MXPA99009986A - Microparticles useful as ultrasonic contrast agents and for delivery of drugs into the bloodstream - Google Patents

Abstract

Microparticles are provided comprising a shell of an outer layer of a biologically compatible material and an inner layer of biodegradable polymer. The core of the microparticles contain a gas, liquid or solid for use in drug delivery or as a contrast agent for ultrasonic contrast imaging. The microparticles are capable of passing through the capillary systems of a subject.

Description

USEFUL MICROPARTICLES AS AGENTS OF ULTRASONIC CONTRAST AND FOR DELIVERY OF MEDICINES WITHIN THE BLOOD TORRENT Background of the Invention Hollow microparticles, sometimes called microbubbles or microspheres, are efficient for backscattering of ultrasound energy. In this way, small microbubbles injected into the bloodstream can improve the reproduction of ultrasonic ultrasound images to help visualize internal structures, such as the heart and blood vessels. The contrast of the ultrasound is achieved when the acoustic impedance between two materials in an interface is different. In this way, the greater the difference in acoustic impedance between the materials, the greater the intensity of an ultrasound echo of that interface. As there is a large difference between the acoustic impedance between a body tissue and the gas, the microparticles that contain gas and circulate within the tissue or blood are strong backscatters of ultrasound energy. For use in the circulatory system, the microparticles must have a diameter of less than about ten microns in order to pass through the capillaries of the circulatory system. The lower limit of a sufficient echogenicity of an icroparticle is about one to two microns. In cardiology, microparticles are useful for intravenous injection, thus providing a contrast in ultrasound in the right chambers of the heart, improving the identification of cardiac structures, the functions of the valve and the detection of intracardiac deviations. However, in order to visualize the left chambers of the heart, the microparticles must first pass through the pulmonary circulation system. These particles must be small enough to pass through the pulmonary capillaries. Otherwise they get trapped inside the lungs. They must also have sufficient structural strength to survive the pressures within the left chambers of the heart. The microparticles also allow the definition of volumes, the movement of the wall and other factors that identify disease states within the heart. The use of contrast agents also facilitates the use of Doppler ultrasound techniques because strong sources of echo that move in the blood stream are much more echogenic than red blood cells, which are the usual sources of echo that are used in Doppler ultrasound techniques. Blood contrast agents can also be used to locate the presence of blood in areas of the body or to identify the absence of blood due to the lack of echogenicity in areas that should be • ecogenic. Examples of these uses are the use of 5 microparticles for the evaluation of perfusion to the myocardium, and for the evaluation of disorders in the coronary septum by the flow of particles through the septum that separates the cardiac chambers. Another example is the use of microparticles to identify vascular embolism as per For example, blood clots and abnormal growths within the vascular chambers due to the absence of ultrasonic contrast. Other uses of contrast agents are to examine the perfusion of organs, as well as to evaluate the damage caused by a heart attack, to examine organs such as the liver, or to differentiate between normal and abnormal tissues, such as tumors and cysts. The present invention offers microparticle contrast agents that are delivered intermittently but that are able to pass through the pulmonary circulation system for an improved examination and diagnosis of both sides of the heart, as well as an examination of other tissues and organs as described above. In addition to the representation of images for the diagnosis, the microparticles according to the present invention are also used for the delivery of medicaments where the drug is released from the particle by diffusion of the microparticle, by degradation of the microparticle or by breaking up the particle using ultrasonic energy. SUMMARY OF THE INVENTION The present invention offers microparticle compositions from which most microparticles have diameters in the range of about one to ten microns, have an outer layer that includes a biologically compatible material and an inner layer that includes a biodegradable polymer . The microparticles can have a hollow core, which contains either a gas or a liquid, or a solid core. The outer layer can be chosen based on the biocompatibility with the bloodstream and the tissues, while the inner layer can be selected based on the desired mechanical and acoustic properties. The materials of both layers can be selected to predetermine the resistance of the microparticle, for example to provide a desired resonant frequency and stability within the diagnostic image reproduction levels of the ultrasound radiation threshold. Methods for forming multi-layer microparticles and use in the reproduction of ultrasonic diagnostic images and delivery of drugs are also provided. The layers can also be selected for their ability to contain and deliver medications. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph of the time course of the intensity of the ultrasound reflected in the left atrium in a test of a contrast agent according to example 7. Figure 2 is a graph of the time course of the intensity of the ultrasound reflected in the left atrium of the contrast agent tested according to example 8. Figure 3 is a graph of the volumetric size distribution of the unfiltered microcapsules performed in example 13, and the distribution of size when the suspension was leaked. Figure 4 shows the resonant frequencies of two microcapsule preparations having different wall compositions that were tested according to example 18. Description of Preferred Embodiments As used herein, the term microparticles has the purpose of including microcapsules, microspheres and microbubbles which are hollow particles that incorporate a core that may be filled with gas or liquid.

