Mustafa Munye - Academia.edu (original) (raw)

Papers by Mustafa Munye

Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationi... more Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes. S ynthetic, non-viral vectors offer advantages over viral vectors for in vivo gene therapy in that they are less immunogenic, have fewer packaging constraints and are safer 1,2. Cationic lipoplexes and polyplexes predominate in the non-viral vector field but increasingly lipopolyplex formulations, which are combinations of lipids with peptides or polymers, are being explored as appreciation develops of their wider range of function-alities and higher transfection efficiencies 3–11. Further detailed functional and structural studies are required to understand the properties of lipopolyplexes, how to formulate components and to develop improved formulations. We are developing a lipopolyplex formulation termed a Receptor Targeted Nanocomplex (RTN), which is a mixture of cationic, receptor-targeting peptides and cationic liposomes with plasmid DNA (pDNA) 12–17. The lipid and peptide components of RTNs feature modular design elements that enable their functionality to be dissected at the molecular level 13. We have shown previously that the peptide mediates DNA packaging and receptor targeting and so the focus of this study was to investigate the function of the lipids and how they contribute to the transfection efficiency of RTN lipopolyplexes. Endosomolysis is a major obstacle to transfection with peptide-DNA formulations and so we hypothesised that addition of the liposome to the peptide might enhance transfec-tion by promoting fusion with the endosomal membrane, leading to improved cytoplasmic release of the DNA 13,18. The neutral lipid DOPE in cationic lipoplex formulations enables higher transfection efficiencies as the conical structure of this lipid promotes the formation of inverted hexagonal structures that rapidly fuse with the endosomal lipid bilayer, independent of charge, thus enabling cytoplasmic release of the DNA. Substitution of DOPE for DOPC, a neutral lipid that favour more stable lamellar structures, greatly reduces lipoplex transfection efficiency 18–22. In this study, we aimed to explore whether the lipid components of the RTN lipopolyplex played a similar role in endosomal membrane fusion to that of lipoplexes and whether this contributed to their improved transfection efficiency. RTN formulations were therefore formulated with peptides mixed with cationic liposomes at the same charge density, containing either the fusogenic neutral lipid DOPE, or the non-fusogenic, neutral lipid DOPC. We hypothesised that if charge density of the liposome component was the more important factor then substituting the neutral lipid component would not affect transfection efficiency whereas if fusogenic properties were more

Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationi... more Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes. S ynthetic, non-viral vectors offer advantages over viral vectors for in vivo gene therapy in that they are less immunogenic, have fewer packaging constraints and are safer 1,2. Cationic lipoplexes and polyplexes predominate in the non-viral vector field but increasingly lipopolyplex formulations, which are combinations of lipids with peptides or polymers, are being explored as appreciation develops of their wider range of function-alities and higher transfection efficiencies 3–11. Further detailed functional and structural studies are required to understand the properties of lipopolyplexes, how to formulate components and to develop improved formulations. We are developing a lipopolyplex formulation termed a Receptor Targeted Nanocomplex (RTN), which is a mixture of cationic, receptor-targeting peptides and cationic liposomes with plasmid DNA (pDNA) 12–17. The lipid and peptide components of RTNs feature modular design elements that enable their functionality to be dissected at the molecular level 13. We have shown previously that the peptide mediates DNA packaging and receptor targeting and so the focus of this study was to investigate the function of the lipids and how they contribute to the transfection efficiency of RTN lipopolyplexes. Endosomolysis is a major obstacle to transfection with peptide-DNA formulations and so we hypothesised that addition of the liposome to the peptide might enhance transfec-tion by promoting fusion with the endosomal membrane, leading to improved cytoplasmic release of the DNA 13,18. The neutral lipid DOPE in cationic lipoplex formulations enables higher transfection efficiencies as the conical structure of this lipid promotes the formation of inverted hexagonal structures that rapidly fuse with the endosomal lipid bilayer, independent of charge, thus enabling cytoplasmic release of the DNA. Substitution of DOPE for DOPC, a neutral lipid that favour more stable lamellar structures, greatly reduces lipoplex transfection efficiency 18–22. In this study, we aimed to explore whether the lipid components of the RTN lipopolyplex played a similar role in endosomal membrane fusion to that of lipoplexes and whether this contributed to their improved transfection efficiency. RTN formulations were therefore formulated with peptides mixed with cationic liposomes at the same charge density, containing either the fusogenic neutral lipid DOPE, or the non-fusogenic, neutral lipid DOPC. We hypothesised that if charge density of the liposome component was the more important factor then substituting the neutral lipid component would not affect transfection efficiency whereas if fusogenic properties were more