Nanoparticle Tracking Analysis Research Articles

Exosomes enriched by miR-429-3p derived from ITGB1 modified Telocytes alleviates hypoxia-induced pulmonary arterial hypertension through regulating Rac1 expression

BackgroundRecent studies have emphasized the critical role of Telocytes (TCs)-derived exosomes in organ tissue injury and repair. Our previous research showed a significant increase in ITGB1 within TCs. Pulmonary Arterial Hypertension (PAH) is marked by a loss of microvessel regeneration and progressive vascular remodeling. This study aims to investigate whether exosomes derived from ITGB1-modified TCs (ITGB1-Exo) could mitigate PAH.MethodsWe analyzed differentially expressed microRNAs (DEmiRs) in TCs using Affymetrix Genechip miRNA 4.0 arrays. Exosomes isolated from TC culture supernatants were verified through transmission electron microscopy and Nanoparticle Tracking Analysis. The impact of miR-429-3p-enriched exosomes (Exo-ITGB1) on hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs) was evaluated using CCK-8, transwell assay, and inflammatory factor analysis. A four-week hypoxia-induced mouse model of PAH was constructed, and H&E staining, along with Immunofluorescence staining, were employed to assess PAH progression.ResultsForty-five miRNAs exhibited significant differential expression in TCs following ITGB1 knockdown. Mus-miR-429-3p, significantly upregulated in ITGB1-overexpressing TCs and in ITGB1-modified TC-derived exosomes, was selected for further investigation. Exo-ITGB1 notably inhibited the migration, proliferation, and inflammation of PASMCs by targeting Rac1. Overexpressing Rac1 partly counteracted Exo-ITGB1’s effects. In vivo administration of Exo-ITGB1 effectively reduced pulmonary vascular remodeling and inflammation.ConclusionsOur findings reveal that ITGB1-modified TC-derived exosomes exert anti-inflammatory effects and reverse vascular remodeling through the miR-429-3p/Rac1 axis. This provides potential therapeutic strategies for PAH treatment.Graphical 1. Identification of Differentially Expressed microRNAs (DEmiRs) in ITGB1 overexpressed TCs.2. Effects of Exo-ITGB1 or miR-429-3p on Hypoxia-Induced PASMCs in vitro.3. Exo-ITGB1 inhibits the hyper-proliferation and migration of PASMCs through regulating miR-429-3p/Rac1 axis in vitro.4. The therapeutic potential of Exo-ITGB1 in hypoxia-induced PAH model in vivo.

Capillary-assisted flat-field formation: a platform for advancing nanoparticle tracking analysis in an integrated on-chip optofluidic environment

Abstract Here, we present the concept of flat-field capillary-assisted nanoparticle tracking analysis for the characterization of fast diffusing nano-objects. By combining diffusion confinement and spatially invariant illumination, i.e., flat-fields, within a fiber-interfaced on-chip environment, ultra-long trajectories of fast diffusing objects within large microchannels have been measured via diffraction-limited imaging. Our study discusses the design procedure, explains potential limitations, and experimentally confirms flat-field formation by tracking gold nanospheres. The presented concept enables generating flat-fields in a novel on-chip optofluidic platform for the characterization of individual nano-objects for fundamental light/matter investigations or applications in bioanalytics and nanoscale material science.

Prediction of severe preeclampsia and intrauterine growth restriction based on serum placental exosome miR-520a-5p levels during the first-trimester.

