A novel bacteriochlorin-styrylnaphthalimide conjugate for simultaneous photodynamic therapy and fluorescence imaging

Article abstract of DOI:10.1039/c7cp04449f

Propargyl-15²,17³-dimethoxy-13¹-amide of bacteriochlorin e (BChl) and a 4-(4-N,N-dimethylaminostyryl)-N-alkyl-1,8-naphthalimide bearing azide group in the N-alkyl fragment were conjugated by the copper(i)-catalyzed 1,3-dipolar cycloaddition to produce a novel dyad compound BChl-NI for anticancer photodynamic therapy (PDT) combining the modalities of a photosensitizer (PS) and a fluorescence imaging agent. A precise photophysical investigation of the conjugate in solution using steady-state and time-resolved optical spectroscopy revealed that the presence of the naphthalimide (NI) fragment does not decrease the photosensitizing ability of the bacteriochlorin (BChl) core as compared with BChl; however, the fluorescence of naphthalimide is completely quenched due to resonance energy transfer (RET) to BChl. It has been shown that the BChl-NI conjugate penetrates into human lung adenocarcinoma A549 cells, and accumulates in the cytoplasm where it has a mixed granular-diffuse distribution. Both NI and BChl fluorescence in vitro provides registration of bright images showing perfectly intracellular distribution of BChl-NI. The ability of NI to emit light upon excitation in imaging experiments has been found to be due to hampering of RET as a result of photodestruction of the energy acceptor BChl unit. Phototoxicity studies have shown that the BChl-NI conjugate is not toxic for A549 cells at tested concentrations (<8 μM) without light-induced activation. At the same time, the concentration-dependent killing of cells is observed upon the excitation of the bacteriochlorin moiety with red light that occurs due to reactive oxygen species formation. The presented data demonstrate that the BChl-NI conjugate is a promissing dual function agent for cancer diagnostics and therapy.

Full text of DOI:10.1039/c7cp04449f

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M. A. Grin, O. Fedorova, M. A. Zakharko, D. A. Pritmov , A. F. Mironov, A. N. Arkhipova, Y. V. Fedorov, G.
Jonusauskas, R. I. Yakubovskaya, N. B. Morozova, A. A. Ignatova and A. Feofanov, Phys. Chem. Chem.
Phys., 2017, DOI: 10.1039/C7CP04449F.
Volume 18 Number
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Physical Chemistry Chemical Physics
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Novel Bacteriochlorin–Styrylnaphthalimide Conjugate for Simultaneous
Photodynamic Therapy and Fluorescence Imaging
a,b,*
c
a,b
a
Pavel A. Panchenko,
Mikhail A. Grin, Olga A. Fedorova, Marina A. Zakharko,
c
c
a
a
Dmitriy A. Pritmov, Andrey F. Mironov, Antonina N. Arkhipova, Yuri V. Fedorov,
d
e
e
Gediminas Jonusauskas, Raisa I. Yakubovskaya, Natalia B. Morozova,
f,g
f,g
Anastasia A. Ignatova and Alexey V. Feofanov
a
A. N. Nesmeyanov Institute of Organoelement Compounds of Russian Academy of
Sciences, 119991, Vavilova str. 28, Moscow, Russia,
Tel.: +7 499 135 80 98, Fax: +7 499 135 50 85, Eꢀmail: pavel@ineos.ac.ru
b
D. Mendeleev University of Chemical Technology of Russia,
1
25047, Miusskaya sqr. 9, Moscow, Russia,
Moscow Technological University, Institute of Fine Chemical Technologies,
19571, Prospect Vernadskogo, 86, Moscow, Russia,
Laboratoire Ondes et Matière d’Aquitaine, UMR CNRS 5798,
Bordeaux University, 33405, 351 Cours de la Libération, Talence, France,
c
1
d
e
Р. Hertsen Moscow Oncology Research Institute – Branch of the National Medical Research
Radiological Centre of the Ministry of Health of the Russian Federation,
nd
1
25284, 2 Botkinskiy pr. 3, Moscow, Russia,
f
Biological Faculty, Lomonosov Moscow State University,
19992, Leninskie Gory 1, Moscow, Russia.
1
g
ShemyakinꢀOvchinnikov Institute of Bioorganic Chemistry of Russian Academy of
Sciences, 117997, MiklukhoꢀMaklaya str., 16/10, Moscow, Russia
Abstract
Propargylꢀ15 ,17 ꢀdimethoxyꢀ13 ꢀamide of bacteriochlorin
2
3
1
e
(BChl) and 4ꢀ(4ꢀN,Nꢀ
dimethylaminostyryl)ꢀNꢀalkylꢀ1,8ꢀnaphthalimide bearing azide group in the Nꢀalkyl fragment
were conjugated by the copper(I)ꢀcatalyzed 1,3ꢀdipolar cycloaddition to produce novel dyad
compound BChlꢀNI for anticancer photodynamic therapy (PDT) combing the modalities of a
photosensitizer (PS) and a fluorescent imaging agent. A precise photophysical investigation of
the conjugate in solution using steadyꢀstate and timeꢀresolved optical spectroscopies revealed
that the presence of naphthalimide (NI) fragment does not decrease the photosensitizing ability
of the bacteriochlorin (BChl) core as compared with BChl, however, the fluorescence of
naphthalimide is completely quenched due to resonance energy transfer (RET) to BChl. It has

