BACKGROUND:Blood-based biomarkers would greatly facilitate the clinical diagnosis of Alzheimer's disease (AD) as minimally invasive measurements. While several blood biomarkers for AD have emerged, the potential of plasma β-amyloid (Aβ) aggregation seeding activity remains underexplored. In this study, we aim to evaluate the ability of this biomarker to distinguish AD and mild cognitive impairment (MCI) due to AD from cognitively unimpaired (CU) individuals and non-AD dementia. METHODS:A total of 549 participants were recruited from Xuanwu Hospital, Capital Medical University, between December 2020 and May 2024. Plasma Aβ aggregation seeding activity was measured using a real-time sonication-based protein misfolding cyclic amplification assay across discovery (n = 120: 30 CU, 30 MCI due to AD, 30 AD, 30 non-AD dementia) and validation (n = 429: 118 CU, 46 MCI due to AD, 141 AD, 124 non-AD dementia) stages. The diagnostic performance of plasma Aβ aggregation seeding activity as a biomarker was assessed using receiver operating characteristic (ROC) curves. RESULTS:In the validation stage, plasma Aβ aggregation seeding activity exhibited high diagnostic accuracy with optimal cutoff values (Thioflavin T fluorescence %) of 42.91 for distinguishing AD from CU (area under the ROC curve [AUC] = 0.93, 95% confidence interval [CI]: 0.91-0.96), 42.02 for AD from non-AD dementia (AUC = 0.91, 95% CI: 0.88-0.94), 43.17 for MCI due to AD from CU (AUC = 0.92, 95% CI: 0.87-0.96), and 43.24 for MCI due to AD from non-AD dementia (AUC = 0.90, 95% CI: 0.85-0.94). Plasma Aβ seeding activity significantly correlated with cognitive functions (Mini-Mental State Examination scores [MMSE]: rs = -0.68, P <0.001; Clinical Dementia Rating scores [CDR]: rs = 0.71, P <0.001). CONCLUSIONS:These findings indicate that plasma Aβ aggregation seeding activity could serve as a promising minimally invasive biomarker for identifying both AD and MCI due to AD. This biomarker potentially facilitates early detection and differential diagnosis of AD at different clinical stages.

While an oral octreotide formulation (Mycapssa®) utilizing transient permeability enhancement has recently been FDA-approved, its clinical utility is often constrained by strict fasting requirements and reliance on the transient disruption of intestinal tight junctions. To develop a more biomimetic and efficient alternative, we developed the first oral formulation of Octreotide (OCT) using donkey-milk exosomes (DME). These naturally derived vesicles are enriched with membrane-fusion and transcytosis proteins that protect biologics from gastric degradation and facilitate endogenous epithelial transport. Exosomes were isolated from donkey milk powder, characterized by nanoparticle tracking analysis, western blotting, and proteomics, and found to possess a mean size of 138.4 ± 4.37 nm, and zeta potential of -42.07 ± 0.99 mV, and abundant transport-associated proteins including PIGR, MFGE8, ANXA2, CD9, CD63, and CD81. OCT was encapsulated using a pH-gradient method, achieving 7.25% entrapment efficiency. Analytical techniques including ATR-FTIR and DSC reveals the characteristic functional groups and thermal transitions of the components, confirming that octreotide was effectively integrated into the DME formulation. In-vitro dissolution studies demonstrated protective release behavior, while MDCK monolayer assays revealed a 37-fold enhancement in permeability compared to free OCT. Pharmacokinetic performance was predicted by GastroPlus™, which projected a rise in fraction absorbed from 8.5% to 88.2%, a reduction in Tmax from 5.5 to 2.2 h, and a four-fold increase in AUC. These predictions were confirmed in-vivo, where oral administration of OCT-exosomes (2 mg/kg) in BALB/c mice achieved a 16-fold increase in systemic exposure (AUC0-24: 435,200 pg·h/mL) compared with free OCT, alongside targeted biodistribution to the liver, spleen, kidney, and intestine. This study provides the first proof-of-concept that donkey-milk exosomes can enable effective oral delivery of octreotide. By combining stable encapsulation, epithelial transport facilitation, predictive modeling, and in-vivo validation, this platform offers a scalable and broadly applicable strategy for transforming injectable peptides and biologics into oral therapeutics.

