Exosome Production Research Articles

Therapeutic Potential of Extracellular Vesicles (Exosomes) Derived From Platelet-Rich Plasma: A Literature Review.

There are exciting advances in the field of exosomes research as a potential regenerative therapy. Platelet-rich plasma represents an abundant source of exosomes. To present evidence on the use of platelet-rich plasma-derived exosomes in dermatology and discuss current technical limitations. A literature search was conducted using Embase (Ovid), Web of Science, and ClinicalTrials.gov in June 2024. Platelet-rich plasma-derived exosomes contain a myriad of growth factors, genetic materials, and lipids that mediate paracrine signaling. Several preclinical studies have demonstrated their potential in wound healing. This includes faster wound contraction in diabetic animal models, as well as enhanced angiogenesis, increased cell proliferation and migration, and protection against hyperglycemic conditions invitro. A case study reported a topical platelet-derived exosome product tried successfully in a patient with persistent scalp wounds. In contrast, there is scarce information on the use of platelet-rich plasma-derived exosomes in hair growth and skin rejuvenation. Isolation techniques, activation methods, and methods of delivery have not been optimized, which warrants further research. Novel delivery methods, such as hydrogel-based preparations, may enable the topical application of exosomes. Exosomes derived from platelet-rich plasma have demonstrated therapeutic potential. More research is needed to standardize its use.

Magnetic Iron Oxide Nanoparticles Enhance Exosome Production by Upregulating Exosome Transport and Secretion Pathways.

Exosomes are cell-released nanovesicles that regulate intercellular communication by transporting a variety of bioactive molecules. They play a crucial role in various physiological and pathological processes, such as the immune response, tissue regeneration, aging, and tumor progression. There has been growing interest in controlling exosome production, which could offer valuable tools for unraveling complex cell communication networks and enabling novel therapeutic applications. Magnetic iron oxide nanoparticles (MNPs), one of the few nanomaterials approved for clinical use, have been shown to remotely modulate cellular activities such as cytoskeleton reorganization, ion channel activation, and cell polarization. In this study, we systematically investigate the effects of MNPs, acting as nanoscale force transducers, on exosome production in two distinct cell types with different responses to mechanical stimuli. Our findings reveal that magnetic force applied to intracellular MNPs induces vesicle relocation and promotes the formation of actin stress fibers. Gene expression analysis further shows that intracellular magnetic force upregulates genes related to exosome transport and secretion as well as other pathways linked to exosome biogenesis. Notably, these forces substantially enhance exosome production, particularly MNP-containing exosomes, which are accompanied by increased intercellular exchange of MNPs. In summary, our study offers valuable insights into MNP-driven exosome production and presents potential strategies for enhancing cell communication and modulating nanoparticle distribution in nanomedicine.

Spheroid-Exosome-Based Bioprinting Technology in Regenerative Medicine.

Since the discovery that exosomes can exchange genes, their potential use as tools for tissue regeneration, disease diagnosis, and therapeutic applications has drawn significant attention. Emerging three-dimensional (3D) printing technologies, such as bioprinting, which allows the printing of cells, proteins, DNA, and other biological materials, have demonstrated the potential to create complex body tissues or personalized 3D models. The use of 3D spheroids in bioprinting facilitates volumetric tissue reconstruction and accelerates tissue regeneration via exosome secretion. In this review, we discussed a convergence approach between two promising technologies for bioprinting and exosomes in regenerative medicine. Among the various 3D cell culture methods used for exosome production, we focused on spheroids, which are suitable for mass production by bioprinting. We then summarized the research results on cases of bioprinting applications using the spheroids and exosomes produced. If a large number of spheroids can be supplied through bioprinting, the spheroid-exosome-based bioprinting technology will provide new possibilities for application in tissue regeneration, disease diagnosis, and treatment.

STAT3 Inhibitor Increases α-Synuclein in PFF-Treated Astroglia Cells by Dysregulating Autophagy and Potentially Affects Exosome Biogenesis.

