BSA-encapsulated cyclometalated iridium complexes as nano-photosensitizers for photodynamic therapy of tumor cells

Article abstract of DOI:10.1039/d1ra01740c

Photodynamic therapy is a promising treatment method. The development of suitable photosensitizers can improve therapeutic efficacy. Herein, we report three iridium complexes (Ir1, Ir2, and Ir3), and encapsulate them within bovine serum albumin (BSA) to f

Full text of DOI:10.1039/d1ra01740c

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BSA-encapsulated cyclometalated iridium
complexes as nano-photosensitizers for
Cite this: RSC Adv., 2021, 11, 15323
photodynamic therapy of tumor cells†
*
Yao Xu, Xiang Wang, Kang Song, Jun Du, Jinliang Liu, Yuqing Miao and Yuhao Li
Photodynamic therapy is a promising treatment method. The development of suitable photosensitizers can
improve therapeutic efficacy. Herein, we report three iridium complexes (Ir1, Ir2, and Ir3), and encapsulate
them within bovine serum albumin (BSA) to form nano-photosensitizers (Ir1@BSA, Ir2@BSA, and Ir3@BSA)
for photodynamic therapy (PDT) of tumor cells. In the structures of Ir(III) complexes, we use the pyrazine
heterocycle as part of the C^N ligands and explore the effect of different ligands on the ability to
generate singlet oxygen (1O2) by changing the conjugation length of the ligand and increasing the
coplanarity of the ligand. Besides, the fabricated nano-photosensitizers are beneficial to improve water
dispersibility and increase cellular uptake ability. Through studying photophysical properties, 1O2
generation capacity, and cellular uptake performance, the results show that Ir1@BSA has the best
photodynamic therapeutic effect on 4T1 tumor cells. This study provides an effective research basis for
the further design of new nano-photosensitizers.
Received 5th March 2021
Accepted 16th April 2021
DOI: 10.1039/d1ra01740c
rsc.li/rsc-advances
have attracted great attention, such as ruthenium
complexes,25,26 platinum complexes,27,28 iridium complexes,29,30
Introduction
etc. Iridium Ir(III) complexes possess higher intersystem
crossing efficiency and better photobleaching resistance than
some organic photosensitizers. More specically, Ir(III)
complexes show a triplet excited-state lifetime from nano-
second to microsecond, benecial for the energy transfer to
react with O2 to generate O2.31 For heteroleptic cationic Ir(III)
complexes, through adjusting their cyclometalated ligands
(C^N ligand) and ancillary ligands (N^N ligand), the ground
state absorption and excited-state absorption of the complexes
are regulated to achieve effective triplet energy transfer, and
ultimately increase singlet oxygen quantum yield.32,33 Although
some studies have been focused on the relationship between
ligand and singlet oxygen quantum yield, and some Ir(III)
complexes have been synthesized based on the modication of
C^N ligand and N^N ligand, there is still no clear enunciation
for the structure–activity relationship between them.
Besides, most Ir(III) complexes are water-insoluble. To make
these Ir(III) complexes become photosensitizers for biological
application, it is necessary to overcome the problem of insuffi-
cient water solubility. There are two methods to solve this
problem. One way is to introduce hydrophilic groups or water-
soluble polymers into an Ir(III) complex by covalent bonding
to improve the solubility.34–36 Another way is to utilize biocom-
patible polymers or protein to encapsulate the Ir(III) complex to
improve the water dispersibility.37,38 These two strategies
provide strategies for the further application of Ir(III) complexes
as photosensitizers.
Photodynamic therapy (PDT) is a noninvasive, photo-controlled
treatment method.1–4 Based on the generation of photo-induced
reactive oxygen species (ROS), this method has been widely
utilized in cancer treatment such as for bladder cancer,
esophageal cancer, lung cancer, and brain cancer, exhibiting
some superiorities over traditional cancer treatments including
operative treatment, radiotherapy, and chemotherapy.5–9 Three
factors inuence the effectiveness of PDT including photosen-
sitizer, oxygen, and light.10 Through producing more ROS to
obtain a better therapeutic effect, the researchers have started
from these three factors and designed multiple methods to
enhance PDT. For example, the nanomaterial is used to carry
oxygen to supply raw materials for PDT.11–14 For another
example, the structure of the photosensitizer is adjusted to
increase singlet oxygen quantum yield.15–18 Besides, the excita-
tion light is regulated to increase the penetration depth of the
light into the tissue for internal tumor treatment.19,20
3
1
Numerous studies have shown that among the three factors,
designing novel photosensitizers through structural optimiza-
tion strategies is a feasible and effective way to enhance the
therapeutic effect.21–24 Benet from the development of chem-
ical synthesis, many types of photosensitizers have been
developed. Among them, noble metal complex photosensitizers
Institute of Bismuth Science, College of Science, University of Shanghai for Science and
Technology, Shanghai 200093, China. E-mail: yhli@usst.edu.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1ra01740c
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Optical properties
The absorption spectra of Ir1, Ir2, Ir3 in CH2Cl2 were shown in
Fig. 1a. Variable concentration experiments conrmed that no
ground state aggregation occurred in the concentration range
from 5 to 50 mM (Fig. S10–S12†). Three Ir(III) complexes
exhibited a wide absorption band from UV to visible region. The
absorption band below 350 nm was due to the 1p,p* transition
of C^N and N^N ligands. The absorption band from 350 to
450 nm was due to the 1p,p* transition and spin allowed singlet
metal to ligand charge transfer (1MLCT) mixed with some
singlet ligand to ligand charge transfer (1LLCT) and singlet
intra-ligand charge transfer (1ILCT). The absorption band larger
than 450 nm was due to the 1MLCT/1LLCT. Especially, three
Ir(III) complexes had obvious absorption at 532 nm, the molar
absorption coefficients were 2225, 4686, and 2425
L
molꢀ1 cmꢀ1, for Ir1 to Ir3 respectively, beneciating to the PDT
induced by green light (532 nm).
