Journal of Controlled Release

Cationic lipid nanoparticles have demonstrated unique potential for extrahepatic mRNA delivery, particularly enabling selective targeting of the pulmonary endothelium. However, their translational development has been hampered by reports of infusion-related immune reactions and innate immune system activation, most notably transient complement activation. Here, we present a case study illustrating the discovery and translational advancement of a selected cationic LNP into non-human primates (NHPs) for initial pharmacokinetic assessment and evaluation of potential immunostimulatory side effects. We show surface charge dependent organ-selective expression of reporter mRNAs from different LNPs in vivo. An mRNA encoding the Tie2 agonist COMP-Angl, was formulated with LNP002, and respective pharmacokinetic and pharmacodynamic readouts were analyzed in two independent non-human primate studies. Notably, dose-dependent transient complement activation could be abrogated by extending the infusion time. Finally, we identified the blood-borne pharmacodynamic biomarker PDGFB for LNP002/mRNA-76 treatment reflecting activated Tie2-signalling in healthy pulmonary endothelium in vivo supported by single cell sequencing and cluster-alignment of downstream effector genes with the same spatial profile as the delivered mRNA.

Stereotactic needle-based delivery remains the standard for local administration of Advanced Therapy Medicinal Products (ATMPs) to the human brain. ATMP administration typically involves multiple trajectories, presenting cumulative risks and prolonging surgery. Reflux-prone, patchy therapy coverage compromises clinical results. We demonstrate a novel approach, deploying a helical delivery catheter via a single access trajectory per target, referred to as SpiraMed. Helix retraction is synchronised with therapy delivery, enabling comprehensive target coverage in seconds. Helix pitch and diameter can be precisely tailored to patient-specific target volume and vascular anatomy, as part of the preoperative stereotactic surgical planning process. Testing in agarose phantoms, live sheep and cadaveric human brain confirms enhanced therapy delivery volume, delivery speed and target coverage, with reductions in reflux and predicted risk of bleeding complication. SpiraMed represents a new paradigm, promising to help deliver on the transformative potential of cell and gene therapies across the spectrum of human CNS disease.

Previously, we demonstrated the biosafety of polyethylene glycol-coated iron oxide nanoparticles (MAG@PEG) in a cellular model of viral oncogenesis, supporting their further preclinical evaluation. Herein, we evaluated the safety and biodistribution of MAG@PEG in tumor-bearing mice and assessed the influence of an external magnetic field on nanoparticle distribution. Animals were randomly assigned to three groups: G1, control (PBS); G2, MAG@PEG (10 mg/kg); and G3, MAG@PEG (10 mg/kg) with magnetic exposure for 1 h. Doses were administered intraperitoneally weekly for 1 month. Results showed that animal behavior and body weight were unaffected by MAG@PEG, while tumor growth was significantly reduced only in G2. Biodistribution studies based on tissue magnetization revealed higher magnetic iron levels in the stomach followed by the spleen, lungs, and tumors, particularly in G2. Histological analysis detected Prussian blue-positive staining mainly in the spleen and in lymphoid-associated structures in the lung, stomach and intestine, while a fraction of MAG@PEG remained within the peritoneal cavity. Serum biochemistry showed no evidence of nephrotoxicity. Furthermore, hemocompatibility studies using human blood samples demonstrated that MAG@PEG was non-hemolytic according to the ASTM F756 standard. Taken together, the biodistribution, histological, biochemical, and hemocompatibility findings provide a comprehensive preclinical assessment of the biosafety of MAG@PEG and identify the route of administration and magnetic targeting as key determinants of nanoparticle biodistribution.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide. This clinical burden is predominantly attributable to atherothrombotic events resulting from platelet hyperactivation, a process integral to the pathophysiology of atherosclerosis. Cilostazol (CLZ) is a clinically established phosphodiesterase-IIIA inhibitor with antiplatelet and vasodilatory activity. However, its extremely low aqueous solubility results in dissolution-limited and variable oral absorption, while dose-dependent adverse effects and cardiovascular safety restrictions may constrain its clinical use. Lipid-polymer hybrid nanoparticles (LPHNPs) represent a highly promising drug delivery system to address these biopharmaceutical limitations. However, their interactions with blood components remain poorly understood, with surface charge among the most controversial physicochemical parameters implicated in platelet toxicity. Here, we applied an integrated ex vivo and in silico screening strategy to design LPHNPs based on poly(lactic acid) and poly(ethylene glycol)-modified lipids displaying positive (amine), nominally neutral (methoxy), and negative (carboxyl) surface charges to systematically identify safe boundaries at the nano-human platelet interface and evaluate their suitability as CLZ carriers. Positively charged nanoparticles precipitated immediately during self-assembly due to uncontrolled ionic pairing and were therefore structurally unviable. Neutral nanoparticles maintained a stable size (~113 nm hydrodynamic diameter) but unexpectedly induced spontaneous platelet aggregation, limiting their applicability. In contrast, negatively charged nanoparticles exhibited exceptional colloidal stability (hydrodynamic size of ~120 nm and zeta potential of -48 mV), achieved significant inhibition of ADP-induced platelet aggregation, and adhered to the platelet surface without inducing activation or membrane disruption. Steered molecular dynamics simulations, performed under identical non-equilibrium pulling conditions, showed the highest interfacial resistance to CLZ extraction for the negatively charged model, qualitatively consistent with the sustained CLZ release profile observed in vitro. These findings highlight the importance of precise surface charge tuning in the development of safe cardiovascular nanomedicines based on LPHNPs, emphasizing the need for comprehensive platelet compatibility testing before further biological evaluation.

