Gas nano / microbubble compositions and uses thereof

Abstract

Provided herein are bubble populations optimized for certain contrast and therapeutic ultrasound uses, methods of making such populations, and methods of use thereof.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of the priority date of U.S. Provisional Application No. 63 / 308,487, filed February 9, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] 2. Background of the Invention Lipid-encapsulated bubbles can be formed by vigorously shaking a container containing a lipid formulation, such as a lipid suspension, and a gas. Bubbles formed in this manner have been used in imaging applications. Summary of the Invention

[0003] Summary of the Invention The present invention relates to optimized populations of lipid-encapsulated bubbles, methods for producing such populations, and methods for using such populations in combination with ultrasound for imaging applications, such as various therapeutic and diagnostic imaging applications. These populations may include nanobubbles and / or microbubbles (for brevity, they are referred to herein as nano / microbubble populations). The use of the nano / microbubble populations provided by the present invention may improve the efficacy of certain therapeutic and / or contrast (e.g., diagnostic imaging) ultrasound applications. The disclosed manufacturing methods include forming selectively enriched nano / microbubble populations using optimized activation criteria and / or unique lipid formulations. Each optimized population may be produced using unique activation parameters, including, for example, activation time and activation rate, and / or unique formulations.

[0004] The nano / microbubble populations provided herein can be used therapeutically with ultrasound. These methods involve the nano / microbubbles absorbing ultrasound energy and then expanding and compressing (e.g., in the case of stable cavitation) or bursting (e.g., in the case of inertial cavitation). The extent to which the nano / microbubbles expand and compress or burst is influenced, in part, by the nano / microbubble size (typically measured by diameter) and composition along with the ultrasound frequency and peak pressure difference. Ultrasonic frequencies and pressures that induce resonance with (in the case of inertial cavitation) or without (in the case of stable cavitation) destruction of the nano / microbubbles can impart disruptive energy to the local environment, which can subsequently induce a variety of intended outcomes, such as thrombus destruction and subsequent blood flow restoration, blood-brain barrier (BBB) ​​destruction, tumor destruction, improved organ blood flow, and the like. Additionally, in the case of inertial cavitation, the nano / microbubbles can be used to deliver therapeutic agents to regions of interest, including, for example, the brain or solid tumors. Lipid-encapsulated gas microbubbles are widely used for contrast-enhanced ultrasound (CEU) imaging. Some of the nano / microbubble populations provided herein, including those enriched for microbubbles having diameters of about 2 to about 6 microns or about 6 to about 10 microns or about 2 to about 10 microns, may also be used with ultrasound in imaging applications where traditional ultrasound contrast agents (UCAs) have been used in the past. These imaging applications include, but are not limited to, cardiography (e.g., using UCAs to opacify the left ventricular cavity and to improve delineation of the left ventricular endocardial border in cases of inadequate echocardiograms). The populations provided herein that are enriched for certain microbubbles (e.g., those having diameters of about 2 microns to about 6 microns or about 6 microns to about 10 microns) may provide better imaging quality than was previously possible due to the higher proportion of these microbubbles.

[0005] The present invention further provides populations of lipid-encapsulated gas nano / microbubbles optimized for therapeutic use in combination with ultrasound, including low frequency ultrasound in the 2.25 MHz range and high frequency ultrasound in the 7.5 MHz range, These optimized populations have different size and concentration characteristics than have been previously produced and used for imaging (e.g., diagnostic) and therapeutic applications.

[0006] Thus, certain imaging and treatment methods provided herein involve the improved use of populations of nano / microbubbles in specific formulations, optionally applied at specific rates and / or doses, along with optimized ultrasound frequency, mechanical index (MI), pulse duration and / or number and / or timing of ultrasound applications.

[0007] Thus, in one embodiment, the present invention provides a composition comprising a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 75% of the lipid-encapsulated perfluorocarbon bubbles are nanobubbles having a diameter ranging from about 100 nanometers to about 1 micron or less, and wherein the lipid-encapsulated perfluorocarbon bubbles comprise DPPC and PEG-DPPE. In one embodiment, at least about 80% of the lipid-encapsulated perfluorocarbon bubbles are nanobubbles having a diameter ranging from about 100 nanometers to about 1 micron or less. The composition contains at least 7.0×10 such nanobubbles per mL. 9 ~2.5×10 10 or 1 x 10 such nanobubbles per mL 10 ~2.5×10 10 It may contain pieces.

[0008] In certain embodiments of this and other aspects, the lipid-encapsulated perfluorocarbon bubbles comprise DPPA, DPPC and PEG-DPPE, optionally in a molar % ratio of about 10 to about 82 to about 8, including 10:82:8, or the lipid formulation comprises DPPE, DPPC and PEG-DPPE, optionally in a molar % ratio of about 10 to about 82 to about 8, including 10:82:8. In certain embodiments of this and other aspects, the PEG-DPPE is PEG5000-DPPE. In certain embodiments of this and other aspects, the PEG-DPPE is MPEG5000-DPPE.

[0009] In some embodiments of this and other aspects, the lipid-encapsulated perfluorocarbon bubbles are lipid-encapsulated perfluoropropane bubbles. In these and other aspects and embodiments, other perfluorocarbon gases can be used as perfluorocarbon gases, including but not limited to perfluoropentane, perfluoroisopentane, perfluorobutane, perfluoropentene and dodecafluoropentane. It should be understood that the various aspects and embodiments described herein in relation to perfluorocarbon gases are equally applicable to other gases, such as, but not limited to, sulfur hexafluoride gas.

[0010] In some embodiments of this and other aspects, the population of lipid-encapsulated perfluorocarbon bubbles is present in a non-aqueous lipid solution comprising DPPA, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water, or the population of lipid-encapsulated perfluorocarbon bubbles is present in a non-aqueous lipid solution comprising DPPE, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water. In some embodiments of this and other aspects, the lipids are present in a total concentration of about 3.75 mg / ml. In some embodiments of this and other aspects, the lipids are present in a total concentration of about 2.5 mg / ml.

[0011] In some embodiments of this and other aspects, the population of lipid-encapsulated perfluorocarbon bubbles is present in an aqueous lipid suspension that includes glycerol and propylene glycol. In some embodiments of this and other aspects, the total lipid concentration of the lipid suspension is about 0.75 mg / ml.

[0012] In certain embodiments of this and other aspects, the population of lipid encapsulated perfluorocarbon bubbles lacks DPPA.

[0013] In another embodiment, the invention provides a method of forming lipid-encapsulated perfluorocarbon bubbles, comprising activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol in the presence of a perfluorocarbon gas at a sufficient agitation speed and time to produce a population of lipid-encapsulated perfluorocarbon bubbles, at least 75% of which are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron. At least about 80% of the lipid-encapsulated perfluorocarbon bubbles can be nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron.

[0014] In certain embodiments of this and other aspects, the lipid formulation comprises DPPA, DPPC and PEG-DPPE, optionally in a molar % ratio of about 10 to about 82 to about 8, including 10:82:8, or the lipid formulation comprises DPPE, DPPC and PEG-DPPE, optionally in a molar % ratio of about 10 to about 82 to about 8, including 10:82:8. In certain embodiments of this and other aspects, the perfluorocarbon gas is perfluoropropane gas.

[0015] In some embodiments, the lipid formulation is activated with an agitation time of about 50 seconds to about 90 seconds and an agitation speed of about 6000 rpm. In some embodiments, the lipid formulation is activated with an agitation time of about 50 seconds, about 60 seconds, about 75 seconds, or about 90 seconds and an agitation speed of about 6000 rpm.

[0016] In some embodiments of this and other aspects, lipid formulation is present in a 2mL Wheaton glass vial with an internal volume of about 3.8ml, and contains about 0.35ml lipid formulation and about 3.45ml gas, or lipid formulation is present in a 2mL Wheaton glass vial with an internal volume of about 3.8ml, and contains about 0.52ml lipid formulation and about 3.28ml gas.In some embodiments of this and other aspects, lipid formulation is present in a 2mL Wheaton glass vial with an internal volume of about 3.8ml, and about 9% of the internal volume is lipid occupied by lipid formulation before activation, or about 14% of the internal volume is occupied by lipid formulation before activation.In these embodiments, lipid formulation can be a non-aqueous lipid formulation.

[0017] In certain embodiments of this and other aspects, the lipid formulation is an aqueous lipid suspension comprising DPPA, DPPC and PEG-DPPE, glycerol and propylene glycol, or the lipid formulation is an aqueous lipid suspension comprising DPPE, DPPC and PEG-DPPE, glycerol and propylene glycol. In certain embodiments of this and other aspects, the total lipid concentration of the lipid formulation is about 0.75 mg / ml.

[0018] In some embodiments, the lipid formulation is activated in the presence of gas for about 90 seconds at a shaking speed of about 4530 rpm. In some embodiments, the lipid formulation is activated in the presence of gas for about 20 seconds to about 25 seconds at a shaking speed of about 6000 rpm.

[0019] In certain embodiments, the lipid formulation lacks DPPA.

[0020] Also provided is a population of lipid encapsulated perfluorocarbon bubbles formed by any of the above methods.

[0021] In another aspect, the invention provides a method of treating a subject having a condition comprising administering to the subject an effective amount of a population of lipid encapsulated perfluorocarbon bubbles, wherein at least 75% of the bubbles in the population are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron, and the lipid encapsulated perfluorocarbon bubbles are DPPC and PEG-DPPE, and applying ultrasound energy to an area of ​​the subject having the condition.

[0022] In another aspect, the present invention provides an improvement in a method of enhancing tissue perfusion in a subject, comprising exposing the subject to therapeutic ultrasound, the improvement comprising administering to the subject a population of lipid encapsulated perfluorocarbon bubbles prior to and / or during exposure to therapeutic ultrasound, wherein at least 75% of the bubbles in the population are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron.

[0023] In certain embodiments of this and other aspects, the condition is the presence of a thrombus / clot, vascular occlusion, retinal vein occlusion, tumor, erectile dysfunction, Alzheimer's disease, Parkinson's disease. In certain embodiments of this and other aspects, the condition is one that would benefit from disruption of the blood-brain barrier (BBB), and optionally, wherein a condition-specific therapeutic agent is also applied to the subject before, during and / or after administration of the population of bubbles and / or before, during and / or after exposure to therapeutic ultrasound.

[0024] In certain embodiments of this and other aspects, the lipid-encapsulated perfluorocarbon gas nanobubbles achieve stable cavitation in vivo following application of ultrasound energy.In certain embodiments of this and other aspects, the lipid-encapsulated perfluorocarbon gas nanobubbles achieve inertial cavitation in vivo following application of ultrasound energy, as optionally detected and / or measured by emitted broadband radio waves.

[0025] Any of the above bubble populations may be used to treat these conditions.

[0026] In another aspect, the present invention provides compositions comprising a population of lipid-encapsulated perfluorocarbon gas bubbles, wherein at least 15% of the microbubbles in the population have a diameter of about 2 microns to about 6 microns, and the lipid-encapsulated perfluorocarbon gas bubbles comprise DPPC and PEG-DPPE. In one embodiment, at least about 20% of the lipid-encapsulated perfluorocarbon gas microbubbles in the population have a diameter of about 2 microns to about 6 microns. These compositions contain at least about 0.24×10 per mL. 9 ~2.44×10 9 Such bubbles may include:

[0027] In some embodiments, the population of lipid-encapsulated perfluorocarbon bubbles is present in a non-aqueous lipid solution comprising DPPA, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water, or the population of lipid-encapsulated perfluorocarbon bubbles is present in a non-aqueous lipid solution comprising DPPE, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water.

[0028] In some embodiments, a population of lipid-encapsulated perfluorocarbon bubbles is present in an aqueous lipid suspension that includes glycerol and propylene glycol.

[0029] In certain embodiments, the population of lipid encapsulated perfluorocarbon bubbles exhibits an acoustic attenuation of ultrasonic frequencies of about 1 or greater, optionally 1.2 or greater, optionally in the range of about 1.2 to about 1.7 of 2.25 MHz ultrasound.

[0030] In another embodiment, the invention provides a method of forming lipid-encapsulated perfluorocarbon gas bubbles comprising activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol in the presence of perfluorocarbon gas at a sufficient agitation speed and time to produce a population of lipid-encapsulated perfluorocarbon gas microbubbles, wherein at least 15% of the microbubbles have a diameter of about 2 microns to about 6 microns or less.

[0031] In some embodiments, about 20% or more of the lipid-encapsulated perfluorocarbon gas microbubbles have a diameter of about 2 microns to about 6 microns or less.

[0032] In certain embodiments, the lipid formulation is a non-aqueous lipid solution comprising DPPA, DPPC and PEG-DPPE, glycerol and propylene glycol and 5% (v / v) or less of water, or the lipid formulation is a non-aqueous lipid solution comprising DPPE, DPPC and PEG-DPPE, glycerol and propylene glycol and 5% (v / v) or less of water.

[0033] In some embodiments, the lipid formulation is activated with an agitation time of about 75 seconds to about 90 seconds and an agitation speed of about 6000 rpm. In some embodiments, the lipid formulation is activated with an agitation time of about 75 seconds or about 90 seconds and an agitation speed of about 6000 rpm.

[0034] In some embodiments, the lipid formulation is present in a Wheaton 2mL glass vial with an internal volume of about 3.8ml, wherein the vial contains about 0.35ml of lipid formulation and about 3.45ml of gas, or the vial contains about 0.52ml of lipid formulation and about 3.28ml of gas.In some embodiments, the lipid formulation is present in a Wheaton 2mL glass vial with an internal volume of about 3.8ml, wherein about 9% of the vial's internal volume is occupied by the lipid formulation before activation, or about 14% of the vial's internal volume is occupied by the lipid formulation before activation.

[0035] In certain embodiments, a solution of propylene glycol and glycerol is combined with the lipid formulation in an equal volume ratio prior to activation.

[0036] In certain embodiments, the lipid formulation is activated for a shaking time of about 60 seconds at a shaking speed of about 6000 rpm.

[0037] In certain embodiments, the lipid formulation is an aqueous lipid suspension comprising DPPA, DPPC, PEG-DPPE, glycerol and propylene glycol or the lipid formulation is an aqueous lipid suspension comprising DPPE, DPPC, PEG-DPPE, glycerol and propylene glycol.

[0038] In certain embodiments, the lipid formulation is activated at an agitation speed of about 6000 rpm for an agitation time of about 30 seconds.

[0039] A population of lipid-encapsulated perfluorocarbon bubbles formed by any of the above methods is also provided.

[0040] In another aspect, the invention provides a method of treating a subject having a condition, comprising administering to the subject an effective amount of a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 15% of the microbubbles in the population have a diameter of from about 2 microns to about 6 microns, and the lipid-encapsulated perfluorocarbon bubbles comprise DPPC and PEG-DPPE, and applying ultrasound energy, optionally at a frequency of about 1 to about 10 MHz, to an area of ​​the subject having the condition.

[0041] In another aspect, the invention provides a method of improving therapeutic or diagnostic ultrasound, the improvement comprising administering to a subject a population of lipid-encapsulated perfluorocarbon gas bubbles, wherein at least 15% of the microbubbles in the population have a diameter ranging from about 2 microns to about 6 microns, optionally at a frequency of about 1 to about 10 MHz, prior to or during exposure to ultrasound. In one embodiment, at least about 20% of the lipid-encapsulated perfluorocarbon gas microbubbles have a diameter ranging from about 2 microns to about 6 microns.

[0042] In certain embodiments of this and other aspects, the condition is cardiac atherosclerosis, myocardial infarction, vascular occlusion, stroke, peripheral arterial disease.

[0043] In certain embodiments of this and other aspects, the lipid encapsulated perfluorocarbon bubbles reach stable cavitation in vivo after application of ultrasonic energy, optionally as detected and / or measured by harmonic frequency emission. In certain embodiments of this and other aspects, the lipid encapsulated perfluorocarbon bubbles reach inertial cavitation in vivo after application of ultrasonic energy, optionally as detected and / or measured by broadband radio waves.

[0044] In certain embodiments of this and other aspects, the population of lipid-encapsulated perfluorocarbon gas bubbles has an acoustic attenuation at a 2.25 MHz ultrasonic frequency of greater than about 1.0. In certain embodiments of this and other aspects, the population of lipid-encapsulated perfluorocarbon gas microspheres has an acoustic attenuation at a 2.25 MHz ultrasonic frequency of about 1.0 to about 1.5 or about 1.0 to about 2.0.

[0045] In another aspect, the invention provides a composition comprising a population of lipid-encapsulated perfluorocarbon gas bubbles, wherein at least 4% of the microbubbles in the population have a diameter of about 6 microns to about 10 microns, and the lipid-encapsulated perfluorocarbon gas bubbles comprise DPPC and PEG-DPPE. In one embodiment, at least about 10% of the lipid-encapsulated perfluorocarbon gas microbubbles in the population have a diameter of about 6 microns to about 10 microns. These compositions contain at least about 0.1×10 8 ~0.32×10 8 may contain such bubbles.

[0046] In some embodiments, the population of lipid encapsulated perfluorocarbon bubbles exhibits an acoustic attenuation at 2.25 MHz ultrasound frequencies in the range of greater than about 1, optionally greater than about 1.2, and further optionally in the range of about 1 to about 1.7.

[0047] In some embodiments, the population of lipid encapsulated perfluorocarbon bubbles lacks DPPA.

[0048] In another aspect, the invention provides a method of forming lipid-encapsulated perfluorocarbon gas bubbles, comprising activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol in the presence of perfluorocarbon gas at a sufficient agitation speed and time to produce lipid-encapsulated perfluorocarbon gas microbubbles, wherein at least 4% of the microbubbles have a diameter of about 6 microns to about 10 microns. In one embodiment, at least about 10% of the lipid-encapsulated perfluorocarbon gas microbubbles have a diameter of about 6 microns to about 10 microns.

[0049] In certain embodiments, the lipid formulation is a non-aqueous lipid solution comprising DPPA, DPPC and PEG-DPPE, glycerol and propylene glycol and 5% (v / v) or less of water, or the lipid formulation is a non-aqueous lipid solution comprising DPPA, DPPC and PEG-DPPE, glycerol and propylene glycol and 5% (v / v) or less of water.

[0050] In certain embodiments, the lipid formulation is present in a 2 mL glass vial manufactured by Wheaton having an internal volume of about 3.8 ml, where the vial contains about 0.35 ml of lipid formulation and about 3.45 ml of gas, or where the vial contains about 0.52 ml of lipid formulation and about 3.28 ml of gas.

[0051] In certain embodiments, the lipid formulation is present in a 2 mL glass vial manufactured by Wheaton having an internal volume of about 3.8 ml, where about 9% of the internal volume of the vial is occupied by the lipid formulation prior to activation, or where about 14% of the internal volume of the vial is occupied by the lipid formulation prior to activation.

[0052] In certain embodiments, the lipid formulation is activated for a shaking time of about 60 seconds at a shaking speed of about 6000 rpm.

[0053] In certain embodiments, the lipid formulation is an aqueous lipid suspension comprising DPPA, DPPC, PEG-DPPE, glycerol and propylene glycol or the lipid formulation is an aqueous lipid suspension comprising DPPE, DPPC, PEG-DPPE, glycerol and propylene glycol.

[0054] In another aspect, the invention provides a method of treating a subject having a condition comprising administering to the subject an effective amount of a population of lipid encapsulated perfluorocarbon bubbles, wherein at least 4% of the microbubbles in the population have a diameter of about 6 microns to about 10 microns, and the lipid encapsulated perfluorocarbon bubbles comprise DPPC and PEG-DPPE, and applying ultrasound energy at a frequency of about 1 to about 10 MHz to an area of ​​the subject having the condition.

[0055] In another aspect, the invention provides a method of improving therapeutic or diagnostic ultrasound, the improvement comprising administering to a subject a population of lipid-encapsulated perfluorocarbon gas bubbles, wherein at least 4% of the microbubbles in the population have a diameter in the range of about 6 microns to about 10 microns, prior to or during administration of ultrasound at a frequency of about 1 to about 10 MHz. In one embodiment, at least 10% of the lipid-encapsulated perfluorocarbon gas microbubbles have a diameter of about 6 microns to about 10 microns.

[0056] In certain embodiments of this and other aspects, the condition is cardiac atherosclerosis, myocardial infarction, vascular occlusion, stroke, peripheral arterial disease.

[0057] In certain embodiments, the population of lipid encapsulated perfluorocarbon bubbles exhibits an acoustic attenuation at a 2.25 MHz ultrasound frequency of greater than about 1.0.

[0058] In certain embodiments, a population of lipid encapsulated perfluorocarbon gas microspheres exhibits an acoustic attenuation at a 2.25 MHz ultrasound frequency of about 1.0 to about 1.5 or about 1.0 to about 2.0.

