MXPA97009717A - Apparatus and method for making vessels filled with opt size gas - Google Patents

Abstract

A method and apparatus for making vesicles suitable for use as contrast agents in which the container containing an aqueous suspension phase and a separated gas phase is agitated using an alternate motion. The reciprocating movement is produced by a stirring arm which moves the container in two substantially perpendicular directions, the movement being in the first direction perpendicular, the movement being in the first direction along an arcuate path. The total trajectory of the movement occurs in a figure pattern at 8. The frequency of agitation is at least about 2800 rpm, the length of the agitator arm is at least about 6 cm, and the angle through which the agitator arm rotates in the first address is at least approximately 3o. The total length of travel around the figure pattern at 8 is at least 0.7 centimeter

Description

APPARATUS AND METHOD FOR MAKING OPTIMUM SIZE GAS-FILLED VESTICLES Related Requests This application is a partial continuation of the United States of America patent application related to serial number 307,305, filed on September 19, 1994, which in turn is a partial continuation of the patent application of the United States of America. United States of America with serial number 159,687 filed on November 30, 1993, which in turn is a partial continuation of the United States of America patent application serial number 076,239, filed on June 11, 1993. , which in turn is a partial continuation of the United States of America patent application with serial number 717,084 and the United States of America patent application with serial number 716,899, both filed on June 18, 1991 , which in turn are partial continuations of the United States of America patent application with serial number 569,828, present on August 20, 1990, which in turn is a partial continuation of the United States of America patent application serial number 455,707, filed on December 22, 1989.

This application is also a partial continuation of the United States of America patent application serial number 160,232, filed on November 30, 1993, which in turn is a partial continuation of the United States patent application of North America with serial number 076,250, filed the one filed on June 11, 1993, which in turn is a partial continuation of the United States of America patent application with serial number 717,084 and the United States patent application of North America with serial number 716,899, both filed on June 18, 1991, which in turn are partial continuations of the United States Patent Application Serial No. 569,828, filed August 20, 1990, which at the same time, it is a partial continuation of the patent application of the United States of North America with serial number 455,707, filed on December 22, 1989. The descriptions of each of these applications in their entirety are hereby incorporated by reference. Field of the Invention The present invention is directed to a method and apparatus for making gas-filled vesicles, especially gas-filled vesicles of the type useful for making ultrasonic images. More specifically, the present invention is directed to a method and apparatus for making gas-filled vesicles by agitation in which the agitation parameters are controlled to provide vesicles of an optimal size in a minimum amount of time.

BACKGROUND OF THE INVENTION Ultrasound is a diagnostic imaging technique that provides numerous advantages over another diagnostic methodology. Unlike techniques such as nuclear medicine and X-rays, ultrasound does not expose the patient to potentially hazardous exposures to ionizing electron radiation that can potentially damage biological materials, such as DNA, RNA, and proteins. In addition, ultrasound technology is a relatively inexpensive modality when compared to techniques such as computed tomography (CT) or magnetic resonance imaging. The principle of ultrasound is based on the fact that sound waves will be differentially reflected outside the tissues depending on the constitution and density of the tissue or vasculature that is being observed. Depending on the composition of the tissue, the ultrasonic waves will dissipate by absorption, penetrate through the tissue, or reflect back. Reflection, referred to as backscattering or reflectivity, is the basis for the development of an ultrasound image. A transducer, which is typically capable of detecting sound waves in the range of 1 MHz to 10 MHz in clinical settings, is used to sensibly detect the return of sound waves. These waves are then integrated into an image that can be quantified. The quantized waves are then converted into an image of the tissue that is being observed. Despite the technical advances of the ultrasound modality, the images obtained are still subject to further refinement, particularly with respect to vasculature and tissue images that are penetrated by a vascular blood supply. Therefore, there is a need for the formulation of agents that will aid visualization of the vasculature and vascular related organs. Vesicles are convenient as contrast agents because the reflection of sound at a liquid-gas interface, such as the surface of a vesicle, is extremely efficient. To be effective as ultrasound contrast agents, the vesicles should be as large and elastic as possible since both of these properties (bubble size and elasticity) are important to maximize the reflectivity sound from the vesicles. Additionally, the vesicles must be stable under pressure, that is, retain more than 50 percent of the gas content after exposure to pressure. It is also very convenient that the vesicles should be re-expanded after the release of the pressure. It is also very convenient to have a high vesicle concentration in order to maximize the reflectivity and, therefore, the contrast. Therefore, the concentration of vesicles is an important factor in determining the effectiveness of the vesicles. In particular, it is convenient to have more than 100 x 106 vesicles per mL and, more preferably, more than 500 x 10"vesicles per mL." Size, however, remains a crucial factor in determining the suitability of the vesicles for the image. In the vesicle regime that can pass safely through the capillary vasculature, the reflected signal (Rayleigh Scatterer) can be a function of the diameter of the vesicles raised to the sixth power so that a vesicle of 4 μm in diameter has 64 times the dispersion capacity of a vesicle of 2 μm in diameter Size is also important because vesicles larger than 10 μm can be dangerous. Large vesicles have a tendency to occlude microvessels following an intravenous or intravascular injection. It is therefore important that the vesicles are as large as possible to efficiently reflect the sound but small enough to pass through the capillaries.

In this regard, it is very convenient that 99 percent of the vesicles are smaller than 10 μm. In addition, the mean vesicle size should be at least 0.5 μm, preferably about 1 μm, and more preferably close to 2 μm for the most effective contrast. In addition, the weighted average volume must be of the order of 7 μm. The elasticity of the vesicles can affect their maximum allowable size, since the greater the elasticity of the vesicle, the greater its ability to "compress" through the capillaries. Unfortunately, numerous factors can hinder the formation of very elastic vesicles, thereby reinforcing the importance of optimizing vesicle size. Although uncoated vesicles have maximum elasticity, they are generally unstable. Consequently, efforts have often been made to increase the stability of the vesicles such as by coating, which has the effect of reducing its elasticity. Furthermore, the use of gas or gas precursors encapsulated in a protein shell has been suggested, the protein being crosslinked with biodegradable crosslinking agents, as well as the use of non-protein vesicles covalently crosslinked with biologically compatible compounds. It can be assumed that the crosslinkers will add a stiffening component to the vesicles, thereby reducing their elasticity.

Although it is known that liposomes can be made by stirring a surfactant solution in a liquid medium (see US Pat.

Number: 4, 684,479 (D'Arrigo)), Has not yet been developed a method for making vesicles that have an optimal size in a minimum amount of time. Accordingly, for all the above reasons, there is a need for a method and apparatus for making vesicles in which the agitation parameters are controlled so as to produce veeiclee of optimum size in a minimum amount of time.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method and apparatus for making vesicles in which the agitation variables are controlled so as to produce optimum-sized veeicles in a minimum amount of time. This and other objects are achieved in a method in which a container containing an aqueous suspension phase and a gas phase are stirred using reciprocating movement. The reciprocating movement is produced by an agitator arm that moves the container in two directions, substantially perpendicular. The movement in the first direction occurs along an arcuate path that has a radius of curvature of at least 6 centimeters and covers an angle of at least 3 °. The global trajectory of the movement occurs in a figure pattern of eight 8. The frequency of agitation is at least 2800 rpm, the amplitude of the agitation is at least 0.3 centimeters and the total length of travel of the container during each cycle is at least 0.7 centimeters. The present invention also encompasses an apparatus for stirring a container containing an aqueous suspension phase and a gas phase using the method described above. Preferably, the apparatus has an agitator arm having a length of at least 6 centimeters that rotates through an angle of at least 3 °.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an elevation of the container portion of the agitator apparatus of the present invention, in which the vesicles are made by the agitation method of the present invention. Figure 2 is an isometric view of the agitation apparatus according to the present invention, without the container. Figure 3 is a longitudinal cross section through the agitator apparatus shown in Figure 2, without the cover, but including the installation of the container shown in Figure 1. Figures 4 and 5 are elevational and plan views, respectively, of the path followed by the container shown in Figure 1 when it is installed in the agitator apparatus shown in Figure 2, having taken Figure 5 along the line VV shown in Figure 4. Figure 6 is a view isometric of the main internal components of the agitator apparatus shown in Figure 2. Figures 7 and 8 are longitudinal cross sections through the agitator apparatus shown in Figure 2 in the vicinity of the region where the agitator arm is mounted on the arrow of the motor, with the position of the agitator arm when the eccentric bushing is in the orientation shown in Figure 8 which is shown in dotted lines in Figure 7. Figures 9 ( a) and (b) are views taken along line IX-IX shown in Figure 7 of the agitator arm, except that in Figures 9 (a) and (b) the eccentric bushing has rotated 90 ° and 270 °, respectively, of its orientation shown in Figure 7. Figure 10 is a cross section taken through the line XX shown in Figure 9 (b), with the orientation of the sleeve when the eccentric hub has been rotated 180 ° shown in dotted lines. Figure 11 is an isometric view of the eccentric bushing mounted on the motor shaft. Figure 12 is a view similar to Figure 9 showing the orientation of the agitating arm when the lower spring tension is employed. Figure 13 is a graph showing the relationship between the frequency of agitation, in rpm, on the one hand, and the length L of the agitator arm, in centimeters, and the angle of transport deflection,, on the other hand, used to obtain the test results shown in Figures 14 - 16. Figures 14 (a) - (c) are graphs showing the percentage of vesicles having a size smaller than 10 μm, the weighted average size of the number, and the particles per mL , against the length of the agitator arm L, in millimeters, according to the length and rpm of the agitator arm are varied according to Figure 13, at a transport deflection angle? Of 6. Figures 15 (a) - (c) are graphs similar to Figures 14 (a) - (c) comparing the results obtained using a transport deflection angle? of 9 ° with those shown in Figures 14 (a) - (c). Figures 16 (a) - (c) are graphs showing the percentage of the vesicles having a size less than 10 μm, the weighted average size number, and the particles per mL, against the total length of the agitation path, In centimetres.

Figure 17 is a graph showing the percentage the relationship between the agitation frequency, in rpm, and the total length of the agitation path, in centimeters used to obtain the test results shown in Figure 16. Figures 18 ( a) - (c) are graphs showing the percentage of the vesicles that have a size less than 10 μm, the weighted average size number, and the particles per mL for three different types of agitation devices.

