Synthesis and Characterization of an Epidermal Growth Factor Receptor-Selective RuII Polypyridyl–Nanobody Conjugate as a Photosensitizer for Photodynamic Therapy

Article abstract of DOI:10.1002/cbic.201900419

There is a current surge of interest in the development of novel photosensitizers (PSs) for photodynamic therapy (PDT), as those currently approved are not completely ideal. Among the tested compounds, we have previously investigated the use of Ru^II<

Full text of DOI:10.1002/cbic.201900419

Accepted Article
Title: Synthesis and Characterization of an Epidermal Growth Factor
Receptor selective Ru(II) Polypyridyl-Nanobody Conjugate as a
Photosensitizer for Photodynamic Therapy
Authors: Johannes Karges, Marta Jakubaszek, Cristina Mari, Kristof
Zarschler, Bruno Goud, Holger Stephan, and Gilles Gasser
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ChemBioChem
10.1002/cbic.201900419
Synthesis and Characterization of an Epidermal Growth
Factor Receptor selective Ru(II) Polypyridyl-Nanobody
Conjugate as a Photosensitizer for Photodynamic Therapy
a,#
a,b,#
c,#
d,#,*
Johannes Karges, Marta Jakubaszek,
Cristina Mari, Kristof Zarschler,
Bruno
b
d
a,
Goud, Holger Stephan, and Gilles Gasser *
a
Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health
Sciences, Laboratory for Inorganic Chemical Biology, F-75005 Paris, France.
Institut Curie, PSL University, CNRS UMR 144, Paris, France.
b
c
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich,
Switzerland.
d
Helmholtz-Zentrum Dresden - Rossendorf, Institute of Radiopharmaceutical Cancer
Research, Bautzner Landstraße 400, D-01328 Dresden, Germany.
#
These authors have contributed equally to the work.
* Email: k.zarschler@hzdr.de, WWW: www.hzdr.de/NanoscalicSystems, Tel. +49 351 260
3678; Email: gilles.gasser@chimieparistech.psl.eu, WWW: www.gassergroup.com, Tel. +33 1
44 27 56 02.
ORCID-ID:
Johannes Karges:
Marta Jakubaszek:
Cristina Mari:
0000-0001-5258-0260
0000-0001-7590-2330
0000-0002-0086-4736
0000-0002-7571-4732
0000-0003-1227-4159
Kristof Zarschler:
Bruno Goud:
1
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ChemBioChem
10.1002/cbic.201900419
Holger Stephan:
Gilles Gasser:
0000-0002-2972-2803
0000-0002-4244-5097
Keywords: Bioinorganic Chemistry, Medicinal Inorganic Chemistry, Metal-based drugs,
Metals in Medicine, Photodynamic Therapy.
2
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ChemBioChem
10.1002/cbic.201900419
Abstract
There is currently a surge for the development of novel photosensitizers (PSs) for photodynamic
therapy (PDT) since those currently approved are not completely ideal. Among the tested
compounds, we have previously investigated the use of Ru(II) polypyridyl complexes with a
[Ru(bipy)
2
(dppz)]2+ and [Ru(phen) (dppz)]2+ scaffold (bipy = 2,2'-bipyridine; dppz =
2
dipyrido[3,2-a:2′,3′-c]-phenazine, phen = 1,10-phenanthroline). These complexes selectively
target DNA. However, since DNA is ubiquitous, it would be of great interest to increase the
selectivity of our PDT PSs by linking them to a targeting vector in view of targeted PDT.
Herein, we present the synthesis, characterization and in-depth photophysical evaluation of a
nanobody-containing Ru(II) polypyridyl conjugate selective for the epidermal growth factor
receptor (EGFR) in view of targeted PDT. Using ICP-MS and confocal microscopy, we could
demonstrate that our conjugate had a high selectivity for the EGFR receptor, which is a crucial
oncological target as it is overexpressed and/or deregulated in a variety of solid tumors.
However, contrary to expectations, this conjugate was found to not produce reactive oxygen
species (ROS) in cancer cells and to be therefore not phototoxic.
Graphical abstract
The synthesis, characterization and in-depth photophysical evaluation of a nanobody-
containing Ru(II) polypyridyl conjugate selective for the epidermal growth factor receptor
(
EGFR) in view of targeted PDT is presented.
3
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ChemBioChem
10.1002/cbic.201900419
Introduction
The use of photodynamic therapy (PDT) has expanded the possible techniques in medicine to
treat various types of cancer (e.g., lung, bladder, oesophageal and brain cancer) as well as
bacterial, fungal or viral infections. Its effect is caused by a combination of an ideally non-toxic
photosensitizer (PS), oxygen and light. Upon light exposure, the PS is able to produce reactive
1
oxygen species (ROS), such as singlet oxygen ( O
2
) or other radicals. Due to the high reactivity
of the latter, these can cause oxidative stress and damage in different cellular compartments
e.g., membrane, nucleus, endoplasmic reticulum, lysosome, mitochondria), leading ultimately
(
to cell death.[1]
Next to the already approved PDT PSs, which are based on a tetrapyrrolic scaffold (i.e.
porphyrins, chlorins, phthalocyanines), the development of Ru(II) polypyridyl complexes as
PDT PSs is receiving more attention due to their ideal photophysical and photochemical
properties, which include, among others, high water solubility, high chemical stability and
1
production.[1a-d, 2] These
photostability, intense luminescence, large Stokes shifts, high O
2
attractive features have allowed one of such complexes, namely TLD-1433, to enter into clinical
[3]
[2f]
trial as a PDT PS against bladder cancer. Phase I has been recently completed.
In this context, our group was able to demonstrate that Ru(II) complexes of the type
[Ru(bipy)
2
(dppz)]2+ (bipy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2′,3′-c]-phenazine) and
2
+
[1a, 2c, 4]
[Ru(phen)
2
(dppz)] (phen = 1,10-phenanthroline) were effective PDT PSs (Figure 1).
As a highlight, we could demonstrate that some of these complexes were non-toxic in the dark
and highly toxic upon light irradiation with IC50 values in the low micromolar range and a
phototoxic index of up to >150.[2c] Based on the extended planar π-system of the dppz ligand,
which is able to intercalate into the base pairs of the DNA, these compounds showed a
preferable nuclear localization. Upon light exposure, these complexes caused oxidative stress,
as well as DNA photocleavage, suggesting that they impaired replication and integrity of the
genetic material.[1a, 2c, 4]
Highly proliferating cells like cancer cells are generally preferably targeted by such compounds
[
5]
over healthy cells, as it is the case for cisplatin. However, other frequently dividing cells in
the organism (e.g. hair follicles, gastrointestinal tract, bone marrow) can be affected, leading to
severe side-effects for the patients.[4a, 6] Thus, it is extremely important to increase the
selectivity of PDT PS, for example, with the development of a suitable delivery system.
