Photoreversible patterning of biomolecules within click-based hydrogels

Article abstract of DOI:10.1002/anie.201106463

Lighting the way: Biochemical cues have been reversibly patterned into hydrogels with full spatiotemporal control by using two photochemical reactions. The hydrogel is conjugated with a peptide by a thiol-ene photoaddition reaction that is initiated by vi

Full text of DOI:10.1002/anie.201106463

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DOI: 10.1002/anie.201106463
Hydrogels
Photoreversible Patterning of Biomolecules within Click-Based
Hydrogels**
Cole A. DeForest and Kristi S. Anseth*
Polymer-based hydrogels have emerged as a unique class of
biomaterials that enable cells to be cultured in three
dimensions within user-defined synthetic microenviron-
ments.[1–3] Based on the specific hydrogel formulation, the
material properties of cell-laden constructs can be precisely
defined to impart different moduli, chemical moieties,
porosity, adhesivity, degradability, and stimuli responsiveness
over nano, micro, and macroscopic scales. Cells have been
engineered to proliferate within, migrate through, and
undergo differentiation inside these materials by tuning the
initial properties of these networks through the incorporation
of physiologically relevant cues .[4]
More recently, hydrogel platforms that permit the intro-
duction of biochemical epitopes at any point in time and space
to affect cell function dynamically after encapsulation have
been developed.[5] Although these techniques have been
successfully utilized to control cell adhesion and motility,[6,7]
promote endothelial tubulogenesis,[8,9] and direct cell out-
growth,[10,11] complementary platforms that enable the intro-
duction and subsequent removal of these signals would be
beneficial. For example, such systems would allow the
dynamic presentation of signaling biomolecules that are
found in the native, temporally variable niche occupied by
stem cells to be recapitulated more closely. Herein, we
demonstrate that the combination of two bioorthogonal
photochemical reactions enables the reversible spatial pre-
sentation of a biological cue, as well as the formation of
mediated addition of a thiol to an alkene, is readily initiated
by visible light (l = 490–650 nm) and an appropriate photo-
initiator (eosin Y).[12,13] The second reaction is the photo-
scission of an o-nitrobenzyl ether to give a nitroso compound
and an acid by-product upon exposure to UV light (l =
365 nm).[14,15] By synthesizing the biological molecule of
interest to contain both the thiol group for the photocoupling
reaction and the photolabile o-nitrobenzyl moiety (Fig-
ure 1a), the thiol-ene and photocleavage reactions can be
used to attach and subsequently remove covalently bound
bioepitopes in hydrogel networks, respectively (Figure 1b).
As these reactions arephotomediated, both the introduction
and subsequent removal of relevant biomolecules can be
explicitly controlled in space and time by exposure to light.
The multifunctional patterning peptide was synthesized
by a combination of standard Fmoc solid-phase methods, in
which the photodegradable azide acid 4-(4-(1-(4-azidobutan-
oyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic
acid[11]
was coupled to the N-terminus of the peptide sequence of
interest. The terminal azide moiety served as a protecting
group during peptide synthesis, which ensured that only one
photodegradable moiety was present per peptide. This azide
group was readily reduced on the resin by the Staudinger
reaction[16] with triphenylphosphine, which liberated the N-
terminal primary amine for further peptide synthesis. The
photoreversible patterning agent Ac-C-(PL)-RGDSK-
(AF488)-NH2 (1, AF488 = Alexa Fluor 488), which is based on
the ubiquitous cell-adhesion ligand RGD, was prepared and
contained both a photoreactive thiol on the cysteine residue,
and an adjacent photodegradable o-nitrobenzyl ether moiety
(PL).
