biomolecules

Article
Alginate Gel Reinforcement with Chitin
Nanowhiskers Modulates Rheological Properties
and Drug Release Profile
Valentina A. Petrova 1 , Vladimir Y. Elokhovskiy 1 , Sergei V. Raik 1 , Daria N. Poshina 1 ,
Dmitry P. Romanov 2 and Yury A. Skorik 1,3, *
1 Institute of Macromolecular Compounds of the Russian Academy of Sciences, Bolshoy pr. V.O. 31,
St Petersburg 199004, Russia
2 Institute of Silicate Chemistry of the Russian Academy of Sciences, Adm. Makarova emb. 2, St.
Petersburg 199034, Russia
3 Almazov National Medical Research Centre, Akkuratova str. 2., St. Petersburg 197341, Russia
* Correspondence: yury_skorik@mail.ru




Received: 24 June 2019; Accepted: 18 July 2019; Published: 19 July 2019


Abstract: Hydrogels are promising materials for various applications, including drug delivery,
tissue engineering, and wastewater treatment. In this work, we designed an alginate (ALG) hydrogel
containing partially deacetylated chitin nanowhiskers (CNW) as a filler. Gelation in the system
occurred by both the protonation of alginic acid and the formation of a polyelectrolyte complex with
deacetylated CNW surface chains. Morphological changes in the gel manifested as a honeycomb
structure in the freeze-dried gel, unlike the layered structure of an ALG gel. Disturbance of
the structural orientation of the gels by the introduction of CNW was also expressed as a decrease
in the intensity of X-ray diffraction reflexes. All studied systems were non-Newtonian liquids that
violated the Cox-Merz rule. An increase in the content of CNW in the ALG-CNW hydrogel resulted
in increases in the yield stress, maximum Newtonian viscosity, and relaxation time. Inclusion of CNW
prolonged the release of tetracycline due to changes in diffusion. The first phases (0–5 h) of the release
profiles were well described by the Higuchi model. ALG-CNW hydrogels may be of interest as soft
gels for controlled topical or intestinal drug delivery.

Keywords: alginic acid; chitin; hydrogel; tetracycline; drug delivery

1. Introduction
Hydrogels based on natural polysaccharides are promising materials for biomedical applications
due to their biocompatibility, biodegradability, and wide range of physical properties [1].
Polysaccharides can be used in the form of films, sponges, and hydrogels for purposes that include
wound-healing and burn coatings [2,3], tissue engineering [4,5], drug and growth factor delivery [6],
and suturing [7].
Hydrogels and composite materials based on the natural polysaccharide alginic acid (ALG) are well
known and widely used in bone tissue engineering [8], drug delivery [9], and cell encapsulation [10,11].
ALG molecules are linear and contain β-d-mannuronic and α-l-guluronic acid residues that are
present in their pyranose forms and are linked by 1–4 bonds. Ionotropic ALG gels are obtained
by adding multiply charged cations (e.g., Ca2+ , Ba2+ , Cu2+ , Al3+ ), which act as crosslinking agents.
These cations interact with the carboxylic groups of the guluronate units of the polysaccharide molecules,
whereas the mannuronate units remain free [12]. ALG does not form ionotropic gels if the mole fraction
of guluronic acid in the polysaccharide is less than 20–25% [13]. In Reference [14], an electrodialysis
method for the preparation of ALG gels crosslinked with Ca2+ was described. Modulation of

Biomolecules 2019, 9, 291; doi:10.3390/biom9070291 www.mdpi.com/journal/biomolecules

Biomolecules 2019, 9, 291 2 of 13

the conditions for electrodialysis created variations in the degree of gel crosslinking. Homogeneous
gels can be created by the method of delayed gelling of ALG [15], which is accomplished by
the decomposition of calcium carbonate with slow acidification by hydrolysis of D-glucono-1,5-lactone.
The polyanionic nature of ALG allows it to interact electrostatically with polycations to form
polyelectrolyte complexes (PEC) [16,17]. The formation and stability of these PEC depend on numerous
factors, including the degree of ionization, charge density, and molecular weight of the polymers;
the nature and position of ionogenic groups; the flexibility of the polymer chains; the concentration
and order of mixing of the polyelectrolytes during the formation of the PEC; and the temperature, ionic
strength, and pH of the medium [18]. This ability to form PEC increases the attractiveness of ALG
as a hydrogel component.
Recently, much attention has been paid to composite hydrogels that show a combination of
the properties of their individual components [19]. The characteristics of a composite hydrogel are
determined by the physicochemical properties of its components and by the structure of the material.
Various hydrogel structures are possible, ranging from structures with a complete separation of polymer
phases to those comprising a matrix with nanoscale inclusions or with continuous phases of both
polymers. The biphasic nature of composite hydrogels, as a rule, determines their advantages when
used for purposes that include superabsorbents, membrane materials, substitutes for living tissues,
carriers of medicinal substances, and materials for making soft contact lenses. Practically all known
methods can be used to produce composite hydrogels based on hydrophilic polymers; one simple
example is the combination of polymers in solution and their binding as a result of various physical
and chemical interactions [20,21].
Several systems based on ALG/chitosan ionic interactions have been studied. PEC hydrogels used
for drug delivery are usually in the form of microparticles or beads. For example, Sarmento et al. [22]
revealed that anionic PEC particles can provide prolonged insulin release and can increase oral insulin
bioavailability. Applications of chitosan and ALG systems for protein and peptide delivery have
been described in a review [23]. Layer-by-layer coating of ALG hydrogel with chitosan has provided
a vehicle for intestinal delivery of probiotics [24]. Moreover, an ALG gel containing crosslinked
chitosan has shown promise for Hg2+ removal from water solutions [25].
The use of nanofillers consisting of polysaccharides with a fibrillar structure, such as chitin
and cellulose, can lead to interesting properties of the composite materials [26–28]. For instance,
chitin nanocrystals have been incorporated into supramolecular cyclodextrin-based hydrogels
as a way to increase the mechanical strength of the hydrogel and its capability for controlled drug
release [29]. The presence of polysaccharide nanocrystals increases the stability of the hydrogel
structure and provides a stable release profile of a biologically active substance without causing
additional cytotoxicity, compared with the original hydrogel.
Chitin nanowhiskers (CNW) have been used as fillers in the preparation of ALG microcapsules
crosslinked with Ca2+ ions [30]. Partially deacetylated CNW contain positively charged amino groups
and are also capable of interacting with negatively charged carboxyl groups of ALG to form PEC. Thus,
CNW can act as natural crosslinking agents that can change the structure and stability of an ALG
hydrogel, alter the mechanical properties, and modulate the controlled release of drugs. Moreover,
the rigid chitin core provides a defined structure for the nanoparticle, while deacetylated chains on
the surface can be chemically modified [31].
The controlled release of drugs or other biologically active substances can solve problems that arise
in situations where a constant concentration of a therapeutically active compound is needed in the blood,
where a predictable rate of release is required over a long period of time, or where an unstable bioactive
compound must be protected [32]. Antibiotics are a class of drugs that present particular challenges
for controlled release, and the development of antibiotic formulations in the form of hydrophilic
gels has been particularly problematic. Typically, antibiotics (e.g., tetracycline and erythromycin)
are applied as ointments composed of a classic hydrophobic base, usually a mixture of petroleum jelly
and lanolin. However, these ointment bases have their drawbacks, primarily that they are susceptible
Biomolecules 2019, 9, 291 3 of 13

