Dexi Liu

Role of liposome size and RES blockade in controlling biodistribution and tumor uptake of GM1-containing liposomes

We have examined the effect of liposome size on liposome circulation time in the blood. Liposomes composed of phosphatidylcholine, cholesterol and ganglioside GM1 were prepared in the various size range. Optimal circulation activity (55% injected dose at 4 h post injection) of GM1-containing liposomes, which correlated with a relatively high uptake of liposomes by EMT6 tumor in mouse, was obtained with a size range of 70 to 200 nm in diameter. Increasing the diameter of liposome to greater than 200 nm resulted in an enhancement of the spleen uptake and decrease of the blood level. For liposomes with a diameter of less than 70 nm, 70% of the injected dose were taken up by the liver, presumably by the parenchymal cells. In contrast, the biodistribution of phosphatidylserine-containing liposomes was relatively insensitive to changes in liposome size; most of the injected dose was found in the liver. The effect of RES blockade on the circulation time of large (d greater than 300 nm), GM1-containing liposomes was also studied. Dextran sulfate 500, a commonly used blockade reagent for Kupffer cells, had no effect. On the other hand, preinjection of a large dose of liposomes with a diameter greater than 500 nm showed variable results depending on the lipid composition of the blocking liposomes. Preinjection of liposomes containing GM1, phosphatidylinositol or (N-polyethyleneglycol) phosphatidylethanolamine effectively reduced the spleen uptake of the large GM1-containing liposomes, whereas liposomes containing phosphatidic acid showed no effect. These results indicate that only spleen homing liposomes can be used as a blocking reagent to prolong the circulation time of the large GM1-containing liposomes.

pH-sensitive, plasma-stable liposomes with relatively prolonged residence in circulation

Acid-sensitive liposomes composed of unsaturated phosphatidylethanolamine (PE) are efficient vehicles for cytoplasmic delivery of the target cells. We have recently shown that liposomes composed of dioleoyl-PE (DOPE) and dipalmitoyl-succinylglycerol (DPSG) retain the acid-sensitivity after exposure to human plasma. In the present work, we have extended these observations to investigate the role of ganglioside GM1 on the blood residence time of these liposomes. Small (d approximately 100 nm) unilamellar liposomes composed of DOPE and DPSG (4:1, molar ratio) became progressively less acid-sensitive when increasing amounts of GM1 were included in the lipid composition. However, partial sensitivity to acid (40-50% release of entrapped contents at pH 4) could be retained up to 5% GM1, even for liposomes which had been exposed to human plasma. Inclusion of GM1 in the lipid composition only slightly increased the release of entrapped contents in the presence of human plasma. The biodistribution of i.v. injected GM1-containing liposomes was studied by following the entrapped 125I-labeled tyraminylinulin marker in Balb/c mice. Inclusion of up to 5% GM1 showed a transient increase in the blood level and a concomitant decrease of liver and spleen uptake of liposomes. Thus, these liposomes are pH-sensitive, plasma-stable and show a relatively prolonged residence time in circulation. They are potentially significant drug carriers in vivo.

Large liposomes containing ganglioside GM1 accumulate effectively in spleen

Large liposomes, with a composition of egg phosphatidylcholine, cholesterol and ganglioside GM1, prepared by an extrusion method, were injected intravenously into mice. After 24 h, up to 50% of injected dose was accumulated in spleen compared with about 15% in spleen for liposomes containing no GM1. The effect of GM1 on spleen accumulation of liposomes was liposome size dependent. Only relatively large liposomes (d greater than 300 nm) showed high accumulation; smaller liposomes were progressively less accumulated. The spleen accumulation increased with increasing injection dose of the liposomes. It was noted that the enhanced uptake by spleen was accompanied by a decrease in the liver uptake, but the total uptake of liposomes by liver and spleen was not dependent on the diameter of liposome or the presence of the ganglioside GM1. Autoradiographs of fixed and sectioned spleen using 125I-labeled tyraminylinulin as a content marker for the liposomes, showed that liposomes localized at the reticular meshwork of the red pulp. These results suggest that larger liposomes containing GM1 are filtered by the spleen during the circulation in blood. The smaller ones with a mean diameter of less than 100 nm are not retained by the filter. The function of GM1 is to prevent liposomes from a rapid uptake by the liver so that liposomes may circulate through the spleen and be filtered. These results, together with the observation that the liposome-entrapped proteins were degraded by the spleen, suggest the potential use of these liposomes for specific drug delivery to the spleen.

