A long-lived cuprous bis-phenanthroline complex for the photodynamic therapy of cancer

Article abstract of DOI:10.1039/c8dt00140e

Copper is an earth-abundant and a biologically essential metal that offers a promising alternative to noble metals in photochemistry and photobiology. In this work, a series of sterically encumbered Cu(i) bis-phenanthroline complexes were investigated for their use in photochemotherapy (PCT). It was found that Cu(dsbtmp)₂⁺ [dsbtmp = 2,9-disec-butyl-3,4,7,8-tetramethyl-1,10-phenanthroline] (compound 3), which possessed the longest excited state lifetime, exhibited significant in vitro photocytotoxicity on A375 (human malignant melanoma) and A549 (human lung carcinoma) cell lines. Fluorescence imaging demonstrated the significant uptake and localization of compound 3 in a perinuclear fashion. A comet assay indicated the induction of DNA damage in the dark. The DNA breaks were significantly amplified upon photoactivation. The light-induced enhancement of cytotoxicity was associated with the formation of reactive oxygen species (ROS), a known intermediate in photodynamic therapy (PDT). This successful demonstration of photocytotoxicity using long-lived cuprous phenanthroline paves the way to exploit this class of photosensitizers for PDT applications.

Full text of DOI:10.1039/c8dt00140e

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10.1039/C8DT00140E.
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Long-lived cuprous bis-phenanthroline complex for the
photodynamic therapy of cancer
Cynthia Al Hageh,a Majd Al Assaad,a Zeinab El Masri,a Nawar Samaan,a Mirvat El-Sibai,a Christian
Khalil,a and Rony S. Khnayzera,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Copper is an earth-abundant and a biologically essential metal agents. On one hand, photodynamic therapy (PDT) entails the
that offers
a promissing alternative to nobel metals in use of light energy to catalytically activate drugs with spatial
photochemistry and photobiology. In this work, a series of and temporal control.6, 7 On the other hand photoactivatable
sterically encumbered Cu(I) bis-phenanthroline complexes were chemotherapy (PACT) relies on the photochemical
investigated for their use in photochemotherapy (PCT). It was transformation of a prodrug into a drug.8, 9 In this perspective,
+
Cu(dsbtmp)2 [dsbtmp=2,9-disec-butyl-3,4,7,8- targeting tumours with PDT or PACT minimizes the damage to
found that
tetramethyl-1,10-phenanthroline] (compound 3), which possessed healthy tissues and organs. In PDT and PACT, variety of
the longest excited state lifetime, exibited significant in vitro transition metal complexes have been investigated especially
photocytotoxicity on A375 (human malignant melanoma) and ones based on noble metal centres such as Ru, Os and Pt.7-9
A549 (human lung carcinoma) cell lines. Fluorescence imaging However, very few studies were aimed at utilizing metal
demonstrated the significant uptake and localization of compound complexes based on earth-abundant copper core for PDT
3 in a perinuclear fashion. Comet assay indicated the induction of applications. Copper is a mineral present in the body with
DNA damage in the dark. The DNA breaks were significantly important physiological functions.10 Cupric and cuprous
amplified upon photoactivation. The light-induced enhancement complexes have shown anticancer activities, and were a topic
12
of cytotoxicity was associated with the formation of reactive of extensive review articles by Santini and coworkers.11,
oxygen species (ROS), a known intermediate in photodynamic Despite the fact that Cu(I) is accepted to be the form
therapy (PDT). This successful demonstration of photocytotoxicity internalized by cells via copper transporter (CTR) protein,13
using long-lived cuprous phenanthroline paves the way to exploit research on Cu(II) complexes had significantly outnumbered
this class of photosensitizer for PDT applications.
Cu(I) as anticancer agents.12 Cu(II) complexes bearing
semicarbazones,14
f]quinoxaline,16
pyrenyl-terpy,15
dipyrido[3,2-a:20,30-c]phenazine,16
dipyrido[3,2-d:20,30-
and
ferrocenyl-terpy17 ligands were successfully exploited for PDT.
