Methylene violet 3RAX-conjugated porphyrin for photodynamic therapy: Synthesis, DNA photocleavage, and cell study

Article abstract of DOI:10.1039/c7ra13176c

A methylene violet 3RAX-conjugated porphyrin has been designed and synthesized as a potential photosensitizer in photodynamic therapy because of its high DNA binding constant and the efficacy in inhibiting cancer cells with subcellular localization.

Full text of DOI:10.1039/c7ra13176c

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Methylene violet 3RAX-conjugated porphyrin for
photodynamic therapy: synthesis, DNA
photocleavage, and cell study†
c
Cite this: RSC Adv., 2018, 8, 4472
Si Yao,‡ac Yunman Zheng,‡b Lijun Jiang,d Chen Xie,d Fengshou Wu,
*
b
de
b
a
*
*
*
Chi Huang,
Xiong Zhang,c Ka-Leung Wong,
Zaoying Li and Kai Wang
Received 9th December 2017
Accepted 16th January 2018
A methylene violet 3RAX-conjugated porphyrin has been designed and synthesized as a potential
photosensitizer in photodynamic therapy because of its high DNA binding constant and the efficacy in
inhibiting cancer cells with subcellular localization.
DOI: 10.1039/c7ra13176c
rsc.li/rsc-advances
An extensive attention has been paid on porphyrin-based
compounds as photodynamic therapy photosensitizers for
Introduction
anticancer in recent years. Porphyrins have long been of interest
because of their high binding affinity to DNA strands.13 There
has been reported that porphyrins linked with some anticancer
drugs can effectively localize in tumour cells and improve the
therapy efficiency through the synergistic effect of porphyrins
and anticancer drugs.14 However, to the best of our knowledge,
few reported the porphyrin–methylene violet 3RAX conjugate
and investigated its application in anti-cancer. To this end, we
rst time designed and synthesized compound 2 by conjugating
porphyrin with synthesized methylene violet 3RAX (1)
(Scheme 1). The binding properties of compounds 1 and 2 with
DNA were characterized through photophysical studies
including absorption and emission spectra. The DNA photo-
cleavage activities of two compounds were investigated by
agarose gel electrophoresis. The generation of singlet oxygen
upon irradiation was determined by using 1,3-diphenyliso-
benzofuran (DPBF) as an indicator. Besides, the subcellular
localization and the photocytotoxicity of compound 2 against
HeLa cells were also evaluated.
Photodynamic therapy (PDT) in vivo or in vitro involves light and
chemicals as photosensitizers and normally depends on the
generation of singlet oxygen.1,2 Singlet oxygen, as a primary
damaging agent, induces the apoptosis of cancer cells in
photodynamic therapy. Specically, it could effectively damage
the tiny blood vessels in the tumour tissue and then cause cell
death and local bleeding. As a result, the tumour tissue will be
completely destroyed aer a period of time.3 Besides, the reac-
tive singlet oxygen is able to manufacture injury through
interacting with biological macromolecules,4 including DNA
and proteins.
Phenazine compounds with heterocyclic structure have been
widely applied in dyes, biomedicine,5–8 pesticide, photoelectric
material,9 and chemical analysis.10 Methylene violet 3RAX, as
one of the typical phenazine derivatives, has the rigid planar
phenazine structure. Methylene violet 3RAX shows high bio-
logical activity in the anti-tumour therapy because it is able to
change the molecular structure of DNA, undermine the module
of DNA, and induce the generation of the reactive singlet oxygen
which can cause cells death by cutting off DNA strands in
tumour cell.11,12
Experimental
NMR spectra were recorded on Varian Mercury-VX400 (400
MHz). The IR spectra (KBr pellets) were recorded on a Shimadzu
FT-IR 3000 (KBr, V cmꢀ1). Mass spectra were obtained from
a Waters ZQ4000 (ESI). Elemental analyses were performed
using an Elementar Vario EL. UV-vis spectra were measured on
a Shimadzu 1901 spectrometer. Fluorescence spectra were
measured on a Perkin Elmer LS-55 spectrometer. Calf-thymus
DNA (ct-DNA) was obtained from Sigma company. An extinc-
tion coefficient of 6600 Mꢀ1 cmꢀ1 at 260 nm was used to
determine the concentration of ct-DNA.15 Silica gel 60 (0.04–
0.063 mm) for column chromatography was obtained from
Chinese medicine Reagent Co., Ltd. Other reagents were not
further puried.
aHubei Collaborative Innovation Center for Advanced Organic Chemical Materials,
College of Chemistry and Chemical Engineering, Hubei University, Wuhan, P. R.
