Exosome Studies Service

    All cells release extracellular vesicles (EVs) as crucial part of their normal physiological activities. There are mainly two types of EVs: ectosomes and exosomes. Ectosomes, including microvesicles, microparticles, and large vesicles in the size range of of 50 nm to 1 μm in diameter, pinch off the surface of the plasma membrane via outward budding. Exosomes, with diameters ranging from 40 nm to 160 nm (average around 100 nm), derive from endosomes. Plasma membranes invaginate to form multivesicular bodies, which intersect with other intracellular vesicles and organelles, enriching the diversity in the constituents of exosomes. EVs, including exosomes, may contain a variety of cellular components such as DNA, RNA, lipids, metabolites, and proteins associated with cell membranes and surfaces. The physiological roles of exosome production are still under investigation, but it is hypothesized that exosomes could help maintain cellular homeostasis by removing excess or unnecessary cellular constituents.

     

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    Figure 1. Structure and Function of Exosomes [1]

     

    Exosomes play role in immune responses, viral pathogenicity, pregnancy, cardiovascular diseases, central nervous system-related diseases, and cancer progression. They transport proteins, metabolites, and nucleic acids to recipient cells, thus modulating these cells' biological responses and potentially influencing disease outcomes. Exosomes show promise in treating various conditions, including neurodegenerative conditions and cancer, and can be engineered to carry therapeutic payloads like short interfering RNAs, antisense oligonucleotides, chemotherapeutic agents, and immune modulators. Their composition affects their pharmacokinetic properties; natural constituents may enhance their bioavailability and minimize adverse reactions. Exosomes also offer diagnostic potential, as they can be detected in biological fluids. Exosome-based liquid biopsy could aid in the diagnosis and determining the prognosis of patients with cancer and other diseases. Multicomponent analysis of exosomes can further illuminate disease progression and response to therapy.

     

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    Figure 2. Targeted Drug Delivery System Based on Exosomes [2]

     

    MtoZ Biolabs provides a comprehensive suite of services for exosome research, including its production, isolation, characterization, and quantitatification, engineered design, and drug loading, as well as functional testing at cellular and animal levels. The platform supports extensive research from exosome large-scale preparation and production to drug delivery. The team employs a range of techniques, such as electron microscopy and particle size analysis, to ascertain exosome quality and purity. It utilizes chemical and biological methods to engineer exosomes, and develops strategies for exosome encapsulating proteins, nucleic acids, and small-molecule drugs. MtoZ Biolabs delivers a one-stop solution for exosome-related challenges, encompassing its preparation, analysis, drug loading design, and data analysis.

     

    1. Structure and Composition of Exosomes

    Exosomes are capable of encapsulating a diverse array of molecules including DNAs, RNAs, lipids, metabolites, and proteins. To date, the ExoCarta exosome database has collected 9769 proteins, 3,408 mRNAs, 2,838 miRNAs, and 1,116 lipids, identified in exosomes derived from various cell types and organisms.

     

    The cell source largely determines the protein secreted by exosomes, yet about 80% of these proteins remain highly conserved among different cells. Key biomarkers such as ALIX, TSG101, heat shock proteins, and tetraspanins (CD63, CD9, and CD81) are crucial, mediating the targeting and selective capability of exosomes. Exosomes are also rich in nucleic acids, encompassing mRNAs, miRNAs, mitochondrial DNA, and piRNAs, lncRNAs, ribosomal RNAs, snRNAs, and tRNAs. Despite being comprised of approximately 200 bp degraded fragments, these nucleic acids can significantly impact protein synthesis in the recipient cells. The lipid composition of exosomes—comprising cholesterol (chol), phospholipids, phosphatidyletha-nolamines, polyglycerols, and diglycerides—differs markedly from that of the cellular plasma membrane. These lipids not only contribute to the structural rigidity and stability of exosomes but also engage in various biological processes, including cellular signaling communication, where they interact with prostaglandin and phospholipases C and D.

