Fabrication of lipid-based nanocarriers
Three lipid-based nanosystems with different diameters were produced by the emulsification method. The lipid phase consisted of OA (1% of the final product, w/v), SPC (1.5%), and mineral oil. The percentage of mineral oil was 1.5%, 5%, and 10% for development of the final products with small (AS), medium (AM), and large (AL) size, respectively. The aqueous phase consisted of Poloxamer 188 (1.5%) and water. Both phases were heated separately at 85°C for 20 min. The water phase was then added in drop form into the lipid phase via high-shear homogenization at 12,000 rpm for 20 min, followed by agitation through a probe-type sonicator (35 W) for 20 min. The nanocarriers were used in the experiments after cooling to room temperature.
The size and zeta potential of the nanocarriers
The average diameter, PDI, and zeta potential of the OA-loaded nanoparticles were measured by a laser-scattering procedure (Nano ZS90, Malvern). The nanoparticles were diluted 100-fold with water before measurement.
Molecular environment of the nanocarriers
The degree of lipophilicity of the nanoparticles was determined by fluorescence spectrophotometry based on the solvatochromism of Nile red . Nile red (1 x 10-3 mg/ml) was incorporated in the lipid phase of the nanosystems. The emission spectra of dye-containing nanosystems were scanned from 550 nm to 700 nm. The excitation wavelength was set at 546 nm.
Human neutrophil purification
The protocol for this purification was approved by the Institutional Review Board of Chang Gung Memorial Hospital. All subjects (20–30 years old) had signed an informed consent. The blood was collected by venipuncture. The neutrophils were isolated using dextran sedimentation before centrifugation in a Ficoll-Hypaque gradient as previously described .
The neutrophil uptake of nanocarriers
The lipid-based nanoparticles were labeled with rhodamine 800 (0.1 mg/ml) as a dye to visualize neutrophil uptake (1.8 x 107 cells/ml). The nanoparticles were treated with the cells for 5 min. The degree of uptake was quantified by measuring the dye fluorescence in flow cytometry. The nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI). The nanoparticle ingestion was observed under confocal microscopy (TCS SP2, Leica).
The neutrophil survival after nanoparticle treatment was measured by LDH release. LDH was analyzed using a commercial kit (CytoTox 96, Promega). The cells (6 x 105 cells/ml) were equilibrated at 37°C for 2 min. Subsequently, the nanocarriers with OA (1–10 μg/ml) were added into neutrophil suspension for 15 min. The OA doses used in this study were 1, 3, and 10 μg/ml. The total LDH leakage was detected after the treatment by Triton X-100.
Superoxide anion production
We used the superoxide dismutase-inhibitable decrease of ferricytochrome c to detect superoxide production . At first, neutrophils (6 x 105 cells/ml) were equilibrated for 2 min after supplementation with ferricytochrome c (0.5 mg/ml) and CaCl2 (1 mM). Then, the nanoformulations were added to the cell suspension for 5 min. The OA doses tested were 0.3, 1, 3, and 10 μg/ml. The cells were activated by fMLF at 100 nM for 10 min. The absorbance with the reduction of ferricytochrome c at 550 nm was quantified by a UV/visible spectrophotometer.
Meo-Suc-Ala-Ala-Pro-Val-p-nitroanide was employed as the substrate of elastase for detecting elastase release . After the incorporation with the substrate (100 μM), the cells were equilibrated for 2 min and then treated with the nanoformulations for 5 min. We used the OA dose of 0.3, 1, 3, or 10 μg/ml for testing elastase inhibition. The neutrophils were activated by 100-nM fMLP. Absorbance at 405 nm was obtained by a UV/visible spectrophotometer.
Intracellular Ca2+ ([Ca2+]i) assay
Furo-3/AM (2 μM) was used to treat the neutrophils (3 x 106 cells/ml) at 35°C for 45 min, followed by centrifugation and resuspension in Hank’s balanced salt solution with CaCl2 (1 mM). The cells were exposed with nanosystems for 5 min at OA dose of 1, 3, or 10 μg/ml. The [Ca2+]i in response to fMLF was detected using a fluorescence spectrophotometer with the excitation and emission wavelength at 488 nm and 520 nm, respectively.
The formation of neutrophil extracellular traps (NETs)
The isolated neutrophils (1 x 106 cells/ml) were incubated with nanocarriers at OA concentration of 3 or 10 μg/ml for 10 min and then stimulated with PMA at 10 nM for 3 h. SYTO Green nucleic acid stain (2.5 μM) was added to the cell suspension for 15 min. The fluorescence intensity was quantified at 485 (excitation) and 535 (emission) nm, respectively .
Male C57BL/6 mice (20-25 g) acquired from the National Laboratory Animal Center (Taipei, Taiwan) were used. All study procedures were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee of Chang Gung University.
Biodistribution of nanocarriers
We employed an NIR bioimaging system (Pearl Impulse, Li-Cor) to monitor the nanoparticle biodistribution. iFluor 790 acid (0.08%) as the NIR dye was incorporated into the nanocarriers. The nanosystems (2 ml/kg) were intravenously injected into the tail vein of the anesthetized mice. The mice were sacrificed after 2 h. The organs were excised to monitor the NIR signal using the Pearl Impulse bioimaging system.
The mice with ARDS-like signs were divided into five groups: ARDS without therapy and ARDS treated with intravenous free OA, AS, AM, or AL. An intratracheal challenge of LPS was carried out as described before . In brief, we injected free OA or OA-loaded nanocarriers into the mice. LPS at 8 mg/kg was administered to the animals after a 30-min injection. The mice were sacrificed 6 h after LPS stimulation. The pulmonary tissue was excised for histological observation and ELISA analysis.
The lung specimen was added into formaldehyde (10%) and embedded in paraffin. The samples were cut to a 3-μm thickness for H&E staining. For the immunohistochemistry, the slices were incubated with anti-MPO or anti-Ly6G antibody for 1 h. Subsequently, the biotinylated donkey anti-goat IgG was used to treat the samples for 20 min. A light microscope was used to visualize the slices.
MPO activity in the lung tissue was measured by the colorimetric assay of combined o-dianisidine HCl and H2O2 as described previously . Cytokines and chemokines in the pulmonary tissue were quantified by ELISA. The tissue was extracted with buffer containing complete protease inhibitors under homogenization (MagNA Lyser, Roche). The homogenate was centrifuged at 11,500 xg for 10 min. The supernatant was taken to measure TNF-α, IL-1β, IL-6, and CXCL-2 employing commercial kits (BioLegend).
The statistical difference in the data of the various treatments was analyzed by the Kruskal-Wallis test. The post hoc test for checking individual differences was Dunn’s test. The 0.05, 0.01, and 0.001 levels of probability were taken as statistically significant.