Targeted ultrasonic contrast agents for molecular imaging and therapy

Targeted Ultrasonic Contrast Agents for Molecular Imaging and Therapy Gregory M. Lanza and Samuel A. Wickline evelopments in molecular science are extending the horizons of noninvasive medical imaging from gross anatomic description to functional cellular and biochemical information. The emerging field of molecular imaging encompasses the noninvasive in vivo diagnosis of complex pathologic processes by detection of unique molecular signatures. Moreover, localization of specific biochemical epitopes with targeted contrast agents affords the opportunity for targeted delivery and deposition of therapeutics. Combining imaging with drug delivery permits verification and quantification of treatment, ie, rational targeted therapy. Potential clinical applications for targeted contrast agents include:

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Detection of microscopic thrombi in vulnerable plaques (myocardial, venous, arterial—particularly carotid) Diagnosis of myocarditis (via certain inflammatory epitopes) Assessment of transplant rejection (heart, liver, kidney) Evaluation of unstable angina/myocardial infarction Early detection and treatment of solid tumors and metastases (colon, breast, prostate, etc) Diagnosis and treatment of inflammation (infection, arthritis, etc) Unlike a blood pool agent, a site-directed contrast agent specifically enhances the signal from pathologic tissue that would otherwise be difficult to distinguish from surrounding normal tissue. In general, assessment of molecular information requires target-specific probes, a robust signal amplification strategy, and a sensitive high-resolution imaging modality. This concept, which has been robustly applied in vitro with techniques such as polymerase chain reactions or immunohistochemistry, is now being extended to noninvasive in vivo applications to

Reprinted with permission from Prog Cardiovasc Dis 2001;44:13-31. Curr Probl Cardiol 2003;28:625-653 Copyright © 2001 by W.B. Saunders Company 0033-0620/01/4401-0002 $35.00/0 doi:10.1016/j.cpccardiol.2003.11.001

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facilitate the detection and visualization of important molecular or cellular moieties present in nanomolar and picomolar concentrations. Successful targeted contrast agents must ideally have: Long circulating half-life (ideally greater than 30 to 60 minutes) Long residence time at targeted site Sensitive and selective binding to epitopes of interest Prominent contrast-to-noise enhancement Acceptable toxicity profile Ease of production and clinical use Applicability with standard commercially available imaging modalities Promise for adjunctive therapeutic delivery (ideal) Molecular imaging is being pursed in all arenas of noninvasive medical imaging, initially with nuclear and magnetic resonance imaging approaches, and more recently, with ultrasound and optical techniques. Each noninvasive modality has unique advantages and disadvantages, and ultimately the best approach is dependent on the question being addressed. For instance, positron emission tomography is frequently used when a target substrate is amenable to positron emitter labeling, such as gancyclovir or FIAU (2⬘-fluoro-5-iodovinyl-1-␤-D-arabinofuranosyl-uracil), an inhibitor of viral thymidine kinase gene expression.1,2 In nuclear medicine, immunoscintigraphy has had limited clinical success, but today radionuclide tagging allows in vivo tracking of miniscule amounts of labeled therapeutics or delivery systems and is streamlining the drug discovery and approval process.3 Molecular imaging with magnetic resonance (MR) is emerging as a particularly advantageous modality given its higher spatial resolutions and the opportunity to extract both anatomic and physiologic information simultaneously. Like ultrasound, early MR contrast agents were blood pool agents that provided passive, temporary tumor or organ accumulation. More recently, we4 and other investigators5,6 have successfully endeavored to develop epitope-specific agents that accumulate and enhance MR detection at specific sites. Acoustic blood-pool contrast agents, first introduced by Gramiak and Shah,7 have become an established tool for enhancing two-dimensional gray scale and Doppler sensitivity. Numerous systemic contrast agents have been described, and several investigators are extending the utility of these particles for either passive (inherent, nonspecific) or active (liganddirected) targeting applications.8-11 Lanza et al12 and others13 have pursued and shown nonmicrobubble-based ligand-targeted acoustic contrast systems. In this review, we discuss various approaches to targeting and review 626

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progress to date on several targeted ultrasonic contrast/therapeutic systems under investigation in academic and industrial settings. In addition, we describe the development of multimodality (ultrasound/MR imaging [MRI]) imaging contrast/therapeutic agents and their unique clinical potential.

Targeting Mechanisms Passive Targeting Taking advantage of the body’s inherent defense mechanisms, passive targeting applications highlight or deliver therapeutics to phagocytic cells responsible for particle clearance. Macrophages are one of the most important components of the immune defense system and play a major role in clearance of particulate contrast media. These cells originate as premonocytes in bone marrow, circulate as monocytes, and localize to connective tissue (histiocytes), liver (Kupffer cells), lung (alveolar and intravascular macrophages), spleen and lymph nodes (free and fixed macrophages), bone (osteoclasts) and bone marrow, the peritoneum and nerve tissue (microglia), creating the monocyte phagocytic system (MPS). Passive phagocytosis by macrophages at all of these sites can occur, but typically, particulate contrast agents are cleared from circulation by cells in the liver, spleen, and bone marrow. On a molecular level, clearance is facilitated by opsonization with blood proteins followed by macrophage uptake of these biologically tagged particles. Opsonins may be immune, immunoglobulins and complement proteins, or nonimmune serum factors, such as fibronectin or thrombus, which bind to foreign particles and promote phagocytosis.14 In general liver sequestration appears to be complement mediated, whereas the spleen removes foreign particulate matter via antibody Fc receptors.15 This nonspecific, nondirected uptake of particles is generally referred to as passive targeting. Investigators using transcutaneous insonification to localize the destruction of microbubbles to specific anatomic regions have extended the passive targeting concept. These efforts use the cavitation of circulating microbubbles doped with therapeutic agents within their membranes, such as lipid-soluble steroids16 and chemotherapeutic compounds,17 or electrostatically bound to particle surfaces, such as antisense oligonucleotides.8,18 Further research using exteriorized spinotrapezius muscle preparations have shown that the process of microbubble cavitation also injures capillaries, destroying their vascular integrity.19 This allows a subset of particles to penetrate into the surrounding tissues and create a Curr Probl Cardiol, December 2003

