Curcumin derivatives as photosensitizers in photodynamic therapy: Photophysical properties and: In vitro studies with prostate cancer cells

Article abstract of DOI:10.1039/c9pp00375d

Photodynamic therapy (PDT) is a minimally invasive approach to treat various forms of cancer, based on the ability of certain non-toxic molecules (photosensitizers) to generate reactive oxygen species (ROS) after excitation by light of a certain wavelength and eventually induce strong phototoxic reactions against malignant cells and other pathogens. Curcumin is one of the most extensively investigated phytochemicals with a wide range of therapeutic properties and has been shown to induce strong photocytotoxic effects in micromolar concentrations against a variety of cancer cell lines. Curcumin (1) is comparatively evaluated with the naturally occurring bisdemethoxy Curcumin (2), which lacks the two methoxy groups, as well as two newly synthesized curcuminoids, the cinnamaldehyde derivative (3) and the dimethylamino one (4), designed to increase the absorption maximum and hence the tissue penetration. The synthetic curcuminoids were successfully synthesized in sufficient amounts and their photophysical properties such as absorption, fluorescence, photobleaching and free radical generation were investigated. Compound 4 exhibited a significant increase in peak absorption (497 nm) and strong fluorescent emission signals were recorded for all curcuminoids. Photobleaching of 4 was comparable to 1 whereas 2 and 3 showed more extended photobleaching but much higher ROS production in very short irradiation times. Compounds 2 and 4 exhibited specific intracellular localization. After dark and light cytotoxicity experiments against LNCaP prostate cancer cell line for all curcuminoids, concentration of 3 μM and irradiance of 6 mW cm^-2 were selected for the PDT application which resulted in remarkable results with very short LD₅₀. Curcuminoids 2 and 4 exhibited a significant dose-dependent PDT effect. The biphasic dose-response photodynamic effect observed for 1 and 3 may provide a strategy against prolonged and sustained photosensitivity.

Full text of DOI:10.1039/c9pp00375d

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Curcumin derivatives as photosensitizers in
photodynamic therapy: photophysical properties
Cite this: DOI: 10.1039/c9pp00375d
and in vitro studies with prostate cancer cells
K. T. Kazantzis,a K. Koutsonikoli,a B. Mavroidi, b M. Zachariadis,c P. Alexiou,b
M. Pelecanou, b K. Politopoulos,a E. Alexandratou *a and M. Sagnou*b
Photodynamic therapy (PDT) is a minimally invasive approach to treat various forms of cancer, based on
the ability of certain non-toxic molecules (photosensitizers) to generate reactive oxygen species (ROS)
after excitation by light of a certain wavelength and eventually induce strong phototoxic reactions against
malignant cells and other pathogens. Curcumin is one of the most extensively investigated phytochem-
icals with a wide range of therapeutic properties and has been shown to induce strong photocytotoxic
effects in micromolar concentrations against a variety of cancer cell lines. Curcumin (1) is comparatively
evaluated with the naturally occurring bisdemethoxy Curcumin (2), which lacks the two methoxy groups,
as well as two newly synthesized curcuminoids, the cinnamaldehyde derivative (3) and the dimethylamino
one (4), designed to increase the absorption maximum and hence the tissue penetration. The synthetic
curcuminoids were successfully synthesized in sufficient amounts and their photophysical properties
such as absorption, fluorescence, photobleaching and free radical generation were investigated.
Compound 4 exhibited a significant increase in peak absorption (497 nm) and strong fluorescent emission
signals were recorded for all curcuminoids. Photobleaching of 4 was comparable to 1 whereas 2 and 3
showed more extended photobleaching but much higher ROS production in very short irradiation times.
Compounds 2 and 4 exhibited specific intracellular localization. After dark and light cytotoxicity experi-
ments against LNCaP prostate cancer cell line for all curcuminoids, concentration of 3 μM and irradiance
of 6 mW cm−2 were selected for the PDT application which resulted in remarkable results with very short
LD50. Curcuminoids 2 and 4 exhibited a significant dose-dependent PDT effect. The biphasic dose–
response photodynamic effect observed for 1 and 3 may provide a strategy against prolonged and sus-
tained photosensitivity.
Received 9th September 2019,
Accepted 8th January 2020
DOI: 10.1039/c9pp00375d
rsc.li/pps
including skin, esophageal, head and neck, lung, and bladder
Introduction
cancers.1,2 PDT causes its cytotoxic effects against harmful or
Photodynamic therapy (PDT), although is one of the most unwanted cells or even pathogens based on the combined
recently introduced therapeutic modalities, has already been action of three elements: a photosensitizing agent, light and
successful in presenting a considerable number of approved oxygen. More specifically, administration or application of the
therapeutic protocols for various applications. It is the rec- photosensitizer (PS) is followed by local irradiation of the
ommended therapeutic strategy to treat age-related macular pathological tissue area. Light exposure of the area occurs at
degeneration and it represents a minimally invasive thera- the appropriate power, duration and most important wave-
peutic approach for the treatment of certain types of cancer length. The latter, should be able to adequately penetrate the
tissue in order to equally affect cells located at deeper tissue
layers. Once the photosensitizer is appropriately light-excited
aLaboratory of Biomedical Optics and Applied Biophysics, School of Electrical and
Computer Engineering, National Technical University of Athens, Zografou Campus,
15780 Athens, Greece. E-mail: ealexan@central.ntua.gr
bLaboratories of Structural Studies of Biomolecules and Pharmaceuticals with NMR,
Institute of Biosciences and Applications, NCSR “Demokritos”, Ag. Paraskevi, 153 10
Athens, Greece. E-mail: sagnou@bio.demokritos.gr
