Computer Methods and Programs in Biomedicine 197 (2020) 105742

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Computer Methods and Programs in Biomedicine

journal homepage: www.elsevier.com/locate/cmpb

A case study of poly (aryl ether sulfone) hemodialysis membrane

interactions with human blood: Molecular dynamics simulation and
experimental analyses
Arash Mollahosseini a, Srija Argumeedi a, Amira Abdelrasoul a,b,∗, Ahmed Shoker c,d
a
Department of Chemical and Biological Engineering, University of Saskatchewan, 57 Campus Drive, Saskatoon S7N 5A9, Saskatchewan, Canada
b
Division of Biomedical Engineering, University of Saskatchewan, 57 Campus Drive, Saskatoon S7N 5A9, Saskatchewan, Canada
c
Nephrology Division, College of Medicine, University of Saskatchewan, 107 Wiggins Rd, Saskatoon, SK S7N 5E5, Canada
d
Saskatchewan Transplant Program, St. Paul’s Hospital, 1702 20th Street West Saskatoon Saskatchewan S7M 0Z9 Canada

a r t i c l e i n f o a b s t r a c t

Article history: Patients with end-stage renal diseases (ESRD) require specific health cares as the accumulation of toxins
Received 22 April 2020 due to the lack of kidney functionality would affect their lives. However, the mortality rate is still high
Accepted 1 September 2020
due to cardiovascular diseases, socks, etc. A majority of patients with chronic kidney disease (CKD) re-
quire hemodialysis services. Blood purifying membranes, as the main component of hemodialysis setups,
Keywords: however, still suffer from lack of optimum biocompatibility, which results in morbidity and mortality
Hemodialysis membrane of hemodialysis service receiving patients. The goal of the present case study is to have an in-depth
Blood understanding of the current blood-hemodialysis membrane interactions occurring during hemodialysis
Biocompatibility sessions using poly (aryl ether sulfone)-poly (vinyl pyrrolidone) (PAES-PVP) membrane. Attenuated total
Interactions
reflectance-Fourier transmission infrared (ATR-FTIR) spectroscopy, Raman spectroscopy, and solid-state
Molecular dynamics simulation
nuclear magnetic resonance (SSNMR) spectroscopy were used to assess the initial chemical structure of
Plasma protein
Aggregation the PAES-PVP membrane along with the variations after with the infections with human blood. Further-
more, scanning electron microscopy (SEM) and Transition electron microscopy (TEM) were used to vi-
sualize the structural variation of the membrane, blood aggregations, and blood clots on the membrane
surface. Besides, Molecular dynamics (MD) simulation was used to assess the interaction of PAES-PVP
with major human blood proteins, in terms of interaction energy, which is a novel contribution to the
area. The macromolecules (human serum albumin (HSA), human serum transferrin (TRF), and human fib-
rinogen (HFG)) were chosen from the plasma protein component. These protein structures were chosen
based on their different molecular size. Three advanced spectroscopy techniques and two advanced vi-
sualization techniques were used for the assessment of the membranes. Spectroscopy studies revealed
amine related peak displacement and intensity shifts as indices for attachment of biological species to
the polymeric membrane surfaces. Raman peaks around 370, 798, and 1299 cm−1, which experienced
significant shifts that were related to carbon-nitrogen and sulfur-oxygen bonds due to protein adhesion.
Visualization techniques illustrated blood protein fouling patterns and extracellular vesicles’ presence in
the pore structures into membranes. The findings highlight the importance of whole structure biocom-
patibility improvement, rather than only focusing on surface modifications of hemodialysis membranes.
Molecular dynamics simulation assessment showed various interaction behaviors for different proteins
suggesting molecular weight and active residues of the protein macromolecules play an important role in
interacting with polymeric structure. FB had the highest interaction (4,274,749.07 kcal/mol) and binding
(10,370.90 kcal/mol) energy with the PAES-PVP structure. TRF owned the lowest interaction energy with
respect to its lower molecular weight and fewer active residue count.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction

∗ End-stage renal diseases (ESRD) affects patients’ lives signifi-

Corresponding author at: Department of Chemical and Biological Engineering,
University of Saskatchewan, 57 Campus Drive, Saskatoon S7N 5A9, Saskatchewan, cantly due to accumulation of metabolic toxins in the body. After
Canada.
the complete failure of the kidney, specific health care is required.
E-mail address: amira.abdelrasoul@usask.ca (A. Abdelrasoul).

