Remarkable enhancement in photocytotoxicity and hydrolytic stability of curcumin on binding to an oxovanadium(iv) moiety

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Remarkable enhancement in photocytotoxicity and hydrolytic stability

of curcumin on binding to an oxovanadium(IV) moiety†

a

b

a

b

Samya Banerjee, Ila Pant, Imran Khan, Puja Prasad, Akhtar Hussain, Paturu Kondaiah* and Akhil

a

5

R. Chakravarty*

Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX

DOI: 10.1039/b000000x

Oxovanadium(IV) complexes of polypyridyl and curcuminꢀbased ligands, viz. [VO(cur)(L)Cl] (1, 2) and

[VO(scur)(L)Cl] (3, 4), where L is 1,10ꢀphenanthroline (phen in 1 and 3), dipyrido[3,2ꢀa:2′,3′ꢀc]phenazine

1

2

0

5

0

(dppz in 2 and 4), Hcur is curcumin and Hscur is diglucosylcurcumin, were synthesized, characterized and

their cellular uptake, photocytotoxicity, intracellular localization, DNA binding, DNA photoꢀcleavage activity

IV

studied . Complex [VO(cur)(phen)Cl] (1) has V N O Cl distorted octahedral geometry as evidenced from its

2

3

crystal structure. The sugar appended complexes show significantly higher uptake into the cancer cells

compared to their normal analogues. The complexes are remarkably photocytotoxic in visible light (400ꢀ700

nm) giving an IC₅₀value of <5 ꢁM in HeLa, HaCaT and MCFꢀ7 cells with no significant dark toxicity. The

green emission of the complexes was used for cellular imaging. Predominant cytosolic localization of the

complexes 1−4 with a lesser extent into the nucleus was evidenced from confocal imaging. The complexes as

avid binders to calf thymus DNA displayed photocleavage of supercoiled pUC19 DNA in red light by

•

generating OH radicals as the ROS. The cell death is via apoptotic pathway involving the ROS. Binding to

2

+

the VO moiety has resulted stability against any hydrolytic degradation of curcumin along with an

enhancement of its photocytotoxicity.

5

6

0

5

0

5

their tunable coordination geometry are suitable for ligating the

metal to various chromophores having pendant tumor targeting

moieties for their photoꢀactivity and enhanced uptake into the

cancer cells for reduced sideꢀeffects to normal cells. The versatile

redox properties of such complexes could provide an alternative

pathway, viz. photoꢀredox pathway to be operative for the

Introduction

Photodynamic therapy (PDT) has emerged as a new modality for

the selective treatment of various types of cancers by killing the

photoꢀexposed cancer cells without harming the unexposed

healthy cells. The FDA approved PDT drug Photofrin is a

hematoporphyrin derivative that requires light and oxygen to

2

3

5

0

5

®

•

generation of different radical species, viz. OH and superoxide

initiate photochemical reaction that leads to generation of highly

radical. The photophysical properties of the complexes with low

energy dꢀd or chargeꢀtransfer (CT) band are of importance as the

complexes could be activated by visible or preferably nearꢀIR red

light making such complexes as the promising alternatives to

1

reactive singlet oxygen ( O ) which causes death of the cancer

2

1–3

cells. Other macrocyclic organic dyes are also known for their

PDT activity. Such dyes generally show severe side effects like

skin photoꢀsensitivity and hepatotoxicity due to oxidative



Photofrin .

4

,5

degradation of the macrocyclic core. These predicaments could

be surmounted by designing transition metal complexes having

bioꢀcompatible photosensitizers like curcumin and related dyes

that are active in the PDT spectral window. Different types of

metalꢀbased photocytotoxic agents are now reported to show

We have shown in a recent communication that curcumin

(Hcur) which is well known for its remarkable medicinal

properties could be successfully used as a novel photosensitizerꢀ

cumꢀcellular imaging agent when bound to an oxovanadium(IV)

11

moiety. Curcumin, 1,7ꢀbisꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ1,6ꢀ

6

–10

significant PDT activity.

Transition metal complexes with

heptadieneꢀ3,5ꢀdione, is used as traditional herbal medicine to

a

70 treat infections, bite, burns and skin diseases besides a wide range

4

0

5

Department of Inorganic and Physical Chemistry, Indian Institute of

Science, Bangalore 560012, India. Fax: +91ꢀ80ꢀ23600683; Tel: +91ꢀ80ꢀ

2932533; Eꢀmail: arc@ipc.iisc.ernet.in

12

of other biological activities. Curcumin as an anticancer drug

stops tumor progression by inhibiting angiogenesis in cultured

2

b

Department of Molecular Reproduction, Development and Genetics,

13

endothelium vascular cells and animal model. Studies with in

Indian Institute of Science, Bangalore 560012, India.

vitro models have revealed that it induces apoptosis via

c †Electronic Supplementary Information (ESI) available: ESIꢀMS and IR

spectra, cyclic voltammograms, energy minimized structures, cellular

data, DNA binding and cleavage data (Fig. S1ꢀS36). For ESI or other

electronic format See DOI: 10.1039/b000000x/

75

mitochondrial pathways involving caspases and Bc1ꢀ2 family of

14, 15

proteins.

It also interferes with the activity of the transcription

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factor NFꢀκB which is known to enhance the activity of the tumor

and HaCaT cells shows mainly cytosolic localization of the

1

6,17

suppressor protein p53.

In addition, curcumin with cisplatin is 60 complexes within 4 h of incubation and this observation is

reported to enhance the reduction of head and neck cancer cell

significant considering nuclear localization could possibly lead to

undesirable mutation and carcinogenesis. The complexes show

photoinduced cell cycle arrest in S and G2/M phase and cell

death via apoptotic pathway. We have observed mitochondrial

18

growth. With all these positive aspects, clinical application of

curcumin (Hcur) as such is severely limited due to its poor

bioavailability, pharmacokinetic profile resulting from its

5

0

5

0

5

0

5

0

5

0

5

hydrolytic instability under physiological conditions, associated 65 localization of complex 2. This is of significance considering that

skin sensitivity and intestinal disorders when taken in its native

form in higher dose. The hydrolytic instability is due to the

the intrinsic pathway of apoptosis largely involves the

mitochondria as is known for the PDT drug Photofrin .

®

1

9

1

2

3

4

5

presence of a highly reactive βꢀdiketone moiety. Microscopic

imaging studies with curcumin alone show complete

disappearance of its fluorescence intensity during the cellular

[Scheme 1]

Results and discussion

1

incubation period. Binding of this βꢀdiketone moiety in its

enolic form to an oxophilic metal centre increases the stability of

curcumin and the resulting complexes show remarkable cytotoxic

70

Synthesis and characterization

Oxovanadium(IV) complexes of curcumin and its derivative,

viz. [VO(cur)(L)Cl] (1, 2) and [VO(scur)(L)Cl] (3, 4), having

1,10ꢀphenanthroline (phen) or dipyrido[3,2ꢀa:2′,3′ꢀc]phenazine

(dppz) were synthesized in good yield from a general synthetic

1

1,20ꢀ23

or photocytotoxic activity.

complexes are thus emerging as a new class of nonꢀporphyrinic

PDT agents. The monoanionic curcumin ligand as

The metalꢀbased curcumin

a

photosensitizer with a visible band near 440 nm and as a 75 procedure in which vanadyl sulfate was first reacted with barium

fluorophore with green emission property is suitable for dual

applications, viz. (i) imaging the cells by fluorescence

microscopy to study cellular localization of the complexes and

chloride in aqueous ethanol (1:5 v/v waterꢀethanol) and the

filtrate after removal of barium sulfate was subsequently reacted

with the respective phenanthroline base in ethanol followed by

addition of curcumin (Hcur) or glycosylated curcumin (Hscur) in

80 acetonitrileꢀethanol mixture. The complexes were characterized

from the spectroscopic and analytical data. Selected

physicochemical data are given in Table 1. The ESIꢀMS of the

complexes showed essentially a single peak corresponding to the

(

ii) damaging the cells on irradiation with a visible light (400ꢀ700

nm).

The present work stems from our interest to increase the

+2

bioavailability of curcumin on complexation with a VO moiety

in a ternary structure having polypyridyl bases as additional

photosensitizers and using the complexes as cellular imagingꢀ

+

[MꢀCl] in both MeCN and aqueous MeCN (Fig. S1ꢀS4, ESI†).

cumꢀphotochemotherapeutic agents. Vanadium being a bioꢀ 85 The mass spectral data suggest the stability of the fiveꢀcoordinate

essential trace element, the oxovanadium(IV) complexes showing

a low energy dꢀd band within the PDT spectral window (630ꢀ800

nm) are suitable as metal based PDT agents considering the

complex as [VO(cur)(L)]Cl in the solution phase. The complexes

gave molar conductance value of ~15 and ~95 S m M in pure

DMF and 10% aqueous DMF, respectively, suggesting their nonꢀ

2

ꢀ1

2

4,25

greater tissue penetration of red light.

In continuation to our

electrolytic behavior in DMF and 1:1 electrolytic nature in an

1

31

previous report, we have now attempted to increase the aqueous 90 aqueous medium due to dissociation of the chloride ligand. The

solubility and cellular uptake of the complexes by derivatization

magnetic moment values of ~1.65 µ indicate the presence of the

B

2

6

of the OH groups of curcumin by glucose. Cancer cells are

known to have higher rates of glucose uptake due to decrease in

V(IV) oxidation state with one unpaired electron. The IR spectra

of the complexes showed three bands near 1590, 1496 and 966

27

ꢀ1

oxidative phosphorylation and enhanced levels of glycolysis.

cm for the C=O, C=C (βꢀdiketonate) and V=O stretching

32

The pendant glucose moieties in the ternary structures are 95 vibrations, respectively (Fig. S5, ESI†). The IRꢀband at ~1590

28ꢀ30

ꢀ1

expected to enhance the cellular uptake of the complexes.

