Carbohydrate Polymers 250 (2020) 116877

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

SYNTHESIS OF IN-SITU SILICA-ALGINATE HYBRID HYDROGELS BY A SOL- T

GEL ROUTE
Aurora C. Hernández-Gonzáleza,*, Lucía Téllez-Juradoa, Luis M. Rodríguez-Lorenzobb
a
Instituto Politécnico Nacional, Depto. de Ingeniería en Metalurgia y Materiales-ESIQIE, CDMX, Mexico
b
Depto. Polymeric Nanomaterials and Biomaterials, ICTP-CSIC, Madrid, Spain

A R T I C LE I N FO A B S T R A C T

Keywords: The preparation of silica-alginate hybrid hydrogels by a sol-gel route is proposed in this work. The in-situ
Silica-alginate hybrids synthesis of silica networks from tetraethoxysilane (TEOS) and aminopropyltriethoxysilane (APTES) precursors
synthesis parameters in an alginate matrix is assayed. The experimental parameters were analyzed in three consecutive stages to
sol-gel process obtain hybrid materials with specific properties. Stage 1: effect of water and catalyst ratios. Stage 2: effect of the
inorganic precursor’s ratio. Stage 3: effect of concentration of the alginate solution. The hydrolysis-condensation
reactions of the silica precursors were regulated by the alginate concentration, the silica precursor nature, and
the HCl/inorganic/H2O ratio. Hybrids prepared with both silica precursors generate longer silica chains. High
alginate amounts increase the storage modulus of synthesized hybrids, and high catalyst ratios reduce their
stability. Compositions with alginate content of 5 and 8 (wt/v)%, TEOS/APTES weight ratio of 74/26, and
without catalyst are suitable to evaluation for their application in tissue engineering.

1. INTRODUCTION organic matrix, leading to the formation of mixed inorganic-organic

Hybrids show the combination of the properties of their inorganic-
organic components blended in a single system at the molecular level
(D. A. Sánchez-Téllez, Téllez-Jurado, & Rodríguez-Lorenzo, 2018). In-
organic-organic networks with different structures and morphologies
can be obtained (Mir et al., 2018). These characteristics, together with a
homogeneous distribution of the components, provide new materials
with novel properties, due to the many possible combinations (Skirtach
et al., 2019). Several factors control the structure and morphology of
the hybrid network: 1) selecting different molecular architectures from
different precursors; 2) modifying the reaction conditions; 3) tuning the
inorganic-organic interaction; 4) altering the polymerization procedure
and 5) grafting the inorganic-organic interphase (Bumajdad, Nazeer, Al
Sagheer, Nahar, & Zaki, 2018; Nonsuwan, Puthong, Palaga, &
Muangsin, 2018).
Sol-gel synthesis allows the mixing of two or more dissimilar phases
to form hybrids under mild reaction conditions. It consists of inorganic
polymerization of molecular precursors (metal alkoxides M(OR)n, M =
Si, Ti, Zr, Al, etc., and OR = OCnH2n+1) at low temperature forming
inorganic frameworks of metal-oxo polymers. The polymerization
process in sol-gel overcomes the difficulty of dispersing inorganic fillers
because inorganic precursors are dispersed at the molecular level in the
polymer networks (Demydova et al., 2020; Gon, Tanaka, & Chujo,
2017).
Inorganic polymerization proceeds in two stages, which occur si-
multaneously to form the gel (Innocenzi, 2016): 1) hydrolysis of the
metal alkoxide forming hydroxyl groups in a sol made of highly reactive
MOH species; 2) condensation of MOH species through alkoxylation,
oxolation and olation mechanisms. The metal-oxo network progres-
sively grows from the solution, leading to the formation of oligomers
and subsequently polymers. These reactions are described as SN2 nu-
cleophilic substitutions and are continuously competing between them
as a function of synthesis parameters like nature of the precursors (the
incoming group nucleophilicity, the M electronegativity, and the out-
going group stability), pH (acid or basic catalysis) and alkoxide/H2O
ratio giving different rates of hydrolysis-condensation reactions and
finally, resulting in hybrids with different compositions and morphol-
ogies. Sol-gel reaction rates are also determined by the polymer matrix
nature and the associated steric hindrance factors (Esposito, 2019).
Therefore, it is not possible to generalize the sol-gel reaction parameters
of each type of system.
Nowadays, several polysaccharides have been considered in appli-
cations ranging from stimulus sensitive materials to simulated extra-
cellular matrices in tissue engineering. Alginate is a highly attractive

⁎
Corresponding authors.
E-mail addresses: acitlalli89@gmail.com (A.C. Hernández-González), ltellezj@ipn.mx (L. Téllez-Jurado),
luis.rodriguez-lorenzo@ictp.csic.es (L.M. Rodríguez-Lorenzob).

