Targeting Photoinduced DNA Destruction by Ru(II) Tetraazaphenanthrene in Live Cells by Signal Peptide

Article abstract of DOI:10.1021/jacs.8b02711

Exploiting NF-κB transcription factor peptide conjugation, a Ru(II)-bis-tap complex (tap = 1,4,5,8-tetraazaphenanthrene) was targeted specifically to the nuclei of live HeLa and CHO cells for the first time. DNA binding of the complex within the nucleus of live cells was evident from gradual extinction of the metal complex luminescence after it had crossed the nuclear envelope, attributed to guanine quenching of the ruthenium emission via photoinduced electron transfer. Resonance Raman imaging confirmed that the complex remained in the nucleus after emission is extinguished. In the dark and under imaging conditions the cells remain viable, but efficient cellular destruction was induced with precise spatiotemporal control by applying higher irradiation intensities to selected cells. Solution studies indicate that the peptide conjugated complex associates strongly with calf thymus DNA ex-cellulo and gel electrophoresis confirmed that the peptide conjugate is capable of singlet oxygen independent photodamage to plasmid DNA. This indicates that the observed efficient cellular destruction likely operates via direct DNA oxidation by photoinduced electron transfer between guanine and the precision targeted Ru(II)-tap probe. The discrete targeting of polyazaaromatic complexes to the cell nucleus and confirmation that they are photocytotoxic after nuclear delivery is an important step toward their application in cellular phototherapy.

Full text of DOI:10.1021/jacs.8b02711

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Article
Targeting Photo-induced DNA destruction by Ru(II)
tetraazaphenathrene in Live cells by Signal Peptide
Christopher S. Burke, Aisling Byrne, and Tia E. Keyes
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b02711 • Publication Date (Web): 16 May 2018
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Targeting Photo-induced DNA destruction by Ru(II) tetraaza-
phenathrene in Live cells by Signal Peptide.
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Christopher S. Burke, Aisling Byrne and Tia. E. Keyes*.
School of Chemical Sciences and National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ire-
land.
KEYWORDS (Word Style “BG_Keywords”). If you are submitting your paper to a journal that requires keywords, provide
significant keywords to aid the reader in literature retrieval.
ABSTRACT: Exploiting NF-κB transcription factor peptide conjugation, a Ru(II)-bis-tap complex (tap = 1,4,5,8-
tetraazaphenanthrene) was targeted specifically to the nuclei of live HeLa and CHO cells for the first time. DNA binding in
the nucleus of live cells was evident from gradual extinction of the metal complex luminescence after it had crossed the nu-
clear envelope, attributed to guanine quenching of the ruthenium emission via photoinduced electron transfer. Resonance
Raman imaging confirmed that the complex remained in the nucleus after emission is extinguished. In the dark and under
imaging conditions the cells remain viable, but efficient cellular destruction was induced with precise spatiotemporal con-
trol by applying higher irradiation intensities to selected cells. Solution studies indicate that the peptide conjugated complex
associates strongly with calf thymus DNA ex-cellulo and gel electrophoresis confirmed that the peptide conjugate is capable
of singlet oxygen independent photodamage to plasmid DNA. This indicates that the observed efficient cellular destruction
likely operates via direct DNA oxidation by photoinduced electron transfer between guanine and the precision targeted
Ru(II)-tap probe. The discrete targeting of polyazaaromatic complexes to the cell nucleus and confirmation that they are
photocytotoxic after nuclear delivery is an important step toward their application in cellular phototherapy.
DNA and protein.18,19 PACT may also be used in tandem
with ROS generation to yield potent dual reactivity, as re-
Introduction
Ru(II) complexes containing phenazine or pol-
yazaaromatic ligands have long been investigated as DNA
sensors or as photodynamic therapy (PDT) agents due to
the well characterized interaction of such compounds with
DNA. For example, type II PDT is possible by exploiting the
long-lived triplet nature of the MLCT state of Ru(II)
polypyridyl complexes to photo-generate toxic singlet oxy-
gen and other reactive oxygen species (ROS) to the detri-
ment of the cell.1–8 A related successful strategy uses Ru(II)
excitation to sensitize a much longer-lived ligand centered
(3LC) excited state, as described by McFarland, Thummel
and coworkers using Ru(II)-pyrene dyads that exhibit ex-
cellent phototherapeutic activity against resistant mela-
noma cells.9,10
ported by Turro and coworkers using photolabile Ru-dppn
complexes, for example.20,21
An important issue in both PACT and ROS mediated cel-
lular destruction is subcellular targeting. Both mechanisms
are efficacious photoactivated strategies but have signifi-
cant potential for uncontrolled off-target effects where the
phototherapeutic is activated outside of locations of its
target. A key objective in therapy is to selectively damage
the genetic material of afflicted cells triggering a response
that results in their death.22 DNA damage may be induced
by ROS or ideally, by direct oxidation via electron transfer
that, if unrepaired, leads to strand breaks and cleavage.23–25
Generally, photoinduced electron transfer (PET) occurs at
guanine residues since it is the most easily oxidized base.26
A limitation of type II phototherapy though is the de-
mand for oxygen as a co-reagent, which is non-specific but
can be limiting in hypoxic environments which are often
characteristic of cancerous tissues.11 Consequently, recent
research has intensified towards oxygen-independent
therapies and photoactivated chemotherapeutics (PACT) is
one of the strategies at the forefront of this field.12–17 The
mechanism of Ru(II) PACT relies on the sensitization of the
thermally accessible distorted 3MC state from which ligand
release can occur under photoirradiation. The toxic impact
of this strategy can be twofold; (i) the controlled release of
a toxic ligand along with (ii) generation of a Ru(II)-aquo
species which is free to metallate biological targets such as
Ru(II) complexes bearing at least two tap ligands pos-
sess an excited state reduction potential sufficiently posi-
tive to oxidize guanine residues of DNA by proton coupled
