Full text
| |
| Author(s): |
Camila Maringolo Ribeiro
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | Araraquara. 0000-00-00. |
| Institution: | Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara |
| Defense date: | 2022-03-18 |
| Advisor: | Fernando Rogerio Pavan; Marlus Chorilli |
| Abstract | |
Tuberculosis (TB) is an infectious disease that affects millions of people annually and a long treatment is necessary for a cure, especially for antibiotic resistant cases. Another particularity of Mycobacterium tuberculosis, the main etiologic agent of TB, is the ability to survive inside macrophages, immune cells in lungs. In a search for an effective, safe treatment that promotes antibiotic delivery at the site of infection and, possibly, contributes to treatment adherence, we proposed a nanotechnological approach based on functionalized liposomes, that is, with the surface modified with an analogue of siderophore (Sid) to carry moxifloxacin (Mox) to treat TB for an intrapulmonary administration. Siderophores are the main strategy of bacteria to take up iron, an essential metal for their metabolism. Four liposomes were developed with soy phosphatidyl, cholesterol and oleic acid: empty liposome (LipV); liposome containing Mox (LipMox); drug-free and Sid-functionalized liposome (LipS) and Sid-functionalized and Mox-containing liposome (LipSMox). The characterization of these liposomes was performed by dynamic light scattering technique (DLS), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). By all methods, we observed spherical particles and approximately 200 nm. The zeta potential was the main difference between them and was approximately -40mV for LipV and LipMox and +15mV for LipS and LipSMox. NTA showed a concentration of ~1011 particles/mL. A method of quantification of Mox in high performance liquid chromatography was developed and validated to determine the encapsulation efficiency (EE) and the release profile (RP). The EE was 51.31% for LipMox and 45.76% for LipSMox and the RP followed the model proposed by Korsmeyer-Peppas, common for liposomes. The minimal inhibitory concentration (MIC) against M. tuberculosis was determined under standard conditions (pH 6.6 and [Fe] = 0.04 µg/mL), with addition of iron (multiples of 2x, 5x and 10x in relation to the commercial medium), pH 7.4 and with addition of 10% fetal bovine serum (FBS). Changing the pH of the medium to 7.4 did not affect the MIC. The MIC of LipMox and LipSMox were less than 0.5%. LipS had MIC close to 10% in all conditions, except in the presence of FBS (MIC >25%), in which there is iron offered, not free, but complexed with ferritin. The time-kill assay showed no significant difference between free and encapsulated Mox, the bactericidal action was concentrationdependent. The cytotoxicity assay was performed for four cell lines: macrophages (J774A.1), fibroblasts (MRC-5), liver cells (HepG-2) and intestinal cells (Caco-2). Heterogeneity was observed in the cytotoxicity index (IC50) results with greater toxicity for liposomes containing Sid. The activity against intramacrophagic M. tuberculosis showed reductions of more than 90% of intracellular bacilli (LipMox 1 and 5%; Mox 1 and 5% and LipSMox 1%). Scanning and transmission electron microscopy confirmed the phagocytosis of liposomal vesicles by macrophages, confirming the possibility of a natural targeting of liposomes to phagocytic cells. Although functionalization with Sid did not improve the biological activity of liposomes, there was still a natural targeting of macrophages, cells of extreme importance in the pathogenesis of TB. Given the advantages of drug delivery of liposomes, an intrapulmonary in vivo administration of this system is proposed for the treatment of TB. (AU) | |
| FAPESP's process: | 18/02344-2 - Evaluation of in vitro and in vivo potential of siderophores-functionalized liposomes containing moxifloxacin against Mycobacterium tuberculosis |
| Grantee: | Camila Maríngolo Ribeiro |
| Support Opportunities: | Scholarships in Brazil - Doctorate |