[0059] In another aspect, the invention provides a method of ultrasound imaging of a subject, comprising administering to a subject any of the populations of lipid-encapsulated perfluorocarbon bubbles described above, or any of the lipid-encapsulated perfluorocarbon bubbles produced by any of the methods described above, applying ultrasound energy at a frequency in the range of about 1 to about 10 MHz, and obtaining an ultrasound image of the subject or a region of the subject.

[0060] In one embodiment, the perfluorocarbon gas is perfluoropropane gas.

[0061] In certain embodiments, the ultrasonic energy is at a frequency of about 1.25 MHz or about 2.25 MHz.

[0062] In another aspect, the present invention provides a method of forming lipid-encapsulated perfluorocarbon gas bubbles, comprising activating a non-aqueous lipid formulation comprising up to 5% (v / v) water, DPPC, PEG-DPPE, glycerol, propylene glycol DPPA or DPPE in the presence of perfluorocarbon gas at a shaking speed of about 6000 rpm for about 30 to about 90 seconds, optionally about 50 seconds, about 60 seconds, about 75 seconds or about 90 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres.

[0063] In certain embodiments, the lipid formulation and gas are present in a vial, optionally a 2 mL vial, and the lipid formulation occupies about 9% or about 14% of the internal volume of the vial.

[0064] In another embodiment, the invention provides a method of forming lipid-encapsulated perfluorocarbon gas bubbles, comprising activating a non-aqueous lipid formulation comprising up to 5% (v / v) of either water, DPPC, PEG-DPPE, glycerol, propylene glycol DPPA or DPPE in the presence of a perfluorocarbon gas at a shaking speed of about 4950 rpm for about 125 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres. In one embodiment, the lipid formulation and gas are present in a vial, optionally a 2 mL vial, and the lipid formulation occupies about 9% or about 14% of the internal volume of the vial.

[0065] In another aspect, the invention provides a method of forming lipid encapsulated perfluorocarbon gas bubbles comprising activating an aqueous lipid formulation comprising DPPC and PEG-DPPE and glycerol and propylene glycol and either DPPA or DPPE in the presence of perfluorocarbon gas at a shaking speed of about 6000 rpm for about 5 to about 90 seconds, optionally about 5 seconds, about 10 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 60 seconds, about 70 seconds, about 80 seconds or about 90 seconds, thereby forming a population of lipid encapsulated perfluoropropane gas microspheres.

[0066] In another aspect, the present invention provides a method of forming lipid-encapsulated perfluorocarbon gas bubbles, comprising activating an aqueous lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol, and either DPPA or DPPE in the presence of perfluorocarbon gas at a shaking speed of about 4530 rpm for about 90 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres.

[0067] In another aspect, the invention provides a method of forming lipid-encapsulated perfluorocarbon gas bubbles, comprising activating an aqueous lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol, and either DPPA or DPPE, in the presence of perfluorocarbon gas at a shaking speed of about 4530 rpm for about 10 to about 20 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres.

[0068] In another aspect, the present invention provides a method of forming lipid-encapsulated perfluoropropane nanodroplets, comprising condensing a population of lipid-encapsulated perfluoropropane gas microspheres by applying sufficient pressure to condense the microbubbles into nanodroplets, wherein the population of gas microspheres is condensed at room temperature.

[0069] In some embodiments, the application of pressure comprises the application of finger pressure, hi some embodiments, the application of pressure comprises the application of pressure in the range of about 6 to about 12 atmospheres.

[0070] In some embodiments, the population of lipid-encapsulated perfluoropropane gas microspheres is provided in a syringe, optionally a 3 mL syringe or a 5 mL syringe, and the application of pressure comprises the application of finger pressure via a plunger. In some embodiments, the population of lipid-encapsulated perfluoropropane gas microspheres is produced by activation of a lipid formulation comprising DPPA, DPPC and PEG5000-DPPE in the presence of perfluoropropane. In some embodiments, the population of lipid-encapsulated perfluoropropane gas microspheres is produced by activation of a lipid formulation comprising DPPC, DPPE and PEG5000-DPPE in the presence of perfluoropropane. In some embodiments, the lipid formulation is a non-aqueous lipid formulation, and optionally the activation of the lipid formulation comprises activation at about 4950 rpm for 45 seconds, or at about 6000 rpm to about 6200 rpm for about 60 seconds, or at about 6600 rpm for about 15 seconds to about 30 seconds. In certain embodiments, the lipid formulation is an aqueous lipid formulation, and optionally activating the lipid formulation comprises activation at about 4530 rpm for 45 seconds.

[0071] In another aspect, the invention provides a method of diagnosing or treating a subject, comprising administering to the subject a population of lipid-encapsulated nanodroplets and applying ultrasound energy to the subject, optionally at a target location, wherein the population of lipid-encapsulated nanodroplets is produced using the methods described herein.

[0072] In some embodiments, the nanodroplets comprise a lipid shell comprised of DPPA, DPPC and PEG5000-DPPE and a liquid center of perfluoropropane. In some embodiments, the nanodroplets comprise a lipid shell comprised of DPPC, DPPE and PEG5000-DPPE and a liquid center of perfluoropropane. In some embodiments, the nanodroplets lack DPPA. In some embodiments, the nanodroplets are about 100 to about 200 nanometers in size.

[0073] In another aspect, the present invention provides a method of treating a subject having erectile dysfunction, comprising administering to a subject in need thereof an effective amount of lipid encapsulated bubbles or nanodroplets and applying ultrasound to the subject's genital area.

[0074] In some embodiments, the subject with erectile dysfunction has mild to moderate erectile dysfunction as assessed by IIEF-5 or IIEF-15 score. In some embodiments, treatment is evidenced by an increase in IIEF-5 or IIEF-15 score after infusion and ultrasound, optionally within 1 to 7 days after infusion and ultrasound.

[0075] In some embodiments, lipid-encapsulated bubbles are administered to a subject, comprising lipid shells that comprise DPPA, DPPC and MPEG5000-DPPE and perfluoropropane gas center, where lipid-encapsulated bubbles are present in activated DEFINITY formulation or activated DEFINITY RT formulation.In some embodiments, lipid-encapsulated bubbles are administered to a subject, comprising lipid shells that comprise DPPC, DPPE and MPEG5000-DPPE and perfluoropropane gas center.In some embodiments, lipid-encapsulated nanodroplets are administered to a subject, comprising lipid shells that comprise DPPA, DPPC and MPEG5000-DPPE and center that comprises perfluoropropane in liquid form, whereby lipid-encapsulated nanodroplets are optionally formed by condensation of lipid-encapsulated bubbles present in activated DEFINITY formulation or activated DEFINITY RT formulation.In some embodiments, lipid-encapsulated nanodroplets are administered to a subject, comprising lipid shells that comprise DPPC, DPPE and MPEG5000-DPPE and center that comprises perfluoropropane in liquid form.

[0076] In certain embodiments, the ultrasound optionally comprises high intensity ultrasound at a frequency in the range of 1 to 2 MHz and further optionally with a mechanical index of about 1 to about 1.5 or about 1 to about 1.3.

[0077] In certain embodiments, the method is performed repeatedly at spaced intervals, optionally the method being performed once or twice weekly for one or more weeks.

[0078] These and other aspects and embodiments of the invention are now described in further detail. [Brief description of the drawings]

[0079] [Figure 1] Photographs of a Norm Jet syringe containing DEFINITY RT activated at 6000 rpm for 60 seconds before application of manual pressure (top), before application of manual pressure and release of such pressure (middle), and after release of pressure by removing the cap (bottom). The figure shows the milky white appearance of activated DEFINITY RT before application of manual pressure and the colorless appearance of activated DEFINITY RT after application of manual pressure. The change in appearance correlates with a selective reduction in the number of bubbles that are >500 nm and continues after removal of the cap and release of pressure from the syringe. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] Detailed Description of the Invention The present invention provides nano / microbubble compositions with improved therapeutic and imaging effects, methods of manufacture, including specific devices used for the manufacture of such compositions, and methods of use in therapeutic and imaging applications. The methods of manufacture result in optimized nano / microbubble compositions for use with ultrasound for various therapeutic applications and / or various imaging applications. Different therapeutic applications may require different optimized nano / microbubble compositions. It has been unexpectedly found that such optimized nano / microbubble compositions can be produced by specific manipulation of lipid formulations, such as lipid solutions and lipid suspensions, and / or specific activation parameters. As an example, certain optimized nano / microbubble compositions provided herein can be produced using specific lipid formulation activation parameters, including activation time and activation speed (or rate), and specific manipulation of lipid formulations.

[0081] Nano / Microbubble Clusters The nano / microbubble populations provided herein are defined by their size and concentration profile, acoustic attenuation and / or production method, and their usefulness in therapeutic ultrasound applications depends in part on their size and / or acoustic attenuation profile.

[0082] As used herein, size profile of a nano / microbubble population refers to the percentage of bubbles in the population having a particular size. A size profile may, in some cases, include the percentage of bubbles of various sizes. Described below are some nano / microbubble populations defined by a size profile.

[0083] Certain embodiments of the invention provide a population of lipid-encapsulated bubbles, wherein about 75% or more of the lipid-encapsulated bubbles are nanobubbles having a diameter ranging from about 100 nanometers to about 1 micron or less (i.e., diameters of about 100 nanometers to about 1 micron or less as contemplated herein). In some cases, the diameters range from about 200 nanometers to about 1 micron or less, or from about 300 nanometers to about 1 micron or less, or from about 500 nanometers to about 1 micron or less. In relevant examples, the upper limit of the diameter range is less than 1 micron or about 950 nanometers or about 900 nanometers or about 850 nanometers or about 800 nanometers or about 750 nanometers or about 700 nanometers. Any combination of these lower and upper limits is contemplated, including about 200 nanometers to about 800 nanometers or about 500 nanometers to less than 1 micron. Thus, by way of example, 75% or more of the lipid-encapsulated bubbles can be nanobubbles having diameters ranging from about 100 nanometers to about 1 micron or less, or ranging from about 200 nanometers to about 800 nanometers. All other combinations of lower and upper limits are contemplated by the present invention.

[0084] Such a population may be said to be enriched in lipid-encapsulated bubbles having diameters in any of the above ranges. Thus, a population of lipid-encapsulated bubbles in which at least about 75% of the lipid-encapsulated bubbles have diameters in the range of about 100 nanometers to about 1 micron is considered to be enriched in lipid-encapsulated bubbles having diameters in the range of about 100 nanometers to about 1 micron. About 75%, about 80%, about 85%, about 90%, about 95% or more of the lipid-encapsulated bubbles in these populations may have any other diameter within the specified range, including a range having a lower limit as above and an upper limit of less than 1 micron or about 950 nanometers or about 900 nanometers or about 850 nanometers or about 800 nanometers or about 750 nanometers or about 700 nanometers, including a range of about 100 nanometers to about 1 micron or less, or a range of about 200 nanometers to about 1 micron or less, or a range of about 300 nanometers to about 1 micron or less, or a range of about 400 nanometers to about 1 micron or less, or a range of about 500 nanometers to about 1 micron or less.

[0085] As described below, nanobubble-enriched populations (compared to prior art populations) can be used in new therapeutic applications or to improve the therapeutic efficacy of existing therapeutic applications.

[0086] Another aspect of the invention provides a population of lipid-encapsulated gas bubbles, wherein about 15% or more of the lipid-encapsulated gas microbubbles in the population have a diameter in the range of about 2 microns to about 6 microns (i.e., diameters of about 1 micron to about 6 microns as contemplated herein). In some cases, the diameters are in the range of about 2.5 microns to about 6 microns or about 3 microns to about 6 microns or about 3.5 microns to about 6 microns. In relevant examples, the upper limit of the diameter range is less than 6 microns or about 5.5 microns or about 5 microns or about 4.5 microns or about 4 microns. Any combination of these lower and upper limits is contemplated. Thus, by way of example, more than 15% of the lipid-encapsulated gas microbubbles in the population may have a diameter in the range of about 2 microns to less than 6 microns or in the range of about 2 microns to about 5.5 microns. All other combinations of lower and upper limits are contemplated by the invention.

[0087] Such populations may be said to be enriched for lipid-encapsulated gas microbubbles having diameters in any of the above ranges. Thus, a population of lipid-encapsulated bubbles in which 15% or more of the lipid-encapsulated gas microbubbles in the population have diameters in the range of about 2 microns to about 6 microns or less is considered to be enriched for lipid-encapsulated bubbles having diameters in the range of about 2 microns to about 6 microns or less. About 15%, about 20%, about 25%, about 30%, about 35% or more of the lipid-encapsulated gas microbubbles in these enriched populations may have diameters in the range of about 2 microns to about 6 microns or less, or may have any other diameter in the specified range, including, for example, the range of about 2.5 microns to about 6 microns or the range of about 3 microns to about 6 microns or the range of about 3.5 microns to about 6 microns or less, including the ranges with the lower limit as above and an upper limit of less than 6 microns or about 5.5 microns or about 5 microns or about 4.5 microns or about 4 microns.

[0088] As described below, enrichment of the microbubble population (relative to prior art populations) in the about 2 to about 6 micron diameter range can be used for new therapeutic or imaging applications or can improve the efficacy of existing therapeutic or imaging applications. Another aspect of the invention provides a population of lipid encapsulated gas bubbles, wherein about 4% or more of the lipid encapsulated gas microbubbles in the population have a diameter in the range of about 6 microns to about 10 microns (i.e., about 6 microns to about 10 microns diameter as contemplated herein). In some cases, the diameter ranges from about 6.5 microns to about 10 microns, or about 7 microns to about 10 microns, or about 8 microns to about 10 microns. In relevant examples, the upper limit of the diameter range is about 9.5 microns or about 9.0 microns or about 8.5 microns or about 8.0 microns or about 7.5 microns. Any combination of these lower and upper limits is contemplated. Thus, by way of example, about 4% or more of the lipid-encapsulated gas microbubbles in a population may have a diameter in the range of about 6 microns to about 10 microns, or in the range of about 6 microns to about 9.5 microns. All other combinations of lower and upper limits are contemplated by the present invention.

[0089] Such populations may be said to be enriched for lipid-encapsulated gas microbubbles having diameters in any of the above ranges. Thus, a population of lipid-encapsulated bubbles in which 4% or more of the lipid-encapsulated gas microbubbles in the population have diameters in the range of about 6 microns to about 10 microns is considered to be enriched for lipid-encapsulated gas bubbles having diameters in the range of about 6 microns to about 10 microns. About 4%, about 5%, about 7%, about 10%, about 12% or more of the lipid-encapsulated gas microbubbles in these enriched populations may have diameters in the range of about 6 microns to about 10 microns or any other diameter in the specified range, including, for example, the range of about 6.5 microns to about 10 microns or the range of about 7 microns to about 10 microns or the range of about 7.5 microns to about 10 microns, including the range with the lower limit as stated above and an upper limit of about 9.5 microns or about 9 microns or about 8.5 microns or about 8 microns or about 7.5 microns.

[0090] As described below, microbubble populations enriched for microbubbles in the about 6 to about 10 micron diameter range (compared to prior art populations) can be used in new therapeutic or imaging applications or can improve the effectiveness of existing therapeutic or imaging applications.

[0091] Lipid formulations, aqueous and non-aqueous The lipid formulations used to form the gas nano / microbubbles of the present invention can be non-aqueous lipid formulations, defined as having 5% or less water (w / w). These non-aqueous lipid formulations are sometimes referred to herein as lipid solutions. Alternatively, the lipid formulations can be aqueous lipid formulations, defined as having more than 5% water (w / w). These aqueous lipid formulations are sometimes referred to herein as lipid suspensions. Both types of lipid formulations can be activated as described herein. Nano / microbubble-enriched populations can be formed using aqueous or non-aqueous lipid formulations with gas.

[0092] Suitable lipid formulations include one, two, three or more lipids. Suitable lipids are provided below. One or more such lipids may be conjugated to polyethylene glycol (PEG).

[0093] One example of an aqueous lipid formulation contains the lipids DPPA, DPPC and MPEG5000-DPPE in a 10:82:8 molar ratio with a combined lipid concentration of about 0.75 mg / ml, as well as propylene glycol and glycerol in aqueous solution. Activation of this aqueous lipid formulation in the presence of perfluoropropane gas (also known as perflutren or octafluoropropane) at 4530 rpm for 45 seconds resulted in a 10 10 nanobubbles and approximately 5 × 10 9 Approximately 66% of the bubbles produced after activation of this aqueous lipid formulation are nanobubbles. Approximately 4% of the microbubbles formed have a diameter in the range of about 2 to about 6 microns.

[0094] Other aqueous lipid formulations are similar to the above formulations, but with different lipid profiles. For example, in some aqueous lipid formulations, DPPA, DPPC and DPPE can be used in molar percentages of about 77 to about 90 mol% DPPC, about 5 to about 15 mol% DPPA, and about 5 to about 15 mol% DPPE, including DPPE-MPEG5000, such as MPEG5000-DPPE. Similar ratios can be used in non-aqueous lipid formulations as well.

[0095] Other examples of aqueous lipid formulations include phosphatidylcholine (PC), phosphatidylethanolamine (PE) and dipalmitoylphosphatidylethanolamine (DPPE), specifically lacking DPPA. The PE can be PE-PEG and / or the DPPE can be DPPE-PEG. For example, the lipid formulation can include the lipids DPPC, DPPE and DPPE-PEG. These can be present in a molar percentage ratio of 82:10:8 (DPPC:DPPE:DPPE-PEG). These lipids can be used in other molar percentages, including about 77 to about 90 mol % DPPC, about 5 to about 15 mol % DPPE and about 5 to about 15 mol % DPPE-PEG. The combined lipid concentration can range from about 0.75 mg / ml to about 1.5 mg / ml. These lipids can be suspended in propylene glycol, glycerol and saline or any combination thereof. These formulations can be activated in the presence of a perfluorocarbon gas, such as perflutren, or any other suitable gas.

[0096] An example of a non-aqueous lipid formulation contains DPPA, DPPC and MPEG5000-DPPE in a 10:82:8 molar ratio and a combined lipid concentration of about 3.75 mg / ml in propylene glycol and glycerol and up to 5% water (w / w). Activation of this non-aqueous lipid formulation in the presence of perfluoropropane gas at 4950 rpm for 45 seconds resulted in a reduction of approximately 3×10 9 Nanobubbles and about 2.5 to 3 × 10 9Approximately 50% of the bubbles produced after activation of this lipid formulation are nanobubbles. Approximately 12% of the microbubbles formed have a diameter in the range of about 2 to about 6 microns or less, and about 1.2% of the microbubbles formed have a diameter in the range of about 6 to about 10 microns.

[0097] Other non-aqueous lipid formulations are described in published PCT application PCT / US2015 / 067615, the entire contents of which are incorporated herein by reference.

[0098] Other examples of non-aqueous lipid formulations include phosphatidylcholine (PC), phosphatidylethanolamine (PE) and dipalmitoylphosphatidylethanolamine (DPPE), optionally specifically lacking DPPA. PE can be PE-PEG and / or DPPE can be DPPE-PEG. For example, the lipid formulation can include the lipids DPPC, DPPE and DPPE-PEG. These can be present in a molar percentage ratio of 82:10:8 (DPPC:DPPE:DPPE-PEG). These lipids can be used in other molar percentages, including about 77 to about 90 mol % DPPC, about 5 to about 15 mol % DPPE and about 5 to about 15 mol % DPPE-PEG. The combined lipid concentration can range from about 0.75 mg / ml to about 1.5 mg / ml. These lipids can be suspended in propylene glycol, glycerol or propylene glycol and glycerol. These formulations can be activated in the presence of a perfluorocarbon gas, such as perflutren, or any other suitable gas.

[0099] Non-aqueous lipid formulations include lipids in propylene glycol or glycerol or propylene glycol and glycerol, so long as the water content is about 5% by weight or less (i.e., weight of water vs. weight of the combination of lipids and propylene glycol and / or glycerol). In some cases, non-aqueous lipid formulations contain less than 5% water (w / w), 1-4% water (w / w), 1-3% water (w / w), 2-3% water (w / w), or 1-2% water (w / w). In some cases, non-aqueous lipid formulations contain less than 1% water (w / w). Water content can be measured at the end of manufacture (and before long-term storage) or after storage, including long-term storage, and immediately prior to use.

[0100] Lipid formulations can also be salt-free (i.e., do not contain any salt other than lipid counterions). More specifically, and as an example, lipids such as DPPA and DPPE are typically provided as sodium salts. As used herein, salt-free lipid formulations can contain such counterions (e.g., sodium when using DPPA and / or DPPE), but do not contain other ions. In some cases, non-aqueous lipid formulations do not contain sodium chloride or chloride.

