WO2007127813A2

WO2007127813A2 - Modulation of signal transduction by site specific ultrasound agent delivery of transmembrane acting compounds

Info

Publication number: WO2007127813A2
Application number: PCT/US2007/067438
Authority: WO
Inventors: Margaret Wheatley; Kelleny Oum
Original assignee: Drexel University
Current assignee: Drexel University
Priority date: 2006-04-25
Filing date: 2007-04-25
Publication date: 2007-11-08
Anticipated expiration: 2008-10-25
Also published as: WO2007127813A3

Abstract

This invention relates to a site specific transmembrane acting ultrasound imaging agent suitable for inducing and modulating a signal transduction cascade in a targeted cells and to methods of inducing and modulating signal transduction in targeted cells together with imaging the site of the attachment of the ultrasound contrast agent.

Description

MODULATION OF SIGNAL TRANSDUCTION BY SITE SPECIFIC ULTRASOUND AGENT DELIVERY OF TRANSMEMBRANE ACTING

COMPOUNDS STATEMENT REGARDING FEDERALLY

SPONSORED RESEARCH OR DEVELOPMENT

This research was supported in part by U.S. Government funds (NIH grant number NIH CA 52823) and the U.S. Government may therefore have certain rights in the invention.

SPECIFICATION

BACKGROUND OF THE INVENTION

1. FIELD OF INVENTION

2. DESCRIPTION OF RELATED ART

In living biological tissues, a coordinated signal transmission between different cells and/or cell compartments, i.e. across membrane structures, is necessary in order to maintain homeostasis and make adaptation to external changes possible. As the inter- and intracellular signal transduction mechanisms mediated by soluble mediators are by now well characterized on the basis of numerous examples, intercellular signals mediated by membrane-associated interaction partners are often still not understood to a large extent. Cell-mediated signals are, however, essential for all multicellular organisms and thus also for maintaining multicellular organ assemblages in higher life forms. Thus, it has emerged that when normal body cells are removed from their tissue assemblage they lose important survival signals. They then die by a process which is referred to as anoikis (isolation), the molecular mechanism of which corresponds to programmed cell death (apoptosis). Knowledge about such survival signals and their molecular mechanisms of action is particularly relevant to the whole area of oncology because tumor cells must bypass or switch off exactly these regulatory systems in order to be able to form malignant tumors.

The problems associated with research into intercellular signal mechanisms are in part purely technical. Thus, for example, in many cases it is impossible or very complicated for methodological or technical reasons to allow two different cell populations to interact in a spatially and temporally controlled manner and to observe their reactions in parallel during this. In addition, such an interaction always triggers a large number of signals which it is scarcely possible, or impossible, to assign to the large number of signal molecules. In order to be able to determine specifically the effect of an individual signaling substance, therefore, in the state of the art there is preparation and investigation of recombinant soluble derivatives of membrane- associated ligands and their receptors. The data obtained in this way may, however, lead to an incorrect estimation of the actual physiological significance of a defined signaling process, because the underlying test system represents a gross simplification in relation to the actual event.

Tumor necrosis factor belongs to the class of cytokines, which are substances which are produced and secreted by a large number of cell types and which regulate, as intercellular mediators, a large number of cellular processes. To date, two different TNF-specific membrane receptors of 55 kDA (TNFRl) and 75 kDA (TNFR2) have been identified. It is known that TNF-alpha oligomers must bind to the membrane receptors for it to be possible to induce at least in physiological solutions some of the various biological activities of TNF-alpha. Although it has been shown that TNF-alpha occurs as oligomer in solution, it has emerged, however, that these non-covalently linked TNF-alpha oligomers are unstable and are converted into inactive forms. It is, however, assumed that the structural changes of TNF-alpha also take place in vivo because monomers and oligomers are found in different proportions in the cerebrospinal fluid of patients with meningitis. TNF-alpha was originally identified as an endotoxin-induced factor able to induce the necrosis of tumors in mice. However, it is known that the antitumor effect of TNF-alpha not only results from the cytoxic effects on tumor cells but is also based on activation of antitumor effector immune cells in the blood, for example macrophages, cytotoxic lymphocytes and neutrophils. The antitumor effect of TNF-alpha is additionally based on a specific damage to tumor blood vessels.

Since TNF-alpha has very low stability and exerts pleiotropic effects in vivo, attempts to date to employ TNF-alpha as systemic antitumor agent have been unsuccessful. It has emerged on administration of TNF-alpha that extremely severe systemic side effects, such as fever and low blood pressure, occur before therapeutically effective doses are reached. Despite the continuing substantial expectations from potential use of TNF-alpha as antitumor agent, the clinical utilizability of this substance therefore continues to be very restricted.

It is generally difficult to employ biologically active proteins such as cytokines for therapeutic purposes because biologically active proteins very often display low stability and short half-lives. The proteins are rapidly removed from the blood by the liver, kidneys and other organs, with the rate of clearance depending on the size of the molecules and the extent of proteolysis. Plasma proteases in particular cause the degradation of such proteins and bring about rapid loss of the biological activity. In order to overcome these problems, in recent years systems for immobilizing biological active proteins on or in carrier systems have been developed. For example, controlled release systems for therapeutic active ingredients or drugs are known, with drugs for example being immobilized by inclusion in a matrix. After administration of such controlled release systems there is then controlled release of the drugs through degradation processes within the living body. The conformation of the active ingredient in the immobilized state is not crucial in these systems. On the contrary, the important point is that the active ingredient is in the active state after release.

U.S. 2004/0265392A1 to Tovar et al. discloses nanoparticles with tumor necrosis factor (TNF) immobilized thereon and challenges associated with delivering an active ingredient in the active state after release.

Examples of ultrasound imaging agent can be found in U.S. Pat. Nos. 6,261 ,537 and 6,680,047 to Klaveness et al., International Patent Publication WO 2007/008220 to Grayburn, U.S. Patent No. 5,585,112 to Unger et al., U.S. Patent No. 5,955,143 to Wheatley et al. and International Patent Publication WO 2002/07861 1, however, these references do not describe inducing and modulating a signal transduction cascade in a targeted cells.

Despite the current developments, there is a need for a contrast agent capable of inducing and modulating a signal transduction cascade in a targeted cells and for methods of inducing and modulating signal transduction in targeted cells together with imaging the site of the attachment of the ultrasound contrast agent. All references cited herein are incorporated herein by reference in their entireties.

