Comparing dendritic and self-assembly strategies to multivalency - RGD peptide-integrin interactions

Article abstract of DOI:10.1039/c1ob05241a

This paper compares covalent and non-covalent approaches for the organisation of ligand arrays to bind integrins. In the covalent strategy, linear RGD peptides are conjugated to first and second generation dendrons, and using a fluorescence polarisation c

Full text of DOI:10.1039/c1ob05241a

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Organic &
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PAPER
Comparing dendritic and self-assembly strategies to multivalency—RGD
peptide–integrin interactions†
Daniel J. Welsh and David K. Smith*
Received 15th February 2011, Accepted 29th March 2011
DOI: 10.1039/c1ob05241a
This paper compares covalent and non-covalent approaches for the organisation of ligand arrays to
bind integrins. In the covalent strategy, linear RGD peptides are conjugated to first and second
generation dendrons, and using a fluorescence polarisation competition assay, the first generation
compound is demonstrated to show the most effective integrin binding, with an EC50 of 125 mM
(375 mM per peptide unit). As such, this dendritic compound is significantly more effective than a
monovalent ligand, which does not bind integrin, even at concentrations as high as 1 mM. However, the
second generation compound is significantly less effective, demonstrating that there is an optimum
ligand density for multivalency in this case. In the non-covalent approach to multivalency, the same
RGD peptide is functionalised with a hydrophobic C12 chain, giving rise to a lipopeptide which is
demonstrated to be capable of self-assembly. This lipopeptide is capable of effective integrin binding at
concentrations of 200 mM. These results therefore demonstrate that covalent (dendritic) and
non-covalent (micellar self-assembly) approaches have, in this case, comparable efficiency in terms of
achieving multivalent organisation of a ligand array.
Introduction
Multivalency, in which moieties with multiple binding ligands are
employed, can be a powerful mechanism for enhancing the binding
of synthetic ligands to biological targets.1 Multivalent ligands
exhibit enhanced binding through (i) a local concentration effect
(in which once a ligand dissociates from its binding partner, there
is a high local concentration to favour ligand rebinding) and/or (ii)
a primarily entropic effect, which after the initial binding event,
favours the binding of a second ligand to a second binding site
on the biological binding partner (Fig. 1). As such, the strategy
is widely employed for the binding of biomolecules with multiple
Fig. 1 Origins of multivalency in biomolecular recognition.
potential binding sites, such as glycoproteins and DNA.2
Integrins are heterodimeric, transmembrane proteins whichplay
As such, understanding the binding of peptides to integrin targets
important roles in cell adhesion and signalling.3 They have been
in detail is an important target in studies of molecular recognition.
of interest in tissue engineering applications,4 and furthermore,
On initial inspection, integrins may appear to be unpromising
given that some integrins are over-expressed on specific cancer
targets for multivalent binding, as they only possess a single
cells, they are an important target for anti-cancer treatments.5 In
ligand binding site. However, in biological systems, integrins are
key work, the tri-peptide Arg-Gly-Asp (RGD) was shown to bind
often clustered in cell membranes. As such, multivalent binding of
to integrins.6 ‘Cilengitide’, a cyclic RGD peptide with enhanced
multiple integrins is a genuine possibility.9 To enhance the binding
integrin binding affinity, is currently under development as an anti-
of RGD peptides to integrins, there has therefore been interest in
angiogenic agent,7 and the X-ray structure of this molecule bound
using multivalent peptide arrays.10
Dendritic molecules,11 which have an inherently branched
to the extracellular segment of integrin avb3, has been published.8
architecture offer a versatile approach for organising covalent
multivalent ligand arrays.12 Pre-formed dendrimers have had
RGD-containing peptides conjugated to their surfaces, and it has
Department of Chemistry, University of York, Heslington, York, YO10 5DD,
UK. E-mail: david.smith@york.ac.uk; Fax: +44 1904 324516
† Electronic supplementary information (ESI) available: Synthetic
been demonstrated that their biological activities in a number
schemes, experimental methodologies and full compound characterisation,
of assays appear to be enhanced, presumably by multivalency.13
methods for Nile Red and Fluorescence Polarisation assays, and TEM
images. See DOI: 10.1039/c1ob05241a
However, it is worth noting that in general, the fundamental
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nature of the interaction between the RGD peptide and the
integrin protein has rarely been explored—with a greater focus on
biological outcome. Liskamp and co-workers recently reported a
dendritic RGD derivative, and carefully assayed its ability to bind
integrin using a competition assay,14 however, the use of cyclic
RGDs led to all compounds showing very strong binding, and
little evidence of multivalent binding. Dumy and co-workers, have
developed template-supported multivalent RGD arrays, and used
cell adhesion assays, to show that multivalency is important when
integrins are supported in cell membranes.15
An alternative approach to multivalency employs self-assembly
to spontaneously organise binding units into a multivalent array.