[0101] Lipid formulations may contain buffer. Buffer can be, but is not limited to, acetate buffer, benzoate buffer, salicylate buffer, diethanolamine buffer, triethanolamine buffer, borate buffer, carbonate buffer, glutamate buffer, succinate buffer, malate buffer, tartrate buffer, glutarate buffer, aconite buffer, citrate buffer, lactate buffer, glycerate buffer, gluconate buffer or tris buffer. Non-phosphate buffer can be preferred in some cases due to its dissolution profile in non-aqueous solvent such as propylene glycol and / or glycerol. In some cases, phosphate buffer can be used (e.g., after or simultaneously with the addition of saline or other aqueous diluent).

[0102] In certain embodiments, the non-aqueous lipid formulation comprises, consists of, or consists essentially of (a) one or more lipids, (b) propylene glycol or glycerol or propylene glycol and glycerol, and (c) a non-phosphate buffer.

[0103] In some cases, the lipid concentration in the lipid formulation may range from about 0.1 mg to about 20 mg per mL, including about 0.6 mg to about 10 mg per mL and about 0.6 mg to about 7.5 mg per mL. In some embodiments, the lipid concentration may range from about 0.75 mg to about 3.75 mg lipid per mL, or about 0.75 mg to about 2.5 mg lipid per mL, or about 2.5 mg to about 7.5 mg lipid per mL, or about 3.75 mg to about 7.5 mg lipid per mL, or about 2.5 mg to about 3.75 mg lipid per mL. In some cases, the lipid concentration is about 2 mg to about 4 mg lipid per mL, or about 2.5 mg to about 3.75 mg lipid per mL, or about 3.75 mg to about 7.5 mg total lipid per mL.

[0104] By way of example, lipid concentrations in non-aqueous lipid formulations can range from about 0.1 mg to about 10 mg lipid per mL of propylene glycol and / or glycerol (total), including about 1 mg to about 5 mg lipid per mL of propylene glycol and / or glycerol (total), or about 2 mg to about 4 mg lipid per mL of propylene glycol and / or glycerol (total), or about 2.5 mg to about 3.75 mg lipid per mL of propylene glycol and / or glycerol (total).

[0105] As another example, the lipid concentration in the aqueous lipid formulation can range from about 0.1 mg to about 10 mg lipid per mL, including about 0.1 mg to about 5 mg lipid per mL, or about 0.3 mg to about 1 mg lipid per mL, or about 0.5 mg to about 1 mg lipid per mL, or about 0.75 mg lipid per mL.

[0106] Prior to administration, the nano / microbubbles may be diluted with an aqueous diluent, which may contain a salt such as, but not limited to, sodium chloride, and thus may be considered a saline solution or saline solution.

[0107] The aqueous diluent may include a buffer, such as a phosphate buffer, and may therefore be considered a buffered aqueous diluent. The aqueous diluent may be a buffered saline solution.

[0108] The non-aqueous lipid formulation may include a buffer, such as a non-phosphate buffer, examples of which are provided herein. Both the lipid formulation and the aqueous diluent may include a buffer. In an exemplary embodiment, either the lipid formulation or the aqueous diluent includes a buffer, but not both. The buffer concentration will vary depending on the buffer used, which will be understood by those skilled in the art and is within the skill of the art to determine. The buffer concentration in the lipid formulation may range from about 1 mM to about 100 mM. In some cases, the buffer concentration may be about 1 mM to about 50 mM, including about 5 mM, or about 1 mM to about 20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 5 mM.

[0109] In some embodiments, the aqueous diluent comprises glycerol, a buffer such as phosphate buffer, salt and water.Such aqueous diluent can be used with non-aqueous lipid formulations that lack glycerol.In some embodiments, lipid formulation further comprises saline (salt and water combined) and glycerol in a weight ratio of 8:1.

[0110] In some embodiments, the aqueous diluent comprises propylene glycol, a buffer such as a phosphate buffer, a salt, and water. Such aqueous diluents may be used with non-aqueous lipid formulations that lack propylene glycol.

[0111] In some embodiments, the aqueous diluent comprises a buffer, such as a phosphate buffer, a salt, and water. Such aqueous diluents may be used with non-aqueous lipid formulations that include both propylene glycol and glycerol.

[0112] The nano / microbubble population administered to a subject, including a human subject, typically intravenously, may have a pH in the range of 4 to 8 or in the range of 4.5 to 7.5. In some cases, the pH may be in the range of about 6 to about 7.5 or in the range of 6.2 to about 6.8. In still other cases, the pH may be about 6.5 (e.g., 6.5±0.5 or ±0.3). In some cases, the pH may be in the range of 5 to 6.5 or in the range of 5.2 to 6.3 or in the range of 5.5 to 6.1 or in the range of 5.6 to 6 or in the range of 5.65 to 5.95. In still other cases, the pH may be in the range of about 5.7 to 5.9 (e.g., ±0.1 or ±0.2 or ±0.3 from either or both ends of the range). In other cases, the pH may be about 5.8 (e.g., 5.8±0.15 or 5.8±0.1).

[0113] Methods for forming and activating nano / microbubble enriched populations The lipid-encapsulated gas nano / microbubbles of the present invention are produced by activation of a lipid formulation, such as a lipid solution or lipid suspension. Activation is a process in which the lipid formulation, contained in a container such as a vial, is physically agitated (e.g., vigorously shaken) in the presence of a gas of interest to form lipid-encapsulated gas nano / microbubbles.

[0114] The present invention provides a method for forming suitable nano / microbubble populations by specifically activating lipid formulations at a sufficient rate and for a sufficient period of time. It has been found that optimized nano / microbubble populations provided herein can be obtained from lipid formulations using specific activation parameters. Some of these specific activation parameters differ significantly from the activation parameters of existing lipid formulations used to form microbubbles for diagnostic ultrasound applications.

[0115] As an example, it has been found that non-aqueous lipid formulations provided herein must be activated at a higher speed (or rate) and for a longer period of time to produce certain enriched nano / microbubble populations of the invention. For example, certain non-aqueous lipid formulations form microbubbles for diagnostic ultrasound applications when activated at 4950 rpm for 45 seconds. However, to produce certain nano / microbubble populations provided herein, certain formulations (examples of which are provided in Example 2) must be activated at significantly faster speeds, including about 6000 rpm, for longer periods of time, such as about 50 to about 90 seconds, including about 75 to about 90 seconds.

[0116] Thus, an enriched nano / microbubble population may be formed by activation of a non-aqueous formulation (such as that described in Example 2) at a shaking speed of 6000 rpm ±10% or ±5% or ±1% and / or a shaking time of about 50 seconds to about 90 seconds at a shaking speed in the range of about 5000 rpm to about 7000 rpm, or about 5500 rpm to about 6500 rpm, or about 5800 rpm to about 6200 rpm, or about 5900 to about 6100 rpm, or about 5950 rpm to about 6050 rpm. In one case, the shaking speed is 6000 rpm.

[0117] Similarly, an enriched nano / microbubble population may be formed by activating the non-aqueous formulation for a shaking time in the range of 40-100 seconds or 45-95 seconds or 46-94 seconds or 47-93 seconds or 48-92 seconds or 49-91 seconds or 40-100 seconds, including but not limited to 40 seconds, 41 seconds, 42 seconds, 43 seconds, 44 seconds, 45 seconds, 46 seconds, 47 seconds, 48 ​​seconds, 49 seconds, 50 seconds, 51 seconds, 52 seconds, 53 seconds, 54 seconds, 55 seconds, 60 seconds, 65 seconds, 70 seconds, 75 seconds, 80 seconds, 85 seconds, 86 seconds, 87 seconds, 88 seconds, 89 seconds, 90 seconds, 91 seconds, 92 seconds, 93 seconds, 94 seconds, 95 seconds and 100 seconds, at any of the above shaking speeds. In other embodiments, the shaking time can be in the range of 70 to 95 seconds, including 71 to 94 seconds, 72 to 93 seconds, 73 to 92 seconds, 74 to 91 seconds, or 75 seconds, 76 seconds, 77 seconds, 78 seconds, 79 seconds, 80 seconds, 81 seconds, 82 seconds, 83 seconds, 84 seconds, 85 seconds, 86 seconds, 87 seconds, 88 seconds, 89 seconds, or 90 seconds. Any range defined by lower and upper boundaries selected from these times is contemplated by the present invention.

[0118] Moreover, it was surprisingly found that if these non-aqueous lipid formulations (such as those described in Example 2) occupied about 9% of the internal volume of the container and the gas occupied about 91% of the internal volume of the container (as described in the Examples and designated as DEFINITY RT 1.5X or DEFINITY RT 1X+0.5X), the optimal shaking speed was about 6000 rpm and the optimal shaking time was about 60 seconds. The shaking speed (or speed, as the terms are used interchangeably) can be in the range of 6000 rpm ±10% or ±5% or ±1% and / or about 5000 rpm to about 7000 rpm or about 5500 rpm to about 6500 rpm or about 5800 rpm to about 6200 rpm or about 5900 to about 6100 rpm or about 5950 rpm to about 6050 rpm. In some cases, the shaking speed is 6000 rpm. Shaking times can range from 55-65 seconds, 56-64 seconds, 57-63 seconds, 58-62 seconds, 59-61 seconds, or can be 60 seconds. The compositions of DEFINITY and DEFINITY RT are provided in the Examples.

[0119] Even more surprisingly, activation of these particular formulations with these parameters produces a population in which 27-56% of the microbubbles have diameters in the range of about 2 microns to about 6 microns or less. Such an enriched microbubble population has not been previously reported without a further separation step.

[0120] Certain activation parameters resulted in enrichment of two microbubble subpopulations. Some of these nano / microbubble populations were enriched for microbubbles having diameters in the range of about 2 to about 6 microns or less and for microbubbles having diameters in the range of about 6 microns to about 10 microns. For example, providing a non-aqueous lipid formulation (e.g., as described in Example 2) occupying about 9% of the interior volume of the container and a gas (as described in the Examples) occupying about 91% of the interior volume of the container for a shaking time of about 60 seconds and a shaking speed of about 6000 rpm resulted in enriched populations for microbubbles having diameters in the range of about 2 to about 6 microns and about 6 to about 10 microns. The shaking speed can be in the range of 6000 rpm ±10% or ±5% or ±1% and / or about 5000 rpm to about 7000 rpm or about 5500 rpm to about 6500 rpm or about 5800 rpm to about 6200 rpm or about 5900 to about 6100 rpm or about 5950 rpm to about 6050 rpm. In some cases, the shaking speed is 6000 rpm or 6200 rpm or 6600 rpm. The shaking time may be in the range of 55-65 seconds, 56-64 seconds, 57-63 seconds, 58-62 seconds, 59-61 seconds or may be 60 seconds.

[0121] Applied to other non-aqueous lipid formulations (such as those described in Example 2), a shaking time of about 90 seconds and a shaking speed of about 6000 rpm resulted in enrichment of the nanobubble population and enrichment of the microbubble population with diameters ranging from about 2 microns to about 6 microns. The shaking speed can be in the range of 6000 rpm ±10% or ±5% or ±1% and / or about 5000 rpm to about 7000 rpm or about 5500 rpm to about 6500 rpm or about 5800 rpm to about 6200 rpm or about 5900 to about 6100 rpm or about 5950 rpm to about 6050 rpm or about 6000 rpm to about 6600 rpm. In some cases, the shaking speed is 6000 rpm or 6200 rpm or 6600 rpm. The shaking time can be in the range of 85 to 95 seconds, 86 to 94 seconds, 87 to 93 seconds, 88 to 92 seconds, 89 to 91 seconds, or can be 90 seconds. As another example, the aqueous lipid formulations provided herein (e.g., those described in Example 1) must also be activated for longer times or at higher speeds (or rates) to produce certain enriched nano / microbubble populations of the invention. For example, certain aqueous lipid formulations produce microbubbles for diagnostic ultrasound applications when activated at 4530 rpm for 45 seconds. However, to produce the nano / microbubble populations provided herein, these aqueous formulations must be activated for shorter periods of time, ranging from about 20 to about 30 seconds, at significantly faster speeds, including about 6000 rpm. Activation of these lipid formulations at about 6000 rpm for about 30 seconds provides an enriched population of microbubbles having diameters ranging from about 2 microns to about 6 microns. Activation of these lipid formulations at about 6000 rpm for about 20 to 25 seconds provides a nanobubble-enriched population. The shaking speed can be in the range of 6000 rpm ±10% or ±5% or ±1% and / or about 5000 rpm to about 7000 rpm or about 5500 rpm to about 6500 rpm or about 5800 rpm to about 6200 rpm or about 5900 to about 6100 rpm or about 5950 rpm to about 6050 rpm or about 6000 rpm to about 6600 rpm. In some cases, the shaking speed is 6000 rpm or 6200 rpm or 6600 rpm.The shaking time may be in the range of 15-35 seconds, 16-34 seconds, 17-33 seconds, 18-32 seconds, 19-31 seconds or may be 20 seconds, 21 seconds, 22 seconds, 23 seconds, 24 seconds, 25 seconds, 26 seconds, 27 seconds, 28 seconds, 29 seconds or 30 seconds.

[0122] Alternatively, these same aqueous lipid formulations (Example 1) can be activated using a lower speed (e.g., about 90 seconds at about 4530 rpm) for a significantly longer time to produce a nanobubble-enriched population. The shaking speed can be in the range of 4930 ± 10% or ± 5% or ± 1% and / or about 4000 to about 5000 rpm or about 4100 to about 4900 rpm or about 4200 to about 4800 rpm or about 4300 to about 4700 rpm or about 4400 to about 4600 rpm or about 4500 to about 4600 rpm or about 4500 to about 4550 rpm or about 4530 rpm. The shaking time can be in the range of 85 to 95 seconds or 86 to 94 seconds or 87 to 93 seconds or 88 to 92 seconds or 89 to 91 seconds or can be 90 seconds.

[0123] These same aqueous lipid formulations (Example 1) can be activated at high shaking speed for a short time (e.g., about 30 seconds at about 6000 rpm) to produce an enriched population of microbubbles having diameters in the range of about 2 microns to about 6 microns. The shaking speed can be in the range of 6000 rpm ±10% or ±5% or ±1% and / or about 5000 rpm to about 7000 rpm or about 5500 rpm to about 6500 rpm or about 5800 rpm to about 6200 rpm or about 5900 to about 6100 rpm or about 5950 rpm to about 6050 rpm or about 6000 rpm to about 6600 rpm. In some cases, the shaking speed is 6000 rpm or 6200 rpm or 6600 rpm. The shaking time can be in the range of 25 to 35 seconds, 26 to 34 seconds, 27 to 33 seconds, 28 to 32 seconds, 29 to 31 seconds, or can be 30 seconds.

[0124] Some of the manufacturing methods provided herein include the use of a 2mL vial as a container for containing lipid formulation and gas. In some cases, the vial contains about 1.76ml of aqueous lipid formulation and about 2.04ml of gas. Thus, the lipid formulation occupies about 46% of the total internal volume (i.e., the volume of lipid formulation as a percentage of the total internal volume), and the gas occupies about 54% of the total internal volume (i.e., the volume of gas as a percentage of the total internal volume).

[0125] Some of the manufacturing methods provided herein include the use of 2mL vials as containers to accommodate small amounts of non-aqueous lipid formulations and gas.For example, a vial with a total internal volume of about 3.8ml can contain about 0.35ml of non-aqueous lipid formulation and about 3.45ml of gas.Thus, the lipid formulation occupies about 9% of the total internal volume, and the gas occupies about 91% of the total internal volume.

[0126] Another example includes a vial having a total internal volume of about 3.8 ml containing about 0.52 ml of non-aqueous lipid formulation and about 3.28 ml of gas. Thus, the comprises about 14% of the total internal volume, and the gas comprises about 86% of the total internal volume. Examples of these latter formulations are described in the Examples and are referred to as DEFINITY-RT 1.5X and DEFINITY-RT 1X+0.5X. This lipid formulation to gas volume ratio (14% to 86%) has surprisingly been found to be useful in producing a bubble population enriched for microbubbles in the size range of about 2 microns to about 10 microns, including about 2 microns to about 6 microns or less and about 6 microns to about 10 microns. Also surprisingly, this effect was apparently lipid concentration independent, as concentrations down to 33% (e.g., from about 3.75 mg / ml to about 2.5 mg / ml) had no effect as long as the lipid formulation volume to gas volume remained constant.

[0127] Thus, depending on the desired enrichment and in accordance with the teachings provided herein, including the examples, the lipid formulations provided herein can be activated in a number of ways and using a number of shaking rates / speeds and shaking time combinations.

[0128] An aqueous lipid formulation, such as, but not limited to, the aqueous lipid formulation described in Example 1, may be activated at about 4530 rpm for about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds, about 70 seconds, about 75 seconds, about 80 seconds, or about 90 seconds.

[0129] Alternatively, the aqueous lipid formulation may be activated for about 5 to 30 seconds, including, but not limited to, about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, or about 30 seconds at about 6000 rpm.

[0130] A non-aqueous lipid formulation, such as, but not limited to, the non-aqueous lipid formulation described in Example 2, may be activated for about 10 to about 90 seconds or about 120 seconds, including, but not limited to, about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds, about 70 seconds, about 75 seconds, about 80 seconds, about 90 seconds at about 4950 rpm.

[0131] A non-aqueous lipid formulation, such as, but not limited to, the non-aqueous lipid formulation described in Example 2, may be activated for about 10 to about 90 seconds, including, but not limited to, about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds, about 70 seconds, about 75 seconds, about 80 seconds, or about 90 seconds at about 6000 rpm.

[0132] A non-aqueous lipid formulation, such as, but not limited to, the non-aqueous lipid formulation described in Example 2 and provided at 1.5 times the fill volume (relative to the fill volume described in Example 2), may be activated at about 6000 rpm for about 40 seconds, 45 seconds, 50 seconds, 55 seconds, 60 seconds, 65 seconds, 70 seconds, 75 seconds, or 80 seconds. In some cases, it is activated at about 6000 rpm for about 60 seconds.

[0133] Non-aqueous lipid formulations, such as, but not limited to, those described in Example 2 and provided with 1 fill volume + 0.5 fill volume of lipid-free matrix (relative to the fill volume described in Example 2), can be activated at about 6000 rpm for about 40 seconds, 45 seconds, 50 seconds, 55 seconds, 60 seconds, 65 seconds, 70 seconds, 75 seconds, or 80 seconds. In some cases, activated at about 6000 rpm for about 60 seconds.

[0134] Another embodiment of the present invention includes the formation of nanodroplets, optionally starting from gas nanobubbles and microbubble populations. Nanodroplets, as used herein, are nanometer-sized (typically less than 500 nm) droplets with a center occupied by gas in liquid form and an outer lipid shell. One example of a gas that can exist in liquid form is a perfluorocarbon gas, such as, but not limited to, perfluoropropane. Other perfluorocarbon gases are contemplated and examples are provided herein. Perfluoropropane has a boiling point of -36.7°C, yet can remain in liquid form at body temperature, which can be utilized to create nanodroplets that can travel through the body more efficiently than larger gas bubbles.

[0135] In certain embodiments, nanodroplets are formed by condensation of the microbubbles. As an example, lipid-encapsulated gas microbubbles in the 0.5-500 micron size range can rapidly condense to form nanodroplets. This can be accomplished by placing the microbubble population into a syringe, such as a 3 mL syringe, and applying manual pressure to the plunger. It has been found that the microbubble suspension changes from a milky white appearance to a clear, colorless appearance upon such application of manual pressure, leading to the conclusion that the milky white appearance correlates with the presence of microbubbles in the 0.5-500 micron size range, and the clear, colorless appearance correlates with a significant reduction in such microbubbles and condensation of the gas into a liquid form. Nanodroplets can be formed without changing (e.g., reducing) the temperature of the microbubble population.

[0136] The nanodroplets may include a shell of one or more lipids, such as phospholipids. The one or more phospholipids may be selected from, but are not limited to, DPPA, DPPC, and DPPE. As with other embodiments of the present invention, one or more of these phospholipids may be PEGylated. In some embodiments, DPPE is present in both PEGylated and non-PEGylated forms. Thus, one nanodroplet population may have a lipid shell consisting of DPPA, DPPC, and DPPE, optionally with DPPE being PEGylated (e.g., including but not limited to, PEG1000-DPPE, MPEG1000-DPPE, PEG2000-DPPE, MPEG2000-DPPE, PEG3000-DPPE, MPEG3000-DPPE, PEG5000-DPPE, or MPEG5000-DPPE, and other examples of PEGylated DPPE provided herein). Another nanodroplet population may have a lipid shell consisting of DPPC, DPPE, and PEGylated DPPE, and may lack DPPA.