BRIEF SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention is a method of modulating a signal transduction cascade in a cell, the method comprising: (1) providing a site specific transmembrane acting ultrasound imaging agent which comprises (a) an echogenic microcapsule or nanocapsule, (b) a signal transduction inducing ligand associated with the echogenic microcapsule or nanocapsule and optionally (c) a therapeutic agent; (2) providing a cell comprising a cell-surface receptor with an affinity to the signal transduction inducing ligand; (3) initiating the signal transduction cascade in the cell by contacting the cell with the site specific transmembrane acting ultrasound imaging agent such that signal transduction inducing ligand selectively binds to the cell-surface receptor and remains associated with the echogenic microcapsule or nanocapsule and thereby modulating the signal transduction cascade in the cell; and (4) detecting selective binding of the site specific transmembrane acting ultrasound imaging agent by using an ultrasonic imaging system.

In certain embodiments, the signal transduction inducing ligand is a cell damage inducing ligand. In certain embodiments, the cell damage inducing ligand is a member of TNF family. In certain embodiments, the cell damage inducing ligand is TRIAL (SEQ. ID. NO. 1). In certain embodiments, the cell damage inducing ligand is HER-2 antibody. In certain embodiments, the site specific transmembrane acting ultrasound imaging agent comprises a therapeutic agent. In certain embodiments, the therapeutic agent is at least one of doxorubicin, docetaxel or herceptin. In certain embodiments, the therapeutic agent is released from the site specific transmembrane acting ultrasound imaging agent by a signal emitted by the ultrasonic imaging system. In certain embodiments, the signal transduction inducing ligand is covalently associated with the echogenic microcapsule or nanocapsule. In certain embodiments, the signal transduction inducing ligand is non- covalently associated with the echogenic microcapsule or nanocapsule.

In certain embodiments, the echogenic microcapsule or nanocapsule comprise (a) an outer surface including (1) a hardened non water soluble polymer and (2) at least one hollow area formed by removal of a non-water soluble sublimable substance; and (b) an inner surface comprising at least one hollow area formed by removal of a water soluble sublimable substance, wherein said echogenic polymer microcapsule or nanocapsule are made from (i) an outer surface forming mixture comprising a non water soluble polymer and the non-water soluble sublimable substance dissolved in one or more volatile non-polar solvents and (ii) an inner surface forming mixture comprising the water soluble sublimable substance dissolved in water. In certain embodiments, the echogenic microcapsule or nanocapsule comprise a gas-containing or gas- generating material. In certain embodiments, the outer surface of the echogenic microcapsule or nanocapsule comprises at least one of poly(lactide), poly(glycolide), a copolymer of lactide and lactone, a poly(anhydride), a poly(styrene), a poly(alkylcyanoacrylate), a poly(amide), a poly(phosphazene), a poly(methylmethacrylate), a poly(urethane), a copolymer of methacrylic acid and acrylic acid, a copolymer of hydroxyethylmethacrylate and methylmethacrylate. In certain embodiments, the site specific transmembrane acting ultrasound imaging agent is administered to a composition of cells, wherein only a fraction of cells comprise cells having the cell-surface receptor with an affinity to the signal transduction inducing ligand and therefore abnormal or tumorous cells can be distinguished from normal cells by the site specific transmembrane acting ultrasound imaging agent selectively binding to the cell-surface receptor of abnormal or tumorous cells.

In certain embodiments, the site specific transmembrane acting ultrasound imaging agent is administered to a mammal in an aqueous carrier.

In certain embodiments, the site specific transmembrane acting ultrasound imaging agent comprises a plurality of signal transduction inducing ligands.

In certain embodiments, a mixture of site specific transmembrane acting ultrasound imaging agents having different signal transduction inducing ligands is administered to a mammal in an aqueous carrier.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS The invention will be described in conjunction with the following drawings in which like reference numerals designate like elements and wherein:

Fig. 1 (prior art) is a scheme demonstrating a process of TRAIL triggering cell death (e.g., apoptosis)

Fig. 2 (prior art) is a scheme demonstrating a process of normal apoptosis induction in a cell.

Fig. 3 is a graph demonstrating dose and time response curves measured for anti-HER2 scFv (C6.5cys)-ligated contrast agent (Example 3).

Fig. 4 is a bar graph demonstrating attachment of C6.5cys scFv HER2 Antibody-PLA microspheres (Example 4). DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the discovery that an ultrasound contrast agent can be used for triggering a specific biological change within a cell, i.e., inducing signal transduction within a cell, when conjugated with a ligand known to induce such change, a signal transduction inducing ligand. In certain embodiments, the ultrasound contrast agent of the invention can induce a cell damage or cell death (e.g., an apoptosis) of particular cells when associated (e.g., by covalent or affinity binding) with a cell damage inducing ligand.

A signal transduction inducing ultrasound contrast agent of the invention comprises an echogenic microcapsule or nanocapsule and a signal transduction inducing molecule associated with the echogenic microcapsule or nanocapsule for example by being covalently attached to the outer surface.

Unexpectedly, conjugation of the signal transduction inducing molecule to a much larger particle such as echogenic microcapsule or nanocapsule did not affect binding capacity or functionality of the signal transduction inducing molecule. Also molecules of a high molecular weight (e.g. TRAIL (SEQ. ID. NO. 1) has molecular weight 34kDa) retained signal transduction activity when covalently attached to microcapsules of contrast agent, even when using methods that used conjugation via a lysine amino group, a group that is located near the active site. Furthermore, it was observed that the inflicted cell death was slower and more global when the cell damage inducing ligand was conjugated to the echogenic microcapsule or nanocapsule as compared to the unconjugated ligand, and that the rate of killing can be controlled by the ligand density attached to the contrast agent.

Advantages of the invention include ability to visualize target cells, focusing energy exactly at the site and at the point of interaction between the signal transduction inducing ligand and the cell, utilization of the interaction between the echogenic contrast agent and the ultrasound energy, and utilizing the force produced by the ultrasound for release of a desired compound (e.g., a therapeutic agent). Previous studies describing utilizing complexes and conjugates of echogenic particles, capsules or bubbles with bioactive agents (e.g., proteins and peptides) did not contemplate modulating the signal transduction cascade in a cell. Signal transduction or a signal transduction cascade involves the binding of extracellular signaling molecules to cell receptors and trigger a cascade of events inside the cell by means of a binding-induced conformational change. Those extracellular signaling molecules include hormones (e.g., melatonin), growth factors (e.g., epidermal growth factor), extra-cellular matrix components (e.g., fibronectin), cytokines (e.g., interferon-gamma), chemokines (e.g., RANTES), neurotransmitters (e.g., acetylcholine), and neurotrophins (e.g., nerve growth factor). The signal transduction cascade involves generation of second messages (e.g., cAMP, or protein kinase activity.)

