Gelatin-based hydrogels through homobifunctional triazolinediones targeting tyrosine residues

Article abstract of DOI:10.3390/molecules24030589

Gelatin is a biopolymer with interesting properties that can be useful for biomaterial design for different applications such as drug delivery systems, or 3D scaffolds for tissue engineering. However, gelatin suffers from poor mechanical stability at phys

Full text of DOI:10.3390/molecules24030589

molecules
Article
Gelatin-Based Hydrogels through Homobifunctional
Triazolinediones Targeting Tyrosine Residues
Roberto Guizzardi 1,†, Luca Vaghi 2,†, Marcello Marelli 3 , Antonino Natalello 1
,
Ivan Andreosso 2, Antonio Papagni 2,* and Laura Cipolla 1,
*
1
Department of Biotechnology and Biosciences, University of Milano—Bicocca, Piazza della Scienza 2,
20126 Milano-IT, Italy; r.guizzardi@campus.unimib.it (R.G.); antonino.natalello@unimib.it (A.N.)
Department of Materials Science, University of Milano—Bicocca, via R. Cozzi 55, 20125 Milano-IT, Italy;
luca.vaghi@unimib.it (L.V.); i.andreosso@campus.unimib.it (I.A.)
CNR, Institute of Molecular Science and Technologies, Via C. Golgi 19, 20133 Milano-IT, Italy;
m.marelli@istm.cnr.it
2
3
*
Correspondence: antonio.papagni@unimib.it (A.P.); laura.cipolla@unimib.it (L.C.);
Tel.: +39-0264483460 (L.C.)
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 17 January 2019; Accepted: 5 February 2019; Published: 7 February 2019
Abstract: Gelatin is a biopolymer with interesting properties that can be useful for
biomaterial design for different applications such as drug delivery systems, or 3D scaffolds
for tissue engineering.
However, gelatin suffers from poor mechanical stability at
physiological temperature, hence methods for improving its properties are highly desirable.
In the present work, a new chemical cross-linking strategy based on triazolinedione
ene-type chemistry towards stable hydrogel is proposed. Two different homobifunctional
1,2,4-triazoline-3,5(4H)-diones, namely 4,40-hexane-1,6-diylbis(3H-1,2,4-triazoline-3,5(4H)-dione)
1
and 4,40-[methylenebis(4,1-phenylene)]bis(3H-1,2,4-triazoline-3,5(4H)-dione)
2 were used as
cross-linkers in different ratio to tyrosine residues in gelatin. The reaction was proved effective
in all experimented conditions and hydrogels featured with different thermal stability were
obtained. In general, the higher the cross-linker/tyrosine ratio, the more thermostable the hydrogel.
The swelling properties are strictly dependent upon the chemical nature of the cross-linker.
Keywords: gelatin; hydrogel; ene-type chemistry; tyrosine; triazolinediones; cyclic diazodicarboxamides;
chemical cross-linking; natural polymers
1. Introduction
Gelatin is a protein mixture obtained from collagen hydrolysis in acid or basic conditions,
with excellent properties in terms of biodegradability, biocompatibility, cell-adhesion features, and
ease of modification, and it is also non-immunogenic. Due to its properties together with its
inexpensiveness and readiness, chemically modified gelatin or gelatin blended with other natural
or unnatural (macro)molecules [
engineering [ ] and for drug delivery [
design concerns its native poor mechanical properties and short degradation times, especially
under physiological conditions [ ], posing difficulties for shaping gelatin into hydrogels and
1–
3
] have been extensively employed as biomaterials for tissue
4,
5
6]. However, the main limitation in gelatin biomaterial
7–9
scaffolds with stable morphologies and the desired mechanical features. In order to overcome
this drawback, gelatin-based biomaterials are usually the result of physical, enzymatic, or chemical
cross-linking [10–13]. In the crosslinking process, the biopolymer functional groups react chemically,
enzymatically or physically interact with the cross-linker of choice, affording a 3D network. Chemical
Molecules 2019, 24, 589; doi:10.3390/molecules24030589
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cross-linking is generally preferred, since it affords stable covalent cross-links and better tuning and
reproducibility of the process. Chemical cross-linking may exploit either the intrinsic reactivity of
functional groups present in the biopolymer (i.e., amino acids side chain) [14
reactivity of functional groups introduced ad hoc in the biopolymer, that can be lately reacted
bio-orthogonally [16]. The second approach often relies on the so-called click-reactions [17 18] based for
example on carbonyl/oxime-hydrazone chemistry [19 20], Staudinger reaction [21 22], Huisgen-type
cycloaddition [23 25], Diels-Alder [26 28], and thiol-ene addition [29 30]. Click-chemistry offers
,15], or the extrinsic
,
,
,
–
–
,
several advantages such as high yield, mild reaction conditions and chemoselectivity. Despite the
very effective chemistry beyond extrinsic bioorthogonal reactions [31], and its broad applicability
to several fields [32], the main limitation is due to the need of a two step process, the first of which
is the introduction of extrinsic functionalities by chemical or enzymatic modification or by genetic
engineering approaches.
Gelatin has been cross-linked by taking advantage of extrinsic functional groups [33
direct cross-linking based on intrinsic amino acid reactivity. The most common cross-linkers used for
gelatin [12] are glutaraldehyde [39 40], 1,4-butanediol diglycidyl ether (BDDGE) [10], genipin [41],
–38] or through
,
citric acid [42], and bisvinyl sulfonemethyl (BVSM) [43]. All of the above mentioned cross-linking
agents target amino, hydroxyl or carboxyl groups in the amino acid side chains. Less exploited
are protein cross-linking techniques targeting the aromatic ring of tyrosine residues. Among them,
oxidative cross-linking of tyrosine phenolic groups has been proposed, mimicking the well-known
natural oxidation process of phenolic moieties [44–46]. In addition to the oxidative coupling affording
zero-length dityrosine adducts, very recently triazolinedione chemistry [47] has been proposed.
Triazolinedione ene-type chemistry recently emerged as a click-reaction for the bioconjugation
to tyrosine residues, mediated by cyclic monofunctional diazodicarboxamides, such as
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) [48–51]. Hetherobifunctional triazolinediones (TADs),
have been used for the synthesis of DNA protein conjugates [52], while homobifunctional TADs have
−
been applied to the cross-linking of synthetic polypetides [53].
In the present work, we propose for the first time the use of homobifunctional TADs for gelatin
cross-linking towards the production of hydrogels and scaffolds.
2. Results and Discussion
2.1. Cross-Linking of Gelatin
Two
cross-linkers,
groups,
different
both
namely
homobifunctional
characterized by
4,40-hexane-1,6-diylbis(3H-1,2,4-triazoline-3,5(4H)-dione)
reagents
two terminals
were
chosen
as
protein
1,2,4-triazoline-3,5(4H)-diones
1
and
and
4,40-[methylenebis(4,1-phenylene)]bis(3H-1,2,4-triazoline-3,5(4H)-dione)
2
(Figure 1a). Reagents 1
2
were synthesized following literature methodologies (see Supplementary Material for details) [54,55].
Figure 1. Cont.

