Cholesterol nanoarchaeosomes for alendronate targeted delivery as an anti-endothelial dysfunction agent.

Sodium alendronate (ALN) is a very hydrosoluble and poorly permeable molecule used as an antiresorptive agent and with vascular anticalcifying capacity. Loaded into targeted nanovesicles, its anti-inflammatory activity may be amplified towards extra-osseous and noncalcified target cells, such as severely irritated vascular endothelium. Here cytotoxicity, mitochondrial membrane potential, ATP content, and membrane fluidity of human endothelial venous cells (HUVECs) were determined after endocytosis of ALN-loaded nanoarchaeosomes (nanoARC-Chol(ALN), made of polar lipids from Halorubrum tebenquichense: cholesterol 7:3 w/w, 166 ± 5 nm, 0.16 ± 0.02 PDI, -40.8 ± 5.4 mV potential, 84.7 ± 21 µg/mg ALN/total lipids, TL). The effect of nanoARC-Chol(ALN) was further assessed on severely inflamed HUVECs. To that aim, HUVECs were grown on a porous barrier on top of a basal compartment seeded either with macrophages or human foam cells. One lighter and one more pronounced inflammatory context was modelled by adding lipopolysaccharide (LPS) to the apical or the apical and basal compartments. The endocytosis of nanoARC-Chol(ALN), was observed to partly reduce the endothelial-mesenchymal transition of HUVECs. Besides, while 10 mg/mL dexamethasone, 7.6 mM free ALN and ALN-loaded liposomes failed, 50 µg/mL TL + 2.5 µg/mL ALN (i.e., nanoARC-Chol(ALN)) reduced the IL-6 and IL-8 levels by, respectively, 75% and 65% in the mild and by, respectively, 60% and 40% in the pronounced inflammation model. This is the first report showing that the endocytosis of nanoARC-Chol(ALN) by HUVECs magnifies the anti-inflammatory activity of ALN even under conditions of intense irritation, not only surpassing that of free ALN but also that of dexamethasone.

Macrophage apoptosis using alendronate in targeted nanoarchaeosomes.

Nanoarchaeosomes are non-hydrolysable nanovesicles made of archaeolipids, naturally functionalised with ligand for scavenger receptor class 1. We hypothesized that nitrogenate bisphosphonate alendronate (ALN) loaded nanoarchaeosomes (nanoarchaeosomes(ALN)) may constitute more efficient macrophage targeted apoptotic inducers than ALN loaded nanoliposomes (nanoliposomes (ALN)). To that aim, ALN was loaded in cholesterol containing (nanoARC-chol(ALN)) or not (nanoARC(ALN)) nanoarchaeosomes. Nanoarchaeosomes(ALN) (220-320 nm sized, ~ -40 mV ξ potential, 38-50 µg ALN/mg lipid ratio) displayed higher structural stability than nanoliposomes(ALN) of matching size and ξ potential, retaining most of ALN against a 1/200 folds dilution. The cytotoxicity of nanoARC(ALN) on J774A.1 cells, resulted > 30 folds higher than free ALN and nanoliposomes(ALN) and was reduced by cholesterol in nanoARC-chol(ALN). Devoid of ALN, nanoARC-chol was non-cytotoxic, exhibited pronounced anti-inflammatory activity on J774.1 cells, strongly reducing reactive oxygen species (ROS) and IL-6 induced by LPS. Nanoarchaeosomes bilayer extensively interacted with serum proteins but resulted refractory to phospholipases. Upon J774A.1 cells uptake, nanoarchaeosomes induced cytoplasmic acid vesicles, reduced the mitochondrial membrane potential by 20-40 % without consuming ATP neither damaging lysosomes and increasing pERK. Refractory to chemoenzymatic attacks, either void or drug loaded, nanoarchaeosomes induced either anti-inflammation or macrophages apoptosis, constituting promising targeted nanovesicles for multiple therapeutic purposes.

Bacterioruberin from Haloarchaea plus dexamethasone in ultra-small macrophage-targeted nanoparticles as potential intestinal repairing agent.

