Lung-Targeted Lipid Nanoparticles Delivery of Wogonin for Pulmonary Fibrosis in Mice via Modulation of Cellular Proteostasis

Introduction

Pulmonary fibrosis (PF) is a progressive and debilitating lung disease characterized by excessive scarring and thickening of lung tissue, leading to reduced elasticity, impaired gas exchange, and gradual decline in respiratory function. With a median survival of approximately 4.5 years, PF is more lethal than many forms of cancer.¹ Clinical studies have shown that PF is frequently accompanied by cardiovascular comorbidities including pulmonary hypertension, cardiac remodeling, and heart failure,² with increasing incidence reported in North America and Europe.³ The pathogenesis of PF is highly complex, and effective therapeutic options remain limited.⁴ While chronic inflammation contributes to disease progression, anti-inflammatory therapies have shown limited clinical benefits. Currently, nintedanib and pirfenidone are recommended for PF patients; however, both are associated with unfavorable adverse effects.^5,6 Thus, there is an urgent need to further elucidate PF pathogenesis and to develop more effective and safer therapeutic strategies.

Natural products have emerged as valuable sources of anti-fibrotic agents. Wogonin, a flavonoid isolated from Scutellaria baicalensis, has demonstrated promising anti-fibrotic effects in preclinical PF models.^7,8 Our previous studies further revealed that wogonin exerts protective effects not only against PF but also against PF-associated cardiac dysfunction,^9,10 potentially through inhibition of CDK9-mediated cell senescence and DNA damage.^11,12 Despite its therapeutic potential, wogonin-- like many natural compounds--exhibits poor tissue specificity, resulting in widespread systemic distribution and off-target effects.¹³ Although the lungs are among the primary organs where wogonin accumulates following intravenous administration, its insufficient enrichment at fibrotic lesions, along with identifying its direct molecular targets and mechanisms of action, is essential to facilitate its clinical translation.¹⁴

Nanoparticle-based drug delivery systems have emerged as powerful tools to enhance drug accumulation at diseased sites.¹⁵ Among these, lipid nanoparticles (LNP), especially liposomes and lipid/nucleic acid complexes, have shown considerable promise in pharmaceutical applications.¹⁶ Liposomes, in particular, were among the first nanomedicine platforms approved for clinical use,¹⁷ with successful examples such as Onpattro,¹⁸ Marqibo,¹⁹ Doxil,²⁰ and Onivvde.²¹ The clinical success of these formulations largely depends on the development of delivery platforms that are both safe and capable of selectively targeting specific tissues or pathological lesions, thereby improving therapeutic efficacy while minimizing off-target toxicity. In this context, tissue-selective carriers are of great significance. Recent studies have showed that ionizable phospholipids (iPhos) with different tail lengths can influence both in vivo efficacy and organ selectivity, as exemplified by iPhos 9A1P9.²² However, the underlying mechanisms governing this selectivity remain incompletely understood.

In this study, we developed a wogonin-loaded lipid nanoparticle system (LNP-Wog) as a targeted therapeutic strategy for PF. LNP-Wog significantly enhanced the cellular uptake of wogonin in vitro and improved lung-specific accumulation in vivo, thereby amplifying its anti-fibrotic efficacy. Treatment with LNP-Wog resulted in marked attenuation of pulmonary fibrosis, improved survival rate, and reduced drug dosage requirement. To elucidate the mechanisms underlying lung targeting and therapeutic activity, we performed a series of multi-dimensional analyses. In vivo imaging and immunofluorescence staining demonstrated rapid and selective pulmonary accumulation of LNP-Wog. Proteomic analysis of the LNP-associated “protein corona” revealed a predominant enrichment of fibrinogen, suggesting a targeting mechanism mediated through interaction with αvβ3 integrin-expressing cells. Furthermore, biotin-affinity pulldown assays identified proteins that directly interact with wogonin. Gene ontology (GO) enrichment analysis indicated that these target proteins are primarily associated with endoplasmic reticulum-related pathways. Mechanistically, wogonin was found to inhibit the phosphorylation of eIF2α, a critical regulatory event in the integrated stress response (ISR), thereby suppressing TGF-β-induced protein synthesis. Collectively, these findings provide new insights into the molecular mechanisms underlying the protective effects of wogonin against pulmonary fibrosis and underscore the therapeutic potential of lung-targeted LNP delivery systems. LNP-loaded wogonin thus represents a promising and effective strategy for the treatment of PF.