Defective clearance of phagocytosed DNA contributes to inflammation, yet the molecular factors governing DNA degradation within phagosomes remain unclear. Here, we present a materials-based platform using engineered microparticles to dissect how DNA is processed inside macrophage phagosomes. Using microcontact printing, we fabricated two classes of DNA-containing microparticles: thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) microspheres encapsulating intercalator-labeled DNA and chromatin-mimetic particles composed of multilayered histone-DNA assemblies with tunable cross-linking. These structures provide precise control over DNA accessibility, protein association, and degradability. Upon phagocytosis by macrophages, DNA embedded within hydrated PNIPAM networks remained intact, indicating restricted diffusion of phagosomal enzymes. In contrast, DNA electrostatically complexed with histone was efficiently degraded but only after proteolytic removal of the histone barrier. When histone was chemically cross-linked, DNA degradation was inhibited. These results demonstrate that proteolysis of DNA-bound proteins is a critical prerequisite for DNase II-mediated cleavage in macrophage phagosomes. This modular microparticle platform offers a reductionist approach for probing the biochemical and physical determinants of DNA degradation within phagocytes and enables a systematic investigation of how protein-DNA interactions, cross-linking, or pathological stabilization of chromatin-like structures influences intracellular DNA persistence and inflammatory signaling.

The blood-brain barrier (BBB) is a mechanically responsive interface that protects the central nervous system. Brain tissue exhibits region-specific stiffness that evolves throughout development and is altered in aging and various neurological diseases. These stiffness changes are increasingly recognized as key modulators of endothelial cell behavior and BBB integrity. However, the mechanisms by which brain endothelial cells sense and adapt to variations in their mechanical microenvironment remain poorly defined. Moreover, how mechanical cues interact with cellular signals from astrocytes and pericytes to modulate endothelial mechanics and junctional organization has been largely unexplored. Here, we demonstrate spatial regulation of subcellular mechanics in human iPSC-derived brain microvascular endothelial cells (iBMECs) in response to physiologically and pathologically relevant substrate stiffness (1-194 kPa). Using atomic force microscopy, we quantified Young's modulus at three distinct cellular regions-tricellular junctions, bicellular junctions, and cell bodies. iBMECs cultured on compliant substrates (1, 2.5, and 15 kPa) exhibited pronounced mechanical polarization, characterized by significantly elevated stiffness at tricellular regions compared with bicellular regions and cell bodies. This spatial organization was lost on supraphysiological stiffness (194 kPa), which reduced overall cell stiffness and eliminated regional distinctions. Co-culture with astrocytes and pericytes decreased global stiffness but preserved the dominant reinforcement at tricellular regions. In contrast, exposure to metastatic breast cancer cells abolished junction polarization at tricellular regions and suppressed stiffness across all regions, particularly on soft substrates. These findings reveal that BBB endothelial mechanics are regulated by both matrix stiffness and BBB cell context in a region-specific manner. This work provides new insight into how physical and cellular cues shape BBB structure and function, with implications for understanding barrier disruption in neurological disease and metastasis.