Solid tumors are known to be refractory to treatment due to a poorly perfused microenvironment that blocks immune cell infiltration and effective drug delivery. Histotripsy is a non-invasive focused ultrasound modality that generates cavitation microbubbles to mechanically disrupt solid tumors, resulting in decreased hypoxia and stimulation of tumor-targeted adaptive immune responses. In this study, we aim to investigate whether post-histotripsy hypoxia reduction and cytotoxic immune cell infiltration are associated with normalization of tumor vasculature and enhanced perfusion. Structural analysis of both treated and contralateral off-target tumors following partial histotripsy revealed significant vascular remodeling, enhanced vascular integrity, and decreased vascular leakage indicative of vascular normalization in multiple in vivo models of melanoma, hepatocellular carcinoma, and pancreatic adenocarcinoma. These effects appeared antigen-specific, as no remodeling was observed in contralateral off-target tumors of discordant pathology. Mechanistically, CXCR3+CD8+ T cells played a role in mediating these vascular changes, as genetic knockout of CD8 and pharmacological antagonism of CXCR3 impaired both remodeling and normalization. Additionally, significant changes in endothelial CXCR4 and Angiopoietin-1 and -2 expressions were observed following histotripsy, indicating their potential involvement in promoting vascular normalization and remodeling. Significant vascular remodeling was accompanied by enhanced tumor perfusion and intratumoral delivery of chemotherapeutic agents and therapeutic monoclonal antibodies. These findings suggest that histotripsy not only creates a more favorable microenvironment for immunotherapy, but also optimizes drug delivery into solid tumors. Combinatorial approaches using histotripsy with chemotherapy or immunotherapy may be a novel therapeutic strategy to overcome current limitations of conventional solid tumor therapies.

Protein replacement therapy is highly effective for treating many protein-deficiency disorders, but its long-term clinical benefit is often limited by anti-drug antibody responses that neutralize therapeutic proteins, compromising safety and efficacy. To address these unwanted immune responses, a lipid nanoparticle-based immunotherapy platform has been developed to induce antigen-specific tolerance. Mice treated with antigen associated with either double-chain phosphatidylserine (PS) or lysophosphatidylserine (LysoPS), a single-acyl-chain phospholipid, showed reduced antibody titers following subcutaneous pre-exposure. However, only LysoPS nanoparticles induced tolerance after oral administration, suggesting structural differences between PS and LysoPS can impact tolerogenic potential, depending on the site of delivery. As an apoptotic "eat-me" signal, PS is recognized by phagocytic receptors such as TIM-4 and is associated with LC3-associated phagocytosis. These nanoparticles have been hypothesized to promote apoptotic mimicry to regulate immunity. However, the contribution of TIM-4 to oral tolerance remains unclear. Here, we investigated the role of TIM-4 in LysoPS-mediated oral tolerance using a knockout mouse model. Our data showed that TIM-4-/- mice induced tolerance to ovalbumin encapsulated in LysoPS comparable to that of wild-type mice. We also found that LysoPS nanoparticles did not follow the same surface engagement and intracellular trafficking pathways as PS nanoparticles. These results reveal that, despite sharing the same headgroup, differences in acyl chain number critically shape nanoparticle-immune cell interactions and provide a mechanistic framework that may explain the unique ability of LysoPS nanoparticles to induce oral tolerance.

