Poly(2-oxazoline) hydrogels crosslinked with aliphatic bis(2-oxazoline)s: Properties, cytotoxicity, and cell cultivation

Article abstract of DOI:10.1002/pola.28009

A series of hydrogels from 2-ethyl-2-oxazoline and three bis(2-oxazoline) crosslinkers - 1,4-butylene-2,2′-bis(2-oxazoline), 1,6-hexamethylene-2,2′-bis(2-oxazoline), and 1,8-octamethylene-2,2′-bis(2-oxazoline) - are prepared. The hydrogels differ by the length of aliphatic chain of crosslinker and by the percentage of crosslinker (2-10%). The influence of the type and the percentage of the crosslinker on swelling properties, mechanical properties, and state of water is studied. The equilibrium swelling degree in water ranges from 2 to 20. With a proper selection of the crosslinker, Young's modulus can be varied from 10 kPa to almost 100 kPa. To evaluate the potential for medical applications, the cytotoxicity of extracts and the contact toxicity toward murine fibroblasts are measured. The hydrogels with the crosslinker containing a shorter aliphatic exhibit low toxicity toward fibroblast cells. Moreover, the viability and the proliferation of pancreatic β-cells incubated inside hydrogels for 12 days are analyzed.

Full text of DOI:10.1002/pola.28009

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Poly(2-oxazoline) Hydrogels Crosslinked with Aliphatic bis(2-oxazoline)s:
Properties, Cytotoxicity, and Cell Cultivation
1
1
2
3
Anna Zahoranov aꢀ , Zuzana Kronekov aꢀ , Miroslav Zahoran, Du ꢁs an Chorv aꢀ t Jr.,
1
1
Ivica Janigov aꢀ , Juraj Kronek
Polymer Institute of the Slovak Academy of Sciences, D uꢀ bravsk aꢀ Cesta 9, 845 41 Bratislava, Slovakia
1
2
Department of Experimental Physics, Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynsk aꢀ Dolina,
8
42 48 Bratislava, Slovakia
3
International Laser Center, Ilkovi cˇ ova 3, 841 04 Bratislava, Slovakia
Correspondence to: J. Kronek (E-mail: juraj.kronek@savba.sk)
Received 29 September 2015; accepted 11 November 2015; published online 17 December 2015
DOI: 10.1002/pola.28009
ABSTRACT: A series of hydrogels from 2-ethyl-2-oxazoline and
contact toxicity toward murine fibroblasts are measured. The
0
three bis(2-oxazoline) crosslinkers—1,4-butylene-2,2 -bis(2-oxa-
hydrogels with the crosslinker containing a shorter aliphatic
exhibit low toxicity toward fibroblast cells. Moreover, the via-
bility and the proliferation of pancreatic b-cells incubated
0
zoline), 1,6-hexamethylene-2,2 -bis(2-oxazoline), and 1,8-octa-
0
methylene-2,2 -bis(2-oxazoline)—are prepared. The hydrogels
inside hydrogels for 12 days are analyzed. VC
2015 Wiley Period-
differ by the length of aliphatic chain of crosslinker and by the
percentage of crosslinker (2–10%). The influence of the type
and the percentage of the crosslinker on swelling properties,
mechanical properties, and state of water is studied. The equi-
librium swelling degree in water ranges from 2 to 20. With a
proper selection of the crosslinker, Young’s modulus can be
varied from 10 kPa to almost 100 kPa. To evaluate the potential
for medical applications, the cytotoxicity of extracts and the
icals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016, 54, 1548–
1559
KEYWORDS: biocompatibility; bis(2-oxazoline); cell cultivation;
cytotoxicity; hydrogels; mechanical properties; poly(2-oxazo-
line); swelling
INTRODUCTION In comparison to conventional flat two-
Moreover, it has been shown that poly(2-oxazoline)s exhibit
5
–7
a good biocompatibility toward various cell lines
as well
dimensional surfaces, three-dimensional matrices represent
1
as fast blood-clearance time and no accumulation in organs
more suitable models for cell cultivation and for studying
8
,9
when administered intravenously in rodents.
the fragile relationship between cells and their environment.
In this context, the development of new convenient materials
for cell cultivation in vitro is of a huge importance. One of
the most common materials used for the cells support are
Various synthetic routes for the preparation of hydrogels
from 2-oxazolines have been described in literature. Chris-
tova et al. presented segmented networks from poly(2-oxazo-
2
hydrogels. Due to their high water content and soft mechan-
10
line) macromonomers and acrylates,
Dargaville et al.
ical properties, they resemble human body tissue quite accu-
rately. Polymers used for the preparation of such hydrogels
have to fulfill strict criteria on biocompatibility as well as on
economic and reproducible preparation.
prepared poly(2-oxazoline)s crosslinked with dithiols using
1
1
12
UV light.
In the work of Farrugia et al.,
the latter
approach was extended for mild conditions in water, suitable
for the encapsulation of fibroblast cells, as demonstrated by
the authors. A different approach involving poly(2-oxazoline)
copolymers containing amino groups crosslinked with epi-
One of the most promising synthetic polymers used for
hydrogels preparation is poly(2-oxazoline). Although they are
structural analogues of poly(amino acids), the tertiary amide
group in poly(2-oxazoline) backbone provides an improved
13
chlorohydrine was shown in the work of Hartlieb et al.
Legros et al. prepared degradable hydrogels and nanogels
from partially hydrolyzed poly(2-oxazoline)s crosslinked by
3
stability in human organism. These polymers can be synthe-
1
,6-hexanediol di-glycidyl ether and its analoque containing
1
4
sized by living cationic polymerization which enables a prep-
disulfid. The same authors prepared aldehyde containing
copoly(2-oxazoline)s, which were subsequently crosslinked
4
aration of well-defined polymers with a low dispersity.
Additional Supporting Information may be found in the online version of this article.
2015 Wiley Periodicals, Inc.
V
C
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1
5
by bifunctional amines and hydrazines.
Preparation of
TCI (Zwijndrecht, Belgium). 2-Ethyl-2-oxazoline (Sigma-
Aldrich, Steinheim, Germany) was dried over KOH during
crosslinked poly(2-oxazoline)s were further summarized
1
6,17
recently in comprehensive reviews.