https://doi.org/10.1016/j.cmpb.2020.105742
0169-2607/© 2020 Elsevier B.V. All rights reserved.
2 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742
However, even such treatment would not save patients ultimately
as a mortality rate equal to 20% is reported (data is related to
the US) [1]. Cardiovascular diseases are reported to be responsi-
ble for half of these deaths [2]. A majority of patients with chronic
kidney disease (CKD) require hemodialysis services. Hemodialysis
(HD) is a life-sustaining treatment for patients whose kidneys have
irreversibly failed and consequently, the biological waste materials
and toxins are being accumulated in the bloodstream [3,4]. One in
ten Canadians have kidney disease, and millions more are at risk,
with an average of 15 people being told that their kidneys have
failed every day. Only 42.7% of hemodialysis patients have sur-
vived their treatment in 2016. Based on the published academic re-
ports, 3.2 million dialysis recipients [5] (2.6 as estimated by others
[6]) around require hemodialysis, and the rate is growing 6% every
year [5]. Exposure of human blood to the hemodialysis membrane
results in several interactions. Adsorption and transformation of
plasma proteins, activation of blood cells, adherence of platelets,
and thrombosis reactions against the membrane can invoke severe
blood reactions causing the increased rate of mortality and mor-
bidity of hemodialysis (HD) patients [7]. A more detailed biolog-
ical reaction pathway is described in previous review articles of
the same authors [8]. Conventionally, there have been two types
of polymeric hemodialysis membranes used, cellulose (cuprophan)
based membranes and synthetic ones [9]. The application of the
cellulose membrane is restricted to some extent by its inadequate
blood compatibility [10]. This was mainly attributed to the hy-
droxyl groups present on the cellulosic membranes [11]. Synthetic
polymers, on the other hand, could be tailored to meet the re-
quirements of the process and thus facilitating reproducibility and
scale-up [12]. Based on the reports released in 2011, More than 90%
of the dialyzers in the world used poly (aryl sulfone)-based mem-
branes (71% were made out of poly (ether sulfone) (PES) and the
22% are of poly (sulfone) (PSF)). Considering the growing rate of
the dialysis demanding patients, membranes with high solute re-
jection rate and a high-water permeability are required. Biocom-
patibility remains a major concern [13,14].
In the hemodialysis field, hemocompatibility is defined as the
ability of the membrane to minimize the biochemical reactivity
between itself and the blood. It is believed when blood contacts
with external materials, protein adsorption on the surface is the
first step of many undesired bio-reactions and bio-responses, fol-
lowed by platelet adhesion and activation of coagulation pathways,
leading to thrombus formation [10]. Such prolonged contact be-
tween the blood and the synthetic polymer surface has resulted in
long-term complications, notably dialysis-induced oxidative stress
(DIOS) and membrane-induced inflammation (MII) [3]. Blood coag-
ulation initiates from the fibrinogen transformation into insoluble
fibrin. Fibrins are turned into fibrin clots with a crosslinked and
steady structure as a result of factor XIII (fibrin stabilizing factor)
secretion, which is activated by thrombin. Platelets will be acti-
vated and aggregated (thrombogenesis), boosting a continuous in-
teraction, which leads to blood clotting. Furthermore, other blood
cells are attracted to the clot and contribute to more fibrin forma-
tion through enzymatic reactions. The formed biological layer or
“protein cake” contains plasma proteins like factor XII, fibrinogen,
vitronectin, kininogens, etc., that could result in further thrombo-
genesis by activation [15–17]. Molecular dynamic simulations in
this study focus on three proteins of the blood protein family, hu-
man serum albumin (HSA), human serum transferrin (HST) and
human fibrinogen (HFG), as they are believed to be responsible for
interacting with external particles and non-human cells [18]. Com-
plement activation as an immune system provocation and leuko-
cyte activation are the other inflammatory responses of the body
to the incompatible membranes [4,8,14].
The adsorption process of the proteins is a believed to be the
initial step of protein provocation and is governed by electrostatic,
van der Waals and hydrogen bonding interactions [19]. Several fac-
tors are believed to affect bloodstream proteins adsorption pro-
cess, including the properties of the proteins, blood’s physiochem-
ical conditions, surface chemistry of the membrane and the dial-
ysis operating conditions. The consecutive-competitive deposition
of the proteins, from the bloodstream onto the membrane, is de-
scribed by Vorman effect: human serum albumin, immunoglob-
ulins, fibrinogen, factor XII and high molecular weight kininogen
would sequentially adsorb onto the membrane surface, one substi-
tuting the other. While Vorman effect describes the high molecular
weight proteins, low to medium molecular weight proteins are also
believed to dynamically adsorb onto the membrane, depending on
the membrane’s transportive and material characteristics [14,20].
More detailed description of the triggered cascades and hemocom-
patibility measures could be found elsewhere [8,14].
Considerations and requirement criteria for enhancing mem-
brane biocompatibility or their degradation by-products should not
trigger an adverse inflammatory reaction, or immune response
once implanted [16]. This is of particular importance in host– im-
plant interactions, as the adsorption of proteins present in physi-
ological fluids, such as albumin, immunoglobulin, fibrinogen, and
fibronectin, dictate the subsequent inflammatory response and the
fate of the implant. While there is tremendous effort attributed to
the hemodialysis and many researchers improved the technology,
there are still many unknown areas and numerous unanswered
questions. Trend assessment during the blood-membrane interac-
tion, behavior of different patients’ blood in membrane systems is
considered the major research gap in the area.
In the area of material science, molecular dynamics (MD) sim-
ulations a growing technique, which is gaining the research atten-
tion and offers microscopic modeling on the molecular scale [21].
MD incorporates step-by-step computational simulation for solv-
ing Newtonian equations numerically, which is efficient in calcu-
lating the system element’s movements and characteristics, usu-
ally in nanoscale levels [22]. Since the method is also efficient
in studying structures, interactions, conformations etc. in biologi-
cal systems [23], interdisciplinary studies of membrane technology
and biological systems would also be a target of academic investi-
gations. Only a few studies have so far targeted computer-assisted
studies for comprehension of hemodialysis membrane materials-
blood components. One of the first studies, which, coupled ex-
perimental and computational approaches, was performed by Kim
et al. [24]. The study targeted Polysulfone (PSF) -polyethylene gly-
col (PEG) modification and used radial function distribution (RDF)
assessment for proving that water molecules create a hydration
layer when the PEG is present in PSF membrane. Modification of
Sasongko et al. reported the interaction of creatinine and urea with
PSF membranes [25]. The paper is written based on PSF as the
major hemodialysis membrane material (while Polyethersulfone or
polyarylethersulfone materials also own a major share of the in-
dustry [26]). The study covers the charge map and the possibil-
ity of the interactions based on density functional theory studies.
Hydrogen bonding intensity was discussed for both creatinine and
urea, and it was reported that urea, despite its smaller size, inter-
acts more strongly with the membrane. Sasongko also reports the
correlation of hydrogen bonding intensity with the ability of the
structures to diffuse through the membrane. Interestingly, stronger
hydrogen bonding energy was mentioned to reflect the higher ca-
pability of species for passing through the membrane [27–29].
However, the correlation is based on the studies over environmen-
tal studies. Blood related researches could comparatively be more
complicated. The blood chemical environment for ESRD patients
could differ for each person depending on the health condition, the
body’s temperature. On the other hand, the dialysis operation con-
dition could also impact the blood-membrane reaction.
 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742
As previously mentioned, experimental studies and MD assess-
ments could be coupled to further facilitate the understanding
of the unknown phenomena. In this study advanced spectroscopy
techniques such as Raman, NMR, and FTIR will be conducted in
this as important tools to offer an in-depth understanding of inter-
actions. While different techniques could analyze different depths
and types of materials, techniques such as Raman and FTIR, how-
ever, the complementary use of the techniques would cover peaks
that might be inactive in the other one [30]. This study intends to
assess the changes imposed on a commonly used membrane (in
Canadian hospitals) during the hemodialysis process experimen-
tally. It also targets the assessment of the selected protein inter-
actions with the polymeric structure of the membrane using an
interaction energy parameter. Accordingly, a comprehensive set of
experimental analyses and molecular dynamic simulations were
performed. As a consequence, this research case study could be a
proper foundation for the academic membrane society researchers,
who are working in hemodialysis area. Besides, this is the first
study offering molecular dynamic simulation studies for hemodial-
ysis membrane assessment.
2. Computational and experimental studies
2.1. Molecular dynamic simulation (MDS)
The interatomic relationships of the structures were defined
using Dreiding force field potential, which is mentioned to be
proper for biological, organic, and main group inorganic structures
[31]. The contributing terms through which the interactions are
assessed could be discussed in contributing forms of stretching
bonds, bond angles, dihedral angles, and van der Waals and elec-
trostatic interactions. The total energy in this simulation is calcu-
lated based on Eq. (1). All the performed simulations involve as-
sessing the interaction energy between two structures, one poly-
mer chain (five monomer chain), and one protein (one molecule,
received from PDB database and protonated). All the simulations
were performed in the absence of water molecules. The interac-
tions could be divided into two classes of bonded and non-bonded
interactions. While bonded interactions would be covered by bond
stretching (r) (Eq. (2)), bond angle (θ ) (Eq. (3)) and dihedral angle
(∅) (Eq. (4)), non-bond interactions are covered by van der Waals
(Eq. (5)) and electrostatic (Eq. (6)) interactions.
Etotal = Ebond (r ) + Eangle (θ ) + Edihedral (∅ ) + Enon−bonding (r ) +
Eelect rostat ic (r )
1
Ebond (r ) = K (r − r0 )2
2 b
1 Where Epolymer chain and Eprotein are the total energies of each
Eangle (θ ) = K ( θ − θ0 )
2
2 θ

