Intratumorally Injected Photothermal Agent-Loaded Photodynamic Nanocarriers for Ablation of Orthotopic Melanoma and Breast Cancer

Article abstract of DOI:10.1021/acsbiomaterials.8b01111

Traditional chemotherapy of cancers may lead to serious adverse reactions due to little drug distribution in tumors. Here, a combination of photothermal therapy (PTT) and photodynamic therapy (PDT) was used for local treatment of orthotopic melanoma and breast cancer via intratumoral (i.t.) injection of photothermal agent-loaded photodynamic nanocarriers. A hydrophobic derivative of indocyanine green, DCC, was synthesized and entrapped into a pH-sensitive photosensitizer-core copolymer, PDCZP, to form DCC@PDCZP. The nanocarriers showed remarkable fluorescence, high singlet oxygen quantum yields, and a strong photothermal effect. Flow cytometry suggested that the nanocarriers were efficiently internalized by cancer cells. Near infrared thermal imaging and fluorescence self-imaging showed that the i.t. injected DCC@PDCZP mainly remained in the tumors, but the intravenous (i.v.) nanocarriers were distributed a little. One i.t. injection of DCC@PDCZP was enough to ablate the orthotopic B16-F10 and 4T1 mouse tumors under 830 and 660 nm irradiation at 4 h postinjection. More importantly, no local recurrences were found, though swabs were formed at 9 days post-treatment. The major anticancer mechanisms included improvement of cancer cell necrosis due to hyperthermia, inhibition of neovascularization, and enhancement of cell apoptosis. The i.t. injection of PTT/PDT nanoformulations is thus a promising local treatment of superficial tumors.

Full text of DOI:10.1021/acsbiomaterials.8b01111

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Controlled Release and Delivery Systems
Intratumorally Injected Photothermal Agent-Loaded Photodynamic
Nanocarriers for Ablation of Orthotopic Melanoma and Breast Cancer
Xiaodong Sun, Bo Zhuang, Mengmeng Zhang, Heliu Jiang, and Yiguang Jin
ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/
acsbiomaterials.8b01111 • Publication Date (Web): 01 Jan 2019
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Intratumorally Injected Photothermal
Agent-Loaded Photodynamic Nanocarriers for
Ablation of Orthotopic Melanoma and Breast
Cancer
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†
,‡
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*,†,‡
Xiaodong Sun, Bo Zhuang, Mengmeng Zhang, Heliu Jiang, and Yiguang Jin
†Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, 27
Taiping Road, Beijing 100850, China, e-mail address: jinyg@sina.com
‡Institute of Pharmacy, Pharmaceutical College of Henan University, Jin Ming
Avenue, Kaifeng 475004, China
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ABSTRACT: Traditional chemotherapy of cancers may lead to serious adverse
reactions due to little drug distribution in tumors. Here, a combination of
photothermal therapy (PTT) and photodynamic therapy (PDT) was used for local
treatment of orthotopic melanoma and breast cancer via intratumoral (i.t.) injection of
photothermal agent-loaded photodynamic nanocarriers. A hydrophobic derivative of
indocyanine green, DCC, was synthesized and entrapped into a pH-sensitive
photosensitizer-core copolymer, PDCZP, to form DCC@PDCZP. The nanocarriers
showed remarkable fluorescence, high singlet oxygen quantum yields, and strong
photothermal effect. Flow cytometry suggested that the nanocarriers were efficiently
internalized by cancer cells. Near infrared thermal imaging and fluorescence
self-imaging showed that the i.t. injected DCC@PDCZP mainly remained in the
tumors but the intravenous (i.v.) nanocarriers were distributed a little. One i.t.
injection of DCC@PDCZP was enough to ablate the orthotopic B16-F10 and 4T1
mouse tumors under 830 nm and 660 nm irradiation at 4 hours post-injection. More
importantly, no local recurrences were found though swabs were formed at 9 days
post-treatment. The major anticancer mechanisms included improvement of cancer
cell necrosis due to hyperthermia, inhibition of neovascularization, and enhancement
of cell apoptosis. The i.t. injection of PTT/PDT nanoformulations is thus a promising
local treatment of superficial tumors.
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KEYWORDS: breast cancer; indocyanine green; intratumoral injection; melanoma;
photodynamic therapy; photothermal therapy; zinc phthalocyanine
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INTRODUCTION
Nanocarriers are widely used as tumor-targeted delivery systems, and intravenous
i.v.) injection of them is a preferred option because the enhanced permeability and
(
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retention (EPR) effect is regarded as the basis of in vivo tumor targeting of nanoscale
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systems. However, the EPR effect is now seriously doubted because it has hardly
been validated in human bodies.2-3 Moreover, several marketed anticancer nanoscale
®
®
formulations (e.g., Doxil , Abraxane ) show little higher survival percentages than
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their free drug solutions. If nanoscale systems have no significant tumor targeting
effect, what’s the prospect of nanocarriers?
Cutaneous melanoma is a severe skin cancer with high incidence rates in western
countries and an estimated 76,380 new cases occurred in the United States in 2016,5
which is the leading cause of death in superficial tumors.6-7 Traditional treatments of
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melanoma include surgical resection, chemotherapy, and radiotherapy. Another
common type of superficial tumors is breast cancer that is the leading malignant
tumor in women with 1.7 million new cases worldwide in 2012. While significant
progress has been made in breast cancer diagnosis and early treatment, this
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malignancy still accounts for over half a million deaths yearly. Recurrence of breast
cancer in the front chest wall after mastectomy and radiotherapy poses a major
problem, as limited therapeutic options remain for clinical application.10 Oral
administration and i.v. injection are the major approaches to chemotherapy. However,
the amount of a drug that makes its way into the tissues of melanoma or breast cancer
is very limited due to wide drug distribution in the body. Moreover, currently
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clinically applied anti-melanoma or breast cancer drugs have no sufficient capacity to
differentiate cancer cells from normal cells, which leads to serious adverse reactions
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among patients. Therefore, an effective treatment with weak or without side effects
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is emergent for therapy of melanoma and breast cancer. Moreover, the monotherapy
of cancer generally does not produce satisfactory results so that a combination therapy
assumes new importance due to various action mechanisms of different treatments to
enhance anticancer efficiency and reduce side effect.12
Recently, photodynamic therapy (PDT) and photothermal therapy (PTT) have been
studied a lot due to their noninvasive property, no drug resistance, significant
therapeutic efficacy, low side effect, and manually control by light irradiation.13-15
PDT is a local treatment of diseases based on light, photosensitizers and oxygen.
