NIR organic dyes based on phenazine-cyanine for photoacoustic imaging-guided photothermal therapy

Article abstract of DOI:10.1039/C8TB01750F

As non-invasive diagnosis and therapy methods, photoacoustic (PA) imaging and photothermal therapy (PTT) have attracted extensive attention. Herein, two new acceptor-donor-acceptor near-infrared organic phenazine-cyanine dyes PH-1 and PH-2 were reported for photoacoustic imaging-guided photodynamic therapy. In the strong donor phenazine molecule, the electron-withdrawing indole salt unit was introduced for absorption to the near-infrared region. To improve water solubility, the two organic dyes were assembled with human serum albumin (HSA) to form nanoparticles of appropriate sizes, i.e., PH-1@HSA and PH-2@HSA, which showed excellent stability in both weakly acidic and weakly basic environments. Moreover, the results showed that PH-1@HSA and PH-2@HSA nanoparticles can effectively transform luminous energy to thermal energy in vitro and in vivo, and they can be utilized for PA imaging. Importantly, PH-1@HSA can accumulate in mice subcutaneous tumors by enhanced permeability and retention (EPR) and damage cancer tissues effectively.

Full text of DOI:10.1039/C8TB01750F

Journal of
Materials Chemistry B
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PAPER
NIR organic dyes based on phenazine-cyanine
for photoacoustic imaging-guided photothermal
therapy†
Cite this: J. Mater. Chem. B, 2018,
6, 7420
Yongchao Yan,a Jiawen Chen,b Zhijuan Yang,b Xiao Zhang,a Zhuang Liu *b and
a
Jianli Hua
*
As non-invasive diagnosis and therapy methods, photoacoustic (PA) imaging and photothermal therapy
(PTT) have attracted extensive attention. Herein, two new acceptor–donor–acceptor near-infrared
organic phenazine-cyanine dyes PH-1 and PH-2 were reported for photoacoustic imaging-guided
photodynamic therapy. In the strong donor phenazine molecule, the electron-withdrawing indole salt
unit was introduced for absorption to the near-infrared region. To improve water solubility, the two
organic dyes were assembled with human serum albumin (HSA) to form nanoparticles of appropriate
sizes, i.e., PH-1@HSA and PH-2@HSA, which showed excellent stability in both weakly acidic and weakly
basic environments. Moreover, the results showed that PH-1@HSA and PH-2@HSA nanoparticles can
effectively transform luminous energy to thermal energy in vitro and in vivo, and they can be utilized for
PA imaging. Importantly, PH-1@HSA can accumulate in mice subcutaneous tumors by enhanced
permeability and retention (EPR) and damage cancer tissues effectively.
Received 4th July 2018,
Accepted 4th October 2018
DOI: 10.1039/c8tb01750f
rsc.li/materials-b
high spatial resolution of ultrasound imaging.6 Compared with
traditional optical imaging, PA imaging provides deeper tissue
Introduction
Nowadays, cancer has become one of the major threats to penetration and higher spatial resolution in vivo because ultra-
human health; thus, it is urgent to develop effective methods sound scatters to a much lesser extent relative to light while
for diagnosis and therapy.1 Chemotherapy is the dominant passing through tissues.
measure to treat cancer; nevertheless, most of the conventional
PTT kills cancer cells by employing agents that convert
chemotherapeutic drugs as single agents have not satisfied luminous energy to thermal energy,7 and it also has some merits
clinical requirements because of multidrug resistance (MDR) including non-invasiveness, low side effects, high selectivity and
and toxic side effects.2 In addition, due to other common no resistance.8–10 The current NIR agent materials include gold-
methods, such as radiotherapy and surgery, cancer patients nanomaterials,11,12 carbon nanomaterials,13 transition-metal
usually suffer pain during the treatment. Recently, noninvasive dichalcogenides,14 organic polymers15,16 and NIR dyes.17 Com-
and mild methods for diagnosis and therapy such as photo- pared to inorganic materials, organic PTT agents show some
acoustic (PA) imaging and photothermal therapy (PTT) have superiorities such as regulable absorption spectrum, biodegrad-
attracted wide attention due to minimal harm to normal tissues ability and rapid elimination in biological tissues.18,19 Due to
and effective tumor lethality.3–5 PA imaging is a non-invasive these reasons, it is essential to design and synthesize NIR
technique that gathers photo-induced ultrasound signals from organic dyes for PA imaging and PTT.
