Two photoactive Ru (II) compounds based on tetrazole ligands for photodynamic therapy

Article abstract of DOI:10.1016/j.jinorgbio.2020.111127

Ru (II) compounds have potential application in photodynamic therapy (PDT). In the current study, two Ru (II) compounds based on the auxiliary ligand 2,2′-bipyridine (bipy) by changing main ligands 5-(2-pyridyl) tetrazole (Hpytz) and di(2H-tetrazol-5-yl) amine (H₂datz) have been successfully synthesized and characterized, [Ru (pytz)(bipy)₂][PF₆] (1) and [Ru(Hdatz)(bipy)₂][PF₆] (2). These compounds can form nanoparticles (NPs) by nano-precipitation. And [Ru(pytz)(bipy)₂][PF₆] NPs with a lower half maximal inhibitory concentration (IC₅₀) of 37 μg/mL on HeLa cells than that of [Ru(Hdatz)(bipy)₂][PF₆] NPs (65 μg/mL). Meanwhile, negligible dark toxicity has been also observed for these NPs even under high concentrations. The results show that [Ru(pytz)(bipy)₂][PF₆] (1) and [Ru(Hdatz)(bipy)₂][PF₆] (2) NPs can inhibit cell proliferation in vitro, and may be potential candidates for photodynamic therapy.

Full text of DOI:10.1016/j.jinorgbio.2020.111127

JournalofInorganicBiochemistry210(2020)111127
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Two photoactive Ru (II) compounds based on tetrazole ligands for
photodynamic therapy
⁎
Jie Yanga,b, Xin Hec, Zhen Kea, Jianjiao Chena, Zhenyuan Zoua, Bo Weib, Dengfeng Zoua,
,
⁎
Jianhua Zoub,
a School of Pharmacy, Guilin Medical University, Guilin 541004, Guangxi, PR China
b Department of Chemistry & Materials Engineering, Jiangsu Key Laboratory of Advanced Functional Materials, Changshu Institute of Technology, Changshu 215500, PR
China
c College of Chemical Engineering, State Key Laboratory of Material-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 210009, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Ru(II)
Ru (II) compounds have potential application in photodynamic therapy (PDT). In the current study, two Ru (II)
compounds based on the auxiliary ligand 2,2′-bipyridine (bipy) by changing main ligands 5-(2-pyridyl) tetrazole
(Hpytz) and di(2H-tetrazol-5-yl) amine (H2datz) have been successfully synthesized and characterized, [Ru
(pytz)(bipy)2][PF6] (1) and [Ru(Hdatz)(bipy)2][PF6] (2). These compounds can form nanoparticles (NPs) by
Tetrazole
HeLa
Photodynamic therapy
nano-precipitation. And [Ru(pytz)(bipy)2][PF6] NPs with a lower half maximal inhibitory concentration (IC50
)
of 37 μg/mL on HeLa cells than that of [Ru(Hdatz)(bipy)2][PF6] NPs (65 μg/mL). Meanwhile, negligible dark
toxicity has been also observed for these NPs even under high concentrations. The results show that [Ru(pytz)
(bipy)2][PF6] (1) and [Ru(Hdatz)(bipy)2][PF6] (2) NPs can inhibit cell proliferation in vitro, and may be po-
tential candidates for photodynamic therapy.
1. Introduction
cytotoxic singlet oxygen (1O2) under light irradiation. As is widely ac-
knowledged, porphyrin and phthalocyanine compounds are recognized
Currently, cancer has become the third leading death source fol-
lowing heart disease and cerebrovascular disease, posing a huge threat
to human health [1]. According to statistics, people in growing numbers
die of cancer every year, so it is of great significance and urgency to
develop new drugs for cancer treatment [2–4]. In this regard, metal
compounds have proven to be effective drugs for the treatment of
various cancers [5–7]. For example, cisplatin or cis-diaminodi-
chloroplatinum (II) has become the most widely used metal-based an-
ticancer drug in clinical practice, but still has strong non-cancer cell
toxicity. It is universally acknowledged that Ru (II) compounds have
raised great interest and have been used to fight against lots of cancer
cell lines, promising them potential candidates for alternative drugs in
cancer phototherapy [8–11]. For example, D. Havrylyuk et.al reported
the photochemical properties and structure-activity relationships of Ru
(II) complexes with pyridyl-benzoazole ligands as promising anticancer
agents [12].
as first and second generation of commercial photosensitizers (PSs),
respectively, and have been developed as diagnostic agents [20–23].
