(232f) Ultrasound Induced Calcein Release From Eliposomes

Ultrasound Induced Calcein Release from eLiposomes

J.R. Lattin¹, W.G.
Pitt, Ph.D.¹, D.M. Belnap, Ph.D.², G.A. Husseini,
Ph.D.³

¹ Chemical
Engineering Department, Brigham Young University, ² Chemistry and
Biochemistry Department, Brigham Young University, ³ Chemical
Engineering Department, American University of Sharjah, Sharjah,           

            Liposomes - bilayer
lipid vesicles - have proven to be effective and versatile drug carriers. 
Targeted drug delivery from liposomes could be used
to further increase their delivery efficiency by more specifically controlling
the site of drug delivery.  One such method of targeting involves using ultrasound
to release sequestered drugs at a specific target site from carriers
such as microbubbles.  However, liposomes are not inherently responsive to
ultrasound.  The goal of the current work is to develop a sub-micron sized
liposomal drug carrier that is responsive to low intensity ultrasound.  The
proposed drug carrier is called an eLiposome, defined as a liposome containing
vaporizable emulsion droplets.  The nano-sized emulsion droplets are formed
from perfluorocarbon liquids with high vapor pressures.  During the low
pressure phase of an ultrasound wave, the local pressure may drop below this
high vapor pressure, allowing the formation and expansion of a vapor phase [1]. 
 As this vapor phase forms and expands, it stretches and disrupts the liposomal
membrane, causing local drug release.  Emulsions were formed from
perfluorohexane (PFC6) and perfluoropentane (PFC5) because of their relatively
high vapor pressures and non-toxicity.  Additionally, both of these
perfluorocarbons have very low solubility in water, enabling nanodroplets to
persist in an aqueous environment. 

            Emulsions were formed
by inserting a 20 kHz ultrasound probe into a cuvette containing 0.2 g of PFC
and 5 mg of dipalmitoyl phosphatidyl choline (DPPC) in 1.5 mL of water.  Large (400
nm) and small (100 nm) emulsion droplets were prepared by varying sonication
time and intensity and/or by extrusion.  Sizes were verified by dynamic light
scattering.  eLiposomes were formed using a lipid sheet refolding technique [2]. 
Interdigitated DPPC sheets were formed from small DPPC vesicles by adding
ethanol.  After sheet formation, excess ethanol was removed from the sheet
suspension by two cycles of centrifugation and washing.  0.2 mL of emulsion was
added to 10 mg of interdigitated sheets along with 0.2 mL of water (for TEM
imaging) or a 30 mM calcein solution (for release experiments).  The solution
was heated to 50°C and stirred with a magnetic stir bar for 30 minutes,
allowing the sheets to fold back into vesicles and trapping nanoemulsion
droplets inside [3]. 
The size distribution of the resulting eLiposomes was controlled by extrusion
through an 800-nm polycarbonate filter at 50°C.  Empty eLiposomes, defined as
liposomes refolded from interdigitated sheets in the absence of emulsion, were
prepared as a negative control.

            The eLiposomes were imaged using cryogenic
transmission electron microscopy (cryoTEM).  The microscope stage was rotated
and the sample was viewed at -45°, 0°, and +45° to verify encapsulation of the
emulsion droplets [3].  The cryoTEM
images verify that PFC6 emulsion droplets have been encapsulated inside the
lipid vesicles using the sheet refolding technique and that this method was
able to encapsulate both large (400 nm) and small (100 nm) emulsion droplets.  Figure
1A shows an example of an 800-nm eLiposome with 3 distinct 100-150 nm emulsion
droplets.  Figure 1B shows an example of an 800-nm eLiposome with one 475-nm
emulsion droplet as well as two smaller droplets.

