Full Paper
Chemistry—A European Journal
doi.org/10.1002/chem.202005332
Hundreds of liposomes per field of view were tethered on
poly(l-lysine)-poly(ethylene glycol) (PLL-PEG)-passivated and
[
23,24]
PLL-PEG biotin-passivated surfaces.
This methodology
maintains the spherical shape and structural integrity of the
[
25]
liposomes. In the membrane of the liposomes were integrat-
ed ATTO488 DOPE and encapsulated in their lumen ATTO655
Carboxy chromophores. Real-time imaging of two microscope
channels (one for each dye) allowed synchronous recording of
[
25]
both the cargo and the liposome membrane. Three different
types of liposomes were prepared in a phosphate-buffered
saline (PBS): one active liposome containing lipid 1·Zn and two
control liposomes. The compositions of the three types of lipo-
somes are listed in Table 2. All liposomes were composed of
DOPC, DOPG, and DSPE-PEG(2000) biotin. The charged DOPG
lipids were incorporated because they have been shown to
render the liposomes unilamellar upon ten cycles of flash-
freezing and thawing while the biotin-conjugated lipids made
surface-immobilization possible, as is illustrated in Figure 3a.
After tethering of the liposomes to the surfaces, a flow of
PBS buffer was used to wash away nonencapsulated dye and
make visualization possible. Figure 3b shows a typical TIRF
image in which hundreds of individual tethered liposomes are
observed. The two-color imaging (see blue and red color
zoom) shows the presence of liposomes in both channels (Fig-
ure 3b), which confirms that ATTO655 Carboxy was successful-
ly encapsulated into the 1·Zn-containing liposomes. Using a
fluidic pump, we exposed the liposomes to a constant flow of
Figure 2. Evaluation of the back-reaction from 1·Zn to 1 on addition of
1
1
Me
6
TREN: a) H NMR spectrum of 1 in [D
3
]acetonitrile. b) H NMR spectrum
2
+
1
of 1 in the presence of Zn ions. 1·Zn was generated in situ. c) H NMR
6
spectrum after addition of Me TREN to the same sample.
ent concentrations. It was found that all three carbohydrate
lipids were able to form aggregates in water with low dispersi-
ty (see Supporting information, Figures S6–S8). The C12 lipid
1
·Zn was even able to form monodisperse aggregates down
À1
to a concentration of 40 mm (43 mgL ); however, at a lower
concentration (4 mm), the dispersity of the aggregates in-
creased, that is, this concentration was below the critical ag-
gregation concentration (CAC). This allowed us to determine
the CAC of the three carbohydrate lipids using the convenient
[
22]
Nile Red method. The CAC values are listed in Table 1. All
lipids have CACs below 100 mm, and the C lipid 1·Zn has the
1
2
À1
lowest CAC of 35 mm (38 mgL ). This fit very well with the ob-
servation made in the preliminary DLS study on the same lipid.
As a trend, it seemed that the CAC increased when shorter
alkyl chains were introduced.
a 10 mm solution of Na EDTA in PBS halfway through the ex-
2
periment and throughout the remaining time of the experi-
Due to the three carbohydrate lipids having almost similar
aggregation properties, we chose to proceed with cargo-re-
lease studies using only the C12 lipid 1·Zn, as this had the
lowest CAC among the three synthesized lipids.
ment. Na EDTA was used in this case due to its high water sol-
2
ubility and high affinity to metal ions. Immediately after intro-
ducing Na EDTA to the liposomes, the ATTO655 chromophore
2
escaped, as shown qualitatively for a few liposomes in the
inset of Figure 3b and in the typical data for individual lipo-
somes in Figure 3c, (see Supporting information, Figure S12
for additional traces). Notably, the intensity of the membrane-
incorporated dye remained at a constant level (Figure 3b and
c), that is, the cargo was released while liposomes remained
bound and maintained their structural integrity. These data
Table 1. Critical aggregation concentration (CAC) and DLS data of the
three carbohydrate lipids 1·Zn, 2·Zn, and 3·Zn.
Lipid
CAC
Hydrodynamic diameter
nm]
Polydispersity
[%]
[
2
+
confirm the hypothesis of Zn -dependent pore formation and
cargo release of liposomes.
1
2
3
·Zn
·Zn
·Zn
35 mm
412.5Æ85.62
327.4Æ58.25
312.6Æ64.50
20
17
23
(
38 mg/L)
95 mm
89 mg/L)
50 mm
48 mg/L)
Several control experiments were performed to verify that
the observed release was not an imaging artifact and to better
understand the mechanism of the EDTA-promoted release. The
liposome only containing DOPC was unaffected by the same
(
(
addition of Na EDTA, and this suggests that lipid 1·Zn is critical
2
for the responsiveness of the lipids (Figure 3d, see Supporting
information, Figure S13 for additional traces). We also prepared
liposomes containing 50% in molar ratio of the inactive lipid 1.
Again, no release of fluorescent cargo was observed (Fig-
ure 3e, see Supporting information, Figure S14 for additional
traces), and this further supports that lipid 1 is only active
when binding Zn ions. Before being immobilized for TIRF mi-
croscopy, the liposomes were analyzed by DLS to evaluate
their hydrodynamic diameter and polydispersity. Interestingly,
both the DLS measurements and the TIRF data showed that
the liposomes incorporated with 1·Zn lipids and the reference
Direct real-time observation of cargo release via EDTA
The cargo-release properties of lipid 1·Zn were evaluated with
a total internal reflection fluorescence (TIRF) microscopy setup.
This setup can not only be used for the visualization of lipo-
somes, but also for monitoring the release of an encapsulated
fluorescent dye from intact liposomes. The general idea
2
+
2
+
behind this assay is illustrated in Figure 3a: Zn
removal
would promote the transient formation of one or more pores,
from which fluorescently labeled cargo would be released out
of the liposomes.
Chem. Eur. J. 2021, 27, 6917 – 6922
6920
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