Materials
4-hydroxymethyl phenylboronic acid pinacol ester (4-PBAP) and N, N'-carbonyl diimidazole (CDI) were purchased from Aladdin (China). Platycodon grandiflorum polysaccharides were supplied by Shanghai Winherb Medical Technology Co., Ltd. 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) was obtained from Beijing Bioss Biotechnology Co. DAPI, Cell Total Protein Extraction kits, Bicinchoninic Acid Assay (BCA) protein assay kit and Membrane Protein Extraction kits were supplied by Beyotime Institute of Biotechnology (China). TNF-α and IL-6 ELISA kit were purchased from Beijing Solarbio Technology Co., Ltd. (China). Antibody CCR2 (anti-rabbit, #ABP53395) special for mouse were purchased from Abbkine (China), with a dilution ratio of 1:1000 for antibody. Antibodies β-Actin (#4967S) and HRP-conjugated anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (USA), with a dilution ratio of 1:1000 for both antibodies during experimental use. All other chemical reagents were supplied by Aladdin (China). All biological reagents, and other cell culture medium were used as received following our previous protocols.
Synthesis and characterization of copolymers.
The synthesis process was carried out according to the relevant literature[35, 38]. Briefly, the ROS reactive groups were first synthesized. The 4-hydroxymethylbenzeneboronic acid pinacol ester (4-PBAP, 5 mmol) and N, N'-carbonyl diimidazole (CDI, 10 mmol) were added to the reaction flask. After added 30 mL of anhydrous dichloromethane to fully dissolve the reaction and stirred at room temperature for 12 h, the unreacted raw material was washed with pure water (3×25 mL) followed by saturated NaCl (3×20 mL), discarded the aqueous layer and dried overnight under anhydrous Na2SO4. The ROS-responsive groups CDI-PBAP were obtained by reduced-pressure drying, and their chemical structures were characterized by NMR as well as IR spectroscopy.
To prepare modified Platycodon grandiflorum polysaccharides (Oxi-PGP), we performed structural modification of PGP by prepared CDI-PBAP, namely PGP (5 mmol) dissolved in formamide (4 mL), added DMAP (10 mmol) dissolved in anhydrous dimethyl sulfoxide (DMSO), and finally added CDI-PBAP (10 mmol), and stirred the mixture overnight at room temperature with a magnetic stirrer. Subsequently, the resulting solution was dialyzed in deionized water and centrifuged at 1500 rpm for 5 min to obtain the supernatant, which was freeze-dried and prepared for use, and the successful synthesis of the modified product Oxi-PGP was verified by a series of characterizations.
Preparation and characterization of ROS responsive PNPs and PNPs@Cur
ROS-responsive nanoparticles were similarly prepared by dialysis. Oxi-PGP (10 mg) was weighed and dissolved in anhydrous DMSO (5 mL), and the resulting solution was dialyzed in deionized water (1000 mL) (MW: 8000–14000 Da) for 36 h, with water replacement at 4 h intervals. The final dialyzed solution was passed through the syringe filter (pore size 0.45 µm) to remove insoluble material to obtain the purified PNPs solution. To validate the nanoparticles for ROS responsiveness, the PNPs were incubated in phosphate-buffered saline (PBS) containing different concentrations of H2O2 (0, 0.1, 0.25, 0.5 and 1 mM) for 6 h. At predetermined time points, the transmittance at 500 nm was measured separately by UV spectrophotometer and the respective percentage of hydrolysis was calculated.
For preparation of PNPs@Cur, we dissolved the lyophilized carrier material with Cur in anhydrous DMSO at a ratio of 6:1 (w/w). The solution was dialyzed in pure water (MW: 8000–14000 Da) to remove the free drug and organic solvents. A portion of the resulting PNPs@Cur solution was taken and stored at 4°C, and the rest was freeze-dried for subsequent experiments. Subsequently, particle size, polydispersity index (PDI) and zeta potential were determined by dynamic light scattering (DLS) with a Delsa Nano C (Beckman Coulter, USA), morphology and size were determined by transmission electron microscopy (TEM, JEM-1400 Plus). Chitosan-based drug delivery nanosystems (CNPs@Cur) were prepared in a similar manner to the above process.
