molecules
Article
The Influence of pH on the Scleroglucan and
Scleroglucan/Borax Systems
Claudia Mazzuca 1 , Gianfranco Bocchinfuso 1 , Antonio Palleschi 1 , Paolo Conflitti 1 ,
Mario Grassi 2 , Chiara Di Meo 3 , Franco Alhaique 3 and Tommasina Coviello 3, *
1
2
3
*
Department of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, Via della Ricerca
Scientifica, 00133 Rome, Italy; [email protected] (C.M.);
[email protected] (G.B.); [email protected] (A.P.);
[email protected] (P.C.)
Department of Engineering and Architecture, University of Trieste, P.le Europa 1, 34127 Trieste, Italy;
[email protected]
Department of Drug Chemistry and Technologies, “Sapienza”, University of Rome, P.le Aldo Moro 5,
00185 Rome, Italy; [email protected] (C.D.M.); [email protected] (F.A.)
Correspondence: [email protected]; Tel.: +39-6-4991-3557
Academic Editor: Derek J. McPhee
Received: 9 January 2017; Accepted: 6 March 2017; Published: 9 March 2017
Abstract: The effects that an increase of environmental pH has on the triple helix of scleroglucan (Sclg)
and on the Sclg/borax hydrogel are reported. Rheological experiments show that the hydrogel is less
sensitive to pH increase than Sclg alone, while at pH = 14 a dramatic viscosity decrease takes place for
both systems. This effect is evidenced also by the reduced water uptake and anisotropic elongation
detected, at pH = 14, by the swelling behaviour of tablets prepared with the Sclg/borax system.
On the opposite, a different behaviour was observed with guar gum and locust bean gum tablets,
tested as reference polysaccharides. The effect of pH on the structure of Sclg and Sclg/borax was
investigated also by means of spectroscopic approaches based on the interaction between Congo red
(CR) and the Sclg triple helix. Obtained results indicated that the CR absorbance maximum is shifted
as a function of pH and by the presence of borax. Principal component analysis allowed very precise
identification of the pH value at which the Sclg helix collapses. Molecular dynamics simulations of
the Sclg/borax–CR complex indicated that, at physiological pH, only a few ordered configurations
are populated, according to the induced circular dichroism (CD) spectrum evidence.
Keywords: scleroglucan; rheology; swelling; Congo red; molecular dynamics simulations; principal
component analysis
1. Introduction
Scleroglucan (Sclg) is a natural polysaccharide produced by fungi of the genus Sclerotium,
extensively studied for various commercial applications (secondary oil recovery, ceramic glazes,
food, paints, cosmetics, etc.) and also for its interesting pharmaceutical applications [1–3]. The polymer
exhibits a backbone build up by (1→3) linked β-D-glucopyranose units with single glucopyranose
side chains linked β-(1→6) (Scheme 1). From a structural point of view, Sclg dissolves in water with
a triple-helix conformation sustained by a network of interchain H-bonds among the hydroxyl groups
linked to the C-2 atoms of the glucose units of the backbone. This H-bond network is responsible for
the high stiffness shown by the polymer.
Furthermore, Sclg, just like other polysaccharides [4], is capable of interacting with borax,
leading to complex systems in which chemical bonds and electrostatic interactions are both involved [5].
In particular, in the case of Sclg, the presence of borax allows the formation of domains where
Molecules 2017, 22, 435; doi:10.3390/molecules22030435
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Molecules 2017, 22, 435
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the interactions among several triple helices (triplexes) of the polysaccharide lead to a hydrogel
network with specific and peculiar properties [6] such as a remarkable anisotropic swelling of tablets,
prepared from the freeze-dried hydrogel [7]. This effect was interpreted as the consequence of
the particular structure present at a microscopic level, enhanced by the compression force applied
Molecules 2017, 22, 435
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during tablet preparation. The presence, within the network, of soft nanochannels capable of playing
with specific
properties
[6] suchwas
as a suggested
remarkable by
anisotropic
swelling
of tablets,
a fundamental
roleand
in peculiar
this unusual
behaviour
means of
molecular
dynamics
prepared from[8].
the freeze-dried
hydrogel
This effect
was interpreted
as the consequence
of the
(MD) simulations
Furthermore,
NMR[7].
studies
evidenced
a mechanism
of hyperdiffusion
of
particular
structure
present
at
a
microscopic
level,
enhanced
by
the
compression
force
applied
during
water molecules along one direction, thus supporting the hypothesis of the presence of well-ordered
tablet preparation. The presence, within the network, of soft nanochannels capable of playing a
nanostructures [9]. These results provided appropriate explanations for the behaviour of Sclg in the
fundamental role in this unusual behaviour was suggested by means of molecular dynamics (MD)
varioussimulations
applications
that have been reported, especially in the field of pharmaceutics [1]. Taking into
[8]. Furthermore, NMR studies evidenced a mechanism of hyperdiffusion of water
accountmolecules
that the Sclg
helix breaks
at pH higher
than 13,ofthe
of the of
present
study was to
alongtriple
one direction,
thusdown
supporting
the hypothesis
theaim
presence
well-ordered
investigate
the effect [9].
thatThese
an increase
of pH may
have on explanations
the overall structure
of the Sclg/borax
nanostructures
results provided
appropriate
for the behaviour
of Sclg in thesystem.
various applications
thatpH
have
reported,
especially inswelling
the field ofofpharmaceutics
Taking
intotested.
In particular,
the effect of
= been
14 on
the anisotropic
Sclg/borax[1].
tablets
was
account
that
the
Sclg
triple
helix
breaks
down
at
pH
higher
than
13,
the
aim
of
the
present
study
was
For this purpose, two approaches were followed: in one case, the tablets were prepared from the
to dissolved
investigate the
effect
that ansolution
increase (pH
of pH= may
havethen
on the
overall in
structure
of the
Sclg/borax
polymer
in an
alkaline
14) and
swelled
distilled
water;
in the other
system. In particular, the effect of pH = 14 on the anisotropic swelling of Sclg/borax tablets was tested.
case, the polymer was first dissolved in water and the prepared tablets were then swelled in the alkaline
For this purpose, two approaches were followed: in one case, the tablets were prepared from the
solution
(pH = dissolved
14). Taking
into
account
that at
pH= 14
helix ofinSclg
is nowater;
longerinpresent
[10–12],
polymer
in an
alkaline
solution
(pH
14)the
andtriple
then swelled
distilled
the other
for an appropriate
comparison,
tablets
with
guar
gum
(GG)
and
locust
bean
gum
(LBG)
(Scheme
case, the polymer was first dissolved in water and the prepared tablets were then swelled in the1) were
also prepared
tested
in=the
environmental
conditions.
alkaline and
solution
(pH
14).same
Taking
into account that
at pH 14 GG
the and
tripleLBG
helixare
of neutral,
Sclg is nowater-soluble
longer
present [10–12],
for an appropriate
guar gum
(GG) and locust bean
gum
galactomannans
consisting
of a linear comparison,
backbone oftablets
β(1→with
4)-linked
D -mannopyranose
units
with the
(LBG)
(Scheme
1)
were
also
prepared
and
tested
in
the
same
environmental
conditions.