Scheme 1 Molecular structures of Ir1 to Ir3 complexes and schematic
of Ir1@BSA to Ir3@BSA, fabrication, and application for photodynamic
therapy.
The emission spectra of Ir1, Ir2, Ir3 in CH2Cl2 were shown in
Fig. 1b. Variable concentration experiments conrmed that no
quenching emission intensity occurred from 5 to 50 mM
(Fig. S10–S12†). Upon excitation at 532 nm, Ir1 exhibited major
photoluminescence peaks at 636 nm, Ir2 exhibited major pho-
toluminescence peaks at 778 nm and 855 nm, and Ir3 exhibited
major photoluminescence peaks at 650 nm. Compared to Ir1,
the Ir2 showed a red-shied emission peak and the emission
peak located in the near-infrared (NIR) region, which indicated
that increasing the conjugation length of the C^N ligand can
signicantly redshi the position of the emission peak. This
result was in line with the reported phenomenon that the
degree of p-conjugation of the C^N ligands impacts the location
of emission.39,40 Further, compared to Ir1, Ir3 exhibited a similar
emission location, indicating that increasing the rigidity of the
C^N ligand cannot change the location of the Ir(III) complex
emission. Due to the long linear alkyl substituents on the N^N
ligand, three complexes presented good solubility and could be
dissolved in different organic solvents such as toluene, tetra-
hydrofuran (THF), CH2Cl2, CH3CN, and CH3OH (Fig. S10–S12†).
Taking the air-sensitive emission, luminous lifetime from
nanosecond to microsecond, and large Stokes shi into
Herein, we reported a series of three bovine serum albumin
(BSA)-encapsulated
Ir(III)-based
nano-photosensitizers
(Ir1@BSA, Ir2@BSA, and Ir3@BSA) for photodynamic therapy
of tumor cells. The three iridium complexes (Ir1 to Ir3) possess
different C^N ligands. Based on the 2,3-diphenylquinoxaline
structure in Ir1, introduce a benzene ring to extend the conju-
gation length of C^N ligand in Ir2 or connect the two phenyl
groups to construct a more plane-rigid C^N ligand in Ir3 to
explore the relationship between these inuencing factors and
singlet oxygen generation ability. Also, to increase the water
dispersibility, we encapsulated BSA on the surface of the
synthesized complexes by self-assembly to construct nano-
photosensitizers. Introducing two long alkyl chains into the
N^N ligand would make an Ir(III) complex that could be better
embedded in the hydrophobic cavity of BSA during self-
assembly to achieve a tighter combination. The study of three
protein-encapsulated nano-photosensitizers could provide
a research foundation with exploring Ir(III) complexes for
photodynamic therapy (Scheme 1).
Results and discussion
Synthesis
The synthesis route from Ir1 to Ir3 was shown in Fig. S1.† These
iridium complexes were synthesized in a three-step method. To
begin with, three pyrazine derivatives (dpqx, dpbq, and dbpz)
were synthesized through the reaction of dehydration between
diketones and diamine compounds. Subsequently, the dimeric
iridium complexes were obtained from the reaction between
these ligands and IrCl3, and the Ir1 to Ir3 complexes were ob-
tained from the reaction between the dimeric iridium
complexes and the alkyl chain decorated bipyridine (N^N)
ligand. The structures of some ligands and nal products were
characterized by 1H-nuclear magnetic resonance (1H-NMR) and
high resolution-mass spectrometry (HR-MS) (Fig. S2–S9†), and
the result showed that the synthesized structures were in line
with its design.
