Rheological Investigation of Thermoresponsive Alginate-Methylcellulose Gels for Epidermal Growth Factor Formulation - MDPI
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cosmetics
Article
Rheological Investigation of Thermoresponsive
Alginate-Methylcellulose Gels for Epidermal Growth
Factor Formulation
Olivia Eskens , Gianna Villani and Samiul Amin *
Laboratory of Cosmetics and Pharmaceuticals, Department of Chemical Engineering, Manhattan College,
Riverdale, New York, NY 10471, USA; oeskens01@manhattan.edu (O.E.); gvillani01@manhattan.edu (G.V.)
* Correspondence: samin01@manhattan.edu
Abstract: Epidermal growth factors (EGF) serve as promising candidates for skin regeneration
and rejuvenation products, but their instability hinders them from widespread use. Protective
immobilization and directed release can be achieved through implementing a hydrogel delivery
system. Alginate and methylcellulose are both natural polymers offering biocompatibility and
environmental sensitivity. This blended gel system was investigated rheologically to understand its
performance in topical applications. Alginate and methylcellulose were found to form a synergistic
gel system that resulted in superior viscosity and thermoresponsiveness compared to the individual
components. Increasing methylcellulose concentration directly enhanced gel elasticity, and higher
viscosities provided better thermal protection of EGF. The addition of EGF at 3.33 mg/mL resulted in
a decrease of viscosity but an increase in viscoelastic modulus. EGF concentration also played a large
role in shear viscosity and thermoresponsiveness of the ternary system. An alginate-methylcellulose
system presents promising rheological tunability, which may provide EGF thermal protection in a
topical delivery format.
Keywords: epidermal growth factor; rheology; thermal response; smart polymers; skin regeneration;
Citation: Eskens, O.; Villani, G.; topical delivery; hydrogel; alginate; methylcellulose
Amin, S. Rheological Investigation of
Thermoresponsive Alginate-
Methylcellulose Gels for Epidermal
Growth Factor Formulation. 1. Introduction
Cosmetics 2021, 8, 3. https://doi.org/
The epidermal growth factor (EGF) is one of the earliest known polypeptide growth
10.3390/cosmetics8010003
factors and the originator of the EGF-like protein family. This class of peptides has highly
similar structural and functional characteristics, promotes mitogenic activity, and typically
Received: 7 December 2020
Accepted: 27 December 2020
uses the same receptors for response triggering [1]. EGF directly affects collagen, elastin,
Published: 30 December 2020
and extracellular matrix biosynthesis, making it a prime candidate for skin regeneration
and rejuvenation. These peptides also have characteristically short half-lives and low
Publisher’s Note: MDPI stays neu- intercellular diffusion rates, allowing them to operate locally in the body but making them
tral with regard to jurisdictional clai- challenging to formulate with. EGF possesses a variety of functional groups and a three-
ms in published maps and institutio- dimensional conformation allowing degradation via chemical and physical pathways such
nal affiliations. as protein denaturation, aggregation, hydrolysis of peptide bonds, or oxidation of amino
acid side chains [2]. The integrity of growth factors is easily compromised, especially in
prolonged storage conditions, by fluctuations in pH, temperature, or complex electrostatic
and hydrophobic interactions [3]. EGF has an optimal pH stability range from 6.0 to 8.0
Copyright: © 2020 by the authors. Li-
with an isoelectric point at 4.6, and an unfolding onset around 40 ◦ C with a transition
censee MDPI, Basel, Switzerland.
midpoint at 55.5 ◦ C [4,5]. Implementing delivery systems for EGF formulation has the
This article is an open access article
potential to reduce the peptide’s instability through diffusion-limiting immobilization and
distributed under the terms and con-
protective encasing [6].
ditions of the Creative Commons At-
tribution (CC BY) license (https://
Polymeric biomatrix characteristics such as water content or crosslink density can
creativecommons.org/licenses/by/
be tailored to optimize the diffusion-controlled EGF retention and release mechanisms.
4.0/).
The physicochemical crosslinking of polymers also leaves them especially responsive to
Cosmetics 2021, 8, 3. https://doi.org/10.3390/cosmetics8010003 https://www.mdpi.com/journal/cosmeticsCosmetics 2021, 8, x FOR PEER REVIEW
Cosmetics 2021, 8, 3 physicochemical crosslinking of polymers also leaves them especially2responsive of 11 to
ronmental changes. In this study, we attempt to tune a hydrogel delivery vehicl
thermoresponsiveness for EGF formulation. The target of the gel system is to effe
immobilize EGF at storage conditions and evenly distribute EGF through therm
environmental shear-induced
changes. In thisgelstudy, we attempt
degradation to tune
upon a hydrogel
topical delivery
application. vehicle with
Hydrogels provide ex
thermoresponsiveness for EGF formulation. The target of the gel system is to effectively
flexibility and hydration for skin compatibility, while extracellular matrix-inspired
immobilize EGF at storage conditions and evenly distribute EGF through thermal and
ery materials utilize the natural growth factor affinity and attachment techniques to
shear-induced gel degradation upon topical application. Hydrogels provide excellent
dermal release. For this reason, alginate is one of the components of the polymer
flexibility and hydration for skin compatibility, while extracellular matrix-inspired delivery
work. Additionally, alginate has previously shown an increase in EGF encapsulatio
materials utilize the natural growth factor affinity and attachment techniques to mimic
ciency as well as inherent regenerative properties [7]. Alginates transform to mat
dermal release. For this reason, alginate is one of the components of the polymeric network.