https://doi.org/10.1016/j.carbpol.2020.116877
Received 31 May 2020; Received in revised form 15 July 2020; Accepted 31 July 2020
Available online 11 August 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.
A.C. Hernández-González, et al.
Table 1
Codes, compositional parameters, and physical description of silica/alginate hybrid hydrogels for Stage I, Stage II, and Stage III.
Stage I
Hybrid code Alg/Si1 Alg/Si2 Alg/Si3
R mol Inorganic/ 0.02
H2O
R mol HCl/inorganic 0 0.15 3 0
ph 8 5 3 8
Physical appearance Viscous Thick Thick emulsion/Zones
emulsion/ emulsion/ of low and medium
gel at 24 h medium viscosity
viscosity
Stage II
Hybrid code Alg/Si4-A Alg/Si4
Rw TEOS/APTES 100/0 81/19
Physical appearance Viscous emulsion Solid gel
Stage III
Hybrid code 8%Alg/Si4+A 5%Alg/Si4+A
Alginate (wt/v)% 8 5
polysaccharide due to its gelling capacity, low toxicity, commercial
availability, and low cost. Alginate hydrogels provide an appropriate
matrix for cell loading. However, they present poor mechanical stability
and must be combined with other materials (Farokhi, Jonidi
Shariatzadeh, Solouk, & Mirzadeh, 2020; Hernández-González, Téllez-
Jurado, & Rodríguez-Lorenzo, 2019; Rinaudo, 2014; Varaprasad,
Jayaramudu, Kanikireddy, Toro, & Sadiku, 2020). The synthesis of al-
ginate-based hybrid hydrogels, through the incorporation of silica-
based domains, represents an attractive option. Silica would limit the
number of alginate hydrophilic molecules, determining their degrada-
tion kinetics and enhancing integrity and mechanical properties. Fur-
thermore, alginate inherent capacity for ionic crosslinking, shear-thin-
ning behavior, and ability to encapsulate bioactive molecules, would
allow its use in combinational tissue engineering strategies (Dodero
et al., 2019; Jeon, Alt, Ahmed, & Alsberg, 2012; Rastogi &
Kandasubramanian, 2019). Silica-alginate hybrids can be obtained by a
sol-gel route introducing the polymer prior to the gelation process.
Shchipunov et al. (Shchipunov & Karpenko, 2004; Shchipunov,
Karpenko, & Krekoten, 2005) reported the preparation of hybrids with
tetrakis (2-hydroxyethyl) orthosilicate (THEOS) and different poly-
saccharides, including alginate. The authors observed that gelling
times, morphology, and viscosity of synthesized hydrogels depended on
the polysaccharide type, charged degree of their macromolecule, and
concentration, which provides a means of manipulating the structural
organization of hybrid polysaccharide-silica nanocomposites. Besides,
Chernev et al. (Chernev, Samuneva, Djambaski, Salvado, & Fernandes,
2006), reported the preparation of hybrid alginate-based materials
using different proportions of the polysaccharide and the tetramethyl
orthosilicate (TMOS) and ethyltrimethoxysilane (ETMS) precursors. In
this paper, the authors analyze the effect of the organic/inorganic re-
lationship on the chemical structure, surface area, and capacity as
carriers for thermostable cell adsorption for use in detoxification of
contaminated sites. From these works, the dependence of the polyionic
nature of the polysaccharide used and the silica precursors are notor-
ious. However, factors such as the water and catalyst ratios, the inter-
action between inorganic precursors, and the growth of the inorganic
network as a function of alginate are still topics barely analyzed. A
careful study of these parameters could lead to a better understanding
of the reactions that promote the hybrid formation and the interaction
between its phases.
In this work, the preparation of silica-alginate hybrid hydrogels is
proposed. The in-situ synthesis of silica networks via sol-gel process
from Tetraethoxysilane (TEOS) and aminopropyltriethoxysilane
(APTES) precursors in an alginate matrix is assayed. The experimental
parameters of the reactions are studied to obtain hybrid materials with
specific properties such as good thermal stability, homogenous organic-
inorganic distribution, and structural stability.
Carbohydrate Polymers 250 (2020) 116877
Alg/Si4 Alg/Si5 Alg/Si6 Alg/Si7 Alg/Si8 Alg/Si9
0.04 0.08
0.15 3 0 0.15 3
5 3 8 5 3
Solid gel Solid gel Viscous gel/low Solid preci- Solid Solid precipitated
continuity pitated precipita-ted and lumps
