Ultra-small, highly negatively charged archaeolipid nanoparticles for active targeting to macrophages of the inflamed mucosa

HIGA L, JEREZ H, ROMERO E, MORILLA MJ.

Basilea

Conferencia; 9th CLINAM; 2016

IntroductionInflammatory bowel diseases (IBD) such as Crohn?s disease and ulcerative colitis, are chronic relapsing disorders of the gastrointestinal tract, charac¬terized by chronic inflammation and epithelial injury induced by the uncontrolled activation of the mucosal immune system. Dendritic cells of lamina propia and macrophages are key antigen-presenting cells in the inflamed mucosa. Following activation, which occurs in response to components of the commensal microbiota and Toll-like receptor signalling, these cells produce large amounts of pro-inflammatory cytokines, such as IL 1β, IL 6, IL 8 and TNF. The imbalance between pro-inflammatory and anti-inflammatory cytokines that occurs in IBD impedes the resolution of inflammation, leading instead to disease perpetuation and tissue destruction. The current treatment is symptomatic, but the frequent oral intake of anti-inflammatory and immunosuppressant drugs or systemic administration of biological agents such as the anti-TNF antibody infliximab is poorly effective and its serious adverse effects deteriorate the patient?s quality of life.We hypothesize that more efficacious and safer therapies could rely on developing macrophages-targeted drug delivery systems capable of specifically delivering high doses of anti-inflammatory drugs with minimal exposure of healthy or distant tissues.The inflamed mucosa is characterized by increased of permeability, thinner mucus layer, a reduced pH and accumulation of positive charged proteins such as transferrin. Besides the ?holes? at the epithelial line following cells loss, greatly enhance the entry of nanoparticles into mucosa, that once into the tissue, the small size enhances its retention at the target site. In this work, we report the development of lipid nanoparticles decorated with polar archaeal lipids (archaeolipidnanoparticles, aLN) extracted from the halophilic archaebacteria Halorubrum tebenquichense, for macrophage targeting of the corticosteroid dexamethasone (Dex). Polar archaeal lipids consist of saturated isoprenoid chains linked via ether bonds to the glycerol carbons at the sn2,3 position. We have previously shown that polar archaeal lipids are ligands for macrophages scavenger receptors class A (SRA). Macrophages and bone marrow dendritic cells expressing SRA, avidly uptake vesicles containing archaeal lipids. Besides, vesicles containing archaeal lipids present high superficial negative charge and are more resistant against chemical, physical and enzymatic attacks than classical vesicles made of phospholipids.Archaeolipid nanoparticles (aLN) made of compritol 888 ATO (glyceryl behenate, compritol) stabilized by archaeolipids, soybean phosphatidylcholine (SPC) and Tween 80 (4; 0.9; 0.3; 3% w/w) were prepared by homogenization-ultrasonication. Dex was incorporated in the core of the nanoparticles by dissolving the drug in compritol. Conventional lipid nanoparticles (cLN) made of compritol, SPC and Tween 80 (4; 1.2; 3% w/w) were prepared as control. Nanoparticles were characterized in terms of particle size, zeta potential, morphology, crystallinity and colloidal stability upon storage. The toxicity of nanoparticles on macrophages(J774.A1 cells) and human epithelial colorectal adenocarcinoma cells (Caco-2 cells) was determined by MTT assay in the absence and presence of a mucin layer.The mucopenetration of nanoparticles was measured as the diffusion of coumarin-6 labeled nanoparticles through a mucus layer using the transwell system. Besides, size and zeta potential of nanoparticles upon incubation with mucins was followed for 4 h. In vitro duodenal lipolysis study was performed by quantification of alkaline compensation in simulated intestinal fluid using pancreatic lipase. Then, the uptake of coumarin-6 label nanoparticles by J774 and Caco-2 cells was measured by flow cytometry in the absence and presence of a mucin layer. Finally, the anti-inflammatory potential of nanoparticles was measured as the capacity to reduce pro-inflammatory cytokine secretion in lipopolysaccharide (LPS) stimulated macrophages.ResultsArchaeolipid nanoparticles showed a significantly decreased in the mean size from 288 ± 60 nm to 73.05 ± 10.7 nm, in the polydispersity index(PDI) from 0.4 to 0.3 and in the zeta potential from - 8 ± 5 mV to -43.8 ± 7.3 mV, respect to conventional nanoparticles. Dex incorporation into aLN resulted in an 8 fold-increase in drug solubility with an encapsulation efficiency of 60 %. The transmission electron microscopy images showed spherical particles in the nanometer range while cryo-transmission electron microscopy images showed circular, ellipsoidal or elongated edged structures of high contrast. There was no significant change in mean particle size, PDI, zeta potential and drug content upon one-month storage at 4°C.The differential scanning calorimetry thermogram of aLN with and without Dex showed a decrease of melting point of compritol from 74.2°C to 66°C. This fact can be explained by the Kelvin effect, where a reduced particle size and increased surface area led to a decrease in the melting enthalpy compared to the bulk lipid. The Dex melting endothermic peak of loaded aLN disappeared, indicating the existence of amorphous Dex or that it has been molecularly dispersed within the compritol matrix.Mucus permeation studies showed that aLN diffused significantly more than cLN, and upon 4 h incubation no increase in mean size was observed. The combination of small size and negative superficial charge could be responsible of the high mucopenetration of aLN. The lipolysis kinetics expressed as alkaline consumption showed that at the end of 20 minutes, the lipolysis of aLN was 45%, which indicated that aLN possessed a resistance to pancreatic lipase compared to cLN (75%). Viability studies revealed no cytotoxicity of aLN in the range of 40- 400 µg/ml of compritol upon 24 hours of incubation with J774 and Caco-2 cells.The uptake of aLN by J774 and Caco-2 cell were 6 and 2.4 folds higher than that of cLN upon 5 h incubation at 37 ºC. The uptake was significantly reduced in the presence of a mucin layer for cLNand aLN in both cell types; however, the uptake of aLN by J774 cells was still 3.5 folds higher than that of cLN. Finally, anti-inflammatory activity of nanoparticles showed that while free Dex significantly reduced the production of IL-6 (75%), cLN-Dex reduced the production of TNF-α (25%) and IL-12 (80%), but aLN-Dex significantly reduced production of TNF-α (50%), IL-6 (75%) and IL-12 (33%).ConclusionsThe ultra-small, highly negatively charge archaeolipid nanoparticles and, no conventional lipid nanoparticles, resulted highly stable under gastrointestinal conditions, highly up taken by macrophages and reduced the secretion of the pro-inflammatory TNF-α, IL-6 and IL-12 from J774 cells stimulated with LPS. Further studies will be done to test the activity of aLN in a more advanced in vitro model based on co-culture of macrophages, dendritic cells and enterocytes that allow simulating the inflammation.