Subphthalocyanines: Addressing water-solubility, nano-encapsulation and activation for optical imaging of B16 melanoma cells

Article abstract of DOI:10.1039/c4cc05503a

Water-soluble disulfonato-subphthalocyanines (SubPcs) or hydrophobic nano-encapsulated SubPcs are efficient probes for the fluorescence imaging of cells. 20 nm large liposomes (TEM and DLS) incorporated about 13% SubPc. Moreover, some of these fluorophores were found to be pH activatable.

Full text of DOI:10.1039/c4cc05503a

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Subphthalocyanines: addressing water-solubility,
nano-encapsulation, and activation for optical
imaging of B16 melanoma cells†
Cite this: Chem. Commun., 2014,
50, 13975
Received 16th July 2014,
Accepted 15th September 2014
Yann Bernhard,a Pascale Winckler,b Remi Chassagnon,c Philippe Richard,a
a
b
a
´
Elodie Gigot, Jean-Marie Perrier-Cornet and Richard A. Decreau*
´
DOI: 10.1039/c4cc05503a
www.rsc.org/chemcomm
Water-soluble disulfonato-subphthalocyanines (SubPcs) or hydro- of SubPcs (emission at around 570 nm, fluorescence quantum yield
phobic nano-encapsulated SubPcs are efficient probes for the fluores- FF = 0.25 and above depending on the nature of alpha and beta-
cence imaging of cells. 20 nm large liposomes (TEM and DLS) substituents),3 but surprisingly none have been examined in the
incorporated about 13% SubPc. Moreover, some of these fluoro- context of OI so far. SubPcs have been widely reported in photo-
phores were found to be pH activatable.
voltaics,3,4 whereas reports on their use in biomedical research are
scarce (PDT on bacteria).5–7 Herein, we report the use of subphthalo-
Molecular imaging allows the diagnosis of various diseases at an cyanines 1a–c as fluorescent probes for OI in vitro.
early stage. There is a need to develop new imaging techniques,
This study presents several aspects of fluorophores 1a–c: the
which may eventually overcome the problems associated with syntheses and optical properties (Fig. 1 and 2), and four new
known methods (MRI and PET).1 Optical imaging (OI) is used in facets: water-solubility, entrapment in a lipidic nanoparticle
preclinical studies and seldom used in clinics. It is appealing (Np) with thorough characterization of the SubPc-containing-Np
because it is non-invasive and uses wavelengths in the optical (Fig. 3), pH-induced fluorescence activation of the amino-containing
window.2 Hence, there is a need for new fluorophores (a) with probes (Fig. 3) and finally SubPc fluorescence imaging of melanoma
satisfactory optical properties and (b) that are activatable, i.e. which B16 cells by both confocal and biphotonic microscopies. The
could be switched ON and OFF reversibly (smart-probes). This study nature of the R group on the apical phenoxy part was addressed
presents a family of fluorescent molecules exhibiting such proper- in order to increase water solubility of the probes and/or to get
ties, subphthalocyanines (SubPcs), which have never been examined pH-induced fluorescence.
as optical probes in OI so far. They are 14-p electron aromatic
macrocycles having C3 symmetry that combine three aza-bridged
isoindole units around a 4-coordinate boron atom. SubPcs have a
convex p surface, which makes them peculiar fluorophores, com-
pared to almost all known fluorophores, which are planar. We view
such a structural feature as a possible asset to prevent p–p stacking
(that leads to subsequent fluorescence quenching). Moreover, the
apical position is another feature that helps prevent aggregation, but
more importantly provides a convenient handle for SubPc mono-
functionalization. There are numerous reports on the fluorescence
a
´
´
Institut de Chimie Moleculaire de l’Universite de Bourgogne (ICMUB),
´
UMR 6302 CNRS – Universite de Bourgogne, BP 47870, F-21078, Dijon Cedex,
France. E-mail: Richard.Decreau@u-bourgogne.fr
b
c
´
Universite de Bourgogne, AgroSup Dijon, Dimacell Imaging Ressource Center,
UMR A 02.102 PAM, F-21000 Dijon, France
´
Laboratoire Interdisciplinaire Carnot Bourgogne, UMR CNRS 6303 – Universite de
Bourgogne, F-21078 Dijon, France
† Electronic supplementary information (ESI) available: Experimental procedures,
spectroscopic details, crystallographic data, additional figures and references.
CCDC 1014064. For the ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c4cc05503a
Fig. 1 Top (left to right): general structure of hydrophobic/hydrophilic
activatable SubPcs 1a, 1b and 1c. Bottom left: incorporation of hydrophobic
SubPc in the liposome structure.
