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Mitochondria-Modulating Porous [email protected]2 Nanoparticles Provide Resistance to Oxidative Injury in Airway Epithelial Cells: Implications for Acute Lung Injury

Authors Wang M, Wang K, Deng G, Liu X, Wu X, Hu H, Zhang Y, Gao W, Li Q

Received 28 November 2019

Accepted for publication 10 March 2020

Published 31 March 2020 Volume 2020:15 Pages 2287—2302

DOI https://doi.org/10.2147/IJN.S240301

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 3

Editor who approved publication: Dr Yan Shen

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Muyun Wang,1,* Kun Wang,1,* Guoying Deng,2,* Xijian Liu,3 Xiaodong Wu,1 Haiyang Hu,4 Yanbei Zhang,5 Wei Gao,1 Qiang Li1

1Department of Pulmonary and Critical Care Medicine, Shanghai East Hospital, Tongji University, Shanghai 200120, People’s Republic of China; 2Department of Orthopedics, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201620, People’s Republic of China; 3College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, People’s Republic of China; 4Department of Cardiothoracic Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201620, People’s Republic of China; 5Department of Geriatric Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Anhui Medical University, Anhui 230022, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Wei Gao; Qiang Li Email [email protected]; [email protected]

Background: Mitochondrial dysfunction played a vital role in the pathogenesis of various diseases, including acute lung injury (ALI). However, few strategies targeting mitochondria were developed in treating ALI. Recently, we fabricated a porous [email protected]2 nanoparticles (NPs) with antioxidant properties.
Methods: The protective effect of [email protected]2 NPs was assessed using confocal imaging, immunoblotting, RNA-seq, mitochondrial respiratory chain (MRC) activity assay, and transmission electron microscopy (TEM) in airway epithelial cell line (Beas-2B). The in vivo efficacy of [email protected]2 NPs was evaluated in a lipopolysaccharide (LPS)-induced ALI mouse model.
Results: This study demonstrated that [email protected]2 NPs significantly increased the resistance of airway epithelial cells under oxidative injury and shifted lipopolysaccharide-induced gene expression profile closer to the untreated controls. The cytoprotection of [email protected]2 was found to be achieved by maintaining mitochondrial function, activity, and dynamics. In an animal model of ALI, pretreated with the NPs improved mitochondrial dysfunction, thus reducing inflammatory responses and diffuse damage in lung tissues. Additionally, RNA-seq analysis provided evidence for the broad modulatory activity of our [email protected]2 NPs in various metabolic disorders and inflammatory diseases.
Conclusion: This study brought new insights into mitochondria-targeting bioactive NPs, with application potential in curing ALI or other human mitochondria-related disorders.

Keywords: mitochondrial dysfunction, porous [email protected]2 nanoparticles, acute lung injury, anti-oxidative injury, anti-inflammation

 

Introduction

Mitochondria are elongated double-membrane-bound organelles with independent self-replicating genome and can be universally found in the cytoplasms of almost all eukaryotic cells. As the most capital organelle, mitochondria are able to “energize” cells with adenosine triphosphate (ATP), as well as involve in various physiological processes including redox signaling, programmed cell death (apoptosis) and calcium flux.1,2 It has been well appreciated that mitochondrial dysfunction played an essential role in the pathogenesis of multiple metabolic and age-related disorders, such as diabetes, obesity, hypertension, and neurodegenerative diseases.3 Recently, studies claimed that mitochondrial dysfunction also contributed to the development of several inflammatory lung diseases, including acute lung injury (ALI), or its more severe stage, acute respiratory distress syndrome (ARDS).4,5

ALI/ARDS is a common respiratory critical syndrome with clinical manifestations of acute, progressive respiratory distress, and multiorgan dysfunction.6 Despite significant progress in intensive care medicine and organ supportive therapy, the mortality of ALI/ARDS remained over 40% throughout the world.7 During the pathogenesis of this disorder, overwhelming uncontrolled innate immune system released large amounts of inflammatory cytokines, which induced mitochondrial dysfunction, manifesting as membrane destabilization or rupture, calcium disorder, oxidative phosphorylation inhibition, and reactive oxygen species (ROS) overload. These changes eventually led to microvascular endothelial–alveolar epithelial barrier disruption, neutrophil infiltration, pulmonary edema formation and gas exchange impairment.6,8 Airway epithelium is continuously considered the first line of host defense against noxious insults. Bundles of studies observed mitochondrial dysfunction of airway epithelial cells (AECs) in ALI/ARDS.5,9,10 Moreover, rescuing AECs from mitochondrial dysfunction through mitochondria transferring relieved LPS-induced ALI.11 A more recent report also affirmed that compensation for mitochondrial impairment by enhancing cellular glycolytic activity could protect airway epithelium from acute injury.10 Therefore, preventing AECs from mitochondrial impairment might provide a promising therapeutic target for ALI/ARDS.

