Liposomes encapsulated iridium(III) polypyridyl complexes enhance anticancer activity in vitro and in vivo

Article abstract of DOI:10.1016/j.jinorgbio.2020.111014

Three iridium(III) complexes [Ir(ppy)₂(CPIP)](PF₆) (Ir-1, ppy = 2-phenylpyridine, CPIP = 2-(4-chlorophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline), [Ir(ppy)₂(DCPIP)](PF₆) (Ir-2, DCPIP = 2-(3,4-dichlorophenyl)-1H

Full text of DOI:10.1016/j.jinorgbio.2020.111014

JournalofInorganicBiochemistry205(2020)111014
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Liposomes encapsulated iridium(III) polypyridyl complexes enhance
anticancer activity in vitro and in vivo
⁎
Lan Baia,1, Wei-Dong Feib,1, Yi-Ying Gua, Miao Hea, Fan Dua, Wen-Yao Zhanga, Lin-Lin Yangc,
,
⁎
Yun-Jun Liua,
a School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China
b Department of Pharmacy, Women's Hospital, Zhejiang University School of Medicine, Hangzhou 310006, PR China
c Department of Pediatrics, Guangdong Women and Children Hospital, Guangzhou 510000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three iridium(III) complexes [Ir(ppy)2(CPIP)](PF6) (Ir-1, ppy = 2-phenylpyridine, CPIP = 2-(4-chlorophenyl)-
1H-imidazo[4,5-f][1,10]phenanthroline), [Ir(ppy)2(DCPIP)](PF6) (Ir-2, DCPIP = 2-(3,4-dichlorophenyl)-1H-
imidazo[4,5-f][1,10]phenanthroline) and [Ir(ppy)2(TCPIP)](PF6) (Ir-3, TCPIP = 2,3,5-trichlorophenyl)-1H-
imidazo[4,5-f][1,10]phenanthroline) were synthesized and characterized. The complexes Ir-1, Ir-2 and Ir-3
were encapsulated in liposomes to form Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo. Morphology, size distribution, and
zeta potential of liposomes were examined by transmission electron microscopy (TEM) and Zetasizer. The cy-
totoxic activity in vitro of Ir-1, Ir-2 and Ir-3 against cancer A549, HTC-116, HepG2, BEL-7402, Eca-109, B16,
HeLa SGC-7901 and normal NIH3T3 cells was evaluated by 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetra-
zolium bromide (MTT) method. Ir-2 and Ir-3 show no cytotoxic activity against the selected cancer cells, and Ir-
1 displays moderate cytotoxic effect on the cell growth in A549 cells. However, Ir-1, Ir-2 and Ir-3 were en-
capsulated in liposomes, the cytotoxic activity was greatly enhanced. In particular, Ir-1-Lipo and Ir-2-Lipo can
Iridium (III) complexes
Liposomes
Cytotoxic activity in vivo
Microtubules
Cytochrome c
effectively inhibit the cell growth in A549 cells with a low IC50 value of 3.1
0.3 and 1.2
0.4 μM. The
apoptosis was assayed by flow cytometry. Ir-1, Ir-2 and Ir-3 reveal weak apoptotic effect, whereas Ir-1-Lipo, Ir-
2-Lipo and Ir-3-Lipo induce an apoptotic percentage of 55.6%, 69.3% and 16.7% in A549 cells, respectively.
Specially, in the assay of antitumor activity in vivo, the inhibiting percentage of tumor growth induced by Ir-2 is
27.65%, while inhibiting percentage of tumor growth caused by Ir-2-Lipo is 57.45%. Obviously, the liposomes
can enhance anticancer activity in vitro and in vivo compared with the complexes. The results show that the
iridium(III) complexes encapsulated liposomes induce apoptosis in A549 cells through ROS-mediated lysosome-
mitochondria dysfunction pathway and target the microtubules.
1. Introduction
an alternative of cisplatin. Over the past decades, a large number of
metal complexes have been extensively investigated in vitro and in
Cancer has become one of the most common life-threatening dis-
eases with rising incidence and mortality. Therefore, it is very essential
to develop effective methods for cancer therapy [1]. One of the defining
features of cancer is the rapid creation of abnormal cells that grow
beyond their usual boundaries, which can then invade adjoining parts
of the body and spread to other organs. To date, cisplatin and its ana-
logues are some of the most effective chemotherapeutic agents in
clinical use [2]. Due to its low solubility in water and severe side ef-
fects, the use of cisplatin in the clinical application is limited [3,4].
These limitation stimulate researchers to find other metal complexes as
vivo, and some of them are at different stages of clinical studies. Among
these metal complexes, iridium(III)-based complexes exhibit high ac-
cumulation in cancer cells and high anticancer activity [5–25]. Com-
plex [Ir(bzq)2(L)](PF6) (bzq = benzo[h]quinolone, L = 1-(3-(1H-imi-
dazo[4,5-f][1,10]phenanthrolin-2-yl)phenyl)guanidine hydrochloride)
shows high cytotoxic activity against HeLa cells with a low IC50 value of
8.96
0.34 μM [26]. Aoki reported that tris-cyclometalated iridium
(III) complex interacts with anionic molecules and/or membrane re-
ceptors on the cell surface to trigger an intracellular Ca2+ response,
resulting in the induction of cell death, accompanied by membrane
⁎ Corresponding authors.
E-mail addresses: yanglinlin@gzhmu.edu.cn (L.-L. Yang), lyjche@gdpu.edu.cn (Y.-J. Liu).
1 These authors contribute equally.
https://doi.org/10.1016/j.jinorgbio.2020.111014
Received 29 November 2019; Received in revised form 22 January 2020; Accepted 27 January 2020
Availableonline31January2020
0162-0134/©2020ElsevierInc.Allrightsreserved.

