Nanoparticles of water-soluble dyads based on amino acid fullerene C60 derivatives and pyropheophorbide: Synthesis, photophysical properties, and photodynamic activity

Article abstract of DOI:10.1016/j.saa.2021.119885

Synthesis, spectral properties, and photodynamic activity of water-soluble amino acid fullerene C₆₀ derivatives (AFD) and four original AFD-PPa dyads, obtained by covalent addition of dye pyropheophorbide (PPa) to AFD, were studied. In aqueous solution, these AFD-PPa dyads form nanoassociates as a result of self-assembly. In this case, a significant change in the absorption spectra and strong quenching of the dye fluorescence in the structure of the dyads were observed. A comparison of superoxide or singlet oxygen generation efficiency of the studied compounds in an aqueous solution showed the photodynamic mechanism switching from type II (singlet oxygen generation of the native dye) to I type (superoxide generation of dyads). All dyads have pronounced phototoxicity on cells Hela with IC₅₀ 9.2 μM, 9.2 μM, 12.2 μM for dyads Val-C₆₀-PPa, Ala-C₆₀-PPa and Pro-C₆₀-PPa, respectively. Such facilitation of type I photodynamic mechanism could be perspective against hypoxic tumors.

Full text of DOI:10.1016/j.saa.2021.119885

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119885
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Nanoparticles of water-soluble dyads based on amino acid fullerene C60
derivatives and pyropheophorbide: Synthesis, photophysical properties,
and photodynamic activity
A.Yu. Belik a, A.Yu. Rybkin a, N.S. Goryachev a,b, A.P. Sadkov a, N.V. Filatova a, A.G. Buyanovskaya c,
V.N. Talanova c, Z.S. Klemenkova c, V.S. Romanova c, M.O. Koifman d, A.A. Terentiev a,b, A.I. Kotelnikov a,b
a Institute of Problems of Chemical Physics, Russian Academy of Sciences, Chernogolovka, Moscow Oblast 142432, Russia
b Lomonosov Moscow State University, Moscow 119991, Russia
c A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow 117813, Russia
d Ivanovo State University of Chemistry and Technology, Ivanovo 153000, Russia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
Three amino acid-fullerene[60]-pyro
pheophorbide a dyads (AFD-PPa)
were synthesized.
ꢀ
ꢀ
AFD-PPa dyads form nanoassociates
in water as a result of self-assembly.
Photodynamic mechanism switching
from type II of dye to I type of dyad
was shown.
ꢀ
All dyads have a pronounced
phototoxicity on HeLa cancer cells.
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis, spectral properties, and photodynamic activity of water-soluble amino acid fullerene C60
derivatives (AFD) and four original AFD-PPa dyads, obtained by covalent addition of dye pyropheophor-
bide (PPa) to AFD, were studied. In aqueous solution, these AFD-PPa dyads form nanoassociates as a result
of self-assembly. In this case, a significant change in the absorption spectra and strong quenching of the
dye fluorescence in the structure of the dyads were observed. A comparison of superoxide or singlet oxy-
gen generation efficiency of the studied compounds in an aqueous solution showed the photodynamic
mechanism switching from type II (singlet oxygen generation of the native dye) to I type (superoxide
generation of dyads). All dyads have pronounced phototoxicity on cells Hela with IC50 9.2 mM, 9.2 mM,
12.2 mM for dyads Val-C60-PPa, Ala-C60-PPa and Pro-C60-PPa, respectively. Such facilitation of type I pho-
todynamic mechanism could be perspective against hypoxic tumors.
Received 20 November 2020
Received in revised form 22 April 2021
Accepted 25 April 2021
Available online 30 April 2021
Keywords:
Fullerene
Water-soluble amino acid fullerene
derivatives
Pyropheophorbide
Fluorescence quenching
Photochemical activity
Photosensitizer
Ó 2021 Published by Elsevier B.V.
1. Introduction
photoexcitation, free radicals are generated – singlet oxygen,
superoxide, hydroxyl radical, and other reactive oxygen species
Photodynamic therapy is an actively developing field of medi-
cine, based on the selective effect on a tumor or microorganisms
of low-toxic molecules of dyes – photosensitizers when excited
into a triplet state with the light of a certain wavelength. Under
(ROS), which suppress the growth of tumors or microorganisms
[1,2]. The effectiveness of the photosensitizer depends on the
chemical structure of the dye and the wavelength of the exciting
light.
Dyes from the class of porphyrins, phthalocyanines, and chlo-
rins are widely used nowadays in medical practice, which have a
E-mail address: alryb@icp.ac.ru (A.Yu. Belik)
https://doi.org/10.1016/j.saa.2021.119885
1386-1425/Ó 2021 Published by Elsevier B.V.

A.Yu. Belik, A.Yu. Rybkin, N.S. Goryachev et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119885
high triplet state quantum yield and absorb in the red spectral
region since the light of these wavelengths penetrates the body tis-
sues best [1–4].