Examples of cellular responses to extracellular stimulation that require signal transduction include gene activation, metabolism alterations, continued proliferation and death of a cell, and stimulation or suppression of locomotion.

Since the protein products of many of the activated genes include transcription factors themselves, gene activation causes further cellular effects. Transcription factors produced as a result of a signal transduction cascade can in turn activate more genes. Therefore, an initial stimulus can trigger the expression of an entire set of genes, which in turn can lead to the activation of a number of complex physiological events. These events include, for example, the increased uptake of glucose from the blood stream stimulated by insulin.

In this invention, cell receptors are preferably cell-surface receptors, however, ligand gated ion channel receptors can be involved also. Examples of cell-surface receptors include G- protein coupled receptors (e.g., chemokine receptors), receptor tyrosine kinases (RTKs) (e.g., growth factor receptors), integrins, and toll-like receptors (TLRs). Examples of ligand gated ion channel receptors include Cys-loop receptors, ionotropic glutamate receptors, and ATP-gated channels. The term "a signal transduction inducing molecule" or "a signal transduction inducing ligand" are used interchangeable herein and encompass molecular species which, when interacting with cell surface receptors, trigger an intracellular cascade of events, for example, TNF-related apoptosis-inducing ligand (APO2L), epinephrine and norepinephrine, glucagon, luteinizing hormone, follicle stimulating hormone, thyroid-stimulating hormone, calcitonin, parathyroid hormone, antidiuretic hormone, insulin, growth hormone, prolactin, oxytocin, erythropoietin, angiotensin II, antidiuretic hormone, gonadotropin-releasing hormone, thyroid- releasing hormone, atrial naturetic hormone, nitric oxide, active fragments thereof such as the 19KDa fragment of TRAIL known as sTRAIL, and synthetic species such as monoclonal antibody engineered through biotechnology for example Herceptin^® or synthetic drugs such as Gleevec^® which has been implicated in dendritic cell activation.

The term "a cell damage inducing ligand" as used herein denotes a ligand capable of triggering cell damage or cell death, both apoptic and non-apoptic. Non-limiting examples of such cell damage inducing ligands include ligands to receptors on cancer cell surfaces such as, for example, (a) an antibody to a receptor on a particular cell surface, the receptor capable of stimulating the cell to divide and grow (e.g., Her-2 specific antibody to Her-2 protein found on the surface of certain cancer cells such as MDA-MB 231) and (b) tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL), which is a member of the TNF family of cytokines that promotes apoptosis; TRAIL induces apoptosis via death receptors (DR4 and DR5) in cancer cells but not in normal cells. The term "ultrasound imaging agent" is used interchangeably with "contrast agent (CA)" and includes the terms "echogenic nanocapsule" and "echogenic microcapsule". The term "echogenic microcapsules" is used interchangeably with the term "microbubbles." Ultrasound Contrast Agent

Any ultrasound contrast agent can be used for covalent modification with a signal transduction inducing ligand and specifically, for modification with an apoptosis inducing ligand, if it has accessible functional groups of its outer surface with can react with corresponding functional groups of such ligands. For example, maleimide groups on a surface of a contrast agent can react with a thiol group of a cystine and therefore a thioester linkage will be formed between the contrast agent and the ligand. A person skilled in the art would readily ascertain a combination of functional groups needed for formation of a covalent bond between the two entities. Any ultrasound contrast agent can be used for surface adsorption modification with a signal transduction inducing ligand and specifically, for modification with an apoptosis inducing ligand, if it has accessible functional groups of its outer surface with can react electrostatically or by hydrophobic-interaction with corresponding functional groups of such ligands. Any ultrasound contrast agent can be used for modification with a signal transduction inducing ligand and specifically, for modification with an apoptosis inducing ligand by incorporation during fabrication, if it he fabrication process does not destroy the activity of such ligands.

Examples of suitable ultrasound imaging agent can be found in U.S. Pat. Nos. 6,261 ,537 and 6,680,047 to Klaveness et al., International Patent Publication WO 2007/008220 to Grayburn, U.S. Patent No. 5,585,1 12 to Unger et al., U.S. Patent No. 5,955,143 to Wheatley et al. and International Patent Publication WO 2002/07861 1 and references described therein.

In a preferred embodiment, the ultrasound imaging agent is made based on methods described in US 2004/0161384 Al to Wheatley et al. Such echogenic microcapsule or nanocapsule comprise (a) an outer surface including (1) a hardened non water soluble polymer and (2) at least one hollow area formed by removal of a non-water soluble sublimable substance; and (b) an inner surface comprising at least one hollow area formed by removal of a water soluble sublimable substance, wherein said echogenic polymer microcapsule or nanocapsule are made from (i) an outer surface forming mixture comprising a non water soluble polymer and the non-water soluble sublimable substance dissolved in one or more volatile non-polar solvents and (ii) an inner surface forming mixture comprising the water soluble sublimable substance dissolved in water. The echogenic microcapsules or nanocapsules comprise a gas-containing or gas-generating material.

The outer surface of the echogenic microcapsule or nanocapsule is made of liposomes, lipid coatings, and polymers. Lipids which may be used to create lipid microspheres include but are not limited to: lipids such as fatty acids, lysolipids, phosphatidylcholine with both saturated and unsaturated lipids including dioleoylphosphatidylcholine; dimyristoylphosphatidylcholine; dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine; distearoyl phosphatidylcholine; phosphatidylethanolamines such as dioleoylphosphatidylethanolamine; phosphatidylserine; phosphatidyl glycerol; phosphatidylinositol, sphingolipids such as sphingomyelin; glycolipids such as ganglioside GMl and GM2; glucolipids; sulfatides; glycosphingolipids; phosphatidic acid; palmitic acid; stearic acid; arachidonic acid; oleic acid; lipids bearing polymers such as polyethyleneglycol, chitin, hyaluronic acid or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, oligo- or polysaccharides; cholesterol, cholesterol sulfate and cholesterol hemisuccinate; tocopherol hemisuccinate, lipids with ether and ester-linked fatty acids, polymerized lipids, diacetyl phosphate, stearylamine, cardiolipin, phospholipids with short chain fatty acids of 6-8 carbons in length, synthetic phospholipids with asymmetric acyl chains (e.g., with one acyl chain of 6 carbons and another acyl chain of 12 carbons), 6-(5-cholesten-3.beta.- yloxy)-l-thio-.beta.-D-galactopyranoside, digalactosyldiglyceride, 6-(5-cholesten-3.beta.- yloxy)hexyl-6-amino-6-deoxy-l-thio-.beta.-D-galacto pyranoside, 6-(5-cholesten-3.beta.- yloxy)hexyl-6-amino-6-deoxyl- 1 -thio-.beta.-D-mannop yranoside, 12-(((V- diethylaminocoumarin-3-yl)carbonyl)methylamino)-octadecanoic acid; N-[12-(((V- diethylaminocoumarin-3-yl)carbonyl)methyl-amino) octadecanoyl]-2-aminopalmitic acid; cholesteryl)4'-trimethyl-ammonio)butanoate; N-succinyldioleoylphosphatidylethanol-amine; l,2-dioleoyl-sn-glycerol;l,2-dipalmitoyl-sn-3-succinylglycerol; l,3-dipalmitoyl-2- succinylglycerol;l-hexadecyl-2-palmitoylglycerophosphoet hanolamine; and palmitoylhomocysteine; and/or combinations thereof. The liposomes may be formed as monolayers or bilayers and may or may not have a coating.

