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acid were found to result in the decomposition of polymerized liposomes as judged
by dye leakage studies. Optimized conditions, obtained through conjugation with
fluorogenic azidocoumarin, included copper sulfate (0.3 mM), ascorbic acid (6 mM),
and ligand, in which conjugation was complete within 1 hour. The yield of this
reaction was calculated to be 23%, although this could be an underestimation due
to fluorescence quenching, and is also limited since only lipids on the outer leaflet
of the bilayer should react. With these conditions, CuAAC was used to introduce
both an RGD peptide as an integrin-targeting ligand, through reaction with
8
,as
well as a fluorescent label onto the polymerized liposomes, and fluorescence imaging
demonstrated successful delivery to cell membranes.
In an effort to pursue alternate bioorthogonal conjugation strategies, Sun and
coworkers evaluated the Staudinger ligation for derivatization of intact liposomes,
which is beneficial as it does not require additional catalyst for conjugation [57].
To do so, PE analog
9
was devised in which the traditional Staudinger reagent, a
triphenylphosphine bearing a proximal ester for covalent trapping during reaction
with azide, was appended (Fig. 4.3e). The authors noted that the synthesis of
9
was
complicated by phosphine oxidation when this compound was treated with aque-
ous methanol, but that this problem could be counteracted through reduction or
avoided altogether when using only organic solvents. Next, the Staudinger ligation
was evaluated for conjugation of azide-tagged lactose analog
10
onto PC/cholesterol
liposomes containing
9
(5 mol%). During the reaction, DLS and dye leakage assays
were performed, which both demonstrated that membrane integrity was maintained.
In addition, the introduction of lactose onto liposomal surfaces was quantified using
the phenol-sulfuric acid test, which indicated that 80% of
9
on the outer leaflet
of the membrane (40% total) were modified. Finally, a lectin-binding assay using
-galactose-binding lectin with detection of aggregation via DLS was used to show-
case the accessibility of lactose units on the surface.
Our group has also been interested in developing bioorthogonal methods for label-
ing intact membranes that circumvent the copper catalyst required for CuAAC, for
which we recently reported an approach involving copper-free click chemistry in
collaboration with Popik and coworkers [58]. While a variety of strained cyclooc-
tynes have been shown to be effective for click chemistry in the absence of copper
catalyst, we exploited the aza-dibenzocyclooctyne (ADIBO) system for this purpose
due to the robust nature, desirable kinetic properties, and ease of synthetic prepa-
ration of these reagents [49, 50]. To assess conjugation, we chose to implement an
assay involving liposome immobilization onto streptavidin-coated microplates. This
was performed by incorporating azido-TEG-glycerolipid
11
along with a fluores-
cent NBD-PE conjugate into liposomes, followed by incubation with ADIBO-TEG-
biotin reagent
12
(Fig. 4.3f). Following washes, the resulting microplate-immobilized
biotinylated liposomes could be detected in high-throughput fashion using a fluores-
cence plate reader. In these studies, a dose-dependent relationship was observed
between the amount of added ADIBO-TEG-biotin and the resulting fluorescence
signal, indicating successful liposome derivatization. Controls, including the replace-
ment of the ADIBO reagent with an acyclic alkyne, the use of liposomes lacking
11
and preincubation of the microplate with biotin, all resulted in no signal, indicating
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