Nanoscale Advances

Inflammation is a fundamental immune response but, when dysregulated, contributes to the pathogenesis of numerous inflammatory disorders. Although there are several conventional anti-inflammatory drugs which are effective, their long term use is often associated with adverse side effects, which highlights the need for safer alternative therapeutic drugs. Probiotic derived membrane vesicles (MVs) have recently emerged as biologically active nanostructures capable of modulating host immune responses. In the present study, MVs isolated from Lactobacillus acidophilus MTCC 10307 were evaluated for their anti-inflammatory efficacy and safety profile using in vitro and in vivo models. In RAW 264.7 macrophages, L. acidophilus MVs significantly attenuated lipopolysaccharide induced expression of the pro-inflammatory mediators Il-1{beta}, Il-6, and iNOS, accompanied by reduced nitric oxide and reactive oxygen species production which was abolished in the proteinase K treated MVs. The protein levels of NF{kappa}B and IL1{beta} were also reduced in the treatment groups. Repeated dose oral toxicity studies revealed no adverse effects, as evidenced by body weight and histopathological evaluation of major organs. The anti-inflammatory properties of L. acidophilus MVs were further validated in an in vivo hind paw edema model, which shows inflammation resolution demonstrated by molecular and histological analysis. Proteomic analysis using LC-MS/MS identified the presence of surface-layer protein A (SlpA) which is a potential bioactive component which might contribute to the observed immunomodulatory effects. Collectively, these findings demonstrate that L. acidophilus MVs exert potent anti-inflammatory activity while maintaining an excellent safety profile using integrated in vitro and in vivo models.

Environmental degradation and accumulation of plastics results in micro- and nanoplastics (MNPLs) that are small enough to cross biological barriers, including the blood-brain barrier. Microglia, resident immune cells of brain, are critical regulators of neuroimmune homeostasis and represent a cellular target of nanoplastic exposure. In this study, we assessed the neurotoxic effects of two sizes of polystyrene nanoplastics (PS-NPs; 100 nm and 500 nm) using integrated in vivo and in vitro exposure and washout paradigms. In vivo exposure in mice (60 days; 0.15 or 1.5 mg/day) showed the accumulation of both PS-NP sizes in the cerebral cortex without histopathological damage. However, cortical microglia showed pronounced morphological remodeling, observed as increased expression of Iba1 and GFAP. Transcriptomic profiling of cortical tissue revealed a strong size-dependent response. The 100 nm PS-NP group revealed 18 DEGs (|log2FC| [&ge;] 2, padj < 0.05), whereas the 500 nm PS-NPs showed more than 4,000 DEGs, including upregulation of immune- and microglia-associated genes (CCL5, CXCL10, LCN2, LYZ2) and downregulation of synaptic and neuronal signaling genes (GRIN2B, SYN1, STX1B, MAP1B, ITPR1/2). In vitro assessment, using BV2 microglia cells, showed internalization of PS-NPs via the endolysosomal pathway, with strong co-localization to Rab7- and LAMP2-positive compartments and prolonged intracellular retention following exposure washout. Also, microglial activation markers (Iba1, CD68) exhibited a transient, size- and concentration-dependent increase, correlated with intracellular particle burden rather than cumulative exposure. Overall, these findings demonstrate that PS-NPs accumulate in brain, driving size-dependent microglia activation and transcriptomic reprogramming, even after cessation of exposure to PS-NPs. HighlightsO_LIPS-NPs (100 nm and 500 nm) reach mouse cerebral cortex following 60-day oral exposure. C_LIO_LIPS-NPs were internalized by microglia; accumulated in endolysosomal compartments. C_LIO_LIPS-NP exposure induced transient microglial activation without sustained cytotoxicity. C_LIO_LIMicroglial activation was correlated with intracellular PS-NPs burden. C_LIO_LITranscriptomics revealed disruption of neuroimmune and microglial regulatory pathways. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/712727v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1aba3eaorg.highwire.dtl.DTLVardef@1967641org.highwire.dtl.DTLVardef@12da637org.highwire.dtl.DTLVardef@1fb8441_HPS_FORMAT_FIGEXP M_FIG C_FIG

