Enzyme-responsive amphiphilic PEG-dendron hybrids and their assembly into smart micellar nanocarriers

Article abstract of DOI:10.1021/ja413036q

Enzyme-responsive micelles have great potential as drug delivery platforms due to the high selectivity of the activating enzymes. Here we report a highly modular design for the efficient and simple synthesis of amphiphilic block copolymers based on a line

Full text of DOI:10.1021/ja413036q

Communication
pubs.acs.org/JACS
Enzyme-Responsive Amphiphilic PEG-Dendron Hybrids and Their
Assembly into Smart Micellar Nanocarriers
Assaf J. Harnoy,†,§,⊥ Ido Rosenbaum,†,§,⊥ Einat Tirosh,‡,§ Yuval Ebenstein,‡,§ Rona Shaharabani,‡,§
Roy Beck,§,∥ and Roey J. Amir*,†,§
†Department of Organic Chemistry, School of Chemistry, Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel
‡Department of Physical Chemistry, School of Chemistry, Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel
§Tel Aviv University Center for Nanoscience and Nanotechnology, Tel-Aviv University, Tel-Aviv 69978, Israel
∥School of Physics and Astronomy, Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel
S
* Supporting Information
Here we report a modular design for the efficient synthesis of
ABSTRACT: Enzyme-responsive micelles have great
potential as drug delivery platforms due to the high
selectivity of the activating enzymes. Here we report a
highly modular design for the efficient and simple
synthesis of amphiphilic block copolymers based on a
linear hydrophilic polyethyleneglycol (PEG) and an
enzyme-responsive hydrophobic dendron. These amphi-
philic hybrids self-assemble in water into micellar nano-
containers that can disassemble and release encapsulated
molecular cargo upon enzymatic activation. The utilization
of monodisperse dendrons as the stimuli-responsive block
enabled a detailed kinetic study of the molecular
mechanism of the enzymatically triggered disassembly.
The modularity of these PEG-dendron hybrids allows
control over the disassembly rate of the formed micelles by
simply tuning the PEG length. Such smart amphiphilic
hybrids could potentially be applied for the fabrication of
nanocarriers with adjustable release rates for delivery
applications.
enzyme-responsive amphiphilic hybrids composed of linear
polyethyleneglycol (PEG) and a stimulus-responsive dendron
with enzyme-cleavable hydrophobic end groups. These
amphiphilic PEG-dendron hybrids are expected to self-
assemble in water into micelles with a hydrophilic PEG shell
and a hydrophobic core and potentially can be utilized to
encapsulate hydrophobic cargo molecules.9 In the presence of
the activating enzyme, the hydrophobic end groups will be
cleaved from the dendron, making it more hydrophilic. This
change in amphiphilicity should result in destabilization of the
micellar aggregates, leading to their disassembly and release of
soluble PEG-dendron hybrids and the encapsulated cargo
(Figure 1).
timuli-responsive micelles that can disassemble and release
Stheir encapsulated cargo upon external stimuli have gained
increasing attention due to their potential utilization as
nanocarriers for drug delivery.1 These responsive materials
are inspired by the ability of many supramolecular assemblies in
Nature to alter their structures and activity in response to
changes in their environment. Most reports in the literature
usually describe stimuli-responsive polymers that respond to
changes in pH,2 temperature,3 irradiated light,4 or their
combination.5 While these approaches offer great control
over the triggering of the disassembly processes, substantial
advantages could be achieved by utilizing enzymes as stimuli.
As many diseases are characterized by imbalances in the
expression and activity of specific enzymes in the diseased
tissue, this overexpression could potentially be translated into
the selective activation of advanced drug delivery platforms.6
However, to date, little has been reported on enzyme-
responsive synthetic micellar nanostructures7 and systems are
often based on breaking the amphiphilic block copolymer into a
soluble hydrophilic polymer and an insoluble hydrophobic
block.8
Figure 1. Schematic representation of the encapsulation of hydro-
phobic guests in the hydrophobic core of a smart micellar nanocarrier.
Upon enzymatic cleavage of the hydrophobic end groups, the
nanocarrier disassembles and the guest molecules are released.
The synthetic methodology combines the utilization of the
PEG chain as a soluble support with thiol-yne chemistry,
yielding an accelerated dendron synthesis. Based on this
synthetic approach, we have synthesized a series of three PEG-
dendron hybrids, 1a−c, with PEG chains of three molecular
weights (2, 5, and 10 kDa) and a second-generation dendron
with four end groups. We chose to use phenyl acetamide as end
Received: December 22, 2013
Published: February 25, 2014
© 2014 American Chemical Society
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groups that can be cleaved by the enzyme penicilin G amidase
(PGA).10 The hybrid block copolymers were synthesized
utilizing monomethyl ether PEG-amine, 2a−c, as starting
materials. Conjugation with an active ester of 3,5-bis(prop-2-
yn-1-yloxy)benzoic acid, 3,11 yielded PEG-diynes, 4a−c. The
latter were further modified by thiol-yne reaction12 with N-Boc
cysteamine, 5, to give tetrafunctionalized PEG-dendrons, 6a−c.
