Accessing lipophilic ligands in dendrimer-based amphiphilic supramolecular assemblies for protein-induced disassembly

Article abstract of DOI:10.1002/chem.201102727

Supramolecular nanoassemblies that respond to the presence of proteins are of great interest, as aberrations in protein concentrations represent the primary imbalances found in a diseased state. We present here a molecular design, syntheses, and study of

Full text of DOI:10.1002/chem.201102727

FULL PAPER
DOI: 10.1002/chem.201102727
Accessing Lipophilic Ligands in Dendrimer-Based Amphiphilic
Supramolecular Assemblies for Protein-Induced Disassembly
Volkan Yesilyurt, Rajasekharreddy Ramireddy, Malar A. Azagarsamy, and
S. Thayumanavan*[a]
Dedicated to Professor Seth R. Marder on the occasion of his 50th birthday.
Abstract: Supramolecular nanoassem-
blies that respond to the presence of
proteins are of great interest, as aberra-
tions in protein concentrations repre-
sent the primary imbalances found in a
diseased state. We present here a mo-
lecular design, syntheses, and study of
facially amphiphilic dendrimers that re-
spond to the presence of the protein,
immunoglobulin G. It is of particular
interest that the ligand functionality,
utilized for causing the binding-induced
disassembly, be lipophilic. Demonstra-
tion of binding with lipophilic ligands
greatly expands the repertoire of bind-
ing-induced disassembly, since this
covers a rather large class of ligand
moieties designed for proteins and
these provide specific insights into the
mechanistic pathways that are available
for the binding-induced disassembly
process. Here, we describe the details
of the binding induced disassembly, in-
cluding the change in size of the assem-
bly in response to proteins, concurrent
release of noncovalently encapsulated
guest molecules, and the specificity of
the disassembly process.
Keywords: dendrimers · disassem-
bly · micelles · proteins · supramole-
cules
Introduction
diseases, notably cancer, due to the enhanced permeability
and retention (EPR) effect.[8] This has spurred interest in
molecular assemblies, based on surfactants,[9] lipids,[10] poly-
mers,[2,11] and dendrimers.[12] While small molecule surfactant
based amphiphilic assemblies exhibit the ability to sequester
lipophilic molecules in aqueous media, these suffer from
low stability and high critical aggregation concentrations
(CACs). On the other hand, phospholipid based liposomes
exhibit enhanced stability and low CACs.[10] However, their
application is largely limited to the delivery of hydrophilic
molecules or lipophilic molecules that can be rendered
water soluble.[10,13] Considering the fact that most drug mole-
cules are lipophilic, it is desirable that systems possessing
lipophilic microenvironments, to accommodate guest mole-
cules, are used. In this context, amphiphilic polymers have
been widely studied for drug delivery applications due to
their ability to form micelles and encapsulate lipophilic
guest molecules. These efforts have resulted in some excel-
lent contributions to the field.[14] However, dendrimers pro-
vide a distinct advantage in fundamentally understanding
the structural factors that control amphiphilic supramolec-
ular assemblies and stimuli-responsive disassemblies. This is
mainly due to the excellent control over their size and, thus,
the perfectly monodisperse nature of these macromole-
cules.[12,15]
Stimuli-responsive supramolecular systems have gained sig-
nificant attention in recent years, as these systems have im-
plications in a variety of areas, especially in drug delivery.[1]
Similarly, amphiphilic assemblies have been attractive be-
cause of the potential to encapsulate water-insoluble drug
molecules within their interior, which can then be released
at a specific location in response to a trigger.[2] In this
regard, amphiphilic systems that respond to physical or
chemical internal or external stimuli have been widely ex-
plored. Systems that respond to change in pH,[3] tempera-
ture,[4] redox environment,[5] and light[6] are popular in this
area. Location-dependent variations in these factors could
be considered to be secondary imbalances in biology. The
primary imbalances in biology that result in a diseased state
involve variations in protein concentrations. Therefore, it is
interesting to design supramolecular assemblies that respond
to proteins, an area that is relatively under-explored.[7]
Among responsive supramolecular assemblies, nanosized
structures have been of great interest, since these have the
propensity to accumulate in the inflamed tissues of certain
[a] V. Yesilyurt, R. Ramireddy, M. A. Azagarsamy,
Prof. S. Thayumanavan
With all these scaffolds, a limited number of reports exist
on protein-sensitive assemblies.[7,16] These reports, however,
are mainly based on the enzymatic activity of the protein
that causes the disassembly rather than a specific ligand–
protein interaction. Designing systems that respond to a
protein-binding event will bring the large class of nonenzy-
Department of Chemistry, University of Massachusetts
Amherst, MA 01003 (USA)
Fax : (+1)413 545 4490
E-mail: thai@chem.umass.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201102727.