In this regard, numerous “drugs” have been documented over the years ranging from mitochondria-targeted cation to triphenylphosphonium (TPP)-conjugated antioxidants.3,12-14 Among them, mitochondria-targeting nanoparticles (NPs) emerged as potential therapeutic alternations due to their improved cell targeting capabilities, desired biodistribution, and better biocompatibilities.1517 Despite the growing evidence of therapeutic outcomes by inducing mitochondrial-mediated tumor cell apoptosis,2,18 the application of nanodevices on the maintenance of mitochondrial homeostasis was quite limited. TPP-conjugated ceria (TPP-ceria) NPs have been developed to mitigate amyloid beta-induced mitochondrial morphology damage and excessive oxidative stress, thus providing potential therapeutic candidate for Alzheimer’s disease.19 However, few studies were about the development of “nano-drug” which could modulate mitochondrial homeostasis and further alleviate ALI/ARDS so far.

Selenium (Se), an essential trace nutrient in humans with multiple biological functions, has been considered a stable and economic antioxidant.20 However, Se may also exert cytotoxicity through generating superoxide along with catalyzing thiols oxidation.21 Rapid advances in nanotechnology have provided unconventional solutions to overcome the obstacles that hinder the clinical use of Se-containing medicine. We have recently developed a novel class of porous [email protected]2 nanoparticles (NPs) with extremely low cytotoxicity and preferred biocompatibility. The novel nanodevice released Se slowly and sustainably and was reported to attenuate oxidative stress in several disease models.2224 As is well known, mitochondrial dysfunction is the major source of ROS overload.4 Therefore, we hypothesized that the porous [email protected]2 NPs might alleviate oxidant injury in ALI/ARDS via modulating mitochondrial dysfunction.

In the present study, we found that the porous [email protected]2 NPs largely enhanced the resistance of AECs to lipopolysaccharide (LPS)-induced oxidative injury through maintaining mitochondrial ROS-scavenging activity and ameliorating mitochondrial dynamics. The NPs also presented potential therapeutic application against LPS-induced ALI in a classical mouse model. Furthermore, Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis provided additional evidence for the broad modulatory activity of the [email protected]2 NPs and their potential for clinical translation in various metabolic disorders and inflammatory diseases.

Materials and Methods

Additional details are provided in the Supplementary Material - Methods.

Synthesis and Characterization of the Porous [email protected]2 NPs

The porous [email protected]2 NPs were synthesized according to the methods used in our previous research.23 The porous [email protected]2 NPs were prepared as schematically illustrated in Scheme 1. Characterization of the NPs was achieved by using a transmission electron microscopy and a D/max-2550 PC X-ray diffractometer.

Cell Culture and Processing

Airway epithelial cell line (Beas-2B) was purchased from Shanghai Institutes for Biological Sciences, China Academy of Science (Shanghai, China). Details are shown in the Supplementary Material - Cell culture and processing.

In vitro Cytotoxicity of the [email protected]2 NPs

Details are shown in the Supplementary Material - In vitro cytotoxicity of the [email protected]2 NPs.

Detection of Intracellular ROS and Mitochondrial ROS

Intracellular and mitochondrial ROS were detected using CellROX™ deep red reagent and MitoSOX™ Red probe, respectively. More details are shown in the Supplementary Material - Detection of intracellular ROS and mitochondrial ROS.

Evaluation of Mitochondrial Respiratory Chain (MRC) Activities

The activities of mitochondrial respiratory chains were measured by MRC Complex I–V Activity Assay Kit according to their manufacturer’s instructions. More details are shown in the Supplementary Material - Evaluation of mitochondrial respiratory chain (MRC) activities.

Western Blotting (WB) Analysis

Details are shown in the Supplementary Material - Western blotting (WB) analysis.

Measurement of Mitochondrial Morphology and Activity

Details are shown in the Supplementary Material - Measurement of mitochondrial morphology and activity.

RNA-Sequencing and Data Analysis

Total RNAs were extracted using TRIzol-based method and the following procedures were conducted by Genergy Biotechnology Co. Ltd. (Shanghai, China). Details are shown in the Supplementary Material - RNA-Sequencing and data analysis.

Establishment of Acute Lung Injury Murine Model

ALI mice model experiments were conducted following the protocols of Shanghai Committee for the Accreditation of Laboratory Animal. All processes have been approved by Shanghai General Hospital Institutional Review Board (the Permit Number, 2018KY201. Shanghai, China). All the operations were performed after sodium pentobarbital anesthesia with all efforts made to minimize mice suffering. Details are shown in the Supplementary Material - Establishment of acute lung injury murine model.

