Endothelium-targeted delivery of dexamethasone by anti-VCAM-1 SAINT-O-Somes in mouse endotoxemia

Microvascular endothelial cells play a pivotal role in the pathogenesis of sepsis-induced inflammatory responses and multiple organ failure. Therefore, they represent an important target for pharmacological intervention in the treatment of sepsis. Glucocorticosteroids were widely used in the treatment of sepsis but vast evidence to support their systemic use is lacking. The limited effects of glucocorticoids in the treatment of sepsis may be explained by differential effects of drug initiated NF-κB inhibition in different cell types and insufficient drug delivery in target cells. The current study aimed therefore to investigate the effects of an endothelial targeted delivery of dexamethasone in a mouse model of endotoxemia induced by two consecutive i.p. injections of lipopolysaccharide (LPS). To achieve endothelial cell specific delivery of dexamethasone, we modified SAINT-O-Somes, a new generation of liposomes that contain the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl) methyl-pyridinium chloride, with antibodies against vascular cell adhesion molecule-1 (VCAM-1). In LPS challenged mice, the systemic administration of free dexamethasone had negligible effects on the microvascular inflammatory endothelial responses. Dexamethasone-loaded anti-VCAM-1 SAINT-O-Somes specifically localized at VCAM-1 expressing endothelial cells in the microvasculature of inflamed organs. This was associated with a marginal attenuation of the expression of a few pro-inflammatory genes in kidney and liver, while no effects in the lung were observed. This study reveals that, although local accumulation of the targeted drug was achieved, endothelial targeted dexamethasone containing anti-VCAM-1 SAINT-O-Somes exhibited marginal effects on inflammatory endothelial cell activation in a model of endotoxemia. Studies with more potent drugs encapsulated into anti-VCAM-1 SAINT-O-Somes will in the future reveal whether this delivery system can be further developed for efficacious endothelial directed delivery of drugs in the treatment of sepsis

On the size-dependent disintegration of small unilamellar phosphatidylcholine vesicles in rat plasma. Evidence of complete loss of vesicle structure.

The destruction of small unilamellar egg phosphatidylcholine vesicles in rat plasma was monitored by measuring release of encapsulated 125I-poly(vinylpyrrolidone) or carboxyfluorescein and by determining transfer of radiolabelled phosphatidylcholine to plasma lipoproteins by means of gel filtration. The susceptibility of the vesicles to the destructive action of plasma increased with decreasing vesicle size, as observed by incubating plasma with individual fractions constituting the small-vesicle peak on Sepharose CL-2B. This results in selective destruction of small vesicles when heterogeneous vesicle populations are incubated with plasma. Samples of homogeneous vesicle populations were incubated with a wide range of plasma concentrations, which resulted in extents of solute and phospholipid release ranging from 10 to 90%. When the extents of solute release were plotted against the extents of lipid release a linear, virtually 1:1, relationship was found, for both carboxyfluorescein and poly(vinylpyrrolidone) as the solute. This suggests that the release of solutes from small unilamellar phosphatidylcholine vesicles as a result of their interaction with plasma (lipo)proteins involves the total destruction of a fraction of the vesicles, the magnitude of which is determined by the vesicle: plasma ratio. Our results argue against a previously presented view suggesting that the interaction between such vesicles and plasma results in the formation of pores through which encapsulated solutes diffuse at Mr-dependent rates [Kirby & Gregoriadis (1981) Biochem. J. 199, 251-254]. The discrepancies between the two studies in observations as well as in interpretation are discussed

INVIVO FATE OF LARGE UNILAMELLAR SPHINGOMYELIN-CHOLESTEROL LIPOSOMES AFTER INTRAPERITONEAL AND INTRAVENOUS-INJECTION INTO RATS

