Targeted inhibition of ATP synthase subunit c by pp10-loaded inhalable albumin nanoparticles ameliorates airway inflammation in asthma

Abstract

The persistent opening of the mitochondrial permeability transition pore (mPTP) plays a critical role in bronchial asthma pathogenesis. The ATP synthase c subunit (c subunit) constitutes a core component of mPTP. A novel c subunit inhibitor, 1,3,8-triazaspiro [4.5] decane derivatives (PP10), effectively suppresses pathological mPTP opening without impairing ATP synthesis. Although intraperitoneal PP10 administration mitigates airway inflammation in asthmatic mice, its hydrophobicity hinders inhaled delivery to airway epithelial mitochondria, requiring penetration of the mucus layer, cell membrane, and mitochondrial outer membrane. To overcome this, we developed inhalable human serum albumin-triphenylphosphine-polyethylene glycol-PP10 nanoparticles (HSA-TPP-PEG-PP10 NPs). These NPs demonstrated efficient mucus penetration, high drug loading, mitochondria-targeting capability, and biosafety. They suppressed house dust mite/lipopolysaccharide (HDM/LPS)-induced mPTP opening, inhibited the mitochondrial DNA (mtDNA)-cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, reduced inflammation in human bronchial epithelial (HBE) cells, and alleviated airway inflammation in asthmatic mice upon inhalation.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04494-z.

Introduction

Bronchial asthma is a common chronic airway inflammatory disease that imposes a substantial economic burden [1]. Among them, severe asthma accounts for 3%-10% of asthmatic patients, characterized by poor control and serving as a major cause of disability and mortality in asthma [2, 3]. Airway epithelial cells, as the first line of defense against external harmful stimuli, play a critical role in the development of asthmatic airway inflammation and airway remodeling, making them important target cells for asthma treatment [4–6]. Mitochondrial dysfunction is involved in the pathophysiology of asthma [7], including airway remodeling, eosinophilic inflammation, cell apoptosis, necrosis, and autophagy [8, 9]. Our study has found that interleukin-13 (IL-13) and lipopolysaccharide (LPS) can induce increased reactive oxygen species (ROS) production, abnormal mitochondrial morphology, and excessive secretion of inflammatory factors in human bronchial epithelial (HBE) cells [10]. Recent studies have revealed that mitochondrial dysfunction is a critical factor mediating the abnormal opening of the mitochondrial permeability transition pore (mPTP) [11, 12]. However, it remains unclear whether mitochondrial dysfunction in asthma also involves abnormal opening of the mPTP, and how to regulate such abnormal mPTP opening. These are critical scientific questions that need to be addressed.

The mPTP is a protein complex situated in the mitochondrial inner membrane, functioning as a non-selective channel [13]. Subsequent to abnormal mPTP opening, mitochondrial DNA (mtDNA) is released into the cytoplasm, thereby activating the cyclic GMP-AMP synthase (cGAS)-interferon response cGAMP interactor (STING) signaling pathways and triggering inflammatory responses [14, 15]. Although the mPTP has been studied for more than 50 years, its molecular structure remains unclear [16, 17]. Notably, researchers have attempted to regulate abnormal mPTP opening by specifically intervening with specific molecules in the basic structure of mPTP [18, 19]. Among these, cyclosporine A (CsA), a specific inhibitor of cyclophilin D located in the mitochondrial matrix, is currently the most commonly used drug for intervening in abnormal mPTP opening [20–24]. However, it has not demonstrated significant efficacy in clinical trials for patients with acute myocardial infarction [25, 26].

Recent studies have revealed that adenosine triphosphate synthase (ATP synthase), in addition to its primary role of synthesizing ATP, also serves as a second function by acting as the aberrantly opened mPTP [16, 27]. The c subunit of ATP synthase, located in the F0 region within the mitochondrial inner membrane, forms a ring structure embedded with lipid plugs [16]. Inhibition or silencing of the c subunit has been shown to suppress mPTP abnormal opening, restore mitochondrial membrane potential, and improve mitochondrial function [28, 29].

Morciano et al. first reported 1,3,8-triazaspiro [4.5] decane derivatives (PP10) as a small-molecule inhibitor targeting the c subunit for mitigating reperfusion injury in myocardial infarction, which is characterized by selectively suppressing abnormal mPTP opening without impairing ATP synthesis [30]. Our previous findings demonstrated that intraperitoneal injection of PP10 significantly alleviated airway inflammation in asthmatic mice, suggesting that PP10 may provide a novel strategy for asthma treatment [31]. However, the hydrophobic nature of PP10 makes it difficult for inhaled PP10 to reach the mitochondria of airway epithelial cells, which needs to overcome multiple barriers, such as the airway mucus layer, epithelial cell membrane, and mitochondrial outer membrane [32–38]. Despite this, airway inhalation remains the mainstream administration method for asthma treatment, primarily due to its advantages of rapid, non-invasive direct delivery to the local airway site; low drug dosage with high local pulmonary concentration; minimal systemic adverse reactions; and high patient compliance [39–41]. Thus, the critical challenge to be addressed in this study is how to targeted deliver the c subunit inhibitor PP10 via the airway to the mitochondria of airway epithelial cells.

Nanotechnology has enabled the development of advanced drug delivery systems through the rational design of functional nanomaterials, achieving precise biodistribution, enhanced therapeutic efficacy, and targeted drug release. Among these, human serum albumin (HSA) has emerged as a particularly promising nanocarrier due to its favorable physicochemical properties and exceptional biocompatibility, including excellent biodegradability, prolonged circulation half-life, minimal toxicity, and low immunogenicity [42]. Notably, HSA-based formulations such as paclitaxel-bound albumin have already gained clinical approval for use [43]. Unlike conventional polymers, albumin-based biopolymers provide a biologically relevant platform for the systematic study of engineered macromolecules with high precision. Capitalizing on these advantages, we focus on developing albumin-based nanotherapeutics for targeted drug delivery [44–47]. This study proposes to encapsulate PP10 within the hydrophobic pockets of albumin and design an HSA-based nanoparticle delivery system. Due to the mucus layer on the airway surface-particularly the highly secretory mucus in the airways of severe asthma-which impedes PP10 from reaching the airway epithelial cells at the lesion site [48], this study will modify the surface of HSA loaded with PP10 with polyethylene glycol (PEG), a FDA-approved polymeric material, to assist PP10 in penetrating the airway mucus barrier [49]. Additionally, the functional site of PP10 is located on the inner mitochondrial membrane. Given the highly negative potential of the mitochondrial membrane, this study will functionalize the surface of PP10-loaded HSA with the mitochondrial-targeting moiety triphenylphosphine (TPP), a lipophilic cationic compound, to facilitate PP10 crossing of the mitochondrial outer membrane barrier [50].