We

cm (C=O stretching) indicates bidentate coordination mode of

have chosen planar phenanthroline bases (L), viz., 1,10ꢀ

phenanthroline (phen) and dipyrido[3,2ꢀa:2′,3′ꢀc]phenazine

the βꢀdiketone (cur/scur) ligand to the metal center. The

oxovanadium(IV) complexes showed a broad and weak dꢀd band

within 720–750 nm in 10% aqueous DMF (Fig. 1 (a)). The

100 curcumin complexes (1 and 2) displayed an intense curcumin

centered visible band at ~434 nm due to π→π* transition of the

(dppz), for their intercalative DNA binding properties and the

dppz ligand having a phenazine moiety for its photoactivity.

Herein, we present the synthesis, characterization, DNA

binding, red lightꢀinduced DNA cleavage activity and visible

lightꢀinduced cytotoxic properties of four oxovanadium(IV)

complexes, viz. [VO(cur)(L)Cl] (L = phen, 1; dppz, 2) and

33

curcumin ligand with a shoulder at around 453 nm. This band

was observed at 424 nm with a shoulder at around 442 nm in

diglucosylcurcumin complexes. This band could be utilized to

[VO(scur)(L)Cl] (L = phen, 3; dppz, 4), where L is 1,10ꢀ 105 photoꢀactivate the complexes by visible light (400ꢀ700 nm) for

phenanthroline (phen), dipyrido[3,2ꢀa:2′,3′ꢀc]phenazine (dppz),

cur is monoanionic curcumin and scur is monoanionic

diglucosylcurcumin (Scheme 1). Complex [VO(cur)(phen)Cl] (1)

has been structurally characterized by Xꢀray crystallography and

generation of ROS which leads to cell death. The dppz ligand in

the complexes showed an additional band near 360 nm assignable

to the n–π* transition involving the phenazine moiety. The other

34

ligandꢀcentered electronic spectral bands were observed in the

the structural details are published in our preliminary 110 UV region.

1

communication.

Significant results of this study include

[Fig. 1]

remarkable enhancement in the photoꢀcytotoxicity and hydrolytic

stability of curcumin on binding to an oxovanadium(IV) moiety.

The complexes are lessꢀtoxic in dark. Cellular imaging of HeLa

The complexes in 10% aqueous DMSO on excitation at 420

nm exhibited green fluorescence at 510 nm with quantum yield

2

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(

φ) values in the range of 0.01ꢀ0.02 (Fig. 1(b)). The curcumin

medium. The absorption spectral study showed a gradual

ligand alone gave a φ value of 0.04 under similar conditions. The 60 decrease in the absorption intensity of curcumin (Hcur) alone in a

2

+

curcumin based fluorescence is retained in the complexes and the

emission spectral properties are suitable to study the cellular

uptake and localization of the complexes inside the cells using

confocal fluorescence microscopy. Glycosylation of curcumin

solution phase, while its VO complexes showed no apparent

spectral change even after 48 h (Fig. S11, ESI†). The results

indicate that coꢀordination of curcumin to the VO moiety

significantly increases its hydrolytic stability. This also provides

2

+

5

0

5

0

5

0

5

0

5

0

5

has led to a blueꢀshift in both the absorption and emission band of 65 a convenient way to deliver the drug into the target cells without

the respective complexes in comparison to the curcumin

complexes. The redox active complexes showed a reduction peak

its degradation under physiological conditions. This is likely to

enhance the therapeutic potential of curcumin. The confocal

images of curcumin alone showed its degradation over time

resulting complete loss of its emission intensity. The metal

1

2

3

4

5

assignable to the V(IV)/V(III) couple near ꢀ1.2 V vs. SCE in

n

4

2

0% DMFꢀH O with 0.1 M [Bu N](ClO ) (TBAP) as the

2

4

supporting electrolyte (Table 1, Fig. S6, ESI†). The complexes 70 complex, however, showed no apparent change in the emission

did not show any oxidative response. Redox stability of the

complexes over a large potential window is expected to reduce

intensity of the metalꢀbound curcumin ligand (Fig. S12, ESI†).

35

their chemical nuclease activity and dark cellular toxicity.

Computational studies were performed for the complexes

Cellular incorporation assay

3

6,37

using B3LYP/LANL2DZ level of DFT.

The energy optimised

75

Cellular uptake studies were carried out to determine the

incubation time of the complexes for inꢀvitro studies before

exposure to visible light. Uptake of the complexes 2 and 4 in

HaCaT, HeLa and MCFꢀ7 cells at 2 and 4 h time points was

studied by FACS (Fluorescence Activated Cell Sorter) analysis in

structures of the complexes showed a distorted octahedral

structure with chelating N,Nꢀdonor phen or dppz, O,Oꢀdonor

curcumin or diglucosylcurcumin monoanion and a chloride

ligand (Fig. 2(a), Fig. S7, ESI†). The axial V=O bond distance is

~

1.61 Å, while the other VꢀO bond distances are ~1.97 Å. It is 80 which the fluorescence of the curcumin complexes were

evident from the optimised structure that the chloride ligand

being at the trans position of the V=O is labile with a long VꢀCl

bond distance of ~2.61 Å. From the molecular orbital pictures it

is observed that the HOMO is concentrated mostly over the

monitored (Fig. 3). The number of events in the upper left

quadrant of the figure represents the population of cells in which

the complex was not taken up and that in upper right quadrant

represents the cell population with complex inside. The

curcumin moiety whereas the LUMO is located over the 85 percentage incorporation of the complexes in the cells is also

polypyridyl rings of the complexes 1, 2 and 4 (Fig. 2(a), Fig.

S8,S9, ESI†). Complex 3 has the HOMO based on curcumin with

significant contribution from the choride ligand and LUMO is

located over the curcumin moiety (Fig. S10, ESI†). Complex 1

shown (Fig. S13, ESI†). On 2 h of incubation, uptake of the

complexes was found to be more in HaCaT cells than in HeLa

and MCFꢀ7 cells. The results indicate different rates of cellular

uptake of the complexes

2 and 4. The uptake of the

was structurally characterized earlier by singleꢀcrystal Xꢀray 90 diglucosylcurcumin complex 4 is significantly higher (~100% on

1

crystallography (Fig. 2(b)). The oxovanadium(IV) complex has

similar coꢀordination mode as observed in the DFT optimised

structure. The V=O bond distance is ~1.54 Å. The VꢀCl bond

trans to the V=O group is very long (~2.78 Å) and this labile

bond is likely to get dissociated in an aqueous solution as is

evidenced from the ESIꢀMS and molar conductivity data (Table

4 h of incubation) compared to its curcumin analogue 2 (~83% at

4 h). This could be due to preferential internalization of the

carbohydrate appended complex in cancer cells by the receptor

38

mediated endocytosis.

95

[Fig. 3]

We also performed experiments to demonstrate the active uptake

of glucose functionalized complexes using 2 and 4. Cells were

first incubated with 10 mM glucose for 2 h followed by addition

of the complexes. The results indicate that the percentage cellular

2

).

[Fig. 2]

Solubility and stability

100 uptake of the sugar appended complex 4 was significantly

compromised from ~99.5% to ~40% upon incubation with excess

glucose, whereas the percentage cellular uptake of the nonꢀsugar

complex 2 remained almost unaltered (Fig. 4(a), Fig. S14, ESI†).

Moreover, the mean fluorescence intensity which demonstrates

The complexes showed good solubility in DMF and DMSO,

moderate solubility in water, methanol, ethanol and acetonitrile,

and poor solubility in hydrocarbons. The diglucosylcurcumin

complexes 3 and 4 having carbohydrate moiety were soluble in 105 the mean compound uptake in cells was also significantly

water. Complex 1 showed solubility up to ~3.3 mg per ml of

DMSOꢀ10% DMEM medium (1:99 v/v, similar to cellular

medium) at 25 °C. Complexes 2ꢀ4 in the same solvent

composition gave solubility up to ~3.8, ~9.9 and ~10.2 mg per

compromised for the sugar appended complex 4 in the presence

of the added excess glucose while remaining same for complex 2

(Fig. 4(b), Fig. S15, ESI†). The enhanced uptake of complex 4 is

indeed due to the introduction of the carbohydrate moiety and the

ml, respectively. The complexes were stable in the monocationic 110 internalization of complex 4 in the cancer cells is mainly through

form on dissociation of the chloride ligand and the solution

stability of the resulting cationic species was evidenced from the

ESIꢀMS and the molar conductivity data. Curcumin alone is

the receptor mediated endocytosis.

[Fig. 4]

1

1,19

known to undergo hydrolysis in an aqueous buffer.

We have

Cytotoxicity studies

observed remarkable enhancement in the aqueous stability of 115

2

+

curcumin as ligand on binding to the VO moiety in a buffer

The anticancer activity of the oxovanadium(IV) complexes

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1

ꢀ4, curcumin and its derivative was tested on HeLa and MCFꢀ7

To further confirm the apoptotic cell death induced by complex

cancer cells as well as on immortalized HaCaT cells in dark and 60 4, we stained the cells that were pretreated with 3 ꢁM of the

upon irradiation with a broadband visible light source (400ꢀ700

complex for HeLa cells and 1 µM of the complex for HaCaT cells

in dark and light with AnnexinꢀV FITC and PI. AnnexinꢀV, a

calcium dependent protein, binds to phosphatidylserine exposed

on the outer surface of the cells undergoing apoptosis due to

ꢀ

2

nm; Luzchem photoreactor, 10 J cm ; irradiation time, 1 h). The

complexes were incubated for 4 h and then irradiated with the

visible light. Dark controls were used to measure the PDT effect.

