WO2010030779A1 - Isolation of microbubbles of selected size range from polydisperse microbubbles - Google Patents

Abstract

In one aspect of the disclosed subject matter, a method for isolating target microbubbles having a predetermined size range from polydisperse microbubbles is disclosed. The method includes applying a first centrifugal field having a first field strength to a suspension comprising the polydisperse microbubbles for a first duration of time, thereby forming a first infranatant comprising at least a portion of target microbubbles and a first supernatant cake comprising microbubbles having a greater size than the target microbubbles; removing the first supernatant cake; applying a second centrifugal field having a second field strength to the first infranatant for a second duration of time, the second field strength being greater than the first field strength, thereby forming a second supernatant cake comprising at least a portion of the target microbubbles and second infranatant comprising microbubbles having a smaller size than the target microbubbles; and isolating the second supernatant cake. In another aspect of the disclosed subject matter, a method for performing high frequency ultrasonic imaging using isolated microbubbles is provided.

Description

ISOLATION OF MICROBUBBLES OF SELECTED SIZE RANGE FROM POLYDISPERSE MICROBUBBLES

RELATED APPLICATIONS The present application claims priority to U.S. Provisional Application

Serial No. 61/095,933, filed on September 10, 2008, the disclosure of which is incorporated by reference herein in its entirety.

BACKGROUND Microbubbles are being employed for several biomedical applications, including contrast enhanced ultrasound, drug and gene delivery, and metabolic gas delivery. Microbubbles can react strongly to ultrasonic pressure waves by virtue of their compressible gas cores, which resonate at the MHz-frequencies used by current clinical scanners. Oscillation of the gas core allows re-radiation (backscatter) of ultrasound energy to the transducer at harmonic frequencies and nonlinear modes, thus providing exquisite sensitivity in detection with current contrast-enhanced pulse sequences and signal processing algorithms. Additionally, microbubbles can cavitate stably or inertially to facilitate drug release and extravascular delivery within the transducer focus. Current commercially available microbubble formulations can be polydisperse in size. In some cases, the size distribution is broad over a range of submicron to tens of μm in diameter. This can be problematic because microbubble behavior depends on size. For example, increasing the microbubble diameter from 1 to 5 μm will change the resonance frequency of an unencapsulated microbubble from 4.7 to 0.72 MHz. Microbubble size can also impact biodistribution and pharmacodynamics after intravenous injection, bioeffects during ultrasound insonification, gas release profile, and other related behaviors. Therefore, microbubbles of a specific size with low polydispersity are desired for advanced biomedical applications. Techniques for producing or isolating monodisperse microbubbles have been in development. For example, microfluidic technologies have been used to engineer monodisperse microbubble suspensions. These techniques include flow focusing, T-junctions and electrohydrodynamic atomization. While these techniques can provide for low polydispersity, they may be slow at generating microbubbles. Using flow focusing, for example, can require several hours to produce microbubbles at sufficient numbers for even a single small-animal trial (~ 0.1 mL x 10 mL^" ). Additionally, dust particles can plug micro-channels, thus requiring fabrication and calibration of a new device. Mechanical agitation is one method to create encapsulated microbubbles for biomedical applications. It is an emulsification procedure in which a hydrophobic phase (i.e., gas) is dispersed within an aqueous surfactant solution by disruption of the interface. Acoustic emulsification (sonication), for example, can generate large quantities of microbubbles (e.g., 100 mL x 10¹⁰ mL^"1) rapidly and reproducibly within just a few seconds. Shaking a serum vial with a device similar to a dental amalgamator produces a sufficient dose of microbubbles (2 mL x 10¹⁰ mL^"1) for a single patient study, at the bedside in less than a minute. However, the size distributions of the microbubbles generated by mechanical agitation are highly polydisperse. Accordingly, there is a need for an efficient method for isolating selected size fractions of microbubbles of interest with sufficient yield from polydisperse microbubbles for biomedical applications.

SUMMARY The disclosed subject matter provides techniques for isolating target microbubbles having a predetermined size range from polydisperse microbubbles, as well as for performing ultrasonic imaging using the size-isolated microbubbles.

In one aspect of the disclosed subject matter, a method for isolating target microbubbles having a predetermined size range from polydisperse microbubbles is provided. The method includes: applying a first centrifugal field having a first field strength to a suspension comprising the polydisperse microbubbles for a first duration of time, thereby forming a first infranatant comprising at least a portion of target microbubbles and a first supernatant cake comprising microbubbles having a greater size than the target microbubbles; removing the first supernatant cake; applying a second centrifugal field having a second field strength to the first infranatant for a second duration of time, the second field strength being greater than the first field strength, thereby forming a second supernatant cake comprising at least a portion of the target microbubbles and second infranatant comprising microbubbles having a smaller size than the target microbubbles; and isolating the second supernatant cake.

In some embodiments of the above method, the applied first centrifugal field and the first duration of time can be sufficient to cause substantially all the microbubbles in the polydisperse microbubbles having a greater size than the target microbubbles to form the first supernatant cake. In these embodiments, the applied second centrifugal field and the second duration of time can be sufficient to cause substantially all the target microbubbles to form the second supernatant cake.

The isolated second supernatant cake can be further redispersed into a new dispersion, which can be subjected to a third centrifugal field having a third field strength for a third duration of time, thereby forming a third supernatant cake comprising at least a portion of the target microbubbles, and then the third supernatant cake is isolated.

In some embodiments of the method, the polydisperse microbubbles include microbubbles having sizes from smaller than about 1 μm to larger than about 10 μm. hi certain embodiments, the target microbubbles have a size range of about 4 μm to about 5 μm. In other embodiments, the target microbubbles have a size range of about 1 μm to about 2 μm. hi some embodiments of the method, the polydisperse microbubbles are coated at least in part with lipids. In other embodiments, the polydisperse microbubbles are coated at least in part with polymeric surfactants.

In some embodiments of the method, the core of the polydisperse microbubbles comprises perfluorobutane. hi some embodiments of the method, the polydisperse microbubbles are obtained by sonication. hi certain embodiments, the polydisperse microbubbles have a multimodal size distribution. hi some embodiments of the method, the microbubble suspension can be placed in a syringe when being subjected to a centrifugal field. The syringe has a longitudinal axis, a length, a cap, and a drainage portion. The drainage portion of the syringe is positioned further away from the central axis of the centrifugal field relative to the cap.

