PEGylated micelle nanoparticles encapsulating a non-fluorescent near-infrared organic dye as a safe and highly-effective photothermal agent for in vivo cancer therapy

Article abstract of DOI:10.1002/adfm.201301045

Photothermal therapy (PTT), as a minimally invasive and highly effective cancer treatment approach, has received widespread attention in recent years. Tremendous effort has been devoted to explore various types of photothermal agents with high near-infrared (NIR) absorbance for PTT cancer treatment. Despite many exciting progresses in the area, effective yet safe photothermal agents with good biocompatibility and biodegradability are still highly desired. In this work, a new organic PTT agent based on polyethylene glycol (PEG) coated micelle nanoparticles encapsulating a heptamethine indocyanine dye IR825 is developed, showing a strong NIR absorption band and a rather low quantum yield, for in vivo photothermal treatment of cancer. It is found that the IR825-PEG nanoparticles show ultra-high in vivo tumor uptake after intravenous injection, and appear to be an excellent PTT agent for tumor ablation under a low-power laser irradiation, without rendering any appreciable toxicity to the treated animals. Compared with inorganic nanomaterials and conjugated polymers being explored in PTT, the NIR-absorbing micelle nanoparticles presented here may have the least safety concern while showing excellent treatment efficacy, and thus may be a new photothermal agent potentially useful in clinical applications. Copyright

Full text of DOI:10.1002/adfm.201301045

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PEGylated Micelle Nanoparticles Encapsulating a Non-
Fluorescent Near-Infrared Organic Dye as a Safe and Highly-
Effective Photothermal Agent for In Vivo Cancer Therapy
Liang Cheng, Weiwei He, Hua Gong, Chao Wang, Qian Chen, Zhengping Cheng,*
and Zhuang Liu*
invasiveness.[1,2] In typical applications
of PTT, photothermal agents with strong
optical absorbance, preferably in the
tissue-transparent near-infrared (NIR)
window (700–900 nm), are generally
indispensable. Ideal photothermal agents
should exhibit strong and stable NIR
absorbance and, thus, are able to effec-
tively convert the absorbed NIR light
energy into heat. In addition, the agents
used in PTT should be nontoxic and show
high tumor-homing ability, to improve
therapeutic efficacy without rendering
toxic side effects. In recent years, a variety
of NIR-absorbing inorganic nanomate-
rials, such as different gold nanostruc-
tures,[3–9] carbon nanomaterials,[2,10–12]
palladium nanosheets,[13,14] copper sulfide
nanoparticles,[15,16] and tungsten oxide
nanowires[17] have been widely explored
by many research groups including ours
as photothermal agents for PTT abla-
tion of cancer cells in vitro and in vivo.
Although many of the above materials
Photothermal therapy (PTT), as a minimally invasive and highly effective
cancer treatment approach, has received widespread attention in recent
years. Tremendous effort has been devoted to explore various types of
photothermal agents with high near-infrared (NIR) absorbance for PTT cancer
treatment. Despite many exciting progresses in the area, effective yet safe
photothermal agents with good biocompatibility and biodegradability are still
highly desired. In this work, a new organic PTT agent based on polyethylene
glycol (PEG) coated micelle nanoparticles encapsulating a heptamethine
indocyanine dye IR825 is developed, showing a strong NIR absorption band
and a rather low quantum yield, for in vivo photothermal treatment of cancer.
It is found that the IR825–PEG nanoparticles show ultra-high in vivo tumor
uptake after intravenous injection, and appear to be an excellent PTT agent
for tumor ablation under a low-power laser irradiation, without rendering any
appreciable toxicity to the treated animals. Compared with inorganic nano-
materials and conjugated polymers being explored in PTT, the NIR-absorbing
micelle nanoparticles presented here may have the least safety concern while
showing excellent treatment efficacy, and thus may be a new photothermal
agent potentially useful in clinical applications.
