Enzymatically triggered self-assembly of block copolymers

Article abstract of DOI:10.1021/ja9060917

(Figure Presented) The polymerization of vinyl monomers with cleavable enzymatic substrates has been shown to lead to water-soluble double-hydrophilic block copolymers which, upon enzymatic activation of the diblock copolymers, become amphiphilic and undergo self-assembly into colloidal nanostructures. The ability to change the chemical and physical characteristics of polymeric materials by an enzymatic reaction opens the way for novel and exciting applications such as enzymatic-triggered activation of surfaces and formation of nanostructures in vivo in a highly controlled manner. Copyright

Full text of DOI:10.1021/ja9060917

Published on Web 09/09/2009
Enzymatically Triggered Self-Assembly of Block Copolymers
,†
‡
‡
,†
Roey J. Amir,* Sheng Zhong, Darrin J. Pochan, and Craig J. Hawker*
Materials Research Laboratory and Departments of Materials, Chemistry and Biochemistry,
UniVersity of California, Santa Barbara, California 93106, and Department of Materials Science
and Engineering and Delaware Biotechnology Institute, UniVersity of Delaware,
Newark, Delaware 19716
Received July 29, 2009; E-mail: ramir@mrl.ucsb.edu; hawker@mrl.ucsb.edu
The increasing interest in nanostructures for biomedical and
polymers 1a and 1b, respectively (Scheme 1). Full cleavage of
material science applications has motivated the development of
controlled methods for the fabrication and assembly of various
molecular systems into functionalized nanostructures. One of
the most successful strategies is the assembly of micelles and
other supramolecular structures from amphiphilic block co-
the benzyl groups was confirmed by the disappearance of the
1
corresponding peaks in the H NMR and by shifts of ca. 8 ppm
3
1
to lower field in P NMR (see Figures S3 and S4 in the
Supporting Information).
1
-5
polymers.
The next major challenge in this rapidly expanding field is
the triggered formation of such nanostructures. Several research
groups have recently reported triggering of stimuli-responsive
5
-18
block copolymers to self-assemble into micelles.
These
reports describe the use of copolymers that are generally
composed of a hydrophilic block and a stimuli-responsive block
and are often regarded as “smart” polymers. Upon an external
stimulus the character of the functional groups of the responsive
block changes (e.g., from hydrophilic to hydrophobic) and the
polymer can be switched from double-hydrophilic to amphi-
6
-9
philic.
Altering the solubility of such polymers can control
the size and shape of the resulting nanostructures. To date,
Figure 1. Schematic presentation for enzymatic activation of a water-
soluble diblock copolymer to give an amphiphilic diblock copolymer
and subsequent self-assembly into colloidal nanostructures (Note: full
cleavage of the solubilizing moieties is not required for self-
assembly).
1
0-12
13-15
16
thermal,
tions,
pH dependent,
and most recently chemically responsive activation
photochemical, combina-
1
7,18
18
of the self-assembly process have been reported. While these
approaches offer great control over the triggering process, they
all lack the high degree of selectivity that can be achieved by
enzymatic activation.
Polymers 1a and 1b were freeze-dried to give white powders,
which were directly dissolved into DI water to yield aqueous
stock solutions. These solutions were further diluted into buffer
In this report we introduce the concept of enzymatically
triggered self-assembly of block copolymers under physiological
conditions. This approach is based on incorporating vinyl
monomers that contain an enzymatic activated substrate, which
renders the monomer water-soluble. The polymerization of such
monomers with a polyethylene glycol (PEG) macroinitiator
yields water-soluble block copolymers. Upon cleavage of the
enzymatic substrates by the activating enzyme, the solubilizing
moieties are removed from the vinyl polymer backbone to yield
a hydrophobic block. Consequently, the polymer becomes
amphiphilic and undergoes self-assembly to form colloidal
nanostructures in situ (Figure 1).
2
3
solution at pH 5 (the optimal pH for the activating enzyme).
As both block copolymers 1a and 1b were found to give similar
results, only 1a is discussed below with comparable data for 1b
available (Supporting Information).
Scheme 1. Synthetic Approach for the Enzymatic and Chemical
Preparation of Amphiphilic Diblock Copolymers
Block copolymers 1a and 1b, containing phosphate moieties
1
9
that can be cleaved by acid phosphatase (APase)
were
2
0
synthesized by nitroxide-mediated polymerization (NMP) of
dibenzyl 4-vinylphenyl phosphate monomer with a monomethyl
ether 5 kDa PEG macroinitiator 2 to yield the dibenzyl-
protected block copolymer 3a (MW (NMR) ) 10 kg/mol) and
2
1
3
b (MW (NMR) ) 16 kg/mol). Deprotection by trimethylsilyl
22
bromide, in the presence of collidine, then gives the desired
†
University of California, Santa Barbara.
University of Delaware.
‡
10.1021/ja9060917 CCC: $40.75  2009 American Chemical Society
J. AM. CHEM. SOC. 2009, 131, 13949–13951 9 13949