Bronchoalveolar Lavage Fluid (BALF) Acquisition and Analysis

Details are shown in the Supplementary Material - Bronchoalveolar lavage fluid (BALF) acquisition and analysis.

Histological Analysis of Lung Injury

All sections were assessed by two independent physicians who were blinded to the treatment group according to international standards.25 Additional details are shown in the Supplementary Material - Histological analysis of lung injury.

Lung Wet-to-Dry (W/D) Weight Ratio

Details are shown in the Supplementary Material - Lung wet-to-dry (W/D) weight ratio.

Statistical Analysis

The results were analyzed by the use of GraphPad Prism 7 software. Data were managed through One-Way ANOVA and Bonferroni’s post hoc test (for equal variance) or Dunnett’s T3 post hoc test (for unequal variance). Quantitative data were described as mean ± standard error (± SEM). The difference was defined statistically significant only when p < 0.05.

Results

Characterization of the Porous [email protected]2 Nanoparticles

The porous [email protected]2 NPs were prepared as schematically illustrated in Figure 1A. The phase structure of the NPs was assessed by the X-ray diffractometer (XRD) pattern (Figure 1B). Various definite characteristic peaks in [email protected]2 NPs, including (100), (011), (012) and (021), suggested their hexagonal phase, using the standard Se phase (JCPDS card No.65–1,876) as a reference. Attributed to their irregular silica coating, the XRD pattern demonstrated a significant increase in the low angle region of our [email protected]2 NPs (Figure 1B). Additionally, we adopted a transmission electron microscopy (TEM) method to observe the morphology and size of [email protected]2 NPs. As shown in Figure 1C and D, the average diameter of NPs was about 55 nm, in which lots of irregular quantum dots (less than 5 nm) distributed from center to the surface. After treating with the hot water, [email protected]2 NPs presented the porous structure (Figure 1E). Coated with PVP, the Se quantum dots on the surface of or inside silica shell twined around them and could be released slowly along with the entrance of PVP into aqueous solution. Besides, we also examined the size distribution and zeta potential by the use of dynamic light scattering (DLS). The hydrodynamic diameter of [email protected]2 NPs was ∼127 nm, which a little larger than that of TEM. Meanwhile, their zeta potential was ∼-19.0 mV due to the OH groups on the surface of the silica. The physiological stability and Se quantum dots release of the [email protected]2 NPs have been studied in our previous research.23

Figure 1 Characterization of the porous [email protected]2 nanoparticles. (A) Fabrication of porous [email protected]2 nanoparticles (NPs). (B) The XRD pattern of porous [email protected]2 NPs as well as the typical hexagonal phase of selenium (JCPDS card No. 65–1,876). (C and D) Low- (C) and high-magnified (D) TEM images of the NPs before heat water treatment. (E) TEM images of porous [email protected]2 NPs after the treatment with heat water at 95°C. (F) The effect of various concentrations of NPs on the cell viability in cultured airway epithelial cells (the Beas-2B cell line). (G) The effect of LPS stimulation (10 ng/mL) and NP treatments (10 μg/mL) on the cell viability in Beas-2B cells. **p < 0.01, ***p < 0.001. Abbreviations: DIW, deionized water; TEOS, tetraethyl orthosilicate.

Subsequently, we determined the cellular toxicity of [email protected]2 NPs on Beas-2B cells using the CCK-8 assay (Figure 1F and G). Cells were co-cultured with various concentrations of [email protected]2 NPs, ranging from 0 μg/mL to 160 μg/mL. Compared to the control group, cellular viability of Beas-2B cells was not affected by the [email protected]2 NPs under 40 μg/mL (Figure 1F). Choosing the concentration of 10 μg/mL, we further demonstrated that the NPs did not show significant inhibitory effect on cell viability combined with LPS (10 ng/mL) (Figure 1G).

The Porous [email protected]2 NPs Enhanced the Resistance of Airway Epithelial Cells to LPS-Induced Oxidative Injury

In previous studies, porous [email protected]2 NPs have been reported to combat oxidative stress in several disease models due to the slow-released Se.22,24,26 Herein, we assessed the effect of 10 μg/mL [email protected]2 on LPS-induced oxidative damage in Beas-2B cells firstly. Cells pretreated with NPs produced less ROS in response to 10 ng/mL LPS than those without [email protected]2 pretreatment (Figure 2A). This was associated with decreased phosphorylation of the NF-κB subunit p65, p38 signaling and extracellular-signal regulated kinase (ERK1/2) signaling in the mitogen-activated protein kinase (MAPK) pathways (Figure 2B and see Supplementary Material-Figure S1A-C). Furthermore, [email protected]2 significantly reversed the inhibition of LPS on expression of nuclear factor erythroid 2-related factor 2 (NRF2) and the downstream target NAD(P)H quinone dehydrogenase-1 (NQO1), indicating an enhanced endogenous antioxidant response in Beas-2B cells (Figure 2C and see Supplementary Material-Figure S1D-E). Meanwhile, the expression of tight junction marker zonula occludens-1 (ZO-1) and the adherence junction marker E-cadherin (E-Ca) was also upregulated by [email protected]2 NPs compared with LPS group (Figure 2C, see Supplementary Material-Figure S1F-G). These suggested that the increased antioxidant activity and cellular tight junction followed [email protected]2 treatment could prevent AECs from LPS-induced damage.