have already shown high efficacies for PTT treatment of can-
cers in pre-clinical animal experiments, most of these currently
used inorganic photothermal agents are non-biodegradable and
would retain in the body for long periods of time, preventing
their further application in clinical cancer treatment due to the
potential long-term toxicity concern.[18]
1. Introduction
Photothermal therapy (PTT) is
peutic approach that employs heat generated from optical
energy to kill cancer cells with high efficiency and minimal
a
hyperthermia thera-
Recently, significant attentions have been paid to the devel-
opment of NIR-absorbing organic materials as photothermal
agents in PTT cancer treatment. A number of conjugated poly-
mers with appropriate aromatic structures exhibit rather large
NIR absorption cross-sections. In several recent studies, we
and others have developed NIR-absorbing nano-agents based
on conjugated polymers, such as polyaniline, polypyrrole, and
poly-(3, 4-ethylenedioxythiophene):poly (4-styrenesulfonate)
(PEDOT:PSS) which, after appropriate surface modification,
could be used for highly effective in vivo PTT treatment of
cancer in animal experiments.[19–22] Despite the encouraging
PTT therapeutic effects obtained using those polymeric nano-
particles, the biodegradation behaviors of conjugated polymers
remain unclear. Porphyrin-lipid, or porphysomes, is another
type of new PTT nano-agent currently under investigation.[23]
However, the absorbance peak of porphyrin compounds
Dr. L. Cheng, H. Gong, C. Wang, Q. Chen
Prof. Z. Liu
Jiangsu Key Laboratory for Carbon-Based
Functional Materials & Devices
Institute of Functional Nano &
Soft Materials Laboratory (FUNSOM)
Soochow University
Suzhou, Jiangsu 215123, China
E-mail: zliu@suda.edu.cn
Mr. W. He, Prof. Z. Cheng
Department of Polymer Science and Engineering
Jiangsu Key Laboratory of Advanced Functional
Polymer Design and Application
College of Chemistry, Chemical Engineering and Materials Science
Soochow University
Suzhou, Jiangsu 215006, China
E-mail: chengzhenping@suda.edu.cn
DOI: 10.1002/adfm.201301045
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locates in the red light region (660–670 nm), which exhibits
much lower tissue penetration ability compared to NIR light
due to the existence of porphyrin-containing hemochrome in
red blood cells.[24] In a few latest reports, several other organic
nanomaterials, such as Prussian blue nanoparticles and dopa-
mine-melanin colloidal nanospheres, also showed their possi-
bility to be used as PTT agents.[25,26] Although those materials
could absorb NIR light, their peaked absorption is not in the
NIR region.
Small organic fluorescent dyes have been extensively
studied in past decades, mainly as labeling probes in optical
imaging. For example, indocyanine dyes, which could be
engineered to acquire strong NIR absorption and emission,
have been synthesized and widely used for in vivo fluores-
cent imaging.[27–29] Among them, indocyanine green (ICG)
has been approved by federal drug administration (FDA) for
a number of clinical applications.[28,30–32] Besides being used
in fluorescent imaging, ICG is also under investigation as a
photodynamic therapy agent,[28,31] which produces reactive
oxygen species instead of heat to damage cancer cells under
light exposure, although the use of ICG for photothermal
cancer treatment has also been demonstrated in several
recent reports.[33]
Organic dyes with high quantum yields (QY) are gener-
ally preferred in fluorescent optical imaging. However, their
photothermal efficiency could be low because a part or even a
majority of the absorbed optical energy is emitted from those
dye molecules as fluorescence instead of heat. Organic dyes
with high NIR absorption co-efficients but low QYs, on the
other hand, may offer the best photothermal efficiency, since
the thermal effect would prevail in the energy dissipation pro-
cess of those molecules after light excitation. Therefore, in
this work, we synthesize a NIR-absorbing heptamethine indo-
cyanine dye, IR825, which shows a super high NIR absorption
peak at ≈825 nm and a rather low QY (<0.1%). A polyethylene
glycol (PEG) grafted amphiphilic polymer is used to solubilize
IR825, forming IR825–PEG micelle nanoparticles with great
solubility in water and other physiological solutions. Com-
pared with ICG, IR825–PEG exhibits stronger photothermal
effect under 808-nm NIR laser irradiation, and more impor-
tantly, dramatically improved photothermal stability. Without
showing any appreciable toxicity to cells, IR825–PEG could
serve as an effective photothermal agent to destruct cancer
cells under NIR light. In vivo animal experiments uncover
the ultra-long blood circulation half-life of IR825–PEG. As
the result, highly efficient passive tumor accumulation of
IR825–PEG is observed, owing to the enhanced permeability
and retention (EPR) effect of cancerous tumors. In vivo photo-
thermal therapy is then carried out in a mouse tumor model
by intravenous (i.v.) injection of IR825–PEG nanoparticles
and laser irradiation of tumors at a low optical power density
(0.5 W cm−2 ), achieving an excellent therapeutic efficacy with
100% of tumor destruction. Moreover, no obvious signal of
toxicity is observed on IR825–PEG injected mice as evidenced
by both hemological and histological data. Our work presents
a new photothermal agent based on nanoparticles of an NIR-
absorbing organic dye, which is safe and highly effectively for
photothermal cancer treatment.
2. Results and Discussion
A heptamethine indocyanine dye, IR825, which is a lipo-
philic cation with strong adsorption at 825 nm (in meth-
anol) (Figure 1a,b), was synthesized following the procedure
described in the experimental section (Scheme 1). The struc-
1
ture of IR825 was confirmed by H-NMR and high-resolution
mass spectrum (HRMS) (Supporting Information, Figure S1).
The molar absorption coefficient of IR825 at the peak of 825
nm in methanol was calculated to be 1.145 × 105 −1 cm−1 . On
M
the other hand, the fluorescence QY of IR825 dye was deter-
mined to be <0.1% in methanol, which was much lower than
that of ICG[34] (Supporting Information, Figure S2).
