Polymeric Encapsulation of Novel Homoleptic Bis(dipyrrinato) Zinc(II) Complexes with Long Lifetimes for Applications as Photodynamic Therapy Photosensitisers

Article abstract of DOI:10.1002/anie.201907856

The use of photodynamic therapy (PDT) to treat cancer has received increasing attention over the last years. However, the clinically used photosensitisers (PSs) have some limitations that include poor aqueous solubility, hepatotoxicity, photobleaching, aggregation, and slow clearance from the body, so the design of new classes of PSs is of great interest. We present the use of bis(dipyrrinato)zinc(II) complexes with exceptionally long lifetimes as efficient PDT PSs. Based on the heavy-atom effect, intersystem crossing of these complexes changes the excited state from singlet to a triplet state, thereby enabling singlet oxygen generation. To overcome the limitation of quenching effects in water and improve water solubility, the lead compound 3 was encapsulated in a polymer matrix. It showed impressive phototoxicity upon irradiation at 500 nm in various monolayer cancer cells as well as 3D multicellular tumour spheroids, without observed dark toxicity.

Full text of DOI:10.1002/anie.201907856

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: Polymeric Encapsulation of Novel Homoleptic Bis(dipyrrinato)
Zinc(II) Complexes with Long Lifetimes for Applications as
Photodynamic Therapy Photosensitisers
Authors: Johannes Karges, Uttara Basu, Olivier Blacque, Hui Chao,
and Gilles Gasser
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201907856
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10.1002/anie.201907856
Angewandte Chemie International Edition
RESEARCH ARTICLE
Polymeric Encapsulation of Novel Homoleptic Bis(dipyrrinato)
Zinc(II) Complexes with Long Lifetimes for Applications as
Photodynamic Therapy Photosensitisers
Johannes Karges,[a] Uttara Basu,[a] Olivier Blacque,[b] Hui Chao,*[c] and Gilles Gasser*[a]
Abstract: The use of photodynamic therapy (PDT) to treat various
types of cancer has received increasing attention over the last years.
However, the clinically used photosensitisers (PSs) have some
limitations that include poor aqueous solubility, hepatotoxicity,
photobleaching, aggregation and slow clearance from the body,
leading to photosensitivity. To circumvent these drawbacks, the
design of new classes of PSs is of high interest. Herein, we present
the first example of the use of bis(dipyrrinato)zinc(II) complexes with
exceptionally long lifetimes as efficient PDT PSs. Using the heavy
atom effect, we could promote the intersystem crossing (ISC) of these
complexes to change the character of the excited state from singlet to
a triplet state, enabling singlet oxygen (1O2) generation. To overcome
the limitation of quenching effects in water as well as to improve water
solubility, the lead compound of this study 3 was encapsulated in a
polymer matrix. While having no observed dark toxicity, it showed an
impressive phototoxicity upon irradiation at 500 nm in various
monolayer cancer cells as well as 3D multicellular tumour spheroids
(MCTS).
the translational status of PDT still remains unsatisfactory. Ideal
photosensitizers must be chemically pure, stable in biological
fluids while being efficient reactive oxygen species (ROS)
generators.[1]
PDT relies on the photoactivation of a preferably non-toxic PS in
the presence of oxygen to create ROS. During this process, the
PS is excited to a singlet state (1PS) from which it undergoes
intersystem crossing (ISC) to a longer-lived triplet state (3PS). In
a type I mechanism, an electron or proton is transferred from or
to the excited 3PS to or from the biological environment involving
•
-
•
•
commonly O2 , OOH or OH radicals. In contrast, in a type II
mechanism, the energy of the excited 3PS is transferred to
molecular oxygen (3O2) to generate singlet oxygen (1O2). These
species are highly reactive and can directly react with its biological
surroundings to trigger cell death. Porphyrins, chlorins and
pthalocyanines form the basic structure of many PSs, which are
not only difficult to synthesise and to purify but also suffer from
other drawbacks, including poor aqueous solubility, aggregation,
slow clearance from the body and hepatotoxicity.[2] It would be
therefore of high interest to develop new PSs based on a half-
porphyrin unit. This design would retain the excited state
properties of porphyrins while being synthetically less challenging.