Tetrazole-based compounds are investigated, so far. These ligands are
usually capable of binding metal ions in a variety of coordination
modes. Some examples of relevant compounds based on the tetrazole-
based ligands have already been studied [24–29].
However, studies on Ru (II)-tetrazole compounds are far from sa-
tisfactory, especially for anticancer application. In previous research,
Ru (II) compounds based on 5-(2-pyrazinyl) tetrazole were reported to
show high phototoxicity, low dark toxicity and excellent bio-compat-
ibility for photodynamic therapy [30]. Inspired by the observations
above, we are devoted to investigating Ru(II)-tetrazole compounds
derived from 5-(2-pyridyl) tetrazole (Hpytz) and di(2H-tetrazol-5-yl)
amine (H2datz). As a result, two compounds are obtained, [Ru(pytz)
(bipy)2][PF6] (1) and [Ru(Hdatz)(bipy)2][PF6] (2) (bipy = 2,2′-bipyr-
idine). Stagni, S. et.al have reported the photophysical characterization
and the reactivity toward electrophiles of compound 1 and ignore its
biological properties [31]. Nano-precipitation was used to prepare the
nanoparticles (NPs) of these compounds for better water dispersity. The
in vitro cytotoxicity study showed that [Ru(pytz)(bipy)2][PF6] (1) NPs
As an emerging technology in recent years, photodynamic therapy
(PDT) is widely used with low systemic toxicity and non-invasiveness,
and is expected to be used in cancer treatment [13–19]. The key of PDT
is to use an effective photosensitizer to convert triplet oxygen (3O2) to
⁎ Corresponding authors.
E-mail addresses: zdf1226@126.com (D. Zou), zoujh93@126.com (J. Zou).
https://doi.org/10.1016/j.jinorgbio.2020.111127
Received 8 February 2020; Received in revised form 25 May 2020; Accepted 25 May 2020
Availableonline01June2020
0162-0134/©2020ElsevierInc.Allrightsreserved.

J. Yang, et al.
JournalofInorganicBiochemistry210(2020)111127
are superior to that of [Ru(Hdatz)(bipy)2][PF6] (2) NPs because of the
lower IC50, which may be ascribed to difference of the ligand. Both NPs
can be up taken by HeLa cells and capable of generating reactive
oxygen species (ROS) in vitro with 2,7-dichlorodihydrofluorescein dia-
cetate (DCF) as a probe. Besides, cell migration across a 2D artificial
gap indicated that these NPs could inhibit the migration of cells, sug-
gesting the potential for the inhibition of tumor metastasis in vivo.
NMR (500 MHz, DMSO) 8.72–8.65 (m, 4H), 8.14 (s, 1H), 8.10–7.95 (m,
6H), 7.76 (d, 2H, J = 8.0 Hz), 7.59 (t, 2H, J = 8.0 Hz), 7.36 (t, 2H,
J = 8.0 Hz) ppm.; 13C NMR (125 MHz, DMSO) 163.5, 162.8, 158.4,
157.9 (C-quaternaries), 152.2, 151.2, 136.8, 136.6, 127.3, 126.8,
124.0, 123.7 ppm. Elemental analysis: calculated: C: 67.96, H: 4.61, N:
27.43 %, found: C: 67.86, H: 4.67, N: 27.51 %. MS (ESI) for
−
C22H19N13RuPF6, c m/z 570 [M-PF6
]
+; IR (KBr, cm−1): 3352 (s),
1623 (s), 1572 (s), 1536 (s).
2. Experimental section
2.4. Preparation of nanoparticles (NPs) of compounds 1 and 2
method of co-precipitation with Polyethylene glycol−5000
2.1. Materials and apparatus
A
The ligands were prepared according to the literature methods
[32,33]. All chemical reagents are analytical grade reagents and can be
used without further purification. The 1H NMR and 13C NMR spectra
were performed on Bruker DRX NMR spectrometer (500 MHz) in
DMSO‑d6 at 298 K as the internal standard. Elemental analyses (C, H
and N) were performed with a PE2400 elemental analyzer. Fluores-
cence spectra were collected on a Hitachi F-4600 spectrofluorometer.
UV–visible spectrum was obtained on a spectrophotometer (UV-3600,
Shimadzu, Japan). The dynamic light scattering (DLS) was recorded on
a 90 Plus particle size analyzer. Scanning electron microscope (SEM)
was measured on a FEI quanta 200F microscope.
