Materials

N-acetylcysteine (NAC) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (China). Soybean phosphatidylcholine (SPC), cholesterol (Chol), sodium deoxycholate (SDC) and lipopolysaccharides (LPS) were purchased from Shanghai Macklin Biochemical Co., Ltd. (China). 1, 2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) was available from Corden Pharma Switzerland LLC. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), L-glutamine, sodium pyruvate, PBS buffer and penicillin-streptomycin (P/S) were obtained from ThermoFisher Scientific Inc. (USA). All other solvents were of analytical grade or specific HPLC grade. Recombinant murine M-CSF was purchased from PeproTech, Inc. (USA). Bleomycin (BLM) was obtained from Hanhui Pharmaceuticals Co., Ltd. (China).

Cell lines and animals

Bone marrow derived macrophages (BMDMs) were harvested and differentiated from 6-week-old C57BL/6 mice [23]. Briefly, bone marrow was flushed from mouse femurs with cold PBS. After filtering using cell strainers of 100 μm (BD Biosciences), cells were harvested by centrifugation at 800 rpm for 10 min and then red blood cells were lysed for 10 min on ice. Cells were cultured for 7 days in 6 cm Petri dishes using DMEM medium containing 10% FBS, P/S (100 µg/mL) and M-CSF (50 ng/mL) at a density of 106 cells/mL. The obtained BMDMs were used as M0 macrophages. M1 macrophages were obtained by further 12 h incubation with 500 ng/mL LPS [24]. The RAW264.7 cells and mouse lung epithelial 12 (MLE‑12) cells obtained from ATCC were incubated in complete medium (DMEM with 10% FBS) at 37 °C and 5% CO2.

Male BALB/C and C57BL/6 mice (6–8 weeks of age) were purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China) and housed at the Animal Center of Jinan University. All experimental procedures involving animals were approved by the Jinan University Laboratory Animal Ethics Committee. Maximum care was taken to limit the number of animals used in this study. Mice were anesthetized with 1% sodium pentobarbital (45 mg/kg) via intraperitoneal injection to ensure free of pain for any invasive operations.

Preparation and characterization of PSLipos and PSLipos-NAC

Two kinds of apoptotic-cell-inspired PS-containing nano-liposomes with low and high modulus (PSLipos-L and -H) were prepared through the combined method of the thin-film hydration, ultrasonication and extrusion [25]. Briefly, SPC, DPPS and Chol were dissolved in chloroform/ethanol solution (5:1, v/v) in round-bottomed flasks with molar ratios of 6.5:1.5:2. Then organic solvents were removed using rotary evaporator at 37 °C. For preparing PSLipos-L or -H, the lipid film was then hydrated in the SDC solutions (PBS buffer, pH 7.4, with the molar ratio of Chol to SDC = 1:0.4) or PBS buffer (pH 7.4) by vigorous shaking and vortex mixing for 30 min at 52 °C. The total lipid concentration was 10 mM. The resulting liposomes were probe-sonicated (150 W) for 3 min under ice bath condition, then homogenized by successive extrusion through a 100 nm pore size polycarbonate membrane (Nucleopore®, Whatman Inc) at 52 °C for 3 times.

The loading of NAC into PSLipos was performed by pH gradient method [26]. Briefly, SPC, DPPS and Chol were dissolved in chloroform/ethanol solution (5:1, v/v) in round-bottomed flasks with molar ratios of 6.5:1.5:2, and then evaporated in a water bath at 37 °C under reduced pressure until the formation of even thin film was observed. Thereafter, SDC/NaHCO3-NaOH buffer solution (pH 10.4) with the molar ratio of Chol to SDC = 1:0.4 was added, and the mixture was incubated again in a water bath at 52 °C for 30 min, sonicated and homogenized through a 100 nm polycarbonate membrane to obtain the pre-formed blank liposomes (pH 10.4), which were further dialyzed in FLOAT-A-LYZER G2 dialysis tubing (MWCO 10 kDa, Spectrum) in PBS (pH 7.4) overnight at 4 °C to adjust the external pH of liposomes to certain pH 7.4. Then, NAC solution (10 mg/mL) was added into the liposome dispersion and incubated for 2 h at 52 °C to obtain the PSLipos-L-NAC with 10 mM of the final total lipid concentration. And all drug-loaded liposomes were purified by the ultrafiltration/centrifugation technique (MWCO 10 kDa, Millipore) at 3000 g for 10 min. PSLipos-H-NAC was made using the similar procedure without the addition of SDC.