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Physical Chemistry Chemical Physics
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been shown that conjugate BChlꢀNI penetrates into human lung adenocarcinoma A549 cells,
accumulates in the cytoplasm where it has a mixed granularꢀdiffuse distribution. Both NI and
BChl fluorescence in vitro provides registration of bright images showing perfectly intracellular
distribution of BChlꢀNI. The ability of NI to emit light upon excitation in imaging experiments
has been found to be due to hampering of RET as a result of photodestruction of energy acceptor
BChl unit. Phototoxicity studies have shown that conjugate BChlꢀNI is not toxic for A549 cells
at tested concentrations (<8 ꢁM) without lightꢀinduced activation. At the same time, the
concentrationꢀdependent killing of cells is observed upon the excitation of bacteriochlorin
moiety with the red light that occurs due to reactive oxygen species formation. The presented
data demonstarte that conjugate BChlꢀNI is a promissing dual function agent for cancer
diagnostics and therapy.
Keywords
Photodynamic therapy, naphthalimide, bacteriochlorin, click chemistry, fluorescent imaging,
photosensitizer, resonance energy transfer
Graphical Abstract
Textual abstract for the table of contents entry
Photosensitizing and fluorescence imaging ability of bacteriochlorin–naphthalimide conjugate is
studied

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1. Introduction
Photodynamic therapy (PDT) has emerged as an important treatment modality for a variety of
1,2
cancers in recent years. PDT involves three necessary components: light, photosensitizer (PS),
and tissue molecular oxygen. Upon irradiation by light of appropriate wavelength, PS is
3
promoted to a short lived singlet state which rapidly converts to excited triplet state ( PS*)
3
through the process of intersystem crossing. Then, PS* transfers its excess energy to nearby O
2
1
molecules to form reactive oxygen species such as singlet oxygen ( O
2
) and free radicals, which
3–6
are toxic to malignant cells and tissues.
Among various photosensitizing agents, porphyrinꢀbased compounds possess unique
advantages related to their ability to be retained in tumors and to produce cytotoxic singlet
oxygen. Photofrin, porphyrinꢀtype PS of the first generation, has achieved some clinical efficacy,
but at the same time it suffered from several drawbacks such as chemical heterogeneity, low
depth of tissue treatment caused by limited light penetration due to absorption wavelength (630
nm) laying outside "phototherapeutic window" (650 – 1350 nm), low molar extinction
coefficient at 630 nm, and prolonged cutaneous photosensitivity caused by its slow elimination
7
in normal tissue. Compared to porphyrins, chlorinꢀ and bacteriochlorinꢀtype PSs in which one or
two pyrrole units (diagonal to each other) are reduced respectively exhibit intense absorption in
the near infrared region (≥ 650 nm), relatively low dark toxicities, rapid clearance from normal
8–13
tissue and low cutaneous phototoxicity.
Despite smaller synthetic availability, diꢀ and
tetrahydroporphyrins could be considered as one of the most promising candidates for creating
efficient secondꢀgeneration drugs for PDT of cancer.
An important characteristic of most of the porphyrinꢀbased photosensitizers is their
ability to fluoresce. This property has been extensively explored in preꢀclinical and clinical
studies for fluorescenceꢀimage guided PDT. Unfortunately, porphyrins, chlorins, and
bacteriochlorins display small Stokes shift between their longer wavelength absorption and
1
4–16
emission bands
and, therefore, their fluorescence is difficult to filter out of the scattered
excitation light. This property of the PS fluorescence complicates imaging of deeply seated and
large tumors. On the other hand, excitation of the porphyrinꢀbased photosensitizer ultimately
1
4–17
leads to the production of reactive oxygen species and related toxicity.
To overcome this difficulty a new approach which consists in the construction of
bifunctional systems combining modalities of PS and diagnostic agent (so called theranostics)
18–22
has been developed.
In such systems, fluorescent moiety can be excited selectively and
independently from the photosensitizer unit enabling fluorescence imaging without toxic effects.
Thus, Pandey and coworkers have used cyanine dyes as fluorescent labels to prepare conjugates
1
8–20
with PSs.
A distinct feature of the emission spectra of cyanine dyes is that their fluorescence

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23
can easily reach nearꢀinfrared (NIR) region upon increasing the length of polymethine chain.
However, polymethine cyanine dyes are not so easy to synthesize and to modify, their photoꢀ
stabilities are relatively low and their Stokes shift is usually less than 25 nm, which may cause
selfꢀquenching as well as the increase of measurement error due to detection of scattered
excitation, thus decreasing the detection sensitivity to a great extent as it is in the case of using
24,25
simple porphyrin sensitizers. Some examples of BODIPY–phthalocyanine
and rhodamine–
2
6,27
phthalocyanine
conjugates have also been described in literature. Despite the higher
photostability of BODIPY and rhodamine derivatives as compared with cyanine dyes, these
types of fluorophores also demonstrate small difference between absorption and emission
maxima. Therefore, NIR dyes with a larger Stokes shift are very promising for the development
of bifunctional conjugates for simultaneous photodynamic therapy and fluorescent imaging.
1
,8ꢀNaphthalimide derivatives are famous organic fluorophores which generally exhibit
high thermo and photostability and are known to act as fluorescent brighteners and dyes for
28,29
30,31
32–34
polymer fibers,
laser active media,
electroluminescent materials
and optical memory
3
5–37
devices.
Because of its intense fluorescence, large Stokes shifts along with the relative ease
of synthetic operations for targeted modification of the molecular structure, this type of
compounds has found application in the construction of fluorescent chemosensors for
38–40
biologically relevant cations and anions,
labels or probes for proteins, cells, lysosomes and
41–43
44–46
other acidic organelles.
In our recent papers,
we have described photophysical properties
th
of naphthalimides containing substituted styryl fragment as an electron releasing group at the 4
position of naphthalene ring. It has been found that the presence of 4ꢀ(N,Nꢀdimethylamino)styryl
group extends the πꢀsystem of the parent chromophore and results in the long wavelength
intramolecular charge transfer (ICT) absorption and emission, which is preferable for
fluorescence imaging. Herein, we report on the synthesis, spectroscopic investigation and
evaluation of PDT activity of novel conjugate of bacteriochlorin photosensitizer and 4ꢀ(N,Nꢀ
dimethylamino)styrylꢀ1,8ꢀnaphthalimide (BChlꢀNI, Scheme 1). The results and experimental
details of our study of optical properties of this molecular system in solution and in living cells
are presented. We report on distinct photoꢀinduced cytotoxicity of BChlꢀNI for human
adenocarcinoma A549 cells as well as redꢀtoꢀgreen conversion of BChlꢀNI fluorescence that can
be useful to control integrity of photosensitizer moiety during irradiation of cancer cells.

