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International Journal of Pharmaceutics: X logoLink to International Journal of Pharmaceutics: X
. 2025 Aug 20;10:100379. doi: 10.1016/j.ijpx.2025.100379

Hesperidin-β-Cyclodextrin inclusion complexes: A novel approach for preventing and treating acute lung injury caused by seawater drowning

Jingjing Hou a,b,1, Mengdi Zhang a,c,1, Zheyi Han a,1, Wanmei Wang a, Haiying Qiu a, Jingwei Yuan a, Fang An b,⁎⁎, Yan Wu a,⁎
PMCID: PMC12398246  PMID: 40895347

Abstract

Seawater drowning-induced acute lung injury (ALI) presents a significant challenge due to the lack of effective prevention and treatment strategies. Hesperidin (Hep) possesses diverse biological activities, including potent antioxidant and anti-inflammatory effects. However, its clinical utility is hindered by poor solubility and limited bioavailability. Therefore, there is an urgent need for the modification of hesperidin to enhance its water solubility and expand its therapeutic potential. In this study, an inhalable formulation of Hep-β-cyclodextrin inclusion complexes (Hep-β-CD) was developed as a promising approach for the management of seawater drowning-induced ALI. The cytotoxicity assessment in BEAS-2B cells revealed minimal adverse effects associated with Hep-β-CD. The administration of Hep-β-CD via the pulmonary route has been found to be highly effective in preventing seawater drowning-induced ALI in mice, achieved through modulation of key inflammatory mediators and a reduction in oxidative stress. The study demonstrated that Hep-β-CD administration significantly decreased the levels of pro-inflammatory cytokines such as Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-6 (IL-6), which are known to contribute to the pathogenesis of ALI. Additionally, the levels of malondialdehyde (MDA) were decreased and the levels of Superoxide Dismutase (SOD) were increased. In summary, the pulmonary delivery of Hep-β-CD was identified as a promising therapeutic strategy for preventing seawater drowning-induced ALI due to its ability to directly distribute the drug to the lungs, where it exerts a dual action of modulating the immune response to reduce inflammation and enhance the antioxidant defense mechanism.

Keywords: Seawater drowning-induced acute lung injury, Hesperidin, Pulmonary administration, Β-Cyclodextrin, Inclusion complexesn

Graphical abstract

The pulmonary delivery of Hep-β-CD was identified as a promising therapeutic strategy for preventing seawater drowning-induced ALI due to its ability to directly distribute the drug to the lungs, where it exerts a dual action of modulating the immune response to reduce inflammation and enhance the antioxidant defense mechanism.

Unlabelled Image

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1. Introduction

The escalating global economy and societal developments have significantly amplified the frequency of overseas travel, maritime operations, and naval exercises, consequently fostering a notable surge in seawater drowning incidents (Qiu et al., 2020). Drowning has ascended to one of the primary three causes of accidental mortality worldwide, as highlighted by the World Health Organization's data, which indicates an annual toll surpassing 500,000 lives (Sun et al., 2019; Zhang and Xie, 2023; Zhang et al., 2022a). Following seawater drowning, the intrusion of high-osmolarity seawater into lung tissues incites severe irritation and damage to alveoli and capillaries, precipitating inflammatory responses, oxidative stress, and apoptosis. These pathological processes lead to conditions such as pulmonary hemorrhage, edema and hypoxia, collectively manifesting as seawater drowning-induced lung injury, which can progress to acute respiratory distress syndrome (ARDS) (Jin and Li, 2017; Liu et al., 2014a; Gaggar and Patel, 2016; Bao et al., 2018; Xiaoyu et al., 2017). Notably, in recent years, the increase in maritime activities has led to a corresponding rise in seawater drowning-induced lung injury incidence. Regrettably, despite this escalating trend, specific and efficacious treatments for this condition remain elusive. Hence, it is an important and urgent task to investigate effective methodologies to treat seawater drowning-induced ALI.

Hesperidin, a flavonoid abundant in various citrus fruits, possesses a wide array of pharmacological activities. Numerous experimental and clinical studies have reported its diverse biological properties, particularly its antioxidant and anti-inflammatory effects (Hegazy et al., 2023; Aksu et al., 2021; Ferreira de Oliveira et al., 2020; Jin et al., 2021). However, its clinical application is often hindered by its poor solubility in water and limited bioavailability in vivo. To address this challenge, β-Cyclodextrin (β-CD), a cyclic oligosaccharide comprising seven glucose units, has been reported as a viable solution (Wang et al., 2024; Cirri et al., 2021; Li et al., 2023). With its hydrophobic internal cavity and hydrophilic exterior, β-CD can encapsulate drug molecules within its tubular structure, forming clathrates. This complexation process enhances drug solubility, stability and bioavailability while also reducing potential toxicity concerns.

Inhalation therapy is widely acknowledged as a primary route for drug delivery, offering numerous advantages over conventional methods. It allows for achieving comparable or enhanced therapeutic effects with lower medication concentrations, bypassing first-pass metabolism and reducing the likelihood of side effects. Dry powder inhalers (DPIs) represent a portable and solid powder delivery system known for maintaining drug stability better than aerosols and nebulizers, while also providing improved convenience and compliance for patients.