Chem. Eur. J. 2012, 18, 223 – 229
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223

matic proteins into the realm of stimuli-responsive supra-
molecular nanoassemblies and, thus, greatly expand the rep-
ertoire of these systems for applications, such as drug deliv-
ery and sensing.
A potential limitation of the above finding is that the pre-
sumed mechanism (Figure 1), allows for the binding-induced
disassembly to be executed with hydrophilic ligands. Howev-
er, most of the custom-designed ligands target the hydro-
phobic binding pockets of proteins; thus, most promising lig-
AHCTUNGTREGaNNUN nds are lipophilic in nature. When these lipophilic ligands
Results and Discussion
are incorporated into our proteins, these functionalities will
be buried within the interior of the assembly and thus might
not be available for binding to complementary proteins
(Figure 2, top).
Molecular design and synthesis: Recently, we introduced a
new class of facially amphiphilic biaryl dendrimers in which
every repeating unit in the dendritic backbone contains both
lipophilic and hydrophilic functionalities.[4a,12e] As a result of
the orthogonal placement of these mutually incompatible
units, these molecules exhibit a unique assembly switch, de-
pending on their solvent environment. In other words, these
dendrimers are able to form micelle-type assemblies in an
aqueous milieu and inverted micelle-type assemblies in
apolar solvents, such as toluene.[12e,17] In contrast to classical
amphiphilic dendrimers,[12a,d] the micellar assemblies from
our dendrimers are formed through aggregation of several
dendrimer molecules. This feature presents a unique oppor-
tunity for stimuli-induced disassembly in these dendrimers
through disaggregation.
Certain hydrophilic–lipophilic balance (HLB) is required
for these facially amphiphilic dendrimers to self-assemble in
the aqueous phase. Therefore, we envisaged the possibility
of disturbing the HLB of a dendrimer, through interaction
with a protein, to cause disassembly and afford a protein-
sensitive amphiphilic nanoassembly. Our basic premise for
the hypothesis is that a typical ligand is a relatively small
moiety that can be incorporated on to a dendrimer side
chain. This functionality makes a certain contribution to-
wards the overall HLB of the dendron and thus the stability
of the assembly. When a protein binds to this ligand moiety,
the ligand is masked and a rather large protein is presented
to the solvent surface. Since water-soluble globular proteins
exhibit a hydrophilic surface, the small ligand functionality
Figure 2. Binding-induced disassembly with lipophilic ligand containing
dendrons.
We hypothesized that an alternate mechanistic possibility
in these types of supramolecular assemblies might allow for
binding-induced disassembly with lipophilic ligands. Here,
we describe and test whether this alternate pathway is avail-
able for binding-induced disassembly. Note that noncovalent
amphiphilic assemblies are in equilibrium with their corre-
sponding monomers (illustrated by the equilibrium in
Figure 2, left). If binding between the ligand and the protein
is favored in the monomeric dendron, then binding can pref-
erentially occur at the monomeric stage. In this case, the
equilibrium would ultimately cause disassembly due to the
Le Chatlier-type effect (Figure 2, bottom). If this pathway is
available for binding-induced disassembly, then the versatili-
ty of this approach will greatly increase, as this approach
would then not be limited to just hydrophilic ligands.
To test our hypothesis, we chose dinitrophenyl (DNP)
moiety as the ligand functionality, because: 1) it is lipophilic
and will be buried within the interior of the amphiphilic ag-
gregate and thus the only reasonable pathway available for
binding involves the equilibrium-driven disassembly;
2) DNP has been shown to bind to rat anti-DNP immuno-
globulin G antibody (IgG) with subnanomolar binding affin-
ity,[18] and 3) synthetic modification of the 2,4-DNP group is
relatively straightforward and allows for the proof-of-con-
cept to be achieved with relative ease. Structures of the tar-
geted dendrons are shown as G1–DNP and G2–DNP. In
these dendrons, the decyl chain acts as the lipophilic unit,
and pentaethylene glycol (PEG) as the hydrophilic unit.
