Dioleoylmelittin as a Novel Serum-Insensitive Reagent for Efficient Transfection of Mammalian Cells

Jy, Legendre; Trzeciak, Arnold; Bohrmann, Bernd; Deuschle, Ulrich; Kitas, Eric; Supersaxo, A.

doi:10.1021/bc960076d

Cited by 67 publications

(64 citation statements)

References 31 publications

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“…The reported DNA transfer activities of polylysine and other cationic peptides suggest that DNA transfer can be achieved with relatively short oligomers (7)(8)(9)(10). Little effort has been made to systematically optimize the structures of these compounds.…”

Section: Resultsmentioning

confidence: 99%

“…A wide variety of cationic polymers have been shown to mediate in vitro transfection, ranging from proteins [such as histones (4) and high mobility group (HMG) proteins (5)] and polypeptides [such as polylysine (3,6), short synthetic peptides (7,8), and helical amphiphilic peptides (9,10)] to synthetic polymers [such as polyethyleneimine (11), cationic dendrimers (12,13), and glucaramide polymers (14)]. Although the efficiencies of gene transfer vary with these systems, a large variety of cationic structures are effective.…”

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confidence: 99%

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A combinatorial approach to the discovery of efficient cationic peptoid reagents for gene delivery

Murphy

Uno²,

Hamer³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

215

185

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A family of N-substituted glycine oligomers (peptoids) of defined length and sequence are shown to condense plasmid DNA into small particles, protect it from nuclease degradation, and efficiently mediate the transfection of several cell lines. The oligomers were discovered by screening a combinatorial library of cationic peptoids that varied in length, density of charge, side-chain shape, and hydrophobicity. Transfection activity and peptoid-DNA complex formation are shown to be highly dependent on the peptoid structure. The most active peptoid is a 36-mer that contains 12 cationic aminoethyl side chains. This molecule can be synthesized efficiently from readily available building blocks. The peptoid condenses plasmid DNA into uniform particles 50-100 nm in diameter and mediates the transfection of a number of cell lines with efficiencies greater than or comparable to DMRIE-C, Lipofectin, and Lipofectamine. Unlike many cationic lipids, peptoids are capable of working in the presence of serum.Viral and nonviral gene transfer systems have been under intense investigation, as interest in the potential of gene therapy for the treatment and prevention of disease is greater than ever (1). Nonviral systems potentially offer many advantages over viral systems, such as ease of manufacture, safety, stability, lack of vector size limitations, low immunogenicity, and the modular attachment of targeting ligands (2). Most nonviral gene delivery systems are based on cationic compounds-either cationic lipids (2) or cationic polymers (3)-that spontaneously complex with a plasmid DNA vector by means of electrostatic interactions, yielding a condensed form of DNA that shows increased stability toward nucleases. Although cationic lipids have been quite successful at delivering genes in vitro, the success of these compounds in vivo has been modest, often because of their high toxicity and low transduction efficiency.A wide variety of cationic polymers have been shown to mediate in vitro transfection, ranging from proteins [such as histones (4) and high mobility group (HMG) proteins (5)] and polypeptides [such as polylysine (3, 6), short synthetic peptides (7, 8), and helical amphiphilic peptides (9, 10)] to synthetic polymers [such as polyethyleneimine (11), cationic dendrimers (12, 13), and glucaramide polymers (14)]. Although the efficiencies of gene transfer vary with these systems, a large variety of cationic structures are effective. Unfortunately, it has been difficult to study systematically the effect of polycation structure on transfection activity.To understand more fully the structure-activity relationship of these cationic polymer delivery systems, we examined a set of cationic N-substituted glycine oligomers (NSG peptoids) of defined length and sequence. Peptoids are a large family of synthetic oligomers that were originally developed for the combinatorial synthesis of compound libraries for drug discovery (15). Recent optimization of the solid-phase coupling chemistry has allowed the synthesis of long oligomers...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

A combinatorial approach to the discovery of efficient cationic peptoid reagents for gene delivery

Murphy

Uno²,

Hamer³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

215

185

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“…One approach to improve the interaction of melittin with DNA has been to incorporate both molecules into cationic liposomes, by coupling melittin via an N-terminal cysteine to a neutral lipid moiety (29). The hybrid molecule dioleyl-melittin binds DNA via the cationic peptide, whereas the lipid component enhances hydrophobic collapse of the DNA.…”

Section: Influence Of Cell Density and Cycling Status On The Efficienmentioning

confidence: 99%

Melittin Enables Efficient Vesicular Escape and Enhanced Nuclear Access of Nonviral Gene Delivery Vectors

