Targeting ZBP1-mediated PANoptosis: inflammation responsive selenized chitosan nanoparticles loaded with Moringa A for anti-viral pneumonia therapy

Targeting ZBP1-mediated PANoptosis: inflammation responsive selenized chitosan nanoparticles loaded with Moringa A for anti-viral pneumonia therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Targeting ZBP1-mediated PANoptosis: inflammation responsive selenized chitosan nanoparticles loaded with Moringa A for anti-viral pneumonia therapy Wenhui Wu, Ruidong Li, Chunmei Lv, Dandan Yang, Shunqiang Song, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6582119/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Viral pneumonia poses a major global public health challenge, where excessive inflammatory responses contribute to tissue damage and respiratory failure. Inflammation-responsive nanoparticles can target inflamed areas, improving drug delivery while minimizing side effects. Chitosan, a biocompatible polysaccharide with anti-inflammatory and immunomodulatory properties, gains enhanced antioxidant and anti-inflammatory capabilities when combined with selenium. This study developed selenium-chitosan nanoparticles loaded with MoringaA (MA), a natural antiviral compound from Moringa oleifera seeds. These nanoparticles target lung inflammation, releasing MA to suppress viral replication and infection while reducing inflammatory responses. Additionally, selenium-chitosan nanoparticles mitigate oxidative stress, regulate immunity, and inhibit PANoptosis—a cell death pathway that exacerbates inflammation. By blocking core proteins in this pathway, they further curb inflammatory factor release. This approach offers a promising therapeutic strategy for viral pneumonia, combining targeted drug delivery, antiviral action, and inflammation control with reduced side effects. Viral pneumonia Inflammation-responsive nanoparticles MoringaA Selenium Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Viral pneumonia poses a serious threat to public health. It is caused by a variety of viruses, such as influenza virus, respiratory syncytial virus, coronavirus, and so on. It is extremely harmful to infants, the elderly, and people with immunodeficiency(Cilloniz et al. 2024 ). It can not only cause symptoms like fever, cough, and breathing difficulties, but may also lead to severe complications such as respiratory failure, liver failure, and heart failure, and even endanger lives. However, current treatments face numerous challenges. On the one hand, new mutant viruses, like SARS-CoV-2, keep emerging, bringing difficulties to prevention, control, and treatment. On the other hand, there is a lack of broad-spectrum antiviral drugs, making it difficult to deal with multiple viral infections. Moreover, existing drugs may have issues such as drug resistance and side effects, and the development of precise treatment plans is hindered by individual differences and viral diversity(Watkins 2022 , Zhang H. et al. 2025 ). Research shows that viral infections may trigger an over - activation of the body's immune system, leading to a pathological state known as a "cytokine storm." This abnormal immune response causes a large - scale release of inflammatory mediators such as IL-6 and TNF-α in a short period, thereby triggering systemic inflammation. This uncontrolled inflammatory cascade not only directly damages alveolar tissue, leading to the occurrence of acute respiratory distress syndrome (ARDS), but may also affect organs such as the heart, liver, and kidneys, ultimately resulting in multiple organ dysfunction(Cabler et al. 2020 , Jiang et al. 2022 ). Therefore, while combating viruses, the control of inflammation cannot be ignored. Moringa seeds are the seeds of the M oringa oleifera tree , belonging to the genus Moringa Adans of the family Moringaceae . They have extensive edible and medicinal values(Gautier et al. 2022 ). In our previous research, we conducted isolation and screening of the active ingredients in Moringa seeds with the guidance of antiviral activity. A novel - structured compound with remarkable anti - influenza virus activity was obtained from the acetone - extracted phase of the methanol extract of moringa seeds, and it was named Moringa A (MA)(Xiong et al. 2022 ). Its chemical structure is shown in Fig. 1 A. Previous studies have found that MA can exert a broad - spectrum anti - influenza virus effect by regulating the autophagy - lysosome pathway, making it a highly promising small - molecule compound against influenza virus(Xiong et al. 2021 ). However, the low solubility of MA limits its bioavailability and efficacy. Due to its poor solubility, its absorption in the gastrointestinal tract is restricted, resulting in a reduced amount of the drug entering the systemic circulation and a decrease in bioavailability. At the same time, the low solubility also delays the release and onset time of MA. It may be difficult for the blood drug concentration to reach an effective level, thus weakening the therapeutic effect. To improve its antiviral effect, this project aims to use a chitosan - based nano delivery system to improve the solubility of MA, enhance its bioavailability, and increase its efficacy. The chitosan nanoparticle drug - delivery system can enhance the stability of drugs, control drug release, and improve the cellular uptake ability of drugs. It is an excellent nano - delivery carrier. Selenium nanoparticles (SeNPs) is a kind of particles with a unique nanostructure. SeNPs can not only effectively scavenge oxygen free radicals, but also significantly reduce the secretion level of pro-inflammatory factors such as IL-6 by regulating the activity of immune cells, thus showing a powerful anti-inflammatory effect(Sun et al. 2023 , Wang S. et al. 2023 ). Its mechanism of action involves activating antioxidant enzymes (such as glutathione peroxidase) to enhance the antioxidant capacity of cells and further alleviate the inflammation caused by oxidative stress. Its remarkable efficacy in multiple inflammatory disease models indicates that SeNPs has broad application potential in anti-inflammatory treatment. However, due to its high surface energy and chemical activity, SeNPs are prone to aggregation and oxidation reactions. These reactions can lead to changes in their structure and surface properties, thereby affecting their stability and function(Bian et al. 2024 , Yang et al. 2022 ). This instability may cause SeNPs to lose their unique nanoscale properties during storage or application, restricting their applications in fields such as biomedicine and catalysis. To improve the stability of SeNPs, it is often necessary to protect them through methods such as surface modification, coating, or treatment with dispersants. To overcome the stability problem of SeNPs, we selected chitosan as the carrier to prepare selenized chitosan. Selenized chitosan is a functional composite material formed by combining selenium with chitosan, which combines the biocompatibility and biodegradability of chitosan with the antioxidant and anti - inflammatory properties of selenium. The selenium element in its structure exists in the form of covalent bonds or coordination bonds, which significantly improves its stability and biological activity(Gao et al. 2020 ). Selenized chitosan not only retains the antibacterial and repair - promoting functions of chitosan but also enhances its antioxidant and immunomodulatory abilities. Research shows that selenium is embedded in the polymer network in a stable coordination form, and this molecular - level synergy endows the material with multiple advantages in fields such as drug controlled release and tissue repair(Gao et al. 2020 ). Hyaluronic acid (HA) is an acidic mucopolysaccharide naturally present in human tissues. It has multiple important physiological functions in the body and is widely used in many different medical fields. From a chemical structure perspective, hyaluronic acid is composed of alternating glucuronic acid and N-acetylglucosamine units. This special molecular arrangement gives it excellent biocompatibility and biodegradability, which provides great advantages when it is used in medical applications such as drug carriers(Lei et al. 2021 , Zhang X. et al. 2021 ). In the field of respiratory diseases, hyaluronic acid has shown potential value in targeted treatment of pneumonia. The core therapeutic mechanism of hyaluronic acid lies in its ability to specifically recognize the abnormally elevated CD44 receptor in an inflammatory state. When pneumonia occurs in the lungs, the expression level of the CD44 receptor on the cell surface increases significantly, and hyaluronic acid can specifically bind to it, providing an ideal target - binding basis for the targeted treatment of pneumonia(Marinho et al. 2021 , Salathia et al. 2023 ). In this study, selenized chitosan is used as the carrier to encapsulate MA, and then HA is used to modify the surface of the selenized chitosan loaded with MA. A nano - targeted drug responsive to the pulmonary inflammatory micro - environment is designed to deliver MA to the pulmonary inflammatory region, enhance its antiviral pneumonia activity, and integrate the inhibitory effect of nano - selenium on pulmonary inflammatory factors and the antiviral effect of MA. 2. Materials and methods 2.1. Materials Moringa A (MA): Lab-synthesized (in-house preparation). Sodium selenite (Cat. No. SLCN8615; Merck Life Science Technology Co., Ltd). Ascorbic acid (Cat. No. RH561123), hyaluronic acid (Cat. No. RH44689), and chitosan (Cat. No. RH482374) were purchased from Guangzhou Ronn Biological Technology Co., Ltd. Fetal bovine serum (Lot No. 10099-141) and DMEM High Sugar Medium (Lot No. C11995) were purchased from Gibco. Cellular Protein Extraction Kit (Item No. E-BC-E002), purchased from Wuhan Eliot Bio-Tech Co. Mice IL-1β (Cat no. E-EL-M0037), TNF-α (Cat no. E-EL-M0048), IL-18(Cat no. E-EL-M0730), IL-6(Cat no. E-EL-M0043), IL-10(Cat no. E-EL-M1210 46), SOD(Cat no. E-EL-H6188) and Glutathione peroxidase(GPx, Cat no. E-EL-R2491) Elisa kit were purchased from Elabscience Biotechnology Co., Ltd. 2',7'-Dichlorofluorescin diacetate (Cat no. ab273640), anti ZBP1 antibody (Cat. no. sc-271483), RIPK3 antibody (Cat no. sc-374639), anti-MLKL antibody (Cat no. sc-293201), GSDMD antibody (Cat no. se-393581), anti-Caspase-7 antibody (Cat no. se-56066), anti-Caspase-3 antibody (Cat no. se-81655) were purchased from Santa Cruz Biotechnology, Inc. Anti-NF-kB p65 antibody (Cat no. ab32536) and anti-GAPDH (Cat no. ab8245) were purchased from Abcam. Horseradish enzyme-labeled goat anti-rabbit IgG (Cat no. SA0012) and BCA protein concentration determination kit (Cat no. PC0020) were purchased from Beijing Solebao Technology Co., Ltd. LPS was purchased from Sigma. 2.2. Virus and Cell lines Influenza A virus A/Puerto Rico/8/34, obtained from the American Type Culture Collection (ATCC). The mouse lung epithelial cell line MLE-12 and RAW 264.7 were purchased from Shanghai Binsui Biotechnology Co., Ltd. 2.3. Preparation and characterization of HA-modified selenized chitosan nanoparticles loading MA(Se@CS@MA@HANPs) The preparation of HA - modified selenized chitosan nanoparticles loading MA referred to the preparation method of nano-selenium by previous reports(Wang W. et al. 2021 , Wu et al. 2022 ). Briefly, under magnetic stirring, 2 mL of a 0.5% (mass fraction) chitosan (CS) solution was thoroughly mixed with 2 mL of a 0.1 M sodium selenite (Na₂SeO₃) solution, and the volume was adjusted to 10 mL with purified water. An appropriate amount of a 0.5 M ascorbic acid solution (Vc) was added dropwise to the mixture, and the volume was adjusted to 20 mL with water. The mixture was stirred at a speed of 500 rpm for 60 min, and then dialyzed for 48 h using a dialysis bag with a molecular weight cut - off of 8,000chang-14,000 (10000 kDa). The dialysate was replaced every 12 h. The Se@CSNPs were collected and stored at 4°C for later use. 10 mg of MA was weighed and dissolved it in 10 mL of a 0.5 mg·mL⁻¹ sodium tripolyphosphate (STPP) solution. Then, 3 mL of the above-mentioned MA solution was added to the selenized chitosan carrier solution. After stirring at 800 rpm for 30 min, a certain amount of hyaluronic acid solution was added so that the drug accounted for a volume ratio of 1:8 of the total volume. The Se@CS@MA@HANPs (abbreviated as MA + Se NPs) were collected and stored at 4°C for later use. The schematic illustration of MA + Se NPs synthesis was shown in Fig. 1 A. The prepared nanoparticles were characterized by using a laser particle size analyzer, Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), Fourier Transform Infrared Spectroscopy (FT-IR), and X-Ray Diffraction (XRD). 2.4. Determination of cumulative release rate In this experiment, the dialysis method was used to conduct in vitro drug release studies. The temperature of the water-bath shaker was set at 37°C, and the oscillation frequency was maintained at a constant speed of 100 r·min⁻¹. Three parallel samples were set for each group. MA powder was added to PBS solution to prepare a liquid equivalent to 0.6 mg·mL⁻¹ of MA. 1 mL of the liquid was taken and placed in a dialysis bag, which was then immersed in 10 mL of PBS (pH = 7.4) solution. Samples of 1 mL were taken at time points (0.33, 0.67, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72 h), and 1 mL of the release medium at the same temperature was added. The released samples were injected for analysis under the chromatographic conditions described in 2.1.2.1 of Chap. 1. The peak areas were recorded, and the cumulative release rate Q was calculated. In the same way, a PBS solution of MA + Se NPs test samples with the same concentration was used as a control for the in vitro release test. The peak areas were recorded, and the cumulative dissolution rate was calculated. 2.5. Drug stability investigation The Se@CS NPs, Se@CS@MA NPs, and Se@CS@MA@HA NPs were stored at 4°C for 30 days. The particle size and zeta potential of the nanoparticles were measured every 5 days using a laser particle size analyzer to evaluate their stability. 2.6. Cellular uptake Coumarin6(C6) was used as a probe to investigate the uptake of nanoparticles by cells. For RAW264.7 cells, three groups were set up: the Free C6 group, the Se@CS@C6NPs group, and the Se@CS@C6@HANPs group, with 3 replicate wells in each group. 500 µL of cell suspension with a density of 1×10⁴ cells per well was inoculated into 24 - well plates containing cover slips and cultured in an incubator for 24 h. After aspirating and discarding the old culture medium, three types of culture media containing coumarin 6 were added respectively. After incubation in the incubator for 2 h, the old culture medium was aspirated and discarded, and the cells were washed 3 times with cold PBS. Then, the cells were fixed with 4% paraformaldehyde for 20 min and washed 3 times with cold PBS again. The cover slips were taken out and mounted on slides with anti - fluorescence quenching mounting medium containing DAPI for 10 min. Subsequently, each slide was placed under a laser confocal scanning microscope to observe the cell uptake of each coumarin 6 preparation group. For LPS-induced M1 macrophages, the Se@CS@C6NPs group and the Se@CS@C6@HANPs group were set up, and the cell uptake experiment was carried out in the same way. 2.7. Cell culture and treatments The MLE-12 cell line was maintained in phenol red-free high-glucose DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Following a 24-hour incubation at 37°C under 5% CO₂, the cells were allocated into five experimental groups (six replicates per group): Normal (Nor): Untreated cells, Virus (Vir): H1N1-infected cells (MOI = 5), MA + Se NPs (1 µg/mL Se@CS@MA@HANPs), MA NPs (1 µg/mL CS@MA@HANPs) and Se NPs (1 µg/mL Se@CS@HANPs). All groups except Con were exposed to H1N1 at an MOI of 5 to induce infection, while Con and Vir received blank medium. Post-treatment, cells were cultured for 48 h (37°C, 5% CO₂), with daily monitoring of cytopathic effects (CPE). Cell viability was assessed using the CCK-8 assay. The levels of IL-6, TNF-α, IL-1β and IL-10 in the supernatants of cells from each experimental group were detected by ELISA. 