Search Results (375)

Our research outlines a method for creating chemosensors utilizing hybrid nanostructures derived from TiO₂ ceramic nanotubes alongside conducting polymers, with embedded gold nanoparticles. The method used to create hybrid nanostructures from ceramic nanotubes and conducting polymers was drop-casting. AFM analysis highlighted an increased roughness, particularly for PANI-EB, exhibiting a significantly larger grain size exceeding 3.5 μm, with an increased inclusion of gold and uniform arrangement on the surface. The Rku parameter values being around three suggested that the layers primarily exhibited peaks rather than depressions, showing a Gaussian distribution. A chemiresistor was created by using an ink-jet printer and a multilayer metallization was achieved with commercial silver ink for printed electronics. Based on the experimental calibration curve, which exhibits adequate linearity over a wider range of H₂S concentrations in air up to 1 ppm, the detection limit was established at 0.1 ppm, a threshold appropriate for recognizing oral diseases. The sensor is a simple, affordable, and durable device designed for individual use, offering significant benefits for patients by enabling improved tracking of the syndrome’s advancement or treatment success. Full article

Exosomes, extracellular vesicles known for their stability, low immunogenicity, and excellent tissue penetration, are employed as delivery vehicles. These exosomes can traverse the tumor barrier and deliver therapeutic agents directly into pancreatic cancer cells. Targeted exosome vectors containing gene fragments to inhibit Kirsten rat sarcoma viral oncogene homolog (KRAS) activity are crucial for treating pancreatic tumors. Therefore, the content of the exosomes is critical. This study aims to compare the function of exosomes released by HEK-293T cells when exposed to ultraviolet A/B and ultraviolet C irradiation to determine its impact. HEK-293T cells were irradiated with ultraviolet A/B, and ultraviolet C for various indicated times, after which the cell count and exosome secretion were measured. Exosomes derived from HEK-293T cells were isolated through differential centrifugation and identified using four methods: cell counting, Bradford assay, nanoparticle tracking analysis (NTA), and Western blot analysis. Preliminary studies demonstrated that the cell count and Bradford assay expression were reduced in ultraviolet C compared to the control, with similar levels observed for ultraviolet A/B and the control. Exosome expression in Western blot analysis showed ultraviolet C, but a higher amount of ultraviolet A/B compared to the control. We introduce a comprehensive approach to ultraviolet irradiation, including ultraviolet A/B and ultraviolet C, which enhanced the secretion of exosomes by HEK-293T targeted vectors for KRAS inhibition in pancreatic cancer. Full article

Background: Thyroid cancer (TC) is the most prevalent endocrine malignancy, and is categorized into well-differentiated and aggressive anaplastic types. Novel therapeutic modalities are needed for TC. Nanomedicine is a promising strategy for the development of precision medicine. In this context, we investigated the use of nanogels (NGs) to deliver agents with different physicochemical properties, specifically the hydrophilic agent doxorubicin (DOX) and the hydrophobic compound curcumin (CUR), in TC cell lines. Methods: Nα-9-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) peptide-based NGs loaded with DOX and CUR were formulated using the solvent-switch method. DOX-loaded NGs were previously characterized. CUR-loaded NGs were characterized through rheology, scanning electron microscopy (SEM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and Fourier transform infrared (FT-IR) spectroscopy. Confocal microscopy, q-RT-PCR, and ATP lite assays were performed to evaluate the uptake and delivery of DOX- and CUR-loaded NGs on TC cell lines. Results: CUR-loaded NGs exhibited a mean diameter of approximately 204.3 nm and a zeta potential of −34.6 mV, indicative of a good stability. In vitro release studies revealed a sustained release profile of CUR over 72 h. Functional analyses demonstrated that Fmoc-FF-loaded NGs were internalized into TC cell lines. They were primarily localized in the cytoplasm rather than in early endosomes, thereby ensuring intracellular stability. Furthermore, Fmoc-FF NGs reduced the nuclear uptake kinetics of DOX in TC cells, suggesting a potential reduction in dose-limiting toxicity. Comparative studies with CUR-loaded NGs revealed similar internalization and delayed nuclear uptake, highlighting the efficacy of Fmoc-FF NGs in delivering hydrophobic agents. Conclusions: Overall, the data suggest that Fmoc-FF NGs represent a promising strategy for delivering agents with diverse physicochemical properties in TC, enhancing their efficacy and safety and warranting further investigation. Full article