GG
and
LBG
presence of randomly attached α(1→6)-linked galactopyranose units as side chains. GG and LBG,
water-soluble galactomannans consisting of a linear backbone of β(1→4)-linked
unlike are
Sclg,neutral,
dissolve
in aqueous solution as random coils. These two polysaccharides were chosen
D-mannopyranose units with the presence of randomly attached α(1→6)-linked galactopyranose
because, according to previous results, GG/borax and LBG/borax tablets also showed an anisotropic
units as side chains. GG and LBG, unlike Sclg, dissolve in aqueous solution as random coils. These
swelling
when
soaked in distilled
water
[13,14].
By means
of rheological
measurements,
the influence
two
polysaccharides
were chosen
because,
according
to previous
results, GG/borax
and LBG/borax
of pH on
flowalso
curves
andan
mechanical
Sclg/borax
was
also evaluated.
ForBya means
deeperofinsight
tablets
showed
anisotropic spectra
swellingofwhen
soaked in
distilled
water [13,14].
rheological
measurements,
the influence
of pH
on flow curves
and mechanical
spectra
of Sclg/borax
about the
effect of
pH on the structure
of the
Sclg/borax
complex,
Congo red
(CR)—a
well-known
was
also
evaluated.
For
a
deeper
insight
about
the
effect
of
pH
on
the
structure
of
the
Sclg/borax
probe capable of interacting with biomacromolecules, including polysaccharides [15,16]—was used
complex, Congo
red (CR)—a well-known
capable
of interacting
with
biomacromolecules,
for a spectroscopic
characterization
(UV and probe
circular
dichroism
(CD)). In
particular,
CR is useful for
including polysaccharides [15,16]—was used for a spectroscopic characterization (UV and circular
following structural transitions in macromolecules (in our case, Sclg triple-helix denaturation) because
dichroism (CD)). In particular, CR is useful for following structural transitions in macromolecules
changes
in environmental conditions induce a shift in the maximum of its absorption spectrum [17].
(in our case, Sclg triple-helix denaturation) because changes in environmental conditions induce a
Principal
component
analysis
(PCA),
allowed
a clear
distinction
to be madeanalysis
between
the allowed
CR spectra in
shift
in the maximum
of its
absorption
spectrum
[17].
Principal component
(PCA),
the presence
Sclg alone
and
Sclg/borax,
and
was possible
to identify
the
pHand
values
at which the
a clear of
distinction
to be
made
between the
CRitspectra
in the presence
of Sclg
alone
Sclg/borax,
and itcollapses.
was possible
to identify
pH values at which
the triple
collapses.
Although
the
triple helix
Although
the the
abovementioned
techniques
arehelix
suitable
for the
characterization
abovementioned
techniques
are suitable
for thea characterization
of polysaccharides,
they
arethis
unable
of polysaccharides,
they
are unable
to provide
structural picture
of the systems.
For
purpose,
to provide a structural picture of the systems. For this purpose, a combined approach of experimental
a combined approach of experimental and computational data can be particularly useful. Thus, in the
and computational data can be particularly useful. Thus, in the present paper, MD simulations
present paper, MD simulations allowed the acquisition of a detailed description about the structure of
allowed the acquisition of a detailed description about the structure of the complex that is formed
the complex
that
formed
between CR and Sclg/borax.
between
CRis
and
Sclg/borax.
Scheme 1. Repeating units of scleroglucan (Sclg); guar gum (GG); and locust bean gum (LBG).
Scheme 1. Repeating units of scleroglucan (Sclg); guar gum (GG); and locust bean gum (LBG).
Molecules 2017, 22, 435
3 of 14
Molecules 2017, 22, 435
3 of 14
2. Results and Discussion
2. Results and Discussion
2.1. Rheology
2.1. Rheology
Figure 1a,b indicates that all the studied systems, with the exception of those carried out at
Figure 1a,b indicates that all the studied systems, with the exception of those carried out at pH = 14,
pH = 14, exhibit the same general characteristics, as far as the shear viscosity trend is concerned.
exhibit the same general characteristics, as far as the shear viscosity trend is concerned. Indeed, all of
Indeed, all of them show the typical shear-thinning behaviour where the upper Newtonian plateau
them show the typical shear-thinning behaviour where the upper Newtonian plateau (around
(around 1000 Pa·s) is connected to the lower Newtonian plateau (around 0.03 Pa·s) by an almost
1000 Pa·s) is connected to the lower Newtonian plateau (around 0.03 Pa·s) by an almost linear trend
linear trend (in logarithmic scale). However, despite this general similarity, the systems differ for
(in logarithmic scale). However, despite this general similarity, the systems differ for the stress τ at
thewhich
stressthe
τ atviscosity
which the
viscosity occurs.
breakdown
occurs. For
for Sclg
up viscosity
to pH = 13,
breakdown
For example,
forexample,
Sclg solutions,
upsolutions,
to pH = 13,
viscosity
breakdown
occurs
around
9
Pa,
while
at
pH
=
14
no
breakdown
is
detectable
as
the system
breakdown occurs around 9 Pa, while at pH = 14 no breakdown is detectable as the system
is
◦ C (1 m·Pa·s).
is essentially
essentially Newtonian
Newtonianwith
witha aviscosity
viscosityapproximately
approximately
4
times
that
of
water
at
25
4 times that of water at 25 °C (1 m·Pa·s).
Actually,
at such
high
pHpH
values,
thethe
integrity
of of
thethe
polymeric
network
is is
seriously
compromised
Actually,
at such
high
values,
integrity
polymeric
network
seriously
compromiseddue
to due
the Sclg
collapse
[18] and
chemical
degradation
of the
chains.
to thetriple-helix
Sclg triple-helix
collapse
[18] to
and
to chemical
degradation
of the
chains.
Figure 1. Cont.
Molecules 2017, 22, 435
Molecules 2017, 22, 435
4 of 14
4 of 14
Figure
Flow
curvesofofSclg
Sclg(a)
(a)and
andSclg/borax
Sclg/borax (b)
viscosity
at at
Figure
1. 1.
Flow
curves
(b) samples
samplesatatdifferent
differentpH
pHvalues;
values;(b’):
(b’):
viscosity
stress
τ
=
16
Pa
(η
16) for Sclg and Sclg/borax samples at different pH values. Mechanical spectra of Sclg
stress τ = 16 Pa (η16 ) for Sclg and Sclg/borax samples at different pH values. Mechanical spectra of
(c)(c)
and
Sclg/borax
(d)(d)
at different
pHpH
values.
In In
Figure
(e),(e),
thethe
data
forfor
both
samples
areare
reported
Sclg
and
Sclg/borax
at different
values.
Figure
data
both
samples
reported
together
with
the
loss
modulus
G”
values
recorded
at
pH
=
14
(polymer
concentration,
cp
= 0.7%,
together with the loss modulus G” values recorded at pH = 14 (polymer concentration, cp
= 0.7%,
T = 25
°C). In Figure (b–e), for an appropriate comparison, the data of Sclg are also shown.
◦
T = 25 C). In Figure (b–e), for an appropriate comparison, the data of Sclg are also shown.
