PNExo™ Exosome-Apple(PNE-FA18)

Benefits of Apple: Bioactive Components and Functions

Apples (Malus domestica) are rich in a diverse array of bioactive compounds, particularly concentrated in the peel and flesh, making them one of the most scientifically examined fruits. These compounds include polyphenols, triterpenoids, dietary fibers, and essential vitamins. Their biological functions span antioxidant activity, inflammation modulation, metabolic regulation, and cellular protection.

• Polyphenols (e.g. Quercetin, Catechin, Chlorogenic Acid): Principal antioxidants that neutralize free radicals, inhibit NF-κB-driven inflammation, and reduce lipid peroxidation. Quercetin supports glycemic regulation by enhancing insulin sensitivity, while chlorogenic acid is associated with cardiovascular and metabolic benefits.
• Dihydrochalcones (e.g. Phloridzin): Unique to apples, these compounds inhibit intestinal glucose transporters (e.g. SGLT1), thereby modulating postprandial glucose uptake and potentially influencing lipid storage and adipogenesis.
• Anthocyanins (in red apple varieties): Natural pigments with reported osteogenic potential, shown to promote osteoblast activity and support bone matrix mineralization in cellular models.
• Triterpenoids (e.g. Ursolic Acid, Oleanolic Acid): Found mainly in apple peel, these compounds activate the Nrf2 pathway to boost endogenous antioxidant defenses and exhibit broad-spectrum antimicrobial and anti-inflammatory effects.
• Vitamins and Micronutrients (e.g. Vitamin C, Vitamin A, Folate): These nutrients reinforce antioxidant defense systems, aid in collagen synthesis, and support immune modulation, often working synergistically with polyphenolic compounds.

What Are Apple-Derived Exosomes?

Apple-derived exosomes are nano-sized extracellular vesicles naturally secreted by apple (Malus domestica) cells. These vesicles carry bioactive molecules and have been studied for their roles in intercellular communication and cross-species regulation. Key features of apple-derived exosomes include:

• Size and structure: Typically 80 to 250 nanometers in diameter, spherical in shape, and negatively charged.
• Isolation methods: Commonly extracted using differential centrifugation, filtration, and ultracentrifugation.
• Molecular content: Rich in plant microRNAs, lipids (mainly glycerophospholipids), proteins, and small metabolites.
• Notable miRNAs: Frequently include mdm-miR482a-5p and mdm-miR396, among others.
• Biological activity: Can be efficiently taken up by mammalian cells, where they may modulate cellular signaling and gene expression.

These characteristics make apple-derived exosomes promising natural carriers for studying molecular transport and plant-to-animal communication in research settings.

Characterization of Apple-Derived Nanovesicles (ADNVs). (A) Size distribution profile of isolated ADNVs analyzed by tunable resistive pulse sensing (qNano), showing an average diameter of 152 ± 32.3 nm. (B) Transmission electron microscopy (TEM) image illustrating the uniform, round morphology of ADNVs with diameters ranging from 80 to 250 nm. (Trentini M, et al., 2022)

Advantages of Apple-Derived Exosomes

Apple-derived exosomes combine natural origin, functional activity, and delivery potential, offering unique advantages in research applications.

• Biocompatibility and Safety: They show no cytotoxicity in tested cell lines and exhibit low immunogenicity due to their plant origin, reducing the risk of immune-related interference.
• Functional Bioactivity: These vesicles possess anti-inflammatory and antioxidant properties, regulate key signaling pathways such as NF-κB and JNK, and promote anti-aging responses by enhancing collagen synthesis and reducing matrix degradation.
• Delivery Capability: They naturally avoid rapid clearance, support targeted transport through surface protein engineering, and efficiently carry small molecules, RNAs, or proteins.
• Stability and Permeability: Apple-derived exosomes are stable in serum and demonstrate strong skin permeability, making them suitable for both systemic and topical delivery research.

Potential Applications of Apple Exosomes

Inflammation and Immune Modulation

Exosomes from apples have been shown to suppress inflammatory signaling by downregulating the NF-κB pathway and reducing cytokine expression. In macrophage models, apple-derived miR-146 promoted an anti-inflammatory phenotype, indicating potential for studying chronic inflammation mechanisms and immune regulation in diseases such as inflammatory bowel disease.

