A bimodal MRI and NIR liposome nanoprobe for tumor targeted molecular imaging

Article abstract of DOI:10.1039/c5tb01160d

The combination of complementary MRI and NIR imaging methods evolved to provide an even more powerful bioimaging tool. Herein, a novel bimodal MRI/NIR nanoprobe GCF-HDA was prepared via a facile self-assembly approach of three types of amphiphilic structures in aqueous solution. The Stokes shift of the NIR moiety increased from 30 to 150 nm and fluorescence quantum yield increased from 1.5 to 8% after conjugation with electron-rich hexadecylamine (HDA) to organic dye Cy7. The photostability of the nanoprobe GCF-HDA was dramatically improved after involving the newly synthesized dye. Molecular dynamics simulation demonstrated that the GCF-HDA is composed of 2.0-3.5 nm clusters and in each cluster the head groups of the amphiphilic molecules assemble together and the tail groups point outwards. The r₁ and r₂ relaxivities of GCF-HDA were found to be 11.87 and 19.91 mM^-1 s^-1 per Gd(iii) chelate at 0.5 T, respectively. In vitro cellular imaging with human glioma U-87 MG cells showed that the GCF-HDA was able to enter the cells and accumulate in the cytoplasm. The targeted GCF-HDA resulted in higher MR contrast enhancement and stronger fluorescence intensity than the corresponding non-targeted probe GC-HDA in the tumor tissue 96 hours post injection. Ex vivo fluorescence imaging and histological analysis of the tumor tissue further confirmed the specific binding ability of the GCF-HDA.

Full text of DOI:10.1039/c5tb01160d

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ARTICLE
A bimodal MRI and NIR liposome nanoprobe for tumor targeted
molecular imaging
Received 00th January 20xx,
Accepted 00th January 20xx
a,b
§
a,b
§
d
a,c
a,b
a,b
Huihui Wang , Hao Wu ,Hujun Shen , Shaote Geng , Beibei Wang , Yanfang Wang ,
a
d
a,c*
Xiaojun Ma , Guohui Li and MingqianTan
DOI: 10.1039/x0xx00000x
The combination of complementary MRI and NIR imaging methods evolved to provide an even more powerful bioimaging
tool. Herein, a novel bimodal MRI/NIR nanoprobe GCF-HDA was prepared via a facile self-assembly approach of three
types of amphiphilic structures in aqueous solution. The Stokes shift of NIR moiety increased from 30 to 150 nm and
fluorescence quantum yield increased from 1.5 to 8 % after conjugation with electron-rich hexadecylamine (HDA) to
organic dye Cy7. The photostability of the nanoprobe GCF-HDA was dramatically improved after involving the newly
synthesized dye. Molecular dynamics simulationdemonstrated that the GCF-HDA is composed of 2.0-3.5 nm clusters and in
each cluster the head groups of the amphiphilic molecules assemble together and the tail groups point outwards. The r1
www.rsc.org/
-1 -1
and r2 relaxivities of GCF-HDA were found to be 11.87 and 19.91 mM s per Gd(III) chelate at 0.5T, respectively. In vitro
cellular imaging with human glioma U-87 MG cells showed that the GCF-HDA was able to enter the cells and accumulate in
the cytoplasm. The targeted GCF-HDA resulted in higher MR contrast enhancement and stronger fluorescence intensity
than the corresponding non-targeted probe GC-HDA in the tumor tissue 96 hours post injection. Ex vivo fluorescence
imaging and histological analysis of the tumor tissue further confirmed the specific binding ability of the GCF-HDA.
complementary optical imaging method, has been established
as a powerful imaging tool for in vivo molecular imaging of
specific targets with high sensitivity. The combination of these
1
. Introduction
Accurate detection and diagnosis are crucial aspects to
consider in the successful treatment of cancer at an early
stage. Recently, non-invasive imaging techniques for cancer
two imaging modalities paves the way for utilizing a single
bimodal probe to achieve high spatial resolution and high
sensitivity in a simultaneous fashion, without the need for a
1
diagnosis including optical imaging , magnetic resonance
2
imaging(MRI) , X-ray computed tomography(CT) , positron
6
, 7
3
radiation source.
For instance, the organic NIR dye Cy5.5
4
5
attached to iron oxide nanoparticles cross-linked by dextran as
a MRI/NIR bimodal nanoprobe (NP) has already been
employed for the preoperative visualization of brain tumor
with MRI and NIR imaging in an effort to discriminate
emission tomography (PET) and ultrasonic imaging have led
to rapid advances in our understanding of human anatomy and
physiology. Among these modalities, MRI is able to non-
invasively visualize deep tissue areas in the human body with
high spatial resolution and is therefore a widely used imaging
tool in clinical practice. However, the sensitivity of MRI in
cancer diagnosis is constrained by the ubiquitous presence of
protons (i.e. water) in virtually all tissue areas. Common
amplification strategies involve the use of smart contrast
agents. Near-infrared (NIR) fluorescence imaging, an excellent
8
tumorous from normal tissues. In order to increase the
sensitivity and accumulation behavior of the probe in tumor
regions, the peptide Cltx that selectively targets glioma tumor,
together with the NIR fluorescent dye Cy5.5 were conjugated
by poly(ethylene glycol)(PEG) onto the surface of iron oxide
9
nanoparticles for glioma tumor imaging. A tumor targeting
micelle based on a poly( ε -caprolactone)-b-poly(ethylene
glycol) block copolymer (PCL-b-PEG) functionalized with
3
+
diethylenetriamine pentaacetic acid-gadolinium (DTPA-Gd )
on the outer shell with T contrast ability as well as a NIR dye
1
present in the micelle core, has already been reported
featuring high longitudinal relaxivity for bimodal tumor
1
0
imaging.
