Tetranuclear gadolinium(III) porphyrin complex as a theranostic agent for multimodal imaging and photodynamic therapy

Article abstract of DOI:10.1021/ic500238s

We describe herein the elaborate design of a Gd(III)-porphyrin complex as a theranostic agent for multimodal imaging and photodynamic therapy. Far-red-emitting (665 nm) and high relaxivity (14.1 mM^-1 s ^-1) with 107% increase upon binding to HSA (human serum albumin) (29.2 mM^-1 s^-1) together with efficiently generating singlet oxygen upon exposure to far-red light irradiation at 650 ± 20 nm demonstrate that this Gd(III)-porphyrin complex with four Gd(III)-DTTA units bound to tetraphenylporphyrin acts as a potentially theranostic agent with excellent performance for magnetic resonance imaging, optical imaging, and photodynamic therapy.

Full text of DOI:10.1021/ic500238s

Article
pubs.acs.org/IC
Tetranuclear Gadolinium(III) Porphyrin Complex as a Theranostic
Agent for Multimodal Imaging and Photodynamic Therapy
Jian Luo, Li-Feng Chen, Ping Hu, and Zhong-Ning Chen*
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences,
155 Yangqiao Road West, Fuzhou 350002, China
S
* Supporting Information
ABSTRACT: We describe herein the elaborate design of a Gd(III)−porphyrin complex
as a theranostic agent for multimodal imaging and photodynamic therapy. Far-red-emitting
(665 nm) and high relaxivity (14.1 mM−1 s−1) with 107% increase upon binding to HSA
(human serum albumin) (29.2 mM−1 s−1) together with efficiently generating singlet
oxygen upon exposure to far-red light irradiation at 650 20 nm demonstrate that this
Gd(III)−porphyrin complex with four Gd(III)−DTTA units bound to tetraphenylpor-
phyrin acts as a potentially theranostic agent with excellent performance for magnetic
resonance imaging, optical imaging, and photodynamic therapy.
functions of Gd(III)-based chelate in MRI and porphyrin in
PDT are likely achieved in a combined compound, complex 6
(Scheme 1) is elaborately designed as a novel theranostic agent with
much improved function relative to the individual precursors.
INTRODUCTION
■
Diagnosis and therapy are traditionally regarded as two separate
issues in medical care. To get an optimal curative effect for
many diseases, especially for cancer, they have been combined
mutually and synergistically.1,2 Theranostic agents provide a
feasible approach to accurately locate the tumor tissues and
monitor the therapeutic responses to individual treatments.3
With the rapid development of nanomedicine, many theranostic
agents based on nanomaterials have been reported,3−7 although
their physiological toxicity remains to be clarified. Nevertheless,
most of the clinically used diagnostic and therapeutic drugs are
organic or coordination compounds, in which their physiological
action has been well understood. Therefore, development of
metal-coordination or organometallic compounds as theranostic
agents that combine both diagnostic and therapeutic functions is
more appealing in clinical application.
Scheme 1. Structure of Complex 6
Among multiple diagnostic imaging techniques, MRI (magnetic
resonance imaging) as a noninvasive diagnosis mode can provide
perfectly whole body images with excellent spatial and anatomical
resolution.8−12 Photodynamic therapy (PDT) is a clinically thera-
peutic modality using a photosensitizer (PS) and light irradiation
to eradicate cancer tissues.13−15 Upon irradiation with the proper
wavelength of light, the PS interacts with molecular oxygen to
generate cytotoxic singlet oxygen (1O2) for killing cancer cells.
Furthermore, the inherent photoluminescence of the PS can also
be used for fluorescence imaging to locate the disease, which is
often referred to as photodynamic diagnosis (PDD).3,16,17
Gd(III)-based chelates are widely utilized as contrast agents
of MRI in clinical diagnosis.8−12,18−22 Porphyrins are clinically
used in PDT for cancer treatments because of their capability
to efficiently generate singlet oxygen and the tendency to pre-
ferentially accumulate in tumor tissue.13−17 Considering that both
As shown in Scheme 1, four Gd(III)−DTTA chelate moieties
are incorporated to tetraphenylporphyrin (TPP) through covalent
Received: January 29, 2014
Published: April 2, 2014
© 2014 American Chemical Society
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a
Scheme 2. Synthetic Routes for TPP(H4DTTA)4
a
(i) Ethyl bromoacetate, K2CO3, 18-crown-6, room temperature for 1 day. (ii) Zn(OAc)2·2H2O, refluxed for 4 h. (iii) Tris(2-aminoethyl)amine,
refluxed for 2 days. (iv) Bromoacetic acid ethyl ester, NaHCO3, 50 °C for 2 days. (v) Trifluoroacetic acid, room temperature for 1 day.