Description of the Preferred Modality In accordance with the method of the present invention, vesicles of optimal size are made by first placing an aqueous suspension 34, which preferably comprises lipids, within a container 9, as shown in Figure 1. As shown in FIG. used herein, the term "vesicle" refers to a spherical entity that is characterized by the presence of an interior void. Preferred vesicles are formulated from lipids, including the various lipids described herein. In any given vesicle, the lipids may be in the form of a monolayer or bilayer, and mono- or bilayer lipids may be used to form one or more mono- or bilayers. In the case of more than one mono-or two-layer, the mono- or bilayers are generally concentric. The vesicles described herein are sometimes called bubbles or microbubbles and include entities commonly called liposomes and micelles, and the like. In this case, lipids can be used to form a unilamellar vesicle (comprised of a monolayer or bilayer, an oligolamellar vesicle (comprised of approximately two or approximately three monolayers or bilayers) or a multilamellar vesicle (comprised of more than approximately three monolayers or bilayers). The internal void of the vesicles can be filled with a liquid, including, for example, an aqueous liquid, a gas, a gaseous precursor, and / or a solid or dissolved material, including, for example, a meta ligand and / or a "Liposome" refers to a generally spherical group or aggregate of antipathetic compounds, which include lipid compounds, typically in the form of one or more concentric layers, more preferably the gas filled liposome is constructed from a single layer (that is, unilamellar) or a single layer of lipid A wide variety of lipids can be used to make liposomes including foefol lipids and non-ionic surfactants (for example, niosomes). More preferably the lipids comprising gas-filled liposomes are in the gel state at physiological temperature. The liposomes can be crosslinked or polymerized and can carry polymers such as polyethylene glycol on their surfaces. Meta ligands directed to the endothelial cells bind to the surface of the gas filled liposomes. A meta ligand is a substance that binds to a vesicle and directs the vesicle to a particular cell type such as, but not limited to, tissue and / or endothelial cells. The meta ligand can be attached to the vesicle by covalent or non-covalent linkages. Liposomes can also be called in the present lipid vesicles. More preferably the liposomes are substantially free of water in the interior. "Micelle" refers to colloidal entities that are formed from lipid compounds when the concentration of lipid compounds, such as lauryl sulfate, is above a critical concentration. Since many of the compounds that form micelles also have surface-active properties (ie, low surface tension capacity and attraction of both water and fat, hydrophilic and lipophilic domains), these same materials can be used to stabilize bubbles. In general, micellular materials prefer to adopt a monolayer or hexagonal phase H2 configuration, yet can also adopt a bilayer configuration. When a micellular material is used to form a gas filled gallbladder, the compounds will generally adopt a radial configuration with the aliphatic (lipophilic) fractions oriented towards the vesicle and the hydrophilic domains oriented away from the surface of the vesicle. In order to direct the target, the ligands can be attached to the micellar compounds or to the amphipathic materials mixed with the micellar compounds. Alternatively, the meta ligands can be adsorbed to the surface of the micellar matter by stabilizing the vesicles. A gas phase is employed on the aqueous suspension phase 34 in the remaining portion, or in the space of the upper part 32, of the container 9. The introduction of the gas phase can be achieved by purging the container 9 with a gas, if a gas other than air is to be used for the gas phase, so that the gas occupies the space of the upper part 32 on the aqueous suspension 34. Thus, before shaking, the container 9 contains an aqueous suspension phase and a gas phase. The container 9 is then installed in the agitator arm 7 of the stirrer device 1 of the present invention, a preferred embodiment, which is shown in Figures 2, 3 and 6-ll, and stirred for a period of time sufficient to form the vesicles. desired. Although filters can be used to further refine the size distribution of the vesicles after agitation, the focus of the present invention is on the control of the agitation parameters in order to produce vesicles of optimal size before any subsequent filtration. the agitation. For this purpose, the inventors found that the size of the vesicles produced by agitation is in principle a function of four variables: (i) the composition of an aqueous suspension phase, (ii) the composition of the gas phase in the space of the upper part, (iii) the volume of the container and the relative volume of the space of the upper part that is occupied initially by the gas phase, and (iv) the definition of the primary agitation parameters --ie, the shape of the trajectory traveled by the container during the agitation, the amplitude of the agitation movement, and the duration and frequency of agitation. According to the method of the present invention, each of these variables must be adjusted in a process to make vesicles to obtain an adequate distribution and concentration of veeicle size, the preferable vesicle size distribution being one in which the vesicles have an average size of at least about 0.5 μm and in which at least 95 percent of the vesicles, and more preferably at least 99 percent of the veeiclee, have a diameter less than 10 μm, and the concentration of vesicles produced is at least 100 x 104 vesicles per mL and, preferably, when menoe 500 x 106 vesicles per mL. Consequently, in sections I-IV below, each of these four variables is given individually. In section V, a preferred apparatus for practicing the method of the present invention is described. Section VI discusses some applications of the vesicles made in accordance with the present invention.

I. THE COMPOSITION OF THE AQUEOUS SUSPENSION PHASE A wide variety of bubble coating agents can be employed in the aqueous phase of the melt. Preferably, the coating agents are lipid. The lipids may be saturated or unsaturated, and may be in straight or branched form, as desired. These lipids may comprise, for example, fatty acid molecules containing a wide range of carbon atoms, preferably between about 12 carbon atoms and about 22 carbon atoms. Hydrocarbon groups which are formed in isoprenoid units, prenyl groups, and / or sterol fractions (eg, cholesterol, cholesterol sulfate, and analogues thereof) can also be used. The lipid can also carry polymer chains, such as amphipathic polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP) polymers or derivatives of the miemos (to target the target in vivo), as described in the Patent of the United States. United States of America Number: 4,310,505 or glycolipids (for in vivo targets), or antibodies or other peptides and proteins (for in vivo targets), etc., as desired. The target or linking compounds can be simply added to the aqueous lipid euepension phase or chemically bound specifically to the lipids. The lipids may also be anionic or cationic lipids, as desired, so that they themselves may be able to bind other compounds such as pharmaceuticals, genetic material, or other therapeutics. Examples of suitable lipid clades and specific suitable lipids include: phosphatidylcholines, such as diolepholphosphatidylcholine, dimyristoylphosphatidyl-dichololine, dipalmitoyl-phe- phipidylcholine (DDPPC), and distearoyl-phosphatidylcholine; phosphatidylethanolamine, talee as dipalmitoylphophthaldylethanolamine (DPPE), dioleoylphosphatidylethanolamine and N-succinyl-dioleoylphosphatidylethanolamine; phosphatidylserines; phosphatidylglycerols; eefingolipidoe, glycolipids, such as ganglioside GM1; glycolipids; sulfatides; glycosphingolipids; phosphatidic acid, talee dipalmatoylphosphatidic acid (DPPA) palmitic fatty acids, - stearic fatty acids; arachidonic fatty acids; lauric acid and fatty acids; fatty acids myrhetic; fatty acids lauroleicoe; fatty acids fisetéricoe, - fatty acids miristoleicoe, - palmitoleic fatty acids; petroselinic fatty acids; oleic fatty acids; isoláuric fatty acids; isomiristic fatty acids; Acid isopalmíticos graeos; isoeetearyl fatty acids, - coleeterol and cholesterol derivatives, such as cholesterol hemisuccinate, cholesterol sulfate, and cholesteryl- (4'-trimethylammonio) -butanoate; esters of polyoxyethylene fatty acid; alcohols of polyoxyethylene fatty acids; ethers of polyoxyethylene fatty acid alcohol; esters of polyoxyethylated sorbitan fatty acids; polyethylene glycol glycol oxalate; glycerol ricinoleate glycol polyethylene esterolee de eoya ethoxylated; ethoxylated castor oil polymers of polyoxyethylene-polyoxypropylene fatty acid stearates of polyoxyethylene fatty acids; 12- (((71-diethylaminocoumarin-3-yl) -carbonyl) -methylamino) octadecanoic acid; N- [12 - (((7'-diethylamino-coumarin-yl) -carbonyl) -methylamino) octadecanoyl] -2-amino-palmitic acid; 1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol; 1,3-dipalmitoyl-2-euccinyl-glycerol; and l-hexadecyl-2-palmitoyl-glycerophenoethanolamine and palmitoylhomociethein; lauryltrimethylammonium bromide (lauryl- = dodecyl-); cetyltrimethylammonium bromide (cetril- = hexadecyl-); Miriethyltrimethylammonium bromide (myristyl- = tetradecyl-); alkyldimethylbenzylammonium chlorides, as in those where the alkyl is 12, 14 or 16 carbon atoms; benzyldimethyl-decylammonium bromide; benzyldimethyldodecylammonium chloride, benzyldimethylhexadecylammonium bromide; benzyldimethylhexadecylammonium chloride; benzyldimethyltetradecylammonium bromide; benzyldimethyltetradecylammonium chloride; Cetyl dimethyl ethyl ammonium bromide; cetildi ethylethylammonium chloride; cetyl pyridinium bromide; cetylpyridinium chloride; N- [1-2,3-dioleoyloxy) -propyl] -N, N, N-trimethylammonium chloride (DOTMA); 1,2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP); and 1,2-dioleoyl-e- (4'-trimethylammonium) butanoyl-en-glycerol (DOTB). As will be apparent to those skilled in the art, once armed with the present descriptions, the above lipid bed is only exemplary, and other useful lipids, fatty acids and derivatives and combinations thereof, can be employed, and these are additional compounds. it is intended to be within the scope of the term lipid, as used herein. As experienced technicians, they will recognize that lipids and / or combinations thereof can, upon shaking the container, form liposomes (ie, lipid spheres having an internal void) that trap gas from the gas phase in their internal void. The liposomes may be comprised of a single lipid layer (a lipid monolayer), two lipid layers (one lipid bilayer) or more than two lipid layers (one multilayer lipid). In general terms, it is preferred that the lipids remain in the gel state, that is, below the traneition temperature (Tm) of the lipid material, particularly during agitation. The temperature of trance from the gel to the crystalline liquid state phase are well known. Those temperatures can also be easily calculated using well-known techniques. Table 1, followed by Derek Marsh, "CRC Handbook of Lipid Bilayers", page 139 CRC Preee, Boca Raton, Florida (1990), for example, the main chain fae transition temperatures for a variety of lipids of representative saturated phosphocholine.

TABLE 1 Diacyl-cn glycero- (3) -saturated phosphocholines: Main chain fusion transitions.

In a preferred embodiment of the invention, the aqueous lipid phase further comprises a polymer, preferably an amphipathic polymer, and preferably one that is directly linked (ie chemically bound) to the lipid. Preferably, the amphipathic polymer is polyethylene glycol or a derivative thereof. The most preferred combination is the lipid dipalmitoylphosphatidylethanolamine (DPPE) bound to polyethylene glycol (PEG), especially polyethylene glycol of an average molecular weight of about 5000 (DPPE-PEG5000). The polyethylene glycol or other polymer can be attached to the DPPE or other lipid by covalent bonding, such as via an amide, carbamate or amine linkage. Alternatively, ether, ether, thioeter, thioamide or disulfide (thioester) linkages can be used with the polyethylene glycol or other polymer to bind the polymer to, for example, coleeterol or other phospholipids. A particularly preferred combination of lipids is DPPC, DPPE-PEG5000 and DPPA, especially in a proportion of about 82 percent: 8 percent: 10 percent (mole percent), DPPC: DPPE-PEG5000: DPPA. Other coating agents that can be used alternatively or in addition, in the aqueous suspension fae include polymers such as proteins, natural and semi-natural carbohydrates and synthetic polymers. A variety of different proteins can be used in the invention to produce the gas filled vesicles. These proteins include albumin of natural (human and animal) and recombinant origins, fibrin, collagen, antibodies and elastin. Natural polysaccharides include starch, cellulose, alginic acid, pectin, dextran, heparin and hyaluronic acid. Semi-natural polysaccharides include methyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose and hydroxyethyl starch. Synthetic polymers include polyvinylpyrrolidone, copolymers of ethylene and propylene glycol (for example Pluronic F-68 and the other Pluronics), polyethylene glycol, polyvinyl alcohol, polylactic acid, lactic and glycolic acid copolymers, polymethacrylate and double ester polymers. Inorganic media such as hydroxyapatite and calcium phosphate can also be used in this invention. In all cases, the bubble coating agents are suspended in the aqueous phase in a container with an upper space of the previously selected gas and then agitated. This results in the formation of the coated, coated vesicles. As one skilled in the art can recognize, once armed with the invention's deciphering, a wide variety of different stabilizing agents and agents can be used to make vesicles according to the principles of the invention. In an experiment with human serum albumin, BRL-Life Technologies, Gaithersburg, Maryland, a 10 milliliter flask containing an albumin solution and a perfluoropropane gas top space (volume of liquid - 6 milliliters, 5 milligrams per milliliter of albumin solution) was stirred for 2 minutes at 2800 rpm with an ig-L-Bug ™ to produce albumin-coated perfluoropropane vesicles having an average diameter of 5 microns, with a concentration of 50 million particles per milliliter. In addition, the use of the invention is compatible with a variety of solvents and / or viscosity agents. The phrase suspending agent, as used herein, denotes a compound that helps to provide the contrast medium with relative uniformity or homogeneity. Numerous of these agents are available including xanthan gum, acacia, agar, alginic acid, aluminum monoeterate, baeorin karaya, gum arabic, unpurified bentonite, purified bentonite, magma bentonite, carbomer 934P, calcium carboxymethyl cellulose, carboxymethyl cellulose sodium, sodium carboxymethyl cellulose 12, carragahen, cellulose (microcrystalline), dextran, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxymethyl cellulose, magnesium aluminum silicate, methyl cellulose, pectin, caffeine, gelatin, polyethylene oxide, polyvinyl alcohol, povidone, propylene glycol , alginate, eylium dioxide, colloidal silicon dioxide, sodium alginate and other alginates as tragacanth. As those skilled in the art would recognize, broad ranges of suspending agents can be used in the contrast medium of the invention as needed or desired.

