Therapeutic methods of use of gas bubble compositions

Abstract

Provided herein are gas bubble populations optimized for certain imaging and therapeutic ultrasound uses, methods of preparing such populations, and methods of use thereof.

Description

[0001]THERAPEUTIC METHODS OF USE OF GAS BUBBLE COMPOSITIONS RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 520,085, filed August 16, 2023, entitled “THERAPEUTIC METHODS OF USE OF GAS BUBBLE COMPOSITIONS”, the entire contents of which are incorporated herein by reference. BACKGROUND Lipid-encapsulated gas bubbles can be formed by vigorous shaking of a container housing a lipid formulation such as a lipid suspension and a gas. The gas bubbles so formed have been used in imaging applications. SUMMARY This disclosure contemplates methods of use of optimized populations of lipid- encapsulated gas bubbles, including in combination with other therapeutic modalities, e.g., radiotherapy (i.e., radiation therapy). The gas bubbles may comprise nanobubbles or a mixture of gas nanobubbles and gas microbubbles. The disclosed methods involve the use of these gas bubble populations in the treatment of disease, e.g., solid tumors (or solid masses) such as vascularized solid tumors (or masses) including solid tumor cancer, as well as treatment of thrombus, embolism, etc., together with ultrasound energy, optionally as an adjunct to other treatment modalities such as but not limited to radiotherapy. Without being bound by any particular theory or mechanism of action, it is contemplated that these gas bubble populations are useful in targeting and penetrating the solid tumor microvasculature, or thrombus or embolism, and thus acting to augment the effects of conventional therapy such as radiotherapy in the treatment of solid tumors or thrombolytic agents in the treatment of thrombi or embolisms. In some embodiments, the disclosed methods use the gas bubble populations provided herein therapeutically with ultrasound and radiation therapy. In some related embodiments, the methods are used to treat a subject having a solid tumor that may be a carcinoma such as a hepatocellular carcinoma. 12700017.1 This disclosure contemplates that the gas bubbles absorb ultrasound energy and then expand and compress (e.g., in the case of stable cavitation) or rupture (e.g., in the case of inertial cavitation). The degree to which the gas bubbles expand and compress, or rupture, is influenced in part by the ultrasound (wave) frequency and peak pressure differences together with the gas bubble size (typically measured in terms of diameter) and composition. Ultrasound frequencies and pressures that induce resonance in the gas bubbles with destruction (in the case of inertial cavitation) or without destruction (in the case of stable cavitation) can impart disruptive energy into the local environment that in turn can trigger a variety of intended consequences including mechanical, biochemical or gene expression sequalae. Further, in the case of inertial cavitation, the gas bubbles may be used to deliver a therapeutic agent to a region of interest including a solid tumor such as a solid tumor cancer. One aspect of this disclosure provides a method of treating a subject having a solid tumor comprising administering an effective amount of a population of lipid-encapsulated perfluorocarbon gas bubbles to a subject having a solid tumor and applying an effective amount of ultrasound energy to the solid tumor (or region surrounding and including the solid tumor). In another aspect, this disclosure provides a method of using a composition comprising a population of lipid-encapsulated perfluorocarbon gas bubbles to treat a subject having a solid tumor and also receiving a conventional therapy such as radiation therapy. In another aspect, this disclosure provides an improvement to radiation therapy comprising administering, to a subject having a solid tumor such as a solid tumor cancer, a population of the foregoing lipid-encapsulated gas bubbles and exposing the subject to ultrasound energy that causes a subset of the gas bubbles to cavitate in vivo, stably and / or inertially. One aspect of this disclosure provides a method of treating a subject having a solid tumor, the method comprising: (a) administering an effective amount of a population of lipid-encapsulated perfluorocarbon gas bubbles to a subject having a solid tumor and receiving another therapy for the solid tumor such as radiation therapy; and (b) applying an effective amount of ultrasound energy to the solid tumor. Another aspect of this disclosure provides a method of enhancing radiotherapy treatment, the method comprising: 12700017.1 (a) administering an effective amount of a population of lipid-encapsulated perfluorocarbon gas bubbles to a subject having a solid tumor cancer; (b) applying an effective amount of ultrasound energy to the solid tumor cancer; and (c) applying radiation therapy to the subject. The solid tumor cancer is a carcinoma such as a hepatocellular carcinoma. Thus, in another aspect of this disclosure provides a method of treating a subject having hepatocellular carcinoma, the method comprising: (a) administering an effective amount of a population of lipid-encapsulated perfluorocarbon gas bubbles to a subject having hepatocellular carcinoma; (b) applying an effective amount of ultrasound energy to the hepatocellular carcinoma; and (c) applying radiation therapy to the subject. In still another aspect, this disclosure provides a method of using a composition comprising a population of lipid-encapsulated perfluorocarbon gas bubbles to treat a subject having a thrombus and / or an embolism, wherein the subject may also be receiving a conventional therapy such as a thrombolytic agent. The method may involve administering the thrombolytic agent before, during, or after administration of the gas bubble population and ultrasound. The thrombolytic agent may be administered at a regular interval (e.g., daily) and the gas bubble population / ultrasound therapy may be performed at a different interval (e.g., every 2 or every 3 days, or every week). The frequency of either treatment may be reduced with time or as the treatment shows efficacy. Thrombolytic (or anti-thrombotic) agents include but are not limited to anti-coagulant agents, fibrinolytic agents and inhibitors of platelet function. The anti-coagulant may be selected from the group consisting of glycosaminoglycans (e.g., heparins) and vitamin K antagonists. The fibrinolytic agent may be selected from the group consisting of plasminogen activators such as tissue plasminogen activator (tPA), streptokinase and urokinase, plasmin and plasminogen. The inhibitor of platelet function may be selected from the group consisting of aspirin, abciximab, clopidogrel and dipyridamole. Specific examples include tissue plasminogen activator (tPA or Alteplase), streptokinase, urokinase, tenecteplase, reteplase, recombinant alteplase, and the like. Depending upon the embodiment, the gas bubbles / ultrasound therapy of this disclosure may be administered before, simultaneously with 12700017.1 or following administration of the thrombolytic agent. Similar methods may be used to treat vascular disease or vascular complications and are contemplated by this disclosure. Other useful categories of such agents include but are not limited to anti-inflammatory agents, anti-platelet agents, lipid-reducing agents, direct thrombin inhibitors, glycoprotein IIb / IIIa receptor inhibitors, agents that binds to cellular adhesion molecules and inhibit the ability of white blood cells to attach to such molecules, calcium channel blockers, beta- adrenergic receptor blockers, cyclooxygenase-2 inhibitors, and angiotensin system inhibitors. The following embodiments apply equally to the foregoing aspects. In some embodiments, about 75% or more of the lipid-encapsulated perfluorocarbon gas bubbles in the population are nanobubbles having a diameter in the range of about 100 nanometers to less than or about 1 micron (or in the range of about 100 nanometers to about less than or about 500 nanometers, or in the range of about 500 nanometers to less than or about 1 micron). The lipid-encapsulated perfluorocarbon gas bubbles may comprise DPPC and PEG- DPPE. The lipid-encapsulated perfluorocarbon gas nanobubbles having a diameter in the range of about 100 nanometers to less than or about 1 micron (or in the range of about 100 nanometers to about less than or about 500 nanometers, or in the range of about 500 nanometers to less than or about 1 micron) may be present in a concentration range of about 7.0 x 109to about 2.5 x 1010nanobubbles per mL, or about 1 x 1010to about 2.5 x 1010nanobubbles per mL. In some embodiments, about 80% or more of the lipid-encapsulated perfluorocarbon gas bubbles are nanobubbles having a diameter in the range of about 100 nanometers to less than or about 1 micron (or in the range of about 100 nanometers to about less than or about 500 nanometers, or in the range of about 500 nanometers to less than or about 1 micron). In some embodiments, the lipid-encapsulated perfluorocarbon gas bubbles comprise (a) DPPA, DPPC and PEG-DPPE or (b) DPPE, DPPC and PEG-DPPE, each optionally in a mole % ratio of about 10 to about 82 to about 8 including 10:82:8. In some embodiments, PEG-DPPE is MPEG5000-DPPE. In some embodiments, the lipid-encapsulated perfluorocarbon gas bubbles are lipid-encapsulated perfluoropropane gas bubbles. In some embodiments, the population of lipid-encapsulated perfluorocarbon gas bubbles is present in a non-aqueous lipid solution comprising (a) DPPA, DPPC and PEG-DPPE or (b) DPPE, DPPC and PEG-DPPE, together with glycerol, propylene glycol, and less than or equal to 5% water (v / v). In some embodiments, the lipids are present in the non-aqueous lipid solution at a combined concentration of about 2.5 mg / ml or about 3.75 mg / ml. In some 12700017.1 embodiments of this and other aspects, the population of lipid-encapsulated perfluorocarbon gas bubbles are made through activation of a non-aqueous lipid solution comprising (a) DPPA, DPPC and PEG-DPPE or (b) DPPE, DPPC and PEG-DPPE, together with glycerol, propylene glycol, and less than or equal to 5% water (v / v). In some embodiments, the population of lipid-encapsulated perfluorocarbon gas bubbles is present in an aqueous lipid suspension comprising (a) DPPA, DPPC and PEG-DPPE or (b) DPPE, DPPC and PEG-DPPE, together with glycerol and propylene glycol, and greater than 5% water (v / v). In some embodiments, the aqueous lipid suspension has a combined lipid concentration of about 0.75 mg / ml. In some embodiments of this and other aspects, the population of lipid-encapsulated perfluorocarbon gas bubbles are made through activation of a aqueous lipid suspension comprising (a) DPPA, DPPC and PEG-DPPE or (b) DPPE, DPPC and PEG-DPPE, together with glycerol, propylene glycol, and greater than 5% water (v / v). In some embodiments, the population of lipid-encapsulated perfluorocarbon gas bubbles lacks DPPA. In some embodiments, the population of lipid-encapsulated perfluorocarbon gas bubbles is formed by activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol, in the presence of a perfluorocarbon gas, at a shaking rate and shaking time sufficient to yield a population of lipid-encapsulated perfluorocarbon gas bubbles wherein 75% or more of the gas bubbles are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron (or in the range of about 100 nanometers to about less than or about 500 nanometers, or in the range of about 500 nanometers to less than or about 1 micron). In some embodiments, the lipid formulation is a non-aqueous lipid solution comprising (a) DPPA, DPPC and PEG-DPPE or (b) DPPE, DPPC and PEG-DPPE, together with glycerol, propylene glycol, and less than or equal to 5% water (v / v). In some embodiments, such a lipid formulation is activated at a shaking rate of about 6000 rpm for a shaking time of about 50 seconds to about 90 seconds. In some embodiments, the lipid formulation is activated at a shaking rate of about 6000 rpm for a shaking time of about 50 seconds, about 60 seconds, about 75 seconds, or about 90 seconds. In some embodiments, the lipid formulation is activated at a shaking rate of about 6000 rpm for a shaking time of about 60 seconds. In some embodiments, the non-aqueous lipid formulation is present in a Wheaton 2 mL glass vial having an internal volume of about 3.8 ml, and comprising (a) about 0.35 ml of lipid formulation and about 3.45 ml of gas or (b) about 0.52 ml of lipid formulation and about 3.28 ml 12700017.1 of gas. In some embodiments, the lipid formulation is present in a Wheaton 2 mL glass vial having an internal volume of about 3.8 ml, and (a) about 9% of the internal volume is occupied by the lipid formulation prior to activation, or (b) about 14% of the internal volume is occupied by lipid formulation prior to activation. In some embodiments, the non-aqueous lipid formulation or non-aqueous solution has a combined lipid concentration of about 2 to about 5 mg / ml, or about 3 to about 4 mg / ml, or about 3.75 mg / ml. In some embodiments of this and other aspects, the lipids are present in a combined concentration of about 2 to about 3 mg / ml, or about 2.5 mg / ml. In some embodiments, the lipid formulation is an aqueous lipid suspension comprising (a) DPPA, DPPC and PEG-DPPE or (b) DPPE, DPPC and PEG-DPPE, together with glycerol, and propylene glycol. The aqueous lipid formulation may have a combined lipid concentration of about 0.75 mg / ml. In some embodiments of this and other aspects, the population of lipid-encapsulated perfluorocarbon gas bubbles is present in (or are made through activation of) an aqueous lipid suspension comprising glycerol and propylene glycol. In some embodiments, the aqueous lipid formulation or aqueous solution has a combined lipid concentration of about 0.5 to about 2 mg / ml, or about 0.5 to about 1.5 mg / ml, or about 0.5 to about 1 mg / ml, or about 0.75 mg / ml. In some embodiments, the aqueous lipid formulation is activated in the presence of gas at a shaking rate of about 4530 rpm for a shaking time of about 90 seconds, or at a shaking rate of about 6000 rpm for a shaking time of about 20 seconds to about 25 seconds. In some embodiments, the lipid-encapsulated perfluorocarbon gas bubbles experience stable cavitation in vivo after application of the ultrasound energy. In some embodiments, the lipid-encapsulated perfluorocarbon gas bubbles experience inertial cavitation in vivo after application of the ultrasound energy, optionally as detected and / or measured by emitted broadband radiofrequency. In some embodiments, the lipid-encapsulated perfluorocarbon gas bubbles are administered systemically. In some embodiments, the lipid-encapsulated perfluorocarbon gas bubbles are administered intravenously. In some embodiments, the lipid-encapsulated perfluorocarbon gas bubbles are administered locally. In some embodiments, the effective amount of ultrasound energy causes stable cavitation of a subset of the lipid-encapsulated perfluorocarbon gas bubbles in vivo. In some embodiments, the effective amount of ultrasound energy causes inertial cavitation of a subset of 12700017.1 the lipid-encapsulated perfluorocarbon gas bubbles in vivo. In some embodiments, the effective amount of ultrasound energy causes stable and inertial cavitation of a subset of the lipid- encapsulated perfluorocarbon gas bubbles in vivo. In some embodiments, the radiation therapy is applied before administration of the gas bubbles and / or after administration of the gas bubbles, optionally wherein the radiation therapy is applied locally to the solid tumor cancer. In some embodiments, the radiation therapy is applied before and / or after applying ultrasound energy to the subject, optionally wherein the radiation therapy is applied locally to the solid cancer. In some embodiments, the population of lipid-encapsulated perfluorocarbon gas bubbles is administered to the subject, then ultrasound energy is applied to the solid tumor, and then radiation therapy is applied to the solid tumor. These populations of lipid-encapsulated perfluorocarbon gas bubbles have a different size and concentration profile than those made and used previously for imaging (e.g., diagnostic) and / or therapeutic applications. In some embodiments of this and other aspects, the lipid-encapsulated perfluorocarbon gas bubbles are lipid-encapsulated perfluoropropane gas bubbles. In these and other aspects and embodiments, other perfluorocarbon gases may be used as the perfluorocarbon gas including but not limited to perfluoropentane, perfluoroisopentane, perfluorobutane, perfluoropentene, and dodecafluoropentane. It is to be understood that the various aspects and embodiments described herein in relation to perfluorocarbon gas are equally applicable to other gases such as but not limited to sulfur (sulpher) hexafluoride gas. The lipid-encapsulated perfluorocarbon gas bubbles may be formed by activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol, in the presence of a perfluorocarbon gas, at a shaking rate and shaking time sufficient to yield a population of lipid-encapsulated perfluorocarbon gas bubbles wherein 75% or more of the gas bubbles are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron. About 80% or more of the lipid-encapsulated perfluorocarbon gas bubbles may be nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron. The therapeutic methods may also include the step of