The particle size, polydispersity index (PDI) and Zeta potential of liposomes were determined by Zetasizer (Nano-ZS, Malvern Instruments, Malvern, UK). The encapsulation efficiency (EE) and drug loading efficiency (DLE) of NAC were calculated by equations: EE% = (the mass of total fed NAC - the mass of unentrapped NAC) / (the mass of total fed NAC) × 100%, and DLE% = (the mass of total fed NAC - the mass of unentrapped NAC) / (the mass of total NAC-loaded liposomes) × 100%, respectively. The content of NAC was measured by UV-Vis spectrophotometry (Persee TU-1810SPC, China) at 207 nm [27]. The morphology of the liposomes was observed by transmission electron microscopy (TEM) [28]. Briefly, one drop of the liposome dispersion was placed onto a holey formvar-coated copper grid (230 mesh, round fields). The excess liquid was sucked away by filter paper. After stained with 2% sodium phosphotungstate solution at room temperature for 2 min and air-dried, the TEM images were obtained on a JEM-1400flash microscopy (JEOL, Japan). The Young’s modulus of liposomes was determined using an atomic force microscope (AFM, Bruker Dimension FastScan) based on the Hertz model according to our previous work [13].

In vitro drug release and stability of PSLipos-NAC

In order to investigate the drug release behavior of liposomal formulations in the medium similar to the extracellular environment of the lung, the Gamble’s solution (its chemical composition was shown in Table S2) which simulates the interstitial lung fluid found within the deep lung was used as the drug release medium [29,30,31]. The in vitro release of NAC from PSLipos-L-NAC or PSLipos-H-NAC was performed using dialysis method. The free NAC solution and PSLipos-NAC dispersion were individually placed into a dialysis tubing (MWCO 10 kDa). The dialysis devices were separately sunk in Gamble’s solution (pH 7.4) at 37 °C under stirring at 100 rpm. At indicated time points, the 2 mL of the release media was collected and equal volume fresh media was added. The NAC concentration in the collected samples was determined by UV-Vis spectrophotometry (Persee TU-1810SPC, China) at 207 nm.

Additionally, we also performed the long-term and accelerated physical stability testing on the liposomes [26, 32]. The fresh as-prepared liposomal suspensions were subjected to centrifugation (3200 g for 1 h at 25 °C) or horizontal mechanical stirring (180 beats/min for 48 h at 37 °C) for accelerated testing, and stored at 4 °C for 30 days for long-term stability testing. The macroscopic appearance, pH change, particle size and PDI, as well as zeta potential of the liposomes were monitored.

In vitro cytotoxicity, anti-inflammatory and pro-healing assaysCytotoxicity evaluation

The cytotoxicity of samples with respect to BMDMs and MLE‑12 cells was measured by Cell Counting Kit-8 (CCK-8, KeyGEN, China). The BMDMs and MLE‑12 cells were seeded into 96-well cell culture plates at the density of 5000 and 3000 cells per well, respectively. After 24 h cultivation, the cells were incubated with different concentrations of free NAC, blank PSLipos and PSLipos-NAC with different modulus for 48 h. The relative cell viability was determined through the CCK-8 assay with following formula Eq. (1):

$$Viability=\frac_}\text_}_}\text_}\times 100\text$$

(1)

Macrophage capture and intracellular distribution

Overall, 104 RAW264.7 cells were seeded in each well of a 96-well plate and allowed to attach overnight. The cells were incubated with 0.5 µmol/mL total lipids of fluorescein-DHPE-labeled PSLipos (molar ratio, fluorescein-DHPE: total lipids = 6:1000) in a DMEM medium for 24 h. Next, the cells were washed with PBS three times and the fluorescence was quantified using a Flow Cytometer (BD FACSCanto). The mean fluorescence intensity (MFI) calculated from at least three independent experiments was used to determine macrophage capture efficiency.