2.8. ROS Detection Intracellular reactive oxygen species (ROS) levels were quantified using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). MLE-12 cells were plated in 96-well plates at a density of 1×10⁴ cells per well and allowed to adhere for 24 hours. Following this incubation period, cells were treated with either curcumin or DMSO vehicle control for 48 hours. As a positive control, tert-butyl hydroperoxide (tBHP) was administered for 4 hours prior to staining.For the detection procedure, culture medium was first removed and cells were washed with 100 µL of 1X assay buffer per well. After buffer removal, cells were incubated with 100 µL of 10 µM DCFH-DA solution at 37°C for 45 minutes under light-protected conditions. Following dye removal, fluorescence intensity was measured immediately using a microplate reader with excitation/emission wavelengths set at 485/535 nm. Quantitative image analysis was performed using ImageJ software, with fluorescence intensities normalized to control group values. 2.9. Detection of H1N1 NP protein in cells The expression of the H1N1 viral NP protein in cells from each experimental group was detected and co-localized using immunofluorescence and confocal laser microscopy to assess the changes in viral particles within host cells after drug treatment. 2.10. Animal experiments SPF KM mice were obtained from Hunan Silek Jingda Laboratory Animal Co., Ltd. and acclimatized for seven days under standardized conditions. Then mice were randomly divided into five groups according to their body weight: the Normal group (Nor), the Model group (Mod), MA + Se NPs Group (treated by Se@CS@MA@HANPs), MA NPs Group (treated by CS@MA@HANPs), and Se NPs (treated by Se@CS @HANPs Group), with eight mice in each group. On the first day, all mice except those in the Nor group, were intranasally infected with ×10 MLD 50 of H1N1 viruses in a 50 µL volume. Mice in the treatment group were administered the corresponding nanoparticles intranasally at a dose of 5 ml/kg daily, while the mice in the Nor and Mod groups received equivalent volumes of deionized water daily. The body weight and survival rate of the mice in each group were monitored daily. On Day 14 post-infection (p.i.), mice were euthanized by cervical dislocation, and lungs were dissected out. Here, all of the animal experimental protocols were approved by the Ethics Committee of Zunyi Medical University (Permit No. ZMU24-2418-02). All animal care and experimental procedures were performed in accordance with the Laboratory Animal Welfare and Ethics Committee of China. 2.10.1. Lung index and Hematoxylin and eosin (H&E) staining Under sterile conditions, the lung tissues was rapidly excised, washed with PBS to remove residual surface blood, and then placed on sterile filter paper to remove excess moisture. The weight of the lung tissue was measured, and the lung index was calculated (lung tissue weight/body weight × 100%). The degree of pulmonary edema in the lungs of mice in each group was analyzed. Besides, lung tissues were fixed. The samples were dehydrated in ethanol, embedded with paraffin and sliced. Then the lung tissues were deparaffinized in xylene for 10 min, stained with H&E for histopathological examination of lung inflammation. 2.10.2. Lung tissue cytokines analysis 100 mg of lung tissue from each experimental group of mice was taken, homogenized in a homogenizer after adding PBS (lung tissue:PBS = 1:9), and then centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected, and the levels of IL-18, IL-1β, TNF-α, IL-6, SOD and GPx in the lung tissue homogenate were detected using the ELISA method. 2.10.3. Immunohistochemical Detection Mice lung tissues were taken and fixed with 4% paraformaldehyde, then dehydrated with gradient ethanol, cleared with xylene, and embedded in paraffin. The tissue was cut into sections 4 –5µm thick. For antigen retrieval, the sections were placed in 10 mM sodium citrate buffer (pH 6.0) and heated in a microwave for 8–15 minutes. After blocking with 3% BSA-PBS at room temperature for 30 minutes, NP antibody (diluted at 1:1000) was added and incubated overnight at 4℃. Following PBS washes, goat anti-rabbit IgG was added and incubated at room temperature for 1 hour. The sections were then treated with streptavidin-horseradish peroxidase (SA-HRP) complex and developed with DAB (3,3'-diaminobenzidine). The sections were counterstained with hematoxylin and mounted with neutral gum. Finally, the sections were observed under an optical microscope, and the expression of NP in the mouse lung tissue was assessed based on the staining results. 2.11. QRT-PCR Approximately 15–20 mg of pulmonary tissue was homogenized in 300 µL of lysis buffer using a high-speed tissue disruptor to generate a uniform cell suspension. The extracted total RNA was quantified via ultra-micro spectrophotometry and subsequently reverse-transcribed into complementary DNA (cDNA). For quantitative analysis, SYBR Green-based real-time PCR (Qiagen, Hilden, Germany) was employed to assess mRNA expression. The primer sequences targeted the murine housekeeping gene PPIA and the influenza viral M segment, designed as follows:Viral M gene: Forward: 5′-CTTCTAACCGAGGTCGAAAC-3′, Reverse: 5′-CGTCTACG CTGCAGTCCTC-3′, PPIA (internal control): Forward: 5′-CGCTTGCTGCAGCCATGGTC-3′, Reverse: 5′-CAGCTCGAAGGAGACGCGGC-3′. The relative quantification of influenza virus M gene copies was computed using the 2 −ΔΔCT method for comparative threshold cycle analysis. 2.12. Effect of Se-MA NPs on PANoptosis of MLE-12 cell The MLE-12 cell line was maintained in phenol red-free high-glucose DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Following a 24-hour incubation at 37°C under 5% CO₂, the cells were allocated into three experimental groups (six replicates per group): Normal (Nor): untreated cells, Virus (Vir): H1N1-infected cells (MOI = 5) and MA + Se NPs (1 µg/mL Se@CS@MA@HANPs). All groups except Con were exposed to H1N1 at an MOI of 5 to induce infection, while Con and Vir received blank medium. Post-treatment, cells were cultured for 48 h (37°C, 5% CO₂). Cells were lysed using RIPA lysis buffer to extract total protein, and the protein concentration was determined using the BCA kit or other methods. Based on the protein concentration, an appropriate amount of protein sample was mixed with loading buffer, boiled to denature the proteins, and then subjected to SDS-PAGE electrophoresis. The gel concentration was selected according to the molecular weight of the target proteins to achieve separation. The separated proteins were transferred from the gel to a PVDF membrane using the wet transfer method, with appropriate voltage and time settings to ensure complete transfer. The membrane was blocked with 5% skimmed milk powder at room temperature for 1 hour. Specific primary antibodies against ZBP1, RIPK3, MLKL, GSDMD, Caspase-3, and Caspase-7 (diluted at a ratio of 1:1000) were added and incubated overnight at 4℃. The membrane was then washed 3–5 times with TBST for 5–10 minutes each time. HRP-conjugated secondary antibodies matching the primary antibodies were added and incubated at room temperature for 1 hour. The membrane was washed again 3–5 times with TBST to remove unbound secondary antibodies. Finally, ECL chemiluminescent substrate was added, and the membrane was imaged using a chemiluminescent imaging system in a dark room to analyze the expression levels of the target proteins. 2.13. Evaluation of the biosafety of MA + SeNPs in mice Mice were divided into two groups (n = 10 per group): the PBS group and the MA + SeNPs group. Both groups received daily intranasal administration of PBS or MA + SeNPs for 14 consecutive days. At the end of the experiment, all mice were humanely euthanized, and major organs (heart, liver, spleen, lungs, kidneys, and brain) were collected for histopathological examination using H&E staining. 2.14. Statistical analysis The data was expressed as mean ± standard error of mean (SEM). Statistical analysis was conducted using one-way ANOVA with Duncan's multiple comparison test on SPSS 27.0 software. All difference with p < 0.05 were considered to indicate significant. 3. Results 3.1. Characterization of S Se@CS@MA@HANPs SEM and TEM analyses of the prepared nanoparticles revealed that Se@CS@MA@HANPs exhibited a quasi-spherical structure with good dispersibility (Fig. 1 B). Based on SEM and TEM results, energy-dispersive X-ray spectroscopy (EDS) was further employed to analyze the elemental composition and distribution of Se in Se@CS@MA@HANPs, with the results shown in Fig. 1 C. In the prepared nanoparticles, selenium accounted for 45.31%, carbon for 6.48%, and oxygen for 21%. The signal of selenium was primarily concentrated within the signal regions of carbon and oxygen, showing a highly overlapping distribution with these elements. This suggests that selenium is likely uniformly encapsulated in the form of nanoparticles within an organic matrix rich in carbon and oxygen, indicating a good binding or encapsulation relationship between selenium and the organic components. Additionally, particle size analysis revealed that on the basis of the spherical structure of Se@CSNPs (with a particle size of 66.32 ± 0.68 nm), the particle size of Se@CS@MANPs increased to 113.49 ± 1.40 nm upon drug loading. After modification with hyaluronic acid (HA), the particle size of Se@CS@MA@HANPs further increased to 208.80 ± 6.46 nm, with a zeta potential of 12.85 ± 2.46 mV, as shown in Figs. 1 C and 8D. Further surface modification of Se@CS@MANPs with HA was confirmed by SEM, as illustrated in Figs. 8E and 8F. FTIR was further employed to characterize different nano-selenium particles as well as MA and HA, as shown in Fig. 1 E. The broad peak at 3400 cm⁻¹ corresponds to the characteristic absorption of O-H stretching vibrations, indicating the presence of intramolecular or intermolecular hydrogen bonds. The peaks near 2920 cm⁻¹ and 1612 cm⁻¹ in Se@CSNPs (a), Se@CS@MANPs (b), and Se@CS@MA@HANPs (c) are attributed to the C-H stretching vibrations of methyl and methylene groups from residual sugar moieties and the carbonyl C = O stretching vibrations, respectively. The absorption peak at 1384 cm⁻¹ is typically associated with the symmetric deformation vibration of -CH₃ (methyl) groups. The C-O stretching vibration absorption peaks are observed at 1300 cm⁻¹ and 1070 cm⁻¹, while the peak near 897 cm⁻¹ corresponds to the characteristic absorption of the β-glycosidic bond in chitosan. All these absorption peaks exhibited varying degrees of redshift upon the addition of MA and HA. The peak near 1450 cm⁻¹ in Se@CS@MANPs (b), Se@CS@MA@HANPs (c), and MA (d) is characteristic of aromatic rings, confirming the successful dispersion of MA on the surface of nano-selenium. The peak near 1662 cm⁻¹ in Se@CS@MA@HANPs (c) and HA (e) corresponds to the C = O stretching vibration in secondary amides, indicating the successful encapsulation of nano-selenium particles by HA. The intensity and sharpness of X-ray diffraction peaks can reflect the crystalline nature of selenium to some extent. As shown in Fig. 1 F, the X-ray diffraction pattern of Se@CSNPs (a) exhibits two sharp characteristic peaks at 2θ = 24° and 30°, along with broad diffraction peaks in the range of 20° to 40°, indicating that SeNPs exist in an amorphous form. HA (e) shows broad diffraction peaks in the range of 10° to 40°, indicating its amorphous nature. MA (d) has a characteristic peak at 42°. The diffraction patterns of Se@CS@MANPs (b) and Se@CS@MA@HANPs (c) not only retain the characteristic peaks of Se@CS but also preserve the 42° peak of MA, suggesting that the combination of Se@CSNPs with MA maintains their respective structural regularities. 3.2. The stability of the drug Figure 2 illustrates the changes in particle size and zeta potential of nano-selenium over a period of 30 days. As shown in the figure, freshly prepared Se@CSNPs (A), Se@CS@MANPs (B), and Se@CS@MA@HANPs (C) all exhibited an orange-red color and were clear and transparent. Subsequently, Se@CS gradually turned dark red, while the other two showed no significant color change. It is speculated that Se@CS might have been oxidized, leading to the gradual transformation of nano-selenium into elemental selenium. In contrast, the other two formulations remained relatively stable after drug loading. From the particle size changes depicted in the figure, we can observe that the particle size of Se@CSNPs increased rapidly from 66 ± 0.68 nm within the first 10 days and eventually stabilized around 99 ± 2.2 nm. The particle sizes of the other two formulations changed little, which also indicates that the stability of the nanoparticles was enhanced after drug loading. The zeta potential values shown in the figure for Se@CSNPs (A), Se@CS@MANPs (B), and Se@CS@MA@HANPs (C) were 22.53 ± 3.01 mV, 19.23 ± 2.01 mV, and 12.85 ± 2.46 mV, respectively. All three exhibited a downward trend over the 30-day period, eventually stabilizing around 8 mV. The zeta potential of Se@CS decreased the most, a result consistent with the particle size observations. In summary, we can conclude that the stability of Se@CS was significantly improved after drug loading. 