Glial cells play essential roles in maintaining neural tissue homeostasis and limiting the spread of injury-induced neuroinflammation, yet their dysfunction can also exacerbate neurodegenerative, neuroimmune, and neurodevelopmental disorders. Therapeutic targeting of specific glial cell populations would open up new treatment possibilities, but currently available viral and non-viral delivery vehicles are limited by carrier toxicity as well as poor delivery efficiency and specificity. To work towards new glia-targeted, non-viral delivery vehicles, we performed an in silico analysis on neural cell specific RNA-Seq datasets in mice to identify surface molecules differentially enriched on neurons, astrocytes, and microglia. We identified Slc2a5 (Glut5) as a microglia-enriched transporter and validated its microglia specific expression in healthy murine neural tissue using RNA in situ hybridization. By employing a sequential thiol-ene and enzymatic catalyzed reaction scheme, we synthesized polyelectrolytic dendrimers with multivalent presentation of fructose, the natural ligand for Glut5, and formulated nano-sized polyionic complexes (PICs) loaded with protein or nucleic acid cargo by mixing oppositely charged dendrimers. Fructose functionalized PICs lacking cationic surface charge enabled fructose-dependent intracellular protein delivery to Glut5-expressing human breast cancer cells (MCF-7) and mouse fibroblasts, while restricting uptake by Glut5-negative mouse astrocytes, confirming transporter-dependent selectivity in vitro. Fructose functionalized PICs resulted in minimal neuroinflammation when injected into mouse striatum at 1 mg/ml but elicited concentration-dependent neural tissue toxicity at higher doses. Fructose-PICs failed to achieve microglia specific intracellular delivery of functional proteins when injected into healthy neural tissue but aided in directing protein delivery to microglia and astrocytes at ischemic stroke lesions. Together, our findings demonstrate proof-of-principle for using in silico RNA-seq screening workflows to identify cell specific surfaceome targets and incorporating that insight into a modular biomaterial platform for targeted drug delivery.

Gas-bubble-enhanced focused ultrasound (FUSGB) is advancing clinically across a myriad of applications, including targeted drug delivery through transient blood-brain barrier opening (BBBO). Its broader translation requires a better understanding of the bioeffects that enable reproducible enhancement of barrier permeability while avoiding vascular injury, underscoring the need to clarify the relationship between acoustic exposure, bubble activity, and the biological response central to treatment optimization. These interactions are difficult to isolate in vivo, and acoustic reflections, geometry, and field distortion in conventional cultureware hinder quantitative in vitro studies. Herein, we present a theoretically and experimentally characterized ultrasound-transparent organ-on-chip device with >99.9% US transparency that enables quantitative in vitro modeling of the FUSGB procedure and study of BBBO. Furthermore, submicron bubbles (sMB, ~800nm) were fabricated and characterized. Acoustic dose-dependent subharmonic and broadband cavitation dose curves (SCD and BCD), as well as temporal responses, were studied. A 3-day accelerated Caco--2 surrogate barrier with a high TEER (~1200xcm2) was used to demonstrate US dose-dependent targeted barrier opening, visualized using an in vitro Evans Blue assay. Together, this study and its findings establish a foundation for quantitative in vitro modeling of FUS BBBO, as well as other US interactions in microphysiological systems, supporting systematic evaluation of procedural regimens, large-parameter-space optimization, acoustic safety window, and exposure conditions for brain drug delivery, and additionally enabling mechanistic insight into FUSGB BBBO.

