The telodendrimer PEG^5kCA₄Rf₄ was used to encapsulate SsnB. For nomenclature, PEG^5k represents a polyethylene glycol with a molecular weight of 5 kDa and CA₄Rf₄ indicates that four cholic acids and riboflavin molecules were conjugated on α-amino and ε-amino groups respectively on the periphery of dendritic polylysine. PEG^5kCA₄Rf₄ was prepared following our previous procedure via peptide chemistry (Guo et al., 2017; Shi et al., 2015). SsnB was encapsulated within a telodendrimer (TD) nanocarrier, PEG^5kCA₄Rf₄, using a thin-film hydration method as previously described (Guo et al., 2017; Shi et al., 2015). Briefly, SsnB solution in DMSO was added into TD solution in CHCl₃/MeOH (10:1 v/v) mixture at a TD/SsnB weight ratio of 10:1 (w/w). The solvents were evaporated on a Rota evaporator, resulting in a thin, homogeneous film of SsnB-TD mixture coated on the flask wall. The film was further dried under high vacuum for 1 h. Subsequently, the film was hydrated in PBS. The particle size distributions of the drug-loaded nanoparticles (NPs) were determined using dynamic light scattering (DLS) Zetasizer (Malvern).

The RAW264.7 macrophage cell line was obtained from American Type Culture Collection (Rockville, MA). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco, Gaithersburg, MD) and supplemented with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA), 1 mM sodium pyruvate (Gibco) and 100 U/mL penicillin G and 100 μg/mL streptomycin at 37°C in a humidified incubator with 5% CO₂ (Meng et al., 2024; Guo et al., 2024). The growth medium was refreshed every other day. A phase contrast microscope was used to monitor the RAW264.7 cells daily.

For cell viability study, Raw264.7 cells were seeded in a 96-well plate with the cell density of 4 × 10³ cells per well. After an overnight incubation, the cells were treated with different concentrations of free SsnB or nSsnB, and blank TD. After 72 h incubation, Cell titer 96 aqueous cell proliferation reagent, which is composed of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS, Cat. #: G3582, Promega) and an electron-coupling reagent phenazine methosulphate (PMS), was added to each well according to the manufacturer’s instructions and further incubated for 1–4 h at 37°C. The cell viability was determined by measuring the absorbance at 490 nm using a microplate reader (BioTek Synergy H1). Untreated cells served as a control. Results were shown as the average cell viability of triplicate wells via an equation: Cell viability % = [(OD_treat-OD_blank)/(OD_control-OD_blank) × 100%].

For examining the effect of nSsnB on inflammation, RAW264.7 6-well plate with the cell density of 3 × 10⁵ cells per well. After an overnight incubation, the cells were treated with LPS (1 μg/mL) ± nSsnB (32 μg/mL) for 24 h. The culture medium and cells were collected for detection of inflammatory cytokines and protein analysis, respectively.

Fresh blood was obtained from a healthy human volunteer and mixed with a PBS solution containing 20 mM EDTA. Red blood cells (RBCs) were then isolated by centrifugation at 1,000 rpm for 10 min. Subsequently, the RBCs were washed three times with PBS and resuspended in PBS (Guo et al., 2024; Guo et al., 2020; Wang et al., 2019). Free SsnB or nSsnB were added to 200 µL of RBC solutions at concentrations of 10, 100, and 200 μg/mL, followed by gentle mixing and incubation at 37°C for 30 min, 4 h, and overnight, respectively. Afterward, the samples were centrifuged at 1,000 rpm for 5 min, and the hemoglobin in the supernatant was measured using a NanoDrop spectrophotometer at 540 nm. PBS and Triton X-100 (2%) were included as negative and positive controls, respectively, by incubating them with RBCs. Hemolytic toxicity was determined using the following equation: Hemolysis% = [(OD_sample - OD_PBS)/(OD_triton - OD_PBS)] × 100%.

Male and female C57BL/6 and HIV-LTR/Luciferase (HLL) mice, aged 8 weeks, were purchased from Jackson Laboratories (Bar Harbor, ME). The mice were housed in a controlled environment with a temperature of 22°C and a photoperiod of 12 h light and 12 h dark, with ad libitum access to food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of SUNY Upstate Medical University (IACUC #344 and #437) and conducted in compliance with the National Institutes of Health and ARRIVE guidelines for the use of laboratory animals.

