Delivery of coumarin-containing all-: Trans retinoic acid derivatives via targeted nanoparticles encapsulating indocyanine green for chemo/photothermal/photodynamic therapy of breast cancer

Article abstract of DOI:10.1039/c8nj00578h

Development of chemo/photothermal/photodynamic therapy with nanoplatforms offers a promising strategy for effective cancer treatment. Recently, all-trans retinoic acid (ATRA), as a potential antitumor drug, has attracted great attention due to its antitumor activity. In this study, a novel coumarin-containing ATRA (AC) and indocyanine green (ICG) dye-loaded nanoparticles with the targeted ligand cyclic (Arg-Gly-Asp-d-Phe-Lys) (cRGD) peptide were fabricated by self-assembly and used as a new theranostic nanoplatform for chemo/photothermal/photodynamic therapy. The as-formed nanoparticles (AC/ICG-TNPs) had a diameter of around 133 nm with uniform monodispersity. Additionally, AC/ICG-TNPs showed marked stability under normal physiological conditions. However, it could rapidly release drugs in a mild acidic microenvironment. Moreover, confocal microscopic observations confirmed that the uptake of AC/ICG-TNPs increased in the breast cancer cells, particularly in MDA-MB-231 cells, probably mediated by cRGD via specific recognition of the overexpressed integrin α_vβ₃. Moreover, free AC exhibited stronger cytotoxic effects than free ATRA in the MTT assay, and AC/ICG-TNPs were demonstrated to possess excellent antitumor efficacy when exposed to NIR irradiation through the combination therapy. Hence, the therapeutic method designed in this study is a good candidate for improved bioactivity of ATRA and site-specific combinational therapy against breast cancer.

Full text of DOI:10.1039/c8nj00578h

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PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Delivery of coumarin-containing all-trans retinoic acid derivative via
targeted nanoparticles encapsulating indocyanine green for
chemo/photothermal/photodynamic therapy of breast cancer
a
a
a
b
Jia Jiao , Hongshuai Wu , Fanghui Chen , Renjie Chen , Baiwang Sun
a,*
,
a,*
Mingliang Wang
a
School of Chemistry and Chemical Engineering, Southeast University, Nanjing
210089, PR China. Fax: +86 25 52090614, Tel: +86 25 52090614, E-mail address:
chmsunbw@seu.edu.cn
b
Affiliated Hospital 2, Nanjing Medical University, Nanjing 210011, PR China
Abstract: Developing the chemo/photothermal/photodynamic therapy with
nanoplatforms offers a promising strategy for effective cancer treatment. Recently,
all-trans retinoic acid (ATRA), as a potential antitumor drug, has drawn great attention
in improving its antitumor activity. In this study, a novel coumarin-containing ATRA
(AC) and indocyanine green (ICG) dye loaded nanoparticles, with the targeted ligand
cyclic (Arg-Gly-Asp-D-Phe-Lys) (cRGD) peptide, were fabricated by self-assembling,
as a new theranostic nanoplatform for chemo/photothermal/photodynamic therapy.
The formed nanoparticles (AC/ICG-TNPs) had a diameter of around 133 nm with
uniform monodispersity. Additionally, AC/ICG-TNPs showed marked stability under
the normal physiological conditions. However, it could rapidly release drug under the
mild acidic microenvironment. Moreover, confocal microscopy observations
confirmed that the uptake of AC/ICG-TNPs increased in the breast cancer cells,
especially in MDA-MB-231 cells, probably mediated by cRGD via specific
recognition of the overexpressed integrin αvβ3. Meanwhile, free AC exhibited stronger
cytotoxic effects than free ATRA in MTT assay, and AC/ICG-TNPs were
demonstrated to possess the excellent antitumor efficacy when exposed to NIR
irradiation through the combination therapy. Hence, this designed therapeutic method
is a good candidate for the improved bioactivity of ATRA and the site-specific

