Synthesis and In Vitro Biological Evaluation of Psoralen-Linked Fullerenes

Article abstract of DOI:10.1111/php.13138

Photodynamic therapy (PDT) is a widely used medicinal treatment for the cancer therapy that utilizes the combination of a photosensitizer (PS) and light irradiation. In this study, we synthesized two novel C₆₀ fullerene derivatives, compounds 1 and 2, with a psoralen moiety that can covalently bind to DNA molecules via cross-linking to pyrimidine under photoirradiation. Along with several fullerene derivatives, the biological properties of several novel compounds have been evaluated. Compounds 1 and 2, which have been shown to induce the production of hydroxyl radicals using several ROS detecting reagents, induced DNA strand breaks with relatively weak activities in the in vitro detection system using a supercoiled plasmid. However, the psoralen-bound fullerene with carboxyl groups (2) only showed genotoxicity in the genotoxicity assay system of the umu test. Compound 2 was also seen to have cytotoxic activities in several cancer cell lines at higher doses compared to water-soluble fullerenes.

Full text of DOI:10.1111/php.13138

DR. TAKEJI TAKAMURA (Orcid ID : 0000-0002-6740-2915)
Article type : Research Article
Synthesis and In Vitro Biological Evaluation of Psoralen-Linked
Fullerenes
Akiko Hashimoto1, Takeji Takamura-Enya*2, Yoshimitsu Oda3
1Department of Applied Chemistry and Biotechnology, Graduate School of Engineering,
Kanagawa Institute of Technology
2Department of Applied Chemistry, Kanagawa Institute of Technology
3Institute of Life and Environmental Sciences, Osaka Shin-Ai College
*Corresponding author’s e-mail: takamura@chem.kanagawa-it.ac.jp (T. Takamura-Enya)
ABSTRACT
Photodynamic therapy (PDT) is a widely used medicinal treatment for the cancer therapy that
utilizes the combination of a photosensitizer (PS) and light irradiation. In this study, we
synthesized two novel C60 fullerene derivatives, compounds 1 and 2, with a psoralen moiety
that can covalently bind to DNA molecules via cross-linking to pyrimidine under
photoirradiation. Along with several fullerene derivatives, the biological properties of
several novel compounds have been evaluated. Compounds 1 and 2, which have been shown
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doi: 10.1111/php.13138
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to induce the production of hydroxyl radicals using several ROS detecting reagents, induced
DNA strand breaks with relatively weak activities in the in vitro detection system using a
supercoiled plasmid. However, the psoralen-bound fullerene with a carboxyl group (2) only
showed genotoxicity in the genotoxicity assay system of the umu test. Compound 2 was also
seen to have cytotoxic activities in several cancer cell lines at higher doses compared to water
soluble fullerenes.
INTRODUCTION
C60 fullerene, a nanocarbon material comprising 60 sp2 carbon atoms, has been studied
widely for its unique properties that derive from its hydrophobic structure, which is similar to
a soccer ball, and enable it to act as a drug and gene carrier. It has been already shown that
paclitaxel complexed with fullerene can efficiently release drugs in vivo and that a gene
delivered by aminofullerene can reduce cancer size (1, 2). Fullerene itself can act as an
antiviral drug toward human immunodeficiency virus (HIV-1) and hepatitis C virus, causing
the inhibition of protease and/or RT polymerases by fitting to the active site of these enzymes
(3, 4).
Fullerenes are also known to be promising photodynamic reagents that induce
reactive oxygen species (ROS) under UV-vis illumination with a quantum yield of
approximately 1.0 (5). In general, ROS induction is classified into two mechanisms: type I,
which involves electron and/or hydrogen abstraction process; and type II, which is
accompanied by energy transfer (6, 7). The former process induces the hydroxyl radical and
superoxide anion radical, and the latter induces singlet oxygen. All these species of ROS
attack biological components, which induces oxidative stress in cells and leads to cell death
(8, 9). Owing to these properties, photodynamic therapy (PDT) using fullerene derivatives is
now considered to be a promising cancer surgical treatment and also a treatment for viral
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infection (10–13). The relative stability of the fullerene derivatives to light irradiation and
their high absorption coefficient make them attractive for PDT. Moreover, the relative ease
with which functional groups can be added to fullerene makes it possible to produce various
fullerene derivatives as candidate reagents for PDT (14–16).