Drug encapsulation and release in vitro
The drug content in PNPs@Cur was measured by HPLC (Agilent 1260, USA)). The measurement was performed using 0.5% acetic acid aqueous solution and acetonitrile (35:65, V%) as the mobile phase with a detection wavelength of 425 nm. Then, the loading content (LC, %) and encapsulation efficiency (EE, %) are calculated according to the following equations 1 and 2, respectively.
The release behavior of Cur in vitro was investigated using dialysis method. For the examination of the release of PNPs@Cur in response to ROS, PNPs@Cur (5 mg) dissolved in deionized water (4 mL) were placed in dialysis bags in PBS (40 mL) containing different concentrations of H2O2 (0, 0.5 and 1 mM). At predetermined time points, 1 mL of the medium was taken separately for HPLC analysis and 1 mL of fresh medium was reinjected into the corresponding concentration of PBS. The cumulative release rate of Cur was calculated using the following Eq. 3.
where Mt is the weight of drug released at time t and M0 is the weight of loaded Cur. The release of the experiments was performed in triplicate.
Preparation of the MM
By utilizing the Membrane Protein Extraction Kit[36, 39], we obtained purer cells membrane materials. Briefly, the collected RAW264.7 cells were resuspended in membrane protein extraction buffer for 10 min under ice bath conditions, followed by transferring the cell suspension to a glass homogenizer and homogenizing about 20 times to ensure cell fragmentation. After centrifugation at 2000×g and 4°C for about 10 min, the supernatant was further centrifuged at 14000×g for about 30 min to collect the cell membrane precipitate and repeated twice to obtain purified cell membranes. Finally, the total protein content in the purified MM was analyzed by the bicinchoninic acid assay (BCA) protein assay. The prepared membrane material was stored at -80°C for future studies.
Preparation of characterization MNPs and MNPs@Cur
MNPs and MNPs@Cur were prepared by membrane extrusion. In brief, MM vesicles were mixed with PNPs and PNPs@Cur (1:5, w/w), followed by substantial sonication for 2 min using a bath sonicator at 40 kHz and 100 W power. Finally, nanoparticles with MM coating were obtained by repeated extrusion through 400, 200 nm polycarbonate porous membranes sequentially for at least 30 times by a micro-extruder (LiposoEasy LE-1, USA).
Then, particle size, PDI and zeta potential were also measured by DLS, as well as morphology by TEM. For further validation of the successful coating of cell membranes, the particular membrane proteins on the surface of macrophages were characterized by Western blotting. Total protein was extracted from macrophages, MM and MNPs by using a protein extraction kit, respectively, and protein concentration was determined by BCA protein assay. Approximately 25 µg of protein was subsequently added to protein loading buffer, boiled for 10 min, and then electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to PVDF membranes. Primary and secondary antibodies were added sequentially after closure with 5% skim milk powder, incubated for 2 h at room temperature, and specific bands were observed with an enhanced chemiluminescence detection kit.
Cytotoxicity and hemolysis assay
The cytotoxicity of PNPs and MNPs were investigated by incubation in RAW264.7 and HUVECs (human umbilical vein endothelial cells) cells. Simply, cell densities of approximately 8000 cells per well were inoculated in 96-well plates, respectively, and after overnight incubation, the medium was replaced with fresh medium containing different concentrations of PNPs or MNPs. After continued incubation for 48 h at 37°C in a cell incubator containing 5% CO2, MTT (5 mg/mL) reagent was added and incubated for another 4 h. Then, the supernatant was removed and DMSO was added to dissolve the bottom residue. The absorbance of each solution was read by multi-wall plate reader at 570 nm to obtain the corresponding cell viability.
As previously reported, direct contact method was used to detect the hemolytic properties of PNPs and MNPs in vitro. Briefly, 2% erythrocyte suspensions were prepared by taking blood from the orbits of mice. Then, 0.5 mL of the red cell suspension was mixed well with saline, purified water, PNPs and MNPs solutions (0.5 mL, 2 mg/mL), respectively. These specimens were then incubated continuously at 37°C for 2 hours. The absorbance of the supernatant was measured at 570 nm using a microplate reader to determine the hemoglobin released from the lysed erythrocytes. Saline and pure water were used as negative and positive controls, respectively.
