Probiotic-derived vesicles based sonosensitizer for PET/CT/NIRF imaging guided the inhibition of atherosclerosis via targeting and regulating macrophages

Introduction: Macrophages play a vital role in the initiation and progression of atherosclerosis (AS). Targeting and regulating macrophages is of great significance for the treatment of AS. Probiotic-derived vesicles (OMVs) have been reported to alleviate inflammation by modulating macrophages. In this study, we engineered a OMVs-based sonosensitizer for PET/CT imaging and treatment of AS and explore the possible underlying mechanisms of OMVs inhibiting AS.

Methods: OMVs were isolated from Lactobacillus supernatants. N₃ was modified on the surface of OMVs for pretargeting-based PET/CT imaging. ICG was loaded into OMVs for NIRF imaging and sonodynamic therapy. Then flow cytometry and confocal microscopy were used to analyze the cell-binding ability of OMVs based sonosensitizer to macrophages or foam cells. PET/CT and near-infrared fluorescent (NIRF) imaging was used verified that OMVs could target macrophages in atherosclerosis. In order to evaluate the therapeutic effects of OMVs on atherosclerosis, ApoE^-/- mice were randomly divided into a control group and an OMV group for in vivo experiments. RNA-Seq and molecular biology experiments were also performed to explore the possible underlying mechanisms of OMVs on atherosclerosis.

Results: OMVs were successfully isolated from probiotics and the sonosensitizer was constructed. Flow cytometry and confocal microscopy displayed better cell-binding ability of OMVs to foam cells. PET/CT/NIRF imaging could visualize atherosclerotic plaque lesions. Compared with the control group, PET/CT/NIRF imaging, aortic oil red staining or hematoxylin and eosin staining were all showed the reduction of plaque area in the OMV group. Bioinformatics analysis revealed that OMVs regulate AS by promoting lipid efflux and macrophages polarization. Gene set enrichment analysis (GSEA) showed significant changes in the "lipid and atherosclerosis" pathway after OMVs-treated. Differentially expressed genes involved in the 'lipid and atherosclerosis' pathway intersect with genes associated with lipid efflux, and define ABCA1 was found to promote lipid efflux. The expression of ABCA1 increased proportionally with OMV concentration. The differentially expressed genes intersected with transcription factors that regulate ABCA1 e.g., NR1H3 was found to upregulate the expression of ABCA1. Western blot and quantitative polymerase chain reaction (q-PCR) confirmed this hypothesis. In addition, the expression of M1 macrophages markers and cytokines such as CD86, NOS2 and TLR4 were downregulated. M2 macrophages markers and cytokines such as CD206, CD209 and IL10 were upregulated.

Conclusions: In this study, a novel and operable sonosensitizer was engineered for theranostic of AS and the sonosensitizer has been proved to have good therapeutic effect on AS. We further demonstrate that OMVs could promote lipid efflux and macrophages polarization for inhibiting AS. This work was supported by China Postdoctoral Science Foundation (2022M721262).