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2. Experimental procedures
Synthesis of conjugate BChlꢀNI. For the synthesis of BChlꢀNI, we used the wellꢀknown
copper(I)ꢀcatalyzed click reaction of 1,3ꢀdipolar cycloaddition between naphthalimideꢀ
containing alkylazide NI6 and triple bond derivative of bacteriochlorin e BChl (Scheme 1). The
latter was afforded from methyl ester of bacteriopheophorbide a BPheid and propargylamine.
Preparation of BPheid starting from biomass Rhodobacter capsulatus was carried out according
4
7,48
to described method.
The first step of the synthesis of naphthalimide NI6, in which
commercial starting material 4ꢀbromoꢀ1,8ꢀnaphthalic anhydride was reacted with ethanolamine,
49
was performed conveniently in EtOH at reflux. Next, 4ꢀbromnaphthalimide NI1 was subjected
5
0
to the Heck coupling reaction with 4ꢀN,Nꢀdimethylaminostyrene under catalyst Pd(OAc)
2
followed by the nucleophilic substitution of hydroxyl by azide group in the hydroxyethyl
fragment via subsequent treatment with POCl and NaN . Details of synthetic procedures and
3
3
identifications are shown in the Supplementary Information. Synthesis of Nꢀbutylꢀ4ꢀ
styrylnaphthalimide NI4 which was used for comparative analysis of spectral characteristics has
44
been described earlier.
Scheme 1. Synthesis of BChl, NI1–6 and BChl–NI
Steadyꢀstate optical measurements. The absorption spectra were taken on a Varianꢀ
Cary 5G spectrophotometer. The fluorescence quantum yield measurements were performed
using a VarianꢀCary 5G spectrophotometer and a FluoroLogꢀ3 spectrofluorimeter. Spectral
measurements were carried out in airꢀsaturated acetonitrile solutions (acetonitrile of
spectrophotometric grade, water content <0.005%, Aldrich) at 20 ± 1 ºC; the concentrations of
–5
studied compounds were of about 0.5–2.0ꢂ10 M. All measured fluorescence spectra were
fl
corrected for the nonuniformity of detector spectral sensitivity. Coumarin 481 in acetonitrile (
ϕ
5
1
=
0.08) was used as a reference for the fluorescence quantum yield measurements. The
5
2
fluorescence quantum yields were calculated by the Eq.(1),

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ꢄ (1 − 10ꢅꢆꢇ)ꢈ
ꢉ
(1)
ꢀꢁꢂ
= ꢀ ꢃꢁ ꢂ
∙
ꢅꢆ
ꢉ
ꢄꢃ (1 − 10 )ꢈ
ꢃ
wherein ꢀꢁꢂ and
ꢀ
ꢁꢂ
ꢃ
are the fluorescence quantum yields of the studied solution and the standard
and ꢊꢃ are the absorptions of the studied solution and the standard
and ꢄꢃ are the areas underneath the curves of the fluorescence spectra of the
studied solution and the standard respectively; and and ꢈꢃ are the refraction indices of the
compound respectively;
respectively;
ꢊ
ꢄ
ꢈ
solvents for the substance under study and the standard compound.
Quantum yields of singlet oxygen generation. The quantum yields of singlet oxygen
(
(
Φꢋ) were estimated in acetone by using tetraphenylporphirin (TPP) as a reference compound
Φ
ꢋꢌ ꢍꢍ= 0.7)53,54 and 1,3ꢀdiphenylisobenzofuran (DPBF) as a O
1
2
trap. In a typical experiment,
solutions (2.5 ml) containing both PSs (TPP and PS under study) and DPBF were placed into 1
cm rectangular spectroscopic cells and irradiated by monochromatic light (510 nm) using the
excitation unit (a xenon lamp and excitation monochromator) of the FluoroLogꢀ3 fluorimeter.
The initial concentration of DPBF corresponded to absorption of about 1 at 414 nm (~40 mM).
Identical initial DPBF concentrations were used for the reference solution of TPP and the
samples. The change in DPBF absorption at 414 nm was recorded as a function of irradiation
time via UV/Vis spectrometry. The values of Φꢋ were calculated by the Eq.(2),
ꢅꢆꢇ
ꢎ 1 − 10
(2)
ꢃ
ꢋ
Φ = Φ
ꢋ
∙
ꢎꢃ 1 − 10ꢅꢆ
wherein
reference compound ( ꢃ
nm versus irradiation time;
ꢎ
and ꢎꢃ are the bleaching rates of the solutions containing PS under study (
ꢎ
) and
ꢎ
) which were found from the slopes of linear plots of absorption at 414
and ꢊꢃ are absorption values at excitation wavelength (510 nm) of
ꢊ
the solutions containing studied PS and reference compound (TPP) respectively. The accuracy of
Φꢋ estimation was about 10%.
Subpicosecond transient absorption setup. A Ti:sapphire laser system output (0.6 mJ
and 30 fs at 800 nm and 1 kHz pulse repetition rate (Femtopower Compact Pro)) was split in two
parts, producing the pump and the probe beams. 80% of pulses were used to pump an optical
parametric generator (TOPAS (Light Conversion)) to generate the wavelength tuneable pump
pulse. Following TOPAS, the harmonic generation or frequency mixing in a nonlinear crystal
produced the excitation pulses in the range 250 – 2600 nm. The probe was a white light
continuum pulse, extending from 390 nm to 900 nm, generated by focusing the 800 nm pulses
2
(~5 ꢁJ per pulse) into a 5 mm thick D O cell. The variable delay time between excitation and
probe pulses was obtained by using a delay line with 0.1 mm resolution. The solutions were
placed in a 1 mm circulating cell. Whitelight signal and reference spectra were recorded with a
twoꢀchannel fiber spectrometer (Avantes Avaspecꢀ2048ꢀ2). A homeꢀwritten acquisition and

