The affinity of lipid-coated microbubbles to maturing spinal cord injury sites.

OBJECTIVE: This laboratory has demonstrated that lipid-coated microbubbles (LCMs) effectively aggregate and deliver chemotherapeutic drugs into rat brain tumor cells and antigliosis agents into maturing rat brain injury sites. In this study, we report the affinity of tail vein-injected LCMs to the injured rat spinal cord by a compressive lesion to the upper thoracic region. METHODS: The accumulation of LCMs in the injured spinal cord was analyzed by labeling it with a lipid-soluble fluorescent dye, 3,3'-dioctadecyloxacarbocyanine perchlorate. Indices of glial fibrillary acidic protein were measured concomitantly with 3,3'-dioctadecyloxacarbocyanine perchlorate-labeled LCMs using confocal microscopy. RESULTS: There was no aggregation of LCMs accumulated 1 and 6 hours after injury; however, when given 2, 4, and 7 days after injury, LCMs showed a clear affinity for the injured region. LCM aggregation shifted from the central necrotic area of the injury on postinjury Day 2 and postinjury Day 4 to the white matter among glial fibrillary acidic protein-positive astrocytes by postinjury Day 7. CONCLUSION: Affinity of LCMs for spinal cord injury sites may be mediated in the early stages after injury by proliferating macrophages in the necrotic center, and then in later stages by glial fibrillary acidic protein-positive astrocytes in adjacent white matter. These findings suggest a potential for using LCMs as a delivery vehicle to concentrate lipid-soluble agents in spinal cord injury sites.

The use of lipid-coated microbubbles as a delivery agent of 7beta-hydroxycholesterol in a radiofrequency lesion in the rat brain.

OBJECTIVE: This laboratory has previously described the aggregation of intravenously administered lipid-coated microbubbles (LCM) around tumors and areas of injury. 7Beta-hydroxycholesterol has been used to inhibit astrocytic proliferation in nervous system injury models. The compound has been given by direct infusion, by epidural catheter, or in liposomes (delivered stereotactically to the injury site). In this article, we report the use of LCM to deliver 7beta-hydroxycholesterol to a radiofrequency injury site in the rat cerebrum. METHODS: First, the ability of LCM to target the thermal lesion in the rat brain was characterized using a lipid-soluble fluorescent dye 3,3-dioctadecyloxacarbocyanine perchlorate. Then, the effectiveness of this delivery system in suppression of glial proliferation was measured by glial fibrillary acidic protein immunoreactivity. RESULTS: Glial fibrillary acidic protein immunoreactivity was significantly reduced when 7beta-hydroxycholesterol was administered via LCM but not alone, suggesting that astrocytic proliferation would correspondingly be diminished. CONCLUSION: LCM were assessed as a delivery vehicle for 7beta-hydroxycholesterol in a rat brain radiofrequency lesion and found to be efficient in reducing astrogliosis, as measured by glial fibrillary acidic protein immunoreactivity.

Evaluation of lipid-coated microbubbles as a delivery vehicle for Taxol in brain tumor therapy.

OBJECTIVE: This work evaluates the potential of lipid-coated microbubbles (LCM) as a delivery vehicle for lipid-soluble antineoplastic agents. We have shown, in rats, the selective affinity of intravenously administered LCM for tumor cells. They are internalized by the tumor cells both in vitro and in vivo. The specificity of LCM for tumors and subsequent incorporation into the cytoplasm could significantly reduce the systemic effects of an agent incorporated into the bubbles, such as Taxol. METHODS: The in vitro methods were as follows. C6 cells (10(5) cells) were treated with Taxol-LCM (6 micrograms/ml), Taxol-Cremophore (6 micrograms/ml), or LCM alone for 8 or 24 hours. Cell death was determined by staining the cells with nuclear staining. Abnormalities of microtubule structures were ascertained by confocal microscopy. The in vivo methods were as follows. Two rat tumor models (C6 and 9L) were used. Rats were treated with single bolus injections or with repetitive (two or three) treatment courses, with respective control animals. Each course consisted of one daily tail vein injection for 5 consecutive days and then 2 days of rest. RESULTS: When compared with either a saline control group or a group receiving Taxol in an oil vehicle, Taxol-LCM reduced tumor progression in Fischer 344 rats inoculated with 9L glioma. The most profound effect was observed with rats treated with three treatment cycles (five daily injections/cycle) separated by two rest periods (2 d/period). CONCLUSION: Both in vitro and in vivo data indicate that Taxol can be incorporated into LCM, can be delivered to the tumor site, and can exert a measurable antitumor biological effect.