The concentrations of these agents will vary depending on the stabilizing means of the bubble that are selected and the agitation parameters can also vary depending on the biological compatibility, low toxicity, availability as pure materials and pharmaceutical grade are the coating agent for lae gas-filled vesicles of the present invention. To prepare the aqueous fae, the lipids, or other coating agents, may be combined with water (preferably distilled water), normal saline (physiological), phosphate buffered saline, or other water-based solution, as will be apparent for loe experts in the art. As one skilled in the art would recognize, once armed with the substance of the present disclosure, various additives may be employed during the aqueous phase of the invention to stabilize this phase, or to stabilize the gallbladder vesicles in the agitation. If necessary, these additives can be added to the aqueous suspension phase before stirring, or they can be added to the composition after the stirring and the resulting preparation of the gas filled vesicles. The use of these additives will, depending on the particular application intended for the vesicles filled with re-gassing gas, as will be readily apparent to those skilled in the art. Numerous stabilizing agents that can be used in the present invention are available, including xanthan gum, acacia, agar, agarose, alginic acid, alginate, sodium alginate, carragahen, dextran, dextrin, gelatin, guar gum, tragacanth, locust bean, basorin , karaya, gum arabic, pectin, casein, bentonite, unpurified bentonite, purified bentonite, magma bentonite, colloidal, cellulose, cellulose (microcrystalline), methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, sodium carboxymethyl cellulose 12, as well as other natural or modified natural celluloses, polysorbate, carbomer 934P, magnesium aluminum silicate, aluminum monostearate, polyethylene oxide, polyvinyl alcohol, povidone , polyethylene glycol. propylene glycol, polyvinylpyrrolidone, silica dioxide colloidal silica. Also, compounds such as perfluorooctyl bromide (PFOB), perfluorooctyl iodide, perfluorotripropylamine, and perfluorotributylamine can be used in the lipid phase as stabilizing agents. Perfluorocarbons with more than 5 carbon atoms will generally be liquid at body temperature, and also perfluorocarbons are very preferred as stabilizing agents. Suitable perfluorocarbons include perfluorohexane, perfluoroheptane, perfluorooctane, perfluorodecalin, and perfluorododecalin. In addition, perfluorinated lipids or partially fluorinated lipids can also be used to aid stabilization. As will be apparent to those skilled in the art, a wide variety of perfluorinated and partially fluorinated analogs of the lipid described in the present invention can be used. Due to their relative hydrophobic nature with respect to hydrocarbon lipids, these perfluorinated or partially fluorinated lipids can still provide an advantage in terms of stability. Examples of perfluorinated or partially fluorinated lipids are F ^ Cu phosphatidylcholine (PC) and F8C5PC. Eeoe analogous, for example, in Santaella and collaborator, Federation of European Biochemical Societies (FEBS). Vol. 336, No. 3, pp. 418-484 (1993), whose descriptions are incorporated herein by reference in their entirety. A wide variety of biologically compatible oils can also be used to help stabilize, such as peanut oil, barley oil, olive oil, safflower oil, corn oil, almond oil, seed oil cotton, Persian oil, sesame oil, soybean oil, mineral oil, light mineral oil, ethyl oleate, myristyl alcohol, isopropyl myristate, isopropyl palmitate, octidodecanol, propylene glycol, glycerol, squalene, or any other known oil as ingestible. These may also include lecithin, sphingomyelin, cholesterol, cholesterol sulfate, and triglycerides. Stabilization can also be effected by the addition of a wide variety of viecosity modifiers (ie, viscosity modifying agents), which can act as a stabilizing agent in accordance with the present invention. This class of compounds includes, but in no way is it reetringed to: 1) carbohydrate and its phosphorylated and sulphonated derivatives; 2) polyethers with molecular weight ranges between 400 and 8000; 3) di- and trihydroxy alkanes and sue polymere in the molecular weight range between 800 and 8000. Liposomes can also be used together with emulsifiers and / or solubilizing agents which may consist of, but are in no way limited to, acacia, cholesterol , diethanolamine, glyceryl monostearate, lanolin alcohols, lecithin, mono- and diglycerides, monoethanolamine, oleic acid, oleyl alcohol, poloxamer, polyoxyethylene etherate 50, polyoxyl 35 castor oil, polyoxyl 10 oleyl ether, etherate 40 polyoxyl, polyeorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycol diacetate, propylene glycol monostearate, eodium lauryl sulfate, sodium stearate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, eorbitan monostearate, stearic acid, triolamine, emulsifying wax, Pluronic F61, Pluronic F64 and Pluronic F68. Other agents that may be added include tale tonicity agents such as polyalcohols such as glycerol, propylene glycol, polyvinyl alcohol, polyethylene glycol, glucose, mannitol, sorbitol, sodium chloride and the like. If desired, antibacterial and / or preservative agents may be included in the formulation. These agents include sodium benzoate, all quaternary ammonium salts, sodium azide, methyl paraben, propyl paraben, sorbic acid, potassium sorbate, sodium sorbate, aecorbil palmitate, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, dehydroacetic acid, ethylenediamine acid tetraacetic acid (EDTA), monothioglycerol, potassium benzoate, potassium metabieulfite, potassium sorbate, eodium bisulfite, sulfur dioxide, and organic mercury salts. If desired, an osmolarity agent may be used to control the oil. The osmotically active materials include the fieiologically compatible compounds such as sugar monosaccharides, disaccharide sugars, sugar alcohols, amino acids, and various synthetic compounds. Suitable sugar sugar monoeaccharides or sugar alcohols include, for example, erythrose, threose, riboea, arabinoea, xylose, lyxose, alose, alose, glucoea, manoea, idoea, galactose, taloea, trehalose, ribulose, fructose, sorbitol, mannitol, and sedoheptulose, the preferred monosaccharides being fructose, mannose, xyloea, arabinoea, mannitol and eorbitol. Suitable disaccharide sugars include, for example, lactose, sucrose, maltose and cellobiose. Suitable amino acids include, for example, glycine, serine, threonine, cietein, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, Usin, arginine and histidine. Synthetic compounds include, for example, glycerol, propylene glycol, polypropylene glycol, ethylene glycol, polyethylene glycol, and polyvinyl pyrrolidone. Various other osmotically active suitable materials are well known to those skilled in the art, and are intended to be within the scope of the term osmotically active agent as used herein. A variety of polymers may also be added, such as those mentioned above for a variety of different purposes and uses. As those skilled in the art would recognize, a wide range of additives may be employed in the aqueous suspension phase of the invention, such as the suspeneion agents described above, as needed or desired. depending on the particular end use. These additives can generally comprise from 0.01 volume percent to about 95 volume percent of the contrast agent resulting from the formulation, although larger or smaller amounts may be employed. As a general guide, a suspending agent is typically present in an amount of at least about 0.5 volume percent, more preferably at least about 1 volume percent, even more preferably at least 10 percent by volume. Generally the suspending agent is typically present in an amount of less than about 50 volume percent, more preferably less than about 40 volume percent, still more preferably less than about 30 volume percent. A typical amount of suspending agent could be about 20 volume percent, for example. Also, typically, to achieve generally preferred ranges of osmolarity, less than about 25 grams / liter, more preferably less than about 20 grams / liter, still more preferably less than about 15 grams / liter, and still more preferably less than about 10 gram / liter of the material is osmotically active, and in some cases osmotically non-active materials are used. The most preferred range of osmotically active materials is generally between about 0.002 grams / liter and about 10 grams / liter. Aetos, as well as other suitable ranges of additives will be readily apparent to those skilled in the art once in possession of the present invention. A wide variety of therapeutic and / or diagnostic agents can also be incorporated into the aqueous phase by simply adding the desired therapeutic or diagnostic agents to that phase. Suitable therapeutic and diagnostic agents, and suitable amounts thereof, will be readily apparent to those skilled in the art once armed with the present disclosure. These agents can be incorporated into or on the lipid membrane, or encapsulated in the resulting liposomes. To further increase the magnetic effect of the resulting gas-filled vesicles for magnetic resonance imaging (MRI), for example, one or more MRI contrast enhancing agents may be added, such as paramagnetic or superparamagnetic contrast enhancing agents. The useful MRI contrast enhancing agents include paramagnetic ions such as metals in transition, including iron (Fe + 3), copper (Cu + 2), and manganese (Mn + 2) and lanthanide such as gadolinium (Gd + 3) and dysprosium (Dy + 3), nitroxide, iron oxides (Fe304), iron sulfuroe and paramagnetic particles such as hydroxyapatites are substituted by manganese (Mn + 2). As well as agents such as chromium (Cr "1" 5), nickel (Ni "'"' '), cobalt (Co + 2) and europium (Eu + 2) are other examples of paramagnetic ions that can be used. Other contrast enhancing agents such as nitroxide radicals or any other atom that maintains an unpaired electron spin, with paramagnetic properties, can be used. Ideally the contrast enhancing agent is added to the aqueous suspension phase before stirring, and is designed so that after stirring, the contraete enhancing agent is incorporated in or on the surface of the replenishing gas-filled vesicles, although addition is also possible after preparation of the vesicles. The resultant vesicles filled with gae may have an improved relaxation capacity, providing a contrast agent especially effective for magnetic resonance imaging. By way of example, manganese (Mn-4-9) will itself be incorporated into the front groups of the lipid when phosphatidylcholine or phosphatidylserine is used in the aqueous lipid phase. If desired, metals can be chelated using liposolublee compounds as shown, for example in Unger et al., U.S. Patent Number: 5,312,617, the disclosure of which is hereby incorporated by reference, in its entirety. . These fat-soluble compounds are very useful, since they will easily be incorporated into the liposome membrane.

The iron oxides and other particles should generally be small, preferably less than 1 μm, more preferably less than about 200 nm, and more preferably less than about 100 nm, to achieve optimal incorporation into or on the surface of the liposome. For improved incorporation, iron oxides coated with aliphatic or lipophilic compounds can be used, since these will tend to be incorporated into the lipid envelope of the bubble surface. It is also within the scope of the present invention that the aqueous suspension phase may contain an ingredient to cause gelation, such as an ingredient that will cause gelation with polymers of lipids and metals that do not gel spontaneously, or that will increase gelation. Gelling agents such as polyvalent metal, sugar and polyalcohol cations can be used. The polyvalent metal catione as well as the gellant agents include calcium, zinc, manganese, iron and magnesium. Useful sugars include monosaccharides such as glucose, galactose, fructose, arabinose, alose and altrose, disaccharides such as maltose, sucrose, cellobiose and lactose, and polysaccharides such as starch. Preferably, the sugar is a simple sugar, ie monosaccharide or a disaccharide. The gelling agents of polyalcohols useful in the present invention include, for example, glycidol, inositol, mannitol, sorbitol, pentaerythritol, galacitol and polyvinyl alcohol. More preferably, the gelling agent employed in the present invention is sucrose and / or calcium. Particular gelatin agents that can be employed in the various formulations of the present invention will become readily apparent to one skilled in the art, once armed with the present disclosure. Combinations of lipids, for example, phosphatidic acid with calcium or magnesium salts and tamale polymers such as alginic acid, hyaluronic acid or carboxymethyl cellulose, can be used to stabilize the lipids. It is hypothesized that divalent cations form metal bridges between lipids and polymers to stabilize gas-filled liposomes within lipid / polymer systems. Similarly, suspensions containing mixtures of chitosan (or materials based on chitin), polylysine, polyethylene imine and alginic acid (or its derivatives) or hyaluronic acid can be prepared. It was discovered that the different materials within the aqueous phase can be important to control the size of the resulting gas-filled veeiclee. Table 2 shows the sizes of the liposomes produced by shaking sterile containers filled with an aqueous phase and a space in the euperior part of nitrogen. In all the caeoe, the size of the liposome was measured by a Particle Sizing System Model 770 particle size light meter (Particle Sizing Systems, Santa Barbara, CA). As the information reveals, the proportion of lipids in the aqueous phase affects the size distribution of the resulting gas-filled liposomes. Specifically, Table 2 below shows the effect of the lipid composition on the average size of the liposome.

TABLE 2 Effect of Lipid Composition on Average Liposome Size * The proportions of dipalmitoylphosphatidylcholine: dipalmitotoylphosphatidic acid: dipalmitoylphosphatidyl-ethanolamine-polyethylene glycol 5000, in molar percentage.

Table 3 demonstrates the dependence of the concentration of a defined lipid composition mixture on the average size of the liposome. As shown in Table 3, variations in the total lipid concentrations are also important to affect the size of the liposome after agitation. In these experiments the proportion of the three different lipid components remained constant and the lipid concentration varied between 0.5 and 5.0 mg ml "1 in the aqueous phase, the gas used was nitrogen, the optimal size of the vesicles for ultrasonic diagnosis with a perfluorobutane top space, occurred when the concentration of lipids in the aqueous phase was 1.0 mg ml 1 TABLE 3 Effect of Lipid Concentration on Average Liposome Size * The concentration of lipids for all samples was based on a molar percentage ratio of dipal itoyl-phosphatidylcholine: dipalmitoylphosphatidic acid: dipalmitoyl-phosphatidylethanolamine-polyethylene glycol 5000 of 82: 10: 8. The gas used was nitrogen. The size of the vesicles may also depend on the concentration of the stabilization media, for example, lipids. For example, it has been found that a lipid concentration of 1.0 mg ml "1 produces gas filled liposomes of approximately the same diameter when nitrogen is used, as the concentration of 5.0 mg ml" 1 of lipids with perfluorobutane. However, it has been found that the higher concentration can result in a slightly larger distribution to larger gas filled liposomes. This phenomenon tends to reflect the increased stability of the liposomes at a higher concentration of lipids. Therefore it is believed that the higher concentration of lipids contributes to stability by acting as a stabilizing agent in the aqueous phase or, the higher concentration of lipids provides more sheets around the gas making it more stable, and thus allowing a persistent greater proportion of the larger liposomes. It is also believed that the euphemial hold on the gae-filled veeicle interface and the aqueous medium is an additional factor that determines the final size of the gas-filled vesicle, when taken into account along with the other variable.

