1-2-dioleoyloxy-3-(trimethylammonium)propane and dimethyldioctadecylammonium

Self-amplifying RNA (saRNA) is a promising biotherapeutic tool that has been used as a vaccine against both infectious diseases and cancer. saRNA has been shown to induce protein expression for up to 60 days and elicit immune responses with lower dosing than messenger RNA (mRNA). Because saRNA is a large (~9500 nt), negatively charged molecule, it requires a delivery vehicle for efficient cellular uptake and degradation protection. Lipid nanoparticles (LNPs) have been widely used for RNA formulations, where the prevailing paradigm is to encapsulate RNA within the particle, including the first FDA-approved small-interfering siRNA therapy. Here, we compared LNP formulations with cationic and ionizable lipids with saRNA either on the interior or exterior of the particle. We show that LNPs formulated with cationic lipids protect saRNA from RNAse degradation, even when it is adsorbed to the surface. Furthermore, cationic LNPs deliver saRNA equivalently to particles formulated with saRNA encapsulated in an ionizable lipid particle, both in vitro and in vivo. Finally, we show that cationic and ionizable LNP formulations induce equivalent antibodies against HIV-1 Env gp140 as a model antigen. These studies establish formulating saRNA on the surface of cationic LNPs as an alternative to the paradigm of encapsulating RNA.

Topics: Animals; Cations; env Gene Products, Human Immunodeficiency Virus; Fatty Acids, Monounsaturated; Female; HEK293 Cells; HIV Antibodies; Humans; Lipids; Luciferases, Firefly; Mice; Mice, Inbred BALB C; Nanoparticles; Particle Size; Quaternary Ammonium Compounds; Ribonucleases; RNA Stability; RNA, Messenger; Transfection

The immunostimulatory capacities of cationic liposomes are well-documented and are attributed both to inherent immunogenicity of the cationic lipid and more physical capacities such as the formation of antigen depots and antigen delivery. Very few studies have however been conducted comparing the immunostimulatory capacities of different cationic lipids. In the present study we therefore chose to investigate three of the most well-known cationic liposome-forming lipids as potential adjuvants for protein subunit vaccines. The ability of 3β-[N-(N',N'-dimethylaminoethane)carbomyl] cholesterol (DC-Chol), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and dimethyldioctadecylammonium (DDA) liposomes incorporating immunomodulating trehalose dibehenate (TDB) to form an antigen depot at the site of injection (SOI) and to induce immunological recall responses against coadministered tuberculosis vaccine antigen Ag85B-ESAT-6 are reported. Furthermore, physical characterization of the liposomes is presented. Our results suggest that liposome composition plays an important role in vaccine retention at the SOI and the ability to enable the immune system to induce a vaccine specific recall response. While all three cationic liposomes facilitated increased antigen presentation by antigen presenting cells, the monocyte infiltration to the SOI and the production of IFN-γ upon antigen recall was markedly higher for DDA and DC-Chol based liposomes which exhibited a longer retention profile at the SOI. A long-term retention and slow release of liposome and vaccine antigen from the injection site hence appears to favor a stronger Th1 immune response.

Topics: Adjuvants, Immunologic; Animals; Antigen Presentation; Cholesterol; Fatty Acids, Monounsaturated; Female; Liposomes; Mice; Mice, Inbred C57BL; Quaternary Ammonium Compounds; Vaccines

Despite considerable interest and investigations on cationic lipid-DNA complexes, reports on lipid-RNA interaction are very limited. In contrast to lipid-DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid-tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid-tRNA interactions with G-C and A-U base pairs as well as the backbone phosphate group with overall binding constants of K(Chol) = 5.94 (+/- 0.8) x 10(4) M(-1), K(DDAB) = 8.33 (+/- 0.90) x 10(5) M(-1), K(DOTAP) = 1.05 (+/- 0.30) x 10(5) M(-1) and K(DOPE) = 2.75 (+/- 0.50) x 10(4) M(-1). The order of stability of lipid-tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations.