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Figure 1.
(
a) The gelatin cross-linkers hetherobifunctional triazolinediones (TADs)
1 and 2 and the
corresponding reduced forms
3
and 4, respectively; ( ) TADs stability in different solvents (DMSO, 1:1
b
DMSO/H2O, 1:1 TRIS Buffer solution-pH = 7.4/acetonitrile). The disappearance of the fuchsia color
indicates the degradation of 1 and 2.
Gelatin can be easily dissolved in aqueous solutions above 37 ◦C; however, TADs are reported
to undergo degradation in water [50], despite a certain stability of some TADs in aqueous medium
reported in bioconjugation reactions by PTAD [56] or in cross-linking reaction of a synthetic Lys-Tyr
polypeptide in TRIS buffer solution by difunctional TAD
2 [53]. In our hands both 1 and 2 almost
immediately decomposed in any type of aqueous environment, as clearly indicated by the disappearing
of their characteristic fuchsia color in a few seconds (Figure 1b). Thus, suitable reaction conditions
should be found for effective gelatin cross-linking by TADs, in order to allow the cross-linking agents
to react with tyrosine residues present in the protein, affording the desired chemical reaction towards
network formation. Gelatin can be dissolved in DMSO, after vigorous stirring for 3 h at 37 ◦C at a
concentration of 12 mg/mL; in addition, DMSO has proven to be fully compatible with the use of
TADs [50]: DMSO solutions of
1 and 2 are stable for several hours since the characteristic fuchsia color
is maintained, confirming the stability of the two cross-linking agents in this medium (Figure 1b).
Thus DMSO could be the solvent of choice for the gelatin cross-linking reaction. Given the amino acid
composition of porcine gelatin, tyrosine is expected to be present from 3 to 4 mmol per 100 g of dry
gelatin [57]. In order to check the efficacy of the cross-linking, different TADs/tyrosine ratios were
◦
used. In a typical experiment, 100 mg of gelatin are dissolved in DMSO (8 mL) at 37 C; after complete
dissolution, the solution is cooled to r.t. and kept in the dark, due to the thermal and photochemical
instability of 1 and 2 [58], and 0.5:1, 1:1, 2:1, 5:1 TADs/tyrosine molar ratio was added. The reaction
mixtures slowly discolored over 30 min of stirring, indicating the progress of the reaction. Upon
completion of the reaction (indicated by the disappearance of the TAD color), cross-linked gelatin was
recovered by precipitation (adding methanol in the case of 1, and acetone in the case of 2).
Cross-linked gelatin hydrogels (Figure 2) were characterized by their thermal resistance at pH 7.4
and 37 ◦C, swelling properties, FT-IR spectroscopy, and SEM. All the collected data demonstrated the
effectiveness of the cross-linking methodology. Cross-linked gelatin shows improved thermal stability
as TADs/tyrosine ratio increases, as a consequence of increased reticulation.

