Light controlled protein release from a supramolecular hydrogel

Article abstract of DOI:10.1039/c002565h

Using the inclusion complex of trans azobenzene and cyclodextrin as a photo-switchable crosslinker, a dextran based photo-responsive hydrogel system has been constructed and employed for a light controlled protein release system.

Full text of DOI:10.1039/c002565h

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Light controlled protein release from a supramolecular hydrogelw
Ke Peng, Itsuro Tomatsu and Alexander Kros*
Received 8th February 2010, Accepted 4th May 2010
First published as an Advance Article on the web 13th May 2010
DOI: 10.1039/c002565h
Using the inclusion complex of trans azobenzene and cyclo-
dextrin as a photo-switchable crosslinker, a dextran based
photo-responsive hydrogel system has been constructed and
employed for a light controlled protein release system.
polymers can be used as building blocks of a supramolecularly
crosslinked hydrogel for a light controlled release of proteins.
Upon mixing these two polymers, an inclusion of trans AB in
CD induces the gel formation when the ABs are in the trans
configuration. The inclusion complexes dissociate upon
trans–cis isomerization of the ABs after irradiation with UV
light (365 nm) resulting in a dissolution of the hydrogel.17,18
Furthermore, proteins can be physically entrapped in this
supramolecular gel matrix simply by dissolving it into the
polymer solutions before the gel preparation. Using green
fluorescent protein (GFP) as a model protein the light
controlled in vitro release was demonstrated. These characteristics
of the current system will be beneficial for the future drug
screening technology using zebrafish embryos as these are
transparent for the 365 nm UV light used in this study.19
Azobenzene carrying dextran (AB–Dex) was prepared by
the thiol–maleimide reaction of 3-[4-(phenylazo)-phenoxy]-
propane-1-thiol (1) and maleimide functionalized dextran
(Mal–Dex) in DMSO. The reaction was allowed to proceed
for 12 hours at room temperature, and the resulting AB
modified dextran was isolated by ultrafiltration and lyophilization
(Fig. 1a).
In the past few years, a number of therapeutic proteins
have been developed against a broad range of diseases such
as cancers, autoimmune diseases and metabolic disorders.1
However, most of these effective therapeutic proteins are
prevented from clinical use by fundamental technical hurdles
particularly with regard to delivery.2
Hydrogels are an ideal candidate for protein delivery, as
they contain large amounts of water in the polymer network in
a way similar to body tissues. Thus it allows to retain the
proteins in the protective 3-D network in their active forms
and prevents them from denaturation during administrations.3
Recently, sustained delivery of proteins using hydrogel systems
composed of a highly biocompatible biopolymer dextran4 or
other polymers5 has been reported, in which the loaded
proteins are released from the hydrogel matrix in a time
dependent manner regulated by the change of mesh size due
to the erosion of the hydrogel network.6
The conjugation of AB to dextran was confirmed by
1H NMR. On the spectra, besides signals attributed to dextran,
peaks at d = 7.2, 7.6 and 7.9 due to the AB were observed. The
reaction was further confirmed by the disappearance of the
peak of maleimide at d = 6.9. The degree of substitution
(DS: defined as the number of substituents per 100 anhydro-
glucosidic rings) was determined to be 6.
On another front, stimuli-responsive hydrogels have been
developed using supramolecular crosslinkers, whose network
can be eroded responding to temperature,7 pH,8 light,9
and other stimuli.10 These stimuli-responsive hydrogels are
potentially beneficial for an efficient drug delivery system
because it can be employed to transport the entrapped drug
to the target site and to trigger the release by a stimulus at the
desired point and time.11 Among these stimuli, light is a
particularly interesting option as it is a remote stimulus that
can be controlled spatially and temporally with great ease and
convenience.12 More importantly, the light irradiation does
not have a harmful effect on the activity of most proteins.13
Previously, we have functionalized dextran with maleimide
moiety, which can react rapidly with thiols via the thiol–
maleimide ‘‘click’’ reaction.14 We have shown that it can be
used for in situ hydrogel formation15 and the resulting nanogel
particles can be used as a drug carrier for hydrophobic drugs
in zebrafish embryos exhibiting an excellent biocompatibility.16
Taking the advantage of the efficient thiol–maleimide reaction,
in the present work, we functionalized dextrans with either
azobenzene (AB) or b-cyclodextrin (CD) moieties. These
b-Cyclodextrin modified dextran (CD–Dex) was also
obtained by the thiol–maleimide reaction of mono-6-thio-b-
cyclodextrin (2) and Mal–Dex in water. The reaction mixture
was stirred for 4 hours at room temperature. The resulting
CD–Dex was obtained after ultrafiltration and lyophilization.
The conjugation of CD was confirmed by the 1H NMR with a
peak at d = 5.1 attributed to the anomeric protons of CD and
disappearance of the maleimide peak at d = 6.9. The DS of
CD–Dex was determined to be 5 in the same way as AB–Dex.
The photoisomerization of AB–Dex was confirmed by
1H NMR (see ESIw). Before irradiation with UV light, peaks
ascribable to the aromatic protons of trans-AB were observed
with a trace amount of cis-AB indicated by the small peaks at
d = 6.8 and 7.3. After 4 hour irradiation with UV light, the
peaks due to trans-AB moiety were disappeared and the peaks
ascribable to the aromatic protons of cis-AB were observed.
From the ratios of the integrals in these spectra, fractions of
trans configuration were determined to be B95 and B0%
before and after UV irradiation, respectively.
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502,
2300 RA Leiden, The Netherlands.
E-mail: a.kros@chem.leidenuniv.nl; Fax: +31 71 527 4397
