Encapsulation of DNA in negatively charged liposomes and inhibition of bacterial gene expression with fluid liposome-encapsulated antisense oligonucleotides

Fillion, Patrick; Desjardins, Annie; Sayasith, Khampoune; Lagacé, Josée

doi:10.1016/s0005-2736(01)00392-3

Cited by 75 publications

(64 citation statements)

References 32 publications

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“…Thus, this strategy may raise the bar for infecting strains to develop resistance, potentially reducing future burdens of antimicrobial-resistant P. aeruginosa. Delivery of antisense oligonucleotides (AS-ODNs) via anionic liposomes has been successful in several bacterial species (72)(73)(74) and may provide a novel means for targeting sRNAs, such as PrrF and PrrH, to block virulence. Future work determining how each of the ⌬prrF1,2 mutant phenotypes contributes to P. aeruginosa virulence attenuation will be critical for the development of AS-ODN molecules that effectively block pathogenesis.…”

Section: Discussionmentioning

confidence: 99%

The prrF -Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

et al. 2015

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Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part of P. aeruginosa's iron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem in P. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ⌬prrF1,2 mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identified phuS, encoding a heme binding protein involved in heme acquisition, and vreR, encoding a previously identified regulator of P. aeruginosa virulence genes, as novel targets of prrF-mediated heme regulation. Finally, we showed that the prrF locus encoding the PrrF and PrrH sRNAs is required for P. aeruginosa virulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ⌬prrF1,2 deletion mutant protects against future challenge with wild-type P. aeruginosa. Combined, these data demonstrate that the prrF-encoded sRNAs are critical regulators of P. aeruginosa virulence. Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium and versatile opportunistic pathogen. Iron is required for P. aeruginosa virulence (1-6) and is obtained through several mechanisms. In anaerobic environments, iron in its ferrous form is freely diffusible through the outer membrane (OM) and transported into the cytoplasm by the Feo inner membrane transport system (7,8). However, the insolubility of ferric iron in aerobic environments limits accessibility to this nutrient. Moreover, the sequestration of iron by host proteins creates a substantial barrier to infection (9, 10). To overcome this barrier, P. aeruginosa synthesizes and secretes two siderophores, pyoverdine and pyochelin, which scavenge ferric iron (1-4). P. aeruginosa can also acquire heme, an abundant source of iron in the human host (11). Once internalized, heme is sequestered by the cytosolic PhuS heme chaperone (12). PhuS transfers heme to the iron-regulated HemO heme oxygenase, which degrades heme to biliverdin, releasing carbon monoxide and iron (13,14). Several studies have shown that iron acquisition is essential for P. aeruginosa virulence (1-5) and biofilm formation (15-17), demonstrating the central role of this element in P. aeruginosa pathogenesis.Despite its essentiality, iron and heme can be toxic due to their ability to catalyze the formation of reactive oxygen species. Thus, to maintain iron homeostasis, P. aeruginosa must be able to not only acquire iron but also regulate uptake of iron and heme from the environment, as well as iron use and storage. Expression of genes encoding iron and heme uptake systems in P. aeruginosa is ...

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Section: Discussionmentioning

confidence: 99%

The prrF -Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

et al. 2015

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“…Anionic lipids (ALs) occur naturally in eukaryotic cell membranes, and DNA delivery systems based on ALs have recently been examined as an alternative to CLs because of their low cytotoxicity (11)(12)(13). ALs can be complexed with anionic DNA via interaction with multivalent cations such as Ca 2ϩ and have been shown to successfully transfer oligonucleotides.…”

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confidence: 99%

Polymorphism of DNA–anionic liposome complexes reveals hierarchy of ion-mediated interactions

Liang

Harries

Wong

2005

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

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Self-assembled DNA delivery systems based on anionic lipids (ALs) complexed with DNA mediated by divalent cations have been recently introduced as an alternative to cationic lipid-DNA complexes because of their low cytotoxicity. We investigate AL-DNA complexes induced by different cations by using synchrotron small angle x-ray scattering and confocal microscopy to show how different ion-mediated interactions are expressed in the selfassembled structures and phase behavior of AL-DNA complexes. The governing interactions in AL-DNA systems are complex: divalent ions can mediate strong attractions between different combinations of the components (such as DNA-DNA and membrane-membrane). Moreover, divalent cations can coordinate nonelectrostatically with lipids and modify the resultant membrane structure. We find that at low membrane charge densities AL-DNA complexes organize into a lamellar structure of alternating DNA and membrane layers crosslinked by ions. At high membrane charge densities, a new phase with no analog in cationic lipid-DNA systems is observed: DNA is expelled from the complex, and a lamellar stack of membranes and intercalated ions is formed. For a subset of the ionic species, high ion concentrations generate an inverted hexagonal phase comprised of DNA strands wrapped by ion-coated lipid tubes. A simple theoretical model that takes into account the electrostatic and membrane elastic contributions to the free energy shows that this transition is consistent with an ion-induced change in the membrane spontaneous curvature, c 0. Moreover, the crossover between the lamellar and inverted hexagonal phases occurs at a critical c0 that agrees well with experimental values.colloids ͉ like-charge attraction ͉ membrane ͉ self-assembly ͉ x-ray G ene therapy using either viral or synthetic vectors is currently one of the most promising strategies for developing cures for many hereditary and acquired diseases. Protocols have been approved for cancer, hemophilia, cystic fibrosis, neuromuscular disorders, and others (1). Although synthetic nonviral systems such as cationic liposomes generally transfect less efficiently than viruses, they have a number of advantages, such as high DNA packaging capacity and low immunogenicity. Cationic lipid (CL)-DNA complexes have emerged as one of the major nonviral DNA delivery platforms (1-7) and have been used to transfect a broad range of cell types and to deliver to cancer vaccines (8-10).Anionic lipids (ALs) occur naturally in eukaryotic cell membranes, and DNA delivery systems based on ALs have recently been examined as an alternative to CLs because of their low cytotoxicity (11-13). ALs can be complexed with anionic DNA via interaction with multivalent cations such as Ca 2ϩ and have been shown to successfully transfer oligonucleotides. An outstanding problem of this approach is the inefficient association between the ALs and DNA molecules, which is attributed to their like-charge electrostatic repulsion.Rational design of AL-DNA vectors requires a coherent understanding of...

