Evaluation of fluorophore-tethered platinum complexes to monitor the fate of cisplatin analogs

Article abstract of DOI:10.1007/s00775-015-1290-2

The platinum drugs cisplatin, carboplatin, and oxaliplatin are highly utilized in the clinic and as a consequence have been extensively studied in the laboratory setting, sometimes by generating fluorophore-tagged analogs. Here, we synthesized two Pt(II) complexes containing ethane-1,2-diamine ligands linked to a BODIPY fluorophore, and compared their biological activity with previously reported Pt(II) complexes conjugated to carboxyfluorescein and carboxyfluorescein diacetate. The cytotoxicity and DNA damage capacity of Pt-fluorophore complexes was compared to cisplatin, and the Pt-BODIPY complexes were found to be more cytotoxic with reduced cytotoxicity in cisplatin-resistant cells. Microscopy revealed a predominately cytosolic localization, with nuclear distribution at higher concentrations. Spheroids grown from parent and resistant cells revealed penetration of Pt-BODIPY into spheroids, and retention of the cisplatin-resistant spheroid phenotype. While most activity profiles were retained for the Pt-BODIPY complexes, accumulation in resistant cells was only slightly affected, suggesting that some aspects of Pt-fluorophore cellular pharmacology deviate from cisplatin.

Full text of DOI:10.1007/s00775-015-1290-2

J Biol Inorg Chem (2015) 20:1081–1095
DOI 10.1007/s00775-015-1290-2
ORIGINAL PAPER
Evaluation of fluorophore‑tethered platinum complexes
to monitor the fate of cisplatin analogs
Justin C. Jagodinsky1 · Agnieszka Sulima2 · Yiqi Cao1 · Joanna E. Poprawski1 ·
Burchelle N. Blackman2 · John R. Lloyd3 · Rolf E. Swenson2 ·
Michael M. Gottesman1 · Matthew D. Hall1
Received: 4 June 2015 / Accepted: 1 August 2015 / Published online: 1 September 2015
© SBIC (outside the USA) 2015
Abstract The platinum drugs cisplatin, carboplatin,
and oxaliplatin are highly utilized in the clinic and as a
consequence have been extensively studied in the labora-
tory setting, sometimes by generating fluorophore-tagged
analogs. Here, we synthesized two Pt(II) complexes con-
taining ethane-1,2-diamine ligands linked to a BODIPY
fluorophore, and compared their biological activity with
previously reported Pt(II) complexes conjugated to car-
boxyfluorescein and carboxyfluorescein diacetate. The
cytotoxicity and DNA damage capacity of Pt–fluoro-
phore complexes was compared to cisplatin, and the Pt–
BODIPY complexes were found to be more cytotoxic with
reduced cytotoxicity in cisplatin-resistant cells. Micros-
copy revealed a predominately cytosolic localization, with
nuclear distribution at higher concentrations. Spheroids
grown from parent and resistant cells revealed penetra-
tion of Pt–BODIPY into spheroids, and retention of the
cisplatin-resistant spheroid phenotype. While most activ-
ity profiles were retained for the Pt–BODIPY complexes,
accumulation in resistant cells was only slightly affected,
suggesting that some aspects of Pt–fluorophore cellular
pharmacology deviate from cisplatin.
Keywords Accumulation · Trafficking · Platinum ·
Resistance
Introduction
Cisplatin (1, cis-[PtCl2(NH3)2]), carboplatin ([Pt(O,O′-cdbca)
(NH3)2], cbdca = cyclobutane-1,1-dicarboxylate, Fig. 1) and
oxaliplatin ([Pt(ethanedioato-O,O′)(chxn)], chxn = 1R,2R-
cyclohexane-1,2-diamine) are used in combination with other
agents to treat a range of cancers. However, with the excep-
tion of most cases of testicular cancer, the treatment does
not effectively eliminate tumors because they develop resist-
ance to the drug. After entering cells, the platinum drugs exert
their toxicity by binding to DNA and inducing apoptosis.
Resistance is conferred through multiple cellular changes,
including reduced uptake, reduced apoptotic signaling, up-
regulated DNA damage repair mechanisms, altered cell cycle
checkpoints, and disrupted assembly of the cytoskeleton [1].
Although the pleiotropic mechanisms underlying cisplatin
resistance are well documented, some mechanisms are still
poorly understood and the clinical relevance of many mecha-
nisms of cisplatin resistance remains unknown. There remains
the need for further studies of mechanisms of action of plati-
num drugs, both alone and in combination with other thera-
peutics, to provide an understanding of why some new plati-
num agents such as BBR3464, picoplatin and satraplatin have
not succeeded in clinical trials [2].
J. C. Jagodinsky and A. Sulima contributed equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-015-1290-2) contains supplementary
material, which is available to authorized users.
* Michael M. Gottesman
mgottesman@nih.gov
1
Laboratory of Cell Biology, National Cancer Institute,
Center for Cancer Research, National Institutes of Health, 37
Convent Drive, Rm. 2108, Bethesda, MD 20892, USA
2
Imaging Probe Development Center, National Institutes
of Health, Rockville, MD, USA
A persisting challenge when studying Pt-based drugs in
biology is that there are few spectroscopic handles available
for monitoring them, as there are for organic-based drugs [3].
3
Advanced Mass Spectrometry Facility, National Institute
of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, MD 20892, USA
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H3N
H3N
Cl
Cl
Bierbach and colleagues have reported a post-labeling system
for determining sub-cellular localization of modified Pt com-
plexes [11], and fluorescent sensors that can be applied to Pt-
treated cells to image Pt species [12–14] have also emerged.
However, these techniques require sample fixation and, there-
fore, live-cell temporal imaging is not possible.
Pt
O
O
O
O
O
O
O
Cisplatin (1)
O
O
O
H2
N
Cl
Cl
O
O
O
Pt
N
O
H2
O
[PtCl2(en)] (2)
NH
O
To address these challenges, a number of Pt complexes
containing tethered fluorophores that retained the traditional
structure–activity requirements for Pt(II) anticancer complexes
(square planar complexes with cis leaving groups and amines)
have been generated [15]. Reedijk and co-workers reported
the synthesis of a Pt(II) complex conjugated to carboxy-
fluorescein diacetate (CFDA) through an ethane-1,2-diamine
(en) ligand, in which the CFDA became fluorescent upon
hydrolysis of the acetate groups to carboxyfluorescein (CF)
[16]. Howell and co-workers subsequently used this complex
(termed FDDP, fluorescent cisplatin, numbered 5 in the pre-
sent paper) to show that the molecule was trafficking to vesic-
ular compartments, with little detectable nuclear localization
[17, 18]. Farrell and co-workers reported a cis-ammine/amine
cisplatin analog tethered to N-(7-nitro-2,1,3-benzoxadiazol-
4-yl)ethane-1,2-diamine (NBD), which undergoes lysosomal
localization and was shown to be cytotoxic, but about tenfold
less so than cisplatin [19]. There is also a commercial ‘univer-
sal linkage system’ (ULYSIS) for DNA labeling based on the
affinity of platinum for nucleobases [20]. These complexes
do not conform to platinum structure–activity rules and con-
tain only a single leaving group, having the general formula
[PtCl(en)(NH2R)]+, with the primary amine tethered to a fluo-
rophore (R), of which a number have been produced including
cyanines, fluorescein, and rhodamine [21]. While not biologi-
cally active, the ULYSIS AlexaFluor-488 dye has been used
to demonstrate defective trafficking of Pt in cisplatin-resistant
cell lines [22]. As examples of more recent work, complexes
tethered to BODIPY (described below) have been reported
[23], including a cationic Pt complex for imaging mitochon-
drial localization [24]. Platts and co-workers reported plati-
num trimethyl bipyridyl thiolates that could emit in the near
infrared for cellular imaging [25].