Materials and Methods

Preparation of Lipid Nanoparticle-Wogonin (LNP-Wog)

Previous studies have demonstrated that LNP containing ionizable phospholipids, such as 9A1P9-DDAB formulations, possess the lung-targeting capabilities. In this study, a 9A1P9-DDAB-based LNP was prepared using the ethanol dilution method.²² Briefly, a lipid mixture containing 9A1P9, dioctadecyl dimethylammonium bromide (DDAB), cholesterol and DMG-PEG was prepared at a mass ratio of 10 mg:6.4 mg:5.24 mg:0.2 mg, respectively, and dissolved in 500 μL ethanol. Separately, wogonin (2 mg, SW8020, Solarbio, 98%) was dissolved in methanol (350 μL), sonicated, and then mixed with sterile water (1150 μL). The lipid solution was rapidly combined with the aqueous wogonin using sterile syringes at a volumetric aqueous-to- ethanol ratio of 3:1. The resulting mixture was sonicated and extruded using an Avanti liposomal extruder to achieve uniform particle size distribution. Methanol was subsequently removed by dialysis using Pur-A-Lyzer (6–8 KDa molecular weight cutoff, MWCO) against 500-fold volume of PBS (pH 7.4) at 4 °C for 2 h.

Characterization of LNP-Wog

Particle size distribution, polydispersity index (PDI), and zeta potential of LNP-Wog were measured by dynamic light scattering (DLS) using a Zetaszier Nano-ZS90 (Malvern, UK) at 25°C. All LNP formulations exhibited a PDI below 0.3, indicating a uniform and narrowly distributed particle size. The encapsulation efficiency of wogonin within the LNP was determined by UV-visible spectrophotometer. Standard wogonin solutions, with blank solvent as control, were scanned in the range of 250–500 nm, and the maximum absorption wavelength of wogonin was identified at 275 nm (Supplementary Figure 1A). A standard calibration curve was generated using wogonin dissolved in methanol (100 μg/mL) (Supplementary Figure 1B). The morphological characteristics of LNP-Wog were further analyzed using transmission electron microscopy (TEM, Hitachi HT7800, Japan).

Release Behavior of LNP-Wog in vitro

The in vitro release profile of LNP-Wog was assessed as previously described.²³ LNP-Wog was loaded into dialysis bag (MWCO: 8–14 kDa) and immersed in 500 mL phosphate buffered saline (PBS, pH 7.4) containing 0.5% (w/v) Tween-80. The system was maintained at 37°C with continuous stirring (100 rpm) for 48 h. At predetermined time points, 5 mL of release solution was collected and replaced with an equal volume of fresh release medium to maintain a constant volume. The concentration of wogonin was determined using a UV-visible spectrophotometer at the wavelength of 275 nm. In-vitro release experiments were performed in triplicate.

In vitro Cellular Uptake

Cellular uptake was assessed as previously described.²⁴ Cy5-labeled wogonin (Wog-Cy5) was synthesized and encapsulated into LNP to obtain LNP-Wog-Cy5. Mouse Lung Epithelial cells (MLE-12) were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin -streptomycin solution (100 U/mL each). Cells were treated with DMSO (control), free Wog-Cy5, or LNP-Wog-Cy5 at a final Wog-Cy5 concentration of 1 μM for 12 h. After treatment, cells were washed with PBS, collected, and analyzed by flow cytometry and confocal microscopy. For flow cytometry, 1×10⁴ events were acquired per sample using a BD FACSCanto system (BD Biosciences, CA), and mean fluorescence intensity was quantified. For imaging, cells were stained with DAPI and visualized using an AX confocal microscope (Nikon, Japan).

