Journal: Advanced Science

Article Title: Acid‐Responsive Nanobot‐Integrated Core‐Shell Microneedles Reprogram the Degenerative Annulus Fibrosus Microenvironment Through Epigenetic Suppression of Ferroptosis

doi: 10.1002/advs.77829

Figure Lengend Snippet: Schematic diagram of the nanobot‐integrated core‐shell microneedle system for sequential annulus fibrosus repair. In this work, we developed a nanobot‐integrated core‐shell microneedle system (TMH/QG MN) for annulus fibrosus repair, which presents a transformative strategy that couples self‐driven microenvironmental normalization with epigenetic modulation. Specifically, the shell layer contains metal‐phenolic nanobots that react with pathological H + to generate O 2 , thereby alleviating hypoxia and acidosis while providing enzyme‐mimetic and photothermal‐enhanced antioxidant activity. The core layer sustains the release of quercetin, which exerts anti‐ferroptotic and anti‐inflammatory effects. Mechanistically, TMH/QG MN suppressed ferroptosis in annulus fibrosus cells by epigenetically silencing ATF3 through DNMT3A‐dependent promoter methylation, thereby relieving ATF3‐mediated repression of SLC7A11 and restoring GPX4‐dependent antioxidant defense. Collectively, these actions alleviated ferroptotic injury, restored matrix homeostasis, enhanced biomechanical resilience, and ultimately achieved annulus fibrosus repair.

Article Snippet: Following blocking with protein‐free rapid blocking solution (Servicebio, G2052‐500ML), the membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution) against ACSL4 (proteintech, 22401‐1‐AP), GPX4 (Abclonal, A1933), MMP3 (proteintech, 17873‐1‐AP), MMP13 (Abclonal, A11148), COL1 (proteintech, 14695‐1‐AP), DNMT1 (proteintech, 24206‐1‐AP), DNMT3A (proteintech, 20954‐1‐AP), DNMT3B (proteintech, 26971‐1‐AP), LaminB1 (proteintech, 12987‐1‐AP), ATF3 (Abclonal, A13469) and β‐actin (BOSTER, BA2305).

Techniques: Antioxidant Activity Assay, Methylation

Journal: Advanced Science

Article Title: Acid‐Responsive Nanobot‐Integrated Core‐Shell Microneedles Reprogram the Degenerative Annulus Fibrosus Microenvironment Through Epigenetic Suppression of Ferroptosis

doi: 10.1002/advs.77829

Figure Lengend Snippet: Degenerative AF is characterized by redox imbalance and ferroptosis activation. (A) Schematic illustration of single‐cell RNA‐seq in human degenerative annulus fibrosus tissues. (B) The tSNE plot visualizes six distinct clusters of human AF cells from intervertebral disc samples. (C) Dot plot displaying the marker genes specific to each AF cell sub‐cluster (mild = Pfirrmann II‐III, severe = Pfirrmann IV‐V). (D) The scores of ferroptosis suppressors and drivers were calculated using the AddModuleScore function. (E) Scores for the pentose phosphate pathway, glutathione metabolism, and peroxisome function were computed to assess the metabolic and antioxidant status of AF cells. (F) Fatty acid degradation and phospholipase D signaling pathway activities were evaluated to examine lipid metabolism and membrane remodeling in AF cells. (G) The volcano plot depicts 6284 DEGs between mild ( p < 0.001) and severe ( p < 0.001) AF samples. (H,I) Enrichment analysis of DEGs was performed to identify significantly enriched biological pathways in mild and severe AF samples. (J) MRI scans of human intervertebral discs from mild and severe degeneration groups. (K,L) Relative protein and mRNA expression levels of the key ferroptosis regulators ACSL4 and GPX4 in human mild and severe degenerative disc tissues. The corresponding uncropped blot images are provided in Table . (M‐N) IHC staining and quantification of positive cells for ACSL4 and GPX4 in mild and severe degenerative IVD samples. Scale bar: 20 µm. (O–Q) GSH content, MDA content, and Fe 2+ levels in mild and severe degenerative IVD samples. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using two‐tailed Student's t ‐test [(D), (E), (F), (K), (L), (M), (O), (P), and (Q)].

Article Snippet: Following blocking with protein‐free rapid blocking solution (Servicebio, G2052‐500ML), the membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution) against ACSL4 (proteintech, 22401‐1‐AP), GPX4 (Abclonal, A1933), MMP3 (proteintech, 17873‐1‐AP), MMP13 (Abclonal, A11148), COL1 (proteintech, 14695‐1‐AP), DNMT1 (proteintech, 24206‐1‐AP), DNMT3A (proteintech, 20954‐1‐AP), DNMT3B (proteintech, 26971‐1‐AP), LaminB1 (proteintech, 12987‐1‐AP), ATF3 (Abclonal, A13469) and β‐actin (BOSTER, BA2305).

