PNExo™ Exosome-Grapefruits(PNE-FG11)

Active Components and Effects of Grapefruits

Grapefruit (Citrus paradisi) is a highly regarded citrus fruit known for its distinctive flavor and rich nutritional value. Extensive scientific literature reports that grapefruits are rich in various compounds with potential biological activity. Research has identified several major classes of active components in grapefruits, including:

• Flavonoids: Flavonoids are one of the primary classes of active compounds in grapefruits, particularly rich in flavanones such as naringin and naringenin. Numerous studies have investigated their diverse biological activities. In both cell and animal models, these compounds have been shown to influence cell proliferation and apoptosis through pathways such as STAT3 and NF-κB. Flavonoids also exhibit notable antioxidant and anti-inflammatory properties by scavenging free radicals, reducing oxidative stress, and modulating the expression of inflammation-related genes, including cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Furthermore, research suggests potential benefits for metabolic health, including lipid-lowering effects (e.g., reducing cholesterol levels) and improved glucose metabolism (e.g., enhancing insulin sensitivity).
• Furanocoumarins: Grapefruits also contain furanocoumarins, such as bergamottin, which have attracted attention in both pharmacology and cell biology. These compounds have been found to influence drug pharmacokinetics by inhibiting enzymes and transporters like CYP3A4 and P-glycoprotein, thereby altering the metabolism of certain medications—an important consideration in drug interaction studies. Additionally, their effects on cellular activity have been examined in vitro, with some studies indicating a role in osteoblast differentiation and other cellular processes.
• Carotenoids: Especially lycopene and beta-carotene in red and pink grapefruits, these are important antioxidants relevant to cardiovascular health research.
• Limonoids: These compounds have shown antiviral and anticancer activities in cell studies.
• Organic Acids: Such as ascorbic acid (Vitamin C) and citric acid, which are important nutrients. Research also suggests their potential role in modulating gut microbiota and immune function.

What are Grapefruit-derived Exosomes?

Grapefruit-derived exosomes are nanoscale extracellular vesicles (typically ranging in diameter from approximately 30 to 210 nm) isolated from grapefruits. Based on research, they possess the following characteristics:

• Biogenesis: Their formation involves the endocytic pathway, leading to the creation of multivesicular bodies (MVBs) that are eventually released into the extracellular space.
• Composition: Grapefruit-derived exosomes carry a variety of biomolecules from their parent cells, including:
• 1. Lipids: Such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin, which contribute to membrane structure and are potentially involved in signaling.
• 2. Proteins: Including heat shock proteins (HSP70, HSP90) and Aquaporins, which are associated with cellular stress responses and transport mechanisms.
• 3. Nucleic Acids: They can carry genetic material like miRNAs and mRNAs, suggesting potential involvement in gene expression regulation.
• Functional Advantages: Research is exploring the inherent properties of Grapefruit-derived exosomes that make them interesting for various studies:
• 1. Biocompatibility: As natural plant-derived vesicles, they are considered to have low immunogenicity. Studies are investigating their potential for uptake via different routes, including oral or transdermal pathways.
• 2. Potential for Targeted Delivery: Through interactions between their surface components (proteins or lipids) and specific receptors, Grapefruit-derived exosomes are being studied as a potential natural carrier system for the delivery of various molecules to recipient cells in research settings.

Surface-Functionalizable Grapefruit-Derived Extracellular Vesicles for Targeted Drug Delivery. (Moon K, et al., 2023)

Potential Applications of Grapefruit-derived Exosomes

Research into the unique properties of grapefruit-derived extracellular vesicles (GEVs) suggests their potential in various research fields, including:

• Grapefruit-Derived Exosomes as Delivery Vehicles

  Due to their nanoscale size and stable lipid bilayer structure, GEVs are being explored as potential natural carriers for molecule delivery. Targeted Delivery: Studies suggest the potential for delivering various molecules via routes like oral or intranasal administration using GEVs. For example, intranasal delivery of GEVs loaded with miR-17 has been investigated for its potential to influence brain tumor progression in research models. Loading Capacity: GEVs can encapsulate diverse molecules, including small molecules, siRNA, and miRNA, enabling potential targeted delivery to specific cell types or microenvironments, such as the gut immune system or tumor microenvironment, in research settings. Stability & Safety: Compared to synthetic carriers, GEVs have shown stability in the gastrointestinal environment and demonstrated low immunogenicity or toxicity in studies.

• Anti-Inflammatory Benefits of Grapefruit Exosomes

  GEVs are being investigated for their effects on gut barrier function, microbiota, and immune response, showing promise in studies related to inflammatory bowel conditions like IBD and colitis. Gut Barrier Support: GEVs contain components like phosphatidic acid (PA) and phosphatidylethanolamine (PE), which have been studied for promoting intestinal epithelial cell proliferation and maintaining barrier integrity in models. Immune Modulation: GEVs are explored for influencing gut immune responses by affecting the balance of pro-inflammatory (e.g., IL-6, TNF-α) and anti-inflammatory factors (e.g., IL-10). Their carried miRNAs may also play a role in regulating macrophage polarization in research. Microbiota Influence: Studies indicate GEVs can impact gut microbiota composition by increasing short-chain fatty acid (SCFA) levels, promoting beneficial bacteria (e.g., Lactobacillus), and influencing pathogen levels, contributing to improved gut microecology in research models.