to contamination by pathogenic microorganisms. Hydrophilic bases, including cellulose derivatives

(e.g., methylcellulose, sodium carboxymethylcellulose, etc.) and, more rarely, crosslinked polymers of
acrylic acid or ALG, can also be used as carriers for antibiotics. These bases are resistant to microbial
contamination, are nontoxic, and do not trigger allergic reactions [33,34].
The purpose of the present work was to obtain hydrogels based on ALG and partially deacetylated
CNW. We hypothesized that cationic CNW may act as an active nanofiller that can change the hydrogel
structure. Therefore, we investigated the rheological and structural properties of ALG-CNW hydrogels
and their influence on the tetracycline release profile. The obtained results may be useful for both
topical and intestinal tetracycline delivery.

2. Materials and Methods

2.1. Materials
In this work, we used sodium alginate with a molecular weight (MW) of 1.3 × 105 (Qingdao Bright
Moon Seaweed Group Co. LTD, China).
CNWs were obtained by partial deacetylation of α-chitin with a particle size of 0.1–0.2 mm,
as previously reported [30,31]. The degree of deacetylation (DDA) was determined by conductometric
titration (DDA 0.40 ± 0.03) and by elemental analysis (DDA 0.40 ± 0.02) [31]. The size of the CNWs
(thickness 6–15 nm, length 100–500 nm) was estimated by scanning electron microscopy [31].
Tetracycline hydrochloride was provided by JSC Vertex (St. Petersburg, Russia). Other reagents
and solvents were of reagent grade and were used without further purification.

2.2. Preparation of ALG-CNW Hydrogels

To obtain ALG-CNW hydrogels, concentrated solutions of ALG were mixed with a 0.5% CNW
aqueous dispersion, and the prescribed amount of water was then added to obtain 4% ALG solution.
The resulting ALG-CNW dispersion (10 g) was homogenized for 1 h with mechanical stirring,
followed by addition of 0.2 mL 2% acetic acid solution under vigorous stirring. Hydrogels were
obtained with an ALG concentration of 4% and a mass fraction of CNW of 0, 2.5, 7.5, and 14.5% relative
to ALG. The pH values of the obtained hydrogels were 4.3 ± 0.2. The hydrogels were kept at room
temperature for 1 day and then stored in a refrigerator at 4 ◦ C for 2 days.
Hydrogels containing tetracycline were obtained by mixing an aqueous solution of tetracycline
and ALG. First, 60 mg of tetracycline was dissolved in water and mixed with the ALG solution.
This mixture was then mixed as described above to obtain the ALG-CNW hydrogels. The final
tetracycline concentration in the gel was 1.5 mg/g.

2.3. Isolation of ALG-CNW Microgels

To isolate the ALG-CNW microgels, the ALG-CNW hydrogel was diluted with water to destroy
the physically linked gel. The microgels were separated from the ALG solution by centrifugation
(MPW-380R, Poland) at 4500 rpm, washed with water, and freeze-dried. For measurements of
the hydrodynamic radii and ζ-potential, the microgels were redispersed in water at 1 mg/mL and then
stirred for 24 h. The large aggregates were then separated by centrifugation (2 min, 2000 rpm)
and the suspension was diluted to 0.1 mg/mL.

2.4. General Methods

The rheological properties of the hydrogels were studied at 25 ◦ C with a Physica MCR301
rheometer (Anton Paar GmbH, Graz, Austria) in a CP25-2 cone-plate measuring system for the shear
and oscillatory tests.
X-ray diffraction analysis was performed with a DRON-3M (Burevestnik, St. Petersburg, Russia)
instrument using Ni-filtered Cu Kα radiation (λ = 1.5418 Å).
Biomolecules 2019, 9, 291 4 of 13

The surface morphology was captured by reflected light at 100× magnification using a Levenhuk
D870T optical microscope (Levenhuk Ltd., Long Island City, NY, USA) equipped with a digital camera.
The hydrodynamic radii and ζ-potential of the ALG-CNW microgels and CNW were measured
with a Photocor Compact-Z device (Photocor Ltd., Moscow, Russia) with a 659.7 nm He–Ne laser
at 25 mV power and a detection angle of 90◦ .
Elemental analysis was performed using a Vario Micro Cube analyzer (Elementar Analysensysteme
GmbH, Langenselbold, Germany).

2.5. Tetracycline Release Kinetics

The release of tetracycline from hydrogels containing tetracycline was determined using
the following procedure: 1 g of a gel containing 1.5 mg of tetracycline was placed in a plastic
tube with a dialysis membrane fixed to the end and the tetracycline-containing gel evenly distributed
over the surface of the dialysis membrane. The tube was immersed in a vessel containing 30 mL
saline (0.9% NaCl solution). The release was promoted by constant stirring at 30 ◦ C. At specific
time intervals, 1 mL of the solution was removed, combined with 0.3 mL 1 M NaOH, and used to
determine the concentration of tetracycline spectrophotometrically with an Ocean Optics USB4000
spectrophotometer (Ocean Optics Inc., Largo, FL, USA) using a calibration curve (380 nm, 0–0.05 mg/mL;
R2 = 0.998). The sampled volume was replaced with 1 mL of saline.