The change of redox properties of cupric compounds through
the introduction of redox active ligands was proven to be
advantageous for PDT application.18 The lack of detailed
studies on Cu(I) could be due to the instability of the majority
of four coordinate complexes in basic solvents such as water.12
The redox properties of a number of Cu(I) complexes was
related to their ability to produce reactive oxygen species
(ROS) in the dark.19, 20 Notably, some Cu(I) complexes showed
selectivity to specific cancer cells with no activity detected on
the healthy cells.21 Importantly, previously investigated
cuprous phenanthroline complexes showed capacity to
interact and sometimes cleave extracted DNA.22, 23 Moreover,
[Cu(phen)2]+ (phen = 1,10-phenanthroline) was found to act as
artificial nuclease,23-26 and [Cu(dmp)2]+ (dmp = 2,9-dimethyl-
1,10-phenanthroline) demonstrated the ability to bind with
DNA through surface association at the minor groove of
DNA.22, 27, 28 Additionally, [Cu(bcp)2]+ (bcp = 2,9-dimethyl-4,7-
Introduction
Cancer is currently associated with large mortality rates due to
its late prognosis.1 Despite significant improvements achieved
in prevention, early detection and treatment of patients,
cancer is still one of the leading causes of death worldwide.1
Cisplatin is a common chemotherapeutic drug with a platinum
noble metal centre.2 Nevertheless, its inherent cytotoxicity
and lack of selectivity lead to side effects and development of
cellular resistivity.3 Since its discovery and effective use in
clinical settings, research on cisplatin analogues and their
4,
5
mechanism of action is on the rise.
The undesirable
characteristics of cisplatin have triggered interest to
investigate new generations of more specific anticancer
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diphenyl-1,10-phenanthroline) was believed to intercalate via bathocuproine or 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline),
22, 27, 28
the major groove.
However, to the best of our [Cu(dsbtmp)2]+ (3) (dsbtmp = 2,9-disec-butyl-3,4,7,8-tetramethyl-
knowledge, these Cu(I) bis-phenanthroline complexes were 1,10-phenanthroline), [Cu(dpphen)2]+ (4) (dpphen = 2,9-diphenyl-
not tested on living cells, and their study was limited to 1,10-phenanthroline), Scheme 1. The hexafluorophosphate salts
interaction with extracted DNA.22, 27-30 In this study we aim at of cuprous compounds
1, 2, and 4 were synthesised using a
studying the in vitro cytotoxicity under dark and slightly modified literature method36 with yield ranging from
photoactivated conditions of these complexes among others 82-93%. Two equivalents of phenanthroline derivative ligands
on cancer cell lines.
were dissolved in 10 mL of methanol. The resultant solution
In general, Cu(I) bis-phenanthroline complexes absorb visible was degassed with argon for 1 hour then added by cannula
light that is attributed to metal-to-ligand charge transfer while stirring to solid Cu(CH3CN)4PF6 under argon atmosphere.
31
(MLCT) transition.22,
Upon photoexcitation, an electron is An orange color was immediately observed. The solution was
promoted from a d10 HOMO ground state to a π* LUMO orbital left stirring under argon for 1 hour. The precipitation was done
residing on one of the ligands, formally resulting in a transient by adding the resultant solution to a vigorously stirring 50 mL
Cu(II) d9 electronic configuration. In the excited state, of diethyl ether. The solid was filtered through a glass frit then
molecules can undergo a “flattening” distortion towards a D2 washed with diethyl ether. The products were recrystallized
configuration. In this flattened geometry the transient Cu(II) from methanol by slow addition of diethyl ether. The solids
centre is coordinatively unsaturated and prone to Lewis base were stored in air until use. The hexafluorophosphate salts of
(solvent) attack.32 This excited state pseudo Jahn-Teller compound
distortion decreases the lifetime of Cu(I) complexes and limits method.34
their use for bimolecular photochemistry. One of the most
efficient strategies used to prevent this Cu(I) geometrical
distortion flattening of complexes in solutions is the
introduction of steric hindrance in the coordination sphere
using bulky ligands.29 Sterically congested Cu(I) complexes
bearing diimine and/or phosphine based ligands possessed
improved excited state lifetimes and photoluminescence
quantum yields.33 The 2,9 substituents on the phenanthroline
have been proven to extend the lifetime of the excited state of
Cu(I) complexes with record values recently reported by
Castellano and coworkers at the microsecond time scale.34
Substituents on the 2,9 positions were also found to increase
the longevity of the complexes by protecting the Cu(I) core in
coordinating solvents environment.33 In this work, a series of
Cu(I) bis-phenanthroline was investigated and the
photocytotoxicity was assessed in vitro. The compounds
utilized here possessed excited state lifetimes ranging from
nanosecond up to a microsecond, allowing a correlation
between the photophysical properties and their application in
PDT.
3 was synthesized according to a literature
Experimental Methods
Materials. DMEM culture media was used with 10% FBS (fetal
bovine serum) and 1% antibiotics. DCFDA (2′,7′-
dichlorofluorescin diacetate) cellular ROS detection assay kit
Scheme 1. Chemical structures of compounds 1-4.
for microplate assay were purchased from Abcam. The comet
assay kit was purchased from Trevigen. 2,9-diphenyl-1,10-
phenanthroline was synthesized according to a published
procedure.35 The remaining ligands, salts, and solvents (HPLC
grade) were purchased from Aldrich and used without further
purification.
