Nucleus-Targeted Organoiridium–Albumin Conjugate for Photodynamic Cancer Therapy

Article abstract of DOI:10.1002/anie.201813002

An organoiridium–albumin bioconjugate (Ir1-HSA) was synthesized by reaction of a pendant maleimide ligand with human serum albumin. The phosphorescence of Ir1-HSA was enhanced significantly compared to parent complex Ir1. The long phosphorescence lifetime and high ¹O₂ quantum yield of Ir1-HSA are highly favorable properties for photodynamic therapy. Ir1-HSA mainly accumulated in the nucleus of living cancer cells and showed remarkable photocytotoxicity against a range of cancer cell lines and tumor spheroids (light IC₅₀; 0.8–5 μm, photo-cytotoxicity index PI=40–60), while remaining non-toxic to normal cells and normal cell spheroids, even after photo-irradiation. This nucleus-targeting organoiridium-albumin is a strong candidate photosensitizer for anticancer photodynamic therapy.

Full text of DOI:10.1002/anie.201813002

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Nucleus-targeted organoiridium-albumin conjugate for
photoactivated cancer therapy
Authors: Pingyu Zhang, Huaiyi Huang, Samya Banerjee, Guy
Clarkson, Chen Ge, Cinzia Imberti, and Peter J. Sadler
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201813002
Angew. Chem. 10.1002/ange.201813002
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Nucleus-targeted
organoiridium-albumin
conjugate
for
photodynamic cancer therapy
Pingyu Zhang,[a,c] Huaiyi Huang,*[b,c] Samya Banerjee,[c] Guy J. Clarkson,[c] Chen Ge,[a] Cinzia Imberti,[c]
Peter J. Sadler*[c]
Abstract: A novel organoiridium-albumin bioconjugate (Ir1-HSA) was
synthesized via reaction of a pendant maleimide ligand with human
serum albumin. The phosphorescence of Ir1-HSA was enhanced
significantly compared to parent complex Ir1. The long
phosphorescence lifetime and high 1O2 quantum yield of Ir1-HSA are
highly favourable properties for photodynamic therapy. Ir1-HSA
mainly accumulated in the nucleus of living cancer cells and showed
remarkable photocytotoxicity against a range of cancer cell lines and
tumor spheroids (light IC50; 0.8-5 M, photo-cytotoxicity index PI = 40-
60) while remaining non-toxic to normal cells and normal cell
spheroids, even after photo-irradiation. This nucleus-targeting
organoiridium-albumin is a strong candidate photosensitizer for
anticancer photodynamic therapy.
functionalized metal complexes (e.g. Pt, Ru, Os) have been
developed for cancer therapy, illustrating that HSA plays a key
role in augmenting anticancer activity.[10]
Here we have conjugated
a
maleimide-functionalized
octahedral organo-iridium(III) complex (Ir1, Figure 1a) to HSA,
giving rise to a large enhancement in the phosphorescence of Ir1-
HSA compared to Ir1. Ir1-HSA is notably nontoxic in the dark, but
exhibits potent photo-cytotoxicity with significant selectivity for
cancer cells and cancer cell spheroids over normal cells. Ir1-HSA
appears to be the first example of an HSA-functionalized iridium
conjugate which allows targeting of cell nuclei, as well as being
an efficient photosensitizer for PDT.
The octahedral organo-iridium(III) complex Ir1, containing two
chelated phenylpyridine ligands and two monodentate pyridines
functionalized with a maleimide substituent, was synthesized and
fully characterized as described in the Supporting Information. It
was highly stable in phosphate-buffered saline (PBS) solution for
12 h in the dark and photostable after 1 h irradiation with blue light
(465 nm; Figure S1).
To investigate the reactivity of the C=C bond of the pendant
maleimides of Ir1, the complex was reacted with cysteine (Cys) in
a molar ratio of 2[Cys]: 1[Ir1] in DMSO-d6/D2O (2/1 v/v) at 298 K
for 30 min. 1H NMR peaks for the vinyl protons of the maleimide
groups at 6.62 ppm disappeared upon the addition of Cys, and
new peaks appeared between 2.9 ppm and 3.9 ppm assignable
to conjugated Cys (Figure S2). The intensities of the peaks
indicated that Cys reacted with each of the two pendant
maleimides of Ir1. The conjugate was further characterised by
ESI-MS (Figures S3-S4).