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experimentꢀcontrol program in LabView made it possible to record transient spectra with an
–
3
average error of less than 10 of O.D. The temporal resolution of our setup was better than 60 fs.
Temporal chirp of the probe pulse was corrected by a computer program with respect to a
Lawrencian fit of a Kerr signal generated in a 0.2 mm glass plate used in a place of the sample.
Nanosecond transient absorption setup. A frequency tripled Nd:YAG amplified laser
system (30 ps, 30 mJ @1064nm, 20 Hz, Ekspla model PL 2143) output was used to pump an
optical parametric generator (Ekspla model PG 401) producing tunable excitation pulses in the
range 410 – 2300 nm. The residual fundamental laser radiation was focused in a high pressure
Xe filled breakdown cell where a white light pulse for sample probing was produced. All signals
were analyzed by a spectrograph (Princeton Instruments Acton model SP2300) coupled with a
high dynamic range streak camera (Hamamatsu C7700, 1nsꢀ1ms). Accumulated sequences
(sample emission, probe without and with excitation) of pulses were recorded and treated by
HPDTA (Hamamatsu) software to produce twoꢀdimentional maps (wavelength versus delay) of
transient absorption intensity in the range 300 – 800 nm. Typical measurement error was better
–3
than 10 O.D.
Fluorescent imaging and procedure of photodynamic treatment of tumor cells.
Human lung adenocarcinoma A549 cells were grown (37 °C, 5% CO ) in Eagle’s minimum
2
essential medium with phenol red, 8% fetal calf serum, 2 mM Lꢀglutamine (so called complete
medium). The cells were subcultured twice per week. On the day prior to an experiment, an
exponentially growing cells were plated on a round cover glasses placed in 24ꢀwell plates (for
microscopic experiments) or seeded directly in 96ꢀwell plates (for cytotoxicity measurements).
4
Sowing density was 5×10 cells per ml.
For microscopic experiments cells were incubated with 1–8 ꢁM of conjugate BChlꢀNI
for 1–3 h in a complete medium at 37 °C. Detailed intracellular distribution of conjugate was
studied with the Leica SP2 confocal laser scanning microscope (Leica, Germany). Conjugate
fluorescence was excited with 488 nm (fits well to the absorption band of NI chromophore) and
5
14 nm (fits well to the absorption band of BChl chromophore) light. An emission in the 500–
00 nm spectral range corresponding to NI unit was registered with a photomultiplier. An
6
emission in the range 730–1000 nm corresponding to PS was registered with a highly sensitive
avalanche photodiode.
Intracellular formation of photoinduced reactive oxygen species (ROS) was measured
using 2',7'ꢀdichlorofluorescein diacetate (DCFDA). Cells were incubated with 1 µM BChlꢀNI for
−
2
3
h and during last 30 min with 25 µM DCFDA. Then cells were irradiated (22 ± 2 mW cm )
with a halogen lamp (500 W) through a water filter (thickness of 5 cm) and a bandꢀpass filter
transmission 660–1000 nm) for 15 min and analyzed by confocal microscopy. Fluorescence was
(

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excited with 488 nm and registered in the 500–540 nm (DCFDA) and 600–650 nm (NI) regions.
Cells incubated with DCFDA and BChlꢀNI without irradiation as well as cells incubated with
BChlꢀNI and irradiated without DCFDA were used as a control.
To clarify the cellular mechanisms of BChlꢀNI transport, cells were thoroughly washed
with a warm medium (37 °C) without serum, preꢀincubated with chlorpromazine (30 ꢁM for 30
min), or methylꢀβꢀcyclodextrin (4 mM for 30 min) in a serumꢀdeprived medium (37 °C) and
further incubated in the presence of BChlꢀNI (4 ꢁM for 70 min) at 37 °C. To study influence of
temperature, cells were incubated with BChlꢀNI (4 ꢁM for 70 min) at 8 °C.
In the confocal microscopy measurements, lateral and axial resolutions were ca. 0.2 and
1.5 µm respectively. Confocal microspectroscopy studies were performed at the 488 nm
55
excitation using an experimental setup described elsewhere.
For cell survival studies, conjugate BChlꢀNI was added to cells (0.04–8 ꢁM with a twoꢀ
fold increment). Control cells were incubated with equivalent concentrations of Cremophor EL.
The cytotoxicity was estimated after incubation of cells with conjugate or Cremophor EL alone
for 6 h in the dark. The photoinduced cytotoxicity was measured on cells incubated with
conjugate or Cremophor EL alone for 3 h and irradiated as described above. After irradiation the
cells were further incubated for 3 h and examined for viability. All the experiments were
performed in duplicate. To evaluate cell viability, cells were stained with Hoechst 33342 (stains
all cells) and propidium iodide (stains dead cells) and analyzed with a fluorescence microscope
56,57
as described elsewhere.
Irradiation of control cells without BChlꢀNI did not induce any
cytotoxicity at the irradiation conditions used.