functions of skin cells and organs, which eventually leads to healing of the affecte
Additionally, alginate has previously shown an increase in EGF encapsulation efficiency as
on the body [8]. Sodium alginate is an anionic, hydrophilic polysaccharide that is ext
well as inherent regenerative properties [7]. Alginates transform to match the functions
from the cell walls of brown algae and various strains of bacteria. The molecular str
of skin cells and organs, which eventually leads to healing of the affected area on the
of sodium alginate is a linear alginic acid copolymer with linked 1-4 glucosidic
body [8]. Sodium alginate is an anionic, hydrophilic polysaccharide that is extracted from
These glucosidic bonds are formed by the homopolymeric blocks of ߚ-D-mannuron
the cell walls of brown algae and various strains of bacteria. The molecular structure of
sodium alginate is aߙ-L-guluronic
and linear alginic acid, outlined inwith
acid copolymer Figure 1. Alginate
linked that contains
1-4 glucosidic bonds.a These
high level of G
glucosidic bonds are formed by the homopolymeric blocks of β-D-mannuronic acidwhile
content results in stiff and brittle gels that tend to be more stable, and high M
content leads to soft, elastic gels with high water adsorption
α-L-guluronic acid, outlined in Figure 1. Alginate that contains a high level of G-block and ion exchange [8].
due to the attractive preferential forces of ions to the extended
content results in stiff and brittle gels that tend to be more stable, while high M-block G-block residues
content leads tothe alginate
soft, elasticstructure.
gels with [9] The
high properties
water of alginate
adsorption and iongels can be[8].
exchange adjusted
This iswith mol
due to the attractive preferential forces of ions to the extended G-block residues withinconstituent
weight, concentration, G-block distribution, cation availability, and the in
tions [8].
alginate structure [9]. The properties of alginate gels can be adjusted with molecular weight,
concentration, G-block distribution, cation availability, and constituent interactions [8].
M-block G-block
(1-4)-linked β-D-mannuronate (1-4)-linked α-L-guluronate
Figure 1. The chemical structure
Figureof1.alginate.
The chemical structure of alginate.
Methylcellulose is the other component selected for the interpenetrated polymer
Methylcellulose is the other component selected for the interpenetrated polym
network due to its thermoresponsive behavior. Methylcellulose is a natural, heterogeneous
work due to its thermoresponsive behavior. Methylcellulose is a natural, heteroge
polymer that forms reversible gels upon heating, and its chemical structure is outlined in
polymer that forms reversible gels upon heating, and its chemical structure is outli
Figure 2. The highly substituted hydrophobic groups within methylcellulose form attractive
Figure 2. The highly substituted hydrophobic groups within methylcellulose form
complexes at increased temperatures, causing gelation [10]. At semi-dilute conditions, as
tive complexes at increased temperatures, causing gelation [10]. At semi-dilute cond
seen in our system, the viscosity of methylcellulose solutions decreases up to a critical
as seen in our system, the viscosity of methylcellulose solutions decreases up to a c
value then increases as a turbid gel develops [10,11]. Mixing alginate with methylcellulose
value then increases as a turbid gel develops [10,11]. Mixing alginate with methylce
creates a complex gel system where hydrophobic interaction, hydrogen bond formation
creates a complex gel system where hydrophobic interaction, hydrogen bond form
between carboxyl and hydroxy groups, and ionic crosslinking are synchronous [12]. The
betweenof
mild gelling conditions carboxyl and hydroxy
both polymers, groups,
along with and
their ionic crosslinking
immunogenic nature, are
makesynchronous
them [12
ideal candidatesmild gelling
for the conditions
preservation of both polymers,
of sensitive along with their
active pharmaceutical immunogenic
components [8,12]. nature,
This study aims to characterize the thermo-rheological behavior of the fluid gel system and comp
them ideal candidates for the preservation of sensitive active pharmaceutical
[8,12].
the effect of EGF whenThis study aims
introduced into to
thecharacterize the thermo-rheological behavior of the flu
polymeric network.
system and the effect of EGF when introduced into the polymeric network.Cosmetics 2021, 8, 3 3 of 11
Cosmetics 2021, 8, x FOR PEER REVIEW 3 of 12
Figure 2. The chemical structure of methylcellulose.
Figure 2. The chemical structure of methylcellulose.
2. Materials and Methods
2. Materials and Methods
Alginic acid sodium salt from brown algae with medium viscosity was obtained from
Alginic acid
Sigma-AldrichsodiumInc. salt(St.
from brown
Louis, algae
MO, withMethylcellulose
USA). medium viscosity wasAlfa
from obtained
Aesarfrom(Ward Hill,
Sigma-Aldrich
MA, USA) with a defined 2% aqueous solution viscosity of 400 cP at 20 ◦Hill,
Inc. (St. Louis, MO, USA). Methylcellulose from Alfa Aesar (Ward C was used.
MA, USA) with a and
Alginate defined 2% aqueous were
methylcellulose solution viscosity of 400
simultaneously cP atinto
stirred 20 °C was used.ofAl-
a solution deionized
ginate and methylcellulose were simultaneously stirred into a solution
water at room temperature. To investigate composition-dependent gel properties, of deionized waterfirst, the
at room alginate
temperature. To investigate
concentration composition-dependent
was held constant at 1% w/v while gel properties, first, theconcentration
methylcellulose algi-
nate concentration
varied between was held1%,constant
1.5%, and at 1%
2%w/v w/v.while methylcellulose
Epidermal concentration
growth factor var- from
was obtained
ied between
Skin1%, 1.5%,Scientific.