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combinational therapy against breast cancer.
KEYWORDS: nanoparticles, therapeutic efficacy, photothermal therapy,
photodynamic therapy, ATRA, coumarin, active targeting
1. Introduction
Breast carcinoma is a devastating malignancy and represents the primary driver of
cancer-related death among women worldwide.1, 2 At present, breast cancer treatments
have been performed on the basis of clinical and pathologic staging and still far from
optimal, which are mainly restricted to chemotherapy, radiation and surgery.3 Recent
decades have witnessed that nanotechnology provides a plethora of promising
avenues for cancer therapy. The nanocarriers (liposomes, polymeric micelles, metal or
oxide based nanoparticles), as an emerging class of therapeutics, can not only prolong
plasma circulation time but also alleviate systemic toxicities and improve the
water-solubility of hydrophobic small molecules.4-6 In addition, the surface of
nanocarriers functionalized with ligands can yield high affinity and specificity for
target sites.7-9 These significant features have drawn increasing interest in nanocarriers
for improving therapeutic efficacy compared with the conventional chemotherapy.10-12
In both clinical practice and exploratory studies, it has been demonstrated that the
monotherapy is inefficient to combat the tumor, which is mainly due to the reason that
the subpopulations of cancer cells in heterogeneous tumor tissue have resistance to
single treatment modality. Therefore, combination therapies with synergistic effect
have obtained tremendous attention.5, 13, 14 Indocyanine green (ICG), a near infrared
dye, is able to simultaneously serve as a potential photosensitizer and photothermal
conversion agent (PTCA) due to its capacity of generating reactive oxygen species
(ROS) and the excellent photothermal conversion efficiency after irradiated by NIR
laser.15 Photothermal therapy (PTT) and photodynamic therapy (PDT), minimally
invasive therapies, utilize the heat or ROS generated from the absorbed optical energy
by photothermal conversion agents (PTCAs) or photosensitizers to damage malignant
tumors.16 Nevertheless, the limited tissue penetration of NIR light and the upregulated
expression of heat shock protein in cancer cells greatly impede the performance of
PTT and PDT. In view of these inadequacies, the combination of PTT, PDT and

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chemotherapy is considered as an effective strategy to achieve the optimized
performance.17
ATRA, the major active metabolite of vitamin A, participates in multiple biological
processes, such as cell proliferation and differentiation.18 ATRA has been shown to be
potential work in prevention and treatment of cancers including breast cancer.
Increasing evidence indicates that ATRA can block tumor growth through damaging
tumor cell DNA and inhibiting tumor cell differentiation.19 However, the utilization of
ATRA is often hindered due to its limited toxicity and therapeutic efficacy.
Consequently, many studies are focused on increasing its bioactivities via structural
modification.20-24 Coumarin, namely benzopyrone, is a versatile nucleus of various
natural compounds with diverse physiological activities. It is reported that coumarin
and its derivatives have anticancer activity against tumors including breast cancer.25-27
On the basis of the above, we synthesized a novel cytotoxic molecule,
coumarin-containing all-trans retinoic acid derivative (AC), through linking ATRA
with 7-hydroxy-4-trifluoromethyl coumarin (HTCM) by ester bond for the first time,
which enhanced toxicity against tumor cells. Furthermore, AC and ICG were
simultaneously loaded into one nanocarrier modified with cRGD peptide, one could
effectively increase the accumulation of drugs in the breast cancer tumors, which
overexpressed cell integrin αvβ3. And it is demonstrated that the designed
nanoplatform could remarkably realize the synergistic effect of chemotherapy, PTT
and PDT. All of the results indicated that the prepared nanosystem is a potential
therapeutic strategy in clinical application for breast cancer therapy.
2. Materials and methods
2.1. Materials
DSPE-PEG2000-COOH, cRGD and ICG were purchased from Shanghai Pengshuo
Biological technology Co., Ltd. (Shanghai, China). Resorcinol, ATRA,
2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU),
4,4,4-Trifluoro-3-oxobutyric
acid
ethyl
ester,
4-Methylmorpholine,
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI) and
N-hydroxysuccinimide (NHS) were bought from Aladdin Reagent Co. Ltd. (Shanghai,