Alg/Si4+A Alg/Si4-T
74/26 0/100
Solid gel Viscous gel
3%Alg/Si4+A 1.5%Alg/Si4+A
3 1.5
2. EXPERIMENTAL METHODOLOGY
2.1. Hybrid materials synthesis
Silica-alginate hybrid hydrogels were prepared using alginic acid
sodium salt (Mw: 8945 g/mol, mannuronate/guluronate ratio of 0.63,
Sigma-Aldrich) (alginate characterization in Supplementary material 1)
as the organic component; tetraethylorthosilicate (TEOS, Sigma-
Aldrich) and 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich) as
the silica source; hydrochloric acid (HCl, Sigma-Aldrich) was the cat-
alyst and 2-propanol was the common solvent.
The experimental procedure was as follows. Alginate was homo-
geneously dissolved in 5 mL of deionized water. After that, TEOS and
APTES were added dropwise to the solution at intervals of 5 min. Then,
1 mL of water with the specified amount of catalyst was added dropwise
for 10 min. The mixture was homogenized for 5 minutes. Finally, the sol
was kept closed in a plastic container at room temperature until gelling
and aging. All the procedure was maintained at 37 °C with mechanical
stirring and pH control. Inorganic/water and HCl/inorganic molar ra-
tios were (0, 0.15, and 3) and (0.02, 0.04, and 0.08), respectively. The
alginate solutions (wt/v)% were 1.5, 3, 5, and 8. TEOS/APTES weight
ratios were 100/0, 81/19, 74/26, and 0/100. Table 1 shows the codes
and compositions of the obtained samples (Alg/Si hybrid materials
images available in Supplementary material 2). Ionic crosslinking of
selected silica-alginate hybrid hydrogels was performed by completely
immersing the disc-shaped materials (diameter = 40 mm and height =
10 mm) in a 1.5 (wt/v)% solution of CaCl2 for 24 h (CaCl2 Sigma-Al-
drich).
2.2. Characterization techniques
FT-IR spectra were obtained in a PerkinElmer Spectrum Two spec-
trometer coupled with an Attenuated Total Reflection (ATR-FTIR) ac-
cessory. Spectra were recorded in transmittance mode in the range from
4000 to 600 cm-1 with 16 scans and a resolution of 2 cm-1.
The thermogravimetric analysis was performed in a TA instrument,
model TGA Q5000. Samples were heated from 25 °C to 800 °C at a
heating rate of 10 °C/min, keeping a constant N2 flow of 10 mL/min.
SEM analyses were recorded in a JEOL Scanning Electron
Microscope, model JSM6701 with a voltage of 15 kV, and a working
distance of 8 mm. Samples were covered with a gold-palladium layer.
29
Si-NMR analyses were developed in a Bruker Advance TM 400WB
spectrometer equipped with a wide-mouth superconducting magnet (89
mm) operating at 9.4 T at a frequency of 400.14 MHz.
Rheological measurements in wet hybrid hydrogels were carried out
in stress-controlled oscillatory rheometer ARG2 TA Instruments using
parallel plate geometry of 40 mm. Dynamic oscillatory frequency
2
A.C. Hernández-González, et al.
sweeps (FST) in the range from 0.1 to 100 rad/s were carried out at 37
°C with a normal force at ∼0.25 N, setting the oscillatory torque at
∼10 μN.m to determine the storage (G´, elastic behavior) and loss (G”,
viscous behavior) moduli of hybrid hydrogels.
3. RESULTS AND DISCUSSION
3.1. Stage I. Effect of inorganic/H2O and HCl/inorganic molar ratio
In this stage, the inorganic/water and the catalyst/inorganic ratios
effect on the molecular structure and physico-chemical properties of the
obtained hybrids were studied. The hybrids were synthesized using 5
(wt/v)% of alginate solution and TEOS/APTES weight ratio of 74/26.
Nine hybrids were obtained (Table 1).
The molecular structure, microstructure, pore morphology, and
physicochemical properties of the final materials depend on the extent
of the hydrolysis-condensation reactions in the sol-gel process
(Innocenzi, 2016) that are affected by the nature of the precursors, the
catalyst and the molar ratios of alkoxide/H2O/HCl (Milea, Bogatu, &
Duta, 2011; Mota, Gomes, Palhares, Nunes, & Houmard, 2019) (see
Table 1). The hydrolysis reactions of TEOS and APTES form silanol (Si-
OH) groups, which condense to form long and interconnected siloxane
chains giving the silica matrix. Fig. 1(a) shows the infrared spectra of
precursors and hybrids with different inorganic/H2O and HCl/in-
