All materials were analytical grade reagents and were used without further purification. Poly (diallyldimethylammonium chloride) (PDADMAC) of a molar mass of 101 kg mol− 1 (number averaged) and 160 kg mol− 1 (weight averaged) and heparin sodium salt (HP) for physicochemical analysis, applied as polycation and polyanion, were purchased from PSS Polymer Standards Service GmbH (Germany) and Merck (Germany), respectively.
Carrier-free recombinant human BDNF (248-BDB-250/CF), referred in text to as BDNF, was purchased from R&D Systems (Canada). BDNF was dissolved in a PBS buffer and stored no longer than 48 hours at the temperature 4˚C.
Sodium chloride (NaCl) was supplied by Avantor Performance Materials Poland S.A. (formerly POCH S.A., Gliwice, Poland), and PBS fluid (balanced salt solution for laboratory use) was purchased from Biomed Lublin (Poland).
PDADMAC and HP powders were dissolved in NaCl of the ionic strength (I) of 0.01 M and pH 5.8 in order to obtain the polyelectrolyte solutions of a constant bulk concentration of 5 mg L− 1 before each adsorption experiment. The polyelectrolyte solutions were filtered before using by disposable syringe filter of pore size 0.20 µm (Equimed, Poland).
Streaming potential measurements (SPM)
The zeta potentials of the successive polyelectrolyte layers, the BDNF layer and the layer stabilities against time were determined by SPM. In the experiments, a homemade streaming potential (SP) cell was applied, described in detail in Refs. 30,31. The SP cell consisted of two polished Teflon blocks having two inlet and outlet compartments. Two silicon-covered silica (Si/SiO2) plates were placed on the blocks separated by a Teflon gasket serving as a spacer. The silicon wafers (plates) were the commercial product (Siegert Wafer GmbH, Germany) and were used as a substrate for polyelectrolyte multilayer formation in streaming potential measurements (SPM).
The silicon plates were cleaned by immersing them for 30 minutes in piranha solution, which is a mixture (1:1 ratio) of 95% sulfuric acid and 30% hydrogen peroxide. After the cleaning, the wafers were thoroughly washed with deionized water and immersed in the 80 ºC water for 30 minutes. The wafers prepared in this way were stored in ultrapure water for no longer than 48 hours.
The parallel plate channel was formed by clamping together the blocks with two Si/SiO2 plates and the spacer, using a press under constant torque conditions. The whole system was placed inside the earthen Faraday cage to avoid any disturbances stemming from external electric fields. The streaming potential ΔEs, occurring when a pure electrolyte flows through the SP cell under-regulated and constant hydrostatic pressure difference ΔP, was measured using the two Ag/AgCl electrodes. Applying the high resistance electrometer (Keithley 6512) allowed for performing the potential measurements at practically zero current conditions.
A series of streaming potential measurements were done for four various pressures forcing the flow through the cell. It allowed obtaining the slope of the dependence ΔEs on ΔP, necessary for determining the zeta potentials of the subsequent layers using the Smoluchowski Eq. 32.
The experimental procedure of the zeta potential evaluation consisted of measuring the zeta potential of Si/SiO2, forming the desired number of polyelectrolyte layers (seven and eight layers with PDADMAC and HP on top, respectively) on Si/SiO2 and adsorption of BDNF either on 7th (PDADMAC-terminated) or 8th (HP-terminated) films. After the adsorption of each layer, the dependence ΔEs on ΔP was determined, and the zeta potential was evaluated using the Smoluchowski formula.
The desorption of the polyelectrolyte chains and BDNF molecules from the multilayers was also studied. After completing the desired number of layers, terminated either by PDADMAC, HP or BDNF, the zeta potential of the multilayer was assessed at specified time intervals (usually every 30 minutes).
The initial bulk concentration of the polyelectrolytes (PDADMAC and HP) was 5 mg L− 1, whereas BDNF was adsorbed from the solution of 0.1 and 1 mg L− 1, respectively. pH and ionic strength of the polyelectrolyte solutions were defined and equal to 5.8 and 0.01 M NaCl, respectively. BDNF was adsorbed from PBS fluid of the ionic strength of 0.15 M and pH 7.4. The adsorption process occurred inside the SP cell for a fixed period (20 and 25 minutes for polyelectrolytes and BDNF, respectively) under flow-controlled transport conditions (flow velocity, Vflow, was 0.02 ml s− 1 for the polyelectrolytes and 0.01 ml s− 1 for BDNF). The adsorbed polyelectrolyte films were rinsed by pure PBS fluid before BDNF adsorption.
