Adsorption/desorption transition of BDNF molecules at/from PAMAM dendrimers (Figure 1) was studied in PBS buffer using the dynamic light scattering, electrophoresis, solution depletion techniques, enzyme-linked immunosorbent assay and atomic force microscopy. This allowed us to precisely determine maximum loading of BDNF molecule at PAMAM-based nanoparticles under in situ conditions. Afterwards, we compared desorption kinetics of BDNF from PAMAM-based nanoparticles as well as PEG-ylated -PAMAM nanoparticles in PBS buffer and in neuron-like differentiated SH-SY5Y cells environment to quantitatively assess in real time cellular internalization by neuroblastoma cells PAMAM-based nanoparticles (using spectrofluorimetry and confocal microscopy evaluation).
2.1. Nanoparticles Synthesis
Filtered (centrifree ultrafiltration device, Merck Group, Darmstadt, Germany) stock solutions of carrier free recombinant human BDNF (248-N4-250/CF, R&D Systems, Canada) of known concentrations (typically 250 mg L-1) in the phosphate buffered saline (PBS) pH 7.4 +/- 0.2 (Biomed, Lublin, Poland) were prepared to remove aggregates and provide constant, free form protein molecules concentration in the solvent. To minimize errors in concentration measurements, two complementary spectrophotometric techniques were used: the BCA (protein quantification bicinchoninic acid assay, kit for low concentration, Abcam, kraj) and UV absorbance at 280 nm measured with microplate spectrophotometer (BioTek Epoch, United States). Prior to each measurement, the stock solution was diluted to a desired bulk concentration, typically 0.01-2 mg L-1. The exact concentration of these solutions after membrane filtration was determined by commercially available ELISA assay (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). The temperature of experiments was kept at a constant value equal to 298 ± 0.1K.
The suspension of PAMAM G5.5 ethylenediamine core five and a half generation dendrimers with sodium carboxylate surface groups, (536784, Sigma Aldrich, kraj) was used as colloid carrier for BDNF. The stock suspension was diluted prior to each adsorption experiment to a desired mass concentration, equal to 10 mg L-1.
For detection of protein – PAMAM nanoparticles in differentiated neuroblastoma SH-SY5Y cells by confocal microscopy, a detectable fluorescent molecule i.e. PAMAM-AF488 was needed. We used Alexa Fluor 488Ò (AF 488)-NHS esters (InvitrogenTM, kraj) ester as fluorescent label. PAMAM at 5 mg ml-1 in pH 8.0 sodium bicarbonate buffer reacted with 1 mg ml-1 amine reactive dye dissolved in dimethylformamide as described in the manufacturer’s protocol, to give fluorescently labeled PAMAM-AF488 conjugates. These conjugates were purified by extensive dialysis against pH 7.4 to remove unreacted label. After dialysis, we further purified conjugates through centrifugal filtration until filtrate absorbance at 490 nm reached background levels.
BDNF-PAMAM dendrimer nanoparticles:
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 bulk concentration (varied between 0.002 – 0.4 mg L-1), with nanoparticle suspension of the bulk concentration 20 mg L-1, (3) the electrophoretic mobility of BDNF-PAMAM nanoparticles was measured and the corresponding zeta potential was calculated. Experiments were performed at 7.4 pH, ionic strength 0.15M, 20oC temperature. In all cases, for adsorption time 2000 seconds BDNF should be irreversibly adsorbed onto the surface creating monolayer as a result of short-range electrostatic interaction between BDNF and PAMAM, since the adsorption process is mediated by electrostatic interactions. The time scale for formation of additional layers is much longer than that of monolayer formation. The adsorption time of BDNF at PAMAM dendrimers was prolonged up to 6 hours in the primary adsorption experiments. To determine unbounded protein concentration after adsorption, we performed ultrafiltration with a 50kDa cutoff membrane (Millipore, Billerica, MA, USA), allowing the separation of BDNF-PAMAM (more than 80kDa) complex and BDNF molecule (28kDa). Protein concentrations were determined using ELISA protein assay.
PEG-ylated BDNF-PAMAM dendrimer nanoparticles:
Poly(ethyleneglycol) with molecular weight 4 kDa, (1546569, Sigma Aldrich) was used for encapsulation of BDNF-PAMAM dendrimer nanoparticles. PEG chains were conjugated to the nanoparticle surfaces via amide bonds formation between PEG amino groups and PAMAM carboxyl groups. An equal volume of before prepared BDNF-PAMAM and PEG (50 mg L-1, pH 7.4, 0.15M PBS) solutions was stirred at room temperature for 1 h. Further, PEG-ylated BDNF-PAMAM solution was ultrafiltered with membrane 10 kDa cutoff (Millipore, Amicon) to remove unconjugated PEG chains.
