Effects of multivalent histamine supported on gold nanoparticles: activation of histamine receptors by derivatized histamine at subnanomolar concentrations

Article abstract of DOI:10.1039/c5ob01354b

Colloidal gold nanoparticles with a functionalized ligand shell were synthesized and used as new histamine receptor agonists. Mercaptoundecanoic acid moieties were attached to the surface of the nanoparticles and derivatized with native histamine. The multivalent presentation of the immobilized ligands carried by the gold nanoparticles resulted in extremely low activation concentrations for histamine receptors on rat colonic epithelium. As a functional read-out system, chloride secretion resulting from stimulation of neuronal and epithelial histamine H₁ and H₂ receptors was measured in Ussing chamber experiments. These responses were strictly attributed to the histamine entities as histamine-free particles Au-MUDOLS or the monovalent ligand AcS-MUDA-HA proved to be ineffective. The vitality of the tissues used was not impaired by the nanoparticles.

Full text of DOI:10.1039/c5ob01354b

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Effects of multivalent histamine supported on gold
nanoparticles: activation of histamine receptors by
derivatized histamine at subnanomolar
concentrations†
Cite this: DOI: 10.1039/c5ob01354b
Friederike Gasiorek,a Ervice Pouokam,b Martin Diener,*b Sabine Schlechta and
Mathias S. Wickleder*a
Colloidal gold nanoparticles with a functionalized ligand shell were synthesized and used as new hista-
mine receptor agonists. Mercaptoundecanoic acid moieties were attached to the surface of the nano-
particles and derivatized with native histamine. The multivalent presentation of the immobilized ligands
carried by the gold nanoparticles resulted in extremely low activation concentrations for histamine recep-
tors on rat colonic epithelium. As a functional read-out system, chloride secretion resulting from stimu-
lation of neuronal and epithelial histamine H1 and H2 receptors was measured in Ussing chamber
experiments. These responses were strictly attributed to the histamine entities as histamine-free particles
Au–MUDOLS or the monovalent ligand AcS–MUDA–HA proved to be ineffective. The vitality of the
tissues used was not impaired by the nanoparticles.
Received 3rd July 2015,
Accepted 5th August 2015
DOI: 10.1039/c5ob01354b
www.rsc.org/obc
particles and applied the concept of multivalency for the inter-
action with biological tissue. Multivalent presentation of
Introduction
During the past 20 years, the number of applications of nano- immobilized ligands is a common concept in ligand–receptor
and microdevices in the fields of biotechnology and biomedi- interactions and can result in high affinities of ligands that
cine has been increased drastically.1,2 Most promising tools possess only moderate binding constants in a similar concen-
are nanoparticles.3 They represent nanoscale materials with tration range when they are presented in their monovalent
unique chemical and physical properties due to their size. form.6–8 Simplifying binding structures reduces the synthetic
These are for example optical absorption (metal nano- complexity of the molecules immobilized on the nanoparticles
particles), photoluminescence (semiconductor quantum dots) and has the advantage of being synthetically well accessible.
or magnetic behaviour (iron oxides). Their applicability derives Multivalent carriers can either inhibit or activate biological
not only from the fact that their dimension range is in the size processes in a different way than the monovalent analogue.
of some biomolecules like proteins or receptors, but also that Usually the effective concentration is much lower.9 There are
through simple surface modification a huge variety of inter- only few examples where this concept is applied on receptor–
actions can be achieved. The basic requirement for every appli- ligand interactions with neurotransmitters. Paolino et al. syn-
cation is the proper surface functionalization of the particles, thesized dendrimeric tetravalent ligands for the activation of
which determines their interaction with the environment. In ligand-gated ion channels.10 Indeed they found activation con-
this study we chose gold nanoparticles as a template because centrations in the low picomolar range. Saada et al. syn-
of their well-known synthesis and functionalization with small thesized gold nanoparticles coated with histamine and applied
molecules.4,5 Moreover they are biocompatible. We took these the conjugates for enzyme activation.11 They also found an
enhancement of the activation properties for these conjugates.
Mediators such as histamine are key regulators of physiologi-
cal processes such as ion transport across epithelial barriers.
Chloride secretion is one of these processes occurring for
example at the colonic epithelium.12,13 Such a secretion can be
activated via stimulation of histamine H1 and/or H2 receptors
as previously shown.14–16 The receptor activity can also be
blocked by inhibitors, which are well-known for the histaminic
receptors. Mediators or neurotransmitters represent an inter-
aInstitute of Inorganic and Analytical Chemistry, Justus-Liebig-University,
Heinrich-Buff-Ring 58, 35392 Giessen, Germany.
E-mail: friederike.gasiorek@anorg.chemie.uni-giessen.de
bInstitute for Veterinary Physiology and Biochemistry, Justus-Liebig-University,
Frankfurter Straße 100, 35392 Giessen, Germany.