Cerebral ischemic stroke, caused by interrupted cerebral blood flow, remains a leading cause of mortality and long-term disability worldwide. Current FDA-approved therapies-intravenous tissue-type plasminogen activator (tPA) and mechanical thrombectomy-are constrained by narrow time windows (4.5-24 h) and limited accessibility. Mesenchymal stem cells (MSCs) have emerged as promising candidates for neurorestoration, yet their therapeutic efficacy is hindered by poor blood-brain barrier (BBB) penetration and systemic entrapment. Increasing evidence indicates that MSCs exert their therapeutic effects primarily through paracrine mechanisms mediated by extracellular vesicles (EVs), which regulate inflammation, apoptosis, neurogenesis, and angiogenesis. However, translation of EV-based therapies from bench to bedside remains limited, largely due to inefficient delivery and the invasiveness of existing routes. Intranasal (IN) administration offers a minimally invasive approach to bypass the BBB and achieve direct, repeated delivery to the brain. This review synthesizes the mechanistic foundations, preclinical progress, and translational potential of intranasal delivery of MSCs and their EVs for ischemic stroke therapy. We highlight comparative analyses of biodistribution, cellular targets, and functional outcomes across administration routes, emphasizing how route optimization governs therapeutic efficacy. Collectively, these insights establish intranasal delivery as a practical platform for next-generation, cellfree regenerative therapies targeting ischemic brain injury. Statement of Significance: Despite extensive investigation of stem-cell-based interventions for ischemic stroke, the influence of administration route on therapeutic outcomes remains poorly defined. This review integrates preclinical and early-phase clinical findings to delineate how delivery pathways shape biodistribution, mechanistic engagement, and neurorepair efficacy of human mesenchymal stem cells (hMSCs) and their derived extracellular vesicles (EVs). By contrasting conventional intravenous and intra-arterial approaches with the emerging intranasal route, this article emphasizes a non-invasive strategy capable of bypassing the blood-brain barrier, supporting multidose regimens, and sustaining localized repair. Beyond summarizing outcomes, this work clarifies mechanistic drivers-angiogenesis, neurogenesis, and immunomodulation-that can be fine-tuned through delivery design. The synthesis provides a framework for rationally optimizing cell-free hMSC-EV therapeutics and underscores the translational promise of intranasal delivery for clinical stroke management.

Human induced pluripotent stem cells (hiPSCs) can differentiate into various types of central nervous system organoids which are valuable for applications in tissue engineering and injury repair. The secreted extracellular vesicles (EVs) of organoids, in particular the small-sized EV subset referred as exosomes (30-200 nm), have emerged as novel therapeutics in regenerative medicine. This study investigated the encapsulation and controlled release of human spinal cord organoid (hSCO)-derived EVs in viscoelastic hyaluronic acid (HA) hydrogels and assessed their impact on organoid patterning. A series of pH-responsive hydrogels were fabricated, leading to sustained EV release regulated by viscoelastic properties. The pH of these hydrogels decreased from 9 to 7 during incubation, which altered hydrogel viscoelasticity, thereby modulating EV release kinetics. In addition, EV-loaded hydrogels regulated key hSCO patterning markers such as DBX1 and ISL1. Furthermore, these EVs in hydrogels can cross a modeled blood-spinal cord barrier and provide cross-barrier capability for delivery. Taken together, the organoid-secreted EVs in viscoelastic HA hydrogels can be released at a controlled rate and have potential to regulate spinal cord organoid patterning. This study advances our knowledge of regulating intercellular communication and developing EV-based therapies for treating neurological disorders such as spinal cord injury.