cBioimaging and Cell analysis, Material and Chemical Characterisation Facility,
University of Bath, Claverton Down, Bath BA2 7AY, UK
in the presence of oxygen, a series of energy transfers and
photochemical reactions is triggered, resulting in the pro-
duction of singlet oxygen (1O2) and other highly reactive
oxygen species (ROS).1,3 These products initiate a domino of
biochemical phenomena which can eventually cause signifi-
cant toxicity and lead to cell death via apoptosis, necrosis or
autophagy.4,5 The advantages of PDT, such as high cure rates
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with minimal toxicity in healthy tissues, specific targeting and degradation profile and the singlet oxygen production.19 In
selectivity, low side effects, potential use as an adjuvant terms of its photochemical properties, curcumin is character-
therapy combined well with all other tumor interventions such ized by a broad absorption spectrum between 300 and 500 nm
as chemotherapy, surgery, radiotherapy or immunotherapy with relatively high extinction coefficient. It can induce strong
and finally its excellent cosmetic results, make it a very promis- phototoxic reactions in micromolar concentrations.20,21 As a
ing option in the treatment of cancer.1,2
result, curcumin appears as a promising photosensitizer in the
An ideal photosensitizer should be able to address as many treatment of local superficial infections and cancers. It would,
as possible of the following criteria: show strong absorption therefore, be of great interest to investigate the photodynamic
with a high extinction coefficient in the red/near infrared potential of curcumin derivatives characterized by higher
region of the electromagnetic spectrum (600–850 nm) to allow absorption maximum and/or higher extinction coefficient
deeper tissue penetration; be an effective generator of singlet anticipating that these characteristics will contribute to
oxygen and other reactive oxygen species; exhibit favorable increased 1O2 generation potential and hence photodynamic
photophysical characteristics; have minimum dark toxicity; toxicity.22
accumulate more in diseased/target tissue rather than the
The present work presents the results on the photophysical
healthy cells; be a single, well-characterized compound, with a and photodynamic investigation of curcumin 1 and three syn-
known and constant composition, stable in solution, serum or thetic curcumin derivatives, 2–4 (Fig. 1) as potential photo-
plasma with a simple and stable drug formulation; have an sensitizers in cancer photodynamic therapy. Curcumin, com-
economical production route with feasible multi-gram pound 1, is the primary curcuminoid in turmeric and all the
scaling.6
commercially available extracts, accounting for at least 70%
Curcumin, the hydrophobic polyphenol found in the of the mixture, and it is the compound for which most
rhizome of turmeric (Curcuma longa L.), has been widely used studies have been reported predominantly using the commer-
in food coloring, drugs and cosmetics. A wide range of thera- cial mixture and in very few cases as the pharmacologically
peutic properties and many exciting pharmacological effects pure compound. Bisdemethoxycurcumin, compound 2,
have been reported for curcumin, including anti-inflammatory occurs also in the naturally extracted mixture but in a very
and antioxidant, chemopreventive and chemotherapeutic small amount (approx. 2%). It is, therefore, important to
potential.7,8 The photobiological and photokilling potential of evaluate the activities of the two individual curcuminoids
curcumin and curcuminoids have been of great scientific inter- separately, and not as part of the extracted mixture.
est since 1987,9,10 investigating the mechanism of the photo- Curcuminoid 3 is a curcumin derivative with no substitution
sensitization potential of such molecules.9,11–15 Curcumin on the aromatic ring and an additional double bond bilateral
seems to adequately meet most of the prerequisites for a desir- to the diketone moiety and, hence, extensive conjugation,
able, highly promising and “drugable” photosensitizer. It is while the p-dimethylamino substitution of 4 is anticipated to
biologically safe even at doses up to 12 g kg−1 day−1 and it can act as an electron donor to the aromatic ring and the conju-
be produced affordably in gram-scale amounts with easy hand- gated diketone. The photophysical properties of the four cur-
ling and storage requirements.11,16 Many cancer cell lines cuminoids such as absorption, fluorescence, photobleaching
in vitro show preferential uptake of a curcumin composite, in and free radical production have been investigated.
comparison to healthy cell lines.17 Its photosensitisation Subsequently, their effect on cell viability in LNCaP prostate
mechanism is not yet fully known, but it has been observed to cancer cells, in the absence of light (dark toxicity) was evalu-
require oxygen.9,10,18 Attention has been drawn on curcumin- ated. Moreover, the cell uptake and localization of the sub-
derived photolytically produced species of active oxygen and stances in LNCaP cells was monitored and their photo-
more specifically singlet state oxygen, hydrogen peroxide and dynamic efficiency was also evaluated. To the best of our
hydroxyl radicals.10,18 Photobleaching experiments to a curcu- knowledge, this is the first time that curcuminoids 3 and 4
min-based composite revealed a clear relationship between its are evaluated as potential PDT photosensitizers.
Fig. 1 Chemical structure of the curcuminoids investigated.
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1
52%. H NMR (500 MHz, DMSO-d6) δ 7.54 (d, J = 15.3 Hz, 2H),
7.31 (s, 2H), 7.14 (d, J = 7.84 Hz, 2H), 6.81 (d, J = 8.0 Hz, 2H),
6.74 (d, J = 15.8 Hz, 2H), 6.06 (s, 2H), 3.83 (s, 6H). 13C NMR
Materials and methods
Chemicals
Unless otherwise stated, all reagents and solvents were (500 MHz, DMSO-d6) δ 183.1, 149.4, 147.9, 140.7, 130.3, 126.3,
obtained from commercial sources and used without further 123.1, 121.0, 115.7, 111.3, 100.8, 55.7. MS (m/z) [M + H]+ calc.
purification. Air sensitive chemistries were performed in dry for [C21H21O6]+ 369.13, found: 369.25.