     

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    Figure 3. Structure and Composition of Exosomes [3]
     

    2. Isolation of Exosomes

    The burgeoning field of exosome research is continuously unveiling their potential applications, necessitating effective isolation and enrichment techniques to explore their biological functions. The inherent heterogeneity of exosomes, in terms of size, content, function, and origin, complicates their isolation. For various purposes and applications, several methods are available, including ultracentrifugation (UC), size-based isolation techniques, polymer precipitation, and immunoaffinity capture techniques.

     

    (1) UC

    Widely recognized as the gold standard for exosome extraction and separation, UC leverages differences in size and density among solute components to segregate large-dose sample components with significant differences in sedimentation coefficient. The process typically involves a series of continuous low-medium speed centrifugation to remove dead cells, cell debris and large-size extracellular vesicles, and then to separate exosomes at a higher speed with a centrifugal force of 100,000 × g, the exosomes were washed with PBS to remove impurities such as contaminating proteins. Factors such as centrifugation time, centrifugal force, rotor type, and settings crucially influence yield and purity of target exosomes. This label-free approach avoids cross-contamination but is time-consuming, costly, and can cause structural damage and aggregation, hindering downstream analysis.

     

    Density gradient centrifugation, often combined with ultracentrifugation, enhances exosomal purity. Among the types utilized, sucrose-based gradients are common but cannot effectively separate exosomes from similarly size and density of retroviruses. Different sedimentation velocities in the Iodixanol gradients have proven effective in separating exosomes from HIV-1-infected cells and achieving high-purity isolations. While offering superior purity, the high viscosity of sucrose solutions can reduce the sedimentation rate of exosomes, resulting in a longer time.

     

    (2) Size-Based Isolation Techniques

    Techniques such as UC and size exclusion chromatography (SEC) rely on the size difference between exosomes and other sample components. SEC operates on the principle that macromolecules can not enter gel pores and are washed out by the mobile phase, whereas smaller molecules are retained in the gel pores and later eluted. Commercial products like qEV separation columns, EVSecond purification columns, and Exo-spin exosome purification columns facilitate this method. SEC is noted for its speed, simplicity, and cost-effectiveness, delivering structurally complete and uniformly sized exosomes without significantly altering their biological properties. However, co-elution of similarly sized particles can compromise purity of target exosomes.

     

    UC employs ultrafiltration membranes with different molecular weight cutoffs (MWCO) to aid in selective separation, serving a supportive role in exosome research. While cost-effective and efficient, this method faces challenges with purity and non-specific binding membrane interactions that can reduce recovery rate.

     

    (3) Polymer Precipitation Method

    Polymer precipitation, typically using polyethylene glycol (PEG), decreases exosome solubility to facilitate recovery via centrifugation. Initially developed for virus isolation, this method exploits the similar biophysical characteristics of viruses and exosomes for scientific separation and purification. Comparisons between UC, modified polymer co-precipitation (ExtraPEG), and commercial kits indicate superiority of the first two, with ExtraPEG offering greater cost-effectiveness and better purity and recovery. Despite its simplicity and suitability for large scale samples, this method can produce lower purity and yield false positives, with residual polymers hindering subsequent functional analysis.

     

    (4) Immunoaffinity Chromatography (IAC)

    IAC harnesses the specific binding between antibodies and ligands to isolate desired substances from heterogeneous mixtures. The binding efficiency depends on the biological affinity pairs, elution conditions and matrix carriers. High-abundance proteins on the surface of exosome membranes, such as four-transmembrane protein superfamily, and ESCRTcomplex-related proteins, are typical biomarkers. IAC, requiring smaller sample volumes compared to UC, also facilitates qualitative and quantitative exosome analysis, offering high specificity, sensitivity, purity, and yield. Techniques like ELISA are employed for enriching exosomes in fluids such as serum, plasma, and urine. Although yields of IAC are comparable to those of UC, IAC demands harsh storage conditions and is less suited for large-scale separations due to potential non-specific matrix interference adsorption.

