Search Results (348)

Background: Amlodipine besylate (AML) is recognized as a calcium channel blocker curative for hypertension. However, the drug emerged recently as an antibacterial cure that competently prevails over resistant strains. Methods: Incorporating amlodipine into zein nanoparticles was employed to innovate a suitable carrier for loading and targeting deep corneal infection. The Box–Behnken design was adopted to produce various formulations of amlodipine-loaded zein nanoparticles (AML-ZNs) with diversity in composition concentration (% w/v), comprising zein, Labrafac, and poloxamer 407. Results: Relying on the optimization criterion, the chosen preference formulation concentration (% w/v) consists of 2.068 for zein, 0.75 for Labrafac, and 1.0 for Poloxamer. Morphological micrography of AML-ZNs showed regular spherical particles in the nanometric scale, and physicochemical characterization procedures confirmed system suitability. While tracking eyedrop optimum features, sodium alginate was selected for coating nanoparticles to improve stability and system viscosity. Both pH and sterility were also considered and maintained. Comparative studies were conducted pre- and post-coating, and the assessed features for the final selected formulation were 349.9 ± 5.8 nm, 0.2186 ± 0.0271, −55.45 ± 1.84 mV, 81.293 ± 0.9%, and 19.3 ± 0.19 cp for size, PDI, surface charge, entrapment, and viscosity, respectively. The AML-ZNs-Alg formulation demonstrates a more controlled pattern of release of roughly 40% of the drug released after 48 h, while the permeation profile shows 37 ± 3.52% permeated after 24 h, confirmed visually. In vitro microbial assay alongside the corneal in vivo microbial and histological pathology evaluation proved the efficacy of amlodipine as an antibacterial agent. Conclusions: These findings highlighted that the prepared AML-ZNs-Alg eyedrop can be a promising system as an antibacterial therapy. Full article

Glottic insufficiency, often caused by laryngeal nerve injury, impairs voice quality and breathing. Current treatments, such as hyaluronic acid injection, require frequent reapplication every 3–6 months. This study aimed to investigate the therapeutic potential of small extracellular vesicles (sEVs) derived from Wharton’s Jelly mesenchymal stem cells (WJMSCs) incorporated into genipin-crosslinked gelatin hydrogels (GCGHs) for promoting vocal fold fibroblast (VFFs) regeneration in vitro. WJMSCs were isolated from umbilical cords, expanded to passage 4, and used for sEV isolation via tangential flow filtration (TFF). The sEVs (585.89 ± 298.93 µg/mL) were characterized using bicinchoninic acid assay (BCA), nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blot. Seven concentrations of sEVs were tested on VFFs to evaluate cytotoxicity and proliferation, identifying 75 µg/mL as the optimal dose. GCGHs were then combined with WJMSCs and sEVs and evaluated for physicochemical properties, degradation, biocompatibility, and immune response. The hydrogels were injectable within 20 min and degraded in approximately 42 ± 0.72 days. The optimal sEV concentration significantly enhanced VFFs proliferation (166.59% ± 28.11) and cell viability (86.16% ± 8.55, p < 0.05). GCGH-MSCs showed the highest VFFs viability (82.04% ± 10.51) and matrix contraction (85.98% ± 1.25) compared to other groups. All hydrogel variants demonstrated minimal immune response when co-cultured with peripheral blood mononuclear cells (PBMCs). GCGH is a promising scaffold for delivering WJMSCs and sEVs to support VFF regeneration, with demonstrated biocompatibility and regenerative potential. Further in vivo studies are warranted to validate these findings. Full article

This study characterized extracellular vesicles (EVs) isolated from medicinal herb Centella asiatica tissue culture and investigated their therapeutic properties using in vitro assays and a ultraviolet (UV)-induced damage mouse model. EVs were isolated from C. asiatica tissue culture and characterized by nanoparticle tracking analysis, and cytotoxicity, antioxidant, anti-melanin, and anti-inflammation properties were evaluated by in vitro assays. C. asiatica EVs were found to contain high levels of polyphenols and mitigate hydrogen peroxide-induced intracellular reactive oxygen species (ROS). The EVs were further able to reduce intracellular melanin content and tyrosinase activity. They exhibited anti-inflammatory effects by downregulating the expression of pro-inflammatory genes, COX2, as well as nitric oxide production. In the UV-induced photodamage mouse model, gels with or without EVs were applied to the UV-damaged site, skin appearance was observed daily, and skin histopathology was analyzed on day 7. In mice with UV-induced skin damage, the daily application of C. asiatica EV gel reduced skin epidermis thickness and inflammation compared to UV-only or blank gel at seven days after UV irradiation. The beneficial effects of C. asiatica EVs on skin quality warrant further studies as promising agents in skin care applications. Full article

