Journal: Theranostics

Article Title: NGR-modified nanovesicles target ALKBH5 to inhibit ovarian cancer growth and metastasis

doi: 10.7150/thno.107766

Figure Lengend Snippet: Preparation and Characterization of NGR-Modified BNVs. Note: (A) Schematic diagram of the preparation process of NGR-ALKBH5-siRNA-BNVs (BNVs: biomimetic nanovesicles; ALKBH5-siRNA-BNVs: biomimetic nanovesicles encapsulating ALKBH5 siRNA; NGR-ALKBH5-siRNA-BNVs: NGR-modified biomimetic nanovesicles encapsulating ALKBH5 siRNA); (B) Particle size distribution of BNVs, ALKBH5-siRNA-BNVs, and NGR-ALKBH5-siRNA-BNVs; (C) Transmission electron microscope (TEM) images of BNVs, ALKBH5-siRNA-BNVs, and NGR-ALKBH5-siRNA-BNVs (scale bar: 50 μm); (D) Fluorescence qPCR results of ALKBH5@BNVs; (E) UV-visible spectrophotometry analysis of the absorbance values of NGR peptide and NGR-BNVs (NGR peptide exhibits an absorption peak at a specific wavelength, which, combined with a standard curve, enables calculation of peptide concentration and modification efficiency); (F) HPLC chromatograms showing characteristic peaks of NGR peptide, BNVs, and NGR-BNVs; (G) Zeta potential analysis of BNVs, ALKBH5-siRNA-BNVs, and NGR-ALKBH5-siRNA-BNVs; (H) Flow cytometry analysis of drug accumulation and targeting capability of BNVs and NGR-BNVs in SKOV3 and OVCAR-3 cells. All experiments were repeated three times.

Article Snippet: Log-phase SKOV3 OC cells (ATCC, USA) were suspended in pH 7.4 PBS and adjusted to a concentration of 2×10 6 cells/100 μL.

Techniques: Modification, Transmission Assay, Microscopy, Fluorescence, Spectrophotometry, Concentration Assay, Zeta Potential Analyzer, Flow Cytometry

Journal: Theranostics

Article Title: NGR-modified nanovesicles target ALKBH5 to inhibit ovarian cancer growth and metastasis

doi: 10.7150/thno.107766

Figure Lengend Snippet: Impact of NGR-Modified BNVs on OC Cell Proliferation and Apoptosis. Note: (A) Schematic representation of the process of NGR-modified BNVs treatment of OC cells; (B) CCK-8 assay evaluating the proliferative capacity of SKOV3 cells in different treatment groups. Compared to the control group, both the NGR-modified nanovesicle group and the ALKBH5 siRNA nanovesicle group significantly inhibited cell proliferation, with the NGR-modified ALKBH5 siRNA nanovesicle group showing the most significant inhibitory effect. Rescue experiments assessed the proliferative capacity of SKOV3 cells after treatment with NGR-modified ALKBH5 siRNA nanovesicles. The results indicated that upon the overexpression of ITGB1 plasmids, the cell proliferation capacity partially recovered, suggesting the crucial role of ITGB1 in the modulation of cell proliferation by NGR-modified ALKBH5 siRNA nanovesicles. (C) CCK-8 assay assessing the proliferative capacity of OVCAR-3 cells in different treatment groups. Both the NGR-modified nanovesicle group and the ALKBH5 siRNA nanovesicle group significantly suppressed cell proliferation, with the NGR-modified ALKBH5 siRNA nanovesicle group demonstrating the most pronounced effect. Rescue experiments on OVCAR-3 cells evaluated the proliferative and apoptotic rates after treatment with NGR-modified ALKBH5 siRNA nanovesicles. The addition of ITGB1 overexpressing plasmids led to a partial recovery in cell proliferation capacity, further confirming the critical role of ITGB1. (D) Flow cytometry analysis of apoptosis rates in different treatment groups of SKOV3 cells. The NGR-modified ALKBH5 siRNA nanovesicle group induced the highest apoptosis rate, significantly higher than other treatment groups. Rescue experiments on SKOV3 cells treated with NGR-modified ALKBH5 siRNA nanovesicles showed a significant decrease in apoptosis rate upon the addition of ITGB1 overexpressing plasmids, indicating the important role of ITGB1 in the induction of cell apoptosis by NGR-modified ALKBH5 siRNA nanovesicles. (E) Flow cytometry analysis of apoptosis rates in different treatment groups of OVCAR-3 cells (Q1 represents necrotic cells, Q2 represents late apoptotic cells, Q3 represents early apoptotic cells, and Q4 represents viable cells). Similarly, the NGR-modified ALKBH5 siRNA nanovesicle group induced the highest apoptosis rate, significantly higher than other treatment groups. Rescue experiments on OVCAR-3 cells after treatment with NGR-modified ALKBH5 siRNA nanovesicles demonstrated a significant decrease in apoptosis rate upon the addition of ITGB1 overexpressing plasmids, further validating the critical role of ITGB1. The experiments were repeated three times, and the data in the figures are presented as mean ± SD. ANOVA was used for statistical analysis, with * indicating p < 0.05 and *** indicating p < 0.001.