Actives and 2% w/v. TheEpidermal
peptide gels growth
were factor was formulated
originally obtained from Skinmg/mL
at 3.33 Ac- and
tives Scientific. The peptide
incorporated prior togels were originally
polymeric formulated
dissolving. After the at 3.33
initialmg/mL and incorpo-
investigation, the alginate
rated prior to polymeric
concentration dissolving.
was varied whileAftermethylcellulose
the initial investigation,
and peptidethe alginate concentra-
concentrations were held
tion wasconstant
varied while methylcellulose and peptide concentrations were
at 2% w/v and 3.33 mg/mL, respectively. Finally, the peptide concentration held constant at was
2% w/v and 3.33inmg/mL,
varied the 1% respectively. Finally, the peptide
alginate–2% methylcellulose concentration
gel system was varied
with additional in the
concentrations of
1% alginate–2%
1.67 andmethylcellulose
6.67 mg/mL. All gelsolutions
system with additional
maintained concentrations
a near-neutral pH.ofThe 1.67viscosity,
and 6.67complex
mg/mL. moduli,
All solutions maintained a near-neutral
and thermoresponsiveness of thepH. The viscosity,
resulting gel systemscomplex
were moduli, and
investigated.
thermoresponsiveness
The Discovery of theHybrid
resulting gel systems
Rheometer were investigated.
(DHR-3) from TA Instruments was employed to
Thedetermine
Discoverythe Hybrid Rheometer
rheological (DHR-3)
properties of thefrom TA Instruments wassystems
alginate-methylcellulose employed bothtowith and
determine without EGF. A stainless
the rheological steel, of
properties 40 mm cone plate with a 2-degreesystems
the alginate-methylcellulose angle was bothusedwithto conduct
all rheological
and without experiments
EGF. A stainless steel, on
40 the
mmtemperature-controlled
cone plate with a 2-degree Peltier plate.
angle wasFlowusedsweeps
to were
conduct carried out between
all rheological the shearonrate
experiments therange of 0.1 to 5000 s−1 . Peltier
temperature-controlled This encompasses
plate. Flow storage,
spreading,
sweeps were carriedpumping,
out between andtherubbing processes
shear rate range during
of 0.1 toproduct
5000 s-1. utilization.
This encompasses An amplitude
sweep was carried out over 0.01% to 50% oscillation
storage, spreading, pumping, and rubbing processes during product utilization. An am- strain with a constant angular fre-
quency of 10 rad s −1 on each gel system to define their individual linear viscoelastic regions.
plitude sweep was carried out over 0.01% to 50% oscillation strain with a constant angular
frequency Frequency
of 10 rads sweeps were gel
-1 on each conducted
system within
to definethetheir
established
individuallinear viscoelastic
linear region with
viscoelastic
− 1
regions. angular
Frequency frequency
sweepsranging from 0.1 towithin
were conducted 100 radthes established
. All of these outlined
linear experiments
viscoelastic re- were
gion with performed isothermally
angular frequency at 25 ◦from
ranging C, and0.1measurements
to 100 rads-1.were taken
All of these under steady-state
outlined experi- sensing
ments wereconditions.
performed Temperature
isothermally sweeps
at 25 evaluated the thermosensitivity
°C, and measurements were taken as under
the gels approach and
steady-
surpasses the skin’s surface temperature of 37.5 ◦ C. A constant shear rate of 10 s−1 was
state sensing conditions. Temperature sweeps evaluated the thermosensitivity as the gels
approach applied to the sample,
and surpasses the skin’sand the temperature
surface temperaturewas increased
of 37.5 by 1◦ C per
°C. A constant shearminute
rate ofbetween
25 ◦ C and 50 ◦ C. All experiments were run a minimum of twice to ensure reproducibility.
10 s-1 was applied to the sample, and the temperature was increased by 1°C per minute
betweenThe 25 °Cdataand presented
50 °C. Allinexperiments
this article are therun
were mean values of of
a minimum each sample’s
twice to ensurevarious
repro-trials, and
across
ducibility. The all
data experiment
presented and sample
in this articletypes,
are thea relative
mean valuesstandard deviation
of each sample’s of 10% or less was
various
trials, and across all experiment and sample types, a relative standard deviation of 10% or for the
observed. Error bars for individual values were excluded in the following graphs
less was sake of clarity.
observed. Error bars for individual values were excluded in the following graphs
for the sake of clarity.
3. Results and Discussion
3. Results andThe deformation and flow behavior of the fluid gel system is important to characterize
Discussion
the storage and release mechanisms of the EGF-loaded product when temperature and
Theshear
deformation
stressorsand
areflow behavior
applied. of thesolutions
Peptide fluid gel are
system
oftenis important to character-
stored at cool temperatures to
ize the storage
prolong and releaselife,
product mechanisms
and a thickof the EGF-loaded product
interpenetrated polymerwhen temperature
network and limit
should further
shear stressors are applied. Peptide solutions are often stored at cool temperatures
EGF deactivation. As the product is topically applied, temperature increases to match to pro-
long product life, and
the body, andahigh
thickshear
interpenetrated
stresses arepolymer
imposed, network
resultingshould further limit
in structural EGF
breakdown and
deactivation. As the product is topically applied, temperature increases to match
pseudoplastic behavior. We aimed to introduce the impact of gel composition on the the body,
and highstructure-property
shear stresses are imposed, resulting
relationship in structural
of the ternary breakdown and pseudoplastic
system.
behavior. We aimed to introduce the impact of gel composition on the structure-property
relationship of the ternary system.Cosmetics
Cosmetics 8,8,
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3.1. Dynamic Viscosity
3.1.The viscosity
Dynamic profile of the fluid gel is linked to the polymeric network and strength
Viscosity
of constituent interactions.