Moreover, supramolecular conjugates like
fluorescent paramagnetic bimodal liposomes have also been
developed in an effort to enhance the cellular penetration
ability of Gd-based agents into tumorous tissues for tumor
1
1-14
imaging.
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NIR dyes with large Stokes shift and MRI contrast agents
2
. Materials and methods
with high relaxivity are highly desirable components for the
development of a MRI/NIR bimodal probe. NIR organic dyes
2
.1 Materials and Reagents
(HDA),
nm can result in deep tissue penetration for in vivo optical dicyclohexylcarbodiimide(DCC)
with unique absorption wavelengths in the range of 600-900 Hexadecylamine
folic
acid
and
(FA),
N-
imaging due to the fact that in this range the tissue absorption hydroxysuccinimide(NHS) were purchased from Aladdin and
1
5-
is weak and the auto-fluorescence background is negligible.
1
Sigma-Aldrich
(Shanghai,
China).
Tert-
7
However, the Stokes shift of the majority of NIR dyes like butyloxycarbonyl1,4,7,10tetraazacyclododecane-1,4,7,10-
indocyanine green (ICG) used in clinical applications is less tetraacetic acid (DOTA) was purchased from Synpartner
than 30 nm, leading to significant overlap of the PharmTech Co., Ltd (Zhejiang, China). 2,3,3-trimethyl-indole
excitation/emission spectra. This often causes considerable and trifluoroaceticacid (TFA) were purchased from Zahn
interferences in optical imaging, as well as fluorescence Chemical Inc. (Shanghai, China). Gadolinium acetate hydrate
18
quenching. Peng and co-workers reported the synthesis of a was purchased from Alfa Aesar Chemical Inc (Tianjin, China).
series of heptamethine cyanine dyes obtained through Iodine propionate was purchased from Beijing
J
& K
nucleophilic substitution reactions at the central position of Cy Technology Inc (Beijing, China). Column chromatography was
dyes to obtain a large Stokes shift (>140 nm) and higher performed using 200 to 400 mesh silica gel from Qingdao
1
9
quantum yield. This finding could be useful for the Ocean Chemical Inc (Qingdao, China).All the organic solvents
production of more efficient MRI/NIR bimodal probes. On the were analytical grade, and used without further purification.
other hand, small molecular paramagnetic contrast agents are Human glioma cells U-87 MG cells were purchased from
often used in a clinical setting in order to improve the MRI American Type Culture Collection (ATCC). Female BALB/c-nu
contrast ratio by shortening the relaxation times of the water mice were purchased from the Animal Center of Dalian
1
20
H NMR resonance. Unfortunately, such contrast agents have Medical University (Dalian, China).
been proved to be tissue nonspecific, extracellular compounds 2.2 Instrumentation and Characterization
and their efficiency in molecular imaging has been limited due
All synthesized compounds were characterized by matrix-
assisted laser desorption ionization time-of-light (MALDI-TOF)
to their lower specificity and lower
r
1
relaxivity.
Supramolecular liposomes can not only provide greater and
prolonged contrast enhancement at a generally lower dosage,
but are also capable of carrying a large number of gadolinium
chelates and targeting moieties. The results from MR
angiography and tumor angiogenesis imaging suggest that
supramolecular liposomes provide an optimal platform for the
development of target-specific MRI/NIR bimodal contrast
mass spectroscopy (5800, AB SCIEX) with
α -cyano-4-
hydroxycinnamic acid as a matrix. The UV-vis absorption
spectra were recorded on a UV-2550 spectrophotometer
(
Shimadzu, Japan) and the NIR fluorescence emission spectra
were measured by a fluorescence spectrophotometer system
LS-55, PerkinElmer, USA). The particle size distribution and
(
stability were operated by the laser particle size analyzer and
zeta potential analyzer. High-resolution transmission electron
microscopy (HRTEM) observations were obtained with a JEM-
2
1, 22
agents.
In this work, we report a method based on a simple
hydrophobic/hydrophilic interaction mechanism for the
2
100 transmission electron microscope operating at 200KV.
preparation of
a folate-receptor (FR) targeted MRI/NIR
The samples for HRTEM were prepared by drying sample
droplets from water dispersion onto a 230-mesh Cu grid
coated with a lacey carbon film and negative stained by
phosphotungstic acid. The contents of Gd element in the
solution were measured by inductively coupled plasma-optical
bimodal probe through self-assembly of small molecule
amphiphilic liposomes. Notably, a novel heptamethine cyanine
dye with large Stokes shift has been prepared as the NIR
imaging moiety and an amphiphilic gadolinium(III) complex,
3
+
1
namely DO3A-Gd , serves as T MR contrast agent. The
emission spectroscopy (ICP-OES 7300DV, PerkinElmer
The cells were imaged by confocal microscope system
FV1000MPE, Olympus, JAP). MRI experiments were
，JAP).
prepared MRI/NIR bimodal probe proves to be of irregular
spherical shape with a size less than 50 nm. Furthermore, the
probe features excellent fluorescent and relaxation properties
with good water solubility under physiological conditions. The
formation mechanism of the self-assembled probe was studied
using a Coarse-Grained (CG) model of a molecular dynamics
(
conducted on a 0.5T magnet scanner (MiniMR-Rat, Shanghai
Niumag Corporation, Shanghai, China). NIR experiments were
performed on a maestro animal in vivo imaging system
(MK50101-EX, CRI, USA). The histological sections were
(
MD) simulation. The large Stokes shift of the NIR feature
analyzed by confocal laser scanning microscope (Leica, TCS-
SP2, Germany) with an inverted microscope (Leica, DMIRE2,
Germany).
combines the characteristics of deep tissue penetration and a
reduced overlap of the corresponding excitation/emission
spectra in tissues, expected to significantly enhance the signal-
to-noise ratio. Presumably, the bimodal probe presented in
this work is able to combine the functions of FR targeted, NIR
fluorescence and MR imaging into one nano-system. Its
feasibility for both in vitro and in vivo imaging has also been
investigated.