3
Edihedral (∅ ) = Ci (cos∅ )i
i=0
 12  σ 6 
σ the structures were moved to the vicinity of each other so they
Enon−bonding (r ) = 4ε − , r < rc
r r
C qi q j
Eelect rostat ic (r ) = r < rc
εr
Where, Kb and Kθ are defined as bond length and bond angle
stiffness constants, respectively, r0 is the equilibrium bond length,
θ 0 is the equilibrium bond angle, Ci variable covers the dihedral
multi-harmonic coefficient, r is the distance between to atoms, ∅
is the zero-energy distance, ɛ is the depth of energy well in van
der Waals interaction and dielectric constant in electrostatic in-
teractions, C is the energy conversion constant, qi and qj are the
3
charges of the two atoms, and rc is the cut-off distance defined to
be 10 Å.
The copolymer block was built using Avogadro software, and
minimization was performed using GAMESS software using the
adiabatic connection method-Becke three-parameter and applying
the density functional theory (DFT) method with Lee–Yang–Parr
(B3LYP) functional and no symmetry constraints [32]. Three blood
protein structures, namely human serum albumin (HSA), human
fibrinogen (FB), and human serum transferrin (TRF)), were chosen
to assess as candidates of blood constituents. The proteins’ struc-
tures were received from the protein data bank. The protonation
of the protein structures was conducted using Autodock software.
Tables 1 and 2 summarize the data related to the mentioned pro-
teins. MD simulations were performed using LAMMPS software.
Five monomers were attached to each other to create a chain of
five-membered polymer to minimize the computational run length.
The simulations were conducted in 3 random positions between
each polymer and each protein, so that different sides of the struc-
tures could interact with each other, and the binding energies were
calculated by taking the average of the three simulations. Further-
more, the influence of long polymeric chains of a ten-membered
PAES-PVP chain on the interaction was investigated and compared
with the five-membered chains on the supplementary information.
The error calculated for all the three proteins were less than 7%,
as presented in Table S.1, Figure S.1 and Figure S.2. The valida-
tion of the polymeric chain model was performed using density
calculations (consecutive npt ensemble; 500 ps NPT run at 1 bar,
100 ps run at 100 bar, ps run at 10,0 0 0 bar and final value af-
ter 500 ps run at 1 bar). The calculated equilibrium density was
achieved to be 1.22 gm cm−3 which is near the real reported den-
sity for polyarylethersulfone (1.37 gm cm−3 [33]). The simulations
were performed using NVE ensemble at 298 K without control-
ling the pressure (the temperature was kept constant at 298 K us-
ing Langevin thermostat). All the interaction simulations included
only one protein and one polymer chain and no further water or
charged ion was used. The number of the atoms and molecules
were 2 molecules and 12,893 atoms PAES-PVP-FB, 2 molecules,
10,557 atoms for PAES-PVP-HSA, and 2 molecules and 6516 atoms
for PAES-PVP-TRF simulations. The structures were simulated sep-
arately to calculate individual energies for each protein and the
polymeric chain. The number of atoms in each individual simula-
tion (which only included one molecule) was 12,583 for FB, 10,247
for HSA, 6206 for TRF, and 310 for PAES-PVP. The interaction en-
ergy was reported as the total energy of the simulation box con-
(1) taining both protein and the polymer. Binding energy was calcu-
lated using Eq. (7).
(2) Binding energy = E polymer + E protein − E polymer (7)
chain and protein
(3)
polymer and protein which was separately calculated trough an in-
dividual MD simulation, and Epolymer and protein is the total energy of
MD simulations at the stage where structures are at the closest
(4) distance. In the binding energy simulation, structures were kept
far away initially, and the simulation was run for 10 0 0 steps. Then
(5) could interact. The simulation was run for 10 0,0 0 0 steps. The en-
ergy value at the 10 0,0 0 0th step was used as Epolymer and protein in
(6) Eq. (7). The structures were moved back to their initial location
and the simulation was run for another 10,0 0 0 steps. Accordingly,
the trend of interaction energies between each pair of polymeric
chain and protein, as well as binding energy between each pair,
are the parameters assessed and discussed in this paper. All the
simulations were performed three times with random locations for
the polymer at the vicinity of the protein. The reported values are
the average values of the three simulations so that the effect of
conformation would be considered in the study. Table 3 presented
4 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742

Table 1
Chemical structure of the sample proteins chosen for interaction study.

Human serum albumin (C2903 H4534 N776 O879 S41) Transferrin C1612 H2471 N437 O485 S21 Fe Fibrinogen C9848 N2729 O3133 S102
(65,684.22 g/mol) (36,461.62 g/mol) (209,902.41 g/mol)

PDB databank ID
RMSD
0.007 0.007 0.038
PDB ID
1AO6 1D3K 3GHG
Amino acid active sites
Ala 258 Gly 459 Asp 159a
Ala 261 Ala 453 Phe 160a
Leu 260 Thr 452 Tyr 190a
Ile 264 Arg 456 Ser 161a
Arg 257 Ala 458 Tyr 189a
Val 241 Thr 457 Asp 224a
Ser 287 Val 533 Leu 192a
Leu 238 Tyr 517 Ser 225a
Ile 290 Lys 534 Ser 226a
His 242 Asp 392 Phe 231a
Tyr 150 Tyr 426 Arg 216b
Ala 291 His 585 Arg 214b
Ala 215 Tyr 122b
Phe 211 Asn 215b
Leu 219 Ser 213b
Arg 222 Ser 121b
Lys 199 Val 157b
Glu 292 Ser 123b
Trp 214 Glu 220b
Arg 218 Met 124b
Gln 196 Asp 217b
Arg 197 Ala 218b
Ser 192
Ser 193
Leu 198
Lys 195
Ala 194
Ala 191
Asn 458
Val 455
Gln 459

Fig. 1. Molecular dynamics simulation study sample for interaction energy assess-
ment between polymeric structure and proteins.

the molecular dynamic simulation parameters and units. The en-
ergy term calculated in the simulation is the total interaction en-
ergy imposed from the macromolecule protein to the polymeric
structure. Fig. 1 illustrates a sample of MDS conducted for polymer
structure-protein interaction.
2.2. Materials and experimental methods
Fig. 2. PAES-PVP monomer structure (a) schematic chemical structure, (b) simu-
lated strucrture using Avogadro software.
a molecular weight cut off of 55 kDa. The surface charge of the
membrane was calculated using a zeta potential analyzer with an
average charge value of −68 mV.

