Y.E. Rahman

The effects of charge and size on the interaction of unilamellar liposomes with macrophages

The effect of the positive surface charge of unilamellar liposomes on the kinetics of their interaction with rat peritoneal macrophages was investigated using three sizes of liposomes: small unilamellar vesicles (approx. 25 nm diameter), prepared by sonication, and large unilamellar vesicles (100 nm and 160 nm diameter), prepared by the Lipoprep dialysis method. Charge was varied by changing the proportion of stearylamine added to the liposomal lipids (egg phosphatidylcholine and cholesterol, molar ratio 10:2.5). Increasing the stearylamine content of large unilamellar vesicles over a range of 0-25 mol% enhanced the initial rate of vesicle-cell interaction from 0.1 to 1.4 microgram lipid/min per 10(6) cells, and the maximal association from 5 to 110 micrograms lipid/10(6) cells. Cell viability was greater than 90% for cells incubated with large liposomes containing up to 15 mol% stearylamine but decreased to less than 50% at stearylamine proportions greater than 20 mol%. Similar results were obtained with small unilamellar vesicles except that the initial rate of interaction and the maximal association were less sensitive to stearylamine content. The initial rate of interaction, with increasing stearylamine up to 25 mol%, ranged from 0.5 to 0.7 microgram lipid/min per 10(6) cells, and the maximal association ranged from 20 to 70 micrograms lipid/10(6) cells. A comparison of the number and entrapped aqueous volume of small and large vesicles containing 15 mol% stearylamine revealed that although the number of large vesicles associated was 100-fold less than the number of small vesicles, the total entrapped aqueous volume introduced into the cells by large vesicles was 10-fold greater. When cytochalasin B, a known inhibitor of phagocytosis, was present in the medium, the cellular association of C8-LUV was reduced approx. 25% but association of SUV increased approx. 10-30%. Modification of small unilamellar vesicles with an amino mannosyl derivative of cholesterol did not increase their cellular interaction over that of the corresponding stearylamine liposomes, indicating that cell binding induced by this glycolipid may be due to the positive charge of the amine group on the sugar moiety. The results demonstrate that the degree of liposome-cell interaction with macrophages can be improved by increasing the degree of positive surface charge using stearylamine. Additionally, the delivery of aqueous drugs to cells can be further improved using large unilamellar vesicles because of their greater internal volume. This sensitivity of macrophages to vesicle charge and size can be used either to increase or reduce liposome uptake significantly by this cell type

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.

Evidence of a membrane-bound phospholipase a in rat liver lysosomes

Abstract A phospholipase a with optimal activity at pH 7.0–8.0 was found in the membranes of rat liver lysosomes. This enzyme is activated by Ca ++ , and is specific for the fatty acid at the 2-position. The presence of this enzyme in lysosomes is not due to mitochondrial contamination.

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.

Studies on rat liver ribonucleases: II. Zonal centrifugation of acid ribonuclease; implications for the heterogeneity of lysosomes

Abstract Results obtained by zonal gradient centrifugations demonstrated that rat liver lysosomes are heterogeneous in terms of their enzyme contents. It is suggested that acid phosphatase and cathepsin C on one hand, and acid ribonuclease and cathepsin D on the other, belong to two different classes of “lysosomes”.

Studies on rat liver ribonucleases: IV. Liver ribonucleases in developing, 2-acetylamino-fluorene fed and partially hepatectomized rats

Abstract A relationship was found between the activity of three ribonucleases and liver growth. All three liver ribonucleases showed high activity in fetuses 3 days before birth, in new-borns and in suckling rats; these enzyme activities decreased gradually and attained the adult level between 30 to 40 days of age. Acid phosphatase was relatively constant during the developing stage of the rat. Acid ribonuclease (i.e., ribonuclease I) increased soon after feeding 2-acetyl-aminofluorene while acid phosphatase decreased. Ribonuclease II and ribonuclease III increased during the first 5–6 weeks and decreased subsequently for approx. 10 weeks before returning to the control level. In regenerating liver, all three ribonucleases were increased soon after partial hepatectomy with a maximal increase at about 4 h after the operation. Ribonuclease II activity was significantly decrease from 8 h to 24. Ribonuclease I and ribonuclease III remained increased up to 72 h after partial hepatectomy. The possible implications of the relationship between ribonuclease activities and phenomena of liver growth is discussed.

Acid Phosphatase and -Glucuronidase Activities of Thymus and Spleen of Rats after Whole-Body X-Irradiation.

Summary Acid phosphatase and β-glucuronidase activities of thymus and spleen from rats after whole-body x-irradiation were studied. The increases found in specific activities 24 and 48 hours after a single dose of 200 r and 1000 r could be interpreted on the basis of a selective nitrogen retention in the tissues after radiation, but the increase of acid phosphatase activity in the thymus and spleen 2 hours after 1000 r remained unexplained. The percentage of free activities of these 2 enzymes did not seem to be affected by radiation.