Synthesis of nitric oxide-releasing gold nanoparticles

Article abstract of DOI:10.1021/ja052027u

We report the synthesis of nitric oxide-releasing gold nanoparticles formed by place-exchange reaction of hexanethiol monolayer-protected clusters with diamine nitric oxide donor precursor molecules, which are subsequently converted to N-diazeniumdiolate NO donors. The nitric oxide release from the N-diazeniumdiolate-modified gold nanoparticles is tunable by varying the number and/or the chemical structure of the exchanged amine ligands. The size and stability of NO-releasing nanoparticles may prove useful for a range of biomedical and pharmaceutical applications. Copyright

Full text of DOI:10.1021/ja052027u

Published on Web 06/08/2005
Synthesis of Nitric Oxide-Releasing Gold Nanoparticles
Aaron R. Rothrock, Robert L. Donkers, and Mark H. Schoenfisch*
Department of Chemistry, The UniVersity of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599
Received March 30, 2005; E-mail: schoenfi@email.unc.edu
The astounding pace of discovery on the bioregulatory roles of
nitric oxide (NO)1 demands new methods for generating NO in a
controlled manner to facilitate both an improved understanding of
NO’s function in physiology and the development of NO-associated
therapies.2 N-Diazeniumdiolate NO donors, small molecules syn-
thesized by the reaction of amines with NO at elevated pressure,
have proven particularly useful for spontaneously generating NO
in aqueous solution.3 Indeed, the rate of NO generation is easily
tuned by varying the amine precursor structure, pH, and/or
temperature. As such, polymers have been modified to release NO
via doping or covalent attachment of the NO donor whereby low
levels of NO release from the polymer interface mimics the
endothelium of healthy blood vessels, preventing platelet adhesion/
activation.4,5 Such polymers have been employed successfully in
the preparation of more thromboresistant sensors and extracorporeal
heart bypass circuits.6 The synthesis of NO-releasing fumed silica
particles (amorphous silicon dioxide, 0.2-0.3 µm) was recently
reported as an effective strategy for generating NO from within a
polymer film.7 The advantage of using N-diazeniumdiolate-modified
fumed silica was the ease with which such particles could be
embedded in a given polymer matrix and their ability to serve as
both a reinforcing filler and a NO donor.
Monolayer-protected cluster (MPC) gold nanoparticles, or MPCs,8
have received much attention due to their unique size (1-5 nm),
stability, and highly functional design.9 The exterior of MPCs is
easily altered by place-exchanging in other thiols containing the
desired functional groups.10 Such modification has enabled the
potential for employing gold nanoparticles as drug delivery vehicles
and contrast agents.9 Herein, we report on the synthesis of gold
nanoparticles designed to controllably release NO. The unique
functionality of these nanoparticles may represent a new platform
for the targeted delivery of NO in vivo.
Gold nanoparticles were synthesized by the reaction of hydrogen
tetrachloroaurate salt with hexanethiol in the presence of sodium
borohydride.11 After 30 min, the reaction was quenched with water.
The nanoparticles were collected by filtration, washed with aceto-
nitrile, and then functionalized with bromo-terminated alkanethiols
by the place-exchange method (Figure 1).10 Specifically, 11-bromo-
1-undecanethiol12 was added (3:1 ratio of bromo- to methyl-
terminated alkanethiol) to a solution of gold nanoparticles in
methylene chloride and stirred for 30 min. The solvent was removed
by rotary evaporation, and the gold nanoparticles were purified with
acetonitrile. The extent of ligand exchange, monitored by NMR,
was easily controlled by varying the reaction time and/or concentra-
tion of bromoalkanethiol. The bromo-functionalized gold nanopar-
ticles were then dissolved in toluene or methylene chloride and
reacted with ethylenediamine, butylamine, hexanediamine, or
diethylenetriamine. The disappearance of the -CH2Br peak in the
NMR spectra of the functionalized nanoparticles indicated the
completion of the reaction (Supporting Information). The amine-
functionalized gold nanoparticles were then suspended in a solution
of methanol and sodium methoxide base, ultrasonic mixed for 1
Figure 1. Synthesis scheme for preparing NO-releasing gold nanoparticles.
min, and pressurized to 5 atm NO for 3 d with constant stirring to
facilitate the synthesis of diazeniumdiolate NO donors13 and prevent
aggregation. The N-diazeniumdiolate-modified MPCs were filtered,
washed (methanol), and stored at -4 °C until use.
