Chemically-selective surface glyco-functionalization of liposomes through Staudinger ligation

Article abstract of DOI:10.1039/b822420j

An efficient and chemoselective liposome surface glyco-functionalization method has been developed based on Staudinger ligation, in which the carbohydrate derivative carrying an azide spacer is conjugated onto the surface of preformed liposomes bearing a

Full text of DOI:10.1039/b822420j

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Chemically-selective surface glyco-functionalization of liposomes
through Staudinger ligationw
Hailong Zhang, Yong Ma and Xue-Long Sun*
Received (in College Park, MD, USA) 15th December 2008, Accepted 19th March 2009
First published as an Advance Article on the web 28th April 2009
DOI: 10.1039/b822420j
An efficient and chemoselective liposome surface glyco-
functionalization method has been developed based on Staudinger
ligation, in which the carbohydrate derivative carrying an azide
spacer is conjugated onto the surface of preformed liposomes
bearing a terminal triphosphine in PBS buffer (pH 7.4) at room
temperature.
amide10 and thiol–maleimide coupling,11 as well as imine12 or
hydrazone linking,13 has been achieved. However, in many
cases, there is a lack of specificity, resulting in the uncontrolled
formation of covalent bonds between liposomes and the
biomolecules of interest. Most recently, Cu(I)-catalyzed
[3 + 2] cycloaddition, namely ‘‘click’’ chemistry, which can
occur efficiently in aqueous media at room temperature and
selectively between azides and alkynes,14 has been investigated
as a novel generic chemical tool for the facile in situ surface
modification of liposomes.15 However, the key limitation of
click chemistry is the use of a Cu(I) catalyst, which results in
residual copper in the targeted liposome preparations, which
could be a potential concern. Normally, it is difficult to
completely remove copper from the resultant liposome15
and thus it will affect the liposome’s subsequent biological
applications.
Liposomes, spherical closed self-assembled (phospho)lipids,
have been extensively studied as models of cell membranes
and carriers for drug/gene delivery applications.1,2 Liposome
surface functionalization facilitates an enormous potential
application of liposomes.3,4 Cell surface carbohydrates
are attractive models for liposome surface modification
with oligosaccharides due to their protein-rejecting ability,
biodegradability, low toxicity and especially their cell targeting
ability through specific binding interactions with receptors
expressed at the targeted cell’s surface. For example,
monosialoganglioside (GM1) can enhance the circulation
lifetimes of liposomes compared to that observed for PEG.5
Sialyl lewis X decorated liposome has been demonstrated to
target the delivery of drugs to endothelial cells based on the
site-specific expression of E- and P-selectin in blood vessels
during inflammation.6 On the other hand, carbohydrate-
decorated liposomes have been used as a multivalent platform
of carbohydrates to inhibit carbohydrate-mediated cell
adhesion. For example, sialic acid-decorated liposome shows
strong inhibitory activity against influenza hemagglutinin and
neuraminidase.7
Staudinger ligation, in which an azide and a triphosphine
selectively react to form an amide, has been used for the
chemically-selective modification of recombinant proteins
under native conditions.16 Recently, Staudinger ligation has
been successfully performed in living animals without
physiological harm, suggesting the potential for applications
in cell surface functionalization.17 In particular, the reaction is
known to occur in high yield at room temperature in aqueous
conditions and without any catalyst, and is compatible
with the unprotected functional groups of a wide range of
biomolecules. Herein, we report efficient and chemically-
selective liposome surface functionalization through Staudinger
ligation, with lactose as a model carbohydrate (Fig. 1). The
high specificity and high yield, as well as the biocompatible
reaction conditions, of the Staudinger ligation approach
makes it an attractive alternative to all of the current protocols
for liposome surface functionalization.
