Targeted inhibition of PARP-1 in pulmonary epithelial cells and macrophages via SPA-functionalized microparticles attenuates sepsis-induced lung injury

TL;DR

A new targeted therapy using PARP-1 inhibitors delivered via specialized microparticles reduces lung damage and improves survival in sepsis.

Contribution

A cell-specific, lung-targeted nanotherapeutic using PARP-1 inhibition is developed and shown to effectively treat sepsis-induced lung injury.

Findings

Pulmonary epithelial cells and macrophages are identified as key pro-inflammatory hubs in septic lungs.

OLA@SPA MPs significantly reduce lung injury, cytokine storm, and improve survival in sepsis models.

The therapy reverses sepsis-associated gene signatures, particularly in NOD-like receptor and TNF signaling pathways.

Abstract

Sepsis-induced acute lung injury (ALI) is a life-threatening condition with limited therapeutic options, driven by a dysregulated inflammatory response within the pulmonary microenvironment. Although hyperactivation of poly (ADP-ribose) polymerase-1 (PARP-1) is recognized as a key contributor to inflammation and cellular injury, its cell type–specific roles in sepsis and strategies for targeted inhibition remain insufficiently explored. In this study, we first identified pulmonary epithelial cells and macrophages as major pro-inflammatory hubs in the septic lung using single-cell RNA sequencing. Based on these findings, we engineered a lung-targeted nanotherapeutic by encapsulating the PARP-1 inhibitor olaparib (OLA) into surfactant protein A (SPA)-functionalized microparticles (OLA@SPA MPs). The OLA@SPA MPs exhibited enhanced pulmonary accumulation and efficient internalization by target cells, resulting in robust suppression of PARP-1 activation. In murine models of sepsis, treatment with OLA@SPA MPs markedly reduced vascular leakage, modulated the cytokine storm, attenuated lung histopathological damage, and significantly improved survival. Mechanistically, transcriptomic analyses revealed that OLA@SPA MPs reversed sepsis-associated gene expression signatures, particularly by downregulating key pro-inflammatory pathways such as NOD-like receptor and tumor necrosis factor (TNF) signaling. Collectively, this work establishes a targeted therapeutic paradigm that translates mechanistic insights into an effective intervention for sepsis-induced lung injury.

Image 1

•Single-Cell Guided Target Identification: Single-cell RNA sequencing of the septic lung microenvironment identified pulmonary epithelial cells and macrophages as central pro-inflammatory hubs, revealing their specific upregulation of PARP-1 and providing a foundational rationale for cell-specific targeting.•Innovative Biomimetic Nanotherapeutic: A novel lung-targeted delivery system was engineered by encapsulating the PARP-1 inhibitor Olaparib into surfactant protein A (SPA)-functionalized microparticles (OLA@SPA-MPs), which demonstrated enhanced pulmonary accumulation and efficient internalization by the target epithelial cells and macrophages.•Potent Therapeutic Efficacy and Survival Benefit: In murine sepsis models, OLA@SPA-MPs treatment robustly attenuated lung injury, modulated the systemic and local cytokine storm, and most importantly, conferred a significant survival advantage, outperforming non-targeted formulations and free drug.•Comprehensive Mechanistic Elucidation: The therapeutic action was mechanistically linked to the potent inhibition of PARP-1 hyperactivation and the downstream parthanatos pathway in target cells, coupled with a transcriptomic reprogramming that reversed sepsis-associated gene signatures, particularly in key pro-inflammatory pathways like NOD-like receptor and TNF signaling.

Single-Cell Guided Target Identification: Single-cell RNA sequencing of the septic lung microenvironment identified pulmonary epithelial cells and macrophages as central pro-inflammatory hubs, revealing their specific upregulation of PARP-1 and providing a foundational rationale for cell-specific targeting.

Innovative Biomimetic Nanotherapeutic: A novel lung-targeted delivery system was engineered by encapsulating the PARP-1 inhibitor Olaparib into surfactant protein A (SPA)-functionalized microparticles (OLA@SPA-MPs), which demonstrated enhanced pulmonary accumulation and efficient internalization by the target epithelial cells and macrophages.

Potent Therapeutic Efficacy and Survival Benefit: In murine sepsis models, OLA@SPA-MPs treatment robustly attenuated lung injury, modulated the systemic and local cytokine storm, and most importantly, conferred a significant survival advantage, outperforming non-targeted formulations and free drug.

Comprehensive Mechanistic Elucidation: The therapeutic action was mechanistically linked to the potent inhibition of PARP-1 hyperactivation and the downstream parthanatos pathway in target cells, coupled with a transcriptomic reprogramming that reversed sepsis-associated gene signatures, particularly in key pro-inflammatory pathways like NOD-like receptor and TNF signaling.

Figures

Identification of the pro-inflammatory characteristics of macrophages and epithelial cells in the pulmonary immune microenvironment of CLP mice using single-cell sequencing database ([GSE207651](GSE207651)). (A) UMAP plot displaying the major cell types distribution in the pulmonary immune microenvironment of CLP mice. (B) Violin plot showed marker genes' expression levels in each cell type. (C) Identification of the differences in gene expression of relevant pro-inflammatory cytokines between pulmonary epithelial cells from CLP mice and those from healthy individuals. (D) Identification of th

Identification in a mouse model of sepsis, and preparation of OLA@SPA-MPs. (A, B) Representative H&E-stained images of lung tissue and corresponding lung injury scores in Control and LPS-treated mice. Scale bar = 100 μm, (n = 6). (C) Wet-to-dry (W/D) weight ratio of lung tissue in each group (n = 6). (D) Protein content in bronchoalveolar lavage fluid (BALF) determined by BCA assay (n = 6). Representative Western blots showing PARP-1 and PAR protein levels in MLE-12 cells (E), MH-S cells (F) and lung tissue (G) from Control and LPS groups. (H-I) ELISA quantification of inflammatory cytokines i

Evaluate the protective effects of OLA@SPA MPs against LPS-induced injury in pulmonary epithelial cells and macrophages in vitro. (A) Cell viability of MLE-12 cells exposed to increasing concentrations of LPS (0–1500 μg mL^−1^) assessed by the CCK-8 assay (n = 4). (B) Protective effect of OLA (0–80 μg mL^−1^) on MLE-12 cell viability following 600 μg mL^−1^ LPS exposure (n = 4). (C, D) CCK8 assay to detect the viability of MLE-12 (C) and MH-S (D) cells after indicated treatments (n = 4). (E, F) Flow cytometric analysis of cellular uptake efficiency in MLE-12 (E) and MH-S cells (F) incubated wi

Assessment of the in vivo targeting effects of OLA@SPA MPs on lung epithelial cells and macrophages and their therapeutic effects on the septic mice model. (A) Representative IVIS images showing fluorescence signals in major organs at 6, 12 and 24 h post-injection after i.v. Injection of indicated treatment. (B) Fluorescence distribution specifically within the lungs at the same time points for the OLA@MPs group and OLA@SPA MPs group. (C) Quantitative comparison of radiant efficiency to evaluate indicated microparticle accumulation. (D, E) Representative immunofluorescence images of lung tissu

Identification of the mechanism of OLA@SPA MPs in treating a sepsis mouse model. (A) Protein concentrations in BALF determined by BCA assay across experimental groups (n = 6). (B) Wet-to-dry (W/D) weight ratios of lung tissue in each group (n = 6). (C–F) Serum levels of the pro-inflammatory cytokines IL-6 (C), TNF-α (D), IL-1β (E) and the anti-inflammatory cytokine IL-10 (F) at 24 h post-LPS injection (n = 6). (G–J) Corresponding cytokine concentrations in BALF at 24 h after LPS administration (n = 6), including IL-6 (G), TNF-α (H), IL-1β (I) and the anti-inflammatory cytokine IL-10 (J). (K, L