As-synthesized IR825 was not water-soluble. We therefore
used PEG-grafted poly (maleicanhydride-alt-1-octadecene)
(C18PMH–PEG), a biocompatible amphiphilic polymer used
in our previous studies to functionalize a variety of inorganic
nanomaterials,[35–37] to solubilize IR825 (Figure 1a), forming
PEGylated IR825 micelle nanoparticles (IR825–PEG) with
excellent stability in physiological solutions (Supporting Infor-
mation, Figure S3). Compared with IR825 in methanol, IR825–
PEG nanoparticles in water showed broadened but slightly
reduced NIR absorbance (Figure 1b), and completely quenched
fluorescence that was close to the noise level (Supporting Infor-
mation, Figure S2), likely owing to the intermolecular interac-
tions between IR825 molecules inside the micelle core. Based
on images of atomic force microscopy (AFM) and transmis-
sion electron microscopy (TEM), the average micelle diameter
was ≈25 nm (Figure 1c; Figure S4, Supporting Information),
consistent to that measured by dynamic light scattering (DLS)
(Figure 1d), even though AFM and TEM imaged nanoparticles
in the dried form, while DLS measured the hydrodynamic sizes
of nanoparticles in the aqueous solution. With time, the diam-
eter of IR825–PEG nanoparticles in phosphate buffered saline
(PBS) remained unchanged, indicating the great stability of
those nanoparticles without aggregation (Figure 1d).
We then studied the photothermal properties of our
PEGylated IR825 nanoparticles. IR825–PEG solutions with
various concentrations at 0.05, 0.1, 0.2, and 0.5 mg mL−1
were exposed to an 808-nm NIR laser at a power density
of 0.5 W cm−2 . Obvious concentration-dependent tempera-
ture increases of IR825–PEG solutions were found under
laser irradiation, while pure water showed little change
(Figure 1e). Compared with ICG, the FDA-approved NIR dye
widely explored in photodynamic and photothermal thera-
pies,[28,31,33] IR825–PEG showed an absorbance peak located
in the longer wavelength (Figure 1g). Under the 808-nm laser
irradiation, the temperature rise of the IR825–PEG solution
was higher than that of ICG at the same concentration (Figure
1f). More importantly, IR825–PEG exhibited outstanding pho-
tostability under constant laser exposure, without losing its NIR
absorbance after 10 min of irradiation (808 nm, 0.5 W cm−2 ),
which, in marked contrast, resulted in nearly complete elimina-
tion of ICG NIR absorbance (Figure 1g). The strong and stable
photothermal performance of IR825–PEG makes it an encour-
aging nano-agent for PTT cancer treatment.
To explore the applications of IR825–PEG nanoparticles in
biomedicine, we first tested their potential toxicity to several
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Figure 1. IR825-PEG characterization. a) A scheme showing the composition of IR825–PEG nanoparticles. b) UV-vis-NIR absorbance spectra of IR825
in methanol and IR825–PEG in water. c) An AFM image of IR825–PEG nanoparticles. d) DLS measured diameters of IR825–PEG after being incubated
in PBS for different periods. Error bars were based standard deviations (SD) of three samples per time point. e) The heating curves of pure water and
IR825–PEG solutions with different concentrations (0.05, 0.1, 0.2, and 0.5 mg mL−1 ) under 808-nm laser irradiation at the power density of 0.5 W cm−2
.
f) The heating curves of IR825–PEG and ICG solutions at the same concentration of 0.1 mg mL−1 under laser irradiation (808 nm, 0.5W cm−2 ).
g) UV-vis-NIR absorbance spectra of IR825–PEG (0.01 mg mL−1 ) and ICG molecules (0.01 mg mL−1 ) before and after 10 min of laser irradiation
(808 nm, 0.5 W cm−2 , 10 min). Inset: Photos of IR825–PEG and ICG solutions before and after laser irradiation.
up to 0.2 mg mL−1 (Figure 2a,b). To further look for any poten-
tial cell damage caused by IR825–PEG nanoparticles, release of
lactate dehydrogenase (LDH), an indicator of cell membrane
damage, was also examined. It was found that the levels of
released LDH from IR825–PEG incubated cells were normal
compared to that of untreated cells, indicating no obvious
cell membrane damage induced by IR825–PEG nanoparticles
(Figure 2c). Reactive oxygen species (ROS)
types of cells. The standard methyl thiazolyl tetrazolium (MTT)
assay was carried out to determine the relative viabilities of
4T1 murine breast cancer cells, HeLa human epithelial carci-
noma cells, and 293T human embryo kidney cells after they
were incubated with IR825–PEG at various concentrations for
24 h and 48 h. No significant cytotoxicity of IR825–PEG was
observed for all three types of cells even at high concentrations
such as ·O2−, ·OH, and H2O2 can damage
biomacromolecules such as DNA, proteins,
and lipids, resulting in a high degree of cyto-
toxicity. Herein, intracellular peroxide and
superoxide levels in three types were assessed
using a dihydroethidine (DHE) probe. No
significant increase in the percentage of DHE
positive cells after treatment with different
concentrations of IR825–PEG nanoparticles
was noticed, suggesting minimal oxidative
stress induced to cells after IR825–PEG treat-
ment (Figure 2d). In addition, we further col-
lected primary mouse macrophage cells from
Scheme 1. A scheme showing the synthetic procedure for IR825.
