Nuclear Targeting with an Auger Electron Emitter Potentiates the Action of a Widely Used Antineoplastic Drug

Article abstract of DOI:10.1021/acs.bioconjchem.5b00466

We present the combination of the clinically well-proven chemotherapeutic agent, Doxorubicin, and ^99mTc, an Auger and internal conversion electron emitter, into a dual-action agent for therapy. Chemical conjugation of Doxorubicin to ^99mTc afforded a construct which autonomously ferries a radioactive payload into the cell nucleus. At this site, damage is exerted by dose deposition from Auger radiation. The ^99mTc-conjugate exhibited a dose-dependent inhibition of survival in a selected panel of cancer cells and an in vivo study in healthy mice evidenced a biodistribution which is comparable to that of the parent drug. The homologous Rhenium conjugate was found to effectively bind to DNA, inhibited human Topoisomerase II, and exhibited cytotoxicity in vitro. The collective in vitro and in vivo data demonstrate that the presented metallo-conjugates closely mimic native Doxorubicin.

Full text of DOI:10.1021/acs.bioconjchem.5b00466

Article
pubs.acs.org/bc
Nuclear Targeting with an Auger Electron Emitter Potentiates the
Action of a Widely Used Antineoplastic Drug
Sebastian Imstepf,† Vanessa Pierroz,†,‡ Paula Raposinho,§ Matthias Bauwens,∥ Michael Felber,†
Thomas Fox,† Adam B. Shapiro,⊥ Robert Freudenberg,# Celia Fernandes,§ Sofia Gama,§ Gilles Gasser,†
́
Felix Motthagy,∥ Isabel R. Santos,§ and Roger Alberto*,†
‡
†Department of Chemistry and Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057
Zurich, Switzerland
§Centro de Cien
̂ ́
cias e Tecnologias Nucleares, Instituto Superior Tecnico, Universidade de Lisboa, Estrada Nacional 10 km 139.7,
PT-2695-066 Bobadela LRS, Portugal
∥Department of Nuclear Medicine, MUMC+, P. Debeyelaan 25, NL-6229 Maastricht, Netherlands
⊥Bioscience Department, Infection Innovative Medicines, AstraZeneca R&D Boston, Waltham, Massachusetts 02451, United States
#Universitatsklinikum Carl Gustav Carus Dresden, Fetscherstrasse 74, D-01307 Dresden, Germany
̈
S
* Supporting Information
ABSTRACT: We present the combination of the clinically well-proven
chemotherapeutic agent, Doxorubicin, and 99mTc, an Auger and internal
conversion electron emitter, into a dual-action agent for therapy.
Chemical conjugation of Doxorubicin to 99mTc afforded a construct
which autonomously ferries a radioactive payload into the cell nucleus. At
this site, damage is exerted by dose deposition from Auger radiation. The
99mTc-conjugate exhibited a dose-dependent inhibition of survival in a
selected panel of cancer cells and an in vivo study in healthy mice
evidenced a biodistribution which is comparable to that of the parent
drug. The homologous Rhenium conjugate was found to effectively bind
to DNA, inhibited human Topoisomerase II, and exhibited cytotoxicity in
vitro. The collective in vitro and in vivo data demonstrate that the presented metallo-conjugates closely mimic native
Doxorubicin.
to show any effect. Since nuclear DNA is the primary
radiosensitive target, Auger emitters must accumulate in the
cell nucleus and integrate in or attach to the double helix to
inflict radiation damage in the form of irreparable double strand
breaks (DSBs). In previous studies, conjugation of Auger
emitters to nucleobases,3−6 (anti-)hormones,7,8 or intercala-
tors9 effected this damage. Reviews by Hofer10 and Kassis11
comprehensively describe the biophysical aspects of Auger
emitters.
INTRODUCTION
■
Auger and internal conversion electron therapy is a particular
form of radionuclide therapy (RNT), which uses low energy
electrons from radionuclides to destroy malignant cells.
Importantly, the delivery of therapeutic radiation doses in
Auger electron therapy occurs over molecular to subcellular
dimensions. The path lengths of Auger electrons contrast those
of α- and β-particles, which deposit their energy in the form of
evenly distributed ionizations over submillimeter to millimeter
ranges, thereby exposing multiple cells along their tracks to
radiationa phenomenon referred to as the “cross-fire effect”.1
As tissue has a particularly high stopping power for the low
energies carried by Auger electrons (eV to low keV range),
their total energy is deposited along ultrashort particle tracks.
