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.
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.
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.
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.
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.
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.
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 Biochemistry by Author "Ariatti, Mario."