ESI-MS (CH2Cl2): m/z 1023.6 ([M + H]+). 1H NMR (CDCl3,
linkages. The chelating character of the DTTA moiety ensures
400 MHz, TMS, ppm): δ −2.74 (s, 2H), 1.45 (t, J = 7.28 Hz, 12H), 4.45
(m, 8H), 4.94 (s, 8H), 7.32 (d, J = 6.88 Hz, 8H), 8.15 (d, J = 6.86 Hz,
8H), 8.88 (s, 8H). 13C NMR (CDCl3, 400 MHz, ppm): δ 14.30, 61.56,
65.80, 112.99, 114.21, 119.51, 132.02, 135.59, 157.78, 169.06.
thermodynamic stability, in vivo safety, and fast water exchange
of complex 6.22 Attachment of four Gd−DTTA units to a rigid
TPP moiety results in a much higher relaxivity (14.1 mM−1 s−1)
per Gd(III) than those of MRI contrast agents (3.0−6.0 mM−1 s−1)
in clinical diagnosis.9,19 The four negative charge of complex 6
induces excellent water solubility and HSA (human serum
albumin) binding character.19,23 Upon binding to HSA, the
relaxivity is remarkably enhanced from 14.1 to 29.2 mM−1 s−1.
Because of much better water solubility compared with free
TPP, the capability of complex 6 to generate singlet oxygen
is significantly improved upon irradiation with a far-red light
(650 20 nm). The superior far-red excitation (650 nm) and
emission (665 nm) ensure complex 6 is applicable to PDT
and PDD in deep tissue.5,24
meso-5,10,15,20-Tetrakis[4-((ethoxycarbonyl)methoxy)-
phenyl]porphyrinatozinc(II) (3). A mixture of 2 (0.75 g, 0.73 mmol)
and Zn(OAc)2·2H2O (0.22 g, 1.0 mmol) in CHCl3 (30 mL) was
refluxed in argon atmosphere for 4 h. After cooling, the reaction mixture
was washed with water for three times. The organic layer was dried over
Na2SO4 and concentrated. The product was purified by chromatography
on a silica gel column using CH2Cl2/CH3OH (100:1) as eluent to give
a purple solid. Yield: 93% (0.74 g). ESI-MS (CH2Cl2): m/z 1085.5
1
([M + H]+). H NMR (CDCl3, 400 MHz, TMS, ppm): δ1.43 (t, J =
7.50 Hz, 12H), 4.42 (m, 8H), 4.91 (s, 8H), 7.31 (d, J = 8.76 Hz, 8H),
8.15 (d, J = 8.13 Hz, 8H), 8.98 (s, 8H). 13C NMR (CDCl3, 400 MHz,
ppm): δ 14.29, 60.37, 65.81, 112.86, 120.52, 131.94, 135.40, 136.27,
150.45, 157.62, 169.09.
Porphyrin 4. Compound 3 (0.54 g, 0.5 mmol) and tris-
(2-aminoethyl)amine (0.73 g, 5.0 mmol) were refluxed in 40 mL of
ethanol for 2 days in argon atmosphere. The solvent was evaporated,
and the solid residua was washed with H2O for five times. The water-
insoluble solid was dried in vacuum to give a deep green solid (0.46 g).
It was used in the next step without further purification.
EXPERIMENTAL SECTION
■
All chemicals were reagent grade and purchased from commercial
sources. All reactions were carried out under a dry argon atmosphere
using Schlenk techniques and vacuum line systems unless otherwise
specified. Solvents were dried, distilled, and degassed prior to use. The
5,10,15,20-tetrakis(4′-hydoxylphenyl)porphyrin (1) was synthesized
as described previously.25
Porphyrin 5. A mixture of 4 (0.46 g) and bromoacetic acid ethyl
ester (1.16 g, 6.0 mmol) in 30 mL of CH3CN was stirred in the presence
of NaHCO3 (0.76 g, 9 mmol) at 50 °C for 2 days in argon atmosphere.