II. THE COMPOSITION OF THE GASEOUS PHASE A wide variety of different gases may be employed in the gas phase of the present invention. Preferably the gases are substantially insoluble in the aqueous suspension phase. By customarily insoluble, it is understood that the gas maintains a solubility in water at 20 ° C and 1 atmosphere of pressure equal to, or less than, about 18 milliliters of gas per kilogram of water, as such, substantially insoluble gases, they have a solubility that is less than the solubility of nitrogen gas. Preferably, the solubility is equal to or less than about 15 milliliters of gas per kilogram of water, more preferably equal to, or less than about 10 milliliters per kilogram of water, at 20 ° C and 1 atmosphere of pressure. In a preferably gas class, the solubility is between about 0.001 and about 18 milliliters of gas per kilogram of water, or between about 0.01 and about 15 milliliters of gas per kilogram of water, or between about 0.1 and about 10 milliliters of gas per kilogram. kilogram of water, or between approximately 1 and approximately 8 milliliters of gas per kilogram of water, or between approximately 2 and 6 milliliters of water per kilogram of water, at the temperature and pressure mentioned above. Loe gaees of perfluorocarbon and fluorinated gas sulfur hexafluoride are, for example; less soluble of 10 milliliters of gas per kilogram of water, at 20 ° C and 1 atmosphere of pressure, and therefore are preferred. Gaees that are not substantially insoluble, as defined herein, are referred to as soluble gases. Other suitable non-soluble or soluble gases include, but are not limited to, hexafluoroacetone, isopropylacetylene, tetrafluoroallene, boron trifluoride, 1,2-butadiene, 1,3-butadiene, 1,2,3-trichlorobutadiene, 2-fluorocarbon. 1,3-butadiene, 2-methyl-1,3-butadiene, hexafluoro-1,3-butadiene, butadiene, 1-fluorobutane, 2-methylbutane, decafluorobutane (perfluorobutane), decafluoroisobutane (perfluoroisobutane), 1-butene, 2-butene, 2-methyl-1-butene, 3-methyl-1-butene, perfluoro-1-butene, perfluoro-1-butene, perfluoro-2-butene, 4-phenyl-3 -butene-2-one, 2-methyl-l-butane-3-yne, butylnitrate, 1-butyne, 2-butyne, 2-chloro-l, 1, 1,4,4,4-hexafluoro-butyne, 3 -methyl-l-butyne, perfluoro-2-butyne, 2-bromo-butyraldehyde, carbonyl eulide, crotononitrile, cyclobutane, methylcyclobutane, octafluorocyclobutane (perfluorocyclobutane), perfluoroobutane, 3-chlorocyclo pentene, cyclopropane, 1,2-dimethylcyclopropane, 1,1-dimethylcyclopropane, ethyl cyclopropane, methylcyclopropane, diacetylene, 3-et-il-3-methyldiaziridine, , 1,1-trifluorodiazoethane, dimethylamine, hexafluorodimethylamine, dimethylethylamine, bie- (dimethyl foefin) amine, 2,3-dimethyl-2-norbornane, perfluoro-dimethylamine, dimethyloxonium chloride, 1,3-dioxolan-2-one, , 1, 1, 1, 2-tetrafluoroethane, 1,1,1-trifluoroethane, 1,1, 2, 2-tetrafluoroethane, 1,2-trichloro-1,2,2-trifluoroethane, 1,1-dichloroethane , 1,1-dichloro-1,2,2,2-tetrafluoroethane, 1,2-difluoroethane, 1-chloro-1,1,2,2,2-pentafluoroethane, 2-chloro-l, 1-pentafluoroethane, -chloro-l, l-difluoroethane, 1-chloro-l, 1-difluoroethane, 1-chloro-l, 1,2,2-tetrafluoro-ethane, 2-chloro-l, 1-difluoroethane, chloroethane, chloro-pentafluoroethane , dichloro-difluoroethane, fluoroethane, nitropentafluoroethane, nitrosopentafluoroethane, p erfluoro-ethane, perfluoroethylamine, ethyl vinyl ether, 1,1-dichloro-ethylene, 1,1-dichloro-l, 2-difluoro-ethylene, 1,2-difluoroethylene, methane, methanesulfonyl-chloride-trifluoro, methanesulfonyl -fluoride-trifluoro, methane (pentafluorothio) -trifluoro, methano-bromo-difluoro-nitroso, methano-bromo-fluoro, methano-bromo-chloro-fluoro, methano-bromo-trifluoro, methano-chloro-difluoro-nitro, methano- chloro-fluoro-dinitro, methane-chloro-fluoro, methane-chloro-trifluoro, methane-chloro-difluoro, methane-dibromo-difluoro, methane-dichloro-difluoro, methane-dichloro-fluoro, methane-difluoro, methane-difluoro-iodo , methane disilane, methane-fluoro, methane-iodomethane-iodine-trifluoride, methane-nitro-trifluoro, methane nitroeo-trifluoro, methane-tetrafluoro, methane-trichloro-fluoro, methane-trifluoro, methanesulfenyl-chloride-trifluoro, 2-methylbutane , methyl ether, methyl-ieopropyl ether, methyl lactate, methyl nitrite, methyl eulphide, methylvinyl ether, neopentane, nitrogen (N2), nitroeoxide, 2-hydroxytryl ester l-2, 3-nonadecantri-carboxylic acid, l-nonen-3-ina, oxygen (O2). oxygen 17 (0 ^), 1,4-pentadiene, n-pentane, dodecafluoropentane (perfluoro-pentane), tetradecafluorohexane (perfluorohexane), perfluoro-isopentane, perfluoroneopentane, 2-pentanon-4-amin-4-methyl, 1-pentene , 2-pentene. { cis } , 2-pentene. { trans} , l-pentene-3-bromo, 1-pentene-perfluoro, tetrachlorophthalic acid, piperidin-2, 3,6-trimethyl, propane, propan-1, 1, 2, 2, 3-hexafluoro, propane-1, 2-epoxy, propan-2, 2-difluoro, propan-2-chloro, propan-heptafluoro-1-nitroso, perfluoropropane, propene, propyl-1, 1, 2, 3, 3-hexafluoro-2, 3- dichloro, propylene-1-chloro, propylene-chloro-. { trans} , propylene-2-chloro, propylene-3-fluoro, propylene-perfluoro, propino, propin-3, 3, 3-trifluoro, styrene-3-fluoro, sulfur hexafluoride, sulfur (di) -deca-fluoro (S2F10) / toluene-2-4-diamino, trifluoroacetonitrile, trifluoromethyl peroxide, trifluoromethyl sulfide, tungsten hexafluoride, vinylacetylene, vinyl ether, neon, helium, krypton, xenon, (especially rubidium enriched with hyperpolarized xenon gas), carbon dioxide, helium and air. Fluorinated gases (ie, a gas containing one or more fluorine molecules, such as sulfur hexafluoride), fluorocarbon gases (ie, a fluorinated gas which is a carbide or fluorinated gas), and gas perfluorocarbons are preferred. (that is, a fluorocarbon gas that is completely fluorinated, such as perfluoropropane and perfluorobutane).

Although virtually any gas can theoretically be employed, in the gaseous phase of the present invention, a particular gas must be chosen to optimize the desired properties of the resulting contrast medium and to match the particular diagnostic application. It has been seen, for example, that certain gases become more stable to gas-filled vesicles with the agitation that others gas, and eeee gaeee are preferred. It has also been found that certain gases provide better imaging results in the diagnostic image such as ultrasound or magnetic resonance imaging. As an example of increasing the stability of gas filled vegetables, it has been seen that gasses: carbon dioxide < oxygen < air < nitrogen < "Neon < helium < perfluorocarbon. For example, as other reasons, fluorinated gases, particularly gas perfluorocarburoe, prefer. Also, although in some cases the soluble gases will function properly as the gas phase in the present invention, inequatably the gasses tend to be more stable than gases with higher solubility, particularly with the creation of the contrast agent in the agitation. It will also be easier to maintain a gaseous phase with those gases that are substantially insoluble from the aqueous suspension phase before stirring, according to the present invention. In this case, substantially insoluble gases, as defined above, are preferred. The quality of the ultrasound images and the duration of these images also correlate with the solubility of the gas in the aqueous medium. The decrease in gas solubility, in general, offers a better resolved image of longer duration in ultrasound. Additionally, it has been generally objected that the size of the vesicles filled with gae produced by agitation correlates with the solubility of the gas in the aqueous medium, resulting in loe gaee of greater solubility, give veeiclee filled with larger gas. It is also believed that the size of the vesicles can be influenced by the interaction of the gas with the inner wall of the vesicles. Specifically, it is believed that the interaction at the interface affects the tension and, consequently, the outward force of the inner gas on the inner wall of the vesicle. A decrease in tension allows smaller vesicles by decreasing the force exerted by the inner gas, thus allowing the force exerted on the outside of the veeicle by the aqueous medium, to contract the veeicle filled with gas. The solubility of gases in aqueous solvents can be estimated by using Henry's Law, since it is generally applicable for pressures up to approximately 1 atmosphere of pressure and for gases that are slightly soluble (Daniels, F. and Alberty, RA , Phyeical Chemietry, third edition, Wiley &Sons, Inc., New York, 1966). As an example, oxygen has a solubility of 31.6 milliliters per kilogram of water at 25 ° C, atmospheric air has a solubility of 21.36 milliliters in 1 kilogram of water at 25 ° C, nitrogen maintains a solubility of approximately 18.8 ml kg " 1 at 25 ° C. Sulfur hexafluoride, on the other hand, has a solubility of about 5.4 ml kg at 25 ° C. In euma, fluorinated gases, fluorocarbon gases, and perfluorocarbon gases are preferred for reasons of stability, insolubility, and the size of the resulting vesicles Particularly preferred are the fluorinated gae sulfur hexafluoride, and the perfluorocarbon gasses: perfluoropropane, perfluorobutane, perfluorocyclobutane, perfluoromethane, perfluoroethane, and perfluoropentane, especially perfluoropropane and perfluorobutane. that the perfluorocarburoe that have menoe of five carbon atoms eon gaeee at room temperature, perfluoropentane, for example, is a liquid haeta approximately 27 ° C. Above this temperature, will occupy the upper space of the container. It has been shown that pefluoropentane can also be used to fill the top space (that is, the space in the flask above the lipid suspension phase) even at room temperature, however. Selecting a defined value of liquid perfluoropentane calculated to fill the space of the upper part and ng the liquid to the container at low temperature, for example, -20 ° C, and then evacuating the container (effectively removing the air from the euperior space) and then By erecting the container, the perfluoropentane will undergo a transition from the liquid phase to a vapor phase at a temperature below its boiling point at 1 atmosphere. Thus, at room temperature it will occupy some or all of the upper space with gas. As those skilled in the art will recognize, one can estimate the decrease in the liquid phase at the vaporization temperature to the vapor phase with a common "three rule" state, Specifically, for each pressure decrease by half, the Boiling temperature will drop approximately 10 ° C. Alternatively, the decrease in temperature can be calculated as a function of the decreased pressure using relationships based on the law of ideal gae on Boyle's law. Another method to fill the space of the upper part with perfluoropentane is first to evacuate the space of the upper part and then fill the upper space with perfluoropentane gae above 27 ° C. Deede then, this method is not limited to perfluoropentane alone, but applies to all gauges of perfluorocarbon, ae and gases in general, provided that the boiling point of the gas is known. If so, two or more different gases can be used together to fill the top space. A gassing mixture can have numerous advantages in a wide variety of applications of the resulting gas filled fillers (such as applications in ultrasound imaging, magnetic resonance imaging, etc.). It has been found that a small amount of a customarily insoluble gas can be mixed with a soluble gas to provide greater stability than would be expected from the combination. For example, a small amount of perfluorocarbon gas (generally at least about 1 mole percent, for example) can be mixed with air, nitrogen, oxygen, carbon dioxide or other gas, more soluble. The gas-filled vesicle contraceptive agent produced after agitation may then be more stable than air, nitrogen, oxygen, carbon dioxide or other more soluble gas. tionally, the use of a mixture of gases can be used to compensate for the increase in the size of the gas-filled vesicle, which could otherwise occur in vivo if they were veeiclee containing pure perfluorocarbon gae to be injected in vivo. . It has been found that some perfluorocarbon gases may tend to absorb or imbibe other gases such as oxygen. Thus, if the perfluorocarbon gas is injected intravenously, it can pick up oxygen or other soluble gases dissolved in the circulating blood. The resulting vesicles can then grow in vivo as a result of this collection. Armed with a knowledge of this phenomenon, one can then premix the perfluorocarbon gas with a soluble gas, such as air, nitrogen, oxygen, carbon dioxide, thereby saturating the perfluorocarbon with its adsorptive or embedding properties. Consequently, this would retard or even eliminate the expansion potential of the gas-filled vesicles in the eanguineous stream. This is significant in light of the fact that a vesicle could grow to a size greater than 10 μM, potentially dangerous because of embolic events that can occur if it is administered in the blood stream. Filling the space of the upper part with more soluble gases than the perfluorocarbon gas, along with the perfluorocarbon gae, the gas filled vesicles will generally not suffer this increase in size after the injection in vivo. Thus, as a result of the present invention, the problem of embolic events as a result of the expansion of the vesicles can be avoided by producing vesicles where that expansion is sufficiently eliminated or delayed. Thus, according to the present invention, if desired, a substantially insoluble gas can be combined with a soluble gae to efficiently produce highly efficient and stable gae-filled vesicles. Multiple samples of lipid solutions (1 mg per mL); proportions of 82: 10: 8 molar percent of DPPC: DPPA: DPPE-PEG-5000) in proportions of 8: 1: 1 in normal saline solution: glycerol, propylene glycol in 2-milliliter flask (actual size 3.7 milliliter ) Wheaton Induetries (Millville, NJ) were placed on a modified Edwarde Model S04 freeze dryer, with a capacity of four cubic feet and subject to reduced pressure. The outer containers of the vials, which make up 60 percent of the total volume, were instilled with 80 percent PFP, with 20 percent air, 60 percent PFP with 40 percent air, 50 percent PFP with 50 percent air, 20 percent PFP with 80 percent air, or 100 percent air. The percentages of gas in the upper spaces of the different samples were confirmed by gas chromatography on a Hewlett Packard Gas Chromatograph Model 1050L interfaced with Hewlett Packard Chem ™ software. The detection mode was detection of flame ionization. The samples were then shaken at 3,300 rpm for 60 seconds using a standard Wig-L-Bug ™, model 3110B and the sizes and counts of the vesicles were determined by optical particle size measurement. An optical particle meter (Particle Sizing Syeteme, Santa Barbara, CA) was used to analyze the size and total counts of the gas-filled vesicles. A sample volume of 5 microliter was used for each analysis, with four samples used for each determination. The results are shown in Table 4. As shown in Table 4, even though only 20 percent of the gae was PFP (a gae eustancialmente insoluble) and 80 percent of the gas was air (a mixture of soluble gases) , there were 100 times more veeiclee than when air was used alone (0 percent PFP). Even more, when air was released alone (0 percent PFP), vegetables were much less stable and a large fraction was above 10 microns. Vesicles with 20 percent PFP and 80 percent air, however, appeared to be as stable as vesicles with 80 percent PFP and 20 percent air, as well as other concentrationee samples of intermediate PFPs, and 20 percent PFP with 80 percent air produced as many gas-filled vesicles as 80 percent PFP with 20 percent air.

