Receptor selective ruthenium-somatostatin photosensitizer for cancer targeted photodynamic applications

Article abstract of DOI:10.1039/c5cc03473f

The efficient conjugation of a ruthenium complex and the peptide hormone somatostatin is presented. The resultant biohybrid offers valuable features for photodynamic therapy such as remarkable cellular selectivity, rapid cell uptake by receptor-mediated e

Full text of DOI:10.1039/c5cc03473f

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DOI: 10.1039/C5CC03473F
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ARTICLE TYPE
Receptor Selective Ruthenium-Somatostatin Photosensitizer for Cancer
Targeted Photodynamic Applications
a
b
a
a
a
b
Tao Wang,‡ Natalia Zabarska,‡ Yuzhou Wu, MarkusLamla, Stephan Fischer, Katharina Monczak,
a
b
a
David Y. W.Ng,* SvenRau* and TanjaWeil*
5
Received (in XXX, XXX) XthXXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
1
4
The efficient conjugation of a Ruthenium complex and the
PDT, and one of them, TLD-1433 from the group of McFarland,
1
5
peptide hormone somatostatin is presented. The resultant 50 will enter phase I clinical trials. Therefore, Ruthenium(II)
biohybrid offers valuable features for photodynamic therapy
such as remarkable cellular selectivity, rapid cell uptake by
receptor-mediated endocytosis, efficient generation of O2
complexes and their bioconjugates are particularly attractive for
targeted PDT. However, the application of the commonly used
Ruthenium polypyridyl complexes in PDT is still restricted by
1
0
1
1
6
their low cell uptake and poor cell type selectivity. To the best of
our knowledge, Ruthenium conjugates offering dual selectivity,
i.e. selective accumulation into tumor cells or tissue via receptor-
mediated cell uptake via membrane receptors that are
overexpressed within the membrane of certain tumor cells and
spatially selective photoactivation to induce cellular toxicity only
upon irradiation, potent phototoxicity as well as low
cytotoxicity in the “off”-state.
5
6
6
7
7
8
8
9
5
0
5
0
5
0
5
0
Photodynamic therapy (PDT) is a non-invasive modality for the
1
2
2
3
3
4
4
5
0
5
0
5
0
5
treatment of various types of cancers (e.g. lung, esophagus, skin
1
tumors). By utilizing light irradiation, PDT activates nontoxic
1
7-19
1
at the tumor site are still elusive.
The development of
photosensitizers (PS) to generate radicals or singlet oxygen ( O )
2
Ruthenium conjugates with such “dual selectivity” would be
highly attractive for cancer therapy.
Somatostatin receptors (SSTRs), especially SSTR subtype 2
species, which initiates apoptosis and/or necrosis and eventually
2
leads to cell death. Spatiotemporal control over the release of the
active species has resulted in selective destruction of tumor cells
within the irradiated area, potentially reducing the dose-limiting
(SSTR 2) offers tumor cell and neovasculature targeting since
3
, 4
they are overexpressed in various tumor cells and in tumor blood
side effects incurred with conventional chemotherapy. However,
the classical PS usually lack sufficient selectivity for tumor cells
2
0
vessels relative to normal tissues. The endogenous peptide
hormone somatostatin (SST) exerts its biological effects through
5
and cause collateral damage to surrounding healthy cells.
2
0
SSTRs with high binding affinity in the nanomolarrange.
Therefore, it is highly desirable to incorporate tumor-specific
targeting moieties onto PS for dual selectivity, allowing
preferential accumulation of PS in tumor cells while providing
Therefore, SST and its analogues have been widely investigated
for targeted drug delivery into SSTR-expressing cancer cells by
2
1
6
bioconjugation to radionuclides and antineoplastic agents.
spatial irradiation control of the tumor site. Various tumor
For the conjugation of SST, commonly used approaches via non-
specific lysine modification are not applicable as a lysine residue
is located within the SST receptor binding domain. To retain the
binding properties of the SST, N-terminal modification via solid
phase synthesis has been applied successfully although tedious
synthesis, exhaustive purification and low overall yields represent
significant limitation. Herein, the modification of the single
disulphide bond of SST offers a facile synthetic strategy to access
targeting molecules have been investigated e.g. antibodies, folic
7, 8
acid, transferrin, RGD as well as aptamers.