The delivery of small-molecule therapeutics across cellular membranes remains a fundamental challenge in drug development, particularly for compounds whose physicochemical properties limit intracellular bioavailability. Inspired by the low toxicity, biodegradability, and synthetic accessibility of triethanolamine-derived lipids, we investigated a series of C18 aminotriester lipids as excipients for the codelivery of anticancer therapeutics. Four structurally related triester lipids bearing saturated, cis-monounsaturated, trans-monounsaturated, and di-unsaturated C18 tails were synthesized through straightforward esterification reactions with triethanolamine. These lipids were evaluated for their ability to enhance the antiproliferative activity of several anticancer agents, including paclitaxel, doxorubicin, amonafide, homoharringtonine, camptothecin, and SN-38. Across multiple drug classes, the aminotriester lipids exhibited minimal intrinsic cytotoxicity and generally produced limited enhancement of drug potency. However, the fully saturated tris-stearate aminotriester lipid selectively enhanced the antiproliferative activity of amonafide in HCT-116 colorectal cancer cells, particularly at lower concentrations. In contrast, unsaturated lipid analogues displayed negligible enhancement effects, and the activity of the tris-stearate lipid was substantially attenuated in A549 lung adenocarcinoma cells, weakly observed in HT-29 colorectal cancer cells, and absent in CT26 murine colorectal cancer cells. Lactate dehydrogenase release assays indicated that these effects were not associated with membrane lysis or nonspecific cytotoxicity. Further, fluorescence microscopy time course experiments suggest improved membrane trafficking of amonafide when codelivered stoichiometrically with fully saturated C18 aminotriester lipids. Collectively, these findings demonstrate that saturated triethanolamine-based aminotriester lipids may function as selective and biocompatible codelivery excipients for certain small-molecule therapeutics. The observed dependence on both lipid saturation state and cellular context suggests that membrane organization and lipid packing may play important roles in mediating these effects. These results establish a foundation for further mechanistic investigations into aminotriester lipid-assisted small-molecule delivery systems.

Clinical studies have revealed that glioblastoma (GBM) is refractory to immunotherapy, such as immune checkpoint inhibitors (ICIs), due to the immunosuppressive tumor microenvironment (TME) primarily intensified by intratumoral upregulation of transforming growth factor-{beta} (TGF{beta}). Primary GBM tumor bulks are surgically removed in clinic, but invasive tumor cells infiltrated into surrounding normal brain tissue cannot be eradicated by systemic therapy due to the integral blood-brain barrier (BBB), leading to near-inevitable tumor recurrence. To address these translational challenges, we have engineered a TGF{beta}-inhibiting protein nanocage comprising human heavy-chain ferritin protein and TGF{beta} receptor II ectodomain that enables BBB penetration and multivalent TGF{beta} entrapment, respectively. We demonstrate that the nanocage platform, denoted T{beta}R2-HFt, following systemic administration, traverses the BBB, and extensively modulates immunosuppressive TME, both stromal and immune components, thereby potentiating the therapeutic efficacy of a clinically used ICI in multiple mouse glioma models. Notably, a majority of the glioma-bearing animals that received surgical tumor resection, followed by treatment with T{beta}R2-HFt and the ICI, survived the tumor and resisted tumor rechallenge, providing a potential means to control GBM in a durable manner.

Therapeutic siRNAs are a major clinical modality for hepatic gene silencing, but efficient extrahepatic delivery and multi-gene modulation remain significant barriers. Nucleic acid nanotechnology offers programmable assembly, but conventional structures suffer from enzymatic instability in vivo . Here we report fully chemically modified RNA nanostructures (FMRNs): a vehicle-free platform entirely composed of stabilized therapeutic nucleic acids that integrate structural precision with metabolic durability. Following systemic administration, FMRNs exhibit enhanced pharmacokinetics and extrahepatic accumulation, with in vivo behavior influenced greatly by hydrodynamic size rather than molecular weight. Using triangular and square geometries, we demonstrate programmable control over siRNA stoichiometry to achieve sustained, simultaneous silencing of up to four genes from a single entity. Nanostructure assembly alone drives productive delivery, especially to heart and muscle, matching lipophilic conjugation. Furthermore, cerebrospinal fluid administration reveals that larger FMRNs improve central nervous system distribution and retention revealing physicochemical parameters that impact brain exposure. FMRNs establish a modular and effective platform combining improved pharmacokinetics, extrahepatic delivery, and multi-gene silencing for complex diseases.