electron transfer (PCET).27–29 These complexes have been
extensively investigated by the Kirsch-De Mesmaeker
group and collaborators who demonstrated that PCET can
result in formation of unique covalent photoadducts be-
tween the Ru-tap complex and the exocyclic amine of gua-
nine.30–32 This phenomenon has been exploited with anti-
sense oligonucleotide conjugates towards gene therapy
with some success in vitro.33–39 Under photoirradiation, Ru-
tap complexes demonstrate efficient single strand cleavage
of supercoiled plasmids ascribed to direct guanine oxida-
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Page 2 of 15
tion by PCET.29,40 Indeed, electron transfer between
ing a nuclear localizing signal (NLS) sequence of the NF-κB
[Ru(tap)2(dppz)]2+ and guanine has been observed directly
in DNA crystals using TRIR experiments.41
transcription factor peptide, namely VQRKRQKLMP (here-
after NLS), across a number of different Ru(II) polypyridyl
complexes.44,48 NF-κB normally resides in the cytoplasm of
mammalian cells as a complex with IκB inhibitor protein
and undergoes activation in response to stimuli such as UV
light, viral infection, etc, that leads to dissociation of the
complex and exposure of the nuclear localization sequenc-
es that drive NF-κB to translocate to the nucleus. The nu-
clear localization sequences50,51 are believed to be recog-
nised by importin proteins that bind to and translocate the
NLS containing proteins into the nucleus through nuclear
pore complexes.52
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8
Despite their potential for DNA targeted therapy, Ru-tap
complexes have not yet been widely explored in this re-
gard in live cells. Gunnlaugsson and coworkers developed
non-toxic gold nanoparticles decorated with Ru-tap com-
plexes and observed diminished luminescence in nuclear
regions attributed to guanine quenching.42 Kirsch-De
Mesmaeker and coworkers synthesized a Ru(II)-tap Tat-
derived peptide conjugate which did not exhibit any toxici-
ty as although plasma membrane permeable, it did not
penetrate the nucleus.43 These examples indicate that, ad-
vantageously, off-target toxicity is not prevalent for Ru-tap
conjugates, likely due to their poor singlet oxygen quan-
tum yield and particle or peptide vectorization which dic-
tates cellular interactions of the complex. Hence, precision
targeting of the probe to the nucleus where the Ru-tap
complex may intimately bind DNA is a prerequisite to the
successful implementation of Ru-tap complexes for DNA
photodamage in live cells.
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Herein, this NLS is exploited to precision target for the
first time, a Ru-tap complex to the nucleus of live cells.
Uptake and interaction with chromosomal DNA was fol-
lowed using confocal fluorescence and resonance Raman
microscopy. Once localized, we investigated the capacity of
the Ru-tap probe to induce cellular destruction with spati-
otemporal control by photosensitized nuclear DNA dam-
age. Finally, the conjugate interaction with ex-cellular DNA
was investigated and its capacity to cleave plasmid DNA is
discussed in the context of its interactions with the nucleus
in live cells.
Our group have focused on development of peptide-
directed metal complex luminophores for organelle target-
ed cellular imaging and sensing, for example at the ER, the
mitochondria, cellular membranes and integrin protein,
and the nucleus and nucleolus.44–49 We demonstrated that
highly effective precision nuclear targeting is possible us-
N
R = OH : [Ru(tap)2(bpyArCOOH)]2+
N
N
N
Ru-tap-acid
N
N
N
N
Ru2+
N
R = OEt : [Ru(tap)2(bpyArCOOEt)]2+
Ru-tap-ester
R = NH-ahx-RRRRRRRR-CONH2 : [Ru(tap)2(bpyArCONH-ahx-RRRRRRRR-CONH2)]10+
R
Ru-tap-R8
N
O
N
+
+
H2N
HN
NH2
H2N
HN
NH2
+
N
N
N
NH3
N
N
N
N
S
Ru2+
N
O
O
O
O
H
H
H
H
N
H
N
N
N
N
N
N
H
N
H
N
H
N
H
NH
O
O
O
O
O
O
N
O
H2N
O
H2N
O
O
NH2
+
NH3
[Ru(tap)2(bpyArCONH-ahx-VQRKRQKLMP-CONH2)]6+
Ru-tap-NLS
Chart 1 Chemical structures of the Ru-tap compounds studied in this work.
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Results and Discussion
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Photophysical characterization of Ru-tap-ester and
Synthesis and characterization of the complex and
its NLS conjugate. The synthesis of tap from 5,6-
diaminoquinoxaline using glyoxal condensation has been
Ru-tap-NLS. As shown in Figure 1, the absorbance and
emission spectra of the peptide conjugates mirror those of
the parent complex with ligand-centered bpy and tap
based transitions observed in the UV region with a maxi-
mum around 280 nm. A broad MLCT band is evident, re-
solved into two visible maxima at ca. 415 and 460 nm, the
most bathochromic of which is ascribed to a Ru→tap tran-
sition.60 Excitation into the MLCT absorbance leads to a
characteristically Stokes-shifted emission feature centered
at about 630 nm in acetonitrile and 640 nm in water, and
the luminescence quantum yield in air equilibrated water
was determined as approximately 2.8 % for both com-
pounds.
53
described previously and an analogous method was im-
plemented in this work to provide the pure ligand as con-
firmed by NMR spectroscopy. In general, preparation of
Ru(II) complexes bearing two tap ligands proceeds via
[Ru(tap)2Cl2] using the classical synthesis from
RuCl3.3H2O.54,55 Following recent developments towards
efficient synthetic protocols for Ru(II) complexes,56 we
elected instead, to adopt [Ru(DMSO)4Cl2] as starting mate-
rial57,58 which permitted clean and rapid conversion to
[Ru(tap)2Cl2] in 81 % yield in just 15 minutes. This novel
protocol may be useful for the future preparation of Ru(II)
complexes bearing similarly π-deficient ligands.
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[Ru(tap)2Cl2] was found to be relatively unreactive and
required aqueous silver triflate activation to cleave the
chloride ligands by precipitation of insoluble AgCl. The Ru-
aquo intermediate was then treated in situ with the heter-
oligand, bpyArCOOR (R = H, Et); a conjugatable bpy deriva-
tive which has been reported previously by our group.59
Through this approach, the parent complexes;
[Ru(tap)2(bpyArCOOH)]2+
(Ru-tap-acid)
and
[Ru(tap)2(bpyArCOOEt)]2+ (Ru-tap-ester), were obtained
in good yield (> 77 %) following purification by flash
chromatography on silica. Their structures are provided in
1
Chart 1. H NMR and 13C NMR analysis conformed as ex-
pected for [Ru(tap)2(bpyArCOOR)]2+ while HR-MS analysis
- +
returned mass ions corresponding to [M + PF6 ] for both
complexes.
