Dokument

Summary:
The main objective of the current study was to developed an optimized system for enhanced nucleic acid delivery with minimum cytotoxicity. For efficient therapeutic gene delivery, PAMAM dendrimer, with ethylenediamine core was chosen due to its biodegradable and non-immunogenic nature. However, the cytotoxicity and unusual biodistribution by this polycationic polymeric system urged the need of protective shielding. Therefore, a non-covalent interaction of dendriplexes with lipids i.e. liposomal modification was chosen as a technique to overcome the associated drawbacks. The methodology section of the thesis deals with the preparation of liposomes, dendriplexes, lipodendriplexes, their subsequent physicochemical characterization and in vitro, in ovo and in vivo studies. In the results section, the characterization of dendriplexes has been discussed, which was the prerequisite of lipodendriplex formation. Dendriplexes formation was confirmed by gel retardation and fluorescence quenching assay. Dynamic light scattering and laser Doppler anemometry also confirmed the stable complex formation, with a desired size range suitable for cellular internalization. Based on highest pDNA transfection efficiency and appropriate cell viability profile, an N/P ratio of 12 has been chosen for complexation with the liposomal formulation. A broad range of liposomal formulations has been investigated and the influence of liposome to PAMAM ratios on surface charge and size of lipodendriplexes are described in detail. Surface morphology of the liposomes and lipodendriplexes has been analysed using atomic force microscopy and their sizes were compared with the results obtained from dynamic light scattering. Transfection studies have been discussed for different lipodendriplexes formulations. It has been observed that the DPPC:CH-PAMAM lipodendriplexes showed a significant improvement in pDNA transfection as compared to other lipodendriplexes formulations and their parent dendriplexes. The higher gene expression was also confirmed using fluorescence microscopy by GFP expression analysis. Cytotoxicity studies including MTT, ROS, lysosomal disruption and DNA damage assays has been discussed in detail. From the results, it has been depicted that the lipid modification of dendriplexes shields the terminal amino group induced toxicity and consequently improves the cell viability. Biocompatibility studies has been performed to check to the compatibility of the complexes in the presence of blood components. Heparin competition and erythrocyte hemolysis assay revealed the biocompatible nature of the lipodendriplexes. Another objective of the study was to deliver the siRNA to investigate the gene silencing effect. Knockdown experiments showed a pronounced downregulation of luciferase, GFP and MDR1 genes by lipodendriplexes compared to dendriplexes or the control siRNA groups. The role of MDR1 in cell migration and colonization of cancer cells has also been described in detail. Downregulation of MDR1 gene by lipodendriplexes exhibited significant inhibition of tumor metastasis and colonization. The knockdown effect of MDR1 gene was also investigated in 3D cell culture environment. Cell migration and ring closure assays were performed using 3D tumor spheroid and ring bioprinting model. A significant reduction in the cell migration was observed in the case of lipodendriplexes treated group. Next step was to investigate the enhanced intracellular accumulation of tyrosine kinase inhibitor (imatinib mesylate) after the downregulation of P-gp (responsible for drug efflux) and subsequent apoptosis in colon carcinoma. Apoptosis assay was done in 2D and 3D cultures using flow cytometry and live dead staining, respectively. The results of 2D culture were in agreement with the data from 3D cell cultures. Cell cycle analysis also confirmed the imatinib mesylate induced apoptosis, indicated by an arrest of Sub-G1 phase depicting an effective downregulation of P-glycoprotein by lipodendriplexes. In order to minimise the unethical use of animals, an alternative in ovo chorioallantoic membrane model (an in vivo like environment) has been used to determine the efficacy and biocompatibility of the complexes. Lipodendriplexes exhibited successful reporter gene (GFP) expression in the CAM with no toxicity observed within the CAM vasculature. After establishing the improved gene transfection and toxicity profile in in vitro conditions, in vivo biodistribution and toxicity assessment of the complexes has performed in female BALB/c mice and discussed in detail. In vivo biodistribution has revealed that encapsulation of dendriplexes with liposomes has essentially increased the cellular uptake of the complexes, which was confirmed by ex vivo imaging of the dissected organs. Acute toxicity studies showed that the lipodendriplexes treated group did not produce any change in body weight, organ to body ratio and in organ tissue histopathology. Serum biomarkers and hematological studies also revealed the biocompatibility in an in vivo environment. From the findings in this thesis, it can be concluded that development of such non-viral nano carrier system could serve for an efficient gene transfection with better safety profile, both for in vitro and in vivo therapeutics. Further in vivo studies using different pre-clinical models for specific targeted delivery against different types of cancer and genetic disorders will help to realise the therapeutic potential of this system.

Das Dokument ist im Internet frei zugänglich - Hinweise zu den Nutzungsrechten