1
Compound 1, [Cu(dmphen)2](PF6), yield 93%. H NMR (CDCl3,
300 MHz), δ: 2.43 (s, 12H), 7.78 (d, 4H), 8.02 (s, 4H), 8.49 (d,
4H). Anal. Calcd for C28H24N4-CuPF6: C, 53.81; H, 3.87; N, 8.96.
Found: C, 53.90; H, 3.86; N, 8.81.
Compound 2, [Cu(BC)2](PF6), yield 82%. 1H NMR (CDCl3, 300
MHz), δ: 2.60 (s, 12H), 7.60 (m, 20H), 7.75 (d, 4H), 8.04 (d, 4H).
Anal. Calcd for C52H40N4CuPF6: C, 67.20; H, 4.34; N, 6.03.
Found: C, 67.46; H, 4.48; N, 5.97.
Compound 3,
[Cu(dsbtmp)2](PF6), yield 80%. 1H NMR (CDCl3,
300 MHz) 1H δ 8.20 (s,2H), 3.68−3.10 (m, 2H), 2.78 (t, J = 6 Hz,
Synthesis and characterization. In this study four sterically
encumbered cationic Cu(I) phenanthroline derivatives were
synthesized and characterized: [Cu(dmphen)2]+ (1) (dmphen =
2,9-dimethyl-1,10-phenanthroline), [Cu(BC)2]+ (2)
(BC
=
2 | J. Name., 2012, 00, 1-3
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prepared in 2 mL Eppendorf tubes and dissolved in 1 mL of
octanol saturated with water. The compounds were dissolved
for 30 minutes in a sonicator bath set at room temperature,
the solution was subjected to centrifugation for 1 minute at
4300 rpm, followed by pipetting out the supernatant. Then, 1
mL of water saturated with octanol was added and the
mixtures were shaken at 2200 rpm for 1 hour at room
temperature. After that, two phases solutions were formed, an
upper phase with octanol solvent and a lower phase with
water solvent. The syringe was washed with water saturated
with octanol prior to the aqueous phase aspiration, and with
octanol saturated with water prior to the octanol phase
aspiration. The aqueous phase was aspirated using a glass
syringe, air was expelled while the needle was passed through
the octanol phase to prevent any traces of octanol to insert
into the needle. Then, the octanol phase was aspirated. The
UV-Vis spectra of the solution placed in a micro-cuvette was
then performed using Cary 60. The octanol phases were
appropriately diluted to keep the absorbance within linear
range. The log P values were calculated based on the following
formula:
6H), 2.52 (t, J = 12 Hz, 6H), 1.76−1.22 (m, 4H), 1.12−0.76 (m,
6H), 0.21 to −0.18 (m, 6H).
1
Compound 4, [Cu(dpphen)2](PF6), yield 86%. H NMR (CDCl3,
300 MHz), δ: 6.53 (t, 8H), 6.77 (t, 4H), 7.39 (d, 8H), 7.88 (d, 4H),
8.01 (s, 4H), 8.51 (d, 4H). Anal. Calcd for C48H32N4CuPF6: C,
66.02; H, 3.69; N, 6.42. Found: C, 65.99; H, 3.72; N, 6.31.
The water-compatible Cu(I) complexes chloride salts of
compounds 1, 2 and 4 were obtained by dissolving 20 mg of
Cu(NN)2PF6 in 10 mL of water and methanol mixture of
different ratio (between 1:1 to 1:2) for each reaction to obtain
a saturated solution. 5 g of pre-washed dowex-22 chloride
form beads were added to the resultant solution. The mixture
was left stirring for 2 h at room temperature. The solution was
filtered on glass frit and the filtrate was completely dried using
rotary evaporator. The resulting orange solid was re-dissolved
in 10:1 water:methanol solution then filtered on glass frit of
medium porosity. The filtrate was then dried using rotary
evaporator followed by vacuum oven. The orange solid was
then dissolved in minimum amount of methanol and purified
using Sephadex LH-20 column. The solvent of the collected
orange band solution was then removed using the rotary
evaporator and the solid dried using a vacuum oven. The solid
products were stored in air until further use. Attempts to
convert compound 3 from the hexafluorophosphate to chloride salt
were not successful using the same strategy used for the remaining
log ꢀ = log(ꢁꢁ
) where the ratio of concentrations was
ꢂꢃꢄꢅꢆꢂꢇ
ꢈꢅꢄꢉꢊ
photometrically determined.
compounds. The water-compatible triflate salt of compound
3
was synthesized using a published procedure34 but using
tetrakis(acetonitrile) copper(I) triflate instead of the
hexafluorophosphate salt. ESI-MS analysis of the water-
compatible complexes matched the predicted M+ for each
Alkaline comet assay. The comet assay was performed
according to a previously published method38 using Trevigen
comet assay silver staining kit.
compound: Compound
1
ESI-MS: m/z 479.13 (M+), compound
ESI-MS: m/z 759.56
4
ESI-MS: m/z 727.33 (M)+.
