PNExo™ Exosome-Broccoli(PNE-VB13)

Benefits of Broccoli

Rich in vitamins, minerals, and antioxidants, broccoli supports overall health and wellbeing. It contains high levels of vitamin C, vitamin K, iron, and potassium, which are essential for maintaining a healthy immune system, strong bones, and proper cardiovascular function. The presence of powerful antioxidants like sulforaphane and glucoraphanin in broccoli contributes to its cancer-preventive properties, making it a valuable addition to any diet.

What Are Broccoli-Derived Exosomes?

Broccoli-derived exosomes are nano-sized vesicles that are naturally secreted by broccoli cells. These exosomes are composed of a lipid bilayer membrane, proteins, lipids, and various bioactive molecules, including RNAs and metabolites. The unique composition of broccoli-derived exosomes enables them to facilitate intercellular communication and deliver therapeutic payloads to target cells. These exosomes are biocompatible and non-immunogenic, making them ideal candidates for drug delivery and other biomedical applications. Their ability to encapsulate and protect bioactive compounds enhances the stability and bioavailability of these compounds, thereby amplifying their therapeutic potential.

Applications of Broccoli-Derived Exosomes

• Broccoli-Derived Exosomes in Therapeutics

  Broccoli-derived exosomes have shown immense potential in the field of therapeutics. Research has demonstrated that these exosomes can effectively inhibit various types of cancer. For instance, sulforaphane encapsulated in broccoli-derived exosomes has been found to reduce melanoma cell proliferation by promoting metabolite generation and reducing cancer molecular markers. Similarly, exosomes encapsulating astaxanthin significantly inhibit the proliferation of human colorectal adenocarcinoma cells. Furthermore, broccoli-derived exosomes containing therapeutic miRNA have been shown to promote the death of human colorectal adenocarcinoma cells more effectively than free miRNA treatment. Exosomes derived from selenium-rich broccoli, loaded with miR167a, significantly promote apoptosis in human pancreatic cancer cells by regulating the IRS1/PI3K/AKT pathway. These findings highlight the potential of broccoli-derived exosomes as a novel and effective approach to cancer therapy.

  Broccoli-derived exosomes as nanocarriers for exogenous miRNAs. (del Pozo-Acebo L, et al., 2022)

• Broccoli-Derived Exosomes in Diagnostics

  The diagnostic potential of broccoli-derived exosomes is also being explored. Due to their natural origin and biocompatibility, these exosomes can be used as biomarkers for various diseases. Their unique composition allows for the detection of specific bioactive molecules associated with disease states, providing a non-invasive method for early diagnosis. For instance, the presence of certain miRNAs encapsulated within broccoli-derived exosomes can serve as indicators of cancer or other pathological conditions. This capability makes them valuable tools in the development of diagnostic assays and personalized medicine.

• Broccoli-Derived Exosomes in Skin Care Applications

  In addition to their therapeutic and diagnostic applications, broccoli-derived exosomes have promising potential in skincare. Their antioxidant properties help reduce oxidative stress and improve skin health. The encapsulation of antioxidant compounds within these exosomes enhances their stability and penetration into the skin, providing long-lasting effects.
  For example, broccoli-derived exosomes containing sulforaphane can protect the skin from UV-induced damage and reduce inflammation. Moreover, their ability to promote collagen synthesis and improve skin elasticity makes them an attractive ingredient in anti-aging formulations. The use of broccoli-derived exosomes in skincare products offers a natural and effective solution for maintaining healthy and youthful skin.

References

1. del Pozo-Acebo L, et al. Therapeutic potential of broccoli-derived extracellular vesicles as nanocarriers of exogenous miRNAs. Pharmacological Research. 2022. 185: 106472.

Case study 1: Yepes-Molina L, 2021

This study explores the potential of broccoli membrane vesicles (BM-vesicles) as nanocarriers for the bioactive compound sulforaphane (SFN), known for its antioxidant and anticancer properties. The BM-vesicles were characterized using various physicochemical methods and tested for their ability to encapsulate SFN. When applied to SK-MEL-28 cancer cells, the encapsulated SFN exhibited a 41% entrapment efficiency and demonstrated significant antiproliferative activity. The encapsulated SFN not only reduced cancer cell proliferation but also altered gene expression, increasing levels of the aquaporin AQP3, crucial for water transport and cellular homeostasis. These findings suggest that BM-vesicles enhance the cellular uptake and metabolism of SFN, leading to improved anticancer efficacy. Thus, BM-vesicles show promise as effective nanocarriers for drug delivery in cancer treatment.

Figure 1. Phase-contrast microscopy images of SK-MEL-28 cells treated with various treatments. Control, free sulforaphane (SFN) at concentrations of 5, 25, and 100 µM, broccoli membrane vesicles (BM-V) with 0.002% and 0.0002% protein, and SFN encapsulated in BM-vesicles at the same concentrations and protein levels. The scale bars for the images are set at 200 µm.