In some embodiments of the disclosed subject matter, a method for isolating target microbubbles having a predetermined size range from polydisperse microbubbles is provided. The method includes: applying a first centrifugal field having a first field strength to a suspension comprising the polydisperse microbubbles for a first duration of time, thereby forming a first infranatant comprising at least a portion of target microbubbles and a first supernatant cake comprising microbubbles having a greater size than the target microbubbles; removing the first supernatant cake; applying a second centrifugal field having a second field strength to the first infranatant for a second duration of time, the second field strength being greater than the first field strength, thereby forming a second infranatant comprising at least a portion of target microbubbles and a second supernatant cake comprising microbubbles having a greater size than the target microbubbles; removing the second supernatant cake; applying a third centrifugal field having a third field strength to the second infranatant for a third duration of time, the third field strength being greater than the second field strength, thereby forming a third supernatant cake comprising at least a portion of the target microbubbles and third infranatant comprising microbubbles having a smaller size than the target microbubbles; and isolating the third supernatant cake. In some embodiments of the above method, the applied second centrifugal field and the second duration of time can be sufficient to cause substantially all the microbubbles in the first infranatant having a greater size than the target microbubbles to form the second supernatant cake. In these embodiments, the applied third centrifugal field and the third duration of time can be sufficient to cause substantially all the target microbubbles to form the third supernatant cake. The isolated third supernatant cake can be further redispersed into a new dispersion, which can be subjected to a fourth centrifugal field having a fourth field strength for a fourth duration of time, thereby forming a fourth supernatant cake comprising at least a portion of the target microbubbles, and then the fourth supernatant cake can be isolated.

In some embodiments of the above methods, the total volume fraction of the microbubbles in the polydisperse microbubbles suspension and each of the subsequently formed supernatant can be equal to or below about 20%, and at least applying one of the centrifugal fields comprises first determining the centrifugal field strength to be applied using a Stake's equation. The Stoke's equation correlates the rise velocity of a microbubble in a suspension relative to the bulk fluid under creeping flow conditions with the size of the microbubble, the centrifugal field strength to be applied, and the effective viscosity of the suspension. The determination of the respective centrifugal field strengths can be based at least on the duration of time during which the respective centrifugal field is to be applied.

In another aspect of the disclosed subject matter, monodisperse microbubbles having a predetermined size range prepared by the above described methods are provided. hi yet another aspect of the disclosed subject matter, a method of performing high frequency ultrasonic imaging is provided. The method includes: administering monodisperse microbubbles having an number-averaged size of between about 1 to 10 μm to an animal; and performing ultrasonic imaging on the animal at a fundamental frequency of at least about 30 MHz. The monodisperse microbubbles for performing the high frequency ultrasonic imaging can have a size range of about 1 μm to about 2 μm, about 4 μm to about 5 μm, or about 6 μm to about 8 μm.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of example embodiments of the described subject matter and its advantages, reference is now made to the following description, taken in conjunction with the accompanying drawings, in which:

Figure 1 depicts a block diagram illustrating a method for isolating target microbubbles according to some embodiments of the disclosed subject matter.

Figure 2 depicts a schematic diagram of differential centrifugation for the size isolation of microbubbles.

Figure 3 depicts size distributions of freshly sonicated microbubbles according to some embodiments of the disclosed subject matter. Figure 4 depicts microscopy images of initial polydisperse and size- isolated microbubbles according to some embodiments of the disclosed subject matter.

Figure 5 depicts plots illustrating size distribution of initial polydisperse and size-isolated microbubbles determined by flow cytometry according to some embodiments of the disclosed subject matter.

Figure 6 depicts plots illustrating size distributions of initial polydisperse and size-isolated microbubbles determined by light scattering and electrozone sensing, respectively, according to some embodiments of the disclosed subject matter. Figure 7 depicts fluorescence intensity and light scattering profiles for microbubble suspensions after size isolation as determined by flow cytometry according to some embodiments of the disclosed subject matter.

Figure 8 depicts the stability of size-isolated microbubbles according to some embodiments of the disclosed subject matter.

Figure 9 depicts the ultrasound contrast video intensity for a healthy adult mouse kidney using polydisperse microbubbles and size isolated microbubbles according to some embodiments of the disclosed subject matter.

DETAILED DESCRIPTION

In one aspect, the disclosed subject matter provides techniques for isolating a selected size fraction of microbubbles (or target microbubbles) from polydisperse microbubbles. First, a dispersion (or suspension) of microbubbles having broad size distribution can be generated or obtained. These initial microbubbles are then subjected to at least two stages of separation. In the first stage, the portion of the microbubbles having a greater size than the target microbubbles are separated from the initial microbubbles and discarded. In the second stage, the remaining microbubbles are further separated into two portions: a first portion containing substantially all the target microbubbles, and the other portion containing mostly microbubbles smaller than the target microbubbles. The first and the second stage can both be divided into several sub-stages of separation to optimize the separation result.

The presently described techniques allow rapid and robust production of a narrowly distributed (or monodisperse) microbubbles of a desired size range from polydisperse microbubbles that can be generated in large quantities by simple mechanical agitation. Both of the generation of initial polydisperse microbubbles and the size-isolation methods as described herein can be conveniently and inexpensively performed at the site where the target microbubbles are intended to be used.

Referring to Fig. 1, a method for isolating target microbubbles having a predetermined size range from polydisperse microbubbles according to some embodiments of the disclosed subject matter will be explained. At 110, a first centrifugal field having a first field strength is applied to a suspension comprising the polydisperse microbubbles for a first duration of time, thereby forming a first infranatant and a first supernatant cake. The first infranatant is a suspension that includes at least a portion of target microbubbles; and the first supernatant cake includes microbubbles having greater sizes than the target microbubbles. At 120, the first supernatant cake is removed. At 130, a second centrifugal field, which has a strength greater than the first field strength, is applied to the first supernatant cake for a second duration of time, thereby forming a second supernatant cake and a second infranatant. The second supernatant cake includes at least a portion of the target microbubbles, and the second infranatant includes microbubbles having a smaller size than the target microbubbles. At 140, the second supernatant cake is isolated.

The polydisperse microbubbles can be fabricated by any suitable methods. For example, in some embodiments, they can be obtained via mechanical agitation, e.g., by vigorous mixing of a surfactant and/or lipid containing aqueous solution while introducing gas into the suspension, or sonicating a surfactant and/or lipid containing aqueous solution. Accordingly, the polydisperse microbubbles can be coated with lipids, such as phospholipids, with surfactants, such as polymeric surfactants, with proteins, such as albumin, or with any mixtures of the above, among other materials suitable for forming microbubbles. It is noted that polymer coated microbubbles are typically more stable in a high shear stress field, such as a large centrifugal field, but they can be more difficult to prepare. Mixing a small amount of surfactants, such as polymeric nonionic surfactants, with lipids can facilitate the formation of the microbubbles. The core of the polydisperse microbubbles can include air or other gas, such as perfluorobutane, that is suitable for the intended application of the microbubbles. The gas can be incorporated into the core by a gas introducer, such as a gas tube, a sonication tip, or any other suitable means.

The polydisperse microbubbles can have a broad size distribution. The distribution depends on the coating materials of the microbubbles, the methods by which they are initially fabricated, and the condition and/or term of their storage, among other factors. For example, in some embodiments of the disclosed subject matter, the size distribution of the polydisperse microbubbles include a range from submicron, e.g., about 0.2 μm or 0.5 μm in diameter, up to more than about 10 μm in diameter, or more than about 25 μm in diameter, or even larger.

The size distribution of polydisperse microbubbles (or any size- isolated subpopulation thereof) can be characterized by measurement systems that report relative weight of each size channel (which is also referred to as size class, i.e., a very narrow band or "slice" of the size distribution spectrum, for example, a 0.1 or 0.2 μm size interval in a spectrum spanning from, e.g., 0.2 to 20 μm) of microbubbles based on the relative numbers of microbubbles falling in such size channel, for example, by methods that are rooted on light scattering principles. Alternatively, the distribution can be characterized by measurement systems that report relative weight of each size channel of microbubbles based on the relative volumes of microbubbles falling in such size channel, for example, using an electrozone sensing method. Additionally, size characterization can be performed based on fluorescence intensity and optical microscopy.