(PEG−5000) was used to prepare the NPs of the compounds. The pre-
paration of the NPs of compounds 1 and 2 is similar. Taking compound
1 as an example, a mixture of compound 1 (5 mg) and PEG−5000
(10 mg) were dissolved in tetrahydrofuran (1 mL). Then 200 μL of such
solution was added to distilled water (5 mL) under ultrasound at room
temperature. Next, tetrahydrofuran (THF) was removed by purging the
solution with nitrogen for 20 min and then stirred at room temperature
overnight. Finally, NPs of the compounds at a concentration of 200 μg/
mL were obtained.
2.5. Singlet oxygen detection
2.2. Synthesis of [Ru(pytz)(bipy)2][PF6] (1)
Singlet oxygen sensor green (SOSG) was used to detect 1O2 gen-
eration. Briefly, 10 μM NPs and 10 μM SOSG was mixed in distilled
water (1 mL), then the fluorescence spectrum was recorded. The sample
was excited at 490 nm and the fluorescence was collected from 510 to
750 nm. After irradiation with a light emitting diodes (LED) lamp (30
mWcm−2) for different periods of time (1, 2, 3 and 4 min), the fluor-
escence was recorded again.
cis-[RuCl2(bipy)2] (0.52 g, 1 mmol) was dissolved in 35 mL of
ethanol, NH4PF6 (0.539 g, 2.2 mmol) was added, and the mixture was
stirred and refluxed for 5 h, and finally the mixture was filtered. A
mixture of Hpytz (2 mmol, 0.294 g) and ethanol (20 mL) was added to
the filtrate. The dark red solution was stirred at reflux overnight. Next,
20 mL of an aqueous solution containing 1 g of NH4PF6 was added to
the solution, stirred for 10 min, and extracted three times with di-
chloromethane (DCM) (30 mL) until the aqueous phase became color-
less. The organic layer was dried over anhydrous MgSO4. The filtrate
was dried by rotary evaporation and dissolved with a small amount of
acetone to remove the precursor [Ru(pytz)(bipy)2][PF6]. After filtra-
tion, the filtrate was poured into a large amount of ether, stirred and
filtered to obtain a crude product. Recrystallization of the crude pro-
duct caused the formation of [Ru(pytz)(bipy)2][PF6] (1) (Scheme 1).
Yield: 40% based on Ru2+.1H NMR (500 MHz, DMSO) 8.45 (t, 2H,
J = 8.0 Hz), 8.42 (d, 2H, J = 8.0 Hz), 8.23 (d, H3, J = 8.0 Hz),
8.04–7.90 (m, 4H and H4), 7.87 (d, 1H, J = 6.0 Hz), 7.83(d, 1H,
J = 6.0 Hz), 7.77 (d, 1H, J = 6.0 Hz), 7.59 (d, 1H, J = 5.6 Hz), 7.57 (d,
H6, J = 5.6 Hz), 7.43–7.22 (m, 4H and H5) ppm.; 13C NMR (125 MHz,
DMSO) 158.9, 158.8, 158.4, 158.3 (C-quaternaries), 153.2 (2C), 152.6,
152.5 (N-ortho CHs), 138.1, 137.8, 137.8 (N-para CHs), 128.4, 127.3,
124.7, 124.5, 124.2 (N-meta CHs) ppm; Elemental analysis: calculated:
2.6. Cell culture and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay
HeLa cell lines were grown in Dulbecco's modified eagle medium
(DMEM) supplemented with 10% fetal calf serum (FBS). They were
incubated in an incubator at the temperature of 37 °C in a moist in-
cubator and 95% air and 5% CO2. Cell viability of the nanoparticles of
compounds 1 and 2 dissolved in phosphate buffer saline (PBS). Cells at
the exponential growth were diluted to various concentrations with
DMEM, and then seeded in 96-well cell culture, respectively, and in-
cubated for 24 h at 37 °C in 5% CO2. MTT solution in PBS (5 mg/mL,
20 μL) was added. After 3 h incubation, medium was removed from
each cell and 200 μL dimethyl sulfoxide (DMSO) was subsequently
added. The absorbance of all the wells at 490 nm were read. Cell via-
bility was determined by the following equation: viability (%) = mean
absorbance of the group incubated with NPs/mean absorbance of the
control group.
