Desorption Transition of Recombinant Human

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Adsorption / desorption transition of Recombinant Human Neurotrophin 4 – physicochemical characterization. Maria D#bkowska, Malgorzata Iwona Adamczak, Jakub Barbasz, Micha# Cie#la, and Bogus#aw Machali#ski Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00909 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

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Adsorption / Desorption transition of Recombinant Human Neurotrophin 4 – Physicochemical Characterization.

Maria Dąbkowska1, Małgorzata Adamczak2, Jakub Barbasz3, Michał Cieśla4, Bogusław Machaliński5, *.

1Department

of Medical Chemistry Pomeranian Medical University, Rybacka 1, 70-204 Szczecin, Poland [email protected] 2Department

of Pharmacy, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway

4M.

Smoluchowski Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland

5Department

of General Pathology Pomeranian Medical University, Rybacka 1, 70-204 Szczecin, Poland [email protected]

*Corresponding authors,


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Abstract

Bulk physicochemical properties of neurotrophin 4 (NT-4) in electrolyte solutions and its adsorption/desorption on/from mica surface have been studied using dynamic light scattering (DLS), microelectrophoresis, 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 NT-4 in various ionic strength and pH conditions. The size of NT-4 homodimer for the ionic strength 0.15M was substantially independent of pH and equal to 5.1 nm. It has been found that the number of electrokinetic charges per NT-4 molecules 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 surface was examined as a function of ionic strength and pH. The kinetics of neurotrophin adsorption/desorption was evaluated at pH 3.5, 7.4 and 11 by direct AFM imaging and ELISA technique. A monotonic increase in the maximum coverage of adsorbed NT-4 molecule with ionic strength (up to 5.5 mg/m2) was observed at pH 3.5. These results were interpreted in terms of 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 to 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 intra-solvent electrostatic interactions yielding aggregation process. Understanding polyvalent neurotrophins interactions may have impact on reversibility/irreversibility of adsorption, hence they might be useful to obtain well-ordered protein layer, targeting at future development of drug delivery systems for treating neurodegenerative diseases.

Keywords: AFM, neurotrophin 4 (NT-4) adsorption on mica, monolayers of NT-4 on mica, iep of neurotrophin 4, desorption of neurotrophins from mica, zeta potential of NT-4


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1. Introduction Neurotrophins-4s (NT-4s) are structurally and functionally related proteins belonging to a cystine knot growth factor family, generally known overall as the neurotrophins. Neurotrophins play an important role in the development and maintenance of the vertebrate nervous system via promoting survival, migration, proliferation, differentiation and death of neurons. These small peptides expressed throughout the brain2, although the synthesized, secreted concentration of NT-4 and their half-live in the body are limited1,2. Neurotrophins-4s are not available for all neurons; therefore, their delivery from cells to tissues results in the formation of concentration gradients3. Decrease in neurotrophins concentration has been associated with the pathology of several neurodegenerative diseases their physiological symptoms4. Accordingly, neuroprotection by improving administration of exogenous NT-4 has been considered to be a potential novel treatment for neurodegenerative diseases5. Our previous studies suggested that NT-4s promote survival and neuroprotection of retinal neurons during in the course of acute chemical damage to the murine retina.6 Although NT-4s may seem promising as potential therapeutic agents, we have not yet understood adsorption of these protein molecules at solid surfaces. Understanding of this process is the most relevant step in the preparation of wellordered protein layer deposited on biomaterials. The majority of information on the crystal structure of intact human NT-4 comes from electron microscopy studies of its fragments by atomic resolution. The interpretation of electron density map in combination with molecular dynamic simulations allowed to establish that the active beta subunits of NT-4 are cationic and exist exclusively as a small homodimer consisting of two protomers7,8. The tertiary structure of NT-4 protomer resembles nonglobular β-sheet. Upon dimerization the amount of non-polar surface area in NT-4 that is inaccessible for solvent rises to values, which are more typical for globular proteins8. NT-4 molecules exist as non-covalent homodimers, stabilized in solvent mainly by hydrogen bonds and van der Waals interactions. The molar mass of NT-4 calculated from amino acid composition is equal to 14 kDa. Contrary to other neurotrophins, NT-4 is expressed pervasively throughout the central nervous system, and the observed concentration of NT-4 in serum samples may reach 19+/-10 pg/ml9-12.


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In spite of its prospective interaction with engineered nanomaterials and tremendous significance for recovering nervous system, NT-4 adsorption on various surfaces was not thoroughly studied in terms of physicochemical aspect. Numerous studies have focused on the structure of the molecule in crystal state13, 14, but the physicochemical parameters characterizing neurotrophins in the bulk15 or adsorbed state have not been systematically investigated. Owing to its significance, protein adsorption was extensively studied both theoretically16-18 and experimentally using AFM19, reflectometry20, QCM21 therefore in presented work attention is paid to the electrokinetic measurements of homodimer protein molecules and possibility using these data to interpret their adsorption on flat surface. This combination studies both kinetics and equilibrium issues of neurotrophin homodimers adsorption, gaining an insight into transition between irreversibility and reversibility of protein adsorption process. Additionally, using combination of experimental results and RSA model allowed us to reveal quite unexpectedly in many of the above citated works relevant information regarding presence of oligomers in usual neurotrophin sample. In view of this deficit, the aim of this work is to assess the kinetics of NT-4 adsorption/desorption on/from mica surface, representing a model of hydrophilic substrate of well-defined and controlled surface properties22. The determination of adsorption and desorption kinetics of NT-4 as a function of ionic strength and pH provides an insight into binding strength of protein and the density of their monolayers. In this work the AFM and ELISA techniques have been used to point out the nature of transition between irreversibility and reversibility of NT-4 adsorption on the mica surface. The use of mica also allows one to precisely determine the coverage of NT4 via AFM imaging of single protein molecules. Another goal of this work was to determine the diffusion coefficient (hydrodynamic radius), electrophoretic mobility, isoelectric point (iep), uncompensated charge and stability in electrolyte solutions under various conditions of ionic strengths and pH. These parameters are of vital importance for predicting monolayer density of NT-4 at various surfaces. It has been found that physicochemical properties of NT-4 have tremendous influence on neurotrophins interactions with model surfaces and its in vitro stability, since they may yield to the aggregation tendency and limited concentration of existing available free molecules in the bulk.


