Journal: Journal of Extracellular Vesicles
Article Title: Small Extracellular Vesicles Engineered Using Click Chemistry to Express Chimeric Antigen Receptors Show Enhanced Efficacy in Acute Liver Failure
doi: 10.1002/jev2.70044
Figure Lengend Snippet: Validation of the production and targeting of DBCO‐scFv in the HepG2/C3A cell model. (A) Sandwich ELISAs were performed to detect serial dilutions of scFv that bound to ASGR1. scFv was then detected using an HRP‐conjugated anti‐6X His tag antibody, and the absorbance at 450 nm was quantified using an absorbance microplate reader. Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. The figure to the right presents a schematic representation of the sandwich ELISAs for assessing the binding affinity of scFv to ASGR1. (B) Confocal microscopy images (630×) showed that after 30 min, scFv, which was stained with an Alexa Fluor 488‐conjugated anti‐His tag antibody, specifically targeted ASGR1 expressed in the membrane of HepG2/C3A cells. ASGR1 was gradually internalized by the cells as the incubation time increased (0.5, 1, 3, and 6 h). Images of the nuclei (DAPI, blue), cell membrane (PKH26, red), and scFv (green) were merged. Scale bar = 50 µm. (C) Quantification of the mean fluorescence intensity of the scFv targeting ASGR1 in HepG2/C3A cells incubated for different durations (0.5, 1, 3, and 6 h). The mean fluorescence intensity per field was calculated across a total of six fields. The fluorescence intensities are presented as arbitrary units with SEMs. (D) Different molar ratios of galactose to scFv were mixed and added to ELISA wells coated with the ASGR1 antigen. The scFv signal was gradually inhibited concomitant with increasing proportions of galactose. Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. (E) Confocal microscopy images (×400) showing that scFv (1 µM) stained with an Alexa Fluor 488‐conjugated anti‐His tag antibody specifically targeted ASGR1 expressed on the membrane of HepG2/C3A cells and that the targeting was inhibited by preincubation with galactose (500 mM) for 1 h. Images of the nuclei (DAPI, blue), cell membrane (PKH26, red), and scFv (green) were merged. Scale bar = 50 µm. (F) The quantification results revealed that the mean fluorescence intensity of the scFv (1 µM) targeting ASGR1 in HepG2/C3A cells was inhibited after preincubation with galactose (500 mM) for 1 h. The mean fluorescence intensity per field was calculated across a total of six fields. The fluorescence intensities are presented as arbitrary units with SEMs. (G) Schematic representation of DBCO‐scFv production through the conjugation of scFv (S19C) and DBCO‐PEG4‐maleimide by a site‐specific cysteine‐cyclooctyne reaction. (H) Sandwich ELISAs were performed to detect serial dilutions of DBCO‐scFv bound to ASGR1. DBCO‐scFv was detected with biotin‐PEG3‐azide (10 µM) via a click reaction and subsequently interacted with streptavidin HRP and TMB. The absorbance was measured at 450 nm by an absorbance microplate reader. Three replicates per sample were performed. The data are presented as the mean ± SEM. The figure to the right presents a schematic representation of the sandwich ELISAs for assessing the binding affinity of DBCO‐scFv for ASGR1. (I) SDS‐PAGE of DBCO‒scFv conjugates was performed under nonreducing conditions. Coomassie blue staining (left) and fluorescence image (right) of the SDS‐PAGE gel of the DBCO‒scFv conjugates. M: marker ladder. Lane 1: scFv protein control group. Lane 2: Calfluor 488 azide control group. Lane 3: scFv + Calfluor 488 azide control group. Lane 4: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 1:1:1. Lane 5: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 1:2:2. Lane 6: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 1:4:4. Lane 7: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 2:4:4. Lane 8: scFv:DBCO‐PEG4‐maleimide:Calfluor 488 azide = 4:4:4. (J) Confocal microscopy images (×400) showing that after 30 min, DBCO‐scFv, detected by click reaction with Calfluor 647 Azide (10 µM), specifically targeted ASGR1 expressed on the membrane of HepG2/C3A cells. DBCO‐scFv was gradually internalized by the cells after a prolonged incubation time (6 h). Images of nuclei (DAPI, blue), cell membranes (FM1‐43, green), and DBCO‐scFv (red) were merged. Scale bar = 50 µm. (K) The mean fluorescence intensity was significantly higher ( p < 0.01) in the DBCO‐scFv‐treated HepG2/C3A cells than in the control cells without scFv treatment. The mean fluorescence intensity per field was calculated across a total of six fields. The fluorescence intensities are presented as arbitrary units with SEMs. (L) Confocal microscopy images (×630) showing that after 3 h of incubation, DBCO‐scFv, detected by click reaction with Calfluor 647 Azide (10 µM), specifically targeted ASGR1 and was internalized into the cytoplasm by HepG2/C3A cells. Images of nuclei (DAPI, blue), cell membranes (FM1‐43, green), and DBCO‐scFv (red) were merged. Scale bar = 50 µm. All data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01.
Article Snippet: Ac4ManNAz‐tetraacylated (Cat No. 1084), AZDye 488 DBCO (Cat No. 1278), DBCO Amine (Cat No. A103), and Biotin‐PEG3‐Azide (Cat No. AZ104) were purchased from Click Chemistry Tools (Scottsdale, AZ, USA).
Techniques: Microplate Reader Absorbance Measurement, Binding Assay, Confocal Microscopy, Staining, Membrane, Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay, Conjugation Assay, SDS Page, Marker, Control
