Selective manipulation of monodisperse targeted microbubbles for multicolor biomarker detection

Abstract

Selective displacement of two or more distinct monodisperse targeted microbubble (tMB) populations presenting unique surface ligands by matching the incoming ultrasound frequency to the size-dependent resonance frequency of each tMB population thereby selecting their respective translations towards a target cell or tissue.

Description

[0001] SELECTIVE MANIPULATION OF MONODISPERSE TARGETED MICROBUBBLES FOR MULTICOLOR BIOMARKER DETECTION

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This International PCT application claims the benefit of and priority to U.S. Provisional Application No. 63 / 662,368, filed June 20, 2024, the specification, claims and drawings of which are incorporated herein by reference in their entirety.

[0004] STATEMENT OF GOVERNMENT INTEREST

[0005] This invention was made with government support under grant numbers R01CA195051, and R01CA239465, awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] The present invention is generally directed to molecular imaging, specifically systems and methods for simultaneous ultrasonic molecular imaging of multiple biomarkers using cloaked targeted microbubbles.

[0008] BACKGROUND

[0009] Current clinically approved contrast enhanced ultrasound imaging provides anatomical and functional (flow) information but lacks phenotypic information characteristic of molecular imaging modalities. Ultrasound molecular imaging (USMI) techniques using targeted ultrasound contrast agent microbubbles (tMBs) were developed to address this shortfall. For example, Phase I clinical trials of USMI using the peptide-bearing microbubble BR55 (Bracco) targeted to VEGF- R2 have been reported for breast and prostate cancers, demonstrating safety and feasibility in detecting tumor angiogenesis. Despite these advances in targeted MB technology, conventional USMI methods remain somewhat cumbersome. For example, using conventional methods, tMBs are injected and provided a 5-10 min dwell time for tMBs to adhere to receptor-bearing vessels while unbound tMBs are cleared from circulation. The slow image acquisition time precludes multiple agents targeting different receptors, as they must be injected in series, thereby substantially prolonging the imaging time. As a result, traditional USMI systems are monochromatic and identify only a single receptor per scan.

[0010] However, there exists a need to image multiple receptors for diagnostic purposes. For example, in cancer therapy, there is a need to diagnose and assess the status of vascular neogenesis and inflammation within the tumor and its periphery, for example in cancer immunotherapy and radiation therapy. The ability to image two or more different receptors, generally referred to herein as multi-color USMI, has general utility in assessing different disease phenotypes, in the same manner as multi-color fluorescence microscopy has revolutionized our understanding of cellular biology.

[0011] With respect to multi-color molecular imaging, the main technical feature to be solved includes the ability to differentiate the signal of each molecular probe from one another. In fluorescence microscopy, this is done using fluorophores with narrow excitation / emission spectra that are sufficiently separate between the two fluorophores, such that each fluorophore can be individually optically addressed through tuning of the excitation wavelength and emission detection filters on the microscope. This is facilitated by the quantum mechanical nature of the interaction of photons with the electron structure of the fluorophore. However, MB acoustics are based on continuum mechanics, and thus tMBs do not exhibit narrow excitation / emission acoustic spectra. For example, the echoes from microbubbles are typically found at the fundamental, subharmonic and ultra-harmonic frequencies of the transmit center frequency, depending on the ambient pressure, bubble size, and shell material. The microbubble acoustic response is a complex function of both the frequency and peak negative pressure of the ultrasound transmit pulse due to bubble size and shell mechanics. As a result, it is difficult to distinguish the echoes between different MB species.

[0012] To address these limitations within the art, as described generally herein, the present disclosure presents a novel system and methods for a multi-color USMI configured to selectively activate differential ligand-receptor binding by using a transmit frequency tuned to unique resonance frequency of differentially sized tMBs.

[0013] SUMMARY OF THE INVENTION

[0014] The technology described herein enables multi-color USMI within a single imaging scan.

[0015] In one embodiment, the present disclosure describes novel systems and methods to facilitate simultaneous ultrasonic molecular imaging of multiple disease biomarkers. Herein Applicants describe systems, methods, and compositions that selectively manipulate cloaked targeted microbubbles (tMBs) via primary acoustic radiation force (Frad). In one aspect, the disclosure provides methods to acoustically manipulate tMBs of varying sizes concurrently and selectively, allowing simultaneous targeting of at least two or more distinct biomarkers. In another aspect, the disclosure describes the novel utilization of Frad to achieve selective displacement of two distinct monodisperse tMB populations flowing simultaneously. In this preferred aspect, selective displacement of tMBs can be accomplished by matching the incoming ultrasound frequency to the size-dependent resonance frequency of each tMB population, thereby optimizing their respective translations towards the target surface continuing one or more biomarkers, such as a target cell population or tissue, such as the vessel wall of a subject. Again, in this aspect the selective displacement of tMBs is consistently applied based on the tMBs specific Frad, despite the tMB populations having matched gas volumes.

[0016] In certain aspects, the resonance frequencies of each tMB size can be selectively applied for the sequential detection of one or more biomarkers in vitro or in vivo. In another aspect, the disclosure provides for selective targeting of two or more distinct tMB populations, engineered to bind, but not limited to, inflammatory or angiogenic biomarkers. As a result, a highly efficient multicolor molecular detection can be achieved, an accomplishment not previously reported in the art using USMI.

[0017] Additionally, the present disclosure includes novel systems, methods, and compositions for producing monodisperse tMBs that can specifically bind inflammatory molecules like P-, E-, or L-selectin, facilitating precise molecular adhesion targeting. This is accomplished by incorporating dibenzocyclooctyl-PEG (DSPE-PEG2000-DBCO) into the MB shell, enabling efficient click-conjugation with custom azide-containing peptide ligands (Azide-IELLQAR) for selectin binding.

[0018] In one embodiment, the present invention provides a system for multi-color ultrasound molecular imaging (USMI). In a preferred aspect, this system of the disclosure includes a solution containing a first monodisperse population of targeted microbubbles (tMBs) having a cloaked first ligand, and a first resonance frequency, and second monodisperse population of tMBs having a cloaked second ligand, and a second resonance frequency, wherein the first and second resonance frequencies are different. In a preferred aspect, the first tMB population is activated by application of an ultrasound radiation force (USRF) at the first resonance frequency which uncloaks the first ligand thereby allowing it to bind to its cognate receptor, such as a biomarker for angiogenesis. Next, the second tMB population is activated by application of an USRF at the second resonance frequency which uncloaks the second ligand thereby allowing it to bind to its cognate receptor, such a biomarker for inflammation. Additional aspects of the present disclosure will be apparent from the claims, specification, and figures provided herein.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figures 1A-C. (A) Shows the monodisperse size distributions of the 1 and 5 pm MBs. (B- C) Frad-selective targeting exhibits maximal attachment at 4 and 7 MHz, for 5 and 1 pm-RGD MBs in an oivPs-coated phantom. Attachment is also maximized at the same frequencies for (C) IELLQAR-MBS targeting P-selectin-coated micro vessel.

