Cell Culture and Reagents
The CFBE41o- (cystic fibrosis bronchial epithelial cell lines, from Dr. Dieter Gruenert[32, 33]) cells were maintained in MEM Earl's salt L-Glutamine (200 mM L-Glutamine) medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B and 10% fetal bovine serum. MEM and other components were purchased from Invitrogen, Carlsbad, CA. TNF-α (R&D Systems Inc., Minneapolis, MN), nile red (Invitrogen), PS-341 (Millenium Pharmaceuticals, Cambridge, MA), PLGA (Avanti Polar Lipids, Alabaster, AL), DSPE-PEG2000 (Avanti) and Pseudomonas aeruginosa LPS (Sigma, St. Louis, MO) were added to cells or injected in mice as indicated. All other common laboratory chemicals were from Sigma or Fisher Scientific.
We dissolved calculated amounts of PLGA and PS-341 and/or nile red in acetone and injected it in DSPE-mPEG2000 emulsifier dissolved in water or PBS followed by immediate rigorous emulsification by a high power sonicator. This result in the synthesis of PEGylated nanoparticles (PNPs) of PLGA dispersed in the aqueous solution, with the water-insoluble drug (PS-341) or dye (nile red) entrapped in the hydrophobic PLGA matrix. We removed acetone by rotary vacuum evaporation and purified drug-loaded nanoparticles by ultracentrifugation followed by rigorous washing (3x) with water or PBS and resuspension in PBS.
Transmission Electron Microscopy (TEM)
Transmission electron microscopy (TEM) was used to determine the size, shape and dispersion of PLGA-PEGPS341 nanoparticles using a JEOL JEM-100cx microscope at an accelerating voltage of 100 kV. The specimens were prepared by drop-coating the sample dispersion onto a carbon-coated 300 mesh copper grid, which was placed on filter paper to absorb excess solvent.
Dynamic laser scattering (DLS)
Dynamic laser scattering (DLS) was employed to measure the size distribution and colloidal stability of the PLGA-PEGPS341 nanoparticles dispersion in water using a Brookhaven Instrument 90Plus Particle Size Analyzer at a wavelength of 633 nm and scattering angle of 90°. DLS was also used to examine the colloidal stability of nanoparticles dispersed in PBS (pH 7.4) over three days.
Release Kinetics and Proteasome Activity Assay
Release kinetics of nile red from PLGA-PEG nanoparticles was quantified by recording absorption of released dye in resuspension buffer (PBS, 100 μl) at 525 nm using the VERSAMAX plate reader and SoftMax Pro software from molecular devices. Nanoparticle samples were aliquoted and incubated at room temperature in triplicate for indicated time points and analyzed for nile red release. We quantified the release kinetics of PS-341 from PLGA-PEG in resuspension buffer (PBS, 100 μl), once daily for a period of 7 days, using Proteasomal Activity Assay from Drug Discovery (BioMol). We recorded proteasome inhibitory activity of room temperature incubated PLGA-PEGPS341 nanoparticles from day 1 to 7 following the manufacturer's protocol. We similarly quantified the efficacy of drug delivery to CFBE41o- cells by quantifying proteasomal activities of cell lysates after 24 hrs of PLGA-PEGPS341, PLGA-PEG (control) or PS341 treatment as indicated. We also quantified proteasomal activities in murine lungs by immunoprecipitating (IP) proteasome from lung extracts (1000 μg) using the proteasome isolation kit (Calbiochem) following the manufacturer's instructions. The 200 μM Suc-LLVY-AMC (Calbiochem) was used as a substrate to estimate chymotrypsin-like proteasomal activity in a 96-well plate. Fluorescence intensities were measured at 360 nm excitation and 440 nm emission by VERSAMax fluorescence plate reader (Molecular Devices) using the SoftMax Pro software. Recombinant purified proteasome (BIOMOL) was used as a positive control while no IP served as a negative control.
All animal experiments were carried out in accordance with the Johns Hopkins University (JHU) Animal Care and Use Committee (ACUC) approved protocol. To induce inflammatory lung disease in vivo, the age (~16 weeks) and sex matched, B6- 129S6- Cftr-/-(Cftrtm1Kthc-TgN(FABPCFTR)) [34, 35] inbred mice (n = 3) were treated, intratracheally (i.t., 10 μg in 100 μl PBS) or intraperitoneally (i.p., 15 mg/kg/bw in 100 μl PBS) with Pseudomonas aeruginosa (Pa)-LPS, 24 hrs post- PLGA-PEGPS341 nanoparticle (intranasal, 5 μl/nostril of 1 μg/μl) or PS341 (i.p., 0.6 mg/kg/bw in 100 μl PBS for 2 days) administration. Based on a previous report [36, 37] and pilot experiments on the release kinetics and in vivo efficacy of the drug, day-3 time point was selected for evaluating the functional efficacy of the drug. Moreover, we have previously standardized that LPS induced lung inflammation, at the selected dose, is at its peak in Cftr-/- mice at 24 hrs. Serum and total lung protein extracts were isolated at day- 3 after euthanasia in the presence of anesthesia following our JHU ACUC approved protocol. The quantification of protein levels by Western blotting of total lung protein extracts (as described below), and cytokine levels by ELISA of brochoalveolar lavage fluid (BALF)/serum (as described below) was used to identify the changes in pro-inflammatory signaling. For live animal imaging experiments, Cftr+/+ mice insufflated with PLGA-PEGNileRed nanoparticles were imaged from day 1-11 using Xenogen IVIS 200 optical imaging device (Ex 465 nm and Em 525 nm) that was directly connected to automatic anesthesia machine providing constant supply of isoflurane.