The mixture was then filtered, and the filtrate was evaporated. The
residua was purified by column chromatography on silica gel (CH2Cl2/
CH3OH = 10: 1). The product was obtained as a purple solid by removal
of the solvent. Yield: 13% (0.22 g, calculated from compound 3). ESI-MS
meso-5,10,15,20-Tetrakis[4-((ethoxycarbonyl)methoxy)-
phenyl]porphyrin (2). Compound 1 (0.68 g, 1.0 mmol), K2CO3
(1.4 g, 10 mmol), ethyl bromoacetate (1.0 g, 6.0 mmol), and 18-crown-6
(53 mg, 0.2 mmol) were dissolved in 30 mL of dry acetone and stirred at
room temperature for 1 day. Then the mixture was filtered, and the
filtrate was evaporated to generate the crude residue, which was purified
by chromatography on a silica gel column using ethyl acetate/petroleum
ether (v/v = 1: 2) as eluent to give a purple solid. Yield: 87% (0.89 g).
1
(CH2Cl2): m/z 3313.0 ([M + H]+), 1657.4 ([M + 2H]+). H NMR
(CDCl3, 400 MHz, TMS, ppm): δ 1.47 (S, 143H), 2.60−3.70 (m, 85H),
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OES (ICP-OES) spectrophotometer. Elemental analyses (C, H, and N)
were carried out on a Perkin-Elemer model 240C elemental analyzer.
Electrospray ionization mass spectra (ESI-MS) were performed on
a Finnigan LCQ mass spectrometer. Emission and excitation spectra
were recorded on a Perkin-Elmer LS55 luminescence spectrometer with
a red-sensitive photomultiplier type R928. UV−vis absorption spectra
were measured on a Perkin-Elmer Lambda 25 UV−vis spectrometer.
Emission lifetime was determined on an Edinburgh analytical instru-
ment (FLS920 fluorescence spectrometer) using picosecond pulsed
diode lasers of the EPL at 375 nm excitation. The absolute emission
quantum yields (Φem) at room temperature were recorded on an
Edinburgh analytical instrument (FLS920 fluorescence spectrometer)
with integrating sphere. All of the spectra were recorded at 25 °C in a
100 mM HEPES buffer solution at pH = 7.2.
Figure 1. Linear relationship between 1/T1 and the concentrations of
■
Gd(III) in 100 mM HEPES solution ( , pH = 7.2), 100 mM HEPES
●
▲
solution with 0.6 mM HSA ( ), and HS (human blood serum, ).
T1 Measurements. The longitudinal relaxation times (T1) of
complex 6 were measured by a standard inversion−recovery pulse
sequence on a PQ-001 NMR Analyzing & Imaging system (Shanghai
Niumag Corp.) at 0.55 T and 25 °C. In each case, five samples were
prepared separately, in which the concentrations of Gd(III) were 0,
0.0625, 0.125, 0.25, and 0.5 mM in 100 mM HEPES buffer solutions at
pH = 7.2. The concentration of Gd(III) was detected by ICP-OES.
The relaxivity r1 is commonly used to express the ability of proton
relaxation enhancement of a paramagnetic compound. It is defined as
the slope of eq 1 with mM−1 s−1 as units
4.79 (m, 8H), 7.35 (d, J = 8.50 Hz, 8H), 8.15 (d, J = 8.63 Hz, 1H), 8.97
(s, 8H). 13C NMR (CDCl3, 400 MHz, ppm): δ 28.21, 29.73, 52.06,
52.71, 55.43, 56.13, 67.83, 80.97, 81.61, 113.08, 131.91, 135.51, 150.43,
157.45, 170.21, 170.69.
