Bioconjugate Chem. 2005, 16, 9−17
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Ligand-Carrying Gas-Filled Microbubbles: Ultrasound Contrast Agents for Targeted Molecular Imaging† Alexander L. Klibanov* Cardiovascular Division, University of Virginia, Charlottesville, Virginia 22908. Received April 24, 2004; Revised Manuscript Received December 8, 2004 INTRODUCTION
Molecular imaging is heralded as the solution to many problems of current medicine (1). It is supposed to offer convenient, universal, high-resolution, and affordable diagnostic imaging insight into the in vivo biological processes on the molecular and cellular level, aiding with personalized, targeted, and image-guided therapeutic interventions. Contrast agents usually suggested for molecular imaging are radioisotopes, paramagnetic materials, or fluorescent dyes, designed for SPECT/PET, MRI, or infrared optical imaging systems, respectively. Ultrasound as an imaging modality offers a cache of advantages over those techniques; therefore, development of molecular imaging contrast agent for ultrasound will result in many benefits. Ultrasound imaging is (1) a real-time modality (routinely, 20-30 frames/s or more); (2) consistently used in image-guided biopsy and therapy, such as cryoablation, (3) inexpensive and popular; equipment is widely installed worldwide, and (4) portable: equipment can be taken to the bedside, ambulance, battlefield, or private practice office (including laptop and hand-held ultrasound units). The concept of ultrasound contrast agent for molecular (targeted) imaging consists of using contrast particles with a specific ligand to the target receptors that are expressed in the area of disease. Targeted contrast is administered intravenously, circulates in the bloodstream, and accumulates in the area of interest via a ligand-receptor interaction. Free circulating agent clears from the bloodstream, and targeted agent signal is then used to demarcate the site and condition of the target diseased tissue (a simplified picture of such imaging is presented on Figure 1). Ultrasound imaging equipment is widely available. Contrast materials for intravenous use are already approved; therefore, targeted agents capable of molecular imaging represent a logical extension of the existing technology. In this review, we will discuss the chemistry of the preparation of the most efficient targeted ultrasound contrast materials, techniques for ligand attachment, and application of these agents for molecular imaging. In this limited format it is not possible to provide the references * Corresponding author: Alexander L. Klibanov, UVA Cardiovascular Division, Cardiovascular Imaging Center, P.O. Box 800158, Hospital Drive, Cobb Hall, Room 1026, Charlottesville, VA 22908-0158. Telephone: (434)243-9773, telefax (434)9823183, e-mail
[email protected]. † Part of the Special Issue collection on Imaging Chemistry that began in issue 6, 2004. A preliminary description of this work was presented at the Symposium on Chemistry and Biological Applications of Imaging Agents and Molecular Beacons, at the spring 2004 National Meeting of the American Chemical Society.
Figure 1. General outline of targeted ultrasound imaging of ligand-carrying microbubble retained on a receptor-coated target surface.
to all the existing literature in the field; examples will be provided to demonstrate the general development trends. Ultrasound Contrast Particle Design: Physical Considerations and Sensitivity. Ultrasound imaging is performed by signal processing of the reflections of sound waves sent toward the tissues within the body. For general clinical diagnostic imaging 1-10 MHz frequency ultrasound is used, which implies sub-millimeter to millimeter wavelength range and comparable spatial resolution. Higher frequencies, up to 20-50 MHz, provide much higher resolution (down to tens of micrometers) at the expense of penetration depth and are applied for imaging of specific organs, such as in ophthalmology, or in the intravascular probes. Lower frequency ultrasound, ∼1-3 MHz, allows deep penetration within the body. High frequencies provide limited penetration, only several millimeters. Ultrasound scattering by the tissues and borders between tissues is dependent on the mismatch of the acoustic impedance, i.e., difference between the product of the speed of sound in the medium multiplied by the density of that medium (2). Therefore, contrast agent particles which present the highest echo response would be the ones that possess the highest acoustic impedance mismatch between the bulk medium (blood has acoustic impedance quite close to water) and the particle microphase. Several types of contrast particles have been suggested for ultrasound imaging, including liquid-core microemulsions and nanoemulsions (3), liposomes (4), and gas-filled microbubbles, with the average size of several micrometers (5-7). Gas-filled microbubbles offer the highest acoustic impedance mismatch and the highest recorded backscatter signals in ultrasound imaging (2, 8). Liquid
10.1021/bc049898y CCC: $30.25 © 2005 American Chemical Society Published on Web 12/30/2004
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Klibanov
Figure 2. Ultrasound imaging of an individual targeted microbubble immobilized on a Petri dish, using Philips HDI 5000 system, equipped with a P4-2 transducer. (A) Microbubble imaging at MI ) 0.2. (B) Dish after microbubble destruction. Reprinted with permission from ref 11. Copyright 2004 Lippincott, Williams and Wilkins.