Topics: Cations; Cholesterol; Circular Dichroism; Fatty Acids, Monounsaturated; Hydrophobic and Hydrophilic Interactions; Lipids; Microscopy, Atomic Force; Nucleic Acid Conformation; Phosphates; Phosphatidylethanolamines; Quaternary Ammonium Compounds; RNA, Transfer; Spectroscopy, Fourier Transform Infrared

The cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dimethyldioctadecylammonium bromide, with or without the helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or cholesterol, and the cationic polymer polyethyleneimine, were compared for their ability to displace fluorescent dyes from DNA. Differences in displacement of the intercalating dyes ethidium bromide and ethidium homodimer correlate with their relative affinities with DNA, with the extent of ethidium homodimer displacement significantly less. Differences in ethidium homodimer and ethidium bromide displacement as a function of the ratio of polycation to DNA and the charge density of the polycation suggest a greater sensitivity of the former to topological changes in condensed DNA. Marked differences in the ability of these cationic delivery systems to displace the minor groove binding dyes 4',6-diamidino-2-phenylindole and Hoechst 33258 upon interaction with DNA are also apparent, with the majority of Hoechst 33258 remaining bound to DNA. Changes in the spectral properties of Hoechst 33258 were further used to characterize polycation-induced changes in solvent accessibility of the DNA minor groove. Taken together, these studies demonstrate differences in the interaction of various cationic lipids and polyethyleneimine in terms of regional displacement of dyes, polycation-induced structural changes in DNA, as well as polycation-mediated changes in solvent accessibility of the minor groove. The relevance of these studies to current models of the structure and assembly of polycation/DNA complexes are discussed.

Topics: Bisbenzimidazole; Cations; DNA, Superhelical; Drug Carriers; Ethidium; Fatty Acids; Fatty Acids, Monounsaturated; Fluorescence; Fluorescent Dyes; Gene Transfer Techniques; Indoles; Intercalating Agents; Osmolar Concentration; Plasmids; Quaternary Ammonium Compounds; Surface-Active Agents

The effect of electrostatic interactions on the conformation and thermal stability of plastocyanin (Pc) was studied by infrared spectroscopy. Association of any of the two redox states of the protein with positively charged membranes at neutral pH does not significantly change the secondary structure of Pc. However, upon membrane binding, the denaturation temperature decreases, regardless of the protein redox state. The extent of destabilization depends on the proportion of positively charged lipid headgroups in the membrane, becoming greater as the surface density of basic phospholipids increases. In contrast, at pH 4.8 the membrane binding-dependent conformational change becomes redox-sensitive. While the secondary structures and thermal stabilities of free and membrane-bound oxidized Pc are similar under acidic conditions, the conformation of the reduced form of the protein drastically rearranges upon membrane association. This rearrangement does not depend on electrostatic interactions to occur, since it is also observed in the presence of uncharged lipid bilayers. The conformational transition, only observed for reduced Pc, involves the exposure of hydrophobic regions that leads to intermolecular interactions at the membrane surface. Membrane-mediated partial unfolding of reduced Pc can be reversed by readjusting the pH to neutrality, in the absence of electrostatic interactions. This redox-dependent behavior might reflect specific structural requirements for the interaction of Pc with its redox partners.

