Molecular imaging with targeted ultrasound contrast microbubbles.
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Contrast Media↗
Biomedical subjects
A L Klibanov
Publications and source records attributed to A L Klibanov.
Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin.
BACKGROUND: Routine methods capable of assessing tissue inflammation noninvasively are currently not available. We hypothesized that tissue retention of microbubbles targeted to the endothelial cell adhesion molecule P-selectin would provide a means to assess inflammation with ultrasound imaging. METHODS AND RESULTS: Phospholipid microbubbles targeted to P-selectin (MB(p)) were created by conjugating monoclonal antibodies against murine P-selectin to the lipid shell. The microvascular behaviors of MB(p) and control microbubbles without antibody (MB) or with isotype control antibody (MB(iso)) were assessed by intravital microscopy of cremasteric venules of control and tumor necrosis factor (TNF)-alpha-stimulated wild-type mice. Retention of all microbubbles increased (P<0.05) with TNF-alpha treatment because of increased attachment to activated leukocytes. Extensive attachment of MB(p) directly to the venular endothelium or to adherent platelet-leukocyte aggregates was observed in TNF-alpha-stimulated mice, resulting in 4-fold greater (P<0.01) retention of MB(p) than either MB(iso) or MB. Enhanced retention of MB(p) was completely abolished in TNF-alpha-stimulated P-selectin-deficient mice. The ultrasound signal from microbubbles retained in inflamed tissue was assessed by contrast-enhanced renal ultrasound imaging of the kidneys of mice undergoing ischemia-reperfusion injury. In wild-type mice, this signal was significantly higher (P<0.05) for MB(p) (12+/-2 U) than either MB(iso) (6+/-3 U) or MB (5+/-3 U). In P-selectin-deficient mice, the signal for MB(p) was equivalent to that from control microbubbles. CONCLUSIONS: Microvascular retention of microbubbles targeted to P-selectin produces strong signal enhancement on ultrasound imaging of inflamed tissue. These results suggest that site-targeted microbubbles may be used to assess inflammation, tissue injury, and other endothelial responses noninvasively with ultrasound.
Animals↗
p-Nitrophenylcarbonyl-PEG-PE-liposomes: fast and simple attachment of specific ligands, including monoclonal antibodies, to distal ends of PEG chains via p-nitrophenylcarbonyl groups.
We have attempted to simplify the procedure for coupling various ligands to distal ends of liposome-grafted polyethylene glycol (PEG) chains and to make it applicable for single-step binding of a large variety of a primary amino group-containing substances, including proteins and small molecules. With this in mind, we have introduced a new amphiphilic PEG derivative, p-nitrophenylcarbonyl-PEG-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (pNP-PEG-DOPE), synthesized by reaction of DOPE with excess of bis(p-nitrophenylcarbonyl)-PEG in a chloroform/triethylamine mixture. pNP-PEG-DOPE readily incorporates into liposomes via its PE residue, and easily binds primary amino group-containing ligands via its water-exposed pNP groups, forming stable and non-toxic urethane (carbamate) bonds. The reaction between the pNP group and the ligand amino group proceeds easily and quantitatively at pH around 8.0, and remaining free pNP groups are promptly eliminated by spontaneous hydrolysis. Therefore, pNP-PEG-DOPE could serve as a very convenient tool for protein attachment to the distal ends of liposome-grafted PEG chains. To investigate the applicability of the suggested protocol for the preparation of long-circulating targeted liposomes, we have coupled several proteins, such as concanavalin A (ConA), wheat germ agglutinin (WGA), avidin, monoclonal antimyosin antibody 2G4 (mon2G4), and monoclonal antinucleosome antibody 2C5 (mon2C5) to PEG-liposomes via terminal pNP groups and studied whether the specific activity of these immobilized proteins is preserved. The method permits the binding of several dozens protein molecules per single 200 nm liposome. All bound proteins completely preserve their specific activity. Lectin-liposomes are agglutinated by the appropriate polyvalent substrates (mannan for ConA-liposomes and glycophorin for WGA-liposomes); avidin-liposomes specifically bind with biotin-agarose; antibody-liposomes demonstrate high specific binding to the substrate monolayer both in the direct binding assay and in ELISA. A comparison of the suggested method with the method of direct membrane incorporation was made. The effect of the concentration of liposome-grafted PEG on the preservation of specific protein activity in different coupling protocols was also investigated. It was also shown that pNP-PEG-DOPE-liposomes with and without attached ligands demonstrate increased stability in mouse serum.
