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Citation: Eckelt, A.; Wichmann, F.;
Bayer, F.; Eckelt, J.; Groß, J.; Opatz, T.;
Jurk, K.; Reinhardt, C.; Kiouptsi, K.
Ethyl Hydroxyethyl Cellulose—A
Biocompatible Polymer Carrier in
Blood. Int. J. Mol. Sci. 2022,23, 6432.
https://doi.org/10.3390/ijms23126432
Academic Editor: Isabella Russo
Received: 29 April 2022
Accepted: 7 June 2022
Published: 9 June 2022
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International Journal of
Molecular Sciences
Article
Ethyl Hydroxyethyl Cellulose—A Biocompatible Polymer
Carrier in Blood
Anja Eckelt 1, 2, †, Franziska Wichmann 1,† , Franziska Bayer 1, John Eckelt 2, Jonathan Groß 3, Till Opatz 3,
Kerstin Jurk 1,3,4,‡ , Christoph Reinhardt 1, 4, †,‡ and Klytaimnistra Kiouptsi 1,4,*, ‡
1Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes
Gutenberg-University Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany; eckelta@uni-mainz.de (A.E.);
fwichman@students.uni-mainz.de (F.W.); franziska.bayer@kgu.de (F.B.);
kerstin.jurk@unimedizin-mainz.de (K.J.); christoph.reinhardt@unimedizin-mainz.de (C.R.)
2WEE Solve GmbH, Auf der Burg 6, 55130 Mainz, Germany; john.eckelt@wee-solve.de
3Department of Chemistry, Johannes Gutenberg University, 55099 Mainz, Germany;
jgross03@uni-mainz.de (J.G.); opatz@uni-mainz.de (T.O.)
4German Center for Cardiovascular Research (DZHK), University Medical Center of the Johannes
Gutenberg-University, Mainz Parter Site Rhine-Main, Langenbeckstrasse 1, 55131 Mainz, Germany
*Correspondence: klytaimnistra.kiouptsi@unimedizin-mainz.de
† These authors contributed equally to this work.
‡ These authors contributed equally to this work.
Abstract:
The biocompatibility of carrier nanomaterials in blood is largely hampered by their activat-
ing or inhibiting role on the clotting system, which in many cases prevents safe intravascular applica-
tion. Here, we characterized an aqueous colloidal ethyl hydroxyethyl cellulose (EHEC) solution and
tested its effect on ex vivo clot formation, platelet aggregation, and activation by thromboelastometry,
aggregometry, and flow cytometry. We compared the impact of EHEC solution on platelet aggregation
with biocompatible materials used in transfusion medicine (the plasma expanders gelatin polysucci-
nate and hydroxyethyl starch). We demonstrate that the EHEC solution, in contrast to commercial
products exhibiting Newtonian flow behavior, resembles the shear-thinning behavior of human blood.
Similar to established nanomaterials that are considered biocompatible when added to blood, the
EHEC exposure of resting platelets in platelet-rich plasma does not enhance tissue thromboplastin-
or ellagic acid-induced blood clotting, or platelet aggregation or activation, as measured by integrin
αIIbβ3
activation and P-selectin exposure. Furthermore, the addition of EHEC solution to adenosine
diphosphate (ADP)-stimulated platelet-rich plasma does not affect the platelet aggregation induced
by this agonist. Overall, our results suggest that EHEC may be suitable as a biocompatible carrier
material in blood circulation and for applications in flow-dependent diagnostics.
Keywords: polymer; nanomaterial; ethyl hydroxyethyl cellulose; platelets; plasma expanders
1. Introduction
Nanomaterials can be functionalized to serve as nanocarriers or biomodulators for
therapeutic applications [
1
,
2
]. One possible application is their use in targeted cancer
therapy [
3
,
4
]. In addition to the non-toxic nature of nanomaterials, an essential requirement
for the safety of nanomaterial therapeutics in human blood circulation is that they do
not change the rheological properties of blood plasma and neither activate nor inhibit
the clotting system. For example, amphiphilic macromolecule assemblies consisting of a
hydrophilic polyethylene glycol tail and a branched hydrophobic head suppress platelet
adhesion, a property favorable in the application in drug-eluting stents [
5
]. In contrast,
silica nanomaterials, depending on their protein corona, have the capacity to support
platelet activation [6,7].