E-mail: martin.diener@vetmed.uni-giessen.de
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob01354b
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esting class of bioactive molecules and yet there is no study on exchange still providing stability in aqueous media. Often poly
multivalent ligand presentation on biological systems. In this (ethylene glycol) (PEG) is used as a ligand as it provides
paper we report the immobilization and multivalence of the enough colloidal stability and reduces nonspecific adsorption.
biogenic amine histamine on gold nanoparticles as well as its Besides “PEGylation”, ω-functionalized thiol surfactants with
biological function in receptor activation.
free carboxylic acid groups are a versatile type of ligand and
are often used in aqueous solution. They offer an additional
anchor point for further attachments of biological molecules.
Herein we chose 11-mercaptoundecanoic acid (MUDA). On the
one hand, a new bond between Au and S with high affinity is
built upon transfer, ensuring a complete replacement of
Results and discussion
Synthesis of the gold nanoparticles
By attaching histamine derivatives on gold nanoparticles we citrate due to strong binding affinities. On the other hand, the
developed a new conjugate for the interaction with compatible free carboxylic acid can serve as an anchor for further coupling
receptors. Water soluble gold nanoparticles with a terminally
reactions with amines. In addition, the long alkyl chain pro-
functionalized thiol shell were prepared and used as a scaffold vides enough flexibility for the active moiety to interact multi-
for the multivalent presentation of histamine. Gold nano- valently with the receptor. The ligand exchange with MUDA
particles with an average diameter of 14 nm were synthesized
giving NP 2 proceeded completely as it is indicated in the
by a modification of a well-known method developed by Turke- infra-red (IR) spectra (Fig. 2).
vich et al. (Fig. 1).17,18
The final step towards the fully functionalized nanoparticle
was accomplished by peptide coupling with EDC (1-ethyl-3-(3-
In an aqueous solution Au(III) was reduced by sodium
citrate which serves not only as reducing agent but also as a dimethylaminopropyl)carbodiimide) and NHS (N-hydroxy-suc-
ligand to prevent aggregation of the formed particles. Even if cinimide). The free carboxylic acid is activated by EDC and
the solvent is limited to water or polar equivalents like DMSO,
forms with NHS in situ a so called ‘active ester’. This reacts
the resulting size of the particles can be defined by the molar easier with amine moieties of e.g. amino acids or peptides.
ratios of the starting materials, the reaction temperature and Although the prepared NHS ester is sufficiently stable for the
time. By this method, gold nanoparticles with a diameter of
reaction, it hydrolyses within hours depending on the pH of
14 nm (NP 1, Fig. 1) could be obtained. Depending on the size the reaction solution. To ensure the coupling, another equi-
of the particles, the surface is coated with several thousand valent of histamine, EDC and NHS were added after two
ligands with respect to the steric demand of a single ligand.
hours. The excess of coupling agents and byproducts of the
The resulting nanoparticles are stabilized by electrostatic reaction were removed by purification with dialysis against
repulsion of the negatively charged citrate ions adsorbed on water (MWCO 3500, three times). The characterization of the
their surface. This layer can be easily replaced by ligands with
a stronger binding affinity, such as for example thiols.19,20
Thus, in a second step the ligand shell was substituted by a bi-
valent ligand. Bivalent ligands give access to further
functionalization steps at the ligand periphery. One end binds
to the surface and gives stability to the particle whereas the
other end is exposed to the solution. Depending on the free
moiety, the particles possess different stabilities at different
pH values. There is a wide variety of ligands suitable for
synthesized and functionalized nanoparticles NP 1–3 was per-
Fig. 1 Synthetic procedure for gold nanoparticles with a mean dia-
meter of 14 nm (NP 1) followed by ligand exchange with 11-mercapto-
undecanoic acid (NP 2) and functionalization with histamine (NP 3, Au–
MUDA–HA). Double arrows at the Au core represent the ligand.
Fig. 2 IR spectra of (a) Au–Citrate NP 1, (b) Au–MUDA NP 2 and (c) Au–
MUDA–HA NP 3; vibration for the amide bond is found at 1641 cm−1
.
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Fig. 3 (A) UV/Vis spectra of the stepwise functionalization of Au–Citrate NP 1, Au–MUDA NP 2 and Au–MUDA–HA NP 3 nanoparticles. (B) TEM
images and DLS measurements with values for the distribution of the hydrodynamic diameters of (i) Au–Citrate NP 1 d = 14.0 0.9 nm, dhydr = 16.7
3.0 nm, (ii) Au–MUDA NP 2 d = 14.5 1.2 nm, dhydr = 24.1 6.7 nm and (iii) Au–MUDA–HA NP 3 d = 14.3 0.7 nm, dhydr = 19.3 2.9 nm; d =
diameter.
formed with different methods. For the characterization of the ligand periphery did not alter the particles significantly. In
core transmission electron microscope (TEM) images, dynamic addition to these methods, the success of the ligand exchange
light scattering (DLS) measurements and UV/Vis spectroscopy was verified by IR spectroscopy (Fig. 2). Identification of the
were used. The images shown in Fig. 3 indicate that the syn- newly formed amide bond at 1641 cm−1 gives evidence for the
thesized nanoparticles have mostly a spherical shape; the successful coupling. Again the colloidal solution was purified
average diameter determined by TEM is 14.0 0.9 nm. The by dialysis. Unlike the particles with MUDA as ligand shell, the
hydrodynamic diameter of the nanoparticles in solution final functionalization with histamine provides a good stability
obtained by DLS measurements was determined by 16.7
at physiological pH value and in a standard buffer solution for
3.0 nm. The absorption maximum was found at 524 nm. The the Ussing chamber experiments (see below). The number of
position and form of the maximum is also a hint for the size the ligands at the surface determined by TGA is about 10 000
of the nanoparticles, as it derives from a distinctive plasmon for 14 nm gold nanoparticles, however determination is
resonance at the gold surface depending on the shape of the difficult due to small amounts of product. In order to find out
material. The obtained data confirmed that the average dia- whether the size of the nanoparticles could be a limiting
meter of the particles cores and the maxima of absorption factor for their properties, two other sizes were investigated.