Quantitative measurement of cellular Reactive Oxygen
Species (ROS) in cells. 20 mM DCFDA (in DMSO), 10X Buffer,
55 mM tert-butyl hydrogen peroxide (TBHP) were included in
the kit. 1X supplemented buffer made up of 10% FBS in 1X
buffer was considered as negative control. 200 µM TBHP in 1X
supplemented buffer was considered as positive control. 3 µM
2
ESI-MS: m/z 783.44 (M+), compound
3
(M)+ and compound
UV-vis titration. Compound
3 was dissolved in 50% DMSO in
Tris-HCl buffer (5 mM, pH=7.2). The absorbance of the
compound in 50% DMSO was measured using UV-vis (Cary 60)
with initial absorbance of 0.47 at λ=450 nm.
of compound
3 were prepared in 1X supplemented buffer.
Two adherent cell lines were used A375 and A549. Cells were
cultured in media without phenol red and were incubated
overnight. Then cells were harvested, seeded in two dark sided
96-well microplate with 100 µL containing ~25,000 cells per
well and incubated overnight. The following day, cells were
washed with 1X buffer then incubated with 100 µL of 25 µM
1.5 mg of activated calf thymus DNA type XV (Sigma-Aldrich)
was dissolved to 1 mg/mL in Tris-HCl overnight at 4 ͦC to insure
complete dissolution. The DNA concentration was determined
using UV-vis. The DNA was premixed with a solution of
compound
HCl buffer (5 mM, pH=7.2) containing a DNA concentration of
472 µg/mL and compound with absorbance of 0.47. The
same concentration of compound was used in the DNA stock
3 to achieve a stock solution of 50% DMSO in Tris-
DCFDA solution for 45 min at 37 ͦC in the dark then washed
3
with 1X buffer. Cells were treated in triplicates with a) 100 µL
of 1X supplemented buffer, b) 100 µL of 200 µM TBHP c) 100
3
and the initial solution used in the titration to avoid dilution
effects on the UV-vis spectra. The DNA stock solution was
µL of 3 µM compound
3.
Data were measured on a
fluorescence plate reader Varioskan Flash (Thermo Scientific)
at Ex/Em of 485/535 nm immediately after treatment for
baseline assessment. The light-exposed plate was subjected to
blue LED light illumination for 30 min after 3 h of incubation.
Both the light-exposed and dark plates were then measured to
furnish the final data. In all photobiological experiments,
excitation was performed using a home-built blue LED setup as
previously described.39, 40
added gradually onto the solution containing compound
3 and
after each addition, the solution was homogenised and
allowed to equilibrate for 2 min before the absorbance was
measured.
Lipophilicity. Log P value where P is the water-octanol
partition coefficient were measured using a slightly modified
literature procedure37 while taking into consideration the EPA
guidelines (EPA 712–C–96–038). 2 mg of the compounds were
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Cell survival assay. The cytotoxicity assay was performed using concentration of compound reported in this study, DMSO was
an MTS cell proliferation assay kit. Briefly, the assay measures not cytotoxic and its percentage didn’t exceed 3%. IC50 values
mitochondrial dehydrogenase activity in cell systems. In the of all Cu(I) bis-phenanthroline complexes have been acquired
current investigations human derived immortalized cell in the dark after 24 h incubation on 2 different cell lines: A375
cultures (A549 and A375) were exposed to a serial dilution of (human malignant melanoma) and A549 (human lung
the compounds tested in light and dark conditions. The carcinoma). Control experiments with cisplatin showed
compounds were dissolved in DMSO and diluted in media. The marginal toxicity after 24 h incubation, hence this control was
highest concentration of DMSO was 3% which did not lead to acquired after 48 h of incubation, Fig. S1.
cell death under our experimental setting. The investigation in
the dark was conducted in 96 well pates and post compound
exposure cells were further incubated for 24 h prior to
Compound 1
Compound 2
1.0
addition of the MTS reagents. The photoexcitation was
performed 6 h post incubation for 30 min using the blue-LED
system and incubation was resumed for a total of 24 h. The
resulting coloured solution was then collected and read at 492
nm for mitochondrial activity determination. The data was
then utilized to generate dose response curved using Graph
pad prism software. Experiments were performed in triplicates
in each run and runs were repeated three times for error
determination.