2.2.1. PAES-PVP hemodialysis membrane 2.2.2. Advanced spectroscopy techniques

A poly (aryl ether sulfone-vinyl pyrrolidinone) (PAES-PVP)
hemodialysis membrane was used in this study, which is currently
used in Canadian hospitals. The details supplied by the manufac-
turer are summarized in Table 4. The chemical structure of the
PAES-PVP monomer is represented in Fig. 2. The PAES-PVP has
Spectroscopy techniques offer assessments over the structure
of various substances. Different ranges of radiation from visible
light, ultraviolet, infrared and X-rays are utilized in various non-
destructive techniques such as XRD, X-ray photoelectron spec-
troscopy (XPS), infrared (IR), near-infrared (NIR), fourier-transform
 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742
Table 2
Structure of amino acids.
infrared spectroscopy (FTIR), attenuated total reflection fourier-
transform infrared spectroscopy (ATR-FTIR), nuclear magnetic res-
onance (NMR), Raman and UV-spectroscopy to assess the chemi-
cal composition of substances, changes imposed to a structure in
a process and interaction of different molecular structure like lig-
ands, proteins, polymers etc. together. Advanced spectroscopy tech-
niques could offer a better understanding of hemodialysis mem-
branes by analyzing the structure of membranes and assessing the
interaction consequences of blood-polymer contact.
5
In this research Raman and attenuated total reflectance-
Fourier transmission infrared (ATR-FTIR) were performed with the
Renishaw-in Via Raman Microscope on both clean and blood-
contacted membrane samples. The laser power was set at 10% and
at 514 nm for the clean sample. For the blood-contacted sample
the laser power was at 100% and at 785 nm.
The samples quantitatively analyzed using 13 C ssNMR tech-
nique. This research work also shows that the combination of a
selectively 13 C-labeled technique and a high spinning speed of
20 kHz in magic-angle spinning (MAS) NMR experiment can im-
6 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742

Table 3 ing was conducted using a Hitachi SU8010 device. Scanning proce-
Parameters’ units in the MD simulation, unit’s package name: real.
dure was performed on both clean and blood-contacted membrane
No. Parameter Unit samples. The blood contacted samples were fixed using ethanol
1 Time Femtosecond and dried for three days. The blood contacted sample was cut in a
2 Mass gm mole−1 cross-section to observe any blood cells inside the PAES-PVP micro-
3 Distance Angstrom tube membrane. Samples were fixed in ethanol. Blood-contacted
4 Energy Kcal mole−1 membranes were further fixed in 1% osmium tetroxide in 0.1 M
5 Temperature Kelvin
sodium cacodylate (NaCac) for 45 min. Followed by that, treated
6 Pressure Atmosphere
7 Charge Multiple of electron charge (1.0 is a proton) samples were rinsed three times with NaCac, followed by a quick
8 Density gm cm−3 rinse with distilled water. Tubes were cut to expose the internal
9 Electric field Volts. angstrom−1 content and mounted on aluminum stubs with a piece of double-
10 Dipole Charge.angestrom
sided carbon sticky tape. Samples were then coated with 10 nm
gold (Quorum Q150T ES).
TEM scans are more accurate and proper for investigations in
Table 4 lower size materials. TEM analysis is identified as one of the tools
Common Canadian hospitals hemodialysis membrane specification. for characterizing protein-membrane interaction. However, rather
Specification Value than the location and extent of the attachment, there exist restric-
tions for clarifying spatial fouling patterns and depth profiling. The
Blood flow rate 200–600 mL min−1
Dialysate flow rate 300–800 mL min−1 mentioned restrictions could be lifted using chemical markers [37].
UF coefficient in vitro (bovine blood) 54 (mL h−1 .mm Hg−1 ) TEM scanning was conducted using Hitachi HT 7700 TEM de-
thickness 35 μm vice. Blood-contacted membranes were coated with 1% ultrapure
Inner diameter 190 μm low melting point Agarose for fouling stabilization. Furthermore,
ß2 -microgolbulin sieving coefficient 0.7
Albumin sieving coefficient Less than 0.01
the blood contacted samples were fixated using 2% Glutaralde-
hyde in 0.1 M Sodium Cacodylate buffer, pH 7.2 for 3 h in the
fridge. Samples were further fixed with Osmicate in 1% Osmium
in 0.1 M Sodium Cacodylate for 1hour at room temp. Then the
prove the detection sensitivity by nearly 2 orders of magnitude. It samples were washed with Sodium cacodylate and water. Then the
also provides a clear Solid-state (ssNMR) spectra with little peak samples dehydrated through graded ethanol series starting at 30%,
overlaps. All 13 C ssNMR spectra were performed on a Bruker AV up to 100%; infiltrate with LR white resin overnight. Block into
III 400WB spectrometer operating at a 13 C resonance frequency of BEEM capsule with fresh LR white polymerize overnight at 60 de-
100.6 MHz. A 2.5 mm MAS H/F/X triple resonance probe head was grees section on leica ultra-cut ultra-microtome at 90 nm thick,
employed. Samples of 5−10 mg were packed inside a Zirconia MAS collect sections on a 200mesh copper grid observe sections with
rotor with a diameter of 2.5 mm and a vessel cap. 13 C DP/MAS TEM. Electron density created the image on TEM where higher
spectra were acquired with a 200-s recycle delay, a 13 C excitation electron density resulted in darker regions and vice versa. These
(90°) pulse length of 3.9 μs, and 20.0 kHz MAS. Typically, 256 were osmicated, though, so density changes may be due to the ab-
scans were conducted in order to obtain a good signal-to-noise ra- sorption of the osmium.
tio. 13 C isotropic chemical shifts were calibrated on the carbonyl
resonance of an external glycine standard, 176.0 ppm with respect
3. Results and discussion
to TMS (0.0 ppm).
Molecular weight cut-off for the provided membrane samples
3.1. Raman spectroscopy
was assessed using Brunauer–Emmett–Teller (BET) ASAP 2020 (Mi-
cromeritics, Georgia, USA). To conduct the BET experiments, sam-
As stated by Khulbe, Raman and infrared spectroscopies could
ples were degassed at 50 °C for 3 h to remove the moisture con-
be simultaneously beneficial for analyzing a polymeric structure
tent. Inert nitrogen gas was incorporated during the experiments.
as each of the analysis techniques could cover specific bonds that
Surface charge measurements for the hemodialysis membrane
might be active in one and inactive in the other spectroscopy tech-
were conducted using a zeta potential analyzer (Zetasizer-Nano Se-
nique [30]. Fig. 3 shows the Raman spectra of neat and blood con-
ries, Malvern Instruments Ltd., UK, ± 0.01 mV).
tacted PAES-PVP hemodialysis membranes. Table 5 summarizes Ra-
man spectra peaks and its intensity before and after blood interac-
2.2.3. Advanced visualization and imaging techniques tion.
Imaging techniques in micro to nanometer levels could assist The peak around 1600 cm−1 is ascribed to carbon-carbon sym-
several fields by offering detailed pictures and revealing phenom- metric vibration of aromatic rings (phenyl or benzene) [38] and
ena’s consequences at the microscopic level. These images could also aromatic amide group [39,40]. A carbon-oxygen symmetric
also be used for mapping specific elements, quantitative mea- single bond is described by the peak at 1144 cm−1 [41]. Peak
surement, the presence of specific biological contaminations, the The peak at 1105 cm−1 depicts asymmetric stretching vibration
thickness of layers, agglomerations pattern etc. Hemodialysis mem- of sulfur-oxygen double bond while the peak at 1071 cm−1 re-
brane could also take advantage of advanced imaging techniques flects the symmetric stretching vibration type of the same bond
through platelet counts, specific biological attachments mapping in [41]. The peak at 1323 cm−1 gives the characteristic of tangen-
the membrane structure and blood coagulation patterns. tial carbon structure in the polymer [42]. The peak at 1298 cm−1
The first and the simplest usage would be surface visual char- could be designated to the in-plane structure of stretching N–H
acterization, morphological and microstructure observations [34]. and in-plane structure of bending C–N, which has changed sig-
Structural configuration and pore dimensions, as well as the thick- nificantly due to wavenumber displacement and intensity increase
ness of selective layers, could be resulted from SEMs [35]. For more in this peak (3 cm−1 and 41,883 respectively) which means more
quantitative usage, adherent platelet numbers could be driven out CH2 deformation [43,44]. The variation in this peak could also be
in specific magnifications [34,36]. Rather than the count, the at- interpreted into the appearance of specific protein structures (β -
tachment shape of the platelets is analyzed through the scans, sheet) on the membrane [45,46]. The peak at 1579 cm−1 describes
which could be affected by the surface chemistry [34]. SEM imag- carbon-carbon in-plane ring vibration [47]. Another intense peak
 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742 7

Fig. 3. Raman spectra before and after blood interaction.

