Multifunctional nano-platform for precision treatment of acute lung injury via synergistic anti-inflammation and oxidative stress modulation

Abstract

Abstract

Acute lung injury (ALI) remains life-threatening conditions lacking effective disease-modifying therapies. Dysregulated inflammation, oxidative stress, and apoptosis form a self-amplifying pathogenic loop that drives alveolar-capillary barrier disruption, highlighting the urgent need for multifunctional therapeutic strategies. Here, we report a pH-responsive biomimetic nanoplatform, pDA/Esc@ZIF-8, for precision treatment of ALI. Zeolitic imidazolate framework-8 (ZIF-8) was employed to encapsulate the natural anti-inflammatory and antioxidant agent esculin (Esc), while a polydopamine (pDA) coating conferred enhanced colloidal stability, intrinsic reactive oxygen species scavenging activity, and pH-triggered drug release, enabling efficient pulmonary accumulation and sustained local retention. In vitro and in vivo ALI models demonstrated that pDA/Esc@ZIF-8 markedly alleviated lung inflammation, oxidative injury, and epithelial apoptosis, thereby preserving alveolar-capillary barrier integrity and improving respiratory function. Mechanistically, the therapeutic effects were closely associated with modulation of the PI3K/AKT/GSK3β signaling axis, leading to suppression of pro-inflammatory responses, attenuation of oxidative stress, and inhibition of mitochondria-dependent apoptotic pathways. Collectively, this multifunctional nanotherapeutic system integrates pulmonary priority accumulation, microenvironment-responsive release, and pathway-level regulation to interrupt the inflammation-oxidative stress-apoptosis cascade, offering a promising and translatable strategy for the treatment of ALI and related inflammatory lung diseases.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12951-026-04690-x.

Introduction

Acute lung injury (ALI) is a life-threatening clinical syndrome caused by various factors, including infection, trauma, and inhalational injury, and is primarily characterized by diffuse damage to the alveolar epithelium and capillary endothelium, leading to disruption of the alveolar–capillary barrier and refractory hypoxemia [1, 2]. ALI can rapidly progress to acute respiratory distress syndrome (ARDS), and despite advances in modern intensive care, the associated mortality rate remains unacceptably high [3]. Currently, the clinical management of ALI/ARDS relies predominantly on supportive therapies such as mechanical ventilation and fluid management, while safe and effective disease-specific interventions are still lacking, underscoring the urgent need for the development of novel therapeutic strategies [4].

From a pathological perspective, the initiation and progression of ALI represent a complex process involving dysregulation across multiple factors and signaling pathways, with uncontrolled inflammation, excessive oxidative stress, and programmed cell death playing central roles [5, 6]. The cascade release of pro-inflammatory cytokines markedly amplifies local inflammatory responses, whereas excessive generation of reactive oxygen species (ROS) further disrupts alveolar epithelial integrity, induces mitochondrial dysfunction, and triggers apoptosis, thereby forming a positive feedback loop that exacerbates lung tissue damage [7]. Consequently, coordinated modulation of inflammation, oxidative stress, and cellular injury has been recognized as a critical strategy for improving ALI outcomes.

In recent years, advances in nanomedicine have provided new technological avenues for the precision treatment of complex inflammatory diseases. Metal–organic frameworks (MOFs) have attracted considerable attention in drug delivery owing to their high specific surface area, tunable pore size, and favorable biocompatibility [8, 9]. Among them, zeolitic imidazolate framework-8 (ZIF-8) exhibits excellent capacity for small-molecule drug loading and undergoes responsive degradation in inflammation-associated acidic microenvironments, enabling stimulus-responsive drug release [10–12]. However, the limited stability of bare ZIF-8 under physiological conditions and its potential biocompatibility concerns have constrained its further biomedical application [13].

Polydopamine (pDA), a biomimetic coating material, has been widely employed for surface modification of nanocarriers due to its strong adhesion capability and excellent biocompatibility [14, 15]. Notably, the catechol moieties in pDA not only enhance the structural stability of nanoplatforms but also confer intrinsic antioxidant activity by scavenging excessive ROS under pathological conditions, thereby exerting potential protective effects in inflammation-related diseases [16]. Accordingly, the integration of ZIF-8 with pDA represents a promising strategy to construct multifunctional nanocarriers with improved stability, microenvironment-responsive drug release, and synergistic therapeutic functionality [17].

Esculin (Esc), a natural small-molecule compound derived from traditional Chinese medicine, has been demonstrated to possess potent anti-inflammatory and antioxidant activities and has shown therapeutic potential in various inflammation-related disease models [18–20]. Nevertheless, its clinical translation remains hindered by several challenges, including low oral bioavailability, rapid metabolic clearance, and limited accumulation in lung tissues [21]. These limitations substantially restrict the application of Esc in ALI therapy. Therefore, the development of an effective delivery system capable of enhancing pulmonary targeting and prolonging the local retention of Esc is of great importance for fully realizing its therapeutic potential.

Based on these considerations, we constructed a composite nanotherapeutic system, pDA/Esc@ZIF-8, using ZIF-8 as the drug carrier and pDA as a functional coating, aiming to achieve efficient Esc loading, lung priority accumulation, and inflammation microenvironment–responsive release (Scheme 1). Through comprehensive in vitro and in vivo investigations, this study systematically evaluated the physicochemical properties and biosafety of the nanoplatform and elucidated its synergistic regulatory effects on inflammation, oxidative stress, and apoptosis in ALI models, along with the underlying molecular mechanisms. Collectively, this work provides new experimental evidence and theoretical support for precision ALI therapy through the integration of natural small-molecule therapeutics with functional nanomaterials.

Scheme 1.

Schematic illustration of the construction and therapeutic mechanism of the pDA/Esc@ZIF-8 nanoplatform for ALI therapy

Results and discussion

Synthesis and characterization of pDA/Esc@ZIF-8

SEM images showed that the as-synthesized ZIF-8 nanoparticles exhibited a well-defined rhombic dodecahedral morphology with good dispersity and an average particle size of 79.5 ± 9.4 nm (Fig. 1A–B). After pDA coating and Esc loading, the particle morphology transformed from regular polyhedra to quasi-spherical nanoparticles with a relatively rough surface, accompanied by a significant increase in particle size to 174.7 ± 29.5 nm (Fig. 1C–D), indicating the successful introduction of the pDA shell and drug payload. TEM further confirmed the formation of a uniform core–shell architecture (Fig. 1E), in which ZIF-8 served as the core carrier and pDA formed the outer coating. This well-defined structure provides a robust basis for stable drug encapsulation and microenvironment-responsive release. Zeta potential measurements revealed that bare ZIF-8 possessed a positively charged surface, whereas pDA/Esc@ZIF-8 displayed a clear charge reversal to a negative potential (Fig. 1F), suggesting a substantial alteration in surface chemistry upon hybridization. Such a change is beneficial for improving colloidal stability and biocompatibility in physiological media, and may also reduce nonspecific protein adsorption and nonselective cellular uptake [22].

Fig. 1.

