Improving the aqueous solubility of moderately soluble drugs using safe and sustainable solvent systems remains an important challenge in pharmaceutical formulation. In this study, a series of aqueous natural deep eutectic solvents (NADESs) were systematically designed and evaluated to enhance the solubility, stability, and biocompatibility of acetaminophen (ACP). The effects of hydrogen bond donor type, hydrogen bond acceptor type, molar ratio, and NADES concentration were investigated to identify the optimal formulation. Among the tested systems, the aqueous betaine chloride-acetic acid NADES (BeCl-Ace) at a molar ratio of 1 : 4 and a concentration of 70% exhibited the highest solubilization capacity, increasing ACP solubility by 25.28 and 22.14-fold at 25 and 37 °C, respectively, compared with water. Computational analyses based on electrostatic potential mapping, binding energy calculations, and interaction region indicator analysis revealed that the enhanced solubility originates from cooperative hydrogen bonding, electrostatic attraction, and van der Waals interactions between ACP and the BeCl-Ace components, particularly BeCl. In addition, the selected NADES formulation significantly improved ACP stability under thermal stress, retaining 89.13% of the initial drug content after 30 days at 37 °C, compared with 70.20% in water. Cytotoxicity evaluation on HEK-293A cells further confirmed good biocompatibility, with cell viability remaining above 92% across the tested concentration range. These findings indicate that aqueous BeCl-Ace NADESs represent a promising and sustainable solvent system for improving ACP formulation performance.

Liver metabolic zonation enables hepatocytes to carry out diverse, spatially organized functions essential for liver homeostasis and metabolism. However, in vitro recapitulation of zone-specific diversity, particularly in human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs), remains challenging, as these cells typically exhibit an immature phenotype. Here, we report a scalable differentiation protocol for generating 3D organoids from PSCs with defined zone 1 (z1-HLCs) or zone 3 (z3-HLCs) identities. When benchmarked against primary human hepatocytes (PHHs), these HLCs exhibit zone-specific enhanced postnatal metabolic functions. z1-HLCs reveal high albumin and urea production, whereas z3-HLCs show elevated glutamine secretion and cytochrome P450 (CYP) enzyme activity, including CYP3A4, CYP1A2, and CYP2C9. Hallmarks of functional zonation were further confirmed via proteomics, transcriptomics, metabolic assays, and oxygen consumption. z3-HLCs show increased sensitivity to acetaminophen-induced cytotoxicity and distinct immune interactions with primary monocytes. Collectively, the zonated liver organoids provide a promising platform for studying zone-specific liver metabolism, drug metabolism, toxicology, and liver disease, including liver injury.

IntroductionMetastasis occurs in almost 50% of patients with uveal melanoma. In patients who are HLA-A*02:01 positive, first line treatment is the Immune mobilizing monoclonal T cell receptor Against Cancer (ImmTAC), tebentafusp, which increases overall survival significantly at one year compared to single-agent immunotherapy or chemotherapy. Hypersensitivity reactions (HSR) to cancer therapeutics are well established. When these therapies offer significant benefits over alternatives, drug desensitization protocols are utilized to improve clinical outcomes in those with suspected IgE-mediated HSRs.Case reportWe present a case of an individual with metastatic uveal melanoma who suffered an anaphylactic HSR to tebentafusp. Given the lack of available ImmTAC therapeutic desensitization protocols, this report's purpose was to create a tebentafusp desensitization protocol based on accepted desensitization protocols for other chemotherapeutic drugs.Management and outcomeThe desensitization procedure consisted of pretreatment with cetirizine, famotidine, diphenhydramine, methylprednisolone, and acetaminophen prior to infusion. Infusions were each given as two diluted infusions limited to 100 mL of reconstitution volume, with cumulative quantities equivalent to standard treatment regimen dosages. Drug was given over no longer than four hours per manufacturer's recommendations. A total of three cycles of three weekly infusions were completed. The patient had no anaphylaxis recurrence. Tebentafusp treatment was withheld after the third cycle due to disease progression.DiscussionFor patients with HLA-A*02:01 positive metastatic uveal melanoma with suspected IgE-mediated HSR to tebentafusp, desensitization can be completed safely. Given the patient's disease progression within two months of treatment, it is unknown whether desensitization impacts the drug's clinical benefit.