C O M M U N I C A T I O N S
To demonstrate the enzymatic cleavage of the phosphate
groups of the polymers, 31P NMR spectra of polymer 1a (3 mg/
mL) in a citrate buffer pH 5 with and without the activating
enzyme (0.13 mg/mL) were measured at different time points.
Figure 2 shows clear evidence for the cleavage of the phosphate
groups by APase which is apparent from both the decrease of
the peak corresponding to the phosphate ester attached to the
polymer backbone (at -3.5 ppm) and the formation of a new
peak that corresponds to the released phosphoric acid (at 1 ppm).
No cleavage was observed in the absence of APase (data not
shown). The extended reaction times for the NMR experiments
are due to the significantly reduced, relative concentration of
the enzyme. While an increased concentration of the polymer is
required for the NMR study (signal-to-noise ratio), a similar
increase in enzyme concentration is not possible due to its limited
solubility in aqueous solutions (0.14 mg/mL).
therefore DLS could not be used to reproducibly monitor the
activation and self-assembly processes.
To examine the self-assembly process in greater detail, the
2
4
fluorescence spectra of highly diluted pyrene containing (2 µM)
an aqueous solution of 1a incubated with APase were recorded
at different time intervals. Pyrene containing solutions of the
polymers without APase, APase itself, and polymer 4c (Scheme
1) were used as control experiments. Figure 3 presents the ratio
of the vibrational band intensities (I /I ) of pyrene’s fluorescence
I
III
spectra in aqueous solutions of APase, polymer 1a, polymer 1a
and APase, and polymer 4c.
I
Figure 3. Quotient of pyrene’s vibrational band intensities (I /IIII).
I
The lower ratio of the vibrational band intensities (I /IIII) for
the polymer 1a after incubation with APase in comparison to
the polymer itself indicates the formation of hydrophobic regions
in the activated polymer solution. This result is expected, as
the enzyme triggers a change in the chemical and physical
properties of the polystyrene based block. These changes,
transform the block copolymer from a double-hydophilic to an
amphiphilic block copolymer, which self-assembles into colloidal
nanostructures having the PEG hydrophilic chains as an outer
shell and the poly(4-hydroxystyrene) chains as a hydrophobic
core. As pyrene molecules migrate into these hydrophobic
Figure 2. 31P NMR spectra of polymer 1a with APase in citrate buffer
at pH 5. The signals at -3.5 and 1 ppm correspond to the polymer bound
phosphate and cleaved phosphoric acid, respectively.
regions, a decrease of the ratio (I
noted that the ratio (I /IIII) for the PEG-b-poly(4-hydroxystyrene)
c is lower than the ratio (I /IIII) for activated polymer 1a. The
difference in the ratios (I /IIII) supports the P NMR results,
which indicated incomplete activation of all the phosphate groups
along the backbone. As mentioned previously, after the enzyme
starts cleaving the phosphate groups, the polymer becomes
amphiphilic and self-assembles. The presence of phosphate
residues leads to a more polar core when compared to p-
hydroxystyrene, 4c. This molecular level understanding of the
enzymatic activation process shows that the desired self-assembly
process does occur under physiological conditions.
An interesting feature observed in the NMR and pyrene
fluorescence experiments is the significant disparity in time
frames. This is due to the sensitivity difference between the two
techniques which necessitates major changes in the relative molar
ratios of polymer (substrate) and enzyme. Greater than an order
of magnitude difference is observed when going from the NMR
experiment (2700 phosphate residues/1 Apase) to the fluores-
cence study (150 phosphate residues/1 Apase).
I
/IIII) is observed. It should be
The 31P NMR experiments reveal that some amount of
phosphate remains bound to the polymer even after long
incubation times (see Supporting Information). We assume that
after the enzyme starts cleaving the phosphate groups, the
polymer then becomes amphiphilic and self-assembles before
all phosphate moieties have been cleaved. Encapsulation of
unreacted phosphates inside the core of the colloidal nanostruc-
tures leads to steric isolation, and the residual phosphates become
less accessible to the activating enzyme. This assumption is
supported by the fluorescence data that are discussed later.
I
4
I
31
I
To demonstrate the concept of enzymatic triggered self-
assembly of block copolymers, polymer 1a was incubated with
Apase in a buffer solution of pH 5. Upon enzymatic cleavage
of the phosphate groups, a hydrophobic backbone consisting of
4
-hydroxystyrene and the corresponding phosphoric acid repeat
units develops (Figure 2); the polymer (4a) becomes amphiphilic
and undergoes self-assembly into nanostructures. Dynamic light
scattering of aqueous solutions of polymer 1a (0.125 mg/mL)
showed no light scattering, indicating that the polymer is soluble
at this concentration. However, the limited solubility of APase
gave aqueous solutions that showed unstable baselines, and
To further demonstrate this novel process and to acquire
information on the shape and size of the self-assembled
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3950 J. AM. CHEM. SOC. 9 VOL. 131, NO. 39, 2009

C O M M U N I C A T I O N S
nanostructures, direct visualization with TEM was used. TEM
micrographs of the nanostructures were obtained from pH 5
buffer solutions of polymer 1a in the presence of the enzyme,
and a representative example is shown in Figure 4. No structures
were observed for the control solutions of the enzyme sample
and polymer without the enzyme (data not shown). In direct
contrast, spherical colloidal nanostructures with an average
diameter of 90 nm were observed for a solution of polymer 1a
after incubation with the activating enzyme (Figure 4). This
clearly shows that enzymatic activation is achieved and required
for nanostructure formation. Similar structures were observed
for the chemically synthesized control polymer 4c (Supporting
Information).
Kriss for their help in acquiring TEM images and Matt Kellum
for his help with GPC analysis.
Supporting Information Available: Synthesis of the monomer,
polymerization procedures, characterization details, samples prepara-
1
13
31
tion, and H NMR/ C NMR/ P NMR/GPC/HRMS/Fluorescense/
TEM data. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Acknowledgment. This material is based upon work sup-
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ꢀ
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JA9060917
J. AM. CHEM. SOC. 9 VOL. 131, NO. 39, 2009 13951