Clickosomes - Using triazole-linked phospholipid connectors to fuse vesicles

Article abstract of DOI:10.1039/c2cc16827h

Two complementary artificial diether phospholipids were synthesized that can undergo a Cu(i)-catalyzed Huisgen-Sharpless click reaction. The resulting lipid can bridge the membranes of large unilamellar vesicles and cause their aggregation and ultimately their fusion.

Full text of DOI:10.1039/c2cc16827h

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COMMUNICATION
Clickosomes—using triazole-linked phospholipid connectors
to fuse vesicleswz
´
Frederic Loosli, David Alonso Doval, David Grassi, Pierre-Leonard Zaffalon,
France Favarger and Andreas Zumbuehl*
´
´
Received 3rd November 2011, Accepted 23rd November 2011
DOI: 10.1039/c2cc16827h
Two complementary artificial diether phospholipids were synthe-
sized that can undergo a Cu(I)-catalyzed Huisgen–Sharpless click
reaction. The resulting lipid can bridge the membranes of large
unilamellar vesicles and cause their aggregation and ultimately
their fusion.
also noted using standard, non-modified liposomes and Cu(II)19
or Zr(IV) salts20 or by simply mixing positively charged and
negatively charged vesicles.21
Recently, we have published the use of benzyloxy-dichloro-
phosphine (BODP) as a convenient reagent for the synthesis of
phospholipids. Here, BODP was used to phosphorylate 1,3-
bis(hexadecyloxy)propan-2-ol 2 (see Scheme 1). In the same
pot, the intermediate was reacted with an alkyne-terminated
amino alcohol to give the protected phosphate 3. After
debenzylation the amine was quaternized with dimethylsulfate
in order to give the 1,3-dipalmitic ester phospholipid Pet–
PCalkyne–Pet (5, for the nomenclature see ref. 22) in 40%
overall yield. In a similar fashion the azido phospholipid 8 was
synthesized (see Scheme 2). Here, a short ethyleneglycol linker
was introduced in the head group of the phosphate 6. After
debenzylation, a substitution of the terminal halogen with an
azido functionality provided the artificial phospholipid Pet–
PTEGazide–Pet (8) in 25% overall yield.
Seldom has a synthetic concept found such wide acceptance as
fast as the improved Cu(I)-catalyzed 1,3-dipolar cycloaddition
of alkynes and azides to form triazoles.1 Only 3 years after the
seminal paper by Sharpless et al. the technique was used on a
phospholipid in order to decorate it with a fragment of a
human prion protein.2 The power to rapidly and reliably
access a cytocompatible membrane label led to many important
contributions in this field, beautifully summarized in a recent
review.3 In parallel, phospholipids were developed that could
be tuned for drug delivery such as the recent example of
polymerized vesicles with multiple docking sites from which
a drug might be cleaved by a change in pH or by oxidation.4
We set out to explore the 1,3-dipolar cycloaddition reaction
to link together several liposomes. Such vesicle aggregates
might ultimately serve as a new type of medical biomaterial
containing a personalized aggregate of liposomes filled with
different drugs. In order to form these materials one typically
introduces modified lipids into a phospholipid liposome
preparation. The head group of the artificial lipid can then
be linked to a lipid in a second liposome. Various interactions
have been used in the literature such as those involving biotin–
streptavidin,5 concanavalin A–a-glucopyranosides,6 phosphate–
guanidinium,7 2-ureido-4[1H]-pyrimidinone with itself,8 cyanuric
acid–melamine,9,10 barbituric acid–triaminopyrimidine,11 and
barbituric acid–adenine.12 Also the use of lipid anchored
ligands was well studied, bridging vesicles with metal ligands
such as Fe(II),13 Co(II),14 Ni(II),15,16 or Cu(II).16–18 Furthermore,
In order to test the concept, we reacted the Pet–PCalkyne–
Pet (5) and the Pet–PTEGazide–Pet (8) under typical Cu(I)-
catalyzed 1,3-dipolar cycloaddition conditions (see Scheme 3).
A single isomer of the corresponding triazole-bridged phospho-
lipid (9) was isolated in 83% yield.
We then set out to test if it was possible to aggregate vesicles
using Cu(I)-catalyzed Huisgen–Sharpless click chemistry.
Unfortunately, neither Pet–PCalkyne–Pet (5) nor Pet–
PTEGazide–Pet (8) formed vesicles when a thin film of the
pure lipid was hydrated with buffer. Only when mixed with a
natural phospholipid, such as eggPC, large unilamellar vesicles
(LUVs, 100 nm diameter) could be formulated by extrusion.23
Several vesicle populations were made containing up to 50 mol%
of either Pet–PCalkyne–Pet (5) or Pet–PTEGazide–Pet (8). The
vesicles were then mixed in appropriate ratios and submitted to
typical Huisgen–Sharpless click reaction conditions. Unfortu-
nately, neither using Cu-catalysis nor heating led to the formation
of vesicle aggregates. We therefore hypothesized that we first
needed to overcome vesicle repulsion in order to let the click-
partners come close enough for a reaction.
a
maleimide substituted cholesterol was crosslinked with
externally added dithiol.18 And finally, vesicle aggregation was
Department of Organic Chemistry, University of Geneva,
1211 Geneva 4, Switzerland. E-mail: andreas.zumbuehl@unige.ch;
Fax: +41 22 379 3215; Tel: +41 22 379 6719
w Electronic supplementary information (ESI) available: Synthetic
details and characterization of all compounds. See DOI: 10.1039/
c2cc16827h
The two types of liposomes were additionally equipped
with oppositely charged lipids inspired by the protocol of
Menger et al.21 One population of vesicles was thus formulated
from 80 mol% eggPC, 10 mol% Pet–PCalkyne–Pet (5), and
10 mol% of (di-n-dodecyl)dimethylammonium bromide (Mix A).
z This article is part of the ChemComm ‘Emerging Investigators 2012’
themed issue.
c
1604 Chem. Commun., 2012, 48, 1604–1606
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Scheme 1 Synthesis of Pet–PCalkyne–Pet (5).
Scheme 2 Synthesis of Pet–PTEGazide–Pet (8).
Scheme 3 Proof-of-concept that Pet–PCalkyne–Pet (5) and Pet–PTEGazide–Pet (8) indeed formed a triazole-bridged phospholipid (9) under
Cu(I) catalysis.
The other vesicle population was formulated from 90 mol%
eggPC, and 10 mol% of the already negatively charged Pet–
PTEGazide–Pet (8) (Mix B). The two vesicle populations
showed surface charges of +75.3 mV ꢀ 1.41 mV and
ꢁ48.3 mV ꢀ 0.6 mV, respectively.
Equimolar amounts of Mix A and Mix B were diluted into
pure H2O with and without addition of CuBr24 and shaken for
a prolonged time. Similarly, Mix B and Mix C were combined.
Fig. 1A shows that after 1 day, both Mix A + B and
Mix B + C led to the formation of vesicle aggregates in the
presence of CuBr, as measured by dynamic light-scattering. No
aggregation between vesicles was found in the absence of CuBr.
After 4 days the aggregated vesicles fused (see Fig. 1B). This
effect was clearly dominant in Mix A + B where up to 67
vesicles fused (relative to the vesicle surface area that was
calculated from the vesicle diameter, see Fig. 1D). Mix B + C
In order to address the question to what degree the addition
of the copper salt could cause vesicle aggregation, a third,
negative control was introduced in the form of a vesicle
containing 90 mol% eggPC, and 10 mol% of (di-n-dodecyl)-
dimethylammonium bromide but no click-reaction partner,
showing a surface charge of +63.8 mV ꢀ 3.9 mV (Mix C).
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1604–1606 1605