All mice received non-invasive tracheal instillation using an aerosolizer with nSsnB (0.625 mg/kg) or vehicle 2 h prior to inducing lung injury with LPS (2.5 mg/kg) or saline, as previously described (Ortiz-Munoz and Looney, 2015; Meng et al., 2022). Briefly, mice in the septic and control groups were anesthetized via intraperitoneal injection of a combination of ketamine (80 mg/kg) and xylazine (8 mg/kg). After induction of anesthesia, mice were positioned in the supine position on an intubating platform, with their incisors suspended and a fiber-optic illuminator positioned over the trachea. Tongue retraction and upward-leftward positioning were performed using forceps to visualize the larynx, aided by hands-free binocular magnifiers. The MicroSprayer Aerosolizer (Cat. #: YAN30012, Shanghai Yuyan Instruments Co., Ltd.) was then inserted into the tracheal lumen, and LPS, nSsnB, and saline solution (not exceeding a volume of 70 µL per mouse) were instilled. Mice were maintained in the same position on the intubating platform for at least 30 s before being placed prone on a heating pad for recovery. All surviving mice were euthanized under anesthesia at 24 h post-LPS administration, and blood (in EDTA), lung tissue (fixed with 10% formalin for histology and frozen for protein analysis), and bronchoalveolar lavage fluid (BALF) were collected. The IT administration of LPS (2.5 mg/kg) in this study did not result in mortality among any of the experimental groups.

BALF was collected from the mouse lung and lavaged with three aliquots of 0.5 mL sterile saline, followed by centrifugation at 250 × g for 10 min. The resulting pellet was resuspended in 1 mL of sterile saline. A 100 µL portion of the cell suspension was then centrifuged using a cytospin centrifuge (Hettich ROTOFIX 32A) at 1,000 rpm for 3 min to adhere the cells to a slide (Meng et al., 2022). The slide was air-dried and stained with Hema-3 (Cat. #: 23-122929, 23-122937, 23-122952, Fisher Scientific, Kalamazoo, MI) for subsequent analysis. Neutrophils and macrophages were quantified in 20 high-power fields (HPF) by blinded reviewers utilizing a Nikon Eclipse TE2000-U research microscope (Nikon, Melville, NY). Protein concentration in BALF was measured using the BCA micro assay kit from Thermo Scientific (Cat. #: 23235) according to the provided guidelines.

Lung tissue was homogenized in RIPA buffer, and the extracted protein was utilized for Western blot analysis (Meng et al., 2023). A total of 20 µg of protein was separated by SDS-PAGE gel electrophoresis, followed by transfer to PVDF membranes (Cat. #: IPVH00010, Millipore Co., Ltd. United States). The membranes were blocked with 5% non-fat milk (Cat. #: 1706404XTU, Bio-Rad Laboratories) in Tris-buffered saline plus 0.5% Tween-20 (TBS-T) from Fisher Scientific (Cat. #: BP337-500) for 1 h at room temperature, and then incubated overnight at 4°C with primary antibodies purchased from Santa Cruz Biotechnology, including MyD88 (Cat. #: sc-74532, 1:200 dilution) and phosphorylated-NF-κB (p65) (Cat. #: sc-136548, 1:200 dilution). Subsequently, the membranes were probed with a secondary antibody conjugated to horseradish peroxidase (HRP) obtained from Santa Cruz Biotechnology (Cat. #: 516102, 1:1,000 dilution) for 1 h at room temperature. Antibody-antigen complexes were visualized using enhanced chemiluminescence (ECL, Cat. #: 34580, Thermo Scientific) according to the manufacturer’s instructions. Quantitative analysis of the immunoreactive bands was performed using densitometry with ImageJ software, and the relative density of the bands was normalized to the density of the corresponding β-actin bands.