It also includes particles in which the core can be a solid material. It is not necessary that the microparticles are precisely spherical, although they will generally be spherical and are described as having an average diameter. If the microparticles are not spherical, then the diameters refer to or are linked to the diameter of a corresponding spherical microparticle which has the same mass and which incorporates approximately the same volume of interior space as a non-spherical microparticle. The microparticles according to the present invention have a two-layer cover. The outer layer of the cover will be a biologically compatible material or a biomaterial as it defines the surface that is exposed to blood and tissues within the body. The inner layer of the shell will be a biodegradable polymer, which may be a synthetic polymer, and which may be adapted to provide the desired mechanical and acoustic properties to the shell or offer drug delivery properties. To be used as ultrasound contrast agents, the nuclei of the microparticles contain gas, usually air or nitrogen. However, for drug delivery purposes, the core can "be either a liquid or a solid material different from the shell layers." To make the microparticles disintegrate with a low-intensity ultrasound energy, however, they must contain a gas to allow acoustic coupling and oscillation of particles Microparticles are constructed in the present such that most of those prepared in a composition will have diameters in the range of about one to ten microns in order to pass Through the capillary system of the body, since the microparticles have an outer and inner layer, the layers can be adapted to serve different functions.The outer covering that is exposed to blood and tissues serves as a biological interface between the microparticles and the body. In this way, it will be constituted by a biocompatible material that is usually amphili or, that is, it has both hydrophobic and hydrophilic properties. Generally, materials compatible with blood are preferred. These preferred materials are biological materials that include proteins such as collagen, gelatin or serum albumins or globulins, either derived from humans or with a structure similar to human protein, glucosaminoglycans such as hyaluronic acid, heparin and chondroitin sulfate and combinations or derivatives of the above. Synthetic biodegradable polymers such as polyethylene glycol, polyethylene oxide, polypropylene glycol and combinations or derivatives can also be used. The outer layer is usually amyphilic, and also has a chemistry that allows charge and chemical modification. The versatility of the surface allows modifications such as the alteration of the load of the external cover, for example selecting a type A gelatin that has an isoelectric point above the physiological pH, or using a type B gelatin with an isoelectric point below the physiological pH . The external surfaces can also be chemically modified to improve biocompatibility, for example by PEGylation, succinylation or amidation, as well as by being chemically bonded to the target half of the surface to bind to the selected tissues. The target moieties may be antibodies, cell receptors, lectins, selects, integrins or chemical or analogous structures of the target receptor of the aforementioned materials. The mechanical properties of the outer layer can also be modified, for example by cross-linking, to make the microparticles suitable for passage to the left ventricle, to provide a particular resonant frequency for a selected harmony of the diagnostic imaging system, or to offer stability for a reproduction level of diagnostic images of ultrasound radiation threshold. The inner cover will be a biodegradable polymer, which can be a synthetic polymer. An advantage of the inner cover is that it offers additional mechanical or delivery properties of medicaments to the microparticle that are not provided or that are inadequately provided by the outer layer, or improve the mechanical properties that are not sufficiently provided by the outer layer , without being limited by the surface property requirements. For example, a biocompatible outer layer of a cross-linked proteinaceous hydrogel can be physically supported using a high modulus synthetic polymer as the inner layer. The polymer can be selected from its modulus of elasticity and elongation, which defines the desired mechanical properties. Typical biodegradable polymers include polycaprolactone, polylactic acid, polylactic-polygluconic acid copolymers, copolymers of lactides and lactones, such as epsilon-caprolactone, delta-valerolactone, polyalkyl cyanoacrylates, polyamides, polyhydroxybutrirates, polydioxanones, polyethamine ketones, polyanhydrides, poly-Corto) esters, polyamino acids, such as polyglutamic or polyaspartic acids or esters of polyglutamic and polyaspartic acids. References on various biodegradable copolymeters are cited in Langer, et al. (1983) Macromol. Chem. Phys. C23, 61-125. The inner layer allows the modification of the mechanical properties of the microparticle shell that are not provided by the outer layer by itself. In addition, the inner layer can offer a transport capacity of medicament and / or delivery of medicament that is neither sufficient nor provided by the external layer itself. To be used as an ultrasonic contrast agent, the inner layer will usually have a thickness that is not greater than that necessary to meet the minimum properties of transport / delivery of medicine or mechanical, in order to increase the volume of gas inside the microparticle . The greater the volume of gas within the microparticle, the better the echogenic properties. The combined thickness of the outer and inner layers of the microparticle shell will depend in part on the mechanical and transport / delivery properties of the microparticle required, but generally the total thickness of the shell will be in the range of 25 to 750 nm. The microparticles can be prepared by an emulsification process to control the sequential interfacial deposition of the shell materials. Due to the amphiphilicity of the material forming the outer layer, stable oil / water emulsions can be prepared with a ratio of the internal phase to the external phase that approaches 3: 1 without the phase inversion which can be dispersible in water for form drops of the stable organic phase without the need for sulfactants, viscosity enhancers or high cut percentages. Two solutions are prepared, the first is an aqueous solution of the external biomaterial. The second is a solution of the polymer that is used to form the inner layer, in a relatively volatile water-immiscible liquid that is a solvent for the polymer, and a relatively non-volatile water-immiscible liquid that is a non-solvent for the polymer. The relatively volatile immiscible solvent in water is usually a C5-C7 ester, such as an isopropyl acetate. The non-volatile water immiscible solvent is usually a C6-C20 hydrocarbon such as decane, undecane, cycloexane, cyclooctane and the like. In the second solution containing the polymer for the inner layer, the polymer in the water-immiscible solvents are combined in such a way that the polymer completely dissolves and the two solvents They are miscible with agitation. The polymer solution (organic phase) is slowly added to the biomaterial solution (aqueous phase) to form a liquid foam. Generally about three parts of the organic polymer solution having a concentration of about 0.5 to 10% of the polymer are added