Figure 2 Porous [email protected]2 NPs enhanced the resistance of AECs to LPS-induced oxidative injury. (A) Representative confocal images showing the effect of [email protected]2 NPs on LPS-induced oxidative stress in Beas-2B cells; the intracellular ROS was stained with CellROX™ deep red reagent, shown in red, and the nucleus was stained with Hoechst, displayed in blue. All the images were caught at the same magnifications. The scale bar represents 50 μm. (B and C) WB showing the effect of [email protected]2 NPs on the increased expression of p-p65, p-ERK1/2 (MAPK), and p-p38 (MAPK) (B), as well as the reduced expression of NRF2, NQO1, ZO-1, and E-Ca (C) after 24 h of LPS stimulation. (D and E) Impact of porous [email protected]2 NPs on the RNA-Seq transcriptome analysis of LPS-challenged human AECs; (D) The differential expression profiles of the top 50 genes. (E) the PCA plot. NP concentration = 10 μg/mL, LPS = 10 ng/mL.

To comprehensively evaluate the protective activity of porous [email protected]2 NPs, we used RNA-Seq method to study the global effects of the NPs on LPS-mediated transcriptome transformation of human AECs (Beas-2B cells). A gene expression heat map (Figure 2D) and principal component analysis (PCA) of all differentially expressed genes (Figure 2E) demonstrated a distinct separation between the control and LPS-stimulated group. Moreover, NPs treatment globally shifted LPS-induced gene expressions profile closer to the untreated controls, with more up-regulated genes than down-regulated genes (see Supplementary Material-Figure S2). As shown in Figure 2D, top 50 differentially expressed genes with [email protected]2 pretreatment displaying a similar pattern to the control group. Among them, many were associated with the morphological integrity and functional stability of mitochondria.2735

Porous [email protected]2 NPs Maintained Mitochondrial ROS-Scavenging Activity in an ALI Cell Model Induced by LPS

As mentioned above, mitochondria served as the capital organelle in the regulations of both energy and free radical metabolism. Mitochondria dysfunction has long been considered playing a crucial part in oxidative injury and contributing to various pathological processes, including ALI/ARDS. Herein, we speculated that the increased resistance of epithelial cells to LPS-induced oxidative injury might be associated with the stable mitochondrial ROS-scavenging activity modulated by porous [email protected]2 NPs. In this study, we adopted a living cell-permeable indicator, MitoSOX to analyze mitochondria-specific ROS by a confocal microscope or microplate reader. As shown in Figure 3A, 24 h exposure of LPS elevated mitochondrial ROS levels in Beas-2B cells, while pretreatment with [email protected]2 NPs significantly reduced the mitochondrial ROS. NPs addition alone had no effect on mitochondrial ROS compared to the control group. Such results were also confirmed by a microplate reader quantificationally (Figure 3B).

Figure 3 Porous [email protected]2 NPs maintained mitochondrial ROS-scavenging activity in LPS-induced human AECs. (A) Representative confocal images showing the effect of [email protected]2 NPs on LPS-induced mitochondria-specific ROS in Beas-2B cells; the mitochondrial ROS was stained with MitoSOX™ Red probe, displayed in red, and the nucleus was stained with Hoechst dye, displayed in blue. All the images were caught at the same magnifications. The scale bar represents 50 μm. (B) Quantification of mitochondrial ROS level by a microplate reader. (CE) The effect of LPS and [email protected]2 NPs on the activity of mitochondrial respiratory chain (MRC) complexes I (C), III (D) and V (E) in Beas-2B cells. NP concentration = 10 μg/mL, LPS = 10 ng/mL. *p < 0.05, **p < 0.01.

To further examine the effects of [email protected]2 NPs on mitochondrial ability responsible for cellular homeostasis, we detected the activities of several redox enzymes within mitochondria, MRC complex I–V in AECs. LPS exposure for 24 h decreased the activity of MRC complex I (Figure 3C). While [email protected]2 NPs addition significantly elevated the activities of MRC complex I, III, and V after LPS stimulation (Figure 3CE). These data indicated an important role of [email protected]2 NPs in maintaining mitochondrial ROS-scavenging function in ALI cell model induced by LPS.