The addition of borax does not significantly affect the upper and lower Newtonian plateau, but
The addition
does
not significantly
affect the upper
Newtonian
it shifts
forward of
theborax
viscosity
breakdown
up to approximately
20–30and
Pa. lower
This suggests
that plateau,
borax
butpromotes
it shifts the
forward
the
viscosity
breakdown
up
to
approximately
20–30
Pa.
This
suggests
that
formation of linkages among Sclg chains, leading to networks more resistant to
borax
promotes When
the formation
of linkages
among
Sclg chains,
leading
networks
resistant
deformations.
pH is raised
to 14, the
Sclg–borax
solution
againtoacquires
themore
Newtonian
to behaviour
deformations.
pHclose
is raised
toof14,
thesolutions.
Sclg–borax
again acquires
the Newtonian
with aWhen
viscosity
to that
Sclg
Thissolution
further confirms
the damages
induced
behaviour
with
a viscosity
to(breakdown
that of Sclg
Thisand
further
confirms
damages
by high pH
values
to the Sclgclose
chains
of solutions.
the triple helices
hydrolysis
of thethe
glycosidic
linkages).
1b’values
sums up
of pH (breakdown
on flow properties
to the systems
with and of
induced
by Figure
high pH
to the
the effects
Sclg chains
of therelated
triple helices
and hydrolysis
borax.
Indeed,Figure
Figure1b’
1b’sums
reports
dependence
the properties
system viscosities
at
thewithout
glycosidic
linkages).
upthe
thepH
effects
of pH onofflow
related evaluated
to the systems
τ
=
16
Pa
(η
16
:
this
value
corresponds
to
the
beginning
of
the
second
Newtonian
plateau
in
Figure
1a).
with and without borax. Indeed, Figure 1b’ reports the pH dependence of the system viscosities
While inatthe
of borax-free
systems,
regardlessto
ofthe
pH,beginning
η16 is almost
constant
and
less than 0.1
Pa
evaluated
τ =case
16 Pa
(η16 : this value
corresponds
of the
second
Newtonian
plateau
s, in the1a).
caseWhile
of Sclg/borax
systems,
η16 increases
up to
pH 12, then
at pH
14almost
it abruptly
falls down
in Figure
in the case
of borax-free
systems,
regardless
of pH,
η16= is
constant
and less
below
0.01
Pa
s.
This
result
further
supports
the
different
structures
between
the
borax
free
and
the
than 0.1 Pa·s, in the case of Sclg/borax systems, η16 increases up to pH 12, then at pH = 14 it abruptly
Sclg/borax
systems.
Also,
stress
sweep
tests
(see
Table
1)
support
the
results
obtained
from
the
flow
falls down below 0.01 Pa·s. This result further supports the different structures between the borax free
curve experiments. All systems, regardless of the presence of borax, and at pH lower than 14, show
and the Sclg/borax systems. Also, stress sweep tests (see Table 1) support the results obtained from
the typical weak gel behaviour, where the storage modulus G’ is clearly higher than the loss modulus
the flow curve experiments. All systems, regardless of the presence of borax, and at pH lower than 14,
G’’ (G’ is about 3 times G’’) and both moduli are constant with the applied stress up to the linear
show the typical weak gel behaviour, where the storage modulus G’ is clearly higher than the loss
viscoelastic threshold. On the other side, when pH = 14, only G’’ is detectable, indicating the typical
modulus G” (G’ is about 3 times G”) and both moduli are constant with the applied stress up to the
behaviour of a solution. This means that, due to pH, the breakage of Sclg chains leads to the
linear
viscoelastic
the other
side,towhen
pH = 14,
G”ofispH
detectable,
destruction
of thethreshold.
polymeric On
network.
In order
discriminate
theonly
effect
and boraxindicating
addition onthe
typical
behaviour
of
a
solution.
This
means
that,
due
to
pH,
the
breakage
of
Sclg
chains
leads to the
the weak gel systems, Table 1 reports, for the different systems, the limit of the linear viscoelastic
destruction
of the
network.(i.e.,
In order
to discriminate
thestorage
effect of
and borax addition on
field in terms
of polymeric
critical parameters
the values
of the critical
(G’pH
c) and loss (G’’c) moduli
theand
weak
gel
systems,
Table
1
reports,
for
the
different
systems,
the
limit
of
the
linear
viscoelastic
field
the values of the critical stress (τc) and deformation (γc)). It can be observed that
Sclg critical
in terms
of critical
parameters
(i.e., the
values
of the
the value
criticalofstorage
lossreduction
(G”c ) moduli
and the
c ) and
parameters
are almost
unaffected
by pH
up to
12, and(G’
only
a small
of critical
values
of the critical
deformation
(γc(while
)). It can
observed
that constant),
Sclg critical
parameters
parameters
G’c, G’’stress
c, and(ττ
occurs
at pH = 13
γc be
remains
almost
due
to the
cc) and
areconnectivity
almost unaffected
by polymeric
pH up to the
valueThe
of 12,
and only
a small
reduction
of critical
loss of the
network.
addition
of borax
increases
the gel
strengthparameters
and, up
pHc ,13,
G’τc,cG’’
c, andat
τc pH
are higher
in comparison
to those
ofconstant),
the samples
without
At the same
G’cto
, G”
and
occurs
= 13 (while
γc remains
almost
due
to theborax.
connectivity
loss of
as in the
case of the
Sclg
systems,
13 is reached,
reductionand,
of G’up
c, G’’
and13,
τc occurs
thetime,
polymeric
network.
The
addition
ofwhen
boraxpH
increases
the gela strength
toc,pH
G’c , G”c ,
and
increase
of pH to
upthose
to 14of
leads
the complete
breakdown
of the
the same
three-dimensional
and
τc the
are further
higher in
comparison
the samples
without
borax. At
time, as in the
polymeric
network,
also
related
to
the
chemical
degradation
of
the
chains.
case of the Sclg systems, when pH 13 is reached, a reduction of G’c , G”c , and τc occurs and the further
increase of pH up to 14 leads the complete breakdown of the three-dimensional polymeric network,
also related to the chemical degradation of the chains.
Molecules 2017, 22, 435
5 of 14
Table 1. Linear viscoelastic threshold for the Sclg and Sclg–borax systems (cp = 0.7%). G’c and
G”c are the critical storage and the loss moduli; τc and γc are the critical stress and the critical
deformation, respectively.
Scleroglucan
pH
G’c (Pa)
G”c (Pa)
τc (Pa)
γc (−)
5.5
12.8
4.8
5.6
0.41
10
-
12
14.4
4.8
5.6
0.37
13
9.7
3.6
4.4
0.42
14
0.0
0.0
0.0
0.0
13
10.0
3.7
5.6
0.52
14
0.0
0.0
0.0
0.0
Scleroglucan–Borax
pH
G’c (Pa)
G”c (Pa)
τc (Pa)
γc (−)
9.0
16.1
5.1
9.1
0.54
10
14.8
5.0
7.2
0.46
12
20.0
5.2
9.1
0.44
Frequency sweep tests (Figure 1e) confirm that, at pH = 14, regardless of the presence of borax,
the storage modulus cannot be detected and only the loss modulus appears due to the breakage of the
three-dimensional polymeric network. Additionally, at pH < 14, frequency sweep tests (Figure 1c,d)
confirm the weak gel nature of Sclg and Sclg–borax systems, as G’ is always 2–3 times G” and both of
them are slightly dependent on angular frequency ω. Moreover, it can be noted that the addition of
borax does not lead to a significant increase of G’ and that, up to pH = 12, all the systems (with and
without borax) show very similar behaviours. The mechanical spectra seem to be, at a first glance,
in contrast with the flow curves and the stress sweep test, where the effect of borax was always
detectable and well evident. However, the overall results from the rheological tests can be explained
by taking into account that:
(i)
(ii)
the presence of borax affects the polymeric network architecture mainly at long range,
the condition actually tested by flow curve and stress sweep experiments, carried out also
beyond the linear viscoelastic threshold;
in the frequency sweep experiments, the systems are explored only at short range, with small
deformations imposed in the linear viscoelastic range, thus less affected by the presence of borax.