The hydrogel was formed by the copper-free, strain-
promoted, azide–alkyne cyclooaddition (SPAAC) click reac-
tion between poly(ethylene glycol) (PEG), tetracyclooct-
yne[17,18] (2), and bis(azido), allyloxycarbonyl (alloc)-pro-
tected polypeptide N3-RGK(alloc)GRK-N3 (3) in an aqueous
medium (Figure 1a).[7,11,19] The resulting idealized SPAAC-
based network is homogenously populated with alloc func-
tionalities that contain photoreactive alkenes. These alkenes
serve as anchor points for the introduction of biochemical
cues by the thiol-ene photoconjugation reaction. As a side
note, this reaction is fully cytocompatible,[7,20] which means
that cells can be readily encapsulated and cultured in these
gels.
complex, well-defined, biomolecular gradients within
a
hydrogel. The results of this study highlight how the
regulation of the biochemical environment can be used in
the development of more sophisticated cell culture substrates.
The reversible patterning strategy is based on the
combination of two orthogonal, biocompatible photoreac-
tions.[11] The thiol-ene reaction, which involves the radical-
[*] Dr. C. A. DeForest, Prof. K. S. Anseth
Chemical & Biological Engineering, University of Colorado
and the Howard Hughes Medical Institute
424 UCB, Boulder, CO 80309-0424 (USA)
E-mail: kanseth@colorado.edu
Homepage: http://www.colorado.edu/che/ansethgroup/
[**] We thank Dr. A. Kloxin and M. Tibbitt for useful discussions on
photopatterning. Fellowship assistance to C.A.D. was awarded by
the US Department of Education Graduate Assistantships in Areas
of National Need. This work was supported financially by the
National Science Foundation (DMR 1006711) and the Howard
Hughes Medical Institute.
Peptide 1 was swelled into the hydrogel network and the
degree of thiol-ene photoconjugation was controlled by
varying the exposure time of the irradiation with visible
light (10 mWcmꢀ2, 0–120 s) or the concentration of the
eosin Y (2.5, 5, or 10 mm, Figure 1c). Eosin Y absorbs light
between l = 450 and 550 nm, with a maximum absorbance at
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201106463.
Re-use of this article is permitted in accordance with the Terms and
Conditions set out at http://angewandte.org/open.
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approximately 515 nm. The patterned peptide concentrations
ranged from 0–1.0 mm. This concentration range is biologi-
cally relevant as numerous groups have used concentrations
of peptides that range from mm–nm to manipulate the binding
of integrins or to sequester growth factors, which ultimately
directs critical cellular functions.[5,21] Subsequently, gels that
contained approximately 1.0 mm of patterned Ac-C-(PL)-
RGDSK(AF488)-NH2 (1) were exposed to UV light at differ-
ent intensities (5, 10, or 20 mWcmꢀ2) for various time periods
(0–600 s) to cleave the o-nitrobenzyl ether group, which
absorbs strongly between l = 325–415 nm, and photorelease
the patterned peptide (Figure 1d). As the photodegradable o-
nitrobenzyl ether moiety absorbs light and cleaves at wave-
lengths less than 415 nm, both the addition and cleavage
photoreactions can be performed independently by irradiat-
ing the system with different light sources. Furthermore, each
of these reactions can be initiated with multiphoton light (l =
860 nm for addition, 740 nm for cleavage), which offers the
opportunity to independently control these reactions in 3D, as
well as the ability to dynamically tune the pericellular region
to assay how cells respond to biochemical changes in their
local environment.
The photocleavage reaction was found to be a first order
degradation with a rate constant (k) given by:
ꢀeI
NAhn
ð1Þ
k ¼
where f is the quantum yield (0.020),[11] e is the molar
absorptivity (4780mꢀ1 cmꢀ1 at l = 365 nm),[11] I is the intensity
of the incident light, NA is the Avogadro constant, h is the
Planck constant, and n is the frequency of light. The value of
the rate constant indicates that the patterned peptide is
almost fully removed (more than 97%) after 10 min of
irradiation with 20 mWcmꢀ2 of UV light, or in real time with
multiphoton light (l = 740 nm). This time scale is relevant for
many typical cell-culture applications, such as the migration
and differentiation of cells, in which cellular responses are on
the order of several hours to days. From this analysis, the
precise amount of peptide that is photoreleased can be readily
predicted for a given light exposure (lines in Figure 1d).
