Animals in the study
8–12-week-old female C57BL/6J mice were purchased from the SPF (Beijing) Biotechnology Co., Ltd., China. Experimental procedures using mice in this work were reviewed and approved by the ethical review board of Guangdong Medical University, and all the experiments were performed in accordance with relevant guidelines and regulations of Animal Ethics Committee of Guangdong province, China.
Preparation of Nar-NPs and biomimetic Nar-NPs (CM@Nar-NPs)
The Nar-NPs were synthesized using emulsification-and-evaporation method as previously described [14] with a little modification. Briefly, 20 mg Naringin (Shanghai Yuanye Bio-Technology Co., Ltd.) and 60 mg PLGA-PEG (lactide:glycolide 50:50, Sigma, USA) were co-dissolved in 5 mL of dichloromethane (Tianjin Damao Chemical Reagent Factory, China) as oil phase (O), 20 mL of PVA (1%, w/w, Sigma) was as external water phase (W). Firstly, the O phase was ultrasonic for 40 seconds on ice bath to form the first emulsification. Then, the first emulsification was dropped in W phase and ultrasonicated for another 40 seconds to form the second emulsification. After that, the second emulsification was added to 100 mL water and stirred for 6 hours for organic regent evaporation and nanoparticle hardening. Finally, the nanoparticles were harvested by centrifuging at 12,000 rpm for 20 minutes and washed 3 times using ultrapure water. The harvested NPs were lyophilized for 48 hours for storage in powdered form.
Preparation of cell membrane cloaked CM@Nar-NPs
Bone marrow-derived mesenchymal stem cells were isolated according to previous published protocols [15]. The extraction of cell membranes followed Shi’s protocol [16], briefly, 5 × 107 cells were collected and suspended in 3 mL of hypotonic lysing buffer containing 50 mM Tris, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 10% (v/v%) benzylsulfonyl fluoride. Then, the cells were kept in an ice bath for 15 − 30 min, followed by 3-round of freeze − thaw processes (frozen in the − 80℃ and thawed at 37°C). The cell suspension was then centrifuged at 800g for 15 min. The supernatant was collected and further centrifugated at 18 000g, 4°C for 60 min. The final precipitate was collected as cell membranes, and the protein content was analyzed by bicinchoninic acid protein assay. Finally, the mixture was extruded via a Hand Extruder to obtain uniform particles.
Characterization of Nar-NPs
The size distribution and zeta potential of the nanoparticles were assayed using a Nano Particle Analyzer SZ-100 (Horiba Scientific, USA). Morphology of NPs were visualized by scanning electron microscopy (Philips Co, Holland).
The encapsulation of Nar. The free Nar, Nar-NPs and CM@Nar-NPs were full wavelength scanned using a UV-vis spectrum(UV 6000), and the data showed that the UV absorption peak of free Nar and Nar-NPs was at 285 nm and 282 nm, respectively (shown in Fig.S1 A). The absorption peak shift is not significant. It is supposed that Nar was successfully encapsulated into NPs. The standard cur of Nar at 285 nm was shown in Fig. S1B. According to this curve, the encapsulation of Nar in NPs was calculated using the absorption values of Nar-NPs and CM@Nar-NPs.
To measure the drug (Nar) loading rate of Nar-NPs, 10 mg lyophilized nanoparticles were dissolved in 1 mL of methanol, and then the amount of Nar in solution was determined by High Pressure Liquid Chromatography (HPLC). HPLC detection was performed using a C18 column (5 µm, 250 mm × 4.6 mm). Whereas the mobile phase, consisting of methanol and 0.1% acetic acid (88:12) (v/v), was maintained at a flow rate of 1.0 mL/minute. The ultraviolet detector wavelength was 280 nm and the injection volume was 20 µL. The loading of Nar was calculated the concentration of Naringin based on the standard curve.
Hemolysis assay
To determine the in vivo biosafety of the compounds, hemolysis of red blood cells treated with the designated compounds was determined [17]. Erythrocytes were originated from normal healthy C57BL/6J mice through washing the anticoagulant whole blood with PBS at 3,500 rpm for 5 minutes. Then, a 4% red blood cell suspension (v/v, in PBS) was blended with different formulations of Nar. The erythrocyte sample lysed using pure water was used as positive control, and erythrocyte sample diluted using PBS was used as a negative control group. After incubating with 100 µg/mL of compounds at 37°C for 4 hours, all the samples were centrifuged at 3,500 rpm, 4°C, for 5 minutes to collect the supernatant and measure its absorbance value at 550 nm by a microplate reader. Also, the red blood cells were added into a 12-well plate and imaged using a Living cell imaging system (EVOSFL Auto, Invitrogen, USA).