2. Experimental section
2.1. Materials
The possibility of a significant increase in the photodynamic
activity was previously demonstrated due to the formation of
non-covalent complexes of the water-soluble pentacationic fuller-
ene derivative with the photodynamic preparation Fotosens, with
xanthene dyes fluorescein, eosin, erythrosine [5–8], as well as
covalent conjugates of polysubstituted fullerene derivatives with
the antitumor antibiotic Ruboxil, dyes fluorescein and chlorin e6
[9–12]. These works showed that the generation of superoxide
anion radical was enhanced by electron transfer or excitation from
a singlet-excited dye to fullerene and then to an oxygen molecule.
The generation efficiency of Oꢀ2- depended on the presence of elec-
trostatic charges on the dye and fullerene. Similar effects were
observed for other fullerene-dye dyads [12–21].
2.1.1. Reagents
The following reagents were used in the studies: fullerene C60
(Fullerene Center, Nizhny Novgorod, 99.5%), L-Valine, L-Proline,
L-Alanine (Sigma), DPBF (1,3-Diphenylisobenzofuran, Sigma),
methyl iodide (Sigma, 99%), NADH (nicotinamide adenine dinu-
cleotide, Sigma), NBT (nitro blue tetrazolium chloride, Sigma),
EDTA (ethylenediamine-tetraacetic acid, Sigma), MTT (3-(4,5-dime
thyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma)
Dichlorobenzene («Acros organics», 99%) and pyridine («Acros
organics», 99%) were distilled and dehydrated before usage. Other
solvents (purity ~ 99.9%) were used without purification.
In addition to spectral properties, an important characteristic of
compounds for photodynamic therapy is their ability to penetrate
biological membranes and accumulate in tumors. There are sug-
gestions that compounds with a minimum charge have the most
effective permeability through biological membranes [22]. In this
regard, in addition to studying fullerene-dye dyads based on pen-
tasubstituted fullerenes derivatives, it seems interesting to analyze
the possibility of creating photoactive water-soluble fullerene-dye
dyads and nanoparticles (NPs) based on monosubstituted amino
acid fullerenes derivatives (AFD) (Fig. 1).
2.2. Synthesis of pyropheophorbide a, C60-amino acid derivatives
and dyads
Synthesis of pyropheophorbide a. The synthesis of
pyropheophorbide a (PPa, Fig. 1) was carried out according to the
previously described method [57] from methylpheophorbide a
(Fig. S1, Supplementary). Product yield: 87.5% mg. The ESI-ms,
NMR, and UV–VIS-spectra of pyropheophorbide a coincide with
those in [58].
An effective and technologically advanced method for creating
such water-soluble AFDs that form stable aqueous solutions with
a concentration of 10–30 mg/ml has been previously developed
[23,24]. According to this technique, fullerene is modified by
attaching an amino acid to the fullerene core by breaking the dou-
ble bond of the fullerene and forming a bond with the amino group
of the amino acid. This method allows one to obtain a wide range
of biologically active derivatives of fullerene, which showed
membrane-active, antioxidant, and immunomodulatory properties
with low toxicity, antivirus, antitumor, and neuroprotective activ-
ities [25–32]. Theoretical calculations showed the thermodynamic
stability of such amino acid fullerene derivatives [33,34].
In various versions, this technique was reproduced by other
researchers [35,36,45,37–44]. It is possible to significantly
expand the possibilities of creating hybrid nanoparticles based
on amino acid derivatives of fullerenes by attaching the second
addendum to the fullerene core by replacing the ‘‘acidic” proton
of the amino acid fullerene derivative. (Fig. 1) [28,46,47]. Thus, it
is possible to create a wide range of biocompatible hybrid struc-
tures using a combination of two different addends: one of
them, the amino acid, gives the fullerene core water solubility,
Synthesis of Zn-substituted pyropheophorbide a (PPa(Zn),
Fig. 1) was carried out according to the previously described
method [59] (Fig. S1, Supplementary).
Synthesis of C60-amino acid derivatives. Amino acid fullerene
derivative (AFD) 1, 2, and 3 (Fig. 1) were synthesized according
to the method described earlier [23] (Fig. S1, Supplementary).
Synthesis of AFD-PPa dyads. Compounds Val-C60-PPa, Ala-C60
-
PPa, Pro-C60-PPa (6, 7, and 8, Fig. 1) were synthesized by attaching
PPa to AFD with replacing the ‘‘acidic” proton of the amino acid full-
erene derivative according to the following procedure (Fig. S1, Sup-
plementary): PPa (0.0288 mmol) was dissolved in 2 mL abs.
chloroform, then with stirring and cooling in an ice bath, a 1.5-fold
excess of thionyl chloride (0.0433 mmol, 5.15 mg) was added to it,
and then it was boiled with stirring for 2 h. After cooling, 2 mL of pyr-
idine was added and the excess thionyl chloride was distilled off. For
synthesis of Val-C60-PPa (as example), a 1.5-fold excess of Val-C60
methyl ester (0.0433 mmol, 38 mg) in 2 mL abs. pyridine was added
to this solution and stirred at room temperature for one day.