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subjected to two sets of purifications by chromatography (silica,
dichloromethane/methanol gradient), and then by semi-
preparative reverse-phase chromatography (Dionex, C18, using
a CH3CN/H2O gradient, with 0.1% TFA), affording 1c in modest
yield (12%).
The 11B-NMR spectrum (11B: S = 3/2) of boron-containing
subphthalocyanines shows a singlet, which is 30 nm upfield shifted
in aryloxy-containing species 1a–c compared to chlorine-containing
species 2 (Fig. S2, ESI†). The 1H-NMR spectrum of the SubPcs shows
the classical multiplet at around 8 and 8.8 ppm corresponding to the
indole moieties and the shifted doublet of the phenoxy picket, and
the 13C-NMR spectrum of SubPcs shows aromatic carbons in the
118–158 ppm window (Fig. S3–S6, ESI†). Mass spectroscopy analysis
(MALDI-TOF or ESI-Q) shows the molecular isotopic in all cases, and
sometimes a signal corresponding to the cleavage of the B–O bond,
Fig. 2 Syntheses of 1a–c. (i) BCl3, p-xylene, Ar atm, reflux, 9% (ii) excess of
p-nitrophenol, toluene, reflux, 78% for 1a (iii) (a) BCl3, p-xylene, Ar atm,
reflux, (b) evaporation (c) excess of para-substituted phenol, toluene,
reflux: 29% for 1a, 16% for 3a and 19% for 3b; (iv) H2, Pd/C, 88%; (v) MeOH,
NH3, or NaHCO3; (vi) 1,3-propanesultone, DMF, 50 1C, 12%.
+
(C24H12BN6 : 395.12) (Fig. S7 and S8, ESI†). X-ray diffraction studies
showed the classical domed structure in 1b (Fig. 3; Fig. S9 and
Table S1, ESI;† CCDC 1014064), i.e. a convex p surface.
Liposome. Species 1a is hydrophobic (no solubility in water
whatsoever), hence it was entrapped in a lipidic nanoparticle
(Np). Liposomes were prepared according to a modified version
of the Batzri and Korn’s injection method, using DPPC as the
phospholipid and PBS as the medium.9–13 Subsequent purification
on Hitrap was achieved (Rt = 8 min) affording a pure suspension of
liposomes, free of unbound SubPc fluorophores. Subsequent char-
acterization of the construct based on size and the dye content was
achieved. The size of the SubPc-containing Np was given by: (a) the
hydrodynamic diameter determined by dynamic light scattering
(DLS): dH = 21 nm (Fig. 4A), (b) which correlates with the mean
Fig. 3 X-Ray diffraction of 1b (CCDC 1014064) showing the characteristic
domed structure of subphthalocyanines.
Synthesis. Cyclotrimerization (30 min/150 1C) of dry dicyano- diameter determined by transmission electron microscopy (TEM):
benzene in the presence of 1 equiv. of boron trichloride (under dTEM = 20 nm (Fig. 4B) (using the negative staining approach,
an inert/anhydrous atmosphere) led to subphthalocyanine 2 which was carried out by mixing a drop of liposome suspension
bearing an axial chlorine atom (SubPc-Cl), which could be isolated with a solution of ammonium molybdate, and subsequent
in 9% yield after chromatography (silica/dichloromethane) (Fig. 2).8
The former was heated in toluene in the presence of an excess
of p-nitrophenol to afford phenoxy-substituted subphthalo-
cyanine 1a in substantial yield (78%, i.e. 7% overall yield from
dicyanobenzene). Alternatively, a one pot approach was con-
ducted: upon cyclotrimerization, a subsequent quick removal
(distillation) of excess BCl3 (and p-xylene) was achieved, then
the crude mixture was reacted with a given phenol derivative
affording SubPcs 1a, 3a, and 3b, respectively, in 16–29% overall
yield. SubPc 1a was subsequently reduced under a H2 atm. in the
presence of activated Pd on charcoal (2.5 equiv.), and purified
by chromatography (silica, dichloromethane/MeOH) to afford
SubPc-NH2 1b in high yield (88%). Attempts to remove the
acetyl and trifluoroacetyl protecting groups in species 3a and 3b
(upon treatment with either sodium bicarbonate or ammonia/
MeOH solutions) led to substantial decomposition of SubPc,
and 1a was barely obtained. The reaction of 1b with propane
sultone (5 equiv. in DMF/50 1C, 72 h) led to a statistical mixture
that was examined by analytical HPLC (Fig. S1, ESI†) and appeared
to contain the starting SubPc 1b and the hydrophilic mono- and
bis-alkylsulfonate species 1c (each peak was subjected to MS).