organic molar ratios. FT-IR spectra show absorption bands of inorganic
and organic components. The band at 1641 cm-1 corresponds to the
COO- of alginate, and the bands from 1127 to 781 cm-1 to the silica
network. The bands at 2945, and 942 cm-1 correspond to the CH2rocking
from TEOS and APTES. At 108 cm-1 is observed a low-intensity band of
Si-OC bond from silica precursors. It is worthy to remark the band at
1045 cm-1 assigned to the Si-OH groups from the hydrolysis of silica
precursors and the band at ∼873 cm-1 of their copolymerization to
form Si-O-Si groups (Pena-Alonso, Rubio, Rubio, & Oteo, 2007; Téllez,
Rubio, Peña-Alonso, & Rubio, 2004; Velasco, Rubio, & Oteo, 2001).
Both absorption bands indicate the progress of hydrolysis-condensation
reactions of silica precursors, and the ratio between their intensities and
those of their precursors allows to establish and compare the selectivity
of these reactions in the sol-gel process. Fig. 1(b) shows the infrared
spectra, in the range of 2000 to 700 cm-1, of hybrids with different
inorganic/H2O molar ratio.
Fig. 1. FT-IR spectra (a) APTES, TEOS, alginate, and hybrid materials and (b) hybrid materials with inorganic/H2O molar ratios of 0.02 (left), 0.04 (center) and 0.08
(right) in the range of 2000-700 cm-1. *Black, red and blue arrows: 1083/1045 cm-1 bands ratio. Green arrows: 1045/873 cm-1bands ratio. Yellow arrows: 873 cm-1
band.
3
Carbohydrate Polymers 250 (2020) 116877
The analysis of 1083/1045 (Si-OC/Si-OH) ratio indicates the degree
of hydrolysis reached by the formation of silanol groups from silica
precursors (Téllez et al., 2004). At a higher HCl/inorganic ratio, the
intensity of the 1083 cm-1 band of Si-OC bonds) decreases in compar-
ison with the band at 1045 cm-1 of Si-OH groups (longer blue arrows
compared to red and black arrows, Fig. 1(b)). Hence, hydrolysis reac-
tions are favored at higher HCl/inorganic ratio (pH of the sol ∼3) and
are not affected by the inorganic/H2O ratio.
Otherwise, the kinetics of the sol-gel reactions must have equili-
brium between the hydrolysis/condensation reactions to obtain mate-
rials with a stable structure (Innocenzi, 2016). Alginate is a polyelec-
trolyte used as a thickening, gelling or stabilizing agent. It tends to
absorb large amounts of water due to its hydrophilic character, af-
fecting the hydrolysis reactions in the sol-gel synthesis. Thus, the in-
organic/H2O molar ratio, required to hydrolyze the silica precursors, is
affected by the alginate presence. This parameter influences the final
structure of the hybrids due to the dynamic between hydrolysis-con-
densation reactions, as observed in the FT-IR spectra, in which higher
inorganic/H2O ratios (less H2O amount) yield bands at 1082 and 942
cm-1 (Si-OC bonds of alkoxy groups) with lower intensity. Otherwise,
the band at 873 cm-1, due to the polymerization of silanol groups, is
higher (yellow arrow) indicating that the condensation reactions at
reduced water disposition are mostly favored. Nevertheless, the avail-
able H2O is enough to hydrolyze the silica precursors in the inorganic/
H2O ratios used. However, the physical appearance is affected (Table 1
and Supplementary material 2), and this can be related to the Si-O-Si/
Si-OH bands intensity ratio (green arrows), due to the condensation
degree of silanol groups forming the silica matrix.
On the other hand, alginate may be depolymerized by the presence
of HCl (Haug & Larsen, 1966; Jensen, Larsen, & Engelsen, 2015) so that,
the alginate structural integrity is preserved in the absence of the cat-
alyst (Bridelli, 2017).
The hybrids´ thermal behavior shows six stages (Fig. 2): (1) 30-75
°C: with 5% weight loss due to hybrid dehydration. (2) 75-200 °C: with
∼4% weight loss due to alginate denaturation and the release of sol-
vents and water evaporation from the hybrid structure. (3) 200-250 °C:
with ∼12% weight loss due to the alginate depolymerization. (4) 250-
500 °C: with ∼12% weight loss due to the silanol polycondensation as a
consequence of the thermal stimulus (Carvalho, Suzana, Santilli, &
Pulcinelli, 2014) and also to the onset alginate degradation. (5) The
A.C. Hernández-González, et al. Carbohydrate Polymers 250 (2020) 116877