TABLE 4 Effect of perfluoropropane percentage In Table 4, Des. its T. = standard deviation, and CV = Coefficient of variance. Also in Table 4, E + debita and b exponent to a certain power, for example, 4.45E + 05 = 5.45 x? O5- In summary, it has been found that only a small amount of a relatively insoluble gas (such as PFP) is needed to stabilize the vesicle, with a soluble gae making up most of the gas. Although the effective solubility of the combination of two or more gases, as calculated by the formula below: (solubility gas A) x (molar percent gas A) + (solubility gas B) x (molar percent eas E) 100 it may be only slightly different than the solubility of the soluble gas, there is still a high count of gae-filled vesicles and gas-filled vesicleability with only a small amount of insoluble gas added inside. While not intending to adhere to any theory of operation, it is believed that substantially insoluble gas is important for a membrane stabilizing effect. Undoubtedly, it is believed that the essentially insoluble gae (as PFP) acts as a barrier against the lipid membrane, possibly effectively forming a layer on the inner surface of the membrane, which retards the egregion of the soluble gas (such as air, nitrogen, etc) . This discovery is both surprising and useful, since it permits the use of only a small amount of the naturally unsolvable gas (for example, a perfluorocarbon or other fluorinated gas) and mainly a biologically more compatible gas (potentially toxic menoe) such as air or nitrogen, to comprise most of the volume of vesicles. The amount of substantially insoluble gasses and the soluble gasses in any mixture can vary widely, as will be recognized by one skilled in the art. However, typically, at least about 0.01 percent, even more preferably, at least about 1 percent, and more preferably, when it is less than about 10 percent. The convenient ranges of variation of the naturally insoluble gas vary, depending on several factors such as the soluble gas to be used additionally, the type of lipid, the particular application, and so on. Exemplary ranges include between about 0.01 percent to about 99 percent of substantially insoluble gas, preferably between about 1 percent and about 95 percent, more preferably between about 10 percent and about 90 percent, and most preferably between about 30 percent and approximately 85 percent. For uses beyond the formation of images for ultrasound diagnosis, such as the uses in the formation of images for magnetic resonance imaging (MRI), loe gaeee paramagnetic as strongly paramagnetic oxygen 17 gae (1702), neon, xenon, helium, argon (especially hyperpolarized xenon gas enriched with rubidium), or oxygen (which is still, though less strong, paramagnetic), for example, are preferably used to fill the upper space although other gases can be used. More preferably, gas 02, neon, hyperpolarized xenon gas enriched with rubidium, or oxygen gas is combined with a customarily insoluble gas such as, for example, a perfluorocarbon gas. The paramagnetic gaees are well known in the art and the convenient paramagnetic gasses will be readily apparent to those skilled in the art. The preferred gae mae for magnetic imaging diagnostic imaging application MRI, either alone or in combination with another gas, is 02. Using a combination of gases, 02 or another paramagnetic gas provides the optimal contrast and perfluorocarbon stabilizes gas 1702 within the trapped gas after agitation. Without the addition of perfluorocarbon gas, gases such as 1702 are generally much less effective, since due to their solubility they diffuse out of the lipid trap after intravenous injection. In addition, gas 1702 is quite expensive. The combination of perfluorocarbon gas with gas 1702 greatly increases the efficiency of the product and decreases the coefficient through the most efficient cost of gaso 02. In a similar way, another gas with paramagnetic properties, such as neon, can be mixed with the gases of the gas. perfluorocarbon. As Table 5 below reveals, a wide variety of different gasses can be used in magnetic resonance imaging application. In Table 5, the R2 (l / T2 / mmol / L.eeg "1) is shown for different gaee in the gas filled vesicles, as shown in Table 5, there are notable differences in the relaxation capacity of the different lae. gas-filled vesicles, the highest R2 relaxation values indicate that the veeicles are more effective as magnetic resonance imaging agents.From the gases shown, the air has the highest R2 value.The air is believed to be the most Due to the paramagnetic effect of oxygen in the air, however, pure oxygen is somewhat less effective, probably due to the greater oxygen solubility and the oxygen balance in the aqueous medium surrounding the veeiclee. the air is about 80 percent nitrogen) helps to stabilize the oxygen inside the vesicles.Nitrogen has much less solubility in water than air.As noted above, the PFP or other perfluorocarbon gaees they can be mixed with a magnetically more active gas such as air, oxygen, 1702 or hyperpolarized xenon enriched with rubidium. By doing so, they can prepare stable, highly active, gas-filled vesicles magnetically.

TABLE 5 Size distribution and relaxation capacity The euperior space of the container can be filled with the gas at ambient pressure, reduced or increased, as desired. In the container of the invention, the gas phase is usually separated from the aqueous suspension phase. By substantially separate it is meant that less than about 50 percent of the gas is combined with the aqueous suspension phase, before agitation. Preferably, it is less than about 40 percent, more preferably less than about 30 percent, still more preferably less than about 20 percent, and preferably less than about 10 percent of the amount combined with the suspension factor. watery The gas phase is maintained substantially apart from the aqueous suspension phase, until approximately the time of use, at that moment the container is agitated and the fae gaeeoea and the aquatic euepeneion fae combine to form an aqueous suspension of gas-filled vesicles. . In this way, ee produces an excellent contrast agent for ultrasound or magnetic resonance imaging. Moreover, since the contrast agent is prepared immediately before use, shelf life problems are minimized.

III. CONTAINER VOLUME AND HIGHER SPACE It has been found that the size of the upper space for the gas can also be used to affect the size of the gas filled veeicles. Since a larger upper space contains proportionally more gas relative to the size of the aqueous phase, the larger spaces will generally produce vesicles larger than the larger spaces of smaller size. Therefore, the upper space, expressed as a percentage of the total volume of the container, should not exceed a maximum value. Moreover, too small a space will not allow enough room for the fluid to move during agitation to efficiently form vesicles. For example, it is a discovery of this invention that when using vials of 3.7 milliliter real volume (Wheaton 300 borosilicate glass, Wheaton Industriee, Millville, NJ, referred to as a nominal size of 2 milliliters, diameter x height = 15 millimeters x 32 millimeters), the volume of the upper space containing the gae is preferably between about 10 percent and about 60 percent of the total volume of the jar. Generally the upper space containing the gas in a fraeco is between approximately 10 percent and approximately 80 percent of the total volume of that flask, although depending on the particular circumstances and the desired application, the appropriate may be more or less gas. More preferably, the headspace comprises between about 30 percent and about 70 percent of the total volume. In general, it has been found that the most preferred volume of upper space containing gas is about 60 percent of the total volume of the container.

IV. OPTIMAL VALUES FOR THE AGITATION PARAMETERS A. Form of the path of travel and amplitude of the agitation. As discussed above, in addition to the compositions of the aqueous suspension phases and gaeeoea, the specific manner in which the vessel containing these faeces is stirred will affect the size distribution of the veeiclee. The conditioning conditions of agitation can be defined with reference to four parameters - the shape of the path traveled by the container during agitation, the amplitude of the agitation movement, the frequency of agitation, and the duration of agitation. It has been found that the path traveled by the container during agitation is especially significant for the formation of vesicles of adequate size. In particular, it has been found that small vesicles can be produced in a minimum amount of time when the agitation takes the form of reciprocating motion. Other types of agitation, as in vortex, it can also produce small vesicles. However, alternative agitation greatly reduces the duration of agitation that is necessary to achieve a high concentration of small vesicles. The inventors have found that small-sized vesicles are obtained in a relatively short period of time, ie 2 minutes or less - when the agitation amplitude - specifically, the length C of the alternative path traveled by the container during the agitation - is when it drops to 0.3 centimeters. In general, the greater the amplitude of the agitation, the smaller the vesicles will be. However, as discussed below, the frequency of agitation is also an important parameter. Since practical considerations associated with agitation equipment will typically result in a drop in the frequency of agitation to undesirably low levels when the amplitude of the agitation is increased beyond a certain maximum amount, the amplitude should be kept low enough to assure that the frequency of agitation remains adequate. For the Wig-L-Bug ™ model 3110B agitation device, this maximum amplitude is approximately 2.5 centimeters. The inventors have also found that it is preferable that the reciprocating movement occurs along an arcuate path 20, as shown in Figure 4, where the amplitude of the agitation movement is denoted C, since the agitation movement at High frequency is carried out more easily in this way. In the preferred embodiment of the invention, the arcuate path 20 is defined by a radius of curvature L, which is formed by an agitator arm of length L. Preferably, the agitator arm 7 has a length L of at least 6 centimeters and rotates through an angle? at least 3 °. As discussed further below, according to the preferred embodiment of the invention, the angle of rotation of the agitator arm? is achieved by using a bushing that has an angle of displacement equal to? . Further, the length L of the stirring arm is defined as the distance from the center line of an eccentric bushing 40 on which the bearing 50 of the stirring arm 7 is mounted, as discussed further below, towards the center line of the container 9, as shown in Figure 3. The length of the longitude of the shaker arm with longer and angles of rotation? larger will increase the amplitude of the agitation and, therefore, will generally reduce the size of the vesicle. However, as discussed above, the maximum values for the length L of the agitator arm and the angle of rotation? employees should be limited to ensure that the amplitude of the agitation C does not become so long that it results in an inadequate agitation frequency.