and inert conditions under an atmosphere of argon. All reac-
1,7-Bis(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione (2, curcu-
tions were routinely checked by TLC on Silica gel Merck 60 min III). Acetylacetone reacted with 4-hydroxybenzaldehyde
F254 and compounds were purified by column chromato- according to the general method. The crude product was
graphy on silica gel using the appropriate solvent systems or recrystallized in ethanol to afford the final product as yellow
1
by preparative HPLC. The purity of the biological tested com- powder. Yield = 34%. H NMR (500 MHz, DMSO-d6) δ 7.57 (d,
pounds was determined by analytical HPLC and was found to J = 8.1 Hz, 4H), 7.55 (d, J = 15 Hz, 2H), 6.82 (d, J = 8.3 Hz, 4H),
be greater than or equal to 95. The purity analysis was per- 6.67 (d, J = 15.9 Hz, 2H), 6.04 (s, 1H). 13C NMR (500 MHz,
formed on
a HPLC Shimadzu 2010EV (Column: Merck DMSO-d6) δ 183.7, 160.3, 140.8, 130.8, 126.3, 121.3, 116.4,
Purospher RP-C8, 250 × 4.6 mm, 5 μm particle size), equipped 101.4. MS (m/z) [M + H]+, for [C19H17O4]+ 309.11. Found:
with a SPD-20A UV/Vis detector. Characterization of target 309.10.
compounds was established by a combination of ESI-MS and
(1E,3E,8E,10E)-1,11-Diphenylundeca-1,3,8,10-tetraene-5,7-dione
1
NMR spectrometry techniques. H and 13C NMR spectra were (3). Acetylacetone was reacted with cinnamaldehyde according
recorded on a Bruker Avance DRX spectrometer (500 MHz) at to the general method. The crude product was recrystallized by
25 °C. Chemical shifts are presented in ppm (δ) with internal dichloromethane/hexane to afford the final product as yellow
1
TMS as standard. MS were obtained by the mass detector of powder. Yield = 63%. H NMR (500 MHz, DMSO-d6) δ 7.59 (d,
the Shimadzu 2010EV HPLC system.
J = 7.4 Hz, 4H), 7.50–7.30 (m, 8H), 7.23–7.07 (m, 4H), 6.38 (d, J
Stock solutions of curcuminoids 1–4 were freshly prepared = 15.1 Hz, 2H), 6.09 (s, 2H). 13C NMR (500 MHz, DMSO-d6)
by dissolving each powder in DMSO. Dulbecco’s phosphate- δ 182.93, 141.06, 140.39, 136.13, 129.11, 128.92, 127.77,
+
buffered saline (DPBS), without CaCl2 and MgCl2, pH 7.4, 127.38, 127.29, 101.39. ESI-MS (m/z) [M + H]+ calc. [C23H21O2
]
dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)- 329.15, found 328.70.
2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained
(1E,6E)-1,7-Bis(4-(dimethylamino)phenyl)hepta-1,6-diene-
from Sigma. RPMI 1640 medium was obtained from LGC 3,5-dione (4). Acetylacetone reacted with 4-dimethylamine
Biomaterials. Fetal Bovine Serum, FBS, antibiotic–antimitotic benzaldehyde according to the general method. The crude
and t-octylphenoxypolyethoxyethanol (Triton X-100) were pur- product was recrystallized by dichloromethane/hexane to
chased from Gibco.
afford the final product as deep red powder. Yield = 42%.
1H NMR (500 MHz, DMSO-d6) δ 7.62–7.41 (m, 6H), 6.74 (d, J =
8.5 Hz, 4H), 6.59 (d, J = 15.3 Hz, 2H), 5.97 (s, 2H), 2.99 (s,
Synthesis of curcumin derivatives24
All curcuminoids were synthesized in the lab to ensure the 12H). 13C NMR (500 MHz, DMSO-d6) δ 182.89, 151.63, 140.48,
purity of the samples.
134.60, 131.03, 128.92, 122.15, 118.62, 111.88, 100.39, 39.35.
+
ESI-MS (m/z) [M + H]+ calc. for [C23H27N2O2 ] 363.21, found
General procedure for synthesis of curcuminoids (1–4)
363.15.
Acetylacetone (2.06 mL, 20 mmol) was added to a solution of
boric anhydride (0.70 g, 10.0 mmol) and stirred at 80 °C for
0.5 h. To the reaction mixture the corresponding benzaldehyde
Spectroscopic methods
Absorption. UV-Vis absorption spectra were recorded using
(20 mmol) was added followed immediately by the addition of 4.5 ml volume cuvettes in the PerkinElmer Lambda 35 UV/VIS
tributyl borate (21 mL, 80 mmol). The reaction mixture was Spectrometer. The wavelength range was 300–700 nm and the
stirred at 60 °C for 0.5 h. Subsequently, a solution of n-butyla- scan speed was set at 480 nm min−1. The absorbance of each
mine (0.8 mL, 10.0 mmol) was added dropwise over 30 min at curcuminoid in DMSO (3 ml samples) was measured at a final
60 °C, and stirred overnight at 80 °C. Hydrochloric acid (1 N, concentration of 20 μM, 10 μM, 5 μM, 1 μM and 0.5 μM. The
60 mL) was added, and the mixture was stirred for another 2 h measurements were performed in triplicates for each concen-
at 60 °C. The organic layer was separated, and the aqueous tration point. Moreover, the absorbance of curcuminoids at
phase was extracted with ethyl acetate (3 × 30 mL). The com- 10 μM concentration was recorded in various solvents, namely
bined organic layers were washed with water, brine, dried over DMSO, ethanol and PBS.
Na2SO4, filtered and concentrated under reduced pressure.
All absorption measurements were carried out at room
The crude product was treated accordingly to afford the final temperature. The samples were freshly prepared just before
curcuminoids as is described below.
measurements.
1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione
Fluorescence. Fluorescence spectra were obtained in DMSO
(1, curcumin I). Acetylacetone reacted with vanillin according solutions (0.3 μM and 0.5 μM) using 4.5 ml volume cuvettes in
to the general method. The crude product was recrystallized in the PerkinElmer LS 45 Luminescence Spectrometer. Each cur-
ethanol to afford the final product as yellow powder. Yield = cumin derivative sample (3 mL) was excited at the wavelength
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that corresponds to its maximum absorption, i.e. 435 nm for 1 Germany) multiphoton confocal microscope equipped with an
and 3, 425 nm for 2 and 497 nm for 4. The slit width was set at Argon laser (excitation lines at 458, 476, 488, 496 and 514 nm),
10 nm both for excitation and emission. Scan speed was a DPSS 561 laser (excitation line at 561 nm) and an IR MaiTai
adjusted to 480 nm min−1
.