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potential drug depot. The optimal ultrasound parameters (eg, frequency, peak negative pressure, etc) for maximizing such deposition is unclear. In contradistinction to ligand-targeted contrast drug delivery systems, the clinical utility of focused ultrasonic destruction of microbubbles is predicated on the a priori knowledge of the treatment site and its ready accessibility to adequate insonification.

Active Targeting Active targeting refers to ligand-directed, site-specific accumulation of contrast and/or therapeutic agents. A wide variety of ligands, including antibodies, peptides, polysaccharides, aptamers, and drugs alone or in combination, may be used to specifically bind to cellular epitopes and receptors. These ligands may be attached covalently (direct conjugation) or noncovalently (indirect conjugation) to the acoustic particle surface. Avidin-biotin interactions are extremely useful noncovalent targeting systems that have been incorporated into many biologic and analytic systems and selected in vivo applications. Avidin has a high affinity for biotin (10-15M), facilitating rapid and stable binding under physiologic conditions. Targeted contrast systems using this approach are administered in 2 or 3 steps, depending on the formulation. Typically, a biotinylated ligand, such as a monoclonal antibody, is administered first and pretargeted to the unique molecular epitopes. Next, avidin is administered, which binds to the biotin moiety of the pretargeted ligand. Finally, the biotinylated acoustic agent, for example a microbubble or nanoparticle, is added and binds to the unoccupied biotin-binding sites remaining on the avidin, thereby completing the ligand-avidin-contrast sandwich. The avidinbiotin approach avoids accelerated, premature clearance of acoustic contrast agents by the MPS system secondary to the presence of targeting antibody on its surface. Additionally, avidin, with 4 independent biotin binding sites, provides signal amplification and improves detection sensitivity. Although avidin-biotin interactions have been used extensively for targeted imaging, particularly in the field of nuclear medicine,20-22 it has met with limited success for several reasons. First, the prerequisite multistep method is time-consuming and cumbersome to implement in a clinical environment. Next, endogenous biotin competes with the biotinylated ligand for avidin binding sites and must be overcome by a marked excess of avidin. Avidin and its natural analogs are immunogenic foreign proteins derived from egg white or bacterial sources that may have clinical implications, particularly when used repeatedly over time. Furthermore, avidin, a cationic macromolecule, rapidly binds and concen628

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trates at anionic sites within the renal glomerulus basement membrane, forming in situ immunocomplexes.23 Thus, avidin-biotin conjugation techniques are ideally suited for in vitro and limited in vivo contrast research. Robust clinical applications require a simpler, one-step covalently conjugated ligand system. Targeting ligands may be chemically attached to the surface of acoustic particles by a variety of methods, depending on the nature of the particle surface. Conjugations may be performed before or after the acoustic particle is created, depending on the ligand used and its tolerance to the chemical processing conditions required to produce the ultrasonic agent. Direct chemical conjugation of ligands to proteinaceous agents often take advantage of numerous amino groups (eg, lysine) inherently present within the surface. Alternatively, functionally active chemical groups such as pyridyldithiopropionate, maleimide, or aldehyde may be incorporated into the surface as chemical “hooks” for ligand conjugation after the particles are formed. Another common postprocessing approach is to activate surface carboxylates with carbodiimide before ligand addition. The selected covalent linking strategy is primarily determined by the chemical nature of the ligand. Monoclonal antibodies and other large proteins may denature under harsh processing conditions, whereas the bioactivity of carbohydrates, short peptides, aptamers, drugs, or peptidomimetics often can be preserved. To ensure high ligand binding integrity and maximize targeted particle avidity, flexible polymer spacer arms, eg, polyethylene glycol or simple caproate bridges, can be inserted between an activated surface functional group and the targeting ligand. These extensions can be 10 nm or longer and minimize interference of ligand binding by particle surface interactions.

Potential Ligands Monoclonal Antibody and Fragments Rapid expansion of the monoclonal antibody industry has prepared the stage for the clinical success of site-targeted contrast agents by providing a plethora of ligands that can be directed against a wide spectrum of pathologic molecular epitopes. Immunoglobin-␥ (IgG) class monoclonal antibodies have been conjugated to liposomes, emulsions, and other microbubble particles to provide active, site-specific targeting. These proteins are symmetric glycoproteins (molecular weight about 150,000 d) composed of identical pairs of heavy and light chains. Hypervariable regions at the end of each of 2 arms provide identical antigen-binding domains. A variably sized branched carbohydrate domain is attached to Curr Probl Cardiol, December 2003

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complement activating regions, and the hinge area contains particularly accessible interchain disulfide bonds that may be reduced to produce smaller fragments. Bivalent F(ab⬘)2 and monovalent F(ab) fragments are derived from selective cleavage of the whole antibody by pepsin or papain digestion, respectively. Elimination of the Fc region greatly diminishes the immunogenicity of the molecule, diminishes nonspecific liver uptake secondary to bound carbohydrate, and reduces complement activation and resultant antibody-dependent cellular toxicity. Complement fixation and associated cellular cytotoxicity can be detrimental when the targeted site must be preserved or beneficial when recruitment of host killer cells and targetcell destruction is desired (eg, antitumor agents). Most monoclonal antibodies are of murine origin and are inherently immunogenic to varying extents in other species. Humanization of murine antibodies through genetic engineering has led to development of chimeric ligands with improved biocompatibility and longer circulatory half-lives. The binding affinity of recombinant antibodies to targeted molecular epitopes can be occasionally improved with selective sitedirected mutagenesis of the binding idiotype.

