(M1130-06-36) Characterization of Blueberry-Derived Exosomes: New Insights into a Potential Nanomedicine

Purpose: Blueberries have demonstrated notable health-promoting properties in ameliorating oxidative stress, augmenting cognitive function, and conferring neuroprotection. Although efficacies of dietary bioactives have been demonstrated, poor stability and/or untargeted delivery often limit their development and clinical efficacy. Exosomes, the nanoscale vesicles released from cells, have been shown to possess prominent characteristics of enhancing drug stability and targeting the treatment of various diseases. Diet-derived exosomes contain endogenous bioactives and have improved stability for therapeutic applications. Exosomes enter cells via transcytosis to release loaded bioactives, which improve cellular uptake and retention as compared to dietary bioactives alone. This study seeks to characterize exosomes, the nanoscale vesicles derived from blueberries, and assess their feasibility as enhanced carriers for bioactive molecules, thereby facilitating superior stability and cellular uptake.

Methods: Blueberry juice was extracted using a blender and filtered, followed by sequential centrifugation at varied speeds and durations to remove large particles and debris. The resultant supernatant was centrifuged at 100,000 × g to obtain exosomes. The morphology of exosomes was first observed via scanning electron microscopy (SEM) and then their sizes and polydispersity index (PDI) were further assessed using a Delsa^TM Nano C system. Total protein and RNA within the exosomes were analyzed via bicinchoninic acid assay (BCA) and NanoDrop spectrophotometry. Exosome-specific biomolecules were detected using the Exo-Check array and RNA sequencing. The uptake of fluorescence-labeled exosomes by normal colon CCD841 CoN cells was assessed under a fluorescence microscope.

Results: Exosomes exhibited a nanosized and sphere-shaped morphology revealed by SEM analysis, aligning with a size of 82.7±6.4 nm as measured by particle sizing analysis (Figures 1A and B). They contained measurable amounts of proteins and RNAs (Table 1). The Exon-Check array detected exosome-specific markers such as GM130, ALIX, TSG10, ICMA-1, ANXA5, FLOT, EpCam, CD63, and CD81 (Figure 1C). RNA sequencing data analysis showed differential expression of miRNAs and their target gene pathways including inflammation regulation and neurotrophic signaling (Figure 2). The uptake of exosomes in cells increased in a dose-dependent manner with the degree of severe inflammation, as triggered by lipopolysaccharide (LPS) concentrations (Figure 3).

Conclusion: Blueberry-derived exosomes are nanosized carriers of protein and RNA biomolecules. Their lipid bilayer structure and nanosize potentially contribute to targeted inflammation. The highly expressed miRNA may serve to reduce oxidative stress and inflammation.

Figure 1. (A) Scanning electron microscope (SEM) image depicting the morphology of blueberry-derived exosomes, (B) Size distribution of blueberry-derived exosomes as measured by the particle-sizing system, and (C) Expression profile of surface proteins on blueberry-derived exosomes as detected by the Exo-Check array.

Figure 2. (A) Length distribution of microRNAs (miRNAs) contained in blueberry-derived exosomes and (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis illustrating categories enriched in the specific target genes of these miRNAs.

Figure 3. EVOS imaging showing the uptake of ExoGlow™-labeled blueberry-derived exosomes by normal colon CCD841 CoN cells under stimulation by varying concentrations of lipopolysaccharide (LPS). The scale bar represents 200 μm.