Nanostructured archaeolipid carriers with anti-inflammatory and antioxidant activity for oral treatment of inflammatory bowel diseases.

HIGA LH, SCHILRREFF P, JEREZ HE, BRISKI A, ROMERO EL, MORILLA MJ.

Basilea

Conferencia; 10 th CLINAM; 2017

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 and macrophages are key cells in the inflamed mucosa, which produce large amounts of pro-inflammatory cytokines. The imbalance between pro-inflammatory and anti-inflammatory cytokines impedes the resolution of inflammation, leading to disease perpetuation and tissue destruction. On the other hand, oxidative stress is considered as one of the etiologic factors involved in several signals and symptoms of IBD that include diarrhea, toxic megacolon and abdominal pain. Once the uncontrolled activation of the immune system occurs, oxidative stress is a major contributing factor to tissue injury and fibrosis. The treatment of IBD is symptomatic, and depending on the stage of the disease, ranges from oral aminosalicylates, anti-inflammatory and immunosuppressant drugs, to endovenous biological agents such as the anti-tumor necrosis factor (TNF)- antibody infliximab. These treatments have limited benefits, because of their systemic adverse effects displayed during their long-term use. More efficacious and safer therapies could rely on developing macrophages-targeted drug delivery systems capable of specifically delivering high doses of anti-inflammatory drugs and antioxidants with minimal exposure of healthy or distant tissues via oral administration. Here we report the development of nanostructured archaeolipid carriers (NAC) for oral targeted delivery of natural antioxidants and the anti-inflammatory dexamethasone (Dex) to macrophages. NAC have a core of neutral and a shell of polar archaeolipids extracted from the halophilic archaebacteria Halorubrum tebenquichense. Polar archaeolipids are a mixture of saturated isoprenoid chains linked via ether bonds to the glycerol carbons at the sn 2,3 position. In contrast to conventional phospholipids, polar archaeolipids are hydrolytic, oxidative and enzymatic attack resistant. Besides, polar archaeolpids are ligands for the macrophages scavenger receptors class A. We have recently reported that ultra-small solid archaeolipid nanoparticles combine high resistance to gastrointestinal conditions with extensive uptake by macrophages (Higa et al., 2017). Neutral archaeolipids, on the other hand, are a mixture of carotenoids with C50 that have higher antioxidant activity than those extracted from algae, plants, yeast and cyanobacteria.NAC made of a core of a mixture of solid (compritol) and liquid (neutral archaeal) lipids stabilized by a shell of polar archaeolipids and Tween 80 (2-2-1.2-3 % w/w) loaded with dexamethasone (NAC-Dex) were prepared by homogenization-ultrasonication.NAC-Dex were characterized in terms of particle size, zeta potential, morphology, crystallinity and colloidal stability upon storage. The toxicity of NAC on macrophages (J774 cells) and human epithelial colorectal adenocarcinoma (Caco-2) cells was determined by MTT assay.The in vitro scavenging capacity of NAC against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2?-azinobis (3-ethylbenzothiazoline-6 sulfonicacid) (ABTS) radicals was measured. Stability of NAC incubated in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), was determined in terms of size retention and lipolytic percentage, respectively.Finally, the anti-inflammatory and antioxidant activity of NAC were measured in a model of inflamed mucosa using co-cultured Caco-2 and differentiated into macrophages human monocytes (THP-1 cells) stimulated with lipopolysaccharide (LPS). The anti-inflammatory activity was determined by measuring the release of pro-inflammatory cytokines. The capacity of NAC to reduce the generation of reactive oxygen species (ROS) by macrophages was measured using the carboxy-H2DCFDA dye.ResultsNAC resulted small, homogeneous, negatively charge and stable: NAC showed a mean particle size of 85.1 ± 23.3 nm with polydispersity index of 0.3 and zeta potential of -32.4 ± 5.8 mV. Dex incorporation into NAC resulted in a 7-fold increase in drug solubility with an encapsulation efficiency of 50 %. 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 thermograms of NAC showed a decrease of melting point of compritol from 74.2°C to 56.7°C and lower crystallinity index compared to the bulk lipid, indicating the liquid lipid was dispersed within the compritol matrix.NAC showed antioxidant activity in vitro: The DPPH radical scavenging test of NAC showed a Trolox equivalent antioxidant capacity of 35 ± 7 mM Trolox/g lipids, and the concentration of NAC that reduced 50 % of the radicals (IC50) was 0.70 mg/ml. Besides, at that concentration, NAC produced 60% of inhibition of the ABTS radical. NAC were resistant to in vitro digestion: No modification in NAC mean size was revealed upon 4 h of incubation in simulated gastric fluid with respect to initial time. Lipolysis of NAC was relatively low, up to 60 min the accumulative lipolytic percentage was 45%.NAC were not cytotoxic: Viability studies revealed no cytotoxicity of NAC in the range of 40- 200 µg/ml of compritol upon 24 hours of incubation with Caco-2 cells and J774 cells. NAC reduced the intracellular ROS levels: The subcellular ROS levels of J774A.1 cells significantly decreased upon co-incubation with LPS and NAC.NAC reduced the production of pro-inflammatory cytokines on a model of inflamed mucosa: The stimulation of THP-1 cells in the co-culture with lipopolysaccharide was followed by a decrease in transepithelial electrical resistance, which is a marker of the integrity of the Caco-2 monolayer and an increase in TNF- production by THP-1 cells and IL-8 by Caco-2 cells. NAC showed high anti-inflammatory activity as measured by reduced production of TNF- and IL-8 in LPS stimulated co-cultures.ConclusionsThe highly stable under gastrointestinal conditions, nanostructured archaeolipid carriers that combines anti-inflammatory and antioxidant activity could be a new strategy to improve the oral treatment of inflamed mucosa.