Animals↗
Noninvasive ultrasound imaging of inflammation using microbubbles targeted to activated leukocytes.
BACKGROUND: Lipid microbubbles used for perfusion imaging with ultrasound are retained within inflamed tissue because of complement-mediated attachment to leukocytes within venules. We hypothesized that incorporation of phosphatidylserine (PS) into the microbubble shell may enhance these interactions by amplifying complement activation and thereby allow ultrasound imaging of inflammation. METHODS AND RESULTS: In 6 mice, intravital microscopy of tissue necrosis factor-alpha-treated cremaster muscle was performed to assess the microvascular behavior of fluorescein-labeled lipid microbubbles with and without PS in the shell. Ten minutes after intravenous injection, microbubble attachment to leukocytes within inflamed venules was greater for PS-containing than for standard lipid microbubbles (20+/-4 versus 10+/-3 per 20 optical fields, P<0.05). The ultrasound signal from retained microbubbles was assessed in the kidneys of 6 mice undergoing renal ischemia-reperfusion injury and in 6 control kidneys. The signal from retained microbubbles in control kidneys was low (<2.5 video intensity units) for both agents. After ischemia-reperfusion, the signal from retained microbubbles was 2-fold higher for PS-containing than for standard lipid microbubbles (18+/-6 versus 8+/-2 video intensity units, P<0.05). An excellent relation was found between the ultrasound signal from retained microbubbles and the degree of renal inflammation, assessed by tissue myeloperoxidase activity. CONCLUSIONS: -We conclude that noninvasive assessment of inflammation is possible by ultrasound imaging of microbubbles targeted to activated leukocytes by the presence of PS in the lipid shell.
Animals↗
Microbubble persistence in the microcirculation during ischemia/reperfusion and inflammation is caused by integrin- and complement-mediated adherence to activated leukocytes.
BACKGROUND: Albumin microbubbles that are used for contrast echocardiography persist within the myocardial microcirculation after ischemia/reperfusion (I-R). The mechanism responsible for this phenomenon is unknown. METHODS AND RESULTS: Intravital microscopy of the microcirculation of exteriorized cremaster muscle was performed in 12 wild-type mice during intravenous injections of fluorescein-labeled microbubbles composed of albumin, anionic lipids, or cationic lipids. Injections were performed at baseline and after 30 to 90 minutes of I-R in 8 mice and 2 hours after intrascrotal tumor necrosis factor-alpha (TNF-alpha) in 4 mice. Microbubble adherence at baseline was uncommon (<2/50 high-power fields). After I-R, adherence increased (P<0.05) to 9+/-5 and 5+/-4 per 50 high-power fields for albumin and anionic lipid microbubbles, respectively, due to their attachment to leukocytes adherent to the venular endothelium. TNF-alpha produced even greater microbubble binding, regardless of the microbubble shell composition. The degree of microbubble attachment correlated (r=0.84 to 0.91) with the number of adhered leukocytes. Flow cytometry revealed that microbubbles preferentially attached to activated leukocytes. Albumin microbubble attachment was inhibited by blocking the leukocyte beta(2)-integrin Mac-1, whereas lipid microbubble binding was inhibited when incubations were performed in complement-depleted or heat-inactivated serum rather than control serum. CONCLUSIONS: Microvascular attachment of albumin and lipid microbubbles in the setting of I-R and TNF-alpha-induced inflammation is due to their beta(2)-integrin- and complement-mediated binding to activated leukocytes adherent to the venular wall. Thus, microbubble persistence on contrast ultrasonography may be useful for the detection and monitoring of leukocyte adhesion in inflammatory diseases.
Albumins↗
Broadband time-domain reflectometry measurement of attenuation and phase velocity in highly attenuating suspensions with application to the ultrasound contrast medium Albunex.
We describe a technique for broadband measurements of the attenuation coefficient and phase velocity of highly attenuating liquid suspensions. To validate the technique we apply it to the ultrasound contrast agent Albunex at concentrations ranging from 0.69 x 10(6) particles/mL to 364 x 10(6) particles/mL. These longitudinal wave measurements were performed on Albunex suspensions maintained at 37 degrees C in a special time-domain reflectometer designed and constructed in our laboratory. The frequency-dependent attenuation coefficients and phase velocities obtained in the reflectometer are compared to broadband through-transmission measurements of these same quantities, which were also performed in our laboratory. Although comparison data between the two techniques are only available at lower concentrations, the agreement is quite good and serves to validate the methods described in this paper.
