Ultrabright Green-Emitting Nanoemulsions Based on Natural Lipids-BODIPY Conjugates

Nanoemulsions (NEs) are water-dispersed oil droplets that constitute stealth biocompatible nanomaterials. NEs can reach an impressive degree of fluorescent brightness owing to their oily core that can encapsulate a large number of fluorophores on the condition the latter are sufficiently hydrophobic and oil-soluble. BODIPYs are among the brightest green emitting fluorophores and as neutral molecules possess high lipophilicity. Herein, we synthesized three different natural lipid-BODIPY conjugates by esterification of an acidic BODIPY by natural lipids, namely: α-tocopherol (vitamin E), cholesterol, and stearyl alcohol. The new BODIPY conjugates were characterized in solvents and oils before being encapsulated in NEs at various concentrations. The physical (size, stability over time, leakage) and photophysical properties (absorption and emission wavelength, brightness, photostability) are reported and showed that the nature of the lipid anchor and the nature of the oil used for emulsification greatly influence the properties of the bright NEs.

Phosphoglycerol-type wall- and lipoteichoic acids are enantiomeric polymers differentially cleaved by the stereospecific glycerophosphodiesterase GlpQ

ABSTRACTThe cell envelope of Gram-positive bacteria generally comprises two types of polyanionic polymers, either linked to peptidoglycan, wall teichoic acids (WTA), or to membrane glycolipids, lipoteichoic acids (LTA). In some bacteria, including Bacillus subtilis strain 168, WTA and LTA both are glycerolphosphate polymers, yet are synthesized by different pathways and have distinct, although not entirely understood morphogenetic functions during cell elongation and division. We show here that the exo-lytic sn-glycerol-3-phosphodiesterase GlpQ can discriminate between B. subtilis WTA and LTA polymers. GlpQ completely degrades WTA, lacking modifications at the glycerol residues, by sequentially removing glycerolphosphates from the free end of the polymer up to the peptidoglycan linker. In contrast, GlpQ is unable to cleave unmodified LTA. LTA can only be hydrolyzed by GlpQ when the polymer is partially pre-cleaved, thereby allowing GlpQ to get access to the end of the polymer that is usually protected by a connection to the lipid anchor. This indicates that WTA and LTA are enantiomeric polymers: WTA is made of sn-glycerol-3-phosphate and LTA is made of sn-glycerol-1-phosphate. Differences in stereochemistry between WTA and LTA were assumed based on differences in biosynthesis precursors and chemical degradation products, but so far had not been demonstrated directly by differential, enantioselective cleavage of isolated polymers. The discriminative stereochemistry impacts the dissimilar physiological and immunogenic properties of WTA and LTA and enables independent degradation of the polymers, while appearing in the same location; e.g. under phosphate limitation, B. subtilis 168 specifically hydrolyzes WTA and synthesizes phosphate-free teichuronic acids in exchange.

Lipid-anchor display on peptoid nanosheets via co-assembly for multivalent pathogen recognition

A facile route to a diversity of functionalized two-dimensional bionanomaterials was developed based on the aqueous co-assembly of lipidated small molecules and nanosheet-forming peptoids.

Soluble versions of outer membrane cytochromes function as exporters for heterologously produced cargo proteins

This study reveals that it is possible to secrete truncated versions of outer membrane cytochromes into the culture supernatant and that these proteins can provide a basis for the export of heterologously produced proteins. Different soluble and truncated versions of the outer membrane cytochrome MtrF were analyzed for their suitability to be secreted. A protein version with a very short truncation of the N-terminus to remove the recognition sequence for the addition of a lipid anchor is secreted efficiently to the culture supernatant, and moreover this protein could be further truncated by a deletion of 160 amino acid and still is detectable in the supernatant. By coupling a cellulase to this soluble outer membrane cytochrome, the export efficiency was measured by means of relative cellulase activity. We conclude that outer membrane cytochromes of S. oneidensis can be applied as transporters for the export of target proteins into the medium using the type II secretion pathway.