C: 67.96, H: 4.61, N: 27.43%, found: C: 67.86, H: 4.67, N: 27.51 %. MS
+; IR (KBr, cm−1): 3348
−
(ESI) for C26H21N9RuPF6, m/z 560 [M-PF6
]
(s), 1616 (s), 1556 (s), 1529 (s).
2.3. Synthesis of [Ru(Hdatz)(bipy)2][PF6] (2)
2.7. Cellular uptake and fluorescence imaging of cellular ROS
cis-[RuCl2(bipy)2] (0.52 g, 1 mmol) was dissolved in 35 mL of
ethanol, NH4PF6 (0.539 g, 2.2 mmol) was added, and the mixture was
stirred and refluxed for 5 h, and finally the mixture was filtered. A
solution of H2data (2 mmol, 0.306 g) in ethanol (20 mL) was added to
the filtrate. The dark red solution was stirred at reflux overnight. Next,
20 mL of an aqueous solution containing 1 g of NH4PF6 was added to
the solution, stirred for 10 min, and extracted three times with DCM
(30 mL) until the aqueous phase became colorless. The organic layer
was dried with MgSO4, The crude product was dissolved in a minimum
amount of acetone and poured into a large amount of diethyl ether.
Recrystallization of the crude product caused the formation of [Ru
(Hdatz)(bipy)2][PF6] (2) (Scheme 1). Yield: 35% based on Ru2+.1H
HeLa cells were incubated with NPs for 24 h in the dark. Then the
cells were washed 3 times with 1 mL PBS. Finally, 1 mL of poly-
oxymethylene was added for 20 min. Then polyoxymethylene was re-
moved and the cells were washed with PBS three times. The samples of
NPs were incubated with 10 mM 2,7-dichlorodihydrofluorescein dia-
cetate (DCF-DA) for an additional 25 min and were washed with 1 mL
PBS three times. This sample was illuminated with an LED lamp for
3 min. The fluorescence of compounds was obtained with the confocal
laser scanning microscopy. The samples were incubated for 24 h and
excited at 405 nm. Then fluorescence spectrum of 500 to 700 nm was
recorded. The sample incubated under DCF-DA irradiation were excited
with a 488 nm laser and fluorescence was recorded at 490 to 600 nm.
2

J. Yang, et al.
JournalofInorganicBiochemistry210(2020)111127
Scheme 1. Illustration of preparation of [Ru(pytz)(bipy)2][PF6] (1) and [Ru(Hdatz)(bipy)2][PF6] (2). a: RuCl3·3H2O, LiCl, DMF; b: NH4PF6, Hpytz and ethanol; c:
NH4PF6, H2datz and ethanol.
2.8. An in vitro assay of 2D cell migration across an artificial gap
(2-pyridyl)pyrazole anion) but are similar to that of [Ru(pztz)(bipy)2]
[PF6] [30]. (Table 1).
The ability to study cell movement is determined by in vitro cell
migration assays. The HeLa cells were incubated with the above-men-
tioned NPs with different concentrations (8 μg mL−1) for 24 h. Then a
wound incision was made into a confluent layer of cells using a yellow
tip. When the cells moved in and filled the damaged area, the open gap
at the incision site was observed under a microscope. Micrographs were
taken at 0 and 24 h after the wound was made, and they were observed
under an inverted microscope (Leica, German). The degree of wound
closure is indicated by measuring the distance between the sides of the
wound.
The morphology and diameter of these NPs were characterized
using scanning electron microscope (SEM) and dynamic light scattering
(DLS). The NPs can self-assemble to form hexagon morphology with
size distributing from 70 to 175 nm for [Ru(pytz)(bipy)2][PF6] (1) NPs,
and 64 to 127 nm for [Ru(Hdatz)(bipy)2][PF6] (2) NPs, respectively
(Fig. 1c and d).
High singlet oxygen (1O2) generation is indispensible for a photo-
sensitizer to be used as photodynamic agent. Singlet oxygen sensor
green (SOSG) was used for the detection of [Ru(pytz)(bipy)2][PF6] and
[Ru(Hdatz)(bipy)2][PF6] NPs. (Fig. 2) It can be observed the fluores-
cence intensity of SOSG was enhanced in the presence of [Ru(pytz)
(bipy)2][PF6] and [Ru(Hdatz)(bipy)2][PF6] NPs, respectively, showing
that these NPs can be used as 1O2 initiator. In addition, the fluorescence
intensity of SOSG in the presence of [Ru(pytz)(bipy)2][PF6] NPs with
irradiation is 4.1 times than the original one, which is superior to that
of [Ru(Hdatz)(bipy)2][PF6] NPs (2.9 times), which indicates the singlet
oxygen generation ability of [Ru(pytz)(bipy)2][PF6] NPs is stronger
than that of [Ru(Hdatz)(bipy)2][PF6].
