Bilateral efforts to improve SERS detection efficiency of exosomes by Au/Na7PMo11O39 Combined with Phospholipid Epitope Imprinting.

Detection of cancer-related exosomes in body fluids has become a revolutionary strategy for early cancer diagnosis and prognosis prediction. We have developed a two-step targeting detection method, termed PS-MIPs-NELISA SERS, for rapid and highly sensitive exosomes detection. In the first step, a phospholipid polar site imprinting strategy was employed using magnetic PS-MIPs (phospholipids-molecularly imprinted polymers) to selectively isolate and enrich all exosomes from urine samples. In the second step, a nanozyme-linked immunosorbent assay (NELISA) technique was utilized. We constructed Au/Na7PMo11O39 nanoparticles (NPs) with both surface-enhanced Raman scattering (SERS) property and peroxidase catalytic activity, followed by the immobilization of CD9 antibodies on the surface of Au/Na7PMo11O39 NPs. The Au/Na7PMo11O39-CD9 antibody complexes were then used to recognize CD9 proteins on the surface of exosomes enriched by magnetic PS-MIPs. Lastly, the high sensitivity detection of exosomes was achieved indirectly via the SERS activity and peroxidase-like activity of Au/Na7PMo11O39 NPs. The quantity of exosomes in urine samples from pancreatic cancer patients obtained by the PS-MIPs-NELISA SERS technique showed a linear relationship with the SERS intensity in the range of 6.21 × 107-2.81 × 108 particles/mL, with a limit of detection (LOD) of 5.82 × 107 particles/mL. The SERS signal intensity of exosomes in urine samples from pancreatic cancer patients was higher than that of healthy volunteers. This bidirectional MIPs-NELISA-SERS approach enables noninvasive, highly sensitive, and rapid detection of cancer, facilitating the monitoring of disease progression during treatment and opening up a new avenue for rapid early cancer screening.

Deep learning-assisted monitoring of trastuzumab efficacy in HER2-Overexpressing breast cancer via SERS immunoassays of tumor-derived urinary exosomal biomarkers.

Monitoring drug efficacy is significant in the current concept of companion diagnostics in metastatic breast cancer. Trastuzumab, a drug targeting human epidermal growth factor receptor 2 (HER2), is an effective treatment for metastatic breast cancer. However, some patients develop resistance to this therapy; therefore, monitoring its efficacy is essential. Here, we describe a deep learning-assisted monitoring of trastuzumab efficacy based on a surface-enhanced Raman spectroscopy (SERS) immunoassay against HER2-overexpressing mouse urinary exosomes. Individual Raman reporters bearing the desired SERS tag and exosome capture substrate were prepared for the SERS immunoassay; SERS tag signals were collected to prepare deep learning training data. Using this deep learning algorithm, various complicated mixtures of SERS tags were successfully quantified and classified. Exosomal antigen levels of five types of cell-derived exosomes were determined using SERS-deep learning analysis and compared with those obtained via quantitative reverse transcription polymerase chain reaction and western blot analysis. Finally, drug efficacy was monitored via SERS-deep learning analysis using urinary exosomes from trastuzumab-treated mice. Use of this monitoring system should allow proactive responses to any treatment-resistant issues.

In Situ Sprayed Exosome-Cross-Linked Gel as Artificial Lymph Nodes for Postoperative Glioblastoma Immunotherapy.