forming the population of lipid- encapsulated perfluorocarbon gas bubbles prior to its use in vivo, as described herein. The therapeutic methods provided herein involve use of the improved gas bubble populations in particular formulations, optionally administered at particular rates and / or doses, 12700017.1 together with optimized ultrasound frequency, mechanical index (MI), pulse duration and / or number and / or timing of ultrasound applications. For example, the lipid-encapsulated gas bubbles are contemplated for use with ultrasound. The ultrasound energy applied may be defined using one or more parameters, including for example an ultrasound frequency in a range of about 0.25 to about 10 mHz, or about 0.5 to about 5 mHz, or about 1 to about 2 mHz, or about 1.5 mHz; a peak negative pressure (PNP) in the range of about 0.125 to about 1.5 MPa, or in the range of about 0.5 to about 1.2 MPa, or in the range of about 0.7 to about 1.0 MPa, or about 0.8 MPa, or about 1.0 MPa, or about 1.2 MPa; a pulse repetition in the range of about 1 to about 30 Hz, or in a range of about 5 to about 15 Hz, or about 5 Hz, or about 10 Hz, or about 20 Hz, or about 30 Hz; a pulse duration in the range of about 50 to about 500 ms, or in the range of about 100 to about 300 ms, or in the range of about 150 to about 250 ms, or about 150 ms, or about 200 ms, or about 220 ms; a mechanical index (MI) in the range of about 0.1 to about 1.2 MI, or in a range of about 0.5 to about 1.0 MI, or about 0.8 MI, or about 1.0 MI, or about 1.2 MI. These and other aspects and embodiments of this disclosure will be discussed in greater detail herein. DETAILED DESCRIPTION This disclosure provides methods of using gas bubble compositions for therapeutic purposes. Different therapeutic applications may require different optimized gas bubble compositions. Certain gas bubble compositions may be generated by particular manipulation of lipid formulations such as non-aqueous lipid solutions and aqueous lipid suspensions and / or particular activation parameters. These novel gas bubble compositions can be used therapeutically for example as an adjunct to another treatment modality such as but not limited to radiation therapy (e.g., in the treatment of a solid tumor cancer). It is contemplated that the gas bubbles provided herein may augment treatment of solid tumors including cancerous solid tumors (also referred to herein as solid tumor cancers), and in particular solid tumors having a tumor microenvironment that is often vascularized with microcapillaries. Such tumors may be cancerous or non-cancerous. Accordingly, some aspects herein provide a method of supplementing radiotherapy, the method comprising administering a population of lipid encapsulated gas bubbles to a subject 12700017.1 having a solid tumor such as a solid tumor cancer, wherein the subject is also receiving radiotherapy treatment. The methods provided herein contemplate the use of gas nanobubble populations to be used as an adjunct to another therapy such as radiation therapy. The gas nanobubble populations (together with ultrasound energy) can augment (or enhance or improve) the efficacy of the other therapy (e.g., radiation therapy). Such improvement may be manifested as a greater reduction in the solid tumor size (volume or diameter) compared to the reduction achieved with the conventional therapy alone. The reduction may be on the order of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% as compared to conventional therapy (e.g., radiation) alone. The solid tumor size may be measured using any standard imaging technique including but not limited to MRI and PET imaging. Similar impact is contemplated for benign solid tumors as well as for thrombi and embolism treatment. Typically, the impact of both the nanobubble / ultrasound therapy and the conventional (e.g., radiation) therapy is more than additive of the individual effects of these modalities. The conventional therapy contemplated by this disclosure is the typical standard of care and may be dictated by the type of solid tumor (or other condition) being treated and, in the context of a solid tumor whether such solid tumor is cancerous or non-cancerous. For example, the radiation therapy contemplated by this disclosure is the typical standard of care and may be dictated by the type of solid tumor cancer being treated. Radiation doses may be delivered once or multiple times including for example at regularly spaced intervals (e.g., daily, every other day, weekly, every 10 days, etc.). Conventional external beam radiation (EBRT) of liver may range from a maximum dose of about 1000 cGy to about 7500 cGy, in some instances. As another example, full liver treatment using EBRT may comprise 2100 cGy spread over multiple daily fractions (e.g., seven fractions), or 3000 cGy spread over twenty fractions. Partial liver treatment may comprise a dose of 5000 cGy to treat a third of the liver volume. Alternatively, partial liver treatment may comprise a dose of up to 7260 cGy for less than a third of the liver volume. Subjects having hepatocellular carcinoma may manifest a lower radiation tolerance and a dose of 2800 cGy using 200 cGy fractions may be warranted. Stereotactic body radiation (SBRT) may be used when higher and potentially ablative doses are prescribed, such as when the tumor is inoperable. For 12700017.1 example, doses in the range of (a) 3000-5400 cGy using a median fractional dose of 600 cGy or (b) 3750-4000 cGy at 800-1250 cGy per fraction may be used. EBRT doses for head and neck cancer may comprise daily fractions of 1.8 – 2.0 Gy, up to total doses of 66-70 Gy over 6-7 weeks. EBRT doses for breast cancer may comprise 40 Gy in 15 fractions or 42.5 Gy in 16 fractions. EBRT doses for high grade glioma may comprise daily fractions of 180 cGy to 200 cGy. Variations on these protocols are known in the art and embraced by this disclosure. The gas bubble population administration and ultrasound treatment may be performed before and / or during and / or after the conventional (e.g., radiation) therapy. In some instances, the conventional (e.g., radiation) therapy may be performed, for example on day zero, and the gas bubble / ultrasound therapy may be performed within hours and / or with days and / or within weeks of day zero. More specifically, the gas bubble / ultrasound therapy may be performed within 12 hours, or within 6 hours, or within 4 hours of the conventional (e.g., radiation) therapy, and / or it may be performed about 1 week and / or about 2 weeks after conventional (e.g., radiation) therapy. The conventional (e.g., radiation) therapy may be performed repeatedly and the gas bubble / ultrasound therapy may also be performed repeatedly. In still other instances, the gas bubble / ultrasound therapy may be administered on day zero and the conventional (e.g., radiation) therapy may be performed thereafter, either on day zero within hours (e.g., within 12 hours, within 6 hours, or within 4 hours) following the conventional (e.g., radiation) therapy, and / or within days or weeks following the conventional (e.g., radiation) therapy. Solid tumors, including solid tumor cancers This disclosure contemplates treating subjects having solid tumors whether such solid tumors are cancerous (and thus capable of metastasis) or non-cancerous. The solid tumors or either type may be vascularized. This disclosure also contemplates treating subjects having conditions characterized by abnormal (including excessive) vasculature. Certain aspects of this disclosure contemplate treating solid tumors or conditions characterized by abnormal (including excessive) vasculature using the gas bubbles and ultrasound combination described herein as the single therapeutic agent (i.e., not as an adjunct to a conventional therapy). Thus, in some instances, the subject has a solid tumor or a condition characterized by abnormal (including excessive) vasculature and is treated by administration of the gas bubble population (including systemic administration) and application of ultrasound energy (including localized application to the tumor and surrounding region). 12700017.1 In some instances, the methods provided herein allow for lower doses of conventional therapy to be used to treat the target condition, including lower radiation doses. In some instances, the methods provided herein are associated with fewer or less severe side effects than other treatment protocols. Solid tumor cancers may be grouped into three categories based on their tissue of origin: melanomas, which initiate in melanocytes (present in the skin, eyes, and mucous membranes); sarcomas, which initiate in the bones or connective tissue; and carcinomas, which initiate in the epithelium. Carcinomas are the most prevalent type of solid tumor cancer (or solid cancer, as the terms are referred to herein interchangeably) and account for the majority of cancer cases worldwide. In some embodiments, a subject has a carcinoma. Common types of carcinomas include adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, transitional cell carcinoma, renal cell carcinoma, ovarian carcinoma, endometrial carcinoma, thyroid carcinoma, pancreatic carcinoma, cervical carcinoma, esophageal carcinoma, lung carcinoma, prostate carcinoma, gastric carcinoma, head and neck squamous cell carcinoma, anal carcinoma, small intestine carcinoma, vulvar carcinoma, penile carcinoma, and hepatocellular carcinoma (HCC). In some embodiments, a subject has hepatocellular carcinoma (HCC). HCC is the most common type of primary liver cancer (that is, liver cancer not caused by metastases from other tissues) and is sometimes associated with underlying liver conditions, such as chronic viral hepatitis (e.g., hepatitis B or hepatitis C) or cirrhosis, such as that caused by heavy alcohol use or non-alcoholic fatty liver disease. Non-cancerous solid tumors (or solid masses) include fat tissue tumors such as lipomas, lipoblastomas, and hibernomas; fibrous tissue tumors such as fibromas, elastofibromas, superficial fibromatosis, desmoid-type fibromatosis, and deep benign fibrous histiocytomas; muscle tissue tumors such as leiomyomas and rhabdomyomas; blood and lymph vessel tumors such as hemangiomas, glomus tumors, and lymphangiomas; nerve tissue tumors such as neurofibromas, schwannomas, and granular cell tumors; and various other tumors such as myxomas and perivascular epithelioid cell tumors (PEComas). Conventional therapies to treat solid tumor cancers include approaches that target the tumor or tumor cells, such as surgery (to resect the tumor), chemotherapy, immunotherapy, cryotherapy, and radiation therapy. The chemotherapy may be an anti-angiogenic therapy. 12700017.1 Therapeutic Applications This disclosure contemplates use of gas bubble populations and, in some instances, nanodroplet populations, in therapeutic applications. These populations may be used in vivo in human or non-human subjects (e.g., companion animals such as dogs or cats, or agricultural animals such as a cattle or swine, or horses) or in vitro. The gas bubble populations and nanodroplet populations provided herein may be used for therapeutic purposes in subjects having a solid tumor, such as a solid tumor cancer, or having a condition characterized by abnormal (including excessive) vasculature, or having a thrombus or an embolism. In some aspects, the gas bubble populations or nanodroplet populations are used as single agent therapies. In other aspects, the gas bubble populations or nanodroplet populations are used in combination with another therapy. For example, in some aspects, the disclosure provides a method of supplementing radiotherapy, the method comprising administering a population of gas bubbles to a subject having a solid tumor cancer, wherein the subject is also receiving radiation treatment. In some aspects, the disclosure provides a method of supplementing a thrombolytic therapy, comprising administering a population of gas bubbles to a subject having a thrombus or an embolism, wherein the subject is also receiving a thrombolytic therapy. The gas bubbles are intended for use with ultrasound energy which causes the bubbles to cavitate (either stably or inertially). In the case of inertial cavitation, this process may be referred to as ultrasound-triggered nanobubble destruction (UTND). Ultrasound-triggered cavitation of the gas nanobubbles occurs when the gas nanobubbles are exposed to appropriate ultrasound frequencies, which cause them to oscillate between expansion and contraction before ultimately collapsing. In some embodiments, UTND is induced using a low- to medium- frequency ultrasound pulse (e.g., 500 kHz). In some embodiments, UTND is induced using a high-frequency ultrasound pulse (e.g., 15-20 mHz, including 18 mHz). Higher ultrasound frequences have shorter wavelengths and are absorbed more easily and can be especially useful in more superficial areas of the body, while lower ultrasound frequencies can penetrate deeper structures. Without wishing to be bound by any particular theory, cavitation of the gas bubbles may act on a solid tumor (or vasculature or thrombus or embolism) by exerting mechanical stress on the endothelium as the bubbles expand, contract, and collapse, thus compromising endothelial integrity. Cavitation may also upregulate ceramide, a mediator of radiation-induced endothelial 12700017.1 cell death. In some embodiments, the combination therapy methods involve administering the gas bubbles to the solid tumor, isonifying the tumor in parallel or shortly after bubble administration, and subsequently subjecting the solid tumor to radiation. Similar methods are contemplated that use nanodroplets in place of the gas bubbles. Still other methods are contemplated that use the gas bubbles or nanodroplets to treat conditions characterized by abnormal (including excessive) vasculature, or a thrombus or an embolism, together with ultrasound, as a single agent therapy or in a combination therapy. The anti-tumor efficacy of ultrasound-triggered nanobubble destruction (UTND) can be assessed by a variety of metrics, including cell composition of the tumor microenvironment (TME) (e.g., immune cell infiltration, incidence of tumor and / or endothelial cell death) and structural and / or biochemical properties of the tumor(s), such as vascularity, including microvessels density, oxygenation, and ceramide production. Tumor vascularity can be assessed using ultrasound imaging (e.g., Power Doppler) or by detecting CD31 (platelet endothelial cell adhesion molecule, PECAM-1) expression on endothelial cells (e.g., using immunohistochemistry or fluorescent activated cell sorting). CD31 is a transmembrane glycoprotein expressed on vascular endothelial cells and is therefore a marker to assess tumor vascularity, and in particular microvessel density. A decrease in CD31 labeling (and thus CD31 expression or presence) indicates a reduction in the microvessels and other vasculature of the TME including the tumor. Without being bound by any particular mechanism or theory, it is contemplated that cavitation of nanobubbles in the TME can cause damage to endothelial cells and also result in increased ceramide production by tumor cells. These changes, in turn, can sensitize endothelial cells to radiotherapy and can facilitate the killing of endothelial cells in the tumor vasculature following radiation treatment. Thus, decreased CD31 expression in the TME can be indicative of enhanced radiosensitivity of endothelial cells following UTND. CD31 expression can be assessed in situ via, for example, immunohistochemical labeling of a tumor sample, such as a tumor biopsy, and imaging (e.g., identifying the percentage of CD31+ cells for a given area of the tumor). Tumor samples can also be dissociated and CD31 expression can be analyzed via flow cytometry. Again, without being bound by any particular theory or mechanism, it is contemplated that destruction of the tumor vasculature may decrease tumor oxygenation, creating a hypoxic environment for tumor cells and potentiating their death. A decrease in tumor oxygenation can therefore further indicate endothelial cell death and a potentially enhanced sensitivity of 12700017.1 endothelial cells to radiotherapy. Tumor oxygenation can be measured using photoacoustic imaging, blood oxygen level dependent (BOLD) magnetic resonance imaging (MRI), positron emission topography (PET), electron paramagnetic resonance spectroscopy (EPR), as well as other suitable methods. The TME can be further analyzed by labeling tumor samples with antibodies or other affinity compounds that bind specific proteins or cellular components. In some embodiments, immune cell infiltration into the TME is assessed using the pan-immune marker CD45 to label immune cells in tumor samples. In some embodiments, immune cells in the TME are further characterized using other markers to identify specific immune cell populations, such as CD3 for T cells, CD20 for B cells, CD56 for NK cells, CX3CR1 for mononuclear phagocytes, etc. Cells in the TME may also be analyzed for incidence of cell death via expression of caspase-3, an effector protein of apoptosis. Increases in caspase-3+ tumor cells and / or caspase-3+ endothelial cells (e.g., CD31+ cells) indicate a positive response to the anti-tumor therapies described herein. Tumor samples can be fixed and labeled to identify cells in situ or they can be dissociated and analyzed via flow cytometry, Western blot, qPCR, and / or other techniques. Ceramide levels in the TME can also