DeepSee Ti:Sapphire laser (Spectra-Physics, Santa Clara, CA,
All fluorescence measurements were carried out at room USA) for multiphoton applications.23 Images were acquired
temperature. The samples were freshly prepared just before with the LAS X software (Leica Microsystems CMS GmbH,
measurements.
Wetzlar, Germany) and are presented without any post-
processing.
Irradiation device
Cell viability evaluation
Irradiation was performed using a custom-made illumination
device developed in the Laboratory of Biomedical Optics and Cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-
Applied Biophysics, National Technical University of Athens. It 2-yl)-2,5-diphenyl-2H-tetrazolium bromide; Sigma] colorimetric
consisted of a Bridgelux Power LED 10 W light source, emitting assay, which measures the capacity of mitochondrial dehydro-
at 430 10 nm, coupled to special optics in order to provide genase to reduce MTT to purple formazan crystals. The MTT
uniform, circular illumination. The device was also equipped test assesses the number of surviving cells.
with a potentiometer in order to adjust power output and a
The calibration curve of the LNCaP cells absorbance was
cooling base with a build-in fan. Irradiation power was made prior to the experiments. 24 h after photodynamic treat-
assessed using a power meter.
ment, the medium was removed and the MTT solution was
added to each dish. The cells were kept in the humidified
incubator for 3 h to allow metabolism of MTT. After incu-
Photobleaching
Each curcuminoid sample (0.5 μM for 1 and 4, 0.2 μM for 2 bation, the formazan crystals were solubilized by adding the
and 3) was irradiated at 12.5 mW cm−2. Fluorescence spectra MTT solvent (10% Triton X and 0.1 N HCl in isopropanol).
were acquired every 1 min between 0–10 min and every 5 min Absorbance was measured using
a computer controlled
between 10–30 min. The fluorescence emission maximum was PerkinElmer Lambda 35 UV/VIS Spectrometer. Spectra were
then recorded in order to evaluate photobleaching.
collected for a wavelength range between 380 and 700 nm. All
measurements were carried in triplicate and data were
expressed as mean standard deviation. From each spectrum,
a pyridine the absorbance at 690 nm was subtracted (as noise) from the
ROS production
Nicotinamide Adenine Dinucleotide (NADH),
nucleotide and biologically active form of nicotinic acid, is a absorbance at 565 nm (maximum peak of formazan absorp-
coenzyme necessary for the catalytic reaction of certain tion spectrum). From these values of absorption and the cali-
enzymes that absorbs at 340 nm. Following the decrease in bration curve, cell viability percentages of treated and control
absorbance at 340 nm, NADH consumption and its conversion samples were calculated.
to NAD+ was assessed and hence the existence of free radicals
in the solution.
Dark cytotoxicity
Freshly prepared solutions consisting of 10 μM of each cur- For determination of dark cytotoxicity, LNCaP cells were incu-
cumin derivative, 100 μM NADH and 0.1 mM EDTA, were irra- bated for 24 h in the dark at a concentration range of 1 μM,
diated at 4.70 mW cm−2 for 20 min. Every 1 min, samples were 3 μM and 5 μM of each derivative. When the incubation time
returned to the spectrometer and the absorption spectra were was completed, the cells were washed with 1 mL DPBS, fol-
acquired. The absorption peak at 340 nm was recorded.
lowed by addition of fresh medium and incubation for 24 h.
Cellular viability was measured with the MTT viability test. All
measurements were carried out in triplicate. All data were
Cell culture conditions
The human prostate cancer cell line LNCaP was obtained from expressed as mean standard deviation.
the American Type Culture Collection (ATCC). The cells were
Light cytotoxicity
cultivated in 75 cm2 culture flasks (Corning) in
RPMI-1640 medium supplemented with 10% heat inactivated For determination of light dependent cytotoxicity, cells were
Fetal Bovine Serum, FBS, and 0.1% antibiotic–antimitotic. irradiated with 3 mW cm−2, 6 mW cm−2 and 9 mW cm−2. The
Cells were kept at 37 °C in a 5% CO2 humidified incubator, exposure time was 60 s. Irradiation occurred in the presence of
trypsinized and re-seeded into fresh medium every 3–5 days.
300 μL DPBS for each Petri dish. Fresh medium was added
when the irradiation was completed. 24 h post irradiation, cel-
lular survival was measured with the MTT assay. All measure-
Intracellular localization
LNCaP cells 10 × 105 were seeded on Nunc™ glass base dishes ments were carried out in triplicate and data were expressed as
(ThermoFisher Scientific, Rochester, NY, USA) and incubated mean standard deviation.
overnight in 2 mL of DMEM complete medium in the same
Photodynamic treatment
conditions as previously described. Cells were subsequently
treated with curcuminoids (3 μM final concentration) for 1 h. Cells were incubated with 3 μM freshly prepared solution of
Specimens were examined on a Leica TCS SP8 MP (Wetzlar, each curcuminoid in enriched medium for 1 h. Before light
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Table 1 Wavelength of maximum absorption and extinction coefficient
exposure, the culture medium containing the photosensitizers
was removed from the dishes and PBS was added (300 μL)
until the cell monolayer was slightly covered. Irradiation was
performed at room temperature with the 430 nm LED-based
illumination device. Irradiance was adjusted to 6 mW cm−2
and the exposure times were 60 s, 120 s and 180 s yielding 360,
720 and 1080 mJ cm−2 fluence rates, respectively. Following
(
SD) for the four synthetic derivatives of curcumin, at concentration
20 μM in DMSO
Curcumins
λmax (nm)
ε (cm−1 M−1
)
1 (curcumin I)
2 (curcumin III)
3
435
426
435
497
42 200 14
50 180 21
61 300 13
51 700 39
irradiation, fresh medium was added and the cells were main- 4 (di-NCH3 curcumin)
tained in the humidified incubator for 24 h. Finally, cellular
survival was measured with the MTT assay. All measurements
were carried out in triplicate. All data were expressed as means
standard deviation.
investigated herein. Curcumin I exhibited the lowest extinction
coefficient of 4.2 × 104 cm−1 M−1 whereas derivative 3 was
found to have the highest extinction coefficient of 6.1 × 104
cm−1 M−1. Compounds 2 and 4 showed comparable extinction
coefficients of a value around 5 × 104 cm−1 M−1
.