In addition to the notable cytotoxic ROS inducing ability of fullerene under the light
condition (17, 18), relatively rapid removal from the treated bodies was observed when
experimental animals were injected with fullerene derivatives owing to the rigid carbon
structure (19). For C60 fullerene derivatives that are efficient ROS producers, several
researchers reported that C60 and C70 fullerenes and their derivatives exerted ROS-producing
ability to cleave DNA (7, 20, 21).
A likely disadvantage of fullerene as a photosensitizer is its low solubility in polar
solvents. Several water-soluble fullerenes, such as dendritic fullerene and fullerene adducted
with three malonate functional groups, have photosensing properties and photocytotoxic
activities in Jurkat cells irradiated by UVA or UVB (22), although the quantum yield of
functional fullerene is expected to be lower than that of the original fullerenes (23). Cationic
functional group-modified fullerene derivatives exert phototoxic effects on three mouse
cancer cell lines: mouse macrophage J774, Lewis lung carcinoma (LLC), and colon
adenocarcinoma CT26 (14).
8-Methoxypsoralen and its analogs are widely used drugs for psoralen with UVA
treatment (PUVA) therapy, which is effective for several skin diseases (24–26). Recently,
the application of photochemotherapy with psoralen for T cell lymphoma was reported (27).
The mechanism of phototherapy of psoralen derivatives is the [2+2] cross adduct formation
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of psoralen to pyrimidine bases in DNA, which leads to cell death (28–31). As noted above,
PUVA is effective for lymphoma cells, but less effective for several carcinoma cells when
compared with other cancer treatment drugs. To overcome these advantages, the conjugation
of psoralen with several photosensitizers has been shown to be lethal to many carcinogenic
cells (32).
In this study, we synthesized fullerene derivatives modified with psoralen that were
able to covalently bind to DNA molecules under UVA irradiation and evaluated their
biological activities. In these types of compounds, the fullerene moiety was located close to
the DNA molecules. Therefore, it is expected that the production of ROS, caused by light
irradiation, and the fullerene combine to efficiently attack DNA molecules and lead to cell
death (Fig. 1).
MATERIALS AND METHODS
Materials. Proton nuclear magnetic resonance (1H-NMR) spectra were recorded by using a
JNM−ECX 400 MHz instrument (JEOL Ltd., Tokyo, Japan). The absorbance and
fluorescence intensity measurements were conducted by using an Infinite F200 microplate
reader (TECAN Group Ltd., Männedorf, Switzerland). Mass spectrometric analysis was
performed by using a MALDI micro MX spectrometer (Waters Corporation, MA, USA).
UVA irradiation was delivered from a Blak-Ray UV Bench Lamp XX-15BLB (Analytik Jena
US, An Endress+Hauser Company, CA, USA) or an CL-1000 Ultraviolet Crosslinker
(Analytik Jena US, An Endress+Hauser Company, CA, USA). Gels were analyzed by using
the LAS-3000 (Fujifilm Corporation, Tokyo, Japan).
Singlet Oxygen Sensor Green was obtained from Molecular Probes, Inc. (OR, USA).
C60 was purchased from SES Research, Inc. (TS, USA). BBr3 solution in CH2Cl2 and L-di-
tert-butyl tartrate were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
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BES-So was purchased from Wako Pure Chemical Corporation (Osaka, Japan).
Aminophenyl fluorescein, , hydroxyphenyl fluorescein, and perinaphthenone (PhO) were
purchased from Sigma-Aldrich Co. LLC. (MO, USA). Supercoiled pBR322 plasmid was
purchased from Thermo Fisher Scientific Inc. (MA, USA). The cell counting reagent SF was
obtained from Nacalai Tesque (Kyoto, Japan)
The tested compounds are shown in Fig. 2. C60 was purchased from SES Research,
Inc. (TS, USA). C60 pyrrolidine tris-acid (C60 tris), [6,6]-phenyl C61 butyric acid methyl ester
3, 8-methoxypsoralen were from Sigma-Aldrich Co. LLC. (MO, USA). Nanom spectra D100
(C60(OH)n) was purchased from Frontier Carbon Corporation (Tokyo, Japan). Compounds 1,
2 and 4 were synthesized in our laboratory（vide infra）.
All other chemicals were obtained from commercially available sources unless stated.
