Combination of ICP-MS, capillary electrophoresis, and their hyphenation for probing Ru(III) metallodrug–DNA interactions
Combination of ICP-MS, capillary electrophoresis, and their hyphenation for probing Ru(III) metallodrug-DNA interactions
Lidia S. Foteeva 0 1 2 3
Magdalena Matczuk 0 1 2 3
Katarzyna Pawlak 0 1 2 3
Svetlana S. Aleksenko 0 1 2 3
Sergey V. Nosenko 0 1 2 3
Vasily K. Karandashev 0 1 2 3
Maciej Jarosz 0 1 2 3
Andrei R. Timerbaev 0 1 2 3
0 Saratov State University , Astrakhanskaya St. 83, 410012 Saratov, Russian Federation
1 Chair of Analytical Chemistry, Faculty of Chemistry, Warsaw University of Technology , Noakowskiego St. 3, 00-664 Warsaw , Poland
2 Vernadsky Institute of Geochemistry and Analytical Chemistry , Kosygin St. 19, 119991 Moscow, Russian Federation
3 Institute of Microelectronics Technology and High-Purity Materials , Acad. Ossipyan St. 6, 142432 Chernologolovka, Russian Federation
Determination of the DNA-binding reactivity and affinity is an important part of a successful program for the selection of metallodrug candidates. For such assaying, a range of complementary analytical techniques was proposed and tested here using one of few anticancer metal-based drugs t h a t a r e c u r r e n t l y i n c l i n i c a l t r i a l s , i n d a z o l i u m trans-[tetrachloridobis(1H-indazole)ruthenate(III), and a DNA oligonucleotide. A high reactivity of the Ru drug was confirmed in affinity capillary electrophoresis (CE) mode, where adduct formation takes place in situ (i.e., in the capillary filled with an oligonucleotide-containing electrolyte). To further characterize the binding kinetics, a drug-oligonucleotide mixture was incubated for a different period of time, followed by ultrafiltration separation into two different in molecular weight fractions (>3 and <3 kDa). The time-dependent distribution profiles of the Ru drug were then assessed by CEinductively coupled plasma mass spectrometry (ICP-MS), revealing that at least two DNA adducts exist at equilibrium conditions. Using standalone ICP-MS, dominant equilibrium amount of the bound ruthenium was found to occur in a fraction of 5-10 kDa, which includes the oligonucleotide (ca. 6 kDa). Importantly, in all three assays, the drug was used for the first time in in-vitro studies, not in the intact form but as its active species released from the transferrin adduct at simulated cancer cytosolic conditions. This circumstance makes the established analytical platform promising to provide a detailed view on metallodrug targeting, including other possible biomolecules and ex vivo samples.
Anticancer metallodrugs; DNA; ICP-MS; Capillary electrophoresis
Anticancer metallodrugs seem to have a great many targets
such as DNA, proteins, membranes, etc. However, the true
lesion responsible for the biological activity of a drug is
difficult to determine. This is not only because metal complexes
are naturally diversely reactive species. There are at least two
additional obstacles. First, analytical techniques and tools
being used to map interactions with different pertinent
biomolecules are not always of the metallomic origin and, as such,
incapable to identify, characterize, and quantify metal species
stemmed from binding. Then the path taken by a metal
complex in reaching its target and accompanying changes in metal
speciation are typically downplayed.
In an attempt to resolve these challenges, the present study
was designed around an experimental anticancer
rutheniumbased drug, indazolium
trans-[tetrachloridobis(1Hindazole)ruthenate(III), and DNA, representing a fruitful
target for metal complexes [1–3]. The selection of this
metallodrug was governed not only by the advanced status
of its development [4, 5] but also by a great deal of
accumulated knowledge regarding the chemistry behind a drug’s
delivery, uptake, and cell processing, including activation and
targeting [6–9]. While DNA is thought to be not the main
target for this (as well as any other) Ru drug [10, 11], no other
cell component is unambiguously proven to play this role. To
simplify the metal–bio system under scrutiny, a DNA
oligonucleotide rich of GMP, the major nucleotide binding
metallodrugs (including the ruthenium drug of interest [12,
13]), was employed in binding experiments (as a DNA
sequence simpler than the more heterogeneous genomic DNA).