This large linear energy transfer (LET) causes ionizations and
electron showers which lead to massive radiation damage along
path lengths of subcellular dimensions. Hence, Auger and
internal conversion electrons exhibit a relative biological
effectiveness (RBE) similar to that of heavy charged particles.2
Vice versa, these ultrashort trajectories have implications
regarding the application of these emitters as decay events
must take place right at the target (or in its immediate vicinity)
99mTc is a widely used radionuclide in nuclear medical
SPECT imaging with a half-life time of 6 h.12,13 Besides the
emission of a 140 keV γ-quant (yield = 89%), it concomitantly
ejects an average of four Auger electrons and ∼0.11 internal
conversion electrons per decay. The Auger electrons of 99mTc
with the highest theoretical probability of inducing DSBs in
DNA are shown in Table 1.14
Earlier reports experimentally confirmed the potential of
99mTc for Auger electron therapy, in cell free systems,15,16 and
Received: August 25, 2015
Revised: October 9, 2015
© XXXX American Chemical Society
A
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imaging with a small animal microSPECT and an ex vivo
biodistribution study in nude NMRI mice were carried out to
elucidate the behavior of radioactive complex 1 in vivo.
Table 1. Average Energy, Yield/Decay and Tissue
Penetration Range of the Most Probable Electrons Emitted
by 99mTc for the Induction of DSBs
average energy [eV]
yield per decay
tissue penetration range [nm]
RESULTS AND DISCUSSION
■
116
226
33
0.75
1.1
6
10.5
2
Design and Synthesis of the ADR-Conjugates.
Conjugates 1 and 2 (Scheme 1) were designed according to
the bifunctional chelator (BFC) concept:43 The biological
vector (ADR) is conjugated via a linker to the dipicolylamine
chelator, which binds strongly to the fac-{M(CO)3}+ core (M =
Re/99mTc). ADR, a strong intercalator, was chosen to spatially
fix the radionuclide to DNA in order for the decay to induce
DSBs via energy transfer by emitted Auger and internal
conversion electrons. The positive charge on the complex
should induce ionic interactions with the negatively charged
phosphate backbone to bring the radionuclide close to DNA.
Chelator C1 was synthesized by reductive amination of ethyl
4-aminobutyrate hydrochloride with an excess of picolinalde-
hyde and sodium triacetoxyborohydride (STAB) as reducing
agent.44 After basic hydrolysis of C1-OEt, bifunctional ligand
L1 was obtained in 44% overall yield (3 steps, SI Charts S1−
S3), by a HBTU mediated amide bond formation with
commercially available ADR·HCl. L1 was fully characterized
by detailed NMR studies (see Charts S4−S6) and high-
resolution mass spectrometry (HR-ESI+).
1.98
in vitro, employing intercalators such as pyrene17 or anthracene
derivatives.18
Doxorubicin (ADR), a natural, broadband chemotherapeutic
agent, is an anthracene derivative, known to intercalate strongly
between DNA nucleobases.19−21 ADR is clinically well-proven
and shows extensive activity against a variety of cancers.22−24 Its
mode of action is ascribed to the inhibition of Topoisomerase
II and to the resulting damage during DNA replication,
inducing apoptosis, the programmed form of cell death.25 Like
many anticancer drugs, ADR causes serious off-target adverse
effects; immunosuppression hampers the quality of life of
patients and the dose dependent, acute cardiotoxicity greatly
reduces therapeutic margins.26,27 To improve the efficacy of
ADR, strategies to enhance delivery and reduce toxicity have
been exploited, e.g., by delivery with (pegylated) lip-
osomes28−30 or in hydrogels,31,32 derivatization of nano-
particles,33−37 and direct administration of modified ADR
derivatives38 or prodrugs.39−42
Inspired by the clinical relevance of ADR, we present in this
study the first well characterized Doxorubicin−99mTc conjugate
(1, Scheme 1) which was scrutinized for radiotoxic effects in
multiple cancer cell lines. In situ, the characterized “cold”
rhenium surrogate 2 was found to bind to DNA and to inhibit
human Topoisomerase II α and β. Furthermore, the model Re
complex retained cytotoxicity toward a human cancer cell line
in vitro. Cellular localization and nuclear targeting were studied
with confocal fluorescence microscopy, and the subcellular
distribution of the ADR-conjugates was corroborated by two
individual quantification methods: radioactivity and inductively
coupled plasma mass spectrometry (ICP-MS). Moreover,
For characterization of 99mTc products at the tracer level,
coinjection with the “cold” rhenium homologue is an accepted
procedure for the unambiguous authentication of a new
radioproduct. Consequently, rhenium complex 2 was prepared
by reacting L1 with the standard precursor (NE-
t4)2[ReBr3(CO)3] in MeOH. Temperatures above 60 °C and
prolonged heating led to methanolysis of the α-glycosidic bond,
resulting in the ADR-aglycone (Doxorubicinone) and meta-
lated daunosamine sugar moiety (Scheme 2). Both degradation
products and their respective masses were identified via UPLC-
ESI+ (see Chart S7). After optimization of the conditions, 2 was
obtained in 63% yield after purification and fully characterized
by NMR studies (see Charts S8−S12).
