Integrin β4-Enriched Small Extracellular Vesicle as Drug Delivery Vehicle for Targeting Pulmonary Metastasis of Hepatocellular Carcinoma

Abstract

Small extracellular vesicles (sEVs) hold significant promise for targeted drug delivery, owing to their unique ability to target and accumulate in specific tissues. The organotropism of sEVs is primarily determined by the presence of integrins on their surface. In this study, sEVs with enriched integrin β4, designated as XP-ITGβ4-sEV, are engineered to enhance lung-targeting capabilities. The therapeutic efficacy of doxorubicin-loaded XP-ITGβ4-sEV (XP-ITGβ4-sEV/Dox) is evaluated in targeting pulmonary metastasis of advanced hepatocellular carcinoma (HCC) using a murine lung metastasis model. Remarkably, treatment with XP-ITGβ4-sEV/Dox effectively suppresses tumor cell colonization in the lungs compared to an equivalent dose of free doxorubicin. Histological analyses reveal a reduction in lung metastatic foci, inhibition of proliferation, and an increase in apoptosis of HCC cells. Notably, XP-ITGβ4-sEV/Dox exhibits a superior therapeutic efficacy with an improved safety profile compared to a higher dose of free doxorubicin that demonstrates similar efficacy. These findings collectively underscore the potential of integrin β4-enriched sEVs as a targeted drug delivery system for addressing pulmonary metastasis of HCC.

Keywords: drug deliveries; extracellular vesicles; hepatocellular carcinoma; integrins; lung metastases.

Figure 1

Lung accumulation of sEVs increases along with ITGβ4 expression. A) Immunoblots showing expression of ITGβ4, positive‐ and negative‐sEV markers in total cell lysate (TCL) and sEV from normal liver cells (MIHA) and various HCC cells (Huh7, MHCC97L and MHCCLM3). B) Representative electron micrographs of indicated sEV labelled by anti‐CD63 antibody and 10 nm gold‐conjugated secondary antibody. C) Size distribution of indicated sEV. D) Near infrared (NIR) signal from various organs collected from nude mice (n = 3) injected with NIR‐labelled sEV. Signal of NIR was quantified. Data were expressed as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, compared by two‐sided Student's t‐test.

Figure 2

Knockdown of ITGβ4 diminishes biodistribution of sEVs to lung tissues in animal. A) Immunoblots showing expression of ITGβ4 and sEV markers in MHCC97L‐sEV after mild proteinase K digestion. B) Flow cytometry detection of beads‐bound sEV upon labelling with anti‐IgG, anti‐ITGβ4 or anti‐CD9 antibody followed by Alexa Fluor 488‐conjugated secondary antibody and quantification of fluorescence signal (n = 3). C) Immunoblot showing expression of ITGβ4, positive‐ and negative‐sEV markers in total cell lysate (TCL) and sEV from indicated cells. D) Representative electron micrographs of indicated sEV labelled by anti‐CD63 antibody and 10 nm gold‐conjugated secondary antibody. E) Size distribution of indicated sEV. F and G) Near infrared (NIR) signal from various organs collected from nude mice (n = 3) injected with NIR‐labelled sEV. Data were expressed as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, compared by two‐sided Student's t‐test.

Figure 3

ITGβ4 plays a role in directing sEVs to lung tissues in animal. A) Immunoblots showing expression of ITGβ4, positive‐ and negative‐sEV markers in total cell lysate (TCL) and sEV from indicated cells. B) Representative electron micrographs of indicated sEV labelled by anti‐CD63 antibody and 10 nm gold‐conjugated secondary antibody. C) Size distribution of indicated sEV. D) Immunoblot showing expression of ITGβ4 and sEV markers in 293FT‐XP‐ITGβ4‐sEV after mild proteinase K digestion. E) Flow cytometry detection of beads‐bound sEV upon labelling with anti‐IgG, anti‐ITGβ4 or anti‐CD9 antibody followed by Alexa Fluor 488‐conjugated secondary antibody and quantification of fluorescence signal (n = 3). F) Near infrared (NIR) signal from various organs collected from nude mice (n = 3) injected with NIR‐labelled sEV. Data were expressed as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, compared by two‐sided Student's t‐test.

