Toward multifunctional "clickable" diamond nanoparticles

Article abstract of DOI:10.1021/acs.langmuir.5b00643

Nanodiamonds (NDs) are among the most promising new carbon based materials for biomedical applications, and the simultaneous integration of various functions onto NDs is an urgent necessity. A multifunctional nanodiamond based formulation is proposed here. Our strategy relies on orthogonal surface modification using different dopamine anchors. NDs simultaneously functionalized with triethylene glycol (EG) and azide (N₃) functions were fabricated through a stoichiometrically controlled integration of the dopamine ligands onto the surface of hydroxylated NDs. The presence of EG functionalities rendered NDs soluble in water and biological media, while the N₃ group allowed postsynthetic modification of the NDs using "click" chemistry. As a proof of principle, alkynyl terminated di(amido amine) ligands were linked to these ND particles.

Full text of DOI:10.1021/acs.langmuir.5b00643

Article
pubs.acs.org/Langmuir
Toward Multifunctional “Clickable” Diamond Nanoparticles
†
†,‡,§
†
§
§
Manakamana Khanal, Volodymyr Turcheniuk,
Alexandre Barras, Elodie Rosay, Omprakash Bande,
§
‡,∥
⊥
†
,†
Aloysius Siriwardena, Vladimir Zaitsev, Guo-Hui Pan, Rabah Boukherroub, and Sabine Szunerits*
†Institute of Electronics, Microelectronics and Nanotechnology (IEMN), UMR-CNRS 8520, Universite
́ ́
Lille 1, Cite Scientifique,
5
9655 Villeneuve d’Ascq, France
‡
Taras Shevchenko University, 60 Vladimirskaya str., Kiev, Ukraine
§
Laboratoire de Glycochimie des Antimicrobiens et des Agroressources (LG2A) (FRE 3517-CNRS), Universite
Verne, 33 Rue St Leu, 80039 Amiens, France
́
de Picardie Jules
∥
Chemistry Department, Pontifical Catholic University of Rio de Janeiro, Rua Marques de Sao Vicente, 225-Gavea, Rio de Janeiro,
22451-900, Brazil
⊥
State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics, Fine Mechanics and Physics,
Chinese Academy of Sciences, 3888 Dong Nanhu Road, Changchun 130033, China
ABSTRACT: Nanodiamonds (NDs) are among the most
promising new carbon based materials for biomedical applications,
and the simultaneous integration of various functions onto NDs is
an urgent necessity. A multifunctional nanodiamond based
formulation is proposed here. Our strategy relies on orthogonal
surface modification using different dopamine anchors. NDs
simultaneously functionalized with triethylene glycol (EG) and
azide (−N ) functions were fabricated through a stoichiometrically
3
controlled integration of the dopamine ligands onto the surface of
hydroxylated NDs. The presence of EG functionalities rendered
NDs soluble in water and biological media, while the −N group
3
allowed postsynthetic modification of the NDs using “click”
chemistry. As a proof of principle, alkynyl terminated di(amido
amine) ligands were linked to these ND particles.
1. INTRODUCTION
lately that hydroxylated NDs (ND−OH) can be directly
10
functionalized by dopamine derivatives. Motivated by the
ease by which functional groups like azide and others can
be introduced via the dopamine terminal amine groups, we
investigate in this work the possibility of using dopamine
chemistry for the formation of multifunctional NDs. To validate
the concept, a model material carrying triethylene glycol and
Integrating biological components with nanomaterials for
delivery of chemotherapeutic drugs or genetic material has
1
−5
been widely explored using a spectrum of approaches.
In
many areas of biomedical research targeting molecules, drugs and
imaging agents need to be simultaneously integrated on the
nanostructure for the convenience of detection and targeted
treatment. However, the implementation of multiple function-
alities onto the surface of a nanoparticle is a difficult task and
fraught with the possibility of nonselective cross reactions.
azide modified dopamine anchors (dop−EG and dop−N )
3
(
Figure 1A) was fabricated. The functional azide moieties
associated with the surface of the NDs could be furthermore
chemoselectively conjugated to alkynyl terminated di(amido
amine) ligands via a Cu(I) catalyzed cycloaddition reaction. The
concept of “click” chemistry, introduced by K. B. Sharpless, is
based on the copper(I) catalyzed triazole formation through the
classic Huisgen 1,3-dipolar cycloaddition between azides and
6
Multifunctionalization strategies for gold or magnetic particles
7,8
have been recently reported. Among the nanomaterials
9
currently being considered are nanodiamonds (NDs). One of
the advantages of NDs over other carbon-based materials such as
fullerenes and carbon nanotubes is that they are completely inert,
optically transparent, and biocompatible and can be function-
alized in many ways depending on their intended ultimate
2
8,29
alkynes.