Results
The absorption spectra of the four curcuminoids (10 μM) in
different solvents, DMSO, ethanol (EtOH) and PBS are depicted
in Fig. 3. When organic solvents were used such as DMSO or
ethanol, the compounds present the characteristic absorption
spectrum. DMSO is considered as a hydrogen acceptor solvent
whereas alcohols can act as either hydrogen donors or accep-
tors depending on the molecular structure of the solute. There
was no significant shift or any other alteration in the absorp-
tion spectrum of four derivatives when changing from DMSO
to ethanol solutions, despite the difference in polarity.
In the case of water-based solution such as PBS, which was
used as a biologically relevant solvent, there is a significant
decrease in absorption and a shift in the wavelength of
maximum absorption to lower wavelengths, which may be
related to the tendency of curcuminoids to form aggregates in
aqueous media.
Fluorescence spectra. The fluorescence spectra of the four
curcumin derivatives in DMSO are presented in Fig. 4.
Compounds 1–3 exhibit similar fluorescence emission
maxima, whereas curcuminoid 4 fluoresces at longer wave-
lengths, as expected based on its higher excitation wavelength.
Compound 2 exhibits the strongest fluorescence emission, as
it was necessary to reduce its concentration to 0.3 μM, to
acquire an emission signal of comparable intensity to the rest
of the compounds for which emission was recorded at a con-
centration of 0.5 μM whereas 1, showed the weakest fluo-
rescence emission.
The desired symmetrical curcuminoids 1–4 were successfully
synthesized according to the literature with slight modifi-
cations.24 The reaction of boric anhydride with acetylacetone is
initially employed to form an acetone-boric oxide complex
aiming to eliminate the reactivity of the central methylene
group towards Knovenagel condensation. In this way, aldol
condensation takes place in the presence of tributyl borate as
a desiccant, between the corresponding benzaldehyde and the
side methyl methyl moieties, instead of the more reactive
central methylene group. The final treatment with 1 N hydro-
chloric acid released the β-diketone moiety from the borate
complex to afford the desired product.
Photophysical properties
Absorption spectra. The absorption spectra of curcuminoids
1–4 in DMSO are shown in Fig. 2. Three of them, 1–3, show
similar spectral characteristics with a peak at around 430 nm,
whereas compound 4 exhibits according to our design, a sig-
nificant and desirable red shift with an absorption peak at
497 nm.
Table 1 summarises the wavelengths of maximum absorp-
tion and the extinction coefficient for the four curcuminoids
Fluorescence emission maximum presented
a linear
relationship with concentration, in the range of 0.01 μM to
0.5 μM, for all the tested substances (data not shown).
Photobleaching by measuring absorbance and fluorescence
Changes in both absorbance and fluorescence emission
maximum of the four curcumin derivatives, relative to the time
of illumination, were monitored. In Fig. 5, changes in fluo-
rescence intensity as percentage of the initial fluorescence
before irradiation were presented.
In the case of 4, the drop in fluorescence emission
maximum is approached linearly in relation to irradiation
time, while in all other cases there is an exponential decrease.
Fig. 2 Absorption spectra of curcuminoids 1 (curcumin I), 2 (curcumin
III), 3, 4 in DMSO (20 μM final concentration).
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Fig. 3 Absorption spectra of the four curcumin derivatives (10 μM) in different solvents: DMSO (solid line), ethanol (dots) and PBS (dashes).
Fig. 5 Fluorescence emission maximum intensity changes as a function
Fig. 4 Fluorescence spectra of curcuminoids, 1 (0.5 μM, λexc = 435 nm),
of illumination time for the four curcuminoids solutions in DMSO at
2 (0.3 μM, λexc = 425 nm), 3 (0.5 μM, λexc = 435 nm), 4 (0.5 μM, λexc
497 nm) in DMSO.
=
concentrations of 0.5 μM for compounds 1 and 4 or 0.2 μM for com-
pounds 2 and 3, after irradiation at 12.5 mW cm−2
.
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As it can be seen, derivative 4 is characterized by a signifi-
cant resistance to photobleaching, compared to the other
three derivatives, making it the most photostable of the four
curcumin derivatives that were examined. The highest photo-
bleaching was observed for compound 2 (curcumin III).
Absorption values were reduced in a similar way with illu-
mination time (data not shown).
Production of free radicals
The relationship between the absorption peak of NADH at
340 nm and the time of irradiation is represented in Fig. 6.
The drop in the absorption peak at 340 nm, is indicative of the
formation of the oxidized NAD+, resulting from the production
of reactive oxygen species (ROS).
In case of derivative 4, the drop of the absorption peak is
linear. For curcuminoids 2 and 3, the reduction is logarithmi-
Fig. 7 Dark toxicity results of the four derivatives at various concen-
trations (1, 3 and 5 µM) in LNCaP cells. After 24 h treatment with each
cally approached, whereas 1 presents an exponential decrease compound, the cells were incubated in growth medium for another 24 h
period. The data are expressed as mean of three experiments. Error bars
present standard deviation.
with irradiation time.
It is obvious from the graph, that Compounds 2 and 3
produce the largest amount of free radicals with very short
irradiation times, a result considered positive for potential
photosensitizers.
Light cytotoxicity
Light dependent cytotoxicity, in the absence of any test
Dark cytotoxicity
compound, is presented in Fig. 8. Irradiance of 3 mW cm−2
,
The results on LNCaP cell viability after incubation in the dark
with curcumin derivatives at concentrations of 1 μM, 3 μM,
5 μM, are shown in Fig. 7. Cellular viability was evaluated by
means of the MTT assay 24 h post incubation with the poten-
tial photosensitizers.