NaCO3 formation (∼5% weight loss), and (6) its thermal degradation
(∼5% weight loss) (Soares, Santos, Chierice, & Cavalheiro, 2004). The
hybrids´ thermal stability is affected by the HCl/inorganic molar ratio,
due to the alginate chains breakdown (by the catalyst presence),
causing rapid depolymerization with subsequent thermal degradation
so that, at a higher HCl/inorganic ratio lower thermal stability in hy-
brids.
The hybrids morphology was analyzed by SEM (Fig. 3). Hybrids
show particles or agglomerated particles with semispherical mor-
phology due to silica polymerization. Such morphologies depend on the
synthesis parameters (Sun, Zhou, & Zhang, 2016). Hybrids prepared
without catalyst (pH ∼ 8) show grape-shaped clusters formed with
nanoparticles of ∼50 nm of diameter. Hybrids with HCl/inorganic =
0.15 and 0.3 ratios (pH < 6) show particles with > 250 nm size. At
higher HCl/inorganic ratio, the presence of agglomerates of particles is
higher because the hydrolysis reaction prevails, according to the FT-IR
and TGA analysis.
Then, it is considered that the dynamic of hydrolysis/condensation
reactions controls the hybrids morphology through two possible pro-
cesses in which nuclei yield silica networks: the nuclei grow to produce
large particles (“particle growth process”) or the smaller particles are
agglomerated to construct larger ones (“aggregative growth model”)
(Bogush & Zukoski Iv, 1991). The highest HCl/inorganic ratio promotes
the “particle growth process” while the absence of HCl promotes the
“aggregative growth model” (S. Chen et al., 2009; S. Chen et al., 2008).
However, it is possible that other factors, like electrostatic interactions
between alginate and silanols formed during the sol-gel process, influ-
ence the network structure and the materials particle size (Shchipunov
& Karpenko, 2004). Coradin et al. (Coradin & Livage, 2003) suggest that
alginate does not inhibit the nucleation of silica particles, but it inter-
feres in the assembly of pre-formed particles. Also, authors like Cai
et al. (Cai et al., 2020) and Schchipunov et al. (Shchipunov et al., 2005)
suggest that the polysaccharide generates a catalytic effect in the silica
precursor in the sol-gel process.
On the other hand, particles with sizes > 100 nm are observed for
hybrids synthesized at the lowest inorganic/H2O molar ratio. And ag-
glomerates of nanoparticles with sizes ≤100 nm are formed for the
highest inorganic/H2O ratio. The production of discrete particles based
on the solubility of intermediate species favors the formation of smaller
diameters. However, the accelerated condensation of these discrete
particles may result in fractional structures, as in hybrids with the
lowest inorganic/H2O ratio. Otherwise, alginate is not observed in the
SEM images, even though it affects the dynamic of hydrolysis/con-
densation reactions and contributes to hybrids structure (FT-IR and
TGA analysis). The physical appearance of the hybrid is also affected.
Hybrid materials with high catalyst proportions result in sectioned
materials with heterogeneous zones (Table 1 and Supplementary ma-
terial 2), which is closely related to alginate depolymerization at low
pH. This “sectioning effect” was observed by Witoon et al. (Witoon,
Chareonpanich, & Limtrakul, 2009) who hypothesized that the growth
of silica nanoparticle clusters is restricted by the incorporation of
polysaccharide chains, in agreement with Coradin et al. (Coradin &
Livage, 2003) who suggested that the silica particle assembly may be
controlled by its limited diffusion in the viscous alginate solutions. Base
on the above, the conception of hybrid materials is as follows. Silica
nuclei grow through the surface and inner face of the alginate matrix to
form nanoparticles. Then they lump together giving particle agglom-
erates whose growth and size are restricted by the alginate chains
(Metwalli, Haines, Becker, Conzone, & Pantano, 2006). According to IR
and TGA analysis, the catalyst causes partial alginate depolymerization
(Haug & Larsen, 1966; Jensen et al., 2015) producing discontinuity of
Fig. 2. Thermograms of hybrids with inorganic/H2O molar ratio of (a) 0.02, (b)
both (silica and alginate) networks in the hybrid matrix (Witoon et al.,
0.04 and (c) 0.08.
2009) and forming silica agglomerates zones with a higher size (SEM).
The catalyst absence favors an interpenetrated silica/alginate network
in which the alginate structure is preserved, and the polycondensation
of Si-OH groups takes place within this structure. As a result, the growth