The role of nanoparticles in the blood circulation is strongly dependent on their surface
properties and the chemistry of the nanomaterial [
8
]. Silica nanoparticles, for example,
Int. J. Mol. Sci. 2022,23, 6432. https://doi.org/10.3390/ijms23126432 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022,23, 6432 2 of 11
disturb the vascular barrier, and are therefore unsuitable for intravascular applications.
They down-regulate endothelial junction proteins, thus disturbing the blood–brain bar-
rier [
9
]. Furthermore, they disrupt VE–cadherin interactions, inducing endothelial leakages
that support cancer extravasation and metastasis [
10
]. Silica nanoparticles interact with
red blood cells [
11
] and have been found to promote platelet microaggregation [
12
–
14
].
Although the interactions of silica nanoparticles with blood cells are largely unfavorable,
the effects of other nanomaterials on human platelets remain ill-defined.
The interference of nanomaterials with platelets and the resulting enhancement of
platelet aggregation behavior may strongly hamper their use in therapeutic applications,
blood analytics, and diagnostics [
15
]. The use of nanomaterials as carriers in the blood-
stream requires that these materials minimally interfere with the clotting system. This
requirement is, for instance, relatively well met by plasma expanders such as gelatin poly-
succinate (Gelafusal
®
) and hydroxyethyl starch (HES) (Volulyte
®
or Vitafusal
®
), which are
routinely used in clinics to increase oncotic pressure to prevent shock caused by severe
blood loss [
16
]. In particular, gelatin solutions (Gelafusal
®
) have been shown to have
minimal effects on blood clotting in whole blood thromboelastography analyses [
17
]. In
addition, studies on platelet activation have demonstrated that HES affects platelet activa-
tion or aggregation at resting and activating conditions [
18
–
20
]. However, to functionalize
nanomaterials as carriers, new nanomaterials are needed that ideally reflect the rheological
properties of human blood without activating the hemostatic system [
21
]. Our goal is to
examine whether ethyl hydroxyethyl cellulose affects ex vivo clot formation and platelet
function, thus considering it as a potential candidate to substitute the plasma expanders
currently in clinical use.
2. Results
Comparing ethyl hydroxyethyl cellulose (EHEC) with other biopolymers with ap-
plication in the blood circulation (Gelafusal
®
, Volulyte
®
, and Vitafusal
®
) (Table 1), we
here propose the use of EHEC as a biocompatible material that does not interfere with the
clotting system while resembling the rheological properties of human blood. The aim of
our study was to investigate whether EHEC solutions have a blood-like rheology profile
and whether they affect clot formation and platelet aggregation behavior. This is critical
because any effect on those parameters will likely preclude their use as a biocompatible
carrier material for future intravascular therapeutic applications.
Table 1. Composition of various plasma expander solutions.
Gelafusal®Volulyte®Vitafusal®
w (g) in 1000 mL Compound w (g) in 1000 mL Compound w (g) in 1000 mL Compound
40.000 Gelatine polysuccinate 60.000 Hydroxyethyl starch 60.000 Poly(O-2-hydroxyethyl) starch
3.675 Sodium acetate trihydrate 4.630 Sodium acetate trihydrate 3.700 Sodium acetate trihydrate
4.590 Sodium chloride 6.020 Sodium chloride 6.000 Sodium chloride
0.403 Potassium chloride 0.300 Potassium chloride 0.400 Potassium chloride
0.133 Calcium chloridedihydrate 0.300 Magnesium chloride
hexahydrate 0.134 Calcium chloride dihydrate
0.203 Magnesium chloride
hexahydrate 0.200 Magnesium chloride
hexahydrate
Polymers are characterized by their molar mass distribution. The molar mass distribu-
tion of EHEC, quantified by size exclusion chromatography, is shown in Figure 1A. The
viscosity of human blood exhibits a shear-thinning behavior, i.e., the viscosity decreases
with increasing shear rate (Figure 1B). This property can be well mimicked by EHEC solu-
tions. In contrast, established blood plasma expanders exhibit Newtonian behavior, i.e.,
constant viscosity with changing shear rates. Consequently, the velocity of EHEC solutions
is comparable to that of human blood, even if the diameter of the blood vessel changes.