This cycloaddition reaction is irreversible and
proceeds in quantitative yields and high selectivity. It is tolerant
to a variety of solvents (including water) and can be performed in
the presence of many other functional groups. “Click” chemistry
related strategies have shown also to be very appealing for the
1
0−16
application.
Although in vivo toxicity of nanoparticles is
dependent on specific surface characteristics, ND particles did
1
7−20
not induce significant cytotoxicity in a variety of cell lines
and were used in various biomedical applications.
4
,5,14,15,21−23
While there have been diverse surface engineering strategies,
Received: February 18, 2015
Revised: March 17, 2015
currently, there are only three reports for the formation of
24−27
orthogonally functionalized NDs.
Our group has shown
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.langmuir.5b00643
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Figure 1. (A) Synthesis of multifunctional clickable NDs. (B) Synthetic routes to (a) azide-modified dopamine (dop−N ) and (b) triethylene glycol-
3
modified dopamine (dop−EG).
1
0,15,25−27,30−32
modification of NDs.
“
We have shown that, by
display a primary average particle size of 4.0 nm. N,N′-Dicyclohex-
ylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), 4-dimethylami-
nopyridine (DMAP), copper(II) sulfate pentahydrate (CuSO ·5H O),
L-ascorbic acid, 4-pentynoic acid, ethylenediaminetetraacetic acid
(EDTA), triethylamine (TEA), acetonitrile, dichloromethane (DCM),
methanol (MeOH), ethanol (EtOH), tetrahydrofuran (THF), hexane
clicking” mannose ligands to NDs, the initial interaction of
Escherichia coli (E. coli) with biotic as well as abiotic surfaces, a
critical step in surface colonization and biofilm formation by this
bacterium, can be inhibited.
derivative to azide-terminated NDs gave ND particles which are
4
2
1
5,32
The “clicking” of a boronic acid
(Hex), ethyl acetate (EtOAc) and dimethylformatide (DMF) were
able to reduce Hepatitis C viral infectivity through their effective
obtained from Sigma-Aldrich and used without further purification.
2
1
33
blocking of viral entry. NDs with “clicked” fluorescent tags and
cyclic RGD peptide were recently reported by Cigler and co-
workers to allow selective targeting of integrin α β receptors on
CuI(PPh ) was synthesized as described in the literature. In short, a
3
solution of triphenyl phosphine (0.69 g, 2.63 mmol) in 10 mL of
acetonitrile was added to a solution of CuI (0.5 g, 2.63 mmol) in the
same solvent (50 mL). A complex started to precipitate after a few
seconds. The mixture was stirred for 1 h, and then, the precipitate was
filtered, washed with acetonitrile, and vacuum-dried (yield 80%).
The alkynyl terminated di(amido amine) ligand was synthesized as
v
3
25
glioblastoma cells with high internalization efficacy. Krueger
and co-workers attached a carbon monoxide delivery agent
via “click” chemistry to NDs and studied the photoactivated
CO release to biological systems. Zhao et al. reported on
the interest of multifunctional NDs where targeting RGD
peptide and Pt-based drugs were clicked onto polyglycerol
coated NDs for the selective killing of U87MG cells over
3
1
3
4
described in the literature.
