Dual targeting improves capture of ultrasound microbubbles towards activated platelets but yields no additional benefit for imaging of arterial thrombosis.

Platelets can be found on the surface of inflamed and ruptured atherosclerotic plaques. Thus, targeting of activated platelets may allow for molecular imaging of vulnerable atherosclerotic lesions. We here investigated microbubbles (MB) functionalized with the selectin ligand sialyl Lewisa individually (MBsLea) or dually with sLea and an antibody targeting ligand-induced binding sites of the activated GPIIb/IIIa receptor (MBDual). Assessed by in vitro flow chamber, targeted MB exhibited increased adhesion to platelets as compared to MBControl. While MBsLea rolled slowly on the platelets' surface, MBDual enhanced the percentage of firm adhesion. In vivo, MB were investigated by ultrasound in a model of ferric chloride induced non-occlusive carotid artery thrombosis. MBsLea and MBDual revealed a higher ultrasound mean acoustic intensity than MBControl (p < 0.05), however MBDual demonstrated no additional increase in mean signal intensity as compared to MBsLea. The degree of carotid artery stenosis on histology correlated well with the ultrasound acoustic intensity of targeted MB (p < 0.05). While dual targeting of MB using fast binding carbohydrate polymers and specific antibodies is a promising strategy to support adhesion to activated platelets under arterial shear stress, these advantages seem not readily translatable to in vivo models.

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.

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.

Incorporation of hydrophilic protein modified with hydrophobic agent into liposome membrane.

The hydrophilic protein-enzyme, alpha-chymotrypsin, can be bound to the liposomal membrane after the preliminary increase in hydrophobicity induced by acylation of protein amino groups with palmitic chloroanhydride. The efficacy of binding depends on the degree of modification. The bound enzyme almost completely preserves its catalytic properties and the ability to interact with a high molecular weight inhibitor. Binding can be performed during both the process of liposome formation and the incubation of a modified enzyme with preformed liposomes. According to ESR and fluorescence spectroscopy, the hydrophobic tail of the modified enzyme is incorporated into the membrane and the protein globule is located on the surface of the membrane. Protein incorportation causes an increase in the amorphous nature of the membrane, and the bound protein is not as mobile as the free protein. The approach discussed can be useful in binding soluble hydrophilic proteins to artificial membranes.

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.

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.

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.

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.

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.

A new hydrophobic anchor for the attachment of proteins to liposomal membranes.

The model enzyme alpha-chymotrypsin covalently modified by a phosphatidylethanolamine derivative has been attached to liposomal membranes in high yield. A maximal protein/lipid ratio of 5.4 X 10(-3) mol enzyme/mol lipid was achieved.

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.

Protein immobilization on the surface of liposomes via carbodiimide activation in the presence of N-hydroxysulfosuccinimide.

A method of the covalent immobilization of proteins on the surface of liposomes, containing 10% (by mol) of N-glutaryl phosphatidylethanolamine, is described. Carboxylic groups of liposomal N-glutaryl phosphatidylethanolamine were activated in the presence of water-soluble carbodiimide and N-hydroxysulfosuccinimide and reacted subsequently with protein amino groups. The liposome-protein conjugates formed contained up to 5 x 10(-4) mol protein/mol lipid. Lectins (RCA1 and WGA) upon immobilization on liposomes retained saccharide specificity and the ability to agglutinate red blood cells. The immobilization of mouse monoclonal IgG in a ratio of 3.5 x 10(-4) mol IgG/mol lipid was achieved. The liposome activation in the absence of N-hydroxysulfosuccinimide resulted in a 2-fold decrease of protein coupling yields.

Immobilization of alpha-chymotrypsin on sucrose stearate--palmitate containing liposomes.

Stable liposomes have been prepared from lipid mixture containing sucrose stearate-palmitate. 1.2 X 10(-4) mol of model enzyme alpha-chymotrypsin per mol of lipid have been coupled to prepared liposomes activated by periodate oxidation of sucrose units.

Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes.