Astrocytes are the most abundant cells in the brain and show neuroprotective function in CNS and reactive astrocytes are characteristic in neurodegenerative diseases. The JAK2-STAT3 pathway plays a crucial role in the process of astrocyte activation. However, as a hallmark of Parkinson's disease, the change in α-syn under the influence of STAT3 inhibitor and the underlying cellular mechanisms remain elusive. Here, we show that PFF can induce an increase in α-syn in SVG p12 cells, which is further enhanced after the inhibition of STAT3. The underlying mechanisms involve the downregulation of autophagy levels and a concurrent decrease in lysosomal function after inhibition of STAT3. Additionally, we observed inhibition of STAT3 resulted in reduced exosome secretion in SVG p12 cells. This is attributed to alterations in SNARE, leading to impaired fusion between MVBs and the cell membrane, subsequently causing the accumulation of intracellular MVBs. Taken together, our data demonstrates that inhibition of STAT3 decreases both the autophagy and lysosome function, which may increase MVBs production. However, we found a potentially decreased exosome production that may be implicated with SNARE complex and need further exploration.

Dynamic reorganization of multivesicular bodies and exosome production impacted by sonoporation

Naturally occurring cell-derived extracellular vesicles (EVs) have emerged as attractive nanocarriers for drug delivery. However, production of large quantities of EVs for clinical applications in a scalable manner remains a significant challenge. This study investigated at the single cell level how sonoporation, or membrane poration produced by ultrasound-induced microbubble cavitation, impacts EV production using mouse macrophage RAW 264.7 cells stably expressing CD63-GFP as a model system. Real-time fluorescence videomicroscopy detected rapid changes in CD63-GFP, a tetraspanin family member highly enriched in intraluminal vesicles tagged with GFP, to track changes in multivesicular bodies (MVBs), which are the cellular compartments where exosomes originate within the cells. Our results revealed distinct dynamic changes in CD63-GFP intensity and distribution in RAW 264.7 cells in terms of response time and duration depending on whether the cells were directly or indirectly impacted by sonoporation, suggesting reorganization of MVBs in response to direct and indirect mechanisms resulted from the mechanical impact of ultrasound pulse on the cells. Analysis of the supernatant from sonoporation-treated RAW 264.7 cells expressing CD63-GFP demonstrated a delayed and sustained increase in the production of CD63-GFP-positive EVs. These results show the robust and detailed effect of sonoporation and reveal insights into sonoporation-induced EV release useful for guiding the application of sonoporation to enhance large-scale EV production.

The Immobilization of an FGF2-Derived Peptide on Culture Plates Improves the Production and Therapeutic Potential of Extracellular Vesicles from Wharton's Jelly Mesenchymal Stem Cells.

The skin is an essential organ that protects the body from external aggressions; therefore, damage from various wounds can significantly impair its function, and effective methods for regenerating and restoring its barrier function are crucial. This study aimed to mass-produce wound-healing exosomes using a fragment of the fibroblast growth factor 2 (FGF2)-derived peptide (FP2) to enhance cell proliferation and exosome production. Our experiments demonstrated increased cell proliferation when Wharton's jelly mesenchymal stem cells (WJ MSCs) were coated with FP2. Exosomes from FP2-coated WJ MSCs were analyzed using nanoparticle-tracking analysis, transmission electron microscopy, and Western blotting. Subsequently, fibroblasts were treated with these exosomes, and their viability and migration effects were compared. Anti-inflammatory effects were also evaluated by inducing pro-inflammatory factors in RAW264.7 cells. The treatment of fibroblasts with FP2-coated WJ MSC-derived exosomes (FP2-exo) increased the expression of FGF2, confirming their wound-healing effect in vivo. Overall, the results of this study highlight the significant impact of FP2 on the proliferation of WJ MSCs and the anti-inflammatory and wound-healing effects of exosomes, suggesting potential applications beyond wound healing.

Exosomes Derived from Rejuvenated Stem Cells Inactivate NLRP3 Inflammasome and Pyroptosis of Nucleus Pulposus Cells via the Transfer of Antioxidants.