Human milk-derived extracellular vesicles (HMEVs) are key components in breast milk, promoting infant health and development. Maternal conditions could affect HMEV cargo; however, the impact of SARS-CoV-2 infection on HMEVs remains unknown. This study investigated the influence of SARS-CoV-2 infection during pregnancy on postpartum HMEV molecules. The median duration from SARS-CoV-2 test positivity to milk collection was 3 months. After defatting and casein micelle disaggregation, HMEVs were isolated from milk samples of nine mothers with prenatal SARS-CoV-2 and six controls by sequential centrifugation, ultrafiltration, and qEV-size exclusion chromatography. The presence of HMEV was confirmed via transmission electron microscopy. Nanoparticle tracking analysis demonstrated particle diameters of <200 nm and yields of >1 × 10¹¹ particles per mL of milk. Western immunoblots detected ALIX, CD9, and HSP70, supporting the presence of HMEVs in the isolates. Cargo from thousands of HMEVs were analyzed using a multi-omics approach, including proteomics and microRNA sequencing, and predicted that mothers with prenatal SARS-CoV-2 infection produced HMEVs with enhanced functionalities involving metabolic reprogramming, mucosal tissue development, and immunomodulation. Our findings suggest that SARS-CoV-2 infection during pregnancy boosts mucosal site-specific functions of HMEVs, potentially protecting infants against viral infections. Further prospective studies should be pursued to reevaluate the short- and long-term benefits of breastfeeding in the post-COVID era. Full article

Chagas disease remains a significant public health concern, with limited treatment options and an urgent need for novel preventive strategies. Extracellular vesicles (EVs) from Trypanosoma cruzi have been shown to modulate host immune responses, often favoring parasite persistence. In this study, we characterized EVs derived from the non-pathogenic trypanosomatids Trypanosoma rangeli and Phytomonas serpens and evaluated their potential as immunogens capable of inducing cross-protection against T. cruzi infection. Isolated EVs were characterized by Nanoparticle Tracking Analysis (NTA) and electron microscopy. A comparative proteomic analysis of EVs was performed using Mass Spectrometry-Based Proteomic Analysis (LC-MS/MS). The effects of EVs on immunomodulation and T. cruzi infection were assessed through in vitro and in vivo assays, using peripheral blood mononuclear cells (PBMCs) and BALB/c mice. The proteomic analysis identified shared proteins between the EVs of T. rangeli, P. serpens, and T. cruzi, including immunogenic candidates such as calpain-like cysteine peptidase and elongation factor 2. In vitro, pre-stimulation with the T. rangeli EVs reduced infection rates of the host cells by T. cruzi. In vivo, immunization with the EVs from T. rangeli and P. serpens led to a significant reduction in parasitemia in the BALB/c mice challenged with T. cruzi, though this did not translate into improved survival compared to controls. Interestingly, the EVs from T. cruzi also reduced parasitemia but did not confer protection against mortality. These findings suggest that while non-pathogenic trypanosomatid EVs exhibit potential immunogenic properties and can reduce parasitic load, their efficacy in preventing disease progression remains limited. Further research is needed to explore the mechanisms underlying these effects and to optimize EV-based strategies for protective immunity against Chagas disease. Full article