Phage Display Phage display techniques are now used to produce recombinant human monoclonal antibody fragments against a large range of different antigens without involving antibody-producing animals. In general, cloning creates large genetic libraries of corresponding DNA (cDNA) chains deducted and synthesized by means of the enzyme reverse transcriptase from total messenger RNA (mRNA) of human B lymphocytes. Immunoglobulin cDNA chains are amplified by polymerase chain reaction (PCR), and light and heavy chains specific for a given antigen are introduced into a phagemid vector. Transfection of this phagemid vector into the appropriate bacteria results in the expression of a scFv immunoglobulin molecule on the surface of the bacteriophage. Bacteriophages expressing specific immunoglobulin are selected by repeated immunoadsorption/ phage multiplication cycles against desired antigens (eg, proteins, peptides, nuclear acids, and sugars). Bacteriophages strictly specific to the target antigen are introduced into an appropriate vector, (eg, Escherichia coli, yeast, cells) and amplified by fermentation to produce large amounts of human antibody fragments with structures very similar to natural antibodies. Although this technology is still in an early stage of development, it has already permitted the production of unique ligands for targeting and therapeutic applications.24-27 630

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Peptides Peptides, like antibodies, may have high specificity and epitope affinity for use as vector molecules for targeted contrast agents. These may be small peptides (5 to 10 amino acids) specific for a unique receptor sequences (eg, the arginine-glycine-aspartate (RGD) epitope of the platelet GIIb/IIIa receptor)28 or larger, biologically active hormones such as cholecystokinin.29 Smaller peptides potentially have less inherent immunogenicity than nonhumanized murine antibodies.

Asialoglycoproteins and Polysaccharides Asialoglycoproteins (ASG) have been used for liver-specific applications because of their high affinity for ASG receptors located uniquely on hepatocytes.30,31 ASG-directed agents (primarily MR agents conjugated to iron oxides) have been used to detect primary and secondary hepatic tumors as well as benign, diffuse liver disease such as hepatitis.32,33 The ASG receptor is highly abundant on hepatocytes, approximately 500,000 per cell, rapidly internalizes and is subsequently recycled to the cell surface. Polysaccharides such as arabinogalactan may also be used to localize contrast agents to hepatic targets. Arabinogalactan has multiple terminal arabinose groups that display high affinity for ASG hepatic receptors.31,34

Aptamers Aptamers are high-affinity, high-specificity RNA- or DNA-based ligands produced by in vitro selection experiments (systematic evolution of ligands by exponential enrichment, SELEX).34 Aptamers are generated from random sequences of 20 to 30 nucleotides, selectively screened by absorption to molecular antigens or cells, and enriched to purify specific high-affinity binding ligands. To enhance in vivo stability and utility, aptamers are generally chemically modified to impair nuclease digestion and to facilitate conjugation with drugs, labels, or particles. Other, simpler chemical bridges often substitute nucleic acids not specifically involved in the ligand interaction. In solution, aptamers are unstructured but can fold and wrap around target epitopes, providing specific recognition. The unique folding of the nucleic acids around the epitope affords discriminatory intermolecular contacts through hydrogen bonding, electrostatic interaction, stacking, and shape complementarity. In comparison with protein-based ligands, aptamers are stable, are more conducive to heat sterilization, and have lower immunogenicity. Aptamers are currently used to target a number of clinically relevant pathologies including angiogenesis, activated platelets, and solid tumors, and their use is increasing. The clinical effectiveness of aptamers as targeting ligands for Curr Probl Cardiol, December 2003

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acoustic particles may be dependent on the impact of the negative surface charge imparted by nucleic acid phosphate groups on contrast clearance rates. Previous research with lipid-based particles suggests that negative zeta potentials markedly decrease liposome circulatory half-life, whereas neutral or cationic particles have similar, longer systemic persistence.

Microbubbles-Based Technology ImaRx Corporation Unger et al35,36 have produced a liposome-based microbubble formulation based on the demand for stable, robust blood pool ultrasonic contrast agents, originally called MRX-115 or Aerosomes (ImaRx, Tucson, AZ) and now called Definity (Dupont-Merck, North Billerica, MA). This agent, initially a nitrogen-filled liposome, later a perfluorobutane-based agent, has been shown effective for cardiac blood pool imaging and Doppler enhancement, and potentially for myocardial perfusion imaging. As is well known and extensively reviewed, microbubbles are excellent ultrasonic reflectors because of their marked acoustic impedance mismatch with the surrounding blood or tissues. In their purest form, they show a damped oscillatory behavior, comparable with a simple spring/mass system. The spring constant is analogous to the compression of the bubble gas; the mass is equivalent to the effective mass of liquid surrounding the particle; and damping is caused by the thermal and viscous effects of the gas and surrounding media. Minimum useful bubble size is dependent on the insonification frequency, surrounding background, scan depth, bubble shell characteristics, etc. Although bubbles down to 0.5 ␮m could be well visualized at 7.5 MHz, their instability would lead to almost instantaneous collapse. Typical microbubble particle sizes are in the range of 3 to 10 ␮m. As a result of this relatively large particle size, circulation half-life has been typically limited to a few minutes. Given the increasing recognition of thrombus of cardiac origin as an important etiology for stroke, the sensitive detection of intracardiac thrombi, particularly thrombi in the left atrial appendage (LAA) or left ventricular apex (mural thrombi), and microthrombi from microfissures of vulnerable carotid and aortic atherosclerotic plaques has garnered significant clinical interest. MRX 408(A1), a thrombus-targeted liposome microbubble, has been reported to enhance the acoustic detection of LAA thrombus in vivo.28,37,38 This targeted agent incorporated onto the vesicle surface a small GPIIb/IIIa-specific binding peptide with high affinity for the RGD domain of the fibrinogen receptor on activated platelets. Microscopic studies showed that the targeted microbubbles adhered to 632