4
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ChemBioChem
10.1002/cbic.201900419
2+
2+
N
N
N
N
N
N
N
N
N
R1
R2
N
N
N
N
Ru
N
Ru
N
N
R
1
2
R = NH2
R = H, R = CN
CH NH2
CH NH2
2
2
OMe
OAc
OH
1
2
R = R =
OMe
OH
CH OH
2
CH Cl
2
CN
Figure 1. Structures of [Ru(bipy)
2
(dppz)]2+ and [Ru(phen) (dppz)]2+ complexes as PSs
2
developed by our group.[1a, 2c, 4]
So far, the examples of Ru(II) polypyridyl complexes for targeted PDT are scare, if we do not
take into account polymer encapsulation/nanoparticle attachment.[4a, 7] The group of Lilge could
recently demonstrate that the premixing of TLD-1433 with transferrin was able to increase the
extinction coefficient, prolongs the absorption range, reduced photobleaching, cellular uptake
[
8]
as well as overall toxicity of the compound. Our group previously demonstrated the efficiency
of the coupling of a metal-based PDT PS to peptides, which are known to bind specifically to
abundant molecular targets on malignant cells. More precisely, in those studies, bombesin, that
is known to target the human gastrin-releasing peptide receptor as well as a nuclear localization
signal peptide that facilitates the intracellular transport into the nucleus were coupled to Ru-
based PDT PSs. We were able to demonstrate an increased uptake of the conjugate in the
receptor-expressing cells in comparison to the free complex.[4a] The groups of Weil and Rau
were able to link the peptide hormone somatostatin to a PS and could show an 100-fold
increased efficiency for somatostatin receptor-expressing cells compared to the free PS.[7a]
Recently, the authors described a macromolecular plasma protein serum albumin–PS conjugate
with several Ru complexes bound to the protein surface. Using the protein as a nanocarrier, the
PSs were delivered selectively to the mitochondria, where it showed an impressive
phototoxicity with IC50 values in the nanomolar range.[7c] Worthy of note, a variety metal
complexes as for example Re(I), Pt(II), Ru(II) or Ir(III) compounds have been successfully
coupled to peptides to increase receptor selectivity.[9]
Among the different established classes of delivery systems[10] (e.g. oil-dispersions,
encapsulation in polymeric particles/lysosomes, targeting peptide-PS conjugates, polymer-PS
conjugates), the conjugation of PS to monoclonal antibodies (mAb) takes advantage of the
5
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ChemBioChem
10.1002/cbic.201900419
excellent target specificity of the latter. However, despite their clinical success, the concept of
utilizing mAb-PS conjugates is afflicted with several important drawbacks. These vector
molecules are known for their high stability and prolonged serum half-life, slow
pharmacokinetics and clearance from the body. This leads to an increase of the absolute level
of the mAb-PS conjugate in the tumor alongside with an increased non-specific uptake in non-
target tissues.[11] Additionally, the treatment of solid tumors is limited due to penetration
problems of the large conjugate into the tumor caused by poor vascularization, drainage,
interstitial pressure and dense stroma.[12] An attractive strategy to circumvent these limitations
is the use of smaller oncotropic vector molecules like antibody fragments or nanobodies
(
NBs).[13] NBs represent the antigen-binding domain of heavy-chain-only antibodies that occur
in species belonging to the family of Camilidae. Their small size, stability, solubility, fast
pharmacokinetics as well as high specificity and affinity for their cognate antigens make them
powerful targeting agents for diagnostic imaging and targeted therapy.[14] Noteworthy in this
context, Caplacizumab, a bivalent anti-von Willebrand factor NB, is currently in Phase III
clinical trials against acquired thrombotic thrombocytopenic purpura.[15]
A recent study has highlighted the high tumor uptake, rapid blood clearance and low liver
uptake of a 99mTc-labed NB as an imaging probe for epidermal growth factor receptor (EGFR)
positive tumors.[16] This receptor, which is involved in many cellular processes such as
proliferation, differentiation and cell survival, represents a crucial target in oncology as it is
overexpressed and/or deregulated in a variety of solid tumors, including head and neck, breast,
non-small-cell lung and pancreatic cancer. Therefore, EGFR is a major target for cancer
therapy.[16-17] Worthy of note, the successful conjugation of the PS IRDye700DX–maleimide
to nanobodies for hepatocyte growth factor receptor targeted PDT was recently
demonstrated.[18]
With this in mind, we report herein the design, synthesis, characterization and in-depth
biological evaluation of a NB-containing Ru(II) polypyridyl conjugate. The conjugate consists
of three building blocks: 1) a [Ru(phen)
(dppz)]2+ complex, which is known to have an
2
excellent phototoxicity[1a, 2c, 4], 2) a 7C12 NB, which is known for specific binding to EGFR
expressing cells[16, 19] and 3) a peptide chain with a poly-glycine unit, which is necessary for an
efficient and site-specific conjugation by a sortase A (SrtA)-mediated trans-peptidation reaction
leading to an 1:1 NB:PS ratio.[20] To the best of our knowledge, we report herein the first NB-
containing Ru(II) polypyridyl conjugate as a PDT PS for EGFR-targeted PDT. As can be seen
2
+
below, thanks to this design, a highly selective NB-containing [Ru(phen) (dppz)] conjugate
2
Ru-NB could be unveiled.
6
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ChemBioChem
10.1002/cbic.201900419
Results and discussion
Synthesis of the [Ru(phen)
2
(dppz-7-maleimidemethyl-S-Cys-(Ser)
2
(Gly)
5
-NH
3
)] complex
The synthetic strategy for the synthesis of the [Ru(phen)
2
(dppz-7-maleimidemethyl-S-Cys-
3
+
(
Ser)
2
(Gly)
5
-NH )] complex is described in Scheme 1.
3
Scheme 1. Total synthesis of [Ru(phen)
2
(dppz-7-maleimidemethyl-S-Cys-(Ser)
2
(Gly) -
5
NH
3
)](TFA)
3
. a) EtOH, reflux 3 h, DMSO, 150°C 2 h; b) 1,10-phenanthroline, LiCl, DMF,
7
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ChemBioChem
10.1002/cbic.201900419
reflux overnight under N
under N atmosphere; d) EtOH, 80°C 3 h under N
atmosphere; f) acetic acid, CH
CN, RT, overnight under N
NH , MeOH, reflux overnight under N
2
atmosphere; c) 1,10-phenanthroline, KBr, H
2
SO
, THF, 60°C 1 h under
, DMF,
, DMF, RT, overnight; i)
atmosphere; j) maleic anhydride, AcOH, reflux
-(Ser) -Cys-CONH )(TFA), CH CN:H O 1:1, RT,
4
, HNO
3
, 90°C 3 h
2
2
atmosphere; e) LiAlH
4
N
2
3
CN, reflux 1 h under N
2
atmosphere; g) (COCl)
2
CH
3
2
atmosphere; h) Phthalimide, K
2
CO
3
NH
2
2
2
1
0 h under N
2
atmosphere; k) (NH
3
-(Gly)
5
2
2
3
2
3
0 h.
The [Ru(phen)
2
(dppz-7-aminomethyl)](PF
6
)
2
complex was synthesized as previously reported
(dppz-7-maleimidemethyl)](PF
in nine synthetic steps.[4a] The synthesis of the [Ru(phen)
)
6 2
2
complex is already published but, in this study, a slightly different experimental procedure was
employed.[4a] The maleimide-containing Ru(II) complexes was prepared by reacting the
[Ru(phen)
2
(dppz-7-aminomethyl)](PF
6
)
2
complex with maleic anhydride. [Ru(phen)
2
(dppz-7-
maleimidemethyl)](PF
6 2
)
was coupled to the poly-glycine chain via a thio-Michael addition
reaction. As recently highlighted, this bioconjugation presents important advantages such as
synthetic accessibility, excellent reactivity and, importantly, biocompatibility.[21] Following
this synthetic strategy, the thiosuccinimide product [Ru(phen)
Cys-(Ser) -(Gly) -NH
)]3+ was prepared by reacting thiol of the (NH
CONH )(TFA) peptide chain with the [Ru(phen) (dppz-7-maleimidemethyl)](PF
2
(dppz-7-maleimidemethyl-S-
-(Gly) -(Ser) -Cys-
complex.
