Gadolinium(III) chelated conjugated polymer as a potential MRI contrast agent

Article abstract of DOI:10.1016/j.polymer.2009.04.003

Nuclear magnetic resonance imaging (MRI) has become a powerful technique in clinical diagnostics. In this work, a new MRI contrast agent by covalently linking Gd(III) chelates to the side chain of conjugated polymer (PF-Gd) is synthesized by Suzuki cross-

Full text of DOI:10.1016/j.polymer.2009.04.003

Polymer 51 (2010) 1336–1340
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Gadolinium(III) chelated conjugated polymer as a potential MRI contrast agent
,
,
,
,
,
,
*
Qingling Xu a 1, Litao Zhu b, Minghui Yu a 1, Fude Feng a 1, Lingling An a 1, Chengfen Xing a 1, Shu Wang a
a Beijing National Laboratory for Molecular Science, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun North First street,
Beijing 100190, PR China
b State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, Beijing 100875, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nuclear magnetic resonance imaging (MRI) has become a powerful technique in clinical diagnostics. In
this work, a new MRI contrast agent by covalently linking Gd(III) chelates to the side chain of conjugated
polymer (PF–Gd) is synthesized by Suzuki cross-coupling reaction. The PF–Gd exhibits a higher relaxivity
and a pronounced enhancement in contrast than that of (NMG)2–Gd–DTPA widely used for clinical
diagnosis. This work should be feasible to potentially lead to a new class of imaging contrast agents.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 19 November 2008
Received in revised form
19 March 2009
Accepted 3 April 2009
Available online 12 April 2009
Keywords:
Conjugated polymers
Synthesis
MRI contrast agent
1. Introduction
molecules [5,6]. They are characterized by a delocalized electronic
structure, where the multiple and single bonds appear in turn along
Nuclear magnetic resonance imaging (MRI) has become
a powerful technique in clinical diagnostics [1]. The image is based
on the NMR signal from the protons of water, where the signal
intensity depends on the water concentration and relaxation times
(T1 and T2). The ability of contrast agents to reduce the longitudinal
relaxation time (T1) of water protons is evaluated by relaxivity (R1)
[1]. Paramagnetic Gd(III) complexes [2], such as Gd(III)–DTPA,
Gd(III)–DOTA and their derivatives, are widely used as contrast
agents for MRI to enhance image contrast due to their abilities to
shorten the relaxation times. However, their relaxivities are still
relatively low to effectively image diseased tissues. To improve the
relaxivity, a strategy has been demonstrated to reduce the molecule
tumbling by attaching Gd(III) chelate to macromolecules [3].
However, due to the internal rotation of flexible chain, the linear
polymer-based contrast agents improve relaxivity slightly.
Although poly(amidoamide) (PAMAM) dendrimers can increase
relaxivity greatly, relaxivity per gadolinium levels off for a limited
water exchange rate at higher generation [4]. It’s necessary to find
new macromolecular structures to get more insight into increasing
the relaxivity.
the backbone to make CPs more rigid than flexible polymers. To the
best of our knowledge, there is no report that takes advantage of
the CPs to improve the relaxivity of Gd(III) complex. In this paper,
we introduce the first CP–Gd(III) conjugate (PF–Gd, see Scheme 1
for the chemical structure) as contrast agent that is designed to
improve the relaxivity.
2. Results and discussion
Scheme 2 shows the synthetic entry into the monomer 7 and the
polymer PF–Gd. Reaction of 2,7-dibromo-9,9-bis(60-bromohexyl)-
fluorene (1) with potassium acetate in DMSO provided 2,7-
dibromo-9-(60-bromohexyl)-9-(60-hydroxylhexyl)fluorene (2) in
37% yield. Compound 3 was obtained by reaction of 2 with 2-
(benzylideneamino)phenol in the presence of 18-crown-6 and
K2CO3 in acetone in 74% yield. After reaction of 3 with ethyl bromo-
acetate in the presence of N,N-diisopropyl-ethylamine (DIEA) and
NaI in anhydrous DMF, the ester 4 was obtained in 65% yield. The
hydroxy group of 4 was bromized by CBr4/PPh3 to get 5 in 83% yield,
which was treated with cyclen in CHCl3 to afford compound 6 in
58% yield. The monomer 7 was obtained by reaction of tert-butyl
bromoacetate with 6 in acetonitrile in 83% yield. The ester-pro-
tected copolymer PF-ester was prepared by Suzuki cross-coupling
reaction [7] between 7, 8 and 9 (with a molar feed ratio of
0.2:0.8:1.0) in the presence of 2.0 M K2CO3 aqueous solution and
Pd(PPh3)4 in THF. The PF-ester was treated with KOH solution in
CH3OH followed by Boc-deprotecting with trifluoroacetic acid (TFA)
Conjugated polymers (CPs) have been used as optical platforms
in highly sensitive bioassays for proteins, nucleic acids and small
* Corresponding author. Tel./fax: þ86 10 6263 6680.
