BDNF-loaded PDADMAC-heparin multilayers: a novel approach for neuroblastoma cell study

Abstract

Biomaterial science has contributed tremendously to developing nanoscale materials for delivering biologically active compounds, enhancing protein stability, and enabling its therapeutic use. This paper presents a process of formation of polyelectrolyte multilayer (PEM) prepared by sequential adsorption of positively charged polydiallyldimethylammonium chloride (PDADMAC) and negatively charged heparin sodium salt (HP), from low polyelectrolyte concentration, on a solid substrate. PEM was further applied as a platform for the adsorption of a brain-derived growth factor (BDNF), which is a protein capable of regulating neuronal cell development. The multilayers containing BDNF were thoroughly characterized by electrokinetic (streaming potential measurements, SPM) and optical (optical waveguide lightmode spectroscopy, OWLS) techniques. It was found that BDNF was significantly adsorbed onto polyelectrolyte multilayers terminated by HP under physiological conditions. We further explore the effect of established PEMs in vitro on the neuroblastoma SH-SY5Y cell line. An enzyme-linked immunosorbent assay (ELISA) confirmed that BDNF was released from multilayers, and the use of the PEMs intensified its cellular uptake. Compared to the control, PEMs with adsorbed BDNF significantly reduced cell viability and mitochondrial membrane polarization to as low as 72% and 58%, respectively. HPLC analysis showed that both PDADMAC-terminated and HP-terminated multilayers have antioxidative properties as they almost by half decreased lipid peroxidation in SH-SY5Y cells. Finally, enhanced formation of spheroid-like, 3D structures was observed by light microscopy. We offer a well-characterized PEM with antioxidant properties acting as a BDNF carrier, stabilizing BDNF and making it more accessible to cells in an inhomogeneous, dynamic, and transient in vitro environment. Described multilayers can be utilized in future biomedical applications, such as boosting the effect of treatment by selective anticancer as adjuvant therapy, and in biomedical research for future development of more precise neurodegenerative disease models, as they enhance cellular 3D structure formation.

Figure 1

The zeta potential of Si/SiO₂ as the function of the number of adsorbed layers determined for (PDADMAC/HP)₃/PDADMAC supplemented by BDNF (A); The stabilities of (PDADMAC/HP)₃/PDADMAC and BDNF adsorbed on (PDADMAC/HP)₃/PDADMAC (B); The zeta potential of Si/SiO₂ as the function of the number of adsorbed layers determined for the (PDADMAC/HP)₄ supplemented by BDNF (C); The stabilities of: (PDADMAC/HP)₄ and BDNF adsorbed on (PDADMAC/HP)₄ (D). Polyelectrolyte adsorption conditions: 5 mg/L, I = 0.01 M, pH = 5.8, adsorption time 20 min. BDNF was adsorbed from the bulk concentration of 1 mg/L and I = 0.15 M, pH 7.4 for 25 min. The points denote SPM results obtained in the NaCl of pH 5.8, 0.01 M NaCl (polyelectrolytes) and in PBS of pH 7.4, 0.15 M (BDNF). Lines represent the fit of experimental data.

Figure 2

Kinetics of BDNF adsorption on silica expressed as the dependence of the coverage (Γ) on time. Bulk protein concentration was 5 mg/L, pH 5.6, NaCl concentration was 0.15 M, flow rate 2.5 × 10^–3 cm³/s. The arrow shows the beginning of the desorption run.

Figure 3

Kinetics of BDNF adsorption on PEM, determined by OWLS, expressed as the dependence of the coverage (Γ) on the time. Bulk protein concentration was 1 mg/L, pH 5.6, NaCl concentration: 0.15 M, and flow rate: 2.5 × 10^–3 cm³/s. Part (A) BDNF adsorption on (PDADMAC/HP)₃/PDADMAC, part (B) BDNF adsorption on (PDADMAC/HP)₄.

Figure 4

Effect of BDNF, PDADMAC/HP, and PDADMAC/HP/BDNF multilayers on cell viability. SH-SY5Y cell line was exposed to various concentrations of BDNF (0.1 mg/L or 1 mg/L), (PDADMAC/HP)₃/PDADMAC, (PDADMAC/HP)₃/PDADMAC/BDNF (1 mg/L) (PDADMAC/HP)₃/PDADMAC/BDNF (0.1 mg/L), (PDADMAC/HP)₄, (PDADMAC/HP)₄/BDNF (1 mg/L), (PDADMAC/HP)₄/BDNF (0.1 mg/L) layers for up to 24 h. As a control, cells were not exposed to BDNF and/or multilayers, (A) Column graph showing mean ± SD of cell viability in relation to control in each setting. For a detailed table containing the statistical significance of tested variables, see Supporting Information Table 1. Statistical analysis was determined using Kruskal–Wallis, one-way ANOVA and Two-Way ANOVA: ****p < 0.0001, ***p < 0.001, ** < 0.01, *p < 0.05.