to a part of the aqueous biomaterial solution having a concentration of about 1 to 20% of the biomaterial. The relative concentrations of the solutions and the ratio of the organic phase to the solid phase used in this step usually determine the size of the final microparticle and the thickness of the wall. After careful mixing of the liquid foam, it disperses in the water and is usually heated to approximately 30-35 ° C with gentle agitation. Although it is not intended to adhere to a particular theory, it is believed that the biomaterial in the foam is dispersed within the hot water to stabilize an emulsion of the polymer in the organic phase encapsulated within a biomaterial liner. To make the water of the biomaterial liner insoluble, a crosslinking agent, such as glutaraldehyde, is added to the mixture to react with the biomaterial liner and make the water insoluble, stabilizing the outer shell. Other cross-linking agents can be used, including the use of cross carbodiimide linkers. As at this point the inner core contains a solution of a polymer, a solvent and a non-solvent with different volatilities, as the more volatile solvent evaporates, or is diluted, the polymer is precipitated in the presence of the less volatile non-solvent. This process forms a film of the precipitate at the interface with the inner surface of the biomaterial cover, thereby forming the inner shell of the microparticle after the more volatile solvent has been reduced in its concentration either by dilution, evaporation or otherwise. Similary. The core of the microparticle contains this • predominantly organic non-solvent way. The microparticles can then be isolated by centrifugation, washed, formulated in a buffer system, if desired, dried. In general, drying by lyophilization removes not only the non-solvent liquid core, but also the waste water to produce hollow microparticles filled with gas. It may be convenient to further modify the surface of the microparticle, for example, in order to neutralize the surfaces against macrophages or the reticuloendothelial system (RES) in the liver. This can be achieved, for example, by chemically modifying the surface of the microparticle so that it is negatively charged, since the negatively charged particles seem to better evade recognition by the macrophages and the RES that the • positively charged particles. Also, hydrophobicity of the surface can be changed by joining the hydrophilic conjugates, such as polyethylene glycol (PEGylation) or succinic acid (succinylation) to the surface, either alone or together with the modification of the charge. The surface of the biomaterial can also modified to provide signaling characteristics for the microparticle. The surface can be labeled by known means with antibodies or biological receptors. For example, if the microparticle was treated to signal tumors and was hollow, it could be used for ultrasound detection to improve the diagnosis of tumors. If the microparticles were filled with drugs they could be used to signal tumors where the drug could be preferentially released at the target site, for example, by increasing the ultrasonic energy to disintegrate the particles at the appropriate time and place. The microparticles can also be measured or processed after manufacturing. This is an advantage over lipid-like microparticles that can not be subjected to mechanical processing after they are formed due to their fragility. The final formulation of the microparticles after the preparation, but before use, is in the form of a lyophilized cake. Late reconstitution of the microparticles can be facilitated by lyophilization with grouping agents that provide a cake having a high surface area and porosity. The grouping agents can also increase the percentage of drying during lyophilization by providing channels for solvent vapor and water to be removed. This also provides a larger surface area that can aid in the subsequent reconstitution. Typical grouping agents are sugars such as dextrose, mannitol, sorbitol and sucrose, and polymers such as PEG and PVP. It is not suitable for the microparticles that are added, either during the formulation or during the subsequent reconstitution of the lyophilized material. Aggregation can be reduced by maintaining a pH of at least one to two pH units higher or lower than the isoelectric point (P1) of the biomaterial that forms the outer surface. The charge on the surface is determined by the pH of the formulation medium. Thus, for example, if the surface of the biomaterial has a P, of 7 and the pH of the formulation medium is less than 7, the microparticle will possess a net positive surface charge. Alternatively, if the pH of the formulation medium is greater than 7, the microparticle would have a negative charge. The maximum potential for aggregation exists when the pH of the formulation medium approaches the P1 of the biomaterial used in the outer shell. Therefore, by maintaining a pH of the formulation medium at least one to two units above or below the P1 of the surface, aggregation of microparticles will be reduced. Alternatively, the microparticles can be formulated on or near the Pi with the use of surfactants to stabilize against aggregation. In any case, the buffering systems of the final formula that must be injected into the subject must be physiologically compatible. The grouping agents used during lyophilization of the microparticles can also be used to control the osmolality of the final formula for injection. An osmolality other than physiological osmolality may be desirable during lyophilization to reduce aggregation. However, when formulating the microparticles for use, the volume of liquid used to reconstitute the microparticles must take this into account. Other additives may be included in order to avoid aggregation or to facilitate the dispersion of the microparticles after the formulation. Surfactants can be used in the formulation, such as poloxomers (polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymers). Water-soluble polymers can also assist in the dispersion of microparticles, such as medium molecular weight polyethylene glycols and medium molecular weight polyvinylpyrrolidones. If the formulation contains a core including medicament, the microparticles can be immersed in a solution of the medicament whereby the solution is spread within the interior. In particular, the use of microparticles with two layers where the inner cover has a porous characteristic, allows a rapid diffusion of a drug solution into the hollow core. The microparticles can be removed again by, for example, lyophilization to produce a drug-containing microparticle filled with gas. The combination of the drug with prefabricated particles allows one to avoid the processing that can lead to the degradation of the drug. To provide microparticles having a solid core containing a medicament, during the formation of the microparticles, the thickness of the inner layers can be increased to occupy more or all of the interior volume. Subsequently, after soaking in the solution containing the medication, the internal solid core will absorb the medication and provide a solid deposit for the medication. Alternatively, the drug can be dissolved in the organic phase with the biopolymer during the microparticle formation process. The evaporation of the organic solvents causes the drug to be coprecipitated with the biopolymer inside the microparticle. We will realize that various modifications of the aforementioned processes can be