2
.3 Synthesis of MRI lipid Gd-DOTA-HDA (1)
The MRI lipid Gd-DOTA-HDA was synthesized by three
sections. Firstly, tert-butyloxycarbonyl DOTA acid (1.0026g,
1
.75
hydroxysuccinimide NHS(0.2448g, 2.13mmol) were added to a
dry round-bottomed flask followed by the addition of CH Cl
mmol),
DCC(1.8087g,
8.77mmol)
and
N-
2
2
2
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(
10 mL). The reaction was stirred at room temperature for 2 h (15mL) and Et
3
N (673ꢀμL, 4.8275mmol). The mixture was then
4, Scheme S1, ESI). heated to 70°C under stirring for 10h in dark. The solvent was
to obtain active DOTA ester (compound
Then hexadecylamine (HDA) (0.5065g, 2.1mmol) and then removed by rotatory evaporation, and the crude mixture
triethylamine (Et N) (732 L, 5.25mmol) were added into the was purified by flash column chromatography (eluted with
mixture solution under room temperature with stable stirring (CH Cl /MeOH/H O 40:20:1), v/v) to yield the compound
for another 12 h. The solvents were removed by rotatory (Scheme S2, ESI) as a dark green powder (211mg, 23%yield). R
evaporation and the crude mixture was purified by adding [CH Cl /MeOH/H O 40:20:1 v/v] 0.6. The final product was
appropriate volumes of diethyl ether. The precipitation was characterized by MOLDI-TOF [(m/z, [M-I] ): 804.57
3
μ
2
2
2
2
f
2
2
2
+
2
+
collected and dried under vacuum, yielding tert-Butyl- (calculated),
804.6874(observed);
, Scheme S1, ESI) as a 827.57(calculated), 826.8386(observed)].
was dissolved in a 1:1 2.5 Synthesis of FR-targeted lipid FA-HDA (Compound3)
(m/z,
[M-I+Na] ):
protected DOTA-HDA (compound
yellow powder. The compound
5
5
2 2
mixture of TFA/CH Cl and stirred at room temperature for 2 h.
FR-targeted lipid FA-HDA (Compound
3, Scheme S3, ESI) was
The solution was concentrated by a rotavapor, and the mixture
resuspended in petroleum ether and freeze-dried to yield a
synthesized in two steps. Firstly, theꢀγ-COOH group of FA
(
1.0034g, 2.3mmol) was activated by DCC/NHS catalyst system
molar ratio of FA:DCC:NHS = 1.1:5.5:1.2) with Et N (1.57mL,
1.3mmol) in anhydrous dimethyl sulfoxide (DMSO, 12 mL)
yellowish oil DOTA-HDA (
6
) (Scheme S1, ESI) (621mg, 57%
(621mg, 0.990mmol) was
(
3
yield). Finally, the compound
6
1
dissolved in 30 mL of deionized water in a round-bottom flask
and pH value was adjusted to 5-6 with 0.1 M NaOH solution. A
under the continuous stirring for 12 h in the dark at room
temperature. Secondly, HDA in 8 mL ethyl acetate was added
dropwisely into the above solution and the reaction was
allowed to proceed in the dark with stirring for 24 h. After
removing the insoluble white precipitate, the solvent was
removed through a rotary evaporator and the crude product
was yielded by precipitating in diethyl ether and dried under
3 3 2
stoichiometric amount of (CH COO) Gd.H O (0.3481 g, 0.988
mmol) was also added to the above solution and the reaction
was stirred in a 90°C oil bath for 2 h. After freeze-drying, a buff
powder was yield (523 mg, 68% yield). R
f
3
[CH
2
Cl
4.5:9:1 v/v] 0.23. No detectable free Gd was measured by
the Xylenol orange test. MALDI TOF mass spectroscopy [(m/z,
2 2
/MeOH/H O
+
3
vacuum (515.1mg, 37% yield). The final product was also
+
[
M+H] ):783.36(calculated),
783.5862(observed);
(m/z,
+
[M+H] ):
characterized
65.7993(observed), [M]: 664.41(calculated)].
.6 Preparation of MRI/NIR bimodal probe
by
MOLDI-TOF
[(m/z,
+
M+Na] ): 805.36 (calculated), 805.5742(observed)].
[
6
2
2.4 Synthesis of NIR lipid Cy7-HDA (2)
With three functional amphiphilic molecules in hand, liposome
NPs were prepared with defined molar ratios of each
individual lipid. Targeted NPs consisted of Gd-DOTA-HDA/Cy7-
HDA/FA-HDA (GCF-HDA) with a molar ratio of 5:5:1. Briefly,
appropriate volume of Gd-DOTA-HDA and Cy7-HDA in
methanol and FA-HDA in tetrahydrofuran (THF) were placed in
a round-bottom flask and stirred to ensure totally mixing of
the lipids. The solvents were removed slowly by a rotatory
evaporator, then appropriate volume of ultra-pure water was
added and the resulting solution was sonicated for 40 min to
obtain NPs. Then, the solution was filtered using 0.8
μmꢀMillipore filters to remove large particles and the final
solution was stoked at 4°C. The size of the NPs was measured
by dynamic light scattering (DLS). For in vivo experiment, the
Compound 7:Phosphoryl chloride(POCl
3
10mL, 0.109 mol) was
added drop wisely into a solution of DMF/DCM(20mL, 1:1,v/v)
under stirring in ice/water bath, followed by adding
cyclohexanone (2.63mL, 25.5mmol). The solution was then
heated to 80°C and refluxed for 3 h. The mixture was
transferred into a beaker and 100 mL of water was added
under ice/water bath, yielding a solid yellow product. After
collecting with filtration and washing with ice-cooled water,
the compound
7 (Scheme S2, ESI) was dried under vacuum
(1.459g, 33.3% yield).