Blood samples, BALF and lung tissue were collected to measure levels of TNF-α (Cat. #: 50-112-8800, Invitrogen) and IL-6 (Cat. #: 50-112-8863, Invitrogen) by ELISA. Serum from blood and BALF were used directly for ELISA following appropriate dilution. Lung tissue required homogenization in protein lysis buffer (RIPA) to extract the supernatant, followed by protein concentration measurement using a BCA assay. The protein solution then was diluted to a final concentration of 1 mg/mL before ELISA analysis. All cytokines were quantified using commercial ELISA kits in accordance with the manufacturer’s instructions.

The lungs were fixed for histological examination by instilling 0.5 mL of 10% neutral formalin through the trachea. Following fixation, the lungs were embedded in paraffin, and 5 μm sections were prepared for staining with Hematoxylin and Eosin (H&E). Histopathological analysis was conducted in a blinded manner by two independent pathologists. Assessment of acute lung injury was performed using a scoring system ranging from 0 to 2, as described in a previous study (Matute-Bello et al., 2001; Meng et al., 2022). This scoring system evaluated parameters such as neutrophil presence in the alveolar and interstitial spaces, hyaline membrane formation, proteinaceous debris in airspaces, and septal thickening. A lung injury score was calculated by summing the scores of each parameter, which were then normalized to the number of fields evaluated. Three representative fields per slide were assessed in 20 high-power fields (HPF) by blinded reviewers using light microscopy at ×400 magnification.

HIV-LTR/Luciferase (HLL) mice are genetically modified mice that are used to study the regulation of gene expression IVIS, particularly in response to NF-κB-mediated signaling, as luciferase activity can serve as a reporter for the activation of this pathway (Blackwell et al., 2000). All bioluminescent data were collected and analyzed using in vivo Imaging Systems (IVIS) at Upstate medical university core research facilities.

The data were expressed as either mean ± SEM or median ± interquartile range following tests for normal distribution using SPSS 22 (SPSS, Chicago, United States). The sample size for each experimental group (n = 3–7) is indicated in the figure legend. Group differences were assessed using either one-way analysis of variance (one-way ANOVA) with Bonferroni’s multiple comparisons test (for normally distributed data) or Kruskal-Wallis test with Dunn’s multiple comparison test (for non-normally distributed data). Statistical significance was considered at P < 0.05. All data were obtained from three or more independent experiments.

Given the ultra flat aromatic structure of SsnB, we have screened our telodendrimers with aromatic building blocks as reported in our previous study (Shi et al., 2015) for SsnB encapsulation. Majority of TD nanocarriers fail to encapsulate SsnB, either for poor loading capacity or stability. As a result, PEG^5kCA₄Rf₄ showed a stable SsnB loading, owing to the aromatic riboflavin building block. In addition, both cholic acid (CA) and riboflavin are amphiphilic, which dramatically stabilize drug loading and nanoparticle stability. As shown in Figure 1A, nSsnB has a uniform and small particle size around 17 nm, which is stable upon storage for weeks. Although SsnB was reported to be a potent TLR4/MD2 inhibitor, it was also shown to have anticancer (Liu et al., 2021) and antiangiogenic (Bateman et al., 2013) activity. Indeed, free SsnB showed significant cytotoxicity upon murine macrophage cell line Raw 264.7 in cell culture with an IC50 of 28 ng/mL (Figure 2B), which is significantly reduced by nSsnB for about 500 fold to 11.4 ug/mL. At the same time, free SsnB has very high hemolytic properties in the in vitro whole blood incubation with almost instant hemolysis after 30 min incubation at 10 ug/mL. While nSsnB is safe without significant hemolysis even overnight incubation at 200 ug/mL (Figure 1C).

We further examined the anti-inflammatory effect of nSsnB in immune cell culture (RAW264.7) in the presence of LPS co-incubation. LPS significantly induced cytokine productions in the culture medium as detected by ELISA, e.g., TNF-α (Figure 2A) and IL-6 (Figure 2B), in comparison to the control. The nSsnB significantly inhibited cytokine productions induced by LPS. And it was shown that nSsnB does not have an immunostimulatory effect without cytokine induction. We further examined the activation of NF-κB in immune cells with Western blot assay. As shown in Figure 2C, the phosphorylated NF-κB P65 (p-NF-κB P65) (P < 0.05 vs. Control) induced by LPS can be effectively inhibited by nSsnB. While nSsnB had no effects on this inflammatory signaling in the comparison to the control group. These results clearly demonstrated that nSsnB possesses an anti-inflammatory effect by inhibiting the LPS-TLR-4/NF-κB pathway.