Lipids bearing hydrophilic polymers such as polyethyleneglycol (PEG), including and not limited to PEG 2,000 MW, 5,000 MW, and PEG 8,000 MW, are particularly useful for improving the stability and size distribution of the gaseous precursor-containing liposomes. Polymers useful for this invention are preferably biodegradable. Preferably, outer surface of the echogenic microcapsule or nanocapsule comprises at least one of polylactide, polyglycolide, a copolymer of lactide and lactone, a polyanhydride, a polystyrene, a polyalkylcyanoacrylate, a polyamide, a polyphosphazene, a poly(methylmethacrylate), a polyurethane, a copolymer of methacrylic acid and acrylic acid, a copolymer of hydroxyethylmethacrylate and methylmethacrylate. Signal Transduction Inducing Ligand

Examples of signal transduction inducing ligands include TNF-related apoptosis- Inducing ligand (A.K.A. APO2L), epinephrine and norepinephrine, glucagon, luteinizing hormone, follicle stimulating hormone, thyroid-stimulating hormone, calcitonin, parathyroid hormone, antidiuretic hormone, Insulin, growth hormone, prolactin, oxytocin, erythropoietin, Epinephrine and norepinephrine, angiotensin π, antidiuretic hormone, gonadotropin-releasing hormone, thyroid-releasin hormone, Atrial naturetic hormone, nitric oxide, active fragments thereof such as the 19KDa fragment of TRAIL known as sTRAIL, and synthtic species such as monoclonal antibody engineered through biotechnology for example Herceptin^® or synthetic drugs such as Gleevec^® which has been implicated in dendritic cell activation. It has been observed that binding of certain ligands to a cell surface receptor initiates a series of events which leads to generation of so-called second messengers within the cell (the ligand is the first messenger). The second messengers then trigger a series of molecular interactions that alter the physiologic state of the cell. Another term used to describe this entire process is signal transduction. Examples of second messengers include Cyclic AMP, Protein kinase activity, calcium and/or phosphoinositides and Cyclic GMP.

In all cases, the seemingly small signal generated by ligand binding its receptor is amplified within the cell into a cascade of actions that changes the cell's physiologic state.

An example of the signal transduction inducing ligand is a cell damage inducing ligand, a ligand capable of inducing a cell death of a particular cell when bound to a receptor on the cell surface. Non-limiting examples of such cell damage inducing ligands include ligands to receptors on cancer cell surfaces such as, for example, (a) an antibody to a receptor on a particular cell surface, the receptor capable of stimulating the cell to divide and grow (e.g., Her-

2 specific antibody to Her-2 protein found on the surface of certain cancer cells such as MDA-

MB 231) and (b) tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL). TRAIL induces apoptosis via death receptors (DR4 and DR5) in cancer cells but not in normal cells.

Tumor necrosis factor belongs to the class of cytokines, which are substances which are produced and secreted by a large number of cell types and which regulate, as intercellular mediators, a large number of cellular processes. Cytokines include, for example, lymphokines, interleukins, monokines and growth factors. Tumor necrosis factor (TNF, also called TNF-alpha or cachectin) is the eponymous member of a large gene family of cytokines which are structurally homologous and have diverse biological properties, some of which overlap. The members known at present include TNF itself and, inter alia, lymphotoxin alpha (LT-alpha) which, like TNF, binds to the same receptors (TNFRl and TNFR2) and displays very similar signaling properties, and LT-beta, FasL, CD27L, CD30L, CD40L, TWEAK, OX40L, EDA, AITRL, VEGI, LIGHT, 4- 1 BBL, APRIL, BLYS , RANKL and TRAIL, for each of which one or more specific membrane receptors exist (Locksley et aL, Cell, 104:487-501, 2001). The molecules of the tumor necrosis factor (TNF) ligand family are involved via their complementary receptors, which are collected into the TNF receptor family in a large number of in particular immunoregulatory processes. Apart from very few exceptions, the members of the TNF ligand family are membrane proteins of type II (Locksley et al., see above). However, it is also possible in many cases to derive soluble forms from these membrane-associated forms by specific proteolysis or alternative splicing. In some cases insoluble and membrane-associated forms of members of the TNF ligand family may differ considerably in their bioactivity (Grell et ah, Cell, 1995). In contrast to lymphotoxin (LT-alpha), which is a genuine secreted protein, however, TNF is, like most other members of the TNF family, produced by the producing cell always initially as membrane-associated TNF from which the classical soluble cytokine TNF is formed only on proteolytic cleavage. This membrane TNF is a genuine transmembrane protein which exists in trimeric form and is biologically active. It has been possible to show in this connection that membrane TNF has a special range of cellular effects which are not attained by soluble TNF and by LT-alpha (Grell et ah, Cell, 83 ( 1995), 793-802). It is of interest that other members of the TNF family behave similarly, i.e., they are bioactive in the normal, membrane- associated form, and, as molecules which are soluble after proteolytic processing or occur in soluble form otherwise, either entirely biologically inactive or show, as in the case of TNF, a restricted range of effects. Apart from TNF, this has been shown for example also for FasL, TRAIL and CD40L (Wajant and Pfizenmaier, Onkologie, 24:6-10, 2001). TNF is formed primarily by macrophages/monocytes, lymphocytes and mast cells and influences inflammations, sepsis, lipid and protein metabolism, blood formation, angiogenesis, wound healing and immune defenses and exerts cytotoxic or cytostatic effects on certain tumor cells. Lymphotoxin a likewise has cytotoxic effects on certain tumor cell lines. Relatively little is known about the molecular basis of the diverse effects of TNF-alpha in vivo and in vitro. Analysis of the crystal structure of the recombinant soluble TNF-alpha molecule has revealed that this substance is an oligomeric protein consisting of three identical subunits each of 17.5 kDA. To date, two different TNF-specifϊc membrane receptors of 55 kDA (TNFRl) and 75 kDA (TNFR2) have been identified. It is known that TNF-alpha oligomers must bind to the membrane receptors for it to be possible to induce at least in physiological solutions some of the various biological activities of TNF-alpha. Although it has been shown that TNF-alpha occurs as oligomer in solution, it has emerged, however, that these non-covalently linked TNF-alpha oligomers are unstable and are converted into inactive forms. It is, however, assumed that the structural changes of TNF-alpha also take place in vivo because monomers and oligomers are found in different proportions in the cerebrospinal fluid of patients with meningitis.