Silver (Ag+) ions are known to be toxic to bacteria, cells, organisms and living systems; yet its impacts on the locomotion of surface-crawling organisms remain poorly quantified. Here we investigated the short-term (0-6 hours) effects of Ag+ ions on the locomotion of Drosophila melanogaster larvae on flat agarose surfaces containing Ag+ ions at different concentrations (0, 1, 10, and 100 mM). By quantifying their locomotion, we found that Drosophila larvae showed shorter accumulated distances and reduced crawling speed. Additionally, we quantified the go/stop dynamics and peristalsis of the larvae and observed that Ag+ ions disrupted the normal, rhythmic, peristaltic contraction of the larvae and "trapped" them in the stop phase. Such toxic effects were dependent on Ag+ concentration and exposure duration.

A programmable 4-input cascade DNA logic gate utilizing toehold mediated strand displacement (TMSD) was implemented on a 3D printed hybrid paper-polymer vertical flow device (3D HPVF) for on/off sensitive and specific fluorescence detection of platelet derived growth factor BB (PDGF BB). Polypropylene was 3D printed directly on paper and thermally cured to create micro paper analytical devices ({micro}PADs). The 3D HPVF comprised of three layers of {micro}PADs enclosed in a casing that clamped each {micro}PAD securely to ensure seamless and efficient wicking between layers. In the presence of PDGF BB, a partially complementary strand to a PDGF B aptamer (PDGF B Apt), cApt, was liberated from a PDGF B Apt/cApt duplex in solution. The solution was then deposited on the 3D HPVF with a dimeric g-quadruplex hairpin. The 4-nucleotide toehold region on the cApt started the hybridization reaction with the dimeric g-quadruplex hairpin (dGH) opening it up allowing formation of a dimeric g-quadruplex structure that binds with thioflavin T (ThT) with enhanced fluorescence intensity at room temperature. The 3D HPVF exhibits a pico molar range of detection from 10pM to 100pM with a 10pM limit of detection (LOD) for PDGF BB concentrations relevant for pregnant women predisposed to early-onset preeclampsia with clear differentiation when compared to similarly competing analytes PDGF AA and AB.

Nanoplastics generated from plastic waste in our ecosystems are becoming increasingly prevalent as bulk plastics exposed to natural factors like water and sunlight fragment to the nanoscale over time. These incidental nanoplastics span a wide range of physicochemical properties, which makes studying nanoplastic interactions in biological systems difficult. Here, we characterized the behavior of incidental nanoplastics generated through mechanical abrasion within coacervate droplets to probe the surface properties of the nanoplastics. We used elastin-like polypeptides (ELPs) to create hydrophobic or charged coacervate microenvironments. Using optical microscopy and fluorescence quantification, we observed that nanoplastics made from polyethylene terephthalate (nPET), nylon 6 (nPA), and polystyrene (nPS) exhibited distinct partitioning behavior with more favorable interactions with hydrophobic droplets. This indicated that the hydrophobic polymer backbone was the predominate surface feature despite exposed functional groups of the incidental nanoplastics, in contrast to findings with model carboxylated latex nanospheres (nPS-COOH). Furthermore, the selective partitioning of incidental nanoplastics into the hydrophobic droplets was able to capture over 80% of nPET in solution, and after recovery of the protein droplet, was able to cumulatively capture over 75% of the nPET feedstock across multiple cycles. This work explores the nuanced surface characteristics of incidental nanoplastics, expands the application of coacervates as chemical probes, and demonstrates a biopolymer approach for effective nanoplastic removal.