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China). Phosphate buffered saline (PBS), DMEM, RPMI1640 medium, sodium
bicarbonate, penicillin-streptomycin antibiotics, fetal bovine serum (FBS),
trypsin-EDTA and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) were provided by Yucheng Biotechnology Co., Ltd. (Nanjing, China).
4',6-diamidino-2-phenylindole (DAPI), reactive oxygen species assay kit and Annexin
V-FITC apoptosis detection kit were obtained from Sigma-Aldrich (St Louis, MO,
USA). All other chemical reagents were purchased from Sinopharm Chemical
Reagent Co., Ltd.
2.2. Preparation of AC/ICG-TNPs
In brief, DSPE-PEG2000-COOH (10mg), DSPE-PEG2000-cRGD (2mg), AC (2mg)
and ICG (0.6 mg) were dissolved in dichloromethane (20 mL) and then the solvent
evaporated under reduced pressure to form a thin film. After 10 mL of deionized
water added into the mixture, the solution became suspension gradually as the
formation of nanoparticles under sonication for 8 min via an ultrasonic processor. To
remove unencapsulated drug molecules, the suspension was centrifuged at 3000 rpm
and then filtrated with 0.22 µm filter membrane. The nanoparticles was obtained by
lyophilization and finally stored at 4 °C for further use. To serve as control, other
nanoparticles including non-targeted nanoparticles encapsulating AC and ICG
(AC/ICG-NPs), targeted nanoparticles without encapsulating AC (ICG-TNPs) and
empty nanoparticles with or without targeted ligand (TNPs or NPs), were all prepared
through the similar process.
2.3. Characterization of nanoparticles
The size and polydispersity (PDI) of nanoparticles were estimated by Dynamic
Light Scattering (DLS). Briefly, the prepared nanoparticles were analyzed by a
Brookhaven A8530 apparatus in triplicate at room temperature and the data were
analyzed using Brookhaven Dispersion Technology Software. The zeta potential was
detected by a Malvern Zetasizer Nanoseries (Malvern Instruments Zen 3600, Malvern,
U.K.) and calculated using Brookhaven Dispersion Technology Software. The surface
morphology of nanoparticles was further investigated by JEM-100CXII transmission
electron microscopy (TEM). The samples were prepared for TEM by evaporating

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water under weak light irradiation after dropped on a copper grid coated with carbon.
2.4. Entrapment efficiency (EE) and drug loading capacity (DLC)
The EE and DLC of AC and ICG loaded in nanoparticles were measured by UV-vis
spectrometer (UV-2450, Shimadzu, Japan) using a standard curve method as
previously described.12, 28 The entrapment efficiency and drug loading capacity were
calculated according to the following equations:
ꢉꢊꢋꢌℎꢍ ꢄꢎ ꢏꢅꢐꢌ ꢋꢑ ꢑꢒꢑꢄꢓꢒꢅꢍꢋꢔꢕꢊꢖ
ꢁ
ꢈ
ꢀꢀ %, ꢂꢃ ꢄꢅ ꢆꢃꢇ =
× 100%
ꢉꢊꢋꢌℎꢍ ꢄꢎ ꢎꢊꢊꢏꢋꢑꢌ ꢏꢅꢐꢌ
ꢉꢊꢋꢌℎꢍ ꢄꢎ ꢏꢅꢐꢌ ꢋꢑ ꢑꢒꢑꢄꢓꢒꢅꢍꢋꢔꢕꢊꢖ
ꢁ
ꢈ
ꢗꢘꢃ %, ꢂꢃ ꢄꢅ ꢆꢃꢇ =
× 100%
ꢉꢊꢋꢌℎꢍ ꢄꢎ ꢍꢄꢍꢒꢕ ꢏꢅꢐꢌ ꢕꢄꢒꢏꢊꢏ ꢑꢒꢑꢄꢓꢒꢅꢍꢋꢔꢕꢊꢖ
2.5. Stability of nanoparticles
The AC/ICG-TNPs were selected to be kept at 4 °C for up to 28 days. The stability
of nanoparticles was detected by Dynamic Light Scattering (DLS) and UV-vis
spectrometer on day 0, 7, 14, 21 and 28 to monitor the changes in size, PDI and UV
absorption of nanoparticles. At each point in time, the suspended nanoparticles were
centrifuged at 3000 RCF for 10 min to ensure that the impurities could be sedimented
and then took out the certain volume of supernate to measure.
To further investigate the stability of AC/ICG-TNPs under physiological conditions,
the prepared nanoparticles were evaluated by DLS in PBS with the presence of 10%
FBS at different times.
2.6. Drug release
The triggered release behaviors of AC from nanoparticles were studied at different
physiological pH through the dialysis method.9 Briefly, the prepared nanoparticles
were dispersed in phosphate buffer (3.0 mL, pH 5.0, 6.0 or 7.4) and the suspension
was placed into a dialysis bag (MWCO = 3500 Da). Then the dialysis bag was
immersed in buffer medium at pH 5.0, 6.0 or 7.4, and gently shaken at 37 °C. At a
preselected time, the dialysate (2 mL) was withdrawn to determine the amount of
released drug utilizing UV-vis spectroscopy through comparing the absorption curve
of pure AC, while a same amount (2 mL) of fresh buffer medium was added back to
compensate the volume loss. Each sample was tested three times.
2.7. Evaluation of photothermal conversion efficiency

