Therapeutic Efficacy of an ω-3-Fatty Acid-Containing 17-β Estradiol Nano-Delivery System against Experimental Atherosclerosis.

Atherosclerosis and its consequences remain prevalent clinical challenges throughout the world. Initiation and progression of atherosclerosis involves a complex, dynamic interplay among inflammation, hyperlipidemia, and endothelial dysfunction. A multicomponent treatment approach targeted for delivery within diseased vessels could prove beneficial in treating atherosclerosis. This study was undertaken to evaluate the multimodal effects of a novel ω-3-fatty acid-rich, 17-β-estradiol (17-βE)-loaded, CREKA-peptide-modified nanoemulsion system on experimental atherosclerosis. In vitro treatment of cultured human aortic endothelial cells (ECs) with the 17-βE-loaded, CREKA-peptide-modified nanoemulsion system increased cellular nitrate/nitrite, indicating improved nitric oxide formation. In vivo, systemic administration of this nanoemulsion system to apolipoprotein-E knock out (ApoE-/-) mice fed a high-fat diet significantly improved multiple parameters related to the etiology and development of occlusive atherosclerotic vasculopathy: lesion area, circulating plasma lipid levels, and expression of aortic-wall inflammatory markers. These salutary effects were attributed selectively to the 17-βE and/or ω-3 polyunsaturated fatty acid components of the nano-delivery system. At therapeutic doses, the 17-βE-loaded, CREKA-peptide modified nanoemulsion system appeared to be biocompatible in that it elicited no apparent adverse/toxic effects, as indexed by body weight, plasma alanine aminotransferase/aspartate aminotransferase levels, and liver and kidney histopathology. The study demonstrates the therapeutic potential of a novel, 17-βE-loaded, CREKA-peptide-modified nanoemulsion system against atherosclerosis in a multimodal fashion by reducing lesion size, lowering the levels of circulating plasma lipids and decreasing the gene expression of inflammatory markers associated with the disease

<i>In vivo</i> histological evaluation of systemic administration of 17-βE as a solution and a CREKA-peptide-modified nanoemulsion system on plaque size and lipid content.

<p>(A) H & E staining of the aortic valves and plaque area analysis quantified using Image J (n = 4 independent animals per group). The asterisk represents statistical significance between the untreated treatment group and the different 17-βE solution, 17-βE nanoemulsion and blank nanoemulsion treatment groups (<i>P</i> < 0.05). (B) Oil-red-O staining of the aortic valves and plaque lipid analysis quantified using Image J (n = 4 independent animals per group). The asterisk represents statistical significance between the untreated treatment group and the different 17-βE solution, 17-βE nanoemulsion and blank nanoemulsion treatment groups (<i>P</i> < 0.05).</p

<i>In vivo</i> histological evaluation of systemic administration of 17-βE as a solution and a CREKA-peptide-modified nanoemulsion system on plaque elastin and smooth muscle cell content (A) Elastin staining of the aortic valves and plaque elastin analysis quantified using Image J (n = 4 independent animals per group).

<p>There was no significant difference in the elastin content across treatment groups. (B) SMA staining of the aortic valves and plaque SMC content analysis quantified using Image J (n = 4 independent animals per group). There was no significant difference in the elastin content across treatment groups.</p

Plasma aspartate transaminase (AST) and alanine transaminase (ALT).

<p>Plasma aspartate transaminase (AST) and alanine transaminase (ALT).</p

Characterization of the uptake of the 17-βE-loaded CREKA-peptide-modified nanoemulsion system and nitric oxide production by human aortic ECs in culture.

<p>(A) Uptake and distribution of the rhodamine-123 dye labeled CREKA-peptide modified nanoemulsion system in cultured human aortic ECs. (B) Transmission electron microscopy of CREKA-peptide modified 17-βE loaded nanoemulsion formulation. The nanoemulsion is spherical with diameter of ~150nm. Scale bar is 500 nm. (C) Effect of 17-βE solution and nanoemulsion on nitric oxide production (nitrate plus nitrite) by cultured human aortic ECs. Data reported as mean ± S.D. for n = 4 independent samples. The asterisk represents statistical significance (<i>P</i> < 0.05) between untreated control cells and the different solution and CREKA-peptide-modified nanoemulsion-based treatments indicated.</p

<i>In vivo</i> safety profile evaluation of systemic administration of 17-βE as a solution and a CREKA-peptide-modified nanoemulsion system.

<p>(A) Body weights of control mice fed a high-fat diet and mice fed a high-fat diet and treated with either the blank nanoemulsion, 17-βE in solution, or the 17-βE-loaded CREKA-peptide-modified nanoemulsion. (n = 8 independent animals per group). (B) Histology of liver and kidney tissues isolated from the control (untreated) and specified treatment groups. (n = 6 independent animals per group).</p

Systemic administration of 17-βE as a solution and a CREKA-peptide-modified nanoemulsion system alters atherosclerosis-related gene expression within the aorta and improves plasma lipid profile.

<p>(A) The effect of 17-βE in solution, 17-βE in nanoemulsion, and the blank nanoemulsion on relative gene expression in ApoE^-/- mice receiving a high-fat diet measured relative to gene expression in wild-type mice. The asterisk indicates statistical significance (<i>P</i> < 0.05) relative to the baseline gene expression measured in the wild-type mice. (n = 4 independent animals per group). (B) Total plasma cholesterol levels measured in the untreated and the different treatment groups at the beginning and end of the efficacy study. Values are represented as means ± SD (n = 6 independent animals per group). The asterisk represents significance between the untreated animals and the different treatment groups (<i>P</i> < 0.05). (C) Total plasma triglyceride levels measured in the untreated and the different treatment groups at the beginning and end of the efficacy study. Values are represented as means ± SD (n = 6 independent animals per group). The asterisk represents significance between the untreated animals and the different treatment groups (<i>P</i> < 0.05).</p