The platycodon secondary saponins (PSS) GP-682 and GPA-696 used in this work were synthesized in our laboratory. N,N-dimethylformamide (DMF) was purchased from Concord Technology Co., Ltd. (Tianjin, China). Nile Red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, was purchased from Aladdin (Beijing, China). FITC was purchased from MedChemExpress (New Jersey, USA). The Cytotoxicity Detection Kit (LDH Activity) was purchased from Roche (Basel, Switzerland). Glutaraldehyde (2.5%) was purchased from GenMed Scientific Inc. (MA, USA). Lev, one of the broad-spectrum antibiotics of quinolone, and terazosin hydrochloride were purchased from Shanghaiyuanye Bio-Technology Co., Ltd. (Shanghai, China). The Pseudomonas aeruginosa PA 14 strain was obtained from associate professor Bai Fang of Nankai University (Tianjin, China). A Pseudomonas aeruginosa antibody (1001/214) [Alexa Fluor® 488] was purchased from Bio-Techne China Co. Ltd. (Shanghai, China). The concentrations of human IL-6, IL-8 and TNF-α were detected using ELISA kits according to the manufacturer's instructions (Lanpai, Shanghai, China). All cell culture reagents were purchased from Gibco BRL Life Technologies (NY, USA).
Separation and purification of PSS
The PSS of GP-682 and GPA-696 were isolated and purified from a total saponin extract of platycodi radix . The preparation process was based on our previously published paper . The purified PSS were identified using HPLC and NMR methods. The detailed data are shown in Additional file 1 (Fig. S1-S9).
Preparation and characterization of PSS micelles
GP-682 (0.05-4.00 mg) and GPA-696 (0.20 mg) were dissolved in 400 μL of DMF solvent and diluted to 0.125 mg/mL to 10 mg/mL solutions. The solution was slowly added into 2 mL of pure water under ultrasonic conditions (200 W, SB-25-12DT ultrasonic oscillator, Ningbo Xinzhi Biotechnology Co., Ltd.). After DMF was removed via dialysis against 5Χ 250 mL water for 12 h, the micelle solution was filtered through a 0.45-μM microporous filter membrane (MCE syringe filter) to obtain a self-assembled micellar solution of PSS. The solution was collected, placed within a -80°C refrigerator for 30 min to freeze, then placed on a freeze dryer (FZ-1C-50, Beijing Boyikang Experimental Instrument Co., Ltd.) for 24 h at 0°C to obtain the solid PSS micelles. A Zetasizer (Nano ZS, Malvern Co. Ltd, UK) was used to analyze the particle size, size distribution and the zeta potential of GP-682 micelles scattered in d.d. H2O. GP-682 micelles were dropped into a copper mesh (Beijing Zhongjingkeyi Technology Co., Ltd., 200 mesh common carbon support membrane) with hydrophilic treatment. Uranium acetate was used to dye the sample. After air-drying at room temperature, the morphology of GP-682 micelles was observed using TEM with an accelerating voltage of 120 kV (Talos F200C, FEI, USA).
The stability of GP-682 micelles in 10% FBS, water and PBS was evaluated using dynamic light scattering (DLS). GP-682 micelles were dispersed in the three solutions for different time durations, then the stability of GP-682 micelles was detected using DLS. Fluorescence spectrophotometry was applied to determine the CMC value of GP-682 micelles with Nile Red as a probe . GP-682 micelle solutions at concentrations from 0.1 to 1000 μg were prepared. Nile Red (10 μL) in tetrahydrofuran was added to 1 mL of GP-682 micelle solutions. The final concentration of Nile Red was 10-6 mol/L. After sonication for 30 min, the fluorescence emission spectra were measured at 560–700 nm with an excitation wavelength of 550 nm. Emission intensity at 633 nm was plotted against the log of GP-682 concentration.
Human normal lung epithelial cells (BEAS-2B cells) were purchased from the American Type Culture Collection (Rockville, MD) and cultured in RPMI medium 1640 containing 10% FBS, 4.5 g/L glucose, L-glutamine and sodium pyruvate. The cells were cultured at 37°C with 5% CO2 in a humidified incubator. When cells achieved approximately 80% confluence, they were used for the following experiments.
Investigation of membrane permeability
BEAS-2B cells were cultured in small confocal dishes (NEST, 801001). Different concentrations of GP-682 micelles (10, 50, 100 μg/mL) or GPA-696 micelles (100 μg/mL) were cocultured with cells for 30 min in the culture medium at 37°C. The FITC (1×10-6 mol/L) was then added to cells for 10 min at 37°C. After washing with precooled PBS, the cells were fixed with 4% paraformaldehyde, and 200 μL of a 4',6-diamidino-2-phenylindole (DAPI) solution was added to the cells for 10 min. Finally, a confocal microscope (Leica TCS SP8) was used to investigate the entry of FITC into the cells. The excitation wavelength was 488 nm, and the emission wavelength ranged from 600 nm to 670 nm. The fluorescence intensity of FITC was detected by ImageJ software.
