experimentally simple approach, based on sequential steps of
chemical self-assembly, offers a straightforward, nonlitho-
graphic method to modify large flat or curved substrates and
it may, therefore, be used for the realization of functional
nanopatterned devices.
branched, globular at higher generations, and exposes 48
peripherial hydroxy end groups. The development of a facile
and robust synthetic route that results in complete carbox-
ylation of all 48 OH groups was required to introduce the
negative charge. While a simple ring-opening reaction of
succinic anhydride was utilized in earlier reports to obtain
carboxylated polymers,[8] the monodisperse structure of the
dendrimers requires unprecedented control over the reaction
conditions. The synthesis of the anhydride-activated 4-(ben-
zyloxy)-4-oxobutanoic acid 8 was therefore targeted since the
benzyloxy-protected carboxylic group can efficiently react
with the dendritic moiety through typical anhydride reactions
(Scheme 1).[9] The anhydride was produced on a 200 g scale,
Charge-stabilized gold nanoparticles [radius a = (5 Æ
0.5) nm] were synthesized by reduction of a gold salt[4] with
citrate and tannic acid and subsequent adsorption under
electrostatic control to gold substrates premodified with a
self-assembled monolayer of octanedithiol (1) as described
earlier.[5] By keeping a constant NP concentration of 5 ꢀ
10À8 m, but altering the ionic strength of the gold sol, NPs
will bind on the surface at a distance corresponding to
approximately two times the double layer thickness kÀ1 from
each other. In our experience, this strategy can be used to
position particles with a 5 nm radius at distances ranging from
5 to 80 nm. For the fabrication of polymer arrays, NPs were
adsorbed under two different conditions, 10 mm and 0.625 mm
citric buffer at a pH 4, which correspond to ka = 1.84 and ka =
0.48, respectively. The mean center-to-center interparticle
distances were determined by scanning electron microscopy
(SEM) to be 17 and 28 nm, respectively, by calculation of
radial distributions g(R), where R is the distance between two
particle centers (Figure 1b). These values correspond to a
nanoparticle coverage q of approximately 30 and 10%,
respectively.
The thiolated area between the NPs was pegylated with
malemide-functionalized polyethylene glycol (M-PEG, aver-
age molecular mass 5000 gmolÀ1) 3 to generate protein and
cell-repellent properties. The double bond of the malemide
group undergoes an alkylation reaction with the free sulf-
hydryl group to yield a stable thioether bond. The chemical
reaction was verified with a quartz crystal microbalance with
dissipation (QCM-D; see the Supporting Information) and
tapping mode (TM) AFM (Figure 2). The NPs were made
positively charged by using cysteamine (4) prior to immobi-
lization of two carboxy-functionalized macromolecules with
similar charge but with distinctly different architectures.
Polyacrylic acid (PA-COOH) with an average molecular
weight of 5100 gmolÀ1 and 54 carboxy groups was chosen as a
linear polymer with a random coil conformation. In contrast,
the nontoxic, water-soluble, and commercially available
fourth generation bis(methylol)propionic acid (bis-MPA)
dendrimer (PFD-TMP-G4-OH, Figure 3)[6] was identified as
a second polymer scaffold.[7] This dendrimer is highly
Scheme 1. Synthesis of 4-(benzyloxy)-4-oxobutanoic acid 8. 1) Succinic
anhydride and 4-dimethylaminopyridine (DMAP) in dichloromethane.
2) Dicyclohexylcarbodiimide (DCC) in CH2Cl2 at 08C.
and successfully reacted with PFD-TMP-G4-OH. The iso-
lated benzyloxy-protected dendrimer intermediate was fur-
ther deprotected using a Pd/C-catalyzed hydrogenolysis
reaction. The progress of the reaction was monitored
continually by NMR spectroscopy and MALDI-TOFanalysis,
and the final carboxylated product—the fourth generation
bis-MPA dendrimer PFD-TMP-G4-COOH48
5
(M =
10163 gmolÀ1, also denoted G4-COOH)—was isolated in a
high total yield of 93% (Figure 3a).
The electrostatic immobilization of the dendrimer as a
function of NP coverage was monitored using QCM-D
(Figure 3b). The adsorbed mass, around 60 ngcmÀ2 for 30%
NP coverage, corresponds adequately to a 1:1 stoichiometric
ratio between the NPs and immobilized dendrimer. No
adsorption of the dendrimer could be observed on the
negative control surface (M-PEG) or on NPs not treated
with cysteamine 4. The presence of the dendrimer was further
verified by monitoring the concentration of a molecular mass
fragment at m/z = 117—which could be assumed to be rather
specific because of the molecular structure of the dendrimer
(Figure 3c)—by chemical mapping using time-of-flight sec-
ondary ion mass spectrometry (TOF-SIMS).
Primary human saphenous vascular endothelial cells
(PHSVEC) were employed to evaluate the effect of anionic
nanodomains expressed by two inherently different polymer
architectures. This cell type lines the inside of blood vessels
and when viable expands laterally, thereby acquiring a
cobblestone-like appearance. After 24 h of incubation, the
cells were fixed with formaldehyde and stained with 4’,6-
diamidino-2-phenylindole (DAPI), Vinculin, and Alexa 488
conjugated phalloidin. Representative fields of view as
observed by confocal laser scanning microscopy (CLSM)
are shown in Figure 4a–f. A trend of decreasing numbers of
cells and decreasing size of cells with larger interparticle
separation was observed (Figure 4g,h). Similar trends have
been observed with RGD-presenting arrays, but morpholog-
Figure 2. TM-AFM height image prior to and after reaction with M-
PEG.
Angew. Chem. Int. Ed. 2011, 50, 3450 –3453
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3451
Lundgren, Anders