Fig. 4 Characterization and properties of 1a-containing nano-vesicles:
(A) DLS; (B) TEM and (C) absorption in THF, water/THF (97 : 3 vol), and
liposome; (D) fluorescence of 1b in DMF with addition of H2SO4 (inset:
fluorescence max as a function of volume (mL) of 0.1% H2SO4 solution
Under these conditions, the quaternized tris-alkylated ammonium
species was not obtained. The mixture was subsequently function of pH (2.6 to 9 in chosen buffers, see ESI†).
added) (E) fluorescence of 1a and 1b in liposome and 1c solution as a
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deposition on a grid and drying (see ESI†)). These values con- which was expected based on our previous observations on free
curred with those found for small unilamellar vesicles (SUVs) SubPc 1b in solution (Fig. 4D and E). Upon lowering the pH, a
prepared by the injection method.9 Finally, the stability of the gradual fluorescence emission was measured. This is because
liposome suspension over time was monitored by DLS, and of protons diffusing through the bilayer leading to the proto-
results showed that the liposomes were reasonably stable, i.e. nation of 1b to form 1b-H+. A gradient of pH was used which led
the hydrodynamic diameter was found to be 40 nm (size doubled to an increase in fluorescence: at 50% FF, the pH was found to
after 24 h at RT, under agitation). The optical properties of free/ be 3.5 (Fig. 4D), which is consistent with average pKa values of
nano-encapsulated SubPc 1a are reminiscent of those of por- primary aniline groups.
phyrin/porphyrinoid species, i.e. a Soret band (303–315 nm) and
a Q band (562–566 nm) (Fig. 4C; Tables S2 and S3, ESI†). The SubPc species 1c is totally water-soluble. The UV/Vis spectrum of 1c
fluorescence quantum yield of 1a was found to be FF = 14% (lex displays sharp absorption bands (Fig. S15, ESI†) comparable with
Water-solubility. Unlike 1a and 1b, alkylsulfonato-containing
=
488 nm, ref. Rhodamine 6G; lem = 570–574 nm). A comparison of that of 1a and 1b in organic solvents (Fig. 4C; Fig. S11 and S13,
the absorption and emission spectra in pure THF, water–THF ESI†). Fluorescence studies with 1c (Fig. S16, ESI†) were performed
(97 : 3 vol) and in liposome suspension (Fig. 4C; Fig. S10 and in pure water or buffer, the fluorescence quantum yield, FF = 4%
Table S3, ESI†) gives an evidence of the location of SubPcs within (at pH o 6; phosphate–citrate buffer) was found to be lower than
the bilayer (as shown for other probes): the UV/Vis spectra of that of 1a in solution but comparable with that of 1a in liposome
SubPcs display sharp peaks upon SubPc incorporation in lipo- (Tables S1 and S2, ESI†). The same activation processes may be
somes, whereas broadened and shifted features are observed for invoked with tertiary amine 1c (Fig. 4E and Fig. S17, ESI†), although
SubPc in water, which is characteristic of aggregation of hydro- the ICT and/or PET events may be affected going from tertiary
phobic species in an aqueous environment. The pure suspension aniline (in 1c) to primary aniline (in 1b). Moreover, even if
of 1a-containing-liposomes (i.e. after purification on Hitrap) was protonation-induced fluorescence occurs, the fluorescence increase
analyzed by UV/Vis spectroscopy, which indicates an average 13% from a deactivated to an activated probe is not as pronounced
encapsulation in 20 nm liposome vesicles (Fig. S11, ESI†).
(by 18–20 for 1b against 3 for 1c). The gradient of pH allows the
Activation. Interestingly, hydrophobic species 1b turned out determination of a pKa value of 6.5. This protonation was examined
to be non-fluorescent under the conditions described for 1a. with respect to the pKa of an anilinium ion, i.e. the conjugate acid
Further fluorescence studies conducted with 1b in various media of aniline. But an aniline is protonable depending on the nature of
showed a change in the fluorescence quantum yield (FF) from the substituents: although the pKa is within 3–4, it jumps as high
o1% (DMF or CHCl3) to 12–15% (DMF + H2SO4 or CHCl3 + TFA) as 7 with N-tBu; 6.5 in the case of N,N-diethylaniline.18 Hence,
(Fig. 4C and E, Fig. S11–S13 and Tables S2 and S3, ESI†). Hence, pKa obtained for 1c is consistent with these values, considering
the fluorescence of this species may be switched-on upon treat- propylsulfonate groups as ethyl groups. In fact 1c is always partially
ment with an acid. Studies show that the phenomenon is turned ON (equilibrium between the amine/sulfonic acid form and
reversible upon addition of a base. A control experiment carried the ammonium/sulfonate zwitterionic form), as a result the activa-
out with 1a (always ON species) showed no fluorescence change tion threshold is not as obvious as for 1b.
