An enzyme-assisted nanoparticle crosslinking approach to enhance the mechanical strength of peptide-based supramolecular hydrogels

Article abstract of DOI:10.1039/c3cc45127e

In this work we reported an enzyme-assisted nanoparticle crosslinking (EANC) strategy to enhance the mechanical stability of peptide-based supramolecular hydrogels by more than 3000 times. This journal is

Full text of DOI:10.1039/c3cc45127e

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An enzyme-assisted nanoparticle crosslinking approach
to enhance the mechanical strength of peptide-based
supramolecular hydrogels†
Cite this: Chem. Commun., 2013,
49, 8653
Received 8th July 2013,
Accepted 31st July 2013
Ying Li,*a Yin Ding,b Meng Qin,*c Yi Caoc and Wei Wang*c
DOI: 10.1039/c3cc45127e
www.rsc.org/chemcomm
In this work we reported an enzyme-assisted nanoparticle crosslinking foot byssus, which is made of protein complexes with nanosized
(EANC) strategy to enhance the mechanical stability of peptide-based granular structures through coordinate bonds mediated mainly by
supramolecular hydrogels by more than 3000 times.
3,4-dihydroxyphenylalanine (DOPA), a non-natural amino acid.16 DOPA
is well known for its ability to adhere clad, metal, and even organic
Peptide-based supramolecular hydrogels have been widely used as surfaces and crosslinking upon oxidation.17 The interactions mediated
useful biomaterials for various biomedical applications, including by DOPA confer the high toughness of marine mussel foot byssus. We
biosensors, drug delivery, tissue engineering, and so on.1–5 Especially, proposed that if DOPA was engineered into peptide hydrogelators, the
due to the high biocompatibility and low cytotoxicity, peptide hydro- engineered hydrogels could be crosslinked via DOPA and nanoparticles
gels have been widely used to culture cells for regenerative medicine. (NPs), mimicking the structure of marine mussel foot byssus. The
Recently, increasing evidence suggests that the mechanical properties resulting hydrogels could therefore possess high mechanical stability.
of the hydrogels are important for cell adhesion, migration, division, Indeed, studies on polymer hydrogels showed that introducing
and even stem cell differentiation.6 However, generally, peptide-based DOPA crosslinking into hydrogels could significantly enhance the
hydrogels are mechanically weak with storage moduli in the range of mechanical stability.18,19 However, practically applying the DOPA
10–1000 Pa.1,7 For the engineering of load-bearing soft tissues, such as based crosslinking to peptide-based supramolecular hydrogels is
cartilage, muscle, blood vessels, and tendon, hydrogels with much challenging, because the strong DOPA–nanoparticle interactions
higher mechanical strength are required, which challenges the may also interfere with the self-assembly of the peptide hydro-
current synthetic strategies. A few efforts have been made towards gelators, leading to inhomogeneous aggregates instead of hydro-
enhancing the mechanical strength of peptide-based hydrogels, gels. To avoid such unwelcome side effects, here we introduced an
including the addition of macromolecules,8,9 incorporation of specific enzyme-assisted nanoparticle crosslinking (EANC) strategy for the
ligand–receptor interactions,10,11 crosslinking,12 formation of hybrid engineering of hydrogels with high mechanical strength.
materials,13 kinetic control,14 and so on.5,15 However, the highest
A schematic illustration of the EANC strategy is shown in Fig. 1.
mechanical stability that the peptide-based hydrogels can reach is still We used the well-studied tyrosine containing peptide, 2-NapGFFY as
below 50 kPa at physiological pH, and the available strategies to the hydrogelator precursor. The stacking of the N-terminal 2-naphtha-
enhance the mechanical strength are also very limited.
lenyl group (2-Nap) as well as the diphenylalanine motif provides the
Here, we report a novel approach to enhance the mechanical driving force for the self-assembly of the peptide into fibrous networks
stability of peptide-based hydrogels with storage moduli of more than of hydrogels.2,4,8,11 The hydrogel is formed in the presence of various
0.2 MPa. This method is inspired by the structure of marine mussel amounts of silica nanospheres. After the hydrogel is formed, a desired
amount of mushroom tyrosinase is added to the hydrogel to convert
a Jiangsu Key Laboratory of Atmospheric Environment Monitoring & Pollution
Control, College of Environmental Science & Engineering, Nanjing University of
Information Science & Technology, 219 Ningliu Road, Nanjing, Jiangsu, 210044,
P. R. China. E-mail: yingliubc@gmail.com; Fax: +86 25 58731090;
Tel: +86 13382024479
b State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry
and Chemical Engineering, Nanjing University, 22 Hankou Road, Nanjing, Jiangsu,
210093, P. R. China
c National Laboratory of Solid State Microstructure, Department of Physics,
Nanjing University, 22 Hankou Road, Nanjing, Jiangsu, 210093, P. R. China.