Characterization and functional evaluation of pDA/Esc@ZIF-8 NPs. (A) SEM images of ZIF-8 NPs. (B) Particle size distribution of ZIF-8. (C) SEM images of pDA/Esc@ZIF-8. (D) Particle size distribution of pDA/Esc@ZIF-8. (E) TEM images of pDA/Esc@ZIF-8. (F) Zeta potential of pDA/Esc@ZIF-8. (G) XRD pattern of pDA/Esc@ZIF-8. (H) UV–vis absorption spectrum of pDA/Esc@ZIF-8. (I) FT-IR spectrum of pDA/Esc@ZIF-8. (J) XPS spectra of pDA/Esc@ZIF-8. (K) Suspension stability of pDA/Esc@ZIF-8 in different solutions. (L) Drug release behavior of pDA/Esc@ZIF-8 in solutions with different pH values. (M) EPR spectra of pDA/Esc@ZIF-8. Data are expressed as mean ± SD, n = 3

X-ray diffraction (XRD) patterns of pDA/Esc@ZIF-8 exhibited characteristic diffraction peaks highly consistent with those of pristine ZIF-8, without apparent peak shifts or pronounced intensity attenuation (Fig. 1G), demonstrating that the crystalline framework of ZIF-8 was well preserved during pDA coating and Esc loading. As shown in Fig. 1H, the characteristic UV–vis absorption peak of Esc was markedly attenuated after its incorporation into the ZIF-8 framework and subsequent coating with a PDA shell. This reduction in absorption intensity is likely attributable to the confinement of Esc molecules within the porous structure of ZIF-8 and the additional shielding effect provided by the PDA coating, both of which decrease the effective exposure of Esc chromophores to incident light [23, 24]. Moreover, the encapsulation process may promote intermolecular interactions, including hydrogen bonding and π–π stacking, as well as interactions between Esc and the ZIF-8/PDA matrix, thereby altering the electronic environment of Esc and affecting its π–π electronic transitions [25, 26]. In addition, PDA exhibits broad-band optical absorption over the UV–visible region, which may partially overlap with and mask the intrinsic absorption features of Esc [27].

Fourier transform infrared (FT-IR) analysis further verified the structural integrity of the composite nanoparticles (Fig. 1I). The spectrum of pDA/Esc@ZIF-8 contained characteristic bands from ZIF-8 (e.g., C = N stretching and Zn–O/Zn–N vibrations), along with signals attributable to Esc (O–H and C–O stretching vibrations) and pDA (O–H/N–H stretching, C = O, and C–N vibrations), confirming successful integration of multiple components. X-ray photoelectron spectroscopy (XPS) analysis indicated that the nanoparticle surface primarily consisted of C, N, O, and Zn elements (Fig. 1J and Fig. S1), further corroborating the successful construction of pDA/Esc@ZIF-8 and providing a solid material basis for subsequent drug-loading performance and biological functions.

Drug loading capacity and pH-responsive in vitro release

To evaluate the drug-loading performance of pDA/Esc@ZIF-8, the encapsulation efficiency (EE) and drug loading content (DLC) were first determined to be 42.5% and 23.18%, respectively (Table S1), indicating the excellent loading capacity of ZIF-8 for Esc. The colloidal stability of pDA/Esc@ZIF-8 was evaluated in PBS and DMEM for 14 days. As shown in Fig. 1K, the hydrodynamic diameter of the nanoparticles showed only a slight increase during incubation, without evident macroscopic aggregation. This result indicates that the nanoplatform maintained favorable dispersion stability under physiologically relevant conditions, which may be attributed to the PDA coating that improves interfacial stability and reduces nonspecific particle aggregation [28].

Esc quantification was performed based on the UV–vis calibration curve of Esc at different concentrations (Fig. S2). The in vitro release profiles revealed a distinct pH-dependent release behavior (Fig. 1L). At pH 7.4, approximately 25% of Esc was released during the initial 4–6 h, after which the release curve reached a plateau with limited additional release up to 48 h. This restrained release behavior suggests that the ZIF-8 framework remains structurally stable under neutral conditions, while the PDA shell further acts as a diffusional barrier to limit premature drug leakage [23, 29]. In contrast, under acidic conditions at pH 5.5, Esc release increased rapidly and reached approximately 80% within 16 h, followed by a relatively stable plateau. This accelerated release under acidic conditions is consistent with the pH-responsive degradation behavior of ZIF-8, which undergoes protonation-induced framework disassembly in acidic environments [25]. As a result, the encapsulated Esc can be released more efficiently from the nanocarrier. The initial burst release is likely attributable to the rapid liberation of surface-adsorbed or loosely encapsulated Esc during the early stage of framework decomposition [30]. The subsequent plateau phase may reflect the depletion of readily releasable Esc, together with the retention of a small fraction of drug molecules that remain more strongly associated with the carrier matrix through intermolecular interactions [31].

To further verify the structural basis of this pH-responsive behavior, TEM analysis was performed after incubation under different pH conditions (Fig. S3). The nanoparticles largely retained their core–shell morphology at pH 7.4, whereas obvious structural disruption and loss of the original architecture were observed at pH 5.5. Collectively, these results demonstrate that pDA/Esc@ZIF-8 integrates colloidal stability under physiological conditions with acid-responsive intracellular release, which is advantageous for controlled drug delivery in inflammatory injury models.

In vitro antioxidant activity of pDA/Esc@ZIF-8

To evaluate the in vitro antioxidant capacity of pDA/Esc@ZIF-8, electron paramagnetic resonance (EPR) spectroscopy was employed to systematically assess its scavenging ability against multiple reactive radicals. As shown in Fig. 1M, pDA/Esc@ZIF-8 exhibited pronounced scavenging effects toward ·OH, ·O₂, DPPH·, and ·NO radicals, indicating a broad-spectrum antioxidant activity. This antioxidant performance can be attributed to the cooperative effects of the electron-donating catechol groups in the pDA coating, the phenolic hydroxyl groups of Esc, and the ZIF-8-mediated controlled release behavior, which together contribute to an integrated antioxidant effect [13, 32]. These properties provide strong experimental support for the potential application of pDA/Esc@ZIF-8 in the treatment of oxidative stress–associated inflammatory diseases.

pDA/Esc@ZIF-8 alleviates oxidative stress and apoptosis in LPS-injured MLE-12 cells

To determine whether pDA/Esc@ZIF-8 could protect ALI-relevant cell types from inflammatory injury, we established an in vitro LPS-induced injury model using MLE-12 cells and RAW264.7 macrophages (Fig. 2A). We first evaluated the cytocompatibility of the nanoplatform and its individual components. CCK-8 assays showed that ZIF-8, Esc, and pDA/Esc@ZIF-8 did not induce evident cytotoxicity in either MLE-12 or RAW264.7 cells within the tested concentration range (Fig. 2B, C). In the LPS-injured MLE-12 model, pDA/Esc@ZIF-8 restored cell viability in a concentration-dependent manner (Fig. 2D). Considering the balance between protective efficacy and material exposure, 50 µg/mL was selected for subsequent functional studies.

Fig. 2.

In vitro protective effects of pDA/Esc@ZIF-8 against LPS-induced injury in MLE-12 cells and RAW264.7 macrophages. (A) Schematic illustration of the in vitro LPS-induced cells injury model. MLE-12 and RAW264.7 cells were stimulated with LPS (1 µg/mL) for 12 h, followed by treatment with different formulations. (B) Cell viability of MLE-12 cells after incubation with different formulations at the indicated concentrations. (C) Cell viability of RAW264.7 cells after incubation with different formulations at the indicated concentrations. (D) Viability of LPS-injured MLE-12 cells after treatment with different concentrations of pDA/Esc@ZIF-8. (E) Representative fluorescence images of ROS in MLE-12 cells stimulated by LPS after different treatments. Scale bar: 400 μm. (F) Quantitative analysis of ROS fluorescence intensity in MLE-12 cells. (G) Representative JC-1 staining images showing mitochondrial membrane potential in MLE-12 cells after different treatments. Scale bar: 200 μm. (H) Representative Annexin V-FITC/PI flow cytometry plots of apoptosis in MLE-12 cells following different treatments. (I) Quantitative analysis of apoptotic MLE-12 cells. (J–L) ELISA analysis of IL-1β, IL-6, and TNF-α levels in the culture supernatants of MLE-12 cells. (M–O) ELISA analysis of IL-10, IL-6, and TNF-α levels in the culture supernatants of RAW264.7 cells. (P) Representative fluorescence images of ROS in RAW264.7 cells stimulated by LPS after different treatments. Scale bar: 100 μm. (Q) Representative live/dead staining images of RAW264.7 cells after different treatments. Scale bar: 100 μm. Data are expressed as mean ± SD, n = 3. ns: no significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Oxidative stress is a central upstream driver of epithelial dysfunction in ALI. Upon LPS stimulation, MLE-12 cells exhibited a marked increase in DCFH-DA fluorescence, reflecting excessive intracellular ROS accumulation and disrupted redox homeostasis. Treatment with free Esc or pDA@ZIF-8 partially attenuated ROS levels, whereas pDA/Esc@ZIF-8 exerted the most pronounced antioxidant effect among all groups (Fig. 2E, F). This enhanced efficacy likely arises from the combined antioxidant properties of Esc and PDA: Esc, a natural coumarin derivative, participates in redox regulation and mitigates oxidative injury [33], while PDA contains catechol/quinone motifs capable of electron donation and radical scavenging [28]. Additionally, the ZIF-8-based nanostructure facilitates efficient intracellular delivery of Esc, enabling the antioxidant components to function more effectively within the injured cellular microenvironment [34].