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Figure 2.
(a) cross-linking reaction between gelatin and 1 or 2; (b) recovered hydrogels; (c) dried
cross-linked gelatin hydrogels.
In order to demonstrate that cross-linking occurs through covalent bonds formation (chemical
cross-linking), instead of non-bonding interactions (i.e., hydrogen bonds, physical cross-linking),
gelatin was also treated with the reduced form (urazole) of the TADs (compounds
in a 5 fold excess in respect to tyrosine for 30 min (as for the treatment with and
worked up. Given that effective cross-linking renders gelatin insoluble in water at 37 C, water
3 and 4, Figure 1a),
1
2
), and identically
◦
solubility assay is an immediate and easy way to check the cross-linking. Thus gelatin treated with
3
◦
◦
and
4 was soaked in water at 37 C; the specimen promptly dissolved in water at 37 C, indicating that
physical cross-linking did not occur. This assay demonstrates that chemical covalent cross-linking is
actually occurring with TADs 1 and 2.
2.2. Characterization of Cross-Linked Gelatin
2.2.1. Thermal Stability
Freeze-dried cross-linked gelatin specimens were rehydrated with 1 mL of PBS buffer (pH = 7.4),
◦
placed in a 37 C chamber to test their thermal stability (Figure 3), and compared with pristine
gelatin samples. As expected, pristine gelatin dissolved almost immediately; cross-linked gelatin with
TADs/tyrosine 0.5:1 ratio displayed better resistance when compared to untreated gelatin, dissolving
in about 1 h when reacted with TAD 1 and 2 h with TAD 2, respectively. In general, it was observed that
the increase of TADs/tyrosine ratios affords better performing cross-linked gelatin. For TAD/tyrosine
ratio 5:1, cross-linked gelatin was stable over a month.