contrast emulsion particles offer lower acoustic impedance mismatch but enhanced stability and prolonged circulation time (3). Multilamellar liposome-based agents were initially hypothesized to provide acoustic response due to their multilamellar lipid structure (4); later, it was shown that their acoustic response was caused by incorporated pockets of gas phase (9). Such liposomes offer acoustic response that is several orders of magnitude lower than that of microbubble-based agents (10). Overall, microbubbles offer the best detection sensitivity and some of these contrast agents are already approved for blood pool indications. Hence, the main focus of this review will be the discussion of the microbubble-based contrast materials, the specific chemistry of their preparation, ligand coupling, and actual molecular imaging applications. Sensitivity of the detection of gas-filled microbubbles by ultrasound is quite high, even in comparison with other modalities suggested for molecular imaging. Typical approved intravenous human dose of ultrasound contrast microbubble material is at sub-milligram level. Actual detectable amount is considerably less. Experimental detection of individual microbubbles with diagnostic imaging clinical ultrasound systems has been reported (8, 11). An estimated mass of the material contained in the shell and gas phase of an average microbubble is about a picogram; it can be clearly observed by ultrasound imaging of targeted microbubble in an in vitro model system (Figures 1 and 2). Within a certain microbubble concentration range, ultrasound backscatter response demonstrated linear dependence on concentration. Therefore, one can expect successful delineation of the areas of microbubble accumulation in the target tissues even if the number of actual targeted tracer particles is low (few thousands of selectively retained microbubbles definitely could be detected at the target area). Additional imaging and postprocessing schemes can be applied to improve detection of targeted microbubbles on the background of normal tissue (the latter may be actually useful to pinpoint the location of the targeted bubbles in relation to anatomical markers and body structures). One important approach is image subtraction, when an image frame is acquired prior to contrast administration, and next one is obtained when targeted contrast accumulates in the area. Frames are aligned properly, and images are subtracted, so that the targeted contrast signal that constitutes the difference between the frames can be recognized easily. To aid in this
procedure, targeted bubble destruction at high ultrasound intensity is often used (12). This way, there is no need to wait for many minutes from obtaining the original blank image to acquiring the “targeted” image, with the probe fixed at the same spot. After the “targeted” frame, all the targeted accumulated bubbles can be cleared by ultrasound irradiation, and the control frame is taken just a few seconds later (Figure 3A vs Figure 3C provide an in vitro model comparison). Another advantage of high-intensity destructive ultrasound imaging is a considerably higher acoustic response achieved in these conditions (Figure 3B). When nondestructive imaging of microbubble contrast is performed at a low ultrasound mechanical index (Figure 3A), the signal from targeted microbubbles is relatively low (arrow on the images pointing toward microbubble location), but quite visible and detectable as compared with control image where bubbles were destroyed, Figure 3C. When bubbles are irradiated with a higher-intensity ultrasound pulse, they are destroyed. Actual intensity and frequency of ultrasound pulses applied for bubble destruction and imaging may have to take into account the potential bioeffects of ultrasound (see below). During destruction, bubbles emit a bright acoustic flash at various frequencies. This signal can be detected by the imaging system very efficiently (Figure 3B). The microscopy image of the plate prior to insonation showed that only a small fraction (several percent) of the target surface is covered with targeted bubbles; just a few bubbles as observed optically (Figure 3D) are sufficient to obtain the high signal-to-noise ultrasound images. Overall sensitivity of the ultrasound for the detection of targeted contrast should be sufficient for molecular imaging. Ultrasound Contrast Particle Design: Microbubble Preparation and Storage