Sulfatide-containing Liposomes Research Articles

Sulfatide-tenascin interaction mediates binding to the extracellular matrix and endocytic uptake of liposomes in glioma cells.

Tenascin-C is an extracellular matrix glycoprotein, whose expression is highly restricted in normal adult tissues, but markedly up-regulated in a range of tumors, and therefore serves as a potential receptor for targeted anticancer drug or gene delivery. We describe here a liposomal carrier system in which the targeting ligand is sulfatide. Experiments with tenascin-C-expressing glioma cells demonstrated that binding of liposomes to the extracellular matrix relied essentially on the sulfatide-tenascin-C interaction. Following binding to the extracellular matrix, the sulfatide-containing liposomes were internalized via both caveolae/lipid raft- and clathrin-dependent pathways, which would ensure direct cytoplasmic release of the cargoes carried in the liposomes. Such natural lipid-guided intracellular delivery targeting at the extracellular matrix glycoproteins of tumor cells thus opens a new direction for development of more effective anticancer chemotherapeutics in future.

Intracellular drug delivery by sulfatide-mediated liposomes to gliomas

We described here a liposomal carrier system in which the targeting ligand was sulfatide, a glycosphingolipid known to bind several extracellular matrix (ECM) glycoproteins whose expression was highly up-regulated in many tumors. In vitro experiments with human glioma cell lines demonstrated that robust intracellular uptake of the liposomes depended specifically on the presence of sulfatide as the key liposomal component. Significant amount of the liposomes remained largely intact in the cytoplasm for hours following their internalization. When anticancer drug doxorubicin (DOX) was encapsulated in such liposomes, most of the drug was preferably delivered into the cell nuclei to exert its cytotoxicity. Use of this drug delivery system to deliver DOX for treatment of tumor-bearing nude mice displayed much improved therapeutic effects over the free drug or the drug carried by polyethylene glycol (PEG)-grafted liposomes. Our results demonstrate a close link between effective intracellular uptake of the drug delivery system and its therapeutic outcome. Moreover, the sulfatide-containing liposomes (SCL) may represent an interesting ligand-targeted drug carrier for a wide spectrum of cancers in which sulfatide-binding ECM glycoproteins are expressed.

Lysine residue 117 of the FasG adhesin of enterotoxigenic Escherichia coli is essential for binding of 987P fimbriae to sulfatide.

The FasG subunit of the 987P fimbriae of enterotoxigenic strains of Escherichia coli was previously shown to mediate fimbrial binding to a glycoprotein and a sulfatide receptor on intestinal brush borders of piglets. Moreover, the 987P adhesin FasG is required for fimbrial expression, since fasG null mutants are nonfimbriated. In this study, fasG was modified by site-directed mutagenesis to study its sulfatide binding properties. Twenty single mutants were generated by replacing positively charged lysine (K) or arginine (R) residues with small, nonpolar alanine (A) residues. Reduced levels of binding to sulfatide-containing liposomes correlated with reduced fimbriation and FasG surface display in four fasG mutants (R27A, R286A, R226A, and R368). Among the 16 remaining normally fimbriated mutants with wild-type levels of surface-exposed FasG, only one mutant (K117A) did not interact at all with sulfatide-containing liposomes. Four mutants (K117A, R116A, K118A, and R200A) demonstrated reduced binding to such liposomes. Since complete phenotypic dissociation between the structure and specific function of 987P was observed only with mutant K117A, this residue is proposed to play an essential role in the FasG-sulfatide interaction, possibly communicating with the sulfate group of sulfatide by hydrogen bonding and/or salt bridge formation. Residues K17, R116, K118, and R200 may stabilize this interaction.

Effect of Adriamycin Entrapped in Sulfatide-Containing Liposomes on Colon Tumor Cells Metastasized.

