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

HIGA L; SCHILLREFF P; JEREZ H; BRISKI, A; EDER L. ROMERO; MORILLA MJ

Basel

Congreso; CLINAM 2017; 2017

CLINAM

Nanostructuredarchaeolipid carriers with anti-inflammatory and antioxidant activity for oral treatmentof inflammatory bowel diseases. Higa LH, SchilrreffP, Jerez HE, Briski A, Romero EL, Morilla MJ.Nanomedicine Research Program,Universidad Nacional de Quilmes, Buenos Aires, ArgentinaIntroductionInflammatory bowel diseases (IBD) such as Crohn?sdisease and ulcerative colitis are chronic relapsing disorders of thegastrointestinal tract, charac­terized by chronic inflammation and epithelialinjury induced by the uncontrolled activation of the mucosal immune system.Dendritic cells and macrophages are key cells in the inflamed mucosa, whichproduce large amounts of pro-inflammatory cytokines. The imbalance betweenpro-inflammatory and anti-inflammatory cytokines impedes the resolution ofinflammation, leading to disease perpetuation and tissue destruction. On the other hand, oxidative stress is considered asone of the etiologic factors involved in several signals and symptoms of IBD thatinclude diarrhea, toxic megacolon and abdominal pain. Once the uncontrolledactivation of the immune system occurs, oxidative stress is a majorcontributing factor to tissue injury and fibrosis. The treatment of IBD is symptomatic, and depending on the stage of thedisease, ranges from oral aminosalicylates, anti-inflammatory and immunosuppressantdrugs, to endovenous biological agents such as the anti-tumor necrosis factor(TNF)-a antibody infliximab. These treatments have limited benefits, because oftheir systemic adverse effects displayedduring their long-term use. Moreefficacious and safer therapies could rely on developing macrophages-targeteddrug delivery systems capable of specifically delivering high doses ofanti-inflammatory drugs and antioxidants with minimal exposure of healthy ordistant tissues via oral administration. Here wereport the development of nanostructured archaeolipid carriers (NAC) for oraltargeted delivery of natural antioxidants and the anti-inflammatory dexamethasone(Dex) to macrophages. NAC have a core of neutral and a shell of polar archaeolipidsextracted from the halophilic archaebacteria Halorubrum tebenquichense. Polararchaeolipids are a mixture of saturated isoprenoid chains linked via ether bonds to the glycerolcarbons at the sn 2,3 position. In contrast to conventional phospholipids, polararchaeolipids are hydrolytic, oxidative and enzymatic attack resistant.Besides, polar archaeolpids are ligandsfor the macrophages scavenger receptors class A. We have recentlyreported that ultra-small solid archaeolipid nanoparticles combine highresistance to gastrointestinal conditions with extensive uptake by macrophages (Higaet al., 2017). Neutral archaeolipids, on the other hand, are a mixture ofcarotenoids with C50 that have higher antioxidant activity than those extractedfrom algae, plants, yeast and cyanobacteria.NACmade 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) wereprepared by homogenization-ultrasonication.NAC-Dexwere characterized in terms of particle size, zeta potential, morphology, crystallinity and colloidalstability upon storage. The toxicity of NAC on macrophages (J774 cells) andhuman epithelial colorectaladenocarcinoma (Caco-2) cells was determined by MTTassay.The in vitro scavenging capacity of NAC against1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2?-azinobis(3-ethylbenzothiazoline-6 sulfonicacid) (ABTS) radicals was measured. Stability of  NACincubated 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 inflamedmucosa 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 therelease of pro-inflammatory cytokines. The capacity of NAC to reduce thegeneration of reactive oxygen species (ROS) by macrophages was measured usingthe carboxy-H2DCFDA dye.ResultsNAC resultedsmall, homogeneous, negatively charge and stable: NAC showed a mean particle sizeof 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 drugsolubility with an encapsulation efficiency of 50 %. Transmission electron microscopy images showedspherical particles in the nanometer range while cryo-transmission electronmicroscopy images showed circular, ellipsoidal or elongated edged structures ofhigh contrast. There was no significant change in mean particle size, PDI, zetapotential and drug content upon one-month storage at 4°C. The differentialscanning calorimetry thermograms of  NAC showeda decrease of melting point of compritol from 74.2°C to 56.7°C and lowercrystallinity index compared to the bulk lipid, indicating the liquid lipid wasdispersed within the compritol matrix.NACshowed antioxidant activity in vitro: TheDPPH radical scavenging test of NAC showed a Trolox equivalent antioxidantcapacity of 35 ± 7 mM Trolox/g lipids, and the concentration of NAC thatreduced 50 % of the radicals (IC50) was 0.70 mg/ml. Besides, at thatconcentration, NAC produced 60% of inhibition of the ABTS radical. NAC were resistant to invitro digestion: Nomodification in NAC mean size was revealed upon 4 h of incubation in simulatedgastric 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 therange of 40- 200 µg/ml of compritol upon 24 hours of incubation with Caco-2cells and J774 cells. NAC reducedthe intracellular ROS levels: The subcellular ROS levels of J774A.1 cells significantlydecreased upon co-incubation with LPS and NAC (figure 1). Fig.1 Decreased generation of reactive oxygen species (ROS). Fluorescenceintensity of carboxy-DCFDA on J774A.1 cells after 24 h co-incubation with 1 μg/mlLPS and NAC. Statistical differences are represented by ***p < 0.001 comparedto positive control. NAC reduced the production ofpro-inflammatory cytokines on a model of inflamed mucosa:The stimulation of THP-1 cells in the co-culture with lipopolysaccharide wasfollowed by a decrease in transepithelial electrical resistance, which is amarker of the integrity of the Caco-2 monolayer and an increase in TNF-aproduction by THP-1 cells and IL-8 by Caco-2 cells. NAC showed highanti-inflammatory activity as measured by reduced production of TNF-aand IL-8 in LPS stimulatedco-cultures.ConclusionsThe highlystable under gastrointestinal conditions, nanostructured archaeolipid carriers thatcombines anti-inflammatory and antioxidant activity could be a new strategy toimprove the oral treatment of inflamed mucosa. ReferencesUltra-smallsolid archaeolipid nanoparticles for active targeting to macrophages of the inflamed mucosa. Higa L,Jerez H, de Farias MA, PortugalRV, Romero EL, Morilla MJ.Nanomedicine. Future Medicine.  2017