China. E-mail: kaiwang@hubu.edu.cn
bCollege of Chemistry and Molecular Sciences, Wuhan University, Wuhan, P. R. China.
E-mail: chihuang@whu.edu.cn
cKey Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute
of Technology, Wuhan, P. R. China. E-mail: wfs42@126.com
dDepartment of Chemistry, Hong Kong Baptist University, Hong Kong, P. R. China.
E-mail: klwong@hkbu.edu.hk
eChangshu HKBU Technology Company Limited, Jiangsu Province, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra13176c
‡ These authors contributed equally to this work.
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Scheme 1 Synthetic route of compound 1 and 2.
General procedure for the synthesis of methylene violet 3RAX amount of DMSO (<5% total volume) and then dispersed in
(1): to a solution of 4-amino-N,N-diethylaniline (5 g, 30.5 mmol), buffer media (pH ¼ 7.4, 0.05 M Tris–HCl, 0.1 M NaCl). UV-vis
aniline (5.8 g, 61.0 mmol) and concentrated hydrochloric acid (8 absorption spectra of compound 1 and 2 were recorded within
mL) in acetic acid/sodium acetate buffer solution (200 mL) was a range of 450–740 nm. Fluorescence spectra of compound 1
added a large excess of potassium dichromate (19.7 g, 67.1 and 2 were recorded between 450 nm and 700 nm. Induced CD
mmol). The reaction mixture was stirred for 3 h at 105 ꢁC through spectra were recorded within a range of 400–700 nm.
protecting from light. Aer cooling to room temperature, the
1,3-Diphenylisobenzofuran (DPBF) was used as a selective
mixture was ltered and sodium chloride (120 g) was added to singlet oxygen (1O2) acceptor,17 which was bleached upon
the ltrate, yielding the precipitate (1). Yield 2.3 g (35%). 1H NMR reaction with O2.18 The compounds with DPBF in DMSO
1
(400 MHz, CDCl3) d: 1.09 (t, J ¼ 7.1 Hz, 6H), 3.33 (q, J ¼ 7.1 Hz, (50 mM) were prepared in the dark. Each sample container was
4H), 5.63 (s, 1H), 5.99 (s, 1H), 7.13 (d, J ¼ 7.8 Hz, 1H), 7.32 (d, J ¼ covered with aluminium foil with a yellow lter (with cut-off
7.8 Hz, 2H), 7.69–7.80 (m, 4H), 7.85 (d, J ¼ 8.9 Hz, 1H); IR (KBr), n/ wavelength <500 nm) on one side. The samples were then
cmꢀ1: 3058, 1649 (NH2), 1339, 1198 (C–NH2, N–H), 1601, 1524, exposed to light (50 watts) through the lter. The normalized
1484 (C]C, C–N), 1069, 1006, 991, 874, 849, 812, 776, 704 (C–H), absorbance of DPBF at 418 nm in these samples was reported as
1392 (–CH3) cmꢀ1; UV-vis (DMSO), lmax/nm: 560; ESI-MS, m/z: a function of the photo-irradiation time. By plotting the rela-
343.58 [M]+. Elemental analysis: C22H23N4 calcd: C 69.74, H tionship between absorbance intensity and irradiation time, the
+
6.12, N 14.79. Found: C 69.69; H 6.23; N 14.70.
rates of 1O2 production of compound 1 and 2 were determined.
The DNA photocleavage activities of compound 1 and 2 were
General procedure for the synthesis of methylene violet
3RAX-conjugated porphyrin (2): to a solution of methylene measured using the plasmid DNA relaxation assay. Briey, the
violet 3RAX (0.1 g, 0.26 mmol) in dry chloroform (20 mL) was plasmid DNA (pBluescript, 0.5 mg), enriched with the covalently-
added 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin (0.2 g, closed circular or supercoiled conformer (Form I), and the one-
0.32 mmol),16 DMAP (0.03 g, 0.26 mmol) and EDCI (0.1 g, 0.53 phor-all plus buffer (10 mM Tris–acetate, 10 mM magnesium
mmol). The solution was heated to 60 ꢁC for 10 h with stirring. acetate, 50 mM potassium acetate, pH 7.5) was vortexed.
