Nebulized inhalation of novel baicalein liposome based on phospholipid complex to alleviate acute lung injury

ABSTRACT

Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are high-mortality respiratory diseases manifested by excessive inflammation and oxidative stress. Current therapies for ALI are limited by uncertain efficacy, severe side effects, restricted administration routes, or inadequate targeting capacity. Baicalein, a natural flavonoid with significant anti-inflammatory and antioxidant activity, shows promise for ALI. Unfortunately, its clinical application is limited by its low solubility and bioavailability. In this study, a novel baicalein liposome (BAPC-DLP) based on a baicalein‒phospholipid complex (BAPC) was constructed for nebulized inhalation. Comparative studies demonstrated that BAPC-DLP outperformed other baicalein-loaded liposomes in colloid stability, mucus penetration ability, and in vivo pulmonary deposition. Further, BAPC-DLP enhanced the baicalein loading capacity and exhibited excellent aerosolization performance. Moreover, it effectively suppressed lipopolysaccharide (LPS)-induced inflammatory responses and scavenged excessive ROS in macrophages and alveolar epithelial cells. Ex vivo fluorescence imaging confirmed its efficient lung deposition and prolonged lung retention. In an LPS-induced ALI mouse model, inhaled BAPC-DLP exerted a superior protective effect to free baicalein by alleviating pulmonary edema, lowering the level of proinflammatory cytokines, decreasing inflammatory cell infiltration, and significantly ameliorating tissue injury. This study presents a promising preventive pharmacotherapy strategy for ALI/ARDS and a potential nebulized inhalation delivery platform for insoluble natural products, highlighting the significant potential for clinical translation.

Graphical abstract

1. Introduction

Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are critical respiratory diseases associated with high morbidity and mortality (Bellani et al. 2016; Beitler et al. 2022). This process involves pathologic changes such as alveolar epithelial cell and pulmonary capillary endothelial cell injury, inflammatory cell infiltration, and pulmonary edema, and critically ill patients may further develop respiratory failure (Hughes and Beasley 2017; Luyt et al. 2020). Extensive evidence suggests that an overwhelming inflammatory cascade response, known as a cytokine storm, and excessive reactive oxygen species (ROS) play a significant role in the progression of ALI (Yin et al. 2023; Ge et al. 2025). Therefore, it is expected to be an effective strategy to alleviate ALI and prevent the progression of ALI by suppressing the excessive inflammatory response at an early stage.

Currently, the clinical management of ALI/ARDS relies primarily on protective lung ventilation and immunosuppressive agents such as glucocorticoids (Sweeney and McAuley 2016). Protective lung ventilation may mitigate mechanical lung injury by restricting tidal volume, but long-term therapy may exacerbate alveolar injury (Matthay et al. 2019). Although glucocorticoids have been investigated as a therapeutic option, their ability to improve survival has not been confirmed (Peter et al. 2008). The potential risks of secondary infection and metabolic dysfunction severely limit their clinical application (Caplan et al. 2017). As a result, effective drugs for the treatment of ALI are lacking. In addition, existing therapeutic methods generally fail to sufficiently target the lungs maintain the effective drug concentration, thereby limiting the therapeutic effect (Steinberg et al. 2006). Therefore, there is an urgent need to develop novel therapeutic approaches for enhancing the therapeutic effect of ALI/ARDS.

In recent years, natural products have been increasingly studied for ALI/ARDS treatment owing to their advantages of low toxicity and multi-target regulation of pathological processes (inflammation, oxidative stress, etc.) (He et al. 2021). Baicalein (BA), a natural flavonoid isolated from the dried roots of Scutellaria baicalensis, is one of the major active components of Scutellaria baicalensis that exerts therapeutic effects (Wei et al. 2015). Extensive studies have indicated that baicalein displays multiple pharmacological activities, including anti-inflammatory, antioxidant, antiviral, and antibacterial effects (He et al. 2015; Patwardhan et al. 2016; Tsou et al. 2016; Luo et al. 2020; Chi et al. 2025). In a previous study, baicalein alleviated ALI by regulating Drp1-induced mitochondrial damage, suppressing inflammatory responses, and attenuating the severity of lung injury (Jiang et al. 2022). In addition, baicalein prevents TLR4-MD2 complex formation by competitively binding to MD2, thereby blocking TLR4-MD2 dimerization and downstream activation of the MAPK and NF-κB pathways, resulting in the inhibition of inflammatory damage in lung tissue and improved survival in ALI mice (Chen et al. 2019). These studies suggest that baicalein is a potentially therapeutic agent for ALI.

However, the clinical application of baicalein is critically constrained by its extremely poor solubility (17.5 μg/mL), low permeability, erratic oral absorption, and rapid metabolic clearance following intravenous administration (Zhu et al. 2017). Although various advanced formulation techniques (e.g. solid dispersion, cyclodextrin complex, nanoemulsion, and polymeric micelle) have been employed to refine drug properties, the enhancement in solubility and bioavailability remains limited (Zhou et al. 2013; Zhou et al. 2017; Yin et al. 2017; Zhang et al. 2020). Moreover, the existing routes of administration of baicalein were mainly studied in oral or injectable routes with insufficient pulmonary targeting, thereby limiting its efficacy against ALI.

To this end, developing a therapeutic strategy that can deliver drugs directly to the lungs is especially imperative. As a non-invasive route of administration, inhalation therapy has emerged as the preferred treatment strategy for respiratory diseases owing to its superior patient compliance and lung-targeting capability (Yue et al. 2023). By direct deposition in the respiratory tract, inhalation administration increases the local drug concentration, thereby lowering the dose and systemic toxicity (Wang et al. 2022; Li et al. 2024). Conventional inhalation dosage forms typically consist of metered-dose inhalers, nebulizers, and dry powder inhalers (Du et al. 2024). Nebulizers are the most commonly used in clinical practice because of the absence of propellants, simple administration operation, sustained high-dose delivery, and low respiratory tract irritation (Yan et al. 2025).

However, there are multiple critical challenges to the development of nebulizers. First, over 70% of small-molecule drugs and 90% of novel chemical entities under development are poorly water soluble, resulting in significant dose limitations for aerosol delivery, especially for drugs requiring high doses (Lou et al. 2024). Furthermore, the stringent biocompatibility requirements for inhaled excipients and the limited number of approved pulmonary-safe excipients make it challenging to balance the physicochemical stability and biosafety for poorly soluble drugs such as baicalein. Moreover, the positive correlation between the nebulization time and the volume of the drug solution creates a paradoxical relationship between high drug-loading demands and patient compliance. Nebulization time can be shortened by increasing the drug concentration, whereas the ensuing technical challenges regarding solubility limitations and formulation stability are not usually easily resolved. Notably, undissolved drugs are readily cleared by the mucosal ciliary system in the conducting zone or phagocytosed by alveolar macrophages in the respiratory zone after inhalation, and long-term retention of insoluble particles may also cause lung irritation and inflammation (Velaga et al. 2018; Borm and Driscoll 2019; Zhou et al. 2020).

Nanobased pulmonary drug delivery has attracted considerable attention for its advantages in improving solubility, prolonging the lung retention time, enhancing therapeutic efficacy, and reducing systemic toxicity (Chang et al. 2024; Yao et al. 2025; Zhu et al. 2025; Zhang et al. 2025). Among the various pulmonary delivery carriers, liposomes exhibit outstanding biocompatibility and biodegradability because their phospholipid bilayer structure is similar to that of pulmonary surfactants (Mehta et al. 2020). Furthermore, the feasibility of the clinical translation of liposomes has greatly increased with the development of scale-up production technologies, rendering liposomes the only clinically available pulmonary drug delivery vehicle to date (Peng et al. 2024). For example, Amikacin liposomal inhalation suspension (Arikayce®) has been approved by the FDA for the treatment of Mycobacterium avium complex lung infections, significantly extending the pulmonary residence time and providing guidance for the development of complicated inhalation formulations (Shirley 2019).

In our previous study, we successfully constructed a phospholipid complex platform that significantly improved drug lipophilicity. Based on this, a variety of drug delivery systems with better compatibility between the drug and the carrier material have been developed, which have favorable effects on improving the drug loading, stability, permeability, and bioavailability of the delivery system (Zhou et al. 2017; Liao et al. 2019; Dong et al. 2020a, 2020b). In particular, we developed two baicalein-loaded nanoparticle systems based on phospholipid complex technology: mucoadhesive chitosan‒phospholipid nanoparticles (BA-CS NPs) and mucus-penetrating poloxamer-based nanoparticles (BA-F127 NPs), both of which were designed for nebulized inhalation therapy (Dong et al. 2020a, 2020b). The clinical translation, however, is challenging because of the limitations of these systems in terms of drug loading and biocompatibility.

Herein, we innovatively integrated a baicalein‒phospholipid complex (BAPC) with liposomes to construct a novel baicalein-loaded liposome (BAPC-DLP, BAPC liposomes with Tween 80) for nebulized inhalation, which significantly enhanced baicalein's solubility and pulmonary delivery efficiency. We first conducted a comparative study to examine the superiority of BAPC-DLP over BA-LP (free BA liposomes without Tween 80) and BAPC-LP (BAPC liposomes without Tween 80) in terms of critical physicochemical properties, mucus penetration ability, and in vivo pulmonary deposition and distribution to validate the design rationale of the delivery system. Next, we evaluated the physicochemical properties and aerosolization performance of BAPC-DLP. Next, we studied the uptake behavior in macrophages and in vitro anti-inflammatory activity. To investigate the superiority of inhaled BAPC-DLP, we further employed fluorescence imaging techniques to analyze the distribution behaviors of DiR-labeled BAPC-DLP compared to free DiR via different administration routes. A murine lipopolysaccharide (LPS)-induced ALI model was established to systematically evaluate the beneficial protective effect of inhaled BAPC-DLP on ALI. Finally, the preliminary biosafety of inhaled BAPC-DLP was evaluated by histopathology and hemolysis test (Scheme 1).