Antineoplastic effect of adriamycin entrapped in sulfatidecontaining liposomes on colon tumor cells metastasized to the liver and preventive effect of the liposome-entrapped adriamycin on liver metastasis were studied by use of an in vivo murine model for liver metastasis made by portal injection of cells of colon 26, a murine colon cancer cell line. The levels of adriamycin in the liver at 24 and 72h after intravenous administration of the drug entrapped in sulfatide-containing liposomes were 68.2±8.7 and 47.5±12.0 nmol/g liver tissue, respectively. In the case of free adriamycin, those levels were 3.1±1.0 and 2.4±1.0nmol/g tissue. These results indicate that upon entrapment of adriamycin in the liposomes, a higher level of the drug was maintained in the liver for a longer time than the free drug. Administration of 6mg/kg of adriamycin entrapped in liposomes at 7 days after the injection of colon 26 cells brought about a significant reduction in the number of colonies on the liver surface counted at 14 days after cell injection compared with the free drug (p<0.05). The pretreatment with adriamycin entrapped in liposomes resulted in the absence of colonies in 4 of 6 mice and significantly prolonged their survival time (p<0.05). These results indicate that liposomal adriamycin is effective in the treatment of liver metastasis.

Endocytosis of sulfatides by macrophages: relationship to the cellular uptake of phosphatidylserine.

These studies initially examined the effect of sulfatides on the endocytosis of phosphatidylserine (PS) liposomes in J774 macrophages employing liposomes composed entirely of PS and the nonexchangeable radiolabel [3H]cholesteryl hexadecyl ether. Bovine brain sulfatides significantly inhibited the uptake of PS liposomes by macrophages to a level of approximately 15% of control values. To examine whether macrophages were also capable of recognizing and internalizing sulfatides, sulfatide-containing liposomes were prepared using phosphatidylcholine (PC), cholesterol, and sulfatides (6:2:4 molar ratio) incorporating the same radiolabel. The sulfatide-containing liposomes were found to be avidly endocytosed by macrophages. Uptake of the sulfatide-containing liposomes by macrophages was significantly greater than the uptake of liposomes made without sulfatides. When the macrophages were incubated with the anionic compounds dextran sulfate and fucoidin, both the binding of the liposomes to the macrophages at 4 degrees C and the internalization of the liposomes at 37 degrees C were inhibited to approximately 10% of control values. The negatively charged phospholipids phosphatidylglycerol and cardiolipin significantly inhibited the uptake of sulfatide-containing liposomes, and PS was not effective in reducing the cellular uptake of these liposomes. Both oxidized low-density lipoprotein (LDL) and acetylated LDL reduced the uptake of the sulfatide-containing liposomes; the uptake observed in the presence of acetylated LDL and oxidized LDL was approximately 70% and 40%, respectively, of control values. These findings demonstrate that macrophages can efficiently endocytose both sulfatides and negatively charged phospholipids to remove them from the circulation.

Pharmacodynamics and tumoricidal effect of adriamycin entrapped in ceramide sulfate-containing liposomes.

Ceramide sulfate (N-acylsphingosine-1-O-sulfate), which lacks the galactose residue of sulfatide, was examined as a possibly preferable constituent of liposomes for drug delivery. Multilamellar vesicles prepared from phosphatidylcholine, cholesterol, and N-lignoceroylsphingosine-1-O-sulfate in a molar ratio of 5:4:1 efficiently entrapped adriamycin, and the retention of the drug in the liposomes in saline at 4 degrees C for 8 d was nearly 100%. In terms of entrapment efficiency and retention of the drug in liposomes, N-lignoceroylsphingosine-1-O-sulfate was superior to N-stearoylsphingosine-1-O-sulfate. A pharmacodynamic study revealed that the blood level of adriamycin was far higher with the drug encapsulated in N-lignoceroylsphingosine-1-O-sulfate-containing liposomes than with the free drug. The drug level in the heart was remarkably reduced with the liposome-entrapped drug, which is advantageous in reducing the cardiotoxicity of this drug. The effect of N-lignoceroylsphingosine-1-O-sulfate-containing liposomes on the blood level of adriamycin was superior to that of sulfatide-containing liposomes, though the effect of the former on the heart level was comparable to that of the latter. The tumoricidal effect on ascitic P388 leukemia and Lewis lung carcinoma was higher with adriamycin entrapped in N-lignoceroylsphingosine-1-O-sulfate-containing liposomes than with the free drug.