remained unchanged after ligand exchange reaction and Following the same synthetic procedure as described above
functionalization. The slight shift can be explained due to the but with different reaction conditions, 25 nm nanoparticles
new ligand. The affinity to a gold surface is higher for thiolated were obtained. Also, 7 nm nanoparticles were prepared using
ligands than for citrate and therefore effects on the plasmon dodecanethiol-protected particles from the Stucky method as
resonance of the particle. Nonetheless, neither the exchange of basic material.21
the ligands nor the purification via dialysis had an effect on
As a negative control for the biochemical application, gold
the agglomeration state. Also the functionalization at the nanoparticles NP 4 (Fig. 4) with a sulfated ligand shell were
Fig. 4 (A) Synthetic procedure for the preparation of Au–MUDOLS NP 4, (B) UV/Vis spectra of Au–Citrate NP 1 and Au–MUDOLS NP 4, (C) TEM
images and DLS measurements with values for the distribution of the hydrodynamic diameters of (i) Au–Citrate NP 1 d = 14.0 0.9 nm, dhydr = 16.7
3.0 nm, (ii) Au–MUDOLS NP 4 d = 14.3 1.3 nm, dhydr = 18.6 2.5 nm; d = diameter.
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synthesized. We already reported from sulfated ligands for the Functionality of the histamine-nanoparticles
inhibition of selectin binding.22 A structurally similar and
From all the synthesized nanoparticles of different sizes, only
simple ligand is sulfated 11-mercaptoundecanol (MUDOLS),
which can be immobilized on gold nanoparticles in the same
way as mentioned before. Again citrate-stabilized gold nano-
particles NP 1 served as basic material.
The sulfated ligand was synthesized separately and then
attached to the surface via ligand exchange reaction. TEM
images, DLS measurements and UV/Vis spectra (Fig. 4) indi-
cated that the colloidal solution was intact and the nano-
particles were stable. Sulfated nanoparticles showed excellent
stability over a wide range of pH value due to interparticle
repulsion through a negatively charged ligand shell. Unlike the
unsulfated Au–MUDA particles they are perfectly stable at
physiological pH.
the 14 nm nanoparticles yielded consistent effects on short-
circuit current (Isc) when they were administered to rat colonic
mucosa mounted in Ussing chambers. Therefore, this type of
particles was used for all subsequent experiments. To investi-
gate the functionality of the Au–MUDA–HA nanoparticles,
their ability to induce chloride secretion was compared with
that of native histamine, which induces a strong chloride
secretion across rat colonic epithelium.15,16 Indeed, the hista-
mine-conjugated nanoparticles induced
a concentration-
dependent increase in Isc, which is a measure for net ion trans-
port across the epithelium (Fig. 6).
These changes in Isc reflect a secretion of chloride ions as
they were abolished in Cl−-free buffer solution (Fig. 6).
However, repetitive administration of these particles led to a
desensitization of the tissue (Table 1). Thus in all subsequent
experiments, each tissue was only exposed once to a single
concentration of the Au–MUDA–HA nanoparticles (10−11 mol
l−1 at the serosal side). The tissue conductance (Gt) was not
significantly changed by the nanoparticles (10−11 mol l−1 at
the serosal side); Gt amounted to 14.4 1.87 mS cm−2 before
and 14.7 1.96 mS cm−2 after administration of the particles
(n = 6; difference not significant). Viability of the tissue was
also not impaired by the Au–MUDA–HA nanoparticles as both
the Ca2+-dependent secretagogue carbachol (5 × 10−5 mol l−1
at the serosal side), i.e. a stable derivative of acetylcholine,23 as
well as the cAMP-dependent secretagogue forskolin (5 × 10−6
mol l−1 at the mucosal and the serosal side), which stimulates
the intracellular production of cAMP,24 induced a prompt rise
in Isc in all tissues (Fig. 6). As the response to histamine evokes
When looking at multivalent effects, the monovalent ligand
3 (Fig. 5) also has to be investigated. Thus the complete ligand
was synthesized separately. To prevent the formation of a di-
sulfide bond between two molecules, the thiol moiety was pro-
tected with an acetyl group.
Fig. 5 Synthesis of the monovalent analogue AcS–MUDA–HA (3) start-
ing from 11-bromoundecanoic acid 1. Reaction conditions: (a) KSOAc,
DMF, 2 h, rt, (b) NHS, DCM, 18 h, rt, (c) EDC, HA·2HCl, NEt3, DCM, 6 h, rt.
