The effect of serum protein fractions on liposome-cell interactions in cultured cells and the perfused rat liver.

We have studied the interactions of liposomes with human skin fibroblasts and mouse P815Y mastocytoma cells in culture, and the perfused rat liver, with the following findings. 1. In all the systems studied serum was found to cause an increase in the uptake of a [14C]cholesterol label into cells from anionic and neutral liposomes and a decrease in the uptake of the label from cationic liposomes. 2. Evidence suggests that albumin enhances the exchange/transfer of [14C]-cholesterol between liposomes and cultured cells. 3. With [14C]cholesterol in the liposome bilayer and [3H]methotrexate entrapped in the aqueous spaces of the liposome, the alpha- and beta-globulin fractions of serum decreased the transfer of both labels from cationic liposomes into cultured cells and the perfused rat liver. The beta-globulin fraction caused increased leakage of methotrexate from fluid liposomes of all charges. 4. The alpha- and beta-globulin fractions of serum appear to enhance the uptake of anionic liposomes into the perfused rat liver.

The effect of reticuloendothelial blockade on the blood clearance and tissue distribution of liposomes.

The blood clearance and tissue distribution of liposomes have been studied in mice subjected to reticuloendothelial blockade with dextran sulphate or carbon. The liposomes have been labelled in the lipid membranes with [3H]-cholesterol, [14C]phosphatidylcholine and/or 99mTc and the content with [14C]inulin. Reticuloendothelial blockade has been shown to slow the rate of clearance of neutral, positively and negatively charged liposomes and of both small unilamellar vesicles and large multilamellar vesicles. In normal animals, the liver uptake accounted for only 20-55% of the total injected radioactivity, the amount varying with the charge and size of the liposomes. Following blockade, the liver uptake of charged and neutral multilamellar liposomes was depressed. This was also true for negatively charged small unilamellar vesicles. The degree of depression of hepatic uptake was between 25-50%, which contrasts with the 80-90% reduction in uptake of a wholly phagocytosed particle (sheep red cells). This difference suggests that mechanisms other than Kupffer cell phagocytosis are also responsible for the normal uptake of liposomes into the liver. In the case of neutral and positively charged small unilamellar vesicles, delayed clearance due to blockade was not associated with 'depressed' hepatic uptake. The site of action of blockading agents for these preparations is not clear. With all preparations of liposomes, blockade produced a slight and variable increase in uptake in the lung and spleen. The alteration of distribution of liposomes by reticuloendothelial blockade is therefore not great and the value of the technique in modifying the tissue distribution of substances within liposomes may be limited.

Inhibitory effect of cholesterol on the uptake of liposomes by liver and spleen.

The effect of cholesterol content of small unilamellar (SUV) and reverse phase (REV) liposomes on blood clearance and tissue distribution has been studied. [14C]Inulin has been used as an aqueous marker of liposomes to represent the uptake of intact liposomes in tissues. The blood clearance of the intravenously-injected SUV and REV liposomes depends on the cholesterol content of liposomes. The cholesterol-free (0 mol%) liposomes are cleared more readily from the circulation than the cholesterol-poor liposomes (20 mol%) and the cholesterol-poor are cleared more rapidly than the cholesterol-rich (46.6 mol%) liposomes. This clearance pattern of liposomes from the circulation is not attributed to the change of size of liposomes due to the increase in cholesterol content of liposomes. However, poor stability of cholesterol-free or cholesterol-poor liposomes in the circulation is partly responsible, but the predominant factor responsible for the observed blood clearance pattern is the inhibitory effect of cholesterol on the uptake of liposomes by reticuloendothelial-rich tissues liver and spleen. Uptake of liposomes by these organs is decreased with increasing cholesterol content of vesicles. It is suggested that to produce liposome preparations with a long circulating half life in vivo it is necessary to inhibit their uptake by liver and spleen.

Improved radioimmunodetection of tumours using liposome-entrapped antibody.