Nanoparticle albumin-bound paclitaxel (Abraxane) was among the first clinically approved nanomedicines and is a first-line chemotherapeutic. It enhances therapeutic efficacy and reduces side effects of paclitaxel. Despite its extensive clinical use, mechanisms of Abraxane's therapeutic effects are the subject of ongoing study. This work posits a new mechanism for Abraxane's effects in lung cancers and metastasis. Tracing the albumin component of Abraxane with radioisotopes or fluorophores, we find that Abraxane homes to the lungs. Abraxane's lung tropism is elevated by inflammation and is eliminated in complement protein C3 knockout mice, showing that complement pathway drives the uptake. Flow cytometry and histology show that neutrophils are the dominant cell type taking up Abraxane in the lungs. We demonstrate Abraxane's tropism to pulmonary neutrophils in mice and in human donor lungs. We note previously undocumented side effects associated with Abraxane's lung tropism, including pro-thrombotic and pro-inflammatory responses in acute lung inflammation. We show that Abraxane's efficacy against lung metastases is affected when lung uptake is eliminated by C3 knockout. Our results provide insight into why Abraxane is effective in lung cancer and metastases. These findings may be used to improve outcomes in patients receiving Abraxane and help design next-generation cancer nanomedicines.

Biological systems such as extracellular vesicles (EVs) are highly sophisticated and functionally diverse. Despite growing recognition of EVs' roles in health and disease through mediating intercellular communication, their inherent heterogeneity and challenges in purity and yield hinder their mechanistic research and clinical translation. To address this gap, we sought to translate essential EV properties into a controllable and simplified platform by engineering biomimetic synthetic vesicles (SVs) incorporating defined proteins and lipids. Using microfluidics, we generated SVs loaded with bulk membrane and cytosolic proteins from cancer cells, as well as individual organotropism-associated integrins. Bulk membrane protein incorporation into SVs reduced surface charge, modified lipid profiles, and enhanced cellular uptake, while integrin incorporation altered biodistribution. To investigate protein loading, we profiled input materials and SVs by mass spectrometry, identifying features linked to high incorporation, such as low molecular weight, positive charge, high isoelectric point, and predicted stability, globularity, lipidation, and amphipathic helices. This work presents a synthetic platform for dissecting the roles of EV-associated features and cargo, while providing insights into protein properties relevant to the optimization of delivery systems.

A broad-spectrum antiviral is required to enable rapid intervention against current, newly emerging and mutating viruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), Sindbis virus (SINV) and chikungunya virus (CHIKV) all engage negatively charged moieties on host cells, such as heparan sulfate (HS) and sialic acid, to facilitate attachment for entry and infection. We designed HS-mimetic nanoparticles in the form of polymer vesicles (polymersomes) by blending membrane-forming and sulfonated copolymers. These biocompatible polymersomes successfully inhibited host cell infection of HS-dependent SARS-CoV-2 and HS-independent IAV in vitro with sub-nanomolar potency. Multivalent interaction of several virus ligands with a single polymersome as quantified by fluorescence correlation spectroscopy likely contributes to their potency. The sulfonated polymersomes also functioned in vivo as demonstrated by a reduction in the spread of SINV, an alphavirus model for CHIKV, in zebrafish embryos, which led to a significantly increased survival. Hence, our polymersomes could form the basis for an urgently required multifunctional broad-spectrum antiviral nanomedicine.

Therapeutic strategies based on increasing uncoupled respiration (browning) in white adipose tissue (WAT) have great potential for treating obesity-related disorders such as diabetes and steatotic liver disease, but have not had a clinical impact because safe and efficient methods for browning WAT have not been developed. Lipid nanoparticles (LNPs) offer a promising platform for adipose-directed siRNA delivery; however, conventional formulations often exhibit limited activity in adipocytes and can induce inflammatory responses that may restrict repeated administration. Here we demonstrate that LNPs that contain ionizable lipids with three lipid tails and siRNAs targeting TLE3 and ZFP423 can efficiently transfect mature adipocytes and induce browning of WAT in vitro and in vivo while providing the biocompatibility needed for repeated subcutaneous injections. Repeated local administration of BLNPs in diet-induced obese mice resulted in sustained target gene silencing, increased expression of thermogenic markers, altered energy metabolism, and decreased hepatic lipid accumulation despite minimal effects on body weight relative to scramble siRNA controls. Collectively, these findings establish BLNPs as a promising platform for adipose-directed RNA delivery and targeted modulation of thermogenic pathways, providing a strategy for investigating how localized adipose tissue remodeling influences metabolic physiology.