An obvious limitation to adding a fluorophore to a
small inorganic complex is that it may significantly alter
the physicochemical properties and biological activity of
the complex. There has been no systematic assessment
of the pharmacology of these analogs, and it is not clear
whether the fluorescent analogs are an appropriate model
for the cellular behavior of cisplatin and its congeners. For
instance, none of the Pt–fluorophore complexes studied
have reported significant nuclear localization.
A significant number of studies exist reporting the syn-
thesis of Pt complexes conjugated to a cytotoxic ligand,
such as a DNA intercalator that happens to also be fluo-
rescent, in an attempt to generate a more toxic or DNA-
specific experimental therapeutic. These constructs are by
NH
H2
N
BocHN
Cl
Cl
H2N
Cl
NH2
Pt
Pt
BocHN
N
Cl
H2
CFDA-Pt (5)
CFDA-Boc (4)
Pt-Boc (3)
O
O
O
O
HO
CO2H
HO
CO2H
O
O
HN
HN
H2N
Cl
NH2
Pt
NHBoc
Cl
CF-Boc (6)
CF-Pt (7)
N
F
N
B
F
N
N
F
N
N
B
F
B
O
F
F
HN
O
O
HN
HN
NH
O
NHBoc
H2N
Cl
NH2
H2N
Cl
NH2
Cl
Pt
Pt
Cl
BODIPY-Boc (8)
BODIPY-Pt (9)
BODIPY-propanamide-Pt (10)
Fig. 1 Structures of the compounds used in this study. For each plati-
num–dye complex, the Boc-protected dye was used as a control for
comparison
For example, none of the Pt drugs are fluorescent in a way
that is amenable to microscopy. In addition, radiolabeling
with [3H] or [14C] is not feasible because the 6 protons on the
ammine ligands of cisplatin exchange rapidly in water, and it
contains no carbon. Carboplatin can be labeled on the ‘leav-
ing’ cbdca ligand, limiting its utility [4, 5], but oxaliplatin’s
chxn ligand can be labeled [6]. While isotopes of 15N and
195Pt are useful for mechanistic studies in solution (for exam-
ple, by HSQC NMR) [7], these are not amenable to cellular
studies. The main technique employed is an atomic detection
technique such as GF-AAS or ICP-MS to measure bulk levels
of Pt in cells because Pt is not endogenous to biology. How-
ever, little information about speciation and cellular locali-
zation of compounds can be gained. X-ray absorption spec-
troscopies such as X-ray absorption near-edge spectroscopy
(XANES, for oxidation state) and X-ray fluorescence (XRF
or SRIXE for cell localization) [8, 9], and nano-scale second-
ary ion mass spectrometry (NanoSIMS) are possible [10].
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definition not appropriate for studying Pt localization as
the fluorophore itself is not ‘innocent’ by design, and the
fluorophore may strongly influence the cellular localization
of the Pt center [3, 26]. Therefore, we synthesized several
complexes reported in the literature, along with two new
boron-dipyrromethene (BODIPY)-conjugated complexes
that differ in the linker employed (amide and propanamide).
It should be emphasized that probe molecules such as those
described in this paper are not candidate experimental ther-
apeutics, though there is also a rich literature describing Pt
complexes tethered to intercalators [26]. The inherent fluo-
rescence of many intercalators means that the cellular fate
of these complexes can be visualized, but they are designed
to modify the biological behavior of the Pt and as such do
not represent candidate probes for the fate of Pt [3].
In this study, we assessed these complexes for solvent
stability in dimethylsulfoxide (DMSO) and dimethylforma-
mide (DMF), lipophilicity (distribution coefficient, logD),
cytotoxicity against parental and cisplatin-resistant cancer
cell lines, and cellular accumulation. Cellular localization
was assessed using live-cell confocal microscopy at 1 and
24 h, and DNA damage was inferred by assessing phospho-
rylation of the histone H2A family member H2A.X in fixed
cells. Cisplatin and a BODIPY-conjugated complex were
also assessed for their activity and distribution in three-
dimensional (3D) culture in Matrigel, a gelatinous mixture
of proteins derived from mouse sarcoma cells which is
often used as a basement membrane matrix in 3D cell cul-
ture [27]. While this manuscript was in preparation, Miller
et al. reported the synthesis of other BODIPY-conjugated
cisplatin and carboplatin analogs, and reported their cyto-
toxicity and DNA damage activities. A comparison with the
activity for complexes synthesized in this study is made,
and we qualify the conclusions in the studies of Miller et al.
by reporting that the activity of BODIPY–Pt is inactivated
by the solvent DMSO [23]. By evaluating the efficacy of
these fluorophore-conjugated Pt complexes as analogs for
cisplatin, this study holds implications for further in vivo
studies of platinum-based compounds for tumor treatment.
purchased from Alpha Aesar. Analytical HPLC analysis was
performed on an Agilent 1200 Series instrument equipped
with multi-wavelength detector and Evaporative Light Scat-
tering Detector (ELSD) using an Agilent Eclipse Plus col-
umn (4.6 × 50 mm, 3.5 µm) with a flow rate of 1 mL/min.
Solvent A was water, solvent B was ACN, and a linear gradi-
ent of 5 % B to 95 % B over 10 min was used. APCI mass
spectrometry was performed on a 6130 Quadrupole LC/MS
Agilent Technologies instrument equipped with diode array
detector. 1H-NMR spectra were recorded with a Varian spec-
trometer operating at 400 MHz. Chemical shifts are reported
in parts per million (δ) and are referenced to tetramethylsi-
lane (TMS). 195Pt-NMR spectra were acquired at 86 MHz
(400 MHz 1H) on a Bruker AV III (Bruker-Biospin, Switzer-
land) spectrometer using a spin echo (SE) sequence with a
10 μs echo time and a 24 kHz B1 field. Except for increased
spectral quality, there was no discernible difference between
the SE and simple 90-pulse-acquire spectra. Chemical shifts
are referenced to K2[PtCl4] in D2O.