To investigate whether the free Cys34 thiol of HSA can similarly
react with the C=C in maleimide, Ir1 (30 M) was incubated with
various amounts of HSA (0-120 M) for 1 h and the products were
separated by RP-HPLC. The peak for Ir1 gradually disappeared
with increasing amounts of HSA, with complete reaction observed
at 120 M HSA (Figure 1b). This molar ratio of [HSA]:[Ir1] of 4:1,
is consistent with the free thiol content of the HSA used,
determined via reaction with 2,2’-dithiodipyridine (2,2’-DTDP)
using a slightly modified literature approach.[11] This gave a thiol
content of 0.27  0.1 mol SH per mol HSA (Figure 1c). Hence, the
concentration of free -SH groups from 120 M HSA was 32.4 
1.2 M, which reacted with 30 M Ir1, suggesting formation of a
1:1 Ir1:HSA adduct. As Cys34 is in a crevice, it is likely that the
second maleimide group is not accessible to a second HSA. RP-
HPLC studies on the time-dependent binding showed that the
reaction was relatively rapid with a half-life of ca. 20 min (Figure
S5).
Photodynamic therapy (PDT) is a non-invasive cancer therapy,[1]
which uses photosensitizers and light to convert cellular triplet
oxygen (3O2) to highly reactive and cell-damaging singlet oxygen
(1O2).[2] Examples of clinical photosensitizers include
hematoporphyrin derivatives (Photofrin) and aminolevulinic acid
(ALA, a porphyrin precursor).[3] In recent years, metal complexes
with high luminescence have emerged as promising photo-
theranostic candidates due to their superior photochemical and
photophysical properties.[4] An octahedral tris-N,N-chelated RuII
complex (TLD1433) recently entered clinical trials for bladder
PDT,[4a] and WST 11 (TOOKAD® Soluble), a square-planar PdII
bacteriochlorophyll derivative, has been approved for vascular-
targeted PDT.[4b]
Human serum albumin (HSA) conjugates can be used to
delivery anticancer drugs. HSA is abundant in blood serum
(ca.0.6 mM), rich in histidine, and contains a free thiol residue at
cysteine-34.[5] Moreover, HSA functions as a physiological
antioxidant,[6] and binds a wide range of biologically and clinically
important molecules.[7] The effectiveness of HSA-coupled
anticancer drugs has been established clinically for doxorubicin
(INNO206; aldoxorubicin),[8] and HSA-based nanoparticle-
encapsulated paclitaxel (Abraxane®).[9] Recently, HSA-
[a]
[b]
[c]
Dr. P. Zhang, C. Ge
College of Chemistry and Environmental Engineering
Shenzhen University
Shenzhen, 518060, China.
Dr. H. Huang
School of Pharmaceutical Science (Shenzhen), Sun Yat-sen
University, Guangzhou, 510275, China
E-mail: huanghy87@mail.sysu.edu.cn
Dr. H. Huang, Dr. S. Banerjee, Dr. G. J. Clarkson, Dr. C. Imberti,
Prof. P. J. Sadler
Department of Chemistry, University of Warwick
Coventry, CV4 7AL, UK
E-mail: P.J.Sadler@warwick.ac.uk
Supporting information and the ORCID identification numbers for the
authors of this article can be found under:https://doi.org/xxxxxx
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Figure 2. (a) Emission intensity of HSA (100 M) after reaction with 10 mol
equiv cystine (giving disulfide formation at Cys34) in PBS (pH = 7.4) for 24 h at
277 K, followed by treatment with Ir1 for 30 min; Inset: Images of the reaction
mixture compared to Ir1-HSA under UVA irradiation showing the decrease in
phosphorescence. (b) Emission intensity of Ir1 (4 M) in the presence of various
amino acids (100 M) in PBS solution. (c) Emission intensity at 515 nm of Ir1 in
the presence (I) vs. the absence (I0) of amino acids according to (b). (d)
Structure of HSA (PDB:5IJF); Cys34 and His39 are labelled
.
Figure 1. (a) Scheme for the conjugation of Ir1 to HSA. (b) Reaction of Ir1 (30
M) with various concentrations of HSA (0, 60, 90 and 120 M, concentrations
of Cys34 free thiol group were 0, 16.2, 24.3, 32.4 M, respectively) studied by
RP-HPLC (UV detection at 280 nm). (c) Variation of absorbance at 340 nm at
various HSA:2,2’-DTDP ratios ([HSA] = 80 M; [DTDP] = 0-110 M) and
determination of the thiol content of HSA. (d) Emission spectra of Ir1 (4 M) in
the presence of increasing concentrations of HSA (0-30 M, [Cys34 free thiol]
of Ir1 with histidine resulted in an emission enhancement of ca.