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
disruption [27]. [Ir(mppy)2(NC-CH3)](OTf) (mppy = 2-phenyl-6-me-
thylpyridine, OTf = trifluoromethanesulfonic acid) is able to antag-
onize the BRD4-acetylated histone protein–protein interaction (PPI) in
vitro, and binds BRD4 to down-regulate c-myc oncogenic expression in
cellula [28]. In recent years, various strategies for cancer therapy have
been researched [29–32]. Some metal complexes show low or no cy-
totoxic activity, but the complexes are capsulated in liposomes, the
cytotoxic activity in vitro and in vivo increases. Mao et al. reported that
[Ru(phen)2(dppz)]2+ (phen = 1,10-phenanthroline, dppz = dipyrido
[3,2-a:2′,3′-c]phenazine) has no cytotoxic activity against MDA-MB-231
cells with an IC50 value more than 200 μM. However, [Ru
(phen)2(dppz)]2+ was encapsulated in the liposome of Ruthenium
(Lipo-Ru), Lipo-Ru displays high cytotoxic activity with an IC50
value < 4.0 μM [33]. In our previous work, we found that [Ir
2. Materials and methods
All reagents and solvents were purchased commercially and used
without further purification unless otherwise noted. Ultrapure MilliQ
water was used in all experiments. DMSO was purchased from Sigma.
1,10-phenanthroline was obtained from the Guangzhou Chemical
Reagent Factory. HepG2 (human hepatocellular carcinoma), HTC-116
(human colon cancer), HeLa (human cervical cancer), A549 (human
lung carcinoma), BEL-7402 (human hepatocellular carcinoma), SGC-
7901 (human gastric adenocarcinoma), Eca-109 (human esophagus
cancer cell), B16 (mouse melanoma cells) and normal LO2 (human liver
cell) cells were purchased from the American Type Culture Collection.
IrCl3·3H2O was purchased from the Kunming Institution of Precious
Metals. DSPE-MPEG2000, cholesterol (CHO-HP) and egg yolk lecithin
(PC-98T) were purchased from Shanghai A.V.T. Pharmaceutical L.T.D.
(Shanghai, China). RPMI 1640, acridine orange (AO), ethidium bro-
mide (EB), propidium iodide (PI), RNAse A and Vitamin E were ob-
tained from Sigma-Aldrich (St. Louis, MO). 3-(4,5-Dimethyl-tiazol-2-
ly)-2,5-diphenyl-tetrazoliumbromide (MTT) was purchased from
Shanghai Beyotime Institute of Biotechnology (Shanghai, China).
Microanalyses (C, H, and N) were obtained with a Perkin-Elmer 240Q
elemental analyzer. Electrospray ionization mass spectra (ESI–MS) were
recorded on an LCQ system (Finnigan MAT, USA) using acetonitrile as
mobile phase. The spray voltage, tube lens offset, capillary voltage and
capillary temperature were set at 4.50 kV, 30.00 V, 23.00 V and 200 °C,
respectively, and the quoted m/z values are for the major peaks in the
isotope distribution. 1H and 13C NMR spectra were recorded on a
Varian-500 spectrometer with DMSO‑d6 as solvent and tetra-
methylsilane (TMS) as an internal standard at 500 MHz at room tem-
perature.
(ppy)2(FBPIP](PF6) (FBPIP
= 2-(4-formyl)benzeno[4,5-f][1,10]phe-
nanthroline) [34] and [Ir(ppy)2(THPDP)](PF6) (THPIP = 11-(6,7,8,9-
tetrahydrophenazin-2-yl)dipyrido[3,2-a:20,30-c]phenazine) [9] ex-
hibits no cytotoxicity against SiHa, HepG2, BEL-7402, A549, SGC-7901
and HeLa, whereas these complexes are encapsulated in liposomes, the
cytotoxicity may be greatly increased. The IC50 values of complexes [Ir
(ppy)2(HPIP)](PF6) (HPIP = 2-(4-hydroxy)phenylimidazo[4,5-f][1,10]
phenanthroline) and [Ir(ppy)2(BHPIP)](PF6) BHPIP = 2-(3-bromo-4-
hydroxy)phenylimidazo[4,5-f][1,10]phenanthroline) against B16 cells
are > 200 μM, but they are capsulated in the liposomes and the IC50
values are 5.2
0.8 and 10.8
1.5 μM [5]. Obviously, liposome as a
drug carrier, it can enhance anticancer activity of the metal complexes.
Therefore, to obtain more information on anticancer properties of this
kind of metal complexes encapsulated in the liposomes, in this paper,
we synthesized and characterized three iridium(III) complexes [Ir
(ppy)2(CPIP)](PF6) (ppy
=
2-phenylpyridine, CPIP
=
2-(4-chlor-
[Ir(ppy)2
ophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline,
(DCPIP)](PF6) (DCPIP
Ir-1),
=
2-(3,4-dichlorophenyl)-1H-imidazo[4,5-f]
2.1. Synthesis of ligands, complexes and liposomes
[1,10]phenanthroline, Ir-2) and [Ir(ppy)2(TCPIP)](PF6) (TCPIP
=
2,3,5-trichlorophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline, Ir-3,
(Scheme 1). Unexpectedly, Ir-2 and Ir-3 exhibit no cytotoxicity against
A549, HTC-116, HepG2, BEL-7402, Eca-109, B16, HeLa and SGC-7901
cells with IC50 values > 200 μM. However, these complexes were en-
capsulated in liposomes Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo, the bioac-
tivity of the complexes has been improved. The liposomes which consist
of dipalmitoylphosphatidylcholine (DPPC) and cholesterol are functio-
nalized with polyethylene glycol (PEG) to increase hydrophilicity, de-
crease immunological recognition. Thus, the liposomes show high in-
hibitory effect on the above cell growth. In particle, Ir-1-Lipo and Ir-2-
2.1.1. Synthesis of CPIP
Phenthroline-5,6-dione (0.315 g, 1.5 mmol) [35], 2-chlor-
obenzaldehyde (0.210 g, 1.5 mmol), NH4Ac (2.31 g, 30 mmol) and
glacial acetic acid (20 mL) were refluxed for 2 h. The cooled solution
was diluted with water and neutralized with concentrated aqueous
ammonia. The precipitate was washed with water (20 mL) and pale
yellow powder was obtained. The precipitate was collected and purified
by column chromatography on silica gel (60–100 mesh) with ethanol as
eluent to give the compound as a brown yellow powder. Yield: 82%.
Anal. Calcd for C19H11N4Cl: C, 68.99; H, 3.35; N, 16.94%. Found: C,
69.11, H, 3.46, N, 16.85%. IR (KBr, cm−1): 3078, 1605, 1542, 1516,
1476, 1352, 1276, 1100, 1070, 1014, 834, 805, 738. ESI-MS: m/
z = 331.0 [M + 1].
Lipo reveal high cytotoxic activity with low IC50 values of 3.1
0.3
and 1.2
0.4 μM toward A549 cells. Surprisingly, Ir-1-Lipo, Ir-2-Lipo
and Ir-3-Lipo have no effect on the cell growth in normal NIH3T3 cells.
Additionally, the apoptosis, intracellular ROS level, the change of mi-
tochondrial membrane potential, the lysosomal permeabilization, Bcl-2
family proteins and in vivo antitumor activity were also investigated in
detail.
2.1.2. Synthesis of DCPIP
DCPIP was synthesized in a manner identical to that described for
CPIP, with 3,4-dichlorobenzaldehyde in place of 2-chlorobenzaldehyde.
Scheme 1. Structures of complexes Ir-1, Ir-2 and Ir-3
2