Synthesis
Compounds 3–10 were examined for biological activity in
this study. Compounds 3–9 were previously reported as out-
lined below, and 10 is novel. Figure 2 illustrates the method
of synthesis of the three BODIPY-containing compounds
8 (BODIPY–Boc), 9 (BODIPY–Pt) and 10 (BODIPY–
prop–Pt). The Pt-free Boc-protected fluorophore derivatives
4 (CFDA–Boc), 6 (CF–Boc) and 8 (BODIPY–Boc) were
obtained in the reaction of N-Boc-ethylenediamine with the
corresponding N-succinimidyl ester of dye in the presence of
base. To synthesize the Pt-containing BODIPY compounds,
the precursors 11 and 12 were synthesized as described pre-
viously [16, 28]. The [(2,3-diaminopropionic acid)(dichloro)
platinum(II)] complex 14 was synthesized in a one-step proce-
dure [29]. NMR, MS and IR structural characterization are in
agreement with the published data for all previously reported
compounds. The BODIPY FL–Pt analog 9 (BODIPY–Pt) was
obtained by the reaction of 12 with BODIPY FL succinimidyl
ester in the presence of base. Compound 10 was prepared by
reaction of 14 with an amine derivative of BODIPY FL (15)
using a standard coupling procedure (Fig. 2). The fluorescein-
containing Pt complexes 5 (CFDA–Pt) and 7 (CF–Pt) were
synthesized as previously reported [16, 17].
Materials and methods
Materials
All reagents and solvents were obtained from commer-
cial sources and used as received unless otherwise noted.
5(6)-Carboxyfluorescein N-succinimidyl ester (CF SE),
cisplatin, dimethylsulfoxide (DMSO), dimethylformamide
(DMF), and 1-octanol were purchased from Sigma-Aldrich.
6-Carboxyfluorescein diacetate N-succinimidyl ester (CFDA
SE) was purchased from Berry & Associates, and BODIPY
LF SE was obtained from Life Technologies. [PtCl2(en)] was
tert‑Butyl (2‑(3‑(5,5‑difluoro‑7,9‑dimethyl‑5H‑4λ4,5
λ4‑dipyrrolo[1,2‑c:2′,1′‑f] [1,3,2] diazaborinin‑3‑yl)
propanamido)ethyl)carbamate; BODIPY–Boc (8)
To a solution of BODIPY FL SE (30 mg, 0.077 mmol) in
anhydrous dichloromethane (10 mL) N-Boc-ethylenedi-
amine (14 μL, 0.084 mmol) was added followed by trieth-
ylamine (12 μL, 0.084 mmol). The reaction was allowed to
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Fig. 2 Scheme of syntheses
for the BODIPY-containing
compounds 8 (BODIPY–Boc),
9 (BODIPY–Pt), and 10
(BODIPY–prop–Pt)
stir for 20 min and then was transferred to a separator fun-
nel. The organic layer was washed with water (2 × 6 mL)
and brine (1 × 6 mL). The solvent was evaporated yielding
(25 μL, 0.178 mmol) was added dropwise at room tempera-
ture. The reaction mixture was stirred for 2 h. Progress of the
reaction was followed by ELSD-HPLC. The reaction mix-
ture was diluted with deionized water (50 mL) and freeze-
dried. Ice-cold water (7 mL) was added to the resulting pow-
der and the mixture was stirred for 5 min. The precipitate
was filtered off and washed with ice-cold water (3 × 5 mL),
ethanol (2 × 5 mL) and diethyl ether (2 × 5 mL) affording 1
as dark orange solid (40.5 mg,Y 72 %). 1H-NMR (400 MHz,
DMF-d7): δ 8.16 (t, 1H, J = 5.48 Hz), 7.72 (s, 1H), 7.14
(d, 1H, J = 3.91 Hz), 6.42 (d, 1H, J = 3.92 Hz), 6.33 (s,
1H), 5.61 (d, 1H, J = 6.65 Hz), 5.36 (bs, 2H), 5.09 (t, 1H,
J = 9.39 Hz), 3.62–3.68 (m, 1H), 3.48–3.53 (m, 2H), 3.24
(t, 2H, J = 7.63 Hz), 3.07–3.08 (m, 1H), 2.65–2.70 (m, 3H),
2.54 (s, 3H), 2.31 (s, 3H). 195Pt-NMR (86 MHz, DMF-d7): δ
−2274. HRMS calculated for C17H24BCl2F2N5OPt (M + H)
628.1, found 628.1.
1
28 mg (Y 84 %) of dark orange solid. H-NMR (400 MHz,
CDCl3): δ 7.07 (s, 1H), 6.87 (d, 1H, J = 3.91 Hz), 6.27 (d,
1H, J = 3.91 Hz), 6.18 (bs, 1H), 6.11 (s, 1H), 4.91 (bs, 1H),
3.23–3.31 (m, 4H), 2.60–2.65 (m, 4H), 2.55 (s, 3H), 2.23 (s,
3H), 1.41 (s, 9H). LC/MS (APCI) m/z: 415 (M+-F)+.
[(3‑(5,5‑difluoro‑7,9‑dimethyl‑5H‑4λ4,5λ4‑dipyrro
lo[1,2‑c:2′,1′‑f] [1,3,2] diazaborinin‑3‑yl)propan)
aminomethyl)‑1,2‑ethylenediamine}dichloroplatinum(II)];
BODIPY–Pt (9)
To a solution of 12 (35 mg, 8.9 × 10−2 mmol) in deion-
ized water (3 mL) a solution of BODIPY FL SE (35 mg,
8.9 × 10−2 mmol) in DMF (6 mL) followed by triethylamine
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J Biol Inorg Chem (2015) 20:1081–1095
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[(2,3‑Diamino‑N‑(2‑(3‑(5,5‑difluoro‑7,9‑dimethyl‑5H‑4λ
4,5λ4‑dipyrrolo[1,2‑c:2′,1′‑f] [1,3,2] diazaborinin‑3‑yl)
propanamido)ethyl)propanamide)dichloroplatinum (II)];
BODIPY–prop–Pt (10)
The desolvation gas was purified nitrogen. All test solutions
were made fresh and incubated at room temperature in the
dark prior to analysis. The MS data were analyzed using
Waters MassLynx 4.1 software.
A solution of BODIPY FL SE (5 mg, 1.28 × 10−2 mmol) in
dichloromethane (1.5 mL) was added dropwise to a solution
of ethylenediamine (1.28 µL, 1.92 × 10−2 mmol) in dichlo-
romethane (1.5 mL) and the mixture was stirred for 30 min.
Progress of the reaction was monitored by ELSD-HPLC.
The solvent was removed under reduced pressure and the
resulting dark orange residue was dried under high vacuum
for 36 h. The crude product 15 (4.3 mg, quantitative) was
Distribution coefficient (log D) measurement
Distribution coefficient measurements were performed by
making 200 µM solutions of compounds in 3 mL PBS, lay-
ered with 3 mL 1-octanol, vortexed briefly, and were shaken
in the dark for 4 h. Mixtures were then allowed to settle at
room temperature. Fluorescence in the 1-octanol and aque-
ous layers was determined using an Ibidi optical-bottom
96-well plate fluorimeter. All samples were run in triplicate
and logD was calculated as log([oct]/[aq]). Data are reported
as mean SD.
1
used in the next step of the synthesis. H-NMR (400 MHz,
DMF-d7): δ 7.94 (bs, 1H), 7.71 (s, 1H), 7.12 (d, 1H,
J = 3.92 Hz), 6.42 (d, 1H, J = 3.92 Hz), 6.32 (s, 1H), 3.21–
3.26 (m, 4H), 2.60–2.64 (m, 4H), 2.53 (s, 3H), 2.313 (s, 3H).