Lung tissues were lysed by sonication (three 5 sec pulses) on ice in cold room using the T-PER (Pierce Biotech. Inc., Rockford, IL) protein lysis buffer containing protease-inhibitor cocktail (Pierce). The protein extracts were suspended in Laemmli's sample buffer (Invitrogen) containing β-mercaptoethanol (Invitrogen), resolved by 4-10% SDS-PAGE 12-well gel (lane- 1, marker; 2-4, control; 5-7, LPS; 8-9, PLGA-PEGPS341 loaded in duplicate to accommodate all samples in single 12-well gel; 10-12, LPS + PLGA-PEGPS341) and transferred to a 0.45 μm pore size nitrocellulose membrane (Invitrogen). The β-actin (Sigma) and NFκB (Santa Cruz Biotech Inc., Santa Cruz, CA) primary antibodies, and anti-rabbit-HRP secondary antibody (Amersham, Piscataway, NJ) were used for immunoblotting.
Six-week-old mice (n = 3 per genotype) were euthanatized as described above and lungs were collected. Lung was fixed in 1 ml 10% neutral buffered formalin overnight (Fisher Scientific, Pittsburgh, PA), embedded in paraffin, sectioned, and prepared for immunostaining. Macrophages and neutrophils were immunostained with the rabbit polyclonal Mac-3 or NIMP-R14 (2 μg/ml) primary antibody (Abcam, Inc., Cambridge, UK), respectively, followed by a secondary goat anti-rat Alexa Fluor 488, 5 μg/ml (Molecular Probes, Eugene, OR) antibody. Nrf2, NOS2 and NFκB levels were similarly quantified using polyclonal antibodies from Santa Cruz Biotech Inc. Negative controls consisted of identical treatments with the omission of the primary antibody. Hoechst dye, 1 μg/ml (Molecular Probes, Invitrogen) was used for nuclear staining. The slides were then mounted (Vectashield; Vector Laboratories Inc., Burlingame, CA), and images were captured as described below. Nuclei were detected by Hoechst (Invitrogen) while H&E was used to evaluate lung morphology and inflammatory state. Images were captured by Axiovert 200 Carl Zeiss Fluorescence microscope using the Zeiss Axiocam HRC camera and Axiovision software with appropriate filter settings for FITC and DAPI. All fluorescent images were captured at room temperature with oil (63X, fluorescence) and air (20X and 40X) as the imaging medium. The magnifications for the fluorescence microscope were LD Plan- Achroplan (20X/0.40 Korr Phz), Neo Fluar (40X/0.6X Phz Korr) and Achromat (63X/1.4 oil), respectively with 1.6X optivar.
IL-1β, IL-6 and MPO Immunoassay
At the indicated time points, BALFs or serum were collected from each mouse as reported earlier [38–40] and stored at -80C until use. BALF or serum IL-1β levels were measured using solid-phase ELISA (R&D Biosystems, Minneapolis, MN). Standards, and high and low cytokine controls were included. The plates were read at 450 nm on 96-well microplate reader (Molecular Devices, Sunnyvale, CA) using SOFT-MAX-Pro software (Molecular Devices). The mean blank reading was subtracted from each sample and control reading. The amount of substrate turnover was determined calorimetrically by measuring the absorbance, which is proportional to IL-1β concentration. A standard curve was plotted and an IL-1β concentration in each sample was determined by interpolation from standard curve. The data represents the mean ± SD of triplicate samples. The IL-6 cytokine and myeloperoxidase (MPO) levels were similarly quantified using an ELISA system (R&D Biosystems and Hycult Biotech, Canton, MA) as described before.
NFκB or IL-8 Reporter Assay
CFBE41o- cells were transfected with NFκB- or IL-8- firefly luciferase promoter (pGL-2) and renila luciferase (pRLTK) control. Cells were induced with 10 ng/ml of TNF-α and/or 100 ng/ml PLGA-PEGPS341 nanoparticles and luciferase activities were measured after overnight treatment. Dual-Luciferase® Reporter (DLRTM) Assay System (Promega) was used to measure NFκB- or IL-8- reporter (firefly luciferase) and renila luciferase activities from CFBE41o- cell extracts. Data was normalized with internal renila luciferase control for each sample and the changes in reporter activities were calculated.
Representative data is shown as the mean ± SD of at least three experiments. The one-way ANOVA with a Dunnett planned comparison was run for each sample versus control. A * p < 0.05 was considered to have statistical significance. The murine and human microscopy data was analyzed by densitometry (Matlab R2009b, Mathworks Co.) and spearman's correlation coefficient was used to calculate the significance among the indicated groups.