Phosphonodithioester–Amine Coupling as a Key Reaction Step for the Design of Cationic Amphiphiles Used for Gene Delivery

A convergent synthesis of cationic amphiphilic compounds is reported here with the use of the phosphonodithioester–amine coupling (PAC) reaction. This versatile reaction occurs at room temperature without any catalyst, allowing binding of the lipid moiety to a polar head group. This strategy is illustrated with the use of two lipid units featuring either two oleyl chains or two-branched saturated lipid chains. The final cationic amphiphiles were evaluated as carriers for plasmid DNA delivery in four cell lines (A549, Calu3, CFBE and 16HBE) and were compared to standards (BSV36 and KLN47). These new amphiphilic derivatives, which were formulated with DOPE or DOPE-cholesterol as helper lipids, feature high transfection efficacies when associated with DOPE. The highest transfection efficacies were observed in the four cell lines at low charge ratios (CR = 0.7, 1 or 2). At these CRs, no toxic effects were detected. Altogether, this new synthesis scheme using the PAC reaction opens up new possibilities for investigating the effects of lipid or polar head groups on transfection efficacies.

Transgene expression in mice of the Opa1 mitochondrial transmembrane protein through bicontinuous cubic lipoplexes containing gemini imidazolium surfactants

Background Lipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, also known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. The polar head can carry a large variety of chemical groups including amines as well as guanidino or imidazole groups. In particular, gemini cationic lipids consist of two positive polar heads linked by a spacer with different length. As for the hydrophobic aliphatic chains, they can be unsaturated or saturated and are connected to the polar head-groups. Many other chemical components can be included in the formulation of lipoplexes to improve their transfection efficiency, which often relies on their structural features. Varying these components can drastically change the arrangement of DNA molecules within the lamellar, hexagonal or cubic phases that are provided by the lipid matrix. Lipofection is widely used to deliver genetic material in cell culture experiments but the simpler formulations exhibit major drawbacks related to low transfection, low specificity, low circulation half-life and toxicity when scaled up to in vivo experiments. Results So far, we have explored in cell cultures the transfection ability of lipoplexes based on gemini cationic lipids that consist of two C16 alkyl chains and two imidazolium polar head-groups linked with a polyoxyethylene spacer, (C16Im)2(C4O). Here, PEGylated lipids have been introduced to the lipoplex formulation and the transgene expression of the Opa1 mitochondrial transmembrane protein in mice was assessed. The addition of PEG on the surface of the lipid mixed resulted in the formation of Ia3d bicontinuous cubic phases as determined by small angle X-ray scattering. After a single intramuscular administration, the cubic lipoplexes were accumulated in tissues with tight endothelial barriers such as brain, heart, and lungs for at least 48 h. The transgene expression of Opa1 in those organs was identified by western blotting or RNA expression analysis through quantitative polymerase chain reaction. Conclusions The expression reported here is sufficient in magnitude, duration and toxicity to consolidate the bicontinuous cubic structures formed by (C16Im)2(C4O)-based lipoplexes as valuable therapeutic agents in the field of gene delivery. Graphical Abstract

The exploration of Thermococcus barophilus lipidome reveals the widest variety of phosphoglycolipids in Thermococcales.

One of the most distinctive characteristics of Archaea is their unique lipids. While the general nature of archaeal lipids has been linked to their tolerance to extreme conditions, little is known about the diversity of lipidic structures Archaea are able to synthesize, which hinders the elucidation of the physicochemical properties of their cell membrane. In an effort to widen the known lipid repertoire of the piezophilic and hyperthermophilic model archaeon Thermococcus barophilus, we comprehensively characterized its intact polar lipid (IPL), core lipid (CL), and polar head group compositions using a combination of cutting-edge liquid chromatography and mass spectrometric ionization systems. We tentatively identified 82 different IPLs based on five distinct CLs and 10 polar head group derivatives of phosphatidylhexoses, including compounds reported here for the first time, e.g., di-N-acetylhexosamine phosphatidylhexose-bearing lipids. Despite having extended the knowledge on the lipidome, our results also indicate that the majority of T. barophilus lipids remain inaccessible to current analytical procedures and that improvements in lipid extraction and analysis are still required. This expanded yet incomplete lipidome nonetheless opens new avenues for understanding the physiology, physicochemical properties, and organization of the membrane in this archaeon as well as other Archaea.

Molecular basis of UHRF1 allosteric activation for synergistic histone modification binding by PI5P

Chromatin marks are recognized by distinct binding modules many of which are embedded in multidomain proteins or complexes. How the different protein functionalities of complex chromatin modulators are regulated is often unclear. Using a combination of biochemical, biophysical and structural approaches we delineated the regulation of the H3unmodified and H3K9me binding activities of the multidomain UHRF1 protein. The phosphoinositide PI5P interacts with two distant flexible linker regions of UHRF1 in a mode that is dependent on the polar head group and the acyl part of the phospholipid. The associated conformational rearrangements stably position the H3unmodified and H3K9me3 histone recognition modules of UHRF1 for multivalent and synergistic binding of the H3 tail. Our work highlights a novel molecular function for PI5P outside of the context of lipid mono- or bilayers and establishes a molecular paradigm for the allosteric regulation of complex, multidomain chromatin modulators by small cellular molecules.