5

0

5

0

5

The extent of cytotoxicity was measured from the IC₅₀values 65 membrane flipping in cells undergoing apoptosis. We have

(

50% inhibitory concentration) obtained from the MTT cell

observed that complex 4 inducing apoptosis only on irradiation

with visible light as the cells stained positive for AnnexinꢀV

indicating membrane flippage (Fig. 7). As shown in the figure,

48ꢀ65% of the cells were in an early apoptotic stage (positive for

11,39

viability assay (Table 3, Fig. 5, Fig. S16ꢀS21, ESI†).

complexes were found to be remarkably photocytotoxic to all the

cells studied and remained essentially nontoxic in dark. The IC₅₀

The

1

2

values of complex 1 were in the range of 6.4–10.0 ꢁM. The 70 Annexin V staining), whereas the population of cells in necrosis

values for complex 2 in light were within 2.3–4.7 ꢁM. The higher

photocytotoxicity of 2 than 1 is due to the presence of

dipyridophenazine as an additional photoactive moiety in 2.

Complex 3 with a curcumin derivative having two sugar moieties

mode (stained with PI) is significantly lower which again

suggests the overall apoptotic mode of cell death.

[Fig. 7]

gave IC₅₀values within 3.5–5.8 ꢁM. The reason for the higher 75 DCFDA assay for ROS

photocytotoxicity of complex 3 than complex 1 could be due to

higher cellular uptake in the presence of the sugar moieties.

Complex 4, the most photoactive one, showed photoꢀcytotoxicity

at nanomolar concentration giving IC₅₀values within 0.63–2.4

The generation of any reactive oxygen species (ROS) by

complex 4 was examined using visible light of 400ꢀ700 nm from

dichlorofluorescein diacetate (DCFDA) assay in both HaCaT and

ꢁM, while being less cytotoxic in dark. A comparison of the IC₅₀80 HeLa cells. DCFDA is a cell permeable fluorogenic probe used

values of the complexes, the ligands, viz. curcumin and sugarꢀ

appended curcumin and other compounds is made in Table 3.

for the detection of ROS in cells. It forms DCF on oxidation

40

42,43

showing an emission maximum at 528 nm.

The fluorescence

While curcumin showed moderate photocytotoxicity, its sugar

derivative is not cytotoxic. Complexes showing photocytotoxicity

of DCF could be quantified from flow cytometry analysis. The

cells treated with 4 in dark did not show any formation of DCF.

in subꢀmicromolar concentration with minimal dark toxicity are 85 However, cells treated with 4 on exposure to visible light for 1 h

of significance in the chemistry of PDT. The observed

showed significant shift in the emission band towards right which

indicates an increase in the intensity of emission resulting from

the generation of DCF by the ROSꢀmediated oxidation of

DCFDA (Fig. 8). The assay data in HaCaT and HeLa cells

90 indicate the generation of ROS on exposure to visible light and

this reactive species possibly causing cell apoptosis. There was

no ROS formation observed in dark.

photocytotoxicity of the complexes 2–4 is comparable to that of



41

30

35

40

45

50

55

the clinically approved PDT drug Photofrin . The present

complexes having curcumin and its sugar derivative are

potentially suitable for further studies in PDT.

[

Fig. 5]

Apoptosis: DNA ladder assay

[Fig. 8]

Fluorescence microscopy

DNA laddering is a convenient way to detect apoptotic cell 95 Fluorescence microscopy experiments were conducted for the

death which results from the activation of endogenous

endonucleases leading to degradation of genomic DNA into interꢀ

nucleosomal fragments of about 180 base pairs and its multiples.

The DNA fragmentations can be analyzed by agarose gel

complexes after 2 and 4 h incubation time in HeLa and HaCaT

cells (Fig. 9). Dual staining was done to study any localization of

the complexes inside the cell nucleus using propidium iodide (PI)

which stains the nucleus. Merged images suggest mainly

electrophoresis giving a “ladder” like pattern at ~180 base pairs 100 cytoplasmic localization of 1ꢀ4 in the HeLa and HaCaT cells

intervals. The alternate way of cell death is necrosis or direct

damaged/fragmentation of genomic DNA by ROS and this is

usually characterized by random DNA fragmentation giving

(Fig. 9, Fig. S22, ESI† panel (c)). This observation is of

significance considering nuclear localization of the drug being

undesirable in PDT to avoid any potential damage to the nuclear

DNA leading to mutation and carcinogenesis. It is apparent from

“smear” pattern on agarose gels. The DNA laddering experiments

were done for complexes 2 and 4 in HeLa and HaCaT cells. As 105 the microscopy data that the complexes enter into the cells within

shown in Fig. 6, it is evident that the complexes show ladder like

2 h and uptake of the complex increases in 4 h (Fig. S23, ESI†).

As mentioned above, free curcumin in HeLa cells showed only

little green fluorescence in 2 h and there was no visible

pattern only after photoꢀirradiation in visible light of 400ꢀ700 nm

ꢀ

2

(10 J cm ; photoꢀirradiation time, 1 h) indicating apoptosis due to

fragmentation of the genomic DNA as is reported for

fluorescence intensity after 4 h (Fig. S12, ESI†). This suggests

3

3(b)

curcumin.

However, no such DNA ladder was observed for 110 degradation of curcumin (Hcur) in a cellular medium. The

the cells treated with the complexes in dark indicating that the

lightꢀinduced cell death proceeds via apoptotic pathway.

[Fig. 6]

stability of curcumin gets significantly enhanced on binding to

the oxovanadium(IV) moiety as observed from the microscopy

data of the complexes (Fig. 9). The intraꢀcellular fluorescence

intensity of the sugar appended complexes 3 and 4 is significantly

115 higher compared to their normal analogue.

Apoptosis: annexin V-FITC/PI assay

[

Fig. 9]

4

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To visualise the PDT effect, complexes 2 (5 ꢁM) and 4 (3 ꢁM)

and cleavage activity of the complexes are thus of importance to

were added to the HaCaT cells and after incubation for 4 h, cells 60 understand their celluar activity. The binding affinity of the

were exposed to visible light of 400ꢀ700 nm. Fluorescence

images after 1 h light exposure displayed irregular nuclear

morphology on staining with PI (Fig. 10, panels (c)). Chromatin

condensation of the nucleus was observed indicating apoptotic

complexes to calf thymus (ct) DNA was studied by spectral and

hydrodynamic methods (Table 1). The absorption spectral

technique was used to determine the intrinsic binding constant

5

0

5

0

5

0

5

0

5

0

5

(K ) value of the complexes by monitoring the decrease in the

b

cell death as bright condensed nuclei were seen in the cells 65 absorption of the curcumin centered bands near 430 nm. The K

b

4

5

ꢀ1

(panels (c)). The PI staining revealed that the nuclear morphology

values range from 6.1(±0.4) x10 to 7.2(±0.2) x 10 M giving an

order of the DNA binding strengths: 4 > 2 > 3 > 1 (Fig. S27,

ESI†). The planarity of the dppz ligand makes its complexes

better binders to ctꢀDNA than their analogues. DNA melting

70 studies in phosphate buffer (pH 7.2) showed stabilizing

interaction of the complexes with ctꢀDNA (Table 1, Fig. S28,

ESI†). The ꢀT_mvalue for curcumin (Hcur) is 0.9 °C while the

values for the complexes lie within 6.9 to 2.0 °C. It is 10.6 °C for

remained unchanged in dark indicating nonꢀcytotoxic nature of

the complexes in dark. The microscopy data suggest that the

complexes remain harmless inside the cells in dark and damage

the cancer cells only upon photoꢀirradiation.

1

2

3

4

5

[

Fig. 10]

Targeting mitochondria

4

5

the classical DNA intercalator ethidium bromide (EB). The

With the knowledge that the complexes get accumulated 75 DNA melting data gave a similar order indicating intercalative

primarily in the cytoplasm of the HeLa and HaCaT cells, we

probed for any specific localization of the complexes to the

cellular organelle. For this purpose, we have chosen complexes 2

and 4 giving green fluorescence and did dual staining with

mode of binding of the dppz complexes to the ctꢀDNA. A similar

order is also obtained from the viscosity study. EB as a DNA

intercalator and Hoechst dye as a groove binder were used as

1/3

references. The plots of (η/η₀) vs. [complex]/[DNA] ratio

mitotracker deep red which stains only the mitochondria. The 80 showed partial intercalative binding mode of the complexes 2 and

data showed complexes

2

and

4

coꢀlocalizing with the

3 to ctꢀDNA (Fig. S29, ESI†). Complex 1 showed primarily

groove binding propensity, while complex 4 having scur and

dppz ligands exhibited a similar plot as observed for the EB

suggesting significant intercalative nature of DNA binding of this

mitotracker in a significant manner on 4 h of incubation as is

evident from the merged images shown in panels (c) of Fig. 11.