TPP(H4DTTA)4. To a CH2Cl2 (5 mL) solution of 5 (0.17 g,
0.05 mmol) was added trifluoroacetic acid (5 mL). After stirring at
ambient temperature for 1 day, the solution was evaporated and the
residue redissolved in CH3OH. To the solution was added dropwise
cold diethyl ether to precipitate the product as a green solid, which
was filtered and dried in vacuo. Yield: 90% (0.11 g). ESI-MS (DMSO):
(1/T) = (1/T) + r [M]
(1)
1 obs
1 d
1
1
m/z 2375.0 ([M + Na]+). H NMR (d6-DMSO, 400 MHz, TMS,
ppm): δ −2.89 (S, 2H), 2.6−3.8 (m, 81H), 4.80 (s, 8H), 7.43 (s, 8H),
8.14 (s, 8H), 8.85 (s, 8H). 13C NMR (D-DMSO, 400 MHz, ppm):
δ 38.58, 52.46, 56.05, 60.22, 68.73, 108.44, 113.21, 120.71, 136.60,
138.85, 142.57, 157.53, 161.45, 169.91, 171.08.
where (1/T1)obs and (1/T1)d are the observed values in the presence
and absence of the paramagnetic species and [M] is the concentration
of the paramagnetic species.
T1-Weighted MRI Phantom Images. Phantom images were
performed using a MiniMR-60 NMR Analyzing & Imaging system
(Shanghai Niumag Corp.). Instrumental parameters were set as
follows: a 0.55 T magnet, section thickness = 5.0 mm, TE = 20 ms,
TR = 200 ms.
Fluorescent Displacement Experiments. Warfarin and dansyl-
glycine are well-known probes for site I and site II, respectively.26 Free
warfarin and dansylglycine exhibit weak fluorescence emissions. Upon
binding to HSA, their fluorescence emissions centered at 380 and
490 nm were greatly enhanced. If the binding of the probes to HSA
was displaced by other species, the fluorescence of the probes
will be remarkably decreased. To 100 mM HEPES buffer solutions
(pH = 7.2) containing 5 μM of the fluorescent probe and HSA were
added 40, 20, 10, 5, 2.5, 1.25, 0.625, and 0 μM of complex 6. Emission
Complex 6. To an aqueous solution of TPP(H4DTTA)4 (82 mg,
0.04 mmol) was added dropwise 1 M NaOH solution until pH = 7.0.
An aqueous solution of GdCl3·6H2O (0.06 g, 0.16 mmol) was slowly
added. To maintain the pH at 7.0, 1 M NaOH solution was fur-
ther added. Upon stirring at room temperature for 1 day, an amount
of cold acetone was added carefully to precipitate the product
as a white solid. Yield: 92% (0.12 g). ESI-MS (CH3OH): m/z 3020.3
([M + 3H2O]−). Anal. Calcd for C108H134Gd4N20Na4O48 (Na4TPP-
(DTTAGd)4·8H2O): C, 40.52; H, 4.22; N, 8.75; Gd 19.65. Found: C,
40.82; H, 4.38; N, 8.58; Gd 19.87.
Physical Measurements. 1H and 13C NMR spectra were recorded
at 400 MHz on a Bruker Avance III NMR spectrometer. Gadolinium
content was determined on an Ultima 2 inductively coupled plasma
Figure 2. (A) T1-weighted phantom MR images for complex 6 with a concentration of 0−50.0 μM. (B) T1-weighted phantom MR images for
complex 6. (a) 100 mM HEPES buffer solution (pH = 7.2); (b) 100 mM HEPES buffer solution with 0.6 mM HSA; (c) HS; (d) 31.25 μM complex
6 in 100 mM HEPES solution; (e) 31.25 μM complex 6 in 100 mM HEPES solution with 0.6 mM HSA; (f) 31.25 μM complex 6 in HS.
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Figure 3. (a) Fluorescence spectral changes of HSA binding dansylglycine (1:1) upon displacement by complex 6. (b) Fluorescence spectral changes
of HSA binding warfarin (1:1) upon displacement by complex 6. (c) Fluorescence intensity changes of HSA binding dansylglycine/warfarin (1:1) at
380 (warfarin) or 490 nm (dansylglycine) upon gradual addition of complex 6.
Figure 4. (a) Absorption spectra of 5 μM complex 6 in HEPES buffer solution (solid line) and 5 μM TPP in DMSO/water (1%) (dashed line).