In this study, we aimed to develop inhalable Hesperidin-β-cyclodextrin inclusion complexes (Hep-β-CD) for the prevention of seawater drowning-induced acute lung injury (ALI) by administering Hep-β-CD intratracheally (i.t.) to corresponding mouse models to elucidate its effects and underlying mechanisms against this condition. Although Hep-β-CD was widely described (Corciova et al., 2015; Wdowiak et al., 2022; Moriwaki et al., 2021), it was first applied to treat seawater drowning-induced acute lung injury. It expands the applications of hesperidin and offers a novel solution for seawater drowning-induced acute lung injury. The drug was delivered directly to the lungs via pulmonary inhalation route, which enhanced targeting, reduced systemic side effects, and improved local therapeutic efficacy compared to traditional oral administration.Overall, this investigation represents the first exploration of inhaled natural products for addressing seawater drowning-induced ALI, providing important insights for future therapeutic considerations.

2. Material and methods

2.1. Materials

Hesperidin and β-cyclodextrin were purchased from Beijing Ouhe Technology Co., Ltd. (Beijing, China). Seawater with an osmolality of 1300 mmol/L and pH of 8.2 was prepared for experimentation. Each liter of seawater contained the following components: sodium chloride, magnesium sulfate, magnesium chloride, calcium chloride, potassium chloride, sodium bicarbonate, and sodium bromide in amounts of 26.518 g, 3.305 g, 2.447 g, 1.141 g, 0.725 g, 0.202 g, and 0.083 g, respectively. The composition was based on data provided by the Chinese Ocean Bureau (https://www.nmdis.org.cn/). ELISA kits for rat TNF-α, IL-6, SOD, and MDA were bought from Beijing Neobioscience Technology Co., Ltd. (Beijing, China).

2.2. Animals

Eight-week-old male BALB/c mice weighing 18–22 g were purchased from Beijing SiPeiFu Biotechnology Co., Ltd. (license number: SCXK 2019–0010). They were allowed a 5-day acclimatization period to adapt to the laboratory environment. They had free access to standard rodent feed and water. All experimental procedures were conducted in compliance with relevant regulations and institutional ethical guidelines.

2.3. Phase solubility study

Phase solubility diagrams were prepared to determine the ratio of the drug to β-CD in solution and how the solubility of the drug changes when a complex is formed. Excess quantity of hesperidin was added to the same volumes of different concentrations of β-CD water solutions (2.0–10.0 mM), and samples were placed in a constant temperature shaker OLB-200B (Shandong Boke Scientific Instrument Co., Ltd.) for 72 h at 37.0 ± 1 °C, then the unreacted hesperidin was removed through filtering, using a 0.45 μm, nylon disc filter. The concentration of hesperidin was determined using high-performance liquid chromatography at 283 nm. The phase solubility diagram was drawn by plotting the concentration of hesperidin versus the concentration of β-CD. All samples were analyzed in triplicate. The concentration of hesperidin in each solution was determined using suitable constructed standard curves. The complex efficiency (CE), which indicates an index of affinity between the drug and β-CD, was calculated from the following equal using the slope a of each phase solubility diagram (Ahad et al., 2022).

CE=α1−α

2.4. Preparation and characterization of Hep-β-CD

Hep-β-CD was prepared using the dielectric grinding method. Briefly, a Hep solution in tert-butanol was combined with an aqueous solution of β-CD in a 1:1 ratio (mol:mol), followed by continuous grinding for 4 h. The resulting suspension was freeze-dried for 48 h using a lyophilizer (Lab-1 A-50, Beijing Boyikang Experimental Instrument Co., Ltd., Beijing, China). The microscale morphologies of Hep, β-CD, their physical mixture (Hep:β-CD, 1:1, mol:mol), and Hep-β-CD powders were examined using a scanning electron microscope (SEM, CUBE2, EmCrafts, Gyeonggi-do, South Korea). The crystalline characteristics of these samples were analyzed by X-ray powder diffraction (XRD, D8 Advance, Karlsruhe, Germany).

For the extraction of Hep from Hep-β-CD, methanol was used as the solvent, and the resulting solution was filtered through a 0.22 μm filter membrane before analysis via high-performance liquid chromatography (HPLC). The HPLC system consisted of an i-Series Plus setup with a Venus II XBP C18 column (150 mm × 4.6 mm, 5 μm) maintained at 30 °C. The mobile phase comprised a mixture of acetonitrile and 0.1 % phosphate water (20/80, v/v), flowing at a rate of 1.0 mL/min. The sample injection volume was 10 μL, and detection occurred at a wavelength of 283 nm (Liu et al., 2014b). The drug-loading (DL) capacity was calculated with encapsulated Hep/total Hep-β-CD × 100 %.

2.5. Evaluation of Hep-β-CD release in Vitro

Hep-β-CD (containing 1 mg hesperidin) and 1 mg bulk drug hesperidin were separately introduced into dialysis bags with a molecular weight cutoff of 10,000 Da. These bags were then placed into closed glass flasks containing 250 mL of the mixture of phosphate buffered solutions (PBS, pH 7.4)/ethanol (2:1, v:v). The system was maintained under constant and moderate agitation in a water bath (37 °C ± 0.1 °C). At predefined intervals (0.5, 1, 2, 4, 6, 8, 10, 12, 24 and 48 h), a 1 mL aliquot of the media was withdrawn and replaced with an equal volume of fresh medium. Following filtration through a 0.45 μm cartridge filter, the mass concentration of hesperidin and inclusion compound was determined, and the cumulative release rate was calculated. (Ge et al., 2018).