E-mail address: wangshu@iccas.ac.cn (S. Wang).
1
Tel./fax: þ86 10 6263 6680.
0032-3861/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymer.2009.04.003

Q. Xu et al. / Polymer 51 (2010) 1336–1340
1337
conjugated polymers [15] with good biocompatibility is underway
in our group.
4. Experimental section
4.1. Material and instrumentation
All chemicals were purchased from Arcos or Alfa Aesar, and used
as received. 2,7-Dibromo-9,9-bis(60-bromohexyl)fluorene [10], 2-
(benzylideneamino)phenol [11], cyclen [12] and compound 9 [13]
were prepared according to the procedures in literatures. Pulmonary
adenocarcinoma cell (A549) was purchased from cell culture center
of Institute of Basic Medical Sciences, Chinese Academy of Medical
Sciences (Beijing, China) and grown in a humidified atmosphere
containing 5% CO2 at 37 ꢁC. The concentration of Gd(III) was deter-
mined by ICP spectrometer. Magnetic resonance imaging (MRI) was
performed on a Siemens 3T scanner (MAGNETOM Trio, A Tim
System) with a TxRx Head coil at Imaging Center for Brain Research
of Beijing Normal University. T1 values were measured using an
Inversion Recovery TSE (IR-TSE) imaging sequence with varying IR
time. The 1H NMR and 13C NMR spectra were recorded on a Bruker
Avance 400 MHz spectrometer. Mass spectra were recorded on
Bruker Biflex MALDI-TOF spectrometer. Elemental analyses were
carried out with a Flash EA1112 instrument. The gel permeation
chromatography (GPC) measurements were performed on Water-
410 system against polystyrene standard with THF as the eluent.
Scheme 1. The chemical structure of PF–Gd.
to give water-soluble PF. The 1H NMR spectroscopy of PF is given in
Fig. 1b. On comparing with that of ester-protected PF-ester (Fig. 1a),
we could find that the proton peaks of –COOCH2CH3
(
dCH ¼ 3:99 ppm, dCH ¼ 1:08 ppm) and Boc (dCH ¼ 1:35 ppm)
2
3
3
groups in PF-ester disappeared. These results indicated that the
protected ethyl and Boc groups were gotten rid of entirely upon
treatments with KOH and TFA, respectively. The PF was mixed with
GdCl3 in DMSO/H2O solution to afford target PF–Gd complex,
where the Gd(III) content was analyzed using ICP spectrometer.
The ability of PF–Gd to enhance the contrast in MRI images was
studied by measuring T1 of water protons as a function of Gd(III)
concentration in aqueous solution. In these experiments the gado-
pentetate dimeglumine ((NMG)2–Gd–DTPA) [8] which is widely
used in clinical diagnosis, was used for comparison. As shown in
Fig. 2a, the T1-weighted images showed that PF–Gd could more
efficiently enhance the contrast in comparison with (NMG)2–Gd–
DTPA at the same Gd(III) concentration. The relaxivity (R1) was
obtained by taking the slope of a plot of Tꢀ11 versus Gd(III) concen-
tration (Fig. 2b). The R1 value of PF–Gd (12.57 mMꢀ1 sꢀ1) was 3.5
times higher than that of (NMG)2–Gd–DTPA (3.64 mMꢀ1 sꢀ1). The
rigidity of the conjugated polymer backbone may result in an
increase in the rotational correlation time and subsequently an
increase in relaxivity. It was noted that the formation of aggregation
[9] due to the amphiphilic characteristic of PF–Gd could also play
roles in the increase of relaxivity.