GP-682 micelles (10, 50, 100 μg/mL) diluted in the culture medium were used to pretreat cells for 30 min at 37°C, then 1×10-6 mol/L FITC was added for the next 30 min. After the administration, 500 μL trypsin was added to cells for digestion. The trypsin was discarded, the cells were suspended in PBS buffer, the cell suspension was centrifuged at 800 r/min for 3 min, the supernatant was discarded, and 1 mL PBS was added to resuspend the cells. A 40 μm nylon cell sieve was used to filter the cell suspension into a flow sample tube, and 104 cells were collected to obtain the intensity data with a fluorescence flow cytometer (BD FACSCalibur System). The excitation wavelength was 490 nm, and the emission wavelength was 530 nm. FCS Express V3 software was used to analyze the results of the flow cytometry experimentation.
Transepithelial Electrical Resistance (TEER)
The BEAS-2B cells were transferred to a cell culture plate (Corning, 3460) with a transwell chamber at a density of 2.5×104/cm2. RPMI 1640 medium (500 μL) was added to the upper chamber, and 1000 μL was added to the lower chamber. RPMI 1640 medium with 10% FBS was added to cultured cells 24 h later to make the cell replication state consistent. 100 μg/mL GP-682 micelles were added to cells to incubate for 90 min or removed at 30 min. TEER at a different time was detected by an EVOM2 Transmembrane cell resistance meter (World Precision Instruments Inc., Florida, USA). The standardized resistance of the transwell chamber equation is sTER=TER×S.
Morphological observations using TEM
GP-682 micelles (100 μg/mL) were added to cells at 37°C for 30 min. BEAS-2B cells without any treatment were used as the control group. The cells were collected and digested with trypsin, and then centrifuged at 1,000 rpm for 10 min. After discarding the supernatant, the cells were washed with precooled normal saline. Cells were then fixed with 2% (v/v) precooled glutaraldehyde and 1% osmium tetroxide. Cells dehydrated with ethanol solutions were embedded with LR white and used for preparing ultrathin sections. The cells were desiccated to the critical point and shadowed with platinum. A transmission electron microscope (TEM) (Hitachi HT7700, Japan) was used to observe the cells.
Endocytic pathway analysis
To check the endocytosis mediated uptake, BEAS-2B cells were seeded in small confocal dishes and pretreated with different endocytosis inhibitors, including sucrose (clathrin-mediated uptake, 1 μM), methyl-β cyclodextrin (caveolae-mediated uptake, 1 μM) and amiloride (micropinocytosis, 1 μM) in serum-free DMEM for 1 h. To visualize the distribution of GP-682 micelles, Cy5.5 labeled GP-682 was synthesized to prepare GP-682-Cy5.5 micelles. The detailed synthesis process of GP-682-Cy5.5 and the preparation of GP-682-Cy5.5 micelles are shown in Additional file 1 (Fig. S10-S13). DAPI was added to cells for 10 min at room temperature. A confocal microscope (Leica TCS SP8) was used to investigate the entry of GP-682-Cy5.5 micelles through endocytosis by the cells. The excitation wavelength was 633 nm, and the emission wavelength was between 653 nm and 700 nm.
Lactate Dehydrogenase (LDH) release assay
2×104 BEAS-2B cells were seeded in RPMI 1640 medium with 10% FBS for 48 h, and a series of GP-682 micelles (10 to 500 μg/mL) were added for 30 min, or fixed-dose GP-682 micelles (50 μg/mL) were added for a range of time durations (30 to 90 min) in RPMI 1640 medium without FBS. The supernatant (100 μL) was collected and incubated with a mixture of diaphorase/NAD+ and iodotetrazolium chloride (100 μL) for the LDH release detection. After 1 h incubation, the absorbance was tested at 500 nm (Spark 10M, TECAN, CH). Triton X-100 was added to cells to obtain the maximum release of LDH (Hc) as a positive control. The LDH release of cells without any treatment was set up as a low level-control (Lc). Percentage with regard to control was expressed as: [(treated mean - Lc)/(Hc - Lc)] × 100.