Purification of the reaction products in the synthesis of AFD-PPa
dyads was carried out by gel chromatography on a 3x70 cm glass
column with Bio Beats SX1 (cross-linked polystyrene) with pyri-
dine as eluent. The fractions yield was recorded using an LKB
Bromma 2151 densitometer at a wavelength of 415 nm. The anal-
ysis of the obtained chromatographic fractions was carried out by
UV–VIS spectrophotometry and fluorescence spectroscopy. During
chromatography, the target product AFD-PPa comes out as the first
main peak, and the unattached excess of PPa comes out as a subse-
quent extended peak.
The synthesis of methyl ester Val-C60-PPa(Zn) (9, Fig. 1) was
carried out using ethylene chlorohydrin according to the following
procedure (Fig. S1, Supplementary): to 0.0655 g (0.1217 mmol) of
PPa(Zn) dissolved in 5 mL of DMF, 0.0098 g (0.1217 mmol) of ethy-
lene chlorohydrin, 0.0251 g (0.1217 mmol) of dicyclohexylcarbodi-
imide were added and left at room temperature for 2 days. To this
solution was added 0.0852 g (0.1 mmol) of N-(monohydro)
fullerenyl valine methyl ester in 5 mL of pyridine and stirred for
24 h at room temperature. Determination of the content of Zn
atoms in the composition of nanoparticles based on Val-C60-PPa
(Zn) was carried out by X-ray fluorescence spectral analysis using
the VRA-30 spectrometer from Karl Zeiss, Jena (Germany).
and the second
photodynamic.
– additional biological properties, including
It should be noted that AFD, even with the addition of one
amino acid, has a sufficiently high solubility in water, up to
30 mg/ml. However, studies show that in this case, NPs are formed,
which often complicates the establishment of the molecular struc-
ture of newly synthesized compounds [48–50]. Specific interac-
tions arise between the AFD and the solution, which leads to a
change in the density of the solution, melting point, surface ten-
sion, conductivity, the dissociation constant, and the effect of
dynamic light scattering [51–56]. Based on the above methods, in
this work, with the aim of creating new photodynamic compounds,
we investigated methods for creating covalent AFD dyads with dye
pyropheophorbide a (PPa) (AFD-PPa). It was shown that these
dyads form NPs in aqueous solutions as a result of self-assembly.
Spectral, photophysical properties of these dyads and NPs based
on them were studied. Photodynamic action was investigated in
an aqueous medium with singlet oxygen and superoxide anion
radical probes, as well as on cell culture HeLa.
2

A.Yu. Belik, A.Yu. Rybkin, N.S. Goryachev et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119885
Fig. 1. Structures of AFDs, PPa, chlorin e6, and AFD-PPa dyads.
2.3. Preparation of water-soluble nanoparticles based on dyads
state spectra of the dyes under study were recorded by a Cary-
Eclipse fluorescence spectrophotometer. Dynamic light scattering
(DLS) measurements were performed at a detection angle of 90°
with a Photocor Complex (Photocor Instruments Inc., USA) setup
equipped with a TEC stabilized diode laser (k = 790 nm).
The photochemical activity of the studied compounds in water
was investigated with NBT and DPBF probes for the detection of
superoxide anion radical and singlet oxygen by the methods
described in [12]. Table 1 shows both the values of the comparative
efficiency of ROS generation by all compounds normalized to the
photodynamic activity of the Ce6 dye and the observed quantum
yield expressed in the number of Oꢀ2- or 1O2 molecules per absorbed
quantum of light detected in the model system. Normalization was
carried out, taking into account the absorbance spectrum of the
compound, transparency spectrum of used cut-off glass filter, and
xenon lamp emission spectrum at k > 630 nm.
To obtain water-soluble dyads 10-fold excess of NaOH in water
was added to dyad in pyridine and stirred for 4 h at room temper-
ature. The solution of AFD-PPa NPs was dialyzed against aqueous
solution using a 14,000 Da membrane (Sigma, D9277) for 3–4 days
with frequent replacement of the aqueous solution to remove
excessive pyridine and unconjugated PPa. The concentration of
AFD-PPa dyads in the resulting solution was estimated from an ali-
quot that was dried by the known molecular weight of the dyad.
2.4. Computational simulation of dyads structures
The spatial structures of AFD-PPa dyads (Fig. 3) were geometri-
cally optimized with a semi-empirical quantum chemistry method
using the MOPAC2016 package [60] with PM7 and COSMO param-
eterization methods to account for the surrounding water [61].