3. Results and Discussion

3.1. Preparation of ALG-CNW Hydrogels

The formation of the ALG-CNW hydrogel started immediately upon acidification. A slow increase
in viscosity was observed until the formation of a hydrogel capable of keeping its shape. Presumably,
the gel-forming centers in the ALG-CNW hydrogels were the positively charged CNW, which are
capable of forming PEC with negatively charged ALG molecules. Further gelation is associated
with the formation of a physical ALG gel, possibly due to the conversion of a part of the ALG to
the protonated form or due to the formation of hydrogen bonds between the hydroxyl and carboxyl
groups of the pyranose rings of L-guluronic acid in neighboring polymer chains. The primary
action that leads to the formation of physical gels is molecular entanglements, in addition to ionic
and hydrogen bonding and hydrophobic interactions.
We believe that the ALG-CNW hydrogels are formed both by electrostatic interaction (i.e., formation
of a PEC due to the interaction of the positively charged amino groups of CNW and the negatively
charged carboxyl groups of ALG) and by other physical interactions (molecular entanglements of
ALG chains, hydrogen bonding) and thus represent a two-phase system. When water is added to
the hydrogel, the physical gel is slowly destroyed, while the main part of the ALG goes into solution
(Figure 1).
The molar ratio between the monomeric units of ALG and CNW in the microgels can be estimated
using the elemental analysis data and the following equation:

ωC ω
" #
1 MWN
  
[ALG] : [CNW ] = − C = 3.2, (1)
x ωN ALG−CNW ωN CNW MWC

where x is the number of C atoms in the ALG monomeric units (x = 6); ω is the mass fraction of
the corresponding element (CNW: C 43.09%, N 6.98%; ALG-CNW microgels: C 36.08%, N 1.60%);
and MW is the corresponding molecular weight.
Elemental analysis showed that the microgels represent a PEC formed between CNW and ALG
(with a triple excess of ALG). Unlike the positively charged CNW (ζ potential +20 ± 2 mV) with Rh
of 300 ± 10 nm, the microgels isolated from the ALG-CNW hydrogel had a negative ζ-potential of
-51 ± 1.7 mV and Rh of 725 ± 60 nm (pH of the microgel dispersion was 5.0). Particles with ζ-potential
of more than 30 mV (either positive or negative) are usually considered stable.
Biomolecules 2019, 9, x FOR PEER REVIEW 5 of 13

Biomolecules 2019, 9, x FOR PEER REVIEW 5 of 13

Biomolecules 2019, 9, 291 5 of 13

Figure 1. Scheme of the formation of alginate (ALG) hydrogel containing partially deacetylated
chitin nanowhiskers (CNW).
Figure 1. Scheme of the formation of alginate (ALG) hydrogel containing partially deacetylated chitin
nanowhiskers (CNW).
Figureand
3.2. Structure 1. Morphology
Scheme of the formation of alginate (ALG) hydrogel containing partially deacetylated
of Hydrogels
chitin nanowhiskers
3.2. Structure and Morphology(CNW).
of Hydrogels
The X-ray diffractogram of the ALG-CNW microgels isolated from a hydrogel (Figure 2-2)
The
indicated
3.2. X-ray
the diffractogram
retention
Structure ofof the
of the structure
and Morphology ALG-CNW microgels
of CNW (Figure
Hydrogels 2-1), isolated
except forfrom a hydrogel
a signal (Figure
broadening 2-2)
at 2Ɵ =
indicated
23°. the retention of the structure of CNW (Figure 2-1), except for a signal broadening at 2θ = 23 ◦.
The X-ray diffractogram of the ALG-CNW microgels isolated from a hydrogel (Figure 2-2)
indicated the retention of the structure of CNW (Figure 2-1), except for a signal broadening at 2Ɵ =
23°.

Figure 2. X-ray diffraction patterns: 1—CNW; 2—lyophilized ALG-CNW microgel, isolated from
the ALG-CNW (7.5%) hydrogel; 3—lyophilized ALG hydrogel; 4—lyophilized ALG-CNW (7.5%).
 Figure 2. X-ray diffraction patterns: 1—CNW; 2—lyophilized ALG-CNW microgel, isolated from the
ALG-CNW (7.5%) hydrogel; 3—lyophilized ALG hydrogel; 4—lyophilized ALG-CNW (7.5%).
Biomolecules 2019, 9, x FOR PEER REVIEW 6 of 13
The diffractogram of the lyophilized ALG hydrogel (Figure 2-3) had reflexes at 2Ɵ = 13° and 23°,
Biomolecules
Figure2019, 9, 291 diffraction patterns: 1—CNW; 2—lyophilized ALG-CNW microgel, isolated from the 6 of 13
2. X-ray
which is also characteristic of ALG itself. The diffractogram of the lyophilized ALG-CNW (7.5%)
ALG-CNW (7.5%) hydrogel; 3—lyophilized ALG hydrogel; 4—lyophilized ALG-CNW (7.5%).
(Figure 2-4) was characterized by a significant decrease in the reflex at 2Ɵ = 13° and a weakly
pronounced reflex at 2Ɵ
The diffractogram = 23°,
of the which are
lyophilized ALGalso characteristic
hydrogel (Figureof ALG.
2-3) Thus, the
had reflexes = 13◦ and
analysis
at 2θ 23◦a,
shows
The diffractogram of the lyophilized ALG hydrogel (Figure 2-3) had reflexes at 2Ɵ = 13° and 23°,
differentalso
which structure of the ALG-CNW and ALGThe hydrogels.
which isis also characteristic
characteristic of of ALG
ALG itself.
itself. The diffractogram of
diffractogram of the
the lyophilized
lyophilized ALG-CNW
ALG-CNW (7.5%)(7.5%)
(FigureExamination
(Figure2-4)
2-4)was
was
of the surface
characterized
characterized
morphology
by aby
significant of thin
decrease
a significant
sections of
in the reflex
decrease 2θ = 13at and
lyophilized
in theatreflex
◦ hydrogels
2Ɵ =a weakly
also revealed
13° andpronounced
a weakly
a
different
reflex
pronounced = reflex
at 2θstructural
◦
23 , whichorganization
at 2Ɵare
of the hydrogels
also which
= 23°, characteristic (Figure 3).the
of characteristic
are also ALG. Thus, Foranalysis
the ALG
of ALG. Thus,
hydrogel,
shows we observed a
thea different
analysis structure
shows a
layered
of the structure,and
ALG-CNW andALGfor the ALG-CNW hydrogel, the structure was of the honeycomb type.
hydrogels.
different structure of the ALG-CNW and ALG hydrogels.
Examination
Examination of the surface
of the surface morphology
morphologyof ofthin
thinsections
sectionsofoflyophilized
lyophilizedhydrogels
hydrogels also
also revealed
revealed a
adifferent
differentstructural
structuralorganization
organizationofofthe thehydrogels
hydrogels(Figure
(Figure 3). For the ALG hydrogel, we
3). For the ALG hydrogel, we observed a observed
alayered
layeredstructure,
structure,and andfor
forthe
theALG-CNW
ALG-CNWhydrogel,
hydrogel,thethestructure
structurewaswasofofthe
thehoneycomb
honeycombtype.type.

