Adsorption/Desorption Transition of Recombinant Human Neurotrophin 4: Physicochemical Characterization

Abstract

Bulk physicochemical properties of neurotrophin 4 (NT-4) in electrolyte solutions and its adsorption/desorption on/from mica surfaces have been studied using dynamic light scattering (DLS), microelectrophoresis, a solution depletion technique (enzyme-linked immunosorbent assay, ELISA), and AFM imaging. Our study presents a determination of the diffusion coefficient, hydrodynamic diameters, electrophoretic mobility, and isoelectric point of the NT-4 under various ionic strength and pH conditions. The size of the NT-4 homodimer for an ionic strength of 0.015 M was substantially independent of pH and equal to 5.1 nm. It has been found that the number of electrokinetic charges per NT-4 molecule was equal to zero for all studied ionic strengths at pH 8.1, which was identified as the isoelectric point (iep). The protein adsorption/desorption on/from mica surfaces was examined as a function of ionic strength and pH. The kinetics of neurotrophin adsorption/desorption were evaluated at pH 3.5, 7.4, and 11 by direct AFM imaging and the ELISA technique. A monotonic increase in the maximum coverage of adsorbed NT-4 molecules with ionic strength (up to 5.5 mg/m) was observed at pH 3.5. These results were interpreted in terms of the theoretical model postulating an irreversible adsorption of the protein governed by the random sequential adsorption (RSA). Our measurements revealed a significant role of ionic strength, pH, and electrolyte composition in the lateral electrostatic interactions among differently charged NT-4 molecules. The transition between adsorption/desorption processes is found for the region of high pH and low surface concentration of adsorbed neurotrophin molecules at constant ionic strength. Additionally, results presented in this work show that the adsorption behavior of neurotrophin molecules may be governed by intrasolvent electrostatic interactions yielding an aggregation process. Understanding polyvalent neurotrophin interactions may have an impact on the reversibility/irreversibility of adsorption, and hence they might be useful for obtaining well-ordered protein layers, targeting the future development of drug delivery systems for treating neurodegenerative diseases.

<p>Electrostatic complex of neurotrophin 4 with dendrimer nanoparticles: controlled release of protein in vitro and in vivo</p>

“…It was determined by DLS that for a PBS concentration of 0.15 M at pH 7.4, diffusion coefficients of NT4 and PAMAM molecules were 10.8×10 −7 ±0.1 cm 2 · s −1 and 6.3×10 −6 ±0.1 cm 2 ·s −1 (at 298 K), respectively, which is in agreement with our previous results 47. Based on the known diffusion coefficient, the hydrodynamic diameters of NT4 and PAMAM were calculated using the Stokes–Einstein relationship:…”

“…Under these conditions, double-layer forces were remarkably weak, and thus interactions between NT4 molecules increased compared to the same electrostatic interaction at lower pH. Because of that, protein aggregation occurred, ie, aggregates consisting of two or three NT4 molecules assembled on the dendrimer surface 47. The large number of NT4 aggregates in the bulk formed due to the decrease in the range of lateral electrostatic interactions (double-layer thickness of 0.79 nm) among adsorbed NT4 molecules at 0.15 M ionic strength.…”

The Role of the Electrokinetic Charge of Neurotrophis-based Nanocarriers: Protein Distribution, Toxicity and Oxidative Stress in in Vitro Setting

“…It has been shown by various theoretical calculations performed using the chemical composition of proteins that the surface charge of the molecule is heterogeneously distributed in the form of positive and negative patches, existing over a wide range of pH. This hypothesis is supported by analogous results reported in the literature on proteins: HSA 43 , IgG 44 , brinogen 45 , NT-4 34 and lysozyme 46 .…”

“…We carried out BDNF adsorption at PAMAM dendrimers in high ionic strength (0.15M) solution, because the reduction of the repulsive interactions between the protein and polymers has been previously reported for different proteins, including polyclonal rabbit immunoglobulin (IgG) 47 , brinogen 45 , albumin 43 and NT-4 34 . By applying this approach, we obtained higher adsorbed amount of BDNF which makes our nanocarriers more biocompatible.…”

“…Concentration of unbounded BDNF protein was higher in the case of BDNF-PAMAM5.5 than BDNF-PAMAM6 nanoparticles, which correlates with the dendrimer charge. As discussed elsewhere 34 , knowing the electrophoretic mobility one can calculate the average number of free charges per protein molecule expressed in coulombs from the Lorenz−Stokes relationship under physiological conditions. The value of the uncompensated charge per BDNF molecule reaches 0.9e (the BDNF molecule acquired a net positive charge which is around three times smaller than for HSA).…”