indicate a positive response to anti-tumor therapy, as cavitation of nanobubbles can upregulate ceramide production by tumor cells. Ceramides are a family of lipids that are produced endogenously in the epidermis but can also be produced by tumor cells upon UTND. Ceramides are a mediator of radiation-induced endothelial cell death, and thus increased ceramide production by tumor cells may further promote the destruction of microvessels that vascularize the TME. In some embodiments, lipid formulations such as the aqueous and non-aqueous formulations provided herein, may be activated to form a sufficient number, sufficient proportion and / or sufficient diameter of nanobubbles, which in turn are optionally diluted into a larger volume, and administered in one or more bolus injections or by a continuous infusion. Administration is typically intravenous injection. Alternatively, the activated formulation may be subjected to pressure to form nanodroplets, as described herein, and the nanodroplets may be administered, optionally in one or more bolus injections or by a continuous infusion. Therapeutic ultrasound is then performed concurrently with administration or shortly thereafter. The ultrasound can be directed to the target region of the body (i.e., the region intended for treatment) including but not limited to the kidney, heart, blood vessels, cardio vasculature, liver, and the head. 12700017.1 Subjects of the disclosure include but are not limited to humans and animals. Humans are preferred in some instances. Animals include companion animals such as dogs and cats, and agricultural or prize animals such as but not limited to bulls and horses. The gas bubble populations and nanodroplet populations are administered in effective amounts. An effective amount is that amount that facilitates or brings about the intended in vivo response and / or application, whether when used as a single agent or when used in combination with another agent. The therapeutic methods provided herein are intended to treat, partially or completely, a condition. In some embodiments, the effective amount is one that causes a reduction in the size of the target solid tumor, or that causes an increased reduction when used in combination with another therapy compared with the reduction achieved using the other therapy alone. In the context of thrombi or embolisms, the effective amount may be the amount that is required to reduce the thrombus or embolism size or to ablate the thrombus or embolism, or to reduce the number and / or size of thrombi or embolisms in a subject. Ultrasound has previously been used or contemplated for use in therapeutic applications. Combining ultrasound with gas bubble populations or nanodroplets, as provided herein, may provide additional benefit, potentially with less adverse effects of the ultrasound. When used alone, ultrasound is hypothesized to have a number of different mechanisms of action, including for example heating of target tissues, mechanical disruption, and the like. The heating of tissues, in some embodiments, may be an undesirable effect. The methods of this disclosure may harness the therapeutic benefit of ultrasound, potentially using lower doses of ultrasound energy than have been previously used. Gas-filled nanobubbles or nanodroplets used together with ultrasound may also have a number of mechanisms of action. One particular mechanism of action is cavitation. Cavitation occurs when administered nanobubbles or nanodroplets absorb the ultrasound energy and in turn undergo a number of mechanical changes. In some instances, the energy absorbed by the nanobubbles or nanodroplets is released from the nanobubbles or nanodroplets, and then absorbed by the surrounding tissue. In the case of stable cavitation, the nanobubbles such as microspheres or the nanodroplets may undergo one or more cycles of swelling and contracting, without ultimate rupturing. In the case of inertial cavitation, the nanobubbles such as microspheres or the nanodroplets rupture. Whether stable or inertial cavitation occurs depends on a number of factors including the energy level of the ultrasound, the location of intended use, and the nature or composition of the nanobubbles or nanodroplets. 12700017.1 For some therapeutic applications, stable cavitation is sufficient to effect the desired clinical benefit. For other therapeutic applications, inertial cavitation is required to effect the desired clinical benefit. As an example, if the nanobubbles or nanodroplets are used as delivery vehicles, then for maximum effect they should rupture in order to release their cargo in vivo. The ultrasound energy may be applied externally or internally. Externally applied ultrasound is preferable in most situations because it is less (if at all) invasive. Internally applied ultrasound may be used in some instances where the target tissue is a deep tissue and / or externally applied ultrasound might cause adverse effects in intervening tissue. Ultrasound may be applied externally by placing an ultrasound transducer in close proximity to an outer surface of a subject, such as the skin or an outwardly facing cavity such as the oral cavity, etc. Ultrasound may be applied internally by introducing an ultrasound transducer into the body of a subject, typically on a probe. An example may be a cardiovascular probe capable of traveling through the circulatory system. Conditions that could benefit from the combined use of gas bubbles such as nanobubbles or nanodroplets and ultrasound energy are described herein. Certain ultrasound parameters are provided herein. In some embodiments, other ultrasound frequencies may be used including for example in the range of about 1-6 MHz for deeper tissues such as the kidney, heart, and liver and 7-10 MHz for superficial tissues such as the carotid artery and breast. Sonoporation refers to the use of ultrasound to increase permeability of cell membranes, vasculature, and the blood-brain-barrier (BBB), typically in a reversible manner. By co- administering gas bubbles such as nanobubbles or nanodroplets with a therapeutic agent, or administration of gas bubbles such as nanobubbles or nanodroplets conjugated to a therapeutic agent and applying ultrasound, it is possible to increase the permeability of a membrane, barrier, or vasculature in order to deliver the therapeutic agent to specific regions of the body. Conditions that would benefit from this combined approach include cardiovascular disease, thrombus or embolisms, and solid tumors such as carcinomas and others listed above. In some instances, nanobubbles and nanodroplets have the added advantage of being capable of passively and preferentially extravasating into tissues such as solid tumors from the circulation. Additionally, tumors typically have leaky vasculatures. Gas bubbles such as nanobubbles and nanodroplets may be used at off-resonant frequencies such as 1-10 MHz. Sonoporation can also be used to sensitize hypoxic tumors to radiation therapy, as described in more detail above. 12700017.1 Still other applications include delivery of oxygen to tumors in order to render such tumors sensitive to radiation treatment. 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), an approved ultrasound thrombolytic device. Nanobubble Populations The gas bubble populations provided herein are lipid-encapsulated gas bubbles. The nanobubble provided herein are lipid-encapsulated gas nanobubbles. The gas may be a perfluorocarbon gas such as perfluoropropane gas. The lipids may comprise DPPC and DPPE, and the DPPE may be used in pegylated. In some embodiments, the lipids comprise DPPC, DPPE and PEG-DPPE such as PEG5000-DPPE. In some embodiments, the lipids comprise DPPA, DPPC and PEG-DPPE such as PEG5000-DPPE. The nanobubble populations provided herein are defined by their size and concentration profiles, their acoustic attenuation properties, and / or their preparatory methods. Their utility in therapeutic ultrasound applications, e.g., the augmentation of radiotherapy for solid tumor treatment, including solid tumor cancer treatment, depend in part on their size and / or acoustic attenuation profiles. As used herein, the size profile of a nanobubble population means the proportion of bubbles in the population that have a particular size. The size profile may include the proportions of bubbles of different sizes, in some instances. The following describes certain nanobubble populations that are defined by their size profile. The gas bubble populations to be used in the methods of this disclosure may be exclusively or predominantly nanobubbles. Such nanobubbles may range in size (i.e., diameter) from about 100 nanometers to about 1 micron, or from about 100 nanometers to about 900 nanometers, or from about 100 nanometers to about 800 nanometers, or from about 100 nanometers to about 700 nanometers, or from about 100 nanometers to about 600 nanometers, or from about 100 nanometers to about 500 nanometers, or from about 100 nanometers to about 400 nanometers, or from about 100 nanometers to about 300 nanometers, or from about 100 nanometers to about 200 nanometers, or from about 200 nanometers to about 1 micron, or from about 300 nanometers to about 1 micron, or from about 400 nanometers to about 1 micron, or from about 500 nanometers to about 1 micron, or from about 600 nanometers to about 1 micron, 12700017.1 or from about 700 nanometers to about 1 micron, or from about 800 nanometers to about 1 micron, or from about 900 nanometers to about 1 micron. The gas bubble populations to be used in the methods of this disclosure may comprise a mixture of nanobubbles and microbubbles. The mixture may be defined by the proportion of nanobubbles (e.g., those bubbles having a diameter in the range of about 100 nanometers to about 1 micron) out of the total number of gas bubbles (e.g., those bubbles having a diameter in the range of about 100 nanometers to about 6 microns). Using the foregoing definition, the proportion of nanobubbles (e.g., having a diameter in the range of about 100 nanometers to about 1 micron) out of the total number of gas bubbles (e.g., having a diameter in the range of about 100 nanometers to about 6 microns) in the population may be about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more, with the remainder of the gas bubbles being in the range of greater than 1 micron to about 6 microns. One aspect of this disclosure provides a population of lipid- encapsulated gas bubbles, wherein about 75% or more of the lipid-encapsulated gas bubbles are nanobubbles having a diameter in the range of about 100 nanometers to less than or about 1 micron (i.e., a diameter of about 100 nanometers to less than or about 1 micron, as intended herein). In some instances, the diameter is in the range of about 200 nanometers to less than or about 1 micron, or about 300 nanometers to less than or about 1 micron, or about 500 nanometers to less than or about 1 micron. In related instances, the upper end 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 ends is contemplated, including but not limited to about 200 nanometers to about 800 nanometers or about 500 nanometers to less than 1 micron. Thus, as an example, 75% or more of the lipid-encapsulated gas bubbles may be nanobubbles having a diameter in the range of about 100 nanometers to less than or about 1 micron, or in the range of about 200 nanometers to about 800 nanometers. All other lower and upper ends combinations are contemplated by this disclosure. Such a population may be referred to as being enriched for lipid-encapsulated gas bubbles having diameters in any of the afore-mentioned ranges. Thus, a population of lipid- encapsulated gas bubbles, wherein about 75% or more of the lipid-encapsulated gas bubbles have a diameter in the range of about 100 nanometers to less than or about 1 micron, is considered enriched for lipid-encapsulated gas bubbles having a diameter in the range of about 12700017.1 100 nanometers to less than or about 1 micron. About 75%, about 80%, about 85%, about 90%, about 95% or more of the lipid-encapsulated gas bubbles in these enriched populations may have a diameter in the range of about 100 nanometers to less than or about 1 micron, or in any other of the specified ranges including for example a range of about 200 nanometers to less than or about 1 micron, or a range of about 300 nanometers to less than or about 1 micron, or a range of about 400 nanometers to less than or about 1 micron, or a range of about 500 nanometers to less than or about 1 micron, including ranges having the afore-mentioned lower ends and upper ends 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. As discussed herein, nanobubble-enriched populations (relative to prior art populations) can be used in new therapeutic applications or can improve the therapeutic efficacy of existing therapeutic applications. Lipid-encapsulated gas bubble populations that contain both microbubbles and nanobubbles may be beneficial for the treatment of heterogenous tumor populations with a wide range of capillary diameters. Lipid Formulations: Aqueous and Non-Aqueous The lipid formulations used to form the gas bubbles provided by the present disclosure may be non-aqueous lipid formulations, defined as having less than or equal to 5% water (w / w). These non-aqueous lipid formulations may sometimes be referred to herein as lipid solutions. Alternatively, the lipid formulations may be aqueous lipid formulations, defined as having greater than 5% water (w / w). These aqueous lipid formulations may sometimes be referred to herein as lipid suspensions. Both types of lipid formulations may be activated as described herein. The nanobubble enriched populations may be formed using aqueous lipid formulations or non-aqueous lipid formulations, together with a gas. Suitable lipid formulations include one, two, three or more lipids. Suitable lipids are provided below. One of more such lipids may be conjugated to polyethylene glycol (PEG). One example of an aqueous lipid formulation comprises lipids DPPA, DPPC and MPEG5000- DPPE in a 10:82:8 mole % ratio, and a combined lipid concentration of about 0.75 mg / ml, as well as propylene glycol and glycerol in an 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 yields about 1010nanobubbles and about 5x10912700017.1 microbubbles. About 66% of the bubbles produced following activation of this aqueous lipid formulation are nanobubbles. About 4% of the microbubbles formed have a diameter in the range of about 2 to about 6 microns. Other aqueous lipid formulations are similar to the afore-mentioned formulation but differ in their lipid profiles. For example, in some aqueous lipid formulations, DPPA, DPPC and DPPE may be used in molar percentages of about 77 to about 90 mole % DPPC, about 5 to about 15 mole % DPPA, and about 5 to about 15 mole % DPPE, including DPPE-MPEG5000 such as MPEG5000-DPPE. Similar ratios may be used in non-aqueous lipid formulations as well. Another example of an aqueous lipid formulation comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE), and dipalmitoylphosphatidylethanolamine (DPPE) and specifically lacks DPPA. The PE may be PE-PEG and / or the DPPE may be DPPE-PEG. For example, the lipid formulation may comprise lipids DPPC, DPPE and DPPE-PEG. These may be present in a mole percent ratio of 82:10:8 (DPPC:DPPE:DPPE-PEG). These lipids may be used in other molar percentages including about 77 to about 90 mole % DPPC, about 5 to about 15 mole % DPPE, and about 5 to about 15 mole % DPPE-PEG. The combined lipid concentration may range from about 0.75 mg / ml to about 1.5 mg / ml. These lipids may be suspended in propylene glycol, glycerol and saline, or any combination thereof. These formulations may be activated in the presence of a perfluorocarbon gas such as perflutren, or any other suitable gas. One example of a non-aqueous lipid formulation comprises DPPA, DPPC and MPEG5000-DPPE in a 10:82:8 mole % ratio, and a combined lipid concentration of about 3.75 mg / ml, in propylene glycol and glycerol and less than or equal to 5% water (w / w). Activation of this non-aqueous lipid formulation in the presence of perfluoropropane gas at 4950 rpm for 45 seconds yields about 3x109nanobubbles and about 2.5-3x109microbubbles. About 50% of the bubbles produced following activation of this lipid formulation are nanobubbles. About 12% of the microbubbles formed have a diameter in the range of about 2 to less than or about 6 microns, and about 1.2% of the microbubbles formed have a diameter in the range of about 6 to about 10 microns. Other non-aqueous lipid formulations are described in published PCT Application PCT / US2015 / 067615, published as WO2016 / 109400, the entire contents of which are incorporated by reference herein. 