Enzymatic- and light-degradable hybrid nanogels: Crosslinking of polyacrylamide with acrylate-functionalized Dextrans containing photocleavable linkers

Article abstract of DOI:10.1002/pola.25845

Enzymatically cleavable and light-degradable hybrid nanogels were prepared by free radical inverse miniemulsion copolymerization of acrylamide (AAm) with a newly synthesized functional dextran crosslinker containing acrylate moieties attached to the backbone via a photolabile linker, that is, dextran-photolabile linker-acrylate (Dex-PL-A). The Dex-PL-A/AAm feed ratio was systematically varied to investigate the influence of the particle composition on the gel properties. The resulting hydrogel nanoparticles were examined with regard to their degradation behavior upon the appliance of the two orthogonal stimuli by turbidity measurements in combination with dynamic light scattering. Although continuous photolytic cleavage of the photolabile linkers between polyacrylamide chains and dextran molecules was found to proceed fast and quantitatively yielding completely disintegrated networks, stepwise irradiation resulted in partial degradation of crosslinking points. Thus, nanogels of a desired specific degree of swelling (DGS) can be obtained by adjusting the irradiation time accordingly. Partial enzymatic cleavage of the dextran backbones of the Dex-PL-A crosslinking molecules resulted in an increase in the DGS of the nanogels up to a constant value. Subsequent irradiation of those swollen hydrogel particles was used to fully degrade the network structure in a second step. Hence, a two-step degradation profile was realized by the combination of the two orthogonal stimuli.

Full text of DOI:10.1002/pola.25845

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Enzymatic- and Light-Degradable Hybrid Nanogels: Crosslinking of
Polyacrylamide with Acrylate-Functionalized Dextrans Containing
Photocleavable Linkers
Daniel Klinger, Katharina Landfester
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
Correspondence to: K. Landfester (E-mail: landfester@mpip-mainz.mpg.de)
Received 4 November 2011; accepted 7 November 2011; published online 28 November 2011
DOI: 10.1002/pola.25845
ABSTRACT: Enzymatically cleavable and light-degradable hybrid
nanogels were prepared by free radical inverse miniemulsion
copolymerization of acrylamide (AAm) with a newly synthe-
sized functional dextran crosslinker containing acrylate moi-
eties attached to the backbone via a photolabile linker, that is,
dextran-photolabile linker-acrylate (Dex-PL-A). The Dex-PL-A/
AAm feed ratio was systematically varied to investigate the
influence of the particle composition on the gel properties. The
resulting hydrogel nanoparticles were examined with regard to
their degradation behavior upon the appliance of the two or-
thogonal stimuli by turbidity measurements in combination
with dynamic light scattering. Although continuous photolytic
cleavage of the photolabile linkers between polyacrylamide
chains and dextran molecules was found to proceed fast and
quantitatively yielding completely disintegrated networks, step-
wise irradiation resulted in partial degradation of crosslinking
points. Thus, nanogels of a desired specific degree of swelling
(DGS) can be obtained by adjusting the irradiation time accord-
ingly. Partial enzymatic cleavage of the dextran backbones of
the Dex-PL-A crosslinking molecules resulted in an increase in
the DGS of the nanogels up to a constant value. Subsequent
irradiation of those swollen hydrogel particles was used to
fully degrade the network structure in a second step. Hence, a
two-step degradation profile was realized by the combination
C
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of the two orthogonal stimuli. 2011 Wiley Periodicals, Inc. J
Polym Sci Part A: Polym Chem 50: 1062–1075, 2012
KEYWORDS: biopolymers; crosslinking; degradation; enzymes;
microgels; photochemistry; swelling
INTRODUCTION Nanogels as particulate structures in the
submicron size range consist of chemically or physically
intermolecularly crosslinked (hydrophilic) networks, which
are swollen in a suitable solvent. They represent a highly
interesting class of materials because they combine the char-
acteristics of macroscopic hydrogels (e.g., structural integrity
in combination with fluid-like transport characteristics) with
those of colloidal dispersions (e.g., good colloidal stability,
facile synthesis, and control over particle size).1,2 Stimuli-re-
sponsive micro- or nano-gels exhibit a response of their re-
spective network structure to changes in the (chemical) envi-
ronment thus rendering those materials ‘‘smart.’’ This
behavior gives rise to a broad variety of applications includ-
ing the fields of drug or gene delivery, chemical sensing, trig-
gered catalysis, and optics.1,2
transition is governed by an imbalance between repulsive
and attractive forces acting in the particles and can be influ-
enced by different triggers such as pH,5,6 temperature,7,8 or
ionic strength.9
On the other hand, the incorporation of labile crosslinking
points is a different approach to stimuli-responsive gel par-
ticles. In this case, the triggered degradation of the crosslink-
ing molecules leads to the disintegration of the network
structure yielding freely soluble polymer chains. Such
degradable nanogels offer the potential to release entrapped
compounds from the network upon the appliance of an
external trigger. Here, an initial mesh size of the gel smaller
than the hydrodynamic diameter of the respective substance
to be released prevents diffusion from the gel until triggered
cleavage of crosslinking points increases the pore sizes
resulting in the release.3,10 This characteristic cannot only be
applied to release applications; a triggered accessibility of,
for example, catalytic substances (metal nanoparticles,
enzymes) is imaginable and of high interest in materials sci-
ence. Investigated approaches to realize the described con-
cept are based on, for example, pH-sensitive crosslinkers
On one hand, a response to a specific stimulus is often char-
acterized by a change in the physicochemical parameters of
the network resulting in a variation of the degree of swelling
(DGS). Realization of this concept is achieved by the incorpo-
ration of functional stimuli-responsive moieties into the gel-
forming polymers.3,4 The resulting reversible volume phase
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SCHEME 1 Synthetic pathway to Dex-PL-A. Reagents and conditions: (a) methyl-4-bromobutyrate, K2CO3/DMF (anhyd.), 25 ꢀC,
16 h, quant.; (b) acetic anhydride/nitric acid (1:2, v/v), 0 ꢀC, 3 h, 53%; (c,d) NaBH4/MeOH/THF, 25 ꢀC, 16 h, NaOH, 25 ꢀC, 7 h, 89%;
(e) acryloyl chloride, NEt3, DCM, 0 ꢀC to RT, 16 h, 65%; (f) oxalyl chloride, DMF (cat.), DCM (anhyd.), 3 h, quant.; (g) detxtran (6k),
NEt3, LiCl, DMF (anhyd.), RT, 16 h, 60 ꢀC, 1 h, 58%.
containing various acid-labile moieties such as tertiary esters
or acetals for the preparation of degradable microgels in
both aqueous11,12 and organic media.13
cally degradable dextran chain.20,21 The resulting microgels
were shown to be degradable due to the hydrolysis of the
labile spacer bonds under either basic or acidic conditions.22
The combination of these microgels with a semipermeable
shell structure finally led to self-exploding capsules as a very
interesting new delivery system.23 Here, hydrolysis of the
degradable microgel core is accompanied by the diffusion of
solvent into the core, thus creating a pressure in the capsule
which finally leads to a burst of the membrane and the
release of compounds from the interior.
