Masters Degrees (Biochemistry)

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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.
The comparative effects of acetylated and deacetylated galactose derivatives in liposomal gene delivery.
(2014) Mokhosi, Seipati Rosemary.; Singh, Moganavelli.; Ariatti, Mario.
The use of cationic liposomes remains the most attractive non-viral approach in gene therapy as these gene carriers provide for ease and versatility in design. In targeted gene delivery, liposomes are coupled to ligands tailored to possess desired characteristics for improved cell-specificity. Carbohydrates have been established as useful targets for the asialoglycoprotein (ASGP) receptor in liver-directed delivery. The main purpose of this study was to comparatively evaluate physicochemical characteristics, DNA-binding interactions and in vitro transfection activities of hepatocyte-targeted liposomes bearing acetylated and deacetylated galactosides in ASGP receptor-mediated gene delivery. Furthermore, in silico studies were carried out to assess ligand-receptor interactions for both galactosides. Novel targeted cationic liposomes conjugated with galactosyl ligands viz. cholest-5-en-3-yl 2-[4-(β-D-galactopyranosyl-1-oxymethyl)-1H-1,2,3-triazol-1-yl]ethylcarbamate (Sc6) and cholest-5-en-3-yl 2-[4-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-1-oxymethyl)-1H-1,2,3-triazol-1-yl]ethylcarbamate (Sc9) were formulated with cytofectin 3β[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) and the neutral co-lipid dioleoylphosphatidyl ethanolamine (DOPE), using the thin film hydration–sonication method. Characterisation of lipoplexes by cryo-transmission electron microscopy (cryo-TEM) showed unilamellar liposomes, and lipoplexes ranging between ~80 – 140 nm. DNA was fully liposome-bound at N:P ratios 2.5:1 – 3:1. Upon inclusion of polyethylene glycol 2000 -distearoylphosphatidyl ethanolamine (DSPE-PEG₂₀₀₀) in liposome formulations, vesicles were more compacted due to steric stabilisation. UnPEGylated lipoplexes achieved better condensation of DNA as determined in band shift and ethidium bromide displacement assays. Nuclease digestion assays revealed suitable protection of cargo DNA by some formulations, with the least protection afforded by the acetylated SM3 derivatives. Cytotoxicity studies in the HEK293 and HepG2 cell lines revealed good cell viabilities under transfection conditions for all liposomes. Transfection efficiency was assessed using the luciferase reporter gene assay. Higher transfection activities were observed in the ASGP receptor-positive HepG2 cell line than the ASGP receptor-negative HEK293 cells line for all lipoplexes. While the acetylated unPEGylated derivative (SM3) demonstrated better transgene expression levels compared to other derivatives, this was not found to be significant. High transfection levels were attributed to favourable size and surface charge, as well as galactoside ligand accessibility to the receptor. In the presence of excess asialofetuin, a marked decrease in transfection efficiencies was observed for all targeted derivatives. Docking scores further confirmed good binding affinity for the deacetylated Sc6 ligand and acetylated Sc9 ligand at ˗6.7 and ˗5.5 kCal/mol, respectively. The acetylated SM3 however, achieved avidity to the binding site through hydrogen bonding via the triazine linker. Overall transfection efficiency results were corroborated by outcomes from molecular studies as both galactoside ligand-conjugated liposomes presented similar binding affinities and transfection efficiency results. It is thus concluded that both these galactosides, with further optimization could present the potential for hepatocyte-specific delivery via ASGP receptor-mediated endocytosis.
A comparative study on three unique galactosylated cationic liposomes with their steically stablized counterparts, in HepG2 cells.
(2013) Govender, Dhineshree.; Singh, Moganavelli.; Ariatti, Mario.; Masola, Bubuya.