Int. J. Mol. Sci. 2022,23, 6432 3 of 11
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 11
Figure 1. Characterization of ethyl hydroxyethyl cellulose. (A) Molar mass distribution. (B) Flow
curves of blood (black) and the different biocompatibles: EHEC (blue), Volulyte (yellow), Gelafusal
(purple), and Vitafusal (red). (C) Clotting time and (D) clot formation time of whole blood and
whole blood diluted with different NaCl (vehicle) and EHEC concentrations (n = 5). Coagulation
Figure 1.
Characterization of ethyl hydroxyethyl cellulose. (
A
) Molar mass distribution. (
B
) Flow
curves of blood (black) and the different biocompatibles: EHEC (blue), Volulyte (yellow), Gelafusal
(purple), and Vitafusal (red). (
C
) Clotting time and (
D
) clot formation time of whole blood and whole
Int. J. Mol. Sci. 2022,23, 6432 4 of 11
blood diluted with different NaCl (vehicle) and EHEC concentrations (n= 5). Coagulation was
initiated by tissue thromboplastin. (
E
,
F
). represent rotational thromboelastometry (ROTEM) graphs
of whole blood diluted with 5% NaCl (
E
) and 5% EHEC (
F
) activated by tissue thromboplastin
(EXTEM). Clotting time appears in green and clot formation time in pink. (
G
) Clotting time and
(
H
) clot formation time of whole blood and whole blood diluted with different NaCl (vehicle) and
EHEC concentrations (2.5%, 5% and 10%) (n= 5). Coagulation was initiated by ellagic acid (INTEM).
(
I
,
J
) represent rotational thromboelastometry (ROTEM) graphs of whole blood diluted with 5% NaCl
(
I
) and EHEC (
J
) activated by ellagic acid (INTEM). Clotting time appears in green and clot formation
time in pink. Black circle: whole blood, white square: whole blood+NaCl, black square: whole
blood+EHEC. All data are expressed as the means
±
SEM. Statistical comparisons were performed
using a one-way ANOVA. * p< 0.05, ** p< 0.01, *** p< 0.001, n.s not significant.
Rotational thromboelastometry (ROTEM) is widely used to rapidly assess the vis-
coelastic changes that occur during clotting and is an essential part of patient blood man-
agement [
22
]. Here, we used two different tests to trigger coagulation: EXTEM, in which
coagulation is initiated by a reagent containing tissue thromboplastin (tissue factor), and
INTEM, in which clot formation is triggered by ellagic acid. Whole blood with different
concentrations of EHEC or the vehicle (NaCl) (2.5%, 5%, and 10%) did not enhance tis-
sue thromboplastin-induced clot formation (Figure 1C–F). The supplementation of whole
blood with 10% EHEC even resulted in prolonged clot formation time when compared
with either whole blood or whole blood with the vehicle (Figure 1D). Similar observations
were made for the initiation of clotting by the contact phase (INTEM) (Figure 1G–J). Thus,
supplementation with EHEC does not enhance clot formation in whole blood but rather
prolongs clotting at higher concentrations.
Since the exposure of human platelet-rich plasma (PRP) to certain nanomaterials is
known to result in quantitative platelet aggregation, we exposed EHEC solution (
0.38% w/w
)
to PRP and performed light transmission aggregometry experiments. Because the addition
of EHEC was comparable to the addition of physiological NaCl solution (0.9% w/w), we did
not detect a significant increase in platelet aggregation under resting conditions (Figure 2A).