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These systematic studies performed for well-defined systems and using mostly direct, in situ methods are particularly needed to understand NT-4 adsorption mechanisms.

2. Materials and Methods In our studies, recombinant human neurotrophin 4 (NT-4) was supplied as an aqueous stock suspension with the concentration of 1000 µg/ml (purchased from Abcam), as well as a lyophilized form from R&D Systems. These stock solutions of NT-4 were diluted to the desired bulk concentration (usually 1-500 mg/L) prior to each experiment. The protein suspensions were additionally subjected to ultrafiltration (centrifree ultrafiltration device, Merck Millipore) in order to remove aggregates and provide constant, free form protein molecules concentration in the solvent. The bulk concentration of NT-4 after purification was determined by commercially available immunoassay method (ELISA kit for NT4, Abcam). Ruby muscovite mica supplied from Continental Trade was used as a substrate for NT-4 adsorption. The solid pieces of mica were freshly cleaved to thin sheets prior to every experiment. Water was purified using a Millipore Elix 5 apparatus. Chemical reagents (sodium chloride, hydrochloric acid, PBS buffer) were commercial products of Sigma-Aldrich, and used without further purification. The experimental temperature was kept constant at 293 K. The diffusion coefficient of NT-4 was determined by dynamic light scattering (DLS), using the Zetasizer Nano ZS Malvern instrument at protein concentration varying between 200 and 500 µg/ml as described previously23-25. The microelectrophoretic mobility of protein solutions was measured using the Laser Doppler Velocimetry (LDV) technique with the aid of the abovementioned Malvern device. Furthermore, the isoelectric point of NT-4 was determined by isoelectric focusing (IEF) in a homogenous polyacrylamide gel containing carrier Bio-Lythe ampholytes (pH range 3-9), and detected by a silver staining kit (BioRad). The deposition of NT-4 molecules on mica sheets was carried out in the diffusion cell containing protein solution in various concentrations, over controlled time. After incubation the mica samples were rinsed with ultrapure water for 1min to remove any nonadsorbed molecules in the fluid film and air-dried before the analyses. Thereafter,


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surface concentration of NT-4 in monolayer adsorbed on mica was determined by AFM imaging in air using the NT-MDT Solver BIO device with the SMENA SFC050L scanning head and independently by NanoWizard® AFM (JPK Instruments AG, Berlin, Germany) for coverages below 10%, accounting for 1mg/m2 of NT-4. We were not able to precisely count the coverage of NT-4 adsorbed on mica surface over 10% (equals 5000 molecules/µm2 of NT-4) using AFM technique hence we used solution depletion technique (ELISA) to study adsorption kinetics for coverage higher than 1.2 mg/m2. We measured protein concentration change in the bulk solution before and after adsorption by ELISA method and calculated per total area available for protein adsorption Γ = ∆cp V/Stot where V is the total volume of protein solution, Stot is the total area available for adsorption26-28. The adsorption of NT-4 molecules on mica sheets was conducted in two parallel ELISA experiments for initial coverage range from 10%, and AFM method for lower coverage. It was followed by desorption of NT-4 molecules carried out by immersing mica sheets covered with adsorbed proteins in pure electrolytes in identical increments of time. Afterwards, the concentration of the desorbed protein solution was measured in the bulk by ELISA, while mica sheets were studied in terms of NT-4 surface concentration (number of protein molecules adsorbed per unit area) by AFM. All AFM measurements were performed in semicontact mode by using high resolution silicon probes (NT-MDT ETALON probes, HA NC series, polysilicon cantilevers with resonance frequencies 240 kHz +/- 10% or 140 kHz +/- 10%, force constants 9.5 N/m +/- 20% or 4.4 N/m +/- 20%). We have captured all images in randomly chosen areas. The images of adsorbed NT-4 molecules were recorded within the scan area of 0.5 µm x 0.5 µm as well 1 µm x 1 µm. Usually 1000 NT-4 molecules were counted over a few randomly chosen areas over the mica sheet with relative precision below 6%. We have determined the protein surface concentration using softwares the Loco’s Shire program v.2.8.24.0 as well ImageJ. To establish a sufficient accuracy in the particle positioning we decided to determine the number and coordinates protein molecule manually. Counting and distribution of protein molecules were based on making comparisons between original image and exactly the same picture altering by digital


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image filters by cutting off the picture background. In this way we verified correctness of analysis of collected particle by using abovementioned softwares. The AFM measurements were performed under air conditions, therefore considerable attention was drawn to elaborate an appropriate experimental procedure of drying the neurotrophins adsorbed on mica. The water film can be evaporated from such hydrophilic monolayer under controlled humidity and temperature in a continuous way without forming drops. The minimized the action of meniscus forces and probable deformation of monolayers.