Compound 3
Compound 4
0.8
0.6
0.4
0.2
0.0
Fluorescence imaging and nuclear localization. These studies
were performed as previously described.39 Briefly, A549 and
A375 cells were cultured in DMEM medium supplemented
with 10% FBS and 100 U penicillin/streptomycin at 37˚C and
300
400
500
600
700
Wavelength (nm)
5% CO2. The cells were plated on cover slips 24 h prior to the Fig. 1 Normalized UV-vis spectra of compounds 1-4 of the hexafluorophosphate
salts in acetonitrile (straight lines) and the water-compatible chloride/triflate
experiment. Cells were then treated with compound
3 for 2 h
salts in DMSO (dashed lines). Data were normalized to the MLCT band of each
compound.
and 6 h respectively. After treatment, cells were fixed with 4%
paraformaldehyde for 10 min and blocked with 1% BSA in PBS
for 1 hour. Samples were then stained with DAPI for 10 min.
Fluorescent images were taken using a 60X objective on Axio
observer Z1 fluorescent microscope (from Zeiss).
DMSO was not added to cisplatin samples to avoid
deactivation of the drug. Each value reported in Table 1 was
the average of three independent experiments along with the
standard error on the mean. The cationic compounds 1-4
exhibited dark toxicity with IC50 ranging from ~2 to 26 µM (Fig.
S2 and Fig. 2). Among the most lipophilic compounds,
Results and discussion
complexes
2 and 3 (log P = 2.21 and 2.64 respectively), with
In this study four sterically encumbered cationic Cu(I)
methyl or aryl groups on the 4,7 and/or 3,8 positions of the
2,9-disubstituted phenanthroline, exhibited lower cytotoxicity
phenanthroline derivatives (compounds
based on slightly modified literature methods, characterized
and tested for potential photocytotoxicity.
1-4) were synthesized
on both cell lines when compared to compounds
Compound , the least lipophilic (log P = 1.02) of the tested
Cu(I) compounds, was the most potent (IC50 ~ 1-2 µM).
Compound , albeit displaying a high log P value of 2.18, has
1 and 4.
1
Hexafluorophosphate salts of Cu(I) bis-phenanthroline complexes
were not water soluble and precipitated out of solution at high
percentage of water that is necessary for biological testing.
Compounds 1, 2 and 4 were converted to chloride salts through a
counter-ion exchange procedure40 (see experimental section for
more details) whereas compound 3 was used as a triflate salt to
afford water-compatible complexes. UV-vis spectra (Fig. 1) of the
hexafluorophosphate salts in acetonitrile and the chloride or
triflate salts in DMSO were compared and matched to
reported data.33, 34 Absorption spectra show a distinct metal-
to-ligand charge transfer (MLCT) absorption band between
400-500 nm and an intra-ligand (π to π* transition) band
between 250-325 nm.
4
phenyl groups that are involved in intramolecular π-stacking
interactions41. The variation in the dark cytotoxicity of
compounds 1-4 could not be solely correlated to lipophilicity
since cellular localization and mechanisms of action is
expected to change with the substituents as previously
reported in Ru(II) complexes bearing 4,7-diphenyl-1,10-
42
and Ru(II) arene
phenanthroline ligand framework,39,
complexes43.
Compounds
1 and 4 have previously demonstrated different
types of interactions with extracted DNA, the former was
found to associate at the minor groove of DNA and the latter
to intercalate at the major groove.22, 27, 28 However, detailed
structure-activity relationship in the dark is beyond the scope
of this work. Photocytotoxicity of compounds 1-4 was assessed
on A375 and A549 cells. Cells were incubated with each
Cytotoxicity of water-compatible compounds 1-4 was assessed
by MTS cell proliferation assays. Stock solutions of Cu(I)
compounds were prepared in DMSO and diluted in cell culture
media to achieve the desired concentrations. At the highest
4 | J. Name., 2012, 00, 1-3
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compound for 6 h, photoactivated for 30 min under 460
Research Article
nm/blue LED illumination, then incubated until cell viability Due to plate-to-plate variability, PI below 3 were assumed to
was assessed. The total incubation time with each compound indicate non-photoactivatable compounds. The cytotoxicity of
was 24 h allowing direct comparison between data. The IC50 complexes
was measured and a phototoxicity index (PI = IC50 dark/IC50 whereas complex
light) was calculated for each complex, Fig. 2, Fig. S2 and Table (PI = IC50 dark/IC50 light) of 13 and 83 on A375 and A549 cells
1,
2, and
4
was not enhanced upon photoexcitation
3
possessed significant phototoxicity indices
2.
