A soft supramolecular carrier with enhanced singlet oxygen photosensitizing properties

Article abstract of DOI:10.1039/c2sm27353e

Herein we report the self-assembly of a supramolecular singlet oxygen photosensitizing system from an adamantane-functionalized, hexaanionic water-soluble zinc(ii) phthalocyanine (PC) and β-cyclodextrin vesicles (CDV). Characterisation of the designed PC, which was synthesized by an asymmetric statistical condensation, was carried out by several analytical techniques such as MALDI-HRMS, NMR, IR, UV/vis as well as steady state and time resolved fluorescence spectroscopy. The influence of the docking of the PC to the CDVs on the PC photoluminescence as well as on the singlet oxygen photoproduction quantum yields was investigated. The results indicate that the host-guest interaction of the photosensitizer and the CDVs significantly prevents the formation of inactive aggregates, and enhances the photosensitizing ability of the PC. The supramolecular assembly constitutes a biocompatible photoactive platform for the design of phototherapeutic agents.

Full text of DOI:10.1039/c2sm27353e

Soft Matter
View Article Online
View Journal | View Issue
PAPER
A soft supramolecular carrier with enhanced singlet
oxygen photosensitizing properties†
Cite this: Soft Matter, 2013, 9, 2453
Jens Voskuhl,a Ulrike Kauscher,a Malte Gruener,b Hendrik Frisch,a Birgit Wibbeling,a
b
a
*
*
Cristian A. Strassert and Bart Jan Ravoo
Herein we report the self-assembly of a supramolecular singlet oxygen photosensitizing system from an
adamantane-functionalized, hexaanionic water-soluble zinc(II) phthalocyanine (PC) and b-cyclodextrin
vesicles (CDV). Characterisation of the designed PC, which was synthesized by an asymmetric statistical
condensation, was carried out by several analytical techniques such as MALDI-HRMS, NMR, IR, UV/vis as
well as steady state and time resolved fluorescence spectroscopy. The influence of the docking of the PC
to the CDVs on the PC photoluminescence as well as on the singlet oxygen photoproduction quantum
yields was investigated. The results indicate that the host–guest interaction of the photosensitizer and
the CDVs significantly prevents the formation of inactive aggregates, and enhances the photosensitizing
ability of the PC. The supramolecular assembly constitutes a biocompatible photoactive platform for the
design of phototherapeutic agents.
Received 11th October 2012
Accepted 17th December 2012
DOI: 10.1039/c2sm27353e
www.rsc.org/softmatter
In the last few years, the rst efforts were made to use organic
and inorganic transporter systems for PCs, such as liposomes,
Introduction
Photodynamic therapy (PDT) constitutes a non-invasive method
for the inactivation of cancer cells or bacteria.1 In this approach,
irradiation of a photosensitizer (PS) with harmless red or near
infrared light (l > 650 nm) leads to the formation of cytotoxic
singlet oxygen (1O2) by energy transfer from the lowest excited
triplet state of the PS to molecular oxygen (3O2).2 Since the
introduction of phthalocyanines (PCs)3,4 and porphyrins5–7 as PS
surfactants and zeolites, to avoid aggregation and secondary
interactions with tissues.8,9 On the other hand, Mazzaglia et al.
described the absorption of photosensitizers such as porphyrins
in cyclodextrin (CD) nanoparticles, which consist of amphiphilic
CDs bearing C6 alkyl chains at the primary face and ethylene
glycol units with amine functions at the secondary face. In this
case, the negatively charged 5,10,15,20-tetrakis-(4-sulfonato-
phenyl)-21H,23H-porphyrin (TPPS) binds to the positively
charged CD nanoparticles due to electrostatic interactions.15–17
In 2000, Ravoo and Darcy reported another class of amphiphiles
1
of O2, several potential carrier systems for these rather hydro-
phobic molecules have been proposed.8,9 PCs are known to be
highly insoluble in aqueous environments, and consequently,
only limitedly available in vivo. Several approaches to increase
their water solubility have been investigated, which include
among others the introduction of amine,10,11 carboxylate12 and
hydroxyl13 functions. Furthermore, it is well known that PCs
tend to form inactive aggregates due to hydrophobic interac-
tions. This behaviour decreases their ability to photosensitize
1O2, as the stacked molecules release the absorbed energy
mainly as heat.14 In this sense, the introduction of charges on
the periphery of the PC is expected to have a positive inuence
on the solubility while avoiding aggregation by electrostatic
repulsion, thus enhancing their bioavailability and 1O2
photoproduction.11,12
based on CDs.18 By the introduction of alkyl chains (C12 or C16
)
on the secondary face of the CD and oligoethylene glycol units on
the primary face, amphiphiles were obtained that form stable
aOrganic Chemistry Institute and CeNTech, Westfalische Wilhelms-Universitat
¨
¨
¨
¨
Munster, Corrensstrasse 40, 48149 Munster, Germany. E-mail: b.j.ravoo@
uni-muenster.de
bPhysics Institute and CeNTech, Westfalische Wilhelms-Universitat Munster,
¨
¨
¨
¨
Heisenbergstrasse 11, 48149 Munster, Germany. E-mail: ca.s@uni-muenster.de
† Electronic supplementary information (ESI) available: Details of reactions and
Fig. 1 Schematic representation of cyclodextrin vesicles decorated with phtha-
locyanine photosensitizer (8).