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The nanoparticle suspensions diluted to various concentrations (40 µg/mL, 60
µg/mL, 80 µg/mL or 100 µg/mL) were exposed to 808 nm laser with a power of 1.5
W/cm2 for 5 min. The changes of temperature of ICG mediated nanoparticles were
recorded by thermal imager.
2.8. Cell culture
The human breast cancer cells MCF-7 cultured in RPMI 1640 medium and
MDA-MB-231 maintained in DMEM were purchased from the Cell Bank of the
Chinese Academy of Sciences (Shanghai, China). Both media were supplemented
with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin
solution. The entire cells were cultured in a humidified atmosphere containing 5%
CO2 at 37 °C.
2.9. Cellular uptake study
MCF-7 or MDA-MB-231 cells were seeded in the confocal petri dish at a density
of 2.0×105 cells per dish allowed to adhere for 24 h under 5% CO2 atmosphere at
37 °C. After the culture medium removed, the cells were washed three times with
PBS. Then an appropriate amount of AC/ICG-TNPs or AC/ICG-NPs suspension with
the same concentration of ICG was added into the cell culture medium and further
incubation for different time durations (1, 4 or 12 h). After the cells fixed by 4%
paraformaldehyde for 30 min at 37 °C, DAPI was added and the cells were stained for
15 min. Finally, the resulting samples were visualized with Confocal Laser Scanning
Microscopic (CLSM).
2.10. Intracellular ROS Detection
The generation of intracellular ROS from AC/ICG-TNPs uptake by MDA-MB-231
cells after NIR laser irradiation were monitored with DCFH-DA probe.
MDA-MB-231 cells were seeded in a 6-well plate at 1×105 cells per well for 24 h and
subsequently incubated with AC/ICG-TNPs for 12h. After removing the medium with
nanoparticles, the cells were washed three times with PBS and incubated with
DCFH-DA for another 20 min. Then the cells were washed with PBS and irradiated
with 808 nm laser (1.5 W/cm2) for 5 min. The fluorescence images were obtained
using a confocal microscopy with 488 nm excitation and emission of 520 nm. ROS

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generated in cells were also quantitatively measured by flow cytometry on a FACS
Calibur (BD Biosciences, USA).
2.11. Cytotoxicity assays
Cytotoxicity of AC/ICG-TNPs was evaluated on MCF-7 and MDA-MB-231 cells
by the MTT colorimetric method29. In brief, the cells were seeded in a 96-well plate
with the density of 1×104 cells per well. 24 h post-seeding, the cells were treated with
various concentrations of the prepared nanoparticles. After incubation for 12 h, the
medium was replaced with fresh one and the cells were then irradiated with 808 nm
laser (1.5 W/cm2) for 5 min. The cells were further cultured by 12 h at 37 °C.
Thereafter, 150 ꢀL of MTT solution (5 mg/mL) was added into each well and
incubated for another 4 h. Afterward, the medium was replaced by 150 ꢀL of DMSO
to dissolve the purple formazan and the absorbance at 490 nm was measured by a
microplate reader. The cytotoxicity assay was performed thrice and the average value
was taken from the three measurements.
2.12. Cell apoptosis
MCF-7 or MDA-MB-231cells were seeded in the 6-well plate at 1×105 cells per
well and cultured for 24 h in an incubator. Then the cells were exposed to different
concentrations of the prepared nanoparticles and incubated for 12 h, while the cells
without treatment were used as a control. The complete medium was then replaced
with fresh one and the cells were irradiated with 808 nm laser (1.5 W/cm2) for 5 min.
After that, the cells were cultured for additional 12 h. Both floating and attached cells
were collected and washed thrice with PBS for quantitative measurement of apoptosis.
After that, the cells were stained with Annexin V-FITC (5 ꢀL) and propidium iodide
(3 ꢀL) for 15 min without light. Finally, the samples were analyzed by flow cytometry
on a FACS Calibur (BD Biosciences, USA).
3. Results and discussion