a
Scheme 1. Synthesis of ADR Conjugates 1 (M = 99mTc) and 2 (M = Re)
a
(i) 2-Pyridinecarboxaldehyde, STAB, TEA, CH2Cl2, 25 °C, 86%; (ii) 0.1 M NaOH, H2O, 85 °C, 84%; (iii) ADR·HCl, HBTU, DIPEA, DMF, 25
°C, 61%; (iv) 1: [99mTc(OH2)3(CO)3]+, H2O, 60 °C, 70% radiochemical yield (RCY), 2: (NEt4)2[ReBr3(CO)3], MeOH, 60 °C, 63%. STAB =
sodium triacetoxyborohydride, TEA = triethylamine, DIPEA = N,N-diisopropylethylamine, HBTU = N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-
yl)uronium hexafluorophosphate, DMF = N,N-dimethylformamide.
B
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Scheme 2. Solvolysis of L1 during the Synthesis of Conjugates 1 (M = 99mTc) and 2 (M = Re) to the ADR Aglycone
(Doxorubicinone) and the Metalated Daunosamine Sugar by Coordination to fac-[M(CO)3]+ (M = 99mTc/Re) in MeOH or
H2O
The α-glycosidic bond is indeed the most sensitive part of
ADR, and cleavage of the daunosamine sugar is part of the
metabolic pathway of the parent drug.24,45,46 In vivo, this
hydrolysis is proton-catalyzed, whereas under our reaction
conditions, the Lewis-acidic fac-{Re(CO)3}+ fragment is likely
to cause methanolysis. We note that labeling experiments,
under fully aqueous conditions, with 99mTc showed a similar
behavior (vide infra). Metal derivatives of ADR are relatively
scarce but have been reported for Fe(III),47−50 Mn(II),51
Cu(II),52 Pt(II),53 Co(II),54 and Pd(II).55 Most of these
complexes, however, are formed in situ and are poorly
characterized. Complex 2 is, to the best of our knowledge,
the first Doxorubicin−metal conjugate.
During labeling of L1 with the fac-{99mTc(CO)3}+ core, a
significantly more hydrophilic side product (yield ∼10−15%)
was observed at elevated temperatures. By analyzing the
aqueous degradation products of surrogate 2 and coinjection,
we attributed this impurity to the labeled daunosamine-chelator
Figure 1. Normalized HPLC coinjection after purification. Top: γ-
trace of 1 (19.72 min, RCP > 98% as determined by integration).
Bottom: UV-trace of rhenium complex 2 (19.41 min).
construct (see Scheme 2). Since most unlabeled L1 remained
intact, the side product likely forms at the tracer level by α-
glycosidic bond cleavage of 1. Side product formation was
emitters located at increasing distances from the DNA central
axis.9,57,58 Since ADR and related anthracycline drugs are strong
suppressed to some extent by acidic quenching of the kit-
intercalators,21 incorporation of this targeting moiety in
contained sodium boranocarbonate and subsequent readjusting
of the [99mTc(OH2)3(CO)3]+ precursor solution to pH 6−7
before labeling of L1. The pH window to label ADR in aqueous
media is narrow: Below pH 6, glycosidic cleavage is promoted
and above pH 7, L1 precipitated from the aqueous solution.
Under optimized conditions, conjugate 1 was obtained in
excellent turnovers from [99mTc(OH2)3(CO)3]+ and in a
radiochemical purity (RCP) of around 85−90% (see Chart
S13). For biological evaluations, the crude reaction mixtures
were purified by HPLC to afford a RCP ≥ 95%. Co-injection
and comparison of the HPLC retention time with homologue 2
confirmed the identity of 99mTc-complex 1 (Figure 1, difference
due to detector separation). Of note, an attempt to directly
radiolabel ADR (no pendent chelator) with 99mTc has been
reported before by stannous(II) chloride reduction of
compound 1 should afford 99mTc decays in the intimate
proximity of DNA, resulting in an efficient energy transfer. The
binding affinity of 2 for double-stranded DNA is therefore a
decisive parameter. Since ADR’s fluorescence is quenched upon
intercalation, the changes in the emission spectrum of complex
2 were monitored upon titration with calf-thymus DNA
(ctDNA).59 The spectroscopic data allowed Fbound (fraction
of intercalator bound to DNA) to be calculated and was fitted
to the equation by Bard et al. to obtain the binding constant
(Kb) and the size of the interaction site (s).60,61 The Kb and s
values for 2 are reported in Table 2 along with free ADR as a
reference (see also Charts S14 and S15). The initial
fluorescence of derivative 2 in PBS (pH 7.4) is maximal in
the absence of ctDNA and is strongly quenched at increasing
DNA concentrations. No shift in the emission maximum (593
nm) was observed. Evidently, the DNA binding constant of 2 is
one order of magnitude smaller than that of free ADR. We
attribute this decrease to the derivatization at the daunosamine-
NH2, which is known to be critical for DNA interaction.62
Nevertheless, the value obtained indicates a strong interaction
of 2 with DNA.