48 h and distilled over CaH under reduced pressure. Benzo-
2
nitrile (Sigma-Aldrich, Steinheim, Germany) was distilled
under reduced pressure prior to use. Potassium hydroxide,
sodium sulfate, and dichloromethane were purchased from
Mikrochem (Bratislava, Slovakia). Methanol (AFT Bratislava,
Slovakia), ethanol (Centralchem, Slovakia), and tetrachloro-
methane (Lachema, Czech Republic) were distilled prior to
use. Deionized water (Milipore) was used for swelling
experiments.
The ’’one-pot’’ copolymerization of monofunctional 2-
oxazolines with bis(2-oxazoline) as a crosslinker resulting in
a formation of a crosslinked network was presented for the
1
8
first time by Seagusa et al. Thanks to its simplicity and
good reproducibility this method has attracted a lot of atten-
1
9
tion in a wide range of applications. Rueda et al. have pre-
pared hydrogels by copolymerization of 2-methyl-2-oxazoline
0
and 1,4-butylene-2,2 -bis(2-oxazoline) initiated by styrene
macroinitiators. A series of poly(2-oxazoline)s hydrogels was
prepared by copolymerization of hydrophilic 2-ethyl-2-
oxazoline, hydrophobic 2-phenyl-2-oxazoline, and 1,4-phenyl-
Synthesis of Crosslinkers
Bis(2-oxazoline) crosslinkers were prepared according to the
2
5
procedure described by N ꢀe ry. Briefly, for the preparation of
0
0
ene-2,2 -bis(2-oxazoline) as a crosslinker in the work of Kelly
1,4-butylene-2,2 -bis(2-oxazoline) (ButBisOx), adipoyl chlo-
2
0
et al. Recently, poly(2-oxazoline)s networks crosslinked by
ride (3.97 ml, 27.3 mmol) dissolved in tetrachloromethane
(60 ml) was added dropwise into the mixture of KOH (9.2 g,
163.9 mmol) and 2-chloroethylamine hydrochloride (7.9 g,
0
1
,4-butylene-2,2 -bis(2-oxazoline) and modified by peptide
sequence RGD (arginine-glycine-aspartic acid) were prepared
2
1
by Schenk et al.
When ground, the gels showed an
68.3 mmol) in distilled water (50 ml) at 0 8C. The mixture
increased adhesion toward cancer cells in comparison to the
was stirred overnight at room temperature. The white pre-
cipitate was filtered out and dried under reduced pressure
at 40 8C (yield 6.34 g, 86.3%). For the cyclization, N,N’-bis(2-
chloroethyl)adipamide (10 g, 37.16 mmol) was dissolved
with KOH (5.21 g, 92.9 mmol) in methanol (250 ml) and
refluxed for 6 h. KCl was filtered out and methanol was
evaporated. The product was dissolved in chloroform, and
then washed four times with brine (50 ml). Chloroform was
evaporated and the product was dried for 2 days under
reduced pressure at 40 8C. Product was obtained as a yel-
lowish powder (yield 4.27 g, 59%).
2
2
adhesion toward endothelial cells. Hartlieb et al. intro-
0
duced poly(2-oxazoline) hydrogels with 1,4-phenylene-2,2 -
bis(2-oxazoline) as a crosslinker for long-term stabilization
2
3
of coagulation factor VIII. Li et al. studied thermorespon-
sive behavior of hydrogels prepared from 2-isopropyl-2-
0
oxazoline and 1,4-phenylene-2,2 -bis(2-oxazoline).
In order to adjust the swelling properties of such hydrogels
various strategies have been implemented: (i) simultaneous
copolymerization of monofunctional monomers of various
1
8,21
alkyl chain lengths with bis(2-oxazoline) crosslinker
; (ii)
aliphatic and aromatic comonomers crosslinked with bis(2-
0
1
,6-Hexamethylene-2,2 -bis(2-oxazoline) (HexBisOx) and 1,8-
2
0
19,24
oxazoline) crosslinker ; (iii) usage of macroinitiator
;
0
octamethylene-2,2 -bis(2-oxazoline) (OctBisOx) were pre-
pared analogously, starting from suberoyl chloride and seba-
coyl chloride, respectively. For NMR and FTIR spectra of
monomers, see Supporting Information Figures S1 and S2.
The crosslinkers were used without any further purification.
and (iv) adjustment of the ratio of a monomer to a cross-
2
0,21
linker.
To the best of our knowledge, the control over
the properties of 2-oxazoline-based hydrogels by varying the
length of aliphatic chain of the crosslinker, as proposed here,
has not yet been presented in literature.
1
ButBisOx: H NMR (400 MHz, DMSO-d
6
, d) 4.10 (t, J 5 9.4 Hz,
In this article, a preparation of 2-oxazoline-based hydrogels
with adjustable properties is presented. The control over
hydrogel properties is achieved by (i) changing the aliphatic
chain length of bis(2-oxazoline) crosslinker and by (ii)
changing the molar ratio of the monomer to the crosslinker.
Such hydrogels were subjected to freeze-drying process in
order to obtain a porous structure. The swelling properties
in various solvents, the mechanical properties, state of water,
and cytotoxicity of the samples are analyzed. Finally, selected
hydrogels are used as substrates for cultivation of pancreatic
b cells.
4
1
1
H; CH -O), 3.64 (t, J 5 9.4 Hz, 4H; CH -N), 2.15 (m, 4H; CH ),
2 2 2
.60–1.45 (m, 4H; CH ). Anal. cacld. for C H N O : 61.2 C,
2
10 16 2 2
4.27 N, 8.22 H; found: 60.2 C, 14.2 N, 8.3 H. M.p. 54 8C.
1
HexBisOx: H NMR (400 MHz, DMSO-d , d) 4.10 (t, 4H; CH -
O), 3.64 (t, 4H; CH -N), 2.14 (t, 4H; CH ), 1.48 (m, 4H; CH ),
1.25 (m, 4H; CH ) Anal. cacld. for C H N O : 64.26 C,
12.49 N, 8.99 H; found: 63.43 C, 12.32 N, 8.84 H. M.p. 65 8C.
6
2
2
2
2
2
12 20 2 2
1
OctBisOx: H NMR (400 MHz, DMSO-d , d) 4.10 (t, 4H; CH -
O), 3.64 (t, 4H; CH -N), 2.14 (t, 4H; CH ), 1.48 (m, 4H; CH ),
6
2
2
2
2
1.22 (m, 8H; CH -CH ). Anal. calcd. for C H N O in %:
2 2 14 24 2 2
66.63 C, 11.10 N, 9.59 H; found: 64.42 C, 10.71 N, 9.42 H.
EXPERIMENTAL
M.p. 56 8C.