Fig. 1 (a) Absorption and (b) emission spectra of Ir1, Ir2, and Ir3 in
1
CH2Cl2 (lex ¼ 532 nm), (c) O2 emission spectra of Ir1, Ir2, and Ir3 in
CH3CN (lex ¼ 405 nm), time-dependent absorbance plot at 410 nm of
(d) Ir1, DPBF, and Ir1 + DPBF, (e) Ir2, DPBF, and Ir2 + DPBF, (f) Ir3, DPBF,
and Ir3 + DPBF in CH3CN (lex ¼ 532 nm, 50 mW cmꢀ2).
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consideration (Fig. S13–S15, Table S1†), we conrmed that the performance, and also revealed that increasing the conjugation
emission of these complexes was phosphorescence. Then, the length of the C^N ligand or increasing its planar rigidity cannot
position of the phosphorescence emission peak and the lumi- effectively increase the singlet oxygen quantum yield.
nescence quantum efficiency did not show regular changes with
the increase of solvent polarity. Based on these considerations,
Fabrication and characterization of Ir1@BSA to Ir3@BSA
the emission of Ir1 to Ir3 was ascribed to the triplet metal to
ligand charge transfer (3MLCT) mixed with triplet ligand to
Next, to improve biocompatibility, these iridium complexes
ligand charge transfer (3LLCT). The study of these optical
were self-assembled with BSA to form nano-photosensitizers by
an emulsify method. To weaken the electrostatic interaction by
properties showed that the luminescence of these complexes
adjusting the pH of the aqueous solution to make BSA around
could be applied for cell phosphorescence imaging (Table 1).
the isoelectric point, these iridium complexes could be well
encapsulated with BSA through hydrophobic–hydrophobic
Singlet oxygen generation evaluation
interaction. The structures of Ir1@BSA to Ir3@BSA were char-
Based on the ROS generation mechanism, the triplet excited
acterized by transmission electron microscope (TEM). In
state lifetime (sT) of the Ir(III) complex is a key factor in the
Fig. 2a–c, many monodisperse balls were observed in the TEM
1
generation of O2. The longer the triply excited-state lifetime,
images. The particle size statistics showed that the particle sizes
the more benecial it is to increase the probability of the
of the three nano-photosensitizers were 61.1 ꢁ 9.7 nm, 68.5 ꢁ
complex colliding with oxygen. The transient absorption (TA)
spectra of Ir1, Ir2, and Ir3 in degassed CH3CN were shown in
Fig. S16.† The sT of Ir1 to Ir3 were 336 ns, 144 ns, and 309 ns at
the maximum triple excited absorption wavelength of 430 nm,
9.1 nm, and 56.6 ꢁ 8.7 nm for Ir1@BSA to Ir3@BSA, respec-
tively. This showed that the emulsication method was
a general encapsulation method, and the prepared nano-
materials have similar particle sizes. In Fig. 2d–f, dynamic light
scattering (DLS) results showed larger hydration particle sizes
and the sizes were 135.7 nm, 148.6 nm, and 151.2 nm for
Ir1@BSA to Ir3@BSA, respectively, which was due to the
formation of a hydration layer. Furthermore, we analyzed the
510 nm, and 460 nm, respectively, indicating Ir1 qualied the
longest sT. Then, two ways were utilized to evaluate the 1O2
generation ability of three complexes.
First, 1O2 presented the unique emission at 1270 nm. In
Fig. 1c, we monitored the unique emission of 1O2 for three
stability of Ir1@BSA to Ir3@BSA by hydrated particle size. The
1
complexes. Through 405 nm light excitation, the O2 emission
results showed that the hydrated particle size of them did not
at 1270 nm of Ir1, Ir2, and Ir3 in CH3CN was observed, and the
change signicantly aer 15 days, which maintained a good
luminescence intensity trend of 1O2 was Ir1 > Ir3 > Ir2, indi-
nano-shape and showed good nano-stability (Fig. S18†). At last,
cating three complexes could catalyze the production of 1O2,
and Ir1 had the highest catalytic efficiency. Subsequently, 1,3-
disphenylisobenzofuran (DPBF) as a 1O2 trap could observe the
rate of 1O2 production and calculate the singlet oxygen
quantum yield (FD). In Fig. 1d–f, under the irradiation of
532 nm light, when DPBF was mixed with the solutions of the
three complexes, the absorption at 410 nm decreased rapidly
with the extension of the irradiation time, and the absorption
decreased dramatically within 30 s. Aer the DPBF solution and
the complex solution alone were irradiated with light, almost no
decrease in absorbance was observed. This showed that the
DPBF was also utilized to measure the 1O2 generation ability of
Ir1@BSA to Ir3@BSA in H2O. In Fig. 2g–i, similar to the result of
Ir1 to Ir3, the absorption of DPBF was sharply decreased when it
was mixed with Ir1@BSA to Ir3@BSA. The calculated 1O2
quantum yields were 0.20, 0.06, and 0.23 in H2O (Fig. S19†), and
the rate of singlet oxygen generation has a linear relationship
with the excitation light power and the concentration of the
Ir1@BSA to Ir3@BSA (Fig. S20†). These results showed that the
three Ir(III) complexes could still show good 1O2 generation
ability aer being wrapped by BSA to form nano-
photosensitizers.