In vitro binding and intracellular drug release
The targeting ability of MNPs was further investigated employing in vitro cellular uptake assays. Cellular escape ability was studied first in normal macrophages. An appropriate number of cells were seeded in 12-well plates, cultured overnight, and the original medium was replaced with fresh medium containing free Cur, PNPs@Cur, and MNPs@Cur. After incubation for 1–4 hours, respectively, the cells were washed three times with PBS, then fixed with 4% paraformaldehyde for 15 minutes, then washed three times with PBS, and finally stained with DAPI for 10 minutes and washed three times before observation by microscopy cell uptake. To imitate the inflammatory cell model, macrophages as well as HUVECs were activated with lipopolysaccharide (LPS,100ng/mL), respectively, and the subsequent steps were performed as described above to observe the targeting ability of MNPs@Cur in inflammatory cells. Likewise, in order to verify the potential role of PGP as priming components of Platycodon grandiflorum, the ability of PNPs@Cur and CNPs@Cur was compared respectively in terms of their uptake by macrophages in different states.
To explore further the intracellular drug release behavior, another fluorescent probe, NR, was selected to be loaded into the nanoparticles. NR did not fluoresce on its own, but excited intense fluorescence when it came into contact with intracellular lipids. The activated macrophages were obtained by LPS pretreatment, and the uptake was observed by replacing the original medium with medium containing PNPs@NR and MNPs@NR after 1, 2, 4 and 8 hours of continuous incubation.
Animals
Female BALB/c mice (6–8 weeks old, 16–18 g) were purchased from Jinan Pengyue Laboratory Animal Breeding Co. All mice were acclimatized to their environment for at least 7 days prior to the experiments. A protocol for animal use was followed by the China Animal Protection Commission and Yantai University for all animal-related procedures. All experiments on animals complied with ethical guidelines.
Animal model induction and treatment
Acute lung injury (ALI) mouse models were established according to the relevant literature. Mice were injected intraperitoneally with LPS (10 mg/kg), and after 5 h of successful modeling, randomly grouped (n=3) and treated with intravenous saline, free Cur, PNPs@Cur and MNPs@Cur, respectively, and euthanized at 24 h after treatment for subsequent experimental analysis.
In vivo targeting to inflammatory site
After establishment of ALI mice, the mice were randomly grouped and injected intravenously with fluorescent dyes DiR (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide), CNPs@DiR, PNPs@DiR and MNPs@DiR, respectively, and normal mice were used as the control group, and the fluorescence distribution of organs in each group was observed by imaging the collected organs at predetermined times with the in vivo imaging system (IVIS).
Cytokine assay
The levels of cytokines (IL-6 and TNF-α) in cell supernatants and serum samples of different treatment groups were measured according to the ELISA KIT instruction manual.
Evaluation of oxidative stress in lung tissue
The lung tissues were collected from different treatment groups for weighing and then 10% lung tissue homogenates were prepared. The levels of MPO and MDA were measured according to the procedure of the relevant kit.
Lung wet/dry ratios
The lung tissues from the mice in the different treatment groups were removed and weighed immediately to obtain wet weights, followed by drying the tissue samples in an oven at 50°C to a constant weight. Finally, the degree of pulmonary edema was quantified by the ratio of wet weight to dry weight.
Histopathological analysis
For histological analysis, major organs (heart, liver, spleen, lung and kidney) were collected after different treatments, fixed overnight in 4% paraformaldehyde and paraffin embedded, then sectioned and stained with hematoxylin-eosin (H&E) for tissue evaluation.
Statistical analysis
The results obtained are expressed as mean ± SD, all statistical analyses were acquired by using GraphPad Prism version 8.0 software (GraphPad, USA). One-way ANOVA and two-way ANOVA were utilized for statistical analysis. Significance levels were set at *P < 0.05, **P < 0.01 and ***P < 0.001. Expression of all fluorescence intensities in the experiments were further calculated by Image J software.