12700017.1 Another example of a non-aqueous lipid formulation comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE), and dipalmitoylphosphatidylethanolamine (DPPE), and optionally specifically lacks DPPA. The PE may be PE-PEG and / or the DPPE may be DPPE- PEG. For example, the lipid formulation may comprise lipids DPPC, DPPE and DPPE-PEG. These may be present in a mole percent ratio of 82:10:8 (DPPC:DPPE:DPPE-PEG). These lipids may be used in other molar percentages including about 77 to about 90 mole % DPPC, about 5 to about 15 mole % DPPE, and about 5 to about 15 mole % DPPE-PEG. The combined lipid concentration may range from about 0.75 mg / ml to about 1.5 mg / ml. These lipids may be suspended in propylene glycol, glycerol, or propylene glycol and glycerol. These formulations may be activated in the presence of a perfluorocarbon gas such as perflutren, or any other suitable gas. The non-aqueous lipid formulations may comprise lipids in propylene glycol, or glycerol, or propylene glycol and glycerol, provided the water content is less than or about 5% by weight (i.e., weight of water to the weight of the combination of lipids and propylene glycol and / or glycerol). In some instances, the non-aqueous lipid formulation comprises 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 instances, the non-aqueous lipid formulation comprises less than 1% water (w / w). The water content may be measured at the end of manufacture (and prior to long term storage) or it may be measured after storage, including long term storage, and just before use. The lipid formulations also may be salt-free (i.e., they do not contain any salts other than lipid counter-ions). More specifically, and as an example, lipids such as DPPA and DPPE are typically provided as sodium salts. As used herein, a salt-free lipid formulation may comprise such counter-ions (e.g., sodium if DPPA and / or DPPE are used) but they do not contain other ions. In some instances, the non-aqueous lipid formulation is free of sodium chloride or chloride. The lipid formulation may comprise a buffer. The buffer may be an acetate buffer, a benzoate buffer, a salicylate buffer, a diethanolamine buffer, a triethanolamine buffer, a borate buffer, a carbonate buffer, a glutamate buffer, a succinate buffer, a malate buffer, a tartrate buffer, a glutarate buffer, an aconite buffer, a citrate buffer, a lactate buffer, a glycerate buffer, a gluconate buffer, or a tris buffer, although it is not so limited. Non-phosphate buffers may be preferred in some instances due to their dissolution profiles in the non-aqueous solvents such as 12700017.1 propylene glycol and / or glycerol. In some instances, a phosphate buffer may be used (e.g., following or concurrent with addition of saline or other aqueous diluent). In some 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. In some instances, the lipid concentration in the lipid formulations 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 7.5 mg lipid per mL, including 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 instances, the lipid concentration is about 2 mg to about 4 mg per mL, or about 2.5 mg to about 3.75 mg per mL, or about 3.75 mg to about 7.5 mg of total lipid per mL. As an example, the lipid concentration in non-aqueous lipid formulations may range from about 0.1 mg to about 10 mg lipid per mL of propylene glycol and / or glycerol (combined), including about 1 mg to about 5 mg lipid per mL of propylene glycol and / or glycerol (combined), or about 2 mg to about 4 mg lipid per mL of propylene glycol and / or glycerol (combined), or about 2.5 mg to about 3.75 mg lipid per mL of propylene glycol and / or glycerol (combined). As another example, the lipid concentration in aqueous lipid formulations may 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. Prior to administration, the gas bubbles may be diluted with an aqueous diluent, and such aqueous diluent may comprise salts such as but not limited to sodium chloride, and thus may be regarded as saline or a saline solution. The aqueous diluent may comprise a buffer such as a phosphate buffer, and thus may be regarded as a buffered aqueous diluent. The aqueous diluent may be a buffered saline solution. The non-aqueous lipid formulation may comprise a buffer such as a non-phosphate buffer, examples of which are provided herein. The lipid formulation and the aqueous diluent may both comprise a buffer. In typical embodiments, either the lipid formulation or the aqueous diluent comprises a buffer, but not both. The buffer concentration will vary depending on the 12700017.1 type of buffer used, as will be understood and within the skill of the ordinary artisan to determine. The buffer concentration in the lipid formulation may range from about 1 mM to about 100 mM. In some instances, the buffer concentration may be about 1 mM to about 50 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, including about 5 mM. In some embodiments, the aqueous diluent comprises glycerol, a buffer such as phosphate buffer, salt(s) and water. Such an aqueous diluent may be used with a non-aqueous lipid formulation that lacks glycerol. In some embodiments, the lipid formulation further comprises saline (salt(s) and water combined) and glycerol in a weight ratio of 8:1. In some embodiments, the aqueous diluent comprises propylene glycol, a buffer such as phosphate buffer, salt(s) and water. Such an aqueous diluent may be used with a non-aqueous lipid formulation that lacks propylene glycol. In some embodiments, the aqueous diluent comprises a buffer such as phosphate buffer, salt(s) and water. Such an aqueous diluent may be used with a non-aqueous lipid formulation that comprises both propylene glycol and glycerol. The gas bubble population to be administered, typically intravenously, to a subject including a human subject may have a pH in a range of 4-8 or in a range of 4.5-7.5. In some instances, the pH may be in a range of about 6 to about 7.5, or in a range of 6.2 to about 6.8. In still other instances, the pH may be about 6.5 (e.g., 6.5 + / - 0.5 or + / -0.3). In some instances, the pH may be in a range of 5 to 6.5 or in a range of 5.2 to 6.3 or in a range of 5.5 to 6.1 or in a range of 5.6 to 6 or in a range of 5.65 to 5.95. In still another instance, the pH may be in a range of about 5.7 to about 5.9 (e.g., + / - 0.1 or + / - 0.2 or + / - 0.3 either or both ends of the range). In another instance, the pH may be about 5.8 (e.g., 5.8 + / - 0.15 or 5.8 + / - 0.1). Methods of Forming Nanobubble Enriched Populations (Activation) The lipid-encapsulated gas nanobubbles of this disclosure are prepared by activating a lipid formulation such as a lipid solution or a lipid suspension. Activation is a process whereby the lipid formulation, housed 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 nanobubbles. In some embodiments, this disclosure provides methods for forming suitable nanobubble populations by uniquely activating lipid formulations at sufficient rates and for sufficient times. It has been found that the optimized nanobubble populations provided herein are obtained from 12700017.1 lipid formulations using particular activation parameters. Some of these specific activation parameters are markedly different from the activation parameters of existing lipid formulations used to form microbubbles for diagnostic ultrasound applications. As one example, it has been found that non-aqueous lipid formulations provided herein must be activated at higher speeds (or rates) for longer time periods time to generate certain enriched nanobubble populations of this disclosure. For example, certain of these non-aqueous lipid formulations generate microbubbles for diagnostic ultrasound applications when activated at 4950 rpm for 45 seconds. However, in order to generate certain of the nanobubble populations provided herein, these formulations, examples of which are provided in the Examples, must be activated at significantly higher rates, including at about 6000 rpm, for longer times, ranging from about 50 to about 90 seconds, including about 60 seconds, or about 75 to about 90 seconds. Certain enriched nanobubble populations therefore may be formed by activating non- aqueous formulations, such as described in the Examples, for a shaking time of about 50 second to about 90 seconds at a shaking rate of 6000 rpm + / - 10%, or + / - 5%, or + / - 1%, and / or at a shaking rate 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 some instances, the shaking rate is 6000 rpm. Similarly, certain enriched nanobubble populations may be formed by activating non- aqueous formulations at any of the foregoing shaking rates for a shaking time in the range of 40 to 100 seconds, or 45 to 95 seconds, or 46 to 94 seconds, or 47 to 93 seconds, or 48 to 92 seconds, or 49 to 91 seconds, or 40 to 100 seconds, including without limitation 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, and 100 seconds. In other embodiments, the shaking time may 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, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 seconds. Any range defined by lower and upper boundaries selected from these times is contemplated by this disclosure. Moreover, it was surprisingly found that when these non-aqueous lipid formulations, such as those described in the Examples, occupied about 9% of the internal volume of the container with gas occupying about 91% of the internal volume of the container (as is described in the Examples and denoted as DEFINITY RT 1.5X or DEFINITY RT 1X + 0.5X) then the optimal shaking speed was about 6000 rpm and optimal shaking time was about 60 seconds. 12700017.1 The shaking speed (or rate, as the terms are used interchangeably) could range from 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 instances, the shaking rate is 6000 rpm. The shaking time could range from 55 to 65 seconds, 56 to 64 seconds, 57 to 63 seconds, 58 to 62 seconds, 59 to 61 seconds, or it could be 60 seconds. The compositions of DEFINITY and DEFINITY RT are provided in the Examples. A shaking rate of about 6000 rpm for a shaking time of about 90 seconds applied to another non-aqueous lipid formulation, such as that described in the Examples, caused enrichment of the nanobubble population. The shaking rate could range from 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 instances, the shaking rate is 6000 rpm or 6200 rpm or 6600 rpm. The shaking time could range from 85 to 95 seconds, 86 to 94 seconds, 87 to 93 seconds, 88 to 92 seconds, 89 to 91 seconds, or it could be 90 seconds. As another example, it has been found that aqueous lipid formulations provided herein, such as those described in the Examples, also must be activated for longer periods of time or at higher speeds (or rates) to generate certain enriched nanobubble populations of this disclosure. In order to generate the gas bubble populations provided herein, these aqueous formulations must be activated at significantly higher rates, including at about 6000 rpm, for shorter times, ranging from about 20 to about 30 seconds. Activation of these lipid formulations at about 6000 rpm for about 20 to 25 seconds provides nanobubble-enriched populations. The shaking rate could range from 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 instances, the shaking rate is 6000 rpm or 6200 rpm or 6600 rpm. The shaking time could range from 15 to 35 seconds, 16 to 34 seconds, 17 to 33 seconds, 18 to 32 seconds, 19 to 31 seconds, or it could be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 seconds. Alternatively, these same aqueous lipid formulations (described in the Examples), can be activated for significantly longer times using the lower rates (e.g., about 4530 rpm for about 90 seconds) in order to generate nanobubble-enriched populations. The shaking rate could range 12700017.1 from 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 could range from 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 it could be 90 seconds. Some of the preparatory methods provided herein involve the use of a 2 mL vial as a container housing the lipid formulation and the gas. In some instances, the vial contains about 1.76 ml of an aqueous lipid formulation and about 2.04 ml of gas. Accordingly, the lipid formulation occupies about 46% of the total internal volume (i.e., 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., volume of gas as a percentage of the total internal volume). Some of the preparatory methods provided herein involve the use of a 2 mL vial as a container housing a smaller volume of a non-aqueous lipid formulation as well as the gas. For example, the vial having a total internal volume of about 3.8 ml may contain about 0.35 ml of non-aqueous lipid formulation and about 3.45 ml of gas. Accordingly, the lipid formulation occupies about 9% of the total internal volume and the gas occupies about 91% of the total internal volume. Another example involves a vial having a total internal volume of about 3.8 ml that contains about 0.52 ml of non-aqueous lipid formulation and about 3.28 ml of gas. Accordingly, the lipid formulation occupies about 14% of the total internal volume and the gas occupies about 86% of the total internal volume. Examples of these latter formulations are described in the Examples and denoted as DEFINITY-RT 1.5X and DEFINITY-RT 1X+0.5X. Accordingly, depending on the enrichment desired, and in keeping with the teachings provided herein including the Examples, the lipid formulations provided herein may be activated in a number of ways and using a number of shaking rate / speed and shaking time combinations. Aqueous lipid formulations, such as but not limited to the aqueous lipid formulation described in the Examples, may be activated at about 4530 rpm for about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 75, about 80, or about 90 seconds. Aqueous lipid formulations alternatively may be activated at about 6000 rpm for about 5- 30 seconds including without limitation about 5, about 10, about 15, about 20, about 25, or about 30 seconds. 12700017.1 Non-aqueous lipid formulations, such as but not limited to the non-aqueous lipid formulation described in the Examples, may be activated at about 4950 rpm for about 10 to about 90 seconds including without limitation about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 75, about 80, about 90 seconds, or about 120 seconds. Non-aqueous lipid formulations, such as but not limited to the non-aqueous lipid formulation described in the Examples, may be activated at about 6000 rpm for about 10 to about 90 seconds including without limitation about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 75, about 80, or about 90 seconds. Non-aqueous lipid formulations, such as but not limited to the non-aqueous lipid formulation described in the Examples and provided at a 1.5 x fill volume (relative to the fill volume recited in the Examples) 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 instances, it is activated at about 6000 rpm for about 60 seconds. Non-aqueous lipid formulations, such as but not limited to the non-aqueous lipid formulation described in the Examples and provided at a 1 x fill volume plus a 0.5 x fill volume of lipid-less matrix (relative to the fill volume recited in the Examples) 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 instances, it is activated at about 6000 rpm for about 60 seconds. Other embodiments of this disclosure involve the formation of nanodroplets, optionally starting from a population of gas nanobubbles and microbubbles. Nanodroplets, as used herein, are nanometer-sized (less than 500 nm, typically) droplets having a center occupied by gas in its liquid form and an outer lipid shell. One example of a gas that may be present in its 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 temperatures and this can be exploited to create nanodroplets that may travel through the body more efficiently than larger gas bubbles. In some embodiments, the nanodroplets are formed by condensing microbubbles. As an example, lipid encapsulated gas microbubbles ranging in size from 0.5 – 500 microns may be readily condensed to form nanodroplets. This may be accomplished by placing the microbubble population in a syringe, such as a 3mL syringe, and applying hand pressure on a plunger. It was found that the microbubble suspension changed from a milky white appearance to a clear 12700017.1 colorless appearance upon application of such hand pressure, leading to the conclusion that the milky white appearance correlated with the presence of microbubbles having a size in the range of 0.5 – 500 microns and that the clear, colorless appearance correlated with a significant decrease in such microbubbles and the condensation of gas to its liquid form. The nanodroplets may be formed without changing the temperature of the microbubble population (e.g., without decreasing the temperature). The nanodroplets may comprise a shell of one or more lipids, such as phospholipids. The one or more phospholipids may be selected from DPPA, DPPC and DPPE, although they are not so limited. As with other embodiments in this disclosure one or more of these phospholipids may be PEGylated. In some embodiments, the DPPE is present in both PEGylated and un-PEGylated forms. Accordingly, some nanodroplet populations may have a lipid shell made of DPPA, DPPC and DPPE, optionally where the DPPE is 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). Other nanodroplet populations may have a lipid shell made of DPPC, DPPE and PEGylated DPPE, and may lack DPPA. The manual (or hand) pressure to be applied to the microbubble preparation present in a syringe such as a 3 mL syringe may be in the range of 6-12 atm. A sufficient manual pressure will be achieved and will be apparent by the conversion of the milky white appearance of the preparation into a clear, colorless appearance. The nanodroplets have a size of about less than 500 nm, including in the range 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 the nanodroplets to access sites in the body which are inaccessible to the larger microbubbles, the nanodroplets may be used for a wider or different variety of in vivo applications as compared to microbubbles. Accordingly, this disclosure contemplates and provides methods that involve administering such nanodroplets to a subject for diagnostic or therapeutic benefit. As an example, a nanodroplet preparation may be administered to a subject and then may be induced to expand by converting its liquid center to a gaseous center. In doing so, the nanodroplet converts into a nano gas bubble and it may cause expansion in the tissue or vasculature where it is present. 