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“…As a result, levels of encapsulation of 48.9% and 43.5% were found in the purified plasmid and oligonucleotide "antisense," respectively. 20 Other studies showed that, in dioctadecyldimethylammonium bromide bilayers, the preference of the monomeric amphotericin B was for the gel phase to be incorporated by 40%, compared to 23% in the fluid phase. 21 As in the DODAC/PHO-S liposomal formulation, the concentrations of PHO-S are not greater than the DODAC concentration.…”

Section: Discussionmentioning

confidence: 99%

“…In another liposomal formulation, researchers proposed the use of a negatively charged liposomal fluid to encapsulate an oligonucleotide "antisense" (anti-β-galactosidase), as well as the plasmid "pUC18." 20 In the study, the liposomes were formulated with synthetic phospholipids dipalmitoylphosphatidylcholine, and dimyristoyl phosphatidylglycerol in a molar ratio of 10:1, in the presence of a solution of a monovalent salt (NaCl, KCl, NaHCO 3 , or phosphate buffer), and the rate of encapsulation and activity "antisense" were measured by Escherichia coli CSH36. As a result, levels of encapsulation of 48.9% and 43.5% were found in the purified plasmid and oligonucleotide "antisense," respectively.…”

Section: Discussionmentioning

confidence: 99%

Potential antitumor activity of novel DODAC/PHO-S liposomes

Luna

Saraiva²,

Filho³

et al. 2016

IJN

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In recent studies, we showed that synthetic phosphoethanolamine (PHO-S) has a great potential for inducing cell death in several tumor cell lines without damage to normal cells. However, its cytotoxic effect and selectivity against tumor cells could increase with encapsulation in cationic liposomes, such as dioctadecyldimethylammonium chloride (DODAC), due to electrostatic interactions between these liposomes and tumor cell membranes. Our aim was to use cationic liposomes to deliver PHO-S and to furthermore maximize the therapeutic effect of this compound. DODAC liposomes containing PHO-S (DODAC/PHO-S), at concentrations of 0.3–2.0 mM, prepared by ultrasonication, were analyzed by scanning electron microscopy (SEM) and dynamic light scattering. The cytotoxic effect of DODAC/PHO-S on B16F10 cells, Hepa1c1c7 cells, and human umbilical vein endothelial cells (HUVECs) was assessed by MTT assay. Cell cycle phases of B16F10 cells were analyzed by flow cytometry and the morphological changes by SEM, after treatment. The liposomes were spherical and polydisperse in solution. The liposomes were stable, presenting an average of ∼50% of PHO-S encapsulation, with a small reduction after 40 days. DODAC demonstrated efficient PHO-S delivery, with the lowest values of IC 50% (concentration that inhibits 50% of the growth of cells) for tumor cells, compared with PHO-S alone, with an IC 50% value of 0.8 mM for B16F10 cells and 0.2 mM for Hepa1c1c7 cells, and without significant effects on endothelial cells. The Hepa1c1c7 cells showed greater sensitivity to the DODAC/PHO-S formulation when compared to B16F10 cells and HUVECs. The use of DODAC/PHO-S on B16F10 cells induced G 2 /M-phase cell cycle arrest, with the proportion significantly greater than that treated with PHO-S alone. The morphological analysis of B16F10 cells by SEM showed changes such as “bleb” formation, cell detachment, cytoplasmic retraction, and apoptotic bodies after DODAC/PHO-S treatment. Cationic liposomal formulation for PHO-S delivery promoted cytotoxicity more selectively and effectively against B16F10 and Hepa1c1c7 cells. Thus, the DODAC/PHO-S liposomal formulation presents great potential for preclinical studies.

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Encapsulation of DNA in negatively charged liposomes and inhibition of bacterial gene expression with fluid liposome-encapsulated antisense oligonucleotides

Cited by 75 publications

References 32 publications

The prrF -Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

The prrF -Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

Polymorphism of DNA–anionic liposome complexes reveals hierarchy of ion-mediated interactions

Potential antitumor activity of novel DODAC/PHO-S liposomes

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