Journal: Advanced Science

Article Title: In Situ Genetic Engineering Facilitates MMP‐9‐Responsive TIMP1 Release Through Proteolytic Cleavage to Boost Diabetic Wound Healing

doi: 10.1002/advs.202524328

Figure Lengend Snippet: Characterization of the MMP9‐responsive protein release system. (A) Schematic illustration of TIMP1 membrane anchoring and three candidate cleavage peptide sequences (by FigDraw). (B) Representative confocal microscopy images showing the cellular localization of the GPI anchor and three candidate peptide sequences fused to ssEGFP (EGFP with secretion signals): ssEGFP (free ssEGFP), ssEGFP‐P1/P2/P3 (ssEGFP fused with three candidate peptide sequences). CellMask (red, cell membrane); Hoechst 33342 (blue, cell nucleus). Scale bars: 20 µm. (C) Detection of SEAP activity from three SEAP‐peptide fusion proteins with or without MMP9 (1000 ng/ml) treatment at the indicated time points. Statistical analyses were performed between the MMP9‐treated group and the untreated control for each fusion protein at each time point. n = 3. (D) Detection of SEAP activity from three SEAP‐peptide fusion proteins at 6 h post‐treatment with different concentrations of MMP9. Statistical analysis was performed among different concentrations of MMP9 for each fusion protein. n = 3. (E) Comparison of the responsiveness of the three SEAP‐peptide fusion proteins to MMP2 and MMP9 enzymes at the indicated time points. Statistical analysis was performed between the MMP2‐treated group and the MMP9‐treated group for each fusion protein at each time point. n = 3. (F) Detection of SEAP activity at different time points in various cell lines after transfection with SEAP‐P2 fusion protein. Statistical analyses were performed among different time points for each cell line. n = 3. (G) Representative confocal microscopy images of ssEGFP‐P2 fusion protein localization following 6 h of MMP9 treatment. CellMask (red, cell membrane), Hoechst 33342 (blue, cell nucleus). Scale bars: 20 µm. (H) Quantitative analysis of ssEGFP fluorescence intensity at the cell membrane. The fluorescence intensity was normalized to the signal intensity on the cell membrane. n = 3 independent experiments. (I) Dynamic changes in SEAP activity in the supernatant following periodic MMP9 treatment of cells transfected with SEAP‐P2 fusion protein. The SEAP activity of the MMP9 treatment group was normalized to that of the corresponding non‐MMP9 treatment group. n = 3. (J) Detection of TIMP1 concentration in the supernatant from cells transfected with TIMP1‐P2 fusion protein at the indicated time points after treatment with MMP9. n = 3. (K) Detection of MMP9 activity in the culture medium using a fluorescent substrate of MMP‐9 (MOCAc‐PLGL(Dpa)AR). Control and TIMP1‐P2 fusion protein (TIMP1 M9RR )‐transfected cells were incubated in the medium containing the indicated concentrations of MMP9 for 12 h. n = 3. Data are presented as mean ± s.e.m.; Two‐way ANOVA with Fisher's LSD test (C, D, E, F, I, K); Unpaired T‐test (H); One‐way ANOVA with Fisher's LSD test (J); ns, not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001; Values sharing the same letter are not significantly different (p > 0.05).

Article Snippet: The primary antibodies used were: TIMP1 antibody (1:200, ABclonal, A1389), MMP‐9 antibody (1:200, ABclonal, A25299), collagen I antibody (1:200, ABclonal, A24112), and collagen III antibody (1:200, ABclonal, A3795).

Techniques: Membrane, Confocal, Microscopy, Activity Assay, Control, Analysis, Comparison, Transfection, Fluorescence, Concentration Assay, Incubation

Journal: Advanced Science

Article Title: In Situ Genetic Engineering Facilitates MMP‐9‐Responsive TIMP1 Release Through Proteolytic Cleavage to Boost Diabetic Wound Healing

doi: 10.1002/advs.202524328

Figure Lengend Snippet: On‐demand TIMP1 delivery rescues MMP9‐induced impairment in vitro. (A) Representative images (left) and quantification analysis (right) of the EdU assay in control, MMP9, and MMP9+ TIMP1 M9RR HaCaT cell groups. Scale bars: 40 µm; n = 3. (B) Representative images (left) and quantification analysis (right) of the EdU assay in control, MMP9, and MMP9+ TIMP1 M9RR BJ cell groups. Scale bars: 40 µm; n = 3. (C) Representative images of the scratch‐wound assay in control, MMP9, and MMP9+ TIMP1 M9RR HaCaT cell groups. Scale bars: 200 µm. (D) Quantitative analysis of the scratch wound coverage in different HaCaT cell groups. Statistical analysis was performed in different groups at each time point. n = 3. (E) Representative images of the scratch‐wound assay in control, MMP9, and MMP9+ TIMP1 M9RR BJ cell groups. Scale bars: 200 µm. (F) Quantitative analysis of the scratch wound coverage in different BJ cell groups. Statistical analysis was performed in different groups at each time point. n = 3. (G) Heatmap of representative differentially expressed genes in different HaCaT samples. Colors represent relative expression levels (hotter colors indicate higher expression). (H) GO analyses of the upregulated (left) and downregulated (right) biological processes (BP) in MMP9+ TIMP1 M9RR ‐treated HaCaT groups compared to MMP9‐treated HaCaT samples. (I) Heatmap of the representative differentially expressed genes in different BJ samples. Colors represent relative expression levels (hotter colors indicate higher expression). (J) GO analyses of the downregulated biological processes (BP) in MMP9+ TIMP1 M9RR ‐treated BJ groups compared to MMP9‐treated BJ samples. (K‐L) qPCR analysis of target gene expression in HaCaT (K) or BJ (L) cells under different treatments. Cells were either transfected (control) or transfected with empty lipid nanoparticles (Empty LNP), LNP‐TIMP1, or LNP‐TIMP1 M9RR , followed by a 24‐h treatment with MMP9 at the indicated concentrations n = 3. Data are shown as mean ± s.e.m.; One‐way ANOVA with Fisher's LSD test (A, B, K, L); Two‐way ANOVA with Fisher's LSD test (D, F). P < 0.05, ** p < 0.01, and *** p < 0.001; values sharing the same letter are not significantly different (p > 0.05).

Article Snippet: The primary antibodies used were: TIMP1 antibody (1:200, ABclonal, A1389), MMP‐9 antibody (1:200, ABclonal, A25299), collagen I antibody (1:200, ABclonal, A24112), and collagen III antibody (1:200, ABclonal, A3795).

Techniques: In Vitro, Analysis, EdU Assay, Control, Scratch Wound Assay Assay, Expressing, Targeted Gene Expression, Transfection

Journal: Advanced Science

Article Title: In Situ Genetic Engineering Facilitates MMP‐9‐Responsive TIMP1 Release Through Proteolytic Cleavage to Boost Diabetic Wound Healing

doi: 10.1002/advs.202524328

Figure Lengend Snippet: TIMP1 M9RR promotes wound healing in db/db mice. (A) Timeline for in vivo experiments in db/db mice. (B) Gross view (left) and simulation plots of the wound closure areas (right) of wounds from mice in different groups as indicated on days 0, 3, 7, and 14 post‐surgery. Scale bars: 2 mm. (C) Quantitative analysis of wound closure rates over time for each group. n = 6. (D) Representative images of H&E staining of wound sections in different groups at the indicated time points. Scale bar: 500 µm. (E‐F) Quantitative measurements of wound width (D) and granulation tissue thickness (E). n = 6. (G) High‐magnification images showed epithelial tongues in different groups on day 3 and day 7. Scale bars: 250 µm. (H) Quantitative analysis of epithelial tongue length. n = 6. (I) Representative images of Masson's trichrome staining of wound sections in different groups at the indicated time points. Scale bar: 50 µm. (J) Quantification analysis of the collagen‐occupied area percentage. n = 6. (K) Representative images of Sirius Red‐stained wounded skin (left), birefringent collagen under polarized light (middle), and color‐coded map of collagen fiber orientation (right) from different groups. BF, bright field; PL, polarized light; 0° corresponds to the horizontal axis of the image. Scale bars: 100 µm. (L) Representative histogram showing the distribution of collagen fiber orientations. The orientation analysis was performed using OrientationJ software. Data are shown as mean ± s.e.m.; Two‐way ANOVA with Fisher's LSD test (C, E, H, J); One‐way ANOVA with Fisher's LSD test (F); p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The primary antibodies used were: TIMP1 antibody (1:200, ABclonal, A1389), MMP‐9 antibody (1:200, ABclonal, A25299), collagen I antibody (1:200, ABclonal, A24112), and collagen III antibody (1:200, ABclonal, A3795).