Highly efficient PDT of cancer includes two key steps: photosensitizers enter cancer
cells and then are excited by an appropriate light. Reactive oxygen species (ROS) and
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singlet oxygen ( O) are produced and cancer cells are killed via apoptosis or
necrosis.16-17 Because singlet oxygen has a short half-life of 40 ns and a short
diffusion distance of 10−20 nm, cell uptake of the photosensitizers is critical to
PDT.18-19 Hyperthermia is an alternative way for efficient therapy for cancer and has
20
already been documented. As another phototherapeutic method, PTT is based on
photothermal agents which strongly absorb near infrared (NIR) light and convert it
into heat to destroy surrounding cancer cells, such as indocyanine green (ICG), gold
nanoparticles, or carbon nanotubes. PTT has been demonstrated as a noninvasive,
harmless and highly efficient therapeutic technique.21-22 In order to achieve the best
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effect of PTT, photothermal agents of an appropriate dose need to reach the tumor
tissues after systemic or local administration. High tumor targeting or distribution of
drugs is difficult to achieve by systemic administration, though a lot of nanocarriers
has been applied. Actually, only less than 1% of drugs may be delivered to tumor
tissues via nanocarriers.23 Against this context, intratumoral (i.t.) injection which
might increase the concentration of drugs in tumor tissues and reduce systemic
toxicity could be a better means of administration for PDT and PTT.24-25
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Here, a hydrophobic derivative of ICG was prepared as a photothermal agent,
which was entrapped into a pH-responsive zinc phthalocyanine copolymer
nanocarrier. Hydrophobic photosensitizers may be well entrapped in this copolymer.
However, the in vivo toxicity of most of them has not been enough explored, such as
quantum dots,26 polyaniline,27 polypyrrole,28 and so on. ICG is a not only
water-soluble clinically applied fluorescent contrast agent and but also acts as a
photothermal agent. Its in vivo safety has been ensured and approved by the FDA.29
However, ICG is not stable in solutions and is rapidly eliminated from the blood
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circulation with only 2−4 min of t , leading to limited clinical application. We
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synthesized the derivative of ICG, i.e., DCC, after introducing a chloride atom, a rigid
circle and an ethyloxycarbonylethyl group to the ICG fluorophore, to achieve cellular
toxicity, high stability, and hydrophobicity. Hydrophobicity makes it easy for DCC to
get entrapped in the cores of nanoparticles or polymeric micelles. PDCZP is a
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pH-sensitive copolymer synthesized in our lab. It can release the entrapped drugs in
the environment at low pH due to the hydrophobic-hydrophilic transition of the pDEA
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group. Moreover, PDCZP would likely release entrapped drugs in the low pH
environment of tumor tissues and from the lysosomes after internalization by cancer
cells. Therefore, the combination of DCC and PDCZP would likely take high
entrapment of photothermal agents, high stability, and rapid release in the tumors. The
drug-loaded nanocarriers were injected into the tissues of orthotopic mouse melanoma
and breast cancer. The i.t. injection of the nanocarriers resulted in ablation of
melanoma and breast cancer and no local recurrences were observed compared to the
control at 9 days post-injection. The highly efficient therapy was explored and the
mechanisms were analyzed.
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EXPERIMENTAL SECTION
Materials. The pH-responsive photosensitizer-core four-armed star-shaped
copolymer, i.e., [methoxy-poly(ethylene glycol)-poly(2-(N,N-diethylamino)ethyl
methacrylate)-poly(ε-caprolactone)] -zinc
β-tetra-(4-carboxyl
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(PDCZP) was synthesized in our previous research,31
benzyloxyl)phthalocyanine
except for the replacement of methoxy-poly(ethylene glycol) 2000 with
methoxy-poly(ethylene glycol) 750. A copolymer with a short PEG chain, i.e.,
(mPEG -pDEA-pCL) -ZnPC , PDCZP in this study, was finally obtained.
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Mitoxantrone chloride was purchased from Beijing Yikang Co., Ltd., China. Other
reagents, including 2,3,3-trimethylbenzoindolenine, ethyl 4-bromobutyrate (EBB) and
ICG, were purchased from Beijing Ouhe Technology Co., Ltd.
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,10-Dimethylanthracene (DMA) and zinc phthalocyanine (ZnPc) were from TCI
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(Tokyo, Japan). Organic solvents were of analytical grade and other chemicals were
of reagent grade. Fetal bovine serum, RMPI 1640, DMEM and trypsin-EDTA were
purchased from Gibco Life Technologies (USA). DAPI and Lyso-tracker red markers
were purchased from Beyotime Biotechnology (Shanghai, China). Purified water was
prepared using the Heal Force Super NW Water System (Shanghai Canrex Analytic
Instrument Co., Ltd., Shanghai, China). Dialysis bags were purchased from Union
Carbide Corporation (New Jersey, USA).