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human clots and that thrombus detection was enhanced in an in vitro flow-through phantom. Continuous intravenous infusion of the lipidencased microbubbles by (2 mL/min or about 2 ⫹ 109 particles/min) helped compensate for the inherent rapid systemic destruction of microbubbles and allowed a significant enhancement of thrombus based on videodensimetric analysis to be shown in vivo. The persistence and stability of this acoustic enhancement to continuous insonification and systemic metabolism was unreported. Incident pressure from routine ultrasound imaging pulse can induce unstable oscillations and destruction of microbubbles. This apparent disadvantage has been turned to an advantage for the focused delivery of therapeutic agents and genetic material. Unger et al39 have shown that ultrasound-directed destruction of therapeutic liposomes can result in increased concentrations of drugs at desired sites relative to untreated contralateral locations. To date, this group has reported the local delivery of dexamethasone39 and paclitaxel17 by this method. Essentially, therapeutic microbubbles circulate and are selectively destroyed by ultrasound as they pass through the beam focused at a predetermined location. Drug entrapped within or on vesicles becomes more available with the destruction of the particles in the blood stream. Although most of the drug will travel with the flowing blood from the site, local uptake is enhanced by the mass-action of the higher local free concentration of compounds. Moreover, ultrasonic disruption of capillaries allows blood constituents including microbubbles to penetrate partially into extravascular spaces.19 The success of this technique is clearly dependent on knowing the precise location for drug delivery, the density of the microvasculature at the treatment site, the therapeutic threshold of drug required for clinical effectiveness, and the accessibility of the pathologic site to insonifcation. Cationic liposomes are well established vectors for genetic transfection,40,41 presumably because their positive surface charge facilitates the noncovalent attachment of phosphate-rich nucleic acids to the particle surfaces and their electrostatic interaction and sequestering to cellular glycocaylx carbohydrates.42 Ultrasound-mediated transfection of mammalian cells for human gene therapy has been reported. Sonoporation involves the use of ultrasound energy at higher levels to facilitate gene delivery and transfection based on stimulation of vascular penetration by bubbles or their remnants.43 Unger et al18 have extended these reports and shown the production of acoustically reflective cationic liposomes capable of enhancing the transfection of cultured cells with 1 MHz continuous-wave ultrasound at power levels of 0.5 watts/cm2 for 30 seconds or less. Higher-energy exposure levels were cytotoxic. Curr Probl Cardiol, December 2003

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Fig 1. Relative position of the transducer with respect to the vessel in B and C. (B) Fluorescently labeled contrast agents flowing evenly distributed in a 50-␮m mouse cremaster arteriole. (C) The velocity of the agents before insonification was approximately 7.5 mm/sec. Insonification at 5 MHz, 800 kPa, and 10 kHz PRF produces radiation forces that displace the contrast agents to the right vessel wall, induces bubble aggregation, and decreases velocity to 1 mm/sec. (Reprinted by permission of Elsevier Science from Acoustic radiation force in vivo: A mechanism to assist targeting of microbubbles, by Dayton P, Klibanov A, Brandenburger G, et al, Ultrasound in Medicine in Biology, Vol 25, pp 1195-1201, Copyright 1999 by World Federation of Ultrasound in Medicine and Biology.46)

Mallinckrodt Corporation Klibanov et al44 have prepared protein-encapsulated microbubbles that have been targeted in vitro using noncovalent avidin-biotin interactions44 and direct conjugation of anti–ICAM-1 antibody.45 In early experiments, these investigators used an inverted avidin-coated Petri dish surface as a model target and biotin as a site-specific ligand to show effective particle attachment. Later, they showed directed displacement of microbubbles using an in vivo flow model and the utility of radiant pressure from the ultrasound beam to force microbubbles against capillary walls (Fig 1).46 Recognizing that early atherogenesis is heralded by the expression of ICAM-1 adhesion molecules on the cell surface, anti–ICAM-1 antibodies directly conjugated to microbubbles were shown to attach selectively to interleukin-1␤–activated endothelium in vitro while resisting detachment with shear rate up to 1000 s-1 (Fig 2).45 Although the circulatory half-life of microbubbles is brief, Klibanov points out that only a few nanograms worth of contrast material weight (ie, mostly shell) can clearly mark the presence of the bubble on the target surface.47 Sensitive tagging of the targeted surface could be achieved with only a small portion of the site being covered. In addition to ligand-directed targeting, these investigators and their collaborators are using passive targeting. As previously discussed, the 634

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Fig 2. Fluorescent micrographs of endothelial cells exposed toeither nonspecific IgG-conjugated bubbles (A) oranti-ICAM-1– conjugated bubbles (B). (Adapted and reprinted withpermission from Villaneuva FS, Jankowski RJ, Klibanov S, et al, Microbubbles targeted to intracellular adhesion molecule-1 bind toactivated coronary artery endothelial cells. Circulation, Vol. 98, pp 1-5, 1998.45)

natural history of circulating particles is phagocytosis by various elements of the MPS system as well as other phagocytic leukocytes. Such clearance is enhanced by opsonin attachment to particle surfaces. Microbubbles may persist in the microcirculation of ischemic-reperfused myocardium secondary to ␤2-integrin and complement-mediated binding to activated leukocytes adherent to venular walls (Fig 3). Further work has shown that inflammatory cells (Fig 4) can phagocytose these particles and be detected to oscillate with ultrasound (Fig 5). Therefore, the potential exists to track these cells and identify regions of inflammation or to deliver therapeutic agents directly to phagocytic cells. This approach is analogous to prior efforts with liposomal particles passively targeted to treat parasitic disease within the MPS, to enhance vaccination efficiency, or to pharmacologically activate macrophages into tumoricidal, microbicidal, or virucidal states.48-50 In selected instances, marked improvements in therapy have been reported. In fact, some drugs can survive macrophage uptake and be released back into the circulation to therapeutic advantage. Unfortunately, most targeted compounds are not intended for these tissues and are rapidly metabolized through leukocyte endosomal digestion.

