WO1996040277A2

WO1996040277A2 - Spray dried polymeric microparticles containing imaging agents

Info

Publication number: WO1996040277A2
Application number: PCT/US1996/008378
Authority: WO
Inventors: Edith Mathiowitz; Jules S. Jacob; Donald E. Chickering, Iii
Original assignee: Brown University Research Foundation
Priority date: 1995-06-07
Filing date: 1996-06-03
Publication date: 1996-12-19
Also published as: AU6378096A; WO1996040277A3

Abstract

Methods are provided for the synthesis of synthetic polymeric delivery systems consisting of microparticles which contain a diagnostic imaging agent using spray drying of dilute synthetic polymer solutions, typically between 0.2 and 20 w/v% polymer. In a preferred embodiment, the imaging agent is a gas, the polymer is biodegradable, the polymer solution is saturated with gas and formed into an emulsion, then spray dried. Microparticles produced by this method typically have an average diameter of between about one to seven microns, and therefore freely pass through the pulmonary capillaries. Additional, or alternative, imaging agents can be incorporated within the microparticles. Larger microparticles can also be manufactured for administration to mucosal membranes or oral administration. Adhesion of these microparticles can be enhanced through the selection of bioadhesive polymers. Targeting can also be achieved by selection of the polymer or incorporation within or coupling to the polymer of ligands which specifically bind to particular tissue types or cell surface molecules. Additionally, ligands may be attached to the microspheres which affect the charge, lipophilicity or hydrophilicity of the particle. The polymeric microparticles are useful in a variety of diagnostic imaging procedures including ultrasound imaging, magnetic resonance imaging, fluoroscopy, X-ray, and computerized tomography. The microspheres may be used in a variety of imaging applications including cardiology applications, as well as for organ and peripheral vein imaging.

Description

SPRAY DRIED POLYMERIC MICROPARTICLES CONTAINING IMAGING AGENTS

Background of the Invention

The present invention is generally in the area of diagnostic imaging agents.

Diagnostic ultrasound is a powerful, non-invasive tool that can be used to obtain information on the internal organs of the body. The advent of grey scale imaging and color Doppler have greatly advanced the scope and resolution of the technique. Although techniques for carrying out diagnostic ultrasound have improved significantly, there is still a need to enhance the resolution of the imaging for cardiac perfusion and cardiac chambers, solid organs, renal perfusion; solid organ perfusion; and Doppler signals of blood velocity and flow direction during real-time imaging. Traditional, simple ultrasonic echograms reveal blood vessel walls and other echo-producing structures. However, since echoes from blood normally are not recorded, identifying which echoes are from which blood vessels is usually difficult. For example, echoes from the far wall of one blood vessel can be confused with the near wall of an adjacent blood vessel, and vice versa. Ultrasonic contrast agents can be used to increase the amount of ultrasound reflected back to a detector. Ultrasonic contrast mediums fill the entire intraluminal space with echoes and readily permit identification of the correct pair of echoes corresponding to the walls of a particular blood vessel. Ultrasonic contrast agents are primarily used in high-flow systems in which the contrast enhancement can be quickly evanescent. For echocardiography, a full display of bubble agents, ranging in size from 2-12 μm, and persisting from two or three to 30 seconds, has been used. For other applications, such as neurosonography, hysterosalpingography, and diagnostic procedures on solid organs, the agent must have a lifetime of more than a few circulation times and concentrate in organ systems other than the vascular tree into which it is injected. It must also be small enough to pass through the pulmonary capillary bed (less than eight microns).

Aqueous suspensions of air microbubbles are the preferred echo contrast agents due to the large differences in acoustic impedance between air and the surrounding aqueous medium. After injection into the blood stream, the air bubbles should survive at least for the duration of examination. The bubbles preferably should be injectable intravenously and small enough to pass through the capillaries of the lungs.

The simplest suspension of air bubbles has been obtained by hand agitation of 70% dextrose or sorbitol solutions. However, this method produces large bubbles with an average diameter of greater than 15 μm that exhibit a very limited in vitro stability (less than 1 minute). Feinstein, S.B. et al. , J. Am. Coll. Cardiol.. Vol. 3, pp. 14-20 (1984); Keller, M.W. et al , J. Ultrasound Med.. Vol. 5, pp. 493-498 (1986). Smaller bubbles (usually approximately 5 μm in diameter) have been obtained by sonicating solutions of 50% or 70% dextrose or Renografin-76 (diatrizoate meglumin 66%) but their in vitro persistence still seldom exceeds a few minutes (Feinstein, J. Am. Coll. Cardiol. 11, 59-65 (1988), and Keller, (1988)), and their in vivo persistence only a few seconds. This short lifetime may be appropriate for some applications in cardiology but may not be sufficient for organ imaging.

A variety of natural and synthetic polymers have been used to encapsulate imaging contrast agents, such as air. Research efforts in this area have to date primarily focused on gelatin and alginate as the encapsulating polymers. Alginate can be ionically cross-linked with divalent cations, in water, at room temperature, to form a hydrogel matrix, as described by Wheatley et al , Biomaterials 11, 713-718 (1990) and Kwok, K.K. et al , Pharm. Res.. Vol. 8(3) pp. 341-344 (1991). Wheatley et al. discloses the production of ionically crosslinked microparticles less than

10 microns in diameter, formed of alginate, and encapsulating air, for use in diagnostic ultrasound. Kwok discloses the production of microparticles in the range of 5 to 15 μm by spraying a sodium alginate solution from an air-atomizing device into a calcium chloride solution to effect crosslinking, and then further crosslinking the resulting microparticles with poly-L-lysine. Schneider et al , Invest. Radiol.. Vol. 27, pp. 134-139 (1992) describes three micron, air-filled polymeric particles. These particles were reported to be stable in plasma and under applied pressure. However, at 2.5 MHz, their echogenicity was low.

Another type of microbubble suspension has been obtained from sonicated albumin. Feinstein et al , J. Am. Coll. Cardiol.. Vol. 11, pp. 59-65 (1988). Feinstein describes the preparation of microbubbles that are appropriately sized for transpulmonary passage with excellent stability in vitro. However, these microbubbles are short-lived in vivo, having a half life on the order of a few seconds (which is approximately equal to one circulation pass) because of their instability under pressure. Gottlieb, S. et al , J. Am. Soc. Echo.. Vol. 3, pp. 328 (1990), Abstract; and Shapiro, J.R. et al, J. Am. Coll. Cardiol.. Vol. 16, pp. 1603-1607 (1990).

Gelatin-encapsulated air bubbles have been described by Carroll et al. (Carroll, B.A. et al, Invest. Radiol.. Vol. 15, pp. 260-266 (1980), and Carroll, B.A. et al , Radiology. Vol. 143, pp. 747-750 (1982)), but due to their large sizes (12 and 80 μm) they would not be likely to pass through pulmonary capillaries. Gelatin-encapsulated microbubbles have also been described in PCT/US80/00502 by Rasor Associates, Inc. These are formed by "coalescing" the gelatin.