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
Yield: 85%. Anal. Calcd for C19H10N4Cl2: C, 62.49; H, 2.76; N, 15.34%.
Found: C, 62.55, H, 2.85, N, 15.55%. IR (KBr, cm−1): 3068, 1607,
1556, 1455, 1408, 1256, 1187, 1072, 1050, 980, 862, 805, 739. ESI-
MS: m/z = 366.0 [M + 1].
130.51, 129.14, 127.66, 125.12, 124.89, 123.03, 122.59, 119.59. IR
(KBr, cm−1): 3045, 1606, 1584, 1506, 1478, 1424, 1352, 1268, 1164,
1080, 839, 809, 754. ESI-MS (CH3CN): m/z 898.8 ([M-PF6]+).
2.2. Preparation of Ir-1, Ir-2, Ir-3-loaded PEGylated liposomes (Ir-1-Lipo,
Ir-2-Lipo and Ir-3-Lipo)
2.1.3. Synthesis of TCPIP
TCPIP was prepared in a manner identical to that described for
CPIP, with 2,3,5-trichlorobenzaldehyde in place of 2-chlor-
obenzaldehyde. Yield: 84%. Anal. Calcd. for C19H9N4Cl3: C, 57.11; H,
2.27; N, 14.02%. Found: C, 57.45, H, 2.46, N, 13.89%. IR (KBr, cm−1):
3029, 1607, 1554, 1507, 1468, 1448, 1368, 1297, 1260, 1191, 1126,
1072, 1031, 973, 887, 804, 737. ESI-MS: m/z = 401.6 [M + 1].
Egg yolk lecithin (75 mg), cholesterol (25 mg), DSPE-MPEG (25 mg)
and iridium(III) complex (4.0 mg) were added to 2 mL of CH3CH2OH.
The resulting mixed solution was dispersed in 10 mL of ultrapure water
and stirred at 55 °C for 30 min. The residual ethanol was removed
through vacuum-rotary evaporation and the mixture was diluted with
water to 10 mL. Then ultrasound was used to reduce the particle size,
followed by filtration with millipore filter (0.2 μm) to obtain crude
product. Subsequently, the supernatant was collected after crude pro-
duct was centrifuged (5000 rpm, 10 min) at 4 °C to remove the dis-
sociative complexes.
2.1.4. Synthesis of [Ir(ppy)2(CPIP)](PF6) (Ir-1)
A mixture of cis-[Ir(ppy)2Cl]2 [36] (0.268 g, 0.25 mmol) and CPIP
(0.165 g, 0.5 mmol) in a mixture of dichloromethane and methanol
(2:1 v/v; 30 mL) was refluxed under argon for 6 h to give a clear yellow
solution. After cooling, a yellow precipitate was obtained by dropwise
addition of saturated aqueous NH4PF6 with stirring at room tempera-
ture over 2 h. The crude product was purified by column chromato-
graphy on neutral alumina with a mixture of CH2Cl2–acetone (1:1, v/v)
as eluent. The solvent was removed under reduced pressure to give a
yellow powder. Yield: 72%. Anal. Calcd. for C41H27N6ClIrPF6: C, 50.44;
H, 2.79; N, 8.61%. Found: C, 50.56, H, 2.68, N, 8.53%. 1H NMR
(DMSO‑d6, 500 MHz, Fig. S1A(a), supporting information) δ 9.13 (d,
2Ha,f, J = 6.5 Hz), 8.27 (dd, 4Hc,d,2,2′, J = 6.0, J = 8.0 Hz), 8.13 (d,
2H6,6′, J = 4.0 Hz), 8.03 (s, 2Hg,j), 7.95 (d, 2Hh,i, J = 7.5 Hz), 7.87 (t,
2.3. Characterization of liposomes
The iridium(III) liposome was prepared by an ethanol injection
method. The prepared liposome (1 mL) was diluted ten times with pure
water at room temperature. Taken the diluted sample (3 mL) into a
cuvette and placed the cuvette into the laser particle size analyzer (ZEN
3690, Malvern) and the size distribution, polydispersity index (PDI) and
ζ-potential of the liposome were measured.
2H9,9′, J = 7.0 Hz), 7.66 (d, 2H7,7′, J = 6.5 Hz), 7.52 (d, 2H8,8′
,
2.4. Drug release from liposomes
J = 5.0 Hz), 7.08–6.95 (m, 6H4,4′,b,e,5,5′), 6.30 (d, 2H3,3′, J = 7.5 Hz).
13C NMR (DMSO‑d6, 125 MHz, Fig. S1A(b), supporting informa-
tion):172.22, 166.98, 152.43, 150.51, 149.19, 148.29, 144.16, 144.10,
138.78, 134.81, 132.28, 131.31, 130.35, 129.32, 128.96, 128.31,
127.01, 125.14, 123.92, 122.47, 120.05. IR (KBr, cm−1): 3043, 1607,
1583, 1563, 1478, 1451, 1421, 1362, 1305, 1268, 1164, 1064, 1031,
844, 759. ESI-MS (CH3CN): m/z 830.9 ([M-PF6]+).
In vitro release of Ir-1, Ir-2 and Ir-3 from Ir-1-Lipo, Ir-2-Lipo and
Ir-3-Lipo suspension was performed using dialysis method. Briefly, Ir-
1-Lipo, Ir-2-Lipo and Ir-3-Lipo (200 μg) was sealed in cellulose dialysis
bags (MWCO = 8–10 kDa) and immersed in 200 mL (pH 7.4) PBS
containing 1% SDS (w/v). Afterwards, the flask was kept on continuous
vibrating at 100 rpm at 37 °C. At 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72 and
96 h, 2 mL of dialysate was extracted and the same volume of buffer
was supplemented. The amount of Ir-1, Ir-2 and Ir-3 in dialysate was
quantified by HPLC and the cumulative release was calculated.
2.1.5. Synthesis of [Ir(ppy)2(DCPIP)](PF6) (Ir-2)
This complex was synthesized in a manner identical that described
for Ir-1, with DCPIP in place of CPIP. Anal. Calcd. for
C
41H26N6Cl2IrPF6: C, 48.72, H, 2.59, N, 8.31%. Found: C, 48.87, H,
2.5. Cell viability assays
2.67, N, 8.22%. 1H NMR (DMSO‑d6, 500 MHz, Fig. S1B(a), supporting
information) δ 9.17 (d, 2Ha,f, J = 8.5 Hz), 8.29 (s, 1Hg), 8.09 (d, 3Hc,d,j
J = 5.0 Hz), 7.91 (d, 2H2,2′, J = 8.5 Hz), 7.70 (t, 4H6,6′,9,9′, J = 7.0 Hz),
7.63 (dd, 2H7,7′, J = 5.0, J = 5.0 Hz), 7.40 (d, 2H8,8′, J = 5.5 Hz), 7.24
Inhibition of cell proliferation by Ir-1, Ir-2, Ir-3, Ir-1-Lipo, Ir-2-
Lipo and Ir-3-Lipo was measured by 3-(4,5-dimethylthiazole-2-yl)-2,5-
biphenyl tetrazolium bromide (MTT) assay [37]. The cells were placed
in 96-well microassay culture plates (8 × 103 cells per well) and grown
overnight at 37 °C in a 5% CO2 incubator. The tested compounds were
then added to the wells to achieve final concentrations ranging from
10−6 to 10−4 μM. The control wells were prepared by addition of
culture medium (100 μL). The plates were incubated at 37 °C in a 5%
CO2 incubator for 48 h. Upon completion of the incubation, stock MTT
dye solution (20 μL, 5 mg/ mL) was added to each well. After 4 h, buffer
(100 μL) containing dimethylformamide (50%) and sodium dodecyl
sulfate (20%) was added to solubilize the MTT formazan. The optical
(t, 3H4,4′,i J = 7.0 Hz), 7.06 (t, 2Hb,e, J = 8.5 Hz), 6.96 (t, 2H5,5′
,
J = 6.5 Hz), 6.86 (t, 2H3,3′, J = 6.0 Hz). 13C NMR (DMSO‑d6, 125 MHz,
Fig. S1B(b), supporting information): 177.34, 168.32, 150.89, 148.68,
147.69, 144.30, 143.84, 138.08, 134.82, 133.78, 133.25, 133.02,
132.10, 131.91, 131.05, 130.99, 129.31, 126.88, 125.98, 125.86,
125.02, 123.20, 122.85, 119.77. IR (KBr, cm−1): 3044, 1607, 1583,
1508, 1478, 1446, 1423, 1351, 1268, 1163, 1076, 1031, 844, 756. ESI-
MS (CH3CN): m/z 864.9 ([M-PF6]+).
2.1.6. Synthesis of [Ir(ppy)2(TCPIP)](PF6) (Ir-3)
density of each well was measured with a microplate spectro-
This complex was synthesized in a manner identical that described
for Ir-1, with TCPIP in place of CPIP. Anal. Calcd. for C41H25N6Cl3IrPF6:
C, 47.11, H, 2.41, N, 8.04%. Found: C, 47.02, H, 2.55, N, 8.16%. 1H
NMR (DMSO‑d6, 500 MHz, Fig. S1C(a), supporting information) δ 9.23
(dd, 2Ha,f, J = 8.5, J = 8.0 Hz), 7.98 (dd, 2Hc,d, J = 5.0, J = 5.0 Hz),
photometer at a wavelength of 490 nm. The IC50 values were de-
termined by plotting the percentage of cell viability versus concentra-
tion on a logarithmic graph and reading off the concentration at which
50% of cells remained viable relative to the control. Each experiment
was repeated at least three times to obtain mean values.
7.87 (d, 2H2,2′, J = 8.0 Hz), 7.83 (d, 2H6,6′, J = 7.5 Hz), 7.69 (d, 2H9,9′
,
J = 7.0 Hz), 7.64 (t, 2H7,7′, J = 6.5 Hz), 7.57 (dd, 2H8,8′, J = 5.0,
J = 5.0 Hz), 7.39 (d, 1Hj, J = 2.5 Hz), 7.36 (d, 2H4,4′, J = 6.0 Hz), 7.24
(s, 1Hk), 7.04 (t, 2Hb,e, J = 7.5 Hz), 6.93 (t, 2H5,5′, J = 6.5 Hz), 6.76 (t,
2H3,3′, J = 6.0 Hz). 13C NMR (DMSO‑d6, 125 MHz, Fig. S1C(b),
Supporting information): 168.33, 151.70, 148.79, 146.11, 143.92,
143.76, 137.83, 134.34, 132.53, 132.27, 132.17, 130.89, 130.81,
2.6. Colony formation assays
A549 cells were inoculated into a six-well plate to make the cell
density of each well of 5 × 104. After 24 h, complexes Ir-1, Ir-2, Ir-3
and liposomes Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo were added into the
wells. After A549 cells were exposed to the complexes for 24 h, the
3