[0137] Digital (or hand) pressure applied to a microbubble preparation present in a syringe, such as a 3 mL syringe, can range from 6 to 12 atmospheres. Sufficient digital pressure is achieved and is evident by the transformation of the milky appearance of the preparation into a clear, colorless appearance.

[0138] Nanodroplets have a size less than about 500 nm, including ranges of about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 200 nm, or about 100 nm to about 200 nm. Given the ability of nanodroplets to access body sites inaccessible to larger microbubbles, nanodroplets may be used for a broader or different variety of in vivo applications compared to microbubbles. Thus, the present invention contemplates and provides methods that include administering such nanodroplets to a subject for diagnostic or therapeutic benefit. As an example, a nanodroplet preparation can be administered to a subject, and then expansion can be induced by converting the liquid center to a gaseous center. By doing so, the nanodroplets can be transformed into nanobubbles and cause expansion within existing tissues or vasculature.

[0139] These nanodroplets are intended for use in any of the diagnostic and therapeutic uses provided herein.

[0140] Choudhary et al., J American Society Echocardiography, 30(2)189-197, 2017; and Hayward et al., Scandantri J Rheumatology, 40(5): 379-382, 2011.

[0141] therapeutic use In addition to the intended use of certain nano / microbubble and nanodroplet populations of the present invention for contrast imaging enhancement, the present invention also contemplates the use of these nano / microbubble and nanodroplet populations in therapeutic applications. These populations may be used in vivo or in vitro in human or non-human subjects. Thus, the nano / microbubble and nanodroplet populations provided herein may be used for imaging purposes, therapeutic purposes, or combined diagnostic and therapeutic purposes.

[0142] The lipid formulation can be activated as described herein to form a sufficient number of nano / microbubbles, which can then be optionally diluted to a larger volume and administered by one or more bolus injections or continuous infusion. Administration is typically intravenous. Alternatively, the activated formulation can be pressurized to form nanodroplets as described herein, and the nanodroplets can be optionally administered by one or more bolus injections or continuous infusion. Therapeutic ultrasound is then performed simultaneously with or shortly after administration. Ultrasound can be directed to any area of ​​the body, including but not limited to the heart, blood vessels, cardiovascular system, liver, kidneys, penis, and head.

[0143] Subjects of the present invention include, but are not limited to, humans and animals, with humans being preferred. Animals include companion animals such as dogs and cats, as well as farm or sport animals, such as, but not limited to, bulls and horses.

[0144] The nano / microbubble and nanodroplet populations are administered in an effective amount, which is an amount that facilitates or enables the intended in vivo response and / or application. The therapeutic methods provided herein are intended to treat, in part or in full, a condition.

[0145] The present invention further contemplates the use of enriched nano / microbubble or nanodroplet populations provided herein with ultrasound in therapeutic applications, which have previously been used or are intended for use in therapeutic applications. The combination of ultrasound and nano / microbubbles or nanodroplets provided herein may provide additional benefits, possibly with fewer adverse effects of ultrasound.

[0146] When used alone, ultrasound has been hypothesized to have several different mechanisms of action including, for example, heating of target tissue, mechanical destruction, etc. Heating of tissue may be an undesirable effect in certain embodiments.

[0147] Gas-filled nano / microbubbles or nanodroplets used with ultrasound may also have several mechanisms of action. One particular mechanism of action is cavitation. Cavitation occurs when the administered nano / microbubbles or nanodroplets absorb ultrasound energy, causing several mechanical changes. In some cases, the energy absorbed by the nano / microbubbles or nanodroplets is released from the nano / microbubbles or nanodroplets and then absorbed by the surrounding tissue. In the case of stable cavitation, the nano / microbubbles, such as microspheres or nanodroplets, may undergo one or more cycles of expansion and contraction without eventual rupture. In the case of inertial cavitation, the nano / microbubbles, such as microspheres or nanodroplets, rupture. Whether stable or inertial cavitation occurs depends on several factors, including the energy level of the ultrasound, the intended location of use, and the nature or composition of the nano / microbubbles or nanodroplets.

[0148] In some therapeutic applications, stable cavitation is sufficient to exert the desired clinical benefit. For other therapeutic applications, inertial cavitation is necessary to exert the desired clinical benefit. As an example, if nano / microbubbles such as microspheres or nanodroplets are used as a delivery vehicle, they must burst to release the cargo in vivo for maximum effectiveness.

[0149] Ultrasound energy may be applied externally or internally. Externally applied ultrasound is preferred in most situations because it is less (if at all) invasive. Internally applied ultrasound may be used in some cases where the target tissue is a deep tissue and / or externally applied ultrasound would cause adverse effects on intervening tissue. Ultrasound may be applied externally by placing an ultrasound transducer near an external surface of the subject, such as the skin or an outward facing cavity such as the oral cavity. Ultrasound may be applied internally by introducing an ultrasound transducer into the subject's body, typically on a probe. An example may be a cardiovascular probe that can travel through the circulatory system.

[0150] Conditions that may benefit from the use of a combination of nano / microbubbles or nanodroplets and ultrasonic energy are described below.Typical ultrasound frequencies range from about 1-6 MHz for deep tissues such as the kidney and heart, and 7-10 MHz for superficial tissues such as the carotid artery and breast.

[0151] Sonothrombolysis refers to the dissolution or destruction of thrombus using ultrasound. The effect of ultrasound can be enhanced by combining with the nano / microbubbles or nanodroplets of the present invention. Thromboembolic diseases that can benefit from sonoporation include acute myocardial infarction (e.g., acute ST-segment elevation myocardial infarction), pulmonary thromboembolism, deep vein thrombosis, retinal vein occlusion (with ocular ultrasound), peripheral arterial disease, cardiac atherosclerosis, microvascular occlusion or thrombosis, and stroke. The combination therapy provided herein can also be used to treat kidney stones.

[0152] Patients experiencing a thromboembolic event may be treated with a combination of ultrasound, nano / microbubbles or nanodroplets and a therapeutic agent such as urokinase or t-PA.

[0153] The nano / microbubbles or nanodroplets used for these indications may be targeted to specific regions of the body or to specific structures, such as thrombi. As an example, the nano / microbubbles or nanodroplets may be targeted to thrombi by conjugation with Arg-Gly-Asp (RGD) peptides, which specifically bind to platelet glycoprotein IIb / IIIa. See Ma et al. Bioconjugate Chemistry, 2020, 31:369-374 for a description of labeling methods. The nanobubble-enriched and microbubble-enriched populations provided herein and the nanodroplets provided herein may be used in this therapeutic application. The ultrasound frequency may be about 1.3 MHz. The method may be performed using an ultrasound treatment probe C5-1s.

[0154] Sonoporation refers to the use of ultrasound to increase the permeability of cell membranes, vasculature and the blood-brain barrier (BBB), typically in a reversible manner. By co-administration of nano / microbubbles or nanodroplets with therapeutic agents or administration of nano / microbubbles or nanodroplets conjugated to therapeutic agents and application of ultrasound, it is possible to enhance the permeability of membranes, barriers or vasculature to deliver therapeutic agents to specific regions of the body. Conditions that would benefit from this combinatorial approach include cardiovascular disease, solid tumors such as glioblastoma and neurodegenerative diseases such as Alzheimer's and Parkinson's. Both nano- and microbubbles and nanodroplets are suitable for these applications. Nanobubbles and nanodroplets may have the added advantage that, as a function of their small size, they can be passively extravasated from the circulation into tissues such as tumors. Additionally, tumors typically have a leaky vasculature. Nanobubbles and nanodroplets may be used at non-resonant frequencies such as 1-10 MHz. Sonoporation can also sensitize hypoxic tumors to radiation therapy.

[0155] Drug delivery approaches are also contemplated by the present invention. As mentioned above, therapeutic agents may be administered before, during or after nano / microbubble or nanodroplet population administration and ultrasound application. These agents may be separate from the nano / microbubbles or nanodroplets, or may be carried in the internal cavity of the nano / microbubbles or nanodroplets, or may be bound to the surface of the nano / microbubbles or nanodroplets, or may be embedded in the lipid shell of the nano / microbubbles or nanodroplets. Thus, some of these drug delivery applications may be targeted applications and some may be non-targeted. The agents delivered include nucleic acids, proteins, cells, cell fragments such as exosomes, etc.

[0156] Yet other uses include the delivery of oxygen to tumors in order to sensitize such tumors to radiation treatment.

[0157] Further applications include connective tissue disorders such as, but not limited to, Peyronie's disease and tendonitis, bursitis. Other conditions treated include uterine fibroid ablation, cataract removal, surgical tissue resection and hemostasis, bone fracture treatment, Meniere's disease (by vestibular nerve destruction), glaucoma, laparoscopic tissue ablation. The combined use of ultrasound with gas bubbles or droplets, such as the nanodroplets provided herein, may be used as part of neurological surgery, ultrasound-assisted immunotherapy, and ultrasound-assisted neuromodulation.

[0158] Other conditions characterized by reduced blood flow or reduced tissue perfusion in large or small blood vessels may also benefit from the combined use of ultrasound with the enriched bubble populations or nanodroplets provided herein. Examples include retinal vein occlusion, coronary artery occlusion, cardiac atherosclerosis, peripheral arterial disease (and associated limb salvage) and other conditions that would benefit from increased peripheral blood flow (e.g., sickle cell disease), atherosclerosis, including removal or destruction of arteriosclerotic plaque, arteriosclerosis, erectile dysfunction, stenosis, and restenosis, e.g., after balloon angiography. These methods may also be used to maintain vascular access, e.g., for long-term dialysis procedures (e.g., removal or destruction of clots, thrombus, stenosis, etc.), including, inter alia, the creation and / or maintenance of arteriovenous fistulas in the cephalic vein, including brachiocephalic fistulas.

[0159] The bubbles or droplets containing the nanodroplet populations provided herein may also be used to enhance high intensity focused ultrasound (HiFu).

[0160] Imaging applications include deep tissue imaging, such as, but not limited to, cardiac imaging, renal imaging, liver imaging, etc. Imaging applications also include breast imaging, any imaging of tissue or area of ​​the body suspected of having a tumor or other growth. These imaging applications may also include enhanced Doppler ultrasound imaging.

[0161] Clinical ultrasound parameters and suitable systems are known in the art. Examples include Philips EPIQ, Philips Sonos 7500, EKOS System (EKOS Endo Wave Infusion Catheter System, EKOS Corporation), approved sonothrombolysis devices.

[0162] Erectile dysfunction In yet other embodiments, the present invention provides methods of using a combination of ultrasound and bubbles or droplets, such as the nanodroplets provided herein, for the treatment of erectile dysfunction (ED), including vascular ED. The bubbles can be enriched populations provided herein or can be microbubble populations used in diagnostic ultrasound, such as those present in activated DEFINITY (i.e., perflutren lipid microspheres having an average diameter range of 1.1 microns to 3.3 microns, with 98% of the bubbles less than 10 microns in diameter).

[0163] Erectile dysfunction (ED) is the inability to achieve or maintain an erection sufficient for sexual activity, at least for any period of time (Yafi, et al, Nat. Rev. Disease Primers, 2, Article number: 16003 (2016)). ED is positively correlated with age; the same study found that 6.5 percent of men aged 20-29 reported experiencing ED, while 77.5 percent of men over 75 reported experiencing ED.

[0164] ED occurs to different degrees in affected individuals; severity can be assessed using the International Institute of Erectile Function (IIEF-5), which is a five-item questionnaire (Rhoden, International Journal of Impotence Research, 14, 245-250 (2002)). An IIEF-5 score of 1-7 indicates severe ED, 8-11 indicates moderate ED, 12-16 indicates mild to moderate ED, 17-21 indicates mild ED, and 22-25 indicates no ED. Severity can also be assessed using the IIEF-15, which is a 15-item questionnaire. An IIEF-15 score of 1-10 indicates severe ED, 11-16 indicates moderate ED, 17-21 indicates mild to moderate ED, 22-25 indicates mild ED, and 26-30 indicates no ED.

[0165] Physiological causes of ED can be divided into endocrine and non-endocrine causes. Endocrine causes are related to hormonal problems, especially reduced serum testosterone. The most common non-endocrine causes are vasculogenic, that is, disorders related to the blood supply.

[0166] ED in subjects with vascular risk factors (including, but not limited to, coronary artery disease (CAD), hypertension, dyslipidemia, smoking, and diabetes) and no risk factors for psychogenic, genitourinary, endocrinological, or neurological causes of ED is considered vascular ED (Yavuzgil, et al, Int. J. Cardiol. (2005)103: 19-26). Blood flow can also be assessed by penile Doppler ultrasound (Koca, et al, J Sex Med. (2010) 7(12): 3997-4002).

[0167] The combined foam / ultrasound treatment may be provided together with the administration of a therapeutic agent used to treat ED.One common pharmaceutical treatment, PDE5 inhibitors, such as sildenafil, vardenafil, tadalafil and avanafil, inhibit PDE5, allowing for the enhancement of signals that promote penile erection.PDE5 inhibitors are effective, but may interfere with other medications (e.g., nitrate-containing medications).When used together with the combined foam / ultrasound treatment, these PDE5 inhibitors may be used in subtherapeutic doses.

[0168] Thus, in some embodiments, the present invention provides a method of treating ED in a subject in need of treatment. In some embodiments, the subject is identified based on a score on the IIEF-5 or IIEF-15 questionnaire. In some embodiments, prior to treatment, the subject is scored or has been scored to indicate severe ED (e.g., IIEF-5 1-7 or IIEF-15 1-10). In some embodiments, the subject is scored or has been scored to indicate severe or moderate ED (e.g., IIEF-5 11 or less or IIEF-15 16 or less). In some embodiments, the subject is scored or has been scored to indicate severe, moderate, or mild to moderate ED (e.g., IIEF-5 16 or less or IIEF-15 21 or less). In some embodiments, the subject is scored or has been scored to indicate at least mild ED (e.g., IIEF-5 21 or less or IIEF-15 25 or less).

[0169] In some embodiments, the subject to be treated is characterized by having mild to moderate ED in any test. Treatment can be measured by improvement in IIEF-5 or IIEF-15 scores. The subject to be treated according to this embodiment will score an increase of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 points on the IIEF-15 test after treatment. An increase in the score on the IIEF-5 test is also evidence of the subject's treatment.

[0170] In some embodiments, the subject scores higher on the IIEF-5 score after treatment (indicating less severe ED). In some embodiments, the subject scores 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or more than 10 points higher on the IIEF-5 score.

[0171] The subject is then administered a bubble population, such as any of the enriched populations present in activated DEFINITY or described herein, or the nanodroplets provided herein. These bubble or droplet preparations may be administered as one or more bolus doses or as a continuous infusion. Intravenous administration is preferred.

[0172] The subject may be subjected to localized (or targeted) ultrasound of the genital areas, including the corpus cavernosum, vulva, or bulbocavernosus muscle area.

[0173] Ultrasound may be applied during instillation of the bubbles or droplets or after instillation of the bubbles or droplets, including but not limited to, after 2-20 minutes, 2-10 minutes, 5-10 minutes, 5-15 minutes, or 5-20 minutes. In some embodiments, DEFINITY microbubbles are used because they may remain in the circulation longer than other microbubble formulations.

[0174] In some embodiments, the subject is treated once. In some embodiments, the subject is treated twice. In some embodiments, the subject is treated at least three times. The subject may receive multiple periods (e.g., 4-8 periods) at spaced intervals, optionally at regular intervals. The treatment may last for one week or more or one month or more. The subject may be re-treated every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. High energy (or high intensity) ultrasound may be used. Ultrasound energy may be applied using, for example, a phased array probe in conjunction with an ultrasound system, such as an iE33 Philips ultrasound system, with a mechanical index of 1.0, using a 2.0 MHz centered ultrasound. In other embodiments, ultrasound is performed over the genital area and optionally specifically in the corpus cavernosum, at 1.3 MHz, with a mechanical index of 1.3, and a pulse interval of 3 seconds. In yet another embodiment, ultrasound can be performed using a harmonic power Doppler (Sonos 5500, Philips ultrasound) at 1.3 MHz, with a pulse repetition frequency of 9.3 kHz, mechanical index of 1.3, and acoustic focus set at the cavernous level. Other variations are contemplated and can be determined by those skilled in the art.

[0175] In some embodiments, the subject is further identified by the presence of vascular risk factors.In some embodiments, the subject has coronary artery disease (CAD), hypertension, dyslipidemia, smoking or diabetes.In some embodiments, the subject has two or more disorders selected from coronary artery disease (CAD), hypertension, dyslipidemia, smoking and diabetes.

[0176] activation, general Activation can be achieved by vigorously shaking the lipid formulation in the presence of gas. Vigorous shaking as used herein refers to the movement of stirring the formulation, whether aqueous or non-aqueous, so that gas is introduced into the formulation from the local surrounding environment in the container (e.g., vial). Any type of movement that stirs the formulation and results in the introduction of gas can be used for shaking. Shaking must be strong enough to allow the formation of foam after a certain period of time.

[0177] Activation is typically performed immediately prior to administration to a subject and is therefore performed by the end user or intermediary, rather than the supplier or manufacturer of the lipid formulation. Lipid formulations are typically provided in vials that minimally contain the lipid formulation and gas in the headspace. In the presence of gas, vigorous shaking of the lipid formulation results in the formation of lipid-encapsulated gas-filled nano / microbubbles that can be used in therapeutic applications. As used herein, "gas-filled" means that the nano / microbubbles contain a gas in the interior cavity, such as a perfluorocarbon gas, including but not limited to perflutren (or perfluoropropane) gas. The lipid shell that encapsulates the gas may be arranged as a monolayer or bilayer, including a monolayer or multilayer bilayer.

[0178] Lipid formulations are typically provided in a container such as a vial with a desired headspace gas. The container can be made of any material, including but not limited to glass or plastic. The glass can be pharmaceutical grade glass. The container can be sealed with a stopper such as a rubber stopper.

[0179] As will be understood from the description of this disclosure, the internal volume of the container may be occupied by the lipid formulation and gas. A container may be defined based on a liquid fill volume, which typically refers to the volume of liquid (e.g., lipid formulation) placed in the container. The liquid fill volume can be in different forms and is typically lower than the entire internal volume of the container. When a container is defined herein by a volumetric measurement, this should be understood to refer to the liquid fill volume. For example, a 0.5-10 mL container is a container having a liquid fill volume of 0.5-10 mL. Examples of suitable containers and their corresponding liquid fill and internal volumes are: Schott 2 mL (liquid fill volume) vial with 2.9 mL internal volume; Schott 3 mL (liquid fill volume) vial with 4.5 mL internal volume; and Wheaton 1 mL (liquid fill volume) v-vial with 1.2 mL internal volume.

[0180] In some embodiments, the container is a 0.5-10 mL container, such as a 1-5 mL container or a 1 mL or 2 mL container. The container can be a vial, such as a glass vial. An example of a suitable vial is a 2 ml glass vial from Wheaton (e.g., commercially available from Nipro, Cat. No. 2702, B33BA, 2 cc, 13 mm, Type I, flint tubing vial), with an actual internal volume of about 3.8 ml.

[0181] The vial may be pre-filled with the gas of interest, such as perflutren gas, prior to introduction of the lipid formulation, or the lipid formulation may be placed in the vial first, followed by introduction of the gas (e.g., via a process referred to as headspace gas exchange). The container is preferably sterile and / or sterilized after introduction of the lipid formulation and / or gas, as described in published PCT application WO99 / 36104.

[0182] In some embodiments, the container is a flat-bottom container, such as a flat-bottom vial. Suitable vials are flat-bottom borosilicate vials, including Wheaton vials. In some embodiments, the container is a non-flat-bottom container or vial. In some embodiments, the container is a V-bottom container, such as a V-bottom vial. In some embodiments, the container is a round-bottom container, such as a round-bottom vial. In some embodiments, the container has converging walls, such that the bottom surface area (or bottom surface diameter) is smaller than the top (opening) surface area (or diameter) or any diameter (e.g., body diameter) therebetween. For clarity, a V-bottom container or vial has converging walls, and its bottom surface area is significantly smaller than either the top or body surface area.

[0183] In some embodiments, the container is a syringe. The lipid formulation can be provided in a pre-filled syringe, optionally in physical contact with gas. In some embodiments, the container is a single-chamber container, such as a vial.

[0184] Activation should be at least 1 x 10 per ml of lipid formulation. 7 Microspheres, at least 5 x 10 per ml of lipid formulation 7 Microspheres, at least 7.5 x 10 per ml of lipid formulation 7 Microspheres, at least 1 x 10 per ml of lipid formulation 8 Microspheres, at least 1 x 10 per ml of lipid formulation 9 At least 5 x 10 per ml of microsphere or lipid formulation 9 Microspheres, at least 10 per ml of lipid formulation 10 At least 1.5 x 10 per ml of microsphere or lipid formulation 10 Microspheres can be produced.