Microbubbles stabilized by microcrystals of galactose (SHU 454 and SHU 508) have also been reported by Fritzsch et al. Fritzsch, T. et al. , Invest. Radiol. Vol. 23 (Suppl 1), pp. 302-305 (1988); and Fritzsch, T. et al , Invest. Radiol.. Vol. 25 (Suppl 1), 160-161 (1990). The microbubbles last up to 15 minutes in vitro but less than 20 seconds in vivo. Rovai, D. er al , J. Am. Coll. Cardiol.. Vol. 10, pp. 125-134 (1987); and Smith, M. et al , J. Am. Coll. Cardiol.. Vol. 13, pp. 1622-1628 (1989).

European Patent Application No. 91810366.4 by Sintetica S.A. (0 458 745 Al) discloses air or gas microballoons bounded by an interfacially deposited polymer membrane that can be dispersed in an aqueous carrier for injection into a host animal or for oral, rectal, or urethral administration, for therapeutic or diagnostic purposes. The microballoons are prepared by the steps of: emulsifying a hydrophobic organic phase into a water phase to obtain an oil-in-water emulsion; adding to the emulsion at least one polymer in a volatile organic solvent that is insoluble in the water phase; evaporating the volatile solvent so that the polymer deposits by interfacial precipitation around the hydrophobic phase in the water suspension; and subjecting the suspension to reduced pressure to remove the hydrophobic phase and the water phase in a manner that replaces air or gas with the hydrophobic phase. There are some major disadvantages of this process. First, only polymers that have very specific solubility profiles can be used to prepare the microbubbles, i.e., they must be "interfacially depositable" on a hydrophobic phase in an aqueous medium, and soluble in a volatile organic solvent that is water-insoluble. This method relies on the interfacial deposition of a polymeric solution around an already formed emulsion, is successful under only very limited interfacial conditions, and requires the use of waxes in each fabrication process.

WO 92/18164 by Delta Biotechnology Limited describes the preparation of microparticles by spray drying under very controlled conditions as to temperature, rate of spraying, particle size, and drying conditions, of an aqueous protein solution to form hollow spheres having gas entrapped therein, for use in imaging.

None of these describe microparticles which can be detected using other methods for detection, such as x-ray, positron or photon emission tomography, or magnetic resonance imaging. WO 93/25242 describes the synthesis of microparticles for ultrasonic imaging consisting of a gas contained within a shell of polycyanoacrylate or polyester. U.S. Patent Nos. 5,334,381, 5,123,414 and 5,352,435 to Unger describe liposomes for use as ultrasound contrast agents, which include gases, gas precursors, such as a pH activated or photo-activated gaseous precursor, as well as other liquid or solid contrast enhancing agents. The gas containing liposomes are fabricated by pressurizing a vessel containing the liposomes with a gas to cause the gas to pass through the liposome membranes and to form gas bubbles within the liposomes. The liposomes also can be sonicated while being pressurized. Solid and liquid contrast agents for use in the liposomes included magnetite and solid iodine particles, as well as solubilized iodinated contrast agents useful for visualizing intravascular flow and detecting tumors in the liver and spleen. U.S. Patent No. 5,393,524 to Quay discloses the use of agents for enhancing the contrast in an ultrasound image. The agents consist of extremely small bubbles, or microbubbles, of selected gases, which exhibit long life spans in solution and are small enough to traverse the lungs, enabling their use in ultrasound imaging of the cardiovascular system and other vital organs. WO 92/21382 discloses the fabrication of microparticle contrast agents which include a covalently bonded matrix containing a gas, wherein the matrix is a carbohydrate.

It is an object of the invention to provide microparticles made from synthetic polymers which contain an imaging agent. It is another object of the invention to provide microparticles containing an imaging agent that can persist for more than a few circulation times in vivo. It is a further object of the invention to provide microparticles containing imaging agents that are targeted to specific regions of the body. It is still another object of the present invention to provide methods for making microparticles having imaging agents entrapped therein. Summary of the Invention

Methods are provided for the production of synthetic polymeric delivery systems consisting of microparticles which contain a diagnostic imaging agent using spray drying of dilute synthetic polymer solutions, typically between 0.2 and 20 w/v% polymer. In a preferred embodiment, the imaging agent is a gas, the polymer is biodegradable, the polymer solution is saturated with gas and formed into an emulsion, then spray dried. Optionally, the solution can be sonicated prior to or during spray drying of the solution. The resulting microparticles encapsulating the gas are useful in ultrasound applications. Microparticles produced by this method typically have an average diameter of between about one to seven microns, and therefore freely pass through the pulmonary capillaries. Additional, or alternative, imaging agents can be incorporated within the microparticles. The microparticles have a large void volume, e.g. , 5- 98%, within the sponge-like interior. Larger microparticles can also be manufactured for administration to mucosal membranes or oral administration. Adhesion of these microparticles can be enhanced or reduced through the selection of bioadhesive polymers. For example, adhesion can be enhanced in the case where the polymer is used for oral administration. Targeting can also be achieved by selection of the polymer or incorporation within or coupling to the polymer of ligands which specifically bind to particular tissue types or cell surface molecules. Additionally, ligands may be attached to the microspheres which effect the charge, lipophilicity or hydrophilicity of the particle. The polymeric microparticles are useful in a variety of diagnostic imaging procedures including ultrasound imaging, magnetic resonance imaging, fluoroscopy, x-ray, and computerized tomography. The microspheres may be used in a variety of imaging applications including cardiology applications, blood perfusion applications as well as for organ and peripheral vein imaging. Brief Description of the Figures

Figure 1 is an electron micrograph microparticles formed by spray drying a polyethyleneglycol-poly(lactic acid-co-glycolic acid) copolymer solution. Figure 2 is an electron micrograph of microparticles formed by spray drying a solution of poly(lactic acid) and sorbitan trioleate.

Detailed Description of the Invention

Methods are provided for the synthesis of polymeric delivery systems consisting of microparticles which contain a diagnostic imaging agent. The microparticles are useful in a variety of diagnostic imaging applications, particularly in ultrasound procedures such as blood vessel imaging and echocardiography.

Selection of Polymers and Solvents Polymers Both non-biodegradable and biodegradable matrices can be used for delivery of imaging agents, although biodegradable matrices are preferred, particularly for intravenous injection. Non-erodible polymers may be used for oral administration. These may be natural or synthetic polymers. Synthetic polymers are preferred due to more reproducible degradation. The polymer is selected based on the time required for in vivo stability, i.e., that time required for distribution to the site where imaging is desired, and the time required for imaging. In one embodiment, microparticles which exhibit an in vivo stability of at least approximately 1 to 10 minutes are fabricated, for example, for use in echocardiography applications. In another embodiment, microparticles with an in vivo stability of between about 20 to 30 minutes or more may be fabricated, for example for use in applications such as echocardiography, neurosonography, hysterosalpingography, and diagnostic procedures on solid organs. The in vivo stability of the contrast agent-encapsulated microparticles can be adjusted during the production by using polymers such as polylactide co-glycolide copolymerized with polyethylene glycol (PEG). PEG if exposed on the external surface may elongate the time these materials circulate since it is very hydrophilic.