The discrimination of radioimmunodetection of tumours is reduced by the presence of circulating radiolabelled antibody (primary antibody). We have prepared liposomes containing an antibody to the primary antibody (secondary antibody), with the intention of complexing and delivering to the liver primary antibody which is not associated with the tumour. In mice bearing xenografts of human tumours which secrete the marker carcinoembryonic antigen (CEA), liposomally entrapped secondary antibody was able to reduce the blood levels of 125I-labelled anti-CEA within 2 h, without reducing the amount of anti-CEA bound to the tumour. We therefore suggest that the use of liposomally entrapped secondary antibody would improve the diagnostic potential of radioimmunodetection of tumours and their metastases.

Prolonged hypoglycemic effect in diabetic dogs due to subcutaneous administration of insulin in liposomes.

The biologic action of insulin entrapped in liposomes (phospholipid vesicles) has been investigated following subcutaneous injection to dogs made diabetic with a combination of alloxan and streptozotocin. The fats of the liposomally entrapped material was determined by injecting rats subcutaneously with either 125I-insulin or the labeled polysaccharide 14C-inulin, incorporated in liposomes labeled with 3H-cholesterol. Injection of liposome insulin (0.75 U/kg) to five diabetic dogs resulted in a mean (+/- SEM) blood glucose fall from 16.4 +/- 0.8 to 2.9 +/- 0.4 mmol/L. The glucose level had still not returned to baseline after 24 h and, correspondingly, immunoreactive insulin (IRI) could still be detected in frozen and thawed plasma 24 h after injection. In contrast, the hypoglycemic effect of the same dose of free insulin with or without empty liposomes virtually ended within 8 h and IRI levels returned to baseline by 3 h after injection. In experiments on rats with liposomally entrapped 125I-insulin or 14C-inulin the proportion of the injected dose of tracer recoverable by excision of the injection site remained constant after about 1 h and 70% of the dose was still fixed in subcutaneous tissue for at least 5 h thereafter. When the plasma collected 3 h after subcutaneous injection of labeled liposomes containing 125I-insulin was passed through a column of Sepharose 6B, 50-75% of the 125I-activity was found in the fractions associated with intact liposomes. One possibility for the persistence of the hypoglycemic effect and of measurable IRI following injection of liposome insulin could be the presence of intact liposomes in the circulation for many hours after adsorption had ceased.

Possible tumor localization of Tc-99m-labeled liposomes: effects of lipid composition, charge, and liposome size.

The possible in vivo distribution of liposomes after they have been directly labeled with Tc-99m has been studied in rats bearing the Walker 256 carcinoma. The importance of lipid composition, charge, and size of liposome were studied with respect to possible tumor-localizing properties. Tumor uptake was best with small, fluid-membrane, negatively charged liposomes, as indicated by the distribution of the Tc-99m label. The uptake was visualized on scintigrams.

Liposome toxicity in the mouse central nervous system.

This study has shown that while some liposomes are highly toxic to the central nervous system, others, of different composition, are tolerated well in the dosage used (0.02-0.05 ml = 4-12 mg of lipid/inoculum). Those composed of lecithin-cholesterol-dicetyl phosphate or lecithin-cholesterol-stearylamine produced generalised epileptic seizures and some deaths due to respiratory failure immediately after injection,and a subsequent widespread tissue necrosis. However liposomes composed of lecithin-cholesterol-phosphatidic acid, or dipalmitoyl lecithin only, produced minimal morphological changes and by the sixth day post-injection the pathology was limited to the mechanical trauma caused by the injection. It is concluded that liposomes of appropriate composition may be sufficiently benign to use as carriers of therapeutic agents into the CNS.

Lytic effect of heparin on liposomes: possible mechanism of lysis of red blood cells by heparin.