100 μm 100 μm
a b

100 μm
Figure 3. Micrographs (100×) of thin sections of freeze-dried hydrogels (a) ALG, (b) ALG-CNW 100 μm
(7.5%).
a b
3.3. Rheological Properties of Hydrogels
Figure 3.3.Micrographs
Figure Micrographs(100×)
(100×)ofof
thin sections
thin of freeze-dried
sections hydrogels
of freeze-dried (a) ALG,
hydrogels (b) ALG-CNW
(a) ALG, (7.5%).
(b) ALG-CNW
The rheological properties of the hydrogels were studied by varying the content of CNW in the
(7.5%).
3.3. Rheological Properties of Hydrogels
ALG gel in a range from 0 to 14.5% CNW (relative to ALG).
3.3. Rheological
The Properties
The rheological
rheological testsofofHydrogels
properties the of the hydrogels
hydrogels were studied
were performed usingbyshear
varying
testingthewith
content of CNW
a decrease in
in the
the ALG
shear The gel
rate in a range
(Down
rheological from 0from
SRproperties
mode) toof14.5%
100 CNWto the(relative
the shydrogels
−1 lowest to ALG).by
were possible
studied value (usually
varying 0.0001 s of). CNW
the content
−1 A highinshear
the
rate The rheological
destroys the tests
structure of the
of the hydrogels
gel, thereby
ALG gel in a range from 0 to 14.5% CNW (relative to ALG). were performed
eliminating using
the shear
influence testing
of the with
stressinga decrease
history. inIn
the −1 −1
thisshear
test, arate (Downin
decrease
The rheological SR
tests mode)
the the from
shear
of rate 100 s were
results
hydrogels to athe
in lowestofpossible
growth
performed the
using value
structure.
shear (usually
testing with0.0001 s ). Ainhigh
a decrease the
shear rate
The destroys
dependence the structure
of viscosity of the
and gel,
shear thereby
stress eliminating
on the shear the influence
rate (Figure
shear rate (Down SR mode) from 100 s to the lowest possible value (usually 0.0001 s ). A high shear
−1 of
4) the stressing
indicates
−1 thathistory.
all the
In this
tested test, a decrease
compositions in
are the shear
non-Newtonianrate results
liquidsin a growth
with a of the
structure structure.
characteristic
rate destroys the structure of the gel, thereby eliminating the influence of the stressing history. In of gels.
The
Thedependence
this test, ashear
decrease inofthe
test in viscosity
the Top SR
shear and
rate shearwas
mode
results stress on theout
in carried
a growth sheartherate
ofwith an(Figure
increase
structure. 4) indicates thatrate
in the shear all the tested
from the
compositions
minimum to are
the non-Newtonian
maximum liquids
possible. with a structure characteristic of
The dependence of viscosity and shear stress on the shear rate (Figure 4) indicates that all thegels.
tested compositions are non-Newtonian liquids with a structure characteristic of gels.
The shear test in the Top SR mode was carried out with an increase in the shear rate from the
minimum to the maximum possible.

a b
Figure 4. Dependence of viscosity (a) and shear stress (b) on the shear rate in the shear test
(Down SR mode). Dots represent experimental values, lines were fitted using the Cross equation.
1—ALG, —ALG-CNW a (2.5%), 3—ALG-CNW (7.5%), 4—ALG-CNW (14.5%). b
Biomolecules 2019, 9, 291 7 of 13

The shear test in the Top SR mode was carried out with an increase in the shear rate from
the minimum to the maximum possible.
Dynamic measurements in the oscillatory mode were also conducted by decreasing the angular
frequency from 100 to 0.1 rad/s (Down F mode) and by increasing from the minimum value of
the circular frequency to 100 rad/s (Top F mode).
The shear test in the Down SR mode assumes the most destroyed gel structure, where the system
behaves like a structured liquid (Figure 4) that can be described by the Cross equation with yield stress:
.
. . (η0 − η∞ )γ
τ γ = τ0 + η∞ γ +  . p (2)
1 + θγ