PEG was chosen as a charge-neutral hydrophilic functionali-
ty to reduce nonspecific interactions between dendrimer and
is essentially changed to
a large hydrophilic moiety
(Figure 1). We hypothesized that such an alteration should
result in a drastic change of HLB and cause disassembly.
Indeed, we have recently shown that disassembly of these
amphiphilic aggregates can be achieved with a specific
ligand–protein interaction, in which the ligand is hydrophilic
and thus is displayed on the surface of the amphiphilic ag-
gregate.[7c] This ligand display provides a convenient access
for protein to bind to the ligand and thus cause the binding-
induced disassembly (Figure 1).
Figure 1. Binding-induced disassembly with hydrophilic ligand containing
dendrons.
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Protein-Induced Disassembly
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protein. The target dendrons are designed in such a way
that these dendrimers are uniformly amphiphilic over the
entire dendritic structure. To achieve this, the dendrons
were constructed from a biaryl monomer, represented by
structure 3 in Scheme 1. The biaryl monomer has several
unique features: 1) it has the AB2 functional groups re-
quired for dendritic growth; 2) dendrimers constructed from
this biaryl building block are fully amphiphilic because of
the ability of the biaryl monomer to carry both lipophilic
and hydrophilic functional groups. Thus, once the dendron is
constructed every repeating layer in the dendritic backbone
contains amphiphilic functionalities; 3) hydrophilic ligands
can be placed in the dendrons by replacing the PEG unit
with the ligand functionality, while the lipophilic ligand is
placed by replacing the decyl moiety within a monomeric
repeat unit.
We were interested in having the syntheses of the den-
drons modular, as this will allow for the incorporation of
other lipophilic ligands on to the dendritic building block.
We have previously utilized the 1,3-dipolar Huisgen cycload-
Scheme 1. Synthesis scheme for G1–DNP dendron.
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225

S. Thayumanavan et al.
dition reaction, the so-called click reaction, as the last step
in our syntheses to install the ligand functionalities. Thus,
the target building block unit for our dendron syntheses in-
volves the presence of acetylene functionality at the lipo-
philic side of the dendron (Scheme 1, 3). The key step in the
synthesis of 3 involves the formation of the biaryl bond.
Thus, synthesis of biaryl compound 3 was achieved by using
Stille coupling as the key step, from the aryl stannane 1 and
bromo-aryl ester 2 (Scheme 1). Reaction between the re-
ported peripheral amphiphilic unit 4[4a] and biaryl building
block 3[16b] in the presence of potassium carbonate afforded
the G1 dendron, 5, in 88% yield. Similarly, the correspond-
ing G2 dendron was synthesized from 3 and the brominated
version of the G1 dendron 5.[4a] Once the first and second
generation dendrons were assembled from the biaryl mono-
mer 3, the 2,4-DNP ligand moiety was attached to these
dendrons through the Huisgen reaction with 8 to obtain the
targeted G1–DNP and G2–DNP dendrons. This reaction
was chosen to install the DNP ligand to the dendrons, as it
allows for easier future ligand variations. All dendrons were
characterized by 1H and 13C NMR spectroscopy, and
MALDI-ToF mass spectrometry, and details of the syntheses
and characterizations are outlined in the Supporting Infor-
mation.
&
^
Figure 3. a) Sizes of G1–DNP ( ) and G2–DNP ( ) dendrons; b) size
changes in G1–DNP and G2–DNP dendron based amphiphilic assemblies
Self-assembling properties of dendrimers: We first studied
the assembly properties of the dendrons G1–DNP and G2–
DNP using Nile red as a fluorescent probe. Nile red is a li-
~
^
due to the presence of anti-DNP IgG; , G1–DNP; &, G2–DNP; , G1–
&
*
DNP+IgG; , G2–DNP+IgG; , IgG.
ACHTUNGTRENNUNGpophilic dye and is not soluble in water unless it is accom-
modated in a hydrophobic pocket of a micelle-like assembly.