provided without departing from the scope and scope of the invention. For example, the thickness of the wall of both the outer and inner layers can be adjusted by varying the concentration of the components in the solutions that form the microparticles. The mechanical properties of the microparticles can be controlled, not only by the total thickness of the wall and the thicknesses of the respective layers, but also by the selection of materials used in each of the layers by their modulus of elasticity and elongation, and cross-linking of the layers. Under certain conditions involving rapid deposition of the internal polymer or very low internal polymer content, the porosity of the inner polymer shell is observed. The pores vary from approximately 0.1 to 2 microns in diameter in the manner observed under electron microscopy. The mechanical properties of the layers can also be modified with plasticizers or other additives. The adjustment of the resistance of the cover can be modified, for example, by the internal pressure inside the microparticles. The precise acoustic characteristics of the microparticle can be achieved by controlling the mechanical properties of the cover, the thickness, as well as the narrow size distribution. The microparticles can be disintegrated by ultrasonic energy to release the gases trapped within the microparticles in the bloodstream. In particular, by properly adjusting the mechanical properties, the particles can be constituted to remain stable at the power of the representation of diagnostic images of the threshold, and at the same time they are disintegrable by an increase in power and / or by being exposed to their resonant frequency. The resonant frequency can be established within the range of the transmitted frequencies of the body image reproduction systems for diagnosis or there can be a harmony of these frequencies. During the formulation process the microparticles can be prepared to contain various gases, including soluble or insoluble gases in the blood. A feature of the invention is that the microparticle compositions can be made with a resonant frequency greater or equivalent to 2 MHz, and generally greater or equivalent to 5 MHz. The typical diagnosis or therapeutic targets for the microparticles of the invention are the heart and the tumors. The following examples are provided by way of illustration but are not intended to limit the invention in any way. Example 1 Preparation of gelatin polycaprolactone microparticles A solution of 1.0 gms of gelatin (275 bl, isoelectric point of 4.89) dissolved in 20 ml of deionized water was prepared at about 60 ° C. the native pH of the solution was 5.07. Separately, 1.0 gms of polycaprolactone (M.W. 50,000) and 6.75 ml of cyclooctane were dissolved in 42 ml of isopropyl acetate with stirring at about 70 ° C. After cooling to 37 ° C, the organic mixture was incorporated little by little into the gelatin solution maintained at 30 ° C and under moderate mixing using a rotary mixer. Once the organic phase was fully incorporated, the mixing speed was increased to 2500 rpm for 5 minutes and then stirred at low cutting for an additional 5 minutes. The o-emulsion was subsequently added by stirring to 350 ml of deionized water maintained at 30 ° C and containing 1.2 ml of 25% gluteraldeide. Immediately after the addition of the emulsion, the pH of the bath was adjusted to 4.7. After 30 minutes, the pH was adjusted to 8.3. The low cut mixing continued for about 2% hours until the isopropyl acetate was completely volatilized. Subsequently, polyoxamer 188 was dissolved in an amount of 0.75 gm inside the bath. The resulting microparticles were recovered by centrifugation and washed twice in a 0.25% polyoxamer 188 aqueous solution. Microscopic inspection of the microparticles revealed spherical capsules with a thin-walled polymer coating that encapsulated a liquid organic core. By staining the slide preparation with coomassie blue G indicated the presence of an outer protein layer that uniformly surrounds the polymer shell. The spectrum of the particle size was determined using a Micro Malvern. The average diameter was 4.78 microns with a spectrum interval of 0.94. Example 2 Preparation of the contrast agent formulation A quantity of microparticles prepared in a manner similar to that of Example 1 was suspended in an aqueous solution of 25 mM glycine, pluronic 0.5% f-127, 1.0% sucrose, 3.0% and PEG-3400 at 5.0%. The suspension was freeze-dried subsequently. The resulting dry powder was reconstituted in deionized water and examined under the microscope to reveal that the microparticles now contained a gaseous core. The dyeing of the preparation with blue commassie G confirmed that the outer protein layer surrounding the capsule was intact and had survived the lyophilization process. The echogenicity was confirmed by insopating both at 2 ^ and 5 MHz a quantity of lyophilized microparticles dispersed in 120 ml of deionized water. The measurement was taken at least 15 minutes after the dispersion of the microparticles to ensure that the backscattered signal was due solely to the gas contained within the microparticles. The display of mode B showed a high contrast indicating that the microparticles were full of gas. Example 3 Preparation of gelatin polylactide microparticles A solution of 1.2 gm of gelatin (225 efflorescence, isoelectric point of 5.1) dissolved in 20 ml of deionized water was prepared at about 50 ° C. The pH of the solution was adjusted to 6.1 using 1 M NaOH. Separately, 0.07 gms of paraffin, 4.5 ml of decane, and 0.69 gms of DL-lactide poly (inherent viscosity of 0.69 dL / gm in CHC12 @ 30 C) were dissolved in 37 ml of isopropyl acetate. The organic mixture was subsequently incorporated little by little into the gelatin solution which was maintained at 30 ° C under moderate cutting mixing using a rotary mixer. Once the organic phase was fully incorporated, the mixing speed was increased to 2000 rpm for 2 minutes and then reduced to approximately 1000 for 4 minutes. The resulting liquid foam was mixed in 350 ml of deionized water maintained at 30 ° C and subsequently 1 ml of 25% glyoteraldeide was added in the form of drops. Rotary mixing continued for about 3 hours until the isopropyl acetate was volatilized. The resulting microparticles were recovered by centrifugation and washed twice in an aqueous pluronic solution f-127 at 0.25%. Microscopic inspection revealed hollow spherical microparticles with an outer protein layer and an internal organic liquid core. The microparticles were lyophilized and tested in a manner similar to Example 2. The results confirmed that the microparticles contained a gaseous core and were strongly echogenic. Example 4 Preparation of Gelatin Polycaprolactone Microparticles A solution of 1.0 gm of gelatin (225 efflorescence, isoelectric point of 5.1) dissolved in 20 ml of deionized water was prepared at about 60 ° C. the pH of the solution was 4.8. Separately, 0.57 gms of polycaprolactone (M.W. 50,000) was dissolved in 1.72 ml of tetrahydrofuran. To this, a mixture of 0.07 g of paraffin, 0.475 g of triethyl citrate, 4.5 ml of cyclooctane and 42 ml of isopropyl acetate was added with stirring. The organic mixture was then incorporated little by little into the gelatin solution which was maintained at 30 ° C and under moderate mixing using a rotary mixer. Once the organic phase was fully incorporated, the mixing speed was increased to 4,700 rpm for 2 minutes and then reduced to 2,000 rpm for 4 minutes. The resulting