Compound 8: To10 mL of toluene was added 2,3,3-trimethyl-
indole (2.225g, 13.9mmol) and iodine acid (1.875g, 9.4mmol)
and the solution was stirred and refluxed in a 100°C oil bath
for 3 h. After adding 15mL of diethyl ether, the solvent was
removed by vacuum evaporation, yielding a red solid product.
The product was then dissolved and precipitated in a mixture
of ether and acetone (1:1, v/v) for several times to yield
liposomes NPs were diluted by saline to obtain
3
a 5
+
mMGd solution. For non-targeted NPs preparation, the
procedure was the same without adding FA-HDA molecules.
2
.7 Molecular dynamics (MD) simulation based on coarse-grained
CG) model
(0.532g, 3.09 mmol) and compound The Gay-Berne electric multipole (GBEMP) parameters for the
(2.204g, 6.14mmol) were added to the solvent of toluene bond stretching, angle bending and torsional potentials were
5mL) and DMF(12mL) and stirred in a 160°C oil bath for 10 h obtained by fitting to the atomistic profiles of the potentials of
in dark. The solvent was removed by rotatory evaporation and mean force (PMFs) constructed from atomic configurations
compound
5.3% yield).
Compound 9: Compound
8 (Scheme S2, ESI) after lyophilization (2.204g,
(
6
7
8
(
2
3
the crude product was purified by precipitation in diethyl ether (OPLS force field) of liquid organics (Cy7-HDA, Gd-DOTA-HDA
and water solution. After washing with ethanol, the NIR and FA-HDA). All bonded parameters are given in Supporting
organic dye compound
701.1mg, 31.4% yield).
Compound 2 (Cy7-HDA)
compound
9
(Scheme S2, ESI) was obtained Information. The GBEMP MD simulation protocol used in this
study is described as follows. The atomistic structure of each
(
:
HDA (2.333g, 9.669mmol) and system was converted into its CG representation in Euler
9
(0.7011g, 0.9655mmol) were added into DMF coordinates containing three Cartesian coordinates (x, y and z)
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Journal Name
and three Euler angles (φ, θ and ψ) of the centers of mass. All transferred to a 96-well plate and the absorbance values at a
GBEMP simulations were carried out in the Generalized wavelength of 450 nm in each well were measured using a
2
4
Kirkwood (GK) implicit solvent
in the TINKER-based Synergy H1 hybrid reader (BioTek Instruments, Inc, USA).
“
GBEMP” program and the equation of motion was integrated
using the Euler’s rigid body integrator with an integration step 2.10 In vivo MRI and NIR study
of 10 fs. The non-bonded interaction cut off was set to 16 Å
Female BALB/c-nu mice (5-6 weeks) were purchased from the
6
with a truncate scheme, and the Gay-Berne and electrostatic
interactions of 1-2 and 1-3 neighbors were scaled with a factor
of 0.003 and 1.0, respectively. Each nucleic acid system was
minimized and subsequent runs were carried out under the
temperature of 300 K for 6 -12 ns.
Animal Center of Dalian Medical University. 2×10 U-87 MG
cells in PBS/Matrigel mixture (1:1 v/v) were inoculated into the
right flank of the nude mice. When the tumors reached 0.3-0.5
cm in diameter, the mice were randomly divided into groups
with four mice for each group. The bimodal probes tumor-
targeted GCF-HDA or non-targeted GC-HDA saline solution
2.8 MRI analysis of bimodal NPs
Relaxation rate measurements of the bimodal NPs was carried were injected from tail vein at a dose of 0.05 mmol-Gd/kg. The
out by using the stoke solution (targeted and non-targeted) mice were anesthetized with 2% isoflurane and scanned with a
diluted with water to obtain five different gadolinium 0.5T permanent magnet MRI scanner at pre-injection and at
concentration in the range of 0.0625-1.0 mM [Gd]. T
1
and T
2
24, 48, 72, 96, 120 and 144 h post-injection using a SE
relaxation time of each sample were measured with an sequence with the parameters TR=500ms, TE=19ms,
2
inversion-recovery (IR) pulse sequence and a Carr-Purcell- FOV=100
Meiboom-Gill (CPMG) sequence, separately, using a 0.5T MRI gap=0.5mm, average=4, matrix size=256
system. The r or r relaxation rate was defined as the slope of enhancement of MRI images was analyzed using Osirix
the line generated from a graph of 1/T (1/T or 1/T ) vs the software. A region of interest was drawn around the whole
atomic concentration of Gd element (shown in equation 1). tumor and the mean signal intensity was captured as a directly
Additionally, the T -weighted axial images were acquired using reflection of the longitudinal relaxation time at each time
×
100mm , slices=12, slices width=1.8mm, slice
×
192. In vivo contrast
1
2
1
2
1
the Spin Echo (SE) sequence. The parameters for relaxation point. Following MRI imaging at each time point, the spectral
rate analysis were SW = 200, RFD = 0.010 ms, TW = 7000 ms, NIR images were acquired by a Cri maestro EX in vivo imaging
RG1 = 20, DRG1 = 3, NS = 4, DL1 = 0.275, NECH = 10000. system using the appropriate filter for probe (excitation: 605
Parameters for MRI images were TR = 500 ms, TE = 19 ms, FOV nm; emission: 645 nm long-pass filter; 10 nm steps).Exposure
2
100 mm , slices = 3, slices width = 2.5 mm, slice gap = times were automatically calculated and kept the same for
=
1
100×
.0 mm, average = 4, matrix size = 256
× r + 1⁄ꢁꢂ 1⁄ꢁꢃ
where r is the relaxivity rate; C is the Gd concentration values; significance at p < 0.05.
and T are relaxation time of the pure solvent and the 2.11 Ex vivo NIR imaging and histological analyses.
aqueous solution containing bimodal NPs, respectively.