Mitochondria are highly sensitive to oxidative stress and serve as a major source for further amplification of ROS-mediated injury. JC-1 staining (Fig. 2G) showed that LPS treatment decreased the mitochondrial membrane potential in MLE-12 cells, as evidenced by reduced red fluorescence and increased green fluorescence, indicating mitochondrial depolarization and functional impairment. After treatment with pDA/Esc@ZIF-8, the mitochondrial membrane potential was partially restored, suggesting that this nanoplatform may help maintain mitochondrial homeostasis by alleviating oxidative stress burden. Together, these results link ROS scavenging to subsequent alterations in cell fate, indicating that the epithelial protective effect of pDA/Esc@ZIF-8 is not limited to a single antioxidant event, but may involve modulation of the ROS–mitochondrial injury positive feedback loop.

Flow cytometric analysis further demonstrated that LPS markedly increased apoptosis in MLE-12 cells (Fig. 2H, I). Treatment with pDA/Esc@ZIF-8 reduced both early and late apoptotic cell populations, exhibiting a significantly stronger effect than either Esc or pDA@ZIF-8 alone. In combination with the observed recovery of mitochondrial membrane potential, these findings suggest that pDA/Esc@ZIF-8 may mitigate mitochondrial dysfunction, thereby attenuating the initiation of mitochondria-mediated apoptotic signaling. Live/dead staining (Fig. S4) further supported this conclusion, indicating that pDA/Esc@ZIF-8 decreases LPS-induced cell death while maintaining a higher proportion of viable cells.

ELISA results showed that LPS significantly elevated IL-1β, IL-6, and TNF-α levels in MLE-12 cell culture supernatants, whereas pDA/Esc@ZIF-8 treatment effectively reduced these pro-inflammatory cytokines, with effects superior to Esc or pDA@ZIF-8 alone (Fig. 2J-L). These results indicate that pDA/Esc@ZIF-8 not only alleviates oxidative damage and apoptosis in epithelial cells but may also diminish their contribution to the inflammatory cascade.

Collectively, the findings in MLE-12 cells demonstrate that pDA/Esc@ZIF-8 exerts protective effects against LPS-induced alveolar epithelial injury in vitro by modulating multiple interrelated processes, including oxidative stress, mitochondrial function, apoptosis, and pro-inflammatory cytokine release.

pDA/Esc@ZIF-8 mitigates inflammatory and oxidative responses in LPS-injured RAW264.7 macrophages

Macrophages are important effector cells in the inflammatory progression of ALI. Their excessive activation can generate large amounts of pro-inflammatory cytokines and ROS, thereby exacerbating epithelial injury, barrier disruption, and inflammatory cell recruitment [35, 36]. As shown in Fig. 2M–O, LPS stimulation significantly induced the secretion of IL-6 and TNF-α in RAW264.7 cells, while disrupting the expression balance of the anti-inflammatory cytokine IL-10. After treatment with pDA/Esc@ZIF-8, IL-6 and TNF-α levels were decreased, whereas IL-10 levels were increased. Notably, this regulatory effect was significantly stronger than that achieved with Esc or pDA@ZIF-8 alone, suggesting that pDA/Esc@ZIF-8 can more effectively attenuate the LPS-induced pro-inflammatory activation state.

Consistent with the cytokine results, DCFH-DA staining showed that LPS treatment markedly increased intracellular ROS levels in RAW264.7 cells, whereas pDA/Esc@ZIF-8 treatment reduced the ROS fluorescence signal (Fig. 2P). These findings indicate that this nanoplatform also exerts antioxidant activity in macrophages. Given that macrophage-derived ROS can further amplify inflammatory signaling and damage surrounding epithelial cells, the ability of pDA/Esc@ZIF-8 to alleviate oxidative stress in macrophages may help reduce oxidative pressure within the inflammatory microenvironment. Live/dead staining of RAW264.7 cells further showed that pDA/Esc@ZIF-8 alleviated LPS-induced macrophage injury and preserved cell viability (Fig. 2Q).

Therefore, the protective effects observed in both LPS-Injured MLE-12 cells and RAW264.7 macrophages support a broader cellular mechanism by which pDA/Esc@ZIF-8 simultaneously protects structural alveolar cells and restrains inflammatory immune-cell activation.

Collectively, the in vitro results indicate that pDA/Esc@ZIF-8 does not act through a single downstream endpoint. Instead, it coordinately suppresses ROS accumulation, preserves mitochondrial function, inhibits epithelial apoptosis, reduces pro-inflammatory cytokine release, and promotes anti-inflammatory cytokine production in macrophages. This multi-level regulation is highly consistent with the pathological architecture of ALI, in which oxidative stress, mitochondrial dysfunction, apoptosis, and inflammatory amplification are interconnected and form a self-reinforcing injury cascade. By targeting multiple nodes within this cascade, pDA/Esc@ZIF-8 provides a mechanistic basis for its therapeutic efficacy in subsequent in vivo ALI models.

Cellular uptake and in vivo biodistribution characteristics of pDA/Esc@ZIF-8

To evaluate the intracellular internalization efficiency and lung-preferential accumulation of the pDA/Esc@ZIF-8 nanoplatform, Cy5.5-labeled pDA/Cy5.5@ZIF-8 was first used for in vitro cellular uptake and in vivo biodistribution studies.

The in vitro cellular uptake results showed that pDA/Cy5.5@ZIF-8 was internalized by MLE-12 cells in a time-dependent manner. Red fluorescence signals were detectable in the cytoplasm after 30 min of incubation and became markedly stronger at 2 h, with the fluorescence mainly distributed in the cytoplasmic region, suggesting efficient cellular entry of the nanoparticles (Fig. 3A). Similar rapid uptake kinetics were also observed in RAW264.7 macrophages (Fig. 3B).

Fig. 3.