Fig. 6 Au–MUDA–HA (10−11 mol l−1 at the serosal side, arrow) induced an increase in Isc (A), which was almost abolished in the absence of chloride
(0 Cl−; black bar, (B). At the end of the experiments, the secretagogues carbachol (5 × 10−5 mol l−1 at the serosal side, white bars) and forskolin
(forsk; 5 × 10−6 mol l−1 at the mucosal and the serosal side, grey bars, (A and B), known to induce chloride secretion, were administered. Line inter-
ruptions are caused by omission of time intervals in order to synchronize the tracings of individual records to the administration of drugs. Values are
means (symbols) SEM (parallel continuous lines), n = 6–8.
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histamine H2 receptor blocker, cimetidine (10−4 mol l−1 at the
serosal side, Table 1). Mepyramine is a substituted ethylamine
derivative, whereas cimetidine has an imidazole ring system
similar to native histamine.26 Both drugs act as competitive
inhibitors of the G-protein coupled histamine receptors type
126 and type 2,27 respectively.
Table 1 Effects of additive concentrations of Au–MUDA–HA 14 nm on
Isc and H1 and H2 receptors
Increase in short-circuit current ΔIsc
(µEq h−1 cm−2
)
Au–MUDA–HA 14 nm
1.09 0.43b
0.61 0.11a,b
0.13 0.033a
0.42 0.11a,b
0.13 0.02a
Viability of the tissue was not altered, as forskolin, the acti-
vator of adenylate cyclase(s),24 evoked an increase in Isc of
11.90 1.10 µEq h−1 cm−2 (n = 6) in the absence and of 10.72
1.18 µEq h−1 cm−2 (n = 6, difference not significant) in the
presence of the histamine receptors antagonists.
No inhibitor
Mepyramine +
cimetidine
0.083 0.041a
Cumulative administration of gradually increased concentrations
(10−11, 5 × 10−11, 10−10 mol l−1, at the serosal side) of Au–MUDA–HA
induced an increase in Isc concomitant with an obvious
desensitization of the tissue as indicated by the reduced Isc response
after repeated administration. Given is the control response in the
absence of any inhibitors and the response to Au–MUDA–HA in the
combined presence of mepyramine (10−4 mol l−1 at the serosal side)
and cimetidine (10−4 mol l−1 at the serosal side). Values are given as
difference to the baseline in Isc just before administration of the
corresponding agonist (ΔIsc) and are means SEM, n = 6. Different
letters (a, b, c) indicate statistically homogenous groups (analysis of
variances followed by post hoc test of Tukey).
Controls with histamine-free nanoparticles
In order to make sure that the effects of the nanoparticles are
not unspecific artefacts resulting from the assembly com-
ponents of the particles, controls were performed either with
the conjugator AcS–MUDA–HA or with the sulfated moiety Au–
MUDOLS. As shown in Fig. 7, these components failed to
induce a rise in Isc. Furthermore, Au–MUDOLS impaired the
viability of the epithelium as it prevented the secretion
induced by a subsequent administration of forskolin (5 × 10−6
chloride secretion in part via stimulation of histamine recep- mol l−1 at the mucosal and the serosal side).
tors located on submucosal neurons, the effect of the inhibi-
tory neurotoxin tetrodotoxin, which inhibits the propagation
of action potentials via blockade of voltage-dependent Na+
Potency of the Au–MUDA–HA nanoparticles
channels, on the Isc evoked by the Au–MUDA–HA nano-
particles was investigated.16,25 Ideed, in the presence of tetro-
dotoxin (10−6 mol l−1 at the serosal side), the Isc evoked by Au–
MUDA–HA (10−11 mol l−1 at the serosal side) only amounted to
0.69 0.20 µEq h−1 cm−2 (n = 6) in comparison to 2.92 0.64
µEq h−1 cm−2 in untreated control tissues (P < 0.05, n = 6).
The response to native histamine is mediated by stimu-
lation of histamine H1 and histamine H2 receptors.16 In
accordance with that observation, the increase in Isc evoked by
Au–MUDA–HA (10−11 mol l−1 at the serosal side) was strongly
inhibited by a combination of the histamine H1 receptor
blocker, mepyramine (10−4 mol l−1 at the serosal side), and the
The highest changes in the Isc or the Gt induced by the nano-
particles were obtained at the concentration 10−11 mol l−1. For
the Isc, the change amounted to 1.09 0.42 µEq h−1 cm−2
.
When comparing this response with a concentration-response
curve of native histamine (Fig. 8) it turned out that a compar-
able increase in Isc (1.05 0.24 µEq h−1 cm−2) was obtained
with histamine in a concentration of 10−5 mol l−1 (Fig. 8B).
Assuming a maximum of 10 000 histaminic units at the
surface determined by TGA, the relative potency is still some
magnitudes higher. In other words, the conjugation to the
14 nm nanoparticles strongly enhanced the potency of hista-
mine to evoke anion secretion.