The viscosity profile of Figure 3 explores
the fluid the influence
gel is linked of methylcellulose
to the polymeric network andconcentra-
strength of
tion on the shear viscosity. The flow behavior of sodium alginate-methylcellulose
constituent interactions. Figure 3 explores the influence of methylcellulose concentration systems
has been previously characterized best by the non-Newtonian Hershel–Buckley
on the shear viscosity. The flow behavior of sodium alginate-methylcellulose systems fluid
model [13]. previously
has been The dominant solution interactions
characterized best by the of alginate are charge
non-Newtonian repulsions between
Hershel–Buckley fluid
dissociated
model [13]. carboxyl groups solution
The dominant and the formation
interactions of of
hydrogen
alginatebonds between
are charge carboxylic
repulsions ac-
between
ids and ionized
dissociated carboxyl
carboxyl groups.
groups andMethylcellulose
the formationsolution interactions
of hydrogen bonds rely heavily
between on hy-
carboxylic
drophobic
acids andchain grouping.
ionized carboxyl Both alginate
groups. and methylcellulose
Methylcellulose solutionsolutions haverely
interactions a distinctive
heavily on
pseudoplastic
hydrophobic chain grouping. Both alginate and methylcellulose solutions havein
behavior at neutral pH. Some hydrophobic sections appear alginate
a distinctive
chains upon protonation
pseudoplastic behavior atof neutral
dissociated carboxyl
pH. Some groups [13].
hydrophobic This gives
sections appear rise
into an increase
alginate chains
inupon
hydrophobic
protonationassociation between
of dissociated alginategroups
carboxyl and methylcellulose
[13]. This gives in rise
the interpenetrated
to an increase in
polymer network.
hydrophobic The resultbetween
association is a synergistic
alginatecombination that has a in
and methylcellulose higher viscosity than
the interpenetrated
either individual
polymer network.component
The result while shear-thinning
is a synergistic is preserved,
combination that and
has athis trendviscosity
higher is also seen
than
ineither individual component while
an alginate-methylcellulose mixtureshear-thinning is preserved, and
studied by Mehmandoost et this trendThis
al. [13]. is also seen
is not
in an alginate-methylcellulose
uncommon as enhanced stabilitymixture studied
of physical andby Mehmandoost
mechanical et al. [13].
properties This is not
of biopolymer
uncommon
blends has been as appreciably
enhanced stability
reported of[14–18].
physical and mechanical properties of biopolymer
blends has been appreciably reported [14–18].
Figure 3. The viscosity vs. shear rate behavior of alginate (blue), methylcellulose (yellow), and
Figure 3. The viscosity vs. shear rate behavior of alginate (blue), methylcellulose (yellow), and
mixtures of the two (green) with varying methylcellulose concentration (%w/v). The circular symbols
mixtures of the two (green) with varying methylcellulose concentration (%w/v). The circular sym-
appearing
bols appearingin in
thethe
plots
plotsrepresent
representthe
themean-values
mean-values of discrete measurements
of discrete measurementsevenly
evenlyacquired
acquiredatat
logarithmicintervals
logarithmic intervalsunder
understeady-state
steady-stateconditions,
conditions,while
whilethe
thecontinuous
continuous lines
lines are
are guides
guides for
for the
the eye.
MC:
eye. methylcellulose;
MC: methylcellulose; Alg: alginate.
Alg: alginate.
Theimpact
The impactofofEGF
EGFaddition
additionononthe
theshear
shearviscosity
viscosityofofthe themixtures
mixtureswith
withvarying
varying
methylcellulose concentration can be observed in Figure 4. When
methylcellulose concentration can be observed in Figure 4. When EGF was introducedEGF was introduced
into
into the
the system,
system, therewas
there was a marginalviscosity
a marginal viscositydrop
dropacross
acrossallallsamples.
samples.The
Thelow
low molecular
molecular
weight polypeptide may disrupt the polymer structural entanglement
weight polypeptide may disrupt the polymer structural entanglement slightly, but slightly, butthe
the
synergistic and pseudoplastic nature of the fluid gel was still retained. It
synergistic and pseudoplastic nature of the fluid gel was still retained. It is expected that is expected
that
the hightheviscosity
high viscosity immobilizes
immobilizes EGFtopical
EGF while while application
topical application
begins tobegins to breakdown
breakdown the gel
the gel structure. At neutral pH, EGF carries a negative charge and
structure. At neutral pH, EGF carries a negative charge and may demonstrate repulsivemay demonstrate
repulsive interactions with the anionic alginate chain. EGF is also minorly composed of
interactions with the anionic alginate chain. EGF is also minorly composed of hydropho-
hydrophobic residues, which possibly complicates the hydrophobic relationship between
bic residues, which possibly complicates the hydrophobic relationship between the long-
the long-chain polymers.
chain polymers.Cosmetics 2021, 8, 3 5 of 11
Cosmetics 2021, 8, x FOR PEER REVIEW 5 of 12
5000
1.0% Alg – 1.0% MC
1.0% Alg – 1.5% MC
Viscosity (cP)
1.0% Alg – 2.0% MC
500
1.0% Alg – 1.0% MC + EGF
1.0% Alg – 1.5% MC + EGF
1.0% Alg – 2.0% MC + EGF
50
1 10 100 1000
Shear Rate (1/s)
FigureFigure 4. Shear
4. Shear viscosity
viscosity behavior
behavior of alginate-methylcellulose
of alginate-methylcellulose mixtures(green)
mixtures (green)with
withvarying
varying methylcellulose
methylcellulose concentra-
concentrations
(%w/v)tions
and(%w/v) and epidermal
epidermal growth growth factor (EGF)
factor (EGF) addition
addition at 3.33
at 3.33 mg/mL(red).
mg/mL (red). The
Thecircular
circularsymbols appearing
symbols in theinplots
appearing the plots
represent the mean-values of discrete measurements evenly acquired at logarithmic intervals under steady-state condi-
represent the mean-values of discrete measurements evenly acquired at logarithmic intervals under steady-state conditions,
tions, while the continuous lines are guides for the eye.
while the continuous lines are guides for the eye.