[0021] Figure 2A-B. Representative fluorescent micrographs illustrating dual selective targeting of 1- and 5- IELLQAR- and RGD-MBs, labeled with Vybrant DiO (green) and Vybrant Dil (red), respectively, against an (A) avp3- and P-selectin-coated micro vessel phantoms are presented. Quantification of MB attachment when the cocktail was pushed at 4 or 7 MHz to target a B,a) avp3- or B,b) P-selectin-coated micro vessel. The opposite frequency and ligand-bearing tMB were used as controls. Data represents mean ± standard deviation for three independent experiments. * / ) ().05, **p<0.01. Scale bar = 10 m.

[0022] Figure 3. Schematic of exemplary tMB presented in a unform flow and subject to primary acoustic radiation force (Frad) which selectively deflect size-selected MBs towards the vessel when the driving frequency is matched to the MB resonance. The schematic shows an in vitro testing procedure consisting of avp3- and P-selectin-coated hollow microfibers suspended in a tank with an ultrasound phased array transducer to drive the MBs and a fluorescent microscope to observe their targeted adhesion.

[0023] Figure 4A-B. (A) The exposed-ligand architecture (ELA) allows complement activation through C3b binding to the peptide, whereas the buried-ligand architecture (BLA) blocks C3b. (B) High-speed streak image shows oscillation and displacement of ~2 pm diameter microbubble under ultrasound radiation force. BLA also reduces the MB complement activation and immunogenicity.

[0024] Figure 5. Results from in vivo rat xenograft tumor studies showing enhanced imaging of av03 integrin on neo-vessels using BLA-RGD MBs with USRF versus BLA-RAD control. USRF increased adhesion of target BLA-RGD microbubbles, but not nontargeting BLA-RAD controls. Figure 6A-B. (A) Selective USRF displacements of smaller (0.5-1 pm radius) MBs on- resonance (7 MHz) compared to off-resonance (3 MHz). (B) Selective USRF displacements of larger (2-2.5 pm radius) MBs on-resonance (3 MHz) compared to off-resonance (7 MHz).

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] The following detailed description is provided to aid those skilled in the art in practicing the various embodiments of the present disclosure, including all the methods, uses, compositions, etc., described herein. Even so, the following detailed description should not be construed to unduly limit the present disclosure, as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present discoveries. The present disclosure is explained in greater detail below. This disclosure is not intended to be a detailed catalog of all the different ways in which embodiments of this disclosure can be implemented, or all the features that can be added to the instant embodiments. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which variations and additions do not depart from the scope of the instant disclosure. Hence, the following specification is intended to illustrate some embodiments of the disclosure, and not to exhaustively specify all permutations, combinations, and variations thereof.

[0027] The present invention provides for multi-color ultrasound molecular imaging (USMI). In a preferred embodiment, the system described herein utilized discrete endangered microbubble, and specifically targeted microbubbles (tMBs) that have been engineered to be decorated with biomolecular ligands that bind specifically to biomarkers on various cell types. In this embodiment, the ligands are cloaked by a buried-ligand architecture (BLA), which is described in U.S. Application No. 17 / 044,266, filed April 3, 2024, the specification, claims and drawings related to BLA being incorporated herein by reference.

[0028] As noted above, engineered tMBs can be selectively activated for ligand-receptor binding using a transmit frequency tuned to its unique resonance frequency. The method relies on two novel design features: First, tMBs can be pushed in the direction of the propagating acoustic wave through the phenomenon of the primary ultrasound radiation force (USRF). The displacement is maximal at the MB resonance frequency. Second, the BLA on the MB surface allows the targeting ligand to be cloaked and concealed from the receptor until the MB is activated by the USRF pulse (See Fig. 4). Targeted BLA-MBs bind when driven by USRF at their resonance frequency (as described below). Thus, Applicants can differentiate two or more bubble species by manipulating the transmitted USRF pulse rather than by analyzing the received echo. In this manner, by “pushing” two different MB species at their unique resonance frequencies, Applicants can distinguish the two, even if they have a similar acoustic emission. To enable this approach, the present technology includes the design and use of engineered MBs with unique resonances.

[0029] In one embodiment, the disclosure provides asystem for multi-color ultrasound molecular imaging (USMI). In this preferred embodiment, the system includes a solution containing a first monodisperse population of targeted microbubbles (tMBs) having a cloaked first ligand, and a first resonance frequency, and second monodisperse population of tMBs having a cloaked second ligand, and a second resonance frequency, wherein the first and second first resonance frequencies are different. Notably, in different embodiments, the multi-color ultrasound molecular imaging (USMI) can include a solution having a plurality of monodisperse populations of targeted microbubbles (tMBs), each having a different cloaked ligand, and a unique resonance frequency. As such, the disclosure herein explicitly describes a multi-color ultrasound molecular imaging (USMI) system that can detect and image two or more tMBs populations.

[0030] The solution continuing two or more tMBs populations can be contacted with a target, which can include an in vitro or in vivo target, and preferably a target cell population of cells or tissue in a human subject. In certain embodiments, a therapeutically effective amount of the solution can be administered, for example, intravenously to a subject in need thereof.

[0031] The two or more tMBs populations can further be activated by the application of differential ultrasound radiation force (USRF). Specifically, in a preferred embodiment an ultrasound radiation force (USRF) at the first resonance frequency is applied to the tMB populations which activates the first tMBs thereby uncloaking the first ligand allowing it to bind to its cognate receptor. Next, a second USRF at the second resonance frequency is applied to the tMB populations which activates the second tMBs thereby uncloaking the second ligand allowing it to bind to its cognate receptor.

[0032] As used herein, the term microbubble (MB) refers to vesicles which are generally characterized by the presence of one or more membranes or walls or shells surrounding an internal void that is filled with a gas or precursor thereto. In some embodiments, the microbubbles comprise one or more lipids. The term lipids include agents exhibiting amphipathic characteristics causing it to spontaneously adopt an organized structure in water wherein the hydrophobic portion of the molecule is sequestered away from the aqueous phase. As described below, a microbubble may also contain target ligands, or other therapeutic agents, and / or other functional molecules. As further used herein, a targeted MB or tMB includes a MB that has been engineered to display a ligand that binds with its cognate receptor, and in a preferred embodiment is cloaked by a BLA on the surface of the MB.