Fig. 7 Au–MUDA–HA (10−11, 5 × 10−11, 10−10 mol l−1, administered cumulatively at the serosal side, arrows) induced an increase in Isc. (B) AcS–
MUDA–HA (10−7, 5 × 10−7, 10−6 mol l−1, at the serosal side, arrows) was ineffective. (C) Au–MUDOLS (10−12, 5 × 10−12, 10−11 mol l−1, at the serosal
side, arrows) failed to change the Isc. All particles except Au–MUDOLS preserved the secretory function of the tissues as they did not impair the
effect of the secretagogue forskolin (forsk; 5 × 10−6 mol l−1 at the mucosal and the serosal side, grey bars). Values are means (symbols) SEM (paral-
lel continuous lines), n = 4–8.
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Among the three sizes of particles tested (7 nm, 14 nm and
25 nm), only the intermediate size 14 nm showed consistent
effects on transepithelial Cl− secretion, indicating a size-
dependence. The smaller nanoparticles exhibited unspecific
interactions whereas the bigger particles evoked no signal in
the Ussing chamber experiments. The observed size depen-
dence might correlate with the number of histamine mole-
cules at the surface. The density of immobilized histamine
moieties at 14 nm core size and the spatial arrangement are
both essential for the activation of histaminic receptors bound
on an epithelium. For a detailed insight into the mechanisms
of receptor activation further experiments will be conducted
on the ligand–receptor-complex which is formed during the
activation process. But also the length of the spacer between
the particle core and the active moiety influences the interplay,
as it was shown in one of our previous studies on multivalent
interactions of gold nanoparticles in selectin binding.22
Further investigations shall be conducted on this influence. In
summary the present work is another example for the influ-
ence of multivalent interactions in biological systems. This
concept can also be transferred to other receptor antagonists
and to different neurotransmitters.
Fig. 8 (A) Histamine induced a concentration-dependent increase in
Isc. (B) Shows the potency of Au–MUDA–HA versus histamine; an equi-
valent potency between Au–MUDA–HA and histamine was obtained
with 10−11 mol l−1 and 10−5 mol l−1, respectively. Values are given as
increase in Isc above baseline in short-circuit current (ΔIsc) just before
administration of the corresponding drug and are means
SEM, n =
6–8.
Conclusion
The gold nanoparticles with an average diameter of 14 nm
were loaded with the histamine derivative and showed excel-
lent activation properties in the Ussing chamber experiments.
Compared to native histamine, the nanoparticles were more
effective at extremely low concentrations. It was found that the
newly developed conjugate was able to induce a concentration-
dependent chloride secretion across rat colonic epithelium via
stimulation of histamine H1 and H2 receptors. These recep-
tors, which are composed of 487 or 359 amino acids, respect-
ively, belong to the class of G-protein coupled receptors with
seven transmembrane spanning regions. Upon binding of the
agonist, these receptors induce the exchange of guanosine
diphosphate (GDP) against guanosine triphosphate (GTP) at
the a-subunit of trimeric GTP-binding proteins. The conse-
quence is the dissociation of the G-protein in the GTP-binding
a-subunit and a b/g-complex and the activation of intracellular
enzymes such as phospholipase C or adenylate cyclase, which
then activate intracellular second messenger pathways.28 An
explanation for this stimulation might be the high local
density of the immobilized ligand. The molecular recognition
between the receptors on the surface and ligands bound to the
nanoparticles is strengthened through the geometric arrange-
ment given by the spherical shape. The density of the binding
moieties allows simultaneous binding resulting in a lower acti-
vation concentration.29 The interaction can be blocked with
same inhibitors used for native histamine. To ensure the
response originated only from multivalent histamine sup-
ported on gold nanoparticles and not from any unspecific
interactions with the biological tissue, the monovalent ligand
AcS–MUDA–HA or the gold nanoparticles with a simple sul-
fated moiety from 11-mercaptoundecanol were synthesized
and tested in parallel. Indeed, no chloride secretion could be
observed. All particles tested in this study preserved the basic
functions of the tissues except for Au–MUDOLS. Only the par-
ticles with the sulfated moiety harmed the tissues irreversibly.
Experimental section
All reactions of nanoparticles in aqueous solution were carried
out in purified Millipore water. Organic solvents were distilled
before use. All chemicals were purchased from commercial
sources and used as received. The ligand exchange reactions
and coupling reactions were done under inert conditions. All
glass vessels were washed with aqua regia and distilled water
prior to use. NMR spectra were recorded on Bruker BioSpin
Avance III AV600 (600 MHz), AV400 (400 MHz) or AV200
(200 MHz) spectrometers using tetramethylsilane (TMS) as an
internal standard with chemical shifts given in ppm relative to
1
TMS (δ = 0.00 ppm) or the respective solvent peaks. H NMR
data are reported as follows: chemical shifts (multiplicity
[ppm], coupling constants [Hz], integration, classification).