Integrin
αIIbβ3
is the major platelet receptor responsible for fibrinogen bridge forma-
tion and subsequent platelet aggregation. Platelet activation induces a conformational
change in integrin
αIIbβ3
, which can be detected by flow cytometric analysis using the
antibody clone PAC-1 [
23
]. To test whether exposure to EHEC triggers platelet activation,
we analyzed the activation state of
αIIbβ3
and the cell surface exposure of the activation
marker P-selectin in platelets exposed to different concentrations of EHEC. Consistent with
the aggregometry data (Figure 2A), platelet exposure to different EHEC concentrations
did not result in platelet integrin
αIIbβ3
activation (Figure 2B) or the surface exposure of
P-selectin (Figure 2C).
To exclude the possibility that EHEC materials prevent or stimulate agonist-induced
platelet aggregation, we next compared EHEC solution in PRP that was activated with the
platelet agonist adenosine diphosphate (ADP), which promotes human platelet aggregation
via the G protein-coupled receptor P2Y12. Importantly, in the dynamic range of ADP
concentration, we did not detect an altered ADP-triggered aggregation response when
EHEC was present in PRP (Figure 3A). We also did not detect differences in platelet
response after incubation with different EHEC concentrations when stimulated with a
threshold concentration of ADP that resulted in secondary platelet activation (Figure 3B).
We applied the same experimental setup in flow cytometry to test whether the exposure of
the platelets to different EHEC concentrations would lead to increased ADP-stimulated
αIIbβ3
activation (Figure 3C) and surface exposure of the activation marker P-selectin
(Figure 3D). Collectively, our results demonstrate that EHEC exposure had no effect on
ADP-stimulated platelet activation.
Int. J. Mol. Sci. 2022,23, 6432 5 of 11
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 11
was initiated by tissue thromboplastin. (E,F). represent rotational thromboelastometry (ROTEM)
graphs of whole blood diluted with 5% NaCl (E) and 5% EHEC (F) activated by tissue thromboplas-
tin (EXTEM). Clotting time appears in green and clot formation time in pink. (G) Clotting time and
(H) clot formation time of whole blood and whole blood diluted with different NaCl (vehicle) and
EHEC concentrations (2.5%, 5% and 10%) (n = 5). Coagulation was initiated by ellagic acid (INTEM).
(I,J) represent rotational thromboelastometry (ROTEM) graphs of whole blood diluted with 5%
NaCl (I) and EHEC (J) activated by ellagic acid (INTEM). Clotting time appears in green and clot
formation time in pink. Black circle: whole blood, white square: whole blood+NaCl, black square:
whole blood+EHEC. All data are expressed as the means ± SEM. Statistical comparisons were per-
formed using a one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s not significant.
Figure 2. The effect of ethyl hydroxyethyl cellulose (EHEC) on platelet function under resting con-
ditions. (A) Aggregometry histograms and representative curves of resting platelets (black) and
platelets incubated with EHEC (blue) or NaCl (grey) (n = 10). Mean fluorescence intensity of (B)
FITC-conjugated PAC-1 antibody and (C) APC-conjugated P-selectin antibody of unstimulated
platelets or platelets exposed to different EHEC concentrations (the shades represent the concentra-
tion increase) (n = 5). All data are expressed as the means ± SEM. Statistical comparisons were per-
formed using a one-way ANOVA. *** p < 0.001, **** p < 0.0001, n.s not significant.
Figure 2.
The effect of ethyl hydroxyethyl cellulose (EHEC) on platelet function under resting
conditions. (
A
) Aggregometry histograms and representative curves of resting platelets (black) and
platelets incubated with EHEC (blue) or NaCl (grey) (n= 10). Mean fluorescence intensity of (
B
) FITC-
conjugated PAC-1 antibody and (
C
) APC-conjugated P-selectin antibody of unstimulated platelets or
platelets exposed to different EHEC concentrations (the shades represent the concentration increase)
(n= 5). All data are expressed as the means
±
SEM. Statistical comparisons were performed using a
one-way ANOVA. *** p< 0.001, **** p< 0.0001, n.s not significant.