Figure 5

Effect of BDNF, PDADMAC/HP, and PDADMAC/HP/BDNF multilayers on mitochondrial membrane potential. The SH-SY5Y cell line was exposed to various concentrations of BDNF (0.1 mg/L or 1 mg/L), (PDADMAC/HP)₃/PDADMAC, (PDADMAC/HP)₃/PDADMAC/BDNF (1 mg/L), (PDADMAC/HP)₃/PDADMAC/BDNF (0.1 mg/L), (PDADMAC/HP)₄, (PDADMAC/HP)₄/BDNF (1 mg/L), (PDADMAC/HP)₄/BDNF (0.1 mg/L) layers for up to 24 h. As a control, cells were not exposed to BDNF and/or multilayers. JC-1 fluorescence ratio was calculated as the J-aggregates to J-monomers ratio of setting in relation to the J-aggregates to J-monomers ratio of the control. Column graph showing mean in relation to control ± SD of JC-1 fluorescence ratio in relation to control in each setting. For a detailed table containing the statistical significance of tested variables, see Supporting Information Table 2. Statistical analysis was determined using Kruskal–Wallis, one-way ANOVA, and Two-Way ANOVA: ****p < 0.0001, ***p < 0.001, ** < 0.01, *p < 0.05.

Figure 6

Effect of BDNF, PDADMAC/HP, and PDADMAC/HP/BDNF multilayers on BDNF release to medium. SH-SY5Y cell line was exposed to various concentrations of BDNF (0.1 mg/L or 1 mg/L), (PDADMAC/HP)₃/PDADMAC, PDADMAC/HP)₃/PDADMAC/BDNF (1 mg/L), PDADMAC/HP)₃/PDADMAC/BDNF (0.1 mg/L), (PDADMAC/HP)₄, (PDADMAC/HP)₄/BDNF (1 mg/L), (PDADMAC/HP)₄/BDNF (0.1 mg/L) layers with or without adhered SH-SY5Y cells for up to 24 h. (A) Column graph showing mean ± SD of BDNF concentration (pg/mL) in each setting. For a detailed table containing the statistical significance of tested variables, see Supporting Information Table 3. Data is presented as the mean ± SD (n = 3). Statistical analysis was determined using Kruskal–Wallis, one-way ANOVA, and Two-Way ANOVA: ****p < 0.0001, ***p < 0.001, ** < 0.01, *p < 0.05.

Figure 7

The effect of PDADMAC/HP and PDADMAC/HP/BDNF multilayers on BDNF uptake to SH-SY5Y cells. Previously, the SH-SY5Y cell line was exposed to various concentrations of BDNF (0.1 mg/L or 1 mg/L) and the (PDADMAC/HP)₃/PDADMAC or the (PDADMAC/HP)₄ multilayer for 24 h. As a control, cells were not exposed to BDNF and/or multilayers. After incubation, cell lysate was collected, and BDNF concentration was measured in 1 μg total cell lysate with the ELISA test. (A) Column graph showing mean ± SD of BDNF concentration (pg/mL) in 1 µg of cell lysate in each setting (n = 3). For a detailed table containing the statistical significance of tested variables, see Supporting Information Table 4. Statistical analysis was determined using Kruskal–Wallis, one-way ANOVA, and Two-Way ANOVA: ****p < 0.0001, ***p < 0.001, ** < 0.01, *p < 0.05.)

Figure 8

Assessment of MDA concentration in cell supernatants obtained after adsorption on BDNF, PDADMAC/HP, and PDADMAC/HP/BDNF. The SH-SY5Y cell line was exposed to various concentrations of BDNF (0.1 mg/L or 1 mg/L), (PDADMAC/HP)₃/PDADMAC, (PDADMAC/HP)₃/PDADMAC/BDNF (1 mg/L), (PDADMAC/HP)₃/PDADMAC/BDNF(0.1 mg/L), (PDADMAC/HP)₄, (PDADMAC/HP)₄/BDNF (1 mg/L), (PDADMAC/HP)₄/BDNF (0.1 mg/L) layers with adhered SH-SY5Y cells, for up to 24 h. MDA concentration was normalized to the total protein concentration of individual supernatant samples. Column graph showing mean ± SD of MDA concentration (nmol/mg protein) in each setting (n = 12). For a detailed table containing the statistical, significance of tested variables, see Supporting Information Table 5. Statistical analysis was determined using Kruskal–Wallis, one-way ANOVA, and Two-Way ANOVA: ***p < 0.001, ** < 0.01, *p < 0.05.

Figure 9

The effect of PDADMAC/HP and PDADMAC/HP/BDNF multilayers on SH-SY5Y cells’ morphology. As previously, SH-SY5Y cell line was exposed to various concentrations of BDNF (0.1 mg/L or 1 mg/L) and (PDADMAC/HP)₃/PDADMAC, (PDADMAC/HP)₃/PDADMAC/BDNF (1 mg/L), (PDADMAC/HP)₃/PDADMAC/BDNF (0.1 mg/L), (PDADMAC/HP)₄, (PDADMAC/HP)₄/BDNF (1 mg/L), (PDADMAC/HP)₄/BDNF (0.1 mg/L) layers and incubated for 8 days. As a control, cells were not exposed to BDNF and/or multilayers. After incubation, pictures of the cells were taken using a Progres Gryphax BETRIA camera (Jenoptic) from under the Leica DMIL LED microscope at 10×/0.25 PH1 and 20×/0.35 PH1(Leica) magnification.