[
99mTcO4]− in the presence of ADR. Yet, authenticity of the
product has not been determined.56
Biological Data. To establish an in situ and in vitro profile,
the cold rhenium conjugate 2 was subjected to a variety of
biological assays the results of which are summarized in Table
2.
Interaction with DNA. DNA is the primary target of Auger
radiation. Hence, the radionuclide needs to sojourn closely
around DNA over the time of its decay, as theoretical
calculations indicated precipitous dose reductions for Auger
Inhibition of Cellular Proliferation (Chemotoxicity)
and Topoisomerase II. The chemotoxicity of ADR-conjugate
2 was investigated by a fluorometric cell viability assay
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Table 2. In Situ and In Vitro Biological Data of Rhenium Complex 2 and ADR
a
b
c
DNA binding
Kb (M−1 per nucl) × 106
Human Topoisomerase II inhibition IC50
Cytotoxicity after 48 h IC50
s (bp)*
n/a
hTOPOII α (μM)
hTOPOII β (μM)
HeLa (μM)
B16F1 (μM)
Cisplatin
ADR
2
n/a
n/a
n/a
9.6 1.1
0.093 0.02
19.7 2.1
20.6 1.6
0.095 0.01
11.4 4.9**
4.98 0.45
0.23 0.03
2.00 0.04
2.20 0.15
1.6 0.2
1.1 0.02
4.5 0.6
9.7 1.2
a
b
Kb: affinity constant for DNA; s: binding site size; *bp: base pairs. Values indicate means standard deviations of triplicate experiments made for
the enzymes side by side. Values indicate means standard deviations of triplicate experiments; cisplatin was used as internal reference; **duplicate
c
experiment only.
(Resazurin). The IC50 of complex 2 toward the standard human
cervical cancer (HeLa) cell line was determined to be 19.7 μM
after 48 h of incubation (Table 2). This inhibitory potency is
significantly reduced compared to native ADR which exhibited
an IC50 in the high nanomolar range. It is a known
phenomenon that amine group derivatization reduces cytotox-
icity, which is currently under investigation for new drug
delivery mechanisms via bio-orthogonal prodrug activation.41,63
On the other hand, the IC50 of 2 was determined to be in the
same range as cisplatin (internal reference), exemplifying that
the metal-conjugate retains a potentially useful cytotoxicity in
vitro. Of note, in recent years fac-{Re(CO)3}+-complexes were
shown to have interesting properties as anticancer agents.64−67
With optimizations, ADR could indeed be a lead structure for
inorganic drugs or delivery thereof, possibly exploiting new
cytotoxic mechanisms.
While different mechanisms of action are discussed in the
Figure 2. Confocal fluorescence microscopy of ADR conjugate 2 (60
μM) in HeLa cells after 2 h incubation. Left: ADR complex
fluorescence (excitation at 488 nm, emission above 600 nm), center:
literature, it is accepted that ADR is a strong inhibitor of human
topoisomerase II (hTopoII).68 It was hence of interest to
investigate if 2 would follow the same mechanism, despite the
chemical derivatization of the parent pharmacophore. Accord-
ingly, the ability of 2 to inhibit hTopoII α and β was
determined by the method of Shapiro et al.69,70 This assay
exploits the preferred binding of a fluorescently labeled
oligonucleotide (TTC)3T to double-stranded plasmid DNA,
containing the triplex forming sequence (TTC)9, after
relaxation by hTopoII α or β versus the supercoiled plasmid.
Changes in fluorescence anisotropy were monitored in the
presence of inhibitor 2 at increasing concentrations to calculate
the %-inhibition. The IC50 was determined by nonlinear least-
squares fit of the data points to an adaptation of the Hill
equation. SI Chart S16 shows the concentration-dependent
inhibition of hTopoII α and β by complex 2. As indicated in
Table 2, ADR-conjugate 2 inhibits hTopoII α and β in
concentrations comparable to the parent drug, and the slight
selectivity of inhibiting hTopoII β more effectively than the α
isoform is equally conserved. The marginally reduced inhibitory
potency of 2 in comparison to free ADR is consistent with the
reduced cytotoxicity of 2 in HeLa cells compared to the parent
drug (vide supra).
Cellular Localization and Distribution. Effective nuclear
accumulation is crucial for an Auger emitter since only decays
in the nucleus are radiotoxic.71−74 ADR’s fluorescence proper-
ties (λabs = 488 nm, λem = 592 nm) allow for determination of
cellular distribution and localization of 2 by confocal
microscopy. Figure 2 displays the fluorescence distribution of
2 (60 μM) after 2 h of incubation in HeLa cells. Fluorescence
was observed in the cytoplasm and in the perinuclear region.