In vivo Biodistribution of LNP-Wog

To evaluate the biodistribution of LNP in vivo,²⁴ C57BL/6J mice were intravenously injected via the tail vein with either free Wog-Cy5 or LNP-Wog-Cy5 at a dose equivalent to 1 mg/kg of Wog-Cy5. At 0, 2, 12, and 24 hours post-injection, mice were euthanized, and major organs--including the brain, heart, liver, spleen, lungs, and kidneys--were collected to examine the dynamic distribution of formulations. Fluorescence intensity of Wog-Cy5 in each organ was measured using an AniView100 imaging system (Bolu Teng Biotechnology, Guangzhou, China) to assess temporal and spatial distribution of LNP-Wog.

Animal Experiments

Pulmonary fibrosis was induced in mice by a single intratracheal instillation of bleomycin (BLM) at a dose of 1.5 U/kg (Macklin, Shanghai, China), as previously described. Control mice received an equal volume of sterile saline.²⁵ To evaluate the therapeutic effects of free wogonin and LNP-Wog in the BLM-induced fibrosis model, male C57BL/6J mice (9–12 weeks old) (Animal license No. SCXK (ZHE) 2024–0001) were randomly assigned to four groups: control, BLM, BLM+free wogonin, and BLM+LNP-Wog. Free wogonin (10 mg/kg) was administered intravenously every other day starting from day 8 post-BLM for 2 weeks. LNP-Wog was administrated using the same schedule at a wogonin-equivalent dose at 2 mg/kg based on its encapsulated wogonin content. Mice were sacrificed on day 21. All animal procedures were approved by the Ethics Committee of the First Affiliated Hospital of Xinxiang Medical University and conducted in accordance with the National Act on the Use of Experimental Animals (China) (No. EC-024-33).

Histological and Immunohistochemical Analysis

To assess tissue morphology, mice from each group were humanely sacrificed, and lung tissues were harvested, rinsed with PBS to remove residual blood, and fixed in 4% paraformaldehyde. The samples were then processed through standard dehydration, paraffin embedding, and sectioned at 4 μm thickness for subsequent analysis. Hematoxylin-Eosin (H&E) staining (G1120, Solarbio, Beijing, China) and Masson’s trichrome staining (G1346, Solarbio, Beijing, China) were performed following the manufacturer’s protocols to evaluate tissue structure and collagen deposition, respectively. Fibrosis was quantified using the Ashcroft scoring method.²⁶ For immunohistochemistry, lung sections were deparaffinized, rehydrated, and incubated overnight at 4°C with primary antibody against fibronectin (15613-1-AP, Proteintech, Wuhan, China). Nuclei were counterstained with hematoxylin. Positive staining was quantified using ImageJ software.

Isolation of Protein Corona

The isolation of protein corona was performed as previously described.²⁷ Briefly, mouse plasma was collected and centrifuged at 12,000×g for 5 min at 4 °C to remove protein aggregates, and the supernatant was reserved for further use. LNP-Wog (400 μL) was placed into 1.5 mL microcentrifuge tubes and mixed with an equal volume of mouse plasma to mimic in vivo protein concentrations. The samples were incubated at 37 °C with gentle shaking for 2 hours, followed by centrifugation at 13,000×g for 30 min to isolate LNP-Wog coated with the protein corona. The resulting pellets were washed three times with cold PBS to remove unbound proteins. Protein samples were then mixed with 1× loading buffer, separated by SDS-PAGE, and analysed by mass spectrometry for protein identification.

Western Blotting

MLE-12 cells were lysed, and proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% BSA and incubated with the following primary antibodies: anti-puromycin (clone 12D10, MABE343, Sigma, Germany), eIF2α (11170-1-AP, Proteintech), p-eIF2α (ab32157, Abcam, UK), and GAPDH (10494-1-AP, Proteintech) as a loading control. Protein bands were visualized using an Amersham Imager 600 (GE Healthcare) with chemiluminescence detection. Band intensities were quantified by densitometric analysis using ImageJ software (NIH, USA).

