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| Author(s): |
Vicky Vanessa Ortega Gaona
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2016-08-26 |
| Examining board members: |
Eneida de Paula;
Maria Isabel Noguiera Cano;
Cíntia Maria Saia Cereda
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| Advisor: | Selma Giorgio; Eneida de Paula |
| Abstract | |
Visceral Leishmaniasis (VL) is a fatal and neglected disease, considered, by the WHO, as one of the six endemic diseases with major relevance in the world. It is a zoonosis that have domestics dogs as main urban reservoir for the parasite L. infantum, and the infection in domestic dogs is named as Canine Visceral Leishmaniasis (CVL). Dogs are considered strategic targets for the disease control measures. The canine infection usually precedes the onset of human cases being more prevalent than VL in humans. The most dogs with positive serology do not exhibit clinical signs, but may, as reservoirs, infect sand flies. CVL is a complex disease with high prevalence rates, up to 80 % of the canine population in endemic areas. Most studies of candidates to be drugs and treatments for CVL systems were performed in vivo without any control over the experimental conditions. In consequence, the use of dog macrophage lineages for in vitro drug toxicity studies are necessary. Furthermore, considering that, there is no treatment to guarantee the parasitological cure for CVL, it is necessary to develop pharmaceutical formulations to improve the effectiveness of leishmanicidial drugs and treatments, increasing this activity and reducing toxicity. The aim of this work was to, develop, characterize and to assess a system to treat DH82 macrophage infection with L.infantum (in vitro), a sustained relase liposomal formulation for the meglumine antimoniate (AME), the main drug used in VL treatment, thus, introducing a new therapeutic approach for the treatment of CVL. Large unilamellar lipossomes (LUVs) composed by egg-phosphatidylcholine, phosphatidylserine and cholesterol (4:0.04:3,molar ratio) were prepared by extrusion at pH 7.4. The liposomal formulation was characterized regarding the encapsulation efficiency of AME (23 %). The formulations was followed for 180 days of storage at 4 ºC. The size, polydispersity index and zeta potential of the vesicles were measuared. The mean diameter of encapsulated liposomes was found to be 359 +/- 3.1 nm and the zeta potential was negative (-61.5 mV), by the presence of negatively charged lipid (PS), in the formulation. Peroxidation levels did not exceed 1 % of the total lipids in the formulation. Citotoxicity studies using DH82 cells demonstrated that free AME induced cell death in a concentration dependent manner, showing a reduction of 50 % of cell viability in 96 hours at 0.005 M; the reduced citotixicity of lipossomal-AME (L-AME), (which could not achieve the 50 % reduction of cell viability at any of the concentration tested), revealed lower toxic potential for the proposed formulation in macrophages. The evaluation tests of leishmanicidal activity revealed the ability of L-AME system to decrease the amount/load of parasites in dogs macrophages by 30 times compared to the infected but not treated controlled macrophages and also showed a 49 % decrease of the parasites when compared with free AME treatment in its maximum concentration (0.01 M), after 96 hours of treatment. Finally it was found colocalization and internalization of the liposome LUV (labeled with NBD-PE) and the parasite L.infantum-mcherry within macrophages DH82 with a superior number of 3000 colocalizações at 8 h of treatment indicating that as the liposomes and the parasite has the same site of action, help us to understand the results obtained in this study as the leishmanicide effect of liposomal system (AU) | |
| FAPESP's process: | 14/12443-7 - Development of a double loading system for meglumine antimoniate and its in vitro evaluation in macrophages infected with L. infantum |
| Grantee: | Vicky Vanessa Ortega Gaona |
| Support Opportunities: | Scholarships in Brazil - Master |