Synthesis of 8-hydroxypsoralen (33). A solution of BBr3 in CH2Cl2 (12 mL, 12 mmol) was
added to 8-methoxypsoralen (430 mg, 2 mmol) solution in CH2Cl2. The reaction mixture was
stirred and intermittently checked by TLC with (hexane:ethyl acetate [2:1]). After
confirmation of the end of the reaction by TLC analysis, 2 M NaOH was added to form a
homogenous solution. The solution was acidified with 2 M HCl and extracted with ethyl
acetate. The organic layer was washed with brine and dried over anhydrous Na2SO4
1
overnight. The resultant mixture was filtered and dried in vacuo. Yield: 98%; H-NMR
(CDCl3) δ: 7.79 (1H, d, J = 9.6 Hz), 7.70 (1H, d, J = 2.3 Hz), 7.26 (1H, s), 6.80 (1H, d, J =
2.3 Hz), 6.36 (1H, d, J = 9.6 Hz), 6.08 (1H, s).
Synthesis of [6,6]-phenyl C61 butyric acid 4 (34). To the solution of [6,6]-phenyl C61 butyric
acid methyl ester (90 mg, 0.101 mmol) in toluene (15 mL), concentrated HCl (6 mL) was
added. Acetic acid was then added in order to produce a homogenous solution, which was
then stirred under reflux. The end of the reaction was checked with TLC (toluene) and the
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mixture was centrifuged and decanted. Precipitates were washed twice with methanol and
dried in vacuo. The obtained material was used in the next step without further purification.
Synthesis of 8-OH-psoralen-[6,6]-phenyl C61 butyric acid ester 1. The mixture of 8-
hydroxypsoralen (101 mg, 0.5 mmol) and 4 (88 mg, 0.1 mmol) was added to CH2Cl2 (30 mL)
and then subjected to ultrasonication. To this mixture, N,N’-dicyclohexylcarbodiimide (103
mg, 0.5 mmol) at 0°C was added and stirred for 5 h at room temperature. The reaction
mixture was dried in vacuo and the crude material was purified by open column
chromatography (SiO2, 15 g; hexane:ethyl acetate [10:1–1:1]). Yield: 31%; MS (MALDI-
1
TOF, matrix: 2,5-dihydroxybenzoic acid) m/z 1080 [M+H+]; H-NMR (CDCl3) δ: 7.78 (2H,
d, J = 9.6 Hz), 7.69 (1H, d, J = 2.3 Hz), 7.67 (1H, d, J = 2.3 Hz), 7.46 (1H, t, J = 7.3 Hz),
6.84 (1H, d, J = 1.8 Hz), 6.80 (1H, d, J = 2.3 Hz), 6.36 (2H, t, J = 9.4 Hz), 4.10 (2H, m)
3.13–3.09 (2H, m), 3.00 (2H, t, J = 7.1 Hz).
Synthesis of 8-(4'-bromobutoxy)psoralen 5. To DMF (50 mL), 8-hydroxylpsoralen (2.1 g,
10.3 mmol), potassium carbonate (2.8 g, 20 mmol), and 1,4-dibromobutane (10 mL) were
added, and the mixture was heated at 90°C for 16 h. The reaction mixture was extracted with
organic solvent (hexane:ethyl acetate [4:1]) and washed with water and brine. The organic
layer was dried over anhydrous Na2SO4 in vacuo. The crude material was purified on open
1
column chromatography (SiO2, 300 g, chloroform). Yield: 85%; H-NMR (CDCl3) δ: 7.75
(1H, d, J = 9.6 Hz), 7.67 (1H, d, J = 2.3 Hz), 7.35 (1H, s), 6.81 (1H, d, J = 2.3 Hz), 6.36 (1H,
d, J = 9.6 Hz), 4.51 (2H, t, J = 5.9 Hz), 3.56 (2H, t, J = 6.6 Hz), 2.22–2.19 (2H, m), 2.04–2.00
(2H, m).
Synthesis of 8-(4'-(4’’-nitrophenoxy)butoxy)psoralen 6. To DMF (30 mL), 5 (8.8 mmol), p-
nitrophenol (5.4 g, 39 mmol) and potassium carbonate (5.4 g, 39 mmol) were added, and the
mixture was heated at 90 °C for 7 h. The reaction mixture was added to water, filtered, and
1
dried in vacuo. Yield: 100%; H-NMR (CDCl3) δ: 8.17 (2H, dd, J = 6.9, 2.3 Hz), 7.75 (1H,
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d, J = 9.6 Hz), 7.66 (1H, d, J = 2.3 Hz), 7.36 (1H, s), 6.96 (2H, dd, J = 6.9, 2.3 Hz), 6.81 (1H,
d, J = 2.3 Hz), 6.36 (1H, d, J = 9.6 Hz), 4.56 (2H, t, J = 5.9 Hz), 4.23 (2H, t, J = 6.2 Hz),
2.20–2.14 (2H, m), 2.07–2.05 (2H, m).
