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USE OF CHITOSAN COATED LIPOSOME WITH HIGH MUCOADHESIVE PROPERTIES FOR ORAL DELIVERY OF SCFA (BUTYRIC ACID AND PROPIONIC ACID) IN HEPATIC STEATOSIS TREATMENT.

Preparation and chemical characterization of chitosan coated liposomes: Liposomes will be prepared by reversed-phase evaporation method with a specific molar ratio between phospholipids and cholesterol. Sodium butyrate (SB) will be dissolved in PBS (pH 6,5) at 10% w/v and loaded into liposomes by sonication and specific magnetic stirring, as reported in literature. Finally, chitosan-coated liposomes will prepared by adding a solution of chitosan to the homogenized liposomal suspensions (with a volumetric ratio 1:1) and subsequent incubation at 10 ºC for at least 1 h. Final products will be purified by dialysis method. Z potential, average hydrodynamic size and Polydispersion index (PI) will be calculated by using Dynamic Light Scattering (DLS). Moreover, considering the need to image a liposome in its hydrated state witch is the ideal scenario for visualization of these dynamic lipid structures, we will utilize environmental scanning electron microscopy (ESEM) with its ability to image wet systems without prior sample preparation. For biological imaging by CLSM (Confocal Laser Scanning Microscopy) will be prepared fluorescently, chitosan-coated liposomes by preparing a solution of 0,1 mg/mL of Fluoresceineamine in PBS and successful added to a lipid solution before preparing liposomes as described in literature. In vitro: hepatocytes cells culture trough evaluation of cell viability compared to different doses and time of incubation with chitosan coated liposomes of butyric acid vs free butyric acid. The ability of hepatocyte to intake free fatty acids will be studied. Cellular imaging by CLSM will be done by using fluorescent,chitosan-coated liposomes; It will be evaluated the kinetics of liposome cellular internalization in human enterocytes models (HT29 cell line) and hepatocytes. Cellular and endosome-lysosome staining will be done by appropriate dyes, in order to study the interaction of liposomes with both cellular compartiments in time. In vivo: young male rats (average body weight around 100 g) will be randomized into seven treatment groups (n=6/group): 1) a control group receiving chitosan coated liposomes per os by gavage; 2) group receiving butyrate free at 10 mg/Kg/die per os by gavage 3) group receiving butyrate free at 20 mg/Kg/die per os by gavage or 4) group receiving butyrate free at 40 mg/Kg/die per os by gavage 5) group receiving chitosan coated liposome loaded with butyrate at a final dose of 10 mg/Kg/die per os by gavage 6) group receiving chitosan coated liposome loaded with butyrate at a final dose of 20 mg/Kg/die per os by gavage; 7) group receiving chitosan coated liposome loaded with butyrate at a final dose of 40 mg/Kg/die per os by gavage. Specifically, rats will be fed with Standard fat diet (SFD) and high fat diet( HFD) for 6 weeks and treatments started together with the HFD and continued for 18 months. We will use a nutritional model of insulin resistance (IR) in non-genetically modified animals that after 6weeks, induced the early events of NAFLD due to fat overnutrition in young animals, excluding age and gender influences. Blood sample will be collected by cardiac puncture and serum obtained. Liver and white adipose tissue will be excised and immediately frozen. It will be studied histologically liver by Ematosillin/ Eosin and Oil Red and mRNA of Occludin and ZO-1 will be evaluated on colon tissue. About plasmatic markers study, it will be evaluated Triglycerides, Glucose, Insulin, HOMA index, AST-ALT, Cholesterol HDL, LDL. In liver extracts, it will be studied relative expression of IL1, IL6, PPARa and PPARy, GLUT-2, PTP1B, SOCS3, CD14. TLR2, TLR4, HMGB-1 TLR9, p50 NFkB, iKba. Attempted results: oral administration of chitosan coated liposomes of butyrate leads to an improvement of intestinal absorption and consequent liver accumulation of the payload obtaining an optimization of the pharmacological efficacy of the butyrate with an improvement of the steatosis.