Albumins↗
Targeting and ultrasound imaging of microbubble-based contrast agents.
Preparation and characterization of targeted microbubbles (ultrasound contrast agents) is described. Specific ligands were attached to the microbubble shell, and ligand-coated microbubbles were selectively attached to various targets, using either an avidin biotin model system or an antigen-antibody system for targeting to live activated endothelial cells. Firm attachment of microbubbles to the target was achieved. Forces necessary to detach microbubbles from the target were estimated to exceed dozens of pN. Microbubbles were bound to the target even in the rapidly moving stream of the aqueous medium. Down to 20 ng of the ultrasound contrast material on the target surface could be detected by the ultrasound imaging with a commercial medical imaging system. At high bubble density on the target surface, strong ultrasound image attenuation was observed.
Antigen-Antibody Reactions↗
Targeting of ultrasound contrast material: selective imaging of microbubbles in vitro.
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Contrast Media↗
Direct video-microscopic observation of the dynamic effects of medical ultrasound on ultrasound contrast microspheres.
RATIONALE AND OBJECTIVES: Ultrasound can cause destruction of microbubble contrast agents used to enhance medical ultrasound imaging. This study sought to characterize the dynamics of this interaction by direct visual observation of microbubbles during insonification in vitro by a medical ultrasound imaging system. METHODS: Video microscopy was used to observe air-filled sonicated albumin microspheres adsorbed to a solid support during insonation. RESULTS: Deflation was not observed at lowest transmit power settings. At higher intensities, gas left the microparticle gradually, apparently dissolving into the surrounding medium. Deflation was slower for higher microsphere surface densities. Intermittent ultrasound imaging (0.5 Hz refresh rate) caused slower deflation than continuous imaging (33 Hz). CONCLUSIONS: Higher concentrations of microbubbles, lower ultrasound transmit power settings, and intermittent imaging each can reduce the rate of destruction of microspheres resulting from medical ultrasound insonation.
Adsorption↗
Targeting of ultrasound contrast material. An in vitro feasibility study.
PURPOSE: It would be beneficial to design a targetable microbubble ultrasound contrast agent that would selectively bind to the areas of interest in the body and enhance the target organ in the ultrasound examination. MATERIAL AND METHODS: We have studied the feasibility of targeting in a model system. We used avidin and biotin as a model ligand-receptor pair. Avidin was adsorbed on the surface of polystyrene, and biotin derivative was attached to microbubble shells. After removal of unincorporated biotin from the microbubbles, they were allowed to come in contact with avidin-coated or albumin-coated plastic. Unbound bubbles were washed by a stream of water. RESULTS: Binding of microbubbles to the surface occurred selectively in the areas where avidin layer was deposited. Binding of microbubbles to avidin layer was dependent on the amount of biotin incorporated in the microbubble shell. Presence of free biotin blocked targeting completely. Acoustic studies were performed using a custom-built ultrasound measurement apparatus and an ultrasound medical imaging system. Microbubble-coated areas of the plastic dish were clearly visualized with ultrasound imaging. A strong backscattered signal was obtained for microbubble surface densities as low as 3%. CONCLUSION: Microbubbles have been selectively targeted via a ligand-receptor system in vitro. Firm binding of microbubbles to avidin-coated surface has been achieved. Microbubbles deposited on the target were visualized with ultrasound imaging systems.
Animals↗
Avidin attachment to biotinylated amino groups of the erythrocyte membrane eliminates homologous restriction of both classical and alternative pathways of the complement.
Lysis of avidin-coated biotinylated sheep red blood cells (RBC) via the classical pathway of homologous (sheep) and heterologous (guinea pig) complement has been studied. The minimal surface density of avidin inducing antibody-dependent lysis via the classical pathway is smaller than that inducing antibody-independent lysis via the alternative pathway. Heterologous lysis via the classical pathway does not depend on the mode of avidin attachment: both biotinylation of membrane amino groups and insertion of biotinyl-lipid into the membrane provide the same lysis of avidin-coated RBCs by guinea pig serum in the presence of anti-avidin antibody. Avidin-free sheep RBC sensitized with hemolytic anti-RBC antibody were lysed by guinea pig, but not by sheep serum, confirming high efficiency of homologous restriction of the complement. However, avidin-coated RBCs were lysed by homologous serum in the presence of anti-avidin antibody at low surface density of avidin attached. The elimination of the homologous restriction depends on the mode of avidin attachment: biotinylation of membrane amino groups provides antibody-mediated lysis via the classical pathway of homologous complement, while insertion of biotinyl-lipid does not provide lysis.