In this study, we optimized the physicochemical properties of PP10 and successfully constructed HSA-TPP-PEG-PP10 NPs, which demonstrated superior mucus-penetrating ability, high drug encapsulation efficiency, robust mitochondrial targeting, and excellent biosafety. These NPs effectively inhibited the abnormal opening of mPTP induced by house dust mite (HDM)/LPS, then suppressed the activation of the mtDNA-cGAS-STING pathway, and attenuated the expression of inflammatory factors in HBE cells. Importantly, airway administration of these NPs significantly ameliorated airway inflammation in HDM/LPS-induced asthmatic mouse model.

Results

PP10 alleviates HDM/LPS-induced asthma airway inflammation in mice via intraperitoneal injection, not intranasal delivery

To evaluate the therapeutic efficacy of PP10 in vivo, we established an HDM/LPS-induced mouse model of asthma and administered PP10 via intranasal or intraperitoneal injection (Fig. 1B). PP10 had no effect on mouse growth and development (Fig. 1C). Compared to the Saline group, the HDM/LPS model exhibited increased inflammatory cell infiltration and mucin secretion (Fig. 1D-G), confirming successful model establishment. Intraperitoneal PP10 reduced inflammatory cell infiltration and inhibited mucin secretion, while intranasal administration slightly decreased mucin secretion. BALF analysis revealed elevated the counts of inflammatory cells (excluding macrophages) (Fig. 1H) and cytokines (IL-6, CXCL1, IL-25, IL-33) in the HDM/LPS group (Fig. 1I-L), which were alleviated by intraperitoneal PP10 but not by intranasal administration.

Fig. 1.

(A) Challenges associated with intranasal administration of PP10. (B) Schematic illustration of the generation process of the mouse model. (C) Mice were weighed on the day of modelling (day 0), on the day of drug administration (day 1), and on the day of the end of drug administration (day 4) to assess the effect of PP10 (2 µg/g, i.n. or i.p.) on growth and development (n = 4). (D-E) HE staining was performed on lung sections in the indicated groups. Scale bar = 20 μm. Quantification of inflammatory infiltration was made using inflammation scores (n = 4). (F-G) PAS staining was performed on lung sections in the indicated groups. Scale bar = 20 μm. Quantification of hyperplasia of goblet cells was made using PAS scores (n = 4). (H) Statistical analysis of the neutrophils, eosinophils, lymphocytes, macrophages and total inflammatory cells in BALF (n = 4). (I-L) ELISA-based measurement and statistical analysis of CXCL1(I), IL-6 (J), IL-25 (K) and IL-33 (L) in BALF (n = 4). Data are expressed as mean ± SEM, one-way ANOVA. Compared with Saline group: ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001. Compared with HDM/LPS group: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001. ns: no significance. PP10, 1,3,8-triazaspiro [4.5] decane derivatives; i.n., intranasal; i.p., intraperitoneal

Furthermore, we investigated PP10’s mechanism in treating airway inflammation by measuring lipid peroxidation, mtDNA, and cGAS-STING pathway proteins in mouse lung tissues. Compared to the Saline group, the HDM/LPS model showed increased lipid peroxidation (Fig. S1A, B, D), mtDNA (Fig. S1C), and cGAS/STING expression (Fig. S1E-H). Intraperitoneal PP10 improved these pathological changes, while intranasal administration did not. These findings demonstrate that PP10 indeed inhibits the activation of the HDM/LPS-induced mtDNA-cGAS-STING signaling pathway, thereby holds potential for ameliorating airway inflammation in mice. However, the hydrophobic nature of PP10 combined with airway mucus barriers restricted its intranasal administration, mandating the development of effective delivery systems.

Synthesis and characterization of HSA-TPP-PEG-PP10 NPs

The synthesis of HSA-TPP-PEG-PP10 was illustrated in Fig. 2A. HSA was initially conjugated with TPP via amide bond formation to form HSA-TPP. Subsequently, PEG modification was conducted to yield HSA-TPP-PEG, followed by the encapsulation of PP10 into the hydrophobic domain of HSA, resulting in HSA-TPP-PEG-PP10.

Fig. 2.

(A) The nanoparticles modified with TPP and PEG possess good water solubility, strong mucus-penetrating ability, and high mitochondrial targeting property. (B) MALDI-TOF characterization of HSA-TPP. (C-D) SDS-PAGE analysis of proteins (HSA, HSA-TPP, HSA-TPP-PEG). (E) Size distribution of HSA, HSA-TPP NPs, HSA-TPP-PEG NPs, and HSA-TPP-PEG-PP10 NPs. (F) TEM images of HSA, HSA-TPP NPs, HSA-TPP-PEG NPs, and HSA-TPP-PEG-PP10 NPs. Scale bar = 200 nm. (G) Zeta potential of HSA, HSA-TPP NPs, HSA-TPP-PEG NPs, and HSA-TPP-PEG-PP10 NPs. (H) Mucosal penetration of different drugs was assessed (n = 3). (I) The absorbance of the gelatin layer (bottom layer) at 595 nm was measured using a microplate reader (n = 3). Data are expressed as mean ± SEM, one-way ANOVA. ^**P < 0.01. HSA, human serum albumin; TPP, triphenylphosphine; PEG, polyethylene glycol; PP10, 1,3,8-triazaspiro [4.5] decane derivatives; NPs, nanoparticles

MALDI-TOF analysis revealed molecular weights of 66.32 KDa for HSA and 78.14 KDa for HSA-TPP (Fig. 2B), confirming ~ 29 TPP molecules conjugated per HSA molecule. SDS-PAGE demonstrated gradual molecular weight increases for HSA-TPP and HSA-TPP-PEG (Fig. 2C, D), validating TPP and PEG modifications. Dynamic light scattering (DLS) analysis indicates that the particle size of PEG-modified nanoparticles (HSA-TPP-PEG) decreases, attributed to PEG reducing the aggregation of HSA-TPP (Fig. 2E). TEM showed that HSA-TPP NPs exhibited increased particle sizes and aggregation, while PEG-modified NPs were spherical and uniformly distributed (Fig. 2F). Additionally, HSA-TPP-PEG-PP10 NPs exhibited a larger particle size and higher zeta potential than HSA-TPP-PEG NPs (Fig. 2E, G), and this difference is presumably attributed to the loading of PP10. HPLC analysis revealed that PP10 achieved an average drug encapsulation efficiency of approximately 89.26%. To evaluate the mucus penetration of nanoparticles, we constructed an in vitro artificial mucus model using the method described by Wu et al. [51]. As shown in Fig. 2H, I, PEG modification significantly enhanced the mucus penetration ability of nanoparticles. This is attributed to the increased water solubility of PEG-modified nanoparticles, reduced aggregation, and smaller particle size.