3.3 The uptake of nanoparticles by cells In this study, Coumarin 6 was used as a model probe to investigate the uptake of the prepared nanoparticles by RAW264.7 cells. As shown in Fig. 3 A, RAW264.7 cells exhibited weak green fluorescence in the cytoplasm, indicating that the uptake of free Coumarin 6 by cells was minimal. Compared with cells treated with free Coumarin 6, cells treated with Se@CS@C6NPs and Se@CS@C6@HANPs showed significantly enhanced green fluorescence, with the strongest fluorescence observed in the Se@CS@C6@HANPs group. This suggests that the prepared nanoparticles possess good characteristics for transmembrane transport across cell membranes. To further investigate the specific affinity of Se@CS@HANPs for inflammatory cells, this study used LPS (lipopolysaccharide)-induced M1-type macrophages differentiated from RAW264.7 cells as a model to observe the uptake of Se@CS@C6@HANPs and Se@CS@C6NPs by these cells within 2 hours. The results showed that M1 macrophages treated with Se@CS@C6@HANPs exhibited significantly higher fluorescence intensity compared to those treated with Se@CS@C6NPs, indicating that nanoparticles modified with hyaluronic acid (HA) have better affinity for inflammatory cells, as shown in Fig. 3 B. 3.4 In vitro antiviral and anti-inflammatory effects of nanoparticles To investigate the synergistic advantages of Se and MA combination therapy in antiviral and anti-inflammatory efficacy, MLE-12 cells infected with H1N1 for 24 hours were treated with SeNPs, MANPs, or inflammation-responsive nanoparticles co-loaded with Se and MA (MA + Se NPs) for 48 hours (Fig. 4 A). The results showed that SeNPs, MANPs, and MA + Se NPs all alleviated cytopathic effects (CPE) to varying degrees and inhibited H1N1-induced cell death. Notably, MA + Se NPs demonstrated significantly superior effects in mitigating CPE and enhancing cell viability compared to Se or MA alone (Fig. 4 B). Further analysis of pro- and anti-inflammatory cytokines in host cells revealed that H1N1 infection triggered a substantial increase in TNF-α, IL-1β, and IL-6 levels, while the anti-inflammatory cytokine IL-10 was reduced. After treatment with the three nanoparticles, TNF-α, IL-1β, and IL-6 levels in MLE-12 cells decreased significantly, whereas IL-10 levels increased. Importantly, MA + Se NPs exhibited markedly stronger reversal effects on these cytokines than Se or MA alone (Fig. 4 C– 4 F). These findings indicate that co-delivery of Se and MA exerts synergistic anti-inflammatory effects. Studies have shown that ROS can activate signaling pathways such as NF-κB and the NLRP3 inflammasome, promoting the expression and release of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6). Excessive ROS also induces oxidative stress, exacerbating inflammatory responses. Influenza virus infection further amplifies ROS production in host cells, triggering inflammation(Muhammad et al. 2022 ) z(Muhammad et al. 2022 ). In our study, H1N1-infected MLE-12 cells exhibited higher ROS levels than uninfected cells (Fig. 4 G). Treatment with SeNPs, MANPs, or MA + Se NPs reduced intracellular ROS, with SeNPs and MA + Se NPs showing the most pronounced effects, underscoring the critical role of Se in scavenging virus-induced ROS. Additionally, the combination of Se and MA exhibited a synergistic inhibitory effect against H1N1 virus. Immunofluorescence and qRT-PCR analyses of viral NP protein and M gene expression revealed that all three nanoparticles suppressed H1N1 viral NP protein and M gene to varying degrees, with MA + Se NPs demonstrating the most potent inhibition (Fig. 4 H– 4 J). 3.5 In vivo antiviral and anti-inflammatory effects of nanoparticles To evaluate the protective efficacy of MA + Se NPs against H1N1 infection in mice, intranasal delivery was employed. Mice infected with H1N1 were administered MA NPs, Se NPs, or MA + Se NPs, followed by monitoring of survival rates and body weight changes over 14 days. As illustrated in Fig. 5 A, by day 8 post-infection (dpi), the H1N1-challenged group exhibited a 20.12% reduction in body weight. In contrast, the MA NPs, Se NPs, and MA + Se NPs treatment groups demonstrated significantly lower weight losses of 11.6%, 9.66%, and 8.37%, respectively. Similarly, survival rates varied markedly among groups (Fig. 5 B). While only 12.5% of the virus-infected control mice survived, the MA NPs, Se NPs, and MA + Se NPs groups showed survival rates of 45%, 60%, and 90%, respectively. Notably, mice treated with three types of NPs displayed a modest reduction in weight loss between 2 and 8 dpi compared to the H1N1-infected group. By 8 dpi, surviving animals in the nanoparticle-treated groups began regaining weight, whereas the untreated infected mice showed minimal recovery. Control mice remained unaffected, displaying no clinical symptoms, weight decline, or mortality. These findings indicate that nanoparticles intervention can mitigate H1N1-induced weight loss and enhance survival in infected mice.At the same time, these three types of nanoparticles can also significantly reduce the lung index of mice (Fig. 5 C), and can improve the lung injury in mice caused by the H1N1virus to a certain extent (Fig. 5 D). It is worth noting that the combined use of MA and Se has a significantly better improvement effect on the above-mentioned indicators than when they are used alone. What’s more, the combined use of MA and Se has an important impact on the balanced regulation of immunity and inflammation in mice with viral pneumonia. As shown in Fig. 5 E, after being infected with the H1N1 virus, a large amount of inflammatory factors IL-1β, IL-18, IL-6 and TNF-α were produced in the lung tissues of mice, while the antioxidant factors SOD and GPx were significantly reduced, exhibiting oxidative inflammatory damage in lung tissue. However, after the mice were treated with MA and Se, the levels of IL-1β, IL-18, IL-6 and TNF-α in the lung tissues of the mice were effectively controlled, and the levels of SOD and GPx were effectively restored. Moreover, the reversing effect of the combined use of MA and Se on these cytokines was significantly better than that of using a MA and Se alone. Further detection by immunohistochemistry and qRT-PCR revealed abundant NP protein-positive cells (Fig. 5 F and 5 G) and viral M gene expression (Fig. 5 H) in the lung tissues of H1N1-infected mice. Following treatment with the three types of nanoparticles, both NP protein-positive cells and viral M gene levels were significantly reduced. Among them, MA + SeNPs exhibited the strongest antiviral efficacy, followed by MANPs, while SeNPs showed the weakest effect. These results fully demonstrate that the combination of MA and Se exerts a synergistic antiviral effect. To investigate the oxidative stress status in the lung tissues of viral pneumonia mice, we detected ROS levels in mouse lungs. As shown in Fig. 5 I, H1N1 infection induced massive ROS production in lung tissues. Studies indicate that excessive ROS can damage pulmonary cells (including epithelial and endothelial cells), leading to cell membrane lipid peroxidation, protein oxidative modification, and DNA damage, ultimately causing cellular dysfunction or even death(Zheng D. et al. 2022 ). This process further exacerbates pulmonary inflammatory responses by promoting the release of inflammatory cytokines, creating a vicious cycle of inflammation. Consequently, lung tissue structure and function become impaired, manifesting as pathological changes such as increased alveolar-capillary permeability, pulmonary edema, and fibrosis—severely compromising normal respiratory function(Ding et al. 2021 , Wiegman et al. 2020 ). Notably, nanodrug treatment significantly reduced ROS levels in lung tissues, particularly in the Se NPs and MA + Se NPs groups. These results clearly demonstrate the critical role of Se in scavenging excess ROS induced by viral infection. The above findings indicate that the co-delivery of MA and Se achieves coordinated control of viral load and inflammation in the lungs of viral pneumonia mice, while also effectively mitigating influenza virus-induced excessive oxidative stress in lung tissues. 3.6 MA + Se NPs inhibits influenza virus-induced PANoptosis in MLE-12 Cells Influenza virus infection exacerbates lung injury through the induction of PANoptosis, a process involving the activation of the host protein Z-DNA binding protein 1 (ZBP1) by viral RNA(Basavaraju et al. 2022 ). This activation triggers the rupture of the cell membrane via the RIPK3/MLKL pathway and the amplification of inflammation in a caspase-8-dependent manner. The death of alveolar epithelial and vascular endothelial cells leads to the collapse of barrier functions, resulting in the massive release of inflammatory factors (such as IL-6 and TNF-α) and damage-associated molecular patterns (DAMPs), which in turn trigger cytokine storms and immune cell infiltration(Cheng et al. 2025 ). PANoptosis acts in concert with pyroptosis (mediated by Gasdermin D) and apoptosis, further aggravating alveolar structural destruction, microthrombus formation, and hypoxia. The excessive inflammation and imbalance in tissue repair ultimately lead to acute respiratory distress syndrome (ARDS) and multi-organ failure, which are core pathological mechanisms underlying the severity of influenza. Targeted inhibition of ZBP1 or RIPK3 may serve as potential therapeutic strategies. The mechanism of PANoptosis is illustrated in Fig. 6 A. After influenza virus infection, the PANoptosis-related proteins ZBP1, RIPK3, MLKL, GSDMD, Caspase-3, and Caspase-7 were significantly upregulated in MLE-12 cells, as shown in Fig. 6 B- 6 H. indicating that the H1N1 virus triggers pyroptosis, apoptosis and necroptosis simultaneously via the ZBP1 pathway, which is defined as PANoptosis. However, after intervention with MA + Se NPs, these proteins were significantly downregulated, demonstrating the inhibition of PANoptosis. This may be an important mechanism by which MA + Se NPs suppress the cytokine storm in mouse lung tissues. 3.7 The biosafety evaluation of MA + Se NPs After 14 consecutive days of intranasal administration, we found that MA + Se NPs did not exhibit significant toxic effects on the heart, liver, spleen, lungs, or kidneys of mice, as shown in Fig. 7 . Histopathological examination of these vital organs revealed no observable cellular damage, inflammatory responses, or other abnormal pathological changes. These results demonstrate that MA + SeNPs possess excellent biocompatibility and low toxicity risks in vivo, indicating favorable biosafety profiles. 4. Discussion Virus-induced lung injury typically results from the dual effects of rapid viral replication and excessive host immune responses. While antiviral drugs alone can reduce viral load, they fail to promptly suppress inflammatory spread; conversely, anti-inflammatory therapy alone may lead to disease relapse and exacerbation due to incomplete viral clearance. Dual intervention, by simultaneously inhibiting viral replication and modulating inflammatory pathways, not only disrupts pathogen transmission but also mitigates tissue damage, thereby breaking the "virus-inflammation" positive feedback loop. This approach significantly reduces the risk of acute lung injury progressing to ARDS and improves patient survival rates. The synergistic mechanism of antiviral and anti-inflammatory actions provides a critical theoretical foundation for this study. MA, an antiviral compound, was combined with anti-inflammatory SeNPs to achieve multi-target antiviral effects, offering a novel nano-delivery strategy for the treatment of viral pneumonia. In viral pneumonia, ROS exacerbate lung injury through a dual mechanism. On the one hand, viral replication induces mitochondrial dysfunction and the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, leading to an excessive production of ROS. This triggers lipid peroxidation, protein inactivation, and DNA damage, directly destroying alveolar epithelial and vascular endothelial cells(Deng et al. 2024 ). On the other hand, ROS amplifies the inflammatory response by activating signaling pathways such as NF-κB and mitogen-activated protein kinases (MAPK), promoting the release of inflammatory cytokines (such as IL-6 and TNF-α) and forming a "cytokine storm"(Alva et al. 2023 ). Meanwhile, ROS collaborate with cell death pathways such as PANoptosis and pyroptosis. Through oxidative stress, ROS enhance the activity of the ZBP1/receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like (MLKL) pathway, exacerbating cell membrane rupture and the release of damage-associated molecular patterns (DAMPs), resulting in the collapse of the alveolar-capillary barrier, microthrombus formation, and hypoxia. Excessive ROS can also inhibit the antioxidant system (such as SOD and glutathione), creating a vicious cycle of oxidative stress, ultimately promoting the development of ARDS and multiple organ failure(Yuan et al. 2024 ). Regulating ROS levels is an important therapeutic strategy for controlling viral pneumonia. After infection with the H1N1 virus, we detected a large number of viral particles and ROS in the lung tissues of mice. In addition, inflammatory cytokines such as TNF-α, IL-1β, and IL-6 were highly active, while antioxidant factors such as SOD and GPx were decreased. This is clearly the direct cause of the high mortality rate of mice in the infected group and the severe pulmonary inflammation. After treatment with MA + Se NPs, the survival rate of the mice increased significantly. The viral particles and ROS in the lung tissues were significantly reduced. The lung index and the levels of pulmonary inflammatory cytokines were significantly decreased, and the inflammatory damage to the lung tissues was significantly alleviated. This indicates that MA + Se NPs have successfully achieved the synergistic effect of antiviral and anti-inflammatory pharmacodynamics. As an important intracellular pattern recognition receptor, ZBP1 can precisely recognize dangerous signals such as nucleic acids released during influenza virus infection(Zhang T. et al. 2020 ). Once it recognizes the molecular patterns associated with the influenza virus, ZBP1 rapidly activates its own functions, recruits key proteins such as RIPK1/3 and MLKL, and initiates the PANoptosis signaling pathway(Zheng M. and Kanneganti 2020 ). This pathway integrates multiple cell death pathways, including necroptosis, apoptosis, and pyroptosis, which prompts cells to undergo intense inflammatory death reactions and release a large number of DAMPs, further exacerbating the pulmonary inflammatory response. ZBP1 plays a crucial role in the lung tissue damage and disease progression caused by influenza virus infection. In our study, we also found that after MLE-12 cells were infected with H1N1, the expression of ZBP1 was significantly increased, which further activated the ZBP1-RIPK3-MLKL-Caspase signaling axis. At the same time, it induced apoptosis, necroptosis, and pyroptosis, and triggered PANoptosis, leading to the death of MLE-12 cells and the exacerbation of inflammation. After the cells were intervened with MA + Se NPs, the PANoptosis of MLE-12 cells was inhibited. We speculate that there may be two pathways. Firstly, after MA inhibits the virus, the expression of ZBP1 decreases, because the expression of ZBP1 is positively correlated with the number of viruses. The decrease in ZBP1 is equivalent to turning off the core initiating sensor of PANoptosis. On the other hand, the scavenging of ROS by Se is also crucial for inhibiting PANoptosis. ROS can activate ZBP1 through oxidative modification, and then trigger necroptosis. ROS can also activate Caspase-1 or Caspase-11. These cysteine proteases are able to cleave Gasdermin D (GSDMD) to form pore-forming proteins, resulting in the rupture of the cell membrane and triggering pyroptosis(Chen S. et al. 2025 ). In addition, ROS can activate Caspase-8 through oxidative modification, and then initiate the apoptosis pathway. The activation of Caspase-8 will further amplify the inflammatory response and promote the release of inflammatory factors(Chen Y. C. et al. 2022). In summary, through in-depth analysis of the pathological mechanisms of viral pneumonia, we have successfully developed an innovative lung-targeting inflammation-responsive nanodrug. This sophisticated nanoplatform integrates MA and Se with exquisite design: MA exerts its outstanding antiviral activity to precisely inhibit viral replication in pulmonary cells, while Se potently scavenges excess ROS through its robust antioxidant capacity, effectively suppressing oxidative stress. This dual-functional combination achieves synergistic antiviral and antioxidant effects. Comprehensive safety evaluations have confirmed the excellent biosafety profile of this nanodrug. Mechanistically, we pioneered a novel research perspective by investigating PANoptosis inhibition. Through systematic cellular experiments and animal infection models, we demonstrated that the nanodrug can effectively modulate the ZBP1-RIPK3-MLKL-Caspase signaling axis, significantly inhibiting apoptosis, necroptosis and pyroptosis processes. Consequently, it blocks PANoptosis activation, reduces excessive release of inflammatory cytokines