2023

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http://dspace.husson.edu/handle/20.500.14298/2

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Browsing 2023 by Author "Dunham, Kaitlyn"

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    CHARACTERIZATION AND ANALYSIS OF BIOACTIVES IN BLUEBERRY-DERIVED EXOSOMES: NEW INSIGHTS INTO A POTENTIAL ANTIOXIDATION NANOMEDICINE
    (2023-04-20) Dunham, Kaitlyn; Canham, Spencer; Palmer, Emilie; Bai, Shuhua; Yang, Tianzhi
    Blueberries provide evident benefits of reducing oxidative stress and inflammation, improving cognitive function, and protecting against neurodegenerative diseases as a “super” fruit. However, high levels of bioactive molecules in blueberries, such as anthocyanin, have poor stability and absorption, leading to low bioavailability in the brain. Exosomes attract strong interest as an important vehicle of intercellular communication and as a delivery carrier of bioactive molecules. Herein, the study focuses on characterizing blueberry-derived exosomal nano-vesicles that contain bioactive molecules, and determining if they have better stability and are more readily taken up by cells. Blueberry juice was directly extracted with an electric blender and passed through filter papers. The collected juice was sequentially centrifuged at 1,000 × g for 10 min, 3,000 × g for 20 min, and 10,000 × g for 40 min at 4 °C to remove large particles and debris. The final supernatant was centrifuged at 100,000 × g for 30 min to obtain exosomes. The exosome morphology was observed with scanning electron microscopy (SEM) and the particle size was determined with a Nano Sizing Analyzer. The exosomes had a size of 82.7±6.4 nm and appeared as individually sphere-shaped morphology as shown in SEM images. Exosomes were analyzed and quantified for total proteins and RNAs. Well-known exosome-unique markers, including Cis-Golgi matrix protein GM130, adaptor protein and sort cargo ALIX, tumor susceptibility gene TSG10, intercellular adhesion molecule 1 (ICAM-1), apoptosis ANXA5, integral membrane protein FLOT, epithelial cell adhesion molecule (EpCam), and transmembrane tetraspanin family CD63 and CD81, were detected by an Exo-Check array. MicroRNA sequencing analysis revealed that the intersection between differentially expressed genes and miRNAs contained in exosomes could unveil a set of candidate target genes. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) study demonstrated that the exosomes showed cytotoxicity in neural cancerous cells. The characterized exosomes with biomolecules may deliver therapeutic molecules in the brain and target neural cells, leading to improved efficacy in the treatment of neurodegenerative diseases.
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    NANOVESICLES IN BROCCOLI SPROUTS AS A NATURAL POTENTIAL MEDICINE FOR THE TARGETED TREATMENT OF INFLAMMATORY BOWEL DISEASE
    (2023-04-20) Palmer, Emilie; Kaserman, Elise; Dunham, Kaitlyn; Kuhn, Brian; Yang, Tianzhi; Bai, Shuhua
    Inflammatory bowel disease (IBD) is an incurable small intestine and colon disorder. While exerting anti-inflammatory effects, bioactives in broccoli sprouts cannot treat IBD well probably due to their instability in the gastrointestinal (GI) tract and/or inadequate transit into colon cells. The study aimed to investigate if broccoli sprout-derived exosome (BSDExo) nanovesicles can protect bioactives from the upper GI extreme environments and confer selectively targeted delivery of bioactives to inflamed colon cells in IBD. Microparticles with 1198.3±86.2 nm and nanovesicles with 40.1±17.2 nm were isolated from broccoli sprout juice using differential centrifugations and measured by a DelsaTM Nano C nanosizing system. They showed different morphologies under a scanning electron microscope (SEM). Total protein and RNA were characterized in both broccoli sprout-derived microparticles and nanoparticles. Exosomes expressed the highest level of transmembrane protein CD63 evaluated by an Exo-Check array. These nanosized and identified proteins are known to be associated with exosomes. BSDExo protected bioactive sulforaphane for 2 hours in stomach-mimicking acid and 24 hours in intestine-mimicking conditions. BSDExo with a total protein level of 25 ng/mL could promote the proliferation of human normal colon epithelial CCD841 CoN cells with a cell viability of 154±5% (p<0.05). Both colon CCD841 CoN and Caco-2 cellular uptake of fluorescence-labeled BSDExo significantly increased with more severe inflammation stimulations. Interleukin 8 (IL-8) secretion from inflammation-stimulated normal colon cells was significantly reduced by the BSDExo treatment (p<0.05). BSDExo also significantly recovered the decreased transepithelial electrical resistance (TEER) values caused by inflammations in Caco-2 cells (p<0.05). Overall, the isolated BSDExo improved the stability, inflammation-targeting, and therapeutic efficacy of bioactives in colon epithelial cells via a naturally formed nanostructure. Demonstrating that BSDExo interacts with the targeting gut inflammatory cells and regulates anti-inflammatory responses would be a significant step forward in treating IBD.