a pulsed laser irradiating tissue, combining high-contrast
In previous studies, PTT agents based on NIR dyes were
and spectroscopic-based specificity of optical imaging with almost cyanine,20 BODIPY,21,22 diketopyrrolopyrrole (DPP)23,24
and benzobisthiadiazole25,26 derivatives. These dyes usually
exhibit a donor–acceptor–donor (D–A–D) structure, in which
BODIPY, DPP and benzobisthiadiazole are electron-withdrawing
groups attached to different electron donors. Stronger donor
group and acceptor group can decrease the energy gap between
HOMO and LUMO, which causes a red shift.27,28 However,
reports on A–D–A structured NIR dyes for photoacoustic
imaging-guided photodynamic therapy are few. Reduced phenazine,
which possesses many modification sites, strong electron-donating
a Key Laboratory for Advanced Materials and School of Chemistry and Molecular
Engineering, East China University of Science and Technology, 130 Meilong Road,
Shanghai 200237, China. E-mail: jlhua@ecust.edu.cn
b Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory
for Carbon-Based Functional Materials & Devices, Soochow University,
Suzhou 215123, China. E-mail: zliu@suda.edu.cn
† Electronic supplementary information (ESI) available: Characterization data for
all compounds, additional spectroscopic data, MS titrations. See DOI: 10.1039/
c8tb01750f
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Scheme 1 Structures of PH-1 and PH-2.
ability and excellent stability, has been used to design fluorescent
probes.29–31 When nitrogen atoms were linked with n-butyl groups,
the phenazine group changed to an electron-rich donor group from
initial electron-deficient group. Meanwhile, indole salt derivatives
are common electron-withdrawing groups because of the presence
of positive charges. In this study, a butyl phenazine donor and alkyl
indole salt acceptor were employed to design and synthesize NIR
phenazine-cyanine dyes PH-1 and PH-2 for PA imaging and PTT, in
Fig. 2 (a) UV-visible absorption spectra of PH-CHO, PH-1 and PH-2 in
DMSO:PBS solution (v:v = 3:7) at 10 mM. (b) Absorption normalization of PH-1
and PH-1@HSA. (c) Absorption normalization of PH-2 and PH-2@HSA.
(d) Photograph of PH-CHO, PH-1 and PH-2 in solution. (e) Photograph of
which intramolecular charge transfer (ICT) occurred from alkyl PH-1 (right) and PH-1@HSA (left) in PBS. (f) Photograph of PH-2 (right) and
PH-2@HSA (left) in PBS.
phenazine to the alkyl indole salt. Unlike the acceptor–donor–
acceptor (A–D–A) structure of PH-2 with two alkyl indole salt groups
on both sides of phenazine, the retained carbaldehyde moiety on
The important intermediate PH-CHO was obtained by Vilsmeier–
PH-1 resulted in an acceptor–donor–another acceptor (A–D–A0)
Haack reaction. The asymmetric cyanine dye PH-1 was obtained
structure with two different routes of the ICT process. We hope
through the reaction of n-butyric acid indole iodide salt with the
to adjust the absorption spectrum by changing the electron-
corresponding phenazine aldehyde in piperidine at 80 1C. The
withdrawing group. We have described the synthesis as well as
symmetric cyanine dye PH-2 was synthesized by Knoevenagel
the optical properties of two phenazine-cyanine dyes PH-1 and
reaction of PH-CHO and butyl indole iodide salt in acetic acid at
PH-2 (Scheme 1), characteristics of PH-1@HSA and PH-2@HSA
70 1C. These main compounds were characterized by 1H NMR,
and the preliminary investigations of PA images and PTT in
13C NMR and MS. The details of the structural identification
nude mouse based on PH-1@HSA.
spectra are given in the ESI.†
Optical properties of PH-CHO, PH-1 and PH-2
Results and discussion
Synthesis of PH-1 and PH-2
As shown in Fig. 2a, the maximum absorption wavelength of
PH-CHO was 430 nm (e = 1.65 ꢀ 104 Mꢁ1 cmꢁ1). When an
The synthetic route for phenazine-cyanine dyes is shown in
aldehyde group was replaced by alkyl indole salt, the maximum
Fig. 1. Phenazine was first reduced by sodium hydrosulfite.