and DAMPs, thereby effectively alleviating pulmonary inflammation and tissue damage. These findings provide groundbreaking theoretical evidence for elucidating the therapeutic mechanisms of MA + Se NPs against viral pneumonia. Declarations CRediT authorship contribution statement WenhuiWu: Writing – original draft, Investigation, Formal analysis, Data curation. Ruidong Li: Investigation, Formal analysis, Data curation. Chunmei Lv: Investigation, Formal analysis, Data curation. Dandan Yang: Methodology, Investigation. Shunqiang Song: Methodology, Investigation. Min Yang: Conceptualization,Writing – review & editing and Validation. Yongai Xiong: Writing – original draft, Funding acquisition, Formal analysis, Data curation. Declaration of competing interest All authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors highly appreciate for resources provided by Guizhou Provincial Key Laboratory of Innovation and Manufacturing for Pharmaceuticals and Key Laboratory of Basic Pharmacology of Guizhou Province and School of Pharmacy in Zunyi Medical University. Ethics statement All animal studies were approved by the Ethics Committee of Zunyi Medical University (Permit No. ZMU24-2408-09) and performed in accordance with the Laboratory Animal Welfare and Ethics Committee of China. Funding This study was financially supported by the National Natural Science Foundation of China (NO. 82260723) and the he Zunyi Science and Technology Project [ZunshikeheHZ(2024)304]. References Alva R, et al. 2023. Oxygen toxicity: cellular mechanisms in normobaric hyperoxia. Cell Biol Toxicol 39:111-143. Basavaraju S, Mishra S, Jindal R, Kesavardhana S. 2022. Emerging Role of ZBP1 in Z-RNA Sensing, Influenza Virus-Induced Cell Death, and Pulmonary Inflammation. mBio 13:e0040122. Bian Y, Zhao K, Hu T, Tan C, Liang R, Weng X. 2024. A Se Nanoparticle/MgFe-LDH Composite Nanosheet as a Multifunctional Platform for Osteosarcoma Eradication, Antibacterial and Bone Reconstruction. Adv Sci (Weinh) 11:e2403791. Cabler SS, French AR, Orvedahl A. 2020. A Cytokine Circus with a Viral Ringleader: SARS-CoV-2-Associated Cytokine Storm Syndromes. Trends Mol Med 26:1078-1085. Chen S, Wu GD, Li T, Jiang J, Zhong Y, Sun D, Qian F, Huang LS. 2025. Targeting GSDMD JX06 inhibits PANoptosis and multiple organ injury. Biochem Pharmacol 233:116765. Chen YC, Yang CW, Chan TF, Farooqi AA, Chang HS, Yen CH, Huang MY, Chang HW. 2022. Cryptocaryone Promotes ROS-Dependent Antiproliferation and Apoptosis in Ovarian Cancer Cells. Cells 11. Cheng X, Zeng T, Xu Y, Xiong Y. 2025. The emerging role of PANoptosis in viral infections disease. Cell Signal 125:111497. Cilloniz C, Dy-Agra G, Pagcatipunan RS, Jr., Torres A. 2024. Viral Pneumonia: From Influenza to COVID-19. Semin Respir Crit Care Med 45:207-224. Deng D, Zhao M, Liu H, Zhou S, Liu H, You L, Hao Y. 2024. Xijiao Dihuang decoction combined with Yinqiao powder promotes autophagy-dependent ROS decrease to inhibit ROS/NLRP3/pyroptosis regulation axis in influenza virus infection. Phytomedicine 128:155446. Ding W, Zhang X, Zhang Q, Dong Y, Wang W, Ding N. 2021. Adiponectin ameliorates lung injury induced by intermittent hypoxia through inhibition of ROS-associated pulmonary cell apoptosis. Sleep Breath 25:459-470. Gao J, Zhao Y, Wang C, Ji H, Yu J, Liu C, Liu A. 2020. A novel synthetic chitosan selenate (CS) induces apoptosis in A549 lung cancer cells via the Fas/FasL pathway. Int J Biol Macromol 158:689-697. Gautier A, Duarte CM, Sousa I. 2022. Moringa oleifera Seeds Characterization and Potential Uses as Food. Foods 11. Jiang Y, Rubin L, Peng T, Liu L, Xing X, Lazarovici P, Zheng W. 2022. Cytokine storm in COVID-19: from viral infection to immune responses, diagnosis and therapy. Int J Biol Sci 18:459-472. Lei C, Liu XR, Chen QB, Li Y, Zhou JL, Zhou LY, Zou T. 2021. Hyaluronic acid and albumin based nanoparticles for drug delivery. J Control Release 331:416-433. Marinho A, Nunes C, Reis S. 2021. Hyaluronic Acid: A Key Ingredient in the Therapy of Inflammation. Biomolecules 11. Muhammad W, et al. 2022. ROS-responsive polymer nanoparticles with enhanced loading of dexamethasone effectively modulate the lung injury microenvironment. Acta Biomater 148:258-270. Salathia S, Gigliobianco MR, Casadidio C, Di Martino P, Censi R. 2023. Hyaluronic Acid-Based Nanosystems for CD44 Mediated Anti-Inflammatory and Antinociceptive Activity. Int J Mol Sci 24. Sun Y, Liang L, Yi Y, Meng Y, Peng K, Jiang X, Wang H. 2023. Synthesis, characterization and anti-inflammatory activity of selenium nanoparticles stabilized by aminated yeast glucan. Int J Biol Macromol 245:125187. Wang S, et al. 2023. Triple Cross-linked Dynamic Responsive Hydrogel Loaded with Selenium Nanoparticles for Modulating the Inflammatory Microenvironment via PI3K/Akt/NF-κB and MAPK Signaling Pathways. Adv Sci (Weinh) 10:e2303167. Wang W, Liu M, Gao W, Sun Y, Dong X. 2021. Coassembled Chitosan-Hyaluronic Acid Nanoparticles as a Theranostic Agent Targeting Alzheimer's β-Amyloid. ACS Appl Mater Interfaces 13:55879-55889. Watkins RR. 2022. Using Precision Medicine for the Diagnosis and Treatment of Viral Pneumonia. Adv Ther 39:3061-3071. Wiegman CH, Li F, Ryffel B, Togbe D, Chung KF. 2020. Oxidative Stress in Ozone-Induced Chronic Lung Inflammation and Emphysema: A Facet of Chronic Obstructive Pulmonary Disease. Front Immunol 11:1957. Wu H, Guo T, Nan J, Yang L, Liao G, Park HJ, Li J. 2022. Hyaluronic-Acid-Coated Chitosan Nanoparticles for Insulin Oral Delivery: Fabrication, Characterization, and Hypoglycemic Ability. Macromol Biosci 22:e2100493. Xiong Y, Rajoka MSR, Mehwish HM, Zhang M, Liang N, Li C, He Z. 2021. Virucidal activity of Moringa A from Moringa oleifera seeds against Influenza A Viruses by regulating TFEB. Int Immunopharmacol 95:107561. Xiong Y, Riaz Rajoka MS, Zhang M, He Z. 2022. Isolation and identification of two new compounds from the seeds of Moringa oleifera and their antiviral and anti-inflammatory activities. Nat Prod Res 36:974-983. Yang L, Cui Y, Liang H, Li Z, Wang N, Wang Y, Zheng G. 2022. Multifunctional Selenium Nanoparticles with Different Surface Modifications Ameliorate Neuroinflammation through the Gut Microbiota-NLRP3 Inflammasome-Brain Axis in APP/PS1 Mice. ACS Appl Mater Interfaces 14:30557-30570. Yuan T, et al. 2024. Scutellarin inhibits inflammatory PANoptosis by diminishing mitochondrial ROS generation and blocking PANoptosome formation. Int Immunopharmacol 139:112710. Zhang H, et al. 2025. Antiviral treatment for viral pneumonia: current drugs and natural compounds. Virol J 22:62. Zhang T, et al. 2020. Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis. Cell 180:1115-1129.e1113. Zhang X, Wei D, Xu Y, Zhu Q. 2021. Hyaluronic acid in ocular drug delivery. Carbohydr Polym 264:118006. Zheng D, Liu J, Piao H, Zhu Z, Wei R, Liu K. 2022. ROS-triggered endothelial cell death mechanisms: Focus on pyroptosis, parthanatos, and ferroptosis. Front Immunol 13:1039241. Zheng M, Kanneganti TD. 2020. The regulation of the ZBP1-NLRP3 inflammasome and its implications in pyroptosis, apoptosis, and necroptosis (PANoptosis). Immunol Rev 297:26-38. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6582119","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452292024,"identity":"f7d85e5b-46df-452f-93ec-701938a131f6","order_by":0,"name":"Wenhui Wu","email":"","orcid":"","institution":"Chongqing Traditional Chinese Medicine Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenhui","middleName":"","lastName":"Wu","suffix":""},{"id":452292026,"identity":"2381b5af-6eae-417f-a191-3adaae300d97","order_by":1,"name":"Ruidong Li","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ruidong","middleName":"","lastName":"Li","suffix":""},{"id":452292027,"identity":"0fd88890-f715-436c-b11b-3fdf6cf19b48","order_by":2,"name":"Chunmei Lv","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chunmei","middleName":"","lastName":"Lv","suffix":""},{"id":452292028,"identity":"511de1e1-4c75-4d0a-934c-5e8d606d0e05","order_by":3,"name":"Dandan Yang","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Yang","suffix":""},{"id":452292030,"identity":"785c0710-a9e0-4631-bfc0-be91f4da3255","order_by":4,"name":"Shunqiang Song","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shunqiang","middleName":"","lastName":"Song","suffix":""},{"id":452292031,"identity":"5d2dd6f5-3a10-447f-a94b-339098354294","order_by":5,"name":"Min Yang","email":"","orcid":"","institution":"Chongqing Traditional Chinese Medicine Hospital","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Yang","suffix":""},{"id":452292033,"identity":"37c04fa2-2261-4b22-820c-de0e45da66da","order_by":6,"name":"Yongai Xiong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYJCCA0AsB2GykaDFmDQtIJDYQLQWg+NnDA983FGbPn/aGQOGD2WHGfhnNxDQciYt4eDMM8dzG2fnGDDOOHeYQeLOAQJaDiQfOMzbdiy3WTrHgJm37TCDgUQCAS3nHzaAtKSzgbT8JUrLDbAtNQk8IC2MxGiRvPEM6Je2A4YzpNMKDvacS+eRuEFAC9/5HOMPH9vq5OVnJ2988KPMWo5/BgEtCgfA1GEwCWLz4FcPBPINYKqOoMJRMApGwSgYwQAAPcZHsOkgId4AAAAASUVORK5CYII=","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yongai","middleName":"","lastName":"Xiong","suffix":""}],"badges":[],"createdAt":"2025-05-03 05:53:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6582119/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6582119/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82134635,"identity":"95a14560-554f-488d-b250-7f56fe80ff3e","added_by":"auto","created_at":"2025-05-07 06:04:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":690482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreparation and characterization of Se@CS@MA@HA NPs (Se+MA NPs)\u003c/strong\u003e. (A) The schematic illustration of Se+MA NPs synthesis. (B) SEM(a) and TEM(b) images of Se+MA NPs. (C) Elemental analysis of Se+MA NPs. (D) Particle size distribution (a) and potential distribution (b) of Se+MA NPs. (E) FTIR spectrum. (F) XRD spectrum.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6582119/v1/35dc7ad4e2a19538fb7fcadd.png"},{"id":82134636,"identity":"ea05a7a8-4fb0-4e4e-82b8-3f0ac46fe797","added_by":"auto","created_at":"2025-05-07 06:04:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":150750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParticle size and Zeta potential of nanoparticles.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Se@CSNPs, (B)Se@CS@MANPs, (C)Se@CS@MA@HANPs\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6582119/v1/d605717e22fa6f2a99ffef5f.png"},{"id":82134638,"identity":"bcb31f68-da2f-4512-b247-d9deb6bd6028","added_by":"auto","created_at":"2025-05-07 06:04:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":607834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular uptake of nanoparticles encapsulating Coumarin 6.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cellular uptake of Coumarin 6 by RAW264.7 cells.(B) Cellular uptake of Coumarin 6 by M1 macrophages.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6582119/v1/bd74d69790fef63f81b9fe6b.png"},{"id":82136362,"identity":"27622d89-5442-4a2a-a751-7ce5b9c4134c","added_by":"auto","created_at":"2025-05-07 06:12:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2068406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e antiviral and anti-inflammatory effects of MA+SeNPs. \u003c/strong\u003e(A) experimental grouping and drug treatment timeline.(B)\u003cstrong\u003e \u003c/strong\u003ecytopathic effect (CPE) and cell viability. (C-E) Levels of inflammatory cytokines (TNF-α, IL-1β and IL-6). (F) Levels of anti-inflammatory cytokine (IL-10). (G)\u003cstrong\u003e \u003c/strong\u003efluorescent probe detection of ROS in MLE-12 cells.(H) immunofluorescence detection of influenza virus NP protein in MLE-12 Cells. (I) NP protein signal intensity. (J) M gene level of influenza virus detected by QRT-PCR. (a) Nor group. (b) Vir group. (c) MA NPs group. (d) Se NPs group. (e) MA+ Se NPs group. The results represent the mean ± SD of three independent experiments. \u003cem\u003e*p\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05, *\u003cem\u003e*p\u003c/em\u003e\u0026nbsp;\u0026lt; 0.01\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. the Vir group.\u0026nbsp;\u003csup\u003e△\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05,\u0026nbsp;\u003csup\u003e△△\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt; 0.01\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. the MA NPs group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05,\u0026nbsp;\u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt; 0.01\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. the MA+Se NPs group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6582119/v1/c3eb60caabab0815ac7dbab5.png"},{"id":82137790,"identity":"513d7262-6013-4c5e-9300-a483f4d647b1","added_by":"auto","created_at":"2025-05-07 06:20:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1811782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e antiviral and anti-inflammatory effects of MA+SeNPs. \u003c/strong\u003e(A) Comparison of body weight changes among different experimental groups of mice. (B) Effect of MA+SeNPs on the survival rate of H1N1-infected mice. (C) Lung index. (D) H\u0026amp;E staining of lung tissue sections. (E) Levels of SOD, GPx, TNF-α, IL-6, IL-1β and IL-18 in mice lungs. (F) Immunohistochemical detection of NP protein in mice lung tissues. (G) ratio of NP protein positive cells. (H) M gene level of influenza virus detected by QRT-PCR. (I)\u003cstrong\u003e \u003c/strong\u003efluorescent probe detection of ROS in mice lungs. The results represent the mean ± SD of six independent experiments. \u003cem\u003e*p\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05, *\u003cem\u003e*p\u003c/em\u003e\u0026nbsp;\u0026lt; 0.01\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. the Vir group.\u0026nbsp;\u003csup\u003e△\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05,\u0026nbsp;\u003csup\u003e△△\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt; 0.01\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. the MA NPs group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt; 0.05,\u0026nbsp;\u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt; 0.01\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. the MA+Se NPs group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6582119/v1/16e35966060616a256bc1767.png"},{"id":82136365,"identity":"fddbdf2e-f05a-4884-9398-c1dd7ce017c9","added_by":"auto","created_at":"2025-05-07 06:12:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":244233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of MA+Se NPs on H1N1-induced PANoptosis in MLE-12 cell\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(A) mechanism and key effector proteins of PANoptosis. (B) western blot detection of PANoptosis-associated key proteins. (C-H) expression of ZBP1, RIPK3, MLKL, GSDMD, Caspase-3 and Caspase-7. The results represent the mean ± SD of three independent experiments. \u003cem\u003e*p\u003c/em\u003e \u0026lt; 0.05, *\u003cem\u003e*p\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e. the Vir group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6582119/v1/48a39fba53d4d367123b32e8.png"},{"id":82137794,"identity":"6744118e-4de8-4b14-a5a8-8d6b3e2ca16d","added_by":"auto","created_at":"2025-05-07 06:20:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2787505,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe safety evaluation of MA+Se NPs treatment \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6582119/v1/707dd5ea39f3ae012a8e9e72.png"},{"id":82142043,"identity":"4fe2b3e0-2d89-488c-b966-89e572d5e9e1","added_by":"auto","created_at":"2025-05-07 06:36:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8600621,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6582119/v1/1d2ed1f3-f426-4db2-8949-4b0c95251a10.