Browsing by Author "Singh, Moganavelli."

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Anti-c-myc cholesterol-based lipoplexes: development, characterisation and evaluation as Onconanotherapeutic agents in vitro.
(2018) Habib, Saffiya.; Singh, Moganavelli.
Strategies aimed at inhibiting the expression of the c-myc oncogene could provide the basis for alternative cancer treatment. In this regard, silencing c-myc expression using small interfering RNA (siRNA) is an attractive option. However, the development of a clinically viable, siRNAbased, c-myc silencing system is largely dependent upon the design of an appropriate siRNA carrier that can be easily prepared. Nanostructures formed by the electrostatic association of siRNA and cationic lipid vesicles represent uncomplicated, well-recognised siRNA delivery systems. Therefore, this study has focused on traditional cationic liposomes as the foundation for the development of a simple, but effective anti-c-myc onconanotherapeutic agent. Novel liposome formulations contained equimolar quantities of the cytofectin, N,Ndimethylaminopropylamidosuccinylcholesterylformylhydrazide (MS09), and cholesterol (Chol); with or without 2 mol % pegylation. Liposomes which contained dioleoylphosphatidylethanolamine (DOPE) as the co-lipid were included for comparative purposes. Pegylated and non-pegylated MS09/Chol (1:1) suspensions were reproducibly prepared by lipid film hydration to give unilamellar vesicles that were stable for at least 10 months at 4 ˚C. Liposomes successfully bound siRNA to form lipoplexes of less than 200 nm in size, with zeta potentials between -16 and -44 mV. These assumed globular and bilamellar structures in which siRNA was partially protected. Although all formulations were well tolerated at ≤14 nM siRNA, pegylation severely inhibited siRNA delivery in cancer cell lines, MCF-7 and HT-29, which overexpress c-myc. The non-pegylated MS09/Chol (1:1) lipoplex, at the MS09:siRNA (w /w) ratio of 16:1, was most effectively taken up by MCF-7 and HT-29 cells, with negligible effect in non-transformed cells when applied at 12 nM siRNA. Lipoplexes directed against the c-myc transcript (anti-c-myc siRNA), mediated a dramatic reduction in c-myc mRNA and protein levels. This was accompanied by a loss of migratory potential and apoptotic cell death. Moreover, oncogene knockdown and anti-cancer effects were superior to that of a commercially available transfection reagent, Lipofectamine™ 3000. Although the DOPE-containing counterpart performed with iii comparable efficacy under standard in vitro conditions, it was incapable of siRNA delivery at physiological serum concentration. Hence, the anti-c-myc MS09/Chol (1:1) lipoplex reported exemplifies a straightforward anti-cancer agent that warrants further investigation in vivo.
The application of layered double hydroxide nanoparticles (LDHs) as potential anticancer drug delivery systems.
(2016) Mncwabe, Zoleka.; Singh, Moganavelli.