2.6. Phototoxicity on HeLa cell line
2.5. Photophysical and photochemical studies
The influence of the compound under study on cell line HeLa
viability was evaluated with staining with MTT. Conditions of cell
incubation and irradiation were the same as in [12].
Absorption spectra were measured using a Cary-60 spectropho-
tometer equipped with a thermostated cell. Fluorescence steady-
3

A.Yu. Belik, A.Yu. Rybkin, N.S. Goryachev et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119885
Table 1
A comparison of the photophysical properties and photodynamic activity of covalent conjugates AFD-PPa.
Ce6
6
7
8
Hydrodynamic radius in water, nm
Q band kQmax, nm
Fluorescence kmax, nm
Relative fluorescence quantum yield
27–80
654
659
0.22*
1
1
1
1
1
20–25
680
23–30
673
32–42
672
667
670
660
4.6ꢂ10-3
3.5ꢂ10-3
9.4ꢂ10-3
Relative efficiency of Oꢀ2- generation in water
Relative efficiency of Oꢀ2- generation in water, absorption normalized
Relative efficiency of Oꢀ2- generation in liposomes
Relative efficiency of Oꢀ2- generation in liposomes, absorption normalized
Relative efficiency of 1O2 generation in liposomes
Relative efficiency of 1O2 generation in liposomes, absorption normalized
2.4
3.6
2.7
4.8
3.5
6.9
10.2
4.0
11.3
0.108 (1/9.3)
0.30 (1/3.3)
12
3.7
3.9
5.2
0.13 (1/7.6)
0.18 (1/5.6)
8.2
0.087 (1/11.5)
0.17 (1/5.9)
8.6
1
0.83
Phototoxicity on HeLa cells IC50
,
l
M
*
Fluorescence quantum yield value of chlorin e6 trisodium salt (Ce6) was taken from work [4].
ene core, which was confirmed by ESI-MS and IR spectra (Figs. S2,
S3, Table S1, Supplementary).
3. Approach justification
In the IR spectra of all synthesized AFD, there was an intense
In recent years, special attention has been paid to the creation
of new highly effective photodynamic compounds based on hybrid
structures «fullerene-dyes». In these structures, the fullerene core
generates reactive oxygen forms by transferring energy or an elec-
tron upon photoexcitation of a dye. In addition, the presence of a
hydrophobic fullerene core ensures membranotronic properties
of such hybrid structures, which can also enhance the photody-
namic effect. Membranotropic properties of such hybrid structures
can vary significantly due to the addition of polar addends to the
fullerene core. Earlier, we studied the photodynamic activity of
hybrid structures based on pentanionic fullerene derivatives hav-
ing a solubility in water>100 mg/ml [62]. At the same time, it
has been suggested that compounds with a lesser charge more
effectively interact with membranes [22].
(strong, s) band near 1707–1726 cmꢁ1
(mC = O), associated with
COOH group. In these spectra the group of 3 peaks exhibit near
1100–1108 cmꢁ1 (strong, s), 951–962 cmꢁ1 and 837–886 cmꢁ1
(middle, m), and weak (w) band near 1250 cmꢁ1. These bands were
found in the IR spectra of all amino acid fullerene derivatives, and
they are associated with fullerene fragment vibrations of these
molecules [46]. There are bands in the spectrum of methyl ether
Val-C60, which could be assigned to vibrations of the amino-acid
fragment: 3315 s, broad (
2878 and 2851w ( CH3), 1707 s (
tion), 1238, 1178 – (carbon fragment vibrations), 1044 cmꢁ1
m
NH +
m
m
OH), 2972 and 2925 m (
C = O), 1360 m (CH3 deforma-
C-
mCH),
m
(m
OH). The IR spectrum of methyl ether Pro-C60 demonstrated the
disappearance of the band near 3300 cmꢁ1 associated with the
stretch mode of the NH bond in proline.
The presence of a C-N bond between the fullerene cage and the
amino group of the amino acid in such derivatives was previously
demonstrated by solid-state NMR [64]. The fact of monoaddition of
amino acids to fullerene in AFD was also shown earlier by the
method of amino acid analysis [65].
To purify the reaction products and to prove the covalent
attachment of PPa and AFD, the method of gel chromatography
was used in this work, followed by analysis of chromatographic
fractions by spectrophotometry, luminescence, and X-ray fluores-
cence analysis. Since the absorption spectra of PPa moiety in the
composition of the AFD-PPa conjugate vary greatly, and the PPa
fluorescence is quenched, the determination of the PPa content
in the AFD structure was carried out by X-ray fluorescence analysis
by recording Zn atoms which were specially introduced into the
PPa. Since PPa(Zn) was unstable under the conditions of the syn-
thesis of 8 using thionyl chloride, the synthesis of 9 was carried
out under milder conditions: ethylene chlorohydrin was used as
a linker, which leads to an increase in the length of the linker
(Fig. S1, Supplementary).