To test whether the biocompatibility of EHEC solution was similar to materials al-
ready in clinical use as plasma expander solutions (Gelfusal
®
, Volulyte
®
, Vitafusal
®
), we
compared the EHEC solution to these solutions in the PRP of the same donor. Indeed,
the EHEC solution was comparable to Gelfusal
®
and Vitafusal
®
and, as expected, EHEC
showed a reduced platelet aggregation response at resting conditions relative to Volulyte
®
(
Figure 4A) [24]
. Furthermore, when comparing the potential role of EHEC to other clin-
ically applicable materials (i.e., Gelfusal
®
, Volulyte
®
, Vitafusal
®
) in ADP-activated PRP,
there was neither an impairment nor an enhancement of ADP-induced platelet aggre-
gation (Figure 4B). Altogether, these data imply that EHEC does not interfere with the
physiological platelet aggregation response of the hemostatic system.
Int. J. Mol. Sci. 2022,23, 6432 6 of 11
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 11
Figure 3. The effect of ethyl hydroxyethyl cellulose (EHEC) on ADP-stimulated platelets. (A) Ag-
gregometry histograms and representative curves of ADP-stimulated platelets with high ADP con-
centrations (green), resting platelets incubated with EHEC (blue) and ADP-stimulated platelets in-
cubated with EHEC (dark blue) (n = 10). (B) Aggregometry histograms and representative curves of
ADP-stimulated platelets with threshold ADP concentrations (green) and ADP-stimulated platelets
Figure 3.
The effect of ethyl hydroxyethyl cellulose (EHEC) on ADP-stimulated platelets. (
A
) Aggregom-
etry histograms and representative curves of ADP-stimulated platelets with high ADP concentrations
(green), resting platelets incubated with EHEC (blue) and ADP-stimulated platelets incubated with
EHEC (dark blue) (n= 10). (
B
) Aggregometry histograms and representative curves of ADP-stimulated
Int. J. Mol. Sci. 2022,23, 6432 7 of 11
platelets with threshold ADP concentrations (green) and ADP-stimulated platelets incubated with
different EHEC concentrations (the shades represent the concentration increase). (
C
) Mean fluores-
cence intensity of the FITC-conjugated PAC-1 antibody and (
D
) APC-conjugated P-selectin antibody
of ADP-stimulated platelets incubated without or with different EHEC concentrations (the shades
represent the concentration increase) (n= 5). All data are expressed as means
±
SEM. Statistical
comparisons were performed using a one-way ANOVA. **** p< 0.0001, n.s not significant.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 11
incubated with different EHEC concentrations (the shades represent the concentration increase). (C)
Mean fluorescence intensity of the FITC-conjugated PAC-1 antibody and (D) APC-conjugated P-
selectin antibody of ADP-stimulated platelets incubated without or with different EHEC concentra-
tions (the shades represent the concentration increase) (n = 5). All data are expressed as means ±
SEM. Statistical comparisons were performed using a one-way ANOVA. **** p < 0.0001, n.s not sig-
nificant.
To test whether the biocompatibility of EHEC solution was similar to materials al-
ready in clinical use as plasma expander solutions (Gelfusal®, Volulyte®, Vitafusal®), we
compared the EHEC solution to these solutions in the PRP of the same donor. Indeed, the
EHEC solution was comparable to Gelfusal® and Vitafusal® and, as expected, EHEC
showed a reduced platelet aggregation response at resting conditions relative to Volulyte®
(Figure 4A) [24]. Furthermore, when comparing the potential role of EHEC to other clini-
cally applicable materials (i.e., Gelfusal®, Volulyte®, Vitafusal®) in ADP-activated PRP,
there was neither an impairment nor an enhancement of ADP-induced platelet aggrega-
tion (Figure 4B). Altogether, these data imply that EHEC does not interfere with the phys-
iological platelet aggregation response of the hemostatic system.
As EHEC did not interfere with platelet aggregation ex vivo, our data argue for the
applicability of EHEC solution as a biocompatible material for human blood plasma.