Techniques: In Vivo, Analysis, Staining, Tissue, Software

Journal: Advanced Science

Article Title: In Situ Genetic Engineering Facilitates MMP‐9‐Responsive TIMP1 Release Through Proteolytic Cleavage to Boost Diabetic Wound Healing

doi: 10.1002/advs.202524328

Figure Lengend Snippet: TIMP1 M9RR modulates immune response, collagen remodeling, and tissue regeneration in diabetic wounds. (A) Heatmap of the representative DEGs in different BJ samples. Colors represent relative expression levels (hotter colors indicate higher expression). (B‐C) GO analyses of the upregulated (B) and downregulated (C) biological processes (BP) in TIMP1 M9RR groups compared to untreated db/db samples. (D) Representative images showing the expression of IL‐10 (green) and iNOS (red) in wound regions from mice in different groups on day 3 and day 7. Nuclei are stained with Hoechst 33342 (blue). Scale bars: 50 µm. (E) Quantitative analysis of IL‐10 and iNOS positive area percentage on day 3 and day 7. n = 6. (F) Representative immunohistochemical images showing the expression of collagen I and collagen III in the wound region from mice in different groups on day 7 and day 14. Scale bars: 100 µm. (G) Quantitative analysis of collagen I and collagen III positive area percentage on day 7 and day 14. n = 6. (H) Representative immunofluorescent staining of CD31 (red), β3‐Tubulin (TUBB3, green), and Cytokeratin 18 (K19, yellow) in the wound regions of mice in different groups on day 14. Nuclei are visualized with Hoechst 33342 (blue). Scale bars: 50 µm. (I) Quantitative analysis of CD31, TUBB3, and K19 positive area percentage on day 14. n = 6. Data are shown as mean ± s.e.m. One‐way ANOVA with Fisher's LSD test (E, J, I). Values sharing the same letter are not significantly different (p > 0.05).

Article Snippet: The primary antibodies used were: TIMP1 antibody (1:200, ABclonal, A1389), MMP‐9 antibody (1:200, ABclonal, A25299), collagen I antibody (1:200, ABclonal, A24112), and collagen III antibody (1:200, ABclonal, A3795).

Techniques: Tissue, Expressing, Staining, Analysis, Immunohistochemical staining

Journal: Advanced Science

Article Title: In Situ Genetic Engineering Facilitates MMP‐9‐Responsive TIMP1 Release Through Proteolytic Cleavage to Boost Diabetic Wound Healing

doi: 10.1002/advs.202524328

Figure Lengend Snippet: Fabrication and characterization of Cryo‐Microneedles (CryoMNs). (A) Schematic illustration of CryoMNs and their application mode to skin wounds. (B) The force‐displacement curve of CryoMNs loaded with bFGF and lipid nanoparticles‐TIMP1 M9RR (CryoMNs@bFGF/TIMP1 M9RR ). The red circle indicates the mechanical failure of the CryoMNs@bFGF/ TIMP1 M9RR . (C) Gross view (up) and H&E staining image (down) of mouse skin with micropores formed after applying CryoMNs. Scale bars: 50 µm. (D) Melting behavior of CryoMNs at room temperature (25°C) over time. (E) Cargo penetration simulated by cryo‐microneedles loaded with Rhodamine B. Scale bars: 3 mm. (F) Quantitative analysis of the penetration area over time as shown in (E, down‐Longitudinal view). (G) Representative fluorescence images of EGFP in HaCaT cells transfected by lipid nanoparticles‐EGFP mRNA (LNP‐EGFP) and CryoMNs loaded with LNP‐EGFP (LNP‐EGFP+CryoMNs). Scale bars: 50 µm. (H) Quantitative analysis of transfection efficiency of LNP‐EGFP and LNP‐EGFP+CryoMNs in HaCaT cells. n = 3. (I) Representative fluorescence images of EGFP in BJ cells transfected with lipid nanoparticles‐EGFP mRNA (LNP‐EGFP) and CryoMNs loaded with LNP‐EGFP (LNP‐EGFP+CryoMNs). Scale bars: 50 µm. (H) Quantitative analysis of transfection efficiency of LNP‐EGFP and LNP‐EGFP+CryoMNs in BJ cells. n = 3. (K) In vivo imaging of C57 mice 24 h without and with application of CryoMNs loaded with lipid nanoparticles‐luciferase mRNA. (I) Comparison of average radiance [p/s/cm2/sr] between different groups. n = 4. Data are shown as mean ± s.e.m.; unpaired T‐test (H, J); multiple unpaired T‐test (L); ns, not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The primary antibodies used were: TIMP1 antibody (1:200, ABclonal, A1389), MMP‐9 antibody (1:200, ABclonal, A25299), collagen I antibody (1:200, ABclonal, A24112), and collagen III antibody (1:200, ABclonal, A3795).