No nuclear accumulation of fluorescence could be detected
while reference experiments with free ADR exhibited the red
fluorescence exclusively in the nucleus (see Chart S17). Closer
scrutiny of the images revealed a weak fluorescence signal in the
DAPI nuclear stain, right: merge of complex 2 and DAPI staining.
DAPI = 4′,6-Diamidin-2-phenylindol. White arrows indicate fluo-
rescence in nucleoli.
nucleoli (white arrows in Figure 2), indicating that conjugate 2
crossed the nuclear membrane. Since nucleoli contain RNA, we
hypothesized that no intercalation takes place in these
organelles and fluorescence is not quenched. Conversely,
DNA intercalation may completely quench the fluorescence
in the nucleus if 2 is fully intercalated. According to the
literature and in our own experience (see Chart S14), ADR’s
fluorescent properties indeed depend on their microenviron-
ment.75 Thus, fluorescence microscopy might not always
display the accurate cellular distribution of compounds, which
dynamically alter their luminescence properties according to
their surroundings. A clear picture can only be obtained by a
complementary analytical technique such as determination of
the radioactivity inside cellular compartments, which allows for
a very sensitive quantification of metal traces.
A small panel of cancer cell lines, namely, human cervix
carcinoma (HeLa), human squamous carcinoma (A431), and
murine melanoma (B16F1), were incubated with radio-
conjugate 1. After removal of unbound compound by washing
with PBS, nuclei and mitochondria were isolated from whole
cells. The radioactivity in the nucleus and mitochondria pellets
was then determined by a dose calibrator and expressed relative
(%) to the activity in the whole cell pellet. In contrast to the
results of fluorescence microscopy, these experiments showed
that 99mTc complex 1 accumulates mainly in the nuclei (∼75−
80%) and to a minor extent in mitochondria (∼1−2%).
Moreover, no significant differences in the distribution of the
conjugates were observed between the three cell lines. These
distributions directly opposed the insights from confocal
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microscopy and demanded confirmation by a third analytical
method. Rhenium homologue 2 was therefore incubated
separately, in the same cell lines and the Re content in the
nuclei and mitochondria determined by ICP-MS, after
digestion of the fractions by aqua regia. Figure 3 summarizes
Radiotoxicity and Mode of Cell Death. Cellular
distribution and localization evidenced that 1 and 2 closely
mimic the parent drug’s in vitro behavior. The predominant
nuclear accumulation of the conjugates and strong DNA
binding indicated that Auger electrons from 1 could indeed
effect radiologic damage. Accordingly, HeLa, A431, and B16F1
cell lines were incubated with different activity concentrations
of 1 ranging 0−9.25 MBq/mL. For comparison with cold
rhenium, the highest activity concentration corresponds to a
solution which is about 1 nM in 99mTc+99Tc, the latter resulting
from the 99mTc transition to the ground state. As a negative
control, [99mTcO4]− was incubated at the maximum activity
concentration, since the [99mTcO4]−-ion is not efficiently
internalized into cells and should not exhibit a radiotoxic
effect.17,79 After 36 h, the fraction of viable cells was determined
by the MTT colorimetric assay to determine the cell line’s
radiosensitivity toward Auger electrons from 1. As evident from
Figure 4, complex 1 induced an activity-dependent inhibition of
Figure 3. Relative uptake of 1 and 2 into nucleus and mitochondria
compared to whole cell uptake for B16F1 (gray), A431 (light gray),
and HeLa (white) cell lines. Quantification was done by a dose
calibrator for 1 and ICP-MS for 2. Values indicate means and error
bars the standard deviations of six replicates for 1 and three replicates
for 2.
these results. The cellular distribution of 2, according to ICP-
MS, coincides very well with the distribution obtained for
radioconjugate 1: About 70−80% accumulates in the nucleus,
whereas only a minor portion of rhenium is found in the
mitochondrial fraction (∼2%).
The quantitative data from ICP-MS and gamma-counting
(absolute uptake values, see Chart S18) diametrically
contrasted those of confocal microscopy, as fluorescence did
not imply a major accumulation in the nuclei (vide supra). Both
of the latter analyses clearly support the hypothesis that ADR
conjugate 2 indeed accumulates mainly in the nuclei but cannot
be visualized due to fluorescence quenching. According to ICP-
MS, the concentration of 2 in the cell nucleus can be calculated
to be about 33 μMthree orders of magnitude lower than the
nuclear concentration of DNA base pairs. Since the
fluorescence signal of ADR is quenched >95% at a roughly
50-fold molar excess of DNA base pairs, the fluorescence of 2
must be completely quenched under the conditions prevailing
in the nucleus. Vice versa, the nuclear concentration of free
ADR can reach up to 340 μM,76 sufficient to cause an excess
fluorescence signal, as observed in the reference experiments
with the parent ADR. Notably, the nuclear uptake of 2 is about
10-fold lower as compared to native ADR. Also, this
observation is in agreement with the reduced potency of
conjugate 2 to inhibit cell survival, as shown above. We do
emphasize the remarkable agreement of relative uptake values
of 1 (99mTc) and 2 (Re), determined by different quantification
modalities. Whereas the chemical matched-pair behavior of
99mTc and Re is frequently quoted,77,78 our results demonstrate
for the first time a biological matched-pair relation of rhenium
and technetium. This largely equivalent in vitro behavior
corroborates the homology of the two elements not only in
chemistry, but also in biology.