Background/Objectives: Over the past twenty years, there has been a rapid increase in studies aimed at comprehending how cells communicate with each other via Extracellular Vesicles (EVs), accompanied by a heightened interest in plant-derived extracellular vesicles due to their potential relevance in dietary supplementation and therapeutic applications. However, there is a limited amount of research on extracellular vesicles derived from mushrooms (MDEVs). Among edible mushrooms, Pleurotus eryngii is peculiar due to its flavor and interesting nutritional profiling. It also produces a wide array of secondary metabolites including biologically active compounds with many health-promoting benefits such as anticancer, antioxidant, antitumor, antiviral, antibacterial, antidiabetic, and anti-hypercholesteremic activities. The aim of this work has been to isolate EVs from the fruiting body and mycelium of P. eryngii in order to investigate their potential applications as nutraceuticals. Methods: MDEVs were isolated by differential and density gradient centrifugation, characterized by Nanoparticle Tracking Analysis (NTA), Scanning Electron Microscopy (SEM) and immunoblotting, and subjected to metabolomic and phenolic profiling. Their antioxidant potential was assessed through in vitro radical scavenging (DPPH, ABTS) and metal-reducing (CUPRAC, FRAP) assays. Results: The findings suggest that mycelium-derived EVs may represent a valuable source of high-quality MDEVs, which exhibited promising antioxidant properties in all assays conducted, particularly in radical scavenging assays. Conclusions: These results highlight the potential of P. eryngii mycelium-derived EVs as a novel natural source of bioactive compounds, paving the way for future applications in nutraceutical and therapeutic fields. Full article

Nanotheranostics combines therapeutic and diagnostic functions within multifunctional nanoparticle platforms to enable precision medicine. This review outlines a comprehensive framework for engineering nanotheranostic systems, focusing on core material composition, surface functionalization, and stimuli-responsive drug delivery. Targeting strategies—from ligand-based recognition to biomimetic interfaces—are examined alongside therapeutic modalities such as chemotherapy, photothermal and photodynamic therapies, gene silencing via RNA interference, and radio sensitization. We discuss advanced imaging techniques (fluorescence imaging FI), magnetic resonance imaging (MRI), positron emission tomography (PET), and photoacoustic imaging for real-time tracking and treatment guidance. Key considerations include physicochemical characterization (e.g., article size, surface charge, and morphology), biocompatibility, in-vitro efficacy, and in-vivo biodistribution. We also address challenges such as rapid biological clearance, tumor heterogeneity, and clinical translation, and propose future directions for developing safe, adaptable, and effective nanotheranostic platforms. This review serves as a roadmap for advancing next-generation nano systems in biomedical applications. Full article

Time-lapse microscopy of mesenchymal stem cell (MSC) cultures allows for the quantitative observation of their self-renewal, proliferation, and differentiation. However, the rigorous comparison of two conditions, baseline (A) versus perturbation (B) (the addition of molecular factors, environmental shifts, genetic modification, etc.), remains difficult because morphology, division timing, and migratory behavior are highly heterogeneous at the single-cell scale. MSCs can be used as an in vitro model to study cell morphology and kinetics in order to assess the effect of, for example, gene therapy and prime editing in the near future. By combining static, frame-wise morphology with dynamic descriptors, we can obtain weight profiles that highlight which morphological and behavioral dimensions drive divergence. In this study, we present A/B Cells Policy: a modular, open-source Python package implementing a robust cell tracking pipeline. It integrates a YOLO-based architecture as a two-stage assignment framework with fallback and recovery passes, re-identification of lost tracks, and lineage reconstruction. The framework links descriptive statistics to a transferable system, opening up avenues for regenerative medicine, pharmacology, and early translational pipelines. It does this by providing an interpretable, measurement-based bridge between in vitro imaging and in silico intervention strategy planning. Full article