Then, hydrogen linked to nitrogen atoms was substituted by
n-butyl group in an alkaline environment to obtain compound 2.
absorption wavelength of PH-1 was about 690 nm (e = 1.25 ꢀ
104 Mꢁ1 cmꢁ1). Compared to the observation for PH-CHO, the
maximum absorption of PH-1 red-shifted by 255 nm due to the
ICT process from alkyl phenazine to the alkyl indole salt. When
another alkyl indole salt was introduced, the maximum absorp-
tion of PH-2 further increased to 720 nm (Fig. 2a, blue line,
e = 4.25 ꢀ 104 cm2 molꢁ1), which showed about 30 nm red-shift
compared to that of PH-1. Significant differences among three
dyes could be observed (shown in Fig. 2d) by the naked eye, in
which the solution changed from orange-yellow, green to deep
green. Furthermore, PH-1 and PH-2 showed much better photo-
stabilities compared with ICG (Fig. S1, ESI†). When exposed to
white light for 50 min, PH-1 almost stayed the same, whereas
ICG showed a decrease to B10%. The absorption spectra of
both PH-1 and PH-2 were more than 800 nm, suggesting that
they have potential applications in PA imaging and PTT.
Characteristics of PH-1@HSA and PH-2@HSA
Poor water solubility limits the biological applications of NIR
Fig. 1 Synthetic routes of PH-CHO, PH-1 and PH-2. Reagents and con-
ditions: (i) Na2S2O4, EtOH, H2O, 80 1C. (ii) NaOH, tetrabutylammonium
dyes.32 Recently, human serum albumin (HSA), a principal
protein in plasma, was employed as a drug carrier due to its
improved water solubility, reduced immunogenicity, low levels
bromide, iodobutane, DMSO, H2O, 40 1C (iii) POCl3, 0 1C, DMF, 60 1C.
(iV) Piperidine, MeCN, 80 1C. (V) CH3COONH4, CH3COOH, 70 1C.
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values of Britton–Robinson (B–R) buffer and then, the average
sizes and zeta potentials were measured. As shown in Fig. 3c
and f, two nanoparticles showed appropriate stability in both
weakly acidic and weakly basic environments. Form pH 5.5 to
8.0, the sizes of PH-1@HSA NPs and PH-2@HSA NPs showed
almost no variation, which manifested that nanoparticles could
be stable with negligible aggregation in both normal tissues
and tumors. However, when the pH value was less than 5.5, the
size of NPs increased abruptly. Meanwhile, the zeta potential
changed to a positive value from negative value, which was a
possible reason for NP aggregation. In the weakly basic micro-
environment, the zeta potentials of two NPs were negative,
which resulted in stability in the serum.
Fig. 3 (a) DLS examination for size of PH-1@HSA in PBS buffer, pH = 7.4.
(b) TEM image of PH-1@HSA NPs. (c) Average diameter and zeta potential
of PH-1@HSA in different pH value B–R buffers. (d) DLS examination for
size of PH-2@HSA in PBS buffer, pH = 7.4. (e) TEM image of PH-2@HSA
NPs. (f) Average diameter and zeta potential of PH-2@HSA in different pH
value B–R buffers.
Photothermal in vitro
To investigate the therapeutic effect, different concentrations of
PH-1@HSA (0, 5, 10, 20, 40 and 80 mM) in solutions were tested.
