Exosome Preparations Research Articles

Circulating Exosomal miRNA Profiles in Non-Small Cell Lung Cancers.

A growing number of studies have shown that microRNAs (miRNAs) can exert oncogenic or tumor suppressor activities in a variety of cancers, including lung cancer. Given their presence in exosome preparations, microRNA molecules may in fact participate in exosomal intercellular transfers and signaling. In the present study, we examined the profile of 25 circulating exosomal microRNAs in ostensibly healthy controls compared to patients with squamous cell lung cancers (SQCLC) or lung adenocarcinomas (LUAD). Eight miRNAs, namely, miR-21-5p, miR-126-3p, miR-210-3p, miR-221-3p, Let-7b-5p, miR-146a-5p, miR-222-3p, and miR-9-5p, were highly enriched in the cohort and selected for further analyses. All miRNAs were readily detected in non-small cell lung cancer (NSCLC) patients of both sexes at all cancer stages, and their levels in exosomes correlated with the clinicopathological characteristics of tumors. Thus, the presence of these miRNAs in circulating exosomes may contribute to the regulation of oncogenic activity in patients with NSCLC.

Exosomes, peptides and peri-procedure management – comparative investigations

Introduction: Exosome technology is a promising new advancement in therapeutic options but is still in its early stages, leading to uncertainties. A small pilot study compared traditional peptide technology (Regenerating Skin Nectar with TriHex Technology® and TriHex Technology® peptide + Octapeptide) to two leading exosome products (Stem cell derived Lyophilized exosomes and Platelet derived exosomes) for post-procedure use. These studies were carried out on pre-clinical (ex vivo) and clinical front to investigate effects on the extracellular matrix, dermo-epidermal junction, and skin tolerance as post-procedure outcomes. Methods and materials: The ex vivo model study was conducted by 3D Genomics using photodamaged skin from facelift patients. The skin was divided, processed under BSL2 conditions and cultured in transwells with specific media. Treatments, including peptides and platelet-derived products, were applied daily for 7 days, while stem cell-derived exosomes were applied as a single application as per the described usage. One set was left untreated as a control. Skin samples were treated and analyzed using immunohistochemistry staining for tropoelastin and CD44. RNA was isolated after treatments and sequenced to assess changes in gene expression. The study aimed to compare the effects of different treatments on skin regeneration and tolerance. Clinically, the participants underwent microneedling of the face and applied exosomes and peptides on each side of the face as comparisons. Participants completed questionnaires on Day 0. On Days 0,1,2,3 and 4 participants completed their self-healing ratings and were also assessed by an assessor on their healing rating on a 5-point scale. Results: The plated exosome products exhibited significant toxicity in the ex vivo model and neither of the exosome products produced relevant changes in gene expression or regenerative effects in this model. Participants in the small clinical series experienced unpleasant burning and stinging in keeping with the effects seen in the ex vivo model. In contrast, the peptide technology showed favorable gene expression upregulation and histological changes in the extracellular matrix and was well-tolerated and comfortable for post-procedure use, as has been demonstrated in multiple clinical trials. Conclusion: The overall impression from these pilot studies suggests that peptide formulations are better tolerated and more effectively target ECM regenerative effects. Conversely, exosome preparations appear to be prone to skin reactions and lack clear targeting of skin regenerative pathways.

Designing a Multiple-Epitope Vaccine Candidate against Leishmania major and Leishmania infantum for Monocyte-Derived Exosome Preparation.