An alternative concept of degradable (micro-)gels focuses on
the utilization of dextrans as naturally occurring polysaccha-
rides, which can be cleaved upon the incubation with dextra-
nase. Covalent functionalization of dextran chains with either
polymerizable vinyl groups14,15 or thermal initiators16
enables the formation of hydrogels by free radical (co)poly-
merization in aqueous solutions.17 The resulting networks
can then be degraded by the addition of dextranase, thus
enabling the release of embedded active compounds.18 Enzy-
matically degradable microgels for the delivery of immuno-
globulin G as a model protein were prepared in the group of
Hennink by the simultaneous embedding of the protein and
dextranase into the network.19
Concerning either degradation- or swelling-induced release
systems based on enzymatic or pH-sensitive materials both
require the addition of protons/enzymes or the localization of
the carrier system in specific milieus. In comparison, the
release of a payload upon applying an external trigger
without
a change in the chemical composition of the
surrounding media bears the advantage of an on-demand
delivery. Among different stimuli fulfilling this criterion, light
represents an outstanding position because it offers the
possibility to change the network properties in very confined
spaces and time scales. Furthermore, it can be applied in a
very precise manner by selecting suitable wavelengths,
Regarding the versatility of such delivery systems, the ability
to respond to multiple orthogonal triggers is generally of
high interest. In this context, the concept of degradable
microgels based on dextran as crosslinkers was further
developed by introducing
a hydrolytically labile spacer
between the polymerizable vinyl groups and the enzymati-
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FIGURE 1 Schematic representation of the stimuli-induced degradation of hybrid p(AAm-co-Dex-PL-A) nanogels.
polarization directions, and intensities in
a
noncontact
dextran-photolabile linker-acrylate (Dex-PL-A). Multiple sub-
stitution of glucopyranose units in the dextran chain with
polymerizable acrylate units enables the utilization of the
functionalized polysaccharide as crosslinker in the copoly-
merization. Enzymatic degradation of the resulting nanogels
can then be achieved by cleavage of the dextran backbone
upon the addition of dextranase. Even though this sensitivity
depends on a change in the chemical composition of the re-
spective surrounding media, it gives rise to a potential
release of compounds in aqueous media of neutral pH, there-
fore representing an alternative approach to predominantly
used acid-labile crosslinkers. The light sensitivity can be real-
ized by the introduction of a photolabile linker molecule
between the polymerizable acrylate units and the dextran
chain. Irradiation of the resulting nanogels leads to
approach. The concept of light cleavable crosslinkers was
recently described for the preparation of photodegradable
macroscopic hydrogels by Kloxin et al.24 Transferring this
approach to the nanometer size range, the preparation of UV-
degradable poly(methyl methacrylate) (PMMA) microgels has
recently been reported by our group.25
The aim of this work is the preparation of double stimuli-
responsive nanogels to be degradable upon the addition of
dextranase or the irradiation with light as an orthogonal sec-
ond trigger. Our approach to realize the described concept is
based on free radical inverse miniemulsion copolymerization
of acrylamide (AAm) with a newly synthesized functional
dextran crosslinker containing acrylate moieties covalently
attached to the backbone via a photolabile linker, that is,
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photolytic cleavage of the covalent linkage of the polyacryl-
amide (PAAm) chains to the dextran chains and is assumed
to yield freely soluble polyacrylamide and dextran chains.
Both stimuli can be applied either separately or subse-
quently, thus enabling a good and versatile control over the
swelling and degradation behavior.
can be applied in a very precise manner—for example, by
selecting suitable wavelengths—and in very confined spaces
in
a noncontact approach, it represents an orthogonal
approach to the enzymatic sensitivity. In this context, o-nitro-
benzylic esters are well-known for their good photolytic
properties,26 such as fast and quantitative reactions27 in a
broad variety of environments.28,29
Synthesis and Characterization of
RESULTS AND DISCUSSION
Dextran-Photolabile Linker-Acrylate
Enzymatically and photolytically degradable hybrid nanogels
were designed to alter their network structure upon appli-
ance of two different stimuli; that is, the addition of dextra-
nase or the irradiation with UV light. The integration of the
double stimuli-responsive character can be achieved by using
a dextran molecule containing radically polymerizable acry-
late groups covalently bound to the polysaccharide backbone
via a photolabile linker molecule (Dex-PL-A). Copolymeriza-
tion with AAm in an inverse miniemulsion process leads to
hybrid nanogels. Here, the functionalized Dex-PL-A molecules
act as cleavable crosslinkers due to multiple acrylate moi-
eties attached to a single dextran chain. Network degradation
is based on either the enzymatic degradation of the dextran
backbone or the photolytic cleavage of the covalent linkage
of the polyacrylamide chains to the dextran chains as shown
in Figure 1. Both stimuli can be applied either separately or
subsequently, thus enabling a good and versatile control
over the swelling and degradation behavior.
Dex-PL-A molecules were synthesized according to Scheme 1
in order to combine the enzymatic degradability of the dex-
tran biopolymer with the photolytic properties of the o-nitro-
benzyl ester groups in the photolabile linker between acry-
late moieties and the polysaccharide backbone.
The carboxylic acid and hydroxyl functionalized photolabile
chromophore (5) was synthesized according to the procedure
described earlier.25 In brief, the ether formation of (1) with
methyl-4-bromobutyrate was followed by nitration of the aro-
matic core with acetic anhydride/nitric acid. Reduction of the
keto group of (4) and subsequent ester hydrolysis yielded (5)
in good yields. In a next step, the hydroxyl group was reacted
with acryloyl chloride to attach the polymerizable unit
through a photolabile o-nitrobenzyl ester bond. The carboxyl
group of (5) was then activated by forming the respective car-
boxyloyl chloride with oxalylchloride. Finally, the crosslinking
molecule Dex-PL-A (8) was obtained by the reaction of
hydroxyl groups of dextran with the activated carboxyl group
of (5), thus covalently attaching the acrylate moieties to the
dextran backbone via the photolabile linker. The obtained
crosslinker was investigated with regard to the degree of sub-
In general, one way to incorporate potential water soluble
functional compounds into polymeric nanogels is their
embedding already during the network formation.19,22 This
approach can be achieved by inverse miniemulsion copoly-
merization of a monomer and a crosslinker in the presence
of the respective substance. A requirement, however, are
water soluble monomers and crosslinkers. Regarding
delivery applications, a release of the respective functional
compound is generally determined by diffusion from the
1
stitution (DS) by means of H NMR measurements. Calculation
of the peak area ratio of the 6 protons (aromatic, olefinic, and
tertiary benzylic) of the photolabile linker to the one anomeric
proton of dextran afforded a DS ¼ 0.17. In other words, on
one dextran chain consisting of ꢁ34 glucopyranosyl groups,
5–6 of these groups are functionalized. Although a low DS is
assumed to decrease the crosslinking efficiency, functionaliza-
tion of the dextran with too many acrylate side groups could
network and governed by the mesh size of the gel.3
A
specific molecular design of the network structure allows
the incorporation of stimuli-sensitive properties, thus ena-
bling a release controlled by external triggers. As one exam-
ple, the stimuli-responsive behavior can be achieved by the
utilization of labile crosslinking points. Triggered (partial)
degradation of the latter leads to either highly swollen gels
of increased mesh sizes or complete particle degradation,
thus enabling the release of functional compounds.11
influence the enzymatic degradation behavior due to
a
reduced accessibility of the dextran backbone as a result of
sterical hindrance. The medium DS of 0.17 should allow a suf-
ficient crosslinking upon copolymerization with AAm while
maintaining the enzymatic degradability.