Oral delivery of water-repelling drugs for acute liver failure (ALF) often runs into problems like poor solubility, instability, and low absorption. In this work, we found that extracellular vesicles derived from honey (HEVs) can serve as a natural nanocarrier that works orally and targets the liver on its own. After isolating and characterizing these vesicles, we saw they are membrane-bound nanoparticles roughly 160 nm in size with a surface charge around -32 mV, and they contain proteins and RNAs. Oral HEVs mainly ended up in the liver and showed some anti-inflammatory activity without any drug loaded. To get better therapeutic effects, we loaded curcumin (Cur), a hydrophobic anti-inflammatory compound, into HEVs to make Cur@HEVs. These loaded vesicles greatly improved how well curcumin is absorbed and released in a sustained manner. In acetaminophen (APAP) caused ALF mice, oral Cur@HEVs improved liver function and survival much more than plain curcumin or empty HEVs did. Specifically, Cur@HEVs simultaneously put the brakes on pyroptosis (via the NLRP3/GSDMD/caspase-1 route) and necroptosis (through the RIPK1/RIPK3/MLKL pathway), which in turn cut down the release of inflammatory cytokines and kept liver cells from dying. Overall, HEVs come across as a safe, food-based nanocarrier that can be taken orally, and Cur@HEVs offer a bioactive platform for dually targeting inflammatory cell death in ALF. Our findings suggest that dietary extracellular vesicles warrant further investigation as potential therapeutic biomaterials, though additional translational studies are needed.

N-acetyl-p-aminophenol (APAP), or acetaminophen, is the most prescribed analgesic and antipyretic, even used in association with other drugs. Because it is easily bought in drugstores, much evidence about its toxic effects is known due to high doses or continuous use, which can lead to death in cases of intoxication. The present study aimed demonstrate the ability of APAP to change the brain and heart electrical activities, as early as significant increase in liver enzymes. Thus, a toxic dose of 1 g/kg was administered orally, and the animals were assessed every 3 h until 12 h for electroencephalography, electrocardiography and liver function data. The results showed that a toxic dose of APAP was capable of reducing brain electrical activity for the delta, theta, beta and gamma waves; however, the alpha wave activity was increased. In the heart, a reduced electrical activity was observed synchronously with the electroencephalogram. During the 12-h follow-up period, a significant increase in liver enzymes was observed. Taken together, we conclude that high doses of APAP can alter frequency brain oscillations and promote cardiac changes independently of liver damage in the first 12 h.

Oxidative stress is a central driver of acute liver injury, and protein carbonylation and lipid aldehyde formation are among its most direct chemical signatures. Most carbonyl-reactive fluorescent probes have been characterized only in two-dimensional culture, and their behavior in three-dimensional tissue is often unclear. Here we describe a protocol for evaluating carbonyl-reactive fluorescent probes in mouse hepatic organoids grown in suspension in the CERO 3D bioreactor, using acetaminophen (APAP) overdose as a well-established model of acute oxidative liver injury. We demonstrate the assay with P7-Hz, a hydrazine-based probe that reacts with carbonyl groups on oxidatively damaged proteins and lipids to form the fluorescent hydrazone product P7-Hz-Ali. The protocol covers organoid culture and maintenance, APAP dosing to induce injury, probe incubation (labeling), and imaging-based quantification of the fluorescence signal as a readout of carbonylation, together with co-staining viability to distinguish probe-specific signal from cytotoxicity. Although P7-Hz is used as the demonstration probe, the same workflow accommodates other carbonyl-reactive probes whose hydrazone (or analogous) product is spectroscopically distinguishable from the unreacted probe, providing a reproducible three-dimensional platform for probe validation.

Acetaminophen (APAP) overdose is a leading cause of liver failure in Western countries and a major clinical challenge because effective treatment options remain limited for patients who present after the optimal early therapeutic window. Iron is distributed among several subcellular compartments, such as cytosol, mitochondria, ferritin, and endosomes/lysosomes. Emerging evidence indicates that intracellular iron homeostasis and organelle function may be critical during the regeneration phase for repairing liver damage. Our recent work identified the metalloreductase six-transmembrane epithelial antigen of the prostate 4 (STEAP4) as a critical regulator of lysosomal iron homeostasis and membrane integrity during the regeneration phase following APAP overdose. Hepatic STEAP4 deficiency promoted lysosomal iron accumulation and impaired hepatocyte proliferation, whereas deferiprone-mediated iron chelation restored lysosomal function and promoted liver regeneration. In this perspective, we discuss the potential of targeting lysosomal iron to promote liver repair after APAP overdose-induced liver injury, as well as the limitations and challenges.