Fluorescent magnetic nanoparticles based on a ruthenium complex and Fe 3O4

Article abstract of DOI:10.1039/c0jm03119d

A fluorescent ruthenium (Ru) complex is coupled to magnetic Fe ₃O₄ nanoparticles (NPs) via 3-(3,4-dihydroxyphenyl) propanoic acid (DHPPA) and O,O′-bis(2-aminopropyl) polypropylene glycol-block-polyethylene glycol-block-polypropylene

Full text of DOI:10.1039/c0jm03119d

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COMMUNICATION
www.rsc.org/materials | Journal of Materials Chemistry
Fluorescent magnetic nanoparticles based on a ruthenium complex
and Fe3O4†
a
a
b
a
Pinxian Xi,ab Kai Cheng, Xiaolian Sun, Zhengzhi Zeng and Shouheng Sun
*
*
Received 16th September 2010, Accepted 16th November 2010
DOI: 10.1039/c0jm03119d
A fluorescent ruthenium (Ru) complex is coupled to magnetic Fe3O4
nanoparticles (NPs) via 3-(3,4-dihydroxyphenyl) propanoic acid
(DHPPA) and O,O0-bis(2-aminopropyl) polypropylene glycol-
block-polyethylene glycol-block-polypropylene glycol (PPG-PEG-
PPG-diamine). The resultant Ru–Fe3O4 NP conjugate shows
excellent colloidal, photochemical and magnetic stability, and is
promising as a dual functional probe for biological imaging
applications.
Incorporation of optically active components onto magnetic nano-
particles (NPs) has attracted great interest for developing multi-
functional imaging probes for biomedical applications.1–3 The optical
components studied thus far have been organic dyes due to their
strong fluorescent emission and the wide variety of molecules.
However, these organic dyes often suffer from quenching and pho-
tobleaching in biological solutions and are unsuitable for use in the
extended period of imaging.4–8 In searching for new imaging agents
with long-term optical stability, ruthenium (Ru)-based inorganic
complexes have drawn much attention. These complexes are able to
emit intense light via a metal-to-ligand charge transfer (MLCT)
process and can serve as long-living dyes in various aqueous solu-
tions.9 By coupling an optically active Ru complex to a magnetic NP,
one would expect to have a robust dual functional probe as both
a long-living dye and a contrast enhancement agent for simultaneous
optical imaging and magnetic resonance imaging applications.
Here, we report the synthesis of a fluorescent magnetic NP
conjugate of Fe3O4–Ru and demonstrate that it is optically and
magnetically stable for cell imaging applications. The Ru complex,
Fig.
1 A schematic illustration of (A) the structure of Ru-
(dcbpy)2(NCS)2 and (B) the conjugate of Fe3O4 and Ru(dcbpy)2(NCS)2
via DHPPA and PPG-PEG-PPG-diamine.
glycol (PPG-PEG-PPG-diamine) before coupling Ru(dcbpy)2(NCS)2
to PPG-PEG-PPG via the reaction between the amine (-NH2) and
isothiocyanate (-NCS) (Fig. 1B). The presence of PPG-PEG-PPG
between the Ru(dcbpy)2(NCS)2 and Fe3O4 NPs serves two purposes:
to make the whole conjugate stable in biological solutions without
serious toxic effects,16,17 and to eliminate the close-range quenching of
Ru(dcbpy)2(NCS)2 by the Fe3O4 NPs.