(b) Fluorescence spectra of 20 μM complex 6 in HEPES buffer solution (solid line) and 20 μM TPP in DMSO/water (1%) (dashed line) excited at 441 nm.
spectra of the eight samples were measured at 25 °C. Excitation and
emission wavelengths used for dansylglycine were 365 and 490 nm,
and those for warfarin were 320 and 380 nm.
Table 1. Spectral Properties of Complex 6
λex (nm)
Φ (%)
393
441
522
561
610
5.45
5.28
4.28
5.33
7.52
4553
0.34
Singlet Oxygen Detection by ADPA. Generation of singlet
oxygen was detected chemically according to the literature using the
disodium salt of 9,10-anthracenedipropionic acid (ADPA, Sigma) as a
singlet oxygen sensor.5,27,28 ADPA is bleached by singlet oxygen to its
corresponding endoperoxide (Figure 6). The reaction was monitored
spectrophotometrically by recording the decrease in optical density at
378 nm. A D2O solution of 10 μM complex 6 and 300 μM of ADPA
were used to measure the UV−vis absorption spectra. 5,10,15,20-
Tetraphenylporphyrin (10 μM) and ADPA (300 μM) dispersed in
D2O solution containing 1% DMSO were used as control samples.
ε (M−1 cm−1
)
30 551 52 025 8077
10 900
0.58
a
brightness (mM−1 cm−1
)
1.67
2.75
0.35
b
lifetime
8.54 ns
a
Brightness determined as the product of extinction coefficient ε
and quantum yield Φ.24 Lifetime was measured upon excitation
at 375 nm.
b
A D2O solution of 5 μM TPP and 25 μM ADPA containing 1%
DMSO were used as control samples.
Solutions were saturated with oxygen and exposed to 650
20 or
Singlet Oxygen Detection by 2,5-Dimethylfuran. 2,5-
Dimethylfuran was also used to detect generation of singlet oxygen.29
In the presence of singlet oxygen, 2,5-dimethylfuran undergoes a
[4 + 2] cycloaddition reaction to give an ozonide, which dimerizes
to give a peroxide product as a single diastereomer (Figure 7). Photo-
oxidation of 2,5-dimethylfuran was accomplished through 650 20 nm
light irradiation. Complex 6 (9.0 mg, 3.0 μmol) and 2,5-dimethylfuran
(0.5 μL, 0.45 mmol) were dissolved in 1.0 mL of d6-DMSO.
475−720 nm light irradiation, and their optical densities at 378 nm
were recorded every 10 or 2 min in a UV−vis spectrophotometer.
The light source was obtained from a 500 W tungsten lamp with
650 20 nm narrow-band or 475−720 nm wide-band filters.
After being saturated with oxygen and irradiated with 650 20 nm
band irradiation, fluorescence spectra of a D2O solution containing
5 μM complex 6 and 25 μM of ADPA were also recorded every 5 min.
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Figure 5. Confocal fluorescence images in human lung cancer cells H1299. (a) Cells without being incubated with complex 6. (b) Bright-field image of
a. (c) Overlay image of a and b. (d) Cells incubated with 20 μM complex 6 in PBS buffer for 4 h (excited by a 405 nm laser). (e) Bright-field image of d.
(f) Overlay image of d and e. (g) Same sample as d but excited by a 568 nm laser source. (h) Bright-field image of g. (i) Overlay image of g and h.
Upon saturation with oxygen, the solution was irradiated and the
photo-oxidation progress was monitored using NMR spectroscopy.
Fluorescence Imaging Experiment. A human lung cancer
cell line H1299 was purchased from commercial sources. Cells were
grown in MEM (Modified Eagle’s Medium) supplemented with 10%
FBS (Fetal Bovine Serum) at 37 °C and 5% CO2. Cells (5 × 108 /L)
were plated on 18 mm glass coverslips and allowed to adhere for 24 h.
Cells were mounted for direct microscopic observation at 37 °C.
Confocal fluorescence images of cells were performed with an
Olympus Fluo View FV1000 laser-scanning microscope. A 60× oil-
immersion objective lens was used. Excitation was carried out with a
semiconductor laser at λ = 405 or 568 nm, and emission was collected
in the range λ = 600−700 nm, including the maximum emission of
complex 6 at 665 nm. Lung cancer cells H1299 were incubated with a
PBS solution of complex 6 (20 μM) for dye loading for 4 h at 37 °C.