Leishmania is a vector-borne protozoon, which causes visceral, cutaneous and mucocutaneous leishmaniosis in human and animals. Monocyte-derived exosome vaccines can be used as prophylaxis and immunotherapy strategies. The aim of this study was to design a multiple-epitope candidate vaccine using leishmaniolysin (GP63) and rK39 proteins against Leishmania major and L. infantum for monocyte-derived exosome preparation. This study was carried out in Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, 2023-2024. Effective immunodominant epitopes were selected from two antigenic proteins of GP63 and rK39 using various immunoinformatics and bioinformatics approaches. Vibrio cholerae β-subunit was used as an adjuvant to stimulate immune responses. Then, appropriate linkers were selected for the fusion of epitopes. The 3D model of candidate vaccine was predicted and validated. This designed candidate vaccine could effectively be used as a prophylaxis strategy against leishmaniosis. A candidate vaccine was designed using bioinformatic and immunoinformatic studies with virtual acceptable quality; however, effectiveness of this vaccine should be verified through further in-vitro and in-vivo studies.

Standardized Reporting of Research on Exosomes to Ensure Rigor and Reproducibility.

Significance: The study of extracellular vesicles (EVs), especially exosomes, has unlocked new avenues in understanding cellular communication and potential therapeutic applications. Recent Advances: Advancements in EV research have shown significant contributions from the International Society for Extracellular Vesicles (ISEV), in establishing methodological standards. The evolution of the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines from 2014 to 2023 reflects enhanced research rigor and reproducibility. The launch of EV-TRACK platform promotes uniformity and reproducibility by providing a centralized repository for data sharing and standardization practices. Furthermore, databases like EVpedia and ExoCarta have facilitated data sharing and collaboration within the scientific community. Concurrently, exosome-based therapies have emerged as a forefront area within regenerative medicine and targeted drug delivery, showcasing the potential of exosomes in promoting tissue regeneration. Critical Issues: Despite advancements, the field grapples with challenges such as vesicular heterogeneity, EV isolation complexity, and standardization. These issues impact research reproducibility and clinical applications. The inconsistency in exosomal preparations in clinical trials poses significant challenges to therapeutic efficacy and safety. Future Directions: The review outlines critical areas for future research, including the need for technological innovation in EV isolation and characterization, the establishment of standardized protocols, and a deeper understanding of exosome biology. The review also highlights the need to reassess guidelines, develop new EV isolation and characterization technologies, and establish standardized protocols to overcome current limitations. Emphasis is placed on interdisciplinary research and collaboration to address the complexities of EV biology, improve clinical trial design, and ultimately realize exosome's therapeutic and diagnostic potential. Continued evaluation and rigorous scientific validation are essential for successful exosome integration.

Exosomes as Conduits: Facilitating Hepatitis B Virus-Independent Hepatitis D Virus Transmission and Propagation in Hepatocytes.

A number of research studies, including ours, have spotlighted exosomes as critical facilitators of viral dissemination. While hepatitis B virus (HBV) transmission through exosomes has been studied, the focus on its satellite virus, the hepatitis delta virus (HDV), has been unexplored in this context. HDV, although being a defective virus, can replicate its genome autonomously within hepatocytes, independently of HBV. Investigations on Huh7 cells revealed an intriguing phenomenon: the HDV proteins, S-HDAg and L-HDAg, are transmitted between cells without a complete viral structure. Detailed analysis further revealed that the expression of these proteins not only bolstered exosome secretion but also ensured their enrichment within these vesicles. Our experimental approach utilized transfection of various plasmids to examine the role of HDV RNA and proteins in the process. One salient finding was the differential propagation of the HDV proteins S-HDAg and L-HDAg, suggesting intricate molecular mechanisms behind their transmission. Notably, the purity of our exosome preparations was monitored using markers such as TSG101 and CD81. Importantly, these exosomes were found to carry both HDV RNA and proteins, highlighting their role in HDV dissemination. This novel study underscores the role of exosomes in mediating the transmission of HDV components between hepatocytes independent of HBV. These revelations about the exosomal pathway of HDV transmission provide a foundation for the development of innovative therapeutic strategies against HDV infections.