     

    (5) Other Separation Techniques

    Various commercial kits, such as the exoEasy Maxi Kit (QIAGEN), MagCapture™ Exosome Isolation Kit PS (Wako), and Minute™ Hi-Efficiency Exosome Precipitation Reagent (Invent), are based on the above traditional separation methods. While these kits offer time saving, high yields, good integrity and so on, their effectiveness varies, and no kit yet excels in isolating ideal exosomes from complex samples. Moreover, the kits can be costly, and their exosome yield and purity often are not high.

     

    3. Characterization of Exosomes

     

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    4. Storage of Exosomes

    Exosomes are an innovative cell-free therapy, but their long-time storage poses significant challenges. The primary storage techniques employed are freezing, freeze-drying, and spray-drying, each catering to maintaining the stability and functionality of exosomes under extended storage conditions.

     

    (1) Freezing

    Freezing preserves biological particles by reducing the temperature to inhibit biochemical activities, commonly applied at 4°C, -80°C, and -196°C. Cryoprotectants are crucial; permeable types like dimethyl sulfoxide (DMSO) and ethylene glycol penetrate cell membranes to avert ice crystal formation, while non-permeable types such as trehalose and sucrose stabilize water molecules through hydrogen bonding, enhancing preservation.

     

    (2) Freeze-Drying

    Freeze-drying involves cooling water-containing materials and freezing to a solid below freezing point, then sublimating the ice directly under vacuum. This three-stage process—pre-freezing, sublimation drying stage, and analytical drying stage—minimizes storage conditions by preserving the exosomes' original activity and reducing potential damage to their biological structures. The resultant freeze-dried exosomal powder effectively reduce storage conditions while maintaining bioactivity.

     

    (3) Spray Drying

    Spray drying systematically dries materials by atomizing EVs into fine droplets within a drying room, and the moisture quickly vaporizes in contact with the hot air to obtain dry powders. The process's efficiency hinges on atomization pressure and outlet temperatures. Offering a one-step, continuous powder production, spray drying is noted for its economical value, scalability, and the ability to fine-tune particle sizes, making it an alternative to freeze-drying for large-scale exosome storage solutions.

     

    5. Exosome Drug Delivery

    Exosomes are small in size, allowing them to evade mononuclear macrophage phagocytosis and freely traverse vascular walls and extracellular matrices. Their surfaces express CD55 and CD59, which inhibit the activation of opsonin and coagulation factors, ensuring their stable and widespread distribution in the biofluids. As derivatives of human cells, exosomes naturally exhibit superior biocompatibility and lower immunogenicity compared to liposomes and other nano-delivery systems. Due to exosomal heterogeneity, they carry various surface proteins that facilitate their entry into cells through mechanisms such as receptor-mediated endocytosis, a crucial pathway for exosome-target tissue communication. This process not only enhances drug encapsulation and cellular internalization but also supports the stable and efficient transport of contents in the blood. Additionally, exosomes possess the innate ability to target specific tissues or cells and penetrate biological barriers like the blood-brain barrier, positioning them as ideal carriers for gene therapies, traditional Chinese medicines, western medicine and so on. However, natural exosomes often face challenges like weak targeting and rapid clearance in the body, leading to poor treatment effects. To address these issues, exosomes are frequently engineered to improve targeting and load with therapeutic agents or modified.

     

    (1) Drug Loading Methods

    Drug loading into exosomes can be categorized into pre-secretory and post-secretory methods:

    ① Pre-Secretory Loading: Therapeutic agents are derived from parent cells to secrete engineered exosomes. This straightforward method, however, offers limited control over drug loading efficiency and may disrupt the natural physiological functions of membrane proteins.

    ② Post-Secretory Loading: This method involves directly incorporating therapeutic agents into exosomes using techniques such as incubation, ultrasound treatment, freeze-thaw cycles, surface treatment, hypotonic dialysis, and pH gradients. While effective, this technique may cause issues like exosome aggregation, membrane damage, and low yields.