Full text

Introduction

1

Sepsis is defined as a dysregulated host response to infection that can result in life-threatening organ dysfunction [1,2], with the lung being one of the most frequently and severely affected organs. The pathogenesis of sepsis-induced ALI is characterized by an uncontrolled inflammatory cascade, increased vascular permeability, and extensive infiltration of immune cells into pulmonary tissues [[3], [4], [5]]. Within this complex microenvironment, resident alveolar epithelial cells and macrophages are not merely passive bystanders but active drivers of the inflammatory storm [6], although their specific roles and interactions have yet to be fully elucidated at the single-cell level [[7], [8], [9]]. Current clinical management of sepsis remains largely supportive, underscoring an urgent need for therapeutic strategies that directly target the cellular mediators of disease pathology (see Scheme 1).Scheme 1. We developed a targeted nanotherapeutic by engineering surfactant protein A (SPA)-functionalized microparticles loaded with the PARP-1 inhibitor Olaparib (OLA@SPA MPs). This biomimetic system efficiently homes to the lung and is internalized by target cells, where it potently inhibits PARP-1 hyperactivation. This intervention suppresses the cytokine storm, attenuates parthanatos, and mitigates lung injury, significantly improving survival in a murine sepsis model, thereby presenting a novel targeted strategy for treating sepsis-induced lung injury.Scheme 1

poly (ADP-ribose) polymerase-1 (PARP-1), the predominant isoform of the ADP-ribosylating enzyme family, plays a central role in regulating DNA repair, gene transcription, and cell death, and mounting evidence suggests that its activation is a critical mediator of the pathological processes underlying acute lung injury (ALI) [[10], [11], [12], [13]]. Activation of PARP-1 represents a pivotal cellular response to stress and injury [[14], [15], [16]]. During sepsis, oxidative stress–induced DNA damage leads to excessive PARP-1 activation [17], resulting in aberrant poly(ADP-ribosyl)ation (PARylation), depletion of intracellular NAD^+^ and ATP, and ultimately a distinct form of programmed cell death known as parthanatos [[18], [19], [20]]. This process further amplifies inflammation and tissue damage. Although pharmacological inhibition of PARP-1 has demonstrated therapeutic potential in preclinical inflammatory models, its clinical translation is hampered by off-target effects and inefficient delivery to specific pathogenic cell populations within injured organs.

Recent advances in nanomedicine provide promising strategies to overcome these limitations [[21], [22], [23]]. Biomimetic drug delivery systems, particularly those functionalized with endogenous targeting ligands, have shown the ability to enhance tissue-specific accumulation and improve cellular selectivity [24,25]. Surfactant protein A (SPA), a collectin predominantly expressed in the lung [26,27], plays an essential role in innate immune defense and interacts with receptors expressed on alveolar epithelial cells and macrophages [28,29]. These characteristics make SPA an attractive ligand for the design of lung-targeted therapeutics. However, to date, a delivery platform that simultaneously achieves precise targeting of pathogenic pulmonary cells and effective inhibition of a central inflammatory driver such as PARP-1 has not been established for sepsis.

In this study, we employed single-cell RNA sequencing to systematically dissect the pulmonary immune microenvironment in sepsis, identifying epithelial cells and macrophages as primary pro-inflammatory contributors. Guided by these insights, we developed a targeted nanotherapeutic by loading the PARP-1 inhibitor olaparib into SPA-modified microparticles (OLA@SPA MPs). We comprehensively evaluated the targeting efficiency, therapeutic efficacy, and underlying mechanisms of OLA@SPA MPs both in vitro and in vivo. Our findings demonstrate that this targeted strategy effectively alleviates PARP-1–mediated lung injury and inflammation, reprograms the septic transcriptomic landscape, and significantly improves survival outcomes, thereby offering a novel and promising therapeutic approach for sepsis.

Results

2

Single-cell transcriptomic profiling identifies pulmonary epithelial cells and macrophages as pro-inflammatory hubs in septic lungs

2.1

To systematically characterize the pulmonary immune microenvironment in sepsis, we performed a comprehensive reanalysis of a publicly available single-cell RNA sequencing dataset (GSE207651) derived from lung tissues of cecal ligation and puncture (CLP)-induced septic mice and healthy controls. This analysis aimed to identify key cellular populations and molecular mechanisms driving pulmonary inflammation during sepsis. First, the overall cellular landscape of the lung immune microenvironment was visualized using uniform manifold approximation and projection (UMAP). As shown in Fig. 1A, major cell populations—including macrophages, epithelial cells, T cells, neutrophils, and other immune and structural cells—were clearly delineated, providing a global framework for subsequent analyses. The identities of these clusters were further validated by examining the expression of established marker genes using violin plots (Fig. 1B), confirming the accuracy of cell-type annotation and ensuring the reliability of downstream analyses. Given the central role of dysregulated inflammation in sepsis pathogenesis, we next focused on the pro-inflammatory characteristics of pulmonary epithelial cells and macrophages. Comparative analysis revealed that pulmonary epithelial cells from CLP mice exhibited a significant upregulation of multiple pro-inflammatory cytokine genes relative to healthy controls (Fig. 1C). Similarly, pulmonary macrophages isolated from septic lungs displayed a markedly enhanced pro-inflammatory gene expression signature (Fig. 1D), indicating that both cell types acquire a pronounced inflammatory phenotype during sepsis.Fig. 1. Identification of the pro-inflammatory characteristics of macrophages and epithelial cells in the pulmonary immune microenvironment of CLP mice using single-cell sequencing database (GSE207651). (A) UMAP plot displaying the major cell types distribution in the pulmonary immune microenvironment of CLP mice. (B) Violin plot showed marker genes' expression levels in each cell type. (C) Identification of the differences in gene expression of relevant pro-inflammatory cytokines between pulmonary epithelial cells from CLP mice and those from healthy individuals. (D) Identification of the differences in gene expression of relevant pro-inflammatory cytokines between pulmonary macrophage cells from CLP mice and those from healthy individuals. (E) Characterize the expression profile of Parp1 in various cell subpopulations of the pulmonary microenvironment from healthy controls and CLP mice. (F) Analysis of the expression of pulmonary surfactant protein (SPA) related receptors in different cellular subpopulations of the pulmonary microenvironment in CLP mice. (G) KEGG pathway enrichment of the DEGs of pulmonary epithelial cells from CLP mice and those from healthy individuals. (H) KEGG pathway enrichment of the DEGs of pulmonary macrophage from CLP mice and those from healthy individuals. (I) Comparison of significant ligand-receptor pairs between pulmonary epithelial cells and other cell type. (J) Comparison of significant ligand-receptor pairs between pulmonary macrophage and other cell type. The color of the dots reflects the communication probability, and the size of the dots represents the calculated P-value. Blank cells indicate zero communication probability. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)Fig. 1

We further examined the expression of Parp1, a key mediator of inflammatory responses and cellular stress, across different lung cell subpopulations. As illustrated in Fig. 1E, Parp1 expression was notably elevated in specific cell types within the septic pulmonary microenvironment, particularly in epithelial cells and macrophages, suggesting its potential involvement in sepsis-associated lung pathology. Given the critical role of pulmonary surfactant in maintaining lung homeostasis and its disruption during lung injury, we next analyzed the expression patterns of surfactant protein A (SPA)-related receptors. Fig. 1F shows the distribution of SPA-associated receptor expression across different lung cell populations in CLP mice, highlighting epithelial cells and macrophages as potential SPA-responsive targets during sepsis. To gain mechanistic insights into the inflammatory programs activated in these cells, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on differentially expressed genes (DEGs) from pulmonary epithelial cells (Fig. 1G) and macrophages (Fig. 1H) of septic versus healthy mice. Both cell types exhibited significant enrichment in inflammation- and immune-related pathways, including NF-κB signaling, TNF signaling, cytokine–cytokine receptor interaction, and NOD-like receptor signaling, confirming their active participation in pro-inflammatory signaling networks during sepsis. Finally, to investigate alterations in intercellular communication within the septic lung microenvironment, ligand–receptor interaction analysis was conducted. Fig. 1I compares communication probabilities between pulmonary epithelial cells and other cell types under septic and healthy conditions, revealing enhanced and specific interactions during sepsis. Likewise, Fig. 1J demonstrates altered communication patterns involving pulmonary macrophages. In these dot plots, color intensity reflects communication probability, while dot size indicates statistical significance, collectively identifying key ligand–receptor pairs that are selectively strengthened during sepsis and may contribute to sustained inflammatory amplification.