Potential of Exosomes as Therapeutics and Therapy Targets in Cancer Patients

After an initial positive response to chemotherapy, cancer patients often acquire chemoresistance and tumor relapse, which makes cancer one of the most lethal diseases worldwide. Exosomes are essential mediators of cell-to-cell communication by delivering their cargo, such as proteins, RNAs and DNA, from cell to cell. They participate in cancer progression, metastasis, immune response and therapy resistance. Their ability to shuttle between cells makes them efficient drug delivery systems. As drug transporters, they provide novel strategies for cancer therapy by advancing targeted drug therapy and improving the therapeutic effects of anti-cancer medications. In this review, a comprehensive overview of the potential of exosomes as therapeutic agents and targeted molecules in the treatment of cancer patients is given. The current challenges of preparation of exosomes loaded with drugs and delivering them to the recipient tumor cells as well as a consequent exosome-mediated cancer therapy are also discussed.

Potential of Exosomes as Therapeutics and Therapy Targets in Cancer Patients

After an initial positive response to chemotherapy, cancer patients often acquire chemoresistance and tumor relapse which make cancer to the most lethal diseases worldwide. Exosomes are essential mediators of cell-to-cell communication by delivering their cargo, such as proteins, RNAs and DNA from cell to cell. They participate in cancer progression, metastasis, immune response and therapy resistance. Their ability to shuttle between cells make them to efficient drug delivery systems. As drug transporters, they provide novel strategies for cancer therapy by advancing targeted drug therapy and improving the therapeutic effects of anti-cancer medications. With this review, a comprehensive overview on the potential of exosomes as therapeutics agents and targeted molecules in the treatment of cancer patients is given. The current challenges of preparation of loading exosomes with drugs and delivering them to the recipient tumor cells as well as a consequent exosome-mediated cancer therapy are also discussed.

Engineered Exosome for Drug Delivery: Recent Development and Clinical Applications.

Exosomes are nano-sized membrane vesicles that transfer bioactive molecules between cells and modulate various biological processes under physiological and pathological conditions. By applying bioengineering technologies, exosomes can be modified to express specific markers or carry therapeutic cargo and emerge as novel platforms for the treatment of cancer, neurological, cardiovascular, immune, and infectious diseases. However, there are many challenges and uncertainties in the clinical translation of exosomes. This review aims to provide an overview of the recent advances and challenges in the translation of engineered exosomes, with a special focus on the methods and strategies for loading drugs into exosomes, the pros and cons of different loading methods, and the optimization of exosome production based on the drugs to be encapsulated. Moreover, we also summarize the current clinical applications and prospects of engineered exosomes, as well as the potential risks and limitations that need to be addressed in exosome engineering, including the standardization of exosome preparation and engineering protocols, the quality and quantity of exosomes, the control of drug release, and the immunogenicity and cytotoxicity of exosomes. Overall, engineered exosomes represent an exciting frontier in nanomedicine, but they still face challenges in large-scale production, the maintenance of storage stability, and clinical translation. With continuous advances in this field, exosome-based drug formulation could offer great promise for the targeted treatment of human diseases.

Optimizing Exosome Preparation Based on Size and Morphology: Insights From Electron Microscopy.

Extracellular vesicles (EVs), including exosomes, are crucial in intercellular communication, but differentiating between exosomes and microvesicles is challenging due to their similar morphology and size. This study focuses on multivesicular bodies (MVBs), where exosomes mature, and optimizes exosome isolation using transmission electron microscopy (TEM) for size information. Considering that EVs are nanocolloidal particles, a salt-free Bis-Tris buffer is found to maintain EV integrity better than phosphate-buffered saline (PBS). Dynamic light scattering (DLS) and TEM analysis confirm that intact exosome fractions under the salt-free Bis-Tris buffer condition exhibit polydispersity, including a unique population of <50 nm vesicles resembling intraluminal membrane vesicles (ILVs) in MVBs, alongside larger populations. This <50 nm population disappears in PBS or Bis-Tris buffer with 140 mM NaCl, transforming into a monodisperse population >100 nm. Immunoelectron microscopy also validates the presence of CD63, an exosome biomarker, on approximately 50 nm EVs. These findings provide valuable insights into exosome characterization and isolation, essential for future biomedical applications in diagnostics and drug delivery.