The size and stability of the gold nanoparticles were characterized
using thermal gravimetric analysis (TGA), UV-vis spectroscopy,
and transmission electron microscopy (TEM). The organic content
of hexanediamine-modified gold nanoparticles was determined to
be ∼22%, a value consistent with previous reports for hexanethiol
MPCs composed of 140 gold atoms (core) protected by 53 thiol
ligands.11 Since NO is highly reactive and might disrupt gold-
sulfur bonds,3 the stability of the hexanethiol MPCs after exposure
to high pressures of NO was evaluated using TGA and UV-vis
spectroscopy to ensure that the conditions necessary for diazeni-
umdiolate formation did not compromise nanoparticle integrity.
Both the organic content of the nanoparticles (as studied by TGA)
and the UV-vis spectra (in toluene such that the absorbance was
1.0 at 400 nm) did not change following NO exposure, indicating
negligible influence on monolayer stability. Transmission electron
microscopy images further confirmed that the core diameter of the
nanoparticles remained constant (2.1 ( 0.9 nm) regardless of amine
derivatization or diazeniumdiolate formation. These studies suggest
that the structural integrity of the MPC gold nanoparticles was not
compromised by the conditions necessary to synthesize the NO
donor and introduce NO-release capability.
Nitric oxide release was measured in phosphate-buffered saline
solution at physiological temperature and pH using a Sievers NOA
chemiluminescence nitric oxide analyzer (Boulder, CO). A constant
stream of nitrogen bubbled through the PBS (200 mL/min) was
used to carry the NO to the analyzer. As shown in Figure 2, the
NO release for diazeniumdiolate-modified MPCs was tunable by
varying the number and/or chemical structure of the substituted
amine ligands. Increasing the concentration of ethylenediamine
ligand from 14 to 21% led to a corresponding increase in total NO
9
9362
J. AM. CHEM. SOC. 2005, 127, 9362-9363
10.1021/ja052027u CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
enetriamine likely accounts for increased NO donor formation (and
release capability) relative to ethylenediamine, even though the
length of the alkyl chain separating the nitrogens remains short
(two methylene units). Butylamine-modified MPCs, a secondary
monoamine derivative, were characterized by the lowest total NO
release of all the amine-modified MPCs studied. Such behavior
was expected, however, as diazeniumdiolate formation is facilitated
by the additional amine.13,14 Notably, the diazeniumdiolate conver-
sion efficiency for the amine-modified MPCs was calculated to be
<1%, regardless of amine structure. Current efforts are focused
on enhancing the conversion of diamines to NO donor to increase
the amount and duration of NO release.
The synthesis of ∼2 nm NO-releasing gold nanoparticles
represents an important step toward the development of a NO-
delivery system that bridges small-molecule diazeniumdiolates and
diazeniumdiolate-modified fumed silica particles (∼200 nm). The
control over the type and amount of amine used during preparation
of the nanoparticles allows for a range of NO-release properties.
Further functionalization of the nanoparticles with receptor mol-
ecules to enable specific antibody-antigen or ligand-receptor
interactions may allow for the targeting of specific tissues or cells.
The size and stability of NO-releasing MPC gold nanoparticles may
prove useful for a range of biomedical and pharmaceutical
applications, including in vivo sensor design and topical creams to
enhance wound healing and/or dilate blood vessels below the skin.
Future studies will include determining the influence of amine
precursor distance from the gold core on diazeniumdiolate formation
and dissociation to NO.
Figure 2. Nitric oxide-release profiles from gold nanoparticles derivatized
with (a) 0% ethylenediamine, (b) 14% ethylenediamine, and (c) 21%
ethylenediamine (varying the number of ligands), and (d) 21% ethylene-
diamine, (e) 21% diethylenetriamine, and (f) 21% hexanediamine (varying
the structure of ligands). Release profiles were reproducible to within 10%.