As well as controlling the introduction and removal of
functional groups in the network by varying the patterning
conditions, spatial control of both the photoaddition and
photoremoval reactions was achieved by selectively exposing
Figure 1. a) Hydrogels were formed from PEG tetracyclooctyne
(Mn =ca. 10000 Da, (2)) and N3-RGK(alloc)GRK(N3)-NH2 (3). The
alloc groups were functionalized with the fluorescent peptide Ac-C-
(PL)-RGDSK(AF488)-NH2 (1) through thiol-ene photoreaction and
unfunctionalized through o-nitrobenzyl ether photocleavage. b) Repre-
sentation of the thiol-ene conjugation reaction (top), the o-nitrobenzyl
ether cleavage reaction (middle), and the overall hydrogel structure
and reversible patterning approach (bottom). c) Concentrations of
peptide 1 patterned by thiol-ene reaction as a function of the initiator
~
*
concentration ( =2.5 mm, & =5 mm, =10 mm) and visible light
exposure time. d) Experimentally-determined and predicted concentra-
tions (lines) of 1 based on the photocleavage kinetics from Eq. (1) as
*
a function of UV light intensity ( =5 mWcmꢀ2, & =10 mWcmꢀ2
=20 mWcmꢀ2) and exposure time.
,
~
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subvolumes of the hydrogel to light. By using traditional
photolithographic techniques, whereby the sample is exposed
to masked light, a 2D pattern of shapes (200 mm lines spaced
200 mm apart) was transferred throughout the thickness of a
gel. This pattern was then partially removed upon exposing
the gel to UV light through a second photomask to obtain a
series of checkered parallel lines (Figures 2a,c). By focusing
pulsed laser light within the hydrogel volume, the function-
alization of the network was controlled in 3D to first create a
double helix, and then to remove part of the structure to form
a single helix (Figures 2b,d). The patterning resolution was
limited by the optical limits of the equipment to approx-
imately 1 mm in the x-y plane for both photolithographic and
multiphoton-based techniques, and to approximately 3–5 mm
in the z-plane for the multiphoton technique. As these
distances are smaller than a typical mammalian cell, these
reactions should prove useful for many applications that are
focused on modifying the local microenvironment around
individual encapsulated cells.
As well as the presentation of discrete signals, graded
biochemical signaling cues are essential for many biological
processes, and can be used to screen the effects of various
morphogens rapidly, over a wide range of concentrations.
Graded signaling can also be used to study chemotaxis and
other phenomena. After patterning the gel with a fixed
amount of 1 (ca. 1 mm) with visible light, non-linear gradients
were formed within the material by exposing the patterned
substrate to a gradient of UV light (10 mWcmꢀ2). The
gradient was generated with a moving opaque photomask[22]
that covered the sample at a rate of 0.4, 0.8, and 1.6 mmminꢀ1.
For continuous gradients of light exposure, this approach
resulted in exponential gradients in the final concentration of
1 with predictable decay constants of 0.175 minꢀ1/(rate of
sample coverage). By photoreleasing peptides that were
Figure 2. Thiol-ene patterning of the fluorescent peptide Ac-C-(PL)-
RGDSK(AF488)-NH2 (1) into the hydrogel a) in 2D throughout the gel
after exposure to masked visible light and b) in 3D after exposure to
focused pulsed laser light. False coloring is used for enhanced visual-
ization. c) and d) Subsets of the pre-patterned cues were removed by
exposure to UV light to modify the original chemical pattern and yield
new 2D and 3D patterns. Scale bars=200 mm.
initially present in a uniform fashion throughout the bulk of
the network (Figure 3a,d) or within line patterns (Fig-
ure 3c,e), intricate exponential gradients were formed over
millimeter-scale distances within the material environment.
