WO1994026299A1

WO1994026299A1 - Liposomes incorporating density media

Info

Publication number: WO1994026299A1
Application number: PCT/US1994/005188
Authority: WO
Inventors: Joanne Haller Hasskamp
Original assignee: Becton Dickinson And Company
Priority date: 1993-05-13
Filing date: 1994-05-10
Publication date: 1994-11-24
Also published as: CA2157796A1; EP0678032A4; AU6909594A; EP0678032A1

Abstract

Liposomes incorporating a density medium and having a defined density. The liposomes are useful as control reagents for monitoring density gradient centrifugation procedures, especially in centrifugal hematology and in immunoassay procedures. The density of the liposomes can be adjusted by the practitioner to correspond to the density of any component of a sample expected to band in a particular position in a density gradient centrifugation procedure.

Description

LIPOSOMES INCORPORATING DENSITY MEDIA

FIELD OF THE INVENTION

The present invention relates to liposomes and methods for their production. The 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 equilibrium with an aqueous solution. Polar lipids such as phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, cardiolipins, phosphatidic acids, cerebrosides and combinations of fatty acids form such membranes when placed in an aqueous environment.

The bimolecular lipid sheets of the membrane are intercalated by aqueous spaces, and these structures can persist even in the presence of excess water. Liposomes are therefore useful vehicles for incorporating 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 immunoassays. PERCOLL beads have also been encapsulated in liposomes for use as markers in spleen tissue for visualization by electron microscopy (EM)

(Cudd and Nicolau. 1986. J. Microencapsulation 3(4):275-282). These authors did not 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 distinguished 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 density markers to calibrate or monitor density gradients in such applications as density gradient centrifugation. These materials in general have taken the form of solid beads of defined buoyant density, such as the Density Marker Beads (cross-linked dextran) available from Pharmacia LKB Biotechnology, Uppsala, Sweden. POLYBEAD PMMA (polymethyl methacrylate) 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 used in immunoassays. However, these commercial density markers and defined density particles may not be manufactured with the 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 instrumentation have been developed which allow hematological analysis by centrifugation of a blood tube to provide hematocrit (HCT), platelet count (PLT), white blood cell count (WBC), and a partial differential cell count (Wardlaw and Levine. 1983. JAMA 249(5):617-620). A commercially available instrument and method based on this technology is the QBC centrifugal hematology analyzer from Becton Dickinson Primary Care Diagnostics (Sparks, MD). The QBC method uses differential metachromatic fluorescence of acridine orange treated blood cells and density gradient cell layering within the bufiy coat to measure the separated packed volumes of red blood cells, white blood cells and platelets. The QBC instrument makes electro-optical measurements of the cell layers and computes the hematocrit, platelet count, WBC and subgroup counts of granulocytes and lymphocytes/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 to increase their density and improve separation from granulocytes. A float is fitted in the QBC capillary tube and settles within the bufify coat during centrifugation, thereby axially expanding the stained white cell and platelet layers approximately 10-fold.

The blood tube is centrifuged to separate the cell types into layers or bands within the tube. It is then illuminated by blue-violet light in the QBC reader instrument to visualize the interfaces between packed and expanded 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 performance 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 Primary Care Diagnostics sells such reagents under the name QBC Centrifugal Hematology Control. The normal and abnormal QBC controls contain stabilized human erythrocytes, mammalian leukocytes and simulated platelets in a plasma-like fluid.

The present invention provides a means for generating particles of a desired density appropriate 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 density markers for monitoring or calibrating density gradients and centrifugal hematology instrumentation, as they can be prepared to have a buoyant density corresponding to various cell types, viruses, bacteria, etc SUMMARY OF THE INVENTION

The present invention provides liposomes of defined density. These liposomes are produced by incorporation of a density medium at a concentration (w/v) which provides a 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 prepared to correspond to the density of a particular cell type, virus, bacterium or molecule. In one embodiment, the liposomes may be produced such that their buoyant density is approximately 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 THE INVENTION