Perfluorocarbon Exposed Sonicated Dextrose Albumin Microbubbles Like other microbubble formulations previously described, perfluorocarbon exposed sonicated dextrose albumin microbubbles (PESDA) Curr Probl Cardiol, December 2003

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Fig 3. Transillumination (left panels) and fluorescent epi-illumination (right panel) fluorescein-labelled albumin microbubbles in venules after ischemia reperfusion and TNF-␣ inflammation. Twenty-five percent adhesion of microbubbles after ischemia reperfusion increased to 70% with TNF-␣. (Reprinted with permission from Lindner JR, Coggins MP, Kaul S, et al, Microbubble persistence in the microcirculation during ischemia/reperfusion and inflammation is caused by integrin- and complement-mediated adherence to activated leukocytes. Circulation, Vol. 101, pp 668-675, 2000.85)

Fig 4. Images obtained on light microscopy showing lipid microbubble attachment to the surface of activated neutrophils at 3 minutes and phagocytosis of microbubbles by 15 minutes. At 30 minutes, microbubbles remain intact intracellularly. Scaling bars ⫽ 2.5 mm. (Reprinted with permission from Lindner JR, Dayton PA, Coggins MP, et al, Noninvasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles. Circulation, Vol 102, pp 531-538, 2000.86)

microbubbles can be used as a blood-pool agent to enhance cardiac anatomy and Doppler signals and to aid in the detection of myocardial perfusion abnormalities. Recently, Porter et al8 have electrostatically bound anionic oligonucleotides to the protein surface of PESDA particles, presumably complexed to exposed amino groups.8 This binding is reported to be better for perfluorocarbon-filled particles rather than air 636

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Fig 5. Radial oscillations of free and phagocytosed microbubbles in response to a 3-cycle sinusoidal pulse at 2.25 MHz, 500 kPa. A-C show the response of a free (nonphagocytosed) phospholipidshelled microbubble, and D-F show the response for a microbubble of the same type phagocytosed by a neutrophil. C and F are “streak” images, which are radius-time curves of the bubble produced with a high-speed camera with 10 nanoseconds temporal resolution. A, B, D, and E show the bubbles before insonification and during maximum expansion, and correspond to times during the streak image as indicated by the arrows. (Data from Dayton et al81)

microbubbles. One might speculate that the hydrophobic nature of the perfluorocarbon core encourages an outward orientation of hydrophilic functional groups, eg, amino groups, and an inward rotation of hydrophobic protein regions. In vitro experiments have shown that antisense oligonucleotides bound to PESDA are dispersed on ultrasonic microbubble destruction. Ultrasound-facilitated myocardial deposition of antisense nucleotides to fibroblast growth factors bound to PESDA has been shown in a Langendorf preparation.51 Additional early in vivo studies further have illustrated the concept of PESDA-facilitated oligonucleotide uptake into hepatocytes.52 Antisense to cytochrome P450 3AW, an inhibitor of cytochromemediated benzodiazepine metabolism, passively conjugated to PESDA was shown to extend sleep time response of rats given midazolam (0.06 mg/kg) versus animals treated with an equivalent intravenous dose of the native oligonucleotide.52 Further provocative studies by this group have shown the inhibition of c-myc, an early response gene regulator of vascular smooth muscle proliferation and collagen synthesis,53 after carotid and coronary balloon overstretch injury. In an initial study involving carotid balloon injury of 21 pigs, the effectiveness of c-myc antisense PESDA, given intravenously with supplemental ultrasound (20 kHz), was compared with c-myc alone and negative controls. At 30 days postinjury, pigs receiving antisense PESDA had significantly larger vessel lumens and less intimal hyperplaCurr Probl Cardiol, December 2003

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sia than controls.54 However, in the coronary circulation, supplemental 20-kHz ultrasound was detrimental and significantly provoked intimal hyperplasia after 30 days. The physiologic basis of these results is unclarified, but the investigators suggest that ultrasonic cellular damage by the low frequencies incited an enhanced inflammatory response to the overstretch injury.55 Continuing studies suggest that intravenous c-myc antisense PESDA microbubbles without additional ultrasound may have the greatest potential for inhibiting intimal hyperplasia after coronary balloon injury.56