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
medium was discarded and new medium was added, the cell colonies
were grown for 9 days, then the medium was discarded, the cells were
washed with phosphate buffered saline (PBS) three times and fixed with
paraformaldehyde solution for 20 min, and then stained with crystal
violet (0.1%, w/v) for 20 min, the cells were imaged under light mi-
croscope.
2.12. Assay of change of mitochondrial membrane potential
A549 cells in 12-well plates were treated with different concentra-
tions of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo for 24 h and then washed
three times with cold PBS. The cells were detached with trypsin–EDTA
solution. The collected cells were stained with 1 μg/mL of JC-1
(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanineiodide,
λex = 585 nm, λem = 590 nm) for 20 min at 37 °C in the dark, then
immediately centrifuged to remove the supernatant. The cell pellets
were suspended in PBS and imaged under a fluorescence microscope.
2.7. Cell cycle arrest by flow cytometry
A549 cells were seeded into six-well plates (Costar, Corning Corp,
New York, USA) at a density of 2 × 105 cells per well and incubated for
24 h. The cells were cultured in Roswell Park Memorial Institute (RPMI)
1640 supplemented with 10% of fetal bovine serum (FBS) and in-
cubated at 37 °C in 5% CO2. The medium was removed and then re-
placed with fresh medium (final DMSO concentration, 0.05% v/v)
containing Ir-1, Ir-2, Ir-3, Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo. After
incubation for 24 h, the cell layer was trypsinized, washed with cold
PBS and fixed with 70% ethanol. 20 μL of RNAse (0.2 mg/mL) and
20 μL of propidium iodide (0.02 mg/mL) were added to the cell sus-
pensions, which were then incubated at 37 °C for 30 min. The samples
were analyzed with an FACSCalibur flow cytometer.
2.13. Reactive oxygen species (ROS) detection
A549 cells were seeded into 12-well plates (Costar, Corning Corp,
New York, USA) at a density of 2 × 105 cells per well and incubated for
24 h. Then the cells were cultured in RPMI 1640 supplemented with
10% FBS and incubated at 37 °C in 5% CO2. The medium was removed
and replaced with fresh medium (final DMSO concentration, 0.05% v/
v) containing different concentrations of Ir-1-Lipo, Ir-2-Lipo and Ir-3-
Lipo, after 24 h, the cells were stained with 20 μM DCFH-DA in PBS for
30 min in the dark. Finally, the cells were washed twice with PBS, then
the cells were imaged under a fluorescent microscope. The fluorescence
intensity of DCF was determined.
2.8. Apoptosis studies by flow cytometry
A549 cells (2 × 105) were exposed to Ir-1, Ir-2, Ir-3, Ir-1-Lipo, Ir-
2-Lipo and Ir-3-Lipo and cultured in RPMI 1640 with 10% of FBS and
incubated at 37 °C in 5% CO2 for 24 h. The cells were washed with ice-
cold PBS, and fixed with formalin (4%, w/v). The cell nuclei were
counterstained with Annexin V-FITC (1 mg/ mL) in the dark for 15 min.
The cells were analyzed by flow cytometry.
2.14. Induction of autophagy
A549 cells were incubated with the fluorescent probe MDC
(monodansylcadaverine, 50 mM, λex = 355 nm, λem = 512 nm) to
quantify the induction of autophagy. A549 cells were seeded onto
chamber slides in 12-well plates and incubated for 24 h. The cells were
cultured in RPMI 1640 supplemented with 10% FBS and incubated at
37 °C in 5% CO2. The medium was removed and replaced with fresh
medium (final DMSO concentration, 0.05% v/v) containing Ir-1, Ir-2,
Ir-3, Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo, then incubated for 24 h. The
medium was removed again, and the cells were washed twice with ice-
cold PBS. The cells were stained with MDC for 10 min, then washed
twice with PBS. The cells were observed and imaged under a fluores-
cence microscope. The effect of the complexes on the expression of
Beclin-1 and LC3 proteins was assayed by Western blot.
2.9. Location of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo at the lysosomes
A549 cells were placed in 12-well microassay culture plates
(4 × 104 cells per well) and grown overnight at 37 °C in a 5% CO2
incubator. Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo were added to the wells at
37 °C in a 5% CO2 incubator for 6 h and further co-incubated with
LysoTracker Red (150 nM, λex = 577 nm, λem = 590 nm) at 37 °C for
0.5 h. Upon completion of the incubation, the wells were washed three
times with ice-cold PBS. After discarding the culture medium, the cells
were imaged under a fluorescence microscope.
2.15. Release of cytochrome c
2.10. Detection of lysosome membrane permeabilition
A549 cells were seeded onto chamber slides in 12-well plates and
incubated for 24 h. The cells were cultured in RPMI 1640 medium
supplemented with 10% FBS and incubated at 37 °C in 5% CO2. The
medium was removed and replaced with medium (final DMSO con-
centration, 0.05% v/v) containing Ir-1-Lipo, Ir-2-Lipo or Ir-3-Lipo for
24 h. The medium was removed again, and the cells were washed with
PBS twice. Then cells were fixed with stationary liquid overnight at
4 °C. Then the cells were incubated with confining liquid for 1 h, and
washed with PBST for three times. And then the cells were incubated
with primary antibody of human cytochrome c for 24 h. After washing,
incubated with fluorescein conjugated anti rabbit secondary antibody
and mounting were done with paramounting media containing DAPI,
scored and analyzed under the Molecular Devices.
A549 cells were placed in 12-well microassay culture plates
(4 × 104 cells per well) and grown overnight at 37 °C in a 5% CO2
incubator. Ir-1-Lipo (1.5 μM), Ir-2-Lipo (0.6 μM) and Ir-3-Lipo
(7.0 μM) were added to the wells at 37 °C in a 5% CO2 incubator for
24 h and stained with AO (1 μg/mL) at 37 °C for 0.5 h. Upon completion
of the incubation, the wells were washed three times with ice-cold PBS.
After discarding the culture medium, the cells were imaged under a
fluorescence microscope.
2.11. Location assays of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo at the
mitochondria
A549 cells were placed in 12-well microassay culture plates
(4 × 104 cells per well) and grown overnight at 37 °C in a 5% CO2
incubator. Ir-1-Lipo (1.5 μM), Ir-2-Lipo (0.6 μM) and Ir-3-Lipo
(7.0 μM) were added and the cells were kept at 37 °C in a 5% CO2
incubator for 6 h and then stained with Mito Tracker ® Deep Red FM
(100 nM, λex = 579 nm, λem = 599 nm) at 37 °C for 1 h. Upon
completion of the incubation, the wells were washed three times with
ice-cold PBS. After discarding the culture medium, 200 μl PBS was
added into every well and the cells were imaged under a fluorescence
microscope.
2.16. Matrigel invasion assays
A BD Matrigel invasion chamber was used to investigate the cell
invasion according to the manufacturer's instructions. A549 cells
(4 × 104) in serum free media with 1.5, 0.6 and 7.0 μM of Ir-1, Ir-2, Ir-
3 or Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo were seeded in the top chamber
of the two chamber Matrigel system. RPMI-1640 (20% FBS) was added
as a chemo-attractant into the lower chamber. Cells were allowed to
invade for 24 h. After incubation, non-invading cells were removed
from the upper surface and the cells on the lower surface were fixed
4