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Synthesis of the Photothermal Agent DCC. The photothermal agent, diethyl
chloro-cylcohexenyl cypate, i.e., DCC, was synthesized as follows (Figure S1 in the
supporting information). Two intermediates, 1 and 2, were prepared according to the
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literature with slight modifications. A solution of POCl (9.5 mL, 42.9 mmol) in
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dichloromethane (DCM, 19.5 mL) was slowly added to an ice-cooled solution of
N,N-dimethyl foramide (DMF, 10 mL, 103.2 mmol) in DCM (10 mL). Then,
cyclohexanone (2.5 g, 6.25 mmol) was added to the reaction solution and refluxed for
3
hours at 50 °C. The mixture was cooled with ice. Ice pieces (50 g) were slowly
added in the stirred mixture followed by agitation for 30 min. The DCM phase was
collected and the water phase was extracted with additional DCM. The DCM
solutions were combined and passed through an MgSO column. The solvent was
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removed from the solution under vacuum. The crude product was purified with a
silica gel column and an eluting solvent of DCM:MeOH (30:1, v/v), to obtain
2-chloro-1-formyl-3-(hydroxymethylene)cyclohex-1-ene (1) as a yellow crystalline
solid. Nuclear magnetic resonance (NMR) spectra and mass spectra were recorded
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using a JNM-ECA-400 NMR spectrometer and a Perkin-Elmer Sciex API-3000 MS
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spectrometer, respectively. H NMR (CDCl , 400 MHz) δ ppm: 1.66−1.74 (m, 2H,
3
CH ), 1.77−2.11 (m, 4H, 2CH ), 2.71 (s, 1H, CH), 2.92 (d, 2H, CH , J = 4.2 Hz), 3.02
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(s, 1H, OH), 10.02 (s, 1H, CH=O); ESI-MS (+), m/z: 173 (M+).
,3,3-Trimethylbenzoindolenine (0.74 g, 3.5 mmol) and EBB (3.7 g, 18.7 mmol)
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were together refluxed at 120 °C for 2 hours, and diethyl ether (300 mL) was added to
the system. The mixture was stirred at room temperature for 13 hours. The precipitate
was recrystallized from DCM and ethyl acetate. The crude product was purified
through a silica gel column by eluting with DCM:MeOH (30:1, v/v).
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-Ethyloxycarbonylethyl-2,3,3-trimethylbenzoindoleninium bromide (2) was obtained
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as a purple solid. H NMR (CDCl , 400 MHz) δ ppm: 1.24 (s, 6H, 2CH ), 1.87 (s, 3H,
3
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CH ), 2.05 (s, 2H, CH ), 2.70 (s, 2H, CH ), 3.27 (s, 3H, CH ), 3.49 (s, 2H, CH ), 4.14
3
2
2
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2
(
t, 2H, CH , J = 3.36Hz), 7.65−7.76 (m, 2H, ArH), 7.93−8.12 (m, 2H, ArH), ESI-MS
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(+), m/z: 324.20 (M+).
Compounds 1 (78.5 mg, 0.5 mmol), 2 (354 mg, 1.0 mmol) and sodium acetate
(41.0 mg, 0.5 mmol) were dissolved in acetic anhydride (10 mL) and stirred at room
temperature for 2 days. The dark green solution was poured into ethyl ether (200 mL)
and filtered. The collected precipitate was purified by the silica gel chromatography
(DCM:MeOH, 99:1−95:5, v/v) to obtain DCC as a dark green solid. The synthetic
procedure and NMR spectrum of DCC are in the supporting information.
UV-Vis Absorption and Fluorescence of DCC and PDCZP. DCC solutions in
methanol and PDCZP solutions in tetrahydrofuran (THF) were scanned in the range
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of 200−900 nm on a TU-1901 spectrophotometer (Beijing Purkinje General
Instrument Co., Ltd., Beijing, China). A DCC solution in dimethyl sulphoxide
(DMSO) was prepared with the absorbance of ~0.7 at the maximal wavelength of 835
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nm, and then 100-fold diluted with DMSO. The dilution was scanned using a
fluorescent spectrophotometer (Cary Eclipse, VARIAN, USA) to find the maximal
excitation and emission wavelengths, i.e., λex, λem.
Quantification of DCC, PDCZP and Mitoxantrone. A DCC-contained sample
was dissolved in methanol and DCC was determined using a spectrophotometry at
830 nm. PDCZP was a conjugate of three polymer blocks and a photosensitizer core,
i.e., PEG, PDEA and PCL and ZnPC . These polymer blocks had no absorption
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though ZnPC had a maximal absorption wavelength of 673 nm. Therefore, PDCZP
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was determined using a spectrophotometry at 673 nm after the sample was dissolved
in THF in the dark. Mitoxantrone was determined using the high-performance liquid
chromatography (HPLC) with reference to the China Pharmacopeia as follows: an
Agilent 1260 HPLC instrument, a Dikma Diamonsil C18(2) HPLC column (5 μm,
250 mm × 4.6 mm) at 30 °C, a mobile phase of acetonitrile/sodium
1
-hepatanesulphonate solutions (30:70, v/v) at the flow rate of 1 mL/min, the injected
volume of 20 μL, and the detection wavelength of 244 nm. A sodium
1-hepatanesulphonate solution was prepared by dissolving 2.2 g of sodium
1
-hepatanesulphonate into water, adding 3.2 mL of acetic acid, and diluting the
mixture to 365 mL with water. The retention time of mitoxantrone was about 24 min.
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Measurement of DCC Photostability. A DCC solution in methanol was prepared
with an absorbance in the range of 0.8−1.0. The photostability of DCC was measured
at 830 nm after continual 250 mW/830 nm light irradiation on the DCC solution with
an interval of 2 min up to 32 min. An 830 nm diode laser emitter (Lasever Inc.,
Ningbo, China) was used. Light intensities were measured using Nova II (OPHIR,
Israel).