Deprotection of the Boc yielded PEG-tetra-amines, 7a−c. In
the last step of the synthesis, 4-nitrophenyl ester of phenyl
acetic acid, 8, was used to introduce the enzyme cleavable
hydrophobic end groups. PEG-dendron hybrids, 1a−c, were
obtained as off-white solids with overall yields of 76%, 86%, and
93%, respectively, demonstrating the very efficient and simple
synthetic methodology (Scheme 1). The synthesized polymers
Table 1. CMCs and Micelles Diameters for PEG-Dendron
Hybrids 1a−c
hybrid
1a
1b
1c
a
CMC
micelle diameter
7.2 μM
12.4 μM
21.7 μM
b
11 nm
14 nm
18 nm
a
b
Calculated by using Nile red. Diameters of the micelles as measured
by DLS.
Scheme 1. Synthesis of Amphiphilic PEG-Dendron Hybrids
Figure 2. Schematic representation of PEG-dendron hybrids, 1a−c,
and their micellar assemblies (left). Size of the micelles by DLS at a
hybrid’s concentration of 160 μM before (in blue) and after (in red)
the addition of the activating enzyme (right).
PEG-dendron hybrids in D2O, which showed only the peaks of
the PEG’s protons (Figures S12−S14).
After the self-assembly of the three PEG-dendron hybrids
1a−c into micelles was confirmed, we studied their response to
enzymatic activity using DLS, fluorescence spectroscopy, and
HPLC. The enzymatic cleavage of the hydrophobic phenyl
acetamide end groups should decrease the hydrophobicity of
the dendron and destabilize the micelles, leading to their
disassembly into the corresponding monomeric hybrids. DLS
measurements of solutions of the amphiphilic hybrids in the
presence of PGA clearly indicated the decrease in the height of
the peaks that correlate with the larger micellar aggregates and
the formation of new peaks that correlate with the smaller sizes
of the nonassembled monomeric chains and the enzyme
(Figure 2).
The enzyme-responsive disassembly was further supported
by the change in the fluorescence of encapsulated Nile red dyes.
As the dye molecules are released into the aqueous environ-
ment upon the disassembly of the micelles, their fluorescence
intensity is expected to decrease.14 As anticipated, time-
dependent decreases in fluorescence were observed for all
three PEG-dendron hybrids, 1a−c, in the presence of PGA,
indicating that the Nile red molecules were released from the
and hybrids were characterized by 1H and 13C NMR, GPC, IR,
and MALDI in order to confirm their structures, and the
experimental values are in good agreement with the theoretical
ones (Supporting Information).
To evaluate the ability of the amphiphilic hybrids, 1a−c, to
self-assemble into micelles, the PEG-dendron hybrids were
dissolved directly in aqueous buffer (PBS, pH 7.4). Self-
assembly was first examined by utilizing solubilization experi-
ments with the solvachromic hydrophobic dye Nile red.13
These measurements indicated the formation of micelles with
critical micelle concentrations of 7.2, 12.4, and 21.7 μM for
hybrids 1a−c respectively (Table 1, Figures S18−S20).
Next, we studied the self-assembled structures using dynamic
light scattering (DLS) and transmission electron microscopy
(TEM). These analyses indicated the formation of spherical
nanostructures with diameters of 11, 14, and 18 nm for hybrids
1a−c, respectively (Figures 2 and S26, Table 1). Further
support for the formation of micelles with a PEG-shell and
dendron-based core was obtained from 1H NMR spectra of the
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hydrophobic cores of the micelles as they disassembled upon
the addition of the activating enzyme (Figures 3, S27, and S28).
As control experiments, micelles based on hybrid 1b were
incubated either in the absence of PGA or with an esterase that
cannot break amide bonds (PLE), in order to examine the
selectivity of the enzymatic activation. In both cases the micellar
assemblies were found to be stable and no disassembly or
degradation was observed by fluorescence spectroscopy and
HPLC (Figures S30, S31, and S37). In addition, micelles based
on amphiphilic Boc protected hybrid 6b were incubated with
PGA and were also found to be stable (Figures S24, S29, and
S36). These control experiments further demonstrate the
selectivity of the reported hybrids. Furthermore, the presence
of a PEG shell gives the micelles stealth properties and helps to
avoid nonspecific activation due to binding to proteins.