Synthesis of 8-(4'-(4''-aminophenoxy)butoxy)psoralen 7. To formic acid (30 mL), 6 (8.8
mmol) and granular zinc (3 g) were added and heated at 90 °C for 1 h. Ethyl acetate was
added to the reaction mixture and filtered. The organic layer was washed with aqueous
NaHCO3, dried over anhydrous Na2SO4, and dried in vacuo. The oily residues thus obtained
were used for the next step without purification.
Synthesis of 8-(4'-(4''-(tert-butylglycinate)aminophenoxy)butoxy)psoralen 8. To THF (30
mL), 7, tert-butyl bromoacetate (3 mL, 18 mmol), and triethylamine (3.6 mL) were added
and stirred for 3 days. The reaction mixture was extracted with ethyl acetate and the organic
layer was dried over anhydrous Na2SO4. The organic layer was dried in vacuo and purified
by open column chromatography (SiO2, 300 g; hexane:ethyl acetate [9:1–1:1]). Yield: 27%;
1H-NMR (CDCl3) δ: 7.66 (1H, d, J = 9.6 Hz), 7.58 (1H, d, J = 2.3 Hz), 7.24 (1H, s), 6.72–
6.71 (3H, m), 6.49 (2H, d, J = 8.7 Hz), 6.26 (1H, d, J = 9.6 Hz), 4.47 (2H, t, J = 5.9 Hz), 3.93
(2H, t, J = 5.7 Hz), 3.69 (2H, s), 1.99–1.96 (4H, m), 1.42 (9H, s).
Synthesis of tert-butyl glyoxylate (35). To methanol (9 mL) solution containing L-di-tert-
butyl tartarate (315 mg, 1.2 mmol), aqueous sodium periodate solution (385 mg, 1.8 mmol,
dissolved in 4.5 mL of water) was added. The reaction mixture was stirred for 80 min at 0°C
and the reaction was quenched by the addition of ethyl acetate and washed twice with water
and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and dried in vacuo
to give a clear oil. The oil was used next step without purification.
Synthesis
of
2,5-di-tert-buthoxycarbonyl-N-(4'-(4''-(8'''-OH-
psoralen)butoxy)phenoxy)pyrroridine[60]fullerene 9. C60 (864 mg, 1.2 mmol), 8 (1.15 g, 2.4
mmol) and tert-butyl glyoxylate were added to toluene and refluxed for 5 h. The reaction
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mixture was dried in vacuo and the crude material was purified by open column
1
chromatography (SiO2, 200 g; toluene:ethyl acetate [1:0–92:8]). Yield: 12%; H-NMR
(CDCl3) δ: 7.77 (1H, d, J = 9.6 Hz), 7.69 (1H, d, J = 2.3 Hz), 7.37 (1H, s), 7.29 (2H, d, J =
9.1 Hz), 7.02–7.00 (2H, m), 6.83 (1H, d, J = 2.3 Hz), 6.38 (1H, d, J = 9.6 Hz), 6.30 (2H, s),
4.60 (2H, t, J = 5.9 Hz), 4.14 (2.0H, t, J = 5.7 Hz), 2.15–2.10 (4H, m), 1.36 (18H, s).
Synthesis
of
2,5-dicarboxy-N-(4'-(4''-(8'''-OH-
psoralen)butoxy)phenoxy)pyrroridine[60]fullerene 2. Compound 9 (133 mg, 0.1 mmol) was
dissolved in mixture of toluene/dichloromethane (1:2) and boron trifluoride diethyl ether
complex (600 µL) was added to the solution. The obtained solution was left to stand for 2
days. The mixture was centrifuged at 5000 rpm for 5 min and decanted. The precipitate was
washed with methanol (5 mL), resuspended, centrifuged at 5000 rpm for 5 min, and decanted.
This process was repeated three times and the precipitate was dried in vacuo. Yield: 66%;
1
MS (MALDI-TOF, matrix: 2,5-dihydroxybenzoic acid) m/z 1199 [M+]; H-NMR (DMSO-
D6) δ: 8.16 (1H, d, J = 9.6 Hz), 8.12 (1H, d, J = 2.3 Hz), 7.70 (1H, s), 7.34 (2H, d, J = 9.1
Hz), 7.12 (1H, d, J = 2.3 Hz), 7.04 (2H, d, J = 8.7 Hz), 6.50 (2H, s), 6.46 (1H, d, J = 9.6 Hz),
4.52 (2H, d, J = 5.9 Hz), 4.10 (2H, d, J = 5.9 Hz), 1.99 (4H, d, J = 6.4 Hz).