To a solution of 15 (4.7 mg, 1.27 × 10−2 mmol) in DMF
(0.7 mL), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (2.4 mg, 1.27 × 10−2 mmol), 1-hydroxyben-
zotriazole hydrate (1.7 mg, 1.27 × 10−2 mmol) and triethyl-
amine (4.5 µL, 3.2 × 10−2 mmol) were added, and the mix-
ture was stirred for 15 min at room temperature. A solution
of 10 (4.7 mg, 1.27 × 10−2 mmol) in DMF (1 mL) was then
added and the reaction mixture was stirred for 2 h. The mix-
ture was diluted with deionized water (30 mL) and freeze-
dried. The resulting residue was then treated with ice-cold
water (4 mL), stirred for 5 min and filtered off. The solid
was subsequently washed with ice-cold water (3 × 2 mL),
ethanol (2 × 1.5 mL) and diethyl ether (2 × 1.5 mL) afford-
Cell lines and cell culture
This study used the KB-3-1 human cervical carcinoma cell
line (a subline of HeLa), and its cisplatin-resistant sublines
KB-CP.5 and KB-CP20. Originally, KB-CP.5 and KB-CP20
cells were selected in a single step in 0.5 µg/mL (1.6 µM)
and in multiple steps up to 20 µg/mL (0.103 mM) cisplatin,
respectively, in our laboratory as described previously [30].
All cell lines were thawed immediately before experimen-
tation, and cell lines are characterized by NCI using short
tandem repeat profiling. The cisplatin stock solutions used
for culturing CP.5 and CP20 cells were prepared in PBS.
The cisplatin-resistant cells were maintained in the pres-
ence of cisplatin, which was removed from growth medium
3 days prior to all experiments. All cell lines were grown
as monolayer cultures at 37 °C in humidified 5 % CO2,
using DMEM with 4.5 g/L glucose (from Invitrogen), sup-
plemented with l-glutamine, penicillin, streptomycin, and
10 % FBS (BioWhittaker).
1
ing 2 as dark orange solid (5.3 mg, Y 61 %). H-NMR
(400 MHz, DMF-d7): δ 8.44-8.56 (m, 1H), 7.97–7.99 (m,
1H), 7.72 (s, 1H), 7.14 (d, 1H, J = 3.99 Hz), 6.42 (d, 1H,
J = 3.99 Hz), 6.33 (s, 1H), 5.87–5.90 (m, 1H), 5.45–5.51
(m, 2H), 4.95–5.01 (m, 1H), 3.66–3.67 (m, 1H), 3.20–3.38
(m, 5H), 2.79–2.82 (m, 2H), 2.59–2.63 (m, 3H), 2.54 (s,
3H), 2.32 (s, 3H). 195Pt-NMR (86 MHz, DMF-d7): δ −2269.
HRMS calculated for C19H27BCl2F2N6O2Pt (M + H) 686.1,
found 686.1.
Cytotoxicity
Cell survival was measured by the CellTiter-Glo (Pro-
mega) assay. Cells were seeded at a density of 1000 cells
per well in 96-well opaque plates and incubated at 37 °C
in humidified 5 % CO2 for 24 h. Compound was serially
diluted 1:3 (DMEM used as diluent) and added to plates to
achieve the intended final concentrations. Solvent (DMSO
and DMF) tolerance testing up to 8.3 % under identical
conditions at 24, 48, and 72 h incubation time points con-
firmed growth of all cell lines was unaffected. Cells were
then incubated an additional 72 h, and the CellTiter-Glo
assay was performed according to the manufacturer’s
instructions (Promega). Bioluminescence values were
measured on a Victor3V spectrophotometer (PerkinElmer).
All CellTiter-Glo assays were performed in triplicate. IC50
Mass spectrometry
Solutions of BODIPY–Pt were prepared using DMSO or
DMF at a concentration of 10 µM. Solutions were kept at
room temperature in the dark for 48 h prior to analysis.
Accurate mass data were obtained on a Waters Premiere
LCT ESI time-of-flight mass spectrometer operated in the
positive ion W-mode at 10 K resolution. The high-perfor-
mance liquid chromatography solvent pump was operated
at 200 µL/min and the solvent composition was 50:40:10
water:methanol:acetonitrile. Samples were flow injected via a
sample loop and ionized in the electrospray ion source. The
electrospray capillary was operated at 3.5 kV and at 350 °C.
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values were defined as the drug concentrations required to
reduce cellular proliferation to 50 % of the untreated con-
trol well. Statistical analysis was performed using Prism
6 Prism 6 (GraphPad Software). All data are expressed as
mean SD.
Confocal microscopy
For two-dimensional confocal localization imaging,
KB-3-1 and KBCP.5 cells were seeded at a density of
5 × 104 cells per well in 8-well chamber slides and allowed
to attach at 37 °C in humidified 5 % CO2 for 24 h. For
three-dimensional confocal localization imaging, each
chamber was coated with 200 μL of 2.5 mg/mL Matrigel.
Then 2000 cells in 200 μL of 2.5 mg/mL Matrigel were
seeded in each well and were cultured for 8 days at 37 °C
in humidified 5 % CO2. Compound at a concentration of
10 µM was added directly to each well and cells were incu-
bated for either 1 or 24 h for two-dimensional culture and
1 h for three-dimensional culture. Hoechst nuclear stain
was added to each two-dimensional culture well (1 µg/mL)
1 h prior to imaging. Media were aspirated and cells were
rinsed with PBS before imaging on a Zeiss 710 NLO con-
focal microscope.
Three‑dimensional cell culture
Each well of a white 96-well plate was coated with 40
µL of 5 mg/mL Matrigel and allowed to gel for 30 min
at 37 °C. Cells were then seeded over this base layer at
a density of 1000 cells per well in 100 µL of 2.5 mg/
mL Matrigel (Corning Matrigel Matrix) and incubated
at 37 °C and 5 % CO2 for one day to culture single-
cell suspensions, or 4 days to allow spheroid formation.
Cells were then treated with compound as previously
described. After drug incubation cytotoxicity was deter-
mined using CellTiter-Glo assays as previously described
and according to the manufacturer’s directions.
H2AX immunohistochemical staining
Results and discussion
KB-3-1 cells were fixed and stained with Phospho-His-
tone H2A.X (Se139; 20E3) Rabbit mAb (Alexa Fluor 647
Conjugate; all components from Cell Signaling Technol-
ogy) following the manufacturer’s instructions and as
previously described [31]. Cells were seeded at 5 × 104
cells per chamber in 8-chamber coverslips and incu-
bated for 24 h. Cells were then incubated with Pt com-
plexes for 24 h, after which they were washed with PBS,
fixed in 4 % paraformaldehyde, and blocked with block-
ing buffer (PBS/5 % normal horse serum/0.3 % Triton
X-100) for 1 h. Diluted antibody was then applied, and
cells were incubated overnight at 4 °C. Cells were then
washed and stained with DAPI nuclear stain (2 µg/mL for
1 h) and imaged using a Zeiss LSM 710 NLO confocal
microscope.