Affinity Pulldown Assay

To identify potential protein targets of wogonin, biotin-labeled wogonin (Bio-Wog) was synthesized by Chongqing Yusi Pharmaceutical Technology (Chongqing, China). The biotin-affinity pulldown assay was performed as previously described.^28,29 Briefly, MLE-12 cell lysates were precleared and incubated with 20 μM Bio-Wog or biotin alone (negative control) at room temperature for 2 h. The mixtures were then incubated with streptavidin magnetic beads for 4 h with gentle rocking. After magnetic enrichment, the beads were washed thoroughly with PBS to remove nonspecifically bound proteins and resuspended in 1× loading buffer. MLE-12 cell lysates were used as input controls. Protein complexes were resolved by 10% SDS-PAGE, visualized by Coomassie Brilliant Blue staining, and subjected to mass spectrometry for protein identification. The identified proteins were analyzed by Gene Ontology (GO) enrichment analyses to identify associated biological processes (BP).

Biosafety Evaluation

To assess the biosafety of LNP-Wog, healthy C57BL/6J mice were intravenously administered LNP-Wog (10 mg/kg) or an equal volume of saline as a control. Blood samples were collected via retro-orbital puncture 48 h post-injection and centrifuged at 1200×g for 10 min at 4 °C. Plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were measured using commercial assay kits (Nanjing Jiancheng Institute, Nanjing, China) to evaluate hepatic and renal function. Additionally, 48 h after treatment, mice were euthanized and major organs (heart, liver, spleen, lungs, and kidneys) were harvested, washed with PBS, fixed in 4% paraformaldehyde, and processed for H&E staining to assess potential tissue damage or toxicity.

Protein Synthesis Assay

To assess protein synthesis in vitro, MLE-12 cells were stimulated with TGF- β (10 ng/mL) with or without wogonin for 12 h. Puromycin (10 μg/mL) was then added to the culture medium for 20–60 min to label nascent proteins.³⁰ After incubation, cells were washed with PBS to remove excess puromycin and lysed for analysis. Protein lysates were subjected to Western blot using anti-puromycin antibody (12D10, Sigma-Aldrich, USA) to detect puromycin- incorporated proteins. Protein synthesis levels were quantified by measuring the total signal intensity across each lane, with background correction based on vehicle only control samples. Results were expressed as fold changes relative to the vehicle-treated control group.

Synthesis of Cy5-Labeled Wogonin (Cy5-Wog)

Wogonin (1.0 equiv.) was added to a reaction flask, followed by the addition of Cy5-COOH (0.7 equiv)., EDC (1.5 equiv)., and DMAP (0.1 equiv.) dissolved in DMF. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then concentrated under reduced pressure and purified by gradient elution using acetonitrile: water (0.1% TFA) ranging from 1:19 to 1:1 over 40 min. The desired product-containing fractions were collected and concentrated under reduced pressure. The residue was redissolved in a small volume of ethanol and precipitated with diethyl ether. This precipitation step was repeated twice with diethyl ether to obtain the final product Cy5-wogonin. The product was characterized by mass spectroscopy (Supplementary Figure 2).

Statistics

All data in this study were presented as mean ± SEM. Group comparisons were performed using one or two-way ANOVA, as appropriate. A post-hoc Tukey’s test was performed to isolate differences. A p-value of <0.05 was considered statistically significant. Analyses were conducted using GraphPad Prism 9.0 software.

Results and Discussion

Synthesis and Characterization of LNP-Wog

We first synthesized LNP-encapsulated wogonin (LNP-Wog), as illustrated in Figure 1A. Transmission electron microscopy (TEM) revealed that LNP-Wog particles exhibited a uniform spherical morphology with an average diameter of approximately 150 nm (Figure 1B and C). LNP smaller than 200 nm are generally associated with prolonged circulation time, likely due to reduced immune recognition and clearance in the body.³¹ In addition to particle size, colloidal stability in aqueous environments is another crucial parameter for LNP formulations. Dynamic light scattering (DLS) analysis showed that LNP-Wog possessed a zeta potential of +37.3 mV in pure water (Figure 1D), exceeding the ±30 mV threshold typically indicative of good colloidal stability.³² The encapsulation efficiency of wogonin within the LNP was 84.6%, confirming a high loading capacity of the formulation. In vitro release studies further demonstrated that more than 60% of wogonin was released within 24 h (Supplementary Figure 1C). Collectively, these results demonstrate the successful synthesis of LNP-Wog with favorable physicochemical properties for biological applications.