In a basic metallomic approach, a highly sensitive
metalspecific method, inductively coupled plasma mass
spectrometry (ICP-MS), is used in on-line combination with a
highthroughput separation technique. Capillary electrophoresis
(CE) constitutes a powerful method to resolve mixtures of
metallodrugs, their metabolites, and adducts with
biomolecules, including DNA binding blocks (such as GMP [12,
13]), prior to ICP-MS detection [14–16]. In situations where
more accurate binding information is requested, ICP-MS
quantification following fractionation of a drug–biomolecule
mixture by ultrafiltration is preferable  (though at the
expense of on-line configuration). Whichever individual
metallomic (or complementary) technique is in use, their
combined application would help gain a deeper understanding of
biotransformations for a given complex. This strategy was
adopted here to develop a versatile analytical platform for
characterization of different metallodrug–DNA systems.
Materials and methods
The lyophilized powder of DNA oligonucleotide (5'- GTC
GTA CTG ATA CAT GAG CC –'3; 6117 Da) was the product
of Genomed (Warsaw, Poland) or Syntol (Moscow, Russia). It
was used as an aqueous solution prepared in accordance with
the producer recommendations and stored at –20 °C no longer
than 1 month. Indazolium
trans-[tetrachloridobis(1Hindazole)ruthenate(III)] was synthesized at the University of
Vienna (see the Electronic Supplementary Material (ESM) for
chemical formula). The stock solution of the Ru drug (1 mM)
was prepared daily in 100 mM NaCl. Human transferrin
(>98%), glutathione (>98%), and L-ascorbic acid (>99.5%)
were purchased from Sigma-Aldrich (St. Louis, USA).
Sodium chloride, sodium dihydrogen phosphate, disodium
hydrogen phosphate, also from Sigma-Aldrich (St. Louis,
USA), were of analytical reagent grade. Sodium hydroxide
and citric acid (>99.5%) were obtained from Fluka (Buchs,
Switzerland). High-purity water (18 MΩ cm–1) used
throughout this work was obtained from a Milli-Q water purification
system (Millipore Elix 3; Saint-Quentin, France).
A X-7 Thermo Elemental and an Agilent 7500a mass spec
trometers were used for off-line and hyphenated ICP-MS
measurements, respectively. In case of standalone
operation, the instrument was equipped with a standard
lowvolume glass spray chamber (Peltier, cooled at 3 °C), and
a concentric glass PolyCon nebulizer operating at a sample
uptake rate of 0.8 mL min–1. Before analysis, the instrument
was tuned to achieve maximum sensitivity and 115In served
as internal standard. For coupling to CE, a torch with a
smaller inlet (1.5 mm) was utilized to minimize the
influence of plasma backpressure on the electrophoretic flow,
and a platinum shield was installed into the torch to improve
sensitivity. Fitted with a microconcentric nebulizer CEI-100
(CETAC, Omaha, NE, USA), the mass spectrometer was
interfaced with an Agilent HP3D CE system (Waldbronn,
Germany). Affinity CE was performed using a CAPEL
105 (Lumex, St. Petersburg, Russia).
Fused-silica capillaries of inner diameter of 75 μm and total
length as specified below were obtained from CM Scientific
Ltd. (Silsden, UK) or BGB Analytik (Schlossboeckelheim,
Germany). Prior to the first use, the capillary was flushed at
1 bar with 1 M NaOH and water (30 min each). The capillary
cassette and sample tray were thermostatted at 37 °C. Samples
were introduced into the capillary hydrodynamically. A high
voltage with a positive polarity placed at the inlet end of the
capillary was applied to generate separations. Instrument
control and data analysis were performed using ChemStation
(Agilent) or Elforan (Lumex) software. The main instrumental
and operational parameters are presented in Table 1.
A Bandelin Sonorex ultrasonic bath (model 1210;
Walldorf, Germany) was used for degassing the solutions.