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Scheme 1. Schematic illustration of AC/ICG-TNPs for targeted and
chemo/photothermal/photodynamic therapy.
3.1. Preparation and characterization of nanoparticles
In this study, several forms of nanoparticles were prepared through thin-film
hydration method under sterile conditions. The schematic diagram of preparation
process was presented in Scheme 1. As depicted in Figure 1A, the suspension of
TNPs exhibited colorless, while the ICG-TNPs and AC/ICG-TNPs suspension
displayed green and yellow green colors, respectively, which demonstrated ICG and
AC were encapsulated successfully. And the AC/ICG-TNPs suspensions presented
evident Tyndall phenomenon. Obviously, the absorption spectrum of AC/ICG-TNPs

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showed a blue-shifted absorption peak of AC from 373 nm to 326 nm (Figure 1A,
Figure S6A). Meanwhile, the absorption spectrum exhibited a peak at 792 nm, which
was attributed to the ICG content (Figure 1A).
The surface morphology, size and size distribution of the nanoparticles were
investigated by TEM and DLS (Figure 1B, Figure 1C, Table 1). It was noticed that
the average diameter of AC/ICG-TNPs was around 133 nm with almost regular
spherical morphology and good monodispersity. Moreover, the size had no significant
difference between NPs and TNPs, while the size of AC/ICG-TNPs is slightly larger
than that of TNPs. The increase of nanoparticle size should be attributed to the
successful encapsulation of AC and ICG and the results were consistent with the
statistical charts inseted in TEM images (Figure 1C). It was reported that
nanoparticles with a size 30-200 nm could favorably accumulate in cancer tissues via
EPR.11, 30 Therefore, the prepared AC/ICG-TNPs had the potential of enhanced
accumulation in tumor sites. The tested results for the zeta potential demonstrated that
after the introduction of targeting ligand cRGD peptide into nanoparticles, the surface
charge of nanoparticles significantly increased from -40.2 mV to -22.9 mV (Figure
1D, Figure S6, Figure S6F). This phenomenon might result from the presence of
positively charged amide and guanidine groups in cRGD peptide.
UV-vis absorption spectra and DLS of nanoparticles in phosphate buffer at pH 7.4
are shown in Figure 1E and Figure 1F to prove the stability of nanoparticles. After
stored at 4 °C for four weeks, the suspension of AC/ICG-TNPs remained clear
without obvious precipitation and the PDI parameters kept at a favorable level.
Additionally, the particle size and the value of UV absorption peak showed little
change. To further confirm the stability of the AC/ICG-TNPs under physiological
conditions, the nanoparticles were measured by DLS in PBS with the presence of 10%
FBS at 37 °C.15 As shown in Figure 2A, the PDI remained below 0.3 and the size
kept stable. These results suggested that the AC/ICG-TNPs would remain excellent
stability under physiological conditions and may be used in the bloodstream with a
long circulation time.
UV-vis spectroscopy was used for quantitative analysis of the content of ICG and