The affinity of lipid-coated microbubbles for maturing brain injury sites.

The availability of a vehicle to deliver lipid soluble agents to a brain injury site may be of potential value in management of brain injury. This work describes the aggregation of intravenously administered Lipid-Coated Microbubbles (LCM) in the injury site following an experimental radiofrequency rat brain lesion. The bubbles can be identified around the region of the injury after the lesion has matured at least 48 h. The greatest bubbles density is evident after the lesion has matured for 10 days. This bubble density, reflecting "affinity," decreases to a plateau level from the second to the third week after injury. In order to investigate the potential relationship of bubble influx to posttraumatic astrocytosis and to cell turnover in the region, we utilized dual-channel laser-scanning confocal microscopy to track both bubble influx into the region and concomitant Glial Fibrillary Acidic Protein (GFAP) expressing astroctyte cell distribution. Cell turnover was assayed in separate sections using immunohistochemical staining of Proliferating Cell Nuclear Antigen (PCNA). We suggest a relationship between the LCM affinity and reactive astrocytes, but found no affinity of LCM for cells which stained positive with PCNA.

Internalization of microbubbles by tumor cells in vivo and in vitro.

Lipid-coated microbubbles (LCM) administered intravenously (i.v.) to rats bearing brain tumor, specifically enhance tumor visualization by ultrasound [1]. In order to understand the basis for this observation, we have examined the interactions of LCM with glioblastoma (C6) and gliosarcoma (9L) tumor cells in vivo and in vitro. LCM and LCM labeled with the fluorescent lipophilic dye 3,3'-dioctadecyloxacarbocyanine perchlorate (diO) were administered to rats bearing brain tumor. LCM and diO-labeled LCM were found principally at the tumor site with no evidence of label in the surrounding normal brain tissue. Analysis of the tumor by confocal laser scanning microscopy revealed that labeled LCM were inside the tumor cells. Similar analysis of LCM interactions with C6 and 9L cells in culture showed that LCM first adsorb at the surface of the cells, and with time became localized inside the cells. Binding and internalization proceeded faster at 37 degrees C than at room temperature (RT). Staining of live cells with N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl) methylamine dihydrochloride (DAMP), a dye that recognizes acidic compartments, showed that the majority of internalized LCM was associated with compartments containing DAMP. If the same uptake mechanism were operative in vivo, it would indicate that a portion of LCM bypasses the reticuloendothelial system and become endocytosed directly by tumor cells.

Detection of experimental rat liver tumors by contrast-assisted ultrasonography.

RATIONALE AND OBJECTIVES: The authors characterized the effect of intravenous lipid-coated microbubbles (LCMs) on the echogenicity of malignant liver tumors. METHODS: Novikoff hepatoma cells were inoculated into the livers of 16 anesthetized Sprague-Dawley rats. Sonograms were obtained weekly after tail-vein injection with either 0.2 mL/kg LCMs or saline control. RESULTS: A statistically significant difference in the signal-to-noise ratio (SNR) was observed between the group that received LCMs (10 rats) and the group that received saline (6 rats) (P < .01). The effect persisted for 30 minutes after contrast injection. Selective leakage and accumulation of LCMs into the tumor tissue itself was confirmed histologically using lipid-specific counterstains. CONCLUSIONS: Intravenous injection of the LCM contrast agent produces a rapid increase in the echogenicity of the experimental Novikoff tumor in the rat liver.

Applications of lipid-coated microbubble ultrasonic contrast to tumor therapy.