.2. Synthesis of Different Ligands. 2.2.1. Synthesis of 1-(6-{[2-
3,4-Dihydroxyphenyl)ethyl]amino}-6-oxohexyl)triaza-1,2-dien-2-
2
(
ium (3). Synthesis of 6-Azidohexanoic N-Hydroxysuccinimide Ester
(2). A mixture of bromohexanoic acid (1) (4 g, 0.02 mmol) in DMF and
2
6
HeLa ones.
sodium azide (NaN , 2.77 g, 0.04 mmol) was heated to 95 °C under
3
stirring overnight. The solvent was evaporated under reduced pressure,
and the product was diluted with water and extracted using DCM to give
6-azidohexanoic (2) as yellow oil. To the solution of 6-azidohexanoic
acid (2) (1.95 g, 0.1 mmol) in dichloromethane was added
dicyclohexycarbodiimide (2.56 g, 0,01 mmol) and N-hydroxysuccinimide
2
. EXPERIMENTAL SECTION
2
.1. Materials. Hydroxyl-terminated nanodiamond particles
(
ND−OH) and amine-terminated nanodiamonds (ND−NH ) were pur-
2
chased from International Technology Center (Raleigh, NC, USA) and
B
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Table 1. Physico-Chemical Properties of Different NDs Used in This Work
a
sample
ND−OH
ND−dop−EG+N3
d (nm)
PI
ζ (mV)
C 1s
O 1s
N 1s
79 ± 13
120 ± 19
130 ± 19
0.246 ± 0.002
0.358 ± 0.012
0.357 ± 0.012
22 ± 2
25 ± 1
43 ± 1
88.4
77.4
70.8
10.1
12.7
12.9
1.5
9.9
ND−dop−EG+(NH2)2
16.3
a
Polydispersity index; mean ± SD, n = 3.
(
1.43 g, 0.01 mmol) and the mixture stirred overnight at room tem-
2H), 2.58−2.52 (m, J = 19.8 Hz, 1H), 2.47−2.27 (m, 3H), 2.09−2.00
13
perature. The solution was filtrated and the filtrate evaporated under
reduced pressure to give 6-azidohexanoic N-hydroxysuccinimide ester (2)
(m, 2H), 1.92−1.85 (m, 1H). C NMR (75 MHz, MeOD) δ 170.50
(d), 168.60 (d), 70.17 (s), 68.77 (s), 63.35 (s), 39.00 (s), 34.09 (s),
32.72 (s), 29.49 (s), 25.16 (s, succinic C−C), 20.55 (s), 19.69 (s). MS
1
(
2.8 g). H NMR (300 MHz, CDCl ) δ 3.35−3.27 (t, 2H), 2.82 (s, 4H,
3
+
succinimide), 2.65−2.58 (t, 2H), 1.82−1.76 (m, 2H), 1.65−1.60 (m, 2H),
1
(ESI): m/z (%) = 367 [M + Na] .
+
.54−1.49 (m, 2H). MS (ESI): m/z (%) = 255 [M + Na] .
Synthesis of N1-(3,4-Dihydroxyphenethyl)-N5-{2-[2-
(2hydroxyethoxy)ethoxy]ethyl}pentane Diamide (7) (dop−EG). To
a solution of dopamine hydrochloride (104.7 mg, 0.76 mmol) and TEA
(0.137 mL, 0.98 mmol) in MeOH was added compound 6 (200 mg, 0.76
mmol), and the mixture was stirred at room temperature for 24 h under
argon. Purification by flash column chromatography using EtOAc:-
Synthesis of 1-(6-{[2-(3,4-Dihydroxyphenyl)ethyl]amino}-6-
oxohexyl)triaza-1,2-dien-2-ium (3). Dopamine hydrochloride (162.5 mg,
0
.86 mmol) was mixed with triethylamine (0.155 mL, 1.12 mmol) in
methanol. 6-Azidohexanoic N-hydroxysuccinimide ester (2) (200 mg,
.86 mmol) was added and then stirred overnight at room temperature
0
under argon. The product was extracted using dichloromethane and
MeOH:H O = 5:1:0.5 as an eluent gave compound 7 (160 mg; 69%) as
a brown-yellow oil. H NMR (300 MHz, MeOD) δ 6.70−6.67 (m, J =
2
1
washed with water. The organic layer was dried (MgSO ) and evaporated
4
