(T1030-01-02) Blueberry-Derived Exosomes as a Natural miRNA Delivery System for the Treatment of Neurodegenerative Diseases

Purpose: MicroRNA (miRNA)-based therapies offer a groundbreaking approach to treating neurodegenerative diseases by targeting key pathological pathways such as neuroinflammation. However, challenges such as poor stability, inefficient delivery to the brain, and potential off-target effects have hindered clinical translation. Blueberries are rich in bioactive molecules known to mitigate oxidative stress and inflammation, key factors in neurodegenerative diseases. However, their therapeutic potential is limited by poor stability and brain bioavailability. Blueberry-derived exosomes (BBDExo) are emerging as promising nanocarriers for delivering bioactives, including microRNAs (miRNAs), due to their stability, biocompatibility, and ability to cross the blood-brain barrier (BBB). This study aims to harness the potential of miRNAs encapsulated within BBDExo to develop a targeted, stable, and efficient delivery system for combating neurodegenerative disease pathology.

Methods: Blueberry-derived exosomes (BBDExo) were isolated from fresh blueberry juice using sequential ultracentrifugation. The juice was first passed through filter paper to remove pulp and debris, followed by centrifugation at 1,000 × g for 10 minutes, 3,000 × g for 20 minutes, and 10,000 × g for 40 minutes at 4°C to eliminate larger particles. The final supernatant was ultracentrifuged at 100,000 × g for 30 minutes to collect exosomes. The size, morphology, and concentration of the exosomes were characterized using dynamic light scattering (DLS) and scanning electron microscopy (SEM). Protein markers on the exosomes were identified and validated using the Exo-Check™ Exosome Antibody Array, while total RNA was extracted for further analysis. High-throughput sequencing was performed to profile microRNAs (miRNAs) and identify candidates targeting inflammatory pathways. Exosome stability was tested under simulated gastrointestinal conditions over a period of 4 hours. Cellular uptake of fluorescence-labeled exosomes was evaluated in brain endothelial cells (bEND.3) using imaging and flow cytometry. The expression of 84 genes related to oxidative stress pathways in BBDExo-treated neuronal SH-SY5Y cells was analyzed using the RT² Profiler™ Human Oxidative Stress Pathway Plus PCR Array. Finally, the therapeutic efficacy of BBDExo in repairing neuronal damage was assessed using Caenorhabditis elegans exposed to methylmercury (MeHg).

Results: BBDExo exhibited a nanosized, spherical morphology with an average size of 82.7 ± 6.4 nm, as confirmed by dynamic light scattering (DLS) and scanning electron microscopy (SEM) (Figure 1A). MicroRNA (miRNA) sequencing identified several neuroprotective candidates targeting inflammatory pathways (Figure 1B). No significant changes in particle size or protein content were observed over 24 hours in a stomach-like environment (pH = 1.5, 37°C). The cellular uptake of BBDExo increased in a dose-dependent manner in brain endothelial bEND.3 cells, suggesting its potential for transport across the blood-brain barrier (Figure 2A). Additionally, BBDExo significantly promoted the proliferation of SH-SY5Y neuronal cells, achieving a maximal increase of 158.8 ± 1.8% at a concentration of 25 µg/mL. Treatment with BBDExo also significantly upregulated antioxidant gene expression in SH-SY5Y cells. In a Caenorhabditis elegans model, BBDExo treatment significantly improved dopaminergic function compared to the PBS control group, with outcomes nearly matching those observed in undamaged neurons (Figure 2B).

Conclusion: This study introduces an innovative miRNA-based therapeutic strategy for neurodegenerative diseases, leveraging the unique properties of BBDExo. By addressing critical barriers in miRNA therapy, including stability, delivery, and BBB crossing, BBDExo offer a transformative platform for advancing precision nanomedicine in neurodegenerative disease management.