Techniques: Activation Assay, Single Cell, RNA Sequencing, Marker, Membrane, Analysis, Expressing, Immunohistochemistry, Two Tailed Test

Journal: Advanced Science

Article Title: Acid‐Responsive Nanobot‐Integrated Core‐Shell Microneedles Reprogram the Degenerative Annulus Fibrosus Microenvironment Through Epigenetic Suppression of Ferroptosis

doi: 10.1002/advs.77829

Figure Lengend Snippet: TMH/QG+NIR attenuates ferroptosis and matrix degradation in TBHP‐treated AFCs. (A) Schematic illustration of AF cells co‐culture system. (B) DCFH‐DA fluorescence staining images and quantitative analysis of TBHP‐pretreated AFCs after different treatments. Scale bar: 50 µm. (C) Intracellular ROS levels in TBHP‐pretreated AFCs measured by flow cytometry analysis and quantitative results. (D,E) Quantitative analysis of MDA and GSH levels in TBHP‐pretreated AFCs co‐cultured with QG, TMH and TMH/QG MN (with NIR irradiation). (F,G) Representative images and quantification of labile iron pool by FerroOrange staining and lipid peroxidation by C11‐BODIPY staining in TBHP‐pretreated AFCs co‐culture with QG, TMH and TMH/QG MN (with NIR irradiation). Scale bar: 20 µm. (H) Western blot analysis and corresponding quantitative statistics of extracellular matrix (COL1), remodeling markers (MMP3, MMP13), and ferroptosis‐related proteins (ACSL4, GPX4). The corresponding uncropped blot images are provided in Table . (I) Immunofluorescence staining and fluorescence intensity quantification of COL1 and MMP3 expression in TBHP‐pretreated AFCs co‐cultured with QG, TMH, and TMH/QG MN (with NIR irradiation). Scale bar: 20 µm. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using one‐way ANOVA with Tukey's multiple comparisons test [(B) to (I)].

Article Snippet: Following blocking with protein‐free rapid blocking solution (Servicebio, G2052‐500ML), the membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution) against ACSL4 (proteintech, 22401‐1‐AP), GPX4 (Abclonal, A1933), MMP3 (proteintech, 17873‐1‐AP), MMP13 (Abclonal, A11148), COL1 (proteintech, 14695‐1‐AP), DNMT1 (proteintech, 24206‐1‐AP), DNMT3A (proteintech, 20954‐1‐AP), DNMT3B (proteintech, 26971‐1‐AP), LaminB1 (proteintech, 12987‐1‐AP), ATF3 (Abclonal, A13469) and β‐actin (BOSTER, BA2305).

Techniques: Co-culture Assay, Fluorescence, Staining, Analysis, Flow Cytometry, Cell Culture, Irradiation, Western Blot, Immunofluorescence, Expressing

Journal: Advanced Science

Article Title: Acid‐Responsive Nanobot‐Integrated Core‐Shell Microneedles Reprogram the Degenerative Annulus Fibrosus Microenvironment Through Epigenetic Suppression of Ferroptosis