Reactive Deep Eutectic Solvents (RDESs): A New Tool for Phospholipase D-Catalyzed Preparation of Phospholipids

The use of Reactive Deep Eutectic Solvents (RDESs) in the preparation of polar head modified phospholipids (PLs) with phospholipase D (PLD)-catalyzed biotransformations has been investigated. Natural phosphatidylcholine (PC) has been submitted to PLD-catalyzed transphosphatidylations using a new reaction medium composed by a mixture of RDES/buffer. Instead of exploiting deep eutectic solvents conventionally, just as the reaction media, these solvents have been designed here in order to contribute actively to the synthetic processes by participating as reagents. RDESs were prepared using choline chloride or trimethyl glycine as hydrogen-bond acceptors and glycerol or ethylene glycol, as hydrogen-bond donors as well as nucleophiles for choline substitution. Specifically designed RDES/buffer reaction media allowed the obtainment of PLs with optimized yields in the perspective of a sustainable process implementation.

Enterococcus faecalis Readily Adapts Membrane Phospholipid Composition to Environmental and Genetic Perturbation

The bacterial lipid membrane, consisting both of fatty acid (acyl) tails and polar head groups, responds to changing conditions through alteration of either the acyl tails and/or head groups. This plasticity is critical for cell survival as it allows maintenance of both the protective nature of the membrane as well as functioning membrane protein complexes. Bacteria that live in fatty-acid rich environments, such as those found in the human host, can exploit host fatty acids to synthesize their own membranes, in turn, altering their physiology. Enterococcus faecalis is such an organism: it is a commensal of the mammalian intestine where it is exposed to fatty-acid rich bile, as well as a major cause of hospital infections during which it is exposed to fatty acid containing-serum. Within, we employed an untargeted approach to detect the most common phospholipid species of E. faecalis OG1RF via ultra-high performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS). We examined not only how the composition responds upon exposure to host fatty acids but also how deletion of genes predicted to synthesize major polar head groups impact lipid composition. Regardless of genetic background and differing basal lipid composition, all strains were able to alter their lipid composition upon exposure to individual host fatty acids. Specific gene deletion strains, however, had altered survival to membrane damaging agents. Combined, the enterococcal lipidome is highly resilient in response to both genetic and environmental perturbation, likely contributing to stress survival.

Role of Intermolecular Forces on Surfactant-Steam Performance into Heavy Oil Reservoirs

Summary This study investigates the role of polar fractions of heavy oil in the surfactant-steamflooding process. Performance analyses of this process were done by examination of the dipole-dipole and ion-ion interactions between the polar head group of surfactants and the charged polar fraction of crude oil, namely, asphaltenes. Surfactants are designed to reduce the interfacial tension (IFT) between two immiscible fluids (such as oil and water) and effectively used for oil recovery. They reduce the IFT by aligning themselves at the interface of these two immiscible fluids; this way, their polar head group can stay in water and nonpolar tail can stay in the oil phase. However, in heavy oil, the crude oil itself has a high number of polar components (mainly asphaltenes). Moreover, the polar head group in surfactants is charged, and the asphaltene fraction of crude oils carries reservoir rock components with charges. The impact of these intermolecular forces on the surfactant-steam process performance was investigated with 10 coreflood experiments on an extraheavy crude oil. Nine surfactants (three anionic, three cationic, and three nonionic surfactants) were tested. Results of each coreflood test were analyzed through cumulative oil recovery and residual oil content. The performance differences were evaluated by polarity determination through dielectric constant measurements and by ionic charges through zeta potential measurements on asphaltene fractions of produced oil and residual oil samples. The differences in each group of surfactants tested in this study are the tail length. Results indicate that a longer hydrocarbon tail yielded higher cumulative oil recovery. Based on the charge groups present in the polar head of anionic surfactants resulted in higher oil recovery. Further examinations on asphaltenes from produced and residual oils show that the dielectric constants of asphaltenes originated from the produced oil, giving higher polarity for surfactant-steam experiments conducted with longer tail length, which provide information on the polarity of asphaltenes. The ion-ion interaction between produced oil asphaltenes and surfactant head groups were determined through zeta potential measurements. For the most successful surfactant-steam processes, these results showed that the changes on asphaltene surface charges were becoming lower with the increase in oil recovery, which indicates that once asphaltenes are interacting more with the polar head of surfactants, then the recovery rate increases. Our study shows that the surfactant-steamflooding performance in heavy oil reservoirs is controlled by the interaction between asphaltenes and the polar head group of surfactants. Accordingly, the main mechanism that controls the effectiveness of the process is the ion-ion interaction between the charges in asphaltene surfaces and the polar head group of crude oils. Because crude oils carry mostly negatively charged reservoir rock particles, our study suggests the use of anionic surfactants for the extraction of heavy oils.