Figure 1 Preparation and characterization of LNP-Wog. (A) Schematic diagram showing the synthesis of LNP-Wog. (B) Representative TEM images of LNP-Wog. (C) The particle diameter of LNP-Wog. (D) The zeta potential of LNP-Wog.

Enhanced Cellular Uptake of LNP-Wog

To evaluate cellular uptake efficiency of LNP-Wog in vitro, Cy5-labeled wogonin (Wog-Cy5) was synthesized (Figure 2A) and analyzed by confocal microscopy and flow cytometry. MLE-12 cells were cultured in 6-well plates or confocal dishes for 24h, and then treated with vehicle, free Wog-Cy5, or LNP-Wog-Cy5 at a final Wog-Cy5 concentration of 1 μM. After 12 h of incubation, cells were washed thoroughly with PBS to remove any unbound compounds and subsequently harvested for analysis. Confocal imaging revealed clear intracellular fluorescence in both treatment groups, confirming successful cellular uptake (Figure 2B). Notably, cells treated with LNP-Wog-Cy5 exhibited markedly stronger fluorescence compared to those incubated with free Wog-Cy5 (Figure 2B). Flow cytometry analysis further demonstrated that the mean fluorescence intensity of Cy5 in cells treated with LNP-Wog-Cy5 was 4.4-fold higher than that in the free Wog-Cy5 group (Figure 2C and D). These results indicate that LNP encapsulation significantly enhances the intracellular delivery and uptake of wogonin in MLE-12 cells, supporting the potential of LNP as an efficient nanocarrier for pulmonary-targeted drug delivery.

Figure 2 The enhanced cellular uptake of LNP-Wog. (A) Synthesis of Cy5 labeled wogonin (Wog-Cy5). (B) Cellular uptake of LNP-Wog-Cy5 was assessed 12 h post-treatment using confocal microscopy. MLE-12 cells were treated with vehicle control (DMSO), free Wog-Cy5, or LNP-Wog-Cy5 at a final Wog-Cy5 concentration of 1 µM. Scale bar: 100 µm. (C) Flow cytometry histogram. (D) Relative quantification of flow cytometry. **p < 0.01, compared with control groups; ##p < 0.01, compared with Wog-Cy5 groups, n=6/group. All data are presented as means ± SEM, one-way ANOVA with a post hoc Tukey’s test.

Targeted Accumulation and Prolonged Retention of LNP-Wog in Mouse Lungs

To assess the biodistribution of LNP-Wog in vivo, mice were intravenously injected with either free Wog-Cy5 or LNP-Wog-Cy5 via the tail vein at a dose of 1 mg/kg body weight. Fluorescence intensity in major organs was monitored at various time points using an in vivo imaging system. Free Wog-Cy5 showed peak accumulations in the lungs, kidneys and liver at 2 h post-injection, followed by rapid clearance within 24 h (Figure 3A and C). In contrast, LNP-Wog exhibited gradual accumulation in the lungs starting at 2 hours, with strong and sustained fluorescence signals exclusively persisting in lung tissue for more than 24 hours. Importantly, the mean fluorescence intensity in the lungs of LNP-Wog-Cy5 group was 23-fold higher than that of the free Wog-Cy5 group (Figure 3B and D). This prolonged retention suggests a controlled release profile of LNP formulation. Overall, these findings indicate that LNP-Wog-Cy5 achieved significantly greater and longer-lasting accumulation in the lungs compared to free Wog-Cy5, highlighting its enhanced lung-targeting capability.

Figure 3 Targeted accumulation and prolonged retention of LNP-Wog in mouse lungs. Fluorescence imaging was performed to assess the biodistribution of Wog and LNP-Wog in major organs of mice. Representative fluorescence images showing indicated the biodistribution of Wog-Cy5 (A) and LNP-Wog-Cy5 (B) at 0, 2, 12, 24 hours post-injection. Quantification of fluorescence intensity in various organs over time of Wog-Cy5 (C) and LNP-Wog-Cy5 (D).

