Supramolecular subphthalocyanine complexes-cellular uptake and phototoxicity

Article abstract of DOI:10.1039/d0cc03065a

In this communication we report on the synthesis and application of axially functionalized boron-subphthalocyanines (SubPC) which are able to form host-guest complexes with cyclodextrins. Here, a tert-butylphenyl substituted SubPC was investigated concerning its complexation with β-cyclodextrin (β-CD) and a β-cyclodextrin polymer. NMR-titrations showed the formation of a 1: 1 complex with β-CD. These assemblies were analyzed for their cellular distribution as well as their phototoxicity towards HeLa cells.

Full text of DOI:10.1039/d0cc03065a

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Supramolecular subphthalocyanine
complexes–cellular uptake and phototoxicity†
Cite this:DOI: 10.1039/d0cc03065a
b
Justin Dubbert,‡a Alexander Hoing, ‡b Nathalie Riek,a Shirley K. Knauer
and
¨
a
Received 28th April 2020,
Accepted 30th May 2020
Jens Voskuhl
*
DOI: 10.1039/d0cc03065a
rsc.li/chemcomm
In this communication we report on the synthesis and application intensity, going along with a highly decreased singlet oxygen
of axially functionalized boron-subphthalocyanines (SubPC) which production.5 Another challenge is the introduction of target-
are able to form host–guest complexes with cyclodextrins. Here, a specific moieties, which are exclusively present on either patho-
tert-butylphenyl substituted SubPC was investigated concerning its gens or at least highly overexpressed in certain cell types
complexation with b-cyclodextrin (b-CD) and a b-cyclodextrin poly- compared to the healthy tissue to keep collateral damage to a
mer. NMR-titrations showed the formation of a 1 : 1 complex with minimum.6,7 First examples to overcome the stacking tendency
b-CD. These assemblies were analyzed for their cellular distribution were to introduce bulky groups with a high steric demand
as well as their phototoxicity towards HeLa cells.
including branched alkyl chains8 or polyaromatics.9 Besides
this, the use of charges introduced by quaternary amines,10
Drug resistance continues to be the main cause for therapy carboxylates,11,12 sulfonates13 as well as phosphates14 was
failure in cancer treatment.1 It is therefore imperative to further studied, since an electrostatic repulsion between the photo-
develop and improve strategies that do not rely on chemother- sensitizers forces the compound to be present in a mainly
apeutics. Due to tumour heterogeneity, even one solid tumour monomeric fashion leading to a higher photosensitizing per-
can contain different sub-populations of cells harbouring varying formance. Another approach to induce a steric separation
resistance, which leads to partial survival and later on recurrence between the photosensitizers is supramolecular host–guest
of the tumour after an initially successful therapy.2 One approach complexation. The macrocyclic hosts encapsulate the photo-
allowing to overcome these obstacles is the use of so called sensitizer or a part of it, to break non-emissive, inactive p–p-
photosensitizers such as phthalocyanines (PC) and porphyrins interaction driven aggregates. Here cucurbit[n]urils (CB[n]),15,16
(POR) in photodynamic therapy (PDT).3 Due to their absorption in cyclodextrins (CD)17,18 as well as CD vesicles,19,20 and calixar-
the near infrared region and their emission between 650 nm and enes (CA)21,22 have been used. For example, Leng et al. followed
750 nm, these macrocyclic aromatic compounds are able to an approach by encapsulation of metal free or zinc containing
undergo an intersystem crossing into a triplet state. This long phthalocyanines (PC) in two b-CD’s to form a 2 : 1 host–guest
lived state is able to convert harmless triplet oxygen into highly systems in alkaline aqueous media, which led to a drastic
reactive and toxic singlet oxygen.4 The mode of action of singlet increase in the production of singlet oxygen due to steric
oxygen involves oxidation processes of virtually all organic struc- separation of the PC monomers.23 Another approach was
tures inside a cell leading to immediate cell death or the induction shown by Ravoo and co-workers, by immobilizing asymmetric
of apoptosis. However, one major drawback of these planar aro- water soluble adamantane functionalized zinc(II) phthalocya-
matic structures is their high tendency to stack in poor solvents nines on the surface of b-CD vesicles which led to an increased
such as water, which under the formation of p–p interactions lead photogeneration of singlet oxygen (from 5% to 20%).19 In the
to the formation of non-emissive stacks which decrease emission same time, Menting et al. went one-step further and combined
CD modified subphthalocyanines (SubPC) and PC with water
a
soluble tetrasulfonated POR to a ternary host–guest com-
plex which was used to induce efficiently excitation energy
transfer.24 Besides CD’s, studies were also carried out with
other host molecules, like pillar[n]arenes by Wu et al. They formed
supramolecular vesicles by self-assembly of a pyridinium con-
taining pyropheophorbide-A with water soluble pillar[5]arenes,
which lead to the encapsulation of the pyridinium-unit.25 This
¨
Institute of Organic Chemistry, University of Duisburg-Essen, Universitatsstrasse 7,
45117, Essen, Germany. E-mail: jens.voskuhl@uni-due.de
b Department of Molecular Biology II, Center of Medical Biotechnology (ZMB),
¨
University of Duisburg-Essen, Universitatsstrasse 5, D-45117 Essen, Germany
† Electronic supplementary information (ESI) available: Synthesis, NMR- and
mass spectra, photophysical characterisation, NMR titrations, fluorescence
microscopy, phototoxiycity. See DOI: 10.1039/d0cc03065a
‡ These authors contributed equally.