4.2. Synthesis of 2,7-dibromo-9-(60-bromohexyl)-9-
(60-hydroxylhexyl)fluorene (2)
A mixture of 2,7-dibromo-9,9-bis(60-bromohexyl) fluorine (1)
(4.46 g, 6.86 mmol) and potassium acetate (0.0672 g, 0.685 mmol)
in 50 mL DMSO was stirred at 80 ꢁC for 4 h. After cooling to room
temperature, 50 mL of H2O was added and the mixture was
extracted with CH2Cl2 (40 mL ꢂ 3). The organic layer was washed
with H2O (50 mL ꢂ 3) and then was dried over anhydrous MgSO4.
The solvent was removed at reduced pressure and the resulting
residue was purified by silica gel column chromatography with
petroleum ether/ethyl acetate 4:1) as eluent to yield 2 as a white
solid (1.47 g, 37%). 1H NMR (400 MHz, CDCl3)
d: 7.52 (d, 2H), 7.47–
7.43 (m, 4H), 3.53 (t, 2H), 3.29 (t, 2H), 1.92 (m, 4H), 1.67 (m, 2H), 1.38
(m, 2H), 1.20 (m, 2H), 1.11 (m, 6H), 0.59 (m, 4H); 13C NMR (100 MHz,
CDCl3) d: 152.22, 139.02, 130.25, 126.08, 121.51, 121.18, 62.75, 55.54,
40.03, 33.82, 32.57, 29.52, 28.91, 27.71, 25.26, 23.55, 23.44; MS
(MALDI-TOF): 588.0 (M þ Hþ); C25H31OBr3: Calcd. C 51.13, H 5.32;
Found C 51.15, H 5.33.
Because of the importance for in vivo use, the biocompatibility of
PF–Gd was studied by typical MTT assay method, in which the
pulmonary adenocarcinoma cell (A549) was incubated with PF–Gd.
The conversion of soluble MTT into formazan is directly related to
mitochondrial activity and subsequently to cell viability [14]. As
shown in Fig. 3, the cell viability decreases down to 37% as the Gd3þ
4.3. Synthesis of compound 3
A mixture of 2 (1.47 g, 2.5 mmol), 2-(benzylideneamino)phenol
(0.59 g, 3 mmol), 18-crown-6, and K2CO3 (0.41 g, 3 mmol) in 10 mL
acetone was refluxed for 1.5 days. After cooling to room tempera-
ture, the solvent was removed under reduced pressure, and then
15 mL of water was added. The mixture was extracted with CH2Cl2
(10 mL ꢂ 3). The organic layer was washed with 20% NaOH aqueous
solution (10 mL ꢂ 6), then dried over anhydrous Na2SO4. The solvent
was removed at reduced pressure and the resulting residue was
purified bysilica gel column chromatography with petroleum ether/
ethyl acetate 4:1) as eluent to afford 3 as sticky oil (1.14 g, 74%). 1H
concentration increases from 0.1 to 3.2 mM with IC50 value of 2.6 mM.
The results show that the PF–Gd is cytotoxic to the A498 cell.
3. Conclusions
In summary, we have developed a new MRI contrast agent by
covalently linking Gd(III) chelates to the side chain of conjugated
polymer. The new agent exhibits a higher relaxivity and a more
pronounced enhancement in contrast than that of (NMG)2–Gd–
DTPA widely used for clinical diagnosis. This work should be
feasible to potentially lead to a new class of imaging contrast
agents. The drawback of this new agent is its cytotoxicity to the cell
and the lack of biodegradability, which restricts its in vivo appli-
cation. The design of other new MRI contrast agent based on
NMR (400 MHz, CDCl3) d: 7.52 (d, 2H), 7.46 (d, 4H), 6.78–6.67 (m,
4H), 3.87 (t, 2H), 3.53 (t, 2H), 2.05–1.91 (m, 4H), 1.63 (t, 2H), 1.38 (t,
2H), 1.26–1.21 (m, 2H), 1.13 (d, 6H), 0.62 (t, 4H); 13C NMR (100 MHz,
CDCl3) d: 152.32,146.65,139.05,136.20,130.25,126.11,121.51,121.20,
120.90, 118.42, 115.03, 111.44, 68.00, 62.76, 55.59, 40.05, 32.56,
29.55, 29.19, 25.74, 25.29, 23.62, 23.58; MS (MALDI-TOF): 616.2

1338
Q. Xu et al. / Polymer 51 (2010) 1336–1340
Scheme 2. Synthesis of PF–Gd: (a) KOAc, DMSO, 80 ꢁC, 37%; (b) 2-(benzylideneamino)phenol, 18-crown-6, K2CO3, acetone, reflux, 74%; (c) ethyl bromoacetate, DIEA, NaI, DMF,
100 ꢁC, 65%; (d) PPh3, CBr4, CH2Cl2, 0 ꢁC, 83%; (e) cyclen, CHCl3, room temperature, 58%; (f) tert-butyl bromoacetate, K2CO3, acetonitrile, room temperature, 83%; (g) 2.0 M K2CO3,
Pd(PPh3)4, THF/water, 80 ꢁC; (h) first step: KOH, CH3OH, DMSO, 80 ꢁC, second step: TFA, room temperature; (i) GdCl3, DMSO/water, room temperature.