Accumulating evidence supports the potential of exosomes as a promising therapeutic approach for intervertebral disc degeneration (IDD). Nevertheless, enhancing the efficiency of exosome treatment remains an urgent concern. This study investigated the impact of quercetin on the characteristics of mesenchymal stem cells (MSCs) and their released exosomes. Exosomes were obtained from quercetin pre-treated MSCs and quantified for the production based on nanoparticle tracking and western blot analysis. The molecules involved in the secretion and cargo sorting of exosomes were investigated using western blot and immunofluorescence analysis. Based on the in vitro biological analysis and in vivo histological analysis, the effects of exosomes derived from conventional or quercetin-treated MSCs on nucleus pulposus (NP) cells were compared. A significant enhancement in the production and transportation efficiency of exosomes was observed in quercetin-treated MSCs. Moreover, the exosomes derived from quercetin-treated MSCs exhibited a greater abundance of antioxidant proteins, specifically superoxide dismutase 1 (SOD1), which inhibit the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome in NP cells. Through in vitro and in vivo experiments, it was elucidated that exosomes derived from quercetin-treated MSCs possessed enhanced anti-inflammatory and antioxidant properties. Collectively, our research underscores an optimized therapeutic strategy for IDD utilizing MSC-derived exosomes, thereby augmenting the efficacy of exosomes in intervertebral disc regeneration.

Activating PKA signaling increases exosome production and attenuates cerebral ischemia-reperfusion injury by regulating Cx43 expression

BackgroundConnexin 43 (Cx43) plays a crucial role in mediating intracellular communication and facitating the interaction between exosomes and recipient cells. This study investigates whether the activation of cAMP/protein kinase A (PKA) can regulate exosomal Cx43 expression and contribute to the functional recovery following ischemia-reperfusion (I/R) injury. MethodsAn intraluminal vascular occlusion was performed on Lewis rats to simulate I/R injury. Concurrently, a PKA activator (8-Bromo-cAMP, 5 mg kg-1) or PKA inhibitor (H 89 2HCl, 20 mg kg-1) was administered intravenously via the tail vein (n = 10). Exosomes were isolated from cerebrospinal fluid, and the expression of exosomal markers (CD63 and CD81) and Cx43 was analyzed using Western blot. The expression of CD63 and CD81 in astrocytes was measured to assess exosome uptake. Spatial learning and memory capability were evaluated using the Morris water maze test. Results8-Bromo-cAMP significantly increased exosome release in cerebrospinal fluid, accompanied by elevated Cx43 expression. Additionally, 8-Bromo-cAMP enhanced exosome uptake by astrocytes, alleviated blood-brain barrier damage and edema, and improved cognitive function. ConclsionsPKA activation enhances exosome production, promotes cognitive function recovery, and attenuates cerebral I/R injury by up-regulating exosomal Cx43 expression.

3D-Cultured MC3T3-E1-Derived Exosomes Promote Endothelial Cell Biological Function under the Effect of LIPUS.

Porous Ti-6Al-4V scaffold materials can be used to heal massive bone defects because they can provide space for vascularisation and bone formation. During new bone tissue development, rapid vascular ingrowth into scaffold materials is very important. Osteoblast-derived exosomes are capable of facilitating angiogenesis-osteogenesis coupling. Low-intensity pulsed ultrasound (LIPUS) is a physical therapy modality widely utilised in the field of bone regeneration and has been proven to enhance the production and functionality of exosomes on two-dimensional surfaces. The impact of LIPUS on exosomes derived from osteoblasts cultured in three dimensions remains to be elucidated. In this study, exosomes produced by osteoblasts on porous Ti-6Al-4V scaffold materials under LIPUS and non-ultrasound stimulated conditions were co-cultured with endothelial cells. The findings indicated that the exosomes were consistently and stably taken up by the endothelial cells. Compared to the non-ultrasound group, the LIPUS group facilitated endothelial cell proliferation and angiogenesis. After 24 h of co-culture, the migration ability of endothelial cells in the LIPUS group was 17.30% higher relative to the non-ultrasound group. LIPUS may represent a potentially viable strategy to promote the efficacy of osteoblast-derived exosomes to enhance the angiogenesis of porous Ti-6Al-4V scaffold materials.