Representative synthetic polymers are: poly (hydroxy acids) such as poly (lactic acid), poly(glycolic acid), and poly (lactic acid-co-gly colic acid), poly gly colides, polylactides, polylactide co-glycolide, polyanhydrides, polyorthoesters, polyamides, polycarbonates, poly alky lenes such as polyethylene and polypropylene, poly alkylene glycols such as poly (ethylene glycol), polyalkylene oxides such as poly (ethylene oxide), polyalkylene terepthalates such as poly (ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides such as poly (vinyl chloride), polyvinylpyrrolidone, polysiloxanes, poly (vinyl alcohols), poly (vinyl acetate), polystyrene, polyurethanes and co-polymers thereof, derivatized celluloses such as alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxy lethyl cellulose, cellulose triacetate, and cellulose sulphate sodium salt (jointly referred to herein as "synthetic celluloses"), polymers of acrylic acid, methacrylic acid or copolymers or derivatives thereof including esters, poly (methyl methacrylate), poly (ethyl methacrylate), poly(butylmethacrylate), poly (isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly (lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referred to herein as "polyacrylic acids"). As used herein, "derivatives" include polymers having substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art. Examples of preferred non-biodegradable polymers include ethylene vinyl acetate, poly (meth)acry lie acid, poly amides, copolymers and mixtures thereof.

Examples of preferred biodegradable polymers include polymers of hydroxy acids such as lactic acid and glycolic acid polylactide, polyglycolide, polylactide co glycolide, and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone). In general, these materials degrade in vivo by both non-enzymatic and enzymatic hydrolysis, and by surface or bulk erosion.

Bioadhesive polymers of particular interest include polyanhydrides, polyacrylic acid, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly (isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly(isopropyl acrylate), poly (isobutyl acrylate), and poly(octadecyl acrylate).

Solvents

As defined herein, the polymer solvent is an organic solvent that is volatile or has a relatively low boiling point and which is acceptable for administration to humans in trace amounts, such as methylene chloride. Other solvents, such as ethyl acetate, acetone, ethanol, DMSO and chloroform also may be utilized, or combinations thereof. In general, the polymer is dissolved in the solvent to form a polymer solution having a concentration of between 0.2 and 10% weight to volume (w/v), more preferably between 0.5 and 3%.

Imaging Agents

Sterilized air or oxygen is preferred for making echogenic microparticles, but other gases such as argon, nitrogen, perfluorocarbons, carbon dioxide, nitrogen dioxide, xenon and helium may be utilized as the imaging agent. Other imaging agents can be incorporated in place of a gas, or in combination with the gas. Imaging agents which may be utilized include commercially available agents used in positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI). Microparticles loaded with these agents can be detected using standard techniques available in the art and commercially available equipment.

Examples of suitable materials for use as contrast agents in MRI include the gatalinium chelates currently available, such as diethylene triamine pentacetic acid (DTP A) and gatopentotate dimeglumine, as well as iron, magnesium, manganese, copper and chromium.

Examples of materials useful for CAT and x-rays include iodine based materials for intravenous administration, such as ionic monomers typified by diatrizoate and iothalamate, non-ionic monomers such as iopamidol, isohexol, and ioversol, non-ionic dimers, such as iotrol and iodixanol, and ionic dimers, for example, ioxagalte. Other useful materials include barium for oral use and non-soluble salts such as ZnAc.

In the preferred embodiment, the imaging agent(s) is added to the polymer solution, which is then emulsified. This can be varied, however, so that the agent is added to the polymer solution after emulsification or even during processing, as described below. Other agents can be added with the polymer to the solvent, as well as during spraying. The amount of imaging agent which is added is determined by the application. In the case of ultrasound imaging, the amount of gas is that which yields particles having gas filled voids forming between 2 and 98% of the microparticle, more preferably between 5 and 95% of the microparticle, which are echogenic after administration orally or intravenously. The other agents are added in an amount which can be detected using standard techniques.

If the imaging agent is a gas, the polymer solution is sparged with the gas, and then may be sonicated or homogenized to make sure the gas is well mixed in the polymer solution prior to spraying. If the imaging agent is a solid, the agent may be encapsulated as solid particles which are added to the polymer solution prior to spraying, or the imaging agent can be dissolved in an aqueous solution which then is emulsified with the polymer solution prior to spraying, or the solid may be cosolubilized together with the polymer in an appropriate solvent prior to spraying. Spray Drying Methods and Apparatus The microparticles are produced by dissolving a biocompatible polymer in an appropriate solvent, dispersing a gaseous imaging agent and/or another imaging agent into the polymer solution, and then spray drying the polymer solution, to form microparticles containing the imaging agent. As defined herein, the process of "spray drying" a solution of a polymer and an imaging agent refers to a process wherein the solution is atomized to form a fine mist and dried by direct contact with hot carrier gases. Using spray drying apparatus available in the art, the polymer solution may be delivered through the inlet port of the spray drier, passed through a tube within the drier and then atomized through the outlet port. The temperature may be varied depending on the gas or polymer used. The temperature of the inlet and outlet ports can be controlled to produce the desired products.

The size of the particulates of polymer solution is a function of the nozzle used to spray the polymer solution, nozzle pressure, the flow rate, the polymer used, the polymer concentration, the type of solvent and the temperature of spraying (both inlet and outlet temperature) and the molecular weight. Generally, the higher the molecular weight, the larger the capsule size, assuming the concentration is the same.

In the preferred embodiment for making echogenic microparticles, gas is dispersed in the polymer solution to form a saturated solution. The gas can be dispersed by bubbling, and/or sonication of the solution. Other means are known to those skilled in the art. Gas may be bubbled through the solution before and/or during the spray drying step. Alternatively, or additionally, the solution may be sonicated before and/or during the spray drying step, to promote distribution of the gas throughout the solution. Typically, ultrasonic frequencies of between 5,000 and 30,000 Hz are utilized to generate the gaseous solution. Microparticles produced by this method are typically smaller than seven microns. Microparticles may be produced which have a porous sponge-like structure, as illustrated in Figures 1 and 2, which are electron micrographs of microparticles produced as described in Examples 14 and 3 respectively.

In the case where non-gaseous imaging agents are utilized, the particles of the imaging agent can be dispersed or dissolved in the solution of the polymer. The solution of the imaging agent and the polymer can be sonicated or homogenized prior to and/or during the spray drying step, to assist in the formation of a homogeneous distribution of the imaging agent and the polymer in the solution. Typical process parameters for a mini-spray drier are as follows: polymer concentration = 0.01-0.05 g/ml, inlet temperature = 40 °C, outlet temperature = 24°C, polymer spray flow = 10-30 ml/min., and nozzle diameter = 1.0 mm ID. Microspheres ranging in diameter between one and ten microns can be obtained with a morphology which depends on the selection of polymer, concentration, molecular weight and spray flow.