Heparin causes lysis of the multilamellar liposomes of all three charges, positive, neutral, and negative, and thus releases the entrapped [3H]glucose or chromate. The lytic effect of heparin is also observed in liposomes prepared from the lipids extracted from human red blood cells. Heparin is found to interact with the phospholipid bilayers, which suggests that the reported lytic effect of heparin on the red blood cells may be mediated through the membrane phospholipid components of these cells.

Lysosomal localization of -fructofuranosidase-containing liposomes injected into rats.

Yeast beta-fructofuranosidase (invertase) or (131)I-labelled albumin were entrapped into liposomes composed of phosphatidylcholine, cholesterol and phosphatidic acid. Of the beta-fructofuranosidase activity in the liposomal preparations 96-100% was latent. The following observations were made in experiments with rats injected with protein-containing liposomes. 1. After injection of beta-fructofuranosidase-containing liposomes (220 units or 1.5mg of beta-fructofuranosidase and 17.5mg of lipid), beta-fructofuranosidase activity in blood retained its latency but the activity declined to 50% of the injected dose in 1h. Within 6h much of this activity was recovered in the liver and spleen (respectively 45% and 10% of that injected). For up to 21h after injection, the mitochondrial-lysosomal fraction was the principal location of the hepatic beta-fructofuranosidase activity. 2. Lysosomal localization of liposomal protein was supported by the observed increase in the trichloroacetic acid-soluble radioactivity during incubation of the lysosome-rich fraction of the liver of rats injected with liposomes containing (131)I-labelled albumin. 3. Association of liposomal protein with lysosomes was demonstrated on subfractionation of the mitochondrial-lysosomal fraction of the liver of rats injected with beta-fructofuranosidase-containing liposomes in a Ficoll-mannitol gradient. beta-Fructofuranosidase, lysosomal and mitochondrial enzyme marker activities were found to exhibit similar distribution patterns along the gradient. However, in similar experiments with rats previously injected with Triton WR-1339 or dextran (known to alter the specific gravity of lysosomes), only beta-fructofuranosidase and lysosomal marker moved along the gradient, in strikingly similar patterns. 4. The lysosomal localization of injected liposome-entrapped material can probably be utilized in the treatment of certain disorders in man.

Effect of liposomally trapped antitumour drugs on a drug-resistant mouse lymphoma in vivo.

A TLX-5 mouse lymphoma which was resistant to 1-ß-D-arabinofuranosyl cytosine (AraC) was used in vivo to study the possibility of using liposomes as drug-delivery vehicles in order to overcome drug resistance.The effects of free drugs (AraC, AraCTP and methotrexate) and the liposomally associated drugs on the survival time of tumour-bearing mice were determined.As a more sensitive measure of cell survival, (125)IUdR was incorporated into the DNA of the ascites TLX-5 cells before i.p. injection. Cell survival and the cytotoxic effects of the drugs on the tumour cells were determined by using a double-headed gamma counter to measure the retention of the (125)I label.Both AraC and AraCTP, either as the free drugs or liposomally associated, had no effects on the tumour. Due to the lack of response of tumour cells to these drugs, further studies were initiated with free and liposomally associated methotrexate (MTX), a drug to which the cells were known to be sensitive. It was found that the liposomally associated MTX, at a 5-10-fold lower dose than the free drug, was (a) more effective in prolonging the survival of tumour-bearing mice and (b) as effective as the free drug in killing tumour cells (as measured by the (125)I retention).In vivo MTX was more effective in the liposomally associated form, whereas liposomally entrapped AraC and AraCTP were ineffective. It is proposed that in vivo liposomally associated drugs may be acting not by actively localizing in the tumour cells, but by the liposomes providing a slow-release drug depot, improving the pharmacokinetic properties of MTX.

Liposomally trapped AraCTP to overcome AraC resistance in a murine lymphoma in vitro.