Finally, at pH = 13, (Figure 1c,d), although both systems (with and without borax) show G’ values
higher than those of G”, they undergo a clear decrease in comparison to the samples at pH < 13.
Furthermore, the greater frequency dependence that is evident for the storage moduli clearly indicates
the shifting of the systems towards a sol condition.
2.2. Water Uptake and Dimensional Increase Studies
It has been reported that tablets, prepared with the Sclg/borax and GG/borax freeze-dried
hydrogels, when soaked in aqueous media, undergo an anomalous swelling, essentially along
one direction. The same behaviour was observed in the case of LBG, although to a much lesser
extent [6–9,13,14,19,20]. In order to test the possible effect of high pH values on the swelling behaviour
of Sclg/borax tablets, two different types of experiments were carried out:
(i)
(ii)
Sclg was first dissolved at pH = 14 before the addition of borax and the tablet preparation.
The swelling experiments were then carried out in distilled water (at 37 ◦ C).
Sclg was first dissolved in distilled water before the addition of borax and the tablet preparation.
The swelling experiments were then carried out in aqueous solutions at pH = 14 (at 37 ◦ C).
For an appropriate comparison, the same tests were performed using tablets obtained with
GG/borax and LBG/borax following the same procedures above reported.
Molecules 2017, 22, 435
6 of 14
The2017,
results,
Molecules
22, 435 reported
in Figure 2, clearly show an interesting behaviour, which differs
6 offor
14
the three kinds of tablets. First of all, it must be pointed out that, in all cases, the presence of
NaOH reduced
significantly
bothuptake
water uptake
and elongation
in comparison
to theobtained
data obtained
reduced
significantly
both water
and elongation
in comparison
to the data
when
whendistilled
only distilled
was used
for tablet
preparations
swelling
experiments
Figure
only
water water
was used
for tablet
preparations
and and
swelling
experiments
(see(see
Figure
3 in3
in
Reference
[13]).
In
fact,
when
the
polymers
were
dissolved
at
high
pH
values
(preparation
Reference [13]). In fact, when the polymers were dissolved at high pH values (preparation i) thei)
the hydrolysis
of glycosidic
bonds
simultaneousdrastic
drasticreduction
reductioninin molecular
molecular weights;
weights;
hydrolysis
of glycosidic
bonds
ledled
to to
a asimultaneous
consequently, the tablets, when
when soaked
soaked in
in water, showed
showed only a slight water uptake and a slight
Sclg, aa mass
mass decrease
decrease had
had already
already occurred
occurred after 15 min, and
and for
for
anisotropic elongation. In the case of Sclg,
this reason an “apparent negative
negative elongation”
elongation” was
was recorded.
recorded. Actually,
Actually, the high pH values promote
this
a depolymerisation reaction but also induce aa conformational
conformational transition helix–coil, thus destabilising
destabilising
Also in
in the
the case of the two tested galactomannans
the original very stable triple-helix conformation. Also
the effect was evident, although
although to a lesser extent than that obtained with Sclg. In
In fact,
fact, it is known
the
that
the
anisotropic
elongation
is
due
to
the
borax
interaction
with
the
glucose
side
chains
of the
that the anisotropic elongation is due to the borax interaction with the glucose side chains of the
polymers,
polymers,
to an of
alignment
of the macromolecules.
As a consequence,
LBG—because
which
leadswhich
to anleads
alignment
the macromolecules.
As a consequence,
LBG—because
of the lower
of
the
lower
number
of
side
chains
(branching
degree
1:4),
when
compared
with
GG
(branching
number of side chains (branching degree 1:4), when compared with GG (branching degree 1:2)—always
degree a1:2)—always
shows
a moreof
evident
reduction
of the
induced
the borax,
as reported
shows
more evident
reduction
the effects
induced
byeffects
the borax,
as by
reported
in Figure
2c,d.
in Figure 2c,d.due
Furthermore,
due to the
dissolution
of the
foritGG
and
LBG it to
was
possible
to
Furthermore,
to the dissolution
of the
tablets, for
GGtablets,
and LBG
was
possible
carry
out the
carry
out
the
experiments
for
a
maximum
of
1
or
2
days.
A
quite
different
behaviour
was
observed
experiments for a maximum of 1 or 2 days. A quite different behaviour was observed when the
when the were
polymers
were dissolved
in water
and the
tablets
then
NaOHsolutions
aqueous
polymers
dissolved
in water and
the tablets
were
thenwere
soaked
in soaked
NaOH in
aqueous
solutions
(pH = 14) (preparation
ii). In
Sclg,was
thedramatic
effect was
dramatic
and very
rapid:loss
the
(pH
= 14) (preparation
ii). In the case
of the
Sclg,case
theof
effect
and
very rapid:
the weight
weight
loss was
alreadyafter
recorded
after
min.
case
of LBG,
GG and
the hydrolysis
was
was
already
recorded
45 min.
In45
the
caseInofthe
GG
and
theLBG,
hydrolysis
reactionreaction
was rather
rather slower.
The hydrolysis
occurred
in a heterogeneous
system
due
to presence,
the presence,
at the
beginning,
slower.
The hydrolysis
occurred
in a heterogeneous
system
due to
the
at the
beginning,
of
of
the
polymeric
chains
as
a
solid
phase
(tablets).
Consequently,
the
weight
loss
was
appreciated
after
the polymeric chains as a solid phase (tablets). Consequently, the weight loss was appreciated after
only 1 day.
Figure 2.2. Relative
increase
of tablet
heights
for Sclg/borax
(a); Sclg/borax,
GG/borax,
and LBG/borax
Relative
increase
of tablet
heights
for Sclg/borax
(a); Sclg/borax,
GG/borax,
and (c);
tablet
water (c);
uptake
forwater
Sclg/borax
and LBG/borax
symbols:(d).
tablets
LBG/borax
tablet
uptake(b);
for Sclg/borax,
Sclg/borax GG/borax,
(b); Sclg/borax,
GG/borax,(d).
and(Full
LBG/borax
prepared
in water
andprepared
swelled in
at water
pH = and
14; empty
prepared
at pH
= 14prepared
and swelled
(Full symbols:
tablets
swelledsymbols:
at pH = tablets
14; empty
symbols:
tablets
at in
water.
T and
= 37 swelled
°C).
pH = 14
in water. T = 37 ◦ C).