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3. Results and discussion
Steadyꢀstate spectroscopic properties. Absorption and emission characteristics of
conjugate BChlꢀNI and monochromophoric derivatives BChl and NI4 are presented in Fig.1 and
Table 1. As it can be seen from Fig.1a,c, absorption spectrum of BChlꢀNI shows the presence of
four bands with maxima at 354, 481, 515 and 747 nm corresponding to electronic transitions in
naphthalimide (481 nm) and bacteriochlorin (354, 515 and 747 nm) moieties. Although the
position and intensity of characteristic Q and Soret bands of bacteriochlorin core in the conjugate
are essentially the same as those in equimolar mixture of BChl and NI4 (Fig.1c), naphthalimide
ICT absorption is observed at slightly higher wavelengths. This red shift could be attributed to
incorporation of electron withdrawing triazole group in the Nꢀbutyl substituent of compound
NI4, which enhances the ICT.
0
0
0
0
,3
,2
,1
,0
a
b
1
0
0
0
0
,0
,8
,6
,4
,2
BChL
BChL
NI4
A
NI4
0,0
550
3
00
400
500
600
700
800
600
650
700
750
800
Wavelength / nm
Wavelength / nm
0,4
0,3
0,2
0,1
0,0
c
d
6
6
6
6
6
6
6
x10
BChl + NI4
BChl + NI4
BChL-NI
5x10
BChl-NI
A
4x10
3x10
2x10
1x10
0
5
3
00
400
500
600
700
800
50
600
650
700
750
800
Wavelength / nm
Wavelength / nm
Fig.1. UV/Vis absorption (a,c) and fluorescence emission (b, d) spectra of compounds BChl, NI4, BChlꢀNI and
equimolar mixture of BChl and NI4 (denoted as «BChl + NI4») in acetonitrile. Excitation wavelength is 460 nm for
–
6
NI4, BChlꢀNI, BChl+NI4 and 515 nm for BChl. Concentration of all compounds – 4.7ꢂ10 M.
a
Table 1. Photophysical characteristics of compounds BChl, NI and BChlꢀNI in acetonitrile.
Intramolecular RET in BChlꢀNI
Förster theory
Experiment
ꢑꢓꢔ
ꢁꢂ
ꢀꢁꢂ
–1
–1
ꢏ
/nm
ꢏ
(ꢏ )/nm
ꢗꢃꢖꢌꢘ/ s
Φꢃꢖꢌꢘ ꢗꢃꢖꢌꢘ/ s
Φꢃꢖꢌꢘ Φꢃꢖꢌꢉ Φ (λex/nm)
ꢋ
ꢐꢑꢒ
ꢐꢑꢒ ꢕꢒ
NI4
462
353; 515;
46
BChlꢀNI 354; 481;
743 (460)
0.032
0.016
–
–
–
–
–
–
–
–
–
–
–
BChl
755 (515)
0.79 (510)
0.82 (510)
7
1
2
12
755 (460)
0.023
0.57ꢂ10
0.995
1.89ꢂ10
0.999 0.981
5
15; 747
a
Quantum yield of generation of singlet oxygen (Φ ) is measured in acetone.
ꢋ

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Fluorescence emission maxima of BChl and NI4 appear at close λ values, however,
naphthalimide spectrum is much more broad and substantially drops into IR region (Fig.1b). In
the case of equimolar mixture, excitation with 460 nm light which is mostly absorbed by NI4
results in the emission band having the same feature as that of individual naphthalimide dye but
interfered to some extent by the sharp collateral peak of the BChl fluorescence (Fig.1d).
Interestingly, the spectrum of conjugate BChlꢀNI where two photoactive units are covalently
linked is found to be different (Fig.1d). Under the excitation at 460 nm, BChlꢀNI demonstrates
only the characteristic band of bacteriochlorin, whereas naphthalimide fluorescence is
completely quenched. This observation implies that in the conjugate, efficient energy transfer
from the naphthalimide to bacteriochlorin fragment may occur. To get deeper insight into the
nature of processes in the excited state of BChlꢀNI molecule we further studied transient
absorption (TRABS) spectra of this system.
b
0
.00
ꢀ0.05
ꢀ0.10
ꢀ0.15
ꢀ0.20
ꢀ0.25
ꢀ0.30
0.53 ps
4
.1 ps
ꢀ
1
0
1
2
3
4
5
6
7
8
9
Time delay / ps
Fig.2. TRABS spectral map with subpicosecond time resolution (a) and TRABS kinetics at 750 nm (b) of BChlꢀNI
in acetonitrile. Excitation wavelength – 470 nm.
Transient absorption spectroscopy. A representative result of a pumpꢀprobe experiment
for BChlꢀNI, i.e. a map of absorbance changes after the laser pulse in delayꢀwavelength
coordinates is displayed in Fig.2a. The excitation of naphthalimide chromophore with 470 nm
light produced the negative signal at 750 nm. In accordance with the position of the long
wavelength absorption band of BChl (Fig.1a), this signal can be ascribed to the ground state
*
bleaching of bacteriochlorin in the conjugate. Another feature of the TRABS map shown in
Fig.1a is a broad positive band stretching from 500 to 680 nm. Probably, this spectral region
1
covers the area where absorption signals of singlet (S ) states of both chromophores are located.
*
Apparently, the contribution of stimulated emission to the negative signal at 750 nm is relatively small, because of
a high value of radiative lifetime (ꢙ ) of bacterioclorin. Radiative lifetime is known to be the ratio of excited singlet
ꢚ
state lifetime to the fluorescence quantum yield. From the data presented in Fig.2 and Table 1, ꢙ = 2ꢜns⁄0.016 =
ꢛ
1
25ꢜns. Such high ꢙ value explains low radiative rate constant (ꢗ = 1/ꢙ ) and low propability of stimulated
ꢚ
ꢚ
ꢚ
emission as a result.