2.6. Stability test

The specific amount of Hep and Hep-β-CD were placed into closed EP tubes, dividing them into three groups for cultivation at light, temperature of 60 °C and relative humidity of 85 %. Each group contained three samples. The concentration of the samples were measured at 283 nm to determine the degradation rate of the inclusion compounds over a designated period.

2.7. Aerodynamic equivalence study

The simulated lung deposition of Hep-β-CD was explored on the Next Generation Impactor (NGI, TPK 2000R, Copley, Nottingham, UK) with an inspiratory flow rate of 60 L/min. Approximately 20 mg of Hep-β-CD powders were filled in hydroxypropyl methylcellulose (HPMC) hard capsules (Capsugel VR, Type 3, Suzhou Capsule Ltd., Suzhou, China) and placed into a DPI device (Type 006, Shanghai Huarui Aerosol Co., Ltd., Shanghai, China) for the deposition test. Deposition was repeated with 10 capsules. The deposited powders were collected from each stage and separately dissolved in methanol for HPLC measurement of Hep. The fine particle fraction (FPF), mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD) were calculated using the software (Copley Inhaler Testing Date Analysis Software, Version 3.10, Copley Scientific). The lung deposition or pulmonary delivery efficiency of particles is determined by their aerodynamic diameter.

2.8. Cell Culture and Treatment

A normal bronchial epithelial cell line (BEAS-2B) was purchased from Meisen CTCC (Zhejiang, China). Briefly, the cells were maintained in a controlled environment at 37 °C with 5 % CO2 and cultured using Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (1:1, D/F-12). BEAS-2B cells were seeded either in 96-well plates at a concentration of 1.5 × 104 cells/mL or in 6-well plates at a concentration of 1 × 105 cells/mL and utilized for experiments following overnight incubation.

The experimental group was cultured in a D/F-12 medium supplemented with seawater at concentrations of 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 % and 80 %. Following a culture period of 6 h, the cells were examined to assess the impact of seawater concentration on BEAS-2B cells. Upon identification of the optimal seawater concentration, the cells were treated with a final concentration of 40 % seawater (0.4 mL in a total volume of 1 mL) for 2, 4, 6, 8 and 10 h. The control group comprised cells cultured in a complete medium devoid of seawater or drugs. The subsequent experiments involved cells treated with a final concentration of Hep-β-CD (100 μmol/L) for 6 h before seawater exposure, as well as those only exposed to seawater at a concentration of 40 % for 6 h (SW group). Cell viability was evaluated using Cell Counting Kit-8 (CCK-8) kits (Ding et al., 2018; Dokumacioglu et al., 2019; Zhang et al., 2011).

2.9. Cytotoxicity assay

BEAS-2B cells were seeded at a density of 1.5 × 104 cells per well in 96-well plates and incubated for 24 h at 37 °C. Hep-β-CD and Hep were separately dissolved in dimethyl sulphoxide (DMSO) and then diluted with culture medium to generate a range of concentrations. These solutions were subsequently added to the wells, ensuring a final DMSO concentration below 0.1 %. The cells were treated with suspensions of Hep-β-CD and Hep in a culture medium containing hesperidin concentrations ranging from 1 μmol/L to 1000 μmol/L, followed by a 24-h incubation period. Afterward, the plates were washed once with sterile phosphate-buffered saline (PBS). A solution of 10 % cell counting kit-8 (CCK-8, Beijing RX Biotechnology, Co., Ltd., Beijing, China) was added to each well and incubated for 2 h. The optical density at 450 nm was measured using a microplate reader (Synergy H1, BioTek Instruments, Winooski, VT, USA), and cell viability was calculated using the formula: [(OD sample – OD blank) / (OD control - OD blank)] × 100 %.

2.10. Western Blot analysis

RIPA lysis buffer (Beyotime Institute of Biotechnology) was used to extract total proteins from the cells. Protein quantification was performed using the BCA assay. After centrifugation at 12,000 ×g for 10 min at 4 °C, 50 μg of protein was denatured in a loading buffer. Protein separation was performed on 10 % SDS-polyacrylamide gels, followed by electrotransfer onto PVDF membranes. Membrane blocking was performed by incubating with 5 % non-fat milk in TBS for 1 h at 4 °C. For the detection of specific proteins, the membrane was incubated overnight at 4 °C with polyclonal rabbit antibodies against β-actin (diluted 1:1000; cat. no. 21244–1-AP; Boster Biological Technology Co., Ltd), TNF-α (1:1000; cat. no. 123966; Shanghai Absin Biotechnology Co., Ltd), IL-6 (1:1000; cat. no. 120201; Shanghai Absin Biotechnology Co., Ltd), and IL-1β (1:1000; cat. no. 26048–1-AP; Proteintech Group, Inc.). Next, the membranes were washed with TBST and then incubated with HRP-conjugated goat anti-mouse secondary antibodies (diluted 1:5000; cat. no. Bs-40296G-HRP; Beijing Biosynthesis Biotechnology Co., Ltd.) for 2 h at 4 °C, following which the relative protein content was analyzed using the ChemiDoc MP Imaging system (BIO-RAD) post-secondary antibody incubation.

