BACKGROUND

A. Field

This disclosure relates to imaging and therapy techniques comprising nanodroplets. More particularly, embodiments of the disclosure relate to the use of nanodroplets to inflate nanobubbles or microbubbles to provide improved imaging and/or therapeutic aspects.

B. Related Art

Various techniques have been developed to image and provide therapy to conditions that require medical treatment, including for example, cancerous tumors. Certain techniques incorporate the use of gaseous bubbles to increase the visibility of regions of interest during ultrasound imaging. Acoustic droplet vaporization (ADV) requires acoustic waves to decrease local pressure of liquid perfluorocarbon (PFC) below its vapor pressure to trigger its phase transition from liquid to gas. Although promising, this field remained stagnant despite the introduction of novel formulations of low-boiling point (<0° C.) perfluorocarbon (PFC) nanodroplets (NDs) that are superheated at body temperature. This is most likely due to the larger ND dose needed compared to microbubbles (MBs), the inability to vaporize all NDs, the amount of phospholipid surfactant that is insufficient to properly stabilize newly formed MBs that are five times larger, and the spontaneous uncontrolled vaporization of ND that could lead to potential toxicity.

Neat perfluorocarbon (PFC) and emulsions of PFC exhibit exceptional properties such as high thermal stability and chemical inertness owing to the unique characteristics of fluorine (F) and the carbon-fluorine (C—F) linkage—the strongest covalent bond in organic chemistry. PFC in its liquid state is often considered gas-like fluid as it displays low intermolecular cohesiveness due to the low polarizability of F, which results in lower Van der Waals (VdW) interactions between pairs of CF2as opposed to VdW interactions between pairs of CH2. More importantly, PFC have additional interrelated key properties that are critical for clinical translation, including biocompatibility, high purity, low surface tensions, high fluidity as well as high vapor pressure and gas solubility compared to hydrocarbons of similar molecular mass (1). In addition, low molecular weight PFC are rapidly excreted by exhalation for PFC with no side effects.

Owing to their unique ultrasound (US) backscattered signal, single PFC gas-filled microbubbles (MBs) (2, 3) or single MB-labeled cells (4) can be detected in vivo using a clinical US system. This unique US response of PFC gas-filled MBs has prompted the development of a variety of new US contrast agents and has increased the possibility of diagnostic imaging worldwide. However, other than altering ligands on the MBshell to target specific tissues, translatable MB-based discoveries as US contrast agents have been stagnant for the past 2 decades, mostly because their micron size limits their distribution to the intravascular space where they can only interact with the endothelial surface (5, 6), circulating cells (7), or blood clots (8, 9).

Nanoscale particles exhibit several advantages for molecular imaging over MBs. Because of their 100-300 nm size, PFC nanodroplets (NDs) are able to leak into the tumor extracellular space (1). In addition, their larger particle count, greater resilience to US and longer in vivo dwell time are expected to improve targeting efficiency of NDs over MBs to intravascular sites in addition to extravascular sites. However, the key impediment of NDs is their lesser echogenicity and thus lesser US contrast compared to MBs due to their limited reflectivity and compressibility (10), requiring large doses that potentially increase side-effects (11). While their backscatter is indistinguishable from tissues, NDs are able to phase change into MBs through a process known as of acoustic droplet vaporization (ADV) resulting in improved detection by US. ADV was introduced almost two decades ago as a potential tool to induce vascular occlusion by locally triggering local embolization on downstream tissues using micron-size perfluoropentane (PFP) filled droplets (12). However, using micron-size PFP-filled droplets potentially leads to spontaneous droplet vaporization, particularly during injection due to shear stress and cavitation, which is a concern for clinical translation (13). In addition, occlusion was accomplished exclusively in the larger arteries, resulting in undesired non-discriminant downstream embolization and side effects (14-16) or incomplete embolization for tumors still fed from smaller arteries.