Cell viability was not significantly affected upon incubation
in the dark at 1 μM and 3 μM concentrations of the test com-
pounds in a statistically significant manner. Incubation with
5 μM concentration of the photosensitizers, however, caused a
considerable decrease in cell survival. Consequently, the
photodynamic treatment experiments were performed using
3 μM of the curcuminoid concentration.
for 60 s irradiation time, not only is not toxic to the cells, but
also caused a slight increase in cell viability compared to non-
irradiated cells. On the contrary, when irradiance of 9 mW
cm−2 was used, there was a 25% decrease in cellular viability.
For irradiance of 6 mW cm−2, there was no significant
effect on cell survival, and consequently this irradiance value
was used in the photodynamic treatment experiments.
Intracellular localization
Intracellular localization of the four curcumins was studied
using confocal fluorescence microscopy. Fig. 9 includes indica-
Fig. 6 The variation of the absorbance peak of NADH at 340 nm in
relationship with time of irradiation. The diagrams refer to solutions of
each derivative at concentration of 10 μM in DMSO. Each solution con-
tains 100 μM NADH and 0.1 mM EDTA.
Fig. 8 Light-induced cell cytotoxicity in LNCaP cells, without any
photosensitiser, after irradiation for 60 s with irradiance set to 3 mW
cm−2, 6 mW cm−2 and 9 mW cm−2
.
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Fig. 9 Confocal fluorescence microscopy on live LNCaP cells incubated for one hour with 3 μM of each curcumin derivative; left column: images
acquired with confocal microscope – for compounds 1 and 2 λex = 458 nm whereas for compounds 3 and 4 λex = 488 nm; middle column: brightfi-
eld images of the cells; right column: merged brightfield and confocal images.
tive images of LNCaP cells incubated with 3 μM of each syn- resulted in significantly more intense fluorescence
thetic curcumin derivative for one hour. Bright field images imaging of the cells. Finally, the cell fluorescence uptake
showing the cell morphology are presented in the middle observed after treatment with 3 and 4 was of the highest
column, whereas the corresponding fluorescence images are intensity.
shown on the left. On the right column, merged images of
optical and confocal mode are displayed.
Fluorescence images showed that none of the substances
affected the cell structure. There was no nuclear localization
Upon 1 h incubation with 3 μM of the compounds, fluo- exhibited by any of the test compounds. However, some peri-
rescence images of the cells revealed that photosensitizers nuclear accumulation could be noticed. Furthermore, this
have entered the cells sufficiently. The lowest cellular fluo- cytoplasmic or perinuclear localization did not seem to be
rescence intensity was observed upon treatment with 1, evenly distributed and some more intensely fluorescent spots
curcumin I, whereas cellular incubation with compound 2 could be identified.
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in this study curcumin 1, was comparatively evaluated in terms
of physicochemical stability and photodynamic potential to
the natural curcumin, 2, which lacks the two methoxy groups
at the meta-position to the hydroxyl moiety of 1, as well as two
newly synthesized curcuminoids. Compound 3, had no substi-
tution on the aromatic ring but possessed an extra double
bond between the aromatic ring and the β-diketone which was
anticipated to increase electron delocalization. Finally, curcu-
minoid 4 was designed to possess the dimethylamine- to
replace the para-phenolic groups of 2, as potential electron
donor moieties.
Indeed, all curcumin derivatives at different concentrations
maintained a characteristic absorption spectrum for the whole
range of concentrations that were used and the absorbance
intensity changed linearly relative to the concentration in all
cases in accordance with results reported from similar
studies.18,34–36 Compounds 1, 2, and 3 exhibited significant
absorption between 350 and 480 nm, whereas 4 was red
shifted absorbing between 400 and 550 nm. This had been
anticipated according to the design of the molecule, based on
the electron donor character of the dimethylamino- group at
the para-position to the unsaturated chain. Bearing in
mind that longer excitation absorption corresponds to deeper
tissue penetration, compound 4 exhibits a favorable and
advantageous property for PS applications.
In addition, all curcuminoids tested showed relatively high
extinction coefficient (ε) in the wavelength of their maximum
absorbance (Table 1), ranging from 42 200 to 61 300 M−1 cm−1
compared to some of the clinically tested photosensitizers
such as Photofrin® (∼3000 M−1 cm−1 at 632 nm), PPIX (∼5000
M−1 cm−1 at 632 nm) and m-THPC (∼35 000 M−1 cm−1 at
652 nm). Molecules with a high extinction coefficient (ε) are
more desirable as PS since they may require a smaller amount
to be administered to the cells and cause significant damage
upon light activation without any undesirable side effects
which may occur due to excessive PS dosage.37,38 The curcumi-
noids scaffold seems to be a privileged structure in that
respect. It is worth mentioning that the additional double
bond of 3 seemed to favor even further the high extinction
coefficient profile of the compounds, exhibiting the highest ε
of all four molecules. Our results are in good agreement with
previous reports39 which investigated and related the physico-
chemical and photochemical properties of curcuminoids with
the keto–enol tautomeric form as well as the substitution
Fig. 10 The photodynamic efficiency of the four synthetic curcumi-
noids on LNCaP cells 24 h post irradiation at 451 nm. The initial photo-
sensitizer concentration was 3 μM and the cells were treated for 1 h
prior to photoactivation.
Photodynamic treatment
LNCaP cells were incubated with 3 μM of each of the four cur-
cuminoids for one hour. Subsequently, the cells were irra-
diated and the induced phototoxicity was assessed 24 h post
PDT by the MTT assay. Fig. 10 presents the results obtained
for the effect of the test photosensitizers on cell viability for
various irradiation times and consequently various irradiation
energy doses.