In this regard, this publication presents studies on the creation
of new photodynamic hybrid structures based on monoamino acid
fullerene derivatives (AFDs) and
a
chlorin derivative
–
pyropheophorbide a and NPs based on them. AFDs 1, 2, and 3 were
used to create photodynamic hybrid structures with one negative
charge (in the form of the sodium salt of amino acid moiety) or
have no charge (in the methylated form of amino acid moiety). It
was shown that such amino acid derivatives are highly soluble in
water, have low toxicity, and exhibit various biological activities:
they
have
pronounced
membranotropic,
antioxidant,
immunomodulating, and antiviral activity, as well as the high
stereospecificity of interaction with membranes and proteins
[25–32,63].
In this work, we propose a new method for creating hybrid pho-
todynamic structures using the original method of attaching
pyropheophorbide to amino fullerene derivatives, which was pre-
viously used to create various biologically active fullerene deriva-
tives [28]. AFDs are known to have limited solubility in water up
to 20–30 mg/mL and tend to form NPs in the aqueous phase. The
addition of hydrophobic PPa should further reduce their solubility
in water. In this regard, in this paper, the possibility of the forma-
tion of NPs based on new conjugates AFD-PPa by their self-
assembly in an aqueous solution was investigated. To solve these
problems, this paper proposes original methods for creating dyads
6, 7, and 8 with pronounced photodynamic activity.
It was determined by X-ray fluorescence analysis that after
covalent attachment of PPa(Zn) to Val-C60, the Zn content of 9 is
3.8% with a calculated value of 4.16%. This proves that the AFD:
PPa(Zn) ratio in the 9 conjugate is 1:1.
4.2. Formation and characterization of water-soluble nanoparticles of
AFD-PPa
4. Results and discussion
To create AFD-PPa water-soluble dyads, the carboxyl groups of
AFD were converted from the methylated form to the form of
sodium salt, and then AFD(Na)-PPa, were transferred to the aque-
ous phase by dialysis as described in section 2.3. The resulting con-
jugates 6, 7, and 8 exhibit amphiphilic properties; therefore, they
are capable of dissolving in water but are also prone to form
4.1. Synthesis and characterization of AFDs and covalent dyads AFD–
PPa
The synthesis conditions were chosen to obtain AFD conjugates
in which one amino acid and one dye were attached to the fuller-
4

A.Yu. Belik, A.Yu. Rybkin, N.S. Goryachev et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119885
nanoassociates. Water-soluble nanoparticles based on them
formed a transparent colored solution and practically did not pre-
cipitate during storage of the solution for several months.
excited singlet state of the PPa dye in the structure of the dyad
as a result of electron or excitation transfer to the fullerene core.
Analysis of the absorption spectra of water-soluble nanoparti-
cles based on AFD-PPa dyads in an aqueous solution showed that
all dyads had pronounced Soret bands in the range of 408–
415 nm and Q-band maximum in 673 – 680 nm range (Fig. 5A
and Table 1). It should be noted that in the wavelength range of
350 – 500 nm, the absorption can also be due to the significant
contribution of the AFD absorption spectrum and light scattering
of nanoparticles (Fig. 5A and Table 1). Given this contribution,
we can conclude that in all dyads, the intensities at the absorption
Q-bands reduced by 1.5–3 times as compared with the spectrum of
the water-soluble analog of PPa – chlorin e6 sodium salt (Ce6). This
effect was previously observed in dyads based on fullerene and Ce6
[12,68]. Moreover, the absorption intensity of AFD-PPa NPs in the
Q band region is close to the individual dyes (Fig. 5A).
A study of the steady-state fluorescence spectra of covalent
AFD-PPa NPs in comparison with the Ce6 spectra in an aqueous
solution showed that the fluorescence quantum yields of conju-
gates 6, 7 and 8 were significantly lower compared to Ce6 by 90,
233, and 30 times, correspondingly (Fig. 5B and Table 1). It is
known that the quantum yield of fluorescence of chlorin e6 in
water is 0.22 [4]; for PPa in dimethylformamide it is 0.31 [69].
Based on these data, the fluorescence quantum yield of these con-
jugates could be calculated (Table 1). The observed result indicates
effective quenching of the excited singlet state of the dye as a
result of electron transfer or excitation to the fullerene core.
It is well known that fluorescence quenching of the dye in the
structure of the dyads occurs due to electron or the energy transfer
from the excited dye to the fullerene. Due to the low overlap inte-
gral between donor fluorescence and acceptor absorbance spec-
trum, according to Forster’s theory, it can be expected that
energy transfer is highly unlikely in the fullerene-chlorin system
[66,70].
4.3. Estimation of the size of AFD-PPa NPs by dynamic light scattering
method
As can be seen from Fig. 2.1., the dynamic light scattering
method didn’t show the formation of the NPs for Val-C60-PPa (com-
pound 6) in pyridine. Nevertheless, as will be discussed further, we
believe that fullerene derivatives under study form NPs even in the
pyridine solution, but their size less than the sensitivity of the
applied method (size < 5 nm).