These peculiar differences observed between Sclg (from one side) and GG and LBG (from the
other side) have to be ascribed to different conformations of the three polymers. Sclg is a triple helix
and the effect of NaOH is revealed to be extremely rapid and dramatic in terms of chain rigidity and
tablet anisotropic elongation. On the other side, GG and LBG, which are flexible chains, in the
Molecules 2017, 22, 435
7 of 14
These peculiar differences observed between Sclg (from one side) and GG and LBG (from the
other
side)
have
to be ascribed to different conformations of the three polymers. Sclg is a triple helix
Molecules
2017,
22, 435
7 of 14
and the effect of NaOH is revealed to be extremely rapid and dramatic in terms of chain rigidity and
presence
of borax
show self-healing
[21,22]
that which
counterbalance,
leastinpartially,
the
tablet
anisotropic
elongation.
On the othereffects
side, GG
and LBG,
are flexible at
chains,
the presence
hydrolysis
induced
by NaOH.
The
effectthat
of NaOH
is particularly
evident
when
swellinginduced
data at
of
borax show
self-healing
effects
[21,22]
counterbalance,
at least
partially,
the the
hydrolysis
pHNaOH.
14 are compared
with
thoseis obtained
in distilled
Figure 3 data
in Reference
Actually,
by
The effect of
NaOH
particularly
evident water
when (see
the swelling
at pH 14[13]).
are compared
the interaction
of Sclg
from onewater
side and
and3 LBG
from the[13]).
otherActually,
side with
takes place
in
with
those obtained
in distilled
(see GG
Figure
in Reference
theborax
interaction
of Sclg
a
different
way:
in
the
case
of
Sclg,
the
borax
promotes
mixed
(chemical
and
physical)
interactions
from one side and GG and LBG from the other side with borax takes place in a different way: in the
between
thethe
triple
helices
(at least
for (chemical
the samples
distilled water)
while,
the helices
case of
case
of Sclg,
borax
promotes
mixed
andprepared
physical)ininteractions
between
the in
triple
GGleast
andfor
LBG,
borax prepared
forms chemical
bridges
between
chains
means
of linkages
that
reversible.
(at
thethe
samples
in distilled
water)
while,
in thebycase
of GG
and LBG,
theare
borax
forms
During
the
swelling
in
NaOH,
the
labile
nature
of
the
borax
cross-links
in
the
GG,
and
to
a
lesser
extent
chemical bridges between chains by means of linkages that are reversible. During the swelling
in
in the LBG,
making
the interchain
interactions
to undergo
rearrangement
in order
to
NaOH,
the labile
nature
of the borax
cross-linksable
in the
GG, andthe
to aneeded
lesser extent
in the LBG,
making
keep,
for a while,
the tendency
toundergo
give the the
anisotropic
elongation of the
tablets.toTo
better
the
interchain
interactions
able to
needed rearrangement
in order
keep,
forillustrate
a while,
thetendency
data reported
in the
Figure
2, the pictures
of Sclg/borax
tablets
prepared
in distilled
water
(a) or in
in
the
to give
anisotropic
elongation
of the tablets.
To better
illustrate
the data
reported
NaOH2,(b),
swelled
in NaOH (a)
or in prepared
distilled water
(b), for
different
of (b),
time,
areswelled
shown
Figure
theand
pictures
of Sclg/borax
tablets
in distilled
water
(a) orperiods
in NaOH
and
inNaOH
Figure (a)
3. or in distilled water (b), for different periods of time, are shown in Figure 3.
in
(a)
t=0
t = 30 min
t = 15 min
t = 45 min
(b)
t=0
t = 60 min
t = 15 min
t = 30 min
t = 45 min
Figure 3. Pictures of Sclg/borax tablets, prepared in water and swelled in NaOH (pH = 14) (a) or prepared
Figure 3. Pictures of Sclg/borax tablets, prepared in water and swelled in NaOH (pH = 14) (a) or
in NaOH (pH = 14) and swelled in water (b), after swelling, for different periods of time, at 37 °C.
prepared in NaOH (pH = 14) and swelled in water (b), after swelling, for different periods of time, at
37 ◦ C.
2.3. Spectroscopic and Chemometric Analysis
The different
behaviours
of the
Sclg and Sclg/borax solutions as a function of pH was examined
2.3. Spectroscopic
and
Chemometric
Analysis
also by characterising the CR–polysaccharide interaction using UV–vis absorption and CD spectroscopy.
The different behaviours of the Sclg and Sclg/borax solutions as a function of pH was
CR is a probe widely used to follow conformational transitions in biomacromolecules, including
examined also by characterising the CR–polysaccharide interaction using UV–vis absorption and CD
polysaccharides [15,17,23–25]. Changes in the conditions surrounding the macromolecules (i.e., the
spectroscopy. CR is a probe widely used to follow conformational transitions in biomacromolecules,
interaction of the tested polymer with other molecules) can induce a well-detectable shift in the
including polysaccharides [15,17,23–25]. Changes in the conditions surrounding the macromolecules
maximum of the CR absorption spectra. As reported in Figure 4, the absorption maximum of CR in
(i.e., the interaction of the tested polymer with other molecules) can induce a well-detectable shift in
solution was red-shifted in the presence of Sclg by increasing pH up to 13.1; at higher pH values, the
the maximum of the CR absorption spectra. As reported in Figure 4, the absorption maximum of CR
absorption maxima decrease abruptly at almost constant values. This behaviour correlates well with
in solution was red-shifted in the presence of Sclg by increasing pH up to 13.1; at higher pH values,
the known Sclg transition, which occurs roughly at pH 13 from an ordered (triple helix) to a single-chain
the absorption maxima decrease abruptly at almost constant values. This behaviour correlates well with
conformation, thus indicating that CR is a useful probe to follow this structural rearrangement.
the known Sclg transition, which occurs roughly at pH 13 from an ordered (triple helix) to a single-chain
A very similar behaviour was observed with the Sclg/borax complex. However, in this case, the
conformation, thus indicating that CR is a useful probe to follow this structural rearrangement. A very
transition pH occurred at pH 13.5 (Figure 4) because of the stabilising effect of borax on the triple-helix
similar behaviour was observed with the Sclg/borax complex. However, in this case, the transition pH
structure [25].
occurred at pH 13.5 (Figure 4) because of the stabilising effect of borax on the triple-helix structure [25].
Molecules 2017, 22, 435
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8 of 14
Figure
4. Absorbancespectra
spectra of
red
(CR)
in water
solution
of Sclg (A);
and Sclg/borax
at different(B) at
Figure
4. Absorbance
ofCongo
Congo
red
(CR)
in water
solution
of Sclg
(A); and (B)
Sclg/borax
Figure 4. Absorbance spectra of Congo red (CR) in water solution of Sclg (A); and Sclg/borax (B) at different
pH values.
different
pH values.
pH values.
The result of PCA on the absorption spectra is reported in Figure 5. The analysed experimental
The
of
isisreported
in
5.5. The
experimental
The result
result
of PCA
PCA
on the
theabsorption
absorption
spectra
reported
inFigure
Figure
The
analysed
experimental
data
are
projected
in on
the
space
of the first spectra
two
principal
components
that take
intoanalysed
account more
than
data
are projected
projected
inthe
the
spaceofof
the
first
two
principal
components
into
account
data99%
in
space
first
two
principal
components
thatthat
take
intoand
account
moremore
than
of the original
variance.