One key challenge in postoperative glioblastoma immunotherapy is to guarantee a potent and durable T-cell response, which is restricted by the immunosuppressive microenvironment within the lymph nodes (LNs). Here, we develop an in situ sprayed exosome-cross-linked gel that acts as an artificial LN structure to directly activate the tumor-infiltrating T cells for prevention of glioma recurrence. Briefly, this gel is generated by a bio-orthogonal reaction between azide-modified chimeric exosomes and alkyne-modified alginate polymers. Particularly, these chimeric exosomes are generated from dendritic cell (DC)-tumor hybrid cells, allowing for direct and robust T-cell activation. The gel structure with chimeric exosomes as cross-linking points avoids the quick clearance by the immune system and thus prolongs the durability of antitumor T-cell immunity. Importantly, this exosome-containing immunotherapeutic gel provides chances for ameliorating functions of antigen-presenting cells (APCs) through accommodating different intracellular-acting adjuvants, such as stimulator of interferon genes (STING) agonists. This further enhances the antitumor T-cell response, resulting in the almost complete elimination of residual lesions after surgery. Our findings provide a promising strategy for postsurgical glioma immunotherapy that warrants further exploration in the clinical arena.

Advancing Tissue Damage Repair in Geriatric Diseases: Prospects of Combining Stem Cell-Derived Exosomes with Hydrogels.

Geriatric diseases are a group of diseases with unique characteristics related to senility. With the rising trend of global aging, senile diseases now mainly include endocrine, cardiovascular, neurodegenerative, skeletal, and muscular diseases and cancer. Compared with younger populations, the structure and function of various cells, tissues and organs in the body of the elderly undergo a decline as they age, rendering them more susceptible to external factors and diseases, leading to serious tissue damage. Tissue damage presents a significant obstacle to the overall health and well-being of older adults, exerting a profound impact on their quality of life. Moreover, this phenomenon places an immense burden on families, society, and the healthcare system.In recent years, stem cell-derived exosomes have become a hot topic in tissue repair research. The combination of these exosomes with biomaterials allows for the preservation of their biological activity, leading to a significant improvement in their therapeutic efficacy. Among the numerous biomaterial options available, hydrogels stand out as promising candidates for loading exosomes, owing to their exceptional properties. Due to the lack of a comprehensive review on the subject matter, this review comprehensively summarizes the application and progress of combining stem cell-derived exosomes and hydrogels in promoting tissue damage repair in geriatric diseases. In addition, the challenges encountered in the field and potential prospects are presented for future advancements.

Visible-Light Cross-Linkable Multifunctional Hydrogels Loaded with Exosomes Facilitate Full-Thickness Skin Defect Wound Healing through Participating in the Entire Healing Process.

The management of severe full-thickness skin defect wounds remains a challenge due to their irregular shape, uncontrollable bleeding, high risk of infection, and prolonged healing period. Herein, an all-in-one OD/GM/QCS@Exo hydrogel was prepared with catechol-modified oxidized hyaluronic acid (OD), methylacrylylated gelatin (GM), and quaternized chitosan (QCS) and loaded with adipose mesenchymal stem cell-derived exosomes (Exos). Cross-linking of the hydrogel was achieved using visible light instead of ultraviolet light irradiation, providing injectability and good biocompatibility. Notably, the incorporation of catechol groups and multicross-linked networks in the hydrogels conferred strong adhesion properties and mechanical strength against external forces such as tensile and compressive stress. Furthermore, our hydrogel exhibited antibacterial, anti-inflammatory, and antioxidant properties along with wound-healing promotion effects. Our results demonstrated that the hydrogel-mediated release of Exos significantly promotes cellular proliferation, migration, and angiogenesis, thereby accelerating skin structure reconstruction and functional recovery during the wound-healing process. Overall, the all-in-one OD/GM/QCS@Exo hydrogel provided a promising therapeutic strategy for the treatment of full-thickness skin defect wounds through actively participating in the entire process of wound healing.

Recent advances in the applications of DNA frameworks in liquid biopsy: A review.