upon addition of acid. Hence, it suggests that protonation of 1b
Biology. Nano-encapsulated hydrophobic SubPc 1a and hydro-
(leading to 1b-H+) occurs at the amine site, and turns the philic bis-alkylsulfonato-SubPc 1c were subsequently incubated
fluorescence ON. The fluorescence switch-on occurs upon pro- (1 h) with B16–F10 melanoma cells (in de-supplemented RPMI).
tonation of the nitrogen at acidic pH through the suppression of Neither compound 1a nor 1c was found to be cytotoxic against
internal charge transfer (ICT) and/or photoinduced electron B16 cells (upon incubation of solutions up to 10 mM for 1 h,
transfer (PET).15–17 In the non-protonated form the one electron followed by the MTT test, i.e. addition of 0.5 mM MTT, followed
hopping from the electron-rich macrocycle may occur, which by incubation for 2 h, subsequent removal of the medium
results in a formal fluorescence extinction. Although the amine and solubilisation in DMSO). For imaging studies, the same
is not conjugated to the cycle, ICT/PET may still occur within 5 Å, concentrations/incubation times were used; cells were sub-
resulting in fluorescence extinction, encountered in other amine- sequently rinsed (with PBS) and fixed (with cold (À30 1C)
containing dyes. Moreover, parallel experiments were conducted methanol left on cells for 5 min at 4 1C). It is expected that
to examine the possible protonation of the azomethine bridges in liposome incorporation into the cell results in the fusion of
SubPc 1a (Fig. S11, ESI†). Previous studies on phthalocyanines membranes, which ought to leave subphthalocyanine 1a in a
¨
suggest a weak Bronsted basicity of the azomethine bridges, membrane (bilayer) environment. On the other hand, several
hence the weak acidic conditions employed here may not be mechanisms have been classically proposed for the cell membrane
sufficient to protonate them. The absorbance bands of SubPc 1a crossing of free molecules (i.e., molecules not incorporated in
were observed by UV/Vis spectroscopy upon gradual addition of liposomes, such as water-soluble species 1c), one being a passive
acid in different quantities (Fig. S11, ESI†). This suggests that even diffusion process. Optical imaging studies were subsequently
in pure trifluoroacetic acid, protonation of all aza-bridges may not achieved by fluorescence imaging using both biphotonic and con-
be complete. The protonation-induced fluorescence has also been focal microscopies (as optical and non-linear optics of SubPcs
examined upon incorporation of 1b in liposomes (Fig. S14 and have been reported).7,14 B16 cells were clearly observed, using a
Table S3, ESI†). At pH 7 the fluorescence emission from a set of filters (DAPI, FITC, etc.), and upon comparison with
suspension of nano-encapsulated 1b in PBS buffer is low, cells which were not incubated with a solution of SubPc
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wavelength of maximum absorption lmax = 570 nm, which
brings SubPc 1a in the range of other known fluorophores,
such as propidium, SNARF-1, and FMN).25
RAD, YB thank the Burgundy Regional Council, CRB (FABER
Program), RAD thanks CNRS for Chaire d’Excellence (CdE).
FABER and CdE fundings were used to fully set up a cell culture
lab at ICMUB. This work was supported by the 3MIM agreement
´ ´
(CNRS, uB and CRB). Julien Boudon, Nadine Millot and Jeremy
Fig. 5 Fluorescence microscopy images of B16–F10 melanoma cells upon
incubation for 1 h with fluorescent SubPc probes and fixation (methanol):
(A) biphotonic images of cells incubated with a liposomal suspension of 1a;
(B) biphotonic images of cells incubated with a solution of 1c in RPMI (exc.
720 nm). (C) Confocal imaging of cells incubated with 1c (exc. 561 nm).
Paris (uB, ICB Institute) for use and training on DLS. Lionel
Apetoh (INSERM, U866) for a generous gift of B16F10 cells.
Notes and references
(Fig. 5A–C; Fig. S18 and S19, ESI,† i.e. transmission, super-
position and fluorescence images of B16 cells). Both strategies,
nano-encapsulation and formation of water-soluble SubPcs
turned out to be equally efficient for the imaging of B16 cells
by fluorescence microscopy.
In summary this is the first successful report on the use of
subphthalocyanines as fluorophores for the in vitro fluorescence
imaging of cells (achieved by confocal and biphoton microscopy).
Concentrations used were relevant for imaging (10 mM), with no
cytotoxicity toward B16F10 melanoma cells. Two strategies were
equally successful for the delivery to cells: (a) nano-encapsulation
in 20 nm SUV (DPPC) liposomes for hydrophobic species 1a and
1b and (b) increasing water-solubility by the introduction of alkyl
sulfonate chains; in the case of bis-sulfonated species 1c fluores-
cence was observable in pure water or buffers. Species 1c is one
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