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Scheme 1 Schematic presentation of [1] used in this study as well as their
complexation with b-CD and b-CD-polymer.
supramolecular encapsulation led to decreased aggregation
and hence increased photogeneration of singlet oxygen. These
examples showed the main benefits of supramolecular com-
plexation of photosensitizers leading to a boost of the photo-
physical performance.
Up to now, no example exists where a guest compound for
CD’s is axially attached to a SubPC leading to a supramolecular,
compact host–guest complex with b-CD. Furthermore, a commer-
cially available b-CD polymer was used as host platform, to form a
multivalent SubPC polymer. These constructs were investigated
concerning their phototoxicity as well as their cellular uptake
(Scheme 1). The water soluble subphthalocyanine was axially
equipped with a tert-butylphenol group, which is known to form
stable host–guest complexes with b-CD’s.26 The synthesis of the
SubPC derivate was carried out by using a cyclotrimerization with
boron trichloride of the corresponding substituted phthalonitrile,
starting from 4-nitrophthalonitrile, which was converted over a
Fig. 1 (A) Photographs of cuvettes with different molar fractions of [1] and
b-CD (lex = 365 nm) c[1] = 100 mM, (B) change in emission intensity upon
addition of increasing amounts of b-CD to [1], (C) plot of the emission
intensity at 610 nm versus the b-CD concentration, (D) NMR signals of H-1
of b-CD with varying molar fractions of [1] and b-CD, (E) Job plot based on
the signals shown in (D).
´
decrease in p–p stacking which resulted in a fluorescence
increase due to a more molecular dissolved state of [1]. An
increase in emission up to a stoichiometry of 1 : 1 (Fig. 1B and
C) was observed. In contrast, higher molecular ratios resulted in
decreased emission. This could be attributed to secondary non-
specific interactions, such as electrostatic attraction between the
hydroxyl-groups of the CD’s and the quarternized pyridine that
lead to the formation of weaker emissive assemblies. This first
hint for a 1 : 1 complex was further investigated by a NMR-
titration technique using variable molar fractions (Job method).
The H-1 signal of the CD’s was chosen due to its strong shift and
its clear differentiation from other signals. As obvious from
Fig. 1D the selected proton shifts (Fig. S15, ESI†) and Job plot
analysis revealed a maximum at a molar fraction of 0.5 confirm-
ing the formation of a 1 : 1 complex between compound [1] and
b-CD (Fig. S13, ESI†). This behaviour was also confirmed by
using a signal of [1] instead of the H-1 CD signal, also revealing
the existence of a 1 : 1 complex (Fig. S14, ESI†). To get deeper
insights into the complexation thermodynamics, an additional
static NMR titration was performed. Analyses revealed that
compound [1] binds to b-CD with an association constant of
around 2100 MÀ1 which is nearly one magnitude lower26 than
two-step synthesis into 4-iodophthalonitrile via Bechamp-
reduction and Sandmeyer-like reaction with potassium iodide
(Scheme S1, ESI†). After completion of the cyclotrimerization, the
axial halide atom of the resulting subphthalocyanine was sub-
stituted efficiently with 4-tert-butylphenolate as nucleophile, in
a two-step methodology, according to an already described
procedure.14 In the first step the chlorine atom was substituted
using silver trifluoromethanesulfonate to increase the electro-
philicity of the boron atom, then 4-tert-butylphenol was added
together with the non-nucleophilic base DIPEA to complete the
substitution. The next step was the further functionalization of
the peripheral groups using a palladium-catalyzed Sonogashira
coupling with 3-ethynylpyridine. Finally, the pyridine units
were quaternized with methyl iodide in DMF to increase the
water solubility of SubPC [1]. In an initial experiment, a fixed
concentration of [1] in water (100 mM) was mixed with different
equivalents of b-CD (Fig. 1A–C) and its emission intensity was
measured by fluorescence spectroscopy. As expected, upon
complex formation between the two compounds, a change in
emission was observed by the bare eye (Fig. 1A) and upon
fluorescence emission determination (Fig. 1B). It was expected
that the encapsulation of [1] inside the b-CD cavity induced a
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the free tert-butylphenol. This might be explained by a less deep interaction of [1] with the cavity of the individual b-CD as well
penetration of the CD cavity going along with decreased hydro- as the b-CD-polymer, driven by hydrophobic interactions, over-
phobic interactions due to steric hindrance (Fig. S16 and S17, comes the known stack effect of photosensitizers, which is
ESI†). To evaluate the photophysical properties of [1], the driven by the formation of p–p interactions. With these well
quantum yield of the emission and the singlet oxygen generation characterized complexes and assemblies in stock, we aimed for
were determined using an integrating sphere (for FF) or a cellular studies, using HeLa cells. Here, we used confocal laser
fluorescence reporter for the generation of ROS (for FD) in scanning microscopy (CLSM) to check for membrane perme-
addition to absorption, excitation and emission spectra ability and intracellular localization of the compounds in HeLa
(Fig. S18–S22, ESI†). Singlet oxygen quantum yields (FD) were cells. [1] showed the highest fluorescence intensity and was
determined using 9,10-anthracenediyl-bis(methylene)dimalonic distributed evenly in the cytoplasm with a distinct accumula-
acid (ADMADM) in aqueous solution as a fluorescence indicator tion at the nuclear membrane. Remarkably, the compound
(lexc = 380 nm), which loses its emission properties in the presence of accumulated strongly at selected structures inside the nucleus,
reactive oxygen species (ROS) by the formation of an endoperoxide.