Aer cooling to room temperature, the reaction mixture was Aliquots of the DNA were pipetted into different Eppendorf
poured into saturated brine (15 mL) and then extracted with tubes. Various amounts of autoclaved water (control sample) or
chloroform (3 ꢂ 10 mL). The combined organic layer was dried compounds 1, 2 were added into the Eppendorf tubes to give
using anhydrous sodium sulfate and concentrated in vacuum. a nal volume of 20 mL in each sample tube. The sample
The residue was puried by silica gel column with chloroform/ mixtures were then photo-irradiated at 400–500 nm for 60 min
methanol (v/v 10 : 1) as an eluent. The second fraction was using a transilluminator (Vilber Lourmat) containing 4 ꢂ 15 W
collected. Yield 0.2 g (85%). 1H NMR (400 MHz, CDCl3) d: ꢀ2.91 light tubes (Aqua Lux) with maximum emission at 435 nm.
(s, 2H), 0.90 (t, J ¼ 7.1 Hz, 6H), 3.44 (q, J ¼ 7.1 Hz, 4H), 5.44 (s, During the irradiation procedure, the compounds did not
1H), 7.49 (d, J ¼ 8.1 Hz, 1H), 7.75–7.84 (m, 16H), 8.21 (m, 9H), undergo any photodegradation because of the low power
8.64–8.85 (m, 11H); IR (KBr), n/cmꢀ1: 2922 (C–H), 1675 (C]O), density of light. Aer photo-irradiation, 2 mL of the 6ꢂ sample
1598, 1508 (Ph-H), 1072, 997, 868, 798, 696 (C–H); UV-vis dye solution (which contained 20% glycerol, 0.25% bromo-
(DMSO), lmax/nm: 419, 516, 551, 588, 647; ESI-MS, m/z: 984.95 phenol blue and 0.25% xylene cyanol FF) was added to each
[M + H]+. Elemental analysis: C67H51N8O+ calc. C 81.77, H Eppendorf tube and mixed well by centrifugation. The sample
5.22, N 11.39. Found: C 81.84; H 5.32; N 11.45.
mixtures were loaded onto a 0.8% (v/v) agarose gel (gel dimen-
The spectral measurements were performed at room sion: 13 cm ꢂ 10 cm), with 1 ꢂ TBE buffer (89 mM Tris–borate,
temperature. The compounds were rst dissolved in a small 1 mM EDTA, pH 8) used as supporting electrolyte, and
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electrophoresed at 1.3 V cmꢀ1 for 3 h using a mini gel set (CBS
Scientic Co., Model No. MGU-502T). Aer electrophoresis, the
gel was stained with 0.5 mg mLꢀ1 ethidium bromide solution for
30 min and then destained using deionized water for 10 min.
The resulting gel image was viewed under 365 nm and captured
digitally using a gel documentation system (BioRad).19
The MTT viability assay was performed according to the
standard methods.20 In brief, HeLa cells (3 ꢂ 103 per well) were
seeded in 96-well plates 24 hours prior to exposure to drugs. The
cells were treated with samples overnight in the dark. The
cytotoxicity was determined by the MTT reduction assay. The
cell monolayers were rinsed twice with phosphate-buffered
saline (PBS) and then incubated with 50 mL MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solu-
tion (0.5 mg mLꢀ1) at 37 C for 3 hours. Then the media were
ꢁ
removed, and 100 mL of DMSO solubilizing reagent was added
and shook for 30 minutes to dissolve the formed formazan
crystals in living cells. The absorbance was measured at a dual
wavelength, 540 nm and 690 nm, on a Labsystem Multiskan
microplate reader (Merck Eurolab, Switzerland). Each dosed
concentration was performed in triplicate wells and repeated
twice for the MTT assay.
Fig. 1 UV-vis absorption spectra of compound 1 (a) and compound 2
(c) with the titration of ct-DNA; maximal absorption intensity of
compound 1 (b) and 2 (d) with the titration to ct-DNA. The concen-
tration of compound 1 and 2 is 4 mmol Lꢀ1. Buffer solution (pH ¼ 7.4,
0.05 mol Lꢀ1 Tris–HCl, 0.1 mol Lꢀ1 NaCl).