While a few groups have successfully vaporized liquid low boiling point droplets of PFP (17), or perfluorobutane (PFB) (18-20) to MBs in animals, their clinical translation and widespread use has been limited by critical efficacy and safety challenges. Firstly, the acoustic energy required to vaporize droplets, particularly NDs, in humans is expected to be higher than the current FDA limit (12) because of the high Laplace pressure inside NDs, the increased interstitial pressure in tumors and the US attenuation in deep tissues (21). Secondly, the required dose of droplets or NDs is high, which potentially leads to side-effects (11). Thirdly, the inability to vaporize all NDs due to tissue attenuation, and uncontrolled spontaneous vaporization of sub-micron droplets at body temperature (13) raises safety concerns as it could lead to unwanted microvascular occlusion. Finally, red blood cell extravasation occurs following ADV-triggered capillary rupture due to initial MB expansion or most likely to the violent MB collapse associated with inertial cavitation at insonation (22). In addition to these safety concerns, MBs formed by ADV might not be stable enough for effective gas embolotherapy as they originate from NDs that are 5 times smaller (23), and thus do not have enough phospholipid surfactants to be properly stabilized. Although the presence of MBs near NDs decreases the ADV threshold (24), the same challenges remain for the clinical translation of ADV.

SUMMARY

Embodiments of the present disclosure relate to the field of medical diagnostics and therapeutics, and more specifically to the development of a new class of imaging and theranostic agents obtained by the combination of perfluorocarbon (PFC) gas filled microbubbles (MBs) and nanobubbles (NBs) with PFC liquid core nanodroplets (NDs). Particular aspects present a novel strategy for improved detection and treatment of diseased tissue. Certain aspects comprise using ultrasound contrast agents (MBs or NBs) to target intravascular and/or extravascular sites, followed by a second injection of non-targeted NDs or NDs targeted to the pre-injected MBs or NBs. In specific aspects, the PFC liquid pool of NDs may then transfer to the MBs or NBs to inflate them. Inflating MBs or NBs may increase ultrasound signal easing detection. However, inflation may be used to speed blood clot dissolution, induce tumor vascular occlusion, aid in gene delivery and transfection, and more.

The inventors have found that NDs act as a PFC and phospholipid reservoirs that transfer to the adjacent NBs or MBs, which triggers their growth. Interestingly, while both NBs and NDs individually exhibit very limited ultrasound signal on B-mode imaging when exposed to low acoustic power, their combination results in a rapid and dramatic increase in signal visible on standard ultrasound imaging. The inventors hypothesize that the low but non-negligible PFC solubility in water and high vapor pressure of PFC allow condensed PFC from the NDs to dissipate and diffuse away into the aqueous phase to reach the NB core and initiate expansion. This innovation is different from the use of NDs as phase change contrast agent (PCCA) because no sound waves are necessary to inflate the NBs or MBs but result from the large vapor pressure in the superheated nanodroplets. Such a driving force does not exist between two microbubbles with the same Laplace pressure.

The inventors have demonstrated rapid NB expansion when mixed with a concentrated solution of fluorescein-labeled NDs (˜10:1 ND/NB ratio) by microscopy and ⋅ultrasound. The inventors showed evidence that the phospholipids from the ND shell were incorporated with the newly formed microbubbles (MBs) by fluorescence microscopy.

Embodiments of the present disclosure also relate to the field of in vitro rare cell isolation. More specific aspects describe a new platform that may improve cell detection sensitivity and isolation using a combination of perfluorocarbon (PFC) gas filled nanobubbles (NBs) or microbubbles (MBs) targeted to any cell surface receptor of interest, and PFC liquid nanodroplets (NDs). Particular aspects include a novel strategy to better detect and isolate cells in vitro using buoyancy. Certain aspects comprise adding targeted NBs or MBs to a cell suspension, followed by the addition of NDs that could be non-targeted or targeted to the MBs or MBs. The liquid PFC from superheated PFC NDs can then transfer to the NBs or MBs to inflate them, increase buoyancy and the floatation of MB-cell construct to ease or speed their recovery using mild or no centrifugation.