Moreover, to overcome the limitations of the current PSs, the use
of metal complexes has emerged as an interesting alternative due
to their attractive photo-photophysical properties (e.g.,
photostability, high 1O2 production), high water solubility and
chemical stability. In this field, most well-studied transition metal
complexes are those of Ru(II)[3], Os(II)[4], Rh(III)[4b, 5] and Ir(III)[3l, 4c,
6]. Such complexes have shown some extremely promising results
with one of such having completed phase I clinical trial.[3c]
However, the metal ions employed in these complexes are not
abundant and are often seen unfavourably by industrial partners
due to potential toxicity. It is therefore essential to develop
complexes based on cheaper, abundant, biologically-relevant
metals that have similar or superior photophysical and
pharmacological properties.
Introduction
Photodynamic Therapy (PDT) is a minimally-invasive medical
technique, which has received increasing attention in the recent
years with applications in oncology, dermatology and
ophthalmology. Photofrin® was the first clinically approved
photosensitiser (PS). It is used to treat various types of cancer
(i.e., non-small lung, bladder, oesophageal, or brain cancer). The
second generation of PSs including, for example, Temoporfin
(Foscan®), Motexafin lutetium, Palladium bacteriopheophorbide
(Tookad® soluble), Purlytin®, Verteporfin (Visudyne®), Talaporfin
(Laserphyrin®) are either clinically approved or were/are currently
undertaking clinical trials. Despite several advanced research and
preclinical studies using the first and second generation of PSs,
[a]
J. Karges, Dr. Uttara Basu, Dr. Gilles Gasser
During the last decade, the use of compounds with a 4,4-difluoro-
4-bora-3a,4a-diaza-s-indacene core commonly called boron-
dipyrromethene (BODIPY) have emerged as promising PDT
agents. BODIPY complexes are well known to be excited to a
singlet state (1PS) and to relax from there in very high quantum
yields causing a strong fluorescence. This allows them to be
majorly known as fluorescence imaging probes.[7] However, this
feature is undesirable for PDT as the majority of the absorbed
energy does not cross to the required triplet state (3PS). A
comprehensive review on the development of these dyes as
potential PDT agents discusses in detail the strategies that have
been adopted to attenuate their fluorescence and enhance their
triplet excited state lifetime.[8] These compounds were found to be
amenable to extensive modification around the central core to
Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for
Life and Health Sciences, Laboratory for Inorganic Chemical Biology,
75005 Paris, France.
gilles.gasser@chimieparistech.psl.eu; www.gassergroup.com
[b]
[c]
Dr. O. Blacque
Department
of
Chemistry,
University
of
Zurich,
Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.
Prof. Hui Chao
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry,
School of Chemistry, Sun Yat-sen University, 510275 Guangzhou,
People’s Republic of China.
ceschh@mail.sysu.edu.cn
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201907856
Angewandte Chemie International Edition
RESEARCH ARTICLE
feature many attractive properties including high extinction
coefficients, resistance to photobleaching, high cellular uptake as
well as low toxicity for PDT applications.[8-9] For use in PDT, the
dipyrrin core structure can be modified by utilising the spin-orbit
coupling of heavy atoms, such as iodine, to promote the ISC.[8-9,
positions 2 and 6 were iodinated using a mixture of iodine and
iodic acid. Finally, the complexes 1-4 were synthesised by
.
complexation of the dipyrrin ligands with Zn(OAc)2 2H2O using
Et3N as a base. All complexes were analysed using 1H, 13C-NMR,
HRMS and the purity of the compounds were verified by
elemental analysis. Additionally, the complexes 1-3 have been
characterised using single crystal X-ray crystallography. Details
on the synthesis and characterisation of the complexes can be
found in the SI (Scheme S1, Table S1, Figure S1-S15).
10]
Importantly, a recent study has shown that the position of
substitution on the BODIPY core has a drastic effect on the ISC.
For example, iodination at positions 3 and 5 increases the
fluorescence process.[10c] To promote the ISC, the heavy atom is
typically placed without the disruption of the planarity and
conjugation of the system.[8] Very importantly, the introduction of
heavy atoms also shifts the absorption of the PS to longer
wavelengths, which can be used to achieve deeper tissue
penetration making them potentially be applicable for treating
deep-seated tumours or large tumours.[11] Worthy of note, the
majority of investigated metal complexes for PDT applications use
blue or UV-A light, which limits the penetration inside the tissue.[12]
Apart from boron, the dipyrrin scaffold can coordinate to other
metals including Ni(II), Zn(II), Cu(II), Fe(III), Rh(III) and Co(III).