Additives to Facilitate Microparticulate Formation A variety of surfactants may be added during the synthesis of the image agent-containing microparticles. Exemplary emulsifiers or surfactants which may be used (0.1-5% by weight) include most physiologically acceptable emulsifiers, for instance egg lecithin or soya bean lecithin, or synthetic lecithins such as saturated synthetic lecithins, for example, dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline or distearoyl phosphatidyl choline or unsaturated synthetic lecithins, such as dioleyl phosphatidyl choline or dilinoleyl phosphatidyl choline. Emulsifiers also include surfactants such as free fatty acids, esters of fatty acids with polyoxyalkylene compounds like polyoxpropylene glycol and polyoxyethylene glycol; ethers of fatty alcohols with polyoxyalkylene glycols; esters of fatty acids with polyoxyalkylated sorbitan; soaps; glycerol-polyalkylene stearate; glycerol- polyoxyethylene ricinoleate; homo- and copolymers of polyalkylene glycols; polyethoxylated soya-oil and castor oil as well as hydrogenated derivatives; ethers and esters of sucrose or other carbohydrates with fatty acids, fatty alcohols, these being optionally polyoxyalkylated; mono-, di- and triglycerides of saturated or unsaturated fatty acids, glycerides or soya-oil and sucrose. Other emulsifiers include natural and synthetic forms of bile salts or bile acids, both conjugated with amino acids and unconjugated such as taurodeoxycholate, and cholic acid. This can, for example, stabilize microbubbles generated prior to spray-drying. Microparticle Size In a preferred embodiment for the preparation of injectable microparticles capable of passing through the pulmonary capillary bed, the microparticles should have a diameter of between approximately one and seven microns. Larger microparticles may clog the pulmonary bed, and smaller microparticles may not provide sufficient echogenicity. Larger microparticles are useful for administration by routes other than injection, for example oral (for evaluation of the gastrointestinal tract), application to other mucosal surfaces (rectal, vaginal, oral, nasal) or by inhalation. The preferred particle size for oral administration is about 10-50 μm. Useful pharmaceutically acceptable carriers include saline containing glycerol and Tween 20. Particle size analysis can be performed on a Coulter counter by light microscopy, scanning electron microscopy, or transmittance electron microscopy. Targeting The microparticles can be targeted specifically or non-specifically through the selection of the polymer forming the microparticle, the size of the microparticle, and/or incorporation or attachment of a ligand to the microparticles. For example, biologically active molecules, or molecules affecting the charge, lipophilicity or hydrophilicity of the particle, may be attached to the surface of the microparticle. Additionally, molecules may be attached to the microparticles which minimize tissue adhesion, or which facilitate specific targeting of the microspheres in vivo. Representative targeting molecules include antibodies, lectins, and other molecules which are specifically bound by receptors on the surfaces of cells of a particular type.

Inhibition of Uptake by the RES

Uptake and removal of the microparticles can also be minimized through the selection of the polymer and/or incorporation or coupling of molecules which minimize adhesion or uptake. For example, tissue adhesion by the microparticle can be minimized by covalently binding poly (alkylene glycol) moieties to the surface of the microparticle. The surface poly(alkylene glycol) moieties have a high affinity for water that reduces protein adsorption onto the surface of the particle. The recognition and uptake of the microparticle by the reticulo-endothelial system (RES) is therefore reduced.

In a further embodiment, the terminal hydroxyl group of the poly(alkylene glycol) can be used to covalently attach biologically active molecules, or molecules affecting the charge, lipophilicity or hydrophilicity of the particle, onto the surface of the microparticle. Methods available in the art can be used to attach any of a wide range of ligands to the microparticles to enhance the delivery properties, the stability or other properties of the microparticles in vivo. Diagnostic Applications

Microparticles are typically combined with a pharmaceutically acceptable carrier such as phosphate buffered saline or saline, then an effective amount for detection administered to a patient using an appropriate route, typically by injection into a blood vessel (i.v.) or orally. Microparticles containing an encapsulated imaging agent may be used in vascular imaging, as well as in applications to detect liver and renal diseases, in cardiology applications, in detecting and characterizing tumor masses and tissues, and in measuring peripheral blood velocity. The microparticles also can be linked with ligands that minimize tissue adhesion or that target the microparticles to specific regions of the body in vivo as described above. The microparticles can also be administered orally.

The microparticles encapsulating an imaging agent other than gases can be detected using a variety of diagnostic imaging methods including positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-rays, fluoroscopy, and magnetic resonance imaging (MRI).

The gas-containing microparticles are detected in vivo using an ultrasonic imaging devices available in the art. To detect a diagnostic imaging agent administered to a patient by ultrasonic imaging, a wave is generated by a transducer external to the body of the patient, and the reflected wave is detected by the same transducer. The gas bubbles are detected by the transducer which produces pulses of ultrasonic waves, having predetermined frequency characteristics. A first pulse has an increasing frequency with time, and a second pulse has a decreasing frequency with time. Imaging arrangements produce images of the region within the specimen after exposure to the first and second pulses. The conventional technique for determining the presence of bubbles in the blood stream uses a Doppler shift in the frequency of the ultrasonic acoustic energy which is reflected by the blood. The amplitude of the Doppler bubble signal increases nearly proportionally with increases in the radius of the bubble.

In Vitro Ultrasonic Imaging Tests

The microparticles may be tested using the following in vitro assay to predict whether the microparticles have a sufficient in vivo stability to be useful for in vivo imaging. Ultrasonic imaging of the microparticles is performed, for example, with an HDI 3000 (ATL, WA) using a C7-4 high resolution transducer. Air containing microparticles, for example, at a concentration of 2 mg/ml, are reconstituted in a suitable vehicle, such as 0.9% NaCl, 1 % glycerol and 0.1 % Tween 20, and resuspended in a 15 ml polystyrene tube. The tubes containing the microparticles then are suspended in a 500 ml high density polyethylene bottle filled with room temperature water. The transducer is placed vertically on the side of the 500 ml bottle which is coated with coupling medium. B-mode images are established for tube filled with vehicle alone and vehicle containing polymeric microparticles. The B-mode image gives two dimensional images of a slice through the scanned tube. The gray scale gain is adjusted to make the 15 ml centrifuge tube anechoic when filled with vehicle alone. B-mode sector images of tubes filled with the microparticles then are generated. B-mode images then are examined to determine the echogenicity of the particles. This method can be used to determine the in vitro stability of the diagnostic agent- loaded microspheres, which is reasonably predictive of their in vivo stability, as illustrated below in the Examples.

The methods and compositions described above will be further understood with reference to the following non-limiting examples. Examples 1-25 demonstrate the formation of microparticles having a gas incorporated therein. In the examples, recovery of polymer refers to recovery of polymer in the form of microparticles. Apparatus

Apparatus used in the following examples are as follows: Spray drying was conducted using a Labplant SD4 equipped with a 1.0 mm nozzle. Sonication of the solutions was conducted using a Cole Palmer

CPX600 Probe Sonicator equipped with standard probe. SEM was conducted using a Hitachi S2700 SEM. Coulter counter analysis was conducted using a Coulter Counter Multisizer Accucomp. Differential Scanning Calorimetry (DSC) was conducted using a

Perkin Elmer DSC7. Polymers with lot numbers that begin with RG or L were obtained from Boehringer Ingelheim Co. of Ingleheim, Germany. Polymers with lot numbers beginning with BPI were obtained from Birmingham Polymers, Inc., Birmingham, Alabama. Span 80 (sorbitan monooleate, Lot 19189L) with HLB of 4.3 and Span 85 (sorbitan trioleate, Lot 13962) with HLB of 1.8 were obtained from ICI Specialty Chemical Co. of Wilmington, DE.

Example 1: Preparation of Air-Containing Polylactide Polymer Microspheres. A 5% (w/v) polylactide solution (PLA, MW=2 kDa) in methylene chloride was bubbled with air and sonicated while being spray-dried. The inlet temperature was 30°C and the outlet temperature was 20°C. A 1.0 mm spray nozzle was used. The microspheres had a porous morphology. The average diameter of the particles ranged from 1 to 10 microns. Example 2: Preparation of Formulation EJ1.

