Note: Descriptions are shown in the official language in which they were submitted.
WO 94/26299 21~ 7 7 9 ~ PCT/US94/05188
C LIPOSOMES INCORP~ENSlTY MEDIA
.~
FIELD OF TH~ INVENIION
?
The present invention relates to liposomes and methods for their production. The10 invention further relates to uses of liposomes in analytical methods.
BACKGROUND OF THE INVENTION
Liposomes are vesicles or sacs having closed membranes of amphiphilic molecules in
15 equilibrium with an aqueous solution. Polar lipids such as phosphatidylcholines,
phosphatidylethanol~mines~ ,go",yelins, cardiolipins, phosphatidic acids, cerebrosides and
conlbillalions of fatty acids form such membranes when placed in an aqueous el~vi~o~ ent.
The bimolecular lipid sheets of the membrane are intercalated by aqueous spaces, and these
structures can persist even in the ~,esence of excess water. Liposomes are the,effi,t; useful
20 vehicles for inco,~o,aling active agents for delivery to a desired therapeutic site and as model
systems for cellular processes. Liposomes have also been used in the art for encapsulation of
dyes and used as tracers in imm~nnassays. PERCOLL beads have also been ~nc~rs~ ted in
liposomes for use as markers in spleen tissue for vi~ li7~tion by electron microscopy (EM)
(Cudd and Mcolau. 1986. J. Microencapsulation 3(4):275-282). These authors did not
25 attempt to make liposomes of defined density, as the goal was only to produce an electron
dense marker for EM studies which could be readily rli~tin~li~hed from subcellular organelles.
They also observed that PERCOLL seemed to render the liposomes less stable.
Materials having a specific, reproducible buoyant density have been used in the art as
30 density markers to calibrate or monitor density gradients in such applications as density
gradient centrifugation. These materials in general have taken the ffirm of solid beads of
SUBSTITUTE SHEET (RULE 26~
WO 94/26299 2~5~ ~ 9 ~' PCT/US94/05188
defined buoyant density, such as the Density Marker Beads (cross-linked dextran) ava(~le
from Pharmacia LKB Biotechnology, Uppsala, Sweden. POLYBEAD PMMA (polymethyl
m.oth~crylate) Monodisperse Particles available from Polysciences, Inc. (Warrington, PA) have
a defined buoyant density of 1.19 which allows more rapid separation of the particles when
5 used in immunoassays. However, these commercial- density markers and defined density t
partides may not be m~n-lf~r,t~lred with thèi buoyant density required for a particular
application and cannot easily be modified to obtain the buoyant density desired. For example,
if a density marker for a particular blood cell type is required, density marker beads may not be
commercially available with the correct buoyant density.
The complete blood cell count (CBC) is used extensively in hematological analysis and
is frequently performed to obtain general information about the status of a patient. Recently,
rapid methods and instn-mt?nt~tion have been developed which allow hematological analysis by
centrifugation of a blood tube to provide hematocrit (HCT), platelet count (PLT), white blood
15 cell count ~WBC), and a partial differential cell count (Wardlaw and Levine. 1983. JAMA
249(5):617-620). A coll~.llelcially available ill~7llulll~ and method based on this technology is
the QBC centrifugal hematology analyzer from Becton Dickinson Primary Care Diagnostics
(Sparks, MD). The QBC method uses di~-~ Lial met~cl~lo~ lic fluorescence of acridine
orange treated blood cells and density gradient cell l~e,ing within the buffy coat to measure
20 the sepal~led packed volumes of red blood cells, white blood cells and pl~t~let~ The QBC
instrument makes electro-optical measurements of the cell layers and computes the hematocrit,
platelet count, WBC and subgroup counts of granulocytes and Iymphocytes/monocytes.
Hemoglobin concentration is derived from the hematocrit and measurements of red cell
density.