Stability. When microbubbles were first proposed as contrast agents, they did not carry a shell; such nonstabilized bubbles could survive only while they were surrounded by the aqueous medium, either normal saline (13) or viscous X-ray contrast (14). Such bubbles would fuse with each other and with the headspace air above the volume of the aqueous medium in the vial and rapidly disappear even prior to administration in the patient. An inadequate level of storage stability required in situ bubble generation in the immediate proximity to the patient location. Improvement of microbubble formulation allowed the preparation of microbubbles stabilized with a lipid shell, typically, a lipid monolayer. Most often, lipids applied for microbubble coating possess a high transition tem-
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Reviews
Figure 3. Ultrasound contrast imaging of biotinylated bubbles targeted to a streptavidin-coated Petri dish. A. Imaging at low ultrasound intensity (MI ) 0.1). B. Imaging at high ultrasound intensity destructive to microbubbles (MI ) 1.5). C. Imaging at low ultrasound intensity (MI ) 0.1) after microbubble destruction. Location of microbubbles on the dish marked by arrow. D. Photomicrograph of targeted microbubbles on a dish prior to destruction. Table 1. Commercially Available Ultrasound Contrast Microbubbles Currently in Clinical Practicea agent name (manufacturer) Levovist (Schering AG) Albunex (MBI/Mallinckrodt) Optison (Mallinckrodt/ Amersham) Definity (Bristol Myers Squibb Medical Imaging) Imagent (Alliance/ Photogen) SonoVue (Bracco) a
gas core
shell composition
design
air
palmitic acid
dry galactose precursor matrix
air
albumin
octafluoropropane
albumin
octafluoropropane
phospholipids (DPPC, MPEG-DPPE, DPPA) phospholipid (DMPC) phospholipids (DSPC, DPPG, palmitic acid)
albumin shell bubbles stored in albumin aqueous saline albumin shell bubbles stored in albumin aqueous saline precursor: aqueous liposomal solution in a vial with C3F8 headspace dry precursor matrix
N2/perfluorohexane vapor sulfur hexafluoride
dry precursor matrix
Contrast agent names are trademarks by respective manufacturers.
perature (e.g., DPPC, 42 °C, or DSPC, 56 °C). Monolayers of such lipids are in gel phase and do not fuse as easily as do monolayers of fluid-phase lipids. Lipid-stabilized microbubbles possess somewhat improved stability over shell-free bubbles. To improve microbubble storage stability even further, we need to prevent direct contact between individual microbubbles and thus reduce the rate of shell fusion and bubble coalescence. This is achieved either by adding electrostatically charged molecules as a part of the microbubble shell (typically as 5-15 mol % of a negatively charged lipid within the bulk zwitterionic lipid) (15) or by grafting a surface brush of hydrophilic polymer, such as poly(ethylene glycol) (PEG) on the microbubble surface (16). PEG brush is similar to the coating of long-circulating liposomes, an established drug delivery system with prolonged circulation time and reduced uptake to Kupffer cells and macrophages. PEGcoated microbubble preparations can be stored for up to several years without a substantial loss of ultrasound contrast response, in our experience. Actual microbubble preparation is typically accomplished by high-shear (such as probe-type sonication) dispersing of gas in the aqueous medium that contains lipids in a micellar or liposomal form. A most primitive but efficient dispersion apparatus consists of two disposable polypropylene syringes, partly filled with gas and
aqueous lipid mixture and connected to each other via a partially closed stopcock (17). Liquid and gas are forced from one syringe to another by back and forth movement of the plungers; high shear and resulting microbubbles are generated in the narrow connector orifice. The size of the manufactured bubbles is dependent on the shear flow and also on the composition of the stabilizer lipid. A more sophisticated and widely applied method consists of sonication of the aqueous medium with a probe-type ultrasound disintegrator in the atmosphere of a desirable gas. When the gas is dispersed in the aqueous micellar medium, freshly generated contact surface between the gas and liquid phases is immediately coated and stabilized with the lipid monolayer to reduce surface tension. As individual microbubbles are closed up, they can no longer fuse with their neighbors due to the presence of the newly deposited shell. Typically, instead of air that was used in the early generation bubble preparations, fluorine-containing inert insoluble gases (such as decafluorobutane, octafluoropropane, or sulfur hexafluoride) are predominantly applied in the second generation microbubble materials (see Table 1 for the characteristics of some microbubble contrast agents available). Commercially developed (nontargeted) microbubbles applied as the markers of circulating blood are stored usually as precursors, mostly dry preparations that