2.11. Reverse transcription-quantitative polymerase chain reaction

The purity and concentration of RNA were assessed using a UV5Nano Ultramicrospectrophotometer (METTLER TOLEDO, Inc.) at an optical density of 260/280 nm. Total RNA extraction from cultured cells was performed using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions (Han et al., 2023). Then, cDNA was synthesized using a Reverse Transcription Kit (Takara, Dalian, China), followed by PCR performed on the SmartCycler® II System (Cepheid Inc., Sunnyvale, CA, USA). GAPDH was used as the reference gene. The thermocycling conditions were as follows: Initial denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Relative quantification of transcripts was determined using the 2−ΔΔCt method. The sequences of the IL-6 primers were 5’-CACTTCACAAGTCGGAGGCT (forward) and CTGCAAGTGCATCATCGTTGT-3′ (reverse; universal), while the sequences of the IL-1β primers were 5’-GGCCCTAAACAGATGAAGTGCT (forward) and TGCCGCCATCCAGAGG-3′ (reverse; universal).

2.12. Detection of intracellular ROS

The cells were inoculated onto 6-well plates and then randomly divided into three groups: control group (Ctl), seawater drowning group (SW), and drug administration group (Hep-β-CD). After the treatment was completed, the cell culture medium was discarded, and the wells were washed three times with sterile PBS. Subsequently, 1 mL of DCFH-DA probe solution, diluted with serum-free D/F-12 medium at a ratio of 1:1000 to achieve a final concentration of 10 μmol/L, was added. The cells were then incubated at 37 °C for 30 min in the dark. After incubation, the cells were washed with a serum-free medium once or twice to remove any residual DCFH-DA that did not enter the cells (Ishola et al., 2019). An appropriate amount of PBS was added to prevent cell death due to drying during the observation process, and then the cells were observed and photographed under a fluorescent inverted microscope.

2.13. Hoechst 33342 staining

The cells were inoculated onto 6-well plates and subsequently divided into three groups: control group (Ctl), seawater drowning group (SW), and drug administration group (Hep-β-CD). Following treatment, the supernatant was discarded, and the cells were gently washed twice with PBS. An appropriate volume of Hoechst 33342 cell staining solution (final concentration: 1×) was uniformly added to the culture medium, and the cells were incubated at a suitable temperature for cell culture for 10 min. Then, the staining solution was aspirated, and the cells were washed with culture medium or PBS two to three times before being examined under a fluorescence microscope (Pyrzynska, 2022).

2.14. AO/EB staining

The cells were seeded onto 6-well plates and then randomly assigned to three groups: control group (Ctl), seawater drowning group (SW), and drug administration group (Hep-β-CD). Each group of cells was treated separately according to its assigned group. Before use, the AO solution and EB solution were mixed in a 1:1 ratio to prepare the working solution, which was used immediately. Following treatment and incubation, the culture medium was removed, and the cells were washed twice with PBS to eliminate residual medium and unattached cells. Fresh PBS or medium was then added, and 20 μL of the working solution per mL of medium or PBS was applied. The cells were incubated at room temperature for 2–5 min and then examined under an inverted fluorescence microscope (Zhang et al., 2022, Zhang et al., 2022b).

2.15. Seawater drowning model and animal treatments for pharmacodynamic study

Male BALB/c mice weighing 18–22 g were purchased from Beijing SiPeiFu Biotechnology Co., Ltd. (Beijing, China). They were allowed a 5-day acclimatization period to adapt to the laboratory environment. All experimental procedures were conducted in compliance with relevant regulations and institutional ethical guidelines. AIC-induced seawater drowning in mice was established using a previously described method. Briefly, the mice were securely placed in a holder and submerged for 25 s in a water bath containing seawater with a depth of 6 cm and a temperature maintained at 25 ± 2 °C (Ahmadi and Shadboorestan, 2016; Bekdash, 2021; Xu et al., 2018; Zhang et al., 2018).

The mice were randomly divided into six groups (n = 5 mice/group): the control group, the model group, the BUD-treated group (drowning followed by intratracheal administration of budesonide, BUD), and the Hep-β-CD-treated group (drowning followed by intratracheal administration of Hep-β-CD). In the model group, saline was intratracheally administered to mimic the administration process. The Hep-β-CD-treated group was further subdivided into low dose (L-Hep-β-CD, 20 mg/kg), medium dose (M-Hep-β-CD, 40 mg/kg), and high dose (H-Hep-β-CD, 80 mg/kg) groups. Specifically, the low dose group received an Hep-β-CD saline suspension (concentration 8 mg/mL) at a dose of 20 mg/kg (containing 7.04 mg of Hep per kg), the medium dose group received a Hep-β-CD saline suspension (concentration 16 mg/mL) at a dose of 40 mg/kg (containing 14.08 mg of Hep per kg), and the high dose group received a Hep-β-CD saline suspension (concentration 32 mg/mL) at a dose of 80 mg/kg (containing 28.16 mg of Hep per kg). All treatments in the experiment were administered via the lung administration route. The specific procedure was as follows: 2 h after modeling, the mice were anesthetized, their mouths were opened using a laryngoscope, the trachea was exposed, a mouse lung administration needle was inserted into the trachea, and the drug was sprayed into the trachea. Immediately after administration, the mice were placed in cages and received treatment once daily for 3 days (Wang et al., 2022; Chen et al., 2022; Wanmei et al., 2023).