Design and synthesis of platinum–fluorophore
conjugate
Our aim was to synthesize Pt–fluorophore complexes and
compare them with cisplatin (1) and [PtCl2(en)] (2) (all
structures shown in Fig. 1). This latter compound was par-
ticularly relevant as the ethane moiety serves as the point
for functionalization (3) and conjugation of fluorophores
(synthetic schemes for BODIPY compounds shown in
Fig. 2). Based on previous literature, we prepared Boc-pro-
tected versions of CFDA (termed here CFDA–Boc, 4) and
CF (termed here CF–Boc, 6), as well as CFDA (CFDA–Pt,
5) and CF (CF–Pt, 7) conjugated to Pt via an amide linker
[17, 18]. We synthesized a Boc-protected BODIPY fluoro-
phore (BODIPY–Boc, 8) and two BOPDIPY-conjugated
Pt complexes that differ in the linker used: one with an
amide linker (BODIPY–Pt, 9) and one with a propanamide
linker (BODIPY–prop–Pt, 10). While this manuscript was
in preparation, BODIPY–Pt (9) was reported by Miller
et al. [23], using an identical synthetic strategy (Fig. 2) that
proceeded via reaction of BODIPY FL succinimidyl ester
(BODIPY FL SE, Fig. 2) conjugated to the terminal amine
of 12. The complexation to Pt did not result in altered exci-
tation and emission maxima (not shown), consistent with
previous reports [23]. 195Pt NMR spectra of BODIPY–Pt
and BODIPY–prop–Pt (Supplementary Figure 1B,1C,
respectively) revealed a single peak in the range consist-
ent with a PtN2Cl2 coordination sphere reported in the lit-
erature [32, 33], and distinct from that of K2[PtCl4] (Sup-
plementary Figure 1A). It should be noted that an earlier
report of ‘bodipy-cisplatin’ exists (prior to Miller et al.) in
Flow cytometry
Cells were trypsinized and 200,000 cells were added to
5-mL FACS tubes. Cells were then centrifuged, washed
twice with IMDM supplemented with 5 % fetal bovine
serum, and resuspended in compound diluted in IMDM
supplemented with 5 % fetal bovine serum (final concen-
tration 25 µM, only fluorescent compounds were exam-
ined). Cells were incubated at 37 °C for 45 min, then
washed as above and resuspended in PBS. Tubes were
immediately placed on ice and analyzed by flow cytom-
etry. FL-1 fluorescence intensities were measured using a
FACSCanto flow cytometer equipped with a 488 nm argon
laser for a total of 10,000 events per sample. Gating analy-
sis was performed using FlowJo flow cytometry software.
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Table 1 Cytotoxicity
Compound
Solvent
Percent mass
increase
LogD
IC50 (μM)
(IC50, μM) of compounds
against parental KB-3-1 and
cisplatin-resistant KB-CP.5
and KB-CP20 cell lines.
Distribution coefficients (logD)
of each compound and percent
increase in molecular weight
compared with cisplatin shown
for Pt complexes
KB 3-1
KB-CP.5
KB CP20
Cisplatin (1)
Saline
–
–
[−2.28 0.046]a
[−2.30 0.019]a
2.5 0.2
1.8 0.1
25.1 3.4
23.9 5.3
>100
DMF
124.7 31.5
[PtCl2(en)] (2)
Saline
2.7 1.9
1.0 0.6
94.9 8.3
>100
>500
DMF
150.0 11.8
Pt–Boc (3)
DMF
133 %
–
ND
16.7 0.9
116.1 8.9
71.6 29.5
>500
172.4 28.7
104.1 2.4
276.1 38.4
>500
431.9 96.9
131.6 5.7
440.3 40.9
>500
CFDA–Boc (4)
DMF
−0.063 0.014
−2.09 0.011
−2.35 0.037
−2.66 0.009
2.15 0.023
0.98 0.04
CFDA–Pt (5)
DMF
308 %
–
CF–Boc (6)
DMF
CF–Pt (7)
DMF
265 %
>500
>500
>500
BODIPY–Boc (8)
DMF
BODIPY–Pt (9)
–
86.7 0.8
86.9 4.4
53.6 2.9
DMF
222 %
35.7 1.5
103.0 17.0
459.7 113.2
226.5 47.2
>500
DMSO
391.7 35.2
BODIPY–prop–Pt (10)
DMF
251 %
0.66 0.011
21.5 5.8
44.6 2.7
192.5 50.3
a
Hall et al. [35]
the literature but no details of synthesis, characterization or
structure were included in that report [34].
create stock solutions of agents. We recently reported the
deleterious effects of DMSO on the biological activity of
platinum(II) complexes [31], caused by ligand replacement
and coordination to DMSO via its sulfur [36]. A number of
alternative solvents have been utilized in the literature for
drug dissolution for biological experiments, one of which
is DMF.
There is surprisingly little information on the inherent
cytotoxicity of DMF. The highest percentage DMF uti-
lized in cytotoxicity assays is 0.25 % in cell culture media,
with subsequent 1:3 serial dilutions of drug reducing it fur-
ther (see “Materials and methods”). We have previously
shown that DMF had no deleterious effect on cisplatin
cytotoxicity when used as the stock solution solvent,
but that DMF did cause cell death at higher concentra-
tions than would be utilized in in vitro assays (3 %) [31].
To determine whether DMF could be utilized as a general
solvent for Pt complexes in biological assays, its effect on
cellular viability was examined to ensure solvent toxic-
ity would not exclude its use. KB-3-1 cells were seeded,
then exposed to varying concentrations of DMF from 8.3
to 0.001 % (1.1 mM–164 μM). After 4, 24, 48 and 72 h,
cellular viability was determined, with longer incubation
Lipophilicity of fluorescent compounds was assessed
by distribution coefficient ([octanol]:[phosphate-buffered
saline, pH 7.2] partition, log D) using fluorescence meas-
urements and compared to literature data for the parent Pt
complexes cisplatin (1) and [PtCl2(en)] (2) (Table 1) [35].
The CFDA–Pt and CF–Pt complexes partitioned pref-
erentially to the aqueous layer (both logD values~−2),
similar to 1 and 2. The Pt complexes with BODIPY both
had positive logD values (BODIPY–Pt logD = 0.98,
BODIPY–prop–Pt logD = 0.66). The slightly lower logD
of BODIPY–prop–Pt can be accounted for based on the
second hydrophilic amide incorporated into the propana-
mide linker. Miller et al. reported a logP value of 0.58 for
BODIPY–Pt, which is slightly lower than our report, but
as their partition was into unbuffered water (which they
defined as logP) our discrepancy is not unreasonable.
Excipient interactions and utility
Given the limited aqueous solubility of the platinum–fluo-
rophore complexes, an organic solvent was required to
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times producing toxicity at higher percentages (Supple-
mentary Figure 2). No effect on viability occurred at 4 h,
and at 24 and 48 h concentrations lower than 1 % did not
affect viability. After 72 h, 0.34 % DMF had little effect on
cell viability, with 1 % DMF inhibiting cell growth almost
completely. This indicates that at the percentages of DMF
utilized in biological assays, there should be no contribut-
ing effect from DMF.
sub-lines, KB-CP.5 and KB-CP20, which show low- and
high-grade resistance, respectively. The Boc-protected dyes
were also assessed as controls to ascertain whether the dyes
possessed biological activity independent of Pt. Cisplatin
and [PtCl2(en)] were tested as positive controls. Consist-
ent with previous results for cisplatin, CP.5 cells showed
resistance to cisplatin (tenfold) or [PtCl2(en)] (40-fold).