Preparation and assay of GP-682/Nile Red micelles
The GP-682/Nile Red micelles were prepared using the same ultrasound method as for GP-682 micelle preparation. The proportion of GP-682 to Nile Red was 10:1. Different forms of Nile Red were produced, including Nile Red dissolved in 0.1% DMSO and GP-682/Nile Red micelles. The final concentrations of Nile Red added to cells were identical (1 μg/mL). BEAS-2B cells were cultured in small confocal dishes and divided into three groups: Nile Red group, GP-682/Nile Red micelles group and GP-682 micelles + Nile Red group. In the Nile Red and GP-682/Nile Red micelles groups, the cells were treated with Nile Red or GP-682/Nile Red micelles only. In the GP-682 micelles + Nile Red group, cells were treated with 100 μg/mL GP-682 micelles for 30 min in advance, and the same dose of Nile Red (1 μg/mL) was added. A confocal microscope (Leica TCS SP8) was used to investigate the entry of Nile Red into the cells. The excitation wavelength was 561 nm, and the fluorescence emission spectra were measured at 580–700 nm.
Distribution analysis of lung tissue using HPLC
Ten male Kunming mice (18-22 g) of SPF grade were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After one week of regular rearing, the mice fasted for 12 h before the experiment. The mice were randomly divided into two groups: the Lev administration group (80 mg/kg), and the GP-682 micelles (5 mg/kg) preadministration for 30 min before Lev administration (80 mg/kg) group. The administration method was intraperitoneal injection. The mice were sacrificed at 0.08, 0.25, 0.5, 1.0, 1.5 and 2 h after Lev injection. The lung tissue of the mice was separated after rinsing with normal saline via heart perfusion. One gram of the lung tissue was homogenized with 3 grams of a normal saline buffer. After centrifugation at 3,000 rpm for 10 min, 100 μL of the supernatant was added to 100 μL of an internal standard solution (50 μg/mL terazosin hydrochloride methanol solution), followed by 200 μL of methanol. The solution was mixed thoroughly by vortexing and centrifuged at 10,000 rpm for 15 min. The supernatant (320 μL) was removed and blow-dried with nitrogen. The residue was reconstituted with 100 μL of methanol and centrifuged at 10,000 rpm for 15 min. The content of Lev-L in the supernatant was determined using high-performance liquid chromatography (HPLC). The HPLC method was performed with a Shimadzu HPLC (lc-20a) coupled with a fluorescence detector (RF-20A, Shimadzu, Japan). The following chromatographic conditions were used: Phenomenex Luna C18 column (150 mm × 4.6 mm, 5 μm); 10 mmol/L phosphate buffer mobile phase (containing 0.01% triethylamine, pH 3)-acetonitrile (82:18); 1 mL/min flow rate; 295 nm excitation wavelength, 490 nm emission wavelength; 35°C column temperature; and 20 μL injection volume. GraphPad Prism 8 was used for data processing and analyses. The methodological investigation is detailed in Additional file 1 (Figs. S14-S15, tables 1-3).
Acute lung injury model
Male KM mice (18-22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed under standard specific pathogen-free conditions with 12/12-h light/dark cycles at 23 ± 2°C and free access to water and food. A total of 105 mice were randomly divided into seven groups (15 mice per group): Model group (Mod); GP-682 micelles group (5 mg/kg GP-682 micelles); Lev administration groups (Lev-H, 52 mg/kg Lev; Lev-M, 26 mg/kg Lev; Lev-L, 13 mg/kg Lev); and GP-682 micelles preadministration for 30 min groups (GP-682 micelles + Lev-M, 5 mg/kg GP-682 micelles+26 mg/kg Lev; GP-682 micelles + Lev-L, 5 mg/kg GP-682 micelles+13 mg/kg Lev). Mice were anesthetized via an intraperitoneal injection of a 4% chloral solution (4 μL/g). Activated P. aeruginosa PA 14 bacteria (1×108/20 μL in PBS) were dropped into the nasal cavity to induce an acute lung infection. The mice were immediately given an antibiotic intervention, except in the Model and GP-682 micelles groups. Survival was recorded 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h after challenge with the PA-14 bacteria.
Another 48 mice were divided into eight groups, as described in the survival experiment, except that one control group (Con) was added. A mild infection model was used to investigate the effect of combination therapy. Activated PA 14 bacteria were used at 1×107/20 μL in PBS for nasal cavity infection. Mice were anesthetized 24 h later via inhalation of ether. Bronchoalveolar lavage (right lung) was performed via the administration of 1 mL of 0.9% saline through a tracheal cannula, and the fluid was collected for cytokine assays. The left lung tissues were eviscerated and fixed in a formaldehyde solution (10%) for hematoxylin-eosin (H&E) staining and bacterial immunofluorescence detection.
The results were reported as the mean values ± SD. Analysis of multiple groups was performed using analysis of variance (one-way ANOVA), and significant differences between two groups were assessed using t-tests. The log-rank test was used to analyze the significant differences in the survival rate experiments. Differences of p < 0.05 were considered statistically significant.