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One can also see that the appearance of negative band on a time scale occurs not immediately
after the excitation pulse and proceeds with a characteristic time of 0.53 ps as obtained from the
†
TRABS time profile (Fig.2b). Hence, the initially formed naphthalimide excited state should
participate in a kind of a fast nonꢀradiative process more likely to be the resonance energy
transfer (RET). The rate constant of RET (ꢗꢃꢖꢌꢘ) can be estimated from the known characteristic
time as 1/0.53 ∙ 10ꢅꢘꢉꢜs (see Table 1 for the value of ꢗꢃꢖꢌꢘ). Quantum efficiency of
intramolecular RET (Φꢃꢖꢌꢘ) is expressed as the ratio of ꢗꢃꢖꢌꢘ to the sum of rate constants of all
other possible processes in the S
1
state of a donor:
ꢗꢃꢖꢌꢘ
ꢗꢃꢖꢌꢘ
ꢗꢃꢖꢌꢘ + ꢙꢞ,ꢟꢅꢘ
(3)
Φꢃꢖꢌꢘ
=
=
ꢗꢃꢖꢌꢘ + ꢗ + ꢗ
ꢚ
ꢝꢚ
In the Eq.(3) ꢗꢚ is the radiative rate constant of naphthalimide chromophore, and ꢗꢝꢚ describes
its nonꢀradiative relaxation which is not related to energy transfer. To estimate the sum (ꢗꢚ +
ꢗꢝꢚ) we used the value inversely proportional to the fluorescence lifetime of compound NI4
44
(
ꢙꢞ,ꢟ = 0.38ꢜns), where RET process is not realized. The obtained value of Φꢃꢖꢌꢘ was found
to be close to unity (Table 1) indicating highly efficient RET in the conjugate BChlꢀNI.
5
8
Additionally, calculations of ꢗꢃꢖꢌꢘ and Φꢃꢖꢌꢘ by the Förster model testify to the same result.
The details of these calculations are shown in Supplementary Information.
Further evolution of TRABS signals of bacteriochlorin unit in BChlꢀNI can be followed
by using the spectral map with nanosecond resolution (Fig.3a). The horizontal cuts of this map at
a given time delay give us the TRABS spectra at these delays. The spectrum immediately after
excitation is shown blue in Fig. 3b. In this spectrum, we can see broad absorption band of
bacteriochlorin singlet state (several S
1
n
→S transitions could contribute) in the range of 400ꢀ650
‡
nm formed as a result of energy transfer and negative features at 515 and 700 nm corresponding
to the ground state bleaching. Kinetics of absorption at 600 nm (Fig.3c) clearly shows that S
state relaxes within 2 ns at which it converts to some longꢀlived species presumably regarded as
triplets. Thus, spectrum after 40 ns (Fig.3b, red line) shows us absorption of T state.
1
1
The lifetime of bacteriochlorin triplet state was found to be dramatically affected by the
presence of oxygen. Whereas the degased solution exhibited single exponential decay with the
time constant of 19 ꢁs ( ꢌ
ꢙ
), in the case of air saturated sample, the observed lifetime was 0.355
ꢠꢡ
ꢌ
ꢠꢡ
ꢌ
ꢁs (ꢙ
) (Fig.3c,d). Considering the difference in ꢙꢌ and
ꢙ
values, one can conclude that the
†
Characteristic time ꢙ = 4.1ꢜps shown in Fig.2b describes the relaxation of the singlet excited state of
ꢉ
1 n
bacteriochlorin. Probably, positive signal of S →S absorption has nonꢀzero intensity at around 750 nm which tends
to decrease upon increasing of time delay. Thus, negative signal at 750 nm is becoming more and more negative
even after fast process with ꢙ = 0.53ꢜps is completed.
ꢘ
‡
Energy transfer process is not visible in this map; it can be perceived with the aid of the subpicosecond experiment
described above.

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b
0
ns
0
0
0
0
.15
.10
.05
.00
4
0 ns
ꢀ0.05
4
00
450
500
550
600
650
700
Wavelength / nm
d
c
oxygenated solution
exponential fit
degased solution
1.0
0.8
0.6
0.4
0.2
0.0
degased solution
exponential fit
lg ꢀA
2
ns
1
9 ꢁs
1
9 ꢁs
0
.01
0
.355 ꢁs
0.001
0
10
20
30
40
0
20
40
60
80
100
120
Time delay / ns
Time delay / ꢁs
Fig.3. TRABS spectral map (a) with nanosecond time resolution, TRABS spectra at different time delays (b) and
TRABS kinetic curves at 600 nm in the presence and absence of oxygen (c, d) of BChlꢀNI in acetonitrile.
Excitation wavelength – 440 nm. Spectral map has negative perturbed signal at 440 nm induced by the filter
rejecting this wavelength.
excitation energy is transferred from PS unit to molecular oxygen upon collisions which leads to
1
formation of O
2
and PS in the ground state. The efficiency of this process given as Φꢃꢖꢌꢉ was
calculated by the Eq.(4).
ꢠꢡ
ꢌ
ꢙ
(4)
Φꢃꢖꢌꢉ = 1 −
ꢙꢌ
As evident from the obtained value of Φꢃꢖꢌꢉ (0.981, see Table 1), bacteriochlorin in BChlꢀNI
1
works as an excellent photosensitizer allowing generation of O
2
with a high quantum yield.
1
Photosensitizing activity. Singlet oxygen ( O
2
) is thought to be a key cytotoxic agent in
5
9,60
the photodynamic inactivation of living cells.
It is formed as a result of energy transfer from
1
photosensitizer triplet state to molecular oxygen. Photosensitized O
2
formation was studied
1
using 1,3ꢀdiphenylisobenzofuran (DPBF) as a singlet oxygen trap. Interaction of DPBF with O
2
53,54
is known to be purely chemical, resulting in formation of colorless endoperoxides.
This
process causes bleaching of the main maximum of DPBF at 414 nm. Fig. 4 illustrates one of the