liquid foam was subsequently added with stirring to 350 ml of deionized water at 30 ° C. for the crosslinking of the gelatin, 1 ml of 25% glutaraldehyde was added in drops. Mixing continued for about 3 hours until the isopropyl acetate was volatilized. The resulting microparticles were recovered by centrifugation and washed twice in a 0.25% f-127 pluronic solution. Microscopic inspection revealed microparticles of the discrete hollow spherical polymer with an outer protein layer and an internal organic liquid core. The microparticles were lyophilized and tested in a manner similar to Example 2. The results confirmed that the microparticles contained a gaseous core and were strongly echogenic. Example 5 Preparation of gelatin pollicaprolactone microparticles with cardodiimide cross-linking A solution of 1.0 grams of gelatin (225 efflorescence, isoelectric point of 5.1) dissolved in 20 ml of deionized water was prepared at about 60 ° C. the pH of the solution was adjusted to 5.5 with 1M NaOH. Separately, 0.85 gms of polycaprolactone (M.W. 80,000) was dissolved in 2.5 ml of tetrahydrofuran. To this was added with stirring a mixture of 0.07 gms of paraffin, 4.5 ml of cyclooctane and 42 ml of isopropyl acetate. The organic mixture was subsequently incorporated little by little into the gelatin solution which was maintained at 30 ° C and under moderate mixing using a rotary mixer. Once the organic phase was fully incorporated, the mixing speed was increased to 3500 rpm for 6 minutes and then reduced to 3000 rpm for 4 minutes. The resulting liquid foam was subsequently dispersed with low cutting mixing in 350 ml of a 0.5 M solution of NaCl maintained at 30 ° C. the crosslinking of the gelatin was achieved by the slow addition of 200 mg of l-ethyl-3- (3-dimethylamino-propyl) cabodiimide dissolved in 3.0 ml of deionized water. Mixing continued for about 3 hours until the isopropyl acetate was volatilized. The resulting microparticles were recovered by centrifugation and washed twice in an aqueous solution of Pluronic f-127 at 0.25%. Microscopic inspection revealed discrete hollow spherical polymer microparticles with an outer protein layer and an internal organic liquid core. Example 6 Preparation of PEGylated surface microparticles The microparticles were prepared in a manner similar to Example 1. After centrifugation, the cream was recovered and dispersed (approximately 15 ml) in a solution of 65 ml of deionized water, 0.50 gms of methoxy-PEG-NCO (N.W. 5000) and 0.50 ml of triethylamine. After allowing the mixture to react overnight at room temperature and with gentle agitation, the capsules were recovered by centrifugation and washed 3 times in a neutral buffer solution of Pluronic f-127 at 0.25%. Example 7 Canine Echogenicity Study A vial of lyophilized microparticles prepared in Example 2 was reconstituted using water. A transesophageal ultrasound probe was placed in the esophagus of an anesthetized dog in such a way that a view of the four chambers of the heart was obtained. The suspension of microparticles was injected into the femural vein of the dog. The appearance of the contrast agent was clearly seen in the ultrasound image of the right chambers of the heart. Subsequently, the agent was observed in the left chambers of the heart indicating the passage through the capillary system of the lungs. The time course of the ultrasound intensity reflected in the left atrium was determined by video densitometry. It was observed that the agent persisted in the left chambers of the heart for approximately 6 minutes (Figure 1). Example 8 Canine Study of Echogenicity Using PEGylated Microparticles A vial of lyophilized microparticles prepared in Example 6 was reconstituted using water. A transesophageal ultrasound probe was placed in the esophagus of an anesthetized dog in such a way that the view of the four chambers of the heart was obtained. The suspension of microparticles was injected into the femural vein of the dog. The appearance of the contrast agent was clearly seen in the ultrasound image of the right chambers of the heart. Subsequently, the agent was observed in the left chambers of the heart indicating the aso through the capillary system of the lungs. The time course of the ultrasound intensity reflected in the left atrium was determined by video densitometry. It was observed that agent persisted in the left chambers of the heart for approximately 16 minutes (Figure 2) after which no further data was collected. Example 9 Preparation of albumin polycaprolactone microparticles A 6% aqueous solution was prepared from a 25% solution of USP grade human serum albumin (Alpha Therapeutic Corp) by dilution with deionized water. The solution was adjusted to a pH of 3.49 using 1 N of HCL. Separately, 8 parts by weight of polycaprolactone (M.W. 50,000) and 45 parts of cyclooctane were dissolved in 300 parts of isopropyl acetate at about 70 ° C. Once the solution was completed, the organic solution was allowed to cool to 37 ° C. With gentle agitation, 42.5 gm of the prepared organic solution was incorporated little by little into 25.0 gm of the albumin solution while maintaining the mixture at 30 ° C. The resulting coarse o-w emulsion was subsequently circulated through a sintered stainless steel filter element with a nominal pore size of 7 microns. The recirculation of the emulsion continued for 8 minutes. Subsequently the emulsion was added with stirring to 350 ml of deionized water maintained at 30 ° C and containing 1.0 ml of 25% gluteraldeide. During the addition, the pH of the bath was monostorered to ensure that it remained between 7 and 8. The final pH was 7.1. The low cut mixing continued for about 2% hours until the isopropyl acetate was completely volatilized. Subsequently, poloxamer 188 was dissolved in an amount of 0.75 gm inside the bath. The resulting microparticles were recovered by centrifugation and washed twice in an aqueous 0.25% poloxamer solution. Microscopic inspection of the suspension revealed spherical particles with a thin-walled polymer shell and an outer protein layer, and an organic liquid core. The diameter punctured as determined by the Micro Malvern particle size analyzer was 4.12 microns. The suspension was then lyophilized in a manner similar to that described in Example 2. The resulting dry cake was reconstituted with deionized water and examined under the microscope to reveal that the microparticles were spherical, discrete and contained a gaseous core. EXAMPLE 10 Container of Microparticle Proteins The microparticles were prepared according to Example 9. After centrifugation, about 1 ml of the microparticle containing cream was recovered and diluted 10 to 1 using deionized water. From the diluted cream, 20 microliter samples were prepared in triplicate at lx, 2x and 4x dilutions with deionized water. The protein content of the samples was determined using a Pierce colorimetric BCA assay and a standard of bovine serum albumin. The average total protein of the diluted cream was determined as 0.441 mg / ml. To determine the total dry weight of the diluted cream, 2 ml were dried in an oven at 40 ° C until no subsequent weight changes were observed (approximately 16 hours). The average weight of 4 replicates was 6.45 mg / ml. The percentage dry weight of the protein that can be used as a measure of the ratio of the outer layer of the protein to the inner layer of the polymer of the microcapsule wall can be determined with the following formula. Average total protein / ml - dry weight / ml x 100%.