3.2. Viscoelastic Modulus
The dynamic modulus further explores the system interactions and performance by
3.2. Viscoelastic Modulus
evaluating gel stiffness. The storage modulus (G’) of pure alginate is consistently lower
The
than dynamic
the modulus
loss modulus further
(G”) under explores
a variety theconditions,
of gel system interactions
and the same and performance by
development
evaluating
is seen ingel stiffness.
Figure 5 [19].The storage
While modulus
the viscous (G’) of
property of pure alginate
alginate remainsis consistently
dominant, thelower
dif- than
theference
loss modulus
between(G”) under
the two a variety
moduli of gelatconditions,
decreased higher angularandfrequencies
the same development
revealing a de-is seen
in Figure
crease in5 [19].
phaseWhile the viscous
angle and property
a fluid-like of alginate
viscoelastic behavior.remains
On thedominant,
other hand,the difference
methyl-
cellulose
between theviscoelastic
two moduli behavior resembled
decreased moreangular
at higher of an elastic gel system
frequencies where G’aisdecrease
revealing typi- in
cally greater than G”, and the change in phase angle is smaller over
phase angle and a fluid-like viscoelastic behavior. On the other hand, methylcellulosethe range of angular
frequency.behavior
viscoelastic The viscoelastic transition
resembled moreofofthe analginate-methylcellulose
elastic gel system where systems
G’ isistypically
better vis-
greater
ualized in Figure 5c. The G’/G” crossover took place at higher angular
than G”, and the change in phase angle is smaller over the range of angular frequency. frequencies as
methylcellulose concentration increased. The elastic component of the system took
The viscoelastic transition of the alginate-methylcellulose systems is better visualized in
stronger dominance as methylcellulose concentration increased, and gel stiffness was en-
Figure 5c. The G’/G” crossover took place at higher angular frequencies as methylcellulose
hanced. While the variance of total gel concentration between samples generates some
concentration
inconclusively,increased.
this outcomeTheiselastic component
similarly embodied by ofanthe system took stronger dominance
alginate-carboxymethylcellulose
as system,
methylcellulose concentration
as studied by Zheng et al. [19]. increased, and gel stiffness was enhanced. While the
variance of total gel concentration between samples generates some inconclusively, this
outcome is similarly embodied by an alginate-carboxymethylcellulose system, as studied
by Zheng et al. [19].
Recalling that EGF caused a slight decrease in shear viscosity behavior, the impact on
dynamic modulus should give more insight into the molecular interactions happening in
the system. The influence of EGF addition on each gel system with varying methylcellulose
concentration is explored in Figure 6. The strength of the gel does not appear to be
jeopardized, suggesting a more complex interrelationship. It was observed that the change
in phase angle decreases with increasing methylcellulose concentration, but the viscoelastic
transition is not as dependent on methylcellulose concentration when EGF is in the system.
In every instance measured, EGF increased gel stiffness signifying that the loss in viscosity
is not necessarily due to a loss of structure. This is conversely related to the impact of
silicone in a silicated hydroxypropylmethylcellulose-alginate gel network that hindered
effective alginate chain interactions and ultimately reduced gel stiffness [18]. Although
elastic modulus increases with the addition of EGF, the relaxation times shift to higher
frequencies meaning shorter relaxation times. This may signal a new network structure
forming between EGF and methylcellulose due to hydrophobic interaction. The complexity
of the ternary interdependence needs to be more substantially defined in order to fully
interpret viscoelastic properties.Cosmetics 2021, 8, 3 6 of 11
Cosmetics 2021, 8, x FOR PEER REVIEW 6 of 12
100
10
Complex Modulus (Pa)
1
1% Alginate G'
1% Alginate G"
0.1
1% Methyl Cellulose G'
1% Methyl Cellulose G"
0.01
1% Alg – 1% MC G'
1% Alg – 1% MC G"
0.001
0.0001
0.1 1 10 100
Angular Frequency (rad/s)
(a)
100
10
Complex Modulus (Pa)
1
1% Alginate G'
1% Alginate G"
0.1
2% Methyl Cellulose G'
2% Methyl Cellulose G"
0.01
1% Alg – 2% MC G'
1% Alg – 2% MC G"
0.001
0.0001
0.1 1 10 100
Angular Frequency (rad/s)
Cosmetics 2021, 8, x FOR PEER REVIEW 7 of 12
(b)
100
10
Complex Modulus (Pa)
1% Alg – 1% MC G'
1
1% Alg – 1% MC G"
1% Alg – 1.5% MC G'
0.1
1% Alg – 1.5% MC G"
1% Alg – 2% MC G'
0.01 1% Alg – 2% MC G"
0.001
0.1 1 10 100
Angular Frequency (rad/s)
(c)
Figure 5. Viscoelastic moduli G’ () and G” () of the resulting gel systems: (a) 1% w/v alginate (blue), 1% w/v methyl-
Figure 5. Viscoelastic moduli G’ (•) and G” (N) of the resulting gel systems: (a) 1% w/v alginate (blue), 1% w/v methylcellulose
cellulose (yellow), and the mixture of the two (green); (b) 1% w/v alginate (blue), 2% w/v methylcellulose (yellow), and the
(yellow), and the mixture
mixture of the twoof(green);
the two (c) (green); (b) 1% w/v alginate
Alginate-methylcellulose mixtures (blue), 2% w/v
with varying methylcellulose
methylcellulose (yellow),
concentration (w/v).and
The the mixture
circular and
of the two (green); (c) triangular symbols appearing inmixtures
Alginate-methylcellulose the plots represent the mean-values
with varying of discrete measurements
methylcellulose concentration evenly ac- The circular
(w/v).