[0033] In some embodiments, the tMBs comprise one or more gases inside a lipid shell. In some embodiments, the lipid shell comprises one or more polymerizable lipids. In some embodiments, the invention provides gas-fdled microbubbles substantially devoid of liquid in the interior. In some embodiments, the microbubbles are at least about 90% devoid of liquid, at least about 95% devoid of liquid, or about 100% devoid of liquid. The tMBs included in this description may contain any combination of gases suitable for the diagnostic or therapeutic method desired. For example, various biocompatible gases such as air, nitrogen, carbon dioxide, oxygen, argon, xenon, neon, helium, and / or combinations thereof may be employed. Other suitable gases will be apparent to those skilled in the art, the gas chosen being only limited by the proposed application of the microbubbles. In some embodiments, the microbubbles contain gases with high molecular weight and size. In some embodiments, the microbubbles contain fluorinated gases, fluorocarbon gases, and perfluorocarbon gases. In some embodiments, the perfluorocarbon gases include perfluoropropane, perfluorobutane, perfluorocyclobutane, perfluoromethane, perfluoroethane and perfluoropentane, especially perfluoropropane. In some embodiments, the perfluorocarbon gases have less than six carbon atoms. Gases that may be incorporated into the microbubbles include but are not limited to: SF6, CF4, C2F6, C3F6, C3F8 C4F6, C4F8, C4F10, C5F10, C5F12, C6F12, (1- trifluoromethyl), propane (2-trifluoromethyl)-l,l,l,3,3,3 hexafluoro, and butane (2- trifluoromethyl)-l,l,l,3,3,3,4,4,4 nonafluor, air, oxygen, nitrogen, carbon dioxide, noble gases, vaporized therapeutic compounds, and mixtures thereof. The halogenated versions of hydrocarbons, where other halogens are used to replace F (e.g., Cl, Br, I), would also be useful.

[0034] In some embodiments, tMBs containing gases with high molecular weight and size are used for ultrasound imaging purposes. Without intending to be limited to any theory, gases with high molecular weight and size enhance ultrasound scattering. In some embodiments, innocuous, low boiling liquids which vaporize at body temperature or by the action of remotely applied energy pulses, like CeFu, are also usable as a volatile confinable microbubble component in the present invention. In some embodiments, the confined gases may be at atmospheric pressure or under pressures higher or lower than atmospheric; for instance, the confined gases may be at pressures equal to the hydrostatic pressure of the carrier liquid holding the gas filled microspheres.

[0035] The resonance frequency of the monodisperse population of tMBs can further be modulated, for example by changing the composition of the MB components. In one embodiment, one or both tMBs can comprise different phospholipids, such as l,2-dipalmitoyl-sn-glycero-3- phosphocholine, l,2-distearoyl-sn-glycero-3-phosphocholine, l,2-diarachidoyl-sn-glycero-3- phosphocholine, l,2-dibehenoyl-sn-glycero-3 -phosphocholine, l,2-dilignoceroyl-sn-glycero-3- phosphocholine, or a combination of the same, or a combination of the same with different acyl chains, such as two chains with different carbon acyl chain lengths, or an unsaturated acyl chain, or a combination with mixed acyl chains, or a combination with other phospholipid headgroups such as phosphate, phosphoethanolamine, phospho-(l'-rac-glycerol), phospho-L-serine, cardiolipin, bis(monoacylglycero)phosphate, ether lipids, oxidized lipids, sterol-modified lipids, inverted headgroups, can modulate the elasticity of the tMB structure resulting on a different resonance frequency.

[0036] In some embodiments, the tMBs of the invention comprise a conjugated target ligand or conjugated ligand - the terms being generally interchangeable. A conjugated ligand may include a molecule, macromolecule, or molecular assembly which binds specifically to a biological target.

[0037] In some embodiments, a ligand may be one or more molecules which specifically bind to receptors, moieties, or markers found on vascular or cancerous cells. In some embodiments, targeting agents are molecules which specifically bind to receptors, moieties or markers found on cells of angiogenic neovasculature or receptors, moieties or markers associated with tumor vasculature. The receptors, moieties or markers associated with tumor vasculature can be expressed on cells of vessels which penetrate or are located within the tumor, or which are confined to the inner or outer periphery of the tumor.

[0038] In one preferred embodiment, a ligand that may be conjugated to a MBs, forming a tMB may include a molecule, macromolecule, or molecular assembly which may be coupled to a MB, and preferably a cloaked microbubble, through a Cu-free click chemistry strain-promoted [3+2] azide-alkyne cycloaddition (SPAAC) “click” chemistry mechanisms. In another preferred embodiment, a conjugated ligand may include a peptide which may be coupled to a MB, forming a tMB and preferably a cloaked microbubble, through a SPAAC click chemistry mechanism.

[0039] In some embodiments, a tMB shell comprises polyethylene glycol) (PEG) polymers tethered to a lipid monolayer. Without intending to be limited to any theory, the PEG polymers tethered to the lipids provide colloidal stability against aggregation and steric effects to block binding of opsonizing plasma proteins, which leads to increased lifetime in blood circulation. In some embodiments, the present invention describes microbubbles conjugated with one or more target ligands. In one preferred embodiment, such conjugated ligands may include PEG-lipid tethered ligands and may be part of a monodisperse population of microbubbles.

[0040] In one embodiment, the invention includes the generation and application of bimodal -brush tMBs, and preferably a hydrated polymer brush architecture, which may include a bimodal PEGylated surface architecture. In this preferred embodiment, the surface of a tMB may be modified with a polymer, such as, for example, PEG. This PEG layer may be bimodal in nature wherein the first population of PEG polymers is of a discrete length, and a second population of PEG polymers is of a different discrete length. One or more ligands may be conjugated with a polymer, such as a PEG polymer that is tethered to a microbubble lipid monolayer. In a preferred embodiment, one or more ligands may be conjugated with a polymer, such as a PEG polymer, that is shorter in length than a second polymer, which may also be of the same, equivalent or different material.

[0041] In one preferred embodiment, a bimodal-brush microbubble may include a shorter PEG polymer tether, in this instance being of -2000 Da molecular weight that may tether the target ligand to an anchoring lipid monolayer. Notably, the -2000 Da PEG chains (PEG2000) extend approximately 4 nm above the microbubble’s lipid monolayer.

[0042] The bimodal-brush microbubble may further include a longer polymer, such as a PEG polymer, that may surround the tethered ligand. In this this embodiment demonstrated in figure 1, a tethered ligand may be surrounded by longer PEG chains of -5000 Da that, in order to maximize entropy, stratify into an overbrush that conceals the ligand from blood components and other unwanted chemical or molecular reactions. In this manner, the tethered ligand is “cloaked” by the larger polymers that form the overbrush. Notably, the -5000 Da PEG chains (PEG5000) extend approximately 9 nm above the surface of the microbubble’s lipid monolayer. In one embodiment, the said first and second ligands, or a third or fourth etc... are functionalized to form azido-functionalized ligands. As noted herein, azido-functionalized ligands are conjugated to the polymer tethers on their respective tMBs through a click chemistry reaction to form a bioconjugate polymer, and preferably a 1,2, 3 -triazole linked bioconjugate formed via strain-promoted [3+2] azide-alkyne cycloaddition (SPAAC). In a preferred embodiment, the polymer tether of the disclosure can include: l,2-distearyol-sn-glycero-3-phosphoethanolamine- N-[methoxy (polyethylene glycol) 5000] (DSPE-PEG5000); and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[dibenzocyclooctyl (polyethylene glycol) 2000] (DSPE-PEG2000- DBCO).