To assess the predictive capabilities of serum exosomal levels of micro-RNA-520a-5p (miR-520a-5p) concerning the occurrence of severe preeclampsia (sPE) and fetal growth restriction (FGR) during the first trimester of pregnancy. During the period spanning from October 2020 to October 2021, serum samples were procured from the first trimester and subsequently preserved by freezing at -80 ℃. These samples were obtained from 105 pregnant women in a nested case-control study. This cohort consisted of individuals who later developed sPE (sPE group, n = 35) and FGR (FGR group, n = 35) during the third trimester. Additionally, 35 women with normal blood pressure were denoted as normal pregnancy group. Serum samples from the first trimester were retrieved from all groups for further analysis after thawing. Exosomes were extracted from the serum samples collected during the first trimester and examined using transmission electron microscopy, western blot, and nanoparticle tracking analysis. Additionally, the determination of their placental origin was also established during the course of the study. Exosome miR-520a-5p levels were measured using real-time quantitative polymerase chain reaction assays, primarily involving quantitative reverse transcription polymerase chain reactions. Fetal placental tissues from the 3 groups were collected shortly after birth, and miR-520a-5p expression was measured using real-time quantitative polymerase chain reaction. Serum placental exosomes and fetal placental tissues were compared for miR-520a-5p levels. Placental trophoblasts were identified as the source of serum exosomes in all 3 groups. It was found that serum placental exosomes exhibited lower levels of miR-520a-5p in both the sPE and FGR groups when compared to the normal pregnancy group. This finding was consistent with observations made in postpartum placental tissues. The predictive accuracy for sPE using miR-520a-5p levels in serum placental exosomes during the first trimester was notably higher (area under the receiver operating characteristic curve = 0.806, P <.05) compared to the prediction of FGR (area under the receiver operating characteristic curve = 0.628, P <.05). Placenta-derived exosomes can be extracted from maternal serum during the first trimester of pregnancy and miR-520a-5p detected from the exosomes. The downregulation of miR-520a-5p serves as a more predictive indicator for the subsequent development of sPE compared to predicting FGR.

Changes in the Transcription of Proliferation- and Apoptosis-Related Genes in Embryos in Women of Different Ages under the Influence of Extracellular Vesicles from Donor Follicular Fluid In Vitro.

We studied the influence of extracellular vesicles from the follicular fluid of a young donor on gene expression (MKI67, MYBL2, CCNB1, CCND1, CCNE1, CALM2, BAX, NDRG1, TP53I3, VEGF, VCAN, HAS2, CTSL2, PIBF1, RPL37, PFKP, GPX3, and AQP3) in embryos of women of different ages. According to nanoparticle tracking analysis data, the concentration of extracellular vesicles was 3.75±0.47×1011 particles/ml and the mean particle size was 138.78±9.90 nm. During co-culturing of the follicular fluid extracellular vesicles with blastocysts of young women, we observed significantly increased expression of mRNA for genes CTSL2, CCND1, CCNE1, VEGF and reduced expression of BAX gene mRNA in comparison with embryos in women of late reproductive age. We hypothesized that addition of extracellular vesicles of the oocyte follicular fluid from a young donor to the culture medium of embryos could slow down apoptosis process typical of blastocyst cells in women above 36 years.

Comprehensive characterization of extracellular vesicles produced by environmental (Neff) and clinical (T4) strains of Acanthamoeba castellanii.

We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of A. castellanii EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for Acanthamoeba-related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of A. castellanii EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of Acanthamoeba-related diseases.

Effect of In Vitro Enzyme Digestion and Bile Treatment on Milk Extracellular Vesicles Stability.

Milk extracellular vesicles (EVs) are nanosized particles with potential immune bioactivities. This study examines their fate during in vitro infant gastrointestinal digestion (GI). Bovine milk is digested using the in vitro INFOGEST method, adjusted for the infant. To unravel the contribution of digestive enzymes from bile, milk is treated with digestive enzymes, bile, or a combination of both. EVs are collected posttreatment using differential ultracentrifugation. EVs characterization includes electrophoresis, immunoblotting, nanoparticle tracking analysis, and atomic force microscopy. EVs protein markers programmed cell death 6-interacting protein (ALIX), tumor susceptibility gene 101 (TSG101), cluster of differentiation 9 (CD9), and xanthine dehydrogenase (XDH) are detected after gastric digestion (G60), but their signal intensity is significantly reduced by intestinal conditions (p<0.05). Enzyme digestion, compared to bile treatment (I60+bile), results in a significant reduction of signal intensities for TSG101 and CD9 (p<0.05). Nanoparticle tracking analysis shows a significant reduction (p<0.05) of EV numbers at the end of the intestinal phase. EVs are detected by atomic force microscopy at the end of the intestinal phase, showing that intact EVs can survive upper gut digestion. Intact EVs can be found at the end of the intestinal phase. However, digestive enzymes and bile reduce the quantity and characteristics of EVs, with digestive enzymes playing a larger role.

Nanoplastics in Water: Artificial Intelligence-Assisted 4D Physicochemical Characterization and Rapid In Situ Detection.