Optical waveguide lightmode spectroscopy (OWLS)
The OWLS sensors (MicroVacuum Ltd., Hungary) made of a glass support (refractive index nS = 1.52578) coated with 170 nm Si0.78Ti0.22O2 (refractive index nF =1.8) and an additional layer (10 nm) of pure SiO2 were applied in the optical waveguide lightmode spectroscopy (OWLS).
The OWLS sensor chips were thoroughly cleaned prior to each experiment. The sensors were sonicated in Hellmanex solution (3%) in an ultrasound bath for 15 minutes. Next, they were rinsed, and sonicated several times in Milli-Q water. After rinsing, sensors were dried with nitrogen flow and placed in a UV cleaner for 15 minutes. Then they were rinsed again with Mili-Q water and dried with a gentle stream of nitrogen. The sensors were immediately used after cleaning.
The surface mass densities of adsorbed polyelectrolytes and BDNF were determined in real-time measurements using OWLS apparatus. OWLS detects refractive index changes 100–200 nm above the sensor’s surface, giving quantitative information on near-surface kinetic processes.
A standard procedure in situ OWLS experiment started with the flow of pure electrolyte solution in order to condition the surface and to achieve a stable baseline (Δm < 15 ng cm− 2 per 1 hour). As silica covered sensor is negatively charged 33, the first polyelectrolyte layer, was formed by supplying a solution of positively charged PDADMAC solution over the sensor surface, which resulted in a signal shift. Upon adsorption onto the sensor’s surface, the effective refractive indexes shift to higher values, allowing for in situ monitoring of the kinetics of adsorption processes. After a rinsing step with electrolyte solution, which was applied to remove the loosely bound molecules from the surface, the measuring cell was filled with negatively charged HP solution, and the kinetics of HP adsorption were monitored. The experiment ended with a rinsing phase. After achieving a stable final signal the whole procedure was repeated 3–4 times in order to obtain the desired number of layers (6–8).
Cell culture
SH-SY5Y neuroblastoma cell line (human, ECACC; Sigma Aldrich, St. Louis, MO, USA) was grown using a 1:1 mixture of Ham's F-12 Nutrient Mixture (Thermo Fisher, Waltham, MA, USA) and Minimum Essential Medium (MEM) (Sigma Aldrich, St. Louis, MO, USA), supplemented with streptomycin (100 U/mL), penicillin (100 µg/mL), L-glutamine (2 mM), and 10% heat-inactivated fetal bovine serum (FBS) (all reagents are Gibco, Thermo Fisher Scientific, Waltham, MA, USA) referred to as “proliferation medium” in text. Reduced serum (1% FBS) differentiation medium was used in the experiments in order to slow down the proliferation and allow for eventual cell differentiation. Cells were kept at 37°C in a saturated humidity atmosphere containing 5% CO2. Every 2–3 days proliferation medium was changed, and the cells were passaged after they had achieved 80% confluence.
Preparation of PEMs for in vitro studies
PEMs, consisting of positively charged PDADMAC and negatively charged HP layers, were used as carriers of BDNF molecules. Before adsorption, polyelectrolyte solutions (1000 mg L− 1, 0.01 M NaCl, pH 5.8) were diluted to 5 mg L− 1 using 0.01 M NaCl, pH 5.8. A 96-well plate (Thermo Fisher Scientific, Waltham, MA, USA) was coated with either 7 or 8 layers of polyelectrolytes using the LbL method. The adsorption time of both PDADMAC and HP was 20 minutes. After each adsorption step, the wells were rinsed three times by 0.01 M NaCl, pH 5.8. This process was repeated until the desired number of layers was obtained. Finally, the wells were washed three times with PBS fluid (I = 0.15 M, pH 7.40).