Batch-to-batch reproducibility and robust quality assessment are crucial for producing cell-free biologics, such as conditioned medium (CM) derived from mesenchymal stem/stromal cells (MSCs). This study investigated the effects of freezing CM at −80 °C prior to concentration, a step that could occur in large scale pipelines, compared to freshly processed CM. Quality assessment included total protein quantification; extracellular vesicle evaluation using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and cytofluorimetry; and biochemical analysis using Raman spectroscopy. The freezing process resulted in a 34% reduction in total protein content, as confirmed for selected bioactive mediators, and significant depletion of specific particle types, particularly larger ones. Interestingly, the total particle concentration and polydispersity remained stable. Alterations in Raman spectra highlighted changes in protein, lipid, and nucleic acid content. These findings demonstrate that even routine steps like freezing can alter CM composition, likely due to temperature-induced structural changes in biological molecules. Careful consideration of pre- and intra-processing handling temperatures is critical for preserving the integrity of CM and ensuring consistent quality. This study emphasizes the importance of refining manufacturing protocols in the production of cell-free biologics. Full article

Telocytes (TCs) are distinctive cells widely localized in the stromal compartment of several human organs, including the skin. By means of their peculiar prolongations named telopodes, skin TCs are organized in networks interconnected with a variety of adjacent cells, being thus supposed to take part in skin homeostasis through both cell-to-cell contacts and the release of extracellular vesicles. A disarrangement/loss of the TC network was shown in human fibrotic skin as well as in the murine model of bleomycin-induced cutaneous fibrosis, but whether such TC alterations may represent just a consequence or a trigger of the fibrotic process still remains to be clarified. Thus, we investigated the effects of skin TC secretome as conditioned medium (TC-CM) on the transition of skin fibroblasts into myofibroblasts promoted by the master profibrotic cytokine transforming growth factor β1 (TGFβ1). Primary cultures of both adult human skin TCs and fibroblasts were obtained by means of immunomagnetic cell separation. Nanoparticle tracking analysis was carried out to measure extracellular vesicles in TC-CM. The combination of multiple morphological, gene/protein expression, and functional assessments demonstrated that TC-CM was able to significantly prevent TGFβ1-induced fibroblast-to-myofibroblast transition. TC-CM did not influence cell viability, while it effectively inhibited TGFβ1-induced fibroblast proliferation, migration, and morphological changes. Indeed, TC-CM was able to reduce TGFβ1-mediated skin fibroblast phenotypic and functional differentiation into myofibroblasts, as shown by a significant decrease in FAP, ACTA2, COL1A1, COL1A2, FN1, and CTGF gene expression, α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein levels, and collagen gel matrix contraction. Furthermore, TC-CM significantly lowered TGFβ1-mediated ERK1/2 signaling pathway activation. This in vitro study proves for the first time that TCs may play an important role in skin homeostasis through the prevention of fibroblast-to-myofibroblast transition via paracrine mechanisms and affords the necessary basis to investigate in the future the feasibility of TC secretome as an innovative antifibrotic therapeutic tool. Full article

Extracellular vesicles (EVs) secreted by umbilical cord-derived mesenchymal stem cells (UC-MSCs) hold significant promise as therapeutic agents in regenerative medicine. This study investigates the effects of UC-MSC-derived EVs on dermal fibroblast function, and their potential in wound healing applications. EVs were characterized by nanoparticle tracking analysis and transmission electron microscopy, revealing a mean size of 118.6 nm, consistent with exosomal properties. Dermal fibroblasts were treated with varying concentrations of EVs (25–100 µg/mL), and their impacts on cellular metabolism, mitochondrial activity, reactive oxygen species (ROS) production, wound closure, inflammatory cytokine secretion, growth factor production, and extracellular matrix (ECM) gene expression were evaluated. At lower concentrations (25–50 µg/mL), EVs significantly enhanced fibroblast metabolic and mitochondrial activity. However, higher concentrations (≥75 µg/mL) increased ROS levels, suggesting potential hormetic effects. EVs also modulated inflammation by reducing pro-inflammatory cytokines (IL-6, TNF-α) while promoting pro-regenerative cytokines (IL-33, TGF-β). Treatment with 50 µg/mL of EVs optimally stimulated wound closure and growth factor secretion (VEGF, BDNF, KGF, IGF), and upregulated ECM-related gene expression (type I and III collagen, fibronectin). These findings demonstrate that UC-MSC-derived EVs exert multifaceted effects on dermal fibroblast function, including enhanced cellular energetics, stimulation of cell migration, regulation of inflammation, promotion of growth factor production, and increased ECM synthesis. This study highlights the potential of EVs as a novel therapeutic strategy for wound healing and tissue regeneration, emphasizing the importance of optimizing EV concentration for maximal therapeutic efficacy. Full article