measurements conducted. See DOI: 10.1039/c2sm27353e
This journal is ª The Royal Society of Chemistry 2013
Soft Matter, 2013, 9, 2453–2457 | 2453

View Article Online
Soft Matter
Paper
unilamellar cyclodextrin vesicles (CDV) in aqueous media. The
Aer deprotection in acidic conditions with triuoroacetic
cavities of the CDs embedded in the surface of these vesicles are acid (TFA), the free amine was reacted with hydroxyl phenyl-
available for complexation with hydrophobic guests such as carboxylate as described above (vide supra). The deprotected
adamantane,19–23 tert-butylbenzene24 and azobenzene25,26 units hydroxyl group was then coupled to 1,2-dichlorophthalonitrile.
so that the CDV can be decorated with functional guest mole- The hydrophobic adamantane provides the ability to bind
cules simply by mixing the host vesicles with the desired guests. divalently to the surface of the CDV.20 The phthalocyanine (5)
Herein we describe the use of such supramolecular so colloids and (1) were nally condensed statistically in a ratio of 3 : 1
for the immobilisation and transport of a tailored PS (Fig. 1). We employing DBU (1,8-diazabicyclo[5.4.0.]undec-7-ene) and
synthesized a PC-adamantane derivative (8) in order to combine anhydrous Zn(OAc)2 in reuxing pentanol, yielding aer puri-
the photosensitizing capacity of the macrocycle with the binding cation by semipreparative HPLC the dark blue zinc(II) PC (7)
ability to the CDV vesicles, which effectively prevents the (2.9% yield, Scheme 2). During the condensation, a trans-
formation of inactive aggregates.
esterication of the methylesters to pentylesters was observed,
which has been previously described by other authors.12 The
identity of the product was conrmed by high resolution mass-
spectrometry, and 1H-NMR and IR spectroscopy. In order to
obtain the water soluble Zn(II) PC (7), the ester groups were
removed by saponication with NaOH. Aer neutralization with
HCl, a green precipitate was obtained which was found to be the
desired asymmetrical, water soluble Zn(II) PC which was
conrmed by MALDI-HR-MS. 1H-NMR spectroscopy only
showed broad, unstructured signals due to stacking of the PC
(8), both in water and in DMSO (see ESI, Fig. S2†).
CDVs were prepared from an amphiphilic b-cyclodextrin
substituted with 7 n-dodecylthiol units on the primary side and 7
oligo(ethylene glycol) units on the secondary side. These
amphiphilic macrocycles form unilamellar bilayer vesicles upon
dispersion and extrusion in aqueous solution. The synthesis of
the amphiphilic cyclodextrin and the preparation and charac-
terization of vesicles have been described in the literature.19 In
order to assess the inuence of (8) on the structure of the CDVs,
sizes and z-potentials were determined by dynamic light
Results and discussion
The synthesis of the PC derivative was carried out by using a
statistical condensation of substituted phthalonitriles, starting
from 1,2-dichlorophthalonitrile, which was converted into the
tetraester (1) by aromatic nucleophilic substitution with an
adequately substituted phenol derivative in the presence of
K2CO3 (Scheme 1). The X-ray diffractogram of crystals of the
resulting tetraester (1) was obtained. The crystal structure
reveals the two nitrile groups as well as the ester moieties in the
ꢀ
periphery, and corresponds to the space group P1 (no. 2) with 2
molecules per unit cell (see ESI, Fig. S1†). A second dinitrile
derivative bearing two adamantane moieties was also synthe-
sized starting from adamantane carboxylate. This was coupled
via an amide bond to mono Boc-protected ethylendiamine
using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI),
ethylcyano (hydroxyimino)acetate (Oxyma Pureꢀ) and N-meth-
ylmorpholine (NMM) as coupling agents. Full details of the
synthesis are provided in the ESI.†
Scheme 1 Synthesis of substituted phthalonitriles (I): K2CO3, DMF, 24 h, rt, (II):
EDCI, Oxyma Pureꢀ, NMM, DMF, 14 h, rt, (III): TFA, CH2Cl2, 4 h, rt, (IV): EDCI,
Oxyma Pureꢀ, NMM, DMF, 14 h, rt, (V): K2CO3, DMF, 24 h, rt.