RATIONALE:Ischemic stroke is sexually dimorphic. Biological sex can influence injury progression and response to treatment. Extracellular vesicles (EV) derived from three-dimensional (3D) human mesenchymal stem cell aggregates (3D-EV) are a promising candidate as a treatment, but their efficacy across these biological variables and in vivo behavior needs to be characterized. This study evaluated whether 3D-EV therapy enhances recovery following ischemic stroke in female and male models using ultra-high-field MRI and their influence on structural, ionic, and metabolic recovery. METHODS:A preclinical model of transient middle cerebral artery occlusion was used to longitudinally evaluate the efficacy of ultrasmall superparamagnetic iron oxide (USPIO)-labeled 3D-EV or saline at reperfusion through intra-arterial injection. MRI was performed at 21.1 T, which included T2-weighted, diffusion-weighted imaging, gradient-recalled echo imaging, and ²³Na chemical shift imaging. Proton magnetic resonance spectroscopy (¹H-MRS) was used to quantify changes in lactate, N-acetylaspartate, creatine, and choline within peri-infarct tissue. Imaging and behavioral outcomes were assessed over 21 days. RESULTS:USPIO-labeled 3D-EV resulted in localized hypointense contrast in the ischemic striatum, indicating delivery of treatment. T2-weighted MRI showed progressive lesion reduction, with a trend toward better recovery in females. ²³Na MRI revealed reduced sodium accumulation, with earlier ionic normalization in 3D-EV-treated animals. Diffusion recovery was observed with sex-dependent trajectories. ¹H-MRS showed lower lactate concentrations and preservation of other metabolites in EV-treated females. Behavioral differences were not significant. CONCLUSIONS:3D-EV therapy showed trends toward structural, ionic, and metabolic recovery following an ischemic insult. Ultra-high-field MRI and MRS can provide sensitive biomarkers to resolve these differences and support 3D-EV as a potential cell-free therapeutic candidate for ischemic stroke.

Peripheral neuropathy (PN) is a debilitating condition characterized by chronic pain, numbness, and motor dysfunction, with limited treatment options. Ischemic stroke can cause central neuropathy, which may also induce PN. Human mesenchymal stem cells (hMSCs) have shown promise in therapeutic applications, but limitations in cell viability, immune response, and efficacy persist. Extracellular vesicles (EVs), which facilitate cell-free intercellular communication, offer a promising alternative for nerve regeneration. Electrical stimulation (ES) has emerged as a method to enhance EV secretion, and this study investigates its potential for promoting EV production from human adipose tissue-derived mesenchymal stem cells (hASCs) and human Schwann cells (hSCs). In this study, hASCs, hSCs, and lipopolysaccharide (LPS)-induced inflamed hSCs were subjected to one hour of low-frequency direct current (DC) electrical stimulation (100 mV/mL) for 7 days. EVs were isolated using differential ultracentrifugation and characterized through nanoparticle tracking analysis (NTA). Gene expression was analyzed via qRT-PCR to evaluate markers associated with EV biogenesis as well as pro- and anti-inflammatory cytokines. Our results demonstrate that ES significantly increases EV secretion from both hASCs and hSCs, with a notable upregulation of genes involved in both the endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways of EV biogenesis. Additionally, ES modulates inflammation-related markers, promoting anti-inflammatory gene expression and reducing pro-inflammatory gene levels. Notably, LPS-induced hSCs exhibited a phenotype shift from myelinating to non-myelinating cells, producing EVs capable of modulating the inflammatory microenvironment. However, prolonged exposure to ES led to a decrease in EV secretion and changes in EV size distribution, suggesting potential cellular adaptation or membrane stress. This study highlights the potential of ES as a scalable, cell-free strategy to enhance EV production, offering new insights into its therapeutic applications for peripheral neuropathy and nerve regeneration.