which resemble intra-nuclear domains like the nucleoli, while
Irradiation of air-saturated solutions of methylene blue (MB, the rest of the nucleus was almost spared (Fig. 2 and Fig. S29,
FD = 0.52) as reference compound and both, the pure SubPC [1] ESI†). We could prove this co-localization using a fusion
and the host–guest systems with b-CD ([1] : b-CD = 1 : 1) and b-CD protein consisting of GFP and the known nucleolar protein
polymer ([1] : b-CD polymer = 1 : 4, based on the individual b-CD nucleophosmin (NPM) (Fig. S26, ESI†). [1] + b-CD basically
units in the polymer) led to a decrease of the ABMDMA emission showed the same subcellular distribution pattern while the
over time with increasing irradiation duration (Fig. S20, ESI†). fluorescence was a little weaker (Fig. 2 and Fig. S30, ESI†).
The ratio of 1 : 4 (based on the b-CD content – see ESI†) for the b- However, the fluorescence signal in the nucleus was more
CD polymer was used to ensure enough space for a full com- granular and a higher magnification revealed that the cytoplas-
plexation of [1] with the CD cavity.
mic portion of the compound is not as evenly distributed
By comparing the slope of the fluorescence decays and using cytoplasm, as compared to [1], but seems to be packed partially
the eqn (S1) (ESI†), it was possible to determine FD (Fig. S21, in vesicular structures. [1] + b-CD-polymer showed a similar
ESI†). This method was applied due to the fact that the phos- fluorescence intensity as [1] (Fig. 2 and Fig. S31, ESI†). Strik-
phorescence of 1O2 is not observable in an aqueous environment, ingly, the nucleus was completely void of compound with no
since it is known that the hydroxyl groups of CD’s extinguish the accumulation at the nucleoli. In addition, cells treated with
phosphorescence of 1O2, thus preventing quantitative deter- [1] + b-CD-polymer showed heterogeneous distribution patterns
mination in the near infrared region of the electromagnetic in contrast to the other compounds. One striking pattern that
spectrum.13 We discovered that, compared to [1], the supramole- reproducibly occurred in most cases was a strong accumulation
cular complexes showed increased photoluminescence (FF) and at one region close to the nucleus, resembling the position of
singlet oxygen (FD) quantum yield (Table S4, ESI†). Here especially the golgi apparatus (Fig. 2 and Fig. S31, ESI†). We therefore
the increase of 65% (from 17% to 28%) in FD for the complex of used a well-established golgi marker (YFP-1, 4-GT) and could
[1] with the b-CD-polymer, compared to uncomplexed [1], is verify that most of the complex consisting of [1] and b-CD-
noteworthy (Table S4, ESI†). These results showed the induced polymer indeed colocalized with this area or its direct vicinity
steric separation of [1] by supramolecular host–guest interactions, (Fig. S27, ESI†). We therefore assume an endocytotic uptake,
leading to a higher availability of monomeric photosensitizers which might be based on the increased size of the compound.
1
for the photogeneration of O2. Consequently, the host–guest This could as well explain the heterogeneous distribution, since
Fig. 2 Membrane permeability of the compounds [1], [1] + b-CD and [1] + b-CD-polymer and the intracellular distribution was determined by
fluorescence microscopy using HeLa cells. Cells were stained with the nuclear dye Hoechst 33342 for 20 min and then treated with 100 mM of the
respective compound in DMEM for 30 min.
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In sum, we developed a unique system to be used for different
purposes by simple formation of supramolecular assemblies.
The pure compound [1] localized to the nucleoli and might be
applicable in photodynamic therapy as effective photosensitizer,
whereas the supramolecular assemblies can be utilized as versa-
tile platform for bio-imaging purposes (e.g. for golgi staining).
The authors acknowledge the Imaging Centre Campus
Essen (ICCE) and the Center of Medical Biotechnology (ZMB).
Conflicts of interest
Fig. 3 (A) Phototoxicity of 50 mM compound ([1], [1] + b-CD, [1] + b-CD-
polymer) with varying length of irradiation on HeLa cells. (B) Phototoxicity
of different compound concentrations ([1], [1] + b-CD, [1] + b-CD-
polymer) after 60 min of irradiation. Cell viability was assessed using the
CellTiter-Fluor Cell Viability Assay.
There are no conflicts to declare.
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¨
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