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Fluorescence Quantum Yield Measurement of DCC and PDCZP. The area
under the fluorescence emission spectra of DCC and ICG solutions in DMSO was
integrated as SDCC and SICG, respectively. Their absorbance at 835 nm was also
measured, as ADCC and AICG, respectively. The fluorescence quantum yield (ΦF/ICG) of
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was calculated as Eq. (1).34
ICG in DMSO was 0.12. Therefore, the Φ
F/DCC
SDCC AICG
F / DCC  0.12
(1)
ADCC SICG
A PDCZP solution in DMF was prepared with the absorbance of about 0.7 at 673
nm. It was 100-fold diluted with DMF and the fluorescence quantum yield (ΦF/PDCZP
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of PDCZP was measured as Eq. (2) with the ΦF/ZnPc of 0.30.34
SPDCZP AZnPc
F / PDCZP  0.30
(2)
APDCZPSZnPc
Singlet Oxygen Quantum Yield Measurement of PDCZP. The singlet oxygen
quantum yield (Φ) of PDCZP in DMF was measured with the same method in our
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previous research. The test was based on the changes of a singlet oxygen chemical
quencher, 9,10-dimethylanthracene (DMA, TCI, Tokyo, Japan) in DMF. A 100
mW/660 nm laser emitter (YSHINELASER, China) was used. Briefly, light
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irradiation was conducted on a series of solutions containing ZnPc, PDCZP, and/or
DMA. Singlet oxygen quantum yields (Φ) of photosensitizers were calculated
according to DMA oxidation rates. ΦPDCZP was calculated after comparison with the
known ΦZnPc (0.56) of ZnPc in DMF.
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Preparation of DCC@PDCZP. DCC and PDCZP of 1:2 (w/w) were dissolved in
THF and water was injected into the solution under bath ultrasound with a 100-μL
syringe. The organic solvent in the suspension was removed at 37 °C under vacuum to
obtain a DCC@PDCZP suspension containing 250 μg DCC/mL. A PDCZP
suspension and a fluorescein isothiocyanate (FITC)@PDCZP (1:5, w/w) suspension
were also prepared as above.
Photothermal Effect of DCC and DCC@PDCZP. A 250 μg/mL DCC solution
in DMSO was prepared and diluted with water to 1, 2, 5, 10, 25, 50 and 100 μg/mL.
The diluted solutions remained stable within 5 hours without obvious agglomerates.
An aliquot (0.5 mL) of the above solutions was put into a 1.5-mL plastic centrifuge
tube. Vertical laser irradiation was performed on the tube with 830 nm and 1 W/cm2.
The sample temperature was measured with the temperature meter attached to a pH
meter (HI2221, HANNA, GE) at 0, 30, 60, 120, 150, 180, 210, 240, 270 and 300 s. A
DCC@PDCZP suspension in water was prepared, containing 250 μg DCC/mL. It was
diluted with water to the same concentrations as the above DCC dilutions. The
photothermal effect of DCC@PDCZP was also measured as above.
Transmission Electron Microscopy, Size and Zeta Potential of PDCZP and
DCC@PDCZP. PDCZP and DCC@PDCZP were observed under a Hitachi H-7650
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80 kV transmission electron microscope (TEM) after staining with sodium
phosphotungstate solutions (pH 7.4). A dynamic light scattering (DLS) method on
Zetasizer Nano ZS (Malvern, UK) was used for measuring the sizes of PDCZP and
DCC@PDCZP at different pH. The zeta potential of PDCZP and DCC@PDCZP was
also measured using the above instrument at 25 °C.
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Encapsulation and Loading Efficiencies of DCC in DCC@PDCZP.
DCC@PDCZP suspensions were centrifuged through ultrafiltration tubes (cutoff
MW, 10,000, PALL Corporation, USA) at 5,000× g for 10 min. Free DCC in the
filtrate was determined on the UV-vis spectrophotometer at 830 nm. The
encapsulation efficiency (EE) and loading efficiency (LE) of DCC were calculated as
Eq. (3) and (4).
EE = (Encapsulated DCC)/(Totally added DCC)×100%
(3)
(4)
LE = (Encapsulated DCC)/(Total amount of DCC@PDCZP)×100%
Release of DCC from DCC@PDCZP. An aliquot (1 mL) of DCC@PDCZP
suspensions (250 μg DCC/mL) was put into a dialysis bag (cutoff MW, 3,500) that
was then immersed in 10 mL of 37 °C phosphate buffered solutions (PBS, pH 7.4,
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.5, or 5.0) containing 8% Tween 80. The dissolution system was incubated at 37 °C
under vibration at 150 rpm. At the predetermined time points, 1 mL of the media was
withdrawn for the spectrophotometric assay of released DCC at 830 nm after dilution
with methanol (1 mL) and ultrasound for 5 min. An equal volume of media was
supplemented into the media at the same time.
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Hemolysis Assay of DCC and PDCZP. Rat erythrocyte suspensions (2%, v/v) in
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saline were prepared according to the report. A series of samples were prepared,
containing 1% erythrocytes and a series of PDCZP or DCC suspensions including 5,
4, 3, 2 and 1 mg/mL in saline and they were incubated at 37 °C until observation.
Hemolysis was observed with naked eyes against the completely hemolytic sample of
water-mixed erythrocytes. Three hours later, the supernatants were also collected and
the absorbance at 577 nm was measured.
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Phototoxicity of light irradiation at 830 nm and 660 nm. B16-F10 mouse
melanoma cells and 4T1 mouse breast cancer cells were used. Cells were grown in the
DMEM (for B16-F10 cells) or RMPI 1640 (for 4T1 cells) culture media containing
10% fetal bovine serum at 37 °C under 5% CO . The B16-F10 or 4T1 cells were
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seeded into 96-well plates (5×10 cells/well). Phototoxicity on cells was assayed by
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directly light irradiation at 830 nm (1 W/cm ) or 660 nm (200 mW/cm ) for 30, 60,
90, 120, 240 and 300 s. The cells were incubated at 37 °C for 24 hours. The cell
viability of B16-F10 and 4T1 cells was measured using the MTT method. An aliquot
(20 μL) of MTT solutions was added to each well. The plates were maintained at 37
°
C for 4 hours. The culture media were discarded, and DMSO (150 μL) was added to
dissolve dark-blue formazan crystals. Formazan was measured at 490 nm using a
microplate reader (Mul-tiskan MK3, Thermo Scientific, US). Cell viability was equal
to (absorbance of sample)/(absorbance of control) × 100%.