Peptide-conjugation to the NLS sequence, H2N-ahx-
VQRKRQKLMP-CONH2 (ahx is aminohexyl linker), pro-
ceeded through a PyBOP coupling protocol in the presence
of two equivalents of the NLS peptide to drive quantitative
conversion based on Ru(II). The crude material was isolat-
ed as its chloride salt by precipitation from ace-
tone/tetrabutylammonium chloride and, where necessary,
was subjected to reverse phase preparative TLC (C18-
Silica, 0.1 % TFA in CH3CN/H2O) to yield the purified con-
jugate. Purity of the conjugate relative to the parent struc-
ture was confirmed by analytical RP-HPLC wherein the
conjugate peak eluted at 11.2 minutes with no evidence of
residual parent complex which has a characteristic reten-
tion time of 14.1 minutes. The peptide-modified complex,
Figure 1 Absorbance and emission spectra (solid and dashed lines
respectively) of RuꢀtapꢀNLS (10 µM) in CH3CN (MeCN, blue),
H2O (orange) and PBS pH 7.4 (green).
As expected both parent complexes exhibit single expo-
nential luminescent decays in water and acetonitrile. In
line with previous reports on Ru-bis-tap complexes,61 the
luminescence lifetime of Ru-tap-ester and Ru-tap-NLS is
slightly longer-lived in acetonitrile than water under aer-
ated conditions, whereas the complex in organic solvent
was significantly more sensitive to quenching by oxygen
than in water. For example, the luminescent lifetime in
acetonitrile doubles upon de-aeration under N2 purge in
the case of Ru-tap-ester (τ (CH3CN, air) = 680 ns; τ (CH3CN,
N2) = 1332 ns) whereas a comparatively moderate (about
20 %) luminescent lifetime increase upon de-aeration was
observed in aqueous solvent. Emission lifetimes were also
slightly longer for Ru-tap-NLS relative to Ru-tap-ester
which may suggest a protective effect exerted by the con-
jugated peptide perhaps reducing quenching by oxygen.
Such behavior has been observed previously for other pep-
tide-conjugates of Ru(II).45,49
1
Ru-tap-NLS, was characterized by H NMR and exhibited
the expected spectrum with signals attributable to the
Ru(II) core and peptide signals that corresponded to a 1:1
conjugate upon integration. Correspondingly, mass spec-
trometry indicated mass ions assigned to [M]+3 and [M]+6
at 706.4377 (calcd. 706.9925) and 353.7231 (calcd.
353.9999) respectively. To compare the impact of nuclear
targeting on the cellular efficacy of the Ru-tap photo-
probe, Ru-tap-acid was also conjugated to the non-specific
uptake vector octa-arginine to provide Ru-tap-R8 (R8:
H2N-ahx-RRRRRRRR-CONH2). Full synthetic protocols and
characterization data for the complexes and conjugates is
available in the ESI.
Interestingly, in acetonitrile, Ru-tap-NLS displays more
complex luminescence behavior where the emission decay
fit best to a bi-exponential model; containing a long-lived
component at 695 ns, comparable with the emission life-
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time of the parent ester, and a short-lived component of 74 involving proton transfer from the NLS peptide of Ru-tap-
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3
4
ns in air (Table 1). This behavior is similar to that reported
by Rebarz et al., who demonstrated that the excited state of
[Ru(tap)2(phen)]2+ can be quenched by proton transfer
from protonated calix [6]crypturea in acetonitrile.62 We
speculate that a similar mechanism may be operative here
NLS to its Ru-tap cargo since the NLS contains relatively
acidic lysine and arginine residues. This behavior is not
observed in water, presumably because the pKa of the tap
is lower in this solvent.
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7
8
Table 1: Summary of the photophysical characterization data for Ru-tap-NLS and Ru-tap-ester.
9
c
Solventa
λ abs (ε)b
nm (x 103 M-1 cm-1)
λem
nm
τ lum
ns
d
ϕlum
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Aer.
Deaer.
Ru-tap-ester
Ru-tap-NLS
CH3CN
H2O
276 (67.9), 416 (18.1), 456 (14.0).
279 (61.9), 415 (16.7), 459 (12.5).
278 (61.3), 414 (17.3), 459 (13.1).
629
639
639
680 ± 9
607 ± 7
515 ± 1
1332 ± 62
753 ± 8
0.041
0.029
PBS
594 ± 9
CH3CN
275 (68.8), 421 (17.2), 460 (13.7).
631
695 ± 2 (71 %)
74 ± 3 (29 %)
659 ± 1
1015 ± 5 (68 %)
119 ± 15 (32 %)
760 ± 3
H2O
PBS
279 (65.8), 415 (16.7), 460 (12.6).
280 (65.3), 415 (16.8), 460 (12.8).
640
640
0.028
605 ± 1
659 ± 4
Notes: a PBS = commercial Dulbecco’s Phosphate Buffered Saline without modifiers, measured at pH 7.4. b Averaged from triplicate analꢀ
yses. Relative standard deviations (not shown) were typically < 5 %. 450 nm excitation, data fit to tailfit criteria of 0.9 < χ2 < 1.1. Deꢀ
c
aeration by N2 purge for 15 minutes. Averaged data is shown ±SD (n = 3). For biꢀexponential fitting, % relative amplitude values are proꢀ
vided in parentheses. d Quantum yields were averaged from triplicate measurements in aerated solutions. using the slope method (estimated
error ± 10 %) and [Ru(bpy)3]2+ as a reference standard (ϕ(air) = 0.018 (CH3CN); 0.040 (H2O)63).