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require addition of water for reconstitution prior to injection (15) and can only be used within several hours after reconstitution. However, some of the bubble preparations can withstand prolonged (years) storage in the aqueous phase; this requires a thicker shell, such as denatured albumin (6) or a shell composed of gel-phase lipid combined with PEG brush coating (18) to avoid microbubble fusion. Overall, storage stability of ultrasound contrast microbubble agents is now comparable with that of the agents used in other molecular imaging modalities, and cumbersome preparation or express delivery from the manufacturer to the hospital is not required. Ultrasound Contrast Particle Design: Ligand Attachment Strategies. There are two general strategies for the attachment of the targeting ligand to the microbubble shell. In the first technique, the ligand is in a separate procedure coupled to the shell-forming molecule or its precursor (such as a lipid residue), to be used as an anchor to retain the ligand attached to the shell monolayer. The bioconjugate is then mixed with the bulk material suited for shell preparation, and bubbles are generated as described above. This approach works well for small organic molecule ligands, such as peptides, peptide mimetics, carbohydrates, hormones, and vitamins (such as biotin). Preparation of ligand-lipid conjugates has been perfected years ago and applied for ligand attachment to microbubbles either directly (19) or via a PEG spacer arm (18, 20, 21). The main advantage of this approach is the ability to perform the synthesis and product purification in the stepwise manner in the fully controlled conditions of an organic chemistry setup, ensuring high product yield and purity. The second general approach to ligand conjugation with the bubble shell calls for covalent or noncovalent attachment of ligands to preformed microbubbles. This approach is most appropriate for the materials that are unstable in the conditions of continuous sonication, highspeed shear mixing, and resulting high temperatures, that are often associated with the preparation of microbubbles. Large protein molecules, such as antibodies or antibody fragments, are easily denatured in harsh conditions. To avoid denaturation of protein ligands, reactive moieties that are stable enough to withstand bubble preparation conditions can be incorporated in the shell first and then applied for the attachment of the protein ligand to preformed bubbles. The groupings to achieve protein ligand binding can be suitable for covalent or noncovalent attachment. The noncovalent attachment strategies have been well developed, mostly with biotin-streptavidin-mediated techniques (Figure 4) (12). Conveniently, microbubbles can be rapidly purified from excess of free lipid, streptavidin, or biotinylated ligand, by repeated low-speed centrifugal flotation washes (e.g., 400g for several minutes). Respectively, after the first round of centrifugal washes during which the microbubbles are purified from excess lipid and biotin-lipid, microbubbles are dispersed in streptavidin solution. Streptavidin is used at an excess over the amount of available biotins on the bubble surface to avoid bubble cross-linking. After a brief incubation, the excess of free streptavidin is removed by flotation. Biotinylated antibody or other ligand is then added to complete the “sandwich” formation on the bubble surface (Figure 4). Coupling yield for the attachment of biotinylated ligands to microbubbles is reasonably high, and large excesses and losses of expensive ligand can be avoided. Covalent attachment of antibody ligands to preformed microbubbles can be easily accomplished after a carbox-
Klibanov
Figure 4. Design of biotinylated microbubble and attachment of biotinylated ligand via a streptavidin linker and PEG spacer arm.