In summary, this integrated single-cell transcriptomic analysis demonstrates that pulmonary epithelial cells and macrophages undergo profound pro-inflammatory reprogramming during sepsis. This is characterized by increased cytokine expression, elevated levels of injury-related molecules such as Parp1 and SPA-associated receptors, activation of multiple inflammatory signaling pathways, and enhanced intercellular communication. These findings identify pulmonary epithelial cells and macrophages as central drivers of lung inflammation in sepsis and highlight them as promising cellular targets for Microparticles-based targeted therapeutic strategies.

Establishment of a sepsis-induced lung injury model and engineering of SPA-functionalized microparticles for targeted olaparib delivery

2.2

To establish a robust in vivo model for studying sepsis-induced lung injury, mice were subjected to lipopolysaccharide (LPS) challenge [30]. Histopathological examination of lung tissues using hematoxylin and eosin (H&E) staining revealed pronounced structural damage in LPS-treated mice, characterized by alveolar wall thickening, inflammatory cell infiltration, and hemorrhage, changes that were largely absent in the control group (Fig. 2A). Consistently, quantitative lung injury scores were significantly elevated in the LPS group (Fig. 2B). Further evidence of lung injury was provided by a marked increase in the lung wet-to-dry (W/D) weight ratio (Fig. 2C), indicating pulmonary edema, as well as elevated protein levels in the bronchoalveolar lavage fluid (BALF) (Fig. 2D), reflecting increased vascular permeability [31]. Given the central role of poly(ADP-ribose) polymerase-1 (PARP-1) in cellular stress responses and inflammation, we next assessed its activation in this model. Western blot analysis demonstrated a substantial upregulation of PARP-1 and its downstream product poly(ADP-ribose) (PAR) in mouse lung epithelial cells (MLE-12), macrophage cells (MH-S), and whole lung tissue homogenates from LPS-treated mice compared with controls (Fig. 2E–G), confirming hyperactivation of the PARP-1 pathway in key pulmonary cell types during sepsis. Consistent with a systemic inflammatory response, ELISA analysis demonstrated significantly elevated levels of pro-inflammatory cytokines (including IL-1β, IL-6, and TNF-α) and reduced levels of the anti-inflammatory cytokine IL-10 in both the serum (Fig. 2H) and bronchoalveolar lavage fluid (BALF) (Fig. 2 I) of LPS-challenged mice [32]. Collectively, these findings confirm the successful establishment of a sepsis-induced lung injury model accompanied by robust PARP-1 activation.Fig. 2. Identification in a mouse model of sepsis, and preparation of OLA@SPA-MPs. (A, B) Representative H&E-stained images of lung tissue and corresponding lung injury scores in Control and LPS-treated mice. Scale bar = 100 μm, (n = 6). (C) Wet-to-dry (W/D) weight ratio of lung tissue in each group (n = 6). (D) Protein content in bronchoalveolar lavage fluid (BALF) determined by BCA assay (n = 6). Representative Western blots showing PARP-1 and PAR protein levels in MLE-12 cells (E), MH-S cells (F) and lung tissue (G) from Control and LPS groups. (H-I) ELISA quantification of inflammatory cytokines in serum (H) and BALF (I) of each group (n = 6). (J) Schematic overview of the experimental workflow. (K) Determination of electroporation conditions load OLA in MPs-SPA. 100 μg OLA was mixed with 100 μg MPs-SPA in system. (L-M) The most appropriate OLA-to-MPs-SPA ratio for maximizing OLA incorporation was determined by fixing the MPs-SPA mass at 100 μg and quantifying OLA concentration via HPLC. The relative OLA loading efficiency under varying OLA doses with fixed electroporation parameters is presented in (M). (N-O) Zeta potential (N) and particle size (O) of MPs, SPA MPs, OLA@MPs, and OLA@SPA MPs were measured using a Malvern laser particle size analyzer. (P) Representative TEM images of MPs, SPA MPs, OLA@MPs, and OLA@SPA MPs. (Q) Western blot analysis of exosomal markers Alix, CD63, and TSG101 in MPs, SPA MPs, OLA@MPs, and OLA@SPA MPs. (R) Targeting ligand SPA expression was quantified at the mRNA level via qRT-PCR relative to control cells (n = 3). (S) Western blot analysis of SFTPA1 in MPs and SPA MPs. (T) HPLC profile of OLA in OLA@SPA MPs. (U-V) Stability of OLA@SPA MPs in mouse serum was evaluated by incubating the particles at 37 °C for various durations; OLA concentrations were measured and relative levels calculated. Data are presented as mean ± SEM. Statistical analyses were performed using one-way ANOVA and two-tailed Student's t-test. ∗∗P < 0.01 and ∗∗∗P < 0.001.Fig. 2

To develop a targeted therapeutic strategy, we engineered a drug delivery system designed to selectively deliver the PARP-1 inhibitor olaparib (OLA) to lung epithelial cells and macrophages. The overall experimental workflow is illustrated in Fig. 2J. Briefly, a stable mouse lung epithelial MLE-12 cell line expressing pulmonary surfactant protein A (SPA) on the cell membrane was generated, from which SPA-functionalized Microparticles (SPA MPs) were harvested. Olaparib was subsequently loaded into the SPA MPs using electroporation [33]. Optimization of electroporation parameters was first performed to maximize drug encapsulation efficiency (Fig. 2K). By fixing the mass of SPA MPs and varying the amount of olaparib, we determined the optimal drug-to-carrier ratio. High-performance liquid chromatography (HPLC) analysis demonstrated that the amount of encapsulated olaparib increased proportionally with the initial loading dose (Fig. 2L), and the loading efficiency reached its maximum at a ratio of 2:1, which was therefore selected for all subsequent experiments (Fig. 2M).

The physicochemical properties of the engineered Microparticles were then systematically characterized. Dynamic light scattering analysis showed that the zeta potential became more negative following SPA functionalization and olaparib loading (Fig. 2N), while particle size remained comparable among different formulations (Fig. 2O). Transmission electron microscopy (TEM) confirmed a spherical, vesicular morphology of all Microparticle formulations, with no apparent structural alterations induced by SPA modification or drug loading (Fig. 2P). To further validate the vesicular characteristics, the expression of canonical extracellular vesicle markers was examined [34]. Western blot analysis confirmed the presence of Alix, CD63, and TSG101 in all Microparticle groups (Fig. 2Q). Successful SPA engineering was further supported by significantly increased SPA mRNA expression in donor cells (Fig. 2R) and the specific detection of SFTPA1 protein in SPA MPs and OLA@SPA MPs, but not in unmodified Microparticles (Fig. 2S). Drug encapsulation was additionally verified by HPLC, which revealed a characteristic olaparib peak in the OLA@SPA MPs formulation (Fig. 2T). Finally, the stability of OLA@SPA MPs was evaluated under biologically relevant conditions. Incubation in mouse serum at 37 °C demonstrated that the Microparticles maintained structural integrity and exhibited a sustained, slow release of olaparib over time, indicating excellent serum stability (Fig. 2U and V). In summary, we successfully established a murine model of sepsis-induced lung injury with confirmed PARP-1 hyperactivation and developed a well-characterized, stable, SPA-functionalized Microparticle system for the targeted delivery of olaparib.