Table 5
Raman results before and after blood interaction.

Peak wavelength
displacement
PAES-PVP before interaction with blood PAES-PVP after hemodialysis (cm−1 ) Intensity Change

Peak’s wavelength Peak’s intensity Identification Peak’s wavelength Peak’s intensity

370 3022 Weak deformation vibration O=S=O 367 63,709 3 60,687

412 3430 Weak deformation vibration O=S=O 411 62,778 1 59,348
626 9091 Medium deformation vibration O=S=O 626 87,474 N/A 78,383
657 2915 Weak stretching vibration C–S 656 50,274 1 47,359
730 3358 Weak stretching vibration C–S 729 53,073 1 49,715
789 22,497 Stretching vibration C–N 786 164,585 3 142,088
1010 2556 Deformation C–H 1008 43,121 2 40,565
1071 12,367 O=S=O 1070 98,059 1 85,692
1105 7326 O=S=O 1104 70,366 1 63,040
1144 54,267 C-O-C 1143 334,388 1 280,121
1200 3986 O–H vibration 1200 51,175 N/A 47,189
1298 2102 Stretching vibration C–N or stretching 1295 43,985 3 41,883
vibration of in-plane N–H
1323 1569 Tangential carbon bond 1323 42,391 N/A 40,795
1490 777 Stretching vibration C–C or weak stretching 1488 31,256 2 30,479
vibration of C–N
1579 13,502 Strong stretching vibration C–C 1578 104,148 1 90,646
1599 20,514 Strong stretching vibration C–C 1597 140,005 2 119,491
3069 790 Medium stretching vibration C–H 3068 5017 1 4227

displacement occurred for the one at 798 cm−1, which is men-
tioned to be responsible for C–N asymmetric stretching, generally
in tertiary amide [40]. Carbohydrates’ characteristics peaks could
be generally found from 1200 to 1460 cm−1 and the peak here
at 1200 cm−1 O–H vibration [48]. The peak at 1010 cm−1 is com-
monly designated to carbon-hydrogen bond in carbohydrates and
its slight displacement to 1008 cm−1 could be interpreted into
polysaccharide presence and more specifically carbon-carbon and
carbon-oxygen bonds in these structures [43]. The peak around
730 cm−1 could be attributed to carbon-sulfur stretching vibration.
The increase in sulfur-oxygen bond content could be proved by
the shift in the attributed sulfur-oxygen peak of nucleotides from
370 cm−1 to 367 cm−1 [49]. The peak around 626 cm−1 is both as-
signed to carbon-sulfur stretching bond and carbon-carbon twist.
Slight shift might be interpreted into presence of the mentioned
bonds as a result of tyrosine amino acid of protein structures [49].
8 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742
Fig. 4. ATR-FTIR spectra of PAES-PVP hemodialysis membranes before and after contact with blood.
3.2. Attenuated total reflectance-Fourier transmission infrared
(ATR-FTIR)
Fig. 4 depicts the FTIR spectra prior to and after the blood-
membrane contact. Table 6 summarizes FTIR spectra peaks and its
intensity before and after blood interaction. Peaks within 3200–
3400 wavenumbers are attributed to the sulfonic group’s CH bond
[50]. The peak around 1677 cm−1 could both be attributed to the
C=O [51]. The peak is designated to represent amide I and owns
the most intense peak shift and variation in intensity, which ap-
proves interaction of the polymer structure with protein macro-
molecules [52]. The peak around 1575 could be attributed to
the O–C-O asymmetric stretching vibration, N–H bending, or N–C
stretching band [51]. Peaks from 30 0 0 to 310 0 cm−1 describe aro-
matic =C–H stretching vibration [39,53]. This is while peaks be-
tween 2900 and 3000 are ascribed to non-aromatic C–H stretch-
ing vibration [54]. The peak around 1240 is attributed to the
O–C-O asymmetric stretching vibration [52] related to the ether
group of the polymer. Peak with the wavenumber around 690
cm−1 represents the C-S-C deformation vibration, and the peak
at 832 cm−1 describes C–H deformation vibration band [55]. The
peak at 1144 cm−1 is ascribed to oxygen and sulfur double bond
symmetric stretching vibration [56]. The peak experiences a small
shit in wave number equal to 2 cm−1 . The same shift for asymmet-
ric stretching vibration of oxygen and sulfur double bond was also
observed for the peak around 1320 cm−1 . PAES characteristic peak
is located around 1232 cm−1 . The peak represents C–C-O vibration
in the polymeric matrix [57]. Since there is no displacement of the
wavenumber after interaction with blood, the peak could be cor-
rectly represented as PAES-PVP membrane signature. Peaks within
the wavenumber of 1520 to 1550 belong to amide II (N–H + N–C)
bonds [58]. In the case of protein interaction with the membrane’s
structure, this peak could be displaced to lower wavenumbers of
1500 cm−1 or further. The spectra here do not possess this peak
at all. Shifts for the bonds, including carbon, nitrogen, and oxy-
gen, could represent the presence of organic fouling on the poly-
meric membrane. Here, this was approved by small variations im-
posed on oxygen groups attached to sulfur (peak 1144 cm−1 and
1320 cm−1 ), carbons belonging to aromatic bonds, and, most im-
portantly, peak representing amide I at 1677 cm−1 .
3.3. Solid-state nuclear magnetic resonance (ssNMR)
The NMR spectra related to neat and blood contacted mem-
branes are shown in Fig. 5. Characteristic peaks proving the struc-
ture of PAES-PVP are mainly sulfone index peak (S-C) around
128 ppm, C=O bond around 177 ppm, carbon conjugated double
bonds around 130 and160 ppm [59–61].
Hydrogen bond formation is predicted to be with the hydroxyl
of amine groups. [59]. The higher presence of amine groups and
hydroxyl groups are available in the aqueous environment of the
blood. Accordingly, both hydrogen bonding probabilities could be
considered here. The extent of the displacement and intensity vari-
ation in the peaks, however, is strongly related to the amount of
foulant. As the blood containing membrane sample contained a
low amount of blood proteins (in comparison with the polymer
 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742 9

Table 6
ATR-FTIR spectra of PAES-PVP before and after blood contact.