Our findings demonstrated that HSA-TPP-PEG-PP10 NPs exhibited minimal cytotoxicity in HBE cells, as illustrated in Fig. 3B. To assess the cellular uptake efficiency of HBE cells for these NPs (Fig. 3A), Cy5-labeled HSA-TPP-PEG-PP10-Cy5 was synthesized. The results indicated that the uptake of NPs by HBE cells was both concentration- and time-dependent, with a significant enhancement observed at a concentration of 50 µg/mL after a 24 h incubation period (Fig. 3D, E). Additionally, laser confocal microscopy analysis revealed that HSA-TPP-PEG-PP10-Cy5 NPs displayed superior mitochondrial targeting capability compared to unmodified TPP NPs (HSA-PP10-Cy5 and HSA-PEG-PP10-Cy5), as shown in Fig. 3C.

Fig. 3.

(A) Schematic diagram of nanoparticle uptake by human bronchial epithelial (HBE) cells. (B) Cell viability was assessed using the CCK-8 test (n = 3). (C) Representative fluorescence microscopy images demonstrating the mitochondrial colocalization of HSA-TPP-PEG-PP10-Cy5 NPs in HBE cells (n = 3). Scale bar = 10 μm. (D) Representative fluorescence microscopy images of HBE cells incubated with different concentrations of HSA-TPP-PEG-PP10-Cy5 NPs for 24 h (n = 3). Scale bar = 50 μm. (E) Representative fluorescence microscopy images of HBE cells incubated with HSA-TPP-PEG-PP10-Cy5 NPs (50 µg/mL) for different time points (n = 3). Scale bar = 50 μm. Data are expressed as mean ± SEM. ns: no significance

HSA-TPP-PEG-PP10 NPs improve mitochondrial function in HBE cells stimulated by HDM/LPS

Whether HSA-TPP-PEG-PP10 NPs can inhibit the abnormal opening of mPTP induced by HDM/LPS in HBE cells and ameliorate mitochondrial dysfunction remains unclear. To this end, HBE cells were pretreated with 50 µg/mL HSA-TPP-PEG-PP10 NPs, followed by the induction of an inflammatory cell model using HDM/LPS. The experimental findings revealed that pretreatment with HSA-TPP-PEG-PP10 NPs effectively prevented the abnormal opening of mPTP triggered by HDM/LPS (Fig. 4A, B) and significantly alleviated mitochondrial dysfunction. This was evidenced by the suppression of HDM/LPS-induced mtROS overproduction (Fig. 4C, D), reduction in mtDNA release (Fig. 4E, F), restoration of mitochondrial membrane potential (Fig. 4G, H), improvement in mitochondrial morphology abnormalities (Fig. 4I, J), and a decrease in intracellular ATP levels (Fig. 4K). Collectively, these findings demonstrate that HSA-TPP-PEG-PP10 NPs are capable of inhibiting the HDM/LPS-induced abnormal opening of mPTP and ameliorating mitochondrial dysfunction in HBE cells.

Fig. 4.

HBE cells were pretreated with PP10 (50 µg/mL) or HSA-TPP-PEG-PP10 NPs (50 µg/mL) for 1 h followed by exposure to HDM (50 µg/ml) and LPS (40 ng/ml) for 24 h. (A) Images showed green when the mPTP was closed. Scale bar = 50 μm. (B) Quantitative analysis of the fluorescence signal of mPTP (n = 3). (C) Images showed the ROS in red. Scale bar = 100 μm. (D) Quantitative analysis of MitoSOX fluorescence signal (n = 3). (E) Immunofluorescence images of dsDNA liberation in HBE cells. Scale bar = 20 μm. (F) Percentage of dsDNA-positive cells (n = 3). (G) The JC1-aggregates fluoresce red and indicate high ΔΨm, while the monomers fluoresce green and indicate low ΔΨm. Scale bar = 50 μm. (H) Quantitative analysis of the fluorescence signal of the J-aggregates (red)/the monomers (green) (n = 3). (I) The mitochondrial morphology in HBE cells of the indicated groups was examined via TEM. Scale bar = 400 nm. Arrowheads in black and red indicate reduced and disappearing cristae and vacuolar degeneration and swelling of mitochondria, respectively. (J) Quantitative analysis of damaged mitochondria (n = 3). (K) Intracellular ATP levels were measured using an ATP assay kit (n = 4). Data are expressed as mean ± SEM, one-way ANOVA. Compared with Control group: ^***P < 0.001, ^****P < 0.0001. Compared with HDM/LPS group: ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001, ns: no significance. mPTP, mitochondrial permeability transition pore. ΔΨm, mitochondrial membrane potential

HSA-TPP-PEG-PP10 NPs inhibit HDM/LPS-induced activation of the mtDNA-cGAS-STING pathway, reducing inflammatory factor expression in HBE cells

To investigate whether HSA-TPP-PEG-PP10 NPs can inhibit the activation of the mtDNA-cGAS-STING pathway induced by HDM/LPS in HBE cells, we pretreated HBE cells with these NPs. The experimental results demonstrated that HSA-TPP-PEG-PP10 NPs significantly suppressed the release of mtDNA into the cytoplasm (Fig. 4E, F), downregulated the expression of cGAS and STING proteins (Fig. 5A-D), and reduced the mRNA expression levels of inflammatory factors, including IL-1β, IL-6, IL-8, IL-33, and TSLP (Fig. 5E-I). These findings indicate that HSA-TPP-PEG-PP10 NPs effectively inhibit the HDM/LPS-induced activation of the mtDNA-cGAS-STING pathway and attenuate the expression of inflammatory factors in HBE cells.

Fig. 5.