(M þ Hþ), 638.1 (M þ Naþ); C31H37O2NBr2: Calcd. C 60.50, H 6.06; N
CDCl3)
8H), 3.83 (t, 2H), 3.51 (t, 2H), 1.93 (m, 4H), 1.60 (m, 2H), 1.37 (m, 2H),
1.23 (m, 8H), 1.11 (b, 6H), 0.61 (b, 4H); 13C NMR (100 MHz, CDCl3)
d: 7.52 (d, 2H), 7.45 (d, 4H), 6.88–6.75 (m, 4H), 4.17–4.10 (m,
2.28; Found C 60.61, H 6.26, N 1.81.
d
:
171.51, 152.29, 150.65, 139.03, 130.23, 126.08, 122.05, 121.49, 121.18,
120.78, 118.90, 112.95, 68.40, 62.72, 60.52, 55.57, 53.60, 53.42,
40.06, 32.54, 29.62, 29.53, 29.07, 25.66, 25.27, 23.65, 23.57, 14.20;
MS (MALDI-TOF): 788.2 (M þ Hþ), 810.2 (M þ Naþ); C39H49O6NBr2:
Calcd. C 59.47, H 6.27; N 1.78; Found C 58.84, H 6.25, N 1.80.
4.4. Synthesis of compound 4
A mixture of 3 (0.97 g, 1.57 mmol), ethyl bromoacetate (0.53 mL,
4.7 mmol), DIEA (0.68 mL, 3.9 mmol) and NaI (0.47 g, 3.14 mmol) in
8.4 mL anhydrous DMF was stirred at 100 ꢁC under N2 atmosphere
for 49 h. After cooling to room temperature, 30 mL of CH2Cl2 was
added, and the organic layer was washed with H2O (15 mL ꢂ 6). The
organic layer was dried over anhydrous MgSO4 and was removed
under reduced pressure. The residue was purified by silica gel
column chromatography with petroleum ether/ethyl acetate 4:1)
as eluent to afford 4 as sticky oil (0.81 g, 65%). 1H NMR (400 MHz,
4.5. Synthesis of compound 5
Tothe solution of 4 (0.76 g, 0.96 mmol)in24 mL anhydrous CH2Cl2
under N2 atmosphere was added PPh3 (0.50 g, 1.92 mmol). After
cooling to 0 ꢁC, CBr4 (0.64 g,1.92 mmol) was added under exclusion of

Q. Xu et al. / Polymer 51 (2010) 1336–1340
1339
Fig. 3. Cell viability as a function of Gd3þ concentrations by typical MTT assay. Cells
were subcultured in 96-well plates the day before the experiment at a density of
6 ꢂ 104 cells/well, and cultured for 24 h. Then cells were treated with PF–Gd with
different concentrations for 24 h respectively. [Gd3þ] ¼ 0.1–3.2
M, [MTT] ¼ 1.0 mg/mL
m
(100 mL/well).
4.6. Synthesis of compound 6
A solution of cyclen (0.16 g, 0.94 mmol) in 3 mL CHCl3 was
passed through neutral alumina, and then 5 (0.32 g, 0.38 mmol) in
1 mL CHCl3 was added dropwise under N2 atmosphere. The
resulting solution was stirred at room temperature for 2 days. The
organic layer was removed under reduced pressure and the residue
was purified by silica gel column chromatography with chloroform/
methanol/aqueous NH4OH (8:2:0.2) as eluent to yield 6 as brown
oil (0.206 g, 58%). 1H NMR (400 MHz, CDCl3)
d: 7.52 (d, 2H), 7.45 (d,
Fig. 1. 1H NMR spectra of PF-ester (a) and PF in DMSO-d6 (b) at room temperature.