4
A.C. Hernández-González, et al.
Fig. 3. Micrographs of hybrids as a function of inorganic/H2O and HCl/inorganic molar ratio.
of the silica network is limited, giving smaller agglomerates of silica
particles. However, it also allows a better distribution in the hybrid
structure and the formation of interpenetrated networks of alginate and
silica, as represented in Fig. 4.
Based on the physicochemical analysis of Alg/Si hybrid materials
from Stage I and the discussion above, Alg/Si4 hybrid composition was
selected to continue the analysis and optimization of the alginate-silica
hybrid composition.
3.2. Stage II. Effect of silica precursors
In this Stage, we studied the effect of silica precursors on the hy-
brids´ physicochemical properties. Therefore, hybrids with different
TEOS/APTES weight ratios (100/0, 81/19, 74/26, 0/100) were syn-
thesized (Table 1). From previous results (Stage I), the selected synthesis
parameters were: inorganic/H2O = 0.04 molar ratio, alginate/in-
organic = 10/90 weight ratio, and no HCl.
The hybrids properties are related to the nature and proportion of
precursors. TEOS is frequently used as a silica precursor due to its re-
activity and size of alkyl substituent (Fidalgo, Ciriminna, Ilharco, &
Pagliaro, 2005). APTES is a silane coupling agent with an NH2 terminal
group that, together with its moderate length, enhances the reactivity
and potential bio-functionality of the materials. The acid-based char-
acter of silanol and amino groups are different, and the surface amino
groups control the electrostatic repulsive forces working the adjacent
particles which are relevant in the silica particle growing process. The
TEOS and APTES combination may confer a further tool in the control
of the homogeneity in the final products (Danks, Hall, & Schnepp,
2016).
The gelling times and physical appearance of hybrids depend on the
TEOS/APTES weight ratio (Table 1 and Supplementary material 2). The
hybrid sample prepared without APTES displays a liquid aspect, even
after one month of the synthesis, while the hybrids obtained without
TEOS and with 81/19 and 74/26 TEOS/APTES weight ratios showed
gelling times of 24 h, 1 h and 0.25 h, respectively. As the APTES content
5
Carbohydrate Polymers 250 (2020) 116877
increases, the gelling time decreases.
29
Si-MAS-NMR spectroscopy (Fig. 5) identifies the species formed
during the sol-gel process and the achieved crosslinking degree of the
network as a function of the TEOS/APTES weight ratio.
The 29Si-NMR spectra shows peaks from -51 to -110 ppm related to
the hybrids´ composition. Hybrid without APTES shows peaks at -102
and -112 ppm due to Q3 and Q4 species of interconnected silica
structure from the condensation of silanol groups (TEOS hydrolysis)
(Munguía-Cortés et al., 2017).
The hybrids with TEOS/APTES ratios of 81/19 and 74/26 show
peaks at -59, -67, -90, -100, and -110 ppm due to (T2, T3) and (Q2, Q3,
Q4) species from APTES and TEOS, respectively. Hybrid without TEOS
shows peaks at -51, -61, and -69 ppm due to the T1, T2, and T3 species
of the formed > SiCH2CH2CH2NH2. The signals of neat APTES (-45
ppm) and TEOS (at -82 ppm) (Kim, Han, Kim, & Lee, 2011) are not
observed. Then, the silica precursors were hydrolyzed in all synthesized
hybrids, and the polycondensation step progressed enough allowing the
T0 and Q1 structures to react (Uhlig & Marsmann, 2008). Table 2 shows
the chemical shifts and peak areas from 29Si-NMR spectra.
Hybrids with TEOS/APTES precursors show a higher displacement
towards Q3 and T3 structures, followed by Q4 units. Thus, the hybrids
are not reaching the maximum coordination number from TEOS poly-
merization, due to the crosslinking with intermediate hydrolyzed
structures from APTES (D. Sánchez-Téllez, Rodríguez-Lorenzo, &
Téllez-Jurado, 2020). Hybrid without APTES presents the pre-
dominance of Q3 units compared to Q4 units, indicating that the
maximum polycondensation degree of silanol groups is not reached,
probably due to the interaction with the alginate matrix. Hybrid
without TEOS yield the highest area for T3 units, indicating that a large
number of APTES molecules reached the maximum condensation de-
gree; also, T2 and T1 units are present in a smaller proportion, de-
creasing the branching structure and reducing the gel stability.
Rheological measurements were used for the elucidation of the
viscoelastic characteristics of the obtained hybrid materials and the
correlation with other physicochemical properties. Fig. 6 shows the
A.C. Hernández-González, et al.
Fig. 4. Schematic representation of hybrids using HCl/inorganic molar ratios of 0.3, 0.15, and 0.
29
Fig. 5. Si-MAS-NMR spectra of hybrids with different TEOS/APTES weight
ratio.
frequency sweep tests (FST) of hybrids with different TEOS/APTES
weight ratios.
Hybrid without APTES shows loss moduli (G”) greater than the
storage moduli (G’), in agreement with its liquid character (Table 1).
The other hybrids display a G’ higher than G” with a gel character. The
highest G’ value are observed for the hybrid with TEOS/APTES ratio of
74/26, while the lowest G’ values are observed were for the sample
without APTES. These results suggest that hybrids obtained with TEOS
and APTES present a higher crosslinking degree giving stronger mate-
rials than those synthesized with TEOS or APTES only, according to the
higher T3 and Q3 signals ratio (29Si-NMR and Table 3). Also, tan (G”/
6
Carbohydrate Polymers 250 (2020) 116877
G’) during the FST test was analyzed. Materials are predominantly
viscous at tan (δ) > 1, and predominantly solid at tan (δ) < 1 (Adibnia,
Taghavi, & Hill, 2017; Tao et al., 2016). Fig. 6(b) shows that the hybrids
with TEOS/APTES ratio of 81/19 (Alg/Si4) and 74/26 (Alg/Si4+A)
present tan (G”/G’) values ∼0.2, decreasing at higher angular fre-
quencies. Hybrid without TEOS shows an initial tan (G”/G’) value, si-
milar to those mentioned hybrids, but at higher angular frequency, it
shows higher values. This is interpreted as follows. When frequency
increases during the assay, the timescale of the input stress of the hy-
brid decreases. Then, the hybrid has less time to recover its initial
configuration, progressively increasing its storage modulus. Besides, G”
is correlated to the stress/dissipated energy ratio and directly related to
the viscous component of the material. When materials have a highly
branched inorganic network, the silica distribution within the hybrid
reduces the formation of viscous zones, and thus, they store more en-
ergy than they dissipate, as a consequence G” is lower, as observed in
hybrids with 81/19 and 74/26 of TEOS/APTES ratios (Adibnia et al.,
2017). These hybrids show a predominance of T3 and Q3 units (29Si-
NMR) with more branched networks.
Hybrid without TEOS, with a low-branching inorganic network,
shows viscous zones that dissipate the mechanical energy meaning that
the G” becomes higher at higher frequencies and the material tends to
flow at lower frequencies (Malkin & Isayev, 2017). Otherwise, hybrids
without APTES show the highest tan (G”/G’) value at low angular fre-
quencies, but it decreases at higher angular frequencies. This negative
slope indicates that the system is predominantly viscous with higher
chains´ mobility at smaller deformations due to a low inorganic poly-
condensation degree, causing liquid mobility.
A.C. Hernández-González, et al. Carbohydrate Polymers 250 (2020) 116877