SDS-PAGE analysis of retention proteins from cell membranes and NPs
The mesenchymal stem cell membrane protein was prepared following the instructions of the Membrane and Cytosol Protein Extraction Kit P0033 (Beyotime, China). Briefly, the cells were scraped off from dishes after washing with pre-cooled PBS buffer and then added the reagents from Extraction Kit. A BCA protein assay kit (Solarbio, China) was used to determine the protein concentration of isolated CM. Stem cell membrane protein samples and CM@Nar-NPs were mixed with loading buffer (Beyotime, China) and denatured at 95°C. 30 µg of protein samples was separated by 10% SDS-PAGE gel, and then, the gel was stained with Coomassie Blue for 2 hours and decolored overnight for imaging.
Cellular uptake to assay the biocompatibility and targeting of the NPs
Due to the weak autofluorescence of Nar, Cumarin 6 (C6) was loaded into the Nar-NPs as a fluorescent marker. Flow cytometry analysis was employed to examine the cell uptake of coumarin 6-loaded Nar-NPs according to a published method [18].
RAW264.7 cells and human bronchial epithelial cells (HBE) were provided by the Cell Library of Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical University. The cells were cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (Gibco) containing 100 µg/mL streptomycin and 100 IU/mL penicillin at 5% CO2 and 37°C. The cells were cultured with C6-Nar-NPs for 4 hours, and then washed twice with PBS to remove unbound nanoparticles followed by trypsinization. Flow cytometry analysis was performed at FITC channel. Results were expressed in terms of mean fluorescence intensity (MFI) of C6 in cells.
CCK8 to assay cell viability
To examine the effects of Nar or Nar-NPs on RAW264.7 cells and HUVEC cells, the CCK8 assay was carried out to assess the cell viability and proliferation. In brief, RAW264.7 cells or HUVEC cells were plated in a 96-well plate at a density of 1×104 cells/well. Cultures were treated with different concentrations of Nar, Nar-NPs and CM@Nar-NPs for 24 hours. Then, CCK8 assay was performed according to the manufactures’ instructions.
DCF-HA kit assay the ROS level in macrophages
The RAW264.7 cells suspension was seeded into 6-well plates (105 cells/well) and cultivated for 24 hours. Next, different formulations of Nar were added to the cells for 24 hours. The cells were harvested to incubate with DCFH-DA for 30 minutes in dark at 37℃ and washed twice with PBS. The resulting mean fluorescence intensity (MFI) of DCF-HA in cells was measured by flow cytometer using FITC channel.
Animal study
8 ~ 12-week-age female C57/6J mice were purchased from SPF biotechnology co., LTD, Beijing, China. Experimental procedures using mice in this work were reviewed and approved by the ethical review board of Guangdong Medical University, and all the experiments were performed in accordance with relevant guidelines and regulations of Animal Ethics Committee of Guangdong province, China.
Biosafety study of different formulations of Nar
To determine the biosafety of Nar in vivo, different formulations of Nar (Free Nar, Nar-NPs and CM@Nar-NPs) were i.n. administrated to normal healthy mice (dose: 10 mg/kg body weight, 5 mice/group) for one week, once per day. The pathological sections of main organs (spleen, liver and kidney) were stained with hematoxylin and eosin (H&E).
LPS-induced ALI mouse model and experimental groups
Sepsis mouse model was induced by intraperitoneal injection (i.p.) of lipopolysaccharide (LPS; Beijing Solarbio Science & Technology Co., Ltd.). The mice were randomly divided into four groups, and intranasally administrated with Nar (10 mg/kg), Nar-NPs (10 mg/kg), CM@Nar-NPs (10 mg/kg) and PBS, respectively. Three hours later, mice were injected i.p. with LPS (5 mg/kg) in 50 µL PBS. After 24 hours, mice were sacrificed and samples (blood, bronchoalveolar lavage fluid, lung tissues, etc.) were collected.
