Elizabeth A. Cerny

Intracellular plutonium: removal by liposome-encapsulated chelating agent.

Chelating agents, such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) were successfully encapsulated within lipid spherules (that is, liposomes). Encapsutlated [14C]EDTA, given intravenously to mice, was retained longer in tissues that nonencapsulated [14C]EDTA. Encapsulated DTPA, given to mice 3 days after pluttonium injection, removed an additional fraction of plutonium in the liver, presumably intracellular, not available to nonencapslulated DTPA. It also further increased urinary excretion of plutonium. Introduction of chelating agents into cells by liposomal encapsulation is a promising new approach to the treatment of metal poisoning

Tissue distribution of EDTA encapsulated within liposomes of varying surface properties

Liposomes containing ethylenediaminetetraacetic acid (EDTA) were prepared with different surface properties by varying the liposomal lipid constituents. Positively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and stearylamine. Negatively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and phosphatidylserine. Neutral liposomes were prepared with phosphatidylcholine alone, dipalmitoyl phosphatidylcholine alone, or with a mixture of phosphatidylcholine and cholesterol. Distribution of 14C-labeled EDTA were determined in mouse tissues from 5 min to 24 h after a single intravenous injection of liposome preparation. Differences in tissue distribution were produced by the different liposomal lipid compositions. Uptake of EDTA by spleen and marrow was highest from negatively charged liposomes. Uptake of EDTA by lungs was highest from positively charged liposomes; lungs and brain retained relatively high levels of EDTA from these liposomes between 1 and 6 h after injection. Liver uptake of EDTA from positively or negatively charged liposomes was similar; the highest EDTA uptake by liver was from the neutral liposomes composed of a mixture of phosphatidylcholine and cholesterol. Liposomes composed of dipalmitoyl phosphatidylcholine produced the lowest liposomal EDTA uptake observed in liver and marrow but modrate uptake by lungs. Tissue uptake and retention of EDTA from all of the liposome preparations were greater than those of non-encapsulated EDTA. The results presented demonstrate that the tissue distribution of a molecule can be modified by encapsulation of that substance into liposomes of different surface properties. Selective delivery of liposome-encapsulated drugs to specific tissues could be effectively used in chemotherapy and membrane biochemistry.

Differential uptake of liposomes varying in size and lipid composition by parenchymal and kupffer cells of mouse liver.

Using liposomes differing in size and lipid composition, we have studied the uptake characteristics of the liver parenchymal and Kupffer cells. Desferal labeled with iron-59 was chosen as a radiomarker for the liposomal content, because Desferal in its free form does not cross cellular membranes. At various time intervals after an intravenous injection of liposomes into mice, the liver was perfused with collagenase, and the cells were separated in a Percoll gradient. It was found that large multilamellar liposomes (diameter of about 0.5 micron) were mainly taken up by the Kupffer cells. For these large liposomes, the rate of uptake by Kupffer cells was rapid, with maximum uptake at around 2 hours after liposome injection. Unexpectedly, small unilamellar liposomes (diameter of about 0.08 micron) were less effectively taken up by Kupffer cells, and the rate of uptake was slow, with a maximum uptake at about 10 hours after liposome injection. In contrast, parenchymal cells were more effective in taking up small liposomes and the uptake of large liposomes was negligible. In addition, liposomes made with a galactolipid as part of the lipid constituents appeared to have higher affinity to parenchymal cells than liposomes made without the galactolipid. These findings should be of importance in designing suitable liposomes for drug targeting.

Liposome-Encapsulated Actinomycin D: Potential in Cancer Chemotherapy

Summary Actinomycin D, when encapsulated within liposomes, is less toxic to mice than the nonencapsulated form. A single dose (0.75 mg/kg) or multiple doses (1 × 0.50, 4 × 0.25 mg/kg) significantly increased the mean survival time of mice inoculated with Ehrlich ascites tumor cells. Liposomes containing actinomycin D were found in tumor cells and cell degeneration and death were subsequently observed.

Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids

Multilameller liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside GM1, monosialoganglioside GM2, monosialoganglioside GM3, disialoganglioside GD1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distributions of 14C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome preparation. Liver uptake up encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides, regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing GD1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing GD1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.

EFFECTS OF ADMINISTRATION OF THIOACETAMIDE OR TESTOSTERONE ON CHROMOSOME-ASSOCIATED PROTEINS IN RAT LIVER AND KIDNEY.

