Preliminary biological evaluation of 18F-FBEM-Cys-annexin V a novel apoptosis imaging agent

Article abstract of DOI:10.3390/molecules20034902

A novel annexin V derivative (Cys-Annexin V) with a single cysteine residue at its C-terminal has been developed and successfully labeled site-specifically with ¹⁸F-FBEM. ¹⁸F-FBEM was synthesized by coupling ¹⁸F-fluorobenzoic acid (¹⁸F-FBA) with N-(2-aminoethyl)maleimide using optimized reaction conditions. The yield of ¹⁸F-FBEM-Cys-Annexin V was 71.5% ± 2.0% (n = 4, based on the starting ¹⁸F-FBEM, non-decay corrected). The radiochemical purity of ¹⁸F-FBEM-Cys-Annexin V was >95%. The specific radioactivities of ¹⁸F-FBEM and ¹⁸F-FBEM-Cys-Annexin V were >150 and 3.17 GBq/μmol, respectively. Like the 1^st generation ¹⁸F-SFB-Annexin V, the novel ¹⁸F-FBEM-Cys-Annexin V mainly shows renal and to a lesser extent, hepatobiliary excretion in normal mice. In rat hepatic apoptosis models a 3.88 ± 0.05 (n = 4, 1 h) and 10.35 ± 0.08 (n = 4, 2 h) increase in hepatic uptake of ¹⁸F-FBEM-Cys-Annexin V compared to normal rats was observed after injection via the tail vein. The liver uptake ratio (treated/control) at 2 h p.i. as measured via microPET correlated with the ratio of apoptotic nuclei in liver observed using TUNEL histochemistry, indicating that the novel ¹⁸F-FBEM-Cys-Annexin V is a potential apoptosis imaging agent.

Full text of DOI:10.3390/molecules20034902

Molecules 2015, 20, 4902-4914; doi:10.3390/molecules20034902
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
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8
Preliminary Biological Evaluation of F-FBEM-Cys-Annexin V
a Novel Apoptosis Imaging Agent
Chunxiong Lu *, Quanfu Jiang , Minjin Hu , Cheng Tan , Huixin Yu and Zichun Hua 2,3,*
1
,
1
2
1
1
1
Ministry of Health & Jiangsu Key Laboratory of Molecular Nuclear Medicine,
Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China;
E-Mails: jiangquanfu@jsinm.org (Q.J.); tangcheng@jsinm.org (C.T.); yuhuixin@jsinm.org (H.Y.)
2
Jiangsu Target Pharma Laboratories Inc., Changzhou High-Tech Research Institute of Nanjing
University, Changzhou 213164, China; E-Mail: huminj98@163.com
3
The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, China
*
Authors to whom correspondence should be addressed; E-Mails: luchunxiong@jsinm.org (C.L.);
huazc@nju.edu.cn (Z.H.); Tel.: +86-510-85514482-3529 (C.L.); +86-25-8332-4605 (Z.H.);
Fax: +86-510-85513113 (C.L.).
Academic Editor: Svend Borup Jensen
Received: 22 December 2014 / Accepted: 6 March 2015 / Published: 17 March 2015
Abstract: A novel annexin V derivative (Cys-Annexin V) with a single cysteine
residue at its C-terminal has been developed and successfully labeled site-specifically with
1
8
18
18
18
F-FBEM. F-FBEM was synthesized by coupling F-fluorobenzoic acid ( F-FBA) with
N-(2-aminoethyl)maleimide using optimized reaction conditions. The yield of
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8
18
F-FBEM-Cys-Annexin V was 71.5% ± 2.0% (n = 4, based on the starting F-FBEM,
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8
non-decay corrected). The radiochemical purity of F-FBEM-Cys-Annexin V was >95%.
18
18
The specific radioactivities of F-FBEM and F-FBEM-Cys-Annexin V were >150 and
.17 GBq/µmol, respectively. Like the 1st generation 18F-SFB-Annexin V, the novel
3
18F-FBEM-Cys-Annexin V mainly shows renal and to a lesser extent, hepatobiliary excretion
in normal mice. In rat hepatic apoptosis models a 3.88 ± 0.05 (n = 4, 1 h) and 10.35 ± 0.08
18
(
n = 4, 2 h) increase in hepatic uptake of F-FBEM-Cys-Annexin V compared to normal rats
was observed after injection via the tail vein. The liver uptake ratio (treated/control) at 2 h
p.i. as measured via microPET correlated with the ratio of apoptotic nuclei in liver observed
using TUNEL histochemistry, indicating that the novel 18F-FBEM-Cys-Annexin V is a
potential apoptosis imaging agent.

