst, red: ZO-1).The experiments were repeated three times independently.

Figure S10 .
Figure S10.The protective effects of pro-DHA and Vorinostat on bEnd.3 cells.a, b) Cell viability of the bEnd.3 cells with different concentrations of (a) pro-DHA or (b) Vorinostat treatments after OGD (n = 3).Data represent mean ± SD of three independent biological replicates.Statistical analyses are performed using one-way ANOVA with Tukey's post hoc test.

Figure S11 .
Figure S11.The proliferative effects of pro-DHA and Vorinostat on bEnd.3 cells.a, b) Cell proliferation of the bEnd.3 cells with different concentrations of (a) pro-DHA or

Figure S12 .
Figure S12.The migratory effects of pro-DHA and Vorinostat on bEnd.3 cells.a) Brightfield images of the wound healing process of bEnd.3 in basolateral well after different concentrations of pro-DHA or Vorinostat treatments.b) Quantification analysis of bEnd.3 fusion rate (n = 3).Data represent mean ± SD of three independent biological replicates.Statistical analyses are performed using one-way ANOVA with Tukey's post hoc test.

Figure S13 .
Figure S13.The promotion of tight junction formation in bEnd.3 cells by pro-DHA and Vorinostat.a, b) Representative images of the expression of (a) Claudin-5 and (b) ZO-1 on bEnd.3 cells with different concentrations of pro-DHA or Vorinostat treatments after OGD.The experiments were repeated three times independently.

Figure S14 .
Figure S14.Cell morphology of bEnd.3 cells with different concentrations of pro-DHA or Vorinostat treatments after OGD.The experiments were repeated three times independently.

Figure S15 .
Figure S15.The transfection efficacy of Nano-plumber using different administration protocols in vivo.a) Representative images of transfection of Nano-plumber using different administration protocols in vivo.b) Quantitative analysis showing the fluorescence intensity of GFP corresponding to (a) (n = 3).Data represent mean ± SD of three independent biological replicates.Statistical analyses are performed using one-way ANOVA with Tukey's post hoc test.

Figure S16 .
Figure S16.The effect of Nano-plumber on promoting the meningeal lymphangiogenesis using different administration protocols in vivo.a) Representative images of meningeal lymphatic vessels (Lyve-1) of using different administration protocols at 28 days after injury (blue: DAPI, green: Lyve-1).b) Quantification of the percent area coverage of Lyve-1 antibody staining (n = 3).c, d) Quantification of the number of (c) loops and (d) sprouts in meningeal whole mounts (n = 3).e) Quantification of the diameters of the meningeal lymphatic vessels.Each data point represents an independent mouse and is an average of 70 measurements along the transverse and superior sagittal sinuses per mouse (n = 3).Data represent mean ± SD of three independent biological replicates.Statistical analyses are performed using one-

Figure S19 .
Figure S19.Quantification of the expression level of Arg-1 in the injury brain at 7 days after injury (n = 6).Data represent mean ± SD of six independent biological replicates.Statistical analyses are performed using one-way ANOVA with Tukey's post hoc test.

Figure S20 .
Figure S20.The long-term anti-inflammatory effect of Nano-plumber in vivo.a, b) Quantification of the expression level of (a) iNOS and (b) Arg-1 in the brain at 28 days after injury (n = 6).Data represent mean ± SD of six independent biological replicates.Statistical analyses are performed using one-way ANOVA with Tukey's post hoc test.

Figure S22 .
Figure S22.Cytotoxicity assess on BV2 cells.a, b) Cytotoxicity assess at different concentrations of (a) pro-DHA or (b) Nano-plumber on BV2 cells (n = 3).Data represent mean ± SD of three independent biological replicates.Statistical analyses are performed using one-way ANOVA with Tukey's post hoc test.

Figure S23 .
Figure S23.Cytotoxicity assess on HT22 cells.a, b) Cytotoxicity assess at different concentrations of (a) pro-DHA or (b) Nano-plumber on HT22 cells (n = 3).Data represent mean ± SD of three independent biological replicates.Statistical analyses are performed using one-way ANOVA with Tukey's post hoc test.

Figure S24 .
Figure S24.Cytotoxicity assess on bEnd.3 cells.a, b) Cytotoxicity assess at different concentrations of (a) pro-DHA or (b) Nano-plumber on bEnd.3 cells (n = 3).Data represent mean ± SD of three independent biological replicates.Statistical analyses are performed using one-way ANOVA with Tukey's post hoc test.

Figure S25 .
Figure S25.H&E staining to assess the toxicity of various formulations on major organs.Scale bar, 100 µm.The experiments were repeated three times independently.