Polymeric Bis(dipyrrinato) Zinc(II) Nanoparticles as Selective Imaging Probes for Lysosomes of Cancer Cells

Article abstract of DOI:10.1021/acs.inorgchem.9b02019

Fluorescence imaging is a powerful tool in biomedical research. It has been frequently used to uncover or better understand physiological mechanisms in disease-related processes such as cancer. The majority of chromophores used for imaging are based on a

Full text of DOI:10.1021/acs.inorgchem.9b02019

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Polymeric Bis(dipyrrinato) Zinc(II) Nanoparticles as Selective
Imaging Probes for Lysosomes of Cancer Cells
Johannes Karges,† Olivier Blacque,‡ Hui Chao,*,§ and Gilles Gasser*,†
†Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical
Biology, 75005 Paris, France
‡Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
§MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, 510275 Guangzhou,
People’s Republic of China
S
* Supporting Information
ABSTRACT: Fluorescence imaging is a powerful tool in biomedical research. It has been frequently used to uncover or better
understand physiological mechanisms in disease-related processes such as cancer. The majority of chromophores used for
imaging are based on a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) scaffold. However, their applications are limited
due to their poor water solubility as well as poor cancer cell selectivity. To circumvent these drawbacks, we present herein the
use of bis(dipyrrinato)zinc(II) complexes. As this class of compounds is associated with a quenching effect of the excited state
in water, the lead compound of this study (3) was encapsulated in a polymer matrix with biotin as a targeting moiety (3-NP).
This encapsulation improved the water solubility, overcame the quenching effects in water, as well as allowed selective
accumulation in the lysosomes with a bright fluorescence signal in monolayer cells as well as 3D multicellular tumor spheroids
(MCTS). As a benefit from the biotin targeting moiety, the nanoparticles were majorly taken up by the sodium dependent
multivitamin transporter (SMVT) which is overexpressed in various cancers cells and selectively accumulated in cancerous cells
over noncancerous cells.
solubility and cancer cell selectivity. To overcome these
limitations, different dyes have been linked to a targeting
moiety or formulations have been investigated to promote
selective drug delivery.10−12
INTRODUCTION
■
The ability to use imaging techniques either on a microscopic
cellular level or even on humans has strongly influenced
modern medical research. Among others, fluorescence imaging
has received increasing attention over the last decades as this
method was found to efficiently monitor physiological
processes. Of special interest is the monitoring of disease-
related organisms or cells.1,2
Among the different classes of imaging compounds
investigated, chromophores based on a 4,4-difluoro-4-bora-
3a,4a-diaza-s-indacene (BODIPY) scaffold are of special
interest. These compounds are associated with a strong UV/
vis absorbance, chemical and photostability as well as sharp
and strong fluorescence. For these reasons, these dyes are
currently being used for numerous cellular applications
including chemosensors, fluorescent switches, as well as in
the labeling of protein or DNA.3−9 However, the applications
of these dyes are generally limited by their poor water
Beside boron complexes, dipyrrin ligands are also able to
coordinate other metal ions such as Ni(II), Zn(II), Cu(II),
Fe(III), Rh(III), and Co(III).13−15 During the past decade,
bis(dipyrrinato)zinc(II) complexes, as an underrepresented
class of compounds, have received increasing attention as
fluorophores (see Figure 1 for an overview of investigated
bis(dipyrrinato)zinc(II) complexes).13,16−22 Unfortunately, the
fluorescence quantum yields (ΦF) of these compounds
reported so far remain inferior to the structurally related
BODIPY derivatives. Therefore, current recent efforts are
made toward improving the luminescent properties of
Received: July 15, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.9b02019
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 1. Overview of reported bis(dipyrrinato)zinc(II) complexes.13,16−22
bis(dipyrrinato)zinc(II) complexes. It was previously demon-
strated that the steric hindrance of the peripheral aryl group of
the dipyrrinato scaffold with methyl groups transformed the
compounds from a weak emitter to a strong fluorescent
chromophore.16 Further studies confirmed this observation
with the introduction of peripheral 1- and 2-naphthyl groups.20
Despite current research efforts, the quenching of the excited
state of this class of compounds in polar solvents remains a
serious problem, limiting their biological applications. This
quenching effect was proposed to be caused by the formation
B
DOI: 10.1021/acs.inorgchem.9b02019
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
of a nonemissive symmetry-breaking charge transfer state from
the emissive excited state, which is not formed in nonpolar
solvents (Figure 2).13,17 Details on the mechanism and the
influence of the solvent polarity have been previously
published.17
environment. To overcome this limitation as well as their poor
water solubility and the quenching effects of the excited state
in water and to make the compounds selective for cancerous
cells, the lead compound of this study, namely complex 3, was
encapsulated inside a polymeric matrix with a biotin end group
to form nanoparticles (3-NP). Biotin is a compound of the
vitamin B family, which is majorly internalized by the sodium
dependent multivitamin transporter (SMVT). Since this
receptor is overexpressed in various cancers and the demand
for biotin in rapidly growing tumors is higher than in normal
tissue, the conjugation of an active molecule to biotin offers the
possibility for specific cancer cell targeting.23−25 The nano-
formulated complex was found to be highly water-soluble and
fluorescent. The particles selectively accumulated in the
lysosomes as shown by the bright fluorescence signal observed
in this organelle as well as being highly luminescent in 3D
multicellular tumor spheroids (MCTS). Importantly, the
particles benefit from the biotin targeting as demonstrated by
a selective accumulation in transfected cancerous cells in
comparison to nontransfected noncancerous human lung cells.
To the best of our knowledge, this is the first report of
bis(dipyrrinato)zinc(II) complexes, which are highly fluores-
cent in an aqueous environment and which are selective for
cancer cells.
Figure 2. Simplified Jablonski diagram demonstrating the solvent
dependency of bis(dipyrrinato)zinc(II) complexes.13,17
EXPERIMENTAL SECTION
■
1
Instrumentation and Methods. H and 13C NMR spectra were
Keeping this in mind, we have designed homoleptic
bis(dipyrrinato)zinc(II) complexes with different aryl groups
in position 8 of the dipyrrin scaffold. Herein, we present the
synthesis and in-depth photophysical and biological inves-
tigation of a series of homoleptic bis(dipyrrinato)zinc(II)
complexes 1−4 (Figure 3) as imaging agents. Complexes 3 and
recorded on a 400 MHz NMR spectrometer (Bruker). Chemical shifts
(δ) are reported in parts per million (ppm) referenced to
tetramethylsilane (δ 0.00) ppm using the residual proton solvent
peaks as internal standards. Coupling constants (J) are reported in
Hertz (Hz), and the multiplicity is abbreviated as follows: s (singlet),
d (doublet), and m (multiplet). Electrospray ionization-mass
spectrometry (ESI-MS) experiments were carried out using a LTQ-
Orbitrap XL from Thermo Scientific (Thermo Fisher Scientific) and
operated in positive ionization mode, with a spray voltage at 3.6 kV.
No sheath and auxiliary gas was used. Applied voltages were 40 and
100 V for the ion transfer capillary and the tube lens, respectively. The
ion transfer capillary was held at 275 °C. Detection was achieved in
the Orbitrap with a resolution set to 100,000 (at m/z 400) and an m/
z range between 150 and 2000 in profile mode. The spectrum was
analyzed using the acquisition software XCalibur 2.1 (Thermo Fisher
Scientific). The automatic gain control (AGC) allowed for the
accumulation of up to 2 × 105 ions for Fourier transform mass
spectrometry (FTMS) scans, maximum injection time was set to 300
ms, and a 1 μs scan was acquired. Ten μL was injected using a
Thermo Finnigan Surveyor HPLC system (Thermo Fisher Scientific)
with a continuous infusion of methanol at 100 μL·min−1. Elemental
microanalyses were performed on a Thermo Flash 2000 elemental
analyzer.