Human mesenchymal stem cells (hMSCs) and their secreted extracellular vesicles (EVs) are promising therapeutics to treat degenerative or inflammatory diseases such as ischemic stroke and Alzheimer’s disease (AD). hMSC-EVs have the coveted ability to contain therapeutically relevant biomaterials; however, EV biogenesis is sensitive to the culture microenvironment in vitro. Recently, the demand for hMSC-EVs has increased dramatically, highlighting the need for scalable bioreactors for large-scale biomanufacturing. In this study, adipose-derived hMSCs were seeded in 2D plates, an ultralow-attachment (ULA) plates as static aggregates, a novel vertical wheel bioreactor (VWBR) as aggregates, and a spinner flask bioreactor (SFB). EV secretion was quantified and compared using ExtraPEG-based ultracentrifugation and nanoparticle tracking analysis. Compared to the 2D group, significantly higher total EV production and cell productivity in the bioreactors were observed, as well as the upregulation of EV biogenesis genes. Furthermore, there was increased EV production in the VWBR compared to the SFB and the static ULA control. Functional assessments demonstrated that EVs, when delivered via culture medium or hydrogel-based systems, significantly attenuated oxidative stress elevation, suppressed proinflammatory cytokine secretion (e.g., TNF-α) and gene expression, and inhibited nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) activation and neurodegenerative markers across in vitro assays. These findings suggest EV-mediated mitigation of oxidative and inflammatory pathways, potentially through modulation of the NF-κB signaling cascade. This study shows the influence of bioreactor types and their microenvironments on EV secretion in hMSCs and their applications in hMSC-EV production and bioengineering. Full article

Inflammatory bowel disease (IBD) is a relapsing–remitting condition resulting in chronic inflammation of the gastrointestinal tract. Present methods are either inadequate or not viable for continuous tracking of disease progression in individuals. In this study, we present the development towards an implantable biosensor for detecting interleukin-6 (IL-6), an important cytokine implicated in IBD. The optimised sensor design includes a gold surface functionalised with a known IL-6-specific aptamer, integrating a recognition sequence and an electrochemical redox probe. The IL-6 aptasensor demonstrated a sensitivity of up to 40% and selectivity up to 10% to the IL-6 target in vitro. Sensors were found to degrade over 7 days when exposed to recombinant IL-6, with the degradation rate rapidly increasing when exposed to intestinal mucosa. A feasibility in vivo experiment with a newly designed implantable gut sensor array confirmed rapid degradation over a 5-h implantation period. We achieved up to a 93% reduction in sensor degradation rates, with a polyvinyl alcohol–methyl acrylate hydrogel coating that aimed to reduce nonspecific interactions in complex analytes compared to uncoated sensors. Degradation was linked to desorption of the monolayer leading to breakage of gold thiol bonds. While there are key challenges to be resolved before a stable implantable IBD sensor is realised, this work highlights the potential of aptamer-based biosensors as effective tools for long-term diagnostic monitoring in IBD. Full article

Cystic fibrosis is a multisystem disorder caused by mutations in the CFTR gene that lead to impaired ion and fluid transport across secretory epithelia. Although the therapeutic impact of CFTR modulators has been extensively studied in airway epithelia, their efficacy in extra-pulmonary tissues, such as the pancreas, has been less explored. This study evaluated the effects of the CFTR modulators, VX770 (ivacaftor), VX661 (tezacaftor), and VX445 (elexacaftor), administered either individually or in combination, on CFPAC-1 cells, a pancreatic ductal epithelial cell line derived from a cystic fibrosis patient harboring the F508del CFTR mutation. The cells were cultured and differentiated onto porous supports, and a panel of functional parameters was assessed. These included transepithelial electrical conductance, fluid reabsorption, apical surface fluid pH, protein concentration, and microviscosity, the latter analyzed with multiple particle tracking. To simulate a pro-inflammatory micro-environment, the cells were preconditioned with lipopolysaccharide (LPS). Treatment with VX661 and VX445 resulted in significant improvement in epithelial function, with the triple combination producing the most pronounced rescue. Pro-inflammatory stimulation by LPS increased the production of cytokine IL6, IL-8, and IL-1β, as well as the protein content of the apical surface fluid. Despite the LPS pro-inflammatory stimulus, CFTR modulators preserved or slightly enhanced their efficacy in restoring CFTR-mediated ion and fluid transport. However, they did not reduce cytokine expression under pro-inflammatory conditions. Collectively, these findings show that CFTR modulators can restore critical aspects of cystic fibrosis pancreatic epithelial physiology in vitro, even under pro-inflammatory stress, supporting their potential relevance beyond the airway disease. Full article