These compounds have been studied since nearly a century in
view of material science for generation of suitable
nanoarchitectures including coordination polymers, one-
dimensional nanowires, two-dimensional nanosheets and metal –
organic frameworks (MOFs).[13] As an unrepresented class of
1
R
7
1
2
2
3
6
5
I
I
8
I
I
N
N
N
N
R =
Zn
3
4
R
compounds,
in
the
recent
years,
the
study
of
Figure 1. Chemical structures of the Zn(II) complexes 1-4 investigated in this
work.
bis(dipyrrinato)zinc(II) complexes have gained interest in view of
their luminescence properties.[13a, 14]
With this knowledge in mind, in this work, we have designed
homoleptic bis(dipyrrinato)zinc(II) complexes as potential PDT
agents. As the atom with the strongest heavy atom effect of the
halogens, we attached iodine in position 2 and 6 to avoid
interfering with the planarity of the conjugated system. To improve
the photophysical properties, peripheral bulky aryl groups were
introduced in position 8 of the dipyrrin core. As shown for BODIPY
complexes as well as bis(dipyrrinato)zinc(II) complexes, these
groups are able to inhibit the internal rotation and highly increase
their photophysical properties.[7a, 13a, 14a] Herein, we present the
synthesis as well as in-depth photophysical and biological
evaluation of a series of homoleptic bis(dipyrrinato)zinc(II)
complexes 1-4 (Figure 1), which were found to accumulate in the
cytoplasm. As the excited state of the bis(dipyrrinato)zinc(II)
complexes is able to be significantly quenched in a polar
environment, the lead compound 3 was encapsulated in a
polymer matrix to overcome this drawback. The complexes as
well as the encapsulated version 3-NP were found to efficiently
generate singlet oxygen upon light exposure causing cell death in
various monolayer cancer cell lines as well as 3D multicellular
tumour spheroids (MCTS), while not showing any dark toxicity. To
the best of our knowledge, these are the first examples of
bis(dipyrrinato)zinc(II) complexes for potential applications in PDT.
The photophysical properties of the investigated compounds are
presented in Table S2. As expected from previous literature, the
complexes have a strong absorbance peak at 516 nm (Figure
S16), which is sligthly red shifted in comparison to other reported
homoleptic bis(dipyrrinato)zinc(II) complexes due to the heavy
14a, 14b, 14d]
atom effect.[13a,
The maximum of the luminescence
signal was measured between 535-550 nm (Figure S17) resulting
in a Stokes shift of 18-34 nm, demonstrating an overlap between
absorption and emission spectra. Compound 4 (1.1%) and
especially compounds 2 and 3 (4.2-4.5%) were found to have high
luminescence quantum yields in comparison to the model
compound 1 (0.3%) due to the hindrance of the internal rotation
of the aryl group in position 8. This effect has been documented
for various BODIPY complexes and other bis(dipyrrinato)metal
13a, 14a]
complexes.[7a,
All complexes were found to have
exceptionally long lifetimes (Figure S18-S21, Table S2) in
comparison to other bis(dipyrrinato)metal complexes in the range
14]
of 207-559 ns.[13a,
These long lifetimes indicate that the
complexes are majorly luminescent due to their longer lived triplet
state, which is being formed due to the heavy atom effect.
Importantly, the presence of air drastically decreased the lifetime,
illustrating that the excited state can interact with a component of
the air. The type of ROS generated was confirmed using electron
spin
resonance
spectroscopy
(ESR)
with
2,2,6,6–
tetramethylpiperidine (TEMP) as a singlet oxygen 1O2 scavenger
and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a •OOH or •OH
radical scavenger. While no signal for the formation of a •OOH or
•OH radical was detected, the formation of 1O2 in MeOH and PBS
was confirmed by the characteristic O2-induced triplet signal of
2,2,6,6-tetramethylpiperidinyloxyl (TEMPO) in the ESR spectrum
(Figure S22-S25). Worthy of note, the majority of clinically used
Results and Discussion
The dipyrrin ligands were synthesised starting from 2, 4-
dimethylmethylpyrrole with the corresponding aldehydes in the
presence of catalytic amounts of trifluoracetic acid and then
oxidised with tetrachloro-p-benzoquinone. Subsequently, the
1
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10.1002/anie.201907856
Angewandte Chemie International Edition
RESEARCH ARTICLE
PSs act by the generation of 1O2.[15] The generation of 1O2 of the
investigated compounds has been quantified by two methods; i)
directly by measuring the phosphorescence signal of 1O2 around
1270 nm upon irradiation at 505 nm and ii) indirectly by capturing
1O2 with a reporter molecule and following its production by
absorbance spectroscopy upon irradiation at 510 and 540 nm.[16]
the commercial dyes MitoTracker® Deep Red, LysoTracker Deep
Red and Hoechst 33342 and their distribution pattern compared.