[0043] As noted above, a cloaked ligand, preferably a ligand that may bind to one or more receptors in a host, can be transiently revealed by the application of ultrasound through the mechanisms of acoustic radiation force displacement of the cloaked microbubble against the receptor-bearing surface and accompanying surface oscillation of the shell. In one embodiment, the frequency of the ultrasound required to transiently uncover the conjugated ligand from a cloaked microbubble may preferably vary from about 3 to 8 MHz, and more preferably between 4 and 7 MHz. In a preferred embodiment, a USRF pulse of approximately 4 MHz will uncloak the ligand of the first tMB, while a USRF pulse of approximately 7 MHz will uncloak the ligand of the second tMB. Notably, the optimal USRF pulse for any tMB can be determined by the size and elasticity among other physical characteristics, as well as pulse length, number of pulse cycles, pulse repetition frequency (PRF), and peak-negative pressure (PNP).

[0044] In another exemplary embodiment, ligands, and in particular azido-functionalized ligands may be conjugated to bimodal-brush microbubbles via SPAAC click chemistry. In one preferred embodiment, such conjugated ligand may include one or more therapeutic molecules, such as small peptides or other inhibitors that may be delivered to a discrete tissue or organ to treat and / or diagnose a disease condition. In one exemplary embodiment, azido-functionalized antagonists for biomarkers aVp3 integrin (cRGD) and p-selectin proteins may be conjugated to bimodal-brush tMB via SPAAC click conjugation. As demonstrated below, in one embodiment, ligand conjugation to a microbubble may be validated by epi fluorescent microscopy, flow cytometry, and Fourier-transform infrared spectroscopy. In yet another embodiment, the sterility of the cloaked microbubble may also be validated on such novel cloaked microbubbles by bacterial culture and endotoxin analysis. A therapeutically effective amount of two or more tMBs having a select conjugated ligand may be administered to a host, such as an animal, and preferably a mammal or human patient. In this embodiment, a host may receive an initial, a repeated, or an escalating dose and may experience no pathologic changes in physical examination, complete blood count, and serum biochemistry profde or coagulation panel. Notably, the two or more tMBs can be administered simultaneously, or sequentially.

[0045] In another embodiment, a therapeutically effective amount of two or more tMBs having unique conjugated ligands, and preferably a therapeutic and / or diagnostic ligand may be delivered to a host, and more specifically a host experiencing a disease condition. In this preferred embodiment, a therapeutically effective amount of two or more tMBs having unique conjugated ligands that may be delivered to a cancer cell or tumor. The cloaked ligand may be introduced to the cell or tumor by the application of ultrasound through the mechanisms of acoustic radiation force displacement of the microbubble against the receptor-bearing surface and accompanying surface oscillation of the shell as described herein.

[0046] In another embodiment, a therapeutically effective amount of differentially sized tMBs having unique conjugated ligands that can bind to unique biomarkers. A biomarker may be associated with a disease condition, for example cancer, as well as a physiological or disease- related process, such as angiogenesis. In one preferred embodiment, a SPAAC click chemistry process may be utilized to generate at least two monodisperse populations of tMBs having or more peptide ligands, such as anti-angiogenesis or inflammation ligands. Specifically, a SPAAC click chemistry process may be utilized generate at least two monodisperse population of tMBs having a Cyclo Arg-Gly-Asp-D-Phe-Cys (RGD) and / or Azide-IELLQARC-OH (lELLQAR)-targeted microbubble against aVp3 integrin and p-selectin, which are known biomarkers expressed on the lumen of neovessels. In this embodiment, the binding of the conjugated peptides can allow enhanced visualization and detection of tumor cells in a host through the improved ultrasound visualizations allowed by the presence of the tMBs at the site of the tumor or cancerous cell. Such enhanced visualization may be accomplished in vivo.

[0047] In some embodiments, the invention provides compositions and methods for the diagnosis and / or treatment of a condition. In some embodiments, at least two monodisperse populations of tMBs may be used with ultrasound, MRI, or other imaging techniques. Ultrasound visualization of tMBs having one or more conjugated ligands may also be used to identify and locate solid tumors, angiogenesis or inflammation activity associated with a disease state such as cancer.

[0048] As used herein, the term “ligand” means any small molecular weight (<5000 Da) molecule that may be functionalized with an azido group and conjugated to the surface of a microbubble and cloaked for molecular imaging.

[0049] “Microbubbles,” “bubbles” “targeted microbubbles,” and their abbreviations are generally used interchangeably herein to refer to a gas core surrounded by a lipid membrane, which can be either a monolayer or a bilayer and wherein the lipid membrane can contain one or more lipids and one or more stabilizing agents. A microbubble may also mean a liposome and / or a micelle. As used herein, a tMB “population” refers to a size-isolated MB (SIMB).

[0050] A “targeted microbubbles,” or a “conjugated microbubble” means a microbubble that is coupled with at least one ligand. A “cloaked microbubble” means a microbubble having buried- ligand architecture (BLA).

[0051] As used herein, the term “ligand” means any small molecular weight (<5000 Da) molecule that may be functionalized with an azido group and conjugated to the surface of a microbubble and cloaked for molecular imaging. In a preferred embodiment, a “target ligand,” or “ligand” can include a molecule or compound that can be chemically modified by addition of an azide or alkynyl group, such as small molecules, natural products, or biomolecules (e.g., peptides or proteins), such as exemplary ligands cRGD and A7R. A “target ligand,” or “ligand” can further mean a molecule or compound that may be conjugated through a SPAAC click chemistry mechanism to a microbubble. Example ligands may include, but not be limited to, drug, a chemical, antibodies, ligands, proteins, peptides, carbohydrates, vitamins, nucleic acids, or combinations thereof.

[0052] A “bioconjugate” or “bioconjugate ligand” means a ligand conjugated with a polymer tether. As used herein, the general term biological marker (“receptors” “biomarker” or “marker” “moi eties”) is a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacological responses to therapeutic interventions, consistent with NIH Biomarker Definitions Working Group (1998). Markers can also include patterns or ensembles of characteristics indicative of particular biological processes. Biomarker measurements can increase or decrease to indicate a particular biological event or process. In addition, if the biomarker measurement typically changes in the absence of a particular biological process, a constant measurement can indicate the occurrence of that process.

[0053] A target molecules or markers, and their corresponding interaction with a cloaked microbubble conjugated with a ligand, may be used for diagnostic and prognostic purposes, as well as for therapeutic, drug screening and patient stratification purposes (e.g., to group patients into a number of “subsets” for evaluation), as well as other purposes described herein, subject

[0054] The present invention includes all compositions and methods relying on correlations between the reported markers, cloaked tMBs, and the therapeutic effect or diagnosis of cells, tissues, or organs. Such methods include methods for determining whether a subject is predicted to respond to administration of a therapy, as well as methods for assessing the efficacy of a therapy. Additional methods may include determining whether a subject is predicted to respond to administration of a therapy. Further included are methods for improving the efficacy of a therapy, such as a cancer therapy, by administering to a subject a therapeutically effective amount of two or more populations of tMBs, each having a unique ligand and resonance frequency that binds to, alters the activity of two or more biomarker, such as an markers such as integrin aV03, of p- selection. In this context, the term “therapeutically effective” is to be understood broadly to include an amount to enable the multi-color ultrasound molecular imaging (USMI) of the disclosure. In one example, a “therapeutically effective amount” means an amount effective to produce a detectable physiological effect, such as two or more ligands binding to their cognate markers in vivo or in vitro or enhancing ultrasound imaging and the like.