Figure 4. Comparison between ethyl hydroxyethyl cellulose (EHEC) and commercial plasma ex-
pander solutions on platelet aggregation. Aggregometry histograms and representative curves of
(A) resting platelets (black) and platelets incubated with EHEC (blue), Gelafusal (purple), Volulyte
(yellow), and Vitafusal (red) (n = 10). (B) ADP-stimulated platelets (green) and platelets stimulated
with ADP and exposed to EHEC (dark blue), Gelafusal (dark purple), Volulyte (brown) or Vitafusal
(dark red) (n = 10). All data are expressed as the means ± SEM. Statistical comparisons were per-
formed using the one-way ANOVA. ** p < 0.01, *** p < 0.001, n.s not significant.
Figure 4.
Comparison between ethyl hydroxyethyl cellulose (EHEC) and commercial plasma ex-
pander solutions on platelet aggregation. Aggregometry histograms and representative curves of
(
A
) resting platelets (black) and platelets incubated with EHEC (blue), Gelafusal (purple), Volulyte
(yellow), and Vitafusal (red) (n= 10). (
B
) ADP-stimulated platelets (green) and platelets stimulated
with ADP and exposed to EHEC (dark blue), Gelafusal (dark purple), Volulyte (brown) or Vita-
fusal (dark red) (n= 10). All data are expressed as the means
±
SEM. Statistical comparisons were
performed using the one-way ANOVA. ** p< 0.01, *** p< 0.001, n.s not significant.
As EHEC did not interfere with platelet aggregation ex vivo, our data argue for the
applicability of EHEC solution as a biocompatible material for human blood plasma.
3. Discussion
Rotational thromboelastometry experiments showed that the supplementation of
blood with different EHEC concentrations does not enhance ex vivo clot formation. This is
in contrast to gelatin-based plasma expanders, which lead to hypercoagulability [
24
]. Based
on platelet aggregometry and flow cytometry analyses with PRP, we conclude that EHEC
does not significantly activate or interfere with platelet function, which is an important
requirement for its potential therapeutic application in the circulation. This is in contrast
to existing polymer-based nanomaterials used in blood, which may have unfavorable
Int. J. Mol. Sci. 2022,23, 6432 8 of 11
properties that affect the clotting system, especially in long-term applications, therefore
virtually precluding their use as nanocarriers [
25
,
26
]. For instance, HES has been described
to enhance fibrinolysis by diminishing the inhibition of plasmin by its serpin inhibitor
α
2-antiplasmin [
27
]. In an ex vivo whole blood thromboelastometry analysis, HES was
demonstrated to have an anticoagulant effect depending on the dilution ratio [
25
], which
may be due to a decay in factor VIII levels [
25
]. Moreover, HES was shown to impair
collagen and epinephrine-induced platelet aggregation in PRP [
28
]. In clinical use, HES
infusion was found to cause a slight but significant decrease in platelet count.
Gelatin-based plasma substitutes also have adverse effects on the clotting system.
Gelatin-based colloidal solutions reduce the clotting capacity of fresh blood, resulting in
clots with decreased weight, mean shear modulus, and increased bleeding times [
29
,
30
].
The analysis of these clots with scanning electron microscopy revealed a less extensive
fibrin mesh [
29
]. Our analyses also show increased clotting times and clot formation times,
but only at the highest EHEC concentrations (10% v/v). In addition to the effects of gelatin
on the coagulation cascade, velocity and ristocetin-induced platelet agglutination were
decreased and thrombin–antithrombin complexes were reduced [
30
]. Taken together, this
highlights the need for new nanomaterials such as EHEC that are inert to platelet function,
i.e., platelet aggregation.
As shown by shear analysis, another favorable property of EHEC nanomaterials for
use as biocompatible carrier polymers in the blood circulation, which clearly distinguishes
this material from other biocompatible polymers, is that the rheological properties of EHEC
solutions resemble the non-Newtonian rheological profile of human blood. This could
enable its use in various medical and analytical applications that require blood-borne
particles. The topical application of EHEC has previously been proposed as a potential
delivery system for local anesthetics [
31
]. Our results suggest that intravascular
in vivo
applications of EHEC-based materials should be investigated, and may turn out to be
useful for targeted chemotherapy approaches to combat tumor growth.