LNP-Wog Significantly Ameliorates Pulmonary Fibrosis in Mice

Our previous studies established that wogonin confers protective effects against PF in vivo at doses ranging from 10 to 50 mg/kg (data not shown). Additionally, in vivo biodistribution results revealed that LNP-Wog-Cy5 accumulated in the lungs at levels 5–30 times higher than free Wog-Cy5 between 12 to 24 hours post injection. Based on these findings, we selected 10 mg/kg of free wogonin and 2 mg/kg of LNP-Wog (based on wogonin content) for this study. Mice were randomly assigned to four groups: control, BLM-induced PF, BLM+free Wog, and BLM+LNP-Wog. The control group received an equivalent volume of saline. BLM administration resulted in approximately 70% mortality, which was reduced to 50% by free wogonin treatment. Notably, LNP-Wog further reduced mortality to 20%, indicating a substantial survival benefit (Figure 4A).

Figure 4 LNP-Wog significantly ameliorates pulmonary fibrosis in mice. (A) Survival rate of mice across different treatment groups. n=10/group. (B) Hydroxyproline content in mouse lung tissues. n=5-7/group. (C) Representative histological images of lung sections stained with H&E, Masson’s trichrome staining and immunohistochemistry for fibronectin. Scale bars: 100 μm. (D) Fibrosis scores based on Masson’s trichrome staining. n=4/group. (E) Quantitative analysis of fibronectin expression based on immunohistochemistry. n=4/group. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, compared with control animals; #p < 0.05, ##p < 0.01, compared with BLM group, two-way ANOVA with a post hoc Tukey’s test.

Hydroxyproline levels, a surrogate marker for collagen deposition, were significantly elevated in mice exposed to BLM but markedly reduced by both treatments, with LNP-Wog showing a more pronounced effect (Figure 4B), highlighting its enhanced anti-fibrotic efficacy. Histological analyses, including H&E, Masson’s trichrome staining, and fibronectin immunostaining, confirmed extensive fibrosis in BLM-treated mice, which was partially alleviated by free wogonin and markedly reversed by LNP-Wog treatment (Figure 4C–E). These results collectively demonstrate that LNP-Wog significantly enhances the therapeutic efficacy of wogonin, offering superior protection against BLM-induced pulmonary fibrosis in vivo.

LNP-Wog Targets Lung Endothelial Cells via Corona Formation

Nanoparticles, such as liposomes, are known to form a “protein corona” upon exposure to biology fluids, which influences their surface properties and in vivo behavior.³³ To investigate this, LNP-Wog was incubated with mouse plasma at 37 °C for 2 h. The resulting protein-coated LNP-Wog particles were isolated by centrifugation, washed with saline, and subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining (Figure 5A) and identified by mass spectrometry.

Figure 5 Proteomics analysis of protein coronas formed on LNP-Wog. (A) SDS-PAGE followed by Coomassie Brilliant Blue staining showing the protein Corona profiles on LNP-Wog, mouse plasma as the input. (B) The 20 most abundant corona proteins were identified and categorized based on their calculated molecular weight and isoelectric point. (C) Immunofluorescence staining of LNP-Wog-Cy5 (red) and CD31-positive endothelial cells (green) in mouse lung tissue. Scale bars: 50 μm.

Proteomic analysis identified 317 proteins associated with LNP-Wog (Supplementary Table 1), with the top 20 most abundant proteins listed in Table 1. These proteins likely play a dominant role in protein corona formation. Notably, 70% of these proteins had molecular weight (MW) below 80 kDa (Figure 5B), indicating an enrichment of low-MW proteins in the corona. Furthermore, at physiological pH 7.4, 75% of proteins exhibited isoelectric points (pI) below 7.4 (Figure 5B), suggesting enrichment of negatively charged proteins. This is consistent with electrostatic interactions between negatively charged plasma proteins and the positively charged surface of LNP-Wog.

Table 1 Top 20 Most Abundant Proteins Identified in the Protein Corona of LNP-Wog

Notably, fibrinogen chain isoforms were the most abundant components in the protein corona, collectively accounting for 66% of the top 20 proteins. Given that fibrinogen interacts with integrins (eg, αvβ3), this suggests a mechanism for endothelial targeting.^34–36 To confirm the binding of LNP-Wog with integrins, membrane proteins isolated from lung tissues by using a cell-membrane cytoplasmic separation kit were incubated with LNP-Wog at 37 °C for 2 h, and isolated by centrifugation, then subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining (Supplementary Figure 3). Mass spectrometry further identified multiple integrins, including intergrin alpha-1, integrin beta-1, integrin alpha-6, integrin beta-2, integrin alpha-3 and integrin alpha-8, as binding partners of LNP-Wog (Supplementary Table 2). Western blotting confirmed the direct interaction between LNP-Wog and Integrin beta-1 (Supplementary Figure 4).