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting ZBP1-mediated PANoptosis: inflammation responsive selenized chitosan nanoparticles loaded with Moringa A for anti-viral pneumonia therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eViral pneumonia poses a serious threat to public health. It is caused by a variety of viruses, such as influenza virus, respiratory syncytial virus, coronavirus, and so on. It is extremely harmful to infants, the elderly, and people with immunodeficiency(Cilloniz et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It can not only cause symptoms like fever, cough, and breathing difficulties, but may also lead to severe complications such as respiratory failure, liver failure, and heart failure, and even endanger lives. However, current treatments face numerous challenges. On the one hand, new mutant viruses, like SARS-CoV-2, keep emerging, bringing difficulties to prevention, control, and treatment. On the other hand, there is a lack of broad-spectrum antiviral drugs, making it difficult to deal with multiple viral infections. Moreover, existing drugs may have issues such as drug resistance and side effects, and the development of precise treatment plans is hindered by individual differences and viral diversity(Watkins \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Zhang H. et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Research shows that viral infections may trigger an over - activation of the body's immune system, leading to a pathological state known as a \"cytokine storm.\" This abnormal immune response causes a large - scale release of inflammatory mediators such as IL-6 and TNF-α in a short period, thereby triggering systemic inflammation. This uncontrolled inflammatory cascade not only directly damages alveolar tissue, leading to the occurrence of acute respiratory distress syndrome (ARDS), but may also affect organs such as the heart, liver, and kidneys, ultimately resulting in multiple organ dysfunction(Cabler et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Jiang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, while combating viruses, the control of inflammation cannot be ignored.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMoringa seeds\u003c/em\u003e are the seeds of the M\u003cem\u003eoringa oleifera tree\u003c/em\u003e, belonging to the genus \u003cem\u003eMoringa Adans\u003c/em\u003e of the family \u003cem\u003eMoringaceae\u003c/em\u003e. They have extensive edible and medicinal values(Gautier et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In our previous research, we conducted isolation and screening of the active ingredients in \u003cem\u003eMoringa seeds\u003c/em\u003e with the guidance of antiviral activity. A novel - structured compound with remarkable anti - influenza virus activity was obtained from the acetone - extracted phase of the methanol extract of moringa seeds, and it was named \u003cem\u003eMoringa A\u003c/em\u003e (MA)(Xiong et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Its chemical structure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Previous studies have found that MA can exert a broad - spectrum anti - influenza virus effect by regulating the autophagy - lysosome pathway, making it a highly promising small - molecule compound against influenza virus(Xiong et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the low solubility of MA limits its bioavailability and efficacy. Due to its poor solubility, its absorption in the gastrointestinal tract is restricted, resulting in a reduced amount of the drug entering the systemic circulation and a decrease in bioavailability. At the same time, the low solubility also delays the release and onset time of MA. It may be difficult for the blood drug concentration to reach an effective level, thus weakening the therapeutic effect. To improve its antiviral effect, this project aims to use a chitosan - based nano delivery system to improve the solubility of MA, enhance its bioavailability, and increase its efficacy. The chitosan nanoparticle drug - delivery system can enhance the stability of drugs, control drug release, and improve the cellular uptake ability of drugs. It is an excellent nano - delivery carrier.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSelenium nanoparticles (SeNPs) is a kind of particles with a unique nanostructure. SeNPs can not only effectively scavenge oxygen free radicals, but also significantly reduce the secretion level of pro-inflammatory factors such as IL-6 by regulating the activity of immune cells, thus showing a powerful anti-inflammatory effect(Sun et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Wang S. et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Its mechanism of action involves activating antioxidant enzymes (such as glutathione peroxidase) to enhance the antioxidant capacity of cells and further alleviate the inflammation caused by oxidative stress. Its remarkable efficacy in multiple inflammatory disease models indicates that SeNPs has broad application potential in anti-inflammatory treatment. However, due to its high surface energy and chemical activity, SeNPs are prone to aggregation and oxidation reactions. These reactions can lead to changes in their structure and surface properties, thereby affecting their stability and function(Bian et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Yang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This instability may cause SeNPs to lose their unique nanoscale properties during storage or application, restricting their applications in fields such as biomedicine and catalysis. To improve the stability of SeNPs, it is often necessary to protect them through methods such as surface modification, coating, or treatment with dispersants.\u003c/p\u003e \u003cp\u003eTo overcome the stability problem of SeNPs, we selected chitosan as the carrier to prepare selenized chitosan. Selenized chitosan is a functional composite material formed by combining selenium with chitosan, which combines the biocompatibility and biodegradability of chitosan with the antioxidant and anti - inflammatory properties of selenium. The selenium element in its structure exists in the form of covalent bonds or coordination bonds, which significantly improves its stability and biological activity(Gao et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Selenized chitosan not only retains the antibacterial and repair - promoting functions of chitosan but also enhances its antioxidant and immunomodulatory abilities. Research shows that selenium is embedded in the polymer network in a stable coordination form, and this molecular - level synergy endows the material with multiple advantages in fields such as drug controlled release and tissue repair(Gao et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHyaluronic acid (HA) is an acidic mucopolysaccharide naturally present in human tissues. It has multiple important physiological functions in the body and is widely used in many different medical fields. From a chemical structure perspective, hyaluronic acid is composed of alternating glucuronic acid and N-acetylglucosamine units. This special molecular arrangement gives it excellent biocompatibility and biodegradability, which provides great advantages when it is used in medical applications such as drug carriers(Lei et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Zhang X. et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the field of respiratory diseases, hyaluronic acid has shown potential value in targeted treatment of pneumonia. The core therapeutic mechanism of hyaluronic acid lies in its ability to specifically recognize the abnormally elevated CD44 receptor in an inflammatory state. When pneumonia occurs in the lungs, the expression level of the CD44 receptor on the cell surface increases significantly, and hyaluronic acid can specifically bind to it, providing an ideal target - binding basis for the targeted treatment of pneumonia(Marinho et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Salathia et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, selenized chitosan is used as the carrier to encapsulate MA, and then HA is used to modify the surface of the selenized chitosan loaded with MA. A nano - targeted drug responsive to the pulmonary inflammatory micro - environment is designed to deliver MA to the pulmonary inflammatory region, enhance its antiviral pneumonia activity, and integrate the inhibitory effect of nano - selenium on pulmonary inflammatory factors and the antiviral effect of MA.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eMoringa A (MA): Lab-synthesized (in-house preparation). Sodium selenite (Cat. No. SLCN8615; Merck Life Science Technology Co., Ltd). Ascorbic acid (Cat. No. RH561123), hyaluronic acid (Cat. No. RH44689), and chitosan (Cat. No. RH482374) were purchased from Guangzhou Ronn Biological Technology Co., Ltd. Fetal bovine serum (Lot No. 10099-141) and DMEM High Sugar Medium (Lot No. C11995) were purchased from Gibco. Cellular Protein Extraction Kit (Item No. E-BC-E002), purchased from Wuhan Eliot Bio-Tech Co. Mice IL-1β (Cat no. E-EL-M0037), TNF-α (Cat no. E-EL-M0048), IL-18(Cat no. E-EL-M0730), IL-6(Cat no. E-EL-M0043), IL-10(Cat no. E-EL-M1210 46), SOD(Cat no. E-EL-H6188) and Glutathione peroxidase(GPx, Cat no. E-EL-R2491) Elisa kit were purchased from Elabscience Biotechnology Co., Ltd. 2',7'-Dichlorofluorescin diacetate (Cat no. ab273640), anti ZBP1 antibody (Cat. no. sc-271483), RIPK3 antibody (Cat no. sc-374639), anti-MLKL antibody (Cat no. sc-293201), GSDMD antibody (Cat no. se-393581), anti-Caspase-7 antibody (Cat no. se-56066), anti-Caspase-3 antibody (Cat no. se-81655) were purchased from Santa Cruz Biotechnology, Inc. Anti-NF-kB p65 antibody (Cat no. ab32536) and anti-GAPDH (Cat no. ab8245) were purchased from Abcam. Horseradish enzyme-labeled goat anti-rabbit IgG (Cat no. SA0012) and BCA protein concentration determination kit (Cat no. PC0020) were purchased from Beijing Solebao Technology Co., Ltd. LPS was purchased from Sigma.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Virus and Cell lines\u003c/h2\u003e \u003cp\u003eInfluenza A virus A/Puerto Rico/8/34, obtained from the American Type Culture Collection (ATCC). The mouse lung epithelial cell line MLE-12 and RAW 264.7 were purchased from Shanghai Binsui Biotechnology Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation and characterization of HA-modified selenized chitosan nanoparticles loading MA(Se@CS@MA@HANPs)\u003c/h2\u003e \u003cp\u003eThe preparation of HA - modified selenized chitosan nanoparticles loading MA referred to the preparation method of nano-selenium by previous reports(Wang W. et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Wu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, under magnetic stirring, 2 mL of a 0.5% (mass fraction) chitosan (CS) solution was thoroughly mixed with 2 mL of a 0.1 M sodium selenite (Na₂SeO₃) solution, and the volume was adjusted to 10 mL with purified water. An appropriate amount of a 0.5 M ascorbic acid solution (Vc) was added dropwise to the mixture, and the volume was adjusted to 20 mL with water. The mixture was stirred at a speed of 500 rpm for 60 min, and then dialyzed for 48 h using a dialysis bag with a molecular weight cut - off of 8,000chang-14,000 (10000 kDa). The dialysate was replaced every 12 h. The Se@CSNPs were collected and stored at 4\u0026deg;C for later use. 10 mg of MA was weighed and dissolved it in 10 mL of a 0.5 mg\u0026middot;mL⁻\u0026sup1; sodium tripolyphosphate (STPP) solution. Then, 3 mL of the above-mentioned MA solution was added to the selenized chitosan carrier solution. After stirring at 800 rpm for 30 min, a certain amount of hyaluronic acid solution was added so that the drug accounted for a volume ratio of 1:8 of the total volume. The Se@CS@MA@HANPs (abbreviated as MA\u0026thinsp;+\u0026thinsp;Se NPs) were collected and stored at 4\u0026deg;C for later use. The schematic illustration of MA\u0026thinsp;+\u0026thinsp;Se NPs synthesis was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003eThe prepared nanoparticles were characterized by using a laser particle size analyzer, Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), Fourier Transform Infrared Spectroscopy (FT-IR), and X-Ray Diffraction (XRD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Determination of cumulative release rate\u003c/h2\u003e \u003cp\u003eIn this experiment, the dialysis method was used to conduct \u003cem\u003ein vitro\u003c/em\u003e drug release studies. The temperature of the water-bath shaker was set at 37\u0026deg;C, and the oscillation frequency was maintained at a constant speed of 100 r\u0026middot;min⁻\u0026sup1;. Three parallel samples were set for each group. MA powder was added to PBS solution to prepare a liquid equivalent to 0.6 mg\u0026middot;mL⁻\u0026sup1; of MA. 1 mL of the liquid was taken and placed in a dialysis bag, which was then immersed in 10 mL of PBS (pH\u0026thinsp;=\u0026thinsp;7.4) solution. Samples of 1 mL were taken at time points (0.33, 0.67, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72 h), and 1 mL of the release medium at the same temperature was added. The released samples were injected for analysis under the chromatographic conditions described in 2.1.2.1 of Chap.\u0026nbsp;1. The peak areas were recorded, and the cumulative release rate Q was calculated. In the same way, a PBS solution of MA\u0026thinsp;+\u0026thinsp;Se NPs test samples with the same concentration was used as a control for the \u003cem\u003ein vitro\u003c/em\u003e release test. The peak areas were recorded, and the cumulative dissolution rate was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Drug stability investigation\u003c/h2\u003e \u003cp\u003eThe Se@CS NPs, Se@CS@MA NPs, and Se@CS@MA@HA NPs were stored at 4\u0026deg;C for 30 days. The particle size and zeta potential of the nanoparticles were measured every 5 days using a laser particle size analyzer to evaluate their stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cellular uptake\u003c/h2\u003e \u003cp\u003eCoumarin6(C6) was used as a probe to investigate the uptake of nanoparticles by cells. For RAW264.7 cells, three groups were set up: the Free C6 group, the Se@CS@C6NPs group, and the Se@CS@C6@HANPs group, with 3 replicate wells in each group. 500 \u0026micro;L of cell suspension with a density of 1\u0026times;10⁴ cells per well was inoculated into 24 - well plates containing cover slips and cultured in an incubator for 24 h. After aspirating and discarding the old culture medium, three types of culture media containing coumarin 6 were added respectively. After incubation in the incubator for 2 h, the old culture medium was aspirated and discarded, and the cells were washed 3 times with cold PBS. Then, the cells were fixed with 4% paraformaldehyde for 20 min and washed 3 times with cold PBS again. The cover slips were taken out and mounted on slides with anti - fluorescence quenching mounting medium containing DAPI for 10 min. Subsequently, each slide was placed under a laser confocal scanning microscope to observe the cell uptake of each coumarin 6 preparation group.\u003c/p\u003e \u003cp\u003eFor LPS-induced M1 macrophages, the Se@CS@C6NPs group and the Se@CS@C6@HANPs group were set up, and the cell uptake experiment was carried out in the same way.