Flexible and biocompatible piezoelectric materials are crucial for next-generation wearable and bio-integrated electronics. In this work, we report a sustainable bio-composite film by incorporating lysozyme, a naturally abundant protein, into a polyvinyl alcohol matrix to achieve efficient electromechanical conversion. The composite exploits the intrinsic molecular dipoles of lysozyme, which are effectively stabilized and aligned within the polymer network. Under applied bending strain and vertical pressure, the film exhibits a pronounced piezoelectric response, as evidenced by time-dependent electrical measurements under forward and reverse bias conditions. The deformation of -helices and other helical structures within lysozyme induces dipole reorientation and charge separation, generating a measurable electrical output. In contrast, pure polyvinyl alcohol films show no detectable response, confirming the essential role of lysozyme in the observed piezoelectricity. Furthermore, the device enables real-time human motion sensing, highlighting its potential for flexible, eco-friendly, and biocompatible electronic applications.

This work develops and characterises a hierachichal oral drug delivery system based on the microencpasulation of drug-loaded amphiphilic nanogels within a mucoadhesive alginate/chitosan shell. Results show a more controlled release and a statistically significant oral half-life with respect to the free drug.

Medulloblastoma (MB) is an aggressive central nervous system (CNS) malignancy that primarily affects children and frequently exhibits metastasis to the leptomeninges of the brain and spinal cord. We developed a {beta}-Cyclodextrin-poly({beta}-Amino Ester) nanoparticle system to deliver the histone deactylase inhibitor (HDACi) Panobinostat to MB by the intrathecal route. Various imaging methods were utilized to study nanoparticle and payload fate following infusion into the cerebrospinal fluid (CSF) of mice via cisterna magna or lumbar access points. Nanoparticles dramatically improved penetration of hydrophobic small molecules into distal regions of the spinal cord. Panobinostat-loaded nanoparticles were effective at treating patient-derived MB, activating pharmacodynamic targets, slowing growth of the primary tumor, decreasing incidence of metastasis at the time of death, and ultimately prolonging survival. These studies provide insight into the mechanisms mediating transport of colloids and therapeutic molecules in the subarachnoid space and highlight new approaches for treating metastatic disease in the CNS.

Polymeric 3D printing of microfluidic devices for biosensing is an appealing fabrication alternative for rapid manufacturing of biosensing devices with complex geometry in a streamlined, repeatable and cost-effective manner without the need for expensive instrumentation such as those employed in photochemical etching and soft lithography. Hybrid 3D printed paper-based microfluidics is an emerging area which harnesses the unique properties of both, merging the construction of microfluidic structures and the inherent capillary-driven flow within paper substrates. In this work, we have fabricated hydrophobic barriers by 3D printing a single layer of machinable wax, thermoplastic polyurethane, polylactic acid and polypropylene directly on chromatography paper to create open microchannels and determine the most suitable material. Characterization of each open microchannel using the four materials revealed polypropylene as the most reliable material with high hydrophobic barrier integrity and resolution. Polypropylene achieved functional microchannels with a resolution of 621 {+/-} 33{micro}m, hydrophobic barrier integrity of (93.75 {+/-} 9.16%), wicking speed of 0.38mm/s and optimal hydrophilicity of channels (51.4 {+/-} 8.36 {degrees}) with minimal embedding during thermal curing. To demonstrate proof of principle, a fluorescence assay demonstrating the formation of a dimeric g-quadruplex structure from a g-rich sequence which significantly enhances fluorescence of thioflavin T was implemented.