Against CP20 cells, [PtCl2(en)] did not achieve an IC50
(up to 500 μM), and were 60-fold resistant to cisplatin. Pt–
Boc was less cytotoxic than its analog [PtCl2(en)] (KB-3-1
IC50 = 16.7 0.9 μM), but cross-resistance profiles were
similar (CP.5 cells tenfold resistant, CP20 cells 26-fold
resistant), indicating that the conjugation to the ethane-
1,2-diamine backbone did not inactivate the complex.
The BODIPY–Pt complex showed activity against
KB-3-1 cells (IC50 = 35.7 1.5 μM), and CP.5 and CP20
cells showed cross-resistance. Miller et al. reported the
IC50 of this complex against two ovarian cancer cell lines
to be >700 μM; however, they dissolved the complex in
DMSO [23].When we dissolved BODIPY–Pt in DMSO,
the IC50 against KB-3-1 was reduced by an order of mag-
To assess DMF’s utility, we examined whether it modi-
fied Pt–Boc. This was examined in two ways. First, Pt–Boc
(3) was dissolved in DMSO-d6 and DMF-d7 and stored in
the dark at room temperature. 1H NMR spectra of the solu-
tions were collected over time (0 h to 1 week, Fig. 3).
As expected based on previous work, DMSO modified
Pt–Boc. The 0-h sample spectrum was collected immedi-
ately after dissolution, and examination of peaks in the NH
region (highlighted in Fig. 3, en ligand at 5.3 and Boc-pro-
tected amine at 6.7) revealed minor peaks already appear-
ing upfield of the parent peaks due to reaction with DMSO.
The reaction with DMSO appeared to be complete at 24 h
because there were no evident subsequent changes to the
spectrum, and at least two products existed in solution,
with little to no parent compound remaining. In contrast,
DMF resulted in no noticeable spectral changes after stor-
age at room temperature for 1 or 10 weeks. Second, these
observations were supported by ESI–MS (positive mode)
spectra of DMF and DMSO solutions of Pt–Boc (10 μM)
collected after 48 h. In DMF, the parent peak corresponded
to unmodified Pt–Boc (m/z = 647.1, Pt–Boc + NH4+), and
no peaks could be assigned to DMF-related adducts (Sup-
plementary Figure 3). The mass spectrum of Pt–Boc in
DMSO revealed a base peak corresponding to the replace-
ment of a Cl ligand by DMSO (m/z = 671.1, [PtCl(DMSO)
(enBoc)]+), consistent with the reaction products of cisplatin
and other platinum(II) complexes previously reported
[31, 37, 38].
We compared the cytotoxicity of cisplatin and
[PtCl2(en)] dissolved in DMF and in saline (consistent with
the clinically formulated solution) towards KB-3-1 cells.
This confirmed that DMF did not deactivate either com-
plex (Table 1). Furthermore, cisplatin-resistant KB-CP.5
cells demonstrated the same degree of cross-resistance to
both complexes in either solvent, but IC50 values were not
reached against the highly resistant KB-CP20 cells. The
maximum concentration tested was lower, as the saline
stock solutions are prepared to 3 mM, while DMF stock
solutions can be prepared at higher concentrations. As
expected, [PtCl2(en)] was less cytotoxic than cisplatin [39].
nitude to 391.7
35.2 μM, confirming that the lack of
activity reported by Miller et al. was probably due to
inactivation by DMSO, rather than the inherent inactivity
of the complex. The BODIPY–prop–Pt complex showed
the greatest cytotoxicity of any Pt–dye complex against
KB-3-1 cells (IC50 = 35.7
1.5 μM), with cross-resist-
ance to CP.5 (twofold) and CP20 (tenfold) cells.
The CFDA–Pt complex showed low-level activity
against KB-3-1 cells (IC50 = 71.6
29.5 μM), and both
CP.5 (fourfold) and CP20 (sixfold) cells showed cross-
resistance. The Boc-protected dye CFDA–Boc also demon-
strated cytotoxicity towards cells (IC50 = 116.1 8.9 μM),
weaker than that of the Pt–CFDA complex, but with equal
activity against the cisplatin-resistant cells. The CF–Pt
complex, representing the hydrolyzed dye form of CFDA–
Pt, did not demonstrate cytotoxicity up to 500 μM, nor did
CF–Boc (Table 1). This is likely due to the negative charge
of the complex conferred by the organic acid groups, and is
reflected in its slightly lower logD value (−2.66).
DNA damage potential
To assess whether the Pt–dye complexes could elicit DNA
damage and cell killing normally elicited by cisplatin [2],
we examined phosphorylation of the histone H2A fam-
ily member H2A.X. H2A.X is phosphorylated at serine
139 (termed γH2A.X) as part of the cellular response to
DNA damage, recruiting DNA-repair proteins, and is also
phosphorylated during apoptosis induced by DNA dam-
age [40]. H2A.X has been shown to be phosphorylated in
cells exposed to cisplatin, carboplatin, and oxaliplatin [41,
42], and to correlate with the cytotoxic potency of platinum
Activity of platinum–fluorophore complexes
Compounds were tested for their inherent cytotoxicity
towards parent KB-3-1 cells, and two cisplatin-resistant
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Fig. 3 Timecourse 1H NMR
spectra of Pt–Boc dissolved in
a DMF and b DMSO. Stabil-
ity of Pt–Boc in solution was
measured at 0, 24, 48, 72 h,
1 week, and in the case of DMF
10 weeks. The NH region of Pt–
Boc is highlighted in the spectra
by a red bar
complexes [31]. Cells were treated with compounds, and
γH2A.X foci were evaluated by immunofluorescence
microscopy after 24 h treatment (Fig. 4, magenta). The
antibody absorbed and fluoresced at different wavelengths
to the compounds to ensure fluorescence spillover was not
responsible for false-positive signals. Cells treated with
cisplatin, [PtCl2(en)], and Pt–Boc elicited a strong DNA
damage response compared with control (untreated) cells.
While CF–Pt and CFDA–Pt did not produce γH2A.X
foci even at high concentrations (100 μM), BODIPY–Pt
and BODIPY–prop–Pt did so at 50 μM. This is sup-
ported by Miller’s observation that fluorescently labeled
53BP1 protein localized to the nucleus in cells treated with
BODIPY–Pt (50 μM). We also examined the ULYSIS 546
platinum complex at 100 μM (Supplementary Figure 4).