. τ0 (η0 − η∞ )
η γ = . + η∞ +  . p (3)
γ 1 + θγ
. .
where τ γ is the shear stress (Pa) as a function of shear rate (s−1 ); τ0 is the yield stress; γ , η0 , η∞
are the effective viscosity, maximum, and minimum Newtonian viscosity, respectively (Pa·s); θ is
the relaxation time (s); p is the power index (for many polymers, this is equal to 2/3).
The contribution of the yield stress at high rates was not significant and appeared at low shear
rates. The calculation was performed by varying the parameters with an accuracy of 1%; the calculation
criterion was the minimum standard deviation (SD) of the viscosity. The lowest Newtonian viscosity is
usually the viscosity of the solvent (in this case, 0.0009 Pa·s, which is the viscosity of the acetic acid
solution at 25 ◦ C).
The tests were carried out over time at a constant shear rate (or angular frequency); therefore,
the gel was structured and the structure grew with time, regardless of the type of test (Figures 5 and 6).
At this point, the Cross formula no longer correctly described the system. This is especially well seen
by the dependences of the shear stress on the shear rate (angular frequency), as shown in Figures 5
and 6. The Cox-Merz rule (i.e., the dynamic viscosity is equal to the shear viscosity when the values of
the angular frequency and shear rate are equal) did not hold for the studied systems.
In the shear test, the assembly and destruction of the gel structure occurred simultaneously
(especially at high strain rates); therefore, the strength of the structure was somewhat lower than in
the dynamic mode. All the ALG-CNW hydrogels shown in Figure 4 behaved similarly.
All the ALG-CNW compositions after the shear test (Down SR mode) went to the gel state, and no
dependence on the shear rate (angular frequency) was observed. The shear stress induced a flow
that depended on the history of stressing and varied over a wide range. ALG gels with CNW had
a stronger structure, with a yield stress reaching 17,000 Pa for the ALG-CNW (14.5%); for the ALG gel,
this value was lower than 1120 Pa. The effect of the structure on the viscosity was noticeable at strain
rates below 0.001 s−1 . The dynamic loss factor (dynamic loss tangent) ranged from 1 to 0.1, which is
typical for the gel.
The rheological properties of the ALG-CNW hydrogels are summarized in Table 1.
 no dependence on the shear rate (angular frequency) was observed. The shear stress induced a flow
that depended on the history of stressing and varied over a wide range. ALG gels with CNW had a
stronger structure, with a yield stress reaching 17,000 Pa for the ALG-CNW (14.5%); for the ALG gel,
this value was lower than 1120 Pa. The effect of the structure on the viscosity was noticeable at strain
rates below 0.001 s−1. The dynamic loss factor (dynamic loss tangent) ranged from 1 to 0.1, which is
Biomolecules 2019, 9, 291 8 of 13
typical for the gel.
The rheological properties of the ALG-CNW hydrogels are summarized in Table 1.

Biomolecules 2019, 9, x FOR PEER REVIEW 8 of 13

a b
Figure 5. Dependence of viscosity (a) and shear stress (b) on the shear rate (angular frequency) of the
Figure 5. Dependence of viscosity (a) and shear stress (b) on the shear rate (angular frequency)
ALG of hydrogel: 1—shear test
the ALG hydrogel: in the Down
1—shear test inSRthemode,
Down2—dynamic test in the Down
SR mode, 2—dynamic F mode,
test in 3—shear
the Down F mode,
test3—shear
in the Top SR mode, 4—dynamic test in the Top F mode.
test in the Top SR mode, 4—dynamic test in the Top F mode.

a b
Figure
Figure 6. Dependence
6. Dependence of viscosity
of viscosity (a) and(a)shear
andstress
shear(b)
stress (b)shear
on the on the shear
rate rate frequency)
(angular (angular frequency)
of the
of the ALG-CNW
ALG-CNW (7.5%) hydrogel:
(7.5%) hydrogel: 1—shear1—shear testDown
test in the in the Down SR mode,
SR mode, 2—dynamic
2—dynamic test
test in in Down
the the Down
F F
mode,
mode, 3—shear
3—shear testtest in the
in the TopTop
SR SR mode,
mode, 4—dynamic
4—dynamic testtest in the
in the TopTop F mode.
F mode.

Table 1. Rheological properties of ALG-CNW hydrogels (η∞ = 0.0009 Pa·s, p = 0.667).

Table 1. Rheological properties of ALG-CNW hydrogels (η∞ = 0.0009 Pa·s, p = 0.667).
CNW Mass Fraction,
Sample CNWRelative
Mass Weight
Fraction, Test Mode τ0 , Pa η0 , Pa·s η0, θ, s Relative SD, %
Relative
%
Sample Test Mode τ0, Pa 𝜽, s
Relative Weight % Down SR 0.079 0.405 Pa∙s 0.015 SD, %
5.7
ALG 0
Down
Down F SR 20 0.079 – 0.405 – 0.015 – 5.7
Top F 1120 – – –
Down F
Top SR 435
20 –
– –
– –
–
ALG 0
Top F
Down SR 5.1
1120– 0.024 –
3.5 12
–
ALG-CNW
2.5 TopFSR
Down 1560 435– – –– – –
(2.5%)
Top
DownF SR 900 5.1 –
3.5 – 0.024 – 12
ALG-CNW Down SR 1.5 6.5 0.15 27
2.5 Down F 1560 – – –
(2.5%)ALG-CNW Down F 2050 – – –
7.5 Top
(7.5%) Top F F 18,000–44,000
900 – – – – – –
Down
Top SR SR 3100 1.5 – 6.5 – 0.15 – 27
ALG-CNW Down
Down SR F 14.42050 114 – 3.4 – 1.5 –
ALG-CNW 7.5 Down F 1820 – – –
(7.5%) 14.5 Top F 18000–44000 – – –
(14.5%) Top F 17,000 – – –
Top
Top SR SR 14503100 – – – – – –
Down SR 14.4 114 3.4 1.5
ALG-CNW
An increase in the content Down F 1820 – – –
14.5 of CNW in the ALG-CNW hydrogel resulted in increases in the yield
(14.5%) Top F 17000 – – –
stress, maximum Newtonian viscosity, and relaxation time (Figure 7). The spread of the yield stress
Top SR growth1450
was almost equal to 100%; this is due to its constant –
during the testing –
process. –