No significant congruency was detected, indicating that the
compounds 1-4 do not majorly localise in these organelles. The
cellular localisation in Hela cells was then quantified in the major
cellular organelles (i.e., nucleus, mitochondria, lysosome,
cytoplasm) using ICP-MS (Figure 2b) and subtraction of the
natural occurring Zn. All complexes 1-4 were found to be present
mostly in the cytoplasm with a small amount of unselective
accumulation in the mitochondria, whereas no signal was
detected inside the nucleus and lysosomes. Interestingly in
previous studies, structurally related Zn(II) porphyrin complexes
were also found to be localising unselectively within the cell with
some interactions with membrane proteins.[18b, 19]
1
Compounds 2-4 were found to have O2 quantum yields (Table
S3) between 43-57% in MeOH and between 2-5% in aqueous
solution. Worthy of note, the generation of 1O2 in an aqueous
environment is much lower as the excited state of the
bis(dipyrrinato) metal complexes is quenched due to the
formation of a symmetry-breaking charge transfer state in a polar
1
environment.[14c] In comparison, the O2 quantum yield of 1 was
drastically lower, which was expected due to its poorer
luminescence properties.
The stability of the complexes in different solvents for up to 48 h
in the dark and upon photo-exposure was studied as it is an
important pharmacological parameter for biological applications.
[17] The complexes were incubated for different times (0, 1, 2, 4, 8,
12, 24, 48 h) in toluene, water and cell culture media (DMEM) and
the corresponding absorbance spectra were recorded. No
significant difference (Figure S26-S37) was observed, indicating
the stability of the complexes under the experimental conditions.
The stability upon irradiation was investigated by constant LED
irradiation at 510 nm and monitoring their absorbance spectra. All
complexes were found with minimal changes in their absorbance
spectra (Figure S38-S41), demonstrating their photostability.
The lipophilicity/hydrophilicity was investigated by determination
of their distribution coefficients between phosphate buffer saline
(PBS) and octanol phases. All complexes 1-4 were found majorly
in the organic phase with logP (Table S4) values between +1.3 -
+2.0. This is expected as the complexes are neutral and bear
large organic lipophilic aromatic systems. This study was followed
by the assessment of the cellular uptake of complexes 1-4 by
determining the amount of Zn after incubating them in human
cervical carcinoma (HeLa) cells for different times (0.5, 1, 2, 4, 8
h) using inductively coupled plasma mass spectrometry (ICP-MS).
Within 4 h, (Figure S42-S45) the maximum metal concentration
was reached and from there on it increased asymptotically.
Worthy of note, structurally related Zn(II) porphyrin derivatives
have been shown to take a much longer incubation time to reach
their uptake maximum.[18] As expected, the comparison of the
Figure 2. a) Confocal luminescence image of HeLa cells incubated with
compounds 1-4 (25 μM, 2% DMSO, v%) for 6 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) Sub-cellular distribution (Cell = Whole
cell, Cyto. = Cytoplasm, Mito. = Mitochondria, Lys. = Lysosome, Nuc. = Nucleus)
of 1-4 (25 μM, 2% DMSO, v%) in HeLa cells after 6 h incubation in the dark
determined after organelle extraction and determination of the amount of Zn
inside each organelle by ICP-MS.
