Liposomes as ultrasound imaging contrast agents and as ultrasound-sensitive drug delivery agents.

Full text

Page 1

CELLULAR & MOLECULAR BIOLOGY LETTERS

LIPOSOMES AS ULTRASOUND IMAGING CONTRAST AGENTS AND
AS ULTRASOUND-SENSITIVE DRUG DELIVERY AGENTS

SHAOLING HUANG1, ANDREW J. HAMILTON2, SUSAN D.TIUKINHOY2,
ASHWIN NAGARAJ2, BONNIE J. KANE2, MELVIN KLEGERMAN3,
DAVID D. MCPHERSON2 and ROBERT C. MACDONALD1*
1Department of Biochemistry, Molecular Biology and Cell Biology, Evanston,
IL 60208. 2Division of Cardiology /Department of Medicine, Northwestern
University, Chicago IL 60611; 3EchoDynamics, Inc., College Park, MD 20642.


Ultrasound imaging contrast agents that exhibit an affinity for specific tissue
types, and especially, disease sites, would have considerable clinical value.
Liposomal dispersions can be prepared such that they both reflect diagnostic
ultrasound [1] and, when conjugated to an appropriate antibody, target
themselves to thrombi in the vascular circulation [2].
The procedure for preparation of ultrasound-reflective liposomes, consisting of
phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol
(PG), and cholesterol (CH), requires sonication to hydrate the lipid thoroughly,
addition of an excipient, lyophilization, and reconstitution. These steps were
examined to generate an optimal preparation [3]. Ultrasound reflectivity was
assessed using a 20 MHz intravascular ultrasound catheter and computer-
assisted videodensitometry. Ultrasound reflectivity was found to be maximal at a
CH concentration of 10 mol %. Variation in PG had little effect, although in the
total absence of PG, aggregation was undesirably high. Optimal acoustic
stability (resistance to loss of reflectivity upon standing following the
reconstitution step) was observed with CH concentrations of 10-15 mol % and
with PG concentrations greater than 4 mol %. Preparations made with 0.2 M
mannitol present during lyophilization were much more ultrasound reflective
than those made with similar concentrations of lactose, trehalose, or sucrose.
The preparations were stable when stored in the lyophilized state, but became
acoustically inactive a few hours after reconstitution at room temperature. Such
preparations could be rejuvenated by lyophilizing a second time. Careful
attention to formulation conditions produced preparations that could be diluted
to 10-50 :g/ml and still produce strong ultrasound reflection. An indication of the
basis of the acoustic activity came from measurements of effects of variations in
ambient pressure; echogenicity was greatly reduced by exposure to 0.5 atm
vacuum or 1.5 atm pressure for 10 s [4]. (Pressure changes of the magnitude
that are present in the arterial circulation had little effect on echogenicity.) Such
a response suggested that the active lipid preparations contained small amounts
of highly dispersed air. Indeed, application of vacuum resulted in the release of
approximately 100 :L of air from a standard preparation of 10 mg lipid in 1 mL
of buffer.

* E-mail: macd@nwu.edu
233

Page 2

CELL. MOL. BIOL. LETT. Vol. 7. No. 2. 2002

234
Maximum ultrasound reflectivity was critically dependent upon the conditions of
lyophilization. Mannitol (0.1-0.2 M) was required during the lyophilization step
and high echogenicity was associated with large-volume freeze-dried cakes and
fusion of liposomes (10X increase in size). Lyophilization from water caused
liposome fusion but the cakes were small and ultrasound reflectivity was weak.
Lyophilization from cryoprotectants (e.g., trehalose) produced large cakes but
little liposome fusion and also resulted in weak ultrasound reflectivity. These
findings indicate that lyophilization from 0.2 M mannitol solution generates a
disrupted array of lipid bilayers that, upon rehydration, fuse and trap small
amounts of air distributed among liposome-size particles. The peculiar
requirement for mannitol rather than a cryoprotective excipient evidently relates
to the unusual freezing behavior of mannitol solutions; extensive crystallization
occurs upon freezing, evidently generating bilayer defects which subsequently
account for both bilayer fusion during freezing and air entrapment during
reconstitution. 50% of the echogenicity originated from particles smaller than 1
micrometers.
The phosphatidylcholine component
ethylphosphatidylcholine, a cationic derivative of PC that is an effective DNA
transfection agent [5], so it became possible to develop a liposome preparation
that could both provide imaging of and gene delivery to a target site. During the
development of such lipid dispersions it was found that transfection of DNA into
cells in vitro was markedly enhanced by irradiation of cells with ultrasound in
the presence of acoustically-active lipid preparations. The procedure was to first
apply DNA-cationic lipid complexes to rat arterial smooth muscle cells in 6-well
plates and then, after 30 min, add a larger portion of cationic, acoustically-active
lipids (without DNA) and immediately subject the cells to 1-MHz ultrasound
(0.5 W/cm2) for a few seconds. Transfection efficiency (and DNA uptake) was
significantly enhanced (up to 5+ fold) by ultrasound exposure.
Ultrasound in conjunction with ultrasound-reflective lipoplexes appears to have
considerable promise for improving gene transfer. Since acoustically-active
liposomes conjugated to antibodies target themselves to vascular disease sites in
vivo, it thus becomes feasible (by using ultrasound) to both identify a disease
site and activate a therapeutic agent in situ.

REFERENCES

1. Alkan-Onyuksel, H., Demos, S.M., Lanza, G.M., Vonesh, M.J., Klegerman,
M.E., Kane, B.J., Kuszak, J. and McPherson, D.D. Development of inherently
echogenic liposomes as an ultrasonic contrast agent. J. Pharm. Sci. 85
(1996) 486-490.
2. Demos, S.M., Alkan-Onyuksel, H., Kane, B.J., Ramani, K., Nagaraj, A.,
Greene, R., Klegerman, M. and McPherson, D.D. In vivo targeting of
acoustically reflective liposomes for intravascular and transvascular ultrasonic
enhancement. J. Am. Coll. Cardiol. 33 (1999) 867-875.
could be replaced by
O-

Page 3

CELLULAR & MOLECULAR BIOLOGY LETTERS


235

3. Huang, S.L., Hamilton, A.J., Nagaraj, A., Tiukinhoy, S.D., Klegerman, M.E.,
McPherson, D.D. and Macdonald, R.C. Improving ultrasound reflectivity and
stability of echogenic liposomal dispersions for use as targeted ultrasound
contrast agents. J. Pharm. Sci. 90 (2001) 1917-1926.
4. Huang, S.-L., Hamilton, A.J., Pozharski, E.V., Nagaraj, A., Klegerman, M.E.
and McPherson, D.D. Physical correlates of the ultrasonic reflectivity of lipid
dispersions suitable as diagnostic contrast agents. J. Ultrasound Med. (2002)
in press.
5. MacDonald, R.C., Ashley, G.W., Shida, M.M., Rakhmanova, V.A.,
Tarahovsky, Y.S., Pantazatos, D.P., Kennedy, M.T., Pozharski, E.V., Baker,
K.A., Jones, R.D., Rosenzweig, H.S., Choi, K.L., Qiu, R. and McIntosh, T.J.
Physical and biological properties of cationic triesters of phosphatidylcholine.
Biophys. J. 77 (1999) 2612-2629.