As shown in Fig. 4a and d, the elevated temperature was related
to the nanoparticles’ concentration and exposure duration. The
temperature of NP solution increased rapidly within 3 minutes
and then rose progressively and remained steady. The tempera-
ture difference of pure PBS buffer was only about 5 1C when
exposed to laser, whereas that of 80 mM PH-1@HSA rose to
about 36.7 1C. This suggested that NPs can absorb light and
transfer luminous energy to thermal energy, which causes
temperature increase. The temperature of PH-1@HSA NP (20 mM)
PBS buffer reached 49.3 1C within 300 s, which is high enough
to kill cancer cells. At the same condition, the temperature of
PH-2@HSA (20 mM) NP PBS buffer reached 52.5 1C. However,
when the concentration of PH-2@HSA was higher than 40 mM,
the temperature curve rose and then descended.
of mononuclear phagocyte system clearance, prolonged circulatory
half-life, improved pharmacokinetic properties, and versatile
roles.33–35 To improve biocompatibility of PH-1 and PH-2, they
were self-assembled with HSA to form nanoparticles named as
PH-1@HSA and PH-2@HSA. As shown in Fig. 2e and f, clear
floccules could be seen when PH-1 was directly added to PBS
solution, which is due to the poor water solubility. Organic
dye nanoparticles showed excellent solubility in pure water.
The same phenomenon could also be observed for PH-2.
Subsequently, the absorption spectra (Fig. 2b and c) of PH-1
and PH-2 before and after assembly were measured. The peak
of PH-1@HSA NPs showed 40 nm red-shift compared to that of
PH-1, which was caused by molecular aggregation. PH-2@HSA
showed no clear red-shift, which may be because of the
improved water solubility of two alkyl indole salts. Besides,
the spectrum of NPs was wider than those of PH-1 and PH-2,
which was ascribed to intermolecular interactions. The
bathochromic-shift and broadening of the spectrum implied
that PH-1 species were indeed self-assembled with HSA to form
nanoparticles. Then, the morphology and particle size of two
nanoparticles were studied by transmission electron microscopy
(TEM) and dynamic light scattering (DLS). As shown in the TEM
image (Fig. 3b), PH-1@HSA exhibited spherical morphology and
there was no distinct aggregation. Furthermore, DLS showed
that the average diameter of PH-1@HSA was about 10 nm, which
was the appropriate size for enrichment in tumor. The shape of
PH-2@HSA NPs was irregular and the average diameter was
about 50 nm.
Then, the photothermal stabilities of PH-1@HSA and PH-2@
HSA were further evaluated by monitoring the variation in the
temperature of the solution (20 mM) during irradiation with
808 nm laser for 3 min, followed by natural cooling for 3 min
Stability of NPs in different pH environments
The micro-environment of tumors is weakly acidic, whereas
that of normal tissues is weakly basic;36 the nanoparticles
should be stable in both weakly acidic and weakly basic
environments. Thus, the pH stabilities of two nanoparticles
were evaluated by DLS. NPs were attenuated with different pH
Fig. 4 Temperature curves of different concentrations of PH-1@HSA
(a) and PH-2@HSA (d) in PBS buffer under 808 nm laser, 1.0 W cmꢁ2
;
PA images of subcutaneous tissue with different concentrations of
PH-1@HSA (b) and PH-2@HSA (e). Relation between intensity of relative
PA signals and concentrations of PH-1@HSA (c) and PH-2@HSA (f).
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after the laser was turned off for five cycles. As shown in Fig. S2 the PA intensity of PH-1@HSA showed 6.2-fold enhancement to
(ESI†), the temperature rose when PH-1@HSA were exposed to PBS, whereas that of PH-2@HSA showed 12.8-fold enhancement.
laser and then declined when the laser was turned off. In the The result was in accordance with photo-thermal data, which
next several cycles, the temperature maximum almost remained suggested that PA conversion efficiency is associated with photo-
unchanged. However, the temperature maximum of PH-2@HSA thermal conversion efficiency. Considering the enhanced PA sig-
decreased gradually (Fig. S3, ESI†). In the first cycle, the highest nals related to NP concentration, PH-1 and PH-2 have potential
temperature was about 52 1C; nevertheless, the highest tempera- applications in PA imaging.
ture in the fifth cycle was just round 42 1C. Furthermore, the
color of PH-2@HSA changed from deep green to yellow green.