To perform the QBC analysis, capillary or venous blood is placed in a glass capillary
tube internally coated with acridine orange and potassium oxalate. The acridine orange stains
white cells and platelets. The potassium oxalate osmotically removes water from the red cells
SUBS~ITUT;E SHEET (RULE ~
WO 94/26299 . ~ l ~i 7 ~ ~ 6 PCT/US94/05188
to increase their density and improve separation from granulocytes. A float is fitted in the
QBC capillary tube and settles within the buffy coat during centrifugation, thereby axially
~Yp~ntling the stained white cell and platelet layers appro~l,la~ely 10-fold.
The blood tube is centrifuged to separate the cell types into layers or bands within the
tube. It is then ilhlmin~ted by blue-violet light in the QBC reader instrument to visualize the
interfaces between packed and e~p~n-led red cell layers and between differentially fluorescing
layers of granulocytes, lymphocytes/monocytes and platelets. Packed cell volumes and test
values (numerical counts and percentages) are computed from the lengths of the five cell
layers, as the length of the layer or band is a reflection of the number of cells present.
The pe,rollllance of the QBC reader and related methods such as that of Wardlaw, et
al., supra, may be monitored by means of a standardized control reagent which upon
centrifugation provides bands having positions in the tube and band lengths which would be
expected upon centrifugation of normal and defined abnormal blood samples. Becton
Dickinson Plhll~y Care Diagnostics sells such l.,age~lls under the name QBC Centrifugal
~em~tology Control. The normal and abnollllal QBC controls contain stabilized human
erythrocytes, m~mm~ n leukocytes and sim~ ted platelets in a plasma-like fluid.
The present invention provides a means for gellelali,l~, particles of a desired density
applupliale for a given application. The particles comprise liposomes incorporating a density
medium such that the particles have the desired buoyant density. Such liposomes are useful as
densit,v markers for monitoring or calibrating density gradients and centrifugal hematology
instr~ ;on, as they can be prepared to have a buoyant density corresponding to various
cell types, viruses, b~ct~-ri~ etc
SUBSTITUTE SHEET (RULE 26
wo 94/26299 S~ PCTIUS94/05188
SU~vIMARY OF THE INVENTION ~
The present invention provides liposomes of defined density. These liposomes areproduced by incorporation of a density merii~lm at a concentration (w/v) which provides a
5 liposome of the desired density. As the density of the defined density liposomes can be easily
adjusted by the practitioner, they may be used to calibrate or monitor density gradients for
identification of the position of a desired band in the gradient or for marking a specific density
point in a gradient. For example, defined density liposomes may be plepa~ed to correspond to
the density of a particular cell type, virus, bacterium or molecule. In one embodiment, the
10 liposomes may be produced such that their buoyant density is app.o;~dmately equal to that of
normal blood granulocytes, thus making the defined density liposome useful in control reagents
for centrifugal hematology systems such as the QBC.
DETAILED DESCRIPTION OF TE~ INVENTION
The liposomes of the invention incorporate a density m~ lm which provides
liposomes having the desired density distribution. The incorporated medium is a density
medium which is coln~a~ible with the Jllelllblalle structure of the liposome, i.e., the me~lillm
does not disrupt the liposome ~em~lane and there is no substantial loss of the m~ lm from
20 the liposome due to me",~ e lç~k~ge. The ~ led density media are particulate media, as
salts and soluble compounds commonly used for density gradient centrifugation may be
inco...pa~;hle with liposome membrane integrity and/or may leak from the liposome. The most
plt;relled density me~ for incorporation comprises polyvinylpyllolidone (PVP) coated
colloidal silica particles, for example PERCOLL (Pharmacia LKB Biotechnology, Uppsala,
25 Sweden). PERCOLL is cc~ nollly used to generate density gradients from 1.0-1.3 g/ml for
use in purification and isolation of cells, viruses and subcellular particles. It can be made iso-
osmotic and is therefore con,pa~ible with the membrane structure of the liposomes when
incorporated. Without wishing to be limited by any particular structure of the defined density
SUBSTITUTE SHEET (RULE 2~
WO 94/26299 ~ ~. 5 7 7 9 ~ PCT/US94/05188
llposomes, Applicant believes that the density m~ lm may be included within the lipid bilayer
as well as encapsulated in the aqueous space. This belief is consistent with the fintling~ of
Cudd and Nicolau, supra. The term "incorporated" and variations thereof are therefore used
herein to include both encapsulated density merlillm and density me(lillm within the lipid
5 bilayer.