To study the intracellular distribution of PSLipos, the cells were pretreated with 0.5 µmol/mL total lipids of fluorescein-DHPE-labeled PSLipos for 3 h in the DMEM medium, replaced with a fresh medium and cultivated for 0 to 24 h. Next, the cells were washed with PBS three times and the fluorescence was quantified using a microplate reader (Cytation3, BioTek). The images at 3 and 12 h was further investigated using a confocal fluorescence microscope. Briefly, the cells were washed with PBS three times and fixed with 4% paraformaldehyde for 15 min. Cell membrane were labeled for 20 min at 25 °C with 10 µM DIL (Invitrogen). Then, the cells were observed under a confocal fluorescence microscope (LSM 880 with AiryScan, Carl Zeiss).

Detection of intra- and extracellular concentrations

106 RAW264.7 cells were seeded in each well of a 6-well plate and induced by 500 ng/mL LPS for 12 h to obtain the M1 phenotype. The cells were pretreated with 61.25 µg/mL NAC and PSLipos-NAC dispersion (0.5 µmol/mL of the total lipid concentration, containing 61.25 µg/mL NAC) for 3 h in the DMEM medium, then replaced with a fresh medium and cultivated for 0 to 24 h. Next, the cell supernatant was collected to determine extracellular NAC concentrations using a NAC ELISA kit (Meimian, Jiangsu, China), and the cells were also collected and lysed to determine intracellular NAC concentrations. To exclude interference from endogenous NAC in experiments as much as possible, background correction was performed for all the measurements to obtain the exogenous extra- and intra-cellular NAC concentrations due to free NAC or PSLipos-NAC treatment by subtracting the endogenous NAC in M1 macrophages at the corresponding time point.

Real‑time quantitative PCR

BMDMs were seeded on 6-well plates (7 × 105 cells/well) and cultured for 12 h, then induced by 500 ng/mL LPS for another 12 h to obtain the M1 polarization state, whereas the cells untreated with LPS were considered as the control group. The M1 macrophages were then treated with 122.5 µg/mL NAC, PSLipos (1mM of the total lipid concentration) and PSLipos-NAC dispersion (1mM of the total lipid concentration, containing 122.5 µg/mL NAC) for 24 h, respectively. Next, the cells were washed with PBS, and the total RNA was isolated and purified from the cultured cells using HiPure Total RNA Mini Kit according to the manufacturer’s instruction. cDNA was prepared using HiScript® II Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, China). Real-time qPCR experiments were performed using AceQ® qPCR SYBR® Green Master Mix (Vazyme, China) in a CFX96 Real-Time PCR Detection System (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous reference gene to normalize the results. Relative gene expressions were calculated by ΔΔCt method relative to the control group. The primer sequences used in the experiments were as follows:

TNF-α: 5'-GGCAGGTCTACTTTGGAGTCATTGC-3' and 5’-ACATTCGAGGCTCCAGTGAATTCGG-3'; IL-1β: 5'-TGCCACCTTTTGACAGTGATG − 3' and 5’-TGATACTGCCTGCCTGAAGC-3'; IL-6: 5'-TTGGTCCTTAGCCACTCCTTC-3' and 5’-TGGAGTCCAGCAGACTCAAT-3'; iNOS: 5'-AGCACAGAATGTTCCAGAATCCC-3' and 5’-GTGAAATCCGATGTGGCCTTG-3'; GAPDH: 5'-AGGAGCGAGACCCCACTAACA-3' and 5’-AGGGGGGCTAAGCAGTTGGT-3'.