1
three complexes could quickly catalyze oxygen to produce O2,
and the catalytic rate was fast. Furthermore, the singlet oxygen
quantum yields of Ir1 to Ir3 complexes were calculated to be
Cellular uptake and cytotoxicity assay
0.84, 0.27, and 0.64 respectively (Fig. S17†). Similarly, Ir1 Benets to the luminescence properties of iridium complexes,
showed the highest singlet oxygen quantum yield. All of these the uptake of nano-photosensitizers by cells could be observed
indicated that Ir1 qualied the remarkable 1O2 generation through phosphorescence imaging. The emission of Ir2@BSA
Table 1 Photophysical properties of Ir1, Ir2, and Ir3
labsa/nm (3/104 L molꢀ1 cmꢀ1
)
lemb/nm (sem/ns, Fem
)
lT1–Tnc/nm (sTA/ns)
FD
d
Ir1
Ir2
Ir3
295 (3.8), 382 (2.2), 480 (0.49)
283 (6.8), 327 (4.7), 413 (2.2), 485 (0.79)
285 (4.2), 350 (0.74), 400 (0.97), 530 (0.23)
636 (817, 0.176)
778, 855 (—e, —)
650 (737, 0.182)
430 (336)
510 (144)
460 (309)
0.84
0.27
0.64
a
Absorption band maxima and molar extinction coefficients in CH2Cl2 at room temperature. b Room temperature emission band maxima, lifetime,
and quantum yield in CH2Cl2 (1 ꢂ 10ꢀ5 mol Lꢀ1). Triplet transient absorption band maxima and lifetime in degassed CH3CN. d Singlet oxygen
c
quantum yield in CH3CN. e Too weak to be measured.
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behind this phenomenon is still unclear, and we will study it in
our following research.
Aerward, to assess the intracellular light-induced 1O2
production, cell viability was analyzed by Cell Counting Kit-8
(CCK-8) assay in Fig. 4. At a concentration of 40 mM, the cell
viability was greater than 90% aer cells were incubated with
Ir1@BSA and Ir2@BSA for 24 h, and even at a concentration of
120 mM, the dark toxicity of Ir3@BSA was weak, indicating that
the dark toxicity of the nano-photosensitizers to cells could be
ignored. Aer exposure to light, we observed different degrees
of phototoxicity of cells. As the concentration of nano-
photosensitizer increases, Ir1@BSA exhibited greater photo-
toxicity, that is, it has a signicant inhibitory effect on cell
viability at a lower concentration. The calculated 50% effective
concentration (EC50) values of Ir1@BSA, Ir2@BSA, and Ir3@BSA
were 5.2 mM, 28.5 mM, and 82.2 mM respectively. The results
presented that the efficiency of PDT was Ir1@BSA > Ir2@BSA >
Ir3@BSA. The difference in therapeutic effect could be
explained by the above-mentioned cellular uptake and singlet
oxygen quantum yield. The uptake of Ir1@BSA by cells was
relatively high, and Ir1@BSA showed a remarkable singlet
oxygen production rate. Therefore, we believe that the dual roles
of Ir1@BSA resulted in the best PDT treatment effect.
Fig. 2 TEM images of (a) Ir1@BSA, (b) Ir2@BSA, and (c) Ir3@BSA (inset:
particle size statistical plot); DLS images of (d) Ir1@BSA, (e) Ir2@BSA,
and (f) Ir3@BSA; time-dependent absorption plots at 415 nm of (g)
Ir1@BSA, DPBF, and Ir1@BSA + DPBF, (h) Ir2@BSA, DPBF, and Ir2@BSA
+ DPBF, (i) Ir3@BSA, DPBF, and Ir3@BSA + DPBF in H2O under 532 nm
light irradiation (50 mW cmꢀ2).
located at the NIR region, which exceeded the detection capa-
bility of the microscope camera, so we only used cell imaging for
Ir1@BSA and Ir3@BSA to observe their uptake. In Fig. 3,
Ir1@BSA showed a strong phosphorescence signal in the cyto-
plasm, and the signal further increased with time, which
showed that the uptake of Ir1@BSA by cells was good. In
comparison, the uptake of Ir3@BSA by cells was poor, that is, as
the incubation time increased, the uorescent signal observed
in the cells was signicantly weaker than Ir1@BSA. Further by
detecting the content of Ir3+ ion in cells, we used the inductively
coupled plasma-mass spectrometry (ICP-MS) to detect the
uptake of the three nano-photosensitizers in tumor cells. ICP-
MS result indicated that the contents of Ir3+ ion in cells were
0.95, 0.67, and 0.66 mg gꢀ1 for Ir1@BSA, Ir2@BSA and Ir3@BSA
respectively, indicating cells had a high uptake rate of Ir1@BSA.