Chemotherapy being one of the principle techniques used in cancer treatment, has been applied in the treatment of a wide spectrum of cancers. However, this mode of treatment is fraught with a myriad of challenges, reducing its effectivity and inducing the need for repeated treatments. Poor drug delivery systems or lack thereof, have led to patients suffering unpleasant side effects that not only cause collateral damage to their bodies but also reduces the quality of their lives. The current array of chemotherapeutic drugs available may be effective in certain cancers, nevertheless the need for their optimization is still necessary for better safety, stability and efficiency of treatment. Thus the current study was designed to investigate the potential of layered double hydroxide (LDH) nanoparticles in the delivery of the broad spectrum anticancer drug, 5-Fluorouracil (5-Fu). Four LDH nanoparticles, MgAl 2:1, MgAl 3:1, ZnAl 2:1and ZnAl 3:1 were successfully synthesized and intercalated with 5-Fu using the calcination reconstruction process to form nanohybrids. The LDHs and their nanohybrids, MgAl 2:1-5-Fu, MgAl 3:1-5-Fu, ZnAl 2:1-5-Fu and ZnAl 3:1-5-Fu were structurally confirmed using XRD, FTIR, UV-Vis, ICP-OES; with size, zeta potential and ultrastructural morphology investigated using nanoparticle tracking analysis (NTA) and electron microscopy (TEM and SEM). LDHs were characteristically hexagonal in shape with sizes ranging from 100 -150 nm, and high zeta potentials enforcing their colloidal stability. The successful intercalation of 5-Fu was confirmed from drug encapsulation efficiency studies to be between 40-60% in the respective LDHs. Furthermore, drug release studies revealed a steady controlled release of the drug over a 7-hour period at pH 4-7, with more than 60% of the drug being released by the end of this period. In vitro MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and SRB (Sulphorhodamine B) cytotoxicity studies on free 5-Fu and LDH bound 5-Fu in human cell lines, breast adenocarcinoma cell line (MCF-7), hepatocellular carcinoma (HepG2), colorectal adenocarcinoma (CaCo-2) and embryonic kidney (HEK293), showed a dose dependent cytotoxicity profile with the free 5-Fu being more toxic to the cells than the bound drug. This was further confirmed in fluorescent apoptotic studies (dual acridine orange and ethidium bromide staining method), where free 5-Fu had a higher apoptotic index than the LDH bound 5-Fu
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.
Cationic liposome mediated transfection with/without a targeting component.
(2005) Singh, Ashika.; Naidoo, Richard.; Singh, Moganavelli.
The transfer and expression of genes in cells is an important technique for basic research and gene therapy of human disease. A model for gene therapy has been investigated making use of a transfection complex consisting of three components, the DNA i.e. the gene to be transferred and expressed; a gene delivery vehicle viz. a cationic liposome and a cell specific targeting ligand, asialoorosomucoid (AOM). Cationic liposomes are positively charged liposomes that have been prepared from synthetic lipids and have been shown to complex or bind to DNA via electrostatic attraction. They have shown potential as an efficient non-viral gene delivery vehicle in human gene therapy. In this investigation, a novel cationic liposome consisting of 3B [N -(N',N'-dimethylaminopropane)carbamoyl] cholesterol (Chol-T), dioleoylphosphatidylethanolamine (DOPE) and biotinylcholesteryl formylhydrazide was prepared and assessed as a mediator of DNA delivery in a mammalian cell culture system viz. the HepG2 cell line. The cationic liposome was synthesised and characterised by electron microscopy. Foreign DNA may be specifically delivered to target cells by a carrier system which makes use of the recognition of the asialoglycoprotein AOM by cognate receptors on the HepG2 cell plasma membrane. The positively charged AOM was biotinylated and due to this biotinylation, binds streptavidin which contains specific binding sites for biotin. The cationic liposome itself contains biotin residues in its bi-Iayer which in turn binds streptavidin resulting in a ternary complex. Further, due to the DNA binding capability of the cationic liposome, a transfection complex is produced consisting of the three components. The experiments were based on the following concepts: (i) Hepatocytes possess a unique receptor that binds to and internalises galactose-terminal asialoglycoproteins by receptor mediated endocytosis. (ii) Due to electrostatic attraction, DNA binds to cationic liposomes forming soluble complexes. (iii) Through the biotin-streptavidin reaction, the biotinylated AOM is attached to the cationic liposome containing biotin forming complexes enabling targeted delivery of the DNA. (iv) DNA containing the pGL3 gene for the luciferase enzyme was used and following transfection experiments, the luciferase assay was performed to ensure successful transfection. The complexes were tested on the hepatocellular carcinoma cell line, HepG2, which possess the asialoglycoprotein receptor. Transfection studies were conducted using a transient expression system, the luciferase assay system. Some degree of success in the transfection of HepG2 cells was observed. Results obtained in this study suggest that transfection using our targeted transfection complex consisting of cationic liposomes and cell specific targeting ligands does in fact transfect cells by receptor mediation.
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 investigation of transgene expression and gene silencing of non-functionalized layered double hydroxides versus amino-acid functionalized hydrotalcites.