respectively. The best of our knowledge, complex 3 represents
the first example of cuprous phenanthroline complex that
displays significant in-vitro photocytotoxicity. The improved
potency of complex
3 upon light activation was attributed to
its extended excited state lifetime. Compounds 1-2 possessed
an excited state lifetime of 100 ns in CH2Cl2 which was
significantly reduced to few ns in weakly coordination solvents
such as CH3CN.29, 33 On the other hand, compound
4 displayed
a lifetime of 310 ns in CH2Cl2 and its sulfonated analogue a
lifetime of 70 ns in aqueous medium at room temperature.29
Table 2. IC50 values of compounds 1-4 on A375 and A549 upon light activation. Three
independent measurements were performed to obtain the arithmetic average and
standard error of the mean. The phototoxicity indices (PI = IC50 dark/IC50 light) were
calculated for each compound. Cells were incubated for 24 h with the compound. Light
activation occurred 6 h post incubation and lasted for 30 min using blue-LED. IC50 values
were obtained using GraphPad Prism software by fitting data into a sigmoidal dose
response function.
Compounds Light
1
2
3
4
IC50 on A375 (µM)
PI on A375
2.1±1.0
0.9
38.9±3.3
0.7
0.56±0.01
12.5
2.63±0.03
1.2
IC50 on A549 (µM)
PI on A549
0.65±0.01 21.3±0.3 0.075±0.003 1.33±0.03
1.3
1
82.9
1.3
70c
Fig. 2 Representative cytotoxicity measurement of compound 3 on A375 (human
malignant melanoma) (a) and A549 (human lung carcinoma) (b) cancer cell lines
plotted as percent viability versus log of the compound concentration in µM in the dark
(black squares) and upon light activated (red circles). Cells were incubated with the
compound for 24 h prior to cytotoxicity assessment. Light activation occurred 6 h post
incubation and lasted for 30 min using blue-LED. IC50 values were obtained using
GraphPad Prism software by fitting data into a sigmoidal dose response function.
Excited state lifetime
(ns)
<10a
<10a
1,200b
a
measured in CH3CN from ref. 29 and 33; b In 1:1 CH3CN:H2O from ref. 34; c in H2O for
the sulfonated analogue from ref. 33.
Compound
CH2Cl2 and 1.2 µs in 1:1 CH3CN:H2O.34 The significant extension
of lifetime in weakly basic solvents of compound relative to
3 exhibited a remarkable lifetimes of 2.8 µs in
3
Table 1. IC50 values of compounds 1-4 on A375 and A549 in the dark. Three
independent measurements were performed to obtain the arithmetic average and
standard error of the mean. Cells were incubated for 24 h with the compounds. Cells
the others can be attributed to shielding the metal from the
approach of coordinating solvents. This protection was
enhanced going from the methyl to phenyl to sec-butyl
substituents on the 2,9-positions of the Cu(I) complex.33,
were incubated for 48
h with cisplatin control. IC50 values were obtained using
45
GraphPad Prism software by fitting data into a sigmoidal dose response function.
Consequently, it can be inferred that efficient bimolecular
energy/electron transfer,31,
46, 47
Compoun
ds Dark
a prerequisite of PDT, was
1
2
3
4
cisplatin
7.7±0.4
21.9±0.4
-2.21a
successfully achieved by extending the triplet excited state
lifetime of Cu(I) bis-phenanthroline complexes. Importantly,
IC50 on
A375 (µM)
1.8±0.5
0.9±0.1
1.02±0.08
3.61b
25.5±0.2
21.8±0.2
2.21±0.19
6.96b
7.0±0.3
6.3±0.1
2.64±0.16
8.16b
3.1±0.1
1.8±0.1
2.18±0.15
6.40b
upon light activation, compound
orders of magnitude smaller than the prototypical cisplatin.
Since only compound displayed high PI, further experiments
3 displayed IC50 that is 1-2
IC50 on
A549 (µM)
3
Log P of
complexes
were performed to elucidate its mechanism of action.
Log P of
ligands
The level of DNA damages were assessed by in-vitro comet
assay. In these gel electrophoresis experiments, the DNA
damage is evaluated at the cellular level. Upon the application
of an electric field, the fragmented DNA migrates outside the
nucleoid. However, the non-denatured DNA stays within the
a value obtained from literature.44
b values for free ligands calculated from ChemDraw Professional (v15.0,
CambridgeSoft)
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nucleoid and subsequently migrates slower on the gel. The the extinction coefficient of the free compound, and εB is the
DNA tail shape, migration pattern, and %tail DNA content extinction coefficient of the fully bound compound.
correlate to DNA damages in the cells.
