Self-assembling ligands for multivalent nanoscale heparin binding

Article abstract of DOI:10.1002/anie.201100019

Supramolecular string of pearls: Polycationic ligands are designed to self-assemble into spherical pseudo-dendrimers that are capable of binding polyanionic heparin with affinities and binding modes similar to covalent nanostructures such as dendrimers an

Full text of DOI:10.1002/anie.201100019

DOI: 10.1002/anie.201100019
Nanostructures
Self-Assembling Ligands for Multivalent Nanoscale Heparin Binding**
Ana C. Rodrigo, Anna Barnard, James Cooper, and David K. Smith*
Heparin is a highly negatively charged sulfated polysaccha-
ride with a heterogeneous mixture of diverse chains with
different lengths that consist of repeating copolymers of 1–4
linked iduronic acid and glucosamine residues in a semi-
random order (Scheme 1).[1] This polysaccharide regulates a
heparin, and were demonstrated to exhibit a spectroscopic
response to heparin.[6] Cationic units have also been attached
to a foldamer in order to bind heparin;[7] also relatively small
cationic molecules, such as surfen, can bind heparin, with the
mode of binding being predominantly electrostatic.[8] In
elegant work, Anslyn and co-workers reported a heparin
receptor based on a trivalent scaffold that displayed proton-
ated amino acids to bind to the anionic charges of heparin in
combination with boronic acids to interact with the diols of
heparin.[9] A similar combination of noncovalent interactions
was employed by Schrader and co-workers to develop
fluorescent polymeric heparin sensors.[10]
Nanoscale structures, which to some extent mimic the
structure of protamine, have also been reported as heparin
binders. For example, spherical cationic dendrimers, such as
polyamidoamines (PAMAMs) have been of particular inter-
est, because they have similar sizes as protamine and bind
heparin by a similar mechanism, displaying multiple cationic
ligands on their polyvalent scaffolds.[11] In some cases, specific
biomimetic units such as arginine-rich peptides have been
attached to dendritic scaffolds and have been shown to bind
heparin.[12] In recent work mimicking this approach, viruslike
particles have been used for the display of multivalent
cationic ligand arrays for heparin binding.[13] In addition,
calix[8]arene has been used as a scaffold for the organization
of multivalent amines, and it was demonstrated that the
conformational flexibility of the calixarene scaffold plays an
important role in heparin binding—in contrast to protamine,
which is conformationally rigid.[14]
We have an active research programme for the develop-
ment of dendritic molecules capable of binding polyanionic
DNA[15] and have recently reported amphiphilic systems that
self-assemble into micellar spherical “pseudo-dendrimers”
prior to DNA binding.[16] We therefore became interested in
the development of protamine mimics that, instead of being
large covalent structures, were held together by noncovalent
interactions between branched ligands. Although such sys-
tems are well known for binding to DNA, there has only been
one previous report about their exploitation for heparin
formulation.[17] Such systems are synthetically straightfor-
ward, with programmed self-assembly of simple building
blocks being used as the key nanofabrication step (Scheme 1),
and can potentially show high-affinity heparin binding—in
analogy to proteins such as protamine. Herein we report our
initial studies of self-assembling heparin binders that act as
protamine mimetics, and uncover their ability to form
organized nanostructured assemblies in the presence of
heparin.
Scheme 1. Structures of heparin (typical repeat unit) and self-assem-
bling ligand G1.
variety of cellular processes, plays important roles in biolog-
ical systems, and interacts with a wide range of protein
targets.[2] Heparin is widely used as an anti-coagulant during
surgery—however, after surgical intervention, it is necessary
to remove heparin in order to allow clotting to take place.[3]
Protamine is currently the only clinically approved heparin
binder, but its use can cause adverse clinical outcomes.[4]
Protamines are highly positively charged proteins with multi-
ple arginine groups on their surfaces, and as a consequence
they bind heparin through electrostatic ion–ion interactions.[5]
Heparin is a fascinating target for supramolecular chemis-
try, and as a consequence, interest has recently developed in
creating synthetic heparin receptors and sensors. There have
been a number of reports in which fluorescent/colored dyes
were functionalized with cationic units capable of binding
[*] A. C. Rodrigo, A. Barnard, J. Cooper, Prof. D. K. Smith
Department of Chemistry, University of York
Heslington, York, YO10 5DD (UK)
Fax: (+44)1904-322-516
E-mail: david.smith@york.ac.uk
Homepage: http://www.york.ac.uk/chemistry/staff/academic/o-s/
dsmith/
[**] We acknowledge the support of MICINN, Spanish Government for a
PhD fellowship (A.C.R.), BBSRC (A.B.), and The University of York
(J.C.). We thank Meg Stark (Department of Biology, University of
York) and Peiyi Wang (Faculty of Biological Sciences, University of
Leeds) for electron microscopy.