For the first time, we present a much-needed technology for the in situ and real-time detection of nanoplastics in aquatic systems. We show an artificial intelligence-assisted nanodigital in-line holographic microscopy (AI-assisted nano-DIHM) that automatically classifies nano- and microplastics simultaneously from nonplastic particles within milliseconds in stationary and dynamic natural waters, without sample preparation. AI-assisted nano-DIHM identifies 2 and 1% of waterborne particles as nano/microplastics in Lake Ontario and the Saint Lawrence River, respectively. Nano-DIHM provides physicochemical properties of single particles or clusters of nano/microplastics, including size, shape, optical phase, perimeter, surface area, roughness, and edge gradient. It distinguishes nano/microplastics from mixtures of organics, inorganics, biological particles, and coated heterogeneous clusters. This technology allows 4D tracking and 3D structural and spatial study of waterborne nano/microplastics. Independent transmission electron microscopy, mass spectrometry, and nanoparticle tracking analysis validates nano-DIHM data. Complementary modeling demonstrates nano- and microplastics have significantly distinct distribution patterns in water, which affect their transport and fate, rendering nano-DIHM a powerful tool for accurate nano/microplastic life-cycle analysis and hotspot remediation.

Abstract PO3-24-11: Impact of Circulating Extracellular Vesicles derived from Obese Women on Pro-Tumor Functions of Stromal Fibroblasts in Breast Cancer

Abstract The incidence of obesity has risen sharply worldwide, and growing evidence shows its impact on several malignancies, including breast cancer (BC). Excessive adiposity and high body mass index (BMI) significantly affect BC incidence, prognosis, and therapeutic response of patients. Indeed, obese women exhibit larger tumor size, limph node involvement, metastatic spread, and poorer survival outcomes. Preclinical and clinical studies indicated that the obese setting provides local and systemic modifications that may stimulate BC progression and influence tumor microenvironment, particularly composed of stromal fibroblasts (SF). The mechanisms behind this interplay are likely to be multifactorial and may involve important adipocyte-derived mediators, such as adipokines and growth factors. Aside from these soluble factors, extracellular vesicles (EVs), nanoscale lipid-bilayer enclosed vesicles released by a variety of cells, are emerging as powerful regulators of cell-to-cell communication able to promote BC initiation, growth, metastatic dissemination and drug resistance. However, the potential impact of obesity-derived EVs in affecting tumor-stimulatory features of SF has not been yet investigated. To this aim, circulating EVs were isolated from the serum of women categorized as normal weight (NW; 18 &amp;gt; BMI &amp;lt; 24.9 Kg/m2) and overweight/obese (OW/Ob; BMI &amp;gt; 25 Kg/m2). Immortalised human mammary control fibroblasts (CF), their cancer-associated counterparts (exp-CAFs), and human CAFs, isolated from biopsies of primary BC, were used as experimental models for stromal fibroblasts. Human MCF-7 and T47D BC cells were employed as experimental cell models for mammary carcinoma in co-culture experiments. Circulating EVs were successfully extracted from the serum of NW and OW/Ob women, and characterized by their shapes, size and protein markers. Transmission Electron Microscopy (TEM), and Nanoparticle Tracking Analysis (NTA) showed that EVs in the isolated fractions were oval or bowl-shaped with a size range between 50-200 nm. In accordance with the Minimal Information for Studies of Extracellular Vesicles (MISEV) 2018, enrichment of the EV markers TSG101, CD81, and CD63 were all detected in the EV isolated fractions. Interestingly, elevated levels of circulating EVs were detected in OW/Ob women compared to NW subjects. Treatment of CF with OW/Ob-EVs resulted in an increased cell motility, invasiveness, contractility along with an up-regulation of CF markers. BC cells cocultured with conditioned medium (CM) derived from OW/Ob-EV-treated CF exhibited higher anchorage-independent growth and migratory capacity than BC cells cocultured with CM derived from NW-EV-treated CF. Moreover, OW/Ob-EVs enhanced tumor-stimulatory activities of exp-CAFs and CAFs, further highlighting how obese-related EVs may have the potential to modulate fibroblast phenotype in BC. Finally, cytokine arrays revealed that treatment of CF with OW/Ob EVs was associated with an enhanced release of Fibroblast Growth Factor 19 and Cystatin C, factors that are closely related to BC growth and development. In conclusion, our data provide first evidence for the functional importance of EVs isolated from obese women in impacting tumor-stroma cross-talk and drive BC progression. Since obesity and its pathophysiological sequelae are on the rise, this new knowledge may help to identify specific biomarkers and innovative targets (i.e. FGF-19, and Cystatin C) that may allow a personalized management of patients affected by BC and obesity. Citation Format: Ines Barone, Piercarlo Del Console, Felice Maria Accattatis, Luca Gelsomino, Giuseppina Augimeri, Ennio Avolio, Sebastiano Andò, Daniela Bonofiglio, Cinzia Giordano, Stefania Catalano. Impact of Circulating Extracellular Vesicles derived from Obese Women on Pro-Tumor Functions of Stromal Fibroblasts in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-11.