To study the efficacy of the complexes 1-4 as PDT PSs, their
cytotoxicity in the dark and upon irradiation at 510 nm (20 min, 5.0
J/cm2) and 540 nm (40 min, 9.5 J/cm2) towards non-cancerous
retinal pigment epithelium (RPE-1), human cervical carcinoma
(HeLa), mouse colon carcinoma (CT-26) and human glioblastoma
astrocytoma (U373) cells was investigated (Table S5-S6). All
complexes were found to be non-toxic in the dark, which is an
important requirement for a PDT agent (IC50 > 100 μM). Upon
exposure to light at both 510 nm or 540 nm, all complexes were
able to generate 1O2 and trigger cell death in all investigated cell
lines with IC50 values in the low micromolar range. Complexes 2
and 3 showed a greater cytotoxicity without any significant
selectivity between cancerous and non-cancerous cells with IC50
values in the low micromolar range (4.3 – 13.4 μM). Complexes 2
and 3 were found to be superior in comparison to 1 and 4 due to
their better photophysical properties including 1O2 production
(Table S3). Under the same conditions, the effect of the
complexes was compared with the anticancer drug cisplatin and
the PS protoporphyrin IX (PpIX). The comparison shows that the
toxicity of the complexes 2 and 3 is in a similar range as cisplatin
whereas PpIX is slightly more toxic upon light exposure. The
phototoxic index (PI) is defined as the ratio between the IC50 value
in the dark and the IC50 value upon light exposure. Complexes 2
and 3, being the most phototoxic complexes, were found to have
PI values between 7.5 - 23.3 in various cell lines.
uptake between 1-4 shows
a slightly better uptake from
compound 4 (4 > 3 ~ 2 > 1, Figure S46) in agreement with the
lipophilicity values. The uptake mechanism was then investigated
by blocking different pathways by pre-incubation with various
uptake inhibitors. Blocking the transport mechanisms with
metabolic (2-deoxy-D-glucose and oligomycin), cationic
transporter (tetraethylammonium chloride) and endocytotic
(ammonium chloride or chloroquine) inhibitors had only a
negligible effect (Figure S47-S50) on the internalisation of the
complexes and therefore the involvement of these pathways was
ruled out. The incubation at lower temperature (4 °C, Figure S47-
S50) showed only a slight decrease in uptake caused by the
slower diffusion processes at lower temperature. Overall, these
results indicate an energy independent cellular uptake through
passive diffusion for all the complexes. The cellular localisation
was then examined using confocal laser scanning microscopy
(Figure 2a) in HeLa cells. The compounds were incubated with
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10.1002/anie.201907856
Angewandte Chemie International Edition
RESEARCH ARTICLE
After evaluating the photocytotoxicity on cell monolayers, the
cytotoxic effect of the complexes was investigated in 3D
multicellular tumor spheroids (MCTS). MCTS are a widely used
tissue model for the assessment of the delivery of drugs as it is a
closer model to clinically treated tumours. It is important to
mention that many investigated anticancer agents have failed
during translation from cancer monolayer cells to in vivo models.
This has been partially attributed to the compromised drug
delivery through the penetration of extracellular barriers. It has
been shown that small MCTS with diameters of 200 μm are able
to mimic intercellular interactions. They can therefore be used to
investigate drug delivery. Recent studies have shown that larger
MCTS can also mimic pathological conditions found in solid
tumours such as hypoxia at the tumour centre and its proliferation
gradients.[20] Consequently, we have chosen to investigate MCTS
with diameters of 800 μm as tumor model. For this purpose, HeLa
For further investigation of the PDT ability in MCTS, the
photocytotoxicity was determined by measurement of the ATP
concentration in the tumor spheroids. All complexes were found
to be non-toxic in dark in the MCTS. (Table S7). On the contrary,
the MCTS treated with compounds 2, 3 or 4 and exposed to light
at 500 nm (16.7 min, 10.0 J/cm2) showed a significant effect on
the cell viability with IC50 values in the micromolar range (IC50 for
2: 26.5 ± 2.3 μM, 3: 23.1 ± 3.9 μM, 4: 89.6 ± 5.4 μM) while complex
1 and H2TPP did not show any significant toxicity (IC50 >100 μM)
under identical experimental conditions. As expected from
experiments in cell monolayers, complexes 2 and 3 showed a
higher phototoxicity than the other investigated compounds with
PI values between 3.8 – 4.3. It was observed that a higher
concentration of the compounds is needed to have the desired
phototoxic effect in MCTS. This was previously observed for
various other drug candidates.[21] We assume that this effect is
due to the hypoxic and reducing microenvironment of MCTS,
which can strongly influence the efficiency of a PDT treatment as
well as the limitation of the light to reach inside the MCTS and
generate the required ROS for the treatment. These factors are
ignored in a 2D monolayer cancer cell model. Therefore,
experiments in the MCTS show a more realistic effect of our
complexes in the 3D MCTS model. To further establish the effect
of the complexes on cell viability, HeLa MCTS were treated with
complexes 1- 4 (50 μM) and stained with calcein AM and EthD-1,
48 h after the light treatment. (Figure 4) This stain can distinguish
between the living (green fluorescence signal) and the dead (red
fluorescence signal) cells. All MCTS treated with the complexes
1-4 in the dark, complex 1 and H2TPP exposed to the light source
showed a strong green fluorescence signal while having no or a
very weak red fluorescence signal indicating that the MCTS were
still intact. The MCTS treated with 4 showed both red and green
signals suggesting that the MCTS is partly intact. Contrary to this,
the light treatment had a drastic effect on MCTS, which were
treated with 2 and 3 where a strong red fluorescence signal was
measured indicating that the MCTS were mostly eradicated.