Table 2
29
Chemical shift, peak areas, and peak areas (%) from Si-NMR spectra of hybrids with different TEOS/APTES weight ratio.
From Unit Chemical shift Peak area Area (%) Peak area Area (%) Peak area Area (%) Peak area Area (%)
(ppm)

A T1 —— —— ——— ——— 0.02 1.41

P T2 58 0.41 10.85 0.25 8.28 0.40 28.17
T T3 67 1.00 26.46 1.03 34.11 1.00 70.42
E
S
T Q2 —— ——— —— 0.13 4.30 0.22 5.82
E Q3 102 1.00 64.81 1.00 33.11 1.32 34.92
O Q4 111 0.54 35.19 0.61 20.20 0.83 21.90
S

hydrolysis-condensation reactions of APTES results in a low poly-

merization degree of silica structures with non-connected silanol groups
into the alginate network (T1 and T2, Table 2), resulting in low storage
and loss moduli, and as a consequence low mechanical integrity.
In TEOS/APTES mixed with alginate, APTES catalyze the sol-gel
reactions through the fast release of -OH radicals (S. Chen et al., 2009);
also, the APTES steric effect decreases with the TEOS interaction.
Therefore, the silanol condensation from the hydrolysis of both silica
precursors generates longer silica chains leading to greater connectivity
and mechanical stability in hybrids (Fig. 6(a)).

3.3. Stage III. Effect of alginate concentration

From the results of the hybrid with higher branched structure and
good rheological behavior, above analyzed, TEOS/APTES = 74/26
molar ratio, inorganic/H2O = 0.04 molar ratio and without HCl, are
the parameters used in this Stage. Fig. 7 shows the 29Si-NMR spectra of
hybrids with different alginate concentration.
The signals at -50 to -120 ppm belong to the silica network struc-
tures. Hybrids show peaks at -59, -67, -90, -100, and -110 ppm due to
(T2, T3) and (Q2, Q3, Q4) structures from APTES and TEOS, respec-
tively. The signal at -90 ppm (Q2) shows lower intensity in hybrids with
3%Alg and 1.5%Alg in which T3 structures show higher areas to the
observed in 5% and 8%Alg hybrids. This may be explained by the low
alginate concentration, which allows the APTES reactions more easily
with a less steric impediment. However, for 8%Alg/Si4+A hybrid, an
increase in the T2 area is observed. A possible explanation is that at

Fig. 6. Graphs of a) storage and loss modulus, and b) tan (loss modulus/storage
modulus) during FST of hybrids with different TEOS/APTES weight ratio.