Variation after
Peak wave blood contact
No. number (cm−1 ) Chemical bond Bond description Intensity
Variation in wave Variation in
number intensity

1 668 S=O Very weak shoulder 0.277643 – 0.001442

deformation vibration
2 698 C–S Very weak shoulder 0.668111 – 0.021121
stretching vibration
3 797 C–S stretching vibration 0.143769 – 0.002302
4 832 C–H Deformation vibration 0.489008 – 0.001204
5 1072 C–H or c-o stretching Deformation vibration 0.379932 – 0.002476
6 1144 O=S=O symmetric stretching 1.22055 2 0.00098
vibration
7 1232 O–C–O Strong asymmetric 0.845202 – 0.013639
stretching vibration
8 1296 C–N or (O=S=O asymmetric stretching stretching vibration 0.348689 - 0.009781
vibration)
9 1320 O=S=O Weak asymmetric 0.258181 2 0.011057
stretching vibration
10 1405 C=C Very weak stretching 0.081202 - 0.002272
vibration
11 1484 C=C Very weak ring vibration 0.729121 - 0.016454
aromatic
12 1575 O–C–O Asymmetric stretching 0.563924 2 0.028642
vibration
13 1677 C=O Stretching vibration 0.034524 22 0.023034
14 2850 C–H Stretching vibration −0.01353 – –
15 2921 C–H Stretching vibration −0.00516 – –
16 3070 C–H Aromatic stretching 0.016023 2 0.007626
vibration
17 3098 C–H Medium aromatic 0.020619 2 0.009485
stretching vibration

Fig. 5. ssNMR spectra of neat and blood contacted PAES-PVP hemodialysis membranes.

content), the shifts are small. Another possibility would be the sta-
ble chemical structure of the membrane in contact with the blood.
Table 7 gives a summary of the peaks and shifts related to ssNMR.
It could be mentioned that the C-S bond, just like two other struc-
tures shown in Table 7, did not experience a peak shift. Accord-
ingly, carbon and sulfur bonds are not broken.
The typical peak variation in ATR-FTIR, Raman and NMR spec-
tra were presented as a result of blood protein attachment. The
C–N bond (1298 cm−1 ) had a slight shift (3) and intensity change
(41,883) in Raman and also, the tertiary amide bonds (798 cm−1 )
shifts along with intensifying variations could be mentioned as
blood particles fouling signs. Intense change in sulfur-oxygen char-
acteristic bond at 370 cm−1 also reflected the protein’s nucleotide
presence over the membrane’s surface. As mentioned in advanced
spectroscopy techniques, this interaction was projected chemically
by displacement of amine shifts and slight changes in symmetric
10 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742

Table 7
ssNMR more important results and shifts in peaks.

PVP/PAES PVP/PAES/Blood

Peak (ppm) Identification Peak (ppm) Shift (ppm)

160 –OC= 161 1

159 –OC= 160 1
137 –CH= 137 N/A
130 –CH=O 130 N/A
122 –SC– 122 N/A

and asymmetric vibration of sulfone and oxygen bonds. There are

different proteins and macromolecular structures in blood with a
vast range of shape and molecular weight, conformational profiles,
and different functional groups and residues. These proteins could
be responsible for variations reflected by spectroscopy analysis.

3.4. Scanning electron microscopy (SEM)

Fig. 6 shows the different magnifications of the neat PAES-

PVP membrane before blood contact at different magnifications.
As seen in the scans, complete microtube (Fig. 6(a)) and conven-
tional surface microstructure of ultrafiltration (Fig. 6 (b and c))
with smooth surface is illustrated.
Fig. 7 offers various clotting and blood particles aggregation il-
lustration over the membrane surface and inside the pores at dif-
ferent magnifications (agglomerated particles in Fig. 7(a) and white
spots on the membrane’s surface in Fig. 7 (b and c). This is, how-
ever, a mild protein attachment to the membrane’s surface. More
intense pore-blocking could occur within the structure of mem-
brane as a result of time pass (later sectors of hemodialysis) or
bioincompatibility affected by the membrane surface chemistry.
Fig. 8 depicts the more intense aggregation and pore-blocking
incidence at different magnifications. Fig. 8(a) shows a cross-
section of microtube completely fouled covered by clots. This
could be observed better in higher magnifications in Fig. 8(b) and
Fig. 8(c). It is believed that the attachment of macromolecules and
followed by that, the middle molecule toxins happens in the pore
structure. Fig. 8 supports clearly supports the idea, and this could
highlight the importance of overall polymeric structure modifica-
tion rather than individual surface chemistry enhancements.

3.5. Transition electron microscopy (TEM)

Fig. 9 shows the TEM cross-section scans of the neat and blood
contacted PAES-PVP hemodialysis membrane. These images clearly
show the porous structure of the membrane. The broader and
more extended support structure of the microtube, which contains
void finger-like channels (exterior surface) and thin microporous
layer (interior surface) are clearly depicted in Fig. 9(a). Fig. 9(b)
illustrates the PAES-PVP membrane with clots being attached to
the reparative surface. The yellow circle indicates an agglomer-
ated clot attached to the membrane. Lighter and more transparent
layer albumin is also covering the clot. This is while the rest of
the surface (yellow rectangles) are covered only with albumin. A
higher magnification, however, indicates small particle attachment
to the surface of the membrane (Fig. 10(a)). More importantly,
Fig. 10 involves the most significant illustration of TEM scans in
this research. Besides the blood-membrane interfacial interactions,
the support layer also experienced attachment of biomolecules. As
identified by yellow circles in Fig. 10(a), white spots, extracellular
vesicles, appeared on and in the pore walls. This is even more fre-
quent in Fig. 10(b). As these were not present in the neat TEM scan
(Fig. 9(a)), the existence of the vesicles could be directly designated
to the blood-polymer interaction. This could be in agreement with
the idea of intra-attachment of biomolecules to the membrane’s
Fig. 6. Different magnifications of neat PAES-PVP membrane surface SEM scans at
different magnifications.
pore walls. The same result was also reported in the SEM section
of this study.
3.6. Molecular dynamics (MD) simulation analysis
The three proteins selected for MD simulations are summarized
in Table 8. Protein structures are commonly produced through the
reaction of amine and carboxyl containing groups which results
in production of polyamide like protein structures and water. The
proteins with higher molecular weights are more likely to have
higher number of functional active sites such as cysteines and sim-
ilar chemical structures which makes the protein more polar. On
the other hand, proteins with lower molecular weight are more
likely to have non-polar structure with less amount of cysteine-
like structure (HSA and TRF). Non-polar structures are more prone
to van der Waals interactions. HSA has a lower molecular weight
 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742 11

Table 8
Average values resulted from different MD simulation between PAES-PVP and different blood proteins.