MCTS
were
incubated
with
complexes
1-4
and
tetraphenylporphyrin (H2TPP) was used as a representative for
the clinically used porphyrin-based compound. The penetration of
the compounds was analysed via Z-stack imaging microscopy.
After 12 h of incubation (Figure S51-S54, Figure 3), all complexes
showed
a luminescence signal at every section depth
corresponding with a complete penetration of the compounds in
the MCTS whereas H2TPP only showed a weak signal.
Figure 3. a) Images of 3D HeLa MCTS after incubation with 3 for 12 h (50 μM,
2% DMSO, v%), from left to right: Brightfield, Zn complex luminescence,
Overlay. b) Excited Z-axis images scanning from the top to the bottom of an
intact spheroid. c) 3D z-stack of an intact spheroid. The investigated Zn
complexes were detected using their luminescence properties (λex = 514 nm,
Figure 4. Representative image of viability assay with HeLa MCTS kept in the
dark and exposed to light. MCTS were treated with compounds 1-4 (50 μM, 2%
DMSO, v%) in the dark for 12 h. After this time, MCTS were exposed to a 500
nm (16.7 min, 10 J/cm2) irradiation. After 2 days, the cell survival was assessed
by measurement of the fluorescence of calcein (λex = 495 nm, λem = 515 nm) or
λ
em = 530 - 610 nm).
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10.1002/anie.201907856
Angewandte Chemie International Edition
RESEARCH ARTICLE
cell death by measurement of the fluorescence of EthD-1 (λex = 495 nm, λem
635 nm).
=
As the excited state of the investigated bis(dipyrrinato)zinc(II)
complexes is significantly quenched in a polar environment due
to the formation of a symmetry-breaking charge transfer state in
a polar environment[14c], the lead compound 3 with the best
photophysical properties as well as phototoxic effect was
encapsulated
with
1,2-distearoyl-sn-glycero-3-
glycol)-2000]
phosphoethanolamine-N-[methoxy(polyethylene
ammonium salt (DSPE-PEG2000-OCH3). Worthy of note,
PEGylated phospholipids are approved by the US Food and Drug
administration (FDA). The generated particles 3-NP were
prepared with a well-defined and average size of 119 nm (Figure
S55). Importantly, the particles were found to be highly water
soluble. Details on the preparation of these particles can be found
in the SI. The drug release kinetics of the generated particles were
studied upon dialysis against a PBS solution at physiological and
low pH. As expected, the particles remained intact at pH 7.4
whereas the majority was released within 4 h at pH 5.0 (Figure
S56). As the absorption and emission spectra of 3-NP and 3 are
identical, it indicates that the photophysical properties of 3 is not
changed after encapsulation. As expected, the luminescence
quantum yield of the particles 3-NP (3.9%) in water were found in
the same range than those of the complex 3 in toluene (4.5%),
overcoming the quenching effect of the excited state in a polar
environment. The cellular localisation was then examined using
confocal laser scanning microscopy (Figure 5a) as well as ICP-
MS after extraction of the corresponding cell organelle (Figure 5b)
in HeLa cells. Interestingly, whereas the pure complex (3) was
mainly localising in the cytoplasm, the generated particles 3-NP
selectively accumulated in the lysosomes. Worthy of note, a small
amount of the particles were found in in the surrounding
cytoplasm. Recent investigations on the encapsulation of large
aromatic systems in the same polymer matrix have also reported
that the generated particles were majorly localising in the
lysosomes.[22] The uptake mechanism was then examined by
preincubation with various inhibitors (Figure S57). As the
incubation at lower temperature (4°C) as well as with metabolic
inhibitors significantly reduced the amount of the complex inside
the cell, an energy depended mechanism is suggested.