Porous [email protected]2 NPs Ameliorated Mitochondrial Dynamics Under LPS Stimulation in Airway Epithelial Cells

As the ROS-scavenging function of mitochondria optimized by [email protected]2 NPs in LPS-stimulated AECs, we next estimated mitochondrial morphology and activity in each group. As shown in Figure 4A, mitochondrial morphology was observed by labeling with MitoTracker probe under a confocal laser microscope. The Beas-2B cells in either control group or NPs-treated group had numerous mitochondria, spread in the perinuclear regions with long lengths. By contrast, mitochondria in LPS-stimulated cells broke seriously, debris increased, and grids decreased. Mitochondrial structure within cells pretreated with [email protected]2 NPs was near to the normal when compared with the model group (Figure 4A). In order to detect mitochondrial activity, we analyzed the fluorescence intensity of MitoTracker in each group. Data showed that relative fluorescence intensity of the mitochondria in LPS-treated group decreased significantly in contrast to the control group. Moreover, the fluorescence of cells in NPs pretreated group displayed much stronger intensity than the model group, suggesting mitochondrial activity elevated following [email protected]2 addition (Figure 4B).

Figure 4 Porous [email protected]2 NPs ameliorated mitochondrial dynamics under LPS stimulation in Beas-2B cells. (A) Representative confocal images showing changes in the mitochondrial morphology of Beas-2B cells; The mitochondria were labeled with MitoTracker Red CMXRos (MTR) staining solution and displayed in red. All the images were caught at the same magnifications. The scale bar represents 50 μm. Yellow boxes indicated local amplification of the mitochondria. (B) Quantification of the red fluorescence intensity in each cell exhibiting the changes in the activity of mitochondria. The relative fluorescence intensity was calculated from more than 40 cells from each group. (C) TEM images showing representative mitochondrial morphologies of LPS and [email protected]2+LPS. Scale bar represents 1 μm. Red boxes indicated the mitochondria and yellow arrowheads indicated [email protected]2 NPs near the mitochondria, respectively. (D) Analysis of mitochondrial FF in each group. (E) Confocal fluorescence images of FITC-labeled [email protected]2 NPs in Beas-2B cells, shown in green; Mitochondria were labeled with MitoTracker Red CMXRos staining solution and shown in red. Yellow arrowheads indicated the distribution of [email protected]2 NPs around mitochondria. Scale bar represents 25 μm. NP concentration = 1 μg/mL. (F) The influence of our [email protected]2 NPs on reduced expression of mitochondrial fusion protein OPA1 and MFN2 upon LPS challenge. NP concentration = 10 μg/mL, LPS = 10 ng/mL. *p < 0.05, **p < 0.01.

Mitochondrial morphology was also analyzed by TEM. Pretreatment with [email protected]2 NPs restored the severe break, cristae disruptions, and vacuolar shape in LPS-challenged AECs (Figure 4C). Furthermore, TEM images of mitochondria were processed by ImageJ software to calculate morphological parameters: the mitochondrial form factor (FF). As shown in Figure 4D, the mitochondrial FF in NPs-treated group increased significantly when compared to LPS group, indicating less fragments and more branches.

Then, we visualized the NPs under TEM and confocal microscope using synthesized fluorescein isothiocyanate (FITC)-conjugated nanoparticles to examine the cellular uptake and location of our [email protected]2 NPs. In TEM images of Beas-2B cells, we observed the uptake of [email protected]2 NPs into cytoplasm and their accumulation near mitochondria (Figure 4C). By use of confocal imaging, we detected similar phenomenon that green fluorescence-labeled NPs distributed beside the red MitoTracker-labeled mitochondria (Figure 4E).

To further investigate the mechanism underlying the changes in mitochondrial activity and morphology, we studied expressions of the mitochondrial fusion protein which regulated mitochondrial function. In the Beas-2B cells, LPS stimulation for 24 h dramatically reduced the expression of mitochondrial fusion protein, represented by optic atrophy 1 (OPA1) and Mitofusin 2 (MFN2) (Figure 4F). However, pretreated with [email protected]2 NPs reversed this downward trend (Figure 4F). The findings suggested that our [email protected]2 NPs were able to ameliorate mitochondrial dynamics, thus mitigating mitochondrial dysfunction in LPS-challenged AECs.

Porous [email protected]2 NPs Showed Potent Therapeutic Activity in an ALI Model in vivo

Since [email protected]2 NPs exhibited strong abilities in ameliorating mitochondrial dysfunction and eliminating excessive oxidative stress in AECs, we anticipated that [email protected]2 might be effective on decreasing LPS-triggered lung injury in vivo. Herein, we adopted a classical mice model of ALI caused by nonlethal dose (10 mg/Kg) of LPS intranasally to induce acute injury within lung tissue. [email protected]2 NPs were given 1 h before LPS stimulation, and the detection was conducted 24 h after LPS challenge (Figure 5A).