Phototoxicity to 4T1 cells
This demonstrated that PH-1 performed better than PH-2 with Subsequently, the cytotoxicity of NPs to 4T1 cells was assessed
respect to photothermal stability. Based on the above reasons, by MTT. The cell viability was measured by an MTT assay using
PH-1 was chosen for further tests.
a microplate absorbance reader (Model 680, Bio-Rad) at
570 nm. In the dark state for 48 h, when the concentration
was lower than 25 mM, the cell viability was more than 50%
(Fig. S4, ESI†), which suggested NPs show suitable toxicity in
the dark state. In the experimental group, 4T1 cells were
exposed to 808 nm laser (0.8 W cmꢁ2) for 20 min in the
presence of PH-1@HSA, and conspicuous photo-toxicity could
be confirmed. When the concentration was increased from 1.25
to 20 mM, the cell viability declined rapidly, and the decrease
was related to the increase in NPs (as shown in Fig. S5, ESI†).
When the concentration was 20 mM, about half of the cells were
killed, which suggested that PH-1@HSA possessed adequate
cytotoxicity in the presence of a laser.
To confirm the PTT effect in vitro, photo-toxicity was also
evaluated by confocal CLSM (confocal laser scanning micro-
scopy) imaging. The Calcine AM (green) and propidium iodide
(red) were utilized to distinguish live and dead cells. Also, 4T1
cells were divided into two groups: control group (laser only)
and experimental group (PH-1@HSA + laser). As shown in
Fig. 5, in the control group, a small red region could be
observed, which suggested that almost all of the cells had
survived. In the experimental group, the red region enlarged
along with irradiating time. When the cells were incubated with
NPs and exposed to laser for 0 min, the phenomenon was
similar with that of the control group, which also indicated
the low toxicity in dark condition. After exposure for 2, 6 and
10 min, the red region became more clear, implying that more
4T1 cells were killed. CLSM imaging confirmed that PH-1@HSA
with measurable concentration indeed damaged cells only in
the presence of a laser.
PA in vivo
Considering that PTT agents are mostly applied for PA imaging,
the performances of PH-1@HSA and PH-2@HSA were verified
using nude mouse (Fig. 4b and e). NPs diluted with PBS buffer
were injected into back subcutaneous tissues of mice, which
were irradiated by 808 nm pulse laser, and the PA signal was
collected by an ultrasonic detector. As shown in Fig. 4b and e, NPs
exhibited a clear PA signal in mice back subcutaneous tissues,
which showed a conspicuous contrast to that of background. The
enhanced intensity of PA signal was associated with the increase
in NP concentration. When pure PBS buffer was injected in mice
back, a small ultrasonic signal could also be detected, which may
be due to hemoglobin. As the NP concentration was about 38 mM,
Fig. 5 Fluorescence confocal image of 4T1 cells treated by laser irradiation
only; incubation with PH-1@HSA and laser irradiation for different times
(808 nm laser: 0.8 W cmꢁ2, concentration = 20 mM, scale bar = 250 mm).
Fig. 6 (a) Thermal image of nude mouse in control group and experimental group at different illuminating times. (b) Relational group of tumor
temperature of mice in control group and experimental group and time. Tumor models were built with 4T1 cells, and PH-1@HSA (200 mL, 500 mM) were
injected in the tail of mice.
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Photothermal in vivo
under argon atmosphere. Then, Na2S2O4 (28.8 g, 16.5 mmol) in
250 mL ultrapure was added into the previous solution. After
adding Na2S2O4, high amount of white precipitate was observed.
The mixture was heated for 1 hour and cooled to room tempera-
ture. After it was filtered, washed with water and dried in vacuo, a
light green solid (2.81 g, 93.6%) was obtained. As it was unstable
in air, compound 1 was not characterized by NMR spectra and
was placed into the next reaction as soon as possible.39
To identify PH-1@HSA photo-thermal effect in vivo, mice experi-
ments were performed. NPs were injected in nude mouse tail; after
4 hours, tumors37 were irradiated by an 808 nm laser (0.8 W cmꢁ2).
As shown in Fig. 5, in the control group, the temperature of tumors
was B37 1C when exposed to laser for 10 min. In the experimental
group, the temperature gradually rose with time and reached
B43 1C after 10 min, which can damage cancer tissues
effectively.38 As shown in Fig. S6 (ESI†), tumor tissue necrosis
could be observed after PTT, and the size of tumor reduced
conspicuously 5 days later. In the control group, tumors enlarged
gradually. Photo-thermal imaging implied that NPs can indeed
accumulate in tumors within 4 h by the EPR effect and generate
heat in vivo. Thus, PH-1@HSA can be utilized for PTT (Fig. 6).