Abstract PO1-25-01: LMTK3 promotes macrophage M2 polarisation and suppresses monocyte infiltration in breast cancer by altering the proteomic cargo of exosomes

Abstract Background: Breast cancer (BC) is a leading cause of cancer death and despite numerous treatment advancements, metastatic BC is incurable. Extracellular vesicles (EVs) are lipid-delimited particles released from cells which contain bioactive protein and nucleic acid cargo. Numerous studies have shown their importance in facilitating communication between cancer cells and the tumour microenvironment, implicating them as mediators of many aspects of carcinogenesis. EV-mediated communication between BC and the innate immune system plays a crucial role in BC progression, metastasis and the maintenance of an immunosuppressive microenvironment. LMTK3 overexpression in BC promotes therapy resistance, disease progression and invasion and metastasis, leading to a poorer prognosis in patients. Analysis of deconvoluted TCGA data indicated that LMTK3 overexpression also correlates with exclusion of M1 macrophages from the tumour microenvironment. This study aimed to explore the role of LMTK3 in EV-mediated communication between BC and immune cells. Methods EVs were collected from conditioned media of wild-type and LMTK3-overexpressing BC cells through differential ultracentrifugation, then characterised by transmission electron microscopy, nanoparticle tracking analysis, quantitative mass spectrometry and Western blotting. Monocytes were treated with EVs and proteomic and phosphoproteomic changes were analysed. 3D coculture models of BC and immune cells were used to assess monocyte and macrophage infiltration, polarisation and anti-cancer activity. Immunofluorescence and flow cytometry was used to assess macrophage polarisation. Bioinformatic analysis of TCGA datasets validated the findings in a clinical setting and the data was also validated in vivo, using syngeneic immunocompetent mouse models. Results LMTK3 overexpression in BC cells promotes the upregulation of a subpopulation of large (80-150nm) CD81+ EVs which are significantly enriched in oncogenic and immunoregulatory proteins, including LDHB, ARHGEF15, PSAT1 and HIC2. EVs from LMTK3-overexpressing but not control BC cells strongly reduce the ability of monocytes to infiltrate BC tumour spheroids and migrate through Matrigel. Proteomics analysis of the monocytes showed an enrichment in M2 polarisation proteins and a downregulation of proteins essential for migration, including S100A8, KLF13 and TRPC3. LMTK3 overexpression in BC also promotes M2 polarisation and exclusion of M1 macrophages in vivo. Summary LMTK3 overexpression in BC correlates with poor prognosis and reduced overall survival. We hypothesise that this is, in part, due to monocyte M2 polarisation and exclusion of M1 macrophages from the TME, mediated by LMTK3-dependent alterations in EV cargo, contributing to tumour immune evasion. Citation Format: Mark Samuels, William Jones, Charlotte Turner, Georgios Giamas. LMTK3 promotes macrophage M2 polarisation and suppresses monocyte infiltration in breast cancer by altering the proteomic cargo of exosomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-01.

Bone marrow mesenchymal stem cell-derived exosomes shuttle microRNAs to endometrial stromal fibroblasts that promote tissue proliferation /regeneration/ and inhibit differentiation