Among them,
tumor-specific peptides offer many attractive features such as
non-immunogenicity, high tissue penetration, high affinity to
cellular biomarkers as well as straight forward conjugation
9
chemistry. Although the coupling of a tumor-specific peptide to
1
0, 11
photosensitizers such as porphyrins has been well studied,
the
conjugation of Ruthenium complexes with tumor-targeting
peptides for targeted PDT has not been described yet.
2
2, 23
defined SST conjugates with conservedbioactivity.
Bis-alkylation reagents have been developed for peptide or
Conventional porphyrin-based PDT agents reveal a number of the
following limitations, such as hydrophobicity, poor light
absorption, lack of specificity, dark toxicity and prolonged skin
2
4
protein modification via disulfide rebridging. They rebridge the
solvent accessible disulfide bonds of peptide or proteins via two
consecutive Michael addition reactions without the loss of their
1
2
sensitivity. Instead, the anticancer activity of Ruthenium(II)
complexes has been extensively investigated since they combine
many attractive features for PDT such as favorable photophysical
properties, facile synthesis, tunable physical properties (i.e.
charge, lipophilicity or redox potential by coordination of the
appropriate ligands), insensitivity of photochemical properties to
2
5, 26
biological activity or structural integrity.
This strategy has
been successfully applied for the modification of peptide
2
3
26, 27
25
hormones,
antibodies,
and therapeutic proteins.
In
addition, disulfide bonds in peptides and proteins are often
unstable under physiological conditions due to disulfide
scrambling. Therefore, the disulfide rebridging provides thioether
1
3
pH-value variations and low toxicity toward healthy tissues.
2
2
conjugates with improved stability.
It also renders the
Ruthenium(II) complexes have demonstrated great potential for
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conjugates responsive to intracellular glutathione and dissociation
occurs in the presence of elevated tumor GSH concentrations thus
recorded in MilliQ water (Fig.1C and 1D). Both Ru-Alkyne 3 and
Ru-SST reveal typical photophysical behaviour of
8
3
2
providing a valuable strategy to control anti-tumor drug release in 45 Ruthenium(II) polypyridine complexes (Table S1-2). The most
2
2
the cytosol of cancer cells.
relevant absorption transition, the metal-to-ligand-charge-transfer
(MLCT) of Ru-SST 8 (458 nm) is identical to 3 (457 nm).
Furthermore, additional characteristic absorption transitions both
5
0
5
Herein, we have designed Ru-SST 8 with attractive phototoxicity
and tumor cell selectivity by combining the unique features of the
2
+
peptide hormone SST and the PS [Ru(bpy) ] . For instance,
for Ru-SST
8 and 3 are presented in Table S1. The
3
potent SSTR2 agonistic activity facilitates transport into SSTR- 50 phosphorescence-type emission is observed at 621 nm for Ru-
2
2
positive cells for tumor cell specific drug delivery and
SST 8 and 631 nm for 3. The hypsochromic shift of the emission
wavelength correlates to the altered nature of the substituent i.e.
the change from alkyne to triazole. For Ru-SST 8, a significantly
increased luminescence intensity compared to Ru-Alkyne 3 is
2
+
1
controlled photoactivation of the nontoxic prodrug [Ru(bpy)3]
induces cytotoxicity only after irradiation in a spatially and
2
8
temporally controlled fashion. Thus, Ru-SST offers many
important features for expanding the anti-tumor features of Ru- 55 observed, which could tentatively be explained by enhanced non-
3
3
complexes such as attractive receptor selective cellular uptake,
light-controllable cytotoxicity, potent anti-proliferative effects
and low systemic toxicity.
radiative deactivation as the MLCT state in 3 is lower in energy.