Biological extracellular vesicles in tear fluids, such as exosomes, are thought to have physiological functions in the management of healthy ocular surface epithelium, including corneal epithelium. However, the physiological roles of tear extracellular vesicles in the ocular surface remain unclear. In this study, we investigated the physiological function of tear extracellular vesicles in mouse tear fluids in the ocular surface epithelium in vitro. Morphological analysis of the isolated extracellular vesicles from mouse tear fluids was performed using nanoparticle tracking analysis and transmission electron microscopy. The identified particles were characterised by immunoblotting for exosomal markers. After confirming the uptake of tear exosomes in cultured corneal epithelial cells, gene expression changes in mouse cultured corneal epithelial cells after tear exosome treatment were analysed. Immunostaining analysis was performed to confirm cell proliferation in the cultured corneal epithelial cells with tear exosome treatment. Tear fluids from mice contain nanoparticles with exosome-like morphologies, which express the representative exosomal markers CD9 and TSG101. The extracellular vesicles can be taken up by cultivated murine corneal epithelial cells in vitro and induce expression changes in genes related to the cell cycle, cell membranes, microtubules, and signal peptides. Treatment with the tear extracellular vesicles promoted cell proliferation of cultured murine corneal epithelial cells. Our study provides evidence that murine tear fluids contain extracellular vehicles like exosomes and they may contribute to the maintenance of the physiological homeostatic environment of the ocular surface. Full article

Extracellular vesicles (EVs), nanoscale membrane-enclosed particles, are natural carriers of proteins and nucleic acids. Microalgae are widely used as a source of bioactive substances in the food and cosmetic industries and definitely have a potential to be used as the producers of EVs for biomedical applications. In this study, the extracellular vesicles isolated from the culture medium of two unicellular microalgae, Chlamydomonas reinhardtii (Chlamy-EVs) and Parachlorella kessleri (Chlore-EVs), were characterized by atomic force microscopy (AFM), cryo-electronic microscopy (cryo-EM), and nanoparticle tracking analysis (NTA). The biocompatibility with human cells in vitro (HEK-293T, DF-2 and A172) and biodistribution in mouse organs and tissues in vivo were tested for both microalgal EVs. An exogenous therapeutic protein, human heat shock protein 70 (HSP70), was successfully loaded to Chlamy- and Chlore-EVs, and its efficient delivery to human glioma and colon carcinoma cell lines has been confirmed. Additionally, in order to search for potential therapeutic biomolecules within the EVs, their proteomes have been characterized. A total of 105 proteins were identified for Chlamy-EVs and 33 for Chlore-EVs. The presence of superoxide dismutase and catalase in the Chlamy-EV constituents allows for considering them as antioxidant agents. The effective delivery of exogenous cargo to human cells and the possibility of the particle yield optimization by varying the microalgae growth conditions make them favorable producers of EVs for biotechnology and biomedical application. Full article

Modified oligonucleotides (oligos) are widely used as convenient tools in many scientific fields, including biomedical applications and therapies. In particular, oligos with lipophilic groups attached to the backbone ensure penetration of the cell membrane without the need for transfection. This study examines the interaction between amphiphilic DNA duplexes, in which one of the chains contains a lipophilic substituent, and several DNA repair proteins, particularly DNA-damage-dependent PARPs, using various biochemical approaches. DNA with a lipophilic substituent (LS-DNA) demonstrates more efficient binding with DNA damage activated poly(AD-ribose) polymerases 1-3 (PARP1, PARP2, PARP3) and DNA polymerase β. Chemically reactive LS-DNA derivatives containing a photoactivatable nucleotide (photo-LS-DNAs) or a 5′ deoxyribose phosphate (dRP) group in the vicinity of double-strand breaks (DSBs) are used for the affinity labelling of PARPs and other proteins in several whole-cell extracts of human cells. In particular, photo-LS-DNAs are used to track the level of Ku antigen in the extracts of neuron-like differentiated SH-SY5Y, undifferentiated SH-SY5Y, and olfactory epithelial cells. In vitro, PARP1–PARP3 are shown to be able to slowly excise the 5′ dRP group at DSBs. LS-DNAs can activate PARP1 and PARP2 for autoPARylation, albeit less effectively than regular DNA duplexes. Full article

Despite remarkable progress in cancer immunotherapy, many agents that show efficacy in murine or in vitro models fail to translate clinically. Zebrafish (Danio rerio) have emerged as a powerful complementary model that addresses several limitations of traditional systems. Their optical transparency, genetic tractability, and conserved immune and oncogenic signaling pathways enable high-resolution, real-time imaging of tumor–immune interactions in vivo. Importantly, zebrafish offer a unique opportunity to study the core mechanisms of health and sickness, complementing other models and expanding our understanding of fundamental processes in vivo. This review provides an overview of zebrafish immune system development, highlighting tools for tracking innate and adaptive responses. We discuss their application in modeling immune evasion, checkpoint molecule expression, and tumor microenvironment dynamics using transgenic and xenograft approaches. Platforms for high-throughput drug screening and personalized therapy assessment using patient-derived xenografts (“zAvatars”) are evaluated, alongside limitations, such as temperature sensitivity, immature adaptive immunity in larvae, and interspecies differences in immune responses, tumor complexity, and pharmacokinetics. Emerging frontiers include humanized zebrafish, testing of next-generation immunotherapies, such as CAR T/CAR NK and novel checkpoint inhibitors (LAG-3, TIM-3, and TIGIT). We conclude by outlining the key challenges and future opportunities for integrating zebrafish into the immuno-oncology pipeline to accelerate clinical translation. Full article