     

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    Figure 4. Common Drug Delivery Modes of Exosomes [4]

     

    (2) Genetic Engineering and Surface Modification 

    Gene-editing approaches, using geneticaly modified parent cells as the main strategy to integrate therapeutic agents into the corresponding exosomes, suitable for RNA or proteins that can not be loaded directly onto the exosomes. Surface modification is a process in which exosomal surface proteins are used as anchoring devices or affinity tags to modify protein or peptide components to the surface of particles. If natural exosomes can not deliver drugs effectively or for targeted applications because of their poor stability and rapid elimination, superficial modification is required. One of the greatest advantages of exosomes is that they are endogenous and can avoid adverse effects such as immune responses. However, the efficiency of exosomal delivery is influenced by both parental and recipient cells. The yield of exosomes from different sources is significantly different, and the targeting of natural exosomes is poor. In order to meet the experimental needs, surface modification of engineered exosomes is a practical application value. Surface functionalization of exosomes with targeted ligands can help them selectively deliver to target cells, enabling exosomes to meet standards in terms of yield and targeted therapy, thus achieving precision therapy of exosomes, so as to accelerate the clinical application of exosomes. In order to precisely target therapeutic drugs to the lesions, the methods commonly used in recent years include chemical linking of targeting peptides, modification of exosomal membranes or progenitor cells by genetic engineering, magnetic nanoparticle technology, electrostatic interaction and post-insertion.

     

    Despite the benefits, engineered exosomes have limitations. Surface modifications do not alter the fundamental structures or molecules of exosomes, and chemical modifications, such as chemically linked targeting peptides, may impact their surface structures. Furthermore, genetic modifications of parental cells are technically complex and may affect the biological activity of membrane proteins. Modifications using virus-derived proteins or peptides necessitate assessments for potential adverse reactions. Additionally, cationic nanomaterials used in electrostatic interactions can induce cytotoxic effects, with generally low loading efficiencies.

     

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    Figure 5. Surface Modification of Engineered Exosomes [3]

     

    Analysis Workflow

    1. Determine the Experimental Procedure Based on Experimental Requirements

    2. Exosome Separation

    3. Exosome Characterization

    4. Exosome Drug Delivery

    5. Testing the Efficiency of Exosome Drug Delivery

    6. Testing at the Cellular/Animal Level

     

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    Service Advantages

    1. Mass Production, Identification, Quantification, and Modification Identification of Exosomes

    2. Design and Separation of Engineered Exosomes

    3. Exosome Drug Loading and Testing of Loading Efficiency

    4. Multilevel Functional Studies of Exosomes

     

    Sample Results

    1. AntiCancer Effects of PMO-miR-146b-5p Delivery via Exosomes Derived from hUC-MSC in Colorectal Cancer

    Antisense oligonucleotides (ASOs) are emerging as a novel platform for targeted cancer therapy. Previous studies highlighted the crucial role of miR-146b-5p in colorectal cancer progression. Yet, developing a reliable method to deliver ASOs to their target RNAs safely and effectively continues to be a significant challenge in translational advances. Exosomes from human umbilical cord mesenchymal cells (hUC-MSC) served as delivery vehicles for anti-miR-146b-5p ASO (PMO-146b). The PMO-146b was covalently bound to the anchor peptide CP05 (P), which specifically bound to the exosomal surface marker CD63. This configuration resulted in the formation of a complex called ePPMO-146b. Treatment with ePPMO-146b involved assessing cell proliferation, uptake ability, and migration, along with in vitro examination of epithelial-mesenchymal transition (EMT) progression. The in vivo anti-tumor efficacy and biodistribution of ePPMO-146b were evaluated using a mouse xenograft model. ePPMO-146b was effectively taken up by SW620 cells, leading to notable suppression of both cell proliferation and migration. Systemically administered, the conjugate showed pronounced anti-tumor effects in a colorectal cancer xenograft mouse model, with significant enrichment of PPMO-146b in the tumor tissue. This study highlighted the potential of hUC-MSC-derived exosomes, anchored with PPMO-146b, as an innovative, safe, and effective vehicle for delivering PMO backboned ASOs.