Samples were incubated at 37 °C in a WB 22 (Memmert,
S c h w a b a c h , G e r m a n y ) o r a T C - 1 5 0 ( B r o o k f i e l d ,
Middleboro, USA) thermostat. For ultrafiltration experiments,
a MPW-350R (JW Electronic, Warsaw, Poland) or a
CM50 M (ELMI Ltd., Riga, Latvia), operating at 10,000 and
1000–10,000 rpm, respectively, and different molecular mass
cut-off filters (Amicon Ultracel; Millipore, Molsheim, France)
A workflow template for sample handling is presented in
Fig. 1. An aliquot of drug stock solution was mixed with
5 × 10–5 M solution of transferrin in 10 mM phosphate
buffer, pH 7.4, containing 100 mM NaCl, to give a 2-fold molar
excess of the drug over the protein in the final solution. The
resultant mixture was incubated for 3 h at 37 °C to ensure
complete adduct formation  and then ultrafiltrated
through a 10 kDa cut-off filter for 30 min (37 °C) to isolate
the adduct. After reverse ultrafiltration, the adduct solution
Operational parameters and settings
Capillary Fused-silica, inner diameter
75 μm, length 70 cm
BGE 10 mM NaH2PO4–10 mM
Na2HPO4, 4 mM NaCl, pH 6.0
Sample introduction 30 mbar for 10 s (injection volume
Voltage 25 kV
Current 28–32 μA
Sheath liquid 1 mM phosphate buffer (pH 6.0),
0.4 mM NaCl, 20 μg L–1 Ge
Fused-silica, inner diameter
75 μm, length 60/50.5 cm
BGE 10 mM NaH2PO4–10 mM
Na2HPO4, 4 mM NaCl, pH 6.0,
2 × 10–5 M oligonucleotide
Sample introduction 10 mbar for 5 s (4 nL)
Voltage 10 kV
Current 47 μA
(~30 μL) was mixed with a solution mimicking cancer cell
cytosol, to give a final adduct concentration of 5 × 10–5 M.
The cytosol solution comprised 10 mM phosphate buffer
(pH 6.0), 4 mM NaCl, glutathione (10 mM), ascorbic acid
(10 mM), and citric acid (100 mM). The mixture was
incubated for 24 h and then ultrafiltrated through a 10 kDa filter
for 40 min. The ultrafiltrate was introduced into the
capillary filled with 10 mM phosphate buffer (pH 6.0),
containing 4 mM NaCl and 2 × 10–7–2 × 10–5 M DNA
oligonucleotide (ACE), or mixed with the DNA oligonucleotide
solution (final concentration of oligonucleotide 2 × 10–6 M).
The mixture was incubated at 37 °C and aliquots were taken
at a specified time (only after 24 h for ICP-MS) for
ultrafiltration (40 min). A consecutive ultrafiltration with different
combinations of filters was then performed. In the case of
CE-ICP-MS, the initial ultrafiltrate was filtrated through a
30 kDa filter and the obtained ultrafiltrate through a 3 kDa
filter, and both fractions (3–30 and <3 kDa) were analyzed.
For off-line ICP-MS, the ultrafiltration sequence was a
three-step in an order of 10, 5, and 3 kDa filter, following
an appropriate dilution of Ru–DNA mixture with 2.5%
nitric acid, so that the four fractions were subject to analysis.
Results and discussion
Intracellular activation of ruthenium drug
As a matter fact, among drug developers there is still no
consensus regarding the mechanism of uptake and activation of
the ruthenium drug of interest [10, 11]. According to the most
often quoted concept of transportation, the drug enters the cell
via transferrin route, being bound to this protein (though not
the main binding partner in the bloodstream). The second
popular hypothesis implies that within cancer cell the drug is
activated to more reactive species, most likely by detaching
transferrin and reduction to some Ru(II) species. A recent
systematic study carried out in our laboratories focused on
intracellular activation chemistry of a given Ru drug,
including cancer cytosol environment [18–20]. Using a
multidimensional metallomic approach, the evolution of a number of low
molecular weight (MW) Ru species was ascertained, in which
glutathione and ascorbic acid as the major bioreductants have
a dominant impact on the drug–transferrin adduct. While the
identification of ruthenium species bears so far a tentative
character, this is not a constraint for the current investigation.
Therefore, for consistency we have used here the same adduct
formation and activation protocol as before (see Sample
When affinity CE (ACE) is the method of choice, chemical
processes comprising a metallodrug (as well as any other
charged or under certain conditions, uncharged species) can
be differentiated kinetically with regard to the time scale of
a typical CE run (tens of minutes) [21, 22]. Rather fast
reactions between the drug and a selected component of the
background electrolyte (e.g., a bioligand) essentially attain
the equilibrium during the time for which the drug stays in
the capillary (more strictly speaking, is transported past the
detector), giving rise to a binding response. With direct
photometric detection, it can be displayed as two novel
signals: a peak of the drug–bioligand adduct and a dip-peak
attributable to a drop in the bioligand concentration (for a
detailed view of all possible response scenarios, see ESM,
Fig. S1). Otherwise, for fairly slow binding processes, one
can expect no binding response (but only the free drug
peak). Notably, such reactions are not discriminated against
the CE assaying but passed to the realm of ordinary CE
Formation of drug–transferrin adduct
Adduct decomposition and release of active Ru species
Separation of the released Ru species
Formation of Ru–DNA adducts
Separation of the adducts and unbound Ru species
Fig. 1 Analytical protocol for formation, isolation, and three-dimensional analysis of Ru–DNA species
mode (using incubation of the reaction mixture for a certain
period of time prior to analysis) .