Cancer is one of the serious threats to public life and health. Early diagnosis, real-time monitoring, and individualized treatment are the keys to improve the survival rate and prolong the survival time of cancer patients. Liquid biopsy is a potential technique for cancer early diagnosis due to its non-invasive and continuous monitoring properties. However, most current liquid biopsy techniques lack the ability to detect cancers at the early stage. Therefore, effective detection of a variety of cancers is expected through the combination of various techniques. Recently, DNA frameworks with tailorable functionality and precise addressability have attracted wide spread attention in biomedical applications, especially in detecting cancer biomarkers such as circulating tumor cells (CTCs), exosomes and circulating tumor nucleic acid (ctNA). Encouragingly, DNA frameworks perform outstanding in detecting these cancer markers, but also face some challenges and opportunities. In this review, we first briefly introduced the development of DNA frameworks and its typical structural characteristics and advantages. Then, we mainly focus on the recent progress of DNA frameworks in detecting commonly used cancer markers in liquid-biopsy. We summarize the advantages and applications of DNA frameworks for detecting CTCs, exosomes and ctNA. Furthermore, we provide an outlook on the possible opportunities and challenges for exploiting the structural advantages of DNA frameworks in the field of cancer diagnosis. Finally, we envision the marriage of DNA frameworks with other emerging materials and technologies to develop the next generation of disease diagnostic biosensors.

Developing Plant Exosomes as an Advanced Delivery System for Cosmetic Peptide.

Peptides are a promising skincare ingredient, but due to their inherent instability and the barrier function of the skin's surface, they often have limited skin absorption and penetration, which can significantly hinder their skincare benefits. To address this, a novel technique called NanoGlow has been introduced for encapsulating peptide-based cosmetic raw materials into engineered nanosized plant-derived exosomes (pExo) to achieve the goal of a healthier and more radiant skin state. In this approach, pExo served as carriers for cosmetic peptides across the intact skin barrier, enhancing their biological effectiveness in skin beauty. The NanoGlow strategy combines chemical activation and physical proencapsulation, boasting a high success rate and straightforward and stable operation, making it suitable for large-scale production. Comprehensive analysis using in vitro cellular absorption and skin penetration models has demonstrated that the nanosized pExo carriers significantly improve peptide penetration into the skin compared to free peptides. Furthermore, in vivo tissue slice studies have shown that pExo carriers efficiently deliver acetyl hexapeptide-8 to the skin's dermis, surpassing the performance of free peptides. Cosmetic skincare effect analysis has also indicated that pExo-loaded cosmetic peptides deliver superior results. Therefore, the NanoGlow technique harnesses the natural size and properties of pExo to maximize the bioavailability of cosmetic peptides, holding great promise for developing advanced peptide delivery systems in both the cosmetic and medical drug industries.

[Exosomes' role in intercellular interactions in different variants of lung injury in fatal cases of COVID-19]. / Rol' ekzosom v mezhkletochnykh vzaimodeistviyakh pri razlichnykh variantakh porazheniya legkikh v letal'nykh sluchayakh COVID-19.

BACKGROUND: Extracellular vesicles are surrounded by a phospholipid bilayer, carrying various active biomolecules and participating in many physiological and pathological processes, including infectious ones. OBJECTIVE: To research the role of exosomes in intercellular interactions in the pathogenesis of various types of lung damage in fatal cases of COVID-19. MATERIAL AND METHODS: We conducted a clinical and morphological analysis of 118 fatal cases caused by coronavirus infection in Moscow. We selected 32 cases with morphological signs of various types of lung lesions for immunohistochemical reaction (IHC) with antibodies against tetraspanin proteins (CD63, CD81), which are involved in the assembly of exosomes, as well as with antibodies against viral proteins: nucleocapsid and spike protein. We determined the main producing cells of extracellular vesicles and cells containing viral proteins, carried out their comparison and quantitative analysis. RESULTS: IHC reaction with antibodies against CD63 showed cytoplasmic granular uniform and subapical staining of cells, as well as granular extracellular staining. We determined similar staining using antibodies against viral proteins. Extracellular vesicles were found in the same cells as viral proteins. The main producing cells of vesicles and cells containing viral proteins were found to be macrophages, type II pneumocytes, and endothelial cells. CONCLUSION: Taking into account the results of the literature, the localization of viral proteins and extracellular vesicles in the same cells indicates the key role of vesicles in the pathogenesis of various forms of lung damage by the SARS-CoV-2 virus, in the dissemination of the pathogen in the organism, which leads to interaction with the adaptive immune system and the formation of immunity.