Figure 5 The inhibitory activity of [email protected]2 NPs in an ALI mouse model induced by LPS. (A) The ALI mouse model with [email protected]2 pretreatments (100 μg/Kg) intranasally (i.n.) 1 h before LPS (10 mg/Kg) challenge; mice were sacrificed 24 h after LPS instillation for further analysis. (B-G) BALF in each group were collected for the detection of total cell amounts (B), neutrophil numbers (C), macrophage counts (D) as well as cytokines IL-1β (E), CCL2 (F), and IL-6 (G) production. (HI) The damage of the alveolocapillary membrane was analyzed by total protein amounts (H) and the lung W/D ratio (I) in BALF. NP concentration = 100 μg/Kg, LPS = 10 mg/Kg, *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviation: ns, not significant.

Firstly, we quantified inflammatory cell infiltration and certain cytokine secretion within bronchoalveolar lavage fluid (BALF) to evaluate airway inflammatory response. Before LPS challenge, the NPs-treated mice showed similar total cells and neutrophils, but higher macrophages numbers in the BALF compared to control group (Figure 5BD). After LPS instillation, it was found that [email protected]2 NPs significantly reduced the total amounts of inflammatory cells as well as neutrophils, but not macrophages within BALF of ALI mice (Figure 5BD). These results suggested that [email protected]2 NPs treatment promoted macrophage infiltration while suppressed neutrophil recruitment. Pretreated with NPs also reduced the levels of proinflammatory cytokines IL-1β, CCL-2, and IL-6 in BALF of the ALI mice (Figure 5EG).

As a key element in the pathogenesis of ALI, we then accessed the permeability of alveolocapillary membrane by measuring total protein in BALF and the wet-to-dry (W/D) weight ratio of the right lung. As shown in Figure 5H and I, [email protected]2 NPs were able to decrease total protein and W/D ratio within lungs of ALI mice. These results demonstrated that our novel NPs were potent in ameliorating the injury of alveolocapillary membrane in early stage of ALI.

In addition to BAL analysis, we also assessed the lung tissue histopathology after LPS challenge with or without [email protected]2 NPs pretreatment. Represented by neutrophils in alveolar space and interstitial space, proteinaceous debris within the airspaces, hyaline membrane formation as well as alveolar septal thickening (Figure 6A), we demonstrated that the total injury score in NPs-pretreated group was apparently decreased in ALI mice (Figure 6B). As for the underlying mechanisms, we discovered reduced phosphorylation of ERK1/2 and p38 as well as increased expression of OPA1, MFN2, NRF2, and NQO1 in ALI lung tissues regulated by [email protected]2 NPs (Figure 6C). These were highly consistent with in vitro experiments. Altogether, we suggested that porous [email protected]2 NPs could improve mitochondrial dynamics, control inflammatory responses as well as decrease diffuse alveolar damage within ALI lungs.

Figure 6 Protective effects of porous [email protected]2 NPs on diffuse lung injuries of ALI mice. (A) The typical histological photos of the lung sections stained with H&E in each group. The scale bar in the left panel represents 1 mm while that in the right panel represents 50 μm. (B) Lung damage in each group was evaluated by five pathophysiological features to get the total score. (C) The impact of [email protected]2 NPs on the expression of several proteins in lung tissues of ALI mice. NP concentration = 100 μg/Kg, LPS = 10 mg/Kg, ns = not significant, **p < 0.01, ***p < 0.001.

GO Annotation and KEGG Pathway Analysis of the Porous [email protected]2 Nanoparticles Treatment in LPS-Stimulated AECs

To further explore the biological processes regulated by [email protected]2 NPs in LPS-treated AECs, we conducted GO enrichment and KEGG pathway analysis. Considering the rich factor and p-value, biological process (BP), cellular component (CC), and molecular function (MF) aspects of the top 10 significant enrichment GO terms are shown in Figure 7A. We identified that the LPS responsive transcripts modulated by [email protected]2 NPs were largely associated with cellular response to DNA damage stimulus (GO: 0006974), regulation of cellular metabolic process (GO: 0031323) and organelle organization (GO: 0006996) (Figure 7A). From the KEGG pathway analysis, we found that NPs-regulated mRNAs mainly participated in infectious diseases (hsa05131, hsa05132, hsa05130), cancers (hsa05205, hsa05225), nervous system (hsa04721), aging (hsa04213), and immune system (hsa04670) (Figure 7B). These data supported the broad modulatory activity of the porous [email protected]2 NPs and their potential for clinical translation in metabolic disorders and inflammatory diseases (e.g., acute lung injury).

Figure 7 GO annotation and KEGG pathway analysis of the porous [email protected]2 NPs treatment in LPS-stimulated AECs. (A) GO enrichment analysis. (B) KEGG pathway analysis.