.9 In vitro fluorescent cell uptake assay and cytotoxicity assay
×
192.
GCF-HDA and GC-HDA NPs. Statistical analysis was performed
C
=
(1)
using one-tailed Student’s t-test, assuming statistical
a
T
w
m
After MRI and NIR imaging at 96 h, the mice were sacrificed
2
and tumor tissue and main organs were isolated for ex vivo NIR
Human glioma cells U-87 MG cells were cultured in high imaging with CRi maestro imaging system. Then the tumor
glucose Dulbecco's Modified Eagle Media (DMEM) (Gibco) tissues were cryosectioned into 6-µm slices and imaged
medium containing 10% fatal bovine serum (FBS) (TransGen immediately at 20X on a confocal laser scanning microscope
Biotech), 1% penicillin/streptomycin, in 35 mm culture dishes equipped with 633 nm leaser source. The images were
4
o
(
2×10 cells per well, 1ml of DMEM medium) at 37 C under 5% processed with the Olympus FV software.
CO . The cells were grown until 80% confluent and 1 mL of
targeted and non-targeted probe (1 mM[Gd]/L) was added,
respectively. After incubation under 5% CO atmosphere at 37
2
3
. Results and discussion
2
o
C for 2 or 4 h, the cells were washed with PBS (pH-7.4) for
three times to remove the extra probes and imaged by
confocal microscope system equipped with an external 633 nm
excitation laser.
3
.1. Synthesis and characterization
Tumor targeted bimodal MRI/NIR liposome GCF-HDA NPs have
been synthesized with three kinds of amphiphilic molecules
through self-assembly in aqueous solution using an
The cytotoxicity of the GCF-HDA was evaluated by Cell
ultrasonicator (Scheme 1). Gd-DOTA-HDA (compound
prepared via the amphiphilic species DOTA-HDA (compound
from the amidation of DOTA-NHS (compound ) with HDA. The
tert-butyloxycarbonyl group on DOTA-HDA has been removed
by TFA to obtain compound with high yield and purity.
1) was
Counting Kit-8 (CCK-8) viability assay. U-87 MG cells were
4
10 per well
5)
seeded in to a 96-well plate with a density of 1 ×
and cultured in the same way mentioned above for 24 h. The
4
medium was replaced with fresh medium containing GCF-HDA
-6
6
nanoprobe at different concentrations (1 ×10 -0.1mM [Gd]).
Afterwards, the DOTA-HDA was complexed with
gadolinium(III) acetate hydrate at pH = 5-6 in aqueous solution
o
at 90 C for 2 hours to obtain the paramagnetic, amphiphilic
After incubating for 6 and 12 h, the medium was removed and
the cells were washed with PBS for three times. Then 150ꢀμL of
CCK-8 (10% in fresh medium) solution was added into the cells
and incubated for another 1 h. 100 μL supernatant was
compound Gd-DOTA-HDA
1 (Scheme S1, ESI). As shown in
Fig.S2, the absorbance of dialyzate is similar to that of the PBS
4
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control. However, the absorbance of Gd-DOTA-HDA and free electron-rich HDA, the Stokes shift of Cy7-HDA increased to
3
+
Gd (5 mM) was 1.51 and 2.32-fold as compared with that of 150 nm, which represents a 5-fold increase compared to that
the dialyzate. This result strongly suggested that the Gd-DOTA- of Cy7. This significant change might be attributed to the intra-
HDA was sable in PBS for storage as long as 7 days.
The NIR organic dye Cy7-HDA (compound
molecular charge transfer (ICT) between the charge donor
1
9
was HDA and the acceptor, the Cy7 dye. The fluorescence
2)
synthesized by reacting intermediate
7 with intermediate 8 at quantum yield of Cy7 was found to be 1.5% and increases to
high temperature followed by conjugation of HDA as an 8% after formation of Cy7-HDA by using ICG as a reference
electron-donating moiety at the central position of Cy7 dye to (Table 1). The latter property is of great advantage for in vivo
obtain the NIR amphiphilic molecule (compound 2) (Fig. 1a and tumor imaging. Finally, the FR-targeted amphiphilic molecules
Scheme S3, ESI). The NIR dye, heptamethine cyanine (Cy7), has FA-HDA were synthesized via amide coupling reaction of HDA
already been extensively used in tumor imaging and cancer with the DCC/NHS activated FA ester (Scheme S4, ESI). All of
therapy in recent years, mostly due to their thermal-stability the intermediates and final compounds have been
2
5-27
and photo-stability.
significant overlap of the excitation/emission spectra due to a ESI).
small Stokes shift (< 30 nm) (Fig. 1b) After conjugation with
However, this NIR dye still exhibits a characterized by MOLDI-TOF mass spectrometry (Fig. S1, S3-4,
.