The target microbubbles have a predetermined size range that is encompassed in the size spectrum of the polydisperse microbubbles. Such a size range can be determined based on the intended use of the target microbubbles. Alternatively, such a size can be determined based on the size distribution of the polydisperse microbubbles. Accordingly, in one embodiment, the above method further includes, prior to applying any centrifugal field, obtaining a size distribution of the polydisperse microbubbles and then selecting a size range from such size distribution as the size range of the target microbubbles.

The polydisperse microbubbles can have a multimodal size distribution, i.e., the distribution contains two or more distinct "peaks" representing a relatively greater weight than other size ranges with similar widths in the entire size spectrum. The multimodal size distribution can be detected using any size characterization techniques, such as those described above. With a knowledge of the features of the multimodal size distribution, e.g., the shapes of the distribution peaks, their positions in the entire size spectrum and their relative positions to each other, appropriate adjustments to the isolation method can be made to improve the yield of the target microbubbles or to increase the efficiency of the isolation method, as will be explained below.

The centrifugal fields in the presently described techniques can be produced by any suitable one or more centrifuge devices, for example, a common bench top centrifuge, such as a bucket-rotor centrifuge having a wide range of spin speeds so as to provide a wide range of centrifugal field strengths. The centrifuge has a central axis located on the spinning axis of the rotor. The field strength of a centrifugal field can be measured by Relative Centrifugal Force (RCF), which denotes the maximum centrifugal acceleration afforded by the centrifuge at a given corresponding spinning speed (e.g., RPM) of the centrifuge rotor. To perform centrifugation, a suspension of the polydisperse microbubbles, or any subsequent infranatant formed, can be placed in a suitable container (herein generally referred to as a flotation column), such as a tube or a syringe. For convenience of operation, a (modified) syringe can be used as the flotation column. The syringe can be tubular and have a longitudinal axis and a length. Additionally, the syringe can have a cap or plunger installed on the top portion that prevents the larger buoyant microbubbles from escaping the syringe (so that they will form a supernatant cake which rests against the cap), and a drainage portion at the bottom to facilitate removal of the infranatant formed as the result of a centrifugation. The drainage portion can be positioned further away from the center axis of the centrifugal field relative to the cap. The syringe can be placed horizontally, which can provide the maximum field strength at a given spin speed, but can also be placed at a certain angle with respect to the axis of the centrifugal field.

The presently described techniques for isolating target microbubbles are based in part on the principles of differential centrifugation where species of particles having different sizes and/or densities are separated in a centrifugation field as a result of their different migration speed in the centrifugal field, as illustrated in Fig. 2.

Determining the appropriate field strength for each of the multiple parts of the size fractionation process according to the disclosed subject matter can be based on a Stoke's equation, which describes the rise velocity of a buoyant particle relative to the bulk fluid under creeping flow conditions as

2(p₂ - _A,) ₂

9η₂ * ^{i 8c} ' ^UJ

where subscript i refers to the particle size class, r_t is the particle radius, p₂ is the density of the liquid medium where the microbubbles are suspended, p_u is the density of the particle, and g_c is the centrifugal acceleration measured in RCF. (g_c = RCF x g, where g is the acceleration in a gravitational field, i.e., 9.8 m/s²). The effective viscosity, η₂ * , of the microbubbles suspension can be calculated using Batchelor and Greene's correlation for the modified fluid viscosity:

- = 1 + 2.5Φ + 7.6Φ² (2) Φ = f>,, (3) where η₂ is the viscosity of the suspension medium, and Φ is total the microbubble volume fraction for N_d size classes. Equations 1-3 can be used to calculate the strength of the centrifugal field (in RCF) for a given initial size distribution of the polydisperse microbubbles, time period and the length of the flotation column. Volume fraction can be assumed to be constant over the entire column, and acceleration/deceleration effects can be neglected. It is noted that the above Stoke' s equation yields more accurate results when the volume fractions of the microbubbles in a suspension subjected to the centrifugal field are equal to or below 20%. A larger volume fraction of microbubbles would cause more significant collision among the migrating particles and accordingly more turbulence in the suspension, thereby making the creeping flow condition assumption less true.

In one embodiment, the appropriate field strengths to be used in the first centrifugal field and the second centrifugal field can be obtained as follows. First, the size distribution of the initial polydisperse microbubbles is measured. The size distribution is then imported into a spreadsheet in order to determine the number density for each size class and the total gas volume fraction. The spreadsheet is used to calculate the relative centrifugal force (RCF) needed for a microbubble size class to rise through the flotation column of length L for a predetermined centrifugation time. The requisite field strengths thus obtained can be tabulated before performing the first centrifugation, and serve as a convenient guide for selecting the appropriate spinning speed (RPM) of the centrifuge to remove fractions of microbubbles larger than the target microbubbles or remove fractions of microbubbles smaller than the target microbubbles. The duration of time for any applied centrifugal field can be predetermined. For example, a duration of time of 1 minute can be chosen for all the centrifugal fields that may need to be applied in order to isolate the target microbubbles. Other values of the duration of time can also be used, for example, 0.5 minutes, 2 minutes, 4 minutes, or any other suitable lengths of time. The duration of time for each of centrifugal field does not need to be the same. For example, a first centrifugal field can be applied for 2 minutes, and a second centrifugal field can be applied for 1 minute. The duration of times can be chosen to be longer than 1 minute in order to minimize transient effects caused by the acceleration and deceleration of the rotor, so that the sample experiences mostly a constant centrifugal field strength.

The suitable field strength of an applied centrifugal field in the described techniques depend on the coating material of the microbubbles, the size distribution of the microbubbles, and the length of the flotation column, among others, and can be between 1 and 500 equivalent gravity. Greater field strength may result in significant microbubble destruction, thereby lowering the yield of the target microbubbles. This range of field strengths can be applied to microbubbles dispersion containing microbubbles in the 1-10 μm diameter range. The specific field strength values can be determined empirically or estimated either by Stokes Law, or through empirical measurements when size measurement is available. The preferred method is to use Stokes Law as an initial estimate and the refine the technique through empirical measurement. One can use Stokes law based on the initial size distribution of the polydisperse microbubbles as outlined above, or a priori without knowledge of the initial size distribution or concentration. For example, one may measure the volume and mass of the microbubble suspension and, based on a comparison of the density of the suspension medium with microbubbles with the density of the suspension medium without microbubbles (i.e., before microbubbles are generated), one may determine the volume fraction of encapsulated gas in computing η₂ * . Obtaining a size distribution of the initial polydisperse microbubbles, prior to subjecting the microbubbles to a centrifugal field, can be useful in a multiple ways:

(1) Selecting a proper strength of a centrifugal field to be applied from the size distribution given a desired or predetermined duration of time during which such a field is to be applied. For example, if one desires to remove all the microbubbles having a greater size than the target microbubbles in one centrifugation, the RCF needed for removing such fractions (as supernatant cake) can be computed based on the above Stoke' s equation given a predetermined time period during which the centrifugal field is to be applied. (2) Validating the initial polydisperse microbubbles. For example, through an examination of the shape of the size distribution, one can determine whether the target microbubbles are present in a sufficient amount in the polydisperse microbubbles to warrant further processing (otherwise the yield of the target microbubbles would not be sufficient for the intended applications). If not, another sample of the polydisperse microbubbles can be obtained and checked for size distribution.