An increase in the content of CNW in the ALG-CNW hydrogel resulted in increases in the yield
stress, maximum Newtonian viscosity, and relaxation time (Figure 7). The spread of the yield stress
was almost equal to 100%; this is due to its constant growth during the testing process.
Biomolecules 2019,9,9,291
Biomolecules2019, x FOR PEER REVIEW 99of
of13
13
Biomolecules 2019, 9, x FOR PEER REVIEW 9 of 13

a
a

b c
b c
Figure 7. Dependencies of the yield stress (a), maximum Newtonian viscosity (b), and relaxation
Dependencies of
Figure 7. Dependencies
Figure of the
the yield
yield stress
stress (a),
(a), maximum
maximum Newtonian
Newtonian viscosity
viscosity (b),
(b), and
and relaxation
relaxation
time (c) of hydrogels on the CNW content. For the yield stress, the minimum and maximum values
time (c)
time (c) of hydrogels
hydrogels on the CNW content. For For the
the yield
yield stress,
stress, the minimum
minimum and
and maximum
maximum values
values
are shown.
are shown.
are
3.4. Release
3.4. Release of
of Tetracycline
Tetracyclinefrom
fromALG-CNW
ALG-CNWHydrogels
Hydrogels
3.4. Release of Tetracycline from ALG-CNW Hydrogels
Tetracyclinewas
Tetracycline was releasedmore
more slowlyfrom
from theALG-CNW
ALG-CNWhydrogels
hydrogelsthan
thanfrom
fromthe
theALG
ALG gel
gel
Tetracycline was released
released more slowly
slowly from the
the ALG-CNW hydrogels than from the ALG gel
(Figure8).
(Figure 8).
(Figure 8).

Figure 8. Tetracycline release kinetics from gels: 1—ALG, 2—ALG-CNW (2.5%), 3—ALG-CNW (7.5%),
Figure 8. Tetracycline release kinetics from gels: 1—ALG, 2—ALG-CNW (2.5%), 3—ALG-CNW
4—ALG-CNW
Figure (14.5%). release kinetics from gels: 1—ALG, 2—ALG-CNW (2.5%), 3—ALG-CNW
8. Tetracycline
(7.5%), 4—ALG-CNW (14.5%).
(7.5%), 4—ALG-CNW (14.5%).
For the ALG CNW hydrogels, a prolonged release of tetracycline was observed for 24 h and was
For the ALG CNW hydrogels, a prolonged release of tetracycline was observed for 24 h and was
dependent on the amount of CNW (Figure 9).
dependent on the amount of CNW (Figure 9).
Biomolecules 2019, 9, 291 10 of 13

Biomolecules
Biomolecules 2019,
2019, 9, x9,FOR
x FOR PEER
PEER REVIEW
REVIEW 10 10
of of
13 13
For the ALG CNW hydrogels, a
prolonged release of tetracycline was observed for 24 h and was
dependent on the amount of CNW (Figure 9).

Figure
Figure9. Dependence of the fraction of released tetracycline on the mass fraction of CNW at different
Figure 9. 9. Dependence
Dependence of of
thethe fraction
fraction of of released
released tetracycline
tetracycline onon
thethe mass
mass fraction
fraction of of
CNWCNW
at at different
different
release times.
release times.
release times.
Assuming a diffusion-controlled release of tetracycline, the cumulative release curves were
Assuminga adiffusion-controlled
Assuming diffusion-controlledrelease
releaseofoftetracycline,
tetracycline,the
thecumulative
cumulativerelease
releasecurves
curveswere
were
linearized according to the Higuchi model [35] (Figure 10a):
linearized
linearized according
according toto the
the Higuchi
Higuchi model
model [35]
[35] (Figure
(Figure 10a):
10a):
√
Q =𝑄 𝑄 a= =
+
𝑎+𝑎 H+
K 𝐾 𝐾√
t,𝑡,√𝑡, (4)
(4)
(4)
where
where QQis is
thethe cumulative
cumulative tetracycline
tetracycline release
release (%);
(%); KHHKis
H is the Higuchi constant; ist is the time (h).
where Q is the cumulative tetracycline release (%); K is the
the Higuchi
Higuchi constant;
constant; tt is the
the time
time (h).
(h).

aa bb
Figure
Figure
Figure 10.10.
10. (a)(a)
(a) Linearized
Linearized
Linearized tetracycline
tetracycline
tetracycline release
release
release curves
curves
curves plotted
plotted
plotted asas
as a cumulative
aa cumulative
cumulative release
release
release vs.vs.
vs. square
square
square root
root
root of of
of
time
time
time forforfor ALG-CNW
ALG-CNW ALG-CNW hydrogels:
hydrogels: 1—ALG,
hydrogels: 1—ALG, 2—ALG-CNW
2—ALG-CNW
1—ALG, 2—ALG-CNW
(2.5%), 3—ALG-CNW(2.5%), 3—ALG-CNW
(2.5%), 3—ALG-CNW
(7.5%), 4—ALG-CNW (7.5%),
(7.5%),
4—ALG-CNW
(14.5%);
4—ALG-CNW (14.5%);
(b) KH (14.5%);
from the(b)(b)
KHKfrom
HiguchiH from
thethe
model Higuchi
for model
0–5 hmodel
Higuchi release for
vs.
for 0–50–5
CNW h content.
release
h release vs.vs.
CNWCNW content.
content.

The
The
The obtained
obtained
obtained curves
curves
curves were
were
were linear
linear for
linear 0–5
forfor
0–50–5h. KHHKisHisis
h. h.
K proportional
proportional
proportional toto
to the
thethedrug
drug
drug diffusion
diffusion
diffusion coefficient
coefficient
coefficient inin
in
the matrix;
thematrix;
the therefore,
matrix;therefore, the
therefore,the release
therelease within
releasewithin the first
withinthe 5 h
thefirst is prolonged due
first5 5h his isprolonged to
prolongeddue limited diffusion,
duetotolimited as K
limiteddiffusion,
diffusion,linearly
H asas KHKH
decreases with increasing CNW content (Figure 10b). This limited diffusion
linearly decreases with increasing CNW content (Figure 10b). This limited diffusion is a result ofof
linearly decreases with increasing CNW content (Figure 10b). This is
limited a result of
diffusion increased
is a resultgel
viscosity
increased and
increased relaxation
gelgel viscosity
viscosity time
andand (Figure 7).time
relaxation
relaxation The
time parameters
(Figure
(Figure 7).7).ofThe
The the Higuchi model
parameters
parameters ofofthe fitting
the aremodel
Higuchi
Higuchi presented
model in
fitting
fitting
Table
are
are 2. Release
presented
presented kinetics
inin Table
Table after
2. 2.
Releasethe
Release 5-h time
kinetics
kinetics point
after
after could
thethe5-h5-hnot
time be
time correctly
point
point could
could described
notnotbebe with the
correctly
correctly Higuchi
described
described
model
with due
the to significant
Higuchi model swelling
due to and a decrease
significant in
swelling the tetracycline
and a decrease concentration.
in the
with the Higuchi model due to significant swelling and a decrease in the tetracycline concentration. After
tetracycline 5 h, the rates
concentration.
After
After 5 h,
5 h, the
the rates
rates ofof tetracycline
tetracycline release
release from
from thethe swollen
swollen ALG-CNW
ALG-CNW hydrogels
hydrogels were
were similar
similar andand
independent from the CNW content
independent from the CNW content (Figure 8). (Figure 8).
Biomolecules 2019, 9, 291 11 of 13

of tetracycline release from the swollen ALG-CNW hydrogels were similar and independent from
the CNW content (Figure 8).