Figure 5. Coupling of amino-containing ligand to carboxylcarrying microbubbles via water-soluble carbodiimide and Nhydroxysulfosuccinimide: the desired and side reactions.
ylated lipid derivative has been incorporated in the bubble shell; typically, carboxylate is positioned on the outer terminus of a PEG polymer spacer, and the other terminus of PEG is grafted to the lipid anchor embedded in the shell (22). Carboxylate can be activated by a watersoluble carbodiimide, such as 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide in the presence of N-hydroxysulfosuccinimide, and resulting active ester can react with an amino group of an antibody or other protein ligand (Figure 5) (23, 24). First, in mildly acidic conditions, the carboxylic acid residue is reacted with the carbodiimide with the formation of an unstable O-acylisourea. It is undesirable for this intermediate to be converted to the stable N-acylisourea, which would then be permanently attached onto the surface of the microbubble. N-Hydroxysulfosuccinimide is therefore added to convert O-acylisourea to N-hydroxysulfosuccinimide ester, which is reasonably stable in mild acidic environments, but would rapidly hydrolyze with the release of the unaltered carboxyl and N-hydroxysulfosuccinimide once the bubble preparation is placed in the mildly alkaline aqueous buffer solution containing the ligand that possesses primary amino groups used for coupling. Irreversible chemical modification of the bubble surface with undesirable N-acylisourea will thus be avoided. The coupling yield of the targeting agent to microbubbles is determined by the rates of two competing
Reviews
Figure 6. Coupling of thiol-carrying ligand to maleimidecarrying microbubbles: the desired and side reactions.
reactions, hydrolysis of N-hydroxysulfosuccinimide ester, and formation of amide bond between the ligand and activated carboxyl. The hydrolysis reaction is pseudofirst-order; the reaction rate constant is dependent on pH (faster at higher pH). The rate of coupling is also dependent on the concentration of primary amine in the deprotonated form (which depends on its pK and pH as well). Ligand coupling to active ester is a second-order reaction; the rate and the ligand coupling yield are both dependent on the concentration of reactants. While this coupling chemistry was successfully applied for antibody coupling to liposomal systems, where carboxylated lipid concentration was in the mM range (24), it is less than perfect for microbubble-based systems. The overall concentration of lipids that are needed to coat the surface of microbubbles is about 2 orders of magnitude less than a typical liposome lipid concentration, as is the concentration of activated carboxyl in the microbubble preparation. To achieve a decent (∼105 antibody per microbubble) level of coating, a large excess of the expensive antibody ligand has to be added (22); most of the antibody is not attached to the bubbles and wasted. To improve coupling yield, one needs to reduce the rate of side reactions of the activated derivative on the bubble surface without reducing the rate of the desired coupling reaction. A different coupling strategy, attachment of thiolcarrying targeting ligands to the particle surface via maleimide (25) or vinyl sulfone, is widely applied in bioconjugate chemistry. Use of maleimide may provide good ligand coupling yield and slow degradation of the reactive group on the bubble surface (Figure 6). Maleimide-PEG-lipid can be easily incorporated in the monolayer shell, and thiol-carrying antibody can be simply added to react with maleimide with a formation of a thioether bond, which is exceptionally stable. The maleimide reactive group possesses much better stability than an NHS ester in the aqueous medium (26), and the rate of unproductive side reactions is greatly diminished; therefore, the yield of ligand coupling to the microbubbles can be improved considerably. An alternative side reaction in this case, oxidation of a thiol with atmospheric oxygen with the formation of disulfide bond, should not be a major problem, because microbubble handling should be performed in the perfluorocarbon atmosphere anyway. An additional benefit from the use of the thiolmaleimide approach, the ability to directionally attach the ligand to the bubbles, should also be taken into account. Protein ligands contain dozens of primary amino groups, and carboxylic acid coupling to those with amide bond formation is quite random. Some of the coupling sites may be located close to the binding site of the ligand, which would greatly reduce the affinity to the target