healthy Blab/c mice, and tested the viabilities
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Figure 2. In vitro cell experiments. a,b) Relative viabilities of 4T1, HeLa, and 293T cells after being incubated with various concentrations of IR825–PEG
a) for 24 h and b) 48 h. c) Levels of released LDH from three types of cells after being exposed to various concentrations of IR825–PEG. d) Generation
of intracellular ROS determined by DHE staining and FACS measurement after various treatments indicated. Error bars were based on SD of three
parallel samples at the minimum. e) Relative viabilities of 4T1 cells after IR825–PEG-induced photothermal ablation at different laser power densi-
ties. Error bars were based on the SD of six parallel samples. f) Confocal fluorescence images of calcein AM/PI co-stained 4T1 cells with IR825–PEG
(25 μg mL−1 ) incubation after being exposed to the 808-nm laser at different power densities. Live and dead cells were stained by Calcein AM and PI,
and presented in white and dark gray colors in those images, respectively.
of those macrophages after incubation with IR825–PEG for 24 h
(Supporting Information, Figure S5). No obvious toxicity of
IR825–PEG was found toward primary macrophages, which
play important roles in the immune system. Our data collec-
tively suggest that IR825–PEG nanoparticles exhibit no appreci-
able toxicity to cells under our tested high concentrations.
Next, we used IR825–PEG nanoparticles as the photothermal
agent for in vitro cancer cell ablation under laser irradiation.
We first studied the cell uptake of fluorescently labeled IR825–
PEG by confocal fluorescence microscope (Supporting Infor-
mation, Figures S6,S7), and observed the efficient uptake of
those nanoparticles by 4T1 cancer cells. For in vitro PTT study,
4T1 cells were incubated with IR825–PEG at the concentra-
tion of 20 μg mL−1 for 4 h and then irradiated by the 808-nm
laser with different power densities. Following laser irradia-
tion, the cells were co-stained by calcine AM and propidium
iodide (PI) to differentiate live and dead cells, respectively, and
imaged by a confocal fluorescence microscope (Figure 2f). An
MTT assay was also performed to quantitatively measure the
relative cell viabilities after PTT treatment under different laser
powers (Figure 2e). It was determined that as the increase of
laser power density, more cells incubated with IR825–PEG
were killed after the laser irradiation. The majority of cells were
destroyed after being incubated with 20 μg mL−1 of IR825–PEG
and exposed to the NIR laser at 0.5 W cm−2 for 5 min. In con-
trast, cells without IR825–PEG incubation were not affected
even after laser exposure at the highest tested power density
(0.5 W cm−2 ). These findings demonstrated that IR825–PEG
nanoparticles hold great promise as an effective photothermal
agent for in vivo tumor therapy.
For animal experiments, we first studied the blood circula-
tion behavior of IR825–PEG nanoparticles. After i.v. injection
of IR825–PEG into female Balb/c mice (1 mg mL−1 , 200 μL per
mouse, or a dose of 10 mg kg−1 ), blood was drown from the
mouse tail at certain time intervals post injection (p.i.), solu-
bilized by a lysis buffer, and then measured by a UV-vis-NIR
spectrophotometer. The NIR absorbance peak of IR825 was
employed to directly determine the IR825–PEG concentrations
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Figure 3. In vivo behavior and photothermal therapy. a) The blood circulation curves of IR825–PEG and IR825–PEG-Cy5.5 after i.v. injection as deter-
mined by measuring IR825 absorbance or Cy5.5 fluorescence, respectively, in the blood at different time points p.i. The unit was the percentage of
injected dose per gram tissue (%ID/g). b) In vivo fluorescence images of 4T1 tumor bearing Balb/c mice at different time points post injection of
IR825–PEG-Cy5.5. c) IR thermal images of 4T1 tumor-bear mice without (upper row) or with (lower row) intravenous injection IR825–PEG (10 mg kg−1
,
24 h p.i.) under the 808-nm laser irradiation taken at different time intervals. The laser power density was 0.5 W cm−2 . Arrows point to the tumors. d)
The growth of 4T1 tumors in different groups of mice after various treatments indciated. The relative tumor volumes were normalized to their initial
sizes. For the treatment group, six mice injected with IR825–PEG at 24 h p.i. were exposed to the 808-nm laser (0.5 W cm−2 , 5 min). Other three
groups of mice were used as controls: untreated (Control, n = 6); laser only without IR825–PEG injection (laser only, n = 6); IR825–PEG injected but
without laser irradiation (IR825-PEG only, n = 6). Error bars were based on SD. e) Survival curves of mice after various treatments as indicated in (d).
f) Representative photos of mice after various different treatments indicated.
in blood samples (Supporting Information, Figure S8). It was
uncovered that the blood circulation of IR825–PEG nanoparti-
cles followed a typical two-compartment model. After a rapid
decay with the first phase (distribution phase, usually a rapid
decline) half-life of 0.99 h 0.26 h, those nanoparticles in the
blood circulation exhibited an ultra-long second phase (elimina-
tion phase, the predominant process for drug clearance) half-
life at 16.6 h 3.5 h (Figure 3a).