1
Fig. 1. Characterization of Ru-SST 8. A. LC-MS spectra of Ru-SST 8,
5
+
4+
2+
5+
m/z= 530 [M ], 663 [M ]. (calcd. exact mass: 530.61486 [M +3H] ,
2+
4+
60
663.01670 [M +2H] , formula:
129 158 28 2
C H N O24RuS ).B. UV-vis
absorption spectra for the photobleaching of ABDA(20 μM) during the
irradiation of Ru-Alkyne 3 and Ru-SST 8 (10 μM) in PBS (1x, pH7.4)
Scheme 1. A. Synthesis of the disulfide rebridging and bioconjugation
reagent 6. a. ethanol/water 3:1, reflux 3 h; b. HBTU, DIEA, DMF,
over a period of 5 min. C. The absorbance spectra of Ru-SST 8 in H O. D.
2
2
0
overnight; c. Oxone, MeOH/H
to receive Ru-SST conjugate 8. d. 2 eq. TCEP for 0.5 h, then compound 6
for 24 h, 50 mM phosphate buffer pH 7.8; e. 2 eq. CuSO
ascorbate, H O, overnight.
2
O 1:1, 24 h. B. Functionalization of SST
The emission spectra of Ru-SST 8 in H O (λ = 460 nm).
2
ex
4
, 4 eq. Na 65 As some Ruthenium(II) polypyridine complexes are known to be
photolabile, their photostability was examined by monitoring the
absorption after irradiation with visible LED light (λ = 470 nm, P
2
A detailed synthetic route for Ru-SST 8 is shown in Scheme 1.
Ru-Alkyne 3 was obtained by refluxing (bpy) RuCl 1 and bpy-
2
34
=
50 ± 3 mW, 50± 3 mW/cm ). Ru-SST 8 and Ru-Alkyne 3
2
3
3
4
5
0
5
0
2
2
retained all their characteristic absorption bands with a slight
decay of 8 % and 7%, respectively. This indicates a significant
improvement in photostability compared to [Ru(bpy) ]Cl , which
Alkyne 2 in ethanol/water 3:1 for 3 h, followed by sephadex
column chromatography purification affording Ru-Alkyne 3 in
7
7
8
8
0
5
0
5
3
2
4
5% yield. Compared to other synthetic methods, this approach
+
featured a decay of 21% under analogous conditions (Fig.S3).
proceeds without the addition of cytotoxic Ag ions, which is
detrimental for biomedical applications in living systems.
3
, 29, 30
Importantly, the consequent application of PDT relies on the
1
production of singlet oxygen. The generation of O of 3 and Ru-
2
Subsequently, Ru-Alkyne 3 was conjugated to SST via extremely
efficient Cu(I) catalysed cycloaddition in water with full
conversion of SST (Scheme 1). Ru-SST conjugate 8 was isolated
in 61% yield after HPLC purification and characterized by HR-
ESI-MS (Fig. S1), HR-MALDI-MS (Fig.S2) and LC-ESI-MS
SST 8 was investigated by applying two different methods. First,
emission quenching caused by oxygen was examined by
monitoring the emission spectra in both oxygen free and oxygen
saturated MilliQ water (Fig.S4). An emission decay of 35% for
Ru-SST 8 and 29 % for Ru-Alkyne 3 was detected. These
quenching values correspond well to the related [Ru(bpy) ]Cl
(
Fig. 1A). The isotopic patterns of the peaks found in HR-MS
3
2
correspond with the theoretical calculations (Fig.S1-2).
3
5
with a quenching value of 41 % in water. This effect is the
result of an energy transfer from the electronically excited
Noteworthy, Ru-SST 8 was observed only as singly charged [M-
+
1]
species in HR-MALDI-MS measurements indicating a
3
Ruthenium(II) complex in its MLCT state to oxygen in its
reduction during the MS measurement. This finding is in
3
3
1
ground state ( O
2
) potentially forming electronically excited
accordance with previous reports in the literature.