Our prior works have demonstrated the therapeutic potential extracellular vesicles (EVs) derived from batch cultures of brain endothelial cells (BECs) owing to their ability to transfer mitochondria to injured recipient cells. However, conventional batch/flask cultures limit scalable and reproducible production of EVs. Here, we evaluated a CELLine AD1000 bioreactor as a platform for sustained production of EVs from mouse b.End3 BECs and compared bioreactor-derived (BR) EVs with EVs obtained conventional batch cultures (BT). Large EVs (lEVs) and small (sEVs) were isolated using a sequential centrifugation protocol and were characterized using dynamic light scattering, nanoparticle tracking analysis, microBCA assay, western blotting, proteomics analysis, and their ability to rescue bioenergetics in oxygen-glucose deprived (OGD) BECs. Bioreactor cultures maintained high cell viabilities (~90%) for up to eight weeks and produced approximately three- to four-fold greater EV protein yields than batch cultures. BR EVs showed reproducible particle characteristics consistent with EV subtypes derived from BT cultures. CD63, CD9 and Arf6 were detected in EV fractions, whereas the mitochondrial outer-membrane protein TOMM20 was enriched in lEVs. Proteomic analyses revealed that bioreactor-derived lEVs retained the mitochondrial cargo signature observed in batch-derived lEVs. Both bioreactor and batch EVs restored relative ATP levels in ODG-exposed BECs. These findings suggest that bioreactor BEC cultures are a scalable approach for generating functionally active BEC-EVs for downstream characterization and therapeutic development.

By decoupling biological function from cellular viability, fixed abiotic cellular materials have emerged as a promising platform for next-generation immunotherapies, preserving essential immunomodulatory activities while enabling unprecedented control over manufacturing, storage, and standardization. Nevertheless, the coupled effects of fabrication history and post-fabrication environmental cues on their biological functionality remain elusive. Here, we show that both preparation route and storage time critically affect the bioactivity of mesenchymal stromal cell (MSC)-derived microtissues. By exploring different devitalization agents, we demonstrate that an ethanol-based strategy provides a green fixation option with comparable preservation of volumetric and fixed cell architecture, as well as immunomodulatory function. Processing protocols did not merely substitute conventional fixation but tailored specific immune-related bioactivity. An unexpected processing protocol and time-dependent enhancement of immunomodulatory performance was observed, with ethanol-fixed microtissues maintaining a stable baseline bioactive profile while increasing functional potency over 8 weeks of storage. We establish a framework for the design of immunomodulatory abiotic cell-derived materials with potential for the treatment of immune-mediated diseases and regenerative therapies, and we hope that the principles uncovered here may contribute to a broader understanding of how devitalized tissues can be engineered, preserved, and functionally tuned, whether lab-made or derived from natural biological systems.

Background: Parkinson's disease (PD) is characterized by the progressive loss of midbrain dopaminergic (DA) neurons and aberrant alpha-synuclein (alpha-syn) aggregation, yet disease-modifying therapies that simultaneously retard neurodegeneration and foster regeneration remain lacking. Methods: In this study, we isolated extracellular vesicles (TH and FOXA2 double-positive cell-derived EVs) from TH and FOXA2 double-positive differentiated neural cells, a distinct midbrain floor-plate-derived neural cell population that co-expresses the floor-plate transcription factor FOXA2 together with TH and should therefore not be equated with conventional mature midbrain dopaminergic neurons, and systematically evaluated their therapeutic potential across three complementary models: A53T transgenic mice, 6-OHDA-lesioned rats, and 6-OHDA-treated human midbrain organoids. Integrative transcriptomic and single-cell sequencing analyses, together with toxicological evaluations in rodents and non-human primates, were performed to investigate the underlying mechanisms and safety profile. These TH and FOXA2 double-positive cells were generated using a fully suspension-based, matrix-free, and chemically defined differentiation system devoid of N2/B27 supplements and fetal bovine serum (FBS). This approach offers a simple, controllable, low-cost, and scalable platform with high batch-to-batch consistency for EV manufacturing. Throughout this study, the term "TH and FOXA2 double-positive cells" refers to this distinct TH and FOXA2 double-positive cell population and should not be interpreted as conventional terminally differentiated dopaminergic neurons. Results: Intranasal administration of TH and FOXA2 double-positive cell-derived EVs in A53T mice and 6-OHDA-lesioned rats significantly ameliorated motor deficits, increased nigral tyrosine hydroxylase (TH)-positive neuron counts, reduced alpha-syn aggregation, and suppressed gliosis. In human midbrain organoids, TH and FOXA2 double-positive cell-derived EVs preserved DA neuronal morphology and reduced GFAP and alpha-syn expression. Notably, when EVs were administered simultaneously with 6-OHDA modeling rather than after modeling was completed, the protective effect was stronger. Multi-omics analysis revealed that TH and FOXA2 double-positive cell-derived EVs reversed pathological signatures of interferon-responsive microglia and oxidative stress-adapted astrocytes, while restoring WNT- and non-canonical WNT-mediated intercellular communications. Safety evaluations showed no discernible organ toxicity. Conclusions: TH and FOXA2 double-positive cell-derived EVs exert neuroprotective effects across multiple PD models by modulating specific glial subpopulations and reinstating development-associated signaling pathways. Together with a fully suspension-based, matrix-free, and N2/B27-free differentiation process that enables low-cost, scalable, and highly reproducible manufacturing, TH and FOXA2 double-positive cell-derived EVs represent a promising therapeutic candidate for Parkinson's disease.