Assessment of the neutrophil-to-lymphocyte ratio (NLR) in blood during LPS-induced lung injury permits investigation of the inflammatory response and immune cell dynamics. By measuring changes in NLR, we can gain insights into the balance between pro-inflammatory (neutrophils) and anti-inflammatory (lymphocytes) responses in the blood circulation following exposure to LPS. The LPS-induced increase in NLR was attenuated by nSsnB (Figure 3A). Consistent with these findings, the LPS-induced increases in plasma TNF-α (Figure 3B) and IL-6 (Figure 3C) were both ameliorated by prophylactic nSsnB administration. Collectively these findings suggest that prophylactic nSsnB ameliorates LPS-induced systemic inflammation.

We measured inflammatory cytokine levels in BALF as an indicator if lung inflammation and total BALF protein as an indicator of increased vascular permeability in ALI. The LPS-induced increase in BALF TNF-α (Figure 3D) and IL-6 (Figure 3E) were significantly reduced by nSsnB (P < 0.05, LPS vs. LPS/nSsnB). Likewise, the increased BALF protein levels observed in LPS-treated mice were significantly reduced by prophylactic nSsnB (Figure 3F). Collectively these finding suggests nSsnB attenuates both systemic and pulmonary inflammation in this model.

Lung injury and inflammation are characterized by increased leukocytes in the BALF. Hema-3 staining was used for cytological analysis of BALF (Figure 4A). Neutrophils (Figure 4B ₁) and macrophages (Figure 4B ₂) were both increased in BALF from LPS-treated mice. In contrast, pretreatment with nSsnB significantly reduced both Neutrophils and macrophages in the LPS/nSsnB group (P < 0.05, LPS vs. LPS/nSsnB). The reductions in BALF leukocytes observed in this experiment support the anti-inflammatory effects of nSsnB on LPS-induced lung inflammation and injury.

Analysis of lung histology is critical to assessing the severity of lung injury, validating animal models, and evaluating potential therapies. LPS-induced lung injury is characterized by polymorphonuclear neutrophils (PMNs) infiltration, alveolar hemorrhage, edema, and the formation of hyaline membranes. Pre-treatment with nSsnB mitigated histological lung injury (Figure 5A) and reduced the lung injury score (Figure 5B) in LPS-induced lung injury (P < 0.05, LPS vs. LPS/nSsnB). The histological findings support the therapeutic efficacy of prophylactic nSsnB in attenuating LPS-induced lung injury.

Myeloid Differentiation Primary Response 88 (MyD88) and Nuclear Factor-kappa B (NF-kB) are crucial components of the Toll-like receptor (TLR) signaling pathway, especially in the context of TLR-4 activation. LPS significantly increased the pulmonary levels MyD88 (Figure 6A) and p-NF-κB P65 (Figure 6B) in C57BL/6 mice (P < 0.05 vs. Control). However, pretreatment with nSsnB significantly reduced the LPS-induced increases in both MyD88 and p-NF-κB P65 (P < 0.05 vs. LPS). LPS-induced activation of NF-κB p65 drives the transcription of pro-inflammatory cytokines, including TNF-α (Figure 6C) and IL-6 (Figure 6D). Our results demonstrate that the elevated levels of TNF-α and IL-6 in lung tissue were significantly attenuated by nSsnB treatment. We also examined pulmonary NF-κB activation using HLL mice to investigate the effects of LPS/nSsnB on NF-κB signaling in vivo (Figure 6E). The signal intensity was significantly elevated in the HLL LPS-treated mice compared to controls. As expected, pretreatment with nSsnB markedly reduced the LPS-induced signal enhancement. Overall, our in vivo and in vitro data validate SsnB as an inhibitor of TLR-2/4 signaling by blocking MyD88 and NF-κB, leading to reductions in inflammation and lung injury.