Incorporation of dioleoyl N-(monomethoxy polyethyleneglycol succinyl)phosphatidylethanolamine (PEG-PE) into large unilamellar liposomes composed of egg phosphatidylcholine:cholesterol (1:1) does not significantly increase the content leakage when the liposomes are exposed to 90% human serum at 37 degrees C, yet the liposomes show a significant increase in the blood circulation half-life (t1/2 = 5 h) as compared to those without PEG-PE(t1/2 less than 30 min). The PEG-PE's activity to prolong the circulation time of liposomes is greater than that of the ganglioside GM1, a well-described glycolipid with this activity. Another amphipathic PEG derivative, PEG stearate, also prolongs the liposome circulation time, although its activity is less than that of GM1. Amphipathic PEGs may be useful for the sustained release and the targeted drug delivery by liposomes.

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.

Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture.

A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells. Upon binding E25 is rapidly internalized and digested intracellularly. Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system. Up to 30% of the cell-adherent liposomal lipid is internalized.

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.

Radioimmunoimaging of lung vessels: an approach using indium-111-labeled monoclonal antibody to angiotensin-converting enzyme.

A murine monoclonal antibody against human angiotensin-converting enzyme was radiolabeled with 111In via diethylenetriaminepentaacetic acid without substantial loss of antigen-binding capacity. This monoclonal antibody designated 9B9 cross-reacted with rat and monkey angiotensin-converting enzyme. Indium-111-labeled 9B9 selectively accumulated 10-20 times greater in the lung than in blood or other organs following intravenous administration in rats. Kinetics of lung accumulation and blood clearance were studied for 111In-9B9-antibody and compared to that of 125I-labeled 9B9 in rat. Highly specific accumulation of 111In-9B9-antibody in the lung of Macaca Rhesus monkeys after intravenous injection was monitored by gamma-imaging. Images of 111In-labeled antibody 9B9 biodistribution in monkey lung noticeably differ from the images of biodistribution of 99mTc-labeled albumin microspheres. This difference may provide information concerning the state of the endothelium of lung capillaries, which is different from the blood flow characteristics determined with routine microsphere technique.

Blood clearance of radiolabeled antibody: enhancement by lactosamination and treatment with biotin-avidin or anti-mouse IgG antibodies.

Methods of rapid blood clearance of 111In-labeled mouse monoclonal antibody 9B9 against angiotensin-converting enzyme were studied. Indium-111-9B9 is specifically accumulated in rat lung, but its blood clearance is relatively slow and target-to-blood radioactivity ratio/g tissue (localization ratio) increases from 11 to 30 only 48 hr postinjection. Injection of second (anti-mouse immunoglobulin) antibodies results in slight (1.8-fold) increase of 9B9 localization ratio. Chemical modification of 9B9 aminogroups with lactose results in enhanced liver uptake and rapid blood clearance of antibody. Blood radioactivity level decreases tenfold, and as a result localization ratio increases threefold (up to 38 in 30 min). Injection of avidin following the injection of biotinylated 9B9 results in rapid clearance of blood radioactivity with increased uptake in liver and spleen. Lung uptake is not changed. Localization ratio increases fivefold over the avidin-untreated animal value. Implications of these approaches for various applications in immunoimaging are discussed.

The antibody-linked chelating polymers for nuclear therapy and diagnostics.

This review deals with the problem of protein modification with chelating polymers. The main purpose of this approach is the preparation of monoclonal antibodies labeled with heavy metal isotopes (alpha-, beta-, and delta-emitting metals and metals used for NMR-tomography). Traditional binding of metals with proteins via chelating agents directly coupled to protein molecule does not allow binding a high number of metal atoms per single protein molecule and can also alter protein specific properties. At the same time, metal-to-protein binding via intermediate chelating polymer makes possible the binding of several dozen metal atoms per single protein without affecting its specific properties. Moreover, the variations in polymer properties and molecular weight allow controlled modified antibody biodistribution and clearance rate. Modified antibodies can be used successfully for nuclear and NMR diagnostics and for nuclear therapy. The following problems are discussed: the chemistry of the coupling of chelating groups to polymer backbone; the binding of chelating polymers to proteins, including monoclonal antibodies; the ability of chelating polymer-to-protein conjugates to bind heavy metals; the influence of the modification on protein conformation and specific properties; the behavior of metal-containing conjugates in vivo; the practical use of conjugates obtained for radioimmunoimaging, radioimmunotherapy, NMR-tomography, and in vitro immunoassays. Future prospects of the approach are also discussed.