under vacuum. Purification of compound 3 was done by flash column
4.8 Hz, 11.6, 2H), 6.56−6.53 (d, J = 8.1 Hz, 1H), 4.26−4.17 (m, J = 26.1
Hz, 1H), 3.75−3.51 (m, J = 70.8 Hz, 6H), 3.42−3.27 (m, J = 43.6 Hz,
6H), 2.69−2.59 (t, J = 30.3 Hz, 2H), 2.37−2.13 (m, J = 73.5 Hz, 6H),
chromatography with EtOAc:Hex = 3:1 as eluent to give compound 3
1
(
150 mg, 68%) as a slightly yellow oil. H NMR (300 MHz, DMSO-d6) δ
13
8
.66 (s, 2H), 7.79−7.76 (t, J = 10.9, 1H), 6.63−6.60 (d, J = 7.9, 1H), 6.55
1.94−1.84 (d, J = 28.7 Hz, 2H). C NMR (75 MHz, MeOD) δ 173.60
(s), 145.95 (s), 144.13 (s), 130.66 (d), 119.69 (s), 115.53 (s), 114.99
(s), 70.12 (s), 69.83 (s), 69.17 (s), 68.76 (s), 63.17 (s), 40.76 (s), 38.92
(s), 34.50 (s), 32.69 (s), 24.88 (s), 21.84 (s), 21.02 (s). MS (ESI):
(
(
1
1
s, 1H), 6.43−6.39 (d, J = 10.1, 1H), 3.29−3.27 (d, J = 6.9, 2H), 3.19−3.12
q, J = 20.5, 2H), 2.05−2.00 (t, J = 14.6, 2H), 1.56−1.43 (m, J = 36.2, 4H),
13
.31−1.23 (d, J = 24.5, 2H). C NMR (75 MHz, DMSO-d6) δ 165,56 (s),
44.35 (s), 143.25 (s), 135.54 (d), 120.34 (s), 117.33 (s), 114.55 (s), 49.79
+
m/z (%) = 421 [M + Na] .
(
s), 40.57 (s), 35.38 (s), 34.41 (s), 29.53 (s), 25.45 (s), 24.92 (s). MS
2.3. Modification of Nanodiamonds. 2.3.1. Functionalization
+
(ESI): m/z (%) = 319 [M + Na] .
with Dopamine Ligands. Hydroxylated nanodiamonds (ND−OH)
−
1
2
.2.2. Synthesis of Ethylene Glycol Terminated Dopamine (dop−
(5 mg mL ) in dry acetonitrile were reacted with dop−N (3), dop−
3
EG) (7). Synthesis of 2-[2-(2-Aminoethoxy)ethoxy]ethanol (5). To a
DMF solution of 2-[2-(2-chloroethoxy)ethoxy]ethanol (4) (4.79 g,
0
reaction mixture was stirred at 90 °C for 24 h. The mixture was filtered,
the solvent evaporated under reduced pressure, diluted with dichloro-
methane, washed with water, dried with MgSO , and evaporated to give
2
NMR (300 MHz, CDCl ) δ 3.75−3.68 (broad, 2H) 3.67−3.63 (m, 6H),
3
EG (7), or a mixture of dop−N (3)/dop−EG (7) (6 mM) in a 1/1 ratio
3
with ultrasonication in an ice bath with a horn-type sonotrode
(amplitude 70%) for 8 h and then left at room temperature for 12 h
under stirring. The dopamine-functionalized ND particles (ND−dop−
N , ND−dop−EG, and ND−dop−EG+N ) were separated by
.031 mmol), sodium azide (2 g, 0.046 mmol) was added and the
3
3
4
centrifugation at 10.000 rpm, purified through four consecutive wash/
centrifugation cycles at 10.000 rpm with acetonitrile and finally oven-
dried at 50 °C overnight.
1
-[2-(2-azidoethoxy)ethoxy]ethanol (4.6 g; 90%) as a yellow oil. H
3
.59−3.57 (m, 2H), 3.39−3.36 (t, 2H). MS (ESI): m/z (%) = 184
2.3.2. Postfunctionalization of ND−dop−N and ND−dop−EG
3
+
[
M + Na] .
+N with Alkynyl-Terminated Di(amido amine) (8). Azide-terminated
3
2
-[2-(2-Azidoethoxy)ethoxy]ethanol (4 g, 0.025 mmol) and Pd/C
NDs (ND−dop−N
3
and ND−dop−EG+N ) were further modified
3
(
1.3 g) in methanol were stirred for 48 h at room temperature under
hydrogen. The Pd/C was filtered off, and the solution was evaporated to
with alkynyl terminated di(amido amine) (8) using “click” chemistry.