Molecules 2015, 20
Keywords: Cys-Annexin V; site-specific labeling; F-FBEM; apoptosis imaging
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8
1
. Introduction
Apoptosis plays an important role, not only in physiology but also in pathology [1,2]. Dysregulation
of apoptosis is associated with many diseases such as cancer, autoimmunity and neurodegenerative
disorders. Therefore, it has significant clinical value of the detection and quantification of apoptosis
in vivo for diagnosis and assessment of therapeutic efficacy. One of the early charateristics of apoptosis
is the externalization of the phospholipid phosphatidylserine (PS) at the cell membrane [3,4]. Annexin
2
+
V, a 36-kDa human protein, shows Ca -dependent binding to negatively charged phospholipid surfaces
and was discovered as a vascular anticoagulant protein [5,6]. The anticoagulant activity is based on the
high-affinity for PS. These characteristics make annexin V derivatives suitable candidates for imaging
of apoptosis. Several annexin V tagged with bifunctional chelators (BFC) have been labeled with 99mTc
for single photon emission computed tomography (SPECT) imaging of apoptosis in vivo [7–11].
However, conjugation of BFC to annexin V for labeling with 99mTc is usually done by targeting an amino
group of one of the 21 lysine residues using BFC, but this method is rather non-specific as any of
the –NH
recombinant expression annexin V can be directly marked with 99mTc, giving derivatives such as
99mTc(CO) -HIS-cys-Anx V [12], 99mTc-annexin V-117 [13] and 99mTc-His10-annexin V [14]. These
2
groups could be targeted. Recent studies have revealed that after structural modification in the
3
new annexin V molecules labeled by site-specific methods greatly improve sensitivity for detecting cell
death in vivo [15]. Our group has reported a site-specific 99mTc labeling method of a novel annexin V
derivative (Cys-Annexin V) with a single cysteine residue at C-terminal [16]. 99mTc-Cys-Annexin V is
a potential SPECT imaging agent of apoptosis. However, because of its higher sensitivity, better spatial
resolution and quantification properties a positron emission tomography (PET) analog would be very
18
desirable. Some groups have reported the labeling of annexin V with N-succinimidy-4- F-fluorobenzoate
(18
F-SFB) for PET imaging of apoptosis [17–19], however this labeling method is non-specific, as the
18F-SFB reacts with any available NH
group in the protein. Thiol-reactive agents such as N-substituted
maleimides can be used to modify proteins on the cysteine group [20]. 18F-N-[2-(4-Fluoro-
2
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benzamido)ethyl]maleimide ( F-FBEM) was used to label thiol-containing proteins as a novel
site-specific labeling prosthetic group [21–23]. We report herein the labeling and preliminary in vivo
1
8
evaluation of the novel F-FBEM-Cys-Annexin V in normal mice and in rat models of apoptosis
induced by cycloheximide. In mice the tracer uptake was studied by dynamic microPET imaging and
microPET in a rat model of hepatic apoptosis. Apoptosis was confirmed in situ on liver slices using the
terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay.
2
. Results and Discussion
2.1. Radiolabeling
Annexin V has been labeled non-specifically with a number of isotopes, including 99mTc, 124I, F and
18
68Ga [11,17,18,24,25]. Site-specific labeling of annexin V can help improve its sensitivity for detecting

Molecules 2015, 20
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cell death in vivo. To take advantage of the higher resolution and more accurate quantification of PET,
labeling annexin V with short half-life positron-emitters such as 18F is of particular interest.
1
8
In this study site-specific labeling of Cys-Annexin V with F-FBEM as prosthetic group is presented.
1
8
As see in Figure 1, Cys-Annexin V and F-FBEM-Cys-Annexin V were eluted at retention times of
1
8
8
.6 min and 9.1 min, respectively, whereas F-FBEM eluted at a retention time of 15.9 min. According
1
8
to HPLC analysis, the radiochemical purity of F-FBEM-Cys-Annexin V was above 95%. The total
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8
18
synthesis time for F-FBEM was about 100 min and 428 ± 65 MBq (n = 4) pure F-FBEM was obtained
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8
18
from 18.5 GBq F-fluoride. 81–262 MBq F-FBEM-Cys-Annexin V was obtained from 111–370 MBq
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8
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F-FBEM as the radiochemical yield was 71.5% ± 2.0% (n = 4, based on the starting F-FBEM,
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8
18
non-decay corrected). The specific radioactivities of F-FBEM and F-FBEM-Cys-Annexin V were
above 150 MBq/µmol and 3.17 GBq/µmol, respectively.
Figure 1. Cont.

