Enzyme-triggered cascade reactions and assembly of abiotic block copolymers into micellar nanostructures

Article abstract of DOI:10.1021/ja501632r

Catalytic action of an enzyme is shown to transform a non-assembling block copolymer, composed of a completely non-natural repeat unit structure, into a self-assembling polymer building block. To achieve this, poly(styrene) is combined with an enzyme-sens

Full text of DOI:10.1021/ja501632r

Communication
pubs.acs.org/JACS
Enzyme-Triggered Cascade Reactions and Assembly of Abiotic Block
Copolymers into Micellar Nanostructures
Jingyi Rao, Christine Hottinger, and Anzar Khan*
Department of Materials, ETH-Zurich, CH-8093 Zurich, Switzerland
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* Supporting Information
ABSTRACT: Catalytic action of an enzyme is shown to
transform a non-assembling block copolymer, composed
of a completely non-natural repeat unit structure, into a
self-assembling polymer building block. To achieve this,
poly(styrene) is combined with an enzyme-sensitive
methacrylate-based polymer segment carrying carefully
designed azobenzene side chains. Once exposed to the
enzyme azoreductase, in the presence of coenzyme
NADPH, the azobenzene linkages undergo a bond scission
reaction. This triggers a spontaneous 1,6-self-elimination
cascade process and transforms the initially hydrophobic
methacrylate polymer segment into a hydrophilic hydrox-
yethyl methacrylate structure. This change in chemical
polarity of one of the polymer blocks confers an
amphiphilic character to the diblock copolymer and
permits it to self-assemble into a micellar nanostructure
in water.
Figure 1. Schematic representation of a polymer building block and its
assembly into a micellar nanostructure upon enzymatic activation in
water.
Scheme 1. Synthesis of Block Copolymer 3, Cleavage of
Azobenzene Linkages upon Enzymatic Action, and
Subsequent 1,6-Self-Elimination Reaction To Yield
Amphiphilic Assembly Precursor 5
eveloping strategies to gain control over formation or
Ddisruption of polymeric nanostructures is a significant
goal of nanotechnology. This research goal has implications in
the areas of drug delivery, biological sensing and imaging, and
tissue engineering applications, among others.1 Therefore, a
large effort is devoted to the construction of nanosized polymer
particles, such as micelles, that can respond to an external
stimulus.2 The nature of the stimuli employed, however, is most
often limited to changes in pH, temperature, radiation, or
electric field.3 Enzymes represent an attractive alternative to the
aforementioned stimuli due to their high selectivity, substrate
specificity, and mild operating conditions.4,5 In this regard,
various examples are reported in which an assembled micellar
nanostructure undergoes a disassembly process through an
enzymatic action.6 The reverse approach, in which an enzyme
triggers assembly of a synthetic polymer into a micellar
structure, however, remains limited to two examples.7 In
addition, these examples are based exclusively on a natural
motif (i.e., hydrolysis or formation of phosphate esters). With a
goal to diversify the available toolbox (i.e., enzyme/substrate
pair) for the preparation of polymer nanostructures and enlarge
the repertoire of the enzymatically activated self-assembling
systems, here, we describe the development of a new strategy
(Figure 1). In this approach, a self-assembly precursor is
designed to have two polymeric segments composed of
completely non-natural repeat units (Scheme 1). One segment
is composed of poly(styrene), which is hydrophobic and
insensitive to the presence of an enzyme. The other segment,
also hydrophobic in nature, carries azobenzene moieties
attached to a hydroxyl methacrylate-based polymer repeat
unit through a benzyl−carbonate linkage. Once brought in
contact with the enzyme azoreductase,8 in the presence of
Received: February 16, 2014
Published: April 10, 2014
© 2014 American Chemical Society
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coenzyme NADPH, the azobenzene linkage undergoes a
cleavage reaction,8,9 which triggers a spontaneous 1,6-self-
elimination cascade process (Scheme 1).10 This transforms the
initially hydrophobic segment into a hydrophilic hydroxyethyl
methacrylate structure. The alteration in chemical polarity of
one of the copolymer blocks imparts an amphiphilic nature to
the diblock copolymer and allows it to assemble into a micellar
nanostructure in water.
The enzyme azoreductase is produced by the microbial flora
of human intestine. Therefore, azoreductase-sensitive systems
are of relevance to colon-related therapeutic applications.11 For
example, a small-molecule drug such as sulfasalazine, in which
cleavage of the azobenzene bond releases the therapeutically
active 5-aminosalicylic acid moiety, is used in inflammatory
bowel disease treatment. Moreover, a variety of azobenzene-
based polymers have been investigated for their colon-specific
delivery applications.9
To establish the aforementioned concept, a careful design of
the enzyme-responsive polymer segment was critical. This was
achieved by placing a carbonate moiety, as a leaving group, at
the benzylic position of the azobenzene functionality in the
hydroxyethyl methacrylate-based monomer 1 (Scheme 1). It
was envisaged that enzymatic cleavage of the azobenzene group
would result in the formation of an aniline group at each
polymer repeat unit.8,9 Such electron-rich aromatic species,
with a leaving group placed at the benzylic position, are known
