Enzyme-triggered disassembly of dendrimer-based amphiphilic nanocontainers

Article abstract of DOI:10.1021/ja906162u

(Figure Presented) We demonstrate a new enzyme-induced disassembly of amphiphilic nanocontainers based on dendrimers. Disassembly and the ensuing release of noncovalently bound guest molecules are of great interest because of their implications in areas s

Full text of DOI:10.1021/ja906162u

Published on Web 09/16/2009
Enzyme-Triggered Disassembly of Dendrimer-Based Amphiphilic
Nanocontainers
Malar A. Azagarsamy, Punidha Sokkalingam, and S. Thayumanavan*
Department of Chemistry, UniVersity of Massachusetts, Amherst, Massachusetts 01003
Received July 23, 2009; E-mail: thai@chem.umass.edu
Stimuli-responsive drug delivery systems have gained sig-
nificant attention in recent years because their controlled release
characteristics have resulted in enhanced efficacy at the site of
action.1 Most of these systems respond to stimuli such as
temperature, pH, light, magnetic fields, and ionic strength.2 An
attractive class of responsive materials would involve molecular
designs that respond to biological stimuli3 such as proteins, since
overexpression of proteins, especially enzymes, has frequently
been implicated in the diseased state of cells. Here, we disclose
our findings on an enzyme-responsive dendrimer assembly.
Dendrimers are interesting as macromolecular structures for a
variety of applications because of their globular shape and the high
degree of control over their size.4 In the area of enzyme-responsive
systems, a novel class of dendrimers called “self-immolative
dendrimers” has been reported, in which all covalently appended
drug/active molecules are released by a single enzymatic trigger at
the dendritic core.5 A useful complement to this approach involves
noncovalently sequestering the guest molecules and releasing them
in response to an enzymatic trigger. This approach is attractive
for two reasons: (i) hydrophobic guests are encapsulated by the
water-soluble dendrimers, and therefore, the hydrophobicity of
the molecule does not affect the fidelity of the approach; (ii)
guest molecules, such as drugs, need not be converted to
prodrugs, and thus, the molecular design is simplified and the
applicability can be rather broad. We report here the enzymatic
disassembly of dendrimer-based micelles. Dendritic micellar
assemblies are also appealing because (i) these macromolecules
exhibit low critical aggregation concentrations (CACs) and high
stabilities relative to their small-molecule counterparts6,7 and
(ii) systematic control over the molecular weight of the dendrimer
through generational variations provides an opportunity for
differential release rates.
Figure 1. Schematic representation of enzyme-induced disassembly of
dendritic micellar assemblies and guest release.
Chart 1. Structures of Ester-Functionalized Amphiphilic Dendrons
We recently reported a distinct class of amphiphilic biaryl
dendrimers that form micelle-like and inverse-micelle-like structures
in polar and apolar solvents, respectively.7 The fact that hydro-
philic-lipophilic balance (HLB) is crucial for the formation of
micellar assemblies of these dendrimers presents an opportunity
for disassembly by disturbance of the HLB in response to an
enzymatic trigger. Unlike classical amphiphilic dendrimers,6 our
facially amphiphilic dendrimers form micellar assemblies by
aggregation of several dendritic molecules as a result of the
orthogonal placement of hydrophilic and lipophilic units in every
repeat unit of the dendrimer. To enzymatically trigger the
disassembly of these assemblies, we installed enzyme-cleavable
ester moieties as lipophilic units. We hypothesized that enzymatic
cleavage of these lipophilic units would render the dendrimer
hydrophilic and cause deaggregation. Since the hydrophobic
container property of these dendrimers in water is predicated
on the micelle-like aggregation, the enzyme-triggered deaggre-
gation event should result in a concomitant release of the
sequestered hydrophobic guest molecules (Figure 1).
The structures of dendrons G0-G3 with a hexyl ester function-
ality as the lipophilic unit and pentaethylene glycol (PEG) as the
hydrophilic unit are shown in Chart 1. PEG was chosen to avoid
nonspecific interactions. The dendrimers were synthesized in a
modular fashion, where the enzyme-sensitive functionalities were
installed using the Huisgen 1,3-dipolar cycloaddition reaction.8 Prior
to testing the disassembly, we investigated the micellar behavior
of these dendrons in water by encapsulating pyrene as a probe.
CACs of these dendrons were determined using the concentration
dependence of the excitation spectrum of pyrene.8 As expected,
the CAC of the small-molecule-surfactant G0 dendron was found
to be in the millimolar range, whereas dendrons G1-G3 exhibited
CACs of 4.3, 0.7, and 0.3 µM, respectively.
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10.1021/ja906162u CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
generation, which is consistent with the DLS data; (ii) the burst
release observed for G0 is also consistent with the DLS results;
and (iii) the maximum dye release observed in the case of dendrons
reached a plateau at ∼50%. We attribute this to the possibility that
the rather hydrophobic backbone of our biaryl dendrimers is capable
of solvating a small amount of pyrene. This was supported by the
significant pyrene encapsulation capability of the hydrolyzed G2
carboxylic acid-containing dendron.8
In summary, we have designed dendrimer-based amphiphilic
assemblies that can noncovalently sequester hydrophobic guest
molecules and release these guests in response to an enzymatic
trigger. This was achieved by incorporating enzyme-sensitive
functionalities at the lipophilic face of the dendrons. This feature
causes a change in the HLB when the enzyme is encountered,
effecting disassembly and guest-molecule release. The noncovalent
nature of the binding and release of the guest molecules is likely
to further increase the repertoire of dendrimers in enzyme-
responsive drug delivery systems and biosensors.
Figure 2. Disassembly of dendritic micellar assemblies upon exposure to
PLE: (a) size evolution of the G1 dendritic assembly using DLS; (b) %
release of pyrene using fluorescence studies.
The enzyme-induced disassembly was first investigated using
dynamic light scattering studies. The assembly size in a 25 µM
solution of dendron G1 was ∼100 nm. Upon addition of a 0.1 µM
solution of porcine liver esterase (PLE), we were gratified to find
a systematic decrease with time in the size of the G1 dendron
assembly, which finally reached ∼10 nm after 16 h (Figure 2a).
The final size (10 nm) is identical to the size of the enzyme. This
indicates that the final unaggregated water-soluble dendrons are
not discernible by DLS. To further test whether the disassembly
occurs solely because of the enzymatic hydrolysis of ester func-
tionalities, PLE was added to a solution of the structurally similar
dendron G1-control (Chart 1)7b that lacks ester functionalities. The
lack of disassembly in this case supports the enzyme-specific
disassembly of the G1 micelle-like assembly.8
Acknowledgment. We thank the NIGMS of the National
Institutes of Health and DARPA for support.
Supporting Information Available: Experimental details and NMR,
DLS, and fluorescence data. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
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We were also interested in the generation dependence of the
disassembly with respect to the relative kinetics of disassembly.
We conceived that the size of the dendrons would have an effect
on this kinetics. For comparison, we maintained identical concen-
trations of the ester functionalities in all generations. Upon exposure
to PLE, the G2 assembly size was reduced to 10 nm in 24 h, while
G3 took 36 h to reach the same size.8 This generation dependence
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