Cellular internalization, preferential pulmonary accumulation, and mitochondrial protective features of pDA/Esc@ZIF-8. (A) Representative fluorescence images showing time-dependent cellular uptake of pDA/Cy5.5@ZIF-8 in MLE-12 cells. Scale bar: 100 μm. (B) Representative fluorescence images showing time-dependent cellular uptake of pDA/Cy5.5@ZIF-8 in RAW264.7 macrophages at different time points. Scale bar: 100 μm. (C) In vivo fluorescence imaging of mice after intravenous administration of Cy5.5-labeled nanoparticles at different time points. (D) Ex vivo fluorescence images of major organs. (E) Quantitative analysis of fluorescence intensity in major organs. (F) Immunofluorescence colocalization analysis of Cy5.5-labeled nanoparticles with the alveolar epithelial cell marker SP-C in lung tissue sections. Scale bar: 20 μm. (G) TEM images showing intracellular nanoparticle localization and mitochondrial ultrastructural changes in MLE-12 cells after different treatments. Red arrows indicate intracellular nanoparticle-associated electron-dense structures. (H) Representative MitoTracker Green staining images showing mitochondrial morphology in MLE-12 cells after different treatments. Scale bar: 10 μm. Data are expressed as mean ± SD, n = 3. * P < 0.05

The in vivo biodistribution study further confirmed the lung-preferential accumulation of pDA/Cy5.5@ZIF-8. After intravenous injection of equivalent doses of Cy5.5, in vivo fluorescence imaging showed that the pDA/Cy5.5@ZIF-8 group exhibited a strong fluorescence signal in the thoracic region as early as 1 h post-injection and remained detectable for 4 h. Compared with free Cy5.5, pDA/Cy5.5@ZIF-8 showed stronger and more sustained retention in the lung region (Fig. 3C). Ex vivo organ imaging and ROI quantification further demonstrated that the fluorescence intensity in lung tissue was significantly higher than that in major organs such as the heart, spleen, and kidney (Fig. 3D, E), confirming the preferential pulmonary accumulation of this nanoplatform.

Immunofluorescence co-localization analysis of lung sections further showed strong Cy5.5 signals in the pDA/Cy5.5@ZIF-8 group, with partial spatial overlap between Cy5.5 fluorescence and SP-C-positive regions, a marker of alveolar type II epithelial cells (Fig. 3F and Fig. S5). This result suggests that the nanoparticles can reach or remain within alveolar epithelial-associated regions, thereby providing a delivery basis for contacting and modulating key structural cells in ALI lung tissue.

The lung-preferential accumulation of pDA/Esc@ZIF-8 may be closely related to its physicochemical properties. The nanoplatform has a particle size of approximately 174.7 ± 29.5 nm, which falls within a suitable range for pulmonary capillary retention and cellular uptake [37]. Its negative zeta potential may help reduce nonspecific protein adsorption and facilitate electrostatic interactions with inflammation-activated endothelial cells [38]. In addition, the pDA shell is rich in catechol groups and possesses strong bioadhesive properties, which may enhance adhesion to pulmonary vascular endothelium and extracellular matrix through hydrogen bonding and π–π interactions, thereby prolonging lung retention [39]. Moreover, the increased pulmonary vascular permeability under ALI conditions may further promote nanoparticle extravasation and local accumulation [40].

Taken together, these results demonstrate that pDA/Esc@ZIF-8 combines efficient cellular uptake with preferential pulmonary distribution, providing an important pharmacokinetic basis for its multi-target protective effects in ALI models.

Organelle-level delivery and mitochondrial homeostasis protection

To further investigate the intracellular trafficking and uptake mechanism of pDA/Esc@ZIF-8, TEM and mitochondrial staining were used to assess nanoparticle localization and mitochondrial integrity in alveolar epithelial cells. TEM analysis revealed that LPS treatment induced pronounced mitochondrial swelling, disrupted cristae structures, and localized vacuolization in MLE-12 cells, indicating oxidative stress–mediated mitochondrial damage (Fig. 3G). In cells treated with pDA/Esc@ZIF-8, electron-dense nanoparticles were observed within intracellular compartments, and mitochondrial structures appeared relatively intact, suggesting that the nanoparticles were effectively internalized and associated with organelles. Notably, cells treated with ZIF-8 alone showed minimal nanoparticle uptake in TEM images, and mitochondrial damage was largely unmitigated. These observations indicate that, in the absence of PDA coating or drug modification, the ZIF-8 framework exhibits low cellular delivery efficiency and cannot effectively reach alveolar epithelial cells to confer protection. In contrast, the combination of PDA coating and Esc incorporation not only improves colloidal stability and surface hydrophilicity but may also enhance cellular uptake through surface chemical modifications, thereby enabling protection of mitochondrial structure and function [28, 29].

MitoTracker staining further showed that mitochondria in untreated MLE-12 cells formed a continuous network, whereas LPS treatment induced mitochondrial fragmentation, resulting in dispersed or rounded morphologies (Fig. 3H). Treatment with pDA/Esc@ZIF-8 partially restored the mitochondrial network, maintaining morphological continuity and structural integrity. These findings are consistent with the observed reduction in ROS levels and recovery of mitochondrial membrane potential, suggesting that pDA/Esc@ZIF-8 can protect mitochondrial homeostasis by alleviating oxidative stress burden.

pDA/Esc@ZIF-8 alleviates oxidative stress and apoptosis and improves respiratory function in ALI mice

To systematically evaluate the in vivo therapeutic efficacy of pDA/Esc@ZIF-8 against LPS-induced ALI, a mouse ALI model was established by intratracheal instillation of LPS at 5 mg/kg. Two hours after model induction, mice were intravenously administered PBS, ZIF-8, Esc, pDA@ZIF-8, or pDA/Esc@ZIF-8 via the tail vein. Histopathological, barrier-function, and inflammation-related analyses were then performed 24 h after LPS challenge (Fig. 4A).

Fig. 4.

pDA/Esc@ZIF-8 alleviates LPS-induced ALI by preserving alveolar–capillary barrier integrity, suppressing inflammation, reducing oxidative stress, and inhibiting apoptosis. (A) Schematic illustration of the LPS-induced ALI mouse model and treatment regimen. For the therapeutic model, ALI was induced by intratracheal instillation of LPS (5 mg/kg), followed by intravenous administration of different formulations 2 h after modeling. For survival analysis, a lethal ALI model was established using high-dose LPS (50 mg/kg). (B) Representative H&E-stained lung sections from different treatment groups. Scale bar: 200 μm. (C) Semi-quantitative pathological injury scores of H&E-stained lung tissues. (D) Total cell counts in BALF from different groups. (E) Total cell counts in BALF from different groups. (F) Lung wet-to-dry weight ratio of mice after different treatments. (G) Representative immunofluorescence images of ZO-1 expression in lung tissues from different groups. Scale bar: 100 μm. (H) Representative flow cytometry histograms showing M1-like and M2-like macrophage populations in BALF after different treatments. (I) Representative immunofluorescence co-staining images of CD86 and CD206 in lung tissues from different groups. Scale bar: 100 μm. Data are expressed as mean ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

H&E staining showed that lung tissues from the ALI group exhibited typical pathological features of acute lung injury, including severe disruption of alveolar architecture, pronounced interstitial edema, extensive inflammatory cell infiltration, and erythrocyte extravasation (Fig. 4B). Semi-quantitative histopathological scoring further confirmed that the lung injury score was significantly higher in the ALI group than in the control group (Fig. 4C). Compared with free Esc or pDA@ZIF-8, pDA/Esc@ZIF-8 treatment resulted in the greatest preservation of alveolar structure, the lowest degree of edema and inflammatory infiltration, and the most pronounced reduction in pathological injury scores.

In ALI, injury to the alveolar epithelium and capillary endothelium increases barrier permeability, leading to protein leakage and pulmonary edema, which are major contributors to hypoxemia and respiratory failure [41, 42]. Consistently, the ALI group showed significantly increased total protein concentration and total cell counts in bronchoalveolar lavage fluid (BALF), together with an elevated lung wet/dry weight ratio, indicating severe alveolar–capillary barrier disruption and pulmonary edema (Fig. 4D–F). pDA/Esc@ZIF-8 treatment markedly reduced BALF protein levels, total cell counts, and the wet/dry weight ratio, and these effects were more pronounced than those observed in the free Esc and pDA@ZIF-8 groups.