Multiplicity is recorded as s = singlet, br s = broadened singlet,
d = doublet, t = triplet, q = quartet, m = multiplet. For 13C
NMR, chemical shifts and structural assignments are reported
where applicable. Because of the rigidity of the alkyl chains in
the thiol shells and the resulting large differences in the relax-
ation times of different protons, no integrals are given for the
proton NMR data of the gold colloids. IR spectra were obtained
on a Bruker IFS48 spectrometer in ATR mode. Electrospray
ionization mass spectra (ESI-MS) were recorded on a Finnigan
LCQDuo spectrometer using methanol solutions of the
respective. TEM measurements were performed on a Philips
CM30 STEM (300 kV, LaB6-cathode) equipped with a GATAN
digital camera. TEM images were recorded using a digital
micrograph. The average particle core sizes were determined
by measuring at least 100 individual particles from recorded
TEM images. The UV/Vis spectra were plotted with an Agilent
8453 spectrophotometer (Agilent Technologies Inc., Santa
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Clara, CA, USA). Dynamic light scattering measurements were µL NEt3 were added to the nanoparticle solution. The reaction
performed with a StabiSizer PMX 200C from Particlemetrix mixture was stirred overnight and subsequently dialysed
(Meerbusch, Germany).
against water three times (3500 MWCO) and stored at 4 °C.
1H NMR (400 MHz, D2O): 7.55 (s, 1H, CH), 6.81 (s, 1H, CH),
2.73–2.86 (m, 2H, CH2(HA)), 2.57–2.71 (m, 3H, CH2(HA) +
Chemical syntheses
Citrate-coordinated gold nanoparticles NP 1 (Au–Citrate Ø NH)), 2.41–2.53 (m, 2H), 2.21–2.34 (m, 4H), 2.10–2.21 (m, 3H),
14 nm). Citrate-coordinated gold nanoparticles were syn- 2.03 (t, J = 7.7, 1H), 1.52–1.68 (m, 1H), 1.36–1.48 (m, 1H), 1.18
thesized by a variation of a procedure first published by Turke- (bs, 8H, CH2), IR (KBr disc, ν/cm−1): 3018.8, 2921.4 & 2850.0
vich. A solution of 50 mg (0.127 mmol) HAuCl4·3H2O in (νC–H), 1641.1 (νCvO, amide), 1596.3 (νCO–NH), 1488.2,
195 mL of Millipore water was heated to reflux for 20 min. 1404.2, 957.1, 948.6, TEM: d = 14.3 0.7 nm, UV/Vis: λmax
Under vigorous stirring a solution of 224 mg (0.52 mmol) of 528 nm, DLS: dhydr = 19.3 2.9 nm.
=
sodium citrate in 5 mL of Millipore water was added quickly.
Sodium 11-mercaptoundecyl sulfate (MUDOLS). In a flame-
The reaction mixture was held at 80 °C for 2 h. Then the solu- dried vessel under argon atmosphere, 353 mg of 11-mercapto-
tion was cooled to 0 °C in an ice bath and filtered (0.2 µm pore undecanol (1.72 mmol, 1 equiv.) was dissolved in 3 ml dry
size). A clear red solution with a particle concentration of DMF. Separately, 395 mg of SO3·DMF complex (2.5 mmol,
2.7 nM was obtained and stored at 4 °C.
1.5 equiv.) were dissolved in 1 mL dry DMF and added to the
1H NMR (400 MHz, D2O): δ/ppm = 2.61 (bs, CH2); IR (KBr solution. After stirring for 1 h at room temperature the solvent
disc, ν/cm−1): 3426.8 (νO–H), 1598.2 (νCvO), 1397.6, 1258.4, was removed under reduced pressure (OPV) and the residue was
618.0; TEM: d = 14.0 0.9 nm; UV/Vis: λmax = 522 nm; DLS: dissolved in 4 mL of 1 M NaOH. A white precipitate appeared.
dhydr = 16.7 3.0 nm.
15 mL of ethanol were added and washed three times. The
11-Mercaptoundecanoic acid coordinated gold nanoparticles product was dried in vacuum. 280 mg of a colorless solid
NP 2 (Au–MUDA Ø 14 nm). An amount of 21 mg 11-mercapto- (0.91 mmol, 53%) could be obtained. The dimer was formed.
undecanoic acid (0.1 mmol, 105 equiv.) was dissolved in
1H NMR (200 MHz, DMSO-d6): δ/ppm = 3.68 (t, J = 6.6 Hz,
8.5 mL Millipore water under the addition of 50 µL tetra- 2 H, CH2), 3.35 (t, J = 6.3 Hz, 2H, CH2), 2.27–2.61 (m, 4H,
methylammonium hydroxide (TMAH). 5 mL of a 2.7 nM solu- CH2), 2.56–2.49 (m, 2H, CH2), 1.67–1.52 (m, 4H, CH2),
tion of citrate-protected gold nanoparticles in water was added 1.36–1.24 (m, 10H, CH2); 13C NMR (200 MHz, DMSO-d6):
dropwise and the solution was stirred for 24 h. The nano- δ/ppm = 65.6, 60.7, 32.5, 29.1, 29.0, 28.9, 28.8, 28.6, 28.5, 27.7,
particles solution was dialysed against 300 mL water three 25.5; ESI-MS: m/z = 633.1593 ([M + Na]+, calcd 633.1612).
times (3500 MWCO). A clear red solution with a particle con-
centration of 1 nM was obtained and stored at 4 °C.