300 ml of 5% (w/v) poly (lactic acid) having a molecular weight of 2,000 ("PLA 2K") /l%(w/v) Span^® 85 (sorbitan trioleate), a detergent available from suppliers such as Aldrich Chemical Co., in methylene chloride was bubbled with nitrogen gas before and during spraying and probe sonicated at 50% amplitude with a Cole Palmer CPX600 probe sonicator using the standard tip for 30 sec before spraying. The loading of Span^® 85 in the PLA microspheres was 16.7% (w/w). The spray drier settings were: compressor =40%; blower=50%; nozzle=1.0 mm; inlet temperature =40 °C; outlet temperature =25 °C. The recovery was 3.2 gms of the original 15 gms of polymer =21.3%. The microspheres were less than 10 microns in size. Example 3: Preparation of Formulation EJ2.

100 ml of 5% (w/v) PLA 2K /2% (w/v) Span^® 85 in methylene chloride was bubbled with nitrogen gas before and during spraying. The loading of Span^® 85 in the PLA microspheres was 28.6% (w/w). The spray drier settings were: compressor =50%; blower =100%; nozzle = 1.0 mm; inlet temperature=50°C; outlet temperature=28°C. The recovery was 0.2 gms of the original 5 gms of polymer =4%. The microspheres were less than 10 microns in size. An electron micrograph of the microspheres is shown in Figure 2. Example 4: Preparation of Formulation EDJ3. 300 ml of 5% PLA 2K (w/v)/l % Span^® 80 (w/v) in methylene chloride was bubbled with nitrogen gas before and during spraying and intermittently probe sonicated at 50% amplitude with a Cole Palmer CPX600 probe sonicator using the standard tip during spraying. The loading of Span^® 80 in the PLA microspheres was 16.7% (w/w). The spray drier settings were: compressor =40%; blower=50%; nozzle= 1.0 mm; inlet temperature = 40 °C; outlet temperature = 25 °C. The recovery was 3.1 gms of the original 15 gms of polymer =23.5%. The microspheres were less than 10 microns in size. Example 5: Preparation of Formulation EDJ4. 300 ml of 5% PLA 2K (w/v)/0.033% Span^® 80 (w/v) in methylene chloride was bubbled with nitrogen gas before and during spraying and intermittently probe sonicated at 50% amplitude with a Cole Palmer CPX600 probe sonicator using the standard tip during spraying. The loading of Span^® 80 in the PLA microspheres was 0.66% (w/w). The spray drier settings were: compressor =40%; blower =50%; nozzle=1.0 mm; inlet temperature =40 °C; outlet temperature = 25 °C. The recovery was about 3 to 4 gms of the original 15 gms of polymer =20-27%.

SEM indicated that the particles were generally spherical with smooth surface structure and size ranging from between 0.5 and 6 microns. Coulter counter analysis yielded the following results: 11.94% of the total was in the size range of greater than 0 to one microns; 56.29% of the total was in the size range of between one and three microns; 31.32% of the total was in the size range of between three and 10 microns; 0.45% of the total was greater than 10 microns in size and the average size was 2.20 microns. Example 6: Preparation of Formulation Al-1.

A formulation containing 1 % PLA 2K (w/v) in 100 ml of methylene chloride with no surfactant was spray-dried. The spray drier settings were: compressor =100%; blower= 100%; nozzle= 1.0 mm; inlet temperature=55°C; outlet temperature=30°C; height= -11 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process. No sonication was used. Approximately 165 mg of the initial 1.0 g of polymer was recovered giving a recovery of 16.5%.

Coulter counter analysis yielded the following results: 5.33% of the total was in the size range between zero and one micron; 59.54% of the total was in the size range between one and three microns; 34.90% of the total was in the size range between three and ten microns; 0.23% of the total was greater than 10 microns in size and the average size was 2.45 microns. Example 7: Preparation of Formulation Al-2.

A formulation containing 2% PLA 2K (w/v) in 287.5 ml of methylene chloride with no surfactant was spray-dried. The spray drier settings were: compressor =100%; blower = 100%; nozzle =1.0 mm; inlet temperature =55 °C; outlet temperature =30°C; height= -11 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process. No sonication was used. Approximately 1.44 g of the initial 5.75 g of polymer was recovered giving a recovery of 25.0%. Example 8: Preparation of Formulation Al-3.

A formulation containing 1.5% PLA 2K (w/v) in 666.7 ml of methylene chloride with no surfactant was spray-dried. The spray drier settings were: compressor =100%; blower=100%; nozzle=1.0 mm; inlet temperature =55 °C; outlet temperature =30 °C; height= -11 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process. No sonication was used. Approximately 3.6 g of the initial 10.0 g of polymer was recovered giving a recovery of 36.0%. Example 9: Preparation of Formulation Al-4.

A formulation containing 1.5% poly(L-lactic acid) ("LPLA") having a molecular weight of 130,000 ("130K") (w/v) in 1000 ml of methylene chloride with no surfactant was spray-dried. The spray drier settings were: compressor =120 degrees counter-clockwise from off; blower=100%; nozzle= 1.0 mm; inlet temperature =40 °C; outlet temperature =21 °C; height= -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 6.0 g of the initial 15 g of polymer was recovered giving a recovery of 40.0%.

SEM indicated donut-shaped microspheres with particles ranging in size from one to 10 microns, which was confirmed by Coulter Counter analysis. Example 10: Preparation of Formulation Cl-1.

A formulation containing a blend of 1.5% LPLA 130K (w/v) and 0.6% PLA 2K (w/v) in 466.6 ml of methylene chloride with no surfactant was spray-dried. The ratio of PLA 130K: PLA 2K was 2.5:1 on a w/w basis. The spray drier settings were: compressor = 120 degrees counter-clockwise from off; blower =100%; nozzle =1.0 mm; inlet temperature =40°C; outlet temperature =21 °C; height= -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 4.56 g of the initial 10 g of polymer (7.0 g PLA 130K+ 3.0 g PLA 2K= 10 g total) was recovered giving a recovery of 45.6%. SEM indicated that the particles were spherical with porous surface structure and size ranging from 2 to 10 microns, which was confirmed by Coulter Counter analysis. Coulter counter analysis yielded the following results: 6.32% of the total was in the size range 0-1 microns; 45.57% of the total was in the size range 1-3 microns; 47.00% of the total was . in the size range 3-10 microns; 1.09% of the total was greater than 10 microns in size and the average size was 2.85 microns. Example 11: Preparation of Formulation Gl-1.

A formulation containing 2.0% PLA:PG (poly(lactic acid - co- glycolic acid) (75:25) 94,000 molecular weight ("94K") (w/v) in 500 ml of methylene chloride with no surfactant was spray-dried. The spray drier settings were: compressor = 120 degrees counter-clockwise from off; blower= 100%; nozzle=1.0 mm; inlet temperature =40 °C; outlet temperature =21 °C; height = -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly).