Complexes localizing in mitochondria are of significance since

mitochondria plays the key role in the intrinsic pathway of 85 complex.

apoptosis. Thus any changes in the cancer cells leading to

The DNA photocleavage activity of the complexes was studied

®

protection from apoptosis could be targeted. Photofrin is known

using supercoiled (SC) pUC19 DNA in TrisꢀHCl/NaCl buffer

upon irradiation with nearꢀIR light of 705 and 785 nm using

diode lasers (Table 4, Fig. S30ꢀS32, ESI†). The choice of these

wavelengths is based on the presence of a dꢀd band within 720ꢀ

750 nm in the electronic spectra. The phen complexes 1 and 3

showed only moderate DNA photocleavage activity due to

presence of monoanionic curcumin as the only photosensitizer.

Complexes 2 and 4 having photoactive phenazine moiety showed

4

to localize in the mitochondria for its activity.

Fig. 11]

[

90

Cell cycle profiling

Cell cycle analysis was performed for complexes 2 and 4 to

study their effect on the cell cycle profile, if any. The analysis

was done in three cell lines, viz., MCFꢀ7, HaCaT and HeLa, by 95 very efficient photocleavage of DNA. A 40 µM solution of these

treating them with the complexes 2 and 4 in dark and light (400ꢀ

00 nm; irradiation time, 1 h). No change in the cell cycle profile

complexes gave essentially complete cleavage of plasmid DNA

(~90%) from its SC to nicked circular (NC) form on 1.0 h photoꢀ

exposure time. Control experiments in dark did not show any

significant DNA cleavage activity of the complexes and the

7

was observed in this visible light with respect to dark control

indicating that this range of light is not inducing any cell cycle

arrest or cell death. Complex 4 showed arrest in S and G2/M 100 ligands. The curcumin dye alone showed ~11% cleavage of SC

phases in MCFꢀ7 and HaCaT cells, while a distinct sub G1

population was obtained in HeLa cells on 7 h of incubation after

light irradiation. Complex 2 arrested MCFꢀ7 cells predominantly

in the S phase. In HaCaT and HeLa cells, S phase and G2/M

DNA in dark. This could be due to generation of reactive oxygen

33

species in an aqueous solution of curcumin. Curcumin as a

photosensitizer showed ~16% cleavage of SC DNA. The

cleavage activity of curcumin gets remarkably enhanced on

arrest could be seen under same experimental conditions (Fig. 12, 105 binding to the metal possibly due to efficient intersystem crossing

46

Fig. S24ꢀS26, ESI†). Cell cycle profiles remained similar to that

of the control for both the complexes in dark in all the three cell

lines.

(ISC). The overall results indicate that the photoꢀexcitation is

metalꢀassisted involving the dꢀd band forming an excited state

21,24

that generates the cleavage active species.

[

Fig. 12]

The DNA groove binding propensity of the complexes was

10 studied using distamycinꢀA as a minor groove binder (50 µM)

and methyl green (25 µM) as a major groove binder (Fig. S33,

1

DNA binding and cleavage

47,48

ESI†).

DistamycinꢀA or methyl green alone showed ~18%

As evidenced from the confocal microscopy study, the

complexes localize mainly in the cytoplasm and significantly in

cleavage of SC DNA in red light of 785 nm for 1.0 h exposure

time. Addition of 1ꢀ4 to the distamycinꢀA bound SC DNA

the mitochondria. This suggests that the mitochondrial DNA 115 showed no apparent decrease in the DNA photoꢀcleavage

could be a potential target of the complexes. The DNA binding

activity. However, the complexes showed significant inhibition in

This journal is © The Royal Society of Chemistry [year]

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View Article Online

DOI: 10.1039/C4DT02165G

5

the DNA cleavage activity for methyl green bound SC DNA

procedure using 1,10ꢀphenanthrolineꢀ5,6ꢀdione as a precursor.

suggesting major groove binding nature of these complexes in 60 The glycosylated curcumin derivative, viz., diglucosylcurcumin

preference to the minor groove. The complexes did not show any

significant photocleavage of DNA in red light under argon as an

inert atmosphere indicating the necessity of molecular oxygen to

(Hscur,

1,7ꢀbis(3ꢀmethoxyꢀ4ꢀβꢀDꢀglucopyranosꢀ1ꢀ

yloxophenyl)heptaꢀ1,6ꢀdieneꢀ3,5ꢀdione), was prepared according

to a literature method (Fig. S36, ESI†). Synthesis of the

complexes was carried out under nitrogen atmosphere using

26

5

0

5

0

5

0

5

generate the cleavage active species (Fig. S34, ESI†). The DNA

3

cleavage reactions involving molecular oxygen ( O ) could 65 Schlenk technique. Tetrabutylammonium perchlorate (TBAP)

2

proceed via two major mechanistic pathways. The excited

electronic state of the complex through efficient intersystem

crossing (ISC) could generate an excited state that can activate

was prepared using tetrabutylammonium bromide and perchloric

acid.

1

2

3

1

molecular oxygen to its reactive singlet ( O , ꢂ ) state by energy

Measurements

2

g

5

,49

transfer in a typeꢀII process. In an alternate pathway, the redox

active photoꢀactivated complex could reduce molecular oxygen to

generate reactive hydroxyl radical species by a photoꢀredox

mechanism. We have observed that singlet oxygen quenchers,

viz. TEMP and sodium azide showed no apparent effect on the

70

The elemental analysis was done using a Thermo Finnigan

FLASH EA 1112 CHNS analyzer. The infrared, electronic

spectra were recorded on PerkinꢀElmer Lambda 35 and Perkinꢀ

Elmer spectrum one 55, respectively, at 25 °C. Molar

50

DNA cleavage activity thus ruling out the possibility of a typeꢀII 75 conductivity measurements were done using a Control Dynamics

singlet oxygen pathway. Significant inhibition in the DNA

(India) conductivity meter. Electrochemical measurements were

made at 25 °C on an EG&G PAR model 253 VersaStat

potentiostat/galvanostat with electrochemical analysis software

270 using a three electrode setup consisting of a glassy carbon

•

cleavage was observed in the presence of OH scavengers, viz.

DMSO, KI and catalase indicating presence of a photoꢀredox

pathway. The involvement of superoxide radical intermediate was

evidenced from the inhibitory role of SOD as an additive. 80 working, platinum wire auxiliary and a saturated calomel

Although the precise mechanistic details are not well understood,

the photoꢀactivated oxovanadium(IV) complexes in red light

seem to reduce the metal ion forming reactive V(III) species that

forms hydroxyl radicals following a Fentonꢀlike mechanism

reference electrode (SCE) in 20% DMFꢀH O. TBAP (0.1 M) was

2

used as

measurements. The electrochemical data were uncorrected for

junction potentials. Electrospray ionization (ESI) mass spectral

a supporting electrolyte for the electrochemical

which is known for the copperꢀdipyridoquinoxaline and ironꢀ 85 measurements were made using Agilent Technologies 6538 UHD

51

bleomycin species. The formation of hydroxyl radicals is also

evidenced from the EPR data using DMPO (5,5ꢀdimethylꢀ1ꢀ

pyrroline Nꢀoxide) as a stabilizer showing the characteristic four

Accurateꢀmass QꢀTOF LC/MS and Bruker Daltonics make

Esquire 300 Plus ESI model mass spectrometers. Room

temperature fluorescence quantum yield measurements were done

using a PerkinꢀElmer LS 55 fluorescence spectrometer using

90 coumarinꢀ153 laser dye as a reference with a known Φ value of

•

line spectra of DMPOꢀ OH adduct upon irradiation of complex 4

5

2

with red light of 785 nm for 1.0 h (Fig. S35, ESI†).

56

0

.56.

Samples were deaerated prior to the spectral

Experimental

measurements. The complexes and the reference were excited at

420 nm. Nearly equal absorbance and the emission spectra were

recorded from 450 to 600 nm. The integrated emission intensity

was calculated using Origin Pro 8.1 software and the quantum

Materials

95

yield was calculated from the equation: (Φ /Φ )

=

S

R

All reagents and chemicals were procured from commercial

sources (s.d. Fine Chemicals, India; Aldrich, USA) and used as

such without further purification. Curcumin (95% curcuminoid

content, 80% curcumin (Hcur)) was purchased from Sigmaꢀ

Aldrich and purified into individual components by following a

2

(

A /A )x((OD) /(OD) )x(n /n ), where, Φ and Φ_Rare the

S R S R S R S

fluorescence quantum yields of the sample and reference,

respectively, A_Sand A_Rare the area under the fluorescence

00 spectra of the sample and the reference respectively, (OD) and

S

40

45

50

55

1

(

OD) are the respective optical densities of the sample and the

5

3

R

reported procedure.

procedures. Supercoiled (SC) pUC19 DNA (cesium chloride

purified) was purchased from Bangalore Genie (India). Trisꢀ

Solvents were purified by standard

reference solution at the wavelength of excitation, and n and n

R

54

S

are the refractive indices for the respective solvents used for the

57,58

sample and the reference.

The NMR spectra were recorded

(hydroxymethyl)aminomethaneꢀHCl (TrisꢀHCl) buffer solution

05 using Bruker Avance 400 NMR spectrometer (400 MHz).

Magnetic susceptibility measurements at 298 K were carried out

with solid sample using MPMS SQUID VSM (Quantum Design,

USA). Vanadium metal was estimated using an Inductively

Coupled Plasma Mass Spectrometer (ICPMS, Thermo X Series

10 II). EPR spectral study was carried out with a Bruker ER200D

spectrometer. Viscometric measurements were performed with a

Schott AVS 310 automated viscometer. Confocal microscopy

was done using Olympus IX 81 fluorescence microscope. Flow

cytometric analysis was performed using FACS Calibur (Becton

15 Dickinson (BD) cell analyzer) at FL2 channel (595 nm).

was prepared using deionized and sonicated triple distilled water.