11-(Acetylthio)undecanoic acid (AcS–MUDA). 500 mg 11-
bromoundecanoic acid (1.89 mmol, 1 equiv.) were dissolved in
1H NMR (400 MHz, D2O): 3.17 (s, 16H, TMAH), 2.76 (t, 70 mL absolute DMF under argon atmosphere. Separately
CH2), 2.16 (t, CH2), 1.73 (quin, CH2), 1.54 (m, CH2), 1.40 (m, 520 mg potassium thioacetate (4.56 mmol, 2.4 equiv.) were
CH2), 1.30 (bs, CH2); IR (KBr disc, ν/cm−1): 3422.8, 3018.5, suspended in 8 mL absolute DMF under argon atmosphere
2920.1 & 2849.8 (νC–H), 1583.5 (νCvO), 1487.9, 1396.2, and added dropwise to the first solution. The reaction mixture
1274.9, 956.8, 948.2, 625.0; TEM: d = 14.5 1.2 nm, UV/Vis: was stirred for 2 hours at room temperature. The precipitate
λmax = 527 nm, DLS: dhydr = 24.1 6.7 nm.
was filtered off, washed several times with water and dried in
Sodium 11-mercaptoundecyl sulfate coordinated gold nano- vacuum. 414 mg of a colorless solid (1.59 mmol, 84%) could
particles NP 4 (Au–MUDOLS Ø 14 nm). An amount of 30 mg be obtained.
11-mercaptoundecanyl sulfate was dissolved in 8.5 mL Milli-
1H NMR (200 MHz, CDCl3): δ/ppm = 2.79 (t, J = 7.17, 2H,
pore water and 50 µL tetramethylammonium hydroxide CH2), 2.27 (t, J = 7.17, 2H, CH2), 2.25 (s, 3H, CH3), 1.61–1.52
(TMAH) was added. 5 mL of a 2.7 nM solution of citrate-pro- (m, 2H, CH2), 1.52–1.44 (m, 2H, CH2), 1.32–1.16 (m, 12H,
tected gold nanoparticles in water was added dropwise and the CH2); 13C NMR (100 MHz, CDCl3): δ/ppm = 196.2 (Cq, CvO,
solution was stirred for 24 h. The nanoparticles solution was acetyl), 179.4, 33.9, 30.7, 29.5, 29.4, 29.3, 29.2, 29.1, 29.1, 29.0,
dialysed against 300 mL water three times (3500 MWCO) and 28.8, 24.7; ESI-MS: m/z = 283.1335 ([M + Na]+, calcd 283.1344).
stored at 4 °C.
2,5-Dioxopyrrolidin-1-yl-11-(acetylthio-)undecanoate
(AcS–
1H NMR (400 MHz, D2O): δ/ppm = 4.00–3.89 (m, CH2), MUDA–NHS). 195 mg NHS (1.7 mmol, 1.1 equiv.) and 700 mg
2.70–2.60 (m, CH2), 1.68–1.51 (m, CH2), 1.37–1.23 (m, CH2); AcS–MUDA (1.55 mmol, 1 equiv.) were dissolved in DCM
IR (KBr disc, ν/cm−1): 2918.7 & 2849.9 (νC–H), 1614.8, 1470.0, under stirring at room temperature. 350 mg DCC (1.7 mmol,
1261.7 & 1205.1 & 1128.1 (ν(R–OSO2–OR), 1068.8, 966.4, 831.8, 1.1 equiv.) were separately dissolved in 5 mL DCM and added
718.9, 628.7, 586.3; TEM: d = 14.3 1.3 nm; UV/Vis: λmax
525 nm; DLS: dhydr = 18.6 2.5 nm.
=
slowly. After 30 min a white precipitate appeared. The reaction
mixture was stirred overnight. The solvent was removed under
Synthesis of histamine functionalized gold nanoparticles NP reduced pressure. 506 mg (1.42 mmol, 92%) of a colorless
3 (Au–MUDA–HA Ø 14 nm). 10 mL of a 1 nM solution Au– powder were obtained.
MUDA were set in a glass vessel washed with aqua regia and
1H NMR (400 MHz, CDCl3): δ/ppm = 2.81–2.77 (m, CH2),
distilled water. 18 mg histamine dihydrochloride (0.1 mmol), 2.53 (t, J = 7.34, 2H, CH2), 2.25 (s, 3H, CH3), 1.71–1.63 (m, 2H,
17 mg EDC (0.11 mmol) and 15 mg HOBt (0.11 mmol) and 50 CH2), 1.52–1.47 (m, 2H, CH2), 1.38–1.17 (m, 12H, CH2); 13C
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NMR (100 MHz, CDCl3): δ/ppm = 196.1 (Cq, CvO, acetyl), tunica muscularis was gently removed in a proximal direction.
169.3 (2 × Cq, CvO), 168.7 (Cq, CvO), 30.9 (CH3), 30.7, 29.5, Two segments of the distal colon of each rat were prepared.
29.3, 29.2, 29.2, 29.1, 29.0, 28.8, 28.7, 25.6 (2 × CH2), 24.6;
ESI-MS: m/z = 380.1502 ([M + Na]+, calcd 380.1508).