The UV/vis absorption and photoluminescence spectra have been
1
36
singlet oxygen ( O ), which is a reactive oxygen species (ROS).
2
2
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1
In order to verify the generation of O , the singlet oxygen sensor
cells was quantified by the normalized single-cell emission
2
9
,10-anthracenediyl-bi(methylene) dimalonic acid(ABDA) was 40 intensity (single-cell emission intensity/area of cell) by using the
1
applied. In the presence of O , an endoperoxide of ABDA is
formed, which decreases the ABDA absorption at 380 nm thus
providing a valuable means of direct monitoring O production.
Ru-Alkyne 3 and Ru-SST 8 (10 µM) were mixed with 20 µM of
ImageJ software (Fig.2B). More than a hundred times higher
uptake was found compared to the control experiment, indicating
significant improvement of cellular transport after conjugation
with SST. The efficient cellular uptake was also detected by
2
1
5
2
ABDA in PBS buffer and then irradiated by a 470 nm LED array 45 emission imaging (Fig.S5). Cell type selectivity was
2
with P = 50 ± 3 mW for 5 min (15 ± 0.9 J/cm ). As shown in
demonstrated by applying Ru-SST 8 on wild type CHO-K1/Ga15
cells and CHO-K1/Ga15/SSTR2 cells overexpressing SSTR2 for
a functional calcium flux assay conducted by GenScript. Ru-SST
8 revealed significant receptor activation and calcium release
already at low concentrations (EC50 of 319.6 ± 1.1 nM) whereas
there was no sign of activation on wild type CHO-K1/Ga15 cells,
indicating high cell selectivity of Ru-SST towards SSTR2 cells
Fig.1B, the generation of singlet oxygen by Ru-Alkyne 3 and Ru-
SST 8 resulted in more than 70% decrease in ABDA absorption
1
0
1
at 380 nm, indicating efficient O generation.
2
5
5
6
6
7
7
8
0
5
0
5
0
5
0
(Fig.2C). To determine the effectiveness of 8 as PDT agents in
cancer cells, its photocytotoxic properties were examined on the
A549 cell line. The dark and light cytotoxicity profiles for Ru-
SST 8 and Ru-Alkyne 3 in A549 cells were screened at a wide
range of concentrations (from 0 to 300 μM) to determine their
potency (IC ). Briefly, the cells were incubated with Ru-SST 8
5
0
or Ru-Alkyne 3 in the dark at different concentrations (from 0 to
300 μM) for 4 h. Subsequently, the cells were washed and
irradiated by a 470 nm LED array with P = 23 ± 3 mW for 5 min
2
(
6.9 ± 0.9 J/cm ). The dark controls were performed in parallel.
The cells were further incubated for 6 h in the dark. The cell
viability was quantified by the Tox-8 assay (Sigma Aldrich)
3
8
according to the manufacturer’s instructions. The Ru-SST
8
1
induced pronounced cytotoxicity with IC50 value of 13.2 ±
.1μM (Fig.2D) after light irradiation, while it remained nontoxic
up to 300 μM (Fig.2E) in the absence of light. Therefore, Ru-SST
displays a high phototoxic index (PI) of greater than 23 (PI is
8
the ratio of the dark and light IC50 values), making it very potent
at very short drug-to light intervals (4 h) and low dosage of
2
visible light (6.9 ± 0.9 J/cm ). In addition, Ru-SST 8 (IC = 13.2
5
0
Fig. 2. A. Confocal microscopy images of 10 μM Ru-SST 8 (left) and Ru-
Alkyne 3 (right) incubated with A549 cells for 4 h. B. The normalized
single cell emission intensity (Single cell emission intensity/area of cell)
was quantified by ImageJ software based on the confocal images (Fig.S6).