Additionally, a comparatively low molecular weight of 6000
g/mol of the used dextran was chosen to enable a good dis-
tribution of the crosslinking points in the resulting gel. Pre-
liminary studies performed on PAAm nanogels crosslinked
with dextran methacrylates confirmed this hypothesis and
are published elsewhere.30
The newly synthesized Dex-PL-A crosslinking molecules are
designed to fulfill both of the above mentioned criteria. As
naturally occurring polysaccharides, dextrans are well-known
for their good water solubility and their enzymatic degrad-
ability upon the treatment with dextranase.14 Hence, the
potential of network formation from aqueous solutions is
combined with the degradation of the gels as a response to
changes of the chemical environment; that is, the presence of
dextranase. Moreover, the introduction of a photolytic cleav-
able linker molecule between the dextran chain and the
polymerizable acrylate groups enables the gel degradation
upon the appliance of light as a second trigger. Because light
Regarding the photolysis of the crosslinking molecules and
the resulting nanogels, a fast and quantitative reaction under
mild conditions is highly favored. The a methyl group on the
benzylic carbon of the o-nitrobenzyl moiety was introduced
into the photolabile chromophore as it is known to increase
the rate of photolysis significantly.31 By considering the
abstraction of a benzylic proton by the photoactivated nitro
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group as the rate limiting step in the photolysis of the
o-nitrobenzyl group, the additional methyl group increases
the acidity of this proton.
Furthermore, Norrish-type side reactions, which primarily
take place for irradiations with wavelengths of k < 300 nm,
should be avoided. In this context, the introduction of alkoxy
substituents in the o-nitrobenzylic core results in a modified
electronic structure of the chromophore which is known to
result in a considerably increased UV absorption for k > 315
nm,32 and thus allows for a more gentle decomposition with
possible benefits for release applications of sensitive
molecules.
Figure 2 presents the UV–vis spectroscopic investigation of
the Dex-PL-A crosslinker. It reveals an absorption maximum
at 260 nm accompanied by the anticipated additional
absorption maximum at 350 nm. Thus, the crosslinker exhib-
its a high absorption in the targeted photolysis wavelength
region of k > 315 nm.
Photolysis of the Cleavable Crosslinker in Solution
To investigate the photolytic performance of the newly syn-
thesized photocleavable crosslinker, time dependent UV–vis
measurements of Dex-PL-A in aqueous solution were per-
formed. The respective spectra are shown in Figure 2.
Irradiation with UV light of the wavelength of k ¼ 365 nm
resulted in a red shift of the absorption maximum at
350 nm and the evolution of two new absorption bands at
235 nm and 270 nm. In addition, absorbance at 305 nm
decreased upon irradiation as shown in magnification in the
inset of Figure 2(a). Along with these light-induced changes
in the spectra, the occurrence of well-defined isosbestic
points over the complete irradiation time scale points
toward a successful cleavage of the chromophore, which is
supposed to be a first-order reaction. As shown in Figure
2(b), the rate of decrease of the absorbance at 305 nm dur-
ing irradiation followed an exponential decay to a constant
level at 176 s. Kinetic analysis was performed by setting the
value of the final absorbance at 245 s as zero. The resulting
kinetic plot of ꢂln [CL]t/[CL]0 versus time shows excellent
linearity, indicating the expected first-order kinetic with
respect to the chromophore concentration. The observed
well-defined photolytic cleavage in short time scales under
mild irradiation conditions in combination with the polymer-
izable acrylate functionalities and the enzymatically degrad-
able dextran backbone renders these newly synthesized mol-
ecules highly interesting as double stimuli-sensitive
crosslinkers for the formation of degradable hydrogels from
aqueous solutions.
FIGURE 2 Irradiation of Dex-PL-A in aqueous solution: (a) time
dependent UV–vis spectra; (b) time-dependent absorbance at k
¼ 305 nm including the exponential fit and kinetic plot of ꢂln
[CL]t/[CL]0 versus time including the linear fit.
nanogels are great potential systems for the embedding and
release of active functional compounds upon the appliance
of one or more orthogonal triggers, nanogel preparation
based on inverse miniemulsion bears a big advantage com-
pared with other methods. In this process, the addition of an
osmotic agent (an ultrahydrophilic compound) such as so-
dium chloride to the dispersed phase prevents diffusion
between the droplets by creating an osmotic pressure which
counteracts the Laplace pressure. Therefore, stable droplets
of the same composition are obtained and can be classified
as ‘‘nanoreactors.’’34 As a result, the composition of the latex
particles after polymerization resembles the composition of
the monomer phase, thus enabling the equal distribution of
all different functionalities in each particle.35 The potential
embedding of functional compounds is therefore highly facili-
tated. Moreover, when compared with precipitation polymer-
ization based approaches, the equal distribution of cross-
linkers in the gel particles enables a better comparability of
the resulting gel properties such as the DGS among the dif-
ferent nanogels prepared.
Preparation and Characterization of Hybrid
p(AAm-co-Dex-PL-A) Gel Nanoparticles by Inverse
Miniemulsion Copolymerization
Nanogel particle formation can be achieved by different
approaches including precipitation (co)polymerization from
aqueous solutions,1,2 crosslinking of preformed polymers in
(mini)emulsions, or copolymerisation of crosslinkers and
monomers in inverse miniemulsion.33 Because degradable
To prepare hybrid p(AAm-co-Dex-PL-A) nanogel particles,
AAm and Dex-PL-A were dissolved in an aqueous sodium
chloride solution and copolymerized in inverse miniemulsion
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TABLE 1 Synthetic Details for the Inverse Miniemulsion Copolymerizations of Hybrid Nanogels
m(Dex-PL-A)/
[m(Dex-PL-A) þ
m(AAm)] (%)
Solvent of Dispersed Phase
AAm
(m/g)
Dex-PL-A
(m/g)
Initiator
Type
Classific.
Set A
Sample
Type (m/g)
HNG-1A
HNG-2A
HNG-3A
HNG-1B
HNG-2B
HNG-3B
HNG-4B
0.950
0.825
0.700
0.950
0.825
0.700
0.400
0.050
0.175
0.300
0.050
0.175
0.300
0.600
5.0
17.5
30
0.5 M NaCl (1.00)
0.5 M NaCl (1.00)
0.5 M NaCl (1.00)
0.5 M NaCl (1.00)
0.5 M NaCl (1.00)
0.5 M NaCl (1.00)
0.5 M NaCl (1.00)
V-59
V-59
V-59
V-70
V-70
V-70
V-70
Set B
5.0
17.5
30
60
by free radical polymerization. Table 1 lists the composition
of the different nanogels in combination with the type of ini-
tiator (see ‘‘Experimental’’ section). Two sets of experiments
were performed to demonstrate the versatility of this prepa-
ration method. Although hybridꢀ nanogels HNG-1A–HNG-3A
of set A were polymerized at 70 C using V-59 as radical ini-
tiator, V-70 was used for the preparation of set B (HNG-1B
to HNG-4B) by initiꢀating the polymerization at a much lower
temperature of 37 C. With regard to a potential embedding
of active compounds, a lower polymerization temperature
holds promise for the embedding of sensitive compounds at
mild conditions.