Article Snippet: Log-phase SKOV3 OC cells (ATCC, USA) were suspended in pH 7.4 PBS and adjusted to a concentration of 2×10 6 cells/100 μL.

Techniques: Modification, CCK-8 Assay, Control, Over Expression, Flow Cytometry

Journal: Theranostics

Article Title: NGR-modified nanovesicles target ALKBH5 to inhibit ovarian cancer growth and metastasis

doi: 10.7150/thno.107766

Figure Lengend Snippet: The Impact of NGR-Modified ALKBH5 siRNA Nanovesicles on Immune Cell Activity Regulating OC Cell Growth. Note: (A) Differential effects of OC cells in various treatment groups on T cell apoptosis in co-culture experiments. The NGR-modified ALKBH5 siRNA nanovesicle group significantly increased the apoptosis rate of OC cells induced by T cells. The rescue experiment assessed the sensitivity of SKOV3 cells to T cells after treatment with NGR-modified ALKBH5 siRNA nanovesicles. Partial restoration of OC cell sensitivity to T cells was observed upon transfection with PD-L1 overexpressing plasmid, indicating the crucial role of ITGB1 in immune evasion. Electron microscopy images showed apoptotic cellular morphology: NGR-modified ALKBH5 siRNA nanovesicles notably intensified the apoptotic morphology of OC cells induced by T cells, including cell shrinkage, chromatin condensation, and formation of apoptotic bodies. (B) The effects of different treatment groups of OC cells on T cell proliferation inhibition in co-culture experiments. The NGR-modified ALKBH5 siRNA nanovesicle group significantly inhibited the proliferation of OC cells. The rescue experiment evaluated the sensitivity of OVCAR-3 cells to T cells after treatment with NGR-modified ALKBH5 siRNA nanovesicles. Partial restoration of OC cell sensitivity to T cells was observed upon transfection with ITGB1 overexpressing plasmid, further confirming the critical role of ITGB1. Electron microscopy of apoptotic morphology: The NGR-modified ALKBH5 siRNA nanovesicle group significantly exacerbated the T cell-induced apoptotic morphology of OC cells, including cell shrinkage, chromatin condensation, and apoptotic body formation. (C) Western blot analysis of IFN-γ secretion levels in the supernatant of co-culture in different treatment groups. The NGR-modified ALKBH5 siRNA nanovesicle group significantly increased the secretion of IFN-γ. (D) Western blot analysis of TNF-α secretion levels in the supernatant of co-culture in different treatment groups. The NGR-modified ALKBH5 siRNA nanovesicle group significantly increased the secretion of TNF-α (Q1 represents necrotic cells, Q2 represents late apoptotic cells, Q3 represents early apoptotic cells, Q4 represents viable cells). All experiments were repeated three times, and the data in the figures are presented as mean ± SD. ANOVA was used for statistical analysis, with * indicating p < 0.05 and *** indicating p < 0.001.

Article Snippet: Log-phase SKOV3 OC cells (ATCC, USA) were suspended in pH 7.4 PBS and adjusted to a concentration of 2×10 6 cells/100 μL.

Techniques: Modification, Activity Assay, Co-Culture Assay, Transfection, Plasmid Preparation, Electron Microscopy, Inhibition, Western Blot

Journal: Theranostics

Article Title: NGR-modified nanovesicles target ALKBH5 to inhibit ovarian cancer growth and metastasis

doi: 10.7150/thno.107766

Figure Lengend Snippet: Regulation of ITGB1 and ALKBH5 Gene and Protein Expression by NGR-Modified ALKBH5 siRNA Nanovesicles. Note: (A) RT-qPCR analysis of ITGB1 gene expression in SKOV3 cells; (B) RT-qPCR analysis of ALKBH5 expression in SKOV3 cells; (C) Western blot analysis of ITGB1 and ALKBH5 protein expression in SKOV3 cells; (D) RT-qPCR analysis of ITGB1 gene expression in OVCAR-3 cells; (E) RT-qPCR analysis of ALKBH5 gene expression in OVCAR-3 cells; (F) Western Blot analysis of ITGB1 and ALKBH5 protein expression in OVCAR-3 cells; (G) Immunofluorescence staining of the co-localization of ALKBH5 and ITGB1 in SKOV3 cells; (H) Immunofluorescence staining in OVCAR-3 cells to assess the co-localization of ALKBH5 and ITGB1 proteins. All experiments were repeated three times, and the data are presented as mean ± SD. Statistical significance was determined by ANOVA, with * indicating p < 0.05 and *** indicating p < 0.001.

Article Snippet: Log-phase SKOV3 OC cells (ATCC, USA) were suspended in pH 7.4 PBS and adjusted to a concentration of 2×10 6 cells/100 μL.

Techniques: Expressing, Modification, Quantitative RT-PCR, Gene Expression, Western Blot, Immunofluorescence, Staining