Nonmicrobubble-Based Technology Nongaseous Acoustically Reflective Liposomes McPherson et al at Northwestern University have developed a nongaseous, site-targeted, acoustically reflective liposome formulation by controlling lipid composition and production method.57 These liposomes typically consist of 60:8:2:30 molar mixtures of phosphatidylcholine (PC): phosphatidylethanolamine (PE): phosphatidylglycerol (PG): cholesterol (CHOL) that are prepared by a dehydration/rehydration method (ie, lyophilization and resuspension). The acoustic particles formed are multilamellar liposomes characterized by entrapment of numerous small irregularly sized vesicles (ie, a raspberry-like appearance), in contrast to nonreflective liposomes that have an onion-like morphology. The unusual morphology of these acoustic vesicles is driven by the presence of phosphatidylethanolamine, which has a propensity for inverted hexagonal conformation,58,59 as well as the rapid reconstitution of the liposomes from a lyophilized mixture of liposomes in mannitol. Although the mechanism of acoustic reflectivity is presently undefined, it appears to be dependent on the final, structural morphology of the lipid vesicles. These particles are less than 1 ␮m in diameter and retain their echogenicity filtered through 0.2-␮m polycarbonate filters, circumstances inconsistent with a stable, gaseous basis of echogenicity. The Northwestern group showed the conjugation of immunoglobulin to these liposomes with retention of echogenicity and bioactivity in vitro. Acoustically reflective liposomes have been targeted to intercellular adhesion molecule-1 (ICAM-1), a molecular endothelial marker of atherosclerotic fatty and fibrofatty plaque.60 In these studies, ICAM-1– targeted liposomes were released by catheter proximate to atherosclerotic carotid or ileofemoral arterial lesions, and binding to plaques was shown with both 20-MHz intravascular and 7.5-MHz transcutaneous ultrasound. Recently, these investigators have extended this research and reformu638

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Fig 6. Acoustic properties of cationic liposomes as compared with water, Albunex, and previously reported echogenic anionic liposomes. (Reprinted with permission from Tiukinhoy SD, Mahowald ME, Shively VP, et al: Development of echogenic, plasmid-incorporated, tissue-targeted cationic liposomes that can be used for directed gene delivery. Invest Radiol 35:732-738, 2000.61)

lated their agent to produce ICAM-1–targeted cationic liposomes for gene delivery applications.61 The component lipids were a 60:10:30 molar mixture of ethyldioleoylphosphatidyl-cholinium triflate (EDOPC): PE: and DC-CHOL [3␤-(N-dimethylaminoethylcarbamate)], essentially 90 mol% cationic lipid plus 10% PE. These cationic particles retain their acoustic characteristics, and conjugation with the ligand did not detract from their ultrasonic reflective properties (Fig 6). Transfection of human umbilical vascular endothelial cells (HUVEC) with a firefly luciferase encoding plasmid electrostatically bound to acoustic cationic liposomes significantly enhanced cell transfection (P ⫽ .007), as compared with plasmid precipitated with calcium phosphate or naked DNA. Transfection efficiency of the acoustic cationic liposomes was similar to Transfectam and significantly better than Tfx-50 reagent, and all were less effective than Lipofectamine (Fig 7).

Perfluorocarbon Emulsion Nanoparticles We have developed a multidimensional targeted nanoparticle platform that addresses many of the issues that have led most targeted imaging and drug delivery approaches to fail. The novel agent is a ligand-targeted, lipid-encapsulated, nongaseous perfluorocarbon emulsion produced through microfluidization techniques, and is robustly stable to handling, pressure, atmospheric exposure, heat, and shear forces. Unlike microbubble formulations that are naturally echogenic, these nanoparticles have poor inherent acoustic reflectivity until concentrated on the surfaces of tissues or membranes (eg, clots, endothelial cells, smooth muscle cells, synthetic membranes, etc). This provides marked improvement in conCurr Probl Cardiol, December 2003

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Fig 7. Gene expression of HUVEC using standard lipid vectors and cationic acoustic liposome. Values for relative light units/well are log 10 units. (Reprinted with permission from Tiukinhoy SD, Mahowald ME, Shively VP, et al: Development of echogenic, plasmid-incorporated, tissue-targeted cationic liposomes that can be used for directed gene delivery. Invest Radiol 35:732-738, 2000.61)

trast signal without increasing the background level. In the early phases of development, we coupled a pretargeted biotinylated ligand to a biotinylated version of the nanoparticle through avidin-biotin interactions (discussed above). Subsequently, we adopted a direct ligand conjugation approach using monoclonal F(ab) fragments to facilitate future clinical implementations (Fig 8). The emulsion nanoparticles have long circulatory half-lives because of their small size and inherent in vivo stability without further modification of their outer lipid surfaces with polyethylene glycol or incorporation of polymerized lipids. Surfactant modifications often detract from targeting efficacy to extend circulatory persistence. The in vivo clearance of the nanoparticles was measured in dogs by quantification of the blood perfluorocarbon content. Nanoparticle distribution and clearance data fit well to a biexponential function with an estimated half-life in vivo of 1 hour. Preliminary data suggest that this novel agent will persist bound to tissue for hours, and dependent on location, even for days. The nongaseous particles are not susceptible to destruction by the incident acoustic pressures that easily cavitate or deform microbubbles during ultrasonic imaging. As previously discussed, intravascular and intracardiac thromboses are important etiologies for stroke and infarction. We have shown marked acoustic enhancement of thrombi created and targeted in vitro (Fig 9) and in vivo in a canine model (Fig 10).12 We have shown that the increased acoustic reflectivity benefits are readily achieved at frequencies typically used for clinical transcutaneous scanning (3.5 to 7.5 MHz) as well as at 640

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Fig 8. Multidimensional platform technology permiting molecular imaging of a wide variety of pathologies. A multiplicity of ligands may be coupled to nanoparticles directly via covalent bonds or indirectly through avidin-biotin interactions. Ultrasonically reflective nanoparticles can be complexed to paramagnetics for MR or radionuclide for nuclear multimodal imaging. Drugs or DNA may be placed on the surface or incorporated within the particle.