2.2. Determination of nanoparticle size distribution
For size determination of BDNF, PAMAM 5.5, BDNF-PAMAM and PEG-ylated BDNF-PAMAM, a dynamic light scattering (DLS) was used.
Suspensions of nanoparticles as well as protein diluted with 0.15M PBS buffer to a suitable concentration were measured in 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.
Ruby muscovite mica (Continental Trade, Poland) was used as a substrate for BDNF, PAMAM and BDNF based nanoparticles, adsorption measurements. Fresh, solid pieces of mica were cleaved into thin sheets prior to every experiment.
The AFM (atomic force microscopy) technique was used to obtain information about the size distribution of BDNF, PAMAM dendrimers and nanoparticles. The nanoparticle as well as BDNF protein were left to deposit on mica sheets placed in the diffusion cell over a controlled time, and then substrate was removed, 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, kraj). 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, BDNF-PAMAM and PEG-ylated BDNF-PAMAM surface dimensions were determined using ImageJ software by gathering the number and coordinates of single protein/nanoparticles molecules. Manual counting of protein/nanoparticles molecules was based on comparing the original image and the same picture altered by digital image filters by cutting off the picture background.
2.3. Nanoparticle zeta potential determination
The electrophoretic mobility of BDNF molecules, PAMAM, BDNF-PAMAM as well as PEG-ylated BDNF-PAMAM nanoparticles was measured at pH 7.4 and 0.15M ionic strength with Laser Doppler Velocimetry (LDV) technique with the aid of the abovementioned Malvern device. Electrophoretic mobility was recalculated to zeta potential using Henry equation valid for higher ionic strength where the polarization of the electric double layer is relevant (the double-layer thickness becomes smaller than the protein dimension).
2.4. 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 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.
2.5. 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 6-Hydroxydopamine (0.1µmol/mL) (6OHD, Sigma Aldrich, St.Louis, MO, USA). Then 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 2.1. Nanoparticles Synthesis) and 80µl MEM without FBS. The last step was to measure toxicity using the MTT assay (Abcam, Cambridge, UK), which protocol 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. 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 OD590nm using Varioskan LUX Multimode Microplate Reader (Thermo Fisher, Waltham, MA, USA). Toxicity was calculated from the equation in the manufacturer's protocol.
2.6. BDNF Quantification
- in PBS
The protein release kinetics from PAMAM as well as PEG-ylated PAMAM nanoparticles 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 1 hour. The BDNF molecules released from nanoparticles 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 simultaneously 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 various protein bulk condition (0.002-1 mg L-1). Afterward, the concentration of desorbed BDNF for different time increment (20 min, 2h, 3h, 5h, 8h, 10h, 24h) was quantified by UV-VIS spectroscopy and calculated according to ELISA standard curve.
- in neuroblastoma cell culture
Concentration of releasing BDNF molecules was determined by exposing differentiated human neuroblastoma cells SH-SY5Y to 6-hydroxydopamine (6-OHDA) as well as nanoparticles with different BDNF concentrations for 24h at 37 oC. At the end of the treatments /incubation, the medium was discarded and collected to quantify BDNF concentration using UV-VIS spectroscopy calculated according to ELISA standard curve.
2.6. Internalization of PAMAM-based nanoparticles
In addition, the cellular internalization of nanoparticles with BDNF by SH-5YSY neuroblastoma cells was further investigated by determination of green fluorescence of PAMAM-AF488 conjugates. Differentiated SH-SY5Y cells and previously treated with 6OHDA (protocol described in 2.5. 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 different time lengths (2min, 5min, 10min, 30min, 1h, 4h and 24h) and were subjected to examinations by spectrofluorimetry evaluation using Varioskan LUX Multimode Microplate Reader.
2.7. Immunofluorescent labeling- nanoparticles imaging in vitro
For immunofluorescence analysis SH-SY5Y cells were plated in 4-well chamber slides at density 5x104/well. After the differentiation and 6OHD treatment (protocols described in sections 2.4 and 2.5, respectively) cells were incubated with BDNF-PAMAM-AF488 and BDNF-PAMAM-AF488-PEG nanoparticles (0.1 μg/mL protein loading) for different time lengths (5min, 10min, 30min, 1h and 24h). At designated time points, cells were washed with PBS and fixed with 70% ethanol for 15min. To visualize surface glycoproteins, cells were stained with wheat germ agglutinin conjugated to Texas Red-X (WGA-Texas Red-X, Thermo Fisher, Waltham, MA, USA) in HEPES buffer for 30min. After DAPI counterstain, the slides were mounted and subjected to z-stack analysis using a LSM700 confocal system (Carl Zeiss, Jena, Germany).
2.8. Statistical analysis
All presented data are expressed as means +/- 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. The data are presented as the mean ± standard deviation (SD).