Calf thymus (ct) DNA, agarose (molecular biology grade),

distamycinꢀA, catalase, superoxide dismutase (SOD), 2,2,6,6ꢀ

tetramethylꢀ4ꢀpiperidone

diacetate (DCFDA), Hoechst 33258, ethidium bromide (EB),

propidium iodide (PI), 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ

(TEMP),

2',7'ꢀdichlorofluorescein

diphenyltetrazolium bromide (MTT), Dulbecco’s modified eagle

medium (DMEM), Dulbecco’s phosphate buffered saline (DPBS)

and fetal bovine serum (FBS) were purchased from Sigma

®

(

USA). MitoTracker Deep Red FM (Cat. no.M22426) (MTR)

was purchased from Invitrogen Bio Services, India. Dipyrido[3,2ꢀ

a:2′,3′ꢀc]phenazine (dppz) was prepared following a literature

6

|

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DOI: 10.1039/C4DT02165G

Syntheses

(54700). Molar conductivity in 10% aqueous DMF at 298 K [Λ /

M

S cm mol ]: 87. ꢁ , ꢁ at 298 K: 1.64.

eff B

2

ꢀ1

60

Vanadyl sulfate (0.16 g, 1.0 mmol) and barium chloride (0.24 g,

.0 mmol) together were dissolved in 15 ml of ethanol and 3 ml

of water. The mixture was then stirred at room temperature for

.5 h under nitrogen atmosphere using Schlenk technique. The

1

X-ray crystallographic procedures

5

0

5

0

5

0

5

0

5

0

5

1

The crystal structure of complex 1 was obtained by using the

mixture was filtered using celite to remove white barium sulphate 65 singleꢀcrystal Xꢀray diffraction technique. Crystals were grown

as the precipitate. The blue filtrate was deaerated and then

saturated with nitrogen. An ethanol solution (10 ml) of the

corresponding phenanthroline base (0.19 g, phen; 0.28 g, dppz,

on slow evaporation of the solvent of an acetonitrile solution of

the complex. Crystal mounting was done on glass fibre with

epoxy cement. All geometric and intensity data were collected at

low temperature (100 K) using an automated Bruker SMART

1

2

3

4

5

(1.0 mmol)) was added to the filtrate. A deep greenish solution

was formed after stirring the mixture for 20 min. To this mixture 70 APEX CCD diffractometer equipped with a fine focus 1.75 kW

was added a deaerated ethanol solution (15 ml) of the βꢀdiketone

ligand (0.36 g, Hcur; 0.69 g, Hscur (1.0 mmol)) which was

sealed tube MoꢀK Xꢀray source (λ = 0.71073 Å) with increasing

α

ω

(width of 0.3° per frame) at a scan speed of 5 sec per frame.

Intensity data, collected using ꢀ2 scan mode, were corrected

for Lorentz–polarization effects and for absorption. The

previously neutralized with Et N (0.10 g, 1.0 mmol). The

ω θ

3

5

9

complexes were precipitated out of the solution spontaneously as

solid after stirring for 1 h. The precipitate was then filtered, 75 structure was solved and refined using SHELXL97 present in the

60,61

isolated and washed with ethanol, THF and chloroform and

finally dried in vacuum over P O

WinGx suit of programs (Version 1.63.04a).

All nonꢀ

hydrogen atoms were initially located in the difference Fourier

maps, and for the final refinement, the hydrogen atoms were

placed in geometrically ideal positions and refined in the riding

4

10.

Complex 1: Yield = 87%. Anal. Calcd for C H N ClO V: C,

3

27

2

7

6

4

0.98; H, 4.19; N, 4.31; V, 7.84. Found: C, 60.79; H, 4.26; N, 80 mode. Final refinement included atomic positions for all the

+

.21; V, 7.91. ESIꢀMS in CH CN: m/z 614.1125 [MꢀCl] . IR

atoms, anisotropic thermal parameters for all the nonꢀhydrogen

atoms and isotropic thermal parameters for all the hydrogen

atoms. The electron density contributions from the highly

3

ꢀ

1

data/ cm : 3064 w, 1590 s, 1491 vs, 1420 s, 1381 m, 1276 s,

1152 m, 1122 m, 1035 w, 966 m, 840 m, 720 m, 555 w, 460 w

(

1

vs, very strong; s, strong; m, medium; w, weak). UVꢀvisible in

disordered solvent molecules were removed using the SQUEEZE

3

ꢀ1

62

0% DMF [λ_m_a_x/nm (

ε

/ dm mol cm )]: 721 (54), 453 sh 85 routine (PLATON). EADP restraint was used on the atoms C25

(

33000), 434 (35600), 265 (30400). Molar conductivity in 10%

and C27 in order to avoid eccentric thermal ellipsoids.

2

ꢀ1

aqueous DMF at 298 K [Λ / S cm mol ]: 104. ꢁ , ꢁ at 298 K:

M

eff

B

1.67.

Crystal data for 1: C H ClN O V, M = 649.96, triclinic, P ī,

3

27

2

7

a = 9.3606(11), b = 12.8152(18), c = 16.3781(19) Å,

Complex 2: Yield = 74%. Anal. Calcd for C H N ClO V: C, 90 102.249(11), = 101.851(10), = 104.124(11)º, U =

α =

β

γ

3

9

29

4

7

3

−3

6

7

2.28; H, 3.89; N, 7.45; V, 6.77. Found: C, 62.41; H, 3.77; N,

1792.1(4) Å , Z = 2, D_c= 1.205 Mg m , T = 293(2) K,

+

.42; V, 6.69. ESIꢀMS in CH CN: m/z 716.1363 [MꢀCl] , IR

reflections collected/unique [R(int)]: 24877/6004 [0.1291],

F(000) = 670, GOOF = 0.912, R1[wR2] = 0.1050 [0.2340] for

3

ꢀ

1

data/ cm : 3070 w, 1587 s, 1490 vs, 1422 s, 1377 m, 1279 s,

2

1

4

7

154 m, 1117 m, 1031 w, 968 m, 810 m, 723 m, 558 w, 463 w,

6004 reflections [I > 2

/ dm mol cm )]: 95 0.1775 (all data)]. The CCDC deposition number is 882736.

31 (64), 454 sh (38600), 434 (43900), 382 (25600), 361

σ

(I)] and 385 parameters [R1(F ) =

3

ꢀ1

35 m. UVꢀvisible in 10% DMF [λ_m_a_x/nm (

ε

(

20300), 268 (46600). Molar conductivity in 10% aqueous DMF

Cellular incorporation assay

2

ꢀ1

at 298 K [Λ / S cm mol ]: 96. ꢁ , ꢁ at 298 K: 1.61.

M

eff

B

Flow cytometric analysis was performed to study uptake of the

Complex 3: Yield = 81%. Anal. Calcd for C H N ClO V: C, 100 complexes 2 and 4 in HeLa, HaCaT and MCFꢀ7 cells.

4

5

47

2

17

6

5

2

5.48; H, 4.86; N, 2.88; V, 5.23. Found: C, 55.61; H, 4.79; N,

Approximately 0.3x10 cells were plated per well of a 6ꢀwell

+

.85; V, 5.26. ESIꢀMS in CH CN: m/z 938.20 [MꢀCl] , IR data/

tissue culture plate in DMEM containing 10% FBS. After 24 h of

3

ꢀ

1

cm : 3310 m (br), 1585 s, 1502 vs, 1421 m, 1361 m, 1255 s,

122 m, 1071 m, 1026 m, 964 m, 816 w, 730 m, 540 w, 469 w

incubation at 37 °C in a CO incubator, complexes 2 and 4 (10

2

1

ꢁM) were added to the cells at different incubation time intervals

3

ꢀ1

ꢀ

(

br, broad). UVꢀvisible in 10% DMF [λ_m_a_x/nm (ε/ dm mol cm 105 (2 and 4 h) in dark. Then the cells were trypsinized, transferred

)

1

]: 752 (55), 439 sh (29600), 420 (36800), 270 (38000). Molar

into 1.5 ml centrifuge tubes, washed once with chilled PBS

centrifuging at 4000 rpm for 5 min at 4 °C. The supernatant was

discarded and the cell pellet was suspended in 200 ꢁl of PBS.

Flow cytometric analysis was performed using FACS Calibur

2

ꢀ1

conductivity in 10% aqueous DMF at 298 K [Λ / S cm mol ]:

M

90. ꢁ , ꢁ at 298 K: 1.69.

eff

B

Complex 4: Yield = 79%. Anal. Calcd for C H N ClO V: C, 110 (Becton Dickinson (BD) cell analyzer) at FLꢀ2 channel. For

5

1

49

4

17

5

6.91; H, 4.59; N, 5.21; V, 4.73. Found: C, 56.83; H, 4.64; N,

confirming that complex 4 was taken up by cells mainly through

glucose receptor mediated endocytosis, HeLa cells were first

incubated with 10 mM glucose for 2 h with subsequent addition

of complexes 2 and 4 for the FACS analysis.

+

.30; V, 4.75. ESIꢀMS in CH CN: m/z 1040.2452 [MꢀCl] , IR

3

ꢀ

1

data/ cm : 3303 m (br), 1588 s, 1503 vs, 1422 w, 1383 w, 1276 s,

1

124 m, 1070 m, 1029 m, 961 m, 818 w, 726 w, 550 w, 467 w.