Oral exosome-like nanovesicles from Phellinus linteus suppress metastatic hepatocellular carcinoma by reactive oxygen species generation and microbiota rebalancing.

The biomedical application of nanotechnology in cancer treatment has demonstrated significant potential for improving treatment efficiencies and ameliorating adverse effects. However, the medical translation of nanotechnology-based nanomedicines faces challenges including hazardous environmental effects, difficulties in large-scale production, and possible excessive costs. In the present study, we extracted and purified natural exosome-like nanoparticles (ELNs) from Phellinus linteus. These nanoparticles (denoted as P-ELNs) had an average particle size of 154.1 nm, displayed a negative zeta potential of -31.3 mV, and maintained stability in the gastrointestinal tract. Furthermore, P-ELNs were found to contain a diverse array of functional components, including lipids and pharmacologically active small-molecule constituents. In vitro investigations suggested that they exhibited high internalization efficiency in liver tumor cells (Hepa 1-6) and exerted significant anti-proliferative, anti-migratory, and anti-invasive effects against Hepa 1-6 cells. Strikingly, the therapeutic outcomes of oral P-ELNs were confirmed in an animal model of metastatic hepatocellular carcinoma by amplifying reactive oxygen species (ROS) and rebalancing the gut microbiome. These findings demonstrate the potential of P-ELNs as a promising oral therapeutic platform for liver cancer treatment.

Target proteins-regulated DNA nanomachine-electroactive substance complexes enable separation-free electrochemical detection of clinical exosome.

Simple and reliable profiling of tumor-derived exosomes (TDEs) holds significant promise for the early detection of cancer. Nonetheless, this remains challenging owing to the substantial heterogeneity and low concentration of TDEs. Herein, we devised an accurate and highly sensitive electrochemical sensing strategy for TDEs via simultaneously targeting exosomal mucin 1 (MUC1) and programmed cell death ligand 1 (PD-L1). This approach employs high-affinity aptamers as specific recognition elements, utilizes rolling circle amplification and DNA nanospheres as effective bridges and signal amplifiers, and leverages methylene blue (MB) and doxorubicin (DOX) as robust signal reporters. The crux of this separation- and label-free method is the specific response of MB and DOX to G-quadruplex structures and DNA nanospheres, respectively. Quantifying TDEs using this strategy enabled precise discrimination of lung cancer patients (n = 25) from healthy donors (n = 12), showing 100% specificity (12/12), 92% sensitivity (23/25), and an overall accuracy of 94.6% (35/37), with an area under the receiver operating characteristic curve (AUC) of 0.97. Furthermore, the assay results strongly correlated with findings from computerized tomography and pathological analyses. Our approach could facilitate the early diagnosis of lung cancer through TDEs-based liquid biopsy.

The sphingolipids change in exosomes from cancer patients and association between exosome release and sphingolipids level based on a pseudotargeted lipidomics method.