2
5
3
3
5
2
2
2
6 2
)
The product was obtained after an overnight reaction at room temperature and isolated via
preparative HPLC. The identity of the obtained complexes was confirmed by HR-MS and the
purity verified by HPLC (Figures S1-S2). We note that S-maleimide adducts[22] have been
found to have some problems of stability and this is the reason why alternative conjugation
techniques are currently sought.
Sortase A-mediated conjugation
Site-specific attachment of the [Ru(phen)
2
(dppz-7-maleimidemethyl-S-Cys-(Ser)
2
-(Gly) -
5
3
+
NH
3
)] complex to the EGFR-specific NB 7C12 by Sortase A requires protein engineering to
endow the desired conjugation site at the C-terminal end of the NB with the unique sortase
recognition motif. To this end, the NB was produced with its C-terminus tagged with a
(
GGGGS)
spacer and a hexahistidine purification tag (His
conjugation leads to the elimination of the His -tag, this design allows the removal of the
3
spacer followed by a Strep-tag, the LPETGG sortase motif, another (GGGGS)
3
6
). As successful sortase A-mediated
6
unreacted NB as well as of the His
6
-tagged enzyme by affinity chromatography (Scheme 2).
8
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ChemBioChem
10.1002/cbic.201900419
Scheme 2. Overview of the Sortase A-mediated site-specific modification of the NB derivative
C12-Strep-Sortag-His with the Ru(phen) (dppz-7-maleimidemethyl-S-Cys-(Ser) (Gly)
NH ) complex resulting in Ru-NB conjugate. The [Ru(phen) (dppz)] complex is highlighted
7
6
2
2
5
-
2
+
2
2
in green, while the engineered NB is drawn in red and the peptide chain with a poly-glycine
unit is depicted in blue. PDB entry of Sortase A from Staphylococcus aureus: 1T2P.[23]
To optimize the sortase-mediated bioconjugation reaction, the molar ratios of SrtA, NB and
3
+
[Ru(phen)
2
(dppz-7-maleimidemethyl-S-Cys-(Ser)
2
(Gly)
5
-NH )] as well as the reaction time
3
were varied (see Figures S3-S4). A 4 h reaction at 30°C with a molar ratio of 1:1:10 was
identified as being ideal (Figure 2).
9
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ChemBioChem
10.1002/cbic.201900419
Figure 2. Course of reaction for the chemoenzymatic conjugation of [Ru(phen)
2
(dppz-7-
3
+
maleimidemethyl-S-Cys-(Ser)
2
(Gly)
5
-NH
3
)] to the EGFR-specific NB 7C12. While the molar
ratio between SrtA and NB was kept constant (1:1), the amount of the Ru(II) precursor was
increased (10-100 nmol) to finally achieve molar ratios of 1:1:1, 1:1:2, 1:1:5 and 1:1:10,
respectively. The reaction was monitored for up to 4 h and aliquots were separated on 15% SDS
polyacrylamide gels. After electrophoresis, gels were imaged with a D-DiGit Gel Scanner (B)
to detect the signal of the Ru(II) complex and subsequently stained with colloidal Coomassie
G-250 (A).
Consequently, these conditions were kept in an upscaled reaction using 2 µmol SrtA, 2 µmol
3
+
sdAb and 20 µmol [Ru(phen)
purification of the reaction mixture by affinity chromatography, the obtained conjugate 7C12-
Strep-[Ru(phen) (dppz-7-maleimidemethyl-S-Cys-(Ser) (Gly) -NH (Ru-NB) was
)]3+
2
(dppz-7-maleimidemethyl-S-Cys-(Ser)
2
(Gly)
5
-NH )] . After
3
2
2
5
3
analyzed by MALDI-TOF MS (Figure S5). The mass spectra of the final purified product Ru-
NB showed a homogeneous population of a single-conjugated NB with a molecular mass of
~17.7 kDa.
Photophysical properties
1
0
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ChemBioChem
10.1002/cbic.201900419
With the conjugate in hand, we performed photophysical measurements to evaluate its potential
as a PDT agent. At first, the absorptions of [Ru(phen) (dppz-7-maleimidemethyl)](PF
Ru(phen) (dppz-7-maleimidemethyl-S-Cys-(Ser) (Gly) -NH )](TFA) and Ru-NB were
2
)
6 2,
[
2
2
5
3
3
measured to investigate if the peptide chain or the NB conjugation had an influence on the
photophysical properties of the Ru(II) polypyridyl complexes. Since the conjugate is insoluble
in CH CN, the measurement of Ru-NB was performed in DMSO. The comparison between the
3
absorption spectra (Figure S6) shows small differences in intensity as well as a small shift of
the absorption band which can be explained by solvent effects. Since all major bands are still
comparable, we assume that the conjugation did not change the photophysical properties of the
Ru(II) polypyridyl complex. As a second experiment, the emission and luminescence of the
conjugate was investigated upon excitation at 450 nm in DMSO. The maximum of the emission
of the complex (Figure S7) was determined to be 633 nm. Consequently, there is a large Stokes
shift which results in minimal interference between excitation and luminescence. The
luminescence quantum yield (Φem) was measured upon excitation at 450 nm by comparison
with the model complex [Ru(bipy)
3
]Cl
2
in CH
CN (Φem = 5.9%).[24] The luminescence quantum
3
yield (Φem) of the conjugate Ru-NB with a value of 3.3% was found to be in the same range
2
+
2+ [2c, 4]
than complexes of the type [Ru(bipy)
2
(dppz)] and [Ru(phen)
2
(dppz)] .
For a deeper
investigation of the excited state, the luminescence lifetimes were determined in degassed and
air saturated DMSO upon excitation at 450 nm to investigate the influence of the presence of
oxygen. As expected, the luminescence lifetime in a degassed solution was much longer (589
ns, Figure S8) than in an aerated solution (134 ns, Figure S9). This shows that oxygen has a
3
significant influence on the lifetime of the excited state and indicates that O
2
can interact with
the triplet state of the complex.
Singlet oxygen generation
Knowing that the triplet excited state of the conjugates are able to interact with oxygen, we
1
were interested in determining the singlet oxygen quantum yield Φ( O
2
) of Ru-NB using two
methods previously described by our group,[25] namely: 1) by direct method by measurement
1
of the phosphorescence of O
2
at 1270 nm. Worthy of note, this method is dependent on the
1
used setup. With the used equipment in our laboratory, we can only detect Φ( O
2
) > 0.20; 2) by
indirect method by measurement of the change in absorbance of a reporter molecule which is
monitored by UV/VIS spectroscopy. Since the measurements were performed in DMSO and
aqueous solution, only rather small values (Table 1) could be measured. This is not surprising
and has already been investigated for several other [Ru(bipy)
(dppz)]2+ and
2
1
1
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ChemBioChem
10.1002/cbic.201900419
[Ru(phen)
2
(dppz)]2+ complex derivatives.[2c, 4a, 4b] In-depth investigations showed that the
excited state of the complex is quenched in an aqueous solution due to hydrogen bonding
interactions between the nitrogen atoms of the dppz ligand and the solvent.[26] Comparison of
the singlet oxygen quantum yield of Ru-NB with the ones obtained for structurally related
Ru(bipy)
(dppz)]2+ complexes , revealed that these values are in the same range. This
[2c]
[
2
strongly suggests that the bioconjugation did not significantly influence this property.