BackgroundHuman bone marrow-derived stem cells (hBMDSCs) are well characterized mediators of tissue repair and regeneration. An increasing body of evidence indicates that these cells exert their therapeutic effects largely through their paracrine actions rather than clonal expansion and differentiation. Here we studied the role of microRNAs (miRNAs) present in extracellular vesicles (EVs) from hBMDSCs in tissue regeneration and cell differentiation targeting endometrial stromal fibroblasts (eSF).MethodsExtracellular vesicles (EVs) are isolated from hBMDSCs, characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) techniques. Extracted total RNA from EVs was subjected to RNA seq analysis. Transfection and decidualization studies were carried out in endometrial stromal fibroblasts (eSF). Gene expression was analyzed by qRTPCR. Unpaired t-test with Welch’s correction was used for data analysis between two groups.ResultsWe identified several microRNAs (miRNAs) that were highly expressed, including miR-21-5p, miR-100-5p, miR-143-3p and let7. MiR-21 is associated with several signaling pathways involved in tissue regeneration, quiescence, cellular senescence, and fibrosis. Both miR-100-5p and miR-143-3p promoted cell proliferation. MiR-100-5p specifically promoted regenerative processes by upregulating TGF-ß3, VEGFA, MMP7, and HGF. MiR-100-5p blocked differentiation or decidualization as evidenced by morphologic changes and downregulation of decidualization mediators including HOXA10, IGFBP1, PRL, PR-B, and PR.Conclusion EVs delivered to tissues by hBMDSCs contain specific miRNAs that prevent terminal differentiation and drive repair and regeneration. Delivery of microRNAs is a novel treatment paradigm with the potential to replace BMDSCs in cell-free regenerative therapies.

Resveratrol Inhibits Hepatocellular Carcinoma Progression through Regulating Exosome Secretion.

Resveratrol is a promising drug for tumor therapy, but its anti-tumor mechanism remains unclarified. The present study aimed to explore the effect of resveratrol on the secretion of exosomes and the role of resveratrol-induced exosomes in the progression of hepatocellular carcinoma. The number and contents of exosomes induced by resveratrol were determined by nanoparticle tracking analysis and high-throughput sequencing in Huh7 cells, respectively. Expression of Rab27a was assessed by western blotting and immunofluorescence. Cell proliferation, migration and epithelial-mesenchymal transition were examined with the stimuli of resveratrol and exosomes, the activity of autophagy and wnt/β-catenin signaling induced by resveratrol-induced exosomes and knockdown of lncRNA SNHG29 were monitored by western blotting and immunofluorescence. It was found that resveratrol might inhibit the exosome secretion by down-regulating the expression of Rab27a, thereby suppressing the proliferation, migration and epithelial-mesenchymal transition of Huh7 cells. Moreover, resveratrol-induced exosomes could also inhibit the malignant phenotype of Huh7 cells via inhibiting the nuclear translocation of β-catenin and the activation of autophagy, which lncRNA SNHG29 might mediate. Resveratrol inhibits hepatocellular carcinoma progression by regulating exosome secretion and contents.

Wound Fluid Extracellular Microvesicles: A Potential Innovative Biomarker for Wound Healing.

Extracellular vesicles, or microvesicles, are a large family of membrane-bound fluid-filled sacs that cells release into the extracellular environment. Extracellular microvesicles (EMVs) are essential for cell-to-cell communications that promote wound healing. We hypothesize a correlation between the concentration of EMVs in wound fluid and the percentage of wound healing in treated chronic, nonhealing, wounds. A prospective, multicenter, randomized, single-blind clinical trial was conducted to evaluate EMV concentration in relation to wound healing percentages. Wound fluid samples were obtained from 16 patients with stage IV trunk pressure ulcers. Patients were divided equally into two groups: (1) control group on negative pressure wound therapy (NPWT) alone and (2) study group with NPWT plus porcine extracellular matrix dressing. NPWT was replaced two times a week, and porcine extracellular matrix applied once weekly for all subjects. An NPWT canister device, called a wound vacuum-assisted closure, containing wound fluid was collected from each patient every 4 weeks. EMVs were isolated and the concentration measured by nanoparticle tracking analysis. The study group's total healing percentage was around 89% after 12 weeks compared with the control group's percentage of about 52% (P ≤ 0.05). Using R programming software, simple linear regression was carried out to investigate the hypothesis. Data demonstrated significant positive correlation (R2 = 0.70; P = 0.05) between EMV concentrations and the healing percentage. There is a positive correlation between EMV concentration and wound healing percentages. Results propose that the EMVs in wound fluid could serve as a biomarker for healing.