Materials. All of the chemicals were obtained from commercial
sources and were used without further purification. Dulbecco’s
Modified Eagles Medium (DMEM), Dulbecco’s Modified Eagles
Medium supplemented with nutrient mixture F-12 (DMEM/F-12),
Fetal Bovine Serum (FBS), Gibco Penicillin-Streptomycin-Glutamine
(Penstrep), and Dulbecco’s Phosphate-Buffered Saline (PBS) were
purchased from Fisher Scientific and Resazurin from ACROS
Organics.
Figure 3. Chemical structures of the bis(dipyrrinato)zinc(II)
complexes investigated in this work.
Synthesis. General synthetic procedure for the synthesis of the
dipyrrin ligand:
4 were previously reported in a study describing the
photophysical properties of bis(dipyrrinato)zinc(II) com-
plexes.17,22 In this work, the ability of these compounds to
be used as imaging probes is investigated. The compounds
were found to selectively accumulate in the cytoplasm, while
being taken up through passive diffusion. These complexes are
highly fluorescent in toluene but undergo quenching in a polar
The aldehyde (2 mmol) and 2, 4-dimethylpyrrole (5 mmol) were
dissolved in a dichloromethane solution (40 mL). Trifluoroacetic acid
(100 μL) was added and the mixture stirred at room temperature
overnight under nitrogen atmosphere. Tetrachlor-p-benzochinon (2
mmol) was added and the reaction mixture stirred another 2 h. After
this time, the solution was filtered and the solvent evaporated. The
crude product was purified by column chromatography on neutral
C
DOI: 10.1021/acs.inorgchem.9b02019
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
alumina with a gradient of dichloromethane/hexane. The fractions
containing the product were united, and the solvent was removed.
The solid was washed with Et2O and dried under vacuum. Yield: 50−
70%.
crystallographic data for these compounds, and they can be obtained
free of charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Spectroscopic Measurements. The absorption of the samples
was recorded on a Lambda 850 UV/vis spectrometer (PerkinElmer)
at room temperature. Emission spectra were recorded on a LS 55
fluorescence spectrometer (PerkinElmer) at room temperature. For
the determination of the luminescence quantum yield, the samples
were prepared in toluene solution with an absorbance of 0.1 at 500
nm. The luminescence quantum yields were calculated by comparison
with the reference Rhodamine B in ethanol (Φem = 0.5)33 applying
the following formula:
General Synthetic Procedure for the Complexation with Zn. The
dipyrrin ligand (1 mmol) was dissolved in a 3:2:3 butanol/ethanol/
H2O (200 mL) mixture to which Zn(OAc)2·2H2O (0.4 mmol) and
Et3N (4 mmol) were added. The reaction mixture was heated at reflux
overnight under nitrogen atmosphere. After completion of the
reaction, the solvent was removed. The crude product was purified
by column chromatography on silica with a gradient of dichloro-
methane/pentane. The fractions containing the product were united,
and the solvent was removed. The solid was washed with Et2O and
dried under vacuum. Yield: 45−63%.
2
Isample i nsample
y
z
F
F
j
reference
j
z
Φ
= Φ
*
*
*j
z
Bis((Z)-2-((3,5-dimethyl-2H-pyrrol-2-ylidene)(phenyl)methyl)-
em,sample
em,reference
j
z
z
j
Ireference nreference
1
sample
k
{
3,5-dimethyl-1H-pyrrole)zinc(II) 1. H NMR (CD2Cl2, 400 MHz):
7.45−7.43 (m, 3H), 7.29−7.27 (m, 2H), 5.92 (s, 2H), 2.34 (s, 6H),
1.29 (s, 6H). 13C NMR (CD2Cl2, 100 MHz): 155.8, 144.3, 142.9,
137.6, 133.9, 127.2, 127.1, 126.8, 121.1, 16.1, 15.2. ESI-HRMS (pos.
detection mode): calcd for C38H38N4Zn [M + H]+ m/z 615.2461;
found: 615.2545. Elemental analysis calcd for C38H38N4Zn (%): C
74.08, H 6.22, N 9.09; found: C 74.12, H 6.33, N 8.89.
F = 1 − 10−A
Φ
em = luminescence quantum yield, F = fraction of light absorbed, I =
integrated emission intensities, n = refractive index, A = absorbance of
the sample at irradiation wavelength.
Stability in Toluene/H2O/DMEM. The stability of the complex
in toluene/H2O/DMEM (2% DMSO, v%) was investigated by UV/
vis spectroscopy. The complex was dissolved with about 0.5
absorption at its absorption maximum and stored at room
temperature in the dark. The absorption spectrum from 210 to 700
nm was recorded with a SpectraMax M2 Microplate Reader
(Molecular Devices) after each time interval (0, 1, 2, 4, 8, 12, 24,
48 h) and compared.
Photostability. The photostability of the complex in toluene (2%
DMSO, v%) was investigated by UV/vis spectroscopy. The complex
was dissolved with about 0.5 absorption at its absorption maximum
and exposed to a constant LED irradiation 510 nm (4.2 mW/cm2)
with an Atlas Photonics LUMOS BIO irradiator. The absorption
spectrum from 210 to 700 nm was recorded with a SpectraMax M2
Microplate Reader (Molecular Devices) after each time interval (0, 5,
10, 15, 20, 25, 30 min) and compared.
Distribution Coefficient. The lipophilicity of a compound was
determined by measuring its distribution coefficient between the PBS
and octanol phase by using the “shake-flask” method. For this
technique, the used phases were previously saturated in each other.
The compound was dissolved in the phase (A) with its major
presence with an absorbance of about 0.5 at 500 nm. This solution
was then mixed with an equal volume of the other phase (B) at 80
rpm for 8 h with an Invitrogen sample mixer and then equilibrated
overnight. Phase A was then carefully separated from phase B. The
amount of the compound before and after the sample mixing was
determined by UV/vis spectroscopy at 500 nm using a SpectraMax
M2 Microplate Reader (Molecular Devices). The evaluation of the
complexes was repeated three times and the ratio between the organic
and aqueous phase calculated.
Cell Culture. Human cervical carcinoma (HeLa), adenocarcinom-
ic human alveolar basal epithelial cells (A549) human lung fibroblasts
(HLF) cells were cultured using DMEM media and retinal pigment
epithelium (RPE-1) cells using DMEM/F-12 with addition of 10%
FBS and 1% penstrep. The cells were cultivated and maintained in a
cell culture incubator at 37 °C with 5% CO2 atmosphere. Before an
experiment, the cells were passaged three times.
Bis((Z)-2-((3,5-dimethyl-2H-pyrrol-2-ylidene)(2,6-
dimethylphenyl)methyl)-3,5-dimethyl-1H-pyrrole)zinc(II) 2. 1H
3
NMR (CD2Cl2, 400 MHz): 7.21 (dd, J = 7.5, 7.5 Hz, 1H), 7.12
(d, 3J = 7.5 Hz, 2H), 5.92 (s, 2H), 2.14 (s, 6H), 2.01 (s, 6H), 1.26 (s,
6H). 13C NMR (CD2Cl2, 100 MHz): 156.4, 143.7, 143.5, 139.6,
136.3, 134.6, 128.4, 128.3, 120.2, 19.5, 16.2, 14.8. ESI-HRMS (pos.
detection mode): calcd for C42H46N4Zn [M + H]+ m/z 671.3087;
found: 671.3079. Elemental analysis calcd for C42H46N4Zn (%): C
75.04, H 6.90, N 8.33; found: C 74.83, H 6.94, N 8.42.