Scheme 1.

Schematic diagrams of the in vivo fate of BAPC-DLP via nebulized inhalation and the protective effect on ALI (image created with BioRender).

2. Materials and methods

2.1. Materials

Baicalein (98% purity) was obtained from Nanjing Zelang Biological Technology Co. Ltd. (Nanjing, China). Soybean phospholipids (S75) were obtained from Shanghai Tywei Pharmaceutical Co. Ltd. (Shanghai, China). Tween 80, DiR and diethylenetriaminepentaacetic acid (DTPA) were purchased from Shanghai Macklin Biochemical Co. Ltd. (Shanghai, China). Egg yolk emulsion was purchased from Qingdao Hopebio Biotechnology Co. Ltd. (Qingdao, China). Dexamethasone 21-phosphate disodium salt (DEX) was obtained from Shanghai Acmec Biochemical Technology Co. Ltd. (Shanghai, China). Gastric mucin was purchased from Shanghai Bide Pharmaceutical Technology Co. Ltd. (Shanghai, China). Coumarin-6 (Cou-6) and LPS from Escherichia coli (O55:B5) were supplied by Sigma–Aldrich (St. Louis, USA). Isoflurane was purchased from Hebei Yipin Pharmaceutical Co. Ltd. (Shijiazhuang, China). A Cell Counting Kit-8 (CCK-8) and a reactive oxygen species assay kit were purchased from Shanghai Beyotime Biotechnology Co. Ltd. (Shanghai, China). IL-6, TNF-α, IL-1β, MCP-1, and IL-10 ELISA kits were purchased from Wuhan Elabscience Biotechnology Co. Ltd. (Wuhan, China). The Pierce™ BCA protein assay kit was manufactured by Thermo Fisher Scientific (Waltham, MA, USA). The polyclonal antibodies against CD68 (28058-1-AP) and MPO (22225-1-AP) were purchased from Proteintech Co. Ltd. (Wuhan, China). All reagents were of analytical or chromatographic grade.

2.2. Preparation of liposomes and confirmation of the formulation composition

2.2.1. Preparation of liposomes

As previously reported in our study with minor modifications (Dong et al. 2020a, 2020b), BAPC was prepared from BA and soybean phospholipids (S75) at a weight ratio of 1:3.5 (w/w) through the solvent evaporation method. In brief, 0.6 g of BA and 2.1 g of S75 were dissolved in 50 mL of ethyl acetate and stirred at 40 °C for 30 min in a 500 mL round-bottom flask. The solvent was then evaporated under vacuum at 40 °C using a rotary evaporator. The resulting residue was collected, dried under vacuum, and stored in a vacuum drying oven at 25 °C until further use.

The conventional thin-film hydration method was applied to prepare liposomes. Briefly, a total of 450 mg of BAPC, 250 mg of S75, and 100 mg of Tween 80 (with a mass ratio of BAPC: total S75: Tween 80 of 1:6:1) were precisely measured and transferred to a 50 mL round-bottom flask dissolved with 10 mL of dichloromethane and 2.5 mL of tetrahydrofuran. After this, the solvent was evaporated at a speed of 80 rpm at 37 °C to generate a thin lipid film, followed by drying under vacuum for 2 h. The film was subsequently hydrated with an appropriate volume of 10 mM phosphate-buffered saline (PBS, pH = 7.4) under constant rotation at 80 rpm for 30 min at 37 °C. The resulting suspension was then sonicated in an ice bath at 150 W for 10 min (5 s pulses and 5 s intervals). Finally, the final volume was adjusted to 10 mL with PBS and then filtered via a 0.22 μm sterile membrane to obtain BAPC-DLP. For comparison, BAPC-LP was prepared only without the addition of Tween 80, and BA-LP was prepared with the addition of 100 mg of BA and 600 mg of S75, followed by the same procedure.

In addition, coumarin-6 (Cou-6) and DiR-labeled liposomes were prepared by dissolving the respective fluorescent dyes in dichloromethane, followed by mixing with 10 mL of dichloromethane and 2.5 mL of tetrahydrofuran. All other steps were performed in the same way.

2.2.2. Particle size distribution and zeta potential of liposomes

After appropriate dilution with pure water, the particle size distribution and zeta potential of the liposomes (BA-LP, BAPC-LP, and BAPC-DLP) were analyzed through dynamic light scattering (DLS) technique, employing a NICOMP 380 zeta potential/particle analyzer (PSS NICOMP, Santa Barbara, CA, USA).

2.2.3. Colloidal stability of liposomes

The colloidal stability of the liposomes (BA-LP, BAPC-LP, and BAPC-DLP) at 4 °C was evaluated with a Turbiscan Tower® stability analyzer (Formulaction, L'Union, France). For analysis, 4 mL aliquots of each formulation were transferred to glass vials and scanned at 4 °C for 24 h. This sophisticated analyzer assesses stability by detecting early subtle changes in transmitted or backscattered light prior to any macroscopic physical changes occurring in nanoscale colloidal dispersions, with the Turbiscan Stability Index (TSI) calculated from transmission light signal variations serving as the key stability parameter.

2.2.4. In vitro drug release profile

The drug release assay was performed by dialysis in PBS (pH = 7.4) with 0.5% Tween 80 and 0.1% ascorbic acid at 37 °C. Briefly, 1 mL of BA suspension, BA-LP, BAPC-LP, and BAPC-DLP (2 mg/mL) were individually transferred into dialysis bags (14 kDa) and immersed in 100 mL of medium with 100  rpm agitation. At predetermined time points (0, 0.5, 1, 2, 4, 8, 12, and 24 h), 2 mL of dialysis medium was withdrawn for high-performance liquid chromatography (HPLC, LC-20 ADXR, Shimadzu Corporation, Japan) analysis, and fresh medium was replenished. The cumulative release was calculated based on the drug concentration at each time point. All the experiments were performed in triplicate. For HPLC analysis, chromatographic separation was performed using an SB-C18 analytical column (4.6 × 250 mm, 5 μm) (Agilent, Santa Clara, CA, USA) under optimized conditions. The mobile phase was 0.05% phosphoric acid and methanol (35:65, v/v) at a flow rate of 1.0 mL/min. The temperature of the column was maintained at 40°C, and the detection wavelength was set at 275 nm. For each analysis, a 10 μL volume of samples was injected into the system.

2.2.5. Mucin stability of liposomes

As mucin is the major component of mucus, a 0.2% (w/v) mucin solution was used to mimic mucus (Gao et al. 2023). To examine mucin stability, 0.25 mL of BA-LP, BAPC-LP, and BAPC-DLP liposomal formulations were incubated with 1 mL of 0.2% (w/v) mucin solution under controlled agitation at 100 rpm at 37 °C. To assess time-dependent changes, particle size and PDI were monitored at predetermined intervals (0, 3, 6, 9, 12, and 24 h) by DLS.

2.2.6. Interactions between mucin and liposomes

The interactions between mucin and liposomal formulations were analyzed using DLS and turbidimetry. For both assays, equal volumes of the liposomal formulations were mixed with 0.2% (w/v) mucin solution at different ratios (1:4, 1:2, 1:1, 2:1, v/v) and incubated for 1 h at 37 °C. In DLS, the particle size and PDI were measured. For turbidimetric analysis, mixed samples were transferred into a 96-well plate, and the measured absorbance at 500 nm was considered the effective absorbance (A_effect). The theoretical absorbance (A_theory) was calculated by measuring and summing the individual absorbances of mucin and liposomes under the same conditions. The interactions between mucin and liposomes are expressed as the difference in absorbance (ΔA), defined by the equation ΔA = A_effect − A_theory.

2.2.7. In vitro mucus penetration behavior of liposomes

To prepare artificial mucus, 250 mg mucin, 250 mg NaCl, 110  mg KCl, 0.295 mg DTPA, 250 μL sterile egg yolk emulsion, and 1 mL RPMI 1640 medium were combined and thoroughly dissolved in 50 mL of distilled water.

As previously reported, the mucus penetration ability of various liposomes was examined using an in vitro Transwell-based artificial mucus model (Peng et al. 2024). Specifically, the apical chamber of the Transwell plate (6.5 mm, 3.0 μm pore size) was layered with 150 μL of prepared artificial mucus, while the basolateral chamber was filled with 800 μL of PBS (pH = 7.4), followed by equilibration for 1 h at 37 °C. Next, 100 μL aliquots of BA-LP, BAPC-LP, BAPC-DLP, and free BA at equivalent concentrations (8 mg/mL) were uniformly applied to the surface of the artificial mucus layer. The system was maintained with orbital shaking at 100  rpm at 37 °C. At predetermined intervals (0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h), 400 μL aliquots were removed from the basolateral chamber and immediately supplemented with the same volume of fresh PBS. The obtained samples were subsequently diluted with ethanol containing 0.02% ascorbic acid and quantified by HPLC. The penetration percentage (%) and apparent permeability coefficient (P_app) of the drug through the artificial mucus layer were calculated to evaluate its penetration efficiency. The P_app of BA was calculated according to the following equation:

$$P_{\mathit{app}}=\mathrm{V}\times \mathrm{dC}/\mathrm{\text{d}}\mathrm{t}\times 1/A\times 1/C_0$$

where V refers to the volume of the basolateral chamber. dC/dt denotes the rate of drug concentration increase in the receptor chamber over time (s), calculated as the terminal receptor chamber drug concentration divided by the transport time. A corresponds to the membrane surface area (cm²), and C₀ represents the inintial drug concentration of the donor chamber.