quired at logarithmic intervals under steady-state conditions, while the continuous lines are guides for the eye. G: storage
and triangular symbols
modulus; appearing
G”: loss modulus. in the plots represent the mean-values of discrete measurements evenly acquired at
logarithmic intervals under steady-state conditions, while the continuous lines are guides for the eye. G: storage modulus;
G”: loss modulus. Recalling that EGF caused a slight decrease in shear viscosity behavior, the impact
on dynamic modulus should give more insight into the molecular interactions happening
in the system. The influence of EGF addition on each gel system with varying methyl-
cellulose concentration is explored in Figure 6. The strength of the gel does not appear to
be jeopardized, suggesting a more complex interrelationship. It was observed that theCosmetics 2021, 8, 3 7 of 11
Cosmetics 2021, 8, x FOR PEER REVIEW 8 of 12
1% Alg – 1% MC G' 1% Alg – 1% MC G"
1% Alg – 1% MC + EGF G' 1% Alg – 1% MC + EGF G"
100
Complex Modulus (Pa)
10
1
0.1
0.01
0.001
0.1 1 10 100
Angular Frequency (rad/s)
(a)
1% Alg – 1.5% MC G' 1% Alg – 1.5% MC G"
1% Alg – 1.5% MC + EGF G' 1% Alg – 1.5% MC + EGF G"
100
Complex Modulus (Pa)
10
1
0.1
0.1 1 10 100
Angular Frequency (rad/s)
(b)
1% Alg – 2% MC G' 1% Alg – 2% MC G"
1% Alg – 2% MC + EGF G' 1% Alg – 2% MC + EGF G"
100
Complex Modulus (Pa)
10
1
0.1
0.1 1 10 100
Angular Frequency (rad/s)
(c)
Figure 6. The impactFigure
of 3.336.mg/mL
The impact of 3.33 mg/mL
epidermal growth epidermal growth
factor addition factor
(red) on addition (red) onmoduli
the viscoelastic the viscoelastic
G’ (•) and G” (N) of
moduli G’ () and G” () of various alginate-methylcellulose mixtures (green) with increasing
various alginate-methylcellulose mixtures (green) with increasing methylcellulose concentration (w/v): (a) 1% alginate–1%
methylcellulose concentration (w/v): (a) 1% alginate–1% methylcellulose; (b) 1% alginate–1.5%
methylcellulose; (b) 1% alginate–1.5% methylcellulose; (c) 1% alginate–2% methylcellulose. The circular and triangular
symbols appearing in the plots represent the mean-values of discrete measurements evenly acquired at logarithmic intervals
under steady-state conditions, while the continuous lines are guides for the eye.Cosmetics 2021, 8, x FOR PEER REVIEW 9 of 12
methylcellulose; (c) 1% alginate–2% methylcellulose. The circular and triangular symbols appear-
ing in the plots represent the mean-values of discrete measurements evenly acquired at logarith-
mic intervals under steady-state conditions, while the continuous lines are guides for the eye.
Cosmetics 2021, 8, 3 8 of 11
3.3. Thermoresponsivity
It is known that composition affects the initial thermal transitions and gelling tem-
3.3. Thermoresponsivity
peratures of compound gel systems [20]. Figure 7a shows the impact of polymer blending
and methylcellulose
It is known that concentration
composition affectson thermoresponsiveness. As expected,
the initial thermal transitions pure alginate
and gelling temper-
showed
atures ofa compound
negligible reaction to increased
gel systems [20]. Figuretemperature,
7a shows while methylcellulose
the impact of polymerdecreased
blending
in
andviscosity until a critical
methylcellulose temperature
concentration is met. The critical temperature
on thermoresponsiveness. As expected, of pure alginate
methyl-
cellulose
showed awas not significantly
negligible reaction toinfluenced by the concentration
increased temperature, range studied.decreased
while methylcellulose As the pol-in
ymers
viscositywere mixed
until together,
a critical thermoresponsiveness
temperature is met. The criticalwas enhanced.ofThe
temperature purehydrophobic gel-
methylcellulose
ling
was mechanism of methylcellulose
not significantly influenced by the is suspected of intensifying
concentration with As
range studied. increasing concentra-
the polymers were
tion,
mixed and hydrophobic
together, interactions from was
thermoresponsiveness alginate enhanceThe
enhanced. thehydrophobic
thermal transition. The
gelling ther-
mecha-
nismreduction
mal of methylcellulose
of viscosityisreveals
suspected of intensifying breakdown
a dual-mechanistic with increasing concentration,
of the prepared gel andnet-
hydrophobic
work during interactions from alginate
topical application. Whenenhance
EGF was theintroduced
thermal transition.
to theseThe thermal
systems, new reduc-
gel
tion of viscositywere
characteristics reveals a dual-mechanistic
observed. Figure 7b shows breakdown of the
a stronger prepared
decrease gel network
in viscosity during
before the
topical gelling
critical application. When EGF
temperature waswas
met.introduced
This may to bethese systems,
attributed newthermal
to EGF gel characteristics
instability
were observed. Figure 7b shows a stronger decrease in viscosity before
and the consequential structural changes impacting the polymeric network. Introduction the critical gelling
temperature was met. This may be attributed to EGF thermal instability
of EGF generally increased the critical temperature in the mixtures studied and proposes and the consequen-
tial structural
that the largelychanges
hydrophilicimpacting theof
structure polymeric network.