The ligand designed for this project (G1) contains
peripheral amines that are protonated at physiological pH
and are hence capable of electrostatic binding to polyanionic
heparin (Scheme 1). These amines are supported on the
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201100019.
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4675

Communications
Scheme 2. Synthesis of self-assembling dendron G1. p-TsOH: p-toluenesulfonic acid, DCC: N,N’-dicyclohexylcarbodiimide, DMAP: 4-dimethylami-
nopyridine, Boc2O: di-tert-butyl dicarbonate, DIPEA: N,N-diisopropylethylamine, MsCl: methanesulfonyl chloride.
degradable, biocompatible scaffold first introduced by Hult,
Frꢀchet, and co-workers.[18] A hydrophobic unit located at the
focal point of the structure drives the self-assembly of the
ligands into a larger nanoscale architecture in aqueous media
as a consequence of the hydrophobic effect. The synthesis of
G1 was achieved using a modular approach (Scheme 2). The
Frꢀchet dendron scaffold was synthesized using their pre-
viously described methodology;[18d] in an early step, though,
modification of the focal point with propargyl alcohol led to
the incorporation of an alkyne group in this position.[19] The
use of p-nitrophenylchloroformate methodology enabled the
incorporation of the N,N-di-(3-aminopropyl)-N-methylamine
surface ligands, with N-tert-butoxycarbonyl (Boc) protecting
groups being used to control the reaction. In the key step,
Cu(I)-catalyzed “click” chemistry was used to attach 1-
Figure 1. Fluorescence intensity of Nile Red in the presence of increas-
azidodocosane 9, synthesized by previously reported method-
ologies from docosan-1-ol.[20] The click chemistry product 10
was purified by gel permeation chromatography. Finally,
deprotection of the surface ligands by using HCl gas in
methanol gave rise to target ligand G1. Full synthetic methods
and characterization data are given in detail in the Supporting
Information.
To probe self-assembly, we performed a Nile Red assay, in
which the solubilization of the hydrophobic dye, as monitored
by fluorescence spectroscopy, acts as a probe for the minimum
concentration at which self-assembly can take place.[21] The
studies were performed in phosphate buffered saline (PBS) at
pH 7.5. As illustrated in Figure 1, Nile Red solubilization
occurred at concentrations above (3.88 Æ 0.25) mm, which
therefore can be regarded as the critical aggregation concen-
tration (CAC). We are confident that self-assembly occurs
ing amounts of G1; Nile Red is used to detect the self-assembly of G1
and determine the critical aggregation concentration (CAC).
even at relatively low concentrations, and that during the
heparin-binding assay (see below), compound G1 will be
present in self-assembled form. Further evidence for self-
assembly was provided by TEM, with spherical aggregates
being observed when an aqueous sample of compound G1
was allowed to settle on the TEM grid, was negatively stained
with uranyl acetate while wet, and was then allowed to settle
for further 10 min before imaging (Figure 2). The diameters
of the spherical aggregates observed were approximately
(8.5 Æ 1.5) nm.
In order to monitor the heparin-binding ability of com-
pound G1, we chose to employ a previously reported
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ratios that indicate the charge ratio between the receptor and
the heparin required for 50% MB displacement.[23] This ratio
indicates how efficiently the receptor uses each of its positive
charges to bind each negative charge of heparin—the smaller
the value, the more effectively the cationic charge of the
receptor is being used. To calculate + /À ratios, we assumed
four positive charges on G1, four negative charges per repeat
unit of heparin, and twenty four positive charges for the
typical protamine sulfate structure. Finally, we also converted
the data into the form of an “effective dose”; expressed in
terms of the mass of the receptor (drug) required to bind a
certain number (100 international units (IU)) of active units
of heparin, this is the way in which protamine activity would
normally be expressed for clinical administration.[24]
Figure 2. TEM image of compound G1 dried from aqueous solution,
indicative of spherical self-assembled nanostructures.