Briefly, the present disclosure provides a novel strategy to achieve the benefits of ADV, including improved detection and tumor microvascular embolization, without the need for ultrasound. Because liquid PFC in NDs is at a much higher partial pressure than its gaseous counterpart in microbubbles (MB), the coexistence of ND and MB leads to PFC vaporization and transfer into MB leading to their expansion. The thermodynamic driving force is the large vapor pressure for the nanodroplets, which are highly superheated. Such a driving force does not exist between two microbubbles with the same Laplace pressure. The extent of the MB expansion was quantified by counting and measuring the size of expanded microbubbles with microscopy and flow cytometry. This MB expansion process was applied to expand poorly echogenic nanobubbles (NBs) to larger MBs and resulted in a 4-fold ultrasound signal enhancement after 30 seconds of incubation at 37° C. The inventors have demonstrated that the rate and extent of expansion were affected by the type of PFC used, MB size, length and type of lipid emulsifier that has an effect on both interfacial tension and the ability of bubbles to expand. Finally, MB inflation was demonstrated in vitro in tubing flow system and in two animal models. First, MBs and ND were both injected subcutaneously in the ear of a mouse to demonstrate MB expansion in vivo using intravital microscopy. In a second animal model, MC38 colon cancer cells were implanted in the flank of a mouse and MC38-targeting MBs and NDs were injected intravenously in sequence to demonstrate tumor targeting and MB inflation at the tumor site using microscopy.

Certain embodiments include a method of imaging a region of interest, where the method comprises: providing bubbles to the region of interest; providing nanodroplets to the region of interest; increasing the average bubble diameter of the bubbles while the bubbles are in the region of interest; and exposing the region of interest to an ultrasound stimulus to generate an image of the region of interest.

Certain embodiments include a method to safely occlude tumor microvessels non-invasively and without the need for ultrasound, where the method comprises: providing bubbles to the region of interest, diameter between 200 nm and 10 μm; providing nanodroplets to the region of interest; increasing the average bubble diameter of the bubbles while the bubbles are in the region of interest.

In particular embodiments, providing the nanodroplets to the region of interest is performed subsequent to providing the bubbles to the region of interest. In some embodiments, the bubbles and the nanodroplets are provided to the region of interest in a composition comprising the bubbles and the nanodroplets. In specific embodiments, the average bubble diameter of the bubbles is increased by an order of magnitude when the bubbles are in the region of interest. In certain embodiments, the average bubble diameter of the bubbles is increased by up to two orders of magnitude when the bubbles are in the region of interest. In particular embodiments, the nanodroplets comprise a perfluorocarbon (PFC).

Specific embodiments include a method of increasing bubble size, where the method comprises: providing bubbles to a first region, wherein the bubbles have an average bubble diameter between 200 nm and 10 μm; providing nanodroplets proximal to the bubbles; and increasing the average bubble diameter of the bubbles while the bubbles are in the first region. In certain embodiments, providing the nanodroplets proximal to the bubbles is performed subsequent to providing the bubbles to the first region. In particular embodiments, the bubbles and the nanodroplets are provided in a composition comprising the bubbles and the nanodroplets. In some embodiments, the average bubble diameter of the bubbles is increased by an order of magnitude when the bubbles are in the region of interest.

Particular embodiments include a method of isolating a cell population in blood or media, where the method comprises: providing bubbles to a region of interest, wherein the bubbles are coupled to targeted cell structures within the region of interest; providing nanodroplets to the region of interest; increasing the average bubble diameter of the bubbles and increasing the buoyancy of the bubbles while the bubbles are coupled to the targeted cell structures within the region of interest; and isolating the bubbles and targeted cell structures within the region of interest from non-targeted cell structures within the region of interest.

In certain embodiments, isolating the bubbles and targeted cell structures within the region of interest from non-targeted cell structures within the region of interest comprises floating the bubbles and targeted cell structures within the region of interest. In particular embodiments, providing the nanodroplets to the region of interest is performed subsequent to providing the bubbles to the region of interest. In some embodiments, the bubbles and the nanodroplets are provided to the region of interest in a composition comprising the bubbles and the nanodroplets. In specific embodiments, the average bubble diameter of the bubbles in increased by an order of magnitude when the bubbles are in the region of interest. In certain embodiments, the nanodroplets comprise a perfluorocarbon (PFC).

Any embodiment of any of the present methods, composition, kit, and systems may consist of or consist essentially of—rather than comprise/include/contain/have—the described steps and/or features. Thus, in any of the claims, the term “consisting of” or “consisting essentially of” may be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb.

The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used in conjunction with the word “comprising” in the claims or specification, denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating certain embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