Engineering exosomes derived from TNF-α preconditioned IPFP-MSCs enhance both yield and therapeutic efficacy for osteoarthritis

BackgroundThe pathogenesis of osteoarthritis (OA) involves the progressive degradation of articular cartilage. Exosomes derived from mesenchymal stem cells (MSC-EXOs) have been shown to mitigate joint pathological injury by attenuating cartilage destruction. Optimization the yield and therapeutic efficacy of exosomes derived from MSCs is crucial for promoting their clinical translation. The preconditioning of MSCs enhances the therapeutic potential of engineered exosomes, offering promising prospects for application by enabling controlled and quantifiable external stimulation. This study aims to address these issues by employing pro-inflammatory preconditioning of MSCs to enhance exosome production and augment their therapeutic efficacy for OA.MethodsThe exosomes were isolated from the supernatant of infrapatellar fat pad (IPFP)-MSCs preconditioned with a pro-inflammatory factor, TNF-α, and their production was subsequently quantified. The exosome secretion-related pathways in IPFP-MSCs were evaluated through high-throughput transcriptome sequencing analysis, q-PCR and western blot analysis before and after TNF-α preconditioning. Furthermore, exosomes derived from TNF-α preconditioned IPFP-MSCs (IPFP-MSC-EXOsTNF−α) were administered intra-articularly in an OA mouse model, and subsequent evaluations were conducted to assess joint pathology and gait alterations. The expression of proteins involved in the maintenance of cartilage homeostasis within the exosomes was determined through proteomic analysis.ResultsThe preconditioning with TNF-α significantly enhanced the exosome secretion of IPFP-MSCs compared to unpreconditioned MSCs. The potential mechanism involved the activation of the PI3K/AKT signaling pathway in IPFP-MSCs by TNF-α precondition, leading to an up-regulation of autophagy-related protein 16 like 1(ATG16L1) levels, which subsequently facilitated exosome secretion. The intra-articular administration of IPFP-MSC-EXOsTNF−α demonstrated superior efficacy in ameliorating pathological changes in the joints of OA mice. The preconditioning of TNF-α enhanced the up-regulation of low-density lipoprotein receptor-related protein 1 (LRP1) levels in IPFP-MSC-EXOsTNF−α, thereby exerting chondroprotective effects.ConclusionTNF-α preconditioning constitutes an effective and promising method for optimizing the therapeutic effects of IPFP-MSCs derived exosomes in the treatment of OA.Graphical

Drug delivery based exosomes uptake pathways

Most cells secrete a material called extracellular vesicles (EVs), which play a crucial role in cellular communication. Exosomes are one of the most studied types of EVs. Recent research has shown the many functions and substrates of cellular exosomes. Multiple studies have shown the efficacy of exosomes in transporting a wide variety of cargo to their respective target cells. As a result, they are often utilized to transport medicaments to patients. Natural exosomes as well as exosomes modified with other compounds to enhance transport capabilities have been employed. In this article, we take a look at how different types of exosomes and modified exosomes may transport different types of cargo to their respective targets. Exosomes have a lot of potential as drug delivery vehicles for many synthetic compounds, proteins, nucleic acids, and gene repair specialists because they can stay in the body for a long time, are biocompatible, and can carry natural materials. A good way to put specific protein particles into exosomes is still not clear, though, and the exosomes can't be used in many situations yet. The determinants for exosome production, as well as ways for loading certain therapeutic molecules (proteins, nucleic acids, and small compounds), were covered in this paper. Further study and the development of therapeutic exosomes may both benefit from the information collected in this review.

Progression in the Relationship between Exosome Production and Atherosclerosis.

Atherosclerosis (AS) is the leading cause of cardiovascular disease, causing a major burden on patients as well as families and society. Exosomes generally refer to various lipid bilayer microvesicles originating from different cells that deliver various bioactive molecules to the recipient cells, exerting biological effects in cellular communication and thereby changing the internal environment of the body. The mechanisms of correlation between exosomes and the disease process of atherosclerosis have been recently clarified. Exosomes are rich in nucleic acid molecules and proteins. For example, the exosome miRNAs reportedly play important roles in the progression of atherosclerotic diseases. In this review, we focus on the composition of exosomes, the mechanism of their biogenesis and release, and the commonly used methods for exosome extraction. By summarizing the latest research progress on exosomes and atherosclerosis, we can explore the advances in the roles of exosomes in atherosclerosis to provide new ideas and targets for atherosclerosis prevention, diagnosis, and treatment.