HBE cells were pretreated with PP10 (50 µg/mL) or HSA-TPP-PEG-PP10 NPs (50 µg/mL) for 1 h followed by exposure to HDM (50 µg/ml) and LPS (40 ng/ml) for 24 h. (A) The expression of cGAS was analyzed by western blotting. (B) Quantitative analysis of Fig. 5A (n = 3). (C) The expression of STING was analyzed by western blotting. (D) Quantitative analysis of Fig. 5C (n = 3). (E-I) The mRNA levels of IL-1β, IL-6, IL-8, IL-33 and TSLP were determined by quantitative real-time PCR in HBE cells (n = 4). Data are expressed as mean ± SEM, one-way ANOVA. Compared with Control group: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. Compared with HDM/LPS group: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001

Intratracheally administered HSA-TPP-PEG-PP10 NPs exhibit uniform distribution in mouse lung tissues

To assess the lung distribution of HSA-TPP-PEG-PP10 NPs in mice, Cy5 red fluorescent-labeled NPs were synthesized and administered via intranasal inhalation. Small animal in vivo imaging demonstrated that HSA-TPP-PEG-PP10-Cy5 NPs were uniformly distributed in the lung tissue of mice both immediately after administration (0 h) and 24 h post-administration (Fig. 6A). Additionally, 24 h after administration, the lung, heart, liver, spleen, kidney, and brain tissues were harvested and analyzed using a small animal in vivo imaging system. Results showed that the nanoparticles were highly retained in lung tissue, and no obvious accumulation was detected in the heart, liver, spleen, kidney, or brain. However, a small amount of nanoparticle accumulation was observed in the bladder of one mouse, suggesting that the nanoparticles may be mainly metabolized and excreted via the urinary system (Fig. 6B). In vitro experiments confirmed that HSA-TPP-PEG-PP10-Cy5 NPs were efficiently internalized by HBE cells and displayed strong mitochondrial targeting. However, whether these NPs specifically target the mitochondria of airway epithelial cells in vivo following inhalation remained unclear. To address this, lung tissue sections were stained 24 h after intranasal administration. Compared with TPP-unmodified nanoparticles (HSA-PP10-Cy5, HSA-PEG-PP10-Cy5), HSA-TPP-PEG-PP10-Cy5 NPs exhibited significantly enhanced mitochondrial targeting efficiency in airway epithelial cells (Fig. 6C).

Fig. 6.

(A) In vivo distribution of HSA-TPP-PEG-PP10-Cy5 NPs (2 µg/g, i.n.) in murine pulmonary tissues was monitored using small animal imaging system at 0 h and 24 h post-administration (n = 3). (B) The distribution of HSA-TPP-PEG-PP10-Cy5 NPs (2 µg/g, i.n.) was assessed in major murine organ systems, including lung, heart, liver, spleen, kidney, brain, and bladder tissues, utilizing small animal in vivo imaging system at 24 h post-administration (n = 3). (C) Representative fluorescence microscopy images demonstrating the mitochondrial colocalization of HSA-TPP-PEG-PP10-Cy5 NPs in the lung tissues of mice (n = 3). Scale bar = 50 μm. (D) Schematic illustration of the generation process of the mouse model. (E) Mice were weighed on the day of modelling (day 0), on the day of drug administration (day 1), and on the day of the end of drug administration (day 4) to assess the effect of PP10 or HSA-TPP-PEG-PP10 NPs (2 µg/g, i.n.) on growth and development (n = 5). Data are expressed as mean ± SEM. ns: no significance

Intratracheal administration of HSA-TPP-PEG-PP10 NPs alleviates HDM/LPS-Induced airway inflammation in a mouse model of asthma

To address the limited efficacy of PP10 in alleviating HDM/LPS-induced airway inflammation in mice, we synthesized HSA-TPP-PEG-PP10 NPs and evaluated their therapeutic potential in an HDM/LPS-induced asthma model (Fig. 6D). Nasal administration of either HSA-TPP-PEG-PP10 NPs or PP10 failed to elicit any discernible effects on the growth and development of mice (Fig. 6E). Compared with mice in the Saline group, those in the HDM + LPS-induced model group exhibited massive inflammatory cell infiltration around the trachea (Fig. 7A, B), a significant increase in goblet cell mucus secretion (Fig. 7C, D), elevated inflammatory cell infiltration in BALF (Fig. 7E), and increased secretion of pro-inflammatory cytokines (Fig. 7F-H). These findings confirm the successful establishment of the mouse asthma model. In contrast, intranasal inhalation of HSA-TPP-PEG-PP10 NPs significantly ameliorated airway inflammation, reduced mucus secretion, decreased inflammatory cell infiltration in BALF, and downregulated pro-inflammatory cytokine expression. However, intranasal inhalation of PP10 alone only partially inhibited pro-inflammatory cytokine secretion (IL-25 and IL-33). Furthermore, HSA-TPP-PEG-PP10 NPs reduced HDM/LPS-induced ROS generation (Fig. 8A, B) and MDA levels (Fig. 8D) in lung tissues. In vivo, these NPs inhibited mtDNA release (Fig. 8C) and downregulated cGAS and STING protein expression (Fig. 8E-H), confirming their ability to inhibit the mtDNA-cGAS-STING pathway and mitigate inflammation. Safety assessments revealed no significant toxicity in major organs (Fig. 8I) or alterations in serum Alanine Aminotransferase (ALT) and Creatinine (Cr) levels (Fig. 8J, K), demonstrating their favorable safety profile. These findings highlight the therapeutic potential of HSA-TPP-PEG-PP10 NPs in treating HDM/LPS-induced airway inflammation.

Fig. 7.

(A) HE staining was performed on lung sections in the indicated groups. Scale bar = 50 μm. (B) Quantification of inflammatory infiltration was made using inflammation scores (n = 5). (C) PAS staining was performed on lung sections in the indicated groups. Scale bar = 20 μm. (D) Quantification of hyperplasia of goblet cells was made using PAS scores (n = 5). (E) Statistical analysis of the neutrophils, eosinophils, lymphocytes, macrophages and total inflammatory cells in BALF (n = 3). (F-H) ELISA-based measurement and statistical analysis of IL-6, IL-25, and IL-33 in BALF (n = 5). Data are expressed as mean ± SEM, one-way ANOVA. Compared with Saline group: ^**P < 0.01, ^****P < 0.0001. Compared with HDM/LPS group: ^##P < 0.01, ^####P < 0.0001. ns: no significance

Fig. 8.