No. polymer-protein interaction Etotal ∗1 (kcal/mol) EVDW ∗2 (kcal/mol) Eelectrostatic ∗3 (kcal/mol) Ehydrogen bonding ∗4 (kcal/mol) nHB∗5 Ebinding ∗6 (kcal/mol)

1 PAES-PVP-FB 4.27E+06 2.40E+05 2.09E+04 −1.94E+03 1.24E+03 1.04E+04

2 PAES-PVP-HSA 9.73E+04 4.84E+03 1.34E+04 −1.66E+03 1.56E+03 −1.66E+03
3 PAES-PVP-TRF 6.38E+04 7.38E+03 6.20E+03 −1.10E+03 7.92E+02 −7.68E+03
4 FB 4.28E+06 4.03E+06 2.04E+04 −1.99E+03 1.23E+03 N/A
5 HSA 9.23E+04 3.14E+03 1.31E+04 −1.66E+03 1.56E+03 N/A
6 TRF 5.28E+04 7.59E+02 6.24E+03 −1.11E+03 7.72E+02 N/A
7 PAES-PVP 3.31E+03 6.99E+00 4.59E+02 −2.02E-01 6.00E+00 N/A
∗
1: Total energy of the protein-polymer pair; ∗ 2: van der Waals energy; ∗ 3: Electrostatic energy; ∗ 4: energy of hydrogen bonding; ∗ 5: number of hydrogen bonding; ∗ 6
binding energy (Eq. (7)).

Fig. 7. SEM scans of blood particles aggregation over the membrane surface at dif- Fig. 8. SEM scans of blood particle aggregation membrane’s surface and pore-
ferent magnifications. blocking at different magnifications.
12 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742

Fig. 9. TEM image of cross-section scans of the neat and blood contacted PAES-PVP
hemodialysis membrane.

Fig. 11. MDS captures between PAES-PVP and three selected blood proteins; (a)
PAES-PVP-FB, (b) PAES-PVP-HSA, (c) PAES-PVP-TRF.