Conventional methods to prepare surface-functionalized
liposomes involve the initial synthesis of the key lipid–ligand
conjugate, followed by the formulation of the liposome with
all the lipid components. In this direct liposome formation
method, some of the valuable ligands inevitably face the
enclosed aqueous compartment and thus become unavailable
for their intended interaction with target molecules. In particular,
it is unrealistic if the targeting ligand is only available in
minimum quantities. Furthermore, lipid–ligand conjugates
normally have a limited solubility and stability in solvents,
or are incompatible with the various stages of their manufacture.
Alternatively, chemical modification methods, which in most
cases involve the coupling of biomolecules to the surface of
preformed vesicles carrying functionalized (phospho)lipid
anchors, have been developed.8,9 So far, variable success using
As a proof-of-concept, we have explored the possibility of
preparing glycosylated liposomes by coupling unprotected
Department of Chemistry, Cleveland State University, 2121 Euclid Ave,
Cleveland, OH 44115, USA. E-mail: x.sun55@csuohio.edu;
Fax: +1 216-687-9289; Tel: +1 216-687-3919
Fig.
1
A schematic illustration of liposome surface glyco-
w Electronic supplementary information (ESI) available: Full experi-
1
mental details, and H and 31P NMR spectra.
functionalization through Staudinger ligation.
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Fig.
3
Dynamic light scattering monitoring of the liposomes
Scheme 1 Synthesis of anchor lipid 5.
A: before conjugation and B: after conjugation.
lactosyl derivatives carrying an ethyl spacer functionalized
with an azide group to the surface of liposomes that incorporate
synthetic anchor lipids bearing a terminal triphosphine via
Staudinger ligation. Firstly, the terminal triphosphine carrying
the anchor lipid, 5, was synthesized by the amidation of
commercially available DPPE with 3-diphenylphosphino-4-
methoxycarbonylbenzoic acid NHS active ester 4, which was
prepared according to the procedure described by Ju et al.18
(Scheme 1). However, it was found that an unwanted
oxidation product, triphosphine oxide 3, easily formed during
the crystallization purification of triphosphine 2 when using
aqueous methanol. Oxidized compound 3 was confirmed by
comparing it to the oxidation product of triphosphine 2 with
hydrogen peroxide in methanol, which gave a typical chemical
shift at 34.2 ppm for a phosphine oxide, while phosphine 2
gave a chemical shift at À3.74 ppm in its 31P NMR spectrum
(Fig. 2A). Fortunately, phosphine oxide 3 could be converted
back to phosphine 2 by reduction with trichlorosilane in
toluene in high yield.19 No oxidized product was formed
during DPPE-triphosphine 5 synthesis and purification when
organic solvents were used. With this oxidation in mind, the
stability of intermediate triphosphine 2 and targeted lipid
triphosphine conjugate 5 were monitored by 31P NMR. As
observed, the oxidation of phosphines 2 and 5 in organic
solvents such as chloroform were very slow (Fig. 2B and 2C).
With anchor lipid 5 in hand, next, small unilamellar
liposomes, composed of saturated phospholipids (DPPC)
and cholesterol (2 : 1 molar ratio), and 5 mol% of anchor
lipid 5, were prepared by sequential extrusion through
polycarbonate membranes with pore sizes of 600, 200
and 100 nm at 65 1C. This produced predominately small
unilamellar vesicles with an average mean diameter of
120 Æ 5 nm, as determined by dynamic light scattering
(DLS). Next, the conjugation of the azide-containing lactose
ligand20 to the preformed liposomes was performed in
PBS buffer (pH 7.4) at room temperature under an argon
atmosphere for 6 h (Fig. 1). DLS was used to verify the
integrity of the vesicles during and after the coupling reaction.
As shown in Fig. 3, there was no significant change in the size
of the vesicles during and after the conjugation reaction.
Therefore, the reaction conditions described above do not
alter the integrity of the liposomes.