Skin Regeneration and Anti-Aging

Research in dermal fibroblast models indicates that apple exosomes can enhance collagen type I production while inhibiting matrix metalloproteinases, supporting their application in skin aging studies. Their integration with hyaluronic acid-based hydrogels has been explored to promote skin repair and support wound healing through fibroblast activation and enhanced cholesterol biosynthesis.

Targeted Drug and Gene Delivery

Due to their stability and biocompatibility, apple-derived exosomes are being investigated as natural delivery vehicles. They can be engineered to carry therapeutic cargos such as siRNA, small molecules, or photosensitizers like ICG, showing promise in targeted cancer therapy and gene silencing studies.

Intestinal Function and Gut Health

Several studies have confirmed the regulatory role of apple-derived exosomes on intestinal transport proteins, such as OATP2B1 and ASBT/SLC10A2. These effects are mediated by specific plant miRNAs, suggesting a potential role in nutrient absorption, bile acid transport, and microbiota-related disease models.

References

1. Fujita D, Arai T, Komori H, et al. Apple-derived nanoparticles modulate expression of organic-anion-transporting polypeptide (OATP) 2B1 in Caco-2 cells. Molecular Pharmaceutics. 2018, 15(12): 5772-5780.
2. Komori H, Fujita D, Shirasaki Y, et al. MicroRNAs in Apple-Derived Nanoparticles Modulate Intestinal Expression of Organic Anion–Transporting Peptide 2B1/SLCO2B1 in Caco-2 Cells. Drug Metabolism and Disposition. 2021, 49(9): 803-809.
3. Trentini M, Zanotti F, Tiengo E, et al. An apple a day keeps the doctor away: potential role of miRNA 146 on macrophages treated with exosomes derived from apples. Biomedicines. 2022, 10(2): 415.
4. Trentini M, Zanolla I, Zanotti F, et al. Apple derived exosomes improve collagen type I production and decrease MMPs during aging of the skin through downregulation of the NF-κB pathway as mode of action. Cells. 2022, 11(24): 3950.
5. Usui S, Zhu Q, Komori H, et al. Apple-derived extracellular vesicles modulate the expression of human intestinal bile acid transporter ASBT/SLC10A2 via downregulation of transcription factor RARα. Drug Metabolism and Pharmacokinetics. 2023, 52: 100512.
6. Sim Y, Seo H J, Kim D, et al. The effect of apple-derived nanovesicles on the osteoblastogenesis of osteoblastic MC3T3-E1 cells. Journal of Medicinal Food. 2023, 26(1): 49-58.
7. Ferroni L, Rubini A, Bargellini P, et al. Apple vescicles: Revolutionary gut microbiota treatment for Inflammatory Bowel Disease. Food Bioscience. 2024, 62: 105052.

Case Study 1: Apple-Derived Nanovesicles Promote Collagen Synthesis and Suppress Inflammatory Pathways in Skin Aging Models (Trentini M, 2022)

A study explored the effects of apple-derived nanovesicles (ADNVs) on human dermal fibroblasts under inflammatory stress. ADNVs were safely internalized without affecting cell viability and significantly enhanced the expression of collagen-related genes (COL3A1, COL1A2), while reducing the expression of MMP1, MMP8, and MMP9, enzymes associated with matrix degradation.

RNA sequencing revealed that ADNVs downregulated pro-inflammatory mediators such as IL-1β, IL-6, and TNFα, and inhibited the NF-κB signaling pathway in TNFα-induced fibroblasts. Oxidative stress was alleviated, with a 5% reduction in ROS-positive cells compared to untreated controls.

When formulated into hyaluronic acid-based hydrogels and MeHA patches, ADNVs demonstrated stable release profiles. These results highlight ADNVs’ dual potential in supporting extracellular matrix restoration and modulating inflammation for skin aging research.

Figure 1. Cytotoxicity and migration analysis of ADNVs. (A) MTT assay of NCTC L929 fibroblasts treated with or without ADNVs shows no significant change in cell viability. (B) Scratch assay of untreated cells over 12 h indicating normal migration. (C) Migration delay observed in TNFα-treated cells, with no notable difference between ADNV-treated and control groups.