Receptor mediated endocytosis allows for the site specific delivery of exogenous DNA via appropriate ligand-receptor interactions. Various ligands have been used to target the asialoglycoprotein receptor (ASGP-R) present on the hepatocyte cell membrane viz. asialofeutin, asialoorosomucoid, lac-BSA, asialolactoferrin, asialotransferrin, asialo-ceruloplasmin and galactose. The high affinity that the receptor displays for the galactose sugar moiety has led to the development of several new galacto-lipids for the incorporation into liposomes intended for hepatocyte targeting. In this study, three cholesteryl derivatives displaying galactose units linked to the sterol skeleton by different spacer elements have been formulated into cationic liposomes with and without polyethylene glycol (PEG) accessories. The three galactosylated liposomal formulations were prepared using near equimolar amounts of MSO9 (N,N-dimethylaminopropylamidosuccinyl-cholesterylformylhydrazide) and DOPE (dioleoylphosphotidylethanolamine) together with the respective galactose derivative (at 10 mole % w/w) viz. Cholesteryl-3β-N-(4-aminophenyl-β-Dgalactopyranosyl) carbamate; Cholesteryl (1-β-D-galactopyranosyl-1,2,3 triazol-4-yl) carbonate; and Cholesteryl-β-D-galactopyranoside. All liposomes displayed DNA binding, nuclease protective capabilities to plasmid DNA, low cytotoxicity (cell viability being within 60-101 %) and an increase in transfection activities, in the human hepatocellular carcinoma cell line HepG2, which expresses the ASGP-R abundantly. The results obtained correlate well with differences in the spacer element in the 3 galactosylated cholesterol derivatives under study and the presence and absence of 2 mole % DSPE-PEG₂₀₀₀ in the liposome formulations. Overall, it was observed that the cationic liposome containing cholesteryl (1-β-Dgalactopyranosyl- 1,2,3 triazol-4-yl) carbonate (with and without PEGylated accessories), which was synthesised chemically using “click chemistry”, afforded the highest in vitro transfection activity, and may be optimised and studied further. The highest levels of transfection activity, in vitro, were attributed to the increased length of the spacer arm between the galactose moiety and the cholesteryl anchor of the targeting component. Two formulations were then subjected to in vivo studies, using male Sprague Dawley rats which yielded little or no transgene expression.
Preparation of chemically modified transferrin proteins and an investigation of their reactions with DNA and other nucleic acids.
(1986) Gordhan, Hasha.; Hawtrey, Arthur O.; Ariatti, Mario.
The molecular biology of human genetic disorders is under intensive investigation at present. In those cases where the disorder is clearly defined in terms of altered gene structure, possibilities may exist for the correction of the disorder by insertion of normal genes through the process of DNA transfection. A possible method for the transfer of genetic material is by attempting to attach DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. By this means one might be able to get DNA into cells. This thesis deals with experimental work on the chemical modification of human serum transferrin by means of water-soluble carbodiimides. The resulting N-acylurea transferrins bind DNA in a reversible manner. Characteristics and properties of the binding interactions are dealt with in detail. N-acylurea derivatives of transferrin were prepared with the water-soluble carbodiimides, N-ethyl-N' -(3-dimethylaminopropyl) carbodiimide and N-ethyl-N' -(3-trimethylpropylammonium) carbodiimide iodide. Reactions were carried out under mild conditions at room temperature for 48-72 hours. [³ H] N-ethyl-N' -(3-trimethylpropylammonium)carbodiimide iodide was used for the determination of covalently attached N-acylurea groups in the protein. Changes in charge properties were determined by agarose gel electrophoresis. Carbodiimide modification of proteins is thought to occur at side chain carboxyl groups of glutamic and aspartic acid residues. This was confirmed by the use of Staphylococcus aureus V8 protease, which cleaves peptide bonds at the carboxyl side of glutamic and aspartic acid residues, but not in the case of substituted side chain carboxyl groups. Through the use of puromycin as a nucleophile it has been shown that other functional groups were not activated upon reaction of transferrin with carbodiimide. The carbodiimide-modified proteins bind various types of DNA and RNA in a reversible manner. Low concentrations of N-acylurea transferrin retarded the migration of pBR322 DNA, M 13 mp 8 single-stranded DNA and Pst 1 restricted lambda DNA on agarose gel electrophoresis, while at higher concentrations the DNA was unable to enter the gel. Nitrocellulose filter binding assays showed that binding of DNA to Nacylurea transferrins was rapid, dependent on concentration of the modified transferrin and sensitive to ionic conditions. Binding was found to occur mainly through electrostatic interactions between phosphate groups of DNA and N-acylurea groups. These conclusions were based on experiments which showed that protein-DNA complexes were dissociated by increasing salt concentrations and by heparin. Non-electrostatic interactions such as hydrophobic interactions and hydrogen bonding are also involved in binding, since half dissociation of complexes, induced by chaotropic salts, KSCN and NaC10₄occurs at lower concentrations of salt than in the case of NaCl. Also RNA polynucleotides inhibit binding of DNA to Nacylurea transferrins to varying extents. The N-acyl urea transferrins have been shown to bind certain specific restriction endonuclease cleavage sites on pBR322 DNA. The N-acylurea transferrin-DNA complexes would thus be suitable for experiments in cell transfections using cells which have transferrin receptors.
Studies on the preparation and interaction of modified transferrin-DNA complexes with HeLa cells.
(1986) Hawtrey, Richard William.; Ariatti, Mario.; Hawtrey, Arthur O.