As discussed in a literature review, AFDs tend to aggregate in an
aqueous medium due to the association of hydrophobic C60 core,
forming nanoparticles. Using dynamic light scattering, it was found
that the amino acid derivative of fullerene 1 and covalent dyads 6,
7, and 8 in aqueous solutions form supramolecular structures: 10–
20 nm as for AFD and 20–42 nm – for the dyads (Fig. 2 and Table 1).
Thus, such water-soluble NPs, obtained by dialysis method, have a
small size (up to 42 nm) with a relatively narrow size distribution.
4.4. Computational simulation of dyads structures
Conformations of dyad 6 as an example of AFD-PPa structures
are presented in Fig. 3. It can be seen that the chlorin fragment
in the dyad is located in a plane perpendicular to the surface of
the fullerene spheroid. The distances between the dye and the full-
erene core in dyad 6 were calculated as 3.8 Å (between the closest
atoms of fullerene and dye) and 8 Å (between the centers of fuller-
ene and dye).
Such a «perpendicular» spatial position of the fullerene core rel-
ative to the dye macrocycle plane could also enhance aggregation
processes, as it was shown for another fullerene-chlorine dyad
with a similar spatial configuration [66].
The goal of computational simulation of dyad structures in the
present work was to make an estimation of a distance between the
fullerene core and the dye and their spatial arrangement. This
rough estimation was made by a relatively simple PM7/COSMO
parametrization method. We want to note that it is possible to cal-
culate absorbance spectra and electron-transfer parameters for
fullerene-dye dyads, although it requires the use of significantly
more complex DFT-based methods that are taking into account
the solvent influence, as was shown for fullerene-porphyrin struc-
tures in [67].
Based on steady-state fluorescence measurements, we can cal-
culate the fluorescence decay lifetime of PPa moiety in the dyads,
given that the lifetime of the original PPa is 1.66 ns [71]. Thus, flu-
orescence decay lifetime of PPa moiety in 6, 7 and 8 were, corre-
spondently, 18, 7.1, and 55 ps. As a result, we can estimate the
electron transfer constants ket in these conjugates, which are in
the range (1.8–14)ꢂ1010 sꢁ1
.
On the other hand, we can estimate the electron transfer con-
stant ket from the formula
ket ¼ k0expðꢁ
aRÞ
ð1Þ
which was obtained as a result of generalizing the electron transfer
data in various molecular structures, including photoexcited mole-
cules [72–76]. Here, k0 = 1013 sꢁ1, R(Å) is the distance between the
4.5. Photophysical properties of AFD–PPa nanoparticles
An analysis of the absorption spectra of 6 dyad dissolved in pyr-
idine shows that upon conjugation of AFD with PPa, a significant
change in the absorption spectrum of the dye fragment in the dyad
occurs, accompanied by an extinction decrease in the Soret band
(407 nm) and Q-band, with a small bathochromic shift of the Q-
band (from 673 nm for PPa to 677 nm for 6, Fig. 4, A). A broad
shoulder was also observed in the dyad spectrum, increasing in
the blue region and in the near UV region, which could be
explained by the contribution of the fullerene fragment. In addi-
tion, in the Q-band region, a second band appears at a wavelength
of 712 nm; a possible reason for this may be the formation of inter-
molecular aggregates of these dyads in a pyridine solution. For
another fullerene-chlorine dyad with a similar spatial configura-
tion, a similar picture of the Q-band peak broadening was shown,
where it was explained by strong aggregation [66].
donor and acceptor (between the
p-orbitals of the dye and fullerene
core), is the parameter characterizing the overlap of the donor and
a
acceptor wave functions due to the superexchange interaction.
Depending on the type of matrix separating the donor and acceptor
(e.g., a saturated hydrocarbon chain, protein structure, or water
molecules), the value
a
can be different: 0.9 Åꢁ1 for saturated
hydrocarbon chains and 1.8 Åꢁ1 for water molecules [74].
The distances between structures of PPa and fullerene core in
dyads, as determined from dyad 6 structure (Fig. 3), are equal to
3.8 A (between the nearest atoms of the conjugated structures)
and 4.2 A (along the linker). Using the formula (1), we can calculate
ket = 2.8 1011 sꢁ1 in the case of electron transfer along saturated
hydrocarbon chains and ket = 1010 sꢁ1 for electron transfer through
the aqueous medium.
Based on these considerations, we can conclude that experi-
mentally estimated electron transfer constants are comparable to
theoretical one, and in dyads under study, electron transfer most
likely occurs, both along the linker chain between PPa moiety
A weak fluorescence signal of the dyad 6 was registered; its
intensity was 21 times lower than the PPa signal (Fig. 4B and
Table 1). The observed effect indicates effective quenching of the
5

A.Yu. Belik, A.Yu. Rybkin, N.S. Goryachev et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119885
Fig. 2. The hydrodynamic radius of NPs: dyad 6 in pyridine (1); AFD 1 in water (2); dyad 6 in water (3); dyad 7 in water (4); dyad 8 in water (5).