1
Table 1. Singlet oxygen quantum yields (Φ( O
2
)) of Ru-NB in DMSO and aqueous solution
determined by direct and indirect method by excitation at 450 nm. Average of three independent
measurements, +-10% (n.d.=not detectable).
Compound
DMSO
direct
n.d.
D
2
O
DMSO
Indirect
9%
PBS
direct
Indirect
4%
Ru-NB
n.d.
In vitro evaluation of EGFR targeting after conjugation
In order to investigate the targeting ability of the functionalized NB, uptake in the human
epithelial cell line A431 originating from an epidermoid carcinoma of the skin was examined
by confocal fluorescence microscopy. These squamous carcinoma cells express approximately
6
[27]
2
x 10 EGFR molecules per cell , which represents a high expression level. Confocal
imaging of A431 cells showed co-localization of Ru-NB with EGFR (Figure 3), thus indicating
the preserved targeting ability of 7C12 after site-specific modification. Noteworthy, Ru-NB
showed a predominant membrane staining even after 48 h of incubation at 37°C, and only very
little intracellular fluorescence was observed. However, it has been shown recently that the free
amine ruthenium complex is characterized by a poor cellular uptake even at high micromolar
concentrations.[4a]
1
2
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ChemBioChem
10.1002/cbic.201900419
Figure 3. Confocal immunofluorescence microscopy images of A431 cells exposed to Ru-NB
for 4, 24 and 48 h at 37°C showing specific binding and co-localization of the single-conjugated
NB with EGFR.
Cellular uptake of the bioconjugates
The presence of a metal ubiquitous in a cellular environment as an essential component of the
PS allows investigating the cellular accumulation of the bioconjugate by inductively coupled
plasma- mass spectrometry (ICP-MS).[28] In order to demonstrate the receptor-specific uptake,
EGFR-positive (A431) and EGFR-negative (MDA-MB-435S) cells were incubated for
different periods of time (4, 24, and 48 h) with different concentrations of the bioconjugate in
the dark at 37°C. The amount of cell-associated ruthenium was determined by ICP-MS and
related to the cellular protein content (Figure 4). Although ruthenium was detectable in the cell
lysate of both cell lines after 24 and 48 h, respectively, the amount of the metal strongly
correlated with the level of EGFR expression. There was more of ruthenium in the EGFR-
overexpressing cell line than in the EGFR-negative one. This finding confirmed that cell
association was primarily mediated by the NB and not by the PS.
1
3
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ChemBioChem
10.1002/cbic.201900419
Figure 4. Amount of cell-associated ruthenium after incubation of EGFR-positive A431 and
EGFR-negative MDA-MB-435S cells with 2 or 20 µM of Ru-NB for up to 48 h at 37°C. The
level of ruthenium in cell lysates of MDA-MB-435S exposed to 2 µM of Ru-NB were below
the analytical limit and are thus not shown.
An identical cell uptake study was performed with the complex [Ru(bipy)
2
(dppz-OMe)](PF
6
2
) ,
resulting in comparable ruthenium levels for the A431 cell line (Figure S10 and Table 2). The
amount of ruthenium detected in MDA-MB-435S cells upon incubation with this non-targeted
Ru-complex was higher at each time point compared to the EGFR-targeting Ru-NB conjugate.
This result is unsurprising as the latter cells lack these receptor proteins at their surface.
Table 2. Head-to-head comparison of uptake of Ru-NB and [Ru(bipy)
2
(DPPZ-OMe)](PF
6 2
)
into A431 and MDA-MB 435S cells. The amount of cell-associated ruthenium [ng/mg protein]
was measured by ICP-MS.
Ru-NB
MDA-MB
53S
Ru(bipy)
2
(DPPZ-OMe) (PF
6 2
)
MDA-MB
453S
A431
A431
4
1
4
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ChemBioChem
10.1002/cbic.201900419
Time
h]
Concentration of substance [µM]
[
2
20
2
20
2
20
2
20
3
.26 ±
11.67 ±
1.70
23.84 ±
1.54
32.87 ±
4.87
1.20 ±
0.28
2.51 ±
0.19
5.75 ±
0.74
8.54 ±
2.23
18.83 ±
2.84
46.94 ±
1.89
1.18 ±
0.14
1.84 ±
0.05
1.92 ±
0.08
5.20 ±
0.90
15.84 ±
2.69
19.93 ±
2.39
4
< LOD
< LOD
< LOD
< LOD
1
.30
.20 ±
.86
1.54 ±
.89
6
1
1
5.52 ±
2.00
5.45 ±
1.32
2
4
8
4
1
To confirm the receptor specificity of the ruthenium accumulation, A431 cells were incubated
in the presence or absence of cetuximab in addition to Ru-NB. The epitope for 7C12 partially
overlaps the cetuximab epitope on domain III of the EGFR extracellular region and an excess
of the mAb can block its interaction with the receptor.[16, 29] After 24 and 48 h of incubation
with 200 nM of Ru-NB at 37°C, 0.77 ng and 2.74 ng ruthenium per mg protein (Table 3),
respectively, were detected in the cell lysates. Upon co-incubation of EGFR-overexpressing
A431 cells with Ru-NB and cetuximab, no cell-associated ruthenium was detectable even after
4
8 h.
Table 3. Amount of cell-associated ruthenium after incubation of EGFR-positive A431 with
00 nM of Ru-NB for 24 or 48 h at 37°C. The level of ruthenium in cell lysates of A431 co-
2
incubated with 1 µM of the EGFR-blocking antibody cetuximab were below the limit of
detection (LOD).
Ru-NB
200 nM
200 nM
1 µM
Cetuximab
-
ng Ru per mg protein
0.77 ± 0.10
2
4
4 h
8 h
< LOD
< LOD
2.74 ± 0.12
These latter findings corroborate the hypothesis that cellular ruthenium association occurs in a
receptor-mediated manner. Overall, Ru-NB targets EGFR specifically. Importantly, the free
water-soluble PS exhibits only poor cell binding capacity and lacks cell line selectivity, until
their conjugation to targeting moieties. These facts together strongly provide the basis for
tumor-specific PDT.
1
5
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ChemBioChem
10.1002/cbic.201900419
Dark cytotoxicity and phototoxicity of Ru-NB
To evaluate the potency of the bioconjugate Ru-NB as a PDT agent, its cytotoxicity in the dark
and upon light irradiation was determined. For these experiments, the A431 cell line had to be
chose due to the strong light sensitivity of the MDA-MB-435S (EGFR negative) cell line that
precluded it from phototoxicity studies. To avoid light sensitivity in A431 cell line, irradiation
at 480 nm was performed in sequences. 6 x 3.5 min of irradiation with 15 min gap in between
-
2
(
(
6.741 J cm ) were used. Dark treatment and surprisingly light irradiation of the A431 cells
48 h incubation with Ru-NB) at 480 nm did not cause any cytotoxic effect (IC50 dark >25 µM,
IC50 light >25 µM, see Figure S11) for Ru-NB. We note that we could not go for higher
concentration due to conjugate precipitation at 50 µM. Adding polyethylene glycol spacers,
changing the ionic strength or the pH could possibly affect the conjugate solubility, and
consequently help solving this problem. Lack of cytotoxicity encouraged us to try to enhance
the internalization of the conjugate into the cells. For that purpose, an additional step was used,
namely temperature change.[30] Cells treated with Ru-NB were incubated for 1 h at 4 ºC. Since
EGFR internalization is an energy-dependent process, incubating cells at 4ºC inhibits the
endocytosis processes but not the binding of Ru-NB to the receptor. A temperature shift to 37ºC
(
for 1 h) allowed then for efficient endocytosis of the receptor with the bound conjugate. This
step enables a higher accumulation of Ru-NB in the cells. Due to conjugate precipitation, the
highest concentration tested was 35 µM. Ru-NB was again found to be non-toxic in the dark
(
IC50 >35 µM.) Unfortunately, light irradiation at 480 nm (6 x 3.5 min with 15 min gap between
irradiations) again did not cause any phototoxic effect (IC50 >35 µM, see Figure S12).