The liposomes of the invention incorporate a density medium which provides liposomes having the desired density distribution. The incorporated medium is a density medium which is compatible with the membrane structure of the liposome, i.e., the medium does not disrupt the liposome membrane and there is no substantial loss of the medium from the liposome due to membrane leakage. The preferred density media are paniculate media, as salts and soluble compounds commonly used for density gradient centrifugation may be incompatible with liposome membrane integrity and/or may leak from the liposome. The most preferred density medium for incorporation comprises polyvinylpyrrolidone (PVP) coated colloidal silica particles, for example PERCOLL (Pharmacia LKB Biotechnology, Uppsala, Sweden). PERCOLL is commonly 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 compatible 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 2Q liposomes, Applicant believes that the density medium may be included within the lipid bilayer as well as encapsulated in the aqueous space. This belief is consistent with the findings of

Cudd and Nicolau, .supra. The term "incorporated" and variations thereof are therefore used herein to include both encapsulated density medium and density medium within the lipid 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 preparation of liposomes and are preferred in the present invention. Multilamellar 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 pressure. 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 a preparation of MLV with a smaller and/or more uniform size. Alternatively, MLV in dispersions can be reduced in size by extrusion through a small orifice under pressure, such as in a French press. If small unilamellar vesicles (SUV) are desired, they may be prepared from a suspension of MLV by sonication under an inert atmosphere. SUV may also be prepared by the solvent injection method. Non-incorporated material may be removed by dialysis, gel filtration, centrifugation or a combination thereof.

Large unilamellar 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 general, lipids are dissolved in organic solvents and the aqueous material to be incorporated is added to the lipid/solvent mixture. The preparation 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 liposomes. Remaining traces of solvent may be removed from the suspension by dialysis or column chromatography, a procedure which reduces the tendency of the vesicles to aggregate. The extrusion method and the reverse phase evaporation method are preferred 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, including gel permeation and electron microscopy. However, the simplest and preferred method for estimating the size distribution is analysis of the light scatter properties of the liposomes. Preferably, sizing is done using a sub-micron particle analyzer such as the Coulter N4MD (Coulter Corporation, Hialeah, FL) which employs photon correlation spectroscopy to size particles by analyzing light intensity fluctuations caused by the Brownian motion of the particles.

In a preferred embodiment of the invention, liposomes of a desired density are produced by incorporation of a particulate density medium. An aqueous preparation of the density medium, most preferably PVP coated colloidal silica, is prepared such that, after addition of any other ingredients to the aqueous phase, the density of the incorporated medium 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 medium, the size of the liposomes and the composition of the lipid bilayer all affect the final density. For example, it has been noted that increased amounts of distearoyl phosphatidylglycerol (DSPG) incorporated in the lipid bilayer result in a lighter particle. The medium the liposome particle is in will also affect its apparent buoyant density. These parameters are easily adjusted by one skilled in the art to obtain liposomes of the desired density using only routine experimentation to vary the parameters.

Optionally, dyes may be included in the density medium for incorporation to facilitate detection of the liposomes. The dyes may be fluorescent or colored dyes and are included in the density medium at a fluorescent or visible concentration as appropriate. For example, sulforhodamine G or sulforhodamine B may be included in the density medium preparation at a fluorescent concentration. Alternatively, lipophilic dyes may be included in the lipid bilayer to facilitate 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 the known procedures described above as long as the procedure does not adversely affect the density medium. In the preferred embodiment, a lipid film is swollen with an aqueous density medium preparation (with or without dye) and extruded through polycarbonate filters to obtain 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 reconstitution and liposome formation at a later time. The resulting liposome preparation 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 preparation may be estimated on a sub- micron particle analyzer, determining the size distribution of the liposomes in the preparation by photon correlation spectroscopy. Electron microscopy is preferred for determining the size more accurately.

In addition to monitoring or calibrating analytical methods and instrumentation based on buoyant density analysis, the defined density liposomes of the invention may also be used in immunoassays. In this embodiment the defined density liposome is derivatized with a ligand appropriate for the immunoassay and is used to generate a detectable immune binding reaction at a defined position or reaction area in a tube after centrifugation. As is known for immunoassays, the ligand may be a capture antibody, an antigen or a hapten noncovalently 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 containing an analyte which is a receptor for the ligand (i.e., the corresponding antigen or antibody) allows the ligand and the analyte to bind and form a complex associated with the defined density liposome.