The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 2.58 g of the initial 10 g of polymer was recovered giving a recovery of 25.8%. SEM indicated that the particles were spherical with porous surface structure and size ranging from 1 to 10 microns, which was confirmed by Coulter Counter Analysis. The particles often collapsed under the electron beam which might be an indication that they are very porous. Coulter counter analysis yielded the following results: 6.41 % of the total was in the size range 0 to 1 microns; 50.42% of the total was in the size range 1 to 3 microns; 41.80% of the total was in the size range 3 to 10 microns; 1.29% of the total was greater than 10 microns in size and the average size was 2.68 microns. Example 12: Preparation of Formulation Bl-1. A formulation containing 1.5% PLA (MW=130K) (w/v) in 1000 ml of methylene chloride with 0.1 % lecithin (w/v) (6.25% w/w) was spray-dried. The spray drier settings were: compressor = 120 degrees counter-clockwise from off; blower = 100%; nozzle =1.0 mm; inlet temperature=40°C; outlet temperature =21 °C; height= -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 7.75 g of solids from the initial 15 g of polymer and 1.0 g of lecithin was recovered giving a recovery of 48.4%.

SEM indicated that the particles were spherical with porous surface structure and unusual fenestrated appearance and size ranging from one to 10 microns, which was confirmed by Coulter counter analysis. Coulter counter analysis yielded the following results: 19.39% of the total was in the size range 0 to 1 microns; 52.43% of the total was in the size range one to three microns; 27.75% of the total was in the size range 3 to 10 microns; 0.43% of the total was greater than 10 microns in size and the average size was 2.00 microns. The particles when dispersed in distilled water did not clump and were uniformly suspended. Example 13: Preparation of Formulation D2-1.

A formulation containing 1.5% polyethylene glycol having a molecular weight of 5,000 ("PEG 5K")-poly(lactic acid-co-glycolic acid) (75:25) ("PLA:PG") (MW=75K) (MW=107.9K, glass transition =30.8 °C) (w/v) in 100 ml of methylene chloride with no surfactant was spray-dried. The spray drier settings were: compressor =120 degrees counter-clockwise from off; blower =100%; nozzle= 1.0 mm; inlet temperature =40 °C; outlet temperature =29 °C; height = +6 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 82 mg of the initial 1.5 g of polymer was recovered giving a recovery of 5.5%. The microspheres were less than 10 microns in size. Exa ple 14: Preparation of Formulation D2-2.

A formulation containing 1.5% PEG (5K)/PLA:PG (75:25, MW=75K) (MW= 107.9K, Mn=27.9K; glass transition =30.8 °C, BPI Lot 412-28-1A) (w/v) in 300 ml of methylene chloride with no surfactant was spray-dried. The spray drier settings were: compressor = 120 degrees counter-clockwise from off; blower=100%; nozzle=1.0 mm; inlet temperature =40°C; outlet temperature =29 °C; height= -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 1.33 g of the initial 4.5 g of polymer was recovered giving a recovery of 29.4%.

SEM indicated that the particles were spherical with porous surface structure and unusual "braided" (patchwork) appearance and size ranging from 1 to 10 microns, which was confirmed by Coulter counter analysis. Coulter counter analysis yielded the following results: 5.59% of the total was in the size range 0 to 1 microns; 49.88% of the total was in the size range 1 to 3 microns; 43.96% of the total was in the size range 3 to 10 microns; 0.60% of the total was greater than 10 microns in size and the average size was 2.70 microns. The particles did not clump excessively when dispersed in distilled water. An electron micrograph of the particles is shown in Figure 1.

Example 15: Preparation of Formulation El-1.

A formulation containing 1.5% PLA:PG (50:50, MW=39K, glass transition=43.3°C, Mn=10.6K, RG503H, Lot No. 34002) (w/v) in 300 ml of methylene chloride with no surfactant was spray-dried. The spray drier settings were: compressor = 120 degrees counter-clockwise from off; blower =100%; nozzle = 1.0 mm; inlet temperature =40 °C; outlet temperature=21°C; height= -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly).

The polymer mixture was bubbled v._th air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 0.85 g of the initial 4.5 g of polymer was recovered giving a recovery of 19.0%.

SEM indicated that the particles were spherical with smooth surface structure and size ranging from 1 to 10 microns. The particles often collapsed under the electron beam, indicating that they might be very porous inside. Coulter counter analysis yielded the following results: 8.63% of the total was in the size range 0 to 1 microns; 55.85% of the total was in the size range 1 to 3 microns; 32.50% of the total was in the size range 3 to 10 microns; 2.99% of the total was greater than 10 microns in size and the average size was 2.46 microns. Example 16: Preparation of Formulation D2-3.

A solution containing 1.5% (w/v) PEG (5K)/PLA:PG (75:25, MW=75K) (total MW=107.9K, glass trar_sition=30.8°C, Lot 412-28-1A) (w/v) in 1000 ml of a mixture of 10% methylene chloride/90% chloroform with no surfactant was spray-dried. The spray drier settings were: compressor =120 degrees counter-clockwise from off; blower= 100%; nozzle=1.0 mm; inlet temperature =40 °C; outlet temperature =21 °C; height= -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 3.6 g of the initial 15.0 g of polymer was recovered giving a recovery of 24.0%. Example 17: Preparation of Formulation D2-4. A formulation containing 1.5% PEG (5K)/PLA:PG (75:25,

MW=75K) (MW=107.9K, glass transition=30.8°C, Lot 412-28-1A) (w/v) in 1000 ml of methylene chloride with no surfactant was spray-dried. The spray drier settings were: compressor =120 degrees counter-clockwise from off; blower=100%; nozzle= 1.0 mm; inlet temperature =40 °C; outlet temperature =21 °C; height = -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 5.37 g of the initial 15.0 g of polymer was recovered giving a recovery of 35.8%.

Coulter analysis indicated that the majority of the particles were in the size range from 1 to 10 microns. Coulter counter analysis yielded the following results: 5.00% of the total was in the size range zero to one microns; 43.70% of the total was in the size range 1 to 3 microns; 49.73% of the total was in the size range 3 to 10 microns; 1.57% of the total was greater than 10 microns in size and the average size was 2.97 microns.

Example 18: Preparation of Formulation Gl-2.

A formulation containing 2.0% PLA:PG (75:25, MW=94K, w/v) in 500 ml of methylene chloride with 0.01 % lecithin (w/v) (0.5% lecithin, w/w) was spray-dried. The spray drier settings were: compressor =120 degrees counter-clockwise from off; blower =100%; nozzle =1.0 mm; inlet temperature =40 °C; outlet temperature =21 °C; height = -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 2.64 g of solids from the initial 10.0 g of polymer and 0.05 g of lecithin was recovered giving a yield of 26.3%.

The particles dispersed moderately well in distilled water with some clumping. Coulter counter analysis yielded the following results: 6.11 % of the total was in the size range zero to one microns; 56.27% of the total was in the size range 1 to 3 microns; 36.55% of the total was in the size range 3 to 10 microns; 1.07% of the total was greater than 10 microns in size and the average size was 2.62 microns. Example 19: Preparation of Formulation Gl-3. A formulation containing 2.0% PLA:PG (75:25, MW=94K, w/v) in 496.5 ml of methylene chloride with 0.05% lecithin (w/v) (2.4% lecithin, w/w) was spray-dried. The spray drier settings were: compressor=120 degrees counter-clockwise from off; blower =100%; nozzle= 1.0 mm; inlet temperature = 40 °C; outlet temperature =26 °C; height = -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 2.69 g of solids from the initial 9.93 g of polymer and 0.248 g of lecithin was recovered giving a yield of 26.4%.

The particles were nicely dispersed in distilled water, with few clumps. Coulter counter analysis yielded the following results: 18.65% of the total was in the size range zero to one microns; 48.20% of the total was in the size range one to three micron; 32.30% of the total was in the size range 3 to 10 microns; 0.85% of the total was greater than 10 microns in size and the average size was 2.16 microns. Example 20: Preparation of Formulation Gl-4.