The correction of human genetic disorders by transfer of genetic material to cells is under intensive investigation in a number of 1aboratories. One possible way of trying to achieve the transfer of nucleic acid is by attaching DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. In order to make use of the ligand protein-receptor approach for DNA transfer, iron-loaded human serum transferrin has been modified with the water soluble carbodiimides N-ethy1-N I -(3-dilllethy1aminopropyl) carbodiimide (CDI) and its quaterary analogue (ECDI) to give modified N-acy1urea transferrins. N-Acy1urea CDI (Fe 3+) transferrin and N-acy1urea CDI (Fe ) transferrin have been found to interact with and bind DNA in a reversible manner which i! dependent on ionic strength. [1251] N-Acy1urea CDI+(Fe3+) transferrin binds to transferrin receptors on Hea cells in culture and undergoes internalization through receptor-mediated endocytosis. Binding of the modified transferrin in the presence of calf thymus DNA to transferrin receptors also takes place. However, although internalization in the presence of DNA doe! appear to take place, the results of the internalization are not fully understood. Transfection studies with N-acy1urea CDI (Fe ) transferrin and plasmid pBR322 DNA as well as plasmid ptkNEO DNA complexes in the HeLa cell system are reported. The results of a number of transfection experiments suggests that N-acy1urea transferrins are capable of transfecting DNA (ptkNEO DNA), carrying genes for resistance to the antibiotic Geneticin (G41S) in the HeLa cell system. However, further development of the transfection system is necessary in order that consistantly reproducible results may be achievd.
Synthesis of DNA - protein conjugates and a preliminary study of their interaction with eukaryotic cell receptors.
(1986) Weiler, Solly.; Hawtrey, Arthur O.; Ariatti, Mario.
Thymidine oligomers were chemically synthesised and linked to available amino functions of transferrin in alternative orientations: (a) A CMP residue attached to the 3' end of (pT)₁₀ with terminal deoxynucleotidyl transferase was oxidised with NaI0 and linked to transferrin via a Schiff base formation. (b) The 5' terminal phosphate group of (pT)₅ was activated with imidazole and reacted with transferrin to form a phosphoramide bond. The (pT)₅ thus attached to the protein was elongated to (pT)₃₀₀ using terminal deoxnucleotidyl transferase and TTP. The latter conjugate was capable of hybridising poly(A) tailed linear PBR322 DNA. The binding of this hybridisation complex to the transferrin receptor on various cell types was investigated.
The use of cholesterol-galacto compounds in liver directed gene delivery.
(2014) Mkhwanazi, Nkosiyethu Knowledge.; Singh, Moganavelli.; Ariatti, Mario.
Gene therapy has to date gained immense interest as a potential method for treating genetic disorders such as Parkinson’s and cystic fibrosis. The liver being a central organ of metabolism is susceptible to several metabolic disorders which could be targeted through liver directed gene delivery. The most common diseases being viral hepatitis and hepatocellular carcinoma towards which this study is focused. Non-viral vectors have gained wide interest as the vector of choice for delivering genes to organs such as the liver, because of their large-scale production potential, easy preparation, low cost and are relatively non-immunogenic to target cells / organs. This study utilized non-viral cationic liposomes targeted to hepatocytes via ligand-receptor recognition. A total of six cationic liposomes (targeted acetylated /non-acetylated, nontargeted, pegylated, non-pegylated) were prepared according to the lipid film hydration method. The liposomes and liposome:DNA complexes were characterized using transmission electron microscope (TEM) and Zeta sizing to determine morphology, lamellarity and size. Results showed spherical, unilamellar cationic liposomes and lipoplexes in the size range of 50-200 nm in diameter. Band shift assays showed that these liposomes have strong DNA binding capabilities which was further confirmed by the ethidium bromide intercalation assays. Nuclease protection assays showed that liposomes were able to protect the integrity of the DNA cargo. From the MTT cell viability assays, low cytotoxicity was observed for all liposomes with cell survival as high as 80 % in most cases. Higher transfection activities were noted in the hepatocellular carcinoma receptor positive cell line (HepG2), for targeted non-acetylated liposomes, compared to the acetylated liposomes. The ligand competition assay and the use of the human embryonic kidney receptor negative cell line (HEK293) confirmed that the complexes entered the cells via receptor mediated endocytosis. Furthermore, it was confirmed that the acetylation of the galactose ligands hindered the process of receptor mediation. Overall, these liposomal formulations are serum tolerant, have low cytotoxicity and are able to selectively target and transfect hepatocytes in vitro. Hence, they have the potential as future non-viral gene delivery vehicles and with further optimisation can be tested in vivo.

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