EGF somewhat Introduction
obstructs of EGF generally
the hydrophobic gelling
increased the critical temperature in the mixtures studied and proposes that the largely
procedure.
hydrophilic structure of EGF somewhat obstructs the hydrophobic gelling procedure.
1% Alg – 1% MC 1% Alg – 1.5% MC
1% Alginate 1% Methylcellulose
1% Alg – 2% MC 1% Alg – 1% MC + EGF
2% Methylcellulose 1% Alg – 1% MC 1% Alg – 1.5% MC + EGF 1% Alg – 2% MC + EGF
1% Alg – 1.5% MC 1% Alg – 2% MC 1600
1600 1400
1400
1200
1200
Viscosity (cP)
Viscosity (cP)
1000 1000
800 800
600
600
400
400
200
0 200
20 25 30 35 40 45 20 25 30 35 40 45
Temperature (°C ) Temperature (°C)
(a) (b)
Figure 7. The
Figure 7. The response of gel
response of gel systems
systems with
with varying
varying methylcellulose
methylcellulose concentration
concentration (w/v)
(w/v) as
as temperature
temperature increased
increased by
by 11 ◦°C
C
per minute: (a) Alginate (blue), methylcellulose (yellow), and the resulting mixtures of the two (green); (b) The impact
per minute: (a) Alginate (blue), methylcellulose (yellow), and the resulting mixtures of the two (green); (b) The impact of of
3.33 mg/mL epidermal growth factor addition (red) on alginate-methylcellulose mixtures (green). Viscosity values were
3.33 mg/mL epidermal growth factor addition (red) on alginate-methylcellulose mixtures (green). Viscosity values were
continuously acquired as a function of increasing temperature.
continuously acquired as a function of increasing temperature.
The
The influence
influence of
of alginate
alginate on
on the
the mixture
mixture was
was investigated
investigated with
with viscosity
viscosity versus
versus tem-
tem-
perature
perature tests visualized in Figure 8a. The shear viscosity of the mixture seemed to
tests visualized in Figure 8a. The shear viscosity of the mixture seemed be
to be
more strongly dependent on the alginate content. This could be due to the amplification
more strongly dependent on the alginate content. This could be due to the amplification
of
of supportive
supportive electrostatic
electrostatic interactions
interactions but
but also
also increased
increased entanglement
entanglement from
from the larger
the larger
overall polymer content. An effective thermal decrease in viscosity is observed
overall polymer content. An effective thermal decrease in viscosity is observed in these in these
samples, but a critical temperature is not met in the testing range. The ratio of the
samples, but a critical temperature is not met in the testing range. The ratio of the compo-compo-
nents
nents manipulates
manipulates the onset of
the onset of hydrophobic
hydrophobic complexation.
complexation. The The pH-dependent,
pH-dependent, anionic
anionic
nature of alginate makes a good delivery system for cationic drugs through electrostatic
interactions [21]. Sodium alginate interacted with lactoferrin through electrostatic attrac-
tion and increased the heat stability of the protein [22]. It is important to consider the
lactoferrin isoelectric point of 8.8, giving it a net positive charge in the neutral alginateCosmetics 2021, 8, x FOR PEER REVIEW 10 of 12
nature of alginate makes a good delivery system for cationic drugs through electrostatic
interactions [21]. Sodium alginate interacted with lactoferrin through electrostatic attrac-
tion and increased the heat stability of the protein [22]. It is important to consider the
Cosmetics 2021, 8, 3 9 of 11
lactoferrin isoelectric point of 8.8, giving it a net positive charge in the neutral alginate gel,
whereas EGF has an acidic isoelectric point. Interestingly, vascular endothelial growth
factor encapsulation and release behavior were not affected by alginate concentration or
G-block gel,
content evenEGF
whereas though
has it
analso hasisoelectric
acidic a basic isoelectric point of 8.5vascular
point. Interestingly, [23]. Bovine serum growth
endothelial
albuminfactor encapsulation
has a similar andpoint
isoelectric release behavior
to EGF, were
and its not affected
release by alginatehydrogel
from a comparable concentration or
networkG-block
at a pH content even
of 7.4 was thoughdependent
strongly it also has ona basic isoelectric
alginate point of[12].
concentration 8.5 [23]. Bovine serum
Addition-
ally, electrostatic behavior within alginate is not influenced by heat treatment [22]. In Fig-hydrogel
albumin has a similar isoelectric point to EGF, and its release from a comparable
ure 8b, itnetwork
can be at a pH of
clearly 7.4 that
seen was strongly dependent
the critical on alginate
temperature concentration
is increased [12]. Additionally,
with higher net-
polymerelectrostatic
and alginate behavior within alginate
concentrations. There isis not
lessinfluenced
irregularity by as
heat
thetreatment
viscosity[22]. In Figure 8b,
of these
EGF-loadedit cangels
be minimizes,
clearly seenindicating
that the critical
bettertemperature is increased
protective capability. with
The higherenhance-
viscosity net-polymer and
ment may alginate
provideconcentrations. There isstructure,
a more immobilized less irregularity as the viscosity
and alginate of these
electrostatic EGF-loaded
behavior and gels
minimizes, indicating better protective capability. The viscosity
hydrogen bonding could increase the EGF affinity to the gel analogous to findings with enhancement may provide
a more immobilized structure, and alginate electrostatic behavior and hydrogen bonding
other proteins
could increase the EGF affinity to the gel analogous to findings with other proteins
Figure 8. The response of gel systems with varying alginate concentration (w/v) as temperature
increased by 1 ◦ C per minute: (a) Alginate (blue), methylcellulose (yellow), and the resulting mixtures
of the two (green); (b) The impact of 3.33 mg/mL epidermal growth factor addition (red) on alginate-
methylcellulose mixtures (green). Viscosity values were continuously acquired as a function of
increasing temperature.Cosmetics 2021, 8, x FOR PEER REVIEW 11 of 12
Figure 8. The response of gel systems with varying alginate concentration (w/v) as temperature
increased by 1 °C per minute: (a) Alginate (blue), methylcellulose (yellow), and the resulting mix-
tures of the two (green); (b) The impact of 3.33 mg/mL epidermal growth factor addition (red) on
Cosmetics 2021, 8, 3
alginate-methylcellulose mixtures (green). Viscosity values were continuously acquired as a func- 10 of 11
tion of increasing temperature.