higher frequencies used for intravascular ultrasonic imaging (12 to 30 MHz).12 Scanning electron micrographs of the in vitro binding of the emulsion nanoparticles to fibrin strands of whole blood clots confirmed the specificity of targeting (Fig 11). We have used advanced techniques in high-frequency acoustic microscopy to establish a first-approximation physical model to elucidate the major principles governing the magnitude of acoustic reflectivity produced by our agent.62 The enhanced acoustic reflectivity of the nanoparticulate emulsion is derived from the collective deposition of the emulsion particles along various tissue planes, creating a layer or layering effect. The nanoparticle emulsion cores show very slow acoustic velocity (about 670 m/sec) and high density (1.7 g/mL) when compared with water and surrounding tissues (about 1,540 m/sec and 1 g/mL, respectively). An acoustic transmission line model (Fig 12) can estimate the magnitude of acoustic enhancement. This model describes the influence of emulsion layer density, speed of sound, and thickness on the increase in acoustic reflectivity. We have characterized the acoustic properties of a wide variety of potential perfluorocarbons and selected and formulated a series of perfluorocarbon compounds of Curr Probl Cardiol, December 2003

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Fig 9. Ultrasonic images (7.5 MHz) of plasma clots pretargeted with antifibrin monoclonal antibody and exposed to control or biotinylated perfluorocarbon emulsion in vitro. The supporting suture (s) and thrombus (t) are clearly delineated. Before incubation with control or biotinylated perfluorocarbon emulsion, the acoustic reflectivity of the clots was low. Application of the control emulsion did not enhance the acoustic reflectivity of the control clots (A); whereas, the biotinylated emulsion greatly increased the echogenicity of the targeted clot (B). The acoustic enhancement imparted by the biotinylated emulsion is appreciated around the surface of the clot, reflecting the inability of the emulsion particles to penetrate into the clot. (Reprinted with permission from Lanza G, Wallace K, Scott M, et al: A novel site targeted ultrasonic contrast agent with broad biomedical application. Circulation 94:3334-3340, 1996.12)

Fig 10. The acoustic enhancement of canine femoral artery thrombus, targeted with biotinylated antifibrin antibody, before (A) and after (B) exposure to biotinylated perfluorocarbon emulsion. The acute arterial thrombus is poorly visualized with a 7.5-MHz linear array, focused transducer. The transmural electrode (a) and the wall boundaries of the femoral artery (f) are clearly delineated. After exposure to the biotinylated emulsion, the thrombus is easily visualized. The anode (a) produces an ultrasonic shadowing effect in the midportion of the contrast-enhanced thrombus. (Reprinted with permission from Lanza G, Wallace K, Scott M, et al: A novel site-targeted ultrasonic contrast agent with broad biomedical application. Circulation, Vol 94, pp 3334-3340.12)

varying acoustic impedance.63,64 Recent studies show that all liquid perfluorocarbon emulsions significantly increase target acoustic reflectivity when bound and that the magnitude of enhancement may be manipulated by formulating with perfluorocarbons of different phase velocity. We have extended our research efforts in ligand-directed ultrasonic 642

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Fig 11. Scanning electron micrographs of fibrin-targeted perfluorocarbon nanoparticles bound to fibrin strands of red blood cell clots. Left, control clot. Middle and right, red blood cell clots after targeted nanoparticle agent. (Adapted and reprinted with permission from Hall CS, Lanza GM, Rose JH, et al, Experimental determination of phase velocity of perfluorocarbons: Applications to targeted contrast agents. IEEE Trans Ultrason Ferroelectr Freq Contr 47:75-84, 2000.87)

Fig 12. Theoretical acoustic transmission line model where Rxy and Txy represent the reflection and transmission coefficients between layers x and y, the wave number of the insonifying wave in layer 2 is k, and d is the dimension of layer 2. (Adapted and reprinted with permission from Lanza G, Trousil R, Wallace K, et al, In vitro characterization of a novel, tissue-targeted ultrasonic contrast system with acoustic microscopy, Journal of Acoustical Society of America, Vol 104, pp 3665-3672, Copyright 1998, Acoustical Society of America.62)

contrast agents and drug delivery to address the “Achilles’ heel” of angioplasty, postprocedural restenosis. We have used targeted acoustic nanoparticles to detect tissue factor, a 43 kD transmembrane glycoprotein responsible for initiating the coagulation cascade65,66 in vivo with intravascular ultrasound (Fig 13).67,68 St Pierre et al69 and others70 have shown that tissue factor is increased in the tunica media of arteries in response to overstretch injury and that specific inhibition of tissue factor–mediated coagulation attenuates neointimal thickening.71 Small Curr Probl Cardiol, December 2003

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Fig 13. High-frequency intravascular ultrasonic images of carotid arteries exposed to tissue factor–targeted or control emulsion nanoparticles after angioplasty. (A) Bright acoustic enhancement in a portion of the vessel wall consistent with nonuniform overstretch injury with the balloon. (B) Lack of acoustic enhancement in the vessel wall after control contrast. (Reprinted with permission67)

size permits the penetration of the contrast nanoparticles through microfractures created by stretch injury, and localized retention of the nanoemulsion creates “acoustic staining” by increasing the ultrasonic backscatter from regions of tissue factor expression. Immunohistochemical analysis showed patterns of tissue factor expression within the injured and uninjured vessels consistent with the acoustic contrast enhancement observed, and scanning electron microscopy confirmed the specific targeting of the nanoparticles to tissue factor constitutively overexpressed by an arterial smooth muscle cell line (Fig 14). Beyond detecting the presence of injury to the arterial wall, molecular imaging of intra-arterial tissue factor expression could uniquely characterize the severity of injury and the magnitude of the response. Accurate profiling of tissue factor expression in injured arterial walls may prove to be an important predictor of subsequent restenosis. We are currently introducing classic lipophilic antiproliferative agents into the nanoparticle formulation and have shown their inhibitory effects on vascular smooth muscle cell proliferation in culture (unpublished). These early results suggest that targeted therapeutic nanoparticles can provide localized prolonged release of potent therapeutic agents. Delivery of minute but effective quantities of drugs may be facilitated by prolonged, intimate contact of the bound nanoparticles with the targeted 644