Synthesis of 5,10-dibutylphenazine (2). 5,10-Dihydrophena
zine (2.81 g, 15.3 mmol), sodium hydroxide (2.34 g, 58.6 mmol),
1 mL water and tetrabutylammonium bromide (0.4 g, 1.2 mmol)
were dissolved in 25 mL DMSO. The mixture was stirred and
heated to 40 1C under argon atmosphere. Then, iodobutane (4.0 mL,
35.2 mmol) was added. The mixture was stirred for another 6 hours.
After cooling to room temperature, the mixture was poured into
water and extracted by CH2Cl2. The organic phase was washed with
water three times. The crude products were obtained by removing
CH2Cl2 and purified by neutral aluminum oxide chromatography
to get pure compound 2 (2.99 g, 66.0%) using petroleum ether as
Conclusions
In summary, two new A–D–A NIR organic phenazine-cyanine
dyes PH-1 and PH-2 were developed for PA imaging and PTT.
The absorption wavelengths of compounds broadened to the
near-infrared region via introducing stronger electron-withdrawing
groups into butylphenazine for structuring strong ICT. The water
solubility of dyes improved by self-assembly with HSA, and NPs
existed steadily in both weakly acidic and weakly basic environ-
ments, which indicated that NPs can work in blood circulation.
Furthermore, PH-1@HSA and PH-2@HSA could transfer luminous
energy to thermal energy effectively in vitro. Importantly, PH-1@
HSA nanoparticles were successfully applied to PA imaging and
PTT in nude mouse. The relatively simple molecular design of
phenazine-cyanine dyes provides a new strategy for efficient photo-
acoustic imaging-guided photothermal therapy materials.
1
an eluent. H NMR (400 MHz, C6D6) d (ppm): 6.64 (m, 4H), 6.21
(m, 4H), 3.11 (m, 4H), 1.42 (m, 4H), 1.07 (m, 4H), 0.74 (t, J = 7.3 Hz,
6H). 13C NMR (101 MHz, C6D6) d (ppm): 137.50, 121.12, 110.96,
45.00, 26.63, 20.15, 13.77. MS (ESI, m/z): calcd for C20H26N2: 294.21;
found: 294.22.
Synthesis of PH-CHO. 5,10-Dibutylphenazine (2) (2.99 g,
10.1 mmol) was dissolved in 20 mL DMF and kept in an ice
bath. The solution was stirred for 10 min under argon atmo-
sphere. Then, phosphorus oxychloride (2.5 mL) was added into
the flask drop by drop, and the solution was stirred for another
30 min. Subsequently, the mixture was heated for 10 hours at
60 1C. Then, the solution was poured into ice water and pH
value was adjusted to weakly basic. The orange precipitate was
filtered and dried. Then, pure product PH-CHO (0.98 g, 27.8%)
was obtained by silica gel chromatography (petroleum ether/
Experimental
Materials and measurements
1
CH2Cl2, v : v = 2 : 1). H NMR (400 MHz, CDCl3) d (ppm): 9.59
(s, 2H), 7.10 (dd, J = 8.1, 1.6 Hz, 2H), 6.72 (d, J = 8.2 Hz, 2H), 3.44
(m, 4H), 1.63 (m, 4H), 1.48 (m, 4H), 1.04 (t, J = 7.3 Hz, 6H). 13
C
Dichloromethane (DCM) was refluxed with calcium hydride and
distilled before use. Ethanol (EtOH) was pre-dried over 4 Å mole-
cular sieves before use. PH-CHO was synthesized according to a
previous literature report.29 All other reagents and reactants were
purchased as commercial products from Energy Chemical or
Sigma-Aldrich and used as received without further purification.