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AC. As shown in Table 1, the AC/ICG-TNPs exhibited higher entrapment efficiency
of ICG than AC, which should be due to the different form of drug-carried. Moreover,
after the conjugation of cRGD peptide, EE and DLC had no obvious changes.
Table 1. Characteristics of AC/ICG-TNPs compared with AC/ICG-NPs.
NPs
Size (nm)
127.89
PDI
0.238
0.277
EE%/DLC% (AC)
34.36/6.64
EE%/DLC% (ICG)
71.84/4.09
Zeta potential (mV)
AC/ICG-NPs
AC/ICG-TNPs
-40.2
-22.9
133.04
34.90/6.82
72.69/4.26
Figure 1. (A) UV-vis spectroscopy of TNPs, ICG-TNPs and AC/ICG-TNPs
suspensions in phosphate buffer (pH 7.4). Inserted images are Tyndall phenomenon of
AC/ICG-TNPs and the appearance of TNPs, ICG-TNPs AC/ICG-TNPs. (B) Size
distribution of NPs, TNPs and AC/ICG-TNPs. (C) TEM images of TNPs and
AC/ICG-TNPs. (D) Zeta potential of AC/ICG-NPs and AC/ICG-TNPs. (E) Changes

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in UV-vis spectroscopy of AC/ICG-TNPs stored at 4 °C in phosphate buffer (pH 7.4)
for 4 weeks. (F) Changes in size and PDI of AC/ICG-TNPs stored at 4 °C in
phosphate buffer (pH 7.4) for 4 weeks.
3.2. Photothermal performance and in vitro release
To evaluate the photothermal efficiency, the AC/ICG-TNPs dispersed in water at
different concentrations were irradiated by 808 nm laser at a power density of 1.5
W/cm2 for 5 minutes, and water was treated as control. As shown in Figure 2, the
temperature changes of AC/ICG-TNPs suspensions with various concentrations were
monitored and recorded by thermal imager at certain exposed time interval. It was
noticed that the increase of temperature in pure water under the NIR irradiation was
almost negligible, while the AC/ICG-TNPs suspensions revealed an evident
concentration-dependent temperature increase. The fastest and greatest temperature
increase was obtained at the highest concentration of AC/ICG-TNPs (100µg/mL), of
which the temperature increased by 48 °C after 5 minutes of irradiation. These results
obtained demonstrated the perfect photothermal conversion capacity mediated by ICG.
It is known that the rapid temperature increase can confer irreversible thermal injury
to cancer cells.
The standard curves of AC and ICG were measured with UV-vis spectroscopy
(Figure S6). At 37 °C, dialysis was used to investigate the release efficiency of AC
from the AC/ICG-TNPs when the pH was 6.0 or 5.0 and the physiological condition
of pH 7.4 was used as the control. As displayed in Figure 2D, the release of AC were
less than 20% after continuous incubation for 48 h at pH 7.4, which further proved the
favorable structural stability and drug loading stability. In contrast, at slightly acidic
conditions of pH 5.0 and 6.0, the corresponding percentages of released AC were up
to approximately 84% and 70% after the same time, respectively. These results
indicated that the AC/ICG-TNPs were relatively stable at physiological pH and also
capable of the accelerated release of AC under the mild acidic condition. Specifically,
the amounts of released AC increased along with a reduction in the pH value, which
might be ascribed to the faster and more thorough disassembly of AC/ICG-TNPs at
some degree in an acidic environment.30 Thus, the accelerated release of drugs at

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lower pH are preferable for the delivery of anticancer drugs.29, 31
Figure 2. (A) Serum stability (against 10% FBS) of AC/ICG-TNPs in PBS at 37 °C.
(B) Thermal imaging images of AC/ICG-TNPs detected by Infrared Camera. (C)
Heating curves of water and various concentrations of AC/ICG-TNPs suspensions. (D)
In vitro AC release profiles of AC/ICG-TNPs in pH 5.0, 6.0 and 7.4 at 37 °C.
3.3. Cell Internalization
Delivering therapeutic agents efficiently into tumor cells is crucial for the
successful cancer therapy. Therefore, the cellular uptake of AC/ICG-NPs and
AC/ICG-TNPs were investigated in MCF-7 and MDA-MB-231 cells using confocal
laser scanning microscopy based on the red fluorescence of ICG. As shown in Figure
3 and Figure 4, it was apparent that the representative red fluorescence was localized
in the cytoplasm and nucleic regions of two cell lines at the 12-hour time-point and
the internalization was found to be time-dependent. Meanwhile, the AC/ICG-TNPs
exhibited stronger red fluorescence at any time-point in MDA-MB-231 cells
compared to AC/ICG-NPs, but it could not observe these significant differences in
MCF-7 cells. These phenomena suggested that the uptake of AC/ICG-TNPs could be
closely related to the interaction of cRGD peptide with the overexpressed αvβ3
integrin of MDA-MB-231 cells, while the weaker uptake efficiency of MCF-7 cells