3. Results and discussions
3.1. 3.1 Absorbance, fluorescence and singlet oxygen generation detection
The absorption spectra of [Ru(pytz)(bipy)2][PF6] (1) and [Ru
(Hdatz)(bipy)2][PF6] (2) in THF and their NPs are shown in Fig. 1a. The
compound 1 exhibits two peaks at 370 nm and 421 nm and compound
2 exhibits two peaks at 469 and 474 nm, respectively. The lumines-
cence of complexes 1 and 2 in THF and their NPs are shown in Fig. 1b.
at room temperature. Compounds 1 and 2 exhibits maximum emission
at 664 nm and 660 nm in THF, respectively. In contrast, compared to
the emission spectra in THF, compounds 1 and 2 NPs exhibit maximum
emission at 672 nm and 619 nm. The difference in absorbance in THF
and water may be attributed to both the aggregation of NPs and the
solvent effect. Compared with the previously reported Ru(II) com-
pounds, the absorbance of [Ru(pytz)(bipy)2][PF6] NPs and [Ru(Hdatz)
(bipy)2][PF6] NPs are similar (between 300 and 400 nm). However, the
fluorescence of the two NPs move to the near infrared (NIR) region,
which is different from [Ru(pztz)(phen)2][PF6] [30] (pztz = 5-(2-pyr-
izinyl)tetrazole anion), [Ru(pmtz)(bipy)2][PF6] [35] (pmtz = 5-(2-
pyrimidyl)tetrazole anion) and [Ru(pypz)(bipy)2][PF6] [36] (pypz = 3-
3.2. In vitro cellular uptake, ROS generation and MTT assay
To investigate the potential of these NPs as cell imaging agents,
confocal imaging was performed. It was found that such NPs can be
used for cell imaging when irradiated with a 405 nm laser (Fig. 3a). On
the other hand, the cell viability was still high in the absence of irra-
diation, indicating the low dark toxicity of the compound. Fig. 3a also
shows the cellular uptake and ROS generation with DCF as a probe of
compounds 1 and 2 in HeLa cells. These NPs can be up taken by HeLa
cells as indicated by the green fluorescence. Further, since fluorescence
can be illuminated, ROS can also be generated upon irradiation at
488 nm.
To investigate the PDT efficacy of [Ru(pytz)(bipy)2][PF6] (1) and
3

J. Yang, et al.
JournalofInorganicBiochemistry210(2020)111127
Fig. 1. (a) Normalized absorbance of compounds 1 and 2 in THF and NPs in water; (b) Normalized fluorescence intensity of compounds 1 and 2 in THF and NPs in
water upon excitation of 400 nm; DLS and SEM of (c) compound 1 NPs; (d) compound 2 NPs.
[Ru(Hdatz)(bipy)2][PF6] (2) NPs, the cytotoxicity was determined by
MTT assay. Different concentrations of NPs of compounds 1 and 2 were
incubated with Hela cells for 24 h. As shown in Fig. 3b and c, the cell
viability tends to decrease as the concentration increases and the IC50
(half maximal inhibitory concentration) of compounds 1 and 2 are
approximate 37 μg/mL (5.2 μM) and 65 μg/mL (9.1 μM), respectively.
A lower IC50 (5.2 μM) was observed in the NPs incubated with com-
pound 1 NPs, demonstrating that compound 1 NPs are superior to
compound 2 ones. IC50 was higher than that of the reported Ru
(II)‑boron dipyrromethene (BODIPY) compound reported by Wang et.al
[34], because the I-substituted BODIPY could undergo more efficient
Table 1
Comparison of absorbance and fluorescence data with other Ru(II) compounds.
Compound
Absorbance
Fluorescence
Reference
[Ru(pztz)(bipy)2][PF6]
[Ru(pztz)(phen)2][PF6]
[Ru(pmtz)(bipy)2][PF6]
[Ru(pypz)(bipy)2][PF6]
[Ru(pytz)(bipy)2][PF6]
[Ru(Hdatz)(bipy)2][PF6]
432,508
422,498
450
473,666
504
30
30
478
35
362,468
370,421
469,474
372
36
672
This work
This work
619
Fig. 2. Fluorescence enhancement of SOSG in the presence of (a) [Ru(pytz)(bipy)2][PF6] NPs and (b) [Ru(Hdatz)(bipy)2][PF6] NPs with irradiation for different
periods of time (0, 1, 2, 3 and 4 min).