tial DGS and a good enzymatic degradability is of high im-
portance. Incorporation of too many crosslinking points is
known to hinder enzymatic degradation of the dextran
chains due to a reduced accessibility caused by too small
mesh sizes of the gel.15 Hence, in every set of nanogels, the
amount of Dex-PL-A crosslinker was varied with the inten-
tion to investigate the influence of the Dex-PL-A/AAm ratio
on the properties of the resulting microgels. For polymeriza-
tions at 70 ꢀC HNG-1A contained 5 wt %, HNG-2A 17.5 wt
%, and HNG-3A 30 wt % of Dex-PL-A, respectively. Analo-
gously, the gel particles HNG-1B–HNG-4B of the second set
contained 5.0, 17.5, 30, and 60 wt % of Dex-PL-A. All poly-
merizations were carried out overnight, and the resulting
dispersions were washed repeatedly with cyclohexane to
remove excess surfactant. The resulting stable dispersions of
hybrid nanogels in cyclohexane were investigated with
regard to the hydrodynamic diameter by dynamic light scat-
tering (DLS) measurements and the particle morphologies
and sizes were determined by scanning electron microscopy
(SEM). Figure 3 shows representative SEM pictures of HNG-
3A in comparison with HNG-3B as well as the respective
Another important factor to be considered during nanogel
preparation is the crosslinking density of the resulting net-
work. Especially in the context of potential carrier applica-
tions, the prepared nanogels should exhibit a low initial DGS
to prevent diffusion of embedded compounds from the net-
work. Even though this parameter can be adjusted by
increasing the Dex-PL-A/AAm ratio (i.e., the amount of cross-
linking points on the dextrane), a balance between a low ini-
FIGURE 3 Representative SEM pictures HNG-3A and HNG-3B nanogels dropcast from cyclohexane dispersion and size distribu-
tions obtained from DLS measurements (number weighted) in comparison with diameters obtained from SEM analysis.
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The transfer of the gel particles to the aqueous phase was
easily achieved by immersing the freeze dried particles over
night in water. Repeated washing of the swollen nanogels
with deionized water removed the soluble fraction of the
crosslinking polymerization reaction. Resulting dispersions of
the nanogels in water were stable without any additional
surfactant due to sterical stabilization of dangling chains of
the swollen outer particle layer. The washing phases and the
aqueous particle dispersions were freeze dried separately
and gravimetric analysis yielded the respective sol/gel con-
tents. The sol fraction consisted of all unreacted monomers
and noncrosslinked polymers and oligomers. Because a
lower sol content reflects a higher crosslinking efficiency, its
determination enables the relative evaluation of the perform-
ance of the crosslinking copolymerization. As mentioned
above, the swelling ratio or the DGS is another important
property of the nanogels. It is determined by calculating the
volume ratio of swollen nanogels in water to nonswollen
dried nanogels via DGS ¼ Vswollen/Vnonswollen. The volume of
the swollen particles was determined by DLS measurements
of the aqueous dispersions, whereas the volume of the non-
swollen particles was calculated from the diameters obtained
from SEM photographs of the dried gel particles from cyclo-
hexane dispersions. Similar to the sol content, this parameter
is an indication for the efficiency of the crosslinking reaction,
because a lower initial DGS is based on a smaller mesh size
of the network caused by the incorporation of more cross-
linking points. Figure 5(a) shows the values for the sol con-
tents and initial DGSs obtained by gravimetric analysis and
DLS measurements. Although determination of either param-
eter does not permit to draw quantitative conclusions
regarding the crosslinking density or the inner morphology
of the nanogels, it nevertheless allows the expedient relative
comparison of the gel properties among the different
samples.
FIGURE 4 Hydrodynamic diameters of hybrid nanogels
obtained from DLS measurements in cyclohexane dispersion
after washing.
number weighted size distributions obtained from DLS and
SEM analysis. Figure 4 shows the hydrodynamic diameters
of the particles in cyclohexane in dependency on the amount
of acrylate groups available for crosslinking.
As can be seen from SEM photographs, inverse miniemulsion
copolymerizations yielded well-defined spherical hybrid gel
particles with nanoscale dimensions. From the hydrodynamic
particle diameters shown in Figure 4, it becomes obvious
that for an increasing amount of polymerizable acrylate
units—realized by increasing the Dex-PL-A/AAm ratio—the
particle size also increases. This effect is assumed to be
based on an increased viscosity of the monomer phase for
higher contents of functionalized dextrans. Because droplet
formation in the miniemulsion procedure derives from a fis-
sion and fusion process upon appliance of high shear forces
by ultrasonication, a higher viscosity of the dispersed phase
hinders the disrupture of bigger droplets of the preemulsion.
When comparing the hydrodynamic diameters obtained from
DLS measurements to the diameters from SEM photographs
(see Fig. 3), it becomes apparent that the values from light
scattering in cyclohexane dispersion are larger than those
obtained from the dried particles. As water was added as a
solvent for the monomer phase, it is still present in the
formed gel particles after polymerization. When compared
with the collapsed dried particles observed by SEM, nanogels
in cyclohexane dispersion consist of swollen networks result-
ing in increased particle diameters.
Figure 5(a) shows the dependency of Dex-PL-A/AAm feed ra-
tio to DGS values and sol contents. As expected, increasing
the feed ratio of Dex-PL-A/AAm [i.e., the theoretical amount
of polymerizable photolabile linker acrylate (PL-A) groups]
for a fixed polymerization temperature, reduces both the sol
content and the initial DGS. Both parameters indicate a
higher copolymerization efficiency.
When comparing the properties of the gel particles polymer-
ꢀ
ized at 70 C to those of the nanogels polymerized at 37 ꢀC
it becomes obvious that for similar feed ratios of Dex-PL-A/
AAm,
a lower polymerization temperature resulted in
increased sol contents and lower degrees of swelling. This
effect can be assigned to comparably lower conversions of
the polymerization reactions. The decreased reaction temper-
ature hinders diffusion of monomers and increases the prob-
ability of termination reactions. Because 1H NMR analysis of
the sol content (data not shown) revealed only negligible
amounts of Dex-PL-A, quantitative incorporation of the cross-
linking molecules is assumed. Based on the ensuing pre-
sumption that the sol content consisted only of unreacted
AAm monomers and oligomers, the effective Dex-PL-A/AAm
ratios were calculated. Figure 5(b) shows the calculated
At very high Dex-PL-A contents (60 wt %), increasing poly-
dispersity and ill-defined morphologies were observed (data
not shown). In this case, the high amount of the functional-
ized dextran influences the stability of the droplets due to
the surface active character of the amphiphilic Dex-PL-A.
Because attempts to form nanogels from pure Dex-PL-A did
not yield stable miniemulsions, the presence of a second
comonomer is a crucial requirement for the formation of
enzymatically and light degradable gel particles by inverse
miniemulsion polymerization.
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values plotted against the theoretical values; that is, the feed
ratios of Dex-PL-A/AAm. It is seen that for the same Dex-PL-
A/AAm feed ratio, higher sol contents for the nanogels poly-
merized at 37 ꢀC correlate with increased effective Dex-PL-
A/AAm ratios compared with the samples polymerized at
ꢀ
70 C.
Plotting the DGS values of the two sets of nanogels against
the effective Dex-PL-A/AAm ratio shows the anticipated
trend of decreasing degrees of swelling with increasing
amounts of actually incorporated crosslinking molecules [Fig.