The normalized single-cell emission intensity with the incubation of Ru-
SST 8 was divided by 1.83, since the Ru-SST 8 has 1.83 times higher
emission than Ru-Alkyne 3. Data are plotted as means ± standard errors
of the means (SEM) using GraphPadPrism5 Software (n=15).C. Calcium
flux induced by Ru-SST 8 (EC50 = 319.6 ± 1.1nM) in SSTR2 expressing
CHO-K1 cells (black) and wild type CHO-K1 cells (red). D. Cytotoxicity
of Ru-SST 8 on A549 cells with light irradiation for 5 min (IC50= 13.2 ±
± 1.1 μM) elicits superior phototoxicity compared to the Ru-
Alkyne complex 3 (IC = 67.5 ± 1.1 μM), underlining
5
0
1
2
5
0
theimportance ofincreased cellular uptake and thus the greater
potential of Ru-SST 8 for PDT (Fig. S7). While existing
Ruthenium-peptide conjugates have been exploited for their
1
8, 39
anticancer activity,
targeting,
transmembrane transport and nuclear
1
6, 40
41
as well as photocontrolled DNA binding, we
demonstrate herein for the first time the potent phototoxicity of
the receptor-targeted Ruthenium heteroconjugate. Our results
clearly demonstrate the great potential of Ru-SST for targeted
PDT due to selective targeting of SST positive cancer cells and
high phototoxicity while maintaining low systemic toxicity.
1
.1μM).E. Cytotoxicity of Ru-SST 8 on A549 cells in the absence of light.
2
3
3
5
0
5
The cellular uptake of 8 into human non-small-cell lung cancer
NSCLC) A549 cells was investigated, since these cells express
(
3
7
SSTR subtypes 1, 2, 4 and5 on the cellular surface. Equal
quantities of Ru-Alkyne 3 and Ru-SST 8 (10 µM) were added to
the culture medium of A549 cells, respectively. After incubation
for 4 h, the cells were washed to remove any conjugates that were
not taken up and the cells were studied by laser scanning confocal
microscopy. Laser excitation at 458 nm was applied that
corresponds to the MLCT absorbance of the metal complex. The
emission images were recorded in the range of 580-707 nm. As
shown in Fig.2A, only minimal emission was observed within the
cells after incubation with Ru-Alkyne 3, while Ru-SST 8 was
transported rapidly and efficiently across the membrane and
accumulated inside the cells. The emission intensity inside the
8
5
Conclusions
We have reported the synthesis, photophysics, cellular uptake and
phototoxicity of the first photostable cancer cell type selective
Ruthenium(II) polypyridine-SST conjugate Ru-SST 8 with great
potential in targeted photodynamic therapy. The functionalization
of SST via disulfide rebridging provides a facile approach to
access defined conjugates with retained activity, compared to
solid phase synthesis. The synthesis route gave high yields under
mild reaction conditions thus offering a convenient strategy to
access even compound libraries of structurally diverse
photodynamic agents, in which the bioactive functionalities are
90
95
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2
+
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biological studies. In addition, conjugation of SST initiates
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taken up into the SSTR expressing cells with 100-fold increased
efficiency compared to Ru-Alkyne 3. Upon light irradiation, Ru-
SST 8 exhibited potent cellular toxicity in the low micromolar
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1
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Institute of Organic Chemistry III, Ulm University, Albert-Einstein-Allee
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DOI: 10.1039/C5CC03473F
Page 5 of 5
ChemComm
Phe Asn Lys Cys Gly Ala
Phe
Trp
Lys
Thr
Phe Thr Ser Cys
5
Dual selectivity
hγ
1O
2
1
1
2
0
5
0
Cell
Death
Ru-SST reveals enhanced selectivity for tumor cells due to cellular
uptake via receptor mediated endocytosis. Upon light irradiation, the
localized conjugate generates an immediate burst of reactive oxygen
species (ROS), which is restricted to the tumor site, resulting in
efficient photo-induced cellular toxicity while potentially avoiding
collateral damage to healthy cells.
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 |5