A formulation containing 2.0% PLA:PG (75:25, MW=94K, w/v) in 500 ml of methylene chloride with 0.1% lecithin (w/v) (2% lecithin, w/w) and 3% BaSO₄ (Tonopaque) (w/v) (58.8% w/w) was spray-dried. The Tonopaque powder was further micronized with a mortar and pestle to reduce particle size and sieved through a 106 micron sieve before adding to the polymer solution. The spray drier settings were: compressor =120 degrees counter-clockwise from off; blower =100%; nozzle= 1.0 mm; inlet temperature =40 °C; outlet temperature =21 °C; height = -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process and was probe sonicated for 10 sec periods at 5 min intervals during spraying as well as before spraying to help disperse the barium particles. Approximately 13.19 g of solids from the initial 10.0 g of polymer, 15.0 g of Tonopaque and 0.5 g of lecithin was recovered giving a yield of 51.7%. Coulter counter analysis yielded the following results: 11.92% of the total was in the size range 0-1 microns; 45.39% of the total was in the size range 1-3 microns; 41.90% of the total was in the size range 3-10 microns; 0.79% of the total was greater than 10 microns in size and the average size was 2.51 microns. Tonopaque appeared to be well encapsulated. Example 21: Preparation of Formulation Ml-1.

A formulation containing 1.5%(w/v) PEG/PLA:PG (75:25, MW= 108kDa, Lot 412-28-lA)/PLAPGA (50:50, MW 39 kDa, Lot 503H) in a 1:3 (w/w) blend in 266.7 ml of methylene chloride was spray-dried. The spray drier settings were: compressor = 120 degrees counter-clockwise from off; blower=100%; nozzle =1.0 mm; inlet temperature =40°C; outlet temperature =24°C; height= -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process. Approximately 1.31 g of solids from the initial 4.0 g of polymer (1.0 g of PEG-PLAPG and 3.0 g of PLAPG) was recovered giving a yield of 32.8%. Coulter counter analysis yielded the following results: 5.44% of the total was in the size range 0-1 microns; 57.41 % of the total was in the size range 1-3 microns; 36.48% of the total was in the size range 3-10 microns; 0.87% of the total was greater than 10 microns in size and the average size was 2.61 microns. Example 22: Preparation of Formulation Ml-2.

A formulation containing 1.5%(w/v) PEG/PLA:PG (75:25, MW=108kDa, Lot 412-28-1A) and PLAPGA (50:50 ,MW 39 kDa, (Lot 503H) in a 1:3 (w/w) blend in 266.7 ml of methylene chloride was spray-dried. Four mg of lecithin (0.1 % w/w) was dissolved in the polymer mixture. The spray drier settings were: compressor =120 degrees counter-clockwise from off; blower =100%; nozzle =1.0 mm; inlet temperature=40°C; outlet temperature =24 °C; height= -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process. Approximately 1.31 g of solids from the initial 4.0 g of polymer was recovered giving a yield of 32.8%.

Coulter counter analysis yielded the following results: 6.28% of the total was in the size range 0-1 microns; 55.97% of the total was in the size range 1-3 microns; 36.95% of the total was in the size range 3-10 microns; 0.80% of the total was greater than 10 microns in size and the average size was 2.62 microns.

Example 23: Preparation of Formulation Ml-3.

A formulation containing 1.5%(w/v) PEG/PLA:PG (75:25, MW=108kDa, Lot 412-28-lA)/PLAPGA (50:50), MW 39 kDa, (Lot 503H) in a 1:3 (w/w) blend in 266.7 ml of methylene chloride was spray-dried. 20 mg of lecithin (0.5% w/w) was dissolved in the polymer mixture. The spray drier settings were: compressor =120 degrees counter-clockwise from off; blower=100%; nozzle=1.0 mm; inlet temperature =40 °C; outlet temperature = 24 °C; height = -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process. Approximately 0.91 g of solids from the initial 4.0 g of polymer and was recovered giving a yield of 22.8%.

Coulter counter analysis yielded the following results: 5.25% of the total was in the size range 0-1 microns; 56.63% of the total was in the size range 1-3 microns; 37.57% of the total was in the size range 3-10 microns; 0.55% of the total was greater than 10 microns in size and the average size was 2.54 microns. Example 24: Preparation of Formulation D2-5. A formulation containing 1.5 %(w/v) PEG(5K)/PLA:PG (75:25,

MW=75K) (MW=107.9K, glass transition=30.8°C, Lot 412-28-1 A) (w/v) in 400 ml of methylene chloride with no surfactant was spray-dried. The spray drier settings were: compressor = 120 degrees counter-clockwise from off; blower= 100%; nozzle=1.0 mm; inlet temperature =40°C; outlet temperature = 27 °C; height= -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 1.29 g of the initial 6.0 g of polymer was recovered giving a recovery of 21.5%.

SEM indicated that the particles were spherical with porous surface structure and unusual "braided" (patchwork) appearance and size ranging from 1-10 microns. Example 25: Preparation of Formulation D2-6.

A formulation containing 1.5% (w/v) PEG (5K)/PLA:PG (75:25, MW=75K) (MW= 107.9K, glass transition =30.8 °C, Lot 412-28-1 A) (w/v) in 400 ml of methylene chloride with no surfactant was spray-dried. The spray drier settings were: compressor =120 degrees counter-clockwise from off; blower= 100%; nozzle=1.0 mm; inlet temperature = 40 °C; outlet temperature = 26 °C; height = -2.5 cm (height is defined as the distance from the bottom of the sample beaker to the top of the head assembly). The polymer mixture was bubbled with air for 5 minutes prior to spraying and during the spraying process, and no probe sonication was used during spraying. Approximately 1.46 g of the initial 6.0 g of polymer was recovered giving a recovery of 24.3%.

SEM indicated that the particles were spherical with porous surface structure and unusual "braided" (patchwork) appearance and size ranging from 1-10 microns. Example 26: Differential Scanning Calorimetry

All of the samples produced in the above Examples were characterized by differential scanning calorimetry (DSC) in order to determine the glass transition and melting point of the polymers before and after fabrication. Thermograms of PLA show glass transitions of 55 °C and melting point of 173.9°C. The addition of lecithin at levels of up to 6% (w/w) decreased the glass transition by 0.5 °C and the melting point by 1 °C, which was probably not significant. Blending low molecular weight PLA with high molecular weight PLA reduced the glass transition to 49°C and the melting point to 169°C, indicating some interaction between the two polymers. Thermograms of a series of PLAPG 75:25 polymers show the glass transition of the polymer before fabrication was 46°C. Blank microspheres loaded with gas had a lower glass transition of 44.7 °C. Addition of lecithin at 0.5% (w/w) reduced the glass transition to 44.5 °C. The glass transition was reduced to 43.8°C at 2.5% (w/w) lecithin. Thermograms of the glass transition temperatures of PEG-PLGA (75:25) show that before spray drying the polymer had a glass transition of 30.8°C, but after gas-loading, spray-dried microspheres had a glass transition of 36.2°C. The glass transition of microspheres sprayed using chloroform as the solvent instead of methylene chloride was the same as the polymer, namely 30.6°C. Example 27: In Vitro Ultrasonic Imaging Tests

All samples were evaluated in vitro using Doppler ultrasound as described above for detecting echogenicity. To determine whether the gas-filled microparticles are useful for in vivo imaging, the microparticles were tested in vitro as follows.

