POLİMERİK NANOPARTİKÜL FLORESAN PROBLARLA BAKTERİYAL FİLMLERİN İN-VİVO GÖRÜNTÜLENMESİ
NOROUZ DIZAJI, Araz
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The aim of this theses is to synthesize polymeric nanoparticle fluorescence probes in order to be used in in-vitro and in-vivo imaging of Staphylococcus aureus and the biofilms produced by these bacteria. 1ml THF solution containing certain amounts of PFBT “[poly(9,9-dioctylfluorene-2,7-diyl-co-benzothiadiazole)]” and DSPE-PEG2000-Maleimid were added to 9ml of DW. The ultra-sonication of this mixture was applied with 12W micro-tip probe sonication for 60s and steered overnight at the room temperature. At the end of this step, packaging of PFBT was done and CPDP-Mal nanocapsules (NCs) were synthetized successfully. Uv-vis spectrophotometery of the NCs was carried out and the results showed the characteristic peak shifting of PFBT from 470nm to 480nm that’s related to successful packaging of PFBT with DSPE-PEG2000-Mals. Zeta-sizer and TEM analysis showed that the prepared NCs have a diameter of 80nm in a narrow distribution. To gain specific targeting properties for obtained NCs, the immobilization of vancomycin (as a ligand) has been carried out in accordance with the protocol. For this purpose, bis-vancomycin molecules containing di-thiol bond were synthetized and Purification of bis-vancomycin was carried out by implementing HPLC and keeping it for three days at freeze-dryer. The LC-MS analysis of bis-vancomycin showed the related characteristic peaks. The di-thiol bonds of bis-vancomycin were cleaved using TCEP in aqueous environment and obtained SH-Van ligands have successfully immobilized onto CPDP-Mal NCs in accordance with the protocol, which was confirmed by the FTIR spectra of the ligands. Antibacterial properties of prepared SH-Van and CPDP-S-Van NCs were investigated by MIC, MBC and DDM. The in-vitro specific targeting properties of the CPDP-S-Van NCs were investigated on S. aureus and E. coli by confocal microscopy. Results showed that the NCs were targeted only to S. aureus. The NCs imaging abilities were examined on the biofilm which was produced in laboratory condition by S. aureus. The results manifested that the CPDP-S-Van NCs demonstrate high-level performance in the imaging of S. aureus biofilms. Cytotoxicity and apoptosis properties of NCs were investigated by MTT/WST-1 and caspase-3 antibody imaging assay. According to the results, cell viability was determined to be over 90% even at the highest dose and no apoptotic properties were detected for NCs. The results manifestly indicate that the prepared NCs can be used for the in-vivo studies. The ICR mouse animal model was used to inspect the specific imaging ability of CPDP-S-Van NCs against S. aureus in in-vivo conditions. Infected animal model was prepared by injecting S. aureus and E. coli bacteria subcutaneously into the right and left backside of the ICR mouse. Specific targeting abilities of NCs were investigated by using non-invasive live animal fluorescence imaging technique by the injection of CPDP-S-Van NCs from tail vein of infected animal model. According to the results the targeting of the prepared NCs has been done successfully.