4

J. Yang, et al.
JournalofInorganicBiochemistry210(2020)111127
Fig. 3. (a) Cellular uptake of compounds 1 and 2 NPs and ROS generation with DCF as probe. MTT assay of (b) [Ru(pytz)(bipy)][PF6] NPs and (c) [Ru(Hdatz)(bipy)2]
[PF6] NPs with or without irradiation.
intersystem crossing (ISC) and produce high singlet oxygen quantum
yields. However, it was superior to that of [Ru(bipy)2(pmtz)]
[PF6]·0.5H2O (pmtz = 5-(2-pyrimidyl)-tetrazole) (21 μM) and [Ru(bi-
py)2(pypz)][PF6] (Hpypz = 3-(2-pyridyl)pyrazole) (12 μM), the struc-
ture of [Ru(bipy)2(pmtz)][PF6]·0.5H2O and [Ru(bipy)2(pypz)][PF6]
were similar to that of compound [35,36].
order to further investigate the influence of [Ru(pytz)(bipy)2][PF6] and
[Ru(Hdatz)(bipy)2][PF6] NPs on cell migration, the 2D artificial gap
was established. And then the two NPs were incubated with HeLa cells
for 24 h. As shown in Fig. 5, a group of HeLa cells show a tendency to
migrate to the other side in the control group after 24 h. In contrast,
almost no cells move to the other side in the group incubated with the
two NPs. Also, the cells did not begin to fill in the 2D gap, either. The
results indicate that that such NPs are cable of inhibiting the cell mi-
gration of HeLa cells, suggesting the potential to interfere with the
metastasis of tumors in vivo.
Low dark toxicity under high concentration is crucial to PDT. Fig. 4
demonstrates that the extreme low dark toxicity of the two kind of NPs
in that the cell viability remained high when the concentration is as
high as 200 μg/mL without irradiation, indicating they are excellent
photosensitizer for PDT.
3.4. Discussion on the relationship between the photophysical and
anticancer property of the NPs
3.3. Cell migration across a 2D artificial gap
Cell migration is important for the metastasis of tumor in vivo. In
In this paper, two nitrogen-rich tetrazole ligands have been selected
Fig. 4. MTT assay of (a) [Ru(pytz)(bipy)][PF6] NPs and (b) [Ru(Hdatz)(bipy)2][PF6] NPs at high concentrations without irradiation.
5

J. Yang, et al.
JournalofInorganicBiochemistry210(2020)111127
Fig. 5. Cell migration of Hela cells incubated with compound 1 and compound 2 NPs for 24 h.
Declaration of competing interest
to construct Ru(II) compounds for photodynamic therapy. First, the
synthesis of [Ru(pytz)(bipy)2][PF6] (1) and [Ru(Hdatz)(bipy)2][PF6]
(2) are similar and easy. Owing to the poor solubility of the two com-
pounds, nanoprecipitation was adopted to prepare the NPs of them by
encapsulating PEG−5000 through a “bottom up” method. The as-pre-
pared NPs have good dispersity in water and the average diameter is
below 100 nm, suggesting the suitability for cellular uptake. High
singlet oxygen generation ability is fundamental to PDT. Therefore,
SOSG, used as 1O2 indicator, demonstrates the stronger 1O2 generation
ability of [Ru(pytz)(bipy)2][PF6] (1) NPs than that of [Ru(Hdatz)
(bipy)2][PF6] (2) NPs with irradiation, which was consistent with the
MTT assay. The IC50 of [Ru(pytz)(bipy)2][PF6] NPs is lower than that of
[Ru(Hdatz)(bipy)2][PF6] NPs. And the NPs can be uptaken by HeLa
cells and act as effective 1O2 initiator in vitro with irradiation.
Eventually, the cell migration experiment shows their ability to inhibit
cell migration, suggesting the potential to inhibit the tumor metastasis
in vivo.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgment
The authors acknowledge financial support from the Natural
Science Foundation of China (Grant. No. 81460119), Natural Science
Foundation of Jiangsu Province (Grant No. BK2012210), the Major
Basic Research Project of the Natural Science Foundation of the Jiangsu
Higher Education Institutions (Grant No. 10KJB430001), Project sup-
ported by the Six Talent Peaks Project in Jiangsu Province (NO. 2018-
SWYY-001) and the Opening Fund of Jiangsu Key.