Ultrasonic imaging of the microparticles was performed with an HDI 3000 (ATL, WA) using a C7-4 high resolution transducer. Air containing microparticles, at a concentration of 2 mg/ml, were reconstituted in vehicle (0.9% NaCl, 1% glycerol and 0.1% Tween 20) and resuspended in a 15 ml polystyrene tube. The tubes containing the microparticles then were suspended in a 500 ml high density polyethylene bottle filled with room temperature water. The transducer was placed vertically on the side of the 500 ml bottle which was coated with coupling medium. B-mode images were established for tube filled with vehicle alone and vehicle contaimng polymeric microparticles. The B-mode image gives two dimensional images of a slice through the scanned tube. W „O_ _ „ 96_-/,4_0_2_.7,*7 PCT US96/08378

-31-

The gray scale gain is adjusted to make the 15 ml centrifuge tube anechoic when filled with vehicle alone.

B-mode sector images of tubes filled with each of the microparticles produced in each of the above examples were generated. 5 B-mode images of each of the individual batches demonstrated highly echogenic particles.

All of the microparticles produced in the above examples displayed positive echogenic behavior. These results correlated well with the in vivo results described below. Since the in vitro and in vivo data showed a

10 high degree of correlation in the working examples, this test is reasonably predictive of the in vivo stability of microparticles. Example 28: In Vivo Echogenic Studies

Two of the formulations prepared in the above examples were tested in vivo as follows.

15 In vivo color Doppler imaging was performed in New Zealand white rabbits weighing approximately 2-2.5 kg. Rabbits were fasted overnight. The rabbits were anesthetized with ketamine (100 mg/ml, 0.7 ml) and rompum (20 mg/ml, 0.5 ml). The microspheres were reconstituted in vehicle (0.9% NaCl, 1% Glycerol and 0.1 % Tween 20)

20 at a concentration in the range of 5-15 mg/ml no more than 30 minutes prior to injection. The dosage form was administered intravenously through a 23 gauge catheter located in the marginal ear vein and was infused over approximately 2-3 minutes. After the administration of the dosage form the catheter was flushed with one ml of normal saline.

25 Ultrasonic imaging of the major blood vessels in the abdomen was performed spleen was performed with an HDI 3000 (ATL) using a C7-4 high resolution transducer. The abdominal aorta was imaged before and after the administration of the agent. Machine settings were adjusted to minimize baseline color Doppler. Enhanced color Doppler signals were

30 detected in the samples as follows:

(a) Formulation Cl-1 (Example 10) was tested in a rabbit by administering 20 mg of the formulation in 2 ml of vehicle over 2 minutes. A significant increase in the color Doppler signal was seen in the abdominal aorta after the first 1.0 ml of contrast agent was administered. The entire aortic lumen was filled with color. One minute post injection completion, the color Doppler enhancement was reduced, but still enhanced over baseline. Five minutes post injection completion the color Doppler level had returned to baseline.

(b) Formulation D2-4 (Example 17) was tested in a rabbit by administering 20 mg of the formulation in 2.0 ml of vehicle. Significant enhancement of the color Doppler was seen after the first 1.0 ml of contrast agent. The whole lumen of the abdominal aorta was filled with color enhancement and increased enhancement was also noted in the inferior venae cavae. The enhancement was observed for at least 22 minutes after which the experiment was stopped.

Claims

We claim:

1. A method of making microparticles for diagnostic imaging of a human or other animal comprising:

(i) dissolving a biocompatible synthetic polymer into a solvent to form a polymer solution;

(ii) adding to the polymer solution an imaging agent; and (iii) spray drying the solution, while simultaneously incorporating a gas into the polymer solution, to form microparticles having an effective amount of imaging agent incorporated therein to be detectable after administration to a patient in need thereof.

2. The method of claim 1 wherein the imaging agent is the gas and the microparticles have a void volume of 5-98 % by volume of polymer.

3. The method of claim 2 wherein the gas is selected from the group consisting of air, oxygen, argon, nitrogen, nitrogen dioxide, perfluorocarbons, xenon and helium.

4. The method of claim 1 wherein the imaging agent is detectable by an imaging technique selected from the group consisting of magnetic resonance imaging, computer tomography, x-ray, positron emission tomography and single photon emission computerized tomography.

5. The method of claim 1 wherein the polymer is biodegradable.

6. The method of claim 1 wherein the polymer is selected from the group consisting of poly (hydroxy acids), polyanhydrides, polyorthoesters, polyamides, polycarbonates, poly alky lenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, poly gly colides, polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene, polyurethanes, synthetic celluloses, polyacrylic acids, poly (butyric acid), poly(valeric acid), and poly(lactide- co-caprolactone).

7. The method of claim 1 wherein the polymer is dissolved in an organic solvent.

8. The method of claim 1 wherein the polymer is a poly(hydroxy acid) combined with a polyalkylene oxide.

9. The method of claim 1 wherein the polymer solution contains between about 0.2 and 20% (w/v) polymer.

10. The method of claim 1 further comprising selecting a polymer which is bioadhesive to tissue.

11. The method of claim 1 further comprising modifying the polymeric matrix to target the microparticles to particular cells or tissues.

12. Microparticles for diagnostic imaging of a human or other animal comprising a biocompatible synthetic polymer having incorporated therein an effective amount of imaging agent incorporated therein to be detectable after administration to a patient in need thereof, wherein the microparticles are formed by

(i) dissolving the biocompatible synthetic polymer into a biocompatible solvent to form a polymer solution;

13. The microparticles of claim 12 wherein the imaging agent is the gas and the microparticles have a void volume of 5-98% by volume of polymer.

14. The microparticles of claim 13 wherein the gas is selected from the group consisting of air, oxygen, argon, nitrogen, nitrogen dioxide, perfluorocarbons, xenon and helium.

15. The microparticles of claim 12 wherein the imaging agent is detectable by an imaging technique selected from the group consisting of magnetic resonance imaging, computer tomography, x-ray, positron emission tomography and single photon emission computerized tomography.

16. The microparticles of claim 12 wherein the polymer is biodegradable.

17. The microparticles of claim 12 wherein the polymer is selected from the group consisting of poly (hydroxy acids), polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalky lenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polygl colides, polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene, polyurethanes, synthetic celluloses, polyacrylic acids, poly (butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone).

18. The microparticles of claim 17 wherem the polymer is a poly (hydroxy acid) combined with a polyalkylene oxide.

19. The microparticles of claim 12 wherein the polymer is bioadhesive to tissue.

20. The microparticles of claim 12 wherein the polymeric matrix is modified to target the microparticles to particular cells or tissues.

21. The microparticles of claim 12, wherein each microparticle has a porous sponge-like structure.

22. A method for imaging tissue in a patient in need thereof comprising administering to the patient the microparticles of any of claims 12 to 21 and detecting the location of the microparticles following administration.

23. The method of claim 1 further comprising sonicating or homogenizing the solution in step (iii), thereby to incorporate the gas into the solution.

24. The microparticles of claim 12 wherein, in step (iii), the solution is sonicated or homogenized, thereby to incorporate the gas into the solution.