37-fold. In contrast, no significant luminescence enhancement
was observed with the other amino acids, including Cys (Figure
2b,c). Although Ir1 binds to Cys34, other factors appear to be
responsible for the enhancement of phosphorescence of Ir1-
HSA. These include interactions with His residues, a strong
candidate being nearby His39 (Figure 2d).
Ir(III) complexes with weakly bound ligands are known to bind
strongly to amino acids/proteins through ligand substitution
reactions, especially to histidine/histidine rich proteins, and their
use in protein staining has been reported.[12] Here it is evident that
histidine can switch on the phosphorescence of Ir1. We recorded
the ESI-MS of a mixture of Ir1 and His; two peaks at m/z 656.2
and 903.2 assignable to His-bound Ir(III) species were detected
(Figure S6). These results suggest that one sterically-hindered
monodentate maleimide ligand is released after binding of Ir1 to
Cys34, being displaced by His39.
0-8.1 M), in PBS (pH = 7.4), reaction time: 20 min at each concentration, ex
=
405 nm; Inset: photos of Ir1 (1) and Ir1-HSA (2) under UVA irradiation. (e)
Dependence of the phosphorescence intensity of Ir1 at = 515 nm on the
concentration of free thiol of HSA, as ratio [Ir1]/[free thiol].
Ir1 (4 M) itself was only weakly emissive in aqueous solution
(ex
=
405 nm), whereas Ir1-HSA showed strong
phosphorescence under the same conditions (inset graph Figure
1d). The phosphorescence of Ir1 gradually increased with
increasing concentrations of HSA (Figure 1d), plateauing at a mol
ratio of ca. 1.0 Ir1:HSA(free-SH); with a phosphorescence
enhancement of ca. 53-fold (Figure 1e).
To remove free thiol groups from HSA (by oxidation to a
disulphide), a 10-fold molar excess of cystine was added to a 100
M HSA solution for 24 h at 277 K. Then this HSA-Cys34-S-S-
Cys product was reacted with Ir1 for 30 min. The observed
phosphorescence was much weaker compared to the conjugate
formed by reaction of HSA-Cys34 and Ir1 (Figure 2a), clearly
suggesting that the free thiol of Cys34 is the binding site of Ir1.
HSA is a large protein (66.5 kDa) with a single-chain of 585
amino acid residues.[5-7] To provide an indication on which
residues of HSA might be involved in the luminescence
enhancement of Ir1, the interaction of Ir1 with various amino acids
was studied by luminescence analysis (Figure 2b,c). Interaction
As Ir1-HSA emitted strong phosphorescence, we evaluated
Ir1-HSA as a potential photosensitizer for PDT. Firstly, Ir1 (0.4
mM) was dissolved in 20 mL MeOH:H2O (1:2 v/v), and HSA (0.4
mM) was added. The reaction mixture was stirred for 1 h, followed
by Ir1-HSA purification by dialysis (10 kDa cut-off filter) to remove
unbound Ir1. The Ir1-HSA conjugate showed a new IR band at
1043 cm-1, characteristic of a new C-S stretch (Figure S7).
Next the localization of Ir1-HSA in living A549 lung cancer cells
was investigated using confocal microscopy. The cells were
incubated with Ir1-HSA ([Ir] = 5 M) for various times. Real-time
imaging showed that Ir1-HSA mainly accumulated in the
cytoplasm within the first 30 min and then migrated to the nucleus
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Figure 3. Confocal microscopy images of living A549 cells incubated with
Ir1-HSA ([Ir] = 5 M) in real time (5, 15, 40, 60, 120 min); ex = 405 nm, em
550  20 nm.
=
Figure 4. Immunofluorescence staining of HSA in cells exposed to Ir1, HSA,
Ir1-HSA ([Ir] = 5 M, 2 h), respectively. ex = 563 nm; em = 580-630 nm.
upon further incubation (60 min-120 min) (Figure 3). In contrast,
the green luminescence of Ir1 alone was observed throughout the
cells, both in cytoplasm and nucleus (Figure S8). This subcellular
localization of Ir1 and Ir1-HSA in A549 cells was also confirmed
by ICP-MS (Figure S9), demonstrating that iridium localized both
in the cytoplasm and nucleus of cells treated with Ir1, whereas it
mainly accumulated in the nucleus of Ir1-HSA treated cells. We
further confirmed that the luminescence of Ir1-HSA was perfectly
co-localized with the blue fluorescence of Hoechst 33258 (a
nucleus dye), with a co-localization efficiency of 0.82 in live A549
cells (Figure S10).