Evaluation of the protective effects of OLA@SPA MPs against LPS-induced injury in pulmonary epithelial cells and macrophages in vitro

2.3

To evaluate the in vitro therapeutic potential of engineered Microparticles, we first established lipopolysaccharide (LPS)-induced injury models in mouse lung epithelial cells (MLE-12) and macrophages (MH-S). Cell Counting Kit-8 (CCK-8) assays demonstrated that exposure to 600 μg mL^−1^ LPS for MLE-12 cells and 150 μg mL^−1^ LPS for MH-S cells for 1 h significantly reduced cell viability (Fig. 3A, Fig. S1A). These concentrations were therefore selected to establish in vitro models of septic injury. We next assessed the intrinsic cytoprotective effect of olaparib (OLA). Pretreatment with OLA dose-dependently restored cell viability in both LPS-injured MLE-12 cells (Fig. 3B) and MH-S cells (Fig. S1B), with 5 μg mL^−1^ identified as an effective and non-cytotoxic concentration. Based on these findings, we proceeded to evaluate the therapeutic performance of the targeted delivery system. Notably, treatment with OLA@SPA MPs conferred the most pronounced protective effect, significantly restoring cell viability in both MLE-12 and MH-S cells compared with the LPS-injured group as well as groups treated with non-targeted OLA@MPs (Fig. 3C and D**)**. This superior efficacy suggested enhanced intracellular delivery of the active drug mediated by SPA functionalization.Fig. 3. Evaluate the protective effects of OLA@SPA MPs against LPS-induced injury in pulmonary epithelial cells and macrophages in vitro. (A) Cell viability of MLE-12 cells exposed to increasing concentrations of LPS (0–1500 μg mL^−1^) assessed by the CCK-8 assay (n = 4). (B) Protective effect of OLA (0–80 μg mL^−1^) on MLE-12 cell viability following 600 μg mL^−1^ LPS exposure (n = 4). (C, D) CCK8 assay to detect the viability of MLE-12 (C) and MH-S (D) cells after indicated treatments (n = 4). (E, F) Flow cytometric analysis of cellular uptake efficiency in MLE-12 (E) and MH-S cells (F) incubated with DiO-labeled MPs, SPA MPs, OLA@MPs, or OLA@SPA MPs, expressed as mean fluorescence intensity (MFI). (G) Comparison of DiO-labeled MPs internalization among L929, DC2.4, MH-S, and MLE-12 cells by using flow cytometry. (H) Western blot analysis of CKAP4 in MLE-12 and MH-S. (I, J) Flow cytometric analysis of cellular uptake efficiency in MLE-12 (I) and MH-S cells (J) incubated with DiO-labeled MPs, SPA MPs, or SPA MPs (CKAP4 blocked), expressed as mean fluorescence intensity (MFI). (K, M) Quantification of dead cells populations in MLE-12 and MH-S cells via Annexin V/PI staining followed by flow cytometry. (L, N) Immunofluorescence images showing PARP-1 and PAR localization in MLE-12 and MH-S cells under different treatment conditions, Scale bar = 20 μm. Data are shown as mean ± SEM, and statistical analysis was performed using one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.Fig. 3

To elucidate the mechanism underlying the enhanced efficacy of OLA@SPA MPs, we next investigated cellular uptake using DiO-labeled MPs. Flow cytometric analysis revealed that the mean fluorescence intensity (MFI) was significantly higher in both MLE-12 and MH-S cells treated with SPA-functionalized MPs (SPA MPs and OLA@SPA MPs) compared with cells exposed to unmodified Microparticles (MPs and OLA@MPs) (Fig. 3E and F). These results were further corroborated by immunofluorescence imaging (Fig. S1C and D). Moreover, comparative uptake studies across different cell types, including fibroblasts (L929) and dendritic cells (DC2.4), demonstrated that DiO-labeled MPs were preferentially internalized by pulmonary-derived MLE-12 and MH-S cells (Fig. 3G). Western blot analysis showed higher expression of CKAP4 in MLE-12 cells than in MH-S cells (Fig. 3H). Pre-blocking CKAP4 significantly reduced the cellular uptake of SPA-MPs, confirming that SPA-mediated targeting is dependent on CKAP4-specific receptor interaction (Fig. 3I and J). Collectively, these findings indicate that SPA modification significantly enhances Microparticle uptake by target lung epithelial cells and macrophages [35]. We next explored the molecular pathways through which OLA@SPA MPs exert cytoprotective effects. Given that PARP-1 hyperactivation is a critical driver of inflammation-associated cell death [36,37], we assessed its activity following treatment. Immunofluorescence staining revealed that LPS stimulation markedly increased the nuclear accumulation of PARP-1 and its enzymatic product poly(ADP-ribose) (PAR) in both MLE-12 and MH-S cells. Treatment with OLA@SPA MPs potently suppressed this effect and was markedly more effective than free OLA or non-targeted OLA@MPs (Fig. 3L–N). Consistently, Western blot analysis confirmed that OLA@SPA MPs most effectively reduced PAR levels in both cell types (Fig. S1E and F).

A key downstream consequence of PARP-1 overactivation is the induction of parthanatos, a regulated cell death pathway characterized by mitochondrial-to-nuclear translocation of apoptosis-inducing factor (AIF) [38,39]. Immunofluorescence analysis demonstrated that LPS exposure triggered pronounced AIF nuclear translocation in both MLE-12 and MH-S cells, whereas this process was markedly attenuated following treatment with OLA@SPA MPs (Fig. S1G and H). In line with these observations, flow cytometric analysis of Annexin V/PI staining revealed that OLA@SPA MPs most effectively reduced the proportion of apoptotic and dead cells in both MLE-12 and MH-S cultures following LPS challenge (Fig. 3K–M). In summary, these in vitro data demonstrate that SPA-functionalized MPs are efficiently internalized by pulmonary epithelial cells and macrophages, resulting in potent inhibition of PARP-1 hyperactivation and its downstream parthanatos pathway. This targeted strategy effectively protects critical lung-resident cells from LPS-induced cytotoxicity, highlighting the therapeutic promise of OLA@SPA MPs for the treatment of sepsis-associated lung injury.

OLA@SPA MPs exhibit superior lung-targeting efficiency and robust protection in septic mice

2.4

Having established the in vitro efficacy of the targeted microparticles, we next evaluated their biodistribution and therapeutic potential in a murine model of sepsis. To assess targeting efficiency, mice were intravenously injected with fluorescently labeled OLA@MPs or OLA@SPA MPs, and particle distribution was monitored using an in vivo imaging system (IVIS). Representative whole-body images, together with subsequent ex vivo organ analyses performed at 6, 12, and 24 h post-injection, revealed distinct biodistribution patterns. Although both formulations were detectable in major organs, the OLA@SPA MPs group exhibited a markedly stronger and more sustained fluorescence signal in the lungs compared with the OLA@MPs group (Fig. 4A and B). Quantitative analysis of radiant efficiency further confirmed significantly higher and prolonged pulmonary accumulation of OLA@SPA MPs over time (Fig. 4C). To further verify cell-type-specific uptake within lung tissue, immunofluorescence staining was performed on lung sections. Consistent with the biodistribution data, OLA@SPA MPs displayed markedly enhanced co-localization with CD326^+^ pulmonary epithelial cells (Fig. 4D) and F4/80^+^ macrophages (Fig. 4E) compared with non-targeted OLA@MPs. These findings demonstrate that SPA functionalization effectively promotes lung homing and cellular internalization of the microparticles by their intended target cells within the septic lung microenvironment.Fig. 4. Assessment of the in vivo targeting effects of OLA@SPA MPs on lung epithelial cells and macrophages and their therapeutic effects on the septic mice model. (A) Representative IVIS images showing fluorescence signals in major organs at 6, 12 and 24 h post-injection after i.v. Injection of indicated treatment. (B) Fluorescence distribution specifically within the lungs at the same time points for the OLA@MPs group and OLA@SPA MPs group. (C) Quantitative comparison of radiant efficiency to evaluate indicated microparticle accumulation. (D, E) Representative immunofluorescence images of lung tissue 24 h after treatment, depicting internalization of OLA@MPs and OLA@SPA MPs by CD326^+^ epithelial cells (D) and F4/80^+^ macrophages (E), scale bar = 200 μm. (F) Survival curves of septic mice following indicated treatments (n = 12). (G) Clinical scores recorded 24 h after indicated treatment (n = 10). (H) Lung injury scores at 24 h post-indicated treatment (n = 6). (I) HE staining of tissues from different groups, scale bar = 100 μm. Data are expressed as mean ± SEM. Statistical significance was assessed by one-way ANOVA and two-tailed Student's t-test: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.Fig. 4

