Preparation and characterization of PAMAM-based nanoparticles with negative/ positive core:
The suspension of ethylenediamine core five and a half generation dendrimers (PAMAM G5.5) with sodium carboxylate surface groups, (536784, Sigma Aldrich, St. Louis, MO, USA) as well as ethylenediamine core six generation dendrimers (PAMAM G6) with amino surface groups, (536717, Sigma Aldrich) were used as a colloid carrier for BDNF. Each stock suspension was diluted prior to each adsorption experiment to a desired mass concentration, equal to 10 mgL-1. Filtered (centrifree ultrafiltration device, Merck Group, Darmstadt, Germany) stock solutions of carrier free recombinant human BDNF (248-BDB-250/CF, R&D Systems, Canada) of known concentrations (typically 50 mgL-1) in the phosphate buffered saline (PBS) pH 7.4 +/- 0.2, 0.15M (Biomed, Lublin, Poland) were prepared to remove aggregates and provide constant, free form protein molecules concentration before adsorption at PAMAM particles. To minimize errors in concentration measurements spectrophotometric techniques the BCA (protein quantification bicinchoninic acid assay, kit for low concentration, Abcam, Cambridge, UK) were used. BDNF adsorption at PAMAM dendrimers was performed employing electrostatic interactions according to the following procedure: (1) the reference electrophoretic mobility of bare PAMAM nanoparticles was measured, (2) BDNF layers were formed by mixing equal volumes of its solutions of the 0.2 mgL-1 bulk concentration, with nanoparticle suspension of the bulk concentration 20 mgL-1, (3) the electrophoretic mobility of BDNF-PAMAM nanoparticles was measured and the corresponding zeta potential was calculated. We obtained BDNF-PAMAM nanoparticles, referred to as “PAMAM-based nanoparticles”. Experiments were conducted at 7.4 pH, ionic strength 0.15M, at room temperature. The whole experimental procedure was performed for adsorption time of 6000 seconds. Afterward, BDNF-PAMAM dendrimer nanoparticles were encapsulated with PEG (poly(ethyleneglycol)) with molecular weight of 4 kDa, (1546569, Sigma Aldrich). We carried out simply mixing BDNF-PAMAM and PEG in aqueous solutions without sonification or other extensive agitation. PEG chains were conjugated to the nanoparticle surfaces via amide or carboxyl bonds depending on type of PAMAM core charge, formation between PEG amino groups and PAMAM surface groups. An equal volume of beforehand prepared BDNF-PAMAM and PEG (50 mgL-1, pH 7.4, PBS) solutions was prepared by mixing at room temperature for 1h. Next, PEG-ylated BDNF-PAMAM solution was ultrafiltered with a membrane of 10 kDa cutoff (Millipore, Amicon) to remove unconjugated PEG chains. This resulted in spontaneous self-assembly of BDNF-PAMAM-PEG complexes that we will further refer to as “PEGylated PAMAM-based nanoparticles”.
The particle size, zeta potential and polydispersity index (PDI) of BDNF, PAMAM 6, PAMAM 5.5, PAMAM-based nanoparticles, PEGylated PAMAM-based nanoparticles were determined with the Zetasizer Nano ZS apparatus (Malvern Instruments, Malvern, UK) equipped with a laser of 633nm wavelengths. Data analysis was performed in automatic mode at 25 ̊C. Measured size was presented as the average value of 20 runs, with triplicate measurements within each run. Particle size distributions were obtained from measured diffusion coefficients.
The size distribution of PAMAM 6, PAMAM 5.5, PAMAM-based nanoparticles, PEGylated PAMAM-based nanoparticles was determined at mica surface by AFM (atomic force microscopy) technique. The nanoparticles were left to deposit on mica sheets (Continental Trade, Poland) placed in the diffusion cell over a 5min, and then substrate was removed and rinsed for half an hour in ultrapure water. The samples were left for air-drying until the next day. Next, the dry sample was placed under 7-10 nm AFM tip. The AFM measurements were carried out under ambient air conditions using the NanoWizard AFM (JPK Instruments AG, Berlin, Germany). The intermittent contact mode images were obtained in the air, using ultrasharp silicon cantilevers (NSC35/AlBS, MicroMash, Spain) and the cone angle of the tip was less than 20o. The images were recorded at the scan rate of 1 Hz for the six randomly chosen places. The images were flattened using an algorithm provided with the instrument. We captured all images in random areas within the scan size of 0.5 x 0.5 µm or 1 x 1 µm. BDNF, PAMAM 5.5, PAMAM6, PAMAM-based nanoparticles and PEGylated PAMAM-based nanoparticles surface dimensions were determined using ImageJ software by gathering the number and coordinates of single protein/nanoparticles molecules.