Scheme 1.(a) Design of the tumor-targeted bimodal GCF-HDA NPs. (b) Schematic diagram of the GCF-HDA NPs as the targeted MRI/NIR bimodal probe for FR-mediated
endocytosis, enabling MRI and NIR imaging of the glioma.
Absorption (black) and emission (blue) spectra of dyes in ethanol (left for
Cy7,ex735nm; right for Cy7-HDA,ex605nm).
Table 1.Photophysical characteristics of NIR dyes in ethanol.
Absorption
Emission
Stokes
QY
dye
ꢄꢅꢆ(nm)
ꢄꢇꢃ(nm)
shift(nm)
(%)
Cy7
780
620
810
770
30
1.5
8.0
Cy7-HDA
150
A bimodal MRI/NIR liposome NP has been constructed through
a self-assembly process of the above mentioned three kinds of
functional amphiphilic molecules, Gd-DOTA-HDA, Cy7-HDA and
FA-HDA (GCF), at a molar ratio of 5:5:1 in aqueous solution
upon ultrasonication. The non-targeted liposomes were also
prepared as a control in this study with a molar ratio of Gd-
DOTA-HDA/Cy7-HDA determined to be 1:1. Upon combination
Fig.1. (a) Synthetic scheme for the amphiphilic heptamethine cyanine dyes. (b)
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of the three functional groups through self-assembly, the was listed in the Supporting Information (ESI). Through
absorption spectrum of GCF nanoparticles is shown in Fig. S5 combination of the simulation results it was revealed that the
(ESI), which exhibits the presence of three kinds of functional blackspots in HRTEM images might be the clusters formed due
amphiphilic molecules, Gd-DOTA-HDA, Cy7-HDA and FA-HDA. to van der Waals (VDW) interactions of adjacent NPs. Similar
The liposomes have been characterized by HRTEM imaging structures have been reported previously involving self-
(Fig. 2a). It was observed that the liposomes are of irregular assembled materials with small molecule amphiphilic
spherical shape with a diameter ranging between 20 and 40 compounds, which have also been studied in detail as
nm (Fig. 2b).Furthermore, some inlayed black spots could be potential candidates for drug delivery systems with controlled
2
8, 29
found with a size of 1.8-3.5 nm (Fig. 2b, inset). The assembly release properties.
process of the liposomes was assessed by molecular dynamics
(
MD) simulations based on a coarse-grained (CG) model
detailed information please see ESI). The initial structure (t = 0
(
ns) of the system consists of a bilayer (Fig. 2c) as well as a
random assembly (Fig. S6, ESI). The potential energies have
been monitored during GBEMP simulations of the two systems
(bilayer and a random assembly). For each system, the energy
landscape became flat after simulation for 9 ns, indicating that
these systems reach their corresponding equilibria. GBEMP
mapping for three self-assembly units is depicted in Fig. S7
(ESI). The CG model of Cy7-HDA (C) is composed of one elliptic
rigid body and three disk-like rigid bodies, that of Gd-DOTA-
HDA (D) is made up of four elliptic rigid bodies and that of FA-
HDA (F) consists of one elliptic rigid body and one disk-like
rigid body. The hydrophobic tail of each model is represented
by one elliptic rigid body that contains one Gay-Berne site and
one non-interacting EMP site. The equilibrium conformation of
the mixture, independent of its starting conformation, shows
similar characteristics: (1) the whole system (C:D:F=5:5:1) is
composed of clusters with various sizes (2.0~3.5 nm in
Fig.2.a) HRTEM images (negative stained by phosphotungstic acid) and b) size-
distribution of GCF-HDA (black histogram) and the clusters (red histogram). c) Potential
energies of the system (the mixture of C, D and F was built in a ratio of 5:5:1) evolving
with MD simulation time. The starting structure (t = 0 ns) of the system was
diameter); (2) in each cluster the head groups of the constructed as a form of bilayer. d) The final structure (t = 9 ns) of the system was
obtained from the end of the GBEMP simulation. The three self-assembly units C, D and
amphiphilic molecules assemble together and the tail groups
F are represented by green, orange and red lines respectively. Gd3+ ion is illustrated as
point outwards. The non-interacting EMP site (denoted by
a van der Waals (VDW) surface. The diameter (d) of each cluster is given in the plot.
orange filled circle) would not involve in electrostatic
interaction but serve just as the connection purpose. In each
In an effort to investigate the photostability of GCF-HDA NPs,
model, the corresponding CG particles of hydrophilic head
the absorption spectra of GCF-HDA NPs in solution have been
group contain one Gay-Berne site and one EMP site which
measured at different time points and compared with that of
both share a same location shown by a red filled circle. In
the ICG solution as control. As shown in Fig. 3a, the
addition, one or two non-interacting EMP sites are included
absorbance of the GCF-HDA NPs solution proved to be almost
into each CG particle in order to connect two different rigid
the same with light exposure for 24 hours, whereas that of the
bodies. The effective energy function of GBEMP model was
ICG solution degraded to 40% with the same exposure time (cf.
calculated as depicted in the section of GBEMP energy
Fig. 3b). The results obtained confirm that the newly
function (please see ESI). In this work, the bond stretching,
synthesized Cy7-HDA shows a higher stability than ICG, which
angle bending and torsional potentials were parametrized by
proves to be an important feature for applications in which a
fitting to the atomistic profiles of the potentials of mean force
long time signal requisition and high tolerance to radiation are
(PMFs) constructed from atomistic conformations of self-
required, e.g. in near-infrared dyes used in bio-imaging.