• Grapefruit Exosomes in Cancer Research

  Research is investigating the potential of GEVs to influence tumor progression through multiple pathways, with effects observed in studies involving models of melanoma, breast cancer, and gastric cancer. Cell Activity Modulation: In vitro studies show GEVs can influence the proliferation and induce cell cycle arrest and apoptosis in certain cancer cell lines (e.g., A375 melanoma, MCF7 breast cancer). Investigated mechanisms include effects on the ERK/AKT pathway and pro-cancer mediators like ICAM1. Tumor Microenvironment Studies: GEVs are being studied for their potential to inhibit the M2 polarization of tumor-associated macrophages (TAMs), potentially influencing the immunosuppressive tumor microenvironment in research models.

• Antioxidant and Anti-Aging Potential of Grapefruit Exosomes

  Rich in antioxidants like flavonoids and Vitamin C, GEVs show potential for scavenging free radicals and reducing oxidative stress in research settings. Skin Research: Plant exosomes (e.g., from grapes, strawberries) have been shown to promote fibroblast proliferation and collagen synthesis, relevant to skin healing studies. GEVs are speculated to potentially act via similar mechanisms for research related to skin regeneration or anti-aging. Neuroprotection Studies: Their antioxidant properties may be relevant for research addressing oxidative stress-related neurological conditions, though further verification is needed.

• Grapefruit Exosomes for Metabolic Support and Liver Health Protection

  GEVs are being investigated for their potential to regulate metabolism, partly through the gut-liver axis. Non-alcoholic Fatty Liver Disease (NAFLD): Oral administration of GEVs has been explored in models of NAFLD for improving gut barrier function and microbiota, potentially reducing endotoxemia and influencing liver inflammation and steatosis in these models. Obesity & Diabetes Studies: Research is exploring the potential of GEVs to modulate gut SCFAs and plasma IgA levels, potentially enhancing host metabolic balance for studies related to obesity and type 2 diabetes.

References

1. Hung W L, Suh J H, Wang Y. Chemistry and health effects of furanocoumarins in grapefruit. Journal of Food and Drug Analysis. 2017, 25(1): 71-83. https://doi.org/10.1016/j.jfda.2016.11.008
2. Igual M, Cebadera L, Cámara R M, et al. Novel ingredients based on grapefruit freeze-dried formulations: Nutritional and bioactive value. Foods. 2019, 8(10): 506. https://doi.org/10.3390/foods8100506
3. Alzahrani F A, Khan M I, Kameli N, et al. Plant-derived extracellular vesicles and their exciting potential as the future of next-generation drug delivery. Biomolecules. 2023, 13(5): 839. https://doi.org/10.3390/biom13050839
4. Huang R, Jia B, Su D, et al. Plant exosomes fused with engineered mesenchymal stem cell‐derived nanovesicles for synergistic therapy of autoimmune skin disorders. Journal of Extracellular Vesicles. 2023, 12(10): e12361. https://doi.org/10.1002/jev2.12361
5. Di Giulio S, Carata E, Mariano S, et al. Plant extracellular vesicles: investigating their utilization as beneficial nutrients in diet. Applied Sciences. 2023, 13(11): 6656. https://doi.org/10.3390/app13116656
6. Moon K, Hur J, Kim K P, et al. Surface‐functionalizable plant‐derived extracellular vesicles for targeted drug delivery carrier using grapefruit. Advanced Materials Interfaces. 2023, 10(22): 2300220. https://doi.org/10.1002/admi.202300220
7. Kilasoniya A, Garaeva L, Shtam T, et al. Potential of plant exosome vesicles from grapefruit (Citrus× paradisi) and tomato (Solanum lycopersicum) juices as functional ingredients and targeted drug delivery vehicles. Antioxidants. 2023, 12(4): 943. https://doi.org/10.3390/antiox12040943
8. Itakura S, Shohji A, Amagai S, et al. Gene knockdown in HaCaT cells by small interfering RNAs entrapped in grapefruit-derived extracellular vesicles using a microfluidic device. Scientific Reports. 2023, 13(1): 3102. https://doi.org/10.1038/s41598-023-30180-3

Case Study 1: Grapefruit Exosomes for siRNA Delivery in Gene Knockdown Studies (Itakura S, 2023)

This research highlights an innovative approach to delivering small interfering RNAs (siRNAs) for gene knockdown studies, addressing the limitations often associated with synthetic delivery vehicles like lipid nanoparticles, such as their potential toxicity and immunogenicity. In this study, grapefruit-derived extracellular vesicles (GEVs) were explored as natural, biocompatible carriers. A novel method was successfully demonstrated for encapsulating siRNAs within these GEVs using a microfluidic device. The efficacy of these siRNA-loaded GEVs was then showcased through their ability to achieve successful gene knockdown in HaCaT human keratinocyte cells, indicating their potential utility as an efficient and natural delivery platform for genetic material in dermatological research and broader biological investigations.

