Wavelength-controlled photocleavage for the orthogonal and sequential release of multiple proteins

Article abstract of DOI:10.1002/anie.201308174

On the right wavelength: Photolabile molecular units that undergo photocleavage under light of different wavelengths can be used for the independent release of different dyes/proteins from a single, preloaded storage hydrogel (see scheme). The controlled release of each protein allowed them to be delivered sequentially and at experimenter-determined times. Copyright

Full text of DOI:10.1002/anie.201308174

Angewandte
Chemie
DOI: 10.1002/anie.201308174
Photoinduced Protein Delivery
Wavelength-Controlled Photocleavage for the Orthogonal and
Sequential Release of Multiple Proteins**
Malar A. Azagarsamy and Kristi S. Anseth*
On-demand manipulation of protein release to alter cellular
phenotypes in real time is of great interest to the fields of drug
delivery, tissue engineering, and regenerative medicine.[1–3]
For such therapeutic and research ventures, hydrogels, owing
to their high water content and mechanical stability, have
emerged as suitable polymeric materials not only to localize
proteins but also to control their release rate on-site.[4,5]
Protein release from hydrogels is typically achieved either
through diffusion or by stimuli. Diffusion-controlled mecha-
nisms rely upon control of the hydrogel mesh size, which
needs to be preprogrammed during synthesis; as a result,
limited regulation of release is only possible afterward. To
complement this strategy, stimuli (e.g., temperature,
pH value, light and proteins)[6,7] sensitive materials have
evolved and become attractive protein-delivery systems, as
they offer opportunities to regulate molecular release using
a specific stimulus. Stimuli controlled mechanisms applied to
hydrogels are typically based on the degradation/swelling of
hydrogel networks, in which noncovalently sequestered
proteins are released in response to increased pore size/de-
cross-linking of networks. Such noncovalent approaches do
not require chemical modification of proteins and have the
potential to precisely control the release of single protein
molecules. However, applying such approaches to control the
release of multiple proteins is quite complex and often
requires either multiple gels or microspheres for encapsula-
tion.[8–12] Thus, a number of studies aimed at directing cellular
processes or disease regulation would benefit from the
delivery of more than one protein and often necessitates
their delivery in varied doses at different time points.[8–11]
Herein, we present an approach that allows precise control
over the release of multiple proteins from a single hydrogel
depot using an external light.
of photocleavage reactions offer unique opportunities to
control material properties more precisely than other classical
stimuli.[14] However, many of these approaches rely on
a single photocleavable unit, and thus, provide limited
opportunities to control different material properties inde-
pendently. Specific examples of wavelength-selective molec-
ular activation by orthogonally functional units were intro-
duced by Bochet,[20] and are now emerging as powerful
strategies in controlling different properties in a sequential
manner. Towards this, del Campo et al. first demonstrated the
utility of such wavelength-selective photocleavable concepts
by the spatial immobilization of multiple particles/fluoro-
phores;[21] where they initially utilized 3,5-dimethoxy benzoin
esters and nitrobenzyl derivatives,[21] a combination of
functionalities coined by Bochet as “orthogonal units”.
Later nitrobenzyl and coumarin derivatives were explored
as a new combination of orthogonal units,[22] which have been
widely exploited for sequential uncaging of bioactive units for
orderly regulation of biological actions.[23–27] More recently,
sequential photoactivation of biomolecular ligands for
spatio–temporal patterning of multiple proteins in a 3D gel
matrix has also been reported.[28–30] Herein, we present two
distinctive photocleavable units, based on: 1) nitrobenzyl
ether (NB) and 2) coumarin methylester (CM), that can be
selectively cleaved at different wavelengths of light and then
harness their wavelength-dependent photodegradable char-
acteristics to regulate the release of multiple proteins at
different times. Specifically, proteins were covalently conju-
gated to hydrogel networks through photodegradable
units[31–34] and the control over their release was achieved by
simply varying the wavelength of light, the intensity and the
time of light exposure.