The formation of protein coronas around engineered nanoparticles (ENPs) in biological environments is critical in nanomedicine, as these coronas significantly influence the biological behavior of ENPs. Despite extensive research on protein coronas, understanding the in situ influence of whole (soft plus hard) protein coronas has remained challenging. In this study, we demonstrate a strategy to assess the in situ effects of whole coronas on the model biosensing of anti-IgG using IgG-conjugated gold nanoparticles (IgG-AuNPs) through fluorescence nanoparticle tracking analysis (F-NTA), which enables the selective tracking of fluorescent particles within complex media. In our approach, anti-IgG and IgG-AuNPs were labeled with distinct fluorescent dyes. The accordance in hydrodynamic diameter distributions observed at two different wavelengths verifies the successful capture of anti-IgG on the IgG-AuNPs. The counting of fluorescent anti-IgG within the size distribution allows for a quantitative assessment of biosensing efficiency. This method was applied to evaluate the effects of four protein coronas—human serum albumin, high-density lipoproteins, immunoglobulin G, and fibrinogen—as well as their mixture across varying incubation times and concentrations. The results suggest that the physical presence of whole protein coronas surrounding the IgG-AuNPs may assist the biosensing interaction in situ rather than screening it. Full article

Probiotic bacteria are normal inhabitants of a healthy human gut, conferring multiple beneficial effects on the gut and beyond. Under various disease states, the abundance and diversity of beneficial bacteria are significantly decreased, a process called dysbiosis. Among the intra- and extracellular components of probiotics, the extracellular vesicles (EVs) secreted by them have recently garnered significant attention as potential mediators of probiotics’ effects on host health. Further, these nanosized particles that encapsulate a wide range of bioactive molecules (proteins, lipids, RNA, and DNA) are standing out as key factors that could mediate gut microbiota–host communication and confer ameliorating effects in experimental inflammatory, metabolic, and cardiovascular disease models. However, a standard protocol of EV isolation from probiotic bacteria, not varying from lab to lab, must be established to achieve consistency in the experimental results in these pre-clinical models. Our current study compared two commonly used methods for EV isolation from biological samples, ultracentrifugation and precipitation, to develop a standard protocol for isolating EVs from the probiotics Lactobacillus acidophilus (LA), a Gram-positive bacterium, and Escherichia coli Nissle (EcN), a Gram-negative bacterium. The ultracentrifugation method gave ~1.5-fold higher EV yield for both LA and EcN compared to the precipitation method. Further, EcN released a higher level of EVs compared to LA. EVs were quantified and characterized by nanoparticle-tracking analysis (NTA) and by measuring the specific surface biomarkers using Western blot. Here, we describe our standardized step-by-step protocol for isolating EVs from probiotic bacteria and their characterization. Full article