The concentration of ULYSIS 546 reagent provided by the
vendor is not disclosed, but using absorption spectrom-
etry a standard curve was generated with AlexaFluor 546
dye to determine the ULYSIS 546 reagent concentration
to be 100 μM. No γH2A.X foci were evident in ULYSIS
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Fig. 4 Assessment of the ability of dyes and platinum–dye con-
jugates to elicit DNA damage. a Control, b cisplatin (5 µM), c
[PtCl2(en)] (25 µM), and d Pt–Boc (25 µM) are non-fluorescent com-
pounds. Fluorescent compounds are shown in e CFDA–Pt (100 µM),
f CF–Pt (100 µM), g BODIPY–Pt (50 µM), and h BODIPY–prop–
Pt (50 µM). Each image shows the fluorescent compound (green),
H2A.X (magenta), and a merge with the DAPI nuclear stain (blue)
546-treated cells. None of the fluorophore–Boc com-
pounds produced foci associated with γH2A.X, indicating
that while they were weakly cytotoxic, the dyes could not
induce DNA damage independent of platinum (Supplemen-
tary Figure 5).
25 % less BODIPY–Pt than parent cells, while
BODIPY–Boc accumulation was unaffected in resistant
cells.
The Pt–CF also showed a small but statistically signifi-
cant 17 % reduction in accumulation, while the CF–Boc
control was unaffected. In contrast, the control CFDA–
Boc dye showed a large 80 % reduction in accumulation
in resistant cells, whereas Pt–CFDA accumulation actually
increased in CP20 cells. The absolute fluorescence meas-
ured in CFDA-treated KB-3-1 cells was two orders of mag-
nitude greater than the CF-treated cells, consistent with the
hydrolytic trapping of CFDA dyes in cells, and the poor
cellular permeability of CF dyes. The weakly resistant CP.5
cell line does not show reduced levels of Pt compared with
KB-3-1 cells, and this was the case for the Pt–dye com-
plexes examined here (not shown).
Cellular distribution of Pt–fluorophore complexes
One feature of many cisplatin-resistant cells is dimin-
ished accumulation compared with parent cells [43]. We
examined the cellular accumulation of fluorophore–Boc
and Pt–fluorophore complexes in KB-3-1 and CP20 cells
using flow cytometry, to examine whether resistant cells
accumulate lower levels of Pt–dye consistent with previ-
ous observations for Pt drugs [22]. Cells were incubated
with compounds for 45 min, then cellular fluorescence
was measured, and values normalized against accumula-
tion in parental cells (Fig. 5). CP20 cells accumulated
We next examined the cellular localization of fluoro-
phore–Boc and Pt–fluorophore complexes (10 μM) in
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1091
fluorophores conjugated to Pt (excitation 488 nm) (Fig. 4).
Cells were treated with 50 μM compound (compared with
10 μM for the live microscopy described above). At this
higher concentration, both BODIPY platinum complexes
demonstrated cytosolic and nuclear localization, as did
the CFDA–Pt complex, while CF–Pt did not produce any
signal (Fig. 4). This is consistent with observations using
XRF to examine cisplatin distribution in A2780 ovarian
carcinoma cells, which showed nuclear and cytosolic pools
of cisplatin [45]. At the same concentration, the dye con-
trols produced minimal cellular signal (Supplementary
Figure 5).
Pt–fluorophore complexes in multicellular systems
We also examined the permeability and activity of
BODIPY–Pt in three-dimensional (3D) culture. There
is surprisingly little literature on the activity of plati-
num complexes in 3D culture compared with 2D and no
literature we could identify examining the behavior of
cisplatin-resistant cell lines grown in 3D [46, 47], despite
the fact that cells growing in multicellular structures have
been shown to have different response patterns to cyto-
toxic agents [48]. These differences stem from factors
such as high interstitial pressure, hypoxia, a large pro-
portion of cells being quiescent (not dividing) and high
metabolite concentrations leading to extracellular acidity
[49]. We established KB-3-1 and KB-CP.5 cells as sphe-
roids by seeding them in Matrigel and allowing growth
for 1 week (until spheroid diameter was approximately
100 μm). Spheroids were incubated with compounds for
1 h, and then confocal microscopy was used to examine
the distribution (Fig. 7). The BODIPY–Boc control dem-
onstrated higher accumulation at the periphery of the
spheroid, supported by the intensity histogram generated
from a cross-section (Fig. 7c, d). This is consistent with
observations for many small molecules, with limited diffu-
sion into spheroids for a range of reasons including lyso-
somal and other trapping mechanisms [50, 51]. In contrast,
BODIPY–Pt did not appear to accumulate at the periph-
ery, with little accumulation occurring in the outer leaflet
of the spheroids but strong fluorescence in the center. This
unusual accumulation feature was not related to cell death,
as spheroids were treated with a low concentration (10 μM
for 1 h). The CF dyes showed little to no accumulation
in spheroids, and while the CFDA–Boc demonstrated a
ring-like fluorescence at the spheroid periphery, CFDA–
Pt demonstrated little to no accumulation in spheroids, in
contrast with monolayer (2D) cells. The CP.5 spheroids
demonstrated similar cell distribution patterns to KB-3-1
spheroids (Supplementary Figure 8).
Fig. 5 Cellular accumulation of the dyes and dye–platinum conju-
gates at 25 µM for 45 min in KB-3-1 and CP.20 cells assessed by flow
cytometry. Values shown are an average of a triplicate and error bars
represent standard deviation, and for each compound uptake is nor-
malized to florescence in KB-3-1 cells. A Student’s T test was per-
formed for each compound between the two cell lines. Comparisons
that are statistically significant are marked (p value < 0.05* < 0.01**
< 0.001*** < 0.0001****)
KB-3-1 cells using live-cell confocal microscopy fol-
lowing incubation for 1 or 24 h (Fig. 6). CF–Boc showed
very low cellular signal, whereas CFDA–Boc, CF–Pt and
CFDA–Pt localized to punctae and ring-like structures
at the cell periphery consistent with previous reports of
endocytosis and vesicular localization of FDDP [17, 18].
CFDA–Pt was distributed evenly through the cell at 1 h,
but at 24 h it was not observed except in occasional cells
that appeared to be dead or dying (rounded). The same
was true for the Boc-protected dye, suggesting elimina-
tion or metabolism of the dye in cells over time. Both
BODIPY–Pt and BODIPY–prop–Pt showed stronger cel-
lular signal at 1 h compared with 24 h, with diffuse sig-
nal observed in the perinuclear space, and no discernible
co-localization with the DNA dye Hoechst. The ULYSIS
546 platinum complex appeared in KB-3-1 cells diffusely
distributed in the perinuclear space at 1 h, and as large
vesicular structures at 24 h (Supplementary Figure 6). In
the resistant CP.5 cells, compounds were strongly local-
ized in large vesicles at 1 h, and in most cases these
were largely absent from cells after 24 h (Supplemen-
tary Figure 7). This is consistent with previous observa-
tions that resistant cells sequester platinum complexes in
vesicular structures [44].