Preconditioning of exosomes derived from human olfactory ensheathing cells improved motor coordination and balance in an SCA3/MJD mouse model: A new therapeutic approach

Exosome therapy is a novel trend in regeneration medicine. However, identifying a suitable biomarker that can associate the therapeutic efficacy of exosomes with SCA3/MJD is essential. In this study, parental cells were preconditioned with butylidenephthalide (Bdph) for exosome preparation to evaluate the therapeutic effect of SCA3/MJD. The therapeutic agent hsa-miRNA-6780–5p was enriched up to 98-fold in exosomes derived from butylidenephthalide (Bdph)-preconditioned human olfactory ensheathing cells (hOECs) compared with that in naïve hOECs exosomes. The particle sizes of exosomes derived from naïve hOECs and those derived from hOECs preconditioned with Bdph were approximately 113.0 ± 3.5 nm and 128.9 ± 0.7 nm, respectively. A liposome system was used to demonstrate the role of hsa-miRNA-6780–5p, wherein hsa-miRNA-6780–5p was found to enhance autophagy and inhibit the expression of spinocerebellar ataxia type 3 (SCA3) disease proteins with the polyglutamine (polyQ) tract. Exosomes with enriched hsa-miRNA-6780–5p were further applied to HEK-293–84Q cells, leading to decreased expression of polyQ and increased autophagy. The results were reversed when 3MA, an autophagy inhibitor, was added to the cells treated with hsa-miRNA-6780–5p-enriched exosomes, indicating that the decreased polyQ expression was modulated via autophagy. SCA3 mice showed improved motor coordination behavior when they intracranially received exosomes enriched with hsa-miRNA-6780–5p. SCA3 mouse cerebellar tissues treated with hsa-miRNA-6780–5p-enriched exosomes showed decreased expression of polyQ and increased expression of LC3II/I, an autophagy marker. In conclusion, our findings can serve as a basis for developing an alternative therapeutic strategy for SCA3 disease treatment using miRNA-enriched exosomes derived from chemically preconditioned cells.

Preparation of Phillygenin-Hyaluronic acid composite milk-derived exosomes and its anti-hepatic fibrosis effect

Liver fibrosis remains a serious problem affecting the health of millions of people worldwide. Hepatic stellate cells (HSCs) are the main effector cells in liver fibrosis and their activation could lead to extracellular matrix deposition, which may aggravate the development of liver fibrosis and inflammation. Previous studies have reported the potential of Phillygenin (PHI) as a hepatoprotective agent to inhibit HSCs activation and fibrosis development. However, the poor water solubility of PHI hinders its clinical application as a potential anti-liver fibrosis therapy. Milk-derived exosomes (mEXO) serve as scalable nanocarriers for delivering chemotherapeutic agents due to their excellent biocompatibility. Here, we developed a PHI-Hyaluronic acid (HA) composite mEXO (PHI-HA-mEXO) drug delivery system, in which DSPE-PEG2000-HA was conjugated to the surface of mEXO to prepare HA-mEXO, and PHI was encapsulated into HA-mEXO to form PHI-HA-mEXO. As a specific receptor for HA, CD44 is frequently over-expressed during liver fibrosis and highly expressed on the surface of activated HSCs (aHSCs). PHI-HA-mEXO can bind to CD44 and enter aHSCs through endocytosis and release PHI. PHI-HA-mEXO drug delivery system can significantly induce aHSCs death without affecting quiescent HSCs (qHSCs) and hepatocytes. Furthermore, we carried out in vitro and in vivo experiments and found that PHI-HA-mEXO could alleviate liver fibrosis through aHSCs-targeted mechanism. In conclusion, the favorable biosafety and superior anti-hepatic fibrosis effects suggest a promising potential of PHI-HA-mEXO in the treatment of hepatic fibrosis. However, detailed pharmokinetics and dose-responsive experiments of PHI-HA-mEXO and the mechanism of mEXO loading drugs are still required before PHI-HA-mEXO can be applied clinically.