Materials
Adipoyl
chloride,
sebacoyl
chloride,
methyl
4-
Synthesis of Hydrogels
nitrobenzenesulfonate, and 2-chloroethylamine hydrochloride
were purchased from Sigma-Aldrich (Steinheim, Germany)
and used as received. Suberoyl chloride was purchased from
The hydrogels were prepared by copolymerization of
2-ethyl-2-oxazoline (EtOx) with bis(2-oxazoline) (ButBisOx,
HexBisOx, and OctBisOx, respectively), using methyl
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4
-nitrobenzenesulfonate (MeONs) (1 mol %) as an initiator
brated for 24 h. Immediately after removing from water, cy-
linders with a diameter of 4 mm were cut from the samples
prior to the measurements of mechanical properties. The ini-
in benzonitrile, with five different molar ratios of BisOx:E-
tOx (2:98, 3:97, 4:96, 5:95, and 10:90). Typically, for the
preparation of 2% ButBisOx hydrogel, ButBisOx (39.2 mg,
tial height H was determined as a deformation in which the
0
0
.2 mmol) and MeONs (21.7 mg, 0.1 mmol) were placed in a
Schlenk flask and dried in vacuum for 45 min. Subsequently,
-ethyl-2-oxazoline (0.99 ml, 0.98 mmol) and benzonitrile
1 ml) were added under nitrogen atmosphere and the mix-
force values started to increase. Mechanical properties were
determined from stress–strain curves, Young’s modulus was
calculated for the small deformations (<15%) as a linear fit
using least squares method. All data are presented as the
mean 6 standard deviation from at least five replicates.
2
(
ture was stirred for 5 h at 110 8C in oil bath. The yellowish
gel was washed in an excess of methanol (23 24 h), ethanol
Differential Scanning Calorimetry
(
13 24 h), and distilled water (43 24 h) and freeze-dried.
Thermal analysis was performed using Mettler-Toledo DSC
All syntheses were done in triplicates.
e
8
21 differential scanning calorimeter (DSC) under a nitro-
gen atmosphere. The thermal behavior of hydrogels samples
swollen in distilled water were studied in the temperature
interval from 245 to 25 8C at a heating rate 5 8C/min.
Characterization
1
13
The H and
C NMR spectra of all compounds were
recorded at room temperature on a Varian VXR-400 (Varian)
in DMSO-d6 solutions using tetramethylsilane (TMS) as an
internal standard. Fourier transform infrared spectroscopy
with attenuated total reflectance (FTIR-ATR) measurements
were performed with FTIR NICOLET 8700 spectrometer
The amount of freezing water was calculated using the
2
6
equation
DH
Wfðwt %Þ5
3100;
(2)
(
Thermo Scientific, UK) using an ATR accessory equipped
DHw
with a Ge crystal. For each measurement, the resolution was
2
1
4
cm and 64 scans.
where DH is the melting enthalpy of water in hydrogel calcu-
lated from the integral of the endothermic peak at 0 8C and
21 27
Scanning Electron Microscopy
DH
w
is melting enthalpy of bulk water (333 Jg ) .
The morphology of the freeze-dried samples was examined
using Tescan TS 5136 MM microscope (Tescan, Czech Repub-
lic) operating at 20 kV in a low vacuum regime (P 5 20 Pa).
Prior to imaging, the dried gels were cut to expose their
inner structure. The scanning electron microscopy (SEM)
images were obtained using backscattered electrons.
The amount of non-freezing water was obtained from the
equation
2
7
Wnfðwt %Þ5EWCðwt %Þ2 Wfðwt %Þ;
(3)
where the equilibrium water content (EWC) in water was
calculated from the weight of the swollen (wsw) and dry
Swelling Properties
For the equilibrium swelling degree (SD) measurements, the
samples were immersed in an excess of solvent [CH Cl ,
(
w ) hydrogel according to eq 4, i.e.
d
wsw2wd
2
2
EWC5
3100:
(4)
CH CH OH, dH O, phosphate-buffered saline (PBS)] and equi-
wsw
3
2
2
librated for 24 h. After this time period, no additional swel-
ling was observed. The samples were then gently dried with
a paper tissue to remove any remaining solvent from the
surface and weighted. SD was calculated using equation
DSC was used also for determination of melting points of
crosslinkers. Measurements were performed in the tempera-
ture interval from 25 to 250 8C at a heating rate 10 8C/min.
wsw2wd
Cell Lines and Growth Media
For the cytotoxicity and cultivation study mice fibroblasts
SD5
;
(1)
wd
3
T3 and mouse insulinoma bTC3 cells were used (both
where wsw is the weight of the swollen sample, wd is the
weight of the dried sample (after freeze-drying). SD is
expressed as the mean 6 standard deviation from hydrogels
from three independent syntheses.
DSMZ, Braunschweig). 3T3 cells were cultivated in Dulbec-
co’s modified eagle medium (DMEM) supplemented with
1
0% fetal bovine serum (FBS), L-glutamine (2 mM), strepto-
mycin (100 mg/ml), penicillin (100 IU/ml). bTC3 cells were
cultivated in DMEM supplemented with 15% horse serum
Mechanical Properties
(
HS), 2.5% FBS, L-glutamine (2 mM), streptomycin (100 mg/
Mechanical properties of hydrogels were determined
employing Texture Analyser TA-XT2i (Stable Micro Systems,
UK) equipped with a force transducer of a resolution of 1
mN and Texture Expert software version 1.16 used for data
acquisition and evaluation. The mobile probe (diameter
ml), penicillin (100 IU/ml), referred further as full DMEM.
All chemicals were purchased from Gibco (Life Technologies).
The cells were cultivated at 37 8C and 5% CO with saturat-
2
ing humidity in CO2 incubator. The cells were trypsinized
and the medium was changed once every 3 days.