Comparison of the disassembly and hydrolysis rates for the
PEG-dendron hybrids 1a−c provided insight into the molecular
mechanism of the disassembly. One might expect that micelles
with thinner PEG shells would be activated faster then those
with thicker ones, as their hydrophobic cores are more
accessible to the enzyme. Interestingly, the opposite trend
was observed and micelles assembled from hybrids with longer
PEG chains disassembled faster (Figure 5).
Figure 3. Fluorescence spectra of Nile red (1.25 μM) in the presence
of PEG-dendron hybrid 1b (160 μM) as a function of time after the
addition of the activating enzyme, PGA (0.14 μM). Fluorescence
intensity decreased as Nile red was released into solution.
We next used HPLC to follow the enzymatic degradation of
the PEG-dendron hybrids, 1a−c. HPLC analyses revealed the
relatively fast disappearance of the amphiphilic hybrids, 1a−c,
upon incubation with the activating enzyme. Furthermore, we
were encouraged to see the formation of only three major
intermediates of increasing polarity (Figures S32−S35). Based
on their relative polarities, rate of formation, the monodisper-
sity, and symmetry of the dendrons, these intermediates are
partially cleaved hybrids with three, two, and one phenyl
acetamide end group (Figures 4, S38, and S39).
Figure 5. Comparison of the disassembly rates (fluorescence assay) of
micelles formed by PEG-dendron hybrids 1a−c.
These rate differences indicate that the cleavage of the
hydrophobic end groups by the enzyme does not occur by
penetration of the enzyme through the PEG shell into the
hydrophobic core (Figure 6: pathway a). Instead, the enzymatic
Figure 4. Change in fluorescence intensity and HPLC analysis of the
enzymatic degradation of the PEG-dendron hybrid 1b. Partially
degraded intermediates are shown schematically.
Figure 6. Schematic representation of two hypothetical enzymatic
activation pathways: (a) the enzyme penetrates through the PEG shell
or (b) equilibrium-based degradation in which the enzyme cleaves the
hydrophobic end groups of the monomeric form of the amphiphilic
hybrids.
Comparisons of the HPLC and fluorescence data showed
good correlations between the decrease in fluorescence and the
disappearance of the tetra-functionalized hybrid (i.e., hybrids
1a−c) and the first intermediate with three hydrophobic end
groups (Figure 4). These correlations indicate that only these
two molecular species contribute to the formation of the
micelles. Once their concentrations decrease, the micelles
disassemble and their fluorescence cargo is released. It is
important to note that at this relatively low enzyme
concentration (0.14 μM), we did not observe the formation
of the fully degraded tetra-amine hybrids, 7a−c, even after 24 h.
However, at the high enzyme concentration of 1.4 μM, the
formation of fully degraded hybrids was observed (Figure S33).
degradation takes place at the “monomeric” form of the
amphiphilic hybrids, which are in equilibrium with the micellar
aggregates (Figure 6: pathway b). This hypothesis of
equilibrium-based activation is in good agreement with other
micellar systems15 and is further supported by the differences in
the CMC values, which indicate that hybrids with the longer
PEG chains are more available than those with shorter chains
(Table 1).
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(8) Rao, J.; Khan, A. J. Am. Chem. Soc. 2013, 135, 14056.
To summarize, a new family of amphiphilic PEG-dendron
hybrids that can self-assemble into enzyme-responsive micellar
nanocarriers were synthesized through a very efficient and
simple synthetic methodology. The utilization of an enzyme
responsive dendron allowed for unprecedented control over the
structure and monodispersity of the stimulus-responsive block
and enabled a detailed mechanistic study. The disassembly
mechanism proceeded through equilibrium dependent enzy-
matic degradation, which is in good agreement with other
enzyme-responsive systems.15 As the disassembly and release
rates depended on the CMC of the amphiphilic hybrids, the
release rates can be simply tuned by adjusting the length of the
PEG block. Such enzyme-responsive hybrids and their self-
assembled micelles have great potential in the field of drug
delivery. Further studies of dendrons of various generations and
end groups are currently in progress.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental information, characterization data, and
control experiments. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
amirroey@tau.ac.il
■
Author Contributions
⊥A.J.H. and I.R. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support has been provided by the Allon fellowship
(The Council for Higher Education of Israel) and TAU Vice-
president seed money. R.B. acknowledges the support of Israel
Science Foundation (Grant 571/11). R.J.A. thanks Prof. Moshe
Portnoy for his support during the establishment of Amir’s
laboratory and Dr. Elizabeth Amir for her helpful comments.
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