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Figure 1. HPLC chromatogram (isocratic, 0.05 mol/L phosphate buffer (pH = 7.0), flow
0
.8 mL/min) of: (A) Cys-Annexin V, t
R
= 8.6 min (UV); HPLC radiochromatograms of
18
18
(B) F-FBEM, t
R
= 15.9 min, (C) reaction mixture ( F-FBEM-Cys-Annexin V, t
R
= 9.1 min,
18F-FBEM, t
= 15.9 min) and (D) F-FEBM-Cys-Annexin V, t
18
= 9.1 min.
R
R
1
8
2
.2. In Vitro Stability of F-FBEM-Cys-Annexin V
To determine the radioactive decomposed side products which may accumulate in non-target
organs, the stability of 18F-FBEM-Cys-Annexin V was studied. The results of the stability of
18F-FBEM-Cys-Annexin V in (A) phosphate buffered saline (PBS, 0.1 mol/L, pH 7.2) and (B) human
1
8
serum, respectively, are presented in Figure 2. The results show that the F-FBEM-Cys-Annexin V is
more stable during biodistribution and PET imaging studies.
B
A
100
1
00
9
8
7
0
0
0
9
8
7
6
5
0
0
0
0
0
60
5
0
0
30 60 90 120 150 180
0
30
60
90 120 150 180
Time (min)
Time (min)
Figure 2. Stability of 18F-FBEM-Cys-Annexin V at different intervals in (A) PBS and
B) human serum.
(
2
.3. Blood Kinetics Studies
Pharmacokinetic parameters, obtained using the DAS 2.1.1 pharmacokinetic calculation program, are
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8
listed in Table 1. Figure 3 shows the blood clearance of F-FEBM-Cys-Annexin V in the mice 2 h
post-injection. Pharmacokinetics of 18F-FEBM-Cys-Annexin V comply with the two-compartment
model with the pharmacokinetic equation of C = 2.359e−0.062t + 3.288e−0.005t (where C is
radiopharmaceutical activity (%ID/g) in blood, t is the time after injection). The half-life of distribution

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phase (t1/2α) and half-life of elimination phase (t1/2β) were 11.261 and 134.62 min, respectively, which
1
8
showed that F-FBEM-Cys-Annexin V can be absorbed quickly and eliminated slowly. Biological
availability was represented by area under concentration-time curve (AUC) and the values of clearance
(CL) and AUC were 0.031 and 643, respectively.
1
8
Table 1. Pharmacokinetic parameters of the F-FEBM-Cys-Annexin V in mice.
Parameter (units)
18F-FEBM-Cys-Annexin V
−
1
K
K
K
12 (min )
0.02
0.038
−
1
21 (min )
−
1
e
(min )
0.009
CL (%ID/g/min)
0.031
T
1/2α (min)
1/2β (min)
11.261
134.62
634.123
T
AUC (%ID/g·min)
6
5
4
3
2
1
0
0
30
60
Time (min)
90
120
1
8
Figure 3. Pharmacokinetic curve for F-FEBM-Cys-Annexin V in mice.
1
8
In the early phase, the blood clearance of F-FEBM-Cys-Annexin V was slow. After 2 h, the
radioactivity concentration of the tracer agent in blood reaches an equilibrium which coincides with the
pharmacokinetic parameters CL, AUC and the pharmacokinetic curves.
2
.4. Dynamic MicroPET Images of Normal ICR Mice
Representative time-activity curves of the major organs (kidneys, liver and heart) were derived from
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6
0-min dynamic microPET scans after intravenous administration of F-FBEM-Cys-Annexin V tracers
(
Figure 4). The radioactivity kinetics were calculated from a region-of interest analysis of the dynamic
small animal PET scans over the heart (squares; mainly representing the cardiac blood pool), kidney
1
8
(
triangles) and liver (diamonds). F-FBEM-Cys-Annexin V was excreted mainly through the kidneys,
as evidenced by the higher renal uptake at early time points and excretion via the bladder.
The kidney uptake reached a peak (11%ID/g) at 13 min after injection and then decreased to 1.43%ID/g
at 60 min p.i.