Interaction of Ru-tap-ester and Ru-tap-NLS with
DNA. As shown in Figure 2, in the presence of calf thymus
DNA (ctDNA), Ru-tap-ester did not exhibit a significant
photophysical response, even under conditions of r = 50 (r
= [DNA]bp/[Ru]). [Ru(tap)2(bpy)]2+, a structural derivative
of [Ru(tap)2(bpyArCOOEt)]2+ (Ru-tap-ester), was shown by
Lecomte et al. to bind DNA with strong concomitant
quenching of luminescence due to a PET to guanine,
whereas in contrast [Ru(Me2tap)3]2+ (Me2tap = 2,7-
dimethyl-1,4,5,8-tetraazaphenanthrene) was sterically
hindered from binding DNA and thus did not demonstrate
a spectroscopic response in its presence.29 Hence, it is
clear, but surprising, that Ru-tap-ester is similarly impeded
from binding DNA presumably due to its pendant aryl-
ester substituent. Importantly, the absence of lumines-
cence quenching does not indicate an excited state reduc-
tion potential that is too low to abstract electrons from
Figure 2 Changes to the absorbance and emission spectra of Ruꢀ
tapꢀester (5 µM, PBS pH 7.4) with increasing r = [DNA]bp/[Ru]
from r = 0 (blue) to r = 20 or 50 (red) as indicated.
guanine because although the luminescence lifetime of Ru-
tap-ester was unchanged in the presence of ctDNA, in a
control
experiment
we
found
that
[Ru(tap)2(bpyArCOOEt)]2+ is quenched by GMP (from τ =
515 ns to τ = 402 ns, see Table 2).
Interestingly, in contrast to Ru-tap-ester, absorbance
hypochromicity (ca. 15 %) and emission quenching was
observed for Ru-tap-NLS in the presence of DNA up to sat-
uration (Figure 3). In addition, like Ru-tap-ester, the pep-
tide conjugate was quenched by GMP (impacting lumines-
cence lifetime which decreased from τ = 605 ns to τ = 483
ns, see Table 2) but not AMP. To evaluate if guanine
quenches luminescence of the ester and conjugate, emis-
sion quenching of each by GMP in PBS was evaluated as a
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function of GMP concentration. From the resulting Stern- be quenched by ctDNA at all, this we attributed to steric
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Volmer plots, quenching rate constants (kq) were deter-
mined from triplicate data as kq = 4.25 x 10-10 M-1 s-1 (R2 =
0.998) and 4.08 x 10-10 M-1 s-1 (R2 = 0.982) for Ru-tap-
ester and Ru-tap-NLS respectively (ESI, Figure S19). These
values are comparable to data reported for related Ru-bis-
tap complexes (for example, [Ru(tap)2(bpy)]2+ quenched
by GMP exhibited kq of 7.4 x 10-10 M-1 s-1).29 The lumines-
cence quenching of Ru-tap-NLS in the presence of ctDNA is
therefore attributed to photoinduced electron transfer
from guanine to the excited Ru as reported for
[Ru(tap)2(bpy)]2+.
inhibition by the aryl ester substituent of Ru-tap-ester of
access of the complex to DNA. In Ru-tap-NLS a similar ste-
ric effect must be active but the evident emission quench-
ing of Ru-tap-NLS can be attributed to the DNA affinity of
its conjugated cationic peptide (+4, pH 7.4) which can elec-
trostatically associate with the polyanionic phosphate
backbone of DNA. This is supported by the absence of
spectroscopic change in the presence of DNA at higher
ionic strength (1 M NaCl in PBS, see Table 2 and ESI Figure
S18). Thus, although guanine is sterically inaccessible to
Ru-tap-ester, strong peptide-mediated electrostatic bind-
ing of Ru-tap-NLS to DNA permits the Ru-tap moiety suffi-
cient proximity to DNA to facilitate PET
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Notably, however, the luminescence intensity of Ru-tap-
NLS was not quenched to the same degree by ctDNA as
other reported Ru-tap complexes under the same condi-
tions.29 And, as described, the Ru-tap-ester does not appear
Table 2: Summary of luminescence lifetime data for the Ru-tap compounds in the presence and absence of biomol-
ecules as indicated.
Compound
τ / free
τ / BSA
τ / ctDNA
τ / GMP
τ / AMP
τ / ctDNA
3h Irradiation
Ru-tap-Ester
515 ± 1
605 ± 1
515 ± 2
564 ± 4
535 ± 1
402 ± 2
483 ± 1
536 ± 1
Ru-tap-NLS
1294 ± 66 (17 %)
482 ± 19 (54 %)
51 ± 5 (29 %)
559 ± 16
544 ± 6 (53 %)
75 ± 5 (47 %)
in PBS
in 1 M NaCl/PBS
582 ± 3 (72 %)
42 ± 11 (28 %)
574 ± 4 (67 %)
31 ± 1 (33 %)
Notes: Errors included as ± SD (n = 3). All fits conformed to tail-fit criteria of 0.9 < χ2 < 1.1. Percentage relative amplitudes of the
decay components are given in parentheses. r = 100 mole equivalents of GMP and AMP, r = 20 mole base pair equivalents of ctDNA
and r = 15 mole equivalents of BSA. All measurements performed in PBS pH 7.4 at room temperature.
To assess binding affinity, an ethidium bromide dis-
placement assay was performed which evaluates the con-
centration of Ru-tap-NLS required to reduce the ethidium
fluorescence intensity by half.64 From triplicate data, an
average apparent binding constant was calculated for Ru-
tap-NLS in buffer at Kapp = 2.26 x 107 M-1. The strong affini-
ty was unsurprising considering the highly cationic nature
of the NLS peptide. Similar binding affinity was calculated
by Brunner and Barton who studied Rh(III)-peptide conju-
gates targeted to DNA mismatches.65 Notably, Ru-tap-ester
did not cause a decrease in ethidium fluorescence, again
underlining its remarkably low DNA affinity (ESI, Figure
S16).