3. Results and Discussion

3.1. NT-4 bulk characteristics

Extensive bulk characteristics of physicochemical properties of NT-4 homodimer for various pH from 3 to 10 and ionic strengths from 0.15 to 10-3 M were performed in this study. Interactions of proteins in aqueous environment are crucial to understanding of their structure, function and assessing the reversibility of adsorption process. The diffusion coefficient of proteins in solutions is indeed strongly related to their size and/or shapes, as well as dynamics of its molecules in solutions, which determines their transport kinetics to interface and conformational alterations. Therefore, determination of these factors can be effectively used for efficient drug design. The diffusion coefficients of NT-4 homodimers for pH = 7.4 and pH = 3.5, the ionic strength of 0.15 M were determined by DLS, at the temperature T = 293 K. The diffusion coefficient of NT-4 homodimer was initially investigated in the bulk concentration. Measured values of the diffusion coefficient were nearly independent of its bulk concentration and approaching value of 10.8 x10-11 m2 s-1 for ionic strength 0.15M and pH 3.5, as well as pH 7.4. Figure 1 presents the time dependence of the diffusion coefficients of NT-4 suspensions for ionic strength 0,15M (pH 3.5 and 7.4).


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14 12 10 8 6 4 2 0 0

200

400

600

800

t [h]

Fig.1. Stability of NT-4 as the dependence of the diffusion coefficient D on the time. The points denote experimental values determined by DLS for 200 µg/ml of NT-4, 0.15M NaCl: 1 – () pH 7.4, 2 – ( ) pH 3.5. The solid line depicts the linear fit of experimental points, that is, D = 10.8 x 10-7 cm2/s. The error bars are calculated as standard deviations of six various measurements. As can be seen, the diffusion coefficients were practically independent from storage time and approached the value of 10.8 x 10-7 cm2/s, which suggested no protein aggregation or specific interaction in protein solution for over a month. It is interesting that almost similar value of 12 x 10-7 cm2 /s was reported by Stroh et al.3 for another neurotrophin, BDNF homodimer (Molar Mass 27 kDa) at pH 7.4. Knowing the diffusion coefficient, the hydrodynamic radius of the protein dH was calculated using Stokes-Einstein relationship: dH =

kT 3πη D

(1)

where dH is the hydrodynamic diameter, k is a Boltzmann constant, T is an absolute temperature, η is the dynamic viscosity of water and D is the diffusion coefficient of NT4. Using the diffusion coefficients obtained for various pH one can calculate from Eq. (1) the hydrodynamic diameters of NT-4 (200 μg/ml, I = 0.15 M). The calculated values are presented in Figure 2.


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dH [nm] 25

20

15

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5

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4

6

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10

12

pH

Fig.2. Hydrodynamic diameter of NT-4 molecule determined by DLS method as a function of pH after ultrafiltration. The points denote experimental results determined experimentally by DLS for 200 µg/ml NT-4, 0.15M NaCl: 1 – () NaCl after ultrafiltration, 2 – ( ) pH 7.4 PBS after ultrafiltration, 3 – ( ) NaCl without ultrafiltration. The lines depict the linear fit of experimental points. The error bars are calculated as standard deviations of four various measurements.

Consistently, the above results indicate that the hydrodynamic diameter of NT-4 homodimer equals almost 5 nm within broad range of pH. Therefore the “hydrodynamic” size of NT-4 can be treated as a good estimate of its geometrical size, given the structural stability of the protein and its compact shape. Moreover, the values obtained at pH 7.4 for NaCl and PBS are very similar, which proved stability of geometrical size of NT-4 for various electrolyte composition. Additionally, DLS study at pH 7.4 suggests that NT-4 homodimer molecules appear in tetramer configuration for samples, which were not subjected to ultrafiltration (Fig.4). These results suggest specific ion and buffer effect on protein-protein interactions what will be discuss later in details. The electrophoretic mobility of NT-4 was studied by microelectrophoretic method. As discussed in ref. 29-31, knowing the electrophoretic mobility, one can calculate the average number of free (electrokinetic) charges Qc per molecule expressed in Coulombs from the Lorenz-Stokes relationship Qc = 3πη d H µe


(2)

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Where dH is expressed in nm, is expressed in µm cm/V s, and, η is the dynamic viscosity of the solvent. Using Qc the average number of elementary charges (e) per NT-4 molecule (Nc) was calculated. It should be mentioned that |e| = 1.602 x 10-19 C. The above equation is most accurate when the double layer thickness (Debye screening  ε kT  length κ −1 = Le =  2  where ε is the permittivity of the medium, k is the Boltzmann  2e I 

constant, I is the ionic strength of the electrolyte solution) is comparable or smaller than the protein characteristic dimension (hydrodynamic diameter). Thereby, using Eq. (2) and the electrophoretic mobility experimental data, for ionic strength of 0.015M, the Nc has ranged from 4.1 e (from Eq.2) to 3.4 e (from Eq.2) for pH from 3.5 to 9.5 respectively. As abovementioned Eq. (2) has some limitations for higher ionic strengths, where double layer thickness becomes less than the protein dimension, thus in order to calculate the electrokinetic charge for ionic strength of 0.15M we applied the following equation27,28 Q c = 2πη d H µ e

1+ κ d H = 2πηd H 1+ κ d H ζ f (κ d H )

(

)

(3)

where f (κ d H ) is Henry’s function, κ-1 is double layer thickness. It should be noted that Eq. (3) is valid for spherical particles and an arbitrary ionic strength. Thereby, using Eq. (3) one can conclude that under physiological condition (pH=7,4 and I=0.15M) the value of uncompensated charge per NT-4 molecule Nc reaches 0.7 e. Ionic strength of 0.15M N c varied between 3.3 e (from Eq.2)/ 26.5 e (from Eq.3) for pH 3.5, and -2.6 e (from Eq.2)/ -27.7 e (from Eq.3) for pH 9.5. The foregoing Nc values are three times smaller than for HSA (determined before using the same method23,32), which indeed enhances the possibility of neurotrophins aggregation for this pH range, in accordance with the above measurements of the hydrodynamic diameter. The experimental data dependence of zeta potential and electrophoretic mobility on pH for various ionic strengths between 0.15M and 10-4M are shown graphically in Figure 3.