4H), 6.84–6.76 (m, 4H), 4.14 (q, 4H), 4.10 (s, 4H), 3.82 (t, 2H), 2.75 (t,
4H), 2.60 (t, 4H), 2.55 (t, 4H), 2.48 (t, 4H), 2.30 (t, 2H), 1.91 (m, 4H),
1.59 (m, 2H), 1.30 (m, 2H), 1.26–1.21 (m, 8H), 1.08 (m, 6H), 0.60 (br,
light and the resulting solution was stirred for 3 h. The organic layer
was removed under reduced pressure and the residuewas purified by
silica gel column chromatography with petroleum ether/ethyl acetate
4:1)aseluent to afford 5 asbrownoil (0.68 g, 83%).1HNMR (400 MHz,
4H); 13C NMR (100 MHz, CDCl3)
d: 171.36, 152.32, 150.55, 138.93,
130.12, 126.00, 121.95, 121.39, 121.15, 120.69, 118.76, 112.87, 68.32,
60.41, 55.53, 54.18, 53.51, 51.36, 47.04, 46.00, 45.22, 40.03, 39.87,
29.54, 28.99, 26.86, 25.58, 23.67, 23.58, 14.16; MS (MALDI-TOF):
942.5 (M þ Hþ), 964.4 (M þ Naþ); C47H67O5N5Br2: Calcd. C 59.93, H
7.17; N 7.44; Found C 58.99, H 7.21, N 6.94.
CDCl3) d: 7.52 (d, 2H), 7.47–7.44 (m, 4H), 6.81–6.76 (m, 4H), 4.14 (q,
4H), 4.10(s, 4H),3.83(t, 2H),3.29(t, 2H),1.93(m, 4H),1.66(m, 2H),1.58
(m, 2H), 1.25–1.19 (m, 10H), 1.10(m, 4H), 0.60 (m, 4H); 13C NMR
(100 MHz, CDCl3) d: 171.49, 152.19, 150.65, 139.03, 130.27, 126.06,
122.06,121.51,121.19,120.78,118.91,112.93, 68.39, 60.52, 55.54, 53.59,
40.10, 39.98, 33.79, 32.56, 29.62, 29.06, 28.91, 27.71, 25.65, 23.63,
23.42,14.20; MS (MALDI-TOF): 850.1 (M þ Hþ); C39H48O5NBr3: Calcd.
C 55.07, H 5.69; N 1.65; Found C 55.00, H 6.04, N 1.99.
4.7. Synthesis of compound 7
To a mixture of 6 (0.1 g, 0.11 mmol) and K2CO3 (0.18 g, 1.3 mmol)
in 5 mL acetonitrile was added a solution of tert-butyl bromoacetate
Fig. 2. (a) T1-weighted magnetic resonance images of PF–Gd (L1) and (NMG)2–Gd–DTPA (L2) at various Gd3þ concentrations. (b) Water proton longitudinal relaxation rate (1/T1) of
PF–Gd and (NMG)2–Gd–DTPA as a function of Gd3þ concentration.

1340
Q. Xu et al. / Polymer 51 (2010) 1336–1340
(0.12 g, 0.62 mmol) in 5 mL acetonitrile slowly at room tempera-
ture. After stirring for 4 h, the mixture was filtered, and the liquid
was concentrated under reduced pressure. The residue was purified
by silica gel column chromatography with CH2Cl2/methanol (47:3)
as the eluent to afford 7 (0.113 g, 83%) as a lightly yellow solid. 1H
polymer PF–Gd with different concentrations for 24 h respectively.