References
4. Conclusions
[1] R.L. Siegel, K.D. Miller, A.J. DVM, CA Cancer J. Clin. 68 (2018) 7–30.
[2] L. Huang, Z. Li, Y. Zhao, Y. Zhang, S. Wu, J. Zhao, G. Han, J. Am. Chem. Soc. 138
(2016) 14586–14591.
In summary, two new Ru (II) compounds based on Hpytz and
H2datz were successfully synthesized. Nanoprecipitation was used to
prepare the NPs of the two compounds. The high singlet oxygen gen-
eration ability of the two NPs guarantees the high phototoxicity. [Ru
(pytz)(bipy)2][PF6] (1) and [Ru(Hdatz)(bipy)2][PF6] (2) NPs have high
phototoxicity as well as negligible dark toxicity. Besides, in vitro study
shows that both NPs of compound 1 and 2 with low IC50 on HeLa cells
can effectively inhibit the migration of tumor cell. The results show that
these compounds may have great potential for photodynamic therapy.
[3] M. Li, Y. Gao, Y.Y. Yuan, Y.Z. Wu, Z.F. Song, B.Z. Tang, B. Liu, Q.C. Zheng, ACS
Nano 11 (2017) 3922–3932.
[4] Z.F. Chang, L.M. Jing, B. Chen, M.S. Zhang, X.L. Cai, J.J. Liu, Y.C. Ye, X.D. Lou,
Z.J. Zhao, B. Liu, J.L. Wang, B.Z. Tang, Chem. Sci. 7 (2016) 4527–4536.
[5] L.L. Zeng, P. Gupta, Y.L. Chen, E.J. Wang, L.N. Ji, H. Chao, Z.S. Chen, Chem. Soc.
Rev. 46 (2007) 5771–5804.
[6] E.R. Jamieson, S.J. Lippard, Chem. Rev. 99 (1999) 2467–2498.
[7] V. Cepeda, M.A. Fuertes, J. Castilla, C. Alonso, C. Quevedo, J.M. Pe’rez, Anti Cancer
Agents Med. Chem. 7 (2007) 3–18.
[8] R. Arakawa, S. Mimura, G. Matsubayashi, T. Matsuo, Photolysis, Inorg. Chem. 35
6

J. Yang, et al.
(1996) 5729.
JournalofInorganicBiochemistry210(2020)111127
[22] M. Göksel, Bioorg. Med. Chem. 24 (2016) 4152–4164.
[9] W. Sun, S.Y. Li, B. Häupler, J. Liu, S.B. Jin, W. Steffen, U.S. Schubert, H.J. Butt,
X.J. Liang, S. Wu, Adv. Mater. 29 (2017) 1603–1608.
[23] Z. Yang, W.P. Fan, W. Tang, Z.Y. Shen, Y.L. Dai, J.B. Song, Z.T. Wang, Y. Liu,
L.S. Lin, L.L. Shan, Y.J. Liu, O. Jacobson, P.F. Rong, W. Wang, X.Y. Chen, Angew.
Chem. Int. Ed. 57 (2018) 14101–14105.
[10] K. Fujita, Y.Y. Tanaka, T. Sho, S.C. Ozeki, S. Abe, T. Hikage, T. Kuchimaru,
S.K. Kondoh, Ta. Ueno, J. Am. Chem. Soc. 136 (2014) 16902–16908.
[11] W. Sun, R. Thiramanas, L.D. Slep, X.L. Zeng, V. Mail-nder, S. Wu, Chem. Eur. J. 23
(2017) 10832–10837.
[24] J. Yang, J. Wang, B. Wei, L. Shen, Y.T. Min, H.J. Cheng, Y. Xu, J. Huang, C.C. Wang,
X. Zhang, M.L. Wu, Q.Y. Li, G.W. Yang, Transit. Met. Chem. 41 (2016) 35–43.
[25] J. Yang, L. Shen, G.W. Yang, Q.Y. Li, W. Shen, J.N. Jin, J.J. Zhao, J. Dai, J. Solid
State Chem. 186 (2012) 124–133.
[12] D. Havrylyuk, D.K. Heidary, L. Nease, S. Parkin, E.C. Glazer, Eur. J. Inorg. Chem.