The Promise of Exosomes as Drug Delivery Systems

Exosomes are small extracellular vesicles that play a role in cell-to-cell communication by transferring bioactive molecules such as proteins, nucleic acids, and lipids between cells. Over the past few years, there have been significant advances in utilizing exosomes as drug delivery systems due to their unique properties, including their ability to target specific cells and tissues. Especially advances in targeted delivery, nanoparticle incorporation, personalized medicine, cargo delivery, imaging, and tracking have excited the pharmaceutical community about their potential use in these areas. I briefly discuss the significance of exosomes in these areas or research interests. Natural Cargo Delivery: Exosomes naturally transport a variety of biomolecules, making them attractive vehicles for delivering drugs, nucleic acids (such as siRNA and miRNA), proteins, and even small molecules. Researchers are harnessing this inherent cargo delivery capability to load exosomes with therapeutic agents. Engineered Exosomes: Scientists are engineering exosomes to enhance their drug delivery capabilities. This includes modifying the surface of exosomes to display targeting ligands that can direct them to specific cell types or tissues, improving their stability, and optimizing their cargo-loading efficiency. Targeted Delivery: One of the key advantages of using exosomes for drug delivery is their potential for targeted delivery. By engineering exosomes to carry targeting molecules on their surface, researchers can selectively deliver therapeutic agents to specific cells or disease sites, minimizing off-target effects. Personalized Medicine: Exosomes can be isolated from a patient's cells (autologous exosomes), loaded with personalized therapies, and then re-administered to the patient. This personalized approach holds promise for tailoring treatments to individual patients' needs. Imaging and Tracking: Exosomes can be labeled with imaging agents to track their distribution and uptake in vivo. This information is crucial for understanding the pharmacokinetics and biodistribution of exosome-based drug delivery systems. Nanoparticle Incorporation: Exosomes can be loaded with nanoparticles, such as liposomes or polymer-based carriers, to increase their cargo capacity and control drug release kinetics. This combination allows for synergistic benefits of both exosome-mediated and nanoparticle-based drug delivery. Clinical Translation: Clinical trials involving exosome-based therapies are underway for various diseases, including cancer and neurodegenerative disorders. These trials provide valuable insights into the safety, efficacy, and challenges associated with exosome-based drug delivery in humans. Regulatory Considerations: The field of exosome-based drug delivery is also evolving in terms of regulatory considerations. Regulatory agencies are working to establish guidelines and standards for developing and approving exosome-based therapies. Exosome Isolation and Purification Techniques: Advances in exosome isolation and purification methods are critical for obtaining high-quality and consistent exosome preparations for drug delivery studies. Improved techniques contribute to the reproducibility and reliability of exosome-based therapies. Disease-Specific Cargo: Exosomes derived from specific cell types or disease models can be isolated and used as delivery vehicles for disease-specific cargo, including diagnostic biomarkers and therapeutic agents. This enables precise delivery to disease-affected cells. Combination Therapies: Researchers are exploring the potential of using exosomes to deliver combination therapies, where multiple therapeutic agents are loaded into a single exosome to achieve synergistic effects. The study of exosomes as drug delivery systems is rapidly advancing and holds great promise for improving the targeted delivery of therapeutic agents, reducing side effects, and enabling personalized medicine approaches. However, scalability, cargo loading efficiency, safety, and regulatory approval challenges still need to be addressed as the field progresses.

Mesenchymal Stromal Cell Exosomes Mediate M2-like Macrophage Polarization through CD73/Ecto-5'-Nucleotidase Activity.