The monodisperse 8 nm Fe3O4 NPs were synthesized via the
reductive decomposition of Fe(acac)3 in the presence of benzyl ether
and oleylamine.18 Fig. 2A shows the transmission electron micros-
copy (TEM) image of the 8 nm Fe3O4 NPs. The NPs have a narrow
size distribution with a standard deviation of diameter of less than
10%. These hydrophobic NPs were made hydrophilic by exchanging
oleylamine with DHPPA- PPG-PEG-PPG-NH2, that was pre-
synthesized via the coupling of the commercially available DHPPA
and PPG-PEG-PPG-diamine.19 In this exchange reaction, two
hydroxyl groups in DHPPA replaced oleylamine and reacted with Fe
cations on the Fe3O4 NP surface, forming a strong Fe(III)–O– chelate
bond. The isothiocyanate (-NCS) group in Ru(dcbpy)2(NCS)2
Ru(dcbpy)2(NCS)2 (dcbpy
¼
4,40-dicarboxy-2,20-bipyridine)
(Fig. 1A) was chosen for the optical study.10,11 It is an inorganic dye
and has been widely used in solar cell12,13 and fluorescent imaging
applications.14,15 In our initial tests, we found that the fluorescence
intensity of the Ru(dcbpy)2(NCS)2 dropped about 75% once it was
coupled directly to the Fe3O4 NPs (Fig. S1, ESI†), which is consistent
with what has been reported.7,8 To avoid this quenching problem, we
functionalized the Fe3O4 NPs with 3-(3,4-dihydroxyphenyl)
propanoic acid (DHPPA) and O,O0-bis(2-aminopropyl) poly-
propylene glycol-block-polyethylene glycol-block-polypropylene
aDepartment of Chemistry, Brown University, Providence, RI 02912,
U.S.A. E-mail: ssun@brown.edu; Fax: (+1) 401 863 9046; Tel: (+1)
401 863 3329
bKey Laboratory of Nonferrous Metals Chemistry and Resources
Utilization of Gansu Province, State Key Laboratory of Applied Organic
Chemistry and College of Chemistry and Chemical Engineering, Lanzhou
University, Lanzhou, 730000, P. R. China. E-mail: zengzhzh@lzu.edu.cn;
Fax: (+86) 931 8912582; Tel: (+86) 931 8610877
Fig. 2 TEM images of (A) the as-synthesized 8 nm Fe3O4 NPs from the
hexane dispersion and (B) the Fe3O4-DHPPA-(PPG-PEG-PPG)1900
-
† Electronic supplementary information (ESI) available: Fig. S1–S6. See
DOI: 10.1039/c0jm03119d
(NH–C(S)-NH)2Ru(dcbpy)2 NPs from water.
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reacted with the amine (-NH2) in an excess of Fe3O4-DHPPA-(PPG-
PEG-PPG)1900-NH2 NPs, forming the Ru–Fe3O4 NP conjugate, as
shown in Fig. 1B. Fig. 2B is the TEM image of the Fe3O4 NPs
modified
with
DHPPA-(PPG-PEG-PPG)1900-(NH–C(S)-
NH)2Ru(dcbpy)2. Comparing with Fig. 2A, we can see that the
exchange reaction did not cause any obvious NP morphology
change.
The Ru-conjugated Fe3O4 NPs were readily dispersed in water.
Inductively coupled plasma-atomic emission spectroscopy (ICP-
AES) analysis on the Fe3O4-DHPPA-(PPG-PEG-PPG)1900-(NH–
C(S)-NH)2Ru(dcbpy)2 dispersion showed that the Ru complex
solubility could reach 23.6 mg Ru/ml. This is an increase of over 2
times compared with the free Ru(dcbpy)2(NCS)2 with a solubility of
10.6 mg Ru/ml. ICP-AES analysis further indicated that in the
aqueous solution containing 23.6 mg Ru/ml, there was 1320.3 mg Fe/
ml. Assuming that each Fe3O4 NP was 8 nm in diameter, we esti-
mated that there were ꢀ36 Ru complex molecules coupled to each
Fe3O4 NP.