In addition, mechanical considerations will also limit the size of the rotation angle of the agitator arm 7. For the Wig-L-Bug ™ model 3110B agitator device, the maximum length of the agitation arm and the maximum angle of rotation to be used are approximately 15 centimeters and approximately 9 °, respectively. Additionally, it is preferred that the agitation device superimposes a movement in a second direction, approximately perpendicular, on the reciprocating movement in the first direction. Preferably, the agitation amplitude in the second direction C is less than about one-tenth of the agitation amplitude in the first direction C. For the fineness of the induction, the first direction of the reciprocating movement will be referred to as the longitudinal direction and the second direction of the alternative movement will be referred to as the transvereal direction. Optimally, the duration of the movements in the longitudinal and tranevereal directions are adjusted so that the sum of the movements in the two directions results in the agitation of the container 9 in the figure pattern in 8. Based on the above, the trajectory Preferred agitation 20 described by the container 9 when attached to the end of the agitator arm 7 of the agitation device 1 of the present invention is shown in Figures 4 and 5. As shown in Figure 5, according to the present invention , the shaker arm 7 imparts movement to the container 9 in the transverse direction as it moves back and forth in the longitudinal direction so that a point in the container 9 runs through the pattern 20 of the figure in 8. The length of the figure in 8 is the amplitude in the longitudinal direction C and the width of the figure in 8 is the amplitude of the agitation in the transverse direction C. When viewed sideways, as shown in Figure 4, the path is arcuate in the longitudinal direction - specifically, an arc having a radius of curvature that is equal to the length L of the agitator arm 7. The length of the arc C is the product of the length L of the agitator arm and the angle? encompassed by the rotation of the agitator arm in the longitudinal plane, expressed in radians - that is, C = L? . Preferably, the pattern of Figure 8 is comprised of approximately two intersecting lines 21 intersecting an angle f and two sections approximately half a circle 22. As discussed further below, in the preferred embodiment of the invention, the angle f formed by the figure pattern at 8 is approximately equal to the angle of rotation of the shaker arm 7 in the longitudinal direction? . As discussed below, this is accomplished by applying sufficient force to the stirring arm 7 from a spring 46 to maintain the stirring arm essentially in a vertical orientation in the transverse plane during agitation, as shown in FIGS. 10. If the tension of the spring is adjusted to allow the stirring arm 7 to rotate through an angle in the transverse plane?, As shown in Fig. 12, then the angle f of the agitation pattern of Fig. 8 experienced by the container 9 will be greater than 0. If f equals 0, then the total distance D traveled in a circuit around the path 20 will be a function of two variables - the length of the stirring arm L and the angle 0 described by the agitator arm as it travels in the longitudinal plane. This distance D can be approximated by the equation: D = 2L [(2 sin 0/2 + II tan2 0/2) / (l + tan 0/2)] Since, preferably, length 1 is at least 6 centimeters and angle 0 is at least 3 °, distance D should preferably be at least about 0.6 centimeters. Also, given that f =? , the amplitude of the agitation in the traneversal direction C will be a function of the amplitude in the longitudinal direction c and the angle 0, and can be approximated by the equation C = (2C tan 0/2) / l + tan 0/2) Since, preferably, the amplitude of the agitation in the longitudinal direction C is at least about 0.3 centimeter and the angle 0 ee when less than 3 °, the amplitude in the transverse direction C should preferably be at less than 0.02 centimeter. The optimal values for the amplitude and shape of the agitation movement discussed above were achieved based on a series of tests, discussed later in subsection CB Frequency and Agitation Duration In addition to the shape and amplitude of the agitation movement, the frequency of agitation is also an important parameter to form vesicles of adequate size.

The frequency is quantified in terms of revolutions per minute ("rpm") experienced by the agitator arm 7 and is defined as the number of times the agitator arm, and, therefore, the container 9 attached to it, passes through completely the path of agitation in one minute. Thus, in the preferred embodiment of the invention, the agitation at a frequency of 3600 rpm means that the container 9 undergoes a stirring motion around the trajectory 20 in the figure of 8 three thousand six hundred times in one minute, or sixty times in one second.

It has been seen that the vesicles can be made using shaking frequencies in the range of 100 rpm to 10,000 rpm. However, it has been seen that there is a frequency of minimal agitation that will result in the production of vesicles of optimum size in a relatively short period of time. As discussed in section C, further on, you have seen that this minimum frequency is approximately 2800 rpm. Although, in general, increasing the frequency of agitation will reduce the size of the vesicle, the limitations of the agitation device will typically set the maximum frequency obtainable. For the Wig-L-Bug ™, the maximum frequency obtainable is approximately 3300 rpm. At frequencies in the range of 2800 to 3300 rpm, the optimum duration of agitation is at least 60 seconds. However, the optimal duration of agitation is related to frequency and may be lower than higher frequencies. Thus, for example, at 4500 rpm the optimum duration of agitation is only 50 seconds. C. Test Results The optimal value for the frequency of agitation, as well as the shape and amplitude of the agitation movement, were developed through a series of tests, as discussed below. A first series of tests was carried out to determine the effect of frequency on the size of the veeicle. Mueetrae of 1 milligram / milliliter of lipid consisting of dipalmitoylphosphatidylcholine (DPPC) (Avanti Polar Lipids, Alabaster, Ala.), Dipalmitoylfoefatidic acid (DPPA) (Avanti Polar Lipide, Alabaster, Ala), and dipalmitoylphosphatidylethanolamine covalently bound to the monomethyl ether of polyethylene glycol of molecular weight = 5000, (DDPE PEG-5000) (Avanti Polar Lipids, alabaster, Ala) in a molar ratio of 82 mole percent: 10 mole percent: 8 mole percent, respectively, were added to a diluent consisting of normal ealine, glycerol (Spectrum Chemical Co., Gardena, Calif.), and propylene glycol (Spectrum Chemical Col, Garden, Calif.), (8: 1: 1, v: v: v). The samples were then heated at 45 ° C for 10 minutes then allowed to equilibrate at room temperature (25 ° C). Lae mortars were then added to nominal vials of 2.0 milliliters of boron eilicate (VWR Scientific, Boeton, Maee) of the type shown in Figure 1 (3.7 milliliter real volume). The bottles were sealed with a butyl rubber stopper and sealed with a gas-tight fit with an aluminum wrap. The upper space in the Fraes was approximately 60 percent of the total volume of the bottles. The samples were then purged with perfluoropropane (Flura Corporation, Nashville, Tenn.) And placed in the agitation device shown in Figure 3, which is further developed in section V.

The containers were stirred for 2 minutes using the movement of figure type at 8 shown in Figures 4 and 5. The length of the agitator arm was 7.7 centimeters and the angle of load deflection 0 and, therefore, the angle of rotation of the agitator arm in the longitudinal plane, was 6 °. Using the relationships discussed above, it was determined that the amplitude of agitation in the longitudinal and transverse directions C and C was approximately 0.8 centimeters and 0.1 centimeters, respectively. Agitation frequencies of 1500 were 2800 and 3300 rpm, measured via a Pistol Grip tachometer Model 08210 of Code-Palmer (Code-Palmer, Nile, 111.). The size was determined by measuring the optical size of small particles in a particle size meter by dark light Particle Sizing System (Santa Barbara, Calif.). Table 6 shows the results of these tests and demonstrates the effect that the frequency of agitation has on the resulting average vesicle size.