3.87 mm) of Texture Analyser in a compression mode was
moving vertically at a constant speed 0.1 mm/s. The defor-
mation was set as 98% of the initial position of the probe.
The hydrogels were swollen in distilled water and equili-
Cytotoxicity Studies
Cell toxicity studies were conducted in accordance with ISO
10993-5 and ISO 10993-12 on mice fibroblast cell line 3T3.
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All experiments were done in hexuplicates, with nontreated
cells as a negative control.
Toxicity of Extracts
For the toxicity of extracts, the dry gels were sterilized by
UV light for 30 min, then immersed in full DMEM medium
and sterilized for another 30 min by UV light. The final vol-
ume of the extraction medium was 1 ml per 0.1 g of dry gel
plus additional volume absorbed by the gel (calculated from
the equilibrium SD). The gels were extracted for 24 h at 37
8
C. Fibroblast cells were seeded on 96-well plate with the
3
seeding density 5 3 10 cells/well and cultivated for 24 h.
The medium was removed from the wells and extracts were
added in three different dilutions—1:1, 1:2, 1:5 (extract:
total volume). The cells were then incubated for additional
2
4 h and the viability was measured by MTT cytotoxicity
5
assay as reported elsewhere.
Direct Contact Toxicity
For the direct contact toxicity, cells were seeded on 24-well
4
plate with the seeding density 2 3 10 cells/well and culti-
vated for 24 h. The dry gels were sterilized by UV light for
FIGURE 1 Synthesis of hydrogels from 2-ethyl-2-oxazoline and
bis(2-oxazoline) crosslinker (2, 3, 4, 5, and 10 mol %). The syn-
thesis was carried out in benzonitrile for 5 h, with 4-methyl
nitrobenzenesulfonate as an initiator. [Color figure can be
viewed in the online issue, which is available at wileyonlineli-
brary.com.]
30 min, and then immersed in DMEM medium with serum,
sterilized by UV light for 30 min, and equilibrated for 24 h.
Cylinders of diameter of 6 mm were cut from the gels and
gently placed on the top of the cell layer. The gels covered
ꢀ
14% of the well surface. The cells were incubated with the
gels for 24 h, then the gels were removed and the viability
was measured by MTT cytotoxicity assay.
RESULTS AND DISCUSSION
Synthesis of Hydrogels
Cell Cultivation
Hydrogels were synthesized by “one-pot” random copolymer-
ization of 2-ethyl-2-oxazoline with bis(2-oxazoline) cross-
linkers (ButBisOx, HexBisOx, and OctBisOx) in benzonitrile
For the cell cultivation studies, three types of hydrogels, 2%
ButBisOx, 2% HexBisOx, and 2% OctBisOx, were selected.
The studies were conducted on murine b-cell line bTC3. Cyl-
inders of diameter of 8 mm were cut from the hydrogels,
swollen in full DMEM medium, and sterilized by UV light.
The cells were injected into the hydrogel cylinders in seed-
18
at 110 8C (Fig. 1). Seagusa et al. used acetonitrile for the
synthesis of crosslinked poly(2-oxazoline)s. Later, in the
19
work of Rueda et al., benzonitrile was employed as a sol-
28
22
vent. In the more recent works of Schenk and Hartlieb,
dichloromethane was used for the preparation of poly(2-
4
ing number 2 3 10 cells/cylinder and hydrogel cylinders
were poured over with the medium. The medium was
changed every 3 days during the experiment. In the selected
time intervals (day 3, day 7, day 12 after seeding), the viabil-
ity of the cells was examined by FDA staining and Resazurin
assay. For FDA staining, the gels were washed in PBS, then
250 ml of FDA solution (0.024 mg/ml in PBS) and 500 ml of
PBS was added to hydrogel cylinder, incubated for 20 min,
and then washed with PBS. The cells in hydrogels were
visualized using confocal laser scanning microscope (LSM
5
4
10 META, Anxiovert 200, Zeiss, Germany, excitation
88 nm, emission 500–550 nm). In case of the Resazurin
staining, the gels seeded with cells were relocated to a new
well, prior to measurement, to avoid the signal from free
unattached cells. Then, 50 ml of stock solution (0.01 mg/ml
in PBS) was added to 500 ml of full DMEM media (without
phenol red), incubated for 2 h, then the absorbance at 595
and 540 nm was measured on the plate reader (Labsystems
Multiskan MS). The experiment was performed in quadrupli-
cates, and the reduction of Resazurin was normalized to the
signal on the day of seeding.
FIGURE 2 Dependence of swelling degree in distilled water
(columns) and gel content (squares) on the polymerization
time for 2% HexBisOx hydrogel. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 3 SEM micrographs of the morphology of freeze-dried samples. ButBisOx (a)–(d), HexBisOx (e)–(h), OctBisOx (i)–(l), per-
centage of crosslinker: 2% column 1; 3% column 2; 5% column 3; 10% column 4; scale bar 200 mm. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
oxazoline) hydrogels. However, due to their relatively low
boiling point, the polymerizations were conducted under
higher pressure in microwave reactor. To overcome this
drawback, we decided to use benzonitrile in our reaction
conditions, due to its higher boiling point, compared to ace-
tonitrile, which enables us to use higher reaction tempera-
tures at ambient pressure. In addition, bis(2-oxazoline)
crosslinkers exhibited good solubility in this solvent. As an
initiator, 4-methyl nitrobenzenesulfonate (MeONs) was used
in our study. MeONs was proved to be a suitable initiator
for living cationic polymerization of 2-oxazolines, with the
propagation rate coefficient slightly lower as that of methyl
reflects additional crosslinking in the gel. After 5 h, no changes
of SD and minimal changes of gel content were observed.
Therefore, we selected 5 h as the polymerization time for all
reactions.
For each crosslinker (ButBisOx, HexBisOx, OctBisOx), five
different molar percentage were selected: 2, 3, 4, 5, and
10%. For 1% of crosslinker, no gelation under our experi-
mental conditions was observed. All syntheses were carried
out in triplicates to ensure the reproducibility of the results.
For 2% ButBisOx, the averaged gel content from three syn-
theses was 83%, for 2% HexBisOx and 2% OctBisOx 86%.
For all other samples, the gel content was higher than 92%.