(A) Immunofluorescence images of ROS expression with DHE in lung tissues from HDM/LPS-induced mouse model of asthma treated with or without PP10 and HSA-TPP-PEG-PP10 NPs. Scale bar = 50 μm. (B) Quantitative analysis of the fluorescence signal of DHE (n = 3). (C) Immunofluorescence images of dsDNA liberation in the lung tissues of mice (n = 3). Scale bar = 20 μm. (D) Mouse lung tissues were homogenized to measure the levels of MDA (n = 4). (E) The expression of cGAS was analyzed by western blotting. (F) The expression of STING was analyzed by western blotting. (G) Quantitative analysis of Fig. 8E (n = 5). (H) Quantitative analysis of Fig. 8F (n = 5). (I) HE staining of the main organs of mice treated with HSA-TPP-PEG-PP10 NPs (n = 3). Scale bar = 20 μm. (J-K) Levels of alanine transaminase (ALT), and creatinine (CR) in mouse serum (n = 5). Data are expressed as mean ± SEM, one-way ANOVA. Compared with Saline group: ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001. Compared with HDM/LPS group: ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001. ns: no significance

Discussion

Mitochondria not only regulate cell death but also initiate inflammatory responses. Mitochondrial dysfunction triggers abnormal opening of the mPTP and release of mtDNA into the cytoplasm, activating the cGAS-STING pathway and inducing inflammation [52]. While the molecular composition of mPTP remains debated, recent studies demonstrate that the c subunit mediates abnormal mPTP opening during mitochondrial dysfunction [16, 19, 53]. PP10, a novel c subunit inhibitor, selectively inhibits mPTP opening without impairing ATP synthesis.

In the HDM/LPS-induced asthmatic mouse model, intraperitoneal injection of PP10 effectively alleviated airway inflammation, reduced mucus secretion, and suppressed inflammatory factor release in asthmatic mouse model. In contrast, intranasal administration of PP10 failed to significantly ameliorate these pathological changes. This discrepancy may be attributed to two key factors: (1) The c subunit, situated on the inner mitochondrial membrane, necessitates PP10 to translocate to the mitochondria of airway epithelial cells for effective inhibition. Nevertheless, intranasal administration encounters multiple barriers, such as the airway mucus barrier, epithelial cell membrane barrier, and mitochondrial outer membrane barrier. Collectively, these barriers result in suboptimal drug concentration and diminished targeting efficiency. (2) Due to the poor water solubility of PP10, solubilizing agents (e.g., DMSO, PEG300, and Tween80) are required in animal studies. However, the high intrinsic viscosity of PEG300 and Tween80 increases the overall viscosity of the PP10 solution, which in turn reduces its absorption efficiency during intranasal administration.

Our study demonstrated that PP10 alleviates HDM/LPS-induced asthma airway inflammation in mice via intraperitoneal injection, not intranasal delivery. However, inhaled medications are the mainstay of treatment of asthma [54], as intraperitoneal injection is unsuitable due to its low bioavailability and associated risks, including infection, bleeding, and necrosis [55, 56]. To address this issue, we innovatively optimized the physicochemical properties of PP10 and successfully developed an inhalable HSA-TPP-PEG-PP10 NPs. These NPs exhibited optimal physicochemical characteristics, including efficient mucus-permeating properties, high drug loading capacity, and excellent biosafety.

To further investigate the uptake capacity of HBE cells for the NPs, we synthesized Cy5-labeled HSA-TPP-PEG-PP10 NPs. The results showed that the uptake of NPs by HBE cells was concentration- and time-dependent, indicating that these NPs enable PP10 to cross the airway epithelial cell membrane barrier. Furthermore, in comparison to TPP-unmodified nanoparticles (HSA-PP10-Cy5, HSA-PEG-PP10-Cy5), HSA-TPP-PEG-PP10 NPs exhibited significantly improved mitochondrial targeting capacity. This result indicates that TPP modification can enhance the mitochondrial targeting ability of nanoparticles. To elucidate the in vitro anti-inflammatory mechanism of HSA-TPP-PEG-PP10 NPs, we pre-treated HDM/LPS-induced HBE cells with these NPs. Our results confirm that in HBE cells, HDM + LPS can induce mitochondrial dysfunction, promote the abnormal opening of mPTP, trigger the release of mtDNA into the cytoplasm, thereby activate cGAS-STING pathway, and ultimately exacerbate inflammatory factor expression. However, pretreatment of HDM + LPS-induced HBE cells with HSA-TPP-PEG-PP10 NPs reversed the aforementioned pathological changes. This result suggests that these NPs can deliver PP10 to the inner mitochondrial membrane for functional exertion, holding promise as a novel strategy for asthma treatment.

Small animal in vivo imaging further demonstrated that inhaled HSA-TPP-PEG-PP10-Cy5 NPs exhibited uniform lung distribution, highlighting their superior pulmonary dispersibility. Consistent with in vitro findings, these NPs also demonstrated robust mitochondrial targeting in airway epithelial cells in vivo. To evaluate the therapeutic efficacy of HSA-TPP-PEG-PP10 NPs in mitigating HDM/LPS-induced airway inflammation, we employed an inhalational delivery approach in an asthmatic mouse model. In comparison to free PP10, these NPs significantly attenuated peribronchial inflammatory cell infiltration, mucus hypersecretion, and levels of inflammatory cytokines in BALF.

To clarify the in vivo anti-inflammatory mechanism of these NPs, we quantified lipid oxidation levels, mtDNA levels, and the expression of key proteins in the cGAS-STING pathway within mouse lung tissues. The results demonstrated that inhalation of these NPs significantly attenuated HDM/LPS-induced elevations in lipid oxidation levels, suppressed mtDNA release, and downregulated the expression of cGAS and STING proteins. From an in vivo experimental perspective, this result further confirms that these NPs can deliver PP10 to the inner mitochondrial membrane to exert its biological function. Collectively, these findings indicate that HSA-TPP-PEG-PP10 NPs mitigate airway inflammation in asthmatic mice by inhibiting activation of the mtDNA-cGAS-STING signaling pathway, holding promise as a novel strategy for asthma treatment.

Our previous study demonstrated that intraperitoneal injection of PP10 suppresses the aberrant opening of the mPTP induced by HDM + LPS, thereby attenuating airway inflammation in a murine asthma model. However, an inhalable delivery strategy targeting this mechanism remains unavailable. To address this gap, we developed an inhalable albumin-based nanocarrier system loaded with PP10. This system exhibits exceptional mucus-penetrating properties, high drug encapsulation efficiency, potent mitochondrial targeting, and favorable biosafety. Importantly, inhalation of HSA-TPP-PEG-PP10 nanoparticles ameliorates airway inflammation in asthmatic mice by inhibiting the mtDNA-cGAS-STING pathway activation, which may provide a novel therapeutic approach for asthma treatment.