Amines↗
Avidin attachment to red blood cells via a phospholipid derivative of biotin provides complement-resistant immunoerythrocytes.
Preincubation of red blood cells (RBC) in an aqueous dispersion of biotin-phosphatidylethanolamine (biotin-PE) provides binding sites for avidin on the surface of these cells (up to 5 x 10(5) avidin molecules per cell). Previously we have shown that biotin covalently attached to the surface of RBC by a chemical reaction with biotin N-hydroxysuccinimide ester permits attachment of avidin to these cells, resulting in the activation of the alternative pathway of complement with subsequent cell lysis. However, avidin attached to RBC via biotin-PE did not cause complement activation. This is not due to the stabilizing action of biotin-PE. In contrast, various phospholipids, including biotin-PE, enhance the lysis of RBC induced by hemolytic antibodies via the classical complement pathway. The potential of avidin-coated RBC to act as activators of the complement alternative pathway depends on the method of biotin attachment to RBC. Complement-resistant avidin-coated RBC can specifically bind biotinylated antibodies. These immunoerythrocytes effectively and specifically bind to the antigen-coated surface and are not lysed by complement even in the presence of soluble antigen. These data extend the possible applications of immunoerythrocytes in drug targeting.
Animals↗
Interaction between oleic acid-containing pH-sensitive and plain liposomes. Fluorescent spectroscopy studies.
The energy transfer method has been applied to study the interaction between pH-sensitive liposomes (phosphatidyl ethanolamine/oleic acid/cholesterol, 4:2:4 molar ratio) and plain liposomes (phosphatidyl choline/phosphatidyl ethanolamine/cholesterol, 4:2:3 molar ratio). It was shown that a slow fusion process occurs between two types of liposomes. Also, the transfer of oleic acid from pH-sensitive liposomes to plain liposomes takes place. This transfer results in the increased permeability of both pH-sensitive and plain liposomes, facilitating the release of liposome-entrapped fluorescent dye. The data obtained were used for a possible explanation of the mechanism of intracytoplasmic drug delivery by pH-sensitive oleic acid-containing liposomes.
Drug Carriers↗
Targeted accumulation of polyethylene glycol-coated immunoliposomes in infarcted rabbit myocardium.
The less than optimal accumulation of immunoliposome-associated reagents at target sites has often been attributed to the rapid in vivo clearance of immunoliposomes from the blood. In an attempt to overcome the drawback of rapid clearance and use the targeting potential of immunoliposomes, we have prepared long-circulating, 111In-labeled immunoliposomes. Targeting properties and enhanced circulation times were demonstrated in a rabbit model of acute experimental myocardial infarct. The specificity of liposomes for newly exposed intracellular cardiac myosin at the necrotic sites was achieved by incorporating monoclonal antimyosin antibody. Extended circulation times were achieved by cocoating the antimyosin-liposomes with polyethylene glycol (PEG). The half-life of the immunoliposomes was 40 min, which increased to 200 min with 4% mol PEG and to approximately 1000 min with 10% mol PEG. The degree of binding of modified immunoliposomes at the target sites was also dependent on the concentration of PEG incorporated at the liposome surface. This study demonstrates the accumulation of long-circulating targeted liposomes at the area of acute rabbit experimental myocardial infarction.
Animals↗
Influence of the steric barrier activity of amphipathic poly(ethyleneglycol) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo.
A series of dioleoyl N-(monomethoxy polyethyleneglycol succinyl)phosphatidylethanolamine (PEG-PE) of different polymer chain length was used in this study. Both the activity of PEG-PE in prolonging the circulation time of liposomes and the relative steric barrier activity of amphipathic polymer, measured by a liposome agglutination assay, were found to be directly proportional to the chain length of PEG-PE (PEG5000-PE greater than PEG2000-PE greater than PEG750-PE). However, PEG5000-PE caused a reduced target binding of immunoliposomes in mice due to its overly strong steric barrier activity. The best PEG-PE species supporting the target binding of immunoliposomes was PEG2000-PE, the activity of which was compatible to that of ganglioside GM1. However, GM1 only showed a weak steric barrier activity, suggesting a different mechanism for this glycolipid.
