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

Gecompliceerde fractuur en antisepsis

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

<p>mRNA expression of pro-inflammatory molecules (cell adhesion molecules and cytokines), vascular integrity related molecules (Tie2, CD31, VE-cadherin, Ang2), blood flow-sensitive transcription factor KLF2 in the liver was determined by real time RT-PCR using GAPDH as housekeeping gene. All data are presented as mRNA level relative to GAPDH. Values are shown as mean ± SD of each group (n = 6). *, P<0.05, free dexamethasone vs. 2xLPS; #, P<0.05, anti-VCAM-1 respectively IgG dexamethasone SAINT-O-Somes vs. 2xLPS.</p

Two consecutive challenges of LPS result in microvascular responses in organs comparable to those induced by a single LPS challenge.

<p>Mice were challenged with LPS by one single i.p. injection (2mg/kg) or two consecutive injections (1mg/kg per injection), with the second injection 3h after the first challenge. 24h after the initial challenge, mRNA expression of endothelial pro-inflammatory molecules, vascular integrity related molecules, blood flow-sensitive transcription factor KLF2 in kidney (A), lungs (B), and liver (C), and kidney damage related markers NGAL and Kim1 (A) were determined by real time RT-PCR using GAPDH as housekeeping gene. All data are presented as fold change relative to saline control. Values are shown as mean ± SD (n = 6 per group). *. P<0.05, 1xLPS respectively 2xLPS vs. control; #, P<0.05, 2xLPS vs. 1xLPS.</p

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

AbVCAM-1 dexamethasone SAINT-O-Somes localization follows the expression patterns of VCAM-1 protein in the microvasculature of organs of LPS challenged mice.

<p>(A) Immunohistochemical detection of VCAM-1 protein and localization of dexamethasone loaded SAINT-O-Somes liposomes in kidney, lung, and liver. LPS was administered intraperitoneally with 3 hours interval, and AbVCAM-1 respectively IgG SAINT-O-Somes administered 2h after the first LPS injection. Mice were sacrificed 24h after the first LPS injection. VCAM-1 expression was detected in double LPS challenged mice without intervention, and the localization of dexamethasone containing AbVCAM-1 respectively IgG SAINT-O-Somes was detected in intervention groups. Black arrows indicate the location of SAINT-O-Somes and liposomes in kidney, lung, and liver. Original magnification: 200x. The images are representative of 3 mice per group.</p

AbVCAM-1 dexamethasone SAINT-O-Somes specifically associate with LPS-activated primary endothelial cells in vitro.

<p>Human umbilical vein endothelial cells (HUVEC) were stimulated twice with LPS. The second stimulation was given 3h after the first challenge. Then quiescent respectively LPS activated HUVEC were incubated with anti-VCAM-1 and non-targeted dexamethasone containing SAINT-O-Somes (dex SOS). (A) Fluorescence microscopy images show the uptake of targeted SAINT-O-Somes by activated HUVEC after 4h incubation. The liposome membrane was labeled with Dil (red) and the nuclei of the cells were stained using Hoechst 33342 (blue). Original magnification 400x. (B) Specificity of AbVCAM-1 SAINT-O-Some association to VCAM-1 was determined by co-incubation of cells with 50 times excess of anti-VCAM-1 monoclonal antibodies together with AbVCAM-1 SAINT-O-Somes. After 3h respectively 21h co-incubation, the association of SAINT-O-Somes with activated HUVEC was quantified by flow cytometric analysis. Data are presented as mean fluorescence intensity (MFI) values ± SD of triplicate samples from one experiment. *, P < 0.05, AbVCAM-1 SAINT-O-Somes vs. non-targeted SAINT-O-Somes; #, P<0.05, association of AbVCAM-1 SAINT-O-Some with LPS stimulated HUVEC vs. unstimulated HUVEC; &, P<0.05, significant difference between with or without excess anti-VCAM-1 antibodies.</p