[0185] The shaking can be swirling (e.g., by vortexing), sideways or up-down. Moreover, different types of motion can be combined. The shaking can occur by shaking a container (e.g., a vial) with an aqueous or non-aqueous lipid solution or shaking the aqueous or non-aqueous solution in a container (e.g., a vial) without shaking the container (e.g., a vial) itself. The shaking is performed mechanically for process standardization. Mechanical shakers are known in the art, and their shaking mechanisms or means can be used in the device of the present invention. An example is an amalgamator, as used in dental applications. The shaking includes at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6100, at least 6200, at least 6300, at least 6400, at least 6500, at least 6600 or more shaking movements per minute.

[0186] The formation of gas-filled microspheres upon activation can be detected by the aqueous or non-aqueous solution turning white and the presence of a foamy material on top of the solution.

[0187] Activation is carried out at a temperature lower than the gel-to-liquid crystal phase transition temperature of the lipid used. "Gel-to-liquid crystal phase transition temperature" refers to the temperature at which a lipid layer (e.g., lipid monolayer or bilayer) transforms from a gel state to a liquid crystal state. This transition is described, for example, in Chapman et al., J. Biol. Chem. 1974 249, 2512-2521. The gel-to-liquid crystal phase transition temperatures of various lipids are readily apparent to those skilled in the art and are described, for example, in Gregoriadis, ed., Liposome Technology, Vol. I, 1-18 (CRC Press, 1984) and Derek Marsh, CRC Handbook of Lipid Bilayers (CRC Press, Boca Raton, Fla. 1990), page 139.

[0188] In view of the present invention, it will be understood by those skilled in the art that the lipid(s) or lipid microspheres may be manipulated before or after being subjected to the methods provided herein. For example, after shaking is completed, the gas-filled bubbles may be extracted from the container (e.g., vial). Extraction may be accomplished by placing a syringe needle or needleless spike (i.e., PINSYNC) into the container, if appropriate, the foam, and withdrawing a predetermined amount of liquid by drawing the plunger into the syringe barrel, or by adding an aqueous liquid, mixing, and withdrawing a predetermined amount of liquid by drawing the plunger into the syringe barrel.

[0189] After activation, the nano / microbubbles may be diluted with saline either in the container or after removal from the container.

[0190] Microsphere diameter is typically measured using instrumentation known and available in the art, including, but not limited to, a Malvern FPIA-3000 Sysmex particle size analyzer and an Accusizer 780.

[0191] device Also provided herein are methods and means (e.g., devices) that facilitate proper and accurate production of the nano / microbubble and nanodroplet populations provided herein. These methods and means reduce the risk of improper production of such populations. Nano / microbubble or nanodroplet populations that are not properly and accurately produced can result in a variety of problems, including, for example, too few gas-filled nano / microbubbles or nanodroplets, thereby reducing therapeutic efficacy. Proper identification and therefore proper activation of lipid formulations can be achieved using devices that can uniquely identify and uniquely activate various lipid formulations.

[0192] Identification of the formulation and / or distinction between various formulations can be achieved in several ways. For example, a device can be used with a scanner that can read the label of the formulation container (e.g., vial). In other cases, identification and / or distinction between various formulations can be achieved using a device that recognizes the shape and size of the container that holds the formulation. These latter devices can include one holder or can include two or more holders.

[0193] In some embodiments, the device receives a container with a formulation, identifies the formulation, and performs different actions depending on the type of formulation detected. The device may, for example, reference an internal or cloud-based lookup table, with an action associated with each formulation. After identifying a formulation, the device performs the action required to activate that formulation, preferably in a fully automated manner.

[0194] Based on the detected formulation, a variety of different actions can be performed. In some embodiments, the device shakes the vial and the formulation accordingly. In some embodiments, the device performs a specific shaking time, pattern and / or speed depending on the detected formulation. Examples of different shaking patterns include, but are not limited to, side-to-side reciprocating motion, up-down reciprocating motion, vibration, spinning motion, figure-of-eight trajectory, circular orbit and tilting back and forth (e.g., rotating the container at an angle and back motion). For example, in one exemplary embodiment, the device associates a shaking time of about 60 seconds with formulation type "A" and about 90 seconds with formulation type "B". When the device detects formulation type "A", the device automatically shakes the formulation for about 60 seconds without the need for the user to input the shaking time. When the device detects formulation type "B", the device automatically shakes the formulation for about 90 seconds without the need for the user to input the shaking time. It should be appreciated that other actions can be associated with the identified and detected formulation.

[0195] In some embodiments, the container containing the lipid formulation includes an indicator indicating the formulation type, and the device includes a detector that reads the indicator to detect the formulation type. The indicator can be machine or device readable. Examples of machine or device readable indicators include magnetic stripes, chips / microchips, barcodes including linear, matrix and 2D barcodes, radio frequency identification (RFID) tags, color labels identifiable by color detection, and the like. Barcodes, such as linear barcodes, can be those that meet or comply with Uniform Code Commission standards or Healthcare Providers Communication Council standards. Such indicators can then be read by devices such as, for example, magnetic stripe readers, chip readers, barcode scanners or readers, RFID tag readers, and the like. Virtually any labeling technology used for authentication and / or "track and trace" purposes can be used with the containers provided herein.

[0196] The indicator may be located on any suitable portion of the sample container, such as the body or cap of the container. In some embodiments, the indicator is integrally formed with or is otherwise a part of the sample container. For example, the indicator may be a physical feature such as a colored cap or a protrusion or indentation on the sample container. In other embodiments, the indicator is attached to the container with, for example, an adhesive, a magnet, a hook-and-loop fastener, a mechanical arrangement such as a sliding indicator behind a locking tab, or any other suitable attachment structure.

[0197] The indicator may provide the end user or intermediate handler of the container with a variety of information including, but not limited to, the name of the company or affiliate that manufactured the formulation and / or components of the formulation, the date of formulation manufacture, the physical location where the formulation was manufactured, the date of container transportation, whether it was stored at a remote location and the conditions and duration of such storage, the date of container shipment, the mode of transportation, the National Drug Code (NDC) as defined by FDA, the contents of the container, and instructions for use, including dosage and route of administration.

[0198] An indicator may serve one or more purposes, including, for example, authentication of the container and the drug product contained therein. Authentication refers to the ability to prove or state that the container originated and was manufactured by an authorized party, making it possible to identify the container and drug product originating from an end user or other unauthorized party. An indicator may also be used for container track and trace. This property may be used to track the container and the drug product contained therein after formulation to the point of administration to a subject. In this regard, the container's movements during that time may be stored in a database, and if desired, such a database may be accessible by the end user to verify the integrity of the drug product.

[0199] The indicator may also be a combination indicator, which is intended to be information that can be read using two different modes. For example, the indicator may include information that is visible to the naked eye (e.g., a written statement of the manufacturer's name) and other information that is machine readable, such as RFID-embedded or barcode-embedded information.

[0200] The indicator may also be a dual-use indicator, intended to serve more than one purpose. For example, the indicator may contain information identifying the formulation and further information identifying the manufacturer and / or date of manufacture. This information may be conveyed using the same format or different formats (e.g., one may be provided by an RFID indicator and the other by a barcode label).

[0201] According to an embodiment, the identification and activation device may include a holder arranged to receive a container containing a lipid formulation and a gas, and a shaking means arranged to shake the holder at at least one speed, the shaking means including a motor driving the movement of the holder via a transmission, where at least a portion of the transmission is arranged to prevent slippage during activation of the UCA formulation.

[0202] The transmission may be arranged to provide friction and / or resistance to motion. The transmission may include at least one of a tooth belt, a drive chain, an O-ring, and a gear box. The transmission may include a gear having first and second gears, the first gear being connectable to the holder and the second gear being connectable to the motor. In such an embodiment, the tooth belt is arranged to engage each of the first and second gears. The first gear may be disposed around a portion of the spindle connectable to the holder and the second gear may be disposed around a portion of the motor, such as a shaft. The first and second gears may be substantially parallel to each other. The first and second gears may be substantially coplanar with each other.

[0203] The transmission may also include first and second O-rings, where the first O-ring is located on a first side of the first gear and the second O-ring is located on a second side of the first gear. In one embodiment, each of the first and second O-rings and the first gear are located around the spindle.

[0204] The transmission can include first and second profile wheels, such as sprockets, having teeth or cogs. As above, the first wheel can be connectable to a holder and the second wheel can be connectable to a motor. In some embodiments, the first and second wheels engage with a perforated or knurled chain, track, or other material. For example, the teeth engage with a perforated or knurled chain, track, or other material.

[0205] Another suitable identification and activation device comprises a holder arranged to receive a container containing a lipid formulation, an identification means for identifying the lipid formulation in the container; and an agitation means arranged to agitate the holder at at least one speed and comprising a motor driving movement of the holder via a transmission, wherein the transmission comprises at least one of a sawtooth, perforated, knurled belt or track, a drive chain, and a gear box.

[0206] The transmission may include a gear having first and second gears, the first gear connectable to the holder and the second gear connectable to the motor. A sawtooth, perforated, knurled belt or track may be disposed to engage with each of the first and second gears. The transmission may include first and second profile wheels, such as sprockets, connectable with the motor and the holder, respectively. A sawtooth, perforated, knurled belt or track or other material (e.g., a drive chain) may be disposed to engage with each of the first and second wheels.

[0207] Each of the apparatuses or devices may further include a housing, and the shaking means is disposed in the housing. It may also include a first identification means disposed to identify the lipid formulation in the vial. The first identification means may include at least one of an RFID reader, a microchip reader, and a barcode scanner. The first identification means may also include at least one antenna.

[0208] The holder may include a shaker arm.The motor may include a direct current (DC) motor, for example a brushless DC motor.

[0209] The at least one speed can be predetermined. The at least one speed can be preset by the device. The at least one speed can be less than about 4530 rpm or can be about 6000 rpm. The device can be configured to shake the holder at two or more speeds, where the two or more speeds are between about 4530 rpm and about 6000 rpm.

[0210] The identification means may be arranged to identify a shaking speed for the lipid formulation in the container. The identification means may be arranged to identify the formulation and / or the shaking speed of the formulation by reading an indicator on the container.

[0211] lipids The lipid formulations provided herein can contain one, and typically more than one, lipid. As used herein, "lipids" or "total lipids" or "combined lipids" refers to a mixture of lipids.

[0212] Lipids may be provided in individual solid state (e.g., powdered) forms. Alternatively, lipids may be provided as lipid blends. Methods for producing lipid blends include those described in US Patent 8,084,056 and published PCT application WO99 / 36104. As used herein, lipid blends are intended to refer to two or more lipids that are blended together to provide a more homogeneous lipid mixture than can be achieved by simple mixing of lipids in individual powdered forms. Lipid blends may be produced via an aqueous suspension-lyophilization process or an organic solvent dissolution-precipitation process using an organic solvent. In an aqueous suspension-lyophilization process, the desired lipids are suspended in water at elevated temperatures and then concentrated by lyophilization. The contents of US Patent 8,084,056 and published PCT application WO99 / 36104 related to methods for producing lipid blends are incorporated herein by reference.

[0213] Alternatively, the lipids may be provided as individual powders that are dissolved together or individually in propylene glycol, glycerol or propylene glycol / glycerol to form a non-aqueous lipid formulation or a precursor to an aqueous lipid formulation.

[0214] The lipids may be cationic, anionic or neutral lipids. The lipids may be of natural, synthetic or semi-synthetic origin, including, for example, fatty acids, fluorinated lipids, neutral lipids, phosphatides, oils, fluorinated oils, glycolipids, surface active agents (surfactants and fluorosurfactants), fatty alcohols, waxes, terpenes and steroids.

[0215] At least one of the lipids can be a phospholipid. As used herein, a phospholipid is a fatty substance that contains an oily (hydrophobic) hydrocarbon chain with a polar (hydrophilic) phosphorus head group. Phospholipids are amphiphilic.

[0216] Preferably, the lipids are all phospholipids, examples of which include, but are not limited to, phosphatidylcholine; 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC); phosphatidic acid; 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA); phosphatidylethanolamine; and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE). DPPA and DPPE may be provided as monosodium salt forms.

[0217] In some cases, one or more of the lipids can be modified.For example, lipids can be conjugated to polymers such as chitin, hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol (PEG).The lipids conjugated to PEG are referred to herein as PEGylated lipids.Preferably, the PEGylated lipid is DPPE-PEG or DSPE-PEG.

[0218] Conjugation of lipids to polymers such as PEG can be achieved by a variety of bonds or linkages, including but not limited to, amide, carbamate, amine, ester, ether, thioether, thioamide and disulfide (thioester) bonds.

[0219] The end groups of PEG include, but are not limited to, hydroxy-PEG (HO-PEG) (or reactive derivatives thereof), carboxy-PEG (COOH-PEG), methoxy-PEG (MPEG) or other lower alkyl groups, such as in iso-propoxy PEG or t-butoxy PEG, amino PEG (NHPEG) or thiol (SH-PEG).

[0220] The molecular weight of PEG can vary from about 500 to about 10,000, including from about 1,000 to about 7,500 and from about 1,000 to about 5,000. In one important embodiment, the molecular weight of PEG is about 5,000. Thus, DPPE-PEG5000 or DSPE-PEG5000 refers to DPPE or DSPE having a PEG polymer having a molecular weight of about 5,000 attached thereto.

[0221] The percentage of PEGylated lipids relative to the total amount of lipids in the lipid solution on a molar basis is from about 2% to about 20%, inclusive. In various embodiments, the percentage of PEGylated lipids relative to the total amount of lipids is from 5 mole percent to about 15 mole percent, inclusive.

[0222] In some embodiments, the lipids are 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, monosodium salt (DPPA) and N-(polyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, monosodium salt (PEG5000-DPPE).PEG5000-DPPE can be MPEG5000-DPPE or HO-PEG5000-DPPE.In some embodiments, the lipids can be one, two or all three of DPPA, DPPC and PEG5000-DPPE. In some embodiments, the lipids can be one, two or all three of DPPC, DPPE and PEGylated-DPPE. In some embodiments, the lipids can be one, two or all three of DPPC, DPPE and PEG5000-DPPE. In these latter embodiments, the lipids can be free of DPPA. PEG5000-DPPE can be MPEG5000-DPPE or HO-PEG5000-DPPE.

[0223] A wide variety of lipids may be used in the present invention, such as those described in U.S. Patent 5,469,854 to Unger et al. Suitable lipids include, for example, fatty acids, lysolipids, fluorinated lipids, such as platelet activating factor (PAF) lipids (Avanti Polar Lipids, Alabaster, CA), including 1-alkyl-2-acetoyl-sn-glycero-3-phosphocholines and 1-alkyl-2-hydroxy-sn-glycero-3-phosphocholines. phosphocholines, including those bound to dioleoylphosphatidylcholine (Ala.); phosphatidylcholines with both saturated and unsaturated lipids, including dioleoylphosphatidylcholine; dimyristoyl-phosphatidylcholine; dipentadecanoylphosphatidylcholine; dilauroylphosphatidylcholine; 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC); distearoylphosphatidylcholine (DSPC); and diarachidonylphosphatidylcholine (DAPC); phosphatidylethanolamines, such as dioleoyl-phosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DSPC), and diarachidonylphosphatidylcholine (DAPC). distearoyl-phosphatidylethanolamine (DPPE) and distearoyl-phosphatidylethanolamine (DSPE); phosphatidylserine; phosphatidylglycerols including distearoylphosphatidylglycerol (DSPG); phosphatidylinositol; sphingolipids such as sphingomyelin; glycolipids such as gangliosides GM1 and GM2; glucolipids; sulfatides; glycosphingolipids; phosphatidic acids such as 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) and distearoylphosphatidic acid (DSPA); palmitic acid; stearic acid; arachidonic acid; and oleic acid.

[0224] Other suitable lipids include phosphatidylcholines, such as dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine; phosphatidylethanolamines, such as dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine, and N-succinyl-dioleoylphosphatidylethanolamine; phosphatidylserines; phosphatidyl-glycerol; sphingolipids. Substances;Glycolipids such as ganglioside GM1;Glucolipids;Sulfatides;Glycosphingolipids;Phosphatidic acids such as dipalmatoylphosphatidic acid (DPPA);Palmitic fatty acids;Stearic fatty acids;Arachidonic fatty acids;Lauric fatty acids;Myristic fatty acids;Lauroleic fatty acids;Mash fatty acids;Myristoleic fatty acids;Palmitoleic fatty acids;Petroselinic fatty acids;Oleic fatty acids;Isolauric fatty acids;Isomyristic fatty acids;Isopalmitic fatty acids;Isostearic fatty acids;Cholesterol and cholesterol Cholesterol derivatives such as cholesterol hemisuccinate, cholesterol sulfate and cholesteryl-(4'-trimethylammonio)-butanoate;Polyoxyethylene fatty acid esters;Polyoxyethylene fatty alcohols;Polyoxyethylene fatty alcohol ethers;Polyoxyethylated sorbitan fatty acid esters;Glycerol polyethylene glycol oxystearate;Glycerol polyethylene glycol ricinoleate;Ethoxylated soybean sterols;Ethoxylated castor oil;Polyoxyethylene Polyoxyethylene fatty acid polymers;Polyoxyethylene fatty acid stearates;12-(((7'-diethylaminocoumarin-3-yl)-carbonyl)-methylamino)-octadecanoic acid;N-[12-(((7'-diethylamino-coumarin-3-yl)-carbonyl)-methyl-amino)octadecanoyl]-2-amino-palmitic acid;1,2-dioleoyl-sn-glycerol;1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinyl-glycerol;and 1-hexadecyl-2-palmitoyl-glycerophosphoethanolamine and palmitoylhomocysteine; lauryltrimethylammonium bromide (lauryl- = dodecyl-); cetyltrimethylammonium bromide (cetryl- = hexadecyl-); myristyltrimethylammonium bromide (myristyl- = tetradecyl-); alkyldimethylbenzylammonium chlorides (e.g., where alkyl is C; 12 , C 14 or C 16 alkyl); benzyl dimethyl dodecyl ammonium bromide; benzyl dimethyl dodecyl ammonium chloride, benzyl dimethyl hexadecyl ammonium bromide; benzyl dimethyl hexadecyl ammonium chloride; benzyl dimethyl tetradecyl ammonium bromide; benzyl dimethyl tetradecyl ammonium chloride; cetyl dimethyl ethyl ammonium bromide; cetyl dimethyl ethyl ammonium chloride; cetyl pyridinium bromide; cetyl pyridinium chloride; N-[1-2,3-dioleoyloxy)-propyl]-N,N,N-trimethyl ammonium chloride (DOTMA); 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP); and 1,2-dioleoyl-e-(4'-trimethylammonio)-butanoyl-sn-glycerol (DOTB).

[0225] In certain embodiments in which DPPA, DPPC and DPPE are used, their molar percentages can be about 77-90 mol% DPPC, about 5-15 mol% DPPA and about 5-15 mol% DPPE (including DPPE-PEG5000). Preferred ratios of each lipid include those described in the Examples section, such as a weight percent ratio of 6.0:53.5:40.5 (DPPA:DPPC:MPEG5000-DPPE) or a molar ratio of 10:82:8 (DPPA:DPPC:MPEG5000-DPPE).

[0226] gas The gas is preferably substantially insoluble in the lipid solutions provided herein. The gas may be a non-soluble fluorinated gas, such as sulfur hexafluoride or a perfluorocarbon gas.

[0227] Examples of perfluorocarbon gases include perfluoropropane, perfluoromethane, perfluoroethane, perfluorobutane, perfluoropentane, and perfluorohexane. Examples of gases that can be used in the microspheres of the present invention are described in U.S. Patent No. 5,656,211, which is incorporated herein by reference. In an important embodiment, the gas is perfluoropropane.