RGD-containing lipopeptides have been demonstrated to insert
into membranes which gain enhanced affinity for integrins,16 and
show improved cell internalisation,17 clustering at the integrin
binding part of the membrane.18 RGD-functionalised lipopeptides
have also been co-formulated with other active units to generate
assemblies capable of enhanced cellular gene delivery.19 Cyclic
RGDs have also been coupled to the surfaces of liposomes,
which have then exhibited enhanced cellular uptake.20 There
have also been studies on the ability of RGD functionalised
lipids to self-assemble in their own right.21 For example, Lee
and co-workers have demonstrated that lipids functionalised with
cyclic RGD self-assemble into nano-ribbons, and can deliver
hydrophobic molecules into cells.22 A number of groups, including
those of Stupp and Ulijn, have demonstrated that incorporating
RGD peptides into self-assembling soft materials can enhance
tissue engineering.23 It is, however, unclear whether self-assembly
enhances the RGD–integrin interaction in thermodynamic terms,
or whether it is simply a useful materials formulation tool.
There has recently been increasing interest in understanding in
fundamental terms how self-assembly can enhance multivalency,
for example in interactions with glycoproteins,24 collagen25 and
DNA,26 but such effects have not really been probed for integrin–
RGD interactions.
The goal of this paper was not to develop high-affinity or
highly selective integrin binding systems— there are a large
number of those already—but rather, to gain insight into different
ways in which ligands can be organised for multivalent binding.
We therefore chose to use linear RGD peptides for ligand
construction. Although they have relatively low affinity binding
and poor selectivity these compounds can be easily synthesised
in multi-gram quantities using straightforward solution-phase
peptide chemistry, and because their integrin binding is less
highly optimised and has relatively low affinity (mM), multivalent
binding effects may be more significant. In this paper, we therefore
report a simple comparison between different RGD peptides.
Monomeric PEG-RGD is compared with first generation dendritic
system G1-RGD3 having three RGD peptide surface groups, and
second generation dendron G2-RGD9 having nine (Fig. 2). This
provides an insight into the effect of covalent multimerisation on
integrin binding. We then compared hydrophilic PEG-RGD with
amphiphilic C12-RGD. The latter compound self-assembles and
can therefore provide insight into the effect of self-assembly on
multivalent ligand organisation. Using this approach, we hoped
to develop an enhanced understanding of the way in which ligands
can be organised to achieve multivalent binding.
Results and discussion
Synthesis and characterisation
The synthesis and characterisation data for all compounds are
provided in the Experimental section and/or ESI.† Dendron
G1-RGD3 was constructed (Scheme 1) from a first generation
‘Newkome-type’ amide/ether scaffold protected at the focal point
Fig. 2 Compounds investigated in this paper.
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Scheme 1 Synthesis of G1-RGD3.
with a benzyl carbamate group, synthesised using previously
published methodology.27 The three peripheral carboxylic acid
groups were then conjugated to the N-terminus of a protected
linear RGD peptide (synthesised via solution-phase peptide cou-
pling methodology—see ESI†) using propylphosphonic anhydride
(T3P) as a coupling agent. Finally, removal of the RGD-peptide
protecting groups using trifluoroacetic acid (TFA) and triiso-
propylsilane (TIS) gave rise to the target compound, G1-RGD3.
Second generation dendritic compound G2-RGD9 was synthesised
in an analogous manner.
Monovalent control, PEG-RGD, and lipopeptide C12-RGD
were constructed by simple conjugation of the N-terminus of
the protected RGD peptide with the appropriate carboxylic acids
mediated by propylphosphionic acid (T3P) and O-(benzotriazol-
1-yl)-N,N,N¢,N¢-tetramethyluronium tetrafluoroborate (TBTU)
respectively, followed by deprotection of the RGD-peptide
(Scheme 2). Negative control, PEG-GGG was synthesised via anal-
ogous solid-phase peptide and protecting group methodologies
(see ESI†). All target compounds were synthesised in good yield,
and fully characterised.