Mesenchymal stem/stromal cell (MSC) exosomes have been shown to alleviate immune dysfunction and inflammation in preclinical animal models. This therapeutic effect is attributed, in part, to their ability to promote the polarization of anti-inflammatory M2-like macrophages. One polarization mechanism has been shown to involve the activation of the MyD88-mediated toll-like receptor (TLR) signaling pathway by the presence of extra domain A-fibronectin (EDA-FN) within the MSC exosomes. Here, we uncovered an additional mechanism where MSC exosomes mediate M2-like macrophage polarization through exosomal CD73 activity. Specifically, we observed that polarization of M2-like macrophages by MSC exosomes was abolished in the presence of inhibitors of CD73 activity, adenosine receptors A2A and A2B, and AKT/ERK phosphorylation. These findings suggest that MSC exosomes promote M2-like macrophage polarization by catalyzing the production of adenosine, which then binds to adenosine receptors A2A and A2B to activate AKT/ERK-dependent signaling pathways. Thus, CD73 represents an additional critical attribute of MSC exosomes in mediating M2-like macrophage polarization. These findings have implications for predicting the immunomodulatory potency of MSC exosome preparations.

A roadmap from research to clinical testing of mesenchymal stromal cell exosomes in the treatment of psoriasis

The most clinically trialed cells, mesenchymal stromal cells (MSCs), are now known to mainly exert their therapeutic activity through paracrine secretions, which include exosomes. To mitigate potential regulatory concerns on the scalability and reproducibility in the preparations of MSC exosomes, MSC exosomes were produced using a highly characterized MYC-immortalized monoclonal cell line. These cells do not form tumors in athymic nude mice or exhibit anchorage-independent growth, and their exosomes do not carry MYC protein or promote tumor growth. Unlike intra-peritoneal injections, topical applications of MSC exosomes in a mouse model of IMQ-induced psoriasis alleviate interleukin (IL)-17, IL-23 and terminal complement complex, C5b9 in psoriatic skin. When applied on human skin explants, fluorescence from covalently labeled fluorescent MSC exosomes permeated and persisted in the stratum corneum for about 24 hours with negligible exit out of the stratum corneum into the underlying epidermis. As psoriatic stratum corneums are uniquely characterized by activated complements and Munro microabscesses, we postulated that topically applied exosomes permeate the psoriatic stratum corneum to inhibit C5b9 complement complex through CD59, and this inhibition attenuated neutrophil secretion of IL-17. Consistent with this, we demonstrated that assembly of C5b9 on purified human neutrophils induced IL-17 secretion and this induction was abrogated by MSC exosomes, which was in turn abrogated by a neutralizing anti-CD 59 antibody. We thus established the mechanism of action for the alleviation of psoriatic IL-17 by topically applied exosomes.

Mesenchymal Stem Cell Exosomes as Immunomodulatory Therapy for Corneal Scarring.

Corneal scarring is a leading cause of worldwide blindness. Human mesenchymal stem cells (MSC) have been reported to promote corneal wound healing through secreted exosomes. This study investigated the wound healing and immunomodulatory effects of MSC-derived exosomes (MSC-exo) in corneal injury through an established rat model of corneal scarring. After induction of corneal scarring by irregular phototherapeutic keratectomy (irrPTK), MSC exosome preparations (MSC-exo) or PBS vehicle as controls were applied to the injured rat corneas for five days. The animals were assessed for corneal clarity using a validated slit-lamp haze grading score. Stromal haze intensity was quantified using in-vivo confocal microscopy imaging. Corneal vascularization, fibrosis, variations in macrophage phenotypes, and inflammatory cytokines were evaluated using immunohistochemistry techniques and enzyme-linked immunosorbent assays (ELISA) of the excised corneas. Compared to the PBS control group, MSC-exo treatment group had faster epithelial wound closure (0.041), lower corneal haze score (p = 0.002), and reduced haze intensity (p = 0.004) throughout the follow-up period. Attenuation of corneal vascularisation based on CD31 and LYVE-1 staining and reduced fibrosis as measured by fibronectin and collagen 3A1 staining was also observed in the MSC-exo group. MSC-exo treated corneas also displayed a regenerative immune phenotype characterized by a higher infiltration of CD163+, CD206+ M2 macrophages over CD80+, CD86+ M1 macrophages (p = 0.023), reduced levels of pro-inflammatory IL-1β, IL-8, and TNF-α, and increased levels of anti-inflammatory IL-10. In conclusion, topical MSC-exo could alleviate corneal insults by promoting wound closure and reducing scar development, possibly through anti-angiogenesis and immunomodulation towards a regenerative and anti-inflammatory phenotype.

Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

Exosomes are secreted nanovesicles with potent signalling activity that are initially formed as intraluminal vesicles (ILVs) in late Rab7-positive multivesicular endosomes, and also in recycling Rab11a-positive endosomes, particularly under some forms of nutrient stress. The core proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) participate in exosome biogenesis and ILV-mediated destruction of ubiquitinylated cargos. Accessory ESCRT-III components have reported roles in ESCRT-III-mediated vesicle scission, but their precise functions are poorly defined. They frequently only appear essential under stress. Comparative proteomics analysis of human small extracellular vesicles revealed that accessory ESCRT-III proteins, CHMP1A, CHMP1B, CHMP5 and IST1, are increased in Rab11a-enriched exosome preparations. We show that these proteins are required to form ILVs in Drosophila secondary cell recycling endosomes, but unlike core ESCRTs, they are not involved in degradation of ubiquitinylated proteins in late endosomes. Furthermore, CHMP5 knockdown in human HCT116 colorectal cancer cells selectively inhibits Rab11a-exosome production. Accessory ESCRT-III knockdown suppresses seminal fluid-mediated reproductive signalling by secondary cells and the growth-promoting activity of Rab11a-exosome-containing EVs from HCT116 cells. We conclude that accessory ESCRT-III components have a specific, ubiquitin-independent role in Rab11a-exosome generation, a mechanism that might be targeted to selectively block pro-tumorigenic activities of these vesicles in cancer.

PERIPHERAL BLOOD EXTRACELLULAR VESICLE LIPIDS AS BIOMARKERS FOR HUMAN INFLAMMATORY BOWEL DISEASE

Abstract BACKGROUND The current paradigm in inflammatory bowel disease (IBD) diagnostics is based on peripheral biomarkers such as C-reactive protein and fecal calprotectin that achieve low sensitivity and specificity for intestinal inflammation. Extracellular vesicles (EVs) are lipid-enveloped particles involved in inter tissue and cell-cell interactions. They also have unique properties reflecting the metabolic and phenotypic nature of the producer cells. Recent data revealed that surface proteins on intestinal epithelial cells-derived EVs can be detected in the peripheral blood. We posit that lipid profiling of circulating EVs (PBEs) can be used to discriminate active IBD from healthy subjects and further classify different stages of IBD (PCT Patent Pending: 13177N/2194P5). METHODS Patients diagnosed with Ulcerative Colitis (UC, n=50) or normal controls (n=50) were recruited at the UK IBD clinic or colonoscopy suite. During their colonoscopy or regular outpatient labs, 2 tubes of blood (20-30 mL) were drawn into K2-EDTA tubes. The blood samples were immediately stored on ice and then centrifuged at 1,500 g for 15 minutes at 4 °C within 30 minutes. The clarified plasma was immediately aliquoted and frozen in liquid N2. PBEs were isolated from plasma using size-exclusion microcolumns. Isolated exosomal preparations were lysed in cold acetonitrile, followed by extraction using a modified Folch method. The lipid fraction was carefully aspirated and dried in a Vacufuge before reconstitution in a chloroform/methanol mixture containing butylated hydroxytoluene. The extracted lipids from active UC and healthy control patient plasma were analyzed using direct infusion ultrahigh resolution Orbitrap mass spectrometry. We used statistical tools LASSO and Random Forest to select informative lipid features and build classification models. The performance of the classifiers was quantified by the receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) based on 10-fold cross-validation. RESULTS Three PBE lipids identified by LASSO were also identified using the Random Forest method along with 7 additional lipids. Discriminating lipid classifiers between active UC and normal patients identified by both LASSO and Random Forest included phosphatidylcholines, plasmalogens, and sphingolipids. An AUC of 0.86 discriminated active UC from normal patients using the Random Forest method and 0.80 using the LASSO method. CONCLUSION These results are the first-ever depiction of harnessing the diagnostic utility of PBEs through lipid profiling. Differences in PBE lipid composition accurately discriminated active UC patients from normal patients paving the way for a diagnostic liquid biopsy for patients with IBD.