(2017) 1687–1694.
[26] J. Yang, Y.T. Min, Y. Ting, M.Y. Chen, J. Wang, H. Tian, L. Shen, Y.S. Zhai,
G.W. Yang, J.H. Zou, Q.Y. Li, H.J. Cui, Inorg. Chim. Acta 419 (2014) 73–81.
[27] L.L. Zheng, H.X. Li, J.D. Leng, J. Wang, M.L. Tong, Eur. J. Inorg. Chem. 2 (2008)
213–217.
[13] J.H. Zou, J.W. Zhu, Z. Yang, L. Li, W.P. Fan, L.C. He, W. Tang, L.M. Deng, J. Mu,
Y.Y. Ma, Y.Y. Cheng, W. Huang, X.C. Dong, X.Y. Chen, Angew. Chem. Int. Ed.
(2020), https://doi.org/10.1002/anie.201914384.
[14] E. Palao, T. Slanina, L. Muchova, T. Solomek, L. Vitek, P. Klan, J. Am. Chem. Soc.
138 (2016) 126–133.
[28] Y.B. Lu, M.S. Wang, W.W. Zhou, G. Xu, G.C. Guo, J.S. Huang, Inorg. Chem. 47
(2008) 8935–8942.
[15] N. Shivran, M. Tyagi, S. Mula, P. Gupta, B. Saha, B.S. Patro, S. Chattopadhyay, Eur.
J. Med. Chem. 122 (2016) 352–365.
[29] X. Xue, X.S. Wang, L.Z. Wang, R.G. Xiong, B.F. Abrahams, X.Z. You, Z.L. Xue,
C.M. Che, Inorg. Chem. 41 (2002) 6544–6546.
[16] J. Wang, Y. Lu, N. McGoldrick, C. Zhang, W. Yang, J. Zhao, S.M. Draper, J. Mater.
Chem. C 4 (2016) 6131–6139.
[30] J. Yang, Y. Xua, M. Jiang, D.F. Zou, G.W. Yang, L. Shen, J.H. Zou, J. Inorg. Biochem.
193 (2019) 124–129.
[17] J. Yang, X.Q. Gu, W.T. Su, X.Y. Hao, Y.J. Shi, L.Y. Zhao, D.F. Zou, G.W. Yang,
Q.Y. Li, J.H. Zou, Mater. Chem. Front. 2 (2018) 1842–1846.
[18] J. Shen, J.J. Chen, Z. Ke, D.F. Zou, L.G. Sun, J.H. Zou, Mater. Chem. Front. 3 (2019)
1123–1127.
[31] S. Stagni, E. Orselli, A. Palazzi, L. D. Cola, S. Zacchini, C. Femoni, M. Marcaccio,| F.
Paolucci, S. Zanarini. Inorg. Chem. 46 (2007) 22.
[32] A. Vogler, Nikol HPure, Appl. Chem. 64 (1992) 1311–1315.
[33] P.C. Ford, A. Vogler, Acc. Chem. Res. 26 (1993) 220–226.
[34] T.J. Wang, Y.J. Hou, Y.J. Chen, K. Li, X.X. Cheng, Q.X. Zhou, X.S. Wang, Dalton
Trans. 44 (2015) 12726–12734.
[19] A. Kamkaew, S.H. Lim, H.B. Lee, L.V. Kiew, L.Y. Chung, K. Burgess, Chem. Soc. Rev.
42 (2013) 77–88.
[20] S.H. Wang, L. Shang, L.L. Li, Y.J. Yu, C.W. Chi, K. Wang, J. Zhang, R. Shi, H.Y. Shen,
G.I.N. Waterhouse, S.J. Liu, J. Tian, T.R. Zhang, H.Y. Liu, Adv. Mater. 28 (2016)
8379–8387.
[35] B. Wei, M.Y. Guo, Y.M. Lu, P.P. Sun, G.W. Yang, Q.Y. Li, Z. Anorg. Allg. Chem. 644
(2018) 6–11.
[36] G.W. Yang, X. Zhang, G.M. Li, J. Yang, L. Shen, D.Y. Chen, Q.Y. Li, D.F. Zou, New J.
Chem. 42 (2018) 5395–5402.
[21] A.M. Garcia, H.A. Weerasekera, S.P. Pitre, B. McNeill, E. Lissi, A.M. Edwards,
E.I. Alarcon, J. Photochem. Photobiol. B 163 (2016) 385–390.
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