Bis((Z)-2-((3,5-dimethyl-2H-pyrrol-2-ylidene)(mesityl)methyl)-
1
3,5-dimethyl-1H-pyrrole)zinc(II) 3. H NMR (CD2Cl2, 400 MHz):
6.95 (s, 2H), 5.92 (s, 2H), 2.34 (s, 3H), 2.10 (s, 6H), 2.01 (s, 6H),
1.29 (s, 6H). 13C NMR (CD2Cl2, 100 MHz): 156.3, 144.1, 143.6,
137.9, 136.7, 135.9, 134.9, 129.1, 120.0, 21.3, 19.4, 16.2, 14.9. ESI-
HRMS (pos. detection mode): calcd for C44H51N4Zn [M + H]+ m/
z 699.3400; found: 699.3402. Elemental analysis calcd for
C44H50N4Zn*0.05 H2O (%): C 75.37, H 7.20, N 7.99; found: C
74.91, H 6.97, N 8.48.
Bis((Z)-2-((3,5-dimethyl-2H-pyrrol-2-ylidene)(anthracenyl)-
1
methyl)-3,5-dimethyl-1H-pyrrole)zinc(II) 4. H NMR (CD2Cl2, 400
3
3
MHz): 8.61 (s, 1H), 8.07 (d, J = 8.5 Hz, 2H), 7.98 (d, J = 8.5 Hz,
2H), 7.51−7.48 (m, 2H), 7.44−7.40 (m, 2H), 5.91 (s, 2H), 2.31 (s,
6H), 0.45 (s, 6H). 13C NMR (CD2Cl2, 100 MHz): 157.3, 144.1,
141.2, 136.9, 134.4, 132.1, 131.3, 128.6, 127.7, 126.7, 125.8, 120.7,
16.7, 15.0. ESI-HRMS (pos. detection mode): calcd for
C54H47N4Zn [M + H]+ m/z 815.3087; found: 815.3089. Elemental
analysis calcd for C54H46N4Zn (%): C 79.45, H 5.68, N 6.86; found:
C 79.60, H 5.73, N 6.72.
X-ray Crystallography. X-ray single-crystal data were collected at
160(1) K on Rigaku OD diffractometers with an Oxford liquid-
nitrogen Cryostream cooler: a XtaLAB Synergy Dualflex (Pilatus 200
K detector) diffractometer for dipyrrin_1, 2, and 4, and a Xcalibur
(CCD Ruby detector) diffractometer for 3. A single wavelength X-ray
source from a microfocus sealed X-ray tube was used with the Cu Kα
radiation (λ = 1.54184 Å)26 for the analyses of dipyrrin_1 and 4 and
with the Mo Kα radiation (λ = 0.71073 Å)26 for the analysis of 2 and
3. The selected single crystals were mounted using polybutene oil on
a loop fixed on a goniometer head and transferred to the
diffractometer. Pre-experiments, data collections, data reductions,
and analytical absorption corrections27 were performed with the
program suite CrysAlisPro.28 Using Olex2,29 the structures were solved
with the SHELXT30 small molecule structure solution program and
refined with the SHELXL2018/3 program package31 by full-matrix
least-squares minimization on F2. Molecular graphics were created
using Mercury 4.0.32 The crystal data collections and structure
refinement parameters are summarized in Tables S1 and S2. CCDC
1937531 (for 3), CCDC 1937532 (for dipyrrin_1), CCDC 1937533
(for 2), and CCDC 1937534 (for 4) contain the supplementary
Cellular Uptake. The cellular uptake of the complex was
investigated by the determination of the Zn content inside the cells.
The complex with a final concentration of 25 μM (2% DMSO, v%)
was incubated at 37 °C on a cell culture dish with a density of ca. 4−6
× 106 cells in 10 mL of media. After this time, the media was removed
and the cells washed with cell media. The cells were trypsinised,
harvested, centrifuged, and resuspended. The number of cells on each
dish was accurately counted. Each sample was then digested using a
60% HNO3 solution for 3 days. Each sample was diluted to a solution
of 2% HNO3 in water. The Zn content was determined using an ICP-
MS apparatus and comparing the results with the Zn references. The
Zn content was then associated with the number of cells.
D
DOI: 10.1021/acs.inorgchem.9b02019
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Cellular Uptake Mechanism. The mechanism of the cellular
uptake was investigated by systematic inhibition of different uptake
pathways and afterward determination of the amount of Zn inside the
cells via ICP-MS. For the experiment, 1 × 106 cells were pretreated
with the corresponding inhibitor.
Control. The cells were incubated with the complex (25 μM, 2%
DMSO, v%) for 1 h at 37 °C. After this time, the cells were washed
with PBS. The cells were detached with trypsin, harvested,
centrifuged, and resuspended. The number of cells on each dish
was accurately counted. Each sample was then digested using a 60%
HNO3 solution for 3 days. Each sample was diluted to a solution of
2% HNO3 in water. The Zn content was determined using an ICP-
MS apparatus and comparing the results with the Zn references. The
Zn content was then associated with the number of cells.
Low Temperature. The cells were incubated at 4 °C for 1 h. The
cells were further incubated with the complex (25 μM, 2% DMSO, v
%) for 1 h at 4 °C. After this time, the cells were washed with PBS.
The cells were detached with trypsin, harvested, centrifuged, and
resuspended. The number of cells on each dish was accurately
counted. Each sample was then digested using a 60% HNO3 solution
for 3 days. Each sample was diluted to a solution of 2% HNO3 in
water. The Zn content was determined using an ICP-MS apparatus
and comparing the results with the Zn references. The Zn content was
then associated with the number of cells.
Metabolic Inhibition. The cells were incubated with 2-deoxy-D-
glucose (50 mM) and oligomycin (5 μM) for 1 h. After the
preincubation, the cells were washed with PBS and further incubated
with the complex (25 μM, 2% DMSO, v%) for 1 h at 37 °C. After this
time, the cells were washed with PBS. The cells were detached with
trypsin, harvested, centrifuged, and resuspended. The number of cells
on each dish was accurately counted. Each sample was then digested
using a 60% HNO3 solution for 3 days. Each sample was diluted to a
solution of 2% HNO3 in water. The Zn content was determined using
an ICP-MS apparatus and comparing the results with the Zn
references. The Zn content was then associated with the number of
cells.
Endocytic Inhibition. The cells were incubated with NH4Cl (50
mM) or chloroquine (100 μM) for 1 h. After the preincubation, the
cells were washed with PBS and further incubated with the complex
(25 μM, 2% DMSO, v%) for 1 h at 37 °C. After this time, the cells
were washed with PBS. The cells were detached with trypsin,
harvested, centrifuged, and resuspended. The number of cells on each
dish was accurately counted. Each sample was then digested using a
60% HNO3 solution for 3 days. Each sample was diluted to a solution
of 2% HNO3 in water. The Zn content was determined using an ICP-
MS apparatus and comparing the results with the Zn references. The
Zn content was then associated with the number of cells.
Cation Transporter Inhibition. The cells were incubated with
tetraethylammonium chloride (1 mM) for 1 h. After the
preincubation, the cells were washed with PBS and further incubated
with the complex (25 μM, 2% DMSO, v%) for 1 h at 37 °C. After this
time, the cells were washed with PBS. The cells were detached with
trypsin, harvested, centrifuged, and resuspended. The number of cells
on each dish was accurately counted. Each sample was then digested
using a 60% HNO3 solution for 3 days. Each sample was diluted to a
solution of 2% HNO3 in water. The Zn content was determined using
an ICP-MS apparatus and comparing the results with the Zn
references. The Zn content was then associated with the number of
cells.