We investigated the fate of intraperitoneally and intravenously injected reverse phase evaporation vesicles of fairly uniform size (100-200 nm) with respect to blood clearance, tissue distribution and integrity in vivo. The vesicles are composed of sphingomyelin and cholesterol in a molar ratio 3 : 2 and contain 125I-labeled poly(vinyl pyrrolidone) in the aqueous compartment. It is shown that following an intraperitoneal injection the vesicles are transported intact, and not associated with cells, from the peritoneal cavity to the blood and are subsequently taken up mainly by liver and spleen, where, particularly in liver, the phospholipid is partially metabolized. After an intraperitoneal injection the rate of vesicle-uptake by liver and spleen is reduced by a factor of 2-3 compared to the rate of vesicle-uptake by liver and spleen following an intravenous injection. The peritoneal cavity functions as a reservoir of vesicles for some hours. The rates of blood clearance and uptake of the vesicles by liver and spleen appear to be slower than that found for vesicles of different lipid composition

Lactosylceramide-induced stimulation of liposome uptake by Kupffer cells in vivo

Incorporation of 8 mol percent lactosylceramide into small unilamellar vesicles consisting of cholesterol and sphingomyelin in an equimolar ratio and containing [3H]inulin as a marker resulted in an increase in total liver uptake and a drastic change in intrahepatic distribution of the liposomes after intravenous injection into rats. The control vesicles without glycolipid accumulated predominantly in the hepatocytes, but incorporation of the glycolipid resulted in a larger stimulation of Kupffer-cell uptake (3.2-fold) than of hepatocyte uptake (1.2-fold). Liposome preparations both with and without lactosylceramide in which part of the sphingomyelin was replaced by phosphatidylserine, resulting in a net negative charge of the vesicles, were cleared much more rapidly from the blood and taken up by the liver to higher extents. The negative charge had, however, no influence on the intrahepatic distributions. The fast hepatic uptake of the negatively charged liposomes allowed competition experiments with substrates for the galactose receptors on liver cells. Inhibition of blood clearance and liver uptake of lactosylceramide-containing liposomes by N-acetyl-d-galactosamine indicated the involvement of specific recognition sites for the liposomal galactose residues. This inhibitory effect of N-acetyl-d-galactosamine was shown to be mainly the result of a decreased liposome uptake by the Kupffer cells, compatible with the reported presence of a galactose specific receptor on this cell type (Kolb-Bachofen et al. (1982) Cell 29, 859–866). The difference between the results on sphingomyelin-based liposomes as described in this paper and those on phosphatidylcholine-based liposomes as published previously (Spanjer and Scherphof (1983) Biochim. Biophys. Acta 734, 40–47) are discussed

THE EFFECT OF LIVER MACROPHAGES ON INVITRO CYTOLYTIC ACTIVITY OF 5FU AND FUDR ON COLON-CARCINOMA CELLS - EVIDENCE OF MACROPHAGE ACTIVATION

While investigating the effects of 5-fluorouracil (5FU) and 5-fluoro-2'-deoxyuridine (FUdR) on the tumoricidal state of rat liver macrophages activated in vitro by means of liposome-encapsulated muramyl dipeptide (MDP), we observed that 5FU in combination with macrophages produced substantially higher extents of cytolytic activity on tumor cells than 5FU alone. In contrast, FUdR failed to produce this effect; rather, at relatively low FUdR concentrations, lytic activity in the presence of macrophages was even significantly diminished as compared with FUdR in the absence of macrophages. Both 5FU and FUdR were able to enhance the cytolytic activity of macrophages activated by liposome-encapsulated MDP. This finding indicates that, rather than inhibiting the activation of macrophages by liposomal MDP, 5FU can act as a stimulator of macrophage activation by itself. This is further supported by the observations that (i) in combination with 5FU, the secretion of TNF induced by liposomal MDP was synergistically enhanced and (ii) that a second treatment of macrophages with the drug, 24 h after the first, fails to produce increased macrophage cytotoxicity. Our results also show that neither 5FU nor FUdR are likely to unfavorably influence the induction of cytotoxic activity of the macrophages. Rather, combinations of 5FU or FUdR and liposomal MDP may result in an additive or synergistic tumoricidal effect