Reagents and chemicals
Poly(L-lactide) (PLA, viscosity approximetely 1.0 dL/g), flurbiprofen and polyvinyl alcohol (PVA) were obtained from Sigma (Steinheim, Germany). Lumogen® F orange 240 was provided by BASF (Ludwigshafen, Germany). All other reagents were of analytical grade and used as received.
PLA nanoparticles were formed by an emulsification-diffusion technique. Briefly, 100 mg of PLA, 10 mg of flurbiprofen and 150 μg of Lumogen® orange were dissolved in 2 ml dichloromethane (DCM). For the control PLA nanoparticles, 100 mg of PLA and 150 μg of Lumogen® orange were dissolved in 2 ml DCM. For both formulations, the organic phase was added to 6 ml aqueous solution of PVA (2%, w/v). This mixture was homogenized in an ice bath for 30 minutes at 24,000 rpm (Ultra Turrax®, IKA, Staufen, Germany) and diluted with 6 ml PVA solution (1%, w/v). DCM was removed by stirring the emulsion over night at room temperature. Finally, the particles were collected by centrifugation at 20,000 g for 10 minutes (Eppendorf, Hamburg, Germany) and washed twice with purified water before lyophilization.
Freeze-drying of the samples
For the lyophilization process a freeze-dryer Epsilon 1–4 (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) was used. Aliquots of the nanoparticles suspension (100 μl) were dispensed into 2 ml Lyovials and diluted with 100 μl trehalose solution (6%, w/v) as a cryoprotective agent. The freeze-drying cycle was performed according to an established protocol. First, the samples were frozen at -40°C for three hours. In the second step, primary drying was performed at a temperature of -34°C for 24 hours and a vacuum of 0.05 mbar, followed by a secondary drying phase for 11 hours at 20°C and a vacuum of 0.025 mbar. At the end of the drying process the vials were sealed and removed.
Nanoparticles were analyzed with regard to particle diameter and polydispersity by photon correlation spectroscopy (PCS) and zeta potential was measured by microelectrophoresis using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). Prior to measurement the samples were diluted with purified water.
Determination of flurbiprofen loading
The amount of flurbiprofen incorporated into the nanoparticles was determined by a HPLC method in which 1 mg nanoparticles was incubated in 1 ml acetonitrile for five minutes at room temperature. The sample was centrifuged (20,000 g for 10 minutes) and the chromatographic separation was carried out using aliquots of the supernatant. The aliquots (20 μl) were injected into a Phenomenex Gemini NX 250 × 4.6 mm, 5 μm particle, C18 column (Phenomenex, Aschaffenburg, Germany). The flow rate was set to 1 ml/minute during the separation, with the mobile phase composed of acetonitrile and 0.1% (v/v) trifluoroacetic acid (57.5: 42.5, v/v). The eluate was analyzed at a wavelength of 245 nm.
In vitro release of flurbiprofen
For each point in time individual samples were prepared as follows: 1 mg nanoparticles were incubated in 1 ml phosphate buffer (0.1 mM, pH = 7.5) at 37°C under constant shaking. At defined points in time (0, 0.5, 1, 3, 5, 7, 24 hours) one sample was centrifuged (20,000 g for 10 minutes). The amount of the released drug was determined in the supernatant by HPLC as described above.
The freeze-dried nanoparticles were always reconstituted prior to the cell culture experiments. Therefore, 40 mg nanoparticles were dissolved in 1 ml purified water and vortexed for two minutes.
The mouse brain endothelial cell line bEnd.3 (ATCC, Manassas, VA, USA) was cultured in DMEM (Gibco, Darmstadt, Germany) high glucose medium containing 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Gibco, Darmstadt, Germany). For the experiments, 5 × 104 cells per cm2 were seeded and the experiments were performed after three days when the cells were post-confluent. APP751 overexpressing CHO cells (7WD10) were cultured in DMEM high glucose medium containing 10% fetal bovine serum, 1 mM sodium pyruvate (Gibco, Darmstadt, Germany), 100 U/ml penicillin/streptomycin and 400 μg/ml geneticin (Calbiochem, Nottingham, UK). For the experiments, 3 × 104 cells per cm2 were seeded, and after 24 hours cells were either treated or co-cultured with the bEnd.3 in the in vitro BBB model.