Fig. 3 A) Nile Red fluorescence emission spectra in the presence
of increasing concentrations of C12-RGD. B) Fluorescence emission
intensities of Nile Red at 635 nm plotted against log[C12-RGD] in order
to determine the Critical Aggregation Concentration (CAC). Excitation
wavelength: 550 nm.
G1-RGD3 and G2-RGD9 showed no evidence of self-assembly at
concentrations <1 mM. Conversely, C12-RGD showed evidence
of self-assembly, with the CAC being calculated as ca. 300 mM in
aqueous phosphate buffered saline (PBS) (pH 7.4).
Transmission electron microscopy (TEM) was then per-
formed on C12-RGD dried from aqueous solution in order
to gain some insight into the morphology of the aggregates.
This revealed that at high concentration (1 mM) small (10–
50 nm diameter) spherical micelles were formed in TRIS
(tris(hydroxymethyl)aminomethane) and PBS buffers. On dilution
to just above the CAC (400 mM), however, larger lamellar
aggregates were observed (see ESI† for TEM images). These
experiments clearly demonstrate that self-assembly of C12-RGD
is taking place.
Scheme 2 Synthesis of C12-RGD and PEG-RGD.
Integrin binding affinity
Self-assembly of RGD peptides
We then probed the ability of these peptides to bind integrin avb3.
To monitor the binding, we employed a competitive fluorescence
polarisation (FP) assay, first established by Li et al.29 This
assay uses the fluorescent probe 5(6)-FL-c[RGDfK] based on a
cyclic RGD peptide30 (Fig. 4, synthesised according to literature
methods—see ESI†). This probe is bound to the integrin, and the
ligand of interest is then titrated into the solution. If binding
occurs between the ligand and the integrin, the fluorescent
probe is displaced, and the FP signal decreases in intensity as
the probe mobility increases. Determining the concentration of
Initially, we monitored whether the RGD peptides aggregated
in aqueous solution, using solubilisation experiments with the
hydrophobic dye Nile Red.28 If self-assembled architectures are
present, Nile Red will be solubilised into the hydrophobic domain
of the aggregates. Varying the concentration of the self-assembling
peptide will lead to increasing levels of dye solubilisation (Fig. 3A)
Plotting the fluorescence emission intensity of Nile Red at 635 nm
versus log[peptide] (Fig. 3B) then allows the determination of crit-
ical aggregation concentrations (CACs). As expected, PEG-RGD,
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Fig.
4
Structure of cyclic RGD-peptide fluorescent probe
5(6)-FL-c[RGDfK].
ligand required for the apparent 50% displacement of the probe
from the integrin binding site yields an EC50 value (effective
concentration) for each ligand of interest. Although there has
been some discussion about the advantages and disadvantages of
fluorescence polarisation assays,31 this approach remains a power-
ful one for comparing related families of ligands and their relative
affinities to bind to the same biological target under equivalent
conditions, and as such, allows us to gain some insight into the
comparative affinities of our family of linear RGD peptides for
integrin avb3.
Fig.
5
Normalised titration curves for the displacement of
5(6)-FL-c[RGDfK] probe (10 nM) from integrin avb3 (280 nM) on the
addition of the synthetic ligands: G1-RGD3 (blue), G2-RGD9 (red),
PEG-RGD (green) and PEG-GGG (purple) after incubating at 29 ◦C for
5 min.‡§
We then investigated G2-RGD9 binding to integrin avb3 using
the same approach. On adding this second generation dendron
to the integrin, there was initially a decrease in the FP signal
as the fluorescent probe was displaced from the RGD binding
site—however, this did not occur at such low concentrations as for
G1-RGD3 (Fig. 5). This indicates that the first generation system is
better able to bind to integrin than the second generation analogue.
Similar effects have previously been reported for saccharide
binding to glycoproteins, where increased steric hindrance at
higher generations can limit the ability of the surface ligands
to effectively bind to their biological target.2a We can therefore
conclude that the first generation system is, in this case, optimal
for integrin binding and we suggest that the surface rigidity and
steric hindrance of the higher generation dendrimer acts to limit
its integrin affinity.
The data above demonstrate the advantage of a dendritic
approach to multivalency, and would suggest it is playing a
significant role in this binding event. At first sight this is surprising,
because integrin only has a single binding site, and the mechanism
by which multivalency may operate is not completely clear.
However, in this assay, the solution-phase integrin is not strictly
‘free’ protein. As a membrane-bound protein, integrin avb3 is
supplied stabilised in Triton X-100 surfactant. This was the form
of integrin used in the assays here. Fig. 6 shows the TEM image of
the Triton X-100-integrin assemblies prior to the addition of G1-
RGD3. This formulation of integrin protein is somewhat similar
to the situation within a cell membrane. As such, we propose
that the dispersion of integrin in the surfactant phase allows
the significant enhancement of binding on the application of a
multivalent, dendritic ligand.