(2017) Nundkumar, Nirasha.; Singh, Moganavelli.; Singh, Sooboo.
Abstract available in PDF file.
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.
Delivery of Fluc-mRNA using functionalised gold nanoparticles in vitro.
(2016) Pillay, Shandré.; Singh, Moganavelli.
Nanomedicine, a branch of nanotechnology which includes the use of nanoparticles, is set to revolutionize the area of gene therapy. Gold nanoparticles (AuNPs) have steadily gained favour as promising gene and drug delivery agents in clinical medicine for diagnostics and the treatment of diseases such as cancer. Cancer is a chronic disease with mortality rates increasing steadily. It has been highlighted as a genetic disease with a rising need for the development of innovative gene therapy strategies and, thus, the development of safe and effective gene delivery vehicles. AuNPs feature unique properties which includes small size, high dispersity, stability and surface plasmon resonance with a capacity for ease-of-synthesis and parametric control via chemical methods. Characteristics which favour gene delivery include their biocompatibility, voluntary cellular uptake and low core toxicity. Functionalisation with cationic polymers enhances the properties of AuNPs in terms of condensation of nucleic acids, cellular internalisation, protection against nucleases and release of the therapeutic gene for expression. Over the years, gene therapy applications have routinely utilized therapeutic plasmid DNA (pDNA) for treatment. However, pDNA expression is associated with many limitations hindering cellular trafficking and nuclear entry. As a promising alternative, mRNA molecules overcome these intracellular barriers with no risk of insertional mutagenesis. In this study, the unique properties of AuNPs, cationic polymers and mRNA molecules were exploited and optimized to provide effective gene delivery vehicles for the intracellular release of mRNA for future cancer gene therapy applications, an area of research which until recently has not been investigated. AuNPs were synthesized and functionalised with chitosan (CS) and poly-l-lysine (PLL), respectively. All nanoparticles and their nanocomplexes were characterized using UV-vis spectrophotometry, ICP, FTIR, transmission electron microscopy (TEM) and Nanoparticle tracking analysis (NTA). Functionalised AuNP:Fluc-mRNA binding, compaction and nuclease protection were assessed using the gel retardation, dye displacement and nuclease protection assays, respectively. The effects of these nanocomplexes in vitro were assessed on three human cell lines, embryonic kidney (HEK293), colorectal adenocarcinoma (Caco-2) and breast adenocarcinoma (MCF-7). The cytotoxicity profile of the nanocomplexes was evaluated using the MTT assay and mRNA transfection efficiency assessed using the luciferase reporter gene assay in the three human cell lines. The AO/EB apoptosis assay was employed to confirm the mechanism of cell death as being either apoptotic or necrotic. Results show that both functionalised nanocomplexes exhibit suitable and favourable properties such as small size (CS-AuNP nanocomplex: 95.0 nm and PLL-AuNP nanocomplex: 89.4 nm), colloidal stability (zeta potential: CS-AuNP nanocomplex: 25.9 mV and PLL-AuNP nanocomplex: -97.1 mV), efficient binding and protection against nucleases, low cytotoxicity (<40%) and significant transgene expression. Furthermore, these functionalised nanocomplexes exhibited apoptosis induction selective to cancer cells when compared to the non-cancer cell line. Functionalised AuNPs proved to be more efficient than their respective cationic polymers by themselves, indicating that AuNPs improve cationic polymer properties and functions. Overall, these characteristics highlight the potential of these AuNPs as suitable carriers of mRNA in vitro and with further studies can be extended to biomedical applications, especially in cancer and immuno-therapy.
The design and synthesis of gold nanoparticles and its interaction with mammalian cells in culture.
(2014) Lazarus, Geraldine Genevive.; Singh, Moganavelli.