Scheme 2 Statistical macrocyclisation of (1) and (5): (I) DBU, Zn(OAc)2$5H2O,
pentanol, 150 ꢀC, 12 h, (II) THF, MeOH, H2O, NaOH, rt, 6 h.
2454 | Soft Matter, 2013, 9, 2453–2457
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Soft Matter
scattering (DLS). Bare CDVs have a characteristic diameter of
around 100 nm aer extrusion of a hydrated lipid lm through a
100 nm polycarbonate membrane. The size distribution of the
CDVs is provided in the ESI.† The CDVs have a slightly negative
z-potential of around ꢁ11 mV, which is due to the preferential
absorption of hydroxide ions by the ethylene glycol chains at the
primary rim of the b-cyclodextrin (Fig. 2, entry 1).19 Upon addi-
tion of (8) (added with 6 eq. of NaOMe), a signicant drop of the
z-potential to ꢁ25 mV was observed, which conrms
the immobilization of the adamantane-functionalized PC on the
CDV surface, since the large number of free carboxylate func-
tions of the guest molecules provide a signicant contribution to
the negative surface potential of the CDVs. Furthermore, the
repulsive forces between the functionalized vesicles play an
important stabilizing role, and no increase in size was observed
upon addition of (8) to the bare CDVs (Fig. 2, entry 2). To verify
that the drop in the z-potential is due to the host–guest
complexation between the hydrophobic adamantane residues of
the PC and the free CD cavities of the vesicles, an excess (10 mM)
of b-CD was added. It was observed that a displacement of the
binding equilibrium occurs and that (8) is released from the
CDVs. The z-potential of bare CDVs was recovered accordingly
(Fig. 2, entry 3). The diameter and size distribution of the CDVs
are not affected by the desorption of (8).
Fig. 3 Absorption spectra of (8) (red line) and (8)-CDV (black line), both recor-
ded in distilled water.
In Fig. 3 the normalized absorption of (8) is shown in
comparison with (8) bound to the vesicles ((8)-CDV), both in
aqueous solution. We note that in each experiment, 6 eq. of
NaOMe were added with (8). The pH of the resulting solutions
(with or without CDV) was slightly above 7. The photophysical
properties of (8) and its derivatives are largely independent of
pH, provided that the acidic or basic functional groups are
decoupled from the photoactive macrocycle.27 The absorption
spectra display characteristic Soret-bands (360 nm) and Q-
bands (685 nm), as previously reported for other zinc(II)
phthalocyanines.28,29 However, free (8) shows a pronounced
shoulder around 620 nm, which diminishes upon attachment
to vesicles, and constitutes distinctive evidence that aggregation
of (8) is inhibited when it is immobilized on the CDVs.
In Fig. 4, the excitation and the emission spectra of (8) and
(8)-CDV are depicted. The emission spectra can be attributed to
Fig. 4 Excitation (red) and emission (black) spectra of (8) (A) and (8)-CDV (B) in
distilled water.
the monomeric species and do not depend on the excitation
wavelength. The absorption and the excitation spectra are
coincident and in good agreement with previously reported
spectroscopic studies of zinc(II) phthalocyanines.30 Clearly, the
peripheral water soluble carboxylic groups do not inuence the
photophysical properties of the macrocycle to any signicant
extent (Table 1).
Singlet oxygen quantum yields (FD) were determined using
9,10-anthracenediyl-bis(methylene)dimalonic acid (ADMADM)
in aqueous solution as a uorescent monitor (lex ¼ 380 nm).
Irradiation of stirred, air saturated solutions of a reference
compound (methylene blue, MB, FD ¼ 0.52) and either (8) or
Fig. 2 Comparison of the size (nm) and the z-potential (mV) of (1) 0.1 mM CDV,
(2) 0.1 mM CDV + 50 mM (8) and (3) 0.1 mM CDV + 50 mM (8) + 10 mM b-CD. The
size distribution of CDVs is provided in the ESI.†
This journal is ª The Royal Society of Chemistry 2013
Soft Matter, 2013, 9, 2453–2457 | 2455

View Article Online
Soft Matter
Paper
Table 1 Photophysical parameters of (8) in solution and bound to CDV in
(Fig. S4 and eqn (S1)†). This method was used in view of the fact
that the phosphorescence of 1O2 was not observable in the
aqueous environment, even when D2O wasemployed as a solvent.