E-mail: qinmeng@nju.edu.cn, wangwei@nju.edu.cn
† Electronic supplementary information (ESI) available: Experimental details and
Fig. 1 Schematic illustration of the EANC strategy.
supporting figures. See DOI: 10.1039/c3cc45127e
c
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Chem. Commun., 2013, 49, 8653--8655 8653

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the tyrosine residue into DOPA. This creates interactions between the weight of 2-NapGFFY, the G0 of the enzyme treated mixture increased
as-formed fibrils and silica NPs. Because the crosslinking is intro- to 250 kPa. Clearly, the EANC strategy successfully increased the
duced after the self-assembly of peptide fibrils, we could effectively mechanical strength of 2-NapGFFY hydrogels by introducing the
minimize the interference between these two processes.
nanoparticle–peptide interactions. Further, the gel recovery experi-
First, we studied the gelation of 2-NapGFFY (peptide purity >95% as ments disclosed that the gel prepared by this method could not fully
in Fig. S1, ESI†). We adopted the recently developed K2S2O8 decom- recover (Fig. S5B, ESI†). Moreover, the storage modulus of 250 kPa is
position method to prepare the hydrogel of 2-NapGFFY (pH 6.6).5 among the highest storage moduli of peptide-based supramolecular
Moreover, the oxygen released from the decomposition of K2S2O8 can hydrogels reported so far.
be used for the subsequent tyrosine oxidation. Similarly, 2-NapGFFY
To confirm that the increase of the mechanical stability of the
hydrogels with silica NPs (B20 nm in diameter) of 20%, 50%, and hydrogels is indeed due to the formation of DOPA, we monitored the
100% of the dry weight of the peptide were also prepared using the change of the storage modulus of 2-NapGFFY in real time after
same procedure by directly adding the nano-dispersed silica NPs to the the addition of tyrosinase. After the hydrogel and tyrosinase were
solution. Transparent hydrogels were formed (Fig. 2A). The mechanical thoroughly mixed, the hydrogel was immediately loaded on a
properties of these hydrogels were similar regardless of the amount of rheometer plate using a spatula and sealed using mineral oil to
the added NPs due to the lack of direct peptide–NP interactions.
avoid water evaporation. As shown in Fig. 3A, without silica NPs, the
Rheology measurements revealed that the storage moduli (G0) of storage modulus increased gradually upon the addition of tyrosinase
these hydrogels were all within 100 Pa at a frequency of 1 Hz, which and reached a plateau of 13 kPa after 10 h. However, in the presence
were higher than the loss moduli (G00) (Fig. 2B), but all lost the gel of 100% weight of NPs, the storage modulus can reach a much
properties at higher frequencies (Fig. S4, ESI†). These studies higher plateau of 32 kPa. It is worth mentioning that the final
confirmed the formation of weak peptide based hydrogels.