Cancer is a disease characterized by accelerated cell growth, resulting in healthy tissue being destroyed by the processes of invasion and metastasis. Non-viral gene delivery approaches have been extensively studied as a basic tool for intracellular gene transfer and gene therapy especially for genetic aberrations including cancer. Gold nanoparticles have attracted strong biomedical interest for drug delivery due to their low toxic nature, surface plasmon resonance and capability of increasing the stability of the payload. In the present study the synthesis of photoluminescent nanoparticles consisting of a gold core coated with polyethyleneimine, poly-L-lysine, cysteine and chitosan is reported. These functionalized gold nanoparticles (FAuNPs) were investigated at different pH values and ionic strength to identify the optimum conditions to produce stable monodisperse nanoparticles. FAuNPs showed good stability at low ionic strength which is important for the flexibility of the polymer chain. All nanoparticle/polymer formulations remained in the size range 11.9-195 nm with narrow particle distributions and low PDI (<1.2). TEM images revealed nanoparticles that were spherical and monodispersed. Nanoparticle and pDNA complexation was efficiently demonstrated in the band shift and ethidium bromide intercalation assays respectively. The serum nuclease digestion assay revealed that the nanoparticles provided partial protection to the complexed plasmid DNA (pCMV-luc). MTT cytotoxicity experiments indicated that the FAuNPs elicited a dose dependent cytotoxic effect with the four mammalian cell lines (HepG2, HEK293, HeLa and Caco2) tested responding differently. Au-PEI/pDNA maintained over 80% cell viability across all cell lines, while the Au-cys/pDNA exhibited a significant 91.8% (p<0.001) in Caco2 cells, Au-Chit/pDNA 126% (p<0.01) in HepG2 cells and Au-PLL/pDNA 104% in Hela cells. Transfection studies were accomplished using the luciferase reporter gene assay. Results showed that the FAuNPs produced greater transgene activity than the cationic polymer/DNA complexes on their own. This was evident for the Au-PEI/pDNA complex which produced a 12 fold increase in the HEK293 cells and a 9 fold increase in the HepG2 cells, compared to the PEI/pDNA complexes alone. The results of this study suggest that FAuNPs low cytotoxicity coupled with the ability to parametrically control particle size and surface properties, make these nanoparticles suitable non-viral gene delivery vectors. However further engineering and modifications of the FAuNPs may be required to enable in vivo gene delivery.
Effects of catharanthus roseus and centella asiatica leaf extracts on enzymes of glutamine catabolism in human colon carcinoma (CACO-2) cell line and in enterocytes from male sprague-dawley rats.
(2015) Dladla, Thobekile Precious.; Masola, Bubuya.; Singh, Moganavelli.
Phosphate-dependent glutaminase (PDG) is a key enzyme in several physiological processes and hence has become a target of anti-cancer drug development because of the role of glutamine in providing energy for rapidly dividing cells. This study investigated the effects of the Centella asiatica and Catharanthus roseus leaf extracts on enzymes of glutamine catabolism in human colon carcinoma (Caco-2) cell line and in enterocytes from Sprague-Dawely rats both treated with plant extracts. Our hypothesis was that these extracts would arrest the growth of Caco-2 cells by inhibiting glutaminase but have less deleterious effects on normal cells due to the former being more avid consumers of glutamine. The 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to assess the cytotoxic effects, and hence the anti-cancer potential of extracts from medicinal plants C. asiatica and C. roseus against Caco-2 and human embryonic kidney (HEK 293) cell lines. These in vitro effects were assessed using various doses of plant extracts (0- 16 mg/ml) and different exposure periods of 24, 48 and 72 h. Results show that the cytotoxicity effect of the C. asiatica extract to the caco-2 cell line is dose dependent whilst C. roseus treatment decreased the Caco-2 cells viability at all the exposure times but this was not dose-dependent. In contrast, both extracts significantly increased cell proliferation of the HEK 293 cells compared to the controls. The PDG and ALT activities were decreased in Caco-2 cells whilst the HEK293 cells were largely unaffected. In the in vivo studies, the activities of phosphate-dependent glutaminase (PDG), glutamate dehydrogenase (GDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined in the enterocytes. Results show that the activities of GDH remained constant whilst there was a decrease in ALT and a slight increase in AST activities in the enterocytes in both the C. asiatica and C. roseus treated groups. The PDG enterocytes activity, on the other hand, was lower in both the treated groups compared to the control group. Expression of both PDG and AST assessed by dot blots in the enterocytes increased in both plant extract treated groups. Induction of apoptosis was also investigated using cytochrome c. The plant extracts were screened for presence of phytocomponents that may be causing changes in Caco-2 cell growth and in enzymes of glutamine catabolism using Gas Chromatography-Mass Spectrometry (GC-MS) and qualitative phytochemical analysis. The extracts revealed the presence of anticancer and many other compounds. We conclude that the both extracts have potent anti-cancer cell activity that leaves normal cells unaffected, in fact stimulating their growth.