TABLE 6 Effect of shaking frequency on average vesicle size As you can see, agitation at a frequency higher than 2800 rpm greatly reduces the average vesicle size obtained after 2 minutes of agitation. A second set of tests was carried out to determine the effect on the size of the vesicle of increasing the length L of the agitator arm and, therefore, the amplitude of agitation in the longitudinal and transverse directions C and C, as well as the stirring distance per cycle D. The tests were carried out in the same way as those previously discussed except that the containers were shaken for 60 seconds with L lengths of shaker arm over the range of 6.7 to 14.8 centimeters. Variations in the agitator arm length resulted in variations in the agitation frequency over the range of 2250 to 3260 rpm, with the frequency of agitation decreasing as the length of the agitator arm increased. The variation in the agitation frequency with the length L of the agitator arm and the rotation angle 0 of the agitator arm is shown in Figure 13. Thus, for example, when a length L of the agitator arm of 6.7 cm was used and an angle of rotation 0 of 6 °, the frequency of agitation was approximately 3200 rpm, while when the length of the agitator arm was increased to 13.8 centimeters, while maintaining the angle of rotation, the frequency low haeta approximately 2700 rpm. The results of this series of tests are shown in Figures 14 (a) - (c). As shown in Figure 14 (a), with an angle of rotation in the longitudinal plane 0 of 6 °, when 98 percent of the vegetables are below 10 microns as long as the length L of the agitator arm is less than 10 microns. 7.7 cm or greater - that is, when the amplitude of agitation in the longitudinal direction C is greater than 0.8 cm. Moreover, the percentage of veeicles below 10 microns reaches a height of about 99 to 99.5 percent at the lengths L of the shaker arm of 9.8 centimeters and greater - that is, when the amplitude of agitation in the longitudinal direction is of 1.0 centimeter and larger. The weighted number of mean vesicle size reaches a height of approximately 2 microns in these conditions, as shown in Figure 14 (b). Although the general effect of increasing the frequency of agitation is to reduce the size of the vesicle when all other variables are kept constant, as discussed previously and shown in Table 6, these data show that increasing the amplitude of agitation by increasing the length The size of the vesicles is reduced in the agitator arm, even though this increase is combined with a reduction in the frequency of agitation, as shown in Figure 13. As shown in Figure 14 (c), more than 400x10 vesicles were obtained by milliliter with todae lae longitudee of the agitator arm and, in fact, the operation of agitator arms in the range of approximately 10 to 12 centimeters results in the production of more than 1000 x 106 vesicles per milliliter. However, as the length of the agitator arm is increased above about 12 centimeters, the particles per milliliter begin to decrease and reach 800xl06 vesicles per milliliter to 14.8 centimeters. Although not shown in Figure 13, with an agitator arm length of 14.8 centimeters and an angle of rotation of the agitator arm of 6o, the determined frequency was only 2550 rpm. Thus, the drop in concentration of the vesicles produced with an arm length of 14.8 centimeters is thought to be due to the drop in the frequency of agitation that accompanies increases in the amplitude of agitation, as discussed previously. Therefore, these data indicate that when using a Wig-L-Bug ™ shaker die, the length of the shaker arm should preferably be less than about 15 centimeters to maximize vesicle concentration. A third test series was carried out using the same material and procedure discussed above except that the angle of deflection of the load and, therefore, the rotation angle of the stirring arm in the longitudinal direction 0 increased from 6 ° to 9 °. , thereby increasing the amplitude of agitation in the longitudinal direction. In addition, the length of the shaker arm was not longer than 11.8 centimeters. The results of these tests are shown in Figures 15 (a) - (c), together with the results of the previously discussed set of tests for comparison. As can be seen in Figure 15 (a), increasing the angle of rotation of the agitator arm 0 from 6 ° to 9 ° reduces the size of the vesicle, although it also has the effect of reducing the frequency of agitation, shown in Figure 13. Thus, with an angle of rotation of the agitator arm of 9 °, even a length of agitator arm of only 6.7 results in more than 99.5 percent of the vesicles are less than 10 microns and have a size medium of approximately 2 microns. In addition, more than lOOOxlO6 vesicle per milliliter was obtained with all the lengths of the agitator arm, as shown in Figure 15 (c). Another series of tests was carried out using the same materials and procedure discussed above except that deflection angles were used and, therefore, the angles of rotation of the stirring arm in the longitudinal direction 0, of 3 °, 5.2 °, 6 ° , 7.8 °, and 9 ° together with lengths L of the shaker arm between 6.7 centimeters and 13.8 centimeters (increasing in increments of approximately 1 centimeter). The total length D of the agitation path 20 at each point was estimated. The frequency as a function of the total path length is shown in Figure 17. The results are shown in Figures 16 (a) - (c) as a function of the length of the total path D. As can be seen , under all tested conditions - that is, a total of trajectory lengths D of 0.7 centimeter and more - plus 95 percent of the vesicles were less than 10 microns and the concentration of vesicles produced was more than 100x10"per In addition, under all conditions in which the length of the total trajectory D was 2.19 centimeters or greater, more than 98 percent of the veeicles were less than 10 microns.This suggests that the total trajectory length of the The agitation should be at least 0.7 centimeters, and more preferably, when less than 2.2 centimeters, the above shows that the small-sized vesicles can be obtained in approximately two minutes or less. or the alternative stirring is carried out so that the stirring frequency is at least 2800 rpm. In addition the movement of agitation should be carried out in two directions suetancialmente perpendicularee, and, most preferably, in a figure pattern at 8. In addition, the agitation amplitude in the main direction should be at least 0.3 centimeters, and more preferably at least 0.8 centimeters, or the total length of the agitation path should be at least 0.7 centimeters and, more preferably, at least 2.2 centimeters.V. THE APPARATUS OF THE INVENTION A. THE PREFERRED AGITATION DEVICE The preferred agitation device 1 of the present invention is shown in Figures 2 and 3. The apparatus is composed of a base 2 and a hinged safety cover 3. A start-stop button 6 and a speed control disc 5 are mounted in a housing 4 that spans the base 2. An arm 7 projects upwardly through an opening 12 in the upper portion of housing 4. Al Turn the disc 5 clockwise increasing the agitation speed, while rotating the disc counterclockwise decreases the speed of agitation. According to the present invention, a mounting fastener 8 is attached to the distal end of the arm 7 which allows the container 9, discussed further below, to be secured to the arm. The bracket 8 is attached with a variety of spring clamps 11 and 12 which hold the container 9 securely in place. Alternatively, a butterfly screw fastener could also be used to provide an even more secure connection of the container 9. As shown in Figure 3, the fastener can be oriented at an angle d with respect to the horizontal. Preferably, the angle d is in the range of -5 ° to + 5 °, and preferably is approximately 0 °. In this case, the container 9 is secured to the fastener 8 and the agitator device 1 operates to agitate the container vigorously along the path shown in FIGS. 4 and 5. Figures 6-12 show the main internal components of the container. agitator device 1 according to the present invention. As you can see, the stirring arm 7 is rotatably mounted on the arrow 42 of an electric motor 44. As best shown in Figures 6 and 9, a cylindrical sleeve 41 is formed at the proximal end of the stirring arm 7. The sleeve 41 houses a bearing 50 that supports a cylindrical eccentric bushing 40. The bushing 40, best shown in Figure 11, is fixedly attached to the arrow 42 - for example, being pressed on or formed integrally with the arrow - and rotates inside the bearing 50. As best shown in Figure 11, one end 43 of the hub 40 is eccentric with respect to the arrow 42 while the other end 45 of the hub is concentric with the arrow. Accordingly, as shown in improvement in Figure 7, the central line of the bushing 40 forms an acute angle 0/2 with the center line of the arrow 42. The angle 0 is known as the load displacement angle. As previously discussed, the charge displacement angle 0 is preferably less than about 3o. As shown in Figures 7 and 8, as the arrow 42 and hub 40 rotate 360 °, the stirring arm 7 rotates back and forth in the longitudinal plane by an angle equal to 0 (when the bushing 40 is in the orientation shown in Figure 8, the position of the shaker arm 7 is as shown in dotted lines in Figure 7). Thus, the rotational movement of the arrow 42 rotates the sleeve 41 in the longitudinal plane and imparts a rectilinear movement along an arcuate path towards the distal end of the shaker arm 7 to which the container 9 is secured, as shown in FIG. Figure 4 Due to the eccentric nature of the bushing 40, the rotation of the arrow 42 also tends to rotate the sleeve 41 of the agitating arm 7 through the angle of load deflection 0 in the transverse direction, as shown in Figure 10 (the position of the agitator arm when the bushing has been turned 180 ° is shown in dashed lines in Figure 10). Thus, if the rotation of the arrow 42 were in the clockwise direction when viewed from left to right in Figure 7, then the orientation of the shaker arm 7 when the eccentric bushing 40 is at 0 ° is shown by the Eolidae line in Figure 7, the orientation when the bushing is at 90 ° from the dotted line in Fig. 10, the orientation when the bushing is 180 ° is shown in Fig. 8, and the orientation when the bushing is 270 ° is shown by the solid lines in Figure 10. Thus, as the eccentric bushing 40 rotates 360 ° inside the bearing 50, the agitating arm 7 imparts a stirring motion to the container 9 in both the longitudinal and transverse directions to achieve the pattern in figure 8. previously discussed. A spring 46 extends from the dead center of the bottom of the sleeve 41 to the bale plate 5 of the agitator housing, as shown in Figure 6. The hold in the reeverto 46 acts to maintain the agitator arm 7 in the pointer. vertical as the bushing 40 rotates. Preferably, the reeving 46 has a sufficient tension so that the stirring arm 7 remains essentially vertically oriented in the transvereal plane, as shown in Figures 9 (a) and (b), although it deviates from the vertical in 0/2. in the longitudinal plane, as shown in Figure 7. Using a spring 46 having a lower spring constant will allow the stirring arm 7 to rotate in the transvereal plane through an angle,, as shown in Figure 12. This has the effect of increasing the amplitude in the transverse direction C. Preferably, the agitating device 1 is assembled by modifying a commercially available agitation device manufactured by Crescent Dental Manufacturing, Inc., 7750 West 47th Street, Lyons, IL 60534 under the name Wig-L-Bug ™ 3110B. Estoe diepositivos Wig-L-Bug ™ employs a pattern of agitation with a type of figure in 8 and is sold having an agitator arm with a length L of 4 centimeters, an angle of deviation of load and, therefore a rotation angle of the agitation arm in the longitudinal direction 0 of 6 °, and operates at a fixed speed of 3200 rpm. In addition, the shaker arm in the Wig-L-Bug has a couple of spoons to hold the samples. Thus, the stirring apparatus of the present invention can be created by modifying a Wig-L-Bug ™ 3110B stirrer to incorporate the container 9, in which the aqueous suepeneum and the gas phases have been added, as previously discussed, on the distal end of the arm 7. Preferably, the Wig-L-Bug ™ agitator is also modified so as to incorporate the mounting clip 8 to secure the container 9 to the agitator arm 7, as shown in Figures 3 and 6. , depending on the composition of the aqueous and gaseous lae faeee, the size of the container, etc., optimum results can be obtained by modifying the Wig-L-Bug ™ in such a way that (i) it provides agitation at a different frequency of 3200 rpm or allows operation over a range of agitation frequencies, (ii) employing a stirrer arm length other than 4 centimeters or (iii) employing an offset load angle 0 other than 6 ° modifying the bushing displacement 40. Other types of alternative agitation diepoeitivoe may also be used in the practice of the present invention, more preferably, devices that impart a agitation movement in figure 8. In addition to the Wig-L-Bug, dietary factors include (FIG. i) the Mixomat, sold by Degussa AG, Frankfurt, Germany, (ii) the Capmix, sold by Espe Fabrik Pharmazeutischer Praeparate GMBH & amp;; Co., Seefeld, Oberay Germany, (iii) Silamat Plus, sold by Vivadent, Liechtenstein, and (iv) Vibros, sold by Quayle Dental, Sussex, England. Figures 18 (a) - (c) show the results of tests on the Mixomat and Capmix compared to the test results obtained from a Wig-L-Bug ™ 3110B using the material and procedures previously discussed with respect to the test results. shown in Figures 13-17, and an agitation duration of 60 seconds, operating the Wig-L-Big ™ at a frequency of 3200 rpm, operating the Mixomat at a frequency of 4100 rpm, and operating the Capmix at a frequency of 4500 rpm. As can be seen, in each example, more than 98 percent of the vesicles were less than 10 microns and more than 800x10"vesicles per milliliter were produced B. THE PREFERRED CONTENDOR According to the present invention, the container that is insured The stirring die 1 can take a variety of different forms.A preferred container 9 is shown in Figure 1 and comprises a body 30 and a gas tight plug 10. When filled, the container 9 forms a gas head space 32. and an aqueous suspension phase 34 substantially separate from one another Alternatively, the container may take the form of a pre-filled syringe, which may, if desired, be adjusted with one or more filters. As in the present case, it includes a syringe, filled with an aqueous phase and an eepacio euperior of a previously selected gae, preferably assembled in the agitator 1 n its long axis oriented in the transverse direction - i.e., perpendicular to the arc length C. After agitation, the gas-filled vesicles are produced in the syringe, ready for use. Regardless of the type of container used, it is preferably sterile, together with its content. Although, in general, the invention is practiced with sterile containers in which the aqueous phase is already present within the container, for selected applications, the stabilization medium can be stored within the container in a dry or lyophilized state. In this case, the aqueous solution, for example, regulated saline solution of ethereal phosphate, is added to the sterile container immediately before stirring. In doing so, the stabilized rehydration medium within the aqueous phase will again interact with the euperior gae space during agitation so as to produce gae-filled vesicles as above. The rehydration of a dried or lyophilized suspension medium necessarily further complicates the product and is not generally desirable, but for certain preparations it may be useful to further extend the shelf life of the product. For example, certain therapeutic agents such as cycloefamide, peptide, and materialee geneticae (such as DNA) could be hydrolyzed in long-term aqueous storage. Rehydration of a previously lyophilized sample to form the aqueous phase and the headspace before agitation may make it practical to produce gas-filled vesicles containing compounds that might otherwise not have a shelf life. A variety of different materials can be used to produce the container, such as glass, borosilicate glass, eilatite glass, polyethylene, polypropylene, polytetrafluoroethylene, polyacrylate, polyethene, or other plastics. Preferred containers are either gas impermeable or wrapped within an exterior gas impermeable barrier before filling with gas. It is then desirable to maintain the integrity of the previously selected gas within the container. Examples of syringe materials that have gae-airtight capabilities may include but are in no way limited to glass eilates or boron silicates, fitted with silicon oxide syringes or syringes of the luer-lock type., and plunger with Teflon tip or Teflon coated. The size of the container, more specifically, its weight, will affect the size of the gas-filled vesicles. Stirring devices will generally agitate more slowly as the container's weight increases beyond a certain level - for example, a Wig-L-Bug ™ 3110B will rapidly stir up a 2 milliliter (real volume 3.7 milliliter) fraeco than a vial of 10 milliliters. Therefore, the volume of the container should not exceed a certain amount depending on the particular agitation diet used. Tests were performed on a Wig-L-Bug using both a 10 milliliter traneparent flask (Wheaton Industries, Millville, New Jersey) and a 2 milliliter amber flask (3.7 milliliter real volume) (Wheaton Induetries, Millville, New Jersey). Again, the agitation speed was measured using a Code-Palmer Pistol Grip tachometer (Code-Palmer, Nile, 111.). Table 7 shows the results, which show that increasing the capacity of the bottle will reduce the frequency of agitation. TABLE 7 Effect of vial size on the Shaking frequency in the Wig-L-Bug TM As you can see, a nominal 2 milliliter provides allows the use of a high frequency of agitation in the Wig-L-Bug. With reference to the dimensions shown in Figure 1, a nominal 2 milliliter container, actual 3.7, preferably has a diameter D of about 1.78 centimeter, and a total height HQ of about 2.54 centimeter.