[0228] Examples of gases are hexafluoroacetone, isopropylacetylene, allene, tetrafluoroarene, boron trifluoride, 1,2-butadiene, 1,3-butadiene, 1,2,3-trichlorobutadiene, 2-fluoro-1,3-butadiene, 2-methyl-1,3-butadiene, hexafluoro-1,3-butadiene, butadiyne, 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-buten-2-one, 2-methyl-1-buten-3-yne, butyl nitrate, 1-butyne, 2-butyne, 2-chloro-1,1,1,4,4,4-hexafluoro-butyne, 3-methyl-1-butyne, perfluoro-2-butyne, 2-bromo-butyraldehyde, carbonyl sulfide, crotononitrile, cyclobutane, methylcyclobutane, octafluorocyclobutane (perfluorocyclobutane) cyclobutane), perfluoroisobutane, 3-chlorocyclopentene, cyclopropane, 1,2-dimethylcyclopropane, 1,1-dimethylcyclopropane, ethylcyclopropane, methylcyclopropane, diacetylene, 3-ethyl-3-methyldiaziridine, 1,1,1-trifluorodiazoethane, dimethylamine, hexafluorodimethylamine, dimethylethylamine, bis-(dimethylphosphine)amine, 2,3-dimethyl-2-norbornane, perfluoro-dimethylamine, dimethyloxonium chloride ide, 1,3-dioxolan-2-one, 1,1,1,1,2-tetrafluoroethane, 1,1,1-trifluoroethane, 1,1,2,2-tetrafluoroethane, 1,1,2-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-1,1-difluoroethane, 1-chloro-1,1,2,2-tetrafluoroethane, 2-chloro-1,1-Difluoroethane, Chloroethane, Chloropentafluoroethane, Dichlorotrifluoroethane, Fluoroethane, Nitropentafluoroethane, Nitrosopentafluoroethane, Perfluoroethane, Perfluoroethylamine, Ethyl vinyl ether, 1,1-Dichloroethylene, 1,1-Dichloro-1,2-difluoro-ethylene, 1,2-Difluoroethylene, Methane, Methane-sulfonyl-chloride-trifluoro, Methane-sulfonyl-fluoride-trifluoro, Methane-(pentafluorothio)trifluoro, Methane-bromo-difluoro-nitroso, Methane-bromo-fluoro, Methane-bromo-chloro-fluoro, Methane-bromo-trifluoro, Methane-chloro-difluoro-nitro, Methane-chloro-dinitro, Methane-chloro-fluoro, Methane-chloro-trifluor Oro, methane-chloro-difluoro, methane-dibromo-difluoro, methane-dichloro-difluoro, methane-dichloro-fluoro, methane-difluoro, methane-difluoro-iodo, methane-disilano, methane-fluoro, methane-iodomethane-iodo-trifluoro, methane-nitro-trifluoro, methane-nitroso-triofluoro, methane-tetrafluoro, methane-trichloro-fluoro, methane-trifluoro, methanesulfenyl chloride-trifluoro, 2-methylbutane, methyl ether, methyl isopropyl ether, methyl lactate, methyl nitrite, methyl sulfide, methyl vinyl ether, neopentane, nitrogen (N2), nitrous oxide, 1,2,3-nonadecanetricarboxylic acid-2-hydroxychrylmethyl ester, 1-nonen-3-yne, oxygen (O2), oxygen 17 (, 17O2), 1,4-pentadiene, n-pentane, dodecafluoropentane (perfluoropentane), tetradecafluorohexane (perfluorohexane), perfluoroisopentane, perfluoroneopentane, 2-pentanone-4-amino-4-methyl, 1-pentene, 2-pentene {cis}, 2-pentene {trans}, 1-pentene-3-bromo, 1-pentene-perfluoro, phthalic acid-tetrachloro, piperidine-2,3,6-trimethyl, propane, propane-1,1,1,2,2,3-hexafluoro, propane-1,2-epoxy , propane-2,2-difluoro, propane-2-amino, propane-2-chloro, propane-heptafluoro-1-nitro, propane-heptafluoro-1-nitroso, perfluoropropane, propene, propyl-1,1,1,2,3,3-hexafluoro-2,3-dichloro, propylene-1-chloro, propylene-chloro-{trans}, propylene-2-chloro, propylene-3-fluoro, propylene-perfluoro, propyne, propyne-3,3,3-trifluoro, styrene-3-fluoro, sulfur hexafluoride, sulfur (di)decafluoride (S2F 10 ), toluene-2,4-diamino, trifluoroacetonitrile, trifluoromethyl peroxide, trifluoromethyl sulfide, tungsten hexafluoride, vinyl acetylene, vinyl ether, neon, helium, krypton, xenon (especially rubidium-enriched hyperpolarized xenon gas), carbon dioxide, helium and air.

[0229] Fluorinated gases (i.e., gases that contain one or more fluorine molecules, such as sulfur hexafluoride), fluorocarbon gases (i.e., fluorinated gases that are fluorinated carbons or gases), and perfluorocarbon gases (i.e., fluorocarbon gases that are fully fluorinated, such as perfluoropropane and perfluorobutane) are preferred.

[0230] Gases such as perfluorocarbon gases are typically present at less than normal concentrations at room temperature due to air entrapment during manufacturing. The concentration of perfluoropropane when present in a vial containing a non-aqueous mixture and gas headspace is expected to be about 6.52 mg / mL at about 1 atmosphere. The concentration of other gases is similarly diluted by air entrapment during manufacturing, as is known in the art.

[0231] A gas, such as perflutren gas, may be injected or otherwise added to a container (e.g., a vial) containing the lipid formulation or to the lipid formulation itself to provide a gas other than air. Gases that are not heavier than air can be added to sealed containers, while gases that are heavier than air can be added to sealed or non-sealed containers.

[0232] It will be appreciated by those skilled in the art that gaseous precursors may also be used after conversion of the precursor to a gas by temperature or pressure changes. EXAMPLES

[0233] Example 1 DEFINITY, an aqueous lipid formulation (i.e., lipid suspension) manufactured by Lantheus Medical Imaging, Inc. (登録商標) The vial contained, prior to activation, 103.5 mg / mL propylene glycol, 126.2 mg / mL glycerol (i.e., glycerin), and the following phospholipids in a matrix of 2.34 mg / mL monosodium phosphate monohydrate, 2.16 mg / mL disodium phosphate heptahydrate, and 4.87 mg / mL sodium chloride in water for injection: 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC; 0.401 mg / mL), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA; 0.045 mg / mL), and N-(methoxypolyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (MPEG5000 DPPE; 0.304 mg / mL). The pH was 6.2-6.8. The combined lipid concentration is 0.75 mg lipid per mL of formulation. The volume of the lipid formulation was approximately 1.76 mL in a 2 cc (2 mL) Wheaton glass vial with an actual internal volume of approximately 3.8 mL and a headspace of approximately 2.04 containing perfluoropropane gas (PFP, 6.52 mg / mL). A commercially available FDA-approved, aqueous-based UCA formulation, DEFINITY (登録商標) (Lantheus Medical Imaging, Inc.), VIALMIX (登録商標) Activation is accomplished by mechanical shaking of the PFP / lipid formulation using a gas-filled microsphere (described in U.S. Patent 6,039,557, the contents of which are incorporated herein by reference and may be used in this process). This entraps the gas in the lipid microspheres, representing an activated product (see DEFINITY® Prescribing Information). The gas-filled bubbles can be analyzed for number and size distribution using a particle size analyzer (AccuSizer 780 or SpetraDyne) upon dilution with saline. Sound attenuation assays were performed using broadband transducers centered at frequencies of 2.25 or 7.5 MHz.

[0234] Example 2 DEFINITY RT, a non-aqueous lipid formulation (i.e., lipid solution) manufactured by Lantheus Medical Imaging, Inc. (登録商標) The vials contained the following phospholipids before activation: 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC; 2.005 mg / mL), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA; 0.225 mg / mL), and N-(methoxypolyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (MPEG5000-DPPE; 1.520 mg / mL) in a matrix of 517.5 mg / mL propylene glycol, 631 mg / mL glycerol (i.e., glycerin), 0.370 mg / mL anhydrous sodium acetate, and 0.030 mg / mL glacial acetic acid. The pH is 5.2-6.4. The combined lipid concentration is 3.75 mg lipid per mL of formulation. Prior to activation, the headspace of the vial contains 6.52 mg / mL octafluoropropane and the viscous solution contains 3.75 mg / mL of the lipid formulation. The volume of the lipid formulation was approximately 0.35 mL in a 2 cc (2 mL) Wheaton glass vial with an actual internal volume of approximately 3.8 mL and a headspace of approximately 3.45 mL containing perfluoropropane gas (PFP, 6.52 mg / mL). Activation is VIALMIX (登録商標) or VIALMIX-RFID (登録商標) This can be performed using a gas trap, whereby gas is entrapped in lipid nano / microbubbles, which represent the activated product. The gas-filled bubbles can be analyzed for number and size distribution using a particle size analyzer (AccuSizer 780 or SpetraDyne) upon dilution in saline. Sound attenuation assays were performed using broadband transducers centered at frequencies of 2.25 or 7.5 MHz.

[0235] Example 3 Aqueous lipid formulations (e.g., DEFINITY (登録商標) , the vial of Example 1) was modified to have various time, time and speed parameters programmable at 4530 rpm, VIALMIX-RFID (登録商標) The samples were shaken using an AccuSizer 780. Samples were taken immediately after shaking and analyzed for bubble size distribution using an AccuSizer 780. Bubbles of various size distributions indicate that maximum formation of 0.5-1 μm microbubbles occurs at approximately 90 seconds of activation (Table 1), 1-2 μm at 45 seconds (data not shown) and 2-6 μm at 10, 20 and 90 seconds (Table 3). Analysis of the samples for acoustic attenuation at each time point showed that the maximum for 7.5 MHz was at 45 seconds, a profile that fits the 1-2 μm microbubble population. Similarly, acoustic attenuation for 2.25 MHz was approximately maximum at 10 and 20 seconds, a profile that fits the 2-6 μm microbubble population (data not shown).

[0236] Example 4 Aqueous lipid formulations (e.g., DEFINITY(登録商標) The vial of Example 1) was modified to have programmable time and rate parameters, VIALMIX-RFID (登録商標) The samples were shaken at 6000 rpm for various times using an AccuSizer 780. Samples were taken immediately after shaking and analyzed for bubble size distribution using an AccuSizer 780. Bubbles of various size distributions showed that maximum formation of 0.5-1 μm microbubbles occurred at approximately 20 and 25 seconds of activation (Table 2), 1-2 μm at 10-15 seconds (data not shown) and 2-6 μm at 5, 25 and 30 seconds (Table 4). Analysis of the samples for acoustic attenuation at each time point showed that the maximum for 7.5 MHz was at 10-15 seconds (data not shown), a profile that fits the 1-2 μm microbubble population. Acoustic attenuation for 2.25 MHz was highest at 5 and 25-30 seconds (data not shown), a profile that fits the 2-6 micron microbubble population.

[0237] Example 5 Non-aqueous lipid formulations (e.g., DEFINITY RT (登録商標) Vials from Example 2) were shaken at 4950 rpm for various times using a 4950 rpm shaker. Samples were taken immediately after shaking and analyzed for bubble size distribution using an AccuSizer 780. Bubbles of various size distributions showed that maximum formation of 0.5-1 μm microbubbles occurred at approximately 250 seconds of activation (data not shown), 1-2 μm at 125 seconds (data not shown) and 2-6 μm at 80 seconds (data not shown). Analysis of the samples for acoustic attenuation at each time point showed that the maximum for 7.5 MHz was between 100 and 150 seconds, a profile that fits the 1-2 μm microbubble population. Similarly, acoustic attenuation for 2.25 MHz was highest at 80 seconds, a profile that fits the 2-6 μm microbubble population (data not shown).

[0238] Example 6 Non-aqueous lipid formulations (e.g., DEFINITY RT (登録商標)The vial of Example 2) was modified to have programmable time and rate parameters, VIALMIX-RFID (登録商標) The samples were shaken at 6000 rpm for various times using an AccuSizer 780. Samples were taken immediately after shaking and analyzed for bubble size distribution using an AccuSizer 780. Bubbles of various size distributions showed that maximum formation of 0.5-1 μm microbubbles occurred at approximately 50-90 s of activation (Table 2A), 1-2 μm at 30-40 s (data not shown) and 2-6 μm at 20-90 s (Table 4). Analysis of the samples for acoustic attenuation at each time point showed that the maximum for 7.5 MHz was at 30 s (data not shown), a profile that fits the 1-2 μm microbubble population. Similarly, acoustic attenuation for 2.25 MHz was highest at 20-90 s (Table 2A), a profile that fits the 2-6 μm microbubble population. Non-aqueous lipid formulations (e.g., DEFINITY RT (登録商標) The vial of Example 2) was modified to have programmable time and speed parameters using a Trinamic brushless motor, VIALMIX-RFID (登録商標) The samples were shaken for various times at speeds of 6200-6600 rpm using an AccuSizer 780. Samples were removed immediately after shaking, inverted 10 times, and analyzed for bubble size distribution using an AccuSizer 780. Bubbles of various size distributions showed that maximum formation of 0.5-1 μm microbubbles occurred at speeds of 6200-6600 rpm for approximately 30-60 s of activation (Table 2B), 1-2 μm at approximately 15 s (data not shown), and 2-6 μm at 15-70 s (data not shown). Data are based on non-aqueous lipid formulations (e.g., DEFINITY RT (登録商標) Example 2) showed that sufficient activation was possible at 6000 rpm, 6200 rpm and 6600 rpm, although the peak times differed. At 6000 rpm and 6200 rpm, the peak time was thought to be about 60 seconds. At 6600 rpm, the maximum effect was seen at about 15 to about 30 seconds.

[0239] Example 7 Non-aqueous lipid formulations (e.g., DEFINITY RT(登録商標) , Example 2) vials were filled with a) unmodified or b) 1.5x the fill volume of non-aqueous lipid formulation (i.e., 1.5x 0.35mL lipid formulation) (referred to as DEFINITY-RT 1.5X fill volume, Table 5) or c) diluted with 0.5x the fill volume of propylene glycol / glycerol (50:50) matrix alone (i.e., 0.35mL lipid formulation and approximately 0.18mL matrix) (referred to as DEFINITY-RT 1X+0.5*, Table 5). Increasing the amount of non-aqueous lipid solution or surprisingly only the matrix increased the number of 2-6μm microbubbles formed. These changes increased the acoustic attenuation at 2.25MHz but not 7.5MHz (Table 5). Bubble size distribution analysis using an AccuSizer 780 showed that the majority of bubbles were smaller than 2μm when shaken at 6000rpm for 60s (data not shown). However, upon dilution with matrix, the distribution became substantially enriched for microbubbles larger than 2 μm, but not smaller than 10 μm, and this distribution enhanced the increase in broadband pulsed acoustic attenuation at 2.25 MHz (Table 5).

[0240] Example 8 Non-aqueous lipid formulations (e.g., DEFINITY RT (登録商標) Vials from Example 1, Example 2) were shaken at 6000 rpm for 60 seconds using a VIALMIX-RFID®. After dilution with saline, samples were inverted 10 times and either (a) taken immediately and analyzed for bubble size distribution using an AccuSizer 780 or (b) transferred to a 3 mL (or 5 mL syringe) Becton Dickinson or Norm Jet syringe with a Luer lock plug inserted and manual plunger pressure applied until the white milky solution became clear (see FIG. 1 for 3 mL syringe). After 10 inversions, the plug was removed and the sample was analyzed for bubble size distribution using an AccuSizer 780. Sufficient manual pressure and release resulted in significant clearance of bubbles larger than 500 nm (see Table 6). Aqueous lipid formulations (e.g., DEFINITY (登録商標)Vials from Example 1) were shaken at 4530 rpm for 45 seconds using a VIALMIX-RFID®. Samples were either (a) taken immediately after shaking, inverted, and analyzed for bubble size distribution using an AccuSizer 780 or (b) transferred to a 3 mL Becton Dickinson or Norm Jet syringe with a Luer lock plug inserted and manual plunger pressure applied until the white milky solution became clear. After 10 inversions, the plug was removed and samples were analyzed for bubble size distribution using an AccuSizer 780. Sufficient manual pressure and release resulted in significant clearance of bubbles larger than 500 nm (see Table 6). Several tests have been done testing the use of devices to assist in applying pressure to the syringe after activation. A caulking gun was adapted to be insertable into the medicated syringe to facilitate and assist in controlling the application of pressure. Additional pressure application devices are contemplated, including mechanically controlled plungers using gears and / or stepper motors with feedback of measured pressure. [Table 1]

[0241] [Table 2A]

[0242] [Table 2B]

[0243] [Table 3]

[0244] [Table 4]

[0245] [Table 5] *(50 / 50 PG / G)

[0246] [Table 6]

[0247] Terms and embodiments Accordingly, the present invention provides several inventive embodiments, which are provided in the form of clauses below: Item 1. A population of lipid-encapsulated perfluorocarbon bubbles, wherein 75% or more of the lipid-encapsulated perfluorocarbon bubbles are nanobubbles having a diameter ranging from about 100 nanometers to about 1 micron or less, wherein the lipid-encapsulated perfluorocarbon bubbles comprise DPPC and PEG-DPPE, and optionally the nanobubbles are about 7.0 x 10 nanobubbles per mL. 9 ~2.5×10 10 Nanobubbles or approximately 1 x 10 per mL 10 ~2.5×10 10 The composition is present in a range of nanobubbles concentrations.

[0248] Item 2. The composition according to Item 1, wherein 80% or more of the lipid-encapsulated perfluorocarbon bubbles are nanobubbles having diameters ranging from about 100 nanometers to about 1 micron or less.

[0249] Item 3. The composition of item 1 or 2, wherein the lipid-encapsulated perfluorocarbon bubbles comprise DPPA, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8, or the lipid formulation comprises DPPE, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8.

[0250] Item 4. The composition according to any one of Items 1 to 3, wherein the PEG-DPPE is PEG5000-DPPE.

[0251] Item 5. The composition according to any one of Items 1 to 4, wherein the PEG-DPPE is MPEG5000-DPPE.

[0252] Item 6. The composition according to any one of Items 1 to 5, wherein the lipid-encapsulated perfluorocarbon bubbles are lipid-encapsulated perfluoropropane bubbles.

[0253] Item 7. The composition according to any one of Items 1 to 6, wherein the population of lipid-encapsulated perfluorocarbon bubbles is present in a non-aqueous lipid solution containing DPPA, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water, or the population of lipid-encapsulated perfluorocarbon bubbles is present in a non-aqueous lipid solution containing DPPE, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water.

[0254] Item 8. The composition of item 7, wherein the lipids are present in a total concentration of about 3.75 mg / ml.

[0255] Item 9. The composition of item 7, wherein the lipids are present in a total concentration of about 2.5 mg / ml.

[0256] Item 10. The composition according to any one of Items 1 to 6, wherein the population of lipid-encapsulated perfluorocarbon bubbles is present in an aqueous lipid suspension comprising glycerol and propylene glycol.

[0257] Item 11. The composition according to Item 10, wherein the total lipid concentration of the lipid suspension is about 0.75 mg / ml.

[0258] Item 12. The composition according to any one of items 1 to 11, wherein the population of lipid-encapsulated perfluorocarbon bubbles lacks DPPA.

[0259] Item 13. A method for forming lipid-encapsulated perfluorocarbon bubbles, comprising: The method includes activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol in the presence of a perfluorocarbon gas at a sufficient agitation speed and time to produce a population of lipid-encapsulated perfluorocarbon bubbles, wherein the bubbles comprise nanobubbles having diameters in the range of about 100 nanometers to about 1 micron.

[0260] Item 14. The method according to Item 13, wherein 80% or more of the lipid-encapsulated perfluorocarbon bubbles are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron.

[0261] Item 15. The method of item 13 or 14, wherein the lipid formulation comprises DPPA, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8, or the lipid formulation comprises DPPE, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8.

[0262] Item 16. The method according to any one of Items 13 to 15, wherein the PEG-DPPE is PEG5000-DPPE.

[0263] Item 17. The method according to any one of Items 13 to 15, wherein the PEG-DPPE is MPEG5000-DPPE.

[0264] Item 18. The method according to any one of Items 13 to 17, wherein the perfluorocarbon gas is perfluoropropane gas, and the lipid-encapsulated perfluorocarbon bubbles are lipid-encapsulated perfluoropropane bubbles.

[0265] Item 19. The method according to any one of Items 13 to 18, wherein the lipid formulation is a non-aqueous lipid solution containing DPPA, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water, or the lipid formulation is a non-aqueous lipid solution containing DPPE, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water.

[0266] Item 20. The method of item 19, wherein the combined lipid concentration of the lipid formulation is about 3.75 mg / ml.

[0267] Item 21. The method according to Item 19 or 20, wherein the lipid formulation is activated at an agitation speed of about 6000 rpm for an agitation time of about 50 seconds to about 90 seconds.

[0268] Item 22. The method of item 19 or 20, wherein the lipid formulation is activated with an agitation time of about 50 seconds, about 60 seconds, about 75 seconds or about 90 seconds and an agitation speed of about 6000 rpm.

[0269] Item 23. The method of item 19, wherein the combined lipid concentration of the lipid formulation is about 2.5 mg / ml.