GMP-compliant extracellular vesicles derived from umbilical cord mesenchymal stromal cells: manufacturing and pre-clinical evaluation in ARDS treatment

Background aimsExtracellular vesicles (EVs) represent a new axis of intercellular communication that can be harnessed for therapeutic purposes, as cell-free therapies. The clinical application of mesenchymal stromal cell (MSC)-derived EVs, however, is still in its infancy and faces many challenges. The heterogeneity inherent to MSCs, differences among donors, tissue sources, and variations in manufacturing conditions may influence the release of EVs and their cargo, thus potentially affecting the quality and consistency of the final product. We investigated the influence of cell culture and conditioned medium harvesting conditions on the physicochemical and proteomic profile of human umbilical cord MSC-derived EVs (hUCMSC-EVs) produced under current good manufacturing practice (cGMP) standards. We also evaluated the efficiency of the protocol in terms of yield, purity, productivity, and expression of surface markers, and assessed the biodistribution, toxicity and potential efficacy of hUCMSC-EVs in pre-clinical studies using the LPS-induced acute lung injury model. MethodshUCMSCs were isolated from a cord tissue, cultured, cryopreserved, and characterized at a cGMP facility. The conditioned medium was harvested at 24, 48, and 72 h after the addition of EV collection medium. Three conventional methods (nanoparticle tracking analysis, transmission electron microscopy, and nanoflow cytometry) and mass spectrometry were used to characterize hUCMSC-EVs. Safety (toxicity of single and repeated doses) and biodistribution were evaluated in naive mice after intravenous administration of the product. Efficacy was evaluated in an LPS-induced acute lung injury model. ResultshUCMSC-EVs were successfully isolated using a cGMP-compliant protocol. Comparison of hUCMSC-EVs purified from multiple harvests revealed progressive EV productivity and slight changes in the proteomic profile, presenting higher homogeneity at later timepoints of conditioned medium harvesting. Pooled hUCMSC-EVs showed a non-toxic profile after single and repeated intravenous administration to naive mice. Biodistribution studies demonstrated a major concentration in liver, spleen and lungs. HUCMSC-EVs reduced lung damage and inflammation in a model of LPS-induced acute lung injury. ConclusionshUCMSC-EVs were successfully obtained following a cGMP-compliant protocol, with consistent characteristics and pre-clinical safety profile, supporting their future clinical development as cell-free therapies.

Exosomal HMGB3 released by glioma cells confers the activation of NLRP3 inflammasome and pyroptosis in tumor-associated macrophages

BackgroundPrevious evidences has highlighted the pivotal role of NOD-like receptor family pyrin domain-containing 3 (NLRP3)-mediated inflammasomes and pyroptosis activation in driving tumor malignancy and shaping the tumor microenvironment. Herein, we aimed to elucidate the impact of high-mobility group box 3 (HMGB3) released in glioma-derived exosomes on macrophage infiltration in gliomas, NLRP3 inflammasome activation and polarization. MethodsTranscripts and protein levels of HMGB3, and cytokines associated with macrophage phenotypes and pyroptosis were assessed in glioma tissues and cell lines (U251, LN229, T98G, A172) using qRT-PCR and/or Western blot analysis. Exosomes secreted from LN229 and NHA cells were isolated via differential ultracentrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and analysis of exosome-related markers. PKH67 staining was employed to examine exosomes uptake by THP-1 differentiated macrophages. Flow cytometry was utilized to assess macrophage pyroptotic rates and polarization-related markers. ResultsHMGB3 expression was elevated in glioma tissues, serum samples and tumor cell lines. Kaplan-Meier curves revealed a positive correlation between higher HMGB3 expression and poor overall survival and recurrence-free survival. Moreover, glioma tissues with increased HMGB3 expression exhibited significant upregulation of M2 macrophages markers (CD68, CD206, Arg1) and NLRP3 inflammasome components (NLRP3, IL-1β, ASC), suggesting that HMGB3 was closely associated with macrophage infiltration and NLRP3 inflammasome activation. Notably, HMGB3 was found to be enriched in glioma cell- secreted exosomes and could be internalized by macrophages. Knockdown of HMGB3 in glioma cell exosomes could restrain M2 macrophage polarization, NLRP3 inflammasome activation and pyroptosis. ConclusionThese findings suggested that glioma cells secreted exosomal HMGB3 could facilitate macrophage M2 polarization, pyroptosis and inflammatory infiltration, indicating HMGB3 might be a poor prognosis factor for glioma.