To address the question as to whether HSA co-migrated with Ir,
we used an immunofluorescence approach to determine the
intracellular localization of HSA. As expected, HSA was not
detected in Ir1 treated A549 cells (Figure 4). However, Ir1-HSA
exposure led to the accumulation of HSA in the cytoplasm and the
nuclear membrane. When the cells were treated with HSA, the
red luminescence was observed as well. This suggests that,
although HSA facilitates delivery of the iridium complex to the
nucleus, it does not itself penetrate past the nuclear membrane,
and is probably released from Ir1-HSA before the Ir(III) complex
migrates into the nucleus.
The phosphorescence quantum yield of Ir1 was very low
(0.001) and its phosphorescence lifetime only 182.7 ns in
methanol:PBS (1:1, v/v; Figure 5a; Table S1) at 298 K. Compared
to Ir1, the quantum yield for Ir1-HSA increased 36x (to 0.036) and
its emission lifetime to 871.8 ns. The long phosphorescence
Figure 5. (a) Phosphorescence lifetimes of Ir1 and Ir1-HSA in methanol:PBS
(1:1 v/v) at 298 K; (b) EPR spectra of Ir1 and Ir1-HSA with TEMP after 20 min
light irradiation (465 nm, 5.76 J/cm2). (c) Fluorescence intensity of ROS in A549
cells treated with Ir1 or Ir1-HSA ([Ir] = 5 M) and ROS probe in the dark or upon
light irradiation (5, 10, 20 min). The excitation wavelength of the ROS probe was
520 nm and the fluorescence was measured at 590-625 nm.
1
lifetime of Ir1-HSA makes it ideal for O2 generation. We used
electron paramagnetic resonance (EPR) spectroscopy with
2,2,6,6-tetramethylpiperidine (TEMP) as a spin trap to detect 1O2
generation by Ir1 and Ir1-HSA under 465 nm irradiation. As
illustrated in Figure 5b, a characteristic 1:1:1 triplet assignable to
2,2,6,6-tetramethylpiperidine-1-oxyl
was
observed
upon
irradiation (20 min). Notably the intensity of the EPR signal
generated by Ir1-HSA was significantly stronger than for Ir1. The
1O2 quantum yield[13] ((1O2)) of Ir1-HSA was 0.83, much higher
than that for Ir1 (0.06) upon 465 nm light irradiation (Table S1).
The long excited-state lifetime and very high 1O2 generation
quantum yield make Ir1-HSA a potential PDT agent. To explore
this, the dark- and photo-antiproliferative activity of Ir1 and Ir1-
HSA was determined against human cancer cells (lung: A549;
hepatoma: Hep-G2; cisplatin resistant lung: A549R) and normal
human cells (lung: MRC-5; liver: LO2). Cells in the ‘light’ plate
were incubated with Ir1 or Ir1-HSA for 2 h in the dark, washed
with PBS, followed by 20 min irradiation using blue LEDs (465 nm,
5.76 J/cm2), while the ‘dark’ plate was kept in the dark. Then all
cells were allowed to recover over 46 h. No cell death was
observed for untreated cells exposed to light (Figure S11). Ir1 was
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Table 1. IC50 (M) values for Ir1 and Ir1-HSA against 2D and 3D (spheroids)
cancer and normal cell lines.
In contrast to other well-studied cyclometalated iridium
complexes, which mainly located in the cytoplasm, Ir1-HSA
appears to be the first reported nucleus-targeting photosensitizer.
There are only a few reports of the transport of albumin to the
nucleus: in response to oxidative stress,[14] and via
permeabilization of cells with digitonin.[15] In our work, it appears
that albumin plays an important role in the transport and delivery
of Ir1 to the cell nucleus.
Importantly, Ir1-HSA exhibited a long phosphorescence lifetime
and remarkably high 1O2 generation quantum yield along with high
photostability, which were essential for efficient PS. Ir1-HSA
exhibited excellent photocytotoxicity against a range of cancer
cell lines and multicellular spheroids with a high photo-cytotoxicity
index while remaining dormant in normal cells/spheroids, even
after photo-irradiation. All these properties confirm that Ir1-HSA
could be an efficient photosensitizer with novel nucleus-targeting
property for potential clinical PDT applications.