2.16. Lung Wet/Dry (W/D) weight measurement

After they were sacrificed, the mice's lung tissues were excised, and their surface fluids were removed using filter paper. The appearance of the lung tissue was observed and photographed. Subsequently, the whole left lung of each mouse was collected, and after wiping off any blood stains, its wet weight was immediately measured. The lung tissue samples were then placed in a 70 °C drying oven for 72 h until a constant weight was achieved, allowing the calculation of the wet-dry ratio (Mathes et al., 2016).

2.17. Histopathological examination

At the end of the pharmacodynamic study, the mice were euthanized, and their entire lung tissues were harvested. The upper lobe of the right lung was submerged in 10 % formalin solution for 24 h. After fixation, the tissues were embedded in paraffin and sectioned into 5 μm slices. These slices were then mounted on silanized slides and stained with hematoxylin and eosin (H&E). Observation of the sections was conducted using an automatic digital slice scanning system (Pannor, 3DHISTECH).

2.18. ELISA analysis

The levels of TNF-α, IL-6, SOD, and MDA in lung tissues were assessed using ELISA kits. To remove any residual blood, the lung tissues were rinsed with PBS at pH 7.4, and excess moisture was blotted with filter paper before weighing. Each tissue sample was then combined with nine times its volume of PBS (e.g., 9 mL of PBS per 1 g of tissue) in a grinding tube and homogenized using a high-speed tissue homogenizer. All procedures were conducted following the manufacturer's instructions.

2.19. Immunohistochemistry

The upper right lung tissues of the mice were excised, embedded in paraffin wax, and subjected to xylene dewaxing followed by ethanol rehydration. Next, the tissues were immersed in an antigen recovery solution (pH 8.0) containing ethylenediamine tetraacetic acid (EDTA), and antigen retrieval was achieved by microwave heating for 15 min. After rinsing with water, the sample was treated with a 3 % H2O2 solution to neutralize endogenous peroxidase activity. The primary antibody NF-κB p65 (diluted 1:100; Cat. no. 10745–1-AP; Proteintech Group, Inc.) was then applied and incubated at room temperature for 30 min. Then, the secondary antibody was applied and incubated for 30 min at room temperature, followed by intermittent washing with PBS for immunohistochemical detection. Positive cells were identified by brown staining, and images were captured using an automatic digital slice scanning system (Kheradmand et al., 2018).

2.20. Statistical analysis

Statistical analysis was conducted using GraphPad Prism software (version 8.0, GraphPad Software Inc., San Diego, CA, USA). Data are expressed as mean ± standard deviation (SD). The one-way analysis of variance (ANOVA) test was used for comparisons among multiple groups, with pair comparisons between groups performed using the t-test. A significance level of P < 0.05 was considered statistically significant.

3. Results

3.1. Phase solubility studies of Hep and β-CD

According to the solubility curve plotted, the inclusion ratio of the hesperidin and β-CD and its type were determined. The solubility curve reveals a regression equation of y = 0.0069× + 0.0233 (R2 = 0.9905), indicating that within the selected concentration range of β-CD, hesperidin concentration increases linearly with β-CD concentration, categorizing the inclusion type as AL type. Therefore, a molar ratio system of 1:1 was chosen for subsequent experiments (Fig. 1A).

Fig. 1.

Fig. 1

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Phase-solubility plot of Hep with β-CD (A). Dissolution profiles of Hep and Hep-β-CD (B). SEM images of β-CD (C), Hep (D), Hep-β-CD (E) and physical mixture (F). XRD graphs of Hep, β-CD, Hep-β-CD, and physical mixture (G).

3.2. Characteristics of Hep-β-CD

Following the inclusion of hesperidin and β-cyclodextrin, the solubility of the resulting compound typically improves compared to Hep. The results indicated that after 48 h, the solubility of Hep-β-CD reached approximately 93 %, which was nine times greater than that of raw hesperidin, demonstrating a significant improvement over hesperidin alone (Fig. 1B).

According to the SEM images, β-cyclodextrin displayed an incomplete and irregular hollow spherical structure with a rough and uneven surface, along with noticeable cracks (Fig. 1C). Hesperidin exhibited large crystals of varying sizes (Fig. 1D). In contrast, the spherical structure of β-cyclodextrin in the inclusion compounds was entirely absent, revealing an irregular, dense, and uniform blocky structure with a smooth surface rather than a rough and uneven one. The morphology resembled small crystals embedded in the plane, with a host-and-object encircling shape. The original form of the two components after inclusion had disappeared, demonstrating differences in size and shape compared to β-cyclodextrin and hesperidin alone (Fig. 1E). The physical mixture exhibited some similarities to the free molecular crystals compared to the original drug, indicating a combination of the two crystals (Fig. 1F).

The presence or absence of characteristic peaks in the spectrum represents clathrate formation, with the corresponding increase or decrease indicating the presence or absence of clathrate. The XRD pattern of Hep displayed characteristic diffraction peaks between 10° and 40°, indicative of its typical crystal structure. The spectra of the physical mixture closely resembled those of hesperidin and β-CD, suggesting minimal interaction between the two components. In comparison, the XRD pattern of Hep-β-CD revealed the disappearance of diffraction peaks at 12.12°, 15.49°, and 19.45° when compared to the physical mixture, confirming the formation of clathrates (Fig. 1G). In addition, the drug-loading (DL) capacity of Hep-β-CD was 28.96 %.