Figure
1. 
CryoTEM images of 800-nm eLiposomes containing A) three
100-150 nm PFC6 emulsion droplets or B) one 475-nm droplet and two smaller
droplets.  Scale bars represent 200 nm.  The straight (B, top left) and mottled
(A, bottom) structures are supporting entities of the holey carbon support film

            Calcein
containing samples were prepared for quantification of release.  During vesicle
formation, calcein was encapsulated inside of the eLiposomes at a concentration
of approximately 15 mM.  At this concentration, the calcein was self-quenched. 
The external calcein concentration was reduced by allowing the sample to settle
at the bottom of the microcentrifuge tube for a few hours to form a gel-like
pellet.  The top phase was removed and replaced with an NaCl solution.  Samples
were further diluted by adding 20 µL of sample to a cuvette along with 2 mL of
NaCl solution in order to leave an external calcein concentration of 1 to 5 µM
so as to be within the linear region of the concentration curve for calcein. 
Sample fluorescence was measured using a QuantaMaster fluorometer (Photon
Technology International, Birmingham, New Jersey) with excitation and emission wavelengths
of 488 nm and 525 nm, respectively. Baseline fluorescence data was collected
for 10 seconds at 4 data points per seconds.  The cuvette was then removed from
the fluorometer and 20-kHz ultrasound was applied using a 3 mm probe (Sonics
and Materials, Newton, CT) inserted directly into the cuvette.  As concentrated
(self-quenched) calcein was released from the interior of the eLiposomes into
the surrounding solution, it was diluted below its self-quenching concentration. 
Fluorescence was again measured after sonication.  Finally 25 µL of 5% Triton
X-100 was added to lyse any remaining liposomes and fluorescence was measured

            Figure
2 shows the results as the time of ultrasound exposure was varied from 100 ms
to 10 seconds at 1 W/cm².  The eLiposome samples demonstrated
increased calcein release compared to conventional liposomes (containing only
calcein) and compared to conventional liposomes with external emulsions,
suggesting that the encapsulated emulsion droplets add an ultrasound sensitive
element to the eLiposomes.  The eLiposomes were less sensitive to ultrasound
when small emulsion droplets were encapsulated.  This is most likely due to the
greater Laplace pressure imposed on the droplets as their size decreases [4]. 
The increased pressure on the droplet adds to the ultrasound intensity required
to vaporize the droplet.  Because Laplace pressure is inversely proportional to
radius, the amount of pressure on the droplet increases as droplets diameter
decreases.  In this study, the effect of droplet size can be best observed with
PFC5 eLiposomes: eLiposomes with large emulsion droplets released approximately
twice as much calcein as those with small droplets after 10 seconds of ultrasound
exposure.  There was also a noticeable difference between eLiposomes formed
with PFC5 and PFC6.  This difference is most likely due to the difference in
vapor pressures; it is easier to promote ultrasonically induced gas formation
with the higher vapor pressure of PFC5.

Figure 2.  Calcein
release from PFC5 eLiposomes (A) and PFC6 eLiposomes (B) when exposed to 20-kHz
ultrasound at 1 W/cm² for varying times.  Data is presented for
eLiposomes with large (n) and small (o)
emulsion droplets, empty control vesicles (l), and empty
vesicles with Large (p) or small (r)
emulsion droplets added to the exterior solution.  Sham experiments were also
performed using eLiposomes with large droplets (u).   Error bars
represent ± 1 standard deviation.

            eLiposomes
formed with PFC5 and PFC6 both released significantly more calcein than
controls, suggesting that the internal emulsions do indeed add an ultrasound
sensitive component to liposomes.

References

1.         Rapoport, N.Y., et al., Controlled and targeted tumor
chemotherapy by ultrasound-activated nanoemulsions/microbubbles. Journal of
Controlled Release, 2009. 138(3): p. 268-276.

2.         Kisak, E.T., et al., The vesosome - A
multicompartment drug delivery vehicle. Current Medicinal Chemistry, 2004. 11(2):
p. 199-219.

3.         Lattin, J.R., D.M. Belnap, and W.G. Pitt, Formation
of eLiposomes as a drug delivery vehicle. Colloids and Surfaces
B-Biointerfaces, 2012. 89: p. 93-100.

4.         Sheeran, P.S., et al., Formulation and Acoustic Studies
of a New Phase-Shift Agent for Diagnostic and Therapeutic Ultrasound.
Langmuir, 2011. 27(17): p. 10412-10420.