Determining stemness and exosome production from nutrient-dependent cells: Influence of the molecular weight cut-off in hollow fiber membrane systems

This study aimed to optimize parameters for cultivating adipose-derived stem cells (ADSCs) in hollow fiber membrane systems. Enhanced ADSC cultivation was achieved in this study by utilizing a higher cell seeding rate (10 mL/min) and fibronectin coating (8 μg/cm²) on the membrane. A pulsatile flow perfusion (30 min on / 3 h off) for 24 h, followed by continuous flow perfusion at a lower rate (<20 mL/min), was employed. Notably, a two-day medium change maintained CD73 expression (79.26 %) compared to scenarios without changes (1.92 %) over eight days. Using a membrane cut-off of 20 kDa and <0.1 μm, a 1.4- and 2.8-fold increase in proliferated cells was observed; however, CD73 expression decreased by 10.07 and 33.63 %, respectively. The membrane with a pore size of <0.1 μm exhibited 1.4-fold higher exosome particle numbers. In conclusion, nutrient supplementation emerged as crucial for optimal ADSC cultivation in hollow fiber systems.

Activation of the Wnt/β-catenin signalling pathway enhances exosome production by hucMSCs and improves their capability to promote diabetic wound healing

BackgroundThe use of stem cell-derived exosomes (Exos) as therapeutic vehicles is receiving increasing attention. Exosome administration has several advantages over cell transplantation, thus making exosomes promising candidates for large-scale clinical implementation and commercialization. However, exosome extraction and purification efficiencies are relatively low, and therapeutic heterogeneity is high due to differences in culture conditions and cell viability. Therefore, in this study, we investigated a priming procedure to enhance the production and therapeutic effects of exosomes from human umbilical cord mesenchymal stem cells (hucMSCs). After preconditioning hucMSCs with agonists/inhibitors that target the Wnt/β-catenin pathway, we assessed both the production of exosomes and the therapeutic efficacy of the optimized exosomes in the context of diabetic wound healing, hoping to provide a safer, more stable and more effective option for clinical application.ResultsThe Wnt signalling pathway agonist CHIR99021 increased exosome production by 1.5-fold without causing obvious changes in the characteristics of the hucMSCs or the size of the exosome particles. Further studies showed that CHIR99021 promoted the production of exosomes by facilitating exocytosis. This process was partly mediated by SNAP25. To further explore whether CHIR99021 changed the cargo that was loaded into the exosomes and its therapeutic effects, we performed proteomic and transcriptomic analyses of exosomes from primed and control hucMSCs. The results showed that CHIR99021 significantly upregulated the expression of proteins that are associated with cell migration and wound healing. Animal experiments confirmed that, compared to control hucMSC-derived exosomes, CHIR99021-pretreated hucMSC-derived exosomes (CHIR-Exos) significantly accelerated wound healing in diabetic mice, enhanced local collagen deposition, promoted angiogenesis, and reduced chronic inflammation. Subsequent in vitro experiments confirmed that the CHIR-Exos promoted wound healing by facilitating cell migration, inhibiting oxidative stress-induced apoptosis, and preventing cell cycle arrest.ConclusionsThe Wnt agonist CHIR99021 significantly increased exosome secretion by hucMSCs, which was partly mediated by SNAP25. Notably, CHIR99021 treatment also significantly increased the exosomal levels of proteins that are associated with wound healing and cell migration, resulting in enhanced acceleration of wound healing. All of these results suggested that pretreatment of hucMSCs with CHIR99021 not only promoted exosome production but also improved the exosome therapeutic efficacy, thus providing a promising option for large-scale clinical implementation and commercialization.Graphical

The role of exosomal molecular cargo in exosome biogenesis and disease diagnosis.

