Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/49—Blood
  • G01N33/491—Blood by separating the blood components
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/5432—Liposomes or microcapsules

Definitions

• the present invention relates to liposomes and methods for their production.
• the invention further relates to uses of liposomes in analytical methods.
• 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.
• 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)
• 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.
• density marker beads may not be commercially available with the correct buoyant density.
• CBC complete blood cell count
• HCT hematocrit
• PHT platelet count
• WBC white blood cell count
• a partial differential cell count Wardlaw and Levine. 1983. JAMA 249(5):617-620.
• 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.
• 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.
• 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
• 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.
• MLV in dispersions can be reduced in size by extrusion through a small orifice under pressure, such as in a French press.
• small unilamellar vesicles SUV
• 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.
• LUV Large unilamellar vesicles
• 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.
• the liposomes 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.
• Coulter N4MD Coulter Corporation, Hialeah, FL
• 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.
• 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.
• 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.
• sulforhodamine G or sulforhodamine B may be included in the density medium preparation at a fluorescent concentration.
• 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.
• 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.
• 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.
• the defined density liposomes of the invention may also be used in immunoassays.
• 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.
• 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.
• analyte which is a receptor for the ligand (i.e., the corresponding antigen or antibody)
• 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.
• the defined density liposomes with associated analyte and tracer conjugate band at a defined position in the centrifuge tube.
• 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.
• the immunoassay may be performed in a competitive assay format.
• 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.
• 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.
• 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.
• 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 dH2 ⁇ . 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.
• 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:
• 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 ⁇ m extruded and 1.0 ⁇ m extruded preparations had pelleted. However, the 0.4 ⁇ m 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 ⁇ m 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 1 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 ⁇ l of liposomes were mixed with 450 ⁇ l 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:
• Venous below lymphocytes with a small "fragment line" between lymphocytes and liposomes, good 13.
• Capillary Bands as a granulocyte.
• 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.
• fragment line between lymphocytes and liposomes.
• Capillary Bands like a granulocyte.
• Capillary Liposomes band just below lymphocytes and mix with them at 14.
• 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.
• Capillary Liposomes mix with lymphocytes but are more concentrated in the upper part of othe lymphocytes, near the platelets.

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