1H NMR and 13C NMR spectra were recorded on a Bruker AM
400 MHz spectrometer, and tetramethylsilane (TMS) was used as
an internal standard. Electrospray ionization and time-of flight
analyzer (ESI-TOF) mass spectra were determined using a Waters
Micromass LCT mass spectrometer. Absorption spectra were
recorded on a Varian Cary 500 UV-vis spectrophotometer. The size
distribution of the nanoparticles was measured using an ALV-5000
laser light scattering spectrometer (DLS). TEM micrographs were
obtained on a JEOL JSM-6360 scanning electron microscope (SEM).
MTT data were measured by a microreader (Model 680, Bio-Rad).
NMR (101 MHz, CDCl3) d (ppm): 189.89, 142.61, 135.27, 130.24,
109.85, 108.29, 45.59, 26.32, 20.13, 13.90. MS (ESI, m/z):
[M + H]+ calcd for C22H27N2O2 : 351.2; found: 351.2.
+
Synthesis of PH-1. PH-CHO (160 mg, 0.45 mmol), 3H-indolium,
1-(2-carboxyethyl)-2,3,3-trimethyl-iodide (162 mg, 0.45 mmol),
15 mL acetonitrile and a drop of piperidine were added into a
flask. The mixture was stirred and heated to 80 1C under argon
atmosphere for 10 hours. Then, the solution was cooled to
room temperature naturally, and acetonitrile was removed. The
black-green product PH-1 (57.1 mg, 18.6%) was obtained by
silica gel chromatography (CH2Cl2/alcohol, v : v
= 12 : 1).
1H NMR (400 MHz, DMSO-d6) d 9.62 (s, 1H), 8.07 (d, J =
15.0 Hz, 1H), 7.77 (d, J = 6.9 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H),
7.54 (t, J = 7.6 Hz, 2H), 7.47 (t, J = 7.4 Hz, 1H), 7.33–7.27 (m, 2H),
6.93 (s, 1H), 6.81 (s, 1H), 6.59 (t, J = 8.7 Hz, 2H), 4.66 (t, J =
5.5 Hz, 2H), 3.67–3.61 (m, 2H), 3.60–3.55 (m, 2H), 1.71 (s, 6H),
1.52 (dd, J = 33.0, 11.2 Hz, 8H), 0.99 (t, J = 7.2 Hz, 6H). 13C NMR
Synthesis of PH-CHO, PH-1 and PH-2
Synthesis of 5,10-dihydrophenazine (1). Phenazine (2.94 g, (101 MHz, DMSO-d6) d 190.04, 189.04, 178.82, 152.37, 142.76,
16.3 mmol) was dissolved in 70 mL ethanol and heated to 80 1C 141.98, 134.37, 133.36, 131.48, 129.13, 128.73, 113.95, 111.13,
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109.45, 108.37, 107.37, 54.70, 50.90, 46.73, 44.18, 43.10, 33.29, 28.91, and exposed to 808 nm laser (0.8 W cmꢁ2) for 0, 2, 6, 10 min; in the
26.17, 21.12, 19.27, 13.80, 13.65. [M]+ calcd for C36H42N3O3 : control group, cells were exposed to 808 nm laser without NPs.
+
564.3226; found: 564.3226.
Cells were further co-stained with 200 nM Calcine AM (AM) and
Synthesis of PH-2. PH-CHO (124 mg, 0.35 mmol), 3H-indolium, 200 nM propidium iodide (PI). CLSM imaging was performed on
1-butyl-2,3,3-trimethyl-iodide (240 mg, 0.7 mmol), ammonium Laser-scanning confocal microscopy (Leica TCS SP5).
acetate (2.31 mg, 0.03 mmol) and 15 mL acetic acid were stirred
Photothermal therapy in vitro
in a flask under argon atmosphere. The mixture was heated to
70 1C for 8 hours and then cooled to room temperature. The solvent
was removed and crude product was purified by silica gel chroma-
tography (CH2Cl2/alcohol, v : v = 8 : 1) to obtain PH-2 (45.3 mg,
The stock solutions of PH-1@HSA and PH-2@HSA were attenuated
with PBS buffer to obtain different (0, 5, 10, 20, 40, 80 mM)
concentration NPs. Then, these solutions were put into tubes with
laser irradiation (808 nm, 1.0 W cmꢁ2) and photographed using
thermal imager per 0.5 minute to record the temperature.