Stained cells were washed three times with PBS buffer. Then the
treated cells were imaged by fluorescence microscopy. For the control
experiment, cells were washed three times with PBS buffer and then
imaged by fluorescence microscopy directly.
mixing the free ligand with GdCl3·6H2O in a 1:4 molar
ratio at pH = 7.0 and characterized by ESI-MS and elemental
analysis.
T1 Relaxivity and in Vitro MR Imaging. The relaxivity
of complex 6 was as high as 14.1 mM−1 s−1 per Gd(III) in
100 mM HEPES buffer solution (25 °C, pH = 7.2, 23 MHz).
Investigated in a buffer solution with 0.6 mM HSA, the
relaxivity of complex 6 was further increased from 14.1 to
29.2 mM−1 s−1 (25 °C, pH = 7.2, 23 MHz) with 107%
enhancement (Figure 1). The signal intensity of T1-weighted
phantom MR image was distinctly improved with the increase
of the concentrations of complex 6 (Figure 2A). In addition,
the signal intensity of T1-weighted phantom MR image was
significantly improved (Figure 2B, e) upon binding to HSA.
Quite high relaxivity (14.1 mM−1 s−1 for per Gd(III)) is
ascribed to the large molecular weight (fw = 3201) and high
molecular rigidity, thus inducing a long rotational tumbling
time (τR).9 In addition, the HSA binding further enhanced
the molecular weight of complex 6 that induces a surprising
increase of relaxivity.
To investigate the relaxivity of complex 6 under physiological
condition, relaxivity measurements were also performed in
human blood serum (HS) (Figure 1). Remarkably, the relaxivity
of complex 6 was estimated as high as 21.2 mM−1 s−1 (32 °C,
23 MHz) when it was measured in HS. The signal intensity of
the T1-weighted phantom MR image in HS (Figure 2B, f) was
much improved relative to that in HEPES buffer (Figure 2B, d).
Interaction between Complex 6 and HSA. There are a
variety of biomacromolecules in the human body such as protein,
glycogen, and nucleic acid, etc. It is likely that the interaction
of contrast agents with macromolecules could slow down the
RESULTS AND DISCUSSION
■
The ligand was synthesized by a five-step route as shown
in Scheme 2. The initial step was nucleophilic substitution
of 5,10,15,20-tetrakis(4′-hydoxylphenyl)porphyrin (1) with four
ethyl bromoacetates in a yield of 87%. Porphyrin 2 was stabilized
by coordination of Zn2+ to give Zn(II) complex 3 in 93% yield.
Because of the poor stability of Zn(II) porphyrin complex in
hot solution and long-term reaction process, the overall yield of
the following two-step amination and carboxymethylation was
only 13%. Free ligand TPP(H4DTTA)4 was then obtained
from an ester hydrolysis reaction in 90% yield in which
coordinated Zn2+ was lost during the hydrolysis process in the
presence of trifluoroacetic acid. Complex 6 was prepared by
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Figure 6. (a) Time-dependent bleaching of ADPA by singlet oxygen generated from complex 6. (b) Change in the absorption of ADPA at 378 nm
with exposure time. (c) Time-dependent fluorescence quenching of ADPA by singlet oxygen generated from complex 6. (d) Change in emission
intensity of ADPA at 453 nm with exposure time. Solutions were exposed to far-red light irradiation at 650 20 nm.
tumbling rate and increase the relaxivity of Gd-based contrast
agents. HSA is the most abundant protein in blood plasma and
cerebral spinal fluid with a molecular weight of 66 kDa and a
concentration between 0.53 and 0.75 mM.19,26 It is known that
HSA can accumulate in a tumor since it is taken up by tumor
cells at a higher level relative to normal cells.19 Upon binding
to HSA, the relaxivity of complex 6 is remarkably enhanced
from 14.1 to 29.2 mM−1 s−1 and the signal intensity of the
T1-weighted phantom MR image was also significantly improved.
