Small Extracellular Vesicles Engineered Using Click Chemistry to Express Chimeric Antigen Receptors Show Enhanced Efficacy in Acute Liver Failure

Abstract

Acetaminophen (APAP) overdose can cause severe liver injury and life-threatening conditions that may lead to multiple organ failure without proper treatment. N-acetylcysteine (NAC) is the accepted and prescribed treatment for detoxification in cases of APAP overdose. Nonetheless, in acute liver failure (ALF), particularly when the ingestion is substantial, NAC may not fully restore liver function. NAC administration in ALF has limitations and potential adverse effects, including nausea, vomiting, diarrhoea, flatus, gastroesophageal reflux, and anaphylactoid reactions. Mesenchymal stromal cell (MSC)-based therapies using paracrine activity show promise for treating ALF, with preclinical studies demonstrating improvement. Recently, MSC-derived extracellular vesicles (EVs) have emerged as a new therapeutic option for liver injury. MSC-derived EVs can contain various therapeutic cargos depending on the cell of origin, participate in physiological processes, and respond to abnormalities. However, most therapeutic EVs lack a distinct orientation upon entering the body, resulting in a lack of targeting specificity. Therefore, enhancing the precision of natural EV delivery systems is urgently needed. Thus, we developed an advanced targeting technique to deliver modified EVs within the body. Our strategy aims to employ bioorthogonal click chemistry to attach a targeting molecule to the surface of small extracellular vesicles (sEVs), creating exogenous chimeric antigen receptor-modified sEVs (CAR-sEVs) for the treatment. First, we engineered azido-modified sEVs (N₃-sEVs) through metabolic glycoengineering by treating MSCs with the azide-containing monosaccharide N-azidoacetyl-mannosamine (Ac4ManNAz). Next, we conjugated N₃-sEVs with a dibenzocyclooctyne (DBCO)-tagged single-chain variable fragment (DBCO-scFv) that targets the asialoglycoprotein receptor (ASGR1), thus producing CAR-sEVs for precise liver targeting. The efficacy of CAR-sEV therapy in ALF models by targeting ASGR1 was validated. MSC-derived CAR-sEVs reduced serum liver enzymes, mitigated liver damage, and promoted hepatocyte proliferation in APAP-induced injury. Overall, CAR-sEVs exhibited enhanced hepatocyte specificity and efficacy in ameliorating liver injury, highlighting the significant advancements achievable with cell-free targeted therapy.

Keywords: acetaminophen; acute liver failure; extracellular vesicles; mesenchymal stromal cells; targeted therapy.

FIGURE 1

Cell‐surface labelling of azido glycans on pcMSCs and pcMSC‐sEVs via metabolic glycoengineering and SPAAC. (A) Scheme for the CAR‐sEVs manufacturing strategy. Following the treatment of pcMSCs with Ac4ManNAz, N₃‐sEVs were subsequently purified from the conditioned medium. These N₃‐sEVs were then subjected to DBCO‐scFv conjugation via click chemistry, resulting in the formation of CAR‐sEVs. (B) Azido groups dose‐dependently expressed on the surface of pcMSCs via metabolic glycoengineering when the cells were treated with increasing concentrations of Ac4ManNAz. Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. (C) The highest expression level of azido groups on the pcMSC surface was observed after 4 days of incubation with Ac4ManNAz (20 µM). Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. (D) The viability of Ac4ManNAz‐treated pcMSCs was analysed by a luminescent cell viability assay. Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. (E) Compared with that of the group without Ac4ManNAz pretreatment, the labelling yield (%) of the group treated with AZDye 488 DBCO (green) to pcMSCs incubated with 20 µM Ac4ManNAz for 4 days significantly increased. Nuclei were stained with DAPI (blue). Scale bar = 50 µm. (F) The mean fluorescence intensity of azido groups on pcMSCs conjugated with AZDye 488 DBCO per field was calculated across a total of six fields. Significant differences (p < 0.001) in mean fluorescence intensity were observed between two groups of pcMSCs, one that was pretreated with Ac4ManNAz and the other that was not. The fluorescence intensities are presented as arbitrary units with SEMs. (G) Illustration of sEVs surface labelling with a fluorescent probe through click chemistry. The labelling strategy involved the reaction of AZDye 488 DBCO with N₃‐sEVs via SPAAC, resulting in the formation of AF488‐sEVs that exhibit a fluorescence signal on their surface. (H) (I) The number of azido groups on the surface of pcMSC‐sEVs gradually increased with increasing doses of AZDye 488 DBCO (0.005, 0.05, 0.5, 5, 10, 20, 40, and 80 µM), reaching a plateau at 10 µM. The labelling yield (%) of AZDye 488 DBCO‐conjugated sEVs was determined by NanoFCM. (J) (K) Compared with that of the group without Ac4ManNAz pretreatment, the labelling yield (%) of AZDye 488 DBCO to N₃‐sEVs purified from the conditioned medium of pcMSCs after 4 days of 20 µM Ac4ManNAz pretreatment significantly increased (p < 0.001). The labelling yield (%) of AZDye 488 DBCO‐conjugated sEVs was determined by NanoFCM. All data are presented as the mean ± SEM. ***p < 0.001, ****p < 0.0001.