Biodistribution of calcitonin encapsulated in liposomes in mice with particular reference to the central nervous system

The biodistribution of [ 125I]porcine calcitonin (pCT) encapsulated in reverse-phase evaporation vesicles (REVs) in mice upon the intravenous administration was examined. It was found that sulfatide significantly improved the stability of REVs in vivo, and altered the relative distribution of [ 125I]pCT encapsulated in liposomes in mice. These sulfatide-containing REVs were able to target [ 125I]pCT into the liver and central nervous system (CNS) reasonably well, with the maximal effect of about 40% and 2% of the injected doses occuring at 30 min and 90 min, respectively, after injection. Neither free [ 125I]pCT, nor sulfatide-free liposome-encapsulated [ 125I]pCT, nor a mixture of free [ 125I]pCT and empty sulfatide liposomes was effective. [ 125I]pCT was widely distributed in the CNS, with predominance in hypothalamus, brainstem, striatum and spinal cord. The result indicate that pCT encapsulated in sulfatide-containing liposomes is able to pass through the blood-brain barrier (BBB), and calcitonin, thus encapsulated, may be applicable to studies on its functions in the CNS.

Antinociceptive activity of liposome-entrapped calcitonin by systemic administration in mice

Porcine calcitonin (pCT) encapsulated in sulfatide-containing reverse-phase evaporation vesicles (REVs) was shown to induce long-lasting and dose-dependent antinociceptive effect by systemic injection in mice. Its antinociceptive activity is found to be dependent on the route of administration. In contrast, neither unencapsulated pCT nor pCT encapsulated in sulfatide-free REVs was effective. These results suggest that pCT entrapped in sulfatide-containing liposomes might be able to cross the blood-brain barrier (BBB) and to produce central antinociception in mice.

Preparation and Some Properties of Cholesterol Sulfate-Containing Liposomes Entrapping Adriamycin.

Cholesterol sulfate (CholSO4) was found to be a useful substitute of sulfatide for the preparation of negatively charged liposomes entrapping adriamycin (ADM). Liposomes composed of egg phosphatidylcholine, cholesterol, and CholSO4 in a molar ratio of 6:3:1 entrapped ADM efficiently and reduced the leakage of the entrapped ADM from the liposomes to the same degree as found for sulfatide-containing liposomes. The novel liposomes induced neither haemolysis nor platelet aggregation

Adriamycin entrapped in sulfatide-containing liposomes

Sulfatide-containing liposomes composed of PC, cholesterol, and sulfatide in a molar ratio of 7 : 2 : 1 entrapped ADM most efficiently among the negatively-charged liposomes tested. A unilamellar vesicle entrapped 123 ADM molecules, of which 6 molecules are localized in the internal space of the vesicle, 4 molecules are embedded into the membrane matrix, and 113 molecules are bound to the inner surface of the liposomal membrane. Highly efficient entrapment of ADM by the liposomes seems to be due to their rigidity. By the experiment using ovarian tumor-bearing nude mice, it was found that the liposome-entrapped ADM was maintained at much higher blood level, at lower concentration in the heart, and at higher concentration in the tumor than the free drug. The antitumor activity of the liposome-entrapped ADM was comparable with that of the free drug. The body weight of the animals was not affected by the former, whereas it was drastically decreased by the latter.

Improved Sulfatide-Containing Liposomes as Carriers of Adriamyein

Various molar ratios of the constituents of sulfatide-containing liposomes (egg phosphatidylcholine, cholesterol, and sulfatide) were examined with respect to liposomal efficiency to entrap adriamycin (ADM) and to the tissue distribution. Among the ratios of the constituents examined, 5:4:1 was the best in terms of entrapment efficiency for both multilamellar vesicles (MLV) and small unilamellar vesicles (SUV). Tissue distribution studies of liposome-entrapped ADM showed that the blood clearance and cardiac uptake of ADM were significantly reduced when the drug was entrapped in the above liposomes, especially in the MLV. Incorporation of ADM into ovarian tumors transplanted into nude mice was increased when the drug was entrapped in the SUV.