Tracking T cells in vivo using MRI is a major challenge due to the difficulty of labeling these non-phagocytic cells with a sufficient contrast agent to generate a detectable signal change. In this study, we explored CD4-Superparamagnetic iron oxide nanoparticles (SPION), which is commonly used in magnetic cell sorting, as a potential receptor-mediated, specific CD4⁺ T cell MRI labeling agent. We optimized the labeling protocol for maximal CD4⁺ cell labeling and viability. Cell health was confirmed with trypan blue assay, and labeling efficacy was confirmed with Prussian blue staining, transmission electron microscopy, and MRI of labeled cell pellets. Key cell functionality was assessed by flow cytometry. Next, CD4-SPION-labeled T cells or unlabeled T cells were delivered via intravenous injection in naïve mice. Liver MRIs pre-, 24 h, and 72 h post-T cell injection were performed to determine in vivo tracking ability. Our results show that CD4-SPION induces significant attenuation of T2 signals in a concentration-dependent manner, confirming their potential as an effective MRI contrast agent. In vitro, analyses showed that CD4⁺ T cells were able to uptake CD4-SPION without affecting cellular activity and key functions, as evidenced by Prussian blue staining and flow cytometric analysis of IL-2 receptor and the IL-7 receptor α-chains, CD69 upregulation, and IFN-γ secretion. In vivo, systemically distributed CD4-SPION-labeled T cells could be tracked in the liver at 24 and 72 h after injection, contrary to controls. Histological staining of tissue sections validated the findings. Our results showed that SPION CD4⁺ T cell sorting coupled with longitudinal MR imaging is a valid method to track CD4⁺ T cells in vivo. This safe, specific, and sensitive approach will facilitate the use of SPION as an MRI contrast agent in clinical practice, allowing for non-invasive tracking of adoptive cell therapies in multiple disease conditions. Full article

Unlike other orchids in the Orchidaceae family, Cymbidium goeringii presents significant challenges for in vitro flowering. In this study, through the screening of different basal media, hormone combinations, and other conditions, we developed efficient rhizome regeneration (micropropagation) and in vitro flowering induction systems from protocorm explants of C. goeringii hybrids. To obtain protocorm explants, seeds were pretreated with either NaOH or NaOCl. Our results indicated that NaOH pretreatment enhanced seed germination more effectively than NaOCl, and Knudson C medium proved more suitable for protocorm induction. The resulting protocorms were then used as primary explants for efficient rhizome micropropagation. An orthogonal design identified the optimal combination for rhizome proliferation: 9.0 mg/L 6-BA, 9.0 mg/L NAA, 3.0 mg/L IBA, and 0.1 g/L activated charcoal (Treatment 9), which achieved a proliferation rate of 35.17%. For rhizome differentiation, MS medium supplemented with 10 mg/L 6-BA, 0.1 mg/L NAA, and 0.1 mg/L AgNO₃ (Treatment 6) achieved a 100% differentiation rate and produced 3.93 buds per explant. Building on this optimized micropropagation system, in vitro flowering was induced directly from rhizomes. The most effective medium was MS (1/3N, 3P) supplemented with 9.0 mg/L 6-BA, 0.1 mg/L NAA, and 0.1–0.3 mg/L TDZ (Treatment 6), resulting in a 36% flower bud induction rate and a 16% normal flower bud formation rate. Orthogonal analysis and ANOVA confirmed that 6-BA was the most significant factor influencing floral transition, with the low-nitrogen and high-phosphorus MS (1/3N, 3P) medium also being a key contributor. Consequently, our study has established an efficient rhizome micropropagation system that enables in vitro flowering induction in C. goeringii hybrids within just six months. This represents a substantial 60–80% reduction in the flowering time (from 6–7 years to 1–2 years), compared to the traditional 6–7-year cultivation period. Future work will focus on ex vitro acclimatization, detailed floral-trait validation, and hormone-regime refinement for fast-tracking breeding programs. Full article