in comparison with FB. It is worth mentioning that proteins could

Fig. 10. TEM image- higher magnified cross-section scans of blood contacted PAES-
PVP hemodialysis membrane.
have different patterns of interaction with polymeric and inorganic
structures. Interaction studies have reported that FB could perfectly
fit in 1:1 interaction models (Langmuir model), while HSA’s inter-
action with the same nanoparticles could completely differ due to
its different chemical structure and induced conformational varia-
tions [62,63]. Rather than the chemical structure of the proteins,
the interaction between blood proteins and the surfaces is highly
dependent on the surface chemistry of the nanoparticle or poly-
mer. The most important factors were mentioned to be the elec-
trostatic charge and hydrogen bonding. Based on the other re-
search findings, hydrophobic surfaces, would own higher binding
tendency to serum proteins in general (in comparison with hy-
drophilic surfaces with the same nature) [64]. The Isoelectric point
of the polymeric structures are also mentioned to be important as
 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742
Fig. 12. The interaction energy between PAES-PVP and blood proteins.
positively charged surfaces could absorb proteins with isoelectric
point lower than 5.5 while negatively charged surfaces absorb the
proteins with isoelectric point higher than 5.5 [65]. While the sur-
face coatings could control the binding tendencies of the serum
proteins and blood-contact materials, the effect of the base ma-
terial (sublayer) could not be neglected [66–68]. The studies are
oriented around adsorption phenomena. However, the case could
be different when hemocompatibility is the main concern. The be-
havior of each protein could be assessed differently, however, in
reality, the blood contacts the surface with all its protein at once.
Accordingly, the case of hemocompatibility could be different, as
different proteins with various characteristics contact one surface
at the same time.
Each protein has a different number of residues and various
types of active sites which are responsible for the different chemi-
cal behavior and related interactions. While there are several active
residues which are between the three proteins (Tyrosine, Alanine),
there are some active sites that exist in two of the proteins (His-
tidine in TRF and HSA, Phenylalanine in FB and HSA), or there are
active sites which exist specifically in one of the proteins (Threo-
nine in TRF or Methionine in FB). Active sites and different func-
tional groups on the surface of the proteins as well as the active
functional groups on the polymeric chains are responsible for the
interactions between each two protein and polymer. Detailed stud-
ies over the effect of each active site and its behavior are com-
monly conduced in molecular docking simulations and DFT stud-
ies. In this study random MD simulations were performed for each
protein-polymer pair and average values were calculated.
Fig. 11 represents each the MD capture at which the pro-
tein reaches the polymer, while Fig. 12 shows the interaction en-
ergy trends during the performed MD simulations. As it could be
seen interaction with FB is much more than the two other struc-
tures. The overall interaction energy could be related to the size
of the protein structures. As a result of higher bond interactions
(all bond, angle and dihedrals) own a greater value. The same
trend could be mentioned for the two other proteins. A better un-
derstanding could be resulted by comparing the binding energy
values. Table 8 reports the interaction energies of each polymer-
protein pair, as well as the binding energies. The interaction with
FB is much more than the two other structures. The final stable
interaction energy values are equal to 4.27E+06, 9.73E+04 and
6.38E+04 kcal/mol for PAES-PVP-FB, PAES-PVP-HSA and PAES-PVP-
TRF, respectively. The overall interaction energy could be related
13
to the size of the protein structures. As a result of higher bond
interactions (all bond, angle and dihedrals) own a greater value.
The same trend could be mentioned for the two other proteins. A
better understanding could be resulted by comparing the binding
energy values. Binding energy, as calculated by Eq. (7) reflects the
interactive values between the two structures involved in the sim-
ulation. Lower binding energy is interpreted as lower interaction
of the polymeric and protein. Different blood proteins own differ-
ent active sites on the surface, which are responsible for different
chemical interactions with outer foreign surfaces. More intensive
(more negative) value of hydrogen bonding is interpreted in higher
hydrophilicity between two structures in MD simulations [69].
TRF has the lowest binding energy to PAES-PVP
(−7.68E+03 kcal/mol). HSA owns slightly less negative bind-
ing energy in comparison with TRF. HSA has lower van der Waals
interaction and higher electrostatic interaction. Higher electrostatic
energy could be the result of active sites’ polar ends interaction
with the polymeric chain. On the other hand, TRF-PAES-PVP sim-
ulation resulted in higher van der Waals and lower electrostatic
interaction, which could be interpreted into a higher contribution
share of nonpolar species when the two structures were close to
each other. Comparison of the two proteins interaction energies
with PAES-PVP polymeric chain reveals that the interaction is both
a function of polymer and protein characterizations.
FB owns the highest amount of binding energy equal to
1.04E+04 kcal/mol. In comparison with HSA, FB-PAES-PVP owns a
fewer number of hydrogen bonding. However, its hydrogen bond-
ing value is more negative. The correlation of stronger hydrogen
bonding and highest interaction energy, had been previously men-
tioned by Sasongko for PSF and blood constituents (creatinine and
urea) [25] and other researchers for environmental applications of
polymers [27–29]. However, hydrogen bonding could not always be
referenced as the main contributor to the binding energy for blood
proteins and metabolites present in the bloodstream. Bloodstream
could be much more complicated than environmental species, such
as natural organic matters. As found by the same authorship (to be
presented in future studies in the same journal), specific proteins
could, such as FB could act differently, when a conventional modi-
fication, such as hydrophilization (which results in more intensive
hydrogen bonding) and there are proteins which could react dif-
ferently. Hydrogen bonding is affected by the electronegative and
electrostatic characteristics of the polymer itself. The fewer num-
ber of hydrogen bonds and more negative hydrogen bonding en-
14 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742
ergy reflects the fact that the more electronegative atoms were in-
volved in hydrogen bonding interaction of PAES-PVP and FB, which
resulted in more negative final hydrogen bonding value. In com-
parison with HSA-PAES-PVP, higher van der Waals and electro-
static energy were observed for FB-PAES-PVP. However, the extent
of bond interaction in individual structures is so that the binding
energy is significantly more than the two other proteins. This re-
flects the different nature of FB.
Higher interaction and binding energy is a result of active
residues on the proteins’ surface and the polymeric chain. FB owns
a higher number of active residues on its surface in comparison
with the two other proteins. Studies over protein residues-carbon
nanotube interaction reported the simultaneous proportional effect
of residues’ molecular weight and their number of amino acids on
interaction energy [18]. This is along with the finding of the cur-
rent paper as fibrinogen has higher interaction and binding energy
with the polymeric chain.
It’s worth noting that DFT and molecular docking simulations
focus on smaller simulations as they focus on interactions between
small chemical moieties. A recently published paper by our re-
search group reveals some aspects of FB interaction with aryl sul-
fone structure [70]. The electrostatic profile of fibrinogen and PES
(by MOE), reflects that one of the O -atoms of the SO2 group cre-
ates a hydrogen bond with NH of Asn215 B (distance = 2.7 Å). The
other oxygen atom of SO2 indicates a hydrogen bond with NH of
Ser123 B (distance = 3.0 Å). It was stated by Saadati et al. that
Arg215 B residue of the FB and oxygen atoms of the ether struc-
ture in poly aryl sulfone structures are capable of forming hydro-
gen bonds with a distance of 4.3 Å. On the other hand, the phenyl
ring of the middle site is near the phenyl group of Tyr122 B, and in
turn might be viewed as a π -stacking structure interaction. In ad-
dition, other phenyl groups sited in both monomer heads indicate
a hydrophobic interaction with Ala218 B and Phe160 A. The study
approves the findings of the current research as high intensity of
hydrogen bonding occurred between FB and PAES-PVP. While the
idea of comprehending the specific small interactions could ben-
efit the better understanding of polymeric structures-proteins in-
teractions, in reality, the protein approaches the membrane with
several active sites and the interactions could affect each other as
well. The dynamic nature of the process of blood filtration could
also push the proteins toward more interactive sites.
DFT studies suggest that when two structures approach to each
other, the interaction site would be somewhere between the most
positively charged on one structure and the most negative one on
the other structure [25]. While the idea is accurate, in the case
of blood filtration in the hemodialysis process, several blood pro-
teins and metabolites contact the membrane. These structures own
different functional groups with a variety of charge structures. Ac-
cordingly, the idea of specific interaction site assessment could not
completely reveal the extent of blood protein-polymer interactions.
The MD approach used in this paper considers the average inter-
action of several sites in different random simulations and reports
it as an average value. Based on the MD results, higher interaction
and binding energies were observed between the polymer and FB.
The simulation of PAES-PVP-FB also resulted in the strongest hy-
drogen bonding value. The more intense hydrogen bonding is in-
terpreted in a higher affinity of the structure to the membrane and
more facilitated pass through the porous structure of the mem-
brane. This was also mentioned for smaller structures in the blood,
such as urea and creatinine. However, as the proteins are compar-
atively larger than the aforesaid structures, the hydrophilic affinity
of the FB protein structure, along with its higher binding energy
value, could be interpreted to its tendency to attach to the mem-
brane, which could result in the provocation and activation of the
cascades. As the hydrogen bonding intensity decreases (to less neg-
ative values) for PAES-PVP-HSA and PAES-PVP-TRF, the binding en-
ergy decreases. However, the binding energy could not only be at-
tributed to the hydrogen bonding intensity. Comparison HSA and
TRF’s energy shares revealed that TRF-PAES-PVP had higher van
der Waals interaction, which was interpreted in more contribution
of non-polar ends of the protein’s active site in the overall inter-
action. On the other hand, HSA-PAES-PVP had higher electrostatic
interaction, which reflected more contribution of active sites’ polar
end of the protein. Another essential fact to consider is the number
of hydrogen bondings that occurred between the polymer and the
two HSA and TRF proteins. While the intensity of hydrogen bond-
ing was more for FB, the number of hydrogen bonding was more
for TRF and HSA. It was noted that frequent weak hydrogen bonds
(as well as π -π interactions) could afford strong interfacial adhe-
sion of two structures [71]. This means rather than comparing the
binding energies of each protein individually with a polymer, in re-
ality, specific proteins such as TRF and HSA might also bind to the
membrane and provoke the patient’s immune system.
As previously mentioned, several factors could contribute to
the interaction between blood protein structures and polymeric
membrane structures. These factors are listed as (but not limited
to) molecular weight, conformational behavior, molecular shape,
chemical formula and functional groups, specific residue’s behav-
iors, surface charge etc. Further analysis regarding this would be
addressed in separate research. The framework offered here could
be used as a tool for a better understanding of hemodialysis mem-
brane assessments and structural changes when polymers are con-
tacted with blood.
4. Conclusion
As the initial step of continuous research over hemodialysis
membranes, the current study, covers experimental and computa-
tional assessment of PAES-PVP membranes with bloodstream con-
stituents. Experimental assessments reflect the fact that carbon-
nitrogen bond of proteins along with their amide structures. The
finding is aligned with simultaneous academic reports of the same
group with molecular docking which suggest NH structure of the
proteins with the polymer. MD studies reflected that PAES-PVP
structure could have the highest binding energy with FB protein
structure. The hydrogen bonding behavior of the three proteins
was also assessed and discussed. The strongest hydrogen bond-
ing interaction occurred between the aryl sulfone polymer and FB.
More provocation and accordingly higher extent of activation of
FB, could be the interpretation of the higher hydrogen bonding in-
tensity along with maximized binding energy. The experimental-
computational framework offered here in the study could be used
as an initial step for the design and modification of hemodialysis
membranes.
Declaration of Competing Interest
The authors have no conflict of interest to declare.
Acknowledgment
The authors would like to acknowledge and express their grati-
tude to Saskatchewan Health Research Foundation SHRF, the De-
partment of Chemical and Biological Engineering at the Univer-
sity of Saskatchewan and Saskatchewan Transplant Program at St.
Paul’s Hospital for the support provided. The authors would also
like to thank the High-Performance Computing Research Facility
(HPCRF), Saskatchewan Structure Science Centre (SSSC), and West-
ern College of Veterinary Medicine Imagine Centre at the Univer-
sity of Saskatchewan for the advanced services and facilities pro-
vided.
 A. Mollahosseini, S. Argumeedi and A. Abdelrasoul et al. / Computer Methods and Programs in Biomedicine 197 (2020) 105742
Research Ethics
Dr. Amira Abdelrasoul, the principal investigator of the project,
has the Research Ethics Approval approved by the Biomedical Re-
search Ethics Board (Bio-REB), and the Operational Approval to
conduct the research in Saskatchewan Health Authority, in Canada.
Computational Data Availability Statement
The raw/processed data required to reproduce these findings
cannot be shared at this time, as the data is critical of the ongoing
research.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.cmpb.2020.105742.
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