TEOS shows slow reaction kinetics in the catalyst absence (Brinker,

1988; S.-L. Chen, Dong, Yang, & Yang, 1996) and with the alginate
presence; a slowdown of condensation reactions giving longer gelling
times is observed. So, the hybrid with TEOS as a unique silica precursor
shows the rheological behavior of a viscous liquid because of the in-
complete condensation reactions.
APTES is easily hydrolyzable (Al-Oweini & El-Rassy, 2009), but the
presence of alginate affects hydrolysis-condensation reactions due to
the basic pH of the solution. At this condition is formed six- or five-
membered intramolecular rings (Van Blaaderen & Vrij, 1993) that re-
duce the APTES condensation degree due to steric effects. The low Fig. 7. 29
Si-NMR spectra of hybrids with different alginate concentration.

7
A.C. Hernández-González, et al.
Fig. 8. FST (a) hybrid materials with different alginate concentration (b) effect
of ionic crosslinking with a 1.5(wt/v)% of CaCl2.
very high percentages of alginate, phase separation is obtained. Zones
rich in silica enable the progressing of the polycondensation reactions.
The 5%Alg/Si4+A hybrid shows the highest percent area for Q4
structures. Considering that the Q4 units indicate the highest poly-
condensation degree of TEOS, 5%Alg/Si4+A hybrid may be the ma-
terial with the highest structural stability. However, as determined in
Stage II study, a higher proportion of Q4 units does not necessarily
indicate a highly branched structure and a homogeneous structure
(Table 2 and Fig. 6). Therefore, it was necessary to perform a rheolo-
gical analysis to elucidate the most stable material.
The G’ and G´´ moduli of all hybrids show gel behavior (Fig. 8(a)).
Besides, at high alginate concentration (8%Alg and 5%Alg), higher G´
and G´´ are observed than for low alginate concentration. The 1.5%Alg
hybrid shows stability in the range of 0.6 to 30 rad/s but at higher
frequencies, the gel breaks down.
In addition, alginate enables the possibility of modifying the hy-
brids´ properties by crosslinking with divalent cations to form ionic
bridges (Hernández-González et al., 2019) (Supplementary material 1).
Fig. 8(b) shows G´ and G´´ of crosslinked hybrids. At lower alginate
concentrations (1.5 and 3%Alg), it is observed that the moduli barely
increase, showing lower structural stability at higher frequencies (∼40
rad/s) than before ionic crosslinking. However, at higher alginate
contents (5 and 8%Alg) G’ and G” increase, maintaining a similar G”/G’
ratio. This indicates stronger gels due to the added bridges from algi-
nate ionic interaction.
Hybrid alginate-silica based hydrogels have been prepared as a
8
Carbohydrate Polymers 250 (2020) 116877
proposal to overcome the limitations of alginate gels described in the
introduction. Parameters affecting the sol-gel reactions and the stability
of the obtained hybrids have been analyzed and the following conclu-
sions achieved.
4. Conclusions
The synthesis of silica-alginate hybrid hydrogels is possible through
an in-situ sol-gel reaction. The synthesis parameters affect the hydro-
lysis-condensation reactions of the silica precursors and change the way
that alginate interacts with them, yielding hybrids with different in-
terconnection degrees in the network and with different mechanical
stability. Polycondensation reactions are favored at high inorganic/H2O
ratios, while hydrolysis reactions at high HCl/Inorganic ratios are fa-
vored. However, the alginate matrix may suffer depolymerization due
to the HCl presence. The crosslinking degree of the inorganic network is
related to the TEOS/APTES ratio. Hybrids synthesized with both TEOS
and APTES precursors generate longer silica chains, higher con-
nectivity, and mechanical stability than hybrids with only APTES or
TEOS. The use of highly concentrated alginate solutions yields hybrids
with higher storage modulus than hybrids synthesized with low con-
centration alginate solutions. Alginates enable the possibility of using
an ionic post-crosslinking method to modulate the features of the ma-
terials. Experimental parameters for compositions with alginate content
of 5 and 8 (wt/v)%, TEOS/APTES weight ratio of 74/26, and without
catalyst are suitable to continue with mechanical and biological testing
for the application in tissue engineering.
CRediT authorship contribution statement
Aurora C. Hernández-González: Conceptualization, Methodology,
Validation, Investigation, Writing - original draft, Visualization. Lucía
Téllez-Jurado: Resources, Writing - review & editing, Visualization,
Supervision, Project administration, Funding acquisition. Luis M.
Rodríguez-Lorenzob: Resources, Writing - review & editing,
Visualization, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
Funding: This work was supported by Instituto Politécnico Nacional,
México (IPN-SIP 20196660 and IPN-SIP 20201294), and AEI/FEDER,
UE (DPI2017-90147-R). Financial support from CONACYT scholarship
is acknowledged.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116877.
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