mechanical strength of the hydrogels prepared in such rheology
Then we tested whether the addition of tyrosinase could experiments is less than those prepared in test tubes due to the
increase the mechanical stability of the hydrogels as proposed. deficiency of the oxygen present for the enzymatic reaction. The
Mushroom tyrosinase (4.2 U mLꢀ1 of final concentration) was added increase of the mechanical strength of the hydrogel was accompa-
to 300 mL of 2-NapGFFY hydrogel (5 mg mLꢀ1) and the mixture was nied by the enzymatic conversion of 2-NapGFFY to 2-NapGFF-DOPA,
allowed to react for 10 h. After the addition of tyrosinase, the which was monitored using the UV-Vis spectroscopy (Fig. 3B). Before
hydrogels became brown. However, as the reaction proceeded, the the addition of tyrosinase, the UV-Vis spectrum of 2-NapGFFY
color became darker and darker, probably due to the oxidation of showed a maximum peak at B275 nm corresponding to the
DOPA to leukochrome and then dopachrome (Fig. 2C, Fig. S2 and S3, absorption peak of tyrosine (Fig. S7, ESI†). As the reaction proceeded,
ESI†).18,20 The mechanical properties of the hydrogels were also the maximum absorption shifted slightly to B280 nm, corre-
measured using rheology at 1 Hz with 0.1% strain based on the sponding to the production of 2-NapGFF-DOPA. Moreover the
linear viscoelastic region (LVE) of the gel (Fig. S5A, ESI†). Clearly, all absorbance of the peak increased gradually because DOPA has a
hydrogels showed much improved mechanical strength. The G0 and much higher extinction coefficient (2.63 and 1.49 ꢁ 103 Mꢀ1 cmꢀ1
G00 of these hydrogels were shown in Fig. 2D and Fig. S6, ESI.† Even for DOPA and tyrosine, respectively). Besides the increase of this
without NPs, the storage modulus of the 2-NapGFFY hydrogel major peak, a shoulder peak in the region between 310 and 380 nm
increased from B60 Pa to B130 kPa at a frequency of 1 Hz. Such was also observed, suggesting the formation of quinone inter-
an increase of mechanical strength may be due to the crosslinking mediates. The successful conversion of tyrosine to DOPA was further
through a poorly understood polymerization from the over-oxidized confirmed using mass spectrometry. The peak of 2-NapGFF-DOPA
quinone product of leukochrome (Fig. S3, ESI†). However, the (739.1423) was clearly detected in the gel after the enzymatic reaction
stabilization effects from the crosslinking between DOPA and silica (Fig. S8, ESI†).
NPs were obvious. The more the NPs, the higher were the storage
To get more insight into the mechanism of the mechanical
moduli. With the increase of nanoparticle contents to 100% of the stabilization of peptide hydrogels using the EANC strategy, we
studied the microscopic structures of the hydrogels using atomic
Fig. 3 Enzymatic reactions monitored using rheology (0.1% strain and 1 Hz)
and UV-Vis spectroscopy. (A) The gelation process tracked with rheology using
Fig. 2 The gel images and rheology measurements (0.1% strain and 1 Hz) of
5 mg mLꢀ1 2-NapGFFY gels at different contents (relative to the weight of the
peptide) of SiO2 NPs before (A and B) and after the enzymatic reactions using
4.2 U mLꢀ1 mushroom tyrosinase (C and D).
oscillation time sweep mode over 5 mg mLꢀ1 2-NapGFFY and 4.2 U mLꢀ1
mushroom tyrosinase with or without 5 mg mLꢀ1 SiO2 NPs, and (B) UV-Vis
spectra of the enzymatic reactions with 1.53 mg mLꢀ1 2-NapGFFY and 4.2 U mLꢀ1
in 50 mM phosphate buffer (pH 6.6).
c
8654 Chem. Commun., 2013, 49, 8653--8655
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Notes and references
1 J. Raeburn, A. Z. Cardoso and D. J. Adams, Chem. Soc. Rev., 2013, 42,
5143–5156.
2 C. Ou, J. Zhang, X. Zhang, Z. Yang and M. Chen, Chem. Commun.,
2013, 49, 1853–1855.
3 Y. Q. Liu, T. Y. Wang and M. H. Liu, Chem.–Eur. J., 2012, 18,
14650–14659; J. H. Collier and T. Segura, Biomaterials, 2011, 32,
4198–4204; Y. F. Tian, J. M. Devgun and J. H. Collier, Soft Matter,
2011, 7, 6005–6011; R. Lin, A. G. Cheetham, P. Zhang, Y. A. Lin and
H. Cui, Chem. Commun., 2013, 49, 4968–4970; S. Sutton, N. L. Campbell,
A. I. Cooper, M. Kirkland, W. J. Frith and D. J. Adams, Langmuir, 2009,
25, 10285–10291; Y. Pan, X. Du, F. Zhao and B. Xu, Chem. Soc. Rev., 2012,
41, 2912–2942; H. Wang and Z. Yang, Nanoscale, 2012, 4, 5259–5267;