Figure 4

Loading of doxorubicin into sEV and characterization. A) Immunoblots showing expression of ITGβ4, positive‐ and negative‐sEV markers in indicated sEVs loaded with or without doxorubicin (Dox). Expressions in respective cells were included for reference. B) Representative electron micrographs showing morphology of indicated sEV loaded with or without Dox. sEV were indicated by arrowhead (red). C) Size distribution of indicated sEV with or without Dox loading. D) Representative chromatograms of 293FT‐XP‐ITGβ4 cells‐derived sEV, Dox‐loaded sEVs (XP‐ITGβ4‐sEV/Dox), Dox (0.4 µg) and no sEV control analyzed by HPLC coupled to photodiode array detector tracking absorption at 480 nm. E) Correlation of the concentration of Dox with peak area obtained from HPLC analysis. F) Drug release profiles of XP‐ITGβ4‐sEV/Dox in PBS of different pH over 48 h. G) murine p53‐/‐;Myc hepatoblast and MHCC97L cells were treated with XP‐ITGβ4‐sEV loaded with Dox (XP‐ITGβ4/Dox; 2 µм Dox equivalence) or same amount of free Dox for 6 h. Flow cytometry analysis to quantify Dox fluorescence in the treated cells. H) Representative fluorescence images of Dox (red) in treated cells. Cell membrane and nuclei were stained with Cell Mask plasma membrane stain (green) and DAPI (blue) respectively. Scale bar, 20 µm.

Figure 5

Efficacy of doxorubicin‐loaded lung‐targeting sEV in targeting lung metastasis in animals. A) Schematic diagram illustrating lung metastasis mouse model (n = 6) and treatment schedule. On Day 0, nude mice were injected intravenously with murine p53‐/‐;Myc hepatoblasts. Starting on Day 1, mice were treated with PBS, XP‐ITGβ4‐sEV, XP‐ITGβ4‐sEV loaded with Dox (XP‐ITGβ4‐sEV/Dox; 2.5 µg Dox per dose), Dox (2.5 µg per dose) or Dox (25 µg per dose) every 3 days for a total of 4 treatments. On Day 11, mice were euthanized for analysis. B and C) Bioluminescence imaging of B) mice and C) excised lungs after treatment. Luciferase signal was quantified. D) Representative H&E‐stained micrographs. Inlets were enlarged and shown. Percentage of colonization (metastatic foci area/total lung area) was quantified. E) Representative micrographs of Ki67 and TUNEL staining. Positive cells were stained using DAB (brown). Backgrounds of Ki67‐ and TUNEL‐stained micrographs were counterstained using hematoxylin and methyl green, respectively. The percentage of Ki67‐positive and TUNEL‐positive cells was quantified. Data were expressed as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. NS, not significant, compared by two‐sided Student's t‐test.

Figure 6

Toxicity evaluation of doxorubicin‐loaded lung‐targeting sEV in animals. Nude mice were treated with PBS, XP‐ITGβ4‐sEV loaded with doxorubicin (XP‐ITGβ4‐sEV/Dox; 2.5 µg Dox per dose) or Dox (25 µg per dose) every 3 days for a total of 4 treatments, and then sacrificed for histological, hematological and biochemical analysis (n = 5). A) Change in body weight of treated mice. B) Representative H&E‐stained micrographs of different organs collected from treated mice. C) Red blood cell and white blood cell counts of treated mice. D) Measurements of plasma ALT, AST, creatinine and CK‐MB level in treated mice. Data were expressed as mean ± SEM. **p < 0.01, *p < 0.05. NS, not significant. Data in A) were analyzed using two‐way ANOVA. Data in C and D) were compared by two‐sided Student's t‐test.