-8.0x10-8
60
50
40
30
20
10
0
1.0
0.8
0.6
0.4
0.2
0.0
A375
A549
-1.2x10-7
-1.6x10-7
-2.0x10-7
4.0x10-5 8.0x10-5 1.2x10-4 1.6x10-4
[DNA] (M)
350
400
450
500
550
600
650
Negative
control
Cmpd 3
Dark
Cmpd 3
Light
Positive
control
Wavelength (nm)
Fig. 3 Bar graph depicting comet assay results expressed in term of tail DNA content (%)
of A375 (red) and A549 (blue) cancer cells under the following conditions: negative
control (cells alone), positive control (cells incubated with KMnO4), cells incubated with
compound 3 in the dark and upon exposure to light.
Fig. 4 UV-vis spectra of compound 3 (50% DMSO in Tris-HCl buffer at pH = 7.2) with
incrementing concentration of calf thymus DNA base pairs. The inset shown a linear fit
of the first 6 data points into equation 1 to extract the intrinsic binding constant (Kb) of
compound 3 to DNA base pairs.
The alkaline comet assay is sensitive, and is used to detect
small amounts of damage including single and double- The qualitative uptake and localization of compound
3 was
stranded breaks. Negative controls contained cells without performed using fluorescence microscopy, Fig. 5.39, 42 Since this
drug whereas positive control contained KMnO4 which is a complex is emissive at room temperature even in the presence
known oxidant that induces DNA damages at the cellular of coordinative solvents,34 this experiment yielded important
level.48 Comet assay data of A375 and A549 cancer cells information on the uptake and cellular localization of
incubated with compound
3 in the dark (Fig. 3) was suggestive compound
3. The compound (12 µM) was incubated with A375
that the latter operated, at least partly, through the induction and A549 cells for 2, 6 and 12 h and then visualized using
of DNA damage. Importantly, the level of DNA single and/or Rhodamine filter on a fluorescence microscope. Cells were also
double-stranded breaks increased in both A375 and A549 stained
cancer cells incubated with compound upon light exposure, dihydrochloride) which is a known fluorescent marker for DNA.
Fig. 3. The positive and negative controls showed identical After 2 h incubation (Fig. 5a and 5b on A375 and A549
results in the absence or presence of light irradiation respectively) compound
showed detectable uptake into cells
with
DAPI
(4′,6-diamidine-2′-phenylindole
3
3
confirming that blue light by itself didn’t change the level of and diffuse cytosolic localization. At that time cells were still
DNA damage. These results are in line with the measured relatively healthy and the compound was not concentrated in
ability of cuprous phenanthroline complexes to interact with the nucleus. At 6 h of incubation, compound
DNA.22, 27, 28 To corroborate the results from comet assay on on A375 and A549 respectively) induced the destruction of the
compound , a spectrophotometric titration experiment in membrane integrity and was partly found in the nucleus and
3 (Fig. 5c and 5d
3
50% DMSO in Tris-HCl buffer at pH = 7.2 was done with calf- predominantly concentrated in the cytosol in a perinuclear
thymus DNA as titrant. It was found that incrementing fashion. After 12 h, cells completely detached from the
amounts of DNA induced
absorbance of compound
binding to DNA base pairs,49, 50 Fig. 4. Using equation 1,50 the Collectively the comet assay, spectrophotometric titration with
intrinsic binding constant of compound to DNA base pairs DNA and fluorescence microscopy experiments indicate that
was found Kb = 3.3 ± 2.5 × 104 M-1 which is comparable to the the main target of compound
is the DNA of cells. This was
a
bathochromic shift in the microscope slides indicating complete cell death at the
3
which is typically observed upon concentration used.
3
3
values obtained for transition metal complexes known to bind not surprising given that metal complexes bearing
to DNA.49, 50
phenanthroline based ligand(s) have demonstrated high
affinity to DNA.39, 50
[DNA]/(εA – εF)= [DNA]/(εB – εF) + 1/Kb(εB – εF)
Eq.1
where εA is the observed extinction coefficient of the
compound after DNA addition (εA = Aobsd/[compound 3]), εF is
6 | J. Name., 2012, 00, 1-3
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fluorescent DCFDA. After diffusion into the cell, DCFDA is
deacetylated by cellular esterases to non-fluorescent
a
compound. If ROS are formed, the latter gets oxidized into
2’,7’-dichlorofluorescein (DCF). DCF is a highly fluorogenic dye
which can be detected by fluorescence spectroscopy with
excitation and emission signals at 495 nm and 529 nm
respectively. Compound
3 was added at 3 µM concentration
and tested in the dark and upon light activation following 4 h
incubation. Control experiments were performed in the
absence of drug (negative control) or upon incubation with
TBHP (tert-butyl hydroperoxide) a known ROS forming agent52.