Athe
clear
distinction
between
the CR spectra
in take
Sclg
Sclg/borax
than
99%
of the
original
variance.
A clear
distinction
between
theCR
CRspectra
spectra
Sclg
and
samples
can
be observed:
the distinction
complexation
of
the polysaccharide
within
borax
a Sclg/borax
strong
99% solution
of the
original
variance.
A clear
between
the
Sclghas
and
Sclg/borax
influence
on the
CRbe
binding,
affecting
both
the first (that
for the 90% ofwith
the variability)
solution
samples
can
observed:
the
of
the
borax
aa strong
solution
samples
can
be
observed:
the complexation
complexation
of accounts
the polysaccharide
polysaccharide
with
borax has
hasand
strong
the
second
principal
component
(that
accounts
for
the
9%
of
the
variability).
Very
important
is
the and
influence
influence on
on the
the CR
CR binding,
binding, affecting
affecting both
both the
the first
first (that
(that accounts
accounts for
for the
the 90%
90% of
of the
the variability)
variability)
and
variation
of pH that
has a remarkable
effect on the
the position
in the
PCs spaceis
the
principal
component
(that
for
the
the
Very
important
the second
second
principal
component
(that accounts
accounts
forabsorption
the 9%
9% of
ofspectra:
the variability).
variability).
Very
important
is the
the
of theof
Sclg
and
thehas
Sclg/borax
sampleseffect
undergoes
a remarkable
shift
for pHthe
higher
than 13.1
andPCs
13.5,space
variation
pH
that
a
remarkable
on
the
absorption
spectra:
position
in
the
variation of pH that has a remarkable effect on the absorption spectra: the position in the PCs space
respectively. This shift is related to the Sclg transition from triple helix to single strand. It must be
of
and the
theSclg/borax
Sclg/borax
samples
undergoes
a remarkable
shift
higher
and
of the Sclg and
samples
undergoes
a remarkable
shift
for for
pH pH
higher
thanthan
13.1 13.1
and 13.5,
also pointed out that at higher pH values, further changes occur in the absence of borax, while such
13.5,
respectively.
This
shift
is
related
to
the
Sclg
transition
from
triple
helix
to
single
strand.
It
must
respectively.
This
is related
to case
the Sclg
transition
from triple
helix to single
strand.
It must be
changes are
notshift
detectable
in the
of Sclg/borax
samples,
thus suggesting
that the
stabilising
be
also
pointed
out
that
at
higher
pH
values,
further
changes
occur
in
the
absence
of
borax,
while
also
pointed
out
that
at
higher
pH
values,
further
changes
occur
in
the
absence
of
borax,
while
such
effects of the borax still remain in the Sclg single strand.
such
changes
are not
detectable
in the
case
Sclg/borax
samples,thus
thussuggesting
suggesting that
that the stabilising
changes
are not
detectable
in the
case
ofofSclg/borax
samples,
stabilising
effects
effects of
of the
theborax
boraxstill
stillremain
remainin
inthe
theSclg
Sclgsingle
singlestrand.
strand.
Figure 5. Projection of the Sclg (filled circles) and Sclg/borax (empty circles) absorption spectra onto
the space spanned by the first two principal components (PCs). The arrows indicate the transition
observed in the spectra at pH = 13.1 (Sclg) and pH = 13.5 (Sclg/borax).
Figure
Projection
the Sclg
circles) and
Sclg/borax
(empty
spectra
onto
The5.CD
spectra of
(Figure
6), (filled
corresponding
to the
absorption
bandcircles)
of CR, absorption
were acquired
at pH
9
Figure 5. Projection of the Sclg (filled circles) and Sclg/borax (empty circles) absorption spectra onto
and
14.
At
pH
9,
a
well-defined
band
is
present
in
both
the
Sclg
and
Sclg/borax
samples.
For
the
Sclg/borax
the space spanned by the first two principal components (PCs). The arrows indicate the transition
the space spanned by the first two principal components (PCs). The arrows indicate the transition
sample
a higher
CD
signal
registered,
due= the
of borax, which affects the mobility
observed
in the induced
spectra at
pH
= 13.1is(Sclg)
and pH
13.5presence
(Sclg/borax).
observed in the spectra at pH = 13.1 (Sclg) and pH = 13.5 (Sclg/borax).
of the lateral domains of Sclg chains [8].
The CD spectra (Figure 6), corresponding to the absorption band of CR, were acquired at pH 9
The
(Figure 6),
corresponding
thethe
absorption
band of CR,
were acquired
at pH 9
and 14. AtCD
pHspectra
9, a well-defined
band
is present in to
both
Sclg and Sclg/borax
samples.
For the Sclg/borax
and
14. aAt
pH 9,induced
a well-defined
band
is presentdue
in both
the Sclg of
and
Sclg/borax
samples.
For the
sample
higher
CD signal
is registered,
the presence
borax,
which affects
the mobility
Sclg/borax
sample
a
higher
induced
CD
signal
is
registered,
due
the
presence
of
borax,
which
affects
of the lateral domains of Sclg chains [8].
the mobility of the lateral domains of Sclg chains [8].
Molecules 2017, 22, 435
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9 of 14
9 of 14
9 of 14
Figure 6. Circular dichroism (CD) spectra of CR–Sclg complex (circles) and CR–Sclg/borax (squares),
Figure 6. Circular dichroism (CD) spectra of CR–Sclg complex (circles) and CR–Sclg/borax (squares),
at pH = 9 (empty symbols) and 14 (filled symbols) in water solution.
at pH = 9 (empty symbols) and 14 (filled symbols) in water solution.
In all samples, with and without borax, the CD signal is lost at pH 14. These data confirm that,
Figure
6. Circular
dichroism
(CD)
spectra
CR–Sclg
complex
(circles)
and
CR–Sclg/borax
(squares),
In
all samples,
with transition
and
without
borax,
the
CD structure
signal is
lost at
pH
14.
data
confirm
at pH
14,
a structural
between
anofordered
(triple
helix)
andThese
a single
chain
takes that,
at
pH
= 9systems.
(empty
symbols) between
and 14 (filled
in water
solution.
both
at pHplace
14, afor
structural
transition
ansymbols)
ordered
structure
(triple helix) and a single chain takes
place for both
systems.
In all samples, with and without borax, the CD signal is lost at pH 14. These data confirm that,
2.4. Molecular Dynamics (MD) Simulations
at pH 14, a structural transition between an ordered structure (triple helix) and a single chain takes
2.4. Molecular
Dynamics
(MD)
Simulations
Thefor
interactions
between
the Sclg/borax and CR were investigated by means of MD simulations.
place
both systems.
Six independent simulations were carried out by starting from random positions of CR with respect
The interactions between the Sclg/borax and CR were investigated by means of MD simulations.