IntroductionHuman mesenchymal stem/stromal cells (hMSCs) hold significant regenerative potential due to their anti-inflammatory and pro-angiogenic secretome. Three-dimensional (3D) hMSC aggregates secrete extracellular vesicles (EVs) with enhanced immunomodulatory properties compared to 2D cultures. However, the clinical translation of hMSC-EVs remains limited by low production yield. This study investigates scalable EV generation from 3D hMSC aggregates in a novel Vertical-Wheel Bioreactor (VWBR), leveraging shear stress-mediated biochemical cues to enhance EV biogenesis and cargo relevant to nerve regeneration.MethodsBone marrow-derived hMSCs were cultured as 3D aggregates in VWBRs and exposed to two different culture media—αMEM/FBS (serum-containing) and DMEM/F12/B27 (serum-free)—under three agitation speeds (25, 40, and 64 rpm). Metabolite analysis and qRT-PCR were performed to assess metabolic activity and EV biogenesis, focusing on ESCRT machinery markers. EVs were isolated and evaluated for yield, size, markers, and microRNA cargo. Functional assays were conducted to measure the effects on EVs on Schwann cells under LPS-induced neural inflammation.ResultsVWBR culture resulted in increased expression of EV biogenesis genes and glycolytic pathway markers compared to static culture. The αMEM/FBS (serum-containing) condition was more robust than DMEM/F12/B27 (serum-free) condition. EV yield (EV number per cell) increased by 3-10 fold (in serum-containing medium) in VWBR compared to static culture, with particle sizes ranging from 120-180 nm and appropriate EV marker expression. microRNA-sequencing showed upregulation of miR-29a-3p, miR-451a, miR-224-5p, miR-16-5p, miR-133a-3p, and miR-143-3p, indicating enhanced EV biogenesis, metabolic reprogramming, and immunomodulatory potential. Functionally, VWBR-derived EVs modulated inflammatory gene expression in Schwann cells exposed to LPS.DiscussionVWBR-driven hydrodynamics promotes EV biogenesis from 3D hMSC aggregates, improving metabolic activity, EV cargo relevance, and functional efficacy. The resulting EVs exhibit therapeutic cargo capable of modulating neural inflammation. These findings advance understanding of dynamic aggregation on metabolic cues and EV production, demonstrating a scalable strategy for generating therapeutically potent hMSC-EVs for neuropathic and regenerative applications.

Therapeutic nanoparticle delivery is crucial for a variety of biomedical applications, such as immunizations, gene and drug delivery, tissue engineering, biomedical implant coating, and other regenerative medicine approaches. The uptake of therapeutic nanoparticles occurs through several endocytosis pathways. However, the uptake mechanism of nanoparticles delivered from a substrate is also regulated by cell-substrate interactions and the ability of cells to mechanosense their surrounding extracellular matrix (ECM). These cell-ECM interactions influence integrin signaling, focal adhesion formation, and cytoskeletal rearrangement to impact nanoparticle uptake. In this study, we investigated the role of ECM and ECM-mimetic coatings─collagen I (COL), fibronectin (FN), laminin (LM), hyaluronic acid (HA), and poly-l-lysine (PLL)─on the uptake of poly(lactic-co-glycolic acid) (PLGA) nanoparticles across three distinct cell types: NIH3T3 fibroblasts, primary rat adipose-derived stem cells (ASCs), and RAW264.7 macrophages, which displayed varying levels of integrin-based focal adhesion formation. Using a quartz crystal microbalance with dissipation (QCM-D) and ellipsometry, we thoroughly characterized ECM coatings, showing variations in coating thickness and mechanical properties. FN and COL coatings significantly enhanced cell proliferation, spreading, and focal adhesion formation, correlating with the highest levels of nanoparticle uptake at longer time points. In contrast, HA and LM coatings resulted in reduced cell adhesion and uptake. Consistent with this, cell types with more mature focal adhesions (ASCs, NIH3T3) showed much higher particle uptake in comparison to cells with limited focal adhesion formation (RAW264.7). Live-cell imaging demonstrated dynamic differences in uptake kinetics with LM coatings showing rapid early uptake, while FN and COL promoted sustained uptake over longer durations. Uptake studies using cytochalasin-D revealed that nanoparticle uptake was highly dependent on the actin cytoskeleton, suggesting the involvement of actin-dependent endocytic pathways. Overall, our findings highlight that ECM-dependent regulation of integrin-based adhesions and cytoskeletal organization modulates nanoparticle uptake in a coating- and cell type-dependent manner. These insights provide a foundation for optimizing substrate-based nanoparticle delivery platforms in regenerative medicine and therapeutic applications.