3

ꢀ1

UVꢀvisible in 10% DMF [λ_m_a_x/nm (ε/ dm mol cm )]: 749 (67), 115

4

44 sh (35600), 426 (41200), 381 (31600), 362 (26500), 267

Cell cytotoxicity assay

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1

RNAse) followed by incubation at 37 °C for 2 h. DNA samples

Photocytotoxicity of the complexes 1ꢀ4 was assessed from 60 were resolved on 1.5% agarose gel at 80 V for ~2 h and

MTT assay in which mitochondrial reductases in viable cells

cleave the tetrazolium rings of MTT forming dark blue

membrane impermeable crystals of formazan which can be

dissolved in DMSO and estimated from spectral measurements at

photographed under UV light.

5

0

5

0

5

0

5

0

5

0

5

Annexin-V FITC and PI assay

39

5

30 nm. The quantity of formazan formed gave a measure of

65

the number of viable cells. HeLa, HaCaT and MCFꢀ7 cells were

used to carry out the MTT assay. About 8000 cells were taken in

each well of a 96ꢀwell culture plate in DMEM containing 10%

FBS. After 24 h of incubation at 37 °C in a CO₂incubator,

To ascertain the apoptotic pathway of cell death, HeLa and

5

ꢀ1

HaCaT cells (4 x 10 cells ml ) were treated with complex 4 (3

ꢁM in HeLa and 1 µM in HaCaT) in 10% DMEM for 4 h,

followed by exposure to visible light for 1 h. The cells were then

1

2

3

4

5

different concentrations (50, 25, 12.5, 6.25, 3.12 and 1.56 µM in 70 cultured for 18 h in complete medium, harvested and washed

HeLa; 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 µM in MCFꢀ7 and

HaCaT) of the complexes dissolved in 1% DMSO ꢀDMEM were

added to the cells and incubation was continued for a period of 4

h in dark. The medium was subsequently replaced with PBS and

twice with chilled PBS at 4 °C. The cells were reꢀsuspended in

200 µl AnnexinꢀV binding buffer (100 mmol HEPES/ NaOH, pH

of 7.4 containing 140 mM NaCl and 2.5 mM CaCl ), stained with

2

AnnexinꢀV FITC and PI, and incubated for 15 min in dark. After

photoꢀirradiation was done in visible light of 400ꢀ700 nm (10 J 75 incubation the cells were analyzed immediately using flow

ꢀ

2

64

cm ; irradiation time; 1 h) using Luzchem Photoreactor (Model

LZCꢀ1, Ontario, Canada) fitted with Sylvania make 8 fluorescent

cytometry.

ꢀ

2

white tubes with a fluence rate of 2.4 mW cm . Post irradiation,

PBS was replaced with DMEMꢀFBS and incubation was

ROS from DCFDA assay

The generation of ROS was probed using 2’,7’ꢀ

3

8

continued for a further period of 20 h in dark. After incubation, a 80 dichlorodihydrofluorescein diacetate (DCFDA) assay. Cell

ꢀ

1

2

5 ꢁl of 4 mg ml of MTT was added to each well and incubation

permeable DCFDA gets oxidized by cellular ROS to generate

fluorescent DCF with an emission maxima at 525 nm. The

percentage of cells generating ROS can be determined by flow

cytometry analysis. To detect any ROS generation, HeLa and

was done for an additional 4 h. The culture medium was

subsequently discarded and 100 ꢁl of DMSO was added to

dissolve the formazan crystals. The absorbance was determined

using a BIORAD ELISA plate reader. The cytotoxicity of the 85 HaCaT cells were incubated with complex 4 (3 ꢁM in HeLa and

complexes was measured as the percentage ratio of the

absorbance of the compound treated with the cells and light

irradiation to that of the irradiated cells in absence of the

compound as a light control. The IC₅₀values of the complexes

1 µM in HaCaT) for 4 h followed by photoꢀirradiation (400ꢀ700

nm) for 1 h in 50 mM PBS. The cells were harvested by

trypsinization and single cell suspension of 1x10 cells ml was

prepared. The cells were then treated with 10 ꢁM DCFDA

6

ꢀ1

were determined by

a

nonlinear regression analysis 90 solution in DMSO in dark for 15 min at room temperature. The

“

log(inhibitor) vs. normalised response (variable slope)” using

distribution of DCFDA stained HeLa and HaCaT cells was

determined by flow cytometry.

6

3

the GraphPad Prism 5.1.

DNA fragmentation analysis

Confocal experiments

95

DNA fragmentation analysis by agarose gel electrophoresis

was performed to determine the cell death mechanism induced by

Uptake of the fluorescent complexes 1ꢀ4 and curcumin (Hcur)

into the cells was visualized using a confocal scanning electron

microscope (Zeiss, LSM510 apocromat). HaCaT and HeLa cells

5

complexes 2 and 4. Briefly, 3.0x10 HaCaT or HeLa cells were

taken in each 60 mm dish. It was grown for 24 h and later treated

with 2 (4 ꢁM in HaCaT and 5 ꢁM in HeLa) and 4 (1 ꢁM in 100 seeding density of 5x10 cells in 1.5 ml of culture medium for 24

HaCaT and 3 ꢁM in HeLa) and incubated for 4 h in dark. One

dish containing the complex was exposed to light for 1 h and

again the cells were left to grow for 4 h along with its dark

control in another dish. After 4 h, cells were trypsinized, washed

were grown on glass cover slips in each 12 well plates at a

4

h. Cells were then treated with the complexes (10 ꢁM) for 2 and 4

h in dark. Cells were fixed and permeabilized with chilled

methanol for 5 min at ꢀ20 °C. Methanol was subsequently

removed followed by washing with 1X PBS. It was later

ꢀ

1

with DPBS and reꢀsuspended in 0.4 ml of lysis buffer (10 mM 105 incubated with propidium iodide (1 mg ml ) for 2 min to stain

TrisꢀHCl; pH, 8.0, 20 mM EDTA, 0.2% tritonꢀX 100) for an

incubation time of 20 min on ice. Lysed cells were centrifuged

for 20 min at 13000 rpm and their supernatant having soluble

chromosomal DNAs including both high molecular weight DNA

the nucleus and visualized under a confocal scanning electron

microscope. To view the PDT effect we performed the

experiments with complexes 2 (5 ꢁM) and 4 (3 ꢁM) by exposing

one of the HaCaT cell plate to visible light for 1 h. The cellꢀ

®

and

Phenol:chloroform (1:1) was added and centrifuged again at

3000 rpm for 20 min to remove the protein present. Later,

nucleosomal

DNA

fragments

was

collected. 110 permeant MitoTracker deep red having a mildly thiolꢀreactive

chloromethyl moiety was used for labeling mitochondria on live

non fixed cells. A 50 nM of MitoTracker Deep Red FM (Cat.

no.M22426) was used for confocal microscopy.

®

1

supernatant was precipitated with 1/10 volume of 3M sodium

acetate (pH, 5.8) and 2 volume of ethanol at −20 °C for

overnight. DNA pellet was washed with 70% alcohol and reꢀ 115 FACS for cell cycle profiling

ꢀ

suspended in TrisꢀEDTA (pH = 8) containing RNAse (100 µg ml

8

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To study the effect of the complexes 2 and 4 on the cell cycle,

3x10 HeLa, HaCaT and MCFꢀ7 cells were plated per well of a

solution.

5

~

6

2

60

ꢀwell tissue culture plate in DMEM containing 10% FBS. After

DNA cleavage experiments

4 h of incubation at 37 °C in a CO incubator, samples with

2

5

0

5

0

5

0

5

0

5

0

5

various concentrations of 2 and 4 in 1% DMSO buffer were

added to the cells and incubation was continued for 4 h in dark.

The medium was subsequently replaced with PBS and photoꢀ 65 using the complexes 1ꢀ4 (40 ꢁM) in 50 mM TrisꢀHCl buffer of

irradiation was done in visible light (400–700 nm) using

Luzchem Photoreactor for 1 h. After photoexposure, PBS was

removed and replaced with DMEMꢀ10% FBS and incubation was

continued for another period of 7 h in dark. After incubation,

The photoꢀcleavage of supercoiled pUC19 DNA (0.2 µg, 50

µM, 2686 baseꢀpairs) was studied by agarose gel electrophoresis

pH 7.2 and 50 mM NaCl containing 10% DMF. The photoꢀ

excitation was carried out in nearꢀIR red light using diode lasers

of 705 nm and 785 nm wavelengths (Model: LQC705ꢀ38E and

LQC785ꢀ100C from Newport Corporation with LD module,

1

2

3

4

5

cells were trypsinized and collected into 1.5 ml centrifuge tubes. 70 continuousꢀwave (CW) elliptical beam and circular beam,

The cells were washed once with chilled PBS of pH 7.4 and fixed

by adding 800 µl of chilled 70% ethanol dropꢀwise with constant

and gentle vortexing to prevent aggregation of cells. The cell

suspension was incubated at ꢀ20 °C for 6 h. The fixed cells were

respectively). The laser powers were 38 and 100 mW,

respectively, measured using Spectra Physics CW laser power

meter (Model 407A). Each sample was preꢀincubated for 1.0 h at

37 °C in dark and then exposed to light. After light exposure,

then washed twice with 1.0 ml of chilled PBS by centrifuging at 75 each sample was further incubated for 1.0 h at 37 °C in dark

4

000 rpm for 5 min at 4 °C. The supernatant was discarded by

followed by addition of the loading dye (25% bromophenol blue,

0.25% xylene cyanol and 30% glycerol (2 ꢁl)), and finally loaded

gently inverting the tube and the cell pellet was suspended in 200

ꢀ

1

ꢀ1

µl of PBS containing 10 ꢁg ml DNAseꢀfree RNAse for 12 h

period at 37 °C for digesting the cellular RNA. After digestion, a

on a 1% agarose gel containing 1 ꢁg ml of ethidium bromide

(EB). The gels were run at 50 V for ~2 h in TAE buffer. The

ꢀ

1

2

0 µl volume of 1.0 mg ml propidium iodide solution was added 80 bands were visualized in UV light, photographed, and the

into the mix and incubated further for 20 min at 25 °C in dark.