HeLa cells (3 ꢂ 103 per well) were incubated in 96-well plates
for 24 hours prior to exposure to drugs. The cells were treated
with samples in the dark overnight. Aerwards, the cells were
exposure to yellow light (1–16 J cmꢀ2) produced from a 400 W
tungsten lamp tted with a heat-isolation lter and a 500 nm
long-pass lter. The uence rate was 6 mW cmꢀ2. Cell viability
was determined by the MTT reduction assay at 24 h post-PDT.21
The cell monolayers were rinsed twice with PBS andꢁ then
incubated with 50 mL MTT solution (0.5 mg mLꢀ1) at 37 C for
3 h. Then the media were removed, and 100 mL of DMSO solu-
bilizing reagent was added and shaked for 30 minutes to
dissolve the formed formazan crystals in living cells. The
absorbance was measured at dual wavelength, 540 nm and
690 nm, on a Labsystem Multiskan microplate reader (Merck
Eurolab, Switzerland). Each dosed concentration at individual
light exposure was performed in quadruplicate wells for the
PDT assay.
that increasing DNA concentration resulted in moderate hypo-
chromism for compound 1 and 2 (H ¼ 20%–30%). Besides,
signicant red shi for compound 1 (Dl ¼ 12 nm) was observed,
which was ascribed to the intercalative interaction between
methylene violet 3RAX moiety and DNA at high compound-to-
base pair ratio,22 and interaction between porphyrins and the
bases of DNA.23 Generally, for a given DNA strand, intercalation
exhibits a large red shi of the Soret band (>15 nm) and
a substantial hypochromicity (>35%), while the small change
normally occurs in the groove binding mode.24 In the presence
of high concentrations of ct-DNA, the absorbance band of
compound 2 exhibited substantial hypochromicity (>20%)
without signicant spectral shi, suggesting that compounds 2
may interact with DNA by groove binding.
The intercalative binding mode was characterized and the
binding constants K of compound 1 and 2 were calculated
respectively according to eqn (1):
HeLa cells (2 ꢂ 105 per coverslip in 6-well plate) were treated
with compound 2 (8 mM) for 6 h at 37 ꢁC. Aer that, the
supernatant was carefully removed and the cells were washed
[DNA]total/|3A ꢀ 3F| ¼ [DNA]total/|3B ꢀ 3F| + 1/(|3B ꢀ 3F|K) (1)
three times with PBS. Subcellular localization of compound 2 where 3A is the ratio of Amax/[1, 2] during the process of titration. 3
was examined using an Olympus FV1000 confocal microscope. corresponds to molar attenuation coefficient. 3B is the ratio of
A diode laser line at 514 nm was used for the excitation of Amax/[1, 2] aer saturated with DNA and 3F is the ratio of Amax/[1, 2]
compounds. Emission band pass lter setting at 600–750 nm without DNA. From a plot of ([DNA]total/|3A ꢀ 3F|) versus [DNA]total
,
was used for compound 2. Pinhole size of 80–100 mm was shown in the Fig. 1b and d, the apparent binding constant can be
selected to exclude uorescence light emitted from out-of-focus obtained by the ratio of the slope to the intercept.25 The values of
planes above and below the focusing plane. A 40ꢂ objective was K obtained for compound 1 and 2 were 7.48 ꢂ 103 Mꢀ1 and
used for image capturing. Images were processed and analyzed 6.48 ꢂ 103 Mꢀ1, respectively. However, these values are lower than
by using the FV10-ASW soware (Olympus).
some of reported compounds: metallo-5-ebselenyl-10,15,20-
triphenylporphyrins with herring sperm DNA,25 and TMPyP
(TMPyP ¼ tetrakis(4-methylpyridiniumyl)porphyrin) with poly
[d(G-C)2].26 The smaller binding constants could partially result
Results and discussions
Effects of the stoichiometric addition of ct-DNA on the from the lack metal ions in the center of the porphyrin rings
compound 1 and 2 were illustrated by absorption spectra because the binding mechanism could be modulated by the
respectively (see Fig. 1a and c). The results in Table S1† showed nature of the metal ions.23
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Fluorescent titration was performed for further investigation to investigate the DPBF inuence. Also in Fig. 2c, the generation
on the binding mode between compound 1, 2 and ct-DNA. As of 1O2 decreased with the addition of compound 1 (see DPBF + 1
shown in Fig. 2a and b, the uorescence intensities of + TPP system), it might be because the compound 1 absorbed or
1
compound 1 and 2 increased with the addition of ct-DNA. The quenched singlet oxygen O2 produced by TPP to some extent.