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was attributed to its low expression of αvβ3.32-34 Interestingly, the AC/ICG-TNPs was
also observed in the proximity of the nuclei and nuclei of MDA-MB-231 cells as
shown by the red fluorescence in merged CLMS images (Figure 4), which was
probably due to the localization of AC/ICG-TNPs or delivery of dye following
endosomal/lysosomal escape.35 Therefore, the above results demonstrated that the
prepared nanoparticles possessed the potential to effectively improve the delivery and
distribution of the drug in cancer cells.
Figure 3. Confocal laser scanning microscopic images of cellular uptake in MCF-7
cells cultured with AC/ICG-TNPs and AC/ICG-NPs for different period time. Nuclei
of cells stained with DPAI (blue) and ICG fluorescence in cells (red). Scale bar: 20
ꢀm.

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Figure 4. Confocal laser scanning microscopic images of cellular uptake in
MDA-MB-231 cells cultured with AC/ICG-TNPs and AC/ICG-NPs for different
period of time. Nuclei of cells stained with DPAI (blue) and ICG fluorescence in cells
(red). Scale bar: 20 ꢀm.
3.4. Photodynamic performance
To prove AC/ICG-TNPs as the PDT agent, the DCFH-DA method was applied to
detect the intracellular ROS of MDA-MB-231 with AC/ICG-TNPs under NIR laser
irradiation. As shown in Figure 5A, in groups where MDA-MB-231 cells treated with
AC/ICG-TNPs for 12 h and followed by being exposed to NIR laser irradiation, an
intensive green fluorescence signal was detected. In contrast, the fluorescence signal
were not significant for the control with laser or free AC with laser or AC/ICG-TNPs
group. These results suggested the perfect capability of AC/ICG-TNPs mediated by
ICG with NIR irradiation in producing ROS. As depicted in Figure 5B, the
quantification of induced ROS intensity was evaluated by flow cytometry. The
fluorescence intensity for the AC/ICG-TNPs with NIR laser irradiation group was
highest than that for the other three groups and there was no obvious difference in
fluorescence intensity for the other three groups, which further confirmed the perfect
capability of AC/ICG-TNPs mediated by ICG with NIR irradiation in generating ROS.
These phenomena indicated that abundant ROS can be generated by AC/ICG-TNPs in
MDA-MB-231 cells with NIR laser irradiation. Therefore, the ICG-mediated

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AC/ICG-TNPs with NIR laser irradiation are suitable candidates in PDT.
Figure 5. (A) CLSM images of singlet oxygen generation in MDA-MB-231 cells
following various treatments as indicated. Green: ROS indicator DCFH-DA; scale bar:
20 ꢀm. (B) Quantification of induced ROS intensity achieved by flow cytometry.
3.5. In vitro cytotoxicity
We investigated the in vitro anticancer effects of the prepared nanoparticles using
MCF-7 and MDA-MB-231 cells by MTT assay. For this purpose, both cell lines were
treated with ATRA, AC, AC/ICG-TNPs and AC/ICG-TNPs with irradiation at
concentrations ranging from 10 ꢀM to 120 ꢀM. As shown in Figure 6, the
cytotoxicity of two kinds of free drugs and AC/ICG-TNPs all exhibited
concentration-dependent profiles in two cell lines. AC displayed the stronger
cytotoxic effects on MCF-7 and MDA-MB-231 cells especially at high concentrations
compared to free ATRA, revealing that utilizing 7-hydroxy-4-trifluoromethyl
coumarin to modificate the structure of ATRA could obviously improve its antitumor
effects. Meanwhile, the cell suppression capacity of AC/ICG-TNPs was slightly
weaker than free AC even if the concentration was up to 120 ꢀM. It might be due to
that AC easily diffused through the cellular membrane and quickly killing the
malignant cells, while the nanoparticles released AC relatively slowly and resulted in
the lower cytotoxicity at the same time.36 However, AC/ICG-TNPs showed the
stronger antitumor efficacy and even more approximately to free AC effect in
MDA-MB-231 than that in MCF-7, which could be attributed to the higher expression
levels of integrin αvβ3 in MDA-MB-231 cells than in MCF-7 cells.32 These results
were also consistent with the cellular uptake study. Futhermore, the cell viability of

