Fig 1: Gln supplementation in 6-month-old 3×Tg-AD mice exhibited inhibitory effects on mild cognitive impairment (MCI) and the induction of oxidative stress and amyloid beta (Aβ) accumulation in the plasma. (A) Dietary groups and experimental schedule. (B) Body weights and (C) food intake were measured weekly. (D) Discrimination index (DI) in the object recognition test (ORT) in wild-type (WT, n = 5), 3×Tg-AD (3×Tg, n = 3), and Gln-diet 3×Tg (3×Tg+Gln, n = 4) mice at 2, 4, and 6 months of age. (E) Change in DI values in the ORT in mice between the age of 2 and 6 months. (F) Reactive oxygen/nitrogen species (ROS/RNS) analysis in plasma. (G) Aβ1–42 content in neuron-derived exosomes (NDEs) isolated from plasma. ELISA results of Aβ1–42 were normalized to those for CD81. Bars represent the means ± SEM. * p < 0.05, *** p < 0.001 vs. WT, and # p < 0.05 vs. 3×Tg in 1-way ANOVA with Tukey’s multiple comparison tests.
Fig 2: CD81‐coated LVs mimic the targeting specificity of EVs. (A) Delivery efficiency of various amounts (10–250 µg) of DiR‐labeled LVs to different immune cells in PBMCs after 1 h incubation. Left: schematic diagram. Right: Flow cytometry quantification of DiR‐positive fluorescence intensity in different immune cells incubated with EVs (n = 3). (B,C) Delivery efficiency of DiR‐labeled LVs coated with various amounts (10–50 µg) of different tetraspanin proteins (CD9/63/81) or GFP control to monocytes (B) or other immune cells (CD81 only, C) in PBMCs after 1 h incubation (n = 3). (D) The DiR positive fluorescence intensity in monocytes treated with different neutralizing antibodies (n = 3). (E) The sequence analysis of human CD81 and CD9. (F) The DiR positive fluorescence intensity in monocytes treated with indicated nanoparticles. Left: representative histograms. Right: representative histogram quantifications (n = 3). (G) Experimental design (left) and representative images (right) of Hepa1‐6‐derived subcutaneous tumors treated with PBS, EVs, or LVs carrying CD81 and/or ACLY via tail vein injection. Macrophage depletion was achieved using clodronate liposomes (n = 5). (H) Weight of harvested tumors from (G) (n = 5). Data are presented as mean ± SD. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one‐way ANOVA with Tukey's HSD test).
Fig 3: CD81‐coated LVs loaded with ACLY inhibitors enhance immunotherapy efficacy and restrain HCC progression. (A) Schematic diagram (left) and representative livers (right) harvested from the HCC model created by hydrodynamically injected Nras‐V12 and myr‐AKT, i.v. treated with LVs loaded without or with CD81, the ACLY inhibitor SB204990, or small interfering RNAs depleting ACLY (siACLY). (B) Representative histogram (upper panel) and quantification (lower panel) of flow cytometry analysis of immunosuppressive TAMs isolated from mouse livers indicated in (A) (n = 5). (C) Measurement of the ACLY enzymatic activity in TAMs and hepatic cells isolated from mouse livers as indicated in (A) (n = 5). (D) Schematic diagram (left) and representative livers (right) harvested from the HCC model created by hydrodynamically injected Nras‐V12 and myr‐AKT, i.v. treated without or with anti‐PD‐L1 antibodies and/or CD81‐coated LVs loaded without or with SB204990. (E) Representative histogram (upper panel) and quantification (lower panel) of flow cytometry analysis of immunosuppressive TAMs isolated from mouse livers indicated in (D) (n = 5). Data are presented as mean ± SD. ns, not significant. ***p < 0.001, ****p < 0.0001 (one‐way ANOVA with Tukey's HSD test).
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