The structural formula of NB, 1 and CM, 2 (See
Supporting Information for syntheses of 1 and 2) utilized in
this work are shown in Figure 1a. Note that both 1 and 2 have
an azide functionality for subsequent functionalization of
these molecules using click chemistry, and furthermore, the
azide functionality of the coumarin molecule 2 is conjugated
to the core ring structure. Under irradiation with UV/Vis
light, nitrobenzyl derivatives, such as 1, cleave to produce
nitrosoacetophenone 3 as a byproduct,[16] while the coumarin
methylester yields the corresponding coumarin methanol
4.[35,36] Prior to utilizing these molecules for protein release,
we first studied the photodegradation kinetics of 1 and 2
(2.5 mm) by separately irradiating them to both at 365 nm
(10 mWcmÀ2) and 405 nm (10 mWcmÀ2), and analyzing the
exposed solutions of 1 and 2 by reverse-phase HPLC. Before
exposing to light, both 1 and 2 exhibited single peaks in the
chromatogram, but exposure to 365 and 405 nm light resulted
in the formation of distinguishable new peaks corresponding
Light-triggered molecular cleavage has received wide-
spread interest in recent years, for activation of so-called
“caged” biomolecular entities,[13–15] alteration of material
properties,[16,17] and to control therapeutic release in real
time.[18,19] Furthermore, user-defined time and spatial location
[*] Dr. M. A. Azagarsamy, Prof. K. S. Anseth
Department of Chemical & Biological Engineering
Howard Hughes Medical institute and the BioFrontiers Institute
University of Colorado at Boulder
596 UCB Boulder, CO 80303 (USA)
E-mail: Kristi.anseth@colorado.edu
[**] We thank the National Science Foundation (DM 1006711) and the
Howard Hughes Medical Institute for funding. We would also like to
thank Dr. Daniel Alge for discussion.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201308174.
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decreases its degradation kinetics and ascribed the relative
higher degradation kinetics of 1 over 2 at 405 nm. In
summary, it appears that both the quantum yield of
degradation and molar extinction coefficient play critical
roles in the degradation behaviors of 1 and 2.
We explored the utility of photodegradable units 1 and
2 for selective, light-controlled release of model com-
pounds, for which we first utilized small-molecular-weight
dyes. Rhodamine B isothiocyanate and fluorescein N-
hydroxysuccinimidyl ester were conjugated to nitrobenzyl
azide 1 and coumarin azide 2, respectively, to afford the
corresponding rhodamine-tethered nitrobenzyl azide 5 and
fluorescein-tethered coumarin azide 6 (Figure 2a). The
dye-conjugated photodegradable azides 5 and 6 were then
covalently bound into poly(ethylene glycol) (PEG) hydro-
gels by copper-free, strain-promoted azide–alkyne click
(SPAAC);[37] this hydrogel formulation was selected as it
does not require any additional reagents or initiators that
might lessen bioactivity of proteins during encapsulation.
4-armed PEG tetra-dibenzocyclooctyne (PEG-DBCO) 7
and 4-armed PEG tetraazide (PEG-N3) 8 were used as
Figure 1. a) Compounds 1 and 2 and their corresponding photocleaved
products 3 and 4. Comparative photodegradation of 1 and 2 at b) 365 nm
and c) 405 nm for different times of light exposure. d) Absorbance spectra
of 1 (0.4 mm) and 2 (0.4 mm) dissolved in acetonitrile/PBS mixture (8:2).
to the degraded products 3 and 4 (see Supporting Information
for HPLC chromatograms). The relative photodegradation of
1 and 2 at different times of exposure to 365 nm light is shown
in Figure 1b, in which 2 shows a very high degradation rate
compared to its nitrobenzyl counterpart 1. The kinetic
constant of degradation (k), determined from the slope of
semi-logarithmic plot of Figure 1b (Table 1) (See Supporting
Table 1: Degradation kinetic constant (k), molar extinction coefficient (e),
and quantum yield of degradation (f). (for error limits see Supporting
Information.)
Compound k365
[ꢀ10À3 sÀ1
k405
e365
[cmÀ1 mÀ1
e405
[cmÀ1 mÀ1
]
f365 f405
]
[ꢀ10À3 sÀ1
]
]
NB 1
CM 2
4
13
2
0.7
4437
2183
935
150
0.16 0.11
0.57 0.15
Information for calculation details and corresponding semi-
logarithmic plots), showed that k of 2 was four times higher
than that of 1 at 365 nm. Conversely, to our surprise, when
exposed to 405 nm (Figure 1c), the degradation trend
reversed, 1 exhibited efficient degradation and its k value
was found to be an order of magnitude higher than that of 2
(Table 1). The results clearly suggested that 1 and 2 undergo
wavelength selective cleavage, that is, at 365 nm light, 2
degrades faster than 1 and vice versa at 405 nm. To better
understand these differences, we calculated the molar extinc-
tion coefficient at the degrading wavelength (e) of 1 and 2 and
their quantum yield of degradation (f) (Table 1 and Fig-
ure 1d), as these parameters directly correlate with k.[17] Even
though the molar extinction coefficient of 1 at 365 nm is twice
as high as 2, we expect that the enhanced degradation of 2 at
365 nm is due to its higher quantum yield of degradation (f).