Upon DNA binding, the luminescence decay from Ru-tap-
NLS fit to a tri-exponential decay model with a long-lived
component of τlong = 1294 ± 66 ns (αlong = 17 %) which was
greatly increased relative to the free compound (τav = 605
ns, Table 2). Since the free probe is quite insensitive to
Figure 3 Changes to the absorbance and emission spectra of Ruꢀ
tapꢀNLS (10 µM, PBS pH 7.4) with increasing r = [DNA]bp/[Ru]
quenching by oxygen and solvent, this enhancement is
probably due to its more rigid positioning in an A-rich sec-
tion of ctDNA which decreases the vibrational deactivation
from r = 0 (blue) to r = 10 (red) as indicated.
rate. An intermediate component was determined as τint
=
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Page 6 of 15
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lifetime of the peptide conjugate in the presence of GMP (τ
= 483 ± 1 ns) may be attributed to complex quenched by
guanine within the DNA helix. Finally, the third component
of the Ru-tap-NLS decay in the presence of ctDNA was
short-lived at τshort = 51 ± 5 ns (αshort = 29 %), suggesting
strong quenching perhaps due to intimate proximity to G-
rich regions of ctDNA. This short component (τshort) is of
the same order of magnitude as the short-lived component
of the lifetime of Ru-tap-NLS observed in acetonitrile (Ta-
ble 1, τ = 74 ± 3) and thus may also arise from protonation
or H-bridging of Ru-tap due to intimate proximity to DNA
upon binding. Notably, a similar tri-exponential decay was
observed in the presence of short oligonucleotides by
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Kirsch-De
Mesmaeker
and
coworkers
using
[Ru(tap)2(phen-TAT)]2+; a Ru-tap complex tethered to a
Tat-derived peptide sequence.43
Ru-tap complexes like [Ru(tap)2(bpy)]2+ can form per-
manent covalent adducts with guanine under irradiation
leading to changes in their absorbance and emission spec-
tra.29,31 Herein, Ru-tap-NLS was subjected to continuous
irradiation in PBS buffer in the presence of ctDNA (Xe-arc,
500 W at source, λ > 355 nm; r = 20, wherein probe should
be fully bound). Over a period of 3 h, the absorbance spec-
trum transformed significantly with strong hyperchomicity
of the MLCT band and new features growing in at approx-
imately 530 nm and 355 nm. These changes are accompa-
nied by significant decreases in emission intensity with up
to 75 - 80 % extinction of emission relative to the free
probe. Correspondingly, as shown in Table 2, the lumines-
cence lifetime also changes following irradiation. The long-
est component of the decay is eliminated and decay kinet-
ics revert to a biexponential process with an approximate
50/50 distribution of lifetimes measured at τav = 544 ns
and τav = 75 ns. These photophysical changes are con-
sistent with the formation of Ru-tap-G photo-adduct as
reported for other Ru-tap complexes.30,66 Importantly, in a
control experiment, we observed that the absorbance and
emission spectra exhibit only minor changes under illumi-
nation in the absence of ctDNA, consistent with high pho-
tostability and low quantum yield of dechelation in aque-
ous buffer (ESI, Figure S17).
Figure 4 Changes to the absorbance (a) and emission (b) spectra
of RuꢀtapꢀNLS (10 µM, PBS pH 7.4) with increasing irradiation
time (500 W) up to t = 3 h. Arrows inserted indicate the direction
of change. Green traces: r = [DNA]bp/[Ru] = 0, t = 0. Blue traces:
r = 20, t = 0. Red traces: r = 20, t = 3 h.
Interaction of Ru-tap-ester and Ru-tap-NLS with
BSA. To assess the impact of non-specific association of
Ru-tap-ester and Ru-tap-NLS to protein, BSA was exploited
as a protein model. BSA is a useful model in this regard
since it is anionic and contains hydrophobic cavities that
could potentially host lipophilic cations like the Ru(II) con-
jugate. However, we found that BSA had little impact,
within error, on the luminescence intensity of Ru-tap-NLS
and Ru-tap-ester up to r = 50 (r = [BSA]/[Ru], see ESI for
spectra). As shown in Table 2, a small decrease in the life-
time was observed for Ru-tap-NLS (τ = 605 ns to 564 ns on
average) but not for Ru-tap-ester which can be ascribed to
a moderate affinity of the cationic conjugated NLS peptide
for BSA. The lifetime decrease may be due to a decrease in
the protecting effect afforded by the tethered peptide to
the Ru-tap moiety from oxygen quenching, or moderate
quenching by tryptophan and tyrosine.67,68 This weak BSA
affinity is important since nuclear uptake of metal com-
plexes can be inhibited by binding serum albumin in cellu-
lo.69 Furthermore, the absence of spectroscopic response
to the presence of protein is useful for probing the selec-
tive response of Ru-tap-NLS to DNA in live cells.
Ru-tap-NLS localizes at the nucleus in vivo. To exam-
ine cellular uptake of Ru-tap-NLS, HeLa and CHO cells were
separately incubated with the conjugate across a range of
concentrations (10−100 μM) in PBS buffer. Using confocal
microscopy, it was found that 100 µM was the optimum
concentration in terms of imaging, and cytotoxicity to-
wards the cells (Figure S20). Interestingly, Ru-tap-NLS was
found to be mildly toxic towards HeLa cells. After 24 h
incubation with 200 µM in the absence of light, from the
Alamar Blue assay, more than 80 % of cells remained via-
ble.
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Figure 5 follows the uptake of Ru-tap-NLS (100 µM) by
whilst the complex was still emitting brightly. DAPI was
found to co-localize strongly with the complex, shown in
Figure 5G, and the corresponding X-Y plot profile (Figure
5H). In order to assess conjugate co-localization, the cells
were imaged after 5 h incubation, a time point by which
the majority of the complex has entered the nucleus but by
which luminescence is not yet extinguished. At this point,
as the complex has not completed localization some emis-
sion from the conjugate in the cytoplasm is evident in Fig-
ure 5G. The punctate appearance of emission from the cy-
toplasm is attributed to the fact that the complex will only
emit from hydrophobic structures, such as membranes
within the cytoplasm.
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HeLa cells incubated at 37°C in the absence of light. Within
2 to 3 hours the complex was observed to cross the mem-
brane and distribute through the cytoplasm, from where it
emits brightly (A and B). The punctate appearance of the
dye distribution suggests it may be contained in endo-
somes, where the average emission intensity was meas-
ured to be 136.8 ± 2.8 a.u. using Image J. Correspondingly,
uptake was found to be temperature dependent, as Ru-tap-
NLS did not cross the cell membrane when incubated at 4
°C (Figure S22), indicating the that the uptake process is
energy dependent, suggesting endocytosis. By 5 h incuba-
tion, the complex has localized to the nuclear region of the
cells, where, under the same imaging conditions, the abso-
lute emission intensity of the probe has decreased, now
with an average emission intensity of 2.6 ± 0.5 a.u. per cell
(Figure 5 C and D). This suggests that Ru-tap-NLS has en-
countered DNA, causing the emission to commence to
switch off. Between 6-9 h incubation, the emission from
the nucleus begins to completely extinguish across the cell
population. This time frame of DNA binding and the emis-
sion switching off may arise as a result of the stage of mito-
sis the cells are going through. The uptake of the polyargi-
nine conjugated complex, Ru-tap-R8 was also assessed in
HeLa cells. Under the same conditions; 100 µM Ru-tap-R8
was incubated with HeLa cells for 5 h, where it was taken
up by the cells, but it remains in the cytoplasm of the cells
and is nuclear excluding. This result indicates that it is the
nuclear-localizing signal peptide that is directing Ru-tap-
NLS to the nucleus of the cells.