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AC/ICG-TNPs under laser irradiation was the lowest among all the groups even
achieving about 3.16% in MDA-MB-231 cells when the concentration was 120 ꢀM
equivalent of AC, suggesting that the chemo/photothermal/photodynamic therapy
provided by AC/ICG-TNPs were highly effective to the breast cancer cells, especially
against MDA-MB-231 cells.
Figure 6. In vitro cytotoxicity of free ATRA, free AC, AC/ICG-TNPs and
AC/ICG-TNPs with the laser irradiation (808 nm, 1.5 W/cm2) for 5 min against
MCF-7 cells (A) and MDA-MB-231 cells (B).
3.6. Cell apoptosis
To further confirm that AC/ICG-TNPs with NIR laser irradiation could induce cell
apoptosis for improving antitumor activity, the annexin V-FITC/PI assays were
employed to analyze early and late apoptosis in MDA-MB-231 cells. As displayed in
Figure 7, the control group with or without NIR laser irradiation both showed no
obvious apoptosis rate (7.8% and 5.83%, respectively), which indicated that the laser
irradiation exhibited negligible influence on MDA-MB-231 cells. Similarly, as for AC
group, the laser irradiation also generated a slight impact on apoptosis rates.
Nevertheless, AC/ICG-TNPs with laser irradiation induced 57.38% cell apoptosis,
while the apoptosis rate was only 36.71% when the MDA-MB-231 cells cultivated
with AC/ICG-TNPs without NIR irradiation. The remarkable elevated apoptosis rates
further demonstrated the significant photothermal and photodynamic damage on
MDA-MB-231 cells. Meanwhile, the apoptosis rate of AC was slightly higher than
that of AC/ICG-TNPs, which was consistent with the results of the MTT assay. The
apoptosis analysis data suggested that the AC/ICG-TNPs under NIR irradiation were a

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promising and effective strategy to induce the apoptosis of MDA-MB-231 cells.
Figure 7. Cell apoptosis of MDA-MB-231 cells after incubation with free AC and
AC/ICG-TNPs at an equivalent AC concentration of 60 ꢀM with or without NIR

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irradiation.
4. Conclusions
In summary, a novel anticancer drug AC was synthesized by conjugating ATRA
with HTCM for enhancing the drug toxicity of ATRA. Then an active targeted
AC-loaded nanoparticles encapsulating ICG dye was designed and prepared as the
vehicle for chemo/photothermal/photodynamic therapy. DLS and TEM showed that
the formed AC/ICG-TNPs had an appropriate diameter with uniform monodispersity.
In addition, it was demonstrated that AC/ICG-TNPs could release AC rapidly under
the mild acidic microenvironment, but it possessed good stability under the normal
physiological conditions. Meanwhile, the encapsulated ICG dye could efficiently
convert the NIR light into heat and generate ROS to accomplish the
photothermal/photodynamic therapy upon NIR irradiation. On the basis of the active
targeting mediated by overexpressed integrin αvβ3 on the breast cancer cells
MDA-MB-231, AC/ICG-TNPs could accumulate in tumor sites with high efficiency
and achieve the enhanced cellular uptake. Thus it exhibited the obvious inhibition for
cancer cells compared with AC/ICG-NPs. Furthermore, the cell apoptosis study
testified that AC/ICG-TNPs showed more effective antitumor performance against the
breast cancer cells after NIR irradiation due to chemo/photothermal/photodynamic
therapy, which was consistent with the results of MTT assay. Taken together, the
present results might afford a promising approach for the improved bioactivity of
ATRA and the site-specific combinational antitumor therapy.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of
China (Grant Nos. 21628101 and 21371031), the International S&T Cooperation
Program of China (No. 2015DFG42240), the Priority Academic Program
Development (PAPD) of Jiangsu Higher Education Institutions and the Technological
Program Foundation of Jiangsu Province (No. BY2015070-09).
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