To confirm the interaction between LNP-Wog and vascular endothelium in vivo, mice were injected with LNP-Wog-Cy5 and sacrificed 12 hours post-injection. Lung tissues were harvested and embedded in OCT. Immunofluorescence analysis showed clear co-localization of LNP-Wog-Cy5 (red) with CD31-positive endothelial cells (green) in lung tissue (Figure 5C), confirming endothelial targeting. These results suggest that fibrinogen-enriched protein corona mediated lung endothelial targeting of LNP-Wog.

Wogonin Inhibits Protein Synthesis via Upregulating eIF2α Phosphorylation

To elucidate the potential molecular mechanism, biotin-labeled wogonin probe (Wog-Bio) was used for affinity pulldown assay as previous described.²⁸ Mice were euthanized using isoflurane and underwent cardiac perfusion with saline to clear circulatory blood. Lung tissues were collected, and cytosolic proteins were isolated using a cell-membrane cytoplasmic separation kit. Wog-Bio was then incubated with cytosolic proteins, followed by enrichment using streptavidin magnetic beads and isolated via magnetic separation, with biotin alone served as the control. Enriched proteins were resolved by SDS-PAGE and visualized by Coomassie-stained (Figure 6A). Mass spectrometry identified 958 interacting proteins in the Bio-Wog treated groups from in-gel enriched samples (Supplementary Table 3). Gene Ontology (GO) analysis revealed significant enrichment in endoplasmic reticulum and nucleic acid metabolism pathways (Figure 6B), both closely related to protein synthesis.

Figure 6 Wogonin inhibits protein synthesis by upregulating eIF2α phosphorylation. (A) SDS-PAGE and Coomassie Brilliant Blue staining of proteins pulled down from MLE-12 cell lysates using Bio-Wog. Biotin alone was used as a negative control; total lysates served as input. (B) Gene Ontology (GO) analysis of candidate proteins identified in the Bio-Wog pull-down. (C) Western blot analysis of the nascent protein synthesis using anti-puromycin antibody; GADPH as a loading control. (D) Western blot analysis of phosphorylated eIF2α (p-eIF2α) and total eiF2α in MLE-12 cells treated with different concentrations of wogonin (0–20 μM) for 24 h. Quantification is shown on the right, n=3/group. **p < 0.01, compared with control groups. (E) Western blot analysis of phosphorylated eIF2α (p-eIF2α) and total eiF2α in lungs of mice treated with or without wogonin. Quantification is shown on the right, n=3/group. *p < 0.05, **p < 0.01, compared with control groups, ##p< 0.01, compared with BLM groups. All data are presented as the mean ± SEM, one-way ANOVA with a post-hoc Tukey’s test.

Using a puromycin-based SUnSET assay,³⁰ we assessed protein synthesis levels across different treatment groups. In MLE-12 cells, treatment with TGF-β upregulated protein synthesis compared to controls, whereas wogonin treatment significantly suppressed this effect (Figure 6C), indicating that wogonin attenuates protein synthesis. Previous studies have demonstrated that phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) at Ser51 suppresses global protein synthesis, a key event in the integrated stress response (ISR) that promotes cellular adaptation and recovery under stress.³⁷ Treatment of MLE-12 cells with wogonin for 12 h resulted in a dose-dependent increase in p-eIF2α levels (Figure 6D). To confirm the expression of p-eIF2α in vivo, mouse lungs of different groups with or without wogonin were harvested. Western blot analysis demonstrated that BLM decreased expression of p-eIF2α, wherever, treated with wogonin reverse these effects, which further indicate that wogonin increases the levels of p-eIF2α (Figure 6E). These results suggest that wogonin alleviates fibrosis by suppressing protein synthesis via activation of eIF2α pathway.