Although the emission spectrum of complex 6 was little shifted
(from 665 to 661 nm), the emission intensity exhibited 26%
enhancement. The noncovalent interaction of HSA with complex
6 originates mostly from electrostatic interaction because the
inner surface of the hydrophobic pocket of HSA is positively
charged whereas complex 6 dispalys four negative charge.
Hydrophobicity and stereospecificity are likely another two
factors that result in the interaction of HSA with complex 6.19
Such noncovalent interactions are of particular significance
because they may afford better accessibility toward low
concentration target with less toxicity and longer half-life of
metabolism in the human body.9
displaced by other species, the fluorescence will be remarkably
reduced. As shown in Figure 3, the fluorescence of both
warfarin and dansylglycine was gradually reduced by addition of
complex 6, implying that complex 6 binds to HSA at both site I
and site II.
Spectral Properties of Complex 6. The TPP moiety is
an intense chromophore with strong Soret band and medium
Q-band absorption. As shown in Figure 4a, relative to free TPP,
the Q-band of complex 6 shows a little red shift due to the
more extended system upon incorporating TPP with four Gd−
DTTA units. This wide range of visible absorption qualifies
complex 6 as a efficient photosensitizer. Upon excitation at
λex > 350 nm, complex 6 shows intense emission at 665 nm,
which is a little red shifted with a much stronger intensity rela-
tive to that of free TPP (655 nm, Figure 4b). The emission of
complex 6 can be excited by a wide range of exciting wavelengths.
Upon excitation at different wavelengths, the fluorescence quantum
yield of complex 6 was estimated as 4.3−7.5% (Table 1). Upon
irradiation by a red light at 610 nm, far-red luminescence occurred
with the maximum at 665 nm and Φem = 7.5%. The far-red
emission triggered by red excitation ensures complex 6 as an
optimal probe that is available for deep tissue imaging and whole-
body imaging.24 Additionally, the fluorescence spectrum was
obviously dependent on pH value. The emission at 665 nm pro-
gressively enhanced with the increase of pH, and there existed a
jump from pH = 5.5 to 6.5 (Figure S3, Supporting Information).
The solution changed from green to purple with the increase of
pH from 3.0 to 10.0.
To determine whether complex 6 binds at Suddlow site I
(subdomain IIA) or Suddlow site II (subdomain IIIA) of HSA,
displacement experiments were performed using warfarin as
a fluorescent probe for site I and dansylglycine for site II.26
Although free warfarin or dansylglycine exhibits weak fluo-
rescence, the emission centered at 380 (warfarin) or 490 nm
(dansylglycine) is greatly enhanced upon binding to HSA.
When the binding site of warfarin or dansylglycine to HSA is
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variation in the absorption of ADPA at 378 nm was unobserved
in the corresponding D2O solution without porphyrin species
under light irradiation (Figure 6b), further confirming that
the bleaching of ADPA in the presence of porphyrin is induced
by singlet oxygen and not by the irradiated light. Upon excita-
tion at 370 nm, ADPA exhibits intense fluorescence at 453 nm.
As depicted in Figure 6c/6d, this fluorescence band was pro-
gressively reduced due to formation of nonfluorescent
endoperoxide through oxidation of ADPA by singlet oxygen
that is generated upon exposing complex 6 to visible irradiation.
In contrast, the absorption (Figure 6a, inner) and fluorescence
(Figure 6c, inner) of complex 6 itself was almost unchanged
upon exposure to visible irradiation, implying that it exhibits
excellent photostability.
1
Generation of O2 was further confirmed by a chemical
trapping method using 2,5-dimethylfuran as a substrate, which
underwent a [4 + 2] cycloaddition reaction with singlet oxygen
and then dimerized rapidly to a peroxide product.29 The photo-
1
oxidation process of 2,5-dimethylfuran was monitored by H
NMR spectroscopy. As shown in Figure 7, the 1H NMR
spectrum of 2,5-dimethylfuran exhibits two singlet peaks at
5.90 (2H, furan) and 2.19 ppm (6H, methyl). When a d6-
DMSO solution of 2,5-dimethylfuran was exposed to red light
(650 20 nm) irradiation in the presence of 1/150 equiv of
complex 6, the signals at 5.90 and 2.19 ppm decreased gradually
and vanished entirely in 4 h. Meanwhile, a group of new signal
peaks occurred at 6.47 and 1.55 ppm and enhanced pro-
gressively due to conversion of 2,5-dimethylfuran to the peroxide
product. Thus, the photo-oxidization experiment further
demonstrates that singlet oxygen is indeed generated upon
irradiation of complex 6 with visible light.