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
with 4% paraformaldehyde and stained with 0.1% of crystal violet. The
cells invading the membranes were photographed and the invading
cells were counted under a light microscope. The mean values from
three independent assays were calculated.
3. Results and discussion
3.1. Synthesis and characterization
The ligands CPIP, DCPIP and TCPIP were prepared by the reaction
of 1,10-phenanthroline-5,6-dione and 4-chlorobenzaldehyde, 3,4-di-
chlorobenzaldehyde and 2,3,5-trichlorobenzaldehyde in the presence of
ammonium acetate and glacial acetic acid. The complexes [Ir
(ppy)2(CPIP)](PF6) (Ir-1), [Ir(ppy)2(DCPIP)](PF6) (Ir-2) and [Ir
(ppy)2(TCPIP)](PF6) (Ir-3) were synthesized by the direct reaction of
CPIP, DCPIP, TCPIP and precursor complex [Ir(ppy)2Cl]2 in a mixture
of dichloromethane and methanol. The complexes were characterized
by elemental analysis, ESI-MS, IR, 1H NMR and 13C NMR. In the IR
spectra, the peaks of 3078 cm−1 for CPIP, 3068 cm−1 for DCPIP,
3029 cm−1 for TCPIP, 3043 cm−1 for Ir-1, 3044 cm−1 for Ir-2 and
3045 cm−1 for Ir-3 are assigned to CeH stretching vibration. The peaks
of 1605 cm−1 for CPIP, 1607 cm−1 for DCPIP, TCPIP, Ir-1, Ir-2,
1606 cm−1 for Ir-3 are assigned to the stretching vibration of C]C. The
peaks of 738 cm−1 for CPIP, 739 cm−1 for DCPIP, 3737 cm−1 for
TCPIP, 759 cm−1 for Ir-1, 756 cm−1 for Ir-2 and 755 cm−1 for Ir-3 are
attributed to the stretching vibration of CeCl. In the 1H NMR spectra of
Ir-1, Ir-2, Ir-3, the chemical shifts of the proton on the nitrogen atom of
the imidazole ring were not observed, because metal coordination
causes electron deficiency in the ligand, as a result, these NH protons
are very active and easy to be exchanged between the two imidazole
nitrogen atoms in solution [39–41]. In the ESI-MS spectra of Ir-1, Ir-2
and Ir-3, the signal of [M-PF6]+ was observed. The measured molecular
weight was consistent with the expected value. The UV–Vis spectra of
10 μM of Ir-1, Ir-2, Ir-3, Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo in PBS so-
lution are shown in Fig. S2 (supporting information). The maximum
absorbance of Ir-1, Ir-2, Ir-3, Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo ap-
pears at 208, 205, 209, 209, 210 and 212 nm, respectively. The com-
plexes and their liposomes can emit luminescence in PBS solution at
ambient temperature (Fig. S3, supporting information), with a max-
imum appearing at 564, 565, 565, 563, 565 and 565 nm for Ir-1, Ir-2,
Ir-3, Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo, respectively. The stability of
the complexes in PBS was also studied by UV–Vis spectra at 0 and 72 h,
no change in the peak shape was observed (Fig. S4), which demon-
strates that the complexes are stable in PBS solution.
2.17. Effects of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo on microtubules
networks
A549 cells were grown at a density of 106 cells/mL and incubated
with Ir-1-Lipo, Ir-2-Lipo or Ir-3-Lipo for 9 h. Subsequently, the cells
were washed twice with PBS, fixed with 2% paraformaldehyde and
incubated with immunostaining blocking solution for 1 h. Then the
cells were then mildly washed with PBS once. Finally, the cells were
incubated with anti-rabbit monoclonal anti-α-tubulin antibody (1:100
dilutions) for 24 h at 4 °C, the cells were washed twice with PBS and
followed by anti-rabbit FITC conjugated IgG antibody (1:500 dilutions)
and DAPI (10 μg/mL). After incubation, the cells were washed once
with PBS and imaged under a fluorescence microscope.
2.18. The expression of caspase and Bcl-2 family proteins
A549 cells (2 × 105) were treated with Ir-1, Ir-2, Ir-3, Ir-1-Lipo, Ir-
2-Lipo and Ir-3-Lipo for 24 h. The cells were harvested in lysis buffer
and centrifuged at 13,000 g for 20 min. The protein concentration of the
supernatant was determined by BCA (bicinchoninic acid) assay. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis was done by loading
equal amounts of proteins per lane. Gels were then transferred to poly
(vinylidenedifluoride) membranes (Millipore) and blocked with 5%
non-fat milk in TBST (20 mM Tris–HCl, 150 mM NaCl, 0.05% Tween
20, pH 8.0) buffer for 1 h. The membranes were then incubated with
primary antibodies at 1:5000 dilution in 5% non-fat milk overnight at
4 °C, and washed four times with TBST for a total of 30 min. After this
the secondary antibodies were conjugated with horseradish peroxidase
at 1:5000 dilution for 1 h at room temperature and then washed four
times with TBST. BeyoECL Star as photographic developer was dropped
into the blots and the blots were imaged.
2.19. Evaluation of antitumor activity in vivo
Liposomes were first proposed as carriers of biologically active
substances in 1971 and it can be used to increase solubility of drug,
prolong drug duration in vivo, target drug delivery and reduce toxicity.
The shape, particle size and zeta potential of the Ir-1-Lipo, Ir-2-Lipo
and Ir-3-Lipo were determined. These liposomes are found to be in-
dividual, spherical or quasi-spherical particles with smooth surfaces.
The particle size, polydispersity index (PDI), zeta potential, en-
capsulation efficiency (EE(%)) and drug loading (DL(%)) of the lipo-
somes are listed in Table 1. The average particle size of Ir-1-Lipo, Ir-2-
All animals were provided by the Laboratory Animal Center of Sun-
Yat Sen University. All animal procedures were approved by the Animal
Ethical Committee of Guangdong Pharmaceutical University. Prior to
each experiment, the mice were fasted overnight and allowed free ac-
cess to water. Different doses of 9.45 mg/kg of Ir-2, 4.725 and 9.45 mg/
kg of Ir-2-Lipo were injected intraperitoneally into mice of different
group (each group contained 6 mice) once a day for ten consecutive
days beginning 24 h after inoculation. This dose was the maximum
tolerated dose based on our preliminary studies. Ir-2 (9.45 mg/kg) was
used as a positive control. The control mice were injected with the
vehicle. Compounds were administered by exact body weight, with the
injection volume being not > 0.2 mL. The weights of the animals were
recorded every 2 days. All animals were sacrificed on the eighteenth
days after tumor inoculation, and the tumors were excised and
weighed. The inhibition rate was calculated as follow:
Lipo and Ir-3-Lipo is 155.1
2.2, 188.7
2.5 and 181.3
2.3 nm,
respectively (Fig. S5, supporting information). Moreover, the size dis-
tribution of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo was narrow with a PDI
value of 0.264, 0.225 and 0.161, which indicated that Ir-1-Lipo, Ir-2-
Lipo and Ir-3-Lipo revealed a comparatively homogeneous composi-
tion without any aggregation (Fig. S6, supporting information). When
the absolute value of zeta potential exceeded 30 mV, the liposomes
were regarded as highly stable, while zeta potential in the range of
[(C T)/C] × 100%
(1)
T is the average tumor weight of the treated group and C is the
average tumor weight of the negative control group [38].
Table 1
The size, zeta potential, EE and DL of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo.
Liposomes Size (nm)
PDI
Zeta potential
EE (%)
DL (%)
2.20. Data analysis
(mV)
Ir-1-Lipo
Ir-2-Lipo
Ir-3-Lipo
155.2
188.7
181.3
2.2 0.26 35.7
2.5 0.23 52.5
2.3 0.16 43.4
2.8
4.1
3.4
96.9
99.1
85.4
2.5 3.1
0.4
0.3
0.3
All data was expressed as means SD. Statistical significance was
evaluated by a t-test. Differences were considered to be significant
when a ⁎P value was < 0.05.
3.1 3.2
1.8 2.8
5

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
value > 200 μM. We infer it is difficult for Ir-2 and Ir-3 to enter into the
cells compared with Ir-1. However, after Ir-1, Ir-2 and Ir-3 were en-
capsulated in liposomes Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo, the cyto-
toxic activity of Ir-1 against A549 cells increases 4.87 times compared
with Ir-1 alone with a low IC50 value of 3.1
0.3 μM. Ir-1-Lipo and
Ir-2-Lipo show high inhibitory effect on the above cell growth. The IC50
values of Ir-2-Lipo toward A549 and HTC-116 cells are 1.2 0.4 and
2.2 0.4 μM. We consider that PEG-loaded long circulation liposomes
can carry the complexes and make them easy to enter into the cells and
exert high cytotoxic activity. Although Ir-3-Lipo shows high release
percentage, after Ir-3 was encapsulated in liposome, Ir-3-Lipo also
exhibits low cytotoxicity compared with Ir-1-Lipo and Ir-2-Lipo. This
may attribute to low cytotoxic efficiency of Ir-3 itself on the selected
cancer cells. Comparing the IC50 values, Ir-2-Lipo exhibits higher cy-
totoxic activity than Ir-1-Lipo, Ir-3-Lipo and cisplatin against the se-
lected cancer cell lines under identical conditions. We also determined
the cytotoxicity in vitro of liposomes and the results show that the li-
posomes have no cytotoxic activity against the selected cancer and
normal cells. The cytotoxicity of Ir-1-Lipo and Ir-2-Lipo is comparable
Fig. 1. Percentage of release of Ir-1, Ir-2 and Ir-3 from Ir-1-Lipo, Ir-2-Lipo and
Ir-3-Lipo.
10–20 mV indicated the liposomes were relatively stable [42]. Ir-1-
Lipo, Ir-2-Lipo and Ir-3-Lipo showed a positive charge with an average
with that of complexes [Ir(ppy)2(adppz)](PF6) (IC50 = 3.2
0.4 μM)
[8] toward A549 cells. Hence, with regard to complexes-encapsulated
liposomes, we think what of two chloride atoms as substituent group
(DCPIP) shows higher cytotoxicity than those of one (CPIP) or three
chloride atoms (TCPIP) as substituent group. Surprisingly, these com-
plexes and their liposomes have no cytotoxicity against normal NIH3T3
cells. Thus, Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo may be potential antic-
ancer drugs.
zeta potential of 35.7
2.8, 52.5
4.1 and 43.4
3.4 mV. Hence,
the Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo liposomes are stable. Ir-1-Lipo,
Ir-2-Lipo and Ir-3-Lipo show high encapsulation efficiency and Ir-2-
Lipo displays the highest drug loading capacity among these liposomes.
3.2. Release in vitro of drug determination
Drug concentration is an important factor to inhibit the cell growth.
The release of Ir-1, Ir-2 and Ir-3 from liposomes Ir-1-Lipo, Ir-2-Lipo
and Ir-3-Lipo was investigated by using the dialysis technique. As
shown in Fig. 1, the liposomes exhibit a slow diffusion rate across the
dialysis bag and the release percentage of Ir-1-Lipo, Ir-2-Lipo and Ir-3-
3.4. Inhibition of colonies
The proliferation of cancer cells is one of the main causes of cancer
patients' difficulty in treatment and death. Therefore, there is an urgent
need to use anti-tumor drugs to inhibit cancer proliferation. To in-
vestigate whether Ir-1, Ir-2 and Ir-3 complexes and liposomes Ir-1-
Lipo, Ir-2-Lipo and Ir-3-Lipo can inhibit cell proliferation in A549
cells, a cell cloning assay was performed using a crystal violet dye
marker. The results of cell proliferation are shown in Fig. 2, compared
with the control group, the number of A549 cells treated with Ir-1, Ir-2
and Ir-3 complexes shows no obvious change. However, we can clearly
find a significant decrease in the number of cells treated with the li-
posomes Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo for 9 days, indicating that
liposomes effectively inhibited cell cloning. Therefore, the above results
demonstrate that the liposomes exhibit excellent ability to inhibit the
cell growth.
Lipo is 8.72
0.72%, 3.87
0.61% and 8.62
0.53% at 0.5 h,
which indicates that no drastic release was observed during the first
30 min. However, the percentage of the free Ir-1, Ir-2 and Ir-3 reached
15.97
1.73%, 19.57
1.63% and 30.77%
2.56% at 96 h, re-
spectively. Ir-1, Ir-2 and Ir-3 were slowly released from Ir-1-Lipo, Ir-2-
Lipo and Ir-3-Lipo, which is probably the result of lipid bilayer-induced
blockage.
3.3. Cytotoxic activity in vitro assays
The cytotoxic activity in vitro of Ir-1, Ir-2, Ir-3 and their liposomes
Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo was investigated employing eight
human tumor cell lines (A549, HTC-116, HepG2, BEL-7402, Eca-109,
B16, HeLa and SGC-7901) and normal NIH3T3 cells by MTT method,
and the cytotoxicity is expressed as half maximal inhibitory con-
centration (IC50) values and presented in Table 2. For comparison,
cisplatin was used as a positive control. Complex Ir-1 shows moderate
cytotoxic activity against the selected cancer cells except BEL-7402 and
3.5. Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo induce G0/G1 cell cycle arrest
Induction of apoptosis and inhibition of cell growth are closely re-
lated to cell cycle, thus, we further examined whether Ir-1, Ir-2, Ir-3, Ir-
1-Lipo, Ir-2-Lipo and Ir-3-Lipo treatment caused any cell cycle-related
events to inhibit the growth of A549 cells. As shown in Table 3, Ir-1
(3.0 μM), Ir-2 (1.2 μM) and Ir-3 (14.0 μM) have no obvious effect on the
cell cycle distribution. The cell cycle phase distribution of cells treated
Eca-109 cells with an IC50 value ranging from 5.7
0.7 to
20.8
2.3 μM. It is unexpected to find that Ir-2 and Ir-3 display no
cytotoxic activity toward the selected cancer cell lines with IC50
Table 2
IC50 values of iridium(III) complexes and liposomes toward selected cancer cell lines.
Complex
A549
HTC-116
HepG2
BEL-7402
Eca-109
B16
HeLa
SGC-7901
NIH3T3
Ir-1
15.1
> 200
> 200
3.1
1.6
5.7
0.7
20.8
> 200
> 200
8.6
2.3
> 200
> 200
> 200
> 200
> 200
> 200
14.7
11.9
> 200
> 200
9.1
1.0
10.1
> 200
> 200
12.0
5.3
1.1
1.3
15.1
> 200
> 200
8.1
1.3
> 200
> 200
> 200
> 200
> 200
> 200
nd
Ir-2
> 200
> 200
6.0
Ir-3
Ir-1-Lipo
Ir-2-Lipo
Ir-3-Lipo
Cisplatin
0.3
0.4
1.8
1.2
0.1
0.4
1.1
0.2
0.6
1.5
1.7
11.0
5.8
0.5
0.2
3.5
1.1
2.4
1.2
0.5
1.2
0.5
1.1
0.7
1.2
2.2
5.7
7.1
0.3
4.9
0.4
2.9
14.1
7.3
7.8
16.2
22.6
28.9
10.4
83.1
11.5
3.3
1.2
12.8
5.2
19.6
6.9
2.4
1.0
11.8
3.6
1.3
nd
0.5
nd: not determination.
6