Preparation of sample stock solutions. All compounds containing 8-MOP, C60, C60(OH)n,
C60 pyrrolidine tris acid (C60 tris), 1, 2, 3, and 4 were dissolved in DMSO to make 1 mM
stock solutions that were further diluted with an adequate amount of distilled water or cell
culture medium. PhO, a well-known efficient singlet oxygen inducer (ca. 100% quantum
1
yield of O2 production in air-saturated organic solvents (36)), was used as a reference
sample.
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Measurement of ROS. Singlet Oxygen Sensor Green reagent (SOSG, 100 µg), which is able
to detect singlet oxygen, was dissolved in methanol (33 mL) to make a 5 µM solution. BES-
So, which can detect the superoxide anion radical, was dissolved in DMSO at the
concentration of 5 µM. Both aminophenyl fluorescein solution (APF) and hydroxyphenyl
fluorescein solution (HPF), which can detect the hydroxyl radical, were further dissolved in
DMF to make a 5 µM solution. To each well of a 96-well plate, the following were added: a
solution of test compounds (final concentrations of 0, 20, 40, and 80 µM), reactive oxygen
detection reagent (final concentration of 0.7 µM), and pure water to make a final volume of
200 µL. The 96-well plate was treated with UVA irradiation (3.3 mW/cm2, 20 min) and the
fluorescence spectrum (excitation, 485 nm; emission, 535 nm) was recorded by using the
TECAN microplate reader. This experiment was performed twice and the values were
averaged.
Measurement of DNA cleaving activity induced by fullerene derivatives. A solution of
pBR322 plasmids (9 µL of 50 µg/mL in TE buffer) and 1 µL sample solution (0, 12.5, 25, 50,
and 100 µM in DMSO) were mixed in each well of a 96-well plate, and then irradiated with
UVA (1.5 mW/cm2, 2 h). Each irradiated sample (5 µL with loading buffer) was
electrophoresed in a 1% agarose gel (14×18 cm) in TBE buffer (50 mM) for 5 h at 80 V.
After electrophoresis, the gels were stained with ethidium bromide for 1 h (0.5 mg/L) and
analyzed with LAS-3000. This experiment was performed three times and the obtained data
were averaged.
Evaluation of genotoxicity of fullerene derivatives using the umu test. Salmonella
typhimurium NM8001 strain, which can detect oxidative damage with high sensitivity, was
used for the umu test (37). Appropriately diluted sample stock solutions (2 µL, the final
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concentrations: 0, 1.25, 2.5, 5 and 10 µM in DMSO) were added to liquid medium containing
Salmonella strains pre-cultured in tryptone-glucose-ampicillin medium for the log growth
phase (200 µL) in a 96-wells microplate, irradiated UVA (1.5 mW/cm2, 20 min), and then
incubated at 37°C for 2 h with shaking. After incubation, the absorbance of each well was
measured at OD595 nm. An aliquot of incubated samples (10 µL) was added to a mixture of Z
buffer (90 µL) and 0.1% SDS solution (50 µL) in a new 96-well microplate, and then
chlorophenol red-β-D-galactopyranoside (10 µL) was added. The microplate was incubated
at 37°C for 1 h. Sodium carbonate solution (1 M, 100 µL) was added to the each well and
then the OD565 nm of the solution was measured. This experiments was performed twice and
the values were averaged. The LacZ units were calculated using the following equation: LacZ
units = OD565 nm/OD595 nm. The ratio of the LacZ units of test compounds to the control value
was calculated and shown as fold induction on the graph.
Evaluation of cytotoxicity by fullerene derivatives. Adequately diluted sample solutions (1
µL) were applied to the cell culture medium containing human cancer cell lines of A549 and
HeLa cells that were pre-incubated in Eagle’s minimal essential medium (200 µL) in a 96-
well microplate for 24 h. After incubation for 1, 3, 6, 12, and 24 h with the test materials, the
medium was changed for Hank’s balanced salt solution and then the 96-well plate was
irradiated under UVA (1.5 mW/cm2, 20 min or 4 mW/cm2, 20 min). After irradiation, the
cell counting reagent SF (10 µL) was added to each well of the 96-well microplate. The cells
were incubated at 37°C for 1 h and the absorbance at 420 nm was measured for calculation of
the cell survival rate.