(3) Improving efficiency of the size-isolation process. Through an examination of the shape of the size distribution, one can identify the shape and position of possible peak(s) in the distribution. For example, if the target microbubbles have a range of about 4 to 5 μm, and the size distribution has a rather focused peak in that range and only a minor tail on the positive side of 5 μm, e.g., a small tail in 5-6 μm range, one can use a centrifugal strength that is only sufficient to remove the fractions of microbubbles having a larger size than 6 μm (instead of a greater centrifugal strength that would remove fractions of microbubbles having a size of larger than 5 μm). This can still obtain a sufficiently monodisperse target microbubbles without suffering from the substantial loss of the target microbubbles, because a greater centrifugal field (sufficient to remove the microbubbles having a size of 5 μm and above) would cause a greater portion of the target microbubbles to collect into the supernatant cake (which will be discarded).

(4) Facilitating the design of a multiple part fractionation process for microbubbles having sizes larger than the target microbubbles. When the initial polydisperse microbubbles have a broad distribution on the positive side of the target microbubbles, a multiple fractioning process can achieve higher isolation efficiency. For example, if the target microbubbles have a range of 1 to 2 μm and the size distribution of the initial polydisperse microbubbles have a significant profile in the range of greater than 2 μm, one can use a first gentle centrifugal field to remove the fractions of microbubbles having a size of greater than 10 μm, and then use a second centrifugal field (stronger than the first one) on the remaining infranatant to remove the fractions of microbubbles having a size of greater than 7 μm. Likewise, a third centrifugal field (stronger than the second one) can be used on the second infranatant to remove the fractions of microbubbles having a size of greater than 4 μm, and so on, until substantially all the microbubbles having a size larger than 2 μm are removed. Such a multiple part size-fractionation process essentially multiplies the length of the flotation column, and therefore can achieve a better separation of different sizes of microbubbles and a higher yield of the target microbubbles as compared with a single centrifugation using a large field strength. A size distribution can also be obtained between the two successive centrifugations to validate the result of the first centrifugation. If necessary, a centrifugation can be repeated to improve the result. Besides, the infranant(s) that contains the target microbubbles which is subject to further centrifugation(s) can be diluted (or redispersed) to the maximum volume allowed in the flotation column before applying a subsequent centrifugation, thereby ensuring that the full length of the flotation column can be utilized to allow a more effective separation of different sizes of microbubbles.

After substantially all the microbubbles having a greater size than the target microbubbles are removed in one or more parts of the process as outlined above (in the form of one or more supernatant cakes), a further centrifugation of the infranatant containing the target microbubbles can be performed using a field strength and duration of time sufficient to cause substantially all of the target microbubbles to form a supernatant cake. Depending on the size distribution of the polydisperse microbubbles, this cake can contain an amount of microbubbles having a size smaller than the target microbubbles that render the cake not as monodisperse as desired. If such is the case, the cake can be further purified. For example, the cake containing the target microbubbles can be redispersed into a new dispersion, and a further centrifugation can be performed on the new dispersion. For quickly improving the purity of the cake (at the expense of a lower yield of target microbubbles), the centrifugal strength for the purification can be selected as approximately the same as or slightly smaller than the one used in the previous step (i.e., the one used to collect substantially all of the target microbubbles to form the supernatant cake). Likewise, the duration of time applied for purification can be selected as similar or slightly smaller than the duration of time last used. The purification can be repeated until an end point is reached, for example, a size distribution of the isolated cake containing the target microbubbles satisfying the needs of the intended application, or by simple visual inspection of the infranant(s) formed, e.g., when the infranant(s) is no longer turbid, indicating the smaller-sized microbubbles are removed from the target microbubbles to a satisfactory degree.

In some embodiments of described techniques of the disclosed subject matter, the initial polydisperse microbubbles have a size range of about 0.5 μm to about 10 μm. In one embodiment, the target microbubbles have a size range of about 4 to about 5 μm. In such an embodiment, a first applied centrifugal field can have a strength of about 70 RCF to remove microbubbles having a size larger than 6 μm, and a second applied centrifugal field can have a strength of about 160 RCF to remove the microbubbles having a size smaller than 4 μm. In another embodiment, the target microbubbles have size range of about 1 μm to about 2 μm. In such an embodiment, a first applied centrifugal field can have a strength of about 270 RCF to remove microbubbles having a size larger than 2 μm, and a second applied centrifugal field can have a strength of about 300 RCF to remove the microbubbles having a size smaller than 1 μm. Depending on the particular processes performed and parameters selected according to the techniques of the disclosed subject matter, the isolated final cake containing the target microbubbles can attain a purity of greater than about 80%, 90%, 95%, 99%, or even higher, meaning that the fractions of microbubbles falling out of the predetermined size range of the target microbubbles can be lower than about 20%, 10%, 5%, 1%, or even lower by volume. In addition, the polydispersity of the target microbubbles can achieve about 0.2 μm, 0.1 μm, or lower in polydispersity index (PI), which is defined as the volume-weighted mean diameter divided by the number-weighted mean diameter. These microbubbles with a narrow size distribution are also referred to as monodisperse microbubbles. In another aspect of the disclosed subject matter, a method for performing high frequency ultrasonic imaging is disclosed. The method includes administering a suspension of microbubbles having a predetermined size range to an animal, and performing the ultrasonic imaging on the animal, e.g., on an organ of the animal, at a fundamental frequency of at least about 30 MHz. The animal can be a mammal, for example, a mouse, a rabbit, a human, or any other suitable mammal. The organ can be an internal organ, for example, a kidney, a liver, and any other suitable organs. The microbubbles can be monodisperse, and can have a size range of, for example, about 1-2 μm, about 4-5 μm, or about 6-8 μm. Microbubbles having different size ranges can be used for different application. For example, microbubbles having a size of about 1-2 μm can be used where negative contrast and shadowing are desired, e.g., for reperfusion studies of very fine capillaries; microbubbles having a size of about 6-8 μm can be used where a large positive contrast and less shadowing are desired. As used herein, the term "about" means that the deviation of the quantity modified by the term can have no more than 10% from the quantity specified or predetermined; in the absence of a specified or predetermined value, the term means that the relative standard deviation of multiple measurements of the same quantity does not exceed 10% of the average of the multiple measurement results.

As used herein, the term "substantially all" means that the portion of the objects modified by this term should be at least 75% of all such objects, and more preferably 85%, 90%, or 95% of all such objects.

As used herein, the term that "have [having] a size range" means that the microbubbles consist of at least 80% of microbubbles in the specified range, and preferably consist of at least 90%, 95%, or 99% of microbubbles in the specified range.

