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

Processing of different liposome markers after in vitro uptake of immunoglobulin-coated liposomes by rat liver macrophages

We compared the metabolic fate of [3H]cholesteryl[14C]oleate, [3H]cholesteryl hexadecylether, 125I-labeled bovine serum albumin and [3H]inulin as constituents of large immunoglobulin-coupled unilamellar lipid vesicles following their internalization by rat liver macrophages (Kupffer cells) in monolayer culture. Under serum-free conditions, the cholesteryl oleate that is taken up is hydrolyzed, for the greater part, within 2 h. This occurs in the lysosomal compartment as judged by the inhibitory effect of the lysosomotropic agents monensin and chloroquin. After hydrolysis, the cholesterol moiety is accommodated in the cellular pool of free cholesterol and the oleate is reutilized for the synthesis mainly of phospholipids and, to a lesser extent of triacylglycerols. During incubation in plasma, however, substantial proportions of both the cholesterol and the oleate are shed from the cells, predominantly in the unesterified form. When the liposomes are labeled with the cholesteryl ester analog [3H]cholesteryl hexadecylether only a very small fraction of the label is released from the cells, even in the presence of plasma. Similar to the label remaining associated with the cells, the released label is identified in that case as unchanged cholesteryl ether. The liposomal aqueous phase marker 125I-labeled bovine serum albumin is also readily degraded intralysosomally and the radioactive label is rapidly released from the cells in a trichloroacetic acid-soluble form. Also, as much as 20% of the aqueous phase marker [3H]inulin that becomes cell-associated during a 2-h incubation with inulin-containing liposomes, is released from the cells during a subsequent 4-h incubation period in medium or rat plasma. The usefulness of the various liposomal labels as parameters of liposome uptake and intracellular processing is discusse

Massive targeting of liposomes, surface-modified with anionized albumins, to hepatic endothelial cells

Human serum albumin (HSA) derivatized with cis-aconitic anhydride was covalently coupled to liposomes with a size of approximately 100 nm [polyaconitylated HSA (Aco-HSA) liposomes]. Within 30 min after injection into a rat, Aco-HSA liposomes were completely cleared from the blood and almost exclusively taken up by the liver, whereas in control liposomes 80% was still present in the blood at that time. Endothelial cells were shown to account for almost two-thirds of the hepatic uptake of the Aco-HSA liposomes, the remainder being recovered mainly in the liver macrophages (Kupffer cells). With fluorescently labeled liposomes it was shown that the Aco-HSA liposomes target a vast majority (>85%) of the cells in the endothelial cell population. Control liposomes were not taken up to a significant extent by the endothelial cells. Uptake of Aco-HSA liposomes by both endothelial and Kupffer cells was inhibited by preinjection with polyinosinic acid, indicating the involvement of scavenger receptors in the uptake process. The uptake of Aco-HSA liposomes by liver endothelial cells was dependent on liposome size; with increasing liposome diameter endothelial cell uptake decreased in favor of Kupffer cell uptake. We have demonstrated that massive in vivo targeting of liposomes to a defined cell population other than macrophages is possible. Aco-HSA liposomes thus may represent an attractive drug carrier system for treatment of various liver or liver endothelium-associated disorders

VCAM-1 specific PEGylated SAINT-based lipoplexes deliver siRNA to activated endothelium in vivo but do not attenuate target gene expression

In recent years much research in RNA nanotechnology has been directed to develop an efficient and clinically suitable delivery system for short interfering RNA (siRNA). The current study describes the in vivo siRNA delivery using PEGylated antibody-targeted SAINT-based-lipoplexes (referred to as antibody-SAINTPEGarg/PEG2%), which showed superior siRNA delivery capacity and effective down-regulation of VE-cadherin gene expression in vitro in inflammation-activated primary endothelial cells of different vascular origins. PEGylation of antibody-SAINTPEGarg resulted in more desirable pharmacokinetic behavior than that of non-PEGylated antibody-SAINTPEGarg. To create specificity for inflammation-activated endothelial cells, antibodies against vascular cell adhesion molecule-1 (VCAM-1) were employed. In TNF alpha-challenged mice, these intravenously administered anti-VCAM-1-SAINTPEGarg/PEG2% homed to VCAM-1 protein expressing vasculature. Confocal laser scanning microscopy revealed that anti-VCAM-1-SAINTPEGarg/PEG2% co-localized with endothelial cells in lung postcapillary venules. Furthermore, they did not exert any liver and kidney toxicity. Yet, lack of in vivo gene silencing as assessed in whole lung and in laser microdissected lung microvascular segments indicates that in vivo internalization and/or intracellular trafficking of the delivery system and its cargo in the target cells are not sufficient, and needs further attention, emphasizing the essence of evaluating siRNA delivery systems in an appropriate in vivo animal model at an early stage in their development. (C) 2014 Elsevier B.V. All rights reserved

Anti-VCAM-1 SAINT-O-Somes enable endothelial-specific delivery of siRNA and downregulation of inflammatory genes in activated endothelium in vivo

The pivotal role of endothelial cells in the pathology of inflammatory diseases raised interest in the development of short interfering RNA (siRNA) delivery devices for selective pharmacological intervention in the inflamed endothelium. The current study demonstrates endothelial specific delivery of siRNAs and down-regulation of inflammatory genes in activated endothelium in vivo by applying a novel type of targeted liposomes based on the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride). To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1). In TNF alpha challenged mice, intravenously administered anti-VCAM-1 SAINT-O-Somes exerted long circulation times and homed to VCAM-1 expressing endothelial cells in inflamed organs. The formulations were devoid of liver and kidney toxicity. Using anti-VCAM-1 SAINT-O-Somes we successfully delivered siRNA to knock down VE-cadherin mRNA in inflamed renal microvasculature, as demonstrated by using laser microdissection of different microvascular beds prior to analysis of gene expression. Using the same strategy, we demonstrated local attenuation of endothelial inflammatory response towards lipopolysaccharide in kidneys of mice treated with anti-VCAM-1 SAINT-O-Somes containing NF kappa B p65 specific siRNA. This study is the first demonstration of a novel, endothelial specific carrier that is suitable for selective in vivo delivery of siRNAs into inflamed microvascular segments and interference with disease associated endothelial activation. (C) 2014 Elsevier B. V. All rights reserved

Effects of endothelial targeted AbVCAM-1 dexamethasone SAINT-O-Somes in kidney of double LPS challenged mice.

<p>mRNA expression of endothelial pro-inflammatory molecules, vascular integrity related molecules, blood flow-sensitive transcription factor KLF2, and organ damage related markers NGAL and Kim1 in kidney was determined by real time RT-PCR using GAPDH as a housekeeping gene. All data are presented as mRNA level relative to GAPDH. Values are shown as mean ± SD (n = 6 per group). *, P<0.05, free dexamethasone vs. 2xLPS; #, P<0.05, anti-VCAM-1 respectively IgG dexamethasone SAINT-O-Somes vs. 2xLPS.</p