Compounds 2 and 4 exhibited a dose-dependent decrease
in cell survival. In the case of curcuminoid 2 even at the lowest
energy dose (360 mJ cm−2) 40–50% of the cell population was
killed 24 h post irradiation whereas, with compound 4, the
LD50 value was achieved at the highest energy dose of 1080 mJ
cm−2. On the other hand, compounds 1 and 3 caused an
initial cell-killing effect at low doses, which seemed to be
reversed at higher energy irradiation doses. It is worth noting
that compounds 1 and 2 had a detrimental effect on cell viabi-
lity even at the lowest energy dose of 360 mJ cm−2
.
Discussion
Curcumin as a photosensitizing agent for PDT has been the pattern of the aromatic ring. In the case of 3 the absence of
subject of a number of studies either in antimicrobial PDT25 any hydroxyl- or amino-groups and the addition of a double
or against various human cancer cells.13,26–28 Some of the bond increases the hydrophobicity of the molecule and mini-
limitations reported for the potential clinical application of mizes the solvent interactions. In the case of the polar organic
curcumin in PDT are its poor solubility, stability and photo- solvents, it is the keto–enol equilibrium, which provides a
stability in aqueous solutions as well as the rapid metabolism sufficient
center
of
solvent–molecule
interactions.
and systemic elimination.29–33 In most studies, commercially Curcuminoids 1 and 2 possess a phenolic OH– group and in
available curcumin is used. In this study pure samples of cur- the case of 4 the nitrogen containing aromatic substitution
cumin (1) synthesized in the lab are employed in the study of may all form intramolecular H-bonds and π-stacking inter-
the PDT related properties. Furthermore, to the best of our actions leading to the formation of aggregates when dissolved
knowledge, there is no study for PDT with curcumin itself or in aqueous media.31–33,40,41 The increased hydrophobicity,
any curcuminoids on human prostate cancer cells. Moreover, poor water solubility which easily leads to aggregated species
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under physiological conditions and drastically lowers the Compounds 2 and 3 exhibited the highest ability to produce
quantum yields of ROS production, has long been one of the free radicals, after irradiation at 430 nm, which is close to
main limitations for the clinical use of PDT. To this end, the the max absorption and corresponds to increased molecular
use of transporters like proteins, liposomes or nanoparticles excitation. Curcumin 1 produced less free radicals and the
has been exploited to protect curcumin and other photosensi- corresponding drop in absorption was approached linearly,
tizers against the undesirable solvent effects.33 This proved to indicating a slower rate of ROS production. Finally, 4
be beneficial to PDT applications. This presents a very interest- revealed a rather small ROS production ability compared to
ing future prospect of this study to enhance the properties of the other three derivatives. It should be noted, however, that
these four curcuminoids and elucidate further their mecha- the irradiation at 430 nm is not sufficiently high to excite
nism of action.
the molecules since the absorption maximum of this com-
In terms of their fluorescence emission and in accordance pound was shifted to almost 500 nm. From a structural
to previous reports12,42 curcumin I (1) and (2) exhibit different point of view, it is evident that the lack of the methoxy-aro-
fluorescent properties which may be ascribed to either the matic substitution in 2 and the additional conjugated
difference in H-bond acceptor/donor properties of the pheno- double bond also with the completely unsubstituted aro-
lic OH, or the difference in strength of the intramolecular matic moiety in 3 favors both photodegradation and free
H-bond in the keto–enol moiety due to the lack of methoxy radical production.
group. All four curcumin derivatives displayed intense fluo-
One of the great advantages of the potential clinical appli-
rescence emission and as a result they can be used not only in cation of curcumin is the fact that being a natural product it is
the application of photodynamic therapy, but also in photo- safe to use even at high daily doses. In accordance, all four
dynamic diagnosis. Especially, compound 4 presented a red compounds were found non-toxic against the LNCaP cell line
shift in both absorption and fluorescence properties, with after a 24 h treatment in the dark, when tested at concen-
emission maximum at 610 nm upon excitation at 497 nm. trations up to 10 μM. There is one literature evidence
Based on these characteristics, compound 4 can be utilized as suggesting that even at concentration of 20 μM of compound 1
a theranostic agent not only for superficial but also for in and 48 h treatment the cell viability of LNCaP was reduced
depth lesions.
only by 30%.44 This low dark toxicity of the compounds pro-
In photobleaching experiments, the fluorescence emission vides the necessary degree of selectivity to their photodynamic
maximum exhibited an exponential decay with respect to application since light activation is a prerequisite for their
irradiation time in the cases of curcuminoids 1, 2 and 3 in cytotoxic action and cell killing effect.
DMSO solutions. Photobleaching of compound 3 has been
Our light-induced cytotoxicity experiments demonstrated
shown to proceed faster than 1 in DMSO, in agreement to our the importance of performing light-only experiments for every
results, and we have similarly identified that 2 undergoes a cell line used in a study. At an irradiance of 3 mW cm−2 and
comparable photodegradation rate with 3. On the contrary, in 6 mW cm−2 for 60 s no or little light toxicity was observed. On
the case of compound 4, the most photostable photosensiti- the contrary, previous studies of our group, with the same cell
zer, the fluorescence decay was approximated linearly and line irradiated with 6 mW cm−2 at a different wavelength sig-
the substance was characterized by resistance to photobleach- nificantly reduced cell viability.45 This finding implies the
ing. Even though in terms of photostability, curcumin meets presence of a wavelength dependence of cell line light toxicity
the standards for a promising PS, however, a reasonable regardless of the irradiation power.