4. Materials and Methods
4.1. Ethyl Hydroxyethyl Cellulose (EHEC)
Ethyl hydroxylethyl cellulose (EHEC)—the commercial product BERMOCOLL EHM
200 (Akzo Nobel Functional Chemicals AB, Stenungsund, Sweden)—was fractionated by
means of a liquid–liquid phase separation. Water was used as solvent, and acetone was
used as non-solvent. At the end, the EHEC had an apparent weight average molar mass
Mn* of 14,700 g/moL, an apparent weight average molar mass Mw* of 492,000 g/mol, and
a dispersity Ð of 33, analyzed by means of size exclusion chromatography (SEC). To prepare
the final EHEC solution, the fractionated polymer was diluted with a concentration of
0.38 % (w/w) in isotonic NaCl solution. The fractionation and preparation were performed
at the WEE-Solve GmbH.
4.2. Size-Exclusion Chromatography (SEC)
The molecular mass distribution of EHEC was determined by means of size-exclusion
chromatography (SEC) using a refractive index detector (RI-71, Shodex, Munich, Germany).
The columns HEMA Bio 40, HEMA Bio 1000, and Suprema 3000 (PSS Polymer Standards
GmbH, Mainz, Germany) were used, and the determination was performed in salt solution
(0.05 mol NaHCO
3
and 0.1 mol NaNO
3
in water) as eluent. The calibration curve was
performed with dextran standards (PSS Polymer Standards GmbH, Mainz, Germany). The
analysis was performed at the WEE-Solve GmbH.
4.3. Rheology Studies
Flow curves were determined by means of an air-bearing rotational rheometer
UDS 200 (Anton Paar, Graz, Austria) in combination with a Paar Physica Viscotherm
V2 thermostat. Z1 cylinder geometry (double gab) was used. 22 mL of the sample was
filled inside the lower cylinder without filtration. The upper cylinder was lowered into the
Int. J. Mol. Sci. 2022,23, 6432 9 of 11
measuring position, surplus material was removed, the samples were allowed to equilibrate
to 37
◦
C for 5 min, and the measurement was started. Measurement profile: shear rate
decreasing from 300 to 10 1/s (for volulyte 6%, gelafusal 4%, and vitafusal 6%, respectively)
and 1000 to 10 1/s (for human blood and EHEC) (5 data points per decade; measuring time:
logarithmic increasing from 1 to 30 s).
4.4. Blood Collection
Whole blood anticoagulated with tri-sodium citrate (109 mM, 1:10) from healthy
donors was collected from the antecubital area after informed consent was retrieved
according to our institutional guidelines and the Declaration of Helsinki.
4.5. Rotational Thromboelastometry (ROTEM)
For the reconstitution of the clotting by the extrinsic and the intrinsic pathway, 300
µ
L
whole blood was added to single-use reagent EX-TEM S or IN-TEM S according to the
manufacturer’s instructions (Werfen GmbH, Munich, Germany). For monitoring the effect
of EHEC on the system, different amounts of 0.38% w/wEHEC solution were added to
300
µ
l whole blood to obtain final concentrations of 96.25
µ
g/mL (5% v/v), 192.5
µ
g/mL
(10% v/v), and 385
µ
g/mL (20% v/v). Additionally equal volumes of isotonic NaCl solution
(154 mM) were used as vehicles. Clotting time and clot formation time were measured by a
whole blood hemostasis analyzer (ROTEM delta, Tem GmbH, Munich, Germany).
4.6. Aggregometry
Whole blood was centrifuged at 200
×
gfor 10 min at room temperature (RT). The
platelet-rich plasma (PRP) was collected and adjusted to 2
×
108 cells/mL with HEPES
buffer (150 mM NaCl, 5 mM KCl, 1 mM MgCl 2, 10 mM D-glucose, 10mM HEPES, pH 7.4).