Figure 4. Surviving fraction (%) as a function of the activity
concentration (0−9.25 MBq/mL) of radioactive ADR conjugate 1 in
■
○
●
HeLa ( ), A431 ( ), and B16F1 ( ) cell lines. Inhibitory potency
(IC50) of 1 in B16F1 cells is 2.7 MBq/mL. Symbols indicate means
standard deviations of eight replicates.
viability and a markedly radiotoxic effect in murine B16F1 cells
which was less pronounced in the human cancer cell lines. At
the maximum activity concentration of ca. 10 MBq/mL, we
found 22.5% (B16F1), 70.0% (A431), and 74.5% (HeLa) of
viable cells. At the same concentration, [99mTcO4]− exhibited
almost no radiotoxic effect: The survival was nearly 90% for all
three cell lines (see Chart S19), reflecting the inability of this
compound to reach the nucleus. This finding underlines the
limited effectiveness of ultrashort path length radiation with
increasing distance from the DNA target. However, the still
non-negligible viability reduction might result in part from a
dose deposition by conversion electrons. Although these
account only for 11% probability, their range is between 200
and 400 μm, which is sufficient to reach the nucleus. We note
that the radiotoxicity of 1 was less pronounced in the human
cell lines, although overall uptake and cellular distribution were
nearly identical. Variations in accumulation of radioactivity can
therefore not be responsible for the observed differences in
viability. Conversely, the response of cells from different tissues
to radiation is known to be heterogeneous and depends on the
intrinsic radiosensitivity, i.e., the ability of cells to detect DNA
damage and the repair pathways associated therein.80,81
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From Figure 4, the IC50 of 1 in B16F1 cells was determined
to be 2.7 MBq/mL, corresponding to a molar concentration of
1.41 × 10−10 M. In a previous study Haefliger et al. indicated an
IC50 of 1.8 × 10−9 M in the same cell line for a pyrene
derivative and proposed that this value could be significantly
lower if a more effective nuclear targeting agent was found.15 In
the present study, this value is now indeed 1 order of
magnitude lower. Since the IC50 of ADR in B16F1 cells is in the
submicromolar range (see Table 2), it is evident that
radiotoxicity can be several orders of magnitude more effective
in inhibiting cell survival than chemotoxicity. By determining
the nuclear radioactivity in the subcellular compartments (vide
supra), only 600 molecules of 1 accumulated in a cell’s nucleus
and are the cause of the observed radiotoxic effects in the
B16F1 cell line. For comparison reasons, about 108 molecules
of native ADR account for the above-mentioned nuclear
concentration of 340 μM. The unique combination of an Auger
emitter with ADR, an effective nucleus targeting vehicle,
therefore significantly potentiates the cell killing ability of the
parent drug.
To investigate the cell death pathway induced by 1, the
B16F1 cell line was subjected to an Annexin V-FITC (An)/
Propidium iodide (PI) assay. Cells were exposed to 1 at
increasing activity concentrations of 1, 2, and 10 MBq/mL or 5
nM Staurosporin, a benchmark drug known to induce
apoptosis.82 After an incubation period of 72 h, the percentage
of apoptotic and necrotic cells was determined by fluorescence-
activated cell sorting (FACS, representative histograms are
shown in Chart S20). As evident from Figure 5, the percentage
Geant4Monte Carlo toolkit84 for the most sensitive B16F1 cell
line. Total dose was determined based on the emission
spectrum of 99mTc reported by Howell85 and calculation of
the energy deposited by disintegrations in the extracellular
compartment, cytosol, and nucleus. Calculated dosimetric S-
values as well as the time-dependent uptake of 1 are given in
Chart S21. Figure 6 depicts the experimentally determined
surviving fractions of the radiotoxicity assay vs calculated doses
in the nucleus and cytoplasm and the mean cellular dose. At the
highest activity concentration of ∼10 MBq/mL (1 nM in 99mTc
and 99Tc), the nuclear dose is 12.5 Gy. This energy transfer can
cause more than 106 ionizations per nucleus in 36 h and
rationalizes the reduced surviving fraction of 22%. The dose in
the cytoplasm is around six times smaller than the dose in the
nucleus at equal activity concentrations (see Chart S22),
reflecting the cellular distribution of the compound. Nearly
identical nuclear doses of a single cell (11.6 Gy) compared to a
cell growing in a monolayer (12.5 Gy) demonstrate the
preferential accumulation of the radionuclide in the cell
nucleus. At ∼10 MBq/mL, the dose in the extracellular
medium is only 0.39 Gy. It is remarkable that more than 99% of
the total radioactivity remaining in the medium inflicts a dose
which is about 30 times smaller than the nuclear dose. This
calculation is further consistent with the survival of 90% for
[
99mTcO4]−, which is not taken up by the cells (vide supra). It
clearly illustrates that Auger and internal conversion electron
emitters like 1, accumulated in the nucleus, lead to a significant
dose and reduced cell survival, whereas the low LET γ-radiation
from disintegrations in the medium fails to inflict such damage.