It is important to point out that ACE analysis of chemical
equilibria in metallodrug–bioligand systems has not yet
become an accepted practice. Binding to proteins is known to
proceed with comparatively slow kinetics (see  for a
summary of kinetic data). For DNA, no published accounts on its
interaction with metal-based drugs assessed by ACE can be
traced in the literature. However, there are at least two reports
by the group of Kane-Maguire and Wheeler [23, 24]
indicative of that interactions between the metal complexes and
DNA may be fast enough to exhibit a binding response in
ACE. When DNA has been used in the electrophoretic buffer,
resolution of the transition metal complexes into their optical
isomers was observed. Such enantioselective effect can be
explained by differences in isomer binding affinity toward
In order to simulate cancer cytosol electrolyte conditions,
10 mM phosphate buffer, pH 6.0, containing 4 mM NaCl, was
chosen as a background electrolyte milieu. (We leave beyond the
scope of this study the issue on the exact point where in-vivo
interaction between active drug forms and DNA occurs, and
cellular electrolyte settings at this point.) The DNA
oligonucleotide concentration in the electrolyte was increased stepwise from
2 × 10–7 to 2 × 10–5 M. As the main parameter affecting analyte
migration velocity and, inversely, the capillary residence (or
reaction) time, the applied voltage was also subject to
optimization. As a result of systematic variations in the range from 6 to
15 kV, a voltage of 10 kV, providing the optimal migration time
and resolution, minimum peak broadening, acceptable running
current, and also a lack of system peaks, was selected.
Upon injection of active drug forms (prepared as described
above), there was no binding response at the lowest
concentration of DNA, while certain changes were noticed to occur at 2 ×
10–6 M; however, peak shape, intensity, and reproducibility were
unsatisfactory. On the other hand, when using a 2 × 10–5 M
DNA-containing electrolyte, the ACE system yielded a
reproducible binding response (Fig. 2, trace C). It should be mentioned
that without incorporating DNA, two well-resolved peaks of
negatively charged Ru species were recorded (Fig. 2, trace B).
This finding is in accord with our previous results , showing
the occurrence of two active drug forms released from the
transferrin adduct in the cytosol-like solution (see also CE-ICP-MS
data below). Comparing traces B and C, it can be inferred that an
additional peak, seen in trace C, is due to an DNA-binding
product evolved in a ca. 20-min timespan.
To gain further insight into the binding phenomenon, the
active forms of the Ru drug were pre-incubated with
oligonucleotide (up to 48 h) and subsequently analyzed by CE-ICP-MS
Fig. 2 Electropherograms proving Ru–DNA binding. Sample: A – water
(blank analysis); B, C – drug (in active form). Concentration of DNA in
electrolyte: A, C – 2 × 10–5 M; B – zero. EOF = electroosmotic flow.
Other ACE conditions, see Table 1
Fig. 3 Various ruthenium species constituting drug’s active forms and
originating from their 24-h interaction with DNA oligonucleotide. Trace
C resulted from blank analysis (without oligonucleotide added). For CE
and ICP-MS conditions, see Table 1
(see experimental for ultrafiltration conditions). The same
electrolyte buffer system was used in these trials as in ACE
but void of oligonucleotide. The prerequisite of analyzing the
two fractions as exemplified in Fig. 3, was that in fraction B
(3–30 kDa) we expected to detect the signals of
Ru–oligonucleotide adducts, while at <3 kDa in fraction A the low MW
species of Ru could be monitored.
From Fig. 3 it is evident that at equilibrium conditions
about 90% Ru are converted into high MW species (for
comparative reasons, Fig. S2 in the ESM shows results of the
CEICP-MS analysis of the same fractions after a shorter
incubation time; note that one anionic form of Ru is still detectable).