12700017.1 These nanodroplets are contemplated for use in any of the diagnostic and therapeutic uses provided herein. Reference may be made to Choudhary et al., J American Society Echocardiography, 30(2): 189-197, 2017; and Hayward et al., Scandanavian J Rheumatology, 40(5): 379-382, 2011. Activation, generally Activation may be achieved by vigorous shaking of the lipid formulation in the presence of a gas. Vigorous shaking, as used herein, refers to a motion that agitates a formulation, whether aqueous or non-aqueous, such that gas is introduced from the local ambient environment within the container (e.g., vial) into the formulation. Any type of motion that agitates the formulation and results in the introduction of gas may be used for the shaking. The shaking must be of sufficient force to allow the formation of foam after a period of time. Activation is typically performed just before administration to a subject and therefore is performed by an end user or an intermediate, but not the supplier or manufacturer of the lipid formulation. The lipid formulations are typically provided in vials that minimally house the lipid formulation and a gas in the headspace. The vigorous shaking of the lipid formulation in the presence of the gas results in the formation of lipid-encapsulated gas-filled gas bubbles that can be used in therapeutic applications. By “gas-filled”, as used herein, it is meant the gas bubbles comprise gas, such as a perfluorocarbon gas including but not limited to perflutren (or perfluoropropane) gas, in their internal cavity. The lipid shell that encapsulates the gas may be arranged as a unilayer or a bilayer, including unilamellar or multilamellar bilayers. The lipid formulations are typically provided in containers such as vials with a headspace gas of interest. The container may be made of any material including but not limited to glass or plastic. The glass may be pharmaceutical grade glass. The container may be sealed with a stopper such as a rubber stopper. As will be understood in the context of this disclosure, the internal volume of a container may be occupied with a lipid formulation and gas. Containers may be defined based on their liquid fill volume, which refers to the volume of liquid (e.g., lipid formulation) typically placed into the container. Liquid fill volume is different from and typically lower than the entire internal volume of the container. When containers are defined by a volume measurement herein, it is to be understood that this refers 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 12700017.1 containers and their corresponding liquid fill and internal volumes are as follows: Schott 2 mL (liquid fill volume) vial having a 2.9 mL internal volume; Schott 3 mL (liquid fill volume) vial having a 4.5 mL internal volume; and Wheaton 1 mL (liquid fill volume) v-vial having a 1.2 mL internal volume. In some embodiments, the container is a 0.5-10 mL container, such as 1-5 mL container, or a 1 mL or 2 mL container. The container may be a vial such as a glass vial. An example of a suitable vial is the Wheaton 2 ml glass vial (commercially available from, for example, Nipro, Cat. No. 2702, B33BA, 2cc, 13 mm, Type I, flint tubing vial), having an actual internal volume of about 3.8 ml. The vial may be pre-filled with gas of interest such as perflutren gas, prior to the introduction of the lipid formulation, or lipid formulation may be introduced into the vial first followed by the gas (e.g., through a process referred to as headspace gas exchange). The containers are preferably sterile and / or are sterilized after introduction of the lipid formulation and / or gas as described in published PCT application WO99 / 36104. In some embodiments, the container is a flat bottom container such as a flat-bottom vial. Suitable vials include 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 round-bottom vial. In some embodiments, the container has converging walls such that its bottom surface area (or bottom surface diameter) is smaller than its top (opening) surface area (or diameter) or smaller than any diameter therebetween (e.g., a body diameter). For clarity, a V-bottom container or vial has converging walls, and its bottom surface area is significantly smaller than any of its top or body surface areas. In some embodiments, the container is a syringe. The lipid formulation may be provided in a pre-filled syringe, optionally in physical contact with the gas. In some embodiments, the container is a single chamber container, such as a vial. The shaking may be by swirling (such as by vortexing), side-to-side, or up and down motion. Further, different types of motion may be combined. The shaking may occur by shaking the container (e.g., the vial) holding the aqueous or non-aqueous lipid solution, or by shaking the aqueous or non-aqueous solution within the container (e.g., the vial) without shaking the container (e.g., the vial) itself. Shaking is carried out by machine in order to standardize the process. Mechanical shakers are known in the art and their shaking mechanisms or means may 12700017.1 be used in the devices of the present disclosure. Examples include amalgamators such as those used for dental applications. Shaking encompasses 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 motions per minute. The formation of gas-filled microspheres upon activation can be detected by the presence of a foam on the top of the aqueous or non-aqueous solution and the solution becoming white. Activation is carried out at a temperature below the gel state to liquid crystalline state phase transition temperature of the lipid employed. By “gel state to liquid crystalline state phase transition temperature”, it is meant the temperature at which a lipid layer (such as a lipid monolayer or bilayer) will convert from a gel state to a liquid crystalline state. This transition is described for example in Chapman et al., J. Biol. Chem. 1974249, 2512-2521. The gel state to liquid crystalline state phase transition temperatures of various lipids will be 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), at p. 139. It will be understood by one skilled in the art, in view of the present disclosure, that the lipid(s) or lipid microspheres may be manipulated prior to or subsequent to being subjected to the methods provided herein. For example, after the shaking is completed, the gas-filled bubbles may be extracted from their container (e.g., vial). Extraction may be accomplished by inserting a needle of a syringe or a needle-free spike (i.e., PINSYNC) into the container, including into the foam if appropriate, and drawing a pre-determined amount of liquid into the barrel of the syringe by withdrawing the plunger or by adding an aqueous liquid mixing and drawing a pre- determined amount of liquid into the barrel of the syringe by withdrawing the plunger. Following activation, the nanobubbles may be diluted with saline, either in their container or following withdrawal from the container. Microsphere diameter is typically measured using instrumentation known and available in the art including but not limited to a Malvern FPIA-3000 Sysmex particle sizer and Accusizer 780. Devices Also provided herein are methods and means (e.g., devices) for facilitating proper and accurate preparation of the nanobubble and nanodroplet populations provided herein. These 12700017.1 methods and means reduce the risk of improper preparation of such populations. Nanobubble or nanodroplet populations that are not properly and accurately prepared can result in various issues including for example having too few gas-filled nanobubbles or nanodroplets, thereby reducing therapeutic efficacy. Proper identification and thus proper activation of a lipid formulation may be achieved using devices capable of uniquely identifying and uniquely activating diverse lipid formulations. Identification of a formulation and / or distinction between different formulations can be achieved in a number of ways. For example, devices may be used with scanners able to read labels on the formulation container (e.g., vial). In other instances, identification and / or distinction between different formulations can be achieved using devices that recognize the shape and size of a container housing a formulation. These latter devices may comprise a single holder or they may comprise two or more holders. According to one aspect, a device receives a container holding a formulation, identifies the formulation and performs different actions depending on the identity of the formulation that is detected. The device associates certain actions with each formulation, for example with reference to an internal or cloud-based look up table. After identifying a certain formulation, the device performs the actions required to activate that formulation, preferably in a fully automatic manner. A variety of different actions can be performed based on the formulation that is detected. In some embodiments, the device shakes the vial and correspondingly the formulation. In some embodiments, the device performs a specific shaking duration, pattern, and / or rate depending on the formulation that is detected. Examples of different shaking patterns include but are not limited to: side to side reciprocation, up and down reciprocation, vibration, a spinning motion, a figure-eight path, a circular path and back-and-forth tilting (e.g., rotating the container by some angle and reversing the action). For example, in one illustrative embodiment, the device associates a shaking duration of about 60 seconds with formulation type “A” and about 90 seconds with formulation type “B.” When the device detects a formulation type “A,” the device automatically shakes the formulation for about 60 seconds without requiring the user to input a shaking time. When the device detects a formulation type “B,” the device automatically shakes the formulation for about 90 seconds. It should be appreciated that other actions can be associated with an identified and detected formulation. 12700017.1 In some embodiments, the container housing the lipid formulation includes an indicator that indicates the formulation type and the device includes a detector that reads the indicator to detect the formulation type. The indicator may be one that is 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 that are identifiable by color detection, and the like. Barcodes such as linear barcodes may be those that comply with or meet Uniform Code Council standards or Health Industry Business Communications Council standards. Such indicators may in turn be read, for example, from a device such as a magnetic stripe reader, a chip reader, a barcode scanner or reader, an RFID tag reader, and the like. Virtually any labeling technology that has been used for authentication and / or “track and trace” purposes may be used in conjunction with the containers provided herein. The indicator may be positioned on any suitable portion of the sample container, such as the body of the container or the cap. In some embodiments, the indicator is integrally formed with or otherwise a part of the sample container. For example, the indicator may be a colored cap or a physical feature such as a protrusion or an indentation on the sample container. In other embodiments, the indicator is attached to the container via, for example, adhesive, magnets, hook-and-loop type fasteners, mechanical arrangement such as sliding the indicator behind holding tabs, or any other suitable attachment arrangement. The indicator may provide the end user or an intermediate handler of the container a variety of information including but not limited to source and / or producer of the formulation contained therein, including for example the name of the company or company subsidiary that made the formulation and / or that produced components of the formulation, the date on which the formulation was made, the physical location where the formulation was made, the date of shipment of the container, the treatment of the container including for example whether it was stored in a remote location and the conditions and length of such storage, the date on which the container was delivered, the means of delivery, the National Drug Code (NDC) as prescribed by the FDA, content of the container, dose and method of use including route of administration, etc. The indicator may serve one or more purposes including for example authentication of the container and the formulation contained therein. Authentication means the ability to identify or mark the container as originating and having been made by an authorized party, and it allows an end user or other party to identify container and formulations originating from another, 12700017.1 unauthorized party. The indicator may also be used to track and trace a container. This feature can be used to follow a container and the formulation contained therein following production and up to the point of administration to a subject. In this regard, the movement of the container during that period of time may be stored in a database, and optionally such a database may be accessible to an end user to ensure the integrity of the formulation. The indicator may also be a combined indicator, intending that it may contain information that is read using two different modes. For example, the indicator may contain information that is apparent and understandable to the visible eye (e.g., it may recite the name of the producer in words) and other information that is machine-readable, such as RFID embedded or barcode embedded information. The indicator may also be a dual use indicator, intending that it may serve two or more purposes. For example, the indicator may contain information that identifies the formulation and further information that identifies the manufacturer and / or date of manufacture. This information may be conveyed in the same format or using different format (e.g., one may be provided in an RFID indicator and the other may be provided in a barcode label). Examples of the foregoing activation devices are provided in in published PCT Application PCT / US2017 / 031150, published as WO2017 / 192910, the entire contents of which are incorporated by reference herein. According to some embodiments, the identification and activation device includes a holder arranged to hold a container comprising a lipid formulation and gas, and a shaking means arranged to shake the holder at least one speed. The shaking means includes a motor arranged to drive movement of the holder via a transmission, wherein at least a portion of the transmission is arranged to prevent slippage during an activation of the UCA formulation. The transmission may be arranged to create friction and / or resistance to movement. The transmission may include at least one of a tooth belt, a drive chain, a O-ring, and a gear box. The transmission may include a gear having first and second toothed wheels, the first toothed wheel being attachable to the holder and the second toothed wheel being attachable to the motor. In such embodiments, a tooth belt is arranged to engage with each of the first and second toothed wheels. The first toothed wheel may be positioned around a portion of a spindle attachable to the holder, and the second toothed wheel may be positioned around a portion of the motor, such as a shaft. The first and second toothed wheels may be substantially parallel to one 12700017.1 another. The first and second toothed wheels also may be substantially co-planar with one another. The transmission may also include first and second O-rings, wherein the first O-ring is positioned on a first lateral side of the first toothed wheel and the second O-ring is positioned on a second lateral side of the first toothed wheel. In some embodiments, each of the first and second O-rings and the first toothed wheel are positioned around the spindle. The transmission may include first and second profiled wheels, such as sprockets, with teeth or cogs. As with the above, the first wheel may be attachable to the holder and the second wheel may be attachable to the motor. In some embodiments, the first and second wheels engage with a chain, track, or another material that is perforated or indented. For example, the teeth are engageable with the chain, track, or other material with the perforations or indentations. Another suitable identification and activation device includes a holder arranged to hold a container comprising a lipid formulation, an identification means arranged to identify the lipid formulation in the container; and a shaking means arranged to shake the holder at least one speed, wherein the shaking means includes a motor arranged to drive movement of the holder via a transmission, wherein the transmission includes at least one of a toothed, perforated, indented belt or track, a drive chain, and a gear box. The transmission may include a gear having first and second toothed wheels, the first toothed wheel being attachable to the holder and the second toothed wheel being attachable to the motor. The toothed, perforated, indented belt or track may be arranged to engage with each of the first and second toothed wheels. The transmission may include first and second profiled wheels, such as sprockets, that are attachable to the motor and holder, respectively. The toothed, perforated, indented belt or track or other material (e.g., a drive chain) may be arranged to engage with each of the first and second wheels. Each of the foregoing apparatuses or devices may further include a housing, the shaking means being disposed in the housing. They also may include a first identification means arranged 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 also may include at least one antenna. The holder may include a shaker arm. The motor may include a direct current (DC) motor, such as a brushless DC motor. 