Therapeutic Agent In certain embodiments, the site specific transmembrane acting ultrasound imaging agent comprises a therapeutic agent. Examples of therapeutic agents can be found in U.S. Pat. Nos. 6,261 ,537, columns 39-60 (incorporated herein). In certain embodiments, the therapeutic agent is at least one of doxorubicin, docetaxel or herceptin.

In certain embodiments, the therapeutic agent is released from the site specific transmembrane acting ultrasound imaging agent by a signal emitted by the ultrasonic imaging system. Therapeutic agents can be added during the stage of making the echogenic particle depending on the solubility of the therapeutic agent and desired formulation, a particular therapeutic agent can be present in the outer surface of the echogenic particle or in the inner surface. Methods of formulating each particular imaging agent with the therapeutic agent would be apparent to a person skilled in the art.

Site Specific Transmembrane Acting Ultrasound Imaging Agent The site specific transmembrane acting ultrasound imaging agent of the invention comprises (a) an echogenic microcapsule or nanocapsule, (b) a signal transduction inducing ligand associated with the echogenic microcapsule or nanocapsule and optionally (c) a therapeutic agent.

Association of the echogenic microcapsule or nanocapsule with the signal transduction inducing ligand can be covalent or non covalent. Both echogenic microcapsules/nanocapsules and ligands have complementary functional groups capable of binding or reacting with each other. Description of covalent and non-covalent attachment is provided in U.S. 2004/0265392A1 to Tovar et al. (incorporated therein) for immobilizing TNF as at each monomer or as a trimer based on interaction of functional groups on the surface of TNF and the nanoparticle surface matrix. It is required that the activity (i.e., ability to affect signal transduction cascade in a cell) of the attached ligand remains substantially comparable to the activity of the free ligand.

Thus, for covalent attachment, it is necessary to have on each of the molecules being joined functional groups capable of reacting with each other to form a covalent bond. Examples of such pairs are maleimido group and thiol group, an amino group and a carboxyl group, a hydrazine/hydrazide group and an alkyl ketone group, etc.

For a non-covalent attachment, an affinity pair can be used with each of the component of the affinity pair being present on each of the molecules being joined, e.g., biotin-avidin affinity pair. The site specific transmembrane acting ultrasound imaging agent of the invention can be made by first making echogenic microcapsule or nanocapsule and then attaching signal transduction inducing ligand either covalently or non-colalently. Covalent attachment is preferred.

Another way of making the site specific transmembrane acting ultrasound imaging agent involves adding the signal transduction inducing ligand to the outer surface making phase such that the ligand.

The method is described in detail in Examples below, e.g., surface ligation of TRAIL to microbubbles (see Examples 4 and 7). The biodegradable polymeric, hard-shelled microbubbles are produced using a double emulsion method that results in a hollow gas-filled sphere which reflects impinging ultrasound beams to enhance ultrasound images. When exposed to the TRAIL-bound microbubbles, cell death was observed in both MCF-7 and MDA-MB-231 human breast cancer cell models over a 48 hour period. Cell death was markedly lower in cells incubated with unmodified microbubbles. The killing abilities of TRAIL were not hindered by its conjugation to the microbubbles. Potential applications of this approach may include the simultaneous ultrasound imaging and treatment of malignant tumors, especially if further adapted to a nano-scale contrast agent platform or the co-localize (on the contrast agent), simultaneous imaging and delivery of drug and Trail, a combination which has been shown to be synergistic. Normal co administration suffers form all the disadvantages of drug toxicity, but co localization within the contrast agent would circumvent this problem. Another embodiment of the method is directed to a Her-2 specific antibody conjugated with microbubble contrast agents (Examples 1-3). Her-2 is a protein found on the surface of certain cancer cells. When human epidermal growth factor attaches itself to Her-2 receptors on the breast cancer cells, it stimulates the cells to divide and grow. BT-474, tumor cell line that overexpresses Her-2, is used to test the antibody conjugated microcapsule contrast agent in vitro.

The invention will be illustrated in more detail with reference to the following

Examples, but it should be understood that the present invention is not deemed to be limited thereto.

EXAMPLES

EXAMPLE 1

Attachment of Diabody anti-HER2 scFv (C6.5cys) to Block ErbB Signaling Pathway

The epidermal growth factor receptor type 2 (ErbB-2 / Her2) tyrosine kinase is a carcinoma-associated receptor whose activation is responsible for tumor cell survival, proliferation, and metastasis in many human cancers. ErbB receptor-binding peptides block the signaling pathway.

To poly(lactic acid) microbubbles (120mg, 34 x 10⁶ bubbles /mg) was added 2-(N- morpholino)ethanesulfonic acid (MES buffer) (8ml) (5OmM) (pH 5.2) and shaken to create a suspension.

To MES buffer (2-(N-morpholino)ethanesulfonic acid) were added PLA (poly (lactic acid)) microbubbles at 15 mg/mL, making a total of 8 mL of microbubble solution in a 15 mL centrifuge tube. BMPH (β-Maleimidopropionic acid hydrazide) was dissolved in ultrapure water to 50 mM (14.86 mg/mL). EDC (l-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride) was dissolved in ultrapure water at 10 mM (19.17 mg/mL). This solution was immediately added to the microbubbles: 1 mL BMPH solution (25 μmol BMPH) and 1 mLEDC solution (50 μmol EDAC). The mixture was incubated for 30 minutes at room temperature with gentle agitation using a rotatory shaker. The reacted microbubbles were purified by centrifugation at 1000 X g for 10 min. The pellet was resuspended in MES buffer. Washing was repeated 2 additional times. In the final wash step, the pellet was resuspended in 4 mL PBS buffer, and labeled "activated microbubble solution".