[0270] Clause 24. The method of clause 19, wherein the lipid formulation is present in a 2 mL Wheaton glass vial having an internal volume of about 3.8 ml and containing about 0.35 ml of lipid formulation and about 3.45 ml of gas, or the lipid formulation is present in a 2 mL Wheaton glass vial having an internal volume of about 3.8 ml and containing about 0.52 ml of lipid formulation and about 3.28 ml of gas.

[0271] Clause 25. The method of clause 19, wherein the lipid formulation is present in a 2 mL Wheaton glass vial having an internal volume of about 3.8 ml, wherein about 9% of the internal volume is lipid occupied by the lipid formulation prior to activation or about 14% of the internal volume is occupied by the lipid formulation prior to activation.

[0272] Item 26. The method according to any one of Items 13 to 18, wherein the lipid formulation is an aqueous lipid suspension containing DPPA, DPPC and PEG-DPPE, glycerol and propylene glycol, or the lipid formulation is an aqueous lipid suspension containing DPPE, DPPC and PEG-DPPE, glycerol and propylene glycol.

[0273] Item 27. The method according to Item 26, wherein the total lipid concentration of the lipid formulation is about 0.75 mg / ml.

[0274] Item 28. The method of item 26 or 27, wherein the lipid formulation is activated in the presence of gas for a shaking time of about 90 seconds at a shaking speed of about 4530 rpm.

[0275] Item 29. The method according to item 26 or 27, wherein the lipid formulation is activated in the presence of gas for a shaking time of about 20 seconds to about 25 seconds at a shaking speed of about 6000 rpm.

[0276] Item 30. The method of item 26 or 27, wherein the lipid formulation is activated in the presence of gas for a shaking time of about 20 seconds or about 25 seconds at a shaking speed of about 6000 rpm.

[0277] Item 31. The method of any one of items 13 to 30, wherein the lipid formulation lacks DPPA.

[0278] Item 32. A population of lipid-encapsulated perfluorocarbon bubbles formed by the method according to any one of Items 13 to 31.

[0279] Item 33. A method for treating a subject having a condition, comprising: administering to a subject having a condition an effective amount of a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 75% of the bubbles in the population are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron, the lipid-encapsulated perfluorocarbon bubbles comprising DPPC and PEG-DPPE; and applying ultrasonic energy to an area of ​​the subject suffering from the condition The method includes:

[0280] Item 34. A method for improving tissue perfusion in a subject, comprising exposing the subject to therapeutic ultrasound, wherein the improvement administering to the subject a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 75% of the bubbles in the population are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron, prior to and / or during exposure of the subject to therapeutic ultrasound. The method includes:

[0281] Clause 35. The method of clause 33, wherein the condition is the presence of a thrombus / blood clot, a vascular occlusion, a retinal vein occlusion, a tumor, erectile dysfunction, Alzheimer's disease, or Parkinson's disease.

[0282] Clause 36. The method of clause 33, wherein the condition is one that would benefit from disruption of the blood-brain barrier (BBB), and optionally, wherein a condition-specific therapeutic agent is also administered to the subject before, during and / or after administration of the population of bubbles and / or before, during and / or after exposure to therapeutic ultrasound.

[0283] Item 37. The method according to any one of Items 33 to 36, wherein the lipid-encapsulated perfluorocarbon gas nanobubbles reach stable cavitation in vivo after application of ultrasonic energy.

[0284] Item 38. The method according to any one of Items 33 to 36, wherein lipid-encapsulated perfluorocarbon gas nanobubbles are optionally detected and / or measured by emitted broadband radio frequency to achieve inertial cavitation in vivo after application of ultrasound energy.

[0285] Item 39. The method according to any one of Items 33 to 38, wherein 80% or more of the lipid-encapsulated perfluorocarbon bubbles are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron.

[0286] Item 40. The method of any one of items 33 to 39, wherein the lipid-encapsulated perfluorocarbon bubbles comprise DPPA, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8, or the lipid formulation comprises DPPE, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8.

[0287] Item 41. The method according to any one of Items 33 to 40, wherein the PEG-DPPE is PEG5000-DPPE.

[0288] Item 42. The method according to any one of Items 33 to 40, wherein the PEG-DPPE is MPEG5000-DPPE.

[0289] Item 43. The method according to any one of Items 33 to 42, wherein the lipid-encapsulated perfluorocarbon bubbles are lipid-encapsulated perfluoropropane bubbles.

[0290] Item 44. The method according to any one of Items 33 to 43, wherein the population of lipid-encapsulated perfluorocarbon bubbles is formed by the method according to any one of Items 13 to 31.

[0291] Item 45. The method according to any one of Items 33 to 43, wherein the population of lipid-encapsulated perfluorocarbon bubbles is the composition according to any one of Items 1 to 12.

[0292] Item 46. A population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 15% of the microbubbles in the population have a diameter of about 2 microns to about 6 microns or less; and The lipid-encapsulated perfluorocarbon bubbles include DPPC and PEG-DPPE, and optionally contain about 0.24×10 microbubbles per mL having diameters of about 2 microns to about 6 microns or less. 9 ~2.44×10 9 exists in the foam concentration range, composition.

[0293] Clause 47. The composition of clause 46, wherein 20% or more of the lipid-encapsulated perfluorocarbon gas microbubbles in the population have a diameter of about 2 microns to about 6 microns or less.

[0294] Item 48. The composition of item 46 or 47, wherein the lipid-encapsulated perfluorocarbon bubbles comprise DPPA, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8, or the lipid-encapsulated perfluorocarbon bubbles comprise DPPE, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8.

[0295] Item 49. The composition according to any one of Items 46 to 48, wherein the PEG-DPPE is PEG5000-DPPE.

[0296] Item 50. The composition according to any one of Items 46 to 49, wherein the PEG-DPPE is MPEG5000-DPPE.

[0297] Item 51. The composition according to any one of Items 46 to 50, wherein the lipid-encapsulated perfluorocarbon bubbles are lipid-encapsulated perfluoropropane bubbles.

[0298] Item 52. The composition according to any one of Items 46 to 51, wherein the population of lipid-encapsulated perfluorocarbon bubbles is present in a non-aqueous lipid solution containing DPPA, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water, or the population of lipid-encapsulated perfluorocarbon bubbles is present in a non-aqueous lipid solution containing DPPE, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water.

[0299] Item 53. The composition of any one of items 46 to 52, wherein the lipids are present in a total concentration of about 3.75 mg / ml.

[0300] Item 54. The composition of any one of items 46 to 52, wherein the lipids are present in a total concentration of about 2.5 mg / ml.

[0301] Item 55. The composition according to any one of Items 46 to 51, wherein the population of lipid-encapsulated perfluorocarbon bubbles is present in an aqueous lipid suspension comprising glycerol and propylene glycol.

[0302] Item 56. The composition according to Item 55, wherein the total lipid concentration of the lipid suspension is about 0.75 mg / ml.

[0303] Item 57. The composition of any of items 46 to 56, wherein the population of lipid encapsulated perfluorocarbon bubbles exhibits an acoustic attenuation at 2.25 MHz ultrasonic frequency in the range of greater than about 1, optionally greater than about 1.2, optionally in the range of about 1 to about 1.7.

[0304] Item 58. The composition of any of items 46 to 57, wherein the population of lipid-encapsulated perfluorocarbon bubbles lacks DPPA.

[0305] Item 59. A method for forming lipid-encapsulated perfluorocarbon bubbles, comprising: 1. A method comprising activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol in the presence of a perfluorocarbon gas at a sufficient agitation speed and time to produce a population of lipid-encapsulated perfluorocarbon gas microbubbles, wherein at least 15% of the microbubbles have a diameter of about 2 microns to about 6 microns or less.

[0306] Clause 60. The method of clause 59, wherein at least 20% of the lipid-encapsulated perfluorocarbon gas microbubbles have a diameter of about 2 microns to about 6 microns.

[0307] Item 61. The method of item 59 or 60, wherein the lipid formulation comprises DPPA, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8, or wherein the lipid formulation comprises DPPE, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8.

[0308] Item 62. The method according to any one of Items 59 to 61, wherein the PEG-DPPE is PEG5000-DPPE.

[0309] Item 63. The method according to any one of Items 59 to 61, wherein the PEG-DPPE is MPEG5000-DPPE.

[0310] Item 64. The method according to any one of Items 59 to 63, wherein the perfluorocarbon gas is perfluoropropane gas, and the lipid-encapsulated perfluorocarbon bubbles are lipid-encapsulated perfluoropropane bubbles.

[0311] Item 65. The method according to any one of Items 59 to 64, wherein the lipid formulation is a non-aqueous lipid solution containing DPPA, DPPC and PEG-DPPE, glycerol and propylene glycol and 5% (v / v) or less of water, or the lipid formulation is a non-aqueous lipid solution containing DPPE, DPPC and PEG-DPPE, glycerol and propylene glycol and 5% (v / v) or less of water.

[0312] Item 66. The method of item 65, wherein the lipid concentration of the lipid formulation is about 3.75 mg / ml.

[0313] Item 67. The method according to item 65 or 66, wherein the lipid formulation is activated at an agitation speed of about 6000 rpm for an agitation time of about 75 seconds to about 90 seconds.

[0314] Item 68. The method of item 65 or 66, wherein the lipid formulation is activated at an agitation speed of about 6000 rpm for an agitation time of about 75 seconds or about 90 seconds.

[0315] Item 69. The method of item 65, wherein the lipid concentration of the lipid formulation is about 2.5 mg / ml.

[0316] Clause 70. The method of clause 65, wherein the lipid formulation is present in a 2 mL Wheaton glass vial having an internal volume of about 3.8 ml, wherein the vial contains about 0.35 ml of lipid formulation and about 3.45 ml of gas, or the vial contains about 0.52 ml of lipid formulation and about 3.28 ml of gas.

[0317] Clause 71. The method of clause 65, wherein the lipid formulation is present in a 2 mL Wheaton glass vial having an internal volume of about 3.8 ml, wherein about 9% of the internal volume of the vial is occupied by the lipid formulation before activation or about 14% of the internal volume of the vial is occupied by the lipid formulation before activation.

[0318] Clause 72. The method of clause 65, wherein a solution of propylene glycol and glycerol is combined with the lipid formulation in an equal volume ratio prior to activation.

[0319] Item 73. The method according to any one of Items 69 to 72, wherein the lipid formulation is activated at a shaking speed of about 6000 rpm for a shaking time of about 60 seconds.

[0320] Item 74. The method according to any one of Items 59 to 64, wherein the lipid formulation is an aqueous lipid suspension containing DPPA, DPPC, PEG-DPPE, glycerol and propylene glycol, or the lipid formulation is an aqueous lipid suspension containing DPPE, DPPC, PEG-DPPE, glycerol and propylene glycol.

[0321] Item 75. The method of item 74, wherein the lipid concentration of the lipid formulation is about 0.75 mg / ml.

[0322] Item 76. The method of item 74 or 75, wherein the lipid formulation is activated at an agitation speed of about 6000 rpm for an agitation time of about 30 seconds.

[0323] Item 77. The method of any of items 59 to 76, wherein the population of lipid-encapsulated perfluorocarbon bubbles lacks DPPA.

[0324] Item 78. A population of lipid-encapsulated perfluorocarbon bubbles formed by the method according to any one of Items 59 to 77.

[0325] Item 79. A method of treating a subject having a condition, comprising: administering to a subject having a condition an effective amount of a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 15% of the microbubbles in the population have a diameter of about 2 microns to about 6 microns or less, and the lipid-encapsulated perfluorocarbon bubbles comprise DPPC and PEG-DPPE; and applying ultrasonic energy, optionally at a frequency of about 1 to about 10 MHz, to an area of ​​the subject suffering from the condition; The method includes:

[0326] Item 80. A method for improving therapeutic or diagnostic ultrasound, the improvement comprising: administering to the subject, prior to or during exposure to ultrasound, optionally at a frequency of about 1 to about 10 MHz, a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 15% of the microbubbles in the population have diameters in the range of about 2 microns to about 6 microns. The method includes:

[0327] Clause 81. The method of clause 79, wherein the condition is cardiac atherosclerosis, myocardial infarction, vascular occlusion, stroke, or peripheral arterial disease.

[0328] Clause 82. The method of clause 79, wherein the lipid encapsulated perfluorocarbon bubbles reach stable cavitation in vivo following application of ultrasonic energy, optionally as detected and / or measured by harmonic frequency emissions.

[0329] Clause 83. The method of clause 79, wherein the lipid encapsulated perfluorocarbon bubbles undergo inertial cavitation in vivo following application of ultrasound energy, optionally detected and / or measured with broadband radio waves.

[0330] Item 84. The method of any one of Items 79 to 83, wherein 20% or more of the lipid-encapsulated perfluorocarbon gas microbubbles have a diameter of about 2 microns to about 6 microns.

[0331] Item 85. The method of any of items 79 to 84, wherein the lipid encapsulated perfluorocarbon bubbles comprise DPPA, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8, or the lipid formulation comprises DPPE, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8.

[0332] Item 86. The method according to any one of Items 79 to 85, wherein the PEG-DPPE is PEG5000-DPPE.

[0333] Item 87. The method according to any one of Items 79 to 85, wherein the PEG-DPPE is MPEG5000-DPPE.

[0334] Item 88. The method according to any one of Items 79 to 87, wherein the lipid-encapsulated perfluorocarbon bubbles are lipid-encapsulated perfluoropropane bubbles.

[0335] Item 89. The method according to any one of Items 79 to 88, wherein the population of lipid-encapsulated perfluorocarbon bubbles is formed by the method according to any one of Items 59 to 77.

[0336] Item 90. The method of any of items 79 to 89, wherein the population of lipid encapsulated perfluorocarbon bubbles has an acoustic attenuation at a 2.25 MHz ultrasonic frequency of greater than about 1.0.

[0337] Item 91. The method of any of items 79 to 89, wherein the population of lipid-encapsulated perfluorocarbon gas microspheres has an acoustic attenuation at a 2.25 MHz ultrasonic frequency of about 1.0 to about 1.5 or about 1.0 to about 2.0.

[0338] Item 92. The method according to any one of Items 79 to 90, wherein the population of lipid-encapsulated perfluorocarbon bubbles is a composition according to any one of Items 46 to 58.

[0339] Item 93. A population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 4% of the microbubbles in the population have a diameter of about 6 microns to about 10 microns, and The lipid-encapsulated perfluorocarbon bubbles contain DPPC and PEG-DPPE, and the microbubbles have diameters ranging from about 6 microns to about 10 microns, with approximately 0.1 × 10 per mL. 8 ~0.32×10 8 Such bubbles are present in a concentration range of composition.

[0340] Item 94. The composition of item 93, wherein 10% or more of the lipid-encapsulated perfluorocarbon gas microbubbles in the population have a diameter of about 6 microns to about 10 microns.

[0341] Item 95. The composition of item 93 or 94, wherein the lipid encapsulated perfluorocarbon bubbles comprise DPPA, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8, or the lipid encapsulated perfluorocarbon bubbles comprise DPPE, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8.

[0342] Item 96. The composition according to any one of Items 93 to 95, wherein the PEG-DPPE is PEG5000-DPPE.

[0343] Item 97. The composition according to any one of Items 93 to 96, wherein the PEG-DPPE is MPEG5000-DPPE.

[0344] Item 98. The composition according to any one of Items 93 to 97, wherein the lipid-encapsulated perfluorocarbon bubbles are lipid-encapsulated perfluoropropane bubbles.

[0345] Item 99. The composition according to any one of Items 93 to 98, wherein the population of lipid-encapsulated perfluorocarbon bubbles is present in a non-aqueous lipid solution containing DPPA, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water, or the population of lipid-encapsulated perfluorocarbon bubbles is present in a non-aqueous lipid solution containing DPPE, DPPC and PEG-DPPE, glycerol, propylene glycol and 5% (v / v) or less of water.

[0346] Item 100. The composition of any of items 93 to 99, wherein the lipids are present in a total concentration of about 3.75 mg / ml.

[0347] Item 101. The composition of any of items 93 to 99, wherein the lipids are present in a total concentration of about 2.5 mg / ml.

[0348] Item 102. The composition of any of items 93 to 98, wherein the population of lipid-encapsulated perfluorocarbon bubbles is present in an aqueous lipid suspension comprising glycerol and propylene glycol.

[0349] Clause 103. The composition of clause 102, wherein the lipids are present in a combined lipid concentration of about 0.75 mg / ml.

[0350] Item 104. The composition of any of items 93 to 103, wherein the population of lipid encapsulated perfluorocarbon bubbles exhibits an acoustic attenuation at 2.25 MHz ultrasonic frequencies in the range of greater than about 1, optionally greater than about 1.2, optionally in the range of about 1 to about 1.7.

[0351] Item 105. The composition of any of items 93 to 104, wherein the population of lipid-encapsulated perfluorocarbon bubbles is devoid of DPPA.

[0352] Item 106. A method for forming lipid-encapsulated perfluorocarbon bubbles, comprising: 1. A method comprising activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol in the presence of a perfluorocarbon gas at a shaking speed and for a shaking time sufficient to produce a population of lipid-encapsulated perfluorocarbon gas microbubbles, wherein at least 4% of the microbubbles have a diameter of about 6 microns to about 10 microns.

[0353] Item 107. The method according to Item 106, wherein 10% or more of the lipid-encapsulated perfluorocarbon gas microbubbles have a diameter of about 6 microns to about 10 microns.

[0354] Item 108. The method of item 106 or 107, wherein the lipid formulation comprises DPPA, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8, or wherein the lipid formulation comprises DPPE, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8.

[0355] Item 109. The method according to any one of items 106 to 108, wherein the PEG-DPPE is PEG5000-DPPE.

[0356] Item 110. The method according to any one of items 106 to 108, wherein the PEG-DPPE is MPEG5000-DPPE.

[0357] Item 111. The method according to any one of Items 106 to 110, wherein the perfluorocarbon gas is perfluoropropane gas, and the lipid-encapsulated perfluorocarbon bubbles are lipid-encapsulated perfluoropropane bubbles.

[0358] Item 112. The method according to any one of Items 106 to 111, wherein the lipid formulation is a non-aqueous lipid solution containing DPPA, DPPC and PEG-DPPE, glycerol and propylene glycol and 5% (v / v) or less of water, or the lipid formulation is a non-aqueous lipid solution containing DPPA, DPPC and PEG-DPPE, glycerol and propylene glycol and 5% (v / v) or less of water.

[0359] Item 113. The method of item 112, wherein the lipid concentration of the lipid formulation is about 3.75 mg / ml.

[0360] Clause 114. The method of clause 112, wherein the lipid formulation is present in a 2 mL Wheaton glass vial having an internal volume of about 3.8 ml, wherein the vial contains about 0.35 ml of lipid formulation and about 3.45 ml of gas, or the vial contains about 0.52 ml of lipid formulation and about 3.28 ml of gas.

[0361] Clause 115. The method of clause 112, wherein the lipid formulation is present in a 2 mL Wheaton glass vial having an internal volume of about 3.8 ml, wherein about 9% of the internal volume of the vial is occupied by the lipid formulation before activation or about 14% of the internal volume of the vial is occupied by the lipid formulation before activation.

[0362] Clause 116. The method of clause 112, wherein a solution of propylene glycol and glycerol is combined with the lipid formulation in an equal volume ratio prior to activation.

[0363] Clause 117. The method of clause 112, wherein the lipid concentration of the lipid formulation is about 2.5 mg / ml.

[0364] Item 118. The method according to any one of Items 114 to 117, wherein the lipid formulation is activated at an agitation speed of about 6000 rpm for an agitation time of about 60 seconds.

[0365] Item 119. The method according to any one of items 106 to 111, wherein the lipid formulation is an aqueous lipid suspension containing DPPA, DPPC, PEG-DPPE, glycerol and propylene glycol, or the lipid formulation is an aqueous lipid suspension containing DPPE, DPPC, PEG-DPPE, glycerol and propylene glycol.

[0366] Item 120. The method of item 119, wherein the lipid concentration of the lipid formulation is about 0.75 mg / ml.

[0367] Item 121. The method of any of items 106 to 120, wherein the population of lipid-encapsulated perfluorocarbon bubbles lacks DPPA.

[0368] Item 122. A population of lipid-encapsulated perfluorocarbon bubbles formed by the method according to any one of Items 106 to 121.