As used herein, all mention of sizes of any microbubbles refers to the diameter of the microbubbles.

EXAMPLES

The following examples are merely illustrative of the presently described subject matter and should not be considered as limiting the scope of the disclosed subject matter in any way.

EXAMPLE 1. Isolation of Microbubbles Having A Predetermined Range of 1-2 Microns and 4-5 Microns And Characterizations Thereof

Materials and Methods

Materials

All solutions were prepared using filtered, 18MΩ deionized water

(Direct-Q, Millipore, Billerica, MA). All glassware was cleaned with 70 vol% ethyl alcohol solution (Sigma- Aldrich; St. Louis, MO) and rinsed with deionized water.

The gas used to form microbubbles was perfiuorobutane (PFB) at 99 wt% purity obtained from FluoroMed (Round Rock, TX). All phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL) and initially dissolved in chloroform (Sigma-Aldrich) for storage. Polyoxyethylene-40 stearate (PEG40S) was obtained from Sigma-Aldrich and dissolved in deionized water. The fluorophore probe 3,3'- dioctadecyloxacarbocyanine perchlorate (DiO) solution (Invitrogen; Eugene, OR) was used to label the microbubbles for part of the experiments.

Generation of Microbubbles

Microbubbles were coated with DSPC and PEG40S at molar ratio of 9:1. The indicated amount of DSPC was transferred to a separate vial, and the chloroform was evaporated with a steady nitrogen stream during vortexing for about ten minutes followed by several hours under house vacuum. 0.01 M phosphate buffered saline (PBS) solution (Sigma-Aldrich) was filtered using 0.2-μm pore size polycarbonate filters (VWR, West Chester, PA). The dried lipid film was then hydrated with filtered PBS and mixed with PEG40S (25 mg/mL in filtered PBS) to a final lipid/surfactant concentration of 1.0 mg/mL. The lipid mixture was first sonicated with a 20-kHz probe (model 250A, Branson Ultrasonics; Danbury, CT) at low power (power setting dialed to 3/10; 3 Watts) in order to heat the pre- microbubble suspension above the main phase transition temperature of the phospholipid (~55 ⁰C for DSPC) and further disperse the lipid aggregates into small, unilamellar liposomes. PFB gas was introduced by flowing it over the surface of the lipid suspension. Subsequently, higher power sonication (power setting dialed to 10/10; 33 Watts) was applied to the suspension for about 10 seconds at the gas-liquid interface to generate microbubbles. For flow cytometry and fluorescence microscopy experiments, DiO solution (1 mM) was added prior to high-power sonication at an amount of 1 μL DiO solution per mL of lipid mixture.

Microbubble Washing & Lipid Recycling

The microbubble suspension was collected into 30-mL syringes (Tyco Healthcare, Mansfield, MA), which were used as the flotation columns. Washing and size fractionation by centrifugation was performed with a bucket-rotor centrifuge (model 5804, Eppendorf, Westbury, NY), which had a radius of approximately 16 cm from the center to the syringe tip and operated between 10 and 4500 RPM. Centrifugation (10 minutes, 300 RCF) was performed to collect all microbubbles from the suspension into a cake resting against the syringe plunger. The remaining suspension (infranatant), which contained residual lipids and vesicles that did not form part of the microbubble shells, was recycled to produce the next batch of microbubbles. All resulting cakes were combined and re-suspended in PBS to improve total yield.

Size and Concentration Measurements

Microbubble size distribution was determined by laser light obscuration and scattering (Accusizer 780A, NICOMP Particle Sizing Systems, Santa Barbara, CA). 2-μL samples of each microbubble suspension were diluted into a 30- mL flask under mild mixing during measurement. Size distribution was also determined using the electrozone sensing method (Coulter Multisizer III, Beckman Coulter, Opa Locka, Fl). A 4-μL sample of microbubble suspension was diluted into a 60-mL flask and stirred continuously to prevent flotation-induced error. A 30-μm aperture (size range of 0.6-18 μm) was used for the measurements. All samples were measured at least three times by either instrument and analyzed for both number- and volume-weighted size distribution.

Optical Microscopy

Direct visual confirmation of microbubble size was performed 48 hours after the samples were prepared using an Olympus 1X71 inverted microscope (Olympus; Center Valley, PA). The microbubble samples were taken directly from the serum vials and imaged at room temperature. Images were captured in both bright-field and epi-fluorescence modes using a high-resolution digital camera (Orca HR, Hamamatsu, Japan) and processed with Simple PCI software (C-Imaging, Cranberry Township, PA). A 4OX objective was used to capture the images of size- isolated microbubbles of 4-5 μm diameter, while a IOOX oil-immersion objective was used for polydispersed microbubbles and size-isolated microbubbles of 1-2 μm diameter. Subsequent image analysis was done using ImageJ 1.4g (available at National Institute of Health website).

Flow Cytometry

A FACScan Cell Analyzer (Becton-Dickinson, Franklin Lakes, NJ) was used to characterize microbubble fluorescence intensity (FL) and light scattering profiles (FSC and SSC). Voltage and gain settings for FSC, SSC and FL were adjusted to delineate the microbubble populations from instrument and sample noise. 10 μL samples were diluted with 3 mL deionized water prior to each measurement. Subsequent data analysis was done using CellQuest Pro (Becton-Dickinson, Franklin Lakes, NJ).

Size Isolation

Following production, microbubbles were collected into 30-mL syringes (length = 8.2 cm) and washed, as above. Production-washing was repeated 3-5 times, each time saving the microbubble cake and recycling the lipid infranatant. The cakes were combined and re-dispersed into 30 mL of filtered PBS. In order to ensure a high yield, the concentration of microbubbles after such re-dispersion should be at least ~1 vol%. Microbubbles of 1-2 μm and 4-5 μm diameter are isolated as follows. At least three separate experimental runs were performed for each isolation, and size distributions were measured at least three times each.

Isolation of 4-5 μm Diameter Microbubbles

Before beginning the isolation process, care was taken to remove large, visible bubbles that can form during production or subsequent handling. Microbubbles of greater than 10-μm diameter were removed by performing one centrifugation cycle at 30 RCF for 1 min. The infranatant consisting of less than 10- μm diameter microbubbles was saved and re-dispersed in 30 mL PBS, while the cake was discarded. Next, microbubbles of greater than 6-μm diameter were removed by performing one centrifugation cycle at 70 RCF for 1 min. The infranatant consisting of less than 6-μm diameter microbubbles was saved and re-dispersed to 30 mL PBS; the cake was discarded. Finally, microbubbles of less than 4-μm diameter were removed by centrifuging at 160 RCF for 1 min. This was repeated about 5-10 times, while each time the infranatant was discarded and the cake was re-dispersed in filtered PBS. Alternatively, 12-mL syringes (L = 6.3 cm) were employed and centrifuged at 120 RCF for 1 min to improve yield. These cycles were repeated until the infranatant was no longer turbid, indicating complete removal of microbubbles less than 4 μm. The final cake was concentrated to a 1-mL volume of 20 vol% glycerol solution in PBS and stored in a 2-mL serum vial with PFB headspace.