Dark Cytotoxicity of DCC, PDCZP and Mitoxantrone. The dark toxicity of
regimens was evaluated on B16-F10 cells that were seeded into 96-well plates (5×103
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cells/well) and cultured overnight. Cells were incubated with a series of PDCZP
(25−200 μg/mL), DCC (0.5−200 μg/mL), and mitoxantrone (0.5−200 μg/mL)
solutions for 24 hours under darkness, respectively. Cell viability was measured using
the MTT method.
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Cell incubation Time Under Darkness. The incubation time under darkness
before photodynamic treatment was investigated. B16-F10 or 4T1 cells were seeded
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into 96-well plates (5×10 cells/well) and incubated for 24 hours. The media were
replaced with a series of DCC or DCC@PDCZP suspensions containing 5−200 μg
DCC/mL, and incubated for 1, 2, 3 and 4 hours, respectively. The cell viability was
measured using the MTT method.
In Vitro Photodynamic and Photothermal Studies of DCC and DCC@PDCZP.
A series of DCC or DCC@PDCZP suspensions were prepared with 20−200 μg
DCC/mL and a series of PDCZP suspensions were prepared with 20−200 μg
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PDCZP/mL. B16-F10 or 4T1 cells were seeded into 96-well plates (5×10 cells/well)
and incubated for 24 hours. The media were replaced with/without the above DCC,
PDCZP and DCC@PDCZP suspensions, respectively. Two hours later, the wells
were light-irradiated or not in the following groups: non-irradiation, DCC, DCC with
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30 nm irradiation, PDCZP, PDCZP with 660 nm irradiation, 830 nm and then 660
nm irradiation, DCC@PDCZP, DCC@PDCZP with 830 nm and 660 nm irradiation,
and blank control. The irradiation conditions included: (1) 830 nm and 1.0 W/cm2
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irradiation for 3 min; (2) 660 nm and 200 mW/cm irradiation for 3 min; (3) continual
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irradiation using (1) and (2) by turns. After irradiation, the cells were incubated for 1
hour under darkness. Cell viability was measured using the MTT method.
In Vitro Cellular Uptake of DCC and DCC@PDCZP and Its Localization in
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cells. B16-F10 mouse melanoma cells (1×10 cells/well) were seeded in 6-well plates
and incubated at 37 ºC for 1 h. An aliquot (100 µL) of FITC@PDCZP was added to
the wells. After incubation for 1, 2, 4, 8 and 12 hours, the cells were washed 3 times
with PBS, processed with trypsin, centrifuged at 4 °C, and finally re-suspended in 0.5
mL of PBS. The fluorescent intensities of FITC in cells were analyzed using a flow
cytometry (BD FACS Calibur, USA). In addition, the cellular uptake of
DCC@PDCZP and free DCC in DMSO (both of them containing 10 µg DCC/mL)
was also investigated after incubation with the cells for 4 hours. The cells were
washed with PBS and homogenated using an ultrasonic homogenizer (80W, HUP-100,
Tianjin Hengao Technology Development Co., Ltd., China) with the power at the 2nd
grade for 20 s (1s each time with 1 s interval). The homogenates were thoroughly
mixed with methanol (3 mL/well) and centrifuged at 5,000 rpm for 5 min. DCC in the
supernatants was determined using a spectrophotometry at 830 nm. The
cell-internalized DCC was calculated.
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The 4T1 cells (2×10 cells/well) were seeded in 35-mm dishes with coverslips at
the bottoms and incubated at 37 °C in 5% CO for 24 hours. The culture media were
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replaced with the DCC@PDCZP suspensions containing 40 μg PDCZP/mL and the
cells were incubated for another 4 hours. The cells were irradiated using the light of
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30 nm and 1.0 W/cm for 3 min or not. The cells on the coverslips were washed three
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times with PBS, stained by the Lyso-tracker red marker for 30 min, fixed by 4%
paraformaldehyde for 15 min, and then stained using DAPI for 5 min. The cells were
washed thoroughly with PBS and mounted on glass slides. The images of cells were
taken using a Nikon TiE-A1 confocal laser scanning microscope (CLSM).
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Animal Models of Orthotopic Melanoma and Breast Cancer. Female BALB/c
nude mice (18−20 g) were purchased from the SPF (Beijing) Biotechnology Co. Ltd,
China. The handling and surgical procedures of animals were conducted in strict
accordance with the Guide for the Use of Laboratory Animals of Beijing Institute of
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Radiation Medicine. B16-F10 cells (0.1 mL, 1×10 cells) or 4T1 cells (0.1 mL, 1×10
cells) were inoculated into the mice under the right forelimb by subcutaneous (s.c.)
injection. Seven days post-inoculation, the mice with an appropriate tumor volume
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ca. 200 mm ) were selected for the studies of tissue distribution and
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pharmacodynamics. Tumor volume (V) was calculated as 푉 = 푎 × 푏 2, where a was
the length of tumor, and b was the width of tumor.
Tumor Temperature Measurement under Light Irradiation. B16-F10 or 4T1
tumor-bearing nude mice were each i.t. injected with 100 μL of DCC@PDCZP
(containing 50 μg DCC/mL). Tumor-bearing mice without treatment were used as the
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control. The tumors were irradiated under 830 nm and 1.0 W/cm laser for 3 min.
Regional infrared thermographic maps were obtained using an infrared thermal
imaging camera (THERMOVISION A40, FLIR, USA) and the temperature was
deduced.36
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In Vivo Imaging of DCC@PDCZP. B16-F10 or 4T1 tumor-bearing nude mice
were randomly divided into two groups (two mice per group), wherein two mice were
i.v. injected with 100 μL of DCC@PDCZP (containing 50 μg DCC/mL) and the
others were i.t. injected. Fluorescence imaging of the B16-F10 tumor-bearing mice
was conducted at 0.5, 1, 2, 4, 6, 8 and 12 hours post-injection using an in vivo
imaging system (IVIS Lumina, USA) at an excitation wavelength of 745 nm and an
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20 nm filter to collect the fluorescence (FL) signals of DCC. The 4T1 tumor-bearing
mice were also treated as above up to 24 h.