−1
The ND suspension (5 mg mL ) in dry DMF (8 mL) was sonicated for
give 2-[2-(2-aminoethoxy)ethoxy]ethanol (5) (3.2 g; 96%) as a yellow
30 min before alkynyl terminated di(amido amine) (8) (20 mg) and a
1
oil. H NMR (300 MHz, CDCl ) δ 3.77−3.73 (m, 3H), 3.70−3.67
catalytic amount of CuI(PPh ) (10 mol %) were added and the mixture
3
3
(
1
m, 5H), 3.65−3.61 (m, 3H), 3.58−3.54 (t, 2H). MS (ESI): m/z (%) =
reacted for 48 h at 80 °C. The functionalized particles were separated by
centrifugation at 10.000 rpm and purified through four consecutive
washing/centrifugation cycles at 10.000 with DMF. Finally, the particles
were washed with an aqueous solution of EDTA (1 mM, three times) to
ensure that all the catalyst was removed and once with MQ water. The
resulting NDs were oven-dried overnight at 50 °C.
2.4. Sample Characterization. X-ray Photoelectron Spectrosco-
py (XPS). X-ray photoelectron spectroscopy (XPS) measurements were
performed with an ESCALAB 220 XL spectrometer from Vacuum
Generators featuring a monochromatic Al Kα X-ray source (1486.6 eV)
and a spherical energy analyzer operated in the CAE (constant analyzer
energy) mode (CAE = 100 eV for survey spectra and CAE = 40 eV for
high-resolution spectra), using the electromagnetic lens mode. No flood
gun source was needed due to the conducting character of the substrates.
The angle between the incident X-rays and the analyzer is 58°. The
detection angle of the photoelectrons is 30°.
+
57 [M + Na] .
Synthesis of 5-[(2,5-Dioxopyrrolidin-1-yl)oxy]-N-{2-[2-(2-
hydroxyethoxy)ethoxy]ethyl}-5-oxopentan Amide (6). 2-[2-(2-
Aminoethoxy)ethoxy]ethanol (5) (3.2 g, 0.02 mmol) and glutaric
anhydride (2.7 g, 0.02 mmol) were mixed in THF overnight at room
temperature. After solvent evaporation, 5-({2-[2-(2-hydroxyethoxy)-
ethoxy]ethyl}amino)-5-oxopentanoic acid (5.2 g, 92%) was obtained as
1
a yellow oil. H NMR (300 MHz, CDCl ) δ 7.35−7.02 (broad, J = 49.9
3
Hz, 1H), 6.81−6.69 (t, J = 34.4 Hz, 1H), 3.80−3.57 (d, J = 70.2 Hz, 9H),
3
2
.48−3.43 (m, J = 15.0 Hz, 1H), 2.79−2.74 (t, J = 13.4 Hz, 2H), 2.47−
.38 (m, J = 26.4 Hz, 3H), 2.34−2.29 (t, J = 16.0 Hz, 1H), 2.08−1.96 (m,
J = 35.6 Hz, 3H), 1.87−1.84 (t, J = 6.6 Hz, 1H). MS (ESI): m/z (%) =
−
2
62 [M − H] .
5
-({2-[2-(2-Hydroxyethoxy)ethoxy]ethyl}amino)-5-oxopentanoic
acid (4.6 g, 0.0174 mmol) and DCC (3.63 g, 0.0175 mmol) were
dissolved in dichloromethane. NHS (2.02 g, 0.0175 mmol) in DCM
was added, and the reaction mixture was stirred overnight at room
temperature. After filtration and solvent evaporation, compound 6
FTIR Spectroscopy. Fourier transformed infrared (FTIR) spectra in
transmission mode were recorded using a ThermoScientific FTIR
−1
instrument (Nicolet 8700) with a resolution of 4 cm . Dried ND
powder (1 mg) was mixed with KBr powder (100 mg) in an agate
mortar. The mixture was pressed into a pellet under 10 tons load for
2−4 min, and the spectrum was recorded immediately. Sixteen
(
5.2 g; 83%) was obtained as a yellow oil. The crude product was used in
1
the next step without further purification. H NMR (300 MHz, MeOD)
δ 4.4.29−4.21 (s, 1H), 3.76−3.36 (m, 8H), 2.88 (s, 4H), 2.79−2.7 (m,
C
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(
EG) and azide-terminated dopamine ligands (dop−EG and
dop−N ) were synthesized according to the synthetic route
3
presented in Figure 1B. Polyethylene glycol, a class of hydrophilic
polymers with excellent water solubility and biocompatibility, is
widely used for the surface capping of nanostructures to impart
35
water dispersibility. The conjugation of dop−EG (7) onto the
surface of ND particles is expected to improve their dispersion in
aqueous media. At the same time, the integration of EG functions
should reduce the uptake of the nanoparticles by the mononuclear
phagocyte system, which is important for their biomedical use.