Cellular ROS production by Ru-NB
The lack of phototoxicity of Ru-NB led us to investigate whether this conjugate could produce
ROS in irradiated cells. For that purpose, we have stained A431 cells with the known ROS
probe DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate). Cells were then treated with Ru-
NB (35 µM) using the receptor internalisation protocol, irradiated (480 nm light for 3.5 min;
-2
1
,124 J cm ) and suspended in PBS buffer. The DCFH-DA signal was detected using flow
cytometry instrument. As can be seen in Figure S13, there was no ROS production in the A431
cells that were treated with Ru-NB and then irradiated, as distinct from the H treated control.
2
O
2
This unexpected result might be caused by the impairment of the internalization of Ru-NB into
the cells. Another explanation would be that the ROS produced are directly reacting with the
1
1
NB itself. However, this hypothesis is unlikely since O
2
was detected during the O production
2
measurements.
1
6
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ChemBioChem
10.1002/cbic.201900419
Conclusion
In summary, in this article, we present the synthesis, characterization and photophysical and
biological evaluation of a novel nanobody containing Ru(II) polypyridine conjugate. As a
benefit of the linkage to a 7C12 nanobody, the conjugate selectively accumulated at the
epidermal growth factor receptor (EGFR). The investigation of the uptake via ICP-MS
indicated that the conjugate has been successfully internalized inside cancerous A431 cells.
Photophysical studies in cuvette suggested that the photophysical properties of the conjugate
remain unchanged in comparison to the compound alone. However, DCFH-DA staining
experiments indicated that no significant ROS was produced inside the cells. Consequently,
photocytotoxicity investigations did not show any significant effect. Focus of future work will
be the successful development of a nanobody-containing Ru(II) polypyridine conjugate with
ROS and photocytotoxicity inside cancerous cells.
Experimental
Materials
All chemicals were obtained from commercial sources and were used without further
purification. Solvents were dried over molecular sieves if necessary. The ligand 5-
(
aminomethyl)-2,2′-bipyridine[31] and the Ru(II) complexes [Ru(bphen)
2
Cl
2
] using the
[
32]
respective ligands , [Ru(phen)
reported.[4a]
2
(dppz-7-aminomethyl)](PF
)
6 2
were synthesized as previously
Instrumentation and methods
1
13
H and C NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer. ESI-MS
experiments were carried out using a LTQ-Orbitrap XL from Thermo Scientific (Thermo Fisher
Scientific, Courtaboeuf, France) and operated in positive ionization mode, with a spray voltage
at 3.6 kV. No Sheath and auxiliary gas was used. Applied voltages were 40 and 100 V for the
ion transfer capillary and the tube lens, respectively. The ion transfer capillary was held at
2
75°C. Detection was achieved in the Orbitrap with a resolution set to 100,000 (at m/z 400) and
a m/z range between 150-2000 in profile mode. Spectrum was analyzed using the acquisition
software XCalibur 2.1 (Thermo Fisher Scientific). The automatic gain control (AGC) allowed
5
accumulation of up to 2*10 ions for FTMS scans, Maximum injection time was set to 300 ms
1
7
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ChemBioChem
10.1002/cbic.201900419
and 1 µscan was acquired. 10 µL was injected using a Thermo Finnigan Surveyor HPLC system
-1
(
Thermo Fisher Scientific) with a continuous infusion of methanol at 100 µL.min . For analytic
and preparative HPLC the following system has been used: 2ꢀx Agilent G1361 1260 Prep Pump
system with Agilent G7115A 1260 DAD WR Detector equipped with an Agilent Pursuit XRs
5
C18 (Analytic: 100Å, C18 5 μm 250ꢀxꢀ4.6 mm, Preparative: 100Å, C18 5 μm 250ꢀxꢀ300 mm)
Column and an Agilent G1364B 1260-FC fraction collector. The solvents (HPLC grade) were
millipore water (0.1% TFA, solvent A) and acetonitrile (0.1% TFA, solvent B). The sample
was dissolved in 1:1 (v/v) CH
3
CN/ H O 0.1% TFA solution and filtered through a 0.2 μm
2
membrane filter. Gradient: 0-3 minutes: isocratic 95% A (5% B); 3- 17 minutes: linear gradient
from 95% A (5% B) to 0% A (100% B); 17-25 minutes: isocratic 0% A (100% B). The flow
rate was 1 mL/min (for preparative purposes: 20 mL/min) and the chromatogram was detected
at 250 nm, 350 nm, 450 nm.
Synthesis
[
Ru(phen)
The synthesis of [Ru(phen)
study, slightly different synthetic route was employed. [Ru(phen)
aminomethyl)](PF (25 mg, 1.0 equiv.) and maleic anhydride (46 mg, 20.0 equiv.) were
suspended in acetic acid (10 mL) under a nitrogen atmosphere. The mixture was refluxed for
0 h. The solution was then cooled down and a sat. aqueous solution of NH PF was added.
The crude product, which precipitated as a PF salt, was collected by filtration and washed three
times with H O and Et O. The product was purified by column chromatography on silica gel
with a CH CN /aq. KNO (0.4 M) solution (10:1). The fractions containing the product were
united and the solvent was removed. The residue was dissolved in CH CN and undissolved
KNO was removed by filtration. The solvent was removed and the product was dissolved in
O. Upon addition of NH PF the product precipitated as a PF salt. The solid was obtained
by centrifugation and was washed with H O and Et O. Yield: 86%. Experimental data fits with
the literature. Purity of the sample was assessed by NMR and HPLC analysis. RP-HPLC: R
6.2 min.