The emission spectra of Nile red, at different G1–DNP and
G2–DNP dendron concentrations in water, indicate that
these dendrons are capable of providing an apolar micro-
binding interaction between the DNP ligand in the dendrons
and the IgG protein will afford a dendron–protein complex,
the HLB of which would be drastically different from that
of the dendron itself. We anticipated that this change would
result in disruption of the micelle-type assembly. In order to
examine if these dendritic micellar aggregates are indeed re-
sponsive to IgG, we monitored the size of the assembly
using DLS. Upon addition of anti-DNP IgG (7.5 mm) to the
solution of G1–DNP (26 mm) a remarkable decrease in the
size of the assembly was observed (Figure 3b). A similar
change in the assembly size was also observed with G2–
DNP. The sizes of the G1–DNP and G2–DNP dendrons de-
creased to approximately 10 nm; this corresponds to the size
of the protein itself, and indicates that the dendritic assem-
bly is disaggregated due to ligand–protein binding.
ACHTUNGTRENNUNGenvironment that sequesters the lipophilic dye molecule
(Figure S1 in the Supporting Information). The emission
spectra at different dendron concentrations were used to
calculate the critical aggregation concentrations (CACs) of
the dendrons. Plotting the emission intensity of Nile red as a
function of dendron concentration afforded an inflection
point, which was taken to be the dendronꢁs CAC (see the
Supporting Information). Using this method, CACs for G1–
DNP and G2–DNP dendrons were found to be 0.035
(18 mm) and 0.020 mgmLÀ1 (5 mm), respectively. Formation
of the micelle-like assembly was further verified with dy-
namic light scattering (DLS) experiments. The dendron sol-
utions (26 mm, above the CACs) were prepared in water and
DLS results showed that 105 and 124 nm sized aggregates
are formed for G1–DNP and G2–DNP dendrons, respective-
ly (Figure 3a). This suggests that these dendrons are indeed
aggregated to form micelle-like nanoassemblies in water,
which are responsible for sequestering Nile red as the lipo-
philic guest.
Although the disassembly of the micellar aggregates pro-
vides good evidence for the anti-DNP IgG sensitivity of the
dendrons, it is necessary to determine whether this dis-
AHCTUNGTREGaNNUN ssembly indeed takes place due to a specific ligand–protein
interaction. Therefore, we examined the effect of the pres-
ence of noncomplementary proteins, pepsin (pI=1.0), cyto-
chrome c (CytC, pI=10.2), and a-chymotrypsin (ChT, pI=
8.8). These proteins were chosen for their diversity in pI
values, since this is often the source of nonspecific interac-
tions. Thus, solutions of G1–DNP and G2–DNP dendrons
were exposed to these three proteins (Figure 4). We were
gratified to find that the size of the dendritic assemblies
Dendrimer–protein interactions: Since the DNP ligand in
the dendrons is known to bind anti-2,4-DNP IgG, we were
interested in testing the effect of the presence of this protein
on the self-assembled structures. Our hypothesis is that the
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Protein-Induced Disassembly
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^
)
Figure 5. a) Guest molecule release from 26 mm solutions of G1–DNP (
and G2–DNP (&) dendrons; b) release from lower G2–DNP concentra-
&
^
(
Figure 4. Size changes: a) G1–DNP
), G1–DNP+pepsin ( ), G1–
^
tions at 13 ( ) and 26 mm (&).
~
*
DNP+a-chymotrypsin ( ), G1–DNP+cytochrome c ( ); b) G2–DNP
~
^
(
), G2–DNP+pepsin (&), G2–DNP+a-chymotrypsin ( ), G2–DNP+
*
cytochrome c ( ).
by increasing the relative ratio of the protein to the den-
dron. The commercial form of the protein imposes an upper
limit on its concentration. Fortunately, however, the lower
CAC of G2–DNP dendron enables us to work with lower
dendron concentration. Thus, when 13 mm G2–DNP dendron
was incubated with the IgG (7.5 mm) for 12 h, we observed
52% dye release, as compared to 45% release with 26 mm of
G2–DNP dendron (Figure 5b).