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Luminescent lifetime imaging microscopy (LLIM) was
performed to determine the lifetime of Ru-tap-NLS in the
cell. Figure 5I shows the false-color lifetime image of a sin-
gle HeLa cell, where Ru-tap-NLS is located outside the nu-
cleus after incubating for 5 h in the absence of light, at 37
°C. When fit to a mono-exponential decay, the luminescent
lifetime of Ru-tap-NLS was found to be 43.1 ± 4.7 ns. As the
luminescence of Ru-tap-NLS switched off when bound to
nuclear DNA, we were unable to measure a lifetime from
within the nucleus. However, comparing the lifetime of Ru-
tap-NLS in solution (Table 2, τ = 605 ± 1), the lifetime has
decreased dramatically by about an order of magnitude
when in the cytoplasm. At such a short lifetime, quenching
is occurring or there is protonation of the complex. In ei-
ther case this does suggest as indicated earlier that the
complex occupies endosomes rather than the cytoplasm
itself, which are typically maintained at acidic pH because
of the activity of ATP-dependent proton pumps in these
organelles.70
To confirm nuclear localization, colocalisation studies
were carried out using the commercial nuclear targeting
dye DAPI (100 nM). HeLa cells were incubated with 100
µM Ru-tap-NLS for 3 h, and DAPI was added at this stage,
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Figure 5 Confocal uptake of RuꢀtapꢀNLS by live HeLa cells after 3 h (A and B), and 5 h (C and D), and RuꢀtapꢀR8 after 5 h (E and F).
Colocalisation of RuꢀtapꢀNLS in the nucleus was confirmed using DAPI (G). HeLa cells were incubated for 3 h in the absence of light, and
DAPI was added 20 minutes prior to imaging RuꢀtapꢀNLS (100 µM) in red (i), DAPI (100 nM) in blue (ii), and the overlay of channels
showing their colocalisation in pink (iii). The crosshair trace across the cell (iv) is represented in the corresponding graph (H), demonstratꢀ
ing colocalisation in the nucleus, analysed using ImageJ. FLIM image of RuꢀtapꢀNLS in HeLa cell at 5 h (I). The corresponding confocal
image can be found in Figure S28. DAPI was excited at 405 nm and emission was collected between 450 – 500 nm. (Note: A,C,E show the
overlay of the RuꢀtapꢀNLS channel and the reflectance, while B,D,F show the RuꢀtapꢀNLS channel only).
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As emission from Ru-tap-NLS evidently switches off af-
1
ter it enters the nucleus, it is challenging but important to
confirm it remains localized there after emission is extin-
guished, to ensure that the loss of emission is not due to
the probe leaving the organelle. Resonance Raman micros-
copy was therefore carried out on live HeLa cell samples
pre- and post-5 hours incubation with the Ru-tap-NLS
complex. An excitation wavelength of 488 nm was em-
ployed to ensure resonant excitation of the MLCT transi-
tion of Ru-tap-NLS. Figure 6 shows the white light image
(bottom, inset) highlighting the nuclear (A) and cytoplas-
mic (B) regions from where the resonance Raman spectra
were collected.
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Using the white light image (Figure 6, inset) to identify
the specific regions of the cell, Raman signals from the nu-
cleus and cytoplasm could be compared. When focused on
the nucleus of HeLa cells post-incubation with the probes,
beyond the point at which the emission had switched off at
the nucleus, an intense resonance Raman signature from
the metal complex was obtained. Whereas, by comparison,
similar but extremely weak metal complex Raman signa-
ture was seen from the cytoplasm. This result confirms
that Ru-tap-NLS has localized at the nucleus of the cells,
and remains present within this organelle once the lumi-
nescence has switched off.
There are notable differences between the cellular spec-
tra and that of Ru-tap-NLS in solution in the absence and
presence of ctDNA (r = 20), also shown in Figure 6. In solu-
tion, the spectrum of Ru-tap-NLS in PBS buffer shows
characteristic tap and bipyridine vibrational modes (1536,
1485, 1277, and 1162 cm-1) consistent with resonance
with MLCT transitions to both ligands under 473 nm exci-
tation. The features are narrower in the cellular spectra
and there appear to be some small shifts to higher fre-
quency (approximately 3 cm-1) for the tap features. How-
ever, most notably, upon DNA binding in solution, tap as-
sociated features appear to diminish in intensity relative to
the bipyridine signals and appear to be absent from the
spectrum of the cellular nucleus resulting in the emer-
gence of an intense new feature at 1481 cm-1 and a shoul-
der centered around 1520 cm-1. The marked differences
between spectra in the presence of DNA in solution, and
particularly in the cell nucleus, are tentatively attributed to
a shifting out of resonance of the tap component of the
spectra upon DNA binding possibly due to protonation or
H-bridging as suggested above in the luminescence lifetime
Figure 6 Top spectra: Resonance Raman (473 nm) of Ruꢀtapꢀ
NLS in PBS (70 µM) in the presence and absence of ctDNA (r =
20). Bottom: Image of a live single HeLa cell, taken using the
CCD camera attached to a Horiba JobinꢀYvon Labram HR inꢀ
strument, using a 50x objective and 300 µm pinhole. Cells were
treated with RuꢀtapꢀNLS (150 µM) for 5 h, and washed x 2 with
supplemented PBS. Raman spectra was collected using a 488 nm
laser from the nuclear region (A) and cytoplasm (B) of the cell.
data. This is supported by a previous study by Marcélis et
al. who reported diminishing resonance Raman intensity
(532 nm) of a Ru-tap photo-adduct with decreasing pH
corresponding to blue-shifting of the MLCT absorbance
shoulder out of resonance upon Ru-tap protonation.71
Nuclear-localized Ru-tap-NLS is capable of photo-
induced cellular toxicity with spatiotemporal control.
