(732b) Eliposomes: Novel Drug Delivery Vehicles

An
eLiposome is a liposome encapsulating an emulsion nanodroplet, and can be used
for drug delivery.  For example, therapeutic agents are encapsulated inside the
eLiposomes, and application of ultrasound can cause the emulsion droplet to
change from liquid to gas, thus increasing the volume inside the vesicle,
causing rupture and release of the drug.

In
this novel method, emulsion droplets were made of perfluorohexane (PFC6) or
perfluoropentane (PFC5) and liposomes were made separately and
then they were mixed using ultrasound; thus we call this the ?ultra? method.
Calcein was encapsulated inside the eLiposomes as a model drug. Dynamic
light scattering and transmission electron microscopy were used to measure the
size of the emulsions, liposomes and eLiposomes. The size of eLiposomes is
appropriate for extravasation into tumors with malformed capillary beds.  We
hypothesize that ultrasound can be used to break open these eLiposomes.
eLiposomes were constructed with folate on their surfaces to induce endocytosis.
eLiposomes were used to successfully deliver calcein as a model drug to HeLa
cells.

      Figure
1 demonstrates that folate was successfully incorporated into PFC5 ultra
eLiposomes, and that the folate was necessary to obtain calcein delivery to the
cytosol of cells.   Parallel experiments were conducted using eLiposomes
prepared with and without DSPE-PEG2000-folate incorporated into the eLiposome
bilayer. 200 uL of each eLiposome sample was added to HeLa cells in 12-well
plates. The plates were incubated at 37°C
for about 2 hours and then 20-kHz ultrasound was applied at 1 W/cm²
for 2 seconds. Finally the cells were washed to remove external calcein and
eLiposomes. Figure 1A shows the resulting calcein fluorescence from eLiposomes
without folate, while figure 1B shows the parallel experiment having folate
incorporated in the eLiposomes. Because the calcein was concentrated (30 mM) in
the eLiposomes, it did not fluoresce until released.  There is very little
calcein signal observable in Figure 1A, suggesting that very few
calcein-containing eLiposomes were endocytosed when folate was absent.  In
Figure 1B, the calcein fluorescence is originating from the cell cytosol, not
the cell surface, suggesting that the folated eLiposomes had been endocytosed
and that the calcein had been released to the cytosol.  Some of these cells
exhibit a granular pattern of calcein fluorescence, perhaps indicating that
some calcein remains in endosomes, and while released from the eLiposome and
diluted sufficiently to fluoresce, some calcein may not have been released
completely from the endosome. 

      While
Figure 1 shows that folate is necessary, Figure 2 shows that emulsions are also
necessary for calcein delivery. Folated ultra eLiposomes with PFC5 emulsions
and conventional liposomes (without emulsions) were prepared, each containing
calcein at 30 mmol. 200 µL of each sample was added to HeLa cells in 12-well
plates. Plates were incubated at 37°C
for about 2 hours and then 20 kHz ultrasound was applied (1 W/cm²
for 2 seconds). Figure 10A shows very little release of calcein to the cells
exposed to folated conventional liposomes (without emulsion) while Figure 10B
shows considerable release of calcein in the parallel experiment employing
folated eLiposomes. Both these images were collected under identical optical
conditions with the same gain and other settings on the confocal microscope.
Most cells show calcein distributed through the cell, but again sometimes
calcein appears more intensely in punctate spots, which might be unruptured
endosomes.

We
propose that our eLiposomes can be used in ultrasonically activated drug
delivery as follows. The eLiposomes could be loaded with drugs, such as
Doxorubicin, using conventional or pH gradient techniques [1, 2].
Upon exposure to ultrasound, the local pressure oscillates above and below
ambient pressure. At sufficiently high acoustic amplitudes, the local pressure
decreases below the vapor pressure of the PFC emulsion inside the eLiposome. 
During this short time, the PFC may start to boil to a gas phase.

      Lipid
bilayers can only sustain about 3% area expansion before they rupture [3, 4]. For the PFC6 and PFC5
of this research, the liquid to gas expansion ratios are about 259-fold and 137-fold,
respectively. For a liposome of 250-nm-diameter eLiposome containing a single
100-nm-diameter PFC6 emulsion droplet, less than 0.2% of the PFC liquid is
required to vaporize to achieve the 3% area expansion for eLiposome lysis. Nucleation
of the vapor phase may or may not occur on the first negative pressure cycle of
the ultrasonic wave.

      Although
any liquid of high vapor pressure could be employed in eLiposomes, PFCs are
useful for making emulsion droplets within eLiposomes because the phospholipids
and PFCs are biocompatible and non-toxic [5].

[1]          Y. barenholz, gilad haran, "Method of amphiphatic
drug loading in liposomes by pH gradient," 1993.

[2]          C. Celia, N. Malara, R. Terracciano, D. Cosco, D.
Paolino, M. Fresta, and R. Savino, "Liposomal delivery improves the
growth-inhibitory and apoptotic activity of low doses of gemcitabine in
multiple myeloma cancer cells," Nanomedicine: Nanotechnology, Biology
and Medicine, vol. 4, pp. 155-166, 2008.

[3]          E. A. Evans, R. Waugh, and L. Melnik,
"Elastic Area Compressibility Modulus of Red-Cell Membrane," Biophysical
Journal, vol. 16, pp. 585-595, 1976.

[4]          R. R. Netz and M. Schick, "Pore formation and
rupture in fluid bilayers," Physical Review E, vol. 53, pp.
3875-3885, Apr 1996.

[5]          M. Nieuwoudt, G. H. C. Engelbrecht, L. Sentle, R.
Auer, D. Kahn, and S. W. van der Merwe, "Non-toxicity of IV Injected
Perfluorocarbon Oxygen Carrier in an Animal Model of Liver Regeneration
Following Surgical Injury," Artificial Cells, Blood Substitutes and
Biotechnology, vol. 37, pp. 117-124, 2009.