Exosomes represent a type of extracellular vesicles derived from the endosomal pathway that transport diverse molecular cargoes such as proteins, lipids, and nucleic acids. These cargoes have emerged as crucial elements impacting disease diagnosis, treatment, and prognosis, and are integral to the process of exosome formation. This review delves into the essential molecular cargoes implicated in the phases of exosome production and release. Emphasis is placed on their significance as cancer biomarkers and potential therapeutic targets, accompanied by an exploration of the obstacles and feasible applications linked to these developments.

Increasing the Survival of a Neuronal Model of Alzheimer's Disease Using Docosahexaenoic Acid, Restoring Endolysosomal Functioning by Modifying the Interactions between the Membrane Proteins C99 and Rab5.

Docosahexaenoic acid (DHA, C22:6 ω3) may be involved in various neuroprotective mechanisms that could prevent Alzheimer's disease (AD). Its influence has still been little explored regarding the dysfunction of the endolysosomal pathway, known as an early key event in the physiopathological continuum triggering AD. This dysfunction could result from the accumulation of degradation products of the precursor protein of AD, in particular the C99 fragment, capable of interacting with endosomal proteins and thus contributing to altering this pathway from the early stages of AD. This study aims to evaluate whether neuroprotection mediated by DHA can also preserve the endolysosomal function. AD-typical endolysosomal abnormalities were recorded in differentiated human SH-SY5Y neuroblastoma cells expressing the Swedish form of human amyloid precursor protein. This altered phenotype included endosome enlargement, the reduced secretion of exosomes, and a higher level of apoptosis, which confirmed the relevance of the cellular model chosen for studying the associated deleterious mechanisms. Second, neuroprotection mediated by DHA was associated with a reduced interaction of C99 with the Rab5 GTPase, lower endosome size, restored exosome production, and reduced neuronal apoptosis. Our data reveal that DHA may influence protein localization and interactions in the neuronal membrane environment, thereby correcting the dysfunction of endocytosis and vesicular trafficking associated with AD.

De novo synthesis of monounsaturated fatty acids modulates exosome-mediated lipid export from human granulosa cells

BackgroundOvarian somatic cells support the maturation and fertility of oocytes. Metabolic desaturation of fatty acids in these cells has a positive paracrine impact on the maturation of oocytes. We hypothesized that the enzyme stearoyl-CoA desaturase 1 (SCD1) in granulosa cells regulates the lipid cargo of exosomes secreted from these cells by maintaining the balance between saturated and unsaturated lipids. We investigated the effect of SCD1 on exosome lipid content in a cumulus-granulosa cell model under physiologically relevant in vitro conditions. MethodsNon-luteinized human COV434 granulosa cells were subjected to treatment with an inhibitor of SCD1 (SCDinhib) alone, in combination with oleic acid, or under control conditions. Subsequently, the exosomes were isolated and characterized via nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. We used liquid chromatography mass spectrometry to investigate the lipidomic profiles. We used quantitative PCR with TaqMan primers to assess the expression of genes involved in lipogenesis and control of cell cycle progression. ResultsA trend toward exosome production was observed with a shift toward smaller exosome sizes in cells treated with SCD1inhib. This trend reached statistical significance when SCDinhib was combined with oleic acid supplementation. SCD1 inhibition led to the accumulation of saturated omega-6 lipids in exosomes. The latter effect was reversed by oleic acid supplementation, which also improved exosome production and suppressed the expression of fatty acid synthase and Cyclin D2. ConclusionThese findings underscore the critical role of de novo fatty acid desaturation in the regulation of the export of specific lipids through exosomes, with potential implications for controlling intercellular communication within the ovary.