To assess the photo-cytotoxicity of Ru-tap-NLS we exam-
ined the impact of the complex on cells after the complex
had reached the nucleus. Figure 7A shows a group of HeLa
cells stained with Ru-tap-NLS for 5 h. A single cell, high-
lighted in the image, was continuously scanned at five-
minute intervals (Ex 470nm, 0.13 mW/cm2), and then im-
aged to assess cell viability in the presence of DRAQ 7, a
nuclear stain that only enters the nucleus of dead cells.
Within 15 minutes of photoirradiation, Ru-tap-NLS emis-
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was observed to the surrounding cells which had not been continꢀ
sion switched off and DRAQ 7 had entered the cell (Figure
7B and C). Importantly, no damage has occurred to the
surrounding cells which remained viable, confirmed by
their impenetrability to DRAQ 7. Similarly, CHO cells were
incubated with Ru-tap-NLS (100 µM) for 7 h, to ensure that
Ru-tap-NLS had bound to nuclear DNA and switched off. A
single cell, shown in Figure 7D, was continuously irradiat-
ed at 5-minute intervals, with DRAQ 7 present. After 10
minutes of photoirradiation, DRAQ 7 had entered the cell
(Figure 7E). In this case, the cell death under irradiation
was faster as Ru-tap-NLS had bound to the nuclear DNA.
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uously irradiated. When RuꢀtapꢀNLS emission had switched off
(T7h) a single CHO cell was irradiated (D) and DRAQ 7 entered
the nucleus after 10 minutes of irradiating (E). DRAQ 7 was exꢀ
cited at 633 nm and emission was collected between 635 – 730
nm. (Note: A and D show the RuꢀtapꢀNLS channel with reflecꢀ
tance, B shows RuꢀtapꢀNLS channel only, and C and E shows the
overlay of the RuꢀtapꢀNLS channel with the DRAQ 7 channel.)
Ru-tap-NLS is capable of singlet-oxygen independent
photo-cleavage of plasmid DNA. In order to further elu-
cidate the phototoxic interactions of Ru-tap-NLS with cel-
lular DNA, photo-induced cleavage studies were carried
out using supercoiled plasmid pUC19. DNA cleavage occurs
when supercoiled plasmid DNA (Form I) relaxes to yield
nicked open-circular (Form II) or linear (Form III) strands
of plasmid DNA. Efficient plasmid cleavage by Ru-tap
complexes has been reported by others using gel electro-
phoresis and AFM experiments and was ascribed to PCET
mechanisms.29,40,72 Here, pUC19 (400 ng) was exposed to
Ru-tap-NLS in a 1:10 ratio of plasmid DNA:Ru. The solution
was irradiated at 458 nm (130 mW) for a duration of 30
seconds up to 30 minutes. After irradiation, the samples
were separated using electrophoresis on a 0.75 % agarose
gel. Figure 8 shows that after only 30 seconds of irradia-
tion, the supercoiled plasmid (Form I) has been nicked
resulting in formation of open-circular form (Form II), in-
dicated by the appearance of a second band in Lane 3 (Fig-
ure 8A), with native supercoiled plasmid remaining evi-
dent on the gel. The band intensities of Form II increase
relative to the intensity of Form I over time, suggesting
more of the supercoiled plasmid is being nicked over ex-
tended irradiation times. However, the plasmid does not
appear to undergo further cleavage, i.e. there is no evi-
dence for linear form (Form III) in Lanes 4-6. Control aga-
rose gel electrophoresis (Figure S27) shows that the irra-
diation process has no damaging effect on the plasmid
when Ru-tap-NLS is not present, nor does Ru-tap-NLS ap-
pear on the gel in the absence of the plasmid, indicating
that the bands present in Figure 8A are a result of plasmid
interactions with Ru-tap-NLS upon irradiation. Consider-
ing the significantly greater irradiation flux required to
yield photoadducts between Ru-tap-NLS and ctDNA (Fig-
ure 4), we speculate that it is unlikely that adduct for-
mation is occurring in the plasmid irradiation experiments
and that the observed changes in Figure 8 are due to cleav-
age processes alone.
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Preliminary bulk cytotoxicity measurements were car-
ried out (supplementary materials) using the Alamar Blue
assay and confirmed the photocytotoxicity of the Ru-tap-
NLS conjugate. As mentioned, in the absence of light Ru-
tap-NLS is minimally toxic towards HeLa cells, with an
IC50 value of 83.4 μM. Whereas even under weak (5
mW/cm2 blue light (440 nm) for 15 minutes the IC50 value
decreased to 51.8 μM.
Further confirmation that the Ru-tap-NLS phototoxicity
is a DNA based process was gleaned from an identical ex-
periment where Ru-tap-R8, the analogous conjugate bear-
ing octa-arginine as a non-specific cell penetrating peptide,
was introduced to the cells. This complex is cell permeable,
distributing throughout the cytoplasm but was found not
to enter the nucleus (Figure 5). Although blebbing of the
cell membrane can be seen under continuous irradiation
with this complex (Figure S25), no DRAQ 7 entered the
nucleus, indicating that the phototoxic effects are a result
of Ru-tap-NLS localizing in the nucleus. In a further control
experiment, under identical irradiation conditions, HeLa
cells with no complex conjugate present also remained
viable and the cell membrane remained intact (Figure
S26). Combined, these results indicate that it is the interac-
tion of Ru-tap-NLS with nuclear DNA that is inducing cell
death upon irradiation. To our knowledge this is the first in
cellulo demonstration of DNA cleavage by a tap complex
and local irradiation using Ru-tap-NLS enables us to image
a single cell, and induce death on a cell-by-cell basis.