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
Fig. 2. Inhibition of colonies of A549 cells (a) treated with Ir-1 (b, 1.5 μM), Ir-2 (b, 0.6 μM), Ir-3 (c, 7.0 μM), Ir-1-Lipo (d, 1.5 μM), Ir-2-Lipo (e, 0.6 μM) and Ir-3-
Lipo (f, 7.0 μM) for 9 days.
with Ir-1-Lipo (3.0 μM), Ir-2-Lipo (1.2 μM) and Ir-3-Lipo (14.0 μM)
was analyzed by flow cytometry after propidium iodide staining.
Table 3 shows that the distribution of G0/G1 significantly increases
after the treatment of A549 cells. Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo
increase 10.7%, 3.5% and 25.9% at G0/G1 distribution compared with
that of the control, in the meantime, a reduction of 8.6% for Ir-1-Lipo,
3.4% for Ir-2-Lipo and 19.6% for Ir-3-Lipo in the cell at S phase
compared with the control was discovered. Although showing the
highest cytotoxic activity against A549 cells, Ir-2-Lipo displays the
lowest effect on the cell cycle arrest among the liposomes. The results
reveal that Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo inhibit the cell growth at
G0/G1 phase.
Lipo and Ir-1-Lipo locate at the intercellular lysosomes. The Pearson's
colocalization coefficient (R) was obtained by analyzing the red and
green fluorescent intensity in 50 cells according to literature [52]. The
R value for Ir-1-Lipo is 0.4601, which indicates a positive correlation.
The lysosomes contain many different acid hydrolases. The change
of lysosome membrane permeabilization (LMP) can release the lyso-
somal constituents, which further trigger cell death. LMP was assessed
by the AO-relocation method. AO (λex = 488 nm, λem = 515 nm) is
weakly alkaline and can accumulate in acidic lysosomal compartments.
And AO is also a metachromatic fluorescent dye, it exhibits red fluor-
escence at high concentrations at acidic pH (in intact lysosomes), but
green fluorescence at low concentrations (when lysosomal contents
diffuse into the cytosol) [53]. As shown in Fig. S7 (supporting in-
formation), a number of red fluorescent points were observed in the
control (a). After A549 cells were exposed to 1.5, 0.6 and 7.0 μM of Ir-
1-Lipo (b), Ir-2-Lipo (c) and Ir-1-Lipo (d) for 24 h, the red fluorescence
decreased and the green fluorescence increased significantly, indicating
that Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo can increase lysosomal mem-
brane permeabilization, which leads to protease infiltrating into the
cytoplasm and further triggers apoptosis pathway.
3.6. Location of the liposomes at the lysosomes and lysosomal
permeabilization
The primary function of lysosomes is to degrade endocytosed and
intracellular material. However, increasing evidences have shown that
lysosomes are involved in other complex and layered activities, such as
intracellular transport, cell antigen processing, and the initiation of
apoptosis [43–46]. In recent years, the studies on lysosomes have been
made great progress [47–51]. To study whether the complexes interact
to lysosomes, A549 cells were treated with Ir-1-Lipo (1.5 μM), Ir-2-
Lipo (0.6 μM) and Ir-3-Lipo (7.0 μM) for 6 h, the cells were stained
with LysoTracker red. As shown in Fig. 3, the lysosomes stained with
LysoTracker red show bright red, the liposomes Ir-1-Lipo, Ir-2-Lipo
and Ir-1-Lipo emit green fluorescence. The merge (yellow) shows the
complete overlap of the red and green, indicating that Ir-1-Lipo, Ir-2-
3.7. Location and change of mitochondrial membrane potential assays
Mitochondrial dysfunction is involved in apoptotic cell death.
Mitochondria play important roles in apoptosis through the release of
pro-apoptotic factors such as cytochrome c and apoptosis-inducing
factor [54]. To evaluate whether the liposomes locate at the mi-
tochondria, A549 cells were exposed to 1.5, 0.6 and 7.0 μM of Ir-1-
Table 3
Cell cycle distribution of A549 cells treated by the complexes and liposomes.
Complex
Control
Ir-1
Ir-2
Ir-3
Ir-1-Lipo
Ir-2-Lipo
Ir-3-Lipo
90.6
G0/G1
S
64.7
25.5
9.8
4.5
2.2
66.2
25.8
8.0
3.8
2.3
63.9
26.2
9.9
4.3
2.3
60.7
28.1
11.2
3.6
1.7
1.4
75.4
16.9
4.2
1.5
68.2
22.1
3.5
2.4
5.5
0.4
0.4
5.9
3.5
G2/M
0.7
1.0
1.1
7.7
0.5
9.7
1.0
7