to undergo an azaquinone methide-mediated spontaneous 1,6-
self-elimination process.10 The driving force for such reactions
comes from an increase in the entropy of the system coupled
with the irreversible formation of the thermodynamically stable
CO2 molecule. In this way, the enzyme-triggered cascade
elimination reaction would transform the hydrophobic
azobenzene methacrylate polymer into a hydroxyl-carrying
hydrophilic polymer segment.
NMR spectroscopy, copolymer 3 displayed aromatic reso-
nances of poly(styrene) (6.3−7.2 ppm) as well as azobenzene
side chains (7.4−8.0 ppm) (Figure S2). The benzylic group
could be observed at 5.1 ppm, and the methylene units located
adjacent to the carbonate and the ester moieties could be
detected in the range of 4−4.5 ppm.
To test the enzyme sensitivity of the prepared polymeric
substrate, block copolymer 3 was suspended in water and then
exposed to a mixture of the enzyme azoreductase and
coenzyme NADPH. The mixture was incubated at 37 °C for
a period of 12 h. Since the polymer was suspended in pure
water that did not contain any cosolvents (typically used in
micellar preparation), elevating the temperature assisted the
micellization process.13 Finally, to remove the cofactor and its
byproducts, the aqueous solution was dialyzed against water.
Examination of the purified aqueous solution by UV−vis
spectroscopy confirmed that the azobenzene linkages were
cleaved, as no absorption signal could be observed at 450 nm
after the enzymatic treatment of copolymer 3 (Figure 3). This
To test the feasibility of the proposed approach, monomer 1
was polymerized through an atom-transfer radical polymer-
ization (ATRP)12 protocol using poly(styrene) macroinitiator 2
(Mn(NMR) = 10400, Mn(GPC) 8500, PDI(GPC) = 1.1). This gave
rise to the targeted block copolymer 3 (Mn(NMR) = 28800,
Figure 3. UV−vis spectra of polymer 3 in chloroform (solid line) and
in an aqueous solution after the enzymatic reaction and purification
(dashed line).
M
n(GPC) = 47500, PDI(GPC) = 1.1). The block copolymerization
aqueous solution was spin-coated onto a silicon substrate for
observation with the help of atomic force microscopy (AFM).
These experiments indicated formation of spherical structures
in the size range of 30−60 nm (Figures 4 and S3−S5).
Transmission electron microscopy (TEM) experiments were
was evidenced by a shift to lower retention time of the
copolymer 3, as compared to the macroinitiator 2, in gel
permeation chromatography (GPC) analysis (Figure 2). In H
1
Figure 2. GPC traces of macroinitiator 2 (solid line), copolymer 3
(dotted line), and copolymer after cleavage of the azo linkages and
subsequent self-elimination reaction (dashed line).
Figure 4. AFM height (left) and phase (right) images (1×1 μm2)
obtained upon spin-coating the aqueous solution after the enzymatic
reaction.
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Communication
carried out by mounting an aqueous drop (∼15 μL) of the
micellar solution onto a carbon-coated copper grid and staining
it with ruthenium tetraoxide vapors. In TEM micrographs, the
periphery of the spherical structures appeared more intensely
stained than the interior (Figures 5, S6, and S7). This suggested
Figure 7. Digital photographs of bulk polymer material before (left)
and after (right) the enzymatic reaction.
Finally, the purified assembling polymer was subjected to
GPC analysis (Figure 2). This examination revealed that the
hydrodynamic volume of the polymer decreased after the
enzymatic reaction. This is most likely due to the fact that
precursor polymer 3 carried carbonate-linked azobenzene side
chains. These bulky side groups were removed from the second
segment of the diblock copolymer upon enzymatic action. This
change in molecular structure of the precursor polymer, 3,
resulted in a decrease in the hydrodynamic volume of the
assembling polymer, 5.
To summarize, the enzyme azoreductase could be employed
as a trigger to assemble a carefully designed polymer with a
completely non-natural chemical composition into a nano-
structured morphology in water. In essence, this work
diversifies the available enzyme/polymer-substrate toolbox for
the preparation of polymer nanostructures and broadens the
range of enzymatically triggered self-assembling systems.
Furthermore, the azoreductase activation capability of the
present concept may present some possibilities to create colon-
related delivery/imaging systems.
Figure 5. TEM micrographs of the aqueous micellar solution (see
Figures S4 and S5 for large area images).
that the micellar shell was composed of the hydrophilic
poly(hydroxyethyl methacrylate) segment known for its high
affinity to ruthenium.14 Dynamic light scattering (DLS) studies
further corroborated that the aqueous solution was indeed
made up of polymer nanoparticles ranging in size from 40 to 90
nm (Figure 6). The difference in the observed size range may
ASSOCIATED CONTENT
* Supporting Information
Synthesis and characterization details. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
anzar.khan@mat.ethz.ch
■
Notes
The authors declare no competing financial interest.
Figure 6. DLS data obtained from the aqueous solution after the
enzymatic reaction.
ACKNOWLEDGMENTS
■
A.K. thanks A. D. Schluter (ETH-Z) for constant support.
̈
come from the fact that AFM and TEM measurements
examined the dry state while DLS measurements reflected on
the solvated state of the material.
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