Oral administration of antioxidant and anti-inflammatory drugs have the potential to improve the current therapy of inflammatory bowel disease. Success of oral treatments, however, depends on the capacity of drugs to remain structurally stable along the gastrointestinal tract, and on the feasibility of accessing the target cells. Delivering anti-inflammatory and antioxidant drugs to macrophages using targeted nanoparticles, could make treatments more efficient. In this work structural features and in vitro activity of macrophage-targeted nanostructured archaeolipid carriers (NAC) containing the high antioxidant dipolar C50 carotenoid bacterioruberin (BR) plus dexamethasone (Dex): NAC-Dex, are described. Ultra-small (66 nm), -32 mV ζ potential, 1200 µg Dex /ml NAC-Dex, consisted of a compritol and BR core, covered by a shell of sn 2,3 ether linked archaeolipids and Tween 80 (2: 2: 1.2: 3 % w/w) were obtained. NAC-Dex were extensively captured by macrophages and Caco-2 cells and displayed high anti-inflammatory and antioxidant activities on a gut inflammation model made of Caco-2 cells and lipopolysaccharide stimulated THP-1 derived macrophages reducing 65 % and 55 % TNF-α and IL-8 release, respectively and 60 % reactive oxygen species production. NAC-Dex also reversed the morphological changes induced by inflammation and increased the transepithelial electrical resistance, partly reconstituting the barrier function. Activity of BR and Dex in NAC-Dex was partially protected after simulated gastrointestinal digestion, improving the chances of BR-Dex joint activity. Results suggest that oral NAC-Dex deserve further exploration as intestinal barrier repairing agent.

Superoxide dismutase in nanoarchaeosomes for targeted delivery to inflammatory macrophages.

Oxidative stress plays an essential role in the pathogenesis and progression of inflammatory bowel disease. Co-administration of antioxidants and anti-inflammatory drugs has shown clinical benefits. Due to its significant reactive oxygen species (ROS) scavenging ability, great interest has been focused on superoxide dismutase (SOD) for therapeutic use. However, oral SOD is exposed to biochemical degradation along gastrointestinal transit. Furthermore, the antioxidant activity of SOD must be achieved intracellularly, therefore its cell entry requires endocytic mediating mechanisms. In this work, SOD was loaded into nanoarchaeosomes (ARC-SOD), nanovesicles fully made of sn 2,3 ether linked phytanyl saturated archaeolipids to protect and target SOD to inflammatory macrophages upon oral administration. Antioxidant and anti-inflammatory activities of ARC-SOD, non-digested and digested in simulated gastrointestinal fluids, on macrophages stimulated with H2O2 and lipopolysaccharide were determined and compared with those of free SOD and SOD encapsulated into highly stable liposomes (LIPO-SOD). Compared to SOD and LIPO-SOD, ARC-SOD (170 ± 14 nm, -30 ± 4 mV zeta potential, 122 mg protein/g phospholipids) showed the highest antioxidant and anti-inflammatory activity: it reversed the cytotoxic effect of H2O2, decreased intracellular ROS and completely suppressed the production of IL-6 and TNF-α on stimulated J774 A.1 cells. Moreover, while the activity of LIPO-SOD was lost upon preparation, gastrointestinal digestion and storage, ARC-SOD was easy to prepare and retained its antioxidant capacity upon digestion in simulated gastrointestinal fluids and after 5 months of storage. Because of their structural and pharmacodynamic features, ARC-SOD may be suitable for oral targeted delivery of SOD to inflamed mucosa.

Ultra-small solid archaeolipid nanoparticles for active targeting to macrophages of the inflamed mucosa.

AIM: Develop nanoparticulate agents for oral targeted delivery of dexamethasone (Dex) to macrophages of inflamed mucosa. MATERIALS & METHODS: Solid archaeolipid nanoparticles (SAN-Dex) (compritol/Halorubrum tebenquichense polar archaeolipids/soybean phosphatidylcholine/Tween-80 4; 0.9; 0.3; 3% w/w) loaded with Dex were prepared. Their mucopenetration, stability under digestion and in vitro anti-inflammatory activity, were determined. RESULTS: Ultra-small SAN-Dex strongly reduced the levels of TNF-α, IL-6 and IL-12 on J774A1 cells stimulated with lipopolysaccharides as compared with free Dex or loaded in ordinary solid lipid nanoparticles-Dex. After in vitro digestion, the anti-inflammatory activity of SAN-Dex was retained, while that of solid lipid nanoparticles-Dex was lost. CONCLUSION: Because of their structural and pharmacodynamic features, SAN-Dex may be suitable for oral targeted delivery to inflamed mucosa.