Elucidation of gene function using RNA interference in a cancer cell culture model.
(2011) Daniels, Aliscia Nicole.; Singh, Moganavelli.; Ariatti, Mario.
RNA interference (RNAi), mediated by small interfering RNA (siRNA), has emerged as a powerful tool for elucidating gene function and also holds great potential for the treatment of acquired and inherited diseases. The delivery of siRNAs still remains a major obstacle for their therapeutic use. Cationic liposomes, a group of positively charged nanovesicles, represent a class of non-viral vectors that have shown the ability to efficiently bind and deliver siRNA. In this study, six cationic liposome formulations containing either cationic cholesterol derivative [N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or N,Ndimethylaminopropylaminylsuccinylcholesterylformyl- hydrazide (MSO9) were prepared with the neutral lipid dioleoylphosphatidylethanolamine (DOPE). Varying amounts of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), (0, 2 and 5 mole percent) were also included in the liposomal formulations as polyethylene glycol is known to improve the lipoplex bioavailability in vivo. We present evidence that siRNA may be delivered to mammalian cells, in vitro, using a novel cationic liposome carrier system and that siRNA binding and transfection efficiency of the cationic liposomes are affected by the degree of pegylation. Cryoelectron microscopy revealed that the liposome vesicles were unilamellar and were in the 30 - 130 nm size range, while band shift assays confirmed the formation of complexes between the siRNA and the liposome preparations. These siRNA lipoplexes were shown to afford protection to their siRNA cargoes against serum nuclease degradation and were also shown to be relatively non-toxic to the HeLa tat luc cell line which stably expresses the firefly luciferase gene. Cryoelectron microscopy revealed that an inverse relationship exists between the lipoplex size and the degree of pegylation. To determine the transfection efficiency of the cationic liposome preparations in the HeLa tat luc cell line, complexes were prepared with anti-luciferase siRNA, which is specific for the firefly luciferase gene, and knockdown of the luciferase gene was monitored in transfected cells. The results show that liposomes containing the cytofectin Chol-T were particularly effective, achieving up to 93.4% gene knockdown at the 30 nM siRNA concentration. The non- pegylated and pegylated cationic liposomes that have been formulated and examined in this study therefore warrant further development to facilitate in vivo studies.
Functionalised gold nanoparticle delivery for c-MYC siRNA in cancer gene therapy.
(2018) Daniels, Aliscia Nicole.; Singh, Moganavelli.; Singh, Sooboo.
Abstract available in PDF file.
Galactosylated liposomes with proton sponge capacity : a novel hepatocyte-specific gene transfer system.
(2012) Habib, Saffiya.; Singh, Moganavelli.; Ariatti, Mario.