Dop−EG (7) is obtained in a multiple step reaction from 2-[2-
(2-chloroethoxy)ethoxy]ethanol (4). The formation of an amide
bond between the terminal amine group of dopamine and the
activated ester of 5-({2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-
amino)-5-oxopentanoic acid (6) resulted in dop−EG. Azide-
terminated dopamine was synthesized in an analogous manner to
dop−EG, through amide bond formation between dopamine and
the activated ester for 6-azidohexanoic acid (2) (Figure 1B).
Before proceeding to the orthogonal surface functionalization
of hydroxylated ND, deagglomeration of the as-received
particles, produced via detonation synthesis, was achieved
through a bead-assisted sonic disintegration process (BASD) as
Figure 2. (A) N 1s high resolution XPS spectra of ND−OH and
ND−dop−EG+N . (B) TEM images of ND−dop−EG+N .
36
reported by Liang et al. The resulting ND−OH particles
showed a hydrodynamic diameter of 79 ± 13 nm (Table 1) and a
zeta potential of ζ = 22 ± 2 mV. The origin of the positive charges
3
3
accumulative scans were collected. The signal from a pure KBr pellet was
subtracted as a background.
is still a point of debate but widely observed for oxygen-
15,37
functionalized NDs.
XPS analysis of the deagglomerated
Transmission Electron Microscopy (TEM). TEM measurements
were performed on an FEI Tecnai G2-F20 microscope.
ND−OH particles indicates the presence of 1.5 atom % nitrogen
(Table 1). The N 1s high resolution core level spectrum of
ND−OH particles shows bands at 402.75 and 399.2 eV assigned
to N−O and C−N bonds present (Figure 2A). This nitrogen
content might be partly responsible for the positive surface
charge of ND−OH particles.
−1
Particle Size Distribution. ND suspensions (20 μg mL ) in water
were sonicated for 1 h. The particle size of the ND suspensions was
measured at 25 °C using a Zetasizer Nano ZS (Malvern Instruments
S.A., Worcestershire, U.K.) in 173° scattering geometry, and the zeta
potential was measured using the electrophoretic mode.
The simultaneous linkage of dopN (3) and dopEG (7)
3
3
. RESULTS AND DISCUSSION
As a preliminary step toward the fabrication of multifunctional,
clickable” diamond particles (Figure 1A), triethylene glycol
to the surface of NDOH was achieved by mixing the particles
with a stoichiometric amount of both dopamine ligands (dop
“
N :dopEG = 1:1) and sonicating the mixtures in an ice bath
3
Table 2. Influence of the Stoichiometry by Using Different Feed Ratios of dop+N (3) and dop−EG (7)
3
a
ratio dop+N (3)/dop−EG (7)
d (nm)
PI
ζ (mV)
C 1s
O 1s
N 1s
3
1/2
1/1
2/1
3/1
105 ± 15
120 ± 19
123 ± 19
121 ± 19
0.346 ± 0.012
0.358 ± 0.015
0.297 ± 0.017
0.324 ± 0.014
23 ± 2
25 ± 1
24 ± 1
26 ± 1
65.7
77.4
68.0
60.8
21.5
12.7
16.5
20.3
12.8
9.9
15.5
18.9
a
Polydispersity index; mean ± SD, n = 3.
−
1
Figure 3. Photograph of suspensions of ND−dop−EG+N (50 μg mL ) in PBS (pH 7.4, 0.1 M) at different time intervals together with a bar diagram
3
of the change in particle size of ND−OH (gray), ND−dop−EG+N (blue), and ND−dop−N (green).
3
3
D
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Figure 4. (A) Postfunctionalization of ND−dop−EG+N with alkynyl-terminated di(amido amine) ligand (8) in a Cu(I) “catalyzed click” reaction.