[0055] In one embodiment, two or more populations of tMBs can be administered via a pharmaceutical composition. As used herein, “pharmaceutical compositions” are compositions that include an amount (for example, a unit dosage) of the disclosed compound(s) together with one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents, and / or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (19th Edition). Pharmaceutical formulations and delivery systems appropriate for the compositions and methods of the disclosure are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20. sup. th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18.sup.th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12.sup.th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 1 l.sup.th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

[0056] Pharmaceutical compositions / formulations are useful for administration to a subject, in vivo or ex vivo. Pharmaceutical compositions and formulations include carriers or excipients for administration to a subject. As used herein the terms “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically compatible formulation, gaseous, liquid, or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery, or contact. As used herein, a “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered composition of the disclosure. The pharmaceutically acceptable carrier may comprise any conventional pharmaceutical carrier or excipient. The choice of carrier and / or excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the carrier or excipient on solubility and stability, and the nature of the dosage form. The term further pertains to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, “Handbook of Pharmaceutical Additives,” 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, N.Y., USA), “Remington's Pharmaceutical Sciences”, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and “Handbook of Pharmaceutical Excipients”, 2nd edition, 1994. Pharmaceutical compositions can be formulated to be compatible with a particular route of administration. Thus, pharmaceutical compositions include carriers (excipients, diluents, vehicles, or filling agents) suitable for administration to any cell, tissue, or organ, in vivo, ex vivo (e.g., tissue or organ transplant) or in vitro, by various routes and delivery, locally, regionally, or systemically. In a preferred embodiment, microbubbles are administered intravenously.

[0057] The target ligands and cloaked microbubble compositions of the invention are useful for determining if a therapy, such as chemotherapy or radiation, may be an effective treatment for cancer or other disease conditions. The target ligands and cloaked microbubble compositions of the invention are useful for predicting the outcome or determining the effectiveness of therapy in multiple cancer types.

[0058] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to.” The term “or” is used herein to mean, and is used interchangeably with, the term “and / or,” unless context clearly indicates otherwise.

[0059] The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

[0060] EXAMPLES

[0061] Example 1: Ultrasound radiation force (USRF) “pushes” targeted MBs and increases their adhesion.

[0062] As noted above, ultrasound radiation force (USRF) “pushes” targeted MBs and increases their adhesion. When excited in an acoustic field, a bubble can travel in the direction of the propagating acoustic wave (Fig. 4B; ultrasound applied from above). The acoustic force that accelerates the bubble is called the primary ultrasound radiation force (USRF, also known as the primary Bjerknes force). The effect is maximal at the bubble resonance frequency. This force can be used to move the MB from the vessel lumen to the wall and enhance ligand-receptor interactions with the target endothelium, or other cell types.

[0063] Example 2: USRF activates BLA-MBs for adhesion to receptor-bearing vessels.

[0064] USRF enhances the adhesion of non-protected molecular imaging agents, as well as activating Applicant’s buried-ligand architecture MBs. The mechanism involves volumetric (and hence dilatational) oscillations that transiently remove the PEG overbrush shielding for a brief period in each acoustic cycle, allowing exposure of the ligand (Fig. 4B). This ligand-revealing (uncloaking) mechanism is superimposed with the forward momentum of the microbubble against an exemplary endothelium cell by USRF. BLA-MBs do not bind appreciably without being activated by USRF. Thus, the combination of USRF and BLA-MBs provides on-demand (ultrasound-gated) binding to the target receptor-bearing surface. As shown in Figure 5 and described further below, Applicant have demonstrated this effect in vivo (Fig. 2), namely BLA- MBs with cyclic-RGD were shown to adhere significantly more to avP? integrin-bearing neovessels in rat angiogenic xenograft tumors when stimulated by USRF (+USRF) compared to passive adhesion (-USRF), and compared to the nontargeting cyclic-RAD peptide.

[0065] Example 3: Microbubble resonance fn.

[0066] For the low USRF pulse pressures to be used in this project (~50 kPa PNP), the microbubble resonance frequency is given by equation where Rois the MB resting radius, is the effective MB shell elasticity, p is the density of the medium (water), and a is a constant that depends on the ambient pressure (~1 atm) and specific heat ratio of the microbubble internal gas. The two parameters available to tune the MB resonance are its size (Ro) and its shell elasticity ( / ). Using a multi -gated spectral doppler technique, we demonstrated selective on-resonance USRF displacements of smaller MBs (1-2 pm diameter) at higher frequency (7 MHz) and of larger MBs (4-5 pm diameter) at lower frequency (3 MHz) (Fig. 6). Applicants have demonstrated the use of two different MB sizes for selective binding in vitro. Example 4: MB size can be used for USRF- and LR-selective binding to two different receptors in vitro.

[0067] Applicants have demonstrated that USRF can be used to selectively bind different MB species with unique resonance frequencies to receptor-bearing surfaces in vitro. Using the results shown in Fig. 6 for selective USRF displacements based on MB size, Applicants designed two MB species: (1) BLA-IELLQAR MBs targeted to p-selectin are 1-pm diameter and resonate at 7 MHz; (2) BLA-RGD MBs targeted to integrin are 5-pm diameter and resonate at 4 MHz. As detailed below, on tubes coated with avPs integrin, appreciable adhesion was only observed for BLA-RGD MBs driven on-resonance (4 MHz). (Fig. 2): On a p-selectin coated tube, only BLA- IELLQAR MBs driven on-resonance (7 MHz) adhered. (Fig. 2): These results show selective targeting using a combination of USRF tuned to the MB resonance frequency and specific ligandreceptor (LR) interactions.

[0068] Example 5: In vitro Acoustic Radiation Force- Selective Microbubble Targeting Ultrasound Molecular Imaging.

[0069] In one embodiment, tMBs have a mean volume-weighted diameter of 1.6 ± 0.3 and 4.9 ± 0.9 pm, the ± generally describing the approximate size herein, with minimal overlap between the size distributions (Fig. 1 A). Frad-assisted targeting showed that 1 pm RGD-MBs exhibited maximal attachment at 7 MHz (1360 ± 242 MBs / mm2), while 5 pm RGD-MBs had increased binding at 4 MHz (1337 ± 296 MBs / mm2). Both sizes exhibited similar RGD-MBs attachment (Fig. IB). In contrast, 1 pm lELLQAR-MBs displayed significantly greater attachment (437 ± 86 MBs / mm2) compared to 5 pm lELLQAR-MBs (171 ± 44 MBs / mm2) when driven at their respective resonance frequencies (Fig. 1C). When the tMB cocktail was infused and pushed at 4 MHz to target an av03- coated micro vessel, the 5 pm-RGD MBs displayed the highest attachment at 507±124 MBs / mm2, in contrast to the nonspecific adhesion of the 1 pm-IELLQAR MBs of 20±35 MBs / mm2(Fig. 2B,a). Conversely, when the tMB cocktail targeted at P-selectin-coated phantom and pushed at 7 MHz, maximal adhesion was observed for 1 pm-IELLQAR MBs, reaching 398±155 MBs / mm2, while attachment was notably reduced for 5 pm-RGD MBs at 2±3 MBs / mm2(Fig. 2B,b). As such, Applicants demonstrate for the first time the Frad-dual selective targeting of two different ligandbearing MBs (RGD or IELLQAR peptides) of two sizes (1 vs. 5 pm) targeting avp3 and P-selectin receptors.