Hepatocyte-directed liposomal gene delivery systems have received much attention in view of the present lack of suitable treatment alternatives for several liver-associated disorders. While targeting of liposomes to the asialoglycoprotein receptor (ASGP-R), nearly-exclusive to hepatocytes, is a well-documented means of achieving cell-specificity, several intra- and extracellular barriers reduce the efficacy of liposomal gene transfer. These include the aggregation and opsonisation of lipoplexes by serum components; and endo/lysosomal degradation of internalised DNA. This study has attempted to address the individual concerns by modifying hepatotropic liposomes with a steric stabilising, polyethylene glycol (PEG) shroud, and an endosomal escape-inducing proton sponge moiety. Novel galactosylated (SH02) and imidazolylated (SH04) cholesterol derivatives were successfully synthesised with the aim of conferring the respective functions of ASGP-R-specificity and proton sponge capability upon cationic liposome formulations. The individual derivatives afforded stable, unilamellar vesicles (< 200 nm, Z-average diameter) when incorporated at 10 % on a molar basis with the cytofectin, 3β[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) and co-lipid, dioleoylphosphatidylethanolamine (DOPE). Modification of these formulations with 1,2-distearoyl-sn-glycero-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000] (DSPEPEG₂₀₀₀), at 5 mol %, gave smaller vesicles (< 110 nm, Z-average diameter) and moderately reduced the instability associated with the combination of both SH02 and SH04 in a single formulation. Individual preparations formed electrostatic complexes with pCMV-luc plasmid DNA, as demonstrated by gel retardation assays and electron microscopy. Furthermore, the liposomes afforded some protection to the DNA cargo against serum nuclease attack during a 4 hour-long exposure to foetal calf serum at 37 °C. However, the DNA-binding and protecting capabilities of the liposomes were reduced upon addition of the PEG coating. Growth inhibition assays showed that lipoplexes derived from individual formulations were well tolerated by human hepatocyte-derived, HepG2, and embryonic kidney, HEK293, cell lines. Expression of the luciferase transgene mediated by non-pegylated formulations containing SH02 was significantly higher in hepatocytes than in the ASGP-R-negative, kidney cells. Furthermore, receptor-mediated internalisation of non-pegylated, galactosylated carriers by hepatocytes was demonstrated by the gross inhibition of transfection in the presence of excess asialofetuin, a natural ligand to the ASGP-R. Liposome acid titration profiles highlighted the endosomal pH-buffering capacity afforded by SH04. However, the imidazolylated lipid enhanced the transfection activity of the non-sterically stabilised Chol-T/DOPE system, but not that of its targeted counterpart, and only with respect to HEK293 cells. Finally, pegylation reduced the transfection capability of liposomes by at least three orders of magnitude in both cell lines. The results suggest that further optimisation of liposome composition is necessary in order to achieve a liposomal system that simultaneously embodies hepatocyte-targeting, proton sponge and long-circulating properties.
An in vitro assessment of functionalized gold nanoparticles in anticancer drug delivery.
(2017) David, Lorenzo Lance.; Singh, Moganavelli.
Abstract available in PDF file.
Layered double hydroxides : synthesis and application in gene delivery to mammalian cells in culture.
(2010) Balcomb, Blake.; Singh, S.; Singh, Moganavelli.