Flow cytometric analysis was performed using FACS Calibur

(Becton Dickinson “Cell Quest Pro” software) cell analyzer at

FL2 channel (595 nm). Data analysis was performed for the

intensities of the bands measured using the UVITECH Gel

Documentation System. Due corrections were made for the trace

amount of NC form present in the SC DNA and the higher

affinity of EB for binding to NC and linear forms of DNA over

68

percentage of cells in each cell cycle phase by using WinMDI 85 the SC form. The error in measuring the band intensities was

65

version 3.1.

~5%. The mechanistic studies were carried out using different

additives (NaN , 0.5 mM; TEMP, 0.5 mM; DMSO, 4 µl; KI, 0.5

3

DNA binding experiments

mM; catalase, 4 units; SOD, 4 units) that were added to the SC

DNA prior to the addition of the complex. For the D O

2

The DNA binding experiments were carried out using calf 90 experiment, this solvent was used to dilute the sample up to 20 µl

thymus (ct) DNA in TrisꢀHCl buffer (50 mM Trisꢀ HCl, pH =

.2) at room temperature by following the procedures reported

final volume. After light exposure, each sample was incubated for

30 min at 37 °C and analyzed for the photocleaved products by

gel electrophoresis.

7

2

4

earlier from our group. The intrinsic equilibrium binding

constants (K ) of the complexes to ctꢀDNA were obtained by

b

McGheeꢀvon Hippel (MvH) method using the expression of Bard

Conclusions

6

6,67

and coꢀworkers.

DNA melting experiments were done by

95

Ternary oxovanadium(IV) complexes of phenanthroline bases

and curcumin or diglucosylcurcumin are presented as novel

curcumin based anticancer agents. The complexes show

remarkable photocytotoxicity in visible light with low dark

toxicity. Coordination of curcumin or its derivative to the

monitoring the absorption intensity of ctꢀDNA at 260 nm at

various temperatures, both in the absence and presence of the

oxovanadium(IV) complexes. Measurements were made using a

PerkinꢀElmer Lambda 35 spectrophotometer equipped with a

Peltier temperatureꢀcontrolling programmer (PTP 6) (±0.1 °C) on

ꢀ

1

100 oxovanadium(IV) moiety significantly increases its hydrolytic

stability and photocytotoxicity. This report also presents a

convenient way to increase the bioavailability of curcumin dye in

a metal bound form in the physiological condition. The

increasing the temperature of the solution by 0.5 °C min . The

viscosity measurements were done using Schott Gerate AVS 310

automated viscometer attached with constant temperature bath at

3

1

7(±0.1) °C. The concentration of ctꢀDNA stock solution was

50 ꢁM (NP) in 5 mM TrisꢀHCl buffer. The complex was added

structurally characterized complexes show DNA cleavage activity

•

gradually on increasing the concentration from 0 to 120 ꢁM, and 105 in nearꢀIR light by generating OH radicals as the ROS. The

the viscosity was measured on each addition. The flow times

were monitored with an automated timer. The relative specific

DNA melting and viscosity data suggest intercalative mode of

DNA binding of the complexes. The carbohydrate appended

complexes get internalized preferentially into the cancer cells

with respect to the nonꢀcarbohydrate analogue. The

1

/3

viscosity of DNA, (η/η₀) was plotted versus [complex]/[DNA],

where η is the viscosity of DNA in the presence of the complex

and η is the viscosity of ctꢀDNA alone in 5 mM Tris buffer 110 photocytotoxicity of 2 and 4 compares well with that of the PDT

0

®

medium. The viscosity values were calculated from the observed

flow time of the ctꢀDNA containing solutions (t), after correcting

drug Photofrin . The complexes result in apoptotic cell death by

forming reactive oxygen species on photoꢀirradiation, while

remaining as inert in dark as evidenced from the DNA laddering,

annexin VꢀFITC/PI and DCFDA data. Confocal microscopy has

for that of the buffer alone (t ), η = (t – t )/t . Due corrections

0

were made for the viscosity of DMF solvent present in the

This journal is © The Royal Society of Chemistry [year]

Journal Name, [year], [vol], 00–00 | 9

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DOI: 10.1039/C4DT02165G

revealed mainly cytosolic localization of the complexes with

14 A. Mukhopadhyay, C. BuesoꢀRamos, D. Chatterjee, P.

Pantazis and B. B. Aggarwal, Oncogene, 2001, 20, 7597ꢀ7609.

15 B. B. Aggarwal, C. Sundaram, N. Malani and H. Ichikawa,

Adv. Exp. Med. Biol., 2007, 595, 1ꢀ75.

16 P. Anand, S. G. Thomas, A. B. Kunnumakkara, C. Sundaram,

K. B. Harikumar, B. Sung, S. T. Tharakan, K. Misra, I. K.

Priyadarsini, K. N. Rajasekharan and B. B. Aggarwal,

Biochem. Pharmacol., 2008, 76, 1590ꢀ1611.

significant mitochondrial uptake as observed for complex 2. This

6

7

8

5

0

5

0



property mimics the PDT activity of Photofrin . In summary,

incorporation of the biocompatible curcumin and its derivative

and photoactive dipyridophenazine base as the photosensitizers to

an oxovanadium(IV) moiety has resulted in a significant

enhancement in the stability thus bioavailability of the curcumin

dye in physiological conditions and the complexes showing

remarkable in vitro photocytotoxicity on cancer cells in visible

light with low dark toxicity could open up a new vistas in the

PDT chemistry.

5

0

5

0

5

1

7

T. Choudhuri, S. Pal, M. L. Agwarwal, T. Das and G. Sa,

FEBS Lett., 2002, 512, 334ꢀ340.

18 R. Wilken, M. S. Veena, M. B. Wang and E. S. Srivatsan, Mol.

Cancer, 2011, 10, 1ꢀ19.

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Med. Biol., 2007, 595, 453ꢀ470; (b) P. Anand, A. B.

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Acknowledgments

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0

A. Hussain, K. Somyajit, B. Banik, S. Banerjee, G. Nagaraju

and A. R. Chakravarty, Dalton Trans., 2013, 42, 182ꢀ195.

S. Banerjee, P. Prasad, I. Khan, A. Hussain, , P. Kondaiah and

A. R. Chakravarty, Z. Anorg. Allg. Chem., 2014, 640, 1195–

We thank the Department of Science and Technology (DST,

Government of India), and the Council of Scientific and

Industrial Research, New Delhi), for financial support

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1

204.

(

SR/S5/MBDꢀ02/2007 and CSIR/01(2559)/12/EMRꢀII/2012).

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(a) A. K. Renfrew, N. S. Bryce and T. W. Hambley, Chem.

Sci., 2013, 4, 3731ꢀ3739; (b) D. Pucci, T. Bellini, A. Crispini,

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Valentini and G. Zanchettab, Med. Chem. Commun., 2012, 3,

A.R.C. thanks the DST for J.C. Bose national fellowship. We are

thankful to the Alexander von Humboldt Foundation for donation

of an electrochemical system and DST for a CCD diffractometer

facility. We are thankful to Sanjoy Mukherjee and Akanksha

Dixit for their help in the computational and fluorescence

microscopy study, respectively. S.B. thanks the University Grants

Commission (UGC), New Delhi, for a research fellowship.

8

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62ꢀ468.

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1.973(5)

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2.783(2)

78.12(18)

94.10(18)

160.3(2)

96.1(2)

N(2)ꢀV(1)ꢀO(3)

N(2)ꢀV(1)ꢀO(4)

N(2)ꢀV(1)ꢀO(7)

N(2)ꢀV(1)ꢀCl(1)

O(3)ꢀV(1)ꢀO(4)

O(3)ꢀV(1)ꢀO(7)

O(3)ꢀV(1)ꢀCl(1)

O(4)ꢀV(1)ꢀO(7)

O(4)ꢀV(1)ꢀCl(1)

O(7)ꢀV(1)ꢀCl(1)

163.4(2)

V(1)ꢀN(2)

92.28(17)

95.0(2)

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100.4(2)

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N(1)ꢀV(1)ꢀO(7)

N(1)ꢀV(1)ꢀCl(1)

83.53(15)

101.96(19)

81.47(14)

174.65(14)

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79.98(15)

55 (a) J. G. Collins, A. D. Sleeman, J. R. AldrichꢀWright, I. 135

Greguric and T. W. Hambley, Inorg. Chem., 1998, 37, 3133ꢀ

3

141; (b) E. Amouyal, A. Homsi, J.ꢀC. Chambron and J.ꢀP.

Sauvage, J. Chem. Soc. Dalton Trans., 1990, 1841ꢀ1845.

J. Guilford II, R. Jackson, C. Choi and W. R. Bergmark, J.

Phys. Chem., 1985, 89, 294ꢀ300.