Suppression of gastric cancer cell proliferation by miR-494-3p inhibitor-loaded engineered exosomes

BackgroundGastric cancer necessitates novel treatments, and exosomes are promising therapeutic carriers. We created miR-494-3p inhibitor exosomes to assess their effects on gastric cancer cells. MethodsWe conducted a comprehensive investigation into the expression of the oncogenic miR-494-3p in gastric cancer tissues from patients. Subsequently, we engineered miR-494-3p inhibitor-loaded exosomes and characterized their morphology and size through transmission electron microscopy and nanoparticle tracking analysis. We next determined the encapsulation efficiency of the miR-494-3p inhibitor within these exosomes and evaluated the exosomes' structural integrity by quantifying the presence of exosomal markers. Following these validations, we co-cultured miR-494-3p inhibitor exosomes with cancer cells and employed PKH26 staining to visualize the efficient endocytosis of engineered exosomes by gastric cancer cells and assess the impact of these modified exosomes on gastric cancer cell proliferation, apoptosis, migration, and invasion. ResultsIncreased expression of miR-494-3p was observed in gastric cancer tissues as compared to controls. Significant low miR-494-3p levels were found within miR-494-3p inhibitor exosomes, signifying effective encapsulation. The incorporation of miR-494-3p inhibitor into engineered exosomes did not alter exosome morphology or size. Finally, PKH26-stained exosomes clearly demonstrated efficient endocytosis by gastric cancer cells, leading to reduced proliferation, migration, invasion, and increased apoptosis. ConclusionOur study identifies elevated miR-494-3p in gastric cancer tissues prompting the development of miR-494-3p inhibitor-loaded exosomes with efficient encapsulation. These engineered exosomes demonstrate successful endocytosis by cancer cells. This highlights their potential for therapeutic use in gastric cancer treatment by suppressing proliferation, migration, and invasion while enhancing apoptosis.

Studying the stability of polymer nanoparticles by size exclusion chromatography of radioactive polymers

The properties of nanomedicines will influence how they can deliver drugs to patients reproducibly and effectively. For conventional pharmaceutical products, Chemistry, Manufacturing and Control (CMC) documents require monitoring stability and storage conditions. For nanomedicines, studying these important considerations is hindered by a lack of appropriate methods. In this paper, we show how combining radiolabelling with size exclusion chromatography, using a method called SERP, can inform on the in vitro degradation of polymers. Using nanoparticles composed of biodegradable poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA), we show that SERP is more sensitive than dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) to detect degradation. We also demonstrate that the properties of the polymer composition and the nature of the aqueous buffer affect polymer degradation. Importantly, we show that minute changes in stability that cannot be detected by DLS and NTA impact the pharmacokinetic of nanoparticles injected in vivo. We believe that SERP might prove a valuable method to document and understand the pharmaceutical quality of polymer nanoparticles.

Harnessing extracellular vesicle membrane for gene therapy: EVs-biomimetic nanoparticles

One of the main concerns in oligonucleotide-based therapeutics is achieving a successful cell targeting while avoiding drug degradation and clearance. Nanoparticulated drug delivery systems have emerged as a way of overcoming these issues. Among them, membrane-coated nanoparticles are of increasing relevance mainly due to their enhanced cellular uptake, immune evasion and biocompatibility. In this study, we designed and elaborated a simple and highly tuneable biomimetic drug delivery nanosystem based on a polymeric core surrounded by extracellular vesicles (EVs)-derived membranes. This strategy should allow the nanosystems to benefit from the properties conferred by the membrane proteins present in EVs membrane, key paracrine mediators. The developed systems were able to successfully encapsulate the required oligonucleotides. Also, their characterisation through already well standardised methods (dynamic light scattering, transmission electron microscopy and nanoparticle tracking analysis) and by fluorescence cross-correlation spectroscopy (FCCS) showed the desired core-shell structure. The cellular uptake using different cell types further confirmed the coating though an enhancement in cell internalisation of the developed biomimetic nanoparticles. This study brings up new possibilities for GapmeR delivery as it might be a base for the development of new delivery systems for gene therapy.

Exosomal lncRNA ROR1-AS1 from cancer-associated fibroblasts inhibits ferroptosis of lung cancer cells through the IGF2BP1/SLC7A11 signal axis