Molecules 2015, 20
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1
1
1
4
2
0
8
6
4
2
0
B
Liver
0
10
20
30
40
50
60
Time (min)
Figure 4. (A) Whole body coronal microPET images of ICR mouse from a 60 min
dynamic scan after injection of 3.7 MBq 18F-FBEM-Cys-Annexin V. (B) Quantified
time-activity curves of major organs (liver, heart and kidney) after injection of 3.7 MBq
18F-FBEM-Cys-Annexin V in normal ICR mice (n = 4).
2
.5. Imaging of Rat Model of Apoptosis
Four rats were treated with cycloheximide to induce liver apoptosis and two rats were used as the control
group. Figure 5 shows the representative coronal microPET images of cycloheximide (CHX)-treated and
normal rats at different times after intravenous injection of 8.2 MBq 18F-FBEM-Cys-Annexin V.
18F-FBEM-Cys-Annexin V tracer uptake in the liver (arrow) was increased with CHX treatment. The
uptake ratios (treated/control) of liver were 3.88 ± 0.05 (n = 4) and 10.35 ± 0.08 (n = 4), respectively, at
1
h and 2 h p.i.

Molecules 2015, 20
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(
A)
(B)
(
C)
(D)
Figure 5. MicroPET images of CHX-treated and normal rats after injection of
18F-FBEM-Cys-Annexin V. (A) CHX-treated at 1 h p.i. (B) Normal at 1 h p.i.
(
C) CHX-treated at 2 h p.i. (D) Normal at 2 h p.i.
(
A)
(B)
Figure 6. Representative TUNEL-stained images of liver specimen in CHX-treated rats (A),
control rats (B). Green color dot represents positive TUNEL staining.

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There were no differences in the blood pool activity between treated and control rats. TUNEL–staining
images of liver sections were shown in Figure 6. These images show that the number of apoptotic nuclei
in CHX-treated liver was more compared to that in non-treatment control rats. The uptake ratio
(
treated/control) of liver at 2 h p.i. as measured via microPET correlated with the ratio (treated/control)
of apoptotic nuclei in liver observed using TUNEL histochemistry.
3. Experimental Section
3.1. General Information
Unless otherwise specified, all reagents were of analytical grade and were obtained from commercial
sources. Cys-Annexin V was supplied by Jiangsu Target Pharma Laboratories Inc. (Changzhou, China).
18F fluoride was obtained from a cyclotron (HM67, Sumitomo Heavy Industries, Ltd, Tokyo, Japan)
1
8
located at the Jiangsu Institute of Nuclear Medicine by proton irradiation of O-enriched water. A
Waters high-performance liquid chromatography (HPLC) system (Waters, Milford, MA, USA)
equipped with a Waters 2998 photodiode array detector (PDA) and a semi-preparative C18 HPLC
1
8
column (250 × 10 mm, 5 μm, CHROM-MATRIX BIO-TECH) was used for F-FBEM purification.
The flow rate was 3 mL/min, and the mobile phase changed from 95% solvent A (0.1% trifluoroacetic
acid in water) and 5% solvent B (0.1% trifluoroacetic acid in acetonitrile) (0–2 min). The mobile phase
was gradually changed to 35% solvent A and 65% solvent B at 32 min. The UV absorbance was
monitored at 218 nm, and the UV spectrum was checked with the PDA detector.
Analytical HPLC was performed on Waters Breeze system with a TSK-GEL column (swG2000SWXL,
3
00 × 7.8 mm 5 µm, Tosoh Bioscience Co., Ltd, Shanghai, China). The absorbance was measured on
the UV detector at 278 nm. Radioanalysis of the labeled compound was conducted using a Cd (Te)
detector. The flow rate was adjusted to 0.8 mL/min and the isocratic mobile phase was 0.05 mol/L
phosphate buffer (pH = 7.0).
A microPET system (Inveon, Siemens Co. Knoxville, TN, USA) and a fluorescence microscope
(
X51, Olympus, Tokyo, Japan) were used. The animal experiments in this study were approved by the
Animal Care and Ethnics Committee of Jiangsu Institute of Nuclear Medicine.
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8
3
.2. Preparation of F-N-[2-(4-Fluorobenzamido)ethyl]maleimide
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8
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F-N-[2-(4-Fluorobenzamido)ethyl]maleimide ( F-FBEM) was prepared as previously described
using a semi-automatic method with some modifications [22,26]. Briefly, the precursor, ethyl
-(trimethylammonium) benzoate trifluoromethanesulfonic acid salt (10 mg, 12 µmol) in anhydrous
4
1
8
acetonitrile (1.0 mL) was heated at 100 °C for 10 min in a sealed vial with 18.5 GBq [ F]fluoride in the
presence of dried Kryptofix2.2.2 (15 mg 6 µmol) and K CO (5 mg, 3.6 mmol). The intermediate was
2
3
hydrolyzed with NaOH (0.5 M, 0.5 mL) at 90 °C for 5 min. After acidification with 7.5 mL 0.1 M HCl,
the solution was loaded onto an activated C18 Sep-Pak column (Waters). The cartridge was
then eluted with 3 mL acetonitrile and the eluate was subsequently evaporated at room temperature
with a stream of nitrogen to obtain 18F-fluorobenzoic acid ( F-FBA). 18F-FBA was treated with
N-(2-aminoethyl)maleimide (MAL, 15 mg, 59 µmol), diethyl cyanophosphonate (20 µL, 99 µmol), and
N,N-diisopropylethylamine (40 µL, 240 µmol) in anhydrous acetonitrile (0.5 mL). The resulting solution
18