w Electronic supplementary information (ESI) available: Experimental
details and additional NMR data of photo-isomerization. See DOI:
10.1039/c002565h
We wondered if the newly synthesised AB functionalized
polymer AB–Dex can also form inclusion complex strongly
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Fig. 3 Photographs of a mixture of AB–Dex (67 mg mLÀ1) and
CD–Dex (100 mg mLÀ1) in PBS; taken after mixing (a) and after
irradiation with UV light (b).
peak between the protons of cis AB and CD. These data
indicate that the inclusion complex formation of AB–Dex with
CD occurs predominantly with the trans configuration and not
with the cis configuration, indicating it can act indeed as a
photoresponsive switch.
A hydrogel was obtained from a mixture of solutions of
trans AB–Dex and CD–Dex. Since trans AB binds inside the
CD cavity as seen in Fig. 2, supramolecular crosslinking points
were formed among the polymers. After UV irradiation, the
gel mixture turned into a solution. This is because trans-AB
moieties were isomerized into the cis configuration, which did
not show an interaction with CD–Dex. It is notable that
the transition from gel to sol is reversible by changing the
wavelength of the light17 (Fig. 3).
Fig. 1 (a) Preparation of azobenzene modified dextran (AB–Dex)
and cyclodextrin modified dextran (CD–Dex) through the thiol–ma-
leimide reaction. (b) Schematic representation of photoresponsive
protein release from the gel composed of trans AB–Dex and CD–Dex.
Upon the UV light irradiation azobenzene moieties isomerise from
trans to cis configurations, resulting in the dissociation of crosslinking
points, and allow the entrapped protein to migrate into the media.
To investigate the potential of the hydrogel as a light-
controlled protein release system, green fluorescent protein
(GFP) was used as a model protein and encapsulated inside
the hydrogel. The GFP carrying gel (18 mg) was placed into a
cuvette with 2 mL of fresh PBS and the fluorescence intensities
of the solution part were measured as a function of time
(Fig. 4). Prior to the UV light irradiation, the emission
intensities of GFP were practically constant, indicating that
the GFP remains entrapped inside the hydrogel network. After
10 minutes of UV light irradiation, the fluorescence intensity
started to increase which was pronounced after 20 minutes.
The release of GFP can be continued; 40% of the loaded
proteins was released in 60 min under UV irradiation and
with CD only when AB is in trans configuration as seen in
other reported systems.17,18 To address this issue, NOESY
spectrum of the mixture of trans and cis AB–Dex was measured
in the presence of CD. As shown in Fig. 2, correlation peaks
between the protons attributed to trans AB and inner protons
of CD were observed which shows the inclusion complex
formation of trans AB and CD. There was no correlation
Fig. 2 NOESY spectrum of a mixture of the isomers of AB–Dex in
D2O in the presence of CD. NOE signals were observed between
the protons attributed to trans AB and the inner (C-3, C-6, C-5)
protons of CD.
Fig. 4 Release of green fluorescent protein (GFP) from the supra-
molecular hydrogel. From the time 0, the sample was placed under UV
light. With a delay of 10 min, GFP was released from the hydrogel
matrix.
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reached ca. 65% in 2 hours. In contrast, a significant increase
in the release of GFP was not observed without UV light
irradiation even after 60 minutes (ca. 10%). Therefore we
conclude that the release of GFP was in a UV light responsive
manner (Fig. 1b).
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The stability of the current gel can be enhanced by using a
dextran polymer with a significant higher molecular weight
and by increasing the number of cyclodextrins and azobenzenes
per dextran chain. These two changes combined would result
in a lowered release of protein before UV-irradiation.
In conclusion, a light responsive hydrogel system composed
of azobenzene functionalized dextran (AB–Dex) and b-cyclo-
dextrin functionalized dextran (CD–Dex) has been prepared
for the light controlled release of proteins. AB–Dex and
CD–Dex were prepared efficiently from maleimide modified
dextran via thiol–maleimide ‘‘click’’ reaction. Using NMR
techniques we confirmed that AB–Dex can be isomerized from
trans to cis upon UV light irradiation and trans isomers can
form inclusion complex with cyclodextrins more firmly than
do cis isomers. Using this photoresponsive supramolecular
interaction as a molecular switch, we constructed a photo-
responsive hydrogel system. Upon UV light irradiation,
trans-AB moieties were isomerized to cis configurations resulting
in the dissociation of the network formed with CD–Dex,
converting the hydrogel into a sol. The light responsive
gel-to-sol transition was successfully employed for the controlled
release of an entrapped model protein, green fluorescent
protein (GFP). This hydrogel system equipped with both
biocompatibility and stimuli-responsivity will contribute to
the future protein administrations where UV irradiation is
applicable such as a light controlled transdermal delivery
system. Currently we are planning to apply this system for
the light controlled protein delivery in transparent zebrafish
embryos, which are relevant for the fast screening of new
potential drugs.19
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16 K. Peng, C. Cui, I. Tomatsu, A. H. Meijer, H. P. Spaink and
A. Kros, Soft Matter, in press.
The authors are grateful to Prof. Dr H. P. Spaink for the
supply of green fluorescent protein. We thank Mr Wim Jesse
for his technical assistance and we also thank Mr Kees
Erkelens and Mr Fons Lefeber for 2D NOESY data. The
authors (A. K. and I. T.) acknowledge the support of the
Smart Mix Programme of the Netherlands Ministry of
Economic Affairs and the Netherlands Ministry of Education,
Culture and Science.
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