The liposomes of the invention may be prepared from a variety of lipids and lipid
mixtures as are known in the art. Reviewed by Szoka and Papahadjopoulos (1980) Ann. Rev.
Biophys. Bioeng. 9: 467-508. Phospholipids are most often used in the pl-el)a,~lion of
10 liposomes and are ~r~felled in the present invention. l~nltil~mpll~r vesicles (MLV) are the
simplest to prepare and may be produced by depositing lipids in a thin film from organic
solvents by rotary evaporation under reduced pres~ e. An aqueous buffer is then added and
the lipids are hydrated with agitation to induce incorporation. Vigorous agitation, brief
sonication or extrusion through polycarbonate membranes may be used to produce a15 ple~ lion of MLV with a smaller and/or more UnirUllll size. Alternatively, MLV in
dispersions can be reduced in size by extrusion through a small orifice under pies~u,e, such as
in a French press. If small l-nil~m~ r vesicles (SUV) are desired, they may be prepared from a
suspension of MLV by sonication under an inert atmosphere. SW may also be prepared by
the solvent injection method. Non-incorporated material may be removed by dialysis, gel
20 filtration, c~ntrifi-~tion or a col,lbillaLion thereo
Large ~lnil~m~ r vesicles (LUV) may be formed from water-in-oil emulsions of lipid
and buffer in an excess organic phase, followed by removal of the organic phase under reduced
pressure. This method is commonly referred to as the reverse phase evaporation technique. In
25 general, lipids are dissolved in organic solvents and the aqueous material to be incorporated is
added to the lipid/solvent mixture. The p,~pa,~lion is then sonicated to form a homogeneous
emulsion. The organic solvents are removed by rotary evaporation until a gel is formed.
Additional buffer is added to the gel and the evaporation vessel is vortexed to suspend the
SUBSTITUTE SHEET (RULE 2~
WO 94/26299 215 ~ 7 ~ ~ PCT/US94/05188
liposomes. Rem~inin~ traces of solvent may be removed from the suspension by dialys~
column chlo,l,a~ography, a procedure which reduces the tendency of the vesicles to aggregate.
The extrusion method and the reverse phase evaporation method are prere,l~d for producing
the defined density liposomes of the present invention.
After the liposomes have been produced, their size distribution may be analyzed. Many
methods are known in the art for size analysis of liposomes, inclllrling gel permeation and
electron microscopy. However, the simplest and prt;r~ d method for estim~tin~ the size
distribution is analysis of the light scatter plope,Lies of the liposomes. Preferably, sizing is
10 done using a sub-micron particle analyzer such as the Coulter N4~ (Coulter Corporation,
Hialeah, FL) which employs photon correlation spectroscopy to size particles by analyzing
light intensity fl~ tions caused by the Bl ow"ian motion of the particles.
In a plert;lled embodiment of the invention, liposomes of a desired density are
15 produced by inco,~uo,~ion of a particulate density medil~m An aqueous p~ lion of the
density ,llP.lill.ll, most plt;rt;lably PVP coated colloidal silica, is plepa~d such that, after
addition of any other ingredients to the aqueous phase, the density of the incorporated mer1illm
will be equal to or greater than the desired density of the liposome. In general, it has been
found that the density of the incorporated medillm, the size of the liposomes and the
20 composition of the lipid bilayer all affect the final density. For eY~mpl~, it has been noted that
increased amounts of di~lcaroyl phosphatidylglycerol (DSPG) incol~o,~ed in the lipid bilayer
result in a lighter partide. The me~ m the liposome particle is in will also afect its appa, e"l
buoyant density. These pa.~nelers are easily adjusted by one skilled in the art to obtain
liposomes of the desired density using only routine ~ nt~tion to vary the pa~a"~elers.