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was heated at 75 °C for 7 min. The reaction was quenched by adding water (8.5 mL) and loaded onto an
activated C18 Sep-Pak column. The cartridge was eluted with 1 mL ethanol which was then loaded on
to the semi-preparative HPLC. The radioactive peak eluting at ~18 min was collected and passed through
a C18 Sep-Pak column which was activated by EtOH/water. The cartridge was washed with 20 mL water
and then eluted with 1 mL CH
2
Cl . The organic layer was evaporated to dryness at room temperature
2
under a stream of nitrogen and utilized for further Cys-Annexin V labeling. The total synthesis time for
1
8
18
F-FBEM was about 100 min and 428 ± 65 MBq (n = 4) radiochemically pure F-FBEM was obtained
1
8
from 18.5 GBq F-fluoride.
1
8
3
.3. Preparation of F-FBEM-Cys-Annexin V
The isolated 18F-FBEM (111–370 MBq) in 10 µL of ethanol was added to a solution of
Cys-Annexin V (50–100 µg in 100 µL, pH = 7.2) PBS (Scheme 1), and the mixture was allowed to react
at room temperature for 15–30 min and loaded onto a NAP-5 column (GE Healthcare, Buckinghamshire,
UK). The NAP-5 column was eluted with 250 µL portions of PBS. The most concentrated
fraction containing the radiolabeled protein (fraction 3, 81–262 MBq) was collected and used for the
biological experiments.
O
OH
Annexian V
O
O
N
N
H
HS
OH
H
HN
O
N
S
N
18F
+
Annexian V
O
H
O
N
18F
O
O
18
1
8
F-FBEM-Cys-Annexin V
F-FBEM
Cys-AnnexinV
18
Scheme 1. Syntheses of F-FBEM-Cys-Annexin V.
1
8
3
.4. In Vitro Stability of F-FBEM-Cys-Annexin V
The in vitro stabilities of freshly prepared 18F-FBEM-Cys-Annexin V were performed in PBS
(
0.1 mol/L, pH 7.2) and human serum, respectively, for different time intervals (0–6 h) at 37 °C in
a water bath.)
18
3
.5. Blood Kinetics Studies of F-FBEM-Cys-Annexin V in Normal Mice
1
8
Five ICR mice were injected via the tail vein with F-FBEM-Cys-Annexin V (0.2 mL) and activity
of approximately 3.7 MBq. Ten μL of blood were taken from tails at 5, 15, 30, 45, 60, 90 and 120 min
after injection. The activity for each sample was determined by a γ-counter and expressed as percentage
of injection dose per gram (%ID/g).