In Vivo Photothermal and Photodynamic Therapy of PDCZP, DCC, and
DCC@PDCZP. Thirty-six B16-F10 tumor-bearing mice were equally divided to nine
groups. The mice in Group a served as controls that were only i.t. injected with PBS.
The mice in Groups b, c, d and e were i.t. injected with PDCZP, DCC, DCC@PDCZP
and mitoxantrone, respectively. The mice in Groups a−e were not irradiated. The mice
in Groups f−i were light-irradiated while the mice in Group f were i.t. injected with
PDCZP and then irradiated with 660 nm laser. The mice in Group g were i.t. injected
with DCC and then irradiated with 830 nm laser. The mice in Group h were injected
with DCC@PDCZP, and then irradiated with 830 nm and sequential 660 nm laser one
hour later. The mice in Group i were irradiated with 830 nm and then 660 nm laser as
the same as Group h but without any administered regimens. All the laser zones
completely covered the tumor xenografts. For Groups a−e, an aliquot (100 μL) of
various regimens, including saline, PDCZP (680 μg/mL), free DCC (100 μg/mL
aqueous suspensions), DCC@PDCZP (containing 100 μg/mL DCC and 680 μg/mL
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PDCZP), and mitoxantrone (200 μg/mL) was i.t. injected on Days 1, 3, 5 and 7. For
Groups f−h, the i.t. injection and light irradiation at 4 hours post-injection were
performed only once, where the mice in Group f underwent the 660 nm and 200
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mW/cm irradiation for 5 min only, those in Group g underwent the 830 nm and 1.0
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W/cm irradiation for 3 min, and those in Group h underwent the above 830 nm and
660 nm irradiation in turn. The mice in Group i underwent the above 830 nm and 660
nm irradiation only once without injection of any regimens. The mice were finally
sacrificed 9 days post-injection. A pharmacodynamic study of 4T1 tumor-bearing
mice was also conducted as above. However, some 4T1 tumor-bearing mice died in
the middle stages of experiments. The remaining mice were sacrificed 8 days
post-injection.
Histopathology and Immunohistochemistry. To further explore the anticancer
mechanisms of PTT and PDT, and their synergistic effect, the excised tumors or scabs
were processed into sections that were stained with hematoxylin and eosin (HE) as in
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our previous research. Briefly, the excised tissues were fixed with 10% formalin
solutions and embedded in paraffin. Sections 5-mm thick were made, HE stained, and
observed under a microscope. Moreover, the tissue sections were dewaxed,
rehydrated, and washed with phosphate buffer solutions (PBS), and endogenous
peroxidase was inactivated with 3% hydrogen peroxide (H O ) for 15 min at room
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temperature. S100 and CD31 in the tissues were measured with the measurement kits
(Wuhan GoodBio Technology Co., Ltd., China) according to the instructions.38-39 The
sections were examined under a microscope. Apoptosis assay was also performed as
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in our previous research.38-39 Briefly, a TUNEL (Roche, Switzerland) assay method
was performed on the sections and incubated for 1 hour at 37 °C. They were
incubated with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature
to detect nucleoli after PBS washing. Images of TUNEL and DAPI fluorescence were
recorded using a fluorescent microscope.
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Statistical Analysis. The results were calculated statistically using the SPSS 16.0
software. All the data was expressed as mean ± standard deviation (SD). One way
analysis of variance (ANOVA) with the LSD test was used to identify differences
(p<0.05 or p<0.01) between the data.
RESULTS AND DISCUSSION
Characteristics and Photostability of DCC. DCC was a dark green solid and its
1
structure was proved by H NMR (Figure S2 in the supporting information). It has a
rigid structure compared to ICG (Figure 1A). The DCC solution in methanol
exhibited a maximal visible absorption wavelength at 830 nm (Figure 1B). The long
absorption wavelength was likely to enhance the biological penetration depth of light
and decrease the interference of Raman scattering, favoring phototherapy in the
body.40-41 More importantly, DCC had strong photostablility with long
photobleaching time. Only 35% reduction of the maximal absorbance of DCC
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solutions occurred after 830 nm and 250 mW/cm irradiation for 32 min (Figure 1C),
indicating a remarkable resistance to photobleaching. Hence, DCC could stay stable
after long-term light irradiation in the next photothermal treatment.
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Figure 1. Molecular structures of ICG and DCC (A), and properties of DCC. (B) the
UV-vis spectrum of DCC solutions in methanol. (C) Visible absorption spectral
changes of DCC solutions in methanol with time dependency. (D) Fluorescent
excitation and emission spectra of DCC solutions in DMSO. (E) Temperature profiles
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of DCC aqueous suspensions after 830 nm and 1.0 W/cm light irradiation with
concentration dependency.
Fluorescent Spectra and Fluorescence Quantum Yield of DCC. The fluorescent
spectrum of DCC solutions in DMSO exhibited a maximal emission wavelength of
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30 nm under the excitation at 750 nm while its excitation spectrum covered a range
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from 610 nm to more than 810 nm with the peaks at 750 and 794 nm (Figure 1D).
The 750-nm laser was selected to excite DCC because no emission lights appeared at
this wavelength. The fluorescence quantum yield (ΦF/DCC) of DCC in DMSO was 0.28,
whereas ICG just had a much lower fluorescence quantum yield of 0.12 than that of
DCC. The fluorescence quantum yields of photosensitizers were related to their
chemical structures. In general, the rigid structure in the molecule would likely
increase its fluorescence quantum yield.42 Compared to ICG, a major molecular
feature of DCC is a rigid bridge ring that is the reason of the high fluorescence
quantum yield. Therefore, DCC had much stronger fluorescence emission ability than
the clinically applied fluorescence imaging agent, ICG. The long fluorescent emission
spectral wavelengths and high fluorescence quantum yield of DCC suggested that
DCC would become a promising fluorescence imaging agent.