emission enhancement may be attributed to the insertion of
DNA photocleavage activities of compound 1 and 2 were
compounds 1 with p-conjugated planar structure into the measured by monitoring the conversion of a supercoiled form
compact double helix groove space of CT-DNA. Subsequently, (form I) to the nicked circular form (form II),29 with the radia-
the compound 1 in the excited state can be effectively protected tion of light at 400–500 nm for 60 minutes. As shown in Fig. 3,
by the strong hydrophobic core of DNA, avoiding the quenching compound 2 at 400 mM had only 20% DNA photocleavage, while
effect of ambient solvents.27 Besides, there were electrostatic compound 1 at 300 mM showed over 60% photocleavage activity
interactions between the compound 1, 2 (with positive charges) towards DNA. Generally, the DNA photocleavage activity of
and phosphate groups of DNA (with negative charges), which organic molecules was mainly depended on their singlet oxygen
enhance the electron transfer from porphyrin to methylene quantum yield and the electrostatic interaction with DNA. Thus,
violet. To this end, the emission from porphyrin part is sup- the signicantly enhanced cleavage of DNA for compound 1
pressed relative to that of methylene violet 3RAX part. The could be due to its relatively stronger electrostatic interaction
bathochromic shi and enhanced uorescence of compound 2 with DNA (higher density of positive charge) and higher 1O2
aer interaction with DNA suggests it may adopt an end-to-end yield of compound 1 comparing with that of compound 2.30
interaction with DNA (J aggregate).28
The cell inhibition ability is one of crucial features for the
1,3-Diphenylisobenzofuran (DPBF) was used as a uorescent conjugated porphyrins serving as anticancer drugs. Therefore,
probe for detection of singlet oxygen (1O2) generated from MTT and PDT assays were performed to determine the cyto-
compound 1 and 2 upon irradiation24 with 5,10,15,20-tetra- toxicity of compound 2 to HeLa cells. As shown in Fig. 4d, there
phenylporphyrin (TPP) as control groups. The relationship was no obvious decrease in cell viability even the incubating
between absorbance ratio of DPBF (A/A0) and illumination time concentration of the compound 2 reached 100 mM aer 24 h
could indirectly reect the 1O2 yield of the compounds. As incubation, so it proves compound 2 with low dark toxicity,
shown in Fig. 2c, the absorbance of DPBF at 418 nm decreased which means compound 2 results in few adverse effects as an
in the presence of either compound 1 or 2 with the increase of anticancer drug. With the light excitation, compound 2 showed
illumination time. According to the slopes of the curves, the obvious photocytotoxicity as in Fig. 4d. With the increase of
relative rates of 1O2 generation for compound 1 and 2 could be concentrations and light dose, the porphyrins exerted high
1
1
compared. The larger slope in values indicated the higher O2 cytotoxicity because of the increasing O2 generation through
quantum yield. Thus, the order of 1O2 quantum yield for PDT mechanism. The light dose required to kill 60% of HK-1
compounds was F(TPP) > F(1) > F(2), indicating the introduc- cells was approximately 1 J cmꢀ2 at 10 mM for compound 2.
tion of porphyrin ring into compound 1 leads to the reduction The dramatic difference between toxicity with/without light
of 1O2 yields. Moreover, we used TPP mixing with compound 1 indicates that compound 2 has great PDT efficiency against
HeLa cells.
Confocal microscopy was used to further investigate the
subcellular localization of compound 2 in HeLa cells. As shown
in Fig. 4a–c, the red uorescence of compound 2 was mainly
localized in the mitochondria of HeLa cells, because methylene
violet 3RAX is a key dye to stain the mitochondria of cells, such
as Janus green B.31 This result suggests porphyrins tailed with
methylene violet 3RAX could be effective in promoting the
Fig. 2 Fluorescence spectra of compound 1 (a) and 2 (b) with the
titration to ct-DNA. Each spectrum was recorded in pH ¼ 7.4,
0.05 mol Lꢀ1 Tris–HCl, 0.1 mol Lꢀ1 NaCl buffer solution. (c) Singlet
oxygen generation of 1 and 2 (50 mM). The change of absorption of Fig. 3 DNA binding profiles of compound 1 and 2 in the one-phor-all
DPBF at 418 nm (A/A0) upon the irradiation time. The spectrum was plus buffer (10 mM Tris–acetate, 10 mM magnesium acetate, 50 mM
recorded in DMSO.
potassium acetate, pH ¼ 7.5).
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