Then the culture media were discarded and MTT (1 mg/mL, 100 L/
well) was added to the wells, which was followed by incubation at
37 ꢁC for 4 h. The supernatant was abandoned, and 150
L DMSO
per well to solubilize the formazan was added and the sample was
shaken for an additional 10 min. This procedure was repeated three
times. The absorbance values of the wells were then read with
a microplate reader at a wavelength of 520 nm. The cell viability
rate (VR) was calculated according to the following equation [14]:
m
m
NMR (400 MHz, CDCl3) d: 7.51–7.48 (m, 2H), 7.44–7.39 (m, 4H),
6.83–6.71 (m, 4H), 4.10 (q, 4H), 4.06 (s, 4H), 3.79 (t, 2H), 3.70–2.00
(m, 22H),1.90 (m, 4H),1.55 (m, 2H),1.46 (m, 2H),1.40 (m, 27H),1.22–
1.10 (m, 16H), 0.55 (m, 4H); 13C NMR (100 MHz, CDCl3)
d: 171.46,
169.86, 152.10, 150.60, 138.98, 130.28, 125.98, 122.02, 121.47, 121.24,
120.74,118.84,112.92, 81.67, 68.36, 60.49, 56.84, 55.49, 55.35, 53.56,
52.89, 52.67, 50.16, 47.66, 40.04, 39.88, 29.58, 29.00, 28.05, 26.21,
25.62, 23.60, 23.45, 14.16; MS (MALDI-TOF): 1284.4 (Mþ).
Aexperimental group
VR% ¼
ꢂ 100%
Acontrol group
where the control group was not treated and the experimental
group was treated with polymer PF–Gd at different concentrations
for 48 h. IC50 was analysed by the statistic software SPSS (Ver. 13.0).
4.8. Synthesis of PF-ester
A mixture of 7 (50 mg, 0.0389 mmol), 8 (82.8 mg, 0.1556 mmol),
4,4,5,5-tetramethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)-1,3,2-dioxaboronane (64.2 mg, 0.1945 mmol) in 5 mL
THF and 1 mL 2 M K2CO3 was degassed, and catalytic amount of
Pd(PPh3)4 was added under nitrogen atmosphere. The resulting
mixture was stirred at 80 ꢁC for 38 h. After cooling to room
temperature, 100 mL of water was added and mixture was extrac-
ted with chloroform. The organic phase was dried over anhydrous
MgSO4. After the solvent was concentrated to a small volume, the
residue was precipitated in ethyl ether. The crude polymers were
purified by precipitation from chloroform into ethyl ether again
and the precipitation was collected and dried under vacuum to get
Acknowledgments
The authors are grateful for the financial supports from the ‘‘100
Talents’’ program of Chinese Academy of Sciences, the National
Natural Science Foundation of China (20725308, 20601027 and
20721061), the National Basic Research Program of China (No.
2006CB806200) and the Major Research Plan of China (No.
2006CB932102).
PF-ester (73.7 mg). 1H NMR (400 MHz, DMSO)
d: 7.94–7.67 (br),
References
7.56 (br), 7.49 (br), 7.37 (br), 7.14–7.05 (br), 6.86–6.62 (br), 4.57 (br),
4.20 (br), 4.13 (br), 3.99 (br), 3.74 (br), 3.65 (br), 3.54 (br), 3.46
(br), 3.41 (br), 3.22–3.10 (br), 2.67–2.61 (br), 2.33 (br), 2.15 (br), 1.67
(br), 1.35 (br), 1.23 (br), 1.08 (br), 0.65 (br); Mw ¼ 4600, PDI ¼ 1.51.
[1] (a) Meade TJ, Taylor AK, Bull SR. Curr Opin Neurobiol 2003;13:597;
(b) Kircher MF, Mahmood U, King RS, Weissleder R, Josephson L. Cancer Res
2003;63:8122.
[2] (a) Caravan P, Ellison JJ, McMurry TJ, Lauffer RB. Chem Rev 1999;99:2293;
(b) Aime S, Botta M, Fasano M, Terreno E. Chem Soc Rev 1998;27:19;
(c) Lee J, Zylka MJ, Anderson DJ, Burdette JE, Woodruff TK, Meade TJ. J Am
Chem Soc 2005;127:13164;
4.9. Synthesis of PF
(d) Werner EJ, Avedano S, Botta M, Hay BP, Moore EG, Aime S, et al. J Am Chem
Soc 2007;129:1870.
To a solution of PF-ester (21.4 mg) in 1 mL DMSO was added
a solution of KOH (19.4) in 1 mL CH3OH, and the resulting solution
was stirred at 80 ꢁC for 24 h. After cooling to room temperature, the
CH3OH was removed under reduced pressure, and then 1 mL of TFA
was added. The resulting mixture was stirred for 12 h at room
temperature, and then the solvent was removed under reduced
pressure. The residue was dialyzed to deionised water for 2 days.
The precipitation was collected and dried under vacuum to give PF
ˇ
´
ˇ
[3] (a) Lebduskova P, Kotek J, Hermann P, Elst LV, Muller RN, Lukes I, et al.