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Cell culture and treatments\u003c/h2\u003e \u003cp\u003eThe MLE-12 cell line was maintained in phenol red-free high-glucose DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Following a 24-hour incubation at 37\u0026deg;C under 5% CO₂, the cells were allocated into five experimental groups (six replicates per group): Normal (Nor): Untreated cells, Virus (Vir): H1N1-infected cells (MOI\u0026thinsp;=\u0026thinsp;5), MA\u0026thinsp;+\u0026thinsp;Se NPs (1 \u0026micro;g/mL Se@CS@MA@HANPs), MA NPs (1 \u0026micro;g/mL CS@MA@HANPs) and Se NPs (1 \u0026micro;g/mL Se@CS@HANPs). All groups except Con were exposed to H1N1 at an MOI of 5 to induce infection, while Con and Vir received blank medium. Post-treatment, cells were cultured for 48 h (37\u0026deg;C, 5% CO₂), with daily monitoring of cytopathic effects (CPE). Cell viability was assessed using the CCK-8 assay. The levels of IL-6, TNF-α, IL-1β and IL-10 in the supernatants of cells from each experimental group were detected by ELISA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. ROS Detection\u003c/h2\u003e \u003cp\u003eIntracellular reactive oxygen species (ROS) levels were quantified using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). MLE-12 cells were plated in 96-well plates at a density of 1\u0026times;10⁴ cells per well and allowed to adhere for 24 hours. Following this incubation period, cells were treated with either curcumin or DMSO vehicle control for 48 hours. As a positive control, tert-butyl hydroperoxide (tBHP) was administered for 4 hours prior to staining.For the detection procedure, culture medium was first removed and cells were washed with 100 \u0026micro;L of 1X assay buffer per well. After buffer removal, cells were incubated with 100 \u0026micro;L of 10 \u0026micro;M DCFH-DA solution at 37\u0026deg;C for 45 minutes under light-protected conditions. Following dye removal, fluorescence intensity was measured immediately using a microplate reader with excitation/emission wavelengths set at 485/535 nm. Quantitative image analysis was performed using ImageJ software, with fluorescence intensities normalized to control group values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Detection of H1N1 NP protein in cells\u003c/h2\u003e \u003cp\u003eThe expression of the H1N1 viral NP protein in cells from each experimental group was detected and co-localized using immunofluorescence and confocal laser microscopy to assess the changes in viral particles within host cells after drug treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Animal experiments\u003c/h2\u003e \u003cp\u003eSPF KM mice were obtained from Hunan Silek Jingda Laboratory Animal Co., Ltd. and acclimatized for seven days under standardized conditions. Then mice were randomly divided into five groups according to their body weight: the Normal group (Nor), the Model group (Mod), MA\u0026thinsp;+\u0026thinsp;Se NPs Group (treated by Se@CS@MA@HANPs), MA NPs Group (treated by CS@MA@HANPs), and Se NPs (treated by Se@CS @HANPs Group), with eight mice in each group. On the first day, all mice except those in the Nor group, were intranasally infected with \u0026times;10 MLD\u003csub\u003e50\u003c/sub\u003e of H1N1 viruses in a 50 \u0026micro;L volume. Mice in the treatment group were administered the corresponding nanoparticles intranasally at a dose of 5 ml/kg daily, while the mice in the Nor and Mod groups received equivalent volumes of deionized water daily. The body weight and survival rate of the mice in each group were monitored daily. On Day 14 post-infection (p.i.), mice were euthanized by cervical dislocation, and lungs were dissected out. Here, all of the animal experimental protocols were approved by the Ethics Committee of Zunyi Medical University (Permit No. ZMU24-2418-02). All animal care and experimental procedures were performed in accordance with the Laboratory Animal Welfare and Ethics Committee of China.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.10.1. Lung index and Hematoxylin and eosin (H\u0026amp;E) staining\u003c/h2\u003e \u003cp\u003eUnder sterile conditions, the lung tissues was rapidly excised, washed with PBS to remove residual surface blood, and then placed on sterile filter paper to remove excess moisture. The weight of the lung tissue was measured, and the lung index was calculated (lung tissue weight/body weight \u0026times; 100%). The degree of pulmonary edema in the lungs of mice in each group was analyzed. Besides, lung tissues were fixed. The samples were dehydrated in ethanol, embedded with paraffin and sliced. Then the lung tissues were deparaffinized in xylene for 10 min, stained with H\u0026amp;E for histopathological examination of lung inflammation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.10.2. Lung tissue cytokines analysis\u003c/h2\u003e \u003cp\u003e100 mg of lung tissue from each experimental group of mice was taken, homogenized in a homogenizer after adding PBS (lung tissue:PBS\u0026thinsp;=\u0026thinsp;1:9), and then centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected, and the levels of IL-18, IL-1β, TNF-α, IL-6, SOD and GPx in the lung tissue homogenate were detected using the ELISA method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.10.3. Immunohistochemical Detection\u003c/h2\u003e \u003cp\u003eMice lung tissues were taken and fixed with 4% paraformaldehyde, then dehydrated with gradient ethanol, cleared with xylene, and embedded in paraffin. The tissue was cut into sections \u003cspan refid=\"Sec28\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;5\u0026micro;m thick. For antigen retrieval, the sections were placed in 10 mM sodium citrate buffer (pH 6.0) and heated in a microwave for 8\u0026ndash;15 minutes. After blocking with 3% BSA-PBS at room temperature for 30 minutes, NP antibody (diluted at 1:1000) was added and incubated overnight at 4℃. Following PBS washes, goat anti-rabbit IgG was added and incubated at room temperature for 1 hour. The sections were then treated with streptavidin-horseradish peroxidase (SA-HRP) complex and developed with DAB (3,3'-diaminobenzidine). The sections were counterstained with hematoxylin and mounted with neutral gum. Finally, the sections were observed under an optical microscope, and the expression of NP in the mouse lung tissue was assessed based on the staining results.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.11. QRT-PCR\u003c/h2\u003e \u003cp\u003eApproximately 15\u0026ndash;20 mg of pulmonary tissue was homogenized in 300 \u0026micro;L of lysis buffer using a high-speed tissue disruptor to generate a uniform cell suspension. The extracted total RNA was quantified via ultra-micro spectrophotometry and subsequently reverse-transcribed into complementary DNA (cDNA). For quantitative analysis, SYBR Green-based real-time PCR (Qiagen, Hilden, Germany) was employed to assess mRNA expression. The primer sequences targeted the murine housekeeping gene PPIA and the influenza viral M segment, designed as follows:Viral M gene: Forward: 5\u0026prime;-CTTCTAACCGAGGTCGAAAC-3\u0026prime;, Reverse: 5\u0026prime;-CGTCTACG\u003c/p\u003e \u003cp\u003eCTGCAGTCCTC-3\u0026prime;, PPIA (internal control): Forward: 5\u0026prime;-CGCTTGCTGCAGCCATGGTC-3\u0026prime;,\u003c/p\u003e \u003cp\u003eReverse: 5\u0026prime;-CAGCTCGAAGGAGACGCGGC-3\u0026prime;. The relative quantification of influenza virus M gene copies was computed using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method for comparative threshold cycle analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Effect of Se-MA NPs on PANoptosis of MLE-12 cell\u003c/h2\u003e \u003cp\u003eThe MLE-12 cell line was maintained in phenol red-free high-glucose DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Following a 24-hour incubation at 37\u0026deg;C under 5% CO₂, the cells were allocated into three experimental groups (six replicates per group): Normal (Nor): untreated cells, Virus (Vir): H1N1-infected cells (MOI\u0026thinsp;=\u0026thinsp;5) and MA\u0026thinsp;+\u0026thinsp;Se NPs (1 \u0026micro;g/mL Se@CS@MA@HANPs). All groups except Con were exposed to H1N1 at an MOI of 5 to induce infection, while Con and Vir received blank medium. Post-treatment, cells were cultured for 48 h (37\u0026deg;C, 5% CO₂).\u003c/p\u003e \u003cp\u003eCells were lysed using RIPA lysis buffer to extract total protein, and the protein concentration was determined using the BCA kit or other methods. Based on the protein concentration, an appropriate amount of protein sample was mixed with loading buffer, boiled to denature the proteins, and then subjected to SDS-PAGE electrophoresis. The gel concentration was selected according to the molecular weight of the target proteins to achieve separation. The separated proteins were transferred from the gel to a PVDF membrane using the wet transfer method, with appropriate voltage and time settings to ensure complete transfer. The membrane was blocked with 5% skimmed milk powder at room temperature for 1 hour. Specific primary antibodies against ZBP1, RIPK3, MLKL, GSDMD, Caspase-3, and Caspase-7 (diluted at a ratio of 1:1000) were added and incubated overnight at 4℃. The membrane was then washed 3\u0026ndash;5 times with TBST for 5\u0026ndash;10 minutes each time. HRP-conjugated secondary antibodies matching the primary antibodies were added and incubated at room temperature for 1 hour. The membrane was washed again 3\u0026ndash;5 times with TBST to remove unbound secondary antibodies. Finally, ECL chemiluminescent substrate was added, and the membrane was imaged using a chemiluminescent imaging system in a dark room to analyze the expression levels of the target proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Evaluation of the biosafety of MA\u0026thinsp;+\u0026thinsp;SeNPs in mice\u003c/h2\u003e \u003cp\u003eMice were divided into two groups (n\u0026thinsp;=\u0026thinsp;10 per group): the PBS group and the MA\u0026thinsp;+\u0026thinsp;SeNPs group. Both groups received daily intranasal administration of PBS or MA\u0026thinsp;+\u0026thinsp;SeNPs for 14 consecutive days. At the end of the experiment, all mice were humanely euthanized, and major organs (heart, liver, spleen, lungs, kidneys, and brain) were collected for histopathological examination using H\u0026amp;E staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data was expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM). Statistical analysis was conducted using one-way ANOVA with Duncan's multiple comparison test on SPSS 27.0 software. All difference with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered to indicate significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of S Se@CS@MA@HANPs\u003c/h2\u003e \u003cp\u003eSEM and TEM analyses of the prepared nanoparticles revealed that Se@CS@MA@HANPs exhibited a quasi-spherical structure with good dispersibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Based on SEM and TEM results, energy-dispersive X-ray spectroscopy (EDS) was further employed to analyze the elemental composition and distribution of Se in Se@CS@MA@HANPs, with the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. In the prepared nanoparticles, selenium accounted for 45.31%, carbon for 6.48%, and oxygen for 21%. The signal of selenium was primarily concentrated within the signal regions of carbon and oxygen, showing a highly overlapping distribution with these elements. This suggests that selenium is likely uniformly encapsulated in the form of nanoparticles within an organic matrix rich in carbon and oxygen, indicating a good binding or encapsulation relationship between selenium and the organic components.\u003c/p\u003e \u003cp\u003eAdditionally, particle size analysis revealed that on the basis of the spherical structure of Se@CSNPs (with a particle size of 66.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 nm), the particle size of Se@CS@MANPs increased to 113.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40 nm upon drug loading. After modification with hyaluronic acid (HA), the particle size of Se@CS@MA@HANPs further increased to 208.80\u0026thinsp;\u0026plusmn;\u0026thinsp;6.46 nm, with a zeta potential of 12.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46 mV, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and 8D. Further surface modification of Se@CS@MANPs with HA was confirmed by SEM, as illustrated in Figs.\u0026nbsp;8E and 8F.\u003c/p\u003e \u003cp\u003eFTIR was further employed to characterize different nano-selenium particles as well as MA and HA, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE. The broad peak at 3400 cm⁻\u0026sup1; corresponds to the characteristic absorption of O-H stretching vibrations, indicating the presence of intramolecular or intermolecular hydrogen bonds. The peaks near 2920 cm⁻\u0026sup1; and 1612 cm⁻\u0026sup1; in Se@CSNPs (a), Se@CS@MANPs (b), and Se@CS@MA@HANPs (c) are attributed to the C-H stretching vibrations of methyl and methylene groups from residual sugar moieties and the carbonyl C\u0026thinsp;=\u0026thinsp;O stretching vibrations, respectively. The absorption peak at 1384 cm⁻\u0026sup1; is typically associated with the symmetric deformation vibration of -CH₃ (methyl) groups. The C-O stretching vibration absorption peaks are observed at 1300 cm⁻\u0026sup1; and 1070 cm⁻\u0026sup1;, while the peak near 897 cm⁻\u0026sup1; corresponds to the characteristic absorption of the β-glycosidic bond in chitosan. All these absorption peaks exhibited varying degrees of redshift upon the addition of MA and HA. The peak near 1450 cm⁻\u0026sup1; in Se@CS@MANPs (b), Se@CS@MA@HANPs (c), and MA (d) is characteristic of aromatic rings, confirming the successful dispersion of MA on the surface of nano-selenium. The peak near 1662 cm⁻\u0026sup1; in Se@CS@MA@HANPs (c) and HA (e) corresponds to the C\u0026thinsp;=\u0026thinsp;O stretching vibration in secondary amides, indicating the successful encapsulation of nano-selenium particles by HA.\u003c/p\u003e \u003cp\u003eThe intensity and sharpness of X-ray diffraction peaks can reflect the crystalline nature of selenium to some extent. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, the X-ray diffraction pattern of Se@CSNPs (a) exhibits two sharp characteristic peaks at 2θ\u0026thinsp;=\u0026thinsp;24\u0026deg; and 30\u0026deg;, along with broad diffraction peaks in the range of 20\u0026deg; to 40\u0026deg;, indicating that SeNPs exist in an amorphous form. HA (e) shows broad diffraction peaks in the range of 10\u0026deg; to 40\u0026deg;, indicating its amorphous nature. MA (d) has a characteristic peak at 42\u0026deg;. The diffraction patterns of Se@CS@MANPs (b) and Se@CS@MA@HANPs (c) not only retain the characteristic peaks of Se@CS but also preserve the 42\u0026deg; peak of MA, suggesting that the combination of Se@CSNPs with MA maintains their respective structural regularities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2. The stability of the drug\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the changes in particle size and zeta potential of nano-selenium over a period of 30 days. As shown in the figure, freshly prepared Se@CSNPs (A), Se@CS@MANPs (B), and Se@CS@MA@HANPs (C) all exhibited an orange-red color and were clear and transparent. Subsequently, Se@CS gradually turned dark red, while the other two showed no significant color change. It is speculated that Se@CS might have been oxidized, leading to the gradual transformation of nano-selenium into elemental selenium. In contrast, the other two formulations remained relatively stable after drug loading. From the particle size changes depicted in the figure, we can observe that the particle size of Se@CSNPs increased rapidly from 66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 nm within the first 10 days and eventually stabilized around 99\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 nm. The particle sizes of the other two formulations changed little, which also indicates that the stability of the nanoparticles was enhanced after drug loading. The zeta potential values shown in the figure for Se@CSNPs (A), Se@CS@MANPs (B), and Se@CS@MA@HANPs (C) were 22.53\u0026thinsp;\u0026plusmn;\u0026thinsp;3.01 mV, 19.