Sodium-Dependent Multivitamin Transporter Inhibition. The
cells were incubated with pantothenic acid (50 μM) or lipoic acid (50
μM) for 1 h. After the preincubation, the cells were washed with PBS
and further incubated with the complex (25 μM, 2% DMSO, v%) for
1 h at 37 °C. After this time, the cells were washed with PBS. The
cells were detached with trypsin, harvested, centrifuged, and
resuspended. The number of cells on each dish was accurately
counted. Each sample was then digested using a 60% HNO3 solution
for 3 days. Each sample was diluted to a solution of 2% HNO3 in
water. The Zn content was determined using an ICP-MS apparatus
and comparing the results with the Zn references. The Zn content was
then associated with the number of cells.
Intracellular Distribution by Confocal Luminescence Imag-
ing. The colocalization of the complex was determined under usage
of its luminescence properties. 1 × 104 cells were seeded on 35 mm
confocal dishes and allowed to adhere overnight. The cells were
incubated with the complex (50 μM, 2% DMSO, v%) for 4 h at 37 °C
in the dark. After this time, the cells were washed with PBS. The cells
were further incubated with Hoechst 33342 (5 μg/mL), LysoTracker
Deep Red (500 nM), and MitoTracker Deep Red (500 nM) for 30
min at 37 °C in the dark. The cells were washed three times with PBS.
Confocal images were taken with a LSM 880 (Carl Zeiss) laser
scanning confocal microscope equipped with a GaAsP detector. The
organelle trackers Hoechst 33342 (λex = 405 nm, λem = 410−470 nm),
MitoTracker Deep Red (λex = 633 nm, λem = 650−720 nm), and
LysoTracker Deep Red (λex = 633 nm, λem = 650−720 nm) were
excited and detected as recommended by the supplier. The
investigated Zn complexes were detected under usage of their
luminescence properties (λex = 514 nm, λem = 530−610 nm).
Intracellular Distribution by ICP-MS. The colocalization of the
complex was determined by measuring the Zn content inside the cell
via ICP-MS. 10 × 106 cells were incubated with the complex (25 μM,
2% DMSO, v%) for 4 h at 37 °C in the dark. After this time, the cells
were detached with trypsin and harvested. The number of cells was
accurately counted. The amount was equally divided into four
portions. In the first portion, the nucleus was extracted using a
nucleus extraction kit (Sangon Biotech) following the manufacturer
recommended protocol; in the second portion, the mitochondria was
extracted using a mitochondria extraction kit (Sangon Biotech)
following the manufacturer recommended protocol; and in the third
portion, the lysosome was extracted using a lysosome extraction kit
(GenMed Scientific) following the manufacturer recommended
protocol. Using the fourth portion, the cytoplasm was extracted.
For this, the cells were detached with trypsin, harvested, and
centrifuged. The cell pellet was resuspended in lysis buffer, and the
cells were lysed. The cell organelles were separated using a vacuum
ultracentrifuge (Optima MAX-XP ultracentrifuge, Beckman Coulter)
for 150 min at 200000g at 4 °C. The supernatant solution was
separated. Each sample was then digested using a 60% HNO3 solution
for 3 days and was diluted to a solution of 2% HNO3 in water. The Zn
content was determined using an ICP-MS apparatus and comparing
the results with the Zn references. The Zn content was then
associated with the number of cells.
(Photo)cytotoxicity on 2D Cell Monolayers. The cytotoxicity
of the complexes was assessed by measuring cell viability using a
fluorometric resazurin assay. The cultivated cells were seeded in
triplicate in 96 well plates with a density of 4000 cells per well in 100
μL of media. After 24 h, the medium was removed and the cells were
treated with increasing concentrations of the complex diluted in cell
media achieving a total volume of 200 μL. The cells were incubated
with the complex for 4 h. After this time, the media was removed and
replaced with 200 μL of fresh medium. For the phototoxicity studies,
the cells were exposed to light with an Atlas Photonics LUMOS BIO
irradiator. Each well was constantly illuminated with either 510 or 540
nm irradiation. During this time, the temperature was maintained
constant at 37 °C. The cells were grown in the incubator for an
additional 44 h. For the determination of the dark cytotoxicity, the
cells were not irradiated and after the medium exchange directly
incubated for 44 h. After this time, the medium was replaced with
fresh medium containing resazurin with a final concentration of 0.2
mg/mL. After 4 h of incubation, the amount of the fluorescent
product resorufin was determined upon excitation at 540 nm and
measurement of its emission at 590 nm using a SpectraMax M2
Microplate Reader (Molecular Devices). The obtained data was
analyzed with the GraphPad Prism software.
Generation and Analysis of 3D Multicellular Tumor
Spheroids (MCTS). A suspension of 0.75% agarose in PBS buffer
was heated inside a high-pressure autoclave. The hot emulsion was
transferred into wells (50 μL per well) of a 96 cell culture well plate.
The plates were exposed for 3 h to UV irradiation to ensure the
E
DOI: 10.1021/acs.inorgchem.9b02019
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 4. Spectroscopic properties of 1−4. (a) Normalized absorption (solid line) and emission spectra (dashed line) in toluene. (b) Fluorescence
quantum yields of 1−4 plotted depending on the solvent (5% DMSO, v%) from nonpolar to polar.
sterility and allow the agarose solution to cool down. After this time,
the agarose was overlaid with a HeLa cell suspension at a density of
3000 cells per well in 150 μL of media. The MCTS were cultivated
and maintained in a cell culture incubator at 37 °C with 5% CO2
atmosphere. The culture media was replaced every 2 days. Within 2−
3 days MCTS were formed from the cell suspension. The formation
as well as integrity and diameter of the MCTS was monitored by an
Axio Observer Z1 (Carl Zeiss) phase contrast microscope. The
luminescence images along the z-axis were captured by (λex = 514 nm,
λem = 530−610 nm) excitation in the z-stack mode with an LSM 880
(Carl Zeiss) laser scanning confocal microscope equipped with a
GaAsP detector.
Preparation of Particles. A solution of 3 mg of 3 in 0.5 mL of
DCM was added to a solution of 6 mg of 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[biotin(polyethylene glycol)-2000] ammo-
nium salt (DSPE-PEG2000-biotin) in 19.5 mL of H2O. The solution
was further treated with ultrasonic pulses with a Scientz-II D
ultrasonic homogenizer using a 2 × 10 min method (tsonication = 2 s,
power = 15%, tbreak = 1 s) while keeping the sample constantly at 25
°C. After this, the DCM was removed by evaporation at 50 °C. The
mixture was filtered through a 0.2 μm syringe filter to remove large
aggregates. After that a clear transparent solution in H2O was
obtained. The amount of encapsulated complex was determined by
ICP-MS as 0.129 mg/mL (Yield: 86%).
Release Kinetics of the Particles. The release kinetics of the
particles were investigated by dialysis against 1× PBS at physiological
pH (7.4) and low pH (5.0) at 37 °C. The particles (200 μL) were
added into a dialysis bag and placed in the PBS bath with gentle
shaking. PBS was changed every 12 h. At various time points (1 h, 2 h,
4 h, 12 h, 24 h, 48 h) the dialysis units were removed and the amount
of compound determined by ICP-MS.
Selectivity Assay between Cancerous and Noncancerous
Cells. Adenocarcinomic human alveolar basal epithelial cells (A549)
were transfected with the fluorescence protein mCherry using a
transfection kit (Beyotime) and following the manufacturer
recommended protocol. Not successfully transfected cells were
removed using the antibiotic G-418. The cells were mixed with an
equal amout of noncancerous human lung fibroblasts (HLF) cells and
allowed to adhere overnight. The cells were incubated with the
complex (10 μM) for 4 h at 37 °C in the dark. After this time, the
cells were washed with PBS. Confocal images were taken with a LSM
880 (Carl Zeiss) laser scanning confocal microscope equipped with a
corresponding aldehydes in the presence of trifluoracetic acid.
