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Preparation and characterization of DoxFILN and their interaction with 4T1 or CTLL‐2 cells. a) Representative schemes and transmission electron microscope (TEM) images of I‐pBMDCN, Dox‐Fn, and DoxFILN in different pH. The samples were stained with 2% phosphotungstic acid. b) Size distribution of DoxFILN and Dox‐Fn in buffers of pH 6.5 or pH 7.4. All data were determined by a Zetasizer. c) The Dox release profiles of Dox‐Fn, DoxFILN −M70 , and DoxFILN in buffers of pH 5.0, pH 6.4, or pH 7.4. All data were presented as mean ± s.d. ( n = 3 samples per group). d) SDS‐PAGE protein analysis (top) and Western blot analysis of <t>IL15Rα</t> protein (bottom) in I‐pBMDCN, I‐pBMDC membrane, and DoxFILN. e) Profiles of the concentration of IL15c on DoxFILN in 72 h in pH 6.5 or pH 7.4. f) Flow cytometry determination of the cellular uptake of FITC‐labelled Fn and Cy5‐labelled membrane protein by 4T1 cells or CTLL‐2 cells after 4 h incubation with FILN −M70 or FILN in buffers of pH 6.5 (IL15c at 10 ng mL −1 equivalent). Data were presented as mean ± s.d. ( n = 3 biological replicates per group) and statistically analyzed using the Student's t ‐test. g) Representative confocal images showing intracellular localization of FITC‐labelled Fn and Cy5‐labelled membrane protein. The cells were stained with Hoechst 33 342 (blue). The scale bars represent a distance of 20 µm. h) Cell viability of 4T1 or CTLL‐2 after being treated by DoxFILN −M70 or DoxFILN in pH 6.5. Data were presented as mean ± s.d. ( n = 3 biological replicates per group) and statistically analyzed using the Student's t ‐test. i) Mechanism of cell‐specific drug delivery by DoxFILN.
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Preparation and characterization of DoxFILN and their interaction with 4T1 or CTLL‐2 cells. a) Representative schemes and transmission electron microscope (TEM) images of I‐pBMDCN, Dox‐Fn, and DoxFILN in different pH. The samples were stained with 2% phosphotungstic acid. b) Size distribution of DoxFILN and Dox‐Fn in buffers of pH 6.5 or pH 7.4. All data were determined by a Zetasizer. c) The Dox release profiles of Dox‐Fn, DoxFILN −M70 , and DoxFILN in buffers of pH 5.0, pH 6.4, or pH 7.4. All data were presented as mean ± s.d. ( n = 3 samples per group). d) SDS‐PAGE protein analysis (top) and Western blot analysis of <t>IL15Rα</t> protein (bottom) in I‐pBMDCN, I‐pBMDC membrane, and DoxFILN. e) Profiles of the concentration of IL15c on DoxFILN in 72 h in pH 6.5 or pH 7.4. f) Flow cytometry determination of the cellular uptake of FITC‐labelled Fn and Cy5‐labelled membrane protein by 4T1 cells or CTLL‐2 cells after 4 h incubation with FILN −M70 or FILN in buffers of pH 6.5 (IL15c at 10 ng mL −1 equivalent). Data were presented as mean ± s.d. ( n = 3 biological replicates per group) and statistically analyzed using the Student's t ‐test. g) Representative confocal images showing intracellular localization of FITC‐labelled Fn and Cy5‐labelled membrane protein. The cells were stained with Hoechst 33 342 (blue). The scale bars represent a distance of 20 µm. h) Cell viability of 4T1 or CTLL‐2 after being treated by DoxFILN −M70 or DoxFILN in pH 6.5. Data were presented as mean ± s.d. ( n = 3 biological replicates per group) and statistically analyzed using the Student's t ‐test. i) Mechanism of cell‐specific drug delivery by DoxFILN.
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Preparation and characterization of DoxFILN and their interaction with 4T1 or CTLL‐2 cells. a) Representative schemes and transmission electron microscope (TEM) images of I‐pBMDCN, Dox‐Fn, and DoxFILN in different pH. The samples were stained with 2% phosphotungstic acid. b) Size distribution of DoxFILN and Dox‐Fn in buffers of pH 6.5 or pH 7.4. All data were determined by a Zetasizer. c) The Dox release profiles of Dox‐Fn, DoxFILN −M70 , and DoxFILN in buffers of pH 5.0, pH 6.4, or pH 7.4. All data were presented as mean ± s.d. ( n = 3 samples per group). d) SDS‐PAGE protein analysis (top) and Western blot analysis of <t>IL15Rα</t> protein (bottom) in I‐pBMDCN, I‐pBMDC membrane, and DoxFILN. e) Profiles of the concentration of IL15c on DoxFILN in 72 h in pH 6.5 or pH 7.4. f) Flow cytometry determination of the cellular uptake of FITC‐labelled Fn and Cy5‐labelled membrane protein by 4T1 cells or CTLL‐2 cells after 4 h incubation with FILN −M70 or FILN in buffers of pH 6.5 (IL15c at 10 ng mL −1 equivalent). Data were presented as mean ± s.d. ( n = 3 biological replicates per group) and statistically analyzed using the Student's t ‐test. g) Representative confocal images showing intracellular localization of FITC‐labelled Fn and Cy5‐labelled membrane protein. The cells were stained with Hoechst 33 342 (blue). The scale bars represent a distance of 20 µm. h) Cell viability of 4T1 or CTLL‐2 after being treated by DoxFILN −M70 or DoxFILN in pH 6.5. Data were presented as mean ± s.d. ( n = 3 biological replicates per group) and statistically analyzed using the Student's t ‐test. i) Mechanism of cell‐specific drug delivery by DoxFILN.
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Preparation and characterization of DoxFILN and their interaction with 4T1 or CTLL‐2 cells. a) Representative schemes and transmission electron microscope (TEM) images of I‐pBMDCN, Dox‐Fn, and DoxFILN in different pH. The samples were stained with 2% phosphotungstic acid. b) Size distribution of DoxFILN and Dox‐Fn in buffers of pH 6.5 or pH 7.4. All data were determined by a Zetasizer. c) The Dox release profiles of Dox‐Fn, DoxFILN −M70 , and DoxFILN in buffers of pH 5.0, pH 6.4, or pH 7.4. All data were presented as mean ± s.d. ( n = 3 samples per group). d) SDS‐PAGE protein analysis (top) and Western blot analysis of IL15Rα protein (bottom) in I‐pBMDCN, I‐pBMDC membrane, and DoxFILN. e) Profiles of the concentration of IL15c on DoxFILN in 72 h in pH 6.5 or pH 7.4. f) Flow cytometry determination of the cellular uptake of FITC‐labelled Fn and Cy5‐labelled membrane protein by 4T1 cells or CTLL‐2 cells after 4 h incubation with FILN −M70 or FILN in buffers of pH 6.5 (IL15c at 10 ng mL −1 equivalent). Data were presented as mean ± s.d. ( n = 3 biological replicates per group) and statistically analyzed using the Student's t ‐test. g) Representative confocal images showing intracellular localization of FITC‐labelled Fn and Cy5‐labelled membrane protein. The cells were stained with Hoechst 33 342 (blue). The scale bars represent a distance of 20 µm. h) Cell viability of 4T1 or CTLL‐2 after being treated by DoxFILN −M70 or DoxFILN in pH 6.5. Data were presented as mean ± s.d. ( n = 3 biological replicates per group) and statistically analyzed using the Student's t ‐test. i) Mechanism of cell‐specific drug delivery by DoxFILN.