Optionally, dyes may be in~l.lded in the density mellillm for incorporation to f~ilit~te
detection of the liposomes. The dyes may be fluolescellL or colored dyes and are incl~lded in
the density medi~lm at a fluorescent or visible conce,.l,~lion as approp,;ate. For example,
SUBSTITUTE SHEET (RULE 26~
215779~
WO 94/26299 PCT/US94/05188
sulforhodamine G or sulforhodamine B may be included in the density mer~ m plel,a,~lion at
a fluolesce~l concentration. Alternatively, lipophilic dyes may be inciuded in the lipid bilayer
to f~ilit~te detection of the liposomes. Such lipophilic dyes are incorporated into the
membrane bilayer upon formation of the liposome vesicle.
Liposomes incorporating the density medium may then be produced using any of theknown procedures described above as long as the procedure does not adversely affect the
density m~di~lm In the pre~lled embodiment, a lipid film is swollen with an aqueous density
medium prepal ~lion (with or without dye) and extruded through polycarbonate filters to obtain
10 the desired size and density distribution of liposomes. Liposome powder as described in
Example 1 may be made in advance and dried for storage, allowing rapid lecon~ tion and
liposome formation at a later time. The resultin~ liposome prep~lion is diluted with an
aqueous buffer, centrifuged to pellet the liposomes, washed and resuspended in an aqueous
buffer. If desired, the size distribution of the liposome plep~Lion may be ~stim~ted on a sub-
15 micron particle analyzer, determining the size distribution of the liposomes in the plepal~lionby photon coll~,lalion spectroscopy. Electron microscopy is p.~;~lled for dete,l"il ing the siz
e
more accurately.
In addition to monitoring or calibrating analytical methods and instrl.",~"l~l;on based
20 on buoyant density analysis, the defined density liposomes of the invention may also be used in
immllno~ ys. In this embodiment the defined density liposome is derivatized with a ligand
applop,iate for the immlmoassay and is used to generate a detect~ble immllne binding reaction
at a defined position or reaction area in a tube after cçntrifi-g~tion. As is known for
imm~lnoaSsayS, the ligand may be a capture antibody, an antigen or a hapten noncovalently
25 associated with the liposome surface, covalently coupled to the liposome surface or
intercalated into the lipid bilayer of the membrane. Exposing the derivatized defined density
liposome to a fluid co,~f ~ g an analyte which is a receptor for the ligand (i.e., the
SUBSTITUTE SHEET (RULE 26~
WO 94/26299 2 lS ~ ~ 9 ~ PCTIUS94/05188
corresponding antigen or antibody) allows the ligand and the analyte to bind and for( a
complex associated with the defined density liposome.
In a sandwich assay format, the liposome is also exposed to a tracer conjugate
5 comprising an antibody or antigen associated with a detect~ble label and specific for the
analyte. The detect~ble label may be a fluorescent compound or a colored absorbing dye. The
tracer conjugate recognizes and binds to the analyte in the ligand/analyte complex on the
liposome surface. Upon centrifugation, the defined density liposomes with associated analyte
and tracer conjugate band at a defined position in the cçntrifi1~e tube. As unbound tracer
10 conjugate is lighter than the liposomes, detectable label above background levels ~vill be
detected in the reaction area only when bound to analyte associated with the defined density
liposomes. The amount of analyte may then be qu~ntit~ted by measuring fluorescence or
absorbance from the detector conjugate in the reaction area. Alternatively, the immlln~assay
may be performed in a competitive assay format. In this embodiment, the derivatized defined
15 density liposomes are exposed to analyte and a col"~eLing tracer conjugate. The competing
tracer conjugate comprises an antigen or antibody which co",peles with the analyte for binding
to the ligand-de~iv~lized liposome. Upon centrifugation, the defined density liposomes with an
amount of associated tracer conjugate inversely proportional to the amount of analyte will
band in the reaction area. A reduction in fluorescence or absorbance in the reaction area may
20 then be used to qu~..l;l~le the analyte. The ro,e~,oi"g centrifugal imm~lno~cs~ys using
derivatized liposomes of defined density may also be pe,~""ed using other particles of defined
density, for example POLYl~.EAD PMMA Monodisperse Particles, with applop,iate
derivatization of the particles ~,vith antigen, antibody or hapten.