5(c)]. The favored incorporation of functionalized dextran for
the lower polymerization temperature (37 ꢀC) is therefore
expected to yield nanogels consisting of networks with
smaller mesh sizes. Additionally, with increasing feed ratios
of Dex-PL-A/AAm, a decreasing deviation of the effective
ratios from the theoretical values is observed for both poly-
merization temperatures. This confirms a higher crosslinking
efficiency if more Dex-PL-A crosslinking molecules are
available.
The systematic variation of the Dex-PL-A/AAm ratio [Fig.
5(a)] revealed HNG-3B nanogels as the most promising can-
didates for potential release applications for three reasons:
(i) The observed relatively low sol content of 29.6 wt %
mainly consisted of AAm monomers and oligomers, therefore
pointing toward a successful crosslinking copolymerization
with an almost quantitative incorporation of Dex-PL-A cross-
linking molecules. (ii) The low initial DGS of 2.5 is poten-
tially effective for the embedding of functional compounds.
(iii) The substantially low polymerization temperature of
ꢀ
37 C renders these synthetic parameters highly effective.
Photolytic and Enzymatic Degradation
Because hybrid p(AAm-co-Dex-PL-A) nanogels HNG-3B exhib-
ited the desired gel properties, their degradation behavior upon
irradiation and treatment with dextranase was investigated to
demonstrate their potential for release applications. Degradation
experiments were conducted in aqueous dispersions with a
solid content of 0.0625% consisting of purified nanogels.
Partial cleavage of crosslinking points by either photolytic dis-
rupture of the connecting bonds between polyacrylamide
chains and dextrans or the enzymatic decomposition of the
latter should result in a loosened network structure with
increased mesh sizes, thus yielding gel particles of an
increased DGS. Further exposure to the respective stimuli is
expected to form completely disintegrated networks consisting
of freely soluble polymer chains and dextran oligomers. Gener-
ally, DLS measurements can be applied to determine increased
particle sizes caused by the higher DGS but are limited to the
partial degradation regime. To investigate the complete degra-
dation behavior, turbidity measurements can be used as they
are capable of visualizing the complete time dependent photo-
lytic and enzymatic particle disintegration as well.
FIGURE 5 Characterization of the gel properties of the hybrid
p(AAm-co-Dex-PL-A) nanogels: (a) DGS and sol content values
in dependency on the feed ratio of Dex-PL-A/AAm. Empty
squares: nanogels of set A polymerized at 70 ꢀC, filled dots:
nanogels of set B polymerized at 37 ꢀC; (b) effective ratio of
Dex-PL-A/AAm in comparison with the respective feed ratio,
the diagonal represents the ideal case of quantitative incorpo-
ration of both compounds and is a guide for the eye; (c) DGS
values in dependency on the effective ratio of Dex-PL-A/AAm;
empty squares: nanogels of set A polymerized at 70 ꢀC, filled
dots: nanogels of set B polymerized at 37 ꢀC.
As shown in a previous publication,25 an increase of the DGS
corresponds to a loosening of the network structure due to the
cleavage of crosslinking points and causes a decreased contrast
in the refractive indices between solvent and particle.36,37 As a
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namic diameters of the irradiated nanogels in comparison
with the relative transmittance of the dispersion.
Irradiation time dependent UV–vis measurements clearly
show the successful cleavage of the photolabile o-nitrobenzyl
ester linkers. When compared with the photolysis of pure
Dex-PL-A crosslinking molecules in solution, the spectra of
the respective nanogels are influenced by scattering of the
dispersion. As seen by turbidity measurements, the scatter-
ing intensity decreases upon irradiation and no isosbestic
points were observed in the raw UV–vis spectra (data not
shown). Nevertheless, the change in absorbance at 270 nm
relative to the absorbance at 300 nm clearly points to a suc-
cessful photoreaction. Because UV–vis spectroscopy of the
irradiation of pure Dex-PL-A exhibited an isosbestic point at
k ¼ 330 nm, normalizing the spectra of the nanogels to the
final absorbance after 165 s of irradiation at this wavelength
yielded the same well-defined isosbestic points as well.
Hence, it was concluded that the photoreaction is not influ-
enced by the gel matrix.
FIGURE 6 Turbidity measurements for the irradiation of an
aqueous HNG-3B nanogel dispersion (0.0625% w/v) with UV
light (k ¼ 365 nm, I ¼ 30 mW/cm2).
DLS measurements of the irradiated samples clearly show
the increase of particle sizes in dependency on the irradia-
tion time. As expected, the light-induced cleavage of covalent
result, the scattering intensity decreases, leading to optically
more transparent dispersions. Quantification of this phenom-
enon can be achieved by calculating the turbidity s(t) as the
intensity ratio of transmitted light of the sample It to transmit-
ted light of pure water I0 as s(t) ¼ ꢂln It/I0. The respective
relative values were then obtained as the ratio of the time
dependent turbidity after exposure to the respective stimulus
to the starting values s(t ¼ 0) as srel ¼ s(t)/s(t ¼ 0)ꢃ100.
First, the photolytic cleavage of the o-nitrobenzylester groups
connecting polyacrylamide chains to dextrans was investi-
gated. Two different experiments were performed: constant
and stepwise irradiation of the sample with UV light. Figure
6 shows the resulting plots of turbidity versus time.
The plot clearly shows a profound decrease of the relative
turbidity down to a constant level of almost 0% after only
ꢁ3 min of continuous irradiation, thus indicating complete
particle degradation in a short time scale under mild condi-
tions. This observation was confirmed since attempts to per-
form DLS measurements on the resulting clear solution did
not yield any results regarding particle sizes. The stepwise
irradiation was performed by repeated cycles of irradiating
the sample for each time 15 s and then keeping the sample
in the dark for 2 min. Turbidity was monitored during the
whole process and was found to decrease only during the
appliance of light. Because turbidity—and therefore the deg-
radation—is not altered in the dark, the photolytic decompo-
sition of the nanogels can be stopped at any desired degree,
thus proving a highly controlled photodegradation profile.
The observed stepwise degradation allowed the detailed
investigation of the photolytic decomposition at fixed time
points of irradiation. Samples were withdrawn after every
irradiation step and analyzed. The chromophore cleavage
was investigted by UV–vis spectroscopy and the particle size
assessed by means of DLS measurements. Figure 7(a) shows
the respective normalized irradiation time dependent UV–vis
spectra, whereas Figure 7(b) depicts the respective hydrody-
FIGURE 7 (a) Irradiation time dependent UV–vis spectra of
HNG-3B nanogels (normalized to k ¼ 330 nm at tirr ¼ 165 s);
(b) irradiation time-dependent hydrodynamic diameters of
HNG-3B nanogels in comparison to the relative transmittance
of the respective aqueous dispersion.
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bonds between polyacrylamide and dextran chains results at
first in highly swollen nanogels characterized by a loosened
network structure and later in completely soluble polymer
chains. Although DLS measurements of irradiated nanogels
up to irradiation times of 90 s yielded well-defined monomo-
dal size distributions, the determination of hydrodynamic
diameters for longer irradiation times exhibited multimodal
distributions due to fragmentation of the gels. For even
longer irradiation times, no results were obtained due to a
too low scattering intensity. As speculated before, the results
confirm that DLS characterization is indeed only possible in
the partial degradation regime. In contrast, the performed
turbidity measurements clearly are highly advantageous to
investigate the full degradation profile. The correlation
between increasing particle sizes and increasing transpar-
ency (i.e., decreasing scattering intensity) is confirmed by
the plot in Figure 7(b).