BDNF adsorption studies in PBS
Protein loading profile at PAMAM-based nanoparticles and PEGylated PAMAM-based nanoparticles with negative charge core as well as positive charge core was conducted by solution depletion ELISA technique (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). Afterwards, to more accurately determine unbound BDNF after adsorption at PAMAM particles, two-step Laser Doppler Velocimetry (LDV) technique with the aid of the abovementioned Malvern device was used. LDV method exploiting the calibrating measurements uses various colloid particles (in our study modelled latex microparticles) for efficient monitoring of desorbed protein molecule concentration in order to determine protein maximum coverage at PAMAM nanoparticles.
BDNF release studies in PBS
The protein release profile from PAMAM-based nanoparticles, PEGylated PAMAM-based nanoparticles with negative charge core as well as positive charge core was assessed by using ultrafiltration method with a 30kDa cutoff membrane (Millipore, Billerica, MA, USA) in PBS at pH 7.4 and 0.15M ionic strength. It was done in two-stage procedure, where first BDNF adsorption process was carried out for 1h. The BDNF molecules released from nanoparticles with various charge core were quantified with ELISA immunoassay method according to the manufacturer’s protocol. Initially, the residual (unbound) BDNF concentration in the filtrate was determined immediately after adsorption at PAMAM nanoparticles by applying sandwich ELISA technique to monitor the maximum concentration of unbound BDNF in the supernatant suspensions. Thus, it was possible to precisely determine concentration of non-adsorbed BDNF molecules at PAMAM as well as PEG-ylated PAMAM nanoparticle surface. These measurements were utilized for determining the maximum coverage of neurotrophin under protein bulk condition (0.1 mgL-1). Afterward, the concentration of desorbed BDNF for a 24h was quantified with UV-VIS spectroscopy and calculated according to ELISA standard curve.
Cell culture and differentiation
SH-SY5Y neuroblastoma cells (human, ECACC; Sigma Aldrich, St. Louis, MO, USA) were used in this study. SH-SY5Y cells were incubated in culture plates in proliferation medium containing Ham’s F-12 Nutrient Mixture (Thermo Fisher, Waltham, MA, USA) and minimum essential medium (MEM) (Sigma Aldrich, St.Louis, MO, USA) mixed in ratio 1:1 and supplemented with streptomycin (100 µg/mL), penicillin (100 U/mL), L-glutamine (2 mM) and 15% heat-inactivated fetal bovine serum (FBS) at 37◦C in saturated humidity atmosphere containing 5% CO2. The proliferation medium was changed every 2-3 days, and the cells were passaged when they reached 80% confluence. After the proliferation step, the cells were transferred into new culture plates and incubated for 24h with MEM supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2mM) and 1% FBS. On the next day, the medium was changed to a differentiation medium consisting of MEM supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2mM), 1% FBS and Retinoic Acid (0.01µmol/mL) (RA, Sigma Aldrich, St.Louis, MO, USA). The differentiation was carried out for 5 days and the medium was changed every 2 days.
6-OHDA and nanoparticles cytotoxicity
Differentiated SH-SY5Y cells were incubated at a density of 3x104 cells/well in 96-well plates for 24h with MEM (without FBS) containing various concentration of 6-hydroxydopamine (20-35µmol/L) (6-OHDA, Sigma Aldrich, St.Louis, MO, USA). 6-OHDA was freshly prepared for each experiment to avoid oxidation. 6-OHDA remains the most widely used neurotoxin in Parkinson’s disease (PD) in vitro models23, due to its structural similarity to dopamine (DA) and high affinity for the DA transporter, which enable it to selectively destroy dopaminergic neurons24. Therefore, for our study we chose to treat human neuroblastoma cell line SH-SY5Y with 6-OHDA, as it has been extensively described in the literature as a proper in vitro model for PD25, 26. Cytotoxicity of 6-OHDA was evaluated by exposing cells to different concentrations of this neurotoxin for 24h at 37oC, and cell viability was estimated by the measuring toxicity using the MTT assay (Abcam, Cambridge, UK), which is based on the conversion of water soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to an insoluble formazan product, which has a purple color. At the end of the treatments, the medium was discarded and new medium containing 0.5 mg/ml was added. Cells were incubated with 50 μL of MTT reagent mixed with 50µl MEM for 3h, then 150µl of detergent solution was added to solubilize the colored crystals. Finally, absorbance was measured at 590 nm using Varioskan LUX Multimode Microplate Reader (Thermo Fisher, Waltham, MA, USA). Toxicity was calculated from the equation provided in the manufacturer's protocol.