5

6

140

Journal Name, [year], [vol], 00–00 | 11

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Page 12 of 15

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DOI: 10.1039/C4DT02165G

75

5

0

5

0

5

0

5

0

5

0

5

0

5

0

Table 1. Selected physicochemical data and ctꢀDNA binding parameters of the complexes 1 ꢀ 4

80

a

ꢀ1

^ꢂ^Tm^h/ °C

1

2

3

4

5

6

7

Complex

IR / cm

ṽ (C=O)

3

ꢀ1

λ_m_a_x/ nm (ε/dm mol

c

d

e

f

g

ꢀ1

λ

f

/ nm [Φ

f

]

µ

eff

Ʌ

M

E

pc / V

K

b

/ mol

ṽ (V=O)

ꢀ1 b

cm )

4

1

1590

966

721 (54)

515 [0.008]

1.67

104

ꢀ1.29

(6.1±0.4) x10

2.0

85

5

2

3

1587

1585

968

964

961

731 (64)

752 (55)

749 (67)

518 [0.01]

506 [0.01]

1.61

1.69

1.64

96

90

87

ꢀ1.22

ꢀ1.34

ꢀ1.15

(5.6±0.5) x 10

(8.4±0.3) x 10

5.4

4.2

6.9

4

90

5

4

1588

509 [0.015]

(7.2±0.2) x 10

a

b

c

d

In solid phase. In 10% DMFꢀwater. In 10% aqueous DMSO.

B 6

µeff in µ using DMSOꢀd solutions of the oxovanadium(IV) complexes at 25 °C.

f

e

2

ꢀ1

Λ

M

, molar conductivity in S cm mol in 10% aqueous DMF at 25 °C. The95complexes are nonꢀelectrolytic in pure DMF. In 20% DMF–H O with

2

ꢀ

1

g

h

0.1M TBAP. The potentials are vs. SCE at a scan rate of 50 mV s . Epc is the cathodic peak potential. Intrinsic ctꢀDNA binding constant (K

b

).

Change in the calf thymus DNA melting temperature.

100

Table 3: IC50 (ꢁM) values of different curcumin complexes and other relevant compounds in different cells

105

Compound

HeLa

HaCaT

MCFꢀ7

(Dark)

a

b

a

b

a

b

(

Light)

(Dark)

(Light)

(Dark)

46±1

43±1

40±2

34±1

28±1

>50

(Light)

1

8.1±0.3

3.3±0.4

4.6±0.2

1.5±0.2

8.2±0.2

>50

6.4±0.3

2.3±0.2

3.5±0.2

0.63±0.02

19±1

10±1

>50

90±5

>50

2

>50

4.7±0.3

5.8±0.2

2.4±0.1

20±2

110

3

46±1

45±1

85±4

4

Curcumin (Hcur)

Hscur

115

>50

c

[

La(pyꢀtpy)(cur)(NO

3

)

2

]

4.6±0.6

>100

d

e

[

(pꢀcymene)Ru(cur)Cl]

19.58 ± 2.37

f

Cisplatin

68.7±3.4 71.3±2.9

1

2

0

a

−2

b

For 4 h incubation in the dark followed by exposure to visible light of 400ꢀ700 nm (10 J cm ) for 1h. For 4 h

c

d

e

f

incubation in the dark. Data from ref. 20. From ref. 23. 72 h incubation in dark. From ref. 40 for an incubation

time of 4 h.

125

Table 4. Selected SC pUC19 DNA (0.2 µg) cleavage data of the complexes 1ꢀ4 and the ligands in red light

a

1

30

Reaction condition

% NC Form

(Red light, 785 nm)

(

In dark)

(Red light, 705 nm)

DNA control

DNA + 1

2

5

3

4

42

90

46

92

15

18

38

87

45

94

16

18

DNA + 2

DNA + 3

10₁₃₅

4

DNA + 4

8

DNA + curcumin (Hcur)

DNA + Hscur

11

13

140

a

In Trisꢀbuffer medium (pH =7.2). Red light from diode lasers. Photoꢀexposure time (t) = 1 h. Concentration of

the complexes (1ꢀ4) and the ligands was 40 µM.

1

2

|

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DOI: 10.1039/C4DT02165G

Fig. 3 Time dependent cellular uptake of complexes 2 (grey bar)

and 4 (black bar) (10 ꢁM) in HeLa, HaCaT and MCFꢀ7 cells as

determined by FACS analysis.

7

8

9

5

0

5

0

5

1

2

3

4

5

6

7

0

5

0

5

0

5

0

5

0

5

0

5

0

Scheme 1. The ligands and the complexes 1ꢀ4 used.

Fig. 4 A comparison of the (a) cellular uptake of complex 4 (10 ꢁM)

at 4 h of incubation with HeLa cells in absence of excess added

sugar and after preꢀincubation with higher glucose concentration (10

mM) as determined from the FACS analysis. (b) Mean fluorescence

intensity demonstrating the mean complex uptake in cells for 2 and

4

under above mentioned condition.

1

00

05

10

15

20

25

30

35

40

Fig. 1 (a) Absorption spectra of curcumin (Hcur) ligand and the complexes

and 4 in 10% aqueous DMF. The inset shows the d–d band of the

oxovanadium(IV) complexes. (b) Fluorescence spectra of Hcur and the

complexes 2 and 4 in 10% aqueous DMSO (λex = 420 nm).

2

Fig. 5 Photocytotoxicity of the complexes 1ꢀ4 in HeLa, MCFꢀ7 and

HaCaT cells on 4 h incubation in dark (black bar) followed by photoꢀ

ꢀ2

irradiation with visible light of 400–700 nm (10 J cm ) (red bar).

Samples tested up to 50 µM concentration are marked as *.

Fig. 2 (a) HOMOꢀLUMO of complex 4 using energy optimized

structure. (b) Reproduced crystal structure of complex 1.

11

Fig. 6 DNA ladder of complexes 2 and 4 showing apoptosis in

HeLa and HaCaT cells caused on photoꢀexposure to visible light

(400ꢀ700 nm): D, in dark; L, in light and M, 100 b.p. DNA ladder.

Journal Name, [year], [vol], 00–00 | 13

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7

8

9

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

1

2

3

4

5

6

7

Fig. 10 Confocal images of complexes 2 (5 ꢁM) and 4 (3 ꢁM) in

HaCaT cells. Panels (a), (d), (g) and (j) show fluorescence of 2. Panels

(a) are for the complex green fluorescence; Panels (b) for PIꢀstaining.

Panels (c) are the merged images. The PDT effect, visible only in light,

is shown by arrows in panels (b) Scale bar: 10 ꢁm.

Fig. 7 FACScan profiles of AnnexinꢀV FITC and PI staining of HeLa

and HaCaT cells undergoing early apoptosis induced by complex 4 in

visible light (400ꢀ700 nm) while remaining nontoxic in dark.

(a)

95

00

05

10

15

1

(b)

Fig. 11 Fluorescence microscopic images of complexes 2 and 4 (10

µM) and Mitoꢀtracker deep red (MTR, 50 nM) in HeLa and HaCaT

cells after 4 h incubation: panels (a) show fluorescence of MTR; panels

b) show fluorescence images of 2 and 4 respectively and panels (c)

show the merged image of MTR and the complexes. Scale bar: 20 µm.

(

Fig. 8 Flow cytometric analysis (FACS) for ROS generation by

complex 4 was performed using DCFDA dye. Generation of ROS is

highlighted by the shift in fluorescence band positions compared to

cells alone in (a) HeLa and (b) HaCaT cells treated with complex 4,

under different experimental conditions as shown by the color

codes. Greater shift implies higher amount of DCF and thus greater

ROS generation.

120

1

25

30

35

Fig. 12 Bar diagram showing %distribution of MCFꢀ7, HaCaT and HeLa

cells treated with complex 2 (5 ꢁM in MCFꢀ7, 3 ꢁM in HaCaT and 4 ꢁM

Remarkable enhancement in

in HeLa) and complex 4 (3 ꢁM in MCFꢀ7, 1 ꢁM in HaCaT and 2 ꢁM in

Fig. 9 Confocal images of HeLa and HaCaT cells (after 4 h) treated

HeLa). UN(D) = untreated dark control, UN(L) = untreated light control,

–

1

with complexes 2–4 (10 ꢁM) and propidium iodide (PI, 1 mg ml ):

p

D

h

=

o

dar

t

k

o

, L

c

=

y

lig

t

h

o

t o

t

f

o

40

x

0ꢀ

i

7

c

00

i

n

ty and hydrolytic

m.

Panels: (a) fluorescence of the complex, (b) fluorescence of PI and

(c) merged image of (a) and (b). Scale bar = 10 µm.

1

4

|

Journal Name, [year], [vol], 00–00

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DOI: 10.1039/C4DT02165G

Remarkable enhancement in

photocytotoxicity and hydrolytic

stability of curcumin on binding to

an oxovanadium(IV) moiety †

7

0

5

75

a

b

Samya Banerjee, Ila Pant, Imran Khan, Puja

a

b

Prasad, Akhtar Hussain, Paturu Kondaiah*,

and Akhil R. Chakravarty*

a

80

1

2

3

4

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5

0

5

0

5

0

5

0

5

0

5

Keywords : Curcumin, Oxovanadium(IV), Apoptotic photocytotoxicity,

Cellular imaging, mitochondria targeting

Synopsis (20 words): Polypyridyl oxovanadium(IV) curcumin

complexes show remarkable hydrolytic stability and visible lightꢀ

induced photocytotoxicity in cancer cells by mitochondria targeting

ROSꢀmediated apoptosis.

85

Pictogram:

Journal Name, [year], [vol], 00–00 | 15

Article Doi

Remarkable enhancement in photocytotoxicity and hydrolytic stability of curcumin on binding to an oxovanadium(iv) moiety

DOI: 10.1039/c4dt02165g

Source and publish data:

Authors:

Article abstract of DOI:10.1039/c4dt02165g

Full text of DOI:10.1039/c4dt02165g

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