Molecular
Dynamics
(MD)
Simulations
to 2.4.
the triple
helix
and, in all
cases,
at a starting distance greater than 1.6 nm. The interaction between
Six independent simulations were carried out by starting from random positions of CR with respect to
CR andThe
triple
helix
takes
place
in
different
ways,
as the triple
helix has
not
a single
binding
site for
interactions
thea Sclg/borax
and CR were
investigated
by nm.
means
of MD
simulations.
the triple
helix
and, in allbetween
cases,
at
starting
distance
greater
than 1.6
The
interaction
between
CRSix
(a independent
different situation
occurs
for
proteins
that
have
only
a
specific
binding
site
for
their
ligands).
simulations were carried out by starting from random positions of CR with respect
CR and
triple
helix
takes
place
in
different
ways,
as
the
triple
helix
has
not
a
single
binding
site for
This
situation
does and,
not allow
a straightforward
cluster greater
analysis
to 1.6
identify
theinteraction
most representative
to the
triple helix
in all cases,
at a starting distance
than
nm. The
between
CR (astructures
different
situation
occurs
for proteins
that have
only a specific
binding
site
forcomplexes
their
ligands).
from
the
sixtakes
independent
simulations.
preliminary
separation
sampled
CR and triple
helix
place in different
ways,Aas
the triple helix
has notofa the
single
binding
site for
This situation
does
not
allow
a
straightforward
cluster
analysis
to
identify
the
most
representative
hasCR
been
carried
out
by
analysing
the
angle
between
the
principal
axis
of
triple
helix
and
CR,
leading
(a different situation occurs for proteins that have only a specific binding site for their ligands).
to This
twofrom
mainthe
representative
families
of structures
in
which
CR to
lays
in a parallel
perpendicular
structures
six
independent
simulations.
A
preliminary
separation
ofmost
theorsampled
complexes
situation
does
not allow
a straightforward
cluster
analysis
identify
the
representative
position
with
respect
to
the
triple
helix
axis.
Two
examples,
representative
of
these
two
families,
are
structures
theanalysing
six independent
simulations.
A preliminary
separation
of the sampled
complexes
has been
carried from
out by
the angle
between
the principal
axis of triple
helix and
CR, leading
to
reported
in
Figure
7.
hasrepresentative
been carried outfamilies
by analysing
the angle between
theCR
principal
of triple helix
and CR, leading
two main
of structures
in which
lays inaxis
a parallel
or perpendicular
position
to two main representative families of structures in which CR lays in a parallel or perpendicular
with respect
to the triple helix axis. Two examples, representative of these two families, are reported in
position with respect to the triple helix axis. Two examples, representative of these two families, are
Figure 7.
reported in Figure 7.
Figure 7. Structures of the complexes sampled during the molecular dynamics (MD) simulations of
the triple helix in the presence of CR. The two structures are the more representative of the complexes
with
perpendicular
(on of
thethe
left)
and parallel
(on theduring
right) orientation
of CR
with respect
the triple-helix
Figure
7. Structures
complexes
sampled
the molecular
dynamics
(MD)to
simulations
of
axis.
Sclg
is
reported
with
a
stick
representation,
the
three
strands
are
coloured
in
red,
blue,
and
green, of
Figure
7.
Structures
of
the
complexes
sampled
during
the
molecular
dynamics
(MD)
simulations
the triple helix in the presence of CR. The two structures are the more representative of the complexes
respectively.
boron
atom
evidenced
instructures
yellow.
are
reported
as
a surface,
the triple
helix
in The
the presence
of is
CR.
The two
areCR
themolecules
more
the
complexes
with
perpendicular
(on the
left)
and
parallel
(on
the
right) The
orientation
of CR representative
with
respect
to theof
triple-helix
byiselements.
axis. Sclg
reported
withleft)
a stickand
representation,
the the
threeright)
strandsorientation
are coloured of
in red,
green, to the
with coloured
perpendicular
(on the
parallel (on
CRblue,
withand
respect
respectively.
The
atomwith
is evidenced
in yellow. The CR
are reported
as a surface,
triple-helix
axis. Sclg
is boron
reported
a stick representation,
themolecules
three strands
are coloured
in red, blue,
coloured by elements.
and green, respectively. The boron atom is evidenced in yellow. The CR molecules are reported as
a surface, coloured by elements.
Molecules 2017, 22, 435
10 of 14
These structures were obtained by performing two independent cluster analyses on the two
families above described. It should be pointed out that the configurations belonging to each one of the
two families (i.e., with CR perpendicular and parallel) are actually very similar. These data support the
spectroscopic evidence that an induced CD on CR can be observed as a consequence of the interaction
with Sclg triple helix. On the other side, a different behaviour was obtained when the complexes
between CR and Sclg single strand (i.e., Sclg conformation at pH 14) were studied [25] and no induced
CD could be detected. These simulations do not allow evaluating which one of these two complexes is
more stable. Anyhow, it can be asserted that, once formed, both complexes were very stable. In the
configuration with a perpendicular orientation of CR, a tighter interaction between the probe and
Sclg/borax takes place, and CR intercalates in a groove of the triple helix. It has to be underlined that
these structures are similar to those observed in the case of the simulation of Sclg (without borax) and
CR [25]. These evidences further support the above-reported statement that the presence of borax does
not significantly influence the CR–Sclg interaction.
3. Materials and Methods
3.1. Materials
Sclg (Actigum CS 11) was provided by Cargill (Minneapolis, MN, USA); GG and LBG by
CarboMer, Inc. (San Diego, CA, USA); borax and NaOH Normex were Carlo Erba (Milano, Italy)
products; and Congo Red (CR) a Sigma Aldrich (St. Louis, MO, USA) product. For the sample
preparations, distilled water was always used.
3.2. Methods
3.2.1. Polysaccharide Purifications
A given amount of each polymer was dissolved in distilled water (polymer concentration, cp = 0.5%
w/v), and then kept under magnetic and mechanical stirring at room temperature for 24 h. In the case
of GG and LBG samples, the dissolution step was carried out at 60 ◦ C. The obtained solutions were
exhaustively dialysed at 7 ◦ C against distilled water and then freeze-dried. The molecular weight cutoff
of the dialysis tubing was 12,000–14,000.
3.2.2. Hydrogel and Tablet Preparation
For the hydrogel preparation, an appropriate amount of each polymer (i.e., Sclg, GG, and LBG)
was magnetically stirred in water for 24 h (pH of the solutions = 5.5). The calculated amount
(i.e., moles of borax = moles of repeating units of polymer) of 0.1 M borax solution was then added and
the system was left under magnetic stirring for 5 min (pH of the solutions = 9.0 because of the borax
buffer effect). The obtained samples (cp = 0.7%, w/v) were kept overnight at 7 ◦ C for gel-setting. For the
preparation of the tablets, 200 mg of each polymer was used, and after the gel-setting the samples
were freeze-dried. Tablets were prepared from the freeze-dried samples with an IR die (PerkinElmer
hydraulic press) using a force of 5.0 kN for 30 s. The weight of tablets was 230 ± 10 mg. The diameter
and the thickness, measured with a screw gauge, were 13.0 ± 0.1 mm and 1.05 ± 0.02 mm, respectively.
Tablets were also prepared starting from pH = 14 aqueous solutions of the polymers.