Case study 2: Wang X, 2023

Pancreatic adenocarcinoma (PAAD) has high morbidity and mortality rates, with broccoli known for its anti-cancer properties but limited by dosage and side effects. This study evaluates the effectiveness of extracellular vesicles (EVs) derived from selenium-enriched broccoli (Se-BDEVs) and conventional broccoli (cBDEVs) in treating PAAD.
Se-BDEVs and cBDEVs were isolated using differential centrifugation and characterized by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). miRNA sequencing, target gene prediction, and functional enrichment analysis were performed to understand their potential functions. The efficacy was then tested on PANC-1 cells.
Both Se-BDEVs and cBDEVs had similar size and morphology. miRNA sequencing revealed their miRNA content, with functional analysis indicating potential roles in pancreatic cancer treatment. In vitro studies showed Se-BDEVs had greater anti-PAAD effects than cBDEVs, attributed to higher miR167a expression. Transfection with miR167a mimics induced significant apoptosis in PANC-1 cells. Bioinformatics analysis identified IRS1, involved in the PI3K-AKT pathway, as a key miR167a target. miR167a in Se-BDEVs demonstrates potential as a novel therapeutic tool for combating PAAD, offering a new approach to cancer treatment.

Figure 1. Effect of BDEVs and Se-BDEVs on PANC-1. Cell viability of PANC-1 was assessed using the CCK-8 assay and expressed as optical density (OD). Panels A and B document the OD values following the exposure of PANC-1 cells to escalating doses (0, 10, 20, and 40 ng/μL) of cBDEVs or Se-BDEVs, highlighting a statistically significant reduction in viability at the highest concentration of 40 ng/μL compared to the baseline (Control), with a p-value of less than 0.001. Panel C presents an amalgamation of the growth trajectories for PANC-1 cells subjected to 40 ng/μL of either cBDEVs or Se-BDEVs, alongside the Control group, which reveals a pronounced divergence in growth patterns, with a p-value of less than 0.001, indicating a superior inhibitory effect of Se-BDEVs over cBDEVs. Panels D and E display the incubation of PANC-1 cells with PKH67-stained cBDEVs or Se-BDEVs over a range of time intervals (30 minutes to 6 hours), with the images calibrated to a uniform scale of 50 µm for consistency in visual assessment.

Figure 2. Effect of miR167a on PANC-1. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to quantify miR167a expression levels, represented by the cycle threshold (Ct) values, in cells treated with Selenium-Bismuth Doped Eosin Y (Se-BDEVs) and control Bismuth Doped Eosin Y (cBDEVs). (B and C) The metabolic activity of PANC-1 cells, post-transfection with miR167a mimics, miR167a inhibitor-coupled mimics, miR167a negative control (NC), and a non-treated control group, were evaluated at 24-hour and 48-hour intervals through optical density (OD) measurements. (D) The apoptotic rate in the aforementioned transfected PANC-1 cells was determined using flow cytometric analysis after a 24-hour incubation period. (E and F) The resulting statistical charts from the apoptosis flow cytometry assay illustrate the proportions of cells in early (annexin V positive, propidium iodide negative) and late (both positive) stages of apoptosis. The asterisks denote highly significant differences at the levels of ***p < 0.001 and **p < 0.01, respectively.

References

1. Yepes-Molina L, Carvajal M. Nanoencapsulation of sulforaphane in broccoli membrane vesicles and their in vitro antiproliferative activity. Pharmaceutical Biology. 2021. 59(1): 1488-1502.
2. Wang X, et al. Selenium biofortification enhanced miR167a expression in broccoli extracellular vesicles inducing apoptosis in human pancreatic cancer cells by targeting IRS1. International Journal of Nanomedicine. 2023. 18: 2431-2446.

• What specific components are found within broccoli-derived exosomes, and how do they contribute to their therapeutic effects?

  Broccoli-derived exosomes contain a rich composition of bioactive molecules, including lipids, proteins, and various types of RNA such as mRNA and miRNA. Key components like sulforaphane and other antioxidants play a crucial role in their therapeutic effects by reducing oxidative stress and inhibiting cancer cell proliferation. Additionally, the presence of specific miRNAs in these exosomes can regulate gene expression in target cells, promoting apoptosis in cancer cells and enhancing the immune response.

• How do broccoli-derived exosomes compare in efficacy to synthetic drug delivery systems for cancer therapy?

  Broccoli-derived exosomes have shown comparable, and in some cases superior, efficacy to synthetic drug delivery systems in cancer therapy. This is primarily due to their natural origin, biocompatibility, and ability to encapsulate and deliver therapeutic molecules directly to target cells. Unlike synthetic systems, broccoli-derived exosomes can naturally evade the immune system, reduce off-target effects, and provide a more targeted and sustained release of therapeutic agents, enhancing their overall efficacy in cancer treatment.

• What are the optimal conditions for storing broccoli-derived exosomes to maintain their stability and bioactivity?

  To maintain the stability and bioactivity of broccoli-derived exosomes, it is essential to store them at -80°C. Cryopreservation helps preserve their structural integrity and functionality over extended periods. Additionally, it is recommended to avoid repeated freeze-thaw cycles, as this can lead to the degradation of exosomal membranes and loss of bioactivity.

• How do you ensure the purity and consistency of broccoli exosomes?

  Each batch undergoes rigorous quality control assessments to confirm size, concentration, and the presence of exosomal markers. Techniques like nanoparticle tracking analysis (NTA) and Western blotting are employed to ensure consistency and purity. NTA provides information about the size distribution and concentration of the exosomes, while Western blotting confirms the presence of specific exosomal markers.

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