Neurological Emergency Treatment Strategy: A Neuron-Targeted Regulation System for Reactive Oxygen Species Metabolism through Ferroptosis Modulation

Figure 1. Validation of preliminary SCI targets for emergency intervention (A) Flowchart of the experiment. (B) Transcriptome KEGG analysis. (C) Transcriptome GSEA analysis. (D) Western blotting showing the expression of TFR, FPN, FTH, and FTL in each group (tissue). (E) Quantitative analysis of TFR protein expression in each group. (F) Quantitative analysis of FPN protein expression in each group. (G) Quantitative analysis of FTH protein expression in each group. (H) Quantitative analysis of FTL protein expression in each group.

Figure 2. Preparation and characterization of the DTLS@CAT liposome. (A) Synthetic route of DSPE-PEG-TPP. (B) Synthetic route of DSPE-PEG-THRre. (C) 1H NMR spectrum of DSPE-PEG-TPP. (D) 1H NMR spectrum of DSPE-PEG-DBCO. (E) 1H NMR spectrum of DSPE-PEG-THRre. (F) DLS measurement of DTLS@CAT and DTLS. (G) TEM image of DTLS@CAT. (H) O2 concentration curves in H2O2 solutions (1 mM) treated with DTLS@CAT and DTLS and measured by a portable dissolved oxygen meter. (I) O2 concentration changes in H2O2 solutions (1 mM) after incubation with DTLS@CAT with different concentrations of CAT (0.06, 0.25, and 0.50 mg/mL, respectively) and PBS. (J) Relative enzymatic activity changes of free CAT and DTLS@CAT under protease K digestion (0.5 mg/mL) for different periods of time. (K) Photograph showing the O2 production of free CAT and DTLS@CAT in H2O2 solutions before and after protease K digestion for 24 h.

Figure 3. Validation of Targeted and Biocompatibility of DTLS@CAT. (A) Immunofluorescence expression of FITC in PC12, BV2, HEK293, and HEPG2 cells in the control, LS, THRreLS, TPPLS, and DTLS groups. Magnification: 20×; scale bar: 100 μm. (B) Immunofluorescence staining of Calcein-AM (green) and PI (red) in PC12 cells in the control, LS, THRreLs, TPPLS, and DTLS groups. Magnification: 20×; scale bar: 100 μm. (C) Isolated imaging of mouse spinal cord with different treatments. (D) Measurement of the fluorescence intensity of materials injected into the organism. (E,F) Material targeting performance statistics. The targeting ratio means the comparison between different materials, and the targeting rate means the fluorescence intensity in the spinal cord/the total intensity of the injected material. *P < 0.05 and ** represents P < 0.01 vs the LS group. #P < 0.05. ##P < 0.01. n.s. stands for no statistical significance. The data are represented as mean ± standard deviation (SD) (n = 3).

Figure 4. Assessment of ROS scavenging efficacy in DTLS@CAT. (A) Immunofluorescence staining of ROS (green) in PC12 cells in the control, FINO2, THRreLS, THRreLS@CAT, and DTLS@CAT groups. Magnification: 20×; scale bar: 100 μm. (B) Flow analysis of ROS in each group. (C) Immunofluorescence staining of mitoSOX (green), MitoTracker (red), and DAPI (blue) in PC12 cells in the control, FINO2, THRreLS, THRreLS@CAT, and DTLS@CAT groups. Magnification: 100×; scale bar: 20 μm. (D) Flow analysis of mitoSOX and MitoTracker in each group. (E) Immunofluorescence staining of FITC (green), MitoTracker (red), and DAPI (blue) in PC12 cells in the THRreLS, THRreLS@CAT, and DTLS@CAT groups. Magnification: 100×; scale bar: 20 μm. (F) Statistical analysis of FITC expression in each group. (G) Statistical analysis of FITC expression in MitoTracker in each group. (H) Immunofluorescence staining of FITC (green), NeuN (red), and DAPI (blue) in the control, THRreLS, THRreLS@CAT, and DTLS@CAT groups at 24 h. Magnification: 20×; scale bar: 100 μm. (I) Immunofluorescence staining of FITC (green), Iba-1 (red), and DAPI (blue) in each group at 24 h. Magnification: 20×; scale bar: 100 μm. *P < 0.05 and **P < 0.01 vs the…