Next, to test whether the experimental conditions used for
the conjugation reaction could provoke some leakage of
the liposomes, we exposed the same type of liposomes
containing encapsulated self-quenching concentrations of
5,6-carboxyfluorescein (CF) to our standard conditions.21
Based on our fluorescence quenching determinations
(Fig. 4), we demonstrated that no apparent leakage was
triggered by the conjugation reaction compared to liposomes
incubated in the absence of the coupling reagents. Therefore,
the conjugation conditions established here are harmless to
liposome surface functionalization.
Furthermore, the grafted carbohydrate on the surface of
the liposomes was quantified by the phenol–sulfuric acid
method.22 Briefly, a phenol solution was added to a solution
of the liposomes with conjugated lactose and mixed. Next,
concentrated H2SO4 was added directly to the solution. The
mixture was then vortexed and allowed to stand for 30 min at
room temperature. The optical density was then recorded at
490 nm. Considering that about 40% of the triphosphine
anchor is oriented towards the interior of the vesicles, 80%
of the out-oriented triphosphines were modified in this
Staudinger ligation modification.
Fig. 4 The kinetics of 5,6-carboxyfluorescein release from liposomes
a: in the presence of lactose-azide and b: in the absence of lactose-azide
(control experiment). The inserts show the changes in the fluorescence
intensity of the liposome reaction suspension and control during the
conjugation and final decomposition with surfactant.
Fig. 2 TLC traces and 31P NMR study of triphosphine derivatives.
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In conclusion, we have developed an efficient and chemo-
selective conjugation method for liposome surface glyco-
functionalization based on Staudinger ligation. The reaction
could be performed under mild conditions in aqueous buffers
without catalyst, in high yields and with reasonable reaction
times. The reaction conditions developed in this work did not
alter the integrity of the bilayers in terms of liposome size and
leakiness, and provided perfectly functional vesicles. This
versatile approach, which is particularly suitable for the
ligation of water soluble molecules and which can accommodate
many chemical functions, is anticipated to be useful for the
coupling of many other ligands to liposomes.
Fig. 5 A: DLS monitoring of the agglutination of glycosylated
liposomes with lectin via B: multivalent interactions.
The authors acknowledge financial support under grants from
American Health Assistance Foundation (AHAF-H2007027)
and the Startup Fund from Cleveland State University. Thanks
go to Dr Dale Ray at CCSB for NMR studies and Dr Xiang
Zhou at CSU for the mass spectrometry studies.
Notes and references
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Fig. 6 A: The stability of liposomes without lactose and B: liposomes
with lactose, as monitored by DLS.
To determine whether the grafted lactose residues
were easily accessible at the surface of the liposomes, a
lectin binding assay was conducted by incubating lactosylated
liposomes in the presence of b-galactose binding lectin
(Arachis hypogae, 120 kDa, Sigma) in PBS (pH 7.4).
After 30 min, visible aggregation was apparent, and was
monitored by a DLS experiment (Fig. 5A). In contrast, neither
aggregation nor size change was observed with the control
liposomes without lactose. Furthermore, the presence of free
lactose (5.0 mM) prevented aggregate formation (not shown),
confirming that the aggregation was due to a specific
recognition of the lactose residues on the surface of the
liposomes by lectin via multivalent interactions (Fig. 5B).
The stability of liposomes over time is an important concern
in drug/gene delivery applications. It is known that GM1
could enhance the circulation lifetimes of liposomes compared
to that observed for PEG.5 In this study, the stability of the
lactosylated liposomes was evaluated by comparing them with
liposomes without lactose at room temperature, as monitored
by DLS. As shown in Fig. 6, both types of liposomes showed
good stability during an 8 d period. However, the liposomes
without lactose began to collapse and aggregate from day 9
(Fig. 6A), while there was no apparent size change for the
lactosylated liposomes (Fig. 6B). This result demonstrates that
the presence of lactose on the liposome surface provides a
steric barrier that prevents liposome aggregation.
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This journal is The Royal Society of Chemistry 2009
3034 | Chem. Commun., 2009, 3032–3034