In a sandwich assay format, the liposome is also exposed to a tracer conjugate comprising an antibody or antigen associated with a detectable label and specific for the analyte. The detectable 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 centrifuge tube. As unbound tracer conjugate is lighter than the liposomes, detectable label above background levels will be detected in the reaction area only when bound to analyte associated with the defined density liposomes. The amount of analyte may then be quantitated by measuring fluorescence or absorbance from the detector conjugate in the reaction area. Alternatively, the immunoassay may be performed in a competitive assay format. In this embodiment, the derivatized defined density liposomes are exposed to analyte and a competing tracer conjugate. The competing tracer conjugate comprises an antigen or antibody which competes with the analyte for binding to the ligand-derivatized 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 then be used to quantitate the analyte. The foregoing centrifugal immunoassays using derivatized liposomes of defined density may also be performed using other particles of defined density, for example POLYBEAD PMMA Monodisperse Particles, with appropriate derivatization of the particles with 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 without departing from the spirit of the invention and without the

SUBSTITUTE SHEET (RULE 26 exercise of inventive skill. These variations and modifications are intended to be included 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 Dil 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 40°C water bath. The film was rotated under 200 mbar of vacuum for 1 hr. The film was then swollen with 150 ml of dH2θ. A tray was pre- cooled on the lyophilizer shelf and the swollen film was poured into the tray and allowed to freeze before turning on the vacuum. The preparation was lyophilized over the weekend using a program in which the preparation was held for 12 hr at -40°C, following which the temperature was ramped up to 25 °C over 8 hr. The shelf temperature was set at 15°C. Following lyophilization, the dry powder was removed from the lyophilizer and scraped out of the tray. The powder was stored in two 50 ml Falcon tubes and referred to as "0.2% Dil Powder."

Saline iso-osmotic PERCOLL solutions were prepared at densities 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:

REFRACTIVE

SOLUTION INDEX TEMP. mOsm

1.123 g/ml 1.3517 21.4°C 313

1.100 g/ml 1.3481 23.0°C 299

1.06 g/ml 1.3425 23.0°C 276 Sixty six mg of 0.2% Dil Powder was weighed into each of three 50 ml round bottom flasks. Ten ml of the PERCOLL solutions was added to each flask to hydrate the powder with each of the density media described above. The powders were swollen by rotating on a rotary evaporator for 30 min. using a 60°C water bath. The swollen mixtures were then extruded in a Lipex extruder at room temperature and passed three times through two stacked 8.0 μm NUCLEOPORE PC membranes (Nucleopore Corp., Pleasanton, CA) on a polyester drain disk. The same set of membranes was used repeatedly for each preparation of liposomes. Three ml of the 8.0 μm extruded mixture was removed and the remainder was extruded through two stacked 1.0 μm NUCLEOPORE PC membranes on a polyester drain disk, passmg through the same set of membranes three times. Three ml of the 1.0 μm extruded mixture was removed. The remainder was extruded through two stacked 0.4 μm NUCLEOPORE PC membranes on a polyester drain disk, passing through the same set of membranes three times.

A solution containing 1% PVP-10, 10 mM MOPSO pH 7.4, 1.05% NaCl (319 mOsm) was prepared ("1% PVP-MBS"). A 10X volume of this solution was added to each aliquot of extrusion mixture and the mixture centrifuged for 30 min. at 1500 xg. After one 30 min. centrifugation the 8.0 μm extruded and 1.0 μm extruded preparations had pelleted. However, the 0.4 μm extruded preparations had to be centrifuged a second time for 30 min. to pellet the liposomes. The supematants were removed and 5X the original volume of 1% PVP-MBS was added. The pellets were resuspended and centrifuged as before. After removing the supematants, the pellets were resuspended in 50 mM MOPSO pH 7.4, 20 mM EDTA, 0.2% NaN3, 1.25% glycerol. The 0.4 μm extruded preparation was resuspended in 0.5 ml and the other preparations were resuspended in 1 ml. The liposome preparations were stored in the refrigerator at 2-8°C. This procedure provided liposome preparations of three different sizes incorporating

PERCOLL solutions of three different densities, i.e., nine different combinations of density and size.

EXAMPLE 2

ASSAYS USING DEFINED DENSITY LIPOSOMES

The nine preparations 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 capillary tubes for banding positions in the QBC instrument. Fifteen μl of liposomes were mixed with 450 μl 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. 13 refers to the interface between the granulocyte and red blood cell layers. 14 refers to the interface between the lymphocyte and granulocyte layers. The results are summarized in the following Table:

LIPOSOME PREPARATION (Density/Size) LIPOSOME BANDING POSITION

1.123/8.0 Venous: Slightly below lymphocytes with streaming into the

RBCs at D. Capillary: Same as above.

1.123/1.0 Venous: below lymphocytes with a small "fragment line" between lymphocytes and liposomes, good 13. Capillary: Bands as a granulocyte.

1.123/0.4 Venous: Slight mixing of liposomes and lymphocytes at 14, good

13. Capillary: Mixing at 14, good 13.

11 SUBSTITUTE SHEET (RULE 2Q 1.100/8.0 Venous: Small "fragment line" between liposomes and lymphocytes. 13 slightly diffuse but not streaming. Capillary: Bands like a granulocyte.

1.100/1.0 Venous: Bands like a granulocyte. Good 13 and 14. Slight

"fragment line" between lymphocytes and liposomes. Capillary: Bands like a granulocyte.

1.100/0.4 Venous: Liposomes mix with lymphocytes at 14 and band just below.

Capillary: Liposomes band just below lymphocytes and mix with them at 14.

1.060/8.0 Venous: Liposomes band below lymphocytes and mix with them.

Capillary: Liposomes band below lymphocytes and mix with them.

1.060/1.0 Venous: Liposomes mix with lymphocytes throughout the lymphocyte band.

Capillary: Liposomes mix with lymphocytes but are more concentrated at the lower part of the lymphocytes.

1.060/0.4 Venous: Liposomes mix with lymphocytes throughout the lymphocyte band.

Capillary: Liposomes mix with lymphocytes but are more concentrated in the upper part of othe lymphocytes, near the platelets.

This experiment demonstrates the banding patterns of liposomes having nine different combinations of incorporated medium density and size. As the experiment illustrates, liposomes having a predefined density and banding pattern may be prepared for use as markers in density gradients.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising liposomes of defined density, the liposomes incorporating a density medium compatible with liposome membrane structure at a concentration which provides liposomes having a selected buoyant density.

2. The composition of Claim 1 wherein the density medium is a particulate medium.

3. The composition of Claim 2 wherein the density medium is polyvinylpyrrolidone coated colloidal silica.

4. The composition of Claims 1 or 3 wherein the liposomes have a buoyant density approximately equal to the buoyant density of a selected cell type.

5. The composition of Claim 4 wherein the liposomes have a buoyant density approximately equal to the buoyant density of granulocytes.

6. The composition of Claims 1 or 3 wherein the liposomes further comprise an incorporated dye.

7. The composition of Claim 6 wherein the dye is a fluorescent dye.

8. A method for monitoring the performance of density gradient centrifugation comprising: a) centrifuging a reagent comprising liposomes of defined density such that the liposomes form a layer according to their buoyant density, the liposomes incorporating a density medium compatible with liposome membrane structure at a concentration which provides liposomes having a selected buoyant density ; b) determining the position of the liposome layer within the centrifuged control reagent, and; c) comparing the position of the liposome layer with a position expected for particles having similar buoyant density.

9. The method of Claim 8 wherein liposomes incorporating a particulate density medium are centrifuged.

10. The method of Claim 9 wherein liposomes incorporating polyvinylpyrrolidone coated colloidal silica are centrifuged.

11. The method of Claim 10 wherein the liposomes form a layer having a buoyant density approximately equal to the buoyant density of a selected cell type.

12. The method of Claim 11 wherein the liposomes form a layer having a buoyant density approximately equal to the buoyant density of granulocytes.

13. The method of Claims 8 or 10 wherein the position of the liposomes in the centrifuged control reagent is determined by detecting a dye incorporated in the liposomes.

14. The method of Claim 13 wherein the dye is detected by detection of fluorescence.

15. An immunoassay method for detecting an analyte comprising: a) contacting the analyte with a composition comprising liposomes of defined density, the liposomes being derivatized with a ligand which is a receptor for the analyte, such that the analyte binds to the ligand to form a liposome/analyte complex; b) centrifuging the complex such that it forms a layer according to its buoyant density, and; c) detecting the layer as an indication of the presence of analyte by means of a detectable label associated with the complex.

16. The method of Claim 15 wherein each one of two or more analytes forms a complex with liposomes which form separately detectable layers upon centrifugation, each liposome/analyte complex being associated with a separately detectable label.