The following experimental examples are provided to illustrate certain embodiments of
the invention but are not intended to limit the scope of the invention as defined by the
appended claims. Variations and modifications of the invention disclosed herein will occur to
those skilled in the art w`ithout departing from the spirit of the invention and without the
SUBSTITUTE SHEET (RULE 26~
WO 94/26299 21~ 7 7 9 ~ PCT/US94/05188
exercise of inventive skill. These variations and modifications are intenrled to be incl~lded
within the scope of the invention.
EXAMPLE 1
PREPARATION OF DEFINED DENSITY LIPOSOMES
Liposomes of defined density were prepared as follows: 1.88 g of lecithin, 0.206 g
DSPG, 1.018 g cholesterol and 10 mg DiI C18(3) (Molecular Probes Inc., Eugene, OR) were
added to a round bottom flask and dissolved in 150 ml of chloroform. The lipid film was
prepared on a rotary evaporator using a 40C water bath. The film was rotated under 200
mbar of vacuum for 1 hr. The film was then swollen with 150 ml of dH2O. A tray was pre-
cooled on the Iyophilizer shelf and the swollen film was poured into the tray and allowed to
freeze before turning on the vacuum. The p~ l a~ion was Iyophilized over the weekend using
a program in which the plep~Lion was held for 12 hr at -40C, following which the
temperature was ramped up to 25 C over 8 hr. The shelf temperature was set at 15C.
Following Iyophilization, the dry powder was removed from the lyol)hili~, and scraped out of
the tray. The powder was stored in two 50 ml Falcon tubes and referred to as "0.2% DiI
Powder. "
Saline iso-osmotic PERCOLL solutions were p.~)~ed at ~l~n~ities of 1.123 g/ml, 1.100
g/ml and 1.06 g/ml. The refractive index was measured using an ABBE Mark II refractometer
(Reichert Scientific Instruments). The osmolality was measured using an Osmette A
instrument (Precision Systems Inc., Natick, MA). The results were as follows:
REFRACTrVE
SOLIJTION INDEX TEMP. mOsm
1.123 g/ml 1.3517 21.4C 313
1.100 g/ml 1.3481 23.0C 299
1.06g/ml 1.3425 23.0C 276
SUBSTITUTE SHEET (RUEE 2~
_ _
WO 94/26299 215 ~ ~ 9 PCT/US94/05188
.
Sixty six mg of 0.2% DiI Powder was weighed into each of three 50 ml round bottom
flasks. Ten ml of the PERCOLL solutions was added to each fiask to hydrate the powder with
each of the density media described above. The powders were swollen by rotating on a rotary
5 evaporator for 30 min. using a 60C water bath. The swollen mixtures were then extruded in
a Lipex extruder at room temperature and passed three times through two stacked 8.0 ~m
NUCLEOPORE PC membranes (Nucleopore Corp. Pleasanton CA) on a polyester drain
disk. The same set of membranes was used repeatedly for each p~a~Lion of liposomes.
Three ml of the 8.0 um extruded mixture was removed and the r~?m~in~er was extruded
10 through two stacked 1.0 ~m NUCLEOPORE PC me,llb,~nes on a polyester drain disk passing
through the same set of membranes three times. Three ml of the 1.0 ~m extruded mixture was
removed. The r~m~in~er was extruded through two stacked 0.4 llm NUCLEOPORE PC
membranes on a polyester drain disk passing through the same set of membranes three times.