1
12.9%). H NMR (400 MHz, DMSO-d6) d 8.22 (d, J = 15.7 Hz, 2H),
7.87–7.81 (m, 4H), 7.69 (d, J = 8.6 Hz, 2H), 7.62–7.57 (m, 2H), 7.57–
7.52 (m, 2H), 7.23 (d, J = 15.9 Hz, 2H), 7.07 (s, 2H), 6.74 (d, J = 8.9
Hz, 2H), 4.62 (t, J = 7.0 Hz, 4H), 3.85 (m, 2H), 3.82–3.78 (m, 2H),
1.78 (s, 12H), 1.66–1.54 (m, 8H), 1.44 (m, 8H), 1.00 (t, J = 7.1 Hz,
6H), 0.95 (t, J = 7.4 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) d 179.54,
171.41, 160.52, 152.34, 143.05, 142.29, 140.97, 133.36, 129.46,
128.94, 127.90, 122.94, 121.74, 117.98, 114.23, 111.92, 107.34,
105.20, 51.32, 30.09, 26.23, 22.49, 19.44, 19.28, 13.88, 13.72. MS
(ESI, m/z): [M]2+ calcd for C52H66N422+: 373.2630; found: 373.2644.
Photothermal therapy in vivo
Fifty mL PBS solution containing 4T1 cells (5 ꢀ 106 mLꢁ1) was
injected in back of nude mice, and these mice were fed till the
tumors grew to B60 mm3. Then, these nude mice were divided
into two groups (2 mice per group): control group and experi-
mental group. In the experimental group, 200 mL PH-1@HSA
PBS solution (500 mM) was injected in the tails of mice. After
4 h, tumors were illuminated by 808 nm laser (0.8 W cmꢁ2) and
photographed by a thermal imaging camera. In the control
group, pure PBS buffer was injected into mice. The nude mice
were purchased from Suzhou Belda Bio-Pharmaceutical Co.
Ltd, and all animal experiments were carried out according to
the protocols approved by the Soochow University Laboratory
Animal Center.
Synthesis of PH-1@HSA and PH-2@HSA
Twenty mg PH-1 was dissolved in 1 mL DMSO to obtain stock
solution. Then, 27 mL solution was added into 1 mL PBS buffer
containing HSA (10 mg mLꢁ1), forming nanoparticles PH-1@
HSA. The mixture was stirred for 24 hours and subsequently
centrifuged for 3 min (14 800 rpm) to remove dye unassembled
with HSA. The synthesis of PH-2@HSA was conducted in a
similar manner.
Conflicts of interest
MTT assay
There are no conflicts to declare.
4T1 cells were seeded to 96 well plate (1 ꢀ 104 per well) and
hatched in DEME culture medium (containing 10% fetal calf
serum) for 24 hours. PH-1@HSA NPs were attenuated with
culture medium to obtain different concentration solutions
(0.7, 1.5, 3.1, 6.3, 12.5, 25, 50 and 100 mM) by replacing previous
medium. 4T1 cells were placed in dark environment for 48 h.
The methyl thiazolyl tetrazolium (MTT) assay was utilized to
evaluate the cytotoxicity of PH-1@HSA. Subsequently, 4T1 cells
were cultured with MTT (25 mL, 5 mg mLꢁ1) for 3 h. The
absorbances at 470 nm were measured after DMSO was added
(100 mL per well).
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (21772040, 21421004, 21372082 and
21572062), the Fundamental Research Funds for the Central
Universities (222201717003) and the Programme of Introducing
Talents of Discipline to Universities (B16017).
Notes and references
Photothermal cytotoxicity of PH-1@HSA
1 A. W. Chris Twelves, M. P. Nowacki, M. Abt, H. Burris III,
A. Carrato, J. Cassidy, A. Cervantes, J. Fagerberg, V. Georgoulias,
Photothermal cytotoxicity of PH-1@HSA was first measured by
MTT assay. 4T1 cells were incubated with different concen-
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into two groups: control group and experiment group. 4T1 cells
were seeded in petri dishes at a density of 2 ꢀ 104 cells per dishes.
In the experiment group, cells were treated by PH-1@HSA (20 mM)
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