Angewandte
Chemie
Abstract: pH-Cleavable cell-laden microgels with excellent
long-term viabilities were fabricated by combining bioorthog-
onal strain-promoted azide–alkyne cycloaddition (SPAAC)
and droplet-based microfluidics. Poly(ethylene glycol)-
dicyclooctyne and dendritic poly(glycerol azide) served as
bioinert hydrogel precursors. Azide conjugation was per-
formed using different substituted acid-labile benzacetal link-
ers that allowed precise control of the microgel degradation
kinetics in the interesting pH range between 4.5 and 7.4. By this
means, a pH-controlled release of the encapsulated cells was
achieved upon demand with no effect on cell viability and
spreading. As a result, the microgel particles can be used for
temporary cell encapsulation, allowing the cells to be studied
and manipulated during the encapsulation and then be isolated
and harvested by decomposition of the microgel scaffolds.
no diffusion limitation because of the small size of the proton
catalyst. Thus, the degradation kinetics depend only on the
chemical bond cleavage and therefore can be precisely
controlled for the release at the given target site.
Recently, acid-cleavable hydrogels have been prepared by
free radical cross-linking[14] and copper(I)-catalyzed azide–
alkyne cycloaddition (CuAAC).[15] However, these proce-
dures are not suitable for the encapsulation of sensitive
biomolecules, because the generation of free radicals and
metal ions can damage them.[16] To achieve homogenous and
efficient encapsulation, however, cells must be encapsulated
during hydrogel formation. Hence, mild cross-linking reac-
tions are required that are 1) orthogonal to the functional
groups of the biomolecules, 2) non-cytotoxic, and 3) fast at
378C.[5] The strain-promoted azide–alkyne cycloaddition
(SPAAC) fulfills all these requirements[17] and has recently
been applied for the encapsulation of living cells into bulk
hydrogels.[18] Although these are promising approaches, cell
release under physiological conditions has not been achieved;
however, this is essential for cell therapy and cellular
biophysics.[19] Additionally, hydrogel particles with micro-
meter-scale dimensions (microgels) are much more useful for
these applications than bulk hydrogels, because they can be
used to control the cellular microenvironment in the culture
very precisely[20] and can be handled by micropipettes and
microsyringes.[21]
Herein we present a new microgel construction kit for the
bioorthogonal encapsulation by SPAAC and the programmed
release of cells triggered by benzacetal hydrolysis. We
combined this chemistry with droplet-based microflui-
dics[19a,22] to generate stimuli-responsive mammalian-cell-
laden microgels (Figure 1). Since we used different substi-
tuted benzacetals as pH-cleavable linkers on the dendritic
building block, we could control the release kinetics in the
interesting pH range between 4.5 and 7.4 without changing
the initial mechanical matrix properties; both parameters are
known to mainly influence cellular fate.[23] This makes our
approach superior to conventionally used enzymatic degra-
dation, in which the kinetics can only be varied by changing
the initial degree of cross-linking.[8]
H
ydrogels represent an important class of biomaterials for
applications in regenerative medicine[1] and controlled deliv-
ery of therapeutic biomolecules,[2] because their mechanical
properties are similar to those of many objects in living
systems. This makes them ideal scaffolds to encapsulate,
stabilize, culture, and deliver living cells,[2a] proteins,[3] and
oligonucleotides.[4] A crucial challenge in these applications is
to release the biological objects with precise control over the
location and release rate.[5] To achieve this goal, the degra-
dation of hydrogels by the cleavage of chemical bonds in the
constituent polymer network, followed by dissolution of the
gel and release of encapsulated guests is a promising strategy.
Among the various hydrogel mechanisms of hydrogel degra-
dation, including photolytic,[6] hydrolytic,[7] and enzymatic
degradation,[8] hydrolysis at acidic pH has great potential for
applications relying on controlled release.[9] Because protons
are known to play a major role in cellular communication,[10]
encapsulated cells can actively degrade their hydrogel matrix,
which has direct impact on cell differentiation,[8] prolifera-
tion,[11] and migration.[12] In contrast to enzymatic degrada-
tion, which is diffusion controlled,[13] acidic hydrolysis shows
[*] D. Steinhilber,[+] T. Rossow,[+] Dr. S. Wedepohl, F. Paulus,
Dr. S. Seiffert, Prof. Dr. R. Haag
We used cytocompatible dendritic polyglycerol (dPG) and
linear poly(ethylene glycol) (PEG) as bioinert hydrogel
building blocks.[24] With these materials, nonspecific hydro-
gel–cell interactions are avoided.[15,25] Thus, we prepared
homo-bifunctional PEG-dicyclooctyne (PEG-DIC) and dPG-
polyazide and used them as macromonomers for the in situ
cross-linking by SPAAC. To achieve microgel degradability at
low pH, the azide was conjugated to dPG through an acid-
labile benzacetal linker. First, hetero-bifunctional linkers
were prepared that bore a terminal azide and dimethylacetal
group (Scheme 1). By using aromatic core segments that carry
either an electron-donating methoxy group in ortho position
to the aldehyde (1a) or a hydrogen atom (1b), it was possible
to fine-tune the kinetics of the acetal degradation, which is
known to strongly depend on the electron density of the
acetal.[26] Our synthesis started from para-hydroxybenzalde-
hydes, which are ideal building blocks because phenolates are
excellent nucleophiles for coupling reactions with electro-
philic azides 2. Since the reactions are high yielding and the
Freie Universitꢀt Berlin
Institute for Chemistry and Biochemistry
Takustrasse 3, 14195 Berlin (Germany)
E-mail: haag@chemie.fu-berlin.de
Dr. S. Seiffert
Helmholtz-Zentrum Berlin, F-ISFM Soft Matter and Functional
Materials, Berlin (Germany)
Dr. S. Seiffert, Prof. Dr. R. Haag
Helmholtz Virtuelles Institut – Multifunctional Biomaterials for
Medicine, Helmholtz-Zentrum Geesthacht
Teltow (Germany)
[+] These authors contributed equally to this work.
[**] This work was financed by the German Research Foundation in the
context of SFB 1112 (D.S.) and SFB 765 (F.P., R.H.), the Fund of the
Chemical Industry (Liebig fellowship; S.S.), and the State of Berlin
(Elsa-Neumann fellowship; T.R.). We thank C. Schlesener and T.
Becherer for GPC and MALDI-TOF measurements and Dr. P.
Winchester for proofreading this manuscript.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2013, 52, 13538 –13543
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim