Am J Transl Res 2022;14(3):1991-2001

www.ajtr.org /ISSN:1943-8141/AJTR0139510

Original Article
ICG-ER: a new probe for photoimaging
and photothermal therapy for breast cancer
Yu Weng1*, Zheng-Jie Wang1*, Teng-Yu Guo3, Wen-Bo Li1, Yi-Yi Cao1, Rui Zuo1, Peng-Fei Xu2, Hua Pang1
1
Department of Nuclear Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing
400016, P. R. China; 2Institute of Clinical Pharmacy & Pharmacology, Jining First People’s Hospital, Jining Medical
University, Jining 272000, P. R. China; 3College of Chemistry, Jilin University, Changchun 130012, P. R. China.
*
Equal contributors.
Received September 29, 2021; Accepted March 3, 2022; Epub March 15, 2022; Published March 30, 2022

Abstract: Breast cancer is common cancer type with high mortality. There are still inperfections in the traditional
diagnosis and treatment methods for cancer. Photoacoustic imaging combines the advantages of high specificity
and deep tissue penetration and is especially suitable for early cancer detection and treatment monitoring. With its
specificity and noninvasiveness; photothermal therapy has become one of the best representative treatment meth-
ods. Indocyanine green (ICG) is a near-infrared imaging reagent approved by the FDA for clinical application, with a
potential application for photothermal therapy. ICG has low targeting specificity. Through the combination of EB and
ICG, the timeliness of ICG circulation in vivo is improved, and the tumor targeting of ICG-E is improved by using RGD.
ICG-ER, an integrated optical probe for diagnosis and treatment, was constructed, and high uptake of ICG-ER by 4T1
cells was observed by flow cytometry and confocal laser scanning microscopy (CLSM). ICG-ER photoacoustic signal
intensity is concentration-dependent. In vivo photoacoustic imaging showed that the ICG-ER concentration time in
the tumor site was long and reached a peak at 42 hours. Under laser irradiation, the temperature of the tumor site
in mice that were injected with ICG-ER reached 56°C. After photothermal treatment, the tumor tissue in the mice
showed obvious necrosis and no tumor recurrence, proving that ICG-ER has a good photothermal effect. Based on
the above results, ICG-ER can be used in breast cancer optical imaging and photothermal therapy, which is expected
to provide new ideas for breast cancer clinical diagnosis and treatment.

Keywords: Breast cancer, indocyanine green, Evans blue, photoacoustic imaging, photothermal therapy

Introduction ment includes surgery, chemotherapy, and

radiotherapy. Although it has specific curative
With the development and progress of science effects, there are problems of trauma, specific-
and technology, human exploration of cancer ity, and systemic toxicity which have always
diagnosis and treatment has also made been confusing [5-7]. Photothermal therapy
advancements. However, as an aggressive type (PTT) improves the management of the above
of cancer, breast cancer has a high mortality issues and has become one of the most repre-
rate. Currently, breast cancer imaging diagno- sentative treatment methods [8-11].
sis methods include X-ray, ultrasound, and
MRI, but X-rays have radioactivity, ultrasound Currently, photothermal conversion agents are
has a strong subjectivity, and MRI has a low mainly inorganic materials and organic dyes
specificity in breast cancer diagnosis [1-3]. [12]. At present, there have been many PTCAs
Photoacoustic imaging (PAI) combines the used in research. Indocyanine green (ICG), as
advantages of high specificity in pure optical the only organic dye approved by clinical prac-
tissue imaging and deep penetration in pure tice, has been widely used in liver surgery [13].
ultrasonic tissue imaging. It can obtain tissue Some studies have shown ICG being used in
images with high resolution and high contrast, breast cancer’s sentinel lymph node (SLN) loca-
avoiding the influence of light scattering, in tion [14-16], which makes it possible to apply
principle [4]. Traditional breast cancer treat- ICG in photoacoustic imaging and photothermal
 New probe for photoacoustic imaging and photothermal therapy

treatment of breast cancer. However, ICG lacks NIR-II in vivo imaging was performed on a small
tumor targeting and has a short half-life in the animal imaging system with fiber-coupled 915
blood, which undoubtedly increases the diffi- nm laser system. Propidium iodide (PI) and a
culty of ICG in diagnosing breast cancer tumors cell counting kit (CCK-8) were made in Dojindo
[17-19]. (Japan). The plasma water was from a Millipore
water purification system. Other reagents were
EB has a long history of being used as a biofuel used without further purification.
and clinical diagnostic reagent [20]. Bobin and
other researchers evaluated the feasibility of Synthesis of ICG-ER
sentinel lymph node identification in 100
patients with breast cancer, which proved Synthesis of ICG-E: EB-Mal was prepared
the dye’s sensitivity in detecting lymph node according to previous work [28]. ICG-NHS (16
metastasis [21]. Integrin αVβ3 receptor is an mg, 1 eq) in 3 mL anhydrous N, N-dimethyl-
essential target for tumor angiogenesis drug formamide (DMF) was stirred with EB-Mal (16
design. Arg-Gly-Asp (RGD) can be recognized by mg, 1.1 eq) with the addition of DIPEA (3 eq).
the integrin αVβ3 receptor [22, 23]. Therefore, Then, the mixture was stirred under an N2
research on RGD-containing peptides as tumor atmosphere at room temperature. The reaction
angiogenesis imaging agents and therapeutic was monitored by HPLC analysis and complet-
drugs is also a current research focus [24-27]. ed in 4 hours. The mobile phases (A) de-
We successfully combined EB with ICG to form mineralized water and (B) acetonitrile were
ICG-E. Then, we combined ICG-E with c(RGDfc) acidified to pH 3 with trifluoroacetic acid.
to obtain ICG-ER (Figure 7), and assume that Gradient elution was performed as follows:
this will make ICG-E more effective in tumor 10% B, 0-3 min; 10-90% B, 3-12 min; 90% B,
targeting. 12-16 min; 90-10% B, 16-18 min; and 10% B,
18-20 min. The product was purified by Pro-
This study combined ICG with EB and RGD to HPLC (Figure S4). MALDI-TOF-MS analysis con-
obtain ICG-ER. ICG is used for optical imaging firmed a mass of 1534.58 [M]+ with an isolated
and photothermal therapy, EB is used to yield of 53% (7.9 mg) (Figures S5, S6).
increase ICG uptake and residence time in
tumors, and RGD is used to target αVβ3 integrin Synthesis of ICG-ER: ICG-E (1 eq, 15 mg) was
overexpressed on tumor angiogenesis endo- dissolved in 0.5 mL of DMSO. RGD-SH (1.1 eq,
thelial cells. ICG-ER has good optical imaging 6.3 mg) was dissolved in 5 mL of degassed
ability and stability. 0.1% sodium ascorbate (w/v) in phosphate-
buffered saline (PBS), and added to the reac-
Material and methods tion solution. The reaction was stirred at RT for
2 h. The reaction mixture was monitored by
Materials and reagents analytical HPLC and purified by Pro-HPLC
(Figure S7). The mobile phases (A) demineral-
All chemical reagents were obtained from ized water and (B) acetonitrile were acidified
the commercial suppliers and used without fur- to pH 3 with trifluoroacetic acid. Gradient elu-
ther purification. MALDITOF-MS spectra were tion was performed as follows: 10% B, 0-3
obtained by an AB SCIEX 4700 TOF/TOF min; 10-90% B, 3-12 min; 90% B, 12-16 min;
System. UV-vis was tested with a Shimadzu 90-10% B, 16-18 min; and 10% B, 18-20 min.
Model UV-1700 spectrometer. FSL1000 fluo- MALDI-TOF-MS analysis confirmed a mass of
rescence spectrophotometer (FSL1000, Edin- 2110.583 [M]- with an isolated yield of 48%
burgh Instruments Ltd.) equipped with the (10.2 mg) (Figure S8).
Quantum Yield measuring accessory and
Report Generator program was used for photo- Cell culture and 4T1 tumor-bearing mice
luminescence (PL) spectra study and Absolute model
Quantum Yield Measurement. Flash chroma-
tography was performed on 200-400 mesh sil- Murine breast cancer: 4T1 breast cancer cells
ica. Thin layer chromatography was performed were purchased from ATCC (Shanghai, China),
using silica gel 60G F254 25 glass plates and and the full medium was prepared with RPMI-
visualized under 254/365 nm ultraviolet light. 1640 medium (Gibco), 10% fetal bovine serum

1992 Am J Transl Res 2022;14(3):1991-2001

 New probe for photoacoustic imaging and photothermal therapy

(FBS), and 1% penicillin streptomycin antibody. measured by CytoFLEX flow cytometry (Beck-
The cells were incubated in an incubator of 5% man Coulter. Suzhou, China).
CO2 and 37°C. When the cells were cultured to
80% confluency, they were passed on at a ratio In vitro and in vivo PA imaging
of 1:3 to carry out cell experiments and con-
struct tumor models. To evaluate the PA imaging effect of ICG-ER, a
gel model with a diameter of 5 mm was pre-
All animals (female BALB/c nude mice, weigh- pared. ICG-ER maximum absorbance in the
ing 18-22 g, aged 6-8 W) were purchased from range of 680 nm-970 nm (interval =5 nm) was
the experimental animal center of Chongqing measured by a VEVO LAZR photoacoustic
Medical University. All experiments and opera- imaging system (VisualSonics Inc., Toronto,
tions were carried out with the approval of Canada). Then, a laser at an excitation wave-
guidelines from the Animal Care and Use length of 805 nm was used (Figure S9).
Committee of the First Affiliated Hospital of Different concentrations (5, 10, 15, 20, and
Chongqing Medical University (Grant Nos. 25, µg/ml) of ICG-ER solution were used for PA
82102093, ethical approval report has been imaging. Then, the region of interest (ROI) of
provided to the Supplementary Data File). The each image was checked with VEVO LAZR soft-
cells were suspended in PBS (100 µl 1×106 ware, and the PA intensity value of the region
4T1 cells per mouse) and then subcutaneously was measured. For in vivo PA imaging, nude
injected into the left hind limb of the female mice bearing 4T1 tumors (n=3) were injected
nude mice. The tumor volume was calculated with ICG-ER (0.35 mg/ml) diluted with 10%
by [π/6 × length × (width)2]. Human serum albumin (HSA) via the tail vein.
PA images were collected at different times
In vitro cytotoxicity and intracellular uptake of (pre, 6 h, 12 h, 24, 36 h, 42 h), and the average
ICG-ER PA intensity of the tumor area was measured.

4T1 cells in logarithmic growth phase were In vitro photothermal effect

seeded in 96-well plates at a density of 2×
104/ml well, and cultured in a cell incubator at To better observe the influence of concentra-
37°C and 5% CO2 until more than 50% adher- tion on ICG-ER temperature under laser irradia-
ent. The experimental group (As) was cocul- tion the following steps were performed. First,
tured with different concentrations of ICG-ER ICG-ER was diluted in different concentrations
(10, 20, 30, 40, and 50 µg/ml) cell culture (10, 20, 40, 80, 160 µg/ml), and normal saline
medium. Pure culture medium was set as the was used as a blank control. Second, diluted
blank group (Ab), sterile culture medium plus ICG-ER and normal saline were added to six
cells as the control group (Ac), and 6 multiple 96-well plates. The 808 nm laser (1.5 W cm-2)
wells were set at each concentration. was used to irradiate for 400 s. Over time, the
temperature changes of ICG-ER and normal
After 24 hours of culture, all cells were washed saline at different concentrations were moni-
with PBS and then incubated with 100 µL of tored with a thermal imager (Fourier 226,
10% CCK-8 reagent for 30 minutes in each China).
well. The multifunctional microplate reader was
set to scan at 450 nm. The cell survival rate In vivo photothermal therapy
was calculated according to the formula [(As-
Ab)/(Ac-Ab)] ×100%. To evaluate whether ICG-ER has the effect of
photothermal therapy, mice with a tumor vol-
To study the uptake of ICG and ICG-ER in tu- ume reaching 50-80 mm3 were randomly
mor cells, 4T1 cells (5×104/ml) were placed in divided into 4 groups (n=3). The first group was
CLSM dishes. After 24 hours, the cells were the control group, and 100 µl of normal saline
cultured in a complete medium containing ICG was injected into the tail vein. In the second
or ICG-ER for 12 hours, and the nuclei were group, 100 µl 0.35 mg/ml ICG-ER solution was
stained with PI. CLSM (Nikon A1, Japan) was injected intravenously. In the third group, 100
used to record fluorescence images directly. In µl of normal saline was injected into the tail
addition, the intracellular uptake of 4T1 cells, vein and irradiated with an 808 nm laser
coincubated with ICG or ICG-ER for 12 h was (1.5 W cm-2) for 10 minutes. The fourth group

1993 Am J Transl Res 2022;14(3):1991-2001

 New probe for photoacoustic imaging and photothermal therapy

Figure 1. A. CLSM images of 4T1 cells coincubated with ICG-ER or ICG for 12 h. B. Flow cytometry analysis of intra-
cellular uptake of ICG-ER and ICG. All images were observed under the CLSM at 200X magnification. The scale bars
are 100 μm.

was the ICG-ER+laser group. One hundred data was analyzed according to Student’s
microliters of 0.35 mg/ml ICG-ER was injected t-test, one-way ANOVA and two-way ANOVA.
intravenously and irradiated with an 808 nm
laser (1.5 W cm-2) for 10 minutes. An infrar- Results
ed thermal imaging camera recorded the tem-
perature change and infrared thermal images. Preparation and characterization of ICG-ER
The tumor volume and weight changes of the
ICG-E was easily synthesized through the direct
four groups of mice were recorded every two
conjugation of ICG-NHS with compound EB-Mal.
days after photothermal treatment. The tumor
The free thiol group of c(RGDfc) was covalently
volume was calculated by V/V0 (V0: initial tu-
attached to the ICG-E maleimide motif to give
mor volume before treatment). After 24 hours
the final ICG-ER product (Figure 7).
of photothermal treatment, blood, main organs
(heart, liver, spleen, lung, kidney), and tumor In vitro cytotoxicity and in vitro cell targeting
tissue were harvested and fixed in 4% parafor-
maldehyde solution (n=3). To observe the bio- After the cells were incubated with ICG-ER for
safety of ICG-ER, ALT, AST, and BUN kits were 24 h, the cell survival rate was calculated
used to detect renal function and liver func- according to the CCK-8 method (Figure S1). The
tion. The main organs were stained with H&E. results showed that at the highest concentra-
Finally, H&E, PCNA, TUNEL, and HSP70 staining tion (50 µg/ml), the cell survival rate reached
were performed analyze the histopathological more than 95%. This indicates that ICG-ER has
changes in tumor tissue. very low biological toxicity.

Statistical analysis To explore the targeting ability of ICG-ER and

ICG on tumor cells, we observed the uptake of
All statistical analyses were performed with ICG-ER and ICG in 4T1 cells by CLSM. The
SPSS 20.0 software. Data were presented as results showed that ICG-ER was concentrated
mean ± standard deviation. The significance of around the nucleus, whereas ICG did not pro-

1994 Am J Transl Res 2022;14(3):1991-2001

 New probe for photoacoustic imaging and photothermal therapy

Figure 2. A. In vitro PA contrast images and PA values of different ICG-ER concentrations. B. In vivo PA images of
tumors in 4T1 tumor-bearing mice after caudal vein injection of ICG-ER at different time points. C. Changes in PA
signal intensities within tumor regions at corresponding time points. The intensity of the PA signal at different time
points before and after ICG-ER injection was analyzed by one-way ANOVA, **P<0.01, ****P<0.0001. (Values are
means ± SD., n=3).

duce such an effect (Figure 1A). Flow cytome- signal in the tumor site was stronger with time
try results showed that the uptake of ICG-ER and reached a peak value at 42 h (Figure 2C).
by 4T1 cells was 99.83%±0.31%, and the This indicates that ICG-ER can be a PA contrast
uptake of ICG was 1.63%±0.15% (Figure 1B). agent in vivo.
Both CLSM and flow cytometry showed that
ICG-ER was more specific to tumor cells than In vitro photothermal effect
ICG. These results provide evidence for ICG-ER
photoacoustic imaging and photothermal ther- To evaluate the photothermal properties of
apy in vivo. ICG-ER, different concentrations of ICG-ER
solution and water were irradiated with an 808
In vitro and in vivo PA imaging nm laser with a power density of 1.5 W cm-2 for
400 s. Through a thermal imager (Fluke Ti32,
PA is a new imaging method, and its sensiti- Fluke Corporation, USA) and infrared imager,
vity is better than that of CT [29]. Because ICG with increasing concentration, the heat gener-
has strong absorbance in the near infrared ated by ICG-ER under laser irradiation also
region, ICG is often used as a PA contrast increased (Figure 3A). At a 160 µg/ml concen-
agent. We will use 10% HSA for ICG-ER diluted tration, the temperature of laser irradiation for
to different concentrations. At the concentra- 240 s reached 65.2°C. These results indicate
tions of 5 µg/ml to 25 µg/ml, the PA signal that ICG-ER can be used for photothermal ther-
was more pronounced. The PA signal intensity apy in tumors (Figure 3B).
increased linearly with increasing ICG-ER con-
centration intensity (Figure 2A). PA images In vivo photothermal therapy
(Figure 2B) and signal values of the tumor site
were recorded before and after ICG-ER was ICG-ER has a good photothermal conversion
injected into the tail vein of nude mice. The PA rate and tumor targeting making ICG-ER feasi-

1995 Am J Transl Res 2022;14(3):1991-2001

 New probe for photoacoustic imaging and photothermal therapy

Figure 3. A. The corresponding IR thermal images of saline and ICG-ER different solution concentrations (10, 20,
40, 80, 160 µg/ml) at a stable power density of 808 nm laser (1.5 W/cm-2). B. Temperature changes of ICG-ER and
normal saline at different concentrations after laser irradiation for 400 s. The higher the concentration of ICG-ER,
the higher the temperature. At a concentration of 160 µg/ml, ICG-ER produced a maximum of 65.2°C after laser
irradiation.

Figure 4. A. IR thermal images of

4T1 tumor-bearing mice in various
groups (control, ICG-ER, laser, ICG-
ER combined with a laser). B. The
thermal imager measured the tem-
perature time curves of tumor sites
in different groups.

ble in photothermal therapy. To verify this, 4T1 The mice injected with normal saline and irradi-
tumor xenografts were established in nude ated with the same laser had little temperature
mice. When the mice tumor volume reached change. In contrast the mice injected with nor-
50-80 mm3, ICG-ER was injected into the mal saline and ICG-ER but not irradiated with
mice’s tail vein, and normal saline was injected laser had no significant temperature change.
into the control group. According to the PA The tumor volumes of mice treated with differ-
imaging results, after injection of ICG-ER and ent methods were recorded (Figure 5A, 5B).
normal saline for 42 hours, 808 nm laser irra- ICG-ER injection alone or laser irradiation had a
diation was carried out. The temperature little therapeutic effect on tumors in mice.
change of the tumor site was recorded by Similarly, the tumors of mice injected with ICG-
an infrared thermal imaging camera (Figure ER and irradiated with laser were eliminated
4A). The mice injected with ICG-ER were irradi- without recurrence. During the 16-day observa-
ated by an 808 nm laser (1.5 W cm-2) and then tion period, there was no significant difference
heated rapidly (statistical temperature, Figure in the bodyweight of mice in each group (Figure
4B). 5C).

1996 Am J Transl Res 2022;14(3):1991-2001

 New probe for photoacoustic imaging and photothermal therapy

Figure 5. A. Photographs of changes in 4T1 tumor-bearing mice of different groups were recorded by cameras every
two days during the 14 days. B. Relative tumor growth curves of the four groups after various treatments during the
observation period. All data were tested for significance by two-way ANOVA. **P<0.01, ****P<0.0001 ICG-ER vs. ICG-
ER+Laser, #P<0.05, ###P<0.001, ####P<0.0001 Laser vs. ICG-ER+Laser, &P<0.05, &&&P<0.001, &&&&P<0.0001 Control
vs. ICG-ER+Laser. (Values are means ± SD, n=3). C. The body weight curves of 4T1 tumor-bearing mice with various
treatments for 14 days.

The histopathology results also verified the Discussion

photothermal effect of ICG-ER. H&E staining
showed that the tumor cells in the treatment Photoacoustic imaging can effectively show
group were necrotic. TUNEL and PCNA showed the structure and function of biological tissue
that apoptosis increased and proliferative cells with the help of a photosensitizer. It provides
decreased in the treatment group. HSP70 an important means to study biological tissue
staining also showed that the expression of morphological structure, physiological charac-
HSP70 in the treatment group was significantly teristics, pathological characteristics, and met-
increased (Figure 6). This indicates that ICG-ER abolic function. Furthermore, it is especially
has a good photothermal effect. suitable for early detection and treatment
monitoring [30]. PTT uses PTCA with high-effi-
To test the biosafety of ICG-ER, one mouse in ciency photothermal conversion. It is injected
each group was taken for H&E staining of the into the human body, uses targeted recognition
main organs (heart, liver, spleen, lung, and kid- technology to gather near tumor tissue, and
ney) (Figure S2). No apparent histopathological converts light energy into heat energy under
lesions were found. These results indicate that near-infrared light (NIR) irradiation to eliminate
ICG-ER has high histocompatibility with the local tumors [31].
human body. At the same time, the blood of
these mice was taken to detect liver and kidney ICG is a tricarbocyanine dye with a near-infra-
function, and the results showed no significant red characteristic absorption peak approved by
change (Figure S3). This indicates that ICG-ER the US Food and Drug Administration (FDA) as a
has high biological safety. clinical near-infrared imaging reagent [32]. ICG

1997 Am J Transl Res 2022;14(3):1991-2001

 New probe for photoacoustic imaging and photothermal therapy

Figure 6. H&E, TUNEL, PCNA, and HSP70 staining of the tumor tissue at 1 day after various treatments. From top
to bottom: H&E-stained cells, TUNEL-positive cells (green), PCNA-positive cells (red), and HSP70-positive cells (red).
The DAPI-labeled nuclei are blue. The scale bars are 50 μm. All images were observed and retained by electron
microscopy at 36.5X magnification.

Figure 7. For the synthesis of ICG-ER, ICG-E

is first prepared by ICG-NHS and EB-MAL,
and the final product ICG-ER is obtained
after c(RGDfc) is covalently attached to
ICG-E.

can strongly absorb light energy and convert it in photoacoustic imaging and photothermal
into heat energy [33], making it suitable for use therapy. In recent years, the research on the

1998 Am J Transl Res 2022;14(3):1991-2001

 New probe for photoacoustic imaging and photothermal therapy

treatment of ICG in breast cancer is in full significantly improve the retention time and
swing, such as modifying ICG with hyaluronic uptake degree of ICG in tumors. Related
acid and metal nanoparticles which can be research has shown that the retention time of
degraded by tumor-specific hyaluronidase, so ICG-RGD in the tumor area is short, and the
that ICG can be targeted by metal nanoparti- photoacoustic signal of the tumor tissue obvi-
cles and can be degraded by tumor hyaluroni- ously declines at the 24th hour [44]. After
dase into the interior of the tumor, so as to using EB, the retention time of ICG-ER in tumor
improve the efficacy of photothermal therapy of tissue was significantly longer than that of ICG-
ICG in breast cancer [34]. However, in this RGD alone, and the photoacoustic signal of
study, the breast cancer tumors were not com- tumor tissue remained at a high level from 12 h
pletely cleared, and the metal nanoparticles to 42 h, which significantly improved the timeli-
were made of rare metal, which made the prep- ness of photothermal therapy of ICG.
aration of the probe more expensive.
We can draw the following conclusion from the
In addition to using metal nanoparticles to above result: ICG-ER is a new albumin binding
enhance the targeting of ICG, it is common to probe that can stably achieve targeted tumor
using RGD to directly modify ICG to enhan- imaging and photothermal therapy. ICG-ER
ce targeting. A study used internalized RGD shows the advantages of low toxicity and high
(iRGD) to modify ICG liposomes to evaluate the targeting at both the cell and animal levels. In
efficacy of photothermal therapy and photody- vivo photoacoustics showed perfect binding
namic therapy. The results show that iRGD- and retention times at the tumor site, which
ICG-LPs has a good targeting for breast cancer, makes ICG-ER a good potential in PA. ICG is a
and the efficacy of photothermal therapy is sig- good PTCA. After the modification of EB and
nificantly stronger than that of non-targeted RGD, the stability of ICG-ER in vivo is enhanc-
ICG-LPs [35]. However, in order to give full play ed, and it has an excellent photothermal con-
to the photothermal effect of ICG, we not only version rate. Related studies have proved that
need to target ICG to the tumor tissue, but also the growth of tumor cells will be inhibited
need to increase the residence time of ICG in when the temperature of tumor site reaches
the tumor tissue, so as to achieve a better cura- 47°C or higher [45]. In the study of photother-
tive effect. mal effects in vitro, ICG-ER increased rapidly
and obviously after laser irradiation, and the
Some studies have shown that the use of albu- temperature reached 65°C at the concentra-
min binder can prolong the blood circulation tion of 160 μg/ml. This advantage gives ICG-
time of drug molecules [36, 37], which will pro- ER a guiding significance in applying targeted
long the time of imaging and therapy, so as to photothermal therapy, and after 16 days of
significantly increase the effective therapeutic photothermal therapy, there is no recurrence in
dose and achieve better therapeutic results. As the tumor site. In general, ICG-ER has potential
a widely used albumin binder, EB is an azo dye in the optical imaging and photothermal treat-
with a high affinity that can reversibly bind to ment of malignant tumors.
human serum albumin in vivo and in vitro [20,
38]. When injected intravenously, EB can Acknowledgements
stably be combined with albumin in the blood.
The authors gratefully acknowledge the
Due to its high water solubility and slow excre-
financial support from the National Natural
tion, EB is widely used in biomedicine, such as
Science Foundation of China (NSFC) [Grant
evaluating blood volume and detecting lymph
Nos. 82102093] and the Natural Science
nodes and tumor location [39-41]. EB can be
Foundation of Chongqing [cstc2021jcyj-msxm-
labeled with many molecular compounds. For
X0006].
example, derivatives such as NOTA-Evans Blue
(NEB), have now been labeled with the nuclide Disclosure of conflict of interest
68
Ga and used clinically [42]. The uptake and
retention time of nuclides in the tumor region None.
was significantly prolonged after EB and radio-
nuclide probe labeling [43], which led us to Address correspondence to: Dr. Peng-Fei Xu,
wonder whether EB combined with ICG could Institute of Clinical Pharmacy & Pharmacology,

1999 Am J Transl Res 2022;14(3):1991-2001

 New probe for photoacoustic imaging and photothermal therapy

Jining First People’s Hospital, Jining Medical [12] Chen R, Wang J and Qiao H. Organic photother-
University, Jining 272000, P. R. China. E-mail: peng- mal conversion materials and their application
feixucsu@outlook.com; Hua Pang, Department of in photothermal therapy. Progress in Chemis-
Nuclear Medicine, The First Affiliated Hospital of try 2017.
Chongqing Medical University, No. 1 Youyi Road, [13] Mielke D, Malinova V and Rohde V. Compara-
tive analysis of endoscopic versus microscopic
Chongqing 400016, P. R. China. E-mail: phua-
ICG-angiography in aneurysm surgery. Surg
1973@163.com
Endosc 2011; 25: 3957-3958.
[14] Tomoharu S, Abdelazeem KK, Megumi T, Ta-
References
kashi H, Kazuhiko Y, Yoshikazu M and Masaka-
zu T. A novel method for sentinel lymph node
[1] Gefferth K. Diagnostic and therapeutic consid-
biopsy by indocyanine green fluorescence
erations of X-Ray hazard and protection in in-
technique in breast cancer. Cancers 2010; 2:
fants and children. 1962.
713-720.
[2] Chang RF, Wu WJ, Moon WK and Chen DR. Au-
[15] Murawa D, Hirche C, Dresel S and Hünerbein
tomatic ultrasound segmentation and mor-
M. Sentinel lymph node biopsy in breast can-
phology based diagnosis of solid breast tu-
cer guided by indocyanine green fluorescence.
mors. Breast Cancer Res Treat 2005; 89:
179-185. Br J Surg 2009; 96: 1289-1294.
[3] Lee CH. Problem solving MR imaging of the [16] Hirche C, Murawa D, Mohr Z, Kneif S and
breast. Radiol Clin North Am 2004; 42: 919- Hünerbein M. ICG fluorescence-guided senti-
34. nel node biopsy for axillary nodal staging in
[4] Valluru KS, Wilson KE and Willmann JK. Photo- breast cancer. Breast Cancer Res Treat 2010;
acoustic imaging in oncology: translational 121: 373-378.
preclinical and early clinical experience. Radi- [17] Shimizu S, Kamiike W, Hatanaka N, Yoshida Y
ology 2016; 280: 332-349. and Matsuda H. New method for measuring
[5] Harbeck N and Gnant M. Breast cancer. Lan- ICG Rmax with a clearance meter. World J Surg
cet 2017; 389: 1134-1150. 1995; 19: 113-118.
[6] Turner NC, Neven P, Loibl S and Andre F. Ad- [18] Hagen A, Grosenick D, Macdonald R, Rinne-
vances in the treatment of advanced oestro- berg H and Schlag PM. Late-fluorescence
gen-receptor-positive breast cancer. Lancet mammography assesses tumor capillary per-
2017; 389: 2403-2414. meability and differentiates malignant from
[7] Early Breast Cancer Trialists’ Collaborative benign lesions. Opt Express 2009; 17: 17016-
Group (EBCTCG), Peto R, Davies C, Godwin J, 17033.
Gray R, Pan HC, Clarke M, Cutter D, Darby S, [19] Poellinger A, Burock S, Grosenick D, Hagen A,
McGale P, Taylor C, Wang YC, Bergh J, Di Leo A, Lüdemann L, Diekmann F, Engelken F, Mac-
Albain K, Swain S, Piccart M and Pritchard K. donald R, Rinneberg H and Schlag PM. Breast
Comparisons between different polychemo- cancer: early- and late-fluorescence near-infra-
therapy regimens for early breast cancer: me- red imaging with indocyanine green--a prelimi-
ta-analyses of long-term outcome among nary study. Radiology 2011; 258: 409-16.
100,000 women in 123 randomised trials. [20] Evans HM and Schulemann W. The action of
Lancet 2012; 379: 432-44. vital stains belonging to the benzidine group.
[8] Cheng L, Wang C, Feng L, Kai Y and Zhuang L. Science 1914; 39: 443-454.
Functional nanomaterials for phototherapies [21] Bobin JY, Zinzindohoue C, Isaac S, Saadat M
of cancer. Chin Clin Oncol 2014; 114: 10869- and Roy P. Original paper tagging sentinel
10939. lymph nodes: a study of 100 patients with
[9] Gnanasammandhan MK, Idris NM, Bansal A, breast cancer. Eur J Cancer 1999; 35: 569-73.
Kai H and Yong Z. Near-IR photoactivation us- [22] Xiong JP, Stehle T, Zhang R, Joachimiak A,
ing mesoporous silica-coated NaYF4: Yb,Er/ Frech M, Goodman SL and Arnaout MA. Crystal
Tm upconversion nanoparticles. Nat Protoc structure of the extracellular segment of integ-
2016; 11: 688-713. rin αVβ3 in complex with an Arg-Gly-Asp ligand.
[10] Karimi M, Sahandi Zangabad P, Baghaee-Rav- Science 2002; 296: 151-155.
ari S, Ghazadeh M, Mirshekari H and Hamblin [23] Yun W, Cai W and Chen X. Near-infrared fluo-
MR. Smart nanostructures for cargo delivery: rescence imaging of tumor integrin αvβ3 ex-
uncaging and activating by light. J Am Chem pression with Cy7-labeled RGD multimers. Mol
Soc 2017; 139: 4584-4610. Imaging Biol 2006; 8: 226.
[11] Chu KF and Dupuy DE. Thermal ablation of tu- [24] Chen X, Park R, Hou Y, Khankaldyyan V, Gonza-
mours: biological mechanisms and advances les-Gomez I, Tohme M, Bading JR, Laug WE
in therapy. Nat Rev Cancer 2014; 14: 199- and Conti PS. MicroPET imaging of brain tumor
208. angiogenesis with 18F-labeled PEGylated RGD

2000 Am J Transl Res 2022;14(3):1991-2001

 New probe for photoacoustic imaging and photothermal therapy

peptide. Eur J Nucl Med Mol Imaging 2004; [36] Heneweer C, Holland JP, Divilov V, Carlin S and
31: 1081-1089. Lewis JS. Magnitude of enhanced permeability
[25] Tsiapa I, Loudos G, Varvarigou A, Fragogeorgi and retention effect in tumors with different
E, Psimadas D, Tsotakos T, Xanthopoulos S, phenotypes: 89Zr-albumin as a model system.
Mihailidis D, Bouziotis P and Nikiforidis GC. J Nucl Med 2011; 52: 625-633.
Biological evaluation of an ornithine-modified [37] Dumelin CE, Trüssel S, Buller F, Trachsel E,
99mTc-labeled RGD peptide as an angiogene- Bootz F, Zhang Y, Mannocci L, Beck SC,
sis imaging agent. Nucl Med Biol 2013; 40: Drumeamirancea M and Seeliger MW. A por-
262-272. table albumin binder from a DNA-encoded
[26] Gellerman G. Novel targeted non-RGD cyclic chemical library. Angew Chem Int Ed Engl
peptide drug conjugates for treatment of hu- 2010; 47: 3196-3201.
man metastatic melanoma. Oncotarget 2017; [38] Oskar S and Igor K. Selective vulnerability of
8: 757-768. the blood-brain barrier in chemically induced
[27] Wang XS, Huang Y, Liu D, Liang L and Liu JP. lesions. J Neuropathol Exp Neurol 1966; 25:
RGD modified docetaxel and suramin loaded 542-59.
liposome for breast cancer targeting therapy. [39] Roller BT, Munson JM, Brahma B, Santangelo
Chinese Journal of Cancer Prevention and PJ, Pai SB and Bellamkonda RV. Evans blue
Treatment 2014; 21: 1783-1793. nanocarriers visually demarcate margins of in-
[28] Xu P, Hu L, Yu C, Yang W, Kang F, Zhang M, Ji- vasive gliomas. Drug Deliv Transl Res 2015; 5:
ang P and Wang J. Unsymmetrical cyanine dye 116-24.
via in vivo hitchhiking endogenous albumin af- [40] Radu M and Chernoff J. An in vivo assay to test
fords high-performance NIR-II/photoacoustic blood vessel permeability. J Vis Exp 2013;
imaging and photothermal therapy. J Nanobio- e50062.
technology 2021; 19: 334. [41] Tsopelas C, Bevington E, Kollias J, Shibli S,
[29] Lee N, Choi SH and Hyeon T. Nano-sized CT Farshid G, Coventry B and Chatterton BE.
contrast agents. Adv Mater 2013; 25: 2641- 99mTc-Evans blue dye for mapping contiguous
2660. lymph node sequences and discriminating the
[30] Liu Y, Teng L, Liu HW, Xu C and Tan W. Recent sentinel lymph node in an ovine model. Ann
advances in organic-dye-based photoacoustic Surg Oncol 2006; 13: 692-700.
probes for biosensing and bioimaging. Science [42] Zhang J, Lang L, Li F, Zhu Z, Niu G and Chen X.
China Chemistry 2019. Clinical translation of an albumin-binding PET
[31] Shanmugam V, Selvakumar S and Yeh CS. radiotracer 68Ga-NEB. J Nucl Med 2015;
Near-infrared light-responsive nanomaterials 1609-1614.
in cancer therapeutics. Chem Soc Rev 2014; [43] Rui T, Orit J, Gang N, Kiesewetter DO, Wang Z,
43: 6254-87. Zhu G, Ma Y, Gang L and Chen X. Evans blue
[32] Moody ED, Viskari PJ and Colyer CL. Non-cova- attachment enhances somatostatin receptor
lent labeling of human serum albumin with in- subtype-2 imaging and radiotherapy. Ther-
docyanine green: a study by capillary electro- anostics 2018; 8: 735-745.
phoresis with diode laser-induced fluorescence [44] Martina C, Francesco B, Giovanni V, Paolo O,
detection. J Chromatogr B Biomed Sci Appl Claudia C, Federica B, Alessia C, Lorena P,
1999; 729: 55-64. Alessandro M and Luisa P. Photoacoustic im-
[33] Yaseen MA. Photothermal and photochemical aging of integrin-overexpressing tumors using
effects of laser light absorption by indocyanine a novel ICG-based contrast agent in mice. Pho-
green (ICG). Proc Spie 2005; 5695: 27-35. toacoustics 2018; 11: 36-45.
[34] Liu R, Hu C, Yang Y, Zhang J and Gao H. Ther- [45] Ghaffari H, Beik J, Talebi A, Mahdavi SR and
anostic nanoparticles with tumor-specific en- Abdollahi H. New physical approaches to treat
zyme-triggered size reduction and drug release cancer stem cells: a review. Clin Transl Oncol
to perform photothermal therapy for breast 2018; 20: 1-20.
cancer treatment. Acta Pharm Sin B 2019; 9:
410-420.
[35] Yan F, Wu H, Liu H, Deng Z, Liu H, Duan W, Liu
X and Zheng H. Molecular imaging-guided pho-
tothermal/photodynamic therapy against tu-
mor by iRGD-modified indocyanine green
nanoparticles. J Control Release 2016; 224:
217-228.

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 New probe for photoacoustic imaging and photothermal therapy

Figure S1. Cell viability of 4T1 cells after incubation with EB-ICG-RGD at different concentrations for 24 h.

Figure S2. H&E staining of the major organs (heart, liver, spleen, lung, and kidney) of 4T1 tumor-bearing mice after
different treatments. The scale bars are 50 μm. All images were observed and retained by electron microscopy at
36.5X magnification.

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 New probe for photoacoustic imaging and photothermal therapy

Figure S3. ALT, AST and BUN of the tumor tissue at 1 day after various treatments. All the indicators are within the
normal range.

Figure S4. HPLC chromatogram (254 nm) of ICG-E.

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 New probe for photoacoustic imaging and photothermal therapy

Figure S5. MALDI-TOF-MS measurement of EB-Mal.

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 New probe for photoacoustic imaging and photothermal therapy

Figure S6. MALDI-TOF-MS measurement of ICG-E.

Figure S7. HPLC chromatogram (254 nm) of ICG-ER.

Figure S8. MALDI-TOF-MS measurement of ICG-ER.

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 New probe for photoacoustic imaging and photothermal therapy

Figure S9. The most optimal wavelength for PA imaging by full-wavelength scanning.