In summary, the experiments demonstrate that the complete
photolytic disintegration of the hybrid p(AAm-co-Dex-PL-A)
network structure upon cleavage of the covalent bonds
between PAAm and dextran can be achieved fast and quanti-
tatively under mild irradiation conditions. Moreover, the
well-defined photoreaction even allows only partial degrada-
tion of crosslinking points and therefore nanogels of a
desired specific DGS can be obtained by adjusting the irradi-
ation time accordingly.
To demonstrate the double stimuli-responsive character of
the hybrid nanogels, further degradation experiments were
conducted to investigate the enzymatic dextran chain cleav-
age upon the addition of dextranase. In principal, the degra-
dation of the dextran backbone in the Dex-PL-A crosslinkers
should lead to a disintegration of the polymeric network and
result in either fully degraded gels or particles exhibiting an
increased DGS (for incomplete dextran chain cleavage).
Investigations were carried out on aqueous dispersions of
the purified HNG-3B gel particles (0.0625% w/v) at 37 ꢀC,
and turbidity of the sample was monitored after the addition
of a dextranase solution. The resulting curve is shown in Fig-
ure 8(a).
FIGURE 8 Enzymatic and subsequent photolytic degradation of
HNG-3B nanogels: (a) turbidity curve; (b) particle size distribu-
tions before and after enzymatic treatment measured by DLS.
the similar particle sizes once again demonstrate the good
correlation between turbidity and degree of degradation (i.e.,
DGS). Moreover, these results point towards a comparable
degradation mechanism including the increase in mesh sizes
by cleavage of crosslinking points.
The initial turbidity decreases as a function of incubation
time until it reached a constant value of 50% after circa 5 h.
Even though no complete particle degradation—correspond-
ing to a relative turbidity of ꢁ0%—was achieved, the drop
in turbidity can be assigned to an increased DGS of the
hydrogel particles. The corresponding increased pore size of
the network results from partial cleavage of the dextran
backbones responsible for the crosslinking of gel. DLS meas-
urements were conducted to confirm this assumption and
the resulting size distributions before and after enzymatic
treatment are shown in Figure 8(b). As expected, the particle
size increased from an initial diameter of 212 nm to
344 nm, thus corresponding to a change in DGS from the
low initial value of 1.4 to the final value of 6.2. When com-
paring the measured hydrodynamic diameter of 344 nm
after enzymatic treatment—corresponding to a turbidity of
50%—to the measured diameter of 364 nm after 60 s of UV
irradiation—corresponding to a similar turbidity of ꢁ50%—
Because any treatment with the enzyme for longer times did
not yield a further decay of the turbidity—that is, breakage
of dextran chains—the final value represents the crucial limit
for enzymatic particle degradation. This observed limitation
can in general be based on various effects.
On one hand Franssen et al. proposed that the enzymatic
degradation of crosslinked dextran methacrylates is influ-
enced by the accessibility of the dextran chains for dextra-
nase.15 Because of the presence of interpenetrating networks
and the fact that the dextran chains are severely strained
this factor is reduced. In addition, the overall amount of
crosslinking points in the network can also play a crucial
role since the diffusion of the enzyme through the hydrogel
matrix is decreased as a result of a screening effect.15 Imag-
ining a high density of crosslinking points (i.e., small mesh
sizes), this would give access to the enzymatic degradation
of the outer sphere due to an enhanced accessibility of the
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surface. The comparably large size of the dextranase enzyme
(44 kDa) may hinder complete particle degradation due to
inaccessible inner parts of the particle core caused by small
mesh sizes.
gels. As shown in Figure 8(a), this pathway could indeed be
used to realize a two-step degradation profile. After the
hybrid nanogel particles reached the maximal DGS upon
treatment with dextranase, irradiation of the swollen gel par-
ticles led to complete particle degradation as demonstrated
by a measured turbidity of ꢁ0%. An additional advantage is
the comparably much faster response to the trigger. Because
enzymatic treatment can induce a continuous but slow deg-
radation profile (ꢁ6 h), the photolytic degradation can be
achieved in less than 2 min.
On the other hand, the interaction between the functional-
ized dextran chains and the active sites of the enzyme was
found to depend on the character of the substitution of the
polysaccharide.14,15 Although Franssen et al. demonstrated
that dextranase is capable of hydrolyzing a bond between a
substituted and an unsubstituted glucopyranose residue of
dextran methacrylates in solution, a cleavage of bonds
between two substituted glucopyranose residues cannot be
achieved by the enzyme. Regarding the case of Dex-PL-A
crosslinkers, the functionalization of the dextran chains with
PL-A moieties is assumed to yield statistically distributed
substitution of glucopyranose residues meaning that func-
tionalization of two neighboring groups in the dextran chain
occurs as well. As in the resulting hydrogel matrix pAAm
chains are also connected by this specific structural unit, the
hindered enzymatic cleavage of the latter results in incom-
plete network degradation.
To verify the results presented above, SEM pictures of HNG-
3B nanogel dispersions at different points of the two-step
degradation were taken and are shown in Figure 9.
The initial nondegraded particles can be identified as flattened
spheres on the silica wafer [Fig. 9(a)]. Even though drying of
the particles from the swollen state resulted in film formation
and flattened structures, the retention of the spherical mor-
phology clearly hints again to the successful crosslinking of
PAAm by Dex-PL-A. After irradiation with UV light, no spheri-
cal structures were observed by SEM but a polymeric film
[Fig. 9(b)], thus indicating complete particle degradation. As
shown in Figure 9(c), after the enzymatic treatment particular
structures were still observed but an increasing film forma-
tion/flattening is visible, caused by the highly swollen state of
the nanogels before drop casting. Finally, in Figure 9(d), no
particular structures were detected and the observed polymer
film results from the completely degraded nanogels upon UV
irradiation subsequent to the enzymatic treatment.
Finally, the overall DS can play an important role as well. Dex-
tran methacrylates with low DS values (and resulting hydro-
gels) can easily be cleaved by dextranase yielding isomaltose
and methacrylated isomaltotriose as the main degradation
products.14 Long unsubstituted chain segments of dextran (i.e.,
18 or more unsubstituted glucopyranose units) are assumed
to have enough conformational freedom to fold correctly in
the binding site of dextranase, thus enabling multiple scissions
by the enzyme with a rate similar to that of native dextran.
For increasing degrees of substitution, the corresponding
shorter unsubstituted chain segments are restricted in their
conformational freedom by neighboring substituted glucopyra-
nose residues. Even though these chain segments can still
bind to the binding site of the enzyme, the affinity and corre-
sponding degradation rate is decreased. As mentioned before,
the DS of 0.17 of Dex-PL-A is related to free chain segments
of ꢁ6 unsubstituted glucopyranose units. As this value was
found to be the lower critical value for successful hydrolysis
of crosslinked dextran methacrylates, the observed partial deg-
radation can be also assigned to a reduced affinity of Dex-PL-
A to the binding sites of dextranase.