Cardiovascular diseases and cancer are leading global causes of morbidity and mortality, necessitating advances in diagnosis and treatment. Doxorubicin (Doxo), a potent chemotherapy drug, causes long-term heart damage due to cardiotoxicity. Small extracellular vesicles (sEVs) carry bioactive molecules—such as proteins, lipids, and nucleic acids—that can modulate gene expression and signaling pathways in recipient cells, including cardiomyocytes. Through the delivery of cytokines, microRNAs, and growth factors, sEVs can influence cell survival, which plays a critical role in the development of cardiotoxicity. This study investigates the role of sEVs derived from breast cancer cells treated or not with Doxo and their potential to induce cardiomyocyte damage, thereby contributing to cardiotoxicity. We isolated sEVs from MCF-7 cells treated or not to Doxo using ultracentrifugation and characterized them through Nanoparticle Tracking Analysis (NTA), Scanning Electron Microscopy (SEM), and Western Blotting (WB) for the markers CD63, CD81, and TSG101. We analyzed cytokine profiles using a Multiplex Assay and Cytokine Membrane Array. We exposed Guinea pig cardiomyocytes to different concentrations of sEVs. We assessed their viability (MTT assay), shortening, reactive oxygen species (ROS–DHE dye) production, mitochondrial membrane potential (JC-1 dye), and calcium dynamics (FLUO-4 dye). We performed statistical analyses, including t-tests, ANOVA, Cohen’s d, and η² to validate the robustness of the results. Treatment of MCF-7 cells with 0.01 μM Doxorubicin resulted in increased sEVs production, particularly after 48 h of exposure (~1.79 × 10⁸ ± 2.77 × 10⁷ vs. ~5.1 × 10⁷ ± 1.28 × 10⁷ particles/mL, n = 3, p = 0.0019). These sEVs exhibited protein profiles in the 130–25 kDa range and 93–123 nm sizes. They carried cytokines including TNF-α, IL-1β, IL-4, IFN-γ, and IL-10. Exposure of cardiomyocytes to sEVs (0.025 μg/mL to 2.5 μg/mL) from both Doxo-treated and untreated cells significantly reduced cardiomyocyte viability, shortened cell length by up to 20%, increased ROS production, and disrupted calcium homeostasis and mitochondrial membrane potential, indicating severe cellular stress and cardiotoxicity. These findings suggest that Doxo enhances sEVs production from breast cancer cells, which plays a key role in cardiotoxicity through their cytokine cargo. The study highlights the potential of these sEVs as biomarkers for early cardiotoxicity detection and as therapeutic targets to mitigate cardiovascular risks in chemotherapy patients. Future research should focus on understanding the mechanisms by which Doxorubicin-induced sEVs contribute to cardiotoxicity and exploring their diagnostic and therapeutic potential to improve patient safety and outcomes in cancer therapy. Full article

Measuring molecular dynamics improves understanding of the structure–function relationships of materials. In this study, we present a novel technique for observing material dynamics using transmission electron microscopy (TEM), in which the gold nanoparticles are employed as motion probes for tracing the polymer dynamics in real space. A thin layer of polymer materials was generated on the 2 μm diameter holes of Quantifoil grids, and gold nanoparticles were dispersed on the membrane surface. By tracking the movement of gold nanoparticles from a series of TEM images taken under continuous temperature control, we obtained mean squared displacement (MSD) curves. The dynamics of poly{2-(perfluorooctyl)ethyl acrylate} (PC₈FA) and poly(stearyl acrylate) (PSA) were analyzed. In the temperature-dependent analysis of the MSD, sharp peaks were observed for both PC₈FA and PSA at positions corresponding to their melting and crystallization temperatures. These results demonstrate the capability of TEM to provide valuable insights into the dynamics of polymer materials, highlighting its potential for widespread application in materials sciences. Full article