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01 mV, and 12.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46 mV, respectively. All three exhibited a downward trend over the 30-day period, eventually stabilizing around 8 mV. The zeta potential of Se@CS decreased the most, a result consistent with the particle size observations. In summary, we can conclude that the stability of Se@CS was significantly improved after drug loading.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3 The uptake of nanoparticles by cells\u003c/h2\u003e \u003cp\u003eIn this study, Coumarin 6 was used as a model probe to investigate the uptake of the prepared nanoparticles by RAW264.7 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, RAW264.7 cells exhibited weak green fluorescence in the cytoplasm, indicating that the uptake of free Coumarin 6 by cells was minimal. Compared with cells treated with free Coumarin 6, cells treated with Se@CS@C6NPs and Se@CS@C6@HANPs showed significantly enhanced green fluorescence, with the strongest fluorescence observed in the Se@CS@C6@HANPs group. This suggests that the prepared nanoparticles possess good characteristics for transmembrane transport across cell membranes. To further investigate the specific affinity of Se@CS@HANPs for inflammatory cells, this study used LPS (lipopolysaccharide)-induced M1-type macrophages differentiated from RAW264.7 cells as a model to observe the uptake of Se@CS@C6@HANPs and Se@CS@C6NPs by these cells within 2 hours. The results showed that M1 macrophages treated with Se@CS@C6@HANPs exhibited significantly higher fluorescence intensity compared to those treated with Se@CS@C6NPs, indicating that nanoparticles modified with hyaluronic acid (HA) have better affinity for inflammatory cells, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4 \u003cem\u003eIn vitro\u003c/em\u003e antiviral and anti-inflammatory effects of nanoparticles\u003c/h2\u003e \u003cp\u003eTo investigate the synergistic advantages of Se and MA combination therapy in antiviral and anti-inflammatory efficacy, MLE-12 cells infected with H1N1 for 24 hours were treated with SeNPs, MANPs, or inflammation-responsive nanoparticles co-loaded with Se and MA (MA\u0026thinsp;+\u0026thinsp;Se NPs) for 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The results showed that SeNPs, MANPs, and MA\u0026thinsp;+\u0026thinsp;Se NPs all alleviated cytopathic effects (CPE) to varying degrees and inhibited H1N1-induced cell death. Notably, MA\u0026thinsp;+\u0026thinsp;Se NPs demonstrated significantly superior effects in mitigating CPE and enhancing cell viability compared to Se or MA alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analysis of pro- and anti-inflammatory cytokines in host cells revealed that H1N1 infection triggered a substantial increase in TNF-α, IL-1β, and IL-6 levels, while the anti-inflammatory cytokine IL-10 was reduced. After treatment with the three nanoparticles, TNF-α, IL-1β, and IL-6 levels in MLE-12 cells decreased significantly, whereas IL-10 levels increased. Importantly, MA\u0026thinsp;+\u0026thinsp;Se NPs exhibited markedly stronger reversal effects on these cytokines than Se or MA alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These findings indicate that co-delivery of Se and MA exerts synergistic anti-inflammatory effects.\u003c/p\u003e \u003cp\u003eStudies have shown that ROS can activate signaling pathways such as NF-κB and the NLRP3 inflammasome, promoting the expression and release of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6). Excessive ROS also induces oxidative stress, exacerbating inflammatory responses. Influenza virus infection further amplifies ROS production in host cells, triggering inflammation(Muhammad et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) z(Muhammad et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In our study, H1N1-infected MLE-12 cells exhibited higher ROS levels than uninfected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Treatment with SeNPs, MANPs, or MA\u0026thinsp;+\u0026thinsp;Se NPs reduced intracellular ROS, with SeNPs and MA\u0026thinsp;+\u0026thinsp;Se NPs showing the most pronounced effects, underscoring the critical role of Se in scavenging virus-induced ROS.\u003c/p\u003e \u003cp\u003eAdditionally, the combination of Se and MA exhibited a synergistic inhibitory effect against H1N1 virus. Immunofluorescence and qRT-PCR analyses of viral NP protein and M gene expression revealed that all three nanoparticles suppressed H1N1 viral NP protein and M gene to varying degrees, with MA\u0026thinsp;+\u0026thinsp;Se NPs demonstrating the most potent inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5 \u003cem\u003eIn vivo\u003c/em\u003e antiviral and anti-inflammatory effects of nanoparticles\u003c/h2\u003e \u003cp\u003eTo evaluate the protective efficacy of MA\u0026thinsp;+\u0026thinsp;Se NPs against H1N1 infection in mice, intranasal delivery was employed. Mice infected with H1N1 were administered MA NPs, Se NPs, or MA\u0026thinsp;+\u0026thinsp;Se NPs, followed by monitoring of survival rates and body weight changes over 14 days. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, by day 8 post-infection (dpi), the H1N1-challenged group exhibited a 20.12% reduction in body weight. In contrast, the MA NPs, Se NPs, and MA\u0026thinsp;+\u0026thinsp;Se NPs treatment groups demonstrated significantly lower weight losses of 11.6%, 9.66%, and 8.37%, respectively. Similarly, survival rates varied markedly among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). While only 12.5% of the virus-infected control mice survived, the MA NPs, Se NPs, and MA\u0026thinsp;+\u0026thinsp;Se NPs groups showed survival rates of 45%, 60%, and 90%, respectively. Notably, mice treated with three types of NPs displayed a modest reduction in weight loss between 2 and 8 dpi compared to the H1N1-infected group. By 8 dpi, surviving animals in the nanoparticle-treated groups began regaining weight, whereas the untreated infected mice showed minimal recovery. Control mice remained unaffected, displaying no clinical symptoms, weight decline, or mortality. These findings indicate that nanoparticles intervention can mitigate H1N1-induced weight loss and enhance survival in infected mice.At the same time, these three types of nanoparticles can also significantly reduce the lung index of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), and can improve the lung injury in mice caused by the H1N1virus to a certain extent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). It is worth noting that the combined use of MA and Se has a significantly better improvement effect on the above-mentioned indicators than when they are used alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhat\u0026rsquo;s more, the combined use of MA and Se has an important impact on the balanced regulation of immunity and inflammation in mice with viral pneumonia. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, after being infected with the H1N1 virus, a large amount of inflammatory factors IL-1β, IL-18, IL-6 and TNF-α were produced in the lung tissues of mice, while the antioxidant factors SOD and GPx were significantly reduced, exhibiting oxidative inflammatory damage in lung tissue. However, after the mice were treated with MA and Se, the levels of IL-1β, IL-18, IL-6 and TNF-α in the lung tissues of the mice were effectively controlled, and the levels of SOD and GPx were effectively restored. Moreover, the reversing effect of the combined use of MA and Se on these cytokines was significantly better than that of using a MA and Se alone.\u003c/p\u003e \u003cp\u003eFurther detection by immunohistochemistry and qRT-PCR revealed abundant NP protein-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) and viral M gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH) in the lung tissues of H1N1-infected mice. Following treatment with the three types of nanoparticles, both NP protein-positive cells and viral M gene levels were significantly reduced. Among them, MA\u0026thinsp;+\u0026thinsp;SeNPs exhibited the strongest antiviral efficacy, followed by MANPs, while SeNPs showed the weakest effect. These results fully demonstrate that the combination of MA and Se exerts a synergistic antiviral effect.\u003c/p\u003e \u003cp\u003eTo investigate the oxidative stress status in the lung tissues of viral pneumonia mice, we detected ROS levels in mouse lungs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, H1N1 infection induced massive ROS production in lung tissues. Studies indicate that excessive ROS can damage pulmonary cells (including epithelial and endothelial cells), leading to cell membrane lipid peroxidation, protein oxidative modification, and DNA damage, ultimately causing cellular dysfunction or even death(Zheng D. et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This process further exacerbates pulmonary inflammatory responses by promoting the release of inflammatory cytokines, creating a vicious cycle of inflammation. Consequently, lung tissue structure and function become impaired, manifesting as pathological changes such as increased alveolar-capillary permeability, pulmonary edema, and fibrosis\u0026mdash;severely compromising normal respiratory function(Ding et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Wiegman et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Notably, nanodrug treatment significantly reduced ROS levels in lung tissues, particularly in the Se NPs and MA\u0026thinsp;+\u0026thinsp;Se NPs groups. These results clearly demonstrate the critical role of Se in scavenging excess ROS induced by viral infection.\u003c/p\u003e \u003cp\u003eThe above findings indicate that the co-delivery of MA and Se achieves coordinated control of viral load and inflammation in the lungs of viral pneumonia mice, while also effectively mitigating influenza virus-induced excessive oxidative stress in lung tissues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6 MA\u0026thinsp;+\u0026thinsp;Se NPs inhibits influenza virus-induced PANoptosis in MLE-12 Cells\u003c/h2\u003e \u003cp\u003eInfluenza virus infection exacerbates lung injury through the induction of PANoptosis, a process involving the activation of the host protein Z-DNA binding protein 1 (ZBP1) by viral RNA(Basavaraju et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This activation triggers the rupture of the cell membrane via the RIPK3/MLKL pathway and the amplification of inflammation in a caspase-8-dependent manner. The death of alveolar epithelial and vascular endothelial cells leads to the collapse of barrier functions, resulting in the massive release of inflammatory factors (such as IL-6 and TNF-α) and damage-associated molecular patterns (DAMPs), which in turn trigger cytokine storms and immune cell infiltration(Cheng et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). PANoptosis acts in concert with pyroptosis (mediated by Gasdermin D) and apoptosis, further aggravating alveolar structural destruction, microthrombus formation, and hypoxia. The excessive inflammation and imbalance in tissue repair ultimately lead to acute respiratory distress syndrome (ARDS) and multi-organ failure, which are core pathological mechanisms underlying the severity of influenza. Targeted inhibition of ZBP1 or RIPK3 may serve as potential therapeutic strategies. The mechanism of PANoptosis is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter influenza virus infection, the PANoptosis-related proteins ZBP1, RIPK3, MLKL, GSDMD, Caspase-3, and Caspase-7 were significantly upregulated in MLE-12 cells, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH. indicating that the H1N1 virus triggers pyroptosis, apoptosis and necroptosis simultaneously via the ZBP1 pathway, which is defined as PANoptosis. However, after intervention with MA\u0026thinsp;+\u0026thinsp;Se NPs, these proteins were significantly downregulated, demonstrating the inhibition of PANoptosis. This may be an important mechanism by which MA\u0026thinsp;+\u0026thinsp;Se NPs suppress the cytokine storm in mouse lung tissues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.7 The biosafety evaluation of MA\u0026thinsp;+\u0026thinsp;Se NPs\u003c/h2\u003e \u003cp\u003eAfter 14 consecutive days of intranasal administration, we found that MA\u0026thinsp;+\u0026thinsp;Se NPs did not exhibit significant toxic effects on the heart, liver, spleen, lungs, or kidneys of mice, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Histopathological examination of these vital organs revealed no observable cellular damage, inflammatory responses, or other abnormal pathological changes. These results demonstrate that MA\u0026thinsp;+\u0026thinsp;SeNPs possess excellent biocompatibility and low toxicity risks in vivo, indicating favorable biosafety profiles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eVirus-induced lung injury typically results from the dual effects of rapid viral replication and excessive host immune responses. While antiviral drugs alone can reduce viral load, they fail to promptly suppress inflammatory spread; conversely, anti-inflammatory therapy alone may lead to disease relapse and exacerbation due to incomplete viral clearance. Dual intervention, by simultaneously inhibiting viral replication and modulating inflammatory pathways, not only disrupts pathogen transmission but also mitigates tissue damage, thereby breaking the \"virus-inflammation\" positive feedback loop. This approach significantly reduces the risk of acute lung injury progressing to ARDS and improves patient survival rates. The synergistic mechanism of antiviral and anti-inflammatory actions provides a critical theoretical foundation for this study. MA, an antiviral compound, was combined with anti-inflammatory SeNPs to achieve multi-target antiviral effects, offering a novel nano-delivery strategy for the treatment of viral pneumonia.\u003c/p\u003e \u003cp\u003eIn viral pneumonia, ROS exacerbate lung injury through a dual mechanism. On the one hand, viral replication induces mitochondrial dysfunction and the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, leading to an excessive production of ROS. This triggers lipid peroxidation, protein inactivation, and DNA damage, directly destroying alveolar epithelial and vascular endothelial cells(Deng et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). On the other hand, ROS amplifies the inflammatory response by activating signaling pathways such as NF-κB and mitogen-activated protein kinases (MAPK), promoting the release of inflammatory cytokines (such as IL-6 and TNF-α) and forming a \"cytokine storm\"(Alva et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Meanwhile, ROS collaborate with cell death pathways such as PANoptosis and pyroptosis. Through oxidative stress, ROS enhance the activity of the ZBP1/receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like (MLKL) pathway, exacerbating cell membrane rupture and the release of damage-associated molecular patterns (DAMPs), resulting in the collapse of the alveolar-capillary barrier, microthrombus formation, and hypoxia. Excessive ROS can also inhibit the antioxidant system (such as SOD and glutathione), creating a vicious cycle of oxidative stress, ultimately promoting the development of ARDS and multiple organ failure(Yuan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Regulating ROS levels is an important therapeutic strategy for controlling viral pneumonia.