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receptor. If a single thiol residue is specifically introduced in a desired portion of the ligand, either by site-directed mutagenesis or by synthesis, oriented coupling to the bubbles will be achieved. The chance of ligand inactivation upon coupling via a single thiol will be greatly reduced. The use of a flexible polymer spacer arm as a linker between the bubble surface and the ligand offers some advantage over a direct attachment of the ligand to the microbubble surface. The microbubble shell, especially one formed from gel-state phospholipids, does not offer rapid ligand mobility along the bubble surface. Therefore, the matching of ligand and receptor pairs along the contact area may require time that is not available during the brief contact between the bubble and the target surface in a flow-through scenario. Use of a flexible polymer tether (such as PEG) allows the ligand to rapidly interrogate the target surface and attach to the target receptors whenever available and in reasonable proximity to the microbubble surface. Additional benefit of placing the ligand on the PEG spacer becomes obvious when microbubbles also carry a grafted PEG polymer brush. If the ligand is attached directly to the lipid anchors, it may be hidden inside the polymer brush layer and become poorly accessible to the target receptor (27). When ligand is placed on the extended tether, especially if the length of the tether exceeds the thickness of the PEG brush, a “tiered brush” structure is created on the surface of the bubbles, where the thick brush layer of shorter PEG provides a stabilization coating, and longer polymer chains with tethered ligand are extended into the surrounding medium for efficient target receptor capture. Ultrasound Contrast Particle Design: Biodistribution and in Vivo Behavior. Initial microbubble contrast preparations were quite unstable in the bloodstream; shell-free microbubbles could not even pass through the lung vasculature, and contrast imaging of myocardium was only possible after intraarterial administration (13). Since that time, ultrasound contrast materials’ circulation time has improved greatly. Modern microbubble contrasts use appropriate lipid, polymer, or protein shells and can persist in the bloodstream for many minutes (up to 1 h for some preparations). This circulation time scale is quite sufficient to perform the task of targeted mapping of the receptors exposed to vascular lumen. For lipid monolayer-based microbubble design, generally, the coating made from longer-chain fully saturated lipid molecules (such as distearoyl phosphatidylcholine as compared with, for example, dimiristoyl phosphatidylcholine) ensures longer circulation time. In the early generation contrast agents, air was used as the gas phase. Use of low-solubility gases, e.g., decafluorobutane, further improves circulation time, because such gases exit the bubble and dissolve in the surrounding blood slower than oxygen or nitrogen. A lipid monolayercoated bubble with decafluorobutane gas core can stay in the bloodstream for many minutes. During the circulation, especially while passing through the lung microcirculation, microbubbles gradually deflate and lose ability to scatter ultrasound; majority of the poorly soluble gas incorporated within the bubbles is exhaled within minutes after intravenous injection, which points to the lungs as the major bubble gas elimination site (28) (actual circulation and exhalation times depend on the specifics of the gas used). Some of the microbubble preparations were retained nonspecifically in the vasculature, especially in the areas of inflammation (29, 30); in part this may be caused by the activation of complement by some of the components
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of the microbubble shell and triggered by the presence of electrostatically charged lipids on the bubble surface (31). Leukocytes, macrophages, and Kupffer cells have a tendency to capture these heavily charged microbubbles, especially the ones carrying phosphatidylserine (32). Presence of the PEG brush on the microbubble surface of charged bubbles reduces complement deposition only partially (31); therefore, it may be desirable to avoid surface charge in the microbubble formulations, especially if reduction of the nonspecific microbubble tissue uptake is desired. In all of the imaging modalities used for targeted and molecular imaging, it is critical to achieve not only a high degree of accumulation of contrast material in the target and low accumulation in normal tissues, but also low level of circulating contrast in the bloodstream. For some of the intravenously injected targeted contrasts used in SPECT or MRI, such as antibodies, long-circulating liposomes, or high molecular mass polymers, it may take up to a day to clear from the bloodstream via liver or kidneys. Therefore, diagnostic imaging cannot be performed rapidly and repeated frequently to monitor treatment efficacy. In the case of