Doctoral Degrees (Biochemistry)

Permanent URI for this collectionhttps://hdl.handle.net/10413/8003

Browse

Now showing 1 - 6 of 6

Gene transfer by receptor-mediated endocytosis : stable expression of NEO following insulin-directed entry into HepG2 cells.
(1989) Huckett, Barbara Isobel.; Hawtrey, Arthur O.; Ariatti, Mario.
Evidence is presented for DNA delivery to cultured HepG2 hepatoma cells via the endocytotic pathway, under the direction of insulin, in a soluble system of transfection leading to stable gene expression. Serum albumin treated at pH 5.5 and 20°C for 48-60h with the water-soluble carbodiimide N-ethyl-N'(3-dimethylaminopropyl) carbodiimide hydrochloride has been found to produce positively charged N-acylurea albumin capable of binding different types of DNA in a reaction which is at least partially electrostatic in nature (Huckett et al, 1986). N-Acylurea albumin, synthesised at an albumin to carbodiimide mole ratio of 1 : 500, resulting in the attachment of 27 Nacylurea moieties per albumin molecule, was covalently conjugated to insulin by glutaraldehyde cross-linkage in order to produce a macromolecule, insulin-[N-acylurea albumin], with the facilities f or both DNA transport and receptor binding. The resultant conjugate, purified by gel filtration through Sephadex G-100, was characterised in terms of molecular size, charge properties and insulin content by polyacrylamide gel electrophoresis, agarose gel electrophoresis and immuno-dotblotting respectively. The conjugated protein was shown by gel band shift and nitrocellulose filter binding assays to bind DNA non-specifically in a reversible reaction which occurs rapidly, is dependent upon protein concentration and the ionic strength of the medium, and involves at least two types of intermolecular interaction. Furthermore, the conjugate was shown by competitive displacement of [ 125I ]insulin to bind specifically and particularly avidly to the HepG2 insulin receptor. When the expression vectors ptkNEO and pAL-8 which incorporate the neo gene were complexed to the conjugate in an in vitro transfection procedure using HepG2 cells, G418 resistant clones developed at frequencies of 2.0 - 5.5 X 10-5, possibly dependent upon vector promoter. Subsequently, a 923bp PstI fragment within the neD sequence was identified by Southern transfer in genomic DNA extracted from transfected cell populations which had been grown on a G418 regime through several subculture passages over a period of 44 days.
Mitochondrial localisation and cellular uptake in vitro using novel ‘mitochondriotropic’ liposomes.
(2016) Narainpersad, Nicolisha.; Ariatti, Mario.; Masola, Bubuya.; Singh, Moganavelli.
Mitochondrial research has made tremendous strides since the 1980/90s when mitochondrial DNA mutations were first identified as a primary cause for human diseases and the organelle’s role in apoptosis was elucidated. These mutations of the mitochondrial genome have been implicated in a spectrum of clinical disorders especially involving the muscle and central nervous system. This makes the mitochondrion a prime candidate for organelle-specific delivery of exogenous materials such as therapeutic DNA and drugs, for therapy of diseases caused by mitochondrial dysfunction. However, reports of mitochondrial targeted delivery systems are limited. Hence vector design and development is of paramount importance. The success of liposomes viz. cationic liposomes, in chromosomal gene therapy make them potential vectors for mitochondrial gene targeting. In this investigation novel ‘mitochondriotropic’ liposomes were synthesised to evaluate their cellular uptake and mitochondrial localisation activity in vitro using four different mammalian cell culture models. Cationic cholesterol derivative, 3β [N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (CHOL-T) was formulated with dioleoylphosphatidylethanolamine (DOPE) to produce cationic liposomes, to which a mitochondrial targeting sequence (MTS) and octaarginine (R8) peptides were attached via two different novel cholesterol-derived cross-linking agents. Size, zeta potential, shape and lamellarity of liposomes and corresponding lipoplexes were assessed by the innovative technique, Nanoparticle Tracking Analysis (NTA) and cryogenic transmission electron microscopy. Their ability to bind, condense and protect plasmid DNA (pCMV-luc), was determined using the band shift, dye displacement and nuclease protection assays repectively. In vitro cytotoxicity and mechanism of cell death prompted by these novel liposomal preparations was determined using the MTT, AlamarBlue® and acridine orange and ethidium bromide (AO/EB) dual staining assays respectively, in the hepatocyte-derived human cell line (HepG2), human embryonic kidney cells (HEK293), the human intestinal cell line (Caco-2) and human cervical carcinoma (HeLa-Tat luc) cells. Fluorescently labelled DNA was used to determine cellular uptake and mitochondrial targeting and localisation ability of these cationic mitochondriotropic liposomal formulations in the target organelles, mitochondria using fluorescence microscopy and the quantitative evaluation of fluorescence in the mitochondrial fraction of cell homogenate cocktails. These mitochondriotropic liposomes successfully bind, condense and protect plasmid DNA in the presence of serum, are fairly well tolerated by all cell lines tested in culture with cell death observed to be apoptotic and not necrotic in nature. The liposomes were shown to successfully enhance cellular uptake in all cell culture models tested. Furthermore, results demonstrate positive mitochondrial targeting and localisation activity facilitated by the presence of MTS peptide and a combination of MTS and R8 peptides on the liposomal surface for all four of these novel liposomal nanovectors.
Novel epidermal growth factor directed cationic lipoplexes promote in vitro hepatotropic gene targeting.
(2014) Sewbalas, Alisha.; Singh, Moganavelli.; Ariatti, Mario.
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.
Receptor targeted gene delivery using folate ligand conjugated cationic liposomes.
(2014) Gorle, Sridevi.; Singh, Moganavelli.; Ariatti, Mario.
