The platycodon secondary saponins (PSS) of 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). 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 (0.2 mg) were dissolved in 400 µL of DMF solvent and configured as 0.125 mg/mL to 10 mg/mL solutions. The solution was slowly dropped 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 and placed to -80 °C refrigerator for 30 min to freeze and 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 in 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 the presence of RPMI 1640 with FBS was evaluated using dynamic light scattering (DLS). GP-682 micelles were dispersed in RPMI 1640 with 10% FBS at 37 °C for different time points, 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 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 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 co-cultured with cells for 30 min in the culture medium at 37 °C. Then the FITC (1 × 10− 6 mol/L) was added to cells for 10 min at 37 °C. After washing by precooled PBS, the cells were fixed with 4% paraformaldehyde, 200 µL 4',6-diamidino-2-phenylindole (DAPI) 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 was 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 pretreated to cells for 30 min at 37 °C, then 1 × 10− 6 mol/L FITC was added for next 30 min. After the administration, 500 µL trypsin was added to cells for digestion. Discarded the trypsin and suspended the cells with PBS buffer, centrifuged the cell suspension at 800 r/min for 3 min, and suspended them again with PBS. Then discarded the supernatant and added 1 mL PBS to suspend 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 get the intensity data of flow cytometer Fluorescence (BD FACS Calibur System). The excitation wavelength was 490 nm, and the emission wavelength was 530 nm. FCS Express V3 software was used to analyze the result of the flow cytometry experiment.
Trans Epithellal Electric 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. Add RPMI 1640 medium 500 µL to the upper chamber and 1000 µL to the lower chamber. RPMI 1640 medium with 10% FBS were cultured to cells 24 h later to make the cell replication state consistent. 100 µg/mL GP-682 micelles were added to cells to incubate until 90 min or removed at 30 min. The TEER at a different time was detected by EVOM2 Transmembrane cell resistance meter (World Precision Instruments Inc, Florida, USA). The standardized resistance of the transwell chamber: sTER = TER × S.
Morphological observations using TEM
GP-682 micelles of 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 1000 rpm for 10 min. After discarding the supernatant, the cells were washed with precooled normal saline. Then cells were fixed with 2% (v/v) precooled glutaraldehyde and 1% osmium tetroxide. Dehydrated cells 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 were 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 endocytosis by the cells. The excitation wavelength was 633 nm, and the emission wavelength was between 653 nm to 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) for a range of time (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 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 GP-682 micelle preparation. The proportion of GP-682 to Nile Red was 10:1. Different forms of Nile Red were made, including Nile Red dissolved in 0.1% DMSO and GP-682/Nile Red micelles. The final concentration of Nile Red added to cells was 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 were fasted for 12 h before the experiment. The mice were randomly divided into two groups: The Lev administration group (80 mg/kg), and GP-682 micelles (5 mg/kg) pre-administration for 30 min before Lev administration (80 mg/kg) group. The administration method was an 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 times 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 l Lev in the supernatant was determined using high-performance liquid chromatography (HPLC). The HPLC method was performed in a Shimadzu HPLC (lc-20a) coupled with a fluorescence detector (RF-20A, Shimadzu, Japan). The following chromatographic conditions were used: column, Phenomenex Luna C18 (150 mm × 4.6 mm, 5 µm); mobile phase, 10 mmol/L phosphate buffer (containing 0.01% triethylamine, pH 3)-acetonitrile (82:18); flow rate, 1 mL/min; excitation wavelength, 295 nm, emission wavelength, 490 nm; column temperature: 35 °C; and injection volume, 20 µL. Graphpad Prism 8 was used for data processing and analyses. The methodological investigation is detailed in Additional file 1 (Fig. S14-S15, table 1–3).
Acute lung injury model
Male KM mice (18–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. 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 pre-administration 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 group. 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 one control group (Con) was added. A mild infection model was used to investigate the effect of combination therapy. Activated PA 14 bacterium was 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 instillation of 1 mL of 0.9% saline through a tracheal cannula, and the fluid was collected for cytokine assays. The mice’s 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 analyzed the significant differences in survival rate experiments. Differences of p < 0.05 were considered statistically significant.