Ogris

Carlisle

Bettinger³

et al. 2001

Journal of Biological Chemistry

211

152

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Entry of exogenously applied DNA into the cytoplasm and subsequent transport into the nucleus are major cellular barriers for nonviral gene delivery vectors. To overcome these barriers, we have covalently attached the cationic peptide melittin to poly(ethylenimine) (PEI). This conjugate condensed DNA into small, discrete particles (<100 nm in diameter), and the membrane lytic activity of melittin enabled efficient release of the DNA into the cytoplasm, as monitored by fluorescence microscopy and flow cytometry. Compared with PEI, the transfection activity was strongly increased within a broad range of cell lines and types tested, including different tumor cell lines but also primary hepatocytes and human umbilical vein endothelial cells. The early onset of gene expression (within 4 h, reaching maximal values after 12 h) and the high reporter gene expression achieved in slowly dividing or confluent cells suggested a further role of melittin after releasing the DNA into the cytoplasm. Intracytoplasmic microinjection of melittin-containing PEI⅐DNA complexes into fibroblasts produced 40% cellular frequency of reporter gene expression that was inhibitable by co-injection of wheat germ agglutinin, whereas simple PEI⅐DNA complexes showed only 10%. These data suggest that melittin enables release of nonviral gene transfer particles into the cytoplasm and also enhances their transport into the nucleus, possibly via the cationic cluster KRKR near the C terminus of the peptide.Nonviral gene delivery systems generally exhibit a superior safety profile compared with viruses, and their use raises fewer ethical concerns; however, their relatively low efficiency of transgene expression is a major drawback. Poor release of the vectors from the endosome into the cytoplasm (1) and, subsequently, inefficient transfer into the nucleus (2) are both thought to contribute to their low transgene expression efficiency. The importance of endosomal escape can be shown using lysosomotropic drugs, such as chloroquine (3), or by incorporating membrane active peptides into the gene transfer complexes (4), which can lead to increases of Ͼ1000-fold in transfection. Similarly, inefficient nuclear transport was demonstrated when Pollard et al. (5) showed by microinjection that only 0.1-1% of free plasmids microinjected into the cytoplasm were transferred into the nucleus. The effects of the cell cycle on the efficiency of transfection using polyelectrolyte complexes was shown by Brunner et al. (6), compatible with the intact nuclear membrane restricting entry of DNA into the nucleus. Inefficient endosomal escape and poor nuclear transfer can both be addressed by incorporation of bioactive peptides into polyelectrolyte vectors. Nuclear localization sequences (NLS) 1 have been coupled either directly to the DNA (7-9) or to a DNA condensing lipid moiety (10). NLS peptides are mainly cationic, and they are easily incorporated into discrete polycation-DNA complexes. In contrast, amphipathic peptides described for pHtriggered lysis of the endosome memb...

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“…Zeta potential measurements show that adding increasing amounts of polycation to a fixed amount of DNA changes the surface charge of polyplexes from negative, through electroneutral, to positive (24,25). The electropositive conjugates nonspecifically bind to the negatively charged cell surface, likely due to interactions with proteoglycans (17).…”

Section: Figmentioning

confidence: 99%

Optimization of Cell Surface Binding Enhances Efficiency and Specificity of Molecular Conjugate Gene Delivery

Schaffer

Lauffenburger

1998

Journal of Biological Chemistry

148

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Molecular conjugates, or polyplexes, are promising synthetic vectors for targeted, in vivo gene delivery, if their efficiency can be improved. Gaining mechanistic information on conjugate gene delivery can potentially yield significant improvements in transfer efficiency by revealing barriers to conjugate transfer from the cell surface to the nucleus. We have developed an experimental system that employs epidermal growth factor as the ligand to direct delivery of DNA encoding the green fluorescent protein to mouse fibroblasts. We report here that the initial step of delivery, binding of the conjugate to the cell surface, is a barrier to gene transfer. We examined the effects of conjugate charge, ligand crosslinker spacer length, and ligand valency on polyplex cell surface binding, internalization, and gene transfer. We find that delivery is both efficient and specific only within a relatively narrow window of conjugate charge, results that correlate with binding and internalization of radiolabeled conjugate. In addition, increasing the cross-linker length can improve binding affinity and delivery. Finally, there is a significant optimum in gene delivery as a function of ligand valency, due to saturation of receptor binding and internalization. Optimizing parameters that affect surface binding therefore improves the efficiency and specificity of molecular conjugate gene delivery.

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Dioleoylmelittin as a Novel Serum-Insensitive Reagent for Efficient Transfection of Mammalian Cells

Cited by 67 publications

References 31 publications

A combinatorial approach to the discovery of efficient cationic peptoid reagents for gene delivery

A combinatorial approach to the discovery of efficient cationic peptoid reagents for gene delivery

Melittin Enables Efficient Vesicular Escape and Enhanced Nuclear Access of Nonviral Gene Delivery Vectors

Optimization of Cell Surface Binding Enhances Efficiency and Specificity of Molecular Conjugate Gene Delivery

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