The percentage dry weight of the protein was calculated as 6.8%. Example 11 Preparation of albumin polylactide microparticles A 6% aqueous solution was prepared from a 25% solution of given USP human albumin by dilution with deionized water. Subsequently, ion exchange resin (AG 501 -X8, BioRad Laboratories) was added to the solution at a ratio of 1.5 gm of resin with 1.0 gm of dry weight of albumin. After three hours the resin was removed by filtration and the pH of the solution was adjusted from 4.65 to 5.5. Separately, 0.41 gm of lactide d-l (0.69 dL / gm in CHCl3: at 30 ° C) and 5.63 gm of cyclooctane were dissolved in 37.5 gm of isopropyl acetate. The organic solution was subsequently incorporated little by little into 25.0 gm of the prepared albumin solution with gentle agitation while maintaining the mixture at 30 ° C. The resulting coarse o-w emulsion was subsequently circulated through a stainless steel sintered metal filter element with a nominal pore size of 7 microns. The recirculation of the emulsion was continued for 8 minutes.

Subsequently, the emulsion was added with stirring to 350 ml of deionized water maintained at 30 ° C and containing 1.0 ml of 25% gluteride. During vision, the pH of the bath is • monitored to ensure it remained between 7 and 8. The final pH 5 was 7.0. The low cut mixing continued for about 2 and 2 hours until the isopropyl acetate was completely volatilized. Subsequently, polyoxamer 188 was dissolved in an amount of 0.75 gm inside the bath. The resulting microspheres were recovered by centrifugation and washed twice in an aqueous 0.25% polyoxamer solution. • Microscopic inspection revealed hollow spherical polymer microparticles with an outer protein layer and an internal organic liquid core. The suspension is formulated with a glycine / PEG 3350 excipient solution and then lyophilized. The resulting dry cake was reconstituted with deionized water and examined under the microscope to reveal that the microparticles were spherical, discrete and contained a gaseous core. Example 12 PEG Modification of the Microparticle Surface The microparticles were prepared in a manner similar to Example 9. After centrifugation, 4 ml of the cream-containing particles (approximately 11 ml of the total production) were suspended again in 31 ml of deionized water. To this was added a 10 ml solution containing 0.3 gm of methoxy-peg-NCO 5000 and the pH adjusted to 8.7. The mixture was allowed to react at room temperature with gentle agitation for 4 and 2 hours. At the end of this period the pH was measured as 7.9. The microparticles were recovered by centrifugation and washed 2 times in a 0.25% polyoxamer 188 solution. The suspension was formulated with a solution of glycine extract / PEG 3350, and then lyophilized. The resulting dry cake was reconstituted with deionized water and examined under the microscope to reveal that the microparticles were spherical, discrete and contained a gaseous core. Example 13 Postfabrication, modification of the size distribution A quantity of microparticles was first prepared in a manner similar to Example 1 with procedures modified to offer a broader spectrum.