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RESULTS AND DISCUSSION
Synthesis of fullerene derivatives 1 and 2:
The psoralen-linked fullerene derivative 1 was synthesized via an esterification reaction of the
corresponding acid 4 using N,N'-dicyclohexylcarbodiimide. Compound 4 was obtained from the
fullerene derivative of [6,6]-phenyl C61 butyric acid methyl ester 3 by the known procedure of
acid hydrolysis of methyl ester (Scheme 1). The acid chloride procedure to gain 1 with the
compound 4 with 8-hydroxypsoralen using acetyl chloride failed, most likely owing to the poor
solubility of the compound 4 in toluene.
For the synthesis of compound 2, 8-hydroxypsoralen was selected as the starting material.
The hydroxyl group was condensed with 1,4-dibromobutane via the SN2 reaction to produce
compound 5. The bromo group of 5 was reacted with p-nitrophenol to produce compound 6, and
the nitro group of 6 was then reduced by metallic zinc in the presence of formic acid and heat.
The obtained amino group of 7 was further reacted with tert-butyl bromoacetate to produce
glycinate derivative 8. The fullerene derivative 10 was obtained from the reaction of compounds
8, 9, and C60; the produced tert-butyl groups tolerated trifluoroacetic acid treatment, but could be
easily removed by treatment with boron trifluoride diethyl ether complex to produce 2. The total
yield based on the starting material was 1.8% (Scheme 2).
Measurement of ROS:
ROS play important roles in PDT owing to their high reactivity and cytotoxicity. A strong ROS-
producing ability under irradiation is therefore required for PS. ROS species induced by the PS
process can be classified into different types. Singlet oxygen is produced by energy transfer from
exciting PS to oxygen molecule in the ground state (Type II) (7). The hydroxyl radical and
superoxide anion radical are produced by electron transfer from the reductant to exciting PS,
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followed by electron transfer from PS to the oxygen molecule in the ground state to generate †he
superoxide anion radical (Type I). Subsequently, the superoxide anion radical reacts with a
proton to produce hydrogen peroxide, which reacts with ferrous ions in vivo, via the Fenton
reaction, to produce the hydroxyl radical (38). We evaluated the production of the
abovementioned four ROS species by fullerene derivatives using each of the fluorescence
detecting reagents (SOSG, APF, HPF, and BES-So reagent) (Fig. S1 and Table 1).
All fullerene derivatives, except C60, were found to produce ROS species; however,
superoxide anion radical formation detected by BES-So (Fig. S1 (g) and (h)) was probably
-
due to preferential formation of O2 in the presence of reducing agents such as NADH (7).
All fullerene derivatives used in this study were able to induce ROS formation, but not all
species of ROS were produced equally: singlet oxygen producing was most prominent for the
compound 4 toward SOSG detection reagents, whereas the hydroxyl radical was favored
produced by C60(OH)n (Fig. S1). C60 has poor water-solubility and also lower solubility in
DMSO, which would be one explanation of the low ROS-producing ability. The efficient
ROS-producing ability of C60 was already reported in well-solubilized conditions using
aprotic solvents such as toluene (5) or even in water, but by using the solubilizing material
PVP (7). In this study, C60 was only suspended in pure water by ultrasonication. Preparation
methods for fullerene solution affect the formation of ROS species; the use of only sonication
was reported not to be effective for the production of ROS, similar to our results (39).
Unexpectedly, the ROS-producing activity of the novel compound 2 was only observed for
the hydroxyl radical detection system of HPF and APF. Moreover, hydroxyl radical
production by 2 was only observed at relatively lower concentrations of approximately 20
μM. Multiple adduction of fullerene with functional groups decreased the quantum yield of
singlet oxygen (23). The presence of the electron-rich moiety of aminophenol in compound 2
may affect the ROS-production ability at higher concentrations.
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Evaluation of DNA cleaving activity of fullerenes:
We designed the novel psoralen-linked fullerenes 1 and 2, which were able to bind to DNA by
using the crosslinking property of psoralen toward pyrimidine moieties in DNA under UV
irradiation To confirm the DNA cleaving ability of fullerene derivatives, gel electrophoretic
analyses of supercoiled pBR322 was performed by the detection of conformational change to the
relaxed form originated from DNA cleaved by ROS and/or psoralen cross-linking. The DNA-
cleaving ability of each fullerene and the positive control of PhO was examined and ratio of
relaxed/supercoiled was shown in Fig. S2 and Table 2.