Initially, the binding of fluorescent probe 5(6)-FL-c[RGDfK]
to integrin avb3 was assessed via FP titration methods and,
in line with the literature,29 as the integrin was added to the
probe (10 nM), the FP signal increased from ca. 35 mP (milli
polarisation units) in the absence of integrin to over 100 mP when
the concentration of integrin was >400 nM. The binding process
was kinetically fast and incubating the samples for longer than
5 min did not have any significant effect on the FP signal.
We then performed competition assays using increasing con-
centrations of our RGD peptides against fixed concentrations
of fluorescent probe (10 nM) and integrin (280 nM). In the
first instance we compared the binding of PEG-RGD, G1-RGD3
and G2-RGD9 in order to investigate whether the covalent den-
dritic strategy to multivalency yielded enhanced integrin binding.
Monovalent ligand PEG-RGD only reduced the normalised FP
signal to ca. 70% of its initial value, and was unable to reduce
the value below 50% (Fig. 5) even at very high concentrations
(ca. 1 mM). As expected, monovalent linear RGD only has weak
affinity for integrin,32 and is therefore unable to fully displace the
strongly binding cyclic RGD fluorescent probe from the protein.
Importantly, PEG-GGG, the negative control, did not displace
5(6)-FL-c[RGDfK] from its integrin binding site at all, with the
FP signal remaining unchanged even at the maximum tested
concentration. This demonstrates that the RGD peptide in PEG-
RGD is directly responsible for the decrease in FP signal, which
can therefore be attributed to weak integrin binding. Compound
G1-RGD3, however, showed enhanced integrin binding (Fig. 5),
reducing the normalised FP signal to <50% of its initial value
at a concentration of ca. 125 mM. Compared with cyclic RGD
peptides, this is still weak binding, but the enhancement in contrast
with the data from PEG-RGD clearly indicates the advantage of a
multivalent strategy. On a per-RGD-peptide basis, the reduction
of FP signal to below 50% of its initial value occurs at 375 mM
for G1-RGD3—significantly below the concentrations assayed for
monovalent PEG-RGD.
However, it should also be noted that on further addition of
all ligands, the FP signal began to rapidly increase in intensity
(Fig. 7). This was particularly notable for G2-RGD9, where the
‡ Fluorescence polarisation data were normalised to 100 mP units in order
to enable comparability between different competition experiments.
§ Data are presented as mean values standard deviations from at least 5
independent scans.
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synthetically more demanding construction of a covalent dendritic
ligand array, such as G1-RGD3. For C12-RGD, the normalised
FP signal was reduced below 50% of its initial value when the
concentration of C12-RGD was increased to ca. 200 mM (Fig. 8).
This makes monovalent C12-RGD a significantly more effective
ligand in comparative terms than the control monovalent ligand
PEG-RGD—a potential multivalent effect. It is interesting to note
that per RGD unit, C12-RGD (200 mM) is a more effective integrin
binder than G1-RGD3 (375 mM). This demonstrates that the self-
assembled system uses its peptide units more effectively in binding
to the integrin than the covalently structured dendritic array. It is
interesting to speculate that the reversible nature of self-assembly
generates ligand arrays with a greater degree of flexibility, and
potentially a better ability to satisfy the binding requirements
of the integrin. This is certainly true when comparing self-
assembling C12-RGD with second generation G2-RGD9 where
the self-assembly approach yields significantly enhanced integrin
binding—perhaps as a result of the better ability of self-assembled
ligands to recombine and better adapt to the binding site of
integrin.
Fig. 6 TEM image of integrin-Triton X-100 assemblies prior to addition
of RGD peptide ligands—scale bar = 500 nm.
Fig. 7 Normalised titration curves at elevated concentrations for the
displacement of 5(6)-FL-c[RGDfK] probe (10 nM) from integrin avb3
(280 nM) on the addition of the synthetic ligands: G1-RGD3 (blue),
G2-RGD9 (red) and PEG-RGD (green) after incubating at 29 ◦C for
5 min.‡§
Fig.