3.3. Stability test

The contents of Hep and Hep-β-CD on day 0 were set as 100 %, respectively. The contents determined on day 5 and 10 were compared with the data of day 0. The results are shown in Table 1. Hep was sensitive to light, temperature and humidity. It was unstable when exposed to high humidity. At 85 % relative humidity, it degrades by about 12 % after 10 days of exposure. In contrast to the free Hep, the inclusion complex significantly enhances the stability of Hep (P < 0.0001). β-CD are capable of protecting the components from degrading.

Table 1.

The effects of light, temperature, and humidity on the stability of Hep and Hep-β-CD. ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to free Hep.

Time (day) Hep (%) Hep-β-CD (%)
Light 0 100.00 ± 0.18 100.00 ± 0.76
5 96.98 ± 0.08 98.14 ± 1.05
10 95.01 ± 0.82 97.59 ± 0.87⁎⁎
Temperature (60 °C) 0 100.00 ± 0.18 100.00 ± 0.76
5 95.76 ± 1.12 97.48 ± 0.32
10 93.10 ± 0.54 97.35 ± 1.26⁎⁎⁎⁎
Humidity (R.H. 85 %) 0 100.00 ± 0.18 100.00 ± 0.76
5 94.24 ± 0.27 97.49 ± 0.41⁎⁎⁎
10 88.41 ± 0.95 96.87 ± 0.29⁎⁎⁎⁎
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3.4. Highly efficient lung deposition of Hep-β-CD

The lung deposition or pulmonary delivery efficiency of particles is determined by their aerodynamic diameter. The MMAD, FPF, and GSD of Hep-β-CD powders were 2.91 ± 0.02 μm, 54.92 ± 0.42 %, and 1.77 ± 0.01 (n = 3), respectively, indicating that Hep-β-CD powders would have effective lung deposition when inhalation (Fig. 2).

Fig. 2.

Fig. 2

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The simulated Hep lung deposition of Hep-β-CD in the NGI. Data are presented as mean ± SD (n = 3). MOC: micro-orifice collector.

3.5. Cell model and cytotoxicity

To establish the cell model of seawater-induced lung injury, BEAS-2B cells were treated with various concentrations of seawater for different durations, and cell viability was assessed using CCK-8. Initially, the control group was cultured in a complete D/F-12 medium, while the experimental groups were cultured in a D/F-12 medium supplemented with seawater at concentrations ranging from 5 % to 80 %. After 6 h of culture, cell viability was measured using CCK-8 (Fig. 3A). Subsequently, BEAS-2B cells were cultured in D/F-12 medium supplemented with 40 % seawater for 2, 4, 6, 8, and 10 h, respectively, and cell viability was assessed using CCK-8. It was observed that cell viability gradually decreased over time compared to the control group, with the survival rate reaching approximately 50 % after 6 h of culture (Fig. 3B). Based on these findings, the D/F-12 medium supplemented with 40 % seawater concentration and a 6-h culture period were selected as the conditions for establishing the cell model of seawater-induced lung injury.

Fig. 3.

Fig. 3

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Cell cytotoxicity. (A)Selection results of seawater concentration. (B) Selection of incubation time in seawater. Safe concentration selection of Hep-β-CD (C) and Hep (D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Concentrations of Hep-β-CD ranging from 1 to 100 μmol/L exhibited no impact on the growth of BEAS-2B cells, remaining within the maximum safe concentration range of Hep for raw materials (Fig. 3C and D). However, as the concentration of Hep-β-CD increased, the cell survival rate decreased. Even at a high concentration of 100 μmol/L, Hep-β-CD showed no adverse effects on the growth of BEAS-2B cells, highlighting its potential safety even at elevated concentrations (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the control group).

3.6. Anti-inflammation effect of Hep-β-CD

To explore the potential impact of seawater on inflammatory cytokines in BEAS-2B cells, we assessed the expression levels of IL-1β, TNF-α, and IL-6 proteins via Western Blot (Fig. 4A), as well as the mRNA levels of inflammatory cytokines (IL-1β, IL-6) using qPCR (Fig. 4B). Our findings demonstrated that compared to the control group, the SW group exhibited elevated expressions of IL-1β, TNF-α, and IL-6. These results indicate that seawater exposure could increase the production of these pro-inflammatory cytokines in BEAS-2B cells.

Fig. 4.

Fig. 4

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Anti-inflammation effect of Hep-β-CD. (A) Representative western blot images levels. (B) The relative mRNA levels of cell proliferation-related cytokines were detected by qPCR. *P < 0.05, **P < 0.01.

3.7. Cell apoptosis staining

In the presence of intracellular ROS, DCFH-DA undergoes oxidation to produce a green fluorescent substance, with fluorescence intensity correlating with intracellular ROS levels. As depicted in the figure, the green fluorescence observed in the model group was more intense compared to both the normal and drug administration groups. Seawater exposure notably elevated intracellular ROS levels in BEAS-2B cells, a phenomenon mitigated by treatment with Hep-β-CD (Fig. 5).

Fig. 5.

Fig. 5

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Cell apoptosis staining. ROS, Hoechst 33342 and AO/EB staining of BEAS-2B. (VS. Ctl *P < 0.05, **P < 0.01, ****P < 0.0001; VS. SW #P < 0.05, ###P < 0.001).