Journal: Advanced Science

Article Title: Interleukin 15‐Presenting Nanovesicles with Doxorubicin‐Loaded Ferritin Cores for Cancer Immunochemotherapy

doi: 10.1002/advs.202409194

Figure Lengend Snippet: Preparation and characterization of DoxFILN and their interaction with 4T1 or CTLL‐2 cells. a) Representative schemes and transmission electron microscope (TEM) images of I‐pBMDCN, Dox‐Fn, and DoxFILN in different pH. The samples were stained with 2% phosphotungstic acid. b) Size distribution of DoxFILN and Dox‐Fn in buffers of pH 6.5 or pH 7.4. All data were determined by a Zetasizer. c) The Dox release profiles of Dox‐Fn, DoxFILN −M70 , and DoxFILN in buffers of pH 5.0, pH 6.4, or pH 7.4. All data were presented as mean ± s.d. ( n = 3 samples per group). d) SDS‐PAGE protein analysis (top) and Western blot analysis of IL15Rα protein (bottom) in I‐pBMDCN, I‐pBMDC membrane, and DoxFILN. e) Profiles of the concentration of IL15c on DoxFILN in 72 h in pH 6.5 or pH 7.4. f) Flow cytometry determination of the cellular uptake of FITC‐labelled Fn and Cy5‐labelled membrane protein by 4T1 cells or CTLL‐2 cells after 4 h incubation with FILN −M70 or FILN in buffers of pH 6.5 (IL15c at 10 ng mL −1 equivalent). Data were presented as mean ± s.d. ( n = 3 biological replicates per group) and statistically analyzed using the Student's t ‐test. g) Representative confocal images showing intracellular localization of FITC‐labelled Fn and Cy5‐labelled membrane protein. The cells were stained with Hoechst 33 342 (blue). The scale bars represent a distance of 20 µm. h) Cell viability of 4T1 or CTLL‐2 after being treated by DoxFILN −M70 or DoxFILN in pH 6.5. Data were presented as mean ± s.d. ( n = 3 biological replicates per group) and statistically analyzed using the Student's t ‐test. i) Mechanism of cell‐specific drug delivery by DoxFILN.

Article Snippet: The primary antibodies used for Western blot were against IL15Rα (R&D, AF551), IL2RG (proteintech, 11409‐1‐AP), Transferrin Receptor (abcam, cat. no. ab214039), Phospho‐Jak1 (Cell Signaling Technology, 74129S), Phospho‐Stat5 (Cell Signaling Technology, 4322T).

Techniques: Transmission Assay, Microscopy, Staining, SDS Page, Western Blot, Membrane, Concentration Assay, Flow Cytometry, Incubation