Although distinguished for their differentiation capacity, human-induced pluripotent stem cells (iPSCs)-derived extracellular vesicles (EVs) have been shown to contribute to functional recovery in the treatment of various traumatic and degenerative diseases. This promising role in therapeutic applications has resulted in considerable attention aimed toward their effective bio-manufacturing. However, traditional culture systems face various insufficiencies. Planar 2D culture results in a lack of scalability, with difficulty in manufacturing clinically relevant doses. Additionally, planar 2D culture lacks the complexity of in vivo biological systems. Although organoids have been proposed to fit this gap by better mimicking in vivo conditions, the traditional generation method of using static culture results in inefficient nutrient and waste transfer. Earlier bioreactor systems, which aim to resolve these issues, also face limitations of homogeneity and stress. Thus, vertical wheel bioreactors (VWBRs) with low shear stress profiles have recently emerged for stem cell organoid cultures, resulting in a more efficient and true-to-form manufacturing process for the secreted EVs. In this chapter, we describe an approach to generate and quantify EVs secreted by iPSC-differentiated human blood vessel organoids (iBVOs) grown in a scalable VWBR. iPSCs are expanded and then differentiated into iBVOs with differentiation media in VWBRs. Their produced EVs are subsequently isolated from the media and quantified using nanoparticle tracking analysis. This culture system should be able to produce a large quantity of the iBVO-derived EVs for the subsequent preclinical and clinical applications.

Organoids offer a promising alternative in biomedical research and clinical medicine, with better feature recapitulation than 2D cultures. They also have more consistent responses with clinical results when compared to animal models. However, major challenges exist in the longevity of culture, the reproducibility of organoid properties, and the development of non-disruptive monitoring methods. Recent advances in materials and microfabrication methods, such as 3D printing and compressive buckling, have enabled three-dimensional (3D) interfaces of microfluidics and bioelectronics to manipulate and monitor these biological models in exciting ways. These advanced systems have great potential for applications in drug delivery, personalized medicine, and disease modelling. We conclude with important future considerations to generate longevity using further technological development in organoid and spheroid models.

Background: Choroid plexus is a complex structure in the human brain that is responsible for the secretion of extracellular vesicles (EVs) in cerebrospinal fluid. Few studies to date have generated choroid plexus (ChP) organoids differentiated from human induced pluripotent stem cells (hiPSCs) and analyzed their secreted EVs. The scalable Vertical-Wheel bioreactors (VWBRs) provide low shear stress and a controlled environment. Methods: This study utilized VWBRs for the differentiation of hiPSCs into ChP organoids and generation of the secreted EVs compared to a static culture. Additionally, this study loaded curcumin into ChP organoid-derived EVs, performed EV lyophilization, and determined the ability of the re-hydrated EVs to alleviate neuro-inflammation. Results: The results demonstrated that the VWBR culture exhibited more aerobic metabolism and active glucose and glutamine consumption than the static control. Consequently, the ChP markers and Endosomal Sorting Complexes Required for Transport-dependent and -independent EV biogenesis genes were significantly upregulated (2–3-fold) in the VWBR, producing four-fold-higher EVs per mL media than the static control. The EVs retained similar size and zeta potential after lyophilization and re-hydration. The cells exposed to amyloid beta 42 oligomers and treated with the curcumin-loaded re-hydrated EVs showed high viability and the reduced inflammatory response determined by TNF-α and IL-6 expression. Conclusions: This study demonstrates a scalable bioreactor system to promote ChP organoid differentiation and generation of EV-based cell-free therapeutics to treat neural inflammation in various neurological disorders.