The conjugation between Fe3O4-DHPPA-(PPG-PEG-PPG)1900
-
NH2 and Ru(dcbpy)2(NCS)2 was characterized by infrared (IR), UV-
Vis absorption and fluorescence spectroscopy. The IR spectrum of
the Ru(dcbpy)2(NCS)2 complex (Fig. S2, ESI†) reveals the
–N]C]S (cumulative double bond) stretching band at 2109 cmꢁ1,
the C]O stretching band of COOH at 1707 cmꢁ1, and the C]N
stretching band of bipyridine at 1615 cmꢁ1. After conjugation to the
Fe3O4-DHPPA-(PPG-PEG-PPG)1900-NH2, the cumulative double
bond disappeared from the IR spectrum and the thioamide bond was
formed with the C]S stretching band red-shifted to 2098 cmꢁ1. The
C]O (from COOH) in the conjugate showed the same absorption at
1707 cmꢁ1 while the C]N (from dcbpy) band appeared at 1623
cmꢁ1. These IR spectrum analyses indicate that the Ru-
(dcbpy)2(NCS)2 binds to PPG-PEG-PPG)1900-NH2 through -NH–
C(S)-NH-, as illustrated in Fig. 1B. The absorption band related to
the free Ru–NCS was not readily found in the IR spectrum of the
conjugate, indicating that both -NCS units in the complex structure
are involved in the coupling reaction and the conjugate shown in
Fig. 1B is the major product.
Fig. 3 (A) The absorption spectra and (B) fluorescence emission spectra
(lex
¼
490 nm) of the Fe3O4-DHPPA-(PPG-PEG-PPG)1900-NH2
(abbreviated as Fe3O4-NH2) and Fe3O4-DHPPA-(PPG-PEG-PPG)1900
-
(NH–C(S)-NH)2Ru(dcbpy)2 (abbreviated as Fe3O4–Ru) NP dispersions
in water (150 mg Fe/ml, 2.68 mg Ru/ml). (C) Fluorescence spectra of
Fe3O4-DHPPA-(PPG-PEG-PPG)1900-(NH–C(S)-NH)2Ru(dcbpy)2 NPs
(2.68 mg Ru/ml) in water (lex ¼ 490 nm) upon addition of H2O2 at the
concentrations of 0, 20, 40 mg mlꢁ1, respectively.
The UV-Vis absorption spectrum of the Fe3O4–Ru conjugate in
water shows two obvious peaks at 312 nm and 500 nm (Fig. 3A). The
absorption at 312 nm comes from the electron transition from
p(bipyridine) to p*(bipyridine) orbitals,20,21 while the 500 nm peak
originates from the metal-to-ligand charge transfer (MLCT) d(Ru)–
p*(bipyridine).20 In the related emission spectrum (Fig. 3B), the
conjugate displays a band centred around 720 nm that arises from the
3MLCT transitions.22,23 As a comparison, the Fe3O4-DHPPA-(PPG-
PEG-PPG)1900-NH2 NPs show very weak UV-Vis absorption and
non-measurable fluorescent emission. The quantum yield of the Ru
complex in the conjugate was measured to be 5.1% (Rhodamine 6G
as the reference). It is slightly reduced from that measured for the free
Ru(dcbpy)2(NCS)2 (6.7%) which is likely due to the partial quenching
by Fe3O4 NPs and the change of the coordination bond character of
the NCS ligand upon its bonding to NH2 in the conjugate.
Fe3O4-DHPPA-(PPG-PEG-PPG)1900-(NH–C(S)-NH)2Ru(dcbpy)2
conjugate. The fluorescent intensity changes were recorded and
shown in Fig. 3C. We can see that with the increased amount of
H2O2 present in the conjugate dispersion, the emission peak of the
conjugate gets stronger and stronger, and has a small blue-shift
due to the enhanced MLCT transitions.24 This proves that the
coupling of Ru(dcbpy)2(NCS)2 to Fe3O4 via the -NH–C(S)-NH-
bond does not change the coordination geometry around the Ru
center.