Topics: Drug Stability; Fatty Acids, Monounsaturated; Fluorescent Dyes; Hot Temperature; Hydrogen-Ion Concentration; Lipid Bilayers; Oxidation-Reduction; Phosphatidylcholines; Plant Leaves; Plastocyanin; Protein Conformation; Quaternary Ammonium Compounds; Spectrophotometry, Infrared; Static Electricity; Thermodynamics; Trees

A model of lipoplex-induced peritonitis was used to characterize the inflammatory response to cationic lipid:DNA lipoplexes with respect to activation of host antitumoral effector mechanisms. Three different cationic lipids were used in these studies: N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(1-[2,3-dioleoyloxylpropyl)-N,N,N-trimethylammonium chloride (DOTAP), and N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA). The DODAC and DOTMA lipoplexes exhibited similar transfection properties in vitro, whereas the DOTAP lipoplexes transfected quite poorly in all cell lines tested. Intraperitoneal injection of cationic lipoplexes into immunocompetent mice resulted in a profound infiltration of inflammatory cells, secretion of interferon-gamma, and increased natural killer activity within the peritoneal cavity. Both DODAC and DOTMA lipoplexes produced similar inflammatory responses, lasting at least 5 days. The inflammation induced by DOTAP lipoplexes peaked by day 3 and resolved to near-control levels by day 5. These data indicate that although cationic lipid DNA complexes may differ in their inflammatory properties, the natural killer activation and interferon-gamma secretion that follow lipoplex administration should provide a functional adjuvant for cancer gene therapies that benefit from immunostimulation.

Topics: Animals; Antineoplastic Agents; Ascitic Fluid; Cations; Cytokines; DNA; Fatty Acids, Monounsaturated; Female; Humans; Killer Cells, Natural; Lipids; Liposomes; Mice; Mice, Inbred C57BL; Peritoneal Lavage; Peritonitis; Quaternary Ammonium Compounds; Transfection; Tumor Cells, Cultured

The association structures formed by cationic liposomes and DNA-plasmids have been successfully employed as gene carriers in transfection assays. In the present study such complexes was studied by cryo-TEM (cryo-transmission electron microscopy). Cationic liposomes made up by DOPE (dioleoylphosphatidylethanolamine) and various amounts of three different cationic surfactants were investigated. The cryo-TEM analysis suggests that an excess of lipid in terms of charge, leads to entrapment of the DNA molecules between the lamellas in clusters of aggregated multilamellar structures. With increasing amounts of DNA free or loosely bound plasmids were found in the vicinity of the complexes. The importance of the choice of surfactant, as reported from many transfection assays, was not reflected in changes of the type of DNA-vesicle association. A tendency towards polymorphism of the lipid mixtures is reported and its possible implications are discussed.

Topics: Cations; Cetrimonium; Cetrimonium Compounds; DNA; Electrochemistry; Fatty Acids, Monounsaturated; Lipid Bilayers; Liposomes; Microscopy, Electron; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Surface-Active Agents; Transfection

Antisense oligonucleotides (ODNs) are promising novel therapeutic agents against viral infections and cancer. However, problems with their inefficient delivery and inadequate stability have to be solved before they can be used in therapy. To circumvent these obstacles, a wide variety of improvements, including phosphorothioate ODNs and liposomes as a carrier system, have been developed. This study was designed to compare the effects of two cationic liposomes on the intracellular delivery and stability of ODNs in CaSki cell cultures. Also the stability of 3'-end phosphorothioate ODNs were investigated. The 3'-modification neither had any effect on the delivery, nor protected the ODNs against degradation. The cellular delivery and stability of ODNs was improved with both cationic liposomes, but a cationic liposomal preparations containing dimethyldioctadecylammonium bromide and dioleoylphosphatidylethanolamine (DDAB/DOPE) was more efficient than commercially available N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniummethylsulf ate (DOTAP). The improved cellular delivery was largely due to the stabilization of ODNs by cationic liposomes. The improved stability in the culture medium indicates that the cationic liposomes per se protect the ODNs from enzymatic degradation. Indeed, intact ODNs were found in the cytoplasm and nucleus only when delivered by cationic liposomes.

Topics: Base Sequence; Cations; Culture Media; Drug Carriers; Drug Stability; Fatty Acids, Monounsaturated; Humans; Liposomes; Molecular Sequence Data; Oligonucleotides, Antisense; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Tumor Cells, Cultured