ROS measurement

ROS production in BMDMs was evaluated using ROS sensitive dye 2’,7'-dichlorodihydrofluorescein diacetate (DCFH-DA) [33, 34]. Cells were seeded in a 24 well plate (5 × 104 cells/well) and cultured in DMEM supplemented with 10% FBS and 1% of P/S for 12 h at 37 °C and 5% CO2. Then cells were stimulated with 500 ng/mL of LPS for 12 h to obtain M1 phenotype. After stimulation, M1 macrophages were incubated with free NAC solution (122.5 µg/mL), PSLipos (1mM of the total lipid concentration) and PSLipos-NAC dispersion (1mM of the total lipid concentration, containing 122.5 µg/mL NAC) respectively for further 24 h. Next, cells were washed twice with PBS and incubated with 10 µM DCFH-DA in Hanks’ balanced salt solution (HBSS) for 30 min. Relative intracellular fluorescence intensity was measured by a microplate reader (Thermo Fisher, USA) with 485 nm excitation and 530 nm emission after washing the cells with HBSS. The high DCF fluorescence signal implies high intracellular ROS.

Anti-inflammatory and pro-healing assays using a transwell coculture system

A co-culture transwell system was established to study the macrophage-mediated anti-inflammatory and pro-healing effects of PSLipos-NAC with different modulus. Briefly, the BMDMs (2 × 104 cells/well) were seeded in the upper chamber and MLE-12 cells (5 × 104 cells/well) were seeded in the lower chamber and cultured for 24 h to reach approximately 80% confluence.

Enzyme-linked immunosorbent assay (ELISA)

The BMDMs in the upper chamber and MLE-12 cells in the lower chamber were stimulated with 500 ng/mL LPS for 12 h, respectively, and free NAC solution (122.5 µg/mL), PSLipos (1 mM of the total lipid concentration) and PSLipos-NAC dispersion (1 mM of the total lipid concentration, containing 122.5 µg/mL NAC) respectively were added to the upper chamber of the transwell for another 24 h incubation. The untreated group without LPS and samples was considered as the control. After that, the supernatants of MLE-12 cells in the lower chamber were collected and the inflammatory cytokines including TNF-α, IL-1β and IL-6 were determined by ELISA Kit according to the manufacturer’s protocol (LunChangShuo Biotech, Xiamen, China).

NO assay

The nitrite concentration in the culture medium was measured as an indicator of NO production by a modified Griess reagent according to the manufacturer’s instruction [35]. Briefly, the culture supernatant of MLE-12 cells in the lower chamber was collected and 100 µL of these supernatants were mixed with an equal volume of Griess reagent in a 96-well plate and incubated at room temperature for 15 min, and the absorbance was measured at 540 nm using a microplate reader. The amount of nitrite in each sample was calculated from a standard curve prepared using known concentrations of sodium nitrite (NaNO2) as the nitrite source.

Scratch-wound healing assay

The MLE-12 cell monolayer in the lower chamber was scratched using a 200 µL pipette tip before washing three times with phosphate-buffered saline (PBS) to clear cell debris and floating cells. After that, BMDMs and MLE‑12 cells were cultivated in 2% FBS DMEM and treated with LPS (500 ng/mL) for 12 h, and the BMDMs (in the upper chamber) were then stimulated with free NAC solution (122.5 µg/mL), PSLipos (1mM of the total lipid concentration) and PSLipos-NAC dispersion (1mM of the total lipid concentration, containing 122.5 µg/mL NAC) respectively. At established time points (0 and 24 h) after the samples adding, cells were photographed using a light microscope at the same position of the wound. The scratch-wound area was quantitatively using ImageJ software and migration rate was determined using the formula (2) shown below:

$$Migration rate\left(\%\right)=\frac_-A}_}_}\times 100\text$$

(2)

A0: The scratch area at 0 h.