Lipid-coated microbubbles (LCM) make an excellent diagnostic ultrasonic contrast agent in experimental tumor systems. LCM have been shown to aggregate in brain tumors and subcutaneous tumors after intravenous administration, and to provide persistent image enhancement for many minutes. In this work, experimental subcutaneous Walker Carcinosarcoma is insonated after the bubbles are given intravenously. Selective necrosis, lymphocyte proliferation and hemorrhage within the tumor can be demonstrated. Preliminary data are given to demonstrate this phenomenon. The mechanism of the effect is discussed in the context of both heating and cavitation.

Quantitative assessment of tumor enhancement by ultrastable lipid-coated microbubbles as a sonographic contrast agent.

We have previously reported that ultrastable lipid-coated microbubbles make a suitable ultrasonic contrast agent in the brain, causing increased intensity of echoes that persists for many hours. We showed that intravenously administered lipid-coated microbubbles accumulate selectively in rat brain gliomas with echogenicity enhancement for up to 1 hour, allowing visualization of the growing lesions 40% (2 days) earlier than can be seen without contrast. This work is a detailed evaluation of the accumulation of the lipid-coated microbubbles in tumor and the effect of the bubbles on the echogenicity of insonified tumors. Using a lipid-specific stain, we measured and characterized the distribution of the bubbles in the brain and tumor. We showed that on the scan, the enhancement of the tumor is accompanied by a change in the signal-to-noise ratio of the echoes from the tumor. We identified characteristic textural changes associated with contrast-enhanced tumor using spectral analysis.

Lipid-coated uniform microbubbles for earlier sonographic detection of brain tumors.

Rapid technological improvements have fostered the continued clinical development of intraoperative neurosonology, despite the fact that no suitable contrast media have been available for ultrasound studies. Because they can be made of uniform size (99% are less than 4.5 mum in diameter) and are thought to cross disruptions in the tumor vessels, artificial lipid-coated microbubbles can fill this gap. Furthermore, these microbubbles are stable in vitro for at least 6 months, with an in vivo halflife of 20 hours or more. This study demonstrated that lipid-coated microbubbles injected intravascularly can intensify echoes from rat brain gliomas. Specifically, when this standardized microbubble contrast agent was injected intravenously daily in rats, the time to visual ultrasonic detection of developing brain tumors (C-6 gliomas) was 4.09 days (n = 11) after tumor inoculation, versus 6.67 days (n = 9) to detection without microbubble injection (Z = -3.71, p = 0.0004).

Bubble production in agarose gels subjected to different decompression schedules.

The relative effectiveness of seven different (military, commercial, and experimental) decompression schedules in reducing bubble formation within aqueous gels has been evaluated quantitatively under rigorously controlled conditions. Specifically, visual counts have been conducted of the bubbles formed in highly purified agarose gels subjected to the different decompression schedules. The order of effectiveness among these schedules in reducing bubble formation in the agarose gel samples was as follows: Model 1 greater than Royal Naval Physiological Laboratory approximately French Ministry of Labor greater than Yount et al. greater than Japanese Department of Labor greater than United States Navy greater than French Navy. It was concluded that the depth at which slow decompression commences is a major factor, along with the total decompression time, in determining the extent of bubble formation.

Screening of membrane surface charges by divalent cations: an atomic representation.

To arrive at a clear and atomically realistic representation of the process of ionic screening, a model with the following necessary and justifiable constraints was devised. 1) The minimum internuclear distance (IND) between a negative site on the membrane and a cation screening the site is equal to the sum of the site's "equivalent" radius (rs) + the diameter of a water molecule (approximately 2.8 A) + crystal radius of the cation (rc). 2) The average value for the dielectric constant (D) over IND is given by D approximately 80 ((IND - rs - rc)/IND). When this simple atomic model for ionic screening is employed in conjunction with equilibrium ion-selectivity theory, it is possible to predict quantitatively, from coulombic energy calculations, the secondary stereospecific actions of certain alkaline-earth cations as well as the predominant screening effect of these divalent cations at the surfaces of different types of membranes. The model also successfully predicts the transition from a predominantly screening situation to a predominantly binding situation, which was observed experimentally when negative surface charge density was decreased in nerve.