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
Fig. 3. Location assay of 1.5, 0.6 and 7.0 μM of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo in the lysosomes after A549 cells were exposed to the liposomes for 6 h. The
excitation and emission wavelengths for red channel are 562 nm and 624 nm, for green channel, the excitation and emission wavelengths are 482 nm and 536 nm,
respectively.
Lipo, Ir-2-Lipo and Ir-3-Lipo for 6 h, the cells were stained with Mi-
toTracker red. As shown in Fig. 4A, the mitochondria were stained red,
liposomes Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo emit green fluorescence.
The merge of the red and green fluorescence suggests that Ir-1-Lipo, Ir-
2-Lipo and Ir-3-Lipo locate at the mitochondria. The Pearson's colo-
calization coefficient value for Ir-1-Lipo is −0.5078, which indicates a
negative correlation.
Lipo (c), Ir-2-Lipo (d) and Ir-1-Lipo (e) for 24 h, JC-1 emits green
fluorescence corresponding to low mitochondrial membrane potential.
The results show that Ir-1-Lipo, Ir-2-Lipo and Ir-1-Lipo can induce a
decrease of mitochondrial membrane potential. To compare the effect
of Ir-1, Ir-2, Ir-3, Ir-1-Lipo, Ir-2-Lipo and Ir-1-Lipo on the mitochon-
drial membrane potential, the ratio of red/green fluorescence intensity
was determined. As shown in Fig. 4C, the ratio of red/green reduces
induced by liposomes Ir-1-Lipo, Ir-2-Lipo and Ir-1-Lipo compared with
Ir-1, Ir-2 and Ir-3, respectively. Moreover, Ir-2-Lipo induces more ef-
fective on the changes of mitochondrial membrane potential than Ir-1-
Lipo or Ir-3-Lipo under the same conditions. This is line with those of
cytotoxicity of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo.
The change of mitochondrial membrane potential was assayed using
JC-1 as fluorescence probe. As shown in Fig. 4B, in the control (a), JC-1
emits red fluorescence with little green fluorescence corresponding to
high mitochondrial membrane potential. After A549 cells (a) were
treated with CCCP (b, positive control) and 1.5, 0.6 and 7.0 μM of Ir-1-
8

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
Fig. 4. (A) Location assay of 1.5, 0.6 and 7.0 μM of
Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo in the mitochon-
dria after A549 cells were treated with the liposomes
for 6 h. (B) The mitochondrial membrane potentials
were assayed after A549 cells (a) were treated with
CCCP (b, positive control) and 1.5, 0.6 and 7.0 μM of
Ir-1-Lipo (c), Ir-2-Lipo (d) and Ir-1-Lipo (e) for
24 h. (C) Ratio of red/green fluorescence intensity
after A549 cells were exposed to different con-
centration of Ir-1, Ir-2, Ir-3, Ir-1-Lipo, Ir-2-Lipo
and Ir-3-Lipo for 24 h. The excitation and emission
wavelengths for red channel are 562 nm and
624 nm, for green channel, the excitation and
emission wavelengths are 482 nm and 536 nm, re-
spectively.
9

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
Fig. 5. DCF fluorescence intensity was determined while A549 cells were treated with different concentration of Ir-1, Ir-2, Ir-3, Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo for
24 h. *P < 0.05 represents significant differences compared with control.
Fig. 6. Apoptosis assays after A549 cells (a) were treated with different concentration of Ir-1 (b), Ir-2 (e), Ir-3 (h), 1.5 (c) and 3.0 μM (d) Ir-1-Lipo, 0.6 (f) and 1.2 μM
(g) Ir-2-Lipo and 7.0 (i) and 14.0 μM (j) Ir-3-Lipo for 24 h.
10

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
Fig. 7. (A) Autophagy was studied after A549 cells (a) were exposed to 1.5, 0.6 and. 7.0 μM of Ir-1 (b), Ir-2 (c) and Ir-3 (d) or Ir-1-Lipo (e), Ir-2-Lipo (f) and Ir-3-
Lipo (g) for 24 h and the cells were stained with MDC. (B) The expression of LC3 and Beclin-1 was assayed by Western blot.
3.8. Intracellular ROS levels
3.9. Apoptosis assays by flow cytometry
A 2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay was used as
fluorescence probe to measure intracellular ROS levels in A549 cells.
Once in the cells, DCFH-DA is hydrolyzed by esterase enzymes to DCF,
which is vulnerable to ROS and can be oxidized to be highly fluorescent
2,7-dichlorofluorescei. As shown in Fig. S8 (supporting information), in
the control (a), no obvious green fluorescence points are observed.
After A549 cells were incubated to 1.5, 0.6 and 7.0 μM of Ir-1-Lipo (b),
Ir-2-Lipo (c) and Ir-1-Lipo (d) for 24 h, a lot of bright green points are
found, which indicates that Ir-1-Lipo, Ir-2-Lipo and Ir-1-Lipo can en-
hance intracellular ROS levels. To quantitatively compare the effect of
different concentrations of Ir-1-Lipo, Ir-2-Lipo and Ir-1-Lipo on ROS
levels, DCF fluorescence intensity was determined. ROSUP was used as
a positive control. For comparison, the effect of Ir-1, Ir-2 and Ir-3 on
ROS was also studied. As shown in Fig. 5, liposomes Ir-1-Lipo, Ir-2-
Lipo and Ir-3-Lipo exhibit more effective on the ROS levels than those
induced by Ir-1, Ir-2 and Ir-3. The results demonstrate that Ir-1-Lipo,
Ir-2-Lipo and Ir-3-Lipo can increase intracellular ROS levels in a con-
centration-dependent manner.
Apoptosis is a form of programmed cell death which occurs through
the activation of the cell-intrinsic suicide machinery. The apoptosis
induced by Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo in A549 cells was in-
vestigated by flow cytometry. Ir-1 (3.0 μM), Ir-2 (1.2 μM) and Ir-3
(14.0 μM) were used as a comparison. As shown in Fig. 6, in the control
(a), the percentage in the early apoptotic cells is 0.57%. Treatment of
A549 cells with Ir-1 (b, 3.0 μM), Ir-2 (e, 1.2 μM) and Ir-3 (h, 14.0 μM)
led to no obvious apoptotic cells. However, A549 cells were incubated
with Ir-1-Lipo (1.5 and 3.0 μM, c and d), Ir-2-Lipo (0.6 and 1.2 μM, f
and g) and Ir-3-Lipo (7.0 and 14.0 μM, i and j) for 24 h, the percentage
in the early apoptotic cells is 2.70% and 55.6%, 4.27% and 69.3%,
6.33% and 16.7%, respectively. At the concentration of IC50 value, the
apoptotic effect of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo follows the order
of Ir-2-Lipo > Ir-1-Lipo > Ir-3-Lipo. This is consistent with those of
cytotoxicity of the liposomes against A549 cells. These data reveal that
the liposomes Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo can effectively induce
apoptosis in A549 cells.
11