40

2.1

20

1.1

0

0.1

-20

-1.1

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-2.1

-40 2

4

6

8

10

pH

Fig.3. Zeta potential/ electrophoretic mobility ( ζ / µe ) of NT-4 as a function of pH at T = 293K. The points denote experimental values determined by DLS: 1 – ( ) 0.15M NaCl, 2 – () 10-2M NaCl, 3 – ( ) 10-3M NaCl. The lines depict the non-linear fit of experimental points. The error bars are calculated as standard deviations of 8 various measurements. For surface potential 30mV the polarization of the double layer is irrelevant. Hence, we used the Henry equation to calculate zeta potential as described elsewhere31,34 . As can be seen, zeta potential of neurotrophin 4 has varied between 36 mV at pH 3.5 and -28 mV at pH 9.5 for 10-3 M NaCl concentration. The electrophoretic mobility of NT-4 for pH 3.5 obtained a positive value of 1.24 μm cm/s V, which corresponds to the zeta potential of 24 mV (calculated using the Henry relationship)23 for 0.15M NaCl. The zeta potential of NT-4 increased with decreasing ionic strength and reached the value of 36 mV for 10-3 M NaCl, which indicates that NT-4 molecule acquired a net positive charge with changing electrolyte concentration. Moreover, the zeta potential of NT-4 molecules vanish at pH = 8.1 for all the studied ionic strengths (Fig.1), which can be identified as the isoelectric point of the protein (denoted by iep).

3.2. Transition of NT-4 Adsorption/Desorption on solid surface

3.2.1.1. NT-4 Adsorption in NaCl solution


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The AFM measurements allowed us to determine the number of NT-4 aggregates adsorbed under diffusion controlled transport condition on mica surfaces at pH 3.5 and ionic strength 0.15 M NaCl. As can be seen, the average NT-4 molecule occupies equivalent of sphere area with diameter of 5.0 nm. According to the above results, the shape of adsorbed single NT-4 molecules resemble globular protein molecule. The size measured by AFM is in a good agreement with DLS bulk measurements. The aggregates appeared as isolated individuals, which enabled an exact determination of their sizes by cutting down possibility of tip convolution artifacts. The average values of distinctive aggregate diameters were 11.1+/-2 nm and 20.2+/-2 nm, which indicate the oligomers distribution of NT-4 in the bulk (Figure 4). a) 45 40 35 30 25

[%] 20 15 10 5 0 0

5

10

15

d [nm]

b)


20

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Number [%]

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30

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Fig.4. Part (a) Histogram of adsorbed NT-4 aggregates (0.15M NaCl, pH 3.5) indicated by direct AFM enumeration is obtained for low surface protein concentration. 30 randomly chosen various areas were taken into account in calculations. Each micrograph of the NT-4 monolayer at the mica surface has size of 1 µm x 1 µm. The inset shows the NT-4 monolayer (semicontact mode) on mica for surface concentration of protein 1200 µm-2. Part (b) Typical size distribution of NT-4 molecules measured in the bulk by DLS (0.15M NaCl, pH 3.5) without ultrafiltration process. The direct AFM imaging method was also used to quantitatively determine NT-4 adsorption kinetics on mica. Neurotrophins monolayers were generated by diffusioncontrolled transport for low bulk concentration of the protein (1 mg/L) and various ionic strength (0.15M and 0.015M), according to the procedure which was described in Ref. 22,21 These experiments were conducted using ultrafiltration protein suspensions before each experiment. The direct AFM enumeration was validated by the theoretical approach, which is based on calculating the coverage using the random sequential adsorption (RSA) approach36-39, including electrostatic screened Coulomb interactions40 between neurotrophin molecules. RSA model was elaborated to understand protein and particle adsorption22,23. Adsorption kinetics was theoretically modelled under an assumption that diffusion is the only mechanism responsible for bringing NT-4 particles close to a surface. The probability of adsorption near the surface, commonly known as a blocking function or


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an available surface function, depends nonlinearly on the surface coverage. When the first particle is put on a surface it blocks specific fraction of it for the subsequent particles. For example, the area blocked by one disk of a radius r is also a disk, but of a radius 2r, because centers of two, non-overlapping disks cannot be closer than 2r. When there are more particles on a surface their exclusion areas can overlap, thus the blocked area is smaller than the product of particles at the surface and the size of a single particle exclusion area. This causes nonlinearity mentioned before. Additionally, when a surface is almost empty, the adsorption probability is close to 1 and therefore, all particles near the surface can be quickly absorbed, which causes rapid decrease in their concentration. Thus, at the beginning, the adsorption rate is limited by diffusion processes that bring subsequent particles from a bulk close to the surface. On the other hand, for almost saturated packing, the probability of adsorption becomes very low; hence the diffusion has enough time to equalize the concentration of particles near the surface and in a bulk. This shows, that precise knowledge of a blocking function is essential to calculate adsorption rate correctly. In order to estimate a blocking function, the deposition was simulated numerically using the random sequential adsorption algorithm36, where the shape of NT-4 particles was modelled as a sphere. The onedimensional diffusion equations with Robin boundary conditions41 were solved using the standard Cranck-Nicholson algorithm. The details of the numerical procedure were described in Ref. 42 It should be mentioned that the specific interactions among protein molecules were neglected in this model. The exact calculations of adsorption kinetics, maximum coverage and structure of NT-4 monolayer are possible since the bulk concentration and the diffusion coefficient of NT-4 are previously obtained from physicochemical measurements. It enables us to adopt the AFM method to directly quantify the amount of adsorbed NT-4 molecules (expressed by N) on mica if the surface concentration (coverage) of neurotrophins is determined as a function of time. Thus, N is expressed hereafter as the number of molecules per square micrometer. Representative micrograph of NT-4 monolayer obtained for pH 3.5 and 0.15M PBS and bulk protein concentration of 1 µg/ml is shown in the inset of Fig.4. As can be seen, the NT-4 molecules spread over solid surface as isolated entities, which enables enumeration by image analyzing softwares. It should be mentioned that