2.2.8. In vivo biodistribution and pulmonary retention behaviors of liposomes

To assess the biodistribution and pulmonary retention behaviors of liposomes, 50 μL of DiR-labeled liposomes (DiR@BA-LP, DiR@BAPC-LP, DiR@BAPC-DLP) were delivered via intratracheal instillation to male BALB/c mice (6−8 weeks old, 20 ± 2 g) with an equivalent DiR dose of 1 mg/kg. At the 24 h post-administration endpoint, the mice were humanely euthanized, and ex vivo images of DiR-labeled liposomes (Ex/Em: 745/800 nm) were captured from excised lungs and major organs (hearts, livers, spleens, kidneys, intestines) using an IVIS Spectrum system (PerkinElmer, MA, USA), with the lungs and livers defined as regions of interest (ROIs) used to evaluate and calculate the fluorescence intensity using Living Image® software for quantitative analysis.

2.3. Characterization of BAPC-DLP

2.3.1. Transmission electron microscopy (TEM) of BAPC-DLP

The morphology of BAPC-DLP was imaged by TEM (JEM-1200EX, JEOL, Tokyo, Japan). BAPC-DLP was subjected to a 10-fold dilution, and 10 μL aliquots were precisely applied onto copper grids. The sample was then subjected to negative staining with 1% (w/v) phosphotungstic acid and allowed to air-dry before examination.

2.3.2. Encapsulation efficiency and drug loading capacity of BAPC-DLP

The encapsulation efficiency (EE%) of BAPC-DLP was evaluated by an ultrafiltration centrifugation method. For total drug quantification, 0.2 mL of BAPC-DLP was diluted to 25 mL with ethanol containing 0.02% ascorbic acid, followed by HPLC analysis to calculate the total baicalein content of each mL of BAPC-DLP (W_total). For free drug assessment, 2 mL of BAPC-DLP was transferred into an ultrafiltration tube and centrifuged for 30 min at a speed of 5000 rpm. The obtained ultrafiltrate (0.1 mL) was diluted with the same solvent system to 5 mL for HPLC measurement (W₁). The ultrafiltration membrane was subsequently rinsed three times with 1 mL of PBS and centrifuged for 5 min at 2500  rpm. The collected wash solutions were combined and diluted to quantify the amount of membrane-adsorbed drug (W₂). The total free drug content (W_free) was calculated as the sum of W₁ and W₂, from which the free drug content of each mL of BAPC-DLP was derived.

EE% was calculated as follows:

$$\mathrm{EE}\mathit{\%}=\left(1-W_{\mathrm{free}}/W_{\mathrm{total}}\right)\times 100$$

Drug loading (DL%) is defined as the ratio of the encapsulated drug mass (mg) to either the total mass (mg) or volume (mL) of the liposomal formulation. which can be calculated through the following equation:

$$\mathrm{DL}\mathit{\%}=\left({W_{\mathrm{total}}-W}_{\mathrm{free}}\right)/W_{\mathit{mass}}\times 100$$

where W_mass refers to the total mass of drug, lipid material, and surfactant in the liposomes.

2.3.3. Storage stability of BAPC-DLP

BAPC-DLP was stored at 4 °C for one week, and the changes in particle size and PDI were monitored daily using DLS.

2.3.4. DPPH∙ scavenging assay

50 μL of gradient concentrations of BA and BAPC-DLP were added to 150 μL of 100 μM DPPH ethanol solution. Following thorough mixing, the mixture was shaken on a horizontal shaker in a dark environment for 30 minutes at a speed of 50 rpm at 37 °C. A microplate reader (SYNERGY H1, BioTek, Santa Clara, CA, USA) was subsequently used to measure the absorbance of the samples at a wavelength of 517 nm.

2.3.5. ABTS·+ scavenging assay

The ABTS·+  scavenging capacity was evaluated using a total antioxidant capacity kit based on the ABTS method. An ABTS·+  solution was prepared by mixing equal volumes of ABTS solution and oxidants, following the provided instructions, and it was stored overnight in the dark. For the working solution, the ABTS·+  solution was diluted 35 times with anhydrous ethanol to reach an absorbance of 0.70 ± 0.02 at 734 nm. Then, 50 μL of gradient concentrations of BA and BAPC-DLP were added to 150 μL of ABTS·+  working solution. Following thorough mixing, the mixture was shaken on a horizontal shaker in a dark environment for 6 min at 50 rpm at 37 °C. A microplate reader was then used to measure the absorbance of the samples at a wavelength of 734 nm.

2.4. Aerodynamic and aerosolization performance evaluation

2.4.1. In vitro aerodynamic performance of BAPC-DLP

A PARI BOY PRO nebulizer was used for the nebulization of the BAPC-DLP. A next-generation impactor (NGI) (HRH-ZJQ-160, Beijing Huironghe Technology, Beijing, China) was used to investigate the in vitro aerodynamic performance of BAPC-DLP. In brief, 2 mL of BAPC-DLP was transferred into the nebulizer device, and aerosolization was initiated. Upon completion of nebulization, drug deposited at each stage were carefully eluted and collected using a specified volume of ethanol containing 0.02% ascorbic acid, followed by HPLC analysis. The acquired data were processed using inhalation formulation evaluation software (version: 2.3.2.5) to calculate critical parameters, including the mass median aerodynamic diameter (MMAD), fine particle fraction (FPF%), and geometric standard deviation (GSD).

In addition, a Spraytec laser diffraction instrument (Malvern Instruments Ltd., Malvern, UK) was employed to analyze the size of the aerosol droplets, which correlates well with the in vivo lung distribution of these droplets. Briefly, 3 mL of BAPC-DLP was added to the nebulizer and then placed at a distance of 2.5 cm from the laser beam of the instrument. Then, the nebulizer was switched on and the size of the aerosol droplets was measured.

2.4.2. Characterization of BAPC-DLP after nebulization

To evaluate the nebulization stability, particle size, PDI, and zeta potential of BAPC-DLP were measured with DLS before and after nebulization. The nebulization characteristics of BAPC-DLP were then investigated, with a detailed analysis of nebulization time, output efficiency, and output rate. The empty nebulizer device was initially weighed precisely (W₀). After adding 3 mL of BAPC-DLP, the total weight was then measured. The actual liposome weight (W₁) was determined by calculating the difference. Nebulization was initiated with simultaneous timing, and the total nebulization duration (T) was recorded when complete. The post-nebulization device weight (W₂) was then measured. Nebulization parameters were calculated as follows:

$$\mathrm{Output}\mathrm{efficiency}\%=W_{\mathit{1}}-\left(W_{\mathit{2}}-W_{\mathit{0}}\right)/W_1\times 100$$

$$\mathrm{Output}\mathrm{rate}=\left[W_{\mathit{1}}-\left(W_{\mathit{2}}-W_{\mathit{0}}\right)\right]/T$$

2.5. Cytocompatibility and cellular uptake behavior of BAPC-DLP

2.5.1. Cell culture

The RAW 264.7, Calu-3, and A549 cell lines were obtained from the Cell Resource Center, Peking Union Medical College (Beijing, China). RAW 264.7 and A549 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Cellmax, China) supplemented with 10% fetal bovine serum (FBS, Excellbio, China). Calu-3 cells were cultivated in DMEM supplemented with 10% FBS and 1% non-essential amino acids (Gibco, USA). All three cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ and 90% relative humidity.

2.5.2. CCK-8 assay

We examined the cytotoxicity of the drugs using the CCK-8 assay. RAW 264.7, Calu-3, and A549 cells were inoculated into 96-well plates at densities of 2 × 10⁴, 5 × 10³, and 5 × 10³ cells per well, respectively. Following 24 h of incubation, the culture medium was substituted with 100 μL of new medium that contained gradient concentrations of BA, BAPC-DLP, or Blank-DLP. After another 24 h of treatment, 10 μL of CCK-8 solution was combined with 90 μL of fresh DMEM to prepare a 100 μL working solution. The cells were then incubated with the CCK-8 working solution for 1 h (RAW 264.7) or 3 h (Calu-3 and A549). The absorbance was subsequently measured at 450 nm with a microplate reader.

2.5.3. Live/dead staining assay

Live/dead staining was conducted according to the protocol of Calcein-AM/PI cytotoxicity assay kit (Beyotime, Shanghai, China). RAW 264.7 cells were seeded in 24-well plates (1 × 10⁵ cells/well) and treated with 250 μM BA, BAPC-DLP, or Blank-DLP (equivalent volume to BAPC-DLP) at 60%–70% confluence. After 24 hours, the cells were washed with PBS, dual-stained with Calcein-AM (live) and PI (dead) at 37 °C for 30 min in the dark, and imaged using a CYTATION5 system (BioTek, Santa Clara, CA, USA).

2.5.4. In vitro cellular uptake

A comparative study of BAPC-DLP cellular uptake was conducted on RAW 264.7 macrophages as a normal cellular model (M0 type) and their LPS-activated counterparts (M1 type) to represent inflammatory conditions. RAW 264.7 cells were seeded into 24-well plates (1 × 10⁵ cells/well) and cultured for 24 hours. To obtain LPS-activated RAW 264.7 cells, 100 ng/mL LPS was added 4 h after inoculation and continued to incubate for 20 h. Thereafter, the cells were exposed to either free coumarin-6 solution (C6 sol) or C6-labeled BAPC-DLP (C6 equivalent concentration: 1 μg/mL) and incubated for 1, 2, and 4 h, respectively. For fluorescence microscopy, the cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, stained nuclei with DAPI containing antifade reagent, and imaged with a CYTATION5 system. Flow cytometric analysis was performed following sequential PBS washing, cell detachment, and PBS resuspension. Quantitative fluorescence profiling was performed on a flow cytometer (FACS Celesta, BD Biosciences, San Jose, CA, USA).