A solution co.. l~ ~.;.. g 1% PVP-10, 10 mM MOPSO pH 7.4, 1.05% NaCl (319 mOsm)
was prepared ("1% PVP-MBS"). A 10X volume ofthis solution was added to each aliquot of
extrusion mixture and the mixture centrifuged for 30 min. at 1500 xg. ~cer one 30 min.
centrifugation the 8.0 ~lm extruded and 1.0 llm extruded p,e~a,~Lions had pelleted. However,
the 0.4 ~m extruded plepa,~ions had to be ce~trifil~ed a second time for 30 min. to pellet the
20 liposomes. The supe~,la~ s were removed and 5X the original volume of 1% PVP-MBS was
added. The pellets were resuspended and centrifuged as before. A~er removing thesupt;llla~ s, the pellets were resuspended in 50 mM MOPSO pH 7.4, 20 mM EDTA 0.2%
NaN3, 1.25% glycerol. The 0.4 ~m extruded plepal~ion was resuspended in 0.5 ml and the
other p~epa~Lions were resuspended in 1 ml. The liposome p,epa,~Lions were stored in the
25 refrigerator at 2-8C.
SUBYlTUTE SHEET (RULE 26
wO 94/26299 215 7 7 9 6 PCT/US94/05188
'~, This procedure provided liposome p~ tions of three different sizes incorporating
O J
PERCOLL solutions of three di~lel-L den~ities i.e., nine di~erel,l cor .I)il~aLions of density and
size.
EXAMPLE 2
ASSAYS USING DEFINED DENSITY LIPOSQMES
The nine pr~lions of liposomes described in Example 1 were mixed with aliquots of
QBC control reagent without granulocytes (R&D Systems, Inc., Minneapolis, MN) and tested
in venous and capillaly tubes for b~n~ing positions in the QBC instrument. Fifteen ~1 of
liposomes were mixed with 450 ,ul of QBC control reagent. Each mixture was tested in
duplicate in each tube type. The spun tubes were viewed under blue excitation using an
Olympus BH2 microscope. I3 refers to the intçrf~ce between the granulocyte and red blood
cell layers. I4 refers to the interface between the Iymphocyte and granulocyte layers. The
results are ~11111111~1 l~ed in the following Table:
LIPOSOME
PREPARATION
(Densitv/Size) LIPOSOME BANDING POSlTION
1.123/8.0 Venous: Slightly below lymphocytes with sLle~l.ing into the
RBC's at I3.
C9p:ll9~y: Same as above.
1.123/1.0 Venous: below Iymphocytes with a small "fragment line" between
Iymphocytes and liposomes, good I3.
~sp~ ry: Bands as a granulocyte.
1.123/0.4 Venous: Slight mixing of liposomes and Iymphocytes at I4, good
I3.
Capilla~y: Mixing at I4, good I3.
SUBSTIME SHEET (RUEE 26
WO 94/26299 215~ 9 ~ PCT/US94/05188
1.100/8.0 Venous: Small "fragment line" between liposomes ~nd
lymphocytes. I3 slightly diffuse but not .7L~ rg.
Capillaly: Bands like a granulocyte.
1.100/1.0 Venous: Bands like a granulocyte. Good I3 and I4. Slight
"fragment line" between Iymphocytes and liposomes.
Capillary: Bands like a granulocyte.
1.100/0.4 Venous: Liposomes mix with Iymphocytes at I4 and band just
below.
CPri~ y: Liposomes band just below Iymphocytes and mix with
them at I4.
1.060/8.0 Venous: Liposomes band below Iymphocytes and mix with them.
Capillary: Liposomes band below Iymphocytes and mix with them.
1.060/1.0 Venous: Liposomes mix with lymphocytes throughout the
Iymphocyte band.
Capilla-y: Liposomes mix with Iymphocytes but are more
concel.ll ~ted at the lower part of the Iymphocytes.
1.060/0.4 Venous: Liposomes mix with Iymphocytes throughout the
Iymphocyte band.
Capillary: Liposomes mix with Iymphocytes but are more
concelll~ ed in the upper part of othe lymphocytes, near the
pl~tplp~tC
This e~y~"illlc~ll dçmon~ les the b~n~ patterns of liposomes hav~ng nine different
combill~lions of incorporated rnç~ -m density and size. As the ~ ye~illlent illustrates,
liposomes having a predçfined density and b~n~li~ pattern may be ylepaled for use as Ill~ke;l,
in density gradients.
SUBSTITUTE SHEET (RULE 26