The difference in the uptake of photosensitizers by cells has
a potential impact on the effect of PDT on cells. The mechanism
Live/dead cell staining
The calcein O,O0-diacetate tetrakis(acetoxymethyl) ester/
propidium iodide (calcein-AM/PI) double stain kit was used to
directly differentiate the living and dead cells aer treatment. In
Fig. 5, when mouse breast cancer (4T1) cells were incubated
with three nano-photosensitizers without 532 light irradiation,
strong green uorescence emitted from calcein-AM was moni-
tored and the observed red uorescence emitted from PI was
negligible, indicating almost all cells were alive. Further, aer
the cells were incubated with photosensitizers and irradiated
with light, the green uorescence decreased and the red uo-
rescence increased, indicating that many cells were dead cells
and the three nano-photosensitizers caused different amounts
of cell death. The live and dead cell staining consolidated that
PDT was effective for 4T1 tumor cell therapy, and Ir1@BSA
presented the best treatment effect.
Fig. 4 Cell viability of (a) Ir1@BSA, (b) Ir2@BSA, and (c) Ir3@BSA for 4T1
cells in the dark or 532 nm light irradiation and EC50 values of (d)
Fig. 3 Luminescence images of 4T1 cells incubated with Ir1@BSA or Ir1@BSA, (e) Ir2@BSA, (f) Ir3@BSA under 532 nm light irradiation (100
Ir3@BSA at 4 h and 24 h. Scale bar, 40 mm.
mW cmꢀ2, 5 min).
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cells incubated with Ir1@BSA showed the strongest green
uorescence, indicating that a large amount of ROS was
produced in the cells. This further showed that Ir1@BSA was an
excellent nano-photosensitizer.
Experimental
General
All solvents and reagents used to synthesis were acquired from
chemical reagent suppliers, such as Alfa-Aesar, Adamas,
Aladdin, and TCI. 1H-NMR spectra were obtained by using
a Bruker 400 MHz NMR spectrometer, and the chloroform-
d (CDCl3) was used as a solvent. HR-MS spectra were obtained
by using an AB SCIEX X500B QTOF electrospray ionization time-
of-ight mass spectrometer.
Synthesis procedures
General synthesis procedure of C^N ligand. Corresponding
raw materials were added into ethanol (20 mL). The mixture was
reuxed for 5 h, then cooled to room temperature (r.t.). Further
cooling in the ice bath resulted in a large amount of sediment in
the mixture. Aer ltering by a vacuum pumping and washing
with ethanol, it was freeze-dried to obtain corresponding C^N
ligand. The crude product is directly used in the next reaction
without purication.
Fig. 5 Fluorescence images of 4T1 cells treated with Ir1@BSA to
Ir3@BSA and then irradiated without (ꢀ) or with (+) 532 nm light, and
further stained with calcein AM/PI (100 mW cmꢀ2, 5 min). Scale bar, 80
mm.
dpqx. Raw materials: benzil (420.5 mg, 2 mmol) and benzene-
1,2-diamine (216.3 mg, 2 mmol); product: white solid (536 mg,
95%).
dpbq. Raw materials: benzil (420.5 mg, 2 mmol) and
naphthalene-2,3-diamine (316.4 mg, 2 mmol); product: bright
yellow solid (565 mg, 85%).
Intracellular ROS detection
To verify that the production of ROS in cells, 2,7-dichlorodihy-
drouorescein diacetate (DCFH-DA) as a cellular ROS indicator
could be used to evaluate the 1O2 generation ability of Ir1@BSA
to Ir3@BSA in 4T1 cells. The generated 1O2 could induce DCFH-
DA to convert to 2,7-dichlorouorescein (DCF), which could
emit green uorescence. In Fig. 6, when cells were incubated
with Ir1@BSA to Ir3@BSA respectively, green uorescence was
observed in three group cells aer 532 nm light irradiation,
while no green uorescence was monitored in the cells without
light irradiation. Through the analysis of the uorescence
intensity, among the three groups of cells exposed to light, the
dbpz. Raw materials: 9,10-phenanthraquinone (416.4 mg, 2
mmol) and benzene-1,2-diamine (216.3 mg, 2 mmol); product:
light yellow solid (527 mg, 94%). 1H-NMR (400 MHz, CDCl3)
d 9.40 (d, J ¼ 7.9 Hz, 2H), 8.56 (d, J ¼ 7.9 Hz, 2H), 8.37–8.28 (m,
2H), 7.90–7.83 (m, 2H), 7.82–7.71 (m, 4H). HR-MS: m/z calcu-
lated for [C20H12N2 + H]+: 281.1073; found: 281.0991.
General synthesis procedure for dimeric iridium complex.