We next investigated the therapeutic efficacy of the targeted delivery system in vivo. Survival analysis revealed that septic mice treated with OLA@SPA MPs exhibited a significantly improved survival rate compared with those receiving saline, free OLA, or non-targeted OLA@MPs (Fig. 4F). This pronounced survival benefit was accompanied by a significant improvement in clinical scores at 24 h post-treatment, indicating an overall attenuation of disease severity (Fig. 4G). Furthermore, evaluation of lung injury confirmed the robust protective effect of OLA@SPA MPs. Consistently, quantitative lung injury scores were significantly lower in mice treated with OLA@SPA MPs (Fig. 4H). Histopathological examination by H&E staining demonstrated that lungs from OLA@SPA MPs–treated mice showed substantially reduced tissue damage, characterized by decreased inflammatory cell infiltration, alleviated alveolar wall thickening, and diminished hemorrhage relative to the other treatment groups (Fig. 4I). In summary, these in vivo results demonstrate that SPA-functionalized MPs efficiently target the pulmonary microenvironment and are preferentially internalized by key pathogenic cell populations. This targeted delivery of OLA translates into superior therapeutic efficacy, leading to marked attenuation of lung injury and, critically, a significant improvement in survival in a lethal murine model of sepsis. In the CLP model, OLA@SPA MPs significantly improved survival compared with untreated CLP mice (Fig. S3A). Histological analysis revealed attenuated alveolar damage and inflammatory infiltration. Lung edema and pro-inflammatory cytokine levels (IL-6 and TNF-α) were markedly reduced, confirming the therapeutic efficacy of OLA@SPA MPs in a clinically relevant sepsis model (Fig. S3B–D). Lungs of mice receiving OLA@SPA MPs showed a substantial alleviation of histopathological damage on H&E-stained sections, as indicated by reduced inflammation, alveolar wall thickening, and hemorrhage relative to other groups (Fig. S3E). This morphological improvement coincided with a significant lowering of the quantified lung injury score (Fig. S3F).

Identification of the mechanism of OLA@SPA MPs in treating a sepsis mouse model

2.5

To elucidate the mechanisms underlying the therapeutic benefits of OLA@SPA MPs, we first evaluated key biomarkers of lung injury and systemic inflammation. Consistent with the observed histological improvement, treatment with OLA@SPA MPs significantly reduced the protein concentration in bronchoalveolar lavage fluid (BALF) (Fig. 5A) as well as the lung wet-to-dry (W/D) weight ratio (Fig. 5B), indicating effective restoration of vascular integrity and marked attenuation of pulmonary edema. We next assessed systemic and local inflammatory responses. ELISA analyses demonstrated that OLA@SPA MPs treatment robustly suppressed serum levels of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β (Fig. 5C–E), while simultaneously inducing a significant elevation in the anti-inflammatory cytokine IL-10 (Fig. 5F). Notably, this shift toward an anti-inflammatory milieu was even more pronounced within the lung. BALF analysis revealed a dramatic reduction in IL-6, TNF-α, and IL-1β levels, accompanied by a substantial increase in IL-10 following OLA@SPA MPs treatment (Fig. 5G–J). These findings indicate that OLA@SPA MPs effectively modulate the dysregulated cytokine storm at both systemic and pulmonary levels.Fig. 5. Identification of the mechanism of OLA@SPA MPs in treating a sepsis mouse model. (A) Protein concentrations in BALF determined by BCA assay across experimental groups (n = 6). (B) Wet-to-dry (W/D) weight ratios of lung tissue in each group (n = 6). (C–F) Serum levels of the pro-inflammatory cytokines IL-6 (C), TNF-α (D), IL-1β (E) and the anti-inflammatory cytokine IL-10 (F) at 24 h post-LPS injection (n = 6). (G–J) Corresponding cytokine concentrations in BALF at 24 h after LPS administration (n = 6), including IL-6 (G), TNF-α (H), IL-1β (I) and the anti-inflammatory cytokine IL-10 (J). (K, L) Immunofluorescence images of PARP-1(K) and PAR (L) expression in CD326^+^ epithelial cells under different treatments, scale bar = 200 μm. (M) Volcano plot of DEGs in the LPS group vs. LPS + OLA@SPA MPs group. (N) Heat maps of DEGs selected based on the results of (M) in the LPS group and LPS + OLA@SPA MPs group. (O) Enrichment analysis of KEGG signaling pathway for DEGs in the LPS group vs. LPS + OLA@SPA MPs group.(P) GSEA enrichment analysis of LPS group and LPS + OLA@SPA MPs group. Data are presented as mean ± SEM. Statistical significance was assessed by one-way ANOVA: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.Fig. 5

Given that Olaparib is a well-characterized PARP-1 inhibitor [40], we next directly evaluated target engagement in vivo. Immunofluorescence staining of lung sections demonstrated that LPS challenge induced a marked increase in PARP-1 expression and its enzymatic activity product, poly(ADP-ribose) (PAR), within CD326^+^ pulmonary epithelial cells (Fig. 5K, L) and F4/80^+^ macrophages (Fig. S2). Importantly, treatment with OLA@SPA MPs almost completely abrogated these signals in both cell populations, an effect that was markedly stronger than that achieved with free OLA or non-targeted OLA@MPs. Following LPS challenge, pronounced PARP-1 hyperactivation in lung tissues triggered pronounced DNA damage (increased γ-H2AX) and concomitant severe depletion of NAD^+^ and ATP. Treatment with OLA@SPA MPs markedly reversed these changes, suppressing γ-H2AX accumulation and preserving NAD^+^/ATP levels (Fig. 4SA–C). Collectively, these results demonstrate that OLA@SPA MPs efficiently deliver Olaparib to lung tissues, thereby suppressing PARP-1-mediated energetic crisis and cellular damage.

To gain an unbiased and comprehensive understanding of the molecular effects of OLA@SPA MPs treatment, we performed RNA sequencing analysis on lung tissues. Volcano plot analysis identified a substantial number of differentially expressed genes (DEGs) between the LPS group and the LPS + OLA@SPA MPs group (Fig. 5M). Hierarchical clustering and heatmap visualization of these DEGs clearly segregated the two groups, indicating that OLA@SPA MPs treatment induces a broad reversal of the sepsis-associated transcriptional profile (Fig. 5N). KEGG pathway enrichment analysis revealed that genes downregulated by OLA@SPA MPs were significantly enriched in multiple critical pro-inflammatory and injury-related pathways, including the NOD-like receptor signaling pathway, TNF signaling pathway, and NF-κB signaling pathway (Fig. 5O). Consistently, Gene Set Enrichment Analysis (GSEA) confirmed significant negative enrichment of gene sets associated with these inflammatory pathways in the OLA@SPA MPs-treated group compared with the LPS group (Fig. 5P).

In summary, these mechanistic investigations demonstrate that OLA@SPA MPs exert potent protective effects in sepsis-induced lung injury by: (1) alleviating pulmonary vascular leakage and edema; (2) reprogramming both systemic and local inflammatory responses toward an anti-inflammatory phenotype; (3) effectively inhibiting PARP-1 hyperactivation within lung epithelial cells and macrophages; and (4) globally reversing the sepsis-associated transcriptomic landscape, particularly through suppression of multiple key pro-inflammatory signaling pathways.

In vitro and in vivo biocompatibility profile of OLA@SPA MPs

2.6

Prior to clinical translation, a comprehensive evaluation of the safety profile of any novel therapeutic is essential. Accordingly, we performed a series of in vitro and in vivo experiments to systematically assess the potential cytotoxicity and systemic toxicity of the OLA@SPA MPs formulation. First, the in vitro cytotoxicity of both the encapsulated drug (OLA@SPA MPs) and free olaparib (OLA) was evaluated in target pulmonary cells (MLE-12 and MH-S), as well as in a non-target fibroblast cell line (L929). After 24 h of treatment, OLA@SPA MPs exhibited a favorable safety profile, with only a mild, concentration-dependent decrease in cell viability observed across all three cell lines at extremely high concentrations (Fig. 6A–C). Notably, the half-maximal inhibitory concentration (IC_50_) values of OLA@SPA MPs were substantially higher than those of free OLA at equivalent doses (Fig. 6D–F). These results indicate that encapsulation of OLA within SPA-modified MPs markedly attenuates its direct cytotoxicity, likely by enabling controlled drug release and reducing acute cellular exposure.Fig. 6Ex vivo and in vivo safety assessment of OLA@SPA MPs.(A–C) Viability of MLE-12 (A), MH-S (B) and L929 (C) cells after 24 h incubation with gradient concentrations of OLA@SPA MPs, assessed by CCK-8 assay (n = 4). (D–F) CCK-8 assay of cell viability in MLE-12 (D), MH-S (E) and L929 (F) after 24 h exposure to varying concentrations of free OLA (n = 4). (G) Histological examination of heart, liver, spleen, lung and kidney in healthy mice, scale bar = 100 μm. (H) Body-weight changes in healthy mice post tail-vein injection with OLA@SPA MPs (equivalent to 10 mg kg^−1^ OLA) or PBS (n = 4). (I, J) Routine hematology and plasma biochemistry analyses in healthy mice 7 days after administration of formulations (n = 6). (K) Blood samples were collected from mice at 7 days after treatment (n = 3). Serum levels of IL-6 and TNF-α were quantified by ELISA. Data are expressed as mean ± SEM. Statistical significance was evaluated by one-way ANOVA: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.Fig. 6