2
(dppz-7-maleimidemethyl)](PF
-dppz-7-maleimidemethyl] is already published but, in this
(dppz-7-
6
)
2
2
+
[4a]
2
a
2
)
6 2
1
4
6
6
2
2
3
3
3
3
H
2
4
6
6
2
2
t
=
1
[
[
(
Ru(phen)
Ru(phen)
Ser) (Gly)
2
(dppz-7-maleimidemethyl-S-Cys-(Ser)
(dppz-7-maleimidemethyl)](PF (16 mg, 1.0 equiv.) and (NH
-NH )(TFA) (11.6 mg, 1.2 equiv.) were dissolved in a 1:1 CH CN/H O mixture (20
2
(Gly)
5
-NH
3
)](TFA)
3
2
6
)
2
2
CO-Cys-
2
5
3
3
2
mL) and stirred in the dark at room temperature. The progress of the reaction was followed via
1
8
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ChemBioChem
10.1002/cbic.201900419
HPLC. After 24 h, additional (NH
2
CO-Cys-(Ser)
2
(Gly)
5
-NH
3
)(TFA) (4.8 mg, 0.5 equiv.) were
added. The reaction mixture was stirred for another 6 h until the complete consumption of the
Ru(II) complex was monitored. The solvent was removed under reduced pressure and the
product was purified by preparative HPLC. The product was isolated as a red TFA salt. Purity
of the sample was assessed by HPLC analysis. Yield: 95%. HRMS (ESI) m/z: Calcd. for
3
+
[C
66
H
62
N
18
O
12RuS-3TFA] : 445.7874, Found: 445.7875; RP-HPLC: R = 14.9 min.
t
E. coli strains and plasmids
Escherichia coli NEB 5-alpha (fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ (lacZ)M15
gyrA96 recA1 relA1 endA1 thi-1 hsdR17) was used in molecular cloning experiments, whereas
E. coli SHuffle® T7 Express (fhuA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt::pNEB3-r1-
R
q
S
cDsbC (Spec , lacI ) ΔtrxB sulA11 R(mcr-73::miniTn10--Tet )2 [dcm] R(zgb-210::Tn10 --
S
Tet ) endA1 Δgor ∆(mcrC-mrr)114::IS10) and E. coli BL21(DE3) (fhuA2 [lon] ompT gal (λ
DE3) [dcm] ∆hsdS) were used for expression of the recombinant proteins. All strains were
purchase from New England Biolabs. The generation of pET-28b:7C12 encoding the EGFR-
specific single-domain antibody 7C12 has been previously described.[33] The plasmid
pGBMCS-SortA was a gift from Fuyuhiko Inagaki (Addgene plasmid # 21931).[34]
Molecular cloning
A DNA fragment coding for a (GGGGS)
3
spacer followed by a Strep-tag, the LPETGG sortase
motif and another (GGGGS) spacer was commercially synthesized including a 5′ restriction
3
site for HindIII and a 3′ restriction site for XhoI, respectively. The ~150-nt fragment was
digested with appropriate restriction endonucleases and ligated in-frame into HindIII/XhoI-
linearized pET-28b:7C12 plasmid.[33] The ligation reactions were transformed into chemically
competent E. coli NEB 5-alpha cells. The DNA sequences of the resulting recombinant
construct pET-28b:7C12-Strep-Sortag-His were checked by Sanger sequencing.
6
Cultivation and expression of recombinant proteins
Freshly transformed E. coli SHuffle® T7 Express or E. coli BL21(DE3) harboring the plasmids
pET-28b:7C12-Strep-Sortag-His
containing 50 μg/mL of kanamycin or 100 µg/mL of ampicillin, respectively, and cultivated at
0°C overnight in an orbital shaker with 50 mm offset and shaking speed of 200 rpm. After
6
or pGBMCS-SortA were inoculated in 10 mL of LB broth
3
that, 5 mL of this pre-culture were transferred into 125 mL MagicMedia™ E. coli Expression
Medium (Life Technologies) in 1000 mL baffled-bottom glass flasks and grown at 30°C for
1
9
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ChemBioChem
10.1002/cbic.201900419
2
4 h. For final harvest, cultures were chilled on ice for 5 min and centrifuged for at least 15 min
at 6,000 × g and 4°C. After removal of the supernatant, cell pellets were either stored at −20°C
or subjected to purification procedure immediately.
Purification of recombinant proteins
A high-capacity Ni-iminodiacetic acid (IDA) resin in combination with an ÄKTA pure
chromatography system (GE Healthcare) was used for purification of hexahistidine tagged
proteins by immobilized metal affinity chromatography (IMAC) under native conditions.
Efficient cell lysis was achieved by addition of 1 mL RIPA cell lysis buffer (G-Biosciences)
supplemented with EDTA-free protease inhibitor cocktail (Roche Diagnostics), 500 µg
lysozyme (Sigma-Aldrich) and 25 U endonuclease (Thermo Scientific Pierce) per 200 mg
bacterial cell pellet. Prior to incubation on ice for at least 15 min, the pelleted cells were
resuspended completely by vortexing or pipetting up and down until no cell clumps remained.
After centrifugation at 10,000 x g and 4°C for 20 min to remove cellular debris, the clarified
supernatant was loaded using an automated sample pump with a flow rate of 0.5 ml/min. IMAC
was performed on a prefilled 5-ml His60 Ni Superflow cartridge (Clontech Laboratories) at a
flow rate of 5 mL/min in equilibration buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5). Before
elution of the hexahistidine tagged proteins by addition of 8 CV elution buffer (50 mM Tris-
HCl, 150 mM NaCl, 500 mM imidazole, pH 7.5), the column was washed with 8 CV
equilibration buffer and 7 CV wash buffer (50 mM Tris-HCl, 150 mM NaCl, 35 mM imidazole,
pH 7.5).Removal of imidazole and buffer exchange after IMAC was achieved by dialysis
against sortase buffer (50 mM Tris-HCl, 150 mM NaCl and 10 mM CaCl , pH 7.5) using a
2
cellulose ester membrane with a molecular weight cut-off of 3.5-5 kDa (Spectrum®
Laboratories).
Gel electrophoresis
Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
carried out according to a standard protocol.[35] For each gel, PageRuler Plus Prestained Protein
Ladder (Thermo Fisher Scientific) was used as molecular weight ladder standard. After
electrophoresis, gels were imaged with a D-DiGit Gel Scanner (LI-COR Biosciences) and
subsequently stained with PageBlue protein staining solution (Thermo Fisher Scientific)
according to the manufacturer’s instructions.
Protein determination
2
0
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ChemBioChem
10.1002/cbic.201900419
Protein concentration was determined with the DC Protein Assay (Bio-Rad Laboratories)
according to the manufacture’s microplate assay protocol using bovine serum albumin in
sortase buffer (50 mM Tris-HCl, 150 mM NaCl and 10 mM CaCl , pH 7.5) as protein standard.
2
Sortase A-mediated conjugation
Small-scale reactions were set up in 100 µL with variable molar ratios of SrtA, 7C12-Strep-
3
+
Sortag-His
different incubation times. The optimal conditions were upscaled and the reaction mixture was
composed of 2 µmol SrtA, 2 µmol NB and 20 µmol [Ru(phen) (dppz-7-maleimidemethyl-S-
)] in sortase buffer (50 mM Tris-HCl, 150 mM NaCl and 10 mM CaCl
6
and
[Ru(phen)
2
(dppz-7-maleimidemethyl-S-Cys-(Ser)
2
(Gly)
5
-NH
3
)] and
2
3
+
Cys-(Ser)
2
(Gly)
5
-NH
3
2
,
pH 7.5). Bioconjugation reactions were incubated at 30°C for up to 6 h in the dark with gentle
shaking.
Purification of conjugation reactions
In the first purification step, all remaining hexahistidine tagged proteins were eliminated from
the reaction mixture by IMAC using prepacked His60 Ni Gravity Columns (Clontech
Laboratories). After collection of the flow-through, the gravity-flow column was washed twice
with equilibration buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5). These wash fractions as
well as the flow-through were analyzed for the presence of the Ru-NB conjugate by SDS-
PAGE. Remaining unconjugated [Ru(phen)
2
(dppz-7-maleimidemethyl-S-Cys-(Ser)
2
(Gly)
5
-
3
+
NH
3
)] was removed in a second purification step by size-exclusion chromatography using
Zeba Spin Desalting Columns (7K MWCO, Thermo Scientific) with elution in PBS. The
purified conjugate was sterile filtered using Whatman Puradisc FP 30 cellulose acetate syringe
filter units with a pore size of 0.2 µm (GE Healthcare Life Sciences) and stored at 4°C.