12700017.1 The at least one speed may be pre-determined. The at least one speed may be pre-set by the apparatus. The at least one speed may be less than about 4530 rpm or it may be about 6000 rpm. The apparatus may be arranged to shake the holder at two or more speeds, wherein the two or more speeds are between about 4530 rpm and about 6000 rpm. Examples of the foregoing activation devices are provided in published PCT Application PCT / US2021 / 044394, published as WO2022 / 031739, the entire contents of which are incorporated by reference herein. The identification means may be further 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 for the formulation by reading an indicator on the container. Lipids The lipid formulations provided herein may comprise one and typically more than one lipid. As used herein, “lipids” or “total lipid” or “combined lipids” means a mixture of lipids. The lipids may be provided in their individual solid state (e.g., powdered) forms. Alternatively, the lipids may be provided as a lipid blend. Methods of making a lipid blend include those described in U.S. Patent No. 8,084,056 and published PCT application WO 99 / 36104. A lipid blend, as used herein, is intended to represent two or more lipids which have been blended resulting in a more homogeneous lipid mixture than might otherwise be attainable by simple mixing of lipids in their individual powdered form. The lipid blend is generally in a powder form. A lipid blend may be made through an aqueous suspension-lyophilization process or an organic solvent dissolution-precipitation process using organic solvents. In the aqueous suspension-lyophilization process, the desired lipids are suspended in water at an elevated temperature and then concentrated by lyophilization. The contents of U.S. Patent No. 8,084,056 and published PCT application WO 99 / 36104 relating to the method of generating a lipid blend are incorporated by reference herein. Alternatively, the lipids may be provided as individual powders that are dissolved together or individually directly into propylene glycol, glycerol or propylene glycol / glycerol to form the non-aqueous lipid formulation or to form a precursor to the aqueous lipid formulation. The lipids may be cationic, anionic or neutral lipids. The lipids may be of either natural, synthetic or semi-synthetic origin, including for example, fatty acids, fluorinated lipids, neutral 12700017.1 fats, phosphatides, oils, fluorinated oils, glycolipids, surface active agents (surfactants and fluorosurfactants), aliphatic alcohols, waxes, terpenes and steroids. At least one of the lipids may be a phospholipid. A phospholipid, as used herein, is a fatty substance containing an oily (hydrophobic) hydrocarbon chain (s) with a polar (hydrophilic) phosphoric head group. Phospholipids are amphiphilic. Preferably all of the lipids are 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. In some instances, one or more of the lipids may be modified. For example, lipids may be conjugated to polymers, such as chitin, hyaluronic acid, polyvinylpyrrolidone, or polyethylene glycol (PEG). Lipids conjugated to PEG are referred to herein as PEGylated lipids. Preferably, the PEGylated lipid is DPPE-PEG or DSPE-PEG. Conjugation of the lipid to the polymer such as PEG may be accomplished by a variety of bonds or linkages such as but not limited to amide, carbamate, amine, ester, ether, thioether, thioamide, and disulfide (thioester) linkages. Terminal groups on the PEG may be, but are not limited to, hydroxy-PEG (HO-PEG) (or a reactive derivative thereof), carboxy-PEG (COOH-PEG), methoxy-PEG (MPEG), or another lower alkyl group, e.g., as in iso-propoxyPEG or t-butoxyPEG, amino PEG (NH2PEG) or thiol (SH-PEG). The molecular weight of PEG may vary from about 500 to about 10000, including from about 1000 to about 7500, and from about 1000 to about 5000. In some important embodiments, the molecular weight of PEG is about 5000. Accordingly, DPPE-PEG5000 or DSPE-PEG5000 refers to DPPE or DSPE having attached thereto a PEG polymer having a molecular weight of about 5000. The percentage of PEGylated lipids relative to the total amount of lipids in the lipid solution, on a molar basis, is at or between about 2% to about 20%. In various embodiments, the percentage of PEGylated lipids relative to the total amount of lipids is at or between 5 mole percent to about 15 mole percent. In some embodiments, the lipids are 1, 2-dιpalmιtoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidic, mono sodium salt (DPPA), and N- 12700017.1 (polyethylene glycol 5000 carbamoyl)-1, 2-dipalmitoyl-sn- glycero-3- phosphatidylethanolamine, monosodium salt (PEG5000-DPPE). PEG5000-DPPE may be MPEG5000-DPPE or HO-PEG5000-DPPE. In some embodiments, the lipids may be one, two or all three of DPPA, DPPC and PEG5000-DPPE. In some embodiments, the lipids may be one, two or all three of DPPC, DPPE and PEGylated-DPPE. In some embodiments, the lipids may be one, two or all three of DPPC, DPPE and PEG5000-DPPE. In these latter embodiments, the lipids may not include DPPA. PEG5000-DPPE may be MPEG5000-DPPE or HO-PEG5000- DPPE. A wide variety of lipids, like those described in Unger et al. U.S. Patent No. 5,469,854, may be used in this disclosure. Suitable lipids include, for example, fatty acids, lysolipids, fluorinated lipids, phosphocholines, such as those associated with platelet activation factors (PAF) (Avanti Polar Lipids, Alabaster, Ala.), including 1-alkyl-2-acetoyl-sn-glycero 3- phosphocholines, and 1-alkyl-2-hydroxy-sn-glycero 3-phosphocholines; phosphatidylcholine with both saturated and unsaturated lipids, including dioleoylphosphatidylcholine; dimyristoyl- phosphatidylcholine; dipentadecanoylphosphatidylcholine; dilauroylphosphatdylcholine; 1,2- dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC); distearoylphosphatidylcholine (DSPC); and diarachidonylphosphatidylcholine (DAPC); phosphatidylethanolamines, such as dioleoyl- phosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) and distearoyl-phosphatidylethanolamine (DSPE); phosphatidylserine; phosphatidylglycerols, including distearoylphosphatidylglycerol (DSPG); phosphatidylinositol; sphingolipids such as sphingomyelin; glycolipids such as ganglioside 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. Other suitable lipids include phosphatidylcholines, such as diolecylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine; phosphatidylethanolamines, such as dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine and N- succinyl-dioleoylphosphatidylethanolamine; phosphatidylserines; phosphatidyl-glycerols; sphingolipids; 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; 12700017.1 physeteric 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 derivatives, such as cholesterol hemisuccinate, cholesterol sulfate, and cholesteryl-(4'-trimethylammonio)-butanoate; polyoxyethylene fatty acid esters; polyoxyethylene fatty acid alcohols; polyoxyethylene fatty acid alcohol ethers; polyoxyethylated sorbitan fatty acid esters; glycerol polyethylene glycol oxystearate; glycerol polyethylene glycol ricinoleate; ethoxylated soybean sterols; ethoxylated castor oil; polyoxyethylene-polyoxypropylene 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)octadecanoy l]-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, such as wherein alkyl is a C.sub.12, C.sub.14 or C.sub.16 alkyl; benzyldimethyldodecylammonium bromide; benzyldimethyldodecylammonium chloride, benzyldimethylhexadecylammonium bromide; benzyldimethylhexadecylammonium chloride; benzyldimethyltetradecylammonium bromide; benzyldimethyltetradecylammonium chloride; cetyldimethylethylammonium bromide; cetyldimethylethylammonium chloride; cetylpyridinium bromide; cetylpyridinium chloride; N- [1-2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA); 1,2-dioleoyloxy- 3-(trimethylammonio)propane (DOTAP); and 1,2-dioleoyl-e-(4'-trimethylammonio)-butanoyl- sn-glycerol (DOTB). In some embodiments where DPPA, DPPC and DPPE are used, their molar percentages may be about 77-90 mole % DPPC, about 5-15 mole % DPPA, and about 5-15 mole % DPPE, including DPPE-PEG5000. Preferred ratios of each lipid include those described in the Examples section such as a weight % ratio of 6.0 to 53.5 to 40.5 (DPPA : DPPC : MPEG5000- DPPE) or a mole % ratio of 10 to 82 to 8 (10 : 82 : 8) (DPPA : DPPC : MPEG5000-DPPE). 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. 12700017.1 Examples of perfluorocarbon gases include perfluoropropane, perfluoromethane, perfluoroethane, perfluorobutane, perfluoropentane, perfluorohexane. Examples of gases that may be used in the microspheres of the disclosure are described in US Patent No. 5,656,211 and are incorporated by reference herein. In an important embodiment, the gas is perfluoropropane. Examples of gases include, but are not limited to, hexafluoroacetone, isopropylacetylene, allene, tetrafluoroallene, 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-methy-1-butene, 3-methyl-1-butene, perfluoro-1- butene, perfluoro-1-butene, perfluoro-2-butene, 4-phenyl-3-butene-2-one, 2-methyl-1-butene-3- yne, butylnitrate, 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), perfluoroisobutane, 3-chlorocyclopentene, cyclopropane, 1,2-dimethylcyclopropane, 1,1- dimethylcyclopropane, ethyl cyclopropane, methylcyclopropane, diacetylene, 3-ethyl-3- methyldiaziridine, 1,1,1-trifluorodiazoethane, dimethylamine, hexafluorodimethylamine, dimethylethylamine, bis-(dimethyl phosphine)amine, 2,3-dimethyl-2-norbornane, perfluoro- dimethylamine, dimethyloxonium chloride, 1,3-dioxolane-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-tetrafluoro-ethane, 2-chloro- 1,1-difluoroethane, chloroethane, chloropentafluoroethane, dichlorotrifluoroethane, fluoroethane, nitropentafluoroethane, nitrosopentafluoro-ethane, perfluoroethane, perfluoroethylamine, ethyl vinyl ether, 1,1-dichloroethylene, 1,1-dichloro-1,2-difluoro-ethylene, 1,2-difluoroethylene, methane, methane-sulfonyl-chlori-detrifluoro, 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-trifluoro, 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, methanesulfenylchloride-trifluoro, 2- 12700017.1 methyl butane, methyl ether, methyl isopropyl ether, methyl lactate, methyl nitrite, methyl sulfide, methyl vinyl ether, neopentane, nitrogen (N.sub.2), nitrous oxide, 1,2,3-nonadecane tricarboxylic acid-2-hydroxycrimethylester, 1-nonene-3-yne, oxygen (O.sub.2), oxygen 17 (.sup.17 O.sub.2), 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)- decafluoro(S.sub.2 F.sub.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. Fluorinated gases (that is, a gas containing one or more fluorine molecules, such as sulfur hexafluoride), fluorocarbon gases (that is, a fluorinated gas which is a fluorinated carbon or gas), and perfluorocarbon gases (that is, a fluorocarbon gas which is fully fluorinated, such as perfluoropropane and perfluorobutane) are preferred. The gas such as the perfluorocarbon gas is typically present below its ordinary concentration at room temperature due to the incorporation of air during production. The concentration of perfluoropropane when present in a vial comprising a non-aqueous mixture and a gas headspace is expected to be about 6.52 mg / mL, at about one atmosphere of pressure. The concentrations of other gases, as known in the art, would be similarly diluted due to incorporation of air during production. The gas, such as perflutren gas, may be injected into or otherwise added to the container (e.g., the vial) comprising the lipid formulation or into the lipid formulation itself in order to provide a gas other than air. Gases that are not heavier than air may be added to a sealed container while gases heavier than air may be added to a sealed or an unsealed container. It will be understood by one skilled in the art that a gaseous precursor may also be used, followed by conversion of the precursor into a gas either by temperature or pressure change. 12700017.1 EXAMPLES The Examples of the present application involve the activation and use of the ultrasound contrast agent (UCA) formulations DEFINITY®and DEFINITY RT®, the specific components of which are described below. Examples 1-6 describe protocols for the activation of the UCA formulations, and Examples 7-10 describe experiments involving the use of DEFINITY®and / or DEFINITY RT®in ultrasound-triggered nanobubble cavitation experiments. DEFINITY®Vials of the aqueous lipid formulation (i.e., lipid suspension) DEFINITY®manufactured by Lantheus Medical Imaging, Inc., prior to activation, contained the following phospholipids: 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) in a matrix of 103.5 mg / mL propylene glycol, 126.2 mg / mL glycerol (i.e., glycerin), and 2.34 mg / mL sodium phosphate monobasic monohydrate, 2.16 mg / mL sodium phosphate dibasic heptahydrate, and 4.87 mg / mL sodium chloride in Water for Injection. The pH is 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 2cc (2 mL) Wheaton glass vial with an actual internal volume of about 3.8 mL and a head space of approximately 2.04 containing perfluoropropane gas (PFP, 6.52 mg / mL). The commercially available, FDA-approved, aqueous based UCA formulation, DEFINITY®(Lantheus Medical Imaging, Inc.) is activated by mechanical shaking (described in US Patent 6,039,557, the content of which is hereby incorporated by reference and may be used in the present process) of the PFP / lipid formulation using a VIALMIX®. This results in incorporation of gas into lipid microspheres and represents the activated product (see DEFINITY® Prescribing Information). Gas-filled bubbles can be analyzed for number and size distribution using a particle sizer (AccuSizer 780 or SpetraDyne) when diluted with saline. The acoustic attenuation assay was performed using a broadband transducer centered on frequencies of either 2.25 or 7.5 MHz. 12700017.1 DEFINITY RT®Vials of non-aqueous lipid formulation (i.e., lipid solution) DEFINITY RT®manufactured by Lantheus Medical Imaging, Inc., prior to activation, contained the following phospholipids: 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 to 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 2cc (2 mL) Wheaton glass vial with an actual internal volume of about 3.8 mL and a head space of approximately 3.45 mL containing perfluoropropane gas (PFP, 6.52 mg / mL). Activation may be performed using VIALMIX®or VIALMIX-RFID®. This results in incorporation of gas into lipid nanobubbles which represent the activated product. Gas-filled bubbles can be analyzed for number and size distribution using a particle sizer (AccuSizer 780 or SpetraDyne) when diluted with saline. The acoustic attenuation assay was performed using a broadband transducer centered on frequencies of either 2.25 or 7.5 MHz. Example 1. Vials of aqueous lipid formulation (e.g., DEFINITY®) were shaken at 4530 rpm for differing time periods using a VIALMIX-RFID®modified to have programmable control of time and speed parameters. Samples were taken immediately after shaking and analyzed for gas bubble size distribution using an Accusizer 780. Bubbles in different size distributions indicate maximal formation of 0.5-1 micron gas bubbles occurred with approximately 90 seconds of activation (Table 1), 1-2 micron at 45 seconds (data not shown) and 2-6 micron at 10 and 20 and 90 seconds (Table 3). Analysis of samples at each time point for acoustic attenuation demonstrated a maximal value of 7.5 MHz at 45 seconds, a profile that matches the 1-2 micron 12700017.1 microbubble number. Similarly, the acoustic attenuation was near maximal for 2.25 MHz at 10 and 20 seconds, a profile that matched 2-6 micron microbubble number (data not shown). Example 2. Vials of aqueous lipid formulation (e.g., DEFINITY®) were shaken at 6000 rpm for differing time periods using a VIALMIX-RFID®modified to have programmable control of time and speed parameters. Samples were taken immediately after shaking and analyzed for gas bubble size distribution using an Accusizer 780. Gas bubbles in different size distributions indicate maximal formation of 0.5-1 micron gas bubbles occurred with approximately 20 and 25 seconds of activation (Table 2), 1-2 micron at 10-15 seconds (data not shown) and 2-6 micron at 5, 25 and 30 seconds. (Table 4). Analysis of samples at each time point for acoustic attenuation demonstrated a maximal value for 7.5 MHz at 10-15 seconds (data not shown), a profile that matches the 1-2 micron microbubble number. The acoustic attenuation was highest for 2.25 MHz at 5 seconds and 25-30 seconds (data not shown), a profile that matches the 2-6 micron microbubble number. Example 3. Vials of non-aqueous lipid formulation (e.g., DEFINITY RT®) were shaken at 4950 rpm for