Presentation Category
Research
Introduction/Context/Diagnosis
Plant cells produce nanometer-sized vesicles for cellular communication, bioactive molecule transport, and modulation of the plant immune response. Extracellular nanovesicles contain proteins, RNA, and other molecules. It has been demonstrated that plant nanovesicles exert bioactive effects on human cells. Anti-inflammatory, anti-cancer, and tissue regenerative properties are significant to dentistry. Other groups have used mechanically disruptive methods to isolate plant nanovesicles. We aim to develop an isolation method for these nanoparticles from various fruits which preserves their structure and function towards their use in cell culture studies aimed at understanding the health benefits of plant nanovesicles in dental treatment and as therapeutics. We selected species known for their potential health benefits. Uniquely, we used a cold press juicer to extract samples. The samples underwent a series of centrifugations to obtain cell- and pulp-free samples. Samples were treated to precipitate nanovesicles, then applied to size exclusion chromatography columns to isolate nanovesicles. Fractions were collected using an automatic fraction collector. The samples were then diluted in phosphate-buffered saline and analyzed by Nanosight nanoparticle tracking analysis (NTA) or Zeta-PALS particle analysis to obtain size range data. Finally the pellet was resuspended in phosphate-buffered saline and placed in -80 ˚C for long term storage. We prepared nanovesicle enriched samples from raspberries, cranberries, limes, blackberries, blueberries, and strawberries. Analysis of blueberry samples by NTA indicate a broad range of particle sizes. Zeta-PALS analysis indicates varying ranges of particles sizes in each sample. Our novel method for nanovesicle extraction is viable and results in vesicle enriched samples. Our use of a cold juicer instead of a blender was effective. Further research will investigate the bioactive effects of these plant-derived nanovesicles on human cells. I would like to thank Drs. Zeitlin and Neelakantan for their mentorship on this project.
Location
Arthur A Dugoni School of Dentistry, 155 5th St, San Francisco, CA 94103, USA
Format
Presentation
Harvesting Nature's Cellular Messengers: Extraction and Isolation of Plant Nanovesicles for Biomedical Applications
Arthur A Dugoni School of Dentistry, 155 5th St, San Francisco, CA 94103, USA
Plant cells produce nanometer-sized vesicles for cellular communication, bioactive molecule transport, and modulation of the plant immune response. Extracellular nanovesicles contain proteins, RNA, and other molecules. It has been demonstrated that plant nanovesicles exert bioactive effects on human cells. Anti-inflammatory, anti-cancer, and tissue regenerative properties are significant to dentistry. Other groups have used mechanically disruptive methods to isolate plant nanovesicles. We aim to develop an isolation method for these nanoparticles from various fruits which preserves their structure and function towards their use in cell culture studies aimed at understanding the health benefits of plant nanovesicles in dental treatment and as therapeutics. We selected species known for their potential health benefits. Uniquely, we used a cold press juicer to extract samples. The samples underwent a series of centrifugations to obtain cell- and pulp-free samples. Samples were treated to precipitate nanovesicles, then applied to size exclusion chromatography columns to isolate nanovesicles. Fractions were collected using an automatic fraction collector. The samples were then diluted in phosphate-buffered saline and analyzed by Nanosight nanoparticle tracking analysis (NTA) or Zeta-PALS particle analysis to obtain size range data. Finally the pellet was resuspended in phosphate-buffered saline and placed in -80 ˚C for long term storage. We prepared nanovesicle enriched samples from raspberries, cranberries, limes, blackberries, blueberries, and strawberries. Analysis of blueberry samples by NTA indicate a broad range of particle sizes. Zeta-PALS analysis indicates varying ranges of particles sizes in each sample. Our novel method for nanovesicle extraction is viable and results in vesicle enriched samples. Our use of a cold juicer instead of a blender was effective. Further research will investigate the bioactive effects of these plant-derived nanovesicles on human cells. I would like to thank Drs. Zeitlin and Neelakantan for their mentorship on this project.
Comments/Acknowledgements
Presentation Category: Research