Then, 500
µ
L of PRP were centrifuged at 2000
×
gfor 10 min at RT to acquire platelet-poor
plasma, which was used as a reference. To examine the nanomaterial effect in PRP, 20
µ
L of
each were added to 180
µ
L PRP, corresponding to final concentrations of 385
µ
g/mL EHEC,
6 mg/mL Volulyte, 4 mg/mL Gelafusal, and 6 mg/mL Vitafusal. Equal volumes of isotonic
NaCl solution (154 mM) were used as vehicles. Where indicated, platelets were stimulated
with 5
µ
M ADP to achieve an 80% aggregation response (Hart biologicals, Hartlepool,
UK). The platelet aggregation was quantified by light transmission aggregometry using an
APACT 4S Plus aggregometer (Diasys Greiner, Holzheim, Germany).
4.7. Flow Cytometry
Platelet-rich plasma (PRP) was diluted 1:5 with HEPES buffer (150 mM NaCl, 5 mM
KCl, 1 mM MgCl 2, 10 mM D-glucose, 10 mM HEPES, pH 7.4). To investigate the effect of
the EHEC in PRP, different amounts of 0.38% w/wEHEC solution were added to diluted
PRP to obtain final concentrations of 96.25
µ
g/mL, 192.5
µ
g/mL, and 385
µ
g/mL. The
samples were incubated with 5
µ
M ADP (Merck KGaA, Darmstadt, Germany) for 5 min at
room temperature. To examine the influence of the nanomaterial in PRP without activation
by ADP, mixtures of diluted PRP with 0.38% w/wEHEC solution were prepared in the
same manner without activation by ADP. Activated platelets and non-activated PRP/EHEC
mixtures were stained with APC (allophycocyanin)-labeled mouse anti-human CD62P
(P-Selectin) (BD Biosciences, San Jose, CA, USA) with FITC (fluorescein isothiocyanate)-
labeled mouse anti-human PAC-1 (BD Biosciences, San Jose, CA, USA) for 15 min at
room temperature. Then, 1ml HEPES buffer was added to the samples and the samples
were analyzed immediately on the flow cytometer. Per sample, at least 10,000 events
were collected. As controls, unstimulated samples were used from the same donors. For
the study, a BD FACSCanto II flow cytometer with BD FACSDiva software (v6.1.3, BD
Biosciences, Heidelberg, Germany) was applied. The histograms were generated using
FlowJo V10 software (FlowJo LLC, Ashland, OR, USA).
Int. J. Mol. Sci. 2022,23, 6432 10 of 11
5. Patents
EP000003072502A1, 25.03.15, “Rheological blood replacement solution and uses thereof”.
Author Contributions:
Conceptualization, C.R. and K.K.; methodology, F.B., K.J. and K.K.; validation,
A.E. and K.K.; formal analysis, A.E., J.G. and K.K.; investigation, A.E., F.W., T.O. and K.K.; resources,
C.R.; data curation, A.E., K.K. and C.R.; writing—original draft preparation, K.K. and C.R.; writing—
review and editing A.E., F.B., K.J., K.K. and C.R.; visualization, A.E., T.O. and K.K.; supervision, K.J.,
K.K. and C.R.; project administration, C.R.; funding acquisition, J.E. and C.R. All authors have read
and agreed to the published version of the manuscript.
Funding:
The project was funded by a WIPANO project grant of the German Ministry of Economic
Affairs and Energy (BMWi; 03THW13L07) to C.R. C.R. was awarded a Fellowship of the Gutenberg
Research College at the Johannes Gutenberg-University of Mainz. K.J, K.K. and C.R. are members of
the DZHK. A.E., K.K., F.W., F.B., J.G., T.O, K.J. and C.R. declare that no competing interests exist.
Institutional Review Board Statement:
The study was conducted in accordance with the Decla-
ration of Helsinki, and approved by the local institutional ethics committee (Ethik-Kommission,
Landesärztekammer Rheinland-Pfalz, Mainz, 837.602.12/2018-13290_1).
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Not applicable.
Conflicts of Interest:
J.E. is the CEO of WEE-Solve GmbH. He is the inventor of the above mentioned
patent, and holds a royalty agreement on the tested EHEC solution. He was solely involved in
funding acquisition. All the other authors declare no conflict of interest.
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