Cell linesa
Ir1
Light
Ir1-HSA
Light
Dark
Dark
A549
89.63.7
83.53.7
75.64.1
90.61.7
89.32.3
100
53.34.5
54.82.6
56.21.5
76.91.6
76.50.9
100
62.32.6
85.63.2
84.95.9
96.46.1
88.63.0
65.65.9
100
1.10.3
2.20.3
2.30.2
78.72.3
66.44.5
4.80.2
100
Hep-G2
A549R
MRC-5
LO2
A549 spheroid
MRC-5 spheroid
100
100
aCells were incubated with the compounds for 2 h in the dark, washed, fresh
medium added, followed by incubation in the dark or irradiation at 465 nm (20
min, 5.76 J/cm2), and a further 46 h incubation. IC50 values for Ir1 and Ir1-HSA
are based on Ir concentration. Under the same experimental conditions, 5-
aminoevulinc acid, a clinical PDT agent and cisplatin gave IC50 > 100 M both
in the dark and upon light irradiation. The IC50 values (concentrations which
caused 50% of cell death) were determined as duplicates of triplicates in three
independent sets of experiments. For each data point, the average and standard
deviation are reported. For 3D toxicity assays, 8 spheroids were selected for
each condition studied.
Acknowledgements
We thank the EPSRC (grants EP/G006792 and EP/M027503/1
for PJS), National Natural Science Foundation of China (NSFC,
21701113), the Science and Technology Foundation of Shenzhen
(JCYJ20170302144346218) and the Natural Science Foundation
of SZU (2018036) for PZ, Newton Fund (NF160307 for HH,
NF151429 for SB) and the Wellcome Trust (209173/Z/17/Z for CI)
for support. We thank Dr. Lijiang Song for excellent assistance
with mass spectrometry; Dr. Ben Breeze with EPR experiments;
and Dr. Ivan Prokes with NMR spectroscopy.
relatively nontoxic toward A549 cells both in the dark (89.6 M)
and light (53.3 M) (Table 1). In contrast, Ir1-HSA was non-toxic
towards A549 cells in the dark (62.3 M), but became highly
cytotoxic upon irradiation (1.1 M) with a high photocytotoxicity
index (PI, PI = dark IC50 /light IC50) of 56.6 (Figure S11). Similar
photodynamic efficiency for Ir1-HSA was also observed for Hep-
G2 and A549R cells. Notably, under the same experimental
conditions, both Ir1 and Ir1-HSA were non-toxic toward normal
cells (MRC-5 and LO2) (Table 1).
We further investigated the photocytoxicity in 3D multicellular
spheroids (MCSs) with semidiameters of ∼400 m. The
cytotoxicities of the complexes toward MCSs were determined by
measurement of ATP concentrations using the CellTiter-Glo® 3D
Cell Viability Assay (Promega). As shown in Table 1, both Ir1 and
Ir1-HSA were non-toxic towards A549 cancer spheroids and
normal cell spheroids in the dark (IC50 > 50 M). However, Ir1-
HSA showed strong phototoxic effects on A549 cancer spheroids
upon light irradiation, with an IC50 value of 4.8 M. We also
studied the effect of Ir1-HSA on the kinetics of 3D MCSs
regrowth. After treatment with Ir1-HSA ([Ir] = 5 M) in the dark,
the diameters of 3D MCSs increased slightly after 48 h. However,
the MCSs treated with Ir1-HSA followed by 20 min light irradiation
decreased in size over time (Figure S12).
A reactive oxygen species (ROS) detection assay kit was used
to determine whether Ir1-HSA produced ROS within the cells
upon irradiation. Cells treated with the red ROS probe and Ir1 or
Ir1-HSA in the dark showed no evident fluorescence and there
was only a very weak fluorescence in the cells treated with Ir1. In
contrast, a strong red fluorescence was shown within the cells
pre-treated with Ir1-HSA following light irradiation (Figure 5c),
suggesting that Ir1-HSA generated ROS efficiently in cancer cells
upon light irradiation.
Keywords:
Organoiridium,
Albumin,
Photosensitizer,
Photodynamic therapy
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Orchid identification numbers
Pingyu Zhang: 0000-0002-2921-9490
Huaiyi Huang: 0000-0002-2091-7954
Samya Banerjee: 0000-0003-4393-4447
Guy Clarkson: 0000-0003-3076-3191
Chen Ge: 0000-0002-4910-6282
Cinzia Imberti: 0000-0003-1187-7951
Peter Sadler: 0000-0001-9160-1941
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10.1002/anie.201813002
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Pingyu Zhang, Huaiyi Huang,* Samya
Banerjee, Guy J. Clarkson, Chen Ge,
Cinzia Imberti, Peter J. Sadler*
Page No. – Page No.
Nucleus-targeted organoiridium-
albumin conjugate for photodynamic
cancer therapy
We report the first example of a nucleus-targeted organo-iridium-HSA conjugate for photodynamic
therapy. Phosphorescence of a weakly emissive maleimide-functionalized Ir complex is greatly
enhanced when conjugated to human serum albumin. The iridium-HSA conjugate targets the
nucleus and generates 1O2 for photodynamic therapy upon irradiation with visible light.
This article is protected by copyright. All rights reserved.