Experimental surgery is one of the most fundamental fields of biomedical research, recreating clinical conditions on living organisms similar to clinical patients, to facilitate the design of novel therapies. For modeling neurological and psychiatric disorders, the majority of operative techniques mandatorily include craniotomy for consecutive interactions with the open brain. The most wide-spread methodology involves stereotactic surgery in rodents, implementing a specific head holder, attached to the coordinate table, providing the precise animal head positioning according to the brain structures topology. Despite the high efficiency, such systems require complex instrumental skills and long procedure duration per animal even for simple implantation protocols. Hence, novel alternative model species could be a valuable supplementary tool for such manipulations. Therefore, the zebrafish (Danio rerio) fits perfectly, due to its low cost, easy husbandry, rapid development, optic transparency, and high genetic and physiological similarity to rodents and humans. Furthermore, the everted zebrafish telencephalon provides excellent accessibility to critical mammalian-like structures involved in emotion processing and cognition. However, conventional avian- or rodent-inspired craniotomies in adult zebrafish often trigger inadvertent disruption of the brain vessels network, resulting in high operative mortality. A novel, geometry-driven, non-lethal cranial window protocol preserving neurovascular integrity and ensuring 90-100% post-operative survival is presented to overcome above mentioned limitations. This rapid, simple, and high reproducible technique opens new avenues for precise region-specific in vivo neurosurgery, high-throughput psychopharmacological screening, and neurostimulation in a powerful non-mammalian model species

The lack of non-invasive, quantitative methods to track the location and proliferation of transplanted cells hampers the clinical translation of cardiac regenerative therapies. Here, we establish a PET reporter for tracking human induced pluripotent stem cell (hiPSC)-derived cardiac cells across complementary two-dimensional (2D) and three-dimensional (3D) cardiac tissue models. Methods hiPSCs were genetically engineered using CRISPR-Cas9 to express a human-derived, anticalin-based, bioorthogonal PET reporter (DTPA-R) that specifically binds radiolabeled DTPA-metal complexes. The engineered hiPSCs were differentiated into ventricular progenitors (HVPs) and cardiomyocytes (CMs). Stability of reporter gene expression and its effects on cellular differentiation and function were evaluated. Uptake of [18F]F-DTPA*Tb by DTPA-R-positive and -negative HVPs was analyzed in vitro, and DTPA-R HVPs were evaluated in ex vivo cultured porcine myocardial slices with and without radiofrequency ablation (RFA) injury. Phantom studies were performed to evaluate PET detectability under clinically relevant large-animal and human imaging conditions and to assess how activity distribution affects signal detection in relation to DTPA-R cardiac cell-equivalent numbers. Results DTPA-R expression was maintained during the differentiation stages of hiPSC-derived cardiac cells and had no measurable impact on migration, differentiation, and functional integration. PET imaging enabled the specific detection of DTPA-R HVPs in intact and injured myocardial slices. At the initial phantom scan, the measured activity corresponded to cell-equivalent numbers of approximately 3.2 x 10^6 HVPs or 5.5 x 10^6 CMs. The compact 200 uL source remained clearly detectable at 6 h, whereas the more dispersed 1,000 uL source progressively approached background activity, indicating that PET detectability depended strongly on local activity concentration and distribution. Conclusion This study establishes a bioorthogonal PET reporter gene platform for quantitative imaging of hiPSC-derived cardiac cells, with no measurable adverse effects on the biological parameters examined, and provides a methodological foundation for future translational and in vivo imaging studies. The approach may also be applicable to other hiPSC-derived cell products beyond cardiac cell lineages.