2
9
triflate. In comparison to methyl triflate, MeONs exhibits
an increased stability during storage and easier synthesis
and purification.
Morphology of Dried Hydrogels
The porosity and the pore size of polymeric material has a
3
0,31
First, the equilibrium SD and the gel content was studied as a
function of the time of polymerization for the 2% HexBisOx
hydrogel (Fig. 2). The gelation was observed already after 30
min of polymerization. After 2 h, the gel content was gradually
increasing from the value 78 to 92% (maximum at 5 h). The
SD decreased from 16.7 for the time 2 h to 12.9. This behavior
strong effect on the behavior and the viability of cells.
In
this work, the morphology of freeze-dried samples was
examined using scanning electron microscopy. For noncon-
ductive polymeric samples, the coating of sample by conduc-
tive (Au) layer before measurement is
a
common
3
2
procedure.
However, the gold sputtering may not be
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efficient method for the thicker 3D structures and charging
tein adsorption resistance, and thus lead to increasing
biocompatibility of polymeric materials. In our study, the
3
3
26
of the surface may occur during the imaging. To avoid this
problem and to simplify the sample preparation, we used
low vacuum regime (P 5 20 Pa) and more energetic back-
scattered electrons to visualize hydrogel structure without
additional coating of the samples. The representative SEM
micrographs are shown in Figure 3.
state of water in hydrogels was evaluated using DSC. The
representative DSC heating curves are shown in Figure 5. It
is noteworthy that a small exothermic peak appears on DSC
curves around 212 8C for the highest percentage of cross-
linker (Supporting Information Fig. S4). From the DSC and
X-ray diffraction study of hydrated poly(2-methoxyethylacry-
late), the exothermic peak at lower temperatures was associ-
In the electron micrographs, the porous inner structure of
hydrogels was revealed. This structure is the consequence of
the freeze-drying process, when the ice crystals disrupt the
hydrogel structure. The comparison of micrograph of freeze-
dried and air-dried samples is shown in Supporting Informa-
tion Figure S3. With the increasing percentage of the cross-
linker, the pores size decreases and the interporous area
increases. The exception is 10% ButBisOx, where the
decreased number of pores but increased size of pores can
be seen, compared to 5% ButBisOx.
35
ated with the formation of hexagonal ice crystals, thus,
with the presence of freezing-bound water. However, the
reported crystallization temperature was around 245 8C.
The shift to the higher temperatures may be due to different
chemistry of poly(2-methoxyethylacrylate) compared to
poly(2-ethyl-2-oxazolines) where hydrogen bound between
water molecules and amidic group can be found. Since this
suggested explanation needs a further study, we limit our
discussion to the freezing and nonfreezing water content.
Swelling Properties
The results from DSC measurements are summarized in
Table 2. For all three types of crosslinkers, we observe a
decreasing amount of free water with the increasing percent-
age of crosslinker. 2% ButBisOx hydrogel contains
The equilibrium SD in various solvents (distilled water,
dichloromethane, ethanol, and PBS) was measured to com-
pare the swelling capacity and polarity between hydrogels
with three types of crosslinkers and with different crosslink-
ing densities. The results are summarized in Table 1. (For
graphical representation, see Fig. 4) The SDs in ethanol, dis-
tilled water, and dichloromethane are comparable for all
hydrogels (except for 2% ButBisOx which swells slightly
less in ethanol, see Table 1). This is in accordance with the
73.2 6 2.4% of free water, in comparison to 29.8 6 3.8% for
10% ButBisOx hydrogel. Interestingly, the percentage of
crosslinker has a stronger influence on the state of water
than the polarity of crosslinker. The opposite observation
36
was made by Kasak et al. for zwitterionic hydrogels, where
the chemistry of crosslinker was the major factor influencing
the state of water.
2
1
results published by Schenk et al. The gels can be, thus,
referred as amphigels and their ability to swell in ethanol
can be further used for the immobilization of therapeutic
compounds. It is noteworthy that in previously mentioned
When comparing the amount of free water in hydrogels with
their morphology (see Section Morphology of dried hydro-
gels), the decreasing free water content with increasing
crosslinking percentage may be explained by the decrease of
the pore size of hydrogels.
2
1
work the lowest percentage of ButBisOx crosslinker was
%, referred to as 150:6 EtOx:crosslinker. The SD of such gel
in distilled water was presented to be 7.3 (in comparison to
.5 in our work). Furthermore, the authors claim that the
hydrogels with even lower crosslinker percentage (up to
%) were also prepared; however, they dissolved during
4
5
Mechanical Properties
2
One of the most important properties of a material used in
contact with cells is its mechanical stiffness. As it was shown
in the pioneering work of Disher et al., the mechanical stiff-
purification.
3
7
When comparing three different types of crosslinker, it is
interesting that despite the observable difference of swelling
between ButBisOx and HexBisOx hydrogels, HexBisOx and
OctBisOx hydrogels swell comparably. The longer aliphatic
chain does not influence the SD of hydrogels. Thus, the swel-
ling ability of hydrogels is controlled mainly by the polarity
of crosslinker.
ness of the substrate influences the behavior of cells. More-
over, the differentiation of the stem cells can be influenced
3
8
by the mechanical properties of their environment. On soft
substrates (Eꢁ1 kPa), the pluripotent stem cells differentiate
toward neural cells, while on rigid surfaces (Eꢁ100 kPa),
the stem cells differentiate toward osteoblasts.
In our work, we have examined the mechanical properties of
hydrogels swollen in distilled water by using Texture Analy-
ser in compression mode. For the sake of clarity, the pre-
sented data are from one synthesis of every type of
hydrogels. The comparison between different syntheses of
the same hydrogel composition is shown in Supporting Infor-
mation Figure S5. The mechanical properties of hydrogels
are summarized in Table 3.
Differential Scanning Calorimetry
In general, three different states of water were described for
3
4
hydrated polymers. The free water, with the freezing tem-
perature around 0 8C, is not bound to the polymeric chains.