L. Q. Chen, J. C. Wu, Y. W. Lihui, T. M. Shu, M. Xu, M. M. Zhang and
T. Yi, Langmuir, 2009, 25, 8434–8438; X. D. Xu, Y. F. Chu, C. S. Chen,
J. X. Chen, S. X. Cheng, X. Z. Zhang and R. X. Zhuo, Small, 2011, 7,
2201–2209; H. Komatsu, S. Matsumoto, S. Tamaru, K. Kaneko, M. Ikeda
and I. Hamachi, J. Am. Chem. Soc., 2009, 131, 5580–5585; J. Li, Y. Gao,
Y. Kuang, J. Shi, X. Du, J. Zhou, H. Wang, Z. Yang and B. Xu, J. Am. Chem.
Soc., 2013, 135, 9907–9914; J. Li, Y. Kuang, Y. Gao, X. Du, J. Shi and
B. Xu, J. Am. Chem. Soc., 2013, 135, 542–545; H. Huang, Y. Ding, X. S. Sun
and T. A. Nguyen, PLoS One, 2013, 8, e59482; T. Y. Cheng, H. C. Wu,
M. Y. Huang, W. H. Chang, C. H. Lee and T. W. Wang, Nanoscale, 2013,
5, 2734–2744; Y. Gao, J. Shi, D. Yuan and B. Xu, Nat. Commun., 2012,
3, 1033; S. E. Wheeler, A. J. McNeil, P. Muller, T. M. Swager and
K. N. Houk, J. Am. Chem. Soc., 2010, 132, 3304–3311; J. Chen, W. Wu
and A. J. McNeil, Chem. Commun., 2012, 48, 7310–7312; S. C. Bremmer,
J. Chen, A. J. McNeil and M. B. Soellner, Chem. Commun., 2012, 48,
5482–5484; A. Wada, S. Tamaru, M. Ikeda and I. Hamachi, J. Am. Chem.
Soc., 2009, 131, 5321–5330; T. Y. Cheng, M. H. Chen, W. H. Chang,
M. Y. Huang and T. W. Wang, Biomaterials, 2013, 34, 2005–2016;
M. Ikeda, R. Ochi, Y. S. Kurita, D. J. Pochan and I. Hamachi, Chemistry,
2012, 18, 13091–13096; M.-O. M. Piepenbrock, G. O. Lloyd, N. Clarke and
J. W. Steed, Chem. Rev., 2010, 110, 1960–2004.
4 L. Lv, H. Liu, X. Chen and Z. Yang, Colloids Surf., B, 2013, 108, 352–357.
5 B. Ding, Y. Li, M. Qin, Y. Ding, Y. Cao and W. Wang, Soft Matter,
2013, 9, 4672–4680.
6 W. Y. Seow and C. A. Hauser, Adv. Healthcare Mater., 2013; T. Dvir, B. P.
Timko, D. S. Kohane and R. Langer, Nat. Nanotechnol., 2011, 6, 13–22.
7 C. Yan and D. J. Pochan, Chem. Soc. Rev., 2010, 39, 3528–3540.
8 D. Li, H. Wang, D. Kong and Z. Yang, Nanoscale, 2012, 4, 3047–3049.
9 J. Wang, H. Wang, Z. Song, D. Kong, X. Chen and Z. Yang, Colloids
Surf., B, 2010, 80, 155–160.
10 Y. Zhang, Z. Yang, F. Yuan, H. Gu, P. Gao and B. Xu, J. Am. Chem.
Soc., 2004, 126, 15028–15029.
11 X. Zhang, X. Chu, L. Wang, H. Wang, G. Liang, J. Zhang, J. Long and
Z. Yang, Angew. Chem., Int. Ed., 2012, 51, 4388–4392.
Fig. 4 AFM images of 5 mg mLꢀ1 hydrogels. (A) Without NPs and enzyme; (B)
with 5 mg mLꢀ1 NPs; (C) with 4.2 U mLꢀ1 enzyme; and (D) with both 5 mg mLꢀ1
NPs and 4.2 U mLꢀ1 enzyme.
force spectroscopy (AFM). As shown in Fig. 4A, the 2-NapGFFY
hydrogel without the enzyme adopted fibril structures of B20 nm,
consistent with the transmission electron microscopy (TEM)
images reported previously.2 The fibrils were branched, forming
interconnecting networks. When silica NPs were added, although
the NPs were attached to the fibrils, the hydrogel network did not
change upon the addition of silica NPs (Fig. 4B). This is probably
due to weak interactions between silica NPs and peptide fibrils.