We then wanted to understand the in vivo distribution of
IR825–PEG in mice. Because of the strong light scattering
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in organ lysate samples, it was impossible to adopt the same
method used for blood sample measurement to determine
the concentrations of IR825–PEG in different organs after i.v.
injection. Therefore, we used amine-terminated C18PMH-
PEG to solubilize IR825, and then labeled IR825–PEG nano-
particles with Cy5.5, a commonly used NIR fluorescent dye
(see Experimental Section for details and Supporting Infor-
mation, Figure S6). The obtained IR825–PEG-Cy5.5 was also
i.v. injected into mice at the same dose. The blood collected
at different time points p.i. was solubilized by a lysis buffer
and measured by a fluorometer to determine the IR825–PEG-
Cy5.5 concentration based on Cy5.5 fluorescence. Different
from directly measuring IR825 absorbance in blood samples,
this fluorescent measurement approach as an indirect method
is in fact measuring Cy5.5 conjugated on the PEG coating of
IR825–PEG. Interestingly, the blood circulation data deter-
mined by these two different methods matched reasonably
well with each other, suggesting that the PEG coating was
stable in IR825–PEG nanoparticles when they were circulating
in the blood after i.v. injection.
Since the Cy5.5 labeling on IR825–PEG nanoparticles was
found to be fairly stable, we next used in vivo imaging approach
to track IR825–PEG-Cy5.5 in mice after injection. Balb/c mice
bearing 4T1 murine breast cancer tumors were intravenously
injected with IR825–PEG-Cy5.5 (200 μL of 1 mg mL−1 solu-
tion for each mouse) and then imaged by a Maestro EX in vivo
fluorescence imaging system (CRi, Inc.). The mouse autofluo-
rescence was removed by spectral unmixing using the Maestro
software, leaving pure Cy5.5 fluorescence shown in Figure 3b. It
was found that IR825–PEG-Cy5.5 tended to be enriched in the
tumor over time, with prominent uptake observed in the tumor
at 24 h p.i. The high tumor accumulation of IR825–PEG nano-
particles could be due to the EPR effect in cancerous tumors
with tortuous and leaky vasculatures, which tend to trap nano-
materials especially those with long blood circulation time.[22,35]
Those mice were then sacrificed at 24 h p.i., with major organs
collected and homogenized by a lysis buffer. Based on the
intensity of Cy5.5 fluorescence, high levels of IR825–PEG-Cy5.5
were observed in the tumor, as well as in reticuloendothelial
systems (RES) such as liver and spleen (Supporting Informa-
tion, Figure S9). The tumor uptake was measured to be as high
as 22.5% ID g−1 . However, it should be aware that the quanti-
tative biodistribution data of IR825–PEG-Cy5.5 based on Cy5.5
labeling may not be entirely accurate, and a precise approach
to directly measure IR825 levels in different organs and tissues
remain to be developed in future studies, as the fluorescence of
IR825 itself is rather weak.
temperature on mice without IR825–PEG injection under the
same irradiation condition showed little change.
Finally, the in vivo therapeutic efficacy of IR825–PEG
induced PTT cancer treatment was studied. Six mice bearing
4T1 tumors with the average volume of ≈60 mm3 on their
back were i.v. injected with IR825–PEG (200 μL, 1 mg mL−1 ).
At 24 h p.i., the tumor of each mouse in the treatment
group was exposed to the 808-nm laser at a power density of
0.5 W cm−2 for 5 min. Three other groups including untreated
mice (control, n = 6), mice exposed to the laser (laser only,
n = 6), and IR825–PEG injected mice without laser irradia-
tion (IR825–PEG, n = 6), were used as the controls (with the
average tumor volume at ≈60 mm3). Tumor sizes were meas-
ured every 2 days after treatment. Remarkably, tumors on
IR825–PEG injected mice were completely eliminated one-day
post NIR laser irradiation (Figure 3d,f). In marked contrast,
neither laser irradiation at this power density nor IR825–
PEG injection by itself would affect the tumor development
(Figure 3d). While mice in the three control groups showed
average life spans of 16–18 days with the tumor volumes
reached up to ≈1.0 cm3, mice in the treated group (IR825–
PEG +Laser) were tumor-free after treatment and survived
over 60 days without a single death (Figure 3e,f). Our results
suggest that IR825–PEG is a powerful agent for in vivo photo-
thermal ablation of cancer.
Despite the absence of obvious in vitro toxicity of IR825–
PEG to a number of different cell lines, its potential in vivo tox-
icity to animals is still an important question to be addressed.
We carefully supervised the behaviors of IR825–PEG injected
(10 mg kg−1 ) Balb/c mice in our experiments after photo-
thermal tumor ablation, and noticed no obvious sign of toxic
effect within 60 days. No abnormalities in body weight (Sup-
porting Information, Figure S10), eating, drinking, grooming,
activity, exploratory behavior, urination, or neurological status
were observed. Mice were then sacrificed at day 60 for careful
necropsy, which uncovered no appreciable abnormality in
major organs. Major organs of mice were sliced and stained by
hematoxylin and eosin (H&E) for histology analysis (Figure 4a),
revealing no noticeable organ damage or inflammatory lesion
in all major organs of mice 60 days after PTT treatment.