The nonfreezing, or bound water, is bound to the polymeric
chains, and therefore exhibits no transition on the DSC heat-
ing curve. The third type is so-called intermediate or
freezing-bound water, for which the cold crystallization peak
below 0 8C on the DSC heating curve is typical. It was sug-
gested that non-freezing water may play a key role in pro-
With increasing crosslinking density, the Young’s modulus of
hydrogels increases for all three crosslinkers. This is a
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TABLE 1 Equilibrium Swelling Degree of Hydrogels in Various
Solvents, Mean 6 SD from Three Independent Syntheses
%
CH
2
Cl
2
CH
3
2
CH OH dH
2
O
PBS
ButBisOx
HexBisOx
OctBisOx
2
3
4
5
18.3 6 1.0 13.2 6 1.5
10.0 6 1.0 7.0 6 0.7
21.3 6 0.9 13.4 6 1.3
8.0 6 0.2
5.5 6 0.3
4.7 6 0.3
2.3 6 0.1
7.7 6 0.7
5.0 6 0.1
4.4 6 0.2
2.4 6 0.1
7.3 6 0.2
5.7 6 0.4
3.4 6 0.2
4.8 6 0.2
4.0 6 0.2
2.1 6 0.2
1
2
3
4
5
1
2
3
4
5
1
0
14.4 6 0.5 10.5 6 0.9
13.2 6 0.1 10.3 6 0.3
7.8 6 0.1
6.2 6 0.2
4.5 6 0.3
5.7 6 0.4
4.1 6 0.3
3.0 6 0.2
6.3 6 0.7
4.5 6 0.3
3.2 6 0.1
2.0 6 0.1
5.7 6 0.5
4.2 6 0.3
3.1 6 0.3
2.1 6 0.0
0
0
3.1 6 0.05 1.9 6 0.1
14.0 6 3.3 10.5 6 0.5
10.8 6 3.6 6.9 6 2.1
12.5 6 0.9 10.7 6 0.1
7.6 6 2.5
4.0 6 0.4
3.2 6 0.1
1.7 6 0.2
6.8 6 1.8
4.0 6 0.2
2.9 6 0.1
1.8 6 0.2
6.2 6 0.3
5.2 6 0.1
3.1 6 0.1
3.9 6 0.3
3.1 6 0.1
1.8 6 0.0
general trend for hydrogels described also in the work of
3
9
Anseth et al. The exception were 10% HexBisOx and 10%
ButBisOx, where the Young’s modulus slightly decreased
(
from 66.7 6 16.8 to 51.4 6 16.2 kPa and from 70.3 6 20.3
to 62.3 6 21.3 kPa, respectively). On the other hand, the
decrease of compression at break with increasing crosslink-
ing density was observed for ButBisOx (from 48 6 6 to
3
2
4 6 11%) and HexBisOx hydrogel (from 44 6 6 to
8 6 4%). The trend was opposite for OctBisOx crosslinker.
The compression at break increased from 32 6 9 to
4
2
3 6 3%. For the comparison of all hydrogels, the stress at
0% (r20) deformation was arbitrary selected. The increase
of r20 with the increase of crosslinked density may be
observed. For the 10% of crosslinker, r20 of OctBisOx hydro-
gel is 44.2 6 7.4 kPa, which is 1.7-fold higher than r20 of
HexBisOx hydrogel (25.9 6 6.4 kPa). However, the SD in
water of 10% HexBisOx (2 6 0.1) does not differ dramati-
cally from the SD of 10% OctBisOx (1.7 6 0.2). The differen-
0
ces in Young s modulus as well as in compression at break
thus could not be explained by the crosslinking densities of
hydrogels. Since the high reaction yields imply successful
incorporation of bis(2-oxazoline) crosslinkers into the poly-
meric chains, we hypothesize that this effect is caused by
sterical reason. Crosslinker with the longest aliphatic (OctBi-
sOx) enables crosslinking between distant polymeric chains
whereas crosslinkers with shorter aliphatic chains (ButBi-
sOx, HexBisOx) reacts preferentially with closer molecules,
leading to the generation of loops and blind ends. These
structures do not contribute to the mechanical stiffness of a
material. A similar behaviour was observed for polyacryl-
amide gels, where the maximum of Young’s modulus was
observed at 5% of the crosslinker. A further increase of the
FIGURE 4 Equilibrium swelling degrees of hydrogels in dis-
tilled water, ethanol, and dichloromethane, mean 6 SD, n 5 3.
[
Color figure can be viewed in the online issue, which is avail-
able at wileyonlinelibrary.com.]
4
0
crosslinker led to a decrease of the modulus.
1
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TABLE 3 Mechanical Properties of Hydrogels
%
E (kPa)
r20 (kPa)
r
br (kPa)
cbr (%)
ButBisOx
2
3
4
5
11.5 6 4.0
26.0 6 3.2
34.2 6 8.5
2.4 6 1.2
4.2 6 1.6
5.4 6 2.1
8.3 6 2.8
10.2 6 4.0
13.9 6 6.0
48 6 6
36 6 7
37 6 10
34 6 5
66.7 6 16.8 11.5 6 3.4 23.0 6 5.4
1
2
3
4
5
1
2
3
4
5
1
0
51.4 6 16.2 19.4 6 7.2 40.1 6 14.4 34 6 11
HexBisOx
21.9 6 3.9
23.2 6 4.7
26.4 6 5.5
5.0 6 0.9
5.5 6 0.9
6.1 6 1.3
18.3 6 6.4
16.2 6 5.3
18.1 6 9.0
44 6 6
39 6 4
37 6 9
70.3 6 20.3 18.1 6 6.7 48.4 6 22.8 31 6 3
62.4 6 21.3 25.9 6 6.4 48.3 6 10.6 28 6 4
0
0
FIGURE 5 The representative DSC heating curves for HexBisOx
OctBisOx
23.7 6 4.2
33.9 6 8.7
5.0 6 1.0
6.7 6 1.7
19.7 6 4.1
19.7 6 3.1
32 6 9
34 6 4
36 6 5
41 6 3
hydrogel with different crosslinker percentage.
The representative stress-deformation curves are depicted in
Figure 6.
39.4 6 11.7 10.1 6 3.8 23.5 6 4.8
65.8 6 14.1 17.3 6 5.0 40.0 6 9.5
85.6 6 17.1 44.2 6 7.4 96.7 6 34.5 43 6 3
Cytotoxicity Studies
E, Young’s modulus; r20, stress at 20% deformation; rbr, stress at break;
br, compression at break; mean 6 SD (nꢂ5).