Table 2. Fitting parameters of the Higuchi model for the tetracycline-containing ALG CNW hydrogels.

CNW Mass Fraction, I Phase (0–5 h)

Relative Weight % R2 a KH
0 0.989 −3.61 34.8
2.5 0.993 −6.79 33.3
7.5 0.988 −5.32 28.7
14.5 0.998 −10.0 23.7

4. Conclusions
Hydrogels were fabricated from ALG and partially deacetylated CNW. The ALG-CNW hydrogels
were formed by various interactions between ALG and CNW polymer chains: electrostatic interactions
upon the formation of PEC, entanglement of ALG chains, and hydrogen bonding. The strength of
the ALG-CNW hydrogels depended on the number of CNW in the gel. The morphology of lyophilized
hydrogels (layered for ALG and honeycomb for ALG-CNW) reflects the features of the structural
organization of the hydrogels. For hydrogels, a more prolonged release of tetracycline was observed
with an increased CNW content in the ALG hydrogel. Release curves correlated well with the Higuchi
model. The mechanism of release prolongation most likely involves the modulation of tetracycline
diffusion in the matrix. This diffusion can be controlled by manipulating the rheological properties
of the gel (viscosity and relaxation time) through changes in the CNW content throughout the ALG
hydrogel. The resulting hydrogels are biopolymers, and they formed simply by the intermolecular
interactions of the polymers used, without the participation of crosslinking agents. These hydrogels
may be of interest as soft gels for prolonged drug delivery.

Author Contributions: Conceptualization, V.A.P.; investigation, V.A.P., V.Y.E., S.V.R., D.N.P., and D.P.R.;
writing—original draft preparation, V.A.P., V.Y.E., S.V.R., and Y.A.S.; writing—review and editing, Y.A.S.;
supervision, Y.A.S.; project administration, Y.A.S.; funding acquisition, Y.A.S.
Funding: This research was funded by the Russian Foundation for Basic Research, project 18-29-17074.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study;
in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish
the results.

References
1. Coviello, T.; Matricardi, P.; Marianecci, C.; Alhaique, F. Polysaccharide hydrogels for modified release
formulations. J. Control. Release 2007, 119, 5–24. [CrossRef] [PubMed]
2. Dai, T.; Tanaka, M.; Huang, Y.Y.; Hamblin, M.R. Chitosan preparations for wounds and burns: Antimicrobial
and wound-healing effects. Expert Rev. Anti-Infect. Ther. 2011, 9, 857–879. [CrossRef] [PubMed]
3. Paul, W.; Sharma, C.P. Chitosan and alginate wound dressings: A short review. Trends Biomater. Artif. Organs
2004, 18, 18–23.
4. Senni, K.; Pereira, J.; Gueniche, F.; Delbarre-Ladrat, C.; Sinquin, C.; Ratiskol, J.; Godeau, G.; Fischer, A.M.;
Helley, D.; Colliec-Jouault, S. Marine polysaccharides: A source of bioactive molecules for cell therapy
and tissue engineering. Mar. Drugs 2011, 9, 1664–1681. [CrossRef] [PubMed]
5. Kiroshka, V.V.; Petrova, V.A.; Chernyakov, D.D.; Bozhkova, Y.O.; Kiroshka, K.V.; Baklagina, Y.G.;
Romanov, D.P.; Kremnev, R.V.; Skorik, Y.A. Influence of chitosan-chitin nanofiber composites on cytoskeleton
structure and the proliferation of rat bone marrow stromal cells. J. Mater. Sci. Mater. Med. 2017, 28, 21.
[CrossRef] [PubMed]
6. Liu, Z.; Jiao, Y.; Wang, Y.; Zhou, C.; Zhang, Z. Polysaccharides-based nanoparticles as drug delivery systems.
Adv. Drug Deliv. Rev. 2008, 60, 1650–1662. [CrossRef] [PubMed]
Biomolecules 2019, 9, 291 12 of 13