At: The scratch area without migrating cells at 24 h.

In vivo assay for treating ALIBleomycin‑induced ALI mouse model

Male BALB/C mice (7–8 weeks, 17–22 g, n = 8) were anesthetized and then instilled intratracheally with 50 µL saline alone or bleomycin (2.5 U/kg) diluted in 50 µL sterile saline [36, 37]. After 2 h, the PBS, NAC solution (1.8 mg/mL), PSLipos (15 mM of the total lipid concentration) and PSLipos-NAC (15 mM of the total lipid concentration, containing 1.8 mg/mL NAC) with different modulus were administered into the lungs via aerosol inhalation using a commercial medical air compression nebulizer (Yuwell, 403D) to form inhaled droplets as shown in Fig. S1, which had a median particle size of about 3.9 μm according to the manufacturer’s guidelines. The amount of sample was fixed at 4 mL per mouse. The mice were sacrificed 24 h after the administration, and the lung tissues and serum were harvested for analysis.

Histopathological analysis

The lung tissue was fixed in a 4% paraformaldehyde solution and embedded in paraffin, and then cut into 5 μm thick sections. The sections were stained with H&E Staining Kit [38], then monitored by NanoZoomer S360 (Hamamatsu Photonics K.K., Japan). A total of 10 fields at 10× magnification (NDP.view 2 software, Hamamatsu Photonics K.K.) were examined for each histologic slice. The indexes of lung injury were evaluated from the five histological features using a scoring system as Gao et al. described [39, 40]: (1) neutrophils in the alveolar space, (2) neutrophils in the interstitial space, (3) hyaline membranes, (4) proteinaceous debris filling the airspaces, and (5) alveolar septal thickening. Each was scored 0, 1, or 2 according to the injury severity. These five independent variables were weighted based on the relevance to ALI, and then were normalized to the number of fields. The final injury score was a continuous value between 0 and 1.

Immunohistochemistry (IHC)

Tissue sections were treated with Anti-iNOS antibody (Abcam) overnight at 4 °C, then washed three times and incubated with a biotinylated secondary antibody in a blocking solution for 1 h. Subsequently, antigen-antibody reactions were detected by staining with diaminobenzidine (Beyotime, China). All sections were imaged using NanoZoomer S360 (Hamamatsu Photonics K.K., Japan). The proportion of iNOS positive area of each field was quantified using Image J software.

Real-time quantitative PCR

Total RNA was isolated and purified from the lung tissue using HiPure Total RNA Mini Kit according to the manufacturer’s instruction. Relative gene expression was calculated as described in 2.5.2. The primer sequences used in the experiments were as follows:

TNF-α: 5'-GGCAGGTCTACTTTGGAGTCATTGC-3' and 5’-ACATTCGAGGCTCCAGTGAATTCGG-3'; IL-1β: 5' -TGCCACCTTTTGACAGTGATG-3' and 5’-TGATACTGCCTGCCTGAAGC-3'; IL-6: 5' -TTGGTCCTTAGCCACTCCTTC-3‘and 5’-TGGAGTCCAGCAGACTCAAT-3'; iNOS: 5'-AGCACAGAATGTTCCAGAATCCC-3' and 5’-GTGAAATCCGATGTGGCCTTG-3'; GAPDH: 5'-AGGAGCGAGACCCCACTAACA-3' and 5’-AGGGGGGCTAAGCAGTTGGT-3'.

Cytokine analysis

The cytokine levels of the serums were quantified using the ELISA kit according to manufacturer’s instructions. The measured cytokines were TNF-α, IL-1β and IL-6.

Statistical analysis

All the data were presented as means ± standard error of mean (SEM) from at least three independent experiments or biological replicates (N ≥ 3). Statistical analysis was performed using GraphPad Prism 8 by unpaired t-tests or one-way ANOVA with Bonferroni post hoc test when applicable. P value less than 0.05 was considered to be statistically significant.