Layered double hydroxides (LDHs) or hydrotalcite-like compounds (HTLcs) are classified as anionic clays in which their structure is based upon brucite and are represented by the following general chemical formula: [MII1-xMIII x(OH)2]x+(An-)x/y.yH2O where MII and MIII represent various possible divalent cations, e.g., Mg2+, Zn2+, Ni2+, Co2+ and Fe2+ and trivalent cations, e.g., Al3+, Fe3+ and Cr3+ respectively. The value x is equal to the stoichiometric ratio of MIII/(MII+MIII) and An- represents exchangeable anions such as CO32-, Cl- and SO42-. It is these exchangeable interlayer anions, which make layered double hydroxide compounds excellent carriers of negatively charged or anionic containing biomolecules, such as DNA and hence can be exploited in their use in gene therapy. In this study, a variety of Mg-Al hydrotalcites (HTs), Zn-Al, Zn-Fe and Mg-Fe LDHs were synthesized using co-precipitation. The synthesized compounds were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, Inductively coupled plasma-optical emission spectroscopy (ICP-OES), Transmission electron microscopy (TEM), Scanning electron microscopy (SEM) and Scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX). XRD patterns for the synthesized HTs and LDHs exhibited characteristic features indicative of an ordered layered material. Elemental analysis of the compounds revealed a measured value of x in the range of 0.25-0.33 for Mg-Al HTs and Zn-Fe LDHs, 0.11-0.16 for Zn-Al LDHs and 0.55-0.58 for Mg-Fe LDHs. FTIR and Raman spectroscopy confirmed the presence of characteristic functional groups and interlayer anions. From electron microscopy, the compounds exhibited classical morphologies typical of HT and LDH compounds and had a lateral size range of 200-300 nm. These compounds were studied in their ability to bind DNA with the use of a gel retardation or band shift assay. This assay confirmed that these compounds are indeed able to bind DNA. The binding mechanism of DNA to the HT and LDH compounds was evaluated and plausible mechanisms were proposed. Furthermore, nuclease digestion assays were carried out in order to evaluate the potential protecting ability that these compounds afford towards the bound DNA in the presence of serum. It was observed that all compounds protected most of the bound DNA. The cytotoxicity of the compounds was evaluated in the HEK293, HepG2 and HeLa mammalian cell lines using the MTS (3-(4,5-dimethylthiazol-2yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salts) assay with a concentration range of 20-100 μg of respective HT/LDH compound. For most of the compounds, cell viability was observed in excess of 80 %. Finally, transfection studies were carried out utilizing green fluorescent protein (GFP) analysis and luciferase gene expression using the same mammalian cells in culture. It was noted that all HTs and LDHs were able to release DNA in a controlled prolonged manner over a period of three days. Green fluorescent protein gene expression commenced after 27 hours and reached a maximum at 72 hours. Efficient luciferase gene expression was observed with luciferase activities for DNA: HTs ranging from 0.05 x 106 - 2.0 x 106 RLU / mg protein and DNA: LDHs ranging from 0.05 x 106 - 16.7 x 106 RLU / mg protein. Highest luciferase activity was recorded as 16.7 x 106 RLU / mg protein.
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 cationic lipoplexes : characterization in cell culture in vitro and in vivo.
(2010) Sewbalas, Alisha.; Ariatti, Mario.; Singh, Moganavelli.; Arbuthnot, Patrick Brian.
Amongst the more promising non-viral DNA vehicles are liposomes, with those derived from cationic lipids showing significant potential, despite moderate transfection levels in vivo. This study has investigated the effect of liposome-anchored ionophore crown ethers on lipoplex-mediated gene transfer in vitro and in vivo. Several liposomes were constructed to include the cytofectin 3β[N(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T), the co-lipid dioleoylphosphatidylethanolamine (DOPE), and 5% (mole/mole) of the cholesteryl crown ethers RUI-128 (aza-18-crown-6) or RUI-129 (aza-15-crown-5). Liposome size and lamellarity were established by transmission electron microscopy. All liposome preparations were shown to bind, condense and protect DNA avidly in the respective band shift, ethidium displacement and nuclease protection assays. Lipoplex targeting to hepatocytes may be achieved via the asialoglycoprotein receptor (ASGP-R), which is abundantly expressed on the human hepatoblastoma cell line HepG2. Therefore six additional liposomes were formulated to include 5% (mole/mole) of the cholesteryl galactosyl RUI-90 (Gal) and cholesteryl glucosyl RUI-92 (Glu) ligands. Their hepatotropic gene delivery was examined in the HepG2 cell line using the pCMV-luc plasmid. Transfection studies in the human embryonic kidney cell line HEK293 (ASGP-R-negative) revealed an increase in transgene activity in lipoplexes displaying the RUI-129 cholesteryl derivative. No ionophore-mediated enhancement of transfection activity was observed in HepG2 cells although Chol-T:DOPE, Chol-T:DOPE:RUI-128 and Chol-T:DOPE:RUI-129 liposomes achieved very high transfection levels, exceeding those of their hepatocyte targeted counterparts. Liposome-anchored crown ethers have been shown to potentiate in vitro transfection activity of lipoplexes in the HEK293 cell line. The novel cholesteryl glycosyl derivatives were, however, unable to enhance the targeted entry of lipoplexes into HepG2 cells. The three most effective preparations from in vitro studies were taken forward for in vivo assessment in NMRI mice at the University of the Witwatersrand Molecular Medicine and Haematology unit. Three groups of mice were employed for the evaluation of Chol-T:DOPE, Chol-T:DOPE:RUI-129 and Chol-T:DOPE:RUI-129-Gal lipoplexes with the Psi-CHECK plasmid. Mice treated with hydrodynamic injection and untreated animals made up two control groups. Luciferase activity was determined on examination of the harvested liver homogenates. All liposomes showed modest, but significant transfection activity (p<0.05) and were well tolerated. The assemblies examined therefore warrant further development.