FIGURE 2

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.

FIGURE 3

Characterization of sEVs and CAR‐sEVs. (A) Transmission electron microscopy (TEM): The morphology and structure of both the sEVs and CAR‐sEVs were characterized as either spherical or cup‐shaped, with diameters ranging from 30 to 150 nm. Scale bar = 200 nm (left). The scale bar for enlarged images is 50 nm (right). (B) Flow cytometry was used to analyse EV surface markers, including CD9, CD63, and CD81. The gating strategy for each marker is shown, with the respective fluorescence intensities on the y‐axis and side scatter on the x‐axis. (C) (D) Nanoparticle tracking analysis (NTA): Quantification and size of sEVs and CAR‐sEVs isolated from conditioned medium of pcMSCs ranging from 80 to 120 nm. The data are presented as the mean ± SEM; n = 6 per group. p value was measured by an unpaired t‐test; ns, non‐significant. (E) (F) The zeta potential (mV) profile and quantification of the sEVs, N₃‐sEVs, and CAR‐sEVs isolated from the conditioned media of the pcMSCs were measured with a Litesizer DLS 500. The surface potential of the vesicles was negatively charged in both groups. The data are presented as the mean ± SEM; n = 6 per group. ns, non‐significant. (G)(i) Representative super‐resolution microscopy images of single pcMSC‐derived native sEVs and modified N₃‐sEVs labelled with surface markers CD9 (green), CD63 (blue), or CD81 (red). The modified N₃‐sEVs were conjugated with DBCO‐Cy5 (red) via click chemistry. Merged images demonstrate the colocalization of these markers on both native and modified sEVs. Scale bars represent 50 nm for individual marker images and 100 nm for merged images. (ii) Quantitative analysis of the percentage of single‐, double‐, and triple‐positive sEVs expressing CD9, CD63, CD81, or DBCO‐Cy5 was conducted using CODI software. The bar charts represent the distribution of sEVs populations in both the native sEV and N₃‐sEV groups. (iii) Clustering analysis strategy for native sEVs and N₃‐sEVs, showing a large field of view with multiple clusters (left panel), a zoomed‐in view of a selected EV cluster (middle panel), and a corresponding graph (right panel) depicting the cluster distribution of CD81/CD63 for native sEVs or DBCO‐Cy5/CD63 for modified N₃‐sEVs. (H) The diagram represents the conjugation of N₃‐sEVs with DBCO‐AF488‐scFv (green) to produce CAR‐sEVs through click chemistry. (I) Representative super‐resolution microscopy image of a single CAR‐sEV particle, labelled with EV surface markers CD9 (red), CD63 (blue), and DBCO‐AF488‐scFv (green). Merged images demonstrate the colocalization of these markers on CAR‐sEVs. (J) Flow cytometry histogram showing the fluorescence intensity of CD9‐positive N₃‐sEVs labelled with DBCO‐AF488‐scFv. The percentage of CAR‐sEVs (CAR+) is calculated to be 45.3% under a 10 µM DBCO‐AF488‐scFv, compared to the control sEVs.

FIGURE 4

CAR‐sEVs significantly enhanced targeting efficacy in the HepG2/C3A cell model. (A) Schematic representation of the conjugation of N₃‐sEVs with DBCO‐Cy5 (red) and DBCO‐AF488‐scFv (green) using click chemistry to generate CAR‐sEVs. (B) Flow cytometry histograms showing the fluorescence intensity of HepG2/C3A cells treated with increasing doses (10⁷, 10⁸, or 10⁹ particles) of CAR‐sEVs or N₃‐sEVs for 18 h. The fluorescence was detected in the Alexa Fluor 488 and PE‐Cy5 channels. Sham treatment (no sEVs) was included as a control. (C) Quantification of targeting efficiency (%) of N₃‐sEVs and CAR‐sEVs at different doses (10⁷, 10⁸, or 10⁹ particles). (D) Confocal microscopy images (630×) showing the cellular uptake of N₃‐sEVs and CAR‐sEVs in HepG2/C3A cells at various time points (0.5, 1, 2, 4, and 6 h). The images illustrate the nuclei (DAPI, blue), sEV particles with azido groups (DBCO‐Cy5, red), and DBCO‐scFv (labelled with Alexa Fluor‐488, green). The merged DIC (differential interference contrast) images to fluorescent images. Scale bar = 10 µm. (E) Flow cytometry was used to measure the mean fluorescence intensity in HepG2/C3A cells treated with N₃‐sEVs (grey) or CAR‐sEVs (red) over 6 h. All data are presented as the mean ± SEM. ***p < 0.001.