We further examined how BAPC-DLP internalization influences inflammation-related functions in M0/M1 macrophages. RAW 264.7 cells were seeded in 24-well culture plates (1 × 10⁵ cells/well) and cultured for 24 h to ensure adherence. The cells were then divided into four groups: the M0 group (no treatment), the M0 + BAPC-DLP group (treated with 25 μM BAPC-DLP), the M1 group (induced with 100 ng/mL LPS), and the M1 + BAPC-DLP group (co-treated with 100 ng/mL LPS and 25 μM BAPC-DLP). After 24 h of treatment, the cell culture supernatants were collected to measure the levels of IL-6 and TNF-α by ELISA, while the cells were harvested and loaded with the redox-sensitive fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) for intracellular ROS detection using flow cytometry and fluorescence microscopy imaging.

2.6. In vitro investigation of anti-inflammatory and cytoprotective effects

2.6.1. In vitro anti-inflammatory effect

The in vitro anti-inflammatory effect was evaluated in both RAW 264.7 macrophages and A549 human alveolar epithelial cells using comparable protocols with minor modifications. RAW 264.7 cells (2 × 10⁵ cells/well in 12-well plates) and A549 cells (2.5 × 10⁴ cells/well in 24-well plates) were seeded and cultured for 24 h to ensure adherence. The cells were pretreated for 2 h and subjected to different treatments: RAW 264.7 cells were treated with BA (25 μM), BAPC-DLP (equivalent to 25 μM BA), or DEX (10 μg/mL), and A549 cells were treated with BA (10 μM), BAPC-DLP (equivalent to 10 μM BA), or DEX (10 μg/mL). After pretreatment, RAW 264.7 and A549 cells were stimulated with LPS at concentrations of 100 ng/mL and 100 μg/mL, respectively, for another 22 h, respectively. Culture supernatants were collected via centrifugation (3500 rpm, 15 min, 4 °C) and analyzed for the levels of proinflammatory cytokines, including TNF-α, IL-6, IL-1β, and MCP-1 by ELISA, according to the manufacturer’s protocols. Untreated cells served as the negative control, and the DEX-treated group was used as the positive control.

2.6.2. Detection of intracellular ROS levels

Intracellular ROS detection was performed in both RAW 264.7 and A549 cells using the same experimental procedures as those described for the anti-inflammatory assay. After 22 h of LPS stimulation, DCFH-DA was added to the culture and incubated for 30 min under standard conditions. The intracellular ROS levels were subsequently assessed through fluorescence microscopy imaging or flow cytometric quantification.

2.6.3. Cytoprotective effect of BAPC-DLP against LPS-induced injury in A549 cells

The protective effect of BAPC-DLP against LPS-induced injury in A549 cells was evaluated using CCK-8 assay and live/dead staining. For the CCK-8 assay, A549 cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and cultured for 24 h to allow adherence. The cells were then pretreated with BA (10 μM), BAPC-DLP (equivalent to BA at 10 μM), or DEX (10 μg/mL) for 2 h prior to 22 h of stimulation with 100 μg/mL LPS. The cells were subsequently incubated with 100 μL of CCK-8 working solution for 3 h, after which the absorbance was measured at 450 nm. For live/dead staining, A549 cells were subjected to the same experimental procedures as those described for the anti-inflammatory assay. After 22 h of LPS stimulation, the cells were washed with PBS and dual-stained with Calcein-AM (live) and PI (dead) at 37 °C for 30 min in the dark and subsequently imaged by fluorescence microscopy.

2.7. In vivo biodistribution of BAPC-DLP

To investigate the superiority of intratracheal instillation of BAPC-DLP, the mice were assigned to four groups: the DiR solution intratracheal instillation group (i.t. DiR), DiR@BAPC-DLP intratracheal instillation group (i.t. DiR@BAPC-DLP), DiR@BAPC-DLP intravenous administration group (i.v. DiR@BAPC-DLP), and DiR@BAPC-DLP oral administration group (p.o. DiR@BAPC-DLP). After the administration of 1 mg/kg DiR or DiR@BAPC-DLP, in vivo imaging was conducted at 0, 1, 4, 8, 12, and 24 h. After the last time point, the lungs and other major organs were harvested for fluorescence analysis. There were three animals in each group.

2.8. In vivo pharmacodynamics of inhaled BAPC-DLP in ALI mice model

2.8.1. LPS-induced ALI mice model

Male SPF BALB/c mice (6−8 weeks, 18−22 g) were randomized into six groups (n = 11/group): a control group and an LPS-induced model group receiving PBS, as well as three preventive intervention groups treated with 1 mg/kg Blank-DLP, BA or BAPC-DLP via intratracheal nebulization using a pulmonary fluid quantification atomizer (YAN30012, Shanghai Yuyan Instrument, Shanghai, China) to ensure uniform pulmonary distribution. As a positive control, a dexamethasone (DEX) group was administered 5 mg/kg via intraperitoneal injection. To evaluate the preventive efficacy, all the treatments were administered 1 h prior to the LPS challenge. The dosage of 1 mg/kg for BAPC-DLP was selected based on systematic dose-ranging studies that identified 0.5−2 mg/kg as the safe therapeutic window, with 1 mg/kg demonstrating optimal anti-inflammatory efficacy in preliminary experiments. The mice were housed under SPF conditions with free access to food and water. First, the mice were anesthetized with isoflurane and placed in the supine position. One hour prior to the LPS challenge, 50 μL of the appropriate therapeutic drug or PBS was administered by intratracheal nebulization to the corresponding groups, while the DEX group was given 0.2 mL DEX (5 mg/kg) intraperitoneally. After 1 h, an ALI model in mice was established in all experimental groups via intratracheal nebulization of 50 μL of LPS (5 mg/kg), except for the control group, which was administered normal saline. The mice were sacrificed by cervical dislocation 24 h after modeling, and samples were collected. In detail, seven mice from each group were randomly selected and subjected to standard bronchoalveolar lavage (BAL) under aseptic conditions. Triplicate lavages were conducted with ice-cold PBS by sequentially instilling and withdrawing three 0.5 mL aliquots via a tracheal cannula. The BAL fluid (BALF) was centrifuged (800 × g, 10 min, 4 °C), and the supernatants were stored at −80 °C. The main organs of the remaining mice (n = 4) were removed intact, and the lungs were weighed. The heart, liver, spleen, kidney and upper lobe of the right lung were fixed in 4% paraformaldehyde for the preparation of paraffin sections. The sample size (n = 4) was determined through preliminary experiments and validated by post-hoc power analysis using G*Power 3.1.9.7, ensuring both statistical reliability with small sample sizes and strict adherence to the 3 R principles of animal research (Kang 2021).

2.8.2. Lung index

Following the acquisition of body weight (g) and lung weight (g), the lung index of the mice were calculated as follows:

$$\mathrm{Lung}\mathrm{index}(\%)=\mathrm{lung}\mathrm{weight}\left(\mathrm{g}\right)/\mathrm{body}\mathrm{weight}\left(\mathrm{g}\right)\times 100$$

2.8.3. Total protein concentration and inflammatory Cytokines in the BALF

The total protein concentration in the BALF was quantified using a bicinchoninic acid (BCA) assay kit according to the manufacturer’s instructions. The absorbance was measured at 562 nm with a microplate reader, and the protein concentration was calculated by interpolation from a bovine serum albumin (BSA) standard curve. Cytokine levels (IL-6, TNF-α, IL-1β, MCP-1, and IL-10) in the BALF were determined using ELISA kits. BALF samples were appropriately diluted prior to analysis. Following the kit protocols, the absorbance was read at 450 nm, and the cytokine concentrations were calculated based on the respective standard curves.

2.8.4. Histological analysis and immunohistochemical staining (IHC staining)

After fixation in paraformaldehyde, the tissues were embedded in paraffin, sliced to approximately 3 µm, and then stained with hematoxylin and eosin (H&E) for histopathological assessment. An independent researcher who was blinded to the group allocation conducted the histopathological analysis. The severity of lung injury was assessed according to the American Thoracic Society scoring system, with scores ranging from 0 (no injury) to 1 (maximal injury) (Matute-Bello et al. 2011). For each specimen, the result was calculated as the average of three randomly selected high-power fields. For immunohistochemical analysis, the deparaffinized sections were incubated with a CD68 polyclonal antibody (1:500, diluted in PBS) and an MPO polyclonal antibody (1:300, diluted in PBS) overnight at 4 °C. After that, the slides were incubated with an HRP-conjugated secondary antibody (1:250, Servicebio, GB23303), followed by color development with diaminobenzidine. All the slides were scanned under a microscope (CI-S, NIKON, Tokyo, Japan) for further analysis.

2.9. Hemolytic assay

Blood was collected from mouse orbits into EDTA tubes, and erythrocytes were isolated by centrifugation for 10 minutes at 3000 rpm, washed with saline until the supernatant was colorless, and then prepared as a 2% erythrocyte suspension. The gradient concentration of BAPC-DLP (200, 100, 20 μg/mL) was mixed with an equal volume of erythrocyte suspension, with ddH₂O as the positive control and normal saline (n.s.) as the negative control. Following incubation for 1 hour at 37 °C, the supernatant was centrifuged (3000 rpm, 5 min), and then the OD values at 540 nm were measured. The hemolysis ratio was calculated to assess blood compatibility. The formula for the hemolysis ratio is as follows:

$$\mathrm{Hemolysis}\mathrm{ratio}(\%)=({\mathrm{OD}}_{\mathit{sample}}-{\mathrm{OD}}_{\mathit{negative}})/{(\mathrm{OD}}_{\mathit{positive}}-{\mathrm{OD}}_{\mathit{negative}})\times 100$$

2.10. Statistical analysis

Quantitative data are expressed as the mean ± SD. Comparisons between two groups of data were analyzed by Student's t-test. One-way or two-way ANOVA followed by Tukey's post-hoc test was used to analyze multiple-group comparisons. All the statistical analyses were conducted with GraphPad Prism (version 9.5.1), with significance thresholds hierarchically denoted as: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3. Results and discussion

3.1. Preparation of liposomes and confirmation of the formulation composition

In this study, a novel baicalein-loaded liposome-based phospholipid complex (BAPC-DLP) was designed to treat ALI via nebulized inhalation using the conventional thin-film hydration method. We systematically optimized the formulation composition, ratio, and amount through a single-factor experimental design, with critical quality attributes as optimization indicators including the particle size distribution, zeta potential, encapsulation efficiency, stability, and relevant substances. Ultimately, we successfully developed a stable formulation of baicalein liposome with high drug loading capacity utilizing a phospholipid complex and Tween 80.