The dose−response fit curve in Figure 5 does not depict a
shoulder at low doses which is indicative of a high LET-like
radiation damage.86 These dosimetry considerations thus relate
the radiobiology of ultrashort path length radiation to the
observed radiotoxicity and connect it to the action of Auger
electrons.
In Vivo Evaluation. The in vivo biodistribution and
stability of ADR conjugate 1 was evaluated in healthy, nude
NMRI mice. Compound 1 was administered intravenously with
activity ranging from 8 to 53 MBq per 200 μL injected volume.
A subgroup of mice (n = 3) was placed under a microSPECT
scanner and imaged in six full body scans, in 10 min frames,
from 0 to 60 min post injection (p.i). All mice (n = 5) were
sacrificed 60 min p.i.
The ex vivo biodistribution analysis (Figure 7) showed a
rapid clearance from the blood pool into organs and a
remainder of less than 4%ID/g in the blood pool at 60 min p.i.
Uptake was most pronounced in the digestive system and
excretion organs while other organs of interest exhibited
generally low uptake (detailed %ID/g numbers, see Chart S23).
The uptake of 1 in the liver suggests a main clearance pathway
via the hepatobiliary system, while uptake in the kidneys also
evidenced a partial renal clearance. Clearance of the compound
into the intestines had already started 1 h p.i., as a large uptake
was visible in these organs. RP-HPLC analysis of the blood
plasma showed >80% intact 1 at 1 h p.i., with minor
contributions from more hydrophilic metabolites (Chart
S24). We tentatively assign the compound at tR ≈ 4 min to a
low-molecular-weight, hydrophilic metabolite that may be
responsible for the uptake in the salivary glands. Blood plasma
analysis evidenced a second metabolite in the region of the
intact compound. Closer examination (Chart S25) strongly
suggested this metabolite to be a reduced species of 1,
containing the Doxorubicinol (Doxol) moiety (Figure 8).
Figure 5. Annexin V (An)/Propidium iodide (PI) assay of 1 in B16F1
cells (72 h incubation time). Staurosporin (5 nM) was used as a
positive control.
of early and late apoptotic cells, after treatment with 1 (17.4−
38.1%), approached the value of Staurosporin (35.5%) in a
dose-dependent fashion, indicating that radiodrug 1 induces
apoptosis. It has been shown before that radiation predom-
inantly implicates this programmed cell death pathway.83
Interestingly, raising the concentration of 1 to 10 MBq/mL
gave rise to a small percentage of necrotic cells. We assume that
at high exposures of radioactivity excessive stress induces to a
partial necrotic cell death pathway.
Microdosimetry. To rationalize the observed radiotoxic
effect and to illustrate the intracellular dose proportions a
microdosimetric evaluation was carried out. The experimental
conditions of the radiotoxicity assay were modeled with the
F
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■
●
Figure 6. Left: Experimentally evaluated surviving fraction in the B16F1 cell line as a function of dose in the nucleus ( ), cytoplasm ( ), and
○
averaged whole cell ( ). Lines represent monoexponential fits to the calculated values. Right: Illustration of the dose proportions (at 9.25 MBq/mL
of 1) in a cell growing in a monolayer.
Figure 7. Ex vivo biodistribution of 1 in healthy, nude NMRI mice (n
= 5, dissection 60 min p.i.). Data is expressed as the mean %-injected
dose per gram of tissue (%ID/g) SEM.
Figure 9. (a) Representative SPECT scan of 1, frame 6:50−60 min p.i.
(b) Overlay of the SPECT scan on a mouse X-ray for better
illustration purposes. Visible are liver (L), part of the intestines (I),
urinary bladder (B), and salivary glands (SG).
pronounced liver uptake may be attributed to the relative
lipophilicity (logD1 = 0.92
0.03) and size of conjugate 1.
Moreover, uptake of a radioactive species into the salivary
glands was also verified by the SPECT scans. Overall, the
collective in vivo data shows close resemblance to the
metabolism of free ADR: predominant biotransformation in
the liver, biliary excretion through the intestines and, to a minor
extent, via the kidneys.90 Similarities between free ADR and
conjugate 1, such as hydrophobicity (logDADR = 0.45 0.03)91
and the “+1” charge at physiological pH, rationalize the
comparable in vivo behavior. Of note, the higher lipophilicity of
the organometallic ADR derivative compared to ADR could
potentially exacerbate the clearance properties of ADR.