To further evaluate systemic safety in vivo, healthy mice were intravenously administered a single dose of OLA@SPA MPs via the tail vein (equivalent to 10 mg kg^−1^ OLA). Histopathological examination of major organs, including the heart, liver, spleen, and kidney, collected 7 days post-injection, revealed no discernible signs of tissue injury, inflammation, or pathological abnormalities compared with the PBS-treated control group (Fig. 6G). In addition, no significant body weight loss was observed in mice receiving OLA@SPA MPs throughout the observation period (Fig. 6H), suggesting the absence of acute systemic toxicity. Comprehensive hematological and plasma biochemical analyses were further conducted to corroborate these findings. No statistically significant differences were detected between the OLA@SPA MPs-treated group and the PBS control group in key indicators of organ function, including alanine aminotransferase (ALT) for hepatic function and creatinine for renal function, nor in standard hematological parameters such as white blood cell, red blood cell, and platelet counts (Fig. 6I and J). To further assess the long-term safety of OLA@SPA MPs, we monitored the serum levels of key pro-inflammatory cytokines, IL-6 and TNF-α, over a 7-day period following intravenous administration. ELISA results showed that both IL-6 and TNF-α remained at baseline levels throughout the observation period, with no significant differences compared to the control group (Fig. 6K). These results indicate that OLA@SPA MPs administration does not induce hepatotoxicity, nephrotoxicity, or hematological abnormalities.

In summary, these comprehensive safety evaluations demonstrate that the OLA@SPA MPs formulation possesses excellent biocompatibility both in vitro and in vivo. Encapsulation within microparticles significantly reduces the intrinsic cytotoxicity of OLA, and a single administration at the therapeutic dose does not result in detectable organ damage or systemic toxicity in healthy mice, thereby supporting its suitability for further preclinical and translational development.

Conclusion

2.7

In this study, single-cell transcriptomic analysis identified pulmonary epithelial cells and macrophages as major inflammatory drivers in septic lungs, providing a pathological basis for cell-targeted intervention. Guided by this insight, we developed a biomimetic delivery system, OLA@SPA MPs, that exploits the abundant expression of surfactant protein A in human lung tissue to achieve preferential pulmonary accumulation and enhanced uptake by SPA-interacting cells. By enabling localized inhibition of PARP-1, this strategy effectively suppressed DNA damage–associated inflammatory signaling, preserved cellular bioenergetics, and alleviated lung injury in both LPS- and CLP-induced sepsis models. Importantly, the use of olaparib, an FDA-approved PARP inhibitor with a well-established clinical safety profile, together with a microparticle formulation compatible with scalable manufacturing, highlights the translational feasibility of this platform. Beyond sepsis-associated acute lung injury, this SPA-guided therapeutic approach may also be applicable to other inflammatory lung diseases characterized by epithelial and macrophage dysfunction, such as acute respiratory distress syndrome and severe pneumonia, warranting further investigation.

Method

3

Materials

3.1

Cell culture media were procured from Gibco Life Technologies, Inc. (Grand Island, NY, USA), including Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) and Roswell Park Memorial Institute (RPMI)-1640 medium. Fetal bovine serum (FBS) was sourced from Zhejiang Tianhang Biotechnology Co., Ltd. (Huzhou, China). Plasmocin was supplied by InvivoGen (Toulouse, France), and penicillin/streptomycin by BioSharp (Hefei, China). Sterile 1 × phosphate-buffered saline (PBS) was also obtained from Gibco Life Technologies, Inc. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). RIPA lysis buffer, a CCK-8 cell proliferation kit, Annexin V–FITC apoptosis detection kit, Hoechst stain , ATP Assay Kit and NAD+/NADH Assay Kit with WST-8 were all provided by Beyotime Biotechnology (Jiangsu, China). Mouse TNF-α, IL-1β, IL-10 and IL-6 ELISA kits were obtained from Dakewe Biotech Co. (Shenzhen, China). Olaparib and dimethyloxalylglycine (DMOG) came from Selleck (Houston, TX, USA). Sucrose for electroporation buffer was acquired from Sinopharm (Beijing, China). Fluorescent dyes DiO and DiD were purchased from Yeasen (Shanghai, China), and PKH26 from MedChemExpress (New Jersey, USA). Protease and phosphatase inhibitors were obtained from Beyotime (Shanghai, China). AIF antibody was sourced from Abcam (Cambridge, UK). For Western blotting. Primary antibodies against PARP1 and SFTPA1 were purchased from ABclonal (Boston, MA, USA); anti-PAR antibody was obtained from Merck Millipore (Billerica, MA, USA); antibodies against β-actin, γ-H2AX, and CKAP4, as well as secondary antibodies (goat anti-mouse IgG H&L-HRP and goat anti-rabbit IgG H&L-HRP), were purchased from Abcam (Cambridge, UK); antibodies against CD63, TSG101, and Alix were obtained from Proteintech Group (Chicago, IL, USA); and all flow cytometry antibodies were purchased from BioLegend (San Diego, CA, USA).

Single-cell transcriptomic profiling

3.2

Publicly available single-cell RNA sequencing data were acquired from the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/) under accession code GSE207651. Initial quality control was implemented through Seurat's subset function, retaining cells expressing 500-6000 genes and containing <25,000 UMIs. This stringent filtering yielded 15,359 high-quality cells for downstream analysis.

Data processing and integration

3.3

The dataset underwent standard preprocessing including expression normalization, identification of 2000 highly variable genes, and scaling to remove technical artifacts. Dimensionality reduction was performed through principal component analysis (50 PCs selected via elbow plot) followed by Harmony integration to correct for batch effects. Cell clustering was achieved through shared nearest-neighbor graph construction and modularity optimization, with final visualization in two-dimensional UMAP space.

Cellular annotation

3.4

Cluster identity was determined using established marker genes from canonical literature, defining 14 distinct populations: T/NK cells (Ptprc, Cd3d, Cd3e, Nkg7, Klrd1), B cells (Ms4a1, Cd19), Neutrophils (Ly6g, Retnlg, Cd14), Macrophages (Cd14, Cd68, Adgre1), monocytes (Cd14, Ly6c2, Cd86), Fibroblasts (Dcn, Fgf7), Epithelials (Epcam, Krt8), Endothelials (Pecam1, Vwf), Pericytes (Pdgfrb, Rgs5), Smooth muscle cells (Cspg4, Acta2) and Cycle cells (Mki67).

Cell to cell communication

3.5

Cell-cell communication patterns were deciphered using CellChat supplemented with CellPhoneDB's ligand-repair database. This integrated approach enabled systematic quantification of signaling probabilities between cell clusters and identification of dominant communication pathways.

Cell Culture

3.6

Murine cell lines, including MLE-12 (mouse lung epithelial), MH-S (mouse alveolar macrophage), L929 (mouse fibroblast), and DC2.4 (mouse dendritic), were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China). All cell lines were maintained in media supplemented with 10% (v/v) fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology, Huzhou, China) and 100 μg mL^−1^ penicillin/streptomycin (Biosharp, Hefei, China). L929 and MLE-12 cells were cultured in DMEM and DMEM/F12, respectively, whereas MH-S and DC2.4 cells were grown in RPMI-1640 medium (Gibco, USA). To eliminate mycoplasma contamination, cells were treated with 25 μg mL^−1^ Plasmocin (InvivoGene, Toulouse, France) for two weeks and confirmed mycoplasma-free using the MycoProbe Mycoplasma Detection Kit (R&D Systems, Minneapolis, MN, USA).