Two cell lines, one sensitive and one resistant to the cytotoxic effects of cytosine arabinoside (AraC) were studied in vitro as a drug-resistance model. The sensitivity of these cell lines, to the effects of free and liposomally trapped AraC and AraCTP as well as empty liposomes alone and mixed with free drug, was studied. This was done by following the inhibition of [3H]-dT incorporation into cellular DNA during exposure to the various drugs and liposomes. Some of the liposomal-lipid compositions inhibited [3H]-dT incorporation at very low concentrations, which made them unsuitable for further study. Liposomes composed of a 7:2:1 molar ratio of phosphatidylcholine:cholesterol:phosphatidic acid were selected as a suitable non-inhibitory carrier. Sensitivity of the two cell lines to free AraC differed by 3 logs, when compared in the [3H]-dT-incorporation assay. The resistant cell line was studied further, and was found to be up to 2 logs more sensitive to AraCTP when given in liposomes than to either the free drug alone or mixed with empty liposomes. It appears from these studies that liposomes are able to help overcome drug resistance in this cell line in vitro.

Clearance of injected radioactively labelled antibodies to tumour products by liposome-bound second antibodies.

Liposomes containing anti-goat immunoglobulin were injected 24 h after administration of 125I-labelled goat antibody against the carcinoembryonic antigen (anti-CEA) to groups of nude mice bearing human tumour xenografts, and normal mice. Controls in each group received radioactively labelled anti-CEA only. In liposome-treated mice, blood 125I levels were lower than those of controls 30 min to 24 h after liposome administration, with corresponding accumulation of 125I activity in the liver and spleen for the first 2 h after liposome injection. [14C]Cholesterol or 99mTc labels in the bilayer were eliminated rapidly from the blood, with uptake in the liver and spleen. In xenograft-bearing mice, 125I activity detected in the tumours up to 6 h after liposome injection was identical to that detected in the tumours of controls. However, 24 h after liposome injection a reduction in the tumour concentration of 125I-labelled anti-CEA was obtained, but the tumour/blood radioactivity was still increased. In two mice given 27 mumol lipid, the blood radioactivity count after 24 h was only 5% of that in the controls. In rabbits, 2 h after administration of liposomes containing anti-goat second antibody, the circulating 125I activity had dropped by 28-40%. The results suggest that administration of liposome-entrapped second antibody approximately 2 h prior to external scintigraphy may clear circulating radioactively labelled primary antibody by up to 50%.

Tissue distribution and tumour localization of 99m-technetium-labelled liposomes in cancer patients.

The possible use of liposomes (phospholipid vesicles) to direct cytotoxic drugs to tumours has led us to investigate the tissue localization of i.v. injected 99m-Tc-labelled liposomes in cancer patients. Twenty mg or 300 mg doses of liposomal lipid (7:2:1 molar ratio of phosphatidylcholine : cholesterol : phosphatidic acid) were used in a study of 13 patients with advanced cancer and one with polycythaemia rubra vera (PRV). In all cases except the patient with PRV the major site of uptake of the label was the liver and spleen. In the patient with PRV the liver uptake was greatly reduced and the major site of uptake was found in regions corresponding to marrow. With the exception of one patient with a primary hepatoma, there was no significant tumour uptake of the label.

Liposomally entrapped second antibody improves tumour imaging with radiolabelled (first) antitumour antibody.

The usefulness of antibodies directed against tumour products for localisation and therapy of cancer is limited by the large proportion of administered antibody which remains non-specifically in the circulation and extravascular space. Liposomally entrapped second antibody (LESA) directed against the first (antitumour) antibody has been used to accelerate clearance of non-tumour-bound first antibody without affecting its clearance from the tumour. Intravenously administered LESA binds the first antibody and LESA is cleared by the reticuloendothelial system. LESA accelerated clearance of radiolabelled antibody directed against carcinoembryonic antigen (CEA) from the circulation in four of five patients with gastrointestinal cancer, enhancing gamma-camera imaging of the tumour in three of them. These results suggest that LESA can improve the sensitivity and specificity of tumour imaging with radiolabelled antitumour antibody. This strategy may also have the potential to improve the therapeutic ratio of some toxic agents linked to antitumour antibody.