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass
spectrometry of purified sdAb-conjugates
2,5-Dihydroxyactetophenone (2,5-DHAP, Bruker Daltonik) was used as matrix for MALDI-
TOF MS. For solubilization of the matrix, 7.6 mg of 2,5-DHAP were dissolved in 375 μL of
absolute ethanol. After this, 125 μL of an 18 mg/mL aqueous solution of diammonium
hydrogen citrate (Sigma-Aldrich) were added. Protein samples were desalted using mixed
cellulose esters membrane filters with a pore size of 0.025 µm and a diameter of 25 mm (MF-
Millipore™ Membrane Filter VSWP, Merck Chemicals). Briefly, the filter was placed on the
water surface of a beaker filled with distilled water. A 2 µL aliquot of the protein sample was
2
1
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ChemBioChem
10.1002/cbic.201900419
carefully pipetted on top of the membrane. After incubation at room temperature for at least
0 min, 2 µL of the dialyzed sample was mixed with 2 µL of 2% TFA solution. After addition
1
of 2 µL of the matrix solution, the mixture was pipetted up and down until the crystallization
starts and the solution became cloudy. Finally, 0.5 µL of the crystal suspension was spotted
onto the ground steel target plate and the droplet was air-dried completely at room temperature.
Spectra were acquired with an autoflex II TOF/TOF (Bruker Daltonik) in positive linear mode
in combination with the flexControl software (Version 3.3, Bruker Daltonik) and analyzed with
the flexAnalysis software (Version 3.3, Bruker Daltonik). Theoretical molecular weights were
calculated using the Compute pI/Mw tool on the ExPASy Server.[36]
Spectroscopic measurements
The absorption of the samples was measured in a cuvette with a Lambda 800 UV/VIS
Spectrometer (PerkinElmer Instruments) or in 96 well plates with a SpectraMax M2
Spectrometer (Molecular Devices). The emission was measured by irradiation of the sample in
fluorescence quartz cuvettes (width 1 cm) using a NT342B Nd-YAG pumped optical
parametric oscillator (Ekspla) at 450 nm. The luminescence was focused and collected at a right
angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i
monochromator. As a detector a XPI-Max 4 CCD camera (Princeton Instruments) was used.
Luminescence quantum yield measurements
For the determination of the luminescence quantum yield, the samples were prepared in a
CH CN solution with an absorbance of 0.1 at 450 nm. This solution was irradiated in
3
fluorescence quartz cuvettes (width 1 cm) using a NT342B OPO pulse laser Nd-YAG pumped
optical parametric oscillator (Ekspla) at 450 nm. The emission signal was focused and collected
at a right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-
2
300i monochromator. As a detector a XPI-Max 4 CCD camera (Princeton Instruments) was
used. The luminescence quantum yields were determined by comparison with the reference
Ru(bipy) ]Cl in CH
CN (Φem=0.059)[24] applying the following formula:
[
3
2
3
ꢎ
ꢋꢇꢀꢈꢀꢇꢀꢉꢊꢀ
Fꢂꢃꢁꢄꢅꢀ
Iꢂꢃꢁꢄꢅꢀ
nꢂꢃꢁꢄꢅꢀ
Φꢀꢁ,ꢂꢃꢁꢄꢅꢀ ꢆ Φꢀꢁ,ꢇꢀꢈꢀꢇꢀꢉꢊꢀ
∗
∗
∗ ꢌ
ꢍ
Iꢇꢀꢈꢀꢇꢀꢉꢊꢀ
nꢇꢀꢈꢀꢇꢀꢉꢊꢀ
F ꢆ 1 ꢏ 10ꢐꢑ
Φem = luminescence quantum yield, F = fraction of light absorbed, I = integrated emission
intensities, n = refractive index, A = absorbance of the sample at irradiation wavelength
2
2
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ChemBioChem
10.1002/cbic.201900419
Lifetime measurements
For the determination of the lifetimes, the samples were prepared in an air saturated and in a
degassed CH CN solution with an absorbance of 0.1 at 450 nm. This solution was irradiated in
3
fluorescence quartz cuvettes (width 1 cm) using a NT342B Nd-YAG pumped optical
parametric oscillator (Ekspla) at 450 nm. The emission signal was focused and collected at a
right angle to the excitation pathway and directed to a Princeton Instruments Acton SP-2300i
monochromator. As a detector a R928 photomultiplier tube (Hamamatsu) was used.
Singlet oxygen measurements
-
Direct evaluation
The samples were prepared in an air saturated DMSO or D
.2 at 450 nm. This solution was irradiated in fluorescence quartz cuvettes (width 1 cm) using
2
O solution with an absorbance of
0
a mounted M450LP1 LED (Thorlabs) whose irradiation, centred at 450 nm, was focused with
aspheric condenser lenses. The intensity of the irradiation was varied using a T-Cube LED
Driver (Thorlabs) and measured with an optical power and energy meter. The emission signal
was focused and collected at a right angle to the excitation pathway and directed to a Princeton
Instruments Acton SP-2300i monochromator. A longpass glass filter was placed in front of the
monochromator entrance slit to cut off light at wavelengths shorter than 850 nm. The slits for
detection were fully open. As a detector an EO-817L IR-sensitive liquid nitrogen cooled
germanium diode detector (North Coast Scientific Corp.) was used. The singlet oxygen
luminescence at 1270 nm was measured by recording spectra from 1100 to 1400 nm. For the
data analysis, the singlet oxygen luminescence peaks at different irradiation intensities were
integrated. The resulting areas were plotted against the percentage of the irradiation intensity
and the slope of the linear regression calculated. The absorbance of the sample was corrected
with an absorbance correction factor. As reference for the measurement in an CH
phenalenone (Φphenaleone=0.95)[37] and for the measurement in a D
O solution [Ru(bipy)
Ru(bipy)3Cl2=0.22)[38] was used and the singlet oxygen quantum yields were calculated using
the following formula:
3
CN solution
2
3
]Cl
2
(
Φ
Sꢂꢃꢁꢄꢅꢀ
Iꢇꢀꢈꢀꢇꢀꢉꢊꢀ
Φꢂꢃꢁꢄꢅꢀ ꢆ Φꢇꢀꢈꢀꢇꢀꢉꢊꢀ
∗
∗
Sꢇꢀꢈꢀꢇꢀꢉꢊꢀ
ꢐꢑ
Iꢂꢃꢁꢄꢅꢀ
I ꢆ I ∗ ꢓ1 ꢏ 10
ꢔ
ꢒ
Φ = singlet oxygen quantum yield, S = slope of the linear regression of the plot of the areas of
the singlet oxygen luminescence peaks against the irradiation intensity, I = absorbance
2
3
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ChemBioChem
10.1002/cbic.201900419
correction factor, I0 = light intensity of the irradiation source, A = absorbance of the sample at
irradiation wavelength.