The dipyrrin scaffold was then oxidized with tetrachloro-p-
benzoquinone. Finally, the complexes 1−4 were obtained by
deprotonation of the dipyrrin ligands with Et3N and
complexation with Zn(OAc)2·2H2O. All complexes were
1
analyzed using H- and 13C NMR and HRMS (Scheme S1,
Figure S1−S12). The purity of the compounds was verified by
elemental analysis.
X-ray Crystallography. The Zn(II) complexes 2, 3, and 4
(Table S1−S2, Figure S13−S16) were characterized with
single-crystal X-ray diffraction studies. The crystal structures of
complexes 3 and 4 were previously reported by Trinh et al.17 in
2014 and Tsuchiya et al.22 in 2016, respectively. The crystal
structure of 4 reported by Tsuchiya et al. was investigated at
113 K and exhibits a ratio of two solvent molecules of
dichloromethane for one Zn(II) molecule. The crystal
structure reported herein was investigated at 160 K and
presents very similar cell parameters and the same C2/c space
group than the previous one, but the solvent molecules of
dichloromethane appeared severely disordered. The SQUEEZE
routine of PLATON34 was used to calculate the solvent
contribution; it suggested a partial occupancy since 580
electrons were attributed to solvent molecules in the P1 cell,
i.e. about 1.47 molecules of dichloromethane per asymmetric
unit for two sets of positions. Despite that little difference, we
can consider that the two crystal structures are equivalent. The
crystal structure of 3 is a polymorph of the previously reported
structure from Trinh et al. The Zn(II) molecules crystallized in
the monoclinic space group P21/n and in the orthorhombic
space group Pbca, respectively. In both structures, no solvent
molecules are present. In our crystal structures, the Zn(II)
complexes 2, 3, and 4 exhibit a distorted tetrahedral
coordination with two substituted dipyrrinato molecules
bound to the central Zn atom. The Zn−N bond distances
are very similar for all complexes; they fall in the range
1.9583(10)−1.9642(10) for 2, 1.9621(11)−1.9690(11) Å for
3, and 1.956(4)−1.967(3) Å for 4. The bite angles N−Zn−N
are all found in the narrow range 94.71(14)−95.35(15)° while
the relative orientation of the bidentate ligands is nearly
perpendicular in the three structures. Based on these structural
parameters, we can assume that there is no significant influence
from the substituent of the dipyrrinato ligand (dimethylphenyl,
trimethylphenyl, and anthracene) on the coordination
geometry of the metal center.
GaAsP detector. The investigated Zn complexes (λex = 514 nm, λem
=
530−610 nm) and the fluorescence protein mCherry (λex = 633 nm,
λem = 650−720 nm) were detected under usage of their luminescence
properties.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of 1−4. The dipyrrin
ligands were prepared by reacting 2,4-dimethylpyrrole with the
Photophysical Characterization of 1−4. The photo-
physical properties (Table S3) of 1−4 were investigated to
F
DOI: 10.1021/acs.inorgchem.9b02019
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 5. (a) Confocal luminescence image of HeLa cells incubated with compounds 1−4 (50 μM, 2% DMSO, v%) for 4 h at 37 °C in the dark.
The investigated Zn complexes were detected using their luminescence properties (Zn, λex = 514 nm, λem = 530−610 nm). (b) Subcellular
distribution (cell = whole cell, Cyto. = cytoplasm, Mito. = mitochondria, Lys. = lysosome, Nuc. = nucleus) of 1−4 (25 μM, 2% DMSO, v%) in
HeLa cells after 4 h of incubation in the dark determined after organelle extraction and determination of the amount of Zn inside each organelle by
ICP-MS.
evaluate their potential as imaging probes. As expected from
previous works on bis(dipyrrinato)zinc(II) complexes,13,16−19
these compounds have a strong and sharp absorption at 493−
495 nm. The different aryl groups in position 8 of the complex
caused neither a significant blue nor a significant red shift. The
maximum of the emission signal was measured to be between
505 and 520 nm, resulting in an overlay between absorption
and emission spectra (Figure 4a) and a small Stokes shift of
12−25 nm. As documented for various BODIPY complexes
and other bis(dipyrrinato)metal complexes,3,13,16 the com-
plexes 2−4, which were designed with a steric hindrance of the
rotation of the aryl groups, were found to have highly increased
fluorescence quantum yields (ΦF) in comparison to the
unsubstituted, model compound 1. As the compound with the
strongest fluorescence signal, 3 was identified as the lead
compound of the series. Interestingly, the ΦF values of
compounds 1−4 vary highly based on the solvent employed
during the measurements (Figure 4b). Fluorescence was found
to be very intense in nonpolar solvents while it was quenched
in polar solvents. Recent investigations have demonstrated that
this effect was caused by the formation of a nonemissive
symmetry-breaking charge transfer state, which is only formed
in polar solvents.17 It is worthy of note that commercially
available dyes were found to have much higher ΦF values than
the compounds investigated in this study while being relatively
insensitive to the polarity of the solvent.3,4,8
Stability of 1−4. As an important parameter for an
imaging agent, its stability in different solvents as well as upon
light exposure was evaluated, as previous investigations showed
that this could be critical for metal complexes.35,36 The
complexes were incubated in toluene, water, and cell culture
media (DMEM) and the corresponding absorbance spectra
monitored over 48 h. As no significant difference (Figure S17−
S28) was detected, the stability of the complexes was
confirmed. In addition, the stability in toluene upon constant
LED irradiation was assessed. Only minimal changes in the
absorption spectra (Figure S29−S32) were recorded, demon-
strating the photostability of the compounds. In comparison,
commercially available dyes are also associated with a high
chemical stability and stability upon light exposure.3,4,8
Determination of the logP Values of 1−4. The
lipophilicity/hydrophilicity of the compounds was assessed
by determination of the logP values between an aqueous
phosphate buffer saline (PBS) phase and an octanol phase
using the “shake-flask” method.37,38 All compounds were
majorly present in the organic phase with logP values between
0.4−1.1 (Table S4), indicative of their lipophilicity. This result
was expected as the complexes are uncharged and bear
lipophilic aromatic systems.
Cellular Uptake and Uptake Mechanism of 1−4. For
further assessment, the cellular uptake of the compounds was
investigated in human cervical carcinoma (HeLa) cells by
measuring the amount of Zn inside the cells after subtracting
the natural amount using inductively coupled plasma mass
spectrometry (ICP-MS). Importantly, the investigations
showed that all compounds were able to be internalized inside
the cells. In agreement with the logP values, compound 4 was
found to be able to reach a slightly higher concentration inside
the cell compared to 1−3 after 4 h of incubation (Figure S33).
Furthermore, the internalization mechanism of the compounds
by blocking different pathways with metabolic (2-deoxy-D-
glucose and oligomycin), cationic transporter (tetraethylam-
monium chloride), and endocytotic (ammonium chloride or
chloroquine) inhibitors was investigated (Figure S35−S38). As
these inhibitors had only a negligible effect on the uptake, the
involvement of these pathways was excluded. The incubation
at lower temperature (4 °C, Figure S34−S37) demonstrated a
slightly lower uptake, which is caused by the temperature
dependency of diffusion processes at a lower temperature.
Overall, these results suggest an uptake by an energy-
independent passive diffusion pathway for all compounds.
Cellular Localization of 1−4. The cellular localization
was determined by confocal laser scanning microscopy in
HeLa cells (Figure 5a) and comparison of the distribution
G
DOI: 10.1021/acs.inorgchem.9b02019
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 6. Characterization of 3-NP. (a) Size distribution determined by DLS. (b) Transmission electron microscopy (TEM) image.