Immunofluorescence staining of lung tissues further showed that the tight junction protein ZO-1 was markedly reduced and disorganized in the ALI group. In contrast, pDA/Esc@ZIF-8 treatment restored a more continuous and linear ZO-1 distribution along the alveolar epithelial barrier (Fig. 4G), indicating effective repair of epithelial barrier integrity. Together with the improved alveolar morphology observed by H&E staining, these results suggest that pDA/Esc@ZIF-8 interrupts the early pathological cycle of structural destruction, edema formation, and respiratory dysfunction by preserving alveolar–capillary barrier integrity.

Inflammatory cell infiltration and cytokine release are major drivers of barrier disruption and tissue injury in ALI. Neutrophil and macrophage count in BALF showed that pDA/Esc@ZIF-8 effectively suppressed inflammatory cell recruitment (Fig. S6). ELISA analysis of BALF further demonstrated that LPS markedly increased IL-1β, IL-6, and TNF-α levels, whereas pDA/Esc@ZIF-8 produced the strongest reduction in these pro-inflammatory cytokines among all treatment groups (Fig. S7). Flow cytometric analysis of macrophage subsets in BALF showed that pDA/Esc@ZIF-8 significantly decreased the proportion of pro-inflammatory M1-like macrophages while increasing the proportion of anti-inflammatory M2-like macrophages (Fig. 4H and Fig. S8). Consistently, immunofluorescence co-staining of lung tissues showed a similar shift in macrophage polarization (Fig. 4I). This macrophage-reprogramming effect may help interrupt the vicious cycle of inflammatory amplification and tissue injury in ALI. Compared with free Esc or pDA@ZIF-8, the superior efficacy of pDA/Esc@ZIF-8 suggests that the anti-inflammatory activity of Esc and the delivery-supporting function of pDA@ZIF-8 may act cooperatively, thereby providing an inflammatory-regulatory basis for subsequent modulation of oxidative stress and apoptosis.

Based on the observed improvements in tissue structure, barrier integrity, and inflammatory regulation, we further evaluated the protective effects of pDA/Esc@ZIF-8 at the levels of oxidative stress, apoptosis, and pulmonary function. Survival analysis was also performed to assess the overall therapeutic relevance of this nanoplatform.

Oxidative stress is a critical driver of ALI progression. ROS fluorescence staining and quantitative analysis of lung tissues showed that the ALI group exhibited markedly increased ROS fluorescence intensity in alveolar epithelial and interstitial regions, whereas pDA/Esc@ZIF-8 treatment significantly reduced ROS accumulation and showed stronger antioxidant efficacy than the other treatment groups (Fig. 5A, B). In addition to amplifying inflammation, oxidative stress can directly induce apoptotic cell death. TUNEL staining showed that the number of apoptotic cells was markedly increased in lung tissues from the ALI group, whereas pDA/Esc@ZIF-8 treatment substantially reduced TUNEL-positive cells, with a stronger protective effect than free Esc or pDA@ZIF-8 alone (Fig. 5C, D). Consistently, immunofluorescence analysis showed that pDA/Esc@ZIF-8 upregulated the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic protein Bax, thereby reducing the Bax/Bcl-2 ratio and helping maintain alveolar type II epithelial cell homeostasis (Fig. S9). The inhibition of apoptosis further explains the preservation of alveolar epithelial integrity and provides a cellular basis for subsequent improvement in pulmonary function.

Fig. 5.

pDA/Esc@ZIF-8 attenuates oxidative stress and apoptosis, improves pulmonary function, and exhibits favorable in vivo biosafety in LPS-induced ALI mice. (A, B) Representative ROS fluorescence staining images and quantitative analysis of lung tissues from different treatment groups. (C, D) TUNEL staining and quantitative analysis of lung tissue sections from different treatment groups. (E-H) Pulmonary function parameters measured by non-invasive whole-body plethysmography. (I) Body weight changes of mice during the 14-day treatment period. (J) Representative H&E-stained sections of major organs from mice after 14 consecutive days of pDA/Esc@ZIF-8 administration. Scale bar: 100 μm. Data are presented as mean ± SD, n = 3. ns: no significant, * P  < 0.05, ** P  < 0.01, *** P  < 0.001

Pulmonary function recovery and survival benefit provide direct functional evidence for the therapeutic efficacy of pDA/Esc@ZIF-8. Non-invasive whole-body plethysmography showed that pDA/Esc@ZIF-8 significantly improved multiple pulmonary function parameters, as evidenced by the normalization of EF50, TV/body weight, EEP, and Penh, indicating restoration of respiratory mechanics and ventilation capacity (Fig. 5E-H).

In a lethal-dose LPS challenge model at 50 mg/kg, the 7-day survival rate was significantly higher in the pDA/Esc@ZIF-8-treated group than in the PBS-treated group (Fig. S10). Further histopathological analysis of lung tissues from this lethal model showed that death in untreated mice was mainly associated with severe lung injury, including alveolar destruction, edema, and inflammatory infiltration, whereas pDA/Esc@ZIF-8 markedly attenuated lung tissue damage (Fig. S11). These functional and survival benefits represent the integrated outcome of structural preservation, inflammatory suppression, oxidative stress reduction, and apoptosis inhibition.

Biocompatibility evaluation

To systematically evaluate the biosafety of pDA/Esc@ZIF-8, in vitro hemocompatibility and in vivo toxicity assessments were conducted (Fig. S12). Hemolysis assays showed that even at a relatively high concentration of 100 µg/mL, the hemolysis rate of pDA/Esc@ZIF-8 remained below 5% (Fig. S13), meeting the established criteria for blood compatibility of biomedical materials and indicating excellent hemocompatibility in vitro.

Systemic toxicity was further evaluated through continuous tail vein administration of pDA/Esc@ZIF-8 (1 mg/kg) for 14 consecutive days. During the treatment period, body weights of mice remained stable without abnormal fluctuations (Fig. 5I). H&E staining of major organs revealed intact tissue architectures without noticeable histopathological abnormalities or inflammatory lesions (Fig. 5J). In addition, hematological parameters and serum biochemical indices related to liver and kidney function showed no significant differences compared with the control group (Fig. S14, A-G). These results collectively demonstrate that pDA/Esc@ZIF-8 exhibits a favorable safety profile and a wide in vivo safety window at the tested dose and administration regimen, providing a reliable foundation for subsequent mechanistic investigations and potential translational applications.

Network pharmacology analysis

To elucidate the potential mechanisms of Esc in ALI from a systems biology perspective, network pharmacology analysis was integrated with RNA-seq and experimental validation to comprehensively characterize its multi-target regulatory properties (Fig. 6A). A total of 67 shared targets between Esc and ALI were identified through database screening (Fig. 6B), which were subsequently used to construct a protein–protein interaction (PPI) network (Fig. 6C) and a “drug–target–disease–pathway” regulatory network (Fig. 6D). Topological analysis revealed that GAPDH, TNF, IL-6 and GSK3β exhibited high degree values, suggesting that these targets may play pivotal regulatory roles in the protective effects mediated by Esc.

Fig. 6.

Network pharmacology analysis reveals the multi-target and pathway-regulatory mechanisms of Esc in ALI. (A) Workflow illustrating the integrated network pharmacology analysis combined with RNA-seq data and experimental validation. (B) Venn diagram showing the shared targets between Esc and ALI. (C) PPI network constructed based on the shared targets of Esc and ALI. (D) Drug–target–disease–pathway regulatory network of Esc in ALI. (E) GO functional enrichment analysis of the shared targets. (F) KEGG pathway enrichment analysis of the shared targets. (G) Key targets involved in the PI3K/AKT signaling pathway identified from KEGG enrichment analysis. (H) Molecular docking analysis showing the binding interactions of Esc with TLR4, AKT1, GSK3β, and PIK3CA etc

Gene Ontology (GO) functional annotation indicated that the potential actions of Esc were mainly associated with biological processes and signaling pathways related to oxidative stress regulation, inflammatory responses, and apoptosis (Fig. 6E). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the PI3K/AKT signaling pathway was the most significantly enriched among all identified pathways (Fig. 6F), with key downstream targets such as GSK3β and CCND1 involved in this pathway (Fig. 6G), suggesting that Esc may exert coordinated regulation of inflammation and cell fate through PI3K/AKT-related signaling. Considering the well-established pro-inflammatory role of TLR4 in ALI, it was hypothesized that Esc may mediate its multi-target therapeutic effects via the TLR4/PI3K/AKT/GSK3β signaling axis (Fig. S15).

