Anno Wagenaar

Synthesis of Pyridinium Amphiphiles Used for Transfection and Some Characteristics of Amphiphile/DNA Complex Formation

Pyridinium amphiphiles have found practical use for the delivery of DNA into cells. Starting from 4-methylpyridine, a general synthesis has been devised for the production of pyridinium amphiphiles which allows variation in both the hydrophobic part and in the headgroup area of the compounds. By means of differential scanning microcalorimetry, zeta potential, particle size measurements and cryo electron microscopy, some characteristics of the pyridinium amphiphile/ DNA complexes have been determined.

Transfection mediated by pH-sensitive sugar-based gemini surfactants; potential for in vivo gene therapy applications

In this study, the in vitro and in vivo transfection capacity of novel pH-sensitive sugar-based gemini surfactants was investigated. In an aqueous environment at physiological pH, these compounds form bilayer vesicles, but they undergo a lamellar-to-micellar phase transition in the endosomal pH range as a consequence of an increased protonation state. In the same way, lipoplexes made with these amphiphiles exhibit a lamellar morphology at physiological pH and a non-lamellar phase at acidic pH. In this study, we confirm that the gemini surfactants are able to form complexes with plasmid DNA at physiological pH and are able to transfect efficiently CHO cells in vitro. Out of the five compounds tested here, two of these amphiphiles, GS1 and GS2, led to 70% of transfected cells with a good cell survival. These two compounds were tested further for in vivo applications. Because of their lamellar organisation, these lipoplexes exhibited a good colloidal stability in salt and in serum at physiological pH compatible with a prolonged stability in vivo. Indeed, when injected intravenously to mice, these stable lipoplexes apparently did not substantially accumulate, as inferred from the observation that transfection of the lungs was not detectable, as examined by in vivo bioluminescence. This potential of avoiding ‘preliminary capture’ in the lungs may, thus, be further exploited in developing devices for specific targeting of gemini lipoplexes.

Synthesis and characteristics of biodegradable pyridinium amphiphiles used for in vitro DNA delivery

Pyridinium amphiphiles have found practical application for the delivery of DNA into eukaryotic cells. A general synthetic method starting from (iso)nicotinoyl chloride has been devised for the preparation of pyridinium amphiphiles based on (bio)degradable esters, allowing structural variation both in the hydrophobic part and in the headgroup area. By means of differential scanning calorimetry, transmission electron microscopy and UV measurements, some characteristics, including hydrolytic behaviour, have been determined. In vitro transfection ability and toxicity have been determined using the eukaryotic COS-7 cell line.

Relationship between molecular structure and supramolecular morphology of DODA-EO2-biotin and related lipids

We have recently reported that a biotinylated lipid molecule, called DODA-EO2-biotin, forms tubular lipid structures upon hydration, which act as a matrix for the formation of ordered helical arrays of streptavidin as well as for secondary macromolecular recognition reactions involving biotinylated structures (Ringler et al., 1997). In the present study, the supramolecular structures formed by the compounds obtained during the synthesis of DODA-EO2biotin and of compounds structurally related to DODA-EO2-biotin were investigated by transmission electron microscopy, with the objective being to understand the relationship between molecular structure and supramolecular morphology. From the eight lipid molecules investigated, only DODA-EO2-biotin formed tubular structures. Several structural parameters were identified as playing a role in the formation of DODA-EO2-biotin tubes, such as the chirality of the biotin moiety, the saturated nature of the lipid chains, the presence of amide bonds and the correct length and structure of the hydrophilic spacer. In addition, helical crystals of streptavidin were only obtained upon binding of streptavidin to the supramolecular assemblies formed by DODA-EO2-biotin.

Gel to liquid crystal transitions for vesicles in aqueous solutions prepared using mixtures of sodium dialkylphosphates (R1O)(R2O)PO2–Na+ and (R3O)2PO2–Na+, where R1= C10H21, R2= C14H29 or C18H37 and R3= C12H25, C14H29, C16H33 or C18H37

The scans recorded by differential scanning microcalorimetry (DSC) for aqueous solutions containing vesicles prepared from mixtures of two sodium dialkylphosphates are complicated where the first surfactant anion (R1O)(R2O)PO2–Na+ has alkyl groups R1 and R2 with different chain lengths, and where the second surfactant anion (R3O)2PO2– has two alkyl groups R3 with the same chain length. For mixed solutions where R1= C10H21, R2= C14H29 or C18H37 and R3= C12H25, C14H29, C16H33 or C18H37, the DSC traces can be understood in geometric terms. Where the chains can be assembled into bilayers with modest mismatch, the DSC traces show well resolved features. With increase in mismatch, the complexities of the DSC traces are consistent with the presence of bilayers in which the domains differ in composition. Poor chain packing and consequent weak intravesicular van der Waals forces between the alkyl chains favour low temperatures for gel to liquid crystal transitions.

Phase transitions in the bilayers of vesicles formed from binary mixtures of symmetric di-n-alkylphosphates in aqueous solutions

Vesicles in aqueous solutions were prepared from binary equimolar mixtures of di-n-alkyl-phosphates (sodium and potassium), (R1O)2PO2–M+ and (R2O)2PO2–M+. When the number of carbon atoms in R1 and R2 differs by two and when R1 or R2= C12H25, C14H29, C16H33 and C18H37 the membranes undergo well defined gel to liquid crystal transitions at characteristic temperatures Tm. The recorded Tms are intermediate between the melting temperatures for vesicles prepared from the respective single di-n-alkylphosphates. Furthermore, the extrema recorded by differential scanning microcalorimetry show that the vesicle membrane is made up of domains that differ in composition. For those vesicles produced from di-n-alkylphosphates where the number of carbon atoms in R1 and R2 differs by more than two the plots recorded by the scanning microcalorimeter are complex. The scans show many extrema, suggesting that the bilayers are formed from many domains having different compositions. In all cases, the scan patterns are essentially repeated through several heat–cool–heat…cycles. The temperatures Tm are increased relative to those of the component surfactants when K+ and Na+ salts are mixed, showing that the counter cations play an important role in determining the thermotropic properties of the vesicles reflecting the importance of electrical interactions in determining the packing within the bilayers.

INTRAMOLECULAR SULFONAMIDE-CARBOXAMIDE REARRANGEMENT

Abstract Several o-carboxy-N,N-dialkylbenzenesulfonamides rearrange intramolecularly to o-chlorosulfonyl-N,N-dialkylbenzamides upon treatment with an excess of thionyl chloride in an apolar solvent.

Efficient intramolecular nucleophilic catalysis in the base-catalyzed hydrolysis of o-(1-hydroxyalkyl)-N,N-dimethylbenzenesulfonamides☆

The title reaction, which proceeds via rate-determining formation of a sultone intermediate, is speeded enormously as a result of the gem-dialkyl effect of o-substituents -C(R1)2OH with R1 = Me, Et and i-Pr.

Orthanilic acid from the reaction of o-nitrobenzenesulfinic acid with sodium iodide

Die o-Nitro-sulfinsaure (I) liefert mit Natriumjodid/Athanol ein Gemisch aus der 0-Amino-sulfonsaure (II),dem Jod-nitrobenzol (III) sowie dem Disulfid (IV).