DCF-HA kits assay the ROS level in lung and liver tissues
The mice were anesthetized and perfused systemic blood before collected tissues. The lungs and livers were removed from the mice and immediately washed with PBS, and then submersed in DMEM medium with 100 µM DCFH-DA for 30 minutes at 37°C in the dark. After washing twice with DMEM medium, the fluorescence intensity of tissues was imaged using a Small Animal Live Imaging System (Eastman Kodak Co. USA).
In vivo biodistribution of NPs
LPS-induced septic mice were intranasally administered with 50 µL of Nar-NPs or CM@Nar-NPs, with indocyanine green (ICG) loaded into the NPs as fluorescent markers. For semiquantitative analysis, the image and fluorescence intensities of mice were collected 2 hours after intranasal administration of NPs and determined using the Kodak Multi Mode Imaging System.
To further analysis the NPs endothelia targeting, the ALI mice were i.n. administrated with coumarin 6-loaded NPs (shown in Fig. 3A, 5 mice/group), after 4 hours, the lungs were collected and isolated all the cells to determine the uptake of NPs using flow cytometry. In addition, healthy mice without LPS treatment were also i.n. with CM@Nar-NPs served as healthy control group.
Measurement of lung vascular leakage
Evans blue-conjugated albumin (EBA) extravasation assay to assess vascular permeability was performed according to the instructions of previous studies [19]. Briefly, EBA (20 mg/kg) was injected retro-orbitally 45 minutes before sacrificing mice. Lungs were perfused with PBS to remove blood. Lung homogenates were incubated with 2 volumes of formalin for 18 hours at 60°C, centrifuged at 5,000 g for 30 minutes, and the optical density (OD) of the supernatant was determined spectrophotometrically at 620 nm. The extravasated EBA in lung homogenate was expressed as µg of EBA per g of lung tissue.
Lung mononuclear cells isolation
Lung mononuclear cells were prepared as previously described [20]. Briefly, lung tissues were sliced into small pieces and incubated at 37°C for 45 min with collagenase IV (1 mg/ml; Life Technologies) in RPMI-1640 medium (HyClone) supplemented with 5% fetal bovine serum (FBS; HyClone), and cells were isolated by gradient centrifugation over 38% Percoll (GE Healthcare Life Sciences). After erythrocyte lysis with ACK lysis buffer (Gibco), the cells were harvested for analyses.
Flow cytometric analysis for macrophage polarization in lung
For surface staining, lung mononuclear cells were labeled with FITC-mouse CD11b(48-0112-82, eBioscience)PE/cyanine7- anti-mouse CD11c (117317; Biolegend), APC-anti-mouse F4/80 (123115; Biolegend) and FITC-anti-mouse CD206 (141703; Biolegend) and the matching control isotype IgG (MCA421; AbD Serotec) in FACS buffer (PBS with 2% FBS) for 30 minutes at 4°C. M1 macrophages were identified as F4/80 + CD11c + CD206– and M2 macrophages were identified as F4/80 + CD11c–CD206 + cells. Flow cytometry data were acquired on BD LSRFortessa X-20 and analyzed using FlowJo software (Tree Star).
Cytokine assay
The whole blood with EDTA originated from septic mice were centrifuged at 3,000 rpm for 10 minutes at 4°C, and the plasma was isolated for ELISA assay of IL-6 and IL-1β levels according to the protocols provided by the manufacture (Thermo Fisher Scientific).
Histology
Lung tissues were fixed by 5 minutes of instillation of 10% formalin through trachea catheterization at a trans-pulmonary pressure of 15 cm H2O, and then fixed in 10% formalin at room temperature for 48 hours. Then lungs were embedded in paraffin, cut into 5 µm sections, and stained with hematoxylin and eosin (H&E).
Survival rate
After lethal dose of LPS (10 mg/kg) challenge, mice were given normal access for water and food, and monitored four times a day for 7 days. Moribund mice were identified and euthanatized using CO2 followed with cervical dislocation. On day 7, all the survived mice were euthanatized.
Statistical analysis
Results are expressed as mean ± SEM Statistical significance was determined by one-way ANOVA with a Games-Howell post hoc analysis for multiple-group comparisons. Two-group comparisons were analyzed by the two-tailed unpaired Student’s t-test.