Four sets of Eppendorf tubes were prepared as follows: a) To (2) scFv C6.5 cys BT-474 : was added 0.3 μL (0.78 mM) C6.5 cys. b) To (2) scFv C6.5 cys MDA-MB468 was added 0.3 μL (0.78 mM) C6.5 cys, c) To (2) scFv C6.5 cys negative control BT-474 was add 50 μL 0.1 N mercaptoacetic acid (5 μmol). d) To (2) Negative Control BT-474 was added 50 μL 0.1 N mercaptoacetic acid (5 μmol). To each eppendorf tube was added 1 mL activated microbubble solution. The tubes were incubated for 2 hr at room temperature on a rotatory shaker. The following additions were made a) To scFv C6.5 cys BT-474: was added 50 μL 0.1 N mercaptoacetic acid (5 μmol). b) To scFv C6.5 cys MDA-MB 231 : was added 50 μL 0.1 N mercaptoacetic acid (5 μmol). c) To scFv C6.5 cys BT-474 Control: was added 0.3 μL (0.78 mM) C6.5 cys and d) to Negative Control BT-474: nothing was added. The mixtures were incubated for 10 minutes at room temperature on a rotatory shaker. Each sample was purified by centrifugation at lOOOXg for 10 minutes. The pellets were re-suspended in PBS buffer. This was repeated 3 additional times. In the final wash step, the pellet was re-suspended in 1 mL phosphate buffered saline (PBS). Samples were tested for echogenicity, and for ability to contact the over-expressing BT-474 cell line by using an in vitro adhesion assay.

EXAMPLE 2 Echogenicity testing of anti-HER2 scFv (C6.5cys)-ligated microbubbles

Dose and time response curves were measured for anti-HER2 scFv (C6.5cys)-ligated microbubbles in an in vitro test apparatus. Antibody gave a peak enhancement at 5 MHz insonation of 17.6 dB at 0.0006 mg/ml, compared to 20 dB prior to conjugation. Antibody-PLA microspheres were tested at 5 MHz for 15 minutes, resulting in a loss of signal of 39%. This is 31.7% more loss than in case of the non-conjugated microspheres (see Fig. 3).

EXAMPLE 3

Assess the targeting ability of anti-HER2-CA to target BT-474 cells in 2-D monolayer

Ligated microcapules were exposed to BT-474 cells growth on cell culture to test for adhesion. BT-474 cells were cultured in T-75 tissue flasks in Dulbeco's Modified Eagle Medium with Earle's salts (DMEM) with Penicillin Streptomycin, Fetal Bovine Serum, and L-

Glutamine. The cells were incubated at 37°C in 95% air and 5% CO2 and passaged regularly at about full confluence. During static attachment studies, the cells were seeded in 12 well culture plates at passage 6. Statistical analyses were performed using the Prism software (San Diego,

CA, Graphpad Software) to analyze the data sets. For analysis within multiple groups, ANOVA analysis was performed using the Newman Keuls multiple comparison tests and p-values were obtained. For analysis of significance within two groups, t-test was performed using Wilcoxon matched pairs-test, where p values were obtained.

The cells were first washed with media and then incubated with the 1 ml of contrast agent containing media for the specified time point (0, 5, 10, 15 min). All studies were done in triplicate, one sample set was pre-blocked for an hour prior to experimentation with the same ligand on the surface of the ligand-modified contrast agent at a concentration of 150 μg/ml in 1 ml of medium, one sample set of non-conjugated PLA, and a control with PLA microspheres that went through the conjugation process but without the linker. At the specified time point, the medium was removed and the cells were washed three times with 1 ml of growth media solution. The cells were then viewed under a phase contrast microscope and pictures were taken.

The static attachment study was performed at time 0, 5, 10, and 15 min at 0.5 mg/ml concentration of conjugated microspheres with 1 ml volume of microcapsule and media mixture in each well of a 12 well plate.

The results of this study were graphed (see Fig. 4). It was observed that as time increases, so did attachment of conjugated microspheres with statistical significance (p<0.05). The max binding was observed in the experimental group at 15 min (2.1 attachment/cell). The results of the controls show minimal attachment, concluding that the binding of the experimental group is due to the presence of scFv antibody, the targeting potential of the scFv antibody to HER2, and specificity of the antibody for HER2 receptor with statistical significance (p<0.05). This statistical significance was found when each negative control group was compared to the experimental. Static attachment of C6.5cys scFv HER2 Antibody-PLA microspheres: as time increases, so does attachment of conjugated microspheres with statistical significance (p<0.05). The max binding is observed in the experimental group at 15 min (2.1 attachment/cell), n=30 and error bars are standard deviation.

EXAMPLE 4 Conjugating of TRAIL onto PLA microcapsules

Dried microcapsules (100 mg) were combined with 5 mg EDC (l-Ethyl-3-[3- dimethylaminopropyl]carbodiimide Hydrochloride) (1 :1 molar ratio of -COOH groups in microcapsules to EDC), 2.7 mg ofNHS (N-hydroxysuccinimide), (1:2 molar ratio to EDC), and 10ml of buffer (0.1 M MES (2-[N-morpholino]ethanesulfonic acid sodium salt), 0.3 M NaCl, pH 6.5,) and shaken on a rotary shaker for 15 min. TRAIL (35μl, 1 :2 molar ratio of COOH groups on the polymer to TRAIL) were then be added and shaken for 3 hours. The microcapsules were then washed 3 times with deionized water followed by centrifugation at 1000 g and lyophilized using a Virtis benchtop freeze dryer.

EXAMPLE 5 Death Induction Study Using Unconjugated TRAIL

Initiation of death was demonstrated by culturing 5x 104 TRAIL-sensitive MCF-7 breast cancer cells in the presence of TRAIL at concentrations ranging from 10ng/ml 50ng/ml for 16 hours at 37°C in a 48 well culture plate. Cell death was measured using the LIVE/DEAD® Reduced Biohazard Viability/Cytoxicity Kit. Components A and B were combined in ImI of HBSS and kept protected from light. The cells were washed with 200μl of HBSS (Hanks' Balanced Salt Solution) and resuspended in lOOμl diluted dye. The cells were then incubated at room temperature in complete darkness for 15 minutes. The dye was aspirated and the cells were resuspended in 50μl in fresh HBSS. A 4% glutaraldehyde solution in HBSS was freshly prepared and added to the cells and incubated at room temperature for 15minutes. Cells were observed under a Nikon Eclipse TE2000-U fluorescent microscope.