The DNA cleavage ratio of PhO and C60(OH)n was 67% and 44% at the maximum
concentration used in this study, respectively. Although other samples showed below 30%
cleavage at the maximum concentration, the obtained results suggested that the ratio of relaxed
DNA form was increased in a dose-dependent manner. The DNA-cleaving ability of fullerene
derivatives modified by acridine or N-vinylpyrrolidone copolymer has been reported previously
(40, 41). A fullerene derivative with one hydroxyl group was unable to cleave DNA, most likely
owing to low solubility in polar solvents (21). C60(OH)n, which is readily soluble in polar
solvents, has potent DNA-cleaving ability compared with other fullerene derivatives. A multiple
amine adducted fullerene derivative was shown to have potent DNA cleaving ability, with over
70% DNA cleaved at 40 mg/L under visible light (42). Given these previous findings, the higher
water solubility of fullerene derivatives confers DNA-cleaving ability in DNA. A carboxylated
fullerene was also reported to cleave DNA under visible light irradiation, and the carboxylated
fullerene tested in this study also enhanced the DNA-cleaving ability under other light conditions.
The DNA-cleaving ability of 1 and 2 was much lower than that of C60(OH)n. Of the fullerene
derivatives, C60(OH)n showed the strongest ROS-inducing ability at the lowest concentration,
especially when the APF hydroxy radical detection system was used; this would make strand
scission of DNA facile.
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Evaluation of genotoxicity using umu test by fullerene derivatives:
The genotoxicity of selected compounds was evaluated through the ability of the photoactive
fullerenes to induce DNA damage in the S. typhimurium NM8001 strain (Fig. 3).
Clear genotoxicity was observed when PhO or 8-MOP was applied to the bacteria. Both
compounds are known to be genotoxic under light irradiation with activity derived from the
different mechanisms (36, 43); that is, the former compound induces genotoxicity through the
induction of ROS and the latter via DNA crosslinking. C60(OH)n had the most potent DNA
cleaving ability using the pBR322 assay, as shown previously, but only a slight induction of
LacZ, which represents genotoxic activity, probably because the most severely toxic effects on
Salmonella were observed to be concentration-dependent. Of the fullerene derivatives tested in
this study, only compound 2 enhanced its LacZ value in a dose-dependent manner. It is
estimated that C60(OH)n caused cell death without damaging DNA, whereas fullerene 2, modified
with psoralen, could attack DNA via psoralen-pyrimidine crosslinking.
Evaluation of cytotoxic activation by fullerene derivatives:
For the purpose of facile comparison of the activity of each compound, the concentration of the
compound and irradiation conditions were initially set at below 10 μM and 1.5 mW/cm2, 20 min,
respectively. Severe cytotoxic effects were observed when PhO, C60(OH)n, C60 tris, and 4 were
used (Fig. S3 and Fig. S4). In this study, the pretreatment time of the compounds to various cell
types varied from 1 h to 24 h. Cytotoxicity of C60(OH)n was apparent with 1 h pretreatment,
whereas the cytotoxicity of C60 tris was only apparent after 6 h of exposure. These trends and
cell viability were not different in either cell type (A549 and HeLa; data not shown). It was
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estimated that these time-dependent cytotoxic effects arose from the different cellar uptake
mechanism of each compound or the difference of biological reaction after cellular uptake of the
compounds (44).
Cell viability of exposed by C60(OH)n and C60 tris were found to decrease by less than
10% after 24 h treatment. In addition, although the activity was not so strong, we found that
compound 4 was slightly cytotoxic.
At higher concentrations of the compounds 1 and 2 (~100 µM), with a higher dose of
irradiation (4 mW/cm2, 20 min), we found that the viability of HeLa and A549 cells with 1 or 2
was clearly decreased by pretreatment for 24 h (Fig. S5). The LD50 values of photo-induced
cytotoxicity of fullerene derivatives are shown in Table 3.