Relationship between CO2 levels and decompression sickness: implications for disease prevention.

Extensive data concerning the incidence of decompression sickness among workers participating in the deepest caisson operation in Japan to date have been collected and analyzed for the period April through August, 1976. When the bottom pressure was between 3.0 and 3.2 ATA, the incidence of decompression sickness was 3.05%; subsequently, the incidence was only 0.96% between 3.2 and 3.4 ATA. The man lock (i.e., decompression chamber) had never been ventilated during the former group of decompressions and the level of CO2 had ranged between 1.8 and 2.3% (v/v); in the latter group of decompressions, the CO2 level ranged between 0.3 and 0.8% with ventilation. All other conditions, including the decompression table used, were the same. Moreover, based upon the nature of the muscular activity required of the caisson workers just prior to decompression, their most common site of affliction was found to lie within the body region where the highest tissue tensions of CO2 would be expected during decompression.

Improved method for studying the surface chemistry of bubble formation.

Bubble formation in agarose gels as a result of rapid decompression following saturation with either N2, CO2, or He has been studied. Bubble number was observed to vary predominantly as a function of decompression magnitude and was virtually independent of the particular gas used. The cavitation threshold (i.e., 1 bubble in 50% of trials) was found to fall between -3 and -4 psig ( approximately -0.25 atm) for all three gases. Modification of the ionic content of the agarose gel medium, either by lowering the pH or including polyvalent cations, had only slight effects, if any, on the number of bubbles produced by a given decompression. From these results, and the work of others briefly reviewed in the paper, it is concluded that the surfactant monolayer surrounding a gas cavitation nucleus is comprised almost exclusively of nonionic surface-active molecules.

Covalent labeling of the tetrodotoxin receptor in excitable membranes.

A photoaffinity labeling technique is described by which a tetrodotoxin analog is covalently bound to receptor sites associated with the sodium pores of excitable membranes. The biological activity of the toxin analog is retained after the covalent binding reaction.

Stabilization of gas cavitation nuclei by surface-active compounds.

Gas bubbles are the primary agent in producing the pathogenic effects of decompression sickness. Numerous experiments indicate that bubbles originate in water, and probably also in man, as pre-existing gas nuclei. This is surprising considering that gas phases larger than 1 micron should rise to the surface of a standing liquid, whereas smaller ones should dissolve rapidly due to surface tension. Several stabilizing mechanisms have been suggested, and each has been refuted on experimental grounds. In this article, we propose a new model that arises out of a systematic study of the earlier theories. We review these theories and conclude that gas cavitation nuclei must be held intact by surface-active skins that are initially permeable. The first quantitative analysis of bubble formation data from supersaturated gelatin is summarized and leads to the further conclusion that skins can become impermeable if the ambient pressure is increased rapidly by a sufficient amount. Our model owes much to Sirotyuk, who "demonstrated experimentally that stabilization of gas bubbles acting as cavitation nuclei in water is always attributable to the presence of surface-active substances in the water".

Structural characteristics of the saxitoxin receptor on nerve.

The effects of uranyl ion (UO22+; at low concentrations binds specifically to phosphate groups) and the cationic dye methylene blue (MB+; binds strongly to carboxyl groups) on saxitoxin (STX) potency in crayfish axon has been studied by means of intracellular microelectrodes. At pH 6.00 +/- 0.05 and 13.5 mM Ca2+, addition of 10.0 muM UO22+ + 5.0 nM STX had only slightly, if any, less effect on the spike's maximum rate of rise [0.79 +/- 0.04 (viz., mean +/- SEM) of control value] than did addition of 5.0 nM STX alone (0.72 +/- 0.05). Under the same conditions of pH and Ca2+ concentration, 1.0 mM MB+ had approximately the same effect: 1.0 mM MB+ + 5.0 nM STX, 0.76 +/- 0.03; 5.0 nM STX alone, 0.70 +/- 0.04. However, at pH 7.00 +/- 0.05 and lower Ca2+ concentrations, 1.0 mM MB+ significantly reduced STX potency. Using 6.0 mM Ca2+: 1.0 mM MB+ + 5.0 nM STX, 0.92 +/- 0.01; 5.0 nM STX alone, 0.68 +/- 0.08. Using 3.0 mM Ca2+, the corresponding values were 0.94 +/- 0.03 and 0.67 +/- 0.04. It is concluded that: (1) In accord with previous suggestions, the ionized acidic group known to exist in the Na channel (and to which a guanidinium group of STX appears to bind) is very likely a carboxyl group and not a phosphate group. (2) The accessible part of the Na channel mouth serving as the saxitoxin receptor probably does not include phospholipid in its structure proper.