differing time periods using a VIALMIX-RFID®modified to have programmable control of time and speed parameters. Samples were taken immediately after shaking and analyzed for gas bubble size distribution using an Accusizer 780. Gas bubbles in different size distributions indicate maximal formation of 0.5-1 micron gas bubbles occurred with approximately 250 seconds of activation (data not shown), 1-2 micron at 125 seconds (data not shown), and 2-6 micron at 80 seconds (data not shown). Analysis of samples at each time point for acoustic attenuation demonstrated a maximal value of 7.5 MHz at 100 to 150 seconds, a profile that matches the 1-2 micron microbubble number. Similarly the acoustic attenuation was maximal for 2.25 MHz at 80 seconds, a profile that matched 2-6 micron microbubble number (data not shown). Example 4. Vials of non-aqueous lipid formulation (e.g., DEFINITY RT®) were shaken at 6000 rpm for differing time periods using a VIALMIX-RFID®modified to have programmable control of 12700017.1 time and speed parameters. Samples were taken immediately after shaking and analyzed for gas bubble size distribution using an Accusizer 780. Gas bubbles in different size distributions indicate maximal formation of 0.5-1 um nanobubbles occurred with approximately 50-90 seconds of activation (Table 2A), 1-2 micron at 30-40 seconds (data not shown) and 2-6 micron at 20-90 seconds. (Table 4). Analysis of samples at each time point for acoustic attenuation demonstrated a maximal value of 7.5 MHz at 30 seconds (data not shown), a profile that matches the 1-2 micron microbubble number. Similarly, the acoustic attenuation was maximal for 2.25 MHz at 20-90 seconds (Table 2A), a profile that matched 2-6 micron microbubble number. Vials of non-aqueous lipid formulation (e.g., DEFINITY RT®, Example 2) were shaken at speeds between 6200 and 6600 rpm for differing time periods using a VIALMIX-RFID® modified to have programmable control of time and speed parameters using a Trinamic brushless motor. Samples were taken immediately after shaking, inverted 10 times, and analyzed for gas bubble size distribution using an Accusizer 780. Gas bubbles in different size distributions indicate maximal formation of 0.5-1 micron nanobubbles occurred with approximately 30-60 seconds of activation with speeds between 6200 to 6600 rpm (Table 2B), 1-2 micron around 15 seconds (data not shown) and 2-6 micron at 15 to 70 seconds (data not shown). The data show that non-aqueous lipid formulations (e.g., DEFINITY RT®, Example 2) can be sufficiently activated at 6000 rpm, at 6200 rpm, and at 6600 rpm, albeit at different peak times. At 6000 rpm and 6200 rpm, peak times appear to be about 60 seconds. At 6600 rpm, maximum effect is seen at about 15 to about 30 seconds. Example 5. Vials of non-aqueous lipid formulation (e.g., DEFINITY RT®) were either a) un- modified or b) contained 1.5 times the fill volume of the non-aqueous lipid formulation (i.e., 1.5 times 0.35 mL lipid formulation) (referred to as DEFINITY-RT 1.5X fill volume, Table 5) or c) was diluted by adding 0.5 times the fill volume with propylene glycol / glycerol (50:50) matrix alone (i.e., 0.35 mL lipid formulation and about 0.18 mL matrix) (referred to as DEFINITY-RT 1X + 0.5*, Table 5). Higher numbers of 2-6 micron microbubbles could be formed by increasing the amount of non-aqueous lipid solution or surprisingly adding only matrix. These changes increased the acoustic attenuation to 2.25 MHz but not at 7.5 MHz (Table 5). Analysis of the 12700017.1 size distribution for gas bubbles using the Accusizer 780 indicated the majority of gas bubbles were less than 2 micron when shaken at 6000 rpm for 60 seconds (data not shown). However, with a dilution of the matrix, the distribution contained substantially more microbubbles above 2 micron but less than 10 um. This distribution produced a pronounced increase in ultrasound attenuation at 2.25 MHz (Table 5). Example 6. Vials of non-aqueous lipid formulation (e.g., DEFINITY RT®) were shaken at 6000 rpm for 60 seconds using a VIALMIX-RFID®. After dilution with saline, samples were inverted ten times and either (a) taken immediately and analyzed for gas bubble size distribution using an Accusizer 780 or (b) transferred to a 3 mL (or a 5 mL syringe) Becton Dickinson or Norm Jet syringe, with a leur-lok plug inserted, and plunger pressure applied by hand until the white milky solution became clear. After ten inversions, the plug was removed and a sample analyzed for gas bubble size distribution using an Accusizer 780. Sufficient hand pressure and release resulted in a marked clearance of bubbles greater than 500 nm (see Table 6). Vials of aqueous lipid formulation (e.g., DEFINITY®) were shaken at 4530 rpm for 45 seconds using a VIALMIX-RFID®. Samples were either (a) taken immediately after shaking and inversion, and analyzed for gas bubble size distribution using an Accusizer 780 or (b) transferred to a 3 mL Becton Dickinson or Norm Jet syringe, with a leur-lok plug inserted, and plunger pressure applied by hand until the white milky solution became clear. After ten inversions, the plug was removed and a sample was analyzed for gas bubble size distribution using an Accusizer 780. Sufficient hand pressure and release resulted in a marked clearance of bubbles greater than 500 nm (see Table 6). Some studies examined the use of a device to aid in pressurizing the syringe after activation. A Caulk gun was adapted to allow insertion of a medical syringe and to facilitate and aid in controlling the pressurization. Additional pressurizing devices involving mechanically controlled plunger using gears and / or a step motor with a measured pressure feedback is contemplated. Example 7. Ultrasound-Triggered Nanobubble Destruction (UTND) and Radiation Therapy in Mice. 12700017.1 Immunodeficient mice are injected with 1 x 106Hu7.5 (also known as Huh7.5 or HuH7.5) hepatocellular carcinoma (HCC) cells, and livers are monitored for tumor growth. Tumors are allowed to grow to 5 mm in diameter before initiation of treatment. Animals are randomized into 1 of 3 groups receiving: (1) nanobubble cavitation alone (ultrasound-triggered nanobubble destruction, UTND, with DEFINITY®or DEFINITY RT®sufficiently activated to generate the gas bubbles disclosed herein), (2) radiation alone (one dose of either 2 or 8 Gy), or (3) nanobubble cavitation (UTND) immediately before radiation. For UTND treatment, animals intravenously receive 1.1 x 109nanobubbles (DEFINITY®or DEFINITY RT®, activated via agitation by VIALMIX-RFID®or as provided herein to generate the gas bubbles disclosed herein) in 90 μL of saline. Final circulating concentrations of nanobubbles are designed to be higher than for diagnostic purposes to ensure sufficient interaction between the nanobubbles and endothelium. UTND treatment is performed by exposing tumor sites to a 16-cycle tone burst at 500-kHz and 3-kHz pulse repetition frequencies with a 10% duty cycle over a 50 ms window. UTND treatments are performed for 5 minutes, resulting in a total of 750 ms exposure. Immediately following ultrasound exposure, mice in the group also receiving radiation are lead-shielded and only the tumor site is exposed to ionizing radiation at doses of 2 or 8 Gy in single fractions using a dose rate of 200 cGy / min. Tumor vascularity and growth is monitored twice weekly using ultrasound for up to 30 days. It is expected that animals receiving tumor-directed radiation in conjunction with UTND will exhibit significantly greater reduction in tumor vascularity and tumor size compared to animals receiving only radiation or only UTND. Example 8. Evaluation of Ultrasound-Triggered Nanobubble Destruction (UTND) and Radiation Combination Therapy on Hepatocellular Carcinoma in Rats. Immunodeficient nude rats are injected with 1 x 106Hu7.5 hepatocellular carcinoma (HCC) cells in 100μL of Matrigel. The right liver lobe of each rat is monitored twice per week via high frequency ultrasound scanning with 32 MHz to monitor tumor growth. When tumors reach a diameter of 5 mm or greater, animals are randomized into 1 of 3 groups receiving: (1) nanobubble cavitation alone (ultrasound-triggered nanobubble destruction, UTND) (DEFINITY®or DEFINITY RT®sufficiently activated to generate the gas bubble populations 12700017.1 disclosed herein), (2) radiation alone (dose of 5 Gy), or (3) nanobubble cavitation (UTND) 3 hours before radiation. For UTND treatment, animals intravenously receive a slow 0.1 mL injection of ultrasound contrast agent (UCA) (DEFINITY®or DEFINITY RT®, activated via agitation by VIALMIX-RFID®) followed by a 0.3 mL saline flush over 10-20 seconds. Following administration of the UCA, animals receive a series of 4 second destructive pulses (Mechanical Index [MI] of 1.35) to cavitate nanobubbles. UTND treatment is performed for 2-3 minutes per animal. Animals receiving radiation treatment are anesthetized with a combination of ketamine and xylazine and given a single 5 Gy dose of radiation. Animals receiving both UTND and radiation are given their radiation dose 3 hours after nanobubble cavitation. Tumors are irradiated with 4 confocal beams fractioned at 1.25 Gy per beam at a dose rate of 245 Gy / min. Tumor vascularity and growth is monitored twice weekly using ultrasound. It is expected that animals receiving tumor-directed radiation in conjunction with UTND will exhibit significantly greater reduction in tumor vascularity and tumor size compared to animals receiving only radiation or only UTND. Example 9. Transarterial Radioembolization (TARE) and UTND Therapy in Individuals with Hepatocellular Carcinoma (HCC) Participants with hepatocellular carcinoma (HCC) are randomized into groups undergoing either (1) transarterial radioembolization (TARE) therapy alone or (2) TARE therapy in combination with UTND. Participants in the TARE-only group receive treatment as is standard in the art. Participants receiving TARE-UTND combination therapy undergo 3 UTND sessions: 1-4 hours after TARE and again approximately 1 week and 2 weeks later. For UTND treatments, 5 mL of ultrasound contrast agent (UCA) (DEFINITY®or DEFINITY RT®, activated via agitation by VIALMIX-RFID®) is suspended in a 50-mL bag of saline and manually reagitated every 2-3 minutes. The UCA / saline solution is infused at a rate of 120 mL per hour. A 4-second UTND sequence (Mechanical Index [MI] = 1.5MHz) is transmitted in 2.3-μsec pulses at a pulse repetition of 100 Hz. The UTND sequence is repeated 3-5 times at the tumor midline and then modified for the remaining UCA / saline infusion. For both groups, TARE treatment includes a radiation dose of no more than 30 Gy. 12700017.1 It is expected that participants receiving TARE treatment in combination with UTND therapy will experience improved tumoral response (e.g., reduction of tumor size) and overall survival. Example 10. Assessment of the Anti-Tumor Efficacy of UTND Therapy Immunodeficient rodents (e.g., immunodeficient mice or immunodeficient nude rats) are injected with 1 x 106Hu7.5 hepatocellular carcinoma (HCC) cells, and livers are monitored for tumor growth. When tumors reach a diameter of 5 mm or greater, animals are randomized into 1 of 5 groups receiving: (1) nanobubble cavitation alone (ultrasound-triggered nanobubble destruction, UTND) (DEFINITY®or DEFINITY RT®sufficiently activated to generate the gas bubble populations disclosed herein) with a low- to medium-frequency ultrasound pulse (e.g., 500-kHz as described in Example 7); (2) nanobubble cavitation alone with a high-frequency ultrasound pulse (e.g., 15-20 mHz, including 18 mHz); (3) radiation alone (dose of 2 Gy, 5Gy, or 8 Gy); (4) nanobubble cavitation (UTND) with a low- to medium-frequency ultrasound pulse 0-3 hours before radiation; or (5) UTND with a high-frequency ultrasound pulse 0-3 hours before radiation. For UTND treatment in mice, animals intravenously receive activated DEFINITY®or DEFINITY RT®, activated via agitation by VIALMIX-RFID®or as provided herein, in 50 to 200 μL of saline. Final circulating concentrations of nanobubbles are designed to be higher than for diagnostic purposes to ensure sufficient interaction between the nanobubbles and endothelium. Tumor sites are exposed to a 16-cycle tone burst at 500-kHz for mice receiving low- to medium-frequency pulses and 18-mHz for mice receiving high-frequency pulses, with or without 3-kHz pulse repetition frequencies, with a 10% duty cycle over a 50 ms window. UTND treatments are performed for 5 minutes, resulting in a total of 750 ms exposure. For low- to medium-frequency pulses and for high-frequency pulses, pulse repetition frequencies, duty cycle, and time may be adjusted depending on the experiment or application. For UTND treatment in rats, animals intravenously receive a slow 0.1 mL injection of ultrasound contrast agent (UCA) (DEFINITY®or DEFINITY RT®, activated via agitation by VIALMIX-RFID®) followed by a 0.3 mL saline flush over 10-20 seconds. Following administration of the UCA, animals receive a series of 4 second destructive pulses (Mechanical Index [MI] of 1.35) to cavitate nanobubbles for rats receiving low- to medium-frequency 12700017.1 ultrasound pulses, while rats receiving high-frequency pulses are exposed to 4 second destructive pulses of 18-mHz. UTND treatment is performed for 2-3 minutes per animal. Mice receiving radiation are lead-shielded and only the tumor site is exposed to ionizing radiation at doses of 2 or 8 Gy in single fractions, using a dose rate of 200 cGy / min. Rats receiving radiation treatment are anesthetized with a combination of ketamine and xylazine and given a single 5 Gy dose of radiation. Mice receiving both UTND and radiation are given radiation immediately following UTND treatment, while rats receiving both UTND and radiation are given radiation 3 hours post-UTND treatment. Following treatment, tumors are monitored at regular intervals until experimental endpoint. Multiple metrics are used to assess anti-tumor efficacy of the treatments, including: (i) Tumor size. Tumor size is monitored using high frequency ultrasound scanning and / or calipers; (ii) Tumor vascularity. Tumor vascularity is measured using ultrasound (e.g., Power Doppler) and / or evaluating CD31 expression via imaging of tumor samples. CD31 is expressed by endothelial cells, and reduction in CD31 expression in tumor samples (e.g., reduction in percentage of area of a tumor that is positive for CD31) is indicative of a reduction in the vascularization (e.g., microvessel density) of a tumor. CD31 expression can be analyzed in situ by labeling tumor samples using anti-CD31 antibodies directly or indirectly (e.g., via a secondary antibody) conjugated to a detectable marker (e.g., a fluorophore) and imaging the samples, and / or by dissociating the tumor and labeling the cells with anti-CD31 antibodies for analysis via flow cytometry. CD31 expression in tumor samples may also be assayed using Western blot or other suitable proteomics analyses; (iii) Tumor oxygenation. Oxygenation of the tumor is likely to be affected by a reduction in the tumor vascularity. Tumor oxygenation is measured using photoacoustic imaging, blood oxygen level dependent (BOLD) magnetic resonance imaging (MRI), positron emission topography (PET), electron paramagnetic resonance spectroscopy (EPR), and / or any other suitable method; (iv) Tumor microenvironment (TME) composition. The type and frequency of cells present in the TME is also indicative of the response to anti-tumor treatment. The TME is analyzed for infiltration of immune cells using the pan-immune marker CD45, and immune cells may be further characterized on the basis of other markers (e.g., CD3 for T cells, CD20 for B cells, CD56 for NK cells, etc.). Tumor samples are assessed for presence of induction of 12700017.1 caspase-3, an effector protein of apoptosis. Apoptotic tumor cells and / or endothelial cells indicate a positive response to the anti-tumor treatments described herein. The TME is also analyzed for ceramide levels. Ceramide is measured by liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), or fast liquid chromatography. Other suitable methods of measuring anti-tumor efficacy of the treatments described herein can also be used. Greater reduction in the tumor in animals treated with both UTND and radiation compared to animals treated with radiation alone may indicate increased susceptibility of the tumor to radiation as a result of UTND treatment. 12700017.1 Table 1 12700017.1 Table 2A 12700017.1 Table 2B 5 12700017.1 Table 3 12700017.1 Table 4 12700017.1 Table 5 12700017.1 *(50 / 50 PG / G) Table 6 12700017.1 EQUIVALENTS 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 function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically 12700017.1 identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” 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” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. 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 terms of exclusivity, 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. As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be 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 within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “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 one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. 12700017.1 It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed 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” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. 12700017.1