The scalable fabrication of stable colloidosomes with controlled permeability and defined multicompartmental architecture remains a critical challenge, limiting their broader use in molecular delivery and environmental remediation. Here, we develop a hybrid lipid-metal-organic framework (lipid-MOF) colloidosome assembled through an interfacial emulsification strategy that integrates the structural rigidity of ZIF-8 particles with lipid-mediated membrane stabilization. During assembly, ZIF-8 particles accumulate at the oil-water interface to form a shell, producing hollow micron-sized spherical colloidosomes. The resulting colloidosomes exhibit excellent colloidal stability in aqueous media for over 30 days with a zeta potential of approximately -50 mV. Nitrogen adsorption measurements reveal a surface area of 45 m2g-1 and an average pore width of 4 nm. Fluorescence imaging shows that hydrophobic Nile red preferentially partitions into the colloidosomal membrane, whereas hydrophilic fluorescein isothiocyanate (FITC) localize predominantly within the aqueous interior, enabling simultaneous encapsulation of molecules with contrasting polarity with loading efficiencies approaching 90%. Furthermore, the colloidosomes demonstrate rapid removal of model pollutants from water, achieving >90% removal of methylene blue and metal ions without stirring. Together, these results introduce lipid-MOF colloidosomes as a new class of hybrid platforms that unify structural stability, multicompartmental encapsulation, and efficient adsorption behavior, opening pathways toward sustainable platforms for drug delivery and environmental bioremediation.

Microneedle (MN) patches have emerged as a highly efficient platform for localized drug delivery, showing great promise in cancer therapy due to their ability to enable precise drug administration. However, conventional MN systems are limited by the low drug-loading capacity of their tips and primarily rely on biologically inert, non-therapeutic matrices for structural support, which restricts further gains in antitumor efficacy. Herein, we present a strategy turning toxicity into therapy by constructing palladium nanoparticle-loaded polyvinyl alcohol/polyethyleneimine (PVA/PEI@Pd) hydrogel microneedles (PPPd-MNs), which exploit the intrinsic cytotoxicity of PEI for synergistic melanoma therapy. The PPPd-MNs efficiently catalyze the deprotection of a doxorubicin prodrug (P-DOX), enabling in situ generation of active doxorubicin (DOX). Notably, the PEI matrix serves a dual function: acting as a robust ligand to stabilize Pd catalysts and functioning as a therapeutic agent that disrupts cancer cell membranes. Both in vitro and in vivo experiments demonstrate that the combination of Pd-mediated bioorthogonal activation of DOX and PEI-induced membrane damage achieves a remarkable synergistic therapeutic outcome in a murine melanoma model, resulting in a tumor inhibition rate of up to 98%. This work repurposes the inherent cytotoxicity of the carrier material as an active therapeutic component, offering a novel paradigm for the design of high-performance bioorthogonal catalytic systems.

BackgroundTo control leishmaniasis, chemotherapy drugs are currently under development. However, these drugs often exhibit poor efficacy and are associated with toxicity, adverse effects, and drug resistance. At present, no specific drug is available for the treatment of leishmaniasis. Meanwhile, vaccine research is ongoing. Recent studies have analysed some experimental vaccines using mathematical models. AimIn previous work, drug targeting was focused on the entire human body rather than specifically addressing infected macrophages and parasites. In our current approach, we aim to eliminate infected macrophages and parasites through nano-drug design. Specifically, we utilise two types of nanoparticles: iron oxide and citric acid-coated iron oxide. Moving forward, we plan to advance this strategy using mathematical modelling of macrophage-parasite interactions. MethodsWe design PDE-based models of macrophages and parasites, incorporating cytokine dynamics, to support nano-drug development. Drug efficacy is estimated using posterior distributions to analyse phenotypic fluctuations of macrophages and parasites during the design phase. We investigate implicit and semi-implicit treatment schemes, focusing on energy decay properties. To model drug flow during treatment, we introduce a three-phase moving boundary problem. Comparative analyses are conducted to evaluate macrophage and parasite behaviour with and without treatment. Finally, the entire framework is implemented within a virtual lab environment. ResultsThe results show that the nano-drug exhibits better efficacy compared to combined drug doses. We analysed and compared two types of nano-drug particles: iron oxide and citric acid-coated iron oxide. We discuss how the drug effectively targets and eliminates infected macrophages and parasites. ConclusionOur models results and simulations will support researchers conducting further studies in nano-drug design for leishmaniasis. These simulations are performed within a virtual lab environment.