Bioconjug Chem 2004;15:881;
(b) Lauffer RB. Chem Rev 1987;87:901;
Rudovsky J, Hermann P, Botta M, Aime S, Lukes I. Chem Commun
2005:2390;
(c) Esfand R, Tomalia DA. Drug Discov Today 2001;6:427;
(d) Kobayashi H, Kawamoto S, Saga T, Sato N,, Ishimori T, Konishi J, et al.
Bioconjug Chem 2001;12:587.
ꢀ
ˇ
´
[4] To´th E, Pubanz D, Vauthey S, Helm L, Merbach AE. Chem Eur J 1996;2:1607.
[5] (a) Thomas III SW, Joly GD, Swager TM. Chem Rev 2007;107:1339;
(b) Liu B, Bazan GC. Chem Mater 2004;16:4467;
(14.6 mg). 1H NMR (400 MHz, DMSO)
d: 7.94–7.65 (br), 7.49 (br),
(c) Achyuthan KE, Bergstedt TS, Chen L, Jones RM, Kumaraswamy S,
Kushon SA, et al. J Mater Chem 2005;15:2648;
(d) Ho HA, Be´ra-Abe´rem M, Leclerc M. Chem Eur J 2005;11:1718;
(e) Bunz UHF. Chem Rev 2000;100:1605;
7.39 (br), 7.16–7.04 (br), 6.95–6.79 (br), 4.57 (br), 4.20 (br), 4.13 (br),
3.86 (br), 3.73 (br), 3.64 (br), 3.54 (br), 3.46 (br), 3.40 (br), 2.80
(br), 2.67 (br), 2.58 (br), 2.33 (br), 2.13 (br), 1.68 (br), 1.49 (br), 1.23
(br), 0.85 (br), 0.65 (br).
(f) Feng F, He F, An L, Wang S, Li Y, Zhu D. Adv Mater 2008;20:2959.
[6] (a) Gaylord BS, Heeger AJ, Bazan GC. Proc Natl Acid Sci U S A 2002;99:10954;
(b) Nilsson KPR, Ingana¨s O. Nat Mater 2003;2:419;
4.10. The preparation of PF–Gd
(c) Pinto MR, Schanze KS. Proc Natl Acad Soc U S A 2004;101:7505;
(d) He F, Tang Y, Yu M, Feng F, An L, Sun H, et al. J Am Chem Soc 2006;
128:6764;
To a solution of PF (11 mg) in 2 mL DMSO was added 3.84 mL of
1 mM GdCl3 aqueous solution, and the mixture was stirred for 24 h
at room temperature. The precipitation was collected and washed
by water, then dried under vacuum to afford PF–Gd (5.9 mg). The
concentration of Gd(III) was determined by ICP spectrometer.
(e) Tang Y, Feng F, He F, Wang S, Li Y, Zhu D. J Am Chem Soc 2006;128:14972.
[7] Miyaura N, Suzuki A. Chem Rev 1995;95:2457.
[8] Arheden H, Saeed M, Higgins CB, Gao DW, Bremerich J, Wyttenbach R, et al.
Radiology 1999;211:698.
´
[9] (a) Andre´ JP, To´th E, Fischer H, Seelig A, Ma¨cke HR, Merbach AE. Chem Eur J
1999;5:2977;
(b) Wang S, Bazan GC. Chem Commun 2004:2508.
[10] Stork M, Gaylord BS, Heeger AJ, Bazan GC. Adv Mater 2002;14:361.
[11] Tauer E, Grellmann KH. J Org Chem 1981;46:4252.
[12] Richman JE, Atkins TJ. J Am Chem Soc 1974;96:2268.
[13] Loewe RS, Tomizaki Ky, Youngblood WJ, Bo Z, Lindsey JS. J Mater Chem
2002;12:3438.
[14] Denizot F, Lang R. J Immunol Methods 1986;89:271.
[15] (a) Wang W, Wang R, Zhang C, Lu S, Liu T. Polymer 2009;50:1236;
(b) Xing C, Shi Z, Yu M, Wang S. Polymer 2008;49:2698.
4.11. Cell viability assay by MTT
Cell viability was assayed using 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT). Cells were subcultured in
96-well plates the day before the experiment at a density of 6 ꢂ 104
cells/well, and cultured for 24 h. Then cells were treated with