Role of cholesterol in the stability of pH-sensitive, large unilamellar liposomes prepared by the detergent-dialysis method

Large unilamellar liposomes prepared by an octyl glucoside-dialysis method were examined for stability at 37 degrees C in the presence or absence of human plasma, using the release of the entrapped calcein as a fluorescence marker. The liposomes were acid-sensitive as they were composed of dioleoylphosphatidylethanolamine, oleic acid and cholesterol. The stability of the liposomes in the absence of plasma was significantly enhanced with increasing cholesterol content. However, the maximal calcein release at pH 5 decreased linearly with increasing cholesterol content of the liposome, indicating that cholesterol had reduced the acid-sensitivity of the liposomes. In the presence of human plasma, calcein release exhibited a biphasic behavior with a fast (plasma-sensitive) and a slow (plasma-resistant) component. Inclusion of cholesterol in the liposomes resulted in an increased proportion of the plasma-resistant component. Liposomes pretreated with human plasma, after removal of excess plasma and the released calcein by gel-filtration, showed a remarkable stability both in the presence and absence of human plasma. The acid-sensitivity of the plasma-treated liposomes with 40% cholesterol was the same as that of the untreated liposomes. These results are discussed in terms of the mechanism by which these liposomes deliver their contents to the cytoplasm of the cells via the endocytic pathway, a known biological activity of the type of liposome described here.

Characterization of plasma-stabilized liposomes composed of dioleoylphosphatidylethanolamine and oleic acid

We have previously reported that small unilamellar liposomes (d less than or equal to 200 nm) composed of dioleoylphosphatidylethanolamine and oleic acid can be stabilized by incubating with normal human plasma (Liu and Huang, Biochemistry 1989, in press). The stabilized liposomes were very stable even under relatively harsh conditions such as extreme pH, high salt and trypsin treatment. Fluorescence depolarization of diphenylhexatriene showed that the stabilized liposome had a high microviscosity in the lipid core, which did not decrease even after the majority of proteins were removed by trypsin. These data suggest that plasma proteins inserted into the lipid bilayer are probably responsible for the stabilization activity. After i.v. injection into mouse, stabilized liposomes showed a relatively low affinity to liver and spleen as compared to a conventional liposome composition.

Size Homogeneity of a Liposome Preparation is Crucial for Liposome Biodistribution in Vivo

AbstractSmall liposomes, prepared by sonication or extrusion, were compared for their biodistribution patterns in mice. Significant difference was observed for a given lipid composition for liposomes prepared by these two different methods. Using gel filtration, negative stain EM, and quasi-elastic light scattering analysis of the liposome preparations, it appeared that such difference was resulted from the heterogeneity in liposome size. Sonicated liposomes, more heterogeneous in size, generally showed a lower uptake in the liver and spleen and higher level in the blood than the less heterogeneous extruded liposomes.

Potentiation of the humoral response of intravenous antigen by splenotropic liposomes

We have recently described that large liposomes composed of egg phosphatidylcholine (PC), cholesterol (chol) and monosialoganglioside GM1 show elevated accumulation in the red pulp of the spleen when they are i.v. administered into mice. Up to 50% of the injected dose was found in spleen at 4 h post injection. In this report, we have investigated the potential application of such liposomes in the stimulation of anti-lysozyme response in mice. Lysozyme entrapped in the splenotropic liposomes composed of PC/chol/GM1 showed higher efficiency in potentiating the humoral response than that of either free lysozyme or lysozyme entrapped in hepatotropic liposomes composed of PC/chol. The results demonstrate that high levels of i.v. antigen delivery by liposomes to the splenic macrophage instead of the liver Kupffer cells is important in the liposomal adjuvanticity. The antibody elicited by the liposome entrapped antigen was mainly IgG1 subtype.

Trypsin-induced lysis of lipid vesicles: Effect of surface charge and lipid composition

We have made a curious observation that the proteolytic enzyme, trypsin, induced a rapid and complete release of the contents of vesicles composed of dioleoylphosphatidylethanolamine (DOPE) and oleic acid (OA). Content release at 37 degrees C, monitored by the release of an entrapped fluorescence marker (calcein), was accompanied by an extensive vesicle aggregation. The lytic activity of trypsin on the vesicles depended on pH and liposome composition. The optimal pH for vesicle lysis was below pH 7.4, which was different from the optimal pH for catalytic activity of trypsin. The lytic activity of trypsin was specific for vesicles composed of DOPE and fatty acids such as OA and palmitoleic acid; vesicles composed of dioleoylphosphatidylcholine, N-methyl-DOPE, and OA, or DOPE combined with other negatively charged lipids such as phosphatidylserine and phosphatidic acid were not sensitive to trypsin. Inhibition of enzyme activity by trypsin inhibitors did not abolish the lytic activity, suggesting that the lytic activity of trypsin is not related to the catalytic activity. However, the lytic activity of trypsin on vesicles composed of DOPE and OA was inhibited in the presence of excess vesicles containing negative charges, or by a pretreatment of trypsin with acylating reagent to reduce the positive-charge content of trypsin. These data demonstrate that vesicle aggregation and lysis are the results of electrostatic interactions of positive charges on trypsin and negative charges on the vesicles. Phase separation and transition to nonbilayer phases of the vesicle lipids are likely involved.