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Strong Photothermal Effect of DCC. Light irradiation remarkably increased the
temperature of DCC aqueous suspensions in a concentration-dependent manner
(Figure 1E). The temperature of a 50 μg/mL DCC suspension rapidly increased from
20 °C to 41.3 °C with a 21.3 °C increase following 150 s light irradiation at 830 nm
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and 1.0 W/cm , while the temperature of the irradiated water hardly changed.
Therefore, DCC had the strong photothermal effect with high temperature above 40
°C after a short time irradiation. However, a little decrease of temperature happened
in the late stages. In the above case, long-time irradiation would leave to temperature
decreasing. For example, after 300-s irradiation, the temperature of solutions
decreased from the top of 41.3 °C to 32.5 °C, i.e., 41% temperature decreasing. The
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experiments were conducted in winter and the room temperature was about 20 °C.
Therefore, the heat loss to the environment could not be neglected. In the other cases,
the maximal temperature points of DCC suspensions depended on concentrations and
the heat loss was unavoidable (Figure 1E, Figure S3). An appropriate concentration
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(100 μg/mL DCC) of DCC@PDCZP and the 830 nm irradiation time of 3 min were
selected for the in vivo pharmacodynamic study. Hyperthermia would lead to
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irreversible damage to the subjected cells, such as cancer cells, so that the strong
photothermal effect of DCC would benefit the treatment of cancer.
Characteristics of PDCZP. PDCZP is a star-shaped copolymer with a zinc
phthalocyanine core and four long arms (Figure 2A). Its solution in THF had the
maximal absorption wavelength of 673 nm (Figure 2B), while the solution in DMF
had the maximal fluorescent excitation wavelength of 666 nm and the maximal
emission wavelength of 681 nm (Figure 2C). The fluorescence quantum yield
(ΦF/PDCZP) of PDCZP was 0.25, a little lower than that (ΦF/ZnPc, 0.30) of its parent
photosensitizer, ZnPc. The singlet oxygen quantum yield (ΦPDCZP) of PDCZP was
0.47, close to that (0.41) of another PDCZP analogue (PEG2000-PDCZP), and a little
lower than that (0.56) of its parent ZnPc and that (0.64) of another amphiphilic
derivative of ZnPc (ZnPc-Brij 58 conjugate, ZPB) previously prepared in our lab.31, 44
The long PDC chains of PDCZP might shield energy transportation or oxygen
diffusion, leading to a decrease of Φ. PDCZP formed a nanoscale particle in water
with a mean hydrodynamic size of 60 nm and a polydispersive index (PDI) of 0.264.
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PDCZP was positively surface-charged in water (neutral, about pH 7) with the zeta
potential of 18.57 mV due to its amine-contained pDEA chains.
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Figure 2. Properties of PDCZP. (A) Molecular structure of PDCZP. (B) The UV-vis
absorption spectrum of PDCZP solutions in THF. (C) Fluorescent excitation and
emission spectra of PDCZP solutions in DMF.
Design and Preparation of DCC@PDCZP. PDCZP is a pH-sensitive copolymer
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nanocarrier, prepared by us and reported previously. Doxorubicin was loaded in the
nanocarriers that expressed the significant in vivo anti-tumor treatment based on their
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long-circulating and pH-sensitive properties after i.v. administration. Moreover, the
pH-sensitive property of PDCZP benefits not only the pH-responsive release of drugs
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in tumors but also the stable entrapment of drugs. Here, we used PEG 750 to replace
previous PEG 2000 to prepare PDCZP because the short PEG chains were satisfied
for preventing the nanocarriers from aggregation with high stability. The pH-sensitive
chain, i.e., pDEA, did not change in this study because its high pH-sensitive property
had been confirmed in our previous research.31, 45-46 Moreover, the DCC@PDCZP
was i.t. injected in this study, leading to the nanocarriers directly exposed to the low
pH environment of tumors and the pH-responsive merit well expressed.
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The injection method was used for preparation of DCC@PDCZP that appeared as
a homogeneously dark green solution that remained unchanged in appearance within
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several days at room temperature. Unlike the usually applied injection method, here
water was slowly injected into the DCC/PDCZP solution in THF. This preparation
process could gradually make the whole system changing from a organic phase state
to a water phase state so that DCC molecules might have sufficient time and space to
enter the hydrophobic core of PDCZP nanocarriers. DCC@PDCZP composed of 1:2
(w/w) DCC/PDCZP had the relatively high EE (65.9%) and LE (25.6%).
pH-Responsive Properties of DCC@PDCZP. The pH-response of anticancer
agent-loaded nanocarriers was expected to facilitate the quick release of the loaded
drugs at the tumor tissues due to low pH.47 PDCZP has been proved to be a
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pH-responsive nanocarrier in our previous work. Here DCC@PDCZP also showed
some pH-responsive properties involving size, zeta potential and DCC release
(Figure 3A, 3B). The pH-sensitive pDEA chain in PDCZP was responsible for this
response.31, 45-46 The lower the pH was, the larger DCC@PDCZP became. Its mean
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size was 126.6, 96.76 and 68.07 nm at pH 5.0, 6.5 and 7.4, respectively (Figure 3A).
The change of sizes was also shown by TEM (Figure 3A). Interestingly,
DCC@PDCZP was positively stained by sodium phosphotungstate at low pH due to
the positive surface charge, where the strong absorption of phosphotungstate on the
surface of PDCZP led to an apparently large size. Furthermore, the surface charge of
DCC@PDCZP changed from the positive zeta potential of 44.2 mV and 28.9 mV at
pH 5.0 and 6.5 to the negative zeta potential of −17.9 mV at pH 7.4 (Figure 3A).
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Figure 3. Properties of DCC@PDCZP. (A) Size and zeta potential of DCC@PDCZP
and the corresponding TEM images with pH dependency (n = 3). (B) DCC release
from DCC@PDCZP with pH dependency (n = 3). (C) Temperature profiles of
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DCC@PDCZP suspensions after 830 nm and 1.0 W/cm light irradiation with
concentration dependency.