AbstractInflammation and oxidative stress are key drivers in the pathogenesis of chronic lung diseases, including asthma, pulmonary fibrosis, and chronic obstructive pulmonary disease. Extracellular vesicles derived from the marine microalga Tetraselmis chuii, referred to as nanoalgosomes, have recently gained attention as natural nanocarriers that possess inherent antioxidant and anti-inflammatory properties. In this study, we investigated the biocompatibility and protective effects of aerosolized nanoalgosomes in a bronchial epithelial-macrophage co-culture model at the air-liquid interface. Co-cultures of CALU-3 epithelial cells and differentiated THP-1 macrophages were primed with aerosolised nanoalgosomes and subsequently exposed to either oxidative stress (tert-butyl hydroperoxide) or an inflammatory stimulus (lipopolysaccharide; LPS). Epithelial barrier integrity and cytotoxicity were evaluated using transepithelial electrical resistance and lactate dehydrogenase release assays, respectively, while intracellular reactive oxygen species levels and cytokine secretion were measured to assess antioxidant and immunomodulatory responses. Nanoalgosomes were non-cytotoxic, preserved epithelial barrier integrity, and significantly reduced oxidative stress. In addition, nanoalgosomes priming attenuated LPS-induced secretion of pro-inflammatory cytokines (IL-1{beta}, IL-6, IL-8, IL-18, TNF-) as well as the anti-inflammatory cytokine IL-10, suggesting a balanced immunomodulatory response. Overall, aerosolized nanoalgosomes maintained epithelial homeostasis and mitigated both oxidative and inflammatory stress, underscoring their potential as a safe, sustainable, and effective therapeutic strategy for chronic inflammatory lung diseases. Given their natural origin, excellent biocompatibility, and suitability for aerosol delivery, nanoalgosomes represent a promising class of inhalable biotherapeutics.

Understanding how airborne particulates disrupt the alveolar barrier requires in vitro systems that recapitulate both the structure and transport properties of the lung air-blood interface. Here, we report a biodegradable lung alveoli-on-a-chip enabled by porous poly(lactic-co-glycolic acid)/polycaprolactone (PLGA/PCL) membranes with an interconnected porous architecture generated via porogen-assisted phase separation process. The membrane exhibits tunable degradation behavior, allowing progressive increases in surface porosity ([~]40%) and reduction in thickness ([~]3 {micro}m) during culture, while PCL maintains mechanical integrity under dynamic conditions. These degradation-driven structural changes regulate membrane transport properties, leading to enhanced permeability and supporting the formation of a functional epithelial-endothelial barrier under air-liquid interface (ALI) culture with breathing-mimetic cycling strain. Primary human alveolar epithelial and microvascular endothelial cells formed confluent, junctional monolayers on opposing membrane surfaces, exhibiting stable barrier function and high viability throughout the culture period. As a functional application, the platform was used to assess diesel particulate matter (DPM)-induced alveolar injury. Apical exposure to DPM induced dose-dependent cytotoxicity, increased barrier permeability, elevated reactive oxygen species, and DNA damage in both epithelial and endothelial layers, demonstrating trans-barrier propagation of particulate-induced injury. Pharmacological modulation with roflumilast-N-oxide (RNO), a phosphodiesterase-4 (PDE4) inhibitor, selectively attenuated oxidative stress and inflammatory responses, with limited effects on barrier integrity. Together, this work establishes degradable PLGA/PCL membranes as tunable interface materials for lung-on-a-chip systems, where structural evolution during degradation directly governs transport and barrier function. The resulting platform provides a physiologically relevant approach for studying particulate toxicity and therapeutic modulation at the alveolar interface.