After choosing certain 6-OHDA concentration, the cells were incubated for 24h with MEM (without FBS) containing 20µmol/L of 6-hydroxydopamine. At the end of the treatments, the medium was discarded and a following solution was added to each well: 20µl of PAMAM-based nanoparticles or PEGylated PAMAM-based nanoparticles with different charged core in PBS (described above in Preparation and characterization of PAMAM-based nanoparticles with negative and positive core section) and 80µl MEM without FBS. The last step was to assess toxicity of various type of nanoparticles on differentiated SH-SY5Y cells using the MTT assay.
BDNF release studied in neuroblastoma cell culture exposed to 6-OHDA
Concentration of released BDNF molecules was determined by exposing differentiated human neuroblastoma cells SH-SY5Y to 6-hydroxydopamine (6-OHDA) as well as nanoparticles with differently charged PAMAM core for 24h at 37oC. At the end of the treatments, the cell supernatant was discarded and collected to quantify BDNF concentration using UV-VIS spectroscopy calculated according to ELISA standard curve. Simultaneously we determined BDNF concentration in differentiated neuroblastoma cell lysate exposed to 6-OHDA what is crucial to enhancing nanoparticle efficacy. To obtain neuroblastoma cells exposed to 20µmol/L, 6-OHDA lysates 3x104 SH-SY5Y cells were trypsinized and washed in medium twice and then followed by four freezes (liquid nitrogen) and thaw (37 °C water bath) cycles. Large particles were removed by centrifugation (2,000 g for 10 min, followed by 13,000 g for 60 min at 4 °C) then lysate was filtered through a 0.22 μm mesh and aliquots were stored at – 80 °C. The protein content was determined by the ELISA assay.
PAMAM-based nanoparticles and PEGylated PAMAM-based nanoparticles behavior in cell culture
In addition, we further investigated the behaviour of our nanoparticles loaded with BDNF in SH-5YSY cell culture by determination of green fluorescence of PAMAM-AF488 conjugates. Differentiated SH-SY5Y cells and previously treated with 6-OHDA (protocol described in 6-OHDA and nanoparticles cytotoxicity) were incubated at a density of 3x104 cells/well in 96-well plates with BDNF-PAMAM-AF488 and BDNF-PAMAM-AF488-PEG nanoparticles (0.1 μg/mL protein loading) for 24h. After the nanoparticles labelled AF488 were removed, and the wells were washed twice with PBS, the cells were subjected to examinations by spectrofluorimetry evaluation using Varioskan LUX Multimode Microplate Reader.
Flow cytometry
To determine a mean fluorescence intensity (MFI) of nanoparticles PAMAM 5.5 and PAMAM 6 conjugated with fluorochrome AF488, the differentiated and treated with 6-OHDA cells SH-SY5Y (protocol described in 6-OHDA and nanoparticles cytotoxicity) were incubated at a density of 3x104 cells/well with PAMAM 5.5-AF488, PAMAM 5.5-AF488-BDNF(0.1μg/mL), PAMAM 5.5-AF488-BDNF(0.1μg/mL)-PEG, PAMAM 6-AF488, PAMAM 6-AF488–BDNF(0.1μg/mL) and PAMAM 6-AF488–BDNF(0.1 μg/mL)-PEG each in 10 separate repetitions for 24h. The cells were then washed with PBS, incubated for 3 min with trypsin, transferred to a cytometric tube, washed twice in PBS and resuspended in 200 µl PBS. Fluorescence was measured and the data were analyzed using a LSRII flow cytometer (BD Biosciences) and the BD FACSDiva software. 10 000 events were acquired to determine the MFI.
Detection of lipid peroxidation
Malondialdehyde (MDA) is the most abundant and stabile individual indicator of the level of the aldehydic products that can be produced from free radical attack on polysaturated fatty acids indicating lipid peroxidation present in cell cultures. The cells were incubated for 24h with different concentration of BDNF, BDNF-PAMAM dendrimer nanoparticles or PEG-ylated BDNF-PAMAM dendrimer nanoparticles, adding 20µl to each well of selected solution prepared in PBS (described in Preparation and characterization of PAMAM-based nanoparticles with negative and positive core) and 80µl MEM without FBS. For this purpose, after completion of 24h of incubation with various type of nanoparticles the cell supernatant was collected from each well (3x104 cells/well) for MDA analysis.