Methods

Ethical statement

All experimental protocols related to animal experiments were approved by the Ethics Committee of Tongji Hospital affiliated with Tongji Medical College of Huazhong University of Science and Technology (TJH-202310017).

Materials

House dust mite (HDM, D.pteronyssinus) was purchased from Greer Laboratory (USA). Lipopolysaccharide (LPS), Dimethyl sulfoxide (DMSO), Human serum albumin (HSA) and Fetal bovine serum (FBS) was purchased from Sigma-Aldrich (USA). 1,3,8-triazaspiro [4.5] decane derivatives (PP10) Tween 80, and PEG300 were purchased from Med Chem Express Co., Ltd. (China). Lipid Peroxidation MDA assay kit, ATP assay kit, and Dihydroethidium (DHE) assay kit were purchased from Beyotime (China). MitoTracker Red CMXRos, DAPI, Hoechst 33,342, RIPA buffer, β-actin (1:2000), HRP-conjugated goat anti-rabbit IgG (1:5000), and HRP-conjugated goat anti-mouse IgG (1:5000) were purchased from Servicebio (China). double-stranded DNA (dsDNA, 1:200) was purchased from Cayman (USA). N, N-Dimethylformamide (DMF) and N-(3-dimethylaminopropyl) N-ethylcarbodiimide hydrochloride (EDC-HCl) were purchased from Energy-chemical (China). N-Hydroxysuccinimide (NHS) was purchased from Solarbio Life Sciences (China). (3-Carboxypropyl) triphenylphosphonium bromide (TPP) was purchased from Aladdin. NH2-PEG-SH was purchased from Ponsure (China). Sulfo-Cy5-SE was purchased from Macklin (China). mPTP Fluorescence Assay Kit and IFKine™ Green Donkey Anti-Mouse IgG (1:200) were purchased from Abbkine (China). MitoSOX and JC1 staining were purchased from Yeasen Biotechnology Co., Ltd (China). Multisciences (Hangzhou, China) provided ELISA kits. Bacinchoninic acid (BCA) assay kit was purchased from Boster Biological Technology (China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) kit was purchased from Sangon Biotech. c subunit (1:2000) was purchased from Abcam (USA). cGAS (1:1000) and STING (1:2000) were purchased from Proteintech (China). Trizol reagent, Takara Prime Script RT Master Mix, and Takara TB Green Premix Ex Taq were purchased from Takara (Japan). ATCC (Manassas, VA, USA) supplied the human bronchial epithelial cells (16HBE). Female BALB/c mice were obtained from Shulaibao Biotechnology Co., Ltd (China).

Animal experiments

Eight-week-old female BALB/c mice (20 g) were housed in an SPF facility with controlled conditions (22 °C ± 2 °C, 55% ± 5% humidity, 12-hour light-dark cycle). After one week of acclimation, 48 mice were randomly divided into six groups of eight. Experimental groups included Saline, PP10 (intranasal or intraperitoneal), HDM/LPS, and HDM/LPS+PP10 (intranasal or intraperitoneal). Some adjustments were made to the asthmatic mouse model induced previously described approach [57] (Fig. 1B). Mice were intranasally sensitized with 50 µg HDM extract daily on days 1–5 and challenged with 25 µg HDM extract daily on days 12–16. LPS (1 µg) was intranasally administered on days 19–22. PP10 (2 µg/g) or vehicle (DMSO+PEG300 + Tween80+Saline) was administered via intranasal or intraperitoneal on days 19–22. Mice were euthanized on day 23 for further analysis.

Collection and cell count of bronchoalveolar lavage fluid (BALF)

BALF was collected using 1 mL syringe, flushing the lungs three times with 0.7 mL PBS. The samples were centrifuged at 500 g for 10 min at 4 °C to isolate the supernatant for cytokine analysis. A cell suspension was prepared with 150 µL RPMI medium, and cytospin slides were calibrated to 10,000 cells per 80 µL. The cell suspensions were centrifuged at 800 g for 5 min in a cytospin (Hettich Universal 320R, Germany) and stained with Liu’s solution, and blindly assessed for differential counts, with counted 200 cells slide per at 400× magnification.

Hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) staining

Lung tissues were sectioned at 4 μm thickness and stained with H&E and PAS. Peri-bronchial inflammation was graded 0–4 based on inflammatory cell layers [10] and PAS-positive cells were scored 0–4 according to their prevalence in airways [10]. Two independent, blinded examiners assessed the samples.

Immunofluorescence staining

Cells and lung sections were stained with MitoTracker Red CMXRos, fixed, permeabilized, and blocked. After antigen retrieval, they were incubated with dsDNA (1:200) overnight at 4 °C, followed by secondary antibodies and DAPI staining. Images were captured using a fluorescent microscope (Olympus BX53, Japan) and analyzed with ImageJ.

Dihydroethidium (DHE) staining

DHE staining assessed ROS release in mouse lungs, following established protocols [58]. A microscope fluorescent (Olympus BX53, Japan) captured images, and ImageJ quantified fluorescence intensity.

Determination of Malondialdehyde (MDA)

MDA in levels mouse lung tissues were determined following homogenization in an ice bath, centrifugation at 10,000 g for 10 min, BCA-based protein quantification, and using measurement a microplate reader at 532 and 562 nm.

Western blot analysis

Western blot was performed to evaluate cGAS and STING expression. Proteins extracted from cells or lung tissues using RIPA buffer were quantified with BCA assays. Samples were separated by 10%-12% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes, and blocked with 5 non%-fat milk in TBST. Membranes were incubated with primary antibodies (c subunit 1:2000, cGAS 1:1000, STING 1:2000, β-actin 1:1000) overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000) at room temperature for 2 h. Proteins using were detected enhanced chemiluminescence and, band densities were analyzed using Image J.

ELISA (Enzyme-linked immunosorbent assay)

ELISA kits were used to measure BALF CXCL1, IL-6, IL-25, and IL-33 according to product recommendations.