Molecular docking analysis further supported this hypothesis. Esc displayed favorable binding affinities with key proteins including TLR4, AKT1, GSK3β, and PIK3CA (Fig. 6H), suggesting that Esc may directly interact with and regulate these core targets, thereby modulating the activity of related signaling pathways.

Transcriptomic analysis reveals the molecular mechanisms of pDA/Esc@ZIF-8

To further investigate the regulatory effects of pDA/Esc@ZIF-8 on gene expression in mouse lung tissues, RNA sequencing (RNA-seq) was performed. Boxplot analysis demonstrated comparable and uniform gene expression distributions between the pDA/Esc@ZIF-8–treated mice and ALI groups without abnormal outliers, indicating high data quality suitable for downstream analysis (Fig. 7A). Principal component analysis (PCA) further revealed clear separation between groups at the transcriptomic level, confirming that the ALI model successfully induced substantial gene expression alterations (Fig. 7B).

Fig. 7.

Transcriptomic analysis reveals the involvement of the PI3K/AKT/GSK3β signaling pathway in the therapeutic mechanism. (A) Boxplot analysis showing the distribution of gene expression levels across different samples. (B) PCA of transcriptomic profiles from different experimental groups. (C) Volcano plot showing DEGs between groups. (D) GO enrichment analysis of the identified DEGs. (E) KEGG pathway enrichment analysis of the DEGs. (F, G) Hierarchical clustering heatmaps of representative genes associated with oxidative stress (F) and inflammatory responses (G). (H) qPCR analysis of key genes involved in the PI3K/AKT/GSK3β signaling pathway. (I-L) Western blot analysis of PI3K, AKT, GSK3β expression levels in lung tissues from different groups. Data are expressed as mean ± SD, n = 3. ns: no significant, * P < 0.05

Differential expression analysis identified a total of 2,594 differentially expressed genes (DEGs), including 1,476 upregulated and 1,118 downregulated genes (Fig. 7C). GO enrichment analysis showed that these DEGs were significantly enriched in biological processes related to the regulation of cellular processes and signal transduction (Fig. 7D). KEGG pathway analysis revealed significant enrichment of the NF-κB and PI3K–AKT signaling pathways (Fig. 7E), suggesting that pDA/Esc@ZIF-8 may exert its therapeutic effects through modulation of these critical pathways. Hierarchical clustering heatmaps further demonstrated that pDA/Esc@ZIF-8 markedly regulated a subset of genes associated with oxidative stress and inflammatory responses (Fig. 7F–G). These transcriptomic findings corroborate the in vitro and in vivo experimental results, indicating that pDA/Esc@ZIF-8 confers pulmonary protection by suppressing inflammatory signaling and alleviating oxidative stress at the gene expression level.

Based on predictions from network pharmacology and RNA-seq analyses, key signaling molecules were further validated by qPCR and Western blot assays (Fig. 7H–L). In the LPS-induced ALI model, TLR4 expression was significantly upregulated, whereas phosphorylation levels of PI3K and AKT were markedly reduced. Concurrently, the pro-apoptotic protein GSK3β was upregulated, and the cell cycle–related protein CCND1 was downregulated, indicating activation of inflammatory signaling, suppression of cell survival pathways, and enhanced apoptotic propensity. Notably, pDA/Esc@ZIF-8 treatment significantly suppressed TLR4 expression, restored PI3K/AKT phosphorylation, downregulated GSK3β, and upregulated CCND1 expression. These results demonstrate that pDA/Esc@ZIF-8 effectively modulates the PI3K/AKT/GSK3β/CCND1 signaling axis, thereby suppressing inflammation, mitigating oxidative stress, and reducing apoptosis.

Collectively, the molecular-level results consistently converge on the PI3K/AKT/GSK3β signaling axis as a central regulatory pathway underlying the protective effects of pDA/Esc@ZIF-8. Activation of PI3K/AKT accompanied by inhibitory regulation of GSK3β provides a unified mechanistic basis for the simultaneous attenuation of inflammatory responses, oxidative stress, and apoptosis. Rather than acting on isolated downstream targets, pDA/Esc@ZIF-8 appears to modulate upstream signaling thresholds, thereby dampening inflammation amplification, reducing ROS accumulation, and promoting cell survival in a coordinated manner. This integrated regulation of the PI3K/AKT/GSK3β pathway highlights a rational material-mediated strategy for orchestrating multi-level cytoprotection in inflammation-associated tissue injury.

Conclusion

In this study, we successfully developed a multifunctional nanotherapeutic system, pDA/Esc@ZIF-8, by encapsulating the anti-inflammatory drug esculin in ZIF-8 and coating it with polydopamine, enabling efficient drug loading, lung- preferential accumulation, and inflammation-responsive release. Both in vitro and in vivo results demonstrated that this nanoplatform significantly alleviated inflammatory cell infiltration and pulmonary edema in ALI models, restored alveolar–capillary barrier integrity, suppressed pro-inflammatory cytokine production and oxidative stress, and markedly improved the respiratory function of ALI mice and the survival rate of severe ALI mice. Mechanistic investigations integrating network pharmacology, RNA-seq, and molecular biology experiments systematically revealed that pDA/Esc@ZIF-8 exerts its therapeutic effects by modulating the PI3K/AKT/GSK3β signaling axis, thereby coordinately suppressing inflammation, mitigating oxidative stress, and reducing apoptosis. Collectively, this study demonstrates that multi-mechanistic integration of natural small-molecule therapeutics with functional nanomaterials can substantially enhance therapeutic efficacy and translational potential, offering a promising strategy for the precision treatment of acute lung injury.

Materials and methods

Synthesis of pDA/Esc@ZIF-8 nanoparticles

Esc was dissolved in DMSO to prepare a stock solution. Zinc nitrate hexahydrate (Zn(NO₃)₂·6 H₂O, 0.3633 g) and 2-methylimidazole (1.626 g) were separately dissolved in 100 mL methanol to obtain the metal precursor solution and ligand solution, respectively. Under magnetic stirring, the zinc precursor solution was slowly added dropwise into the ligand solution, followed by the addition of the Esc stock solution. The reaction was allowed to proceed at room temperature for 12 h. The resulting precipitate was collected by centrifugation at 10,000 × g for 10 min and washed three times with methanol to remove unreacted components, yielding Esc@ZIF-8 nanoparticles.

Subsequently, 4 mg of Esc@ZIF-8 was dispersed in 20 mL Tris–HCl buffer (10 mM, pH 8.5), and dopamine hydrochloride (1 mg) was added. The mixture was stirred for 2 h under dark conditions to allow dopamine self-polymerization on the particle surface. The final product was collected by centrifugation (10,000 × g, 10 min), washed three times with deionized water to remove free monomers, freeze-dried, and stored at − 20 °C in the dark for further use.

Synthesis of fluorescently labeled pDA/Cy5.5@ZIF-8

Fluorescently labeled nanoparticles (pDA/Cy5.5@ZIF-8) were prepared using a protocol identical to that of pDA/Esc@ZIF-8, except that Esc was replaced with Cy5.5 as the encapsulated molecule. The obtained nanoparticles were purified by centrifugation and washing, followed by freeze-drying, and were used for in vitro and in vivo fluorescence imaging to evaluate cellular uptake and biodistribution.