7. Wu, H.; Williams, G.R.; Wu, J.; Wu, J.; Niu, S.; Li, H.; Wang, H.; Zhu, L. Regenerated chitin fibers reinforced
with bacterial cellulose nanocrystals as suture biomaterials. Carbohydr. Polym. 2018, 180, 304–313. [CrossRef]
8. Venkatesan, J.; Bhatnagar, I.; Manivasagan, P.; Kang, K.H.; Kim, S.K. Alginate composites for bone tissue
engineering: A review. Int. J. Biol. Macromol. 2015, 72, 269–281. [CrossRef]
9. Tønnesen, H.H.; Karlsen, J. Alginate in drug delivery systems. Drug Dev. Ind. Pharm. 2002, 28, 621–630.
[CrossRef]
10. Chávarri, M.; Marañón, I.; Ares, R.; Ibáñez, F.C.; Marzo, F.; del Carmen Villarán, M. Microencapsulation
of a probiotic and prebiotic in alginate-chitosan capsules improves survival in simulated gastro-intestinal
conditions. Int. J. Food Microbiol. 2010, 142, 185–189. [CrossRef]
11. Ghidoni, I.; Chlapanidas, T.; Bucco, M.; Crovato, F.; Marazzi, M.; Vigo, D.; Torre, M.L.; Faustini, M. Alginate
cell encapsulation: New advances in reproduction and cartilage regenerative medicine. Cytotechnology 2008,
58, 49–56. [CrossRef]
12. Draget, K.; Bræk, G.S.; Smidsrød, O. Alginic acid gels: The effect of alginate chemical composition
and molecular weight. Carbohydr. Polym. 1994, 25, 31–38. [CrossRef]
13. Grasdalen, H.; Larsen, B.; Smidsrød, O. A PMR study of the composition and sequence of uronate residues
in alginates. Carbohydr. Res. 1979, 68, 23–31. [CrossRef]
14. Shchipunov, Y.A.; Greben, V.; Postnova, I. Preparation of calcium alginate gels by electrodialysis. Russ. J.
Phys. Chem. A 2000, 74, 1168–1171.
15. Shchipunov, Y.A.; Koneva, E.; Postnova, I. Homogeneous alginate gels: Phase behavior and rheological
properties. Polym. Sci. Ser. A 2002, 44, 758–766.
16. Sæther, H.V.; Holme, H.K.; Maurstad, G.; Smidsrød, O.; Stokke, B.T. Polyelectrolyte complex formation using
alginate and chitosan. Carbohydr. Polym. 2008, 74, 813–821. [CrossRef]
17. Tam, S.; Bilodeau, S.; Dusseault, J.; Langlois, G.; Hallé, J.P.; Yahia, L. Biocompatibility and physicochemical
characteristics of alginate–polycation microcapsules. Acta Biomater. 2011, 7, 1683–1692. [CrossRef]
18. Etrych, T.; Leclercq, L.; Boustta, M.; Vert, M. Polyelectrolyte complex formation and stability when mixing
polyanions and polycations in salted media: A model study related to the case of body fluids. Eur. J. Pharm.
Sci. 2005, 25, 281–288. [CrossRef]
19. Dragan, E.S. Design and applications of interpenetrating polymer network hydrogels. A review. Chem. Eng. J.
2014, 243, 572–590. [CrossRef]
20. Kabanov, V.A. Polyelectrolyte complexes in solution and in bulk. Russ. Chem. Rev. 2005, 74, 3–20. [CrossRef]
21. Khutoryanskiy, V.V.; Staikos, G. Hydrogen-Bonded Interpolymer Complexes: Formation, Structure and Applications;
World Scientific: Singapore, 2009.
22. Sarmento, B.; Ribeiro, A.; Veiga, F.; Sampaio, P.; Neufeld, R.; Ferreira, D. Alginate/chitosan nanoparticles are
effective for oral insulin delivery. Pharm. Res. 2007, 24, 2198–2206. [CrossRef]
23. George, M.; Abraham, T.E. Polyionic hydrocolloids for the intestinal delivery of protein drugs: Alginate
and chitosan—A review. J. Control. Release 2006, 114, 1–14. [CrossRef]
24. Cook, M.T.; Tzortzis, G.; Khutoryanskiy, V.V.; Charalampopoulos, D. Layer-by-layer coating of alginate
matrices with chitosan–alginate for the improved survival and targeted delivery of probiotic bacteria after
oral administration. J. Mater. Chem. B 2013, 1, 52–60. [CrossRef]
25. Chang, Y.H.; Huang, C.F.; Hsu, W.J.; Chang, F.C. Removal of hg2+ from aqueous solution using alginate gel
containing chitosan. J. Appl. Polym. Ccience 2007, 104, 2896–2905. [CrossRef]
26. Dai, Q.; Kadla, J.F. Effect of nanofillers on carboxymethyl cellulose/hydroxyethyl cellulose hydrogels. J. Appl.
Polym. Sci. 2009, 114, 1664–1669. [CrossRef]
27. Liu, M.; Huang, J.; Luo, B.; Zhou, C. Tough and highly stretchable polyacrylamide nanocomposite hydrogels
with chitin nanocrystals. Int. J. Biol. Macromol. 2015, 78, 23–31. [CrossRef]
28. Yang, X.; Bakaic, E.; Hoare, T.; Cranston, E.D. Injectable polysaccharide hydrogels reinforced with cellulose
nanocrystals: Morphology, rheology, degradation, and cytotoxicity. Biomacromolecules 2013, 14, 4447–4455.
[CrossRef]
29. Zhang, X.; Huang, J.; Chang, P.R.; Li, J.; Chen, Y.; Wang, D.; Yu, J.; Chen, J. Structure and properties of
polysaccharide nanocrystal-doped supramolecular hydrogels based on cyclodextrin inclusion. Polymer 2010,
51, 4398–4407. [CrossRef]
Biomolecules 2019, 9, 291 13 of 13

30. Lin, N.; Huang, J.; Chang, P.R.; Feng, L.; Yu, J. Effect of polysaccharide nanocrystals on structure, properties,
and drug release kinetics of alginate-based microspheres. Colloids Surf. B Biointerfaces 2011, 85, 270–279.
[CrossRef]
31. Petrova, V.A.; Panevin, A.A.; Zhuravskii, S.G.; Gasilova, E.R.; Vlasova, E.N.; Romanov, D.P.; Poshina, D.N.;
Skorik, Y.A. Preparation of N-succinyl-chitin nanoparticles and their applications in otoneurological pathology.
Int. J. Biol. Macromol. 2018, 120, 1023–1029. [CrossRef]
32. Bajpai, A.K.; Shukla, S.K.; Bhanu, S.; Kankane, S. Responsive polymers in controlled drug delivery.
Prog. Polym. Sci. 2008, 33, 1088–1118. [CrossRef]
33. Bardajee, G.R.; Pourjavadi, A.; Soleyman, R. Novel nano-porous hydrogel as a carrier matrix for oral delivery
of tetracycline hydrochloride. Colloids Surf. A Physicochem. Eng. Asp. 2011, 392, 16–24. [CrossRef]
34. Ueng, S.W.; Lee, M.S.; Lin, S.S.; Chan, E.C.; Liu, S.J. Development of a biodegradable alginate carrier system
for antibiotics and bone cells. J. Orthop. Res. 2007, 25, 62–72. [CrossRef]
35. Siepmann, J.; Peppas, N.A. Higuchi equation: Derivation, applications, use and misuse. Int. J. Pharm. 2011,
418, 6–12. [CrossRef]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).