Fig. 3. Calculated conformation of dyad 6.
6

A.Yu. Belik, A.Yu. Rybkin, N.S. Goryachev et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119885
Fig. 4. Absorption (A) and fluorescence (B) spectra of obtained compounds: PPa (1), 6 (2), and 1 (3). All in pyridine, concentration 10 lM, kex = 417 nm.
Fig. 5. Absorption (A) and fluorescence (B) spectra of obtained compounds: Ce6 (1), 1 (2), 6 (3), 7 (4), 8 (5). All compounds dissolved in water, concentration 10
kex = 410 nm.
lM,
and fullerene core and along the direct shortest line connected the
-orbitals of the dye and fullerene.
ronment can change, which will significantly change the
p
conditions of charge transfer and the efficiency of the photochem-
ical reaction as a whole [78,79]. It was found that all dyads in lipo-
somes had an almost equal activity of Oꢀ2- generation, exceeding
that of Ce6 by 3.5–4.0 times, or 5–11 times per absorbed quantum
of light (Fig. 6B and Table 1).
4.6. Photodynamic activity of nanoparticles in water and in the
structure of liposomes
A study of the singlet oxygen 1O2 generation in liposome solu-
tions showed that the dyad’s photoactivity was significantly lower
(by 8–12 times) than that of Ce6. However, due to the low absorp-
tion in the red region, the normalized photoactivity of 6, 7, and 8
dyads were only 3.3–5.9 times lower (Fig. 7 and Table 1).
Comparing the activity of Oꢀ2- b 1O2 generation in liposome med-
ium by dyads under study, we can conclude that the transfer of
energy from an excited molecule to oxygen occurs mainly by the
electron transfer mechanism, realizing I type of photochemical
reaction. Thus, for all dyads under study, photodynamic mecha-
nism switching from type II (singlet oxygen generation by Ce6) to
I type (superoxide generation by dyads) was demonstrated. Facili-
tation of the type I photodynamic mechanism could be perspective
against hypoxic tumors.
The photochemical activity study of AFD-PPa NPs in water was
evaluated by the generation of superoxide anion Oꢀ2- and singlet
oxygen 1O2 using the NBT and DPBF probes, correspondingly. The
activity of generating ROS conjugates was compared with the cor-
responding activity of chlorin e6, a commonly used PS. PPa and Ce6
both have a pronounced absorption in the Q-band region of the
spectrum and a high quantum yield of singlet oxygen generation
(0.52 for PPa in DMF [69] and 0.7 for Ce6 in water [77]).
It has been shown that water-soluble nanoassociates exhibit
photochemical activity that significantly exceeds the generation
activity of Oꢀ2- by Ce6, while the conjugate 7 is most effective. Its
effect is 3.6 times greater than the effect of Ce6 (Fig. 6). The photo-
chemical activity of the dyads 6 and 8 also exceeds the activity of
free chlorin by 2.4 and 2.7 times (Table 1). It should be noted that
the photochemical activity was compared at the same concentra-
tion of compounds, while their absorption in the excitation region
(at k > 630 nm) is slightly different for dyads under study.
In this regard, the relative photochemical activity was esti-
mated, calculated per one quantum of absorbed light, taking into
account the absorption of compounds in the band 630–800 nm.
It was found that the activity of 7 exceeds the activity of Ce6
10.2 times by one absorbed quantum, while the activity of 6 and
8 – exceeds 4.8 and 3.7 times (Table 1).
4.7. Photodynamic action of NPs on tumor cells HeLa
The photodynamic effect of NPs 6, 7, 8, and Ce6 as a reference
was studied in HeLa cells, as it was described in Section 2.6. It
was found that irradiation in the region > 630 nm alone does not
cause cell death, due to the absence of compounds with a photody-
namic effect, both in the cells themselves and in the DMEM
medium.
A comparison was also made of the photochemical activity of
Oꢀ2- generation for these nanoparticles upon their introduction into
the liposome solution. In this case, due to the lipophilicity of the
dyads, they can enter the liposome structure in the form of individ-
ual compounds, while their structure and the polarity of the envi-
The test substances were applied to a plate with HeLa cells
under a lamp with an orange filter that cuts off light in the region
of the Soret band of dye absorption (<420 nm) in order to eliminate
the manifestation of an additional photodynamic effect from sun-
light or fluorescent lamps. Amino acid fullerene derivative did
7

A.Yu. Belik, A.Yu. Rybkin, N.S. Goryachev et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119885
Fig. 6. Kinetics of superoxide Oꢀ2- formation sensitized by compounds in water (A) and in the liposomes (B): 6 (1), 7 (2), 8 (3), Ce6 (4), 1 (5), control (6). Concentration 2
lM.
