Browsing by Author "Narainpersad, Nicolisha."

Now showing 1 - 2 of 2

Cationic liposome mediated targeted gene delivery with and without pegylated accessories.
(2009) Narainpersad, Nicolisha.; Singh, Moganavelli.; Ariatti, Mario.
As a consequence of safety issues encountered by the use of viral vectors in gene therapy, there has been a steady increase in the development and application of non-viral vectors, especially liposomes. Cationic liposome mediated delivery is one of the most promising nonviral delivery methods. These liposomes are prepared from synthetic lipids, are positively charged and interact favourably with DNA through electrostatic interactions. Cationic liposomes have also shown immense potential in the targeting of specific cell types such as HepG2 (hepatocellular carcinoma) cells, a model in vitro gene delivery system for the study of hepatocyte function. However, these liposomes also have a number of limitations in vivo. In an attempt to overcome these restrictions, a hydrophilic polymer, polyethylene glycol (PEG) is incorporated into the cationic liposome. This covalent attachment of (PEG) to the liposomal surface is thought to sterically stabilise liposomes, promote biological stability, inhibit aggregation, decrease toxicity and immunogenicity, prevent interaction with serum proteins and complement and thus prolonging the circulation time of liposomes in vivo. The versatility and simplicity of cationic liposomes have made them vitally significant non-viral gene delivery vehicles for human gene therapy. In this investigation novel untargeted and targeted glycosylated liposomes with and without PEG were synthesised to evaluate their gene transfer activities in vitro to potentially develop a suitable gene delivery system for future in vivo applications. A constant molar quantity of the cationic cholesterol derivative, 3 [N-(N’, N’-dimethylaminopropane)-carbamoyl] cholesterol (CHOL-T) was mixed with dioleoylphosphatidylethanolamine (DOPE) and a galactose/glucose derivative to produce targeted cationic liposomes. PEG liposomes were prepared in the same way with the addition of distearoylphosphoethanolamine polyethylene glycol 2000 (DPSE-PEG2000), 2% on a molar basis. Supported by transmission electron microscopy characterisation, we present evidence that the pegylation of liposomes affects the DNA binding capability and transfection efficiencies of the cationic liposomes in addition to protecting the plasmid DNA in lipoplexes from serum nuclease degradation. Optimal DNA : liposome binding ratios were obtained from gel retardation studies and confirmed by ethidium bromide intercalation assays. These complexes were then tested on the human hepatoma cell line, HepG2, to determine toxicity and assess transfection efficiencies. From results obtained in this study, it appears that both cationic and pegylated cationic liposomes are well tolerated by cells in vitro. The results further suggest that targeting by use of glycolipids incorporated into the structure of the liposome increases transfection, while pegylation of cationic liposomes marginally decreases the transfection efficiency of the lipoplexes to HepG2 cells in vitro.
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.