Additionally, the incubation with endocytotic inhibitors had a
drastic effect. Overall, an energy depended endocytosis pathway
is suggested for 3-NP.
Figure 5. 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. b) Sub-
cellular distribution (Cell = Whole cell, Cyto. = Cytoplasm, Mito. = Mitochondria,
Lys. = Lysosome, 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.
To study the PDT efficacy of 3-NP, its cytotoxicity in the dark and
upon irradiation at 500 nm (16.7 min, 10.0 J/cm2) on HeLa cells
was investigated. While showing no dark cytotoxicity (IC50 > 100
μM), the particles were found to be highly toxic upon light
exposure with an IC50 value of 1.2 ± 0.3 μM and therefore a PI of
83.3, which is much higher than for 3 itself. This clearly highligths
the success of this encapsulation. In addition, the generated
particles were tested on HeLa MCTS as a 3D model. The
luminescence signal of 3-NP was measurable at every section
depth corresponding with
a complete penetration of the
compounds in the MCTS (Figure S58). Cytotoxicity studies of 3-
NP indicate no observed dark toxicity (IC50 > 100 μM) while being
highly phototoxic with an IC50 value of 5.3 ± 1.2 μM and having
therefore a PI of 18.9. This once again demonstrate the interest
of the encapsulation since the complex alone 3 had an IC50 value
of 23.1 ± 3.9 μM while H2TPP as a representative of the porphyrin-
based compounds sowed no observed effect (IC50 > 100 μM)
under the same conditions.
Conclusion
In summary, we have designed and synthesised homoleptic
iodinated bis(dipyrrinato)zinc(II) complexes with remarkably long
excited state lifetimes as potential PDT PSs. The iodine atoms
can promote spin-orbit coupling thus enabling an ISC process.
ESR spectroscopy confirmed that the excited state of the
complexes 3PS is able to interact with molecular oxygen to
generate singlet oxygen upon light exposure at clinically relevant
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10.1002/anie.201907856
Angewandte Chemie International Edition
RESEARCH ARTICLE
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enter cancer cells within 4 h through passive diffusion where they
accumulate in the cytoplasm. While showing no dark toxicity, they
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cell lines at a low micromolar concentration. Additionally, they
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the excited state of the complexes is quenched in an aqueous
environment, the lead compound 3 was encapsulated in a
polymer matrix. The generated nanoparticles was found to not
only have an improved water solubility and photophysical
properties but also to accumulate selectively in lysosomes,
contrary to the complex itself which accumulated in the cytoplasm.
Upon light exposure, the particles caused cell death at very low
micromolar concentrations in monolayer cancer cells as well as
3D tumour model HeLa MCTS. We strongly believe that these
compounds as well as their corresponding particles have a great
potential for the development of a new class of PDT PSs.
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Acknowledgements
We thank Dr. Philippe Goldner for access to state-of-the-art laser
apparatus. This work was financially supported by an ERC
Consolidator Grant PhotoMedMet to G.G. (GA 681679), 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.).
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Keywords: Bioinorganic Chemistry
• Medicinal Inorganic
Chemistry • Metals in Medicine • Photodynamic Therapy •
Photosensitizers
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10.1002/anie.201907856
Angewandte Chemie International Edition
RESEARCH ARTICLE
RESEARCH ARTICLE
To circumvent limitations of current
photodynamic therary photosensitizers,
Johannes Karges, Uttara Basu, Olivier
Blacque, Hui Chao* and Gilles Gasser*
we
propose
the
use
of
bis(dipyrrinato)zinc(II) complexes. These
compounds were found to have no dark
toxicity, while having an impressive
phototoxicity upon irradiation in various
monolayer cancer cells as well as 3D
multicellular tumour spheroids (MCTS).
Page No. – Page No.
Polymeric Encapsulation of Novel
Homoleptic Bis(dipyrrinato) Zinc(II)
Complexes with Long Lifetimes for
Applications as Photodynamic
Therapy Photosensitisers
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