Figure 8. Structure of the proposed Doxol metabolite of 1.
Doxol is part of the hepatic metabolism of the parent ADR,
which arises due to chemoselective reduction by carbonyl
reductases in vivo.87,88 The metabolite analysis of the urine
(total activity recovered 0.26%ID) revealed a multitude of more
hydrophilic metabolites with only a small remainder of intact 1
(Chart S22).
Figure 9 shows a representative microSPECT scan of
conjugate 1 (frame 6:50−60 min p.i.), which largely reflects
the ex vivo biodistribution. The major hepatic assimilation of
the compound was already visible 10 min p.i., rationalizing the
low blood pool remainder at 60 min p.i. A short blood half-life
is known for the parent ADR.89 Region of interest (ROI)
analysis in the liver throughout the frames indicated a slow
clearance of the compound into the intestines. The fast and
CONCLUSION
■
Doxorubicin (ADR), a routinely applied anticancer pharma-
ceutical, could serve as a lead structure for targeted molecular
imaging or medicinal inorganic drug delivery. We showed that
two ADR−metal conjugates (M= 99mTc, 1 and Re, 2) mimic
the in vitro behavior of ADR. Differences observed in
fluorescence microscopy between ADR and 2 enticed a careful
G
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Article
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study of the cellular distribution which unambiguously
evidenced a major nuclear accumulation. These results advocate
caution when assessing the distribution of luminescent
compounds in general, as a fluorescence signal by itself may
be deceptive and not reflect the true cellular localization.
Furthermore, 99mTc compound 1 exhibited a dose-dependent
reduction of viability of up to 80% in the B16F1 cell line
through its Auger electron emission, making this study an
important step toward a possible treatment strategy with these
low-energy electrons. As demonstrated, Auger emitters may
provoke inhibition of cell survival at concentrations which are
much lower than the ones of their chemotoxic counterparts.
This offers the prospect that conjugates of Auger emitters and
chemotherapeutic agents, like 1, could mitigate dose-dependent
adverse effects, as the subnanomolar concentrations likely do
not elicit pharmacological responses. For drugs with potent side
effects, such as ADR, overcoming these drawbacks could greatly
improve therapeutic margins. Multidrug resistance (MDR)92−94
and the much-dreaded cardiotoxicity95,96 are both concen-
tration-dependent phenomena which can likely be circum-
vented due to the minute radio-drug concentrations employed
in Auger electron therapy. Evidence for this concept has been
shown for MDR.97
The similarities of conjugate 1 to ADR are an incentive for
studies in tumor-bearing mice, allowing for a dosimetric
evaluation in vivo and an assessment of the validity of the
proposed system in the treatment regime. Furthermore,
clonogenic assays will provide insights into long-term viability
of the cells. Such studies, in addition to alternative constructs
with different chelating units, are currently ongoing.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bioconj-
chem.5b00466.
Materials and methods (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: ariel@chem.uzh.ch. Tel.: +41-52-635 46 31.
■
Notes
The authors declare no competing financial interest.
(17) Haefliger, P., Agorastos, N., Renard, A., Giambonini-Brugnoli,
G., Marty, C., and Alberto, R. (2005) Cell uptake and radiotoxicity
studies of an nuclear localization signal peptide-intercalator conjugate
labeled with [Tc-99m(CO)(3)](+). Bioconjugate Chem. 16, 582−587.
(18) Vitor, R. F., Esteves, T., Marques, F., Raposinho, P., Paulo, A.,
Rodrigues, S., Rueff, J., Casimiro, S., Costa, L., and Santos, I. (2009)
Tc-99m-Tricarbonyl Complexes Functionalized with Anthracenyl
Fragments: Synthesis, Characterization, and Evaluation of Their
Radiotoxic Effects in Murine Melanoma Cells. Cancer Biother.Radio-
pharm. 24, 551−563.
ACKNOWLEDGMENTS
■
We gratefully acknowledge M. De Saint-Hubert, I. Pooters, S.
Voo, and R. Wierts from the MUMC+ for their care during the
in vivo evaluation, F. Wild for ICP-MS measurements, and the
Graduate School of Chemical and Molecular Sciences Zurich
(CMSZH) for a travel grant. M. Bauwens is supported by the
Weijerhorst Foundation. This work was financially supported
by the Swiss National Science Foundation (Professorships No
PP00P2_133568 and PP00P2_157545 to G.G.), the University
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Adriamycin (14-Hydroxydaunomycin) a Novel Antitumor Antibiotic.
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Forschung of the University of Zurich (G.G.), and an SBFI
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DOI: 10.1021/acs.bioconjchem.5b00466
Bioconjugate Chem. XXXX, XXX, XXX−XXX