Generation of MLE-12 cells stably expressing SPA: The lentiviral vector carrying Sftpa1 was synthesized and cloned into Stbl3 by General Biosystems (Anhui, China). MLE-12 cells were transduced with lentivirus at a multiplicity of infection (MOI) of 50 in six-well plates using the Hitrans G P transduction enhancer (Genechem, Shanghai, China). Stable cells were selected with 1.5 μg mL^−1^ puromycin and subsequently validated by Western blot analysis and qRT-PCR analysis.

Preparation of MPs

3.7

MLE-12 and MLE-12-SPA cells (6 × 10^6^) were seeded in 10-cm dishes and treated with 800 μM Dimethyloxalylglycine (DMOG) in DMEM/F12 medium to induce vesicle formation. After 48 h, the conditioned medium was harvested and centrifuged sequentially at 1000 g for 10 min and 14,000 g for 2 min to remove cell debris. The microparticles (MPs and SPA MPs) were pelleted by centrifugation at 14,000 g for 60 min at 4 °C and washed twice with sterile 1 × PBS before resuspension for downstream assays. In this study, the term “microparticles (MPs)” refers to engineered, EV-like membrane vesicles used as a drug delivery platform, rather than strictly classified endogenous extracellular vesicles. For both in vitro and in vivo experiments, particle dosing was normalized based on total protein content measured by BCA assay, ensuring consistency and reproducibility across experiments.

Loading of olaparib into MPs via electroporation

3.8

Olaparib (10 mg mL^−1^ in DMSO) was mixed with MPs at a 2:1 mass ratio in 400 mM sucrose buffer. Electroporation was performed using a Gene Pulser Xcell system (Bio-Rad, USA) with an exponential pulse (500 V, 125 μF) in 0.2-cm cuvettes.

Detection and characterization of olaparib-loaded MPs

3.9

Olaparib concentration within the MPs was quantified by high-performance liquid chromatography (HPLC) using an LC-2030C Plus system (Shimadzu, Japan) equipped with a ShimNex C18 column (4.6 × 250 mm, 5 μm, 100 Å). MPs were disrupted by adding a 3 × volume of acetonitrile and chloroform in a 1:2 (v/v) ratio, vortexed, and centrifuged at 10,000 g for 5 min. The lower organic phase was filtered (0.45 μm PTFE) and injected for analysis. A standard solution of Olaparib (10 mg) was prepared in the same solvent mixture. Chromatographic separation was performed at 25 °C with a 1 mL min^−1^ flow rate and a mobile phase composed of methanol (A) and acetonitrile (B); detection wavelength was set at 254 nm. MPs quantification was achieved by measuring their total protein content: MPs were lysed in Precipitation Detection Buffer at 4 °C for 30 min, centrifuged at 12,000 g for 30 min, and the supernatant analyzed using a BCA Protein Assay Kit (Thermo Fisher Scientific). MPs size distribution and morphology were assessed by dynamic light scattering (Malvern Zetasizer Nano ZSP) on 1 mL of 30 ng mL^−1^ MPs, and transmission electron microscopy (TEM) after ddH_2_O wash, deposition onto copper grids and imaging on HT7700-SS/FEI Tecnai G20 TWIN. For in vitro release studies, MPs were incubated in mouse serum at 37 °C for different time points; after retrieval and wash, Olaparib remaining in MPs was determined by HPLC or microplate reader.

Cell viability assay

3.10

MLE-12 and MH-S cells were seeded into 96-well plates at a density of 5 × 10^3^ cells per well. After adhesion, treatments were applied, and following a 1-h incubation the medium was replaced with CCK-8 working solution and incubated for an additional 1.5 h (Cell Counting Kit-8, MeilunBio, Dalian, China). To assess the protective effect of various treatments against LPS-induced damage, cells were allocated into six groups: control, PBS, OLA, MPs, OLA@MPs and OLA@SPA MPs. Each treatment (equivalent to 5 μg mL^−1^ OLA for the relevant groups) was applied for 30 min prior to introducing LPS, followed by viability measurement using the same CCK-8 assay kit.

RT-qPCR assay

3.11

Total RNA was extracted from cells using TRIzol reagent (TaKaRa, Shiga, Japan) in accordance with the manufacturer's protocol. Reverse transcription quantitative real-time PCR (RT-qPCR) was performed to determine the relative expression levels of SPA in MLE-12-SPA cells. Primers were custom-synthesized by AGbio (Changsha, China) or purchased from Thermo Fisher Scientific (Waltham, MA, USA). The qPCR reactions were carried out on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq™ (TaKaRa, Shiga, Japan), and data were analyzed using the 2^−^ΔΔCt method.

Flow cytometry analysis

3.12

MLE-12 and MH-S cells were plated in 6-well plates at a density of 5 × 10^4^ cells per well. After adhesion, cells were subjected to the respective treatments and, following 24 h incubation, harvested, washed with PBS, and stained using the Annexin V–FITC apoptosis detection kit. Apoptotic cell populations were quantified via flow cytometry using an Attune NxT instrument (Invitrogen) and analyzed with FlowJo software.

For assessment of microparticle uptake, various cell lines were seeded in 6-well plates and incubated with DiO-labeled MPs for 3 or 6 h; cells were then collected, washed in PBS, and evaluated by flow cytometry on a Beckman CytoFLEX S system (USA).

For receptor blocking experiments, cells were pre-incubated with a CKAP4 neutralizing antibody (10 μg/mL) for 1 h prior to treatment with fluorescently labeled SPA-MPs. Cellular uptake was quantified by flow cytometry.

Staining

3.13

To assess uptake of MPs by MLE-12 and MH-S cells, each line was seeded and incubated with DiO-labeled MPs for 3 or 6 h. After incubation, cells were washed with PBS, nuclei stained with Hoechst for 10 min, and fixed in 4 % paraformaldehyde for 15 min at room temperature. Images were acquired using a confocal microscope (Suny CSIM110). To evaluate in vivo MP internalization, 100 μL PKH26-labeled MPs were injected into mice; lung tissues were then fixed, cryo-sectioned, blocked with 1 % BSA for 2 h, and incubated overnight at 4 °C with primary antibodies (Alexa Fluor® 647 anti-mouse CD326 and Alexa Fluor® 488 anti-mouse F4/80). Afterwards, nuclei were counter-stained with DAPI for 20 min and visualized by confocal microscopy. For detection of PARP-1, PAR and AIF in cells, treated MLE-12 and MH-S cells were washed, fixed with 4 % paraformaldehyde for 15 min, permeabilised with 0.1 % Triton X-100 for 15 min, blocked with 5 % BSA for 1 h, and incubated overnight at 4 °C with primary antibodies (rabbit anti-mouse PARP-1, mouse anti-mouse PAR, rabbit anti-mouse AIF). Secondary antibodies (Cy5-goat anti-mouse IgG and Cy3-goat anti-rabbit IgG) were applied for 2 h at room temperature, followed by DAPI nuclear staining for 20 min, and confocal imaging. For tissue analysis of lung sections, fixed and frozen lung slices were blocked with 1 % BSA for 2 h, incubated overnight at 4 °C with primary antibodies (Alexa Fluor® 647 anti-mouse CD326, Alexa Fluor® 488 anti-mouse F4/80, rabbit anti-mouse PARP-1, mouse anti-mouse PAR), then treated with the same secondary antibodies for 2 h at room temperature, counter-stained with DAPI for 20 min, and imaged via confocal microscopy.

Western blot

3.14

MPs and cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors at 4 °C for 30 min, and the lysates were cleared by centrifugation at 12,000 g for 30 min at 4 °C. Protein concentrations were measured using a BCA Protein Assay Kit, and equivalent protein amounts were loaded per lane. Samples were then boiled for 5 min, separated by SDS-PAGE, and transferred onto a PVDF membrane. The membrane was blocked with 5% non-fat milk at room temperature for 2 h and incubated overnight at 4 °C with primary antibodies. After several washes in Tris-buffered saline containing 0.05% Tween-20, secondary HRP-conjugated antibodies were incubated at room temperature for 1 h. Signal detection was performed using NcmECL Ultra reagent (P10100, NCM Biotech) and chemiluminescent exposure.