     

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    Figure 6. Schematic Diagram of the Preparation of Extracellular Vesicle Anchor Peptide (CP05) PMO Bound to hUC MSCs Exosomes [5]

     

    2. Therapeutic Potential of Plant-Derived Extracellular Vesicles as Exogenous miRNA Nanocarriers

    EVs, important in cell-to-cell communication, are found in both plant and animal systems. Diets rich in vegetables and fruits, which contain bioactive molecules, have been linked to disease prevention. Plant-derived EVs (PDEVs) resemble mammalian EVs in both biogenesis and morphology and are capable of transporting bioactive molecules, including miRNAs. Despite this, the biological functions of PDEVs remain partially understood, and standardized isolation methods are yet to be established. Recent studies have successfully isolated PDEVs from foods such as broccoli, pomegranate, apple, and orange using techniques like UC and SEC. These studies also evaluated the potential of PDEVs to enhance the transport of synthetic miRNAs. Additionally, the role of food-derived EVs to serve as carriers of dietary (poly)phenols and other secondary metabolites has been investigated. The characterized EVs from the four sources contained four families of miRNAs in both the tissues and the EVs themselves. miRNAs within the EVs from broccoli and fruit sources were notably resistant to RNase degradation and were actively transported into cells. EVs transfected with combinations of specific miRNAs (ath-miR159a, ath-miR162a-3p, ath-miR166b-3p, and ath-miR396b-5p) exhibited toxic effects on human cells, a result also observed with natural broccoli doing to EVs. Moreover, PDEVs were noted for transporting trace amounts of phytochemicals, including flavonoids, anthocyanidins, phenolic acids, or glucosinolates. PDEVs thus demonstrated significant potential as nanocarriers for functional miRNAs, offering promising avenues for RNA-based therapy. Their ability to transport essential bioactive molecules underscored their potential in medical and therapeutic fields.

     

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    Figure 7. Therapeutic Potential of Plant-Derived Extracellular Vesicles as Exogenous miRNA Nanocarriers [6]

     

    3. Exosomes Loaded with Obeticholic Acid Alleviate Liver Fibrosis Through Dual Targeting of the FXR Signaling Pathway and ECM Remodeling

    End-stage of liver fibrosis poses a significant risk of progressing to cirrhosis and hepatocellular carcinoma, making liver transplantation the only effective treatment currently available. Building on previous research that has demonstrated the protective effects of farnesoid X receptor (FXR) agonists like obeticholic acid (OCA) against liver injury, a study proposed a novel therapeutic strategy that also addressed the challenge of side effects. Utilizing exosomes as biological carriers, the estrogen-like FXR agonist OCA was delivered directly to the liver. This approach not only reduced the side effects typically associated with systematic administration but also ensured that OCA accumulated effectively in the liver. Research findings indicated that exosomes carrying OCA significantly improved fibrosis markers and reduced the levels of liver enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum. The mechanism underlying these beneficial effects involved the modulation of hepatic stellate cells (HSCs), remodeling of the extracellular matrix (ECM), and activation of the Fxr-Cyp7a1 signaling cascade. In mouse models of CCL4-induced liver fibrosis, exosomes loaded with OCA demonstrated enhanced anti-fibrotic properties, pointing to a potential therapeutic effect. The use of Exo-loaded OCA not only offered a promising approach for treating liver fibrosis but also exemplified the potential of exosomes as precise drug delivery systems targeting liver tissue. This strategy provided a valuable protective effect against liver fibrosis, highlighting its promise for further development and clinical application.