Isolation of 1-2 μm Diameter Microbubbles The infranatant collected from the 4-5 μm isolation was centrifuged at 270 RCF for 1 min for one cycle in order to remove microbubbles of approximately 3-μm diameter and above by collecting them into the cake. The infranatant consisted mostly of microbubbles 1-2 μm diameter. The target microbubbles were collected into a concentrated cake by centrifuging at 300 RCF for 10 min. The final cake was re-dispersed to a 1-mL volume of 20 vol% glycerol solution in PBS and stored in a 2- mL serum vial with PFB headspace.

Results Polydispersity of Freshly Sonicated Microbubbles

Preparation of microbubbles by sonication of a 50 mL lipid mixture resulted in a polydisperse suspension of approximately 10⁹ to 10¹⁰ particles mL^"1. Particle sizing with the Accusizer and Multisizer showed a distribution ranging from the lower limit of resolution, -0.5 μm, to greater than 15 μm diameter (Fig. 3). A significant portion of the freshly generated suspension contained submicron microbubbles. Submicron microbubbles also have been observed by static light scattering and freeze-fracture transmission electron microscopy. For larger microbubbles, the number-weighted distribution tailed off near 6-8 μm diameter (Fig. 3A). The volume-weighted distribution, however, showed a significant population out to greater than 10 μm diameter (Fig. 3B). As volume is directly proportional to the cube of the diameter, microbubbles with larger diameters tended to skew the volume-weighted size distribution. Median volume-weighted diameters were chosen in order to evaluate the samples during size isolation, since this gives a more rigorous indication of the central tendency than arithmetic mean in a skewed distribution. Interestingly, the Accusizer consistently measured distinct peaks centered on approximately 1-2, 4-5, 7-8 and 9-11 μm diameter for each batch of lipid- coated microbubbles. These peaks were clear from the volume-weighted distribution, but they also could be discerned from the number-weighted distribution. In the laboratory, these peaks were observed for a variety of gas and lipid combinations (data not shown). Size distribution was also measured using a Multisizer III. While the Accusizer measures size based on light obscuration and scattering, the Multisizer utilizes electrical impedance sensing of the volume of electrolyte displaced by the microbubble as it passes through an orifice. The multimodal distribution was not observed on the Multisizer, which gave a broad distribution with a single peak located at ~1 μm for the number-weighted distribution and ~8 μm for the volume-weighted distribution. From this data, it was unclear whether the multimodal distribution was real, and could not be resolved by the Multisizer, or if it was an artifact of the Accusizer. Therefore an improved characterization of the microbubble distribution was sought.

Microscopy allowed direct visual inspection of individual microbubbles from the suspension. Bright-field and epi-fluorescence microscopy images are shown in Fig. 4 (Bright-field images (left) and fluorescence images (right) of the membrane probe DiO; Arrows point to microstructural features of high probe density. The initial, polydisperse microbubble suspensions (A, B) are shown for comparison to the isolated 4-5 μm diameter microbubbles (C, D) and 1-2 μm diameter microbubbles (E, F). Analysis of the bright-field images using ImageJ gave mean diameters of 4.6 ± 0.3 μm and 1.8 ± 0.3 μm for microbubbles seen in Figs. 4C and 4E, respectively). In fluorescence mode, microbubbles appeared as bright rings with dark centers, clearly showing uptake of DiO into the shell, hi bright- field mode, microbubbles appeared as dark spheres with bright centers. Diffraction rings were particularly prevalent for the smaller microbubbles. This confirmed the predominance of gas-filled microbubbles in the suspension. Analysis of the bright- field images using ImageJ indicated that the distribution of the freshly generated microbubbles was multimodal, with a mean diameter of 4.0 ± 3.0 μm for the image shown in Figure 4A.

Flow cytometry was used to further characterize the polydisperse microbubbles, as shown in Fig. 5 (Fig. 5(A) shows the serpentine trend for the initial polydisperse microbubble suspension (cytometer settings: detector Pl, voltage EOl, amp 1.98; detector P2, voltage 287, amp 1.49); Fig. 5(B) shows no serpentine trend for the isolated 4-5 μm microbubbles (cytometer settings: detector Pl, voltage EOl, amp 2.30; detector P2, voltage 173, amp 2.88)). Forward- (FSC) and side- (SSC) light scattering measurements were taken. A serpentine shape was observed on the dot plot of FSC versus SSC for the polydisperse microbubble suspensions, as shown in Figure 5 A. The serpentine shape appeared to correlate with the distinct peaks found by the Accusizer.

The origins of polydispersity in the freshly generated suspension of lipid-coated microbubbles observed here can be explained by the multiple interacting mechanisms occurring during entrainment and cavitation-induced disintegration, as described above. The fact that the microbubbles themselves can be oscillating in the acoustic field and can act as cavitation nuclei adds further complexity to analysis. Additionally, the dynamics of lipid adsorption and spreading and monolayer shell formation are expected to play a role in determining the "apparent surface tension" and, for the lipids used here, can be expected to add additional surface viscosity and elasticity terms. While polydispersity can be unavoidable, the ability of mechanical agitation to rapidly generate large numbers of microbubbles brings this technique to the forefront of current microbubble creation methods. Given the excellent stability of lipid-coated microbubbles and the apparent presence of distinct peaks in the multimodal distribution, size isolation by differential centrifugation appeared to be a feasible approach. In the following, experiments for isolating narrow distributions of target microbubbles and characterization of their size distribution and long-term stability are described.

Size Isolation of Microbubbles

Submicron microbubbles were found to be relatively unstable and therefore were not isolated. Instead, microbubbles in the 1-2 μm and 4-5 μm diameter ranges were isolated. These ranges are interesting for biomedical applications. While both sizes are comparable to that of an erythrocyte, they can yield different biodistributions, resonance frequencies, and acoustically induced bioeffects. In general, the 1-2 μm microbubbles were approximately 100-fold more abundant than the 4-5 μm microbubbles in the initial dispersion.

Microbubbles in the larger diameter range (4-5 μm) were isolated first, while the smaller microbubbles were saved for the subsequent isolation of the 1-2 μm fraction. After repeated centrifugation and re-concentration according to the simple model, microbubbles with diameters of 4-5 μm were successfully isolated from the initial polydisperse suspension, as shown in Fig. 6 (shown are isolated subpopulations at the 1-2 μm (A, B) and 4-5 μm (C, D) diameter size ranges; results are summarized in Table 1). Table 1 : Summary of size distributions.

Multiple centrifugations were needed to expel smaller microbubbles (< 4 μm), which were more abundant in the initial suspension. The final 4-5 μm microbubble suspension typically had a total volume of 1 mL with concentration in the order of 10⁸ to 10⁹ mL^"1, as determined by the Accusizer. Table 1 summarizes both averaged number-weighted and volume-weighted mean and median values for each size fraction. Microbubbles of 1-2 μm diameter were isolated in fewer centrifugations than for the 4-5 μm microbubbles. For instance, separation of microbubbles less than 2 μm diameter in the infranatant was typically completed by a single centrifugation. However, the final part of the process for concentrating microbubbles greater than 1-μm diameter required substantially higher centrifugal force than for the 4-5 μm microbubbles, which is consistent with their lower buoyancy. The final 1-2 μm microbubble suspension typically had a total volume of 1 mL with concentration on the order of 10⁹ to 10¹⁰ mL^"1, as determined by the Accusizer.