The hydroxyl groups of the CDV most likely quench the phos-
phorescence of 1O2, thus preventing its quantitative determina-
tion in the near-infrared region of the electromagnetic spectrum
(1275 nm). The FF and the FD of (8)-CDV are signicantly higher
(by a factor of roughly 3 within the experimental error) than for
pure (8) in aqueous solution (Table 1). These results show the
enhanced availability of monomeric chromophores in the (8)-
distilled water
(8)
(8)-CDV
lmax/nm (Soret-band)
lmax/nm (Q-band)
lmax/nm (emission)
358
682
690
3.24
0.03
0.05
360
682
690
0.63, 2.57
0.07
suo/ns (ꢃ0.05)
FF (ꢃ0.02)
FD (ꢃ0.02)
0.20
1
CDV assembly for the photogeneration of O2. By comparison
(8)-CDV in 10 ꢂ 10 mm quartz cuvettes led to a drop of the
ABMDMA emission intensity over time (Fig. 5 and ESI, Fig. S4†).
Comparison of the slopes of the uorescence decays led to FD
with monomers of other octaoxosubstituted Zn(II) PCs (FF ¼ 0.30
and FD ¼ 0.70), and assuming that a dimer absorbs like two
1
monomers but does not show any uorescence or O2 photo-
production,27 we conclude that about 25% of the photosensitizer
molecules are activeas monomeric species on the CDV. The host–
guest interaction of two adamantane moieties with the cavities of
the CDV compensates the free energy of aggregation. The driving
force of the hydrophobic effect overcomes the stacking effect of
PC, a process that is also driven by hydrophobic exclusion of the
macrocycles. This approach constitutes a successful way to
signicantly avoid aggregation of PCs in aqueous media by
immobilization on the surface of the vesicles.
Conclusion
We have documented a synthetic route for a water soluble zin-
c(II) phthalocyanine bearing adamantane moieties. Vesicles
formed by an amphiphilic b-cyclodextrin allow host–guest
interactions and immobilization of the phthalocyanine in
aqueous media, thus hindering the formation of inactive
aggregates and enhancing the photosensitizing ability. This
supramolecular assembly constitutes a biocompatible photo-
active platform for the design of new phototherapeutic agents,
and the possibility of combining this system with other ada-
mantane-functionalized molecules for enhanced targeting or
uorescent properties opens the way for simultaneous detec-
tion and inactivation of cancer cells or bacteria.
Acknowledgements
The authors gratefully acknowledge nancial support from the
Deutsche Forschungsgemeinscha (DFG grants Ra 1731/1-2 to
BJR and Str1186/1-1 to CAS).
Notes and references
1 T. J. Dougherty, B. W. Henderson, C. J. Gomer, G. Jori,
D. Kessel, M. Korbelik, J. Moan and Q. Peng, J. Natl. Cancer
Inst., 1998, 90, 889–905.
2 C. Schweitzer and R. Schmidt, Chem. Rev., 2003, 103, 1685–
1757.
¨
3 M. Regehly, K. Greish, F. Rancan, H. Maeda, F. Bohm and
¨
B. Roder, Bioconjugate Chem., 2007, 18, 494–499.
4 K. K. Ishii, M. Shiine, Y. Shimizu, S. Hoshino, A. Hisaku,
K. Sogawa and N. Kobayashi, J. Phys. Chem. B, 2008, 112,
3138–3143.
Fig. 5 Decaying emission spectra of ADMADM at different irradiation times: (A)
MB (0 s, 25 s and 50 s) and (B) (8)-CDV (0 s, 240 s, 480 s). (C) Slopes of ADMADM
decay for (8)-CDV (red) and for MB (black).
2456 | Soft Matter, 2013, 9, 2453–2457
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Soft Matter
5 R. K. Pandey, K. M. Smith and T. T. Dougherty, J. Med. Chem., 18 B. J. Ravoo and R. Darcy, Angew. Chem., Int. Ed., 2000, 39,
1990, 33, 2032–2038. 4324–4326.