Measurement of cytotoxicity
The cytotoxicity of free flurbiprofen or PLA-flurbiprofen nanoparticles was assessed using the alamarBlue® reagent (Invitrogen, Karlsruhe, Germany). bEnd.3 cells were seeded on 96-well plates (Greiner, Frickenhausen, Germany) and after reaching post-confluency, cells were treated with increasing concentrations of free or nanoparticulate flurbiprofen, ranging from 25 μM to 750 μM (which corresponded to approximately 32 μg/cm2 to 942 μg/cm2 nanoparticles). The unit μg per cm2 refers to the amount of nanoparticles which are administered to the cells and this unit reflects possible local sedimentation on the surface of the cells, which locally might lead to different concentrations. After 72 hours, cells were incubated for another four hours with 1 × alamarBlue® in medium. The absorbance was measured with an Anthos plate reader 2010 (Anthos Labtec, Salzburg, Austria) using a 570 nm measurement filter and a 600 nm reference filter. The cell viability was calculated as percentage of absorbance in relation to vehicle control treated cells.
Measurement of the transepithelial electrical resistance of endothelial cells
The transepithelial electrical resistance (TER) was used to analyze the toxicity of the nanoparticles for endothelial cells. bEnd.3 cells were seeded on 24-transwell cell culture inserts (ThinCerts™, Greiner Bio-One, Frickenhausen, Germany) and placed into the cellZscope® device . The TER of the cells was measured automatically every hour under physiological conditions by impedance spectroscopy. When cells were post-confluent, equal amounts of drug-loaded and unloaded nanoparticles (approximately 2.4 mg nanoparticles per cm2) were added luminally and the TER was measured (this concentration corresponds to 750 μM nanoparticulate flurbiprofen).
Cellular binding and uptake of nanoparticles
bEnd.3 cells were cultured in 24-well plates (Greiner, Frickenhausen, Germany) and treated with approximately 100 μg/cm2 PLA-flurbiprofen nanoparticles for four hours at 37°C. After the incubation, cells were washed twice with PBS (Invitrogen, Karlsruhe, Germany) and subsequently trypsinized and harvested. Fixing was performed with FACS-Fix (10 g/L paraformaldehyde (PFA) and 8.5 g/L NaCl in PBS, pH 7.4) before flow cytometry analysis. Per sample, 104 cells were counted using FACSCalibur and CellQuest Pro software (Becton Dickinson, Heidelberg, Germany). The fluorescent labeling of the nanoparticles via Lumogen® F Orange 240 allowed a detection at 524/539 nm. To study the endocytotic uptake of the PLA nanoparticles, bEnd.3 cells were grown on glass coverslips (Marienfeld, Lauda-Königshofen, Germany) and treated with approximately 100 μg/cm2 PLA-flurbiprofen nanoparticles at 4°C or 37°C for one hour or four hours. After the incubation, cells were put on ice and washed with PBS pH2 to remove the surface-bound nanoparticles, mimicking the acidic environment of endosomes where ligands dissociate from their receptor after internalization . Cells were fixed with 4% PFA and 0.12 M sucrose in PBS for 10 minutes at room temperature and the cell nuclei were stained with 2 μM DRAQ5™ (Biostatus Limited, Leicestershire, UK) for 10 minutes at room temperature. Samples were embedded in Prolong® Gold antifade reagent (Invitrogen, Darmstadt, Germany) and the confocal laser scanning microscope (CLSM) study was performed with a CLSM equipped with ZEN 2008 software (LSM 710; Zeiss, Jena, Germany).
Treatment of 7WD10 with nanoparticles
To examine the biological activity of flurbiprofen-loaded nanoparticles, 7WD10 were treated with free or nanoparticulate flurbiprofen, ranging from 50 μM to 250 μM flurbiprofen. The administered concentration of the nanoparticles was adjusted to the free flurbiprofen, which corresponds to approximately 65 μg/cm2 to 317 μg/cm2 nanoparticles. After 48 hours, the supernatants were collected and centrifuged at 18,000 g for 20 minutes at 4°C. Levels of Aβ were measured by an Aβ specific ELISA.
Transport assay of nanoparticulate flurbiprofen in an in vitro BBB model
bEnd.3 cells were seeded on 24-transwell cell culture inserts. After reaching post-confluency, bEnd.3 cells were co-cultured with 7WD10 in the lower compartment and bEnd.3 cells were treated with 300 μM free flurbiprofen or nanoparticulate flurbiprofen, ranging from 300 μM to 750 μM flurbiprofen, which corresponds to approximately 380 μg/cm2 to 942 μg/cm2 nanoparticles. After 72 hours, the supernatants of the lower compartment were collected and centrifuged at 18,000 g for 20 minutes at 4°C. Levels of Aβ were measured by an Aβ specific ELISA.