8 Normalised titration curves for the displacement of
5(6)-FL-c[RGDfK] probe (10 nM) from integrin avb3 (280 nM) on the
addition of the synthetic ligands. C12-RGD (orange), PEG-RGD (green)
and SDS (light blue) after incubating at 29 ◦C for 5 min.‡§
increase in FP intensity started at 250 mM, but was also the
case for G1-RGD3, where the increase started at 800 mM and
PEG-RGD, where the increase started at ca. 2 mM. It should
be noted that these linear RGD ligands are being applied at
relatively high micromolar concentrations in this assay—owing to
their low binding affinities for integrins. We therefore propose that
this increase in FP signal is caused by a non-specific interaction
between the RGD ligands and the integrin target. This could be
mediated by terminal carboxyl group on each of the ligands,
leading to non-specific binding (e.g. integrin surface binding)
and subsequent aggregation/precipitation, hence explaining the
increase in apparent FP intensity. This argument is supported
by the observation that the onset of this process occurs at
a concentration of 250 mM for G2-RGD9—an effective ligand
concentration of 2.25 mM (250 mM ¥ 9), for G1-RGD3 the effective
ligand concentration is 2.4 mM (800 mM ¥ 3) and for PEG-RGD
the effect is also observed at an effective ligand concentration of
ca. 2 mM.
We suggest that binding to integrin may actually encourage the
surfactant-like peptide to aggregate, even below its CAC. It is also
plausible that C12-RGD may itself insert into the Triton X-100
assemblies and that the overall combined multivalent aggregate
then binds to integrin proteins in adjacent assemblies. TEM
imaging indicates that the Triton–integrin assemblies do change
morphology somewhat in the presence of C12-RGD, in support
of this latter hypothesis (see ESI†).
We were concerned that C12-RGD may be affecting the FP
signal by interfering with the Triton X-100 surfactant assemblies,
rather than forming specific interactions with integrin. We there-
fore tested the interaction of a control mono-anionic surfactant,
which also contained a twelve carbon atom hydrophobic chain,
sodiumdodecylsulfate (SDS), with the integrin. Detergent SDS
showed no effect at all on the FP assay (Fig. 8), unambiguously
demonstrating that the effect of C12-RGD can be attributed
to specific interactions between the RGD peptide unit of the
lipopeptide and the integrin protein, rather than the disruptive
surfactant-like nature of the compound.
We then investigated the potential of C12-RGD to bind to
integrin, and assess whether self-assembling a multivalent array
of RGD ligands is a plausible replacement strategy for the
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5 (a) P. C. Brooks, R. A. Clark and D. A. Cheresh, Science, 1994, 264,
569–571; (b) P. C. Brooks, A. M. P. Montgomery, M. Rosenfeld, R.
A. Reisfeld, T. Hu, G. Klier and D. A. Cheresh, Cell, 1994, 79, 1157–
1164; (c) C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides and D.
E. Ingber, Science, 1997, 276, 1425–1428; (d) W. Arap, R. Pasqualini
and E. Ruoslahti, Science, 1998, 279, 377–380; (e) K. E. Gottschalk
and H. Kessler, Angew. Chem., Int. Ed., 2002, 41, 3767–3774; (f) K. N.
Sugahara, T. Teesalu, P. P. Karmali, V. R. Kotamraju, L. Agemy, D. R.
Greenwald and E. Ruoslahti, Science, 2010, 328, 1031–1035.
6 M. D. Pierschbacher and E. Ruoslahti, Nature, 1984, 309, 30–33.
7 (a) M. A. Dechantsreiter, E. Planker, B. Matha, E. Lohof, G.
Holzemann, A. Jonczyk, S. L. Goodman and H. Kessler, J. Med.
Chem., 1999, 42, 3033–3040; (b) G. Tabatabai, M. Weller, B. Nabors,
M. Picard, D. Reardon, T. Mikkelsen, C. Ruegg and R. Stupp, Targeted
Oncol., 2010, 5, 175–181 .
8 J.-P. Xiong, T. Stehle, R. Zhang, A. Joachimiak, M. Frech, S. L.
Goodman and M. A. Arnaout, Science, 2002, 296, 151–155.
9 (a) L. J. Kornberg, H. S. Earp, C. E. Turner, C. Prockop and L.
Juliano, Proc. Natl. Acad. Sci. U. S. A., 1991, 88, 8392–8396; (b) S.
Miyamoto, S. K. Akiyama and K. M. Yamada, Science, 1995, 267,
883–885; (c) G. Maheshwari, G. Brown, D. A. Lauffenburger, A. Wells
and L. J. Griffith, J. Cell Science, 2000, 113, 1677–1686; (d) D. C. Worth
and M. Parsons, Int. J. Biochem. Cell Biol., 2008, 40, 2397–2409.