doi: 10.1002/advs.77829

Figure Lengend Snippet: Transcriptomic profiling identifies ATF3 as a key regulator of TMH/QG+NIR‐mediated ferroptosis inhibition in degenerative AFCs. (A) RNA sequencing of AFCs treated with either TBHP+NIR or TBHP+NIR+TMH/QG. (B) Volcano plot identifying 3571 DEGs between TBHP+NIR and TBHP+NIR+TMH/QG groups. (C,D) Functional enrichment analysis of DEGs using GO biological processes and KEGG pathways. (E–G) GSEA plots demonstrating significant enrichment of processes related to extracellular matrix degradation, oxidative stress and ferroptosis. (H) 25 key genes identified from the intersection of ferroptosis driver genes and DEG_Down, 14 key genes identified from the intersection of ferroptosis suppressor genes and DEG_Up. (I) Cluster analysis of DEGs highlighting specific ferroptosis driver and suppressor genes. (J) PPI network of the top 28 hub genes with Atf3 ranked as the top hub gene. (K) 2 key genes ( Atf3 and Trib2 ) identified from the intersection of key genes and DEG_Top50. (L) Western blot analysis of ATF3 and TRIB2 protein expression levels in TBHP+NIR and TBHP+NIR+TMH/QG groups. The corresponding uncropped blot images are provided in Table . (M) Cluster analysis of DEGs showing the Integrated Stress Response between TBHP+NIR and TBHP+NIR+TMH/QG groups. (N) Relative mRNA expression levels of Ddit3 and Chac1 . (O) Representative images and quantitative analysis of C11‐BODIPY staining to assess lipid peroxidation in shAtf3 and LV‐Atf3 groups. Scale bar: 20 µm. (P) Immunofluorescence staining of ATF3 in the TBHP+TMH/QG+NIR+LV‐Atf3 group compared to the TBHP+TMH/QG+NIR+LV‐NC control group. Scale bar: 20 µm. (Q) Western blot analysis of ECM (COL1, MMP3, and MMP13) and ferroptosis (ACSL4 and GPX4) markers in TBHP+TMH/QG+NIR+LV‐Atf3 and TBHP+TMH/QG+NIR+LV‐NC groups. The corresponding uncropped blot images are provided in Table . (R) Flow cytometry analysis of intracellular ROS levels in TBHP+TMH/QG+NIR+LV‐Atf3 and TBHP+TMH/QG+NIR+LV‐NC groups. (S,T) C11‐BODIPY and FerroOrange staining images in TBHP+TMH/QG+NIR+LV‐Atf3 and TBHP+TMH/QG+NIR+LV‐NC groups. Scale bar: 20 µm. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using two‐tailed Student's t‐ test (N).

Article Snippet: Following blocking with protein‐free rapid blocking solution (Servicebio, G2052‐500ML), the membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution) against ACSL4 (proteintech, 22401‐1‐AP), GPX4 (Abclonal, A1933), MMP3 (proteintech, 17873‐1‐AP), MMP13 (Abclonal, A11148), COL1 (proteintech, 14695‐1‐AP), DNMT1 (proteintech, 24206‐1‐AP), DNMT3A (proteintech, 20954‐1‐AP), DNMT3B (proteintech, 26971‐1‐AP), LaminB1 (proteintech, 12987‐1‐AP), ATF3 (Abclonal, A13469) and β‐actin (BOSTER, BA2305).

Techniques: Profiling, Inhibition, RNA Sequencing, Functional Assay, Analysis, Western Blot, Expressing, Staining, Immunofluorescence, Control, Flow Cytometry, Two Tailed Test

Journal: Advanced Science

Article Title: Acid‐Responsive Nanobot‐Integrated Core‐Shell Microneedles Reprogram the Degenerative Annulus Fibrosus Microenvironment Through Epigenetic Suppression of Ferroptosis

doi: 10.1002/advs.77829

Figure Lengend Snippet: DNMT3A‐mediated ATF3 promoter methylation underlies the anti‐ferroptotic effect of TMH/QG+NIR in degenerative AFCs. (A) GSEA plots identify significant enrichment of DEGs in the DNA methylation pathway. (B,C) Methylation‐specific PCR and corresponding quantification of the Atf3 promoter methylation level in degenerative AFCs treated with the TMH/QG+NIR microneedle system. The corresponding uncropped gel images are provided in Table . (D) Western blot analysis of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) in the TBHP+NIR and TBHP+NIR+TMH/QG groups. The corresponding uncropped blot images are provided in Table . (E) ChIP‐qPCR analysis of DNMT1, DNMT3A, and DNMT3B enrichment at the Atf3 promoter region. (F) ChIP‐qPCR analysis specifically quantifying DNMT3A enrichment at the Atf3 promoter. (G) Measurement of MDA content. (H) Western blot analysis of SLC7A11 and ATF3 protein levels in the TBHP+NIR+TMH/QG group with or without DY‐46‐2. The corresponding uncropped blot images are provided in Table . (I) Relative mRNA expression level of Slc7a11 and Atf3 in the TBHP+NIR+TMH/QG group with or without the DNMT3A inhibitor DY‐46‐2. (J) Flow cytometry analysis of intracellular ROS levels. (K) Representative FerroOrange staining images in the TBHP+NIR+TMH/QG group with or without DY‐46‐2. Scale bar: 20 µm. (L) Western blot analysis of key ferroptosis (ACSL4 and GPX4) and extracellular matrix (COL1, MMP3 and MMP13) markers in the TBHP+NIR+TMH/QG group with or without DY‐46‐2. The corresponding uncropped blot images are provided in Table . (M) IHC staining of ATF3, SLC7A11 and DNMT3A expression in human disc tissues from mild and severe degeneration groups. Scale bar: 20 µm. (N) MSP of the ATF3 promoter methylation level in human mild and severe degeneration groups. The corresponding uncropped gel images are provided in Table . (O) Western blot analysis of ATF3, SLC7A11 and DNMT3A protein expression in human disc tissues from mild and severe degeneration groups. The corresponding uncropped blot images are provided in Table . (P) Relative mRNA expression of ATF3 in human mild and severe degeneration groups. (Q–S) Correlation analysis of relative ATF3 expression in AF tissue and relative GPX4 , SLC7A11 and DNMT3A expression. (T) Schematic model illustrating the proposed mechanism. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using two‐tailed Student's t ‐test [(C), (F), (G), (I) and (P)] and one‐way ANOVA with Tukey's multiple comparisons test (E).