SAW. APPLICATIONS OF THE PRODUCED VESICLES ACCORDING TO THE PRESENT INVENTION The following represents several parameters to determine the size of the gas-filled vesicle. Vesicle size is important in terms of maximizing product efficacy and minimizing toxicity. Additionally, the vesicles should be as flexible as possible both to maximize the effectiveness and to minimize adverse interactions of tissue as it lodges in the lungs. The present invention creates vesicles of the desired size with very thin conformal membranes. Because the lae veeiclee laepae are so thin and conformal, for example only 1 mg / ml is necessary to stabilize the membranes, it has been found that larger diameter gas-filled vesicles can be used without causing pulmonary hyperemission. For example, the patient has been given a dose of up to five times the amount needed for diagnostic imaging without any evidence of pulmonary hypertension. In comparison, dosie much smaller than bubble of air covered with albumin of smaller diameter in these animals cauean eevera pulmonary hype. Because the veeicles of the present invention are so flexible and deformable, they easily slip through the capillaries of the lung. Additionally, the coating technologies used with the preend lipids (for example lipids having polyethylene glycol) decrease adverse pulmonary interactions while at the same time increasing the stability in vi tro and in vivo, and the efficacy of the product. The size of the gallbladder filled with gae as a contrast medium for general ultrasound should be as large as possible (without embolic effects) because the backscatter or the ultrasound effect is proportional to the radius at the sixth power when the frequencies are such that the vesicles are in the Rayleigh scattering regime. For magnetic resonance imaging, larger vesicles of the invention are also preferred. The ability of the present invention to prepare and employ larger-sized vesicles with less potential for toxic effects increases their effectiveness relative to other products. An additional parameter that influences the ultrasound contrast is the elasticity of the veeicle membrane. The greater the elasticity, the greater the contrast effect. Because the present vesicles are coated with ultra-thin membranes of lipid elasticity, it is quite similar to naked gas and the reflectivity and contrast effect are maximized. The stirring process of the present invention readily produces vesicles of an aqueous phase and an upper gas space within a sterile container. The invention is sufficient to produce vesicles with very desirable properties for magnetic resonance or ultrasound imaging applications. However, for e-selected applications, a filter can be used to produce vesicles with still more homogeneous size distributions and of desired diameters. For example, to measure in vivo preemption in ultrasound using harmonic veeiclee gas filling phenomena, it can be useful to have very precisely defined vesicle diameters within a narrow range of sizes. This is easily carried out by injecting the vesicles (produced by shaking the container with aqueous phase and upper gas space) through a filter of defined size. The resulting vesicles will not be larger than a very close approximation of the filter pore size in the filter membrane. As you noticed earlier, for many magnetic resonance or ultrasound imaging applications, it is desirable to make the vesicles as large as possible. For certain applications, however, much smaller gas-filled vesicles may be desirable. To direct them, for example, to tumors or other diseased tissues, it may be necessary for the vesicles filled with gae to leave the vascular space and enter the intertice of the tissue. Much smaller gas-filled vesicles can be useful for these applications. These smaller gas-filled vesicles (for example, appreciably below one meter in diameter) can be largely produced by modifications in the compounds in the aqueous phase (composition and concentration) and the upper space (gas composition and composition). volume of the upper space), but also by injection through a filter. Very small gas-filled vesicles of substantially homogenous size can be produced by injecting through, for example, a 0.22 micron filter. The gas-filled vesicles resulting from nanometer sizes can then have desirable properties to be targeted. The above examples of lipid suspensions can also be sterilized by autoclaving and with an appreciable change in the size of the suspensions. The sterilization of the contrast medium can be carried out by autoclaving and / or sterile filtration carried out either before or after the stirring step, or by other means known to those skilled in the art. After filling the containers with the aqueous fae and the euperior space of the previously selected gas, the eelladae bottles can be stored indefinitely. It does not need to have particles to precipitate, vesicles filled with gas to burst or other interactions between the vesicles filled with gas, particles, colloid or emuleionee. The life in the container of the container filled with the aqueous phase and the upper space of gae only depends on the stability of the compounds within the aqueous phase. These shelf-life and sterility-amenable properties confer substantial advantages to the present invention over the prior art. The problem of stability, as with the aggregation and precipitation of particles, which is so common in the field of contraceptive medium for ultrasound, has been discussed here. Gas-filled vesicles that are produced by agitation of the multi-phase container of the invention have been found to have excellent utility as contrast agents for imaging for diagnosis, such as ultrasound imaging or magnetic resonance imaging. The vesicles are useful for imaging a patient in general, and / or for specifically diagnosing the presence of diseased tissue in a patient. The imaging process can be carried out by administering a gae-filled vesicle of the invention to a patient, and the patient can then be scanned by ultrasound or magnetic resonance imaging to obtain an image of an internal region of a patient and / or of any diseased tissue in that region. By region of a patient, the whole patient is understood, or a particular area or portion of the patient. The liposomal contraceptive agent can be used to provide images of the vasculature, heart, liver and spleen, and to image the gaetrointestinal region or other cavities of the body, or in other ways as will be readily apparent to those skilled in the art, as characterization of tissue, image formation of blood depot, etcetera. Any of the ultrasound imaging or magnetic resonance imaging devices can be employed in the practice of the invention, the type or model of the device for the method of the invention not being critical. The gas-filled vesicles of the invention can also be employed to administer a wide variety of therapy for a patient for the treatment of disease-causing diets., be affected, as one skilled in the art will recognize. They can also use magnetically activated vesicles to decrease pressure by magnetic resonance imaging. The vesicles increase the susceptibility of the volume and, in this way, they increase the relaxation T2 but even more for the relaxation T2 *. Because the effects of eeatic field gradients are mainly related to spin echo experiments (under the 180 ° radio frequency refocusing pulse) the effect of the vesicles is less marked on T2 than on weighted pulse sequences T2 * where the effects of the static field are not compensated. The increase in pressure results in the loss of veeiclee or the rupture of veeicles (for more soluble gases) as well as a decrease in the diameter of vesicles. Consistent with this, l / T2 decreases with increasing pressure. After getting rid of the remaining pressure of the remaining particles, they expand again and 1 / T2 increases again slightly. Compound vesicles of approximately 80 percent PFP with 20 percent air show increased stability and a slight fall at 1 / T2 with pressure returning to the baseline after release of the precession (ie, the vesicles are set but have a free preemption effect l / T2). When the images of the gradient echo are obtained and the intensity of the signal is measured, these effects are much more marked. The intensity of the signal increases with the increase in pressure (1 / T2 * decreases with the increase in pressure). Because the experiment is done relatively quickly (it takes less than a tenth of time to make gradient echo images that measure T2). The duration of exposure to pressure is much shorter and the nitrogen-filled vesicles return near the baseline after pressure release (ie there is very little loss of vesicles). Accordingly, a signal strength or gradient echo again falls near the baseline upon return to ambient pressure. For the measurement of pressure by magnetic resonance imaging, the veeiclee can be designed either to separate with increased pressure or to be stable but to decrease the diameter of the vesicle with increasing pressure. Because the radius of the magnetic resonance imaging vesicle affects 1 / T2 *, this ratio can be used to estimate the pressure by magnetic resonance imaging. As one skilled in the art would recognize, administration of the gae-filled vesicles to the patient can be carried out in various ways, such as intravenously or intraarterially by injection, orally, or rectally. The useful dosie to be administered and the particular mode of administration will vary depending on the age, weight and the particular mammal and the region of the member to be explored or treated, and the particular contrast medium or therapy that is going to use. Typically, the dose is initiated at lower levels and is increased until the desired contrast enhancement or therapeutic effect is achieved. The patient can be any type of mammal, but more preferably is a human. The descriptions of each of the patents and publications cited or referred to herein are entirely incorporated in the preamble by reference. For those skilled in the art, various modifications of the invention in addition to those shown and described herein will be apparent from the foregoing description. These modifications are also intended to fall within the scope of the appended claims.

Claims (44)

1. A method for making vesicles, comprising the steps of: a) placing an aqueous euepension phase and a gaseous phase substantially separated from the aqueous suspension phase in a container; and b) shaking the container imparting an alternating motion to the membrane until the vesicles are formed.

2. The method according to claim 1, wherein the amplitude of the reciprocating movement is at least 0.3 centimeters.

3. The method according to claim 2, wherein the amplitude of the reciprocating movement is not greater than about 2.5 centimeters.

4. The method according to claim 1, wherein the total length of the container travel during reciprocating movement is at least about 0.7 centimeters.

5. The method according to claim 1, wherein the frequency of the reciprocating movement is at least about 2800 rpm.

6. The method according to claim 2, wherein the frequency of the reciprocating movement is not greater than about 4500 rpm.

The method according to claim 1, wherein the reciprocating movement occurs in at least one first direction, the movement and the first direction are presented along an arcuate path.

The method according to claim 7, wherein the angle of rotation encompassed by the arcuate path is at least about 3 °.

The method according to claim 8, wherein the angle encompassed by the arcuate path is not greater than about 9 °.

The method according to claim 7, wherein the radius of curvature of the arcuate path is at least about 6 centimeter.

The method according to claim 10, wherein the radius of curvature of the arcuate path is not more than about 15 centimeters.

The method according to claim 1, wherein the reciprocating movement comprises movement in first and second directions that are substantially perpendicular.

The method according to claim 12, wherein the reciprocating movement occurs along a path having approximately a pattern of figure at 8.

The method according to claim 13, wherein the total length of the container's travel around the figure pattern at 8 is at least about 0.7 centimeter.

15. The method according to claim 1, wherein the aqueous suspension phase comprises lipids.

16. The method according to claim 15, wherein the aqueous suspension fae comprises dipalmitoylphosphatidylcholine, dipalmitoylfoephatidic acid, and dipalmitoylfoefatidylethanolamine.

17. The method according to claim 16, wherein the gas phase comprises a gae perfluorocarbon.

18. The method according to claim 1, wherein the gas phase initially occupies at least 10 percent of the volume of the container.

19. The method according to claim 1, wherein the agitation produces vesicles of which 95 percent are less than 10 microns.

20. The method according to claim 19, wherein the duration of the stirring is not greater than about 2 minutes.

21. The method according to claim 1, wherein the agitation produces vesicles having an average size of less than 2.5 microns.

22. The method according to claim 1, wherein the container is a syringe.

23. An apparatus for mixing vesicles, comprising: a) a container containing an aqueous phase and a gaseous phase substantially separated from the aqueous euepeneion phase; b) an element for shaking the container imparting an alternative movement thereto until the vesicles form.

24. The apparatus according to claim 23, wherein the agitating element comprises means for agitating at an amplitude of at least 0.3 centimeters.

25. The apparatus according to claim 24, wherein the element for stirring further comprises an element for stirring at an amplitude no greater than about 2.5 centimeters.

26. The apparatus according to claim 23, wherein the stirring element comprises elements for stirring the container along a path having a total length of at least about 0.7 centimeters.

27. The apparatus according to claim 23, wherein the means for agitating comprises elements for agitating at a frequency of at least 2800 rpm.

28. The apparatus according to claim 27, wherein the agitating element further comprises elements for agitating at a frequency no greater than about 4500 rpm.

29. The apparatus according to claim 23, wherein the stirring element comprises elements for agitating along an arcuate path.

30. The apparatus according to claim 29, wherein the angle of rotation encompassed by the arcuate path is at least about 3 °.

31. The apparatus according to claim 30, wherein the angle encompassed by the arcuate path is not greater than about 9 °.

32. The apparatus according to claim 29, wherein the radius of curvature of the arcuate path is at least about 6 centimeters.

33. The apparatus according to claim 32, wherein the radius of curvature of the arcuate path is not greater than about 15 centimeters.

34. The apparatus according to claim 23, wherein the element for agitating comprises elements for agitating in first and second directions substantially perpendicular.

35. The apparatus according to claim 23, wherein the agitating element further comprises elements for agitating along a path having approximately a pattern of Figure 8.

36. The apparatus according to claim 35, wherein the total length of the figure pattern at 8 ee from when it is about 0.7 centimeters.

37. The apparatus according to claim 23, wherein the aqueous suspension phase comprises lipids.

38. The apparatus according to claim 37, wherein the aqueous suspension phase comprises dipalmitoyl-phosphatidylcholine, dipalmitoylphosphatidic acid, and dipalmitoyl-phemphatidylethanolamine.

39. The apparatus according to claim 38, wherein the faee gaeeoea comprises a gae perfluorocarbon.

40. The apparatus according to claim 1, wherein the gaseous fae initially occupies at least 10 percent of the volume of the container.

41. An apparatus for making a vesicle, comprising: a) a container containing a water-soluble faece and a gas-phase fae usually separated from the aqueous suspeneion phase; and b) a stirring device for stirring the container to form veeiclee in the beam, the stirring die (i) having an arm having a length of at least about 6 centimeters, (ii) an element for imparting an alternative stirring motion. to the arm, and (iii) means to attach the container to the arm.

42. The apparatus according to claim 41, wherein the arm is rotatably mounted in the stirring device.

43. The apparatus according to claim 41, wherein the element for imparting reciprocating movement to the arm comprises an element for rotating that arm through an angle of at least about 3 °.

44. An apparatus for making vesicles, comprising: a) a container containing an aqueous sodium phase and a gas phase substantially separated from the aqueous suspension phase; b) a stirring die to agitate the container to form vesicle in the beam, the stirring die (i) having an arm, (ii) an element for imparting an agitation reciprocating motion to the arm having a frequency of at least about 2800 rpm, and (iii) means for coupling the container with the arm.