Gene therapy has become an important strategy to treat several human diseases, including cancer, viral infections and inherited disorders. In response to this growing trend, a number of gene delivery vectors have been manufactured both to facilitate nucleic acid uptake by target cells and also to promote the transport of genetic materials into the nucleus. The success of gene therapy however depends on the efficient delivery of therapeutic genes into target cells both in vitro and in vivo. Cationic liposomes represent a class of non-viral vectors that have shown the ability to bind and deliver DNA cargo to defective cells efficiently. This study has focused on the development of a novel folate-targeted cationic liposome-mediated gene delivery system. This receptor is overexpressed on numerous cancer cell types and offers a convenient docking point for subsequent cellular uptake of folate decorated liposome-DNA complexes by receptor mediation. In this study, a total of six cationic liposome preparations comprising either cationic cholesterol cytofectin -dimethylpropylamidosuccinylcholesterylformylhydrazide (MSO9) or 3β[N(N1,N1-dimethlaminopropylsuccinamidoethane)-carbamoyl]-cholesterol (SGO4) were formulated by mixing the fusogenic neutral helper lipid, dioleoylphosphatidylethanolamine (DOPE) as a common constituent. DSPE-PEG₂₀₀₀ was also used in formulations for possible in vivo development of PEGylated, targeted liposomes. The targeting ligand folate was appended to the distal end of liposome-anchored DSPEPEG₂₀₀₀, for prominent display and optimal receptor recognition. Transmission electron micrographs revealed liposomes to be unilamellar, spherical shaped vesicles with a narrow size range (50 - 80 nm in diameter). Agarose gel retardation studies demonstrated complex formation between cationic liposomes and plasmid DNA, whilst serum nuclease protection assays showed that the liposome formulations were capable of protecting the complexed DNA in lipoplexes against serum nuclease digestion. Ethidium bromide dye displacement studies yielded information on the compaction or condensation efficacy of the liposomes with respect to the cargo plasmid. In addition, particle sizes determined by dynamic light scattering confirmed the suitability of lipoplexes for future in vivo applications in which extravasation is essential. Importantly, these liposome:DNA complexes were found to exhibit minimal growth inhibition levels in HEK293, HeLa and KB cells. Further investigations were carried out to determine the optimal transfection activity of complexes in the folate receptor-positive cell lines (HeLa and KB). The plasmid containing the transgene firefly luciferase (pCMV-luc) was used in transfection studies. Results showed that folate targeted liposomes, irrespective of cytofectins MSO9 or SGO4 achieved highest transfection activities in vitro, specifically via receptor mediation. Lower transfection activity was observed for by untargeted PEGylated and unPEGylated liposomes compared to that of the folate targeted liposomes, strongly implicating folate receptor-mediation in the uptake of ligand-displaying lipoplexes. This was further confirmed by flow cytometry analysis. Furthermore, zeta potential values obtained for targeted complexes revealed low negative surface charge, thus minimizing the possibility of electrostatic interaction between lipoplexes and target cells. The cytofectin, MSO9, achieved 10 fold greater transfection activity than the cytofectin SGO4 although they are closely related, differing only in their spacer lengths. Competition assays using free folate (200 μM) to confirm folate receptor mediated lipoplex uptake in the HeLa, and KB cells revealed a dramatic decline in transfection activity due to the excess free folate binding to and blocking access to the folate receptors on the cell membrane. The two novel PEGylated lipoplexes designed for folate receptor-mediated uptake by transformed mammalian cells display very favourable physicochemical characteristics, low cytotoxicity and promising transfection profiles in vitro. Therefore further investigation of the cationic liposome formulations examined in this study in vivo is warranted.
Studies on the coupling of DNA to low density lipoproteins (LDL) and the interaction of these complexes with eukaryotic cells.
(1987) Khan, Zainub.; Hawtrey, Arthur O.; Ariatti, Mario.
The application of Molecular Biochemistry for transfection studies in eukaryotic systems is well documented. Of the numerous methods employed for the introduction of foreign DNA into eukaryotic cells, the use of low density lipoproteins (LDL) as carriers of DNA into cells has not been reported. LDL was isolated, characterized with respect to its protein and lipid components, and then variously modified in an attempt to enhance its affinity for DNA. It was found that both unmodified and modified LDL could interact with DNA, at physiological pH. The carbodiimide modified LDL (ECDI - LDL) showed the greatest affinity for DNA. LDL and ECDI - LDL were used to study LDL receptor binding in skin fibroblasts. This was followed by a study of receptor binding activities of both unmodified LDL and ECDI - LDL complexed to DNA (pBR322). Although the extent of binding of ECDI - LDL and ECDI - LDL - DNA complexes to plasma membranes was greater, the internalization and degradation of both modified and unmodified LDL complexes were equivalent. This additional binding was attributed to non - receptor - specific affinity of the carbodiimide modified complexes for the plasma membrane. The transfection of foreign DNA into eukaryotic cells in culture was monitored by assaying for the expression of the cloning vector, pSV2cat, complexed to LDL or ECDI - LDL and introduced into the cells by LDL receptor - mediated endocytosis. Of the cell lines in which the expression of the pSV2cat recombinant DNA was monitored, the human lung fibroblasts showed the greatest activity of the expressed chloramphenicol acetyl transferase enzyme. Although transfection efficiency was lower than that of the calcium phosphate - DNA coprecipitation procedure, the LDL receptor - mediated transfection of eukaryotic cells was carried out under physiological conditions and may be applicable in vivo.
Targeted gene transfer to mammalian systems using liposome constructs containing cholesterol components with or without biotinylated molecular accessories.
(2005) Singh, Moganavelli.; Ariatti, Mario.
Abstract available in PDF file.

Browse

Browsing Doctoral Degrees (Biochemistry) by Author "Ariatti, Mario."