Background: Traumatic brain injury (TBI) occurs in individuals of all ages, predominantly during sports, accidents, and in active military service members. Chronic consequences of TBI include declined cognitive and motor function, dementia, and emotional distress. Small extracellular vesicles (sEVs), previously referred to as exosomes, are nano-sized lipid vesicles that play a role in intercellular communication. Our prior research established the efficacy of sEVs derived from human adipose stem cells (hASC sEVs) in accelerating the healing of brain injuries. The hASC sEVs are a biologic therapeutic and need to be stored at −20 °C or −80 °C. This limits their use in translating to everyday use in clinics or their inclusion in first-aid kits for application immediately after injury. To address this, here we demonstrate that hASC sEVs can be stored at room temperature (RT) for two months post lyophilization. Methods: A transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA) were used to validate the morphology of lyophilized RT sEVs. Using in vitro models of neuronal injury mimicking physical injury, inflammation, and oxidative stress, we demonstrate that lyophilized RT hASC sEVs are viable and promote the healing of neuronal injuries. Results: The lyophilized sEVs maintain their purity, size, and morphology upon rehydration. Lyophilized, RT stored sEVs showed better efficacy after two months compared with −80 °C stored sEVs. Conclusions: RT storage of lyophilized hASC sEVs maintains their efficacy to accelerate the healing of injuries in neuronal cells. This will advance the use of hASC sEVs, bringing them closer to use in clinics, home first-aid kits, and on battlefields by active service members. Full article

The proteomic analysis of serum extracellular vesicles (EVs) could be a useful tool for studying the pathophysiology of Crohn’s disease (CD) and ulcerative colitis (UC), as well as for biomarker discovery. To characterize the proteomic composition of serum EVs in patients with CD and UC to identify biomarkers and molecular pathways associated with pathogenesis and activity. Methods: Serum EVs were enriched and analyzed in patients with quiescent CD, active CD (aCD), quiescent UC, active UC (aUC), and healthy controls (HCs) (n = 30 per group). All groups were matched for age and sex. Disease activity was assessed by ileocolonoscopy and categorized based on the SES-CD (CD) and the endoscopic sub-score of the Mayo Score (UC). EVs were enriched by ul-tracentrifugation, and their size and concentration were determined by nanoparticle tracking analysis. The expression of CD63, CD81, and CD9 was determined using West-ern blotting. Proteomic analysis was performed by label-free nano-LC MS/MS. A total of 324 proteins were identified; 60 showed differential abundance in CD-HC, 34 in UC-HC, and 21 in CD-UC. Regarding disease activity, the abundance of 58 and 32 proteins was altered in aCD-HC and aUC-HC, respectively. Functional analyses revealed that proteins associated with aCD were involved in immune regulation, whereas those linked to aUC were enriched in oxidative stress. We have identified expressed proteins between EVs from patients with CD and UC, depending on the presence of disease, the disease type, and the disease activity. These proteins are potential candidates as disease biomarkers and open new research avenues to better understand these conditions. Full article

This study investigates the tribological effects of nano-sized metal oxides (ZrO₂, CuO, Y₂O₃ and TiO₂) in Group III type base oil containing 0.3% pour point depressant (PPD) and 5% viscosity modifier (VM) to enhance friction and wear performance. The homogenized lubricant samples with varying concentrations of oxide nanoparticles (0.1–0.5 wt%) on a linear oscillating tribometer performed static and dynamic frictional tests. Optical and confocal microscopy surface analysis evaluated the wear of the specimen, and SEM and EDX analyses characterized the wear tracks, nanoparticle distributions, and quantification. The cooperation between PPD and nanoparticles significantly improved friction and wear values; however, the worn surface suffered extensively from fatigue wear. The collaboration between VM and nanoparticles resulted in a nanoparticle-rich tribofilm on the contact surface, providing excellent wear resistance that protects the component while also favorably impacting friction reduction. This study found CuO reduced wear volume by 85% with PPD and 43% with VM at 0.5 wt%, while ZrO₂ achieved 80% and 63% reductions, respectively. Y₂O₃ reduced wear volume by 82% with PPD, and TiO₂ reduced friction by 20% with VM. These nanoparticles enhanced tribological performance at optimal concentrations, but high concentrations caused tribofilm instability, highlighting the need for precise optimization. Full article