Claims

What is claimed is: CLAIMS 1. A method of treating a subject having a carcinoma, the method comprising: (a) administering an effective amount of a population of lipid-encapsulated perfluorocarbon gas bubbles to a subject having a carcinoma and receiving radiation therapy; and (b) applying an effective amount of ultrasound energy to the carcinoma.

2. A method of enhancing radiotherapy treatment, the method comprising: (a) administering an effective amount of a population of lipid-encapsulated perfluorocarbon gas bubbles to a subject having a carcinoma; (b) applying an effective amount of ultrasound energy to the carcinoma; and (c) applying radiation therapy to the subject.

3. The method of claim 1 or 2, wherein the carcinoma is hepatocellular carcinoma.

4. A method of treating a subject having hepatocellular carcinoma, the method comprising: (a) administering an effective amount of a population of lipid-encapsulated perfluorocarbon gas bubbles to a subject having hepatocellular carcinoma; (b) applying an effective amount of ultrasound energy to the hepatocellular carcinoma; and (c) applying radiation therapy to the subject.

5. The method of any one of claims 1-4, wherein 75% or more of the lipid-encapsulated perfluorocarbon gas bubbles are nanobubbles having a diameter in the range of about 100 nanometers to less than or about 1 micron, and wherein the lipid-encapsulated perfluorocarbon gas bubbles comprise DPPC and PEG-DPPE, optionally wherein the nanobubbles are present in a concentration range of about 7.0 x 109to about 2.5 x 1010nanobubbles per mL, or about 1 x 1010to about 2.5 x 1010nanobubbles per mL. 12700017.

16. The method of any one of claims 1-4, wherein 80% or more of the lipid-encapsulated perfluorocarbon gas bubbles are nanobubbles having a diameter in the range of about 100 nanometers to less than or about 1 micron.

7. The method of any one of the foregoing claims, wherein the lipid-encapsulated perfluorocarbon gas bubbles comprise (a) DPPA, DPPC and PEG-DPPE, optionally in a mole % ratio of about 10 to about 82 to about 8 including 10:82:8, or (b) DPPE, DPPC and PEG-DPPE, optionally in a mole % ratio of about 10 to about 82 to about 8 including 10:82:

8.

8. The method of any one of the foregoing claims, wherein PEG-DPPE is PEG5000-DPPE.

9. The method of any one of the foregoing claims, wherein PEG-DPPE is MPEG5000- DPPE.

10. The method of any one of the foregoing claims, wherein the lipid-encapsulated perfluorocarbon gas bubbles are lipid-encapsulated perfluoropropane gas bubbles.

11. The method of any one of the foregoing claims, wherein the population of lipid- encapsulated perfluorocarbon gas bubbles is present in a non-aqueous lipid solution comprising (a) DPPA, DPPC and PEG-DPPE or (b) DPPE, DPPC and PEG-DPPE, together with glycerol, propylene glycol, and less than or equal to 5% water (v / v).

12. The method of claim 11, wherein the lipids are present in a combined concentration of about 2.5 mg / ml or in a combined concentration of about 3.75 mg / ml.

13. The method of any one of claims 1-10, wherein the population of lipid-encapsulated perfluorocarbon gas bubbles is present in an aqueous lipid suspension comprising (a) DPPA, DPPC and PEG-DPPE or (b) DPPE, DPPC and PEG-DPPE, together with glycerol and propylene glycol, and more than 5% water (v / v).

14. The method of claim 13, wherein the lipid suspension has a combined lipid concentration of about 0.75 mg / ml. 12700017.

115. The method of any one of the foregoing claims, wherein the population of lipid- encapsulated perfluorocarbon gas bubbles lacks DPPA.

16. The method of any one of the foregoing claims, wherein the population of lipid- encapsulated perfluorocarbon gas bubbles is formed by activating a lipid formulation comprising DPPC and PEG-DPPE, glycerol and propylene glycol, in the presence of a perfluorocarbon gas, at a shaking rate and shaking time sufficient to yield a population of lipid-encapsulated perfluorocarbon gas bubbles wherein 75% or more of the gas bubbles are nanobubbles having a diameter in the range of about 100 nanometers to about 1 micron.

17. The method of claim 16, wherein the lipid formulation is a non-aqueous lipid solution comprising (a) DPPA, DPPC and PEG-DPPE or (b) DPPE, DPPC and PEG-DPPE, together with glycerol, propylene glycol, and less than or equal to 5% water (v / v), and optionally wherein the lipid formulation is activated in the presence of gas at a shaking rate of about 6000 rpm for a shaking time of about 50 seconds to about 90 seconds.

18. The method of claim 17, wherein the lipid formulation is activated at a shaking rate of about 6000 rpm for a shaking time of about 50 seconds, about 60 seconds, about 75 seconds, or about 90 seconds.

19. The method of claim 16, wherein the lipid formulation is an aqueous lipid suspension comprising (a) DPPA, DPPC and PEG-DPPE or (b) DPPE, DPPC and PEG-DPPE, together with glycerol, and propylene glycol, , optionally wherein the lipid formulation is activated in the presence of gas at a shaking rate of about 4530 rpm for a shaking time of about 90 seconds, or at a shaking rate of about 6000 rpm for a shaking time of about 20 seconds to about 25 seconds, or at a shaking rate of about 6000 rpm for a shaking time of about 20 seconds or about 25 seconds. 20 The method of any one of claims 1-19, wherein the lipid-encapsulated perfluorocarbon gas bubbles experience stable cavitation in vivo after application of the ultrasound energy. 12700017.

121. The method of any one of claims 1-20, wherein the lipid-encapsulated perfluorocarbon gas bubbles experience inertial cavitation in vivo after application of the ultrasound energy, optionally as detected and / or measured by emitted broadband radiofrequency.

22. The method of any one of claims 1-21, wherein the lipid-encapsulated perfluorocarbon gas bubbles are administered systemically.

23. The method of any one of claims 1-21, wherein the lipid-encapsulated perfluorocarbon gas bubbles are administered locally.

24. The method of any one of claims 1-21, wherein the lipid-encapsulated perfluorocarbon gas bubbles are administered intravenously.

25. The method of any one of claims 1-24, wherein the effective amount of ultrasound energy causes stable cavitation of a subset of the lipid-encapsulated perfluorocarbon gas bubbles in vivo.

26. The method of any one of claims 1-24, wherein the effective amount of ultrasound energy causes inertial cavitation of a subset of the lipid-encapsulated perfluorocarbon gas bubbles in vivo.

27. The method of any one of claims 1-24, wherein the effective amount of ultrasound energy causes stable and inertial cavitation of a subset of the lipid-encapsulated perfluorocarbon gas bubbles in vivo.

28. The method of any one of claims 1-27, wherein the radiation therapy is applied before administration of the gas bubbles and / or after administration of the gas bubbles, optionally wherein the radiation therapy is applied locally to the carcinoma.

29. The method of any one of claims 1-28, wherein the radiation therapy is applied before and / or after applying ultrasound energy to the subject, optionally wherein the radiation therapy is applied locally to the carcinoma. 12700017.

130. The method of any one of claims 1-28, wherein the population of lipid-encapsulated perfluorocarbon gas bubbles is administered to the subject, then ultrasound energy is applied to the carcinoma, and then radiation therapy is applied to the carcinoma.

31. A method of treating a subject having a thrombus and / or embolism comprising administering a composition comprising a population of lipid-encapsulated perfluorocarbon gas bubbles to a subject having a thrombus and / or an embolism, wherein the subject is receiving treatment with a thrombolytic agent.

32. The method of claim 31, wherein the thrombolytic agent is administered before and / or during and / or after administration of the gas bubble population and ultrasound.

33. A method of treating a subject having a thrombus and / or an embolism, the method comprising: (a) administering an effective amount of a population of lipid-encapsulated perfluorocarbon gas bubbles to a subject having a thrombus and / or an embolism and receiving thrombolytic therapy; and (b) applying an effective amount of ultrasound energy to the thrombus and / or the embolism.

34. A method of enhancing a thrombolytic treatment, the method comprising: (a) administering an effective amount of a population of lipid-encapsulated perfluorocarbon gas bubbles to a subject having a thrombus and / or an embolism; (b) applying an effective amount of ultrasound energy to the thrombus and / or the embolism; and (c) administering a thrombolytic agent to the subject. 12700017.1

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