Measurement of Aβ species by ELISA
The levels of Aβ40 and Aβ42 peptides were determined using a cell-based sandwich ELISA assay as described . Briefly, the monoclonal antibody IC16 (1:250 in PBS, pH 7.2) raised against amino acids 1 to 15 of the Aβ sequence was used as a capture antibody. To generate standard curves, synthetic Aβ40 and Aβ42 peptides (JPT Peptide Technologies, Berlin, Germany) were used. These Aβ peptides were solubilized in dimethyl sulfoxide (DMSO) at 10 μg/ml and aliquots were stored at -80°C. The capture antibody was inclubated overnight in 96-well high-binding microtiter plates at 4°C. After the capture antibody was removed, conditioned media samples (20 μl for detection of Aβ40 and 100 μl for Aβ42) and freshly diluted Aβ peptide standards (125 to 6,000 pg/ml in PBS containing 0.05% Tween-20, 1% BSA) were added. Subsequently, C-terminal detection antibodies specific for Aβ40 and Aβ42 labeled with horseradish peroxidase (HRP) using the Pierce EZ-Link™ Plus Activated Peroxidase kit (Thermo Fisher Scientific, Rockford, IL, USA) were diluted in PBS containing 0.05% Tween-20, 1% BSA, added to each well, and incubated overnight at 4°C. Plates were washed three times with PBS containing 0.05% Tween-20 and once with PBS. Then, 50 μl of TMB (3,3',5,5'-Tetramethylbenzidin) ELISA Peroxidase Substrate (Interchim, Montlucon cedex, France) was added and incubated for 1 to 10 minutes at room temperature in the dark. The reaction was stopped by adding 50 μl of 2 M H2SO4 and the absorbance was measured using a Paradigm microplate reader (Beckman Coulter, Krefeld, Germany) at 450 nm. The levels of the Aβ40 and Aβ42 peptides were normalized to Aβ total (Aβ40 + Aβ42) and the average of triplicate measurements for each concentration was normalized to the control condition (DMSO or unloaded PLA nanoparticles).
Nanoparticle plasma protein binding assay
To obtain human plasma, blood was taken at the ENT department at the Medical University Mainz from 15 different seemingly healthy donors in k2EDTA coated tubes (Greiner, Frickenhausen, Germany) to prevent blood clotting. The blood samples were labeled anonymously and could not be traced back to a specific donor. Studies were approved by the local ethics committee of the University Medical Center of the Johannes Gutenberg-University of Mainz, and informed consent was obtained in accordance with the Declaration of Helsinki. The PLA nanoparticles were incubated with equal amounts of human plasma for different time points (5, 15, 30 and 60 minutes), loaded onto a sucrose cushion (0.7 M in PBS) and centrifuged through the cushion to separate nanoparticle-protein complexes from plasma (12,000 rpm for 20 minutes at 4°C). Pellets were washed three times with PBS and proteins were eluted from the recovered particles by adding an equal volume of SDS sample buffer (62.5 mM Tris–HCl pH 6.8; 2% (w/v) SDS, 10% glycerol, 50 mM dithiothreitol (DTT), 0.01% (w/v) bromophenol blue) to the pellet and incubated at 95°C for five minutes. Proteins were separated on a 12% SDS-polyacrylamide gel. To visualize the kinetic evolution of the protein corona, the SDS-polyacrylamide gel was stained with Coomassie brilliant blue R-250 (Bio-Rad, München, Germany) and protein quantification was performed using the BioRad Protein Assay (Bio-Rad, München, Germany). To examine the presence of apolipoproteins in the nanoparticle-protein complex, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.01% Tween-20 and the following antibodies were used: α-apoA4 (Cell Signaling, Boston, MA, USA ); α-apoE and rabbit-α-mouse immunoglobulin G (IgG) antibody conjugated with HRP (Santa Cruz, Dallas, TX, USA).
All graphs and statistics were performed using the GraphPad Prism 4 software (GraphPad, La Jolla, CA, USA). Data were analyzed by two-way analysis of variance (ANOVA) coupled to Bonferroni post-tests for multiple comparisons. P <0.05 was considered as statistically significance.