Short-circuit current measurements
S-(11-((2-(1H-Imidazol-4-yl)ethyl)amino)-11-oxoundecyl)
ethanethioate (AcS–MUDA–HA). 97 mg AcS–MUDA
The mucosa–submucosa preparation was fixed in a modified
Ussing chamber bathed with a volume of 3.5 ml on each side
of the mucosa. The tissue was incubated at 37 °C and short-
circuited by a computer-controlled voltage-clamp device (Inge-
nieur Büro für Mess- und Datentechnik Mussler, Aachen,
Germany) with correction for solution resistance. Tissue con-
ductance (Gt) was measured every minute by the voltage devi-
ation induced by a current pulse ( 50 µA, duration 200 ms)
under open-circuit conditions. Short-circuit current (Isc) was
continuously recorded on a chart-recorder. Isc is expressed as
µEq h−1 cm−2, i.e. the flux of a monovalent ion per time and
area, with 1 µEq h−1 cm−2 = 26.9 µA cm−2. At the end of each
experiment, tissue viability was tested by administration of the
cholinergic agonist, carbachol (5 × 10−5 mol l−1 at the serosal
side) and/or the administration of forskolin (5 × 10−6 mol l−1
at the mucosal and the serosal side), an activator of adenylate
cyclase(s), which induce a strong Ca2+-respective cAMP-depen-
dent Cl− secretion.23,24
4
(0.27 mmol, 1 equiv.) were dissolved in DMF to form a clear
solution. 34 mg HA·2HCl (0.30 mmol, 1.1 equiv.) and a few
drops NEt3 were added and the solution was stirred overnight.
Subsequently 50 mL water were added and a white solid
appeared. The precipitate was filtered off and washed three
times with water. The crude product was dissolved in CHCl3,
filtered again and purified via column chromatography
(CHCl3/MeOH 9/1 Rf
= 0.7). After evaporation 70 mg
(0.19 mmol, 70%) of a colorless powder were obtained.
1H NMR (400 MHz, CDCl3): δ/ppm = 7.50 (s, 1H, CH), 6.75
(s, 1H, CH), 6.33 (br s, 1H, NH), 3.52–3.42 (m, 2H, CH2(HA)),
2.79–2.69 (m, 2H, CH2(HA)), 2.53–2.39 (m, 2H, CH2), 2.56 (s,
3H, CH3), 2.10 (t, J = 7.24 Hz, 2H, CH2), 1.59–1.43 (m, 4H,
CH2), 1.26–1.14 (m, 12H, CH2); 13C NMR (100 MHz, CDCl3):
δ/ppm = 196.4 (Cq, CvO, acetyl), 173.7 (Cq, CvO, amide),
134.8 (CH, Car), 127.6 (Cq, Car), 118.6 (CH, Car), 39.2
(CH2(HA)), 36.9 (CH2 (HA)), 30.7 (CH3), 29.5, 29.3, 29.3, 29.2,
29.1, 28.7, 28.4, 26.9, 25.7; ESI-MS: m/z = 354.2221 ([M + H]+,
calcd 354.2210).
Drugs
Au–MUDOLS, Au–MUDA–HA, carbachol, cimetidine, hista-
mine dihydrochloride, and mepyramine were dissolved in
aqueous stock solutions. Forskolin (Calbiochem, Bad Soden,
Germany) was dissolved in ethanol (final maximal concen-
tration 0.25%, v/v). Tetrodotoxin was dissolved in 2 × 10−2 mol
l−1 citrate buffer. If not indicated otherwise, drugs were from
Sigma, (Taufkirchen, Germany).
Animals
Female and male Wistar rats with a body mass of 200–250 g
were used. The animals were bred and housed at the institute
for Veterinary Physiology and Biochemistry of the Justus-
Liebig-University Giessen at an ambient temperature of
22.5 °C and air humidity of 50–55% on a 12 h : 12 h light–dark
cycle with free access to water and food until the time of the
experiment. Experiments were approved by the named animal
welfare officer of the Justus Liebig University (administrative
number 487_M) and performed according to the German and
European animal welfare law.
Statistics
Results are given as means SEM, with the number (n) of
investigated tissues. When means of several groups had to be
compared, an analysis of variance was performed followed by
post hoc Tukey’s-test. For the comparison of two groups, a
Mann-Whitney U-test was applied. P < 0.05 was considered to
be statistically significant.
Solutions
The standard solution for the Ussing chamber experiments
was a buffer solution containing (mmol l−1): NaCl 107, KCl
4.5, NaHCO3 25, Na2HPO4 1.8, NaH2PO4 0.2, CaCl2 1.25,
MgSO4 1 and glucose 12. The solution was gassed with carbo-
gen (5% CO2 in 95% O2, v/v); pH was 7.4. For the Cl−-free
buffer, NaCl and KCl were substituted by Na gluconate and K
gluconate (KGluc), respectively. The Ca2+ concentration in the
Cl−-free buffer was increased to 5.75 mmol l−1 to compensate
for the Ca2+-buffering properties of gluconate.30
Acknowledgements
We wish to acknowledge Anne Schulze (Institute of Inorganic
and Analytical Chemistry, Justus-Liebig-University at Giessen)
for the TEM images.
References
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colon. Briefly, the colon was placed on a small plastic rod with
a diameter of 5 mm. A circular incision was made near the
anal end with a blunt scalpel, and the serosa together with the
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