<Table 3>
Recently, we have already reported the possible cell penetration of the water-soluble
fullerene derivative; however, in the same conditions, cell penetration of a non-water-soluble
fullerene derivative was not observed (45). In this study, the cytotoxicity of fullerene derivatives
could depend partly on the hydrophilic properties of the compounds. Although C60 does not have
ROS-producing ability, the fullerene derivatives 3 and 4 did. However, all three fullerene
derivatives were not showed clearly cytotoxic. These fullerene derivatives with no hydrophilic
groups may agglomerate readily and/or precipitate in the incubation medium. In contrast,
C60(OH)n and C60 tris, which hydrophilic groups (carboxyl or hydroxyl groups), have ROS-
producing ability and cytotoxicity. In addition to the reported cytotoxicity of cationic fullerene
derivatives (14), the hydrophilicity of compounds
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CONCLUSION
We successfully synthesized two fullerene derivatives with a psoralen moiety and evaluated
their biological activities. The fullerene derivatives 1 and 2 have ROS-producing activity and
DNA-cleaving activity under UVA irradiation. In addition, the fullerene derivative 2 induced
genotoxicity toward bacteria under UVA irradiation. The fullerene derivatives 1 and 2
induce cell death at high concentrations and/or strong UVA irradiation. As a result, we
suggest that psoralen-linked fullerenes are potential PDT reagents. A novel synthesized
psoralen-linked fullerene with stronger cytotoxicity than 2 and the study of combined
fullerene derivatives and drug delivery systems are necessary for the application of PDT.
ACKNOWLEDGEMENTS:
This work was partially supported by the Grants-in-Aid for Scientific Research grant awarded
to a doctoral student from the Kanagawa Institute of Technology. This work was supported
by the Ministry of Education, Cultures, Sports, Science and Technology (MEXT) Program
for the Strategies Research Foundation at Private Universities 2015–2019 (grant number S
511019L).
SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section
at the end of the article:
Figure S1. Results of ROS-producing ability using several detection reagents. (a), (b):
SOSG, (c), (d): APF, (e), (f): HPF, (g), (h): BES-so. †: p<0.05 in student’s t-test vs a value at
0 µM.
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Figure S2. DNA-cleaving ability of the test compounds. The bars represent 0, 12.5, 25, 50,
and 100 µM, respectively. Mark of † indicates p<0.05 in student’s t-test vs 0 µM.
Figure S3. Cytotoxicity to A549 cells. This graph shows cell viability after UVA irradiation
(1.5 mW/cm2, 20 min) following exposure to the test compound for 24 h.
Figure S4. Cytotoxicity to HeLa cells. This graph shows cell viability after UVA irradiation
(1.5 mW/cm2, 20 min) following exposure to the test compound for 24 h.
Figure S5. Results of cytotoxicity at high concentration under strong irradiation. These
graphs show the results after irradiation UVA (4 mW/cm2, 20 min) (a) A549 cells exposed to
test samples for 24 h and (b) HeLa cells exposed to test samples for 24 h.
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FIGURE CAPTIONS
Figure 1. Schematic diagram of DNA-cleaving mechanism by psoralen-linked fullerene
Figure 2. Structure of test samples.
Figure 3. Result of umu test. These graphs represent (a), (b) LacZ units, and (c), (d) OD595 nm
.
Scheme 1. Synthesis of fullerene derivative 1
Scheme 2. Synthesis of fullerene derivative 2
Table 1. ROS-producing ability rate per µmol of tested compounds* (relative fluorescence
intensity/µmol of tested compounds).
Tested compounds
PhO
8-MOP
C60
C60(OH)n C60 tris
1
2
3
4
Detection
reagents
SOSG
APF
0.16
0.06
0.06
0.02
0.02
0.00
0.00
0.00
0.02
0.01
0.01
0.00
0.17
0.19
0.42
0.00
0.09
0.13
0.13
0.00
0.18
0.11
0.37
0.00
0.07
0.11
0.85
0.01
0.08
0.12
0.05
0.00
0.26
0.15
0.41
0.00
HPF
BES-So
* The initial slope value was calculated from the dose-dependent curve of each of the test
compounds from Figure S1 in the Supporting Information.
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Table 2. DNA-cleaving ability rate of the tested compounds* (relative DNA cleaving
rate/µmol of tested compounds).
PhO
2.1
8-MOP
0.067
C60
C60(OH)n C60 tris
0.22 0.045
1
2
3
4
0.008
0.040
0.064
0.063
0.042
* The rate value was calculated from the dose dependent curve of Figure S2 in the Supporting
Information.
Table 3. LD50[µM] of fullerene derivatives under UVA irradiation†‡.
1‡
2‡
3†
-
4†
†
†
C60
C60(OH)n C60 tris†
A549
HeLa
-
-
2.6
3.5
3.2
3.0
49.8
6.7
31.0
6.4
17.8
8.8
-
UVA irradiation: †1.5 mW/cm2, 20 min ; ‡4.4 mW/cm2, 20 min.
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