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.
A novel in vitro anticancer drug delivery strategy using platinum encapsulated gold nanoparticles.
(2017) Maney, Vareessh.; Singh, Moganavelli.
Abstract available in PDF file.
Novel siRNA lipoplexes : their targeted and untargeted delivery to mammalian cells in culture.
(2011) Dorasamy, Shantal.; Singh, Moganavelli.; Ariatti, Mario.
The high gene knockdown specificity and efficiency of RNA interference (RNAi) provides a potentially viable avenue for the development of a new class of nucleic acid therapeutics for gene-based disease conditions. However, serum instability, inefficient cellular trafficking and non-specific effects of small interfering RNAs (siRNAs), one of the functional mediators of RNAi, has necessitated the development of carriers to facilitate targeted cell delivery. The decline of viral vectors in human gene therapy as a consequence of safety issues has intensified the importance of non-viral vector development. Among the non-viral vectors available for siRNA delivery, cationic liposomes have emerged as an attractive option owing to their simplicity, versatility, relatively low toxicity and potential for cell-specific targeting. Although existing cationic lipids and liposomes traditionally used for DNA delivery have also been used for siRNAs, there still exists a need to develop cationic lipids tailored towards siRNA transfection for improved gene silencing efficiency. Among the cell specific targets available for RNAi therapeutics, hepatocytes expressing the asialoglycoprotein receptor (ASGP-R) are an ideal choice due to the large number of disease targets present for treatment. In this investigation four novel cationic liposome formulations were prepared from equi-molar quantities of either the cationic cholesterol derivative 3β [N-(N’,N’- dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or 3β [N-(N’, N’, N’- trimethylammoniumpropyl)-carbamoyl] cholesterol iodide (Chol-Q) and DOPE, with and without the hepatotropic ligand, cholesteryl-β-D-galactopyranoside. Electrophoretic gel analysis and SYBR®green displacement assays were employed to determine siRNA binding and condensation efficiencies for all cationic liposomes; while liposome and lipoplex size measurements were made by cryoTEM. SiRNAlipoplex stability in serum was determined by the nuclease protection assay. Cell studies performed on the ASGP-R+ human hepatoma cells, HepG2 and the ASGP-Rembryonic kidney cells, HEK293, to determine lipoplex toxicity and transfection efficiencies were also undertaken. We show that the cationic liposomes formulated for this investigation were able to bind synthetic siRNA optimally at a positive to negative charge ratio of ± 1 : 6. In addition, the cationic liposomes were able to afford siRNA duplexes substantial protection from ribonuclease digestion in serum. From the results obtained in this study, it appears that the cationic liposomes are well tolerated by both the HEK293 and HepG2 cells in vitro. More importantly, the results obtained demonstrated higher transfection efficiencies for the targeted lipoplexes compared with the untargeted controls, strongly supporting the notion that incorporation of the cholestryl-β-D-galactopyranoside into the liposome structure increases transfection efficiency to the targeted HepG2 cells in culture via ASGP receptor mediation. Comparative studies in the HEK293 cell line yielded low transfection effeciences in the order of 20%, with no significant difference being recorded between galactosylated and non-galactosylated lipoplexes.