[0369] Item 123. A method of treating a subject having a condition, comprising: administering to a subject having a condition an effective amount of lipid-encapsulated perfluorocarbon bubbles, wherein at least 4% of the microbubbles in the population have a diameter of about 6 microns to about 10 microns, and the lipid-encapsulated perfluorocarbon bubbles comprise DPPC and PEG-DPPE; and applying ultrasonic energy at a frequency of about 1 to about 10 MHz to an area of ​​the subject suffering from the condition; The method includes:

[0370] Item 124. A method for improving therapeutic or diagnostic ultrasound, the improvement comprising: administering to the subject a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 4% of the microbubbles in the population have diameters in the range of about 6 microns to about 10 microns, prior to or during exposure to ultrasound at a frequency of about 1 to about 10 MHz; The method includes:

[0371] Clause 125. The method of clause 124, wherein the condition is cardiac atherosclerosis, myocardial infarction, vascular occlusion, stroke, or peripheral arterial disease.

[0372] Clause 126. The method of clause 124, wherein the lipid-encapsulated perfluorocarbon bubbles achieve stable cavitation in vivo after application of ultrasonic energy.

[0373] Clause 127. The method of clause 124, wherein lipid-encapsulated perfluorocarbon bubbles undergo inertial cavitation in vivo following application of ultrasonic energy.

[0374] Item 128. The method according to any one of Items 124 to 127, wherein 10% or more of the lipid-encapsulated perfluorocarbon gas microbubbles have a diameter of about 6 microns to about 10 microns.

[0375] Item 129. The method of any of items 124 to 128, wherein the lipid encapsulated perfluorocarbon bubbles comprise DPPA, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8, or the lipid formulation comprises DPPE, DPPC and PEG-DPPE, optionally in a molar ratio of about 10 to about 82 to about 8, including 10:82:8.

[0376] Item 130. The method according to any one of items 124 to 129, wherein the PEG-DPPE is PEG5000-DPPE.

[0377] Item 131. The method according to any one of items 124 to 129, wherein the PEG-DPPE is MPEG5000-DPPE.

[0378] Item 132. The method according to any one of Items 124 to 131, wherein the lipid-encapsulated perfluorocarbon bubbles are lipid-encapsulated perfluoropropane bubbles.

[0379] Item 133. The method according to any one of Items 124 to 132, wherein the population of lipid-encapsulated perfluorocarbon bubbles is formed by the method according to any one of Items 106 to 121.

[0380] Item 134. The method of any of items 124 to 133, wherein the population of lipid-encapsulated perfluorocarbon bubbles has an acoustic attenuation at a 2.25 MHz ultrasonic frequency of greater than about 1.0.

[0381] Item 135. The method of any of items 124 to 133, wherein the population of lipid-encapsulated perfluorocarbon gas microspheres has an acoustic attenuation at a 2.25 MHz ultrasonic frequency of about 1.0 to about 1.5 or about 1.0 to about 2.0.

[0382] Item 136. A method for ultrasound imaging of a subject, comprising: Administer lipid-encapsulated perfluoropropane bubbles prepared by the method according to any one of items 46 to 58, 78, 93 to 105, or 122, or any one of items 59 to 77, or 106 to 121, to the subject; applying ultrasonic energy at a frequency in the range of about 1 to about 10 MHz; and obtaining an ultrasound image of the target or control area. The method includes:

[0383] Clause 137. The method of clause 136, wherein the ultrasonic energy is at a frequency of about 1.25 MHz.

[0384] Item 138. A method for forming lipid-encapsulated perfluoropropane bubbles, comprising: In the presence of gas, a non-aqueous lipid formulation containing 5% (v / v) or less of water, DPPC, PEG-DPPE, glycerol, propylene glycol DPPA, or DPPE is activated at a shaking speed of about 6000 rpm for about 30 to about 90 seconds, optionally about 50 seconds, about 60 seconds, about 75 seconds, or about 90 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres. The method includes:

[0385] Clause 139. The method of clause 138, wherein the lipid formulation and gas are present in a vial, optionally a 2 mL vial, and the lipid formulation occupies about 9% or about 14% of the internal volume of the vial.

[0386] Item 140. A method for forming lipid-encapsulated perfluoropropane bubbles, comprising: A non-aqueous lipid formulation containing up to 5% (v / v) water, DPPC, PEG-DPPE, glycerol, propylene glycol, DPPA, or DPPE is activated in the presence of gas at about 4950 rpm for about 125 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres. The method includes:

[0387] Clause 141. The method of clause 140, wherein the lipid formulation and gas are present in a vial, optionally a 2 mL vial, and the lipid formulation occupies about 9% or about 14% of the internal volume of the vial.

[0388] Item 142. A method for forming lipid-encapsulated perfluoropropane bubbles, comprising: An aqueous lipid formulation containing DPPC and PEG-DPPE and glycerol and propylene glycol and either DPPA or DPPE is activated at a shaking speed of about 6000 rpm for about 5 to about 90 seconds, optionally about 5 seconds, about 10 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 60 seconds, about 70 seconds, about 80 seconds or about 90 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres. The method includes:

[0389] Item 143. A method for forming lipid-encapsulated perfluoropropane bubbles, comprising: Aqueous lipid formulations containing DPPC and PEG-DPPE, glycerol and propylene glycol and either DPPA or DPPE are activated at a shaking speed of about 4530 rpm for about 90 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres. The method includes:

[0390] Item 144. A method for forming lipid-encapsulated perfluoropropane bubbles, comprising: An aqueous lipid formulation containing DPPC and PEG-DPPE, glycerol and propylene glycol, and either DPPA or DPPE is activated at a shaking speed of about 4530 rpm for about 10 to about 20 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres. The method includes:

[0391] Item 145. A method for forming lipid-encapsulated perfluoropropane nanodroplets, comprising: The method includes condensing a population of lipid-encapsulated perfluoropropane gas microspheres by applying sufficient pressure to condense the microbubbles into nanodroplets, wherein the population of gas microspheres is condensed at room temperature.

[0392] Clause 146. The method of clause 145, wherein the application of pressure comprises application of finger pressure.

[0393] Item 147. The method of items 145 or 146, wherein the application of pressure includes application of a pressure in the range of about 6 to about 12 atmospheres.

[0394] Paragraph 148. The method of any of paragraphs 145 to 147, wherein the population of lipid-encapsulated perfluoropropane gas microspheres is provided in a syringe, optionally a 3 mL syringe, and the application of pressure comprises application of finger pressure via a plunger.

[0395] Item 149. The method of any of items 145 to 148, wherein the population of lipid-encapsulated perfluoropropane gas microspheres is produced by activation of a lipid formulation comprising DPPA, DPPC and PEG5000-DPPE in the presence of perfluoropropane.

[0396] Item 150. The method of any of items 145 to 148, wherein the population of lipid-encapsulated perfluoropropane gas microspheres is produced by activation of a lipid formulation comprising DPPC, DPPE and PEG5000-DPPE in the presence of perfluoropropane.

[0397] Item 151. The method of item 149 or 150, wherein the lipid formulation is a non-aqueous lipid formulation, and optionally activating the lipid formulation comprises activation at about 4950 rpm for 45 seconds, or at about 6000 rpm to about 6200 rpm for about 60 seconds, or at about 6600 rpm for about 15 seconds to about 30 seconds.

[0398] Clause 152. The method of clause 149 or 150, wherein the lipid formulation is an aqueous lipid formulation, and optionally activating the lipid formulation comprises activation at about 4530 rpm for 45 seconds.

[0399] Item 153. A method for diagnosing or treating a subject, comprising: 153. A method comprising administering to a subject a population of lipid encapsulated nanodroplets and optionally exposing the subject to ultrasound energy at a target location, wherein the population of lipid encapsulated nanodroplets is produced by a method according to any of paragraphs 145 to 152.

[0400] Item 154. The method of item 153, wherein the nanodroplets comprise a lipid shell consisting of DPPA, DPPC and PEG5000-DPPE and a liquid center of perfluoropropane.

[0401] Item 155. The method of item 153, wherein the nanodroplets comprise a lipid shell consisting of DPPC, DPPE and PEG5000-DPPE and a liquid center of perfluoropropane.

[0402] Item 156. The method of item 155, wherein the nanodroplets lack DPPA.

[0403] Item 157. The method of any one of items 153 to 156, wherein the nanodroplets are about 100 to about 200 nanometers in size.

[0404] Item 158. A method for treating a subject having erectile dysfunction, comprising: administering to a subject in need of treatment an effective amount of lipid-encapsulated bubbles or nanodroplets; and An ultrasound is performed on the genital area of ​​interest The method includes:

[0405] Clause 159. The method of clause 158, wherein the subject with erectile dysfunction has mild to moderate erectile dysfunction as measured by IIEF-5 or IIEF-15 scores.

[0406] Clause 160. The method of clause 158 or 159, wherein treatment is evidenced by an increase in IIEF-5 or IIEF-15 score.

[0407] Item 161. The method of any of items 158 to 160, wherein the subject is administered lipid-encapsulated bubbles comprising DPPA, DPPC and MPEG5000-DPPE and a lipid shell comprising a perfluoropropane gas center, wherein the lipid-encapsulated bubbles are present in an activated DEFINITY formulation or an activated DEFINITY RT formulation.

[0408] Item 162. The method of any of items 158 to 160, wherein the subject is administered lipid-encapsulated bubbles comprising DPPC, DPPE and MPEG5000-DPPE and a lipid shell comprising a perfluoropropane gas center.

[0409] Item 163. The method of any of items 158 to 160, comprising administering to the subject lipid-encapsulated nanodroplets comprising a lipid shell comprising DPPA, DPPC and MPEG5000-DPPE and a core comprising perfluoropropane in liquid form, and optionally forming the lipid-encapsulated nanodroplets by condensation of lipid-encapsulated bubbles present in the Activated DEFINITY Formulation or the Activated DEFINITY RT Formulation.

[0410] Item 164. The method of any of items 158 to 160, wherein the subject is administered lipid-encapsulated nanodroplets comprising a lipid shell comprising DPPC, DPPE and MPEG5000-DPPE and a core comprising perfluoropropane in liquid form.

[0411] Item 165. The method of any of items 158 to 164, wherein the ultrasound optionally comprises high intensity ultrasound with a frequency in the range of 1 to 2 MHz and further optionally with a mechanical index of about 1 to about 1.5 or about 1 to about 1.3.

[0412] Paragraph 166. A method according to any of paragraphs 158 to 165, wherein the method is performed repeatedly at spaced intervals, optionally once or twice weekly for one or more weeks.

[0413] Equivalent While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and such variations and / or modifications are deemed to be within the scope of the inventive embodiments described herein. More generally, those of ordinary skill in the art will readily recognize that all of the parameters, dimensions, materials and configurations described herein are intended to be examples, and that the actual parameters, dimensions, materials and / or configurations will depend on the particular application or applications to which the inventive teachings are applied. Those of ordinary skill in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the inventive embodiments described herein. Thus, it is to be understood that the above embodiments are merely exemplary, and that, within the scope of the appended claims and equivalents thereof, the inventive embodiments may be practiced other than as specifically described and claimed. Inventive embodiments of the present invention relate to each individual feature, system, article, material, kit and / or method described herein. Furthermore, any combination of two or more of such features, systems, articles, materials, kits and / or methods is included within the scope of the invention, unless such features, systems, articles, materials, kits and / or methods are mutually inconsistent.

[0414] All definitions defined and used herein should be understood to take precedence over any dictionary definitions, definitions in any documents incorporated by reference, and / or ordinary meaning of the defined term.

[0415] All publications, patents, and patent applications disclosed herein are hereby incorporated by reference with respect to the subject matter cited, if any, and may be incorporated in their entirety.

[0416] As used in the specification and claims, the terms "a," "an," and "the" should be understood to mean "at least one," unless clearly indicated to the contrary.

[0417] As used herein and in the claims, "and / or" should be understood to mean "either or both" of the elements connected thereby, i.e., in some cases the elements are present jointly and in other cases the elements are present separately. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements are so joined. Other elements may optionally be present other than those specifically identified by the "and / or" clause, whether or not those elements are related to the elements specifically identified. Thus, as a non-limiting example, a description of "A and / or B," when used with an open-ended term such as "comprising," can refer to, in some embodiments, only A (optionally including elements other than B); in other embodiments, only B (optionally including elements other than A); in yet other embodiments, both A and B (optionally including other elements), etc.

[0418] "Or" as used herein and in the claims should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., as the inclusion of at least one, but not only more than one, of a number or list of elements and, optionally, further unlisted items. Only terms clearly indicating the contrary, such as "one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of only one of the elements of a number or list. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of" when used in the claims shall have its ordinary meaning as used in the field of patent law.

[0419] The term "at least one," as used herein and in the claims, referring to one or more elements, is understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and does not exclude any combinations of elements in the list of elements. This definition allows for elements to be present, if desired, other than those specifically identified in the list of elements described with "at least one," whether or not related to the specifically identified elements. Thus, as non-limiting examples, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer, in certain embodiments, to at least one, optionally including more than one, A, and no B (and optionally including elements other than B); in other embodiments, to at least one, optionally including more than one, B, and no A (and optionally including elements other than A); in still other embodiments, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally other elements), etc.

[0420] It should also be understood that in any method that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions are described, unless contrary to purpose.

[0421] In the claims and the above specification, all transitional phrases such as "comprise," "include," "carry," "have," "contain," "involve," "hold," "consisting of," and the like, are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Examining Guidelines, Section 2111.03.

Claims

1. and a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 75% of the lipid-encapsulated perfluorocarbon bubbles are nanobubbles having a diameter ranging from about 100 nanometers to about 1 micron or less, wherein the lipid-encapsulated perfluorocarbon bubbles comprise DPPC and PEG-DPPE, and optionally the nanobubbles are at a density of about 7.0 x 10 per mL. ９ ~2.5 x 10 １０ Approximately 1 x 10 nanobubbles per mL １０ ~2.5 x 10 １０ The composition, wherein the nanobubbles are present in a concentration range of 10 nanobubbles.

2. 1. A method for forming lipid encapsulated perfluorocarbon bubbles, comprising: The method includes activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol in the presence of a perfluorocarbon gas at a sufficient agitation speed and time to produce a population of lipid-encapsulated perfluorocarbon bubbles, wherein the bubbles comprise nanobubbles having diameters ranging from about 100 nanometers to about 1 micron.

3. A population of lipid encapsulated perfluorocarbon bubbles formed by the method of claim 2.

4. 1. A method of treating a subject having a condition, comprising: administering to a subject having a condition an effective amount of a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 75% of the bubbles in the population are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron, the lipid-encapsulated perfluorocarbon bubbles comprising DPPC and PEG-DPPE; and applying ultrasonic energy to an area of ​​the subject suffering from the condition The method includes:

5. 1. A method of improving tissue perfusion in a subject comprising exposing the subject to therapeutic ultrasound, the improvement comprising: administering to the subject a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 75% of the bubbles in the population are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron, prior to and / or during exposure of the subject to therapeutic ultrasound. The method includes:

6. a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 15% of the microbubbles in the population have a diameter of about 2 microns to about 6 microns or less; and The lipid-encapsulated perfluorocarbon bubbles include DPPC and PEG-DPPE, and optionally contain about 0.24×10 microbubbles per mL having diameters of about 2 microns to about 6 microns or less. ９ ~2.44 x 10 ９ exists in a range of concentrations of bubbles, composition.

7. 1. A method for forming lipid encapsulated perfluorocarbon bubbles, comprising:

1. A method comprising activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol in the presence of a perfluorocarbon gas at a sufficient agitation speed and time to produce a population of lipid-encapsulated perfluorocarbon gas microbubbles, wherein at least 15% of the microbubbles have a diameter of about 2 microns to about 6 microns or less.

8. A population of lipid encapsulated perfluorocarbon bubbles formed by the method of claim 7.

9. 1. A method of treating a subject having a condition, comprising: administering to a subject having a condition an effective amount of a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 15% of the microbubbles in the population have a diameter of about 2 microns to about 6 microns or less, and the lipid-encapsulated perfluorocarbon bubbles comprise DPPC and PEG-DPPE; and applying ultrasonic energy, optionally at a frequency of about 1 to about 10 MHz, to the area of ​​the subject suffering from the condition; The method includes:

10. A method for improving therapeutic or diagnostic ultrasound, the improvement comprising: administering to the subject, prior to or during exposure to ultrasound, optionally at a frequency of about 1 to about 10 MHz, a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 15% of the microbubbles in the population have diameters in the range of about 2 microns to about 6 microns or less. The method includes:

11. a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 4% of the microbubbles in the population have a diameter between about 6 microns and about 10 microns; and The lipid-encapsulated perfluorocarbon bubbles include DPPC and PEG-DPPE, and the microbubbles have diameters ranging from about 6 microns to about 10 microns and are present at approximately 0.1×10 per mL. ８ ~0.32 x 10 ８ A range of such bubbles are present, composition.

12. 1. A method for forming lipid encapsulated perfluorocarbon bubbles, comprising:

1. A method comprising activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol in the presence of a perfluorocarbon gas at an agitation speed and for a time sufficient to produce a population of lipid-encapsulated perfluorocarbon gas microbubbles, wherein at least 4% of the microbubbles have a diameter between about 6 microns and about 10 microns.

13. A population of lipid encapsulated perfluorocarbon bubbles formed by the method of claim 12.

14. 1. A method of treating a subject having a condition, comprising: administering to a subject having a condition an effective amount of lipid-encapsulated perfluorocarbon bubbles, wherein at least 4% of the microbubbles in a population have a diameter of about 6 microns to about 10 microns, and the lipid-encapsulated perfluorocarbon bubbles comprise DPPC and PEG-DPPE; and applying ultrasonic energy at a frequency of about 1 to about 10 MHz to an area of ​​the subject suffering from the condition; The method includes:

15. A method for improving therapeutic or diagnostic ultrasound, the improvement comprising: administering to the subject a population of lipid-encapsulated perfluorocarbon bubbles, wherein at least 4% of the microbubbles in the population have a diameter in the range of about 6 microns to about 10 microns, prior to or during exposure to ultrasound at a frequency of about 1 to about 10 MHz; The method includes:

16. 1. A method for ultrasound imaging of a subject, comprising: Administering lipid-encapsulated perfluoropropane bubbles prepared by the method of any one of claims 6, 8, 11 or 13 or any one of claims 7 or 12 to a subject; applying ultrasonic energy at a frequency in the range of about 1 to about 10 MHz; and obtaining an ultrasound image of the target or control area. The method includes:

17. 1. A method for forming lipid-encapsulated perfluoropropane bubbles, comprising: A non-aqueous lipid formulation containing up to 5% (v / v) of water, DPPC, PEG-DPPE, glycerol, propylene glycol DPPA or DPPE is activated in the presence of gas at a shaking speed of about 6000 rpm for about 30 to about 90 seconds, optionally about 50 seconds, about 60 seconds, about 75 seconds or about 90 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres. The method includes:

18. 1. A method for forming lipid-encapsulated perfluoropropane bubbles, comprising: A non-aqueous lipid formulation containing up to 5% (v / v) of either water, DPPC, PEG-DPPE, glycerol, propylene glycol DPPA or DPPE is activated in the presence of gas at about 4950 rpm for about 125 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres. The method includes:

19. 1. A method for forming lipid-encapsulated perfluoropropane bubbles, comprising: An aqueous lipid formulation comprising DPPC and PEG-DPPE and glycerol and propylene glycol and either DPPA or DPPE is activated at a shaking speed of about 6000 rpm for about 5 to about 90 seconds, optionally for about 5 seconds, about 10 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 60 seconds, about 70 seconds, about 80 seconds or about 90 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres. The method includes:

20. 1. A method for forming lipid-encapsulated perfluoropropane bubbles, comprising: An aqueous lipid formulation containing DPPC and PEG-DPPE, glycerol and propylene glycol, and either DPPA or DPPE is activated at a shaking speed of about 4530 rpm for about 90 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres. The method includes:

21. 1. A method for forming lipid-encapsulated perfluoropropane bubbles, comprising: An aqueous lipid formulation containing DPPC and PEG-DPPE, glycerol and propylene glycol, and either DPPA or DPPE is activated at a shaking speed of about 4530 rpm for about 10 to about 20 seconds, thereby forming a population of lipid-encapsulated perfluoropropane gas microspheres. The method includes:

22. 1. A method for forming lipid-encapsulated perfluoropropane nanodroplets, comprising: The method includes condensing a population of lipid encapsulated perfluoropropane gas microspheres by applying sufficient pressure to cause the microbubbles to condense into nanodroplets, wherein the population of gas microspheres is condensed at room temperature.

23. 1. A method of diagnosing or treating a subject, comprising:

23. A method comprising administering to a subject a population of lipid-encapsulated nanodroplets and, optionally, exposing the subject to ultrasound energy at a target location, wherein the population of lipid-encapsulated nanodroplets is produced by the method of claim 22.

24. 1. A method of treating a subject having erectile dysfunction, comprising: administering to a subject in need of treatment an effective amount of lipid-encapsulated bubbles or nanodroplets; and Applying ultrasound to the targeted genital area The method includes:

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