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determining the surface concentration via direct counting does not require information about protein size and shape, which makes this proceeding reliable. The dependence of a surface concentration N of neurotrophins molecules as a function of the square root on adsorption time for pH 3.5 and two various ionic strengths (0.15M and 0.015M) is presented in Fig.5.

6

N [µm-2]

Γ [mg/m2] 5

20000

4 15000 3 10000 2 5000 1

0

0 0

10

20

30

40

t

1/2

1/2

[s ]

Fig.5. The dependence of the surface concentration of NT-4, N (µm-2)/ Γ coverage (mg/m2) on the square root on adsorption time t1/2 (s1/2). The points denote experimental results obtained by the direct AFM enumeration of adsorbed protein molecule for I=0.15M ( ) and for I=0.015M ( ) solution depletion method ELISA () for I=0.15M, cb=1 µg/ml, pH 3.5. The lines show the exact theoretical results obtained by the numerical solution of the diffusion equation using the random sequential adsorption model for 0.15M (upper line) and 0.015M (bottom line). Knowing dependence of N on adsorption time t one can quantitatively validate the theoretical adsorption model for a broad surface concentration range protein adsorption under diffusion-controlled transport condition, using the following relationship24 1/ 2

D N = 2  π 

t1/ 2 nb

(4)

where nb is the bulk concentration of protein expressed in m-3.


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As previously applied to the interpretation of other proteins and nanoparticles adsorption on mica23,24 theoretical approach based on terms of the random sequential adsorption (RSA) model at this point was employed. In this work, attention is paid to the electrokinetic studies which offer a distinct possibility of direct, in situ determination of electric properties of protein and can be conveniently interpreted by the RSA model adjust to the lateral electrostatic interactions between adsorbed molecules. Accordingly to this approach, the bulk transport of proteins is described by the phenomenological continuity equation. Selecting system of well establish physicochemical properties allowed one to combine the dynamic aspect of the interfacial interactions between neurotrophins and homogenous mica surface. As can be observed the surface concentration rises gradually with t1/2 for adsorption time 25 min (t1/2 = 5 min1/2). Furthermore, the value of maximum coverage increases significantly with ionic strength and time due to the dropping range of lateral electrostatic interactions. The figure 5 demonstrates that the theoretical results derived by numerical solution of the governing mass transfer equation well described the experimental data. Therefore the direct AFM enumeration of adsorbed NT-4 molecules could be reasoned procedure for assessing its surface concentration. The maximum coverage obtained for 0.15 M NaCl was equal to 23800 molecules per μm2 and decreased until 1820 molecule per μm2 for 0.015M NaCl. However, it should be taken into account that AFM image resolution for densely packed neurotrophin monolayer adsorbed on mica is particularly restricted for coverage obtained 4000 µm-2 and below that value. As can be concluded, theoretical predictions are compatible with the experimental data both for lower and upper ionic strength presented in this work. These results correspond with the results already published in our previous work. Therefore, adsorption of NT-4 homodimers for surface concentration N higher than 4000 um-2 was conducted by solution depletion method using ELISA test. This is one of the simplest methods to study protein adsorption by measuring any protein concentration change before and after adsorption process. The results were consistent with AFM measurements and are presented in Figure 4. Considering the electrokinetic charge data depicted earlier we noticed that irreversibility of adsorption process should be expected at pH 3.5, where NT-4 molecules and mica bear opposite signs of electrokinetic charge. It also suggests more


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positive side of NT-4 molecules facing the mica surface, which could be related to their conformational changing during adsorption.43 It is useful to express the neurotrophin surface concentration in terms of the coverage (2D density) denoted by Γ in mg/m2. This unit is combined with N in μm-2 through below presented relationship and widely applied in literature.

Γ=

Mw N A

(5)

where A is the Avogadro’s constant and Mw is the molar mass of the protein. When using the above equation, it is important to consider the effective molecular mass of a protein, which depends on unknown degree of hydratation. Therefore, this is a major disadvantage of using Γ compared to the surface concentration N. As can be seen in Figure 3, experimental data are well reflected by the theoretical results calculated by neglecting hydratation for broad range of N up to 23800 μm-2, which corresponds to 0.53 mg m-2. Insufficiency in filtration process could cause certain discrepancy in experimental results presented in the plot. These results allowed us to determine unknown bulk concentration of neurotrophin or its diffusion coefficient in a convenient way via reassigning adsorption kinetic. Moreover, the experimental data are shown monotonically increased in maximum coverage of irreversibly bounded protein, which suggests that this effect is caused by lateral electrostatic interactions between adsorbed NT-4 molecules (positively charged at pH 7.4), whose range decreases proportionally to 1/(κa)1/2 (a is a characteristic protein dimension, κ-1 is the double layer thickness). This hypothesis is supported by analogous results reported in the literature on colloid science (proteins HSA24, IgG23, fibrinogen44), dendrimers45, 46, gold and silver nanoparticles47. Hence, a decrease in the maximum coverage observed for lower ionic strength, analogous to the previous results obtained for HSA and IgG, can be assigned to the increased range of lateral interactions among adsorbed molecules.