Techniques: Staining, Analysis, Fluorescence, Transfection, In Vivo Imaging, Luciferase, Comparison

Journal: Advanced Science

Article Title: In Situ Genetic Engineering Facilitates MMP‐9‐Responsive TIMP1 Release Through Proteolytic Cleavage to Boost Diabetic Wound Healing

doi: 10.1002/advs.202524328

Figure Lengend Snippet: Both TIMP1 M9RR and bFGF enhance the therapeutic effect of bFGF on diabetic wound healing. (A) Gross view (left) and simulation plots of the wound closure areas (right) of wounds from mice in different groups on days 0, 3, 7, and 14 post‐surgery. Scale bars: 2 mm. (B) Quantitative analysis of wound closure rates over time for each group. n = 6. (C) Representative images of H&E staining of wound sections in different groups at the indicated time points. Scale bar: 500 µm. (D‐E) Quantitative measurements of wound width (D) and granulation tissue thickness (E). n = 6. (F) High‐magnification images showed epithelial tongues in different groups on day 3 and day 7. Scale bars: 250 µm. (G) Quantitative analysis of epithelial tongue length. n = 6. (H) Representative images of Masson's trichrome staining of wound sections in different groups at the indicated time points. Scale bar: 50 µm. (I) Quantification analysis of the collagen‐occupied area percentage. n = 6. (J) Representative images of Sirius Red‐stained wounded skin (left), birefringent collagen under polarized light (middle), and color‐coded map of collagen fiber orientation (right) from different groups. BF, bright field; PL, polarized light; 0° corresponds to the horizontal axis of the image. Scale bars: 100 µm. (K) Representative histogram showing the distribution of collagen fiber orientations. Orientation analysis was performed using OrientationJ software. Data are shown as mean ± s.e.m.; Two‐way ANOVA with Fisher's LSD test (B, D, G, I); One‐way ANOVA with Fisher's LSD test (E); p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The primary antibodies used were: TIMP1 antibody (1:200, ABclonal, A1389), MMP‐9 antibody (1:200, ABclonal, A25299), collagen I antibody (1:200, ABclonal, A24112), and collagen III antibody (1:200, ABclonal, A3795).

Techniques: Analysis, Staining, Tissue, Software

Journal: Advanced Science

Article Title: In Situ Genetic Engineering Facilitates MMP‐9‐Responsive TIMP1 Release Through Proteolytic Cleavage to Boost Diabetic Wound Healing

doi: 10.1002/advs.202524328

Figure Lengend Snippet: Both TIMP1 M9RR and bFGF modulate immune response, collagen remodeling, and tissue regeneration in diabetic wounds. (A) Representative images showing the expression of IL‐10 (green) and iNOS (red) in wound regions from mice in different groups on day 3. Nuclei are stained with Hoechst 33342 (blue). Scale bars: 50 µm. (B) Quantitative analysis of the IL‐10 and iNOS positive area percentage on day 3. n = 6. (C) Representative immunohistochemical images showing the expression of IL‐10 (green) and iNOS (red) in wound regions from mice in different groups on day 7. Nuclei are stained with Hoechst 33342 (blue). Scale bars: 50 µm. (D) Quantitative analysis of the IL‐10 and iNOS positive area percentage on day 7. n = 6. (E) Representative images showing the expression of collagen I and collagen III in the wound region from mice in different groups on day 7. Scale bars: 100 µm. (F) Quantitative analysis of collagen I and collagen III positive area percentage on day 7. n = 6. (G) Representative images showing the expression of collagen I and collagen III in the wound region from mice in different groups on day 14. Scale bars: 100 µm. (H) Quantitative analysis of the collagen I and collagen III positive area percentage on day 14. n = 6. (I) Representative immunofluorescent staining of CD31 (red), β3‐Tubulin (TUBB3, green), and Cytokeratin 18 (K19, yellow) in the wound regions of mice in different groups on day 14. Nuclei are visualized with Hoechst 33342 (blue). Scale bars: 50 µm. (J) Quantitative analysis of CD31, TUBB3, and K19 positive area percentage on day 14. n = 6. Data are shown as mean ± s.e.m.; One‐way ANOVA with Fisher's LSD test (E, J, I); Values sharing the same letter are not significantly different (p > 0.05).

Article Snippet: The primary antibodies used were: TIMP1 antibody (1:200, ABclonal, A1389), MMP‐9 antibody (1:200, ABclonal, A25299), collagen I antibody (1:200, ABclonal, A24112), and collagen III antibody (1:200, ABclonal, A3795).

Techniques: Tissue, Expressing, Staining, Analysis, Immunohistochemical staining