EXAMPLE 6 Demonstration of Potency of TRAIL-Ligated Contrast Agent Using the LIVE/DEAD assay from Molecular Probes the amount of dead cells versus live cells was evaluated qualitatively. The basis for the viability test is differential permeability of live and dead cells to a pair of fluorescent stains. SYTO® 10, a green fluorescent nucleic acid stain, is a highly membrane-permeable dye and labels all cells, including those with intact plasma membranes. DEAD Red® is a cell- impermanent red fluorescent nucleic acid stain that labels only cells with compromised membranes. With breast cancer cell line MCF-7, TRAIL was placed on cells at concentrations ranging from 10-50ng/ml and analyzed using the LIVE/DEAD assay. Using TRAIL alone, 95 % of the MCF-7 breast cancer cells stained red, for dead, demonstrating that TRAIL was conjugated onto microcapsules via the method described, with TRAIL at concentrations ranging from 10ng/ml-50ng/ml. One control set of unmodified PLA-COOH microcapsules were also added to a set of MCF-7 cells to check that the TRAIL was inducing death and not the microcapsules themselves. As expected, no red fluorescence was observed in controls. The number of live and dead cells was observed qualitatively. The number of dead cells (red) increased with increased microcapsule-ligated TRAIL concentration, up to about 85%. This demonstrates the ability to create a modified contrast agent that will initiate cell death on cancer cells. Secondly, the concentrations of the ligand can be varied to control the amount of cell death. This can be seen as the concentration of TRAIL on the capsules increases, more of the cells appear red under microscopic examination. When no TRAIL was present cell death did not occur.

EXAMPLE 7 Poly(lactic acid) Ultrasound Contrast Agent with Tumor Necrosis (TNF)-related Apoptosis-

Inducing Ligand Surface Bound Using Thiol-Conjugation Method a) Synthesis of microbubbles

Microbubbles were prepared by a double emulsion (W/O)/W solvent evaporation process. Poly (D,L-lactic acid) (PLA) or Poly (D,L lactic-co-glycolic acid) (PLGA) (0.5Og) was dissolved in methylene chloride (10ml), and ammonium carbamate (ImI) (IM) solution was added. The polymer solution was probe sonicated at 1 1 OW for 30s. The resulting (W YO) emulsion was then poured into cold (4°C), polyvinyl alcohol solution (5%) and homogenized for 5 min at 9000 rpm. The double emulsion was then poured into a 2% isopropanol solution and stirred at room temperature for 1 hour. The capsules were then collected by centrifugation and washed three times with hexane. The capsules were flash frozen, lyophilized for 48 hours, and stored in a desiccator at -20⁰C until used. Ammonium carbamate sublimes in the lyophilizer, leaving a gas-filled void in its place thereby producing echogenic polymeric microcapsules of less than 3 microns in size as confirmed by particle size analysis (Malvern Instruments).

Alternatively, microbubbles were prepared by a double emulsion (W/0)/W solvent evaporation process using camphor (0.05g) and PLA/PLGA (0.50g) dissolved in methylene chloride (10ml), and ammonium carbonate (ImI) (4% (w/v)) solution was added. The polymer solution was probe sonicated at 1 1OW for 30s. The resulting (W/O) emulsion was then poured into cold (4°C), 5% polyvinyl alcohol solution and homogenized for 5 min at 9000 rpm. The double emulsion was then poured into a 2% isopropanol solution and stirred at room temperature for 1 hour. The capsules were then collected by centrifugation and washed three times with hexane. The capsules were flash frozen, lyophilized for 48 hours, and stored in a desiccator at -20⁰C until used. Camphor and ammonium carbonate sublime in the lypophilizer, leaving a void in their place and producing echogenic polymeric microcapsules of less than 3 microns in size. b) Echogenicity of microbubbles

The pulse-echo setup was used to determine acoustic performance of the drug loaded agents. A 12.7 mm diameter, 50.8 mm spherically focused transducer was used with a center frequency of 5 MHz. The transducer had a -6 dB bandwidth of 91 % and a pulse length of 1.2 mm. The transducer was placed in a 25°C water bath with 18.6 MΩ-cm deionized water. The transducer was then focused through an acoustically transparent window in the sample holder at a depth of 14 cm from the top of the surface. A pulser/receiver (5072 PR Panamterics Inc.) was used to generate a pulse repetition frequency of 100 Hz. Received signals were then amplified 40 dB and read in an oscilloscope (Lecroy 9350 A). Data was then stored and analyzed using Lab View 7 Express (National Instruments). c) Thiol conjugation of TRAIL to microbubbles