10 L. Auzzas, F. Zanardi, L. Battistini, P. Burreddu, P. Carta, G. Rassu,
C. Curti and G. Casiraghi, Curr. Med. Chem., 2010, 17, 1255–1299.
11 (a) F. Vo¨gtle, G. Reichardt, N. Werner, and A. J. Rackstraw, Dendrimer
Chemistry -Concepts, Syntheses, Properties, Applications, Wiley-VCH,
Weinheim, 2009; (b) G. R. Newkome, and C. N. Moorefield, Handbook
of Dendrimers: Synthesis, Nanoscience and Applications, Wiley-VCH,
Weinheim, 2009; (c) D. A. Tomalia, Prog. Polym. Sci., 2005, 30, 294–
324.
Conclusions
In summary, this paper reports the synthesis of dendritic RGD
peptides, an RGD lipopeptide and positive and negative control
peptides. Interestingly, the dendritic approach appeared to give op-
timal binding at the first generation, with the more highly branched
second generation system having lower affinity recognition as well
as a greater degree of non-specific ligand–protein interaction.
Endowing the RGD unit with a hydrophobic chain encourages
lipopeptide self-assembly and appears to enhance integrin binding.
Notably, the self-assembled approach is comparable to, and
competitive with the use of a first generation dendritic scaffold
to organise a multivalent ligand array (Fig. 9).
Fig. 9 Comparison of dendritic and self-assembled approaches to
multivalency.
12 (a) O. Rolland, C.-O. Turrin, A.-M. Caminade and J.-P. Marjoral,
New J. Chem., 2009, 33, 1809–1824; (b) U. Boas, J. B. Christensen,
and P. M. H. Heegaard, Dendrimers in Medicine and Biotechnology,
Royal Society of Chemistry, Cambridge, 2006; (c) B. Klajnert, and
M. Bryszewska, Dendrimers in Medicine, Nova Science, UK, 2007;
(d) I. J. Majoros, and J. R. Baker, Dendrimer-Based Nanomedicine, Pan
Stanford Publishing,Singapore, 2008.
13 (a) S. Monaghan, D. Griffith-Johnson, I. Matthews and M. Bradley,
Arkivoc, 2001, 2 Part 8; (b) R. Shukla, T. P. Thomas, J. Peters, A. Kotlyar,
A. Myc and J. R. Baker, Chem. Commun., 2005, 5739–5741; (c) H. Yang
and W. J. Kao, Int. J. Med., 2007, 2, 89–99; (d) E. Hill, R. Shukla, S. S.
Park and J. R. Baker, Bioconjugate Chem., 2007, 18, 1756–1762; (e) C.
A. Boswell, P. K. Eck, C. A. S. Regino, M. Bernado, K. J. Wong, D.
E. Milenic, P. L. Choyke and M. W. Brechbiel, Mol. Pharmaceutics,
2008, 5, 527–539; (f) T. L. Kaneshiro and Z.-R. Lu, Biomaterials, 2009,
30, 5660–5666; (g) C. L. Waite and C. M. Roth, Bioconjugate Chem.,
2009, 20, 1908–1916; (h) Z. Li, P. Huang, X. Zhang, J. Lin, S. Yang, B.
Liu, F. Gao, P. Xi, Q. Ren and D. Cui, Mol. Pharmaceutics, 2010, 7,
94–104; (i) D. Q. McNerny, J. F. Kukowska-Latallo, D. G. Mullen, J.
M. Wallace, A. M. Desai, R. Shukla, B. Huang, M. M. B. Holl and J.
R. Baker, Bioconjugate Chem., 2009, 20, 1853–1859.
As such, the data in this paper indicate that both dendritic
(covalent), and self-assembly (non-covalent) strategies to multiva-
lency can be used in similar ways to enhance the binding of RGD
peptides to integrins, although in this case, the self-assembled
approach appears to give rise to slightly higher affinity integrin
binding. In further work, by modifying the peptide (e.g. RGDS), or
changing the lipophilic group to enhance self-assembly, we believe
that significantly enhanced binding affinities will be achieved, and
alternative nanostructures with higher integrin affinities will be
achieved.
Acknowledgements
We thank the EPSRC and the University of York for funding this
research through the DTA scheme.
14 (a) I. Dijkgraaf, A. Y. Rijnders, A. Soede, A. C. Dechesne, G. W. van
Esse, A. J. Brouwer, F. H. M. Corstens, O. C. Boerman, D. T. S. Rijkers
and R. M. J. Liskamp, Org. Biomol. Chem., 2007, 5, 935–944; (b) C.-B.