Abstract This report deals with the isolation and analysis of liver cell nuclei from rats receiving thioacetamide daily, and of kidney cell nuclei from rats in which the testosterone balance had been altered in various ways. The results indicate that in the enlargement of liver cell nuclei produced by thioacetamide administration, there is a large increase in nuclear RNA and a large increase in “residue” protein. In the enlargement of kidney cell nuclei produced by testosterone administration, there is no increase in nuclear RNA, and there is a slight decrease in “residue” protein per milligram of DNA. Neither treatment produced any quantitative change in the capacity of the chromosomes to bind the “soluble” (saline-extractable) proteins. The values for nuclear RNA, “residue” protein, and “soluble” protein per milligram of DNA are all considerably higher for liver cell nuclei than the corresponding values for kidney cell nuclei.

Studies on the mechanism of erythrocyte aging and destruction I. Separation of rat erythrocytes according to age by ficoll gradient centrifugation

Abstract A 22–34% linear Ficoll gradient is described for use in separating red blood cells of different ages. Newly synthesized, 59Fe-labeled red cells were found in the top fractions of the gradient, and these cells moved down toward the lower part of the gradient as they became older. The Ficoll gradient method gave a higher resolution compared to existing published methods. Red blood cells with increasing density, increasing sphericity and decreasing volume were separated by the Ficoll gradient method. The protein content remained constant in red cells up to about two weeks of age, and then showed a trend to decrease with age. Changes in density, shape, volume, and probably protein content, are characteristics of aging red cells.

Studies on the mechanism of erythrocyte aging and destruction II. Membrane fragmentation in rat erythrocytes after in vitro treatment with lysophosphatides: Scanning electron microscope studies

Abstract Rat red blood cells of differing ages were separated according to density by Ficoll gradient centrifugation. Cells from the top, middle and bottom fractions were incubated with sublytic concentrations of lysophosphatidylethanolamine (LPE) or lysophosphatidylcholine (LPC). The surface transformations of these red cells were observed with a scanning electron microscope. In the presence of low concentrations of LPE, normal red blood cells with their characteristic biconcave-disc shape become irregular with large bulges on the surface. As the concentrations of LPE increase these bulges elongate as pseudopod-like protuberances which transform into narrow spicules. A small globular tip forms at the end of each spicule as a result of the pinching-in of the membrane. Continuing constriction leads to the detachement of the small fragment at the tip and membrane fragmentation occurs. Microfragmentation of the red cell membrane induced by LPE or LPC is an irreversible phenomenon. In the presence of a given concentration of LPE, the red cells from the top fractions of the Ficoll gradient are more resistant than cells from the middle fractions; cells from these latter fractions are in turn more resistant than those from the bottom fractions of the gradient. The sequence of membrane transformation produced by lysophosphatides in vitro can satisfactorily explain most of the in vivo age-dependent changes found in red blood cells.

Radiolytic Generation of Gases from Synthetic Waste, Annual Report: 1991

Annual report of an Argonne National Laboratory Chemistry Division program on radiolytic generation of gases from synthetic waste. This report includes results of studies on simulated waste solutions to measure the presence and absence of organic chelators and their products.

Liposome uptake into human colon adenocarcinoma cells in monolayer, spinner, and trypsinized cultures.

Abstract The purpose of this study was to begin investigating the nature of liposome interactions with colon tumor cells. Thus, experiments were performed to study the uptake and incorporation of multilamellar and of reverse-phase evaporation liposomes of neutral charge into monolayers, suspended spinner cultures, and trypsinized cells of a human colon adenocarcinoma cell line, LSI74T. The results showed that the same tumor cells cultured under each condition exhibited a distinct pattern of vesicle uptake as determined at 0, 15, 30, 60, and 120 min. In monolayer cultures of LS174T cells, the uptake of liposomes bearing [3H]actinomycin D in the lipid bilayers was linear throughout the incubation period. In contrast, in trypsinized and spinner suspension cultures, uptake of liposomes was biphasic. There was a proportional uptake of both liposome (labeled with [3phosphatidylcholine or [14C]cholesterol) and of actinomycin D (trace labeled with 3H) into the cells under all culture conditions, indicating quantitative delivery of the drug with the intact lipid vesicle. Although the amount of actinomycin D presented to tumor cells by the two liposomes was equivalent, reverse-phase evaporation liposomes were more effective than multilamellar vesicles in inhibiting uridine uptake. In the presence of excess liposomes (10 times the uptake studies), saturation of the tumor cell surface occurred by 120 min. However, the liposomes remained accessible to enzymatic removal for 60 min. Liposome-saturated tumor cells remained refractory to further binding of liposomes for at least 2 hr. The results thus revealed that differences in cell uptake were due to the state of the target cells and not the liposome types, or their differential leakage of labels.