Animals↗
Lipophilic polylysines mediate efficient DNA transfection in mammalian cells.
Low molecular weight (Mr approximately 3000) poly(L-lysine) (PLL) conjugated to N-glutarylphosphatidylethanolamine is an effective carrier to promote DNA-mediated transfection in cultured mammalian cells. The conjugates, named 'lipopolylysines', contained an average of two phospholipid groups per molecule of PLL. Similar conjugates of the non-degradable poly(D-lysine) also had a similar transfection activity, indicating that the degradation of the carrier is not required for the activity. Unconjugated polylysines had little activity. The transfection activity of the lipopolylysine has been optimized with respect to the DNA concentration, DNA/carrier ratio, incubation time and the presence of serum in the incubation medium. The binding of lipopolylysine with DNA was measured by the degree of retardation of DNA in agarose gel electrophoresis. It was found that at the optimal DNA/lipopolylysine ratio for transfection, all DNA were found in large complexes which did not enter the gel. The transfection activity of the lipopolylysine, under optimal conditions, was approximately 3-fold higher than that of lipofectin, a widely used commercial reagent. Moreover, lipopolylysine mediated transfection even in the presence of 10% calf serum; whereas the lipofectin lost about 70% of its activity under the same condition. However, unlike lipofectin the transfection activity of the lipopolylysine depended on scraping the treated cells. Furthermore, lipopolylysine only transfected attached monolayer cells, and not suspension cells.
Animals↗
Activity of amphipathic poly(ethylene glycol) 5000 to prolong the circulation time of liposomes depends on the liposome size and is unfavorable for immunoliposome binding to target.
Dioleoyl-N-(monomethoxy polyethyleneglycol succinyl)-phosphatidylethanolamine (PEG-PE) (mol. wt. of PEG = 5000), an amphipathic polymer, can be incorporated into the liposome membrane and significantly prolong the blood circulation time of the liposome. As little as 3.7 mol% of PEG-PE in liposome resulted in maximal enhancement of liposome circulation time. However, this activity of PEG-PE was only seen with relatively small liposomes (d less than or equal to 200 nm); larger liposomes containing PEG-PE showed an unusually high level (approx. 35% injected dose) of accumulation in the spleen. We have tested whether the small, PEG-PE containing liposomes are suitable for immuno targeting by incorporating a lung-specific monoclonal antibody on the liposome surface. While another amphiphile, ganglioside GM1, which is well known for its activity to prolong the liposome circulation time, significantly enhanced the lung binding of the immunoliposomes, PEG-PE incorporation of immunoliposomes resulted in a low level of target binding. To test if the reduced target binding is due to a steric barrier effect of the surface PEG polymer, we have incorporated a small amount of N-biotinaminocaproylphosphatidylethanolamine into the PEG-PE containing liposomes and examined the liposome agglutination induced by the addition of streptavidin. As little as 0.72 mol% PEG-PE in these liposomes completely abolished agglutination. In contrast, incorporation of GM1 in liposomes only reduced the rate, but not the extent, of liposome agglutination. These results strongly support the hypothesis that PEG-PE prolongs liposome circulation time by providing a strong steric barrier which prevents close contact with another liposome or cell. Since GM1 provides only a weak steric barrier effect, its activity to prolong the liposome circulation time must involve another yet unknown mechanism.
Agglutination↗
Targeting of macromolecular carriers and liposomes by antibodies to myosin heavy chain.
Macromolecular carriers and liposomes were covalently coupled to monoclonal antibodies against cardiac myosin heavy chain. Deferoxamine-modified polymers bound tightly with 67Ga and 68Ga radioisotopes. Ternary deferoxamine-polylysine antibody conjugates specifically targeted the radioisotopes to a myosin-coated microplate. Scatchard analysis revealed a high affinity of the conjugate for the target with a Kas of approximately 10(8) M-1. Liposomes that contained immobilized antimyosin antibodies were targeted specifically to the myosin-coated plate. Additional coating of these liposomes with polyethylene glycol reduced specific binding to the target in vitro. However, because of the presence of polyethylene glycol on the surface of liposomes, these liposomes had a long half-life and slowly cleared from the blood-stream after intravenous injection. These immunoliposomes showed up to 16- to 18-fold specific localization to the necrotic areas of the myocardium in rabbits with experimental infarction.
Animals↗