To investigate the function of the phospholipid complex and Tween 80 in the formulation and verify the rationality of its composition, we prepared BAPC-LP (BAPC liposomes without Tween 80) and BA-LP (free BA liposomes without Tween 80) for comparison (Figure 1a). Comparative studies of multiple dimensions were conducted on their physicochemical properties, colloidal stability, in vitro drug release, mucus penetration, and in vivo distribution against BAPC-DLP.

Figure 1.

Preparation of liposomes and confirmation of the formulation composition. (a) Schematic illustration of the preparation of BA-LP, BAPC-LP, and BAPC-DLP (image created with BioRender). (b) Colloidal stability of liposomes measured by Turbiscan. (c) In vitro drug release profiles (n = 3). (d) Increased particle size and (e) PDI of mucin‒liposome mixtures with different mucin to liposome ratios (v/v) (n = 3). (f) The difference in absorbance (ΔA) between the theoretical and effective absorbance (n = 3). (g) Schematic diagram of an artificial mucus penetration experiment (image created with BioRender). (h) Penetration percentage curves of BA and liposomes across artificial mucus in Transwell and (i) P_app at 24 h (n = 3). (j) Schematic representation of liposomes biodistribution and pulmonary retention via intratracheal instillation through an IVIS (image created with BioRender). (k) Ex vivo imaging of the main organs in mice at 24 h. Quantitative analysis of the average radiant efficiency in the (l) lungs and (m) livers of the mice (n = 3) was performed. The data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

First, the particle size distribution and zeta potential of the three obtained liposomes were evaluated by DLS (Figure S1). The particle sizes of BA-LP and BAPC-LP were approximately 115 nm and 124 nm, while BAPC-DLP decreased to approximately 103 nm upon the addition of Tween 80. The PDI of BA-LP was greater than 0.4, whereas those of BAPC-LP and BAPC-DLP were approximately 0.2, indicating that the phospholipid complex enhanced the uniformity and dispersibility of the liposomes. In addition, the zeta potential for all three liposomes was approximately −40 mV, with no significant differences.

We subsequently analyzed the colloidal stability of the three liposomes using Turbiscan Lab®Expert to obtain TSI values, which were negatively correlated with colloidal stability. As depicted in Figure 1b, the TSI value of BA-LP continuously increased over 24 h, while the growth of BAPC-LP significantly slowed. In comparison, BAPC-DLP was always maintained at a lower level, demonstrating the best colloidal stability.

The in vitro drug release results are shown in Figure 1c. The extremely low water solubility of free BA resulted in less than 20% release within 24 h. In contrast, all the liposomal formulations achieved more than 80% release due to a marked improvement in solubility. In addition, the initial release rates of the three liposomes were comparable. After 4 h, however, BA-LP and BAPC-LP still maintained similar release profiles, while the cumulative release of BAPC-DLP was more than 10% lower. This deceleration may be attributed to the Tween 80 in BAPC-DLP, which alters the structure and stability of the membrane (Ji et al. 2012).

Effective mucus penetration and prolonged lung retention are essential to facilitate efficient pulmonary drug delivery. Therefore, we further studied the pulmonary delivery behavior of the three liposomes, focusing on their mucus penetration ability and lung retention characteristics. First, the stability of liposomes in mucus is critical for penetration through the mucus layer. As presented in Figure S2, the particle size of all three types of liposomes increased after incubation with mucin, with no significant change in the PDI. Specifically, BA-LP showed the largest increase in particle size of approximately 40 nm, followed by BAPC-LP at 20 nm, while BAPC-DLP exhibited the best stability, with only a 10 nm increase. Furthermore, the changes in physicochemical properties and turbidity after liposome-mucin mixing were used to assess their interaction. As indicated in Figure 1d‐e, increasing mucin concentration resulted in a gradual increase in particle size and a rapid decrease in PDI for BA-LP and BAPC-LP, with a peak at a ratio of 2:1 mucin to liposome, indicating the strongest interaction at this ratio. Conversely, a further increase in mucin contributed to decreased particle size and increased PDI, probably due to mucin saturation on the liposome surface and vesicle reintegration. In contrast, the size and PDI of BAPC-DLP varied minimally, indicating its weak interaction with mucin. Similarly, the turbidity experiments shown in Figure 1f indicated that BA-LP resulted in the greatest turbidity change, followed by BAPC-LP, while BAPC-DLP resulted in a slight change. These results indicated that BAPC-DLP weakly interacts with mucin, which may be attributed to the steric hindrance effect of Tween 80. This hydrophilic polymer creates a barrier on the surface of BAPC-DLP, preventing mucin contact and inhibiting mucin interaction, thus possibly facilitating liposomal mucus penetration (Huang et al. 2022).

Based on these findings, we further established a Transwell-based in vitro mucus penetration model to assess mucus penetration behavior in artificial mucus (Figure 1g). The results are shown in Figure 1h, where free baicalein weakly penetrated through the mucus layer, while its liposomal formulations considerably enhanced penetration. Specifically, BA-LP and BAPC-LP reached mucus penetration rates of 30.29 ± 3.78% and 30.65 ± 0.41%, respectively, over 24 h, with no significant differences. In comparison, BAPC-DLP achieved a penetration rate of 52.80 ± 1.69%, approximately 1.75 times higher than those of the other liposomal formulations. Furthermore, the calculated apparent permeability coefficients (P_app) further confirmed this, indicating that BAPC-DLP's P_app was significantly higher than those of BA-LP and BAPC-LP (Figure 1i). Taken together, the unique design of BAPC-DLP markedly facilitated drug mucus penetration, demonstrating its superior potential for pulmonary delivery.

To further investigate the in vivo behavior of liposomes, we assessed their biodistribution and pulmonary retention ability with a fluorescence imaging system (Figure 1j). Ex vivo images (Figure 1k) revealed predominant pulmonary accumulation 24 h after intratracheal instillation, although some distribution heterogeneity was observed due to the inherent technical limitations of this administration method. Despite this variability, the quantitative results shown in Figure 1l‐m revealed that BAPC-LP exhibited a slight increase in lung fluorescence intensity compared to BA-LP. Conversely, BAPC-DLP showed a stronger fluorescence intensity. The fluorescence intensity was 2.75 times greater than that of BA-LP and 1.91 times greater than that of BAPC-LP, which suggested that BAPC-DLP had excellent pulmonary retention ability. In addition, there were no significant differences in liver fluorescence among the groups. The introduction of a phospholipid complex and Tween 80 in BAPC-DLP significantly improved stability and slowed drug release in the complicated pulmonary microenvironment while reducing nonspecific interactions with pulmonary biocomponents (e.g. alveolar macrophages and mucins), which prevented rapid clearance and extended residence time in the lungs.

This study comprehensively demonstrated that BAPC-DLP improved liposome uniformity, stability, and sustained release through the synergistic effect of the phospholipid complex and Tween 80. It also resulted in better mucus penetration and lung retention. Based on its excellent performance, BAPC-DLP was adopted as the core formulation for further studies.

3.2. Characterization of BAPC-DLP

After identifying BAPC-DLP as the ultimate formulation, we characterized the physicochemical properties and quality of BAPC-DLP. As shown in Figure 2a, TEM images revealed that BAPC-DLP displayed a spherical morphology with a uniform particle size distribution. DLS measurements indicated that the blank liposomes had an average size of 91.60 nm and a PDI of 0.230. Once BAPC was loaded, the particle size increased to 103.61 nm, with a PDI of 0.210 (Figure 2b). Additionally, the zeta potential changed from −34.32 to −39.42 mV. Notably, particles smaller than 200 nm and those with a negative charge are considered favorable for inhalation therapy, as they can also help to penetrate through the mucus layer more effectively, resulting in improved lung delivery efficiency (Wang et al. 2022). According to Figure 2c, the encapsulation efficiency was 97.43%, and the drug loading capacity was 10.06%. The typical absorption peak of BA at 275 nm in the UV spectrum of BAPC-DLP indicated that its UV absorption characteristics remained unchanged when it was encapsulated in liposomes (Figure 2d). Additionally, the stability results showed that no noticeable changes in particle size and PDI occurred at 4 °C within one week, which suggested the excellent storage stability of BAPC-DLP (Figure 2e). More importantly, the BAPC-DLP we constructed could load nearly 10 mg/mL BA, resulting in a dramatic increase in solubility of approximately 500-fold compared to free BA (Figure 2f). This brilliant achievement is particularly prominent in current studies on enhancing the solubility of baicalein, effectively overcoming the limitations of drug solubility in nebulized inhalation formulations.

Figure 2.