The H2A.X microscopy of fixed cells pre-treated with
compounds also allowed examination of fluorescence, as
the H2A.X antibody (AlexaFluor 647-conjugated, excita-
tion 633 nm) spectral properties do not overlap with the
To determine whether the cisplatin-resistant cells main-
tained their phenotype in 3D, we examined the activity of
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Fig. 6 Confocal images of live KB-3-1 cells in culture after incu-
bation with dye or dye–platinum conjugate at 10 µM for either 1 or
24 h: a CF–Boc 1 h, b CF–Boc 24 h, c CF–Pt 1 h, d CF–Pt 24 h,
e CFDA–Boc 1 h, f CFDA–Boc 24 h, g CFDA–Pt 1 h, h CFDA–Pt
24 h, i BODIPY–Boc 1 h, j BODIPY–Boc 24 h, k BODIPY–Pt 1 h,
l BODIPY–Pt 24 h, m BODIPY–prop–Pt 1 h, and n BODIPY–prop–
Pt 24 h. Each image represents a merge of the Hoechst nuclear stain
(1 µg/mL blue) and the fluorescent compound (green)
cisplatin, [PtCl2(en)] and BODIPY–Pt against cells grown
in Matrigel. One common strategy for assessing response
to cytotoxins in 3D is to grow spheroids on a non-adher-
ent surface such as agarose, and assess cellular response
based on changes in diameter (and, therefore, spheroid
volume) or by a biochemical read-out [52]. Cells can also
grow as spheroids in protein matrices such as Matrigel,
and while viability assays are complicated by 3D archi-
tecture for a number of reasons [53], we utilized CellTiter
Glo viability assays (which assay ATP content) with a
prolonged incubation period (60 min) to ensure all cells
were lysed [54].
Two Matrigel growth conditions were examined. Cells
were plated in Matrigel, and allowed to establish for 24 h
or 4 days before addition of compounds. At 24 h, cells
were considered to be single cells, whereas at 4 days mul-
ticellular spheroids were apparent in Matrigel (observed by
transmission microscopy, not shown). We examined these
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J Biol Inorg Chem (2015) 20:1081–1095
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Fig. 7 Distribution of
BODIPY–Boc and BODIPY–Pt
at 10 µM in KB-3-1 three-
dimensional spheroids grown
in Matrigel. Confocal images
show a BODIPY–Pt and b
BODIPY–Boc distribution. The
optical slice of each image is
4.0 µM. Each image shows the
fluorescent compound (green)
merged with the bright field
image. Graphical representation
of fluorescence intensity plotted
as a factor of distance across
spheroid for c BODIPY–Pt
and d BODIPY–Boc. Arrows
signify the boundaries of the
spheroids
Table 2 Cytotoxicity (IC50, μM) of compounds against parental
KB-3-1 and cisplatin-resistant KB CP.5 cell lines
Matrigel conditions for two reasons. First, we wanted to
assess if differences in drug response were due to multi-
cellularity versus an effect conferred simply by the pres-
ence of Matrigel. For example, it has been shown that
Matrigel, as a dense protein solution, participates in drug–
protein binding [55], though the extent of cisplatin binding
to Matrigel is not known. Second, we wondered whether
the single-cell suspension of cells in Matrigel responded to
cisplatin in a similar manner to 2D cells, which also have
minimal cell–cell contacts.
Culture method
IC50 (μM)
2D
3D 1 Day
3D 4 Day
Cisplatin (1)
KB-3-1
2.5 0.1
4.5 0.7
9.4 0.7
CP.5
21.0 2.8
22.9 4.6
32.1 6.9
[PtCl2(en)] (2)
KB-3-1
4.1 0.2
21.4 1.0
150a
CP.5
107.4 10.9
297.7 21.8
>500
As anticipated based on general literature evi-
dence, the spheroids (3D 4 day) were less sensitive
to cisplatin than monolayer (2D) cells (Table 2, 2D
IC50 = 2.5 0.1 μM, 3D 4 day IC50 = 9.4 0.7 μM).
The same pattern was observed for [PtCl2(en)]. Sphe-
roids formed from CP.5 cells retained a resistant pheno-
type towards cisplatin (3D 4 day: 3.4-fold resistant vs.
2D: 8.4-fold resistant) and [PtCl2(en)], though the degree
of relative resistance was diminished. The sensitivity of
single-cell Matrigel suspension (3D 1 day) was interme-
diate between 2D cells and 3D spheroids. In most cases
CP.5 cells grown in 2D and 3D 1 day showed statisti-
cally equivalent sensitivity. The BODIPY–Pt complex
also showed reduced efficacy against 3D 4-day sphe-
BODIPY–Pt (9)
KB-3-1
36.9 1.3
53.8 7.1
140.9 6.6
CP.5
141.5 12.5
185.7 47.2
299.5 92.2
Cells were analyzed in 2D culture and 3D culture grown for either 1
or 4 days
a
Single data point
Conclusions
In this study, we examined several fluorophore-conjugated
platinum complexes and assessed them in the context of
the mechanism of action of cisplatin. The BODIPY-teth-
ered platinum complexes demonstrated elevated cytotoxic-
ity compared with the CF/CFDA-conjugation complexes,
roids (Table 2, 2D IC50 = 36.9
1.3 μM, 3D 4 day
IC50 = 140.9 6.6 μM), spheroids were cross-resistant.
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cross-resistance to cisplatin-resistant cells and phosphoryl-
ation of H2A.X. At higher concentrations, nuclear locali-
zation of the BODIPY–platinum complexes was observed,
along with cytosolic/perinuclear staining that could be
detected at lower concentrations. This observation is con-
sistent with results from other techniques examining Pt
distribution in cells, as diverse as sub-cellular fractionation
[56, 57] and XRF/SRIXE [45]. Our observations of activity
are consistent with that of Miller et al. who used a locali-
zation assay to demonstrate that 53BP1 relocalized to the
nucleus following BODIPY–Pt treatment, with the qualifi-
cation that DMSO as a solvent inactivates these complexes
as demonstrated here. In other words, while DNA binding
correlates with cytotoxicity of Pt complexes, the majority
of Pt in a cell is not bound to DNA. The CF and CFDA
platinum complexes demonstrated poor cytotoxicity, and
DNA localization and H2A.X phosphorylation was not
observed.
The main discrepancy appears to be related to uptake
defects in cisplatin-resistant cells. While cisplatin-resist-
ant cell lines (CP.5 and CP20) showed cross-resistance
to Pt–fluorophore complexes indicating, the reduction
in accumulation measured by flow cytometry (Fig. 5) for
BODIPY–Pt and CF–Pt is less than that observed for cells
exposed to agents such as [14C]carboplatin, cisplatin, and
dyes used as markers of endocytosis (50–75 % reduction)
[22, 58]. This result suggests that the platinum–fluorophore
complexes must enter the cell through pathways somewhat
different from those used by the compounds listed above.
Exactly what the pathway of these BODIPY–platinum
compounds is within the cell to the nucleus remains to be
determined, but since the cisplatin-resistant cells that we
examined are resistant to these compounds, and their defect
is in uptake and intracellular trafficking of platinum deriva-
tives, it appears likely that they share intracellular traffick-
ing pathways with clinically used cytotoxic platinum com-
pounds. We believe these BODIPY complexes demonstrate
improved potential for understanding the intracellular traf-
ficking of platinum compounds in vitro and in vivo.
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Acknowledgments This research was supported by the Intramural
Research Program of the National Institutes of Health, National Can-
cer Institute. We thank George Leiman for editorial assistance, and
Robert O’Connor (NIDDK) for assistance with NMR acquisition.
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Compliance with ethical standards
Conflict of interest None declared.
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