3.2.1.2. NT-4 Adsorption in PBS solution

Variations in the heterogeneous population (containing different number of NT-4 molecules in aggregate) of adsorbed protein at pH 7.4 (ionic strength 0.15M) for two types of electrolytes, NaCl and PBS, could be correlated with two factors: low density of


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charges on protein molecules and changes in the solutions composition. These electrolytes contain various charged counterions, which may be vital for protein stability because adsorption is partially governed by intra-solvent protein-protein interaction and directly related to the changes in heterogenous population of adsorbed protein. The importance of intra-solvent interactions was suggested by Schneider et al. 48,

who studied attractive interactions between biomacromolecules in the bulk and

taking into account the role of electrolyte (various counterions) in proteins stability. According to Schneider et al.48, H2PO4 ions are able to create more interactions between protein molecules in solution than chloride ions due to the presence of numerous hydrogen bonds.

45 40 35 30 25

[%] 20 15 10 5 0 0

5

10

15

20

d [nm]

Fig.6. Histogram of adsorbed NT-4 aggregates (0.15M pH 7.4: PBS – full bar, NaCl – empty bar) indicated by direct AFM enumeration is obtained for low surface protein concentration. The Figure was created by taking into account of 30 randomly chosen various areas where each micrograph of the NT-4 monolayer at the mica surface has size 1 x 1 µm or 0.5 x 05 µm. The inset shows the NT-4 monolayer on mica for surface concentration of protein 600 µm-2.


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In this case, bulk ions composition can cause formation of multiple hydrogen bonds between protein molecules, encouraging aggregation or enhanced charge heterogeneities leading to localized attraction and repulsion. It was demonstrated in details in second histogram (Fig.6), where the presence of a larger number of oligomers at mica surface in pH 7.4 and ionic strength 0.15M PBS was observed. In comparison to NaCl solutions, numerous aggregates with diameters 11.4+/-2 nm, as well as 22.4+/-2 nm have appeared at mica surface for PBS.

Figure 6 and DLS measurements

(polydispersity index) prove that a probability of NT-4 oligomers appearing in the bulk greatly increases at pH 7.4 in PBS solution. Therefore, one can conclude that buffers with greater valency are more effective in reducing double layer force, reflecting the presence of an additional short-ranged attraction at 0.15 M ionic strength. Moreover, lowering the net charge of NT-4 molecule at pH 7.4 (0.7e) in comparison to pH 3.5 (3.3 e) at constant ionic strength (0.15M) indicates reduction of double layer forces and key role of electrostatic interactions. At pH 7.4 proteins are more prone to aggregation, due to a low positive value of the zeta potential. Considering the first kinetics regime of NT-4 adsorption (Fig.5) we assessed the variations between type and number of aggregates at pH 3.5 (Fig.4) and pH 7.4 (Fig.6) for ionic strength 0.15M. The comparison between the two above depicted histograms of adsorbed NT-4 (Fig. 6) for electrolyte NaCl with pH 7.4 and PBS with 7.4 revealed that ion composition is crucial to understanding protein interaction. The presented results remain in good accordance with Roberts et al.34 work, where the buffer effect on protein-protein interaction was examined. It was found that binding of sulfate ion to protein surface neutralizes some of protein charge compared to the same interactions in sodium chloride. For PBS buffer it was quite distinctive that the number of interactions between protein molecules could have increased and had direct impact on adsorption process on the mica surface, as shown in Fig.7.


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6000 -2

N [µm ] 5000

4000

3000

2000

1000

0 0

5

10

15

20

t

25 1/2

1/2

[s ]

Fig.7. The dependence of the surface concentration of NT-4, N (um-2)/coverage Γ (mg/m2) on the square root on adsorption time t1/2 (s1/2). The points denote experimental results obtained by the direct AFM enumeration of the adsorbed protein molecule for I=0.15M, pH 3.5 ( ), pH 7.4 NaCl ()/ PBS ( ) without ultrafiltration. The lines presents the exact theoretical results obtained by the numerical solution of the diffusion equation using the random sequential adsorption model for homodimer NT-4 molecule at various concentration: solid line for 1 μg/ml, hash line for 0.7 μg/ml, dotted line for 0.5 μg/ml. As can be seen the surface concentration of neurotrophins at mica surface decreased in PBS solution, which could be related to the appearance of higher number of protein oligomers in the bulk. Therefore, the effective concentration of neurotrophin homodimer in PBS buffer before adsorption on mica surface resulted from two times lower bulk concentration of NT-4 than in NaCl solution. These exact determinations of adsorption kinetics of NT-4 molecules are based on RSA model and allowed us to precisely determine concentration of neurotrophins homodimer in the solutions used for adsorption process. The RSA model was used for calculating the kinetics of homodimer of NT-4 molecule, adsorbed at mica surface for various bulk concentrations from 1 to 0.5 μg/ml. The kinetics of NT-4 molecule was approximated in terms of equally sized sphere having diameter of 5.1 nm, forming NT-4 molecule. These molecules were adsorbed on mica surface according to the Monte Carlo simulation scheme.49 The lateral interactions among different molecules were calculated by using


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Yukawa pair potential, physically derived from the screened Coulomb interactions according to Ref.50. Theoretical data obtained from RSA simulations for various concentrations of NT-4 suspensions are shown in Figure 5. Together with the experimental results from DLS and AFM, the RSA data suggest that neurotrophin molecules form aggregates in the bulk at pH 7.4, ionic strength 0.15M, and thereby concentration of single protein molecule in the solution decreases. In this study we proved unequivocally that adsorption of single NT-4 molecules at mica surface in the initial kinetic adsorption region is diffusion-controlled process for both type of electrolytes (NaCl and PBS) and pH 3.5 as well pH 7.4. Therefore the adsorption rate for the above described conditions is indeed first order rate process, hence, it relates to first order in neurotrophin concentration. Moreover, the results shown in Figure 5 suggest that the coverage of single proteins can be precisely determined by AFM method, especially for the lower coverage range below 1 mg/m2.