Characterization of BAPC-DLP. (a) Representative TEM images of BAPC-DLP (scale bar: 100 nm). (b) Particle size and PDI of Blank-DLP and BAPC-DLP. (c) Encapsulation efficiency (EE%) and drug loading capacity (DL%) of BAPC-DLP (n = 3). (d) UV–vis spectrum of free BA, BAPC-DLP, and Blank-DLP. (e) Storage stability examination of BAPC-DLP at 4 °C within one week (n = 3). (f) Formulation technology and methods used by scientists in recent years to improve BA solubility (Zhou et al. 2017; Yin et al. 2017; Schneider et al. 2017; Dong et al. 2020a, 2020b; Yan et al. 2020; Markowski et al. 2023; Singla et al. 2023). (g) DPPH⋅ and (h) ABTS·+ scavenging abilities of various concentrations of BA and BAPC-DLP, respectively (n = 3). The data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Baicalein, recognized as a highly effective antioxidant, has excellent free radical scavenging ability. We assessed the free radical scavenging ability of free BA and BAPC-DLP in vitro via DPPH· and ABTS·+ scavenging assays. As shown in Figure 2g, both BA and BAPC-DLP exhibited improved DPPH·-scavenging ability with increasing concentrations. Within the concentration range of 12.5–50 μM, BAPC-DLP significantly outperformed free BA, indicating that the liposomes enhance antioxidant activity through improved solubility and dispersion. As the concentration continued to increase, the DPPH scavenging abilities of BAPC-DLP and BA began to converge. At 250 μM, the DPPH· scavenging rates for BA and BAPC-DLP reached 93.71 ± 2.80% and 95.44 ± 0.38%, respectively. Similarly, in Figure 2h, BAPC-DLP displayed higher ABTS·+ clearance at lower concentrations, but the difference from BA decreased with higher concentrations. Both assays demonstrated strong antioxidant capacity for BAPC-DLP and free BA. BAPC-DLP exhibited superior performance to BA, possibly owing to its lipid-based protective structure and uniform dispersion in the medium, highlighting its potential as an efficient delivery system for increasing the antioxidant efficacy of BA.

3.3. Aerodynamic and aerosolization performance evaluation

Nebulizers convert drug solutions into aerosols for delivery to the lower respiratory tract. To ensure accurate delivery of BAPC-DLP via nebulization, its aerosolization behavior and performance were investigated immediately. We employed the PARI BOY PRO, a widely used jet nebulizer shown in Figure 3a, as the aerosol generation device to measure the aerodynamic particle size distribution of BAPC-DLP via the NGI. The results shown in Figure 3b revealed that more than 80% of the BAPC-DLP aerosol particles settled in the 3rd to 7th stages of the NGI, with cutoff diameters ranging from 0.98 to 5.39 μm, indicating efficient deposition in the lower respiratory tract, particularly on the surfaces of the trachea, bronchus, and alveoli. Based on three measurements conducted with the NGI, BAPC-DLP was demonstrated to possess excellent nebulization performance, with an average MMAD of 2.94 ± 0.17 μm and an FPF% of 75.40 ± 2.08%, along with a GSD of 2.13 ± 0.07. As illustrated in Figure 3c, the frequency and cumulative distributions of the droplet sizes following the aerosolization of BAPC-DLP indicated a volume mean diameter (VMD) of 3.01 μm, with 82.06% of the droplets under 5 μm in size. The results are consistent with those obtained from the NGI method, which further validates the superior nebulization performance of BAPC-DLP via a PARI BOY PRO jet nebulizer.

Figure 3.

Aerodynamic and aerosolization performance evaluation. (a) Nebulizer for aerodynamic determination of BAPC-DLP. (b) Aerodynamic particle size distribution of BAPC-DLP determined by NGI (n = 3). (c) The droplet size distribution of nebulized BAPC-DLP evaluated by Spraytec laser diffraction analysis. (d) Nebulization time, (e) output efficiency, and (f) output rate of BAPC-DLP in 3 individual batches. (g) Particle size, (h) PDI, and (i) zeta potential of BAPC-DLP before and after nebulization (n = 3). The data are presented as mean ± SD. ****p < 0.0001.

Additionally, we determined the nebulization time, output efficiency, and output rate for three batches of BAPC-DLP to assess its nebulization delivery consistency. According to the results in Figures 3d‐f, the nebulization time was approximately 4.57 ± 0.10 min/mL, and the output efficiency reached 71.66 ± 1.32%, with an output rate of 0.16 ± 0.01  g/min, which were quite high values that highlighted the excellent nebulization performance and delivery uniformity of BAPC-DLP (Makled et al. 2017). To investigate the effect of the aerosolization process on BAPC-DLP stability, we collected and analyzed the suspension after nebulization (Figures 3g‐i). The results indicated that the particle size of BAPC-DLP slightly increased from 87.03 ± 0.12 nm to 113.90 ± 0.57 nm, and the PDI changed from 0.226 to 0.290, while there were no significant changes in the zeta potential. The aerosolization process may induce minor changes in particle size and PDI due to shear forces, potentially causing slight liposome disruption. However, while colloidal stability was marginally reduced after nebulization, the impact remained within acceptable limits. Overall, the colloidal stability of BAPC-DLP was not significantly affected by aerosolization.

Given the results above, BAPC-DLP manifests remarkable abilities concerning aerodynamic particle size distribution, fine particle fraction, delivery consistency, and aerosolization stability, providing a promising opportunity for applications in pulmonary drug delivery.

3.4. Cytocompatibility and cellular uptake behavior of BAPC-DLP

Before investigating the cellular effects of BAPC-DLP, we first examined the cytotoxicity of BA, BAPC-DLP, and Blank-DLP in RAW 264.7 murine macrophages, Calu-3 human bronchial epithelial cells, and A549 human alveolar epithelial cells. The results, as shown in Figures 4a–c, indicated that the cell viability of RAW 264.7 cells remained unaffected by any treatment within the 0–250 μM range, suggesting their good biocompatibility. The live/dead cell staining assays in Figures 4d and S3 further validated this result, which showed that after 24 h of treatment at 250 μM, the proportion of living cells labeled with Calcein-AM (green) approached 100%, with few dead cells stained with propidium iodide (PI) (red). Notably, in Calu-3 cells and A549 cells (Figures S4, S5), free BA decreased cell viability to less than 80% at 50 and 25 μM, respectively. In contrast, BAPC-DLP maintained over 80% viability even at 250 μM. These data demonstrated the superior biocompatibility of BAPC-DLP compared to free BA, indicating its potential for pulmonary applications.

Figure 4.

Cytocompatibility and cellular uptake behavior of BAPC-DLP. (a–c) Cell viability of RAW 264.7 cells after incubation with (a) BA, (b) Blank-DLP, and (c) BAPC-DLP for 24 h (n = 6). (d) Live/dead staining in RAW 264.7 cells imaged at 10 × magnification. (Scale bar: 200 μm). (e) M0 and (f) M1 macrophages uptake of the C6 solution and BAPC-DLP at different time points was analyzed by flow cytometry (n = 3). (g‐h) Fluorescence microscopy imaging of the uptake of the C6 solution, BAPC-DLP by (g) RAW 264.7 cells and (h) LPS-activated RAW 264.7 cells at different times (scale bar: 200 μm). (i‐j) Inflammatory cytokine levels in M0 and M1 macrophages following BAPC-DLP uptake: (i) IL-6, (j) TNF-α (n = 4). (k) Flow cytometric analysis of intracellular ROS levels in M0 and M1 macrophages following BAPC-DLP uptake (n = 4). ****p < 0.0001.

Increasing evidence indicates that the cytokine storm in ALI/ARDS is closely related to the activation of macrophages, which are the predominant immune cells in the lung. LPS, a key component of Gram-negative bacteria, activates macrophages and promotes M1 polarization, triggering an inflammatory response and leading to increases in NO, ROS, and inflammatory cytokines. Therefore, we labeled liposomes with Cou-6 to assess differences in the cellular uptake of BAPC-DLP by LPS-activated (M1) and unactivated (M0) RAW 264.7 macrophages. Flow cytometry analysis (Figure 4e‐f and S4) revealed a marked time-dependent increase in uptake in both cell types. The uptake of BAPC-DLP was markedly greater than that of free Cou-6, indicating effective drug uptake enhancement by liposomes. Moreover, M1 macrophages demonstrated notably increased internalization of BAPC-DLP compared to M0 macrophages. Observations from fluorescence microscopy reinforced that M1 macrophages had significantly higher green fluorescence intensity than M0 macrophages (Figure 4g‐h). The augmentation of uptake by activated macrophages may account for the activation of Toll-like receptors on the surface and the upregulation of various endocytosis-related proteins (Liu et al. 2024).

Following the confirmation of efficient cellular uptake, we further investigated whether BAPC-DLP internalization modulates the inflammatory function of macrophages. Specifically, M0 and M1 macrophages were treated with or without BAPC-DLP, and key inflammatory cytokines (IL−6 and TNF-α) were measured via ELISA, and oxidative stress was evaluated through intracellular ROS level using flow cytometry. As shown in Figures 4i‐k, S7, and S8, BAPC-DLP had differential regulatory effects on the two macrophage types. In M1 macrophages, BAPC-DLP treatment markedly inhibited the production of pro-inflammatory cytokines (IL-6 and TNF-α) and decreased the level of intracellular ROS. In contrast, M0 macrophages treated with BAPC-DLP showed no significant increase in pro-inflammatory factors and ROS, with all the measured parameters remaining comparable to those of the untreated group. These results demonstrated that BAPC-DLP selectively inhibited the excessive activation of M1 macrophages while maintaining M0 macrophage homeostasis, revealing its favorable targeted anti-inflammatory properties. Therefore, the enhanced cellular uptake under inflammatory conditions provides the fundamental basis for the targeted anti-inflammatory effects of BAPC-DLP at inflammatory sites.