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experiments on photosensitized bleaching of DPBF in a solution of the conjugate BChlꢀNI in
acetone under illumination with green (510 nm) light which is absorbed by both chromophores.
Rapid photobleaching of DPBF was also detected in solution of free bacteriochlorin BChl
(Fig.S24, Supplementary Information). This observation is consistent with the results of previous
studies which showed that metalꢀfree bacteriochlorins efficiently photosensitized singlet oxygen
6
1,62
formation in air saturated solutions.
In solution of the free naphthalimide dye,
photobleaching of the trap was not found (Fig.S25, Supplementaty Information). Quantum yields
of singlet oxygen production of BChl and BChlꢀNI under the excitation at 510 nm were found to
be 0.79 and 0.82 respectively (Table 1). Such close values indicate that photosensitizing activity
of the naphthalimide dye in the conjugate is, apparently, caused by efficient energy transfer from
the dye to the bacteriochlorin, which is in a good agreement with the above timeꢀresolved studies
of BChlꢀNI, and the presence of naphthalimide fragment in BChlꢀNI does not decrease the
1
ability of PS core to generate O
2
.
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
0 s
15 s
A
30 s
45 s
6
9
0 s
0 s
120 s
1
2
80 s
40 s
3
50
400
450
500
550
Wavelength / nm
Fig.4. Changes in the UV/Vis absorption spectrum of a mixed solution
–
6
–5
containing the conjugate BChlꢀNI (2.6ꢂ10 M) and DPBF (4.0ꢂ10 M) in
acetone upon irradiation at 510 nm.
In vitro studies of conjugate BChlꢀNI. As confirmed with confocal microspectroscopy
and confocal laser scanning microscopy, conjugate penetrates into cancer A549 cells (Fig.5,6).
Intracellular fluorescence spectra of BChlꢀNI were measured by confocal microspectrosopy with
the 488 nm excitation wavelength, which fitted well to the absorption band of the NI moiety of
the conjugate. Typical intracellular emission spectrum of BChlꢀNI is shown in Fig.5e. Its shape
and longꢀwavelength maximum (757 nm) correspond to the fluorescence of bacteriochlorin
moiety. Intense illumination of a cell with the 488 nm light leads to the changes in intracellular
fluorescence spectra of BChlꢀNI: a band with the 757 nm maximum disappears due to
photobleaching of acceptor (BChl), and a donor (NI) fluorescence is restored, which has a
maximum at ca. 600 nm (Fig.5f). These changes confirm RET between NI and BChl
chromophores in cells and show that polarity of cellular microenviroment is similar to the

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polarity of diethyl ether solution (ε = 4.3), where NI chromophore has fluorescence maximum at
44 §
608 nm.
1
20
00
e
a
c
1
8
6
4
2
0
0
0
0
0
600
650
700
750
800
Wavelength / nm
1
20
00
b
d
f
1
80
60
40
20
0
600
650
700
750
800
Wavelength / nm
Fig.5. Intracellular distribution of BChlꢀNI fluorescence in A549 cells. Distribution of fluorescence in the 550ꢀ650 nm
a, c) and >730 nm (b, d) ranges at the low power 488 nm excitation before (a,b) and after (c,d) the high power
(
prolonged illumination (photobleaching) of cells with the 488 nm wavelength. (e,f) Examples of intracellular
fluorescence spectra of BChlꢀNI (black line) at the 488 nm excitation before (e) and after (f) the high power
prolonged illumination (photobleaching) of cells. Red line – fluorescence spectrum of Bchl in 1% Chremophor EL
solution. Spectra are normalized to the highest intensity. Cells were incubated with 2 ꢁM of BChlꢀNI for 3 h.
In agreement with microspectroscopy studies, confocal laser scanning microscopy reveals
intracellular fluorescence of BChlꢀNI in the range of 730 nm and longer wavelengths (at the
excitation of 488 or 514 nm) and absence of fluorescence in the 500–650 nm range (Fig.5a,b).
As clearly seen (Fig.5b, Fig.6), conjugate accumulates in cytoplasm of A549 cells and does not
penetrate into the nucleus. In cytoplasm, BchlꢀNI has diffuse distribution. The pattern of
intracellular distribution is similar at different extracellular concentrations of BChlꢀNI and
different incubation times of cells with the conjugate (Fig.6). An increase in extracellular
concentration of the conjugate from 2 to 8 µM and incubation time from 1 to 3 h increases
moderately intracellular fluorescence that indicates to saturation of intracellular accumulation of
BChlꢀNI at concentrations higher than 4 µM and incubation time of 3 h.
§
Similar blue shift of naphthalimide emission band was also observed in rabbit blood serum. However, RET
between NI and BChl chromophores remains highly efficient as it is in CH CN (the solvent we used for the
3
photophysical studies, ε = 37.5). This was demonstrated by the comparison of emission spectra of conjugate BChlꢀ
NI in rabbit blood serum with the corresponding spectra of equimolar mixture containing individual photosensitizer
BChl and naphthalimide dye (see Supplementary information, Fig.S26).

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a
b
c
d
e
f
Fig.6. Intracellular accumulation of BChlꢀNI in A549 cells. Confocal fluorescent images describing
the intracellular distribution of the conjugate based on the bacteriochlorin fluorescence (exitation 514
nm, emission >730 nm). Cells were incubated with 2 (a,d), 4 (b,e) ,8 (c,f) ꢁM conjugate for 1 h (a–c)
or 3 h (d–f).
Intense prolonged illumination of cells with 488ꢀnm or 514 nm light leads to the
appearance of fluorescence in the 500ꢀ650 nm range due to photobleaching of BChl
chromophore. In the course of photobleaching, the distribution of intracellular fluorescence
becomes granular (Fig.5c,d) as a result of photodynamic (phototoxic) processes induced in cells.
To study the mechanism of BChlꢀNI penetration into cells, the known inhibitors of
clathrinꢀdependent (chlorpromazine) and caveolaeꢀdependent (methylꢀβꢀcyclodextrin)
63
endocytosis were used. It was found that intracellular penetration of BChlꢀNI was not
inhibited, when cells were preꢀtreated with chlorpromazine (inhibitor, which induces dissociation
of clathrin from a cell surface) (Fig.7b) or with methylꢀβꢀcyclodextrin (compound that depletes
plasma membrane cholesterol) (Fig. 7c). Therefore, clathrinꢀdependent or caveolae dependent
endocytosis pathways are not noticeably involved in BChlꢀNI uptake into A549 cells. A
decrease in incubation temperature from 37 to 8 °C led to a threefold decrease in BChlꢀNI
intracellular accumulation (Fig.7d,e).This effect can be explained by a passive diffusion of
BChlꢀNI through the plasma membrane, which is affected by a decrease in membrane fluidity at
a low temperature.
The studies showed that conjugate BChlꢀNI was not toxic for A549 cells at tested
concentrations (<8 ꢁM, Fig.8) without lightꢀinduced activation. At the same time, the
concentrationꢀdependent killing of cells (Fig.8) was observed upon irradiation with the red light.