At the same time, almost an order of magnitude lower e of 2
over 1 at longer wavelengths (i.e., at 405 nm) drastically
Figure 2. a) Rhodamine B tethered nitrobenzyl azide 5 and fluorescein-
tethered coumarin azide 6; b) PEG polymeric precursors that produce
SPACC hydrogel networks: 4-arm PEG-tetra DBCO 7 and 4-arm PEG-
tetraazide 8; c) Schematic representation of light-wavelength-regulated
selective release of dye molecules from hydrogel networks; Dye-release
upon exposure to light sources of d) 365 nm (10 mWcmÀ2) for 5 min
and e) 405 nm (10 mWcmÀ2) for 20 min. Blue-gray bars indicate the
light exposure.
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precursors to form the hydrogel (Figure 2b). In this case,
a slight molar excess of PEG-DBCO 7 over PEG-N3 8 was
used to allow subsequent covalent tethering of azides 5 and 6.
We tested the light-triggered release capabilities of 5 and 6
by separately incorporating them into different hydrogels and
exposing to 365 nm light (10 mWcmÀ2) for 10 min. A sudden
increase in fluorescence observed within 10 min in both cases
clearly indicated the release of covalently tethered dye
molecules into solution. Then, to investigate orthogonal
release, both 5 and 6 were introduced together at equal
concentrations into a single hydrogel and exposed to both 365
and 405 nm light sources separately (Figure 2c). After 5 min
of irradiation with 10 mWcmÀ2 of 365 nm light (Figure 2d),
almost 70% of coumarinꢀs fluorescein was observed to be
released from the gel in a period of just 20 min after exposure,
while the release of nitrobenzylꢀs rhodamine was only about
26%, presumably because of the enhanced cleavage rate of
coumarin methyl esters over nitrobenzyl ethers. However,
under the same conditions, when gels were exposed to
10 mWcmÀ2 of 405 nm for 20 min (Figure 2e), the release of
rhodamine (60%) was significantly higher than that of
fluorescein (10%), indicating not only an inversion in the
cleavage trend, but also the higher photocleavage rate of
nitrobenzyl over coumarin at 405 nm. Two points are note-
worthy here: 1) The dye-release trend is in agreement with
the cleavage data quantified in solution, photocleavage
studies of 1 and 2 (Figure 1); 2) Conversion of the conjugated
azide of coumarin 2 into its corresponding triazole did not
result in a significant change in its degradation behavior.
Overall, these results demonstrated the feasibility for the
photocleavable units 1 and 2 as orthogonal units to inde-
pendently control the release of dual therapeutic agents after
gel formation.
Scheme 1. Incorporation of photocleavable azides onto BMP-2 and
BMP-7 proteins: a) Traut’s reagent, pH: 7.0, Phosphate buffer (PBS),
room temperature; b) 7 (for BMP-2) or 8 (for BMP-7), PBS, pH: 7.0,
room temperature.
The orthogonal dye-release results then prompted us to
further investigate the effectiveness of 1 and 2 in regulating
the release of two different proteins of biological relevance.
For this purpose, we selected isoforms of the bone morpho-
genetic proteins (BMPs): BMP-2 and BMP-7, which are key
regulatory proteins closely involved in the cascade of events
occur during bone regeneration. Studies have shown that
delivering BMP-2 and BMP-7 in combination or in a sequen-
tial manner results in improved osteogenic differentiation of
mesenchymal stem cells (MSCs).[10,38] Herein, BMPs were
covalently modified with photocleavable azides to enable
their attachment to SPACC hydrogels and later to trigger
their release upon exposure to light of pre-selected wave-
lengths. BMP modification was achieved upon treating the
amine functionality of the proteins with Trautꢀs reagent to
produce an extended free thiol and then exposing the
resultant thiol to maleimide containing photocleavable
azides 7 and 8 (Scheme 1). The resulting modified proteins
BMP-2-NB-N3 (5 ng) and BMP-7-CM-N3 (5 ng) were then
introduced together into the hydrogel by SPAAC. After
leaching out untethered protein molecules for 3 days, the
hydrogels were separately exposed to light of 365
(5 mWcmÀ2) and 405 nm (5 mWcmÀ2) at day 4, and analyzed
using enzyme-linked immunosorbent assay (ELISA) to
monitor the BMP release kinetics. In hydrogels exposed to
365 nm light for either 2, 4, or 6 min independently (Fig-
Figure 3. Daily release of BMP-2 and BMP-7 upon exposure to light of
a) 365 nm (5 mWcmÀ2) and b) 405 nm (5 mWcmÀ2) for varied times;
c) Sequential release of BMP-2 and BMP-7 upon exposing to both 405
and 365 nm light but at different times; d) Relative ALP activity for
hMSCs seeded on hydrogels that contained both BMP-2 and BMP-7 in
equal amounts (10 ngmLÀ1) and exposed to 365 nm (for 6 min) and
405 nm (for 12 min) separately and also to both but sequentially, that
is, 405 nm first for 12 min and then 365 nm for 6 min (see text for
details); native BMPS: cells seeded on PEG hydrogels and exposed to
BMP-2 and BMP-7 (10 ngmLÀ1) but at day 1 and day 4, respectively;
no light: not exposed to any light but gel contained both BMPs; no
BMPs: cells seeded on hydrogel that did not contain any proteins.