Nanodiscs are nanoscale lipid bilayer membrane mimetics surrounded by two membrane scaffold proteins (MSP). They are widely used as soluble cassettes for membrane proteins and lipids in diverse applications. The original MSP1 was derived directly from human apolipoprotein A-1, and novel constructs have been adapted from this original design, including nanodiscs with larger sizes and covalent circularization. Here, we developed MSPs with a range of different fluorescent C-terminal protein tags, including a versatile HaloTag fusion. These fluorescent MSP were purified following typical MSP purification procedures with similar yield. Then, we demonstrate that fluorescent MSPs form nanodiscs with similar structure and stoichiometry to conventional MSP nanodiscs. These fluorescent MSP constructs enable a range of different applications and provide a versatile template for future design of nanodiscs with unique functions. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/716332v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@f85870org.highwire.dtl.DTLVardef@764055org.highwire.dtl.DTLVardef@179b7c5org.highwire.dtl.DTLVardef@ff6a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Dragonfly and cicada wing-inspired titanium nanopillar surfaces show promising bactericidal properties for antibacterial medical implant applications, but the precise mechanisms of bacteria-nanopillar interactions under hydrated conditions remain unclear. Cryo-electron tomography (cryo-ET) enables the visualisation of cellular organelles within their native hydrated cellular environment at molecular resolution. Visualising the bacteria-material interface on nanostructured surfaces by cryo transmission electron microscopy (cryo-TEM) requires the preparation of thin lamellae. Obtaining lamellae of bacteria directly on metal substrates while in a non-fixed and hydrated state requires cryo-focused ion beam (cryo-FIB) milling to isolate the targeted bacteria from the bulk sample. This approach faces additional challenges compared to tissues or cells on TEM grids, as titanium samples require a simultaneous cross-section of soft and hard materials at the same position and require vitrification, which embeds the sample in a thick layer of ice. Nonetheless, we demonstrate how to target a specific bacterium interacting with a titanium nanopillar surface using correlative cryo-fluorescence imaging, and how lamellae can still be prepared from vitrified samples by extracting the targeted bacterium and its surrounding as a small volume and transferring it to a receptor grid for thin lamella preparation, called targeted cryo-lift-out. Here, we outline the workflows and discuss their advantages and limitations for producing lamellae through lift-out techniques under cryogenic conditions, using methods that do not involve gas injection systems (GIS) for the lift-out transfer. These advances enhance cryo-ET applications, enabling in situ investigations of the interface between bacteria and nanopillars to effectively study the bactericidal mechanisms of biomimetic nature-inspired nanotopographies in a hydrated environment.

Micro and nanoplastics are pollutants which concentration in different biotopes increases continuously over time, which poses the question of their potential effects on health. In animals, these micro and nanoplastics are recognized as particulate materials and thus handled by macrophages, which are therefore a key cell type to study. Most studies have used an experimental scheme in which the cells are exposed to a single dose of plastics, with a readout made immediately after exposure. However, this classical experimental scheme does not take into account the impact of biopersistence, nor the potential cellular adaptation that may take place when cells are exposed repeatedly to a low dose of plastics. We thus used a repeated exposure scheme, in order to better take into account these phenomena. Within this frame, we compared the macrophages responses to a persistent nanoplastic, i.e. polystyrene nanoparticles and to a biodegradable nanoplastic, i.e. polylactide, by a combination of proteomic and targeted experiments. Our results show that under this repeated exposure scheme, the proteome changes were of a lesser (for PS) or similar (for PLA) extent than under the acute exposure mode, indicating cell adaptation. However, PLA particles induced mitochondrial dysfunction and depression of response to bacterial molecules perceived as danger signals, such as lipopolysaccharide. Polystyrene nanoparticles also induced a slight alteration of the immune functions of macrophages. This indicates harmful effects even in the repeated exposure scheme.

Human Neutrophil Elastase (HNE) plays a vital role in several inflammatory diseases, however its role in the tumour microenvironment and the potential in cancer treatment is still unrevealed. Considering the potential of {beta}-lactams as HNE inhibitors, the present work describes the development of a synthetic strategy to obtain two different types (Type I and Type II) of quenched activity-based probes (qABPs), using a {beta}-lactam ring as a warhead and BODIPY-FL as a fluorophore. The two types differ in mechanism and relative position between the fluorophore and the quencher moiety. The qABPs synthesized presented IC50 values against HNE lower than 0.5 {micro}M, and high selectivity compared with homologous serine hydrolases. Type II qABPs showed a more efficient turn-on mechanism, and selectively targeted HNE in different cell lysates. The qABP 22 was internalized in U937 cells and in human neutrophils and successfully targeted HNE in both.