Figure 7. Process of photo-oxidation of 2,5-dimethylfuran by singlet
oxygen generated from complex 6, monitored using H NMR spectros-
copy. Solutions were exposed to far-red light irradiation at 650 20 nm.
1
Fluorescence Imaging. As both photosensitizer and
luminophore, the central TPP moiety generates effectively
singlet-oxygen upon excitation with a far-red irradiation (650 nm).
Furthermore, the TPP moiety exhibits a far-red emission
(665 nm) with favorable luminescence efficiency. The superior
far-red excitation and emission ensure complex 6 applicable to
PDT and PDD in deep tissue.5,24
Human lung cancer cell H1299 was used to verify the cellular
uptake of complex 6 (Figure 5). Confocal fluorescence image
experiments upon irradiation with a 405 (Figure 5d−f) or 568
(Figure 5g−i) nm laser revealed that red fluorescence of complex
6 was distributed in the whole inside space of cells, which
was clearly naked-eye observable (Figure 5d or 5g). Bright-field
measurements (Figure 5e or 5h) indicated that the cells were
viable throughout the imaging experiments upon treatment with
complex 6. It is ingested harmlessly by living cells with sufficiently
low cytotoxicity so as to be applied in vivo.
CONCLUSION
■
Complex 6 with four Gd−DTTA moieties appended to a
porphyrin framework was elaborately designed. The longi-
tudinal relaxivity is as high as 14.1 mM−1 s−1 in HEPES buffer
solution (pH = 7.2). Upon interacting with HSA, r1 is increased
to 29.2 mM−1 s−1 with 107% enhancement. Since HSA can be
taken up by tumor cells at a higher level relative to normal cells,
the biological compatibility and targeting are significantly
enhanced upon binding to HSA, whereas the cell toxicity is
much reduced. Fluorescence imaging studies indicate that it can
be ingested harmlessly by living cells. The Gd(III)−porphyrin
species exhibits much higher photodynamic activity than free
TPP, in which singlet oxygen is efficiently generated upon
exposure to far-red light irradiation at 650
20 nm. It is a
potential candidate as a clinical theranostic agent with excellent
functions for multimodal imaging and photodynamic therapy.
Photodynamic Activity of Complex 6. Generation of
singlet oxygen triggered by complex 6 was detected chemically
using the disodium salt of 9,10-anthracenedipropionic acid
(ADPA) as a detector (Figure 6), which is bleached to non-
ASSOCIATED CONTENT
■
5,27
1
fluorescent endoperoxide in the presence of O2.
Figure 6b
S
* Supporting Information
shows the decrease of the absorption at 378 nm as a function
of irradiation time. The sharp decrease in the absorbance of
ADPA with irradiation time demonstrated the generation of 1O2
when a D2O solution of complex 6 was exposed to light irra-
diation at 650 20 nm. The absorbance of ADPA at 378 nm
decreased much more slowly in the case of free TPP (decreased
about 20% in 150 min), indicating that free TPP exhibits a much
Figures giving additional UV−vis and emission spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
1
lower efficiency to generate O2 than complex 6. Incorporat-
*E-mail: czn@fjirsm.ac.cn.
ing tetraphenylporphyrin with four Gd−DTTA moieties thus
1
Notes
improves significantly the capability to generate O2 due to the
much increased water solubility. In striking contrast, distinct
The authors declare no competing financial interest.
4190
dx.doi.org/10.1021/ic500238s | Inorg. Chem. 2014, 53, 4184−4191

Inorganic Chemistry
Article
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ACKNOWLEDGMENTS
■
We are thankful for financial support from the NSFC (20931006,
91122006, and 21390392), the 973 project (2014CB845603)
from MSTC, and the NSF of Fujian Province (2011J01065).
(25) Esch, J. H.; Feiters, M. C.; Peters, A. M.; Nolte, R. J. M. J. Phys.
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