Characterization of the isolated microbubbles

Table 1 gives the average PI value for the freshly generated microbubbles and the size-isolated microbubbles. The initial suspension was highly polydisperse, with PI values as high as 18 but no lower than 6. The PI for the 4-5 μm fraction was only 1.5 ± 0.1, while that of the 1-2 μm fraction was only 1.5 ± 0.2. Bright-field and epi-fiuorescence microscopy images provided direct visual confirmation for the narrow size distribution of size-isolated microbubbles, as shown in Fig. 4. These results are in agreement with the size distributions determined by the Accusizer and Multisizer. Fluorescence images also showed microstructural features within the lipid shell. For example, brighter spots indicating non-uniform fluorophore distribution were often observed (see arrows in Figures 4D and 4F).

Flow cytometry was performed to characterize the size-isolated fractions, as shown in Fig. 7. (For Fig. 7, three different tests (fluorescence intensity FL, forward- FSC and side- SSC light scattering versus particle count) were performed for the same sample as represented by each column of plots. Column 1 (A, D, G) and Column 2 (B, E, H) samples of Fig. 7 had median volume-weighted diameters of 1.8 μm and 4.6 μm, respectively. Column 3 (C, F, I) was a mixture of these two suspensions. FL measurements (A-C) confirmed their relative size distributions with median values of 500, 1114 and 792, respectively. FSC (D-F) and SSC (G-I) measurements showed a similar tend: (FSC) 131, 214 and 194; (SSC) 286, 349 and 376, respectively. Results are summarized in Table 2.)

Table 2. Summary of flow cytometry measurements.

Fluorescence intensity (FL), FSC and SSC measurements were all taken under the same cytometer settings. The serpentine shape was not observed for the size-isolated suspensions, as it was for the polydisperse case. Instead, the data points were found to be clustered in one region of the dot plot. The lack of the serpentine shape in the size-isolated samples indicated that they were indeed subpopulations of the initial multimodal sample. Table 2 lists the median values of three cytometry tests for each microbubble sample. Comparison of the FSC and SSC results for individual, size-isolated fractions and their mixture supported the existence of two distinct microbubble subpopulations. Monomodal distributions were observed for the individual size-isolated suspensions, with a lower median value corresponding to the 1-2 μm microbubbles. When the size-isolated microbubbles were subsequently mixed together, a bimodal distribution appeared with two distinct peaks that agreed with the respective median values for the individual suspensions.

A single peak was observed on the FL histogram for the size isolated microbubbles (Fig. 7). The median FL value for 1-2 μm microbubbles was lower than that for the 4-5 μm microbubbles. Upon mixing, a bimodal distribution was observed with peak median FL values corresponding to those of the individual suspensions. Assuming that each microbubble is a perfect sphere and the fraction of fluorophores in the lipid shell is the same for all microbubbles, regardless of size, the number of fluorophores per microbubble should be directly proportional to the surface area, or the square of the diameter. This was confirmed when comparing the averaged FL values versus microbubble squared diameter. The fluorescence intensity value for the mixture of 1-2 μm and 4-5 μm microbubble samples (775 ± 18) agreed with the average between the FL values measured for each individual monodisperse suspension (510 ± 16 and 1110 ± 35). In brief, the above results demonstrated the effectiveness of the isolation methods for isolating distinct fractions of the microbubbles at the desired size ranges.

Stability of Size-Isolated Microbubbles For biomedical applications, it is desired that the microbubbles be stable for at least 48 hours at their respective size distributions. A test of microbubble stability was performed using samples concentrated to 10¹⁰ mL^"1 for 1-2 μm microbubbles, and 10⁸ mL^"1 or 10⁹ InL^"1 for 4-5 μm microbubbles, in a 1-mL volume of 20 vol% glycerol in PBS and stored in a sealed 2-mL serum vial with PFB headspace, as shown in Fig. 8. (Fig. 8 shows the distributions at various time points for the 1-2 μm (A, B) and 4-5 μm (C, D, E, F) diameter microbubbles. Number- weighted (A, C, E) and volume-weighted (B, D, F) distributions are shown for inspection of polydispersity. The suspensions initially were dispersed in 1-mL volume of PBS with 20 vol% glycerol, with a concentration of ~10¹⁰ mL^"1 for the 1-2 μm diameter microbubbles and either ~10⁸ mL^"1 (C, D) or ~10⁸ mL^"1 (E, F) for the 4-5 μm diameter microbubbles. Each curve is the average of three experiments with three measurements each. Results are summarized in Table 3.) Table 3 : Summary of stability of size-isolated microbubble suspensions.

Table 3 shows the concentration and PI for the 1-2 and 4-5 μm microbubbles at various time points following size isolation. Both size fractions were stable over two days. Microscopy after two days storage also indicated the persistence of intact microbubbles at their isolated size range over this timeframe. However, results indicated that the microbubbles underwent ripening during longer term storage. For 1-2 μm microbubbles, the concentration decreased from by an order of magnitude, and PI nearly doubled over a period of 28 days. For 4-5 μm microbubbles at less than 1 vol % encapsulated gas, the concentration decreased by more than half, and PI nearly doubled over a period of 14 days. Higher microbubble concentrations provided much greater stability, as seen for the comparison of the 4-5 μm microbubbles in Fig. 8. m general, encapsulated gas fractions were found to be greater than 1 vol % were necessary for good stability, particularly when the vial is intermittently opened to the atmosphere as typically occurs for an in vivo study (data not shown).

When measuring the number-weighted distribution with the Accusizer, the monomodal peak for the 4-5 μm microbubbles changed to a bimodal peak during storage. Fig. 8C shows that 4-5 μm microbubbles degraded into 1-2 μm microbubbles as well as larger microbubbles over the test period. The formation of the larger microbubbles is consistent with Ostwald ripening, in which small microbubbles shrink at the expense of larger ones, as a major factor affecting microbubble stability.

EXAMPLE 2. High Frequency Ultrasonic Imaging Using Isolated Microbubbles

Materials and Methods

Initial polydisperse microbubbles were generated using the materials and methods as described in Example 1. Size isolation methods as described in the application were used to isolate microbubbles of 1-2, 4-5, and 6-8 μm diameter from polydisperse microbubbles. These microbubbles are characterized using multiple sizing/counting techniques. Ultrasound imaging of a mouse kidney was performed at 40 MHz (fundamental) using a Visualsonics Vevo 770 during tail-vein, bolus injections of size-sorted and unsorted microbubble suspensions (0.1 niL; 5 x 10 #/mL). Time-intensity ultrasound contrast curves were generated and fit to a single- compartment pharmacokinetic model. Area under the curve (AUC), circulation persistence (t*) and clearance rate (k2) were assessed in the studies.