To study effect of the PPG-PEG-PPG length on the fluorescence
quenching of Ru(dcbpy)2(NCS)2 by Fe3O4 NPs, we chose to func-
tionalize Fe3O4 NPs with different PPG-PEG-PPG-diamine mole-
cules (Mw ¼ 500, 1900 and 3000). Fig. S3, ESI†shows the TEM
images of the Ru–Fe3O4 conjugate with PPG-PEG-PPG in Mw ¼
500, 1900 and 3000, respectively. The average PPG-PEG-PPG
coating thickness around each Fe3O4 NP was characterized with
dynamic light scattering (DLS) that measures the hydrodynamic
diameter of the NPs in their dispersion state. From the measurement
The successful coupling of Ru(dcbpy)2(NCS)2 to Fe3O4-DHPPA-
(PPG-PEG-PPG)1900-NH2 NPs was further confirmed by the fluo-
rescence intensity change of the Ru complex induced by hydrogen
peroxide (H2O2) oxidation. It is known that H2O2 can oxidize
Ru(II) to Ru(III), causing even stronger MLCT transitions.24 In our
test, H2O2 was added at concentrations controlled at 0, 20 and
40 mg mlꢁ1 respectively to the water dispersion of the
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results in water (see Fig. S4A, ESI†), the (PPG-PEG-PPG)500, (PPG-
PEG-PPG)1900 and (PPG-PEG-PPG)3000 coated NPs have average
sizes of 30, 40 and 45 nm, respectively. Fluorescence measurements of
these three NPs show that the conjugate with the (PPG-PEG-
PPG)500 coating was partially quenched and had a 48% intensity
drop, while those with larger PPG-PEG-PPG exhibited only about
6% (for (PPG-PEG-PPG)1900 and 8% (for PPG-PEG-PPG)3000
)
intensity drop compared to the fluorescence intensity of the free
Ru(dcbpy)2(NCS)2 (see Fig. S4B, ESI†).10
The fluorescent stability of the Fe3O4–Ru conjugate with (PPG-
PEG-PPG)1900 was tested and compared with Rhodamine B (RhB)
isothiocyanate conjugated to Fe3O4 NPs via the same chemical
method used for coupling Ru(dcbpy)2(NCS)2 to Fe3O4 (Fig. 1B).
Upon exposure to UV light (365 nm) for 6 h, the fluorescence
intensity of the Fe3O4-DHPPA-(PPG-PEG-PPG)1900-(NH–C(S)-
NH)-RhB NPs decreased 60%, but that of the Fe3O4-DHPPA-(PPG-
PEG-PPG)1900-(NH–C(S)-NH)2Ru(dcbpy)2 NPs did not show any
measurable change (see Fig. S5, ESI†). The dispersion stability of the
Fe3O4–Ru conjugate in different PBS buffer solutions (pH ¼ 5, 6, 7)
plus 10% fetal bovine serum (FBS) was also tested over 50 h at an
Fig. 5 (A) A fluorescent image of the SK-BR-3 cells incubated with the
Fe3O4-DHPPA-(PPG-PEG-PPG)1900-(NH–C(S)-NH)2Ru(dcbpy)2 for
4 h (the conjugate was excited at 488 nm); (B) SK-BR-3 cell viability upon
their incubation with Fe3O4-DHPPA-(PPG-PEG-PPG)1900-(NH–C(S)-
NH)2Ru(dcbpy)2 NPs at different concentrations for 24 h.
The Fe3O4–Ru conjugate as an optical imaging agent was evalu-
ated in SK-BR-3 cells that are Her2-positive breast cancer cells. These
cells were incubated with the conjugate in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% FBS and 1%
penicillin/streptomycin (Gibco BRL, Grand Island, NY) at 37 ꢂC for
4 h. The cells were then washed with PBS and fixed onto a Fluorodish
with 4% paraformaldehyde in PBS for 10 min at room temperature.