BackgroundTargeting ferroptosis is a potential strategy for cancer treatment. Activated cancer-associated fibroblasts (CAFs) can affect the progression of lung cancer through exosomes. This study investigated the mechanism by which exosomal lncRNA ROR1-AS1 derived from CAFs affects ferroptosis of lung cancer cells. MethodsCAFs were identified by western blot and immunofluorescence. Exosomes derived from CAFs (CAF-exo) were analyzed by transmission electron microscope, nanoparticle tracking analysis and western blot. The expression levels of ROR1-AS1, IGF2BP1 and SLC7A11 in lung cancer were analyzed by bioinformatics analysis and detected by qPCR and western blot. The lung cancer cells were treated with Erastin and/or CAF-exo, then cell viability was detected by cell counting kit-8, and the ferroptosis-related indicators were detected by corresponding kit. The relationship between IGF2BP1 and ROR1-AS1 or SLC7A11 was determined by RNA pull down and RNA immunoprecipitation, and their effects on cell ferroptosis were confirmed by rescue experiment. Xenotransplantation experiment was used to determine the effect of CAF-exo on tumor growth and ferroptosis in vivo. Immunohistochemistry was used to identify the Ki-67 and 4-HNE expression. ResultsROR1-AS1, IGF2BP1 and SLC7A11 were upregulated in lung cancer and indicated poor prognosis. LncRNA ROR1-AS1 increases the stability of SLC7A11 mRNA by interacting with IGF2BP1. Exosomal ROR1-AS1 from CAFs inhibited ferroptosis of lung cancer cells in vitro and in vivo. The effect of ROR1-AS1 overexpression or IGF2BP1 overexpression on ferroptosis of lung cancer cells was partially reversed by IGF2BP1 silencing or SLC7A11 inhibition. ConclusionsCAFs secrete exosomal ROR1-AS1 to promote the expression of SLC7A11 by interacting with IGF2BP1, thereby inhibiting ferroptosis of lung cancer cells.

Discovery of a hyperalkaline liquid condensed phase: significance toward applications in carbon dioxide sequestration.

Bicarbonate ion-containing solutions such as seawater, natural brines, bovine serum and other mineralizing fluids have been found to contain hyperalkaline droplets of a separate, liquid condensed phase (LCP), that have higher concentrations of bicarbonate ion (HCO3 -) relative to the bulk solution in which they reside. The existence and unique composition of the LCP droplets have been characterized by nanoparticle tracking analysis, nuclear magnetic resonance spectroscopy, fourier transform infrared spectroscopy, dissolved inorganic carbon analysis and refractive index measurements. Carbon dioxide can be brought into solution through an aqueous reaction to form LCP droplets that can then be separated by established industrial membrane processes as a means of concentrating HCO3 -. Reaction of calcium with the LCP droplets results in calcium carbonate precipitation and mineral formation. The LCP phenomenon may bear on native mineralization reactions and has the potential to change fundamental approaches to carbon capture, sequestration and utilization.

Extracellular vesicles characterization in patients with hypertrophic cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is diagnosed according to the presence of morphological and functional traits of the heart, often in the presence of genetic mutations. A specific biomarker assessing the aetiopathology of this condition is lacking. Extracellular vesicles (EVs), small particles released by all cells into biological fluids, hold promise as diagnostic and prognostic tools for cardiac diseases. We aim at characterising plasma-derived EVs isolated from 18 consecutive HCM patients and 13 healthy volunteers (CTR). Methods: HCM underwent echocardiographic assessment and genetic testing evaluating 200 genes (NGS). EVs were isolated via ultracentrifugation from platelet-free plasma. Quantitative and qualitative assessments of EVs were performed by nanoparticle tracking analysis and transmission electron microscopy. FACS analysis was used to characterize EV subpopulations. Data are expressed as median and interquartile ranges. Results: Most patients were male (70.8% HCM and 54% CTR) with a median age of 61 (52.5-71) years (HCM) and 47 (44-52) (CTR). Missense mutations in the MYH7 gene were the most found in HCM. The median maximum wall thickness in HCM was 16 mm (15-19) vs 8 mm (7-9) in CTR. No differences were found in EV concentrations between HCM and CTR, respectively, 3.6*109 EV/ml/cell count (2*109-5*109) and 5*10⁹ EV/ml/cell count (4*109-6*109). However, EV concentration was positively associated with the sudden cardiac death risk score in HCM (r= 0.63). Among the EVs positive for CFSE (a specific dye for EVs), those released from platelets, progenitor endothelial cells and neutrophils were increased in HCM patients vs CTR, respectively, by 1.7-, 1.5- and 1.1-fold. A strong negative association (r= -0.74) was found between progenitor endothelial cell-derived EVs and the E/E’ ratio of diastolic function, a strong predictor of first cardiac events. Conclusions: HCM patients present a peculiar phenotypic pattern of EVs that associates which diastolic function and sudden cardiac death.