Figure 7. (a) Confocal luminescence image of HeLa cells incubated for 4 h with 3-NP (Zn, 10 μM, λex = 514 nm, λem = 530−610 nm) and
LysoTracker Deep Red (LTR, 500 nM, λex = 633 nm, λem = 650−720 nm) at 37 °C in the dark. The Pearson correlation coefficient of Zn and LTR
was found to be 0.62 and the Mander’s overlap coefficient 0.83. (b) Subcellular distribution (Cell = whole cell, Lys. = lysosome, Cyto. = cytoplasm,
Mito. = mitochondria, Nuc. = nucleus) of 3-NP in HeLa cells after 4 h incubation in the dark determined after organelle extraction and
determination of the amount of Zn inside each organelle by ICP-MS.
pattern inside the cells with the commercial dyes MitoTracker
Deep Red, LysoTracker Deep Red, and Hoechst 33342. As no
significant colocalization was detected, this indicates that the
major localization of the complexes 1−4 was not inside these
organelles. For verification, the localization of the complexes
was also investigated by extraction of the major cellular
organelles (i.e., nucleus, mitochondria, lysosome, cytoplasm)
and quantification of the amount of Zn inside using ICP-MS.
In agreement with the microscopy studies, all complexes 1−4
were found to be majorly present in the cytoplasm (Figure 5b).
It is worthy of note that structurally related bis(dipyrrinato)-
zinc(II) complexes as well as related Zn(II) porphyrin
complexes previously investigated were also found to be
mainly localizing unselectively in the cell or with membrane
proteins.39,40
(Photo)cytotoxicity of 1−4. The cytotoxicity of the
complexes in the dark as well as upon irradiation at 510 nm
(20 min, 5.0 J/cm2) and 540 nm (40 min, 9.5 J/cm2) toward
noncancerous retinal pigment epithelium (RPE-1) and human
cervical carcinoma (HeLa) cells was investigated (Table S5) as
it was previously shown that this could be problematic for
imaging agents.41 All complexes 1−4 were found to be
nontoxic in the dark as well as upon irradiation (IC50 > 100
μM). This is an important requirement for an imaging probe as
these agents should not trigger any kind of stress or toxicity for
the cell.
H
DOI: 10.1021/acs.inorgchem.9b02019
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Nanoparticle Formation (3-NP) and Characterization.
Despite the success of the investigated compounds to enter
HeLa cells with a green fluorescence in the cytoplasm, the
abilities of bis(dipyrrinato)zinc(II) complexes to act as imaging
probes is limited due to (1) the quenching of the excited state
in polar solvents, (2) water solubility problems, as well as (3)
the lack of selectivity of the compounds for cancerous cells. To
overcome these limitations, the lead compound 3 with the
brightest fluorescence was encapsulated in the polymer matrix
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotin-
and minorly in the surrounding cytoplasm. A recent study of
the encapsulation of organic aromatic systems or structurally
related bis(dipyrrinato)zinc(II) complexes with a DSPE-
PEG2000-OCH3 matrix for photodynamic therapy, aggregating
induced emission, and 2-photon imaging also showed them to
be mainly localizing in this organelle.43,44
3D Multicellular Tumor Spheroid Penetration of 3-
NP. For further evaluation of the imaging properties of 3-NP,
its fluorescence in 3D multicellular tumor spheroids (MCTS)
was investigated. It is worthy of note that the application of
many investigated molecules for diagnosis or therapy is
partially limited due to compromised delivery.45−47 Therefore,
in this study, the penetration of 3-NP toward large MCTS with
a diameter of 800 μM was investigated by confocal laser
scanning microscopy. As the luminescence signal was
measurable at every depth (Figure S40), a full penetration of
the particles was confirmed.
Selectivity Assay of 3-NP between Cancerous and
Noncancerous Cells. As 3-NP is mainly taken up through
the SMVT transporter, these nanoparticles could show a
significant cancer cell selectivity. The targeting effect of 3-NP
was investigated by mixing transfected cancerous and non-
transfected noncancerous human lung cells. More precisely,
cancerous adenocarcinomic human alveolar basal epithelial
cells (A549), which have a biotin receptor, were transfected
with the fluorescence protein mCherry and mixed with
noncancerous human lung fibroblasts (HLF) cells. Not
successfully transfected cells were removed using an antibiotic
(G-418). Using confocal laser scanning microscopy (Figure 8),
we could show that 3-NP was only internalized in the A549
cells, which were transfected with mCherry, demonstrating its
ability as a cancer cell selective imaging agent.
(polyethylene glycol)-2000] ammonium salt (DSPE-PEG2000
-
biotin) to generate the corresponding nanoparticle formulation
3-NP with a biotin end group as a targeting moiety. It is
worthy of note that PEGylated phospholipids have been
approved by the US Food and Drug administration (FDA) for
medical applications. The generated nanoparticles 3-NP were
found to be spherical shaped with an average size of 93 nm, as
determined by dynamic light scattering (DLS, Figure 6a), and
104 nm, as determined by transmission electron microscopy
(TEM, Figure 6b). Very importantly, the particles benefit from
a high water solubility. The photophysical evaluation of 3-NP
showed no significant difference in the absorption or emission
spectra between 3 and 3-NP, indicating that the photophysical
properties are not being influenced by the encapsulation. The
nanoparticles were found to have a ΦF value of 0.26, which is
in the same range as the complex alone in an apolar solvent.
Overall, this encapsulation allows overcoming the quenching
effects associated with bis(dipyrrinato)zinc(II) complexes. The
cytotoxicity of 3-NP in the dark as well as upon irradiation at
500 nm (16.7 min, 10.0 J/cm2) in HeLa cells was investigated.
As expected, 3-NP was found to be nontoxic (IC50 > 100 μM),
as requested for an imaging agent. The release of the complex
from the nanoparticle formulation was studied by dialysis
against a PBS solution at physiological pH (7.4) and lowered
pH (5.0). As expected, the formulation remains intact at
physiological pH whereas the complex is released at low pH
(Figure S38).
Cellular Uptake and Uptake Mechanism of 3-NP. The
cellular internalization and uptake mechanism of 3-NP was
investigated by blocking different pathways through preincu-
bation with various inhibitors (Figure S39). In addition to the
pathways investigated for 1−4, the sodium-dependent multi-
vitamin transporter (SMVT) as a potential internalization
mechanism was studied as biotin as well as many biotin
conjugates are majorly taken up through this pathway.42 As the
incubation with pantothenic acid and lipoic acid drastically
decreased the amount of the compound inside the cell, the
uptake through SMVT was assumed to be the primary
pathway. In addition, the incubation with metabolic inhibitors
as well as upon lower temperature (4 °C) decreased the
amount of the compound inside the cell, suggesting an energy-
dependent mechanism. Furthermore, the incubation with
endocytotic inhibitors also decreased the internalization.
Overall, these results suggest that 3-NP is majorly taken up
through the SMVT transporter while minor amounts are taken
up by endocytosis.
Cellular Localization of 3-NP. The imaging properties of
the particles 3-NP were further studied by determination of
their localization within HeLa cells using confocal laser
scanning microscopy (Figure 7a) as well as ICP-MS after the
corresponding organelle extraction (Figure 7b). Interestingly,
whereas complex 3 accumulated in the cytoplasm, its
nanoformulation 3-NP was majorly found in the lysosomes
Figure 8. Confocal luminescence image of a mixture of mCherry (λex
= 633 nm, λem = 650−720 nm) transfected cancerous adenocarci-
nomic human alveolar basal epithelial cells (A549) and noncancerous
human lung fibroblasts (HLF) cells incubated for 4 h with 3-NP (Zn,
10 μM, λex = 514 nm, λem = 530−610 nm) at 37 °C in the dark.