After washing and recovery by centrifugation, approximately half of the microparticles containing cream were diluted in 125 ml with an 0. 25% polyoxamer 188. The suspension was subsequently filtered using a pe membrane filter with 5 micron sieve (Nucleopore) housed in a stirred cell (Amicon). The filtrate was discarded while the permeate was filtered once again using a 3 micron sieve filter in the stirred cell system until the volume of the filtrate reached about 20 ml. The filtrate was diluted to a volume of 220 ml using a 0.25% polyoxamer 188 solution. The filtering process of 3 microns was repeated until the volume of the filtrate was again about 20 ml. Figure 3 provides a comparison of the volumetric size distribution of the suspension of unfiltered microparticles with the permeate of 5 microns and the filtering of 3 microns. The results, derived from a Micro Malvern particle size analyzer, show a gradual narrowing of the size spectrum towards a specific size range defined by the pore size of the filters used. EXAMPLE 14 Canine Study Representative of Echogenicity A 31 kg thoracotomized male crossed dog was injected with 1 cc of reconstituted microparticle composition made in accordance with example 4. This was delivered to the circulation through a peripheral venous injection. The representation of activated harmonic ultrasound images (once each beat) of the left ventricle was performed for 9 minutes. A contrast effect on the myocardium could be observed during the activated image representation. The representation of real-time harmonic ultrasound images (30 Hz) in the next 4 minutes increased the destruction of bubbles. Left ventricular opacification remained persistent for 13 minutes of the imaging period. No adverse hemodynamic effect was observed. In a separate study, 0.1 cc of the reconstituted microparticle agent was administered in a similar manner to a thoracotomized male crossed dog. The image representation of the activated harmonic ultrasound was carried out for 1 minute, followed by 4 minutes of increased particle destruction with real time image reproduction. Once again, no adverse hemodynamic effects were observed, and the left ventricular opacification was apparent and persistent. EXAMPLE 15 - Stopped Locking of Albumin Polylactide Microparticles A lyophilized cake in a 10 ml serum vial, composed of excipient and lactide-containing microparticles was prepared in a similar manner to Example 11 and placed inside a 50 ml centrifuge tube. . Sufficient isopropyl alcohol was added to cover the cake and allowed to soak for 30 seconds. The aqueous F68 pluronic solution (0.25% w / w) was added to fill the tube. After centrifugation, the supernatant was removed and another rinse was carried out. A filtered and saturated solution of rhodamine B was added to the microparticles and allowed to soak overnight. Under the microscope, the microparticles appeared filled with dye solution. A saturated solution of the F68 dye was made to be used as a lyophilization excipient. Four ml of the excipient were combined with about 2 ml of the solution containing microparticles and the resulting mixture was divided between two 10 ml serum bottles. The flasks were frozen at -80 ° C and lyophilized in a FTS trough dryer. Both bottles were purged with perfluorobutane gas by five pumping purge cycles with a vacuum pump. The observation showed some microparticles that were filled with half the red solution and the other half filled with gas. There was no obvious leakage of the dye from these microparticles during the observation. The microparticles were rinsed with four 20 m portions. of solution F68 in a vacuum filter. The microparticles were placed in a test tube, centrifuged and an initial spectrum was taken. The specimen was sonicated in an ultrasonic bath, centrifuged and another spectrum was taken. Abs. Initial (553-800) Abs. Sonicada (553-800) 1.164 1.86 The highest absorption after sonication indicates that the encapsulated dye was released after the insonation of the microparticles. EXAMPLE 16 Preparation of wall-modified albumin polycaprolactone microparticles Albumin-coated microparticles were prepared in a manner similar to Example 9 with the exception that 0.20 gm of paraffin was also dissolved within the organic solution together with the polycaprolactone and the cyclooctane. Microscopic inspection of the finished microparticle suspension revealed spherical particles with a morphology and appearance virtually identical to those prepared without the addition of paraffin. Example 17 Dye load of polycaprolactone microparticles of human serum albumin A lyophilized cake in a 10 ml serum vial, composed of excipient and microparticles containing paraffin was prepared according to example 16 and placed inside a centrifuge tube of 50 mi. The cake was covered with methanol and allowed to soak for 30 seconds. The tube was then filled with an aqueous solution of pluronic F68 at 0.25% (w / w) mixed lightly and centrifuged in order to precipitate the microparticles now filled with fluid. The supernatant was removed and the tubes were again filled with Pluronic solution. The microparticles were resuspended by vortexing and centrifuged again. After removing the solution from the supernatant, two ml of a saturated and filtered solution of brilliant blue dye G in an aqueous solution F68 at 0.25% (w / w) were added. The microparticles were allowed to soak for approximately 72 hours. Microscopic examination revealed that 90-95% of the microparticles were filled with dye solution. A lyophilization excipient was prepared. 4 ml of the excipient was added to the microparticle solution and mixed by vortexing. Two vials of 10 ml serum were filled with 3 ml each of solution and frozen at -80 ° C. The bottles were lyophilized in a FTS flask lyophilizer. Both flasks and a portion of the deionized water were purged with perfluorobutane for 10 minutes. Both flasks were reconstituted with deionized water and rinsed with two 40-rale portions of 0.25% (w / w) F68 solution in a vacuum filter. The resulting microparticle solution was divided into two 3 ml portions. A portion is sonic in an ultrasonic bath to disintegrate the bubbles. Both portions were diluted 1/10 with F68 solution and placed in UV visible specimens. The specimens were centrifuged and the visible spectrum was taken. Absorption (at 605nm-800nm) Sonicate 0.193 Not sonicated 0.136 The highest absorption after sonication indicates that the encapsulated dye was released after the insonation of the microparticles. Example 18 Acoustic resonance of microparticles To demonstrate an acoustic tuning method of microparticle construction, the microparticles prepared according to the procedures described in example 9 and 16 were reconstituted with deionized water and compared for their acoustic properties using the procedures described below: Two coupled 5 MHz transducers were placed in a tank filled with degassed water one in front of the other. The depth of the water was approximately 3 inches. The transducers, one emitter and the other receiver, were placed 6 inches apart to maximize the received signals. A circular chamber 2 inches in diameter and 2 centimeters wide was placed between the two transducers with the position of the intermediate chamber 3 inches from the emitter. The two circular faces of the chamber were covered with a 3 mil polyethylene film and the chamber was then filled with degassed water. Sound waves propagated easily from the emitter through the camera to the receiver. The sound source was programmed for Gaussian Noise with a peak-to-peak amplitude output of 10 Volts from the ultrasonic generator. The signal from the receiver was amplified with a 17 dB preamplifier and an oscilloscope. The digital electronics of the oscilloscope can perform Fast Fourier Transformations (FFT) of the received waveforms and can deploy these distributions. After having made the baseline readings, the microparticle contrast materials of the test were delivered into the chamber through a hypodermic syringe and mixed thoroughly by pumping the syringe. During the post-test evaluation, the FFT data was converted to the Test Agent Transfer Function. The transfer function (TF) is determined by dividing the data of the transmission spectrum of the bubbles between the spectral data without bubbles, ie: Sm bubbles where T (f) is requested by the FFT. ", The contrast agent selectively attenuates sound waves depending on their spectral distribution, that is, more energy is absorbed from the sound at or near the resonance of the bubbles instead of outside the resonance. Therefore the procedure can be used to evaluate the resonant spectral distribution of people. The data derived from the two agents with approximately an identical size distribution but different internal cover thickness were collected the same day with the same equipment programmed in the same settings. Everything else remained constant for a variety of doses of the agent. The normalization of the spectrum was carried out by dividing the spectral arrangement by the minimum value. In this way, the peak value becomes a unit and when plotting on the same graph it is quite easy to differentiate the two graphs. These standardized data are presented in Figure 4. The inspection of the results shown in Figure 4 clearly show that when the concordance with the cover wall increases, the resonant frequency can be changed from 2.3 MHz to 8.9 MHz. , the resonant frequency of an agent can be controlled by controlling the composition and thickness of the wall. Example 19 Effect of acoustic properties on in-vivo echogenicity Two microparticle formulas containing air were evaluated for their in-vivo efficacy. A bottle of lyophilized microparticles was prepared according to that described in examples 1 and 2 (formula a). A second bottle of lyophilized microparticles was prepared in a manner similar to that described in examples 1 and 2, except that the amount of polymer was used four times, producing microparticles with a thick internal wall and therefore a higher resonant frequency (formula B) Both bottles were reconstituted immediately before use. From the particle size analysis, both formulas had an average diameter of the microparticles of about 4 microns and an almost identical microparticle concentration. The in-vitro acoustic characterization showed that formula A had a resonant frequency close to 5 MHz, and formula B had a resonant frequency greater than 10 MHz. A 5 MHz transesophageal ultrasound probe was placed in the esophagus of an anesthetized dog in such a way that a view of the four chambers of the heart was obtained. The reconstituted microparticle suspension (4 cc of formula A) was injected into the femural vein of the dog. The appearance of the contrast people was clearly seen in the ultrasound image of the right and left chambers of the heart. Subsequently, 4 ce of thin-walled microparticle suspension (formula B) was injected into the femural vein of the dog. Even though the appearance of the contrast agent was once again clearly observed in the heart ultrasound image, the contrast effect decreased substantially when compared to the injection of the equivalent volume of formula A. Subsequent injections of the dilutions made to From formula A demonstrated a dose effectiveness greater than four times of formula A that had a resonant frequency near the center frequency of the ultrasound diagnostic system compared to formula B with a resonant frequency of a higher peak.