Stability and Safety of LNP-Wog

To assess the stability and potential in vivo application of LNP-Wog nanoparticles, we stored LNP-Wog and blank LNP in distilled water at 4°C for 7 days. Hydrated particle size measurements on day 7 showed no significant changes, confirming size stability throughout the storage period (Figure 7A). Polymer dispersion index (PDI) and zeta potential analyses also revealed no significant alterations in either LNP-Wog or blank LNP over 7 days (Figure 7B and C). These consistent values indicate strong repulsive forces that prevent particle aggregation, suggesting ideal drug retention in vivo.

Figure 7 Stability and Safety of LNP-Wog in vivo. (A) Hydrodynamic diameter of LNP-Wog. Changes in polydispersity index (B) and Zeta potential (C) of LNP-Wog over 7 days in sterile water. (D) Evaluation of liver and kidney function following treatment with LNP-Wog (10 mg/kg) for 48 h. n=5/group. All data are presented as the mean ± SEM. (E) Representative H&E images of major organs (heart, liver, spleen, lung, and kidney) from healthy mice treated with saline or LNP-Wog. Scale bars: 100 µm.

With safety concern regarding the in vivo use of LNP-Wog, biosafety was assessed in healthy mice. Serum levels of liver and kidney function markers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CR) were measured 48 hours after LNP-Wog administration, control mice were administered the same volume of sterile saline. No significant differences were observed compared to controls (Figure 7D), confirming the absence of liver or kidney toxicity. Furthermore, histological examination of major organs such as heart, liver, spleen, lung, and kidney tissues from treated mice revealed no detectable toxic effects (Figure 7E). Taken together, these results demonstrate that LNP-Wog nanoparticles possess excellent stability and a favorable safety profile for in vivo applications.

Conclusions

In this study, we developed a cationic lipid nanoparticles system (LNP-Wog) as a lung-targeted delivery platform for wogonin to combat pulmonary fibrosis. We demonstrated that LNP-Wog effectively accumulates in mouse lungs and preferentially targets pulmonary endothelial cells in vivo. Notably, LNP-Wog rapidly forms a fibrinogen-rich protein corona upon exposure to the circulation, enabling specific binding to integrin expressed on pulmonary endothelial cells. This interaction promotes lung-selective targeting, prolonged retention, and enhanced local drug exposure.

Mechanistically, LNP-mediated delivery promotes the accumulation of wogonin in lung tissue, where it interacts with endoplasmic reticulum–associated proteins. Biotin-affinity pulldown combined with Gene Ontology enrichment analysis indicated that wogonin primarily targets pathways involved in protein synthesis. Specifically, wogonin enhances phosphorylation of eIF2α, a key regulator of the integrated stress response, thereby suppressing aberrant protein synthesis and attenuating fibrotic progression. Collectively, these features enable LNP-Wog to significantly improve anti-fibrotic efficacy while reducing the required therapeutic doses. Compared with free wogonin, LNP-Wog achieved superior therapeutic outcomes at markedly lower doses.

Importantly, biosafety evaluation demonstrated that LNP-Wog exhibits excellent biocompatibility. Serum biomarkers of liver (AST, ALT) and kidney (BUN, CR) function remained unchanged, and histopathological analysis showed no evidence of organ toxicity.

Despite these promising findings, several challenges remain for clinical translation of LNP-based formulations, including limited shelf life, potential contamination risks, long-term biosafety and immunogenicity, scalability under good manufacturing practice (GMP) conditions, and possible differences in pharmacokinetics and therapeutic efficacy in humans.³⁸

In summary, LNP-Wog achieves efficient lung targeting and enhanced intracellular delivery through protein corona-mediated endothelial interactions. Meanwhile, wogonin exerts its anti-fibrotic effects by regulating eIF2α-mediated proteostasis and suppressing abnormal protein synthesis. The synergistic effect of optimized delivery and precise pharmacological action enables LNP-Wog to achieve potent anti-fibrotic efficacy at reduced dose. These findings not only highlight a promising nanoplatform for targeted delivery of wogonin but also deepen the mechanistic understanding of its anti-fibrotic action. Moreover, this work broadens the therapeutic potential of traditional Chinese medicine-derived monomers and support the further development of LNP-Wog as a viable strategy for the treatment of pulmonary fibrosis.