FIGURE 5

Compared to unmodified sEVs, CAR‐sEVs enhanced the therapeutic effects against the APAP challenge in C3A cells. (A) (i) APAP dose‐dependently reduced the viability of HepG2/C3A cells, with an IC50 of 10 mM APAP. (ii) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with pcMSC‐CM in the presence of APAP (10 mM), as determined by a luminescent cell viability assay. (iii) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with sEVs from pcMSCs in the presence of APAP (10 mM), as determined by a luminescent cell viability assay. (iv) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with pcMSC‐CM in the presence of increasing doses of APAP (0, 10, 20, 40 mM). Three replicates per sample were performed, and the experiment was repeated three times. (v) The viability of HepG2/C3A cells significantly increased after 48 h of incubation with sEVs from pcMSCs in the presence of increasing doses of APAP (0, 10, 20, 40 mM). Three replicates per sample were performed, and the experiment was repeated three times. (vi) Compared with corresponding doses of sEVs, CAR‐sEVs significantly increased the viability of HepG2/C3A cells in a dose‐dependent manner after 48 h of incubation in the presence of APAP (10 mM). Three replicates per sample were performed, and the experiment was repeated three times. The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) The heatmap displays the predicted interactions between pcMSC‐sEVs miRNAs and genes of interest categorized into four major functional groups: anti‐inflammation (TGF‐β1, TGF‐β2, TGF‐β3, IL‐6, IL‐1β, TNF‐α, and NKB1), liver regeneration promotion (PTEN, MKK4, MFAP4), anti‐apoptosis (caspase‐3, caspase‐7, caspase‐8, caspase‐9, Bax and Bim), and anti‐fibrosis (COL1A1 and COL3A1). TargetScanHuman 8.0 was used to calculate the targetability score, with only the top miRNAs having a TargetScore context++ score of ≤ −0.1 selected. The miRNAs were selected using TargetScanHuman 8.0, with a TargetScore context++ score ≤ −0.1. The miRNA expression is shown as log2 RPM (reads per million), with higher values indicating greater miRNA expression. (C) (i) The line chart showing the highly expressed miRNAs significantly involved in specific biological processes of negative regulation of hepatocyte proliferation (GO:2000346) and positive regulation of the apoptotic process (GO:0043065). (ii) The Venn diagram illustrates the overlap of miRNAs involved in two biological processes. The overlapping area represents 15 miRNAs predicted to target genes involved in both processes. (iii) The top miRNAs from this overlap are listed in the table below.

FIGURE 6

Compared with unmodified sEVs, CAR‐sEVs exhibited superior therapeutic efficacy in attenuating APAP‐induced liver injury in an ALF mouse model. (A) Representative liver morphology from different groups at 24 h post‐APAP injection: sham group (NaCl 0.9%), pathological control group (APAP/PBS), and pcMSC, pcMSC‐CM, sEVs (10⁹ particles), and CAR‐sEVs (10⁹ particles) treatment groups (n = 3 per group). The enlarged images are presented at 5× magnification with a scale bar of 5 mm. (B) (C) Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the sham group (n = 9), pathological control group (APAP/PBS) (n = 8), and pcMSCs (n = 5), pcMSC‐CM (n = 8), sEVs (n = 5–8), and CAR‐sEVs (n = 5–8) treatment groups were measured 24 h after saline or APAP injection. (D) (i–iv) Serum levels of inflammatory cytokines (IL‐1β, IL‐6, TNF‐α, and MCP‐1) measured after the administration of pcMSCs, pcMSC‐CM, sEVs (10⁹ particles), or CAR‐sEVs (10⁹ particles) (n = 5–6 per group). (E–G) Representative images of liver sections stained with haematoxylin and eosin (H&E) (E), PCNA staining (F), and TUNEL assay with cleaved caspase‐3 staining (G) from the sham group, pathological control group (APAP/PBS), and groups treated with pcMSCs, pcMSC‐CM, sEVs (10⁹ particles), and CAR‐sEVs (10⁹ particles) 24 h after saline or APAP injection. Images are presented with a scale bar of 50 µm. Quantification was performed by analysing four to five fields per group, including necrotic area (%), PCNA+ cells (%), TUNEL+ cells (%), and cleaved caspase‐3+ cells (%). All the data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns indicates non‐significance.