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Figure 3. Thermal stability of hydrogels at 37 ◦C; results for any TAD/tyrosine ratio are means of 3
independent experiments.
2.2.2. Swelling Properties
Based on thermal stabilities, cross-linked gelatin with TADs/tyrosine 2:1 and 5:1, which resulted
the more stable, were tested for their swelling behavior in water by gravimetric analysis. Gel swelling
properties are usually dependent upon several factors, including pore size of the network, interactions
between the network (polymer chains and cross-linkers) and the solvent, and chain mobility during
the swelling process [59]. The dynamic swelling properties (swelling degree, SD) and the equilibrium
water content (EWC) for Gel_TAD1 and Gel_TAD2 are reported in Figure 4. All of the hydrogel
samples were prepared of same dimensions, approximately (10 mm diameter
described in the Experimental Section.
×
5 mm height), as
Figure 4. Cont.

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Figure 4.
(a) Swelling degree for gelatin hydrogel cross-linked either with TAD
1 or 2 at TAD/tyrosine
molar ratio 2:1 and 5:1; (
b) equilibrium water content; data are average of three independent
experiments, bars indicate standard deviation and statistical analysis was performed with t-student
with ** p < 0.01).
The SD plot (Figure 4a) shows a similar kinetic behavior for all of the considered specimen within
the first 5 h. However, Gel_TAD1 samples have better water retaining properties if compared to
Gel_TAD2, requiring higher times for reaching the equilibrium. This behavior might be ascribed to the
different hydrophilicity of the linkers and eventually to the different conformational freedom that may
have a role in the chain mobility of the network. The diphenyl moiety in TAD
and confers higher conformational rigidity to the cross-linker if compared to the hexyl moiety in TAD
. In addition, the chemical nature of TAD has more relevance in influencing gel properties as a
2 is more hydrophobic
1
2
function of TAD/Tyr ratio: the higher the TAD/Tyr ratio, the lower SD and EWC values.
2.2.3. FT-IR Characterization
Gelatin specimens were analyzed by FTIR measurements in attenuated total reflection (ATR).
The ATR-FTIR absorption spectra of the different gelatin samples display the typical spectral features
of polypeptides and are characterized by the Amide I and Amide II bands and several partially
overlapped components in the fingerprint region around 1500–800 cm−1 (Figure 5, insets a-1 and b-1).
Small spectral changes were observed after TAD 1 and TAD 2 treatments. The intensity variations were
evaluated by second derivative analysis that enable to discriminate among overlapped components of
the absorption spectra. In particular, a significant increase of the 1013 cm−1 and 952 cm−1 peaks was
observed in the 5:1 TAD
also present in the neat TAD
vibrations [60
observed in the 5:1 TAD
1/tyrosine ratio (Figure 5, insets a-2 and a-3). These components, which are
1
cross-linker (Figure 5, inset a-1), were tentatively a−ss1igned to the CN
,
61]. In the case of TAD 2 cross-linker, a strong increase of the 1512 cm component was
2/tyrosine ratio (Figure 5, insets b-2 and b-3). This component is typically
assigned to the CC vibrations of the aromatic ring [61,62]. The FTIR data thus confirm the gelatin
cross-linking through TAD 1 and TAD 2.