ure 3a), we observed significantly increased release of BMP-
7, that is, tethered through coumarin, over nitrobenzyl
tethered BMP-2. As expected, the amount of protein release
increased steadily with increasing light exposure, but in case
of BMP-7, the trend continued only up to 4 min. No
significant increase in cumulative release was observed
upon further light exposure, suggesting that optimal light
exposures can be tailored to achieve maximum release of
BMP-7 while minimizing BMP-2 release. The release data
obtained for hydrogels exposed to 405 nm light for various
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times (Figure 3b) led to the expected opposite trend with
increased BMP-2 release compared to BMP-7 (as observed
with the model compound release studies, Figure 2d and e).
We then sought to investigate the possibility of releasing
both proteins from a single gel, but in a manner that allowed
their sequential and independent release. To achieve this,
hydrogels were first exposed to 405 nm for 12 min at day 3,
which resulted in a substantial and predominant release of
BMP-2, and then exposed to 365 nm for 6 min at day 6 to
trigger the release of the still-tethered BMP-7. Figure 3c
shows the entire daily release of both the proteins from before
exposure to light of 405 and 365 nm to trigger their cleavage
and release from the gel. This result clearly illustrates that the
second exposure to light (365 nm), results in extensive release
of BMP-7 (ca. 40%), suggesting the requisite of higher energy
light for cleavage of coumarin molecules and the resulting
release of tethered BMP-7. Given that the majority of the
BMP-2 (ca. 62%) was released during the first exposure to
light (405 nm), the second exposure at 365 nm resulted in just
10% release of additional BMP-2. Taken together, the
amount of recovered BMP-2 and BMP-7 was in the range
of about 68–72%. Overall, the release results clearly demon-
strated the sequential release of BMP-2 and BMP-7 by an
ordered exposure to light of different wavelengths.
We tested the bioactivity of released BMPs by evaluating
their influence on osteogenic differentiation of human MSCs
(hMSCs), as measured by their alkaline phosphatase (ALP)
activity at day 8. We observed that 1) neither light exposure
nor covalent modification of the BMPs significantly affected
their bioactivity, 2) hMSCs exposed to photoreleased BMP-2
and BMP-7 led to elevated levels of ALP compared to control
cells that were not exposed to either BMPs, and 3) BMP-2
appeared to influence ALP activity more as compared to
BMP-7 (Supporting Information). Then, to test the activity of
sequential release of BMPs, BMP-2 and BMP-7 were first
separately conjugated into different hydrogel formulations,
and hMSCs were seeded on top of the gel. After allowing the
cells to adhere to the gel surface, the gels were exposed to
365 nm light (5 mWcmÀ2) for 10 min. When analyzed at day 8
(see Figure S4 in the Supporting Information), hMSC ALP
activity was increased for photoreleased gels, as compared to
controls (i.e., not exposed to light), but it was also comparable
to the cellular responses obtained for native BMPs delivered
in a soluble form. These results suggest that BMPs released by
light indeed retain a bioactive nature that can locally
influence cellular function.
2) the cellular activity observed during sequential exposure
was comparable to that exposed to native BMP-2 and BMP-7
at day 1 and day 4, respectively. Collectively, these results
clearly suggested that differentiation of hMSCs can be
manipulated by sequential release of BMP-2 then BMP-7
by user-defined exposure to 405 and 365 nm light.
In summary, we have synthesized and studied a new
combination of selectively photocleavable nitrobenzyl- and
coumarin-based molecular entities using light of different
wavelengths. Our study has shown that the coumarin azides 2
cleave better than nitrobenzyls 1 with 365 nm light, but the
cleavage of nitrobenzyl 1 is more efficient than coumarin 2 at
405 nm. We demonstrated the potential utility of the wave-
length-dependent photocleavable characteristics of these
molecular units to selectively release dye/protein (BMP-2
and BMP-7) molecules of any choice from a single hydrogel
depot after gel formation. Selective protein release was
further used to stimulate sequential signaling of hMSCs at
different times by systematically switching the light from one
wavelength to another. Such orthogonally programmed
multiple protein release should allow researchers to tune
the release kinetics of multiple proteins at any time from pre-
loaded protein depots. We expect that this work should
provide basic insight for strategies to synthesize and engineer
new materials for controlled delivery of more than one
protein with various release patterns and profiles and
ultimately have implications in approaches to design materi-
als for applications in the delivery of protein therapeutics
related to tissue regeneration, wound healing, and disease
treatment.
Received: September 17, 2013
Published online: October 31, 2013
Keywords: dyes/pigments · hydrogels ·
.
photocontrolled release · proteins · protein delivery
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