To further understand the potential toxicology of IR825–
PEG nanoparticles in relatively short and long terms, serum
biochemistry assay and complete blood count were carried on
IR825–PEG injected (10 mg kg−1 ) healthy Balb/c mice at 1, 7,
and 40 days p.i. The liver function markers including alanine
aminotransferase (ALT), alkaline phosphatase (ALP), aspartate
aminotransferase (AST), as well as kidney function marker urea
nitrogen (BUN) and the albumin/globin ratio, were all meas-
ured to be normal (Figure 4b–d), suggesting no obvious hepatic
or kidney disorder of mice induced by IR825–PEG treatment.
A complete blood count assay was also carried out. Compared
with the control group, all parameters including white blood
cells (WBC), red blood cells (RBC), hematocrit (HCT), hemo-
globin (Hgb), mean corpuscular volume (MCV), mean cor-
puscular hemoglobin (MCH), mean corpuscular hemoglobin
concentration (MCHC), and platelets, were normal and within
the reference ranges for healthy Balb/c mice (Figure 4e). Our
results collectively evidence that IR825–PEG is not noticeably
toxic in vivo to mice at our tested dose, although future careful
Encouraged by the high tumor accumulation of IR825–PEG
nanoparticles and its strong NIR optical absorption, we then
carried out an in vivo photothermal therapy experiment using
IR825–PEG. After being intravenously injected with IR825–
PEG solutions (1 mg mL−1 , 200 μL for each mouse) for 24 h,
mice bearing 4T1 tumors were anesthetized and exposed to an
808-nm laser at the power density of 0.5 W cm−2 . An infrared
(IR) thermal mapping apparatus was used to record the tem-
perature change in the tumor area under NIR irradiation.
From mice i.v. injected with IR825–PEG, their tumor surface
temperature rapidly increased from ≈30 °C to ≈60 °C within 5
min of laser irradiation (Figure 3c). In comparison, the tumor
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Figure 4. In vivo toxicology study. a) H&E stained images of major organs. IR825–PEG injected mice that survived after PTT (with tumors eliminated)
were sacrificed 60 days after treatment. Untreated healthy mice were used as the control. No noticeable abnormality was observed in major organs
including, liver, spleen, kidney, heart, and lung. b) Serum biochemistry data including liver function markers, c) blood urea nitrogen (BUN) levels, and
d) albumin/globin ratios. Healthy female Balb/c mice i.v. injected with IR825–PEG (dose = 10 mg kg−1 ) were sacrificed at 1, 7, and 40 days p.i. for blood
collection. Untreated healthy mice were used as the control. e) Complete blood counts. Blood levels of WBC, RBC, HCT, Hgb, MCV, MCH, MCHC,
and platelets of control and IR825–PEG treated mice were measured. Gray areas indicate the reference normal ranges for healthy female Balb/c mice.
Statistic was based on 5 mice per data point.[38]
studies are still required to systematically examine any potential
long-term toxicity of our IR825–PEG nanoparticles at various
doses to animals, before any possible clinical use of this new
organic photothermal agent. Moreover, we still need to develop
a reliable approach to extract and quantitatively measure IR825
in different organs of mice after injection, so that we could
understand the possible degradation and excretion behaviors of
IR825–PEG in vivo.
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anhydride-alt-1-octadecene) (C18PMH, Sigma-Aldrich) and 143 mg
(1 eq) mPEG-NH2(5K) (Biomatrik, Jiaxing, China) were dissolved in 5 mL
dichloromethane with 6 μL triethylamine (TEA, Sinopharm Chemical
Reagent Co.) and 11 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC, Fluka) added. After 24 h of stirring, the dichloromethane solvent
was blowing-dried by N2. The leftover solid was dissolved in water,
forming a transparent clear solution, which was dialyzed against distilled
water for 2 days in a dialysis bag with molecular weight cut-off (MWCO)
of 14 kDa to remove unreacted mPEG–NH2. After lyophilization, the
final product was stored at –20 °C for future use.
To synthesize C18PMH–PEG–NH2, 10 mg (1 eq) C18PMH, 95 mg
(0.67 eq) mPEG–NH2(5k), and 48 mg (0.33 eq) NH2–mPEG(5k)–BOC
(Polymere, Germany) were mixed together in dichloromethane under
agitation to form a homogeneous solution, EDC(2 eq) and TEA (8 eq)
were then added under magnetic stirring. After stirring for 24 h at room
temperature, the dichloromethane solvent was blowing-dried by N2.
Subsequently, 2 mL trifluoroacetic acid (TFA, Sinopharm Chemical
Reagent Co.) was added under magnetic stirring for 3 h at room
temperature to de-protect the Boc group. After evaporating the TFA
solvent, the leftover solid was dissolved in water, and dialyzed for 2 days
in a dialysis bag (MWCO 14 kDa) to remove unreacted PEG polymers
and other reagents. After lyophilization, the final product (C18PMH–
PEG–NH2) was stored at –20 °C for future use.
Formation of IR825–PEG Nanoparticles: 20 mg as-synthesized IR825
was first dispersed in 5 mL methanol containing 20 mL TEA. Another
solution of 100 mg C18PMH–PEG or C18PMH–PEG–NH2 polymer in
2 mL methanol was then added to the reaction vessel. The mixture was
stirred for 4 h before it was transferred into 5 mL water. The IR825–PEG
nanoparticles were obtained after dialyzing the solution for 2 days in a
dialysis bag (MWCO 14 kDa) to remove TEA and other reagents. The
encapsulation efficiency of IR825 by C18PMH–PEG was nearly 100%, as
evidenced by the fact that the filtrate was completely colorless even after
a long period of dialysis.
