New strategy for targeting of photosensitizers. Synthesis of glycodendrimeric phenylporphyrins, incorporation into a liposome membrane and interaction with a specific lectin

Article abstract of DOI:10.1039/b816128c

Two glycodendrimeric phenylporphyrins were synthesized and their interaction with phospholipids was studied at the air-water interface and in liposome bilayers; such liposomes bearing glycodendrimeric porphyrin could constitute an efficient carrier for dr

Full text of DOI:10.1039/b816128c

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New strategy for targeting of photosensitizers. Synthesis
of glycodendrimeric phenylporphyrins, incorporation into a
liposome membrane and interaction with a specific lectinw
ab
Severine Ballut, Ali Makky,cd Bernard Loock,ab Jean-Philippe Michel,cd
´
cd
Philippe Maillard*ab and Veronique Rosilio*
´
Received (in Cambridge, UK) 15th September 2008, Accepted 27th October 2008
First published as an Advance Article on the web 19th November 2008
DOI: 10.1039/b816128c
Two glycodendrimeric phenylporphyrins were synthesized and
their interaction with phospholipids was studied at the air–water
interface and in liposome bilayers; such liposomes bearing
glycodendrimeric porphyrin could constitute an efficient carrier
for drug targeting in photodynamic therapy.
biological functions. Due to the weak nature, in the millimolar
range, of interactions between a single specific carbohydrate
and a receptor protein subunit, nature uses cluster carbo-
hydrates in order to obtain biologically meaningful affinities
for the receptors. The cluster effect appears when the multi-
valent carbohydrates interact with more than one receptor
binding site simultaneously and cooperatively, resulting in
better cellular recognition. Several methods of carbohydrate
clustering have been described, including the attachment of
carbohydrates to natural scaffolds, synthetic polymers,
synthetic glycopeptides or simple oligomerization through
organic linkers.9 Among these various ways, dendritic
structures (glycodendrimers) are emerging as ligands for
carbohydrate-binding proteins.10 Due to rapid advances in
this area, promising potential medicinal applications
have appeared in the last ten years, including treatment of
cancers.11–13
The incorporation of carbohydrates on tetrapyrrolic macro-
cyclic cores usable in photodynamic therapy (PDT) continues
to be pursued vigorously by a number of research teams.1 In
our laboratories, efforts have been focused on the preparation
and in vitro evaluation of the phototoxicity of a series of
neutral glycoconjugated tetrapyrrolic macrocycles as potential
photosensitizing agents for photodynamic therapy.2 However,
these compounds are usually poorly water-soluble molecules,
and tend to form aggregates in the aqueous solution. This
affects unfavourably both their formulation and bio-
availability, and limits pharmacologic studies. Glycoconjugation
modifies the amphiphilicity of macrocycles, and can favor
their interaction with the tumor cell surface membrane.3
Concerning this latter property, indications are that glyco-
sylation provides the possibility for specific interaction of the
resulting conjugate with lectin type receptors overexpressed in
certain malignant cells.4 Glycoconjugation can thus be a
potentially effective strategy for targeting photosensitizers
toward tumor cells.5 The identification of the transport
mechanisms through the biological membranes was a crux.
However, it would be advantageous to use these mechanisms
for the optimization of photosensitizer targeting towards
tumor cells. In this context, the use of glycodendrimers as
recognition motifs was very exciting. It has been widely
accepted that carbohydrate–protein interactions play a crucial
role in a large number of biological processes.6 Since most
proteins possess multiple carbohydrate-recognition domains
and typically exist as oligomeric structures, this limitation is
often overcome through multivalency.7,8 Lectin receptors are
multisubunit and multivalent proteins with many important
In a continuation of our work to prepare and study neutral
targeting glycoconjugated photosensitizers, we report the
synthesis, characterization and behavior in liposomes of
glycodendrimers linked to meso-tetraaryl-porphyrins shown
in Fig. 1.
Recently, Ballardini et al. described the synthesis of
two symmetric dendrimers, incorporating tetrasubstituted
porphyrin units as the core and, in one case, four benzyl-
oylated or deprotected and in the other case, twelve acetylated-
b-D-glucopyranosyl- or b-D-glucopyranosyl residues at the
peripheries of the tetrapyrrolic macrocycle.14 These un-
protected tetrasubstituted molecules are very water-soluble.
We have shown that an amphiphilic structure of the glyco-
conjugated photosensitizers induces a better photocytotoxicity
in vitro.5 With the aim of increasing this photoefficiency, we
designed a new family of glycoconjugated photosensitizers
bearing only one glycodendrimer moiety, with variable length
for the spacer linking the carbohydrate to the porphyrin, on
the para position of one meso-phenyl group.
a UMR 176 CNRS/Institut Curie, Institut Curie, Baˆt 110, Univ.
Paris-Sud, F-91405 Orsay, France
b Institut Curie, Section de Recherches, Centre Universitaire, Univ.
Paris-Sud, F-91405 Orsay, France.
E-mail: philippe.maillard@curie.u-psud.fr; Tel: 33 169863171
c Univ. Paris-Sud, UMR 8612, Lab. Physico-Chimie des Surfaces,
´
ˆ
5 rue J-B. Clement, F-92296 Chatenay-Malabry, France
d CNRS, UMR 8612, F-92296 Chaˆtenay-Malabry, France.
E-mail: veronique.rosilio@u-psud.fr; Tel: 33 146835418
w Electronic supplementary information (ESI) available: Further
experimental details. See DOI: 10.1039/b816128c
Fig. 1 Structures of glycodendrimeric porphyrins.
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Scheme 1 Synthesis of glycodendrimeric porphyrins.