Yim, I. Dijkgraaf, R. Merkx, C. Versluis, A. Eek, G. E. Mulder, D. T.
S. Rijkers, O. C. Boerman and R. M. J. Liskamp, J. Med. Chem., 2010,
53, 3944–3953.
15 (a) D. Boturyn, J. L. Coll, E. Garanger, M. C. Favrot and P. Dumy, J.
Am. Chem. Soc., 2004, 126, 5730–5739; (b) E. Garanger, D. Boturyn, J.
L. Coll, M. C. Favrot and P. Dumy, Org. Biomol. Chem., 2006, 4, 1958–
1965; (c) Z.-H. Jin, V. Josserand, S. Foillard, D. Boturyn, P. Dumy,
M.-C. Favrot and J.-L. Coll, Mol. Cancer, 2007, 6, 41; (d) L. Sancey,
E. Garanger, S. Foillard, G. Schoehn, A. Hurbin, C. Albiges-Rizo, D.
Boturyn, C. Souchier, A. Grichine, P. Dumy and J.-L. Coll, Mol. Ther.,
2009, 17, 837–843.
16 (a) B. Hu, D. Finsinger, K. Peter, Z. Guttenberg, M. Barmann, H.
Kessler, A. Escherich, L. Moroder, J. Bohm, W. Baumeister, S. Sui and
E. Sackmann, Biochemistry, 2000, 39, 12284–12294; (b) V. Marchi-
Artzner, B. Lorz, U. Hellerer, M. Kantlehner, H. Kessler and E.
Sackmann, Chem.–Eur. J., 2001, 7, 1095–1101.
17 S. Cressman, I. Dobson, J. B. Lee, Y. Y. C. Tam and P. R. Cullis,
Bioconjugate Chem., 2009, 20, 1404–1411.
Notes and references
1 (a) M. Mammen, S.-K. Choi and G. M. Whitesides, Angew. Chem.,
Int. Ed., 1998, 37, 2754–2794; (b) A. Mulder, J. Huskens and D. N.
Reinhoudt, Org. Biomol. Chem., 2004, 2, 3409–3424; (c) J. D. Badjic,
A. Nelson, S. J. Cantrill, W. B. Turnbull and J. F. Stoddart, Acc. Chem.
Res., 2005, 38, 723–732; (d) J. Huskens, Curr. Opin. Chem. Biol., 2006,
10, 537–543; (e) V. Martos, P. Castreno, J. Valero and J. de Mendoza,
Curr. Opin. Chem. Biol., 2008, 12, 698–706.
2 (a) Y. M. Chabre and R. Roy, Curr. Top. Med. Chem., 2008, 8, 1237–
1285; (b) N. Jayaraman, Chem. Soc. Rev., 2009, 38m, 3463–3483; (c) M.
A. Kostiainen, J. G. Hardy and D. K. Smith, Angew. Chem., Int. Ed.,
2005, 44, 2556–2559; (d) G. M. Pavan, A. Danani, S. Pricl and D. K.
Smith, J. Am. Chem. Soc., 2009, 131, 9686–9694; (e) S. Jones, G. M.
Pavan, A. Danani, S. Pricl and D. K. Smith, Chem.–Eur. J., 2010, 16,
4519–4532.
3 (a) R. O. Hynes, Cell, 1992, 69, 11–25; (b) O. Cleaver and D. A. Melton,
Nat. Med., 2003, 9, 661–668; (c) E. F. Plow, T. A. Haas, L. Zhang, J.
Loftus and J. W. Smith, J. Biol. Chem., 2000, 275, 21785–21788; (d) R.
O. Hynes, Nat. Med., 2002, 8, 918–921.
18 V. Marchi-Artzner, B. Lorz, C. Gosse, L. Jullien, R. Merkel, H. Kessler
and E. Sackmann, Langmuir, 2003, 19, 835–841.
4 L. Perlin, S. MacNeil and S. Rimmer, Soft Matter, 2008, 4, 2331–2349.
4800 | Org. Biomol. Chem., 2011, 9, 4795–4801
This journal is
The Royal Society of Chemistry 2011
©

View Online
19 (a) M. Colin, R. P. Harbottle, A. Knight, M. Kornprobst, R. G. Cooper,
A. D. Miller, G. Trugnan, J. Capeau, G. Coutelle and M. C. Brahimi-
Horn, Gene Ther., 1998, 5, 1488–1498; (b) M. F. M. Mustapha, S. M.