The direct contact toxicity was evaluated on 3T3 mice fibro-
blast cell line after 24 h cultivation in contact with hydrogels
covering approximately 14% of the well surface. Figure 7
represents the percentage of viable cells compared to non-
treated controls (cells cultivated without hydrogels).
c
in a decreased cell viability (e.g., 55.8 6 8.6% for 10% Hex-
BisOx). These effects may be due to the higher stiffness of
hydrogels causing the defects in the cell membranes and/or
by the leakage of low molecular chemical compounds.
The highest viability (100.4 6 8.4%) was exhibited by cells
grown in contact with 2% ButBisOx, which is the softest
and the most hydrophilic hydrogel. Hydrogels prepared by
more hydrophobic crosslinkers (HexBisOx or OctBisOx)
exhibited decrease in cell viability (Fig. 6). In addition, the
increase in the crosslinker concentration (> 2%) also results
To further analyze the toxic effect of low molecular com-
pounds, the toxicity of hydrogel extracts toward murine
3T3 fibroblasts was studied. The gels were extracted for
24 h in full DMEM medium at 37 8C. The toxicity studies
were performed on extracts from all hydrogels (Supporting
TABLE 2 The States of Water of Poly(2-oxazoline) Hydrogels
%
EWC (%)
W
f
(%)
Wnf (%)
ButBisOx
HexBisOx
OctBisOx
2
3
4
5
95.4
89.5
84.5
80.2
72.2
92.9
87.1
81.8
76.8
67.4
92
73.2 6 2.4
52.3 6 12.0
50.7 6 6.1
35.0 6 0.7
29.8 6 3.8
71.8 6 1.7
62.7 6 6.3
49.9 6 4.1
40.2 6 2.1
25.4 6 6.9
75.0 6 2.4
51.2 6 3.1
43.0 6 6.8
40.9 6 3.4
25.6 6 2.4
22.2 6 2.4
37.2 6 12.0
33.8 6 6.1
45.2 6 0.7
42.3 6 3.8
21.1 6 1.7
24.4 6 6.3
32.0 6 4.1
36.6 6 2.1
42.0 6 6.9
17.0 6 2.4
40.1 6 3.1
37.7 6 6.8
35.7 6 3.4
35.1 6 2.4
1
2
3
4
5
1
2
3
4
5
1
0
0
91.3
80.7
76.6
60.7
FIGURE 6 Representative stress-deformation curves of ButBisOx
0
(
orange), HexBisOx (blue), and OctBisOx (black) hydrogels con-
taining 2% (solid) and 10% (dashed) of crosslinker. [Color figure
can be viewed in the online issue, which is available at wileyonli-
nelibrary.com.]
f
EWC, equilibrium water content, W , free water content; Wnf, nonfreez-
ing water; the data are presented as
measurements
a mean 6 SD from three
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FDA staining for 12 days. Nowadays, Resazurin dye is trade-
marked as alamarBlueVR and it is used for the investigation
4
2
of viability and proliferation of mammalian cells.
The
FIGURE 7 Contact toxicity of hydrogels toward 3T3 fibroblasts
evaluated by MTT test, mean 6 SD from hexuplicates. The
hydrogels differ in composition (But-Hex-Oct) and in concentra-
tion (2–10%) of the crosslinker. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
Information Table S6). In Figure 8, the median from three
hydrogels from different syntheses for every concentration
and type of crosslinker is presented. The extracts leading
to the decrease of cell viability to 75% and lower was con-
sidered as toxic in our study. The viability of the cells incu-
bated in extracts prepared from the ButBisOx and
HexBisOx crosslinker is not compromised, and reached
8
7% and higher. The extracts from OctBisOx hydrogels
were not toxic up to 3% of the crosslinker percentage,
however, higher crosslinking density led to the decreased
viability of cells (30.5 6 1.77% for 4% OctBisOx). In gen-
eral, and in agreement with the contact cytotoxicity results,
the lowest cell viability was observed for the cells incu-
bated with extracts of hydrogel made of 4% of crosslinker,
this effect is most pronounced for HexBisOx hydrogels.
This effect may be caused by the presence of low molecu-
lar compounds, monomers and of benzonitrile which are
present in the extracts as demonstrated by HPLC (Support-
ing Information Fig. S7) and which is, in general, difficult
to wash out from denser networks. In addition, benzoni-
trile can interact via intermolecular Van der Waals forces
with more hydrophobic crosslinkers. It was shown that 2-
4
1
oxazoline monomers, as well as concentrations of benzo-
nitrile above 1 mg/ml (Supporting Information Fig. S8) are
toxic. The toxicity of extracts depends on a combination of
several factors. The factors influencing the toxicity include:
(
i) the efficiency of the purification procedure (ii) the SD
of hydrogel in water (the more swollen hydrogel is, the
higher amount of benzonitrile can be entrapped in 1 g of
its weight), and (iii) the release rate of compounds into
the medium.
FIGURE 8 Toxicity of extracts from hydrogels evaluated by MTT
3
Cell Cultivation
test on 3T3 fibroblast cell line (seeding density 5 3 10 cells per
For the cell cultivation study, the hydrogels with 2% of
crosslinker were selected, due to their low toxicity in both
contact and extract studies. The viability of cells cultivated
inside the hydrogels was evaluated by Resazurin assay and
well) after 24 h of incubation, mean 6 SD from hexuplicates.