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Figure 5. Insets a-1 and b-1: ATR-FTIR absorption spectra of untreated gelatin, TADs and cross-linked
−1
gelatin specimens reported in the 1725–800 cm region.; the second derivatives of TAD
1 (inset a-2)
and TAD (inset b-2) cross-linked samples are reported in the spectral regions where the contributions
2
of the TADs moieties can be detected. The intensities of the indicated components were evaluated from
the second derivative spectra (insets a-3 and b-3). Error bars refer to three independent measurements.
Spectra are shown after normalization at the Amide I band area.
2.2.4. Scanning Electron Microscopy Micrographs
Low-vacuum scanning electron microscopy was used in order to investigate the surface
morphological changes induced by the different TADs cross-linkers and ratio experimented. A
porous structure along with the foam-like morphology was recognizable (Figure 6 and Supplementary
Material, Figure S5). As a general trend, moving from 0.5:1 to 5:1 TAD/tyrosine ratio (Figure 6, and
Supplementary Material, Figure S5) the increased cross-linkers amount results in an increase in the
reticulation texture and a less heterogeneity in porosity and pore size due to increased cross-linking,
while pristine gelatin shows a heterogeneous texture with open pores ranging from 5 to 20 µm
(Figure 6a). The addition of the cross-linkers increases the interconnected porosity and the pore size
shrinks down, ranging from 5 to 2 µm.
Figure 6. Representative SEM micrograph of gelatin samples (
1 in (b) 0.5:1 and (c) 5:1 TAD 1/tyrosine ratio respectively.
a) pristine gelatin and cross-linked with

Molecules 2019, 24, 589
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3. Materials and Methods
3.1. General
All reagents and solvents were purchased from commercial sources (Sigma-Aldrich S.r.l., Milan,
Italy and Fluorochem Ltd., Hadfield, United Kingdom) and used without further purification. Gelatin
from porcine skin-Type A was used for hydrogel preparation (Sigma-Aldrich, catalog no. G2500).
1
Tyrosine content in gelatin is reported as 3.4
µ
mol every 100 mg of gelatin [57]. H NMR spectra were
recorded with a Bruker Avance 500 (Bruker corp., Billerica, MA, USA). ATR-FTIR spectra of TAD
1 and
2
and related synthetic intermediates were recorded with a Perkin-Elmer Spectrum 100 (Perkin-Elmer
Waltham, MA, USA); ATR-FTIR spectra of gelatin specimens were collected with a Varian 670-IR
(Varian Australia Pty Ltd., Mulgrave VIC, Australia) spectrometer equipped with the Quest (Specac)
ATR device [63]; Scanning electron microscopy (SEM) analysis were performed with a Philips XL30
ESEM (FEI, Hillsboro, OR, USA).
Cross-linked gelatin was freeze-dried by a Christ alpha 1–2 freeze dryer (Christ, Osterode am
Harz, Germany). Melting points were measured with a Stanford Research Systems Optimelt apparatus.
3.2. Synthesis of Cross-Linking Agents
Cross-linkers
McGrath [54]. The procedure by Mallakpour and co-worker was used for the oxidation of urazoles
and 55]. For sake of completeness synthetic procedures are reported in the Supplementary Material.
1 and 2 were synthesized adapting literature procedures reported by Culbertson and
3
4
[
3.3. Gelatin Cross-Linking
3.3.1. Preparation of TAD 1 Cross-Linked Hydrogel Gel_TAD1
◦
To 100 mg of gelatin, 8 mL of DMSO were added and the suspension heated to 37 C until complete
dissolution. Given tyrosine content, for the 0.5:1 and 1:1 TAD/Tyr hydrogel, 95 L and 191 L of
a freshly prepared 5 mg/mL TAD solution in DMSO were added respectively (corresponding to
1.7 and 3.4 mol of TAD ); for the 2:1 and 5:1 molar ratio, 127 L and 318 L of a freshly prepared
15 mg/mL TAD solution in DMSO were added respectively (corresponding to 6.8 and 17.0 mol
of TAD ). The solutions were reacted at r.t. in the darkness under stirring until the purple solution
µ
µ
1
µ
1
µ
µ
1
µ
1
turned colorless (30 min). Cross-linked gelatin was recovered by precipitation by the addition of 8 mL
of methanol. The suspension was centrifuged (6500 rpm, 45 min) and washed first with methanol
(5 mL) then with deionized water (2 mL
tubes were freeze-dried and used for further characterization.
×
2). The hydrogels formed on the bottom of the centrifuge
3.3.2. Preparation of TAD 2 Cross-Linked Hydrogel Gel_TAD2
As described for Gel_TAD1, for the 0.5:1 and 1:1 TAD/Tyr hydrogel, 63 µL and 126 µL of a freshly
prepared 10 mg/mL TAD
3.4 mol of TAD ) to the gelatin solution; for the 2:1 and 5:1 molar ratio, 126
prepared 20 mg/mL TAD solution in DMSO were added respectively (corresponding to 6.8 and
2
solution in DMSO were added respectively (corresponding to 1.7 and
µ
2
µ
L and 315 L of a freshly
µ
2
17.0 µmol of TAD 2). The solutions were reacted at r.t. in the darkness under stirring until the purple
solution turned colorless (30 min). Cross-linked gelatin was recovered by precipitation by the addition
of 8 mL of acetone. The suspension was centrifuged (6500 rpm, 45 min) and washed first with acetone
(5 mL) then with deionized water (2 mL
tubes were freeze-dried and used for further characterization.
×
2). The hydrogels formed on the bottom of the centrifuge
3.4. Thermal Stability Studies
Three replicas of dried Gel_TAD specimens (ca 90 mg, 10 mm diameter
each tested condition (TAD and Tyr/TAD ratio) were placed in tagged wells of 12 multiwell plates,
×
5 mm height) from