Research progress of exosomes in pathogenesis, diagnosis, and treatment of ocular diseases.

Exosomes are natural extracellular vesicles with a diameter of 30-150nm, which exist in biological fluids and contain biomolecules related to the parent cell, such as proteins, nucleic acids, lipids, etc. It has a wide range of biological functions, and participates in the regulation of important physiological and pathological activities of the body. It can be used as a biomarker for early diagnosis of ocular diseases, a potential therapeutic target, a targeted drug carrier, and has a high potential for clinical application. In this paper, we summarized the genesis mechanism, biological functions, research and application progress of exosomes, focused on the engineering strategy of exosomes, and summarized the advantages and disadvantages of common engineering exosome preparation methods. Systematically combed the role of exosomes in corneal diseases, glaucoma, and retinal diseases, to provide a reference for further understanding of the role of exosomes in the pathogenesis, diagnosis, and treatment of ocular diseases. Finally, we further summarized the opportunities and challenges of exosomes for precision medicine. The extension of exosome research to the field of ophthalmology will help advance current diagnostic and therapeutic methods. Tiny exosomes have huge potential.

One-Step Pharmaceutical Preparation of PEG-Modified Exosomes Encapsulating Anti-Cancer Drugs by a High-Pressure Homogenization Technique.

The use of exosomes encapsulating therapeutic agents for the treatment of diseases is of increasing interest. However, some concerns such as limited efficiency and scalability of conventional drug encapsulation methods to exosomes have still remained; thus, a new approach that enables encapsulation of therapeutic agents with superior efficiency and scalability is required. Herein, we used RAW264 macrophage cell-derived exosomes (RAW-Exos) and demonstrated that high-pressure homogenization (HPH) using a microfluidizer decreased their particle size without changing their morphology, the amount of exosomal marker proteins, and cellular uptake efficiency into RAW264 and colon-26 cancer cells. Moreover, HPH allowed for modification of polyethylene glycol (PEG)-conjugated lipids onto RAW-Exos, as well as encapsulation of the anti-cancer agent doxorubicin. Importantly, the doxorubicin encapsulation efficiency became higher upon increasing the process pressure and simultaneous HPH with PEG-lipids. Moreover, treatment with PEG-modified RAW-Exos encapsulating doxorubicin significantly suppressed tumor growth in colon-26-bearing mice. Taken together, these results suggest that HPH using a microfluidizer could be useful to prepare PEG-modified Exos encapsulating anti-cancer drugs via a one-step pharmaceutical process, and that the prepared functional Exos could be applied for the treatment of cancer in vivo.

Large-scale Isolation of Exosomes Derived from NK Cells for Anti-tumor Therapy.

Exosomes are lipid bilayer-enclosed vesicles, actively secreted by cells, containing proteins, lipids, nucleic acids, and other substances with multiple biological functions after entering target cells. Exosomes derived from NK cells have been shown to have certain anti-tumor effects and potential applications as chemotherapy drug carriers. These developments have resulted in high demand for exosomes. Although there has been large-scale industrial preparation of exosomes, they are only for generally engineered cells such as HEK 293T. The large-scale preparation of specific cellular exosomes is still a major problem in laboratory studies. Therefore, in this study, we used tangential flow filtration (TFF) to concentrate the culture supernatants isolated from NK cells and isolated NK cell-derived exosomes (NK-Exo) by ultracentrifugation. Through a series of characterization and functional verification of NK-Exo, the characterization, phenotype, and anti-tumor activity of NK-Exo were verified. Our study provides a considerably time- and labor-saving protocol for the isolation of NK-Exo.