3.5. In vitro investigation of anti-inflammatory and cytoprotective effects

As previously mentioned, the activation of macrophages and the excessive inflammatory response play crucial roles in the pathogenesis of ALI. The RAW 264.7 cell line, a well-established murine macrophage model, recapitulates the key inflammatory phenotypes and ROS-producing features of primary alveolar macrophages during ALI pathogenesis following LPS stimulation. Therefore, we selected this cellular model to evaluate the in vitro anti-inflammatory and ROS-scavenging effects of BAPC-DLP. We first measured the levels of NO and inflammatory cytokines via the Griess method and ELISA to assess the anti-inflammatory effects of gradient concentrations of BAPC-DLP. The results presented in Figure S9a–c showed that 100 ng/mL LPS significantly increased the levels of NO, TNF-α, and IL-6, confirming the successful establishment of the inflammation model. After intervention with BAPC-DLP at a concentration gradient of 12.5−100 μM, the treatments significantly lowered the levels of NO, TNF-α, and IL-6, effectively inhibiting inflammation. With increasing BAPC-DLP concentration, there was a gradual decrease in the levels of these factors; nevertheless, at a concentration of 25 μM, the rate of decrease markedly slowed, which suggested that a plateau was reached.

Considering the concentration‒effect relationship described above, we selected 25 μM as the working concentration and compared the anti-inflammatory effects of BAPC-DLP and free BA, with dexamethasone (DEX) serving as a positive control. The results shown in Figure 5a‐d revealed that BAPC-DLP significantly outperformed free BA in reducing the levels of inflammatory cytokines, including IL-6, IL-1β, and MCP-1, primarily because of the improved uptake efficiency of the liposomal system. In particular, there was no statistically significant difference in TNF-α regulation between the BAPC-DLP and BA groups, which may be attributed to BA having reached a plateau in anti-inflammatory effects at this concentration, similar to that of BAPC-DLP. In addition, excessive ROS attack intracellular lipids, proteins, and DNA, triggering oxidative damage, such as lipid peroxidation, and exacerbating inflammatory injury (Hu et al. 2024; Wang et al. 2024). We explored the regulatory effect of BAPC-DLP on ROS in LPS-activated macrophages. BAPC-DLP exhibited a dose-dependent effect on ROS scavenging, with a plateau at 25 μM, as demonstrated by flow cytometric analysis of DCFH-DA fluorescence (Figure S9d). At this concentration, BAPC-DLP had a greater ability to scavenge ROS than free BA, with no significant difference compared with the DEX group (Figures 5e and S10). As further confirmed by the fluorescence microscopy results shown in Figure 5f, both the BAPC-DLP and DEX groups presented significant green fluorescence attenuation, which visually indicated their remarkable ROS scavenging capacity.

Figure 5.

In vitro investigation of anti-inflammatory and cytoprotective effects. (a‐d) Inhibitory effect of various treatments with BA, BAPC-DLP and DEX on inflammatory factors produced by RAW 264.7 cells, including (a) IL-6, (b) TNF-α, (c) IL-1β, and (d) MCP-1 (n = 4). (e) Mean fluorescence intensity of ROS production after treatments in RAW 264.7 cells analyzed by flow cytometry (n = 4). (f) Representative images of intracellular ROS after treatments in RAW 264.7 cells stained with DCFH-DA (green). (g) Cell viability of LPS-induced A549 cells after treatments for 24 h (n = 4). (h) Live/dead staining of LPS-induced A549 cells after treatments at 10× magnification (scale bar: 200 μm). (i‐j) Inhibitory effect after treatments on inflammatory cytokine production in LPS-induced A549 cells: (i) IL-6 and (j) TNF-α (n = 4). (k) Representative images of intracellular ROS after treatments in LPS-induced A549 cells stained with DCFH-DA (green) (scale bar: 200 μm). The data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Alveolar epithelial injury represents a fundamental pathological alteration in barrier dysfunction during ALI/ARDS, which directly drives the development of pulmonary edema, inflammatory cell infiltration, and lung tissue injury (Sun et al. 2024). Therefore, we established an LPS-induced injury model using A549 human alveolar epithelial cells to assess the protective effects of BAPC-DLP on the alveolar epithelium. Based on CCK-8 assay results, A549 cells were treated with 10 μM BA or BAPC-DLP within the established safe concentration range. As shown in Figure 5g, LPS treatment significantly reduced A549 cell viability. Following different treatments, BAPC-DLP demonstrated greater efficacy than BA in restoring cell viability. This protective effect was visually confirmed by live/dead staining, as shown in Figure 5h, which revealed a substantial reduction in PI-positive (dead) cells in the BAPC-DLP-treated group. Furthermore, measurement of the IL-6 and TNF-α levels in the supernatants (Figure 5i‐j) revealed that, compared with free BA, BAPC-DLP more effectively attenuated the LPS-induced increase. Consistent with these findings, intracellular ROS levels were significantly reduced following BAPC-DLP treatment, as visualized by fluorescence microscopy (Figure 5k).

To summarize, this study demonstrated that BAPC-DLP has substantial anti-inflammatory and alveolar epithelial protective effects, providing compelling experimental evidence for its potential application in ALI management.

3.6. In vivo biodistribution study of BAPC-DLP

To comprehensively assess the biodistribution characteristics of BAPC-DLP, DiR-labeled BAPC-DLP (DiR@BAPC-DLP) was prepared and delivered to the lungs via intratracheal instillation, with free DiR as a control, followed by in vivo imaging. We also compared the differences in the distribution of DiR@BAPC-DLP when administered via intratracheal instillation with those via intravenous injection and oral administration routes.

Figure 6a revealed that DiR@BAPC-DLP was predominantly distributed in the gastrointestinal tract following oral administration, and the fluorescent signals decreased markedly after 24 h, indicating gradual elimination of the drug over time. After intravenous injection, DiR@BAPC-DLP was distributed rapidly throughout the body via the blood circulation. In the intratracheal instillation groups, the fluorescence intensity of the free DiR group decreased significantly after 8 h, while DiR@BAPC-DLP remained enriched in the lungs, indicating notable sustained release of DiR@BAPC-DLP. Notably, the drug distribution of DiR@BAPC-DLP in the lungs was more pronounced than that in the free DiR solution. In particular, the systemic fluorescence signal of the free DiR group was evidently attenuated after 24 h, while DiR@BAPC-DLP still retained a strong fluorescence intensity, which further confirmed its superior pulmonary retention property. As illustrated in Figure 6b‐d, ex vivo imaging and quantitative analysis indicated that the oral administration group did not show significant fluorescent signals in major organs, while the intravenous injection group displayed a systemic fluorescent distribution. Notably, in the intratracheal instillation group, the fluorescence intensity was specifically concentrated in the lung, and the fluorescence intensity was significantly lower in the liver than that in the intravenous injection group. This evidence suggests that pulmonary delivery can significantly improve lung-targeting efficiency and reduce distribution to non-target organs. Further analysis revealed that the sustained release properties of the DiR@BAPC-DLP led to a notably prolonged pulmonary retention time, with a 1.8-fold increase in the lung fluorescence intensity after 24 h compared to the free DiR group.

Figure 6.

In vivo biodistribution study of BAPC-DLP. (a) In vivo biodistribution of mice after different treatments at 0, 1, 4, 8, 12, and 24 h using IVIS. (b) Ex vivo imaging of the dissected hearts, livers, spleens, lungs, kidneys, and small intestines of the mice at 24 h. (c) Quantitative analysis of the average radiation efficiency in the lung of the mice (n = 3). (d) Quantitative analysis of radiation efficiency in the liver of the mice (n = 3). The data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

In summary, we confirmed the superiority of pulmonary administration of BAPC-DLP from the spatiotemporal dimension in improving pulmonary targeting efficiency, reducing the risk of systemic exposure, and prolonging the lung retention time, which holds good promise for alleviating ALI.

3.7. In vivo pharmacodynamics of inhaled BAPC-DLP in the ALI mice model

Given the excellent in vitro anti-inflammatory effects and in vivo lung targeting and retention properties of BAPC-DLP, we established an LPS-induced ALI model in mice to compare its in vivo preventive protective effects with free BA, with DEX as a positive control. The preventive administration regimen was implemented based on our previous research on baicalein nanoformulation (Liao et al. 2022). As shown in Figure 7a, the treatments were administered 1 h prior to LPS stimulation. All the mice survived through the 24-h period following LPS challenge, and mortality attributable to the model was not observed before the experimental endpoint. After 24 h, the mice presented pathological features of acute lung inflammation, including severe lung tissue damage, cytokine storms, and increased neutrophil and macrophage infiltration. By collecting BALF and lung tissues, we comprehensively assessed the protective effect of BAPC-DLP on ALI in terms of the severity of lung edema, inflammatory inhibition effect, infiltration of inflammatory cells, and alterations in pulmonary histopathology.

Figure 7.

In vivo pharmacodynamics of inhaled BAPC-DLP in the ALI mice model. (a) Schematic diagram of the ALI experimental design in mice. (b) Percentage change in the body weight of the mice following 24-h LPS challenge (n = 4). (c) Lung index (n = 4). (d) Total protein concentration in the BALF was assayed by the BCA method (n = 7). (e–h) The level of the proinflammatory cytokines of (e) IL-6, (f) TNF-α, (g) IL-1β, and (h) MCP-1 in the BALF were determined by ELISA (n = 7). (i) The level of IL-10 in the BALF was determined by ELISA (n = 7). (j) IHC staining of CD68 and MPO in the lungs after various treatments (scale bar: 100 μm). (k) H&E staining of lungs after various treatments (scale bar: 100 μm and zoom-in scale bar: 50 μm). The data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

The pharmacodynamic evaluation was conducted at a dose of 1 mg/kg, which was previously determined as the optimal protective dose through systematic dose-ranging studies. As depicted in Figure 7b, LPS treatment significantly reduced the body weight of the mice, while the weight loss in the BAPC-DLP treatment group was alleviated to a level comparable to that in the DEX group. As shown in Figure 7c, the lung index in the LPS group increased from 0.58 to 0.88 in comparison with that in the control group, suggesting pulmonary edema. After treatment, the lung indices for Blank-DLP, BA, BAPC-DLP, and DEX were reduced to 0.82, 0.79, 0.76, and 0.66, respectively, with notable reductions in the BAPC-DLP and DEX groups. Furthermore, the analysis of the BALF, as illustrated in Figure 7d, revealed that pulmonary delivery of BAPC-DLP resulted in a significant reduction in total protein concentrations.

