Dual-purpose PEG scaffolds for the preparation of soft and biofunctional hydrogels: The convergence between CuAAC and thiol-ene reactions

Article abstract of DOI:10.1039/c3cc42084a

Orthogonally functionalized PEGs displaying alkenes and azides have been prepared and their dual-purpose scaffolding potential was exploited via click chemistry for controlled insertion of biorelevant moieties as well as facile fabrication of soft, non-to

Full text of DOI:10.1039/c3cc42084a

Chemical Communications
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Volume 49 | Number 62 | 11 August 2013 | Pages 6923–7014
ISSN 1359-7345
COMMUNICATION
Michael Malkoch et al.
Dual-purpose PEG scaffolds for the preparation of soft and biofunctional
hydrogels: the convergence between CuAAC and thiol–ene reactions
1359-7345(2013)49:62;1-X

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Dual-purpose PEG scaffolds for the preparation of soft
and biofunctional hydrogels: the convergence
between CuAAC and thiol–ene reactions†
Cite this: Chem. Commun., 2013,
49, 6938
Received 21st March 2013,
Accepted 16th April 2013
a
a
a
b
¨
Kim Oberg, Yvonne Hed, Isabella Joelsson Rahmn, Jonathan Kelly,
Peter Lowenhielmbc and Michael Malkoch*a
¨
DOI: 10.1039/c3cc42084a
www.rsc.org/chemcomm
Orthogonally functionalized PEGs displaying alkenes and azides via a dendritic hybridization approach and their dual-purpose
have been prepared and their dual-purpose scaffolding potential function evaluated for the construction of soft and bioactive
was exploited via click chemistry for controlled insertion of bio- hydrogels.
relevant moieties as well as facile fabrication of soft, non-toxic and
degradable hydrogels.
PEGs, 6 kDa and 10 kDa, were selected as model components
for the introduction of dual functionalities, Scheme 1. For the
bifunctionalization, anhydride activated AB2C-type monomer 1 was
Poly(ethylene glycol) (PEG) based hydrogels are promising 3D used to efficiently produce the desired scaffolds.10,11 Esterification
networks for a range of biomedical applications including reactions of the two PEGs with 4 equimolar excess of 1, followed
tissue engineering,1 drug delivery,2 and as artificial extra cellular by mild acidic deprotection afforded P6k-[G1]-(OH)4-(N3)2 5 and
matrices (aECMs) for studying matrix-influenced cell behaviour.3 P10k-[G1]-(OH)4-(N3)2 6. Both compounds were isolated by
Their biocompatibility along with their hydrophilic and elastic simple purification protocols and their architectural integrity
nature makes them attractive materials for producing high water was supported by NMR and MALDI-ToF MS as can be seen in
content reservoirs that mimic natural ECMs. Traditional PEG Fig. S2, (ESI†). To exploit these scaffolds for the fabrication of
hydrogels are prepared via non-controlled radical initiated chain functional hydrogels, the four terminal hydroxyl groups were
growth polymerization of acrylic end-functional prepolymers. reacted with anhydride 2 and monitored by MALDI. The result-
However, to offer superior properties in terms of uniform network ing P6k-[G1]-(ene)4-(N3)2 7 (Fig. 1) and P10k-[G1]-(ene)4-(N3)2
8
structures and improved mechanical performance, step-growth decorated with four peripheral enes and two azides were isolated
approaches capitalizing on robust chemical reactions such as in excellent yields. The versatility of this concept was further
copper-catalyzed azide–alkyne cycloaddition (CuAAC) click reaction explored to generate functional PEG scaffolds with a higher and
and UV initiated thiol–ene coupling (TEC) reaction are nowadays controlled number of dual-purpose sites. In this case, P6k-[G1]-
preferred.4,5 Furthermore, the inherent inertness of PEGs commonly (OH)4 8 with four terminal hydroxyl groups was treated in a
requires addition of biorelevant moieties to enhance their cell- similar fashion resulting in a dendritic–linear–dendritic (DLD)
interaction efficacy in which bioorthogonal click chemistries block-copolymer P6k-[G2]-(ene)8-(N3)4 12. A pure DLD polymer
have emerged as a powerful tool.6 Strategies allowing unprecedented was obtained in three reaction steps with a total yield of 63% and
control over both presentation of biochemical cues and network displayed two-fold increased orthogonal functionalities i.e. eight
physiochemical properties are highly desired. One elegant strategy to ene and four azide groups, Fig. S3, (ESI†). While these scaffolds
introduce a high degree of functionality within hydrogels while were targeted for the fabrication of functional hydrogels, it is
maintaining precise control over the 3D architecture has been to important to point out that the dual functionalities can accurately
use dendritic–linear–dendritic (DLD) hybrids. In fact, such scaffolds be modified for other multipurpose studies.
have also been evaluated for improved gene transfection,7 ionically
The orthogonal nature of these scaffolds was assessed towards
crosslinked self-healing hydrogels8 and adhesives for corneal chemoselective modifications of the azide functionalities with
wound repair.9 In this report, bifunctional PEGs are prepared biorelevant moieties (BMs) via the robust CuAAC click reaction.12