As indicated in Table 1, G1 shows effective binding of
heparin with an EC50 value of (102 Æ 3) mm. This value
corresponds to a + /À ratio of (1.41 Æ 0.05). This result
indicates that more than one positive charge on the receptor is
required for each negative charge of heparin and hence it is
likely that not all positive charges are directly involved in the
binding of heparin. The effective dose of G1 was calculated to
be (0.48 Æ 0.01) mg/100IU heparin. Under the same assay
conditions, protamine had an EC50 value of (20 Æ 1) mm. This
value is lower than that observed for G1, but it must be
remembered that protamine has a significantly higher mass
than G1 and thus the apparent EC50 is artificially improved.
The + /À ratio leads to a better comparison of the receptors,
and for protamine, the obtained value is (1.64 Æ 0.11), thus
indicating that protamine uses its positive charge a little less
efficiently than compound G1. Furthermore, the dose of
protamine required for 50% MB displacement is (0.46 Æ
0.02) mg/100IU heparin, which is very similar to the effective
dose of G1 under these conditions.
The binding assays were then repeated in the presence of
5 mm aqueous NaCl in order to determine the effect of
electrolytes on heparin binding. Given that receptor–heparin
interactions are predominantly based on electrostatics, it
might be expected that increasing the ionic strength would
decrease the binding affinity. However, Table 2 indicates that
in this assay, both G1 and protamine appear to bind heparin
more efficiently. It must be remembered that this assay is a
competitive one, and we can therefore conclude that the
binding between MB and heparin is more adversely affected
by salt than the interaction between the receptors and
heparin. Evidence for this hypothesis is provided by the
observation that with NaCl concentrations above 5 mm,
binding of the probe dye molecule to heparin was no longer
observed. Comparison of the data for G1 and protamine,
methylene blue (MB) displacement assay.[22] As a cationic
dye, MB forms moderate strength electrostatic interactions
with heparin, and importantly, when bound to heparin, the
UV/Vis spectrum of the dye changes significantly—unbound
MB has a lmax at 664 nm, whereas MB bound to heparin has a
lmax at 568 nm. The addition of a more strongly binding ligand
to a mixture of heparin and MB displaces the MB from
heparin, and consequently leads to changes in the UV/Vis
spectrum of the dye. Hence, in this competition assay, UV/Vis
spectroscopy is capable of indirectly reporting on the
interactions between synthetic receptors and heparin.
The experiment was performed using the optimized
conditions (MB (10 mm) and heparin (72.5 mm) in tris(hydrox-
ymethyl)aminomethane hydrochloride (Tris HCl; 1 mm)).
Heparin concentrations were calculated assuming the struc-
ture shown in Scheme 1, with an apparent “molecular” mass
(Mr) of 605.04 per repeat unit. For comparison, the novel
compound G1 and protamine were both assayed for their
abilities to bind heparin. Protamines are not available in
monodisperse form, because they are mixtures of poly-
arginylated peptides; however, the following typical prot-
amine structure was assumed for purposes of calculation:
NH-Pro-Arg4-Ser-Arg-Pro-Val-Arg5-Pro-Arg2-Pro-Arg2-Val-
Ser-Arg6-Gly-Arg4-COOH. Compound G1 (or protamine)
was added in increasing aliquots to the heparin–MB complex
and normalization of the UV/Vis data against unbound MB
allowed us to calculate the concentration at which 50% of the
MB was displaced from its complex with heparin.