balance should exist between acute and prolonged photosen-
sitivity and post-treatment random excitation of the exhibited cytotoxicity after irradiation. In particular, com-
photosensitizer. pounds 1, 2 and 3 caused at the lowest fluence tested a very
In light activation experiments all four curcuminoids
Absorption was reduced in a similar way with illumination significant reduction in LNCaP cell survival while the
time (data not shown), indicating that the decrease is defi- reduction in cell population was less for curcuminoid 4. This
nitely due to a lower extinction and maybe due to a lower fluo- acute light-induced toxicity may be related to their effective
rescence yield. The observed findings suggest that it is not radical production which was clearly demonstrated in our
photomodification but true photobleaching. In photomodifi- study. Curcumin, 1, has been previously reported to exhibit
cation, the original absorption spectrum diminishes and new such a PDT energy dose dependence. Micromolar concen-
absorption peaks are emerged. In the case of all the com- trations (in the range of 0.4–13.5 μM) of the photosensitizer, in
pounds studied in the current paper, there were no new peaks combination with low light, were shown to be effective for the
observed in the absorption spectra only reduced absorption in treatment of oral cancers, after
the same wavelengths.43
Furthermore, significant reduction of hepatoblastoma
3 h
of incubation.13
a
Another important characteristic of an ideal photosensitizer (HuH6, HepT1) and hepatocellular carcinoma (HepG2,
is its ability to produce Reactive Oxygen Species (ROS). Upon HC-AFW1) cell viability was observed, after their treatment
irradiation, the photosensitizer transforms from its ground with curcumin 1 and exposure to blue light. It was derived that
singlet state to a short-lived excited singlet state of higher curcumin mediated PDT effectively enhanced the anti-tumor
energy which eventually may be converted to a relatively properties in epithelial liver cancer cells by inducing loss of
long-lived excited triplet state via intersystem crossing.1 viability via ROS production.46
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Upon increasing light fluence, however, two distinct pro- lation may be responsible for the observed light energy dose
files were observed regarding their photodynamic effect on dependent PD effect. Additionally, the intracellular localization
LNCaP cells. More specifically, compounds 2 and 4 presented of curcuminoid 2, may have been the reason for a more dra-
an increasing PDT efficacy with increasing energy dose result- matic PD effect against LNCaP cells, since the high ROS pro-
ing in increased cytotoxicity effects. Compounds 1 and 3 duction capacity of 2 was also accompanied by this special
exhibited an inverse dose–response with the lowest fluence accumulation inside the cell, causing possibly more cellular
rate used (360 mJ cm−2) causing the most powerful PDT effect, damage.
whereas higher fluence rates resulted in significantly less cyto-
In conclusion, the four curcuminoids presented promising
toxic effect in a dose-dependent manner. It may be suggested characteristics in terms of PDT efficacy. Pure, synthetically pro-
that at higher fluence rates, increased radical concentration duced materials were used, even for the naturally occurring
may favour radical quenching through intermolecular inter- curcumins 1 and 2, making the correlation of structure to
actions resulting in formation of non-active adducts before the activity direct. All of them showed sufficient ROS production
PD effect may be exerted, or implies fast degradation of cellu- and remarkably short LD50, especially compound 2, making
larly incorporated curcumin.47,48 Tanaka, Hamblin et al. them highly promising photosensitizers. It is the first time
observed a similar behavior when they examined the effect of that these four curcuminoids are tested against this cell line.
Photofrin-mediated photodynamic therapy on bacterial arthri- Curcuminoid 4 achieved a significant red shift in the absorp-
tis in mouse model.49,50 After application of PDT for the treat- tion, which is favorable for PDT due to deeper light pene-
ment of an MRSA arthritis infection in the mouse knee (1 μg tration. Finally, the biphasic dose response observed for com-
Photofrin, 635 nm diode laser illumination), a biphasic light pounds 1 and 3 is of great significance and further investi-
dose response occurred. The greatest reduction of MRSA CFU gation is underway since it may be implicated in reducing the
was seen with a fluence of 20 J cm−2, whereas lower antibacter- prolonged and sustained photosensitivity of such photosensiti-
ial efficacy was observed with fluences that were lower but also sers upon photodynamic treatment.
higher.49,50
A very rapid and very effective PDT action that is reversed
upon longer irradiation time constitutes an exciting property,
which is able to address the limitations of prolonged photo-
Conflicts of interest
sensitivity, a usual PDT drawback.
There are no conflicts to declare.
Taking advantage of their intense fluorescence properties,
their cellular localization and concentration was studied by
means of confocal microscopy. Although, the fluorescence
intensity of the photosensitizer inside the cells depends on
Acknowledgements
both its intracellular concentration and its fluorescence The authors acknowledge support of this work by the projects
quantum yield, it can be considered as a meter of its bio- “Target Identification and Development of Novel Approaches
availability. Intracellular localization of the photosensitizer is for Health and Environmental Applications” (MIS 5002514)
of primary importance as the produced ROS are characterized which is implemented under the Action for the Strategic
by very short lifetimes and their targets should be very close to Development on the Research and Technological Sectors,
their production site.51–53 Therefore the subcellular localiz- and “A Greek Research Infrastructure for Visualizing and
ation of the photosensitizer essentially determines the localiz- Monitoring Fundamental Biological Processes (BioImaging-
ation of the primary targets. There was no specific cellular GR)” (MIS 5002755) which is implemented under the
localization observed for curcumin 1 in LNCaP cells, contrast- Action “Reinforcement of the Research and Innovation
ing previous findings where mitochondrial curcumin localiz- Infrastructure”. Both projects are funded by the Operational
ation was reported in non-small-cell lung cancer A549 cells, Programme
“Competitiveness,
Entrepreneurship
and
and membrane preference was observed in MCF7 breast Innovation” (NSRF 2014–2020) and co-financed by Greece and
cancer cells.54,55 However, it is important to note that higher the European Union (European Regional Development
(10 or 20 μM) curcumin concentrations and longer incubation Fund). B. Mavroidi gratefully acknowledges financial support
times up to 4 h54,55 and 24 h (ref. 56 and 57) were employed in by Stavros Niarchos Foundation (SNF) through implemen-
those studies, that may have indeed promoted the observed tation of the program of Industrial Fellowships at NCSR
cellular localization.
“Demokritos”.
On the other hand, curcuminoids 2, 3 and 4 indeed accu-
mulated intracellularly in specific areas of the cells, which did
not colocalised with Mitotracker or Lysotracker staining,
suggesting that they do not localize in mitochondria or lyso-
somes (data not shown). The apparent cellular localization of 2
and 4 may be associated with their PD effect in LNCaP cells.
Especially, in the case of 4, where a relatively small amount of
ROS was observed, this intense cellular influx and accumu-
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