A microfluidic approach for sequential assembly of siRNA polyplexes with a defined structure-activity relationship

Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid’s transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed “oligomers”) with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure-function relationships. We chose nanoparticle shielding polyethylene glycol (PEG) and cell targeting folic acid (termed “PEG-ligands”) as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azide—alkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core—lipid anchor—PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEG-ligands (relative to ncore oligomer) showed optimal siRNA mediated gene knock-down when containing defined PEG domains of in sum 24 and 36 ethylene oxide repetitions, 12 EOs each from the lipid anchor and 12 or 24 EOs from the PEG-ligand, respectively. These results confirm that transfection efficiency depends on the linker length and stoichiometry and are consistent with previous findings using core—PEG-ligand polyplexes formed by click modification of azide-containing core polyplexes with aforementioned PEG-ligands. Hence, successive microfluidic assembly might be a potentially powerful route to create defined multi-component polyplexes with reduced batch-to-batch variability.

A microfluidic approach for sequential assembly of siRNA polyplexes with defined structure – activity relationship

Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid’s transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed ‘oligomers’) with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure - function relationships. We chose nanoparticle shielding PEG and cell targeting folic acid (termed ‘PEG-ligands’) as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azide – alkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core - lipid anchor - PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEG-ligands (relative to ncore oligomer) showed optimal siRNA mediated gene knock-down when containing defined polyethylene glycol (PEG) domains of in sum 24 and 36 ethylene oxide (EO) repetitions. These results confirm that transfection efficiency depends on the linker length and stoichiometry and are consistent with previous findings using core - PEG-ligand polyplexes formed by click modification of azide-containing core polyplexes with DBCO-PEG-ligand. Hence successive microfluidic assembly might be a potentially powerful route to create defined multi-component polyplexes with reduced batch-to-batch variability.

A microfluidic approach for sequential assembly of siRNA polyplexes with defined structure – activity relationship

Therapeutic nucleic acids provide versatile treatment options for hereditary or acquired diseases. Ionic complexes with basic polymers are frequently used to facilitate nucleic acid’s transport to intracellular target sites. Usually, these polyplexes are prepared manually by mixing two components: polyanionic nucleic acids and polycations. However, parameters such as internal structure, size, polydispersity and surface charge of the complexes sensitively affect pharmaceutical efficiency. Hence a controlled assembly is of paramount importance in order to ensure high product quality. In the current study, we present a microfluidic platform for controlled, sequential formulation of polyplexes. We use oligo-amidoamines (termed ‘oligomers’) with precise molecular weight and defined structure due to their solid phase supported synthesis. The assembly of the polyplexes was performed in a microfluidic chip in two steps employing a design of two successive Y junctions: first, siRNA and core oligomers were assembled into core polyplexes. These core oligomers possess compacting, stabilizing, and endosomal escape mediating motifs. Second, new functional motifs were mixed to the core particles and integrated into the core polyplex. The iterative assembly formed multi-component polyplexes in a highly controlled manner and enabled us to investigate structure - function relationships. We chose nanoparticle shielding PEG and cell targeting folic acid (termed ‘PEG-ligands’) as functional components. The PEG-ligands were coupled to lipid anchor oligomers via strain promoted azide – alkyne click chemistry. The lipid anchors feature four cholanic acids for inserting various PEG-ligands into the core polyplex by non-covalent hydrophobic interactions. These core - lipid anchor - PEG-ligand polyplexes containing folate as cell binding ligand were used to determine the optimal PEG-ligand length for transfecting folate receptor-expressing KB cells in vitro. We found that polyplexes with 20 mol % PEG-ligands (relative to ncore oligomer) showed optimal siRNA mediated gene knock-down when containing defined polyethylene glycol (PEG) domains of in sum 24 and 36 ethylene oxide (EO) repetitions. These results confirm that transfection efficiency depends on the linker length and stoichiometry and are consistent with previous findings using core - PEG-ligand polyplexes formed by click modification of azide-containing core polyplexes with DBCO-PEG-ligand. Hence successive microfluidic assembly might be a potentially powerful route to create defined multi-component polyplexes with reduced batch-to-batch variability.