To poly(lactic acid) microbubbles (120mg) was added 2-(N-morpholino)ethanesulfonic acid (MES buffer) (8ml) (5OmM) (pH 5.2) and shaken to create a suspension. To N-[β- Maleimidopropionic acid] hydrazide (BMPH) ( 14.86mg) was added deionized water (ImI) for a 25umol solution. To l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (19.17mg) was added deionized water (ImI) for a 50 μmol solution. To the suspension of microbubbles and MES buffer was added equal amounts of the 25umol solution BMPH solution (ImI) and the 50 μmol solution EDC solution (ImI). The resulting solution was shaken on a rotary shaker for 30 minutes. Microbubbles were collected via centrifugation (1000xg)(10min), supernatant discarded and pellet re-suspended in MES buffer (8ml). Wash step repeated 2x. After final wash step pellet was re-suspended in phosphate buffer solution (PBS) (4ml). To this solution was added Tumor necrosis (TNF)-related apoptosis-inducing ligand (1:1000 molecular weight ratio of TRAIL:total PLA carboxyl groups). The suspension was shaken end-over-end for 1.5 hours. Resulting TRAIL conjugated microspheres were collected via centrifugation (1000xg)(10 min), flash frozen, and lyophilized for 48 hrs. d) Inducing apoptosis in breast cancer cells via ultrasound contrast agents MCF-7 and MDA-MB-231 cells were cultured in 48 well- plates. Suspensions (0.5 mg/ml) of TRAIL conjugated microbubbles (TRAEL-Mb), and controls of unmodified microbubbles (Mb) and microbubbles coupled to TRAIL using the procedure above with the omission of the chemical crosslinker BMPH and EDC (TRAIL-Mb-no Xlink) were made by adding microbubbles to cellular growth media. To each well ImI of microbubble suspended media was added (n=3). An additional control group was cells incubated with TRAIL (100ng/ml) without any microbubbles (TRAIL). Cells were incubated in a humidified, 5% CO₂ environment. At time points 3hrs, 24hrs, and 48 hrs cells were assayed using Vybrant Apoptosis Assay #2 Alexa Fluor 488 annexinV/propidium iodide (molecular probes) and photographed using an Olympus IX microscope and spot software. To assay the cells for apoptosis, each well was aspirated and washed with cold sterile 500ul PBS). To each well binding buffer (20OuI), annexin V ( 15ul), and propidium iodide (3ul) were added. The cells were incubated at room temperature 15 min, washed with annexin binding buffer (20OuI) and imaged. MDA-MB-231 and MCF-7 cells both showed little apoptosis after 3, 24, and 48 hr incubation with Mb and TRAIL-Mb-noXlink. TRAIL-Mb had significant apoptosis effect on both cell lines starting at 3 hours and continuing for 48 hours. Compared to TRAIL alone on the cells, in which full apoptosis of cell populations was seen starting at 24 hrs, TRAIL-Mb continued to induce significant apoptosis the entire experimental period. While the invention has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. REFERENCES Grell, M., Krammer, P. H. and Scheurich, P. Segregation of APO- I/Fas antigen- and tumor necrosis factor receptor-mediated apoptosis. Eur. J. Immunol. 24, 2563-2566 (1994) Grell et al., Cell 83, 793-802 (1995)

Grell et al., Lymphokine Cytokine Res. 12, 143-148 (1993) Krippner-Heidenreich et al., submitted to J. Biol. Chem. (2002) Meager, A. J. J. A cytotoxicity assay for tumor necrosis factor using a human rhabdomyosarcoma cell line. Immunol. Methods 144(1), 141-143 (1991)

Wuest, T. "Fibroblast activation protein" spezifische rekombinante

Antikorperderivate zur Tumordetektion und Therapie. Dissertation University of Stuttgart, Shaker Verlag, Aachen (2001) Wuest, T., Gerlach, E., Banerjee, D., Gerspach, J., Mossmayer, D., and Pfizenmaier,

K. TNF-Selectokine: a novel prodrug generated for tumor targeting and site-specific activation of tumor necrosis factor. Oncogene 21, 4257-4265 (2002)

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of modulating a signal transduction cascade in a cell, the method comprising: providing a site specific transmembrane acting ultrasound imaging agent which comprises (a) an echogenic microcapsule or nanocapsule, (b) a signal transduction inducing ligand associated with the echogenic microcapsule or nanocapsule and optionally (c) a therapeutic agent; providing a cell comprising a cell-surface receptor with an affinity to the signal transduction inducing ligand; initiating the signal transduction cascade in the cell by contacting the cell with the site specific transmembrane acting ultrasound imaging agent such that signal transduction inducing ligand selectively binds to the cell-surface receptor and remains associated with the echogenic microcapsule or nanocapsule and thereby modulating the signal transduction cascade in the cell; and detecting selective binding of the site specific transmembrane acting ultrasound imaging agent by using an ultrasonic imaging system.

2. The method of claim 1 , wherein the signal transduction inducing ligand is a cell damage inducing ligand.

3. The method of claim 1 , wherein the cell damage inducing ligand is a member of

TNF family.

4. The method of claim 3, wherein the cell damage inducing ligand is TRIAL (SEQ. ID. NO. 1).

5. The method of claim 1 , wherein the cell damage inducing ligand is HER-2 antibody.

6. The method of claim 1 or 2, wherein the site specific transmembrane acting ultrasound imaging agent comprises a therapeutic agent.

7. The method of claim 6 wherein the therapeutic agent is at least one of doxorubicin, docetaxel or herceptin.

8. The method of claim 1 or 2, wherein the signal transduction inducing ligand is covalently associated with the echogenic microcapsule or nanocapsule.

9. The method of claim 1 or 2, wherein the signal transduction inducing ligand is non- covalently associated with the echogenic microcapsule or nanocapsule.

10. The method of claim 1 or 2, wherein the echogenic microcapsule or nanocapsule comprise (a) an outer surface including ( 1 ) a hardened non water soluble polymer and (2) at least one hollow area formed by removal of a non-water soluble sublimable substance; and (b) an inner surface comprising at least one hollow area formed by removal of a water soluble sublimable substance, wherein said echogenic polymer microcapsule or nanocapsule are made from (i) an outer surface forming mixture comprising a non water soluble polymer and the non- water soluble sublimable substance dissolved in one or more volatile non-polar solvents and (ii) an inner surface forming mixture comprising the water soluble sublimable substance dissolved in water.

11. The method of claim 1 or 2, wherein the echogenic microcapsule or nanocapsule comprise a gas-containing or gas-generating material.

12. The method of claim 1 or 2, wherein the outer surface of the echogenic microcapsule or nanocapsule comprises at least one of poly(lactide), poly(glycolide), a copolymer of lactide and lactone, a poly(anhydride), a poly(styrene), a poly(alkylcyanoacrylate), a poly( amide), a poly(phosphazene), a poly(methylmethacrylate), a poly(urethane), a copolymer of methacrylic acid and acrylic acid, a copolymer of hydroxyethylmethacrylate and methylmethacryl ate .

13. The method of claim 1 or 2, wherein the site specific transmembrane acting ultrasound imaging agent is administered to a composition of cells, wherein only a fraction of cells comprise cells having the cell-surface receptor with an affinity to the signal transduction inducing ligand and therefore abnormal or tumorous cells can be distinguished from normal cells by the site specific transmembrane acting ultrasound imaging agent selectively binding to the cell-surface receptor of abnormal or tumorous cells.

14. The method of claim 1 or 2, wherein the site specific transmembrane acting ultrasound imaging agent is administered to a mammal in an aqueous carrier.

15. The method of claim 1 or 2, wherein the site specific transmembrane acting ultrasound imaging agent comprises a plurality of signal transduction inducing ligands.

16. The method of claim 1 or 2, wherein a mixture of site specific transmembrane acting ultrasound imaging agents having different signal transduction inducing ligands is administered to a mammal in an aqueous carrier.

17. The method of claim 6, wherein the therapeutic agent is released from the site specific transmembrane acting ultrasound imaging agent by a signal emitted by the ultrasonic imaging system.