Establishment of a septic mouse model

3.15

Male C57BL/6J mice (6–8 weeks old, weighing 18–20 g) were obtained from SHULAIBAO Biotech (Wuhan, China). The animals were housed in micro-isolator cages under controlled conditions, and all procedures were approved by the Experimental Animal Ethics Committee of Renmin Hospital of Wuhan University (Approval No. 20241202B) in compliance with the relevant ethical guidelines.

Mice were randomly divided into six groups: Control group, LPS group, OLA group (intraperitoneal injection of OLA, 10 mg/kg), MPs group, OLA@MPs group (tail vein injection of OLA@MPs, OLA equivalent dose 10 mg/kg), and OLA@SPA MPs group (tail vein injection of OLA@SPA MPs, OLA equivalent dose 10 mg/kg). After 1 h of treatment, the Control group received an intraperitoneal injection of PBS, while all other groups received an intraperitoneal injection of LPS (20 mg/kg, diluted in PBS). Survival rates were recorded at 0, 12, 24, 36, 48, 60 and 72 h post‐induction.

Sepsis-induced acute lung injury (si-ALI) was induced using a cecal ligation and puncture (CLP) procedure as previously described [15]. Briefly, mice were anesthetized with sevoflurane, and a longitudinal abdominal incision was performed to access the cecum. The cecum was partially ligated at the mid-portion using a 4-0 silk suture, followed by a single perforation with a 20-gauge needle. A small amount of cecal content was gently expressed to ensure successful perforation. Subsequently, the cecum was repositioned into the peritoneal cavity, and the abdominal wall was sutured. Postoperatively, mice received subcutaneous fluid resuscitation with sterile saline (50 μL/g) and were allowed to recover on a warming pad before being returned to standard housing conditions. Sham-operated animals underwent identical surgical procedures without ligation or puncture of the cecum.

Clinical score

3.16

At 24 h post‐LPS stimulation, clinical assessment was performed using the murine sepsis score (MSS) system, which assesses seven parameters: appearance, level of consciousness, activity, response to stimulus, eye condition, respiratory rate, and respiratory quality. Each parameter is scored on a scale from 0 to 4, with higher scores indicating more severe impairment. A composite score is calculated by summing the individual parameter scores. This scoring system provides a comprehensive assessment of the clinical status of mice in experimental sepsis models.

ELISA and BALF protein content assay

3.17

Serum and BALF concentrations of IL-6, TNF-α, IL-1β and IL-10 were determined using corresponding mouse‐specific assay kits, following the manufacturer's guidelines. BALF protein concentrations were determined using the BCA protein assay kit (Thermo Fisher Scientific).

Assessment of DNA damage and bioenergetic status

3.18

Lung tissues were homogenized for Western blot analysis of γ-H2AX. NAD^+^ and ATP levels were quantified using commercial assay kits according to the manufacturers’ instructions and normalized to total protein content.

Transcriptomic profiling and functional analysis

3.19

Total RNA extracted from lung tissues was subjected to library preparation and sequenced on the Illumina platform. Differential gene expression analysis of the bulk RNA-seq data was conducted using DESeq2, which employs a negative binomial model to address read count over-dispersion. Significantly dysregulated genes were defined as those exhibiting |log2FoldChange| > 1 with an adjusted p-value <0.05 (Benjamini–Hochberg correction). To elucidate the biological implications of the differentially expressed genes, KEGG pathway enrichment analysis was performed using the clusterProfiler package. A hypergeometric test was applied to evaluate the statistical representation of the gene set in each KEGG pathway, with a significance threshold set at p*-value <0.05. The results were visualized using dot plots to display enriched pathways based on gene ratio and p-*value. Special emphasis was placed on pathways related to cytokine-related signaling pathways. Gene Set Enrichment Analysis (GSEA) was performed using the clusterProfiler R package to identify biological pathways exhibiting coordinated expression changes. The analysis was conducted on a pre-ranked gene list sorted in descending order by log2foldchange values. This approach enables the detection of pathways where genes show concordant expression shifts without applying arbitrary significance thresholds. Statistically enriched pathways were identified using an adjusted p-value cutoff of 0.05. The results were visualized using the GseaNb function from the GseaVis package, which effectively displays the enrichment profiles and core enrichment genes for significant pathways.

HE staining and histopathological evaluation of lung tissues

3.20

For hematoxylin and eosin (H&E) staining, the heart, liver, spleen, lungs, and kidneys were harvested from mice and fixed in 4% paraformaldehyde at 4 °C for 24 h. After fixation, tissues were dehydrated through a graded ethanol series, embedded in paraffin, and sectioned into 5-μm-thick slices. The sections were deparaffinized, rehydrated, stained with H&E (Beyotime Biotechnology), and examined under a light microscope.

Lung injury was evaluated in a blinded manner by two independent, experienced pathologists who were unaware of the experimental group allocation. A semi-quantitative lung injury scoring system was applied based on previously published criteria. Specifically, four pathological parameters were assessed: alveolar wall thickening, inflammatory cell infiltration, hemorrhage, and alveolar congestion. Each parameter was scored on a scale from 0 to 4 (0, absent; 1, mild; 2, moderate; 3, severe; 4, very severe). The total lung injury score was calculated as the sum of individual parameter scores.

Lung water content was determined using the wet-to-dry (W/D) weight ratio. Briefly, the right lungs were excised immediately after sacrifice and weighed to obtain the wet weight, followed by drying at 60 °C for 48 h until a constant weight was achieved. The dry weight was then recorded, and the W/D ratio was calculated as the ratio of wet weight to dry weight.

Biosafety evaluation

3.21

To assess the biosafety of OLA and OLA@SPA MPs, various concentrations of OLA and OLA@SPA MPs (0, 10, 20, 50, 100, 150, 200, 400 μg mL^−1^, based on OLA concentration) were applied to three types of normal cells: mouse lung epithelial (MLE-12), mouse alveolar macrophage cells (MH-S) and mouse fibroblasts (L929). After 24 h of co-incubation, cell proliferation and viability were evaluated using a Cell Counting Kit-8 (CCK-8) assay kit. Male C57BL/6J mice (6 weeks old, 18–20 g) were randomly assigned to different groups and allowed to acclimate for one week prior to the experiment. After 7 days of treatment, blood samples were collected for hematological analysis, including red blood cell (RBC) count, white blood cell (WBC) count, platelet (PLT) count, hemoglobin (HGB) concentration, alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), blood urea nitrogen (BUN), and creatinine (CREA) levels. Major organs—heart, liver, spleen, lungs, and kidneys—were harvested for histopathological examination. Body weight changes were monitored over an 8-day period.

Statistical analysis

3.22

Data were analyzed using GraphPad Prism 10.0 software. Survival rates between groups were compared using the log-rank (Mantel–Cox) test. For comparisons involving three or more groups, one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test was performed. Differences between two groups were assessed using either a two-tailed unpaired t-test or the Mann–Whitney U test, depending on data distribution. Statistical significance was set at P < 0.05. Data are presented as means ± standard error of the mean (SEM). Statistical significance is indicated as follows: ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Consent for publication

Not applicable.

Funding

This work was supported by 10.13039/501100001809National Natural Science Foundation of China(82572461, 82272226) and 10.13039/501100001809National Natural Science Foundation of China Young Scientist Program (82202409).

CRediT authorship contribution statement

Xinyu Xie: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Miao Wu: Conceptualization, Data curation, Formal analysis, Investigation. Yuanyuan Geng: Investigation, Methodology, Writing – original draft, Writing – review & editing. Jiawei Bai: Project administration, Resources, Software, Supervision, Validation. Chengtai Ma: Investigation, Methodology, Project administration, Resources. Yan Yan: Resources, Software, Supervision, Validation. Lisen Lu: Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Liying Zhan: Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software.

Declaration of competing interest

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