TEM and SEM

An appropriate amount of pDA/Esc@ZIF-8 nanoparticles was dispersed in deionized water and ultrasonicated to ensure uniform dispersion. A drop of the suspension was placed onto an ultrathin carbon-coated copper grid or a clean silicon wafer and air-dried at room temperature. The morphology and structural features of the nanoparticles were observed using a transmission electron microscope (JEM-1200EX, JEOL, Japan) and a scanning electron microscope (Sigma 300, Zeiss, Germany).

UV–visible absorption spectroscopy

ZIF-8, pDA, Esc, and pDA/Esc@ZIF-8 were dispersed in ultrapure water to obtain uniform suspensions at a concentration of 0.1 mg/mL after ultrasonication (100 W, 40 kHz, 15 min). One milliliter of each suspension was transferred into a quartz cuvette with a 10 mm optical path length, and UV–vis absorption spectra were recorded in the range of 200–800 nm using ultrapure water as the blank.

Fourier transform infrared (FT-IR) spectroscopy

Dried powders of ZIF-8, pDA, Esc, and pDA/Esc@ZIF-8 were mixed with spectroscopic-grade KBr, ground thoroughly, and compressed into pellets. FT-IR spectra were recorded in the range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹ to analyze characteristic functional groups.

Dynamic light scattering and zeta potential analysis

The hydrodynamic diameter and surface zeta potential of the nanoparticles were measured using a Zetasizer Nano ZS90 system. Samples were dispersed in deionized water, mildly sonicated, and measured at 25 °C. All measurements were performed in triplicate.

X-ray diffraction (XRD)

The crystalline structure of the samples was characterized using an X-ray diffractometer (D8 ADVANCE, Bruker) with Cu Kα radiation (λ = 1.5406 Å). The operating voltage and current were set at 40 kV and 40 mA, respectively. Diffraction patterns were recorded over a 2θ range of 5–80° with a step size of 0.02°.

X-ray photoelectron spectroscopy (XPS)

Elemental composition and chemical states were analyzed using an ESCALAB 250Xi spectrometer (Thermo Fisher Scientific). Samples were drop-cast onto silicon wafers and dried under vacuum prior to analysis. Measurements were performed using Al Kα radiation (1486.6 eV) at a power of 150 W, with a pass energy of 20 eV and a step size of 0.1 eV. Charge neutralization was applied, and spectra were processed using Shirley background subtraction and Gaussian–Lorentzian peak fitting, focusing on C 1s, N 1s, O 1s, and Zn 2p regions.

Suspension stability evaluation

pDA/Esc@ZIF-8 nanoparticles (1.0 mg) were dispersed in 5 mL DMEM complete medium or PBS (pH 7.4) to obtain suspensions at a concentration of 0.2 mg/mL. Samples were incubated at 37 °C, and at days 1, 3, 5, 7, and 14, the hydrodynamic diameter was measured by dynamic light scattering to assess colloidal stability under simulated physiological conditions.

Drug loading and release studies

The drug loading and encapsulation efficiency of pDA/Esc@ZIF-8 were determined using UV–vis spectrophotometry. After the loading reaction, the nanoparticle suspension was centrifuged at 20,000 rpm for 30 min at 4 °C, and the supernatant was collected. The concentration of free Esc in the supernatant was quantified at its characteristic absorption wavelength using a pre-established standard curve.

In vitro drug release experiments were performed under sink conditions to ensure a constant concentration gradient throughout the release process. Briefly, 1 mg of pDA/Esc@ZIF-8 nanoparticles was dispersed in 10 mL of phosphate-buffered saline (PBS, pH 5.5 or 7.4) and incubated at 37 °C in a thermostatic shaking incubator operating at 100 rpm. At predetermined time intervals (1, 2, 4, 8, 16, 24, and 48 h), the suspension was centrifuged at 20,000 × g for 10 min to separate the nanoparticles from the release medium. Subsequently, 1 mL of the supernatant was collected for the quantification of released Esc by UV–vis spectrophotometry, and an equal volume of fresh prewarmed PBS was immediately added to maintain a constant release volume and sink conditions. The cumulative release of Esc was calculated based on the measured drug concentration at each sampling point.

Electron paramagnetic resonance (EPR) analysis

Hydroxyl (·OH) and superoxide (O₂·⁻) radicals were detected using DMPO as a spin-trapping agent, while DPPH· and NO· were used as standard radical systems. pDA/Esc@ZIF-8 nanoparticles were incubated with 1 mM H₂O₂, and EPR spectra were recorded at different time points to evaluate free radical scavenging capacity.

Cell culture

MLE-12 cells and RAW264.7 macrophages were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂. When reaching 70–80% confluence, cells were detached using 0.25% trypsin-EDTA and passaged at a ratio of 1:3 for MLE-12 cells and 1:4–1:5 for RAW264.7 cells.

LPS-induced oxidative injury model

MLE-12 cells and RAW264.7 macrophages were stimulated with LPS (1 µg/mL) for 12 h to establish an oxidative injury model, followed by treatment with free Esc, ZIF-8, pDA@ZIF-8, or pDA/Esc@ZIF-8 (ZIF-8: 50 µg/mL) for an additional 12 h.

Cytotoxicity assay

MLE-12 cells and RAW264.7 macrophages were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and allowed to adhere overnight. Cells were then incubated with various concentrations (0, 10, 50, 100, 150, and 200 µg/mL) of Esc, ZIF-8, or pDA/Esc@ZIF-8 for 24 h. After treatment, 10% CCK-8 reagent (in serum-free medium) was added to each well, and the plates were incubated for an additional 1 h at 37 °C. Absorbance was measured at 450 nm using a microplate reader. Cell viability was expressed as a percentage relative to the untreated control group.

Cell viability under oxidative stress

MLE-12 cells were seeded at 1 × 10⁵ cells per well and treated with LPS (1 µg/mL) for 12 h to induce oxidative injury. Cells were then incubated with pDA/Esc@ZIF-8 nanoparticles (0–200 µg/mL) for an additional 12 h, followed by CCK-8 assay as described above.

Cellular uptake assay

For cellular uptake studies, MLE-12 cells and RAW264.7 macrophages were seeded onto coverslips in 24-well plates and allowed to adhere overnight. Cells were then incubated with Cy5.5-labeled pDA/Cy5.5@ZIF-8 nanoparticles (50 µg/mL) for 0, 30, 60, and 120 min at 37 °C. After incubation, cells were washed three times with ice-cold PBS to remove unbound nanoparticles, fixed with 4% paraformaldehyde for 15 min, and nuclei were counterstained with DAPI (1 µg/mL) for 5 min. Intracellular fluorescence distribution was observed using a fluorescence microscope.

ELISA

Levels of IL-6, IL-1β, and TNF-α were quantified using ELISA kits according to the manufacturers’ instructions. Absorbance was measured at 450 nm, and cytokine concentrations were calculated based on standard curves.

Intracellular ROS detection

Intracellular ROS levels were detected using the DCFH-DA fluorescent probe. MLE-12 cells and RAW264.7 macrophages were seeded in 24-well plates and subjected to the indicated treatments. After treatment, cells were washed with PBS and incubated with DCFH-DA (10 µM) in serum-free medium for 30 min at 37 °C in the dark. Subsequently, cells were washed three times with PBS to remove excess probe. Fluorescence images were acquired using a fluorescence microscope, and the mean fluorescence intensity was quantified using ImageJ software.

Mitochondrial membrane potential (ΔΨm) measurement

ΔΨm was assessed using the JC-1 dye. Treated cells were incubated with JC-1 for 20 min, washed, and analyzed by fluorescence microscopy based on the red/green fluorescence ratio.