6 F. Schmitt, P. Govindaswamy, G. Suss-Fink, W. H. Ang, 19 P. Falvey, C. W. Lim, R. Darcy, T. Revermann, U. Karst,
¨
P. J. Dyson, L. Juillerat-Jeanneret and B. Therrien, J. Med.
Chem., 2008, 51, 1811–1816.
7 N. Nishiyama, H. R. Stapert, G. Zhang, D. Takasu, D. Jiang,
M. Giesbers, A. T. M. Marcelis, A. Lazar, A. W. Coleman,
D. N. Reinhoudt and B. J. Ravoo, Chem.–Eur. J., 2005, 11,
1171–1180.
T. Nagano, T. Aida and K. Kataoka, Bioconjugate Chem., 20 C. W. Lim, B. J. Ravoo and D. N. Reinhoudt, Chem. Commun.,
2003, 14, 58–66. 2005, 5627–5629.
8 C. A. Strassert, M. Otter, R. Q. Albuquerque, A. Hone, Y. Vida, 21 J. Voskuhl, M. C. A. Stuart and B. J. Ravoo, Chem.–Eur. J.,
¨
B. Maier and L. De Cola, Angew. Chem., Int. Ed., 2009, 48,
7928–7931.
9 Y. N. Konan, R. Gurny and E. Allemann, J. Photochem.
Photobiol., B, 2002, 66, 89–106.
2010, 16, 2790–2796.
22 J. Voskuhl, T. Fenske, M. C. A. Stuart, B. Wibbeling,
C. Schmuck and B. J. Ravoo, Chem.–Eur. J., 2010, 16, 8300–
8306.
10 X. Jiang, S. Yeung, P. Lo, W. Fong and K. P. N. Dennis, J. Med. 23 D. A. Uhlenheuer, D. Wasserberg, C. Haase, H. D. Nguyen,
Chem., 2011, 54, 320–330.
11 H. Li, T. J. Jensen, F. R. Fronczek, H. Vicente and M. Graça, J.
Med. Chem., 2008, 51, 502–511.
J. H. Schenkel, J. Huskens, B. J. Ravoo, P. Jonkheijm and
L. Brunsveld, Chem.–Eur. J., 2012, 18, 6788–6794.
24 S. K. M. Nalluri, J. L. Bultema, E. J. Boekema and B. J. Ravoo,
Chem. Sci., 2011, 2, 2383–2391.
12 W. Liu, T. J. Jensen, F. R. Fronczek, R. P. Hammer, K. M. Smith,
M. Graça and H. Vicente, J. Med. Chem., 2005, 48, 1033–1041. 25 S. K. M. Nalluri and B. J. Ravoo, Angew. Chem., Int. Ed., 2010,
13 M. Hu, N. Brasseur, S. Z. Yildiz, J. E. van Lier and
49, 5371–5374.
C. C. Leznoff, J. Med. Chem., 1998, 41, 1789–1802.
26 S. K. M. Nalluri, J. Voskuhl, J. B. Bultema, E. J. Boekema and
B. J. Ravoo, Angew. Chem., Int. Ed., 2011, 50, 9747–
9751.
´
14 D. A. Fernandez, J. Awruch and L. E. Dicelio, Photochem.
Photobiol., 1996, 63, 784–792.
15 A. Mazzaglia, A. Valerio, N. Micali, V. Villari, F. Quaglia, 27 C. A. Strassert, G. M. Bilmes, J. Awruch and L. E. Dicelio,
`
M. A. Castriciano, L. M. Scolaro, M. Giuffre, G. Siracusano
Photochem. Photobiol. Sci., 2008, 7, 738–747.
28 J. W. Owens, R. Smith, R. Robinson and M. Robins, Inorg.
Chim. Acta, 1998, 279, 226–231.
and M. T. Sciortino, Chem. Commun., 2011, 47, 9140–9142.
16 N. Kandoth, E. Vittorino, M. T. Sciortino, T. Parisi, I. Colao,
A. Mazzaglia and S. Sortino, Chem.–Eur. J., 2012, 6, 1684–1690. 29 A. Ogunsipe, J. Chen and T. Nyokong, New J. Chem., 2004, 28,
17 A. Mazzaglia, N. Angelini, R. Darcy, R. Donohue, 822–827.
D. Lombardo, N. Micali, M. T. Sciortino, V. Villari and 30 A. Ogunsipe, D. Maree and T. Nyokong, J. Mol. Struct., 2003,
L. M. Scolaro, Chem.–Eur. J., 2003, 23, 5762–5769. 650, 131–140.
This journal is ª The Royal Society of Chemistry 2013
Soft Matter, 2013, 9, 2453–2457 | 2457