3.2.3. Water Uptake and Dimensional Increase Studies
The swelling of Sclg/borax, GG/borax, and LBG/borax tablets, prepared from distilled water
solutions, was carried out by soaking the tablets in NaOH at pH = 14 at 37 ◦ C. On the other side,
the swelling of tablets, prepared from the alkaline polymer solutions (pH = 14) was carried out by
soaking the tablets in distilled water at 37 ◦ C. At fixed time intervals, the tablets were withdrawn,
the excess of solvent was removed with soft filter paper for 5 s, and then the corresponding weights
and dimensional variations along the longitudinal axis were determined by means of a screw gauge
Molecules 2017, 22, 435
11 of 14
with an accuracy of ±0.1 mm. No remarkable variations of cross-section dimensions were detected
during the swelling process. All experiments were carried out in triplicate and the obtained values
always lay within 10% of the mean.
3.2.4. Rheology
A rheological characterisation of the polymer solutions and of the gels prepared at different pH
values was performed, at 25 ◦ C, by means of a controlled stress rheometer Haake Rheo-Stress RS150,
using, as a measuring device, a shagreened plate and plate apparatus (PP35 TI: diameter = 35 mm;
gap between plates = 1 mm). This kind of device is needed to avoid possible slippage phenomena
at the wall [26]. To avoid gel shrinking, due to a possible solvent evaporation, the equipment was
kept inside a glass bell with a constant moisture level. Rheological properties were studied under
small and large deformations, as well as in flow conditions, by applying different procedures: stress
(ν = 1 Hz, ω = 2πν = 6.28 rad/s) and frequency (in the linear viscoelastic region; constant deformation
γ = 0.05) sweep, and flow curves (maximum waiting time, 45 s, for the determination of shear viscosity
at each stress).
3.2.5. Conformational Transition Studies
The capability of polysaccharides to adopt the helix conformation was examined by characterizing
the CR–polymer complexes [15,23]. Aliquots of a NaOH stock solution were added to the polymer
samples (1 g/L) containing 10 µM CR, in order to obtain the desired pH values. The solutions
were left to equilibrate until their UV–vis or circular dichroism (CD) spectra did not change with
time (about 30 min). As a reference, similar experiments were carried out on a 10 µM CR solution.
The absorption spectra were recorded with a Cary 100 spectrophotometer (Varian, Palo alto, CA, USA)
while CD spectra were performed on a J-600 apparatus (Jasco, Easton, MD, USA). In both cases, a 1 cm
path length quartz cuvette was used. For these experiments, 1 g/L of Sclg solution, with or without
borax in a 1:1 ratio was used. Results were compared to those obtained with GG and LBG solutions
(1 g/L).
3.2.6. Molecular Dynamics Simulation
MD simulations were carried out using the software package Gromacs v4.6.5 [27]. The force field
parameters for Sclg and borax were those previously used [6,8,28], while for CR, the parameters were
taken from Król et al. [29]. The simulations were carried out on one triple helix, composed of chains of
7 Sclg repeating units, 1 CR molecule, and roughly 17,000 water molecules [30]. These systems were
simulated in a cubic box (8.2 nm for each side) in an isothermal–isobaric (NPT) ensemble. Electrostatic
interactions were calculated with the particle mesh Ewald (PME) algorithm [31] (cutoff 1.4 nm);
sodium ions were added to assure electroneutrality conditions. A double cutoff was used for
the van der Waals interactions (1.0–1.4 nm). Pressure and temperature were kept constant by the
Berendsen algorithm [32]. Six independent starting configurations were prepared with CR at different
random distances (higher than 1.6 nm) and orientations with respect to the polysaccharide. After the
interactions between CR and triple helix reached equilibrium, the trajectories of the systems were
then analysed for 20 ns. The clusterisations were obtained using a homemade script, to evaluate the
angle between CR and the triple helix, and the g_cluster tool of the GROMACS software package with
a cutoff of 0.3 nm. G_cluster was used also to obtain the more representative structure in the clusters
(i.e., the structure with the smallest average root-mean-square deviation from all the other structures
of the cluster). The structural images were obtained using the Chimera software [33].
3.2.7. Chemometric Analysis
The results of UV–vis experiments on the binding of CR with polysaccharides were processed
by means of the chemometric exploratory data analysis technique, principal component analysis
(PCA) [34,35]. Using PCA, a data set is compressed by projecting the experimental data on
Molecules 2017, 22, 435
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a low-dimensional space without losing the relevant information. This goal can be achieved by
defining the axes of this subspace (called principal components) as those along which the variance of
the projected data is maximised, under the constraint of orthogonality. Mathematically, this concept
takes the form of the bilinear model, X = TPT , where X is the matrix of the original experimental data,
T is the matrix containing the coordinates of the samples in the space of the principal components
(scores matrix), and P is a matrix describing the contribution of the original experimental variables to
the definition of the principal component space (loadings matrix). Chemometric analysis was carried
out using the SIMCA (Soft Independent Modeling of Class Analogy, MKS Instruments AB UMEÅ
Sweden) program.
4. Conclusions
From an overview of the above-reported results, it can be concluded that pH has a remarkable
effect on both Sclg and Sclg/borax systems. While in the case of Sclg, flow curves show the stability
of the polysaccharide triple helix up to pH = 13 with a breakdown of the viscosity at 9 Pa, in the
presence of borax, the initial Newtonian plateau is shifted forward and the viscosity breakdown occurs
at approximately 20–30 Pa. On the other side, at pH = 14, for both systems a dramatic decrease of
viscosity takes place because of the breakdown of the triple-helix structure together with the chemical
degradation of the glycosidic linkages, thus leading to a viscosity that becomes only 4 times higher
than that of distilled water. These effects are also clearly evidenced by the swelling behaviour of
tablets prepared with the Sclg/borax system. When the tablets were swelled at pH = 14, reduced water
uptake and anisotropic elongation were detected due to the beginning of the Sclg chain degradation
process, accompanied by the breakdown of the triple helix. When the polymer for tablet preparation
was preliminary dissolved at pH = 14, the obtained dosage form showed almost no water uptake
and no anisotropic elongation because an almost complete Sclg chain degradation and triple-helix
denaturation already occurred during the polymer dissolution (i.e., before the tablet preparation).
These effects were reduced in the case of the other two tested polysaccharides, GG and LBG, due to
the different initial conformation (coil) and a healing effect (peculiar of these galactomannans) that
somehow slow down the chain degradation induced by the high pH value. For a deeper insight about
the effect of pH on the structure of the Sclg/borax complex, the interaction between CR and the Sclg
triple helix was investigated by spectroscopic and chemometric approaches. The information acquired
from these experiments clearly shows that the borax shifts the triple helix denaturation to higher pH
values, as evidenced also by the rheological characterisation.
Finally, in agreement with the observed CR-induced CD, MD simulations allowed identification
of the most representative structures of the CR–triple-helix complexes: the probe was located mainly
along a perpendicular or parallel orientation with respect of the Sclg chain, as already observed in the
simulations previously carried out in the absence of borax.
Acknowledgments: We acknowledge the CINECA award under the ISCRA initiative, for the availability of high
performance computing resources and support. Financial support from University “Sapienza” (UNIV_2015),
Grant No. C26A15MH7C, is also acknowledged.
Author Contributions: C. Mazzuca, G. Bocchinfuso, A. Palleschi and P. Conflitti designed and carried out the
spectroscopic analysis, PCA and molecular dynamics simulation. M. Grassi carried out the experiments and
analysed the rheological data. C. Di Meo, F. Alhaique and T. Coviello prepared the hydrogel systems, carried out
the water uptake experiments and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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