Figure 1. Characterization of Grapefruit-Derived Extracellular Vesicles (GEVs). (a) Cryo-TEM image showing the morphology of GEVs. (b) Size distribution profile of GEVs measured by tunable resistive pulse sensing (TRPS), confirming nanoscale vesicle size.

Figure 2. Cellular Uptake and Intracellular Localization of GEVs. (a) Flow cytometry histogram showing uptake of DiO-labeled GEVs by HaCaT cells at different concentrations and time points. (b) Quantification of mean fluorescence intensity ratios (MFIR). (c) Confocal microscopy images show intracellular localization of DiI-labeled GEVs (red) in HaCaT cells; nuclei (blue) and lysosomes (green) are also visualized.

Case Study 2: Research on Grapefruit Exosomes for Wound Healing (Savc Y, 2021)

This study investigated grapefruit-derived extracellular vesicles (GEVs) as a novel, cell-free agent with potential in advanced wound care strategies. Leveraging the known anti-inflammatory and wound-influencing properties of grapefruit, researchers assessed the capacity of isolated GEVs to support cellular events crucial for wound regeneration. In HaCaT keratinocytes, GEVs significantly enhanced cell viability (up to 64.75% increase at 72 h) and reduced intracellular ROS levels. A scratch assay showed that GEVs accelerated wound closure, achieving 82.38% closure at high concentration versus 34.39% in controls. Expression of key extracellular matrix genes such as COL1A1 and fibronectin was notably upregulated. GEVs also promoted angiogenesis in HUVEC cells, increasing tube formation metrics. These findings support the potential of GEVs as a promising tool for wound healing research.

Figure 1. Effect of GEV Concentrations on HaCaT Cell Viability Over Time. Cell viability of HaCaT cells treated with different concentrations of grapefruit-derived extracellular vesicles (GEVs) was measured at 24, 48, and 72 hours to evaluate dose- and time-dependent effects.

Figure 2. Effect of Grapefruit-Derived EVs on HaCaT Cell Scratch Closure. Scratch assay evaluating the impact of GEVs at 4.00 × 10⁹ and 0.50 × 10⁹ particles/mL on wound closure in HaCaT cells after 24 hours. Micrographs were captured using an inverted light microscope, and scratch area quantification was performed using Zen 2011 software.

References

1. Itakura S, Shohji A, Amagai S, et al. Gene knockdown in HaCaT cells by small interfering RNAs entrapped in grapefruit-derived extracellular vesicles using a microfluidic device. Scientific Reports. 2023, 13(1): 3102. https://doi.org/10.1038/s41598-023-30180-3
2. Savc Y , Krba O K K , Bozkurt B T, et al. Grapefruit-derived extracellular vesicles as a promising cell-free therapeutic tool for wound healing. Food & Function. 2021, 12(11): 5144-5156. https://doi.org/10.1039/d0fo02953j

• Are grapefruit-derived exosomes suitable for RNA delivery studies?

  Yes. Due to their natural content of small RNAs and compatibility with mammalian cells, grapefruit-derived exosomes are increasingly explored as plant-based RNA carriers in non-clinical delivery studies. Their lipid bilayer structure protects nucleic acids from degradation and facilitates cellular uptake, making them valuable for model systems evaluating cross-kingdom communication or gene regulation.

• Do grapefruit-derived exosomes exhibit cytotoxicity in standard cell models?

  No. Published research indicates that grapefruit exosomes are non-toxic across a wide range of concentrations in HaCaT and HUVEC cell lines. Even at high doses, they maintained or enhanced cell viability, supporting their use in cell-based experimental platforms.

• Are grapefruit exosomes compatible with downstream proteomic or transcriptomic analyses?

  Yes. These exosomes can be used in downstream omics workflows, including proteomics, RNA sequencing, and lipidomics. They are isolated using standardized procedures that preserve biomolecular integrity, making them compatible with high-resolution analytical techniques.

• Can grapefruit exosomes be used for therapeutic purposes?

  No. All PNExo™ exosomes are strictly intended for research and industrial manufacturing purposes. They are not approved for therapeutic use or personal DIY applications. Unauthorized use may violate safety and regulatory guidelines.

• How should grapefruit exosome products be stored and handled?

  Lyophilized grapefruit exosomes should be stored at 4°C, while reconstituted or liquid forms should be stored at –20°C to –80°C. Avoid repeated freeze–thaw cycles. Before use, centrifuge to collect particles, gently resuspend in deionized water, and handle carefully to maintain structural integrity.

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