Furthermore, the stability of the hydrodynamic diameters of
the liposome NPs in aqueous solution was also assessed. No
significant change in the hydrodynamic particle size has been
observed and the poly dispersity index (PDI) was found to be
less than 0.3 over 6 days (Fig. 3c). The hydrodynamic diameter
was in the range of 70-90 nm, which is larger than those
obtained by TEM. This probably due to the aggregation of
partial nanoparticles, which contribute great to total particle
size in DLS analysis. It is necessary to mention that by TEM we
image single particles, while DLS gives an average size
estimation, which is biased toward the larger-size end of the
assembly units (Fig. S8, ESI). To parametrize the Gay-Berne
potentials for nucleic acid models shown in Fig. S7 (ESI), the
atomistic energy profiles using OPLS atomistic force field in
this study were constructed for the intermolecular van der
Waals (VDW) interactions between two identical molecular
fragments (homodimer), illustrated in Fig. S9 (ESI). In each
case, diverse configurations were generated with different
orientations (such as, cross, end-to-end, face-to-face, etc.) as
well as at various separations. The initial Gay-Berne
parameters for CG particles were derived through fitting to the
corresponding atomistic energy profiles in gas phase using a
generic algorithm. GBEMP force field for self-assembly units
6
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population distribution. The zeta potential measurements surface of the cell membrane.
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1
8, 30,31
FA is a vitamin, essential
3
0
indicated that the GCF-HDA liposome NPs prove to be for the proliferation and maintenance of all cells. On the
positively charged with a potential of 65.5 mV. Fig. 3d membrane of a majority of malignant tumor cells, FR is often
illustrates concentration-dependent T
GCF-HDA and GC-HDA agents, wherein the GCF-HDA exhibits a in the serum as efficient as possible. This is done to satisfy the
better contrast enhancement. The r and r relaxivities of the uncontrolled proliferation needs of tumor cells. Considering the
liposome NPs have also been measured as shown in Fig. 3e high affinity of tumor cells to FA via over-expressed FR, FA
and 3f. The r and r relaxivities of GCF-HDA were found to be may be applied as a tumor-targeted media in FA-targeting
1.87 and 19.91 mM s per Gd(III) chelate at 0.5T, with 9.27 cancer imaging and therapy.
1
weighted images of found to be over-expressed in order to capture any FA present
3
0
1
2
1
2
-1
-1
32-34
1
The results obtained show that
and 15.53 mM s per Gd(III) chelate for the control agent GC- the GCF-HDA NPs can serve as a fluorescence probe for FR-
HDA, respectively. The r relaxivity of GCF-HDA was slightly positive tumor cell imaging. The viability of cells incubated
higher than that of the corresponding non-targeted GC-HDA, with different concentrations of GCF-HDA NPs was shown in
-1 -1
1
but much higher than that of the clinically approved T
Fig. 4b. We can see that the GCF-HDA NPs displayed low
toxicity (ca. 90%) to U-87 MG cells when the concentration of
probes less than 0.01 mM [Gd] for 6 and 12 h.
1
-1 -1 20
weighted contrast agent, Gd-DOTA (3.66 mM ∙s ).
Fig. 3. Absorbance intensity change of GCF-HDA NPs (a) and ICG (b) over time when the
solutions were exposed to ambient light. c) Monitoring the stability of the targeted and
non-targeted nanoparticle solution at room temperature for 6 days. d) T
images of the targeted and non-targeted groups. Plots of 1/T (r ) and 1/T
1
-weighted
1
1
2
2
(r ) versus
the concentration of the Gd3+ element for the targeted group (e) and non-targeted
group (f).
Fig. 4. a) The in vitro cellular uptake and cancerous cell imaging of bimodal NPs.
Confocal microscope images with the 633 nm excitation wavelength both for FR-
targeted and non-targeted NPs (1 mM [Gd]/mL) after incubation for 2 and 4 h (Scale
bar represents 20 μm ). b) CCK-8 assay of U-87 MG cell viability after treatment with
3
.2 In vitro NIR fluorescence imaging and cytotoxicity
The selective targeting ability of GCF-HDA NPs for FR-
expressing cells was evaluated by FR-positive human glioma U-
-6
GCF-HDA NPs at the concentration of 10 – 0.1 mM [Gd] for 6 and 12 h. Cells treated
87 MG cells via in vitro imaging. As shown in Fig. 4a, the
with fresh medium were used as control.
liposomes were able to enter the cells and accumulate in the
cytoplasm. The latter has been validated by the strong
fluorescence intensity around the perinuclear region. The cell
morphology remains intact, indicating that the cells feature a
normal physiological activity in the presence of NPs. Notably,
only U-87 MG cells treated with GCF-HDA NPs show greater
fluorescence at 2 and 4 hours, respectively. This finding may be
due to the active FR-mediated endocytosis that occurs on the
3
.3 In vivo MR/NIR tumor imaging
Tumor imaging is a valuable tool in the diagnosis and
treatment of various tumor types, providing visual anatomical
and physiological information noninvasively using appropriate
1
probes. The T -weighted 2D MR images of tumor bearing mice
contrast enhanced with the FR-targeted GCF-HDA and non-
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targeted GC-HDA before and after tail vein injection are shown hours, respectively. A greater and more prolonged NIR fluorescence
in Fig. 5a using a U-87 MG xenograft model (0.05 mmol-Gd/kg signal was observed in the tumor area using GCF-HDA compared to
3
+
body weight, calculated for Gd ). The contrast gets gradually GC-HDA for at least 96 hours post-injection, indicating an effective
stronger over time as a result of bimodal probe accumulation binding of the targeting agent in the tumor tissue. Fig. 5d illustrates
in the tumor sites, reaching a maximum at 96 (GCF-HDA) or 72 the statistical analysis of the fluorescence intensity in the tumor
hours (GC-HDA) post-injection. The images become darker region at each time point. Remarkably, the fluorescence intensity of
over time after 96 hours of administration due to elimination the FR-targeted GCF-HDA was stronger than that of GC-HDA,
and excretion of the agents. The quantitative MR indicating that a larger amount of the probe accumulated in the
enhancement in tumor regions before and after injection of tumor site. The latter finding is also consistent with the MR images
the liposome bimodal probes is plotted in Fig. 5b. obtained. Since the fluorescence NIR images obtained at the same
Administration of the targeted GCF-HDA resulted in higher MR time point followed MRI imaging, the quantitative fluorescence
contrast enhancement than that of the corresponding non- intensity (Fig. 5d) indicates a similar profile to that of MR imaging
targeted probe GC-HDA in the tumor tissue at 96 hours (p < (Fig. 5b), further suggesting that the GCF-HDA accumulate in the
0.01). This finding is likely due to the GCF-HDA with the FA tumor at 24 hours and increased progressively up to 96 hours post-
moiety targeting the tumor tissue more readily than the non- injection. In our previously reported study with FA modified
targeted GC-HDA agent without FA. The FA functionalized chitosan probes, we demonstrated that by using the targeting
liposomes can accumulate in tumor cells through receptor- agents, a strong and prolonged tumor enhancement was observed
mediated endocytosis at both high concentration and with significant and localized accumulation in tumor tissues in
35
specificity. The vasculature in the solid tumor is often found to vivo.