To understand if photosensitized generation of singlet
oxygen by the Ru-tap conjugate is responsible for the ob-
served strand nicks i.e. a type II photosensitized reaction,
sodium azide (NaN3) was added to the Ru-tap-NLS plasmid
samples before the irradiation process. Sodium azide is a
specific quencher of singlet oxygen,73 and its presence did
not impact the extent of plasmid cleavage by Ru-tap-NLS
thus indicating that singlet oxygen is not the cause of the
observed photocleavage (Figure 8B). In an analogous con-
trol experiment [Ru(bpy)3]2+, an efficient singlet oxygen
generator,74 was incubated and irradiated with the plas-
mid. Correspondingly, Figure S28 shows that in the ab-
sence of NaN3 [Ru(bpy)3]2+ induced plasmid DNA cleavage
over the irradiation time between 30 seconds to 20
Figure 7 Phototoxic effects were analyzed by irradiating a single
HeLa cell at 470 nm (0.13 mW/cm2) (A) for 5ꢀminute intervals, in
the presence of DRAQ 7. The DRAQ 7 was found to enter the cell
after 15 minutes, where RuꢀtapꢀNLS emission switched off (B)
and DRAQ 7 emission was seen in the nucleus (C). No damage
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minutes (Lanes 1-8) converting the supercoiled Form I to ly operates by a similar mechanism, consistent with the
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Form II, with no supercoiled plasmid remaining after the
process. However, in the presence of NaN3, no damage to
plasmid DNA was observed.
extinction of the emission from the complex as the cell is
destroyed. However, we note that in cells adduct formation
may well remain operative and cannot be ruled out as a
contributor to the observed photo-induced cellular de-
struction. Overall, to our knowledge this is the first
demonstrated PDT effect for a Ru-tap complex in cellulo,
enabled by signal peptide precision targeting of the com-
plex to the nucleus. Potentially, this approach could pre-
sent new opportunities towards the application of such
complexes in PDT.
Thus, we conclude that the origin of plasmid DNA cleav-
age by Ru-tap-NLS is most likely via direct oxidative dam-
age at the guanine base or through a Type I sensitized re-
action mediated by electron transfer to the excited state
complex. This is consistent with previous reports on relat-
ed tap complexes and is supported by the luminescent life-
time data for the Ru-tap-NLS in the presence of DNA or
GMP discussed above. We therefore conclude that the
mechanism of photoinduced cell death by Ru-tap-NLS like-
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Figure 8 Agarose gel electrophoresis. Gel (A) gel electrophoresis of supercoiled (400 ng) pUC19 plasmid DNA exposed to RuꢀtapꢀNLS in
a 1:10 ratio, and irradiated at 488 nm over 30 minutes. The reactions were carried out in a buffer solution made up of 25 mM NaCl and 80
mM HEPES. Lane 1 pUC19 plasmid control Lane 2 pUC19 + RuꢀtapꢀNLS no irradiation Lane 3 30 seconds Lane 4 2 minutes Lane 5 10
minutes Lane 6 30 minutes. Gel (B) Gel electrophoresis of RuꢀtapꢀNLS and pUC19 plasmid DNA (400 ng) in the presence of the singlet
oxygen scavenger sodium azide (5 %). Lane 1 pUC19 only. Lane 2 pUC19 + RuꢀtapꢀNLS No irradiation. Lane 3 30 seconds. Lane 4 2
minutes. Lane 5 10 minutes. Lane 6 20 minutes. Lane 7 30 minutes. Samples were irradiated using a 458 nm argon ion laser (280 mW),
and separated on a 1.2 % agarose gel.
clear excluding and hence under irradiation showed low
photocytotoxicity.
Conclusions
In summary, we exploited a nuclear localizing signal (NLS)
The interaction of the Ru-tap-NLS peptide conjugate was
explored with ctDNA and was found to bind strongly, driv-
en by electrostatic interactions with the cationic peptide.
Photoirradition of Ru-tap-NLS with plasmid DNA con-
firmed by gel electrophoresis that the peptide conjugate
induces singlet oxygen independent photocleavage in the
plasmid. This was taken to indicate that the photocytotoxi-
city observed in cellulo by the Ru-tap-NLS conjugate within
the nucleus may be occurring via direct DNA oxidation by
photoinduced electron transfer with guanine and possibly
also due to other factors such as adduct formation. This is
to our knowledge the first example of discrete targeting of
polyazaaromatic complexes to the cell nucleus. Such tar-
geted nuclear delivery is an important step toward the
application of such complexes in cellular phototherapy and
holds future implications for hypoxic treatments, PDT and
theranostics.
peptide sequence from nuclear factor-kappa B (NF-κB)
transcription to target a DNA photocleaving ruthenium bis
1,4,5,8-tetraazaphenanthrene complex specifically to the
nucleus of mammalian cell lines for the first time. The Ru-
tap-NLS peptide conjugate reliably enters and localizes
within the nuclei of living HeLa and CHO cells from where
it emits briefly before emission is extinguished. Emission
extinction is attributed to binding of the complex to nucle-
ar DNA where photoinduced electron transfer quenches
luminescence. Resonance Raman microscopy confirmed
that the complex was retained, localized in the nucleus
after its emission had switched off. Once in the nucleus
after emission had been extinguished, the complex was
highly photocytotoxic, consistent with quenching by elec-
tron transfer and individual cells could be selectively irra-
diated and destroyed whist surrounding cells were viable.
Conversely, in the absence of high intensity photoirradia-
tion the conjugate showed low cytotoxicity. Similarly,
where the same complex was introduced to cells but con-
jugated instead to the cell permeable peptide octa-
arginine, the complex reached the cytoplasm but was nu-
ASSOCIATED CONTENT
Supporting Information. Full synthesis and structural data,
additional photophysical characterization and cellular data.
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States for Metal–Organic Photodynamic Therapy: Verifica-
tion in a Metastatic Melanoma Model. J. Am. Chem. Soc. 2013,
135 (45), 17161–17175.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
tia.keyes@dcu.ie
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Author Contribution
Authors contributed equally to this work
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
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Based upon works supported by Science Foundation Ireland
under [14/IA/2488)] and [17/TIDA/4930]. The National Bio-
photonics and Imaging Platform, Ireland, was established
through funding received by the Irish Government’s PRTLI,
Cycle 4, and EU Structural Funds Programmes 2007−2013.
C.S.B. and T.E.K. gratefully acknowledge the Irish Research
Council for PhD scholarship funding. Professor R O’Kennedy,
DCU, is gratefully acknowledged for access to cell culture facil-
ities and gel electrophoresis apparatus.
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