BDNF adsorption on PEM
Stock solutions of 1 mg L− 1 and 0.1 mg L− 1 BDNF bulk concentrations in PBS were prepared directly before adsorption on PEM. BDNF was adsorbed at room temperature for 15 minutes. The BDNF excess that did not adsorb on PEMs was collected for determination of residual BDNF concentration using DuoSet ELISA test.
DuoSet ELISA test
All the reagents were purchased from R&D Systems (Canada). Protein release profile from PEMs in bulk/in vitro, residual BDNF concentration, and cellular uptake were determined utilizing ELISA DuoSet assay (DY248) according to the manufacturer’s instructions. Briefly, polystyrene plates (DY990) were covered by ELISA coating buffer (DY006) containing diluted capture antibody (part of the DuoSet kit). After overnight incubation, the plate was washed three times with a washing buffer (WA126). Then, wells were blocked with Reagent Diluent (DY995) for 1 hour, and samples, and standards were added. The next steps involved the addition of detection antibody diluted in Reagent Diluent (part of the DuoSet kit), Streptavidin-HRP (DY998), and Substrate (DY999), respectively, with washing steps in between and incubation times as suggested in the datasheet. All the incubations were at room temperature. Finally, Stop Solution (DY994) was added, and the absorbance was immediately read at λ450 and λ540 (as a wavelength correction) with Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). SkanIt™ Software for Microplate Readers ver. 5.0 was used for the analysis of the data.
Cell viability
SH-SY5Y cells were seeded at a density of 3 × 104 cells per well in 96-well plate in the following experimental variants: no layers no BDNF (as a control), BDNF (0.1mg L− 1 or 1 mg L− 1), 7th (PDADMAC-terminated), 7th (PDADMAC/BDNF (1mg L− 1) - terminated), 7th (PDADMAC/BDNF (0.1mg L− 1) - terminated), 8th (HP - terminated), 8th (HP / BDNF (1 mg L− 1) - terminated), 8th (HP/BDNF (0.1 mg L− 1) - terminated) layers. While adhering, cells were incubated for 2 hours in a reduced serum differentiation medium. After additional 24 hours, the viability of neuroblastoma was assessed using the Alamar Blue® reagent assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instruction. Alamar Blue® is a resazurin-based assay, and its essence is the ability of a healthy cell to convert resazurin into fluorescent resorufin. The brighter the fluorescence, the greater the viability of the cell population. The fluorescence was read using Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). SkanIt™ Software for Microplate Readers ver. 5.0 was used for the analysis of the data.
BDNF bulk and in vitro concentration
Supernatants (medium with 1% FBS serum) from SH-SY5Y cells incubated with PEMs were collected after 24 hours. Every time point was obtained from at least six replicates for each experimental variant. The BDNF concentration was measured with Duoset ELISA test.
Detection of mitochondrial membrane potential
JC1 is a marker of mitochondrial wellbeing. In a healthy cell with high mitochondrial membrane potential, JC-1 forms complexes emitting red fluorescence. In an apoptotic or unhealthy cell with low mitochondrial membrane potential, the dye remains in its monomeric form emitting green fluorescence. SH-SY5Y cells were seeded in a 96-well black plate with clear bottom, coated with multilayers (as described in ‘Preparation of PEMs for in vitro studies’) and BDNF protein in either of the two concentrations (1 mg L− 1 or 0.1 mg L− 1, respectively). Cells were incubated for 24 hours. Subsequently, according to the manufacturer's instructions, cells were stained with JC-1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) for 30 minutes at 37°C in a CO2 incubator. Fluorescence was measured at Ex535nm/Em595nm and Ex485nm/Em535nm using Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).
