• Login
    View Item 
    •   ResearchSpace Home
    • College of Agriculture, Engineering and Science
    • School of Life Sciences
    • Biotechnology
    • Biochemistry
    • Doctoral Degrees (Biochemistry)
    • View Item
    •   ResearchSpace Home
    • College of Agriculture, Engineering and Science
    • School of Life Sciences
    • Biotechnology
    • Biochemistry
    • Doctoral Degrees (Biochemistry)
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Novel epidermal growth factor directed cationic lipoplexes promote in vitro hepatotropic gene targeting.

    Thumbnail
    View/Open
    Thesis (4.177Mb)
    Date
    2014
    Author
    Sewbalas, Alisha.
    Metadata
    Show full item record
    Abstract
    The need for the improvement in protocols for cellular gene delivery has propelled cytofectin based liposomes as suitable non-viral gene carriers. The amenability of cationic liposomes to modification enables research based enhancement of their carrier capability. The liposomes formulated in this study show potential for cancer therapeutics, where effective delivery at the molecular level is essential. Cell specific targeting may be attained through cationic vector manipulation to favourably utilise overexpressed cancer cell specific receptors. This study serves as an evaluation of a hepatocyte-directed liposomal gene delivery system, exploiting the abundant epidermal growth factor (EGF) receptors on hepatocellular carcinoma cells (HepG2) in vitro. The inclusion of polyethylene glycol (PEG) served to limit steric hindrance and to increase stability of the formulations. Four liposomes comprising cytofectins 3ß[N-(N',N'-dimethylaminopropane)-carbamoyl] (Chol-T) and N,N-dimethylaminopropylamidosuccinyl-cholesterylformylhydrazide (MS09) at 50 mol%, were formulated through thin film rehydration with dioleoylphosphatidylethanolamine (DOPE) and PEG to generate liposomes that are cationic and have stealth capability. Hepatotropic lipoplexes were formed from EGF adsorption onto formulated liposomes, prior to characterisation and cell culture studies. All liposomes displayed as nano-sized particles (60 – 181 nm) with varying levels of colloidal stablility and distribution as evidenced by transmission electron microscopy and nanoparticle tracking analysis. Moderate to highly cationic lipid : DNA charge ratios were observed by the mobility shift and ethidium bromide dye displacement assays. Broad range protection of plasmid DNA integrity was identifed, with DSPE-PEG2000-grafted liposomes offering greatest shielding against nuclease attack. In vitro cytotoxicity was determined using the MTT assay, and reporter gene expression, was assayed using the luciferase and green fluorescent protein (GFP) reporter gene assays in the receptor positive HepG2 and the receptor negative Chinese Hamster ovary (CHO-K1) cell line. These novel EGF-tagged cationic liposomes displayed negligible cytotoxicity to both cell lines and were capable of high transgene activity in the HepG2 cells compared to the CHO-K1 cells. The Chol-T-EGF liposome significantly (P<0.0001) potentiated transgene targeting, compared to the commercially available transfection reagent, Lipofectin. Targeting was further confirmed from the YI-12 peptide–EGFR competitive transfection determinations in the HepG2 cell line. Results obtained for the luciferase reporter assay was corroborated by the flow cytometric quantification of GFP expression. The size distribution, physicochemical properties and in vitro studies strongly suggest that these targeted lipoplexes should be optimized for future applications in vivo.
    URI
    http://hdl.handle.net/10413/12473
    Collections
    • Doctoral Degrees (Biochemistry) [76]

    DSpace software copyright © 2002-2013  Duraspace
    Contact Us | Send Feedback
    Theme by 
    @mire NV
     

     

    Browse

    All of ResearchSpaceCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsAdvisorsTypeThis CollectionBy Issue DateAuthorsTitlesSubjectsAdvisorsType

    My Account

    LoginRegister

    DSpace software copyright © 2002-2013  Duraspace
    Contact Us | Send Feedback
    Theme by 
    @mire NV