Discussion

Mitochondria have been widely exploited as therapeutic targets for various cancers due to their significant functional and structural differences between normal and cancer cells.36,37 Over the years, numerous mitochondrial-targeting nanodevices have been designed as antitumor strategies and exhibited remarkable potencies.3840 Notably, Zhang et al18 reported a novel mitochondria and cancer cell dual targeting polyprodrug nanoreactors, which could inhibit cellular respiration, amplify mitochondrial ROS (mtROS) and endow long-term excessive oxidative stress, eventually leading to cancer cell apoptosis. The strategy overcame several major defects that compromised efficiency, such as short lifetime, insufficient intracellular level, and limited action range of endogenous ROS, thus was considered to be promising for theranostics.18 On the other hand, researches on nanodevices application to maintain mitochondrial homeostasis were relatively limited. Recently, Kwon and colleagues synthesized mitochondria-targeting TPP-conjugated ceria nanoparticles, which mitigated mitochondria damage, eliminated mitochondrial ROS and suppressed neuronal death, suggesting a promising strategy for the prevention and cure of Alzheimer’s disease.19 We recently fabricated a novel porous SiO2-coated ultrasmall Se particle ([email protected]2 NPs), which served as a drug delivery and theranostic nanoplatform. This new developed nanodevice has proved to effectively restrain excessive oxidative stress in several disease models.2224,26 Accordingly, we speculated that the antioxidant effect of our [email protected]2 NPs was achieved by modulating mitochondria dysfunction. In the current study, we found the porous [email protected]2 NPs, accumulated beside mitochondria, could maintain their ROS-scavenging activity, as well as ameliorate mitochondrial dynamics, thus protecting AECs from LPS-induced acute injury (Figure 8). In addition, we used RNA-seq method and demonstrated that porous [email protected]2 NPs globally shifted LPS-induced gene expression profile of human AECs closer towards the normal control, among which many were related to the morphological integrity and functional stability of mitochondria. Moreover, [email protected]2 NPs could reduce acute inflammatory responses and attenuate lung tissue injuries in a mouse model of ALI. GO annotation and KEGG pathway analysis indicated a wide application potential for different kinds of disorders, such as infectious diseases, cancers, and nervous system disorders. These data altogether suggested a potential role of our [email protected]2 NPs for clinical applications in treating ALI/ARDS and other mitochondria-related disorders.

Figure 8 Diagram of the modulatory activity of porous [email protected]2 NPs on mitochondrial dysfunction in airway epithelial cells.

The application of nanodevices in modulating mitochondrial dysfunction largely depended on their mitochondrial-targeting efficiency. Nowadays, drug systems that could successfully target mitochondria were mainly achieved by coupling with mitochondria-targeted peptides41,42 or TPPs.12,40 However, the chemical reactions mentioned above were relatively complicated, and the organic solvent during the synthesis process was difficult to completely remove, which might cause damage to the liver, kidney, and nervous system, therefore limiting their clinical transformation prospects. Among different drug delivery systems, silicon nanoparticles attracted interests of researchers and were widely used as theranostic platforms due to their large specific surface area, uniform particle size distribution, as well as favorable biocompatibility and stability.43 Recently, we constructed a novel [email protected]2 nanodevice and validated its mitochondrial-protecting effects in the present study. Meanwhile, we found that these NPs were able to enter the cytoplasm and accumulated near the mitochondria. However, we scarcely observed them inside the mitochondria, indicating the necessity to further optimize their mitochondrial-targeting efficiency. Through attaching cations and forming positively charged surface, nanoparticles could interact with the phospholipid bilayers and change mitochondrial membrane potential, thereby modifying their permeability and entering into mitochondria.44 Accordingly, we would modulate the charge of [email protected]2 NPs by surface modification in order to enhance the aggregation into mitochondria in our future work. Besides, organic functional groups could be incorporated into our silica frameworks to develop organic-inorganic hybrid NPs, since the degradation rate and location of organosilica nanoparticles have been reported to be well controlled by varying the organic groups present.45,46 These may provide solutions to the potential risk of uncontrolled degradation of the silica framework and unwanted in vivo retention.47