Skin‐like robust materials with prominent sensing performance have potential applications in flexible bioelectronics. However, it remains challenging to achieve mutually exclusive properties simultaneously including low interfacial impedance, high stretchability, sensitivity, and electrical resilience. Herein, a material and structure design concept of mixed ion‐electron conduction and mechanical interlocking structure is adopted to fabricate high‐performance mechanical‐bioelectrical dual‐modal composites with large stretchability, excellent mechanoelectrical stability, low interfacial impedance, and good biocompatibility. Flower‐like conductive metal‐organic frameworks (cMOFs) with enhanced conductivity through the overlapped level of metal‐ligand orbital are assembled, which bridge carbon nanotubes (denoted as cMOFs‐ b ‐CNTs). Then, precursor of poly(styrene‐ block ‐butadiene ‐block ‐styrene)/ionic liquid penetrates the pores and cavities in cMOFs‐ b ‐CNTs‐based network fabricated via filtration process, creating a semi‐embedded structure via mechanical interlocking. Thus, the mixed ion‐electron conduction and semi‐embedded structure endow the as‐prepared composites with a low interfacial impedance (51.60/28.90 kΩ at 10/100 Hz), wide sensing range (473%), high sensitivity (2195.29), rapid response/recovery time (60/85 ms), low limit of detection (0.05%), and excellent durability (>5000 cycles to 50% strain). Demonstrations of multifunctional mechanical‐bioelectrical dual‐modal sensors for in vivo/vitro monitoring physiological motions, electrophysiological activities, and urinary bladder activities validate the possibility for practical uses in biomedical research areas. This concept creates opportunities for the construction of durable skin‐like sensing materials.

Glioblastoma (GBM) is the most common malignant tumor of the CNS, accounting for 80% of brain tumors. Recent advances in gene editing and immunology have led to the development of chimeric antigen receptors (CARs), including CARs incorporating chlorotoxin (CLTX) receptor domains for GBM targeting. While CAR-engineered neutrophils have shown promise, their systemic administration can induce severe off-target effects. Extracellular vesicles (EVs), secreted by these cells, retain properties of their parent cells—including CAR expression and tumor-lytic activity—yet their potential has not been explored in CAR Neutrophils. To address this, CAR-CLTX DNA was inserted into the AAV1 safe harbor locus of induced pluripotent stem cells, which were subsequently differentiated into neutrophils. Conditioned media were collected during differentiation and processed using extraPEG-based EV isolation. EVs were quantified via nanoparticle tracking analysis, electron microscopy, and western blotting. Proteomic and microRNA (miRNA) profiling of EV cargo was conducted at days 12 and 21 of differentiation. Cytotoxic effects of EVs were assessed in U87MG and LN229 glioblastoma cells in 2D culture, 3D organoids, and in vivo models. EVs were secreted in high quantities within the 100–200 nm size range, expressed canonical exosomal markers, and exhibited characteristic morphology. Proteomic analysis identified neutrophil-associated proteins involved in migration, chemotaxis, and degranulation. miRNA sequencing revealed high levels of tumor-suppressive miRNAs (e.g., miR-182) implicated in apoptotic signaling, T cell receptor pathways, cytokine-cytokine receptor interactions, Jak-STAT, Hedgehog, and NK cell-mediated cytotoxicity. EV uptake by glioma cells was demonstrated in vitro. Cytotoxic effects of CAR Neutrophil EVs were observed across glioblastoma cell lines, organoids, and in vivo. These findings demonstrate that CAR Neutrophil-derived EVs carry anti-tumor protein and miRNA cargo, are efficiently taken up by GBM cells, and mediate glioma cell cytotoxicity. This supports the feasibility of correlating EV cargo composition with function in GBM treatment.