Results

The results demonstrate that contrast video intensity of the ultrasound image increases with increasing microbubble size and concentration, as illustrated in Fig. 9. (Fig. 9 shows time-intensity curves of strength and duration of the backscattered contrast signal in a mouse kidney, illustrating the effect of size of microbubbles on ultrasound backscattered intensity.) For example, the AUC increased over 4-fold for 6-8 μm diameter microbubbles as compared with for 4-5 μm microbubbles at a concentration of 10 #/mL. Likewise, the AUC increased by 83% from 10 to 10 #/mL for 6-8 μm diameter microbubbles. At high enough concentration, shadowing occurred for all microbubble sizes; the threshold concentration for shadowing decreased with increasing microbubble size. Surprisingly, 1-2 μm diameter microbubbles exhibited only shadowing and did not increase the video intensity for any of the concentrations tested. Contrast persistence was also found to increase with microbubble size and concentration. For example, t* increased from 120 ± 26 sec to 442 ± 102 sec for 4-5 μm vs. 6-8 μm diameter microbubbles at a concentration of 10⁸ #/mL. k2 was found to be independent of microbubble size and concentration.

The above results showed that, for high-frequency imaging in mice, large microbubbles strongly increase the backscattered signal, while small microbubbles mainly attenuate. This is an important finding considering that several commercially available lipid-based contrast agents have a large percentage of microbubbles less than 2 μm in diameter. The results suggest that use of larger microbubbles requires orders of magnitude lower microbubble concentrations without a concomitant decrease in circulation persistence, which has important implications on applications such as ultrasound molecular imaging and ultrasound-assisted drug delivery.

The foregoing merely illustrates the principles of the disclosed subject matter. Various modifications and alterations to the described embodiments will be apparent to those skilled in the art in view of the teachings herein. It will be appreciated that those skilled in the art will be able to devise numerous modifications which, although not explicitly described herein, embody the principles of the disclosed subject matter and are thus within its spirit and scope.

Claims

WHAT IS CLAIMED IS:

1. A method for isolating target microbubbles having a predetermined size range from polydisperse microbubbles, comprising:

applying a first centrifugal field having a first field strength to a suspension comprising the polydisperse microbubbles for a first duration of time, thereby forming a first infranatant comprising at least a portion of target microbubbles and a first supernatant cake comprising microbubbles having a greater size than the target microbubbles;

removing the first supernatant cake;

applying a second centrifugal field having a second field strength to the first infranatant for a second duration of time, the second field strength being greater than the first field strength, thereby forming a second supernatant cake comprising at least a portion of the target microbubbles and second infranatant comprising microbubbles having a smaller size than the target microbubbles; and

isolating the second supernatant cake.

2. A method for isolating target microbubbles having a predetermined size range from polydisperse microbubbles, comprising:

applying a first centrifugal field having a first field strength to a suspension comprising the polydisperse microbubbles for a first duration of time, thereby forming a first infranatant comprising at least a portion of target microbubbles and a first supernatant cake comprising microbubbles having a greater size than the target microbubbles;

removing the first supernatant cake;

applying a second centrifugal field having a second field strength to the first infranatant for a second duration of time, the second field strength being greater than the first field strength, thereby forming a second infranatant comprising at least a portion of target microbubbles and a second supernatant cake comprising microbubbles having a greater size than the target microbubbles; removing the second supernatant cake;

applying a third centrifugal field having a third field strength to the second infranatant for a third duration of time, the third field strength being greater than the second field strength, thereby forming a third supernatant cake comprising at least a portion of the target microbubbles and third infranatant comprising microbubbles having a smaller size than the target microbubbles; and

isolating the third supernatant cake.

3. The method of claim 1, wherein the applied first centrifugal field and the first duration of time are sufficient to cause substantially all the microbubbles in the polydisperse microbubbles having a greater size than the target microbubbles to form the first supernatant cake.

4. The method of claim 2, wherein the applied second centrifugal field and the second duration of time are sufficient to cause substantially all the microbubbles in the first infranatant having a greater size than the target microbubbles to form the second supernatant cake.

5. The method of claim 3, wherein the applied second centrifugal field and the second duration of time are sufficient to cause substantially all the target microbubbles to form the second supernatant cake.

6. The method of claim 4, wherein the applied third centrifugal field and the third duration of time are sufficient to cause substantially all the target microbubbles to form the third supernatant cake.

7. The method of claims 1, further comprising:

redispersing the isolated second supernatant cake into a new dispersion;

applying a third centrifugal field having a third field strength to the new dispersion for a third duration of time, thereby forming a third supernatant cake comprising at least a portion of the target microbubbles; and

isolating the third supernatant cake.

8. The method of claims 2, further comprising:

redispersing the isolated third supernatant cake into a new dispersion;

applying a fourth centrifugal field having a fourth field strength to the new dispersion for a fourth duration of time, thereby forming a fourth supernatant cake comprising at least a portion of the target microbubbles; and

isolating the fourth supernatant cake.

9. The method of claim 1, wherein the total volume fraction of the microbubbles in the polydisperse microbubbles suspension and in the first supernatant is equal to or below about 20%,

wherein at least applying one of the first and the second centrifugal fields comprises first determining the centrifugal field strength to be applied using a Stoke's equation, the Stoke's equation correlating the rise velocity of a microbubble in a suspension relative to the bulk fluid under creeping flow conditions with the size of the microbubble, the centrifugal field strength to be applied, and the effective viscosity of the suspension,

wherein the determination of the respective centrifugal field strengths is based at least on the duration of time during which the respective centrifugal field is to be applied.

10. The method of claim 1, wherein the polydisperse microbubbles include microbubbles having sizes from smaller than about 1 μm to larger than about 10 μm.

11. The method of claim 1, wherein the target microbubbles have a size range of about 4 μm to about 5 μm.

12. The method of claim 1, wherein the target microbubbles have a size range of about 1 μm to about 2 μm.

13. The method of claim 1, wherein the polydisperse microbubbles are coated at least in part with lipids.

14. The method of claim 1, wherein the polydisperse microbubbles are coated at least in part with polymeric surfactants.

15. The method of claim 1, wherein the core of the polydisperse microbubbles comprises perfluorobutane.

16. The method of claim 1, wherein the polydisperse microbubbles are obtained by sonication.

17. The method of claim 1, wherein the polydisperse microbubbles have a multimodal size distribution.

18. The method of claim 1, wherein at least one of the polydisperse microbubbles suspension and the first supernatant is placed in a syringe when being subjected to the respective first or the respective second applied centrifugal field, the syringe having a longitudinal axis, a length, a cap, and a drainage portion, wherein the drainage portion is positioned further away from the center of the centrifugal field relative to the cap.

19. Monodisperse microbubbles having a predetermined size range prepared by any one of the methods of claim 1 or claim 2.

20. A method of performing high frequency ultrasonic imaging, comprising:

administering monodisperse microbubbles having an number-averaged size of between about 1 to 10 μm to an animal; and

performing ultrasonic imaging on the animal at a fundamental frequency of at least about 30 MHz.

21. The method of claim 20, wherein the monodisperse microbubbles have a size range of about 1 μm to about 2 μm.

22. The method of claim 20, wherein the monodisperse microbubbles have a size range of about 4 μm to about 5 μm.

23. The method of claim 20, wherein the monodisperse microbubbles have a size range of about 6 μm to about 8 μm.