Synthesis of HSA-TPP-PEG-PP10 NPs

TPP was labeled to HSA for mitochondria-targeting ability according to the previous literature [59]. Briefly, TPP (2.58 mg), NHS (1 mg), and EDC·HCl (1.30 mg) were dissolved in 0.1 mL DMF and stirred overnight at room temperature under nitrogen. Subsequently, 4 mg HSA (pre-dissolved in PBS, pH 7.80, 1.0 mg/mL) was introduced to the reaction mixture and stirred for 12 h at room temperature. The product was dialyzed at 4 °C (12 h) to eliminate unreacted small molecules. Next, 2.40 mg NHS and 3.20 mg EDC·HCl dissolved in 40 µL DMF were added to 2 mL of the dialyzed HSA-TPP solution, followed by 1 h stirring. Then, 6.4 mg NH2-PEG-SH dissolved in 80 µL PBS (pH 7.80) was incorporated, and the mixture was stirred for 12 h before another 12 h dialysis at 4 °C. PP10 conjugation was achieved by adding 0.5 mL PP10 solution (2 mg/mL) to the mixture, followed by 12 h stirring. Finally, the solution underwent two ultrafiltration cycles (MWCO 30 kDa, 2000 g, 5 min each) to remove residual impurities, followed by freeze-drying (18 h). The final NPs were stored at 4 °C.

Sulfo-Cy5-SE was conjugated to HSA-PP10, HSA-PEG-PP10, and HSA-TPP-PEG-PP10 for fluorescent observation. For the preparation of HSA-PP10-Cy5 NPs, 125 µL PP10 (pre-dissolved in methanol, 2 mg/mL) was added to 0.5 mL HSA (1 mg/mL), stirred for 12 h, ultrafiltered twice (2000 g, 5 min), then mixed with 30 µL Sulfo-Cy5-SE (pre-dissolved in DMSO, 10 mg/mL), stirred in the dark for 12 h, and dialyzed at 4 °C for 12 h. For the preparation of HSA-PEG-PP10-Cy5 NPs, 0.60 mg NHS and 0.80 mg EDC·HCl in 10 µL DMF were added to 0.5 mL HSA, stirred for 1 h, mixed with 20 µL NH2-PEG-SH (80 mg/mL), stirred for 12 h, dialyzed, then combined with 125 µL PP10 (2 mg/mL), stirred for 12 h, ultrafiltered twice (2000 g, 5 min), and finally mixed with 30 µL Sulfo-Cy5-SE (10 mg/mL), stirred in the dark for 12 h, and dialyzed. For HSA-TPP-PEG-PP10-Cy5 NPs, HSA-TPP-PEG-PP10 was synthesized according to the aforementioned procedure, mixed with 30 µL Cy5 (10 mg/mL), stirred in the dark for 12 h, and dialyzed at 4 °C for 12 h. The final NPs were stored at 4 °C.

Characterization of HSA-TPP-PEG-PP10 NPs

The molecular weights of both HSA and HSA-TPP were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, 5800 MALDI TOF, USA). The separation of HSA, HSA-TPP, and HSA-TPP-PEG was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The morphology of the NPs was observed using transmission electron microscopy (TEM, Hitachi 7700, Japan). Dynamic light scattering (DLS; Malvern NanoZS, UK) was used to measure the NPs’ zeta potential and particle size.

Mucus penetration experiment in vitro

A 10% (w/v) gelatin solution was prepared by dissolving gelatin in deionized water using an oven. After adding 1 mL to each vial, the solution was allowed to set at room temperature. Artificial mucus was prepared containing salmon sperm DNA (500 mg), porcine mucin (250 mg), diethylene triamine pentaacetic acid (DTPA, 295 mg), NaCl (250 mg), KCl (110 mg), egg yolk emulsion (250 µL), RPMI-1640 (1 mL), and water (50 mL). The artificial mucus was added to the surface of the gelatine in each vial. Protein solutions (HSA-PP10, HSA-PEG-PP10, HSA-TPP-PEG-PP10) were mixed with Coomassie blue and shaken for 2 h. Stained samples (100 µL) were added to mucus droplets and incubated at 37 °C. Penetration was photographed at 0, 1, and 2 h. After cooling, the mucus layer was removed, and the gelatin was washed, melted (37 °C, 30 min), and mixed. Absorbance at 595 nm was measured using a microplate reader.

PP10 entrapment efficiency of HSA-TPP-PEG-PP10 NPs

The high-performance liquid chromatography (HPLC, LC100, China) system was employed to sequentially measure the peak areas corresponding to gradient concentrations of PP10 standards. A standard calibration curve was subsequently established by plotting PP10 concentrations against their respective peak areas for quantitative analysis of encapsulated PP10. The HSA-TPP-PEG-PP10 complex was prepared using the aforementioned synthesis protocol, wherein ultrafiltration (2000 g, 5 min) was utilized to isolate unbound PP10. Collect the filtrate for HPLC analysis to determine the concentration of unencapsulated PP10. The entrapment efficiency (EE) was calculated according to the following equation: EE (%) = (Mass of encapsulated PP10 / Total mass of PP10 initially added) ×100%.

In vitro cytotoxicity and cell uptake of HSA-TPP-PEG-PP10 NPs

HBE cells were seeded in 96-well plates and cultured in RPMI media supplemented with 10% FBS at 37 °C and 5% CO₂ for 12 h to examine the cytotoxicity of HSA-TPP-PEG-PP10 NPs. HSA-TPP-PEG-PP10 NPs were applied to the cells at concentrations of 5 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL, 100 µg/mL, and 200 µg/mL for 24 h, or 50 µg/mL for 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, and 72 h. The CCK-8 kit was then used to measure cell viability.

HBE cells were seeded in 24-well plates and incubated with HSA-TPP-PEG-PP10-Cy5 NPs at concentrations of 1 µg/mL, 5 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL, and 100 µg/mL for 24 h to observe the uptake of HSA-TPP-PEG-PP10-Cy5 NPs at different concentrations. The cells were treated with 50 µg/mL HSA-TPP-PEG-PP10-Cy5 NPs for 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h to observe the uptake of HSA-TPP-PEG-PP10-Cy5 NPs at various time points. The cells were washed with PBS, stained their Mitochondria with Mito-Tracker Green and their nuclei with Hoechst 33,342. After thoroughly cleaning with PBS, the cells were photographed using a fluorescence inverted microscope (CKX53, Olympus, Japan).