Mitochondrial morphology analysis

Cells were incubated with pre-warmed MitoTracker Green working solution at 37 °C for 30 min in the dark. After washing, mitochondrial morphology and distribution were observed using a confocal microscope.

Live/Dead cell staining

Cell viability was assessed using Calcein-AM/PI double staining. Cells were incubated with Calcein-AM (2 µM) for 30 min in the dark, washed, and further incubated with PI for 5 min in the dark, washed, and observed by fluorescence microscopy.

Flow cytometric analysis of apoptosis

Treated MLE-12 cells were collected, resuspended in binding buffer, and stained with Annexin V-FITC and PI for 15 min at room temperature in the dark. Apoptotic cells were analyzed using a flow cytometer (BD LSR II), and data were processed with FlowJo software.

In vivo imaging

To evaluate the pulmonary accumulation and biodistribution of the nanoplatform, BALB/c mice were randomly divided into two groups. Mice received a single tail vein injection of either free Cy5.5 or pDA/Cy5.5@ZIF-8 at an equivalent Cy5.5 dose of 7 mg/kg. In vivo fluorescence imaging was performed at 1, 2, 4, 8, 16 and 24 h post-injection using the IVIS Spectrum imaging system. At 24 h post-injection, mice were euthanized, and major organs (heart, liver, spleen, lung, and kidney) were excised for ex vivo imaging under identical acquisition parameters. Quantitative analysis of fluorescence intensity in each organ was performed using Living Image software by drawing regions of interest. To ensure the reliability and comparability of the biodistribution data, all in vivo fluorescence images were acquired and processed using identical imaging parameters.

qPCR

Total RNA was extracted from mouse lung tissues using TRIzol reagent. qPCR was performed using SYBR Green Master Mix to quantify the expression levels of target genes, with GAPDH used as the internal reference. Relative gene expression was calculated using the 2⁻ΔΔCt method. Primer sequences are listed in Table S2.

Hemolysis assay

Red blood cells were isolated from anticoagulated mouse blood by centrifugation at 1000 rpm for 5 min, washed three times with PBS, and diluted to a 2% suspension. Equal volumes of red blood cell suspension and pDA/Esc@ZIF-8 solutions (1–200 µg/mL) were mixed and incubated at 37 °C for 1 h. PBS and deionized water served as negative and positive controls, respectively. After centrifugation at 3000 rpm for 15 min, absorbance of the supernatant was measured at 542 nm.

Establishment of the ALI mouse model

Male BALB/c mice aged 6–8 weeks were anesthetized with isoflurane and placed in a supine position at a 45° angle. After tracheal intubation, LPS dissolved in sterile saline was intratracheally instilled at a dose of 5 mg/kg in a total volume of 50 µL to establish the ALI model. Two hours after LPS challenge, mice were randomly assigned to six groups (n = 6 per group): Control, ALI, ZIF-8, Esc, pDA@ZIF-8, and pDA/Esc@ZIF-8. Mice in the treatment groups received a single tail-vein injection of the corresponding formulation at a dose of 1 mg/kg in 200 µL PBS. Mice in the Control and ALI groups received an equal volume of PBS via tail-vein injection. All treatments were administered once only. Lung tissues and bronchoalveolar lavage fluid were collected at the indicated experimental endpoints for subsequent histological, inflammatory, oxidative stress, and molecular analyses.

H&E staining

Twenty-four hours after ALI induction, mice were sacrificed, and lung tissues were fixed, embedded, sectioned, and stained with hematoxylin and eosin. Histopathological changes were examined under a light microscope.

Lung function assessment

Lung function was evaluated using non-invasive whole-body plethysmography (WBP) at 24 h after ALI modeling and treatment. Mice were placed individually in a plethysmography chamber and allowed to acclimate for 10 min in an unrestrained, conscious state. Respiratory parameters, including expiratory flow at 50% tidal volume (EF50), tidal volume normalized to body weight (TV/BW), enhanced pause (Penh), and end-expiratory pause (EEP), were recorded continuously for 5 min using a calibrated system. Data from at least 50 consecutive breaths per mouse were averaged for analysis. All measurements were performed in a quiet environment at consistent temperature and humidity.

Bronchoalveolar lavage fluid (BALF) protein content and cell count

BALF was centrifuged at 3000 rpm for 10 min at 4 °C. Protein concentration was determined using a BCA assay. Cell pellets were resuspended in PBS and counted using an automated cell counter.

Lung wet-to-dry weight ratio

Lung tissues were weighed to obtain wet weight (W), dried at 70 °C to constant weight, and weighed again to obtain dry weight (D). The W/D ratio was calculated to assess pulmonary edema.

Survival analysis

Severe ALI was induced using high-dose LPS (50 mg/kg). Mice were treated with PBS or pDA/Esc@ZIF-8 and monitored for 7 days. Survival data were analyzed using the Kaplan–Meier method, and differences between survival curves were evaluated using the log-rank (Mantel–Cox) test.

Immunofluorescence staining

Lung tissue sections were subjected to antigen retrieval, permeabilization, and blocking, followed by incubation with primary and fluorescent secondary antibodies. Nuclei were counterstained with DAPI, and images were acquired using confocal microscopy.

Target prediction and network pharmacology analysis

Potential targets of Esc were identified using SwissTargetPrediction, SEA, HERB, STITCH, SuperPred, and PharmMapper databases. ALI-related targets were retrieved from GeneCards, OMIM, and HERB databases. Common targets were identified and subjected to further analysis.

PPI network construction and pathway analysis

Common targets were imported into the STRING database to construct a PPI network, which was visualized using Cytoscape. Core targets were identified based on degree values.

GO and KEGG enrichment analysis

GO and KEGG enrichment analyses were conducted using R software and online platforms to identify key biological processes and signaling pathways.

Molecular docking

Ligand and receptor structures were prepared and docked using AutoDockTools. Binding energies were calculated to evaluate interaction stability.

Western blot

Protein samples were extracted, quantified, separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary and secondary antibodies. Protein bands were visualized using ECL reagents and quantified using ImageJ.

RNA sequencing (RNA-seq)

RNA-seq was performed on lung tissues from ALI and pDA/Esc@ZIF-8-treated mice. Library preparation, sequencing, and data processing were conducted by a commercial service provider. Differentially expressed genes were identified using edgeR, followed by GO and KEGG enrichment analyses.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software. All quantitative data are presented as mean ± SD. For comparisons between two groups, a two-tailed Student’s t-test was used when appropriate. Survival curves were generated using the Kaplan–Meier method and compared using the log-rank test. Mice were randomly assigned to treatment groups after ALI induction. Histological scoring was performed in a blinded manner by investigators unaware of group allocation. A value of P < 0.05 was considered statistically significant. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; ns indicates non-significant differences.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

X. D. and Y. W. conceived and designed the study, with X. D. supervising all aspects of the research. Y. X., E. H., Z. H., and Y. W. conducted the experiments, analyzed the data, and prepared the figures. Y.X. and E.H. wrote the manuscript.

Funding

This research was supported by the Joint Project on Regional High-Incidence Diseases Research of Guangxi Natural Science Foundation (No. 2025GXNSFAA069718); the Guangxi Zhuang Autonomous Region Clinical Key Specialty Construction Project (No. 2025073); and the Fund of The Scientific Research Projects of The Second Affiliated Hospital of Guangxi Medical University (No. EFYKY202004).

Data availability

Data will be made available on request.

Declarations

Ethics approval and consent to participate

All animal experiments were conducted in accordance with the regulations of the Institutional Animal Care and Use Committee of Guangxi Medical University (No. 202401032).

Consent for publication

All authors agree to publication.

Competing interests

The authors declare no competing interests.