The lipid based ESCRT-independent mechanism, which contributes to MVB formation, is one of the crucial procedures in exosome biogenesis. n-SMase is a key lipid metabolism enzyme in this mechanism and can induce the hydrolysis of sphingomyelins (SMs) to ceramides (Cers), thereby promoting the formation of ILVs inside MVBs. Therefore, the regulation of n-SMase can realize the alteration in exosome release. According to the fact that cancer-associated cells have a tendency to release more exosomes than healthy cells, lipid extracts in exosomes from healthy volunteers, HCC and ICC patients were analyzed by a novel pseudotargeted lipidomics method focused on sphingolipids (SLs) to explore whether cancer-related features regulate the release of exosomes through the above pathway. Multivariate analysis based on the SLs expression could distinguish three groups well indicated that the SLs expression among the three groups were different. In cancer groups, two species of critical Cers were up-regulated, denoted as Cer (d18:1_16:0) and Cer (d18:1_18:0), while 55 kinds of SLs were down-regulated, including 40 species of SMs, such as SM (d18:1_16:0), SM (d18:1_18:1) and SM (d18:1_24:0). Meanwhile, several species of SM/Cer exhibited significant down-regulation. This substantial enhancement of the SMs hydrolysis to Cers process during exosome biogenesis suggested that cancer-related features may potentially promote an increase in exosome release through ESCRT-independent mechanism. Moreover, differential SLs have a capability of becoming potential biomarkers for disease diagnosis and classification with an AUC value of 0.9884 or 0.9806 for the comparison between healthy group and HCC or ICC groups, respectively. In addition, an association analysis conducted on the cell lines showed that changes in the SM/Cer contents in cells and their exosomes were negatively correlated with the levels of released exosomes, implied the regulation of exosome release levels can be achieved by modulating n-SMase and subsequent SL expression.

Coaxial dual-path electrochemical biosensing and logic strategy-based detection of lung cancer-derived exosomal PD-L1.

Exosomal programmed death ligand-1 (ExoPD-L1) is a vital marker of immune activation in the early stages of tumor therapy and it can inhibit anti-tumor immune responses. However, due to the low expression of ExoPD-L1 in cancer cells, it is difficult to perform highly sensitive assays and accurately differentiate cancer sources. Therefore, we constructed a coaxial dual-path electrochemical biosensor for highly accurate identification and detection of ExoPD-L1 from lung cancer based on chemical-biological coaxial nanomaterials and nucleic acid molecular signal amplification strategies. The measurements showed that the detected ExoPD-L1 concentrations ranged from 6 × 102 particles per mL to 6 × 108 particles per mL, and the detection limit was 310 particles per mL. Compared to other sensors, the electrochemical biosensor designed in this study has a lower detection limit and a wider detection range. Furthermore, we also successfully identified lung cancer-derived ExoPD-L1 by analyzing multiple protein biomarkers expressed on exosomes through the "AND" logic strategy. This sensor platform is expected to realize highly sensitive detection and accurate analysis of multiple sources of ExoPD-L1 and provide ideas for the clinical detection of ExoPD-L1.

Engineered Exosomes with ATF5-Modified mRNA Loaded in Injectable Thermogels Alleviate Osteoarthritis by Targeting the Mitochondrial Unfolded Protein Response.

Osteoarthritis (OA) progression is highly associated with chondrocyte mitochondrial dysfunction and disorders of catabolism and anabolism of the extracellular matrix (ECM) in the articular cartilage. The mitochondrial unfolded protein response (UPRmt), which is an integral component of the mitochondrial quality control (MQC) system, is essential for maintaining chondrocyte homeostasis. We successfully validated the pivotal role of activating transcription factor 5 (ATF5) in upregulating the UPRmt, mitigating IL-1ß-induced inflammation and mitochondrial dysfunction, and promoting balanced metabolism in articular cartilage ECM, proving its potential as a promising therapeutic target for OA. Modified mRNAs (modRNAs) have emerged as novel and efficient gene delivery vectors for nucleic acid therapeutic approaches. In this study, we combined Atf5-modRNA (modAtf5) with engineered exosomes derived from bone mesenchymal stem cells (ExmodAtf5) to exert cytoprotective effects on chondrocytes in articular cartilage via Atf5. However, the rapid localized metabolization of ExmodAtf5 limits its application. PLGA-PEG-PLGA (Gel), an injectable thermosensitive hydrogel, was used as a carrier of ExmodAtf5 (Gel@ExmodAtf5) to achieve a sustained release of ExmodAtf5. In vitro and in vivo, the use of Gel@ExmodAtf5 was shown to be a highly effective strategy for OA treatment. The in vivo therapeutic effect of Gel@ExmodAtf5 was evidenced by the preservation of the intact cartilage surface, low OARSI scores, fewer osteophytes, and mild subchondral bone sclerosis and cystic degeneration. Consequently, the combination of ExmodAtf5 and PLGA-PEG-PLGA could significantly enhance the therapeutic efficacy and prolong the exosome release. In addition, the mitochondrial protease ClpP enhanced chondrocyte autophagy by modulating the mTOR/Ulk1 pathway. As a result of our research, Gel@ExmodAtf5 can be considered to be effective at alleviating the progression of OA.