In normal nuclei, Hoechst 33342 staining revealed a round shape with a light blue hue and deep blue particles. In contrast, apoptotic nuclei exhibited intensified staining, characterized by brighter fluorescence and a rounded or condensed, clumped structure. In addition, nuclear fluorescence in the model group appeared brighter compared to both the normal and drug administration groups, indicative of a higher proportion of apoptotic cells (Fig. 5).

After AO/EB staining, cells with intact membranes exhibited uniform green nuclei. However, cells treated with seawater displayed notable alterations in morphology, including cell fragmentation and orange-stained nuclei with condensed or fragmented chromatin, indicative of apoptotic death. No significant distinction was observed between the treated and normal groups. These findings underscore the significant apoptosis-inducing effect of seawater on BEAS-2B cells (Fig. 5).

3.8. Hep-β-CD alleviated seawater drowning-induced ALI

Seawater drowning-induced acute lung injury (ALI) led to severe symptoms in the mice, including shortness of breath. Upon examination, the excised lungs exhibited a dark-red appearance, significant edema, and substantial bleeding compared to the smooth and red lungs of healthy mice (Fig. 6). From left to right, the groups shown are the normal, model, BUD-treated, low-dose Hep-β-CD-treated, medium-dose Hep-β-CD-treated, and high-dose Hep-β-CD-treated groups.

Fig. 6.

Fig. 6

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The lung appearance of mice, images of H&E staining and lung W/D ratios. **P < 0.01.

In both the BUD and Hep-β-CD groups, all mice exhibited normal physical activity, with minimal hemorrhage and predominantly clear alveolar structures in the lungs, signifying a remarkable alleviation of seawater drowning-induced ALI. Specifically, in the Hep-β-CD low, medium and high dose groups, the lung tissues resembled that of the normal group, characterized by intact alveolar walls, preserved alveolar structure, and absence of inflammatory infiltration. Conversely, in the model group, the lung tissues exhibited structural damage, including alveolar collapse, thickened alveolar walls, alveolar congestion, and severe inflammatory infiltration. While the BUD group showed thinner alveolar walls and some inflammatory infiltration, the extent of normal alveolar structure was greater than that in the model group. Mice treated with Hep-β-CD low dose demonstrated complete lung morphology and structure, with thin alveolar walls and minimal inflammatory infiltration. Although the medium and high dose groups exhibited thicker alveoli, they showed slight improvement compared to the model group. These findings highlight the efficacy of Hep-β-CD in treating lung injury induced by seawater drowning.

Moreover, we used the lung W/D ratios as an indicator to assess the degree of pulmonary edema. Following seawater exposure, there was a notable increase in the lung W/D ratios. However, both Hep-β-CD and BUD administration resulted in a reduction of the W/D ratios, indicating mitigation of pulmonary vascular leakage and pulmonary edema induced by seawater exposure.

3.9. Anti-inflammation and regulating oxidative stress effect of Hep-β-CD

Following seawater drowning, the expression of two prominent pro-inflammatory cytokines in lung tissues, namely TNF-α and IL-6, was found to be significantly increased in the model group. However, after prophylactic pulmonary inhalation, the levels of TNF-α and IL-6 in the low-dose mice of both the BUD and Hep-β-CD groups decreased, showing no difference compared to the normal group. Notably, lung inhalation of Hep-β-CD effectively prevented the escalation of pro-inflammatory factors induced by seawater drowning. Additionally, compared to the normal group, the SOD level decreased significantly while the MDA level increased in the model group. Post-administration, the SOD level in both the BUD and Hep-β-CD low dose groups showed a significant increase, while the MDA level decreased, returning to normal levels. These results suggest that Hep-β-CD could regulate the oxidative stress levels induced by seawater drowning (Fig. 7).

Fig. 7.

Fig. 7

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IL-6, TNF-α, SOD and MDA results of mice. *P < 0.05, **P < 0.01, ***P < 0.001.

3.10. Hep-β-CD regulate immune response

The expression results of NF-κB (p65) protein in lung tissue of mice induced by seawater drowning are shown in Fig. 8. The results indicated that, compared to the normal group, the staining of brown and yellow cells, indicative of NF-κB (p65) protein expression, were significantly increased in the model group. However, the effect of the low dose and Hep-β-CD groups differed significantly from that of the normal group.

Fig. 8.

Fig. 8

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Expressions of NF-κB (p65) in the lung tissues. (A-F) normal group, model group, BUD, Hep-β-CD (20 mg/kg), Hep-β-CD (40 mg/kg), and Hep-β-CD (80 mg/kg).

4. Discussion

Drowning is one of the major causes of unintentional injuries and fatalities. Direct exposure to hypertonic seawater poses a threat to alveolar epithelial cells, resulting in fetal hypoxia, acidosis, and the subsequent development of ALI/ARDS. However, there is a lack of specific and effective treatment for this condition (Shagirtha et al., 2017; Jansook et al., 2018; Yang et al., 2022). The acute inhalation of seawater can inflict direct harm upon alveolar epithelial cells, compromising the integrity of the alveolar epithelial barrier and intercellular connections, consequently increasing capillary permeability. Under the influence of seawater's high osmotic pressure, the extravasation of intravascular fluid escalates, fostering lung tissue edema. Through extensive research, the impact of inflammation, oxidative stress, alterations in effective components, apoptosis, and autophagy on the pathophysiological progression of acute lung injury induced by seawater drowning has progressively unfolded (Zhang et al., 2022, Zhang et al., 2022b; Shankar et al., 2021; Bilodeaux et al., 2023; Reizine et al., 2021).