3.2.2. NT-4 Desorption The two complement methods ELISA and AFM imaging were used to check the reversibility of neurotrophin adsorption. It was done in a two-stage procedure, where first the NT-4 monolayers were formed as mentioned above. Afterwards, the desorption vessel was filled with pure electrolyte solution and mica sheets with defined neurotrophin coverage were immersed for a prolonged time period (up to 24 hours) without any flow. The supernatant was used to determine retrievable bulk protein concentration via ELISA and simultaneously to perform re-adsorption on freshly cleaved mica sheets for AFM study. By knowing the total area available for adsorption we could calculate from ELISA measurements the surface concentrations of NT-4 and thereafter compare them with AFM results. These measurements were utilized for determining the maximum coverage of neurotrophin under various bulk conditions.


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5

21000

Γ [mg/m ] 2

N [µm-2]

4

17000

3

13000

2

8600

1

4300

0 0

200

400

600

800

1000

1200

t[min]

Fig.6. Desorption kinetics of NT-4 molecules from mica surface determined by AFM and ELISA methods. Experimental results expressed as the dependence of protein concentration c (mg/ml) on desorption time t (min). The points denote experimental result obtained for I = 0.15M NaCl, pH 3.5 (, ) and pH 11 ( , ) and I=0.15M PBS. The error bars show the standard deviation resulting from ELISA and AFM measurements uncertainty. As can be seen, the initial coverage of NT-4 decreased with desorption time, attaining final steady-state values after 600 min (Fig.6.). Additionally, relevant decline in the protein concentration was observed after 10 minutes period for monolayer obtained at pH 3.5, as well as pH 11. The starting point of desorption experiment is one minute. As can be noticed, the variations in the recovering bulk concentration for ionic strength 0.15M and pH 3.5 with desorption time were limited to 0,1 μg/ml for longer adsorption times for both types of coverages. This suggests that at pH 3.5 the NT-4 molecule binds to mica surface primarily through electrostatic interactions between positively charged amino acid residues and negatively charged mica surface. Consecutive

observations

showed

significant

discrepancies

between

concentrations of desorbed NT-4 molecule from mica surface at pH 3.5 and pH 11 for two various coverages (surface concentration 3 mg/m2 and 1.2 mg/m2). We found that neurotrophins adsorption at pH 3.5 (3.3 e) when protein molecules bear positive electrokinetic charge is more sufficient than at pH 11 (-4.1 e) where mica and protein gain negative electrokinetic charge. Hence NT-4 adsorption process on oppositely


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charged mica surface could be more effective for bulk condition when pH of protein suspension lay far below isoelectric point. In our case, we should additionally take into account that the zeta potential of mica surface at a constant ionic strength decreased moderately with increasing pH. It means that at pH 11, both mica surface and NT-4 gained more negative zeta potential value. It is interesting that for the lower range of NT-4 coverage below 0.2, the effect of the substrate remains significant, making desorption process more efficient than density packed monolayer. In the initial (diffusion) stage of adsorption process the NT-4 molecules irreversibly bound mica surface hence their population is governed by the lateral interaction range (i.e. by the ionic strength). Afterwards, NT4 molecules are forced to adsorb in-between previously adsorbed molecules. Occuring repulsive electrostatic interactions within the monolayer caused rising binding energy of these molecules, which is more positive than before. As a result, the further population of protein molecules is not tightly bound to the mica surface and may be desorbed. We assumed that the main driving force for protein adsorption/desorption from solid surface is electrostatic interactions, hence physical properties could be a key to understanding transition protein adsorption/desorption process.

4. Conclusions

The kinetic of neurotrophin homodimer adsorption on mica surface was determined using images of single protein molecules obtained by AFM (for coverage range lower than 1.2 mg m-2), and solution depletion technique (for coverage range higher than 1.2 mg m-2). The combination of physicochemical measurements of neurotrophin molecules in the bulk and at model surface allowed us to establish that the mechanism of adsorption was bulk diffusion controlled process, while the surface activation barriers (involving energy barrier of diffusion and electric charge issue) were negligible. It is possible to determine the coverage of neurotrophins under in situ conditions by using RSA model postulating a 3D adsorption of NT-4 molecule as discrete particles. Therefore, adsorption and desorption kinetics of proteins with arbitrary zeta potential can be efficiently established.


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The transition between completely reversible and irreversible regime of adsorbed proteins at fixed ionic strength 0.15M is found for low surface coverage monolayer of NT-4 and high pH of rinsing solution. The transition between reversibility and irreversibility of adsorption could have resulted from heterogeneous (dipolar) charge distribution over adsorbed NT-4 molecule, which induced local attraction and repulsion in the system. The results suggest that NT-4 homodimer adsorption at hydrophilic surface at pH = 3.5 and pH =7.4 and I = 0.15 M is governed by electrostatic interactions, which results in the formation of irreversibly bound protein monolayer. The maximum coverage of protein monolayer was equal to 5.53 mg m-2 (considering hydration), which is in good agreement with experimental data. In view of the experimental results one can conclude that the coverage of NT-4 monolayer can be controlled easily by adjusting the adsorption time, ionic strength of the solution, as well as the protein bulk concentration. Neurotrophin monolayers can be applied in, e.g., designing new therapeutic strategies such as drug delivery systems for treating neurodegenerative diseases.