Summarizing the degradation behavior of the p(AAm-co-Dex-
PL-A) hybrid nanogels three different degradation profiles
were realized. At first, the exclusive photolytic cleavage of
the photolabile linkers between dextran and PAAm chains
was successfully used to fully degrade the nanogels upon
continuous irradiation in a short time span. Partial degrada-
tion of crosslinking points and therefore nanogels of a
desired specific DGS were obtained by adjusting the irradia-
tion time accordingly. Second, partial enzymatic cleavage of
the dextran backbones of the Dex-PL-A crosslinking mole-
cules resulted in an increase of the DGS of the nanogels up
to a constant value. Third, the subsequent irradiation of
those swollen hydrogel particles could be used to fully de-
grade the network structure. Hence, by combining the two
orthogonal stimuli, a two-step degradation profile was real-
ized rendering such hybrid nanogels highly versatile materi-
als for potential release applications.
Summarizing the statements made above, incomplete micro-
gel degradation upon treatment with dextranase is assumed
to be based on: (i) the reduced accessibility of the dextran
chains in the interpenetrating p(AAm-co-Dex-PL-A) network
for the enzyme; (ii) hindered cleavage of two neighboring
substituted glucopyranose residues; and (iii) decreased affin-
ity of the Dex-PL-A to the binding site of the enzyme due to
the relatively high DS. Nevertheless, the observed sixfold
increase in particle volume after treatment with dextranase
holds great potential for the release of functional compounds
from the network triggered by increasing mesh sizes.
EXPERIMENTAL
Materials
All chemicals were commercially available and used without
further purification unless otherwise stated. AAm was
obtained by Fluka chemicals and Lubrizol U was kindly pro-
vided by Lubrizol, France. All other chemicals and solvents
were obtained by Sigma Aldrich.
Having demonstrated partial enzymatic degradation, the
question arises whether complete particle disintegration can
be subsequently achieved by irradiation of the swollen nano-
Instrumentation
1H (300 MHz) spectra were measured using a Bruker spec-
trometer. Particle size distributions were determined by DLS
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FIGURE 9 SEM investigations on the stimuli-induced degradation behavior of HNG-3B: (a) swollen microgels in water; (b) com-
pletely degraded microgels after UV irradiation; (c) highly swollen microgels after treatment with dextranase; (d) completely
degraded microgels after first enzymatic treatment and subsequent irradiation with UV light.
using a NICOMP zetasizer measuring at a fixed scattering
angle of 90ꢀ. The measurements were carried out at 25 ꢀC
on diluted dispersions in the respective solvents. Turbidity
measurements were performed in transmission using a He-
Ne Laser and a photo diode. A Gemini 1530 (Carl Zeiss AG,
Oberkochem, Germany) with an InLens detector was used to
take SEM. The samples were prepared by drop-casting of
diluted dispersions on a silicon wafer. UV–vis spectra of
crosslinkers in solution were determined by using a Perkin
Elmer Lambda 25 UV/VIS spectrometer. The respective irra-
diation time dependent UV–vis spectra of the nanogels were
measured by using a TECAN plate reader and a Hellman 96-
well quartz plate.
extraction with ethyl actetate, subsequent nitration of the
crude product was achieved by the reaction with acetic
anhydride and nitric acid (1:2 v/v). In the next step, the
reduction of the keto group with excess borohydride was
conducted and directly followed by inducing the ester cleav-
age by addition of NaOH in water. Purification by recrystalli-
zation from ethyl acetate/hexane afforded HEMNPBA (5) in
89% yield as a pale yellow solid.
1H NMR (CDCl3): d ¼ 7.41 (s, 1H), 7.24 (s, 1H), 5.37 (q, J ¼
6.2 Hz, 1H), 3.97 (t, J ¼ 6.4 Hz, 2H), 3.82 (s, 3H), 2.37 (t, J ¼
7.2 Hz, 2H), 2.00 (p, J ¼ 6.8 Hz, 3H), 1.35 (d, J ¼ 6.3 Hz,
1H). 13C NMR (CDCl3): d ¼ 174.92, 154.05, 146.56, 139.12,
138.33, 108.97, 108.89, 68.24, 64.95, 56.23, 30.18, 24.84,
24.18. MS (FD) m/z 299.2 (Mþ).
Synthesis of Dextran-Photolabile Linker-Acrylate
Dex-PL-A was obtained by first synthesizing the carboxylic
acid functionalized photolabile linker molecule containing
the acrylate moiety and subsequent coupling with dextran.
Scheme 1 shows the synthetic pathway and the reaction
conditions.
4-(4-(1-(Acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-
butanoic acid (AEMNPBA) (6)
HEMNPBA (1.000 g, 3.34 mmol) was dissolved under argon
in anhydrous dichloromethane (40 mL). Triethylamine
(1.014 g, 10.00 mmol) was added and the solution was
cooled to 0 ꢀC. Acryloyl chloride (0.756 g, 8.35 mmol) was
dissolved in 10 mL of anhydrous dichloromethane and added
dropwise to the reaction vessel. The mixture was stirred
over night and allowed to warm up to room temperature.
Afterward, the solution was washed in every case thrice
with sodium carbonate solution, diluted hydrochloric acid,
and deionized water. The organic solvent was evaporated
4-(4-(1-Hydroxyethyl)-2-methoxy-5-nitrophenoxy)
butanoic acid (HEMNPBA) (5)
As described in a previous publication,25 the product was
prepared based on the synthetic protocols by Holmes.27,38
Briefly, acetovanillone was reacted with methyl 4-bromobuty-
rate in the presence of potassium carbonate to form the cor-
responding keto ester. After removing the inorganic salts by
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under reduced pressure and the resulting solid was stirred
in aceton/water over night. Insoluble contents were removed
by filtration, and the remaining phase was extracted thrice
with dichloromethane. The combined organic layers were
washed with diluted hydrochloric acid and deionized water,
dried over MgSO4, and the solvent was removed. The
remaining yellow solid was purified by column chromatogra-
phy over silica using CHCl3/MeOH 10:1 v/v as eluent and
pure AEMNPBA (0.771 g, 2.18 mmol) was obtained in 65%
yield as yellow solid.
consisting of a solution of the nonionic surfactant Lubrizol U
(100 mg) in 10 g of cyclohexane. The miniemulsion was
formed by first stirring the mixture at 1750 rpm for 1.5 h
and then homogenizing the obtained pre-emulsion by ultra-
sonication for 2 min at 90% intensity (Branson sonifier
ꢀ
W450 Digital, 0.5" tip) at 0 C. After addition of an oil solu-
ble initiator, polymerizations were carried out over night in
an oil bath set at either 70 ꢀC or 38 ꢀC depending on the
used initiator. The respective compositions of the dispersed
phase and the types of initiators of the different reactions
are summarized in Table 1.
1H NMR (CDCl3): d ¼ 7.59 (s, 1H), 7.00 (s, 1H), 6.53 (q, 1H,
J ¼ 6.4 Hz), 6.43 (dd, 1H, J ¼ 1.5 Hz, J ¼ 17.3 Hz), 6.15 (dd,
1H, J ¼ 10.4 Hz, J ¼ 17.3 Hz), 5.87 (dd, 1H, J ¼ 1.5 Hz, J ¼
10.4 Hz), 4.12 (t, 2H, J ¼ 6.2 Hz), 3.92 (s, 3H), 2.61 (t, 2H,
J ¼ 7.1 Hz), 2.18 (m, 2H), 1.65 (d, 1H, J ¼ 6.4Hz). 13C NMR
(CDCl3): d ¼ 178.44, 164.93, 154.04, 147.15, 139.79, 133.34,
131.39, 128.23, 109.10, 108.14, 68.63, 68.04, 56.28, 30.22,
23.97, 22.01.