Hydrogels were crosslinked by ButBisOx (A), HexBisOx (B), and
OctBisOx (C) crosslinker. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
1
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4
FIGURE 9 Cultivation of pancreatic b-cells inside hydrogels, seeding density 2 3 10 cell/hydrogel cylinder, resazurin staining of
cells (A), FDA staining after 7 days (B), and 12 days (C) of incubation. [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
oxidized blue Resazurin is reduced in mitochondria to pink
fluorescent Resofurin, which enables to measure metabolic
activity of the cells. Moreover, as Resazurin does not disrupt
the mitochondrial respiratory chain, it is non-toxic for the
cells, thus, it can be used for a long-time monitoring of cell
viability study. During the first 7 days of cultivation, an
increase in the signal of reduced Resazurin is observed in all
three types of hydrogels indicating the cell proliferation [Fig.
to 103.8 6 21.9% for 2% ButBisOx hydrogels, 93.6 6 4.9%
for HexBisOx hydrogels and 83.6 6 7.7% for OctBisOx
hydrogels. These results are in agreement with the FDA fluo-
rescence staining, where, in comparison to the hydrogels
after 12 days of cultivation [Fig. 9(C)], the hydrogels after 7
days of cultivation contained higher number of viable cells
stained green [Fig. 9(B)]. The highest loss in cell viability is
observed for OctBisOx hydrogels with the longest aliphatic
chain. The decrease in cell viability can be due to the con-
9(A)]. The Resazurin reduction is comparable in all three
4
3
types of hydrogels; 148.6 6 24.6% for 2% ButBisOx hydro-
gels, 146.8 6 8% for 2% HexBisOx hydrogels and
fined space and insufficient nutrients supply. The morphol-
ogy of the cells cultivated in hydrogels was spherical and
cells formed aggregates, what is particularly visible for 2%
ButBisOx. Due to the anti-fouling properties of poly(2-oxazo-
136.8 6 10.2% for 2% OctBisOx hydrogels, normalized to
the value at the beginning of the experiment. However, after
4
4
12 days of cultivation, the reduction of Resazurin decreased
line)s, the cell–cell interactions were promoted over the
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cell-surface interactions, what led to the formation of spheri-
cal aggregates of cells.
8 P. Goddard, L. E. Hutchinson, J. Brown, L. J. Brookman, J.
Control. Release 1989, 10, 5–16.
9
F. C. Gaertner, R. Luxenhofer, B. Blechert, R. Jordan, M.
Essler, J. Control. Release 2007, 119, 291–300.
0 D. Christova, R. Velichkova, W. Loos, E. J. Goethals, F. D.
CONCLUSIONS
1
Poly(2-oxazoline)-based hydrogels have recently been recog-
nized as promising scaffolds for the cultivation of fibro-
Prez, Polym. (Guildf). 2003, 44, 2255–2261.
11 T. R. Dargaville, R. Forster, B. L. Farrugia, K. Kempe, L.
Voorhaar, U. S. Schubert, R. Hoogenboom, Macromol. Rapid
Commun. 2012, 33, 1695–1700.
1
2
45
blast,
as well as cancer cell lines.
Up to date, the
cultivation studies were limited to the poly(2-oxazoline)s
crosslinked by thiol-ene click reaction. In our work, we
focused on the hydrogels prepared by the copolymerization
of (2-ethyl-2-oxazoline) with bis(2-oxazoline) crosslinkers.
1
2 B. L. Farrugia, K. Kempe, U. S. Schubert, R. Hoogenboom,
T. R. Dargaville, Biomacromolecules 2013, 14, 2724–2732.
3 M. Hartlieb, D. Pretzel, K. Kempe, C. Fritzsche, R. M. Paulus,
1
M. Gottschaldt, U. S. Schubert, Soft Matter 2013, 9, 4693–4704.
We synthesized a series of hydrogels by copolymerization of
14 C. Legros, M. C. De Pauw-Gillet, K. C. Tam, S.
Lecommmandoux, D. Taton, Polym. Chem. 2013, 4, 4801–4808.
2
1
-ethyl-2-oxazoline with three different bis(2-oxazoline)s –
0
0
15 C. Legros, M. Pauw-gillet, De, K. Chiu, S. Lecommandoux,
D. Taton, Eur. Polym. J. 2015, 62, 322–330.
,4-butylene-2,2 -bis(2-oxazoline),
1,6-hexamethylene-2,2 -
0
bis(2-oxazoline), and 1,8-octamethylene-2,2 -bis(2-oxazoline).
The bis(2-oxazoline) crosslinkers with the longer aliphatic
chain were used for the preparation of hydrogels for the first
time. Hydrogels showed a comparable SD in distilled water,
ethanol, and dichloromethane. The SD in water varied with
the type and molar percentage of crosslinker from
1
6 A. M. Kelly, F. Wiesbrock, Macromol. Rapid Commun. 2012,
3, 1632–1647.
3
1
7 U. S. Schubert, M. Hartlieb, K. Kempe, J. Mater. Chem. B 2014,
8 Y. Chujo, K. Sada, K. Matsumoto, T. Saegusa, Macromole-
1
cules 1990, 23, 1234–1237.
9 J. Rueda, R. Suica, H. Komber, B. Voit, Macromol. Chem.
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0 A. M. Kelly, A. Hecke, B. Wirnsberger, F. Wiesbrock, Macro-
mol. Rapid Commun. 2011, 32, 1815–1819.
1 V. Schenk, E. Rossegger, C. Ebner, F. Bangerl, K.
1
21.32 6 0.88 for 2% ButBisOx, to 1.72 6 0.16 for 10% Oct-
BisOx. Young’s modulus varied from 11.5 6 4 kPa for 2%
ButBisOx to 85.6 6 17.1 kPa for 10% OctBisOx. The hydro-
gels with lower crosslinking density and a shorter aliphatic
chain of crosslinker were well tolerated by fibroblast 3T3
cells in both contact and extract toxicity studies. The pancre-
atic bTC3 cells were cultivated within the hydrogels for 12
days and formed spherical aggregates. The modification of
hydrogel samples to promote the cell adhesion will be the
subject of the ongoing studies.
2
2
Reichmann, B. Hoffmann, M. H o€ pfner, F. Wiesbrock, Polym.
(Basel) 2014, 6, 264–279.
22 M. Hartlieb, S. Schubert, K. Kempe, N. Windhab, U. S.
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2
5 L. N eꢀ ry, H. Lefebvre, A. Fradet, Macromol. Chem. Phys.
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2
ACKNOWLEDGMENTS
26 T. Morisaku, J. Watanabe, T. Konno, M. Takai, K. Ishihara,
Polym. (Guildf). 2008, 49, 4652–4657.
The authors thank the Slovak Grant Agency, VEGA, for the finan-
cial support in the projects No. 2/0163/15 and 2/0156/15.
A. Zahoranov ꢀa wants to acknowledge I. Chodak and O. Sluciak
for their useful advices and comments.
2
7 M. B. Ahmad, M. B. Huglins, Polym. Int. 1994, 33, 273–277.
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9 M. Glassner, D. R. D’hooge, J. Young Park, P. H. M. Van
(
2
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