Acknowledgements: This work was supported by the National Science Center’s grant, called OPUS 2012/07/B/NZ5/02498 (to BM) and the National Centre for Research and Development grant STRATEGMED1/234261/2NCBR/2014 (to BM). We wish to thank prof. Mirosława El Fray from the Polymer Institute WestPomeranian University of Technology for allowing us to use the equipment.


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(30) Adamczyk, Z.; Bratek, A.; Jachimska, B.; Jasiński, T.;Warszyński, P. Structure of Poly(acrylic acid) in Electrolyte Solutions Determined from Simulations and Viscosity. J.Phys.Chem.B 2006, 110, 22426-22435. (31) Delgado, A. V.; Gonzalez-Caballero, E.; Hunter, R. J.; Koopal, L. K.; Lyklema, J. Measurement and Interpretation of Electrokinetic Phenomena (IUPAC Technical Report). Pure Appl. Chem. 2005, 77, 1753−1805. (32) Santiago, P.S.; Carvalho,F.A.O.; Domingues, M.M.; Carvalho, J.W.P.; Santos, N.C.; Tabak, M. Isoelectric Point Determination for Glossoscolex paulistus Extracellular Hemoglobin: Ologomeric stability in Acidic pH and Relevance to Protein-Surfactant Interactions. Langmuir, 2010, 26(12), 9794-9801. Curtis, R.A.; Ulrich, J.; Montaser, A.; Prausnitz, J.M.; Blanch, H.W. Protein(33) Protein Interactions in Concentrated Electrolyte Solutions: Hofmeister-Series Effects. Biotechnol. Bioeng. 2002, 79, 367-380. (34) Roberts, D.; Keeling R.; Tracka.M.; van der Walle, S.U.; Warwicker, J; Curtis, R. Specific Ion and Buffer Effects on Protein-Protein Interactions of a Monoclonal Antibody. Mol. Pharmaceutics 2015, 12, 179-193. (35) Kujda, M.; Adamczyk, Z.; Cieśla, M. Monolayers of the HSA dimer on polymeric microparticles – electrokinetic characteristic. Coll Surf B: Biointerface 2016, 148, 229-237. Feder, J. Random Sequential Adsorption. J Theor Biol. 1980, 87, 237-254. (36) (37) van Tassel, P.R; Guemouri, L.; Ramsden, J.J.; Tarjus, G.; Viot, P.; Talbot, J. A Particle-Level Model of Irreversible Protein Adsorption with a Postadsorption Transition. J.Colloid Interface Sci. 1998, 207, 317-323. (38) Feder, J.; Giaever, I. Adsorption of ferritin. J.Colloid Interface Sci. 1980, 78, 144-154. (39) Adamczyk, Z.; Barbasz, J.; Cieśla, M. Mechanisms of Fibrinogen Adsorption at Solid Substrates. Langmuir 2011, 27, 6868-6878. (40) Adamczyk, Z.; Warszyński, P. Role of Electrostatic interactions in particle adsorption. Adv. Colloid Interface Sci. 1996, 63, 41-149. (41) Erban, R.; Chapman, S.J. Reactive boundary conditions for stochastic simulations of reaction–diffusion processes. Phys. Biol. 2007, 4, 16. (42) Cieśla, M. Barbasz, J. Modelling of interacting dimers adsorption. Surf. Sci. 2013, 612, 24-30. (43) Etheve, J.; Dejardin, P. Adsorption Kinetics of Lysozyme on Silica at pH 7.4: Correlation between Streaming Potential and Adsorbed Amount. Langmuir 2002, 18, 1777-1785. (44) Bratek-Skicki, A.; Żeliszewska, P.; Adamczyk, Z.; Cieśla, M. Human Fibrynogen Monolayers on Latex Particles: Role of Ionic Strength. Langmuir 2013, 29, 3700-3710. (45) Longtin, R.; Maroni, P.; Borkovec, M. Transition from Completely Reversible to Irreversible Adsorption of Poly(amido amine) Dendrimers on Silica. Langmuir 2009, 25, 2928-2934. (46) Pericet-Camara, P.; Cahill, B.P.; Papastavrouu, G.; Borkovec, B. Nanopatterning of solid substrates by adsorbed dendrimers. Chem. Commun. 2007, 266−268.


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(47) Kooij, E.S.; Brouwer, E.A.M.; Wormeester,H.; Poelsema, B. Ionic Strength Mediated Self-Organization of Gold Nanocrystals: An AFM Study. Langmuir 2007, 18, 7677−7682. (48) Schneider, C.P.; Shukla, D.; Trout, B.L. Effects of Solute-Solute Interactions on Protein Stability Studied Using Various Counterions and Dendrimers. PLoS ONE 2011, 6 (11), e27665. (49) Kujda, M.; Adamczyk, Z.; Cieśla, M. High Density Monolayer of Plasmid Protein on Latex Particles: Experimentas and Theoretical Modeling. J. Stat. Mech. 2015, 1742-5468. (50) Z. Adamczyk "Particles at Interfaces. Vol. 9 - Interactions, Deposition, Structure", Academic Press-Elsevier, New York 2006, 50-743.


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Desorption Transition of Recombinant Human

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