Corresponding raw materials were added into a mixed solvent
of 2-methoxyethanol (15 mL) and water (5 mL). The mixture was
reuxed under argon protection for 24 h. Aer cooling to r.t.,
5 mL of H2O was added. Further cooling in the ice bath resulted
in precipitate in the mixture. The precipitate was recrystallized
with petroleum ether (PE)/CH2Cl2 to obtain solid. The solid is
directly used in the next reaction without purication.
[Ir(dpqx)2]2Cl2. Raw materials: dpqx (124 mg, 0.44 mmol) and
IrCl3$xH2O (70 mg, 0.2 mmol); product: red solid (120 mg,
76%).
[Ir(dpbq)2]2Cl2. Raw materials: dpbq (146 mg, 0.44 mmol) and
IrCl3$xH2O (70 mg, 0.2 mmol); product: dark red solid (128 mg,
72%).
[Ir(dbpz)2]2Cl2. Raw materials: dbpz (123 mg, 0.44 mmol) and
IrCl3$xH2O (70 mg, 0.2 mmol); product: dark red solid (80 mg,
51%).
General synthesis procedure for Ir1 to Ir3. Corresponding
dimeric iridium complex, 4,40-dinonyl-2,20-bipyridine (dnbpy),
Fig. 6 (a) Fluorescence images of 4T1 cells treated with Ir1@BSA to
Ir3@BSA and then irradiated without (ꢀ) or with (+) 532 nm light, and
further stained with DCFH-DA (532 nm, 100 mW cmꢀ2, 5 min). Scale
bar, 40 mm. (b) The corresponding cell fluorescence quantitative
statistical analysis of cells in (a).
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and silver triuoromethanesulfonate (F3CSO3Ag) were added integrating sphere. The emission lifetime was obtained by
into a mixed solvent of CH2Cl2 (20 mL) and CH3OH (10 mL). The a ash photolysis spectrometer (Edinburgh LP980, UK) with
mixture was reuxed under argon protection for 24 h. Aer a 355 nm Nd:YAG laser or a uorescence spectrometer (FS5)
cooling to r.t., NH4PF6 (10 equivalent) was added and the with a 365 nm picosecond laser (Edinburgh, UK).
mixture was stirred for 1 h. Then, the solvent was removed and
the crude product was puried by column chromatography
(silica gel, 100–200 mesh; eluent, CH2Cl2/ethyl acetate, v/v). The
nal product was further recrystallized with petroleum ether
and CH2Cl2.
Singlet oxygen detection
The generated singlet oxygen catalyzed by Ir1, Ir2, and Ir3 in
CH3CN was measured by an FS5 uorescence spectrometer with
an InGaAs detector (lex ¼ 405 nm). The emission range was
recorded from 1100 nm to 1600 nm.
Ir1. Raw materials: [Ir(dpqx)2]2Cl2 (158 mg, 0.1 mmol), dnbpy
(81.6 mg, 0.2 mmol), and F3CSO3Ag (51.4 mg, 0.2 mmol);
column chromatography (100/1, v/v); product: red solid
(117 mg, 44.7%). 1H-NMR (400 MHz, CDCl3) d 8.47 (d, J ¼
Fabrication and characterization of Ir1@BSA to Ir3@BSA
5.7 Hz, 2H), 8.21 (s, 2H), 8.04 (d, J ¼ 0.9 Hz, 1H), 8.02 (d, J ¼ Ir1 (1.3 mg), Ir2 (1.4 mg), or Ir3 (1.3 mg) was dissolved in 1 mL
0.9 Hz, 1H), 7.83 (d, J ¼ 3.9 Hz, 4H), 7.68–7.65 (m, 6H), 7.60 (dd, CH2Cl2 as an oil phase and BSA (7 mg) was dissolved in 5 mL
water (pH ¼ 5) as an aqueous phase. Two phases were emulsi-
ed by using an ultrasonic cell crusher (SCIENTZ-950E, China).
J ¼ 11.2, 3.9 Hz, 2H), 7.49 (dd, J ¼ 5.7, 1.1 Hz, 2H), 7.36 (s, 1H),
7.34 (s, 1H), 7.18–7.11 (m, 4H), 6.78–6.73 (m, 2H), 6.68 (td, J ¼
7.5, 1.1 Hz, 2H), 6.47 (d, J ¼ 7.3 Hz, 2H), 2.82 (dd, J ¼ 14.5, Aerward, CH2Cl2 in the emulsion was removed to obtain a pale
7.3 Hz, 4H), 1.61 (dd, J ¼ 13.6, 7.3 Hz, 4H), 1.26–1.20 (m, 24H), orange transparent solution. Finally, the Ir1@BSA to Ir3@BSA
0.86 (t, J ¼ 6.9 Hz, 6H). HR-MS: m/z calculated for [C68H70F6- was obtained by ltrating with a mixed cellulose ester
IrN6P ꢀ PF6]+: 1163.5286; found: 1163.5194.