After incubation with the conjugate for 4 h, the SK-BR-3 cells were
clearly imaged and the conjugate was seen in the cytoplasm26,28
(Fig. 5A). It is worthy of note that, both Fe3O4-DHPPA-(PPG-PEG-
PPG)1900-NH2 and Fe3O4-DHPPA-(PPG-PEG-PPG)1900-(NH–
C(S)-NH)2Ru(dcbpy)2 showed no measurable toxicity to SK-BR-3
cells29 when the Fe concentration was under 100 mg mlꢁ1 (equal to
0.415 mg Fe3O4 NPs/mL) or the Ru concentration under 3 mg mlꢁ1
(equal to 0.021 mg Ru(dcbpy)2(NCS)2/mL) (Fig. 5B). Furthermore,
ꢂ
incubation temperature of 37 C. DLS was used to track the size
change of the NPs during the incubation period (see Fig. S6, ESI†).
The size was measured to be 38 nm initially and stabilized at about 43
nm after 50 h. The slight increase in size was most likely due to the
absorption of FBS onto the conjugate.
The magnetic properties of the Fe3O4 NPs and the Fe3O4–Ru
conjugate were also investigated with a vibrating sample magne-
tometer. Fig. 4 shows the room temperature hysteresis loops of the
as-prepared Fe3O4 NPs, Fe3O4-DHPPA-(PPG-PEG-PPG)1900-NH2
NPs
and
Fe3O4-DHPPA-(PPG-PEG-PPG)1900-(NH–C(S)-
NH)2Ru(dcbpy)2 NPs. These Fe3O4 NPs are superparamagnetic at
room temperature. The saturated magnetic moment of the Fe3O4-
the
Fe3O4-DHPPA-(PPG-PEG-PPG)1900-(NH–C(S)-
NH)2Ru(dcbpy)2 did not release any detectable amount of Ru under
the current cell culture conditions. This indicates that the Fe3O4–Ru
conjugate is a robust optical probe that can be used for long-time cell
tracking and other imaging applications.
DHPPA-(PPG-PEG-PPG)1900-(NH–C(S)-NH)2Ru(dcbpy)2
NPs
was measured to be 39.8 emu/g NPs, which was lower than that of the
as-synthesized Fe3O4 NPs (62.3 emu/g NPs). This moment reduction
is due to the diamagnetic polymer shell contribution in the conjugate.
The pure Fe3O4 NP cores in these three NPs have a similar magnetic
moment. This indicates that the surface modification does not change
the magnetic properties of the Fe3O4 NPs core and that the Fe3O4–
Ru conjugate should be a good contrast agent for magnetic reso-
nance imaging applications.25
Conclusions
In summary, by coupling fluorescent Ru(dcbpy)2(NCS)2 with
magnetic Fe3O4 NPs via a hydrophilic co-polymer PPG-PEG-PPG,
we have synthesized a new class of dual functional NPs. The Fe3O4–
Ru conjugate is readily soluble in water with the Ru complex solu-
bility reaching 23.6 mg Ru/ml. The polymer coating separates the
Fe3O4 NPs and the Ru complex, preventing the fluorescent Ru
complex from being quenching by the magnetic Fe3O4 NPs. The
Fe3O4–Ru conjugate is colloidally, optically and magnetically stable
in PBS buffers and shows no measurable cytotoxicity to the SK-BR-3
cells. It should serve as a robust dual functional probe for biological
imaging applications.
Acknowledgements
The work was supported partially by the Fundamental Research
Funds for the Central University (Lzujbky-2010-163), the Science
Program of Lanzhou (07-1-32), China Scholarship Council (P.X.)
and the Brown imaging fund.
Fig. 4 Room temperature hysteresis loops of the as-synthesized Fe3O4,
Fe3O4-DHPPA-(PPG-PEG-PPG)1900-NH2 (abbreviated as Fe3O4-NH2)
and Fe3O4-DHPPA-(PPG-PEG-PPG)1900-(NH–C(S)-NH)2Ru(dcbpy)2
(abbreviated as Fe3O4–Ru) NPs.
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