In this study, we utilized hesperidin as the raw material and β-cyclodextrin as the inclusion material to produce Hep-β-CD inclusion compound powder, which significantly enhanced hesperidin's solubility and achieved a particle size suitable for pulmonary inhalation. The resulting Hep-β-CD powders proved suitable for use as dry powder inhalers (DPIs), offering advantageous features such as high drug loads, portability, propellant-free formulation, and robust stability. Our initial investigations indicated that the ground Hep-β-CD particles tended to be larger in size. Consequently, freeze-drying emerged as the preferred method for Hep-β-CD preparation. Additionally, the modified state of hesperidin within Hep-β-CD facilitated its dissolution and release speed, rendering it conducive to prompt prevention and treatment following pulmonary administration. A primary challenge encountered was controlling the particle size of the inclusion compound. To address this, we conducted preliminary experiments exploring various cyclodextrin types and solvent selections, with particle size serving as the key observation parameter. The findings indicated that the requirements for pulmonary inhalation administration could be met using β-cyclodextrin and tert-butanol as the solvent.

The Hep-β-CD inclusion compound could suppress the expression of TNF-α, IL-1β, IL-6, and other pro-inflammatory cytokines in BEAS-2B cells exposed to seawater-induced lung injury, thereby attenuating the inflammatory response and mitigating cell apoptosis. Furthermore, our findings revealed that the Hep-β-CD inclusion compound could downregulate the expression of pro-inflammatory cytokines and decrease the apoptosis ratio of lung tissue cells in seawater-drowned mice. Incorporating hesperidin into Hep-β-CD notably enhanced its anti-inflammatory and anti-apoptotic properties, effectively leveraging the pharmacological potential of hesperidin. The utilization of β-CD inclusion compound powder aerosols represents a promising strategy for enhancing the bioactivity of insoluble drugs such as hesperidin.

In our animal experiments, we observed that Hep exhibited remarkable efficacy in attenuating the inflammatory response and preventing seawater-induced ALI in mice. Specifically, Hep significantly downregulated the expression of NF-κB p65 in lung tissues of ALI mice induced by seawater drowning, effectively suppressing the release of TNF-α and IL-6 and thus ameliorating ALI symptoms. Increasing evidence have shown the diverse pharmacological activities of Hep. For instance, it can regulate the TLR4/NF-κB signaling pathway to prevent neuroinflammation, apoptosis and memory impairment induced by lipopolysaccharides. Moreover, previous studies have reported its ability to mitigate lung damage in ALI mice, potentially through modulation of the MD2 protein. Furthermore, Hep has been demonstrated to protect lung cells from acrolein-induced apoptosis. However, the precise mechanism by which Hep mitigates ALI remains elusive. Our study revealed that Hep significantly alleviated histopathological alterations induced by seawater drowning, suppressed lung inflammation, and reduced the lung W/D ratio. These findings suggest that Hep could shield the lungs from seawater-induced ALI by dampening inflammatory responses and modulating oxidative stress.

5. Conclusions

Seawater drowning often results in severe lung damage, leading to life-threatening conditions such as ALI or ARDS. Unfortunately, effective treatment or prevention methods for such conditions are currently lacking. Hep, a natural flavonoid, possesses notable anti-inflammatory, anti-apoptotic, and antioxidant properties. Previous studies have demonstrated its efficacy in preventing and treating ALI/ARDS in animal and cell models induced by various factors. In this study, we prepared a pulmonary inhalable powder comprising Hep and β-CD, delivering them directly to lung tissue to address seawater drowning-induced lung injury while investigating the underlying mechanisms. Through the establishment of animal and cellular models of lung injury induced by seawater drowning, we observed the protective and therapeutic effects of Hep-β-CD on BEAS-2B cells and mice, elucidating its association with inflammatory factors and oxidative stress. Hence, the pulmonary delivery of Hep-β-CD could be a promising strategy for preventing seawater drowning-induced ALI.

CRediT authorship contribution statement

Jingjing Hou: Writing – original draft, Visualization, Validation, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Mengdi Zhang: Writing – review & editing, Visualization, Resources, Data curation, Conceptualization. Zheyi Han: Writing – review & editing, Formal analysis. Wanmei Wang: Resources, Data curation. Haiying Qiu: Software, Formal analysis. Jingwei Yuan: Supervision, Formal analysis, Conceptualization. Fang An: Visualization, Validation. Yan Wu: Writing – review & editing, Supervision, Project administration, Funding acquisition.

Ethics Statement

All experimental protocols with mice followed the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) and were approved by the Ethics Committee of the Air Force Medical Center (Ethical approval number: 2024–21-PJ01).

Funding information

This work was financially supported by Air Force Medical Center Technology Assistance Program (2022ZTYB16).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Thank to ZYEdit for language editing.

Contributor Information

Fang An, Email: anfanghbnu@123.com.

Yan Wu, Email: wuyan2023@fmmu.edu.cn.

Data availability

Data will be made available on request.

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Data Availability Statement

Data will be made available on request.

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