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
(caption on next page)
12

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
Fig. 8. (A) The release of cyt-c was examined after A549 cells were exposed to 1.5, 0.6 and 7.0 μM of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo for 24 h. (B) The integrated
fluorescent intensity/cell was determined after different concentration of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo treated A549 cells for 24 h. *P < 0.05 represents
significant differences compared with control.
Fig. 9. The percentage of 1.5, 0.6 and 7.0 μM of Ir-1, Ir-2 and Ir-3 or Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo inhibiting cell invasion in A549 cells.
3.10. Effect of the liposomes on autophagy
Autophagy is lysosomal degradation pathway essential for
fluorescence points were found, which suggests a release of cyto c. The
green fluorescence intensity was also examined, as shown in Fig. 8B,
compared with the control, Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo cause an
increase of green fluorescence intensity. Moreover, Ir-2-Lipo shows the
most effective on the release of cyto c among these liposomes.
a
homeostasis under normal conditions. Autophagy is often observed in
cancer cells in response to anticancer treatments [55–57]. To in-
vestigate the effect of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo on autophagy
induction in A549 cells, the cellular morphological changes were ob-
served by monodansylcadaverine (MDC) staining, a marker for autop-
hagic vacuoles [58]. As shown in Fig. 7A, in the control (a) or Ir-1 (b,
3.0 μM), Ir-2 (c, 1.2 μM) and Ir-3 (d, 14.0 μM)-treated cells, weak green
fluorescence points were observed. After A549 cells were treated with
Ir-1-Lipo (e, 3.0 μM), Ir-2-Lipo (f, 1.2 μM) and Ir-3-Lipo (g, 14.0 μM)
for 24 h, a number of bright green fluorescence points were discovered,
which indicated that Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo caused a
marked increase in the number of MDC-positive cells. Up-regulation of
Beclin-1 expression and the conversion from LC3-I to LC3-II (lipidated
form) is a hallmark of autophagy. As shown in Fig. 7B, in the presence
of Ir-1 (3.0 μM), Ir-2 (1.2 μM) and Ir-3 (14.0 μM), no obvious change in
the expression of Beclin-1 and the conversion from LC3-I to LC3-II was
found compared with the control. After A549 cells were exposed to Ir-
1-Lipo (3.0 μM), Ir-2-Lipo (1.2 μM) and Ir-3-Lipo (14.0 μM) for 24 h,
the conversion from LC3-I to LC3-II and the upregulation in the ex-
pression of Beclin-1 protein were observed compared with that of the
control. These results indicate that Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo
can induce autophagy in A549 cells, but Ir-1, Ir-2 and Ir-3 cannot in-
duce autophagy under the same conditions.
3.12. Cell invasion studies
Metastasis, the main cause of death of cancer patients, is the spread
of tumor cells from a primary tumor source to other organs, and re-
mains as one of the ultimate challenges in cancer treatment [60]. Cell
migration and invasion cause metastasis dissemination of tumor cells by
promoting cell penetration into the lymphatic system and blood vessels.
Cells then undergo extravasation into a new site (metastatic site), where
they proliferate, leading to cancer metastasis [61]. To evaluate the
ability of the complexes and liposomes to prevent cell invasion, the cell
invasion in vitro assays were conducted. As shown in Fig. S9 (sup-
porting information) and Fig. 9, although Ir-1, Ir-2 and Ir-3 show low
or no cytotoxic activity against A549 cells, after A549 cells were in-
cubated with 1.5, 0.6 and 7.0 μM of Ir-1, Ir-2 and Ir-3 for 24 h, these
complexes exhibit highly inhibitory effect on the cell invasion. How-
ever, Ir-1 and Ir-2 were encapsulated in liposomes Ir-1-Lipo and Ir-2-
Lipo, the percentage of inhibiting cell invasion increases 17.5 and
27.2%, respectively. The results demonstrate that Ir-1-Lipo, Ir-2-Lipo
and Ir-3-Lipo can effectively inhibit the cell invasion in A549 cells.
3.13. Effect of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo on microtubule networks
3.11. Release of cytochrome c in vitro studies
Microtubules are one of the key structural components of the cy-
toskeleton in eukaryotic cells comprising of α and β-tubulin hetero-
dimers. They play an important role in the fundamental cellular pro-
cesses such as structural and regulatory functions, including the
maintenance of cell shape and intracellular transport machinery as well
as cell growth and mitosis and have emerged as an attractive and viable
target in the development of anticancer drugs [62–65]. To obtain in-
sight into the mechanism of cell growth inhibition induced by the li-
posomes, the morphology of microtubules and cytoskeleton were stu-
died by immunofluorescence staining. As shown in Fig. 10, in the
control (a), A549 cells showed a well-organized microtubule network.
Cytochrome c (cyto c) plays an important role in mitochondrial
electron transport and cell metabolism. Its role in maintaining a normal
mitochondrial membrane potential is well established [59]. Liposomes
Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo induce a decrease in the mitochon-
drial membrane potential and cause mitochondria dysfunction, which
will lead to a release of cytochrome c. Thus, it is necessary to measure
the levels of cyto-c in A549 cells. As shown in Fig. 8A, in the control, no
obvious green fluorescence was observed, which indicated that no cyto
c was released. However, treatment of 24 h of A549 cells with Ir-1-Lipo
(3.0 μM), Ir-2-Lipo (1.2 μM) and Ir-3-Lipo (14.0 μM), a lot of green
13

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
Fig. 10. Assays of microtubules network induced by 3.0, 1.2 and 14.0 μM of Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo for 9 h.
However, A549 cells were exposed to 3.0, 1.2 and 14.0 μM of Ir-1-Lipo,
Ir-2-Lipo and Ir-3-Lipo for 9 h, the cells were shrunk with improper
microtubule bundling, and the rounded morphology of microtubules
were observed, indicating that Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo can
result in microtubules depolymerization and cause cytoskeleton dis-
ruption and inhibition of the cell proliferation. The results indicate that
tubules may be potential target of the antitumor activity of Ir-1-Lipo,
Ir-2-Lipo and Ir-3-Lipo.
1-Lipo, Ir-2-Lipo and Ir-3-Lipo was further supported by the cleavage
of polymerase (PARP), a major caspase-3 substrate, as assessed by
Western blot. Ir-1, Ir-2 and Ir-3 were used as a comparison. As shown
in Fig. 11, in the presence of Ir-1 (3.0 μM), Ir-2 (1.2 μM) or Ir-3
(14.0 μM), no obvious change in the expression of caspase-3 and
cleaved PARP were observed compared with the control. However,
treatment of 24 h of A549 cells with Ir-1-Lipo (3.0 μM), Ir-2-Lipo
(1.2 μM) or Ir-3-Lipo (14.0 μM) led to an increase in the expression of
caspase-3 and cleaved PARP. The proteins of the Bcl-2 family playing a
major role in controlling apoptosis through the regulation of mi-
tochondrial processes and the release of mitochondrial proapoptotic
molecules are important for the cell death pathway [64]. After A549
cells were exposed to Ir-1-Lipo (3.0 μM), Ir-2-Lipo (1.2 μM) or Ir-3-
3.14. Expression of Bcl-2 family proteins
Caspase-3 plays a crucial role in mediating apoptotic DNA frag-
mentation during apoptosis [66–70]. The induction of apoptosis by Ir-
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L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
Fig. 11. The expression of caspase-3, cleaved PARP and Bcl-2 family proteins after A549 cells were treated with 3.0, 1.2 and 14.0 μM of Ir-1 (b), Ir-2 (c), Ir-3 (d) or
Ir-1-Lipo (e), Ir-2-Lipo (f) and Ir-3-Lipo (g) for 24 h.
Fig. 12. The in vivo antitumor activity of Ir-2-Lipo in A549 xenograft model. (A) Tumor volume growth trend of control group, 9.45 mg/kg of Ir-2, 4.725 mg/kg and
9.45 mg/kg of Ir-2-Lipo groups. Tumor volumes were tracked by the mean tumor volume (cm3)
SD (n = 6) and calculated as relative tumor growth rate [T/C%]
SD) mg after the tumor was treated with Ir-2 or Ir-2-Lipo for
values. (B) Photographs of tumor from treatment groups and vehicle group. (C) Tumor weight (mean
10 days. (D) Inhibiting percentage of tumor growth induced by 9.45 mg/kg and different concentrations of Ir-2-Lipo. *P < 0.05 represents significant differences
compared with control.
Lipo (14.0 μM) for 24 h, the expression of Bcl-2 protein was down-
regulation and the expression of Bad and Bax was up-regulated com-
pared with that of control.
used as a model for this study. Xenograft tumors were generated by
subcutaneous implantation of A549 cells into nude mice. Mice bearing
A549 tumors were chosen and randomly divided into four groups. After
the treatment with 4.725 mg/kg and 9.45 mg/kg of Ir-2-Lipo daily for
10 consecutive days, the mouse body weights and relative volume of
tumor were measured and recorded every day. Ir-2 (9.45 mg/kg) was
used as a comparison. As shown in Fig. 12A–D, 10 days treatment of Ir-
3.15. Antitumor evaluation in vivo
Athymic nude mice xenografted with human A549 lung cells were
15

L. Bai, et al.
JournalofInorganicBiochemistry205(2020)111014
2-Lipo resulted in a decrease in the mean weights and relative volume
of tumor compared with those of control or Ir-2. The inhibiting per-
centages of tumor growth induced by Ir-2 (9.45 mg/kg) and 4.725 or
9.45 mg/kg of Ir-2-Lipo are 27.65%, 34.04% and 57.45%, respectively.
Obviously, complex Ir-2 shows low inhibitory effect on the tumor
growth, whereas Ir-2-Lipo displays high antitumor activity. Further-
more, Ir-2-Lipo inhibits tumor growth in a concentration-dependent
manner.
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Three iridium(III) complexes Ir-1, Ir-2, Ir-3 and their long circula-
tion liposomes Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo were synthesized and
characterized. Ir-1 shows moderate or no cytotoxic activity, Ir-2 and Ir-
3 have no any cytotoxic activity against the selected cancer cells.
However, Ir-1, Ir-2 and Ir-3 were encapsulated in liposomes Ir-1-Lipo,
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against A549 cells, IC50 values are 3.1
14.1
0.3, 1.2
0.4 and
1.8 μM, respectively. In particular, Ir-1-Lipo and Ir-2-Lipo
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Liposomes Ir-1-Lipo, Ir-2-Lipo and Ir-3-Lipo interact to lysosomes,
increase intracellular ROS level and induce a decrease of mitochondrial
membrane potential. In addition, the liposomes can release cyto c, in-
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whereas Ir-1, Ir-2 and Ir-3 show weak or no anticancer activity com-
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vivo shows that Ir-2-Lipo can effectively inhibit the tumor growth. In
summary, we think the liposomes induce apoptosis through the fol-
lowing two pathways: (I) ROS-mediated lysosome-mitochondria dys-
function pathway; (II) The liposomes inhibit polymerization of micro-
tubules and further inhibition of the cell growth at G0/G1 phase
pathway. This work will be helpful for designing and synthesizing of
new iridium(III) complexes as potent anticancer reagents.
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Declaration of competing interest
Authors declare no competing interest exists.
Acknowledgements
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This work was supported by the National Natural Science
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1H NMR and 13C NMR (Fig. S1), UV–Vis spectra (Fig. S2),
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