ultrasound, circulating bubbles may clear from the bloodstream completely within a fraction of an hour, or even faster, depending on the particular shell design and gas core. We can speculate that the microbubbles that had attached to the target tissue should maintain their stability much better than the bubbles still in circulation. Targeted bubbles do not repeatedly pass through the lungs where most of gas exchange and bubble deflation seem to take place. In an in vitro setting, targeted bubbles can be maintained on the antigen-coated surface for at least a day. Even if the majority of the bubbles clears from the bloodstream and other tissues, and only a small fraction of the administered bubbles is retained at the target, high detection sensitivity should allow successful target detection and delineation. Thus, we can expect high target-to-blood ratio within 10-15 min after microbubble contrast administration (12), and in this situation further extending contrast circulation time may not bring any benefits for the timely completion of the imaging procedure and successful diagnosis. Potential Safety Concerns Associated with Targeted Microbubble Systems. Contrast agents need to possess an excellent safety profile to be permitted for clinical use. Therefore, potential safety, toxicity, and side effect concerns affiliated with targeted microbubble agents need to be taken seriously and evaluated in detail. There are generally three groups of safety questions concerning microbubble contrast to be applied in molecular imaging: (1) the general reaction of the organism to contrast agent particles; (2) potential generation of immune response to particle components, such as targeting ligands; (3) ultrasound radiation bioeffects potentiation in the presence of microbubble agents in the living tissues. Concerns regarding undesired reactions of the organism to the intravascular administration of particle agents had been raised many times over the past years, especially with liposomes (33). In some patients an undesired systemic hypersensitivity-type reaction to the particle administration might occur; agent infusion is then to be discontinued and antiinflammatory therapy performed (34). One likely explanation for these adverse events is based on the electrostatic surface charge of the particles that results in complement activation leading to the undesired systemic effects (33). The administered dose of ultrasound contrast agents is usually lower than the
Klibanov
dose of therapeutic liposomes; therefore, the resulting undesirable effects are quite rare. For ultrasound contrast particles, addition of a PEG brush on particle surface might diminish complement-mediated events somewhat, but might not fully solve the problem (31) (similarly, liposome therapeutics described above that cause complement activation are PEGylated). The effective solution to this problem would be to formulate the agent without a substantial content of electrostatically charged components, which may be easily achievable for most of the particle systems by careful choice of ligands, coupling chemistries, and coupling anchors. Concerns regarding immune response to targeted particles, i.e., development of antibodies against targeting ligands (such as full size antibody molecules) and other microbubble shell components, have been evaluated for liposome-based drug carrier systems and should not be very different for the targeted ultrasound contrast particles (35). The use of humanized/human antibodies, small antibody fragments, and small molecule ligands is expected to minimize development of secondary antibodies in patients. Monitoring of this issue will ensure safety of repeated clinical application of targeted contrast ultrasound particles. The presence of gas bubbles may enhance the bioeffects generated by ultrasound (36). In clinical practice, the intensity of ultrasound radiation transmitted into the body of the patient by the transducer of the imaging system is strictly controlled and limited; mechanical index (MI) and thermal index quotients are constantly computed and restricted by the equipment according to applicable FDA guidelines (37). In vitro and in animal studies it has been shown that in certain conditions, a combination of microbubbles and ultrasound may cause RBC hemolysis (38), enhance transfection (a term “Sonoporation” was even coined (39)), and in some instances result in the rupture of microvasculature and formation of miniature petechial hemorrhages (40) that may actually be applied for enhancement of angiogenesis (41) and for drug delivery (42). The perceived degree, potential practical importance, and alleged dangers of the ultrasound bioeffects vary. Regarding hemolysis, even if we assume that a billion injected bubbles would in a worstcase scenario damage a billion RBCs (