RNA binding protein RALY facilitates colorectal cancer metastasis via enhancing exosome biogenesis in m6A dependent manner

Tumor metastasis is the leading cause of cancer-related death in patients with colorectal cancer (CRC). Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA-binding proteins, involved in the tumorigenesis and metastasis of various cancers. However, the molecular mechanisms of hnRNPs in CRC metastasis remain unclear. This study aims to uncover the pivotal roles and molecular mechanisms of hnRNPs in CRC metastasis. Clinical database analysis suggested that the expression of hnRNP-Associated with Lethal Yellow (RALY, an important member of hnRNPs) was strongly correlated with the aggressiveness and survival of CRC patients. Gain- and loss-of-function studies demonstrated that RALY promotes the production of exosomes by increasing the formation of multivesicular bodies (MVBs) and enhancing the fusion of MVBs with the plasma membrane. Notably, RALY directly interacts with phospholipase D2 (PLD2) to enable exosome biogenesis, and cooperates with RBM15b to control PLD2 mRNA stability in an m6A-dependent manner. RALY-mediated exosome secretion activates pro-tumor macrophages and further facilitates CRC metastasis, while rescue experiments in vivo further confirmed that RALY-mediated exosome biogenesis facilitates CRC metastasis. Collectively, our findings demonstrate that RALY promotes exosome biogenesis and facilitates colorectal cancer metastasis by upregulating PLD2 and enhancing exosome production in an m6A-dependent manner, suggesting potential therapeutic strategies for combating CRC metastasis.

Large-Area Silicon Nitride Nanosieve for Enhanced Diffusion-Based Exosome Isolation.

Nanoporous membranes have a variety of applications, one of which is the size-selective separation of nanoparticles. In drug delivery, nanoporous membranes are becoming increasingly important for the isolation of exosomes, which are bio-nanoparticles. However, the low pore density and thickness of commercial membranes limit their efficiency. There have been many attempts to fabricate sub-micrometer thin membranes, but the limited surface area has restricted their practicality. In this study, large-area silicon nitride nanosieves for enhanced diffusion-based isolation of exosomes are presented. Notably, these nanosieves are scaled to sizes of up to 4-inch-wafers, a significant achievement in overcoming the fabrication challenges associated with such expansive areas. The method employs a 200nm porous sieve (38.2% porosity) for exosome separation and a 50nm sieve (10.7% porosity) for soluble protein removal. These 300nmthick nanosieves outperform conventional polycarbonate membranes by being 50 times thinner, thereby increasing nanoparticle permeability. The method enables a 90% recovery rate of intact exosomes from human serum and a purity ratio of 3 × 107 particles/µg protein, 4.6 times higher than ultracentrifugation methods. The throughput of the method is up to 15mL by increasing the size of the nanosieve, making it an ideal solution for large-scale exosome production for therapeutic purposes.

Shikonin reduces M2 macrophage population in ovarian cancer by repressing exosome production and the exosomal galectin 3-mediated β-catenin activation

BackgroundShikonin (SK), a naphthoquinone with anti-tumor effects, has been found to decrease production of tumor-associated exosomes (exo). This study aims to verify the treatment effect of SK on ovarian cancer (OC) cells, especially on the production of exo and their subsequent effect on macrophage polarization.MethodsOC cells SKOV3 and A2780 were treated with SK. The exo were isolated from OC cells with or without SK treatment, termed OC exo and SK OC exo, respectively. These exo were used to treat PMA-induced THP-1 cells (M0 macrophages). M2 polarization of macrophages was determined by measuring the M2 specific cell surface markers CD163 and CD206 as well as the secretion of M2 cytokine IL-10. The functions of galectin 3 (LGALS3/GAL3) and β-catenin in macrophage polarization were determined by gain- or loss-of-function assays. CB-17 SCID mice were subcutaneously injected with SKOV3 cells to generate xenograft tumors, followed by OC exo or SK OC exo treatment for in vivo experiments.ResultsSK suppressed viability, migration and invasion, and apoptosis resistance of OC cells in vitro. Compared to OC exo, SK OC exo reduced the M2 polarization of macrophages. Regarding the mechanism, SK reduced exo production in cancer cells, and it decreased the protein level of GAL3 in exo and recipient macrophages, leading to decreased β-catenin activation. M2 polarization of macrophages was restored by LGALS3 overexpression but decreased again by the β-catenin inhibitor FH535. Compared to OC exo, the SK OC exo treatment reduced the xenograft tumor growth in mice, and it decreased the M2 macrophage infiltration within tumor tissues.ConclusionThis study suggests that SK reduces M2 macrophage population in OC by repressing exo production and blocking exosomal GAL3-mediated β-catenin activation.