Establishment of a novel microfluidic co-culture system for simultaneous analysis of multiple indicators of gefitinib sensitivity in colorectal cancer cells.

The therapeutic effect of gefitinib on colorectal cancer (CRC) is unclear, but it has been reported that stromal cells in the tumor microenvironment may have an impact on drug sensitivity. Herein, we established a microfluidic co-culture system and explored the sensitivity of CRC cells co-cultured with cancer-associated fibroblasts (CAFs) to gefitinib. The system consisted of a multichannel chip and a Petri dish. The chambers in the chip and dish were designed to continuously supply nutrients for long-term cell survival and create chemokine gradients for driving cell invasion without any external equipment. Using this system, the proliferation and invasiveness of cells were simultaneously evaluated by quantifying the area of cells and the migration distance of cells. In addition, the system combined with live cell workstation could evaluate the dynamic drug response of co-cultured cells and track individual cell trajectories in real-time. When CRC cells were co-cultured with CAFs, CAFs promoted CRC cell proliferation and invasion and reduced the sensitivity of cells to gefitinib through the exosomes secreted by CAFs. Furthermore, the cells that migrated out of the chip were collected, and EMT-related markers were determined by immunofluorescent and western blot assays. The results demonstrated that CAFs affected the response of CRC cells to gefitinib by inducing EMT, providing new ideas for further research on the resistance mechanism of gefitinib. This suggests that targeting CAFs or exosomes might be a new approach to enhance CRC sensitivity to gefitinib, and our system could be a novel platform for investigating the crosstalk between tumor cells and CAFs and understanding multiple biological changes of the tumor cells in the tumor microenvironment.

Selection of bifunctional RNAs with specificity for arginine and lipid membranes.

The molecular mechanisms of selective RNA loading into exosomes and other extracellular vesicles are not yet completely understood. In order to show that a pool of RNA sequences binds both the amino acid arginine and lipid membranes, we constructed a bifunctional RNA 10Arg aptamer specific for arginine and lipid vesicles. The preference of RNA 10Arg for lipid rafts was visualized and confirmed using FRET microscopy in neuroblastoma cells. The selection-amplification (SELEX) method using a doped (with the other three nucleotides) pool of RNA 10Arg sequences yielded several RNA 10Arg(D) sequences, and the affinities of these RNAs both to arginine and liposomes are improved in comparison to pre-doped RNA. Generation of these bispecific aptamers supports the hypothesis that an RNA molecule can bind both to RNA-binding proteins (RBPs) through arginine within the RBP-binding site and to membrane lipid rafts, thus facilitating RNA loading into exosomes and other extracellular vesicles.

Dual-Aptamer Recognition of DNA Logic Gate Sensor-Based Specific Exosomal Proteins for Ovarian Cancer Diagnosis.

Clinical diagnosis of ovarian cancer lacks high accuracy due to the weak selection of specific biomarkers along with the circumstance biomarkers localization. Clustering analysis of proteins transported on exosomes enables a more precise screening of effective biomarkers. Herein, through bioinformatics analysis of ovarian cancer and exosome proteomes, two coexpressed proteins, EpCAM and CD24, specifically enriched, were identified, together with the development of an as-derived dual-aptamer targeted exosome-based strategy for ovarian cancer screening. In brief, a DNA ternary polymer with aptamers targeting EpCAM and CD24 was designed to present a logic gate reaction upon recognizing ovarian cancer exosomes, triggering a rolling circle amplification chemiluminescent signal. A dynamic detection range of 6 orders of magnitude was achieved by quantifying exosomes. Moreover, for clinical samples, this strategy could accurately differentiate exosomes from healthy persons, other cancer patients, and ovarian cancer patients, enabling promising in situ detection. By accurately selecting biomarkers and constructing a dual-targeted exosomal protein detection strategy, the limitation of insufficient specificity of traditional protein markers was circumvented. This work contributed to the development of exosome-based prognosis monitoring in ovarian cancer through the identification of disease-specific exosome protein markers.

