Journal: Journal of Extracellular Vesicles

Article Title: Unravelling Ligand Conjugation Performance in Extracellular Vesicles: A Quantitative Assessment of Lipid, Protein, and Membrane Modifications

doi: 10.1002/jev2.70174

Figure Lengend Snippet: The impact of ligand conjugation on cell uptake of mEVs‐Tf. (a) Schematic illustration of the principle for evaluating the correlation between ligand conjugation and cell uptake. (b) Confocal fluorescence microscopy analysis of the cell uptake of DiD‐labelled blank mEVs and the transferrin conjugated counterparts in the presence or absence of 50 mM free transferrin, respectively. HepG2 cells were incubated with DiD‐labelled (red) mEV‐Tf for 12 h, and stained with Hoechst 33342 (blue) for cell nuclei imaging. (c) Lysosome colocalization analysis of DiD‐labelled blank mEVs and mEVs‐Tf engineered by different methods. HepG2 cells were incubated with DiD‐labelled (red) mEV samples for 24 h, and stained with Hoechst 33342 (blue) and LysoTracker (green) for cell nuclei and lysosome imaging, respectively. (d) Semi‐quantitative analysis of the colocalization degree between mEVs and lysosomes using the Manders' colocalization coefficients M1 and M2. (e) Representative dot‐plots between particle size and ligand number of the mEVs‐Tf‐CF488 engineered by lipid modification (panel‐i), protein modification (panel‐ii) and membrane insertion (panel‐iii), respectively. The transferrin concentration was 50 µM for all three samples. (f), (g) Percentage of Tf‐CF488 positive particles (f) and ligand density (g) of the mEVs‐Tf‐CF488 engineered by different methods with increasing input concentrations of Tf‐CF488. (h) FCM analysis of the impact of ligand density on the cell uptake degree of the DiD‐labelled mEVs‐Tf‐CF488 engineered by lipid modification (panel‐i), protein modification (panel‐ii) and membrane insertion (panel‐iii), respectively. (i,j) The relationship between cell fluorescence intensity (FL‐DiD) and input concentration of Tf‐CF488 (i), and ligand density (j) for DiD‐labelled mEVs‐Tf‐CF488 engineered by different methods.

Article Snippet: The concentration of accessible transferrin conjugated on mEVs was measured by a human transferrin ELISA kit (MultiSciences).

Techniques: Conjugation Assay, Fluorescence, Microscopy, Incubation, Staining, Imaging, Modification, Membrane, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: Unravelling Ligand Conjugation Performance in Extracellular Vesicles: A Quantitative Assessment of Lipid, Protein, and Membrane Modifications

doi: 10.1002/jev2.70174

Figure Lengend Snippet: Impact of ligand conjugation on MFGE8 accessibility and surface‐ligand distribution homogeneity of transferrin. (a) Schematic illustration of ligand conjugation effects on the cell binding behaviour of mEVs‐Tf. (b) FCM analysis of the MFGE8‐mediated cell uptake of mEVs. HepG2 cells were treated with DiD‐labelled blank mEVs with (blue) or without (red) labelling against MFGE8 antibodies. (c) Relationships between particle size and the immunofluorescence intensity of MFGE8 for blank mEVs (panel‐i) and the transferrin‐conjugated mEVs engineered by lipid modification (panel‐ii), protein modification (panel‐iii) and membrane insertion (panel‐iv), respectively. (d) The percentage of MFGE8‐positive particles (left y‐axis) and the immunofluorescence intensity (right y‐axis) of the mEV samples. (e) Correlation between the fluorescence intensity of CF488 versus CF594 for individual mEVs‐Tf‐CF488 (panel‐i) and mEVs‐Tf‐CF488&Tf‐CF594 engineered by lipid modification (panel‐ii), protein modification (panel‐iii) and membrane insertion (panel‐iv), respectively. (f) The percentage of CF594‐positive particles (left y‐axis) and the relative fluorescence intensity of CF594 to CF488 (right y‐axis) of the mEV samples. Note: In these experiments, the concentrations of sPLD, TCEP and DSPE‐PEG‐MAL for lipid‐, protein‐ and membrane‐based ligand conjugation were 10 U/mL, 2 and 1.25 mM, respectively.

Article Snippet: The concentration of accessible transferrin conjugated on mEVs was measured by a human transferrin ELISA kit (MultiSciences).

Techniques: Conjugation Assay, Binding Assay, Immunofluorescence, Modification, Membrane, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Unravelling Ligand Conjugation Performance in Extracellular Vesicles: A Quantitative Assessment of Lipid, Protein, and Membrane Modifications

doi: 10.1002/jev2.70174

Figure Lengend Snippet: In vivo cancer targeting performance of ligand‐conjugated mEVs. (a) In vivo fluorescence images of the cancer‐bearing mice injected with DiD‐labelled blank mEVs, and mEVs‐Tf engineered by distinct methods at different time points. (b) Time‐dependent variation in radiant efficiency of the tumour site of the mice treated with various mEV formulations. (c) Ex vivo fluorescence images of tumours and other organs excised from the treated mice at 24 h post‐injection. (d) Radiant efficiency of the tumours and other organs based on the ex vivo fluorescence images. (e) Schematic illustration of the principle for characterizing the IgG and IgM adsorption on mEVs after serum incubation. (f), (g) Representative distributions and the bivariate dot‐plots of the particle size and the immunofluorescence intensity of IgG (f) and IgM (g) of the mouse serum‐incubated blank mEVs (panel‐i), and the transferrin‐conjugated counterparts engineered by lipid modification (panel‐ii), protein modification (panel‐iii) and membrane insertion (panel‐iv), respectively. (h)–(j) Immunolabelling ratio (h), immunofluorescence intensity (i), and particle size (j) of the mouse serum‐incubated mEV samples. (k) The ELISA measurement of the accessible transferrin concentration of the mEV samples before and after serum incubation.

Article Snippet: The concentration of accessible transferrin conjugated on mEVs was measured by a human transferrin ELISA kit (MultiSciences).

Techniques: In Vivo, Fluorescence, Injection, Ex Vivo, Adsorption, Incubation, Immunofluorescence, Modification, Membrane, Enzyme-linked Immunosorbent Assay, Concentration Assay