Photothermal Stability Test: Aqueous solutions of IR825–PEG and ICG
(0.1 mg mL−1 ) in two 35-mm cell culture dishes were exposed to the
808-nm laser at the power density of 0.5 W cm−2 for 10 min. Photos
and UV-vis-NIR spectra of those two solutions before and after laser
irradiation were recorded and presented in Figure 1g.
3. Conclusions
We have successfully developed a new generation of photo-
thermal therapeutic agent based on PEGylated micelle nano-
particles of a NIR-absorbing low-QY organic dye, IR825. It is
found that IR825–PEG nanoparticles exhibit excellent compat-
ibility in physiological environments, show no observable tox-
icity to three classes of cells at the tested concentrations, and can
be used as a highly effective photothermal agent with remark-
ably improved photostability in comparison to ICG. With a long
blood circulation half-life, the IR825–PEG nanoparticles show
surprisingly high in vivo tumor uptake by the EPR effect after
i.v. injection, and appear to be an excellent PTT agent for tumor
ablation under a low-power laser irradiation, without rendering
any appreciable toxicity to the treated animals. Although at the
current stage we do not have direct data to study the degradation
behaviors of IR825–PEG nanoparticles in vivo, those PEGylated
micelles encapsulating small organic dye molecules likely could
be slowly degraded in biological systems, and may have much
less concern regarding their potential long-term toxicity in
comparison to various inorganic photothermal agents as well
as organic conjugated polymers. On the other hand, the dose
(10 mg kg−1 ) and optical power density (0.5 W cm−2 ) used in our
work are rather low in comparison to those used in a lot of other
reported photothermal cancer treatment studies by systemic
administration (e.g., laser power densities at 2–4 W cm−2 are
needed for gold nanorods and nanoshells at similar doses),[39,40]
suggesting that our IR825–PEG nanoparticles show compa-
rable, if not better, photothermal therapeutic efficacy compared
to many types of existing photothermal agents. Although fur-
ther careful studies are required to understand the detailed bio-
distribution, pharmacokinetics, and long-term dose-dependent
toxicology of IR825, it is expected the development of organic
photothermal agents such as that presented in this study may
be a reasonable approach to make photothermal therapy a clini-
cally acceptable therapeutic method.
Fluorescent Labeling of IR825–PEG: C18PMH–PEG–NH2 was
used to solubilize IR825 in this case, yielding IR825–PEG–NH2
with amino groups at PEG terminals. The purified IR825–PEG–NH2
(0.5 mg mL−1 ) was reacted with an amine reactive dye, Cy5.5–NHS
(Passkey Technology Co., Ltd. Hunan China) at 0.1 mg mL−1 in a
pH 7.4 phosphate buffer (0.02 M). After overnight stirring at room
temperature in a light-proof container, the obtained IR825–PEG–Cy5.5
was purified by centrifugal filtration through 100 kDa MWCO filters
and repeated water washing until no noticeable Cy5.5 color in the
filtrate solution.
4. Experimental Section
Synthesis of Heptamethine Indocyanine Dye IR825:[41–43] The procedure
of synthesizing IR825 is illustrated in Scheme 1. In brief, 2,3,3-trimethyl-
4,5-benzo-3H-indole (TMBI, 10.0 g, 48 mmol) and alpha-bromo-p-toluic
acid (BTA, 12.3 g, 57 mmol) were dissolved in 40 mL dichlorobenzene
and stirred under 110 °C for 4 h. The solid crude product was then
filtrated and recrystallized in methanol. The purified product as denoted
intermediate 1 was dried under vacuum.
80 mL DMF/dichloromethane mixture (1/1, v/v) was added
dropwisely into a solution of phosphoryl chloride (37 mL) and anhydrous
dichloromethane (35 mL) under stirring in ice/water bath; afterwards,
10 g cyclohexanone was added dropwisely. The ice/water bath was
removed and the solution was then heated and refluxed for 3 h. The
mixture was poured into ice, yielding a solid product which was collected
by filtration and washed with iced diethyl ether. The resulting yellow
product was denoted as intermediate 2 and used directly.
Intermediate 1 (1.64 g, 3.9 mmol) and intermediate 2 (0.34 g,
1.9 mmol) were mixed in 260 mL butanol/toluene (7/3, v/v), and stirred
for 4 h under 115 °C. The solvent was removed by rotatory evaporation,
and the crude product was purified by dissolving in DMF and precipitating
in diethyl ether. After filtration, the product was dried under vacuum.
Synthesis C18PMH–PEG and C18PMH–PEG–NH2: C18PMH–PEG was
synthesized following a literature procedure.[36] 10 mg (1 eq) poly(maleic
Characterization: Fluorescence spectra of different samples were
obtained on a FluoroMax 4 luminescence spectrometer (HORIBA
Jobin Yvon). UV-vis-NIR absorbance spectra were recorded by using
a
PerkinElmer Lambda 750 UV-vis-NIR spectrophotometer. AFM
images were acquired using a Multi-Mode V Atomic forcemicroscopy
(AFM, Veeco). TEM was performed using a FEI 20 TEM operated
at an accelerating voltage of 200 kV. The samples were stained with
phosphotungstic acid (1 wt%) before TEM imaging.