However, after the enzymatic reaction, the shape of the peptide
fibrils was dramatically altered. The fibrils became a bit thinner of
B15 nm (Fig. 4C). Interestingly, the diameter of the fibrils was not
homogeneous. This might be due to the random conversion of
tyrosine residues to DOPA in the peptide fibrils. Obviously, the
fibrils became more entangled after the enzymatic reaction. This
explains the high mechanical strength of the hydrogels after the
enzymatic treatment. Moreover, when silica NPs were added to
the system, the fibrils were further connected through the nano-
particle hubs (Fig. 4D). The multiple valences of NPs effectively
increased the inter-fibril interactions and thus the incorporation
of NPs further enhanced the mechanical strength of the hydrogels
because of the formation of more hydrogen bonds and weak
covalent bonds between DOPA and the hydroxyl groups on the NP
surfaces (Fig. S9, ESI†).
12 J. Gao, W. Zheng, D. Kong and Z. Yang, Soft Matter, 2011, 7, 10443–10448.
13 B. Adhikari, J. Nanda and A. Banerjee, Soft Matter, 2011, 7, 9259–9266;
J. Nanda, B. Adhikari, S. Basak and A. Banerjee, J. Mater. Chem. B, 2012,
116, 12235–12244; J. Nanda, A. Biswas, B. Adhikari and A. Banerjee,
Angew. Chem., Int. Ed., 2013, 52, 5041–5045.
14 J. Boekhoven, J. M. Poolman, C. Maity, F. Li, L. van der Mee,
C. B. Minkenberg, E. Mendes, J. H. van Esch and R. Eelkema, Nat.
Chem., 2013, 5, 433–437; A. R. Hirst, S. Roy, M. Arora, A. K. Das,
N. Hodson, P. Murray, S. Marshall, N. Javid, J. Sefcik, J. Boekhoven,
J. H. van Esch, S. Santabarbara, N. T. Hunt and R. V. Ulijn, Nat.
Chem., 2010, 2, 1089–1094.
15 K. L. Morris, L. Chen, J. Raeburn, O. R. Sellick, P. Cotanda, A. Paul,
P. C. Griffiths, S. M. King, R. K. O’Reilly, L. C. Serpell and D. J. Adams,
Nat. Commun., 2013, 4, 1480; K. J. Nagy, M. C. Giano, A. Jin, D. J. Pochan
and J. P. Schneider, J. Am. Chem. Soc., 2011, 133, 14975–14977.
16 M. J. Harrington, A. Masic, N. Holten-Andersen, J. H. Waite and
P. Fratzl, Science, 2010, 328, 216–220.
17 J. H. Waite, N. H. Andersen, S. Jewhurst and C. J. Sun, J. Adhes.,
2005, 81, 297–317; B. P. Lee, P. B. Messersmith, J. N. Israelachvili
and J. H. Waite, Annu. Rev. Mater. Res., 2011, 41, 99–132; Q. Ye,
F. Zhou and W. M. Liu, Chem. Soc. Rev., 2011, 40, 4244–4258.
18 B. P. Lee, J. L. Dalsin and P. B. Messersmith, Biomacromolecules,
2002, 3, 1038–1047.
In summary, in this work we successfully developed the EANC
strategy to enhance the mechanical stability of peptide-based
hydrogels by more than 3000 times. The mechanical stability of
the hydrogels can be tuned by the amount of nanoparticles
added or the enzymatic reaction time. Investigation of other
nanoparticles and further exploring the biomedical applications
of such hydrogels will be our next endeavor.
This work was supported by the 973 Program of China (no.
2012CB921801), the NSFC, under grant no. 91127026, 81121062,
31170813 and 11074115, and the Priority Academic Program
Development of Jiangsu Higher Education Institutions. Dr Ying
Li was supported by CPSF (2013M531312) granted by China.
19 D. E. Fullenkamp, J. G. Rivera, Y. K. Gong, K. H. Lau, L. He, R. Varshney
and P. B. Messersmith, Biomaterials, 2012, 33, 3783–3791.
20 M. Rolff, J. Schottenheim, H. Decker and F. Tuczek, Chem. Soc. Rev.,
2011, 40, 4077–4098.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 8653--8655 8655