3
(
B) N 1s high resolution XPS spectra of ND−dop−EG+(NH ). (C) FTIR spectra of ND−OH (black), ND−dop−EG+N (blue), and after “click”
2
3
reaction with alkynyl-terminated di(amido amine) ligand (8) (green). (D) Stability of ND−dop−EG+(NH ) in PBS over time.
2
2
with a horn-type sonotrode for 8 h. The successful synthesis of
counts for 8.4 atom % (six nitrogen atoms). The presence of the
NDdopEG+N particles is confirmed by XPS analysis. The
three NHCO linkages and the N group is
3
3
total atomic percentage of nitrogen increased from the initial
furthermore confirmed by XPS analysis of the N 1s region
(Figure 2A). Next to bands at 402.7 and 399.2 eV due to NO
1
.5 atom % (NDOH) to 9.9 atom % for NDdopEG+N3
Table 1). Assuming that the nitrogen component is only from
the nanodiamond itself, the integration of dopamine ligands
(
and CN of NDOH itself, the contribution of the N
3
+
−
+
−
group at 405.2 (NN N ) and 401.9 eV (NN N )
E
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and the presence of the NHCO linkage at 400.6 eV
can be distinguished. The peak ratio of the NHCO band
over the sum of the azide bands at 405.2 and 401.9 eV is 1.05 and
correlates well with the theoretical ratio 1. This suggests that the
The successful integration of ligand 8 was in addition
confirmed by FTIR analysis (Figure 4C). ND−OH particles
show a broad peak at 3400 cm− assigned to the vibration of
surface hydroxyl groups and/or adsorbed water molecules, and
an additional sharper one at 1633 cm− due to the bending mode
δ(OH) of surface hydroxyl groups on the NDs. In addition, the
1
1
transfer ratio of dopEG (7)/dopN (3) ligands onto NDOH
3
is equivalent to the concentration of the ligand in solution.
−1
To prove the stoichiometric controllability of the postsyn-
band at 1107 cm is indicative of the presence of C−O−C−
thetic modification, particles with different feed ratios of dop−N
3
functions of cyclic ethers. The FTIR spectrum of the ND−dop−
−1
(
3) and dop−EG (7) were prepared. Table 2 shows that different
EG+N particles displays a band at 2125 cm characteristic of
3
feed ratios do not have a significant impact on the size and zeta-
potential of the resulting NDs. From XPS quantification of the
nitrogen component and by accounting for the 1.5 atom %
nitrogen content from diamond itself, it becomes clear that a
the νas(N3) stretching mode. In addition, a broad band between
−1
3
000 and 3600 cm assigned to the vibration of surface amino
groups and/or adsorbed water molecules is seen. The peak at
−1
1
625 cm is most likely due to a superposition of the NH
stoichiometric dopamine transfer takes place for dop−N /dop−
3
scissoring mode and the OH deformation mode of adsorbed
water. Similar vibration modes were reported for amine-
terminated ND particles prepared from hydroxyl-terminated ND
EG ratios of 1/2, 1/1, and 2/3 accounting for approximately
8
N, 6N, and 10N, respectively. However, increasing the dop−
3
N /dop−EG ratio to 3/1 results in the integration of some-
5
particles functionalized with 3-aminopropyltrimethoxysilane.
what more dop−PEG over dop−N and the stoichiometry is
3
The CH stretching bands of the organic linker molecules were
2
sacrificed.
−1
−1
observed at 2855 and 2924 cm . The broad band at 1105 cm is
indicative for the C−O−C stretching bonds from the dop−EG
ligand. Successful “clicking” of ligand (8) to ND−dop−
Transmission electron microscopy (TEM) measurements of
ND−dop−EG+N particles are shown in Figure 2B. They reveal
3
the presence of spherical particles with a mean diameter of 12 ±
EG+N particles results in the consumption of azide functions
3
4
nm obtained from the analysis of several thousands of nano-
and their replacement with surface triazole groups. As a
consequence, the νas(N3) stretching mode at 2094 cm− is absent
in the FT-IR spectrum of the clicked product. The size of ND−
dop−EG+(NH ) particles was estimated as 130 ± 19 nm, with a
particles. The surface modified layer could not be observed due
to its high transparency to the electron beam. However, the
1
hydrodynamic diameter of ND−dop−EG+N is determined as
3
2
2
120 ± 12 nm due to partial aggregation in solution (Table 1).
strongly positive zeta potential of 43 ± 1 mV. This might indicate
However, it remained unchanged over days, indicating good
colloidal stability in aqueous media.