Table 1. Nitric Oxide Release Properties of Amine-Derivatized
Monolayer-Protected Gold Nanoparticles
half-life
(min)
release
total NO
ligand
% aminea
longevity (min)
(pmol/mg)
hexane
butylamine
ethylenediamine
ethylenediamine
hexanediamine
diethylenetriamine
2
15
78
88
68
63
5
60
200
300
600
360
400
2 000
9 750
19 300
87 000
38 000
21
14
21
21
21
Acknowledgment. This research was supported in part by the
National Institutes of Health (EB 000708). A.R.R. also gratefully
acknowledges a fellowship from Pfizer. Special thanks to David
Slade and Mary Robbins for valuable discussion.
release (from 9750 to 19 300 pmol of NO/mg of MPC) and NO
release duration (from 200 to 300 min). The elevated NO release
is attributed to enhanced NO-donor formation due to a larger
concentration of amines. A small amount of NO (400 pmol/mg)
was also measured from the hexanethiol MPC controls. This NO
release was negligible at periods >5 min, indicating that NO may
intercalate within the hydrophobic alkyl chains under the conditions
necessary for diazeniumdiolate synthesis (5 atm NO), but the
amount of such NO is small and rapidly released upon solution
immersion. The diazeniumdiolate-modified MPCs also released low
levels of NO under a warm (37 °C) stream of nitrogen gas,
suggesting a possible thermal dissociation mechanism. However,
the level of NO release was greater in solution (data not shown),
indicating that the N-diazeniumdiolate-modified nanoparticles
undergo both thermal and proton-driven dissociation. The diazeni-
umdiolate-modified MPCs retained full NO-release characteristics
when stored under nitrogen at -4 °C through 14 d.
The NO release from diazeniumdiolate-modified MPCs was also
tunable by varying the amine precursor structure. Increasing the
length of the alkyl chain separating the nitrogens from two to six
methylene units led to an increase in the total amount of NO
released (Figure 2d,f) (from 19 300 to 87 000 pmol of NO/mg of
MPC for ethylenediamine- and hexanediamine-modified MPCs,
respectively), suggesting a NO release/diazeniumdiolate structure
relationship. Indeed, the half-life data (Table 1) show that separating
the amines results in a more rapid release of NO as well, analogous
to the dissociation behavior reported for small-molecule diazeni-
umdiolates.13,14 The total amount of NO released from diethylen-
etriamine-modified MPCs (38 000 pmol of NO/mg) was between
that measured for ethylenediamine- and hexanediamine-modified
MPCs. The presence of an additional secondary amine in diethyl-
Supporting Information Available: Synthetic procedure for 11-
bromo-1-undecanethiol and representative 1H NMR spectra for amine-
functionalized gold nanoparticles. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) Marletta, M. A.; Tayeh, M. A.; Hevel, J. M. BioFactors 1990, 2, 219-
225.
(2) Keefer, L. K. Chemtech 1998, 28, 30-35.
(3) Hrabie, J. A.; Keefer, L. K. Chem. ReV. 2002, 102, 1135-1154.
(4) Radomski, M. W.; Rees, D. D.; Dutra, A.; Moncada, S. Br. J. Pharmacol.
1992, 107, 745-749.
(5) Diodati, J. G.; Quyyumi, A. A.; Hussain, N.; Keefer, L. K. Thrombosis
Haemostasis 1993, 70, 654-658.
(6) Frost, M. C.; Reynolds, M. M.; Meyerhoff, M. E. Biomaterials 2005, 26,
1685-1693.
(7) Zhang, H.; Annich Gail, M.; Miskulin, J.; Stankiewicz, K.; Osterholzer,
K.; Merz, S. I.; Bartlett, R. H.; Meyerhoff, M. E. J. Am. Chem. Soc. 2003,
125, 5015-5024.
(8) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem.
Soc., Chem. Commun. 1994, 801-802.
(9) Feldheim, D. L., Foss, C. A., Eds. Metal NanoparticlessSynthesis,
Characterization, and Applications; Marcel Dekker: New York, 2000; p
360.
(10) Hostetler, M. J.; Templeton, A. C.; Murray, R. W. Langmuir 1999, 15,
3782-3789.
(11) Hostetler, M. J.; Wingate, J. E.; Zhong, C.-J.; Harris, J. E.; Vachet, R.
W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Wignall, G.
D.; Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W. Langmuir
1998, 14, 17-30.
(12) Troughton, E. B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G.; Allara,
D. L.; Porter, M. D. Langmuir 1988, 4, 365-385.
(13) Hrabie, J. A.; Klose, J. R.; Wink, D. A.; Keefer, L. K. J. Org. Chem.
1993, 58, 1472-1476.
(14) Davies, K. M.; Wink, D. A.; Saavedra, J. E.; Keefer, L. K. J. Am. Chem.
Soc. 2001, 123, 5473-5481.
JA052027U
9
J. AM. CHEM. SOC. VOL. 127, NO. 26, 2005 9363