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In the used conditions, the photodynamic effect was achieved through the excitation of
bacteriochlorin moiety. Conjugate concentrations that provides 90% and 50% photoinduced cell
death are 0.70 ± 0.05 and 0.33 ± 0.05 ꢁM respectively. These data indicate that the conjugate is
an active photosensitizer, and its further investigation for anticancer photodyanmic therapy is
warranted.
a
b
c
1
0 µm
d
e 12
1
0
8
6
4
2
0
Fig.7 Study of mechanisms of intracellular penetration of BChlꢀNI. (aꢀd) Confocal fluorescent images showing the
intracellular distribution of BChlꢀNI in control cells (a), cells preꢀincubated with 30 ꢁM chlorpromazine (b) or 4
mM methylꢀβꢀcyclodextrin for 30 min (c) or in the cells at 8°C (d). (e) Comparison of average intracellular
fluorescence intensities of BChlꢀNI at incubation regimes depicted in (aꢀd): CPZ, MβCD –treatment with
chlorpromazine or methylꢀβꢀcyclodextrin, respectively. Cells were incubated with 4 ꢁM BChlꢀNI for 70 min at
37 °C (aꢀc) or 8°C (d).
1
00
80
60
40
20
0
0
,1
1
Concentration / ꢁM
Fig.8. Concentration dependent survival of A549 cells after photodynamic treatment with the conjugate (circles).
An abscissa is the photosensitizer concentration in the medium. Cells were incubated with the conjugate for 3 h,
irradiated with light, and their survival was determined 3 h after irradiation. Control cells (triangles) were incubated
with conjugate for 6 h without irradiation.

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a
b
c
DCFDA
d
e
f
NI
2
5 µm
Fig.9. Verification of lightꢀinduced ROS formation in cells using DCFDA. Confocal fluorescence images show
distribution of DCFDA (aꢀc) and NIꢀmoiety (dꢀf) fluorescence in the irradiated A549 cells. Fluorescence was
excited at 488 nm and registered in the 500ꢀ540 nm (DCFDA, green) and 600ꢀ650 nm (NI, red). (a,d) – Cells were
incubated with DCFDA but without BChlꢀNI and irradiated with red light. (b,e) –Cells were incubated with BChlꢀ
NI but without DCFDA and irradiated. (c,f) – Cells were incubated with BChlꢀNI and DCFDA and irradiated.
To confirm the formation of reactive oxygen species (ROS) in cells under irradiation,
cells were incubated with both DCFDA and BChlꢀNI and irradiated with the red light (Fig.9). In
viable cells, DCFDA undergoes deacetylation to nonꢀfluorescent 2',7'ꢀ dichlorofluorescein. This
compound reacts quantitatively with ROS in cells and produces the fluorescent dye 2',7'ꢀ
dichlorofluorescein. DCFDA is known to be very reactive with peroxides but can be also
6
4
oxidized by other ROS. Bright fluorescence of DCFDA and NIꢀmoiety was observed in
cytoplasm after irradiation (Fig.9c,f). Appearance of DCFDA fluorescence indicates ROS
generation during irradiation of cells. No fluorescence of DCFDA and NI was detected in control
cells without irradiation (data not shown). No fluorescence of DCFDA was observed in cells
incubated with DCFDA but without BChlꢀNI and irradiated (Fig.9a) thus confirming again that
BChlꢀNI does produce ROS during irradiation with the red light.

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4. Conclusions
In summary, we have prepared novel bacteriochlorin–naphthalimide conjugate BChlꢀNI. Steadyꢀ
state and timeꢀresolved spectroscopical study of BChlꢀNI in solution has shown that the
presence of NI fragment does not decrease the photosensitizing activity of BChl, however the
fluorescence of naphthalimide is quenched due to efficient resonance energy transfer. In vitro
results revealed high photoinduced cytotoxicity and fluorescent imaging capability that can be
realized using both chromophores. Farꢀred absorption and fluorescence of BChl moiety makes
this conjugate suitable for photodynamic treatment of deeply located tumors as well as for
fluorescent imaging and navigation in vivo. At the same time farꢀred fluorescence of the
conjugate is poorly detectable in cellular studies, since conventional confocal microscopes
equipped with photomultiplierꢀbased detection systems have low sensitivity in the 700–900 nm
**
spectral range. Accordingly, photoinduced magentaꢀtoꢀred conversion of fluorescence can
facilitate visualization of the conjugate in cellular studies. Moreover, this color conversion of
BChlꢀNI fluorescence could be a useful indicator of integrity of photosensitizer moiety during
irradiation of cancer cells and can help to optimize a power and dose of light during
photodynamic treatment. Thus, newly synthesized conjugate BChlꢀNI is a good candidate for
the nextꢀgeneration “bifunctional” photosensitizer allowing PDT and fluorescent diagnostics to
be performed.
Acknowledgements
O.A.F. thanks RSF project № 16ꢀ13ꢀ10226 (synthesis of naphthalimide derivatives, steadyꢀstate
absorption and fluorescence spectroscopy), M.A.G. thanks RSF project № 16ꢀ13ꢀ10092
(synthesis of bacteriochlorin derivatives), G.J. thanks the Région NouvelleꢀAquitaine for
financial support (timeꢀresolved optical measurements).
*
*
We used confocal laser scanning microscopy to visualize the intracellular distribution of the conjugate BChlꢀNI.
Fluorescence was registered with a highly sensitive avalanche photodiode or with a photomultiplier. Both detectors
record intensity of emitted light in each point of scanned specimen and do not recognize color (wavelength) of
detected photons. Color of image is selected by a scientist using palettes, which are available in software. In order to
provide better color reproduction, we chose a red color for NI emission in Fig.5c. Fluorescence of BChl moiety has
wavelengths that are not visible to a human eye. We decided to use dark magenta color for this fluorescence in the
presented images.

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