I
DOI: 10.1021/acs.inorgchem.9b02019
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
CONCLUSIONS
REFERENCES
■
■
(1) Rao, J.; Dragulescu-Andrasi, A.; Yao, H. Fluorescence imaging in
vivo: recent advances. Curr. Opin. Biotechnol. 2007, 18 (1), 17−25.
(2) Leblond, F.; Davis, S. C.; Valdes, P. A.; Pogue, B. W. Pre-clinical
In summary, we have prepared a series of bis(dipyrrinato)-
zinc(II) complexes as imaging probes and evaluated in-depth
their photophysical and biological properties. The compounds
were found to have a strong fluorescence in apolar environ-
ments whereas their fluorescence was quenched in polar
solvents. The complexes were found to majorly accumulate in
the cytoplasm of HeLa cells by passive diffusion. To overcome
the limitation of these compounds (e.g., quenching of the
excited state in water, water solubility problems, and poor
selectivity for cancer cells), the lead compound 3 was
encapsulated in a polymer matrix with a biotin end group.
The generated nanoparticles were found to have a bright
luminescence, to selectively accumulate in the lysosomes of
cancer cells, and to fully penetrate 3D MCTS. As a benefit
from the biotin targeting moiety, the nanoparticles selectively
accumulated in cancerous cells over noncancerous cells.
Overall, we strongly believe that this class of compounds and
their corresponding nanoparticles have a great potential as
bright, selective imaging probes for cancer cells.
́
whole-body fluorescence imaging: Review of instruments, methods
and applications. J. Photochem. Photobiol., B 2010, 98 (1), 77−94.
(3) Loudet, A.; Burgess, K. BODIPY Dyes and Their Derivatives:
Syntheses and Spectroscopic Properties. Chem. Rev. 2007, 107 (11),
4891−4932.
(4) Benniston, A. C.; Copley, G. Lighting the way ahead with boron
dipyrromethene (Bodipy) dyes. Phys. Chem. Chem. Phys. 2009, 11
(21), 4124−4131.
(5) Wagner, R. W.; Lindsey, J. S. Boron-dipyrromethene dyes for
incorporation in synthetic multi-pigment light-harvesting arrays. Pure
Appl. Chem. 1996, 68 (7), 1373−1380.
(6) Yee, M. C.; Fas, S. C.; Stohlmeyer, M. M.; Wandless, T. J.;
Cimprich, K. A. A cell-permeable, activity-based probe for protein and
lipid kinases. J. Biol. Chem. 2005, 280 (32), 29053−29059.
(7) Karolin, J.; Johansson, L. B. A.; Strandberg, L.; Ny, T.
Fluorescence and Absorption Spectroscopic Properties of Dipyrro-
metheneboron Difluoride (BODIPY) Derivatives in Liquids, Lipid
Membranes, and Proteins. J. Am. Chem. Soc. 1994, 116 (17), 7801−
7806.
(8) Boens, N.; Leen, V.; Dehaen, W. Fluorescent indicators based on
BODIPY. Chem. Soc. Rev. 2012, 41 (3), 1130−1172.
ASSOCIATED CONTENT
■
S
* Supporting Information
(9) Rezende, L. C. D.; Emery, F. S. A Review of the Synthetic
Strategies for the Development of BODIPY Dyes for Conjugation
with Proteins. Orbital: Electron. J. 2013, 5 (1).
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b02019.
(10) Villemin, E.; Ong, Y. C.; Thomas, C. M.; Gasser, G. Polymer
encapsulation of ruthenium complexes for biological and medicinal
applications. Nat. Rev. Chem. 2019, 3, 261−282.
(11) Larson, N.; Ghandehari, H. Polymeric Conjugates for Drug
Delivery. Chem. Mater. 2012, 24 (5), 840−853.
(12) Steichen, S. D.; Caldorera-Moore, M.; Peppas, N. A. A review
of current nanoparticle and targeting moieties for the delivery of
cancer therapeutics. Eur. J. Pharm. Sci. 2013, 48 (3), 416−427.
(13) Sakamoto, R.; Iwashima, T.; Tsuchiya, M.; Toyoda, R.;
1
Syntheses and H and 13C NMR, ESI-HRMS, and UV/
vis spectra for 1−4; cell uptake mechanicm studies;
crystal data and structure refinement parameters (PDF)
Accession Codes
CCDC 1937531−1937534 contain the supplementary crys-
tallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
̈
Matsuoka, R.; Kogel, J. F.; Kusaka, S.; Hoshiko, K.; Yagi, T.;
Nagayama, T. New aspects in bis and tris (dipyrrinato) metal
complexes: bright luminescence, self-assembled nanoarchitectures,
and materials applications. J. Mater. Chem. A 2015, 3 (30), 15357−
15371.
(14) Baudron, S. A. Luminescent dipyrrin based metal complexes.
Dalton Trans 2013, 42 (21), 7498−7509.
AUTHOR INFORMATION
■
(15) Wood, T. E.; Thompson, A. Advances in the Chemistry of
Dipyrrins and Their Complexes. Chem. Rev. 2007, 107 (5), 1831−
1861.
Corresponding Authors
*(H.C.) E-mail: ceschh@mail.sysu.edu.cn, Tel. +86
2084110613.
*(G.G.) E-mail: gilles.gasser@chimieparistech.psl.eu; Tel. +33
144275602.
(16) Sazanovich, I. V.; Kirmaier, C.; Hindin, E.; Yu, L.; Bocian, D.
F.; Lindsey, J. S.; Holten, D. Structural Control of the Excited-State
Dynamics of Bis(dipyrrinato)zinc Complexes: Self-Assembling
Chromophores for Light-Harvesting Architectures. J. Am. Chem. Soc.
2004, 126 (9), 2664−2665.
ORCID
(17) Trinh, C.; Kirlikovali, K.; Das, S.; Ener, M. E.; Gray, H. B.;
Djurovich, P.; Bradforth, S. E.; Thompson, M. E. Symmetry-Breaking
Charge Transfer of Visible Light Absorbing Systems: Zinc Dipyrrins.
J. Phys. Chem. C 2014, 118 (38), 21834−21845.
Hui Chao: 0000-0003-4153-5303
Gilles Gasser: 0000-0002-4244-5097
Notes
(18) Kusaka, S.; Sakamoto, R.; Kitagawa, Y.; Okumura, M.;
Nishihara, H. An extremely bright heteroleptic bis (dipyrrinato)
zinc (II) complex. Chem. - Asian J. 2012, 7 (5), 907−910.
(19) Sakamoto, R.; Kusaka, S.; Kitagawa, Y.; Kishida, M.-a.; Hayashi,
M.; Takara, Y.; Tsuchiya, M.; Kakinuma, J.; Takeda, T.; Hirata, K.
Fluorescent azadipyrrinato zinc (ii) complex: hybridisation with a
dipyrrinato ligand. Dalton Trans 2012, 41 (46), 14035−14037.
(20) Lee, S.; Seok, C.-H.; Park, Y.; Lee, A.; Jung, D. H.; Choi, S.-H.;
Park, J. Enforced Effects of Side Group Substitution Position on
Luminescence Properties; Synthesis of Bis(dipyrrinato)zinc Complex
Derivatives. Mol. Cryst. Liq. Cryst. 2010, 531 (1), 365−372.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by an ERC Consolidator
Grant PhotoMedMet to G.G. (GA 681679) and has received
support under the program “Investissements d’Avenir”
launched by the French Government and implemented by
the ANR with the reference ANR-10-IDEX-0001-02 PSL
(G.G.), the National Science Foundation of China (Nos.
21525105 and 21778079 for H.C.), and the 973 Program (No.