Figure 5. Validation of ferroptosis inhibition mediated by DTLS@CAT. (A) Immunofluorescence staining of TFR (green), LysoTracker (red), DAPI (blue), and light field in PC12 cells in the control, FINO2, THRreLS, THRreLS@CAT, and DTLS@CAT groups. Magnification: 100×; scale bar: 20 μm. (B) Statistical analysis of TFR expression in each group. (C) Statistical analysis of TFR expression in LysoTracker cells in each group. (D) Model diagram of material mechanism of action. (E) Western blotting showing the expression of NOX2, GPX4, MitoFT, TFR, and FPN in each group (cell). (F) Western blotting showing the expression of NOX2, GPX4, MitoFT, TFR, and FPN in each group (tissue). (G,I,K,M,O) Quantitative analysis of NOX2, GPX4, MitoFT, TFR, and FPN protein expression in cells. (H,J,L,N,P) Quantitative analysis of NOX2, GPX4, MitoFT, TFR, and FPN protein expression in tissues. *P < 0.05 and **P < 0.01 vs the control group in the comparison of the control, THRreLS, THRreLS@CAT, and DTLS@CAT groups. *P < 0.05 and **P < 0.01 vs the control + FINO2 group in the comparison of the control + FINO2, THRreLS + FINO2, THRreLS@CAT + FINO2, and DTLS@CAT + FINO2 groups. *P < 0.05 and **P < 0.01 vs the control (6…

Figure 6. Validation of the potential mechanism of DTLS@CAT in inhibiting ferroptosis. (A) Transcriptome GO analysis. (B)Transcriptome GSEA analysis. (C) Western blotting showing the expression of CTSD, Parkin, PINK, and UCP2 in each group (cell) at 24 h. (D) Western blotting showing the expression of CTSD, Parkin, PINK, and UCP2 in each group (tissue) at 6 h and 24 h. (E,G,I,K) Quantitative analysis of CTSD, Parkin, PINK, and UCP2 protein expression in cells. (F,H,J,L) Quantitative analysis of CTSD, Parkin, PINK, and UCP2 protein expression in tissue. **P < 0.01 vs the control group in the comparison of the control, THRreLS, THRreLS@CAT, and DTLS@CAT groups. ** P < 0.01 vs the control + FINO2 group in the comparison of the control + FINO2, THRreLS + FINO2, THRreLS@CAT + FINO2, and DTLS@CAT + FINO2 groups. **P < 0.01 vs the control (6 h) group in the comparison of the control (6 h), THRreLS (6 h), THRreLS@CAT (6 h), and DTLS@CAT (6 h) groups. **P < 0.01 vs the control (24 h) group in the comparison of the control (24 h), THRreLS (24 h), THRreLS@CAT (24 h), and DTLS@CAT (24 h) groups. #represents P < 0.05. ## represents P < 0.01. n.s. stands for no statistical significance. The data are…

Figure 7. Validation of DTLS@CAT for promoting axon regeneration and ensuring axon stability. (A) Immunofluorescence staining of NF200 (green), GFAP (red), and DAPI (blue) in the SCI, THRreLS, THRreLS@CAT, and DTLS@CAT groups in 30 d.p.i. Scale bar: 1 mm. (B) Statistical analysis of axon penetration in each group. (C) Statistical analysis of damage area in each group. (D) Immunofluorescence staining of Ace tubulin (green), Tyr tubulin (red), and DAPI (blue) in the SCI, THRreLS, THRreLS@CAT, and DTLS@CAT groups at 24 h. Scale bar: 1 mm. (E) Statistical analysis of microtubule stability area in each group. (F) Statistical analysis of Ace tubulin/Tyr tubulin in each group. **P < 0.01 vs the SCI group. # represents P < 0.05, and ## represents P < 0.01. n.s. stands for no statistical significance. The data are represented as mean ± SD (n = 3).

Figure 8. Validation of tissue architecture and behavioral repair in mice by DTLS@CAT. (A) H&E and Nissl staining of the spinal cord of mice with different treatments. (B) Electrophysiological measurement of each group at Day 30. In the figure, each small space in the horizontal direction represents 1 ms, and each small space in the vertical direction represents 5 mV. (C) BMS score of mice at the corresponding time points. (D) Gait marks of the mice in each group at Day 30. Scale bar: 1 mm. **P < 0.01 vs the SCI group. The data are represented as mean ± SD (n = 3).

Figure 9. Comprehensive analysis of motor function recovery in mice following DTLS@CAT therapy. (A) High-frame rate camera image showing the motion of the mice in each group. The dots represent the leg joints of the mouse, and the white lines are the connections between the joints. (B) Representative color bar charts of motor hind limb movement in mice. (C) Footstep analysis of motor hind limb movement in mice. (D) Quantitative analysis of maximal hip height, maximal toe height, maximal muscle tension, and cycle duration in different treatment mice. *P < 0.05 and **P < 0.01 vs the SCI group. n.s. stands for no statistical significance. The data are represented as mean ± SD (n = 3).

Figure 10. Comparative analysis of the efficacy of DTLS@CAT and clinical hormone pulse therapy. (A) Immunofluorescence staining of NeuN (red), DAPI (blue) in the SCI, TDTLS@CAT, and dexamethasone groups at severe injury, moderate injury, and mild injury. (B–D) Statistical analysis of NeuN expression in each group with different injury degrees. (E–G) Statistical analysis of BMS scores for different degrees of injury in each group. *P < 0.05 and **P < 0.01 vs the SCI group. # represents P < 0.05 and ## represents P < 0.01. The data are represented as mean ± SD (n = 3).