Grosse, L. Kudsiova, M. Elbs, E. A. Raiber, J. B. Wong, A. P. R. Brain,
H. E. J. Armer, A. Warley, M. Keppler, T. Ng, M. J. Lawrence, S. L.
Hart, H. C. Hailes and A. B. Tabor, Bioconjugate Chem., 2009, 20,
518–532.
20 A. P. C. A. Janssen, R. M. Schiffelers, T. L. M. ten Hagen, G. A. Koning,
A. J. Schraa, R. J. Kok, G. Storm and G. Molema, Int. J. Pharm., 2003,
254, 55–58.
21 J. A. Zupancich, F. S. Bates and M. A. Hillmyer, Biomacromolecules,
2009, 10, 1554–1563.
22 (a) Y.-B. Lim, E. Lee and M. Lee, Angew. Chem., Int. Ed., 2007, 46,
3475–3478; (b) Y.-B. Lim, O.-J. Kwon, E. Lee, P.-H. Kim, C.-O. Yun
and M. Lee, Org. Biomol. Chem., 2008, 6, 1944–1948.
25 S. W. A. Reulen, P. Y. W. Dankers, P. H. H. Bomans, E. W. Meijer and
M. Merkx, J. Am. Chem. Soc., 2009, 131, 7304–7312.
26 (a) P. P. Karmali and A. Chaudhuri, Med. Res. Rev., 2007, 27, 696–722;
(b) S. Bhattacharya and A. Balaj, Chem. Commun., 2009, 4632–4656;
(c) N. M. Rao, Chem. Phys. Lipids, 2010, 163, 245–252; (d) P. Posocco,
S. Pricl, S. Jones, A. Barnard and D. K. Smith, Chem. Sci., 2010, 1,
393–404; (e) S. P. Jones, N. P. Gabrielson, C.-H. Wong, H.-F. Chow,
D. W. Pack, P. Posocco, M. Fermeglia, S. Pricl and D. K. Smith, Mol.
Pharmaceutics, 2011, 8, 416–429.
27 (a) G. R. Newkome and X. Lin, Macromolecules, 1991, 24, 1443–1444;
(b) G. R. Newkome, X. Lin and C. D. Weis, Tetrahedron: Asymmetry,
1991, 2, 957–960; (c) J. K. Young, G. R. Baker, G. R. Newkome, K. F.
Morris and C. S. Johnson Jr., Macromolecules, 1994, 27, 3464–3471;
(d) C. M. Cordona, R. E. Gawley and C. Gawley, J. Org. Chem., 2002,
67, 1411–1413.
23 (a) M. O. Guler, L. Hsu, S. Soukasene, D. A. Harrington, J. F. Hulvat
and S. I. Stupp, Biomacromolecules, 2006, 7, 1855–1863; (b) Z. Huang,
T. D. Sargeant, J. F. Hulvat, A. Mata, P. Bringas, C.-Y. Koh, S. I. Stupp
and M. L. Snead, J. Bone Miner. Res., 2008, 23, 1995–2006; (c) M.
Zhou, A. M. Smith, A. K. Das, N. W. Hodgson, R. F. Collins, R. V.
Ulijn and J. E. Gough, Biomaterials, 2009, 30, 2523–2530.
24 (a) B. S. Kim, D. J. Hong, J. Bae and M. Lee, J. Am. Chem. Soc., 2005,
127, 16333–16337; (b) Y. Lim and M. Lee, Org. Biomol. Chem., 2007, 5,
401–405; (c) M. K. Mu¨ller and L. Brunsveld, Angew. Chem., Int. Ed.,
2009, 48, 2921–2924.
28 M. C. A. Stuart, J. C. van de Pas and J. B. F. N. Engberts, J. Phys. Org.
Chem., 2005, 18, 929–934.
29 W. Wang, Q. Wu, M. Pasuelo, J. S. McMurray and C. Li, Bioconjugate
Chem., 2005, 16, 729–734.
30 For original reports of c[RGDfK] see: R. Haubner, R. Gratias, B.
Diefenbach, S. L. Goodman, A. Jonczyk and H. Kessler, J. Am. Chem.
Soc., 1996, 118, 7461–7472.
31 D. M. Jameson and J. A. Ross, Chem. Rev., 2010, 110, 2685–2708.
32 X. Montet, M. Funovics, K. Montet-Abou, R. Weissleder and L.
Josephson, J. Med. Chem., 2006, 49, 6087–6093.
This journal is
The Royal Society of Chemistry 2011
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