[0070] Example 6: Materials and Methods.

[0071] Fluorescently labeled 1 or 5 pm diameter size-isolated MBs with a buried-ligand architecture (DBPC: DSPE-PEGsooo: DSPE-PEG2000-PDP / DBCO) were functionalized with RGD and IELLQAR peptides, as well as RAD and no peptide as controls, respectively. The size distribution of the MB populations was characterized using a Multisizer 3 Coulter Counter. The tMBs were pumped individually through an av3- or P-selectin-coated micro vessel (200 pm ID) at a wall shear stress of 3.5 dyn / cm2. The (pMB was matched at 0.05 pL / mL for both MB sizes. tMBs were acoustically manipulated using Frad at their resonance frequency (7 MHz for 47 cycles for 1 pm MBs, and 4 MHz for 27 cycles for 5 pm MBs; 6.7 ps pulse length, 0.15 MHz PRF, 133 kPa PNP). Then, tMBs of both sizes were simultaneously pumped in a cocktail manner and selectively displaced using Frad at their resonance frequencies against 0^3- or P-selectin-coated micro vessels. After Frad-assisted targeting, z-stack microscopic fluorescent images of 10 different sections of the phantom were captured and analyzed to quantify specific tMB attachment per mm2. Specific interactions were determined by subtracting attached RAD-MBs from RGD-MBs and MBs with no ligand from IELLQAR-MBS.

[0072] Saturated diacyl phosphocholine (PC) lipids can be used to stabilize MBs, PC is a neutral, zwitterionic lipid that is abundant in cell membranes and has the necessary biophysical properties to pack as cylinders hexagonally in the lipid monolayer shell to minimize C3b binding and complement activation. Lipids below their main phase transition temperature are optimal to stabilize MBs colloidally. Increasing acyl chain length increases intermolecular cohesion and therefore increases MB stability and elasticity ( / ). As noted above, manipulating MB elasticity (X) and resonance ( / 0) was done with DSPC (Cl 8:0) (Fig. 8). In additioanl embodiment, MCs can contain core lipid components having shorter and longer acyl chains, such as DPPC (Cl 6:0) and DBPC (C22:0), respectively.

[0073] For MB formation, PEGylated lipid is used for the bimodal brush used in the BLA, which faciltates USRF-activatable tMBs. The PEG brush architecture optimally includes PEG concentratiosn to be sufficiently high such that the grafting distances is less than the Flory radius. In one embodiment, a tMB can include 5 mol% PEG5000 (overbrush) and 5 mol% PEG2000- ligand (ligand tether), for a total of 10 mol% PEG. This PEG can be fixes to DSPE-PEG, as PEG- lipid is well studied, widely available and known to perform well biologically in MB and liposome applications. Examplary PEG-lipids that can l,2-distearyol-sn-glycero-3-phosphoethanolamine- N-[methoxy (polyethylene glycol) 5000] (DSPE-PEG5000), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[PDP (polyethylene glycol) 2000] (DSPE-PEG2000-PDP) and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[dibenzocyclooctyl (polyethylene glycol) 2000] (DSPE-PEG2000-DBCG) can be incorporated into the tMBs structures.

[0074] Size-isolated MBs (SIMBs) containing perfluorobutane are produced by sonication and size-isolated to 2-pm diameter with differential centrifugation. Size distribution can be measured by a Coulter Multi sizer 4e with a 30-pm aperture.

[0075] Microfluidic MBs (MMBs) with perfluorobutane cores can be produced by microfluidics to 2-pm diameter, as previously described. Size distribution can be measured by a Coulter Multisizer 4e with a 30-pm aperture. This is the method used to generate the MBs shown in Fig. 7. Colloidal stability can be assessed by measuring the MB concentration and size distribution by a Coulter Multisizer directly after formulation and then at days 1, 7, 14 and 28 after refrigerated storage in a sealed and capped serum vial (1.0 at 5 * 109MB / mL) with a perfluorobutane headspace. MBs can be resuspended prior to each measurement. T

[0076] Measurements of the MB resonance frequency can be taken from attenuation measurements using the method employed to generate Fig. 7. In one embodiment, engineered SIMBs can be functionalized with either: m f =7 MHZor

[0077] 3) RDAADn: f7oU= 7 MHZ ( ,cont .ronl).

[0078] (Cyclo Arg-Gly-Asp-D-Phe-Cys (RGD) and cyclo Arg-Ala- Asp-D-Phe-Cys (RAD)). The RGD or RAD peptides are dissolved in a 1-mL sterile 2% acetic acid solution to a concentration of 1 mg / mL. The RGD or RAD peptide is then mixed with the SIMB comprising DSPE-PEG2000- PDP. The IELLQAR peptide (Azide-IELLQ ARC-OH) is dissolved in a 1-mL solution comprising acetonitrile and sterile deionized water in a 2: 1 ratio. The IELLQAR peptide is then mixed with SIMB comprising DSPE-PEG2000-DBCO. Each peptide is conjugated at a 15-fold molar excess relative to the PDP or DBCO groups. The conjugation reaction takes place with 250 pL of the stock MBs (concentrations 1-10 x 109MBs / mL) for 1 h at room temperature under gentle agitation. Following incubation, the SIMB solutions are washed via centrifugation at 200*g for 5 min to eliminate excess unconjugated peptides. The prepared SIMBs were then stored at 4°C for future use. As additional controls, SIMBs without peptides (i.e., those with functional PDP or DBCO groups but lacking bound peptide addition) can be investigated.

[0079] Following conjugation, the size and concentration of SIMBs are directly visualized by brightfield microscopy and measured by the Multisizer. The efficiency of ligand binding to the SIMB surface is assessed through a BCA protein quantification assay. This method relies on a colorimetric reaction of cysteines in peptides: RGD, RAD, and IELLQAR. Standard curves are prepared for each peptide by serially diluting concentrations from 0 to 50 pg / mL. Subsequently, 100 pL of each RGD-, RAD- or IELLQAR-SIMB sample undergoes ultrasonic disruption using a Branson 3510 ultrasonic. From each disrupted sample, 25 pL is pipetted into three wells of a 96- well clear flat-bottom microplate, incubated at 60°C for 30 min, and then absorbance is measured at 562 nm using the Synergy Hl Microplate Reader (BioTek). To account for background interference, PBS and no-ligand-MBs are employed as blank and control, respectively. Finally, the binding efficiency was calculated as:

[0080] Binding efficiency (%) =PePtlde detected( s) x 100 JJ\ Peptide added (pg)

[0081] Molecular density is determined by calculating the number of molecules per SIMB and per unit of surface area. This procedure is repeated with three independently prepared SIMB batches for each peptide.