These gradients were either continuous or discrete line
Figure 3. Ac-C-(PL)-RGDSK(AF488)-NH2 (1) was patterned into hydrogels by using unmasked light (panels (a), (b), and (d)) or a mask that
contains 200 mm slits spaced 200 mm apart (panels (c), (e)). The prepatterned samples were subsequently exposed to gradients of UV light that
were generated by a moving opaque photomask (1.6, 0.8, or 0.4 mmminꢀ1 for (a); 0.4 mmminꢀ1 for (b–e)) to induce photodegradation of the PL
moiety and to create exponentially decaying peptide gradients. By shuttering the light (panel (b)) or releasing pre-patterned lines (panels (c), (e)),
unique gradients in the peptide concentration were generated across the network. Solid gray lines are predicted concentrations based on
predetermined photocleavage kinetics from Eq (1). Scale bars=400 mm.
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demand. We expect such approaches for dynamically con-
trolling the properties of materials to be helpful in establish-
ing a more complete understanding of how cells respond to
their extracellular environment, and assist in the rational
design of cell-delivery systems for applications in regener-
ative medicine.
Experimental Section
Complete experimental procedures for peptide and macromolecular
precursor synthesis, formation of the network, photoreversible
patterning, generation of the gradient, and cell culture, seeding, and
visualization are given in the Supporting Information.
Figure 4. a) Parallel lines (200 mm wide) of Ac-C-(PL)-RGDSK(AF488)-
NH2 (1) were patterned into the hydrogel at 1.0 mm by visible thiol-
ene photocoupling. Seeded NIH 3T3 cells only adhered to regions of
the hydrogel surface that were functionalized with 1. b) Localized cell-
detachment was observed upon spatial removal (24 hours post-
seeding) of RGD through masked UV light exposure. Green=RGD (1),
red=F-actin, blue=nuclei. Scale bar=200 mm.
Received: September 13, 2011
Revised: November 16, 2011
Published online: December 8, 2011
Keywords: bioorganic chemistry · hydrogels · peptides ·
.
photochemistry · RGD peptides
patterns. Alternatively, by shuttering the light, more complex
stepped gradients were formed, in which regions of constant
peptide concentration were interspaced with regions of
decreasing concentration (Figure 3b). Each of these gradients
was readily predicted for a given light exposure, and the
predictions correlated well with the experimental findings.
Ultimately, this chemistry is useful as it can be performed
in the presence of cells because the wavelengths, exposure
times, and intensity of light are all cytocompatible.[11,23] To
demonstrate that this patterning approach offers dynamic
control over cell function, mouse embryonic fibroblast
(NIH 3T3) cells were seeded at 8 ꢀ 103 cellscmꢀ2 onto con-
structs that contained patterned islands of 1 (ca. 1 mm). The
initial attachment of the cells was confined to the adhesive
regions of the material that was functionalized with 1 (200 mm
lines of 1 spaced 200 mm apart, Figure 4a). The cells only
adhered to and exhibited a spread morphology on these
regions. Twenty-four hours after seeding, the adhesive ligand
was removed from selective areas of the initial pattern by
rotating the same 200 mm line pattern by 458 clockwise and
exposing the material to UV light. This procedure caused the
cells within these user-defined regions to detach from the
surface of the material within minutes of exposure (Fig-
ure 4b). These cells were subsequently isolated, expanded,
and further cultured. This method provides an efficient way of
potentially sampling a subset of a total cell population for
assaying the proliferation, gene expression, metabolic activity,
and differentiation of stem cells within user-defined regions of
the culture substrate.
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By utilizing multiple, bioorthogonal, light-based reactions,
we were able to exert spatiotemporal control over the
reversible presentation of biologically relevant chemical
cues. The level of control that can be obtained over the
biochemical functionality of the hydrogel platform should
enable dynamic cell functions to be assayed selectively, and
for the cellular microenvironment to be programmed on
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