When compared to the controls for relative fluorescence
intensity,
a turn-on fluorescence was displayed in cells
incubated with photoactivated compound 3, Fig. 6. The blue
light did not cause any increase of ROS level. Thus, it can be
inferred that compound
3 induced the formation of ROS only
upon light activation in both A375 and A549 cells. These
results correlate well with the high phototoxicity indices of
Fig. 5 Fluorescence imaging following the treatment of A375 and A549 cells for 2 h (a
and b respectively) and 6 h (c and d respectively) with 12 µM of compound 3. The
compound
3 on these cancer cell lines, Fig. 2 and table 2. The
orange signal emanated from the intrinsic emission of compound
3 (Rhodamine
induction of ROS formation upon light activation in the
targeted tumours offers an advantage over conventional
chemotherapy.
filter/RH) and the blue emission is from DAPI which stains the nucleus. Cells were fixed
with 4% paraformaldehyde for 10 min, and blocked with 1% BSA, in PBS for 1 hour.
Samples were stained with DAPI for 10 min before taking fluorescent images using a
60X objective on Axio observer Z1 fluorescent microscope (from Zeiss).
Conclusions
Based on the cytotoxicity results in this work (Tables 1 and 2),
The following sterically congested Cu(I) complexes were
synthesized and characterized: [Cu(dmphen)2]+
the mechanism leading to the photoactivation of compound
3
(1
) [Cu(BC)2]+
was postulated to be through quenching of the long-lived
triplet state to produce reactive oxygen species (ROS) which
induce oxidative stress in cells and ultimately cell death, Fig.
6.51
(2 3 4). The cytotoxicity
), [Cu(dsbtmp)2]+ ( ) and [Cu(dpphen)2]+ (
of these homoleptic Cu(I) bis-phenanthroline complexes was
screened on A375 and A549 cancer cells. IC50 values were
obtained in the dark and upon blue light MLCT excitation.
Compounds 1-4 were potent in the dark with IC50 ranging from
2 to 25 µM. However, only compound 3, which exhibited the
longest excited state lifetime, displayed enhanced potency
upon photoactivation. In the latter case, high phototoxicity
indices of ~13 on A375 and ~83 on A549 cancer cells were
obtained which are comparable to ruthenium polypyridine
congeners. This provides a proof-of-principle that cuprous
phenanthroline photosensitizers can display in-vitro
photocytotoxicity and may provide viable alternative to noble
metal complexes in photobiology. Comet assay performed on
6
A375
A549
5
4
3
2
1
0
compound
biological target. DNA damages were detected upon
incubation of cells with compound in the dark and were
further elevated upon visible light excitation. Furthermore,
UV-vis titration of compound with calf-thymus DNA revealed
a hypochromic shift which is suggestive of DNA binding. An
3 was strongly suggestive of DNA being a possible
3
3
Negative
control
Positive
control
Cmpd 3
Dark
Cmpd 3
Light
intrinsic binding constant Kb between complex 3 and DNA base
pairs was found to be Kb = 3.3 ± 2.5 × 104 M-1 that is on the
same order of magnitude to known DNA binding complexes.49,
50
Owing to the photoluminescence properties of complex 3,
Fig. 6 DCFDA (2′,7′-dichlorofluorescin diacetate) fluorescence assay for the detection of
ROS in A375 (red) and A549 (blue) cancer cells performed on buffer (negative control),
tert-butyl hydroperoxide (positive control), compound
photoactivation.
3 in the dark and upon
fluorescence imaging experiments successfully indicated
intracellular uptake and perinuclear localization in A375 and
A549 cells. A loss of membrane integrity was observed 6 h
post-incubation and it proceeded cell death. Finally, a DCFDA
fluorescence assay revealed that the photocytoxicity of
The production of ROS was assessed using DCFDA (2′,7′-
dichlorofluorescin diacetate) fluorescence assay which allows
measurement of hydroxyl, peroxyl and other ROS activity
within the cell. Briefly, cells were incubated with the non-
complex
3
was likely due to the formation of reactive oxygen
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the advantageous utilization of sterically encumbered Cu(I) bis-
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Conflicts of interest
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There are no conflicts to declare.
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Acknowledgements
We thank Prof. Felix N. Castellano and Dr. Catherine E.
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synthesis of Cu(I) complexes. We would like to acknowledge
the School Research and Development Council at the Lebanese
American University for funding this work.
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A table of contents entry:
An earth-abundant cuprous bis-phenanthroline photosensitizer showed potential use in photodynamic therapy of
cancer.
1