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Fig 14. Scanning electron micrographs (12,000x) of porcine aortic smooth muscle cells known to overexpress cell surface tissue factor in culture. (A) Cells exposed to phosphate buffer as a control. (B) Cells exposed to unconjugated control emulsion. (C) Cells exposed to tissue factor–targeted immunoemulsion. (D) Cells pretreated with anti-tissue factor antibody and subsequently exposed to tissue factor–targeted immunoemulsion. (Reprinted with permission from Lanza GM, Abendschein Dr, Hall CH, et al. Molecular imaging of stretch-induced tissue factor expression in carotid arteries with intravascular ultrasound. Invest Radiol 35:227-234, 2000.68)

cells and an exchange of lipids and drug between the lipid surfaces. Prevention of vascular smooth muscle migration and proliferation may reduce neointimal formation and provide a clinically convenient mechanism to address the issue of arterial restenosis after angioplasty. Curr Probl Cardiol, December 2003

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Fig 15. Enhancement of femoral artery thrombus with fibrin targeted ultrasound/MRI.

Multimodality Imaging With Emulsion Nanoparticles In addition to acoustic imaging, we have coupled our acoustic particle with paramagnetic compounds and created a bifunctional ultrasonic contrast agent that can be used in combination with MRI. The potential dual-modality nature of the agent could allow, for example, tumors or lymph nodes initially detected by MRI to be biopsied or surgically resected using ultrasound guidance. The strengths of ultrasound, ie, flexibility, high resolution, portability, and ease of use, create the perfect adjunct to less versatile imaging methods. One can easily envision alternative situations in which ultrasound is used to screen for tumor, such as prostate or breast carcinomas, and MRI is used in individuals with positive test results to confirm, define, and ascertain the stage of disease. Many site-directed MR agents have been previously studied, but virtually all have failed to overcome the partial volume effects of imaging because of inadequate paramagnetic payloads.5 Tens of thousands of paramagnetic atoms can be bound to the surface of each nanoparticle, resulting in dramatic T1-weighted contrast effects for MRI. In our first studies with our targeted MRI agent, we incorporated a lipid-conjugated gadolinium-diethylenetriaminepentaacetic acid (DTPA) complex into the surfactant layer (Fig 15).4 Partially occlusive canine femoral artery 646

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Fig 16. Photomicrograph of rabbit cornea showing vessel growth induced by bFGF pellet. (B) Percent change in mean MR signal intensity versus time for ROI depicted in C and D. Line A: Anti-␣v␤3 antibody conjugated to nanoparticles (DM101) (n ⫽ 4). Line B: Saline control (n ⫽ 5). Line C: Isotype-matched irrelevant antibody-conjugated to nanoparticles (M399) (n ⫽ 6). Line D: Preblocked with anti-␣v␤3 antibody before targeted nanoparticles. C and D are gradient echo T1-weighted MR images of corneal capillary bed before (C) and after (D) ␣v␤3-targeted paramagnetic-acoustic nanoparticles. (Abbreviations: bFGF, basic fibroblast growth factor; ROI, region-of-interest; adapted and reprinted with permission from Anderson SA, Rader RK, Westlin WF, et al: Rapid magnetic resonance contrast enhancement of neo-vasculature with ␣v␤3 targeted nanoparticles. Magn Reson Med 44:433-439. Copyright 2000. Reprinted with permission of Wiley-Liss, Inc, a subsidiary of John Wiley & Sons, Inc.82)

thrombi were exposed to the bifunctional agent in situ. The thrombi were clearly imaged and detected with ultrasound in vivo, then excised, fixed in formalin, and readily detected by MRI. The locations, shapes, and sizes of the MRI and ultrasonic images correlated well with optical images of the same vessels. We have further refined our MRI-acoustic agent in vitro to optimize the concentration of gadolinium borne on the particle surface.72-74 Targeted diagnosis and treatment of solid tumors remain the “Holy Grail” of molecular imaging and treatment. However, the dependence of solid tumor growth on neovascularization75 and the integrin ␣v␤376-80 have created a potential molecular signature for targeting applications. Curr Probl Cardiol, December 2003

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We82 and others83,84 have shown noninvasive detection of the ␣v␤3 integrin expression on neovascular endothelial cells with magnetic resonance (Fig 16). We expect the opportunity for simultaneous ultrasonic detection to provide important flexibility in the diagnosis and treatment of early or metastatic tumors with notable cost-benefit advantages.

Conclusion Targeted contrast agents take several physical forms and can accomplish site-directed imaging or therapy by a variety of active and passive mechanisms. Ligand-targeted particles provide the opportunity to detect the expression of pathognomonic cell-surface molecular signatures present in nanomolar or picomolar quantities. These agents have been conceptually shown to improve the diagnosis of early atherosclerosis, vulnerable plaques, intracardiac thrombus, and angiogenesis, and could be in clinical trial for these applications within the next few years. In addition, the particulate nature of these agents creates an ideal platform for targeted drug delivery of steroids, antineoplastics, and oligonucleotides with the opportunity for rational therapeutic dosing, a feature unique to drug delivery coupled with molecular imaging. The opportunity to synergize ultrasonic imaging with other noninvasive imaging modalities, such as MRI, is creating an exciting clinical opportunity and flexibility unachievable with either modality alone. Multimodal molecular imaging and rational, targeted drug delivery will likely change the future of clinical medicine as these technologies continue to mature.

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