[0082] Active recombinant av03- or p-selectin-coated microvessel phantoms can be made using microdialysis cellulose hollow fibers (200 pm I.D., 216 pm O.D., MWCO: 13 kD. These fibers are cut into 1-inch lengths and employed as the microvessel flow phantoms. First, the micro vessels are manually flushed with ethyl alcohol followed by washing with deionized water. To prevent nonspecific adhesion, the micro vessels are blocked by filling them with casein and then immersed in a casein solution (1 mb). The phantoms are left overnight at 4°C. Subsequently, the micro vessels are rinsed with PBS, filled with avp3 integrin or p-selectin solution at a concentration of 200 pg / mL and incubated for 2h at 4°C. Finally, the phantoms are rinsed with PBS to eliminate unbound avP3 integrins, followed by a slow flush with casein and a final wash with PBS. For ultrasound imaging experiments, a wall-less tissue phantom is made using protocols described in the art, and the same receptor coating protocol is followed as for the microvessel phantoms.

[0083] The tank-on-a-microscope method can assess the selectivity of binding of different tMB species to aVP3 integrin and p-selectin on cellulose micro-dialysis tubes at 3 vs 7 MHz, using the USRF pushing pulses described herein. The avP3- or O-selectin-coated microvessels are immersed and placed horizontally in a water tank that allows simultaneous USRF exposure and microscope observation. An Olympus inverted microscope with a 100x water immersion objective interfaced with a Dhyana 400D camera is used to observe, image and record flowing and attached SIMBs inside the phantom. An ultrasound transducer is immersed in the water tank and positioned parallel to the microvessel at a distance of 5 cm. A clinical, linear array ultrasound probe (LA332, Esaote) with 144 elements covered by a silicone lens with an elevational focus of 23 mm, a 0.245 mm pitch, a 4.6 MHz center frequency and 100% (-6 dB) bandwidth is used to apply USRF pulses. The probe is driven by the 64-channel Ultrasound Advanced Open Platform (ULA-OP, X-Phase). The temperature of the water bath is set to 37°C. During the experiments, the SIMB suspensions are diluted in a 20 mL vial containing 2 mL of a 1% BSA-PBS solution. The gas volume fraction (cpMB) is matched at 0.05 pL / mL for both SIMBs, with concentrations adjusted accordingly. The mixtures are gently stirred and withdrawn using a syringe pump at a shear stress of 3.5 dyn / cn , ensuring a laminar flow and allowing the solution to flow from the vial through the tubing. SIMBs are then pushed towards the vessel wall by administering USRF pulses continuously for a duration of 4 min. These pulses are delivered at frequencies of 3 or 7 MHz, including a pulse length of 10 ps by varying the number of cycles, a 0.1 MHz pulse repetition frequency (PRF), and a peaknegative pressure (PNP) of 50 kPa. The incoming USRF beam is matched to the resonance frequency at which the SIMB species experience the greatest displacement. Following cessation of USRF, a series of z-stack images (10-15 images, 13-20 pm step size) are captured for 10 different sections of the phantom. These images are obtained using both brightfield microscopy and the fluorescence channel for Vybrant Dil or Vybrant DiO (excitation / emission spectra of 549 / 565 or 483 / 501 nm, respectively). Images with merged fluorescent channels are postprocessed to visualize SIMB attachment. All images are processed utilizing Imaged software; the multi-point tool and particle analyzer are employed to count the total number of fluorescently labeled SIMBs attached to the vessel wall. The average number of SIMBs per vessel wall can be determined through the above method and can be expressed in terms of the number of tMBs attached per surface area (mm2). Finally, both SIMB species are simultaneously pumped in a cocktail manner and selectively displaced using USRF at their resonance frequencies against either avP3- or p-selectin-coated micro vessels. Three distinct av03- and p-selectin-coated micro vessels can be analyzed per condition.

[0084] In addition to the microscopy evaluation described above, selectivity of binding of tMB species to aVp3 integrin and p-selectin can be accessed in a tubeless phantom at 3, 5 and 7 MHz using the fundamental mode and CPS imaging pulses developed as described above. In this embodiment, selective targeting of the two MB species at 3 vs 7 MHz can be shown by ultrasound imaging of adherent MBs under physiologic shear flow.

[0085] All data can be expressed as the mean ± standard deviation. Differences between experimental groups can be assessed using unpaired t-tests and one-way ANOVA and Tukey test for multiple comparisons. Data can be evaluated using GraphPad software. A p-value < 0.05 of indicates statistical significance.

Claims

CLAIMSWhat is claimed is1. A system for multi-color ultrasound molecular imaging (USMI) comprising:- a solution containing a first monodisperse population of targeted microbubbles (tMBs) having a cloaked first ligand, and a first resonance frequency, and second monodisperse population of tMBs having a cloaked second ligand, and a second resonance frequency, wherein the first and second first resonance frequencies are different; and- a first ultrasound radiation force (USRF) at the first resonance frequency directed to the solution which activates the first tMBs, thereby uncloaking the first ligand which binds to its cognate receptor, followed by a second USRF at the second resonance frequency directed at the solution which activates the second tMBs thereby uncloaking the second ligand which binds to its cognate receptor.

2. The system of claim 1, wherein the tMBs have buried-ligand architecture (BLA) on their surfaces.

3. The system of claim 2, wherein said BLA comprises a hydrated polymer brush architecture.

4. The system of claim 3, wherein the hydrated polymer brush architecture comprises a bimodal PEGylated surface architecture.

5. The system of claim 4, wherein the bimodal PEGylated surface architecture comprises:- a plurality of shorter polyethylene glycol (PEG) molecule forming a polymer tether that attaches the first and second ligands to an anchoring lipid on their respective tMBs; and- a plurality of longer PEG chains that stratify into an overbrush that cloaks the first and second ligands on their respective tMBs.

6. The system of claim 5, wherein said first and second ligands are functionalized to form azidofunctionalized ligands.

7. The system of claim 6, wherein the azido-functionalized ligands are conjugated to the polymer tether on their respective tMBs through a click chemistry reaction to form a bioconjugate polymer.

8. The system of claim 7, wherein said click chemistry reaction comprises strain-promoted [3+2] azide-alkyne cycloaddition (SPAAC).

9. The system of claim 8, wherein said bioconjugate polymer comprises a 1,2, 3 -triazole linked bioconjugate.

10. The system of claim 9, wherein the polymer tether is selected from: 1,2-distearyol-sn-glycero- 3-phosphoethanolamine-N-[methoxy (polyethylene glycol) 5000] (DSPE-PEG5000); and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[dibenzocyclooctyl (polyethylene glycol) 2000] (DSPE-PEG2000-DBCQ).