Figure 2. Transcriptomic impact of ADNVs on dermal fibroblasts with or without TNFα. (A, B) Volcano plots showing gene expression changes in dermal fibroblasts cultured with (A) or without (B) TNFα. Each plot compares control (no ADNVs) versus ADNV-treated conditions. The ten most significantly altered genes by fold change and statistical relevance are highlighted.

Case Study 2: Immunomodulatory Effects of Apple-Derived Exosomes on Macrophages (Trentini M, 2022)

A study evaluated the immunoregulatory effects of apple-derived nanovesicles (ADNVs) on THP-1-derived macrophages. The nanovesicles, with an average diameter of 152 nm, were efficiently internalized by macrophages and fibroblasts without inducing cytotoxicity, as confirmed by stable LDH release and enhanced mitochondrial activity.

Following ADNV treatment, macrophages adopted an elongated morphology typical of the anti-inflammatory M2 phenotype. This shift was supported by reduced gene expression of IL-1β and IL-8. RNA sequencing revealed upregulation of regulatory microRNAs including miR-146a-5p, miR-125a-3p, and miR-30a-5p, which are known to suppress NF-κB pathway activation.

These results suggest that ADNVs promote macrophage polarization and suppress inflammation through miRNA-mediated signaling, positioning them as a promising natural tool for immune modulation research.

Figure 1. Cellular uptake and immunomodulatory effects of ADNVs on THP-1 macrophages and fibroblasts. (A) Fluorescence microscopy shows internalization of red-stained ADNVs by green-stained THP-1-derived macrophages and fibroblasts (FU). (B) SEM images depict morphological differences between M0 macrophages, control, and ADNV-treated cells, indicating phenotype changes. (C) Immunofluorescence staining shows macrophage cytoskeleton (green), nuclei (blue), and M1 markers iNOS and CD68 (red), confirming uptake and activation.

Figure 2. miRNA network modulation and cytokine expression in cells treated with ADNVs. (A) miRNA-target gene interaction network generated by miRNet software; key miRNAs and metabolic pathway genes are color-coded. (B) Overlapping target genes of miR-146a-5p, miR-125a-3p, and miR-30a-5p visualized in a shared network. (C) Heatmap showing expression profiles of differentially expressed miRNAs in control vs. ADNV-treated THP-1 cells over 48 h. (D, E) RT-qPCR results of IL-1β and IL-8 expression in THP-1 (D) and fibroblasts with/without TNFα stimulation (E).

References

1. Trentini M, Zanolla I, Zanotti F, et al. Apple derived exosomes improve collagen type I production and decrease MMPs during aging of the skin through downregulation of the NF-κB pathway as mode of action. Cells. 2022, 11(24): 3950.
2. Trentini M, Zanotti F, Tiengo E, et al. An apple a day keeps the doctor away: potential role of miRNA 146 on macrophages treated with exosomes derived from apples. Biomedicines. 2022, 10(2): 415.

• How are the apple-derived exosomes isolated and purified?

  The exosomes are isolated using a combination of differential centrifugation, ultracentrifugation, and membrane filtration techniques to ensure high purity and intact vesicle morphology.

• Can PNExo™ Apple exosomes be used as carriers?

  Yes. They show strong potential for drug and RNA delivery research. Their natural membrane structure supports encapsulation of small molecules, proteins, and nucleic acids, and they exhibit good biocompatibility with minimal immunogenicity.

• Is batch-to-batch consistency ensured?

  Yes. Each production batch undergoes rigorous quality control testing for particle size, concentration, RNA content, and sterility to ensure reproducibility and consistency.

• What analytical methods are recommended for characterization after reconstitution?

  Techniques such as nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), transmission electron microscopy (TEM), and Western blotting for EV markers are commonly used to confirm size distribution and integrity.

• Can I request customized apple-derived exosome formulations or isolation services?

  Yes. Creative Biostructure offers custom exosome services including tailored isolation, characterization, miRNA profiling, and formulation based on your specific research needs. Please contact us to discuss project details and receive technical consultation.

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