2015CB856301 for H.C.).
̈
(21) Sakamoto, R.; Iwashima, T.; Kogel, J. F.; Kusaka, S.; Tsuchiya,
M.; Kitagawa, Y.; Nishihara, H. Dissymmetric Bis(dipyrrinato)zinc-
J
DOI: 10.1021/acs.inorgchem.9b02019
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(II) Complexes: Rich Variety and Bright Red to Near-Infrared
Luminescence with a Large Pseudo-Stokes Shift. J. Am. Chem. Soc.
2016, 138 (17), 5666−5677.
evidence of sodium-dependent multivitamin transporter (SMVT). Int.
J. Pharm. 2013, 441 (1−2), 535−543.
(43) Zang, Q.; Yu, J.; Yu, W.; Qian, J.; Hu, R.; Tang, B. Z. Red-
emissive azabenzanthrone derivatives for photodynamic therapy
irradiated with ultralow light power density and two-photon imaging.
Chem. Sci. 2018, 9 (23), 5165−5171.
(44) Karges, J.; Basu, U.; Blacque, O.; Chao, H.; Gasser, G.,
Polymeric Encapsulation of Novel Homoleptic Bis(dipyrrinato)
Zinc(II) Complexes with Long Lifetimes for Applications as
Photodynamic Therapy Photosensitisers. Angew. Chem., Int. Ed.
2019 DOI: 10.1002/anie.201907856. Pluen, A.; Boucher, Y.;
Ramanujan, S.; McKee, T. D.; Gohongi, T.; di Tomaso, E.; Brown,
E. B.; Izumi, Y.; Campbell, R. B.; Berk, D. A.; Jain, R. K. Role of
tumor−host interactions in interstitial diffusion of macromolecules:
Cranial vs. subcutaneous tumors. Proc. Natl. Acad. Sci. U. S. A. 2001,
98 (8), 4628−4633.
(45) Netti, P. A.; Berk, D. A.; Swartz, M. A.; Grodzinsky, A. J.; Jain,
R. K. Role of Extracellular Matrix Assembly in Interstitial Transport in
Solid Tumors. Cancer Res. 2000, 60 (9), 2497−2503.
(46) Friedrich, J.; Seidel, C.; Ebner, R.; Kunz-Schughart, L. A.
Spheroid-based drug screen: considerations and practical approach.
Nat. Protoc. 2009, 4, 309.
(47) Goodman, T. T.; Ng, C. P.; Pun, S. H. 3-D Tissue Culture
Systems for the Evaluation and Optimization of Nanoparticle-Based
Drug Carriers. Bioconjugate Chem. 2008, 19 (10), 1951−1959.
(22) Tsuchiya, M.; Sakamoto, R.; Shimada, M.; Yamanoi, Y.;
Hattori, Y.; Sugimoto, K.; Nishibori, E.; Nishihara, H. Bis
(dipyrrinato) zinc (II) complexes: emission in the solid state. Inorg.
Chem. 2016, 55 (12), 5732−5734.
(23) Maiti, S.; Paira, P. Biotin conjugated organic molecules and
proteins for cancer therapy: A review. Eur. J. Med. Chem. 2018, 145,
206−223.
(24) Collina, S. New perspectives in cancer therapy: the biotin-
antitumor molecule conjugates. Med. Chem. 2014, 8, 1−4.
(25) Roth, K. S. Biotin in clinical medicinea review. Am. J. Clin.
Nutr. 1981, 34 (9), 1967−1974.
(26) Rigaku Oxford Diffraction, 2015.
(27) Clark, R.; Reid, J. The analytical calculation of absorption in
multifaceted crystals. Acta Crystallogr., Sect. A: Found. Crystallogr.
1995, 51 (6), 887−897.
(28) Crys Alis Pro (version 1.171.39.13a), Rigaku Oxford Diffraction,
2016.
(29) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A.
K.; Puschmann, H. OLEX2: a complete structure solution, refinement
and analysis program. J. Appl. Crystallogr. 2009, 42 (2), 339−341.
(30) Sheldrick, G. M. SHELXT−Integrated space-group and crystal-
structure determination. Acta Crystallogr., Sect. A: Found. Adv. 2015,
71 (1), 3−8.
(31) Sheldrick, G. M. Crystal structure refinement with SHELXL.
Acta Crystallogr., Sect. C: Struct. Chem. 2015, 71 (1), 3−8.
(32) Macrae, C. F.; Edgington, P. R.; McCabe, P.; Pidcock, E.;
Shields, G. P.; Taylor, R.; Towler, M.; Streek, J. v. d. Mercury:
visualization and analysis of crystal structures. J. Appl. Crystallogr.
2006, 39 (3), 453−457.
(33) Brouwer, A. M. Standards for photoluminescence quantum
yield measurements in solution (IUPAC Technical Report). Pure
Appl. Chem. 2011, 83 (12), 2213−2228.
(34) Spek, A. L. PLATON SQUEEZE: a tool for the calculation of
the disordered solvent contribution to the calculated structure factors.
Acta Crystallogr., Sect. C: Struct. Chem. 2015, 71, 9−18.
(35) Basu, U.; Karges, J.; Chotard, F.; Balan, C.; Le Gendre, P.;
Gasser, G.; Bodio, E.; Malacea Kabbara, R., Investigation of photo-
activation on Ruthenium(II)-arene complexes for the discovery of
potential selective cytotoxic agents. Polyhedron 2019, DOI: 10.1016/
j.poly.2019.02.041.
(36) Renfrew, A. K.; Karges, J.; Scopelliti, R.; Bobbink, F. D.;
Nowak-Sliwinska, P.; Gasser, G.; Dyson, P., Towards Light Activated
Ruthenium-Arene (RAPTA-type) Prodrug Candidates. ChemBioChem
2019, DOI: 10.1002/cbic.201900236.
(37) Karges, J.; Goldner, P.; Gasser, G. Synthesis, Characterization,
and Biological Evaluation of Red-Absorbing Fe (II) Polypyridine
Complexes. Inorganics 2019, 7 (1), 4.
(38) Karges, J.; Blacque, O.; Jakubaszek, M.; Goud, B.; Goldner, P.;
Gasser, G. Systematic investigation of the antiproliferative activity of a
series of ruthenium terpyridine complexes. J. Inorg. Biochem. 2019,
198, 110752.
(39) Dab̧ rowski, J. M.; Pucelik, B.; Pereira, M. M.; Arnaut, L. G.;
Stochel, G. Towards tuning PDT relevant photosensitizer properties:
comparative study for the free and Zn2+ coordinated meso-
tetrakis[2,6-difluoro-5-(N-methylsulfamylo)phenyl]porphyrin. J.
Coord. Chem. 2015, 68 (17−18), 3116−3134.
(40) Pavani, C.; Uchoa, A. F.; Oliveira, C. S.; Iamamoto, Y.; Baptista,
M. S. Effect of zinc insertion and hydrophobicity on the membrane
interactions and PDT activity of porphyrin photosensitizers. Photo-
chem. Photobiol. Sci. 2009, 8 (2), 233−240.
(41) Alford, R.; Simpson, H. M.; Duberman, J.; Hill, G. C.; Ogawa,
M.; Regino, C.; Kobayashi, H.; Choyke, P. L. Toxicity of Organic
Fluorophores Used in Molecular Imaging: Literature Review. Mol.
Imaging 2009, 8 (6), 341−354.
(42) Vadlapudi, A. D.; Vadlapatla, R. K.; Pal, D.; Mitra, A. K. Biotin
uptake by T47D breast cancer cells: functional and molecular
K
DOI: 10.1021/acs.inorgchem.9b02019
Inorg. Chem. XXXX, XXX, XXX−XXX