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The authors thank the University of Geneva and the Swiss
National Science Foundation (200021-121718) for the finan-
cial support and acknowledge COST Action D43 and the
contributions of the Bioimaging, Mass Spectrometry, and
NMR platforms at the University of Geneva.
Notes and references
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Fig. 1 Analysis of particle-size using dynamic light-scattering (A and
B) and transition electron microscopy (C and D, vesicle age = 4 days).
Vesicles measured in A showed minor aggregate formation after day 1
and larger aggregates after day 4. C and D show the effect of Cu-salt
induced vesicle aggregation (C) and the additional click reaction
between vesicles (D) (scale bar = 1 mm).
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Fig. 2 Proposed mechanism of action: oppositely charged 100 nm
large unilamellar vesicles aggregate and are then tethered together via
1,3-dipolar cycloaddition. The close proximity of the membranes is
promoting vesicle fusion and giant unilamellar vesicles are formed.
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showed fusion of only approx. 7 vesicles (Fig. 1C). These facts
are hinting at a dynamic aggregation–dissociation in Mix B + C
versus a stable chemical linkage in Mix A + B and lead us to
propose the mechanism of action in Fig. 2.
In conclusion, we have synthesized artificial phospholipids
containing either an alkyne or an azido function. In the
presence of CuBr, oppositely charged large unilamellar vesi-
cles formulated from a mixture of eggPC and the artificial
phospholipids would initially aggregate, held together by a
triazole-linked phospholipid connector and then fuse into
large unilamellar structures.
c
1606 Chem. Commun., 2012, 48, 1604–1606
This journal is The Royal Society of Chemistry 2012