DCC@PDCZP is positively-charged at the low pH environment of tumor tissues
so that it could be likely to interact with surrounding cancer cells as i.t. injection of
these carriers. Moreover, once DCC@PDCZP was internalized into the endosomes
and then lysosomes that were generally at pH 5.0 or lower, its highly positive charge
state (i.e., 44.2 mV) would lead to the so-called “proton sponge” effect and then
membrane damage of lysosomes and cargos release to cell plasma,48 favoring
subsequent PTT and PDT.
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DCC release from DCC@PDCZP was also highly dependent on pH (Figure 3B).
pH 7.4, 6.5 and 5.0 may represent the environments of blood circulation and normal
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tissues, tumor tissues and cellular lysosomes, respectively. Here DCC release from
DCC@PDCZP was very little at pH 7.4, but highly improved at pH 6.5 and 5.0,
favoring the treatment of cancer. Our previous studies and other reports showed a
similar conclusion.31, 45-46 More importantly, unlike other research, we performed the
i.t. injection of DCC@PDCZP directly into tumor tissues so that little drug was
wasted.
High Photothermal Effect of DCC@PDCZP. DCC@PDCZP showed the similar
photothermal behavior to DCC, also depending on time and concentrations (Figure
3C). For example, a 50-μg/mL DCC@PDCZP suspension underwent a rapid increase
of temperature from 20 °C to 41.6 °C after 150-s light irradiation at 830 nm and 1.0
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DCC@PDCZP suspensions of different concentrations also showed similar profiles
(Figure S4). However, there was a special case. A 250 μg/mL DCC@PDCZP
suspension maintained a profile of continual temperature increase despite the long
time irradiation for 300 s. Therefore, unlike free DCC, DCC@PDCZP maintained
high temperature for a long time post-irradiation, which might be attributed to
PDCZP-self hindrance of thermal diffusion or heat loss. The long-term maintenance
of hyperthermia should favor ablation of tumors.
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No Hemolysis by DCC and DCC@PDCZP. Neither free DCC nor PDCZP
showed any hemolysis within 3 hours after incubation with erythrocytes even when
the concentration of DCC or PDCZP was up to 5 mg/mL. All these samples’
supernatants showed nearly zero absorbance at 577 nm. Therefore, DCC@PDCZP
had no hemolytic effect and the safety of its injection was ensured. The results of
hemolytic experiments were shown in Figure S5, Table S1 and Table S2 in the
supporting information.
High Dark Cytotoxicity of DCC and Little Dark Cytotoxicity PDCZP. We
detected the effect of light irradiation on the growth of mouse melanoma B16-F10
cells and mouse breast cancer 4T1 cells. The light irradiation, including 830 nm (1
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W/cm ) and 660 nm (200 mW/cm ), did not affect the growth of the two types of cells
even though the irradiation time was extended to 5 min. The cells always kept a high
viability of more than 95%. Dark cytotoxicity of the photosensitizer PDCZP, the
photothermal agent DCC and the anticancer chemotherapeutic mitoxantrone was also
investigated. Mitoxantrone is a synthetic anthraquinone chemotherapeutic drug that
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can intercalate with DNA to inhibit their synthesis and transcription. Mitoxantrone is
used to treat certain types of cancer, including breast cancer and melanoma. The in
vitro/in vivo anti-melanoma effect of mitoxantrone was reported a lot.50-53 B16-F10
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cells showed different viability depending on the types and concentrations of
regimens (Figure 4A). The half inhibitory concentrations (IC ) of PDCZP, DCC and
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mitoxantrone under darkness were 409, 10.3, and 1.46 μg/mL, respectively. Therefore,
PDCZP had hardly any dark cytotoxicity, while DCC and mitoxantrone had a
relatively strong dark cytotoxicity though the effect of mitoxantrone was higher
(Figure 4A). However, these values were obtained at 24 hours post-incubation. In fact,
DCC could not maintain a high concentration for such a long time at the in vivo
targeted site. Therefore, the dark incubation time was further investigated for DCC
and DCC@PDCZP.
Figure 4. Dark cytotoxicity of PDCZP, DCC and mitoxantrone on B16-F10 cells
depending on concentration (A), and DCC and DCC@PDCZP on B16-F10 (B, D) and
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T1 cells (C, E) depending on time and concentration. The dark cytotoxicity of DCC
and mitoxantrone was compared (A). N = 4, * p < 0.05, ** p < 0.01.
Both DCC and DCC@PDCZP showed the similar dark cytotoxicity on B16-F10
and 4T1 cells (Figure 4B−E). Two hours post-incubation, the viability of all the cells
was close to or above 80%, even with highly concentrated DCC (e.g., 100 μg/mL).
However, the growth of cells was highly inhibited by the highly concentrated samples
at 3 hours post-incubation, such as 100 μg/mL DCC, and the cell viability was only
less than 50%. Therefore, phototherapy should be initiated at 2 hours post-dark
incubation.
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High In Vitro Anticancer PDT/PTT Effect of DCC@PDCZP. The in vitro
anticancer PDT/PTT effect of DCC@PDCZP was explored in depth on B16-F10 and
4T1 cells. Compared to the regimens under darkness, the therapeutic effect was highly
enhanced by both the PDT of PDCZP at 2 hours post-incubation and the PTT of DCC,
and more importantly, the synergistic PDT/PTT of DCC@PDCZP (Figure 5).
Moreover, the synergistic PDT/PTT effect of DCC@PDCZP was always the highest
one among all the groups at various concentrations. Even at a low concentration of 20
µg/mL DCC, DCC@PDCZP still showed a high cytotoxicity with the cell viability of
less than 30% under light irradiation, whereas the PTT of DCC showed the cell
viability just close to 70% at the same concentration (Figure 5). The IC of PDCZP
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under 660 nm irradiation and DCC@PDCZP under 830 nm irradiation was 46.13
µg/mL and 41.42 µg/mL for B16-F10 cells, respectively. However, the IC50 of
DCC@PDCZP under 830 nm/660 nm irradiation was as low as 7.92 µg/mL. The
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