Table 1 shows the effective concentration at which 50%
displacement was achieved (EC50 value). It becomes apparent
that the lower the concentration, the more effective the
binding. In addition, we have converted the data into + /À
Table 1: Data from methylene blue (MB) displacement assay.[a]
Table 2: Data from methylene blue (MB) displacement assay.[a]
Receptor
EC50 [mm][b]
+/À Ratio[c]
Effective Dose[d]
Receptor
EC50 [mm][b]
+/À Ratio[c]
Effective Dose[d]
G1
Protamine
(102Æ3)
(20Æ1)
(1.41Æ0.05)
(0.48Æ0.01)
(0.46Æ0.02)
G1
Protamine
(47Æ8)
(14Æ2)
(0.65Æ0.10)
(0.23Æ0.03)
(0.34Æ0.05)
(1.64Æ0.11)
(1.16Æ0.15)
[a] Performed in 1 mm Tris-HCl. [b] EC50 is the concentration required for
50% displacement of MB from its complex with heparin. [c] The +/À
ratio represents the receptor/heparin charge ratio required for 50%
displacement. [d] The effective dose is reported in mg of receptor per
100 IU heparin.
[a] Performed in 1 mm Tris-HCl in the presence of 5 mm NaCl. [b] EC50 is
the concentration required for 50% displacement of MB from its
complex with heparin. [c] The +/À ratio represents the receptor/heparin
charge ratio required for 50% displacement. [d] The effective dose is
reported in mg of receptor per 100 IU heparin.
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however, would indicate that G1 becomes a significantly more
effective heparin binder than protamine in the presence of
electrolyte. Indeed, G1 operates at an effective dose of
(0.23 Æ 0.03) mg/100IU heparin, which is significantly lower
than protamine, for which the dose is (0.34 Æ 0.05) mg/100IU.
Thus, the ability of protamine to compete against MB
increases by a factor of 1.5, while the ability of G1 to compete
with MB more than doubles. This result indicates that salt is
less capable of screening the effective charge of the G1
assemblies, which therefore exhibit better multivalent binding
to heparin.[15g]
In a final experiment, we used TEM to probe the
interaction between G1 and heparin in water (Figure 3). We
observed micellar aggregates on the TEM grid, which
Figure 4. Schematic showing the organization of self-assembled
micelles formed by G1 in the presence of heparin.
effectively behave like pseudo-dendrimers when binding to
heparin, with the individual ligands being combined non-
covalently to give a spherical aggregate. This architecture is
an intriguing form of nano-orientated soft matter, in which
the interactions between a biological polymer and a self-
assembled soft nanostructure are able to control the eventual
nanoscale morphology (Figure 4).
In summary, we can conclude that G1 self-assembles into
a polycationic spherical form that is capable of binding
polyanionic heparin in a multivalent manner. These self-
assembled architectures mimic the behavior of covalently
constructed proteins such as protamine and covalent spherical
macromolecules such as dendrimers in terms of binding
affinity and mode of binding. TEM indicates that binding to
heparin induces nanoscale organization; such simple materi-
als may have potential biomedical applications. In future
work, the ability of these self-assemblies to intervene in
biological processes will be studied, as will higher-generation
dendritic systems and the potential of these systems to
degrade and disassemble, thus leading to controlled heparin
binding and release.
Figure 3. TEM image of self-assembled spherical G1 nanostructures in
the presence of heparin, leading to linear organization of the nano-
structures.
correspond to aggregates of G1. Indeed, the size of these
aggregates was approximately (6.5 Æ 1.0) nm, which is rea-
sonably consistent with the observations in the absence of
heparin (Figure 2). However, remarkably, these micellar
aggregates, unlike those observed in the absence of heparin,
were aligned in an organized manner within domains on the
TEM grid. The micelles appear to be arranged in closely
packed linear assemblies. This heparin-induced nanoscale
organization of micelles was further confirmed by cryo-TEM
methods (see the Supporting Information). We propose that
heparin, as a relatively inflexible polymer, organizes the
micellar aggregates along its backbone as a consequence of
electrostatic interactions (Figure 4). This mode of binding has
often been observed for the binding of spherical cationic
systems to DNA, and is commonly referred to as “beads on a
string”.[25] This model helps explain, why more than one
positive charge on the G1 aggregate is required to effectively
neutralize each anionic charge on the heparin (Table 1), since
some of the positive charges on the micellar structures will be
unable to make effective contact with the linear heparin
polymer and as such will not be useful for the neutralization
of the overall heparin charge. This mode of binding has also
previously been suggested for spherical covalently bound
dendritic macromolecules that bind to heparin,[11c] and the
experimental evidence presented herein therefore clearly
illustrates that our self-assembled nanoscale architectures
Received: January 3, 2011
Revised: March 10, 2011
Published online: April 19, 2011
Keywords: heparin · ligands · nanostructures · self-assembly ·
.
supramolecular chemistry
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