membrane lter (pore ¼ 1 mm, Titan, China). The morphology
Ir2. Raw materials: [Ir(dpbq)2]2Cl2 (178 mg, 0.1 mmol), dnbpy of Ir1@BSA to Ir3@BSA was observed by a transmission electron
(81.6 mg, 0.2 mmol), and F3CSO3Ag (51.4 mg, 0.2 mmol); microscope (HT7800, Hitachi, Japan). Hydrate particle sizes of
Ir1@BSA to Ir3@BSA in aqueous solution were measured by
a laser particle sizer (Malvern Zetasizer Nano ZS90, UK).
column chromatography (50/1, v/v); product: dark red solid
(174 mg, 61.7%). 1H-NMR (400 MHz, CDCl3) d 8.63–8.58 (m,
4H), 8.17 (s, 2H), 7.99 (d, J ¼ 8.4 Hz, 2H), 7.91–7.87 (m, 6H),
7.72–7.68 (m, 6H), 7.64 (dd, J ¼ 5.4, 0.8 Hz, 2H), 7.51 (dd, J ¼ 9.7,
4.7 Hz, 2H), 7.42–7.37 (m, 2H), 7.23 (s, 2H), 7.12 (d, J ¼ 8.3 Hz,
2H), 6.78–6.73 (m, 2H), 6.68–6.63 (m, 2H), 6.56 (d, J ¼ 7.1 Hz,
2H), 2.80 (dq, J ¼ 20.6, 6.8 Hz, 4H), 1.64–1.56 (m, 4H), 1.27–1.18
(m, 24H), 0.85 (t, J ¼ 7.0 Hz, 6H). HR-MS: m/z calculated for
[C76H74F6IrN6P ꢀ PF6]+: 1263.5599; found: 1263.5569.
Singlet oxygen quantum yield measurement
Singlet oxygen quantum yields (FD) of Ir1 to Ir3 and Ir1@BSA to
Ir3@BSA were calculated by using a reference method. DPBF
was utilized to capture 1O2, and [Ru(bpy)3]Cl2 was used as
a reference (FD ¼ 0.56 in CH3CN).41 The FD of materials
depended linearly upon the absorbance degradation rate of
DPBF at 410 nm (in CH3CN) or 415 nm (in H2O) under the same
conditions. In general, DPBF (30 mM) was mixed with Ir1 to Ir3
(in CH3CN) or Ir1@BSA to Ir3@BSA (in 95% H2O containing 5%
ethanol) in various concentration, then the mixture was irra-
diated with 532 nm laser. The absorbance at 410 nm or 415 nm
was recorded and the FD was calculated using the following
equation.
Ir3. Raw materials: [Ir(dbpz)2]2Cl2 (78.6 mg, 0.05 mmol),
dnbpy (40.8 mg, 0.1 mmol), and F3CSO3Ag (25.7 mg, 0.1 mmol);
column chromatography (30/1, v/v); product: dark red solid
(53 mg, 40.6%). 1H-NMR (400 MHz, CDCl3) d 9.45 (d, J ¼ 9.0 Hz,
1H), 8.60 (d, J ¼ 5.1 Hz, 3H), 8.56 (d, J ¼ 8.1 Hz, 1H), 8.39 (d, J ¼
9.1 Hz, 2H), 8.33 (s, 3H), 8.12 (d, J ¼ 7.9 Hz, 1H), 07.91 (t, J ¼
7.0 Hz, 2H), 7.86–7.76 (m, 3H), 7.62 (d, J ¼ 5.7 Hz, 1H), 7.29 (d, J
¼ 5.6 Hz, 3H), 7.22 (d, J ¼ 4.4 Hz, 4H), 7.10 (t, J ¼ 7.7 Hz, 2H),
7.02 (d, J ¼ 5.7 Hz, 1H), 6.45 (d, J ¼ 7.5 Hz, 1H), 2.76–2.70 (m,
4H), 1.73–1.69 (m, 4H), 1.33–1.21 (m, 24H), 0.88–0.85 (m, 6H).
HR-MS: m/z calculated for [C68H66F6IrN6P ꢀ PF6]+: 1159.4973;
found: 1159.4961.