To determine whether dye release is specific only to IgG,
we exposed G1–DNP and G2–DNP dendrons to pepsin,
ChT, and CytC. We indeed found that exposure of G1–DNP
and G2–DNP dendrons to these proteins did not cause any
significant change in the fluorescence of Nile red; this indi-
cates that there is no dye released from dendritic assembly
(Figure 6). It should also be noted that CytC is a metallo-
protein and we have previously shown that addition of posi-
tively charged CytC to negatively charged carboxylate poly-
mers and dendrimers results in quenching of fluorescence of
the noncovalently encapsulated fluorophores; this indicates
the nonspecific binding of CytC to these assemblies.[19] Here,
the lack of fluorescence change of Nile red in the presence
of CytC provides additional support for the lack of nonspe-
cific interactions between the dendritic assemblies and pro-
teins.
were unchanged in the presence of these proteins. These re-
sults support our hypothesis that IgG binding indeed caused
micellar disassembly.
Next, we investigated the possibility of binding-induced
guest release from the micellar interiors. If the size change
observed in DLS was indeed due to the disassembly of the
micellar aggregate, then it is reasonable to anticipate that
the disassembly event should cause a concomitant release of
guest molecules noncovalently encapsulated within the den-
dritic interiors. For this purpose, Nile red encapsulated solu-
tions of G1–DNP and G2–DNP dendrons were exposed to
the anti-DNP IgG. Indeed, we observed Nile red release
due to protein binding, as evident from the decrease in
emission intensity of the solution (Figure 5). Interestingly,
the guest release exhibited a time-dependent behavior,
which is most likely due to the limited accessibility of the
lipophilic ligand functionalities that are buried inside the
cores of the micellar assemblies. Also, note that we observed
only 50 and 45% of the dye molecules to be released within
the total time frame, for G1–DNP and G2–DNP dendrimers,
respectively. The lack of 100% release could be attributed
to the fact that the dendron–protein complex still presents
hydrophobic functionalities, and provides an opportunity for
lipophilic guest molecules to be bound to the complex. Also,
we were interested in finding if the guest release would be
enhanced if the percentage of free dendrons were decreased
To further examine whether the binding events and the
concurrent dye release are specifically due to the DNP–IgG
interactions, we also synthesized a G1-control[4a] dendron
(Figure 7a). This dendron is structurally similar to G1–DNP,
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S. Thayumanavan et al.
Figure 6. Guest molecule release from: a) G1–DNP, and b) G2–DNP den-
~
^
drons, in the presence of IgG ( ), pepsin (&), cytochrome c ( ) and a-
*
chymotrypsin ( ).
except that it lacks the DNP ligand functionality. When the
IgG was added to the solution of Nile red encapsulated G1-
control dendron (26 mm) we observed no guest release from
this assembly (Figure 7b); this indicates that the release ob-
tained with DNP-containing dendrons was indeed due to
the specific ligand–protein interactions.
Figure 7. a) The structure of G1-control dendron; b) guest release from
&
^
G1-control ( ) versus G1–DNP ( ) after binding IgG.
surface of the dendron and provides the opportunity for
binding with the protein while remaining in the assembly.
We do not have a way of discounting this possibility at this
time. Nonetheless, it is clear that the binding-induced disas-
sembly strategy is not limited to hydrophilic ligands. Dem-
onstration of the protein-sensitive disassembly by using lipo-
philic ligands opens up the possibility of utilizing such mo-
lecular design for a variety of applications, particularly in
drug delivery and sensing.
Conclusion
We have designed and synthesized facially amphiphilic
biaryl dendrimers that contain lipophilic ligand functionali-
ties that are complementary to anti-DNP immunoglobulin G
(IgG). These dendrimers were shown to self-assemble into
micelle-like aggregates in aqueous solution and are capable
of encapsulating lipophilic guest molecules, such as Nile red.
We have shown that these aggregates disassemble in re-
sponse to the ligand–protein interaction. We attribute the
disassembly event to the significant HLB change caused by
the ligand–protein interaction. This disassembly process
clearly suggests that the binding event between a protein
and the ligand-containing dendron does not have to occur in
the self-assembled aggregate stage. In this scenario, it is
likely that the equilibrium between the monomeric state of
the dendron and the micellar aggregate provides a viable
pathway for the binding-induced disassembly process. Alter-
nately, it is also possible that there is conformational flexi-
bility within the building block units in the assembly that
allows for transient exposure of the lipophilic ligand on the
Acknowledgements
We thank the NIGMS of the NIH (GM-065255) for support.
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Received: August 31, 2011
Published online: November 30, 2011
Chem. Eur. J. 2012, 18, 223 – 229
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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