Cell Culture Experiments: 4T1 murine breast cancer cells, human
cervical cancer HeLa cells, and 293T human embryo kidney cells were
cultured in standard cell media recommended by American type culture
collection (ATCC), under 37 °C within 5% CO2 atmosphere.
Cells seeded into 96 well plates were incubated with different
concentrations of IR825–PEG for 24 h. Relative cell viabilities were
determined by the standard MTT assay. For in vitro photothermal
therapy, 4T1 cancer cells were incubated with and without IR825–PEG
(25 μg mL−1 ) for 4 h and then irradiated by an 808-nm laser at different
power densities (0.1, 0.3, 0.5, 0.8 W cm−2 ) for 5 min. The cells were
stained with Calcine AM/PI for 30 min, washed with PBS, and then
imaged by a confocal fluorescence microscope (Leica).
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DOI: 10.1002/adfm.201301045

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The LDH leakage was measured using a cytotoxicity detection kit
(Promega Cat. 7891) following the vendor’s protocol. In this experiment,
4T1, HeLa, and 293T cells lysed by 1% Triton-X-100 were used as positive
controls, while the cell-free medium was used as the negative control.
The analysis was performed using a microplate reader (Bio-Rad).
ROS production was evaluated by a hydroethidine (DHE) probe
known be oxidized by various oxidative agents. In brief, cells were treated
with different concentrations of IR825–PEG for 24 h. After trypsination,
cells were centrifuged at 1000 min r−1 for 3 min, re-suspended in cell
culture medium containing 1 μM DHE (sigma), and analyzed using a
flow cytometry (FACS Calibur from Becton, Dickinson and Company).
Tumor Model: Balb/c mice were obtained from Suzhou Belda Bio-
Pharmaceutical Co., Ltd. and used under protocols approved by
Soochow University Laboratory Animal Center. The 4T1 tumors were
generated by subcutaneous injection of 2 × 106 cells in ≈40 μL serum-
free RMPI-1640 medium onto the back of each female Balb/c mice.
In Vivo Fluorescence Imaging: 4T1 tumor bearing mice were
intravenously injected with 200 μL of 1 mg mL−1 IR825–PEG–Cy5.5 and
imaged using the Maestro in vivo fluorescence imaging system (CRi
Inc.), in vivo spectral imaging from 650 nm to 800 nm (10 nm step) was
carried out (central excitation wavelength = 640 nm) with an exposure
time of 100 ms for each image frame. Auto-fluorescence (particularly
from food residues in the stomach and intestine) was removed by using
the spectral unmixing software.
Blood Circulation: Blood circulation was measured by drawing ≈10 μL
blood from the mouse tail vein post injection of IR825–PEG or IR825–
PEG–Cy5.5. Each blood sample was dissolved in 1 mL of lysis buffer
(1% SDS, 1% Triton X-100, 40 mM Tris Acetate) to form a homogenous
clear solution. The concentrations of IR825–PEG in the blood lysates
were determined by the UV-vis spectra IR825 absorbance. A blank blood
lysate sample without IR825–PEG was measured to determine the base-
absorption level from red blood cells, which was subtracted from the
measured IR825 absorbance at 825 nm of blood samples collected from
IR825–PEG injected mice for the concentration calculation (Supporting
Information, Figure S5). The IR825–PEG level in the blood was presented
as the percentage of injected dose per gram tissue (% ID g−1 ).
harvested, fixed in 10% neutral buffered formalin, processed routinely
into paraffin, sectioned at 8 micrometers, stained with hematoxylin
and eosin (H&E) and examined by a digital microscope (Leica QWin).
Examined tissues include liver, spleen, kidney, heart, lung, and intestine.
Blood Analysis: Fifteen healthy Balb/c mice were injected with 200 μL
of 1 mg mL−1 IR825–PEG (a dose of 10 mg kg−1 ). Other five mice were
used as the un-treated control. Mice were sacrificed to collect the blood
(0.8 mL) for blood biochemistry assay and complete blood count at
1 day, 7 days, and 30 days post injection of IR825–PEG (five mice per
group). The serum chemistry data and complete blood count were
measured in Shanghai Research Center for Biomodel Organism.
Supporting Information
Supporting Information of IR825 characterization data, biodistribution
data, and body weight data (after treatment) is available from the Wiley
Online Library or from the author.
Acknowledgements
This work was partially supported by the National Natural Science
Foundation of China (51222203, 51002100, 51132006), the National
“973” Program of China (2011CB911002, 2012CB932601), and a Project
Funded by the Priority Academic Program Development of Jiangsu
Higher Education Institutions. L.C. was supported by a Post-doctoral
research program of Jiangsu Province (1202044C).
Received: March 26, 2013
Revised: April 29, 2013
Published online:
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2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Funct. Mater. 2013,
DOI: 10.1002/adfm.201301045