-
Indirect evaluation
For the measurement in DMSO: The samples were prepared in an air-saturated DMSO solution
containing the complex with an absorbance of 0.2 at the irradiation wavelength and 1,3-
diphenylisobenzofuran (DPBF, 30 µM). For the measurement in PBS buffer: The samples were
prepared in an air-saturated PBS solution containing the complex with an absorbance of 0.2 at
the irradiation wavelength, N,N-dimethyl-4-nitrosoaniline aniline (RNO, 20 µM) and histidine
(
10 mM). The samples were irradiated on 96 well plates with an Atlas Photonics LUMOS BIO
irradiator for different times. The absorbance of the samples was measured during these time
intervals with a SpectraMax M2 Microplate Reader (Molecular Devices). The difference in
absorbance (A -A) at 415 nm for the DMSO solution and at 440 nm for the PBS solution was
0
measured and plotted against the irradiation times. From the plot the slope of the linear
regression was calculated as well as the absorbance correction factor determined. The singlet
oxygen quantum yields were calculated using the same formulas as used for the direct
evaluation.
Cell culture
Cell culture flasks, dishes and plates (CELLSTARS) were supplied by Greiner Bio-One GmbH.
The adherent human tumor cell lines A431 (ATCC® number: CRL-1555) and MDA-MB 435S
(
ATCC® number: HTB-129) were maintained as previously reported.[32, 39] All cell lines were
confirmed to be mycoplasma-negative using the Venor®GeM Advance Mycoplasma Detection
Kit (Minerva Biolabs) and were tested monthly.
Cell uptake studies
A total of 300,000 MDA-MB 435S cells and 450,000 A431 cells were seeded in T25 cell culture
flasks in 5 mL DMEM supplemented with 10% fetal calf serum (FCS), respectively, and
incubated in a humidified atmosphere of 95% air/5% CO at 37°C. After 48 h of incubation,
2
cells were washed twice with warm PBS. The buffer was then replaced by fresh DMEM
supplemented with 10% FCS and different concentrations of the Ru-NB conjugate or
Ru(bipy)
2
(DPPZ-OMe) (PF
6
2
) . Following incubation at 37°C for certain time periods, medium
was removed and the cells washed three times with warm PBS and trypsinized. After
resuspension in warm DMEM with 10% FCS, the pellets were collected by centrifugation at
2
4
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ChemBioChem
10.1002/cbic.201900419
2
00 x g for 5 min and washed once with warm PBS. The cell pellets were resuspended in 500 μL
of PBS, lysed by 10 freeze−thaw cycles, and sonicated in an ice-cold ultrasonic bath for 20 min
SONOREX SUPER 10P digital, Bandelin). After determination of the protein content, the
(
lysates were lyophilized on an Alpha 2-4 LSC plus (CHRIST).
ICP-MS studies
After digestion of samples in distilled ultrapure 65% HNO
3
(Roth) and dillution in 1% HNO ,
3
ICP-MS measurements were performed on an iCap RQ ICP-MS spectrometer (Thermo Fisher
Scientific) equipped with a SC-2DX autosampler (ESI). Calibration was done with Ru single
element standard (Merck 170347). Rh and Sc were used as internal standards. Limit of detection
(LOD) was 50 ng/L Ru.
Confocal microscopy
A total of 100,000 A431 cells were seeded in 35 mm imaging dishes (IBIDI) in 2 mL DMEM
supplemented with 10% fetal calf serum (FCS), and incubated in a humidified atmosphere of
9
5% air/5% CO at 37°C. After 24 h of incubation, media was refreshed and cells were
2
incubated with 100 nM of Ru-NB at 37°C for up to 48 h. Afterwards, cells were washed thrice
with ice-cold PBS, fixed with 4% paraformaldehyde and 2.5% sucrose in PBS, and
permeabilized with 0.25% TritonX-100 in PBS for 10 min. To prevent unspecific antibody
binding, cells were incubated with 10% FCS in PBS overnight at 4°C. Cells were then incubated
with rabbit anti-EGFR (D38B1) Alexa Fluor® 647 monoclonal antibody (Cell Signaling
Technology) and with StrepMAB-Classic Chromeo 488 conjugate (IBA Lifesciences) for 2 h
at RT in the dark. Cells were again washed three times with PBS, and the nuclei were stained
using Hoechst 33258. Fluorescence microscopy was performed with the Fluoview 1000
confocal laser scanning microscope (Olympus) using a 60 × (NA 1.35) oil objective.
Dark cytotoxicity and phototoxicity
The dark and light cytotoxicity of the Ru(II)-containing conjugates was assesed by fluorometric
cell viability assay using resazurin (ACROS Organics). For dark and light cytotoxicity with the
[
40]
EGFR internalisation step , A431 cells were seeded in triplicates in 96 well plates at a density
of 4000 cells per well in 100 µl, 24 h prior to treatment. Cells were then treated with serum free
DMEM media containing 0.3% of BSA for 1 h at 37 ºC. The medium was then replaced with
increasing concentrations of Ru-NB, then cells were incubated on ice for 1 h. After that time,
cells were transferred for 1 h at 37 ºC. The medium was then replaced by fresh complete
2
5
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900419
medium. For the dark and light cytotoxicity without the EGFR internalisation step, A431 cells
were seed in triplicates in 96 well plares at a density of 4000 cells per well in 100 µl, 24 h prior
to treatment. The medium was then replaced with increasing concentrations of Ru-NB for 44 h.
Cells used for the light cytotoxicity experiments with Ru-NB were exposed to 480 nm light for
6
x 3.5 min with 15 min gap in between irradiations or in a 96-well plate using a LUMOS-BIO
photoreactor (Atlas Photonics). Each well was individually illuminated with a 5 lm LED at
-2
constant current (6.741 J cm ). After 44 h in the incubator, the medium was replaced by fresh
-1
complete medium containing resazurin (0.2 mg mL final concentration). After 4 h incubation
at 37°C, the fluorescence signal of the resorufin product was read by SpectraMax M5
mictroplate reader (ex: 540 nm em: 590 nm). IC50 values were calculated using GraphPad Prism
software.
Cellular ROS production
1
0 cm cell culture plates were seeded with A431 cell line and allowed to adhere overnight.
Next, the cells were incubated with a DCFH-DA solution (100 µM) in DMEM media for 30
min at 37 ºC. Cells were then washed and treated with serum free DMEM media containing
0
.3% of BSA for 1 h at 37 ºC. The medium was then replaced in the plates with either Ru-NB
dilution, 0.1 mM H or media. Cells were then incubated on ice for 1 h. After that time, the
2
O
2
cells were transferred for 1 h at 37 ºC. The medium was then replaced by fresh complete
medium. The cells used for the light experiments were exposed to 480 nm light for 3.5 min
-2
using a LUMOS-BIO photoreactor (Atlas Photonics; 1.124 J cm ). All cells were then washed,
collected and gated using Fortessa instrument in Cytometry Platform at the Curie Institute.
Data was analysed using FlowJo 10.5.2 software.
Acknowledgments
We gratefully acknowledge financial support from the ERC (Consolidator Grant PhotoMedMet
(
GA 681679) to G.G.). This work has received support under the program «Investissements
d’Avenir » launched by the French Government and implemented by the ANR with the
reference ANR-10-IDEX-0001-02 PSL (G.G.). We thank Prof. Philippe Goldner for access to
state-of-the-art laser apparatus and S. Beutner and S. Weiss for performing ICP-MS
measurements. This work was supported by the Helmholtz Initiative and Networking Fund
(
Functional Nanomaterials for Multimodality Cancer Imaging (NanoTracking), project ID:
VH-VI-421) and we thank Utta Herzog for excellent technical support during the cell culture
work.
2
6
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ChemBioChem
10.1002/cbic.201900419
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