Molecules 2015, 20
.6. Dynamical MicroPET Images of Normal Mice
Four ICR mice were anesthetized with 1%–2% isoflurane, positioned prone, immobilized, and
4911
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injected via the tail vein with 3.7 MBq F-FBEM-Cys-Annexin V (0.2 mL) and imaged dynamically
for 1 h. The images were reconstructed using a two dimensional ordered-subset expectation
maximization (2D OSEM) algorithm without correction for attenuation or scattering. For each scan,
regions of interest (ROIs) were drawn over the liver and major organs using the vendor-supplied software
(
(
ASI Pro 5.2.4.0) on decay-corrected whole-body coronal images. The radioactivity concentrations
accumulation) within the liver, heart and kidneys were obtained from mean pixel values within the
multiple ROI volume and then converted to megabecquerel per milliliter per minute using the calibration
factor determined for the Inveon PET system. These values were then divided by the administered
activity to obtain (assuming a tissue density of 1 g/mL) an image-ROI-derived percent injected dose per
gram (%ID/g).
3
.7. MicroPET Images of Rat Model of Apoptosis
Four male SD rats (258 ± 2 g) were treated IV with 10 mg/kg cycloheximide to induce liver apoptosis.
Two male SD rats (262 g and 256 g) were treated IV with saline as the control group. 3 h after treatment,
the rats were anesthetized with 1%–2% isoflurane and were injected via the tail vein with 8.2 MBq
18F-FBEM-Cys-Annexin V (0.2 mL). Ten-minute static scans were acquired at 1 and 2 h after injection
with a MicroPET (Inveon, Siemens), respectively, which was from 1 h to 1 h and 10 min or from 2 h to
2
h and 10 min. Immediately after MicroPET imaging, the livers were dissected. Then, using the livers,
formalin-fixed paraffin-embedded specimens were prepared for Terminal deoxynucleotidyl
transferase-mediated nick end labeling (TUNEL) staining.
3.8. TUNEL Staining
Because our imaging studies were designed to determine the uptake and biodistribution of
18F-FBEM-Cys-AnnexinV after chemically induced apoptosis, it was important to confirm apoptosis in
the livers of treated rats by independent methods that provide quantitative results. A marker of apoptosis
was scored by performing a TUNEL assay that measures DNA fragmentation, a characteristic feature of
apoptosis. Terminal deoxynucleotide transferase adds labeled nucleotides to the 3' termini at double-stranded
breaks in the fragmented DNA. TUNEL assays were performed according to the manufacturer’s
instructions, using the fluorescein-conjugated Colorimetric TUNEL Apoptosis Assay Kit (Beyotime
Institute of Biotechnology, Shanghai, China). Briefly, slices were freed of paraffin through xylene and
graded EtOH washes and then incubated with proteinase K (Beyotime Institute of Biotechnology,
2
mg/mL in 10 mmol/L Tris, pH 8.0). After proteinase digestion, the slides were equilibrated in pH 7.4
buffer, the terminal deoxynucleotidetransferase enzyme and Biotin-dUTP labeling mix (Beyotime
Institute of Biotechnology) were added, and the slides were incubated at 37 °C for 1 h in a humid
chamber. The number of TUNEL-positive cells was counted on 10 randomly selected ×100 fields for
each section by use of an Olympus fluorescence microscope.

Molecules 2015, 20
. Conclusions
Cys-annexin V, a novel annexinVderivative with a single cysteine residue at the C-terminal, could be
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site-specifically labeled with F-FBEM in high yields and high radiochemical purity. In normal mice,
18F-FBEM-Cys-Annexin V was excreted mainly through the renal pathway. Hepatic uptake of
18F-FBEM-Cys-Annexin V was significantly increased in the rats treated with CHX compared to
controls, which correlated well with the increase in cell death observed using TUNEL histochemistry.
1
8
These results indicate that the novel F-FBEM-Cys-Annexin V is a potential apoptosis imaging agent
and further study is needed.
Acknowledgments
The authors are very grateful to the Ministry of Health Foundation of China (W201207), Open Fund
Project (KF-GN-201304) of State Key Laboratory of Pharmaceutical Biotechnology and Public
service platform for Science and technology infrastructure construction project of Jiangsu Province
(
BM2012066) for their financial support.
Author Contributions
Chunxiong Lu and Zichun Hua conceived and designed the experiments; Chunxiong Lu,
Quanfu Jiang and Cheng Tan performed the experiments; Chunxiong Lu and Huixin Yu analyzed the
data; Zichun Hua and Minjin Hu contributed Cys-Annexin V; Chunxiong Lu wrote the paper.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Sample of the compound Cys-Annexin V is available from the authors.
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