be vastly abnormal, resulting large gaps of 300-800 nm
3
.4 Stability of the GCF-HAD in fetal bovine serum (FBS) solution.
3
0
between adjacent endothelial cells. This feature is the reason
why NPs are able to accumulate in solid tumors efficiently.
Therefore, the FA-targeting GCF-HDA exhibits a more rapid
accumulation into the tumor sites through synergistic effects
of enhanced permeability and retention (EPR) effect and FR-
mediated endocytosis. On the other hand, the elimination rate
of the targeted GCF-HDA was found to be slower than that of
the non-targeted analogue GC-HDA, most likely due to the FA
functionalization that increases the retention time of the NPs
The stability of the liposome NPs in the organism is important for
bioimaging. The liposome GCF-HAD NPs were added into 50% fetal
bovine serum (FBS) solution and their aggregation behavior was
observed by DLS for 0 h, 0.5 h, 1 h and 24 h. The result in Fig. S10
showed that there was no aggregation, and the size distribution did
not change much within 1 h. Even at 24 h, the liposome NPs
showed a particle behavior with an increased size distribution. On
the other hand, the intensity change of MRI and NIR was always in
consistent with each other during the process of in vivo imaging. If
they disentangled in the organism, they would be metabolic in their
own way and the intensity change of MRI and NIR would become
different. So the GCF-HAD NPs were stable in the organism after
injection.
3
0
in tumorous tissues.
3
.5 Ex vivo fluorescence imaging and histological analysis
Ex vivo fluorescence imaging and histological studies of the
tumor tissue and major organs may provide further organ and
structural information of FA binding on a microscopic level
through fluorescence signal analysis. The ex vivo fluorescence
images of tumors and major organs collected after 96 hours
recorded on a Maestro EX in vivo imaging system are shown in
Fig. 6a. A strong red fluorescence of Cy7-HDA was observed in
the tumor regions of the mice injected with FR-targeted GCF-
HDA, whereas an insignificant fluorescence signal was
observed in the tumor tissue in the mice injected with non-
targeted GC-HDA. Furthermore, NPs were found to
accumulate in the liver and spleen as well with a negligible
signal observed from the heart, lungs and kidneys. The
Fig.5. In vivo MRI/NIR images of U-87MG-bearing mice. a) MRI images (pseudocolor
maps represent tumor area) were obtained at different intervals after tail vein injection
of targeted GCF-HDA and non-targeted GC-HDA NPs, respectively, at 24, 48, 72, 96, 120
and 144 h. b) A quantification and statistical analysis of MRI signal intensity at the
tumor region both for targeted and non-targeted group. c) NIR images acquired at 24, quantitative fluorescence intensity profile shown in Fig. 6b
8, 72, 96, 120 and 144h after tail vein injection of targeted GCF-HDA and non-targeted illustrates that administration of FR-targeted GC-HDA NPs
4
GC-HDA NPs, respectively. d) A quantification and statistical analysis of fluorescence
intensity at the tumor region both for targeted GCF-HDA and non-targeted GC-HDA
group. The data were represented as mean ±SD (n=4, *p<0.05, **p<0.01).
results in approximately a 5-fold increase in the fluorescence
signal compared to GC-HDA at 96 hours. The histological
analysis of the tumor tissue indicates that stronger red
fluorescence was associated with the injection of FR-targeted
GCF-HDA and an insignificant fluorescence signal was obtained
from the tissue section in the mice injected with non-targeted
Fig. 5c shows the fluorescence images of the tumor bearing
mice after tail vein injection of the targeted GCF-HDA and non-
targeted GC-HDA at time points of 24, 48, 72, 96, 120 and 144
8
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GC-HDA (Fig. 6c). This result provides further evidence for the China (2013YQ17046307) and the Nature Science Foundation
hypothesis that the targeting agent GCF-HDA exhibits a specific of Liaoning Province, China (2013020177).
binding ability to the cells in the tumor tissue.
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Statements
The authors state that all animal studies were carried out at
Specific Pathogen Free Animal Center at the Dalian Medical
University according to the animal protocols (No. L2014014)
approved by the Animal Ethics Committee (AEC).
2
2
2
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2
Acknowledgements
This work was financially supported by the National Nature
Science Foundation of China (91227126), National Key
Scientific Instrument and Equipment Development Project of
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