Objective To prepare folic acid (FA)-modified Mojiahuangqi (Astragalus membranaceus, AM) exosome-like nanoparticles loaded with calycosin (Cal) (FA-Exos@Cal) and investigate their inhibitory effect on M1 polarization of macrophages in vitro. Methods AM exosome-like nanoparticles (Exos) were isolated by differential centrifugation combined with sucrose density gradient centrifugation. FA-Exos@Cal was prepared by the thin-film dispersion-incubation method. Transmission electron microscopy (TEM), bicinchoninic acid (BCA) assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering, HPLC, and dialysis bag method were used to characterize the particle morphology, protein properties, particle size, ζ potential, drug-loading characteristics, and in vitro release behavior. Fluorescence microscopy and flow cytometry were employed to evaluate the targeting ability of FA-Exos@Cal to M1-type RAW264.7 cells. Cell counting kit-8 (CCK-8) assay was used to detect the effect of FA-Exos@Cal on macrophage proliferation. Flow cytometry was applied to investigate its influence on the expression level of M1 marker CD86. Enzyme-linked immunosorbent assay (ELISA) and other methods were used to determine the expression levels of inflammatory factors including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6, and nitric oxide (NO). Results FA-Exos@Cal was successfully prepared. TEM showed a typical vesicular structure. The protein concentration, particle size, ζ potential, drug loading capacity, and entrapment efficiency were (3.52 ± 0.26) mg/mL, (127.4 ± 2.6) nm, (-11.14 ± 0.30) mV, (12.14 ± 0.18)%, and (27.21±1.04)%, respectively. There was no significant difference in protein properties before and after modification, and the drug exhibited good sustained-release effect in vitro. FA modification significantly enhanced the cellular uptake efficiency of FA-Exos by M1-polarized macrophages while reducing the cytotoxicity of Cal toward macrophages. Moreover, FA-Exos@Cal could significantly reduce the CD86-positive rate of M1-type macrophages and down-regulate the expression levels of IL-1β, TNF-α, IL-6, and NO. Comparative analysis with Exos, Cal, and the physical mixture of Exos and Cal revealed that the combined use of Exos and Cal exerted a synergistic effect. Conclusion FA-Exos@Cal, an engineered targeted biomimetic nano-delivery system, was successfully prepared. It was confirmed to have excellent targeting ability to M1-type macrophages, and could effectively inhibit M1 polarization and the release of inflammatory factors, which is expected to provide a new strategy for the treatment of inflammatory diseases.

calycosin  /  Astragalus membranaceus exosome-like nanoparticles  /  folate receptor-mediated targeting  /  macrophage M1 polarization  /  anti-inflammation