Mitochondria-targeting efficiency of HSA-TPP-PEG-PP10 NPs

HBE cells were seeded in an 8-well coverglass chamber for 48 h. Then 200 µL of fresh medium with or without NPs (HSA-PP10-Cy5, HSA- PEG-PP10-Cy5, HSA-TPP-PEG-PP10-Cy5, 50 µg/mL) was replaced in each well. After 24 h incubation, the mixture was removed. Then, the cells were washed with PBS, stained their Mitochondria with Mito-Tracker Green and their nuclei with Hoechst 33,342. After thoroughly cleaning with PBS, the cells were photographed using a ZEISS LSM 900 with Airyscan confocal laser scanning microscope (Germany).

Mitochondrial permeability transition pore (mPTP) opening assay

The opening of mPTP in HBE cells from each group was assessed using a mPTP Fluorescence Assay Kit following the manufacturer’s instructions. Fluorescence intensity was quantified using ImageJ after images were rapidly acquired with a fluorescence inverted microscope (CKX53, Olympus, Japan).

ATP measurements

Following manufacturer directions, an ATP assay kit measured intracellular ATP. After that, a luminometer (Agilent, USA) determined relative light unit values.

Measurement of the mitochondrial membrane potential

As directed by the manufacturer, JC1 staining was used to detect the mitochondrial membrane potential (ΔΨm). With the use of ImageJ software, the density of the monomers (green) and J-aggregates (red) was measured after image capture using a fluorescence inverted microscope (CKX53, Olympus, Japan).

Mitochondrial reactive oxygen species measurement

MitoSOX staining was performed in HBE cells according to the manufacturer’s instructions. Fluorescence intensity was measured using ImageJ software from images captured using a fluorescence inverted microscope (CKX53, Olympus, Japan) in a timely manner.

Transmission electron microscopy (TEM)

Cells were fixed with electron microscopy fixative at room temperature for 2 h, followed by refrigeration at 4 °C until use. Following dehydration, the fixed cells were embedded in epoxy resin and incubated at 60 °C for 48 h. Subsequently, ultrathin sections were stained with uranyl acetate and lead citrate. Images were acquired using a Hitachi TEM-7700 electron microscope.

Quantitative real-time Polymerase chain reaction (PCR)

Total RNA was extracted using Trizol Reagent and reverse-transcribed into cDNA using the Takara Prime Script RT Master Mix. PCR was performed using Takara TB Green Premix Ex Taq on a CFX Connect (Bio-Rad) to amplify target genes. The 2^−ΔΔCt method was employed to quantify target gene mRNA expression. Expression levels were presented as fold-changes relative to the control. Primer sequences are listed in Supplementary Table S1.

Distribution analysis of HSA-TPP-PEG-PP10 NPs in mouse lungs

The Cy5-labelled NPs were directly delivered to the lung via intranasal administration. Fluorescence intensity in the mouse lungs was measured using a small animal in vivo imaging system at 0 h and 24 h. At 24 h post-administration, mice were euthanized, and their lung tissues were harvested, embedded in OCT compound, and flash-frozen at -80 °C for cryo-sectioning. Lung sections were stained with Mito-Tracker Green and DAPI. Fluorescence microscopy (Olympus BX53, Japan) was utilized to capture of images the stained sections.

Efficacy assessment of HSA-TPP-PEG-PP10 NPs for treatment of HDM/LPS-induced mouse

The experimental groups included Saline, PP10, HSA-TPP-PEG-PP10 NPs HDM/LPS, HDM/LPS+PP10, and HDM/LPS + HSA-TPP-PEG-PP10 NPs. The asthmatic mouse model was constructed as described in Sect. "PP10 alleviates HDM/LPS-induced asthma airway inflammation in mice via intraperitoneal injection, not intranasal delivery" (Fig. 6D). Mice received intranasal administrations of PP10 (2 µg/g), HSA-TPP-PEG-PP N10Ps (2 µg/g, corresponding to the intraperitoneal reference dose), or vehicle (Saline) from days 19 to 22. All mice were euthanized on day 23 for further analysis, and sample processing was performed as previously described.

Evaluation of HSA-TPP-PEG-PP10 NPs toxicity in major mouse organs

Heart, liver, kidney, and spleen tissues were sectioned into 4 μm slices and stained with hematoxylin and eosin (H&E). Blood samples were collected and centrifuged at 300 g for 15 min. Serum ALT and Cr levels were measured using colorimetric assays to assess liver and kidney functions.

Data analysis

All statistical studies used GraphPad Prism 8.0. All results were reported as means ± standard error of the mean (SEM). Comparison of the variables between groups was made by student’s t-test or one-way ANOVA followed by Tukey’s post-hoc test. Statistical significance was p-value < 0.05.

Fig. 9.

(A) Synthesis of HSA-TPP-PEG-PP10 NPs. (B) Through intranasal administration, PEG-modified NPs were delivered into mice, enabling their penetration through the airway mucus barrier. (C) Allergen-triggered mtROS overproduction in airway epithelia induces mitochondrial dysfunction, causing prolonged opening of mPTP, mtDNA cytosolic release, and subsequent cGAS-STING activation, thereby promoting inflammatory cytokine secretion and exacerbating asthmatic airway inflammation. (D) Inhaled HSA-TPP-PEG-PP10 nanoparticles (NPs) traverse the airway mucus barrier, are internalized by epithelial cells, and release PP10. PP10 targets mitochondrial c subunits, attenuating HDM/LPS-induced mtROS overproduction, restoring mitochondrial function, and inhibiting mPTP-mediated mtDNA release, thereby suppressing cGAS-STING activation and inflammatory cytokine production to alleviate asthmatic inflammation

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Author contributions

D.W. and T.Z. contributed equally to this work. D.W., T.Z., Y.W. and S.X. conceived and designed the experiments. D.W., T.Z., Y.C., C.B., S.Y., C.W., Q.L., C.L., and J.H. performed the experiments. D.W., M.L., Z.L., and X.M. analyzed the data. D.W., Y.Z., and J.S. contributed reagents, materials and analysis software. D.W., T.Z., Y.W. and S.X. wrote and revised the paper. Y.W., and S.X. supervised the studies. All authors approved the final manuscript.

Funding

This study was supported by grants from National Natural Science Foundation of China (81370134), Natural Science Foundation of Hubei Province of China (2012FFB02422), the National Key R&D Program of China (No. 2024YFC3407200), the Scientific Research Innovation Capability Support Project for Young Faculty, and the Fundamental Research Funds for the Central Universities (2024BRA003).

Data availability

Data will be made available on request.

Declarations

Competing interests

The authors declare no competing interests.