TSG6-Exo@CS/GP Attenuates Endometrium Fibrosis by Inhibiting Macrophage Activation in a Murine IUA Model.

Intrauterine adhesion (IUA) is characterized by the formation of fibrous scar tissue within the uterine cavity, which significantly impacts female reproductive health and even leads to infertility. Unfortunately, severe cases of IUA currently lack effective treatments. This study presents a novel approach that utilizes tumor necrosis factor-(TNF) stimulated gene 6 (TSG6)-modified exosomes (Exos) in conjunction with an injectable thermosensitive hydrogel (CS/GP) to mitigate the occurrence of IUA by reducing endometrium fibrosis in a mouse IUA model. This study demonstrate that TSG6-modified Exos effectively inhibits the activation of inflammatory M1-like macrophages during the initial stages of inflammation and maintains the balance of macrophage phenotypes (M1/M2) during the repair phase. Moreover, TSG6 inhibits the interaction between macrophages and endometrial stromal fibroblasts, thereby preventing the activation of stromal fibroblasts into myofibroblasts. Furthermore, this research indicates that CS/GP facilitates the sustained release of TSG6-modified Exos, leading to a significant reduction in both the manifestations of IUA and the extent of endometrium fibrosis. Collectively, through the successful construction of CS/GP loaded with TSG6-modified Exos, a reduction in the occurrence and progression of IUA is achieved by mitigating endometrium fibrosis. Consequently, this approach holds promise for the treatment of IUA.

A highly sensitive and selective fluorescent biosensor for breast cancer derived exosomes using click reaction of azide-CD63 aptamer and alkyne-polymer dots.

Exosomes have gained recognition as valuable reservoirs of biomarkers, holding immense potential for early cancer detection. Consequently, there is a pressing need for the development of an economical and highly sensitive exosome detection methodology. In this work, we present a fluorescence method for breast cancer-derived exosome detection based on Cu-triggered click reaction of azide-modified CD63 aptamer and alkyne functionalized Pdots. The detection threshold for the exosomes obtained from the breast cancer serum was determined to be 6.09 × 107 particles per µL, while the measurable range spanned from 6.50 × 107 to 1.30 × 109 particles per µL. The employed methodology achieved notable success in accurately distinguishing breast cancer patients from healthy individuals through serum analysis. The application of this method showcases the significant potential for early exosome analysis in the clinical diagnosis of breast cancer patients.

Comparison of extracellular vesicle isolation methods for the study of exosome cargo within Toxocara canis and Toxocara cati excretory secretory (TES) products.

Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host's immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.

The Landscape of Exosomes Biogenesis to Clinical Applications.

Exosomes are extracellular vesicles that originate from various cells and mediate intercellular communication, altering the behavior or fate of recipient cells. They carry diverse macromolecules, such as lipids, proteins, carbohydrates, and nucleic acids. Environmental stressors can change the exosomal contents of many cells, making them useful for diagnosing many chronic disorders, especially neurodegenerative, cardiovascular, cancerous, and diabetic diseases. Moreover, exosomes can be engineered as therapeutic agents to modulate disease processes. State-of-art techniques are employed to separate exosomes including ultracentrifugation, size-exclusion chromatography and immunoaffinity. However, modern technologies such as aqueous two-phase system as well as microfluidics are gaining attention in the recent years. The article highlighted the composition, biogenesis, and implications of exosomes, as well as the standard and novel methods for isolating them and applying them as biomarkers and therapeutic cargo carriers.