Detection of lipid peroxidation
Lipid peroxidation is mainly caused by free radicals’ attack on specific compounds such as polyunsaturated fatty acids (oxidative stress). The malondialdehyde (MDA) concentration in cell cultures can determine the intensity of this process. After adhesion to the plate, SH-SY5Y cells (3 × 104 cells/well) were incubated on multilayers (described in “Preparation of PEMs for in vitro studies”) for 24 hours. Experimental details were indicated in previous sections. SH-SY5Y treated with 1000 µM H2O2 were included as an oxidative stress control. After incubation, the cell supernatant was collected from each well. MDA concentration in cell supernatant was determined using reverse-phase, high-performance liquid chromatography (HPLC)-spectrophotometric method (Agilent 1260 Infinity II, Agilent Technologies, Waldbronn, Germany)34, using BDS Hypersil C18 column. 10 mM TEP (1,1,3,3-tetraethoxypropane) was prepared freshly and diluted in water to 0.05, 0.075, 0.1, 0.15, and 0.2 µM TEP as the working calibrants. 100 µl of the supernatant or standard was treated with 200 µl of 5% TCA (trichloroacetic acid) and 10 µl of 0.4% BHT (butylated hydroxytoluene) in absolute ethanol for deproteinization and antioxidant effect. The samples were vortexed, 100 µl of 0.6% TBA(2-thiobarbituric acid) was added and then reacted for 45 minutes at 90°C to form an adduct MDA-(TBA)2, which can be detected at 532 nm. After the final centrifuge at 8 000 × g (10 minutes, 4°C) supernatants were collected and adjusted to pH 7.0 with 1 M NaOH. The elution buffer was determined to be 50 mM KH2PO4 (pH 7.0 fixed with KOH) and CH3OH (70:30, v/v). The sample run was 6.5 minutes, with a flow rate of 0.5 ml minutes− 1, an injection volume of 20 µl, and visible excitation and emission at 528 and 553 nm. The analysis of MDA release by the human neuroblastoma cells SH-SY5Y was performed by integrating the retention times and the peak areas compared with known concentrations of MDA calculated as mM MDA equivalent from the TEP standard calibration (1:1 conversion under acidic conditions).
Protein lysate isolation
SH-SY5Y cells were seeded as previously described in the upper sections. After 24 hours of incubation cells were washed with 1 × PBS (P4417, Sigma Aldrich, St. Louis, Missouri, United States), trypsinized (#25200056, Thermo Fisher Scientfic, Waltham, MA, USA) for 5 minutes at 37°C, followed by centrifugation at 1000 × g for 5 minutes. Pelleted cells were resuspended in the cell lysing mixture of 1 × RIPA Lysis and Extraction Buffer, 1 × Halt™ Protease Inhibitor Cocktail and 1 × Halt™ Phosphatase Inhibitor Cocktail (Cat. No. 89901, 87786 and 78420 respectively, Thermo Fisher Scientfic, Waltham, MA, USA). After 20 minutes. of incubation at 4°C, the remains of the cells were centrifuged at 15 000 × g in 4°C for 15 minutes. Lysates were immediately transferred to -80°C for future analysis.
BDNF uptake in SH-SY5Y
Total protein concentration of the collected cell lysates (n = 6) was quantified with BCA protein assay kit for low concentrations (ab207002, Abcam, Cambridge, United Kingdom), according to manufacturer’s protocol. Briefly, whole cell lysates with unknown protein concentration, and reference protein standard (bovine serum albumin, concentration range 0.5–40 µg/mL) were incubated with green colored BCA Working Solution for two hours at 37°C. After that the total protein concentration was determined by measuring the color change of the sample solution from green to purple which is proportional to the protein concentration. Absorbance was measured against λ562 using Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). SkanIt™ Software for Microplate Readers ver. 5.0 was used for the analysis of the data.
BDNF concentration in 1 µg of the whole cell lysate was subsequently determined using DuoSet ELISA test.
The analysis of cell morphology
Cells were seeded as described in previous sections. After 24 hours, the medium was changed. The cells were further cultured until 7 days after seeding in a complete cell culture medium. The medium was refreshed every 2–3 days. After 8 days, pictures were taken with Progres Gryphax BETRIA camera (Jenoptic) from under the Leica DMIL LED microscope at 10x/0.25 PH1 and 20x/0.35 PH1(Leica) magnification.
Statistical analysis
All of the data are expressed as means ± standard deviations (SD) from a minimum of three separate studies. The Kruskal-Wallis test and one-way ANOVA followed by Dunnett’s multiple comparisons test, performed using GraphPad Prism (version 9.1.1 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com) were used to do statistical analysis between each study group. For analysis comparing experimental groups, a two-way ANOVA was utilized. Statistics were judged significant at *p < 0.05, **p < 0.01, ***p < 0.001.