Evaluation of unintended immunotoxicity represents an important component of nonclinical safety assessment, as perturbation of immune function may increase susceptibility to infection, impair vaccine responses, and disrupt immune homeostasis. Regulatory guidance, including the ICH S8 Immunotoxicity Guideline, recommends a weight-of-evidence approach in which observations from conventional toxicological endpoints are integrated with functional immune assays to support interpretation of immune system effects. The present study applied an integrated immunotoxicity evaluation framework to examine concordance among structural, functional, and cellular immune endpoints in male Sprague-Dawley rats using a well-characterized immunosuppressive reference compound. Hematological evaluation revealed leukopenia characterized primarily by lymphocyte depletion. Reductions in spleen and thymus weights were accompanied by histopathological evidence of lymphoid depletion in multiple immune tissues, including spleen, thymus, lymph nodes, Peyers patches, and bone marrow. Functional immune competence was assessed through hemagglutination antibody response to sheep red blood cells and delayed-type hypersensitivity assays, both of which demonstrated marked suppression of adaptive immune responses. Flow cytometric immunophenotyping further demonstrated substantial reductions in B-cell populations and decreases in CD4 and CD8 T-cell counts, whereas NK cell populations were comparatively less affected. The concordance of hematological alterations, lymphoid tissue changes, impaired functional immune responses, and lymphocyte subset depletion provides integrated evidence of immune system perturbation. These findings demonstrate that complementary immunotoxicity endpoints collectively support hazard characterization of immune system effects under GLP conditions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/713556v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@beaf9dorg.highwire.dtl.DTLVardef@fb9f10org.highwire.dtl.DTLVardef@187ff06org.highwire.dtl.DTLVardef@1780dc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Inflammation serves as a vital physiological process essential for preserving health and countering illness. Yet, persistent inflammation drives osteoclastogenesis and ongoing bone erosion in rheumatoid arthritis (RA), mainly via macrophage activation and overproduction of pro-inflammatory cytokines like TNF-, IL-1{beta}, and IL-6. Limitations of prolonged conventional treatments underscore the need for safer small-molecule inhibitors that address both inflammation and osteoclast formation. Chalcones, natural plant defense compounds, exhibit diverse pharmacological properties including anti-inflammatory, anticancer, antibacterial, antifungal, and antiparasitic actions, owing to their characteristic reactive , {beta}- unsaturated carbonyl moiety. This study assessed chalcone derivative 7a for its anti-inflammatory effects in vitro and in vivo, alongside its capacity to modulate osteoclast differentiation, offering the inaugural demonstration of its dual anti-inflammatory and anti-osteoclastogenic properties. In LPS-stimulated macrophages, 7a substantially curtailed nitric oxide production, curbed pro-inflammatory cytokines (TNF-, IL-1{beta}, IL-6), and concentration-dependently diminished iNOS and COX-2 expression while inhibiting reactive oxygen species levels. In vivo, oral 7a dosing potently alleviated carrageenan-evoked paw swelling and restored serum lactate dehydrogenase and C-reactive protein to normalcy. In LPS-exposed mice, it further lowered systemic cytokines and rectified dysregulated biomarkers such as LDH, ALP, ALT, AST, creatinine, and urea. Moreover, in RANKL-stimulated osteoclast cultures, 7a markedly suppressed osteoclastogenesis by downregulating pivotal markers like tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase-9 (MMP-9). Derivative 7a also enhances antioxidant defense--superoxide dismutase and catalase--via blockade of NF-{kappa}B and MAPK pathways. Overall, chalcone derivative 7a displays robust anti-inflammatory and anti-osteoclastogenic activity, positioning it as a compelling candidate for RA therapy.