It has been demonstrated that [email protected]2 NPs increased the resistance of airway epithelium to LPS-induced oxidative injury and improved outcomes of ALI mice. In fact, several nanodevices have been reported to protect against this disorder through different ways.48,49,50,51 Zhang et al prepared an anti-ICAM-1 antibodies-coated stimuli-responsive NP encapsulated with 2-[(Aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1)] with the ability to target inflammatory endothelia and inhibit neutrophil transmigration, thus restraining the progression of ALI.48 Recently, another anti-inflammatory nanomaterial, the peptide-gold nanoparticle hybrid (P12), has been exploited to specifically target innate immune cells, regulate Toll-like receptor (TLR) signaling and attenuate lung inflammatory responses.49,51 Current nano-based therapies for ALI mainly aimed to inhibit the activation of inflammatory pathways, such as NF-κB and TLR signal transduction. However, over-attenuation or long-term suppression of these signaling might influence immune function in vivo, for instance, induce defects in antibacterial ability.52,53 Mitochondria were suggested to facilitate cellular homeostasis by producing several redox enzymes, such as MRC complex I–III; otherwise, mitochondrial impairment might further cause damage to the entire cell.54 Herein, we demonstrated that [email protected]2 NPs significantly enhanced cellular resistance by promoting mitochondrial morphology and function stable when stimulated with LPS, thereby minimizing the side effects of single-act on immune-inflammatory pathways. In in vivo experiments, we showed that the porous [email protected]2 NPs reduced numbers of total inflammatory cells and neutrophils, with elevated macrophage counts in the BALF of ALI mice. This was consistent with previous understandings that elevated recruitment of alveolar macrophage modulated neutrophil infiltration and protected against LPS-induced ALI55,56 and implied another possible explanation for the therapeutic activity of our NPs. In the near future, we would like to explore the impacts of our [email protected]2 NPs on other cell types within lung tissue to further elucidate mechanisms underlying the protective activity against acute injury and optimize our therapeutic regimen.

As mentioned above, mitochondrial dysfunction involved in various pathological conditions, providing a broad prospect for the clinical application of our nanodevice. For instance, chronic obstructive pulmonary disease (COPD) is defined as a common lung disorder characterized by irreversible and progressive airflow limitation; this disease is induced by chronic exposure to several noxious particles or gases, such as cigarette smoke (CS).57 Oxidant–antioxidant imbalance is one of the leading factors involved in the pathogenesis of COPD58 and mitochondria function of airway epithelium was dramatically disturbed in the presence of cigarette smoke extract (CSE).59 Our preliminary data showed that the porous [email protected]2 NPs also decreased CSE-induced excessive mtROS production in AECs (data were not shown). Therefore, we had good reasons to believe that [email protected]2 NPs would become a promising candidate for the prevention and treatment of COPD. In addition, GO annotation analysis and KEGG pathway analysis also provided us evidence for the global clinical application prospects of our nanodevice. Results showed that NPs-modulated mRNAs mainly participated in infectious diseases, cancers, nervous system, and aging, which were closely related to mitochondrial dysfunction. These evidences reinforced our conclusion and gave us the confidence to continue our research in a category of disease models.

Conclusions

The present study demonstrated that the porous [email protected]2 NPs enhanced the resistance of airway epithelial cells against LPS-induced acute injury. Globally, [email protected]2 NPs shifted the LPS-induced gene expression profile of human AECs closer to the normal control. Possible mechanisms lied in protecting functional stability and preserving the structural integrity of mitochondria in AECs (Figure 8). Furthermore, this nanodevice was able to control airway inflammatory responses and attenuate tissue injuries in an LPS-induced ALI mouse model. GO annotation and KEGG pathway analysis showed broad therapeutic potentials for various disorders. This study brings new insights into mitochondria-modulating bioactive NPs, with application potential in curing ALI/ARDS or other human mitochondria-related diseases.

Abbreviations

ALI, acute lung injury; ARDS, acute respiratory distress syndrome; AEC, airway epithelial cell; NP, nanoparticle; LPS, lipopolysaccharide; ROS, reactive oxygen species; TPP, triphenylphosphonium; TEM, transmission electron microscopy; XRD, X-ray diffractometer; MRC, mitochondrial respiratory chain; CCK-8, Cell Counting Kit-8; BALF, bronchoalveolar lavage fluid; GO, Gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; ATP, adenosine triphosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gels; TBS, tris-buffered saline; MTR, Mitotracker Red CMXRos; FPKM, fragments per kilobase of transcript per million fragments mapped; IL-1β, interlukin-1β; CCL-2, macrophage chemotactic protein-1; IL-6, interlukin-6; H&E, hematoxylin-eosin; W/D ratio, wet to dry ratio; ± SEM, mean ± standard error; ERK, extracellular-signal regulated kinase; MAPK, mitogen-activated protein kinase; NRF2, nuclear factor erythroid 2-related factor 2; NQO1, NAD(P)H quinone dehydrogenase-1; ZO-1, zonula occludens-1; E-Ca, E-cadherin; PCA, principal component analysis; FF, form factor; FITC, fluorescein isothiocyanate; mtROS, mitochondrial ROS; TLR, Toll-like receptor; COPD, chronic obstructive pulmonary disease; CSE, cigarette smoke extract.

Disclosure

The authors declare no conflict of interest in this work.

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