The gating currents of sodium channels: pore-population-size effects.

Sodium-channel behavior has been modeled in order to determine the answer to the following question: How large must a population of "on-off" sodium pores be before the inherently random behavior of the individual channels becomes smoothed to yield the expected gating current-conductance relationships which would be predicted from an infinite pore array? Results of this analysis show that for the "opening" situation, an excellent fit was obtained whenever more than about 10 pores were considered. Significant discrepancies were observed in the "closing" situation, however, for pore arrays of 50 or less. Marked hysteresis is apparent in the behavior of small port populations.

Axonal surface charges: evidence for phosphate structure.

The effect of different extracellular alkaline-earth cations (Ca2+, Mg2+, Sr2+, Ba2+) upon the threshold membrane potential for spike initiation in crayfish axon has been studied by means of intracellular microelectrodes. This was done at the following extracellular concentrations of the divalent uranyl ion (UO2/2+): 1.0 X 10(-6) M, 3.0 X 10(-6) M, and 9.0 X 10(-6) M. At each concentration employed, extensive neutralization of axonal surface charges by UO2/2+ was evidenced by the fact that equal concentrations (50 mM) of alkaline-earth cations did not have the same effect on the threshold potential. The selectivity sequences observed at the different uranyl-ion concentrations were: 1.0 X 10(-6) M UO2/2+, Ca2+ greater than Mg2+ greater than Sr2+ greater than Ba2+; 3.0 X 10(-6) M UO2/2+, Ca2+ greater than Mg2+ greater than Ba2+ larger than or equal to Sr2+; 9.0 X 10(-6) M UO2/2+, Ca2+ approximately Ba2+ greater than Sr2+ greater than Mg2+. These selectivity sequences are in accord with the equilibrium selectivity theory for alkaline-earth cations. At each of the concentrations used, uranyl ion did not have any detectable effect on the actual shape of the action potential itself. It is concluded that many (if not most) of the surface acidic groups in the region of the sodium gates represent phosphate groups of membrane phospholipids, but that the m gates themselves are probably protein-aceous in structure.

Axonal surface charges: binding or screening by divalent cations governed by external pH.

1. The effect of different extracellular alkaline-earth cations (Ca(2+), Mg(2+), Sr(2+), Ba(2+)) upon the threshold membrane potential for spike initiation in crayfish axon has been studied by means of intracellular micro-electrodes. This was done at the following pHs of the bathing saline: 6.00, 5.35, 5.00, and 4.65 +/- 0.05.2. At pH 6.00, the four alkaline earths had essentially the same effect upon the threshold membrane potential.3. At pH 5.35 or lower, it was found that equal concentrations of the alkaline-earth cations did not have the same effect on the threshold potential. The selectivity sequences observed at the different pHs were: pH 5.35, Ca(2+) > Mg(2+) > Sr(2+) >/= Ba(2+); pH 5.00, Ca(2+) approximately Ba(2+) > Sr(2+) >/= Mg(2+); pH 4.65, Ba(2+), Sr(2+) > Ca(2+) > Mg(2+).4. It is shown that the individual selectivity sequences are predicted rather closely by the equilibrium selectivity theory for alkaline-earth cations.5. It is concluded that the only difference between excitable cells which show screening and those which bind divalent cations is the net density of ionized, surface acidic groups in the region of the sodium gates.