Synthesis of the glycodendrimeric porphyrins is shown in
Scheme 1. N-Z-glycine was linked to 3 by EEDQ in ethanol
(yield 86%) to give 4 then the tertio-butyl protection of
carboxylic acid was removed by trifluoroacetic acid in methylene
chloride to give compound 5 (92%). 2-Aminoethoxy-O-a-
peracetyl-D-mannose, and 2-aminoethoxy-ethoxy-O-a-peracetyl-
D-mannose prepared by the protocol described by Dahmen et al.
and Sasaki et al.15 were selected for peptide coupling with triacid
5. HATU16 promoted peptide coupling allowed the preparation
of branched glycopeptides with good yields (6 70%, 7 72%).17
Catalytic hydrogenation was used for selective cleavage
of benzyloxycarbonyl group into amine, in the presence of
p-toluensulfonic acid (H2–Pd/C 10%, MeOH) to afford the key
building blocks with good yields (8 96%, 9 82%). Dendrimeric
moieties 8 and 9 were linked to 5-para-benzoic acid-10,15,20-
triphenyl porphyrin using a mixture of HOBT, EDC and Et3N
with good or acceptable yields to give protected glycodendrimeric
porphyrins 10, and 11 (yield 86% and 57%, respectively) then
´
were quantitatively O-deacetylated under Zemplen’s conditions
to afford glycodendrimeric porphyrins 1 and 2.18
To evaluate the conditions of incorporation of compounds 1
and 2 into a liposome membrane, the two derivatives
were mixed with dimyristoylphosphatidylcholine (DMPC) in
a (1 : 1) ratio and the mixtures were spread at the air–water
interface.19,20 Fig. 2 shows that, whereas the isotherm of the
DMPC-compound 2 mixed monolayer lies between those of
the pure components, that of the mixed DMPC-compound 1
one is located at higher molecular areas and surface pressures.
Obviously in both cases, the phospholipid and porphyrin
derivatives interacted. However, if for compound 2, this
interaction was apparently attractive, for compound 1, it
was most probably repulsive. Thus, DMPC would mix better
with compound 2 than with compound 1.
The incorporation of compound 2 in liposome membranes
led to the formation of larger vesicles than DMPC ones
(Table 1). This is consistent with the p–A isotherms in
Fig. 2, which show an expansion of the phospholipid mono-
layer in the presence of compound 2. For DMPC liposomes
bearing compound 1, vesicles appeared smaller and less stable
with time than those prepared from DMPC or mixtures of
DMPC and compound 2. This is also in agreement with
the results in Fig. 2 that show an unfavourable interac-
tion between DMPC and compound 1, probably due to the
Fig.
2 Interfacial behaviour of mixed monolayers (1 : 1) of
dimyristoylphosphatidylcholine and compounds 1 and 2.
Table 1 Mean diameter of liposomes before and after incubation with Concanavalin A (Con A) at room temperature for 1 hour
Mean diameter before
Con A addition Æ s (nm)
Mean diameter after
Con A addition Æ s (nm)
Liposome composition
Polydispersity index
Polydispersity index
Pure DMPC
DMPC–compound 1
DMPC–Compound 2
185.0 Æ 0.08
178.0 Æ 2.2
218.0 Æ 1.2
0.103 Æ 0.029
0.096 Æ 0.013
0.179 Æ 0.034
187.0 Æ 1.3
210.0 Æ 4.15
2510 Æ 821
0.142 Æ 0.036
0.229 Æ 0.007
0.617 Æ 0.229
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presence of the sugar moieties in the vicinity of phospho-
lipid headgroups. This repulsive interaction between DMPC
and compound 1 could hinder the formation and stabilisa-
tion of vesicles and lead to the separation of DMPC vesicles
on one side and self-aggregated compound 1 molecules on
another.
Notes and references
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the glycodendrimeric phenylporphyrin derivatives were left
in contact for 1 hour with Concanavalin A (Con A), a
mannose-specific lectin (0.5 mg/ml). Their diameters were
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addition of the lectin. Conversely, for liposomes of
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These striking results could originate from (i) the poor
mixing properties of compound 1 with DMPC that would
lead to the low incorporation rate of this porphyrin into
phospholipid bilayers, and thus to a limited interaction of
those liposomes with Con A, (ii) the longer spacer in com-
pound 2 compared to that in compound 1, which would
increase the mobility of mannose moieties and facilitate their
interaction with Con A, and (iii) the existence of Con A dimers
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apparent size.
a dramatic increase in their
In this work, two glycodendrimeric phenylporphyrins
(compounds 1 and 2) were synthesized and their interaction
with phospholipids was studied at the air–water interface and
in liposome bilayers. The expansion of the DMPC-compound
1 mixed monolayer compared to monolayers of the pure
components accounts for an unfavourable interaction that
affected the formation and stabilisation of liposomes. Con-
versely, compound 2 favourably interacted with phospholipid
molecules and formed mixed liposomes, which aggregated in
the presence of a-mannose specific concanavalin A. These
results show that the tetrapyrrolic macrocycle 2 was indeed
embedded into the phospholipid bilayer and that its sugar
moieties protruded into the surrounding aqueous phase. Such
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liposomes
bearing glycodendrimeric phenylporphyrin
could constitute an efficient carrier for drug targeting in
photodynamic therapy.
The authors acknowledge financial support from the
20 L. Berthelot, V. Rosilio, M. L. Costa, S. Chierici, G. Albrecht,
P. Boullanger and A. Baszkin, Colloids Surf., B, 1998, 11, 239;
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‘‘Programme Incitatif et Cooperatif Retinoblastome’’ of
´ ´
Institut Curie, and for A. Makky’s PhD grant, from ARC
(Association pour la Recherche contre le Cancer).
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226 | Chem. Commun., 2009, 224–226