Molecules 2019, 24, 589
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◦
hydrated with PBS (4 mL, pH = 7.4, physiological conditions), and kept sealed at 37 C. The specimens
were periodically visually inspected.
3.5. Swelling Studies
Dynamic swelling measurements were made by gravimetric measurements. Three replicas of
dried 2:1 and 5:1 Gel_TAD specimens (ca 90 mg, 10 mm diameter
distilled water at 25 ◦C. The swollen gel discs were periodically removed from water, blotted with
filter paper, and weighed on an analytical balance (Analytical Balance 220 g 0.1 mg, Radwag AS
220/C/2) and returned to the swelling medium till the equilibrium is reached.
Swelling degree (SD) was calculated from the following equation and reported as a function
×
5 mm height) were soaked in
×
of time:
−1
Swelling degree (SD, g·g−1) = (Wt − W0) × W0
.
(1)
where Wt is the weight of swelling hydrogel at different time and W0 is the dry weight of the gel.
The equilibrium water content (EWC), was calculated from the following equation:
−1
EWC (%) = (We − W0) × We × 100.
(2)
where We is the swelling weight of the sample at equilibrium and W0 is the dry weight of the gel.
3.6. SEM Analysis
Scanning electron microscopy (SEM) analysis were performed working at 8 kV accelerating
voltage and in low vacuum mode (1 Torr). Sample were dried, cut, fixed with conductive carbon
tape to standard SEM stubs and directly analyzed. Working at low vacuum condition, no conductive
coatings were applied in order to preserve the original structure. Samples showed good stability under
electron beam illumination at the operating conditions.
4. Conclusions
A new cross-linking methodology for proteins such as gelatin useful for the preparation of
hydrogels has been proposed through the use of homobifunctional triazolinediones. The reaction
is effective, as demonstrated by several characterization techniques and hydrogel thermostability if
compared to untreated gelatin.
Supplementary Materials: The following are available online, Scheme S1: Synthesis of the cross-linking agents
and experimental procedures, Figure S1: 1H NMR of urazole , Figure S2: 1H NMR of urazole
3 4, Figure S3:
ATR-FTIR of urazole 3, Figure S4: ATR-FTIR of urazole 4, Figure S5: SEM images of hydrogels.
Author Contributions: A.P. and L.C. conceived and designed the experiments; R.G., L.V., A.N., I.A., and M.M.
performed the experiments, and analyzed the data; all authors contributed to paper writing.
Funding: This research received no external funding.
Acknowledgments: L.C. gratefully acknowledges Fondo di Ateneo (FA) 2018.
Conflicts of Interest: The authors declare no conflict of interest.
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