\u003c/p\u003e \u003cp\u003eAfter infection with the H1N1 virus, we detected a large number of viral particles and ROS in the lung tissues of mice. In addition, inflammatory cytokines such as TNF-α, IL-1β, and IL-6 were highly active, while antioxidant factors such as SOD and GPx were decreased. This is clearly the direct cause of the high mortality rate of mice in the infected group and the severe pulmonary inflammation. After treatment with MA\u0026thinsp;+\u0026thinsp;Se NPs, the survival rate of the mice increased significantly. The viral particles and ROS in the lung tissues were significantly reduced. The lung index and the levels of pulmonary inflammatory cytokines were significantly decreased, and the inflammatory damage to the lung tissues was significantly alleviated. This indicates that MA\u0026thinsp;+\u0026thinsp;Se NPs have successfully achieved the synergistic effect of antiviral and anti-inflammatory pharmacodynamics.\u003c/p\u003e \u003cp\u003eAs an important intracellular pattern recognition receptor, ZBP1 can precisely recognize dangerous signals such as nucleic acids released during influenza virus infection(Zhang T. et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Once it recognizes the molecular patterns associated with the influenza virus, ZBP1 rapidly activates its own functions, recruits key proteins such as RIPK1/3 and MLKL, and initiates the PANoptosis signaling pathway(Zheng M. and Kanneganti \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This pathway integrates multiple cell death pathways, including necroptosis, apoptosis, and pyroptosis, which prompts cells to undergo intense inflammatory death reactions and release a large number of DAMPs, further exacerbating the pulmonary inflammatory response. ZBP1 plays a crucial role in the lung tissue damage and disease progression caused by influenza virus infection. In our study, we also found that after MLE-12 cells were infected with H1N1, the expression of ZBP1 was significantly increased, which further activated the ZBP1-RIPK3-MLKL-Caspase signaling axis. At the same time, it induced apoptosis, necroptosis, and pyroptosis, and triggered PANoptosis, leading to the death of MLE-12 cells and the exacerbation of inflammation. After the cells were intervened with MA\u0026thinsp;+\u0026thinsp;Se NPs, the PANoptosis of MLE-12 cells was inhibited. We speculate that there may be two pathways. Firstly, after MA inhibits the virus, the expression of ZBP1 decreases, because the expression of ZBP1 is positively correlated with the number of viruses. The decrease in ZBP1 is equivalent to turning off the core initiating sensor of PANoptosis. On the other hand, the scavenging of ROS by Se is also crucial for inhibiting PANoptosis. ROS can activate ZBP1 through oxidative modification, and then trigger necroptosis. ROS can also activate Caspase-1 or Caspase-11. These cysteine proteases are able to cleave Gasdermin D (GSDMD) to form pore-forming proteins, resulting in the rupture of the cell membrane and triggering pyroptosis(Chen S. et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In addition, ROS can activate Caspase-8 through oxidative modification, and then initiate the apoptosis pathway. The activation of Caspase-8 will further amplify the inflammatory response and promote the release of inflammatory factors(Chen Y. C. et al. 2022).\u003c/p\u003e \u003cp\u003eIn summary, through in-depth analysis of the pathological mechanisms of viral pneumonia, we have successfully developed an innovative lung-targeting inflammation-responsive nanodrug. This sophisticated nanoplatform integrates MA and Se with exquisite design: MA exerts its outstanding antiviral activity to precisely inhibit viral replication in pulmonary cells, while Se potently scavenges excess ROS through its robust antioxidant capacity, effectively suppressing oxidative stress. This dual-functional combination achieves synergistic antiviral and antioxidant effects. Comprehensive safety evaluations have confirmed the excellent biosafety profile of this nanodrug. Mechanistically, we pioneered a novel research perspective by investigating PANoptosis inhibition. Through systematic cellular experiments and animal infection models, we demonstrated that the nanodrug can effectively modulate the ZBP1-RIPK3-MLKL-Caspase signaling axis, significantly inhibiting apoptosis, necroptosis and pyroptosis processes. Consequently, it blocks PANoptosis activation, reduces excessive release of inflammatory cytokines and DAMPs, thereby effectively alleviating pulmonary inflammation and tissue damage. These findings provide groundbreaking theoretical evidence for elucidating the therapeutic mechanisms of MA\u0026thinsp;+\u0026thinsp;Se NPs against viral pneumonia.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWenhuiWu: Writing \u0026ndash; original draft, Investigation, Formal analysis, Data curation. Ruidong Li: Investigation, Formal analysis, Data curation.\u0026nbsp;Chunmei Lv: Investigation, Formal analysis, Data curation. Dandan Yang: Methodology, Investigation. Shunqiang Song: Methodology, Investigation. Min Yang: Conceptualization,Writing \u0026ndash; review \u0026amp; editing and Validation. Yongai Xiong: Writing \u0026ndash; original draft, Funding acquisition, Formal analysis, Data curation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors highly appreciate for resources provided by Guizhou Provincial Key Laboratory of Innovation and Manufacturing for Pharmaceuticals and Key Laboratory of Basic Pharmacology of Guizhou Province and School of Pharmacy in Zunyi Medical University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies were approved\u0026nbsp;by the Ethics Committee of Zunyi Medical University (Permit No. ZMU24-2408-09)\u0026nbsp;and performed in accordance with\u0026nbsp;the Laboratory Animal Welfare and Ethics Committee of China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the National Natural Science Foundation of China (NO. 82260723) and the he Zunyi Science and Technology Project [ZunshikeheHZ(2024)304].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlva R, et al. 2023. Oxygen toxicity: cellular mechanisms in normobaric hyperoxia. Cell Biol Toxicol 39:111-143.\u003c/li\u003e\n \u003cli\u003eBasavaraju S, Mishra S, Jindal R, Kesavardhana S. 2022. Emerging Role of ZBP1 in Z-RNA Sensing, Influenza Virus-Induced Cell Death, and Pulmonary Inflammation. mBio 13:e0040122.\u003c/li\u003e\n \u003cli\u003eBian Y, Zhao K, Hu T, Tan C, Liang R, Weng X. 2024. A Se Nanoparticle/MgFe-LDH Composite Nanosheet as a Multifunctional Platform for Osteosarcoma Eradication, Antibacterial and Bone Reconstruction. Adv Sci (Weinh) 11:e2403791.\u003c/li\u003e\n \u003cli\u003eCabler SS, French AR, Orvedahl A. 2020. A Cytokine Circus with a Viral Ringleader: SARS-CoV-2-Associated Cytokine Storm Syndromes. Trends Mol Med 26:1078-1085.\u003c/li\u003e\n \u003cli\u003eChen S, Wu GD, Li T, Jiang J, Zhong Y, Sun D, Qian F, Huang LS. 2025. Targeting GSDMD JX06 inhibits PANoptosis and multiple organ injury. Biochem Pharmacol 233:116765.\u003c/li\u003e\n \u003cli\u003eChen YC, Yang CW, Chan TF, Farooqi AA, Chang HS, Yen CH, Huang MY, Chang HW. 2022. Cryptocaryone Promotes ROS-Dependent Antiproliferation and Apoptosis in Ovarian Cancer Cells. Cells 11.\u003c/li\u003e\n \u003cli\u003eCheng X, Zeng T, Xu Y, Xiong Y. 2025. The emerging role of PANoptosis in viral infections disease. Cell Signal 125:111497.\u003c/li\u003e\n \u003cli\u003eCilloniz C, Dy-Agra G, Pagcatipunan RS, Jr., Torres A. 2024. Viral Pneumonia: From Influenza to COVID-19. Semin Respir Crit Care Med 45:207-224.\u003c/li\u003e\n \u003cli\u003eDeng D, Zhao M, Liu H, Zhou S, Liu H, You L, Hao Y. 2024. Xijiao Dihuang decoction combined with Yinqiao powder promotes autophagy-dependent ROS decrease to inhibit ROS/NLRP3/pyroptosis regulation axis in influenza virus infection. Phytomedicine 128:155446.\u003c/li\u003e\n \u003cli\u003eDing W, Zhang X, Zhang Q, Dong Y, Wang W, Ding N. 2021. Adiponectin ameliorates lung injury induced by intermittent hypoxia through inhibition of ROS-associated pulmonary cell apoptosis. Sleep Breath 25:459-470.\u003c/li\u003e\n \u003cli\u003eGao J, Zhao Y, Wang C, Ji H, Yu J, Liu C, Liu A. 2020. A novel synthetic chitosan selenate (CS) induces apoptosis in A549 lung cancer cells via the Fas/FasL pathway. Int J Biol Macromol 158:689-697.\u003c/li\u003e\n \u003cli\u003eGautier A, Duarte CM, Sousa I. 2022. Moringa oleifera Seeds Characterization and Potential Uses as Food. Foods 11.\u003c/li\u003e\n \u003cli\u003eJiang Y, Rubin L, Peng T, Liu L, Xing X, Lazarovici P, Zheng W. 2022. Cytokine storm in COVID-19: from viral infection to immune responses, diagnosis and therapy. Int J Biol Sci 18:459-472.\u003c/li\u003e\n \u003cli\u003eLei C, Liu XR, Chen QB, Li Y, Zhou JL, Zhou LY, Zou T. 2021. Hyaluronic acid and albumin based nanoparticles for drug delivery. J Control Release 331:416-433.\u003c/li\u003e\n \u003cli\u003eMarinho A, Nunes C, Reis S. 2021. Hyaluronic Acid: A Key Ingredient in the Therapy of Inflammation. Biomolecules 11.\u003c/li\u003e\n \u003cli\u003eMuhammad W, et al. 2022. ROS-responsive polymer nanoparticles with enhanced loading of dexamethasone effectively modulate the lung injury microenvironment. Acta Biomater 148:258-270.\u003c/li\u003e\n \u003cli\u003eSalathia S, Gigliobianco MR, Casadidio C, Di Martino P, Censi R. 2023. Hyaluronic Acid-Based Nanosystems for CD44 Mediated Anti-Inflammatory and Antinociceptive Activity. Int J Mol Sci 24.\u003c/li\u003e\n \u003cli\u003eSun Y, Liang L, Yi Y, Meng Y, Peng K, Jiang X, Wang H. 2023. Synthesis, characterization and anti-inflammatory activity of selenium nanoparticles stabilized by aminated yeast glucan. Int J Biol Macromol 245:125187.\u003c/li\u003e\n \u003cli\u003eWang S, et al. 2023. Triple Cross-linked Dynamic Responsive Hydrogel Loaded with Selenium Nanoparticles for Modulating the Inflammatory Microenvironment via PI3K/Akt/NF-\u0026kappa;B and MAPK Signaling Pathways. Adv Sci (Weinh) 10:e2303167.\u003c/li\u003e\n \u003cli\u003eWang W, Liu M, Gao W, Sun Y, Dong X. 2021. Coassembled Chitosan-Hyaluronic Acid Nanoparticles as a Theranostic Agent Targeting Alzheimer\u0026apos;s \u0026beta;-Amyloid. ACS Appl Mater Interfaces 13:55879-55889.\u003c/li\u003e\n \u003cli\u003eWatkins RR. 2022. Using Precision Medicine for the Diagnosis and Treatment of Viral Pneumonia. Adv Ther 39:3061-3071.\u003c/li\u003e\n \u003cli\u003eWiegman CH, Li F, Ryffel B, Togbe D, Chung KF. 2020. Oxidative Stress in Ozone-Induced Chronic Lung Inflammation and Emphysema: A Facet of Chronic Obstructive Pulmonary Disease. Front Immunol 11:1957.\u003c/li\u003e\n \u003cli\u003eWu H, Guo T, Nan J, Yang L, Liao G, Park HJ, Li J. 2022. Hyaluronic-Acid-Coated Chitosan Nanoparticles for Insulin Oral Delivery: Fabrication, Characterization, and Hypoglycemic Ability. Macromol Biosci 22:e2100493.\u003c/li\u003e\n \u003cli\u003eXiong Y, Rajoka MSR, Mehwish HM, Zhang M, Liang N, Li C, He Z. 2021. Virucidal activity of Moringa A from Moringa oleifera seeds against Influenza A Viruses by regulating TFEB. Int Immunopharmacol 95:107561.\u003c/li\u003e\n \u003cli\u003eXiong Y, Riaz Rajoka MS, Zhang M, He Z. 2022. Isolation and identification of two new compounds from the seeds of Moringa oleifera and their antiviral and anti-inflammatory activities. Nat Prod Res 36:974-983.\u003c/li\u003e\n \u003cli\u003eYang L, Cui Y, Liang H, Li Z, Wang N, Wang Y, Zheng G. 2022. Multifunctional Selenium Nanoparticles with Different Surface Modifications Ameliorate Neuroinflammation through the Gut Microbiota-NLRP3 Inflammasome-Brain Axis in APP/PS1 Mice. ACS Appl Mater Interfaces 14:30557-30570.\u003c/li\u003e\n \u003cli\u003eYuan T, et al. 2024. Scutellarin inhibits inflammatory PANoptosis by diminishing mitochondrial ROS generation and blocking PANoptosome formation. Int Immunopharmacol 139:112710.\u003c/li\u003e\n \u003cli\u003eZhang H, et al. 2025. Antiviral treatment for viral pneumonia: current drugs and natural compounds. Virol J 22:62.\u003c/li\u003e\n \u003cli\u003eZhang T, et al. 2020. Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis. Cell 180:1115-1129.e1113.\u003c/li\u003e\n \u003cli\u003eZhang X, Wei D, Xu Y, Zhu Q. 2021. Hyaluronic acid in ocular drug delivery. Carbohydr Polym 264:118006.\u003c/li\u003e\n \u003cli\u003eZheng D, Liu J, Piao H, Zhu Z, Wei R, Liu K. 2022. ROS-triggered endothelial cell death mechanisms: Focus on pyroptosis, parthanatos, and ferroptosis. Front Immunol 13:1039241.\u003c/li\u003e\n \u003cli\u003eZheng M, Kanneganti TD. 2020. The regulation of the ZBP1-NLRP3 inflammasome and its implications in pyroptosis, apoptosis, and necroptosis (PANoptosis). Immunol Rev 297:26-38.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Viral pneumonia, Inflammation-responsive nanoparticles, MoringaA, Selenium","lastPublishedDoi":"10.21203/rs.3.rs-6582119/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6582119/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eViral pneumonia poses a major global public health challenge, where excessive inflammatory responses contribute to tissue damage and respiratory failure. Inflammation-responsive nanoparticles can target inflamed areas, improving drug delivery while minimizing side effects. Chitosan, a biocompatible polysaccharide with anti-inflammatory and immunomodulatory properties, gains enhanced antioxidant and anti-inflammatory capabilities when combined with selenium. This study developed selenium-chitosan nanoparticles loaded with MoringaA (MA), a natural antiviral compound from \u003cem\u003eMoringa oleifera\u003c/em\u003e seeds. These nanoparticles target lung inflammation, releasing MA to suppress viral replication and infection while reducing inflammatory responses. Additionally, selenium-chitosan nanoparticles mitigate oxidative stress, regulate immunity, and inhibit PANoptosis\u0026mdash;a cell death pathway that exacerbates inflammation. By blocking core proteins in this pathway, they further curb inflammatory factor release. This approach offers a promising therapeutic strategy for viral pneumonia, combining targeted drug delivery, antiviral action, and inflammation control with reduced side effects.\u003c/p\u003e","manuscriptTitle":"Targeting ZBP1-mediated PANoptosis: inflammation responsive selenized chitosan nanoparticles loaded with Moringa A for anti-viral pneumonia therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 06:04:26","doi":"10.21203/rs.3.rs-6582119/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fc4d973a-a488-4052-a017-23109d01c08e","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-07T06:04:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 06:04:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6582119","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6582119","identity":"rs-6582119","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}