Ultrasensitive and specific detection of DNA is highly important for early cancer screening and diagnosis. Nucleic acid amplification technology is the most commonly used method for oncogene detection, but nonspecific amplification may occur. We designed a nicking endonuclease (NEase)-mediated exponential rolling circle amplification (RCA) that avoids nonspecific amplification for the CRISPR/Cas12a preamplification process. The purpose was to construct a NEase-assisted target recycling (NATR)-triggered no-nonspecific exponential RCA (NER) reaction integrated with a CRISPR/Cas12a (NATR-NER/Cas12a) system, enabling ultrasensitive and high-fidelity target detection. Innovatively, two circular single-stranded DNAs (ssDNAs) with NEase recognition sites were designed as the preprimer and template for RCA. In the presence of the target, the endonuclease Nt.BstNBI cleaves the circular preprimers into linear fragments, triggering the NER reaction. This generates many short ssDNA fragments, which are recognized by CRISPR/Cas12a and generates a fluorescence signal. The proposed strategy exhibited a wide linear range (10 fM-1 nM), a low detection limit (0.77 fM), and specifically recognized single mismatched DNA. In serum samples, this method exhibited good agreement with real-time quantitative polymerase chain reaction (qPCR) results at lower cost. The developed NATR-NER/Cas12a system provides a promising tool for the early screening and clinical diagnosis of cancer in resource-limited areas.

Extracellular vesicles (EVs) derived from human organoids are phospholipid bilayer-bound nanoparticles that carry therapeutic cargo. However, the low yield of EVs remains a critical bottleneck for clinical translation. Vertical-Wheel bioreactors (VWBRs), with unique design features, facilitate the scalable production of EVs secreted by human blood vessel organoids (BVOs) under controlled shear stress, using aggregate- and microcarrier-based culture systems. Human induced pluripotent stem cell-derived BVOs cultured as aggregates or on Synthemax II microcarriers within VWBRs (40 and 80 rpm) were compared to static controls. The organoids were characterized by metabolite profiling, flow cytometry, and gene expression of EV biogenesis markers. EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blotting. Lipidomics provided insights into EV lipid composition, while functional assays assessed the impact of EVs in a D-galactose-induced senescence model. VWBR cultures showed more aerobic metabolism and higher expression of EV biogenesis genes compared to the static control. EVs from different conditions were comparable in size, but the yields were significantly higher for microcarrier and dynamic cultures than static aggregates. Lipidomic profiling revealed minimal variation (< 0.36

Lung cancer remains the leading cause of cancer-related deaths, and there is an urgent need for innovative therapies. MicroRNA (miRNA)-based gene therapy has shown promise, but efficient delivery systems are required for its success. This study investigates the use of extracellular vehicles (EVs) secreted by natural killer (NK) cells as delivery systems for miRNAs targeting PD-L1/PD-1 immune checkpoint and FOXM1, in combination with Carboplatin, to enhance anticancer efficacy in lung cancer models. NK-EVs were isolated from NK92-MI cells and characterized using nanoparticle tracking analysis (NTA), proteomics and Western blotting, confirming their exosomal characteristics. Gene ontology profiling and RNA-seq identified highly expressed miRNAs such as miR-5193 and miR-149-5p, which were loaded into NK-EVs via electroporation. Agarose gel electrophoresis confirmed their entrapment and Quickdrop spectrophotometer was used to estimate the quantity. In vitro, miRNA-loaded NK-EVs demonstrated significant cytotoxicity against Osimertinib-resistant PDX (TM0019, Jackson Labs) and H1975R (with L858R mutations) lung cancer cells, with approximately 1.2 to 1.6-fold (p < 0.01) decrease in cell viability compared to NK-EVs alone. In vivo, the combination of miRNA-loaded NK-EVs and Carboplatin significantly reduced tumor volumes (3.5 to 4-fold, p < 0.001) in PDX and H1975R xenograft models, with the most pronounced effect observed in combination therapies. Western blot analysis showed downregulation of tumor-associated markers: PD-1/PD-L1, FOXM1, Survivin, NF-κB and others vs untreated group, p < 0.001) suggesting immune checkpoint inhibition, apoptosis and anti-inflammatory activity. These findings highlight the potential of NK-EVs as effective carriers for miRNAs in combination with chemotherapy, offering a promising therapeutic strategy for NSCLC with EGFR mutations.