After the polymerization, coagulates were removed by filtra-
tion and the resulting dispersions were centrifuged at
5000 rpm for 30 min to collect the particles. The superna-
tant was removed and replaced by cyclohexane. Redispersion
was carried out using a vortex. To remove excess surfactant,
the dispersions were further washed four times with cyclo-
hexane, following the procedure described above. The puri-
fied dispersions of the MGs in cyclohexane were examined
with regard to the particle size distributions by means of
DLS and SEM. Freeze-drying yielded the crosslinked gel par-
ticles as white to slightly yellow colored powders. The freeze
dried particles were swollen over night at 0.5% (w/v) in
water at room temperature. Two additional washing steps by
centrifugation at 14,000 rpm for 90 min and redispersion in
deionized water were performed to remove the sol content,
which consisted of unreacted monomer and crosslinker, solu-
ble (noncrosslinked) polymers, and oligomers. The purified
particles were freeze dried and redispersed in the respective
media by simple swelling at room temperature for 6 h at the
desired concentration.
Dextran-Photolabile Linker-Acrylate (8)
In the first step, oxalyl chloride (0.333 g, 2.62 mmol) in 5
mL of anhydrous dichloromethane was added dropwise to
an ice cooled solution of AEMNPBA (0.771 g, 2.18 mmol) in
30 mL of anhydrous dichloromethane under argon. Few
drops of DMF were added, and the mixture was stirred at
room temperature for 2.5 h. The solvent was removed under
reduced pressure and the flask was vented with argon. The
resulting oil was dissolved in 5 mL of anhydrous DMF and
added dropwise to a solution of dextran (6000 g/mol,
1.596 g, 0.226 mmol, 9.044 mmol glucopyranosyl groups),
LiCl (4.0 g), and NEt3 (0.572 g, 3.39 mmol) in 50 mL of
anhydrous DMF. The reaction mixture was stirred at room
temperature over night and then heated to 60 ꢀC for 1 h.
After cooling the solution to room temperature, the modified
dextran was obtained by precipitation in isopropanol. The
crude product was dissolved in water, precipitated again in
isopropanol. This purification procedure was repeated for a
total of two times and the functionalized dextran was
obtained in 58% yield as slightly yellow colored powder
from freeze drying of the aqueous solution. The DS was
obtained from 1H NMR spectroscopy by calculating the peak
area ratio of the 6 (olefinic, aromatic, and tertiary benzylic)
protons assigned to the multiplets from 7.70 to 5.60 ppm
relative to the anomeric protons of dextran assigned to the
multiplet at 5.25–4.75 ppm by using the formula:
Photolytic Degradation
Dispersions of purified nanogels in water (0.0625% w/v)
were placed in a quartz cuvette and the sample temperature
was adjusted to 37 ꢀC using a brass heating mantle com-
bined with a cryostat. Turbidity was measured in transmis-
sion by using a He-Ne laser emitting at k ¼ 633 nm. Irradia-
tion was carried out on a 90ꢀ angle relative to the laser
beam by using a UV-LED emitting at k ¼ 365 nm with an
intensity of I ¼ 30 mW/cm2. Transmitted light intensity of the
laser was measured by a photo diode combined with a GG-
385 edge filter to block scattered UV light. After irradiation,
the samples were diluted and the particle sizes were deter-
mined by DLS. In addition, the particle morphologies were
investigated by SEM. For irradiation time-dependent measure-
ments, samples were taken after fixed irradiation intervals.
UV–vis spectra were recorded using plate reader, and particle
sizes were determined from diluted samples by DLS.
A½6HðPL ꢂ AÞ@7:70 ꢂ 5:60 ppmꢄ=6
DSðDex ꢂ PL ꢂ AÞ ¼
A½1HðanomericÞ@5:25 ꢂ 4:75 ppmꢄ
A DS of 0.17 was determined meaning that in a dextran
chain of 34 glucopyranosyl groups every sixth group is func-
tionalized with the photolabile linker-acrylate.
Enzymatic Degradation and Subsequent Irradiation
Sample preparation and instrumentation were analogous to
the photolytic degradation experiments. After temperature
equilibration for 30 min to 37 ꢀC, 10 lL of a dextranase
solution in water (47% w/v) were added to dispersions of
purified nanogels in water (0.0625% w/v; 3 mL) and the
turbidity was monitored. After 6 h of enzymatic treatment,
UV irradiation was carried out for 45 min.
Synthesis of Hybrid Nanogels by Inverse
Miniemulsion Copolymerization
The dispersed phase was prepared by dissolving various
amounts of AAm and Dex-PL-A in a 0.5 M NaCl solution.
Afterward, the mixture was added to the continuous phase
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ARTICLE
9 Eichenbaum, G. M.; Kiser, P. F.; Dobrynin, A. V.; Simon, S.
A.; Needham, D. Macromolecules 1999, 32, 4867–4878.
CONCLUSIONS
In conclusion, a new system for potential enzymatic- and
light-triggered release applications based on degradable
nanogels was successfully developed. Realization of this con-
cept was achieved with newly synthesized water soluble
crosslinking molecules consisting of vinyl functionalized dex-
trans. In addition to the inherent enzymatic cleavability of
the dextran backbone upon incubation with dextranase, light
sensitivity was incorporated into the crosslinking structure
by the covalent attachment of multiple radically polymeriz-
able acrylate units via a photolabile linkers to the polysac-
charide chains. The resulting Dex-PL-A structures are charac-
terized by their good water solubility which—in combination
with multiple vinyl groups per chain—offers the possibility
for the preparation of a broad range of enzymatic- and light-
degradable (nano-)gels by copolymerization with different
vinyl functionalized monomers from aqueous solutions. As a
first model system, AAm was copolymerized with Dex-PL-A
in an inverse miniemulsion. High-crosslinking efficiency of
the resulting p(AAm-co-Dex-PL-A) nanogels was achieved by
systematic increasing the Dex-PL-A/AAm ratio. It was shown
that irradiation with UV light induced either complete parti-
cle degradation or a desired specific DGS by adjusting the
irradiation time accordingly. In addition, a two-step degrada-
tion profile based on the subsequent appliance of the two
orthogonal stimuli was realized by first generating highly
swollen nanogels by partial enzymatic cleavage of the Dex-
PL-A crosslinking molecules and their successive complete
degradation upon irradiation. The facile way of preparation
at ambient temperatures of 37 ꢀC from aqueous solutions
and the observed low initial DGS is promising for the poten-
tial embedding of functional water soluble compounds al-
ready during the polymerization. In combination with the
well-defined degradation profiles, this feature renders these
new materials highly interesting for triggered release appli-
cations in aqueous dispersions.
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D. Klinger acknowledges the International Max Planck
Research School (IMPRS) for financial support.
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U.; Kreiter, M. Adv. Mater. 2008, 20, 4563–4567.
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