The azides were reacted with alkyne functional biotin, L-dopamine
a KTH Royal Institute of Technology, Department of Fibre and Polymer Technology,
Teknikringen 56-68, SE-100 44 Stockholm, Sweden. E-mail: malkoch@kth.se;
(DOPA) and D-mannose (Man) at room temperature under
THF–water conditions in the presence of sodium ascorbate
and copper sulfate. For complete conversion of the azide
Fax: +46 8 790 8283; Tel: +46 8 790 8768
b
¨
Molnlycke Health Care AB, Box 130 80, SE 40252 Gothenburg, Sweden
groups
a [PEG-N3] : [BM-alkyne] : [Cu] : [ascorbate] ratio of
c SP Technical Research Institute of Sweden, Box 857, SE 501 15 Borås, Sweden
† Electronic supplementary information (ESI) available: Details of the experi-
mental procedures and synthesis. See DOI: 10.1039/c3cc42084a
1 : 1.4 : 0.2 : 0.4 was found to be optimal resulting in scaffolds
13, 14 and 15. As shown in Fig. 1, the attachments of dopamines
c
6938 Chem. Commun., 2013, 49, 6938--6940
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Scheme 1 (a) Introducing dual crosslinkable and bioactive functionalities to PEGs and (b) hydrogel fabrication.
Fig. 1 (a) MALDI-ToF and (b) 1H NMR used to follow CuAAC coupling to P6k-G1-ene4-(N3)2 with alkyne functional DOPA.
proceed with high efficacy producing the targeted compound
The motivation for the construction of functional hydrogels
14 in the range of the expected theoretical molecular weights was to mimic body tissues of various Young’s modulus, ranging
and with narrow dispersity. The completion of these reactions from soft brain tissue of 0.1–1 kPa to crosslinked osteoid collagen
were furthermore confirmed using 1H NMR by the disappearance of 25–40 kPa.16 UV initiated TEC chemistry has recently been
of the methylene group (–CH2N3) at 3.24 ppm, Fig. 1b.
shown to be a robust method for the construction of hydrogels
One crucial parameter when utilizing CuAAC for producing with gel fractions of 95% and above.5 As a result, 2 kDa PEG with
biomaterials is the removal of catalytic copper to avoid any copper dithiol functionalities P2K-(SH)2 17 was synthesized and used as a
induced toxicity.13 The concentration of copper in drinking water flexible crosslinker for the fabrication of hydrogels. Four different
should be below 1.3 ppm14 and for in vitro culturing of hepatocyte networks were prepared via UV initiated TEC chemistry in ethanol
cells the TD50 (toxic-dose-50) is in the level of 48 ppm (750 mM).15 (E) or water (W) with a balanced molar ratio between the [ene] and
The initial copper concentration in the crude P6k-[G1]-(ene)2-(Man)2 [SH] of dual-functional azide–ene PEGs 7, 8, 12 and the cross-
15 was calculated to be 34760 ppm, which is over 700 times above linker 17. All hydrogels, H6k-[G1]-(N3)2-E, H10k-[G1]-(N3)-E and
the threshold TD50 levels found in the mentioned in vitro studies. H6k-[G2]-(N3)4-E and H6k-[G1]-(N3)2-W, were efficiently produced
Interestingly, a simple purification protocol was identified which within 10 minutes upon a cytocompatible UV exposure of 365 nm
included purging through a neutral Al2O3 column followed by a (9 mW cmÀ2) and from 30 wt% of dry materials including
dialysis treatment in 0.5% (w/w) EDTA (pH 7.0). Graphite furnace photoinitiator I2959.17 As shown in Fig. 2, both the length of
atomic adsorption spectroscopy (GF-AAS) of the purified compounds PEG and the dendron generation (G1 and G2) have a dramatic
13–16 revealed a substantial [Cu] reduction to acceptable con- effect on the modulus and swelling properties. The Young’s
centrations of 3.5 to 11.5 ppm.
moduli (E) were found to be similar to several body tissues
c
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assay, Fig. 3. In all cases, the LDH levels were in the range of the
control containing only cell culturing medium. Intriguingly, the
exposure of fibroblast cells to all G1 hydrogels resulted in full
structural degradation within 3 days. This phenomenon was
not observed for hydrogels exposed to cell culturing media
without cells under equivalent conditions. The H6k-[G2]-(Man)4
network was found to be more stable and required 7 days
to fully degrade. The detected non-toxicity demonstrates the
feasibility of these hydrogels as temporary aECMs during the
recovery of damaged soft tissue. A comprehensive and in-depth
study of affinity of cells vs. hydrogels decorated with larger
dendrons and higher loading of an array of multivalent BMs,
including ECM adhesive peptides, is on-going and will be
described in future publications.
In summary, a robust methodology for the construction of
bifunctionalized PEGs has been proposed. Their dual-purpose
scaffolding ability via CuAAC click chemistry and UV initiated
TEC chemistry resulted in the fabrication of bioactive hydrogels
with moduli in the range of several body tissues. The apparent
degradability of the networks coupled with the low cytotoxicity
towards dermal human fibroblast cells may render them suitable
as impermanent and aECMs.
Fig. 2 (a) Obtained moduli and (b) water swelling properties.
We acknowledge Vinnova-Sambio and the Swedish Research
Council VR (2011-5358 and 2010-453) for financial support. Dr
Sofia Almqvist is acknowledged for the cell viability study.
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