Apparently these species comprise at least two
oligonucleotide adducts migrating as the overlapping peaks. It is
important to note that in the studies on modeling physiological
conditions, the selection of CE system parameters capable to
enhance the resolution is typically restricted to very few
variables, such as the applied voltage in our case. With regard to
notable peak broadening, this was no great surprise as DNA
oligonucleotides are known to adsorb onto the fused-silica
capillary wall .
As shown in Fig. 3, even after prolonged incubation, a low
MW species of Ru remains unattached to oligonucleotide and,
importantly, migrates ahead of the EOF (i.e., in the migration
Table 2 Results of the ICP-MS
analysis (n = 3; P = 0.95)
Concentration of Ru (×10–6 M)
a LOQ: limit of quantification (1.2 × 10–9 M).
range of positively charged analytes. Very likely the respective
peak seen in trace A is due to [RuIIHind(OH)(H2O)3]+
(Hind = 1H-indazole) identified as one of few (and the only
cationic) ruthenium form released from the transferrin adduct
under simulated cancer cytosol conditions  (cf., trace C).
The fact that fraction A contains no negatively charged species
can be inferred that non-hydrolyzed species, such as
[RuIIHindCl4(GSH)]2– and [RuIIHindCl4(GSSG)]2–  (see
two co-migrating peaks in trace C recorded in the absence of
DNA), tend to interact more strongly with DNA.
It is useful to note that although CE-ICP-MS proved to be
highly practicable as a speciation tool , the technique can
be insufficiently sensitive when dealing with metal–bioligand
species in real samples . Even though this is not the case of
the present study, we considered direct ICP-MS analysis to be
offering viable supplementary information since through a
series of ultrafiltration steps the content of the Ru species could
fall beyond the limit of detection of CE-ICP-MS.
Analytical measurements of ruthenium originating from its
drugs can be hampered by interfering m/z signals. A detailed
investigation by Brouwers et al. showed, however, that neither
ultrafiltrates, being free of proteins, nor the diluents, such as 1%
nitric acid, but biological matrices cause a problem of interfering
peaks at the mass-to-charge ratios of ruthenium . Therefore,
we have limited validation experiments on evaluation of
unresolved spectral interferences to a blank (drug-free) analysis, as
well as the analysis of DNA nucleotide as the only
noncommercial chemical used. Both gave negligible interfering
signals (≤4 cps). As an additional proof of no unanticipated spectral
interferences, matching results were obtained by measuring
101Ru and 102Ru isotopes for all samples shown in Table 2.
On the other hand, it was deemed obligatory to verify that
all the sample preparation steps, particularly ultrafiltration,
made no substantial analyte loss (e.g., associated with the
sorption onto filter membrane or a plastic device). In the
ultrafiltrate obtained after the treatment of the transferrin adduct
with simulated cancer-cytosol solution, the Ru concentration
was found within 10% of the nominal value (based on initial
drug amount), indicating the satisfying recovery.
Table 2 gives a summary on the ruthenium distribution
between fractions different in MW. As expected, the highest
Ru level was found in the fraction bracketing the MW of DNA
nucleotide (ca. 6 kDa). The amount of free ruthenium relative
to the total amount of high MW Ru species conforms to the
CE-ICP-MS data. What was less predictable, a rather high
concentration of Ru exists in the fraction with the MW over
10 kDa. The most plausible explanation is that this is
attributed to the formation of bis-DNA adducts.
In summary, the present work demonstrates a proof of
principle for investigating the interaction of metal-based drugs with
biological targets. With the integrated use of CE and ICP-MS
techniques, the formation of adducts between the
experimental Ru(III) drug (importantly, as its active species to be
possibly released in cancer cytosol) and DNA oligonucleotide has
been confirmed. However, the binding system utilized to
illustrate this platform is quite provisional, as the drug of choice
is believed to primarily attack other biostructures inside the
cancer cell (e.g., proteins like GRP-78), while the DNA model
system selected takes into account only some elements of the
supposed in vivo situation. Yet the binding information
acquired suggests that the analytical approach described above
may indeed be useful for assessing the intracellular fate of
metallodrugs. Subsequent efforts to use this methodology will
focus on different drug–biomolecule systems under
circumstances matching as much as possible a real-world situation.
Acknowledgements The authors gratefully acknowledge the financial
support of Warsaw University of Technology and the Russian Foundation
for Basic Research (grant no. 16-33-60230). M.M. is thankful to Łukasz
Górski for donation of DNA oligonucleotide.
Compliance with ethical standards
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