Fig. 8. Phototoxicity of compounds under study in HeLa cancer cell with 30 min
irradiation of the red light (solid line) and in the dark (dash line): Ce6 (1); 6 (2), 7
(3), 8 (4).
Fig. 7. Kinetics of singlet oxygen 1O2 generation, sensitized by the following
compounds in liposome medium: Ce6 (1), (2), (3), (4), control (5).
Concentration 2 M.
6
7
8
l
5. Conclusion
not exhibit dark toxicity, as well as recorded photodynamic effects
under the experimental conditions, which is associated with its
extremely low absorption in the irradiation region (k > 630 nm),
and, therefore, the weak ability to generate both superoxide and
singlet oxygen or other types of ROS when irradiated.
It is important to note that all conjugates 6, 7, and 8 do not have
any noticeable dark toxicity (Fig. 8), which is quite typical for most
fullerene derivatives [41,80].
A number of water-soluble covalently-linked dyads were syn-
thesized by the original inexpensive method based on the conjuga-
tion of amino acid fullerene C60 derivatives with the dye –
pyropheophorbide a.
With the formation of dyads, a significant decrease in the
absorption of the dye in its structure is observed, as well as strong
fluorescence quenching of the dye. It is shown that these dyads
form photoactive water-soluble nanoparticles.
All the studied dyads have similar phototoxicity, which was sig-
nificant, 10–14.5 times lower than that of Ce6 (Fig. 8 and Table 1).
Given the lower absorption of dyads in comparison with Ce6 in the
excitation region, the decrease in phototoxicity is 4–7 times. Under
the experiment conditions, the photodynamic effect on the cells
could provide only such compounds that either penetrate the cells
or interact with their membranes. If the studied conjugates pene-
trate and accumulate in cells weaker than the comparison com-
pound Ce6, this will significantly reduce their observed
photodynamic activity.
Based on the data on the photodynamic activity of dyads in cells
and the structure of liposomes (Table 1), it can be concluded that
singlet oxygen, rather than superoxide anion radical, exhibits the
greatest cytotoxic effect. As a result, AFD-PPa dyads under study,
that generate ROS predominantly by type I mechanism were less
effective under conditions of oxygen saturation; however, they
could be effective under conditions of hypoxia (e.g. low oxygen
concentration), where type II photochemical reaction is signifi-
cantly hampered.
It was found that, depending on the structure, NPs in liposomes
generate superoxide anion radical 5–11 times more effective, cal-
culated per quantum of absorbed light, than chlorin e6, and at
the same time, their quantum yield of singlet oxygen decreases
by 3–6 times compared to chlorin e6. Various attached amino acids
give the structures a slight water solubility and do not significantly
affect the photophysical properties of nanoparticles.
The obtained nanoparticles have low toxicity in the dark; at the
same time, the phototoxicity of dyads is inferior to the activity of
chlorin 10–14.5 times. Based on the obtained data, we can con-
clude that the newly synthesized dyads predominantly enhance
photodynamic reactions by type I mechanism (superoxide genera-
tion), while under normal conditions, cells are damaged by type II
mechanism (singlet oxygen generation).
Further development of such type of photoactive fullerene-dye
dyads should be aimed at the enhancement of type I mechanism,
reducing the degree of aggregation of the photoactive molecules
in water-soluble nanoparticles and maintaining their absorption
in the Q-band excitation region. We believe that the proposed
8

A.Yu. Belik, A.Yu. Rybkin, N.S. Goryachev et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 260 (2021) 119885
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CRediT authorship contribution statement
activity,
dyepig.2020.108411.
Dye
Pigment
108411
(2020),
https://doi.org/10.1016/j.
A.Yu. Belik: Supervision, Validation, Writing - original draft,
Writing - review & editing. A.Yu. Rybkin: Investigation, Methodol-
ogy, Writing - original draft. N.S. Goryachev: Investigation, Data
curation, Formal analysis. A.P. Sadkov: Investigation. N.V. Fila-
tova: Investigation. A.G. Buyanovskaya: Investigation. V.N. Tala-
nova: Investigation. Z.S. Klemenkova: Investigation, Validation.
V.S. Romanova: Conceptualization, Investigation. M.O. Koifman:
Supervision, Investigation. A.A. Terentiev: Investigation, Valida-
tion. A.I. Kotelnikov: Conceptualization, Methodology, Validation.
[13] D. Kuciauskas, S. Lin, G.R. Seely, A.L. Moore, T.a. Moore, D. Gust, et al., Energy
and photoinduced electron transfer in porphyrinꢁfullerene dyads, J. Phys.
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Declaration of Competing Interest
The authors declare that they have no known competing finan-
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Acknowledgments
Studies were funded by the Ministry of Science and Education
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