+
3
the presence of partly protonated −NH groups. The stability of
the cationic NDs in PBS over time was remarkable, and no
aggregation occurred over a time period of 1 month (Figure 4D).
The colloidal stability of the ND−dop−EG+N particles in
3
phosphate buffer (PBS) was assessed through particle size
measurements and compared to that of ND−OH, and when
1
0
modified entirely with dop−N (ND−dop−N ) (Figure 3).
4. CONCLUSION
3
3
Suspensions of ND−dop−EG+N particles in PBS show excellent
3
In summary, multifunctional, “clickable” diamond nanoparticles
have been successfully designed. The model material was formed
through the integration of triethylene glycol modified dopamine
and azide-functionalized dopamine ligands. Data obtained from
dispersibility and stability over time. In the case of azide particles
without EG units present, the particle size increased from 150 nm
to around 600 nm in the short time span of 3 h. This indicates the
interest of multifunctinal ND−dop−EG+N particles over ND−
3
XPS analysis suggest that the transfer of dop−EG and dop−N
10
3
dop−N ones as prepared previously.
3
ligands onto the surface of ND−OH is equivalent to the
concentration of the ligand in solution. The colloidal stability of
the formed ND−dop−EG+N3 was highly improved and
comparable to ND−OH. Chemoselective conjugation of an
alkynyl terminated di(amido amine) ligand onto the azide
Postfunctionalization of Azide Functions in ND
dopEG+N Particles with Alkyne-Terminated Ligands.
3
For different applications, NDdopEG+N particles need to
3
sustain further chemical modifications without affecting their
colloidal stability. One class of reactions that is very attractive for
38
functions of ND−dop−EG+N particles using Cu(I) catalyzed
3
an efficient grafting of ligands is the so-called “click” chemistry.
“
click” chemistry could be successfully carried out. The
In a proof of principle, the azide functions were reacted using
branched structures such as the alkynyl terminated di(amido
postfunctionalization had no significant effect on the stability
of the nanostructures over time. While an alkynyl terminated
di(amido amine) ligand was used here, the described concept is
generally applicable to any molecule carrying propargyl arms,
such as glycans, fluorescent molecules, etc. The implementation
of other desired functions depending on the sought after
application is possible, following the outlined strategy, making
the concept of wide interest.
34
amine) ligand (8) (Figure 4A). The success of the integration
of ligand 8 onto NDdopEG+N particles was evidenced
3
by the drastic increase in the atomic percentage of the N 1s
(
16.3 atom %) component when compared to NDdop
EG+N particles (9.9 atom %) (Table 1). For comparison, the
3
amount of nitrogen in commercially available NDNH is only
2
2.3 atom %. The high resolution N 1s XPS spectra of the formed
NDdopEG+(NH ) particles (Figure 4B) reveals the
2
2
conversion of the surface azide groups into the corresponding
triazole functions by the absence of the characteristic azide band
at 405.2 eV. The spectrum can be deconvoluted into bands at
AUTHOR INFORMATION
■
*
Corresponding Author
Phone: +33 3 62 53 17 25. Fax: +33 3 62 53 17 01. E-mail:
sabine.szunerits@iri.univ-lille1.fr.
4
02.6 eV (CN), 400.4 eV (NN and NHCO,
incorporating also the band of the NO from diamond itself),
and 399.5 due to protonated NH groups respectively and the
Notes
2
diamond contribution.
The authors declare no competing financial interest.
F
DOI: 10.1021/acs.langmuir.5b00643
Langmuir XXXX, XXX, XXX−XXX

Langmuir
ACKNOWLEDGMENTS
Article
nanodiamond in Xenopus embryos. J. Mater. Chem. 2010, 20, 8064−
■
8
(
069.
Financial support from the Centre National de la Recherche
Scientifique (CNRS), the Universite Lille 1, and the Institut
Universitaire de France (IUF) is gratefully acknowledged.
Support from the European Union through an FP7-PEOPLE-
IRSES (No. 269009) is acknowledged as well as French Ministry
of Foreign Affairs for a doctoral fellowship for M.K.
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DOI: 10.1021/acs.langmuir.5b00643
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