Article Snippet: Following blocking with protein‐free rapid blocking solution (Servicebio, G2052‐500ML), the membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution) against ACSL4 (proteintech, 22401‐1‐AP), GPX4 (Abclonal, A1933), MMP3 (proteintech, 17873‐1‐AP), MMP13 (Abclonal, A11148), COL1 (proteintech, 14695‐1‐AP), DNMT1 (proteintech, 24206‐1‐AP), DNMT3A (proteintech, 20954‐1‐AP), DNMT3B (proteintech, 26971‐1‐AP), LaminB1 (proteintech, 12987‐1‐AP), ATF3 (Abclonal, A13469) and β‐actin (BOSTER, BA2305).

Techniques: Methylation, DNA Methylation Assay, Western Blot, Analysis, ChIP-qPCR, Expressing, Flow Cytometry, Staining, Immunohistochemistry, Tissue, Two Tailed Test

Journal: Advanced Science

Article Title: Acid‐Responsive Nanobot‐Integrated Core‐Shell Microneedles Reprogram the Degenerative Annulus Fibrosus Microenvironment Through Epigenetic Suppression of Ferroptosis

doi: 10.1002/advs.77829

Figure Lengend Snippet: Preclinical evaluation of the TMH/QG+NIR strategy for AF repair. (A) Schematic illustration of the therapeutic strategy for rat tail puncture‐induced IDD using TMH/QG MN under NIR irradiation. (B) Infrared thermographic images of rat tails treated with QG, TMH and TMH/QG MNs under NIR irradiation. (C) Representative X‐ray and T2‐weighted MRI images of rat tails from the indicated treatment groups at 4‐ and 8‐weeks post‐treatment. (D–F) Quantitative assessment of disc degeneration: DHI, MRI grading scores and disc water content at 4 and 8 weeks (n = 5). (G,H) Histological analysis of AF tissues at 4 and 8 weeks. Representative H&E and SO/FG staining images. (I) Methylation‐specific PCR analysis and quantification of the CpG island CGI‐2 region in the rat Atf3 promoter. The corresponding uncropped gel images are provided in Table . (J) Western blot analysis of ATF3, DNMT3A and ferroptosis markers (ACSL4 and GPX4) in disc tissues. The corresponding uncropped blot images are provided in Table . (K) Immunohistochemical staining of ATF3, DNMT3A, ACSL4, GPX4, COL1 and MMP3 expression in AF tissues. (L–N) Biochemical assays of ferroptosis‐related factors in disc tissues: Fe 2+ content, MDA level and GSH level at 8 weeks post‐treatment. (O) Compressive modulus of IVDs from the Con, IDD and TMH/QG+NIR groups. (P) Energy dissipation efficiency (U hys ) of IVDs from Con, IDD and TMH/QG+NIR groups. Data are presented as mean ± SD (n ≥ 3), and p ‐values were calculated using one‐way ANOVA with Tukey's multiple comparisons test [(D), (F), (I), and (L) to (O)] and Kruskal‐Wallis test with Dunn's multiple comparisons test (E).

Article Snippet: Following blocking with protein‐free rapid blocking solution (Servicebio, G2052‐500ML), the membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution) against ACSL4 (proteintech, 22401‐1‐AP), GPX4 (Abclonal, A1933), MMP3 (proteintech, 17873‐1‐AP), MMP13 (Abclonal, A11148), COL1 (proteintech, 14695‐1‐AP), DNMT1 (proteintech, 24206‐1‐AP), DNMT3A (proteintech, 20954‐1‐AP), DNMT3B (proteintech, 26971‐1‐AP), LaminB1 (proteintech, 12987‐1‐AP), ATF3 (Abclonal, A13469) and β‐actin (BOSTER, BA2305).

Techniques: Irradiation, Infrared, Analysis, Staining, Methylation, Western Blot, Immunohistochemical staining, Expressing