Claims (56)

1. A microparticle composition containing microparticles with diameters in the range of about 1 to 10 microns, an outer layer of a biologically compatible material and an inner layer including a biodegradable polymer.

2. A composition according to claim 1, wherein at least a majority of said microparticles have diameters within said range.

3. A composition according to claim 1, wherein said microparticles have hollow cores.

4. A composition according to claim 1, wherein said microparticles include solid cores.

5. A composition according to claim 1, wherein said biologically compatible material is compatible with the blood.

6. A composition according to claim 1, wherein said outer layer includes a chemically crosslinked amyphilil biopolymer.

7. A composition according to claim 3, wherein said cores contain a gas.

8. A composition according to claim 3, wherein said cores contain a liquid.

9. A composition according to claim 1 or 7, wherein said microparticles contain a medicament.

10. A composition that includes gas-filled microparticles of a size capable of passing the capillary circulation system of the body, wherein said microparticles are mechanically adjusted to resonate at a predetermined resonance frequency.

11. A composition according to claim 10, wherein said microparticles are selected to resonate at said frequency by a selection of the particle size.

12. A composition according to claim 11, wherein the diameter of the particle is within the range of about 1 to 10 microns.

13. A composition according to claim 10, wherein the resonant frequency mentioned is > 2 MHz.

14. A composition according to claim 13, wherein the frequency mentioned is > 5 MHz.

15. A composition according to claim 10, wherein the mechanical adjustment is made by selecting the thickness of the wall.

16. A composition according to claim 10, wherein the mechanical adjustment is made by selecting the material of the wall for the mentioned microparticles.

17. A composition according to claim 10, wherein the mechanical adjustment is made by selecting the degree of cross-linking of the wall material of the aforementioned microparticles.

18. A composition according to claim 10, wherein the mechanical adjustment is made by adjusting the internal pressure within said microparticles

19. A composition according to claim 10, wherein said resonant frequency is in the range of the transmitted frequencies of a system for reproducing diagnostic body images.

20. A composition according to claim 10, wherein said resonant frequency is a harmony of a system for reproducing diagnostic body images.

A composition according to claim 10, wherein the microparticles are mechanically adjusted to remain stable when exposed to a diagnostic image reproduction level of ultrasound irradiation power threshold and are disintegrable upon exposure to an increase in the mentioned power.

22. A composition according to claim 10, wherein the microparticles are mechanically adjusted to remain stable when exposed to a diagnostic image reproduction level of the ultrasonic irradiation power threshold and are disintegrable upon exposure to frequencies close to the resonant frequency of the microparticles.

23. A composition according to claim 21 or 22, where the mentioned microparticles contain a medicine.

24. A composition according to claim 21 or 22, wherein said microparticles contain soluble or insoluble gases in the blood.

25. A composition that includes gas filled microparticles of a size capable of passing the capillary circulation system of the body and including target surface halves to join selected tissues.

26. A composition that includes gas-filled microparticles of a size capable of passing the blood capillary circulation system and including conjugated hydrophilic polymers on the surface.

27. A composition according to claim 26 wherein said hydrophilic polymers include polyethylene glycol.

28. A composition according to claim 26 which includes polyethylene glycol, polypropylene glycol, its derivatives and combinations thereof.

29. A composition according to claim 25 or 2-6 which includes a chemically charged outer surface.

30. A composition according to claim 28 wherein said external surface is PEGylated.

31. A composition according to claim 29 wherein said external surface is succinylated.

32. A composition according to claim 25, wherein said target moieties are selected from the group consisting of antibodies, cellular receptors, lectins, selectins, integrins, receptor targets, receptor analogues and active fragments of the foregoing.

33. A composition according to claim 1, wherein said biologically compatible material includes a protein.

34. A composition according to claim 1, wherein said biologically compatible material includes collagen, gelatin, albumin, globulin or glycosaminoglycan.

35. A composition according to claim 1, wherein said biologically compatible material includes albumin

36. A composition according to claim 1, wherein said biologically compatible material includes a biopolymer.

37. A composition according to claim 1, wherein said biologically compatible material includes a gelatin.

38. A composition according to claim 1, wherein said biodegradable polymer includes a synthetic polymer.

39. A composition according to claim 38, wherein said polymer includes polycaprolactone.

40. A composition according to claim 38, wherein said polymer includes polylactide, polyclucolide, or copolymers thereof.

41. A composition according to claim 38, wherein said polymer includes copolymers of caprolactone with lactic or glycolic acid.

42. A composition according to claim 1, wherein said inner layer is porous.

43. A process for forming multi-layer microparticles that can be suspended in a medium suitable for injection, and which includes the steps: a. forming a mixture of a first aqueous dispersion of a biologically compatible material and mixing with a second solution of a biodegradable polymer where the second mentioned solution includes a relatively volatile water-immiscible solvent and a relatively non-volatile water immiscible non-solvent for the aforementioned polymer . b. emulsifying the aforementioned mixture of step (a) to form microdroplets with an interior containing said polymer solution and an outer layer including said biologically compatible material; c. removing the above-mentioned relatively volatile solvent from said microparticles to form an internal layer of said polymer in said biopolymer. d. Dry the aforementioned microparticles to remove the aforementioned non-solvent and residual water.

44. A process according to claim 43, wherein in said step (c) said volatile solvent is removed by evaporation.

45. A process according to claim 43, wherein in said step (c) said volatile solvent is removed by dilution.

46. A process according to claim 43, further including the cross-linking step of said outer layer.

47. A process according to claim 43, wherein in step (d) mentioned a freeze drying is performed.

48. A process according to claim 43, further including the step of loading said microparticles after drying with a gas other than air.

49. A process according to claim 43, further including the step of selecting the microparticle size.

50. A method to generate an image for echogenic inspection that includes the steps of: a. introducing within a subject a composition of gas-filled microparticles capable of passing the capillary circulation system of the aforementioned subject where the aforementioned microparticles have an outer layer that includes a biologically compatible material and an inner layer that includes a biodegradable polymer; b. subjecting the aforementioned subject to adequate ultrasonic radiation; c. detecting the reflected, transmitted, resonant ultrasonic radiation and / or the frequency and / or amplitude modulated by the microparticles mentioned in the aforementioned subject.

51. A method according to claim 50, wherein at least a majority of said microparticles have diameters in the range of about 1 to 10 microns.

52. A method for delivering a pharmaceutically active agent to a subject that includes the steps of introducing into the subject a composition that includes microparticles capable of passing the capillary circulation of the aforementioned subject, with an outer layer that includes a biologically compatible material, a layer internal including a biodegradable polymer and containing the aforementioned pharmaceutically active agent.

53. A method according to claim 52, wherein at least a majority of said microparticles have diameters in the range of about 1 to 10 microns.

54. A method according to claim 52 which further includes the step of applying localized ultrasound energy to the target tissue or organ to release the pharmaceutically active agent at the specifically desired site.

55. A method according to claim 54 wherein the target organ is the heart.

56. A method according to claim 55 wherein the target tissue is a tumor.