Lastly, the impact of EGF concentration on thermoresponsiveness was studied from
Lastly, the impact of EGF concentration on thermoresponsiveness was studied from
Figure 9 for the 1% alginate–2% methylcellulose system. As EGF concentration increased,
Figure 9 for the 1% alginate–2% methylcellulose system. As EGF concentration increased,
the critical temperature of the system decreased, signaling greater thermal instabilities.
the critical temperature of the system decreased, signaling greater thermal instabilities.
Similarly, higher higher
Similarly, concentrations of growth
concentrations factors factors
of growth lead tolead
lowerto encapsulation yields in
lower encapsulation yields in
other alginate delivery systems [8]. It is suspected that at higher EGF concentrations,
other alginate delivery systems [8]. It is suspected that at higher EGF concentrations, the the
protective capability of the gel network is insufficient. With this comes an implied pivotal
protective capability of the gel network is insufficient. With this comes an implied pivotal
ratio between EGF and
ratio between EGFpolymer contentcontent
and polymer where instabilities are effectively
where instabilities minimized.
are effectively The The
minimized.
samplesample
containing the lowest amount of protein demonstrated additional synergy
containing the lowest amount of protein demonstrated additional synergy as the as the
shear viscosity surpassed that of the original alginate-methylcellulose system.
shear viscosity surpassed that of the original alginate-methylcellulose system.
1% Alg – 2% MC 1% Alg – 2% MC + EGF (1.67 ug/mL)
1% Alg – 2% MC + EGF (3.33 ug/mL) 1% Alg – 2% MC + EGF (6.67 ug/mL)
3200
2700
2200
Viscosity (cP)
1700
1200
700
200
20 25 30 35 40 45
Temperature (°C)
Figure 9. The effect of epidermal growth factor concentration (red) on the viscosity of 1% w/v
alginate–2% w/v methylcellulose ◦
Figure 9. The effect of epidermal growthgel mixture
factor (green) when
concentration temperature
(red) increased
on the viscosity byw/v
of 1% 1 C per minute.
algi-
nate–2%Viscosity values were gel
w/v methylcellulose continuously acquired
mixture (green) as atemperature
when function of increased
increasingbytemperature.
1 °C per minute.
Viscosity values were continuously acquired as a function of increasing temperature.
4. Conclusions
4. ConclusionsThere is a wide range of prospective research to further define the structure-property
There is a wide of
relationship thisofalginate-methylcellulose-EGF
range prospective research to furthernetwork.
define theAlginate and methylcellu-
structure-property
lose were found to form a synergistic gel system that resulted in superior viscosity and
relationship of this alginate-methylcellulose-EGF network. Alginate and methylcellulose
thermoresponsiveness compared to the individual components due to an increase in hy-
were found to form a synergistic gel system that resulted in superior viscosity and ther-
drophobic association in the interpenetrated polymer network. The stiffness and elasticity
moresponsiveness compared to the individual components due to an increase in hydro-
of the gel mixture were directly related to methylcellulose concentration. The addition
phobic association in the interpenetrated polymer network. The stiffness and elasticity of
of EGF resulted in a decrease in viscosity but an increase in viscoelastic modulus. A
the gel mixture were directly related to methylcellulose concentration. The addition of
new hydrophobically-associated network is suggested to form with EGF incorporation
EGF resulted in a decrease in viscosity but an increase in viscoelastic modulus. A new
as shorter relaxation times were observed in the ternary system. EGF concentration also
hydrophobically-associated network is suggested to form with EGF incorporation as
played a large role in thermoresponsiveness of the hydrogel mixtures attributed to its
shorter relaxation times were observed in the ternary system. EGF concentration also
inherent thermal instability. Overall, this system presents a great opportunity for a highly
played a large role in thermoresponsiveness of the hydrogel mixtures attributed to its in-
tunable and effective delivery vehicle for EGF with thermoresponsive behavior. The pro-
herent thermal instability. Overall, this system presents a great opportunity for a highly
tectivity of the gel is suspected to be dependent on the ratio of polymer to the peptide as
tunablewell
andaseffective delivery
individual vehicle
polymer for EGF with
composition, but thermoresponsive
this aspect requiresbehavior. The pro-
further investigation.
tectivity of the gel is suspected to be dependent on the ratio of polymer to the peptide as
well asAuthor
individual polymer composition,
Contributions: but and
Conceptualization this methodology,
aspect requires
S.A.;further investigation.
investigation and validation, O.E.
and G.V.; data curation and visualization, O.E.; supervision and analysis, S.A.; writing, O.E. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.Cosmetics 2021, 8, 3 11 of 11
Conflicts of Interest: The authors declare no conflict of interest.
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