The levels of inflammatory factors in the BALF are shown in Figure 7e‐h. Compared to the control group, the levels of proinflammatory cytokines, including IL-6, IL-1β, TNF-α, and MCP-1, were dramatically increased in the LPS group, whereas they were distinctly reduced in the DEX group, confirming the successful establishment of the model. Compared with the LPS group, all the treatment groups presented different extents of reduction in inflammatory factors. Interestingly, these factors, particularly IL-6 and MCP-1, also tended to decrease in the Blank-DLP group. This effect may be attributed to the combined anti-inflammatory and pulmonary protective activities of its phospholipid constituents, which provide both immunomodulation and alveolar stabilization through surfactant replenishment, as documented in previous studies (Srivastava and Thakkar 2020; Alemán et al. 2022; Abouzid et al. 2023; Hou et al. 2025). In comparison with Blank-DLP, the BA and BAPC-DLP groups presented more pronounced decreases in inflammatory factors, with BAPC-DLP outperforming free BA, which was attributed to its enhanced lung deposition, mucus penetration, and synergistic effect of the liposome itself with the drug. Moreover, the level of IL-10, an anti-inflammatory cytokine in BALF, as presented in Figure 7i, demonstrated an increasing trend among all treatment groups, with notable statistical significance in the BAPC-DLP and DEX groups.

Subsequently, immunohistochemical analysis of the expression of CD68 (macrophage marker) and myeloperoxidase (MPO, neutrophil marker) in lung tissue was performed to investigate the extent of immune cell infiltration. As shown in Figure 7j, the proportion of CD68-positive cells was evidently elevated in the LPS group versus the control group, whereas BAPC-DLP treatment led to a significant reduction. As a marker enzyme specific to the primary granules of neutrophils (Shen et al. 2024), the expression level of MPO can reflect the extent of neutrophil infiltration. The LPS group exhibited substantial neutrophil infiltration. However, BAPC-DLP treatment dramatically reduced the MPO levels in the lung tissue, suggesting the effective inhibition of neutrophil infiltration in the lung tissue and alveoli.

The histopathological analysis of hematoxylin and eosin (H&E) staining in Figure 7k demonstrated that BAPC-DLP significantly facilitated lung tissue repair in mice with ALI. Specifically, the control group exhibited uniformly small alveoli and evenly spaced alveolar septa, indicating typical physiological characteristics. In contrast, the LPS group showed extensive damage to alveolar structures, characterized by thickening of the alveolar septa, collapse of the alveolar spaces, and considerable infiltration of neutrophils. After the BAPC-DLP intervention, the alveolar walls became clear, and inflammatory cell infiltration was reduced, significantly restoring LPS-induced pathological changes in the lung tissue. These significant improvements were confirmed by blinded histopathological scoring, as shown in Figure S11, which revealed a marked decrease in lung injury scores in BAPC-DLP treated mice compared to the LPS model group.

BAPC-DLP may alleviate ALI through a synergistic mechanism. On the one hand, baicalein exerts multi-target anti-inflammatory effects by synergistically inhibiting the NF-κB and MAPK signaling pathways, scavenging ROS, and modulating macrophage polarization (He et al. 2015; Wan et al. 2017; Chi et al. 2025). On the other hand, the phospholipid-based liposomal carrier itself provides additional anti-inflammatory benefits. This synergistic combination not only enhances overall anti-inflammatory efficacy while maintaining a favorable safety profile but also presents distinct clinical advantages over conventional therapies. Notably, while the DEX group demonstrated the most potent anti-inflammatory efficacy, with certain BALF inflammatory factors even lower than the control levels in Figure 7e‐h, this potent efficacy coincides with its characteristic immunosuppressive properties, which substantially limit its clinical applicability (Mokra et al. 2019). In contrast, BAPC-DLP demonstrated a favorable safety profile with balanced anti-inflammatory activity through multi-target mechanisms, achieving comparable therapeutic benefits in ameliorating lung pathology without inducing the systemic side effects. These findings position BAPC-DLP as a promising alternative to corticosteroids for ALI management, highlighting its potential as a viable therapeutic strategy.

While the present study provides compelling evidence for the preventive protective efficacy of BAPC-DLP, investigating its therapeutic efficacy following injury establishment represents an important direction for future research. Future work should therefore focus on establishing therapeutic administration protocols to determine its post-LPS efficacy and to elucidate the relevant molecular pathways through integrated spatial pharmacodynamics and multi-omics approaches to characterize its mode of action comprehensively. Further validation of specific molecular targets and detailed signaling mechanisms should be systematically investigated in subsequent studies.

In conclusion, inhaled BAPC-DLP was proven to be a promising and effective therapy for ALI management by alleviating pulmonary edema, calming cytokine storms, and inhibiting the infiltration of inflammatory cells, presenting extensive clinical translation and application potential.

3.8. Preliminary biosafety evaluation of BAPC-DLP

Biosafety is a fundamental prerequisite for effectively combating ALI. In this study, the preliminary biosafety of BAPC-DLP was investigated by hemolysis assay and H&E staining. It has been reported that inhaled nano/micrometer-sized particles, especially ultrafine particles smaller than 0.1 μm, may enter the systemic circulation via the trans-epithelial transport pathway, which may trigger potential hemolytic effects and vascular endothelial dysfunction. Therefore, we assessed the blood compatibility of BAPC-DLP. The results illustrated in Figure 8a‐b demonstrated that within the concentration range of 10−100 μg/mL, the hemolysis ratio for BAPC-DLP was less than 5%, confirming its commendable blood compatibility. According to Figure 8c, histopathological evaluation through H&E staining demonstrated that there were no notable pathological lesions of the major organs (heart, liver, spleen, kidney) of mice following inhalation administration of BAPC-DLP. The tissue structures in all the examined organs were preserved, with no evidence of inflammatory infiltration, cell vacuolation, or necrosis observed, which suggests the good biocompatibility of BAPC-DLP.

Figure 8.

Preliminary biosafety evaluation of BAPC-DLP. (a) Hemolysis ratio of BAPC-DLP at different concentrations (n = 3). (b) Representative images of the hemolysis assay. (c) Representative H&E images of the heart, liver, spleen, and kidney in different groups of LPS-treated ALI mice (scale bar: 200 μm).

4. Conclusion

In summary, the present research successfully constructed a novel liposomal formulation for nebulized inhalation based on baicalein‒phospholipid complex (BAPC-DLP) to enhance the protective effects on ALI. By integrating phospholipid complex technology with the liposome technique, BAPC-DLP dramatically improved the loading capacity of BA and the pulmonary delivery efficiency while exhibiting superior systemic stability, mucus penetration ability, nebulization performance, and biocompatibility. In addition, BAPC-DLP was demonstrated to be more efficiently taken up by activated macrophages and to exert superior inflammatory suppression and ROS scavenging effects than BA. Furthermore, BAPC-DLP exhibited significant protective effects on alveolar epithelial cells, effectively mitigating LPS-induced injury in A549 cells. In vivo biodistribution studies revealed that BAPC-DLP exhibited excellent lung targeting and retention ability via inhalation. In an LPS-induced ALI model in mice, inhaled BAPC-DLP significantly inhibited disease progression, alleviated pulmonary edema, markedly downregulated the expression levels of critical inflammatory factors such as TNF-α, IL-6, IL-1β, and MCP-1, and significantly improved injury to lung tissues. In this study, we explored for the first time nebulized inhalation administration of baicalein for the treatment of ALI, offering an important approach to significantly increase the solubility of poorly soluble drugs and the drug loading capacity of nanocarriers. Therefore, this research not only presents a highly promising drug candidate for ALI/ARDS treatment, but also opens new avenues for the development of nebulized inhalation formulations of poorly soluble natural products.

Supplementary Material

Supplementary material

Supplemental Figures

Funding Statement

This research was supported by the Medical and Health Science and Technology Innovation Project of the Chinese Academy of Medical Sciences (2021-I2M-1-030).

Ethical approval statement

All animal experimental protocols were approved by the Animal Protection and Welfare Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (Approval No. 00001820 for in vivo biodistribution studies and No. 00001833 for the ALI pharmacodynamics model). The use of mice was essential for this study to evaluate the in vivo biodistribution and protective efficacy of inhaled BAPC-DLP. Male BALB/c mice (6−8 weeks, 18–22 g) with SPF grade were provided by Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China), and all the mice were accommodated in the Institute of Materia Medica, Peking Union Medical College, Chinese Academy of Medical Sciences. The mice were housed in standard air-conditioned cages with a 12-h light‒dark cycle at a room temperature of 25 ± 2°C for 7 days before the experiments. The mice were housed under SPF conditions with free access to food and water.

All procedures were conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org) to ensure the highest standards of animal welfare. Anesthesia was induced and maintained using isoflurane (4% for induction, 1.5% for maintenance). At the end of the study, the mice were euthanized via cervical dislocation under anesthesia with 1.5% isoflurane.

Authors contributions

Zhiyang Wen, Yuling Liu, and Xuejun Xia designed the research. Zhiyang Wen carried out the experiments and performed data analysis. Jinghan Yu, Yingying Meng, Simeng Du, Yu Jiang and Yongwei Shi participated in some of the experiments. Zhiyang Wen drafted the manuscript. Jun Ye, Yuling Liu and Xuejun Xia revised the manuscript. All of the authors have read and approved the final manuscript. All the authors agree to be accountable for all aspects of the work.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/10717544.2025.2596168.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data will be made available on request from the corresponding author.