W
WRef
ARef
A
h2
FD ¼ FRDef
ꢂ
ꢂ
ꢂ
2
hRef
W is the DPBF degradation rate caused by 1O2, A ¼ 1 ꢀ 10ꢀOD, A
and OD are the absorptivity and absorbance of Ir1 to Ir3 or
Ir1@BSA to Ir3@BSA at 532 nm, and h is the refractive index of
solvent.42,43
Photophysical studies
The UV-vis absorption spectra of Ir1, Ir2, and Ir3 in different
solvents including toluene, THF, CH2Cl2, CH3CN, and CH3OH
Cellular uptake
were measured by a UV-1900 spectrometer (Shimadzu, Japan) in 4T1 cells were seeded in 24-well plates at the density of 1.2 ꢂ 104
a 1 cm quartz cuvette. The emission spectra (lex ¼ 436 nm) of Ir1 cells per well and cultured for 24 h (37 ꢃC, 5% CO2). Ir1@BSA or
and Ir3 in different solvents were recorded by a uorescence Ir3@BSA (20 mM) were added and further incubated for 4 or
spectrometer (Edinburgh FS5, UK). The emission spectra of Ir2 24 h. All cells were washed with PBS (pH ¼ 7.4). For imaging
in several solvents were recorded by a ber optic spectrometer method, phosphorescence imaging of cells was recorded by
(NOVA-EX, Ideaoptics, China) with a 532 nm laser (China) as an using a CKX41 inverted microscope (Olympus, Japan) with an
excitation light source. The quantum yields of Ir1 and Ir3 were Olympus U-RFLT50 mercury lamp. A blue light was utilized as
obtained by an FS5 uorescence spectrometer with an an excitation source and the emission was collected in a red
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channel. For ICP-MS method, aer incubation for 4 h, the cells > Ir2. Structure–activity relationship studies have shown that
were collected and digested, and then the content of Ir3+ ion was extending the conjugation length or increasing the plane
detected by the ICP-MS spectrometer (iCAP™ RQ ICP-MS, rigidity of C^N ligands cannot effectively increase the produc-
Thermo Fisher, USA).
tion of singlet oxygen. Aer further encapsulation by BSA, Ir1 to
Ir3 can self-assemble to form nanostructures, which are
spherical-like and well dispersed. The diameters are between
50–70 nm and have a stable nano-morphology. Aer forming
the nano-photosensitizers, the singlet oxygen quantum yields of
the Ir1@BSA, Ir2@BSA, and Ir3@BSA were 0.20, 0.06, and 0.23
in H2O, respectively. According to CCK-8 analysis, the three
nano-photosensitizers have lower dark toxicity and stronger
phototoxicity. The EC50 values are 5.2 mM, 28.5 mM, and 82.2 mM
for Ir1@BSA, Ir2@BSA, and Ir3@BSA respectively. We attribute
this to the dual factors of the difference of cell uptake ability
and singlet oxygen generation ability. We furtherly veried the
PDT effects of the three nano-photosensitizers with the live/
dead cell staining method and the intracellular reactive
oxygen staining method, which are consistent with the cell
phototoxicity results by the measurement of CCK-8. Therefore,
we believe that nano-photosensitizers are different from tradi-
tional small-molecule photosensitizers in PDT, which have
important signicance and value for PDT to increase tumor
uptake and biocompatibility in tumor treatment.
Cytotoxicity assay
4T1 cells were seeded in 96-well plates at the density of 5 ꢂ 103
cells per well and cultured for 24 h. Various concentrations of
Ir1@BSA to Ir3@BSA were added into cells and further incu-
bated for 24 h. For dark-toxicity, the cells were washed with PBS.
CCK-8 was utilized to evaluate cell viability. The absorbance at
450 nm was recorded by a BioTek Cytation 3 microplate reader
(USA). For phototoxicity, aer incubation, the cells were irra-
diated with 532 nm light (100 mW cmꢀ2) for 5 min. Aer
another 24 h incubation, the cells were stained with CCK-8 and
the absorbance at 450 nm was measured. The EC50 values of
phototoxicity were calculated by a sigmoidal tting of the
concentration–cell viability curve.
Live/dead cell staining
Furthermore, a calcein-AM/PI double stain kit was used to stain
live and dead cells respectively. The pre-seeded 4T1 cells were
incubated with Ir1@BSA to Ir3@BSA (40 mM) for 24 h, and then
cells were irradiated with or without 532 nm irradiation (100
mW cmꢀ2) for 5 min. Aer another 24 h irradiation, all cells
were co-stained with calcein-AM (10 mM) and PI (5 mM) for
30 min and washed with PBS. The uorescence cell images were
collected by an inverted uorescence microscope (calcein-AM,
blue light excitation, green light channel emission; PI, green
light excitation, red light channel emission).
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work was nancially supported by the Natural Science
Foundation of Shanghai (19ZR1434700), the National Natural
Science Foundation of China (21501121).
Intracellular ROS detection
The uorescence probe DCFH-DA (Beyotime, China) was used
to detect the generation of intracellular ROS. 4T1 cells were
seeded in 24-well plates at the density of 1.2 ꢂ 104 cells per well
and cultured for 24 h. Ir1@BSA to Ir3@BSA (40 mM) was added
and further incubated with cells for 24 h. Subsequently, the cells
were washed with PBS and further incubated with DCFH-DA (10
mM) for 30 min. Aer washing with PBS, cells were irradiated
with a 532 nm light (100 mW cmꢀ2) for 5 min. The intracellular
uorescence was recorded immediately by using a CKX41
inverted microscope. The uorescence intensity was calculated
by the cell uorescence quantitative method via ImageJ
soware.
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