     

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     Figure 8. Exosomes Loaded with OCA Alleviate Liver Fibrosis Through Dual Targeting of the FXR Signaling Pathway and ECM Remodeling [7]

     

    Sample Submission Requirements

    1. Minimize Impurity Contamination

     

    Services at MtoZ Biolabs

    1. Experimental Procedures

    2. Instrument Parameters

    3. Raw Data

    4. Data Analysis Report

     

    Applications

    1. Disease Diagnosis with Exosomes

    Exosomes are commonly used as biomarkers for diagnosing and prognosticating diseases, predominantly used in oncology, with significant advancements in cardiovascular diseases, tuberculosis, and central nervous system-related disorders. Liquid biopsy is a minimally invasive and efficient method for tumor diagnosis. As tumor-derived exosomes facilitate oncogenesis in healthy cells, their high concentration in biological fluids makes them ideal for liquid biopsies. Identifying cancer-specific exosomal biomarkers enhances the accuracy and early detection of tumors, given the differential expression of bioactive molecules carried by exosomes from healthy versus oncological cells.

     

    2. Exosome-Based Targeted Drug Delivery for Disease Treatment

    Exosomes are explored both as therapeutic agents themselves and as carriers for drugs, offering an effective alternative to traditional delivery systems like liposomes. Due to their ability to enter cells efficiently and their lower of immune clearance rate when administered in models like mice, exosomes present a viable method for delivering functional therapeutic agents. Their good tolerability further underscores their therapeutic potential. Additionally, the possibility of receptor-mediated targeting facilitated by ligands on the surface of exosomes allows for precise delivery to affected tissues. Engineered exosomes, with specific ligands, can be designed to manipulate signaling pathways within targeted cells or direct the exosomes to particular cell types, enhancing therapeutic outcomes.

     

    FAQ

    Q1: What are the advantages and disadvantages of the exosome isolation strategy?

     

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     Q2: What are the advantages and disadvantages of exosome drug delivery technology?

     

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    References

    [1] Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020 Feb 7;367(6478):eaau6977. doi: 10.1126/science.aau6977. PMID: 32029601; PMCID: PMC7717626.

    [2] Ferreira D, Moreira JN, Rodrigues LR. New advances in exosome-based targeted drug delivery systems. Crit Rev Oncol Hematol. 2022 Apr;172:103628. doi: 10.1016/j.critrevonc.2022.103628. Epub 2022 Feb 18. PMID: 35189326.

    [3] Liang Y, Duan L, Lu J, Xia J. Engineering exosomes for targeted drug delivery. Theranostics. 2021 Jan 1;11(7):3183-3195. doi: 10.7150/thno.52570. PMID: 33537081; PMCID: PMC7847680.

    [4] Zhang Y, Bi J, Huang J, Tang Y, Du S, Li P. Exosome: A Review of Its Classification, Isolation Techniques, Storage, Diagnostic and Targeted Therapy Applications. Int J Nanomedicine. 2020 Sep 22;15:6917-6934. doi: 10.2147/IJN.S264498. PMID: 33061359; PMCID: PMC7519827.

    [5] Yu S, Liao R, Bai L, Guo M, Zhang Y, Zhang Y, Yang Q, Song Y, Li Z, Meng Q, Wang S, Huang X. Anticancer effect of hUC-MSC-derived exosome-mediated delivery of PMO-miR-146b-5p in colorectal cancer. Drug Deliv Transl Res. 2023 Nov 17. doi: 10.1007/s13346-023-01469-7. Epub ahead of print. PMID: 37978163.

    [6] López de Las Hazas MC, Tomé-Carneiro J, Del Pozo-Acebo L, Del Saz-Lara A, Chapado LA, Balaguer L, Rojo E, Espín JC, Crespo C, Moreno DA, García-Viguera C, Ordovás JM, Visioli F, Dávalos A. Therapeutic potential of plant-derived extracellular vesicles as nanocarriers for exogenous miRNAs. Pharmacol Res. 2023 Nov 19;198:106999. doi: 10.1016/j.phrs.2023.106999. Epub ahead of print. PMID: 37984504.

    [7] Azizsoltani A, Hatami B, Zali MR, Mahdavi V, Baghaei K, Alizadeh E. Obeticholic acid-loaded exosomes attenuate liver fibrosis through dual targeting of the FXR signaling pathway and ECM remodeling. Biomed Pharmacother. 2023 Dec;168:115777. doi: 10.1016/j.biopha.2023.115777. Epub 2023 Oct 31. PMID: 37913732.

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