The MDA analysis in cell supernatant was determined using reverse-phase, high-performance liquid chromatography (HPLC)-spectrophotometric method according to Ref.27. HPLC (varian vista series) was performed on an Agilent Zorbax column (300A). Qualitative and quantitative analyses of MDA release by the differentiated human neuroblastoma cells SH-SY5Y threated 6-OHDA was performed by integrating the retention times and the peak areas compared with known concentrations of MDA standard prepared in the same solvent. 1,1,3,3-tetraethoxypropane (TEP) standards were freshly prepared daily. The solvent blank was absolute ethanol (400 ml/l) in distilled water. The stock standards were 10 mM and 100 mM TEP and the working calibrants were 0.0, 0.25, 0.50, 0.75 and 1.0 mM TEP, prepared from dilution of the 100mM TEP with absolute ethanol (400ml/l). The HPLC calibration was performed for each run. Samples were calculated as mM MDA equivalent from the TEP standard calibration (1:1 conversion under acidic conditions).
Detection of mitochondrial membrane potential
JC-1 (Caymanchem, MI,USA) is a lipophilic, cationic dye that can selectively enter into mitochondria. In healthy cells, JC-1 forms aggregates which display strong fluorescence intensity with excitation and emission at 535nm and 595nm. In apoptotic/unhealthy cells, JC-1 exists as monomers which show strong fluorescence intensity with excitation at 485 nm and emission at 535 nm. Differentiated and treated with 6-OHDA cells SH-SY5Y (protocol described in 6-OHDA and nanoparticles cytotoxicity) were incubated at a density of 3x104 cells/well with PAMAM 5.5, PAMAM 6, PAMAM 5.5-BDNF(0.02μg/mL), PAMAM 6–BDNF(0.02μg/mL), PAMAM 5.5–BDNF(0.02μg/mL)-PEG, PAMAM 6-BDNF(0.02μg/mL)-PEG, PAMAM 5.5-BDNF(0.1μg/mL), PAMAM 6-BDNF(0.1μg/mL), PAMAM 5.5-BDNF(0.1μg/mL)-PEG, PAMAM 6-BDNF(0.1μg/mL)-PEG, PAMAM 5.5-PEG, PAMAM 6-PEG, each in 16 separate repetitions for 24h. The cells were then incubated with JC-1 Staining Solution for 15 min in a CO2 incubator at 37°C according to the manufacturer's protocol. Fluorescence was measured with Ex535nm/Em595nm and Ex485nm/Em535nm using a Varioskan Lux Reader (Thermo Fisher Scientific, MA, USA). The ratio of fluorescence intensity of aggregates to fluorescence intensity of monomers was used as an indicator of cell health.
Confocal microscopy
To determine the morphological changes the SH-SY5Y cells were seeded at a density of 1 x 105 cells/well in a 4-well chamber slide, differentiated with RA and treated with 6-OHDA. Next, the cells were incubated with: PAMAM 5.5, PAMAM 5.5-PEG, PAMAM 5.5-BDNF (0.1µg/mL), PAMAM 5.5-BDNF (0.1µg/mL)-PEG, PAMAM 6, PAMAM 6-PEG, PAMAM 6-BDNF (0.1µg/mL) and PAMAM 6-BDNF (0.1µg/mL)-PEG for 24 hours. Subsequently, the cells were fixed with 70% ethanol for 15 min and washed twice with PBS. For cell membrane staining the slides were incubated with 1:100 wheat germ agglutinin-Texas Red X (ThermoFisher, Waltham, MA, USA) in HBSS buffer for 20 min. After washing the slides were counterstained with DAPI, mounted and examined using Carl Zeiss LSM 700 microscope (Zeiss, Jena, Germany). The cell nuclei dimensions were measured at their widest point using ZEN software. For each specimen at least 200 measurements were taken.
Statistical analysis
All presented data are expressed as means +/- standard deviation (SD) from at least three independent experiments. Statistical analysis among each study group was performed using Kruskal-Wallis test. Two-Way ANOVA was used for analysis between experimental groups. p < 0.05 was considered statistically significant.