11. The system of any of claims 1-10, wherein said first ligand is Cyclo Arg-Gly-Asp-D-Phe-Cys (RGD).

12. The system of claim 11, wherein the cognate receptor for the RGD ligand is av 3 integrin.

13. The system of claim 11, wherein the cognate receptor for the first ligand is different than the second ligand cognate receptor, and selected from: an angiogenesis biomarker, an inflammation biomarker, a cancer biomarker, or a diagnostic biomarker.

14. The system of any of claims 1-10, wherein said second ligand is Azide-IELLQARC-OH.

15. The system of claim 14, wherein the cognate receptor for the Azide-IELLQARC-OH ligand is p-selectin.

16. The system of claim 14, wherein the cognate receptor for the second ligand is different than the cognate receptor for the first ligand, and is selected from: an angiogenesis biomarker, an inflammation biomarker, a cancer biomarker, or a diagnostic biomarker.

17. The system of claim 5, wherein said shorter PEG molecule forming said polymer tether has a molecular weight of -2000 Dalton (Da), and said longer PEG chains forming said overbrush has a molecular weight of -5000 Da.

18. The system of claim 1, wherein the first and second USRF pulse is between approximately 3 and 8 MHz, the first and second USRF pulse being different.

19. The system of claim 1, wherein the first and second USRF pulse is between approximately 4 and 7 MHz, wherein the first and second USRF pulse being different.

20. The system of claim 1, wherein the solution is administered to a subject in need thereof.

21. The system of claim 1, wherein said subject includes a target region comprising a cell or population of cells disposed of in vivo or in vitro.

22. The system of claim 21, wherein said cell or population of cells comprises endothelial cells.

23. The system of claim 1, wherein the first and second tMB populations are between l-5pm in diameter.

24. The system of claim 1, wherein said first tMB population is between 1-2 pm diameter, and said second tMB population is between 4-5 pm diameter.

25. The system of claim 1, further comprising one or more additional monodisperse populations of targeted microbubbles (tMBs) having a unique cloaked ligand, and a unique resonance frequency.

26. A pharmaceutical composition comprising the therapeutically effective amount of the first and second tMBs of any of claims 1-25, and a pharmaceutically acceptable carrier.

27. A kit comprising the pharmaceutical composition of claim 26, a container to hold the composition, and instructions for use.

28. A method for multi-color ultrasound molecular imaging (USMI) comprising the steps:- generating a first monodisperse population of targeted microbubbles (tMBs) having a cloaked first ligand, and a first resonance frequency, and second monodisperse population of tMBs having a cloaked second ligand, and a second resonance frequency, wherein the first and second first resonance frequencies are different;- administering a therapeutically effective amount of the first and second tMBs to a subject in need thereof;- activating the first tMBs by application of an ultrasound radiation force (USRF) at the first resonance frequency thereby uncloaking the first ligand; and- activating the second tMBs by applying a second USRF at the second resonance frequency thereby uncloaking the second ligand.

29. The method of claim 28, wherein said step of generating comprises generating the tMBs having buried-ligand architecture (BLA) on the tMBs surface.

30. The method of claim 29, wherein said step of generating the tMBs having BLA comprises generating the tMBs having a hydrated polymer brush architecture.

31. The method of claim 30, wherein said step of generating the tMBs having a hydrated polymer brush architecture comprises generating the tMBs to have a bimodal PEGylated surface architecture.

32. The method of claim 28, wherein the bimodal PEGylated surface architecture comprises:- a plurality of shorter polyethylene glycol (PEG) molecule forming a polymer tether that attaches the first and second ligands to anchoring lipids on their respective tMBs; and- a plurality of longer PEG chains that stratify into an overbrush that cloaks the first and second ligands on their respective tMBs.

33. The method of claim 28, wherein said first and second ligands are functionalized to form azidofunctionalized ligands.

34. The method of claim 33, further comprising conjugating the azido-functionalized ligands to the polymer tether on their respective tMBs through a click chemistry reaction to form a bioconjugate polymer.

35. The method of claim 34, wherein said step of conjugating comprises the step of conjugating said azido-functionalized ligands to the polymer tether through a process of strain-promoted [3+2] azide-alkyne cycloaddition (SPAAC).

36. The method of claim 34, wherein said step of conjugating comprises the step of conjugating said azido-functionalized ligand to said polymer tether through a strain-promoted [3+2] azidealkyne cycloaddition (SPAAC) conjugation reaction between the polymer tether and the azido- functionalized peptide ligand to form 1,2,3-triazole linked bioconjugate37. The method of claim 36, wherein the polymer tether is selected from: 1,2-distearyol-sn- glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) 5000] (DSPE-PEG5000); and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[dibenzocyclooctyl (polyethylene glycol) 2000] (DSPE-PEG2000-DBCG).

38. The method of any of claims 28-37, wherein said first ligand is Cyclo Arg-Gly-Asp-D-Phe- Cys (RGD).

39. The method of claim 38, wherein the cognate receptor for the first ligand is av|33 integrin.

40. The method of claim 38, wherein the cognate receptor for the first ligand is different than the cognate receptor for the second ligand, and is selected from: an angiogenesis biomarker, an inflammation biomarker, a cancer biomarker, or a diagnostic biomarker.

41. The method of any of claims 28-47, wherein said second ligand is Azide-IELLQ ARC-OH.

42. The method of claim 41, wherein the cognate receptor for the second ligand is p-selectin.

43. The method of claim 41, wherein the cognate receptor for the second ligand is different than the cognate receptor for the first ligand, and is selected from: an angiogenesis biomarker, an inflammation biomarker, a cancer biomarker, or a diagnostic biomarker.

44. The method of claim 32, wherein said shorter PEG molecule forming said polymer tether has a molecular weight of -2000 Dalton (Da), and said longer PEG chains forming said overbrush has a molecular weight of -5000 Da.

45. The method of claim 28, wherein the first and second USRF pulses are between approximately3 and 8 MHz, wherein the first and second USRF pulses are at a different MHz.

46. The method of claim 28, wherein the first and second USRF pulse is between approximately4 and 7 MHz, wherein the first and second USRF pulse are at a different MHz.

47. The method of claim 46, wherein the first USRF pulse is approximately 4 MHz, and the second pulse is approximately 7 MHz.

48. The method of claim 28, wherein said subject comprises a human, or a target cell.

49. The method of claim 28, wherein said subject includes a target region comprising a cell or population of cells disposed of in vivo or in vitro.

50. The method of claim 49, wherein said cell or population of cells comprises endothelial cells.

51. The method of claim 28, wherein the first and second tMB populations are between l-5pm in diameter.

52. The method of claim 28, wherein said first tMB population is between 1-2 pm diameter.

53. The method of claim 28, wherein said first tMB population is between 4-5 pm diameter.

54. The method of claim 28, further comprising one or more additional monodisperse population of targeted microbubbles (tMBs) having a unique cloaked ligand, and a unique resonance frequency.

55. A pharmaceutical composition comprising the therapeutically effective amount of the first and second tMBs of any of claims 28-54, and a pharmaceutically acceptable carrier.

56. A kit comprising the pharmaceutical composition of claim 54, a container to hold the composition, and instructions for use.

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